Bioactive Peptides-Impact in Cancer Therapy

Therapeutic, Probiotic,
and Unconventional Foods
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Therapeutic, Probiotic,
and Unconventional Foods
Edited by
Alexandru Mihai Grumezescu
University Politehnica of Bucharest, Bucharest, Romania
Alina Maria Holban
University of Bucharest, Bucharest, Romania
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Contents
Contributors
Preface
xiii
xvii
1. Introduction in Nutraceutical and
Medicinal Foods
Lia-Mara Ditu, Madalina E. Grigore,
Petronela Camen-Comanescu,
Alina Maria Holban
1. Introduction
2. Nutraceuticals
2.1 Food Nutrients
2.2 Herbals
2.3 Dietary Supplements
3. Functional Foods
4. Medicinal Foods
5. Probiotics, Prebiotics and Synbiotics
5.1 Probiotics
5.2 Prebiotics
5.3 Synbiotics
6. Conclusions
Acknowledgments
References
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Section A
Probiotics and Prebiotics
2. Probiotics: Supplements, Food,
Pharmaceutical Industry
Swathi Putta, Nagendra S. Yarla, Dhananjaya B.
Lakkappa, Sarat B. Imandi, Rama Rao Malla,
Amajala K. Chaitanya, Brahma P.V. Chari, Silas Saka,
Rama Rao Vechalapu, Mohammad A. Kamal,
Vadim V. Tarasov, Vladimir N. Chubarev,
Korada Siva Kumar, Gjumrakch Aliev
1. Introduction
1.1 Prebiotics
1.2 Probiotics
1.3 Synbiotics
2. Types of Probiotics
2.1 Bacteria
2.2 Yeast and Molds
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3.
4.
5.
6.
Functions of Gut Microbiota
Mechanism of Action of Probiotics
Safety and Risk Assessment
Probiotic Therapy
6.1 Diabetes
6.2 Obesity
6.3 Liver Diseases
6.4 Cancer
6.5 Diarrhea
6.6 Allergies
7. Available Probiotic Food
7.1 Yakult
7.2 Kefir
7.3 Yogurt
7.4 Kombucha
7.5 Sauerkraut
References
Further Reading
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3. Selection of New Probiotics: The
Case of Streptomyces
Sneha Hariharan, Selvakumar Dharmaraj
1. Introduction
1.1 Aquaculture Status of the World
1.2 Implementation of Probiotics Usage
in Aquaculture
2. Probiotics
2.1 Definition
2.2 Mode of Action
2.3 Common Microorganisms Used as
Probiotics
2.4 Application of Probiotics in Aquaculture
3. Prospect of Using Marine Streptomyces
as Probiotics
3.1 Life Cycle of Marine Streptomyces
3.2 Taxonomical Classification of Marine
Streptomyces
3.3 Morphological Identification of
Marine Streptomyces
3.4 Applications of Marine Streptomyces
as Probiotics in Aquaculture
3.5 Selection of Efficient Strains of Marine
Streptomyces as Probiotics
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vi Contents
3.6 Possible Limitations in the Usage of
Marine Streptomyces as Probiotics
4. Conclusion
References
Further Reading
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54
4. Development of New Probiotic
Foods—A Case Study on Probiotic
Juices
Asit Ranjan Ghosh
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5. Prebiotics and Their Production
From Unconventional Raw
Materials (Mushrooms)
Hrudayanath Thatoi, Sameer K. Singdevsachan,
Jayanta K. Patra
1. Introduction
2. Nutritional Values of Mushrooms
3. Bioactive Components of Mushrooms
3.1 Low Molecular Weight Compounds of
Mushrooms
3.2 High Molecular Weight Compounds
of Mushrooms
3.3 Mushrooms as a Possible Source of
Prebiotics
4. Mushroom as Potential Source of
Pharmaceuticals
4.1 Antitumor and Immunomodulatory
Properties
4.2 Antioxidant Activity
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6. Probiotics in the Rescue of Gut
Inflammation
Veeranjaneya Reddy Lebaka, Young Jung Wee,
Venkatarami Reddy Narala, Vinod Kumar Joshi,
1. Introduction
2. Probiotic Microorganisms
3. Probiotic Products
3.1 Dairy Products
3.2 Nondairy Products
3.3 Why Fruits are Ideal Choice?
3.4 Preparation of Fruit Juice Probiotics
3.5 Types of Fruit Juice Probiotics
3.6 Yeast Probiotic Juices
4. Challenges
4.1 Survivability and Stability
4.2 Sensory Traits
5. Possible Remedies
5.1 Supplementation of Growth
Promoters and Protectants
5.2 Adaptation
5.3 Induction of Resistance
6. Future Perspectives
References
Further Reading
4.3 Hypoglycaemic/Antidiabetic Activity
4.4 Antimicrobial Activity
5. Conclusion
References
Further Reading
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1.
2.
3.
4.
5.
Introduction
Gut Microbiota, Probiotics, and Dysbiosis
Gut Immunity
Gut-Probiotics Interaction
Dysbiosis is the Cause of Inflammation
at Gut
6. Dysbiosis and Inflammatory Diseases
7. What Makes Probiotic Special for
Reducing Inflammation in the Gut?
8. How Do Probiotics Regulate
Inflammation?
9. Use of Probiotics and Consequences
9.1 Inflammatory Diseases
9.2 Suppression of Histamine Signaling
9.3 Reduction of Appetite and Glucose
Uptake
9.4 Repair of Damaged Epithelial Barrier
9.5 Antimicrobial Peptides and Antagonism
10. Conclusion
References
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7. Probiotics as an Adjunct to
Conventional Treatment in
Vulvovaginitis: Past, Present,
and Future
Princy L. Palatty, Poornima R. Bhat, Ramakrishna P.
Jekrabettu, Thomas George, Sueallen D’souza,
Soniya Abraham, Mohammed Adnan, Michael Pais,
Taresh Naik, Devika Gunasheela, Manjeshwar S.
Baliga
1. Introduction
2. Anatomy of Female Genital System
2.1 External Genitalia
2.2 Internal Genitalia
3. Normal Flora of the Vagina
4. Vulvovaginitis
5. Characteristic Features of Different
Types of Vulvovaginitis
5.1 Bacterial Vaginosis
5.2 Vulvovaginal Candidiasis
5.3 Trichomoniasis
5.4 Treatment of VV
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Contents vii
6. Antibacterial Drugs
7. Antifungal Drugs
7.1 Imidazoles and Triazoles
7.2 Imidazole and Triazoles for Topical Use
7.3 Drugs Used in Resistant Infections
7.4 Probiotics in Vulvovaginitis
7.5 Mechanism of Action of Probiotics
7.6 L. acidophilus
8. Clinical Studies With Probiotics in
Women’s Health
9. Adverse Effects
10. Conclusions
References
Further Reading
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Section B
Therapeutic Foods and Ingredients
Muhammad Kaleem, Asif Ahmad
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Leila Mehdizadeh, Mohammad Moghaddam
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9. Bioactive Peptides—Impact in
Cancer Therapy
Edwin E. Martínez Leo, Armando M. Martín Ortega,
Maira R. Segura Campos
1. Cancer: A Worldwide Inflammatory Disease
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10. Essential Oils: Biological Activity
and Therapeutic Potential
8. Flavonoids as Nutraceuticals
1. Introduction
2. Extraction of Flavonoids
2.1 Conventional Extraction Techniques
2.2 Modern Extraction Techniques
3. Absorption, Metabolism and
Bioavailability of Flavonoids
3.1 Bioavailability of Flavonoids
4. Toxicity of Flavonoids
5. Antioxidant Activity of Flavonoids
6. Flavonoids and Cardiovascular Diseases
6.1 Anti-Inflammatory Activities of
Flavonoids
6.2 Atherosclerosis
7. Antidiabetic Activity of Flavonoids
8. Hepato-Protective Effects of Flavonoids
9. Anticancer Activity of Flavonoids
10. Effect of Flavonoids on Osteoporosis
10.1 Mechanism of Action
10.2 Epidemological studies
11. Antibacterial Effect of Flavonoids
12. Antiviral Activity
13. Conclusion
References
Further Reading
2. Chronic Inflammation and Oxidative
Stress as Potential Triggers of Cancer
2.1 Inflammation and Metastasis
2.2 Inflammation and Tumor Cell Proliferation
3. Functional Food and Dietary Bioactive
Compounds
3.1 Functional Proteins and Bioactive
Peptides
3.2 Proteins and Bioactive Peptides
with Antiinflammatory and
Immunomodulatory Activity
3.3 Protein and Bioactive Peptides with
Anticancer Activity
3.4 Protein and Bioactive Peptides with
Antioxidant Activity
4. Conclusion
References
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1.
2.
3.
4.
Introduction
Applications of Essential Oils
Antimicrobial Activity of Essential Oils
In Vitro Methods for Quantifying
Antimicrobial Activity
4.1 Agar Absorption Assay
4.2 Disc Diffusion Assay
4.3 Well Diffusion Assay
4.4 Agar- and Broth-Dilution Methods
4.5 Vapor Phase Test
5. Percent Inhibition of Mycelia Growth
6. Determination of MIC, MBC, and MFC
7. Efficacy of Antibacterial Activity of
Essential Oils and Components
7.1 Plant Studies
7.2 Human Studies
8. Efficacy of Antifungal Activity of
Essential Oils and Their Components
8.1 Food and Human Studies
8.2 Plant Studies
9. MIC, MBC, or MFC Determination
10. Influenced Factors on the EOs
Antimicrobial Activities
11. Antimicrobial Activity of Essential Oil
Components
12. Mechanism of Action
13. Synergy Between Essential Oils or With
Other Compounds
14. Toxicity of Essential Oils
15. Conclusion
References
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viii Contents
11. Nutritional and Therapeutic
Potential of Spices
12. Novel Nutraceutical Compounds
Asma Afreen, Zaheer Ahmed, Nomana Anjum
Mian K. Sharif, Rebia Ejaz, Imran Pasha
1. Herbs and Spices
1.1 Preamble
1.2 Importance
1.3 Origin
2. Historical Perspective
3. Production and Trade: Global Scenario
3.1 Production
3.2 Spices Trade
4. Overview of Spices
4.1 Cloves
4.2 Black Pepper
4.3 Turmeric
4.4 Coriander
4.5 Cumin
4.6 Flaxseed
4.7 Cinnamon
4.8 Cardamom
4.9 Ginger
4.10 Garlic
5. Therapeutic Impact of Spices on
Human Health
5.1 Antiinflammatory Activity
5.2 Antiemetic Activity
5.3 Antitumor Activity
5.4 Antimicrobial Activity
5.5 Antihypertensive Activity
5.6 Antibilious Activity
5.7 Antispasmodic Activity
5.8 Anticonvulsive Activity
5.9 Antioxidant Activity
5.10 Chemopreventive Activity
6. Clinical Studies: Animal vs Humans
6.1 Animal Studies
6.2 Human Studies
7. Food Applications
7.1 Role of Food Industries
7.2 Value Added Products of Spices
7.3 Artificial vs Natural Colorants
8. Safety Issues in Spices and Their
Management
8.1 Mycotoxins
8.2 Bacterial Contamination
8.3 Pesticide Residues
8.4 Management of Safety in Spices
9. Quality and Safety Standards
9.1 Food Safety and Quality Assurance
9.2 Food Safety and Quality Assurance
Systems
10. Conclusion
References
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1. Introduction
2. Nutraceuticals and Functional Foods
3. Novel Nutraceutical Compounds
3.1 Carotenoids
3.2 Phytosterols
3.3 Polyphenols
3.4 Omega 3-Fatty Acids
4. Future Prospects
References
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13. Nutraceutical and Medicinal
Importance of Seabuckthorn
(Hippophae sp.)
Prakash C. Sharma, Meenu Kalkal
1. Introduction
1.1 Taxonomical Classification
1.2 Geographical Distribution
1.3 Plant Morphology
2. Harvesting
3. Postharvest Processing
3.1 Extraction
3.2 Analytical Techniques for Extract
Analysis
4. Biochemical Profiling of Seabuckthorn
4.1 Berries
4.2 Leaves
4.3 Bark
4.4 Seed and Seed Oil
5. Traditional Applications of Seabuckthorn
6. Nutraceuticals in Seabuckthorn
7. Pharmacological Effects of Seabuckthorn
7.1 Anticancer and Antitumor Activity
7.2 Antiinflammatory Activity
7.3 Antimicrobial Activity
7.4 Hepatoprotective Ability
7.5 Radioprotective Effect
7.6 Antiatherogenic and
Cardioprotective Activity
7.7 Antiulcerogenic Effect
7.8 Effect on Platelet Aggregation
7.9 Hypoglycemic Effect
7.10 Antiaging Potential and Skin Whitening
7.11 Eye diseases
7.12 Immunomodulatory
7.13 Neuroprotective Ability
7.14 Antiobesity
7.15 Antioxidant and Cytoprotective Ability
7.16 Healing Property
7.17 Antistress and Adaptogenic Ability
8. Commercial Products of Seabuckthorn
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Contents ix
9. Conclusion
Acknowledgment
References
Further Reading
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14. Therapeutic Potential of Flaxseed
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15. Pharmaceutical, Nutraceutical and
Therapeutic Properties of Selected
Wild Medicinal Plants: Thyme,
Spearmint, and Rosemary
Muhammad H. Alu’datt, Taha Rababah, Mohammad
N. Alhamad, Sana Gammoh, Majdi A. Al-Mahasneh,
Carole C. Tranchant, Mervat Rawshdeh
1. Introduction
2. Occurrence of Phenolic Compounds in
Medicinal Plants
3. Properties of Extracted Phenolic
Compounds from Medicinal Plants
3.1 Antioxidant Properties of Medicinal
Plants
3.2 Biological Properties of Medicinal
Plants
4. Wild Medicinal Plants
4.1 Bioactive Components of Thyme
4.2 Bioactive Components of Rosemary
4.3 Bioactive Components of Spearmint
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16. Virgin Coconut Oil as Functional Oil
Yashi Srivastava, Anil D. Semwal, Gopal K. Sharma
Ankit Goyal, Ami Patel, Manvesh K. Sihag,
Nihir Shah, Beenu Tanwar
1. Introduction
2. Nutraceutical Components of Flaxseed
2.1 Flaxseed Oil
2.2 Flax Proteins
2.3 Flax Dietary Fibers
2.4 Flax Lignans
3. Health Promoting Effects of
Nutraceutical Components of Flaxseed
3.1 Flaxseed in Cardiovascular Diseases
3.2 Flaxseed in Rheumatoid Arthritis and
Inflammation
3.3 Flaxseed in Hypercholesterolaemia
3.4 Flaxseed in Diabetes
3.5 Flaxseed in Obesity
3.6 Flaxseed in Hypertension
3.7 Flaxseed in Tumor and Cancer Treatment
3.8 Flaxseed in Kidney Diseases
4. Estimated Intakes of Whole and Milled
Flaxseed, ALA and Fibers
5. Flaxseeds for a New Millennium
6. Conclusions
References
Further Reading
5. Conclusions
References
Further Reading
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1. Introduction
2. Standards
3. Methods for the Extraction of VCO and
Preparation of VCM
3.1 Wet Extraction
3.2 Fermentation
3.3 Enzymatic
3.4 Low Temperature/Centrifuge/Super
Critical Carbon Dioxide Extraction
Technique
3.5 Extraction of VCO (Process Optimized
by Central Plantation Crop Research
Institute, Kasargod, Kerela, India)
3.6 Preparation of Virgin Coconut Meal (VCM)
4. Physicochemical Properties and
Therapeutic Use of VCO
5. Value Added Products, Economy, Status,
and Government Policies
6. Other Commercial Utilization of VCO
7. Future Perspective of VCO
8. Conclusion
Acknowledgment
References
Further Reading
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17. Health Effects of Various Dietary
Agents and Phytochemicals
(Therapy of Acute Pancreatitis)
Elroy Saldanha, Ramakrishna J. Pai, Thomas George,
Sueallen D’Souza, Mohammed Adnan,
Michael Pais, Taresh Naik, Reshmina C.C. D’Souza,
Rithesh D’Cunha, Manjeshwar Shrinath Baliga
1. Introduction
2. Clinical Features of AP
2.1 Current Treatment Modality for AP
2.2 Antioxidants and Free Radical
Scavengers in the Prevention of AP
3. Dietary Agents and Phytochemicals in
the Prevention of AP
4. Green Tea
4.1 Emblica officinalis
4.2 Rheum rhabarbarum
5. Grapefruit
6. Curcumin
7. Ellagic Acid
8. Cinnamtannin B-1
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x Contents
9. Capsaicin
10. Alpha-pinene
11. Piperine
12. Zerumbone
13. Lycopene
14. Quercetin
15. Genistein
16. Apigenin
17. Resveratrol
18. Conclusions and Future Directions
References
Further Reading
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Section C
Unconventional Foods and Food
Ingredients
18. Production of Bioactive
Compounds From Waste
Christianah O. Jayeola, Babasola A. Adebowale,
Lateef E. Yahaya, Semiu O. Ogunwolu,
Olayiwola Olubamiwa
1. Introduction
1.1 Types of Agricultural Waste
1.2 Health Implications of Unused Waste
1.3 Way Forward for Converting Waste
to Wealth
1.4 Benefit of Utilizing Waste
1.5 The Case of Agro-Industrial Wastes
in Nigeria
1.6 Bioconversion
2. Cocoa Wastes
2.1 Wealth Out of Cocoa Waste
2.2 Cocoa Sweating/Pulp/Mucilage
2.3 Cocoa Pod Husk
2.4 Cocoa Bean Shell for Animal Diet
3. Cashew Waste
3.1 Value Addition for Cashew Waste
3.2 Cashew Apple
3.3 Cashew Nut
3.4 Cashew Testa
3.5 Cashew Tree Bark Gum
3.6 Cashew Nut Shell Liquid
4. Coffee Waste and Its By-Products
4.1 Coffee Waste
4.2 Coffee Husk/Hull
4.3 Utilization of Coffee Mucilage
4.4 Parchment Charcoal
4.5 Coffee Waste Water
5. Utilization of Waste Kolanut
5.1 Kolanut Waste
5.2 Kola Pod Husk
5.3 Kola Pod Husk in Animal Diets
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5.4 Kola Pod Husk in Fertilizer
5.5 Kola Testa
5.6 Waste Nuts From Defective
Nuts/Weevil Nut
5.7 Kola Nut in Beverages and
Pharmaceutical Industry
6. Tea (Camellia sinensis) Wastes
6.1 Wastes From Tea
6.2 Spent Tea Leaf as Animal Feed
7. Cotton Seed
7.1 Cotton Seed Waste
7.2 Cotton Seed Meal
8. Cassava Byproducts (Peel and Leaf)
8.1 Cassava Paste
9. Water Hyacinth (Eichrornniagrassipes)
9.1 Water Hyacinth
(Eichrornniagrassipes) Wastes
10. Hydrolysed Feather
10.1 Hydrolysed Feather Wastes
11. Plantain Peels
11.1 Plantain Peels Wastes
12. Conclusion
References
Further Reading
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19. Unripe Plantain Flours
Ijeoma A. Olawuni, Florence O. Uruakpa,
Abimbola Uzoma
1. Introduction
2. ACTUAL Problem
3. Justification, Objectives, and Scope of
This Study
4. Agronomy
5. Economic and Social Impact
6. Primary Products
6.1 Boiled Plantain
6.2 Plantain Pastry
6.3 Roasted Plantain
6.4 Fried Plantain
6.5 Plantain Fritters
6.6 Plantain Chips
7. Secondary and Derived Products
8. Production of Plantain Flour
9. Nutritional Content of Plantain
10. Requirement for Export and Quality
Assurance
11. Storage of Plantain
12. Selected Experimental Study
12.1 Production of Unripe Plantain Flours
13. Characterization of Plantain Flour
13.1 Bulk Density
13.2 Swelling Index
13.3 Water and Oil Absorption
Capacities
13.4 Wettability
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Contents xi
13.5 pH (Hydrogen Ion Concentration)
13.6 Gelling Point
13.7 Moisture Content
13.8 Total Starch
13.9 Thermal Analysis
13.10 Drying Characteristics
13.11 Moisture Content
13.12 Moisture Ratio
13.13 Mass Shrinkage Ratio
14. Microbiological Analyses
14.1 Sample Preparation
14.2 Yeast and Molds
14.3 Total Viable Counts
14.4 Salmonellae
14.5 Staphylococci
14.6 Characterization and
Identification of Microbial Isolates
15. Selected Experimental Study Results
15.1 Influence of Maturity and
Pretreatment on the Functional
Characteristics of Unripe Plantain
Flours
16. Effect of the Maturity Time and Drying
Method on the Thermal Properties of
Plant Starch
17. Impact of Drying and Maturity Time
on the Functional Properties of
Plantain Flour
17.1 Oil Absorption
17.2 Swelling Index
17.3 Wettability
17.4 Bulk Density
17.5 Gelling Point
17.6 Water Absorption
17.7 pH (Hydrogen Ion Concentration)
17.8 Mean Hourly Variation of the
Temperatures and Relative Humidity
of a Three-Chamber Solar Dryer
18. Conclusion
19. Recommendation
References
Further Reading
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21. Vegan Nutrition: Latest Boom in
Health and Exercise
Katharina C. Wirnitzer
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1. Introduction
1.1 Life Expectancy
1.2 Longevity
1.3 Vegetarian Trend
1.4 Vegetarian and Vegan Diets: What Do
Veggies Eat?
1.5 Veggies: Who Are They and How Do
They Perform?
2. A Historical Review of Protein: A Myth
That Is Still Kept Alive
2.1 The Myth About Meat
2.2 Protein: The Most Misunderstood and
Misinterpreted Nutrient
2.3 Plant Protein: Inferior Quality?
2.4 Animal Protein: Detrimental to Health
and Sports due to Residues
2.5 Plant Protein: Beneficial to Human
Health
3. Carbohydrate as the Main Fuel
4. Pioneering Work
4.1 Wholesome Nutrition
4.2 Galina Schatalova
5. Lifestyle-Related Impact on Health
5.1 Dietary-Related Impacts on Health
5.2 Sports-Related Impact on Health
6. Vegetarian Diets in Sports and Exercise
6.1 Early Studies
6.2 Some Position Statements (Brief Outline)
6.3 Current Studies About Exercise
Performance at Plant-Based Diets
7. The “Plant-Strong” Athlete: Permanent
Linkage of Vegan Diet to Sports and Exercise
7.1 Vegan Top-Level Athletes
7.2 Advantages to Athletes Resulting From
Predominantly Plant-Based Diets
7.3 Key Nutrients of Vegan Diets
7.4 How to Fuel the Vegan Athlete:
“Functional Food” Allowed
8. Conclusion
Acknowledgments
References
Further Reading
374
Index
455
351
351
351
356
356
356
357
358
358
358
360
360
361
363
363
363
366
20. Dry Beans: Processing and
Nutritional Effects
Rocio Campos-Vega, Priscila Zaczuk Bassinello,
Raquel de Andrade Cardoso Santiago,
B. Dave Oomah
1. Introduction
2. Processing
2.1 Dry Processing
2.2 Food Preparation/Wet Processing
2.3 Germination and Other Processes
Including Fermentation
3. Health Benefits
3.1 Non-Communicable Diseases
3.2 Antioxidant Capacity
4. Concluding Remarks and Future Trends
References
Further Reading
392
393
396
396
397
399
400
401
402
406
406
407
409
410
417
419
420
422
423
428
428
428
431
432
437
437
438
452
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Contributors
Brahma P.V. Chari
Visakhapatnam, India
Numbers in parentheses indicate the pages on which the authors’
contributions begin.
(15),
GITAM
University,
Soniya Abraham (117), Father Muller Medical College,
Mangalore, India
Vladimir N. Chubarev (15), I.M. Sechenov First Moscow
State Medical University, Moscow, Russia
Babasola A. Adebowale (317), Cocoa Research Institute of
Nigeria (CRIN), Ibadan, Nigeria
Rithesh D’Cunha (303), Father Muller Medical College,
Mangalore, India
Mohammed Adnan (117,303), Father Muller Medical
College; Father Muller Research Centre, Mangalore, India
Sueallen D’Souza (117,303), Father Muller Medical College;
Father Muller Research Centre, Mangalore, India
Asma Afreen (201), Allama Iqbal Open University,
Islamabad, Pakistan
Reshmina C.C. D’Souza (303), Father Muller Medical
College, Mangalore, India
Asif Ahmad (137), PMAS-Arid Agriculture University,
Rawalpindi, Pakistan
B. Dave Oomah (367), Pacific Agri-Food Research Centre,
Agriculture and Agri-Food Canada, Summerland, BC,
Canada
Zaheer Ahmed (201), Allama Iqbal Open University,
Islamabad, Pakistan
Mohammad N. Alhamad (275), Jordan University of
Science and Technology, Irbid, Jordan
Gjumrakch Aliev (15), “GALLY” International Biomedical
Research Consulting LLC, San Antonio, TX; University
of Atlanta, Johns Creek, GA, USA; Russian Academy of
Sciences, Chernogolovka, Russia
Majdi A. Al-Mahasneh (275), Jordan University of Science
and Technology, Irbid, Jordan
Muhammad H. Alu’datt (275), Jordan University of
Science and Technology, Irbid, Jordan
Nomana Anjum (201), Allama Iqbal Open University,
Islamabad, Pakistan
Manjeshwar S. Baliga (117), Father Muller Medical
College, Mangalore, India
Priscila Zaczuk Bassinello (367), Embrapa Rice & Beans,
Santo Antônio de Goiás, Brazil
Raquel de Andrade Cardoso Santiago (367), Universidade
Federal de Goiás, Goiânia, Brazil
Selvakumar Dharmaraj (27), Karpagam Academy of
Higher Education, Coimbatore, India
Lia-Mara Ditu (1), Research Institute of the University of
Bucharest, Bucharest, Romania
Rebia Ejaz (181), University of Agriculture, Faisalabad,
Pakistan
Sana Gammoh (275), Jordan University of Science and
Technology, Irbid, Jordan
Thomas George (117,303), Father Muller Medical College;
Father Muller Research Centre, Mangalore, India
Asit Ranjan Ghosh (101), VIT University, Vellore, India
Ankit Goyal (255), Mansinhbhai Institute of Dairy & Food
Technology (MIDFT), Mehsana, Gujarat, India
Poornima R. Bhat (117), Father Muller Medical College,
Mangalore, India
Madalina E. Grigore (1), University Politehnica of
Bucharest; National Research & Development Institute
for Chemistry and Petrochemistry—ICECHIM,
Bucharest, Romania
Petronela Camen-Comanescu (1),
Bucharest, Bucharest, Romania
of
Devika Gunasheela (117), Gunasheela Infertility Hospital,
Bangalore, India
Rocio Campos-Vega (367), Autonomous University of
Queretaro, Querétaro, Mexico
Sneha Hariharan (27), University of Madras, Chennai,
India
Amajala K. Chaitanya
Visakhapatnam, India
Alina Maria Holban (1), Research Institute of the University
of Bucharest, Bucharest, Romania
(15),
University
GITAM
University,
xiii
xiv Contributors
Sarat B. Imandi (15), Krishna University, Machilipatnam,
India
Imran Pasha (181), University of Agriculture, Faisalabad,
Pakistan
Christianah O. Jayeola (317), Cocoa Research Institute of
Nigeria (CRIN), Ibadan, Nigeria
Ami Patel (255), Mansinhbhai Institute of Dairy & Food
Technology (MIDFT), Mehsana, Gujarat, India
Ramakrishna P. Jekrabettu (117), Father Muller Medical
College, Mangalore, India
Jayanta K. Patra (79), Dongguk University, Goyang-si,
Republic of Korea
Vinod Kumar Joshi (55), Dr. Y. S. Parmar University of
Horticulture and Forestry, Solan, India
Swathi Putta (15), University College of Pharmaceutical
Sciences, Andhra University, Visakhapatnam, India
Muhammad Kaleem (137), PMAS-Arid Agriculture
University, Rawalpindi, Pakistan
Taha Rababah (275), Jordan University of Science and
Technology, Irbid, Jordan
Meenu Kalkal (227), Guru Gobind Singh Indraprastha
University, New Delhi, India
Mervat Rawshdeh (275), Jordan University of Science and
Technology, Irbid, Jordan
Mohammad A. Kamal (15), King Abdulaziz University,
Jeddah, Saudi Arabia; Enzymoics; Novel Global
Community Educational Foundation, Hebersham,
NSW, Australia
Silas Saka (15), Krishna University, Machilipatnam, India
Dhananjaya B. Lakkappa
Ramanagara, India
(15),
Jain
University,
Veeranjaneya Reddy Lebaka (55), Yogi Vemana
University, Kadapa, India
Rama Rao Malla (15), GITAM University, Visakhapatnam,
India
Armando M. Martín Ortega (157), Autonomous
University of Yucatan, Yucatán, Mexico
Edwin E. Martínez Leo (157), Autonomous University of
Yucatan, Yucatán, Mexico
Leila Mehdizadeh (167), Ferdowsi University of Mashhad,
Mashhad, Iran
Mohammad Moghaddam (167), Ferdowsi University of
Mashhad, Mashhad, Iran
Taresh Naik (117,303), Father Muller Medical College;
Father Muller Research Centre, Mangalore, India
Venkatarami Reddy Narala (55), Yogi Vemana University,
Kadapa, India
Elroy Saldanha (303), Father Muller Medical College,
Mangalore, India
Maira R. Segura Campos (157), Autonomous University
of Yucatan, Yucatán, Mexico
Anil D. Semwal (291), Defence Food Research Laboratory,
Mysore, India
Nihir Shah (255), Mansinhbhai Institute of Dairy & Food
Technology (MIDFT), Mehsana, Gujarat, India
Mian K. Sharif (181), University of Agriculture,
Faisalabad, Pakistan
Gopal K. Sharma (291), Defence Food Research
Laboratory, Mysore, India
Prakash C. Sharma (227), Guru Gobind Singh Indraprastha
University, New Delhi, India
Manjeshwar Shrinath Baliga (303), Father Muller
Research Centre, Mangalore, India
Manvesh K. Sihag (255), Mansinhbhai Institute of Dairy
& Food Technology (MIDFT), Mehsana, Gujarat, India
Sameer K. Singdevsachan (79), North Orissa University,
Baripada, India
Semiu O. Ogunwolu (317), Cocoa Research Institute of
Nigeria (CRIN), Ibadan, Nigeria
Korada Siva Kumar (15), National Institute of Food
Technology Entrepreneurship and Management
(NIFTEM), Haryana, India
Ijeoma A. Olawuni (341), Federal University of
Technology, Owerri, Nigeria
Yashi Srivastava (291), Central University of Punjab,
Punjab, India
Olayiwola Olubamiwa (317), Cocoa Research Institute of
Nigeria (CRIN), Ibadan, Nigeria
Beenu Tanwar (255), Mansinhbhai Institute of Dairy &
Food Technology (MIDFT), Mehsana, Gujarat, India
Ramakrishna J. Pai (303), Father Muller Medical College,
Mangalore, India
Vadim V. Tarasov (15), I.M. Sechenov First Moscow State
Medical University, Moscow, Russia
Michael Pais (117,303), Father Muller Medical College;
Father Muller Research Centre, Mangalore, India
Hrudayanath Thatoi (79), North Orissa University,
Baripada, India
Princy L. Palatty (117), Father Muller Medical College,
Mangalore, India
Carole C. Tranchant (275), Université de Moncton,
Moncton, NB, Canada
Contributors xv
Florence O. Uruakpa (341), Missouri State University,
Springfield, MO, United States
Abimbola Uzoma (341), Federal University of Technology,
Owerri, Nigeria
Katharina C. Wirnitzer (387), Pedagogical University
Tyrol; Leopold-Franzens University of Innsbruck;
Tyrolean University Conference; The NURMI Study,
Innsbruck, Austria
Rama Rao Vechalapu
Machilipatnam, India
Lateef E. Yahaya (317), Cocoa Research Institute of
Nigeria (CRIN), Ibadan, Nigeria
(15),
Krishna
University,
Young Jung Wee (55), Yungnam University, Gyeongsan,
South Korea
Nagendra S. Yarla
Visakhapatnam, India
(15),
GITAM
University,
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Preface
Novel approaches in food development focus on identifying and applying natural, therapeutic, or alternative components in designing functional foods. This volume aimed to bring together the most recent progress in the field of food
dietary supplements and food products with therapeutic value, empathizing their bioactive components and trends in
obtaining unconventional products. This book contains three sections: Section A, Probiotics and Prebiotics; Section B,
Therapeutic Foods and Ingredients; and Section C, Unconventional Foods and Food Ingredients. In the first section,
readers are introduced to advances made in probiotics and the prebiotics industry and their impact on consumers’ health.
Section B reveals the bioactive and potential therapeutic activities of some food ingredients which are currently being
investigated or already utilized as nutraceuticals, while Section C highlights the nutritional value of some newly adopted
or rediscovered diets.
The volume contains 21 chapters prepared by outstanding contributors from Romania, India, Australia, Russia, United
States of America, Korea, Pakistan, Canada, Nigeria, Mexico, Brazil, and Austria.
In Chapter 1, Introduction in Nutraceutical and Medicinal Foods, Lia-Mara Ditu et al. define and briefly discuss various types of nutraceuticals, functional foods, food supplements, and medicinal foods, highlighting their potential impact on
consumers’ health. Also, an introduction to probiotics, prebiotics, and synbiotics is given in this chapter.
Section A, Probiotics and Prebiotics starts with Chapter 2, entitled Probiotics: Supplements, Food, and Pharmaceutical
Industry, prepared by Swathi Putta and collaborators. This chapter discusses types of probiotics, their known and potential
effects in food and health, and new progress made in food and pharmaceutical industries to develop health-promoting probiotic products.
Chapter 3, Selection of New Probiotics: The Case of Streptomyces, by Sneha Hariharan and Selvakumar Dharmaraj
discusses the selection of new probiotics and presents the properties and potential of Streptomyces species to be utilized as
efficient probiotics, especially in aquaculture and production of marine-related foods.
In Chapter 4, Development of New Probiotic Foods—A Case Study on Probiotic Juices, Lebaka Veeranjaneya Reddy and
colleagues describe the procedure required for the development of new probiotic foods, by exemplifying the development
of fruit juice probiotics. The application of such products on consumers’ health, food design, and new trends in probiotic
products and processes are presented here.
Chapter 5, Prebiotics and Production of New Prebiotics from Unconventional Raw Materials (Mushrooms), prepared
by Hrudaynath Thatoi et al., highlights the information related to prebiotic and nutraceutical values of bioactive substances obtained from mushrooms for developing new and potential prebiotics from such an inexpensive and abundant
source.
Chapter 6, Probiotics in the Rescue of Gut Inflammation, written by Asit Ranjan Ghosh, reveals recent information
regarding the gut-brain axis, explaining the link between digestion, mood, health, behavior, and even way of thinking. This
chapter shows the probiotic attitude of microbiota to maintain homeostasis and the potential of probiotics to downregulate
(the cause of) inflammation.
In Chapter 7, Probiotics as an Adjunct to Conventional Treatment in Vulvovaginitis: Past, Present and Future, Princy
Louis Palatty and collaborators deal with a highly debated subject: the potential impact of probiotics in therapy for genital
tract infections. Since these infections are one of the leading causes of infertility, alternative therapeutic approaches and
prevention strategies are intensively investigated, probiotics representing one of the most successful candidates for such
approaches.
Chapter 8, entitled Flavonoids as Nutraceuticals, written by Muhammad Kaleem and Asif Ahmad, is the first chapter of
Section B, Therapeutic Foods and Ingredients. In this chapter, the use of bioactive compounds as an appropriate alternative
to synthetic medicines is widely dissected. This manuscript highlights the pharmacological importance of flavonoids that
may be supplemented in staple food to develop different nutraceutical products.
xvii
xviii Preface
In Chapter 9, Bioactive Peptides—Impact in Cancer Therapy, Edwin E. Martínez Leo et al. analyze principal biopeptides
that could have a positive effect on the dietary treatment of cancer and its principal alterations, inflammation and oxidative
stress. Research advances in cancer treatment are essential to improve the results in patients affected by this disease. These efforts include the development of more effective and less toxic therapies, such as targeted therapies, immunotherapies, and vaccines for cancer treatments, and the improvement of those that have existed for decades at the pharmacological and dietary level.
Leila Mehdizadeh and Mohammad Moghaddam in Chapter 10, Essential Oils—Biological Activity and Therapeutic
Potential, discuss the biological activity of essential oils, such as their antimicrobial potential, which has been investigated
to reduce the hazardous effects of synthetic fungicidal and bactericidal products. The increasing interest in the possible application of essential oils for pathogen control and preservative-design application has been directed toward investigating
new sources of biologically active natural products in different industries.
Chapter 11, Nutritional and Therapeutic Potential of Spices, prepared by Mian Kamran Sharif et al. describes the properties of various Asian spices along with their therapeutic role and food applications. Furthermore, the multiplicity of potentially beneficial nature of spices is explored, along with possible strategies to obtain maximum health outcomes without
exposure to undesirable side effects.
Asma Afreen and collaborators, in Chapter 12, Novel Nutraceutical Compounds, dissect nutraceutical effects and biomedical applications of phytochemicals including carotenoids, phytosterols, polyphenols, and Omega 3-fatty acids which
are used as preventive medicines because of their established health benefits.
Chapter 13, Nutraceutical and Medicinal Importance of Seabuckthorn (Hippophae sp.), prepared by Prakash C. Sharma
and Meenu covers nutraceutical and medicinal applications of seabuckthorn. Many research studies have reported diverse
medicinal properties of seabuckthorn preparations: antimicrobial, antiulcerogenic, antioxidative, anticarcinogenic, radioprotective, hepatoprotective, antihypertensive, antiinflammatory, and immunomodulatory. These medicinal properties of
seabuckthorn are attributed to the presence of important bioactive compounds in different parts of seabuckthorn plant
mainly in berries, leaves, and seeds.
Ankit Goyal et al., in Chapter 14, Therapeutic Potential of Flaxseed, delineate the recent findings of flaxseeds on
the physiological functionality of nutraceutical formulations along with their applications in foods. Various nutraceutical
preparations of flax and flaxseed oil such as Essentiale, Lipostabil, Efamol, and Essaven are available in the global market
for the treatment of general fatigue, pain, atherosclerosis, eczemas, and other diseases.
Chapter 15, Pharmaceutical, Nutraceutical, and Therapeutic Properties of Selected Wild Medicinal Plants: Thyme,
Spearmint, and Rosemary, by Muhammad H. Alu’datt and collaborators discusses the chemical, nutraceutical, and pharmaceutical properties of selected wild medicinal herbs (thyme, spearmint, and rosemary) and their phenolic constituents.
Numerous studies showed that phenolic extracts have anticancer, antiviral, antiinflammatory, hypolipidemic, antioxidant,
antidiabetic, antihypertensive, and hypoglycemic effects in vivo because of phenolics’ ability to interact with biological
molecules such as DNA, hormones, and enzymes (e.g., angiotensin-I converting enzyme (ACE), α-glucosidase and αamylase), and to modulate cell-signaling pathways and epigenetic modifications.
In Chapter 16, Virgin Coconut Oil as Functional Oil, Srivastava Yashi et al. dissect the health benefits of virgin coconut
oil, such as preventing oxidation of low-density lipoprotein lipids and increasing the production of antioxidant enzymes.
The total polyphenol, antioxidant activity, tocopherol, phytosterol, monoglycerides, and diglyceride content particular to
virgin coconut oil ensures a unique nutraceutical effect.
Chapter 17, Health Effects of Various Dietary Agents and Phytochemicals (Therapy of Acute Pancreatitis), written by
Elroy Saldanha and collaborators, presents the beneficial properties of some dietary agents such as green tea, Emblica
officinalis, grapefruit, rhubarb, and phytochemicals present in various dietary agents (i.e., curcumin, ellagic acid, cinnamtannin B-1, capsaicin, beta-pinene, piperine, zerumbone, lycopene, resveratrol and the flavonoids quercetin, genistein, and
apigenin) in the prevention and treatment of chemical-induced acute pancreatitis.
Section C: Unconventional Foods and Food Ingredients debuts with Chapter 18, Production of Bioactive Compounds
from Waste, prepared by Jayeola Christianah Olayinka et al. This paper highlights the impact of technological innovation
on the bioconversion of some widely-available agro-wastes and their application toward modern agriculture and the sustainable food industry.
Chapter 19, Unripe Plantain Flours, prepared by Florence Ojiugo Uruakpa and collaborators, discusses the impact of
drying methods, time of maturation, and pretreatments on the functionality of plantain flours. New processing methods are
needed to reduce waste and add value to its use in food processing.
In Chapter 20, Dry Beans: Processing and Nutritional Effect, Rocio Campos-Vega and collaborators review the postharvest processing of dry beans essential for ensuring high quality for food preparation, storage, and food safety. Details are also
provided for technologies with minimal energy footprints as well as novel processes for development of functional food ingredients from dry beans in regard to compositional and nutritional changes and their impact on human health and wellness.
Preface xix
Katharina C. Wirnitzer, in Chapter 21, Vegan Nutrition: Latest Boom in Health and Exercise, offers a comprehensive
overview of vegetarian and vegan diets ranging from myths about meat and early studies on the effect of vegetarian diets
upon sports, through the flood of studies published on the health-threatening effects of foods from animal sources, to current studies showing the benefits of predominantly plant-based diets on human health and sports performance.
This book is dedicated to scientists, food researchers, students, and industrial companies who seek scientific evidence
on recent tools and perspectives in functional and unconventional foods, is a resourceful tool for biotechnologists, microbiologists, biochemists, and clinicians, and an interesting and updated reference for any reader interested in learning about
trends and progress in Therapeutic, Probiotic, and Unconventional Foods.
Alina Maria Holban
University of Bucharest, Bucharest, Romania
Alexandru Mihai Grumezescu
University Politehnica of Bucharest, Bucharest, Romania
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Chapter 1
Introduction in Nutraceutical
and Medicinal Foods
Lia-Mara Ditu⁎, Madalina E. Grigore†,‡, Petronela Camen-Comanescu§, Alina Maria Holban⁎
*
Research Institute of the University of Bucharest, Bucharest, Romania, †University Politehnica of Bucharest, Bucharest, Romania, ‡National Research
& Development Institute for Chemistry and Petrochemistry—ICECHIM, Bucharest, Romania, §University of Bucharest, Bucharest, Romania
1.
INTRODUCTION
Modern eating preferences and progress made in the food industry have led to a completely new definition of nutrition and
health through eating. Eating habits have a great impact on humans’ health, environment, industry, and economy. Numerous
diseases have arisen as endemic to modern society, such as obesity, osteoporosis, cancer, diabetes, allergies, and dental
problems, which can occur at an early age and could be related to eating habits and preferences. While developed countries
face problems related to aging populations, high energy foods, and unbalanced diets, developing countries still face malnutrition and limited availability of nutritious foods.
In recent years, new topics in food research, such as nutraceuticals, functional foods, and food supplements have
emerged to mitigate health problems, especially those pertaining to metabolism and the gastrointestinal (GI) tract (Cencic
and Chingwaru, 2010).
At the level of the GI tract, certain gut microbiota and their products (e.g., probiotic microorganisms, prebiotics) play
a significant role in the hosťs health due to their impact on nutritional, immunologic, and physiological functions. The
molecular mechanisms by which nutraceuticals, functional foods, and food supplements might improve the health of consumers are widely unknown, but the potential of such products in health support, and even in the development of efficient
alternative therapies, is huge and supported by numerous studies and empirical observations.
2.
NUTRACEUTICALS
Nutraceutical(s) is a term used to describe any product derived from food sources with extra health benefits in addition to the
basic nutritional value found (Gul et al., 2016). Various confusing and contradictory definitions between terms such as “nutraceuticals,” “functional food,” “herbal remedies,” and “health food” are common. The lack of internationally agreed-upon
definitions and distinctions between them is created by the legal framework of different countries, marketing (market presentation and doses), and public perception (Lockwood, 2007; Aronson, 2017). The Directive 2002/46/EC on food supplements
and novel foods (recently modified by the new European Parliament and Council Regulation (EU) 2015/2283) which defines
new food categories and the classification of food supplements, still does not mention the term “nutraceutical.”
The term “nutraceutical” was introduced in 1989 by Dr. Stephen DeFelice, MD, the founder and chairman of the
Foundation for Innovation in Medicine (FIM), Cranford, NJ. He defined “nutraceutical” as "a food (or part of a food) that
provides medical or health benefits, including the prevention and/or treatment of a disease" (DeFelice, 2002). Given the
fact that this term has often been used as a synonym for functional food or dietary supplements, in order to describe healthpromoting foods or their extracted components, in 2003, Kalra tried to make a distinction between these three terms and
redefined them. So, when functional food aids in the prevention and/or treatment of disease(s) and/or disorder(s) other than
anemia, it is called a nutraceutical (Kalra, 2003).
According to Das et al. (2012), nutraceuticals can be classified depending upon their easier understanding and application, that is, for academic instruction, clinical trial design, and functional food (Das et al., 2012). Thus, nutraceuticals
can be classified on the basis of their natural sources (products obtained from plants, animals, minerals, or microbial
sources), mechanism of action (antioxidation, antibacterial, hypotensive, anti-inflammatory, etc.) or as per chemical nature
of the products (amino acid-based substances, carbohydrates and derivatives, fatty acids and structural lipids, isoprenoid
Therapeutic, Probiotic, and Unconventional Foods. https://doi.org/10.1016/B978-0-12-814625-5.00001-7
© 2018 Elsevier Inc. All rights reserved.
1
2
SECTION | A Probiotics and Prebiotics
d­ erivatives, phenolic substances, microorganisms: probiotics, prebiotics, minerals) (Pandey et al., 2011; Wildman, 2016;
Prakash et al., 2012). Their sources may range from isolated food nutrients (vitamins, minerals, amino acids, and fatty
acids), herbals (herbs or botanical products as concentrates or extracts), dietary supplements (reagents derived from other
sources), and diets to genetically engineered “designer” foods and processed products such as cereals, soups, and beverages
(Malik et al., 2008; Dureja et al., 2003; Chauhan et al., 2013).
2.1
Food Nutrients
Phytonutrients are substances with established nutritional functions. The most widely known are antioxidants, vitamins, and essential minerals but carbohydrates, proteins, amino acids, and fatty acids are also included in this category. Phytonutrients, also
known as phytochemicals, are bioactive compounds accumulated in different parts of plants, especially as a result of secondary
metabolism. In plants they have either defensive or disease protective properties (Prakash et al., 2012). Phytonutrients are not essential nutrients, however they have important properties such as antioxidant activity, antimicrobial effects, modulation of detoxification enzymes, stimulation of the immune system, decrease of platelet aggregation and modulation of hormone metabolism,
and anticancer property (Shiny et al., 2013). The majority of foods, such as whole grains (oats, rice, sorghum, wheat), beans,
fruits (grapes, blueberries, raspberries, blackberries, melons), vegetables (tomatoes, potatoes, garlic, broccoli, kale, parsley,
spinach), and herbs contain phytonutrients. Significant scientific data shows that among the most relevant phytonutrients which
provide health benefits are phenols and other secondary metabolites such as tannins, terpenoids or isoprenoids, and alkaloids.
Amongst the phytochemicals, several groups of polyphenols (anthocyanins, proanthocyanidins, flavanones, isoflavones,
resveratrol, and ellagic acid) are currently used in the nutraceutical industry (Espín et al., 2007). Polyphenols possess
antioxidant, anti-inflammatory, anti-microbial, cardioprotective properties and play a role in the prevention of neurodegenerative diseases and diabetes mellitus (Scalbert et al., 2005). Several studies have reported that polyphenols present
antioxidant properties but it has become clear that the mechanisms of action of polyphenols go beyond the modulation of
oxidative stress (Scalbert et al., 2005; Kim et al., 2012; Lu and Foo, 2000). Also, epidemiological studies have shown an
association between the risk of chronic human diseases and the consumption of polyphenolic rich diet. It is well established that ­polyphenol-rich foods can increase plasma antioxidant capacity (Pandey and Rizvi, 2009). For example, it was
demonstrated that tea polyphenol administration inhibits carcinogen-induced increases in the oxidized DNA base in animal
models (Frei and Higdon, 2003). In another study, it was reported that the tea (Camellia sinensis) consumed in the form
of black tea and green tea has been found to have cancer-preventing activities (Mbuthia et al., 2017). The antioxidant and
anti-inflammatory activities of red wine were also demonstrated (Rice-Evans et al., 1997).
Phenols are secondary metabolites which protect plants from photosynthetic stress, reactive oxygen species and are
involved in defense against herbivores and pathogens, the attraction of pollinators, etc. (Das et al., 2012; Dai and Mumper,
2010). Within scientific literature, surveys indicate that phenolic compounds are the most numerous and structurally diverse
phytoconstituents in most plants. There are approximately 8000 different classes of polyphenols, the most important being
phenolic acids (vanillic acid, caffeic acid), flavonoids (flavonols, flavones, flavanones, anthocyanidins), and polyphenols
(coumarins and tannins) (Saxena et al., 2013). Flavonoids are the most studied group of plant phenols and are reddish pigments found in red grape skins and citrus fruits (Dai and Mumper, 2010).
The major sources of polyphenols are cereals (i.e., soy flour), vegetables (i.e., pepper, onion, beans), oilseeds (i.e.,
rapeseed, canola, flaxseed, and olive seeds), fruits (i.e., apple, apricot, black grape, cherry, berries) and beverages (i.e., red
wine, black and green tea, fruit juices, tea, coffee) (Prakash et al., 2012).
Tannins, water-soluble polyphenols that are present in many plant foods, are a heterogeneous group used as astringents,
diarrhea preventatives, as diuretics, against stomach and duodenal tumors, as anti-inflammatories, antiseptics, antioxidants,
and hemostatic (EFSA, 2008; Chung et al., 1998; Scalbert, 1991; Hagerman et al., 1998). Indoles are found in cruciferous
vegetables, lignans in flaxseed, and isoflavones can be found in peanuts, lentils, soy, and other legumes (Tyug et al., 2010).
Terpenoids or isoprenoids are one of the largest group of secondary metabolites of plants. Under this umbrella are included carotenoids, saponins, tocopherols, and isoprenoid derivatives, as well as isoprenoid derivatives (Wildman, 2016).
Based on the number of isoprene units, they are clustered in: hemiterpenoids (i.e., prenol and isovaleric acid), monoterpenoids (i.e., eucalyptol, limonene, camphor, pinene), diterpenes (i.e., cafestol), sesquiterpenes (i.e., artemisinin), triterpenes
(i.e., limonin, nomilin, cucurbitacin, lanosterol), and tetraterpenoids (i.e., lycopene). They play an important role as signaling compounds and growth regulators in plants, in defense against pathogens and parasites, and in the attraction of specific
insects for pollination (Breitmaier, 2006).
For example, it was reported that the terpenoids isolated from Rhizoma Curcumae present anticancer processes related to the
retardation of cell cycle arrest, the induction of apoptosis, and the inhibition of metastasis or tissue invasion (Lu et al., 2012).
Carotenoids (tetraterpenoids), the most recognizable pigments, are synthesized by plants, algae, and photosynthetic
bacteria and are responsible for the yellow, orange, and red colors of those organisms. They are important in ­photosynthesis
Introduction in Nutraceutical and Medicinal Foods Chapter | 1 3
and photo-protection. In the human diet the most common and important carotenoids are: α-Carotene, β-carotene, βcryptoxanthin, lutein, zeaxanthin, and lycopene (Galanakis, 2016).
Alkaloids are significant for protection and plant survival because they ensure against micro-organisms (antibacterial
and antifungal activities), insects and herbivores (feeding deterrents) and as well as threats from other plants by means of
allelopathically active chemicals (Saxena et al., 2013). The most well-known alkaloids are caffeine and nicotine, which
have stimulant proprieties, analgesic morphine, and quinine, which is used as an antimalarial drug (Imaga, 2013; Almagro
et al., 2015). The semi-synthetic derivatives of dimeric alkaloid vinblastine and vincristine are present at low concentration
levels in Catharanthus roseus plants and are used to treat diseases such as acute leukemia, malignant lymphoma, Hodgkin's
disease, acute erythremia, and acute panmyelosis (Almagro et al., 2015; Zhu et al., 2014). The most recent developments
regarding alkaloids refer to the neurological, anti-cancer and anti-microbial bioactivities (Hotchandani and DesgagnePenix, 2017). A recent study investigated the physical anti-fatigue and exercise performance effects of Actinidia arguta
crude alkaloids extracted with 70% ethanol. In this study, four groups of male Kunming mice (n = 16) were used. AACA
at doses of 0 mg/kg/day (vehicle), 50 mg/kg/day (AACA-50), 100 mg/kg/day (AACA-100), or 200 mg/kg/day (AACA-200)
were administered orally for 28 days. The efficiency of AACA treatment on exercise performance was studied using the
forelimb grip strength experiment and by the measurement of the weight-loaded swimming time. After 28 days, it was
reported that the swimming time of the AACA-100 group was the longest among all groups studied, and the mice in the
AACA-treated groups had decreased levels of lactate, ammonia, and creatine kinase after a physical challenge, compared
with the vehicle group. It was concluded that the alkaloids extracted from A. arguta could be used as a new anti-fatigue and
exercise performance agent with physiological benefits when taken at optimized and reasonable doses (Liu and Liu, 2016).
In another study, researchers examined the antitumor properties of total alkaloids of S. alopecuroides (TASA) against HeLa
cells. After 24 h, it was observed that more than 50% of HeLa cells died using a treatment which involved 8.75 mg/mL of
TASA, and cell death rate increased further with longer incubation. The apoptotic rates of HeLa cells in the experimental
groups were 16.0% and 33.3% at concentrations of 6.25 mg/mL and 12.50 mg/mL, respectively (Li et al., 2016).
2.2
Herbals
Medicinal plants represent one of the most important fields of traditional medicine all over the world (Chanda and Kaneria,
2011). Over time, it has been demonstrated that many plants can be used to prevent disease and that an herbal diet provides
many phytonutrients which can lower the risk of disease, including cancer and cardiovascular diseases (Jhansi and Manjula,
2016). In Europe, more than 1500 species of medicinal and aromatic plants are widely used for biomedical practices.
Nutraceutical plants produce health-promoting phytochemicals and they can be consumed fresh and dried (whole or parts
of plant), as powder, standardized extracts, tinctures, capsules, tablets or teas (Hoareau and DaSilva, 1999).
Herbals have nutritional, antioxidant, antimicrobial, and medicinal properties, the treatment of human diseases using plant
products is increasing, because plants are sources of bioactive molecules that have been shown to be safe and efficient in treating diseases (Jhansi and Manjula, 2016). Antioxidant activity of the phenolic compounds is believed to prevent most of the
major chronic diseases, providing protection against infections and numerous degenerative diseases. Many herbs such as oregano, basil, dill, garlic, ginger, lemongrass, oregano, and parsley have antioxidant activity due to their high levels of phenolic
compounds (Henning et al., 2011). In a study, it was demonstrated that six Chinese medicinal foods such as pagoda tree flower,
Chinese white olive, clove, prickly ash peel, Chinese star anise, and villous amomum fruit are novel, natural, and economical
sources of dietary antioxidants which can be used in the prevention of diseases caused by free radicals (Liu et al., 2008).
Phenolic constituents are closely associated with anti-microbial activity. Scientifically supported studies on Thymus vulgaris
(thyme), Lavandula angustifolia (lavander), Melissa officinalis (lemon balm), Ocimum basilicum (basil), Allium schoenoprasum (chive), Petroselinum crispum (Parsley), Laurus nobilis (laurel), and Rosmarinus officinalis (rosemary) showed the high
potential of these extracts to inhibit bacterial growth and also to limit infections (Dostalova et al., 2014; El et al., 2014; Viswanad
et al., 2011). Various medicinally important herbs have been studied for centuries because of their anti-microbial activity in
the traditional systems of medicine. For example, the investigation of aqueous extracts of five plant species traditionally used
in medicine for in vitro antimicrobial activity against five different Candida species isolated from human oral cavity, by disc
diffusion method. It was reported that the highest toxicity against all the Candida species tested was obtained by using aqueous extracts of the Allium sativum bulb. Aqueous extracts of Azadirachta indica showed weak anti-Candida activity whereas
Ocimum sanctum, Murraya koenigii and Withania somnifera did not display any anti-Candida activities (Pathak, 2012). Another
study tested the antimicrobial potency of aqueous and ethanolic decoction (individual extract) and concoction (mixed extract) of
three common medicinal herbs (turmeric Curcuma longa, Tulsi plant Ocimum sanctum, and neem Azadirachta indica) against
the in vitro growth of A. hydrophila. It was reported that among the decoctions, A. indica exhibited the most potent antibacterial
property against A. hydrophila and among the concoctions, both the aqueous and ethanolic triherbal extracts mixed in the ratio
of 1:1:1 had higher antibacterial activity than the other concoctions and decoctions (Harikrishnan and Balasundaram, 2008).
4
SECTION | A Probiotics and Prebiotics
The common medicinal plants widely used as nutraceuticals are: Allium cepa (onion), Allium sativum (garlic), Avena
sativa (Oat straw), Capsicum annum (Red pepper), Curcuma longa (Turmeric), Ginkgo biloba (Ginkgo), Hypericum perforatum (St.-John’s-Wort), Matricaria chamomilla (Chamomile), Panax ginseng (Ginseng), Urtica dioica (Stinging nettle),
Valeriana officinalis (Valerian), and Aloe vera (aloe).
2.3
Dietary Supplements
Dietary supplements are products that supplement food with different dietary ingredients such as vitamins, minerals, and amino
acids, increasing the total daily intake. In contrast with dietary supplements, nutraceuticals must not only supplement the diet, but
should also contribute in the prevention and/or treatment of disease and/or disorders (Cencic and Chingwaru, 2010). Dietary supplements are regulated by the FDA as food, not as drugs. However, many dietary supplements contain ingredients that have strong
biological effects which may conflict with a particular medicine or even a medical condition; therefore, their use must be carefully
supervised. There are many types of dietary supplements and they may be classified into the following categories: Vitamins,
Dietary mineral, Amino acids (Table 1) and proteins, Essential fatty acids, and enzyme supplements (Katz and Meller, 2014).
Numerous industrial representatives have produced protein-based products utilized mainly as bodybuilding supplements.
However, there are numerous natural sources which could be used in daily food to increase protein uptake. The richest
protein food sources can be found in Table 2.
2.3.1
Fatty Acids
The human body is able to synthesize most of the necessary fats; however, we are not able to produce linoleic and alphalinolenic fatty acids, which are essential and need to be supplemented by food intake. These basic fats, found in plant foods,
are used to build specialized fats called omega-3 and omega-6 fatty acids, which are important in the normal functioning of
all tissues of the body (Thiébaut et al., 2009). Table 3 presents the main essential fatty acids and their food sources.
List of vitamins and minerals utilized for dietary supplements (in alphabetical order)
A
I
Vitamin A
Iodine
B
Iron
Biotin
K
Vitamin B1 (Thiamin)
Vitamin K
Vitamin B12
M
Vitamin B6
Magnesium
C
N
Calcium
Niacin
Choline
P
Chromium
Pantothenic Acid
Copper
Phosphorus
Vitamin C
Potassium
D
R
Vitamin D
Riboflavin
E
S
Vitamin E
Selenium
F
Z
Fluoride
Zinc
Folate
Introduction in Nutraceutical and Medicinal Foods Chapter | 1 5
TABLE 1 List of Essential and Non-essential Amino Acids
Amino Acid
Essential
Non-essential
Alanine
*
Asparagine
*
Aspartic acid
*
Arginine
*
Cysteine
*
Glutamine
*
Glycine
*
Glutamic acid
*
Histidine
*
Isoleucine
*
Lysine
*
Leucine
*
Phenylalanine
*
Methionine
*
Serine
*
Proline
*
Tryptophan
*
Threonine
*
Tyrosine
*
Valine
*
TABLE 2 Food Products With High Protein Content
Type of Food
Product
Protein Amount/Serving
High-Protein Dairy and Eggs
Greek Yogurt
23 g per 8 oz. serving
Cottage Cheese
14 g per 1/2 cup serving
Swiss Cheese
8 g per 1 oz. serving
Eggs
6 g per 1 large egg
Milk, 2%
8 g per 1 cup serving
Whey Protein
24 g per scoop, on average
Steak (Top or Bottom Round)
23 g per 3 oz. serving
Ground Beef (95% Lean)
18 g per 3 oz. serving
Pork Chops (Boneless)
26 g per 3 oz. serving
Chicken Breast (Boneless and Skinless)
24 g per 3 oz. serving
Turkey Breast
24 g per 3 oz. serving
High-Protein Meat
Continued
6
SECTION | A Probiotics and Prebiotics
TABLE 2 Food Products With High Protein Content—cont’d
Type of Food
Product
Protein Amount/Serving
High-Protein Seafood
Yellowfin Tuna
25 g per 3 oz. serving
Halibut
23 g per 3 oz. serving
Octopus
25 g per 3 oz. serving
Sockeye Salmon
23 g per 3 oz. serving
Tilapia
21 g per 3 oz. serving
Anchovies
24 g per 3 oz. serving
Corned Beef
24 g per 3 oz. serving
Light Tuna
22 g per 3 oz. serving
Chicken
21 g per 3 oz. serving
Sardines
21 g per 3 oz. serving
Navy Beans
20 g per 1 cup serving
Dried Lentils
13 g per 1/4 cup serving
Roast Beef
18 g per 3 oz. serving
Canadian Bacon
15 g per 3 oz. serving
Chorizo
21 g per 3 oz. serving
Pepperoni
18 g per 3 oz. serving
Roasted Turkey Breast
18 g per 3 oz. serving
Jerky
13 g per 1 oz. serving
Peanut Butter
8 g per 2 tbsp serving
Mixed Nuts
6 g per 2 oz. serving
Bean Chips
4 g per 1 oz. serving
Smoothie Drinks (i.e., containing whey proteins)
16 g per 1 cup serving
Tofu
12 g per 3 oz. serving
Edamame
8 g per 1/2 cup serving
Green Peas
7 g per 1 cup serving
Frozen Greek Yogurt
6 g per 1/2 cup serving
Wheat Germ
6 g per 1 oz. serving
Soba Noodles
12 g per 3 oz. serving
Quinoa
8 g per 1 cup serving
High-Protein Canned Foods
High-Protein Deli
High-Protein Snacks
High-Protein Produce
High-Protein Frozen Foods
High-Protein Grains
2.3.2
Enzyme Supplements
External enzyme sources which may represent components of dietary supplements are usually represented by animals,
plants and microorganisms. Products containing enzymes have been consumed by man for millennia, the most well-known
examples existing in cheese and bread making, dry aging of meats, and a variety of fermentation processes including
­brewing, wine and vinegar production, and lactic acid fermentations. Yeast has been used not only as a source of vitamins,
but also as a source of beneficial enzymes for various health conditions such as combating constipation and stimulating normal digestion by the action of yeast proteases and amylases. The use of enzymatic digestive promoters or aids continues to
flourish and represents the most important type of enzyme based dietary supplement. Most enzymes encountered in dietary
supplements and their sources are presented in Table 4 (Enzyme Technical Association, 2017).
Introduction in Nutraceutical and Medicinal Foods Chapter | 1 7
TABLE 3 Essential Fatty Acids, Abbreviations and Main Sources
Type
Name
Abbreviation
Source
Omega-6 fatty acids
Linoleic acid
LA
Vegetable oils, meat, poultry, eggs
γ-Linolenic acid
GLA
Dihomo-γ-linolenic acid
DGLA
Arachidonic acid
AA
Adrenic acid
Tetracosatetraenoic acid
Tetracosapentaienoic acid
Omega-3 fatty acids
Docosapentaenoic acid
DPA (n-6)
α-Linolenic acid
ALA
Stearadonic acid
SDA
Eicosatetraenoic acid
ETA
Eicosapentaenoic acid
EPA
Docosapentaenoic acid
DPA (n-3)
Green leafy vegetables, flax and chia seeds, canola,
walnut, soybean oils, oily fish, algae oil, krill oil
Tetracosapentaenoic acid
Tetracosahexaenoic acid
Docosahexaenoic acid
DHA
TABLE 4 Enzymes Found in Dietary Supplements and Their Main Sources
Enzyme
Source
Alpha-galactosidase
Aspergillus niger
Amylase
Aspergillus oryzae, Bacillus subtilis, Bacillus amyloliquefaciens, Aspergillus niger
Amylase (ß-amylase)
(Malt diastase), barley malt
Cellulase
Aspergillus niger, Trichoderma longibrachiatum (reesei)
Invertase
Saccharomyces cerevisiae
Lactase
Aspergillus oryzae, Kluyveromyces lactis
Lipase
Aspergillus oryzae, Aspergillus niger, Rhizopus oryzae, R. japonicus, Arthrobacter ureafaciens, Candida
cylindracea, Rhizomucor miehei, Rhizopus delemar
Pancreatin
Porcine/bovine pancreas
Pancrelipase
Bovine and porcine pancreas
Protease, botanical
Bromelain (Ananas comosus) and Papain (Carica papaya)
Protease, animal
Porcine pepsin, bovine or porcine (trypsin), bovine or porcine (chymotrypsin), bovine (pepsin)
Protease, microbial
Aspergillus niger, Aspergillus oryzae, Bacillus subtilis, Aspergillus oryzae, Aspergillus melleus, Bacillus
licheniformis, Bacillus thermoproteolyticus, Rhizopus niveus
Superoxide dismutase
Bacillus spp.
8
SECTION | A Probiotics and Prebiotics
3.
FUNCTIONAL FOODS
The term functional food was proposed for the first time in 1984 in Japan, and defines food products that contain physiologically functional constituents which take part in preventing diseases being directly involved in the modulation of our
physiological systems such as the immune, endocrine, nerve, circulatory, and digestive systems (Arai, 1996). Unlike nutraceutics, this is considered food, not being commercialized as medical doses, but in the form of oats, wheat, lentils, peas,
beans, modified oil such as bran oil, soy protein (Mortazavian and Meybodi, 2016). Functional foods include whole and
fortified foods and enriched or enhanced dietary components which may reduce the risk of chronic disease and provide a
health-benefit beyond the traditional nutrients it contains (Gul et al., 2016). Most known examples include probiotic yogurts, cholesterol-lowering spreads and foods with added nutrients, such as omega-3 fatty acids (Williamson, 2009).
It was reported that food bioactive compounds exert significant effects on the intestinal environment, modulating the
gut microbiota composition and probably its functional effects on mammalian tissues. In this case, this idea will change the
way the biological roles of functional food components are being investigated because their metabolites and effects may
depend on the gut microbiota and even change from one individual to another (Laparra and Sanz, 2010). One study tested
pomegranate, a fruit native to the Middle East, in relation to a variety of chronic diseases. It reported promising results
against cardiovascular disease, diabetes, and prostate cancer from human clinical trials and the in vitro antioxidant activity
of pomegranate has been attributed to its high polyphenolic content, specifically punicalagins, punicalins, gallagic acid, and
ellagic acid (Johanningsmeier and Harris, 2011).
4.
MEDICINAL FOODS
A medicinal food is formulated to be consumed or administered internally, under the supervision of a qualified physician. Its intended use is a specific dietary management of a disease or condition for which distinctive nutritional requirements are established by the medical evaluation (on the basis of recognized scientific principle) (Mortazavian and
Meybodi, 2016).
Since foods were scientifically proven to significantly impact our health, to prevent or even help treat disease, new concepts and scientific fields have emerged to investigate the specific impact of foods on people’s health.
The expanding field of Nutrigenomics (also called nutritional genomics) is devoted to studying how food influences
gene expressions and contributes to health or longevity, to disease and early death. The principles behind nutrigenomics
can be summarized in several key points; genes play a role in disease development and prevention, a poor diet can be a serious risk factor for many diseases, nutrient deficiencies and toxic chemicals in low-quality foods have an effect on human
gene expressions, each person is different in terms of how much their genes/health are impacted by their diet, and a healthy
but also personalized diet can be used to prevent, mitigate or cure chronic diseases (Axe, 2017). In particular, polyphenols, as part of the central dynamic interaction between the genome and the environment with specificity at physiological
concentrations, are known to affect the mechanisms underlying human health. It seems that numerous dietary compounds
modulate epigenetic mechanisms, therefore the regulation of gene expression including expression of enzymes and other
molecules responsible for drug absorption, distribution, metabolism and excretion in cancer, metabolic syndrome, neurodegenerative disorders and hormonal dysfunction (Remely et al., 2015).
5.
PROBIOTICS, PREBIOTICS AND SYNBIOTICS
The use of probiotics, prebiotics, synbiotics and nutraceuticals in clinical medicine was sustained by the fact that the human body can discriminate between different microbial species, microbial products and bioactive plant species which are
inducing beneficial effects to health (Penner et al., 2005).
5.1
Probiotics
Probiotics are living microorganisms, which, when administered in adequate amounts, confer a health benefit to the host
(Hotel and Cordoba, 2001). Microbial species included in the probiotic category are both yeast (Saccharomyces cerevisiae)
and bacteria, including lactic acid bacteria (LAB; such as species of Lactobacillus, Streptococcus, and Enterococcus),
Bifidobacterium sp., Propionibacterium sp., Bacillus sp., and Escherichia coli (Bernardeau et al., 2006; Borriello et al.,
2003). The main probiotic supplements present on the market utilize lactobacilli, streptococci and bifidobacteria species
which are normal constituents of the human gastrointestinal microbiota (Davis, 2016).
Investigating and understanding the interactions between pathogenic and probiotic bacterial strains could provide
the basis for innovative anti-pathogenic strategies and infection control, with the limitation of the antibiotics use and
Introduction in Nutraceutical and Medicinal Foods Chapter | 1 9
­ odulation of bacterial activities in an eco-friendly manner, thus limiting the chemical antimicrobial substances selecm
tive pressure and the emergence of drug-resistant and highly virulent bacterial strains. In this landscape, probiotics and
nutraceuticals play a major role.
Interaction of viable probiotic bacteria with other species from intestinal microbiota involves competitive exclusion phenomena (competition for nutrients and adhesion sites) and the synthesis of inhibitory molecules. The interaction with the human host occurs regardless of probiotics’ viability and is mediated by the interaction of bacterial
cells/sub-cellular fractions with mucosal associated lymphoid tissue structures, activating both non-specific and
specific immune defense (Kobyliak et al., 2017; Sanders et al., 2010). Even if the beneficial effects of probiotics
are well established, the lack of mechanistic understanding of probiotic activity is a major problem for predicting
the safety of probiotic intervention (De Vrese and Schrezenmeir, 2008). Whereas probiotics are generally reported
to protect the intestinal barrier, there might be conditions where probiotics facilitate translocation and induce sepsis
(Boyle et al., 2006).
The main effects of probiotics on the host are the following:
1. Prevention and/or reduction of duration and complaints of rotavirus-induced or antibiotic-associated diarrhea as well as
alleviation of complaints due to lactose intolerance.
2. Reduction of the concentration of cancer-promoting enzymes and/or putrefactive (bacterial) metabolites in the gut.
3. Prevention and alleviation of unspecific and irregular complaints of the gastrointestinal tracts in healthy people.
4. Beneficial effects on microbial aberrancies, inflammation, and other complaints in connection with inflammatory diseases of the gastrointestinal tract, Helicobacter pylori infection, or bacterial overgrowth.
5. Normalization of passing stool and stool consistency in subjects suffering from obstipation or an irritable colon.
6. Prevention or alleviation of allergies and atopic diseases in infants.
7. Prevention of respiratory tract infections (common cold, influenza) and other infectious diseases as well as treatment of
urogenital infections. Insufficient, or at most preliminary, evidence exists with respect to cancer prevention, a so-called
hypocholesterolemic effect, improvement of the mouth flora and caries prevention or prevention or therapy of ischemic
heart diseases or amelioration of autoimmune diseases (e.g., arthritis).
Several studies have demonstrated the efficiency of probiotics. On study, for example, showed that subcutaneous administration of Lactobacillus salivarius attenuated colitis and pro-inflammatory cytokine production in a mouse model
(Penner et al., 2005). In another study, yogurt was produced from base milk containing three important nutraceuticals
(ω-3-fatty acids, isoflavones, and phytosterols). The cultures employed to make the yoghurts were single probiotic strains
of Lactobacillus gasseri or Bifidobacterium infantis and for a two-stage fermentation procedure, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus was used, providing the rapid acidification. Yogurts containing counts
of >1.0 × 108 cfu mL−1 of the individual probiotics and high counts of the traditional species from yogurt were awarded
overall scores for sensory acceptability >4.0 out of 5.0, and it was reported that the nutraceuticals appeared to have no
adverse effect on flavor (Awaisheh et al., 2005).
5.2
Prebiotics
The term prebiotic was introduced by Gibson and Roberfroid as a non-digestible and selectively fermented ingredient that
beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in
the colon, conferring benefits upon host well-being and health (Gibson and Roberfroid, 1995).
Today, only bifidogenic, non-digestible oligosaccharides (particularly inulin, its hydrolysis product oligofructose, and
(trans) galactooligosaccharides), fulfill all the criteria for prebiotic classification. They are dietary fibers with a wellestablished positive impact on the intestinal microflora. Other health effects of prebiotics (prevention of diarrhea or obstipation, modulation of the metabolism of the intestinal microbiota, cancer prevention, positive effects on lipid metabolism,
stimulation of mineral adsorption and immunomodulatory properties) are indirect, that is, mediated by the intestinal microbiota, and therefore less well proven. In recent years, successful attempts have been reported to make infant formula more
breastmilk-like by the addition of fructo- and (primarily) galactooligosaccharides (Gibson et al., 1995; Saito et al., 1992).
Several in vitro studies were reported. In one case, pure culture species of Bifidobacterium (longum, breve, pseudocatenulatum, adolescentis) were tested for their ability to ferment oligofructose, and it was observed that B. adolescentis was seen
to grow best and was able to metabolize both short- and long-chain oligofructose (Gibson et al., 2007). Another study tested
the ability of Bifidobacterium and Lactobacillus to grow on MRS agar containing oligofructose. It was reported that 7 of 8
Bifidobacterium strains and 12 of 16 Lactobacillus strains were able to grow on agar containing oligofructose (Kaplan and
Hutkins, 2000).
10 SECTION | A Probiotics and Prebiotics
5.3
Synbiotics
The term synbiotic is used when a product contains both probiotics and prebiotics, thus providing the probiotic bacteria
in combination with a prebiotic component that stimulates probiotic bacteria survival and growth in the gastrointestinal
tract (Schrezenmeir and de Vrese, 2001). Symbiotics provoke significant alterations in the composition of the colonic microbiome leading to altered metabolic activity of the organ. It reduces exposure of cytotoxic agents, including mutagens
and carcinogens, to the intestinal lining; decreases cell proliferation in the colonic tissue; and develops mucosal structure
(Raman et al., 2016).
It has been reported in several studies that synbiotic combination of a specific oligofructose-enriched inulin (SYN1) and
Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12 for 12 weeks caused a 16% and 18% increase in the numbers
of Lactobacillus and Bifidobacterium, respectively, and a 31% decrease in the numbers of Clostridium perfringens (Rafter
et al., 2007; Tufarelli and Laudadio, 2016), and also, that synbiotics consumption decreases cancer risk factors in patients
with colon cancer (Tufarelli and Laudadio, 2016). Synbiotics are also reported to show immunomodulatory effects (Raman
et al., 2016). A recent study evaluated the effect of consumption of a symbiotic shake containing Lactobacillus acidophilus,
Bifidobacterium bifidum, and fructooligosaccharides on glycemia and cholesterol levels in elderly people. This study was
conducted on 20 volunteers (10 for the control group and 10 for the symbiotic group), aged 50 to 60 years, and criteria
for inclusion in the study were total cholesterol > 200 mg/dL, triglycerides > 200 mg/dL, and glycemia > 110 mg/dL. After
30 days, it was reported that the symbiotic group showed a nonsignificant reduction in total cholesterol and triglycerides,
a significant increase in HDL cholesterol, and a significant reduction in fasting glycemia. No significant changes were
observed in the control group (Sáez-Lara et al., 2016).
6.
CONCLUSIONS
Alternative medical practices often include food-related products and currently there are numerous scientific proofs to
demonstrate the efficiency of some dietary components in preventing and even treating diseases. Modern society has
­allowed the emergence of numerous metabolic diseases and it is widely accepted that food preferences and alternatives have
changed significantly within the last 100 years. The food industry has also changed to fulfill consumers’ requirements, and
modern technologies allowed production of differently processed foods, with improved aspects such as flavor and lower
costs. Because there is an accepted correlation between modern feeding and the emergence of modern diseases, there comes
the obvious question that foods may be the answer in preventing and even treating these health-threatening conditions. In
order to try answering this question, we first must reveal the intimate impact of considered food products on the specific
physiology and molecular processes of the human body, and recent research fields, such as nutrigenomics, have emerged
in order to investigate these aspects and help develop personalized therapies with particular medicinal or functional foods
to treat and prevent diseases.
ACKNOWLEDGMENTS
This work was supported by Young Researchers Grant of the Research Institute of the University of Bucharest (ICUB), project number
13054/2017, project manager Dr. Ditu Lia-Mara.
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Section A
Probiotics and Prebiotics
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Chapter 2
Probiotics: Supplements, Food,
Pharmaceutical Industry
Swathi Putta⁎, Nagendra S. Yarla†, Dhananjaya B. Lakkappa‡, Sarat B. Imandi§, Rama Rao Malla†, Amajala K.
Chaitanya†, Brahma P.V. Chari†, Silas Saka§, Rama Rao Vechalapu§, Mohammad A. Kamal¶,‖,#, Vadim V. Tarasov⁎⁎,
Vladimir N. Chubarev⁎⁎, Korada Siva Kumar††, Gjumrakch Aliev‡‡,§§,¶¶
⁎
University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India, †GITAM University, Visakhapatnam, India, ‡Jain University,
Ramanagara, India, §Krishna University, Machilipatnam, India, ¶King Abdulaziz University, Jeddah, Saudi Arabia, ‖Enzymoics, Hebersham, NSW,
Australia, #Novel Global Community Educational Foundation, Hebersham, NSW, Australia, ⁎⁎I.M. Sechenov First Moscow State Medical University,
Moscow, Russia, ††National Institute of Food Technology Entrepreneurship and Management (NIFTEM), Haryana, India, ‡‡“GALLY” International
Biomedical Research Consulting LLC, San Antonio, TX, USA, §§University of Atlanta, Johns Creek, GA, USA, ¶¶Russian Academy of Sciences,
Chernogolovka, Russia
1.
INTRODUCTION
1.1
Prebiotics
Prebiotics are the substances which reach the colon in the intact form. They are given to the animals for beneficial microbial growth in the intestine and to assist normal digestion processes. Prebiotics are short-length carbohydrates, such as
fructooligosaccharides, glucooligosaccharides and inulin, which resist digestion in the upper gastrointestinal tract and are
fermented in the colon to produce short-chain fatty acids, such as acetate, butyrate and propionate, and have positive effects
on colonic cell growth and stability.
1.2
Probiotics
Probiotics are the live microorganisms which, when administered in adequate amounts, confer a health benefit on the host.
A live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial
community, by insuring improved use of feed or by enhancing its nutrition, by enhancing the hosťs response to disease, or
by improving the quality of the ambient environment.
1.3
Synbiotics
Synergistic combinations of both pro and prebiotics are called synbiotics because this approach includes a food or food
supplement having both live cells of the beneficial bacteria.
2. TYPES OF PROBIOTICS
The microbes used as probiotics represent different types such as bacteria, yeast or mold. However, there are more common
species of each such as:
2.1
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Bacteria
Lactobacillus: acidophilus, sporogenes, plantarum, rhamnosum, delbrueckii, reuteri, fermentum, lactus, cellobiosus,
brevis, casei, farciminis, paracasei, gasseri, crispatus;
Bifidobacterium: bifidum, infantis, adolescentis, longum, thermophilum, breve, lactis, animalis;
Therapeutic, Probiotic, and Unconventional Foods. https://doi.org/10.1016/B978-0-12-814625-5.00002-9
© 2018 Elsevier Inc. All rights reserved.
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16 SECTION | A Probiotics and Prebiotics
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Streptococcus: lactis, cremoris, alivarius, intermedius, thermophilis, diacetylactis;
Leuconostoc mesenteroides;
Pediococcus;
Propionibacterium;
Bacillus;
Enterococcus;
Enterococcus faecium.
2.2 Yeast and Molds
Saccharomyces cerevisiae, Saccharomyces bourlardii, Aspergillus niger, Aspergillus oryzae, Candida pintolopesii,
Sacaromyces boulardii.
Ideal probiotic strains for this kind of application should be resistant to bile, hydrochloric acid, and pancreatic juice;
be able to tolerate stomach and duodenum conditions and gastric transport; and have the ability to stimulate the immune
system, thereby improving intestinal function via adhering to and colonizing the intestinal epithelium. In addition, probiotic strains must be able to survive during manufacture and storage in order to exert significant and worthwhile healthful
outcomes (Lin et al., 2006). Fig. 1 demonstrates about the minimum qualification of a microbe in order to substantiate it as
a probiotic (FAO/WHO, 2002). Whereas Table 1 demonstrates about the overview of various microbial species recognized
as probiotics.
Potency
Probiotics
Efficacy
Scientifically
validated
health
benefits
Safety
FIG. 1 Substantiation of the word “PROBIOTIC”.
TABLE 1 Description of Probiotic Microorganisms
Species
Nature
Mechanism
Reference
Lactobacillus
species
Gram-positive, facultative anaerobic
or microaerophilic, rod-shaped
Production of enzymes which digest and metabolize
proteins or carbohydrates, synthesize vitamin B complex,
vitamin K and facilitate breakdown of bile salts
Patil and
Reddy
(2006)
Bifidobacterium
species
Gram-Positive, non-motile, often
branched anaerobic bacteria, rods
shaped
Metabolize lactose, generate lactic ions from lactic acid
and synthesize vitamins. They also ferment indigestible
carbohydrates and produce beneficial short chain fatty acids
Galdeano
et al. (2007)
Streptococcus
thermophilus
Gram-positive bacterium, and a
fermentative facultative anaerobe,
chain of spheres
Metabolize lactose, improve lactose intolerance and
antimicrobial activity
Soccol
et al. (2010)
Saccharomyces
boulardii
Gram negative, can grow under
aerobic or anaerobic conditions
The diploid form is ellipsoid-shaped,
haploid form is spherical shaped
Secrete proteases and other substances that breakdown
bacterial enterotoxins and inhibit their binding to intestinal
receptors. They also enhance vitamin production and reduce
serum cholesterol level
Kedar et al.
(2010)
Probiotics: Supplements, Food, Pharmaceutical Industry Chapter | 2 17
The selection of the probiotic must be strain specific, viable and dose specific, must remain stable and viable for long
period of time, have the capacity to survive through the stomach and into the intestine, and thrive in the intestine. The level
of probiotics in the food that serve as delivery systems needs to be high, suggesting a minimum level of live probiotic cells
should be at least 106–107 CFU/mL or CFU/g before consumption (Nualkaekul et al., 2012).
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FUNCTIONS OF GUT MICROBIOTA
Participation in the formation of the intestinal wall.
Resistance to colonization (Cummings and Macfarlane, 1997).
Production of vitamins (Lanning et al., 2005).
Production of short chain fatty acids, metabolites (Resta, 2009).
Modulation of the innate as well as the acquired immune system.
Interactions with the mucosalimmune system (Turnbaugh et al., 2009).
Degradation of xenobiotics (Qin et al., 2010).
Able to stimulate epithelial cell signaling pathways (Stetinova et al., 2010).
Increased production of cytoprotective molecules (Liu et al., 2015).
MECHANISM OF ACTION OF PROBIOTICS
Main mechanisms of action of probiotics (Narwal and Shashi, 2011; Rastogi et al., 2011; Tiwari et al., 2012; Calafiore
et al., 2012; Kumar, 2013) are the following:
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Balances the gut and oral microbial flora thus making its colonization resistant to pathogenic bacteria.
Modifies the structure and function of intestinal epithelium and improves the barrier function.
Improves water quality.
Interacts with phytoplankton.
Provides macro- and micronutrients.
Synthesizes and enhances the bioavailability of nutrients.
Reduces symptoms of lactose intolerance.
Decreases the prevalence of allergy in susceptible individuals.
Inhibits the growth of pathogenic enteric bacteria by decreasing luminal pH.
Produces antimicrobial compounds like hydrogen peroxide, bacteriosin, organic acids and dipicolinic acid which inhibits the growth of pathogenic bacteria.
Decreases production of inflammation-associated molecules.
Prevents cytokines-induced apoptosis.
Alters the host immune response by increasing IL-10, TGF-β, decreasing TNF-α and increasing the IgA production.
Competes for adhesion sites.
Modifies microbial population by biofilm formation.
Increases the calcium and other minerals absorption.
Lowers the toxigenic and mutagenic reaction by its metabolic product.
Increase the turnover of enterocytes by production of butyric acid.
SAFETY AND RISK ASSESSMENT
For safety assessment of microbial species used in food and feed productions, to set priorities for the need of risk assessment.
The assessment is made for a selected group of microorganisms, which if favorable, leads to the “Qualified Presumption of
Safety” status (EFSA). The safety of the potential probiotic should be assessed by the minimum required tests:
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Determination of antibiotic resistance patterns.
Assessment of certain metabolic activities (e.g., d-lactate production, bile salt deconjugation).
Assessment of side effects during human studies.
Epidemiological surveillance of adverse incidents in consumers (after market).
If the strain under evaluation belongs to a species that is a known mammalian toxin producer, it must be tested for toxin
production. One possible scheme for testing toxin production has been recommended by the EU Scientific Committee
on Animal Nutrition (SCAN, 2000).
If the strain under evaluation belongs to a species with known hemolytic potential, determination of hemolytic activity
is required.
18 SECTION | A Probiotics and Prebiotics
6.
PROBIOTIC THERAPY
6.1
Diabetes
The “gut connection” to diabetes received more attention in recent years because there is an intricate relationship between
intestinal microbiota and development of metabolic diseases primarily diabetes.
Lower counts of Bifidobacterium and Faecalibacterium prausnitzii were found in diabetic individuals and both of them
Gram-positive (Furet et al., 2010). Increased counts of Bacteroides ovatus and decreased Bacteroides fragilis were found
in type I diabetics (Goffau et al., 2013). Type I diabetic children showed higher counts of Clostridium, Bacteroides and
Veillonella, followed by lower counts of Bifidobacterium and Lactobacillus than in healthy children (Murri et al., 2013).
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L. casei significantly decreased plasma glucose levels and inhibited the production of β-cells specific CD4 T-cells and
cytokines that are the binding factors for induction of autoimmune diabetes (Matsuzaki et al., 1997).
L. acidophilus CCFM6, L. plantarum CCFM47, CCFM232, and L. rhamnosus GG (LGG) have the ability to affect rat
intestinal α-glucosidase inhibition, short-chain fatty acids production, and utilization of prebiotics, as well as gastrointestinal tract tolerance.
L. acidophilus NCDC14 and L. casei NCDC19 has apparently reduced lipid peroxidation, HbA1c and ameliorated
intestinal transit in diabetic rats; however, without concomitant blood glucose reduction (Yadav et al., 2008). It has also
been shown that L. casei decreased the oxidative stress and suppressed the effector functions of CD4+ T cells, accompanied by reducing the proinflammatory molecules (Villarini et al., 2008).
L plantarum DSM15313 and L gasseri BNR17 is suggested to reduce glycaemia, improve glucose tolerance, and reduce
insulin resistance (Andersson et al., 2010; Yun et al., 2009).
L reuteri GMNL-263 has been shown to reduce glycaemia and HbA1c levels, and to prevent renal fibrosis (Lu
et al., 2010).
L. acidophilus La5 and B. lactis Bb12 noticeably reduce blood glucose levels, glycated hemoglobin and remarkably improve antioxidant status and total serum antioxidant capacity with elevation of erythrocyte superoxide dismutase (SOD)
and glutathione peroxidase (GPX) levels (Ejtahed et al., 2011).
Bifidobacterium adolescentis improves insulin sensitivity by increased production of glucagon-like peptide 1 (GLP-1)
(Chen et al., 2012).
L. acidophilus La5, B. lactis Bb12 decreased fasting blood glucose levels and HbA1c, increased erythrocyte superoxide
dismutase, glutathione peroxidase activities, and total antioxidant status (Ejtahed et al., 2012).
L. acidophilus, L. casei, L. bulgaricus, L. bifidum, B. longam, B. breve and s. thermophilus rise in fasting plasma glucose levels and resulted in a decrease in serum hs-CRP and an increase in plasma total GSH (Asemi et al., 2013).
L. reuteri GMNL-263 have been demonstrated to suppress serum glucose, insulin, leptin, C peptide, glycated hemoglobin, GLP-1 level, inflammatory IL-6 and TNF in adipose tissues and PPAR andGLUT4 gene expression in high
fructose-fed rats (Hsieh et al., 2013).
L. sporogens had significant effects on serum insulin, hs-CRP, uric acid and plasma total GSH levels (Asemi
et al., 2014).
Lb. kefiranofaciens M and Lb. kefiri K prevented the onset of T1D by stimulating the production of GLP-1, regulating
the immune-modulatory reaction, and modifying the microbiota (Wang et al., 2012).
A combination of probiotics with the ability to inhibit α-glucosidase and a prebiotic can be used to mitigate T2D and
also protect the probiotics from adverse conditions of the GIT.
It is, therefore, possible that an oral supplementation of probiotics with antihyperglycemic properties might be beneficial to T2D patients (Lawrence et al., 2015). The administration of probiotics may improve the prognosis of diabetes
through modulation of gut microbiota. Probiotics increase insulin sensitivity and reduce autoimmune response by reducing
the inflammatory response and oxidative stress, as well as increasing the expression of adhesion proteins within the intestinal epithelium, reducing intestinal permeability.
6.2
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Obesity
L. sporogenes experienced a mean 32% reduction in total cholesterol and 35% reduction in LDL cholesterol over a
3-month period (Mohan, 1990).
L. gasseri, abdominal, visceral, and subcutaneous fat areas decreased significantly. Body weight also decreased significantly (Lu et al., 2004).
Probiotics: Supplements, Food, Pharmaceutical Industry Chapter | 2 19
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S. thermophilus and E. faecium resulted in an 84% reduction in LDL and an increase in fibrinogen levels and precipitation of cholesterol with free bile acids, formed in the media because the activity of the bacterial enzyme bile salt hydrolase (Parvez et al., 2005).
L. paracasei Angiopoietin-related protein 4 (Angptl4), a lipoprotein lipase inhibitor which inhibits the uptake of fatty
acids from circulating triglyceride-rich lipoproteins in white adipose and muscle tissues was found to be increased
(Aronsson et al., 2010).
L. acidophilus NCFM and L. gasseri SBT2055 showed a decrease in fat mass (Andreasen et al., 2010).
B. pseudocatenulatum SPM 1204, B. longum SPM 1205, and B. longum SPM 1207 has been shown to decrease body
weight gain and fat accumulation, blood serum levels of total cholesterol, HDL-C, LDL-C, triglyceride, glucose, leptin,
and liver toxicity biomarkers (AST, ALT) (An et al., 2011).
Pediococcus pentosaceus LP28/Lactobacillus plantarum SN13T has been shown to decrease body weight gain, visceral
fat accumulation and liver lipid contents (triglyceride and cholesterol) and hepatic lipid droplet accumulation and adipocyte size (Zhao et al., 2012).
Lactobacillus curvatus HY7601 or Lactobacillus curvatus HY7601 in combination with Lactobacillus plantarumKY1032 effectively suppressed body weight gain and reduced the adipose tissue weight in mice fed a high-fat highcholesterol diet for 9 weeks (Yoo et al., 2013).
Lactobacillus acidophilus, L. bulgaricus, L. bifidum, and L. casei shown to decrease triglyceride, malondialdehyde
(MDA), IL-6 and insulin resistance (Mazloom et al., 2013).
Saccharomyces boulardii Biocodex shown to decrease body weight gain and fat mass, hepatic steatosis and total liver
lipids content, decreases hepatic (50% decrease in CD11c macrophages level, F4/80, IL-1β and MCP-1mRNA) and
systemic inflammation (↓plasma cytokine concentrations of IL-6, IL-4, IL-1β and TNF-α) (Everard et al., 2014).
Lactobacillus rhamnosus GG shown ↑ glucose tolerance ↑ insulin-stimulated Akt phosphorylation and GLUT4 translocation in skeletal muscle ↓ endoplasmic reticulum (ER) stress in skeletal muscle ↓ M1-like macrophage activation in
white adipose tissues ↑ insulin sensitivity (Park et al., 2015).
6.3 Liver Diseases
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Lactobacillus casei Normalized phagocytic capacity, decreased TLR4, sTNFR1, sTNFR2, and IL10 levels (Loguercio
et al., 2005).
Lactobacillus acidophilus, Lactobacillus helveticus, and Bifidobacterium in rats with alcohol pancreatitis-related liver
damage effectively protected against endotoxin/bacterial translocation, as well as liver damage in the course of acute
pancreatitis and concomitant heavy alcohol consumption (Marotta et al., 2005).
Lactobacillus, Bifidobacterium and Bacteroides. These changes reduce the expression of TNF-α, IL-1β, and IL-6, and
attenuate necroinflammation of the liver (Nardone et al., 2010).
Lactulose versus l-ornithine l-aspartate versus probiotics 110 billion colony-forming units twice daily for 3 months
improved minimal hepatic encephalopathy and improve quality of life (Kuczynski et al., 2011).
Lactobacillus rhamnosus, 12 billion CFU/day for 8 weeks, improved transaminases, reduced lipopolysaccharide levels
(Vajro et al., 2011).
Lactobacillus rhamnosus GG shown to reduced plasma ALT, endotoxin level, liver steatosis, and inflammation as increasing HIF-mediated mucosal protecting factors and tight junction proteins, positive modification of gut microflora
and reduction of endotoxemia (Wang et al., 2011).
L. plantarum encapsulated alginate beads induce a dose-dependent reduction of endotoxin level in rats exposed to alcohol. Also, a reduction in liver function test was observed, as well as molecular markers of inflammation (e.g., NF-κβ,
TNF-α and IL-12/p40 subunit) (Murguia-Peniche et al., 2013).
Lactobacillus GG supplementation reduced hepatic inflammation and markedly reduced TNF-expression (Wang et al., 2013).
L. acidophilus and B. longum shown to improve intestinal permeability, attenuated hepatic fat accumulation and
butyrate-­producing probiotic, Clostridium butyricum, decreased hepatic inflammatory indexes, insulin resistance, triglycerides content, and endotoxin level (Endo et al., 2013).
Bifidobacterium adolescentis SPM0212 found increased expression of myxovirus (Mx) resistance A, decreased extracellular surface antigen of HBV level and the gene expression was inhibited by 40% in HepG2.2.15 cells (Lee et al., 2013).
Reduced risk of hospitalization for HE (hepatic encephalopathy), improved CTP (Child-Turcotte-Pugh) and MELD
(model for end-stage liver disease) scores (Dhiman et al., 2014).
Saccharomyces boulardii is able to reduce hepatic steatosis through lowering the hepatic lipid content and low-grade
systemic inflammation (Everard et al., 2014).
20 SECTION | A Probiotics and Prebiotics
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Lactobacillus rhamnosus R0011 and Lactobacillus Acidophilus acts as modulation of the gut-liver axis: reduction of
ALT, TLR4, TNF-, and IL-1 expression, increasing IL-10 expression (Hong et al., 2015).
Lactobacillus rhamnosus and acidophilus mildly decreased intrahepatic lymphocytes and TNF-α expression, as well as
reverse irregular and deteriorated microvilli due to alcohol exposure (Hong et al., 2015).
6.4
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B. longum also plays a role as an immunomodulator and biological response modifier by providing an additional mechanism of tumor suppression. Probiotics also stimulate apoptosis through end-product formation (Okawa et al., 1993).
Bifidobacterium produces lactic acid and lower the intestinal pH and therefore develops a favorable environment which
modulates the bacterial enzymes and has cell wall antitumor activities and induces activation of phagocytes to destroy
growing tumor cells (Sekine et al., 1994).
Lactobacillus rhamnosus GG, Bifidobacterium lactis Bb12 and oligofructose-enriched inulin reduced proliferation and
DNA damage in colonic mucosa and the capacity of fecal water samples to induce necrosis in colonic cells in polypectomized patients. Increased production of interferon (IFN)-γ by peripheral blood mononuclear cells (PBMC) was
observed in the cancer patients (Rafter et al., 2007).
Lactobacillus augments the functions of macrophages, natural killer (NK) cells and T cells and exerts anti-cancer activity (Kato et al., 1988).
Bifidobacterium adolescentis SPM1207 had less fecal water content than did control rats, decreasing colon toxicity, due
to reduced exposure to soluble toxic compounds (Lee et al., 2009).
Lactobacillus rhamnosus GG administration reduced the number of coliforms and significantly elevated the count of
lactobacilli (Bertkova et al., 2010).
L. gasseri OLL2716: LG21 in colorectal cancer patients was shown to decrease alkalosis in stool and cancer markers
(fecal product putrescine synthesis oxidized products from incomplete fermentation) (Apás et al., 2010).
B. longum BB536 and L. johnsonii shown probiotic adherence to the colonic mucosa, decreased pathogens and dendritic
phenotypes CD83-123, CD83-HLADR, CD83-11c (Gianotti et al., 2010).
Lactobacillus acidophilus and Lactobacillus casei were able to increase the apoptosis-induction capacity of 5-­fluorouracil
in colorectal carcinoma cell line LS513, suggesting that these probiotics may be used as adjuvants in anticancer chemotherapy (Baldwin et al., 2010).
L. rhamnosus and P. freudenreichii were able to diminish the amount of cancer proteins as c-myc, bcl-2, cyclin D1 and
rasp-21 and enhance the levels of GSH, SOD, CAT reduce inflammation and infection (Kumar et al., 2011).
Bifidobacterium or Lactobacillus consumption may limit the formation of toxic metabolites by decreasing the dehydroxylation of primary bile acids and reducing fecal deoxycholic acid concentrations (De Preter et al., 2011).
Propionibacterium freudenreichii increased apoptosis, chromatin condensation, formation of apoptotic bodies, cytotoxicity of camptothecin, DNA laddering, cell cycle arrest and ROS formation (Kumar et al., 2013).
6.5
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Cancer
Diarrhea
S. boulardii effectively reduces the incidence of AAD. In a study of 180 hospitalized patients, 1 g daily of S. boulardii
taken during and up to 2 weeks after antibiotic therapy reduced the incidence of AAD by over 55% (9.5% vs. 21.8%)
(Surawicz et al., 1989).
S. boulardii, for which there is a risk of hematogenous dissemination in immunocompromised patients, was effective in
inhibiting the recurrence of episodes of clostridium difficile infection (Castagliuolo et al., 1999).
L. rhamnosus GG (LGG) or a placebo given to 204 malnourished children in Peru (6–24 month old) was associated with
a significantly lower incidence of diarrhea (Oberhelman et al., 1999).
Lactobacillus plantarum reduces incidence of diarrhea in daycare centers when administered to only half of the children
(Vanderhoof, 2000).
Saccharomyces boulardii were able to reduce recurrence of clostridium difficile diarrhea and effects on C. difficile and
Klebsiella oxytoca resulted in decreased risk and/or shortened duration of antibiotic-associated diarrhea and shortened
the duration of acute gastroenteritis (Marteau et al., 2001).
Lactobacillus GG was analyzed in a meta-analysis, as for separate etiologies, it was evident that this probiotic was most
effective for rotavirus diarrhea, where it induced an average reduction of diarrhea is 95% (Szajewska et al., 2007).
Lactobacillus rhamnosus seem to have the capability of reducing the shedding of rotavirus33 in a dose-dependent manner, thus allowing reducing the risk of spreading of the infection (Fang et al., 2009).
Probiotics: Supplements, Food, Pharmaceutical Industry Chapter | 2 21
6.6 Allergies
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Bifidobacteria and Lactobacilli potentiate IgA response to potentially harmful antigens and IgA production in Peyer’s
patches have been shown to be enhanced (Yasui et al., 1992).
Lactobacillus GG influenced the immune response of human by activation of lymphoid cells present in lymphoid tissues of gastrointestinal tract (Majamaa and Isolauri, 1997).
Lactobacillus GG was administered to high risk infants; there was a 50% reduction in observed atopic eczema
(Kalliomaki et al., 2001).
Lactobacillus GG reduced symptom of score, and reduced sensitivity to birch and apple pollen (Helin et al., 2002).
L. fermentum reduced symptoms of atopic dermatitis in infants with moderate-to-severe disease (Weston et al., 2005).
L. rhamnosus HN001 in pregnant women and their newborn infants substantially reduced the cumulative prevalence of
eczema in infants (Wickens et al., 2008).
L. casei DN-114 001 probiotic (with immune-modulating activity) could improve human health and modify the immunological profile of pre-school age children with allergic symptoms to inhalants (Giovannini et al., 2007).
L. casei reduced the number of rhinitis episodes in 64 preschool children with allergic rhinitis (Giovannini et al., 2007).
L. casei CRL431 and B. lactis Bb-12 are used as supplementation for 12 months to 119 infants with cow's milk allergy
showed tolerance in Hol et al. (2008).
Probiotic preparations were also used to treat antibiotic-associated diarrhea, lactose intolerance, dental problems, Helicobacter
pylori infections, irritable bowel syndrome, necrotizing enterocolitis, vitamin production, eczema and bacterial vaginosis.
There are several techniques developed to make a required dosage form of probiotics with high efficacy to increase the
shelf life of probiotics in its powder form. Some techniques to prepare power form of probiotics are encapsulation, extrusion and emulsification with spray drying, freeze drying, fluidized bed drying and gel bead technology.
Tablets and capsules can be easily designed to control the release, enhancing the adhesion and colonization of probiotic
microorganisms to the intestinal epithelium of human host. Several new techniques like PROBIO-TECH, STAR, LIVEBAC,
PROBIOCAP, starch encapsulation of LAB, controlled drug delivery, oil matrix complex, cryotabletting, trisphere, and bioadhesive vaginal tablets were developed to provide resistance to stomach acid, to improve stability, to prevent solubilization
of probiotics, to release the drug only in intestinal pH, and to improve the shelf life of probiotics (Yadav and Bhitre, 2013).
7. AVAILABLE PROBIOTIC FOOD
7.1 Yakult
Yakult is the live (LcS) (Lactobacillus casei Shirota). Yakult contains skimmed milk powder, sugar, glucose, water, and
more than 6.5 billion LcS.
Uses:
1. Prevent digestive disorders such as diarrhea and constipation.
2. Help build immunity and reduce risk of infections.
7.2
Kefir
Kefir is a microbial symbiotic mixture of lactic acid bacteria (108 CFU/g), yeast (106–107 CFU/g), and acetic acid bacteria (105
CFU/g) that stick to a polysaccharide matrix. The predominant species for kefir preparation are Lactobacillus paracasei ssp.
paracasei, Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus plantarum, and L. kefiranofaciens.
Uses:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Antitumoral
Anti-inflammatory
Antimicrobial
Immunoregulatory
Antiallergenic
Wound healing
Antidiabetic
Antimutagenic
Antigenotoxic
22 SECTION | A Probiotics and Prebiotics
7.3 Yogurt
Yogurt is a dairy product obtained through the fermentation of milk, partly skimmed milk or skim milk by the lactic bacteria Lactobacillus bulgaricus and Streptococcus thermophilus with which the lactic bacteria Lactobacillus acidophilus
and Lactobacilluscasei or the bacteria Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium infantis and
Bifidobacterium breve may be combined.
Uses:
1.
2.
3.
4.
5.
6.
7.
8.
7.4
Prevent osteoporosis
Reduce the risk of high blood pressure
Antibiotic-associated diarrhea and acute diarrhea in children
Vaginal yeast and bacterial infections
Urinary tract infections
Lactose intolerance
Helicobacter pylori infections that cause stomach ulcers
Preventing colorectal cancer
Kombucha
Is a mixture of black tea and sugar fermentation. Technically, the fermentation becomes a mixture of yeast and bacteria (i.e.,
Bacterium xylinum, Bacterium gluconicum, Acetobacter ketogenum, and Pichia fermentans).
Uses:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
7.5
Antidiabetic activity
Reduce atherosclerosis
Antihypertensive effect
Anti-inflammatory
Alleviate arthritis, rheumatism, and gout
Hepatoprotective activity
Cure hemorrhoids
Increase body resistance
Enhance the immune system
Relieve bronchitis and asthma
Reduce menstrual disorders and menopausal hot flashes
Improve hair, skin, and nail health
Reduce stress and nervous disturbances, and insomnia
Improve eyesight
Sauerkraut
The cabbage is finely shredded, layered with salt, and left to ferment. Fully cured sauerkraut keeps for several months in an
airtight container stored at 15°C (60°F) or below by a process of pickling called lactic acid fermentation with Lactobacillus
species, including L. brevis and L. plantarum.
Uses:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Increases the bioavailability of nutrients
Promote gut health
Boost circulation
Cardioprotective action
Provide quick energy
Stimulate the immune system
Strengthen bones
Antihyperlipidemic activity
Anti-inflammatory activity
Anticancer property
Improve vision and skin health
Probiotics: Supplements, Food, Pharmaceutical Industry Chapter | 2 23
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FURTHER READING
Coccorullo, P., Strisciuglio, C., Martinelli, M., Miele, E., Greco, L., Staiano, A., 2010. Lactobacillus reuteri (DSM 17938) in infants with functional
chronic constipation: a double-blind, randomized, placebo-controlled study. J. Pediatr. 157, 598–602.
Narbona Lopez, E., Uberos Fernandez, J., Armada Maresca, M.I., Couce Pico, M.L., Rodriguez Martinez, G., Saenz de Pipaon, M., 2014. Nutrition and
Metabolism Group of the Spanish Neonatology Society: recommendations and evidence for dietary supplementation with probiotics in very low birth
weight infants. An. Pediatr. 81 (6), 397.
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­associated to bronchus. Int. J. Immunopathol. Pharmacol. 12, 97–102.
Wong, V.W., Tse, C.H., Lam, T.T., 2013. Molecular characterization of the fecal microbiota in patients with nonalcoholic steatohepatitis—a longitudinal
study. PLoS One 8 (62885).
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Chapter 3
Selection of New Probiotics:
The Case of Streptomyces
Sneha Hariharan⁎, Selvakumar Dharmaraj†
*
University of Madras, Chennai, India, †Karpagam Academy of Higher Education, Coimbatore, India
1.
INTRODUCTION
1.1 Aquaculture Status of the World
Feeding an expected global population of 9.6 billion by 2050 is a daunting challenge that is engaging researchers, technical experts, and leaders the world over. A relatively unappreciated yet promising fact is that fish can play a major role in
satisfying the palates of the world’s growing middle income class while also meeting the food security needs of those with
less income. Already, fish represents 16% of all animal protein consumed globally, and this proportion of the world’s food
basket is likely to increase as consumers with rising incomes seek higher value seafood, while aquaculture steps up to
meet increasing demand. Aquaculture has grown at an impressive rate over the past decades. It has helped to produce more
food fish, kept the overall price of fish down, and made fish and seafood more accessible to consumers around the world
(Word Bank Report, 2013). For this reason, greater investment is needed in the industry for new and safer technologies,
their adaptation to local conditions, and their adoption in appropriate settings. Fisheries and aquaculture are sources of not
just health, but also of wealth. Employment in the sector has grown faster than the world’s population, providing jobs to
tens of millions and supporting the livelihoods of hundreds of millions. Fish continues to be one of the most-traded food
commodities worldwide. It is especially important for developing countries, sometimes worth half the total value of their
traded commodities. Global fish production continues to outpace world population growth, and aquaculture remains one
of the fastest-growing food producing sectors (FAO, 2016). FAO reports concerning aquaculture status of the world for the
years 2015 and 2016 are still in development. The FAO report for the year 2014 is complete and clearly discussed below.
Global aquaculture production has experienced tremendous growth over past 50 years, starting from a production of less
than a million tons in the early 1950s to over 167.2 million tons in 2014 (Fig. 1) and is expected to rise in accordance with
the demands of the world’s growing population. Individually, the inland fisheries and mariculture alone contribute around
47.19 and 53.90 million tons in the year 2014 (Fig. 2). Aquaculture production has increased at an average annual growth
rate of 5.8%, from 44.3 million tons in 2005 to 73.8 million tons in 2014. The value of aquaculture production is estimated
at USD $160.2 billion in 2014, and may continue to rise in the future (FAO, 2016).
World food fish aquaculture production in 2014 consisted of 49.9 million tons of finfish (68%), 16.1 million tons of
mollusks (22%), 6.9 million tons of crustaceans (9%), and 0.9 million tons of other aquatic animal species (1%). Inland
aquaculture produced 43.6 million tons of finfish, representing 59% of world food fish aquaculture in 2014. The above
quantities do not include production of aquatic plants, mostly seaweeds, that reached 28.5 million tons in 2014, of which
aquaculture produced 27.3 million tons (96%). Carrageenan seaweeds (including Kappaphycus alvarezii and Eucheuma
spp.) are the main cultured seaweeds (11 million tons), followed by Japanese kelp (7.7 million tons) (FAO, 2014).
Aquaculture experienced high average annual growth of 10.8% in the 1980s and 9.5% in the 1990s. However, the
growth rate eased to an average of 5.8% in the period of 2005–14. It has been estimated that Asia has the highest aquaculture production (quantitative and value) when compared with rest of the continents (Figs. 3 and 4). The contribution of
aquaculture to total fish production has risen steadily, reaching 44% in 2014. Asia has produced more farmed fish than wild
fish since 2008, with aquaculture reaching a share of 55% of its total production in 2014. In the same year, the contributions
from aquaculture were between 17% and 18% for other continents, except Oceania (13%). In 2014, the top ten aquaculture producers (excluding aquatic plants and nonfood products) were China (45.5 million tons), India (4.9 million tons),
Therapeutic, Probiotic, and Unconventional Foods. https://doi.org/10.1016/B978-0-12-814625-5.00003-0
© 2018 Elsevier Inc. All rights reserved.
27
180
120
Value
Volume
100
140
120
80
100
60
80
60
40
40
20
20
0
2005
2006
2007
2008
2009
2010
2011
2012
2013
0
2014
Year
FIG. 1 Global aquaculture production.
Inland
fisheries
46%
Mariculture
54%
FIG. 2 World aquaculture production status in 2014.
0.21
2.93
1.86
3.36
Africa
America
Asia
Europe
Oceania
92.72
FIG. 3 World aquaculture production quantity (million tons) by continents in the year 2014.
Production value (million US$)
Production quantities (million tons)
160
Selection of New Probiotics: The Case of Streptomyces Chapter | 3 29
1.42
13.62
3.7
19.02
Africa
America
Asia
Europe
Oceania
128.02
FIG. 4 World aquaculture production value (million US$) by continents in the year 2014.
Indonesia (4.3 million tons), Vietnam (3.4 million tons), and Bangladesh (2 million tons), followed by Norway, Chile,
Egypt, Myanmar, and Thailand. They contributed 89% of the world’s production (FAO, 2014).
Many millions of people around the world find a source of income and livelihood in the fisheries and aquaculture sector. In 2014, about 56.6 million people were engaged in the primary sector of capture fisheries and aquaculture and, of this
total, 36% were engaged full time, 23% part time, and the remainder were either occasional fishers or of unspecified status.
For the first time since the period 2005–10, the total engagement in fisheries and aquaculture did not increase (FAO, 2016).
1.2
Implementation of Probiotics Usage in Aquaculture
Although aquatic food production through aquaculture is the fastest growing sector, and vaccines are being developed and
marketed in aquaculture, disease is still a major problem in the aquaculture industry (Bondad-Reantaso et al., 2005). During
the last decades, chemical additives and veterinary medicines, especially antimicrobial agents, were used to prevent and
control diseases that occur in aquaculture (Wang and Xu, 2004; Cabello, 2006; Lupin, 2009). However, the risks associated
with the transmission of resistant bacteria from aquaculture environments to humans, and the introduction in the human
environment of nonpathogenic bacteria, containing antimicrobial resistance genes, and the subsequent transfer of such
genes to human pathogens existed according to FAO (2005). Consequently, serious loss because of the spread of diseases
has been increasingly recorded. This is a significant constraint on aquaculture production and trade, and negatively affects
economic development in many countries.
The utility of antimicrobial agents as a preventive measure has been questioned, given extensive documentation of the
evaluation of antimicrobial resistance among pathogenic bacteria. On the other hand, antibiotics inhibit or kill beneficial
microbiota in the gastrointestinal (GI) ecosystem, but also made antibiotic residue accumulate, resulting in fish products
that are harmful for human consumption (WHO, 2006). Because of the health risks associated with the use of antibiotics
in animal production, there is a growing awareness that antibiotics should be used with more care (Defoirdt et al., 2011).
Resistance mechanisms can arise one of two ways: chromosomal mutation or acquisition of plasmids. Chromosomal
mutations cannot be transferred to other bacteria, but plasmids can transfer resistance rapidly (Lewin, 1992). Several bacterial pathogens can develop plasmid-mediated resistance. Plasmids carrying genes for resistance to antibiotics have been
found in marine Vibrio species and they could be the bacteria found in aquaculture ponds, transfer via plasmids, transduction via viruses, and even direct transformation from DNA absorbed from the particles in the water, or on sediment surfaces,
could all be likely mechanisms for genetic exchange (Moriarty, 1997). For example, transference of multi-drug resistance
occurred in Ecuador during the cholera epidemic (1991–94) in Latin America, beginning among persons who were working on shrimp farms. Although the original epidemic strain of Vibrio cholerae 01 was susceptible to the 12 antimicrobial
agents tested, in coastal Ecuador it became multidrug resistant by the transference of resistance genes of noncholera vibrios
that are pathogenic to shrimp (Weber et al., 1994). In addition, other evidence of the transmission of resistance between
aquaculture ecosystems and human has been demonstrated, when a novel florofenicol resistance gene floR, in Salmonella
typhimurium DT104, which also confers resistance to chloramphenicol and is almost identical by molecular sequence to
the florofenicol resistance gene was first described in Photobacterium damsela, bacterium found in fish (Angulo, 2000).
30 SECTION | A Probiotics and Prebiotics
A significant issue affecting production is the loss of stock through disease. Diseases caused by Vibrio spp. and
Aeromonas spp. are commonly implicated in episodes of mortality. When faced with disease problems, the common response has been to turn to antimicrobial drugs. The livestock and aquaculture industries have experienced widespread use
of antimicrobial drugs in their practices. While the use of such products has an obvious benefit to treat animals infected
by bacterial disease, the use of antimicrobial drugs has been either prophylactic (preventative), or for growth enhancement
(Van den Bogaard and Stobberingh, 2000). Certain antimicrobial drugs have been shown to positively influence growth
of livestock and are used widely (Acar et al., 2000; Phillips et al., 2004). Given this, and the desire to prevent establishment of pathogenic bacteria, it is argued that antimicrobial drugs have been widely overused (Aarestrup, 1999). Schwarz
et al. (2001) provided a good overview of antimicrobial drugs used in animals and the associated potential hazards. In
aquaculture, this was felt most dramatically in the shrimp industry where massive increases in production, overcrowding
of animals and unchecked antibiotic usages led to the emergence of numerous antibiotic resistant bacteria and production
crashes in many Asian countries (Karunasagar et al., 1994; Moriarty, 1999). For example, production figures for shrimp
in the Philippines dropped by 55% in 2 years, from 90,000 to 41,000 tons between 1995 and 1997. In fact, it has never
recovered and, in 2002, a mere 37,000 tons was produced. An industry previously worth USD $760 million is now worth
only USD $240 million (FAO, 2007). Similarly, Thai shrimp production dropped by 40% between 1994 and 1997 due to
disease problems, bacterial pathogens, and shrimp viruses. Within aquaculture, there are numerous reports of antibiotic
resistant bacteria of farm origin. However, the risk is not just the potential loss to the farmer. The emergence of antibiotic
resistant bacteria on aquaculture farms could pose a risk to human health. There are many reports illustrating the transferal of resistant genes between bacteria. This process means antibiotic-resistant bacteria originating from a shrimp farm
could potentially transfer plasmids to bacteria involved in human health problems. Studies point to a farm animal origin
in certain antibiotic-resistant bacteria genes that have made their way into human bacteria. However, recent reports argue
against this phenomenon. The argument is based on the view that, although antibiotic-resistant bacteria have arisen in
animal husbandry through the use of antimicrobials, there is insufficient data to show a link to resistant gene transferal to
humans. They argue in favor of the beneficial role antibiotics play in farming, and caution against premature, unscientific
decisions to restrict antibiotic use. Regardless of which argument is true, governments and organizations have introduced
much tighter restrictions for antibiotic usage in animal production (Molina-Aja et al., 2002; Sahul Hameed et al., 2003;
Alcaide et al., 2005).
In 1997, the European Union (EU) initially banned the use of avoparcin, and in 1999, included virginiamycin, spiramcin, tylosin, and bacitracin as banned growth promoters in animal feed and also implemented a ban on the use of all
nontherapeutic antimicrobials in animal production (Delsol et al., 2005). The United States has been less stringent. There
was a proposal in 2000 to introduce a ban on the use of fluoroquinolone, and there was concern also about the use of
virginiamycin (Nawaz et al., 2001). More recently a bill called “Preservation of Antibiotics for Medical Treatment Act of
2005” was presented in the US congress. This act would see a ban on the nontherapeutic use of any drug intended for human use as well as in the production of feed animals (Martin, 2005). Other countries which currently have less antibiotic
control, such as many of the Asian countries, are likely to be pressured through foreign restrictions, via the export markets
which are being tightly controlled for antibiotic-contaminated products. Despite chloramphenicol being banned in Thailand
since 1999 as a result of worldwide concern over its use in animal production, trace levels are still detected in shrimp from
Thailand, causing a temporary ban of Thai shrimp by the EU (Heckman, 2004). Chloramphenicol has also been detected
in shrimp from Myanmar, India, Pakistan, and Vietnam, highlighting the continued misuse of antimicrobial drugs in Asian
shrimp farming. A leading example in the eradication of antibiotic use can be seen in the Norwegian salmon industry. After
concern about the use of antibiotics in the late 1980s, there has been a 95% drop in usage from 50 to 1 ton annually. During
the same period, salmon production has increased 10-fold from about 5500 to 55,000 tons. The turnaround has been attributed to the use of vaccines, better husbandry, and selective breeding programs (Maroni, 2000). There is a developing social
attitude against unnecessary use of antimicrobial drugs and where possible, farmers now seek to move away from nonessential antimicrobial drug use. Several alternative methods have been considered to improve the quality and sustainability
of aquaculture production (Li et al., 2006).
Science-based knowledge on probiotics and prebiotics has increased in recent years (Subasinghe et al., 2009; FAO,
2006). The use of probiotics or beneficial bacteria, which control pathogens through a variety of mechanisms, is increasingly viewed as an alternative to antibiotic treatment. The use of probiotics in human and animal nutrition is well documented (Rinkinen et al., 2003), and recently, they have begun to be applied in aquaculture (Gatesoupe, 1999; Gomez-Gil
et al., 2000; Irianto and Austin, 2002a,b). While probiotic research in aquaculture first focused on fish juveniles, much
attention has recently been given to larvae of fish, shellfish, and live food organisms in aquaculture as they are easily prone
to diseases, and they provide health benefits in several ways. They are important sources for C, N, and S cycles, and any
imbalance in the microflora of systems leads to pathogenesis (Rengpipat, 1996).
Selection of New Probiotics: The Case of Streptomyces Chapter | 3 31
Now, numbers of preparations of probiotics are commercially available and have been introduced to fish, shellfish,
and mollusk farming as feed additives, or are incorporated in pond water (Prado et al., 2010). According to the producers’
claims, these products are effective in supporting the health of aquatic animals and are also safe. However, there are doubts
with regard to the general concept of probiotics and to these claims. Indeed, the current explanations and principles are
still not enough to describe what probiotics actually are, where they come from, and what they can do (Wang et al., 2008).
Thus, there is clearly a need for increasing our knowledge of aquaculture animals and of effective preparation, technological applications, and safety evaluation of probiotics. The importance of aquaculture products is set to increase dramatically
as a result of overfishing of the world's waters and a continuing increase in demand for seafood. With increasing demand
for environment-friendly aquaculture, alternatives to antibiotic growth promoters in fish nutrition are now widely accepted.
The chapter mainly focuses on the importance of marine Streptomyces which can be an alternative and replacement food.
This chapter also discusses the prospects of using marine Streptomyces as probiotics and their limitations in aquaculture.
2.
PROBIOTICS
2.1
Definition
Probiotics are beneficial microorganisms, or their products, that provide health benefits to the hosts. These probiotics has been
used in aquaculture as disease control agents, or as feed supplements, to improve growth and in some cases, as a means of replacing antimicrobial compounds (Moriarty, 1997; Dharmaraj and Dhevendaran, 2010). Much research has been carried out
in the field of probiotics over the past 30 years, but the original idea was possibly formed by Metchnikoff in the early 1900s.
Metchnikoff (1907) theorized that human health could be aided through the ingestion of fermented milk products. The word
“probiotic” was introduced by Parker (1974), who defined it as “organisms and substances which contribute to intestinal microbial balance.” The term probiotic means “for life,” originating from the Greek words “pro” and “bios” (Gismondo et al., 1999).
According to Browdy (1998), one of the most significant technologies that evolved in response to disease control problems is the
use of probiotics. Probiotics are live microbes that can be used to improve the host intestinal microbial balance and growth performance. Development of probiotics in aquaculture management will reduce the prophylactive use of antimicrobial drugs, as the
recent overdependence on the antimicrobial drugs poses potential hazards to people who consume them (Salminen et al., 1999).
Fuller (1989) proposed the widely accepted definition for probiotics, which he gives as “a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance.” Fuller's definition was a
revision of the original probiotic concept which referred to protozoans producing substances that stimulated other protozoans (Lilly and Stillwell, 1965). Several modifications were put forward to shorten the definition for probiotics (Gram et al.,
1999; Irianto and Austin, 2002a,b). Verschuere et al. (2000) proposed a definition which states that “a live microbial adjunct
which has a beneficial effect on the host by modifying the host associated or ambient microbial community, by ensuring
improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment.” Kesarcodi-Watson et al. (2008) defined it as “it should benefit the host either
nutritionally or by changing its immediate environment.” Current probiotic applications and scientific data on mechanisms
of action indicate that nonviable microbial components act in a beneficial manner, and this benefit is not limited just to
the intestinal region (Salminen et al., 1999). The concept of probiotic activity has its origins in the knowledge that active
modulation of the gastrointestinal tract (GIT) could confer antagonism against pathogens, help development of the immune
system, provide nutritional benefits, and assist the intestinal mucosal barrier (Vaughan et al., 2002).
Today probiotics are quite commonplace in health-promoting “functional foods” for humans, as well as therapeutic,
prophylactic, and growth supplements in animal production and human health (Senok et al., 2005). Multiple ways exist
in which probiotics could be beneficial, and these could act either singly or in combination for a single probiotic. These
include: inhibition of a pathogen via production of antagonistic compounds, competition for attachment sites, competition
for nutrients, alterations of enzymatic activity of pathogens, immune-stimulatory functions, and nutritional benefits such as
improving feed digestibility and feed utilization (Bomba et al., 2002). It is often reported that a probiotic must be adherent
and colonize within the GIT, replicate to high numbers, produce antimicrobial substances, and withstand the acidic environment of the GIT (Dunne et al., 1999; Mombelli and Gismondo, 2000). However, these descriptions are misleading. These
beliefs are based on the understanding that a probiotic must become a permanent member of the intestinal flora. While
bacteria with this capacity are common, and much probiotic research focuses on attachment capacity of bacteria, it has actually been demonstrated that transient bacteria can also exert beneficial effects (Isolauri et al., 2004). Additionally, contrary
to the requisite of being able to attach to mucus and produce antimicrobial substances, a probiotic need only possess one
mode of action. Multistrain and multispecies probiotics have proven that it is possible to provide synergistic bacteria with
complementary modes of action to enhance protection (Timmerman et al., 2004).
32 SECTION | A Probiotics and Prebiotics
2.2
Mode of Action
There are three general modes of probiotic actions that have been classified as follows:
(1) Probiotics might be able to modulate the host’s gut defense mechanism, including the innate as well as the acquired
immune system, and this mode of action is most likely important for the prevention and therapy of infectious diseases,
as well as for the treatment of inflammation of the digestive tract or parts thereof.
(2) Probiotics can also have a direct effect on other microorganisms, commensal and/or pathogenic ones, and this principle
is of importance in many cases for the prevention and therapy of infections and restoration of the microbial equilibrium
in the gut.
(3) Finally, probiotic effects may be based on actions affecting microbial products, host products and food ingredients and
such actions may result in inactivation of toxins and detoxification of host and food components in the gut.
According to the above postulates, all three modes of probiotic actions are likely associated with gut and/or gut microbiota (Oelschlarger, 2010). Therefore, the fact (probiotics may affect the microbial products, host products and food ingredients, which may result in inactivation of toxins, detoxification of host and food components in the gut) that has apparently
dealt with is another “organ,” the so-called “microbiotic canal” with the increased knowledge of the specific activity of
the gut microbiota (Wolf, 2006). In general, the gut microbiota remain relatively stable throughout life once established,
although they can be influenced by several factors such as mode of delivery, hygiene, and the use of antibiotics. The gut
microbiota with the epithelium and mucosal immune system orchestrate a network of immunological and nonimmunological defenses, providing both protection against pathogens and tolerance to commensal bacteria and harmless antigens (Sanz
and Palma, 2009). The important role of commensal bacteria in development of an optimally functioning mucosal immune
system was demonstrated in germ-free animals (Tlaskalová-Hogenová et al., 2004). Therefore, the imbalance of gut microbiota has been linked to several diseases including inflammatory bowel diseases, periodontal disease, rheumatoid arthritis,
atherosclerosis, and allergies. So, probiotics, that is, microbial strains that have beneficial effects on the host, are thought to
benefit this intestinal ecosystem (Julio and Marie-Joséé, 2011).
In addition, some probiotics strains also induce the secretion of multiple antimicrobial materials by intestinal Paneth
cells through cell-autonomous MyD 88-dependent toll-like receptor activation (Vaishnava et al., 2008) and regulate the
alterations of permeability related with infections, stress, and inflammatory conditions (Lutgendorff et al., 2008). As for the
aquatic animals such as fish and shrimp, the colonization of the gastrointestinal tract starts immediately after hatching and
is completed within a few hours to modulate expression of genes in the digestive tract, thus creating a favorable habitat for
them and preventing invasion by other bacteria introduced later into the ecosystem (Balcázar et al., 2006). This is attributed
to competitive exclusion mechanisms and improved immune system development and maturation. Intake of probiotics has
been demonstrated to modify the composition of the microbiota, and therefore assist in returning a disturbed microbiota (by
antibiotics or other risk factors) to its normal beneficial composition (Gómez and Balcázar, 2008). As for the mechanisms
during this physiological process, the production of antimicrobial substances, competition for nutrients or adhesion receptors, inhibition of virulence gene expression, and enhancement of the immune response are all included (Irianto and Austin,
2002a,b; Vine et al., 2004; Kim and Austin, 2006; Balcázar et al., 2007). However, the exact mechanism by which these
probiotics do this is not known. Advances in the understanding of the mechanisms between gut microbiota and probiotics
and how the immune system of aquatic animals generally responds to gut microbiota would be of great help to identify the
molecular targets of probiotics and the biomarkers of their effects, and to provide sounder evidences on their benefits on
physiologic conditions and immune-mediated disorders.
Also proposed are other possible modes of action of probiotics that include the inhibition of a pathogen through the
production of bacteriocin like compounds, competition for attachment sites, competition for nutrients (particularly iron
in marine microbes), alteration of enzymatic activity of pathogens, immuno-stimulatory functions, and nutritional benefits such as improving feed digestibility and feed utilization (Kesarcodi-Watson et al., 2008; Martinez Cruz et al., 2012).
In order to achieve this probiotic status, the microbes have to follow certain number of criteria in terms of their biosafety
and function. The advantageous characteristics of a potential probiotic include (i) should not be destructive to the host,
(ii) proper transportation to the active site and their ability to survive in that environment, (iii) their potential in colonizing
and propagating in the host, and (iv) virulence genes or antibiotic resistance genes should not be expressed (Hai, 2015).
2.3
Common Microorganisms Used as Probiotics
Probiotics currently used in aquaculture industry include a wide range of taxa—from Lactobacillus, Bifidobacterium,
Pediococcus, Streptococcus, Carnobacterium spp., Bacillus, Flavobacterium, Cytophaga, Pseudomonas, Alteromonas,
Selection of New Probiotics: The Case of Streptomyces Chapter | 3 33
Aeromonas, Enterococcus, Nitrosomonas, Nitrobacter, Vibrio spp., and yeast (Sahu et al., 2008; Hemaiswarya et al., 2013).
While using some beneficial probiotic bacteria for fish, some might be highly pathogenic, Vibrio alginolyticus for example, will lead to destructive effects in the aquaculture systems. Therefore, it is necessary to take care in the choice of
probiotic before administration. The best-known probiotic strains such as Bifidobacteria, Lactobacilli, and Streptococcus
­thermophilus are employed as the dietary supplementation in the aquaculture industry and they will increase the efficiency
and sustainability of aquaculture production (Kim et al., 2007).
Typically, the lactic acid bacteria (LAB) have been widely used and researched for human and terrestrial animal purposes, and LAB are also known to be present in the intestine of healthy fish (Hagi et al., 2004). LAB mainly focused on
the fact that they are natural residents of the human gastrointestinal tract (GIT) with the ability to tolerate the acidic and
bile environment. LAB also functions to convert lactose into lactic acid, thereby reducing the pH in the GIT and naturally
preventing the colonization by many bacteria (Klewicki and Klewicka, 2004). The most widely researched and used LAB
is lactobacilli and bifidobacteria (Ross et al., 2005).
Other commonly studied probiotics include the spore forming Bacillus spp. and yeasts. Bacillus sp. have been shown
to possess adhesion abilities, produce bacteriocins (antimicrobial peptides) and provide immune-stimulation (CladeraOlivera et al., 2004; Barbosa et al., 2005). The strains appear to be effective probiotics and commercial products containing
such strains have been demonstrated to improve shrimp production to a level similar to that when antimicrobials are used
(Decamp and Moriarty, 2006). Bacillus spp. hold added interest in probiotics as they can be kept in the spore form and
therefore stored indefinitely on the shelf (Hong et al., 2005). The yeast, Saccharomyces cerevisiae, also has been commonly
studied, whereby immune-stimulatory activity was demonstrated, and production of inhibitory substances was shown (Van
der Aa Kühle et al., 2005).
2.4 Application of Probiotics in Aquaculture
When looking at probiotics intended for an aquatic usage it is important to consider certain influencing factors that were
fundamentally different from that of terrestrial based probiotics (Kesarcodi-Watson et al., 2008; Gatesoupe, 1999). A fairly
constant habitat of resident microbiota in the gastrointestinal tract of terrestrial livestock is important, whereas most microbiota is transient in aquatic animals (Moriarty, 1999). A shift in the intestinal microflora of Atlantic halibut (Hippoglossus
hippoglossus) larvae during first feeding was studied and the results showed the transition from a prevailing Flavobacterium
spp. intestinal flora to an Aeromonas spp./Vibrio spp. Dominant flora occurred when first feeding commenced (Bergh et al.,
1994). It indicated that the gut microbiota of aquatic animals may change rapidly with the intrusion of microflora from water, live food, and an artificial diet. In addition, aquatic animal and microorganisms share the same ecosystem in the aquatic
environment, and it suggested an interaction between the microbiota, including probiotics, and the host is not limited to the
intestinal tract.
Most probiotics used in aquaculture belong to the lactic acid bacteria, of the genus Bacillus, to the photosynthetic
bacteria or to the yeast, bacteriophages, microalgae, although other genera or species have also been mentioned (Lauzon
et al., 2008). The wide applications belong to endospore-forming members of Bacillus genera (Hong et al., 2005), in which
Bacillus subtilis is commonly used in aquaculture. One of the first evaluations of commercial products focused on a bacterial preparation called Biostart that is derived from Bacillus isolates. It was used during the production of cultured catfish
studying the effect of inoculum concentration (Queiroz and Boyd, 1998). In 1998, Moriarty (1998) reported that the use of
commercial probiotic strains of Bacillus spp. increased the quality and viability of pond-raised shrimp. Meanwhile, Chang
and Liu (2002) evaluated the effect of Enterococcus faecium SF68 and Bacillus toyoi isolates present in Cernivet LBC
and Toyocerin, respectively, to decrease the mortality of the European eel because of the edwardsielosis, ensuring greater
efficiency with E. faecium SF68. It is relevant to note that E. faecium has long been known as a probiotic for humans,
whereas B. toyoi has been used with terrestrial animals. Moreover, a B. subtilis strain combined with hydrolytic enzymes to
produce Biogen, was used to supplement the feed of Oreochromis niloticus, obtaining significant increases in productivity
(El-Haroun et al., 2006).
Many studies have reported promising results using a single beneficial bacterial strain as probiotic in the culture of
many aquatic species (Tovar-Ramírez et al., 2010; Wang and Gu, 2010; Zhou et al., 2010; Wang, 2011). It is important to
consider the possibility of using different species, as suggested by Noh et al. (1994) and Bogut et al. (1998). The effect of
probiotics, photosynthetic bacteria (Rhodobacter sphaeroides) and Bacillus sp. (B. coagulans) on growth performance and
digestive enzyme activity of the shrimp, Penaeus vannamei, was investigated and the results showed that the effects were
related to supplementation concentrations of probiotics and thus use of a 10 g/kg (wet weight) supplement of probiotics in
shrimp diet was recommended to stimulate productive performance (Wang, 2007). A mixture of Bacillus probiotic bacteria
(Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus) was also evaluated in the gilthead sea bream (Sparus aurata)
34 SECTION | A Probiotics and Prebiotics
l­ arviculture focusing on their effect on survival, growth, and general welfare (Avella et al., 2010). The data generated in this
study show the benefit of administration of Bacillus probiotic mixture in terms of stress response and growth, and provide
scientific and technical support for the implementation of sustainable development of sea bream aquaculture. Similar results
were also observed in olive flounder supplemented with Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus
sakei, Bacillus subtilis, and Saccharomyces cerevisiae as individual and mixed enriched diet (Harikrishnan et al., 2011a).
Lactobacilli probiotics individually or mixed with a Sporolac-enriched diet were used to enhance the immune status,
thereby improving the disease resistance in lymphocystis disease virus infected olive flounder (Paralichthys olivaceus) and
the results demonstrated better innate immune response and disease resistance were found in groups supplemented with
mixed probiotics (Harikrishnan et al., 2010). However, feeding experiments conducted on O. niloticus using the diets containing single or mixed isolates of probiotic bacteria show different results in survival rates and highest with fish fed diets
supplemented with B. pumilus was observed, followed by a mixture of probiotics (B. firmus, B. pumilus and C. freundii in
equal numbers), and then C. freundii (Aly et al., 2008). This indicates that the beneficial effects of probiotics fed aquatic
animals are associated with probiotic strains, isolation species, culture animals, and water quality. Altogether, the data reported above may well explain the current trend to prefer alternative probiotics for the application in aquaculture.
The human probiotic, Lactobacillus rhamnosus ATCC (American Type Culture Collection, Rockville, MD, USA), was
used in rainbow trout for 51 days to reduce mortality by Aeromonas salmonicida, the causative agent of the fish disease
“furunculosis” (one of the major fish diseases in many parts of world). Mortality was reduced from 52.6% to 18.9% when
109 cells g−1 were administered with feed, when probiotic dose was increased to 1012 cells g−1 of feed the mortality reached
46.3% (Nikoskelainen et al., 2001). Apparently, increasing dosage does not necessarily improve protection. Abasali and
Mohamad (2010) increased the gonadosomatic index and the production of fingerlings in females of reproductive age,
­using mixed cultures consisting of L. acidophilus, L. casei, E. faecium, and B. thermophilum (Primalac).
Additionally, a large number of studies have combined probiotics with prebiotics, a selectively fermented ingredient that
allows specific changes both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon
host well-being and health (Gibson et al., 2004). Thus, the synbiotics, as a combination of probiotics and prebiotics, have
been studied with synergistic effects anticipated. Currently, there are several recognized functional prebiotic oligosaccharides such as fructooligosaccharides (FOS), mannan oligosaccharides (MOS), insulin, ß-glucan, and xylooligosaccharides
(XOS) in use around the world. The effect of dietary application of a commercial probiotic (Bacillus spp.) and MOS, used
singularly and combined, on the survival, growth performance and feed cost-benefit of larval European lobster (Homarus
gammarus) was assessed and the results strongly suggest that the dietary combination of Bacillus spp. and MOS is cost effective when used to promote survival and provides the added benefits of improved growth performance, compared to their
individual supplementation (Daniels et al., 2010). It suggested that the combined application of probiotics and prebiotics
has promising prospects in replacing growth-promoting chemotherapeutics in the aquaculture industry and could be a useful
tool in the rearing of certain aquatic animals. Recently, herbs and probiotics are combined in diet and treated as one of the
promising alternative tools to supplement and supplant antibiotics, chemicals or vaccines (Sahu et al., 2008; Nayak, 2010).
According to Harikrishnan et al. (2011b), administration of probiotics (Lactobacillus sakei BK19) and Chinese skullcap
(Scutellaria baicalensis) can effectively minimize the mortality and restore the altered hematological parameters, enhancing the innate immunity in O. fasciatus against Edwardsiella tarda, which indicates a promising role as a preventative in
disease and disease outbreaks in aquaculture.
Aquatic animals have a much closer relationship with their external environment. Potential pathogens are able to maintain
themselves in the external environment of the animal (water) and proliferate independently of the host animal (Verschuere
et al., 2000). These potential pathogens are taken up constantly by the animal through the processes of osmoregulation and
feeding. A study of Atlantic halibut, Hippoglossus hippoglossus, showed the transition from a prevailing Flavobacterium
spp. intestinal flora to an Aeromonas spp./Vibrio spp. dominant flora occurred when first feeding commenced (Bergh et al.,
1994). This study highlighted the impact that the external environment and feeding had on the microbial status of the fish.
However, the same study also found that the larvae did maintain a specific intestinal flora different from that of the external
tank flora. This showed that, although there were ever-present external environmental factors influencing the microbial
flora inside an aquatic animal, they could still maintain a host specific flora at any given time. It was suggested that this
ability did not apply to bivalve larvae (Jorquera et al., 2001). Their work demonstrated that the transit time of bacteria in
bivalve larvae was too short to allow the establishment of a bacterial population different from that of the surrounding water.
Based on the intricate relationship an aquatic organism has with the external environment when compared with that of
terrestrial animals, the definition of a probiotic for aquatic environments needs to be modified. Apart from the requirement
of the probiotic to be a live culture, this definition is a lengthy way of describing a probiotic as defined by Irianto and Austin
(2002a,b) thus “a probiotic is an entire or components of a microorganism that is beneficial to the health of the host.” The
latter definition is in accordance with that given by Salminen et al. (1999). The nonrequirement of being a live culture
Selection of New Probiotics: The Case of Streptomyces Chapter | 3 35
would allow for certain suggested immunostimulants (Itami et al., 1998; Smith et al., 2003), which are bacterial derivatives
such as peptidoglycan and lipopolysaccharides, to be included as probiotics. Although there is some dispute about what an
aquatic probiotic actually is, all definitions differ from that of Fuller (1989) in that there is no longer the requisite for the
probiotic to be acting in the GIT. Therefore, modes of action such as competition for nutrients and production of inhibitory substances could occur in the culture water. Additional effects of probiotic action should also be considered, given the
modified definition, including change of the water quality and interaction with phytoplankton (Verschuere et al., 2000).
Phytoplankton capable of producing substances toxic to other bacteria could potentially act in a beneficial manner. For example, Skeletonema costatum, a common microalga used in mollusk and crustacean larviculture, has been shown to produce
an organic extract capable of inhibiting the growth of Listonella anguillarum and three other vibrios (Naviner et al., 1999).
Another study has shown a microalga, Caulobacter sp., produced the antibiotic thiotropocin (Kawano et al., 1997).
This compound was shown not only to be inhibitory towards the fish pathogen Lactococcus garvieae, but also had antimicrobial activity against Skeletonema costatum and Heterosigma akashiwo. Perhaps of more importance is the consideration
of what effect adding a probiotic bacterium will have upon phytoplankton. Microalgae are required for most larviculture
in aquaculture and, in fact, certain bacteria can stimulate microalgal growth (Suminto Hirayama, 1996, 1997; Fukami
et al., 1997). Thus, probiotics could be specifically targeted for microalgae production; however, the subsequent effects of
such bacteria towards the larvae must be established. The more realistic approach is using probiotics aimed at improving
the health of the larvae and then determining whether this bacterium has an effect upon the microalgae. It would be very
desirable to discover a probiotic that benefited the larvae and was either beneficial to or did not impair the microalgae.
Consequently, the bacteria could be cocultured with the microalgae as the entry-point into the larviculture system. This
was done by Gomez-Gil et al. (2002), who found the shrimp probiotic C7b could be cocultured with shrimp larvae food,
Chaetoceros muelleri, without affecting the microalga. Similarly, Avendaño and Riquelme (1999) investigated the growth
of seven bacterial strains with Isochrysis galbana. Four of these strains did not affect growth of the microalgae, while coculture significantly improved ingestion of bacterium C33 by larval scallop, Argopecten purpuratus. Probiotics in aquaculture
may act in a manner similar to that observed for terrestrial animals. However, the relationship of aquatic organisms with the
farming environment is much more complex than the one involving terrestrial animals. Because of this intimate relationship
between animal and farming environment, the traditional definition of probiotics is insufficient for aquaculture.
In this sense, Verschuere et al. (2000) suggest a broader definition: “It is a microbial supplement with living microorganism with beneficial effects to the host, by modifying its microbial community associated with the host or its farming
environment, ensuring better use of artificial food and its nutritional value by improving the host's response to diseases and
improving the quality of the farming environment.” The microorganisms present in the aquatic environment are in direct
contact with the animals, with the gills and with the food supplied, and have easy access to the digestive tract of the animal.
Among the microorganisms present in the aquatic environment are potentially pathogenic microorganisms, which are opportunists; in other words, they take advantage of an animal’s stressed situation (high population density, poor nutrition) to
cause infections, worsening in zootechnical performance and even death. For this reason, the use of probiotics for aquatic
organisms aims not only to directly benefit the animal, but the farming environment as well. Bergh et al. (1994) observed
that, when starting its first feeding, the intestinal flora of the Atlantic halibut (Hippoglossus hippoglossus) changed from a
prevalence of Flavobacterium spp. to Aeromonas spp./Vibrio spp. showing the influence of the external environment and
food on the microbial community of this fish. Vibrio spp., Plesiomonas shigelloides, and Aeromonas spp. are the main
causative agents of diseases in aquaculture, and may even cause food infections in humans. The interaction between the
environment and the host in an aquatic environment is complex. The microorganisms present in the water influence the
microbiota of the host's intestine and vice versa. Makridis et al. (2000) demonstrated that the provision of two strains of
bacteria via food directly into the farming water of the incubators of turbot larvae (Scophthalmus maximus) promoted the
maintenance of the bacteria in the environment, as well as the colonization of the digestive tract of the larvae. Changes in
salinity, temperature, and dissolved oxygen variations change the conditions that are favorable to different organisms, with
consequent changes in dominant species, which could lead to the loss of effectiveness of the product. Accordingly, the addition of a given probiotic in the farming water of aquatic organisms must be constant, because the conditions of environment
suffer periodic changes.
Currently, commercial products are available in liquid or powder form, and various technologies have been developed
for improvement in the case of fermentation processes which has been focused on optimizing the conditions to increase the
viability and functionality of probiotics (Lacroix and Yildirim, 2007). The production is generally carried out in batch cultures due to the difficulty of industrial scale operation of continuous systems (Soccol et al., 2010). More recently, systems
have been developed for immobilization of probiotics, especially using microencapsulation. Microbial cells at high density
are encapsulated in a colloidal matrix using alginate, chitosan, carboxymethylcellulose, or pectin to physically and chemically protect the microorganisms. The methods commonly used for microencapsulation of probiotics are the ­emulsion,
36 SECTION | A Probiotics and Prebiotics
extrusion, spray drying, and adhesion to starch (Rokka and Rantamaki, 2010). Focused on the application to ­aquaculture,
Rosas-Ledesma et al. (2012) have effectively encapsulated cells of Shewanella putrefaciens in calcium alginate, demonstrating the survival of encapsulated probiotic cells through the gastrointestinal tract of sole (Solea ­senegalensis). Encapsulation
in alginate matrices protects bacteria from low pH and digestive enzymes; this protection helps to release the probiotic into
the intestine without any significant damage (Morinigo et al., 2008). Currently, the lyophilized commercial preparations
have advantages for storage and transport. However, conditions for reconstitution of these preparations such as temperature,
degree of hydration, and osmolarity of the solution are vital to ensure the viability of bacteria (Muller et al., 2009). It is
important to emphasize that these products must provide a health benefit to the host; for this, it is necessary that contained
microorganisms have the ability to survive storage conditions, and after that, in the digestive tract of aquatic species, remaining viable and stable, and finally improving production (Wang et al., 2008). According to the opinion of the producers,
these preparations are safe to use and effective in preserving the health of aquatic animals (Irianto and Austin, 2002a,b).
Thus, the variety of microorganisms present must therefore be considered in the choice of probiotic to be used in aquaculture. Intensive farming systems utilize high stocking densities among other stressors (e.g., management), which often
end up resulting in low growth and feed efficiency rates, in addition to weakening the immune system, making these animals vulnerable to opportunistic pathogens present in the environment. In this sense, the effect of probiotics on the immune
system has led to a large number of studies with beneficial results on the health of aquatic organisms, although it has not yet
been clarified how they act. In addition, probiotics can also be used to promote the growth of aquatic organisms, whether
by direct aid in the absorption of nutrients, or by the nutrient supply. However, less attention has been put on the use of
Actinobacteria as probiotics in aquaculture despite being widely known as prolific a producer of secondary metabolites,
particularly the genus Streptomyces (Butler, 2008). The genus Streptomyces demonstrated promising results as probiotics
in aquaculture (Das et al., 2010; Dharmaraj and Dhevendaran, 2010; Augustine et al., 2015). This chapter discusses the
prospect of using marine Streptomyces as a probiotic candidate in aquaculture.
3.
3.1
PROSPECT OF USING MARINE STREPTOMYCES AS PROBIOTICS
Life Cycle of Marine Streptomyces
Streptomycetes are highly abundant in nature and remain dormant as spores for long periods until conditions become favorable for growth. The life cycle of a Streptomyces species has been described in the subsequent manner: (1) initial nuclear
division phase, (2) primary mycelium, (3) secondary mycelium (including aerial), and (4) the formation of spores. The
life cycle of Streptomyces is illustrated in Fig. 5. Once a spore encounters conditions favorable for growth, it germinates.
Once a spore settles in a nutrient rich medium, it is stimulated to exit its dormant state and undergo germination and form
Germinating spore
Vegetative mycelium
Vegetative
growth
Aerial
growth
Aerial hyphae
Spores
Life cycle of
Streptomyces
New
generation
Spore chains
FIG. 5 Life cycle of marine Streptomyces.
Aerial and vegetative
mycelium
Selection of New Probiotics: The Case of Streptomyces Chapter | 3 37
germ tubes. This is the first step of several morphological differentiations in its life cycle. A germ tube grows out from the
spore and elongates into long branching filamentous cells during vegetative growth, forming a mesh of hyphae called the
substrate or vegetative mycelium, which grows deep into the solid growing medium. Elongation of hyphae is accomplished
through insertion of new cell wall material at the hyphal tip. Infrequent crosswalls separate the hyphae into cellular compartments. Each compartment contains multiple copies of the chromosome and DNA is spread throughout the whole compartment with little separation of individual nucleoids. When growth of the vegetative mycelium has given rise to a colony,
nutrient limitation and probably cell density signals contribute to trigger formation of an aerial mycelium on the colony
surface (Elliot et al., 2008; Flärdh and Buttner, 2009; Dharmaraj and Dhevendaran, 2016). The aerial hyphae represent reproductive structures and are transformed into pigmented spore chains that mature and eventually release separated spores.
Sporulation in the aerial hyphae is restricted to the apical compartment (also referred to as the sporogenic cell) in which
a high level of DNA replication takes place generating several copies or more of uncondensed, evenly distributed chromosomes (Ruban-Osmialowska et al., 2006). A developmentally controlled form of cell division (sporulation septation)
compartmentalizes the sporogenic cell into prespores. In coordination with this septation, the chromosomes are positioned
and segregated so that each prespore compartment receives one copy of the genome (Flärdh, 2003). After the completion
of septation, nucleoids are condensed, the prespores become rounded or ovoid, synthesize a thick spore wall, pigment, and
the spores are separated.
3.2 Taxonomical Classification of Marine Streptomyces
Actinobacteria represents one of the largest taxonomic units among the 18 major lineages currently recognized within the domain
bacteria, including five subclasses and 14 suborders (Stackebrandt, 2000). Among the five subclasses, actinobacteria—bacteria
belonging to the Order Actinomycetales (commonly called actinomycetes)—account for approximately 7000 of the metabolites
reported in the Dictionary of Natural Products. Actinomycetes have a high GC content in their deoxyribonucleic acid (DNA) and
grow as aerial/substrate mycelia (Yoshida et al., 2008). They are responsible for the production of about half of the discovered secondary metabolites (Bull, 2004; Berdy, 2005; Dharmaraj and Alagarsamy, 2009), notably antibiotics (Strohl, 2004), antitumour
agents (Olano et al., 2009), anticancer compounds (Dharmaraj, 2011a,b), immunosuppressive agents (Mann, 2001) and enzymes
(Pecznska-Czoch and Mordarski, 1988; Oldfield et al., 1998). A large number of actinomycetes have been isolated and screened
from terrestrial habitat in the past few decades (Williams et al., 1989). Recently, the rate of discovery of new metabolites from
terrestrial actinomycetes has decreased, whereas the rate of re-isolation of known compounds has increased (Fenical et al., 1999;
Fenical and Jensen, 2006). Thus, it is crucial that new groups of actinomycetes from unexplored or underexploited habitats be
pursued as sources of novel secondary metabolites.
Indeed, the marine environment is virtually untapped as a source of novel actinomycete diversity (Stach et al., 2003;
Magarvey et al., 2004) and therefore, of new metabolites (Bull et al., 2005; Fiedler et al., 2005). This is partly caused by
the lack of effort spent in exploring marine actinomycetes, whereas terrestrial actinomycetes have been, until recently, a
successful source of secondary metabolites. Furthermore, skepticism regarding the existence of indigenous populations of
marine actinomycetes arises from the fact that the terrestrial bacteria produce resistant spores that are known to be transported from land into sea, where they can remain available but dormant for many years (Bull et al., 2000). Thus, it has been
frequently assumed that actinomycetes isolated from marine samples are merely of terrestrial origin.
Recent data from culture-independent and culture-dependent studies have shown that indigenous marine actinomycetes
indeed exist, and it was confirmed that they are autochthonous flora in the marine environment (Moran et al., 1995; Mincer
et al., 2002; Jensen et al., 2005; Lam, 2006; Das et al., 2008). A series of papers describing the distribution of actinomycetes in the marine environment, published in the dedicated volume of the Antonie van Leeuwenhoek journal (Bull and
Goodfellow, 2005), confirmed the indigenous nature of marine actinobacteria. This view was best supported by the discovery of the first new obligate marine actinomycete genus, Salinispora (formerly known as Salinospora) (Mincer et al.,
2005). While early research estimated low numbers (Jensen et al., 1991) and patchy distribution (Mincer et al., 2002) of
actinomycetes in the marine environment, more recent studies suggested higher abundance and diversity of actinobacteria
with numerous novel taxa (Gontang et al., 2007). Thus, bona fide actinomycetes not only exist in the oceans, but they are
also widely distributed in different marine ecosystems (Lam, 2006; Dharmaraj, 2011a,b).
In the marine environment, the representatives from six families have been listed based on molecular studies. The representative families are Micromonosporaceae, Nocardiaceae, Nocardiopsaceae, Pseudonocardiaceae, Streptomycetaceae,
and Thermonosporaceae. However, greater sequence coverage or improved DNA extraction efficiencies may be required to
detect the rare phylotypes. Besides, new strategies need to be developed for the cultivation of frequently observed but yetto-be-cultured marine actinobacteria (Jensen and Lauro, 2008). Actinomycete genera identified by cultural and molecular
techniques from different marine ecological niches include Actinomadura, Actinosynnema, Amycolatopsis, Arthrobacter,
38 SECTION | A Probiotics and Prebiotics
Blastococcus, Brachybacterium, Corynebacterium, Dietzia, Frankia, Frigoribacterium, Geodermatophilus, Gordonia,
Kitasatospora, Micromonospora, Micrococcus, Microbacterium, Mycobacterium, Nocardioides, Nocardiopsis, Nonomurea,
Psuedonocardia, Rhodococcus, Saccharopolyspora, Salinispora, Serinicoccus, Solwaraspora, ---------Streptomyces,
Streptosporangium, Tsukamurella, Turicella, Verrucosispora and Williamsia (Stach et al., 2004; Jensen et al., 2005; Ward
and Bora, 2006; Das et al., 2006a,b). More actinobacterial genera are expected to be discovered and reported with cultureindependent studies in the years to come. However, regardless of the geographical origin, marine actinomycetes were
shown to follow a well-documented pattern in secondary metabolite production (Jensen et al., 2007). It is surmised that
marine actinomycetes have different characteristics from those of their terrestrial counterparts and therefore, might produce
different types of secondary metabolites. Over the past decade, information on the diversity of actinomycetes in marine
habitats has grown considerably, while the somewhat longer held interest in their ability to produce secondary metabolites
has continued quite strongly (Stackebrandt et al., 1997).
Marine actinomycetes are a prolific source of secondary metabolites and the vast majority of these compounds are
derived from the single genus Streptomyces. Streptomyces species are distributed widely in marine and terrestrial ­habitats
(Pathom-aree et al., 2006) and are of commercial interest due to their unique capacity to produce novel metabolites.
It was also expected that Streptomyces species will have a cosmopolitan distribution, as they produce abundant spores
that are readily dispersed (Antony-Babu et al., 2008). These filamentous bacteria are well adapted to the marine environment and are able to break down complex biological polymers. Streptomyces are classified within the Gram-positive bacteria and form together with the genera Kitasatospora (Omura et al., 1982) and Streptacidiphilus (Kim et al., 2003), the
­family Streptomycetaceae. The latter family constitutes a separate phylogenetic branch within the phylum Firmacutes,
class Actinobacteria, order Actinomycetales, suborder Streptomycineae, based on 16S rRNA gene sequence analysis, with
high DNA G + C content of 69–78 mol%. The taxonomy of Streptomyces is shown in Fig. 6 (Stackebrandt et al., 1997;
Anderson and Wellington, 2001). In fact, the genus Streptomyces alone accounts for a remarkable 80% of the
­actinomycete natural products reported to date; a biosynthetic capacity that remains without rival in the microbial world
(Watve et al., 2001).
Families
Suborders
Micromonosporaceae
Micromonosporineae
Frankiaceae
Acidothermaceae
Sporichthyaceae
Frankineae
Geodermatophilaceae
Microsphaeraceae
Pseudonocardiaceae
Pseudonocardineae
Streptomycetaceae
Streptomycineae
Nocardiaceae
Gordoniaceae
Mycobacteriaceae
Corynebacterineae
Dietziaceae
Tsukamurellaceae
Corynebacteriaceae
Intrasporangiaceae
Dermabacteraceae
Jonesiaceae
Brevibacteriaceae
Micrococcineae
Dermatophilaceae
Micrococcaceae
Promicromonosporaceae
Cellulomonadaceae
Microbacteriaceae
Actinomycetaceae
Actinomycineae
Propionibacteriaceae
Propionibacterineae
Nocardioidaceae
Streptosporangiaceae
Thermomonosporaceae
Streptosporangineae
Nocardiopsaceae
Glycomycetaceae Glycomycineae
Bifidobacteriaceae
Acidimicrobiaceae
Coriobacteriaceae
Sphaerobacteraceae
Rubrobacteraceae
Orders
Actinomycetales
Bifidobacteriales
Acidimicrobiales
Coriobacteriales
Sphaerobacterales
Rubrobacterales
5%
FIG. 6 Phylogenetic positions of the Streptomyces based on 16S rRNA gene sequence analysis. Adapted from Stackebrandt, E., Rainey, F. A.,
Ward-Raine, N. L., 1997. Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int. J. Syst. Bacteriol. 47, 479–491.
Selection of New Probiotics: The Case of Streptomyces Chapter | 3 39
3.3
Morphological Identification of Marine Streptomyces
Marine Streptomyces exhibit different cultural characterization in various types of culture media, which is one of the important criteria in the classical identification. The main characteristics include spore formation, aerial hyphae, with or without
color and the soluble pigment, and different growth conditions on various media. The morphological differentiation of marine
Streptomyces is generally controlled by relevant genes. Marine Streptomyces display the greatest morphological differentiation
among Gram-positive bacteria; however, the cell structures are typical as prokaryotes and totally different from fungi.
The whole structure of a hyphae cell corresponds to bacterial organization: the cytoplasm contains genomic DNA
regions, ribosomes, and various inclusions, presumably reserve substances such as polyphosphates, lipids, or polysaccharides. According to the difference of morphology and function, the mycelia can be divided into vegetative mycelium and
aerial mycelium. Known as vegetative mycelium or primary mycelium, the substrate mycelium grows into the medium or
on the surface of the culture medium. The main function of the substrate mycelium is the absorption of nutrients for the
growth of marine Streptomyces. Under the microscope, the substrate mycelia are slender, transparent, phase-dark, and more
branched than aerial hyphae. The single hyphae is about 0.4–1.2 μm thick, usually does not form diaphragms and fracture,
and is capable of developing branches. The substrate mycelia are white, yellow, orange, red, green, blue, purple, brown,
black, and other colors; some hyphae can produce water-soluble or fat-soluble pigment. The water-soluble pigment can
seep into culture medium, which changes the medium to the corresponding color. The nonwater-soluble (or fat-soluble)
pigment makes the colony the corresponding color. The color of the substrate mycelia, and whether there are soluble pigments, provide important references in the determination of new species.
Aerial mycelium is the hyphae that the substrate mycelium develops to a certain stage and grows into the air. Sometimes,
aerial hyphae and substrate mycelia are difficult to distinguish. It can be easily distinguished by an impression preparation
on a cover slip viewed in a dry system with a light microscope: substrate hyphae are slender, transparent, and phase-dark;
aerial hyphae are coarse, refractive, and phase-bright.
In all kinds of marine Streptomyces, the formation of aerial hyphae is dependent upon the species characteristics, nutritional conditions, or environmental factors. At a certain stage aerial hyphae of marine Streptomyces get differentiated
and can form reproductive hyphae called spore-bearing mycelium. Marine Streptomyces has classical polyspores, which
form long chains frequently having more than 50 spores. The spores of marine Streptomyces are often called arthrospores
(Cross, 1970). The sporulating aerial hyphae of marine Streptomyces can be differentiated into the following main types:
(A) Rectiflexibiles type, straight or flexuous spore chains, partly in fascicles; (B) Retinaculiaperti type, spore chains with
hooks, open loops or short, irregular spirals having 1–4 turns; (C) Spira type, spore chains in spirals demonstrating two
different subtypes: (a) closed, compact spiral and (b) open, loose, and stretched spirals; (D) Verticillati type, spore chains
formed in whorls and branched in umbels.
The length, shape, position, and color of marine Streptomyces spore chains are an important basis for classification.
Spore chains of the marine Streptomyces have various types of spore-bearing structures: straight, flexous, fascicied, monoverticillate (no spirals), open loops (primitive spirals hooks), open spirals, closed spirals, monoverticillate (with spirals),
biverticillate (no spirals), biverticillate (with spirals). Mature spores show a variety of colors such as white, gray, yellow,
pink, lavender, blue, or green, and so on.
Hyphal growth of marine Streptomyces shows much similarity with filamentous fungi (Xiang and Morris, 1999). Marine
Streptomyces apical hyphal growth was observed using fluorescence microscopy (Flärdh, 2003). The apical cell extends its
cell wall only at the tip (green). Once this cell has divided by forming a new hyphal cross wall, the subapical daughter cell
is unable to grow and eventually switches its polarity to generate a lateral branch with a new extending tip. A consequence
of tip growth is that DNA, which replicates along most of the hyphal length, has to move towards the tip and into new
branches by means of a process proposed to designate nucleoid migration. For clarity, few schematic nucleoids are drawn
(red), and they are not meant to reflect the actual number of chromosomes per cell. Furthermore, individual nucleoids are
typically not observed in vivo as separated bodies in growing hyphae (Fig. 7). In general, when nutrients become limited,
a developmental switch occurs during which hyphae start to escape the moist environment and grow into the air. These socalled aerial hyphae can further differentiate into long chains of spores, which can withstand adverse conditions. Following
their dispersal, these spores will reinitiate growth in suitable environments.
Some of the key processes involved in the formation of aerial hyphae by marine Streptomyces appear to be very similar
to fungi. Both groups secrete highly surface-active molecules that lower the surface tension of their aqueous environment
enabling hyphae to grow into the air. In the case of filamentous marine Streptomyces, small peptides (i.e., Sap B and streptofactin) are secreted, while filamentous fungi use proteins known as hydrophobins to decrease the water surface tension.
Although these fungal and bacterial molecules are not structurally related, they can, at least partially, functionally substitute
each other (Wosten and Willey, 2000).
40 SECTION | A Probiotics and Prebiotics
Apical cell
Nucleoids
FIG. 7 Hyphal growth of marine Streptomyces (green areas—apical cell, red areas—nucleoids).
The bld cascade (for bald, meaning unable to form aerial hyphae) controls the checkpoints that (eventually) lead to the
onset of aerial growth, resulting in the formation of surface-active molecules that lower the water surface tension and enable
hyphae to grow into the air. Moreover, the bld cascade seems to potentiate hyphae to undergo full development (Kelemen
and Buttner, 1998; Willey et al., 2006). Another regulatory pathway is the shy pathway (Claessen et al., 2006), which
controls the expression of the chaplin and rodlin genes. These genes encode proteins that assemble into a rodlet layer that
provides surface hydrophobicity to aerial hyphae and spores. Both pathways control the production of structural proteins
that are involved in the formation of aerial hyphae. When hyphal growth is limited, much of the biomass becomes converted
into spores through the extraordinary parasitic growth of a fluffy white aerial mycelium. The syncytial aerial hyphal tips
(which may contain more than 50 copies of the genome) undergo multiple cell divisions to generate a string of unigenomic
compartments, destined to become tough, desiccation-resistant spores (Flärdh et al., 1999). Thus, substantial growth is
interpolated between the first sporulation related decisions, made in the substrate mycelium, and the decisions involved in
the formation and maturation of the spore compartments themselves (Chater, 2001). Additionally, the marine Streptomyces
spore wall synthesizing complex (SSSC) does not only direct synthesis of the peptidoglycan layer but is also involved in
the incorporation of anionic spore wall glycopolymers, which contribute to the resistance of spores. The SSSC also contains
eukaryotic type serine/threonine kinases which might control its activity by protein phosphorylation (Sigle et al., 2015).
3.4 Applications of Marine Streptomyces as Probiotics in Aquaculture
Marine Streptomyces are widely distributed in various biological sources such as fish, mollusks, sponges, seaweeds, mangroves, corals as well as in seawater and sediments. These microorganisms are gaining importance not only for their taxonomic and ecological perspectives, but also have been widely recognized as industrially significant. Their potential for
producing diverse range of novel secondary metabolites includes antibiotics, antitumor agents, antiparasitics, antioxidants,
immunosuppressive agents, enzymes, enzyme inhibitors, and food grade pigments (Dharmaraj et al., 2009; Dharmaraj,
2010; Pimentel-Elardo et al., 2010; Manivasagan et al., 2013; Janardhan et al., 2014; Tan et al., 2015). They have produced
wide-variety chemical compounds and have the advantage of producing antagonistic and antimicrobial compounds that can
be as valuable probiotics in aquaculture. The ability of producing antagonistic compounds may help the probiotics to compete for nutrients and attachment sites in the host. There have been reports that probiotics which were used for aquaculture
also have the ability to synthesize compounds like bacteriocins (Desriac et al., 2010), siderophores (Lalloo et al., 2010),
enzymes [protease, amylase, lipase] (Augustine et al., 2015), hydrogen peroxide (Sugita et al., 2007) and organic acids
(Sugita et al., 1997). Table 1 summarizes all the features and mechanisms of actions of the probiotic effects evidenced in the
marine Streptomyces. Schematic representation detailing the various applications of marine Streptomyces as probiotics in
aquaculture has been shown in Fig. 8. You et al. (2007) reported the activity of marine Streptomyces as a potential organism
against biofilms produced by Vibrio spp. These organisms synthesize siderophores and it has been suggested that their use
can influence the growth of pathogenic Vibrio sp. by competition for iron in the aquatic environment. Probiotics with the
capability of producing siderophores are believed to outcompete the pathogens by limiting the bioavailability of iron (Ahmed
and Holmstrom, 2014), resulting in growth attenuation of the pathogens due to the fact that iron is essential for growth as well
as biofilm formation (Weinberg, 2004). They inhibited the biofilm formation of Vibrio harveyi, Vibrio vulnificus, and Vibrio
anguillarum at a concentration of 2.5% (v/v). They dispersed the mature biofilm and inhibited the quorum sensing system
of V. harveyi by attenuating the signal molecules, N-acylated homoserine lactones’ activity. The strains have the ability to
attenuate the biofilms and also inhibit their quorum-sensing system (Iwatsuki et al., 2008). It is suggested that the strain is
a promising candidate for use in marine aquaculture and is also helpful in the prevention of diseases caused by Vibrio spp.
You et al. (2005) described the potential of marine Streptomyces against the shrimp pathogen Vibrio spp. and recommended
Selection of New Probiotics: The Case of Streptomyces Chapter | 3 41
TABLE 1 The Probiotic Effects Demonstrated by Marine Streptomyces Bacteria Through Different
Mechanism of Actions
Mode of
Actions
Marine Streptomyces
as Probiotics
Growth
enhancing
effect
Outcomes
References
Streptomyces fradiae
and Streptomyces sp.
(1) Improved growth of postlarval shrimp P. monodon and
ornamental fish, Xiphophorus helleri.
(2) Produced growth promoting hormone, indole acetic
acid which enhanced growth of X. helleri.
Dharmaraj and Dhevendaran
(2010, 2013) and Aftabuddin
et al. (2013)
Single cell
protein
Streptomyces sp.
(3) Used as a protein source for host which increased
food conversion rate and food conversion efficiency,
enhanced growth performance
Dharmaraj and Dhevendaran
(2010, 2015), Suguna and
Rajendran (2012), and
Dharmaraj et al. (2013)
In vivo
experiment
Streptomyces strains
(CLS-28, 39 and 45)
(4) Protection of Artemia against V. harveyi—At a
concentration of 106 CFU/mL killed all Artemia nauplii
in 72 h and on addition of Streptomyces strains
[at 1% (v/v)] increased the survival of Artemia nauplii
by 67% and adults by 61% after 72 h of exposure at
same concentrations of cells
(5) Protection of P. monodon against V. harveyi—At a
concentration of 107 CFU/mL killed 55% of P. monodon
after 5 days exposure and on supplementation
Streptomyces CLS-28 incorporated feed increased the
survival of P. monodon by 67% compared
Das et al. (2008, 2010)
Exoenzyme
production
Streptomyces strains
(CLS-28, 39 and 45)
(6) The cultures possess amylolytic, proteolytic, and
lipolytic properties which helps in the feed utilization,
digestion and resulting in increased weight for the
Penaeus monodon
Das et al. (2008, 2010)
Improvement
in water
quality
Streptomyces fradiae
Streptomyces sp.
Streptomyces CLS-28
(7) The strains reduced the level of ammonia in the
culture system and there is an increase in the total
heterotrophic bacterial populations which helped to
accelerate the decomposition of waste materials
Das et al. (2006a,b, 2010) and
Aftabuddin et al. (2013)
Antibacterial
and viral
activity
Streptomyces sp.
(8) The cultures which exhibited probiotic effect have
been initially screened for antiviral activity against
fish and shellfish pathogens (Aeromonas hydrophila,
Serratia sp., and Vibrio spp [V. alginolyticus, V. harveyi,
V. parahaemolyticus]) which cause deleterious effects
Dharmaraj and Dhevendaran,
(2011, 2016)
marine Streptomyces as potential probiotic strains due to their ability to degrade macromolecules such as starch and protein
in culture pond water, to produce antimicrobial agents and form heat and desiccation-resistant spores. More recently, there
have been studies on the possible use of marine Streptomyces in disease prevention against aquatic pathogens.
Besides displaying the inhibitory effect on bacterial pathogens in aquaculture, marine Streptomyces also has been reported to exhibit antiviral activity, specifically against the white-spot syndrome virus. Very few studies have been carried
out on the antiviral property of marine Streptomyces against White Spot Syndrome Virus (WSSV) in penaeid shrimps
(Jenifer et al., 2015). WSSV infection can cause cumulative mortality up to 100% within 3–10 days, thereby causing considerable economic loss to the shrimp farmers. In a report by Kumar et al. (2006), antibiotic extracts were obtained from
the fermentation broth of twenty-five isolates of marine Streptomyces (isolated from the coastal waters off the Southwest
coast of India), incorporated in the formulated feed and supplemented to the postlarvae (PL-20) of the black tiger shrimp
Penaeus monodon for 2 weeks to treat against WSSV. The pattern of posttreatment survival % (PCS %) in the 27 treatments
(25 experimental and two controls) exhibited a wide range of variation from 11 to 83% during the course of the experiment.
PCS % was lowest in the controls (C1-4.3%, C2-5.2%) on day 7. However, six probiotic feeds (SA 2, SA 8, SL 27, SL 33,
SL 39, and SL 85) supplemented to postlarvae shrimp recorded the highest PCS percent ranging between 50% and 83%.
Also, severity of the infection observed on days 3, 4, and 5 in postlarvae shrimp fed with other diets was not visible. In this
case, positive effect was obtained by the antibiotic extract incorporated in the feed against WSSV infected penaeid shrimps.
42 SECTION | A Probiotics and Prebiotics
Antibacterial
Spores
Antifungal
Marine Streptomyces
Antiviral
Antimicrobial compounds
Antibiofilm
Antivirulence
Antiquoram
Mycelium
Siderophore
Arial
Mixed with feed ingredients
mycelia
Substrate mycelia
Intestine
Growth enchancing effect +
Supplemented to fish and prawn
Producing exoenzymes
intestinal enzyme resistance
Low pH tolerance
Good protein source
White spot syndrome virus
FIG. 8 Schematic representation of various applications of Marine Streptomyces as probiotics in aquaculture.
Marine Streptomyces are primarily saprophytic, living in diverse habitats with the development of branching hyphal
filaments (Flärdh and Buttner, 2009). This unique growth adaptation allows marine Streptomyces colonization of the solid
substrates by adhering and penetrating to gain access to insoluble organic materials of the host environment. Different hydrolytic enzymes such as amylase, protease and lipase can be produced by marine Streptomyces to break down the insoluble
organic materials to provide nutrients for the formation of densely packed substrate mycelium which is reused to fuel the reproductive phase of aerial growth in producing chains of spores (Chater et al., 2010). These unique physiological adaptations
of marine Streptomyces are believed to make them potential probiotics, such as the secretion of exoenzymes which may be
helpful in facilitating the feed utilization and digestion once they colonize the host intestine in aquaculture. Das et al. (2010)
demonstrated that the feed incorporated with marine Streptomyces increased the weight of Penaeus monodon shrimp, suggesting that these marine Streptomyces sp. secreted hydrolytic exoenzymes to improve the amylolytic and proteolytic activity
in the shrimp digestive tract for more efficient use of the feed. The feed supplemented with marine Streptomyces fradiae isolated from mangrove sediment was also shown to enhance the growth of the postlarval P. monodon (Aftabuddin et al., 2013).
The formation of enzymatic digestion, sonic vibration and desiccation-resistant spores demonstrated by marine
Streptomyces constitute some of the attractive features for this genus of bacteria to resist the harsh environment conditions,
thereby allowing them to retain longer shelf life in the aquaculture ponds before being taken up or to resist the low pH in
the gastro intestinal tracts of the animals. However, it should be noted that marine Streptomyces spore is only resistant to
moderately high temperature as compared to the highly heat resistant endospores of Bacillus sp. which is compositionally
and physiologically different from the Streptomyces spore (McBride and Ensign, 1987).
Das et al. (2006a,b) reported a preliminary study on the use of marine Streptomyces incorporated feed as a probiotic
source for the growth of black tiger shrimp. Cells of marine Streptomyces were incorporated at different concentrations (0,
2.5, 5.0, 7.5, and 10.0 g/kg feed) in the formulated feeds, supplemented for 25 days and growth was monitored. At a concentration of 10 g, shrimp fed with marine Streptomyces incorporated feed showed high growth in terms of length (15.79%) and
weight (57.97%) when compared with the control [length (4.08%) and weight (32.77%)]. The growth of the tiger shrimp,
Penaeus monodon, also increased with an increase in the concentration of marine Streptomyces in the supplemented feed.
Das et al. (2010) isolated marine Streptomyces strains from the sediment of the shrimp culture system which has the ability
to reach the digestive system of the shrimp, therefore allowing easier establishment and growth of the probiotics in the host.
These findings indicate that the spore-forming capacity of marine Streptomyces with high acidity and bile acids tolerance
makes them a more practical alternative than those bacteria with nonspore forming capability. Further, Das et al. (2010)
Selection of New Probiotics: The Case of Streptomyces Chapter | 3 43
studied the potential of marine Streptomyces as probiotic by conducting in vivo experiment which successfully demonstrated the protection effect of marine Streptomyces on both juvenile and adult Artemia (15 days old) from Vibrio pathogens. The study demonstrated that the marine Streptomyces at 1% concentration (v/v) resulted in higher survival rates than
the untreated control group of Artemia after challenged with V. harveyi or V. proteolyticus at 106 CFU/mL. The protective
response shown by the study suggests that marine Streptomyces could be administrated to target organisms through bioencapsulation in Artemia as a vector for supplementing the beneficial marine Streptomyces as probiotics in aquaculture.
Previously many reports were recorded on the use of bioencapsulated probiotics in live food such as Artemia and rotifers
to be more effective in the delivery of the probiotics to the digestive tract of the target aquaculture organisms (Gatesoupe,
2002; Suzer et al., 2008). The study also further evaluated the efficacy of the marine Streptomyces in protecting the shrimp
P. monodon from the Vibrio pathogens. The feed supplemented with marine Streptomyces sp. CLS-28 for 15 days was
found to exert protective effects on shrimp P. monodon against the 12 h challenge of V. harveyi (LD50 at 106.5 CFU/mL). A
recent study reported that marine S. rubrolavendulae M56 was found to exhibit antagonistic activity against all four Vibrio
species including V. harveyi, V. alginolyticus, V. parahaemolyticus, and V. fluvialis in an in vitro coculture experiment. In
order to confirm the in vitro findings, the bio granules of marine Streptomyces rubrolavendulae M56 resulted in lower
percentage of mortality of P. monodon postlarvae with the reduction of viable Vibrio sp. in the culture system after 28 days
(Augustine et al., 2015).
Besides showing good growth promoting effects in shrimp, all the feeds supplemented with marine Streptomyces were
shown to improve growth performance of the ornamental fish, Xiphophorus helleri (red sword tail fish) after a 50 day feeding trial when compared to control without the marine Streptomyces sp. (Dharmaraj and Dhevendaran, 2010). In the same
study, marine Streptomyces strains exhibited the production of growth-promoting hormone, indole acetic acid, which could
have attributed to the better growth rate of the fish Xiphophorus helleri fed with marine Streptomyces supplemented feeds
(Dharmaraj and Dhevendaran, 2010, 2013). It has also been reported that dietary supplementation of microbial carotenoids
to the ornamental fishes not only resulted in the recovery of colouration to the fishes; it also showed improved growth of
the fishes (Dharmaraj and Dhevendaran, 2011).
The major problem which substantially affects the health of the aquaculture livestock is due to the poor water quality
which results in high level accumulation of metabolic waste by the cultured organisms, particularly the rise of ammonia
and nitrite as well as the decomposition of residual feed. The probiotic marine Streptomyces was also found to regulate the
microflora of the aquaculture water, besides controlling the pathogenic microorganisms, resulted in better pond conditions.
Previous studies showed that the application of probiotic product did not adversely affect the microflora of aquaculture, in
turn they increased the abilities of protein mineralizing and ammonifying bacterial population which help to accelerate the
decomposition process of the accumulated wastes materials (Devaraja et al., 2002). Many reports also exhibited similar
type of results indicating the reduction of ammonia level and an increase in the total heterotrophic bacteria in the ponds/
tanks treated with the probiotic marine Streptomyces as compared to control ponds/tanks (Aftabuddin et al., 2013). These
all establish that marine Streptomyces could be applied as probiotics in aquaculture which may directly or indirectly improve the water quality as well as the growth performance and yield of the particular cultured organisms.
In aquaculture, essential and expensive components of the feed are proteins, especially fish meal. Since the supply of
fish meal has become uncertain and the prices have increased rapidly, the need for cheaper alternative protein sources has
increased. Among unconventional protein sources, microbial origin appears to be a promising substitute for fish meal, replacing up to 25%–50%. In the present contest, microbial single cell protein of marine Streptomyces is one of the alternative
sources of protein and has been utilized and evaluated for better food conversion efficiency and growth for fish and shrimp
(Manju and Dhevendaran, 1997; Suguna and Rajendran, 2012; Selvakumar et al., 2013).
Marine Streptomyces are known to produce secondary metabolites that enhance the growth of juvenile fish, shrimp and
prawn. There have been reports on the application of marine Streptomyces as a SCP source, incorporated in the formulated
feed and supplemented to juvenile prawns weighing about 0.130–0.160 g/body weight. The ingredients of the control feed
consisted of fishmeal (14.76 g), groundnut oil cake (14.76 g) and rice bran (35.24 g) with tapioca flour (35.24 g) as binder,
whereas the SCP incorporated feed consisted of fishmeal + Streptomyces cells (14.76 g + 165 mg), the rest being the same
as the control feed. After 50 days of feeding trials, growth parameters such as feed conversion efficiency, feed conversion
ratio and protein content were analyzed. Prawns fed with marine Streptomyces incorporated feed showed improved growth
(140.54%), feed conversion efficiency (45%), and higher protein content (54.72%), whereas the prawns fed with control
feed showed less growth (89.52%), food conversion efficiency (20%) and protein content of 35.02%. The feed conversion
ratio was less for the SCP fed-prawns (2.217) than the control (5.015). Therefore, among unconventional protein sources,
single cell protein (SCP) of microbial origin appears to be a promising substitute for fishmeal, and can replace up to
25%–50% fishmeal in aquaculture operations (Manju and Dhevendaran, 1997). It has been reported that the use of marine
Streptomyces not only demonstrated beneficial effects as probiotic in aquaculture, the incorporation of Streptomyces in the
44 SECTION | A Probiotics and Prebiotics
feed is a cost-effective approach as the probiotic bacteria replaced up to 30%–40% of the fishmeal used in the feed preparation. Reports clearly proved that marine Streptomyces can be a cheaper alternative protein source in aquaculture feed
(Dharmaraj and Dhevendaran, 2010, 2015; Dharmaraj et al., 2013).
3.5
Selection of Efficient Strains of Marine Streptomyces as Probiotics
To select efficient strains which can be used as probiotics, particularly marine Streptomyces, there are certain steps which
must be followed: (1) preliminary information about sampling collection sites, (2) isolation and identification of probiotic
strains, (3) various methods to be adopted to characterize the strains to be potent probiotics, like survivability tests (resistance to low pH, pepsin, bile, and pancreatin), colonization (autoaggregation, hydrophobicity, and coaggregation) and
safety (antibiotic susceptibility test and nonhemolytic activity), (4) evaluating the potent probiotic strains ability to fight
against pathogens prevailing in a particular environment, (5) assessing the effects of the probiotic strains in the host, (6)
knowledge on cost effective benefits of the probiotics (Gomez-Gil et al., 2000; Sheeja et al., 2011).
Many researchers described isolation of probiotic strains from different sampling sites. Marine Streptomyces strains can
be isolated from samples such as natural seawater where larvae are growing, culture water where larvae are densely reared
in the laboratory, sediments from the marine environment associated with bottom dwelling animals, and from digestive gut
of marine fishes (Maeda et al., 1997). It is quite obvious to recommend the site from the native environment, where the
survivability of the strains is already established. Microbial food networks are an essential part of marine aquaculture and
have a direct impact on yields (Moriarty, 1997). The probiotic strains screened all play an important role in productivity, the
nutrition of the cultured animals, disease control, water quality, and environmental impacts (Wang et al., 2008).
In support of the marine Streptomyces, in particular, as probiotics, the comprehensive screening procedures and experimental observations can be followed, which have been shown in Table 2. Marine Streptomyces usage as a component of
formulated feed ingredients which can be supplemented to fish has been summarized in Fig. 9. Marine Streptomyces can
be mass-cultured, harvested, lyophilized and used to fortify feed (Dharmaraj and Dhevendaran, 2010). In some studies,
the supernatant of the broth culture of the strain has been collected, concentrated, and added to the feed. In this particular
case, the positive effects may be observed, although it may contravene the definition of probiotics, as there is no addition of
live microbial supplement but only cellular components (Kumar et al., 2006). At last the probiotic strains selected should
possess certain properties: (1) it should be nonpathogenic to the host; (2) it should be accepted by the host, may be through
ingestion, potential colonization, and replication within the host; (3) it should reach the location where the effect is required; (4) it should actually work in vivo as similar to in vitro findings; and (5) it should not preferably carry any virulence
resistance genes or antibiotic resistance genes (Verschuere et al., 2000).
3.6
Possible Limitations in the Usage of Marine Streptomyces as Probiotics
There are three possible setbacks in the usage of marine Streptomyces as probiotics in aquaculture as follows:
(1) Difficulties in culturing methods
(2) Risk of lateral gene transfer
(3) Production of awful odor compounds.
Marine Streptomyces occur in different biological sources such as fish, mollusks, sponges, seaweeds and mangroves
as well as seawater and sediments. They are randomly distributed, many difficulties arise in culturing methods, there are
currently no proposed standardized media, the organism itself has a slow growth rate of approximately 14 days, and identification requires tedious laboratory procedures in culture-dependent studies.
There is substantial risk in the use of marine Streptomyces as a probiotic in aquaculture were the lateral gene transfer
of antibiotic resistance genes that produce efflux pumps, ribosomal protection proteins, and modifying enzymes, by which
the organism itself protects from its own antibiotics. It is preferable that the probiotic strains not contain any virulence
resistance genes or antibiotic resistance genes because of the emergence of multidrug-resistant pathogens in aquaculture.
There have been recent reports on the antibiotic resistance developed by most of the commonly used probiotics such as
Lactobacillus sp. (Sharma et al., 2015), Bifidobacterium sp. and Bacillus sp. (Gueimonde et al., 2013). Extensive use of antibiotics in aquaculture has caused antibiotic-resistant bacteria to be widespread (Furushita et al., 2003), which may lead to
the condition that the aquatic environment may serve as a reservoir of antibiotic resistance (Biyela et al., 2004). Antibiotic
resistant genes causing agents like ribosomal protection proteins and antibiotic modifying enzymes have originated from
marine Streptomyces or any other antibiotic producing microbes by lateral gene transfer (Chopra and Roberts, 2001).
Recent studies reported that the antibiotic resistant phenotypes displayed by the probiotic marine Streptomyces strains
TABLE 2 Selection of Suitable Marine Streptomyces as Probiotics in Aquaculture by Some Screening Process and Experimental Observations
Si. No.
Parameters
Methods
Observations
1
Preliminary
screening
(a) Isolation of marine Streptomyces from various marine sources like sediments,
seawater, sponges, seaweeds, mangroves, fish and shellfish by using different
media like Actinomyces isolation agar, Glycerol asparagine agar, Starch casein
agar, Kuster’s agar, and Yeast Malt Extract agar
(b) Preliminary identification and characterization of the strains has to be carried
out using parameters like Morphological (mycelial colouration, soluble
pigments, melanoid pigmentation, spore morphology), Biochemical (carbon
utilization, amino acids influence, sodium chloride tolerance), physiological
(pH, temperature) and chemo-taxonomical
(c) In vitro antimicrobial activity has to be carried out by agar well diffusion and
disc diffusion methods
(d) To test the strains for the extracellular enzymes activities like amylase, protease
(e) The strains which should be supplementation to the fish and shellfish have to be
mass cultured in a suitable medium (it can be broth or solid surface)
(1)
(2)
(3)
(4)
(5)
Marine Streptomyces were found to possess filamentous
hyphae, they form tough and leathery colonies which stick on
to the various media used
Identification of potent strains can be done by these
conventional methods—both species and genus level as
described (Kampfer, 2006)
The strains which exhibited good antagonism against fish and
shellfish pathogens which affect their immune system are to be
selected
The strains which exhibit the maximal enzymatic activities are
to be selected
The growth of the strains can be observed on the surface of the
medium in case of static condition but in broth they grow like a
mat which can be harvested by centrifugation and stored at 4°C
Experimental
screening
(f) In vivo evaluation of probiotic strain:
(1) The cultured strains can be lyophilized and mixed with feed ingredients, if it is
supplemented as live feed
(2) It can be dried and mixed with feed ingredients, can be supplemented as SCP
source
(3) The supernatant extract or lyophilized cells can be added directly to the culture
water
(g) Check for the strains adhering to the gut of the fish or shellfish and colonized.
This can be done by plating the gut extract to the suitable media for the growth
of particular strain
(h) In vitro calibration of probiotic potentials of the strains: screening for
survivability (resistance to low pH, pepsin, bile, and pancreatin), colonization
(autoaggregation, hydrophobicity and coaggregation) and safety (antibiotic
susceptibility and nonhemolytic activity)
(i) Dose optimal: The effective way of introduction and optimum dose to be
determined. Since marine Streptomyces is a very slow grower, the lag period
and the doubling time would also be determined
(j) If the cultures mixed with feed ingredients then proximate compositions like
moisture content, crude protein, crude fat, crude fiber, ash, nitrogen-free extract
has to be assessed in the feed prepared, as well as the excreta and the gut of the
fish and shellfish before and after feed supplementation
(k) Statistical analysis: At least triplicate experiments has to be analyzed using
various tools like one way or two-way ANOVA, Regression, T-test, Duncan’s
multiple range test and Tukey test
(6)
All the growth, survival related parameters have to be analyzed,
for example SGR, RGR, AGR, FCR and FCE. Water quality
analysis has to be carried out and the strains should not
promote algal growth in the culture system
(7) To observe whether the strain is retaining and support the
adhesion properties in the gut and get colonized in the host or
to be supplemented regularly
(8) The isolates should be susceptible to range of different
antibiotics and they should not be exhibiting hemolytic activity.
The cultures should exhibit excellent viability at low pH range
and they should be acid-tolerant, resistance to pepsin, bile and
pancreatic
(9) Marine Streptomyces is nonmotile aerobic, grows as mat in
broth (in static condition) and as clump (in shaker culture).
The harvested cells has to be centrifuged, lyophilized and at
last packed. Supplementation level of the cultures has to be
optimized which can be assessed by MIC protocol. Prevention
of fungal and algal contaminants
(10) The proximate composition value should be of permissive level
prescribed by AOAC (2000). It is assessed accordingly to the
body weight of fish and shellfish
(11) Comparison of the various treatments significance level (P < .05)
3
Postexperimental
screening
(l) Further, confirmation on the molecular identification of the cultures used (gene
sequencing and 16S rRNA phylogenetic analysis)
(m) Parameters for mass scale culture and optimum culture conditions to be
determined since the metabolic processes are controlled by various sources like
carbon, nitrogen, phosphorous, metals, induction, feedback regulation, growth
rate, and enzyme decay
(n) Finally, a cost-benefit analysis will also have to be carried out
(12) This is done either for the confirmation of strains until species
level (if any novel strains) and also deposition of the strains to
the culture centers
(13) These parameters help in regular usage of the strains to possess
the probiotic property and maximal yield effectively
(14) This may help in commercialization of the probiotic product for
the usage in aquaculture
Selection of New Probiotics: The Case of Streptomyces Chapter | 3 45
2
46 SECTION | A Probiotics and Prebiotics
Marine Streptomyces
strains
Inoculated in 1 L conical flask
Starch casein broth
Incubated at room temperature for 7 days
Grew as a mat
(cell mass)
Lyophilized
Mixed with
Formulated feed
ingredients
FIG. 9 Flow chart on usage of marine Streptomyces as probiotics along with feed ingredients and its supplementation to the aquatic fishes.
were generally conferred by their intrinsic resistant properties (Das et al., 2010; Latha et al., 2015). Therefore, systematic
screening for potential antibiotic resistance gene determinants in a potential probiotics genome must be conducted to assess
the potential risks and mobility. Even when marine Streptomyces was not in use as a probiotic, these lateral gene transfer
occur naturally. In addition, most studies to date suggest that the probable cause for antibiotic resistance patterns found in
aquatic microorganisms is due to increased use of synthetic antibiotics and drugs (Dang et al., 2008). Furthermore, remedial strategies can be a valuable tool to remove the genetic elements that harbor the antibiotic resistance from the relevant
probiotic strains (Morelli and Campominosi, 2002; Rosander et al., 2008). Reports on successful establishment of protoplast formation remedial methods were adopted to remove the two resistant plasmids from the parent Lactobacillus reuteri
(ATCC55730) without affecting the probiotic properties of the strain (Rosander et al., 2008). For forward, in-depth studies
can bring to light any significant disputes in the future.
A final limitation is the production of awful odor causing agents such as Geosmin (trans-1,10,-dimethyl-trans-(9)decalol) and MIB or 2-methyl-isoborneol (exo-1,2,7,7-tetramethyl-[2.2.1]heptan-2-ol) by the marine Streptomyces.
Planktonic cyanobacteria, bacteria, and several genera of fungi also produce geosmin and MIB (Wood et al., 2001). In
freshwater aquaculture and in the recirculation water system, these two compounds which are semivolatile terpenoids, impart an earthy-musty taste and odor to the water, as well as to the cultured fish (Klausen et al., 2005; Guttman and Van Rijn,
2008). They reduce the palatability of the cultured livestock and negatively impact aquaculture industries (Auffret et al.,
2011). These off-flavor compounds are known to be absorbed and bioaccumulated in the gills, skin and flesh of fish up to
200- to 400-fold as compared to the ambient concentration, resulting in a lower commercial value of the fish (Howgate,
2004). Many efforts have been carried out for the removal of these earthy odor compounds by the use of powdered activated
carbon, ozonation, and biofiltration (Elhadi et al., 2004). Among these technologies, ozonation is suggested to be effective
in the use of Streptomyces as the probiotics in aquaculture. Ozone has been known to remove odorants such as geosmin
and MIB via oxidation (Gonçalves and Gagnon, 2011). Existing studies reported on the combined effect of ozonation
(at0.3mgO3/L ROC) and Bacillus sp. S11 probiotic diets can protect shrimp P. monodon from Vibrio sp. challenge test
without harm to the shrimp or he probiotic bacteria in their internal system (Meunpol et al., 2003).
Several microorganisms have been implicated in the biodegradation of MIB (Pseudomonas spp., Pseudomonas
aeruginosa, Pseudomonas putida, Enterobacter spp., Candida spp., Flavobacterium multivorum, Flavobacterium
spp., Slaviensisbacillus spp., Bacillus subtilis) and geosmin (Bacillus cereus, Bacillus subtilis, Arthrobacter atrocyaneus, Arthrobacter globiformis, Rhodococcus moris, Chlorophenolicus strain N-1053, Rhodococcus wratislaviensis).
Selection of New Probiotics: The Case of Streptomyces Chapter | 3 47
Cyc2 gene amplified from marine Streptomyces genome by PCR
Marker inserted into Cyc2 gene (cyc2 + R)
Cyc2 + R inserted into plasmid
Plasmid returned to marine Streptomyces
Crossover event occurs and adds cyc2 + R to genome
Marine Streptomyces strain can no longer produce geosmin
FIG. 10 Double crossover event of mutant Cyc2 gene.
Other ways by which geosmin can be eliminated is through the insertion of genetic mutation in marine Streptomyces so that
it can no longer produce geosmin. Recent research with marine Streptomyces has shown that the Cyc2 protein (specifically
the N-terminal domain) is required for geosmin biosynthesis. The Cyc2 gene of marine Streptomyces can be made to be
nonfunctional or even removed entirely by PCR and a double crossover event. This process can be explained via flowchart
(Fig. 10). The ability to mutate Streptomyces genes to prevent the synthesis of geosmin may provide an opportunity to
introduce the organism onto a bio-filter without impacting the removal of metabolic waste products while eliminating the
synthesis of off-flavors.
4.
CONCLUSION
Marine Streptomyces exhibits promising results as a probiotic application in aquaculture but very few studies have been
carried out to establish its efficacy. Further widespread understanding and experimentation is still required to launch marine
Streptomyces as a probiotic in disease prevention and growth enhancement for aquaculture animals. Much research about
the organism has been done relating to the bioactive compound synthesis. Additionally, more detailed research focusing on
the usage of molecular and other techniques in order to clarify the possible underlying mechanism of marine Streptomyces
as probiotics in aquaculture environment. Overall, marine Streptomyces has potential as a candidate for probiotics and as
well as an alternative to antibiotics in maintaining a sustainable aquaculture.
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FURTHER READING
Donia, M., Hamann, M.T., 2003. Marine natural products and their potential applications as anti-infective agents. Lancet Infect. Dis. 3, 338–348.
Chapter 4
Development of New Probiotic
Foods—A Case Study on Probiotic Juices
Veeranjaneya Reddy Lebaka⁎, Young Jung Wee†, Venkatarami Reddy Narala⁎, Vinod Kumar Joshi‡
*
Yogi Vemana University, Kadapa, India, †Yungnam University, Gyeongsan, South Korea, ‡Dr. Y. S. Parmar University of Horticulture and Forestry,
Solan, India
1.
INTRODUCTION
Development of the man-microbe symbiosis during early life is a very intriguing and important biological process.
In ­humans, the intestinal microbiota plays a key role in host physiology and metabolism (Scholtens et al., 2012). The
intestinal microbiota is active during the first years after birth. The infant gastrointestinal tract (GIT) is rapidly colonized
through events related to the process of birthing (Adlerberth and Wold, 2009; Sela and Mills, 2014; Thum et al., 2012).
Exposure to vaginal, fecal, epidermal, and milk microbiota are among the various routes by which microbial inoculation
may occur (Cabrera-Rubio et al., 2012; Sela and Mills, 2014). The past decade has witnessed increasing attention and zeal
being dedicated to explicate the role of the gastrointestinal microbiota in health and diseases as well as explore and exploit
novel ways to investigate and manipulate the gut microbial composition for an improved health and well-being.
The word “probiotic” comes from the Greek word “ρο-βίο” which means “for life”. Probiotics were first introduced in
the twentieth century around the year 1900 by the Russian Nobel Prize winner Elie Metchnikoff, who studied the longevity of Bulgarian farmers and suggested a direct link to their daily consumption of fermented milk products that contained
large amounts of live nonpathogenic bacteria such as Lactobacillus bulgaricus, which can modify human intestinal flora in
favor of microbial species useful to the host organism (Zhang et al., 2005). Later on it was found that yogurt contained the
microorganisms required to guard the intestine from the damaging effects of other harmful bacteria. Different microorganisms have been used since then as probiotics in the last century for their ability to prevent and cure diseases. Kollath in 1953,
first defined the term “probiotic”, when he used the term to denote all organic and inorganic food complexes as “probiotics,” in contrast to harmful antibiotics, for the purpose of upgrading such food complexes as supplements. Vergio, in his
publication “Anti- und Probiotika,” compared the detrimental effects of antibiotics and other antimicrobial substances with
favorable factors (“Probiotika”) on the gut microbiology. Lilly and Stillwell proposed probiotics to be “microorganisms
promoting the growth of other microorganisms” (Vasudha and Mishra, 2013). Probiotics are defined as live microorganisms with a positive influence on their host with the ability to improve the intestinal microbial equilibrium (Guarner and
Schaafsma, 1998). An expert panel was convened in October 2013 by the International Scientific Association for Probiotics
and Prebiotics (ISAPP) to discuss the field of probiotics. The FAO/WHO definition of a probiotic—“live microorganisms
which when administered in adequate amounts confer a health benefit on the host”—was reinforced as relevant and sufficiently accommodating for current and anticipated applications (Hill et al., 2014). The probiotic market was worth 15.7
billion Euros in 2010, and is expected to increase to 22.6 billion euro by 2015 (BCC Research, 2011).
2.
PROBIOTIC MICROORGANISMS
To consider a microorganism as probiotic, the validation of its characteristics, strain identification, health benefits and
other characteristics are required (Kailasapathy, 2010). For a long time, a very limited number of microbial strains, then
used in food products or as supplements, were considered as probiotics based on their relevant properties (Grattepanche
and Lacroix, 2010). The large variety of functional fermented products and modernization of the biochemical and genetic
investigations of microorganisms has led to an increase in the number of microorganisms with probiotic potential. A list of
various types of microorganisms used as probiotics is given in Table 1.
Therapeutic, Probiotic, and Unconventional Foods. https://doi.org/10.1016/B978-0-12-814625-5.00004-2
© 2018 Elsevier Inc. All rights reserved.
55
56 SECTION | A Probiotics and Prebiotics
TABLE 1 List of Microorganisms Used as Probiotics
Lactobacillus Species
Bifidobacterium Species
Yeast and Other Species
L. acidophilus
B. bifidum
Saccharomyces boulardii
L. casei Shirota
B. breve
Saccharomyces cerevisiae
L. delbrueckii spp. bulgaricus
B. infantis
Streptococcus thermophilus
L. johnsonii
B. longum
Enterococcus faecalis
L. reuteri
B. adolescentis
Enterococcus faecium
L. rhamnosus
B. animalis
Pediococcus acidilactici
L. gallinarum
B. lactis
Lactococcus lactis
L. plantarum
Leuconostoc mesenteroides
L. salivarius
Bacillus cereus
L. crispatus
Escherichia coli Nissle 1917
L. gasseri
Propionibacterium freudenreichii
Probiotic microorganisms are usually available as culture concentrates in dried or deep-freeze form as food additives
for industrial or home use. These may be consumed either as food products (fermented or nonfermented) or as dietary
supplements (products in powder, capsule or tablet forms). To be defined as probiotics, these bacteria must also fulfill some
specific criteria listed by the European Union (Becquet, 2003):
●
●
●
●
●
●
●
●
Detailed definition and typing
Lack of pathogenic effects (i.e., production of enterotoxins and cytotoxins, entero invasiveness, adhesion of pathogens,
hemolysis, serological pathogenicity, presence of antibiotic-resistant genes)
Strain reaching its site of action, usually the gut, and thus survive to physiological stress met during its ingestion: acid
stomach and gut pH, presence of biliary salts (Butel, 2014)
Ability to adhere to the intestinal epithelium
Ability to colonize the colon
Proven clinical effect on health
Safety (Gorbach, 2000)
Competitive antagonism against pathogenic bacteria
The beneficial effects of probiotics (food with added live microbes) on human health are being increasingly promoted
by health professionals. It has been observed that probiotics can play a significant role in many metabolic and immunological functions and also could have a significant effect in alleviating infectious disease in children. Disturbed gut microbial
balance has been underscored as an originating factor for various metabolic, lifestyle, and diet-related maladies such as
obesity, endotoxemia, insulin resistance, type 2 diabetes (T2DM), metabolic syndrome (MetS), inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), nonalcoholic fatty liver disease (NAFLD), GIT cancers and more. So far, over
900 investigations with human involvement and numerous review articles have been published on the favorable effect of
probiotics. The studies were conducted with different probiotic strains on different health benefits and on different target
populations (Makinen et al., 2012). Fig. 1 shows the key health benefits bestowed by probiotics.
3.
PROBIOTIC PRODUCTS
Classification and types of probiotic foods are given in Fig. 2.
3.1
Dairy Products
Traditionally dairy fermented products have been considered to be the best probiotics carriers because they are easy to
manufacture. All the dairy industry products (milk, yogurt, cheese, milk proteins, and milk related desserts) have been
Development of New Probiotic Foods—A Case Study on Probiotic Juices Chapter | 4 57
Colonization resistance
Suppression of endogenous pathogens
Effect on intestinal
microbiota composition
Control of irritable bowl syndrome
Control inflammatory bowl diseases
Reduce antibioticassociated diarrhea
Improve immune response and
parasitic infections
Probiotics
Effect on immunity
Urinary tract and upper
respiratory tract infection
Management of allergy
Prevention of cancer
Effect on metabolic
process
Improve lactose metabolism and food digestion
Cholesterol normalization
B vitamins
Alleviate food allergy
Reduce constipation and ulcers
Reduce blood pressure
Diabetes
Antioxidative activity
FIG. 1 Major health benefits bestowed by probiotic microorganisms.
Probiotic foods
Nondairy
origin
Dairy origin
Sour milk, diphilus milk yoghurt,
acido-whey, ice-cream, lassi,
cheese, curd, nonfermented goat’s
milk beverage, frozen synbiotic
dessert, etc.
Plant based
Cereals
Bread and bakery products
Health drinks/beverages
Puddings
Edible film on pan bread
Fruit and
vegetables
Juices
Puree
Pulp
Beverages
Whole fruits
Powdered fruits
Animal based
Soy
Dry meat and
fish
sausages
Frozen desserts
Sausages
Soy curd
Soy yoghurt
Soy milk drink
FIG. 2 Classification and types of probiotic foods.
u­ tilized for probiotification and consumers have accepted the presence of microorganisms in the dairy products they consume (Boza-Méndez et al., 2012). Dairy-based products account for approximately 43% of the functional beverage market, and is mostly comprised of fermented products (Özer and Kirmaci, 2010). Fermented milks, especially yogurt-style
products, are the most popular functional probiotic beverages with kefir in Western Europe and North America and ymer
in Denmark being good examples. Probiotics in dairy products were shown to be very promising features for a functional
58 SECTION | A Probiotics and Prebiotics
food, because they exhibited excellent conditions for maintaining the viability of probiotic bacteria (Buriti et al., 2007a,b;
Souza and Saad, 2009; El-Dieb et al., 2012). When comparing other matrices with dairy matrices, the protective effect of
the latter, especially from milk proteins on probiotics in the digestive system, has been discussed in the literature. Proteins
are sources of bioactive peptide precursors, which resist passage through the digestive tract. Furthermore, milk has a physicochemical composition rich in protein with considerable amounts of lipids resulting in a protective matrix for probiotics.
These characteristics favor the survival of probiotics against adverse conditions of the digestive system. Milk proteins are
utilized as a suitable carrier matrix for probiotic bacteria, suggesting that it is effective in allowing probiotic bacteria reach
their site of action (Ritter et al., 2009).
3.2
Nondairy Products
In recent years, nondairy probiotic delivery has been attracting more attention due to the increased demand from consumers.
This demand is particularly due to an increase in the lactose intolerant population (around 70% in Asia), allergies to milk
proteins and the prevalence of high cholesterol. Increase in the consumer vegetarianism throughout the developed countries
has also increased the demand for vegetarian probiotic products. These are the major drawbacks related to the fermented
dairy products (Heenan et al., 2004; Yoon et al., 2006). Economic and cultural factors may also negatively affect the consumption of probiotic dairy products, since most are fermented foods. Nondairy probiotic beverages are particularly attractive due to their lack of dairy allergens, low cholesterol content and vegan friendly status (Prado et al., 2008). Furthermore,
different substrates can provide different combinations of antioxidants, dietary fiber, minerals and vitamins. This has led
to the success of biofunctional foods and a desire to expand and provide alternative probiotic beverage choices that are not
dairy-based. The non dairy functional/probiotic foods market is projected to have an annual growth rate of 15% between
2013 and 2018 (MarketsandMarkets, 2013). The above fact is highlighted by the trend in the U.S. functional food market,
which is developing in a different fashion from that seen in Europe, with its functional food sector more broadly defined as
neutraceuticals and consumer interest tending to lie more with botanical dietary supplements rather than fortified of foods.
The above mentioned draw backs of dairy probiotics has led to the search for new and alternative carriers for probiotic
microbes. Development of nondairy probiotic products, such as fruits, vegetables and cereals, has been demonstrated as one
of the best choices and demand for nondairy probiotics is increasing (Yoon et al., 2006; Prado et al., 2008; Granato et al.,
2010a,b). The structural characteristics and composition (nutrients such as minerals, vitamins, dietary fibers, and antioxidants including good amount of sugars) of fruits, vegetables and cereals are suitable and ideal substrates for probiotic microbes (Reddy et al., 2015). Demand in the development of fruit juice based probiotics is increasing and is more attractive
due to their taste, nutrient profiles and general acceptance as being healthy and refreshing foods (Nualkaekul et al., 2011).
3.3 Why Fruits are Ideal Choice?
Fruits are among the most important foods of mankind as they are not only nutritive but also play a vital role in maintaining
health. Fruits, both fresh and processed, not only improve the quality of diet, but also provide essential ingredients such as
carbohydrates vitamins, minerals, antioxidants. Fermentation is a viable technique in the development of new products with
modified physicochemical and sensory qualities, especially flavor and nutritional components. Alcohol, acetic and lactic
acid fermentations are important for quality in production. Fermented beverages have been known to humankind from time
immemorial (Reddy et al., 2013).
Fruit juices have good amounts of sugars, minerals, and vitamins, which generally enhances the survival of probiotics
during storage (Ding and Shah, 2008; Sheehan et al., 2007). Fruit juices are also an ideal choice for consumers who are
interested in low cholesterol foods or suffer from lactose intolerance (Granato et al., 2010a,b; Prado et al., 2008). The rate
of lactose intolerance (LI) incidence in different ethnic groups is given in Table 2. Previous studies have revealed that pH,
organic acids levels, dietary fiber, protein, total phenol, and oxygen are the main factors which affect the survival of probiotics in fruit juices (Champagne et al., 2005; Nualkaekul et al., 2011; Saarela et al., 2006; Shah, 2000). It has been suggested
that fruit juice is an ideal medium for carrying functional food ingredients such as probiotics because its residence time in
the stomach is short, therefore the bacteria are not overexposed to the unfavorable acidic conditions of the stomach.
The protein and dietary fiber present in the fruit juice was shown as favorable for the survival of probiotics during storage in fruit juices (Champagne et al., 2011; Ding and Shah, 2008; Nualkaekul and Charalampopoulos, 2011; Saarela et al.,
2006; Sheehan et al., 2007). The cell count of Lactobacillus casei in mare milk (8.59 ± 0.04 log CFU/mL) and in pineapple
juice (8.20 ± 0.01 log CFU/mL) are comparable; the implication of this being that fruits are the best media for probiotic
growth, in addition to being naturally full of essential nutrients and tasting good (Luckow and Delahunty, 2004a,b; Sheehan
et al., 2007; Zhou et al., 2009). Fruit juice is cited as a healthy food product, and is consumed regularly by a large p­ ercentage
Development of New Probiotic Foods—A Case Study on Probiotic Juices Chapter | 4 59
TABLE 2 Lactose Intolerance Rate (LI) Incidence in Different Ethnic Groups
Sl. No.
Ethnicity/Geographic Region
% Population With LI
1
East Asian
90–100
2
Indigenous (North America)
80–100
3
Central Asian
80
4
African American (North America)
75
5
African (Africa)
70–90
6
Indian (Southern India)
70
7
Indian (Northern India)
30
8
Balkans Region
55
9
Latino/Hispanic
51
10
French (Southern France)
65
11
Anglo (North America)
21
12
Italian (Italy)
20–70
13
German (Germany)
15
14
British (UK)
5–15
Data from de Souza Neves Ellendersen, L., Granato, D., Bigetti Guergoletto, K., Wosiacki, G., 2012. Development and sensory profile of a probiotic beverage
from apple fermented with Lactobacillus casei. Eng. Life Sci. 12, 475–485.
of the population. However, the survival of probiotics in fruit-based matrix is more complex than in dairy products because
bacteria usually need protection from the acidic conditions in the media (Shah, 2007). In spite of the challenges involved
in the process of nondairy probiotic foods, it was suggested that fruit juice could serve as a good substrate for probiotics
(Mattila-Sandholm et al., 2002; Tuorila and Cardello, 2002).
Fresh fruits and vegetables contain mostly cellulose which is not digested by the gastrointestinal system. Use of vacuum
impregnation technology for probiotic bacteria were done with apples, showing promising results (Alzamora et al., 2005;
Betoret et al., 2003). Thus, fruit matrices are certainly an important area of research and development with great potential
for the functional food market. According to Kourkoutas et al. (2005), fruits contain requirements necessary for probiotic
adhesion to plant tissue. In this way, fruits such as apples and pears, due to their cellulose content, may exert a protective
effect on the probiotic microorganisms during passage through the intestinal tract. Studies conducted by Martins et al.
(2013) group showed that fruits, such as apple, guava, banana, and melon, have potential as carriers for probiotic bacteria.
The results of scanning electron microscopy showed a positive interaction between the probiotic microorganisms and the
fruity tissues, since bacteria strongly adhered to the fruit surface (Martins et al., 2013). Therefore, the intrinsic characteristics of the plant surface microarchitecture, due to the presence of ridges and natural prebiotic compounds (such as oligosaccharides), protect probiotic microorganisms from the acidic environment of the stomach and are a source of nutrients,
which positively influences bacterial survival (Ranadheera et al., 2014). However, Ijabadeniyi (2010) indicated the need
for further work on the mechanism of internalization of microorganisms in plants. In particular, the way microorganisms fit
into openings naturally present on the fruits’ surfaces, such as stomata and lenticels, and in tissue damaged by processing
(Martins et al., 2013).
Studies demonstrate that the natural sugars that are present in juices can facilitate the growth of probiotic organisms
and taste good. This is true of tomato, pomegranate, pineapple, orange, and cashew-apple juice. These microbes can impact
physiochemical aspects, such as increasing the concentrations of flavanones and carotenoids in orange juice, and have
shown good survival rates during beverage storage. The final acidity of these beverages is quite high after the fermentation
by probiotic Lactobacillus species (Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus
plantarum, and Lactobacillus acidophilus). The enrichment of juices with brewer’s yeast autolysate before fermentation
positively impacts the nutritional quality of the final beverage, raising the feasibility of cofermentation by the appropriate
combination of bacteria and yeast. Examples of commercially available probiotic-containing fruit juices include Biola and
Bioprofit (Priya and Munishamanna, 2013).
60 SECTION | A Probiotics and Prebiotics
3.4
Preparation of Fruit Juice Probiotics
A flow diagram of preparation of probiotic fruit juice is depicted in Fig. 3.
3.5 Types of Fruit Juice Probiotics
To avoid the disadvantages of dairy-based fermented foods and to get appealing tastes and refreshing profiles, several nontraditional, nondairy-based fermented foods (fruit juices) have been developed (Tables 3 and 4). Some fruits, such as apples,
oranges, black currant, banana, blueberry, pineapple, cashew apple, cantaloupe melon, raspberry, pomegranate juice, etc.
(Savard et al., 2003; Yoon et al., 2005; Pereira et al., 2011; Nualkaekul et al., 2012; Fonteles et al., 2013; Anekella and
Orsat, 2013), mixed with vegetable juice (Nosrati et al., 2014) are being employed for the development of probiotic juices.
3.5.1 Apple
Manufacturing of probiotic apple juice by Lactobacillus casei fermentation was studied. For optimum production pH (4.6),
temperature (30°C) inoculum (4.87 log CFU/mL) and incubation period (16 h) were selected as effecting factors. During
fermentation and storage, yellowness increased and redness reduced. The initial pH and temperature was demonstrated to
have an effect on fermentation and the growth of microorganisms varied in accordance to the species and substrate used.
The effect of temperature on the growth of Lactobacillus casei was greater than that of pH value. Initial pH had no significant effect on the biomass. A good number of living cells was observed at a mild temperature (~30°C) as higher temperatures diminished the viability of Lactobacillus casei. The best viability was observed at a pH 6.4 and a temperature of
30°C. Biomass production was increased in apple juice that was inoculated with 7.48 log CFU/mL. Apple juice produced
by using optimum conditions was then refrigerated for 42 d in order to examine the bioavailability of Lactobacillus casei.
After 3 weeks of storage, the viable cell number increased from 8.41 to 8.72 log CFU/mL and then decreased to 8.62 on day
35. The number of viable cells decreased at the end of storage period to a range quite acceptable (8 CFU/mL) for probiotic
products (Gökmen et al., 2003). Optimization of culture conditions to develop the probiotic apple beverage was studied
Fruits
Cleaning, sorting, and
juice preparation
Juice
Inoculation with
probiotic organism
Fermentation
Probiotic fruit juice
Packaging and storage at
optimal conditions
FIG. 3 Schematic flow diagram of preparation of probiotic fruit juice.
Development of New Probiotic Foods—A Case Study on Probiotic Juices Chapter | 4 61
TABLE 3 List of Fruit Based Probiotic Products Developed Recently
Sl. No.
Name of the Fruit
Reference
1
Fermented banana pulp
Tsen et al. (2004)
2
Fermented banana
Tsen et al. (2009)
3
Tomato-based drink
Yoon et al. (2004)
4
Many dried fruits
Betoret et al. (2003)
5
Green coconut water
Prado et al. (2008a)
6
Cranberry, pineapple, and orange juices
Sheehan et al. (2007)
Kiwi
Sheehan et al. (2007)
7
Grape and passion fruit juices
Saarela et al. (2006)
8
Probiotic banana puree
Tsen et al. (2009)
9
Nonfermented fruit juice beverages
Renuka et al. (2009)
10
Blackcurrant juice
Luckow and Delahunty (2004a,b)
Pomegranate
Mousavi et al. (2011)
11
Plum juice
Sheela and Suganya (2012)
12
Cashew apple juice
Pereira et al. (2011)
13
Fruit juices (mango, sapota, grape)
Vijaya Kumar et al. (2013)
Peach
14
Guava
Dipjyoti et al. (2015)
15
Mixture of pineapple, apple, and mango
juices
Mashayekh et al. (2015)
16
Clarified apple juice
Pimentel et al. (2015a)
17
Clarified apple juice with oligofructose or
sucralose
Pimentel et al. (2015b)
using response surface methodology. It was found that 10 h of fermentation at 37°C in Gala apple juice is best. Sensory
evaluation of the prepared-fresh, fermented probiotic apple beverage determined it to have a thick texture and sweet taste
while the probiotic apple beverage stored for 28 days at 7°C showed a thick texture and acidic taste (Table 5). Finally, when
the fermented probiotic beverage was tested by potential consumers, it showed an acceptance index of 96% (de Souza
Neves Ellendersen et al., 2012).
Pimentel et al. (2015a) have studied the physicochemical characteristics, probiotic viability and acceptability of
Lactobacillus paracasei fermented clarified apple juice with oligofructose. No change in the physicochemical characteristics, acceptability or stability in storage, including enhanced probiotic survival, was observed. They also tested the effect
of packaging material (probiotic juice in plastic or glass) on storage (4°C for 28 days) and suggested that viability of the
probiotic culture in glass package was greater than in plastic and also clarified that packaging material (glass or plastic)
have no influence on the physicochemical characteristics and consumer acceptability of juices. In another study Pimentel
et al. (2015b) investigated the effect of adding oligofructose or sucralose as sugar substitutes as well as a probiotic on
sensory quality and acceptance of clarified apple juice fermented with Lactobacillus paracasei. The study showed that
oligofructose (20 g/L) substituted juices were less sweet than those with sucrose (20 g/L). However, both oligofructose and
sucralose contributed to increased acceptance (taste and overall impression) of the pure juices. The probiotic supplementation increased the turbidity of the juice but acceptance (appearance, aroma, flavor, texture, and overall impression) did
not diminish. The above studies demonstrate that it is possible to develop a synbiotic apple juice that has a similar sensory
profile (excepting the presence of particles and turbidity) and acceptance to that of the sucrose-added juice by adding
Lactobacillus paracasei as a probiotic culture and oligofructose as a sugar substitute and prebiotic.
Fruit Juice Composition
Juice %
Probiotic Strain
Brand
Producer and Country
Strawberry, black current, rosehip
Exotic: blend of banana, grape, lime and lemon
20
L. plantarum vv
Pro Viva
Skanemejerier/Sweden
Blend of banana, grape, lime and lemon and enriched with Vit C and minerals
12
L. plantarum vv
Pro Viva Active
Skanemejerier/Sweden
Raspberry, black current, and grape
15
L. plantarum vv 299v
SHOT
Skanemejerier/Sweden
Whey drink with apricot and peach
17
L. ramnosus GG
Gefilus/Gfilac
Valio/Sweden/Finland
Orange/peach juice + prebiotic + Vit C
60
L. ramnosus GG
Gefilus/Gfilac
Valio, Sweden/Finland
Pineapple + carrot + Ca + B-carotene
50 and 10
L. ramnosus GG
Gefilus/Gfilac
Valio, Sweden/Finland
Multifruit (mango, peach, grape, orange passion fruit)
80
L. ramnosus GG
Gefilus/Gfilac
Valio, Sweden/Finland
Apple and grape
100
L. ramnosus GG
Gefilus/Gfilac
Valio, Sweden/Finland
Orange and mango
95
L. ramnosus GG
Biola
Tine BA/Norway
Apple and pear
95
L. ramnosus GG
Biola
Tine BA/Norway
Orange
L. reuteri
“R”
Ingman/Sweden
Orange, pineapple and Ca
L. reuteri
“R”
Ingman/Sweden
Multifruit
L. reuteri
“R”
Ingman/Sweden
Peach-banana
B. lactis
“Its Alive”
UK
Pomegranate, blackberry, cranberrymango, apple, strawberry or lemon ginger
100
L. plantarum vv 299v
GoodBelly
Nextfoods/USA
Orange juice
100
B. animalis
DAWN
Kerry group/Ireland
Ginger-lime, orange-ginger-pineapple, pineapple-lemon-cayenne and raspberry
100
Bacillus coagulans GBI 306086
Probiotic waters
Suja/USA
Data from Mousavi, Z.E., Mousavi, S.M., Razavi, S.H., Emam-Djomeh, Z., Kiani, H., 2011. Fermentation of pomegranate juice by probiotic lactic acid bacteria. World. J. Microbiol. Biotechnol. 27(1), 123–128.
62 SECTION | A Probiotics and Prebiotics
TABLE 4 Examples of Commercial Fruit Juice-Based Probiotic Drinks
Development of New Probiotic Foods—A Case Study on Probiotic Juices Chapter | 4 63
TABLE 5 Sensory Profile Data for the Three Apple Samples (Fresh Juice, Fresh Probiotic Juice and Stored
Probiotic Beverage)
Samples
Attributes
Gala Apple Juice
Fresh Probiotic Apple Beverage
Probiotic Apple Beverage (Stored for 28 days)
7.83a (±0.94)
3.54b (±2.28)
5.84b (±2.10)
7.53a (±0.78)
3.32b (±2.44)
3.80b (±2.07)
Acidicd
4.30a (±2.53)
3.32a (±2.53)
6.13b (±2.57)
Applec
6.49a (±1.40)
3.60b (±2.20)
4.04b (±2.35)
Sweete
6.43a (±2.02)
6.21a (±2.05)
4.56b (±2.45)
0.99a (±2.23)
5.81b (±2.01)
6.31b (±2.40)
Appearance
Caramel colorc
Aroma
Applec
Taste
Texture
Thickd
Means followed by the same letter, on the same line, did not differ significantly from each other (P > .05).
c
Average of 9 tasters.
d
Average of 7 tasters.
e
Average of 10 tasters.
Source: de Souza Neves Ellendersen, L., Granato, D., Bigetti Guergoletto, K., Wosiacki, G., 2012. Development and sensory profile of a probiotic beverage
from apple fermented with Lactobacillus casei. Eng. Life Sci. 12, 475–485.
3.5.2
Banana
Banana (Musa spp.) is an important food crop cultivated widely in tropical and subtropical areas and is one of the major
fruits in the world (Gowen, 1995). It was processed widely for various products including banana puree, banana pulp,
banana figs, banana flour or powder, banana chips, canned banana slices, banana jam, banana vinegar, banana wine, and
banana juice, etc. Application of banana as a medium for LAB fermentation has also been studied (Aegerter and Dunlap,
1980; De Porres et al., 1985). Probiotic banana product with Lactobacillus acidophilus and certain other fruits (as prebiotic)
have been prepared and might provide functional benefits (as synbiotic) (Prajapati et al., 1987). Stability and viability of
free and immobilized Lactobacillus acidophilus by n-carrageenan entrapment was evaluated. Results revealed that entrapment enhanced stability and viability along with fermentation efficiency (Tsen et al., 2004). After the completion of fermentation, more viable cells in gel beads (108 CFU/(mL gel)) than in free cells (106 CFU/mL) were observed. Immobilized
cells survive under adverse conditions and fermented efficiently compared to free cells. Immobilized Lactobacillus acidophilus fermented banana medium was found to possess synbiotic properties and resulted in desirable viable cell counts of
108 CFU/mL in the final product (Tsen et al., 2004). Yogurt with 15% banana marmalade (BM) was also prepared to study
the effect of fermentation conditions on its sensory properties. Results from this study have potentially contributed to the
use of probiotic cultures in fruit-flavored yogurt production. 106 log CFU/g viable cells were observed at 1 week of storage
at 4°C and then began to decrease in both viable cell count and sensory qualities. The best sensory scores were recorded in
the yogurts produced in 15% BM fermented with Bifidobacterium bifidum (Songül et al., 2012).
3.5.3
Blackberry
Rubus fruticosus, commonly referred to as the blackberry, has a high abundance of healthy antioxidants and nutrients
such as anthocyanins, proanthocyanidins and other flavonoids, salicylic acid, ellagic acid, and fiber (Hager et al., 2008a,b;
Rommel et al., 1992; Wang and Lin, 2000). Many of these compounds have been recognized for their anticancer properties. Commercially probiotic blackberry fruit juice is prepared with 108 CFU/mL by Next foods USA (Vattem et al., 2005).
3.5.4
Blueberry
Blueberries species are widely distributed in North America, Europe, Asia, and Africa, and they have the highest antioxidant capacity. Zhu et al. (2016) clearly demonstrated that the combination of blueberry juice and probiotic bacteria has
64 SECTION | A Probiotics and Prebiotics
a synergistic effect against the progression of AFLD. Blueberry juice can reduce the damage and apoptosis of AFLD by
improving the activity of SIRT1 and promote activity of liver cells against oxidative damage by increasing the activities
of related enzymes to remove oxygen free radicals, protect liver cells, effectively prevent lipid peroxidation, and regulate
lipid metabolism.
3.5.5
Black Current Fruit
Luckow and Delahunty (2004a,b) evaluated the consumer’s acceptance for the appearance, aroma, texture, and flavor of
the probiotic fruit juices. Novel blackcurrant juices containing the probiotic cultures were compared with the conventional
blackcurrant juices by means of descriptive analysis. The probiotic juices contained aroma and flavors characteristic of
the functional ingredients. Subsequent testing took place in a local shopping center where the consumers were presented
with two randomly coded blackcurrant juice samples. One of the products was a natural blackcurrant juice and the other
was a commercially processed blackcurrant juice containing probiotic cultures. The consumers were instructed that one of
the juice samples contained “special ingredients” designed to improve their health. They were asked to assess their overall
impression of both the juices and to rate their acceptance of the sensory characteristics. Additionally, based on their overall
impressions and guided by their individual expectations, the consumers were asked to identify the juice they perceived to
be the “healthiest” (e.g., containing the “special ingredients”). The juice preference was dependent on the gender and the
age. In general, the consumers selected their most preferred juice product as the “healthiest” sample.
3.5.6
Cranberry Bush Fruit
Cranberrybush (Viburnum opulus L.) is the fruit of a deciduous shrub, which belongs to Caprifoliaceae family native to
Europe, North Africa and North Asia and also often found in the central zone of western Russia. It is commonly used for
ornamental purposes (Sedat Velioglu et al., 2006; Anonymous, 2008). The bark and fruit of the European cranberrybush
tree are widely used in pharmacology and known for their antispasmodic and antimicrobial properties, relief of asthma,
cold, fever, nervousness, water retention problems, cough, cramps, stomachache, menstrual cramps, uterine infections,
blood pressure, and infertility (Anonymous, 2008; Nellessen, 2006). In Turkey, after harvesting the fruits in autumn, a
fermented juice product is produced via spontaneous fermentation by putting the fruit in plastic jars topped with water and
left for 3–5 months. This juice is not very palatable due to its strong sour flavor. The local people who relish its consumption believe the astringent taste of the juice can be reduced with a longer fermentation (Sedat Velioglu et al., 2006; Sagdic
et al., 2014). 332 isolates of lactic acid bacteria (LAB) strains and Leuconostoc species isolated from traditional fermented
gilaburu fruit juice were evaluated their probiotic potential. Characterization and probiotic potentials of the LAB isolated
from fermented gilaburu (V. opulus) juice were studied just once and further research needs to be done on their qualities in
similar food formulations as a probiotic (Sagdic et al., 2014).
3.5.7
Cashew Apple
Anacardium occidentale L. is a tropical tree native to the northern and northeastern regions of Brazil. Cashew apple is
pseudofruit and is the part of the tree that connects it to the cashew nut, the tree’s true fruit. The cashew apple is very
popular and highly consumed as a drink and as concentrated juice. Cashew apple is rich in fructose, glucose, minerals,
several amino acids and is considered a good antioxidant with high ascorbic acid and phenols. (Zepka et al., 2009; Rabelo
et al., 2009).
Pereira et al. (2011) studied the probiotification of cashew apple juice by Lactobacillus casei NRRL B-442. The optimum conditions for probiotic cashew apple juice production were an initial pH 6.4, a fermentation temperature of 30°C,
an inoculation level of 7.48 log CFU/mL (Lactobacillus casei) and 16 h of fermentation process. It was observed that the
Lactobacillus casei grew during the refrigerated storage. Viable cell counts were higher than 108 CFU/mL throughout the
storage period (42 days). The juice’s lightness and yellow tint increased, resulting in a total color change from the initial
redness of the juice which was substantially reduced during fermentation and storage. The above observations clearly indicate that cashew apple juice is as good a food matrix for Lactobacillus casei growth as dairy products and probiotic cashew
apple juice fermented with Lactobacillus casei is a excellent functional health drink. The probiotic juice was stable up to
42 days of refrigerated storage without considerable viability losses and the characteristic color of the juice (yellowness)
was enhanced along fermentation and storage. Enzyme browning, which is characteristic of cashew apple juice, is usually
prevented using chemical products such as sodium metabissulfite, was avoided with just the application of fermentation
technology. Besides taste, color is an important factor in food acceptance and the maintenance of the characteristic color
without preservatives is a technological advantage. Therefore, cashew apple juice fermented with Lactobacillus casei is a
healthy alternative of functional foods containing probiotics.
Development of New Probiotic Foods—A Case Study on Probiotic Juices Chapter | 4 65
3.5.8
Green Coconut Water
Soccol et al. (2007) developed probiotic green coconut water fermented by Lactobacillus plantarum AC-1, Lactobacillus
plantarum B-7, and Bifidobacterium animalis subsp. lactis BFL-9. The supplementation of the beverage with yeast extract,
hydrolyzed soy protein, and sucrose produced maximum yield to a mixed culture with 8 h of fermentation. After 28 days
of cold storage at 4°C, lactobacilli and Bifidobacterium animalis subsp. lactis presented 8 and 7 log CFU/mL, respectively.
The fermented beverage added sugar and coconut aroma presented good sensorial characteristics and acceptability from
potential consumers. Furthermore, the beverage could be classified as “low calorie” delivering only 33.5 kcal/100 mL.
3.5.9
Dates
An economically cheap medium is critical for industrial scale production of probiotic microorganisms (Oh et al., 1995).
In this context, high amounts of sucrose as well as reducing sugars (especially glucose and fructose) present in date palm
(Phoenix dactylifera) fruit will offers potential for convenient and inexpensive biomass production (Al-Shahib and Marshall,
2003). Date syrup and pits were reported to have positive influence as nutrients for the cultivation of Lactococcus lactis and
consequently, were suggested as a suitable substrate for the cultivation of microorganisms (Khiyami et al., 2008). Date powder was used for the first time as a low-cost substrate during the optimization of culture conditions for the economic production of a probiotic bacterium, Lactobacillus casei ATCC 334. The effect of 11 factors on bacterial growth was investigated
using the Taguchi experimental design and three factors, including palm date powder (38 g/L), tryptone (30 g/L) and agitation
rate (320 rpm), were found to be the most significant parameters by response surface methodology (Shahravy et al., 2012).
3.5.10
Guava
Guava (Psidium guajava L.) is a widely cultivated fruit plant among the Psidium species. It is distributed worldwide in the
tropical and subtropical areas. The fruit has a light yellow or pink pulp, is eaten fresh or as preserves, and is processed for
use in dairy and baked products. It is rich in vitamin C, carbohydrates, proteins, calcium phosphorus, vitamin A, pantothenic acid, riboflavin, and thiamin (Diwan and Shukla, 2004; Reddy and Reddy, 2011).
The suitability of probiotic guava fruit juice beverage fermented with lactic acid bacteria isolated from milk, curd, whey
and Lactobacillus plantarum has been studied by Dipjyoti et al. (2015). In this study the probiotic strains isolated from dairy
products are used. The formulated guava fruit beverage was inoculated with these isolates and incubated at 30°C and measured for pH, acidity, sugar content, and viable cell count changes during fermentation under controlled conditions. The pH
of the guava fruit beverage was initially 5.5 at °C and decreased gradually to a suitable range. The titrable acidity at 30°C
was 0.28%–0.32% and found to increase with further fermentation. The sugar at 30°C was initially 15%–22%. The viable
cell counts at 30°C were estimated, ranging between 5.6–8.9 × 106. The fruit beverage was assessed for acceptability through
sensory evaluation. Molecular confirmation of isolate no. 8 through 16 s rDNA sequence has been done and was found to be
Lactobacillus coryneformis. The final product resulted in suitable pH, acidity, sugar content and ideal number of viable cell
counts. The probiotic guava fruit beverage could serve as a healthy beverage for consumers with dairy allergies, providing
benefits to gut health, prevention of diarrhea and an excellent nutrient source for populations vulnerable to undernourishment.
3.5.11
Mango
Mango (Mangifera indica L.) belongs to the Anacardiaceae family, comprises >70 genera, and is one of the most economically important fruits. According to the historical records, its cultivation began as a fruit tree in India around 4000 years ago.
It is reputedly called the “king of fruits.” Mango occupied fifth rank in the total production of fruit crops worldwide and is
produced in over 90 countries. Asia accounts for approximately 77% of global mango production. Mango is an important
source of antioxidants, vitamins, minerals, and dietary fiber. Unlike many other fruits mangos do not contain any allergic
proteins, and are a healthy alternative to dairy products for probiotification (Reddy et al., 2015; Vijay Kumar et al., 2015).
Probiotification of mango juice was carried out by four lactic acid bacteria (Lactobacillus acidophilus (MTCC10307),
Lactobacillus delbrueckii (MTCC911), Lactobacillus plantarum (MTCC9511) and Lactobacillus casei) fermentation
(Reddy et al., 2015). Mango juice fermentation was performed at 30°C for 72 h under microaerophilic conditions. Microbial
population, pH, titrable acidity, sugar, and organic acid metabolism were measured during the fermentation period and the
viability of the strains was determined under the storage conditions at 4°C for 4 weeks. The four lactic acid bacteria used
in this study showed good growth in mango juice and were found capable of rapidly utilizing the juice for cell synthesis
and lactic acid production without nutrient supplementation. The lactic acid cultures rapidly fermented mango juice and reduced the level of sugar. Lactobacillus plantarum consumed the sugar at a much faster rate than the other three strains. The
lactic acid bacteria reduced the pH to as low as 3.2 from 4.5 within 72 h of fermentation (Fig. 4). Substrate ­concentration
66 SECTION | A Probiotics and Prebiotics
2.25
5
4.5
4
1.75
3.5
pH
Acidity (%)
2
1.5
3
1.25
1
2.5
0
12
24
36
48
60
72
2
Time (h)
FIG. 4 Acid production and pH decrease kinetics during fermentation by L. plantarum, L. delbruekii, L. acidophilus, and L. casei in mango juice.
(Reproduced with permission from Reddy, L.V., Min, J.H., Wee, Y.J., 2015. Production of probiotic mango juice by fermentation of lactic acid bacteria.
Microbiol. Biotechnol. Lett. 43(2), 120–125.)
was reduced to 5.8% (w/v) from 12% (w/v). Lactobacillus plantarum showed the faster utilization of sugar and reduction of pH of the mango juice compared to the other strains used. The viability of cells maintained at 1.0 × 107 CFU/mL
throughout the storage period. The effect of cold storage on the viability of lactic acid bacteria species in fermented mango
juice is presented in Table 6. From this investigation, it can be concluded that mango juice is suitable for the production
probiotic beverage. The total phenolic content (TPC), antioxidant and antimicrobial activity of probiotic mango juice was
investigated by Kumar et al. (2015). TPC was increased significantly in 72 h of fermented probioticated juice and DPPH
radical scavenging activity was significantly higher in probioticated mango fruit juice than in nonprobioticated fruit juice.
No significant difference between the sensory scores of probioticated and nonprobioticated products was noticed and the
influence of fermentation on juice texture, taste, flavor and overall acceptance was not significant. From these results, it can
be concluded that probiotic mango fruit juice can be utilized to deliver probiotic LAB to lactose-intolerant people and those
who are allergic to milk-based products.
3.5.12
Noni Fruit
Noni (Morinda citrifolia) is a tropical and subtropical plant that grows on the Pacific islands. It contains good natural medicinal qualities (Dixon et al., 1999; McClatchey, 2002) and has been cultivated for over 2000 years. All the parts of the
plant (fruit, leaves, bark, and root) have been known to contain active compounds (Chan-Blanco et al., 2006). Most noni
fruit is consumed as fresh juice, which is traditionally made by the natural fermentation of noni fruit in sealed containers for
4–8 weeks at ambient temperature (Wang et al., 2009). Wang et al. (2009) assessed the feasibility of the noni fruit as a substrate for the development of probiotic juice by Lactobacillus casei, Lactobacillus plantarum, and Bifidobacterium longum.
All tested strains grew well in noni juice. After 4 weeks of storage at 4 °C, Bifidobacterium longum and Lactobacillus
plantarum survived under low pH conditions in the fermented juice. Lactobacillus casei produced less lactic acid than
Bifidobacterium longum and Lactobacillus plantarum. After 4 weeks of cold storage at 4°C. They found that Bifidobacterium
longum and Lactobacillus plantarum are optimal probiotics for fermentation with noni juice.
3.5.13
Peach
Peaches (Prunus persica) are rich source of minerals, vitamins and contain a good amount of sugar (Pakbin et al., 2014).
They are rich in phytochemicals, dietary fiber and polyphenols that provide health benefits to the consumers (Magerramov,
2006). Probiotic peach juice was prepared by Pakbin et al. (2014) using three different LAB species (Lactobacillus casei, Lactobacillus delbrueckii, and Lactobacillus plantarum) and it was determined that all species adequately fermented
the peach juice and showed positive growth (reached 1.12 × 109 CFU/mL after 48 h at 30°C). The viable cell counts of
Lactobacillus plantarum and Lactobacillus delbrueckii in fermented peach juice were 7.2 × 105 and 1.7 × 107 CFU/mL after
4 weeks in storage at 4°C. However, Lactobacillus casei was unable to survive at low pH and high acidity conditions and
in 4°C cold storage for 1 week it completely lost cell viability.
Development of New Probiotic Foods—A Case Study on Probiotic Juices Chapter | 4 67
TABLE 6 Effect of Cold Storage on the Viability of Lactic Cultures in Fermented Mango Juice
Viability (CFU/mL)
Time (weeks)
L. acidophilus
9
L. delbrueckii
L. plantarum
L. casei
2.2 ± 0.21 × 10
1.5 ± 0.2 × 109
10.8 ± 1.0 × 108
13.8 ± 1.6 × 108
22 ± 1.4 × 107
10.8 ± 1.1 × 107
7.3 ± 1.2 × 107
12.2 ± 1.0 × 107
5.8 ± .07 × 106
3
52.2 ± 3.4 × 105
4 ± 0.2 × 106
6 ± 0.6 × 106
1.0 ± 1.0 × 106
4
1.5 ± 1.0 × 106
1.6 ± 0.4 × 106
1.0 ± 0.4 × 106
6.5 ± 0.3 × 106
0
1.72 ± 0.2 × 10
2.0 ± 0.24 × 10
1
15.4 ± 1.2 × 108
2
3.5.14
9
9
Pear
The Korean pear (Pyrus pyrifolia Nakai) is one of the most abundantly produced fruits in South Korea and is mainly
composed of 85%–88% water, 10%–13% carbohydrates, 0.3% proteins, and 0.2% lipids (Kim et al., 2010a,b). It is mostly
consumed as a fresh fruit. Kim et al. (2010a,b) have studied the suitability and potential effects of Korean pear puree on
probiotic microorganism Leuconostoc mesenteroides. The pH and titratable acidity of the pear puree were 4.06% and
0.66%, respectively, after 12 h of fermentation. The viable cell count of Lactobacillus mesenteroides 51-3 rapidly increased
to 3.7 × 109 CFU/g after 12 h of cultivation. The content of lactic acid and acetic acid was determined to be 0.138% and
0.162%, respectively, after 12 h of fermentation. When the fermented pear puree was stored at 4°C, the pH, titratable acidity
and viable cell count (109 CFU/g) remained fairly constant for 14 days.
3.5.15
Pineapple
Probiotic pineapple juice beverage was produced from sonicated pineapple juice by Lactobacillus casei NRRL B442 fermentation. Maximum microbial viability was found at 31°C and pH 5.8 (optimized conditions). After 42 days of storage
under refrigeration (4°C), the microbial viability was 6 × 106 CFU/mL in the nonsweetened sample and 4.7 × 106 CFU/mL
in the sweetened sample. The pH of both samples during storage was decreased due to postacidification. Browning of juice
was not observed and color of the juice was maintained throughout the storage period. From this it can be concluded that the
sonicated pineapple juice is suitable for Lactobacillus casei cultivation and as well as good substrate for the development
of an alternative nondairy probiotic beverage.
The survival of probiotic lactic acid bacteria, belonging to Lactobacillus plantarum and Lactobacillus fermentum
species, was monitored on artificially inoculated pineapple pieces throughout storage. The main nutritional, physicochemical, and sensorial parameters of minimally processed pineapples were monitored. Finally, probiotic Lactobacillus
were further investigated for their antagonistic effect against Listeria monocytogenes and Escherichia coli O157:H7 on
pineapple plugs. These results suggested that at 8 days of storage, the concentration of Lactobacillus plantarum and
Lactobacillus fermentum on pineapples pieces ranged between 7.3 and 6.3 log CFU/g, respectively, without affecting
the final quality of the fresh-cut pineapple. The antagonistic assays indicated that Lactobacillus plantarum was able to
inhibit the growth of both pathogens, while Lactobacillus fermentum was effective only against Lactobacillus monocytogenes. This study suggests that both Lactobacillus plantarum and Lactobacillus fermentum could be successfully
applied during processing of fresh-cut pineapples, contributing at the same time to inducing a protective effect against
relevant food borne pathogens.
An examination of the fermented functional drink production was done based on the mixture of pineapple, apple and
mango by Lactobacillus casei PTCC 1608. To produce a probiotic fermented drink based on the mixture of pineapple,
apple and mango, microbe suspension with initial concentration 106, 107 CFU/mL is provided and inoculated to the
concentrated mixture of juice (concentration of 20%, 30%, and 40%) and fermentation process is performed for 72 h at
a temperature of 37°C. During fermentation in all treatments, the population of probiotic bacteria was increased due to
using sugar and nutrients in juice, acidity is increased and sugar is reduced. Based on the results, F2T2 treatment with
concentration of 30% of juice (including 15% pineapple juice, 7.5% apple juice and 7.5% mango juice) and density
107 CFU/mL is the best treatment and with the highest bacteria measured after 28 days. The results of the study suggest
that the mixture of pineapple, apple and mango juice is a good medium for the growth of lactic acid bacteria and functional drink production.
68 SECTION | A Probiotics and Prebiotics
3.5.16
Pomegranate
Pomegranate (Punica granatum, Punicaceae) is a well known tropical fruit produced worldwide. The fresh juice contains
85.4% water and considerable amounts of total soluble solids (TSS), total sugars, reducing sugars, along with bioactive
secondary metabolites (anthocyanins, ellagic acid derivatives, and hydrolyzable tannins) and nondigestible carbohydrates
(prebiotics). These antioxidants are more potent, on a molar basis, than many other antioxidants including vitamin C,
vitamin E, coenzyme Q-10 and alpha-lipoic acid (Aviram et al., 2002). The antioxidant level of pomegranate juice was
found to be higher than green tea and red wine (Gil et al., 2000). It has also been reported that pomegranate contains
considerable health-promoting properties with antimicrobial, antiviral, anticancer, antioxidant and antimutagenic effects
(Negi et al., 2003).
Attempts were made to produce a nondairy probiotic drink based on pomegranate juice which already possesses
many inherent health benefits. Probiotic pomegranate juice was produced through its fermentation by four LAB
strains (Lactobacillus plantarum, Lactobacillus delbruekii, Lactobacillus paracasei, Lactobacillus acidophilus)
(Mousavi et al., 2011). Fermentation was carried out at 30°C for 72 h under microaerophilic conditions. Lactobacillus
plantarum and Lactobacillus delbruekii showed better microbial growth and sharp pH increment at the initial stages
of fermentation in comparison with other strains. The most significant acid present in pomegranate juice is citric
acid which was consumed considerably by all probiotic lactic acid bacteria. Higher viability during the storage time
was observed in Lactobacillus plantarum and Lactobacillus delbruekii. There was no loss in viability of cells up to
2 weeks, but it decreased dramatically after 4 weeks. The characteristic low pH value of pomegranate juice could
be adjusted by mixing with other fruits or by protecting probiotics from acidic environment by microencapsulation
(Mousavi et al., 2011). It can be concluded that pomegranate juice was proven to be a suitable media for the production of probiotic drink.
3.5.17
Sapota
Sapota (Achras sapota L. or Manilkara zapota L.) is one of the major fruit crops in India, Mexico and Venezuela.
Sapota fruit contains significant fermentable sugars as well as protein, phenolics carotenoids, ascorbic acid, minerals
(potassium, copper and iron), and vitamins (A, C, folate, and pantothenic acid). Because of these properties, sapota
is considered a healthy fruit to alleviate micronutrient malnutrition. Recently, it was also reported that a methanolic
extract of sapota fruit inhibits tumor growth (Srivastava et al., 2014). There are about 40 varieties of sapota grown
in different parts and “Kalipatti” is the leading cultivar in the India. Vijay Kumar et al. (2015) developed a probiotic
sapota juice and studied the growth kinetics of LAB, antioxidant, antimicrobial and sensory quality. From this investigation, it can be concluded that sapota juice is also suitable for the production probiotic beverages with a high cell
count (8 × 108 CFU/mL). Total phenolic content (TPC), antioxidant and antimicrobial activity of probiotic sapota juice
was investigated. TPC was increased significantly in 72 h fermented probioticated juice and DPPH radical scavenging
activity was significantly higher in probiotic sapota fruit juice than in nonprobiotic fruit juice (Fig. 5). No significant
difference between the sensory scores of probioticated and nonprobiotic products was noticed and the influence of
fermentation on juice texture, taste, flavor and overall acceptance was not significant. From these results it can be concluded that probiotic sapota fruit juice can be use to deliver probiotic LAB to lactose-intolerant people and those who
are allergic to milk-based products.
3.5.18
Tomato
Yoon et al. (2004) have studied the suitability of the tomato juice as a substrate for the production of probiotic juice by four
different LAB (Lactobacillus acidophilus LA39, Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus
delbrueckii subsp. bulgaricus D7). After 72 h of fermentation the biomass concentration maximally reached to 9 × 109 in
Lactobacillus acidophilus LA39, pH was reduced to 4.1 and acidity was increased to 0.65%. Lactobacillus plantarum consumed the sugar at a much faster rate than the other three LAB. The viability remains in a range of 6–8 log CFU/mL. During
the storage Lactobacillus delbrueckii showed the highest viability compared to other three. The lactic acid cultures significantly fermented tomato juice and reduced the level of sugar. The effect of potential probiotics and autochthonous LAB on
health-promoting and sensory properties of tomato juice was studied by Di Cagno et al. (2013). Compared to unfermented
tomato juice or tomato juice fermented with allochthonous strains, the values of viscosity, color, total antioxidant activity,
concentration of ascorbic acid, glutathione, and total free amino acid increased when the tomato juice was fermented with
autochthonous starters. From these results, it can be concluded that the fermented tomato juice could be utilized as a substrate for probiotification, and the product could serve as a health beverage for consumers like vegetarians and individuals
allergic to dairy products.
Development of New Probiotic Foods—A Case Study on Probiotic Juices Chapter | 4 69
Mango juice
Sapota juice
Probioticated mango juice
Probioticated sapota juice
120
Relative phenolic loss (%)
100
80
60
40
20
0
Appearance
Colour
Flavour
Taste
Overall acceptance
Parameter
FIG. 5 Sensory-evaluation of probioticated and nonprobioticated mango and sapota fruit juice. (Reproduced with permission from Vijay Kumar, B.,
Vijayendra, S.V.N., Reddy, O.V.S., 2015. Trends in dairy and non-dairy probiotic products—a review. J. Food Sci. Technol. 52, 6112–6124.)
3.6 Yeast Probiotic Juices
The probiotic characteristics of the Saccharomyces cerevisiae var. boulardii (S. boulardii) were confirmed by double-blind
studies (Czerucka et al., 2007). In 1920 Henri Boulard isolated it from lychee fruit. S. boulardii was used as a preventative and
therapeutic agent for the curing of diarrhea (McFarland, 2009; Rajkowska et al., 2012). The volume of studies reporting significant numbers of yeast in traditional fermented beverages indicates their importance in these fermentations. Yeasts in dairy
generate desirable aromatic compounds, proteolytic and lipolytic activities and can aid bacterial growth by producing amino
acids, vitamins and other metabolites, and contribute to the final composition of the product by producing ethanol and carbon
dioxide. In particular, studies have demonstrated that yeast can exert a positive effect on the abundance of Lactobacillus in
fermented environments (Gadaga et al., 2001), and this might be a key function in such symbioses, as well as preventing the
growth of undesirable species. In addition the previously mentioned benefits, it also helps protect health like other probiotic
bacteria strains through the production of enzymes like phytase (useful in chelate degradation) and short chain fatty acids, degradation of pathogenic toxins and improves the immune system (Czerucka et al., 2007; McFarland, 2009). It can increase the
availability of nutrients in fermented food systems via change of food components and biofortification of folate (Rajkowska
et al., 2012). These positive effects are dependent on the amount of S. boulardii in the diet. However, above 103 CFU/g causes
an undesirable taste and texture due to alcoholic fermentation. Therefore, S. boulardii is still under research as a food additive and starter culture (Joshi and Thorat, 2011). While yeast only comprise <0.1% of the gut microbiota, they are 10 times
larger than prokaryotes and can thus impede colonization of pathogenic bacteria (Czerucka et al., 2007). Some species of
Saccharomyces and Candida yeasts are common to both fermented beverages and the gut microbiota. As such, these species
could be investigated with a view to their contribution to fermentations and optimizing health-promoting potential. However,
to date Saccharomyces boulardii is the only recognized probiotic yeast. Success has been made in incorporating them in commercial fermented milk products, but excessive gas production during storage can be an issue.
A new functional beverage by the fermentation of probiotic yeast Saccharomyces cerevisiae boulardii was formulated
using tomato juice (Fratianni et al., 2013). The different culturing media did not negatively affect the yeast resistance to
the in vitro passage through acidic and pancreatic environments. Several biochemical parameters of the fermented juice,
its polyphenol and lycopene content, as well as its DPPH free radical scavenging capability, were evaluated during storage.
Fermentation of the juice with S. boulardii caused only a slight decrease of total polyphenols (0.442 mol/L vs. 0.474 mol/L,
respectively) and lycopene (13.38 g/mL vs. 16.76 g/mL, respectively), compared to raw juice. On the other hand, after passage through in vitro double gastric + pancreatic conditions, the amount of such biocomponents was constant during storage
until 28 days, and only decreased after 56 days. The amount of fermented juice required to inhibit the DPPH activity of 50%
increased three times. The developed product might be an alternative for delivering probiotics, especially for individuals
70 SECTION | A Probiotics and Prebiotics
suffering from lactose intolerance to dairy products. Fermentation of pomegranate juice as single or mixed substrate with
orange juice, without addition of extra nutrients, using kefir grains (mixture of yeast and LAB) was studied. During storage
at 4°C for 4 weeks, sugar consumption and ethanol production were monitored as was the survival of lactic acid bacteria.
The results showed that addition of orange juice improved the ability of kefir grains to ferment pomegranate juice and
increased the survival rates of LAB contained in kefir grains during storage. It is worth noting that 75% of cells survived
(6.48 log CFU/mL) after 4 weeks of storage in the fermented substrate (24% pomegranate juice). Lactic acid formation was
observed in all products, being especially high (1.3–1.9 g/L) in mixed substrates, indicating metabolic activity of microbes
during storage. Sensorial test results of the product showed consumer acceptance for all the fermented juices. These results
suggest that there is a possibility to produce low alcoholic nutrient fruit beverages with potential antioxidant (fruit constituents) and probiotic properties.
4.
4.1
CHALLENGES
Survivability and Stability
The health benefits of probiotics mainly depend on their quantity in foods and their survivability in the gastrointestinal
tract. The viability of probiotics is strain-dependent and different from one strain to another (do Espirito Santo et al., 2011;
Tripathi and Giri, 2014). In the final product the number of probiotics should be at least 106 or 107 CFU/mL at the end of
storage which corresponds to 109 CFU per portion (Nualkaekul and Charalampopoulos, 2011).
A good amount of research has been carried out and usable data is available on enhancing the storage stability of probiotics. The interpretation of this data is hindered by certain factors such as the probiotic process conditions prior to storage
and many studies lack kinetic data. Kinetic data, along with storage temperatures and aw, would allow better interlaboratory
comparison of results, and improve predictions of probiotic survival in different storage conditions. Following this train of
thought, it is not enough to merely secure viable cell counts that meet the minimal requirement at end of the storage period
(typically 106–107 cells/g) but also to limit the magnitude of viability loss during shelf life. This could potentially avoid the
overdosing of probiotic cells in the product at initial stage and keep the cost of production at an economically reasonable
level (Makinen et al., 2012).
In addition to the essential nutrients (minerals, vitamins, dietary fibers, antioxidants), juices contain several strong components that could limit probiotic survival in juices (Perricone et al., 2015). According the Tripathi and Giri (2014), these
can divide into three groups:
1. Food parameters: pH, titratable acidity, molecular oxygen, water activity, presence of salt, sugar and chemicals, like
hydrogen peroxide, bacteriocins, artificial flavoring, and coloring agents;
2. Processing parameters: heat treatment, incubation temperature, cooling rate, packaging materials and storage methods,
oxygen levels, volume;
3. Microbiological parameters: strains of probiotics, rate and proportion of inoculation.
pH is one of the very important factors affecting the viability of probiotics. Juices contain a significant amount of organic
acids resulting in the low pH. Consequently the juices could have the combined effect of acidic conditions and the intrinsic
antimicrobial effect of acids. A few major probiotics (Lactobacilli and bifidobacteria, Lactobacilli) are resistant from pH 3.7
to 4.3 and can survive in fruit juices. Bifidobacteria are less acid tolerant, and a pH of approximately 4.6 is detrimental for
their survival (Tripathi and Giri, 2014; Reddy et al., 2015). In contrast, the above probiotics showing good viability in low
pH of fruit juices in these cases pH cannot explain the trends experienced by some probiotics. Nualkaekul et al. (2011) investigated the factors that affected the survival of Bifidobacterium longum in model solutions and in fruit juices (orange, grapefruit, blackcurrant, pineapple, pomegranate, and strawberry). They reported that after storage at 4°C for 6 weeks in orange,
grapefruit, blackcurrant, and pineapple juices, bifidobacteria decreased not <0.8 log CFU/mL and orange and pineapple
juice supported the highest cell count. Furthermore, they found some controversial data on the effects of pH, as the decrease
in grapefruit was only 0.5 log CFU/mL, despite the low pH (3.21) and the high concentration of citric acid (15.3 g/L). On
the other hand, the probiotic viability was below the detection limit in pomegranate after 1 and 4 weeks in strawberry juice.
These results suggest that survival was the outcome of synergistic and antagonistic action of some factors in that phenolic
compounds could play a significant role. Generally, pH exerts a detrimental effect, but protein and dietary fiber could protect cells from acidic stress; the role of citric and malic acids is controversial, as they seemed to protect probiotics, whereas
phenols could cause a strong viability loss (Tripathi and Giri, 2014). Although the pH is a drawback for probiotic survival in
juices, Ranadheera et al. (2014) observed that the incorporation of lactic acid bacteria into fruit juices with low pH enhanced
the resistance of bacteria to subsequent stressful acidic conditions, such as those found in gastrointestinal tract.
Development of New Probiotic Foods—A Case Study on Probiotic Juices Chapter | 4 71
4.2
Sensory Traits
Another critical challenge for probiotification of fruit juices is consumer acceptance (Luckow and Delahunty, 2004a,b; de
Souza Neves Ellendersen et al., 2012). It has been reported that probiotification of fruit juice can result in flavors described
as “dairy,” “medicinal,” “acidic,” “salty,” “bitter,” “astringent,” “artificial,” or “earthy” (Granato et al., 2010a,b; Luckow
and Delahunty, 2004a,b; Saeed et al., 2013). However, it is unclear whether all probiotic cultures give the product the same
flavor at the same levels of intensity (Luckow et al., 2006). The probiotic effects on the sensory characteristics of juices
depend on the type of fruit, probiotic organism, and the storage temperature and supplementation of prebiotics and protectants. Some researchers have shown that probiotics did not affect the overall acceptance of certain fruit juices. For example
Perricone et al. (2015) demonstrated no adverse change in flavor for pineapple juice containing Lactobacillus reuteri; and
Tapia et al. (2007) for a fresh apple beverage fermented by Lactobacillus casei, and de Souza Neves Ellendersen et al.
(2012) using apple juice.
A possible solution for unpleasant flavor outcomes in probiotic juices is masking, in other words, the addition of pleasant aroma and volatile compounds able to “mask” the presence of probiotics. Luckow et al. (2006) reported that the addition of tropical fruit juices such as pineapple, mango or passion fruit (10%, v/v) might positively contribute to the aroma
and flavor of the final product. Finally, Ranadheera et al. (2014) confirmed that some fruit juices could naturally mask the
“medicinal” taste of probiotics. However, the addition of probiotic cultures to fruit juices presents numerous technological
challenges, due to their acidity, the presence of oxygen, and inherent differences among fruits (Saeed et al., 2013; Vasudha
and Mishra, 2013).
5.
POSSIBLE REMEDIES
To overcome initial challenges, many authors have proposed successful strategies to improve the survival of probiotics in
juices. In this section authors provide details of some compelling solutions.
5.1
Supplementation of Growth Promoters and Protectants
An easy way to improve probiotic stability in fruit juice could be the fortification of juice with some growth promoters and
protectants (oligosaccharides, cellulose and dietary fiber) or with some ingredients able to exert a protective effect. Saarela
et al. (2006) reported that apple juice fortified with glucans, for example oat flour (with 20% of β-glucan) could protect
Lactobacillus rhamnosus during refrigerated storage. Oligofructoses could increase the viability of probiotic cultures during processing and storage of the products because they are substrates available for the metabolism of these microorganisms
(Donkor et al., 2007) and, could therefore increase the stability of probiotics in fruit juices during storage. Furthermore, oligofructoses have a sweet taste similar to sucrose and may be used as sugar substitutes (Renuka et al., 2009; Yousaf et al., 2010).
Pimentel et al. (2015a) have produced probiotic apple juice with oligofructose fermented with Lactobacillus paracasei. After fermentation they have evaluated the physicochemical characteristics, probiotic viability and acceptability after
refrigerated storage (4°C for 28 days) in plastic or glass packages. Results suggested that the oligofructose addition did not
change any physicochemical characteristics and storage stability of the products. Rakin et al. (2007) fermented beetroot
and carrot juices with yeast autolysate before lactic acid fermentation with Lactobacillus acidophilus; enhanced growth of
Lactobacillus acidophilus, reduced fermentation time, enriched the juices with amino-acids, vitamins, minerals and antioxidants and a positive effect on the survival of probiotics was observed with the supplementation of autolysate.
Few studies have evaluated the effect of supplementation of nonfermented juices with probiotics (Champagne and
Gardner, 2008; Ding and Shah, 2008; Saarela et al., 2011; Sheehan et al., 2007; Sohail et al., 2012) or prebiotics (Renuka
et al., 2009; Yousaf et al., 2010; Martinez-Villaluenga et al., 2006). It has also been shown that supplementation of a model
fruit juice with green tea extract stabilizes probiotics for 6 weeks of refrigerated storage (Shah et al., 2010). Furthermore,
the addition of oat or barley fibers to fruit juice improved the survival of Bifidobacterium breve added as a frozen concentrate to the juice (Saarela et al., 2011).
5.2 Adaptation
Gobetti et al. (2010) reported that the exposure of probiotics to a sublethal stress conditions could induce resistance.
Perricone et al. (2014) have evaluated the viability of Lactobacillus reuteri DSM 20016 in pineapple, orange, green apple,
and red fruit juices and found that the probiotic experienced a strong viability loss in red-fruit juice, probably due to a
combined effect of low pH and phenols. Prolongation of the viability was achieved by two different strategies: (1) strain
72 SECTION | A Probiotics and Prebiotics
cultivation in a lab medium containing different amounts of red fruit juices (up to 50%); and (2) supplementation of the
medium with vanillic acid (phenol stress) or acidified to pH 5.0 (acid stress). These approaches resulted in Lactobacillus
reuteri by 5 (phenol stress) or 11 days (pH stress). Saarela et al. (2011) improved the survival of Bifidobacterium breve by
UV mutation and with cultivation of probiotic organism at a sub-lethal pH level.
5.3
5.3.1
Induction of Resistance
Mutagenesis
UV light or chemicals have been commonly used to obtain strains with altered characteristics or to study different microbial
processes. This technique has been used successfully in probiotics research for increasing the stability of Bifidobacterium
breve and Bifidobacterium animalis in low pH products (Saarela et al., 2011). This approach has also been used to improve the stability of the product in terms of sensorial attributes, for instance, metabolic activity of Bifidobacteria during
manufacture or storage of food is often not desirable, since the production of large amounts of acetic acid may result in
undesirable flavors. Novel Bifidobacteria strains producing low amounts of acetic acid have been obtained by UV mutagenesis; these strains would make possible the elaboration of stable and organoleptically acceptable products (Sánchez and
Margolles, 2012).
5.3.2
Selective Pressure
Selective pressure (stress factor) has also been employed to obtain resistant probiotic strains. Usually strains obtained using
this technique present stable phenotypes and cross-resistance to other stresses (acid and temperature). Both Lactobacilli
and Bifidobacteria were improved with increased heat, oxygen (Li et al., 2010), or acid (Collado and Sanz, 2006) tolerance (Berger et al., 2010) by selective pressure technique. Though the use of these stress-resistant strains can be useful for
improving stability in industrial processes, care should be taken as the stress adaptation may alter other properties of the
strain (Gueimonde et al., 2007). It was also reported that the use of stress-resistant strains in probiotification do not promote
significant changes in the behavior of starter cultures and the sensory properties of fermented milks (Sánchez et al., 2010).
Hence these adapted strains could be a potential option in developing stable probiotic products.
5.3.3
Genetic Modification of the Strains
Genetic modification is an alternative tool for improving stability and survivability of probiotic microorganisms. However,
this is not viable in all the countries, for example in Europe, GMOs are not well accepted by consumers. There are two different basic approaches that can be pursued:
(1) Homologous expression—modify the expression/production of genes already present on the microorganism.
(2) Heterologous expression—introduce genes from other microbial species.
Examples of both do exist, for first one—over expression of a chaperone in Lactobacillus paracasei was found to increase
the strain stability (Desmond et al., 2004), and heterologous expression of the betaine uptake system (BetL) from Listeria
into Lactobacillus salivarius was found to increase tolerance to acid and high osmolar conditions (Sheehan et al., 2006).
5.3.4
Encapsulation/Microencapsulation
A number of techniques have been developed and evaluated for the reduction of fatal effects of the gastrointestinal system
on probiotic microorganisms. Among these, encapsulation technique is one of the most promising. The technology of
encapsulation of probiotic living cells adopted from the immobilization technology is used for both enzymes and whole
cell culture in the biotechnology industry. It is a process by which bioactive materials are coated with other protective
materials, or their mixtures, and sealed contents can release at controlled rates under the influences of specific conditions.
Microencapsulation protects the bioactive component from environmental stresses such as oxygen, high acidity, and gastric
conditions and can be used for passing through the stomach with little damage (Huq et al., 2013). Protection of the microencapsulated bioactive component when passing through the stomach, could be increased using water insoluble wall materials. In recent years, many studies have been carried out on preservation of probiotic microorganisms by microencapsulation
during food processing and storage (Lapsiri et al., 2012). Proteins, polysaccharides, sugars and their combinations, or some
liquid food matrices can be used to encapsulate probiotics (Chavez and Ledeboer, 2007; Pispan et al., 2013).
Microencapsulation of probiotic organisms is of interest to the probiotic food industry as the best method to maintain
the potency of probiotic microorganisms being delivered to the gastrointestine (Siuta-Cruce and Goulet, 2001). When
considering encapsulation, we need to maintain two things: their size (typically between 1 and 5 μm diameter), which
Development of New Probiotic Foods—A Case Study on Probiotic Juices Chapter | 4 73
i­mmediately excludes nanotechnologies; and the fact that they must be kept alive. Main reasons for using this method for
protection of probiotics are as follows:
●
●
●
●
Improving viability and stability of probiotic cultures during production, storage and passage through the gastrointestinal tract (Kailasapathy, 2002; Krasaekoopt et al., 2003; Sultana et al., 2000)
Providing a controlled and efficient release of probiotic bacteria in GIT (Crittenden et al., 2006; Kailasapathy, 2002)
Easier handling of the cultures (Picot and Lacroix, 2003)
Limited effects on sensory properties of the product containing microcapsules (Picot and Lacroix, 2003)
Encapsulation technology is usually followed a three stages process. The first step consists of incorporating the bioactive component in a matrix which can be liquid or solid. In the case of the core being liquid, incorporation will be a dissolution or a dispersion in the matrix, whereas if the core is solid, the incorporation will be an agglomeration or an adsorption.
For the second step, the liquid matrix is dispersed while a solution is pulverized on the solid matrix. The last step consists
of stabilization by a chemical (polymerization), a physicochemical (gelification) or a physical (evaporation, solidification,
coalescence) process (Picot and Lacroix, 2003).
There are several microencapsulation methods for probiotics including spray drying, freeze drying, fluidized bed drying, extrusion, emulsion, coacervation, and phase separation (Kailasapathy, 2002). However, two widely used encapsulation techniques are extrusion and emulsion (Krasaekoopt et al., 2003). A variety of materials have been used for the
microencapsulation of probiotics including alginate, starch, alginate-starch, cellulose acetate phthalate, κ-carrageenan,
gelatin, xanthan-gellan, chitosan and whey protein (Doleyres and Lacroix, 2005; Krasaekoopt et al., 2003). It is also difficult to achieve viable probiotics at ambient temperatures without effecting the sensory attributes. One means to achieve
stability is through the separation of the bacteria from the fruit juice after the fermentation and then mix them at the point
of consumption. In the case of the Probiotic Straw by BioGaia, dried probiotic cells are immobilized in the inner wall of the
straw. This seems to be a good vehicle for delivering probiotics.
6.
FUTURE PERSPECTIVES
The utilization of nondairy foods, fruit juices, as probiotics carriers presents potential advantages and valuable alternatives
for the food industry. Because several probiotic bacteria observed impressive viability in nondairy foods, there is great
potential in using fruits, as substrate or as active ingredients (prebiotic), during manufacture of probiotic foods. In order to
improve the survival rates of probiotic microorganisms during food production, the process of microencapsulation shows
promise. There are some challenges to be resolved for the safe launch of fruit juice probiotics, in other words the survivability/stability and the effects on the sensory traits; however promising possible solutions exist. Some probiotic juices
are already available to consumers and many other products will be launched on the market in the near future. Ingredients
used in encapsulation materials are recognized as being safe and may be used in food applications. Therefore, there is of
widespread interest in the improvement of the physical and mechanical stability of the polymers use in probiotics encapsulation, to ensure high population of probiotics not only in food during storage but also after gastrointestinal digestion.
Existing probiotics generally belong to the genus Lactobacillus. However, few strains are commercially obtainable for
probiotic function. Gene technology and relative genomics will play a role in upcoming research and the development of
new strains, with gene sequencing allowing for an increase in appropriate mechanisms and the functionality of probiotics.
More research is warranted on the effect of storage on the functional properties of probiotics. It remains to be determined to
what extent the age of the stored cultures influences their health benefits. It is indisputable that fruit and vegetable matrices
will be an important research and development area for future probiotic/functional food markets. Comprehensive analysis
of the effects of these functional foods on human health is highly advantageous.
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Chapter 5
Prebiotics and Their Production
From Unconventional Raw Materials
(Mushrooms)
Hrudayanath Thatoi⁎, Sameer K. Singdevsachan⁎, Jayanta K. Patra†
*North Orissa University, Baripada, India, †Dongguk University, Goyang-si, Republic of Korea
1.
INTRODUCTION
Mushrooms are defined as heterotrophic macrofungi (Ascomycota and Basidiomycota) with characteristic fruiting bodies
that are either epigeous (of fruiting bodies above the ground) or hypogeous (of underground fruiting bodies) (Chang and
Miles, 1992). Mushrooms have been used as food and medicine since ancient times (Chang and Miles, 2004). Currently
both cultivated and wild edible mushrooms are used directly or indirectly as food or medicine (Chang and Miles, 2004).
Edible mushrooms are widely consumed in many countries as food for their appealing taste, aroma and nutritional values
due to high protein, fiber, vitamins, mineral contents and low/no calories and cholesterol (Thatoi and Singdevsachan, 2014).
Approximately 14,000 mushroom species have been identified worldwide, of which 2000 are safe for human consumption
and 650, in addition to being edible, possess medicinal properties (Chang and Miles, 2004). Beside their high nutritional
value, mushrooms are rich in many bioactive metabolites of high medicinal values such as phenolics and polyphenolic compounds, lectins, polysaccharides, terpenoids, sterols, and various volatile compounds (Ferreira et al., 2010; Singdevsachan
et al., 2016). Most of the medicinal mushroom research is in preliminary stages, being based on different tests with crude
extracts of the whole mushroom fruiting bodies or mycelia, or with partially purified bioactive compounds.
Today’s foods are not only projected to mollify hunger and provide essential nutrients, but also prevent diseases and
improve the health of consumers. Such foods are known as functional foods (Siro et al., 2008). According to the Institute of
Medicine’s Food and Nutrition Board, “Functional Foods” are foods or dietary components that provide a health benefit beyond basic nutrition. Prebiotics such as oligosaccharides and polysaccharide (inulin) have emerged to generate great interest
as functional food ingredients because they are able to manipulate the composition of colonic microbiota in the human gut
by inhibition of exogenous pathogens (Rycroft et al., 2001) and thus improve the health of the host (Roberfroid, 2000, 2002).
Recent developments in prebiotics have stressed the need to search for new potential sources of prebiotics. In this context,
mushrooms could serve as a potential source for prebiotics as they contain carbohydrates like chitin, hemicellulose, ß and aglucans, mannans, xylans and galactans. Chitin, a water insoluble polysaccharide, is indigestible to humans and plays a role
as a dietary fiber (Kalac, 2009). Several mushroom polysaccharides are credited with prebiotic effects. Crude polysaccharides
from Ganoderma lucidum, Lentinus edodes, Pleurotus eryngii, and Flammulina velutipes were found to have significant prebiotic activities (Yamin et al., 2012; Chou et al., 2013). However the bioactivities of water insoluble polysaccharides was less
as compared to water soluble polysaccharides (Tao et al., 2006). Most of the polysaccharides from mushrooms are present as
linear and branched glucans with different types of glycosidic linkages such as (1→3), (1→6)-ß-glucans and (1→3)-a-glucans.
However, some of them are true heteroglycans containing arabinose, mannose, fructose, galactose, xylose, glucose, and glucuronic acids as main side chain components or in different combinations. Even though mushrooms polysaccharides are of
different chemical compositions, most of them belong to the group of ß-glucans (Wasser, 2002). Digestive enzymes secreted
by the pancreas of brush border of vertebrates, and of mammals in particular, are unable to hydrolyze ß-glucosidic bonds. This
makes them resistant to acid hydrolysis in the stomach and they remain nondigestible by human digestive enzymes (Van Loo,
2006). The nondigestible property of mushroom carbohydrate enables it to be considered as a potential source of prebiotic
because it meets a part of the definition of prebiotics. However, extensive studies need to be carried out before such a claim
could be made due to the fact that all dietary carbohydrates are not necessarily prebiotics (Gibson et al., 2004).
Therapeutic, Probiotic, and Unconventional Foods. https://doi.org/10.1016/B978-0-12-814625-5.00005-4
© 2018 Elsevier Inc. All rights reserved.
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80 SECTION | A Probiotics and Prebiotics
Mushrooms are not only important as prebiotics, but also for other biological properties such as antibacterial, anticancer, antioxidant, antiviral, and antihypoglycemic immunomodulatory activities, as well as being an active medicine in the
prevention of cardiovascular diseases (Lemieszek and Rzeski, 2012; Thatoi and Singdevsachan, 2014). Polysaccharides
of Lentinus edodes and Pleurotus florida also exhibited antitumor and immunomodulating properties (Yu et al., 2010;
Maity et al., 2011). Further, Lentinus edodes is the most studied species with potential antimicrobial activities (Alves et al.,
2012a). Additionally, a number of mushroom species, namely Coprinus plicatilis, Lentinus tigrinus, Ganoderma applaunatum, Helvella crispa, Agaricus bisporus, Hypsizygus ulmarius, Calocybe indica, and Flammulina velutipes, have been
reported for their antioxidant activity (Babu and Rao, 2013; Thatoi and Singdevsachan, 2014). In a current study, information related to bioactive substances and nutraceutical values of mushrooms for their food and pharmaceutical applications
have been discussed.
2.
NUTRITIONAL VALUES OF MUSHROOMS
Mushrooms serve as a food with rich nutritional value. While some mushrooms have medicinal value and are used as dietary supplements, some have both properties (Wani et al., 2010; Kalac, 2013). They are quite rich in protein and dietary
carbohydrates, with essential amino acids and fiber, poor fat but excellent important fatty acids content (Table 1). In addition, edible mushrooms provide a rich source of vitamins (Mattila et al., 2001; Heleno et al., 2010). Thus, they could be an
excellent source for many different nutraceuticals and could be used directly in human diet for promoting health (Vaz et al.,
2010; Pereira et al., 2012). During recent years, consumers have been about health and nutritional foods. This has ignited
the commercialization of natural foods consumed as dietary supplements. Mushrooms can be considered a functional food
as it is a major source of dietary carbohydrates and proteins (Kalac, 2013; Valverde et al., 2015). Functional foods should
not be claimed as cures for disease however, there is an increasing number of scientific studies that strongly support functional foods, such as mushrooms, as having a role in disease prevention and in some cases, of bringing about suppression
or remission of disease in an individual (Chen et al., 2012a,b; Valverde et al., 2015).
Since ancient times, mushrooms have been treated as a distinctive kind of food. The Greeks believed that mushrooms
provided strength for warriors in battle. Pharaohs regarded mushrooms as a delicacy and the Romans considered mushrooms as a “Food of the Gods” and served them only on special occasions. Chinese treated mushrooms as a healthy food,
the “elixir of life.” Indigenous Mexicans used mushrooms as hallucinogens in religious ceremonies and in shamanism, as
well as for therapeutic purposes (Chang and Miles, 2004). In recent years, fast-growing mushrooms have received a remarkable amount of interest with the realization that they possess high nutritional values with medicinal properties.
The cultivation procedures may influence the chemical configuration and the nutritive potential of various edible mushrooms. Additionally, significant variations occur both among and within species (Reis et al., 2012; Kalac, 2013). Recently
several authors reviewed the nutritional composition of mushrooms and reported that they contain a high moisture percentage that ranges between 80 and 95 g/100 g, approximately (Kalac, 2009, 2013; Thatoi and Singdevsachan, 2014; Valverde
et al., 2015). Furthermore, edible mushrooms are a good source of protein, 200–250 g/kg of dry matter; in which leucine,
valine, glutamine, glutamic, and aspartic acids are the most abundant amino acids. Mushrooms are low-calorie foods since
they provide low amounts of fat. Edible mushrooms contain high amounts of ash, mainly calcium, copper, iron, magnesium, potassium, phosphorus, and zinc. Carbohydrates are found in high proportions in edible mushrooms, 200–800 g/kg
of dry matter, including chitin, glycogen, trehalose, and mannitol; as well as containing fiber, ß-glucans, hemicelluloses;
and pectic substances (Kalac, 2009, 2013; Thatoi and Singdevsachan, 2014; Valverde et al., 2015). Additionally, glucose,
mannitol, and trehalose are abundant sugars in cultivated edible mushrooms, while fructose and sucrose levels are low.
Mushrooms are also a good source of vitamins with high levels of riboflavin, niacin, folates, and traces of vitamin C,
B1, B12, D, and E (Kalac, 2009, 2013; Thatoi and Singdevsachan, 2014; Valverde et al., 2015). Mushrooms are the only
nonanimal food source that contains vitamin D; consequently they are the only natural source of vitamin D available to
vegetarians. Specifically wild mushrooms are an excellent vitamin D2 source because, unlike their cultivated counterparts
which are usually grown in darkness, they receive the UV-B light needed to produce vitamin D2 (Kalac, 2009, 2013; Thatoi
and Singdevsachan, 2014; Valverde et al., 2015).
3.
BIOACTIVE COMPONENTS OF MUSHROOMS
Mushrooms in the 20th century are well known to people all over the world as an important biosource of novel secondary metabolites. Mushrooms contain biologically active compounds (Wasser, 2002). The secondary metabolites of
mushrooms are chemically diverse, with wide variety of biological activities. While applications of these compounds
are already explored in traditional medicines, they are in new targets of modern medicine. In particular, mushrooms
TABLE 1 Nutritional Values of Mushroom Shown in Percentage
Moisture
Protein
Carbohydrate
Lipids/Fats
Ash
Fiber
References
Agaricus bisporus
–
41.06
28.38
2.12
7.01
18.23
Pushpa and Purushothoma (2010)
Agaricus bisporus
–
33.48
46.17
3.10
5.70
20.90
Manikandan (2011)
Agaricus campestris
88.17
18.57
58.16
0.11
23.16
–
Pereira et al. (2012)
Agaricus comtulus
87.94
21.29
50.11
0.46
28.14
–
Pereira et al. (2012)
Agaricus lutosus
87.04
23.24
49.71
1.10
25.96
–
Pereira et al. (2012)
Amanita umbrinolutea
73.60
16.78
47.59
6.77
28.86
–
Pereira et al. (2012)
Armillaria mellea
88.27
16.38
71.28
5.56
6.78
Auricularia auricula
–
4.20
82.80
8.30
4.70
Boletus aereus
–
17.86
72.83
0.44
8.87
Boletus armeniacus
71.50
18.25
68.10
1.56
12.09
Boletus edulis
–
21.07
70.95
2.45
5.53
Heleno et al. (2011)
Boletus erythropus
–
20.92
52.43
0.75
25.9
Grangeia et al. (2011)
Boletus impolitus
88.90
16.01
56.63
2.94
24.43
Boletus reticulatus
–
22.57
55.16
2.55
19.72
Bovista aestivalis
73.23
15.59
52.37
0.18
31.86
–
Pereira et al. (2012)
Bovista nigrescens
76.41
20.94
72.18
3.64
3.24
–
Pereira et al. (2012)
Calocybe gambosa
90.92
15.46
69.82
0.83
13.89
Calocybe indica
–
17.69
64.26
4.10
7.43
3.40
Manikandan (2011)
Calocybe indica
–
21.60
49.20
4.96
12.80
13.20
Pushpa and Purushothoma (2010)
Calvatia utriformis
–
20.37
59.92
1.9
17.81
Chlorophyllum rhacodes
88.28
19.32
65.29
3.29
12.10
–
Pereira et al. (2012)
Clavariadelphus pistillaris
84.22
16.27
62.37
0.59
20.77
–
Pereira et al. (2012)
Clavariadelphus truncatus
90.97
15.98
69.62
1.54
12.86
–
Pereira et al. (2012)
Clitocybe costata
76.92
17.27
70.36
1.50
10.87
–
Pereira et al. (2012)
Clitocybe gibba
72.66
14.59
60.45
4.29
20.68
–
Pereira et al. (2012)
Clitocybe odora
88.49
17.33
70.66
2.46
9.55
Vaz et al. (2011)
19.80
Manikandan (2011)
Heleno et al. (2011)
–
–
Pereira et al. (2012)
Pereira et al. (2012)
Heleno et al. (2011)
Vaz et al. (2011)
Grangeia et al. (2011)
Vaz et al. (2011)
Continued
Prebiotics and Their Production From Unconventional Raw Materials (Mushrooms) Chapter | 5 81
Species
Species
Moisture
Protein
Carbohydrate
Lipids/Fats
Ash
Fiber
References
Coprinus comatus
85.19
15.67
70.35
1.13
12.85
Cortinarius praestans
89.16
14.56
63.98
2.58
18.89
Fistulina hepatica
–
50.09
31.62
1.89
16.4
Flammulina velutipes
–
17.60
73.10
1.90
7.40
3.70
Manikandan (2011)
Flammulina velutipes
90.68
17.89
70.85
1.84
9.42
–
Pereira et al. (2012)
Hygrophorus chrysodon
92.09
15.11
54.51
3.48
26.91
–
Pereira et al. (2012)
Laccaria laccata
–
62.78
12.77
3.76
20.69
Heleno et al. (2009)
Laccaria salmonicolor
–
37.28
37.41
2.03
23.28
Heleno et al. (2009)
Lentinus edodes
–
32.93
47.60
3.73
5.20
28.80
Manikandan (2011)
Lentinus sajor-caju
80.29
28.36
68.24
2.42
4.88
–
Singdevsachan et al. (2013)
Lentinus torulosus
80.97
27.31
64.95
1.36
13.16
–
Singdevsachan et al. (2013)
Leucoagaricus leucothites
85.29
20.51
51.93
1.10
26.46
–
Pereira et al. (2012)
Lycoperdon echinatum
–
23.52
65.83
1.22
9.43
Lycoperdon umbrinum
71.98
14.53
51.96
0.37
33.14
–
Pereira et al. (2012)
Lyophyllum decastes
–
18.31
34.36
2.14
14.20
29.02
Pushpa and Purushothoma (2010)
Pleurotus florida
–
27.83
32.08
1.54
9.41
23.18
Pushpa and Purushothoma (2010)
Pleurotus ostreatus
–
30.40
57.60
2.20
9.80
8.70
Manikandan (2011)
Pleurotus sajor-caju
–
19.23
63.40
2.70
6.32
48.60
Manikandan (2011)
Ramaria aurea
88.52
14.60
77.47
2.26
5.68
–
Pereira et al. (2012)
Russula cyanoxantha
–
16.8
74.65
1.52
7.03
Grangeia et al. (2011)
Russula delica
–
50.59
25.57
0.91
22.93
Heleno et al. (2009)
Russula delica
–
26.25
34.88
5.38
17.92
Russula olivacea
–
16.84
43.39
1.99
37.78
Grangeia et al. (2011)
Suillus mediterraneensis
–
24.32
45.43
2.61
27.64
Heleno et al. (2009)
Suillus variegates
90.77
17.57
63.76
3.31
15.36
Tricholoma imbricatum
–
50.45
41.22
1.88
6.45
Volvariella volvacea
–
37.50
54.80
2.60
1.10
Vaz et al. (2011)
–
Pereira et al. (2012)
Heleno et al. (2009)
Grangeia et al. (2011)
15.42
–
Pushpa and Purushothoma (2010)
Pereira et al. (2012)
Heleno et al. (2009)
5.50
Manikandan (2011)
82 SECTION | A Probiotics and Prebiotics
TABLE 1 Nutritional Values of Mushroom Shown in Percentage—cont’d
Prebiotics and Their Production From Unconventional Raw Materials (Mushrooms) Chapter | 5 83
which could play important roles in modern medicine represent an unlimited source of compounds with different
medicinal properties, including low-molecular-weight (LMW, e.g., amines, cerebrosides, catechols, isoflavones, organic germanium, quinones, triacylglycerols, sesquiterpenes, steroids, and selenium) and high-molecular-weight compounds (HMW, e.g., glycoproteins, glycopeptides, homo and heteropolysaccharides, proteins, RNA-protein complexes)
(Figs. 1 and 2) (Ferreira et al., 2010).
3.1
Low Molecular Weight Compounds of Mushrooms
Mushrooms contain a diversity of complex organic compounds such as phenolic compounds, polyketides, triterpenoids
and steroids derived from secondary metabolism (Wasser, 2002; Zaidman et al., 2005). Such compounds have been
used in the treatment of many health problems, including free radical related diseases, microbial infections and cancer,
to name a few. (Paterson, 2006; Vaz et al., 2010). The secondary metabolites with LMW present in mushrooms include
quinones, cerebrosides, isoflavones, catechols, amines, triacylglycerols, sesquiterpenes, steroids, organic germanium,
and selenium (Ferreira et al., 2010). The naturally occurring 6-(3,4-dihydroxystyryl)-4-hydroxy-2-pyrone (hispidin)
was isolated from the culture broth of Phellinus linteus, Gymnopilus marginatus, G. patriae, G. parvisporus and
Inonotus hispidus. Hispidin is a potent inhibitor of PKCß, a protein kinase which plays an important role in angiogenesis (Zaidman et al., 2005). Hispidin synthesized by Gonindard et al. (1997) possesses cytotoxic activity against human keratinocytes (SLC-1 tumor cell line) and human pancreatic duct cells (Capan-1 tumor cell line). Sterols isolated
from Cordyceps sinensis were found to have the ability to inhibit the proliferation of K562, Jurkat, WM-1341, HL-60,
and RPMI-8226 tumor cell lines (Bok et al., 1999). Ganomycin A and B isolated from Ganoderma pfeifferi, showed
antibacterial activity against B. subtilis, M. flavus and S. aureus (Mothana et al., 2000). Agrocybe aegerita, an edible
mushroom, is an important source of bioactive secondary metabolites such as indole derivatives with free radical scavenging activity, and cylindan with anticancer activity.
FIG. 1 Bioactive components of mushrooms.
84 SECTION | A Probiotics and Prebiotics
OH
Ganomycin A
Ganomycin B
Lentinan
H
O
H3C
O
HO
O
H3C
H
OH
O
OH
O
O
O
H
H
HO
HO
O
O
O
H
O
H
OH
O
H
O
OH
O
H
H
OH
O
O
O
H
OH
O
H
O
H
OH
O
H
O
H
HO
Krestin
H
O
O
O
O
HO
Schizophyllan
O
OH
HO
O
O
H
O
O
HO
H
O
OH
O
H
HO
O
O
O
O
HO
H
O
O
H
O
HO
H
HO
O
H
O
O
O
O
H
HO
OH
O
H
O
O
H
O
O
H
H
O
H
O
H
OH
H
HO
OH
OH
O
O
O
O
H
H
O
O
O
O
HO
O
O
O
OH
HO
OH
OH
OH
HO
HO
OH
O
HO
Hispidin
O
H
N
O
HO
Sterol
N
Agrocybenine
FIG. 2 Selected bioactive compounds with medicinal importance isolated in different mushroom species.
3.2
High Molecular Weight Compounds of Mushrooms
High-molecular weight compounds, including polysaccharides and polysaccharide conjugates, have been isolated from
medicinal mushrooms and developed as cytostatic polysaccharide drugs in Japan and approved in other countries for the
clinical treatment of cancer patients. These are “Lentinan” from the fruiting bodies of Lentinus edodes, “Schizophyllan”
(Sonifilan, SPG) from the culture fluid of Schizophyllum commune, and “Krestin” (PSK), from the cultured mycelium of
Trametes versicolor. Lentinan and schizophyllan are pure ß-glucans, whereas Krestin (PSK) is a protein bound polysaccharide. These three compounds possess immunomodulating properties (Mizuno, 1993; Larone, 2002, 2002; Zaidman et al.,
2005; Zhang et al., 2007).
Prebiotics and Their Production From Unconventional Raw Materials (Mushrooms) Chapter | 5 85
3.3
Mushrooms as a Possible Source of Prebiotics
A prebiotic is a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth of one
or a limited number of bacteria in the colon (Gibson and Roberfroid, 1995). The concept of prebiotics is laid out by certain
criteria such as resistance to gastric acidity, fermentation by intestinal microflora and selective stimulation of the growth,
hydrolysis by mammalian enzymes and gastrointestinal absorption, and/or activity of intestinal bacteria associated with
host well-being and health (Gibson et al., 2004) The critical criteria for a food ingredient as prebiotic is reviewed in Fig. 3.
The first attribute of prebiotics, which is nondigestible or resistant to the upper gut tract, indicates that the prebiotics can
withstand digestive processes before they reach the colon, thus effectively stimulating beneficial bacteria like bifidobacteria and lactobacilli (Gibson and Collins, 1999; Wang, 2009). Prebiotics are found in several vegetables and fruits, and are
considered as functional food components (Jovanovic-Malinovska et al., 2014). The consumption of prebiotics may have
many positive health benefits including but not limited to, improved colonic integrity, immune function, reduced allergic
response, improved digestion and elimination.
The criteria for the classification of a food ingredient as a prebiotic include selective fermentation by potentially beneficial bacteria in the colon (Gibson et al., 2004; Wang, 2009). The effects of this fermentation may lead to an increase in the
appearance or change in the arrangement of short-chain fatty acids, increased fecal weight, reduction in luminal colon pH,
decrease in nitrogenous end products and reductive enzymes, increased expression of the binding proteins or active carriers
associated with mineral absorption, and immune system modulation (Douglas and Sanders, 2008; Slizewska et al., 2012),
which is beneficial to the host health. Selective stimulation of the growth and/or activity of intestinal bacteria potentially
associated with health and well-being is considered as one of the typical criteria for prebiotic classification (Gibson et al.,
2004). Further, prebiotics are markedly suitable for the growth and activities of probiotics such as bifidobacteria and lactobacilli, while suppressing the growth of clostridia and bacteroides (Wang, 2009).
Gibson (2004) found dietary carbohydrates like fibers and oligosaccharides to be important prebiotics. Stowell (2007)
extensively reviewed the existing prebiotics. Inulin, fructooligosaccharides, galactooligosaccharides, lactulose and polydextrose are considered to be “established prebiotics,” whereas isomaltooligosaccharides, xylooligosaccahrides are grouped
under “emerging prebiotics.” Other than the aforementioned prebiotic compounds, mannitol, maltodextrin, raffinose, and
sorbitol are also prebiotics with proven health properties (Mandal et al., 2009; Vamanu and Vamanu, 2010).
Though soybeans (Crittendan and Playne, 1996), chicory root (Roberfroid, 2000), herbs (Guo et al., 2004), oats (Gokavi
et al., 2005), and cereal (Michida et al., 2006) are major source of prebiotics, edible mushrooms are gaining much attention as an alternative source (Guo et al., 2004; Aida et al., 2009). The major components rendering prebiotic function in mushrooms are nondigestible polysaccharides such as glucan, chitin, and hetropolysaccharides. Several mushroom
polysaccharides like pleuran, lentinan, schizophyllan, ß and a-glucans, mannans, xylans, galactans, chitin, inulin, and
hemicelluloses can be credited to promising prebiotic effects. Chitin are water insoluble polysaccharides that are present in some taxonomical groups of Zygo-, Asco-, Basidio-, and Deuteromycetes (Vetter, 2007). Fructans are members
of a larger group of inulins and oligosaccharides. These polysaccharides consist of fructose moieties, which are lined by
glycosidic bonds (Kelly, 2008). These polysaccharides help to increase the population of bifidobacteria and lactobacilli in
FIG. 3 Criteria for prebiotic compounds.
86 SECTION | A Probiotics and Prebiotics
the colon, thereby adding health benefits to the host (Roberfroid, 2007; Olmstead, 2008). d-Glucans are polysaccharides
linked by a or ß-glycosidic linkage (Andriya et al., 2009). These polysaccharides are known to activate the population of
Lactobacillus rhamnosus, Bifidobacterium bifidium, and Enterococcus (Ruthes et al., 2013; Giavasis, 2014). Grifloan are
polysaccharides having a ß-linked glucose molecule with triple helix structure. It has beneficial effects on Bifidobacterium
and Lactobacillus, and adverse effects on Salmonella. It helps in increasing glucose consumption and activity of lysosomal
enzyme, ß-d-glucourinodase, in macrophages (Andrea et al., 1999).
Most of the mushrooms can be used as prebiotics are Agaricus bisporus, Auriculariajudae, Boletus erythropus,
Calocybeindica, Flammulina velutipes, Ganoderma lucidium, Geastrum saccatum, Hericium erianaceus, Lentinusedodes,
and Pleurotus ostreatus. Pleuran from oyster (Pleurotus ostreatus) mushrooms and lentinan from shiitake (Lentinus edodes)
mushrooms are the most frequently used ß-glucans as prebiotics. Both of them are effective against intestine inflammation
(Zeman et al., 2001) and inhibit the development of intestinal ulcers in rats (Nosalova et al., 2001). Lentinan also shows
a positive effect on peristalsis in weaned piglets (Van Nevel et al., 2003). Additionally, it has been demonstrated that sclerotial ß-glucans from mushrooms (Pleurotus tuber-regium, Polyporous rhinocerus, and Wolfiporia cocos) can be utilized
by human colonic bacteria in vitro (Wong et al., 2005). The colonic fermentation of sclerotial ß-glucans isolated from P.
tuber-regium could also enhance the absorption of calcium and magnesium in ovariectomized rats (Wong et al., 2006).
Synytsya et al. (2009) gave a positive overview, citing that mushroom glucans of Pleurotus ostreatus and P. eryngii were
able to stimulate the growth of probiotics—Lactobacillus sp. (four strains: Lac A–D), Bifidobacterium sp. (three strains:
Bifi A–C), and Enterococcus faecium (two strains: Ent A and B)—to a certain degree.
Further, synergetic effects of mushroom polysaccharides have been demonstrated by several researchers for the growth
of probiotics. The research studies have indicated that polysaccharides from Pleurotus spp. (Synytsya et al., 2009), Lentinus
edodes, Tremella fuciformis (Guo et al., 2004), and Agaricus spp. mushrooms (Giannenas et al., 2011) have prebiotic
activity. The effect of three kinds of crude polysaccharides (PSI, PSII, and PSIII) from Agaricus blazei Murill (obtained
by diethyl-amino ethanol cellulose column chromatography) on the growth of lactic acid bacteria have been described
by Lili and Jianchun (2008). Additionally, mushrooms such as Pleurotus ostreatus and Lentinus edodes can significantly
modify intestinal flora composition by promoting the breakdown and propagation of beneficial microorganisms such as
Lactobacilli and Bifidobacteria, as well as by hindering the growth of pathogenic bacteria such as E. coli, Clostridium,
and Salmonella (Zhou et al., 2011). The effect of crude polysaccharides extracted from Ganoderma lucidum on the growth
of probiotics have been studied by Yamin et al. (2012) in batch-culture fermentation of human fecal culture. Growth promotion of Bifidobacterium sp. and Lactobacillus sp. and growth inhibition of Salmonella sp. proved its prebiotic effect.
Prebiotic activity of crude polysaccharides from Lentinula edodes stipe, Pleurotus eryngii base, and Flammulina velutipes
base were also studied (Chou et al., 2013). Recently, the prebiotic property of edible mushrooms (Auricularia auriculajudae, Pleurotus ostreatus, Pleurotus sajor-caju, Pleurotus abalonus, and Volvariella volvacea) was evaluated by Saman
et al. (2016) and found that three selected mushrooms (Pleurotus ostreatus, Pleurotus sajor-caju, and Pleurotus abalones)
stimulate the growths of bifidobacteria (Bifidobacterium bifidum TISTR 2129, B. breve TISTR 2130) and lactobacilli (B.
animalis TISTR 2195 and B. longum TISTR 2194); and could suppress the growth of harmful bacteria in human gut model.
4.
MUSHROOM AS POTENTIAL SOURCE OF PHARMACEUTICALS
4.1 Antitumor and Immunomodulatory Properties
The active components in mushrooms responsible for anticancer activity include lentinan, krestin, lectin, hispolon, calcaelin, illudin S, psilocybin, Hericium polysaccharide A and B (HPA and HPB), ganoderic acid, schizophyllan, laccase, etc.
(Patel and Goyal, 2012). Among the different components, polysaccharides from mushrooms are the best known and most
potent substances with antitumor and immunomodulating properties. A novel water-soluble polysaccharide (POPS-1) was
isolated from the fruiting bodies of the mushroom, Pleurotus ostreatus by hot-water extraction, ethanol precipitation, and
fractionation by DEAE-cellulose ion exchange and Sepharose CL-6B gel filtration chromatography technique by Tong
et al. (2009). Cytotoxicity assay showed POPS-1 presented significantly higher antitumor activity against the HeLa tumor
cell line in vitro, in a dose-dependent manner. However, it exhibited significantly lower cytotoxicity to human embryo kidney 293T cells than HeLa tumor cells compared with anticancer drug 5-fluorouracil. This finding suggests that the soluble
polysaccharide POPS-1 may be considered as a potential candidate for developing a novel low-toxicity antitumor agent
(Tong et al., 2009).
Methanol extract of Phellinus linteus and its fractions, including methylene chloride, ethyl acetate, and n-butanol, have
shown the potential for antiangiogenic effects through the inhibition of human umbilical vein endothelial cell (HUVECs)
proliferation, migration and assembly into capillary-like structures as well as in vivo angiogenesis. The findings of this
Prebiotics and Their Production From Unconventional Raw Materials (Mushrooms) Chapter | 5 87
study indicate the potential role of the mushroom extract in stimulated angiogenesis, such as inflammation and tumor development (Lee et al., 2010). Huang et al. (2011) evaluated the anticancer effect of a mycelial culture of the mushroom P.
linteus, and revealed its potential mechanism in vivo conditions. Mushroom extract from Phellinus linteus, when administered daily for 8 weeks in human hepatoma (Hep3B) cell-transplanted mice, resulted in a significant reduction in tumor size
and increase in T cell numbers; IL-12, IFN-c and TNF-a secretion; NK cell activity and phagocytic ability were observed.
From this, it can be concluded that P. linteus extract exhibits a potential therapeutic use for both antitumor and immunomodulatory effects. Li et al. (2011) studied the possible antitumor effect of glycosylated protein and proteoglycan purified
from P. linteus on human cancer cells and mechanisms involved. Studies on cell inhibition assay showed the antiproliferative effect of proteoglycan on human hepatocellular liver carcinoma (HepG-2), human colon adenocarcinoma (HT-29), human lung cancer (NCIH-460) and human breast adenocarcinoma (MCF-7) cells. There was increase in spleen and thymus
weights, the plasmatic immunoglobulin receptor pIgR and IgA levels when HT-29-bearing mice were treated with 100 mg/
kg proteoglycan. On the other hand, measurement by ELISA showed a significant decrease in plasmatic prostaglandin E2
(PGE2), regenerating islet-derived protein 4 (Reg IV), epidermal growth factor receptor (EGFR), and (protein kinase B)
Akt concentrations. The study suggests the immunopotentiator role of proteoglycan that protects T cells from escaping
PGE2 attack, enhance the mucosal IgA response, and disrupt the Reg IV/EGFR/Akt signaling pathway (Li et al., 2011).
Akiyama et al. (2011) studied the effects of agaritine, a hydrazine-derivative from hot-water extract of the mushroom
Agaricus blazei Murrill, on human leukemic monocyte lymphoma (U937) cells. It was found that Agaritine treatment
induced DNA fragmentation, annexin V expression, cytochrome c release and increase in caspase-3, 8, and 9 activities.
This suggests that agaritine moderately induces apoptosis in U937 cells. Additionally, Agaricus blazei has been used as an
adjuvant in cancer chemotherapy; various types of antileukemic bioactive components have been extracted from it. Apart
from this, lentinan, produced from the fruiting bodies of the shiitake mushroom (Lentinus edodes), is a ß-(1→3), ß-(1→6)d-glucan which showed effective antitumor and immunopotentiating activity (Vannucci et al., 2013).
Glucan from edible mushrooms has shown immunomodulatory activity, encouraging stimulation of macrophages, splenocytes and thymocytes. An immunoenhancing water soluble glucan (Table 2) from an edible mushroom, Pleurotus florida,
cultivar Assam Florida, has been found to stimulate macrophages, splenocytes and thymocytes (Roy et al., 2009). Glucan
(Table 2) from the mushroom, Lentinus squarrosulus consists of (1→3,6)-linked, (1→3)-linked, (1→6)-linked and terminal
ß-d-glucopyranosyl moieties in proportions of approximately 1:2:1:1, and showed optimum activation of macrophages as
well as splenocytes and thymocytes (Bhunia et al., 2011). Other immunostimulating glucan that have been reported are
(1→4)-, (1→6)-branched glucan (Table 2) from Calocybe indica (Mandal et al., 2012), PS-I (1→6-ß-d-glucan) and PS-II
(1→3,6-ß-d-glucan) from Termitomyces robustus var. (Bhanja et al., 2012). Glucan were also reported from an ectomycorrhizal edible mushroom Russula albonigra (Krombh.) Fr. (Nandi et al., 2012) and from Ramaria botrytis (Table 2) (Bhanja
et al., 2013). Glucan (Table 2) isolated from Tricholoma crassum (Berk.) Sacc. showed macrophage activation in vitro by
NO production in a dose dependent manner and strong thymocyte and splenocyte immunostimulation in mouse cell culture
medium (Samanta et al., 2013).
Glucan from hybrid mushrooms also exhibits immunomodulatory activity. A (1→6)-ß-d-glucan from a somatic hybrid
mushroom (PfloVv5FB) of Pleurotus florida and Volvariella volvacea was reported to exhibit significant immunoenhancing properties which could stimulate the macrophages, splenocytes, and thymocytes (Das et al., 2010). Sarkar et al. (2012)
reported another (1→6)-ß-d-glucan from a hybrid mushroom, obtained by backcross mating between hybrid mushroom
PfloVv12 and Volvariella volvacea, which was found to be immunostimulating in nature. Some more examples of such
glucan include a water soluble glucan (comprising terminal (1→3,6)-linked and (1→6)-linked ß-d-glucopyranosyl moieties
in a molar ratio of nearly 1:1:3) isolated from an edible hybrid mushroom (Pfle1r) of Pleurotus florida and Lentinula edode
(Maji et al., 2012) and a (1→3,6)-ß-d-glucan from a hybrid mushroom (PfloVv5FB) (obtained through protoplast fusion
between Pleurotus florida and Volvariella volvacea strains) (Maity et al., 2013).
Several heteroglycans have been reported to impart potential immunomodulatory effects. A heteroglycan (Table 3)
isolated from the hot water extract of the fruiting bodies of an edible mushroom, Lentinus squarrosulus (Mont.) Singer,
contains d-galactose, l-fucose, and d-glucose in a molar ratio of nearly 1:1:5; this polysaccharide is capable of activating macrophages, splenocytes and thymocytes (Bhunia et al., 2010). Heteroglycan (Table 3) from the edible mushroom
Calocybe indica, var. APK2, comprises d-glucose, d-galactose, and l-fucose in a molar ratio of nearly 3:1:1 and has
been proven to show immunoenhancing and cytotoxic activity upon HeLa cell lines (Mandal et al., 2011). Heteroglycans
(Table 3) extracted from the hot aqueous extract of Pleurotus ostreatus cultivar, containing d-glucose and d-galactose in
a molar ratio of nearly 7:1, is also an immunostimulant (Maity et al., 2011). Heteroglycans from hybrid mushrooms also
exhibit immunoenhancing activity. Some examples of these are (a) heteroglycan (Table 3) from an aqueous extract of the
somatic hybrid mushroom (PfloVv1aFB) of Pleurotus florida and Volvariella volvacea, constituted of d-galactose, dmannose, and d-glucose with the molar ratio of nearly 1:1:4 (Patra et al., 2011); (b) heteroglycan (Table 3) from an alkaline
88 SECTION | A Probiotics and Prebiotics
TABLE 2 Repeating Unit of Glucan Isolated From Different Mushroom Species
Mushroom Species
Repeating Unit of Glucan
References
Pleurotus florida
→3)-α-D-Glcp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→6)-β-D-Glcp-(1→
6
↑
1
α-D-Glcp
Roy et al. (2009)
Lentinus squarrosulus
→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→6)-β-D-Glcp-(1→
6
↑
1
β-D-Glcp
Bhunia et al. (2011)
Calocybe indica
→[6)-β-D-Glcp-(1]2→4)-β-D-Glcp-(1→[4)-α-D-Glcp-(1]2→
6
↑
1
β-D-Glcp
Mandal et al. (2012)
Russula albonigra
→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→3)-α-D-Glcp-(1→
6
↑
1
α-D-Glcp-(3→1)-α-D-Glsp
Nandi et al. (2012)
Ramaria botrytis
→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→
3
↑
1
β-D-Glcp-(1→3)-β-D-Glsp
Bhanja et al. (2013)
Tricholoma crassum
→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→
6
↑
1
β-D-Glsp
Samanta et al. (2013)
extract of a somatic hybrid mushroom (PfloVv1aFB) of Pleurotus florida and Volvariella volvacea and consisting of dgalactose, d-mannose, and d-glucose with the molar ratio of nearly 1:1:4 (Bhunia et al., 2012); (c) heteroglycan (Table 3)
isolated from hot aqueous extract of fruiting bodies of an edible hybrid mushroom Pfle1r of Pleurotus florida and Lentinula
edodes and made up of d-glucose, d-mannose, and d-galactose residues in a molar ratio of nearly 1:1:1 (Maji et al., 2013);
and (d) an immunostimulating water-soluble heteroglycan (PS-II) (Table 3) isolated from aqueous extract of the fruiting bodies of a hybrid mushroom, Pfls1h of Pleurotus florida and Lentinus squarrosulus (Mont.) Singer., composed of
(1→6)- and (1→2,4,6)-a-d-galactopyranosyl, terminal ß-d-mannopyranosyl and terminal ß-d-glucopyranosyl residues in a
relative proportion of approximately 1:1:1:1 (Sen et al., 2013). An immunoenhancing water-soluble hetero-polysaccharide
(Table 4) isolated from an alkaline extract of the fruit bodies of an ectomycorrhizal edible mushroom, Tricholoma crassum
(Berk.) Sacc., exhibited splenocyte, thymocyte as well as macrophage activations (Patra et al., 2012). Heteropolysaccharide
(Table 4), having molecular weight ~2.1 × 105 Da, was isolated from hot aqueous extract of the fruit bodies of hybrid mushroom Pfle 1p. The hybrid mushroom Pfle 1p was obtained through intergenic protoplast fusion between Pleurotus florida
and Lentinula edodes. The heteropolysaccharide contained d-glucose, d-galactose, and d-mannose in a molar ratio of
nearly 4:2:1 and showed in vitro macrophage activation by NO production and also stimulated splenocytes and thymocytes
(Maity et al., 2013).
4.2 Antioxidant Activity
Different wild mushroom species were reported to have antioxidant activity, which mainly related to their phenolic compounds
(phenolic acids and flavonoids), followed by tocopherols, ascorbic acid and carotenoids (Ferreira et al., 2009). These molecules
TABLE 3 Repeating Unit of Heteroglycan Isolated from Different Mushroom Species
Repeating Unit of Heteroglycan
References
Lentinus squarrosulus
F
D
A
C
E
→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→6)-α-D-Glcp-(1→3)-β-D-Glcp-(1→4)-β-D-Glcp-(1→
6
4
↑
↑
α-L-Fucp
β-D-Glcp
B
G
Bhunia et al. (2010)
Calocybe indica
→3)-α-D-Galp-(1→4)-β-D-Glcp-(1→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→
6
↑
1
α-L-Fucp
Mandal et al. (2011)
Pleurotus ostreatus
C
E
D
C
E
D
→3)-β-D-Glcp-(1→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→3)-β-D-Glcp-(1→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→
3
3
↑
↑
α-D-Galp
α-D-Glcp
A
B
Maity et al. (2011)
Hybrid mushroom
(PfloVv1aFB)
→6)-β-D-Glcp-(1→2)-α-D-Galp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→
6
6
↑
↑
1
1
α-D-Manp
β-D-Glcp
Patra et al. (2011) Bhunia et al. (2012)
Hybrid mushroom (Pfle1r)
→6)-α-D-Galp-(1→6)-α-D-Glcp-(1→
2
↑
1
β-D-Manp
Maji et al. (2013)
Hybrid mushroom (Pfle1r)
C
β-D-Manp
1
↓
B
2A
→6)-α-D-Galp-(1→6)-α-D-Galp-(1→
4
↑
1
β-D-Glcp
D
Sen et al. (2013)
Prebiotics and Their Production From Unconventional Raw Materials (Mushrooms) Chapter | 5 89
Mushroom Species
90 SECTION | A Probiotics and Prebiotics
TABLE 4 Repeating Unit of Heteropolysaccharide Isolated From Different Mushroom Species
Mushroom
Species
Repeating Unit of Heteropolysaccharide
References
Tricholoma
crassum
D
B
A
→6)-β-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→
3
↑
1
α-D-Galp-(4←1)-β-D-Manp
C
E
Patra et al. (2012)
hybrid mushroom
(pfle 1p)
→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Galp-(1→6)-α-D-Galp-(1→
2
4
↑
↑
1
1
β-D-Glcp
β-D-Manp
Maity et al. (2013)
were quantified in different species, mainly from Finland, India, Korea, Poland, Portugal, Taiwan, and Turkey. Ferreira et al.
(2009) reviewed the values currently available in literature, but expressed in different basis (dry weight, fresh weight, and extract). Helvella crispa from India revealed the highest content of phenolic compounds expressed per gram of extract (34.65 mg/g),
while Sparassis crispa from Korea revealed the highest value expressed in a dry weight basis (0.76 mg/g). Auricularia fuscosuccinea (white) from Taiwan (32.46 mg/g of extract), Agaricus silvaticus (3.23 × 10-3 mg/g of dry weight), and Ramaria Botrytis
(2.50 × 10-4 mg/g of fresh weight) from Portugal, contained the most tocopherols of all the species. Auricularia fuscosuccinea
(brown) from Taiwan (11.24 mg/g of extract), Suillus collinitus from Portugal (3.79 mg/g of dry weight) and Agaricus bisporus
from Poland (0.22 mg/g of fresh weight) revealed the highest levels of ascorbic acid. Lactarius deliciosus from Portugal revealed
the highest content of ß-carotene (0.09 mg/g of extract).
The antioxidant properties of A. brasiliensis were evaluated using methanol, ethanol, dimethyl sulfoxide and water as
solvent; extraction was preceded for a variety of times and temperatures. The best conditions for extraction of antioxidants
is with methanol as solvent at 60°C for 60 min (Mourão et al., 2011). Mourão et al. (2011) showed that average antioxidant
activity for the closed cap group is 24% higher than opened caps. The phenolic composition of methanolic extract from
A. bisporus was analyzed by HPLC and it contains rutin, gallic acid, caffeic acid, and catechin. All these contribute to its
antioxidant activity (Abah and Abah, 2010). Armillaria mellea, commonly known as the honey mushroom, is pathogenic
and grows on living trees and on dead and decaying food material (Zekovic et al., 2010). Armillaria mellea has a strong
symbiotic relationship with Gastrodia elata, known as Tian Ma (Lung and Chang, 2011). The antioxidant activity of
Armillaria mellea ethanol extract is higher than that of Lycoperdon saccatum which is in accordance with its higher content of the two trace elements, selenium and zinc (Zekovic et al., 2010). Methanolic extracts from dried mycelia, mycelia
free broth and hot water extracts from dried mycelia by Armillaria mellea submerged cultures showed low EC50 values
(<10 mg/mL). Their ascorbic acid and total phenol contents are well correlated with the reducing power and the scavenging effect on superoxide anions (Lung and Chang, 2011). A low molecular weight polysaccharide (2.8 × 104 Da) isolated
from the fruiting body of Auricularia polytricha exhibited stronger hydroxyl radical scavenging activity than vitamin C at
the same concentration (Sun et al., 2010). Furthermore, polysaccharides isolated from water, acidic and alkaline solutions
from Lentinus edodes separately showed antioxidant activity through inhibition activity of hydroxyl, ABTS+ radical and
lipid peroxidation (Li et al., 2012). Heleno et al. (2011) have screened antioxidant activities of six different mycorrhizal
mushrooms: Boletus aereus, B. edulis, B. reticulatus; not edible: B. purpureus, B. satanas, B. rhodoxanthus. Among these,
B. aereus showed highest antioxidant activity. Their studies revealed that EC50 value for DPPH scavenging activity
(0.43 mg/mL) of the mushroom B. edulis was lower than that of Indian (1.4 mg/mL), Taiwanian (~1.5 mg/mL) and Turkish
(~0.5 mg/mL) specimens (Heleno et al., 2011). Antioxidant activities of ethanolic extracts of B. edulis and B. auranticus
have been also identified by Vidovic et al. (2010). However, the total phenol as well as hydroxyl radical scavenging activity
of B. auranticus (EC50 0.016 mg/mL) was found to be higher than B. edulis, except reducing power, where B. edulis shows
higher activity. Studies by Vidovic et al. (2010) have reported Variegatic acid in both the extracts.
Water extracts of Pleurotus ostreatus with higher phenolic content possessed better antioxidant activities than ethanol
extract (Chirinang and Intarapichet, 2009). Kim et al. (2009) studied the antioxidant activities of methanolic extracts of oyster mushrooms. The extract from yellow strain (P. cornucopiae) showed the highest radical scavenging activity, r­ educing
Prebiotics and Their Production From Unconventional Raw Materials (Mushrooms) Chapter | 5 91
power, ferrous chelating ability and total phenolic contents over the dark-gray strain (P. ostreatus) and pink strain (P. salmoneostramineus). Yim et al. (2010) have also studied the antioxidative potential and total phenolic content of water extract
of P. ostreatus and found that the ferric reducing power was significantly higher than BHA and ascorbic acid.
Antioxidant and antiradical activity of methanol extracts from six Termitomyces species (T. titanicus, T. aurantiacus, T.
letestui, T. clypeatus, T. microcarpus, and T. eurhizus) growing in Tanzania were studied by Tibuhwa (2012). Results showed
highest ability to decrease DPPH radical by T. microcarpus (EC50 < 0.1 mg/mL) followed by T. letestui (EC50 = 0.14 mg/
mL), while least ability was shown by T. eurhizus (EC50 = 0.36 mg/mL). The study also showed high antiradical activity
(EAU515 1.48) of T. microcarpus followed by T. aurantiacus (EAU515 1.43), while the lowest was observed from T. eurhizus (EAU515 0.7). Additionally, T. microcarpus contained high amount of phenols, flavonoids and ß-carotene (Tibuhwa,
2012). Wang et al. (2012a,b) isolated three polysaccharides from the mushroom Tricholoma lobayense and evaluated their
antioxidant activity. A study by Chatterjee et al. (2011) revealed that alcohol extract of T. giganteum possesses better antioxidant activity than that of water and ethyl acetate extract of the same mushrooms.
4.3
Hypoglycaemic/Antidiabetic Activity
Edible and medicinal mushrooms are a potent source of biologically active compounds with antidiabetic effects (Kiho et al.,
1994a; Hong et al., 2007). Many mushroom species appear to be effective for both the control of blood glucose levels and
the amelioration of the course of diabetic complications. Medicinal mushrooms such as Agaricus bisporus, A. subrufescens,
Cordyceps sinensis, Coprinus comatus, Ganoderma lucidum, Inonotus obliquus, Phellinus linteus, Pleurotus spp., Poria
cocos and Sparassis crispa have been reported to have antihyperglycemic effects (Silva et al., 2012). Additionally, mushrooms are known to contain compounds which help in proper functioning of the liver (Wani et al., 2010), pancreas and other
endocrinal glands, thereby promoting formation of insulin and related hormones, consequently ensuring healthy metabolic
functions (Wasser and Weis, 1999; Zhang and Lin, 2004; Chen et al., 2012a,b). Mushroom polysaccharides such as beta
glucans have the ability to restore the function of pancreatic tissues through increased insulin output by ß-cells, which causes
lowering of blood glucose levels. Polysaccharides have also been shown to improve the sensitivity of peripheral tissues to
insulin. In addition to this, the consumption of mushrooms reportedly decreases the lipid levels, including total cholesterol,
total triglyceride, and low-density lipoproteins, and increases the level of high-density lipoproteins (Lee et al., 2012).
Studies made by Kiho et al. (1994b) showed that Glucuronoxylomannan (AC) from the fruiting bodies of T. fuciformis
exhibited a significant dose-dependent hypoglycemic activity in normal mice, and also demonstrated significant activity in
streptozotocin-induced diabetic mice by means of intraperitoneal administration. Exopolysaccharides (EPS) of the mushroom T. fuciformis has reported to exhibit a considerable hypoglycemic effect and improved insulin sensitivity, possibly
through regulating PPAR-gamma-mediated lipid metabolism, when evaluated for antidiabetic activities in mice models
(Cho et al., 2007). These results indicated that Tremella fuciformis has a potential oral hypoglycemic effect and can be used
as a functional food in the management of Diabetes mellitus (DM). The exo-polymer (GAE) produced by submerged mycelial cultures of Ganoderma applanatum and Collybia confluens have shown hypoglycemic effects in streptozotocin (STZ)induced diabetic rats. The results strongly demonstrated the potential of exopolymer in combating diabetes in experimental
animals (Yang et al., 2007). Water-soluble polysaccharide (FA) from fruiting bodies of A. auricula-judae has been reported
to show a hypoglycemic effect on genetically diabetic mice (KK-Ay).
The fruit body of Agaricus subrufescens is useful as a health promoting food. Studies preformed on murine models
and human volunteers to examine the immune-enhancing effects of the naturally outdoor-cultivated fruit body of Agaricus
brasiliensis KA21 (i.e., Agaricus blazei) have shown antitumor, leukocyte-enhancing, hepatopathy-alleviating and endotoxin
shock-alleviating effects in mice (Liu et al., 2008). In the human study, percentage body fat, percentage visceral fat, blood
cholesterol level, and blood glucose level were decreased and natural killer cell activity was increased (Liu et al., 2008).
Beta-glucans and oligosaccharides (AO) of Agaricus blazei Murill showed antihyperglycemic, antihypertriglyceridemic, antihypercholesterolemic, and antiarteriosclerotic activity indicating overall antidiabetic activity in diabetic rats; AO had about
twice the activity of beta-glucans with respect to antidiabetic activity (Kim et al., 2005). Further supplement of Agaricus
blazei Murill extract has improved insulin resistance among subjects with type 2 DM. The increase in adiponectin concentration after taking Agaricus blazei Murill extract might be the mechanism that brings the beneficial effect (Hsu et al., 2007).
Recent studies have determined that mushrooms such as Hericium spp. may have important physiological functions
in humans, including antioxidant activities, regulation of blood lipid levels and reduction of blood glucose levels (Wang
et al., 2005). Researchers have found that the hypoglycemic effects of feeding the methanol extract of H. erinaceus to
­streptozotocin-induced diabetic rats significantly lowered elevation rates of blood glucose levels (Wang et al., 2005).
Additionally, the culture broth of Inonotus obliquus also possesses significant antihyperglycemic, antilipid peroxidative,
and antioxidant effects in alloxan-induced diabetic mice (Sun et al., 2008).
92 SECTION | A Probiotics and Prebiotics
Crude polysaccharides of Cordyceps sinensis were tested in normal mice and streptozotocin-induced diabetic mice.
Oral administration significantly lowered the glucose level in mice (Kiho et al., 1993). A polysaccharide obtained from
the cultural mycelium of Cordyceps sinensis showed potent hypoglycemic activity in genetically diabetic mice after intraperitoneal administration. The plasma glucose level was quickly reduced in normal and streptozotocin-induced diabetic
mice after intravenous administration (Kiho et al., 1996). Cordyceps, a Chinese herbal medicine with a fruiting body and
stem, has been proposed to have multiple medicinal properties. The diabetic rats had significantly lowered weight gain and
higher blood glucose response in oral glucose tolerance test than the control rats; these changes were significantly reduced
by administrating the fruiting body of Cordyceps. These improvements suggested that the fruiting body of Cordyceps
has a potential to be a functional food for diabetes management (Lo et al., 2004). Another study revealed that isolated
polysaccharide from Cordyceps sinensis (CSP-1) produced a significant drop in blood glucose level in both STZ-induced
diabetic rats and alloxan-induced diabetic mice. This led to the conclusion that CSP-1 may stimulate pancreatic release of
insulin and/or reduce insulin metabolism (Li et al., 2006). Researchers evaluated the antidiabetic effect of an alpha-glucan
(MT-alpha-glucan) from the fruit body of maitake mushrooms (Grifola frondosa) on KK-Ay mice. These data suggest that
MT-alpha-glucan has an antidiabetic effect on KK-Ay mice which might be related to its effect on insulin receptors, that is
increasing insulin sensitivity and ameliorating insulin resistance of peripheral target tissues (Hong et al., 2007).
4.4 Antimicrobial Activity
Certain mushrooms have antimicrobial properties which provide control for many human diseases, are generally safe for
use and are also effective. Several mushrooms are reported to effectively exhibit both antibacterial and antifungal activity against antibiotic resistant pathogens (Sharma et al., 2014). Agaricus bisporus, the most cultivated mushroom in the
world, is in forefront for its antibacterial activity. The methanolic extract revealed MIC = 5 µg/mL against Bacillus subtilis
even lower than the standard ampicillin (MIC = 12.5 µg/mL) (Barros et al., 2008a). It also displayed antibacterical activity against Gram positive bacteria Bacillus cereus, Micrococcus luteus, Micrococcus flavus, Staphylococcus aureus and
Staphylococcus epidermidis (Tambekar et al., 2006; Öztürk et al., 2011; Ozen et al., 2011) and Gram negative bacteria
Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus vulgaris, Salmonella
typhi, and Salmonella typhimurium (Tambekar et al., 2006; Ozen et al., 2011). Other Agaricus species (such as Agaricus
bitorquis) methanolic extracts also showed inhibitory effect upon all the tested Gram-positive bacteria and Gram-negative
bacteria (Öztürk et al., 2011). Agaricus silvicola methanolic extract also revealed antimicrobial properties against Bacillus
cereus (MIC = 5 µg/mL), Bacillus subtilis (MIC = 50 µg/mL), and against Staphylococcus aureus (MIC = 5 µg/mL), lower
than the standard ampicillin (MIC = 6.25 µg/mL) (Barros et al., 2008a). The mycelium of Agaricus cf. nigrecentulus and
Tyromyces duracinus (ethyl acetate extracts) showed activity against only Staphylococcus saprophyticus (Rosa et al., 2003).
Conversely, Agaricus essettei, Agaricus silvicola, Agaricus silvaticus, and Agaricus cf. nigrecentulus did not show any
antibacterial activity against Gram-negative bacteria (Rosa et al., 2003; Öztürk et al., 2011; Barros et al., 2008a).
The ethanolic extracts of Armillaria mellea mycelium showed antibacterial effect against Sarcina lutea, however no
activity was observed upon other Gram-positive bacteria (Kalyoncu et al., 2010). However, ethanolic extract of their fruiting bodies showed broad-spectrum antimicrobial activity (Kalyoncu and Oskay, 2008). Cantharellus cibarius methanolic
extract demonstrated good activity against Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus (Barros
et al., 2008a,b; Ozen et al., 2011) and Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa (Ozen et al.,
2011). Clitocybe alexandri methanolic extract presented significant activity against Bacillus subtilis, Micrococcus luteus,
Enterobacter aerogenes, and Escherichia coli (Solak et al., 2006). Kalyoncu and Oskay (2008) tested antimicrobial activity of chloroform and ethanolic extracts from Clitocybe geotropa, the latter showing significant capacity against Bacillus
cereus and Proteus vulgaris.
The genus Cortinarius is one of the most diverse genera of mushrooms. Ethyl acetate extracts of C. ardesiacus, C.
archeri, C. atrosanguineus, C. austrovenetus, C. austroviolaceus, C. coelopus, C. [Dermocybe canaria], C. clelandii, C.
[D. kula], C. memoria-annae, C. persplendidus, C. sinapicolor, and C. vinosipes, plus an additional 47 collection samples
not identified at the species level, exhibited IC50 values =0.09 of mg/mL against Staphylococcus aureus and P. aeruginosa
(Beattie et al., 2010).
Ganoderma lucidum is one of the most well known traditional medicinal mushrooms. Various extracts have been found
to be equally effective when compared with gentamycin sulfate, the acetone extract being the most effective. The extract
of this mushroom demonstrated strong antibacterial activity, mainly against Klebsiella pneumonia, and moderate inhibition against Bacillus subtilis and Staphylococcus aureus (Quereshi et al., 2010), but in the study reported by Sheena et al.
(2003), its methanolic extract showed poor antimicrobial activity. Other authors described the capacity of aqueous extract
to inhibit 15 types of Gram-positive and Gram-negative bacteria, with the highest inhibition exhibited against Micrococcus
Prebiotics and Their Production From Unconventional Raw Materials (Mushrooms) Chapter | 5 93
luteus (Gao et al., 2005). Ethyl acetate extracts of Phellinus sp., Gloeoporus thelephoroides and Hexagonia hydnoides inhibited Bacillus cereus growth while the same extract of Nothopanus hygrophanus mycelium presented inhibitory activity
against Listeria monocytogenes and Staphylococcus aureus. Irpex lacteus mycelium extract was the most effective, presenting a broad spectrum of activity (Rosa et al., 2003).
The antimicrobial activity of Pycnoporus sanguineus has been known since 1946, when Bose isolated poliporin, a compound active against Gram-positive and Gram-negative bacteria and without toxicity in experimental animals. Rosa et al.
reported inhibition against Listeria monocytogenes and Staphylococcus aureus (Rosa et al., 2003). Smânia et al. (1995,
1997) showed that this mushroom produces cinnabarine, an orange pigment active against Bacillus cereus, Staphylococcus
aureus, Leuconostoc mesenteroides, Lactobacillus plantarum, and several Streptococcus spp. Cinnabarine was more active
against Gram-positive than Gram-negative bacteria (Rosa et al., 2003). Additionally, all the tested gram-positive bacteria
were susceptible to methanolic extracts of Lactarius species and Tricholoma portentosum (Barros et al., 2007a,b; Ozen
et al., 2011). Among Lactarius species (L. piperatus, L. camphorates, L. volemus, L. delicious), L. camphoratus methanolic extract was the one with greater antimicrobial activity (Ozen et al., 2011). Methanolic extracts of Ramaria botrytis
and ethanolic extract of Ramaria flava inhibited the growth of Gram-positive bacteria better than Gram-negative bacteria
(Gezer et al., 2006). The antimicrobial effect of ethanolic extract of Laetiporus sulphureus was tested by Turkoglu et al.
(2007) and strongly inhibited the growth of the gram-positive bacteria tested, including Bacillus subtilis, Bacillus cereus,
Micrococcus luteus, and Micrococcus flavus.
Lepista nuda methanolic extract had a good action on Gram-positive bacteria, more specifically on Bacillus cereus,
Bacillus subtilis, and Staphylococcus aureus (Dulger et al., 2002; Barros et al., 2008b). Ishikawa et al. (2001) reported
the inhibitory activity of Lentinus edodes ethyl acetate extract against Bacillus cereus, Bacillus subtilis, Staphylococcus
aureus, and Staphylococcus epidermidis. This mushroom (aqueous extract), as well as menthol extract of n-BuOH fraction of Phellinus linteus, demonstrated good activity against MRSA (Hur et al., 2004; Hearst et al., 2009). Furthermore,
Streptococcus pyogenes was very sensitive to Lentinus edodes chloroform extract whereas no effect was found against
Escherichia coli, Pseudomonas fluorescens, Klebsiella pneumoniae or Camphylobacter jejuni (Hatvani, 2001). The ability
of L. edodes extracts to improve oral health has also been extensively researched, because it showed a strong bactericidal
effect upon Streptococcus mutans and Prevotella intermedia which are strongly implicated in dental caries and tooth decay
(Hirasawa et al., 1999; Signoretto et al., 2011). Mycelium of Leucopaxillus giganteus (methanolic extract) presented antimicrobial capacity, inhibiting only Gram-positive bacteria and in the decreasing order: Staphylococcus aureus > Bacillus
cereus > Bacillus subtilis (Barros et al., 2007c).
Methanolic extract of Phellinus rimosus and Navesporus floccosa showed moderate inhibition of Bacillus subtilis and
Staphylococcus aureus (Sheena et al., 2003). Pleurotus ostreatus and Meripilus giganteus showed broad-spectrum antimicrobial activity. The maximum effect was shown by ethanolic extracts of Pleurotus ostreatus against Sarcina lutea
(Kalyoncu et al., 2010). Ether extract of Pleurotus sajor-caju showed high antibacterial activity against Staphylococcus
aureus, whereas Staphylococcus epidermidis showed high sensitivity for ethanol extract (Tambekar et al., 2006).
In vitro antimicrobial activity of the acetone and methanol extracts of the mushrooms Amanita rubescens, Cantharellus
cibarius, Lactarius piperatus, and Russula cyanoxantha was studied by Kosanic et al. (2013). The antimicrobial activity was estimated by determining the minimal inhibitory concentration by using micro dilution plate method against five
species of bacteria and five species of fungi. Generally, the tested mushroom extracts had relatively strong antimicrobial
activity against the tested microorganisms, suggesting that mushrooms may be used for pharmaceutical purposes in the
treatment of various diseases. The culture filtrates of 27 edible mushrooms were screened for antimicrobial activity against
plant pathogens (Chen and Huang, 2011). It was found that culture filtrates of Lentinula edodes and Clitocybe nuda were
able to completely inhibit spore germination of Colletotrichum higginsianum. Three culture filtrates that contained substances having the capacity to completely inhibit spore germination of Alternaria brassicicola were Ganoderma lucidum,
L. edodes, and C. nuda. These results suggest that substances from edible mushrooms have the potential to be developed
into biocontrol agents for the control of plant diseases (Chen and Huang, 2011). Menaga et al. (2012) found that bioactive
compounds from Pleurotus florida mushroom extracts could be used as alternate therapeutics to antibiotics. Additionally,
Alves et al. (2012b) have reported that Fistulina hepatica, Russula botrytis, and Russula delica are the most promising
mushroom species as antimicrobial agents. It is important to develop new studies with different mushroom species to address the microorganisms so problematic to human health.
5.
CONCLUSION
Mushrooms are an alternate source of nutrients with low fats and calories. In general, proteins of mushrooms contain all
nine essential amino acids required by humans. Additionally, they are also a relatively good source of nutrients such as
94 SECTION | A Probiotics and Prebiotics
phosphorus, iron, and vitamins, including thiamine, riboflavin, ascorbic acid, ergosterol, and niacin. Mushrooms have also
been reported as therapeutic foods which are required to prevent diseases such as hypertension, diabetes, hypercholesterolemia, and cancer. All these functional characteristics are mainly due to the presence of dietary fibers, bioactive components,
antioxidants, lectins and antimicrobial agents. Furthermore, edible mushrooms are gaining much attention as an alternative
source of prebiotics. The major components rendering prebiotic function in mushrooms are nondigestible polysaccharides
such as glucan, chitin and hetropolysaccharides. Several mushroom polysaccharides, like pleuran, lentinan, schizophyllan,
ß and a-glucans, mannans, xylans, galactans, chitin, inulin and hemicelluloses, can be credited with promising prebiotic
effects. Mushrooms rich in immune-modulating polysaccharides are used as health-promoting food supplements (nutraceuticals). However, the mechanism of action of various secondary metabolites isolated from medicinal and wild edible mushrooms are yet to be discovered. Thus, further research will uncover different applications with respect to the high nutritional
and therapeutic potential of mushrooms, namely as functional foods or as a source of nutraceuticals for maintenance and
promotion of health and a good quality of life.
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FURTHER READING
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Chapter 6
Probiotics in the Rescue of Gut
Inflammation
Asit Ranjan Ghosh
VIT University, Vellore, India
1.
INTRODUCTION
The gut is considered to be the second brain of the body. The gut-brain axis explains the link between digestion, mood,
health, behavior, and even psychological state. The enteric nervous system (ENS) of the gut and central nervous system
(CNS) of the brain are highly connected in the process of signal transduction including the gut flora (Mayer et al., 2014).
Multiple research articles and reviews have refreshed our conceptual understanding regarding the connection of health
in relation to gut flora. The type and number of floral members are found to be instrumental in maintaining good health
(Lozupone et al., 2012). Current research has recently overwhelmingly established that bacterial gut flora are specific to
good health, leading to the bacterial ecology of the gut. This beneficial and health supportive bacterial ecology is sometimes
jeopardized or impaired, leading to a condition called dysbiosis (Maynard et al., 2012).
Inflammation is a pervasive condition which is characterized by significant symptoms including pain, redness, swelling,
heat, and loss of function. Inflammation is an immunological strategy to eliminate the cause of cell injury, clear necrotic
cells, and damaged tissues, and to initiate in tissue repair. However, chronic inflammation appears to be the cause of several
inflammatory diseases such as rheumatoid arthritis, diabetes, various cardiovascular diseases, and obesity (Lescheid, 2014;
Festi et al., 2014). Gut flora in the state of dysbiosis is affected, which results chronic inflammation by activation of T cells
subsets (Th1, Th17, γδT) and suppression of regulatory mechanisms by reducing IgA synthesis, lowering levels of suppressive cytokines IL-10, TGF-β, and reducing Treg cells (Honad and Litman, 2012).
The present treatment of inflammation in the gut (as well as the resultant diseases) is the normalization of gut microbial
population by the introduction of beneficial members of the gut microbiota. Fecal microbiota transplantation (FMT) used in
alleviating many infectious and inflammatory diseases where fecal microbiota of a healthy individual is transplanted strategically to the gut impaired individual (Bakken et al., 2011). By 2013, the Food and Drug Administration (FDA) approved
the legal use of feces as a drug for human health (Ratner, 2014). However, beneficial microbiota of diverse origin also
shows promise in offering beneficial effects to human health. Fermented food and nutraceuticals are also recommended
for reinstating gut flora (Selhub et al., 2014; Dubey et al., 2015, 2016). “Live microorganisms that, when administered in
adequate amounts, confer a health benefit on the host” are probiotics (FAO/WHO) (Marteau, 2006). In the following review, the main focus of discussion is to identify the role played by probiotics in the mitigation of inflammation of the gut.
2.
GUT MICROBIOTA, PROBIOTICS, AND DYSBIOSIS
The gut is a hollow tubular structure into which nutrient-rich food is pushed, processed, and absorbed, then wastes are
expelled as it occurs in even the most primitive hydra. This process occurs sequentially in the buccal cavity, esophagus,
stomach, and intestines in humans. The large intestine offers a home for a large number of microbes called gut microbiota
or/and commensal microflora. In the beginning of life, prenatal maternal exposure plays a vital role in post-natal microfloral colonization, gut-associated lymphoid tissue (GALT) development, and maintenance of integrity of the mucosal barrier
(Daliri and Lee, 2015). In adults, bacterial phyla like bacteroidetes and firmicutes are common with less abundance of
actinobacteria, proteobacteria, and verrucomicrobia (Eckburg et al., 2005; Hakansson and Molin, 2011). The beneficial microbes are called probiotics with special characteristics (Fig. 1) (Pande et al., 2012). The undigested metabolic ingredients
of the host which support growth of probiotics are termed “prebiotics.” Together, probiotics and prebiotics constitute the
Therapeutic, Probiotic, and Unconventional Foods. https://doi.org/10.1016/B978-0-12-814625-5.00006-6
© 2018 Elsevier Inc. All rights reserved.
101
102 SECTION | A Probiotics and Prebiotics
FIG. 1 Probiotics: characteristics and uses. Probiotics are described with multiple characteristics, giving them potential for multiple applications in
restoring good health.
functional food, called synbiotics. The effectiveness of probiotics is strain-specific, and each strain may contribute to host
health through different mechanisms (Pande et al., 2012).
Probiotics commonly stem from the category of lactic acid bacteria (LAB) which are Gram positive, nonspore forming,
catalase negative, acid tolerant, and strictly fermentative with lactic acid as the major end product during sugar fermentation. LAB with probiotic potentials are known to exert positive influence on host health and physiology (Marteau, 2006;
Pande et al., 2012). Probiotics can be bacteria, molds or yeasts, but most are lactic acid bacteria and consist of a ­number
of heterogenous bacterial genera within the phylum Firmicutes. Among all bacteria, genera such as Carnobacterium,
Enterococcus, Lactobacillus, Lactococcus, Lactosphaera, Leuconostoc, Melissococcus, Oenococcus, Pediococcus,
Streptococcus, Tetragenococcus, Vagococcus, and Weissella are recognized as LAB (Lozupone et al., 2012; Lescheid, 2014;
Festi et al., 2014). Some are homofermentative, such as Lactococcus and Streptococcus, as they yield two lactates from one
glucose molecule, whereas others are heterofermentative (Leuconostoc and Weissella) transform a glucose molecule into
lactate, ethanol, and carbon dioxide. All probiotic bacteria are LAB, but all LAB are not probiotic. This phenomenon was
observed in a study by Shruthy et al. (2011). LAB constitute an integral part of the healthy gastrointestinal (GI) micro ecology and is critically involved in host metabolism. Gut microbiota ferment various substrates like lactose, biogenic amines,
allergenic compounds, and convert them into short chain fatty acids (SCFA), organic acids, gases with enzymes, vitamins,
antioxidants, and bacteriocins (Fig. 1) (Pande et al., 2012). In a study by Gowri and Ghosh (2010a,b) several probiotic
strains were shown to produce bacteriocin-like Pediocin from Pediococcus pentosaceus GS4 with a molecular weight
of 9.9 kDa (unpublished data) that inhibits the growth of Staphylococcus aureus (ATCC 25923), Listeria monocytogens
(ATCC 15313) and diarrhoeagenic bacteria including Shigella dysenteriae type1, Shigella sonnei, Salmonella typhimurium,
Vibrio cholerae O1 and O139. Conjugated linoleic acid (CLA) is a natural ligand of PPARγ (peroxisome proliferatoractivated receptor gamma). Some probiotics have the property of biohydrogenation of linoleic acid to CLA and thus, can
regulate inflammation (Dubey et al., 2012, 2015).
Selection criteria for probiotics: Probiotics have attained considerable interest and importance for a variety of medical
conditions, and millions of people around the world consume probiotics daily for perceived health benefits. A probiotic
strain must be able to survive in the extremely harsh conditions of the digestive tract of the host, such as high acidity in
the stomach and concentrated bile found in the small proximal of the intestine. An effective probiotic should be capable of
Probiotics in the Rescue of Gut Inflammation Chapter | 6 103
gastrointestinal tract transition, influencing metabolic activities like cholesterol assimilation, lactase activity and vitamin
production, overcoming effects of peristalsis, and possession the capacity for colonization. In addition, it must also be safe,
commercially feasible, and technologically compatible and must remain viable in storage while maintaining acceptable
sensory attributes (Saarela et al., 2000; Bagad et al., 2012; Dubey et al., 2015, 2016).
Gut microbiota is so unique that it has potential to be distinct in populations and as well as in population units (Lozupone
et al., 2012). It also lays the foundation to establish the state of human health. Research data confer that several factors
like gene, environment, and diet play a major role in having specific gut microbiota (Filippo et al., 2010; Arumugam et al.,
2011; Wu et al., 2011). Diet discrepancy and floral diversity remain a key to observation in human microbiome research.
For example, dominance of Prevotella and the plant based diet of healthy children in Africa differs with Bacteroides and
animal protein-rich diet among healthy adults in the United States (Arumugam et al., 2011; Wu et al., 2011). Differences in
microbial composition have been observed in a disease state (Sun et al., 2010; Knights et al., 2011), with special mention
of antibiotic-associated diarrhea (Young and Schmidt, 2004), ulcerative colitis (UC) (Frank et al., 2007), Crohn’s disease
(CD) (Dicksved et al., 2008), irritable bowel syndrome (IBS) (Carroll et al., 2011), Clostridium difficile-associated diarrhea
(Chang et al., 2008), and with drug (paracetamol) metabolism (Clayton et al., 2009). Therefore, gut microbiota establish
a balance being influenced by several factors, which in turn determine the state of health. Disturbances potentially lead to
dysbiosis when the immune-regulatory network is ruffled.
Dysbiosis brings change in gut flora and becomes the etiology of many inflammatory diseases (Maynard et al., 2012)
like CD, UC, IBS, IBD, necrotizing enterocolitis, and extraintestinal diseases like rheumatoid arthritis, multiple sclerosis,
diabetes, atopic dermatitis, asthma, obesity, and metabolic syndromes (Maynard et al., 2012). Research findings are rather
convincing in establishing these inflammatory pathologies with dysbiosis; so reshaping the floral diversity with the application of beneficial bacteria (probiotics) is in the forefront of medical research.
3.
GUT IMMUNITY
A healthy gut is the natural home of a healthy microbiota. Normal gut function is closely linked with restoring the health of
the mucosal layer lining its length and associated lymphoid tissues. The gut is the site of the largest microbial populations
and the biggest source of immunological stimulations (Delcenserie et al., 2008). It is necessary to understand how the immunological complexity is overcome and restore homeostasis.
The largest mass of lymphoid tissues are assembled in human intestine and immune cells like phagocytes (neutrophils,
monocytes, macrophages, even natural killer—NK cells), dendritic cells, and lymphocytes (T and B) are stored to combat
any attack by innate and adaptive immune systems (Salminen et al., 1998). Together, with immunological function, the
human intestine has three primary physiological functions: digestion of food, absorption of nutrients, and keeping toxins
and toxic elements out of the body (Jakoby and Ziegler, 1990). Failure in any of these functions results in defective energy
production, faulty energy need, and waning of body’s reserve, eventually culminating in a disease. However, the gut epithelia are central to the orchestration of intestinal defenses and possess multitasking abilities. They are composed of five
different types of epithelium: absorptive enterocytes, goblet cells, Paneth cells, M cells, and enteroendocrine cells and are
developed from common stem cells located near the base of the intestinal crypts. Maturation of the intestinal mucosa and
GALT is initiated by microbial colonization. Central to sensing the colonizers of the intestine is the expression of wide
range of germline-encoded pattern recognition receptors (PRRs) by intestinal epithelial cells (IECs) and residential immune
cells in the gut. PRRs, such as toll-like receptors (TLRs) and C-type lectin receptors (CLRs), are found on the cell surface
or in endosome, and cytosolic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) of the intestinal
mucosa can recognize the pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) which are expressed by the
constituents of resident microbiota and pathogens. However, the mechanisms behind the differential recognition between
resident microbiota and pathogens via PRRs-MAMPs interaction are not well understood (Maynard et al., 2012; Koboziev
et al., 2014; Daliri and Lee, 2015; Jakobsson et al., 2015).
In the adaptive immune system, T (CD4+) cells are activated and are differentiated into Th1 or Th2 cells, depending on
the interactomes. The Th1 cells produce proinflammatory cytokines like IFNγ, TNFα, and IL-2 while Th2 cells produce
IL-4, IL-5, IL-6, and IL-13, respectively. The Th1 cells thus stimulate phagocytosis and eliminate pathogenic microbes
while Th2 cells produced IL-4 that induces B lymphocytes to produce antibodies (IgA, IgG) to neutralize and eliminate
pathogens and pathogenic epitopes. In this regard, transcription factors STAT (signal transducer and activator of transcription)-1, STAT-4, and T-bet, along with IL-12, IFNγ, are responsible for Th1 response while STAT-6, GATA-3 transcription
factors, and IL-4, IL-5, and IL-13 are associated with Th2 cells response (Delcenserie et al., 2008; Lescheid, 2014). Another
T cell subset (Th17) may be developed which produces proinflammatory cytokines, IL-17. However, it remains critical to
sustain Th1/Th2 balance. The resultant direction rules the disease pathology. In chronic inflammation, it is Th1 cells that
104 SECTION | A Probiotics and Prebiotics
take the lead, though regulatory T cells (T reg) may intervene in the immune response with the liberation of high levels of
IL-10 and moderate levels of transforming growth factor (TGFβ) (Haller et al., 2000; Delcenserie et al., 2008; Lescheid,
2014; Daliri and Lee, 2015). The GALT is a critical regulator in differentiation of pathogenic microbes while preserving a
tolerance toward residential microbiota and food antigens. Several microbial metabolites and structural components also
participate in this regulation.
4.
GUT-PROBIOTICS INTERACTION
The gut-probiotic interaction is of prime importance in laying a foundation for good health. During the neonatal period,
interaction with early microbial colonizers and growing intestinal epithelial cells (IECs) enable the development of mature
GALT. With time, GALT develops tolerance toward gut microbiota at the early phase of life (Delcenserie et al., 2008). It
appears that microbiota undergo clonal selection and achieve endurance, possibly functioning like an organ. In many cell
line-based studies, it was shown that strain dependent probiotic-IEC interactions induce to produce several cytokines that
can modulate the in vitro expression of pro- and antiinflammatory cytokines (Haller et al., 2000; Ruiz et al., 2005; Tien
et al., 2006; Ogawa et al., 2006; Lescheid, 2014; Kang and Im, 2015). Probiotics, such as Lactobacillus sakei, modulates
production of IL-1β, IL-8, TNF-α by Caco-2 cells (Haller et al., 2000). Bifidobacterium lactis Bb12 induces IEC to produce
IL-6 proinflammatory cytokine, a growth factor of B lymphocytes, and supports production of platelets (Ruiz et al., 2005).
In a separate study, Vinderola et al. (2005) showed that coincubation of primary IEC with probiotic Lactobacillus helveticus
R389 and Lactobacillus casei CRL 431 in mice also stimulated IL-6 expression. Zhang et al. (2005) also demonstrated
TNF-α-induced IL-8 production in Caco-2 cells using live and dead Lactobacillus rhamnosus GG (Adams, 2010) (Table 1).
Besides stimulation in cytokines and chemokines production, many probiotic strains can influence phagocytosis
(Arunachalam et al., 2000), enhance NK cell activity (Ogawa et al., 2006), stimulate IgA production (Park et al., 2002),
suppress lymphocyte proliferation (Sturm et al., 2005), induce apoptosis (Carol et al., 2006) and cell-mediated immunity
(de Waard et al., 2003). Arunachalam et al. (2000) demonstrated the increased phagocytosis activity in PBMC (peripheral
blood mononuclear cells) on consumption of B. lactis HN019, and, more recently, by using probiotic Enterococcus faecium
AL41 (Dvorožňáková et al., 2016).
TABLE 1 Cells-Probiotic Interaction and Cytokine Production
Probiotic Strains Used
Cytokine Production
Cell Lines Used
Outcome
References
L. sakei
IL-1β, IL-8, TNF-α
Caco-2
Stimulation
Haller et al. (2000)
L. johnsonii
TGF-β
Caco-2
Stimulation
Haller et al. (2000)
E. coli Nissle 1917
IL-8
HT29/19A
Stimulation
Lammers et al. (2002)
B. lactis Bb12
IL-6
Murine IEC
Stimulation
Ruiz et al. (2005)
L. rhamnosus GG
IL-8
Caco-2
Stimulation
Zhang et al. (2005)
L. casei CRL 431
IL-6
Murine IEC
Activation
Zhang et al. (2005)
L. helveticus R389
IL-6
Murine IEC
Activation
Vinderola et al. (2005)
B. longum
IL-10, IL-12
Murine IEC
IEC activation
Rigby et al. (2005)
L. gasseri and L. johnsoni
IL-12, IL-18
DC
Activation
Mohamadzadeh et al. (2005)
L. casei subsp. casei
IL-15
Caco-2
Activates NK cells
Ogawa et al. (2006)
L. casei DN-114001
CXCL1, CXCL2, CCL20
Caco-2
Attracts macrophages
Tien et al. (2006)
Bifidobacteria
IL-10
Mononuclear
cells and HT29
Antiinflammatory
Imaoka et al. (2008)
L. delbruekii
and L. fermentum
IL-6
IEC
Antiinflammatory
Hegazy and El-Bedewy (2010)
VSL#3
TNF-α, IL-6, and IL-10
Rat model
Antiinflammatory
activity via PI3k/Akt
and NF-κB pathway
Dai et al. (2013)
Probiotics in the Rescue of Gut Inflammation Chapter | 6 105
The important roles thus played by the probiotics are of being immune-modulators. As per reports, probiotics that play
a beneficial role to the host are antiinflammatory. To demonstrate this property and other mechanisms, in vitro studies are
carried out using human intestinal cells (HT-29 and Caco-2). However, an ex-vivo screening method has also been reported
to identify the antiinflammatory property of probiotic strain in question (Kwon et al., 2010). Mohamadzadeh et al. (2005)
showed that some probiotic strains induce DCs to express high levels of IL-12 and IL-18 while Rigby et al. (2005) demonstrated that Bifidobacterium longum can induce murine colonic DCs to produce IL-10 and IL-12. In practice, immune cells
from mesenteric lymph nodes are collected and cocultured with probiotic strains for 72 h, followed by the simultaneous
measurement of IL-10 and IL-12. A promising antiinflammatory strain shows induction of a high level of IL-10 and low
level of IL-12 expression. Some strains are also proinflammatory and express IL-12, IL-1β, and TNF-α (Table 1). The candidate probiotic strains are antiinflammatory and induce tolerance signaling. In one study, a mixture of five probiotic strains
(IRT5: Bifidobacterium bifidum, L. casei, Lactobacillus acidophilus, Lactobacillus reuteri, and Streptococcus thermophilus) could induce the enhanced upregulation of induced regulatory T cell (iTregs, CD4 + Foxp3+) populations in immune
cells (dendritic cells) of mesenteric lymph nodes more than any combination of 1, 2, or 3 strains (Kwon et al., 2010). It is
evident that subtle balance is regulated between Th1 and Th2 cells which governs the resultant immunological response.
5.
DYSBIOSIS IS THE CAUSE OF INFLAMMATION AT GUT
Dysbiosis is the state of alterations in microbial flora in the gut, directly causing several particular inflammatory diseases.
The gut is the locus of intense activity allochthonus microbial flora and it can be changed with the influence of several internal and external factors (Carding et al., 2015). Influences such as infection (Kamada et al., 2013), antibiotic use (Dethlefsen
et al., 2008), diet (de Filippo et al., 2010), disease condition-obese (Ley et al., 2006; Armougom et al., 2009), and age
(Kalliomäki et al., 2008) are the driving factors, directly and indirectly causing dysbiosis and disease. It is an experimentally
established fact that food born viral pathogens in murine modeling altered the gut microflora and induced both local and
systemic inflammation (Kamada et al., 2013). Antibiotic use has a direct relation to the alteration of gut microbiota and is the
major cause of dysbiosis (Hawrelak and Myers, 2004). It causes the overgrowth of existing microflora, C. difficile, causing
antibiotic-associated diarrhea (Boccardo et al., 2004) as well as reduced SCFA production resulting in electrolyte imbalance and diarrhea (Bengmark, 1996). Diet determines the state of microbiota in a human host. Western diet with high fat
and low fiber induces increased growth of gram-negative bacteria with resultant increase concentration of LPS (Cani et al.,
2007); which in turn interact with TLR-4 and initiate the inflammatory cascade (Wright et al., 1990). Diet, either animal or
plant-rich, influences the gut flora. Experimental data show the abundance of bile-tolerant, amino acid metabolizing flora
like Alistips, Bilophila, and Bacteroides among volunteers with animal-rich diet with decreased levels of members of polysaccharides metabolizing Firmicutes (Roseburia, Eubacterium rectale, and Ruminococcus bromii) (David et al., 2014). Diet
rich in polyphenols may alter the composition of gut flora. Red wine polyphenol consumption caused to increase bacterial
members such as Enterococcus, Prevotella, Bifidobacterium, Bacteriodes, and E. rectale in healthy volunteers. Many studies
have been conducted to find a relationship between several autoimmune disorders and dysbiosis using germ-free mice (GF)
(Carding et al., 2015). Turnbaugh et al. (2006) showed that colonic flora from obese mice colonized in GF mice caused an
increase in body weight and fat in comparison to flora colonized from lean mice. Ingested food after digestion leaves many
undigested organic products, along with digestive enzymes, damaged epithelial cells and mucus, and thus becomes the
source of energy for colonic flora on fermentation. This in turn produces a gamut of metabolites which become determinants
in restoring health and/or disease. Alterations of colonic flora (from obese and lean mice) produce different metabolites and
results pathophysiology (Bermon et al., 2015). A large number of metabolites are produced. Among them, SCFA, long chain
fatty acids (LCFA), branched chain fatty acids (BCFA), CLA, conjugated linolenic acid (CLNA), amines, vitamins, phenolic
compounds, flavonoids, and with metabolites xenobiotic properties are very important (Hawrelak and Myers, 2004; Festi
et al., 2014; Carding et al., 2015). Increased amount of SCFA (propionate) production was found evident in obesity, due to
alteration of gut microbiota (Schwiertz et al., 2010). Diet intake influences the composition of gut flora and health conditions. Azoxymethane-induced mice undergo dysbiosis and develop colon cancer. However, intervention of our probiotic
P. ­pentosaceus GS4 reinstates the colonic flora and enhances the metabolite CLA production to mitigate carcinogenesis
(Dubey et al., 2015, 2016). Alteration in gut microbiota composition was evident among Type2 diabetic subjects where
member bacteria, Bacteriodes and Prevotella, were in higher amounts than Firmicutes and Clostridia (Larsen et al., 2010).
6.
DYSBIOSIS AND INFLAMMATORY DISEASES
Currently a large number of diseases are associated with dysbiosis. Alteration in microbial composition sets the foundation
of pathophysiological changes in humans. The condition which governs the abundance of Firmicutes (Escherichia coli)
106 SECTION | A Probiotics and Prebiotics
and Bacteriodes (Bacterioides fragilis) also rules the availability of abundant lipopolysaccharides (LPS) and other cellularstructural fragments like lipid A, peptidoglycans (PG), which are part of PAMPs. Abundance of gram negative bacteria
and LPS showed a link to a high fat diet, high rate of bile acid secretion resulting damage to the gut barrier, leakage and
LPS-mediated endotoxicity followed by inflammation of the gut (Cani et al., 2008). The hypothesis was validated experimentally in a mouse model inducing type 2 diabetes and obesity (Hakansson and Molin, 2011). The interaction of LPS with
colonic macrophages (CD14) and TLR 4 triggers the release of proinflammatory cytokines like IL-6, IL-1, and TNF-α.
Additionally, cellular fragments of gram positive bacteria like lipoteichoic acid (LTA) can bind to CD14 mediated TLR2,
and induce IEC to release proinflammatory cytokines but reduced IL-12 and IFNγ (Hermann et al., 2002).
Inflammation at the gut refers to diseases like IBD which includes UC and CD. It is characterized by chronic relapsing inflammation afflicting the intestinal mucosa (Hawrelak and Myers, 2004). UC is mostly restricted to the colon and
rectum while CD may happen in any part of the alimentary canal; however severity is observed in the terminal ileum. Both
are different diseases and have separate presentations and pathophysiology. These two constitute IBD, which is majorly a
result of dysbiosis (Mukhopadhyay et al., 2012) and breach of the intestinal barrier (Maloy and Powrie, 2011). In particular,
transmembranous tight junction proteins, claudin 2, is upregulated while claudin 5 and claudin 8 are downregulated, resulting in breach of gut epithelia. Along with claudins, dysregulations of defensins are also involved in the process of dysbiosis
(Peterson et al., 2014). Alteration in microbiota like decrease in members of Firmicutes and corresponding increase in
Bacteroidetes and Enterobacterioceae are evidence of IBD (Hansen et al., 2010). In a separate study, it was shown that
T-bet deficient transgenic mice had an altered microbiota. Transcription factor T-bet deficiency causes the development of
colitogenic mice and fails to develop Th1. Interestingly, microbiota of these mice also induced inflammation in recipient
wild mice, proving that dysbiosis is directly linked with inflammation at gut (Garrett et al., 2007).
Besides, IBS (irritable bowel syndrome) (Gophna et al., 2006), GERD (gastroesophageal reflux disease) (Frank et al.,
2007), celiac disease (Nadal et al., 2007), SIBO (small intestinal bacterial overgrowth), C. difficile-associated disease
(Lozupone et al., 2012), atopy and asthma (de los Angeles et al., 2007; Wagner et al., 2008; Mikami et al., 2009), autism,
schizophrenia (Lyte, 2014), autoimmune disorders (Vaahtovuo et al., 2008; Roesch et al., 2009), and colon cancer (Scanlan
et al., 2008; Dubey and Ghosh, 2013; Dubey et al., 2016) are also in association with dysbiosis. Dysbiosis leads to an increased risk of neoplastic transformation which is found to be directly related to chronic inflammation of the gut and the
development of colon cancer (Greer and O’Keefe, 2011). It can be metaphorically described as “holes in a pot of water”.
Aside from these, several other lifestyle diseases are directly or indirectly involved in developing obesity: T1D, T2D, hypertension, and cardiovascular diseases respectively (Backhed et al., 2007; Olefsky and Glass, 2010; Cani and Delzenne,
2011). The imbalance in the constitution of flora leads to leaky gut formation due to disruption of the gut barrier, damage in
tight junctions and inflow of foreign antigens, proteins (non self) and formation of antibodies (Hänsch, 2012). Many foreign
proteins are homologous to self-proteins (molecular mimicry) of the thyroid and pancreas, which result attacks on its own
protein due to molecular mimicry by formed antibodies, a reaction that leads to autoimmune diseases such as Hashimoto’s
thyroiditis and T1D (type1diabetes) (Wen et al., 2008).
7. WHAT MAKES PROBIOTIC SPECIAL FOR REDUCING INFLAMMATION IN THE GUT?
Probiotics dispense beneficial effects to host to reinstate the intestinal disturbances stemming from changes in gut flora
(dysbiosis) and the release of various metabolites. The potential for this is strain specific. In general, it is not only the live
strains, but also the dead cellular components of probiotic strains which modulate the health (Adams, 2010). Several surface
active compounds like surface layer protein (SLP), lipoteichoic acid (LTA), lipopolysaccharide (LPS), heat killed antigen
(HKA)(Adams, 2010); molecular metabolites like bacteriocin, vitamins, short chain fatty acids (SCFA), long chain fatty
acids (LCFAs)(Mishra et al., 2016), production of antihistamine (Dev et al., 2008), γ-aminobutyric acid (GABA) (Selhub
et al., 2014), citrullination (Chirivi et al., 2013; Daliri and Lee, 2015); biohydrogenation property like to produce conjugated linoleic acid (CLA) (Dubey et al., 2012) or conjugated linolenic acid (CLNA) with properties like antioxidant (Gowri
and Ghosh, 2013), cholesterol assimilation (Daliri and Lee, 2015); production of β-galactosidase; immunomodulation
(Bermon et al., 2015); pathogen suppression (Adams, 2010), antagonism (competition for food and space) and many other
components, give strains probiotic potential (Fig. 2). Probiotics either heat-killed Enterococcus faecalis FK-23 or caused
dead Bifidobacteria-induced immune response in animal studies (Adams, 2010). EC-12, a commercially available dietary
probiotic formula composed of heat-killed E. faecalis could stimulate GALT and combat vancomycin-resistant enterococci
in chick models (Sakai et al., 2006). Likewise, live and dead cells of gram-positive probiotic strains (L. casei, L. helveticus)
were able to stimulate IL-6 production on intervention in murine intestinal epithelial cells (Vinderola et al., 2005). It has also
been demonstrated that heat-killed, whole cell lysate, and SLP preparations of the probiotic strain GS4 stimulated the adaptive immune system and produced likely antibodies (Ghosh and Dubey, 2014). In a separate study, Matsuguchi et al. (2003)
Probiotics in the Rescue of Gut Inflammation Chapter | 6 107
FIG. 2 Metabolic and structural components of a probiotic candidate in the regulation of inflammation. CLA-conjugated linoleic acid is a biohydrogenated metabolite and natural ligand for PPAR gamma (peroxisome proliferator-activated receptor)γ; short chain fatty acid (SCFA) like butyrate
modulate gene expression; surface components like surface layer protein (slp) helps in adherenceaas well as in histone deacetylation inhibition which
suppress the pro-inflammatory cytokines and lipoteichoic acid (LTA) can activate toll-like receptors.
showed that a preparation of six-heat killed Lactobacillus strains were stimulated to secret proinflammatory TNF-α in
mouse spleenocytes with markedly different stimulations among strains (Matsuguchi et al., 2003). Growth of fish cell line
(SAF-1) cultivated in the presence of cytoplasmic extracts of probiotic strains (L.delbrueckii subsp. lactis) was inhibited,
indicating induction of apoptosis (Salinas et al., 2008). Viable cytoplasmic extracts or/and nonviable probiotic strains were
demonstrated to induce immune response and maintain homeostasis.
8.
HOW DO PROBIOTICS REGULATE INFLAMMATION?
Probiotics regulate inflammation in a host by several mechanisms following different pathways. The primary goal of probiotics is to enhance epithelial barrier function (Madsen, 2012). Some probiotic strains can stimulate intestinal epithelial cell
(IEC) signaling pathways such as (1) inhibition of NF-kB activity, (2) alteration of MAPK and ERK pathways, (3) activation of PI3K and Akt, and (4) PPARγ dependent pathway, respectively. Some probiotic strains participate in proteasome
function (Thomas and Versalovic, 2010), others in expression of cytoskeleton anchoring proteins (Anderson et al., 2010),
some can produce cytoprotective heat shock proteins to fortify gut barrier function, and other strains initiate TLRs-PAMPs
interactions to influence activation of DCs and Th1 (Daliri and Lee, 2015).
SCFA is the result of probiotic metabolism of resistant starch and undigested polysaccharides in the distal intestine which
humans have difficulty digesting, in order to harvest energy for colonic cell health. SCFA butyrate is very important as it
has antiinflammatory and anti-cancer activity (Greer and O’Keefe, 2011). Additionally, it inhibits angiogenesis, induces
apoptosis, regulates transcriptional upregulation to detoxify enzymes, and activates glutathione-S-transferase (Scharlau
et al., 2009). SCFA (propionate and acetate) are natural ligands for GPR (G-protein-coupled receptor) 41 and GPR43, respectively, which are widely expressed in the distal intestine, colon, and adipocytes (Xiong et al., 2004; Maslowski et al.,
2009; Ang and Ding, 2016). These signaling molecules bind to respective receptors and regulate inflammatory pathways by
decreasing the inflammatory response. SCFA also participates in antiinflammatory mechanisms by modulating intracellular
levels of calcium in neutrophils; inducing immune cells in inhibiting the expression of adhesion molecules, and chemokine
production which in turn suppressed the recruitment of macrophages and neutrophils (Bermon et al., 2015). Again, SCFA
108 SECTION | A Probiotics and Prebiotics
(butyrate) acts as an inhibitor of histone deacetylases (HDI) and therefore can affect gene expression, arrest growth, induce
antiinflammation and apoptosis (Carding et al., 2015).
PSA (polysaccharide A) is elicited by some probiotic strains of human origin, like B. fragilis, and has the potential to
induce IL-10 expression from regulatory Foxp3 + CD4 + T cells (van Baarlen et al., 2013). The strain GS4 (P. pentosaceus)
has the ability to biohydrogenate linoleic acid (LA) to CLA. Besides substrate LA, it can utilize sesame oil to produce CLA
(Dubey et al., 2012, 2015). Furthermore, the strain was able to modulate the dysbiosis afflicted in mouse models due to
azoxy-methane induction, and participated in reinstating the gut microbiota (Dubey et al., 2015). Experimental evidence
showed that GS4 could also mitigate the inflammation, regulate transcriptional up-regulation to detoxify enzymes and
induce apoptosis (Dubey et al., 2015, 2016). CLA was determined to be a potential probiotic compound for controlling
inflammation of the gut. CLA, it being a natural ligand for PPARγ, arrests the NF-kB pathway and thus stops or modulates
inflammation (unpublished data). Bassaganya-Riera et al. (2012) demonstrated that probiotic bacteria with CLA producing
potential suppressed DSS-induced colitis targeting macrophage PPARγ.
Probiotic strain L. salivarius LS33 has peptidoglycan that was determined to be protective against colitis in mouse models via NLR-peptidoglycan interaction (Fernandez et al., 2011). Strain specific LTA and cell wall bound LTA are also part
of MAMPs which mediated immune response via MAMPs-PRRs interaction (van Baarlen et al., 2013). Surface layer proteins (SLP) of probiotic strains offer an additive property for inhibition of pathogenic strains for adherence to enterocytes
and nutritional translocation. The SLP-minus strains of L. helveticus fb213, L. acidophilus fb116, L. acidophilus fb214
showed a decreased degree of cell adherence to HT29 (Meng et al., 2014). The P. pentosaceus GS4 strain also possesses
SLP of 98 kDa and shows adherence ability to HCT 116. Removal of SLP shows much reduced level of adherence (Dubey
and Ghosh, 2011; Ghosh and Dubey, 2014). It therefore has important role to play in the cell biology and immunology
(unpublished data). Similarly, strain specific potential like pilin, of L. rhamnosus GG (LGG), binds well with mucosa and
modulated IL-8 induction in Caco2 cells. The strain has potential to elicit protein molecules, like p75 and p40, which has
demonstrated protection against colitis in mouse model (van Baarlen et al., 2013).
Probiotics regulate the immunity and inflammatory genes in the gut (Plaza-Diaz et al., 2014). Probiotics and commensals interact with immune cells in the gut, including IEC, M cells, dendritic cells (DC), macrophages, T and B cells,
respectively. Experimental evidence proves that strain specific probiotic has potential to reduce the inflammation of the gut.
In particular, the influence of probiotics leads to antiinflammatory response in cultured cell lines (HT29, HCT116, Caco-2)
by the gene expression of mucin, TLRs, NF-kB, interleukins, and caspases. Surface active antigens also induced downregulation of proinflammatory and upregulation of antiinflammatory genes in vitro experimental conditions (Plaza-Diaz
et al., 2014). Protective and beneficial roles played by probiotics, with the modulation of immunity and inflammatory gene
expression, have been extensively reviewed elsewhere (van Baarlen et al., 2013; Plaza-Diaz et al., 2014). Beyond targeted
metabolites, surface active compounds and genes, recent research reveals many more untapped metabolites of commensals
and probiotics are also participating to impact the health status of a host (Pirhaji et al., 2016).
9.
9.1
USE OF PROBIOTICS AND CONSEQUENCES
Inflammatory Diseases
Irritable bowel syndrome: Irritable Bowel Syndrome (IBS) is the most common functional gastrointestinal disorder with
a reported prevalence in the general population between 6% and 46% (Wikipedia, https://en.wikipedia.org/wiki/Irritable_
bowel_syndrome#Epidemiology). IBS is characterized by a collection of functional gastrointestinal symptoms such abdominal pain, defecatory frequency, and/or constipation. The etiology of IBS is still not clear and numerous factors are
involved in the damage to the mucosa, including microorganisms, psychological factors, and dietary habits (Chey et al.,
2015). In addition, the gut-associated immune system is upregulated as evidenced by increased inflammatory cytokines
such as IL-1, IL-6, and IL-10 (Strober and Fuss, 2011). The upregulated gastrointestinal associated immune tissue is known
to stimulate discharge of enterochromaffin cells and other cells, which release serotonin and/or histamine resulting in GI
symptoms. The type of colonizing microflora may play an important role in regulating immunity (Swidsinski et al., 2005).
IBS patients host an intestinal microflora containing few Lactobacilli and a lowered Bifidobacteria fecal concentration. In
normal conditions, an immunologic tolerance is maintained with respect to the commensal enteric bacteria, which prevents
intestinal inflammation. This controlled homeostatic response is lost in susceptible individuals who then develop a chronic
aggressive cellular immune response at the intestinal level. Oral administration of LGG in patients with Crohn’s disease
resulted in the promotion of intestinal IgA immune response, as well as reduction in the pain and severity in many inpatients
(Lescheid, 2014). The positive role of probiotics in IBS therapy has been established, but the beneficial effect of bacterial
supplementation as an adjunct to treatment is an emerging trend (Swidsinski et al., 2005; Wang et al., 2007) (Table 2).
Probiotics in the Rescue of Gut Inflammation Chapter | 6 109
TABLE 2 Some Examples of Probiotics Used in the Alleviation of Inflammation at Gut
Inflammatory
Diseases
Irritable bowel
syndrome
Ulcerative
colitis
Probiotics Used
Details
Outcome
References
B. infantis 35624
Clinical trials
Remission
Brenner and Chey (2009)
E. coli DSM17252
Clinical trials
Remission
Enck et al. (2009)
Bifidobacterium + Lactobacillus
Clinical trials
Remission
Shadnoush et al. (2013)
VSL#3
Murine model
Remission
Distrutti et al. (2013)
L. acidophilus
Abdin and Saeid (2008)
B. infantis 35624
Clinical trials
Remission
Groeger et al. (2013)
L. rhamnosus OLL2838
Regulates zonula
cccludens-1 and
myosin genes
Remission
Miyauchi et al. (2009)
L. plantarum Lp91
Murine model
Remission
Duary et al. (2012)
VSL#3
Murine model
Remission
Bassaganya-Riera et al. (2012)
Crohn’s Disease
Bifidobacteria, Lactobacilli
Clinical trials
Remission
Fujimori et al. (2007)
Colon cancer
Lactic acid bacteria
Cell lines
Antiproliferation of
colon cancer cells
Thirabunyanon et al. (2009)
VSL#3
Murine model
Remission
Bassaganya-Riera et al. (2012)
Pediococcus pentosaceus GS4
HT-29; Caco-2
and Murine model
Remission and
Antiproliferation of
colon cancer cells
Dubey et al. (2015, 2016)
Irritable bowel diseases (IBD): Probiotics used to treat patients suffering from IBD (LP9-Brenner and Chey, 2009).
However, only two probiotic formulae, VSL#3 and E. coli strain Nissle 1917, are in use with support from experimental
evidence through multiple clinical trials (Hormannsperger and Haller, 2010). VSL#3 is a mixture of eight probiotic strains
(L. acidophilus, Lactobacillus bulgaricus, L. casei, Lactobacillus plantarum, Bifidobacterium breve, Bifidobacterium infantis, B. longum, and S. thermophilus), however, convincing probiotic potential has yet to be established though a large
number experimental and clinical studies (Hormannsperger and Haller, 2010). E. coli Nissle 1917 showed defense against
EHEC O157 infection (Zyrek et al., 2007) and reduced DSS-induced colitis in gnotobiotic mice (Ukena et al., 2007).
However, no proper mechanism was demonstrated in either case. Involvement of cell wall component, LPS-TLR2, was
explained to demonstrate apoptosis in IECs with the liberation of heat shock proteins (hsp 25 and hsp 70) (Hormannsperger
and Haller, 2010) (Table 2).
Diabetes and obesity: A large number of research based reports indicate a distinct link between diabetes and obesity
(Festi et al., 2014). These pathogenecities are the outcome of metabolic disorder due to dysbiosis (Turnbaugh et al., 2006).
Fat enriched diet with increased lipoproteins like chylomicron, low density lipoprotein (LDL), very low density lipoprotein (VLDL), and intermediate densitylipoprotein (IDL) modifies the intestinal microbiota and cause metabolic disorders
which initiate inflammation, insulin resistance and type II diabetes (Amar et al., 2011). These lipoproteins enable transport
of cholesterol and triglycerides within the blood stream. Absorption of cholesterol from the intestine has been reduced
by improving the intestinal microflora via administration of probiotics (Cani et al., 2007; Huey-Shi et al., 2009; Round
and Mazmanian, 2009; Larsen et al., 2010). Amar et al. (2011) illustrated in vivo animal modeling and showed that TNFα
continuously released in the adipose tissue during obesity, to activate protein kinase C and to increase the phosphorylation
of the insulin receptor substrate on serine residue such as ser-307, leading to inactivation insulin signaling molecule and
hence, to insulin resistance. Efficacy of probiotics in reducing serum cholesterol levels demonstrated in vivo models which
subsequently improved insulin resistance.
Colon cancer: Colorectal cancer is one of the leading causes of cancer morbidity and mortality in many countries
(Dubey and Ghosh, 2012) and it is thought that chronic inflammation (Dubey and Ghosh, 2013) is caused by an interaction between dietary factors and genetic predisposition (Klein et al., 2013; Festi et al., 2014). Epidemiological studies have
110 SECTION | A Probiotics and Prebiotics
shown that consumption of fermented milk products containing probiotic bacteria help to reduce the risk of cancer at a
number of sites. L. acidophilus, L. casei Shirota strain and LGG have been shown to have inhibitory properties on chemically induced tumors in animals (Festi et al., 2014) (Table 2). There is some evidence that probiotic P. pentosaceus GS4
can interfere with various stages of the azoxymethane induced colon cancer process in experimental mouse models, such as
degenerative effects on secondary organs like liver, kidney, and intestine (Dubey et al., 2015),with the prevention of DNA
damage in the colon by live bacteria, suppression of preneoplastic changes in the colon and suppression of colon tumors
in animals (Dubey et al., 2016). Preliminary studies on the effect of probiotic consumption in the control of cancer show
promise.
Rheumatoid arthritis: Probiotics are now in use to control rheumatoid arthritis. Remission of symptoms of RA has
been demonstrated in mouse models using Lactobacillus salivarius, possibly resulting in the decrease of IL-10, TNF-α and
an increase in TGF-β levels (Sheil et al., 2004). In a randomized double-blind, placebo controlled clinical trial, Alipour
et al. (2014) showed L. casei to be effective as a supplement in the treatment of 22 patients with rheumatoid arthritis who
received daily 1 capsule containing 108 of colony forming units for 8 weeks. This treatment brought down high sensitivity
C-reactive protein, tender and swollen joints and inflammatory cytokines among treated patients. A similar experiment
was carried out by de los Angeles et al. (2011) using L. rhamnosus GR-1 and L. reureri RC-14 to treat 15 RA patients.
Patients received a capsule daily for 3 months. Results revealed the impressive effect of probiotic use in the alleviation of
RA. The health assessment scores were more improved than the 14 patients who received a placebo. However, there is little
unanimity in the use of probiotics in the treatment of RA and although they are regarded as safe; may pose a threat to the
health of immunocompromised patients. A review of various studies reveals that Lactobacillus GG is the most suitable and
safe probiotic as it has undergone the highest number of extensive safety evaluation experiments, both in vitro and in vivo
(Snydman, 2008).
HIV: Results of the “Probio-HIV” clinical trial published in PLOS One (d’Ettorre et al., 2015) reveals the fact that probiotic intervention reduces inflammatory consequences by improving GI tract immunity among HIV-afflicted individuals.
Patients received combined antiretroviral therapy with probiotic supplements. Probiotic intervention clearly demonstrated
a statistically significant reduction in the activation of CD4+ T lymphocytes with a reduction in the expression of marker
proteins such as lipopolysaccharide binding protein and high sensitivity-C reactive protein (d’Ettorre et al., 2015).
Mental health: Some probiotic strains (L. helveticus and B. longum) have the potential to produce serotonin and influence human mental health (Maynard et al., 2012; Lyte, 2014). Resistance to insulin leads to T2D, and also causes an
increased risk of depression epidemiologically (Nicolau and Masmiquel, 2013). Psychological stress may increase the
permeability of the gut barrier (Selhub et al., 2014). A clinical trial demonstrated that oral probiotic intervention reduced
anxiety, mental stress, and improved positive attitude (Bested et al., 2013).
Besides the implementation of probiotics in certain diseases to mitigate the inflammation, the used of probiotic functionalities may be credited with the following activities beneficial to health:
9.2
Suppression of Histamine Signaling
Some probiotics have the ability to suppress histamine signaling, and are therefore important in the treatment of allergic
diseases (Dev et al., 2008). Different strains of Bifidobacterium spp. (B. infantis and B. longum) showed efficacy in the
treatment of nasal allergy in induced animal models. The experiment demonstrated the suppression of allergy biomarkers
(histamine H1 receptor, histidine decarboxylase (HDC) mRNA expression, HDC activity, and histamine content) with the
dosage of 40 mg/rat for a period of 4 weeks.
9.3
Reduction of Appetite and Glucose Uptake
Falcinelli et al. (2015) reported that probiotic treatment reduces appetite and glucose levels to control obesity and T1D.
Appetite is controlled by many compounds including hormone leptin, and is produced mainly by adipose tissues. An experiment exposed zebrafish larvae to L. rhamnosus for 8 days. Results demonstrated a modulation in the composition of gut
microbiota, with a direct relationship to upregulation of genes related to reduction of glucose uptake and appetite (Falcinelli
et al., 2015).
9.4
Repair of Damaged Epithelial Barrier
The most important anti-inflammatory property offered by several probiotics is their potential in maintaining the intactness of the gut-epithelial barrier (Rao and Samak, 2013). A number of studies, including animal models and clinical
Probiotics in the Rescue of Gut Inflammation Chapter | 6 111
t­rials, demonstrated the damaged epithelial repairing roles of probiotics under diseased conditions (Khailova et al., 2013;
Sindhu et al., 2014; Dubey et al., 2015). In all these experiments, major probiotic species from the genus Lactobacillus
(L. rhamnosus GG, L. plantarum DSM 9834, L. reuteri R2LC, L. paracasei NCC2461), P. pentosaceus GS4, and B. ­infantis,
Saccharomyces boulardii, Saccharomyces cerevisiae UFMG 905, and E.coli Nissle 1917 were used (Lescheid, 2014).
9.5 Antimicrobial Peptides and Antagonism
Similar to restoring of the damaged gut-epithelial barrier, production of antimicrobial peptides (AMP) such as bacteriocins,
nicin (Thomas and Versalovic, 2010) helps to foster innate immunity (Lescheid, 2014) to antagonizing pathogenic strains
(Gowri and Ghosh, 2010a). Some probiotic strains (VSL#3) induce host epithelial cells to release AMP like β-defensin-2
involving NF-kB, AP-1, and MAPK pathways (Schlee et al., 2008).
10.
CONCLUSION
Disruption in the composition of microbiota has been found to be a key factor in the development of diseases in a host,
causing local inflammation. Probiotics, either live or dead, have gained convincing experimental results to modulate the
microbial composition of the gut to restore lost flora diversity and to mitigate inflammation and consequences (Lebeer
et al., 2010). One of the newly evolved therapeutic approaches against different inflammatory diseases is probiotic microflora-mediated therapy. Probiotics are an important component of functional foods. They can modulate gut microflora and
maintain homeostasis in the GI tract; and contribute in normalization of intestinal colonization. Probiotics are beneficial
whether used as single or with multiple strains however, beneficial effects generally depend on specific strains.
Probiotics collectively demonstrate potential to mitigate or eliminate inflammation in the gut by interacting with IECs,
expressing related genes and augmenting GALT. Probiotics, both alive and dead, exert beneficial effects to host. This is
an important feature due to the fact that live probiotics faces several pharmacological difficulties. One study shows that
removal of LTA from potential probiotics may enhance the antiinflammatory activity (Lebeer et al., 2010). Therefore, genetically modified probiotics (GMP) may be used to control inflammation of the gut in the future. Chronic inflammation
causes disturbance to normal behavior and mental health. Besides reducing the inflammation of the gut, probiotics will also
offer solutions in managing psychological well-being with the release of chemical neurotransmitters (Lyte, 2014).
The total inflammatory network developed due to dysbiosis is the root cause of several syndromes and diseases. Again,
dysbiosis is the result of alterations of microbiome and is influenced by both internal and external conditions. Results obtained from several clinical trials provide confidence for the use of probiotics as a living drug for the alleviation of inflammation in the gut. The potential use of probiotics, with prebiotics, is so expansive that they will become a panacea for many
diseases in near future.
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Chapter 7
Probiotics as an Adjunct to Conventional
Treatment in Vulvovaginitis: Past, Present,
and Future
Princy L. Palatty⁎, Poornima R. Bhat⁎, Ramakrishna P. Jekrabettu⁎, Thomas George⁎, Sueallen D’souza⁎, Soniya
Abraham⁎, Mohammed Adnan⁎, Michael Pais⁎, Taresh Naik⁎, Devika Gunasheela†, Manjeshwar S. Baliga⁎
⁎
Father Muller Medical College, Mangalore, India, †Gunasheela Infertility Hospital, Bangalore, India
1.
INTRODUCTION
Women’s health consists of physical, psychological, and social challenges which are considerably different from those
of men of the same age group. There are various factors affecting women’s health through out their lives such as genetic
susceptibility to illness and disease, varying hormone levels, environmental exposures, physiological variations with time,
gender specific social, and other conditions (Senie, 2015). Of all ailments bothering women, the urogenital health is one
of the most neglected aspects in traditional communities. This innocuous disease affects more than one billion women
worldwide annually and many patients suffering from these ailments have scant resources or knowledge for maintaining
their health. In addition to this, urogenital health may have effects on child birth and quality of life. The VV has a negative
impact on woman’s personal confidence and self-worth by causing psychosexual problems like dysparenunia, a sense of
shame, and unworthiness in informing either doctors or the partner (Mastromarino et al., 2013). In lieu of these observations, reproductive tract infections have become a major concern in public health worldwide and studies aim to remedy
them (Reid et al., 2001a,b; WHO, 2003).
2. ANATOMY OF FEMALE GENITAL SYSTEM
The female genital system can be divided into External Genital Organs and Internal Genital Organs (Dutta and Konar,
2014). The external genitalia is also known as vulva or pudendum, and is externally visible. They consist of mons veneris
(mons pubis), labia minora and majora, hymen, clitoris, vestibule, urethra, Skene glands, greater vestibular (Bartholin)
glands, vestibular bulbs, and conventional perineum (Dutta and Konar, 2014). Internal genitalia include vagina, cervix,
uterus, fallopian tubes, and ovaries. They are placed internally in the body and special instruments are needed for examination (Dutta and Konar, 2014).
2.1
External Genitalia
Mons Pubis: It is a pad of subcutaneous adipose connective tissue which is anterio superior to pubic symphysis. This area
will be covered by hair in adults (Dutta and Konar, 2014).
Labia majora: Vulva on either side is bound by two longitudinal folds or elevations composed of subcutaneous adipose
tissue and fat called Labia majora. The size is highly variable in individuals. On the surface they are made up of skin consisting of squamous epithelium, hair follicles, sebaceous glands, and sweat glands. Beneath the skin they contain adipose
tissue and connective tissue with rich venous plexus. They are homologous to the scrotum in males. They begin anteriorly
from the mons and meet medially in front of the anus to form Posterior Commisure. The inner side of labia majora lacks
hair follicles (Dutta and Konar, 2014).
Labia minora: On the either side within the labia majora, two folds of thick skin, devoid of hair follicles are present
called labia minora or nymphae. They are made up of skin, connective tissues, sebaceous glands, erectile muscle fibers,
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nerve endings, and blood vessels. They are exposed only when the labia majora are separated, though this is not the case
with parous women. In the upper part, the clitoris is enclosed by the labia minora in front and behind to form the prepuce
and frenulum, respectively. The labia minora’s lower part fuses to the skin to form the fourchette which is a fold of skin in
the midline. The fossa navicularis is present between the fourchette and vaginal orifice. The labia minora are similar to the
ventral aspect of the penis in males (Dutta and Konar, 2014; Miranda, 2015).
Clitoris: It is a highly erectile organ located below the anterior joining of labia minora. It is homologous to the
penis in males. It is different from penis in that the urethra in females is completely separate from clitoris (Dutta and
Konar, 2014).
Vestibule: It is a triangular space bounded between clitoris, fourchette, and labia minora. It consists of four orifices.
Urethral meatus: It is made up of membranous connective tissue, connecting bladder and vestibule. It is located in the
midline about 1 cm anterior to the vaginal orifice and 1–1.5 cm below the pubic arch. It gives rise to paraurethral or Skene’s
glands, opening bilaterally (Dutta and Konar, 2014; Miranda, 2015).
Skene glands: They secrete lubrication to urethra, opening bilaterally into the posterior wall of urethral opening or
vestibule directly.
Vaginal orifice: It is a posteriorly located space in the vestibule, differing in size. In nulliparous women it may be
bounded by labia minora on either side, and covered by hymen. In parous women it may be exposed (Dutta and Konar,
2014; Berek, 2007; Miranda, 2015).
Hymen: Vaginal orifice is covered by a septum of mucus membrane called hymen, which varies in shape greatly. It will
be usually cresentric or circular in virgins and is ruptured during sexual intercourse. During child birth it gets extremely
lacerated to form cicaterized nodules which are of different sizes, forming carunculae myrtiformis on either side of the
vaginal orifice (Dutta and Konar, 2014).
Bartholins glands: The greater vestibular or Bartholin’s gland are responsible for lubricating the vagina by their alkaline secretion during sexual excitement. They are located on the posterior part of vestibular bulb in the superficial perineal
pouch and open on either side of the posterior part of the vestibule above the hymen (Dutta and Konar, 2014).
Vestibular bulbs: Beneath the mucous membrane of the vestibule, elongated erectile tissues are located bilaterally called
vestibular bulbs. They are incorporated into the bulbocavernosus muscle on either side of the vaginal orifice in front of the
bartholins gland (Dutta and Konar, 2014).
Perineum: It forms the pelvis floor and is formed by muscles and fascia (Berek, 2007).
2.2
Internal Genitalia
(A) Vagina: It is a hollow fibromuscular tube connecting the internal uterine cavity with the exterior vulva. It is located
within the pelvis posterior, extending to the bladder and anterior to rectum. In a dorsal lithotomy position, the axis of the
vagina is almost pointing toward the sacrum, whereas in an upright standing position it is horizontal. It forms an excretory
channel for uterine secretions, namely menstrual blood. It serves as organ for copulation and as the birth canal during labor.
In an erect position, the canal is directed upward and backward forming 45 degree angle with the horizontal plain. The long
axis lies almost parallel to the pelvic inlet and at a 90 degree angle to uterus. The diameter of the canal is approximately
2.5 cm, being narrowest at the introitus and widest at the upper part; it has good distensible properties (Dutta and Konar,
2014; Berek, 2007; Miranda, 2015).
The vagina has four walls: anterior, posterior, and two lateral walls. The anterior and posterior walls are widely spaced,
whereas the lateral walls are stiffer, especially in the center, thus forming an “H-”shaped structure in the transverse section.
The space between the vaginal walls and impending uterine cervix forms fornices. The vaginal walls become blended with
cervix, and the two are inseparable after the fornices. There are four fornix, one anterior, one posterior, and two lateral
fornices. The anterior fornix is narrowest, the upper third is related to the bladder and the lower two-thirds relate to the
urethra. The posterior fornices are deepest, related to the pouch of Douglas in the upper third, rectum in middle, and the
anal canal and perineal body in the lower third. The lateral fornices are related to pelvic cellular tissue, ureter, and uterine
artery in the upper third; levator ani in the middle; and bulbocavernosus muscle, vestibular bulbs, and bartholins glands in
the lower third (Soccol et al., 2010; Senok et al., 2009; Dovnik et al., 2015; Mastromarino et al., 2013; Dutta and Konar,
2014). On the microscopic level, the vagina is composed of three layers (Dutta and Konar, 2014; Berek, 2007; Miranda,
2015; Magowan et al., 2009).
(a) Mucosa: Non stratified keratinised sqamous epithelium without glands. The mucosa has a pattern of transverse
ridges called rugae. Lubrication mainly occurs by secretion of the bartholins gland and cervix by transudation. Mucosa is
hormone sensitive. Estrogen leads to proliferation and maturation of cells. Mixed bacterial flora, with a predominance of
lactobacilli, colonize the vaginal mucosa. Normal pH is slightly acidic 3.5–4.
Probiotics as an Adjunct to Conventional Treatment in Vulvovaginitis: Past, Present, and Future Chapter | 7 119
(b) Muscularis: It consists of connective and smooth muscles packed loosely. They have two layers: the outer longitudinal muscle layer and inner circular muscle layer.
(c) Adventitia: It is an endopelvic fascia which gets attached to muscularis.
(B) Uterus: It is a hollow pyriform fibromuscular organ divided into the upper uterine body or corpus, middle isthmus,
and lower cervix. It lies between the bladder and rectum, in the pelvic cavity.
(a) Uterine body: It consists of a globe-shaped fundus lying above the opening of the fallopian tubes, the body proper
which is a triangular area and the cornua of uterus which lies superio lateral to the body and is attached to the uterine tube,
round ligament, and the ligament of the ovary. Two lateral tube-like extensions of the uterus, one on either side, are called
fallopian tubes; they terminate into the body with finger like projections called fimbria. The ovaries are located nearby, on
either side of the fimbria.
(b) Isthmus: This is a constricted portion between the body and cervix. It is connected to anatomical internal os above
and histological internal os below.
(c) Cervix: It is the lowest or bottommost portion of uterus, separating the body of uterus above, from the vagina below. It is
cylindrical in shape and measures 2.5 cm in length and diameter. It extends from histological internal os to external os opening
into the vagina. It consists of vaginal and supra vaginal parts. In nulliparous women, the external os looks conical whereas in parous women, it has a slit forming anterior and posterior lip. The body of the cervix is made up of outer serous perimetrium, middle
thick bundles of musclular layers and connective tissue called myometrium, and an inner mucosal layer called endometrium.
Microscopically, mucosa contains endocervical glands. The transformation zone between vagina and cervix called the portio
vaginalis is sensitive to hormonal effects and is constantly irritated due to hormones, infections, and trauma. As a result, the portio
vaginalis is more prone to in situ carcinoma. Mucosa of the cervix has endocervical glands producing limited watery secretions.
These secretions are alkaline, with a pH of 7.8, and highly hormone sensitive. Under the influence of estrogen, they provide nutrition and facilitate the ascent of sperm, whereas under influence of progesterone, they prevent their entry. Cervical mucus forms the
bulk of vaginal discharge, sometimes resulting in mucus plugs which block the cervical canal. They have bacteriolytic property.
3.
NORMAL FLORA OF THE VAGINA
Microbiological flora of female genital tract is complex, dynamic, and not well understood. Efforts are being made to isolate the microbiome of the genital tract for many years (Lamont et al., 2011). The normal flora content of the female genital
tract is dependent on various factors such as age, hormones, and pH in the genital tract of the host, to name a few (Davis,
1996). In newborns, the genital mucosa is sterile; they acquire commensals which are varied flora of nonpathogenic organisms from skin, genitalia, and the intestine. Then, under the influence of maternal estrogen, there will be glycogen deposition in vaginal cells and various bacilli may be seen (predominantly Lactobacilli or Deoderlin’) for the first month, similar
to adult commensal flora. After one month of life, glycogen gets depleted and pH goes up to 7 until menarche. Vaginal flora
at prepuberty age is dominated by diphtheroids, Staphylococcus epidermidis, streptococci, and Escherichia. coli and large
varieties of other aerobic and anaerobic organisms. After menarche, under the influence of estrogen, cells get glycogen
deposition and Lactobacillus becomes the predominant species yet again (Todar, 2015), along with, and in lesser numbers,
other varied microbiota including Staphylococcus, Ureaplasma, Corynebacterium, Streptococcus, Peptostreptococcus,
Gardenerella, Bacteroides, Mycoplasma, Enterococcus, Escherichia, Veillonella, Bifidobacterium, and Candida, to name a
few. These normal flora are of immense help in maintaining normal vaginal health and prevention of disease. These resident
flora commonly found at a given place normally are called as commensals. Transient organisms include those nonpathogens or potential pathogens which are present temporarily in the mucosa. Until normal resident flora is intact, transient flora
is harmless; however when normal commensals are disturbed, transient flora can flourish, proliferate, and lead to disease
(Kirmani, 1988). The flora can also be influenced by various other factors like local factors (temperature, moisture), immunity, heat and personal factors like time in the menstrual cycle, pregnancy, infections, methods of birth control, frequency
of sex, number of sexual partners, as well as various habits and practices such as douching and previous use of antibiotics
(Linhares et al., 2010). It is a highly complex and finely balanced ecosystem (Zhou et al., 2004).
4. VULVOVAGINITIS
Vulvovaginitis includes a spectrum of diseases resulting in vaginal or vulvar symptoms such as increased vaginal discharge,
odor, itching, burning, and discomfort. Vulvovaginitis may be attributed to vaginal and vulvar tissue infection or inflammation and change in normal flora (Sobel, 2015a; Hainer and Gibson, 2011). In practice, vulvitis, vaginitis, and vulvovaginitis
are terms used interchangeably to refer to lower genital tract infections in women (Joishy et al., 2005). The fact that it is the
most common reason for women to seek physician advice in the United States indicates that its incidence is not affected by
120 SECTION | A Probiotics and Prebiotics
TABLE 1 Etiology of Different Types of Vulvovaginitis
Type of VV
Etiology of VV
Bacterial vaginosis
Gardenerella Vaginalis, Mycoplasma hominis, anaerobic bacteria
like Prevotella and Mobiluncus
Vulvovaginal candidiasis
Candida albicans, Candida krusei, Candida glabrata
Trichomoniasis
Trichomonas vaginalis
Atrophic vaginitis
Estrogen deficiency
Erosive lichen planus
Unknown
Irritant or allergic contact dermatitis
Contact irritation or allergic reaction
the socioeconomic demographics (Hainer and Gibson, 2011). The CDC estimates that in the USA, 40–45% vulvovaginitis
cases are bacterial vaginosis, 20%–25% cases are vulvovaginal candidiasis and 15%–20% cases are trichomoniasis (CDC,
2006). In India, the incidence of bacterial vaginosis is 45%, vulvovaginal candidiasis 31%, trichomoniasis 2%, gonorrhea
3%, nonspecific urogenital causes 5%, and other causes 14% (Puri et al., 2003).
From an etiological perspective, infectious sources are the most common cause of vulvovaginitis followed by noninfectious sources. The incidence of noninfective vaginosis is less common and caused by myriad abiotic factors (enlisted in
Table 1). The infective vaginosis is proved to be caused by pathogenic microbes and is classified as bacterial vaginosis when
caused by bacteria like Prevotella sp., Mobiluncus sp., Group B streptococcus infection, Gardnerella vaginalis, Ureaplasma,
Mycoplasma, and numerous others (Jahic et al., 2013). The fungal VV is commonly caused by Candida albicans, but could
also be caused by the non C. albicans like Candida glabrata and others, though this is rare. The protozoal VV is caused by
infection with Trichomonas vaginalis (TV) (CDC, 2010). Additionally, reports also indicate that 50% of all infective vaginosis are caused by bacteria, 50% cases by fungi and parasites and that polymicrobial vaginitis is also common (MHFW, 2007).
From a pathogenesis perspective, reports indicate that the vaginal pH plays an important role in maintaining health of the
vagina. Under the influence of estrogen, vaginal cells will have glycogen deposits from puberty to menopause. Normal vaginal
flora is a complexly dynamic and balanced ecosystem which maintains the normal pH in vagina by converting glycogen into
lactic acid. Thus acidic pH is maintained and the environment becomes unfavorable to pathogenic growth. Any of these etiological factors can disturb the estrogen levels, affect the vaginal pH, change normal vaginal flora, and disrupt the balance among
the organisms. As a result pH changes and the environment becomes unfavorable to normal flora and favorable for pathogens to
flourish. After menopause, atrophic changes may be seen in the vagina which can lead to vaginal dryness and other symptoms.
Thus various physiological, physical, personal, medical, and other factors may have a role in disease and its progression (CDC,
2006). Patients with vulvovaginitis present with the following symptoms (Faro, 1993; Shivadas, 2010; Soper, 2015):
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
I
Changes in volume, odor, color, consistency of vaginal discharge
Itching in vulvar region or pruritis
Burning sensation in vagina
Irritation in vagina
Redness or erythema
Dysuria
Dyspareunia or painful sexual intercourse
Spotting/bleeding
Signs found on clinical examination
(a) Redness of introitus or erythema
(b) Excoriation of genital area
(c) Vaginal discharge
(d) Cervical erosions, if associated with cervicitis
(e) Adenexal motion tenderness
Vulvovaginal candidiasis is often marked with intense inflammation including itching and soreness and thick white curdy
odorless discharge. Vulval itching could be one of the associated symptoms in candidiasis. These symptoms could be more experienced during the premenstrual period (Sobel, 2015a). Bacterial vaginosis is associated with thin gray or y­ ellow discharge,
a musty or fishy odor (Puri et al., 2003), minimal inflammation, and comparatively less irritation. Generally speaking, bacterial
Probiotics as an Adjunct to Conventional Treatment in Vulvovaginitis: Past, Present, and Future Chapter | 7 121
vaginosis is never associated with vulvar itching (Anderson et al., 2004; Sobel, 2015a). Trichomoniasis is characterized by
profuse thin malodorous, yellow to green discharge and may be associated with a burning sensation, intense itching, dysuria,
and dyspareunia. These symptoms could be noted more during or after menstrual periods (Sobel, 2015a).
Vaginal dryness and dyspareunia may be signs of atrophic vaginitis (Shivadas, 2010). Some of the considerations for
clinical examinations are the presence of cheesy discharge, itching, signs of inflammation like vulvar or vaginal oedema,
excoriations, and fissures means an increased likelihood of candidiasis. Lack of fishy odor makes diagnosis of bacterial
vaginosis unlikely. Lack of dyspareunia makes diagnosis of trichomoniasis unlikely (Anderson et al., 2004). These considerations may aid in diagnosing the probable cause, however none of these features of patient history or clinical examination may be conclusive in establishing a definitive diagnosis (Hainer and Gibson, 2011). Many times symptoms will be
recurrent, especially in vulvovaginal candidiasis which is known for recurrence. It is estimated that >50% of women above
25 years of age will have vulvovaginal candidiasis a some point in their lifetimes, and 5% of them will develop recurrent
vulvovaginal candidiasis, characterized by having at least >4 episodes of vulvovaginal candidiasis in 1 year, or >3 episodes
of vulvovaginitis candidiasis unrelated to antibiotic use in 1 year (Ringdahl, 2000).
Abnormal vaginal discharge (in terms of quantity, odor, color) is usually indicative of vaginal infections but, in a few
cases, could indicate mucopurulent cervicitis, which could be caused by sexually transmitted infections such as Neisseria
gonorrhoeae or Chlamydia trachomatis. Some of the clues leading to diagnosis of Chlamydial cervicitis are (a) age <24 years,
(b) sexual intercourse with new partner in past 2 months, (c) mucopurulent cervicitis, (d) cervical bleeding by swabbing the
endocervical mucosa, (e) no contraceptives use (Egan and Lipsky, 2000). If more than two following signs are present in
women with white discharge, the possibility of C. cervicitis could be considered. In such situations, cultures for Chlamydia
species and N. gonorrhoeae should be done. Possibility of cervicitis in abnormal vaginal discharge could be considered,
however vaginal discharge is a poor predictor of cervical infections/cervicitis (WHO, 2003). If abnormal vaginal discharge
is associated with other symptoms like abdominal pain, fever, menometrorrhagia, dyspareunia, dysuria, nausea, vomiting,
bleeding, and uterine tenderness on pelvic examination a with abnormal vaginal discharge, then the possibility of pelvic
inflammatory diseases could be considered and relevant investigations should be done accordingly (WHO, 2003).
Patients suffering from BV are at an increased risk for the acquisition of the some STI’s like HIV, N. gonorrhoeae,
C. trachomatis, HSV-2, complications after gynecological surgery, complications of pregnancy, and increased recurrence
of BV. Similarly, TV has association with increased transmission of HIV. In pregnancy TV can be associated with preterm
rupture of membranes and preterm delivery (CDC, 2010). Complications of VVC are rare but can still cause chorioaminionitis in pregnancy, vulvar vestibulitis, and have a persistent disease or common recurrence. The clinical symptoms and signs
associated with different vaginosis are listed in Table 2.
TABLE 2 Clinical Signs of Different Types of Vaginitis
Type
Discharge
Itching
Pain
Vagina
Vulva
Bacterial
Vaginosis
Fishy odor,
thin, white or
gray colored,
homogeneous
discharge
May not be
the primary
complaint
May not be the
primary complaint
Vaginal
inflammation may
be absent
Vulva may not be
affected
Vulvovaginal
Candidiasis
Odorless, cheesy,
curdy, white, thick
discharge
Itching is
commonly
associated
Burning sensation,
dyspareunia, dysuria
are commonly
associated
Redness, fissures,
cracks, signs of
inflammation are
common
Vulval excoriations are
common
Trichomoniasis
Yellow to green
froathy purulent
discharge
May not be
presenting
symptom
Dyspareunia, dysuria
and vaginal soreness
are common
Signs of
inflammation with
strawberry like
inflamed vagina
Vestibular redness may
be present
Atrophic
vaginitis
Yellow or whitegray, odorless thin
discharge
Not commonly
seen
Vaginal dryness,
painful sexual
intercourse may be
present
Vaginal redness and
easily traumatized
Thin and dry vestibule,
loose labia majora with thin
subcutaneous fat. Labia
minora thin and friable.
Erosive lichen
planus
Yellow or gray
Excessive
itching may be
present
Pain, dyspareunia
postcoital bleeding
may be present
Redness with friable
epithelium
Erosions and plaques are
common distinctive from
others
122 SECTION | A Probiotics and Prebiotics
5.
CHARACTERISTIC FEATURES OF DIFFERENT TYPES OF VULVOVAGINITIS
5.1
Bacterial Vaginosis
It is a polymicrobial clinical syndrome due to an imbalance between hydrogen peroxide producing lactobacilli species and
anerobic organisms in the vagina by pathogenic organisms such as G. vaginalis, Mobiluncus species, Mycoplasma hominis,
and Peptostreptococcus species and various other anerobes (Egan and Lipsky, 2000). It is estimated that BV is the most
common cause of vulvovaginitis; one-third to one-quarter of affected women remain asymptomatic (Egan and Lipsky,
2000). High numbers of cases of BV are reported to sexually transmitted disease clinics but the role of sexual transmission
of BV is still unclear. It also afflicts women who are not sexually active. Studies done to investigate the role of treating
male partners for reducing recurrence have no benefits (CDC, 2010). Nonetheless, women with BV have an increased risk
for sexually transmitted diseases like N. gonorrhoeae, C. trachomatis, HIV, HSV-2, etc. (CDC, 2010). Pregnancy, multiple
sexual partners, douching, and use of an IUD pose significant risk for BV. BV in pregnancy can predispose the mother
for premature rupture of membranes, preterm labor, and complications in pregnancy (Egan and Lipsky, 2000). BV can be
recurrent and may require repeated treatments. The persistence of BV could also predispose the afflicted to complications
after gynecological surgeries (Owen and Clenney, 2004). BV could be diagnosed with various clinical criteria like Amsel’s
criteria and gram staining of vaginal discharge. Gram staining is considered the gold standard for diagnosing BV by identifying concentration of gram negative rod-like organisms characteristic of BV (CDC, 2010). Nugents scoring and Spiegel
diagnostic criteria for identifying causative organisms in gram staining could be used in diagnosis of BV (Egan and Lipsky,
2000). The important criteria for clinical diagnosis consist of four points.
1.
2.
3.
4.
Patient should have homogeneous, thin, white vaginal discharge that smoothly coats the vaginal walls
Presence of clue cells on microscopic examination
pH of vaginal fluid >4.5
A fishy or amine odor of vaginal discharge before or after addition of 10% KOH (positive whiff test)
Three out of four criteria must be met to establish accurate clinical diagnosis of bacterial vaginosis in 90% of affected women. Among the four previously listed diagnostic criteria, the presence of “clue cells’ is the most significant
(Egan and Lipsky, 2000). Other tests include the DNA probe test for G. vaginalis. The prolineaminopeptidase card
test and OSOM BV blue test are available. Polymerase chain reaction (PCR) for G. vaginalis could also be used for
to achieve the highest certainty. Culture is not indicated for low specificity and pap smear has no clinical utility in
diagnosis of BV.
5.2 Vulvovaginal Candidiasis
Vulvovaginal candidiasis is caused by C. albicans in 80%–90% of patients. Recently there has been an increased occurrence of C. glabrata and C. tropicalis, probably due to overuse of over the counter antifungal drugs (Horowitz et al., 1992).
It is estimated that about 75% of women will experience VVC some time in their life and about 5% of them have recurrent
VVC (Egan and Lipsky, 2000). More than 50% of asymptomatic women have candida as a part of normal vaginal flora,
hence it is very difficult to establish candida as a definitive cause. Risk factors include use of contraceptive pills, intrauterine
devices, spermicide, diaphragm, immunocompromise, diabetes, pregnancy, taking antibiotics, practicing oral sex, and first
intercourse at a young age, to name a few. However, candidiasis is not known to be sexually transmitted, nor is it related to
the number of sexual partners (Egan and Lipsky, 2000).
Complications of VVC are rare, though it can cause chorioamnionitis in pregnancy, vulvar vestibulitis, and be a persistent and recurrent disease. The latter is the most common. Recurrence is defined as more than four episodes in a 12-month
period, and it is not clearly understood whether persistence of infection, precipitating factors, intestinal reservoir, or sexual
transmission is responsible for the return of disease (Sobel, 1992; Egan and Lipsky, 2000). Non albican candida species like
C. glabrata and others, which are not easily identified on microscopy, are known to cause recurrent vulvovaginal candidiasis
(RVVC). VVC could also be classified as uncomplicated and complicated VVC, and includes recurrent VVC and severe
VVC (CDC, 2010).
Clinical symptoms of white, curdy, thick, odorless vaginal discharge, pruritis vulvae, vaginal irritation, dysuria, and
signs of vaginal inflammation like fissures, excoriation, redness, vulvar edema, and normal vaginal pH could help in clinical evaluation. Demonstration of hyphae, pseudohyphae, budding yeast cells, in saline/KOH wet mount slide and gram
staining could help in diagnosis of candidiasis. When patients are symptomatic but hyphae in wet mount cannot be seen,
culture in Nickerson’s medium or Sabouraud’s dextrose agar should be considered. If the patient is asymptomatic but the
culture is positive, treatment should not be started. In RVVC, unusual species could be expected (Sobel, 2015c).
Probiotics as an Adjunct to Conventional Treatment in Vulvovaginitis: Past, Present, and Future Chapter | 7 123
5.3 Trichomoniasis
Trichomoniasis is caused by protozoan T. vaginalis (Owen and Clenney, 2004). It accounts for about 10%–25% of incidence of vaginitis and is known to be the third most common cause of cases diagnosed. Use of intrauterine contraceptives,
tobacco smoking, and multiple sexual partners are said to be the risk factors for trichomoniasis. Female patients with trichomoniasis present with profuse, thin, green to yellow, purulent malodorous vaginal discharge, dysuria, and increased vaginal
pH. It could be transmitted sexually (Sobel, 2015b) and male partners of infected women can be asymptomatic or may be
having nongonococcal uretheritis (Cudmore et al., 2004). Trichomoniasis has an increased association with other sexually
transmitted diseases and can increase the transmission of human immunodeficiency virus (Owen and Clenney, 2004; CDC,
2010; Egan and Lipsky, 2000). Around 20%–50% women could be asymptomatic with persistent trichomonas infection
(Sherrard et al., 2011). In pregnancy, trichomoniasis may be associated with preterm rupture of membranes and preterm
deliveries (Egan and Lipsky, 2000; Cudmore et al., 2004). Trichomoniasis is not known to infect oral and rectal mucosa. It
is diagnosed primarily via saline wet mount preparation where motile trichomonads could confirm the presence of trichomoniasis. This test, which has a sensitivity of 60%–70%, should be done immediately for best results. In symptomatic,
microscopy negative cases, culture, and microscopy, which has sensitivity of 98% and almost 100% specificity, could be
tried for T. vaginalis. Other tests like DNA probe test, rapid card test (OSOM trichomonas rapid test), Affirm vpIII nucleic
acid probe test and PCR, could also be done to diagnose trichomoniasis (APHL, 2013). Pap smear has low sensitivity in
diagnosis of T. vaginalis (CDC, 2010). In infected male partners, the wet mount test has low sensitivity, so culture and PCR
testing would be more appropriate and helpful (CDC, 2010).
5.4 Treatment of VV
The treatment of VV is specific and depends on the organism associated (Sobel, 2015a). Antimicrobials like nitroimidazoles, lincosamides, macrolides, floroquinolones, cephalosporins, and antifungal drugs are the most commonly used
group of drugs in treatment of vulvovaginitis and associated cervicitis (WHO, 2003). Among these drugs nitroimidazoles
are effective for both bacterial vaginosis and trichomoniasis (Amit et al., 2013). Lincosamides, such as clindamycin, are
other group of drugs shown to be effective in bacterial vaginosis. Most of the topical and oral antifungals available are effective in VVC. Recurrence of infections are common in vulvovaginitis. Concomitant treatment of sexual partners is indicated
in few infections like trichomoniasis, gonorrhea, and chlamydia infections (CDC, 2010). In conventional practice, VV,
especially bacterial VV, is treated with the standard regimens of metronidazole or clindamycin. Fungal VV is treated with
antifungals like fluconazole and clotrimazole. Protozoal VV is treated with metronidazole or its congeners. Treatment could
be administered orally or intravaginally (CDC, 2010). The subsequent sections addresses the pharmacology, usefulness,
and side effects of antibacterials and antifungals used in treatment of VV.
6. ANTIBACTERIAL DRUGS
Nitroimidazoles are a group of antimicrobials with established actions against anaerobic and certain protozoal organisms.
They are effective against a variety of anaerobic and protozoal organisms, inhibiting their growth and survival. These drugs
are the first choice for certain genitourinary anaerobic infections like bacterial vaginosis, and protozoal infections like
trichomoniasis (Amit et al., 2013). Metronidazole forms the prototype of this group, used extensively in treatment of bacterial vaginosis and trichomoniasis. 5-Nitroimidazoles enter the bacterial or protozoal cell as prodrug by passive diffusion.
Inside the bacterial cytoplasm or specific organelle in protozoa where there is lack of drug resistant cells, prodrug is activated by intracellular reduction. In this process, there is a transfer of electron to the nitro group of the drug, forming short
lived nitroso free radicals. These radicals, being cytotoxic, can interact with DNA molecules, leading to extreme injury of
the DNA. They cause inhibition of DNA synthesis, oxidative damage to DNA leading to breakage of strands, degradation,
and finally causing cell death (Löfmark et al., 2010). These are selectively toxic to microorganisms that are anerobic or
microaerophilic and hypoxic or anoxic cells (Kapoor et al., 2003). With respect to toxicity, studies have shown that high
doses metronidazole can cause neurotoxic features in humans. Long-term high dosing of metronidazole is carcinogenic in
rodents; in bacteria screening methods it was found to be mutagenic (Brunton et al., 2011). Additionally, studies have also
revealed that metronidazole is safe in pregnancy irrespective of trimester, but it is generally not advised in the first trimester
(Martindale, 2015). Some of the newer versions of the drugs in this family include tinidazole, secnidazole, ornidazole, and
satranidazole.
Lincosamide: A class of drug that includes lincomycin and clindamycin is very important in the treatment of VV.
Of the two, clindamycin is most commonly used and is a congener of lincomycin. It is an effective alternative for use
124 SECTION | A Probiotics and Prebiotics
in treatment of bacterial vaginosis (CDC, 2010). Clindamycin enters the cell and binds reversibly to 50 S ribosomal
subunit of the sensitive micro organism, thereby inhibiting the protein synthesis in its early stages. Clindamycin is
mainly bacteriostatic, but at high concentrations it may attain bactericidal properties against sensitive strains of organisms. Its mechanism is similar to that of macrolides: chloramphenicol. All these drugs have site of action in close
vicinity on 50 S subunit of the ribosomes (Brunton et al., 2011; Martindale, 2015). Clindamycin is sensitive to most
of the Pneumococci, Streptococcus pyogenes, Viridans streptococci, methicillin sensitive strains of Staphylococcus
aureus; anaerobic bacteria like Bacteroides fragilis, Bacteroides melaninogenicus, Fusobacterium, Peptostreptococcus,
Peptococcus, Clostridium perfringens, Actinomyces israelii, Nocardia asteroides; typical bacteria like Chlamydia,
Pneumocystis jiroveci, and T. gondii (Brunton et al., 2011).
The adverse effects associated with clindamycin include diarrhea, known as pseudomembraneous enterocolitis
(Martindale, 2015). Vaginal use can rarely cause diarrhea. It is estimated that 2%–20% cases of clindamycin are associated with diarrhea. If untreated, complications could be fatal. Other minor gastrointestinal side effects like gastritis, nausea, vomiting, abdominal cramps, and unpleasant taste are frequent (Brunton et al., 2011). Hypersensitivity
reactions ranging from urticaria, rashes, fatal Steven Johsonson’s syndrome, and vesiculobullous exofoliative dermatitis have been reported (Martindale, 2015). Transient leucopenia, agranulocytosis, thrombocytopenia, eosinophilia, musculoskeletal joint inflammation, and liver abnormalities including jaundice and rise in liver enzymes have
been reported. Clindamycin can cause renal dysfunction, though occurrences are rare (Martindale, 2015). Injection
site reactions like thrombophlebitis on intravenous use and sterile abscess from intramuscular use are reported.
Cardiopulmonary arrest can result from too rapid iv infusion. Benzyl alcohol containing clindamycin preparation
should be avoided in neonates, as they are known to cause neonatal gasping syndrome (Martindale, 2015). Topical
use of clindamycin results in local irritation, dry skin, and contact dermatitis as side effects. Intravaginal use may be
associated with local irritation and a burning sensation.
Macrolides: are bacterostatic protein synthesis inhibitor antimicrobials. They consist of erythromycin and its semisynthetic derivatives like clarithromycin and azithromycin. Further semisynthetic derivatives of erythromycin with activity
against macrolide resistant strains are called ketolides, which includes telithromycin. They are mainly sensitive against
gram positive bacteria like Streptococci, Pneumococci, Staphylococci, Corynebacterium, Mycoplasma, Leigonella, C. trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, and a few gram negative bacteria. Azithromycin is highly sensitive
against Chlamydia species, Legionella, and N. gonorrhoeae. They act by binding to 50 S ribosomal subunits of bacteria and
prevent protein synthesis. They prevent translocation of t-RNA from acceptor site to donor site, resulting in a halt of protein
synthesis (Brunton et al., 2011).
Tetracyclines: These are congeners of polycyclic naphthacenecarboxamide. They include chlortetracycline,
oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, and glycyclines, including tigecycline.
Antibiotics in this group are called broad spectrum antibiotics since they are active against aerobic and anaerobic
gram positive, gram negative bacteria, Ricketessia and Chlamydia. Tetracyclines enter bacterial cells either by passive
diffusion porins or by active pumping. Inside the cell they bind to the 30S ribosomal subunit during protein synthesis
and prevent access of amino aceyl t-RNA to the acceptor site on mRNA-ribosomal complex, thus preventing protein
synthesis and death of the organism. The adverse effects associated with use of tetracycline include gastrointestinal
disturbances including nausea, vomiting, epigastric distress, and pseudomembranous enterocolitis is also possible.
Photosensitivity is reported with demeclocycline, doxycycline, and others. Renal toxicity including azotemia, nephrogenic diabetes insipidus, and Fanconi syndrome has been reported with tetracyclines. Hepatotoxicity is a known
adverse drug reaction (ADR) with tetracyclines. Tetracyclines are known to increase susceptibility to liver damage
during pregnancy. Discoloration of teeth in children is due to enamel discoloration. Others side effects include thrombophlebitis raised intracranial pressure, vestibular toxicity, hypersensitivity reactions, hematological abnormalities
including leukocytosis, thrombocytopenic purpura, eosinophilia, fever, and asthma. Tetracyclin is not recommended
during pregnancy (Brunton et al., 2011).
Beta lactam antibiotics: This group of antimicrobials includes penicillins, cephalosporins, and carbapenems.
Lactam antibiotics interfere with bacterial cell wall synthesis. The bacterial cell wall is made of a rigid peptidoglycan
complex consisting of glycan chains cross linked with peptide chains. This cross linking is done by an enzyme called
transpeptidases. Lactam antibiotics inhibit the transpeptidases enzyme, thereby leading to formation of a deficient cell
wall which undergoes lysis and death. This peptidoglycan cell wall is unique to bacteria compared to human cells.
Lactams specifically act only on bacterial cell wall, and do not affect the cell walls of the patient. Due to high rate of
resistance to Neisseria, they are no longer considered as therapy for gonorrhea infections, unless sensitivity of the drug
has been proven as effective in a particular geographical area. However cephalosporins are sensitive and are considered
as a first line of defense in the treatment of gonorrhea (Brunton et al., 2011). Cephalosporins are similar to penicillins
Probiotics as an Adjunct to Conventional Treatment in Vulvovaginitis: Past, Present, and Future Chapter | 7 125
in mechanism of action. The adverse effects associated include pain at the site of injection, diarrhea, hypersensitivity
reactions, nephrotoxicity, bleeding (some cephalosporins cause hypoprothrombinemia), neutropenia, and thrombocytopenia (Brunton et al., 2011).
7. ANTIFUNGAL DRUGS
7.1
Imidazoles and Triazoles
The azole antifungals include the imidazole and triazole, with a similar mechanism of action and sensitivity pattern to
different fungi. They include topical and oral antifungal drugs. Clotrimazole, miconazole, ketoconazole, econazole,
butoconazole, oxiconazole, sertaconazole, and sulconazole are imidazoles; terconazole, itraconazole, fluconazole,
voriconazole, posaconazole, and isavuconazole are triazoles. Triazoles are newer drugs, less toxic and more effective than the imidazoles. This group of the antifungals inhibits the enzyme 14 demethylase a CYP 450-dependent
microsomal enzyme, which converts lanosterol to ergosterol, which is required for the fungal cell wall synthesis.
This results in impaired biosynthesis of ergosterol and accumulation of 14 methyl sterol, which then consequentially
inhibits the growth of microorganisms by impairing certain membrane bound enzyme systems and alters the permeability of the sensitive fungi.
Ketoconazole is the first orally active azole available for systemic use featuring corticosteroid suppression properties. In
addition to fungal steroid inhibition, it also blocks human gonadal and adrenal steroid synthesis (Harvey et al., 2008). It has
been replaced with other newer drugs that trigger fewer ADRs and a wider spectrum of activity. Itraconazole is another drug
with similar mechanism of action like ketoconazole, and is available for oral use. The major benefit of Itraconazole is that,
unlike ketoconazole, it lacks the endocrinological side effects. The adverse effects include nausea, vomiting, taste changes,
diarrhea, abdominal cramps, anorexia, hepatotoxicity, hypertriglyceridemia, hypokalemia, rashes, congestive heart failure, adrenal insufficiency, pedal edema, hypertension, rhabdomyolysis, and anaphylactic reactions (Brunton et al., 2011;
Harvey et al., 2008).
Fluconazole: It is a fluorinated bistriazole with a broad spectrum of activity. The mechanism of action is the same as
other congeners. Unlike ketoconazole, it lacks endocrine adverse effects. It can penetrate both normal and inflamed meninges and attain good concentration in CSF, which make it a good candidate for therapy in treating fungal infections of
the brain. It also attains good concentration in bone marrow and can be used in treating fungal infections of bone marrow
transplant recipients. The adverse effects associated with its use include nausea, vomiting, headache, abdominal pain, diarrhea, skin rashes, hepatic failure, Steven Johson’s Syndrome, reversible alopecia, and teratogenic effects such as skeletal
and cardiac abnormalities if taken during pregnancy.
7.2
Imidazole and Triazoles for Topical Use
Clotrimazole: It is an antifungal imidazole used for topical application in superficial/mucocutaneous candidiasis, dermatophytosis, and pityriasis versicularis. It is metabolized in the liver and excreted in feces and urine. Initially clotrimazole
was also given orally at dose of 200 mg/day but is no longer in use because many newer better drugs are available. The
mechanism of action and sensitivity of the drug is the same as that of other azole drugs. It is useful in curing dermatophyte
infections in 60%–70% cases, 80% in cutaneous candidiasis, >80% in VVC, and almost 100% in immunocompromised
hosts having oral and pharyngeal candidiasis using troches.
The adverse effects include reddening of the skin, stinging sensation, itching, urticaria, edema, vesication, and desquamation on topical application to the skin. On vaginal application, ~1.6% users experience mild burning sensation of the
vagina, skin rashes, lower abdominal cramps, and increased frequency of urination. The sexual partner of woman using
vaginal clotrimazole may experience irritation of penis (Brunton et al., 2011). Intravaginal clotrimazole may damage condoms or diaphragms made up of latex rubber therefore contraceptive failure can occur (Martindale, 2015). If clotrimazole
is taken orally, patients can experience mild gastrointestinal and neurological adverse effects. It also has a porphyrogenic
effect in vaginal use therefore alternative therapies should be considered in vulnerable patients (Martindale, 2015).
7.3
Drugs Used in Resistant Infections
Amphotericin B: is a mixture of derivatives of fungi Streptomyces, belonging to the polyene macrolide group. It can cause
serious adverse effects and is used primarily in resistant life threatening systemic fungal infections like mucormycosis
and candidemia. The amphotericin B molecule binds to fungal ergosterol, disrupting the integrity of cell wall by creating
126 SECTION | A Probiotics and Prebiotics
pores or channels. These channels can cause electrolytes (like intracellular K+) to leak from the fungi and cause cell death.
Amphotericin B specifically binds to ergosterol which is characteristic in fungi and some protozoa. Ergosterol is absent in
bacteria, animals, and humans, as a result it acts specifically on fungi. It can act synergistically with flucytosine. It is sensitive to most of the fungi, particularly Aspergillus and Candida. The adverse effects associated with its use include anaphylaxis, fever with chills, renal impairment like azotemia, hypokalemia, hyponatremia, excess magnesium loss, hypotension,
anemia, thrombophlebitis, thrombocytopenia, hepatic dysfunction, and varied adverse neurological effects. It is a drug with
low therapeutic index.
Prognosis of most of these antimicrobial regimens in vulvovaginal infections is good; however recurrence, failure of
therapy, and development of antimicrobial resistance are also possible. Antimicrobial resistance is encountered quite frequently with the therapy of VV. It is said to be on the rise with different drugs, in different types of VV. Long-term therapy
in recurrent infections and oft-repeated therapy with antecedent adverse effects preclude its use. The recurrence rate of BV
is around 57% at 12 months. Around 5%–15% patients with VVC would have recurrence >4 times in 12 months leading
to recurrent VVC. Around 17% of the patients with TV would have reinfection within 3 months. These infections may be
seen even during pregnancy and in nursing mothers, where some of these drugs could be contraindicated. Furthermore,
many instances of vulvovaginitis afflict patients with other sexually transmitted infections where combinations of regimens
should be used, causing increased chances of drug-drug interactions and adverse events (WHO, 2003). The treatment of
this common disease presents many difficulties and the use of probiotics is being researched and has become the matter of
interest for more successful solutions with fewer adverse side effects.
7.4
Probiotics in Vulvovaginitis
Probiotics by definition are termed as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” as defined by WHO (Soccol et al., 2010). Substances such as nondigestible food
­components increase growth and activity of probiotics and are called “prebiotics.” Prebiotics and probiotics could
be called “­ synbiotics.” Probiotics belong to Lactic Acid Bacillus strains (LAB), and consist of numerous substrains
(Ehrström et al., 2010). Probiotics are gram positive, nonspore forming, facultative, or anaerobic bacilli (Shalev, 2002).
Probiotics are derived from milk and dairy products like yogurt and curd. The historical use of probiotics in promoting human health dates back to ancient times and is mentioned in some of the older texts of Hinduism and early
Christianity (Soccol et al., 2010).
At the beginning of 20th century Illya Ilyich Metchnikoff, the Nobel Prize winner in medicine 1908 at Pasteur institute,
was able to link the benefits of bacteria in yogurt with human health. Later Tissier reported that breastfed infants had microorganisms producing bifidofactor in the gut, which may have role in maintaining flora in intestine and protecting against
infections. This work was followed by numerous studies of probiotics and by the end of the century, researchers were able
to link probiotics with the metabolic, trophic, and protective effects. Metabolic effects included digestions of nondigestible
dietary fats, endogenous mucus, savings of energy, production of vitamin K, and absorption of mineral ions. Trophic effects
included control of epithelial cell proliferation, homeostasis, and regulation of the immune system. Protective functions
include effects against pathogens and barrier functions.
Lactobacilli are distributed throughout the gastrointestinal and genital tract in the human body, forming part of normal
healthy flora. Lactobacilli, especially Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus iners, are the most
commonly found microbes in the vagina of healthy women of reproductive age (Falagas et al., 2006). It is now clear that
disruption of the dynamic equilibrium of vaginal microbiota may lead to excessive colonization of pathogenic organisms
causing VV. Modulation of these bacteria would lead to overgrowth of pathogenic bacteria, therefore reestablishing the normal flora colonization with the supplementation of probiotics would counter the pathogenesis and prevent the development
of vaginitis (Ehrström et al., 2010). For years probiotics have been used as food supplements with excellent safety profiles
(Senok et al., 2009; Dovnik et al., 2015).
Probiotics are said to confer various health benefits such as maintaining the innate immune system of the gut, prevention of antibiotic induced diarrhea, alleviation of constipation, traveler’s diarrhea, inflammatory bowel syndrome,
reduction of hypercholestremia, protection against colon and bladder cancer, osteoporosis, allergic rhinitis, and weight
loss. However few benefits have been clinically evaluated (Soccol et al., 2010). The bacterial strains commonly used as
probiotics are Lactobacillus, Streptococcus, and Bifidobacterium; other organisms are used therapeutically, including
Enterococci and Yeasts (Soccol et al., 2010). Many in vitro studies and clinical studies were conducted to assess the
benefits of probiotics in vulvovaginitis. These studies are summarized in Tables 3–5. Probiotics are found to be effective in the treatment of urogenital tract infections, especially vulvovaginitis caused by BV and VVC, by their varied
mechanisms of action.
TABLE 3 Commonly Used Species of Probiotics
Commonly used species of “Probiotics” (Waigankar amd Patel, 2011)
Lactobacillus species
Bifidobacterium species
Streptococcus species
• L. acidophilus
• L. casei
• L. fermentum
• L. gasseri
• L. johnsonii
• L. lactis
• L. paracasei
• L. plantarum
• L. reuteri
• L. rhamnosus
• L. salivarius
• B. bifidum
• B. breve
• B. lactis
• B. longum
• S. thermophilus
Saccharomyces
(Palatty et al., 2009)
• S. boulardii
• S. cerevisiae
TABLE 4 Few Studies Conducted Regarding Use of Probiotics in BV
Intervention
Disease Studied
Results
References
Comparison of yogurt containing
L. acidophilus 1 × 108 CFU OD × 2 months vs
pasteurized yogurt
BV and RVVC
60% vs 25% cure rate
Shalev (2002)
LR GR1 and LF RC 14 suspended in skimmed
milk given once daily for 14 days
BV, RVVC and
UTI
They have potential to restore
urogenital flora in 7 days
Reid et al. (2001a,b)
Oral capsules containing 108 CFU of
L. rhamnosus GR-1 plus L. fermentum RC-14
or L. rhamnosus, each day for 28 d
BV
Normal vaginal flora was restored
Reid et al. (2004)
LRGR1 109 + LRRC14 109 capsule orally
twice daily for 30 days vs placebo after initial
remission with metronidazole
BV
Resolution of BV 88% vs 40% after
30 days
Anukam et al. (2006)
Oral capsule containing 109 CFU of L.
rhamnosus GR-1 + L. fermentum for 60 days
BV
Probiotics colonized the vagina
properly and the Nugent score
normalized after the treatment
Reid et al. (2004)
Oral capsules containing 2.5 × 109 CFU of
L. rhamnosus GR-1 and L. reuteri RC-14,
14 days vs placebo
BV
Significant reduction of Nugent score
in probiotic group
Petricevic et al. (2008)
Vaginal tablets containing 106 CFU of
L. acidophilus and estriol 0.03 mg, once daily
or twice daily for 6 days
BV
Microbiological cure (Nugent criteria)
and clinical cure were observed on
days 15 and 28 after intervention
Parent et al. (1996)
Vaginal tampons containing 108 CFU of
L. gasseri, L. casei var. rhamnosus, and
L. fermentum, 5 tampons during menstruation
BV
Microbiological cure was observed
based on Nugent score and Amsel
criteria
Eriksson et al. (2005)
Vaginal tablet containing 107 CFU of
L. acidophilus, 0.03 mg of estriol and 600 mg
of lactose, daily for 6 d
BV
Vaginal flora was enhanced significantly
by the probiotic administration in
combination with low-dose estriol
Ozkinay et al. (2005)
Vaginal tablets containing 109 CFU of
L. brevis, L. salivarius subsp. salicinius, and
L. plantarum, for 7 d
BV
All of the patients in the probiotic
group were free of BV, showing a
normal or intermediate vaginal flora
Hemalatha et al.
(2012)
Vaginal capsules containing between 108 and
1010 CFU of L. gasseri LN40, L. fermentum
LN99, L. casei subsp. rhamnosus LN113,
and P. acidilactici LN23, for 5 days, after
conventional treatment of bacterial vaginosis
BV and VVC
LN had a good colonization rate in the
vagina of patients with BV and women
receiving LN
Ehrström et al. (2010)
Vaginal capsule containing 108 CFU of
L. rhamnosus, L. acidophilus, and
S. thermophiles, 21 days, for 7 days on 7 days
off, and 7 days on
BV prophylaxis
Reduction in recurrence
Ya et al. (2010)
128 SECTION | A Probiotics and Prebiotics
TABLE 5 Some Studies Done on Probiotics in VVC
Intervention
Disease Studied
Results
References
Vaginal suppositories with Lactobacillus GG
twice/day for 7 days
VVC
Improvement in symptoms and
culture negative
Hilton et al. (1995)
Group 1 (58 women): vaginal L. acidophilus
weekly
Group 2 (50 women): vaginal clotrimazole
weekly
Group 3 (56 women): placebo
VVC
Cases of VVC (culture-confirmed)
in 21 months (median) of follow
up: group 1: 9/58 (15.5%) group 2:
7/50 (14%) group 3: 18/56(32.1%)
Williams et al. (2001)
Group 1 (32 women): L. rhamnosus GR-1 +
L. fermentum RC-14 orally once/day for 60 days
Urogenital
infections
Improvement of vaginal symptoms:
30% (group 1) vs 12% (group
2) and reduction in vaginal
candidiasis
Reid et al. (2003)
Group 1 (n = 10): GR-1/RC-14 8 × 108/day orally
for 28 days
Group 2 (n = 12): GR-1/RC-14 1.6 × 109/day orally
for 28 days
Group 3 (n = 11): GR-1/RC-14 6 × 109/day orally
for 28 days
Group 4 (n = 9): GG 1010/day orally for 28 days
Vulvovaginitis
Study confirms potential efficacy of
lactobacilli at dose of 108 viable
organisms daily to restore and
maintain normal vaginal flora
Reid et al. (2001a,b)
7.5
Mechanism of Action of Probiotics
Different mechanisms have been postulated to explain the mechanism of probiotics in treatment of vulvovaginitis. The
mechanism of action of probiotics is a debated issue and may be strain or species specific. Often multiple mechanisms are
attributed to a single strain or species, however no individual strain would be expected to have all possible mechanisms
(Soccol et al., 2010). A few of the known mechanisms are listed here:
1. Probiotics replace the normal microbiota of the vagina and prevent colonization of pathogenic organisms by competing for food and other requirements.
2. Probiotics can metabolize the glucose and produce lactic acid, acetic acid, and propionic acid, which causes a rise in
vaginal pH, preventing pH-sensitive pathogenic organisms from thriving or survive in an unfavorable environment
(Shalev, 2002).
3. A few strains like of lactobacillus, like Lactobacillus delbrueckii, Lactobacillus fermentum RC 14, are known to produce hydrogen peroxide which can counteract the pathogens. Lactobacillus rhamnosus GR-1 is known to resist the
killing of normal flora by spermicidal nonoxynol-9 (Reid and Hammond, 2005).
4. Probiotics can secrete some antimicrobial products, like bifido factor and a bacterosin like substance called pentosin
TV35b, which can fight against pathogens (Senok et al., 2009).
5. Some biosurfactants are known to be produced by probiotics, such as Surlactin produced by Lactobacillus acidophilus
RC-14 (Falagas et al., 2006).
6. Some strains can produce collagen-binding proteins which can inhibit pathogen adhesion to vaginal epithelium
(Waigankar and Patel, 2011).
7. Probiotics can coaggregate the pathogens and kill them or prevent them from spreading infections (Cribby et al., 2008).
8. Immunomodulation through the toll-like receptor 9 is possibly responsible for antiinflammatory activity of probiotics
(Waigankar and Patel, 2011).
9. Immune regulation at various levels is also possibly responsible for disease prevention. They are known to increase
production of antiinflammatory cytokines, decrease the production of proinflammatory cytokines, stimulate dendritic
cells, thereby modulating Th1- or Th2-mediated immunity regulation (Suchetha et al., 2015).
10. Reduction of 70kDA heat shock protein production and decrease in vaginal mannose-binding lectin concentration,
reduces the capacity of microbial killing (Cribby et al., 2008).
11. They may play a role in mucous membrane integrity and mucin production (Suchetha et al., 2015).
Probiotics as an Adjunct to Conventional Treatment in Vulvovaginitis: Past, Present, and Future Chapter | 7 129
Probiotics could be considered as a single organism or combinations of multiple strains: “probiotic cocktails” (Palatty
et al., 2009). The various strains of probiotics used are listed in Table 3. The salient features of some important strains of
probiotics are listed here.
7.6
L. acidophilus
It is a heterofermentative organism, lives in acidic medium and ferments lactose into lactic acid, ethanol, carbon dioxide, and acetic acid, which can prevent growth of pathogens. It assists in production of vitamin B complex vitamins,
bile conjugation, and regulation of amino acids by separation from bile. They can assist in improving gastrointestinal
functions in diarrhea, promote immunity, prevent yeast infections of the genitourinary tract, and lower cholestrol
levels (Palatty et al., 2009).
L. rhamnosus GR-1: Adheres strongly to uroepithelium and prevents adhesion of pathogens in the vagina. They could
be found in the vagina after oral administration of the strains.
L. fermentum RC-14: Produce biosurfactants, produce a large amount of hydrogen peroxide and prevent adhesion of
pathogens to genital epithelium. They could be found in the vagina after oral administration of the strains (Mastromarino
et al., 2013).
Lactobacillus brevis, Lactobacillus salivarius FV-2, Lactobacillus plantarum FV-9: These have all the previously mentioned features. Additionally, they reduce vaginal inflammatory cytokines, like IL 1, 6, and have inhibitory action on HSV-2
in cell cultures (Mastromarino et al., 2013).
8.
CLINICAL STUDIES WITH PROBIOTICS IN WOMEN’S HEALTH
Probiotics in pregnancy: Currently no data suggests that the probiotics are unsafe in pregnancy and breastfeeding. Two
studies evaluated probiotics during the first trimester reported that there were no malformations of the fetus. One metaanalysis and eight randomized control trials regarding probiotic use in pregnancy during third trimester (32–36 weeks) have
not reported any increase in fetal anomalies. These studies were done for lactobacillus and Bifidobacterium. It is unlikely
that probiotics could affect the pregnancy and fetal development (Elias et al., 2011).
Probiotics and breastfeeding: Probiotics are not expected to be secreted in breastmilk. There were no reports of increased
adverse effects in infants of lactating women on probiotics. Studies were done only in Lactobacillus and Bifidobacterium
strains; therefore other strains need further evaluation. Probiotics appear to be safe in lactating mothers (Elias et al., 2011).
Probiotics in BV: Many in vitro and clinical studies have been done to evaluate the efficacy of the probiotics, either
orally or intravaginally, alone or as an adjunct to antimicrobial therapy in the treatment of the BV (Cribby et al., 2008).
Many studies have reported beneficial effects of probiotics in treatment of BV and prevention of associated STIs (Ehrström
et al., 2010; Cribby et al., 2008). However a Cochrane review conducted in 2009 evaluating role of probiotics in BV, had
inconclusive results due to variation in methodologies, parameters used to assess, different strains of probiotics used, and
the varied adjunct therapy used in different trails. They opined that oral metronidazole and oral probiotic combination apparently had beneficial effects compared to different regimens they assessed in BV. They suggest that further standardized,
well-designed randomized control trials, with a larger sample size of patients, would be required for conclusive report to
develop recommendations (Senok et al., 2009). A few studies have also been done to assess the efficacy of probiotics in
BV during pregnancy resulting in positive reports (Facchinetti et al., 2013). Nevertheless, BV can pose as a great challenge
in the therapy, being the most common condition in women, associated with morbidities in certain populations, including
greater incidence of post-operative infections, adverse pregnancy outcome, disease recurrence, development of antimicrobial resistance, and increased susceptibility to STI. The use of probiotics for BV therapy has an excellent safety profile; it
should be considered a good alternative to existing treatments.
Probiotics in VVC: VVC, due to its high prevalence and high rates of recurrence, presents a significant challenge in
treatment. Complicated VVC including RVVC has limited therapeutic options. Many in vitro and clinical studies have
reported beneficial effects of probiotics in the treatment of VVC (Xie et al., 2013). In a review done by Falagas et al.
(2006), it was reported that either oral or local probiotic will be an effective option in treatment of VVC; however some
studies had failed to support these findings so the results remain inconclusive. They reported that methodological shortcomings in various studies conducted had resulted in inconclusive assessment of probiotics in VVC. They opine that a
few strains of probiotics, like L. acidophilus, L. rhamnosus GR-1, L. fermentum RC-14, are show potential and could
be used to prevent recurrence in RVVC. However, they agree that better designed, standardized, uniform, large sample
sized clinical trials with specified strains of probiotics and adjunctive therapy would be the needed, in order to develop
a definitive view.
130 SECTION | A Probiotics and Prebiotics
Probiotics in Trichomoniasis: There is not much evidence to support the effects of probiotics in TV. The subject needs
further research and evaluation.
Probiotics in Cervicitis: Probiotics could be helpful in preventing gonococcal and chlamydia infections. The subject
needs further research and evaluation.
Comparison of oral vs intravaginal probiotics in vulvovaginitis:
Probiotics are available for oral consumption as tablets, capsules, powder for suspension and intravaginal application as
pessary, vaginal tablet, and cream. Intravaginal probiotics appear to be a more obvious solution for successful colonization
and local effect in the vagina (Ehrström et al., 2010). However, many studies have claimed that oral probiotic supplementation can also produce similar rates of lactobacilli colonization in the vagina (Reid et al., 2001a, b). Oral ingestion of probiotics could be well accepted by practically most women. There are many barriers for the orally supplemented probiotics
to achieve concentration in vagina. The roadmap for orally supplemented probiotics is as follows: they have to sustain
viability in the gastric acid pH, pass through the bile, survive in the gut, pass through the rectum, and finally ­colonize
the vagina and uroepithelium (Reid et al., 2001a, b). Few studies have reported that oral supplementation of probiotics of
some strains, like L. rhamnosus GR-1, L. fermentum RC-14, are effective equivalents and ascend from the rectum and effectively colonize in the vagina (Reid et al., 2001a, b). There is a conflict in using probiotics in vulvovaginitis in that no
clear consensus exists regarding the preferred route of administration in vulvovaginitis (Senok et al., 2009; Dovnik et al.,
2015; Falagas et al., 2006).
9. ADVERSE EFFECTS
Probiotics are generally considered safe, without any adverse effects. However, there have been a few incidents where probiotic strains have been isolated in infectious wounds. Approximately 0.2% positive blood culture for lactobacillemia have
been reported in Finland during a 5-year period from 1995 to 2000. Infectious endocarditis, fungemia, and abscess of the
liver have been associated with probiotics. Interestingly, in only a few cases probiotics consumption had positive isolates
from infectious sites. Continued vigilance in identifying, typing, and cataloguing all probiotic bacteria associated with
bacteremia would be helpful to ascertain the possible of cause (Borriello et al., 2003). Such adverse effects are seen more
in patients who are immunocompromised or have serious underlying diseases (Falagas et al., 2006). A case of metabolic
acidosis has been reported after oral consumption of L. acidophilus (Ku et al., 2006). Probiotics such as Streptococci and
Enterococci use may give rise to theoretical concerns regarding adverse effects due to the fact that they are pathogens themselves. Pancreatitis has been reported in a study where six different strains were installed directly into the intestine (Cribby
et al., 2008). Properties of individual probiotics are specific to strain and species; therefore adverse effects of individual
probiotics should not be overgeneralized (Martindale, 2015). Probiotics have to be used with caution in patients who are
immunocompromised, preterm neonates, and special populations (Martindale, 2015).
10.
CONCLUSIONS
Studies in the recent past have shown that probiotics are promising in women’s reproductive health and the scientific
evidence for their use in specific clinical scenarios is strong. In this regard, the most important aspect that needs to be considered, with the advent of increasing cases of antibiotic-resistant pathogenic microorganisms, is the use of probiotics for
the treatment of VV in a natural and nontoxic treatment modality. Probiotics are being shown to be help the host overcome
infection, prevent infections of the reproductive tract, restore and maintain a healthy vaginal ecosystem, and thereby improve female health in relation to reproductive health. The other important aspect is that probiotics are a cost-effective in the
treatment of VV and will be of great use with a potential for wide application. Further studies are required to ascertain their
benefits in different disease states and in different age groups in various parts of the world. From a mechanistic perspective,
although much remains to be learned about the use of appropriate probiotic strains, it is evident that benefits can be strain
dependent. In addition to this, studies are also required to understand the underlying mechanistic issues and interactions of
different probiotic organisms.
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Senie RT. Introduction to epidemiology of women’s health, 1st chapter An Overview of Women’s Health: From Past to the Future. [cited 2015 Aug 26].
Available from: http://samples.jbpub.com/9780763769857/Chapter1.pdf.
Senok AC, Verstraelen H, Temmerman M, Botta GA. Probiotics for the treatment of bacterial vaginosis(Review) [Internet]. Cochrane Database of
Systematic Reviews. 2009 [cited 2015 Aug 19]. Available from: http://www.bibliotecacochrane.com/PDF/CD006289.pdf.
Shalev, E., 2002. Ingestion of probiotics: optional treatment of bacterial vaginosis in pregnancy. IMAJ 4, 357–360.
Sherrard J, Donders G,White D. 2011 European (IUSTI/WHO) guideline on the management of vaginal discharge [Internet] [cited on 2015 jul 15].
Available from: http://www.iusti.org/regions/europe/pdf/2011/Euro_Guidelines_Vaginal_Discharge_2011.Intl_Jrev.pdf.
Shivadas A. Vaginitis [Internet]. Cleveland clinic center for continuing education. [Last updated Aug 2010; cited 2015 Aug 1]. Available from: http://www.
clevelandclinicmeded.com/medicalpubs/diseasemanagement/wome ns-health/vaginitis/Default.htm.
Sobel JD.Approach to women with symptoms of vaginitis [Internet]. 2015a [last updated june 5 2015, cited 2015 Jul 31]. Available from: http://www.
uptodate.com/contents/approach-to-women-with-symptoms-of-vaginitis?source=related_link.
Sobel JD. Candida vulvovaginitis [Internet]. [last updated 2015b, june,05; cited 2015, Aug, 6]. Available from: http://www.uptodate.com/contents/
candida-vulvovaginitis.
Sobel, J.D., 1992. Pathogenesis and treatment of recurrent vulvovaginal candidiasis. Clin. Infect. Dis. 14 (Suppl. 1), S148–S153.
Sobel JD. Trichomoniasis [Internet]. [Last updated 2015c Jul 27; cited 2015 Aug 7]. Available from: http://www.uptodate.com/contents/trichomoniasis?s
ource=preview&language=en-US&anchor=H1&selectedTitle=1~58 #H1.
Soccol, C.R., Vandenberghe, L.P.D.S., Spier, M.R., Medeiros, A.B.P., Yamaguishi, C.T., De Dea Lindner, J., et al., 2010. The potential of probiotics: a
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overview-of-vaginitis.
Suchetha, A., Vinayashree, M.P., Apoorva, S.M., Sapna, N., Sravani, K., Darshan, B.M., 2015. Probiotics—a legacy of good health. J. Res. Med. Dent.
Sci. 3 (1), 1–6.
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with HIV. J. Assoc. Nurses AIDS Care 12 (4), 51–57.
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Ya, W., Reifer, C., Miller, L.E., 2010. Efficacy of vaginal probiotic capsules for recurrent bacterial vaginosis: a double-blind, randomized, placebocontrolled study. Am. J. Obstet. Gynecol. 203, 120.e1–120.e6.
Zhou, X., Bent, S.J., Schneider, M.G., Davis, C.C., Islam, M.R., Forney, L.J., 2004. Characterization of vaginal microbial communities in adult healthy
women using cultivation-independent methods. Microbiology 150 (8), 2565–2573.
FURTHER READING
Hillström, L., Pettersson, L., Pálsson, E., Sandström, S.O., 1977. Comparison of ornidazole and tinidazole in single-dose treatment of trichomoniasis in
women. Br J. vener Dis. 53 (3), 193–194.
Vaginitis Introduction—Self-Study STD Modules for Clinicians from CDC [Internet]. Centre for disease control and prevention USA [last updated July
2013, cited 2015 Jul 31]. Available from: http://www 2a.cdc.gov/stdtraining/selfstudy/vaginitis/vaginitis_introduction_self_study_from_cdc.html.
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Section B
Therapeutic Foods
and Ingredients
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Chapter 8
Flavonoids as Nutraceuticals
Muhammad Kaleem, Asif Ahmad
PMAS-Arid Agriculture University, Rawalpindi, Pakistan
1.
INTRODUCTION
Flavonoids are polyphenolic secondary metabolites, which are not required for the survival of a plant, but do impart color
and aroma to flowers, leaves, and fruits. They may also help the plant withstand adverse conditions such as insect attacks
or bacterial and viral infestation. Examining the protective role of flavonoids, many studies have been carried out to investigate their antibacterial, antiviral, antioxidant, and antifungal properties. Additionally, much research has also been
conducted to evaluate their pharmacological properties against different maladies such as diabetes, cardiovascular diseases, cancer, neurodegenerative, osteoporosis (especially post-menopausal), as well as several bacterial, and viral diseases
(Ahmad et al., 2015).
The basic skeletal unit of a flavonoid consists of two aromatic rings (A & B) which are linked to each other via heterocyclic ring C. based on the functional group attached, which is the type of derivatization and position of attachment of B
ring to the C ring. Flavonoids are classified into various groups which include flavonols, flavan-3-ols, flavones, flavanones,
isoflavones, and anthocyanins. Individual compounds present within the subclasses are further characterized by the pattern
of hydroxylation and conjugation (Babu et al., 2013). One of the most prevalent groups of plant metabolites is flavonoids.
A large number of flavonoid compounds have already been explored; research continues to expand this existing knowledge
base. At present, about 9000 flavonoid compounds are known (Ahmad et al., 2015).
Flavonoids are present in almost all parts of the plant. Sources of flavonoids in the human diet include tea, wine,
blackberries, blueberries, propolis, honey, red beans, and nuts. Black rice is an excellent source of anthocyanins, having
116.58 mg/g (Pedro et al., 2016; Tsuchiya, 2010; Ahmad et al., 2015). Vegetables, fruits, berries, and red wine are good
sources of anthocyanidins, while parsley, celery, and herbs are excellent sources of flavones (Crozier et al., 2009). Genistein
and daidzein are major isoflavones which are found in Genista tinctoria, a Chinese medicinal herb, and other leguminous
plants (Veitch, 2013). Excluding fungi and algae, flavonols such as quercetin, kaempferol, isorhamnetin, and fisetin can be
commonly found throughout the plant kingdom (Babu et al., 2013). Hesperidin and naringin are examples of flavanones
mostly present in the citrus fruits. Naringin gives bitter taste to grapefruit (Jung et al., 2006). Sources of anthocyanidins and
proanthocyanidins include cabbage, currants, barley, banana, berries (strawberries, raspberries, cranberries, blueberries,
black berries), chocolate, tea (black and green), wine, beer, spices, onions, plums, peas, grapes, peaches, nuts (walnuts,
peanuts, cashews, pistachio, almonds), mangos, and lentils (Kruger et al., 2014).
Flavonoids are the secondary metabolites of different fruits and vegetables having a critical role in performing numerous metabolic activities. Flavonoids have gained lot of attention because of their effects to beneficial health. The lowest
levels of coronary heart diseases have been reported in France in spite of pervasive spoking and high fat intake. This is due
to frequent consumption of red wine. Flavonoids have recently been identified as the major constituent preventing cancer in
daily diet. Low risk of coronary heart disease and premature aging prevention is also attributed to daily consumption of flavonoid rich food. In addition, flavonoids reveal a lot of biological activities, including antihepatotoxic, anti-inflammatory,
antiviral, antibacterial, antiosteoporotic, and antiulcer actions.
At present, health consciousness has increased, driving the attention of researchers and industrialists towards natural
and nutraceutical foods. This has encouraged fortification of processed foods with flavonoids to boost the immune system. Following high population growth, the food processing industry is expanding at high rate, which makes safe and
healthy products necessary. With the expansion of industries, industrial waste and byproducts of industries are increasing.
These byproducts include such things as seeds and peels. These industrial byproducts happen to be a rich source of certain valuable constituents, including flavonoids, and minerals. Such valuable constituents of industrial byproducts can be
Therapeutic, Probiotic, and Unconventional Foods. https://doi.org/10.1016/B978-0-12-814625-5.00008-X
© 2018 Elsevier Inc. All rights reserved.
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extracted and incorporated into other products to increase their nutraceutical potential. Purification and concentration of these
flavonoids is valuable to pharmaceutical industries as well. There is continued exploration of their potential and utility in
concentrated or purified forms as therapeutic agents (Stalikas, 2007; Routray and Orsat, 2012).
2.
EXTRACTION OF FLAVONOIDS
Flavonoids are collected from dry, frozen, or lyophilized material due to the fact that enzymes act on and destroy flavonoids
when the plant material is fresh or not dried. The plant material is generally dried at low temperatures to minimize the loss
of flavonoids due to heat exposure. After drying, the plant material is usually ground to fine powder. Grinding increases the
surface area of plant material and helps in the efficient extraction of flavonoids. After grinding, the next step is extraction
with a solvent. This is a critical step because there is a huge diversity of flavonoid compounds that vary in structure and
type of derivatization. Flavonoids can be broadly categorized into two groups for the choice of solvent for extraction. One
group of flavonoids is less polar and includes flavanones, flavonols, methylated flavones, and isoflavones. The other groups
of flavonoids are flavonoid glycosides and polar aglycones. Nonpolar or less polar flavonoids can be extracted with ethyl
acetate, chloroform, diethyl ether, and dichloromethane, while polar flavonoids can be extracted with water alcohol mixture or alcohol alone. Factors affecting extraction of flavonoids from plant material include time of extraction, extraction
temperature, pressure, and solvent (Andersen and Markham, 2005).
There are two types of extraction techniques: (1) Conventional extraction and (2) Modern extraction techniques.
2.1
Conventional Extraction Techniques
Conventional extraction techniques include maceration, hydrodistillation, and Soxhlet extraction. Maceration technique has
been used for a long time in extraction of essential oils for medicinal purposes. The process of maceration involves grinding
of material, addition of an appropriate amount of a solvent, followed by straining and pressing to remove occluded residue
from the solution. The efficiency of this process was improved by shaking and by removing the concentrated solution. In
the last step, filtration is carried out to remove impurities (Azmir et al., 2013). Hydrodistillation is another conventional
technique which does not use organic solvents and uses water or steam for the extraction of flavonoids and other bioactive
compounds (Silva et al., 2005). Soxhlet extraction was designed for the separation of lipids, but adapted for the extraction
of other constituents from the food material such as flavonoids (Azmir et al., 2013).
2.2
Modern Extraction Techniques
There are several problems associated with conventional extraction techniques which include longer time of extraction,
high cost of more pure solvent, decomposition of heat sensitive compounds, and less extraction selectivity. To overcome
these problems, several new techniques have been developed which include microwave assisted extraction, ultrasound assisted extraction, pressurized liquid extraction, and supercritical fluid extraction.
In microwave assisted extraction, microwave energy is used for the extraction of important constituents of plant material into liquid solvent. The frequency of microwaves ranges between 300 MHz and 300GHz. In this type of extraction,
electromagnetic energy is converted into heat energy. Microwave assisted extraction takes place in three steps: (1) solute
separation from the sample matrix under high pressure and temperature, (2) solvent diffusion against sample matrix, and (3)
solute is released to solvent from the matrix of the sample. Extraction has been shown to yield an increase of four times the
polyphenol in microwave assisted extraction as compared to conventional techniques. Besides that, it reduces the time of
extraction and reduces the volume of solvent used for extraction (Routray and Orsat, 2012). Microwave assisted extraction,
using comparable concentration of solvent for extraction and temperature, requires much less extraction time as compared
to solvent extraction and ultrasound assisted extraction (Li et al., 2011).
Comparison between different extraction techniques is described in Fig. 1., showing the time required for extraction,
extraction temperature, and the concentration of solvent used for extraction. Factors affecting the microwave extraction
include solvent system, time of microwave application, temperature of extraction, and surface contact area. The choice of
solvent is among the most important factors, especially in the case of microwave extraction. One of the important factors
for the solvent to be considered is the dielectric constant of solvent, which affects its rate of heating and its extraction capability. The properties of solvent can be improved by combining different solvents. For thermo-labile compound extraction, the combination of solvents to be made must have a lower dielectric constant; this ensures the maintenance of lower
temperature, otherwise the desirable compounds may be lost (Casazza et al., 2010). Another factor to be considered in
the choice of solvent is solubility of the desired compound in the solvent. Depending upon the polarity of flavonoid to be
Flavonoids as Nutraceuticals Chapter | 8 139
Time (min)
Temperature (°C)
Solvent concentration (%)
100
80
60
40
20
Solvent concentration (%)
Temperature (°C)
0
Ultasonic
extraction Microwave
extraction
Time (min)
Super
critical
extraction
Solvent
extraction
FIG. 1 Comparison of different techniques used for extraction of flavonoids.
extracted, polar or nonpolar solvent may be chosen. Polar solvents are generally used for flavonoid glycosides, while for
flavonoid aglycones, nonpolar solvents are used. It is mostly reported, with increase in time of extraction, extraction yield
increases, but this only remains true for a certain period, after which extraction yield tends to decrease. In many studies in
which extraction condition was optimized by response surface techniques, it was concluded the effect of extraction time
on extraction yield is quadratic rather than linear. This is described by degradation of flavonoid compounds by prolonged
heating (Xiao et al., 2008; Ghafoor et al., 2009). As temperature increases, the solubility of compounds in solvent increases,
so high temperature extraction can be advantageous over low temperature extraction. High temperature also exerts pressure
on the cellular walls, thus helping in the release of desirable compounds. In addition to this, the viscosity of solvent used
also decreases, thus improving the solubility and mobility. However, it has been reported extraction efficiency increases
with increase in temperature, but this rule only applies up to an optimum level, beyond which it starts to decrease. Effect of
temperature is different for extraction of different compounds; it depends upon the nature of compound. If the compound
is sensitive to heat, it may be extracted at low temperatures. Similarly, prolonged high temperature along with high power
is not desirable because it may degrade the desirable compound (Khajeh et al., 2010). Extraction efficiency increases with
the increase in surface area, so most of the sample preparation steps include milling, grinding, and homogenization. This is
also the case in microwave extraction; greater surface area results in more efficient extraction (Kothari and Seshadri, 2010).
Ultrasound assisted extraction uses ultrasound waves having a frequency of 20 kHz–100 MHz for the extraction of different constituents from the matrix of the substance. UAE improves the extraction efficiency by producing cavitation. Heat
is produced when ultrasound waves pass through the material because kinetic energy is converted into heat energy. Factors
affecting the extraction efficiency of UAE include time, temperature, and frequency of ultrasound used for extraction (Khan
et al., 2010). A study was conducted to compare the Soxhlet extraction with ultrasound assisted extraction for oil and polyphenols from powder of grape seed. It was found that ultrasound assisted extraction reduced the time of extraction from 6 h
to 15 min, with consumption of less extraction solvent (Da Porto et al., 2013).
Supercritical fluid is the state in which gas or liquid is not distinguishable, due to having a density similar to liquid and
viscosity as gas. Supercritical fluids have more diffusivity as compared to liquid in solid material, and possess good transport properties which help to reduce the extraction time. Several advantages associated with supercritical fluid extraction
include improving the extraction efficiency in terms of low extraction time, and greater yield, and it can also be coupled
with chromatographic techniques like supercritical fluid chromatography and gas chromatography (Herrero et al., 2006;
McHugh and Krukonis, 2013).
3. ABSORPTION, METABOLISM AND BIOAVAILABILITY OF FLAVONOIDS
The absorption and metabolism of dietary flavonoids have continued to be a controversial issue for some time. In the past,
the consensus regarding flavonoids was that they were fairly large and polar molecules which could not be absorbed after
ingestion, and were hydrolyzed to corresponding aglycones by bacterial enzymes in the lower part of the intestine. Most
140 SECTION | B Therapeutic Foods and Ingredients
flavonoids occur as glycosides in food (Drzikova et al., 2005). Different glycosidic units are present. Among these, glucose
is the most common. Other glycosidic units include arabinose, rhamnose, and galactose (Cook and Samman, 1996). β
Linkage present in these sugars resist hydrolysis by pancreatic enzymes, so it was thought that intestinal microflora hydrolyze this β linkage in sugars. However β endoglucosidases, which can hydrolyze flavonoid glycosides, have been characterized in the small intestine of humans. These β glucosidases include lactase phlorizin hydrolase and cytosolic enzyme,
which, it is believed, deglycosylate flavonoids create a site for conjugation (Lazaridou and Biliaderis, 2007; Welch, 1995).
Some flavonoid constituents, such as kaemferol-3-glucoside and luteolin-7-glucoside, are absorbed in the small intestine
after hydrolysis, supporting the activity of β-glucosidase. Luteolin-3-glucoside is converted to aglycone during the passage from intestine (Day et al., 1998). Some studies showed anthocyanins are absorbed unchanged (Cao and Prior, 1999).
Absorption kinetics of flavonoids varies from food to food, due to presence of different sugars and the variety of functional
groups around the flavan nucleus. Absorption also depends upon the dose, vehicle of administration, gender and colon microbial population (Hollman et al., 1999; Hollman and Katan, 1999; Erlund et al., 2001). Flavonoids that are absorbed from
the small intestine are metabolized in the liver as compared to compounds which are absorbed by colon.
Due to high molecular weight, flavonoids must to be degraded into smaller molecules to be absorbed across the intestinal epithelium. Procyanidin oligomers that consist of 7 units are not capable of translocating across the small intestinal
epithelium, while procyanidin dimmers and trimmers can translocate across intestinal epithelium (Déprez et al., 1999).
In addition to flavonoid glycoside hydrolysis, cecal microbes degrade polymers and breakdown flavonoids into monophenolic acids. Quercetin metabolism by intestinal microbes produces 3,4-dihydroxyphenylacetic acid and phloroglucinol
through the breakdown of C3-C4 bond of the heterocycle (Winter et al., 1991). Cultured colon flora convert tannins into
aromatic compounds (Kim et al., 1998). Deprez and coworkers incubated C14 labeled proanthocyanidins with human colonic bacteria under anaerobic conditions that were similar to an enteric environment. All the substrate was degraded to
phenylpropionic, phenylvaleric, and monohydroxylatedphenylacetic acids (Hollman et al., 1999). Phenolic acid degradation of catechin produces similar metabolites. Existing data suggest that flavonoids are altered structurally in vivo, but it is
unclear whether it is dominated by phenolic acid or flavonoid isomers. After oral administration of quercetin in humans, it
was recovered in plasma but minimum amount was detected in urine (Olthof et al., 2000).
Genistin and its octylglucosides are dominant isoflavones found in soy foods (Wiseman et al., 2002). Lactase phlorizin
hydrolase mammalian enzyme hydrolyzes ingested isoflavones and released aglycones daidzein, glycitein, and genistein.
Gut microflora act on these constituents and convert them into isoflavan equol, and convert genistein into p-ethyl phenol
(Hur et al., 2000). Isoflavonoids which are unconjugated are quickly absorbed from the upper parts of the small intestine
while conjugated glycoside are slowly absorbed. This is dependent on their hydrolysis in the distal site of the intestine
(Sfakianos et al., 1997; King and Bursill, 1998). Once isoflavonoids are absorbed, they are converted to β-glucuronides. It is
understood that these conjugates circulate in plasma and are excreted through urine because intact glucuronides isoflavones
have been found in urine of human after consumption of soy foods (Clarke et al., 2002).
In vivo study shows that the cell wall of intestines are permeable to dimers and trimmers of proanthocyanidins (Holt
et al., 2002). Proanthocyanidins with a higher degree of polymerization are broken into monomers and dimers in the form
of epicatechin in the stomach. After being broken into monomers and dimers, they are absorbed in the cell wall of small
intestine. Those proanthocyanidins which have a degree of polymerization greater than 10 and are not broken into simpler
molecules, are not absorbed by the small intestine and pass through the small intestine unchanged. In the colon these polymeric proanthocyanidins are degraded by colon microflora which is present in the large intestine (Holt et al., 2002; Deprez
et al., 2001; Kruger et al., 2014). Overall absorption and metabolism of flavonoids is shown in Fig. 2.
3.1
Bioavailability of Flavonoids
The study of bioavailability of dietary flavonoids is of great importance due to their health promoting effects. Absolute bioavailability, which is expressed in the percentage of non nutrient part of food, is estimated from the part of a molecule that gets
absorbed from intestine and found in blood circulating in the system after ingestion, followed by their passage through the liver
(Hollman et al., 1997). Bioavailability is not entirely indicated by the extent of absorption, but other factors as well, such as
distribution and metabolism (bioconversion which takes place in the gut and biotransformation which takes place in the liver)
and also effects bioavailability of non nutrient plant factor following ingestion. It can be concluded that bioavailability quantifies the exposure of the body (not including the liver or the gut) to the non-nutrient plant factor in question (Wiseman, 1999).
Bioavailability of flavonoids is low; consumed constituent in circulation is less than 10% at nanomolar or low micro molar range after few hours of consumption. Isoflavones are the most bioavailable subclass of flavonoids comparatively, while
flavan-3-ols and anthocyanins are least absorbed (Manach et al., 2005; Barnes et al., 2011). Factors which affect the bioavailability of flavonoids include the presence of fiber, micro- and macronutrients, gastrointestinal transit time and gut microflora.
Flavonoids as Nutraceuticals Chapter | 8 141
O
A
B
C
Flavonoid glycosides
Oligomeric units
Stomach
Monomeric units
Dimeric units
Trimeric units
Corresponding
aglycone like
Methylated
aglycone
Anthocyanins
(Unchanged)
Degylcosylate
absorption
in portal
vein
O methylated
sulphates
glucuronides
Duodenum
Jejunum
Ileum
Colon
Flavonoid polymers
Gut microflora
Glucohronides
Mono phenolic acid
Renal excretion
Cell
FIG. 2 Digestion and metabolism of flavonoids.
Flavonoid bioavailability is dependent on the proportion of taken quantity which is absorbed, varying from 0.2% to
0.9% of tea catechins, to 20% for quercetin and isoflavones (Field, 2001). A large proportion of flavonoids remain unabsorbed and a high concentration of these compounds interacts with gastrointestinal mucosa. Absorbed flavonoids are conjugated in the liver by the process of glucuronidation, methylation or sulfation, or are metabolized into smaller compounds
(Avila et al., 1989).
Little information exists regarding the relation of bioavailability of flavonoids to interaction between food constituents
and flavonoids. Some studies show fat improves bioavailability of flavonoid absorption due to micellarization of solubilized
polyphenols (Ortega et al., 2009). Flavonoids that bind to dietary fiber are not accessed by hydrolytic enzymes in the small
intestine, but can be degraded by colonic microbes later on (Pérez-Jiménez et al., 2009). Processing and homogenization
of food may enhance the bioaccessibility of flavonoids, for example the flavanones naringenin from tomato products are
absorbed better when compared to fresh fruits (Porrini and Riso, 2008).
4. TOXICITY OF FLAVONOIDS
Diets containing flavonoids are generally thought to be safe because many foods that are naturally rich in flavonoids have
been consumed for a long time with no ill effects. Studies that are based on observation demonstrate that there is no correlation between flavonoids (normally quercetin, up to 68 mg) as part of a routine diet and various cancers, including cancer of
the breast, lung, respiratory tract, gastrointestinal tract (Harwood et al., 2007). Although these associations provide strong
evidence for the safety of flavonoids, there has actually been little experimental research to track the possibility of adverse
effects (Erdman et al., 2007). Hooper and Colleagues reviewed the effects of different flavonoids subclasses on cardiovascular diseases and other risk factors. They selected those flavonoids subclasses that are normally present in the human diet.
Except from acute rise of blood pressure after black tea consumption that was observed in four studies, and they presume
that this may be due to caffeine, and no other adverse effects were detected among the foods tested (Hooper et al., 2008;
Erdman et al., 2007).
The proceedings of the International Life Sciences Institute North America Flavonoids Workshop describe that if flavonoids are consumed in low amounts, then they are not detrimental to health, however some evidence shows that flavonoids
could cause adverse effects when consumed in high amounts or by some vulnerable groups or populations (Lambert et al.,
2007). The potential risks due to flavonoids in the geriatric population include thyroid toxicity, antinutritional effects, carcinogenicity and genotoxicity (Egert and Rimbach, 2011).
142 SECTION | B Therapeutic Foods and Ingredients
The consumption of a test meal showed acute intake of flavonol rich beverages like coffee, tea, red wine and cocoa
leads to impaired absorption of non heme iron in humans. Intake of beverages containing 20–25 mg flavonoids per serving
reduces the absorption of iron by 50%–70%, while consuming beverages containing 100-400 mg total flavonoids per serving reduces the absorption of iron by 60%–90% (Corcoran et al., 2012). It is evident that high consumption of flavonoid
increases the risk of iron deficiency in marginal iron status populations like the elderly. Because populations in western
countries consume adequate amounts of iron, the chance of developing anemia due to intake of flavonoids is lower (Erdman
et al., 2007).
In vitro studies and animal models show that flavonoids may possess antithyroid and goitrogenic activity. Genistein
possess more pronounced antithyroid effects in animals where iodine status is low. However, no evidence has been provided
to suggest soy foods or isoflavones affect thyroid function adversely in normal humans (Messina and Redmond, 2006).
Additionally, Bitto and colleagues showed no effect of genistein on serum thyroid hormone after three years of treatment
in postmenopausal and osteopenic women. Flavonoid toxicity depends upon the dose, duration of intake and type of flavonoids (Moon et al., 2006). Safety concerns of isoflavones due to their high intake are related to estrogenicity, though prior
exposure duration may have an affect and lead to adverse outcomes. High concentrations of isoflavones that are not readily
achieved through diet result in genotoxicity (Jerome-Morais et al., 2011). In vitro studies show that quercetin is mutagenic
and genotoxic, but this is not confirmed with in vivo studies (Harwood et al., 2007).
Observance to dietary recommendations, recommending greater intake of plant foods to promote health, results in
higher consumption of flavonoids. However, due to the putative health benefits of flavonoids, increased consumption has
been encouraged, not only of naturally occurring flavonoid-rich foods, but also of foods that are fortified with flavonoids
and dietary supplements like phytochemicals (Corcoran et al., 2012). For example, quercetin is marketed as a dietary supplement with dose recommendations exceeding 1000 mg/day, while daily intake of this flavonol from food is anticipated at
10–100 mg. There is no evidence suggesting quercetin toxicity resulting from consuming supplements (Egert and Rimbach,
2011). It is mostly older adults who use supplements and likely take prescribed medicines as well. Therefore the focus of
research ensures safety of high intake of flavonoids specifically in older people (Lambert et al., 2007; Prasain et al., 2010).
5. ANTIOXIDANT ACTIVITY OF FLAVONOIDS
Flavonoids possess several properties, but one of the most important that is their described ability to scavenge free radicals
and act as antioxidants. The difference in the antioxidant capacities of different flavonoids is different, depending on the
type of functional group and its arrangement around the flavonoid skeleton. The number of hydroxyl groups, their configuration and substitution, affect the mechanism by which flavonoids act as antioxidants, like chelation of metal ion or
scavenging free radicals (Pandey et al., 2012). One of the important factors which affect their ability to act as an antioxidant
is the configuration of the hydroxyl group on ring B, because this ring has the ability to donate an electron and hydrogen to
hydroxyl and scavenge reactive oxygen species (Cao et al., 1997).
Several mechanisms are described for the antioxidant activity of flavonoids. They include: scavenging reactive oxygen
species, enzyme inhibition and trace element chelation that contributes in the generation of free radicals. In this way they
suppress formation of reactive oxygen species, antioxidant defense protection and up regulation. The enzymes which are
inhibited by flavonoids and help in the generation of reactive oxygen species include NADH oxidase, mitochondrial succinoxidase, glutathione S-transferase and microsomal monooxygenases (Kumar and Pandey, 2013).
Oxidative stress is associated with peroxidation of lipids. The protective effect of flavonoids against the peroxidation
of lipids is described frequently (Kumar et al., 2013). Reactive oxygen species formation is enhanced by metal ions, the
mechanism involved in this reaction is that hydrogen peroxide is reduced by these metal ions resulting in the generation
of hydroxyl radical which is highly reactive (Mishra et al., 2013a). Among several flavonoids constituents, quercetin is the
most commonly described flavonoid with antioxidant properties. The antioxidant properties of quercetin are due to chelation of iron and iron-stabilizing properties (Kumar and Pandey, 2013).
Because flavonoids mostly occur as flavonoid glycosides, in which a flavonoid molecule is attached to sugar molecule,
its position and the number of attached sugar molecules also effect its antioxidant properties. By comparing the antioxidant
activities of flavonoid aglycones and glycosides, it has been reported that aglycones have greater antioxidant potential. These
findings are supported by the fact that the flavonol glycosides, which are found in tea, decrease with the increase in number
of glycosidic moieties. Although aglycones are stronger antioxidant when compared to glycosides, their bioavailability is low
(Hollman et al., 1999). As the degree of polymerization increases, the antioxidant activity or scavenging of reactive species
in procyanidins is improved. As compared to monomeric procyanidin, dimers, and trimmers of procyanidin are more effective in scavenging superoxide anion. Similarly, the tetramers of procyanidin show greater activity against superoxide and
peroxynitrite mediated oxidation. In the same way, hepta and hexamers are strong antioxidants (Kumar and Pandey, 2013).
Flavonoids as Nutraceuticals Chapter | 8 143
Experiments carried out on animals have shown that flavonoids might be a valuable anti-inflammatory, owing in part
to the fact that they inhibit blood-vessel damage (Harborne and Williams, 2000). Research recommend flavonoids for their
potential to prevent chronic inflammation (Funakoshi-Tago et al., 2011) related to heart disease, arthritis, type 2 diabetes,
Alzheimer’s disease, dementia, and many other diseases. Flavonoids may increase blood sugar metabolism and studies
have shown that flavonoids might have a positive result on abnormal collagen making, a symptom linked to diabetes that
subsidizes poor blood sugar control (Kumar and Pandey, 2013). A study demonstrated that flavonoids showed activities for
inhibiting retinopathy, a diabetic disorder which can occur prior to blindness. (Kumar and Pandey, 2013).
6.
FLAVONOIDS AND CARDIOVASCULAR DISEASES
In the western world, the most common cause of death is cardiovascular diseases (CVD); worldwide about one third of all
the deaths are caused by cardiovascular diseases. There are two types of factors involved in the development of cardiovascular diseases. The primary factors can be modified, like diet, life style, environment, smoking, and exercise. The second factors cannot be modified: genetic factors, gender, history, and age. The common phenomenon involved in the development of
CVD is the atherosclerotic plaque formation which is initiated by endothelium damage. Inflammation and oxidative stress
are the key factors contributing to the damage of endothelium.
Consumption of fruits and vegetables is inversely associated with incidence of CVD, due to the presence of bioactive
compounds like flavonoids. Current research has focused on diet containing bioactive compounds, as an alternative to
pharmaceutical medication, in the maintenance of cardiovascular health,. It can be concluded from the analysis of multiple
studies that as the mean consumption of flavonoids increases, mortality due to cardiovascular diseases decreases. Another
epidemiological study was conducted to determine the effect of blueberry and strawberry intake for 16 years follow up
period on the mortality due to CVD in postmenopausal woman. It was concluded that the result demonstrated a significant connection between the two (Mink et al., 2007). Similarly, another study showed that the cardio-protective effects of
Nigella sativa are due to the presence of phenolic compounds like thymoquinone, by reducing low density lipoprotein, total
cholesterol and triglycerides (Shafiq et al., 2014).
Several mechanisms have been described for the mode of action of flavonoids as cardio protective agents. The ability of
flavonoids to control oxidative stress and act as anti-inflammatory agents is responsible for their cardio-protective properties. The anthocyanins present in black rice grain and the proanthocyanidins found in red rice and grape seeds scavenge
hydroxyl radicals and superoxide ion (Walter and Marchesan, 2011; Kruger et al., 2014). Production of NO is stimulated
by proanthocyanidins which are present in red grapes. The availability of NO in acute oxidative stress like reperfusion/ischemia is protective to cardiomyocytes, because it inhibits the cardiomyocytes apoptosis (Jones and Bolli, 2006). Similarly,
the supplementation of blueberry, which is rich in proanthocyanidins and anthocyanins, improved the endothelial dysfunction and decreased blood pressure in animals which were fed a high cholesterol/high fat diet (Rodriguez-Mateos et al.,
2013). In a human study, cranberry juice consumption for 4 weeks did not affected the function of vascular endothelial as
well as the level of NO generated. It was concluded that anthocyanins might be undetectable in plasma after 12 h due to
rapid clearance. Another reason for no activity observed might be due to a lower concentration of anthocyanins in the juice
(Rodriguez-Mateos et al., 2013). Similar results were also reported by Riso et al. (2013) who studied the effect of wild
berry juice in humans.
Among CVDs, the effect of flavonoids on stroke is not clear. The association between intake of five flavonoid classes
(flavan-3-ols, flavonols, anthocyanidins, flavanones, flavones) and risk of stroke and mortality caused by stroke was studied
by Mursu et al. (2008) in eastern Finish men, age 42–60 years. During the follow up time, 153 deaths occurred due to CVD
and 102 patients suffered from ischaemic stroke. The men consuming the highest amount of flavonol and flavan-3-ol had
low risk for ischemic stroke, which is 0.55 and 0.59 as compared to the lowest quartile. So it was concluded that a greater
intake of flavonoids decreased the chances of ischemic stroke as well as mortality caused by CVD. This reduction in the risk
for ischemic stroke and CVD mortality was significant. Similarly, high intake of cocoa or chocolate reduced the systolic
blood pressure by 5.9 mmHg, at the population level it has the capacity to reduce risk of stroke by 8%, mortality due to
coronary artery disease by 5%, mortality caused by all factors at 4% (de Pascual-Teresa et al., 2010).
6.1 Anti-Inflammatory Activities of Flavonoids
The protective action of tissue in response to the invasion of pathogens, irritation, cell injury and for removal of necrotic
and damaged cells is known as inflammation. For a short period, the process of inflammation helps to maintain the tissue
integrity by minimizing the effect of injury or invasion. But if the inflammation takes place for a long period of time, it
can mediate the development of several chronic diseases such as CVD, cancer, arthritis, neurodegenerative diseases, and
144 SECTION | B Therapeutic Foods and Ingredients
pulmonary diseases (Rubio-Perez and Morillas-Ruiz, 2012). Several studies show the anti-inflammatory activity of flavonoids. Chronic inflammation is caused by the excessive production of chemokines and cytokines. Cytokines and chemokines act as regulatory proteins under normal physiological conditions, but their excessive production disrupts the gradient
balance and more ROS are produced. It has been shown that the grape flavonoids control chronic inflammation by reducing
ROS level and by modulating pathways of inflammation. As flavonoids are natural compounds, they can target multiple
steps in the inflammation pathway as compared to mono-targeted synthetic anti-inflammatory drugs (Sung et al., 2012). In
the same way, proanthocyanidins extracted from the seeds of grapes have been shown to modulate the immune system in
inflammatory conditions and induce production of prostaglandin E2 and nitric oxide (Terra et al., 2007).
6.2 Atherosclerosis
Atherosclerosis is characterized by the plaque formation in large arteries, and it is one of the major factors contributing
to incidence of stroke and myocardial infarction. Atherosclerosis is caused by high level of lipoprotein and cholesterol
in plasma (Hackam and Anand, 2003). Factors which contribute to development of atherosclerosis include hypertension,
diabetes, diet, obesity, smoking, and aging. Beside therapeutic treatment, the risk for cardiovascular diseases persist which
necessitates the search for therapeutic agents that control the risk factors of atherosclerosis. Seeking the French Paradox,
many studies have been carried out to evaluate the potential of flavonoids against atherosclerosis. In addition to the French
Paradox, several epidemiological studies showed negative correlation between the incidence of atherosclerosis and intake
of flavonoids. High intake of fruits and vegetables rich in flavonoids reduces several risk factors for development of atherosclerosis including: high tolerance to glucose, maintaining good body mass index, lowering blood pressure (Mulvihill
and Huff, 2010).
In a mice model study, the effect of pomegranate juice, which is rich in proanthocyanidins and anthocyanidins, decreased the accumulation of macrophage CE and lipid peroxides without affecting the level of cholesterol in plasma.
Supplementation of pomegranate for a period of three months reduced the risk of atherosclerosis up to 44%. In a similar
animal model study, supplementation of resveratrol in the lab chew for four months reduced the total cholesterol level of
plasma along with LDL-C and increased the HDL cholesterol. In addition to this resveratrol prevented the lipid peroxidation and increased cholesterol efflux. Another study showed that risk for atherosclerotic plaque development was significantly reduced by the consumption of resveratrol in mice (Aviram et al., 2008).
Naringenin plays an important role to overcome the metabolic problem that is connected to dyslipidemia and resistance
to insulin. Consequently it was shown to prevent atherosclerosis development in mice fed a high fat diet. Naringenin treatment attenuated the adverse effects caused by hyperinsulinemia and hyperlipidemia which was induced by western style
diet. In mice that were fed a western diet, hyperlipidemia led to development of atherosclerosis in the aortic sinus evidenced
by the development of plaque is that increased 10 times as compared to chow fed animals. Naringenin treatment decreased
the incidence of atherosclerosis by 70% (Mulvihill and Huff, 2010).
7. ANTIDIABETIC ACTIVITY OF FLAVONOIDS
One of the most widely prevalent metabolic disorders is diabetes, which is characterized by hyperglycemia which may be
the result of either no excretion of insulin, or production of non-functional insulin. High levels of sugar in the blood result
in short term protein and lipid metabolism changes and irreversible long term changes in the vascular system (Brahmachari,
2011). The long term manifestation of insulin results in damage and dysfunction of various organs like nerves, kidneys,
eyes, blood vessels, and heart. In the past few decades, it has been observed that there is a rapid increase in the incidence
of coronary artery diseases (CAD) (2010). Several studies reporting anti-diabetic activities of flavonoids are described in
Table 1.
Currently, diabetes is treated by several anti-diabetic agents, which include biguanides, sulfonylureas, glinides, and
α-glucosidase inhibitor, along with insulin to control regulation of blood sugar level. These therapies are associated with
several adverse health effects, so research has been focused to find new therapeutic agents which have minimum or no adverse health effects. Plants are a natural source of drugs; several drugs which are widely used have been obtained directly
or indirectly from plant sources. Several studies have reported that either plant parts or extracts of plant parts possess antidiabetic properties when assessed through experimental trials. This antidiabetic activity of plants is due to the presence of
phytochemicals which are termed as flavonoids (Brahmachari, 2011).
Meta-analysis and several epidemiological studies showed that there is inverse correlation between consumption of
a diet rich in flavonoids and several disorders related to aging, such as osteoporosis, cardiovascular diseases, cancer, and
neurodegenerative diseases (Graf et al., 2005; Arts and Hollman, 2005). Several studies have been conducted that showed
Flavonoids as Nutraceuticals Chapter | 8 145
TABLE 1 Antidiabetic Activities of Flavonoids Reported During the Period of 2010–15
Model of
study
Diabetes
caused by
Pilea
microphylla
Mice
Total flavonoids
Litsea
coreana
Total flavonoids
Flavonoid
Source
Results
Reference
Quercetin, luteolin7-o-glucoside, rutin,
chlorogenic acid,
isorhoifolin
High fat diet and
streptozotocin
induced
diabetes
dipeptidyl peptidase IV was
inhibited with IC50 of 520.4 μg/mL
Bansal et al.
(2012)
Male
SpragueDawley
rats (Type II
diabetes)
Diabetes
induced by
Streptozotocin
Extract treatment increased
sensitivity of insulin, HDL-C and
increased the body weight
Lu et al. (2010)
Selaginella
tamariscina
Rats
Diabetes
induced by high
fat diet and low
concentration
of STZ
Treatment with total flavonoids
decreases the triglycerides,
glycosylated hemoglobin A1C, free
fatty acids, Total cholesterol, fasting
blood glucose level, and LDL-C,
with significant increase in SOD
Zheng et al. (2011)
Total flavonoids
Sanguis
draxonis
Rats
Type II diabetes
induced by high
fat diet and
streptozotocin
Showed hypoglycemic activity
and increased the tissue steatosis
and dyslipidemia. In addition
islet protecting effects was also
observed
Chen et al. (2013)
Fruit extract rich in
flavonoids
Carissa
carandas
Rats
Diabetes
induced by
alloxan
Reduced the high level of glucose
in blood by 48%, by oral intake of
extract at rate of 400 mg/kg
Itankar et al.
(2011)
Rats
Diabetes
induced by
streptozotocin
Intraperitoneal injection at the rate
of 50 mg/kg showed protective
effects against diabetes induced by
(STZ) and decreased the activity of
antioxidant enzymes
Abdelmoaty et al.
(2010)
Rats
Alloxan
Lowered glycosylated hemoglobin
level in blood and improved the
tolerance of insulin and glucose.
Salib et al. (2013)
Quercetin
Extract containing
flavonoids like
chrysoeriol 7-o-β-Dgalactopyranosyl and
luteolin 7-O-[6″-O-αL-rhamnopyranosyl]-βD-galactopyranoside
Hyphaene
thebaica
consumption of flavonoid rich diet regulate digestion of carbohydrates, secretion of insulin and uptake of glucose in insulin
sensitive tissue by regulating several intracellular pathways (Hanhineva et al., 2010).
The anti-diabetic effect of flavon-3-ols are reported by several studies. Epigallocatechin gallate (EGCG), a flavon-3-ol,
at a concentration of 0.1-10 μM improved the viability of β-cells along with improving their insulin secretory function in
rat cells in which glucose toxicity was induced. In addition, the function of mitochondria was also improved by maintaining its functional integrity in pancreatic b-cells exposed to glucose toxicity (Erdman et al., 2007). A high level of free
fatty acids in the plasma plays an important role in impairing insulin resistance and development of type II diabetes. This
insulin resistance induced by fatty acid can by minimized by the consumption of ECG and EGCG (Deng et al., 2012;
Boden et al., 2001). Similar to EGCG and ECG, naringin and hesperidin minimized the oxidative stress and hyper glycemia in male albino rats in which diabetes is induced by streptozotocin, by oral administration at the dose of 50 mg/kg for
a period of 1 month (Mahmoud et al., 2012). In the same way, a diet supplementation of either hesperidin or naringin at
the rate of 200 mg/kg reduced the glucose level in the blood along with increasing leptin and plasma insulin concentration
(Goldwasser et al., 2010; Jung et al., 2003).
146 SECTION | B Therapeutic Foods and Ingredients
Bilberry extract, a rich source of anthocynins, has been studied for anti-diabetic effect in mice with type II diabetes.
Insulin sensitivity and hyperglycemia was improved by bilberry extract, along with down regulation of gluconeogenic
enzyme expression like G6Pase and PEPCK (Takikawa et al., 2010). The seed coat of black soybeans is rich in delphinidin, cyanidin and petunidin. Soybean seed coat extract treatment by gavage ameliorated insulin resistance improved the
insulin concentration in the serum, along with improving tissue glucose utilization in a rat model study (Nizamutdinova
et al., 2009).
8.
HEPATO-PROTECTIVE EFFECTS OF FLAVONOIDS
One of the vital organs of the body is the liver. It regulates several physiological processes and plays an important role in the
vital processes of body like secretion, storage, metabolism and detoxification of exogenous as well as endogenous toxins
(Adewusi and Afolayan, 2010). In addition, the liver takes part in biochemical processes such as growth, nutrient provision,
supply of energy and reproduction. It helps in the metabolism of fats and carbohydrates by the secretion of bile and storing
vitamins (Adewusi and Afolayan, 2010). Due to these vital functions, hepatic disease is one the greatest threats to the world
population. Structural or functional damage to the liver is termed hepatic disease. Liver damage is caused by autoimmune
disorders like primary biliary cirrhosis, immune hepatitis as well as by other biological factors including viruses, bacteria
or other parasites. Liver damage can also be caused by chemicals found in certain drugs such as antituberculosis drugs
and CCl4. Despite enormous achievements in medicine, until now, there are no drugs available which stimulate complete
liver function or help in the regeneration of liver cells. Beside, that there are several side affects as well as adverse health
effects associated with these drugs. Several studies have investigated the efficacy of plant extract against hepatic diseases
and positive results are found. Besides that, consumption of certain foods protects against liver damage. Hepatoprotective
properties of plant based foods are mostly attributed to bioactive compounds like flavonoids. Following these successes,
several studies have been conducted to check the hepatoprotective activities of plant extract rich in flavonoids or individual
flavonoid compounds (Madrigal-Santillán et al., 2014).
Grapefruit is an excellent source of the flavonoid compound naringin which is metabolized as naringenin in the body.
The hepatoprotective activity of naringin was reported in 2004 in rats in which hepatic damage was induced by dimethylnitrosamine (DMN). Oral administration of naringenin at the dose of 20 and 50 mg/kg per day for the period of 4 weeks
protected the mice from damage induced by DMN. This was shown via liver weight as well levels of aspartate transaminase
(ASAT), alanine transaminase (ALAT), and bilirubin. Other studies since then have shown that naringenin has the ability to reduce the serum level of ASAT, ALAT, gamma-glutamyltranspeptidase (GGT), lipid hydroperoxides, ALP, protein
carbonyl content, conjugated dienes, superoxide dismutase (SOD), glutathione-s-transferase (GST), glutathione peroxidase (GPx), catalase (CAT), and alcohol dehydrogenase (Lee et al., 2004). The hepatoprotective activities of flavonoids
extracted from plants are described in Table 2.
As described above berries are good source of flavonoids and are consumed at large scale worldwide. Currently berry
extract is used as a functional food ingredient and dietary supplement. The administration of cranberry at the rate of 7 mg/kg
is effective in minimizing the adverse effects on the liver caused by acute and chronic administration of CCl4, as evidenced
by normalizing the level of ASAT, ALAT, and bilirubin. In addition to this, it also prevented the lipid peroxidation products
from accumulation in the liver of rats, with apparent mitochondrial ultrastructure preservation (Cheshchevik et al., 2012).
The hepatoprotective effects of anthocyanins and proanthocyanidins was investigated by Shin et al. (2010) in rats. Hepatic
damage was induced by DMN followed by the oral intake of anthocyanins and proanthocyanidins at a dose of 20 mg/kg
for a period of 4 weeks. This minimized the adverse effects caused by DMN and reduced the serum level of ALP, ASAT,
ALAT and bilirubin. In addition, it also minimized the accumulation of collagen which is induced by DMN. This was demonstrated by histological analysis of red stained tissue.
Grapes and grape seeds are rich source of flavonoids like resveratrol, proanthocyanidin, anthocyanidins, epicatechins,
and catechins. The hepatoprotective effect and antioxidant activity of grape seeds was observed in rats in which hepatitis
was induced by oxidative stress and assessed by marker enzymes like GGT, ASAT, LDH, ALAT, SOD, GSH, MDA, GPx,
and GST (Dogan and Celik, 2012). The animals were divided into four groups: (I) control, (II) 20% ethanol, (III) 15% grape
seed, and (IV) 20% ethanol +15% GS. The ethanol treated group was observed to contain a significantly high amount of
marker enzymes in the serum as compared to the control group. This level of marker enzymes was significantly decreased
in group IV, which was fed ethanol along with grape seed. This showed that the adverse effects caused by the oxidative
stress of ethanol were minimized by the consumption of grape seed. Several other studies also described the hepatoprotective effect of grape or seeds of grapes (Hassan, 2012; Dogan and Celik, 2012; Oliboni et al., 2011; Liu et al., 2012;
Madrigal-Santillán et al., 2014; Madi Almajwal and Farouk Elsadek, 2015).
TABLE 2 Hepatoprotective Effects of Total Flavonoids/Individual Flavonoid Compounds Described During the Period of 2010–16
Flavonoid
Source
Liver Damage was
induced by
Model of Study
Liver Damaged
Assessed by
Dose of Flavonoid
Supplementation
Reference
Citromitin
Citrus depressa
D-galactosamine
rats
alanine aminotransferase
and aspartate
aminotransferase activities
300 mg/kg body
wt 4 h
Akachi et al.
(2010)
Tangeretin
Citrus depressa
D-galactosamine
rats
alanine aminotransferase
and aspartate
aminotransferase activities
300 mg/kg body
wt 4 h
Akachi et al.
(2010)
Nobiletin
Citrus depressa
D-galactosamine
rats
alanine aminotransferase
and aspartate
aminotransferase activities
300 mg/kg body
wt 4 h
Akachi et al.
(2010)
Hirsutin, quercetin, avicularin
Lespedeza cuneata
G. Don
tert-butyl hyperoxide
HepG2 cells
Cytotoxicity
10 μg/mL
Kim et al. (2011)
Total flavonoids
Rosa laevigata Michx
fruit
Paracetamol
Mice
alanine aminotransferase,
aspartate aminotransferase,
malondialdehyde,
superoxide dismutase, GSH
Total flavonoid
Abelmoschus manihot
(L.) Medic Flowers
CCl4
Mice
ALT, AST, ALP, SOD, GPx,
CAT and GST
125, 250, 500
Ai et al. (2013)
Total flavonoid content
Solanum melongena
tert-butyl hydroperoxide
(t-BuOOH)
human hepatoma
cell lines, HepG2
Improved viability of cells
14.49 ± 1.14% to
44.95 ± 2.72%
Akanitapichat
et al. (2010)
Catechin glycoside, miricitrin3-O-glucoside, astragalin,
isoquercitrin, hyperin,
quercetin-3-O-rhamnoside
Nelumbo nucifera
Gaertn
CCl4-induced liver
toxicity
rats
300 and 500 mg/kg
Huang et al.
(2010)
Luteolin, isorhamnetin
Viola odorata
Paracetamol
Mice
Serum hepatic enzymes
and bilirubin
250 mg/kg and
500 mg/kg
Qadir et al.
(2014)
Quercetin
Convolvulus arvensis
Paracetamol
Mice
Serum hepatic enzymes
and bilirubin
200 mg/kg and
500 mg/kg
Ali et al. (2013)
Liu et al. (2011)
Flavonoids as Nutraceuticals Chapter | 8 147
148 SECTION | B Therapeutic Foods and Ingredients
A flavonoid named Silymarin has three structural components: silydianine, silibinin, and silychristine. These are extracted from the seeds and fruit of milk thistle Silybum marianum (Compositae) and have been reported to stimulate enzymatic activity of DNA-dependent RNA polymerase 1 and subsequent biosynthesis of RNA and protein which results in
biosynthesis of DNA and cell proliferation resulting in liver regeneration in damaged livers (He et al., 2004). The phytochemical properties of this flavonoid are also involved in regulation of permeability, integrity, ROS scavenging, and inhibition of leukotriene and collagen production (Saller et al., 2001; Kumar and Pandey, 2013).
Different flavonoids such as quercetin, rutin, catechin, naringenin, and venoruton have been reported for their hepatoprotective effect (Tapas et al., 2008). Several chronic maladies such as diabetes can cause clinical hepatic manifestations.
Diabetic mice have decreased levels of ROS, glutathione, and ligase catalytic subunits expressions in their liver. As a result,
anthocyanins are gaining attention due to the discovery that increased levels of these antioxidants have a preventive effect
against many diseases. Zhu et al. (2012) explained that anthocyanin increases Gclc expressions by elevating cAMP levels
to activate PKa (Protein kinase), which further increases Gclc transcription resulting in decrease in hepatic ROS levels.
Moreover, C3G treatment reduces hepatic lipid peroxidation, inhibiting release of pro-inflammatory cytokines, and protecting against hepatic steatosis development (Zhu et al., 2012).
Additionally, flavonoids extracted from Laggera alata were also observed for their hepatoprotective effect against
carbon-tetrachloride induced liver damage in rats. Flavonoids fed at 100 μg/ML concentration inhibited cellular leakage of
AST, ALT, and also improved cell viability (Wu et al., 2006). Similarly, flavonoids administrated at 50, 100, and 200 mg/kg
in an in-vivo experiment significantly reduced AST, ALT, albumin, total protein levels, and hydroxyproline levels in liver.
Histopathological observations showed improvement in damaged liver cells. Many clinical experiments have also revealed
the effect of flavonoids on hepatobiliary dysfunctions including feelings of bloating, less appetite, abdominal pain, and
nausea. Hirustrin and avicularin flavonoids extracted from many sources were reported to have protection against induced
hepatotoxicity. Additionally, luteolin and quercetin suppress phosphorylation of VEGF receptor 2 which is induced by
VEGF (Carmeliet and Jain, 2000).
9. ANTICANCER ACTIVITY OF FLAVONOIDS
As flavonoids are a natural product, they are considered a safe and ideal candidate for chemoprevention or the treatment of
cancer (Szliszka et al., 2011; Yoshimizu et al., 2004). The synthetic agents used for the treatment of cancer are highly toxic
and destructive to healthy cells. An ideal anticancer agent is one which has a maximum capacity to inhibit tumor growth or
to kill cancer cells, but causes minimum adverse health effects (Zhao et al., 2012). Because long term consumption of flavonoids is not toxic, and due to their inherent biological activity, flavonoids are ideal future candidates for cancer therapy.
Many studies have shown that cytotoxic effects of flavonoids on cancer cells, with no or minimum adverse health effects.
These findings have simulated the research to develop flavonoid based chemotherapies (Sak, 2014).
Due to the presence of polyphenol aromatic rings in flavonoids, it has been found that flavonoids possess pro and antioxidant properties (Leung et al., 2007). Most studies report the beneficial effect of flavonoids due to their reactive oxygen
scavenging properties, but recent studies show anticancer properties of flavonoids may be due to their pro-oxidant properties (Li et al., 2008; Habtemariam and Dagne, 2010). Higher oxidative stress is observed in the cancerous cells as compared
to normal cells, making them more susceptible to be killed by a substance which enhances reactive oxygen species level
like flavonoids (Valdameri et al., 2011; Yuan et al., 2012). A flavonoid acting as pro-oxidant or antioxidant is dependent on
the concentration, type of cell and culture condition in which it is grown (Pacifico et al., 2010).
One of the important factors that plays a significant role in the prevention of cancer is diet. It has been shown that consumption of vegetables and fruits which are rich in flavonoids prevent the development of cancer (Mishra et al., 2013b). The
consumption of apple and onion, good sources of quercetin, are negatively associated with incidence of prostate, stomach,
lung, and breast cancer. Similarly, it has been observed that wine drinkers are less susceptible to the development of colon,
lung, and stomach cancer (Kumar and Pandey, 2013).
Flavonoids, along with their synthetic analogs, are currently being studied for their therapeutic potential against cervical, breast, ovarian, and prostate cancer. Some flavonoid compounds, like genistein, quercetin, and flavopiridol, are at the
late phase of clinical trials for cancer treatment (Lazarevic et al., 2011). The anticancer activity of flavonoids is reportedly
due to modulation of several factors like epidermal growth factor receptors (EGFRs), protein kinases, vascular endothelial growth factor receptors (VEGFRs), platelet derived growth factor receptors (PDGFRs) and cyclin dependent kinases
(Ravishankar et al., 2013; Singh and Agarwal, 2006).
Cancer is one of the most fatal diseases that exists. The incidence of cancer is increasing continuously, not only in developing countries, but also in developed countries like United States of America, the United Kingdom, France, and Italy
(Ahmad et al., 2015). Cancer incidence and deaths caused by cancer decreased with the use of radiotherapy, chemotherapy,
Flavonoids as Nutraceuticals Chapter | 8 149
and immunosuppressants, however deaths caused by cancer are still greater as compared to cardiovascular diseases in persons having age greater than 85 years (Ahmad et al., 2015).
Several studies have been carried out to check the antiproliferative activity of flavonoids in vitro and in vivo. The
antiproliferative activity of flavonoid constituents isolated from Dracocephalim kotschyi, an ethnobotanical remedy, was
studied in vitro against normal and malignant cell lines. The hydroxyflavone isolates, including apigenin, luteolin, and
isokaempferol, showed antiproliferative effects comparable in both normal and malignant cells. Hydroxyl flavones, which
are methylated like penduletin, calycopterin, cirsimaritin, and xanthomicrol, showed preferential antiproliferative activities
against malignant cells (Moghaddam et al., 2012). This study showed that there might be a structure activity relationship.
Flavonoid constituent may be modified in such a way to enhance their anti-cancer potential without affecting normal cells.
Similarly, in another study six flavonoids were isolated from litchi leaf and studied for anticancer activity. Among six flavonoids which include kaempferol-3-o-β-gluscoside, rutin, procyanidin A2, epicatechin, kaempferol 3 o-a-rhamnoside, and
luteolin, procyanidin A2 showed maximum anticancer activity against human cervical carcinoma HeLa cells and human
hepatoma HePG2 cells (Wen et al., 2014).
Several mechanisms has been described for the anticancer activity of flavonoids which include protein tyrosine kinase
inhibition, phosphatidylinositol-3-kinase inhibition, topoisomerase inhibition, antiangiogenic effects, as well as antioxidant
and pro-oxidant activities. Byun et al. (2010) showed that flavonols quercetin, fisetin, and myrecetin, while the flavone luteolin is the potent inhibitor of protein tyrosine kinase. Among other flavonoids which have been reported to inhibit protein
tyrosine kinase, include apigenin, luteolin, and quercetin (Ravishankar et al., 2013; Huang et al., 1999).
Phosphatidylinositol 3-kinase is also inhibited by quercetin, apigenin, luteolin, and myricetin. In addition to that,
antiangiogenic effect is induced by flavonoids by vascular endothelial growth factor expression regulation and NFkB
(Ravishankar et al., 2013).
10.
EFFECT OF FLAVONOIDS ON OSTEOPOROSIS
Osteoporosis and fragile fracture are common world problems, primarily afflicting aging people. Approximately one in two
women and one in five men suffer from osteoporosis related fractures in their lives (van Staa et al., 2001) and these osteoporosis associated fractures are on the rise (Hardcastle et al., 2011). Diet is one changeable therapy likely play an important
role in osteoporosis development and treatment. Recent research has shown that a healthy diet plan, rich in vegetables and
fruits, is linked with low bone resorption whereas a poor diet plan consisting of processed foods results in less bone mineral
density (Welch and Hardcastle, 2014).
The occurrence of osteoporosis is much more common in women as compared to men. In the female population, incidences of osteoporosis are much higher in postmenopausal women as compared to premenopausal women. This is due to
hormonal imbalance, and in most cases, it is termed estrogen dependent osteoporosis. In the maintenance of bone health,
estrogen plays a vital role by decreasing cytokines while simultaneously increasing the osteoclast apoptosis. Consequently,
estrogen deficiency results in bone loss. In the same way, estrogen deficiency decreases the life span of osteoblast (Chiechi
and Micheli, 2005). There are several flavonoid compounds which are capable of generating estrogen response. Chemicals
capable of producing estrogenic response are referred to as phytoestrogen. Among flavonoids, lignans and isoflavones are
important phytoestrogens. Genistein, which is an isoflavone obtained from soy. Following a regimen of genistein with a
consumption rate of 56 mg/d, results showed a similar effect as by hormone replacement therapy which is currently the
most widely used for treatment of postmenopausal osteoporosis. This treatment increases the bone mineral density insignificantly with the hormone replacement therapy (Morabito et al., 2002). Bone strength, as well as bone mineral density, increases with increasing consumption of isoflavones. It has also been shown that fructooligosaccharides consumption along
with isoflavones, enhances the action of isoflavones. This effect of fructooligosaccharides is due to its prebiotic properties
(Ahmad and Kaleem, 2016; Mathey et al., 2004).
10.1
Mechanism of Action
Bone is in a continuous state of modeling and remodeling to maintain a balance between osteoclast and osteoblast cells.
This balance is responsible for maintaining physiological structure and mineral contents of bone. After the achievement
of peak bone mass, there is gradual loss of bone. This loss of bone mass is higher in women, occurring more rapidly after
menopause (Welch et al., 2004). However, men have more loss of bone mass as compared to women, but this loss is occurs
at a slower rate with age. Mass loss predicts fractures in both men and women (Khaw et al., 2004). This loss with age is
usually due to imbalance in the anabolic and resorptive activities of osteoblasts and osteoclasts respectively. The osteocyte
cells are embedded within mineralized bone and give signal to osteoclasts to start resorption and are then activated to send
150 SECTION | B Therapeutic Foods and Ingredients
signals through mechanical loading and microbial damage (Nishimura et al., 2012). Several signaling pathways maintain
the activities of osteoclasts and osteoblasts which includes RANK ligand and BMP (Bone Morphogenetic Protein) pathways. These signals also maintain formation of osteoclasts and their survival in modeling and remodeling in normal bone.
However, these osteoclasts are negatively regulated by osteoprotegerin (Nishimura et al., 2012). These RANKL pathways
are also regulated by parathyroid hormone. Moreover, inflammatory cytokines may stimulate osteoclast formation and
resorption of bone by inducing expressions of RANKL. Many different reviews have demonstrated the protective effect of
flavonoid phytochemicals on bone protection by reducing RANKL and matrix metalloproteinases which digests bone collagen and reduces osteoblasts activities (Welch and Hardcastle, 2014).
10.2
Epidemological studies
Flavonoids are bioactive polyphenols found in fruit and vegetables. These flavonoids have anti-oxidant and antiinflammatory properties which are strongly associated with bone health. Epidemiological linkages between bone health
and flavonoid-intake have been observed in different studies. A positive link between flavonoid intake and spine, demerol
and neck bone mineral density was observed while negative link was observed between flavonoids intake and markers of
bone resorption in Scottish peri-menopausal women (Welch and Hardcastle, 2014). Moreover, catechins and flavanones
were negatively associated in resorption of bone markers.
In another study, positive association of total flavonoid intake and BMD of spine in women in TwinsUK cohort was
observed. In subclasses of flavonoids, anthocyanins were strongly linked with hip and spine BMD. Flavonols and polymers
were linked with higher BMD of hip (Welch and Hardcastle, 2014). Additionally, tea which is rich in catechins, has also
been reported for providing protection against hip fracture in different epidemiological studies (Welch and Hardcastle,
2014).
11. ANTIBACTERIAL EFFECT OF FLAVONOIDS
The flavonoids extracted from plants are also known to have a strong effect against microbial infection. They have been
found effective against microbes in many in vitro studies. Many flavonoids, such as flavone, flavonol glycosides, flavanones, apigenin, and chalcones, have been studied for their potent antimicrobial effect. However, these flavonoids have
many different cellular targets as site of action except one. The most common molecular action of flavonoids is the formation of complex with proteins through different non-specific forces such as hydrophobic effect, covalent bonding and
hydrogen bonding, so their antibacterial effect might be related with their capability to deactivate microbial adhesions, cell
envelope proteins, enzymes and many others. Beyond that, lipophilic flavonoids have the capability to disrupt microbial
membranes. Moreover, catechins (a class of flavonoid compounds) have been widely studied for their antibacterial effect.
They have been studied for in vitro-antibacterial effect against streptococcus, Vibrio cholera, shigella and many others ailments (Gerdin and Svensjö, 1982). Catechins were found to have a lethal effect against cholera toxin Vibrio cholera and
also inhibit glucosyltransferases in S mutants. Another study explained the inhibitory effect of apigenin, quercetin and
pentahydroxyflavone against E. coli (Ohemeng et al., 1993). Moreover, other flavonoids, such as Naringenin and sophoraflavanone G, were also found effective against streptococci and Staphylococous aureus. This effect of flavonoids might
be due to reduction in membrane fluidity of hydrophilic and hydrophobic regions and inner and outer layers of membranes.
Furthermore, the relationship between the antibacterial concept and membrane interference supports the idea that flavonoids can lower the membrane fluidity of bacteria (Ahmad et al., 2015). The antimicrobial effect of two other flavonoids,
licochalcones A and C extracted from roots of Glycyrrhiza inflata, were also studied against Staphylococcus aureus and
Micrococcus luteus, showing that licochalcone A has a strong inhibitory effect against incorporation of radioactive precursors in proteins, DNA and RNA (Kumar and Pandey, 2013). The antibiotics have a similar effect for inhibition of respiratory
chain, as energy is a primary component for biosynthesis of macromolecules and for absorbance of various metabolites.
Further studies suggested that CoQ and cytochrome are inhibition sites for flavonoids in bacterial electron transport chain.
Several studies support the antimicrobial effects of these phytochemicals extracted from different medicinal and edible
plants (Kumar and Pandey, 2013).
12. ANTIVIRAL ACTIVITY
Natural compounds extracted from different plant sources are gaining importance for the development of different antiviral
medicines due to their having fewer side effects. Flavonoids extracted from these compounds were recognized for antiviral
effects since 1940 and different scientific studies have revealed their efficacy. There is a pressing need to develop effec-
Flavonoids as Nutraceuticals Chapter | 8 151
tive drugs against HIV (human immunodeficiency virus). The effect of these antiviral compounds depend on inhibition of
many enzymes linked with life cycle of these viruses (Gerdin and Svensjö, 1982). Flavan-3-ol was demonstrated be much
more effective than flavonoids and flavones for their inhibition against HIV-1,2. The flavonoid Baicalin, which is extracted
from Scutellaria baicalensis, was also found effective against inhibition of HIV-1 infection and replication. Similarly, catechins were also found to inhibit the DNA polymerases of HIV-1. It has also been demonstrated that flavonoids acacetin,
chrysin, and apigenin, inhibit HIV-1 activation through a unique mode of action which usually involves prevention of viral
transcription (Cushnie and Lamb, 2005). Moreover, the anti-dengue effect of hesperetin, quercetin and daidzein was also
studied at different levels of dengue virus type-2 infection. Among these flavonoids, quercetin was observed most effective against dengue virus type-2 infection. Several other flavonoids, such as dihydrofistein, dihydroquercetin, pelargonidin
and catechin, also revealed activities effective against many types of viruses, including HSV, polio virus and sindbis virus
(Gerdin and Svensjö, 1982). Prevention of viral polymerase binding of viral capsid proteins was suggested as an antiviral
mode of action.
13.
CONCLUSION
The potential of flavonoids to prevent several ailments and diseases is well known. Good sources of flavonoids include
cocoa, tea, berries and several other fruits and vegetables. As a result of mass production and processing of food products,
a large quantity of byproducts such as seeds and peels are obtained which are a rich source of flavonoids. Due to increasing demand of nutraceutical products, and products free from synthetic ingredients, research has been focused towards the
extraction and utilization of flavonoids. The previously mentioned facts highlight the potential of flavonoids as nutraceutical agents for food manufacturing industries. Besides having nutraceutical properties, flavonoids have great potential to be
used as therapeutic agents against various diseases with no or minimum adverse/side effects.
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FURTHER READING
American Diabetes Association, 2010. Diagnosis and classification of diabetes mellitus. Diabetes Care 33 (Suppl. 1), S62–S69.
Bimakr, M., et al., 2012. Optimization of supercritical carbon dioxide extraction of bioactive flavonoid compounds from spearmint (Mentha spicata L.)
leaves by using response surface methodology. Food Bioprocess Technol. 5 (3), 912–920.
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Chapter 9
Bioactive Peptides—Impact in Cancer
Therapy
Edwin E. Martínez Leo, Armando M. Martín Ortega, Maira R. Segura Campos
Autonomous University of Yucatan, Yucatán, Mexico
1.
CANCER: A WORLDWIDE INFLAMMATORY DISEASE
Carcinogenesis is a process of successive steps occurring at a phenotypic and genetic level, identified histologically as
hyperplasia, early adenoma, late adenoma, carcinoma and metastases. Cancer cells have a high rate of mutation. It is hypothesized that “cancer is initiated in a population of rare cells with stem cell properties” (NCI, 2014). All body tissues
are derived from stem cells that are located within a specific area of each organ or tissue and are defined by their ability to
self-renew and differentiate into the cell types that make up each organ.
Stem cells are prone to the accumulation of multiple mutations that are characteristic of carcinogenesis. Stem cells
and tumor cells share important properties: self-renewal capacity, ability to differentiate, telomerase activity, activation of
antiapoptotic pathways, increased transmembrane activity and ability to migrate and metastasise (Lee and Muller, 2010).
The genesis of cancer involves certain genetic alterations that allow excessive and unregulated cell proliferation that
becomes autonomous. The damage to the genetic material leads to a failure in DNA repair, causing mutations that trigger
the expression of genes favoring the development of a cancer phenotype, and altered, uncontrolled cell replication. Any
population of cells that make up a tumor are the result of clonal expansion from a single precursor cell that has suffered
genetic damage; hence, the tumors are clonal (Mathews, 2015).
Cancer is one of the main causes of death worldwide. In 2012 there were 14 million new cases, with 8.2 million deaths
linked to cancer, and it is expected that the number of cases will increase to 22 million over the next two decades (WHO,
2014). Approximately 30% of cancer deaths are due to environmental factors and unhealthy lifestyles, which have become
synonymous with a developing, modern, capitalist, globalized and technological society. Certain factors, such as insufficient consumption of fruits and vegetables, lack of physical activity, smoking, alcohol consumption, stress and high-fat
mass index, are entirely preventable and modifiable (WHO, 2014). This is the reason that most cancer pathophysiology is
focused on the environmental aspect, which leads to disorders in the redox state of the organism and an imbalance in proinflammatory processes linked to the alteration of gene modulation on oncogenes. These factors are potentially reversible,
therefore, finding food strategies that decrease disease risk are now being studied, creating opportunities to progress the
development of potential anticancer foods.
Cancer is a complex disease that involves environmental and multigenic components in which numerous imbalances in
DNA modulation, activation of proto-oncogenes and inhibition of tumor suppressor genes are implicated (Mathews, 2015).
Proto-oncogenes are genes directly or indirectly involved in cell proliferation. Their genetic alteration leads to activation
of oncogenes. Imbalances in modulation signals of oncogenes lead to altered mechanisms of cell growth and proliferation,
which causes the transformation of a normal cell to cancer (NCI, 2014). Mutations of genes that turn into oncogenes can
either be inherited, or result from exposure to environmental substances that cause cancer (NCI, 2014). Gene mutation positively influences carcinogenesis due to an increase in their expression, providing an increased cell proliferation capacity.
There are >100 identified oncogenes, such as ERBB2 in breast and ovarian cancer, members of the RAS family in cancers
of the lung, pancreas and colon, and members of the MYC lymphoma family (Pylayeva-Gupta et al., 2011).
Tumor suppressor genes regulate replication mechanisms, their mutation causes a loss of expression of regulatory molecules, such as p53 and retinoblastoma protein (RB), and a consequent loss of growth control mechanisms, replication and
cell division (Lee and Muller, 2010) that results in cancer progression due to an imbalance between cell proliferation and
death (apoptosis).
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© 2018 Elsevier Inc. All rights reserved.
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Epidemiological, pharmacological and genetic evidence substantiate the association between inflammation and oncogene expression, due to changes in the cellular environment (Mantovani et al., 2008). Recent studies associate the effects of
chronic inflammation on genetic alterations in cancer due to epigenetic modifications, including DNA and histone methylation, which ultimately leads to silencing of tumor suppressor genes.
2. CHRONIC INFLAMMATION AND OXIDATIVE STRESS AS POTENTIAL TRIGGERS
OF CANCER
Inflammation is a complex physiological response of an organism to cell injury, which may have biologically originated
from bacteria or viruses. The chemicals that cells produce are in a state of imbalance, as in the instance of free radicals (FR),
reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Bayarsaihan, 2011). FR formation can be increased
in many pathological situations due to the action of stimulants, such as opsonized bacteria, viruses, immunoglobulins and
chemotactic peptides, among others; and exposure to various deleterious agents, such as ionizing radiation, ozone, nitrogen
dioxide, sulfur dioxide, streptozotocin, doxorubicin (adriamycin), smog, acid rain, pesticides, various radioactive compounds and cigarette smoke (Kisten et al., 2014).
The redox imbalance, generated by an increase in ROS and RNS and/or a decrease in their reduction mechanisms (antioxidant), leads to a chemical cell state called oxidative stress, in which there is an alteration in oxidative-reductive intracellular homoeostasis. There are many natural sources of oxidative stress, for example, exposure to environmental oxidants,
toxins, heavy metals, ultraviolet light (UV) and inflammation. High levels of ROS exert a toxic effect on biomolecules,
particularly DNA, proteins and lipids; leading to oxidative damage in multiple cell locations, subsequently causing various
diseases, including cancer (Reuter et al., 2010).
Normally, 95% of the O2 consumed by human cells is reduced to H2O during the generation of adenosine triphosphate
(ATP) in the mitochondrial electron transport chain. However, about 5% is converted to ROS. ROS are products of normal
cell metabolism and play a vital role in stimulating signaling pathways in animal and plant cells in response to changes
occurring in the intracellular and extracellular environment. Most ROS are generated within the cell, in the mitochondrial
respiratory chain, by the partial chemical reduction of the oxygen molecule (O2). ROS species include hydrogen peroxide
(H2O2), O2– and OH∙ (Cayuela, 2012). Under a sustained stress environment, ROS are produced over a long period of time
and can damage cell structure and function, inducing mutations and neoplastic transformation.
Cancer is an oxidative stress-induced disease. Neoplastic cells inherently produce a greater amount of ROS than healthy
cells. Cancer cells generate ROS and RNS that react with cell membranes by generating reactive aldehydes and lipid peroxidation products, such as malondialdehyde, acrolein, and crotonaldehyde, which react directly with the nitrogenous bases of
DNA, generating exocyclic DNA adducts (propane-DNA adducts, ethene-DNA adducts) (Nair et al., 2007). These adducts
are mutagenic and producers of base substitutions, generating a potentially carcinogenic environment (Kisten et al., 2014).
The direct effects of ROS, generally attributed to its high concentrations at the site of damage, include breaks in DNA
strands, mutations, aberrant DNA crosslinking and mutations in proto-oncogenes and tumor suppressor genes, thus promoting neoplastic transformation. For example, ROS can decrease the expression and enzymatic activity of DNA repair genes
and can increase DNA methyltransferase expression, leading to a global genome hypermethylation; resulting in colonic
adenomatous polyps (APC), cyclin-dependent kinase 2 (CDK2), susceptible breast cancer-1 gene (BRCA1), RB, and the
DNA repair gene, mutL homolog 1 (hMLH1) (Reuter et al., 2010).
Meanwhile, most of the NO− is synthesized by nitric oxide synthase (NOS), usually after receiving an inflammatory or
immune stimulation. The calcium-independent inducible isoform (iNOS) increases the amounts of NO formed and is only
expressed during the inflammatory process. The iNOS are induced by cytokines, such as interferon-γ (IFN-γ), tumor necrosis
factor alpha (TNF-α), interleukin-1 (IL-1), and lipopolysaccharide (LPS). These RNS can induce other reactive species by
excessive lipid peroxidation. Proteins and lipids are primary targets for oxidative attack and modification of these molecules
can increase the risk of mutagenesis (Saavedra et al., 2010). Moreover, the characteristic tumor microenvironment (TME)
in cancer can lead to increased metabolic rate, increased energy demand, and excessive mitochondrial ATP synthesis, which
collectively promote the increased production of ROS in the cell, which subsequently leads to infiltration of inflammatory
cells into the tumor tissue, contributing to oxidative stress and increasing the inflammatory process (Essick and Sam, 2010).
Several studies describe cancer patients as having higher plasma ROS concentrations compared to healthy individuals,
and a decrease in antioxidant responsedirectly related to decreased levels of glutathione peroxidase and superoxide dismutase (SOD) (Khansari et al., 2009; Stachowicz-Stencel et al., 2011).
The association between chronic inflammation, cancer, and oxidative stress is well known (Reuter et al., 2010). The
inflammatory process is an inducer of ROS and RNS and proinflammatory cytokine production (TNF-α, IL-1β, IFN-γ)
that exacerbates the same generation of oxidants, resulting in cell deregulation, prolonged inflammatory processes and
Bioactive Peptides—Impact in Cancer Therapy Chapter | 9 159
decreased antioxidant capacity, thereby facilitating a mutagenic environment (Khansari et al., 2009). However, during
inflammation, important epigenetic changes occur that favor the development of cancer (Barros and Offenbacher, 2009).
Necrosis factor kappa-beta (NF-κβ) over expressed in patients with chronic inflammation has demonstrated patterns of altered histone methylation status, leading to modified cell replication and proliferation processes (Covarrubias et al., 2015).
Several studies show that inflammation increases the risk of cancer and may promote tumor progression. In colon
cancer, chronic inflammation and colonic damage directly cause DNA alterations. Furthermore, chronic inflammation and
loss of protective mucosa increase intestinal permeability to environmental toxins and mutagens which induce mutations in
stem cells, leading to cancer (Grivennikov and Karin, 2010). Inflammation may stimulate proliferation of cells that contain
oncogenic mutations induced by carcinogens, and can result in the production of ROS and RNS by immune cells, as well
as immune stimulation mediated by ROS production in pre-malignant cells, inducing mutagenic enzymes expression and
inactivation of damaged DNA (Zeng et al., 2014). As depicted in Fig. 1, there is ample evidence that inflammation has a
role in tumor promotion (Grivennikov and Karin, 2010).
2.1
Inflammation and Metastasis
The majority (90%) of cancer deaths are due to metastasis. Immune cells are present in all tumors due to molecular changes in
the invasive front of the tumor, and are involved in various forms of direct and indirect interactions with cell metastasis. Indeed,
it has been found that the inflammatory microenvironment influences several key stages of metastasis (Rohan et al., 2014).
The process of epithelial-mesenchymal transition (EMT), which is critical for metastasis to occur, is mediated by various inflammatory cytokines (TGF-β, IL-1, TNF-α and IL-6) and may be a consequence of TNF-κβ activation. Inflammatory
signals also regulate the production and activity of various proteases, which degrade the extracellular matrix and facilitate
the invasion of cancerous cells. Chemokines can directly stimulate the migration of malignant cells through blood vessels,
FIG. 1 Inflammation is important at various stages of tumourigenesis. Inflammation may contribute to tumor initiation by inducing DNA damage
through intermediates like radical oxygen species, and via activation of epigenetic mechanisms, which lead to silencing of tumor suppressor genes. During
tumor promotion, immune and inflammatory cells produce cytokines and chemokines, which facilitate cancer cell survival, proliferation and promote the
angiogenic switch, which results in increased tumor growth (Grivennikov and Karin, 2010).
160 SECTION | B Therapeutic Foods and Ingredients
while cytokines, such as TNF-α, can increase vascular permeability (Laua et al., 2009; Lawrence, 2009). Moreover, cytokines are important for the survival, recruitment, colonization and growth of metastatic cells through the same mechanisms
that affect the growth and survival of primary tumors (Chaffer and Weinberg, 2011).
A high body-mass index (BMI) is associated with increased risk of developing cancer because the tissue contains more
macrophage infiltration and is enriched in gene expression of macrophages associated with proinflammatory pathways,
including IL-6, IL-8, CC chemokine receptor type 5 (CCR5) and peroxisome proliferator-activated receptor (PPAR).
Proinflammatory cytokines promote lipolysis and additional free fatty acid production, leading to a positive feedback for
chronic inflammation (Ito, 2007).
The recruitment of monocytes leads to their differentiation into macrophages, due to exposure to hypoxic conditions,
resulting in tumor-associated macrophages (TAMs) residing in the TME, which has a direct role in the development of angiogenesis, migration and invasion of tumor cells in the metastasis process. Furthermore, TAMs guide a tumor intravasation
outside and inside the tumor vasculature in distant organs of metastasis, including lung and bone cells. The role of TAM in
metastasis is depicted in Fig. 2 (Williams et al., 2016).
2.2
Inflammation and Tumor Cell Proliferation
Tumor growth (tumor promotion) is the sum total of a malignant cell proliferation versus cell death. Both processes are
strongly affected by inflammation and inflammatory cytokines produced by immune cells, which infiltrate the tumor, such
as IL-6 and TNF-α, as they can be used as mitogens and survival factors for premalignant cells (Horng, 2015). This tumor
growth is mediated by cytokines, which activate oncogenic transcription factors, NF-κβ and signal transducer and activator of transcription 3 (STAT3). The activation of these factors are found in 50% of all cancers, and is required to express a
variety of target genes important for tumorigenesis (Valenzuela et al., 2011).
The uncontrolled proliferation of tumor cells requires the regulation of multiple signaling pathways, including signaling
cascades involved in survival, proliferation and cell cycle progression. The most important effects of oxidants in these signaling pathways have been observed in mitogen-activated protein (MAP)/activating protein 1 (AP-1) and NF-κβ. Induction
of redox-sensitive pathways for cell proliferation is required for cell division, which has substantial energy requirements
and produces metabolic energy-generating reactions, which must be buffered to prevent oxidative damage and ultimate cell
death (Grivennikov and Karin, 2010).
Chronic inflammation is present in all stages of cancer, and the oxidative-reductive imbalances lead to increased oxidative stress, increased cell damage and a decrease in the body’s response to carcinogens.
FIG. 2 Role of TAMs and inflammation process in metastasis. TAMs: tumor-associated macrophages; IL 8: interleukin 8; EGRF: epidermal growth
factor receptor; CCR-2: chemokine receptor type 2; CFS-1: colony stimulating factor 1; VEGF: vascular endothelial growth factor (Williams et al., 2016).
Bioactive Peptides—Impact in Cancer Therapy Chapter | 9 161
3.
FUNCTIONAL FOOD AND DIETARY BIOACTIVE COMPOUNDS
Functional foods are defined as “any food in its natural or processed form that, besides its nutritional components, contains
additional components that promote health, physical ability and mental state of a person.” The International Life Sciences
Institute (ILSI) establishes that a food is functional if it can satisfactorily demonstrate that it has a beneficial effect on one or
more specific functions in the body, improving health and well-being and/or decreasing the risk of disease (Howlett, 2008).
According to the American Dietetic Association (ADA), functional foods are “foods that potentially have a beneficial effect
on health when consumed as part of a varied diet on a regular basis at effective levels, including fortified, enriched or enhanced foods” (Hasler et al., 2009). Currently, the ADA categorizes functional foods as conventional, modified, medicinal
and for special dietary uses, as shown in Table 1.
Bioactive compounds are chemicals with potential biological activities and impart functionality to a food beyond its
nutritional aspects. Bioactives are usually of plant origin and are products of the secondary metabolism of plants, and are
therefore commonly referred to as “phytochemicals” (Keith and Miller, 2013). Phytochemicals have demonstrated antioxidant, anticarcinogenic, antiinflammatory, immunomodulating and antimicrobial properties (Heber, 2004).
3.1
Functional Proteins and Bioactive Peptides
Functional proteins and bioactive peptides are proteins and peptides that can to exert specific biological effects on the body,
in addition to their nutritional value as a source of amino acids (Korhonen and Pihlanto, 2003; Rutherfurd-Markwick and
Moughan, 2005). Most bioactive peptides are spontaneously generated during in vivo protein digestion. Currently, the production of bioactive peptides from food proteins is performed by in vitro enzymatic hydrolysis, using commercial enzymes,
such as alcalase, flavourzyme, pepsin and pancreatin (Segura et al., 2013).
Peptides have been isolated from enzymatic protein hydrolysates of milk, sardines, corn, soy, egg, jelly, and beans,
among others (Korhonen and Pihlanto, 2003). The scientific literature shows that bioactive peptides can exert their action
both locally (gastrointestinal tract) and systemically, because they can pass through the intestinal epithelium and reach
peripheral tissues through the bloodstream (Segura et al., 2010).
Various peptides present antioxidant, antihypertensive, antiinflammatory, hypoglycaemic, immunomodulatory and anticancer activity in vitro and in vivo (Herrera et al., 2014a, b). The novel nutritional concept is the use of food-derived proteins and peptides to enhance a biological function and prevent or decrease the risk of disease, due to a demonstrated effect
on the environment of the regulated expression of the human genome (Panchaud et al., 2012).
Biopeptides and several proteins have been proposed to treat dental disease, mineral malabsorption, diarrhea, hypertension, thrombosis and immunodeficiencies. For example, an enteral formula containing TGF-β [Modulen IBD
TABLE 1 Categories of Functional Foods
Functional Food Category
Selected Functional Food Examples
Conventional foods (whole foods)
Garlic
Nuts
Tomatoes
Modified foods
Fortified
Calcium-fortified orange juice
Iodised salt
Enriched
Folate-enriched breads
Enhanced
Energy bars, snacks, yoghurts, teas, bottled water, and other functional foods formulated with
bioactive components such as lutein, fish oils, Ginkgo biloba, Hypericum perforatum (St John’s
wort), saw palmetto, and/or assorted amino acids
Medicinal foods
Phenylketonuria formulas free of phenylalanine
Foods for special dietary use
Infant foods
Hypoallergenic foods, such as gluten-free and lactose-free foods
Weight-loss foods
Source: Hasler, C.M., Brown, A.C., ADA, 2009. Position of the American Dietetic Association: functional foods. J. Am. Diet Assoc. 109, 735–746.
162 SECTION | B Therapeutic Foods and Ingredients
(Nestle, Vevey, Switzerland)] is effective in treating Crohn’s disease, inducing clinical remission and healing mucosa, as a
result of its antiinflammatory effect (Hartman et al., 2009; Afzal et al., 2004).
Commercially, bioactive peptides are fundamental constituents of products or ingredients of functional foods, such as
the dairy product, Calpis® cultured milk (trademark AMEEL®) marketed in Japan by Calpis Co., which has hypotensive
action associated with valyl propyl proline (VPP) and isoleucyl propyl proline (IPP) biopeptides; and the CholesteBlock®
drink marketed in Japan by Kyowa Hakko, with hypocholesterolaemic action associated with isolated soy biopeptides
(Hartmann and Meisel, 2007).
This review examines the effect of antioxidant and anti-inflammatory peptides that could be used as a alternative dietary
cancer treatment.
3.2
Proteins and Bioactive Peptides with Antiinflammatory and Immunomodulatory Activity
Studies of peptides with antiinflammatory activity are limited; however, among the described mechanisms of action, inhibition of the processes that regulate superoxide ion production by neutrophils during the acute phase response in inflammation, is important. This also revealed an important role in the inhibition of leukocyte elastase, which is responsible for the
degradation of collagen and proteoglycans and high phagocytosis, for example, in rheumatoid arthritis, emphysema, cystic
fibrosis, bronchitis, asthma, acute respiratory distress syndrome and other conditions of chronic inflammation (Drago et al.,
2006; Herrera et al., 2014a; Mulero et al., 2011).
Various foods have been proven to decrease the production of certain proinflammatory cytokines important for the development and complication of cancer, such as egg biopeptides (Leea et al., 2009), blueberries (Laua et al., 2009) and the
Momordica grosvenori Swingle fruit (Pana et al., 2009), which exhibit a significant decrease in TNF-α, IL-6, IL-1β, IFN-γ,
IL-8, IL-17, NOS and cyclooxygenase-2 (COX-2) expression.
Peptides present in egg yolk have antiinflammatory activity in LPS-stimulated RAW 264.7 macrophage cells, modulating the expression of proinflammatory cytokines and promoting antiinflammatory cytokine (Xua et al., 2012). Among the
antiinflammatory peptides, those with immunomodulatory activity are of particular interest. The most studied of these are
derived from dairy products. This effect seems to be related to the net positive charge of these peptides, which are structurally organized and cause the formation of ion channels in the membrane of bacterial cells, leading to lysis of the plasma
membrane and cell death (Bellamy et al., 1992).
Whey has been shown to have antioxidant and immunomodulatory activity (Marshal, 2004). The most accepted mechanism of action associates these activities with a high content of cysteine-rich proteins that contribute to the synthesis and,
consequently, increased levels of glutathione, a powerful intracellular antioxidant. Glutathione is also necessary for the
activity and proliferation of immune cells, particularly T-cells (Harris et al., 2015).
Clinical studies have shown that administration of whey protein concentrate, in addition to conventional treatments, was
useful in the treatment of AIDS, hepatitis B and hepatitis C (Bayford, 2010). In accordance with these findings, administration of such concentrates in AIDS patients is reported to potentially offset the common, glutathione deficiency in these
patients (Moreno et al., 2006; Micke et al., 2001). However, results are inconclusive because the duration of treatment and
dose of whey used among studies are inconsistent.
The human milk proteins have shown significant immunomodulatory benefits. This is mainly attributed to the casein
phosphopeptides and lactoferrin contents, which show resistance to digestion and maintained activity after processes such
as pasteurization (Wada and Lönnerdal, 2015).
Studies in animals and cell models suggest that lactoferrin possesses immunomodulatory activity (Siqueiros-Cendón
et al., 2014). These studies indicate that lactoferrin may have dual mechanisms of action, the first involves the inhibition
of the production of various cytokines, such as TNF-α and IL-1β. The existence of lactoferrin receptors on monocytes,
lymphocytes, macrophages, neutrophils and epithelial cells suggest that lactoferrin may directly affect the regulation of
cytokine production by regulating signaling pathways mediated by these receptors. The second mechanism of action could
be related to inhibition of innate immunity stimulation, by binding to lipid A of bacterial LPSs, as well as oligonucleotides
containing unmethylated CpG oligodeoxynucleotides, thereby inhibiting stimulation of Toll-like receptors from macrophages (Onishi, 2011).
Consistent with its immunomodulatory activity, lactoferrin is able to inhibit local inflammatory responses to skin inflammation in humans and modulate the expression of proinflammatory genes in mouse intestine (Wakabayashi et al.,
2006), as well as in animal models of skin inflammation, mediated by allergens and intestinal inflammation with a direct
action on the levels of proinflammatory cytokines and IL-10, after 24 h of administration (Takakura et al., 2006).
Currently, there are transgenic cows expressing the human lactoferrin gene and, therefore, produce human lactoferrin in the milk (Thomassen et al., 2005). The antimicrobial capacity, antiviral or immunomodulatory are not exclusive to
Bioactive Peptides—Impact in Cancer Therapy Chapter | 9 163
l­ actoferrin or ovotransferrin, but include peptides resulting from their digestion, such as lactoferricin (LFcina) (Wong et al.,
2014) or ovotransferrin A, a 92-amino acid (OTAP-92) peptide, which retains this activity (Ibrahim et al., 2014).
LFcina is a peptide corresponding to the amino-terminal of lactoferrin, which has a bactericidal activity as potent as lactoferrin itself. It has been shown that this peptide also possesses antiviral and immunomodulatory activity. Indeed, in addition
to the mechanisms described for immunomodulation lactoferrin, LFcina can inhibit the action of cytokines and released ILs,
such as IL-6. It also has the ability to bind DNA, enter the cell, cross the nuclear membrane and act as a transcription factor.
Finally, lactoferricin has the ability to potentiate the effect of antiviral and antibacterial agents (Wong et al., 2014).
Lysozyme is another protein known for its immunomodulatory activities, which inactivates many microorganisms by
binding to the bacterial wall and breaking the bond between the β-1,4 N-acetylglucosamine and N-acetylmuramic acid.
Lysozyme has immunoregulatory functions, which, when combined with immunotherapy, enhances the immune response
in immunosuppressed cancer patients (Sava, 1996). It has been suggested that the lysozyme produced by immunomodulation may result from stimulation of the phagocytic function, and peptidoglycan hydrolysis products can act as adjuvants or
immunomodulators (Li-Chan and Nakai, 1989).
3.3
Protein and Bioactive Peptides with Anticancer Activity
Several studies have demonstrated, in animal models and in vitro, that whey proteins have anticancer activity. The mechanism of action appears to be related, as previously mentioned, with increase in glutathione synthesis, with consequent stimulation of immunity and antioxidant activity (Xiao et al., 2005). In addition, glutathione is a substrate of two kinds of enzymes:
a selenium-dependent glutathione peroxidase and an enzyme belonging to the glutathione transferase family. Both favor
elimination of mutagens and carcinogens, which can promote development of cancer (Micke et al., 2001). Glutathione has
also been shown to exert iron chelating activity. Iron can act as a mutagen, causing oxidative tissue damage. The anticancer
activity of whey proteins has also been associated with inducing the production of somatostatin, a known antiproliferative
agent in colon cancer (Wada and Lönnerdal, 2015). However, there are very few human clinical studies to corroborate the
results obtained in vitro or in animal models, and the obtained results are ambiguous so further studies are necessary.
The anticancer activity of human lactoferrin has been widely studied. Phase I clinical studies, in which lactoferrin was
administered to patients with refractory solid tumors, proved to be nontoxic with a dose tolerance of 1.5–9 g/day (Hayes
et al., 2006).
In animal models and in vitro it has been shown that bovine lactoferrin has anticancer activity, inhibiting both tumor
growth and metastasis formation. Thus, it has been shown to inhibit carcinogenesis in the colon, esophagus, lung and bladder when administered to rats orally in the post-initial cancer state. Concerning their mechanism of action, intestine bovine
lactoferrin enhances the immune response, inducing caspase-1 activity with consequent production of mature IL-18, resulting in potentiation of antitumor activity T- and NK-cells. Consequently, these cells can produce IFN-γ which, together
with IL-18, can inhibit angiogenesis. Although the mechanism by which lactoferrin results in the activation of caspase-1
and IL-18 is unknown, it may occur by activating specific receptors within the bowel, epithelial and immune cells. In this
regard, it is reported that lactoferrin may induce selective apoptosis of cancer cells by binding to these specific receptors
(Iigo et al., 2005; Mahanta et al., 2015).
Lfcina has antitumor activity, with the ability to inhibit the formation of metastases and induce selective apoptosis of
cancer cells. Thus, the active Lfcina mitochondrial apoptotic pathway is at least partly related to the generation of ROS
mechanisms (Riedl et al., 2015).
3.4
Protein and Bioactive Peptides with Antioxidant Activity
Peptides with antioxidant activity are obtained from various proteins. Protein hydrolysates and peptide fractions can be
used as functional ingredients in food systems to reduce oxidative changes during storage. Antioxidant peptides may limit
oxidative damage in foods (act as natural antioxidants) and protect cells against in vivo oxidation when ingested (Vioque
et al., 2006).
There are many studies describing the antioxidant potential from various peptides in food. These peptides are considered to have the potential to control oxidative processes in the human body (Samaranayaka and Li-Chan, 2011). The possible use of antioxidants during cancer treatment is not conclusive because they might interfere with drug treatment; various
studies point to the role of certain antioxidant compounds in preventing cancer.
Most studies related to the intake of antioxidants are epidemiological, and evaluate the decrease in cancer risk attributable to the intake of foods rich in antioxidant compounds (Sharma et al., 2009). Few studies examine the effects subsequent
to cancer diagnosis.
164 SECTION | B Therapeutic Foods and Ingredients
Preclinical studies have shown that some antioxidants can protect cells from the damaging effects of chemotherapy and
radiotherapy and, therefore, decrease the high toxicity of these treatments. However, this protective effect would also apply to neoplastic cells and may decrease the effectiveness of treatment (Viñas et al., 2012). Peptides can be used to prevent
cancer and other diseases by their ability to modulate antioxidant enzymes as an important regulator of proinflammatory
processes in the organism. Similarly, various peptides have shown an antioxidant effect in extracellular fluids, which helps
balance the redox state of the organism.
Some antioxidant peptides, such as those structurally related to Pro-His-His, may exert a strong synergistic effect with
certain other antioxidants, such as phenolic compounds (Saito et al., 2003). In potato, three biopeptides (Phe-Gly-Glu-Arg,
Phe-Asp-Arg-Arg and Phe-Gly-Glu-Arg-Arg) have been identified with an inhibitory effect on linoleic acid oxidation at
55.3%, 58.5% and 61.7%, respectively, using a β-carotene bleaching test. Oral administration of these peptides at doses
of 100 mg/kg in Wistar rats decreased ethanol-induced damage to the stomach lining by 67.9%, 57.0% and 60.3%, respectively (Kudoa et al., 2009).
In marine organisms, peptides displaying significant antioxidant activity have been found, including the hydrolysates of
tilapia surimi (Sun et al., 2013). These peptide fractions exhibit amino acid sequences rich in Trp, Met, Cys and Tyr. Trials
in liver cancer cell lines (HepG2) treated with H2O2 showed that one of the three protein fractions exhibited a significant
decrease in ROS, compared to controls (Wiriyaphana et al., 2013).
Pinctada fucata-derived peptides demonstrated the ability to restore antioxidant activity in vitro by inducing the activity
of antioxidant enzymes such as SOD, catalase (CAT) and glutathione peroxidase (GSH-Px), and decreasing lipid peroxidation (Wua et al., 2013).
Other foods, such as palm oil (Changa et al., 2015), sweet potatoes (Zhang et al., 2014) and egg (Memarpoor-Yazdia
et al., 2012), also contain peptides with proven antioxidant activity and, in some studies, fractionated and ultrafiltered protein hydrolysates are reported to show differences in antioxidant activity. In corn gluten, fractions <10 kDa exhibited higher
antioxidant activity (Zhuanga et al., 2013), while in Mucana pruriens (velvet bean) increased in vitro antioxidant activity
was most potent for peptides <1 kDa (Herrera et al., 2014a, b).
Although there are still no studies on the role of antioxidant peptides in cancer patients, their mechanisms of action
and potential chemopreventive benefits are expected to be similar to that of other bioactives displaying antioxidant activity. However, unlike other compounds, peptides could favor the modulation of antioxidant enzymes. The development of
research in this area opens the possibility to develop new food and innovative treatments for cancer treatment, providing
robust scientific evidence of their effectiveness on the physiological status of people with cancer can be proven.
4.
CONCLUSION
Chronic inflammation and oxidative stress are directly involved in the development of chronic diseases, such as cancer.
People with cancer have elevated levels of free radicals and proinflammatory cytokines, and low plasma concentrations of
antioxidants. Therefore, a diet based on functionality, rather than nutrition alone, will be crucial to improving the physiological state of cancer patients. The use of functional foods offers patients a wider choice of treatment options. Currently,
the development of bioactive peptides for cancer are an important ally in its prevention and treatment, and directly relates
to their effects on inflammation and oxidative stress. Although more studies are needed to define the effectiveness of dietary peptides in the physiology of cancer, many of their mechanisms of action are already defined and, therefore, these
molecules have a potential therapeutic role in this disease.
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Chapter 10
Essential Oils: Biological Activity and
Therapeutic Potential
Leila Mehdizadeh, Mohammad Moghaddam
Ferdowsi University of Mashhad, Mashhad, Iran
1.
INTRODUCTION
Plants have long been recognized as providing potential sources of different classes of chemical components, known as
phytochemicals, such as terpenoids, alkaloids, phenolics, glucosides, which are effective products for control or treatment
of various diseases. Essential oils and their major compounds, monoterpenes, are among the most promising classes of
natural products that can be used as safer pest and disease control agents (Bassolé and Juliani, 2012).
Essential oils have various biological activities and therapeutic effects, which can be used in different industries and
treat several disorders in humans, animals, plants, and foods. Essential oils (EOs) are oily, hydrophobic, aromatic, and volatile liquids that can be extracted from plants. They may be derived from specialized cells or groups within particular regions
of the plant, such as stems, leaves, the foliage, bark, wood, fruit, seeds, and rhizomes (Miguel, 2010). Essential oils (EOs)
are usually obtained by steam distillation. They are complex mixtures of components, including terpenic derivatives, with
well-known aromatic properties, and contain a range of oxygenated and nonoxygenated terpene hydrocarbons. Different
essential oils of plant species have various therapeutic potential. Some essential oils, such as aniseed, calamus, camphor,
cedar wood, cinnamon, citronella, clove, eucalyptus, geranium, lavender, lemon, lemongrass, lime, mint, nutmeg, orange,
palmarosa, rosemary, basil, vetiver, and wintergreen, have been traditionally used by people for various purposes in different parts of the world (Freires et al., 2015).
Essential oils enter the body in different ways, such as inhalation, absorption via skin and ingestion, and exert their
properties in the body. Different essential oils from plants indicate several useful therapeutic characteristics, such as
analgesic (peppermint, lemongrass, clove, rosemary); antibiotic (tea tree, lavender); antifungal (tea tree, lemongrass);
anti-­inflammatory (yarrow, German chamomile, lavender, clove); antiseptic (tea tree, lavender); antispasmodic (marjoram, rosemary, peppermint, cedarwood); antiviral (melissa, tea tree, lavender, lemon); aphrodisiac (black pepper, jasmine, rose, sandalwood); astringent (cypress, frankincense, sandalwood); carminative (peppermint, fennel, chamomile,
­melissa); ­cytophylactic (­lavender, cedarwood, frankincense); diuretic (juniper, grapefruit, lemon); euphoric (neroli, Roman
­chamomile, jasmine, orange, grapefruit, rose); emmenagogue (rose, geranium, basil, clary sage, chamomile, rosemary,
ginger, marjoram, juniper); expectorant (eucalyptus, thyme, fennel, rosemary, cypress, sandalwood, cedarwood, pine, clary
sage); hormonal (generally) (jasmine, clary sage, fennel, geranium, rose, neroli); laxative (peppermint, black pepper, marjoram, fennel, orange, pine); rubefacient (rosemary, peppermint, black pepper, lemongrass); sedative (lavender, Roman
chamomile, German chamomile, carrot seed, vetiver, valerian); sudorific (basil, cardamom, ginger, rosemary, black pepper);
vasoconstrictor (cypress); vasodilator (black pepper, eucalyptus, marjoram, rosemary); vulnerary (lavender, frankincense);
antiphlogistic (german chamomile); spasmolytic (carway, coriander); antinociceptive (summer savory, polygermander)
(Hajhashemi et al., 2002, Abdollahi et al., 2003); immunomodulatory (ginger, sage, clove) (Carrasco et al., 2009); psychotropic (parsley); acaricidal (lavender); and cancer-suppressing activities (sage) (Price and Price, 2007).
The recent trend of using natural compounds in medicines and food preservation has led to an increasing interest in
EOs application. Essential oils and their components also possess antibacterial, antifungal, antiviral, insecticidal, and antioxidant properties. These activities can be mediated by single compounds or groups of compounds, and these secondary
metabolites have biological functions in the plants from which they originate, such as protection against predators and
microbial pathogens, as well as involvement in defense mechanisms against abiotic stress (Bassolé and Juliani, 2012; Lang
and Buchbauer, 2012).
Therapeutic, Probiotic, and Unconventional Foods. https://doi.org/10.1016/B978-0-12-814625-5.00010-8
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168 SECTION | B Therapeutic Foods and Ingredients
Due to the broad spectrum of biological, antimicrobial, and other beneficial effects, essential oils have been used as a
medicinal agent for over thousands of years. Previously, we studied biological activity and chemical composition of essential oils from different plants. For example, antibacterial activity of Ocimum ciliatum (Moghaddam et al., 2014); antifungal
activity of Echinophora platyloba (Moghaddam et al., 2015) and Mentha piperita (Moghaddam et al., 2013); and the antioxidant activity of Cuminum cyminum (Moghaddam et al., 2015). In addition, we collected the results on antifungal activity
of essential oil obtained from previous research (Moghaddam and Mehdizadeh, 2016).
Primarily, essential oils are used in flavoring and fragrance, such as the food industry to affect the organoleptic quality of the foods, as well as in medicinal productions. Over time essential oils of plants have evoked interest as sources of
natural products. In general, in order to achieve a significant antimicrobial and/or antioxidant activity, a relatively high
concentration of EOs are required (Gutierrez et al., 2008). Thus, plants like oregano, thyme, garlic, bay leaf, rosemary, and
clove, or their EOs, can be used alone or in combination with other preservatives, to improve the shelf life of food products
(Yerlikaya and Gokoglu, 2010).
In this manuscript the biological activity of essential oils, especially their antibacterial and antifungal activities, are
reviewed.
2. APPLICATIONS OF ESSENTIAL OILS
Although the use of plant essential oils have been primarily studied in the medical fields, the natural properties of essential
oils and their effectiveness in new applications have been explored and developed recently, especially in food, cosmetic,
and public health fields. In particular, essential oils have been studied for their potential use as alternative remedies for the
treatment of many infectious diseases(Freires et al., 2015).
Essential oils from various herbs and spices have been widely acknowledged as food flavorings and preservatives agents
because of their inherent aromatic and antimicrobial constituents. They have great potential to be natural preservatives
that can be added in almost all foods, due to their high efficiency at very low concentrations (Roller and Seedhar, 2002).
Moreover, food safety is usually ensured by the addition of antimicrobials that prevent, or considerably retard, microbial
organisms prompting spoilage. A variety of different synthetic fungicides and chemicals, such as benzimidazoles, aromatic hydrocarbons, and sterol biosynthesis inhibitors, with various degree of persistence for many years, are employed
as antifungal agents to inhibit the growth of plant pathogenic fungi. In many areas around the world, the extensive use
of these chemicals has led to the development of resistance. In order to overcome this problem, higher concentrations of
these chemicals were used, but widespread use of pesticides has significant drawbacks including cost, handling hazards,
pesticide residues, and threats to human health and the environment. Moreover, some synthetic pesticides can also cause
environmental pollution, because of their slow biodegradation in the environment, and decline human health (Isman, 2000).
Development of new safe and biodegradable alternatives as natural fungicides has increased. Therefore, there has been a
growing interest in finding safer alternative materials to replace synthetic chemical products, and research of the possible
use of natural products and plant secondary metabolites, such as plant-based essential oils and extracts for pest and disease
control in agriculture, which may be less damaging for pest and disease control.
3. ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS
Plant essential oils have stimulated attention among the naturally-occurring bioactive agents with favorable antimicrobial
activity (Galvão et al., 2012; Bassolé and Juliani, 2012). They are a potentially useful source of antimicrobial compounds. It
is often quite difficult to compare the results obtained from different studies, because the compositions of the essential oils
can vary greatly depending upon the geographical regions, variety or age of the plants, and methods of drying and extraction of the oil (Cakir et al., 2004). The antimicrobial activities of the essential oils of various plant species have been studied
before. For example antimicrobial activities of essential oils from C. cyminum (Naeini et al., 2009); Geranium macrorrhizum (Radulović et al., 2010); Agastache sp. (Ownagh et al., 2010); Hypericum maculatum (Ðorđević et al., 2014); clove
and eugenyl acetate (Vanin et al., 2014); and other plants have already been scrutinized for their antimicrobial properties
and activity.
4.
IN VITRO METHODS FOR QUANTIFYING ANTIMICROBIAL ACTIVITY
Several methods are used to investigate the antimicrobial activity of essential oils. This review will focus on the most important tests, including agar absorption assay, disc and well diffusion assay, agar- and broth-dilution methods, and vapor
phase tests.
Essential Oils: Biological Activity and Therapeutic Potential Chapter | 10 169
4.1 Agar Absorption Assay
Twenty milliliters of nutrient agar (Oxoid) plates invert and allow drying in a 37°C incubator for 30 min. Five hundred
microliters of the selected oil, then pipette onto the surface of the agar. A sterile glass spreader is used to spread the oil
over the surface of the agar plate, and to work the oil into the agar. Plates are subsequently left upright at room temperature
for 30 min. Any plate with visible oil on the surface follows with drying in a 37°C incubator for 30 min. If oil is still visible, the plate is left upright at an angle on the bench overnight, to allow excess oil to drain to one side. The following day,
oil is removed using a sterile disposable 1 mL pipette. Five hundred microliters of the selected bacteria is spread over the
essential oil treated nutrient agar plate. The plate incubates overnight at 37°C and the bacterial colonies are then counted
(Hood et al., 2003).
4.2
Disc Diffusion Assay
The disc diffusion assay is one assay to perform, and is used as a screening tool. In this assay, an agar Petri dish is inoculated with the relevant test organism, then a small amount of oil is placed onto a paper disc, in this case small holes
are punched into the agar surface, or put on a small paper disc, which afterward is placed onto the agar. The diffusion of
the oil through the agar creates a zone adjacent to the agar or disc where microbial growth is prevented due to the high
concentration of oil. The antimicrobial activity can be estimated from the size of the originating inhibition zone (Böhme
et al., 2014).
4.3 Well Diffusion Assay
Nineteen milliliters of molten nutrient agar inoculated with 0.5 mL for an overnight culture of microorganisms. The inoculated agars are then poured into Petri dishes and allowed to set. Four wells are created using a 6 mm cork borer. Hundred
microliters of undiluted oils or nutrient broth is placed into these wells. The plates incubate at 37°C overnight and the zones
of inhibition are recorded the following day (Hood et al., 2003).
4.4 Agar- and Broth-Dilution Methods
Agar- and broth-dilution methods are also commonly used for assessing activity, whereby a series of dilutions of the oil is
performed in the relevant growth medium. After inoculation of the assay and incubation, the presence or absence of growth
is recorded.
4.4.1 Agar Dilution Assay
Fifteen milliliters of molten nutrient agar aliquot is poured into sterile plastic petri dishes and allowed to set. A 5 mL agar
overlay, containing a final concentration of 2% essential oil, with and without 0.02% Tween 80, are added to each plate.
Two control plates are used, one with agar alone, and another with agar plus 0.02% Tween 80 (final concentration). The
plates are then inoculated by streaking a single colony of each bacterium onto the surface of the agar and incubated at 37°C
overnight. Bacterial growth is measured on a scale of zero (no growth) to four (growth of control) (Hood et al., 2003).
4.4.2
Broth Dilution Assay
In the broth dilution test, concentration series of the antimicrobial substance are established using a broth medium,
which is seeded with microorganisms. The oil emulsifies into the aqueous test media by the following method. The
essential oil is added to a sterile Eppendorf tube (Sarstedt) and 1/10 of the oil’s volume of a 10% solution of Tween 80
in water is added, the solution is mixed via vortex. Aqueous solution (e.g., nutrient broth) is then added in 10–20 μL
aliquots, with brief vortexing between each addition. This continues until the ratio of aqueous solution to oil is brought
to 2:1, and a final volume of 4.5 mL. An overnight bacterial culture (500 μL) is then added to each tube. Three control
tubes, one containing 0.02% Tween 80 in nutrient broth, one containing 100 μL of oil, 0.02%Tween 80, and nutrient
broth, and one containing only nutrient broth are also prepared. The test tubes then incubate with shaking at 37°C for
12 h, after which each suspension is wasserially diluted (10-fold) with sterile nutrient broth to a final concentration
of 10−7. Five hundred microliters of the 10−5, 10−6, and 10−7 dilutions are spread out onto nutrient agar plates using
an alcohol flamed glass spreader. The plates then incubate overnight at 37 °C, and the bacterial colonies are counted
(Hood et al., 2003).
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4.5 Vapor Phase Test
Up to now, there is no standardized method available for the vapor phase test. In general, a seeded agar plate is placed
upside down onto a reservoir that comprises a certain amount of volatile oil. In this case, the generated inhibition zone is
considered as criterion for the antimicrobial activity (Morris et al., 1979).
5.
PERCENT INHIBITION OF MYCELIA GROWTH
When the growth of the colony in the control treatment reaches the edge of the plate, the diameters of both treatments and
control are measured. Fungistatic effect is expressed in terms of mycelia growth inhibition (%). The percentage of inhibition of fungal growth is determined on the growth in test plates, and compared to the respective control plates.
The inhibition rate could be estimated according to the formula of previous studies:
I = DT - Dt / DT´100
where I is the inhibition rate of mycelia growth of tested fungal isolates (%), DT is the diameter of the mycelia growth
area in the control medium (mm), and Dt is the diameter of the mycelia growth area in the treated medium (mm) (Jing et al.,
2014; Messgo-Moumene et al., 2015).
6.
DETERMINATION OF MIC, MBC, AND MFC
Agar- and broth-dilution methods allow the minimum inhibitory concentration (MIC) of an oil to be determined. MIC is evaluated in order to determine the antimicrobial potency of the tested substance (Varela et al., 2008). The MIC can be defined as the
lowest amount of essential oil required to inhibit the visible growth of the test organism. A method described by Charles et al.
(1989) for determining the nature of toxicity (fungistatic and/or fungicidal effect) of the essential oils against fungi, to identify
if each essential oil has only fungistatic activity on the pathogens, or if it could also have a fungicidal effect. The inhibited
fungal mycelia plugs of the essential oil treated PDA plates reinoculate into fresh medium; revival of their growth determines
which concentration of essential oil has fungicidal effect on the tested fungi. The minimum concentration of oil required to inhibit mycelia growth of fungi is different. It is evident that the inhibitory effect of oil on mycelia growth of fungi varies among
fungal species, and differences between fungi in their resistance to essential oils has been found (Moghadam et al., 2016).
When a broth dilution method is used, the sampling of each oil dilution to quantify the surviving organisms allows for
minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) to be determined. The MBC and
MFC refer to the minimum concentration of oil required to kill 99.9% of the cells originally inoculated into the assay for
bacteria and fungi, respectively. Like the disc-diffusion assay, dilution methods are also subject to interlaboratory variation,
but because the assay outcome is quantified in terms of oil concentration rather than zone size, values obtained by different
researchers are more easily compared. With these assays, variables, such as the presence of a solubilizing agent (Böhme
et al., 2014) or choice of test organism, may also influence the result. Furthermore, data will be expressed as a percentage
(vol/vol) to enhance the comparability of results (Hammer and Carson, 2011a; Böhme et al., 2014).
7.
EFFICACY OF ANTIBACTERIAL ACTIVITY OF ESSENTIAL OILS AND COMPONENTS
Some essential oils from various plant species have very strong antibacterial potential for food and pharmaceutical industries (Osei-Safo et al., 2010; Cavar et al., 2012; Teixeira et al., 2013). Antibacterial activities of the essential oils from
several plants have been previously investigated. For example, in vitro antibacterial activities of essential oil of Ocimum
basilicum (Wannissorn et al., 2005); oregano and thyme (Fadli et al., 2012) against numbers of gram-negative and positive
bacterial species were reported.
7.1
Plant Studies
The inhibitory effects of O. ciliatum essential oil against phytopathogenic strains, including Ralstonia solanacearum, Pseudomonas
syringae pv. lachrymans, P. syringae pv. syringae, Pseudomonas tolaasii, Xanthomonas oryzae pv. oryzae, Xanthomonas citri,
Brenneria nigrifluens, Pantoea stewartii subsp. indologenes, Agrobacterium vitis, and Rhodococcus fascians, were investigated,
which indicated that the oil had antibacterial activity against all of the tested bacteria (Moghaddam et al., 2014). Additionally, the
Scabiosa arenaria oil was tested against 16 g-positive and gram-negative bacteria. It was found that the oils exhibited interesting
antibacterial activity, comparable to those of thymol, which was used as a positive control (Besbes et al., 2012).
Essential Oils: Biological Activity and Therapeutic Potential Chapter | 10 171
7.2
Human Studies
The antibacterial activities of EOs and their isolated constituents were investigated with a view towards their potential
applicability in new dental formulations. A limited number of clinical trials reported the effects of EOs on caries-­related
streptococci (mainly Streptococcus mutans) and lactobacilli. The most promising species with antibacterial potential
against cariogenic bacteria are: Achillea ligustica, Baccharis dracunculifolia, Croton cajucara, Cryptomeria japonica,
Coriandrum sativum, Eugenia caryophyllata, Lippia sidoides, Ocimum americanum, and Rosmarinus officinalis. In some
cases, the major phytochemical compounds determined the biological properties of EOs. Menthol and eugenol were considered notable compounds, demonstrating an antibacterial potential (Freires et al., 2015).
According to the preceding investigations, A. ligustica All. (ligurian yarrow) (Cecchini et al., 2012), B. dracunculifolia
DC (broom weed), C. sativum L.(coriander), L. sidoides Cham. (rosemary-pepper), Mikania glomerata Sprengel (guaco)
and Siparuna guianenses Aubl. (wild lemon) (Galvão et al., 2012) have shown antibacterial activity against Streptococcus
mutans. Moreover, R. officinalis L. (rosemary) (Bernardes et al., 2010) and E. caryophyllata L. (clove) (Freires et al., 2015)
have demonstrated antibacterial activity against Streptococcus sobrinus. The antibacterial properties of Achillea cretica
justified its use in traditional medicine for the treatment of wounds contaminated through bacterial infections (Küçükbay
et al., 2012).
8.
EFFICACY OF ANTIFUNGAL ACTIVITY OF ESSENTIAL OILS AND THEIR COMPONENTS
The fungal agent contaminates leaves, stems, flowers and fruits of plants, either by direct penetration or through wounds.
High humidity, particularly the available moisture on the plant surface, and low temperatures, can incite infestation of
fungi. Pathogenic fungi infect crops and foods and cause significant yield reduction and economic losses. Numerous studies on the analysis of antifungal action of various essential oils showed that they had varying degrees of antifungal effect
against different phytopathogenic fungi (Bajpai et al., 2013).
Essential oils have exhibited significant antifungal activities. These inhibitory effects have been observed on different
types of fungi, such as dermatophytic and phytopathogenic fungi, molds, and yeasts, which caused different post-harvest
diseases, animal and human disorders (Banihashemi and Abivardi, 2011; Lang and Buchbauer, 2012).Various essential oils
could be useful as alternative substances to replace synthetic fungicides in plant disease management (Gwinn et al., 2010;
Nguefack et al., 2013).
Several studies have been published to confirm the effect of essential oils and their major compounds on pathogenic
fungi. Aromatic plants, such as members of Asteraceae, Lamiaceae, Rutaceae, and Verbenaceae, contain essential oils
which have bioactivity against fungi (Edris, 2007). Reports from previous researches showed that the essential oils of
Thymus vulgaris and M. piperita have antifungal activity against Rhizoctonia solani. In addition, antifungal activities of
the essential oils of thyme (Abdel-Kader et al., 2011) against Macrophomina phaseolina, spearmint against Fusarium
oxysporum f. sp. radicis cucumerinum (Nosrati et al., 2011), Syzygium aromaticum against F. moniliforme, F. oxysporum
(Rana et al., 2011), and Fusarium sp.(Borrego et al., 2012), F. oxysporum, Fusarium verticillioides, and Fusarium avenaceum (Ćosić et al., 2010), Aspergillus sp., Mucor sp., Trichophyton rubrum and Microsporum gypseum (Rana et al., 2011),
Aspergillus niger, Aspergillus clavatus, Penicillium sp. (Borrego et al., 2012), and Botrytis cinerea (Siripornvisal et al.,
2009), Candida, Aspergillus, and dermatophyte species (Thosar et al., 2013) were previously demonstrated.
8.1
Food and Human Studies
Over the last two decades, fungal infections have increased greatly because of the increasing size of the population at risk,
including patients who are immune compromised, receiving parenteral hyperalimentation and/or broad-spectrum antibiotics and intravascular catheter users (Bouza et al., 2008). Many pathogens, such as B. cinerea (gray mold rot), F. oxysporum
(vascular wilt), Colletotrichum capsici (fruit rot), Sclerotinia sclerotiorum (water soaked spot), and Fusarium solani (fruit
rot), reduce the shelf life and market value of foods, render them unfit for human consumption, and cause undesirable
effects on human health. Additionally, the mycotoxins which are produced by fungi cause serious health problems, and
many people in underdeveloped countries are exposed to the harmful effects of these pathogens, such as Fusarium and
Aspergillus sp. (Moghadam et al., 2016). Furthermore, Candida albicans is responsible for the majority of yeast infections
in humans (Cavaleiro et al., 2015).
The antifungal activity of Angelica major oil against clinically relevant yeasts and molds was evaluated, and exhibited a broad-spectrum antifungal activity, including all tested fungi (animal and human pathogenic species or spoilage
fungi): Candida sp., Cryptococcus neoformans, Aspergillus sp., and dermatophytes (Cavaleiro et al., 2015). Previously
172 SECTION | B Therapeutic Foods and Ingredients
the ­inhibition efficacy of A. ligustica essential oils against Bacillus cereus, Streptococcus pyogenes, and C. albicans were
evaluated (Cecchini et al., 2012). In addition, the in vitro antifungal activities of essential oils from C. cyminum and
S. arenaria were investigated in order to evaluate their efficacy against Candida sp., C. albicans, and phytopathogenic fungal strains (Besbes et al., 2012; Safavi-Naeini et al., 2014). Furthermore, as a fumigant in food systems, the essential oil of
C. cyminum provided sufficient protection of food samples against fungal association, without affecting seed germination
(Kedia et al., 2014).
8.2
Plant Studies
In developing countries the pre- and post-harvest losses in world crops may occur due to fungal disease. Medicinal plants
are rich sources of biologically active compounds. There has been an increasing interest in looking at antifungal properties
of natural products from aromatic plants, particularly essential oils. Essential oils are favorable natural antifungal agents,
with potential applications in agro industries, to control phytopathogenic fungi which are causing severe destruction in
crops. Therefore, it is reasonable to expect many essential oils and their constituents are found to exhibit antifungal properties, but the high cost of production of essential oils, and the low concentration of active components, often prevent their
direct use in the control of fungal diseases of plants and animals. In spite of this limitation, currently there is much research
being performed for the development of safer antifungal agents, such as plant-based essential oils, to control phytopathogens in agriculture.
The majority of the works that begun so far have concentrated on the effect of essential oils on inhibition of fungal mycelia growth under in vitro conditions. A previous study demonstrated that the essential oils were potential and promising
antifungal agents, and could be used as biofungicide in protection of plants against phytopathogenic fungi (Soylu et al.,
2010). In previous work, the volatile constituents of the essential oils of different medicinal plants have been shown in vitro
activity, against a number of tomato pathogens (Soylu et al., 2005; Soylu et al., 2006). However, very few studies have
been conducted in vivo conditions to show fungicidal properties of essential oils against plant pathogenic fungi (Oxenham
et al., 2005; Soylu et al., 2007). According to the results of various studies, antifungal activities of essential oils from aerial
parts of aromatic plants belong to the Lamiacea family, such as origanum (Origanum syriacum L. var. bevanii), lavender
(Lavandula stoechas L. var. stoechas), and rosemary (R. officinalis L.); these were investigated against B. cinerea (Soylu
et al., 2010).
Moreover, the efficacy of the essential oil from the flowers of Cestrum nocturnum L. was evaluated, for controlling the
growth of some important phytopathogenic fungi including B. cinerea, C. capsici, F. oxysporum, F. solani, Phytophthora
capsici, R. solani, and S. sclerotiorum (Al-Reza et al., 2010).
Our results from previous studies showed that different plant species have antifungal activities against various phytopathogenic fungi. Previous findings showed that M. piperita oil was found effective against pathogenic fungi including
Drechelera spicifera, F. oxysporum f.sp. ciceris and M. phaseolina. The antifungal activities of the oil increased with
an increase in the concentration. Also, minimum effective concentrations of oil against fungal pathogens were different
(Moghaddam et al., 2013). In a different study, the antifungal activity of the E. platyloba essential oil was evaluated against
some phytopathogenic fungi including Alternaria alternate, Culvularia fallax, M. phaseolina, F. oxysporum, Cytospora
sacchari, and Colletotricbum tricbellum. The antifungal test results indicated that the essential oil displayed great potential
of antifungal activity as a mycelia growth inhibitor against the tested phytopathogenic fungi (Moghaddam et al., 2015). The
essential oil of C. nocturnum had a remarkable effect on spore germination of all the plant pathogens, with concentration
and time-dependency (Al-Reza et al., 2010).
9.
MIC, MBC, OR MFC DETERMINATION
The sensitivity of the various pathogens may depend on the morphological and physiological characteristics of the fungus hyphae. The minimum concentration of oil required to inhibit the mycelia growth of tested fungi was different. It is
evident that the inhibitory effect of the oil on mycelia growth of fungi varied among the fungal species. These results are
in agreement with other studies which found differences between fungi in resistance to essential oils (Moghaddam et al.,
2015; Moghadam et al., 2016). In general, percentages of inhibition depend on the day of observation, dose and fungi. It
has been established that the composition of essential oils will depend on the plant species, the chemotypes and the climatic
conditions; therefore, their antimicrobial activities could be as varied (Shu and Lawrence, 1997). Inhibition of growth of
fungal pathogens may be due to a major component in Salvia oficinalis, such as menthone, menthol, and menthofuran.
Furthermore, it is possible that other minorcomponents may act together synergistically in each oil; this has already been
suggested (Stević et al., 2014).
Essential Oils: Biological Activity and Therapeutic Potential Chapter | 10 173
The antimicrobial activities were evaluated against oral pathogens by detecting MIC and MBC/MFC of five essential
oils such as tea tree oil, lavender oil, thyme oil, peppermint oil, and eugenol oil. The oral pathogens included Staphylococcus
aureus, Enterococcus fecalis, Escherichia coli, and C. albicans (Thosar et al., 2013).
10.
INFLUENCED FACTORS ON THE EOs ANTIMICROBIAL ACTIVITIES
The antimicrobial activities of essential oils depend on factors such as plant species, number of samples, methods used to
extract active compounds, and methods employed for measuring antimicrobial capacity. In addition, the chemical composition of essential oils can vary according to geo-climatic location, growing conditions (season, soil type, amount of water),
and the planťs genetics. Therefore, the antimicrobial activities of essential oils from the same plant species can vary due
to the different types and sources of essential oils. In particular, the antifungal activity of essential oil is associated with
phytochemical components, and some other factors that influence its antifungal activity. In general, percentage of inhibition of mycelia growth depends on various factors, such as the antifungal activity method, the day of observation, screening
methods, using concentration and examined fungal species. Due to preceding findings, minimum effective concentration
of the oil against fungal pathogens was different, and the antifungal activity of essential oil is increased with rising in the
concentration (Moghaddam et al., 2013; Moghaddam et al., 2015b).
Morphological and physiological characteristics of the fungus hyphae can affect the sensitivity of various pathogens.
By comparing the percentage inhibition of mycelia growth for every fungus, a difference between the tolerances against the
oil can be observed. This may be due to production of more enzymes by some fungus, such as M. phaseolina, which catalyzes the oxidation, and thus inactivation of the added oil. Furthermore, inhibition of the growth of these fungal pathogens
may be due to emulsion damage to the cell wall and cell membrane and, to various degrees, due to the different capacity
of each oil to penetrate into the chitin-based cell walls of fungal hyphae (Moghaddam et al., 2013). The mode of action of
the essential oil as antifungal agents may also be due to inhibition of respiration and disruption to the permeable barriers of
cell membrane structures (Cox et al., 2001). In contrast, natural compounds act on the internal mechanisms of the fungus,
leading to malformation of important structures, cytoplasmic granulation, disorganization of cell contents, disruption of
the plasma membrane, and inhibition of fungal enzymes, consequently inhibiting germination, germ tube elongation, and
reduction or inhibition of the mycelia growth. On the other hand, increased fungistatic potential may be triggered by a
mixture of essential oils, due to the combined activities of two or more components of the essential oils (Goni et al., 2009;
Begnami et al., 2010).
11. ANTIMICROBIAL ACTIVITY OF ESSENTIAL OIL COMPONENTS
Monoterpene or sesquiterpene hydrocarbons and their oxygenated derivatives are the major components of plants’ essential
oils, and exhibit potential antimicrobial activities and strongly inhibit microbial pathogens (Moghaddam et al., 2015). It
has been reported that some monoterpenes possess antifungal potential against plant pathogenic fungi, and many fungicidal
(Zhao et al., 2011; Moghaddam et al., 2013; Moghaddam et al., 2015), and bactericidal (Lo Cantore et al., 2009) properties. The natural pesticidal properties of some monoterpenes make them useful as potential alternative pest control agents,
as well as good lead compounds for the development of safe, effective, and fully biodegradable pesticides. Some earlier
papers on the analysis and antifungal properties of the essential oil of different species have shown that they have a varying
degree of growth inhibition effect against some Fusarium, Botrytis, and Rhizoctonia species, due to their different chemical compositions (Al-Reza et al., 2010). Previously the chemical structures of the most potent monoterpenes against plant
pathogenic fungi were tested. Carvone had promising antifungal activity against some potato storage diseases: Fusarium
sulphureum, Phoma exigua var. foveata and Helminthosporium solani (Hartmans et al., 1995). Some studies have considered the inhibitory properties of methyleugenol against phytopathogens. It strongly inhibited the growth of Alternaria
humicola, Colletotrichum gloeosporioides, R. solani and Phytophthora cactorum, showing the best activity against this
latter microorganism, after prompting structural hyphae damage (Meepagala et al., 2002). According to the reports from
different investigations for new antifungal compounds, some monoterpenes, such as camphene, camphor (ketone), carvone (ketone), 1,8-cineole (an ether-containing monoterpene), cuminaldehyde, fenchone, geraniol, limonene (a monocyclic monoterpene hydrocarbon), linalool, menthol (nonaromatic alcohol), myrcene and thymol (an aromatic alcohol),
demonstrated antifungal activities against some tested plant pathogenic fungi like R. solani and F. oxysporum, Penecillium
digitatum and A. niger, which caused damping-off, vascular mold, green mold and black mold, respectively. In addition,
L-carvone strongly inhibited the growth of post-harvest fungi: Colletotrichum musae, C. gloeosporioides and Fusarium
subglutinans f.sp. ananas. Similarly, geraniol showed fungistatic and fungicidal effect against P. digitatum, Penicillium
italicum, and Geotrichum candidum. Potent antifungal properties of thymol against various plant pathogens were also
174 SECTION | B Therapeutic Foods and Ingredients
documented (Marei et al., 2012; Zhou et al., 2014). On the other hand, thymol, limonene and 1,8-cineole have a potential to
be used as fungicides (Marei et al., 2012). Moreover, the findings from previous studies indicated that citrus essential oils
are a mixture of volatile compounds, consisting mainly of monoterpene hydrocarbons, and are widely used in the food and
pharmaceutical industries because of their antifungal activities (Jing et al., 2014).
The in vitro antibacterial activity of terpenes, such as carvacrol and thymol, against a number of gram-negative and
positive bacterial species have already been reported (Nostro et al., 2004; Fadli et al., 2012).
12.
MECHANISM OF ACTION
The mechanisms of antifungal action of monoterpenes are not fully understood. However, several studies concluded that, as
lipophilic agents, they execute their action at the level of the membrane and membrane embedded enzymes (Sikkema et al.,
1994). Phenolic terpenes are the active antimicrobial compounds of essential oils. It would seem reasonable that their antimicrobial mode of action might be related to that of the other compounds. Most of the studies on the mechanism of phenolic
compounds have focused on their effects on cellular membranes. It has been reported that these compounds caused their
action due to a change in the fatty acid composition of cell membrane (Prashar et al., 2003). It is also stated that the activity of monterpenes was attributed to their interactions with cellular membranes. These interactions may result in changes,
such as inhibition of respiration and alteration in permeability (Cox et al., 2001). Actually, phenolic compounds not only
attack cell walls and cell membranes, thereby affecting the permeability and release of intracellular constituents, but they
also interfere with membrane function. Essential oil components have the capability to alter cell permeability by entering
between the fatty acyl chains making up membrane lipid bilayers and disrupt the lipid packing. Due to this, the membrane
properties like membrane flue to permeability and functions may get changed. Thus, active phenolic terpenes might have
several invasive targets which could lead to the inhibition of plant pathogenic fungi (Hammer and Carson, 2011b). Some
monoterpenes had potent inhibitory effects against most of the tested fungal species. In general, the inhibitory action of
monoterpenes on microorganism cells involves cytoplasm granulation, cytoplasmic membrane rupturing and inactivation
and/or synthesis inhibition of intracellular and extracellular enzymes (Cowan, 1999). Essential oils principally acted against
the cell cytoplasmic membrane of microorganisms and monoterpenes, to destroy the cellular integrity by inhibiting the respiration process in the microbial cell. The hydrophobicity of essential oils enables them to accumulate in cell membranes,
disturbing the structures and causing an increase of permeability. Resultant leakage of intracellular constituents and impairment of microbial enzyme systems occur (Bajpai et al., 2013), leading to extensive losses of the cell contents and causing
the cell to die (Lang and Buchbauer, 2012).
The regulation and function of the membrane bound enzymes may also alter the synthesis of many cell wall polysaccharide components, such as glucan, chitin, and mannan, then alter the cell growth and morphogenesis (Sánchez et al., 2004).
The nature of hydrogen bond interactions is still the subject of many current discussions, and its importance in enzymatic
environment has been shown in some drug design studies (Silva et al., 2011).
Pectin methyl esterase (PME) enzyme modifies the degree of methyl sterification of pectins, which are major components of fungi cell walls. Such changes in pectin structure are associated with changes in cellular adhesion, plasticity, pH,
and ionic contents of the cell wall, and influence fungi development, membrane integrity, and permeability. On the other
hand, fungi produce cellulase to degrade cell walls during pathogenesis, and inhibition of this enzyme ultimately affects
the disease development (Goodman et al., 1967). It has been reported that production and activity of cellulase is inhibited
by commercial fungicides (Hammer et al., 2004). Many advances have been made in the knowledge of pathogenic plant
fungi at the cellular and molecular level. The β-glucosidase proteins from fungi are attractive targets for the design of new
antifungal agents to control the Fusarium disease in plants (Chatrchyan et al., 2011).
Some essential oils can cause extensive cellular damage at much lower concentrations, probably due to better penetration and contact. For example, the major components of C. cyminum are terpenes, which have the capability to inhibit
respiration of Candida, and may have adverse effects on mitochondria. It may be able to the cause various morphological
changes and cell death (Uribe et al., 1985).
On the other hand, some investigators reported that the antifungal activity resulted from a direct effect of essential oil
vapors on fungal mycelium. They further postulated that the lipophilic nature of essential oils render them more absorbable
by the fungal mycelia than by agar, due to the highly lipophilic nature of the fungal mycelia and the high water content of
the agar media (Edris and Farrag, 2003).
Contact and volatile phase effects of different concentrations of the essential oils were found to inhibit the growth of different fungi in a dose dependent manner. Volatile phase effects of essential oils were consistently found to be more effective
on fungal growth than contact phase effect. Spore germination and germ tube elongation were also inhibited by the essential
oils tested. Light and scanning electron microscopic (SEM) observations revealed that essential oils cause considerable
Essential Oils: Biological Activity and Therapeutic Potential Chapter | 10 175
morphological degenerations of the fungal hyphen, such as cytoplasmic coagulation, vacuolations, hyphae shriveling and
protoplast leakage and loss of condition (Soylu et al., 2010).
13.
SYNERGY BETWEEN ESSENTIAL OILS OR WITH OTHER COMPOUNDS
Several studies have investigated synergy between essential oils or components. Variation between different outcomes was
observed according to the test organism, antibiotic and essential oil tested. The combination of two antimicrobial agents
result in an antagonistic, indifferent, or synergistic effect, depending on the mechanisms exerted by each agent against the
test organism, the characteristics of the test organism, and any chemical interactions between the two antimicrobial agents.
The possibility that one or more essential oil component interacts synergistically within an essential oil has long been
proposed as a potential factor contributing to the overall antimicrobial activity of an oil. Although the antimicrobial activity of an essential oil is mainly attributed to its major components, the synergistic or antagonistic effect of components in
minor percentage in the mixture has to be considered (Fadli et al., 2012). Minor oil compounds might be important for the
antibacterial effect of the oils, as several recent microbiological studies have revealed that the whole essential oils can have
stronger antibacterial activities than their individual components. Therefore, minor components might be critical for the
activity of the oil and may contribute to synergistic effects (Carović-Stanko et al., 2010).
The type of interaction occurring between essential oil components appears to depend on the organism, components examined and component ratios. This is supported by two studies investigating terpene combinations, which found that interactions
could be synergistic, indifferent, or antagonistic, depending on concentrations and ratios of components (Cox et al., 2001).
In conclusion, chemical composition of the main compounds and precursors could explain their strong antifungal activity (Stević et al., 2014). Synergistic or antagonistic effects between major and minor oil components, and their mutual
interaction, play an important role in the overall activity of the essential oils (Dikbas et al., 2008).
The constituents, structure and functional groups of the oils play an important role in their antifungal activity, with
phenolic groups usually being the most effective components (Holley and Patel, 2005). Therefore, essential oils’ antifungal
action could be attributed to the presence of monoterpenes, sesquiterpenes and oxygenated compounds, such as alcohols,
phenols and aldehydes. According to the results of preceding studies, monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpenes, and phenolic compounds are responsible for inhibiting the mycelia growth of various fungi. In the
antimicrobial action of essential oil components, the lipophilic character of their hydrocarbon skeleton and the hydrophilic
character of their functional groups are of the main importance. The activity rank of essential oil components is as follows:
phenols > aldehydes > ketones > alcohols > ethers > hydrocarbons. The highest activity is reported for phenols (thymol, carvacrol, and eugenol), which is explained by the acidic nature of the hydroxyl group, forming a hydrogen bond with an
enzyme active center (Böhme et al., 2014). Therefore, essential oils with phenols as main compounds express the highest
activity against microorganisms, and their activity spectrum is the broadest (El-Shiekh et al., 2012). In a study concerning
Candida sp., several alkyl phenols showed different MIC values: thymol, methylthymol, eugenol, methyleugenol, anethole,
estragole, and amphotericin, respectively (Fontenelle et al., 2011).
14. TOXICITY OF ESSENTIAL OILS
Despite of being natural compounds, essential oils are far from nontoxic; the majority of essential oils, in high enough
doses, will cause toxic effects. The toxicity of essential oils can be evaluated in laboratory assays, such as cytotoxicity tests,
where the effects of oils on animal cells are examined or in vivo, and are recorded. Some information may also be extracted
from instances where humans have shown toxic effects after essential oil exposure. Furthermore, toxic effects may occur
following ingestion or dermal exposure.
Many essential oils, including lavender (Prashar et al., 2004), Nepeta cataria (catnip) and Melissa officinalis (lemon
balm) (Suschke et al., 2007), have been investigated with tests demonstrating that essential oils show toxicity at very low
concentrations. It is supposed that one of the primary mechanisms of cytotoxicity is membrane damage, similar to that seen
in bacteria and yeasts.
15.
CONCLUSION
EOs and their constituents generally display efficient antimicrobial properties, which have been used as preventatives
against various microbial diseases. More studies should be carried out on synergism and antagonism of constituents in essential oils and foods, before using these substances. Moreover, the degree of toxicity should be first investigated when they
are employed for conservation and therapeutic aims, especially in the fields of cosmetics and food.
176 SECTION | B Therapeutic Foods and Ingredients
In conclusion, due to the mentioned problems, such as side effects of using chemical compounds for controlling pest
and diseases in recent years, there has been a clear tendency towards the utilization of alternative methods for pest and
disease control in order to protect the environment and human health. Research on plant extracts and essential oils, which
may substitute the use of agrochemicals, or which may contribute to the development of new compounds, is extremely
important. Therefore, one such alternative is the use of natural plant protectants with pesticidal activity, such as essential
oils and their major components, since they tend to have low mammalian toxicity, cause less environmental damage and
benefit from wide public acceptance.
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Chapter 11
Nutritional and Therapeutic Potential
of Spices
Mian K. Sharif, Rebia Ejaz, Imran Pasha
University of Agriculture, Faisalabad, Pakistan
1.
1.1
HERBS AND SPICES
Preamble
Spices are seeds, fruits, roots, bark, berries, buds, or vegetable substances, and have been used primarily for flavoring,
coloring, or preserving food since antiquity. Major spices produced and used in the subcontinent, especially India and
Pakistan, are black pepper, cloves, cinnamon, seeds of flax, cardamom, poppy, fenugreek, cumin, sesame, fennel, carom,
coriander, turmeric, tamarind, ginger, onion, garlic, and red chilies. These spices are a hidden treasure of numerous therapeutic components like thymol, eugenol, curcuminoids, linalool, zingiberene, piperine, alpha crocin, coriandrol, cuminaldehyde, and capsaicin, and are helpful therapies against various health disorders. Moreover, these possess pharmacological
activities including antimicrobial, antioxidant, anticarcinogenic, antiemetic, antimutagenic, antihypertensive, antidiabetic,
anticonvulsive, antifungal, antiviral, hypolipidemic, chemoprotective, and prebiotic activities. Spices are widely used in
food products, like syrups, jams, preserves, pickles, snacks, biscuits, and candies. Additionally, modern food technologies
have made possible extraction and utilization of active nutrients from spices for addition to an array of processed foods.
Herbs and condiments are a blend of one or more spices, mostly added in cuisines before serving for flavor enhancement
and aroma. These are natural plant parts, having delicate stems and utilized as crude powder and flavoring after purification of principle components to impart pungency in food items. Herbs are also a subset of spices and considered as flavor
potentiators. These terms date back to ancient times. They are still frequently used for the purpose of preservation, color
enhancement, and aroma activators. Due to their chemical, nutritional, and medicinal attributes, these have been the focus
of recent studies for improving human health (Farkas, 2000).
1.2
Importance
Due to some characteristics of spices and herbs, they are also defined as fragrant plants or plant parts. They have great potential to promote health and act as curative agents for treatment of many physiological disorders. As well as being utilized
as food additives or preservatives, they are used to play the roll of therapeutics, medicines, and pharmaceuticals because
they are comprised of unique biological compounds like antioxidants and phenols. Spices are assumed to play a significant
part in the advancement of nations and civilizations. They are basic supplements for improving sensory attributes, as well
as for creating numerous culinary dishes. Furthermore, spices are considered to retain many pharmacological properties
and new ways for the manufacturing of valuable pharmaceuticals are being discovered. These provide additional nutritional
benefits as well, which make them an essential food item. Some of them act as inhibitors of microorganism growth while
others reduce mycotoxin production. Spices play a significant role in everyday life of humankind, as essential additives in
traditional, and large scale cooking, of beverages and pharmaceuticals. Immediately after picking and extraction, and up to
the point of consumption, spices can be attacked by microorganisms, such as yeast, mold, and bacteria.
1.3
Origin
The most important spices, like pepper, cardamom, cloves, ginger, cinnamon, and turmeric, are generally present all over
the world, especially in tropical regions. Coriander, cumin, mustard, and sesame seeds are the spices grown in nontropical
Therapeutic, Probiotic, and Unconventional Foods. https://doi.org/10.1016/B978-0-12-814625-5.00011-X
© 2018 Elsevier Inc. All rights reserved.
181
182 SECTION | B Therapeutic Foods and Ingredients
areas. Agro-climatic conditions (humidity and temperature) of these environments offer difficulties in the production and
management of spices. Proficient and viable drying systems are mandatory and important to increase the shelf life stability
of food products incorporated with spices in humid areas.
2.
HISTORICAL PERSPECTIVE
Spices are central to the historical cultural and economic background of nations. Marco Polo investigated Asia in 13th
century and determined that Venice as a better place to exchange goods. After that, Vasco de Gama, another explorer, focused his world investigation from India to Africa. He came back with spices, such as pepper, cinnamon, and ginger, and
also focused his efforts on developing and continuing trade with India. In 1942, Columbus arrived in the Americas and
established a direct route for Western Europe to the Spice Islands. He collected red peppers and other aromatic plants to
send to Spain, for further research by his colleagues. During the 15th century on through the 17th century, war between
European countries over the Indonesian Spice Islands was a constant. A war was fought for spice trade between the Dutch
and English in 1780, and eventually the Dutch were defeated, loosing all their spice sources and trade centers. In 1962, the
United Stated entered in the global spice race. United States is currently the major spice importer, followed by Germany,
Japan, and France. Spices are exchanged through various routes throughout the world, and have been the most important
trade item since prehistory, through to present times. Asia is still the largest grower of most spices including pepper, clove,
ginger, and cinnamon. However, a few spices are also cultivated and harvested in the West, which has its own comprehensive assortment of herb and spice crops. The major supplier of pepper is Brazil, a leading producer of cardamom is
Guatemala, and ginger is mostly bred in Jamaica. Europe, the United States, and Canada produce most of sesame seeds and
as well as several herbs.
3.
3.1
PRODUCTION AND TRADE: GLOBAL SCENARIO
Production
Peppers are the most important and valuable spices in the worldwide trade, followed by capsicum. The top three exporting
countries of spices are from tropical environments such as those in the Mediterranean and continental regions. China, India,
and Indonesia are the major spice trading countries, while the most significant buyers are Vietnam, Brazil, and Sri Lanka.
Area and production statistics of spices are limited, as compared to other horticultural crops. Spices were cultivated in an
area of 7587 thousand hectares, with a production of 31,859 thousand tons. The total world export and import of spices and
herbs was 3592 and 3454 thousand tons, respectively (Anon, 2007). India and China are the largest producers of medicinal
spices, and are also converting these herbs and species into powders, essential oils, oleoresins, specialty extracts, and other
numerous blends. India has established Spice Agri Export Zones, as well as crafting advancements in packaging, quality
management, processing, and innovation in production and handling of herbs. The main aromatic plant importer is the
European Union, where approximately 55%–60% of the total produced spices are used for human consumption, 35%–40%
by the processing industries, and 10%–15% for the culinary purposes. United States and Japan are the largest single country
merchants of spices. They represent a high-value commodity and serve as an excellent source for foreign exchange and
commerce. Currently 40–50 spices have global economic and culinary significance.
3.2
Spices Trade
Around the globe, there are 112 types of aromatic plants that are utilized as spices and vegetable seasonings. More than
3 million tons of spices per year are produced by India alone, which comprises about 50% of the entire world export. The
trade distribution structure in spices can be divided into three comprehensive segments: industrial, catering, and retail. It is
predicted that approximately 85% of the global trade of spices is in dried and cleaned form, or utilized as crude spice without further processing. The major international markets of spices are the United States, the European Union, Saudi Arabia,
Japan, Singapore, and Malaysia. China, Indonesia, Vietnam, India, Madagascar, Brazil, Spain, Guatemala, and Sri Lanka
are the principal supplying countries. There was a developing fashion toward the trade of treated herbs and species that
procure greater income. The swelling demand for value-added processing of spices, such as capsicum and ginger, present
business prospects for the foodstuff and processing activities in worldwide markets. The major producers and exporters of
black pepper are Vietnam, Malaysia, Indonesia, Brazil, and India. In Japan more than 50% of ginger consumed is imported
from other countries. The European as well as American Spice Trade Associations have set stringent quality criteria for safe
exports and imports of spices and herbs.
Nutritional and Therapeutic Potential of Spices Chapter | 11 183
4.
OVERVIEW OF SPICES
Most organic compounds obtained from spices do not contribute directly to human development or health. Certain compounds, conventionally termed secondary metabolites, have great significance. Compounds that form volatile and nonvolatile
natural products are commonly presumed to be biologically unimportant because they have complex chemical formation and
biosynthetic pathways. In the past few years, owing to the newly discovered abundance of hidden benefits in spices, scientific
research to explore new hypothesis has led to progress in both chemistry and new synthetic remedies and practices. Most
aromatic plants have bioactive secondary metabolites that possess multipurpose pharmacological and therapeutic properties.
The fields of molecular biology and nanotechnology together can definitely play a role in the exciting new research of the
chemical structure of these compounds. The following spices are mostly produced and consumed in South Asia:
●
●
●
●
●
●
●
●
Clove
Black pepper
Turmeric
Coriander
Cumin
Flaxseed
Cinnamon
Cardamom
Additionally, some spices are used as fresh vegetables and did not require any type of drying. These include:
●
●
●
Ginger
Garlic
Onion
4.1
Cloves
The most precious spice, derived from an evergreen tree having a height of 15 m, is known as the clove tree (Syzygium aromaticum). It has been used around the globe for centuries in preservation of food items and formulation of medicinal items.
Buds are produced by the tree, which are used whole, or ground as a spice. The principle phenolic components of clove are
eugenol, terpenoids, tannins, and gallic acid, which have great potential for pharmaceutical, food, and agricultural applications (Shan et al., 2005). Historically, clove is generally cultivated in Indonesia, but it is now cultured in other parts of the
world, including Brazil. It possesses a higher percentage of antioxidant and antimicrobial activity as compared to several
other fruits, vegetables, and spices, and should deserve special attention (Fig. 1). Dengue, a serious health problem prevalent in Brazil and other tropical countries, is countered by clove, due to its larvicidal activity (Cortés-Rojas et al., 2014).
Organic acids, diterpenes, flavonoids, and volatile acids are the major phenolic compounds in spices and herbs. Among the
spices, clove is one of the richest sources of natural antioxidants.
4.2
Black Pepper
Pepper (Piper nigrum) belongs to the family piperaceae and is known as the king of spices. It requires a specific temperature and rainfall for growth. The part used is small grapelike berries and dried parts and fruits of the perennial pepper
plant. Capsicums, chili peppers, and paprika are also included in this category. The dissimilarity amongst them is not only
due to their color, there are also wide differences in their botanic names and attributes. Contrary to other peppers, red
pepper can be used fresh, ground, or in powdered form. The dried pepper is washed to eliminate stalks, peels, and stem
heads. The white pepper is the product obtained from berries that are fully ripened. The therapeutic potential of black
pepper is given in Fig. 2.
4.3 Turmeric
Turmeric (Curcuma longa) grows naturally in open forests of India and parts of Asia, and require little darkness for growth. It
belongs to the Zingiberaceae family of plants, and provides a yellow color and flavor when dried. Sun drying or assisted drying
is the best possible way for drying turmeric, followed by crushing to make powder which can then be used as a food additive.
It contains several active compounds (3%–5%): curcumin, alpha, and beta tumerone, zingiberene, and curcumol. Turmeric
posses many therapeutic properties, including antihepatotoxic, hypolipidemic, antitumor, and anticancer activities (Fig. 3).
184 SECTION | B Therapeutic Foods and Ingredients
Skin
irritations
Acne,
pimples
Sepsis
Morning
sickness
Bacterial
infection
Pain killer
Parasite
infection
Vermifuge
Analgesic
Anesthetic
Stomachic
Stimulant
Carminative
Antiperspirant
FIG. 1 Therapeutic impact of clove on human.
Heart diseases
Gangrene
Indigestion
Earache
Abdominal
tumors
Insect bites
Constipation
Lung diseases
Sunburm
Oral abscosses
Joint pain
Epilapsy
FIG. 2 Therapeutic impact of black pepper on human health.
Liver disorders
Tooth decay
Nutritional and Therapeutic Potential of Spices Chapter | 11 185
Stress and
tension
Rheumatism
Body ache
Colic
inflamation
Skin diseases
Ulcers
Stomach
disorders
Dental diseases
Intestinal
worms
Leukodema
Fever
Dyspepsia
Urinary
diseases
Hepatic
diseases
FIG. 3 Therapeutic impact of turmeric on human health.
4.4
Coriander
Coriander (Coriandrum sativum) is an annual plant, has a strong aroma and belongs to the family Apiaceae. It is broadly
cultivated in various environments around the globe. In general, coriander falls into two major categories on the basis of
fruit size. This ultimately determines its oil content and use. Cooler regions yield a small fruited type, var. microcarpum
(diameter 1.5–3.0 mm), having an oil content ratio (0.5%–2.0%), and is grown for the extraction of its essential oil. The
larger fruited type, var. vulgare (diameter 3.0–5.0 mm), is grown in humid and subtropical environments, has oil content
(<1%) and is used for grinding and blending. The chief components include coriandrol, alpha pinene, linalool, and limonene. Essential oil of coriander acts as a carminative and spasmolytic agent (Fig. 4).
4.5
Cumin
Cumin seeds (Cuminum cyminum) belong to the family umbelliferae. They are ellipsoid, corrugated, greenish brown
in color, and are used whole or as grounds. They have a subtle odor and flavor. Whole seeds are cooked to impart
flavor to vegetables, rice dishes, and yogurt dressed salads. The plant has thin leathery leaves and the flowers are
white or pink in color. Cumin seed has a pungent and nutty flavor that intensifies during heating and roasting. The
Mediterranean region and the Egypt are the main growing areas of cumin seeds. References to cumin can be found in
the Bible. In the Mediterranean region, it is widely used in cuisine for its aromatic characteristics. Areas having low
atmospheric humidity perfect for growing cumin. Cumin seeds contain a high percentage of oil content (45%) and
protein (23%). It has bitter flavor and warm aroma owing, to its high content of essential oil. The chief constituent and
most abundant compound found in black cumin is thymoquinone (Weiss, 2002). Amino acid profiles include lysine,
leucine, isoleucine, valine, alanine, and glycine; additional components are starches, lignans, alkaloids, organic acids,
and poisonous glucosides. The seeds also contain minor quantities of minerals, such as sodium, iron, zinc, copper,
phosphorus, and calcium, as well as vitamins like vitamin C, vitamin B complex, and folic acid, which impart numerous health benefits to the consumer (Fig. 5).
186 SECTION | B Therapeutic Foods and Ingredients
Renal disorders
Digestive
disorders
Respiratory
disorders
Insomnia
Urinary
disorders
Dizziness
Cystitis
Hay fever
Rashes
Allergies
Sore throat
Cough
Nose bleed
Vomiting
FIG. 4 Therapeutic impact of coriander on human health.
Rhinitis
Tumor
Cough
Cataracts
Hydrophobia
Migraine
Jaundice
Alopecia
Paralysis
Rheumatism
FIG. 5 Therapeutic impact of black cumin on human health.
Headache
Abdominal
disorders
Nutritional and Therapeutic Potential of Spices Chapter | 11 187
4.6
Flaxseed
Flaxseed (Linum usitatissimum) originated from the temperate areas of India and Pakistan, and is rich in dietary fiber
and oil. It is noteworthy for its unique omega-3 profile (Kronberg et al., 2006). It is a good dietary source of omega-3 for
vegetarians who cannot consume fish (Trebunova et al., 2007). Flaxseed is recognized as one of the most important nutraceuticals, and needs more scientific attention (Basch et al., 2003). Flaxseed, and its various forms such as backed products,
possesses many health and nutritional benefits. Canada (35%), Argentina (21.8%), China (18.9%), India (13.8%), and the
United States (11.3%) are the major flaxseed producing countries. The use of flaxseed in cuisines is not high, however,
it is widely used in pharmaceutical industries (Madhusudhan, 2009). Sauces and preserves made from this spice have a
short shelf life, and can be stored only for few days. In spite of this, they are still considered a source of healthy bioactive
amalgams supporting vitality, and commonly thought of as appropriate food for long excursions (Opara and Chohan, 2014).
Volatile oils, lignans, fiber, protein, vitamins, and minerals are the basic components present in flaxseed. The consumption
of about 100 g of dry flaxseed provides 450 kcal energy due to its high percentage of oil (41%), protein (20%), moisture
(8%), ash (4%), and total dietary fiber (27%). When compared with other common protein sources, such as soybean, fish
oil, and corn, it delivers a superior content of fatty acids. Flaxseed is a source of good-quality protein (globulins) which
ranged from 58% to 66% of the total seed protein. Flaxseed is an essential contributor of omega-3 and omega-6 which
provides, 57% and 16% respectively, or fatty acids in the human diet. These omega fatty acids are vital for proper cerebral
function, epidermal health, nervous system health, renal function, and sexual organs health (Peter, 2004). Flaxseed is recommended for premenopausal women to enhance the luteal phase for its antiestrogenic effect. In addition to their weak
estrogenic/antiestrogenic properties, lignans in general have also exhibited antioxidant activity, antiangiogenic activity,
antimitotic activity, and cytotoxic effects on nonestrogen-dependent human breast cancer and promyelocytic leukemic
cell lines (Gulcin, 2005). Flaxseed inhibit platelet aggregation, thereby irritating the action of platelet-activating factor
(PAF). Recently, flaxseed’s therapeutic effects are under consideration as antimalarial, antiviral, antifungal, and antibacterial agents (Fig. 6). Moreover, its beneficial effects are also linked to cures for diabetes, cancer, and cardiovascular diseases
(Penumathsa et al., 2007). Higher blood estrogen levels in Western populations are due to the lower intake of dietary fiber.
This has led the stimulation of tumor cells growth (Prasad, 2009). Flaxseed has proved its potential for reducing cholesterol
and risk of cancer, and heart related illnesses.
Sore throats
Diabetes
Stomach
disorders
Eye and ear
health
Radiations
effective
Infectious
diseases
Stroke
Constipation
Assist fertility
FIG. 6 Therapeutic impact of flaxseed on human health.
Hypertension
188 SECTION | B Therapeutic Foods and Ingredients
4.7
Cinnamon
Cinnamon (Cinnamomum verum) is the hardiest tree among the spices and belongs to Lauraceae family and genus
Cinnamomum. It can tolerate harsh conditions of soil and temperature, and its height is 2–3 m. Ideal high quality seeds
are small, flat, uniform, and yellowish in color. The presence of volatile oils, monoterpenes, sesquiterpenes, and phenyl
propenes, in all parts of cinnamon possess a faint and pleasant aroma. Bark from the cinnamon trunk contains oil contents
ranging from 5% to 75%, and include cuminaldehyde and cinnamyl acetate (Fig. 7).
4.8
Cardamom
Cardamom (Elettaria cardamomum) belongs to family Zingiberaceae. It is a high, budding (<5 m), perennial herb, with
fruit harvested from panicles at the bottom of the plant. The fruit has a trilocular shell that contains 15–20 seeds. It is native
to India and various parts of Europe. The therapeutic benefits of cardamom are given in Fig. 8.
4.9
Ginger
Ginger (Zingiber officinale) belongs to family Zingiberaceae, and is native to Asia. It is one of the most important vegetables spices used, and is consumed whole or as a ground spice. Ginger is closely related to two other cooking spices,
turmeric and cardamom. The odor of ginger is mainly due to its volatile oil constituents, which varies from 1% to 3%. The
pungency is primarily due to gingerols, which are a homologous series of phenols. Zingiberene is an essential component
in ginger, and its concentration varies according to soil conditions. The pungency of ginger powder results from dehydrated
gingerols known as shogaols. Shogaols are formed from high temperature heating of the respective gingerol. Fresh ginger
contains moisture (80.9%), protein (2.3%), minerals (1.2%), fiber (2.4%), and carbohydrates (12.3%). Iron, calcium, and
phosphorous are important minerals present in ginger, followed by vitamins such as thiamine, riboflavin, niacin, and vitamin C. The nutrient profile varies with growing conditions, variety, agronomic applications, preserving methods, and dry or
humid storage conditions. The health benefits associated with the consumption of ginger are given in Fig. 9.
Tumors
Fungal
infection
Organ
indurations
Bacterial
infection
Diaphoresis
Sore throats
Astringent
Spasm
FIG. 7 Therapeutic impact of cinnamon on human health.
Stomachic
Nutritional and Therapeutic Potential of Spices Chapter | 11 189
Heart diseases
Kidney
diseases
Urinary
diseases
Constipation
Bacterial
infection
Snake bite
Teeth infection
Bladder
diseases
Pulmonary
tuberculosis
Sore throat
Digasstive
disorders
Eyelid
inflamation
Asthama
FIG. 8 Therapeutic impact of cardamom on human health.
Diabetes
Inflamation
Sore throats
Fever
Stomach
disorders
Infectious
diseases
Respiratory
disorders
Constipation
Gingivitis
Dementia
Hypertension
FIG. 9 Therapeutic impact of ginger on human health.
Stroke
Arthritis
190 SECTION | B Therapeutic Foods and Ingredients
4.10
Garlic
Garlic (Allium sativum) is a member of the onion family Alliaceae. It has been used since antiquity for domestic, therapeutic, and food applications. Its unique and distinctive flavor is due to presence of sulfur containing compounds which comprise about 1% of its dried weight. l-cysteines and sulfoxides are vital biological constituents of garlic. Additionally, garlic
composition plays a significant role for its potential health benefits, due to the high availability of carbohydrates, proteins,
minerals, and phenolic components, though their quantity are dependent on agro-climatic and crop conditions (Fig. 10).
5. THERAPEUTIC IMPACT OF SPICES ON HUMAN HEALTH
Worldwide, Pakistan, and India have been well known for their enormous production of herbs and spices. It is globally accepted that a wide range of physiological and pharmaceutical benefits can be derived from spices. Many heath disorders,
like degenerative diseases and oxidative stress, are controlled and cured with plant based diets, because they contain numerous beneficial chemical compounds and antioxidants (Carlsen et al., 2010). A number of metabolic ailments and age-related
issues are connected with oxidative mechanisms in the human body. Due to nonhazardous effect of spices on humans, they
are considered safe for use in food without any detrimental side effects. The health benefits associated with the consumption of various spices are briefly discussed below.
5.1 Antiinflammatory Activity
Several health disorders, such as back pain, rheumatism, skin eruptions, paralysis, hemiplegia, and related inflammatory
diseases, can be cured with the topical application of black cumin seed oil. Thymoquinone in black cumin seed oil is responsible for antiinflammatory action by inhibiting the eicosanoid generation and membrane lipid peroxidation, through
the inhibition of cyclooxygenase and 5-lipoxygenase (5-LO) pathways of arachidonate metabolism. The liquid extract obtained from black cumin has been investigated for analgesic, antipyretic, and antiinflammatory activities in mammal models (Parthasarathy et al., 2008). Additionally, the combined effect of both cumin and turmeric are accountable for removal
of certain toxins and poisonous substances, by triggering the action of enzymes that then repair and prevent DNA damage.
Colic pain
Atherosclerosis
Diabetes
Fever
Intestinal worms
Loss of memory
Dysentery
Paralysis
Liver disorders
Tuberculosis
Sinusitis
Bronchitis
FIG. 10 Therapeutic impact of garlic on human health.
Nutritional and Therapeutic Potential of Spices Chapter | 11 191
5.2 Antiemetic Activity
Ginger is an excellent remedy against vomiting (antiemetic), sickness, nausea, and loose BMs. It has direct impact on the
gastrointestinal tract, as opposed to mitigating the pain through the central nervous system. Antimotion action is generally
produced by ginger, due to its fundamental and marginal anticholinergic and antihistaminic attributes. Likewise, nausea
related with motion sickness is moderated by ginger, due to its ability to hinder the growth of gastric dysrhythmias and the
rise plasma vasopressin. The active constituents of ginger inhibit the multiplication of colon bacteria and eliminate flatulence caused by undigested carbohydrates. However, fresh extraction of ginger seems the most compelling, as compared
to aged or dried extracts or ethanolic extracts. Some digestive problems and other illness can be treated easily with the
consumption of black cumin as a diaphoretic, liver tonic, stomach, and diuretic. Symptoms, including loss of appetite, puerperal diseases, dropsy, and vomiting, can be easily cured by taking black cumin seed with buttermilk. Obesity and dyspnea
can also be cured by eating black cumin seed with other herbs and species (Jenkin et al., 1999).
5.3 Antitumor Activity
The extraction of ginger exerts a therapeutic antitumor effect in vitro on certain tissue destroyed with the Epstein-Barr
virus, and also against antioxidant effects, which could have applications in cancer treatment. According to some researchers, ginger also possess the ability to protect nerve cells, and is also helpful in diagnosis of Alzheimer’s disease. Ginger
aggrandizes insulin-sensitivity and is also beneficial for the treatment of acute diseases, such as diabetes. This was shown
in research using mouse adipocyte cell cultures (Sekiya et al., 2004). Similarly, other spices, like onions and garlic, have
proved their potential against the process of carcinogenesis (Tapsell et al., 2006; Krishnaswamy, 2008; Martos et al., 2011).
5.4 Antimicrobial Activity
The ginger extract of 2000 mg/mL has shown antimicrobial action in previous studies. Aspergillus, a mold recognized for
making aflatoxin, a carcinogen, is also inhibited by ginger. At atmospheric temperatures ginger reflects inhibitory action
against Aspergillus niger, Saccharomyces cerevisiae, Mycoderma, and Lactobacillus acidophilus at 4%, 10%, 12%, and
14% respectively. In conventional Chinese medicine ginger is used to increase the flow of body fluids. The antimicrobial
effect of the black cumin extract and its constituents against an extensive range of parasitic organisms, bacterial, and fungal
disease, has been comprehensively explored. The ethanolic abstract was shown to have an anticestodal effect in children
(Basch et al., 2003). Finally, black cumin seed extract, and its main component, which is thymoquinone, was thoroughly
studied for use in treating cells diseased with schistosomiasis.
5.5 Antihypertensive Activity
Ginger also enhances circulation of blood by augmenting cellular metabolic activity. Through this benefit, it can play a role
in the relief of cramps and tension. According to Japanese studies, constituents in ginger are very helpful in mitigating the
blood pressure and reducing cardiac workload.
5.6 Antibilious Activity
Black cumin has antibilious property, and may be administrated internally in sporadic fevers. The aromatic plant is considered a priceless remedy in hepatic and digestive disorders as well, because it is a restorative for a wide range of circumstances connected to mode swings, e.g., it restores and amuses the persons in the feeling of sadness and depression
situation. Black cumin as a remedy has applications in chronic headache and migraines, and is also useful in treating in
sores, mercury poisoning, and leprosy. It alleviates swelling from the hands and feet when soaked in water. Externally, it is
used for afflictions such as in pimples, abscess, alopecia, eczema, freckles, and leukoderma. They can also be used for its
anthelmintic and antibacterial properties (Bhatia et al., 2006).
5.7 Antispasmodic Activity
Black pepper is comprised of a variety of active biological components which exhibit pharmacological activities. These
components include piperine, alkaloids, amides, and other essential volatile oils, which account for 98% of black pepper’s
mass. Bioavailability of specific drugs and nutrients, like beta carotene, was enhanced by the rich alkaloids content of in
192 SECTION | B Therapeutic Foods and Ingredients
pepper. Some spices, because of their medicinal composition, act as tranquilizers and sedative agents, giving relief to pain
or reducing irritation and discomfort (De La Torre et al., 2017). In the southern Asian regions, turmeric has been used in
natural healing against injuries, bone disorders, stomach aches, parasites, inflammation, and other internal malfunctions.
Several studies demonstrate its potential against oxidation, cholekinetic, and inflammation (Names, 2013). Recent animal
and human efficacy trials explored its role against precarcinogenesis and atherosclerotic.
5.8 Anticonvulsive Activity
Remedial effects of spices include antiinflammatory, antihistamine, analgesic, anticonvulsive, antiparacitic, antiinfectious,
and expectorant. Spices act as mucolytic drugs, that penetrate the thick mucus layer made on lungs of persons suffering
from respiratory ailments, and relieve involuntary muscle contractions. The most well known spices having a high percentage of antioxidants and antimicrobials in their composition, are cumin (cuminaldehyde), clove (eugenol), and cinnamon
(cinnamaldehyde). Cardamom and turmeric powder showed therapeutic effects and are traditionally used for the prevention
of inflammation, intestinal disorders, stomach pains, and constipation. Both of these spices improving the taste and mouthfeel of food products, and act as antidiuretic, antioxidant, antimicrobial, and carminative agents. (Peter, 2004). Apart from
culinary benefits, cardamom can be used for relaxation of the nervous system, stimulation of skin fibers, and decreasing
pain. A lot of work was done to access the functional and chemical properties of spices, which revealed significant inhibition of cancer cells and oxidative damage, activation of enzymes responsible for brain stimulation, maintenance of human
body temperature, and topical irritation (Rubio et al., 2013).
5.9 Antioxidant Activity
Efficacy trials that are conducted to develop antioxidants, showed spices as intermediates of disease inhibition. From a
dietary perspective, the functionality of herbs and spices will be revealed through examination of their properties as foods.
Current research on essential oils from herbs focuses or their chemical constituents and therapeutic values. In developed
countries, widespread study is being done to determine the healthy constituents of spices, and discovering their genetic
attributes. Currently, foods which contain a high ratio of herbs and spices are accepted and desired. Hopefully in the near
future, the common use of aromatic plants in the preservation of health and security from disease, will be largely supported
by scientific evidence. The cognitive dysfunction related to aging, and the severe effects of cerebral and cognitive function
on the mental health of a living being, is also reduced using herbs and species. There are very limited studies available on
the use of spices with diabetic patients; the best witnessed effect is that of the ginseng compound on hyperglycemia Tapsell
et al., 2006).
5.10
Chemopreventive Activity
Chemotherapy is a modern treatment in which carcinogens are removed or destroyed at cellular or molecular level. This
type of treatment is promising for patients suffering from cancer. In this process, certain natural and synthetic chemicals, or
their mixture, are employed to decrease, retard or suppress the process of cancer forming cells. Health promoting properties
of cumin and turmeric possess cancer preventing activity, and increase the sensitivity of specific drugs against cancer cells,
increasing the effectiveness of chemotherapy. Likewise, these spices also reduce the progression of other severe neurological or mental disorders, like sclerosis and Alzheimer disease. The activity of the human immunodeficiency virus (HIV)
enzyme integrase causes or increases viral proliferation. These effects were greatly reduced by intake of curcumin, due to
its numerous antiviral attributes (Daood et al., 2006).
6.
CLINICAL STUDIES: ANIMAL VS HUMANS
6.1 Animal Studies
There was an observed substantial increase in glutathione levels and mucin contents, with little reduction in intestinal
histamine concentration of rat stomach, by the intervention of black seed oil, causing demonstrable protection from ulcers
induced by chemicals (Ammon, 2008). In another study, hypotensive activity of the seeds was established. It was found
that crude extract of cumin considerably reduces the high blood pressure of rats, similar to that of the drug Nifedipine.
These studies showed that the plant contains a variety of constituents that can be used for the treatment of hypertension.
Blood homeostasis, body weight, and toxicity of rats were investigated by employing cumin oil and seeds, for the control of
Nutritional and Therapeutic Potential of Spices Chapter | 11 193
h­ ypercholesterolemia and hyperglycemia. According to other research, a progressive increase in hematocrit and hemoglobin concentration was noted, while significant decreases in lipid profiles, including LDL, triglycerides, serum cholesterol,
leukocytes, and platelets, and glucose concentration were observed (Cortés-Rojas et al., 2014). There is evidence that 9%
of cholesterol, and other undesirable compounds, were eliminated from the body by consumption of half a piece of garlic
daily. Garlic possess activity for anticlotting (in vivo studies) and antihypertensive (5.5% decrease in blood pressure). In a
recent finding, it was found that the use of ginger for the treatment of nausea and vomiting during pregnancy was the most
effective, and possibly safest, option. The pharmacological properties of ginger, as an antioxidant and free radical scavenger, have shown a protective and beneficial impact in both animal and human studies. Risk of high blood pressure could
be reduced by giving an effective dose of ginger to person with hypertension. Animal studies have shown that gingerol,
a main active constituent of ginger, has both antiinflammatory and pain relieving effects. Many controlled clinical trials
have reported that risks associated with rheumatoid arthritis, cancer, and headaches, could be reduced by flaxseed intake.
Epidemiological studies showed that rates of certain cancers, particularly hormone-dependent cancers, are predominantly
lower in Asia, Africa, and Eastern Europe, than in Western world. Lower cancer rates are associated with their fiber enriched diet that reduces blood lipids and blood glucose level, and lowers CVD chances.
6.2
Human Studies
Equal portions of men and women were selected for trial from a college. They were given ginger extract and asked to monitor stomach and intestinal functioning. At the same time, a drug dimenhydrinate used to control motion and sickness was
given to another group, and results were recorded (Elgayyar et al., 2001). All individuals using only the drug were more
prone to the disorder. According to health studies, ginger is extensively used for reducing the of diarrhea, loose stools,
and sickness. It was concluded from research that consumption of ginger powder four times a day for sick, weak children
and pregnant women, significantly reduced vomiting, fever, and nausea (Subbulakshmi and Naik, 2002). Further studies
regarding lower blood pressure was due to less production of glucose in liver through gluconeogenesis and showed that by
administration of spices in food may be a good option for control of diabetes. In recent research, it was showed that one
gram of cumin seeds twice daily reduced blood glucose levels in human subjects after 2 weeks of oral intervention. Cumin
grains act as nutraceuticals and have a protective effect against induction of oxidative pressure and cancer, by reducing the
production of nitrogenous compounds. There was an 80% protection from neurological disorders and other types of inflammation. Moreover, in a latest study, normal women consumed black cumin seed oil, and powder. Following analysis examined their hemoglobin profile, such as creatinine kinase, prolactin, red, and white blood cells, platelets, and hemoglobin.
All previously mentioned parameters decreased significantly, except prolactin levels, by incorporating these seed into the
diet. Flaxseed supplemented muffins were prepared by replacing wheat flour, and for 3 weeks, were fed to 29 individuals
(22 men and 7 women) suffering from higher cholesterol levels. One serving consists of 20 g fiber/day which is equal to
50 g flaxseed without fat/day. This dietary administration of flaxseed greatly reduced total cholesterol (1.2%), low density
lipoprotein (1.8%), apolipoprotein B (1.4%), and apolipoprotein A-I (1.9%), however no significant impact on lipoprotein
levels was observed, as compared to the control group who consumed muffins prepared from wheat flour without supplementation of flaxseed (Ballabh et al., 2008).
7.
FOOD APPLICATIONS
Herbs, spices, and their supplements consist of notable antioxidant-rich products. The most common antioxidant-rich
foods are berries, fruits, nuts, chocolate, vegetables, and their products. Herbs and spices are used in amounts that do not
adversely affect the taste and flavor of the final product and are acceptable to the consumer. Large quantities of herbs and
spices are not generally not used in healthy foods. They are incorporated in moderate amounts to substitute, or partially replace, other pleasing food components like like sugar, salt and saturated fat in desserts, marmalades, soups, Mediterranean
cuisines and dressing (Table 1). These spices make vegetable dishes and vegetarian cuisine more sumptuous and delicious
(Tapsell et al., 2006). The consumption of medicinal herbs, spices, and their derived products are increased owing to
their use in pharmaceuticals, food colorants, and flavors, as well as other consumable products, like tea, tablets, capsules,
creams, syrups, and liquids (Aggarwal and Kunnumakkara, 2009).
7.1
Role of Food Industries
The appeal of spice use to the food industry is significant and growing, due to the important role of aromatic compounds
in food products. They are categorized by phytochemical groups, for example: aldehydes, alcohols, amines, esters, ethers,
194 SECTION | B Therapeutic Foods and Ingredients
TABLE 1 Major Flavoring Components of Spices and Their Value-Added Products
Spices
Flavor Components
Value Added Products
Black pepper
Piperine
Dehydrated and freeze-dried pepper, frozen green pepper, white pepper, green
pepper in brine, pepper oil, pepper oleoresin, ground pepper, organic pepper,
sterile pepper, canned tender green pepper
Cardamom
Linalool
Green cardamom, cardamom oil, cardamom oleoresin
Ginger
Gingerol
Ginger oil, oleoresin, candy, preserves, vitaminized effervescent ginger powder,
plain effervescent powder, starch from spent ginger, wine, beer, medicinal
beverages, encapsulated ginger oil, dehydrated ginger
Shogaol
Turmeric
Curcuminoids
Dehydrated turmeric powder, oil, butter, margarine, cream desserts, fruit wine,
bread, biscuit, and cakes
Cumin
Cuminaldehyde
Cumin oil, cumin powder
Cinnamon
Cinnamaldehyde
Cinnamon sticks, oil, powder, bakery products like bagels
Eugenol
Chilli
Capsaicin
Chilli powder, used in sauces and pickle
Clove
Eugenol
Whole or ground cloves, powder, incorporate in beverages
Coriander
Linalool
Coriander powder
Flaxseed
Linatine
Flaxseed meal, oil, jam, flakes, flaxseed balls, flaxseed flour
Glucosides
ketones, terpenes, thiols, and other miscellaneous compounds. Interest in the potential of spices is noteworthy, arising from
the chemical compounds contained in spices, such as phenyl propanoids, terpenes, flavonoids, and anthocyanins. A diverse
range of products are prepared with value and apparent quality or appeal increased through spices, mostly on the level of farm
operations. Basic quality characteristics of spices in food products, like aroma, flavor, pungency, and color, should be properly
preserved through effective preparations. Because of the increasing utilization of herbs and spices in processed foods, cultural
foods, natural scents, and new beverage products, the herbal industry is growing at a rapid speed, and is estimated to be more
than 10 billion dollars, increasing at a rate of 3%–4% per year. Essential liquid extract from plants having volatile elements are
an indispensable part of the production of cosmetics, perfumes, and pharmaceuticals (Charkraverty et al., 2003).
7.2 Value Added Products of Spices
Cumin, clove, and cinnamon have the potential to be used as preservatives in many foods, namely in processed meat, to
replace chemical preservatives. Spices provide beneficial effects, in addition to being a natural alternative to synthetic preservatives (De La Torre et al., 2017). The primary products harvested for spices are divided from portions of the plant, such
as fruits, seeds, leaves, stems, flowers, buds, roots, rhizomes, bark, wood, and resins. Flaxseeds are ground to flour and a
wide range of bakery products, like breads, muffins, and cereals, are prepared to provide a pleasant nutty flavor, as well
as increased nutritional value in the final product. Black pepper has been used for preparation in dehydrated, freeze dried
and frozen foods, and brined and canned tender products. Ginger powder has been used for candy preparation, preserves,
vitamin enriched, and plain effervescent drinks, wine, beer, and medicinal beverages. Spices are also incorporated in dairy
products to form a complete protein food. The amino acid and protein profile of flaxseed is closely related to legumes such
as soybean flour. Cumin seeds can be utilized to form many food items, can be added to tea, coffee, breads, canned food,
extracted for wine or vinegar, mixed with honey or sprinkled on salads. Black cumin oil in capsule form is taken by some
for its therapeutic effects (Heindl, 2003).
7.3 Artificial vs Natural Colorants
Manmade food colorants used in the food sector pose a threat for severe health ailments, such as different types of cancer, asthma, allergic reactions, hyperactivity, and thyroidism. This creates an alarming situation, as compared to natural
Nutritional and Therapeutic Potential of Spices Chapter | 11 195
c­ olorants, and has influenced change in regulations, as well as human inclination. Spices, like red chilies, turmeric, and
saffron, were widely used in food cuisines to improve color and flavor before synthetic colors became available. The Indian
center has created technology for production of accepted food colors, such as red chilies, for safe addition in food, and
without posing any health hazards. Developing countries can benefit from the incredible agricultural prospects of spice
crops as a source of natural flavors. The recovery of essential oil, oleoresin, and aromatic compounds from various spices
are the greatest focus. Several methods, using chemical and physical processes, are used for the extraction of oils and
oleoresins from the spices, such as by the use of steam, hydrocarbons, chlorine, enzymes, various acids, gases, and bacterial cultures. Currently, technologies like recombinant DNA and genetic engineering have been employed for extraction of
natural phenols, ketones, and other flavoring materials. Techniques, like single cell culture and cloning, should support the
food technologist as well as persons who are engaged in flavor enhancing procedures (Gulcin, 2005).
8.
SAFETY ISSUES IN SPICES AND THEIR MANAGEMENT
The microbiological hazards connected with herbs and spices differ drastically, and are mainly dependent on plant type and
the procedure to which it is subjected. This is a major factor impacting the method of production. Bulb and root crops are
literally connected to the soil, and susceptible to tainting because of poor irrigation and watering practices.
8.1
Mycotoxins
Presence of mycotoxins pose danger to health of humans because of the existence of pathogen outgrowth and poison
produced by it. Because of low moisture in herbs and dry spices, the movement of water is frequently under 0.60. As a
result, these commodities are naturally stable during storage. Spices play host to many fungi and microscopic organism
spores. These are mostly mesophilic anaerobes, mesophilic aerobes, and thermophilic aerobes. Spices are most likely to
possess human pathogens like coliforms and Escherichia coli, the life organism known to arise from fecal pollution, and
widespread hygiene problems. Aflatoxin is normally found higher in ginger and chili; the four kinds prevalent being: B1,
B2, G1, and G2. Most hazardous among the four is B1. Aflatoxin, which is not destroyed by cooking, is cancer-causing.
The higher levels in spices and herbs officially acceptable to the European Union are: for G1 + G2 + B1 + B2 (10 ppb),
and for B1 (5 ppb). The European Union is trying to establish the maximum permissible limit for ochratoxin. There is an
increased frequency of occurrence of human pathogens, that is, Bacillus cereus, E. coli, and Salmonella in spices (Pruthi,
1980). Because of low water movement, spices and herbs are inherently impervious to bacterial deterioration. A few spices
possess strong antifungal and antibacterial properties, this is especially the case with such spices as ajowan, clove, and cinnamon. Insect pests frequently found to infest spices are confused flour beetles, cigarette beetles, Indian meal moths, and
saw-toothed beetles. Spices can possess antioxidant (cancer prevention agents) and radio protective agents. These agents,
along with antifungal and antibacterial properties, provide nutraceutical and therapeutic potential to spices. Mycotoxins are
detrimental metabolites delivered by various molds types in favorable environments. Molds can pollute agricultural foodstuffs during gathering, handling, and storage. Most of them are capable of producing auxiliary metabolites, causing a wide
range of ailments to both humans and animals. Amongst these, mycotoxins have the greatest impact overall. Currently, the
ones considered most problematic are fumonisins, aflatoxins, T-2 poison zearalenone, ochratoxin A, and deoxynivalenol.
Among spices and herbs, the most commonly experienced mycotoxins are ochratoxin and aflatoxin. Some mycotoxins being distinguished in herbs and spices were ochratoxin, aflatoxin, and fumonisin. Aflatoxin can be produced in red pepper
both before and after harvest. Reddy et al. (2001) examined 124 specimens with three distinct characteristics in chili pods
and established that aflatoxin contamination might be associated by sample grades. The most prominent concentration of
969 mg/kg AFB1 was determined in one specimen of grade 3 which was of low quality. Scientists studied 36 samples of
ground red pepper acquired from several producers in four districts of Turkey. Levels of Aflatoxin B1 identified in five
examples was 10.5–31.2 mg/kg.
8.2
Bacterial Contamination
Spices are used worldwide as flavoring agents and are a diet staple item. They are exposed to an extensive variety of natural
tainting, during gathering, and preparing, by waste water, dust, humans, and animals, to name a few. Contaminated spices
can be of microbiological concern, provided they are actually consumed. Cuisine using spices may represent a danger for
the health of many because they are frequently used in food with minimal preparation activity or are eaten plain. Spices
are a significant source for microscopic spore-forming organisms in various foods, such as casseroles, gravies, soups,
and stews. Under favorable conditions, they can grow to critical levels. The main microorganisms liable for these are
196 SECTION | B Therapeutic Foods and Ingredients
Clostridium perfringens, B. cereus, Salmonella, toxigenic molds, and E. coli. The number of microorganisms in spices fluctuates significantly, depending on the spice. Turmeric, dark pepper, allspice, and capsicum possess the most notable levels
of microbes, whereas, cinnamon, and cloves have a tendency toward minute levels of contaminants. Microflora of numerous spices is comprised of mostly spore forming mesophiles come from contact with the soil. Scientists conducted experiment on 18 white 4 black pepper samples procured from India. The majority of examined samples were contaminated with
two to four aflatoxins, nonetheless, they remained below the 20 mg/kg limit established by the European Union. Cinnamon
oils can stifle the Aspergillus parasiticus development entirely. Cinnamon samples, gathered from Egypt and studied in the
United States, were polluted by aflatoxin B1. Coriander was contaminated with two sorts of mycotoxins, especially AFB1.
Ginger was contaminated with mycophenolic acid. Large amounts (110 mg/kg) of OTA was found in turmeric (ThirumalaDevi et al., 2001). It was observed in a study that spices, including cardamom and coriander, are tainted by aflatoxin B1
at levels above the resistance threshold set by the World Health Organization. Fungi can taint herbs and spices in the field,
throughout collection, sorting, drying, granulating, handling, storage, and packaging. Preharvest mycotoxin generation
happens in environmental conditions favorable to mold development. Control of contamination needs to occur at all various aspects of postharvest, like, handling, cultivation practices, throughout collecting, techniques, and time used for conditions amid storage, drying, and the seed quality in order to minimize harm. Among spices cinnamon, red cardamom, white
and black peppers, cumin, mustard, peppermint, turmeric, and ginger, were observed to be contaminated with Aspergillus
parasiticus or Aspergillus flavus. Coriander is spice most vigorously polluted with fungi. Clove, because of its microbial
inhibitory influence, was the least contaminated spice. Cinnamon was observed to be tainted with numerous fungi (11 out
of 20) comprised of mycotoxin delivering organisms.
8.3
Pesticide Residues
Pesticides are a collection of compounds intended for control of weeds, insects, disease, or different irritations upon harvests, such as animals or landscape plants. Judicious utilization of pesticides has assumed a basic role in nourishing the
world's growing population by drastically expanding crop production. Nonetheless, safety of pesticides and their influences
on the environment are a great concern. Importing nations will recognize pest contamination in spices and can reject delivery or demand lowered costs due to additional processing needed to ameliorate a contaminated import. Chili and paprika
are not tainted by mold poisons like aflatoxins. One noteworthy issue may be insect pests or rodent infestation, but these are
secondary causes due to poor facilities and storage practices. Pesticide residues found in these items are mostly carbamates
and organophosphorus, and organochlorine mixes. A few pesticides will accumulate in the items unless they are denatured.
8.4
8.4.1
Management of Safety in Spices
Radiation
Radiation presents good opportunity to enhanced shelf life, upgrade microbial safety and quality without any change to the
normal flavor in spices. Presently, this treatment is broadly carried out in North America and Europe to disinfect imported
spices. Similarly, different spice generating nations have also began using this technique to ensure the quality of their produce. Radiation sanitization, alongside good manufacturing and agricultural practices, assist to produce high quality clean
spices, devoid of synthetic and pesticide deposits. Good irradiation for pathogenic control and other measures to reduce organism growth in herbs, spices, and seasonings, has been practiced by the International Consultative Group on food Irradiation
(ICGFI) underneath the aegis of WHO, IAEA, and FAO. The key points of interest, preirradiation treatment, packaging prerequisites, the light medicines, and the doses necessity for purification of radiation, along with edge dosages contributing to
organoleptic changes. Low irradiation doses (<1 KGy) hinder sprouting in ginger, garlic, onion. Medium quantity application
(1–10 KGy) remove contaminating microorganisms and pathogens of food and high dose application (>10 KGy) sterilizes
food for unique prerequisites and for shelf stable food. The vast majority of whole spices are secured by the pericarp and their
naturally occurring antioxidants; they require fewer protections from spoilage compared to ground spices. Radiation treatment
includes controlled utilization of vitality of ionizing radiations, for example, gamma beams, X-beams to food products to accomplish the desired end result, such as, disinfestation, time span of shelf life expansion and disinfection.
8.4.2
Ionizing Radiations
Irradiations are electromagnetic. They possess g short wavelengths with high energy. These radiations launch electrons from
a molecule of food to form electrically charged ions. Various refined systems can distinguish spices or herbs sanitized by
ionizing radiation. A tag carried by the product is the best way for consumers to recognize that product is being irradiated.
Nutritional and Therapeutic Potential of Spices Chapter | 11 197
8.4.3
Food Sterilization
Food sterilization can be utilized successfully to fight molds and different microbes in spices and herbs. Sterilization and
disinfection is commonly by means of warmth, chemicals steam, low temperature utilization, lack of hydration, drying up,
lyophilization, adjustment of acidity, use of concoction additives or irradiation. Steam cleansing or chemical fumigation is
generally thought of as being the best practice for prepared or ground spices and herbs, because these procedures are simple
and inexpensive to complete, particularly contrasted with radiation treatment requiring profoundly complex and expensive
equipment. Chemicals allowed for cleansing are propylene oxide and ethylene oxide.
8.4.4
Fumigation
The most common fumigants are ethylene oxide, methyl bromide, and ethylene dibromide. Thy are utilized to treat herbs
and spices for microbial sanitization and insect disinfestation. Less viable fumigants leave substance deposits on spices and
are unsafe to humans. Numerous countries have forbidden fumigant use. Different nations comprising India are willing to
ban aflatoxin. Storage pests cause significant loss of revenue and investment to ranchers and middlemen.
8.4.5
Pest Control
Pest control is focused on diminishing the loses caused by vermin feeding, which subsequently results in the deterioration,
microorganism intrusion, and contamination of products. Only legitimately prepared workers, wearing proper defensive
garments, (e.g., overalls, gloves, head protectors, goggles, face coverings), ought to apply agrochemicals.
9.
QUALITY AND SAFETY STANDARDS
9.1
Food Safety and Quality Assurance
Food safety is one of the key issues confronting the entire chain of food production worldwide. Aromatic items are hygroscopic in nature and are exceedingly delicate with respect to moisture; absorption of moisture results in caking, discoloration, hydrolytic rancidity, mold development, and insect infestation. As spices contain unstable aromatic qualities, loss of
these qualities, and the retention of foreign odors is the result of ineffective packaging, and may pose major safety issues.
Moreover, warmth and light accelerate deterioration of fragrance and flavor constituents. Spices containing natural colors
need to be safeguarded from light (capsicum, cardamom, turmeric, and saffron). The key oil segments normally exhibited
in a large amounts of the spices, are liable to oxidation by barometrical oxygen, especially at high storage temperatures,
bringing about the onset of off-flavors. Quality has stands out as the most vital and precarious factor in the world, and is
equally important for herbs and spices. National sustenance laws and controls intend to shield consumers from health risks.
Administrations have determined the maximum possible parameters of conceivable impurities for foods. These incorporate
the nature of herbs and their individual flavors (Vasudeven et al., 2000). Contaminants include incidental material, microbial disease, insect invasion, excreta of birds, insects, and animals, pesticide residue, mycotoxin deposits, and heavy metals.
Some importing countries are strict, to the point that food handling security brings levels of contamination so low, as to
be dismissed as a danger. Contaminants by and large make spices and herbs completely lose their chemical and physical
qualities, further contributing to unsafe ailments. Production of quality spices, with no residual pesticides or compounds is
critical in the development of a free global trade. Organic spices get higher costs, about 20%–50%, owing to their production without chemicals and pesticides, and the resulting quality to the consumer is undisputed (McNamara et al., 2005).
Consumers are turning out to be more quality cognizant, and agriculturists, merchants, exporters and processors must
maintain the quality of their products at each phase, from production to end sale. In developed nations, the steps essential
for quality assurance have become a required element of production (Szallasi, 2005).
9.2
Food Safety and Quality Assurance Systems
Quality assurance systems, like Hazard Analysis Critical Control Point (HACCP), have played significant role in the manufacturing of quality spices. The European Spice Association (ESA) has developed quality criteria for spices and herbs, which
function as a standard for different nations of the European Union. Beside this, nations like Germany, the Netherlands, and
the United Kingdom have established their own purity standards for spices. Beside purity requirements, importing countries
demand a limit to measurable contaminants like heavy metals, microbial contamination, pesticide residues, and aflatoxin.
Other countries like Japan, the United States, and countries of the EU, have set MRLs (maximum residue limits) for spices
(Beristain et al., 2001). The International Organization for Standardization (ISO), an overall system of national norms
198 SECTION | B Therapeutic Foods and Ingredients
standard institution working in together, creates specific benchmarks for an extensive variety of items that are exchanged
globally. ISO benchmarks for particular spices and key oils have been defined and received by the specialized boards of
trustees representing the producer countries, and are continually updated. For instance, the conventional strategy for assessing capsicum spiciness HPLC (High execution fluid chromatography) is performed with particular mixes which are distinguished, and levels are measured. In 1995, The Codex Alimentarius Commission established a Code of Hygienic Practice
for Dried Aromatic Plants and Spices. This code points out hygienic necessities in the collecting and production of plants
and spices, and of the foundational configuration and cleanliness of facilities for handling hygienic prerequisites (Ballabh
et al., 2008). The food safety system includes good agricultural practices, quality management systems, good manufacturing practices, International Standards Organization and hazard analysis, and critical control points. All these measures are
well adopted to monitor and mitigate the threats in herbs and spices. Numerous processing units in trading and importing
nations have already been certified under one or more registered quality frameworks. Accreditation under HACCP is mandatory because herbs and spices are food items, and there must be no danger of contamination beyond permissible levels at
any of the critical control points. The essential oil industry has to adopt frameworks specifically recognizing the common
areas of jeopardy to food safety and excellence, in the production and supply chain. By realizing good agricultural practices
on the farm, the yield of agriculture commodities can also improved.
Over 20 national industrial organizations are involved in the Canadian on Farm Food Safety Program (COFFS), including
Canadian Herb, Spice and Natural Health Products. These industrial organizations are developing specific HACCP-based
On Farm Food Safety Programs and Good Agriculture Practices. The mission of the COFFS system is to give assurance
from contamination of the food chain, from the farm to the fork, through the use of HACCP standards. Regardless of the
fact that it is challenging to get rid off all threats to food purity and cleanliness, an accurate detailed on-farm food safety
program using good agricultural practices will curtail incidents of contamination. The herbs and spices standards cover
natural health products, therapeutic, and culinary food stuffs (developed and wild crafted). There is a concern to identify
the prime areas or phases (drying techniques or storage) that can considerably affect the quality result. Employing a hazard
analysis and critical control point (HACCP) framework, focused on hindrance, as opposed to depending on final item testing, appears to be a valuable development for herb and spice safety. Sterilizing procedures, as well as secure packaging,
and storage methods, are an important component in maintaining the natural properties of spices. Spices in their whole-seed
form are of great interest and degrade much more slowly than spices that have been ground or reduced to powder form.
The most ideal approach to limit health hazards in aromatic plants, is to assure that any chemical pesticides employed as
part production, are used as per the maker's guidelines, with adherence to government controls for their application. Herbs
and spices should be grown in soils with low levels of heavy metal concentration, and any foreign contaminating objects or
substances, with continued avoidance during growth and postharvest operations.
10.
CONCLUSION
Spices are an excellent source for a wide range of organic acids, phenolics, essential volatile oils, and aromatic compounds.
The nutritional and pharmacological qualities of spices are the most versatile among all the other food ingredients, and possess numerous health promoting properties, such as antioxidant, antimicrobial, antihypertensive, anticonvulsive, and analgesic and antiulcer activities. Furthermore, spices should be made part of food to increase their therapeutic potential to promote
general health, as well as for treatment of specific health ailments. Advancement in food technologies has developed more
methods and possible products for extraction and utilization of these active nutrients in array of processed food products.
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Chapter 12
Novel Nutraceutical Compounds
Asma Afreen, Zaheer Ahmed, Nomana Anjum
Allama Iqbal Open University, Islamabad, Pakistan
1.
INTRODUCTION
The term Nutraceuticals was first devised by Stephen DeFelice in 1989, famous as the founder for innovation in medicine.
He developed the word through contraction of the words Nutrition and Pharmaceutical. Term was clarified in 1994 with
the following definition; “any substance that may be considered a food or part of a food and provides medical or health
benefits, including the prevention and treatment of disease” (Keservani et al., 2010).
Nutraceuticals range from organic nutrients (proteins, fats, carbohydrates, minerals, and vitamins), dietary supplements
(the product that has one or more of the following nutritional elements: mineral, vitamin, amino acids [proteins]), herbs or
other botanicals. They might also contain a regime (of extracts, distillates, constituents, or metabolites) and diet of genetically contrived ‘designer’ foods, and processed foods, such as beverages, cereals, and soups, that are for express treatment
or disease prevention. Such substances may not be generally recognized as safe (Prabu et al., 2012).
Nutraceuticals in all forms are now being used for treatment and prevention of many chronic diseases such as cardiovascular disease (CVD), cancer, diabetes (Type-II) and many other health problems (Hu et al., 2017b). Epidemiological
investigations continue to focus on the ingestion of vegetables and fruits, and other therapeutic foods, not only for prevention of such chronic diseases, but also for the treatment of other health issues. Change in lifestyle, dietary modification,
and the use of nutraceuticals may be as effective and less costly as any other secondary disease management. This chapter
summarizes the work done on some of the nutraceuticals, their pertinent aspects of disease prevention and their mechanism
of action in the body.
2.
NUTRACEUTICALS AND FUNCTIONAL FOODS
Functional foods and nutraceuticals are the terms used interchangeably for prevention and treatment of diseases. Additional
terms, like medical foods and dietary supplements, are also used for this purpose. Researchers reported the brief description of each term to clarify the terms from different points of view. Functional foods are described as food products that
have specific and strong purposes, and are taken as part of a routine regime for advantageous effects that drive beyond
and are known as traditional nutritional effects. These include milk, cheese, and eggs enriched with omega-3 fatty acids;
yogurt enhanced with live active cultures (probiotics); fruit juices and drinks with increased antioxidant levels; cereals
and grains enriched with dietary fiber; improved fatty acid vegetable oils; vegetable proteins, legumes, and fruit products.
Nutraceuticals are those products that emphasize the expected results of prevention and treatment of diseases, such as
products produced from an animal, plant, or marine source through extraction or purification (e.g., antioxidants present in
elk velvet, fish oils, and blueberries), or derived from dehydrated, processed, or pressed plant substances and revealed to
have a biological benefit, or to provide defense against long-lasting diseases. The author described the concept of nutraceuticals as nutrition required for health and pharmaceutical therapy for injury or sickness, that lead to a preventive medical
approach (McClements et al., 2015). Minerals, vitamins, amino acids, or any other food substances in the form of dietary
supplement are said to have more valuable consequences if supplemented with the diet (Prabu et al., 2012). However the
health claims, or the beneficial effects of nutraceuticals, are not optimally realized owing to low or variable bioavailability
(McClements et al., 2015; Rein et al., 2013; Faraloni and Torzillo, 2017). Many factors are associated with poor bioavailability, such as physiological process, restricted release of nutrients from the food matrix, physicochemical properties of
the food (Moelants et al., 2012), disturbances in gastrointestinal fluids, low permeability of epithelium cells, development
of inexplicable multiplexes in gastrointestinal tract, and molecular transformations in the GIT (Fernández-García et al.,
2012; McClements et al., 2015). For these reasons, food scientists are now focusing on the food delivery systems to make
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© 2018 Elsevier Inc. All rights reserved.
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nutraceuticals more bio-available and keep these factors in mind when preparing artificial nutraceuticals. However, limited
information is available on factors that affect the bioavailability of organic nutraceuticals.
3.
NOVEL NUTRACEUTICAL COMPOUNDS
“Phytochemical” is a word derived from the Greek word phyto meaning plant, and therefore termed as “plant chemicals.”
They are defined as nonnutrient bioactive compounds present in vegetables, fruits, grains, and other plant foods. The work
done so far has established that phytochemicals reduce the risk of major chronic diseases. Around 5000 individual phytochemicals are recognized in vegetables, fruits, and whole grains, but health benefits of few of the phytochemicals have been
reported. They have varied compositions and ratios in different foods, and their mechanisms are often complementary to
one another. Therefore, it is reported and foreseeable that to eat maximum plant-based foods daily, is to get the maximum
health benefits. They are classified into broad categories such as alkaloids, organo‑sulfur compounds, nitrogen-containing
compounds, phenolics, phytosterols, and carotenoids (Liu, 2012). This chapter will focus on carotenoids, phytosterols,
polyphenols, and omega-3 fatty acids used as nutraceuticals and their important health benefits.
3.1
Carotenoids
Carotenoids are abundantly present in plant-based foods; are lipid soluble C-40-based isoprenoid pigments; and categorized by a protracted conjugated π-electron system which can only be manufactured by plants and microorganisms (Biehler
et al., 2012); and are one of the biotechnological and commercially most substantial pigments (Cardoso et al., 2017).
They are very sensitive to oxidation, heat, and light because of the unsaturated chemical structure (Ribeiro et al., 2010).
A few carotenoids in hundreds have been recognized in nature; but Vitamin A includes retinol, retinal, and retinoic acid;
and Carotene include lycopene, lutein, astaxanthin, and cryptoxanthin are the most studied carotenoids. Among the many,
around 40 are present in a normal human diet (Krzyzanowska et al., 2010; Eliassen et al., 2012) and it is suggested that
50 species of carotenoids play a vital role in the human diet (Kaulmann and Bohn, 2014), with an intake of 5–15 mg/day
per capita (Biehler et al., 2012). Only 20 carotenoids are present in human plasma and tissues, represented as lycopene,
β-carotene (highly hydrophobic molecule) (McClements et al., 2015; Faraloni and Torzillo, 2017), lutein, α-carotene, and
β-cryptoxanthin (Krzyzanowska et al., 2010). Four carotenoid, α- and β-carotene, γ-carotene and β-cryptoxanthin, are the
provitamins that are converted to retinol (Sharoni et al., 2012). They belong to the tetra-terpenes family and are characterized by a poly-isoprenoid structure, with a long conjugated double bond chain and a two-sided equilibrium around the
central/principal double bond.
3.1.1
Bioavailability and Mechanism of Action
The bioavailability of carotenoids varies widely because of dependence on the structure of the carotenoid itself in food matrix. In general, astaxanthin, owing to free carotenoid nature, has greater bioavailability than a polar species (e.g., β-carotene
and lycopene). It has been described in literature that astaxanthin from Haematococcus pluvialis (a green microalgae), shows
greater bioavailability than β-carotene from Spirulinaplatensis, and lutein from Botryococcusbraunii. Esters of xanthophyll
seem to have lower bioavailability, but scientific consensus is lacking. It has been suggested that their hydrolyzation occurs
in the human small intestine for further absorption (Dhankhar et al., 2012; Faraloni and Torzillo, 2017). They are partially
absorbed, which involves release from plant cells, and then form micelles with the help of fatty food and bile acids. The
absorption process takes place by passive diffusion via the intestinal brush border membrane of the enterocytes. Xanthophyll
esters like lutein are absorbed with facilitation of class b-type 1 scavenger receptors (SR-B1). Chylomicrons incorporate the
carotenoids and are then released to the lymphatic system. After this, they are combined with lipoproteins in the liver, and
finally absorbed into the blood stream (Gordon, 2011). Passage of carotenoids to the extrahepatic tissues occurs via interaction of lipoprotein substances with receptors, and degradation by lipoprotein lipase. Carotenoid absorption is a comparatively
slow process, having peak plasma concentration up to 24 h after food intake (Leopoldini et al., 2011). Only 3–5 g of fat are
required for absorption of carotenoids and are considered a key factor for their absorption. Only six carotenoids and their
metabolites have been reported to be metabolized in human tissues, particularly in the intestine. Thirty-four carotenoids and
eight metabolites were noticed in the serum of lactating mothers and in breast milk. A schematic flow on the metabolism of
carotenoids in the body is shown in Fig. 1. The absence of bile acids or malfunctioning in the metabolism of fat, like in the
diseases of the pancreas and small intestine, the carotenoids are not properly stored (Tanaka et al., 2012). Evidence from
the literature indicates that thermal treatment and homogenization exerted positive effects on bioaccessibility of these compounds, however, dietary fiber produces a negative effect on their bioavailability (Fernández-García et al., 2012). To make
the nutrients more bioavailable, the food industry has developed encapsulation of carotenoids present in fresh vegetables and
fruits, employing their them in crystal form or bound to protein complexes (Ribeiro et al., 2010).
Novel Nutraceutical Compounds Chapter | 12 203
FIG. 1 Metabolism of carotenoids.
3.1.2
Carotenoids and Beneficial Health Effects
Carotenoids are considered an important component of the human diet, due to bearing potential health benefits. Some of the very
important carotenoids are metabolized to retinol, and act as precursors of vitamin A in animals. Their high ­consumption is also
linked with reduction of chronic disease risk, such as age-related macular degeneration, cardiovascular diseases, diabetes, skin,
tuberculosis, and various cancers such as colon, breast, prostate, and lung cancer (Chen et al., 2016; Cooperstone et al., 2017;
Soh et al., 2017). They also play a vital role in cell communication, scavenging peroxyl radicals, protecting the photo-oxidative
process by process similar to singlet molecular oxygen, and interact synergistically with other antioxidants (Meinke et al., 2010).
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Carotenoids as Antioxidant
Regardless of other potential organic nutrients, carotenoids have been reported to act as an antioxidant and exert many
other beneficial effects. They reached the maximum tissue and plasma concentrations of about 2 μM (Kaulmann and Bohn,
2014) as compared to polyphenol. Their plasma shelf half-life is also long (expanding from days to weeks as compared to
polyphenols, 2–30 h) due to inadequate metabolism, low renal clearance, and fat solubility. Due to these potential capabilities, carotenoids have been associated with the prevention of diabetes, cancer (Ge et al., 2012), and inflammatory bowel
diseases (Xu et al., 2013; Kaulmann et al., 2016). Carotenoids aid in stabilization of unpaired electrons after quenching
radical through an extended π-electron system, a vital component of carotenoids. For this reason, carotenoids are powerful
hunters of peroxyl radicals and singlet oxygen (1O2). They act via electron acceptance, donation hydrogen abstraction, or
physical quenching (Bouayed and Bohn, 2012). Hunting of such oxygen by carotenoids is greatly dependent on their physical reduction and a number of conjugated double bonds where energy is transferred between two molecules (Gruszecki,
2009). Lycopene is regarded as a stronger antioxidant as compared to phytofluene or phytoene, due to the extended πelectron systems. Orientation within biological membranes is also an important role of the carotenoids (Gruszecki, 2009),
as they are present in lipid/water interfaces and lipophilic environment due to lipid soluble origins. Xanthophylls, not more
hydrophobic than carotenes, are found in membranes of the cells at aqueous/lipid border and have the power to scavenge
aqueous and lipid phase radicals (Bouayed and Bohn, 2012). Mostly carotenoids scavenge the radicals at lipid phase due
to their deep location in a polar core of lipid membranes (Kabat et al., 2012), so within the cells, carotenoids are joined
with different membranes, like membranes outside the cell, but carotenoids have also been demonstrated to be affiliated
with mitochondria and cell nucleus (Kaulmann and Bohn, 2014). Liposomes also show the presence of carotenoids, however cytosol does not contain this freely, resulting in protection of various types of cell membranes (Ben-Dor et al., 2005).
Carotenoids’ cellular acceptance from micelles, and the resulting asymmetric apocarotenoids (β-carotene-di‑oxygenase 2,
BCDO2, and symmetric (β-carotene‑oxygenase 1, or BCO1), become very bioactive but this is highly dependent on the administered dosage and genetic factors (Borel, 2012). Astaxanthin derived from marine animals exerts a higher antioxidant
activity due to the presence of hydroxyl (OH) and keto (CO) moieties on each ionone ring—a unique structure that has a
powerful effect on superoxide and hydroxyl radicals, quenching effect on singlet oxygen, a strong scavenging consequence
on hydrogen peroxide, and preventive effect on lipid peroxidation both in vivo and in vitro (Dhankhar et al., 2012).
Carotenoids and the Skin
Carotenoids such as lutein, lycopene, and beta-carotene, are the key components that aid in the reduction of aging of the skin
and the risk of developing cancer. However it has been demonstrated that carotenoids react with radicals that are present free,
which further leads to breakdown resulting in a quenching of the defensive series in the body (Blume-Peytavi et al., 2009). It
is understood from previous studies that oral intake of antioxidative substances raise the antioxidative potential in the body,
therefore oral intake of lutein enhances the skin integrity. However, not much data is available on the uptake of antioxidants,
appearance, and their cleavage in the skin, due to the shortage of noninvasive methods for assessment of antioxidants in the
skin. Consumption of single synthetic substances does not exert the positive effect, however, fruit and vegetable consumption
always revealed a progressive result on the antioxidative potential of the skin (Meinke et al., 2010). Organic extracts available in the form of pills or capsules could be an excellent substitute for getting the maximum benefits of antioxidative agents.
Olive oil, sea-buckthorn oil, and curly kale are those that exert a function of antioxidative capacity (Schmidt et al., 2010). Kale,
rich in lutein—a powerful antioxidant, is a good source of beta-carotene, and phenolic compounds, including flavonoids and
glycosinolates (Schmidt et al., 2010; Meinke et al., 2010). Olive oil also provides a rich amount of tocopherols and phenolic
substances. However, limited information is available on the bioavailability of single carotenoid in the skin, organic extracts, or
fruits that have low carotenoid concentration. In comparison to synthetic antioxidant compounds, the natural mixture of antioxidants in extract exerts positive effect on the antioxidative network more effectively. It was proven in a study done on organic
kale extract or placebo oil administered orally for 4 weeks to well-nourished subjects. It was found that carotenoids levels in the
skin were raised throughout the study period, but less in comparison to blood levels. The delay in skin levels was potentially due
to the dermal area and the carotenoids themselves. However, it was further concluded that after stopping the supplementation,
carotenoid levels decreased much faster in the blood than skin, indicating the possibility of a peripheral buffer role of the skin
for carotenoids (Meinke et al., 2010). Lutein and zeaxanthin present in Hass avocado and astaxanthin from marine animals are
highly bioavailable and help protect skin from visible and UV radiation (Dreher and Davenport, 2013; Dhankhar et al., 2012).
Carotenoids and Pulmonary Health
Pulmonary dysfunction is strongly associated with a high rate of mortality and disability in older adults. Dysfunctioning
in older adults is assessed by forced vital capacity (FVC) and forced expiratory volume in one second (FEV1), and is most
commonly connected with asthma, fibrotic lung disease and chronic obstructive pulmonary disease (Shaheen et al., 2010).
Novel Nutraceutical Compounds Chapter | 12 205
Pathophysiology of the aging of the lung is mainly due to the inflammation, oxidative stress, and low dietary intake of
antioxidant rich foods. Studies indicate that pulmonary health is closely associated with dietary antioxidants, however, the
relation of pulmonary health with specific foods has not been entirely consistent. In the Netherlands, Great Britain, and
Scotland, results from the population studies indicate that a high intake of fruits was associated with raised FEV1 levels, in
comparison to the vegetable consumption, which showed no association. The National Health and Nutrition Examination
Survey (NHANES III) reported low FEV1 level linked to low-grade systemic inflammation (Semba et al., 2012). In the
Japanese population, a case-control investigation reflected that greater consumption of vegetables, as opposed to fruits, was
related to low risk of chronic obstructive pulmonary disease. However, in the US women who consumed both fruits and
vegetables in large amounts had a low risk of pulmonary disease. Similarly, US women who were older, living in cosmopolitan areas, consumed more orange colored plant based foods, dark green leafy vegetables, and fruits, had high serum
α-carotene and β-carotene concentrations with improved pulmonary functions (Semba et al., 2012).
Carotenoids as Antitumor Agents
Carotenoids, being antioxidants, exert antioxidative properties which are considered the principal beneficial effect to attenuate carcinogenesis (Chatterjee et al., 2012). They mediate their effects through other processes: by cell growth regulation, modulation of gene expression, and immune response, gap junction communication, apoptosis, retinoid-dependent
signaling, and act as a modulator of phase I and II drug metabolizing enzymes. A number of studies have been done which
demonstrate the association between the carotenoid intake and cancer risks (Sharoni et al., 2012; Yuan et al., 2011), however some studies showed a negative correlation, or even revealed adverse effects. More intake of tomatoes and tomatobased foods (a rich source of lycopene) by subjects afflicted with cancer of the esophagus, stomach, pharynx, colon, oral
cavity, and rectum, in comparison to controls, showed significant decrease in risk of cancers (Freeman and Reimers, 2010).
Supplementation of βcarotene in the diet, with a quantity of 50 mg per day over a 5 year period revealed no influence on
the incidence of new basal or squamous cell carcinoma in healthy subjects, and earlier recovery for individuals with skin
cancer (Yuan et al., 2011). Furthermore the consumption of 25 mg per day of βcarotene, along with supplementation of
vitamin C, to patients with colorectal adenoma over the course of 5–8 years, did not show any reduction in the incidence
of colorectal adenoma that had adenomas earlier. In a large scale study, patients’ diets were supplemented with either βcarotene of around 20 mg per day, α-tocopherol of dosage 50 mg per day, in combination, or were in a control group, for a
5–8 year period. This name of the trial was the Alpha-Tocopherol, BetaCarotene (ATBC), study. Results were astonishing;
the occurrence of lung cancer was increased by 18% in patients who got βcarotene alone and in those who were given a
dosage combined with α-tocopherol, the mortality rate was increased by 8% (Krzyzanowska et al., 2010). It has also been
reported that individuals who developed lung cancer as a result of smoking and/or asbestos contact, β-carotene did not exert
any protective action and even induced lung pathology. However, in a recent study, higher carotenoid and retinol concentration of blood was found to significantly reduce the risk of lung cancer; a conclusion drawn from metaanalysis of prospective
studies (Abar et al., 2016). Yuan et al. (2011) demonstrated that carotenoid without provitamin-A activity like astaxanthin,
avoid toxicity of retinoid, and deliver protection against lung cancer. 20 years later in a follow-up group study, evidence
pointed to an inverse association between β-carotene and β-carotene present in the diet, lutein/zeaxanthin, and lycopene
intake and colorectal adenoma (Jung et al., 2013). In some studies lycopene consumption is also associated with a reduction
in prostate cancer risk (Sharoni et al., 2012), but some researchers reported insignificant effects of dietary lycopene and serum levels on prostate cancer (Krzyzanowska et al., 2010). In another study done on women, evidence suggested that higher
α and β-carotene, lutein plus zeaxanthin, lycopene, vitamin C, and total carotenoids reduce the breast cancer risk (Eliassen
et al., 2012; Bakker et al., 2016). However, in a recent study authors suggested an inverse association of plasma carotenoid
concentration with the risk of premalignant breast disease among women aged 50 and younger (Cohen et al., 2017). Saffron
derived from the Crocus sativus flower is also a rich source of natural carotenoids; studies suggest that intake of dietary
carotenoids have powerful antitumor effects both in vitro and in vivo. Use of carotenoid without the perspective for conversion to vitamin A, may provide further protection and avoid toxicity (Bolhassani et al., 2014). The highest antitumor
effects of astaxanthin and canthaxanthin (keto-carotenoid astaxanthin, 3,3-dihydroxy-b,b-carotene-4,4-dione) are source
dependent, originating from algae and aquatic animals, which have also been described as effective for various tumor cells
from prostate cancer, oral fibrosarcoma, breast cancer, embryonic fibroblasts, and colon cancer (Dhankhar et al., 2012;
Dreher and Davenport, 2013). Hass avocado, rich in a number of bioactive phytochemicals such as terpenoids, carotenoids,
phenols, d-mannoheptulose, and glutathione, and possesses anticarcinogenic activity in cancer of the pharynx, larynx, and
oral cavity. It is becoming a primary area of cancer investigation (Dreher and Davenport, 2013).
Carotenoids and Eye Health
Visual impairment and blindness are leading causes of age-related macular degeneration (AMD) and cataracts. These conditions are associated with oxidative processes induced by light within the eye. Epidemiological studies have confirmed that
206 SECTION | B Therapeutic Foods and Ingredients
factors related to oxidation are linked to increased danger of blurred vision, drusen formation, AMD, loss of dark vision,
and visual acuity. Similarly, studies demonstrated that high consumption of dietary carotenoids, especially zeaxanthin, and
lutein, reduce the risk for both AMD and nuclear cataracts (Krzyzanowska et al., 2010). These two carotenoid pigments
intensified in the macula of the eye, however, zeaxanthin found in the cone cells of the fovea, the middle of macula, are very
important for ocular health. All of these give protection against free radical impairment, oxidative stress in eyes and quench
the reactive oxygen species (ROS). The concentration and the density of these macular carotenoids vary from individual
to individual, and can be greater than 10-fold. Furthermore, Meyers et al. (2013) reported that macular pigment optical
density (MPOD)—a multifactorial phenotype, is a way to understand the factors that favor the deposition of carotenoids in
the retina. Studies suggest women with higher levels of lutein and zeaxanthin in their diets or serum have higher MPOD.
These causes account for only 8% of the variability in MPOD, reflecting the influence of other factors, such as differences
in the transportation of carotenoids, acceptance, and metabolism of known dietary stimuli on MPOD (Meyers et al., 2013,
2014). Supplementation of 30 mg per day of lutein over a period of 5 months increases the lutein serum levels, thus raising
its levels in the macula. High consumption of lutein and zeaxanthin through diet, over a period of 8 years, showed the risk
of cataract by 19%. Other research on the association between dietary β-carotene consumption and cataracts after 12 years
demonstrated no benefit (Krzyzanowska et al., 2010). Astaxanthin is also reported to be a powerful inhibitor of DHA oxidation (Dhankhar et al., 2012). Hass avocado contains 185 μg of lutein/zeaxanthin per one-half fruit, has greater bioavailability than other fruit and vegetable sources contributing to eye health, and aids in the absorption of carotenoids with the
help of monounsaturated fatty acids (Dreher and Davenport, 2013).
Carotenoid and Cardiovascular Risk Markers
The mortality and morbidity rate of coronary heart disease (CHD) is increasing daily. This is not isolated in Western societies. It is prevalent and increasing dramatically in developing nations as well (Riccioni et al., 2011; Grosso et al., 2014). It is
an established fact that dietary intervention is considered a primary step in the treatment of CHD and other diseases, more
so than any other treatments, therefore phytochemicals, particularly carotenoids, present a significant role in the maintenance of good health, as well as in diseases prevention (Lee et al., 2011; Yamagata, 2017). Literature shows that circulating
concentrations of carotenoids is inversely related to risk of developing cardiovascular diseases (CVDs) (Detopoulou et al.,
2010; Suzuki et al., 2010) by lowering the cholesterol oxidation in blood vessel walls. They stave off atherosclerosis activity
as an antioxidant, thereby reducing lipid peroxidation in low density lipoproteins. Suppression of reactive oxygen species
is generated through reduction of endothelin-1 gene expression by the lycopene, which leads to mitigation of endothelial
dysfunctioning, thus promoting the antioxidative effect directly, and inducing several gene expressions (Yamagata, 2017).
In some clinical studies the effect of nonpolar β-carotene exerted no beneficial effect on cardiovascular disease. However
in some trials, polar astaxanthin had a positive response on myocardial prevention; this still needs to be confirmed in human studies (Fassett and Coombes, 2012). Most adipokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6
(IL-6), elevate the vascular disease, and adiponectin acts as antiinflammatory and antiatherogenic. Decreased levels of adiponectin enhance the oxidative stress. It is carotenoids that are inversely associated with the inflammatory markers, that is
interleukin-6 (IL-6) and highly sensitive C-reactive protein (hs-CRP) (Suzuki et al., 2010). This was investigated among a
cross-section of 437 Japanese test subjects, including 321 women and 116 men. The significant inverse association has been
established between blood levels Vitamin E, adiposity, and carotenoids as well, among Mexican-American children where
the prevalence of excess weight and obesity is high. However, in this group, serum concentration of retinol was positively
associated with adiposity (Gunanti et al., 2014). Another study on children with simple obesity showed that intervention of
mixed carotenoid supplementation decreased the body mass index, waist to height ratio and subcutaneous adipose tissue
(Canas et al., 2017). Another study was done on 95 women to investigate the relationship between intake of vegetables and
fruit consumption, plasma carotenoids values and plasma extracellular superoxide dismutase (SOD) activity in relation to
health and disease status. However, they concluded that plasma extracellular superoxide dismutase action was not directly
related to fruit and vegetable intake and serum carotenoids levels. The disturbances in the extracellular superoxide radical’s
activity may lead to chronic disease such as CVD and diabetes (Zheng et al., 2011).
3.2
Phytosterols
Phytosterols (PS) are plant sterols (or stanols) normal bioactive components, which are extensively disseminated in plants
and plant comprising nutriments. Over 100 phytosterols have been recognized and the most abundant include β-sitosterol,
campesterol, brassicasterol, and delta 5-avenasterol, and stigmasterol (Srigley and Haile, 2015). These bioactive components mainly belong to the Triterpene family and have a methyl or ethyl group at C24, differing from the cholesterol group
functionally and metabolically (Gylling and Simonen, 2015). On the other hand, plant stanols are the saturated derivatives
Novel Nutraceutical Compounds Chapter | 12 207
of phytosterols. Phytosterols are present as hydroxycinnamate stearyl esters, fattyacyl esters, acylated steryl glycosides or
sterylglycosides as free or conjugated forms. Plant sterols/stanols stabilize the plant membranes and synthesize the alkaloids, steroidal saponins, and other steroids. Rich foundations of the PS and their esters are found in vegetable oils, plant
seeds, legumes, and cereals, especially wheat and rye. When following standard western eating habits, a mean daily intake
of about 300 mg of phytosterols and 30 mg of phytostanols is expected (Gylling and Simonen, 2015; Santas et al., 2013).
3.2.1
Bioavailability and Mechanism of Action
Studies revealed that the mechanism of action of PS is similar to the action of cholesterol, due to the analogous structure
with cholesterol. Similar to cholesterol, phytosterols are immersed in the proximal part of the small intestine after incorporation into the mixed micelles, and act within the intestinal lumen, balancing out cholesterol absorption and excretion
of the whole body. This is shown in Fig. 2. However, the absorption rate of PS is low as compared to cholesterol (Aldini
et al., 2014). The rate of absorption of phytosterols is about 5% while 40% to 60% of dietary cholesterol is immersed in the
body, depending on the structural components, that is, the nucleus of sterol and side chain of PS (Santas et al., 2013). The
efficiency rate of phytosterols absorption is <2%, and for phytostanols <0.2% (Gylling and Simonen, 2015).
FIG. 2 Absorption mechanism and metabolism of phytosterols.
208 SECTION | B Therapeutic Foods and Ingredients
Phytosterols and phytostanols are carried by the lipoproteins in circulation, mainly 70%–80% in low-density lipoproteins (LDL) and 20%–30% in high-density lipoproteins (HDL) particles, similar to cholesterol circulation mechanism with
the serum conc. of phytosterols is <24 μmol/L (<1.0 mg/dL), and serum concentration of phytostanol is not even high,
<0.3 μmol/L (<12 μg/dL) (Gylling and Simonen, 2015). An in detail mechanism of action of cholesterol and phytosterols
can be seen in the reference section (Santas et al., 2013).
3.2.2
Phytosterols and Beneficial Health Effects
Numerous studies have delivered reliable evidence on the valuable health effects of phytosterols due to the use of phytosterols as a nutraceutical and dietary supplement (Srigley and Haile, 2015; Yi et al., 2016; Shuang et al., 2016). The beneficial
role of phytosterols, in vitro and in vivo studies, in the prevention of ailments and disease are reported in this chapter.
Phytosterols and Antiinflammatory Effect
The role of PS is usually attributed to modulating cytokine production, but the modulation level is still not clear. Some studies indicate that production of proinflammatory cytokines, such as IL-6 or TNF-a, can be reduced by PS intake (Devaraj
et al., 2011; Santas et al., 2013) but some indicate the opposite effect (Santas et al., 2013; Guthrie et al., 2017). Additionally,
some studies suggested that they induce production of gamma interferon in Jurkat T cells, and cytokines IL-10 and IL-4
do not possess any significant change by the intake of PS (Santas et al., 2013). Another metaanalysis suggested that intake
of PS regularly did not show a significant effect on C-reactive protein, and reduced LDL-cholesterol significantly. It was
further suggested that studies are needed to explore the potential antiinflammatory effect (Rocha et al., 2016). Evidence of
beneficial inflammatory functions of PS has been reported in atherosclerosis, where atherogenesis occurs due to inflammatory processes (Aldini et al., 2014). This antiinflammatory effect is completely independent of the hypocholesterolemic
effect (Othman and Moghadasian, 2011; Aldini et al., 2014). A study done on rats fed with different doses of PS resulted in
significant reduction in atherosclerosis lesion, in terms of severity and size of lesion in arootic roots, with the positive effect
in reduction of total concentrations of plasma LDL, VLDL, and an increase in fecal cholesterol excretion (Moghadasian
et al., 2016). Inflammatory bowel disease refers to Ulcerative Colitis and Chron’s disease, resulting from initiation of the
immune and nonimmune response of the mucosa. Many studies report the antiinflammatory effect of PS of β-sitosterol,
sterol guggulsterone, and phytosteryl ferulates in the treatment of inflammatory bowel disease (Lee et al., 2012). However,
Aldini et al. (2014) reported that PS intervention did not stop the incidence of colitis, but can be attributed to reduction in
disease severity. Another longitudinal crossover trial demonstrated that PS induces reduction in the proinflammatory pathways (Cubedo et al., 2017).
Phytosterols as Antioxidant
Vegetable oils which contain phytosterols and their derivatives are known to exert an antioxidant function against the lipid
metabolism. Derivatives of Ps, that is β-sitosterol, stigmasterol, and campesterol, from natural sources showed the antioxidant effect on oxidation of methyl linoleate. Phytosterols also blocked oxidation and ingestion of alpha-tocopherol in betalinoleoyl-gamma-palmitoyl phosphatidyl choline (PLPC) liposomal membranes; the consequences are more prominent
than with dimyristoyl PC, having similar concentrations (Hu et al., 2017a). Stigmasterol enhances oxidation of both methyl
linoleate in solution and PLPC liposomal membranes in aqueous dispersions. It is attributed to oxidation of allylic hydrogens at the 21 and 24 numbers. Taken together, the study showed that phytosterols are chemically antioxidative uncertain
radical scavengers, and physical stabilizers of membranes (Yoshida and Niki, 2003). Soybean oil comparatively contains
less alpha-tocopherol, and may reduce the plasma levels of lipoproteins and deplete the antioxidant defenses resulting in
functional damage to hepatocytes (Saayman, 2011). However, a study on Atlantic salmon (Salmosalar L.) concluded that
dietary phytosterols at higher levels affect the metabolism of lipids and raise the TAG concentration in liver and plasma
(Liland et al., 2013).
Phytosterols and Atherosclerosis Prevention
Studies have also investigated the antiatherogenic effect of phytosterols in different animal models and have shown the protective effect of PS. These protective effect include reduction of lipid accumulation in arteries, decrease of plague formation
that develops atherosclerosis, reduction of lesion size and even inhibition of existing lesions produced by the cholesterol
lowering action of PS (Ferguson et al., 2017). Calcification and deposits of sterols are key factors for the origination and
progression of atherosclerosis that resulting in atherosclerotic arterial calcification and calcific aortic valve disease. After
the cholesterol, plant sterols, that is campesterol and sitosterol, are present in these lesions (Moore et al., 2013). At ­present,
Novel Nutraceutical Compounds Chapter | 12 209
the only effective treatment of calcific valve disease is surgical replacement of the valve. Detailed investigation on the
role of PS in terms of CVD risk, is still required before future implementation (Schött et al., 2014). It has also been demonstrated that PS may induce inflammation, and cholesterol efflux may be reduced from the macrophages due to PS that
directly resulted in the development of atherosclerosis (Lottenberg et al., 2012). A study conducted by Devaraj et al. (2011)
verified that fortified orange juice, or orange beverage with phytosterols, reduces the circulating cytokine levels or PAI-1
activity, which then reduces the synthesis of atherosclerosis. Additionally, research has shown that PS and β-sitosterol derived from pine also inhibited the cholesterol transport. An experiment was done on HT29-MTX intestine cell model that
formulated the mucus layer similarly to the intestine (Yi et al., 2016).
Phytosterols as Anticancer
High consumption of PS, either alone or in combination, acts as a cancer preventive component, however, sufficient data
on human subjects is still not available. Literature shows that consumption of PS inhibited carcinogen production, angiogenesis, retards the cancer cell growth, and promotes the cell apoptosis. Findings of some of existing research results
(Suttiarporn et al., 2015; Cilla et al., 2015; Llaverias et al., 2013; Yazan et al., 2011; Heuvel et al., 2012; Sureshkumar et al.,
2012) are presented in Table 1. A recent case-control study indicates that intake of campesterol, β-sitosterol, campestanol,
β-sitostanol, campestanol, and total sterols have an inverse relationship with the risk of colorectal cancer (Zhang et al.,
2016).
Phytosterols as Antidiabetic
The role of PS from plant extracts has also been observed in enhancing diabetes or obesity-associated disorders, although
extensive work has not been done on this. It has been observed that PS regulates the hepatic gene expression of gluconeogenic enzymes, that is glucose-6-phosphatase, phosphoenol-pyruvate carboxykinase (Pepck), and up-regulate b-oxidation
enzymes, such as peroxisome proliferator-activated receptor alpha (PPAR-a) (Misawa et al., 2012; Suttiarporn et al., 2015).
Isolated PS from the Aloevera plant (lophenol and cycloartenol) improved hyperglycemia via oral administration to Zucker
diabetic fatty rats (Misawa et al., 2012). Obesity is one of the most serious factors associated with chronic diseases such
as diabetes. Various earlier research estimated the ability of PS in relation to glucose and insulin resistance, by the intake
of PS alone or in combination, for weight loss in male Sprague-Dawley rats and Zucker diabetic rats (Furlan et al., 2013;
Misawa et al., 2012).
Phytosterols and Cholesterol-Lowering Effects
It is well documented that elevated cholesterol levels of plasma increase the risk of development of coronary heart diseases (CHD) (Srigley and Haile, 2015). Worldwide, many functional foods have been innovated for prevention of CHDs.
Among nutraceuticals, phytosterols have become of great interest for prevention of CHD and their associated risk factors
(Rideout et al., 2014; Granado-Lorencio et al., 2014). Since 1950, the role of both phytosterols and phytostanols in reducing the blood cholesterol level has been confirmed in many clinical trials (Srigley and Haile, 2015; Ras et al., 2014; He
et al., 2011; Investigators, 2016). The most recent clinical trials on humans, and other models, with signal or statin interventions, are presented in Table 2. It is evident from the studies that PS demonstrated reduction of cholesterol levels up to
30%–40% after a period of two to 3 weeks, and cholesterol levels remained static for at lease 1 year of continuous daily
consumption of 1.5–2.0 g of phytosterols (Rocha et al., 2011; Bitzur, 2013). LDL-cholesterol can be efficiently reduced up
to 10%–15%, with daily consumption of 0.8–4.0 g of phytosterols (Santas et al., 2013; Merino et al., 2013; Cheung et al.,
2017; Ferreira-Santos et al., 2017). However, a dose >2.0 g/day did not show any further reduction in LDL cholesterol, and
is therefore not generally recommended (Kunces et al., 2013; Zampelas, 2014). In addition to the role of PS in decreasing
the LDL cholesterol, PS also increases the HDL cholesterol, decreases the triglycerides level, and ratio of apo-lipoprotein
B/apo-lipoprotein A1 (Kunces et al., 2013; Kalsait et al., 2011). Use of phytosterols/stanols is also a cost effective remedy
to reduce the risk of cardiovascular diseases. This is proved by Eussen et al. (2011) who did an investigation on the entire
Dutch population, aged 35–75 years old (>8 million people). Results indicated that phytosterols/-stanols as mono and in
combined therapy, are above the thresholds for cost-effectiveness, ranging from €20,000 to €50,000.
No clinical relevance might be dependent on the duration of intervention or treatment on related CHD biomarkers
(Katan et al., 2003). Most studies indicated that adjunctive therapy with statin resulted in a reduction of hypercholesterolemia (He et al., 2011; Zampelas, 2014; Lin et al., 2011; Andrade et al., 2015). Zampelas (2014) also established that inclusion of these substances in the treatment should not be isolated. Males are also sensitive to PS, as compared to females,
although having the same mechanism of action, along with the genetic differences, in sterol metabolism and PS intake
(Weingärtner et al., 2014). Furthermore, an advantageous outcome of phytosterols is dependent on the capability to reduce
TABLE 1 Phytosterols as Anticancer Agent
Phytosterols
Source
Type of
Cancer
Model
10% campesterol:
75% β-sitosterol
Acros organics, NJ
Prostate
Human
24-Methylene-ergosta-5-en-3β-ol,
24-methylene-ergosta-7-en-3β-ol,
fucosterol, gramisterol, campesterol,
stigmasterol, β-sitosterol, and 3
triterpenoids (cycloeucalenol,
lupenone, and lupeol)
Bran extract of the
black rice cv. rice
berry
Leukemic
cell
β-Sitosterol, campesterol, and
stigmasterol
Beverages made of
fruit juice and milk
Mixture and of 2a,3b–dihydroxyolean12-en-28-oic acid
Type of
Cell Line
Dosage
Duration
Effect
Reference
C-3 and
DU145
16 μM
72 h
• Significantly induced
growth-suppression
(P < .05) and apoptosis
• Cell cycle analysis
showed decreased
mitotic subpopulations
and control ell growth
apoptosis
Ifere et al.
(2010)
Mouse
WEHI-3
12.5–1000 μg/mL
24–48 h
• Strong antileukemic cell
proliferation
• Gramisterol is a significant
anticancer compound
Suttiarporn
et al. (2015)
Colon
Human
Caco-2
[β-Cx (3 M),
β-sitosterol (12 M),
campesterol (1 M),
stigmasterol (0.25 M)]
24 h
Anticarcinogenic activity
Cilla et al.
(2015)
Coleus tuberosus
(extract)
Nasopha­
ryngeal
Human
Raji cells
0.7 mg/mL and
0.1 mg/mL
–
Significant antitumor
promoting activity
Mooi et al.
(2010)
20% campesterol, 22% stigmasterol,
and 41% β-sitosterol (supplement)
Dietary phytosterols
supplement
(Lipofoods S.L.,
Gavà, Barcelona,
Spain)
Breast
Mouse
Orally
2% added to the
powdered food
4, 8, or
13 weeks
Reduces development of
mammary hyperplastic
lesions (at age 4 weeks) and
total tumor burden (at age
13 weeks)
Llaverias
et al. (2013)
Phytosterols
Kenaf seeds
oil (Hibiscus
cannabinus)
Ovarian/
colon
Human
CaOV3/
HT29
100–5000 g/mL
72 h
Apoptosis effect, three
extractions were cytotoxic
towards CaOV3 cell line
Yazan et al.
(2011)
Phytosterols
Walnut oil
Breast
Mouse
3T3-L1
85 g of ground whole
walnuts, 34 g of
defatted walnut meat,
51 g of walnut oil, or
5.6 g of walnut skins
1, 2, 4,
and 6 h
postprandial
β-Sitosterol is the most
efficacious activators of
farnesoid X receptor
Heuvel et al.
(2012)
9,19-Cyclolanost-24-en-3-ol,acetate;
campesterol; stigmasterol; gammasitosterol; desmosterol; stigmasta5,24(28)-dien-3-ol,(3.beta.,24Z)-;ergost22-en-3-0l, (3.beta., 5.alpha., 22E,
24R)-;ergost-8,24(28)-dien-3-ol,4,14dimethyl,(3.beta.,4.alpha.,5.alpa)
Calotropis gigantea
Cervical
Human
HPV16
E6
–
–
Shows higher docking
energy.
Maximum potential against
the HPV16 E6 cervical
oncoprotein.
Sureshkumar
et al. (2012)
TABLE 2 Summary of Interventional Studies of Phytosterols Single or Combined With Other Therapies on Cardiovascular Disease Markers
Studied
Parameters
Study Subjects
Cohort study
Nonfasting blood
lipids
35,597 Dutch men
and women
Single
Experimental
Lipid profile
Different Phytosterols preparations
incorporated into milk
Combined
Experimental design
Phytosterols-poor diet,
(126 mg/2000 kcal); phytosterolsabundant diet, (449 mg/2000 kcal)
Combined
Stable statin treatment + low-fat
PS-enriched fermented milk (PS-FM)
(daily intake of 2 g PS-FM)
Phytosterols
Therapy
Study Design
Naturally occurring PSs
Single
Fucosterol, racemosol, stigmasterol,
and stigmasta- 7,22-dien-3β,4β-diol
from Lagenaria siceraria (Molina)
Study
Duration
Conclusions
References
12.2 years
Lowers total cholesterol and
lower LDL-C, particularly
among men
Ras et al.
(2014)
Wistar rats
administered once
daily 30 mg/kg of
phytosterols
5–90 days
Significantly reduces lipid
profiles
Increases HDL
Kalsait et al.
(2011)
Lipid profile
Hypercholesterolemic
13 men, and 7 women
4-weeks
FPS induces significant
decreases in LDL cholesterol.
Reduces vascular inflammation
markers
Kunces
et al. (2013)
Randomized crossover
feeding trial
Lipid and
cholesterol levels
24 subjects
4-weeks
Lowers the cholesterol
Increases fecal cholesterol
excretion
Raises plasma cholesterol/
lathosterol ratio
Lin et al.
(2010)
Combined
Interventional study
Open-label trial
LDL-cholesterol
35 elderly with
LDL-C concentrations
between 3.35 mmol/L
and 4.90 mmol/L
6 weeks
Reduces LDL-C
Inhibit cholesterol absorption in
the intestine
Decreases cholesterol:
cholesterol ratio
Andrade
et al. (2015)
Ezetimibe and phytosterols (low
phytosterols diet plus (1) ezetimibe
placebo phytosterols placebo, (2)
10 mg ezetimibe per day phytosterols
placebo, and (3) 10 mg ezetimibe per
day 2.5 g phytosterols per day)
Combined
Randomized controlled
trial
Cholesterol
metabolism
21
hypercholesterolemic
subjects
3 weeks
Significantly decreases
cholesterol absorption in the
intestine, plasma LDL-C
Increases fecal excretion
Lin et al.
(2011)
Substituting soy or milk proteins for
carbohydrates
Combined
Randomized, doubleblind crossover trial with
3 intervention phases
Blood pressure
parameters
352 healthy adult
8 weeks
Expressively reduces systolic
blood pressure. No significant
effect on diastolic blood
pressure
He et al.
(2011)
High fat supplemented with PS
(2%) + HF diet supplemented with
ezetimibe (EZ)
Combined
Experimental
Triglycerides
levels
Syrian golden
hamsters (12/group)
6 weeks
PS and EZ lower intestinal
cholesterol absorption.
Reduces hepatic cholesterol
triglycerides levels
Rideout
et al. (2014)
Supplemented with 1 × 250 mL milkbased fruit drink/day [β-Cx (0.75 mg/
day) and PS (1.5 g/day)]
Single +
combined
A randomized, doubleblind
Total cholesterol,
c-HDL, c-LDL
and bone
turnover markers
38 postmenopausal
women
4 weeks
β-Cx plus PS considerably
reduces total cholesterol,
c-HDL, c-LDL and bone
turnover markers
GranadoLorencio
et al. (2014)
212 SECTION | B Therapeutic Foods and Ingredients
serum triacylglycerol, as this is attributed to decrease generation of VLDL, which act as the key carriers of these types of
lipids (Andrade et al., 2015). In another review about guidelines on PS, it is stated that, along with the beneficial effects,
supplementation of PS may lead to a paradoxical increase in cholesterol levels in some individuals, and phytosterolemia
may be possible risk factor for cardiovascular diseases (Weingärtner et al., 2014; Rocha et al., 2011). In addition to this, PS
in the form of soya bean oil given as Parenteral Nutrition (PN) may cause cholestasis, as they are not metabolized in the
liver efficiently. Use of phytosterols regularly on animal models lead to reduction in bile flow, hence affected the biliary
secretions. Unfavorable changes have also been reported in patients on soya based PN (Le et al., 2011; Saayman, 2011; El
Kasmi et al., 2013).
3.3
Polyphenols
Polyphenols (PPs) are the secondary metabolites found in plants, which devour defensive mechanism against oxidants,
ultraviolet radiations, and other pathogens. Somewhere between 100,000 and 200,000 of PPs metabolites are assumed
to exist in nature (Zhang and Tsao, 2016). They are categorized into several classifications on the basis of phenol rings
and operational components that bind these rings to one another (Vauzour, 2012). Flavonoids are the most present polyphenols in human food and >4000 different types have been identified. Flavonoids are classified into subclasses: flavonols, anthocyanins, flavanones, flavones, flavanols, isoflavones, and anthocyanins (delphinidin, pelargonidin, malvidin,
cyanidin) (Vauzour, 2012; Yeh et al., 2016). Lignans are diphenoilc components having phytoestrogen activity. Phenolic
compounds are usually a third of the polyphenolic compounds present in food and are categorized into two major classes:
Hydroxybenzoicacid derivatives, including protocatechuic acid, p-hydroxybenzoic acid, gallic acid; and hydroxycinnamic
acid derivatives, including coumaric acid, caffeic acid, Ferulic acid, chlorogenic acid, and sinapic acid (Sileika et al., 2013).
Flavonols are categorized into myricetin, quercetin, kaempferol, and isoflavones (estrogen-like structure compounds include genistein, glycitein, and daidzein). The approximate intake of PPs is estimated to be 01 g per day (Bahadoran et al.,
2013; Vauzour, 2012).
3.3.1 Absorption and Mechanism of Action
It is reported that food preparation processes, digestion, absorption, and metabolism, are highly dependent on the bioavailability of these bioactive compounds. Dietary PPs are hydrolyzed by gastrointestinal enzymes or microflora, which then
must be conjugated in the cells of the intestine, and at the end, in the liver by sulfation, methylation, or glucuronidation
processes (Bahadoran et al., 2013). Consequently, after this PPs penetrate target tissue and exhibit their own biological
properties. PPs, such as flavanols, procyanidins, and quercetin, are rapidly absorbed into the plasma within 2 or 3 h after the
intake. It is also stated that the availability of PPs is low, and microbiota of the gut metabolize them into simpler metabolites which are absorbed efficiently. However, this is highly varied among individuals, which should be kept in mind while
conducting clinical trials (Tomás-Barberán et al., 2016). In vivo clinical studies suggest PPs that have lower metabolite can
reach plasma to have antioxidant and antiinflammatory effects (González-Sarrías et al., 2017).
3.3.2
Polyphenols and Beneficial Health Effects
Various beneficial effects on health have been reported for dietary PPs. Some of the important effects include antidiabetic,
antiviral and antimicrobial, antiinflammatory, antioxidant, antiallergic, anticarcinogenic, ulcer, hypotension, vascular fragility, and hypocholesterolemic capabilities (Kaulmann et al., 2016; Kim et al., 2014; Calabriso et al., 2016; Williamson,
2017; Santino et al., 2017). They are discussed here.
Polyphenols as Antidiabetic
Diabetes mellitus (Type-II) is known as a metabolic disorder caused by a number of factors, such as impaired digestion and
metabolism of dietary carbohydrates, increase in gluconeogenesis, dysfunctioning of β-cell of the pancreas, insulin resistance of peripheral tissues, reduction in glucose storage and defective insulin signaling pathways, lead to a hyperglycemia
state. There are a number of oral antidiabetic drugs available for the management of type-II diabetes, but the importance
of organic foods (recently termed nutraceuticals) cannot be ignored, and their use is considered as long term management.
Among these organic foods, PPs are considered an antihyperglycemic, due to their potential efficacy on the metabolism
of carbohydrates and glucose homeostasis (Bahadoran et al., 2013). PPs mainly reduce the absorption of carbohydrates
in the intestine, modulate the enzyme activities of glucose metabolism, improve the functioning of β-cells and action of
insulin, and stimulate insulin secretion. In one study it was demonstrated that the Mediterranean diet, which is rich in PPs,
is effective in improving the health status of patients with metabolic syndrome, and is considered an important factor in
Novel Nutraceutical Compounds Chapter | 12 213
FIG. 3 Role of dietary polyphenols as antihyperglycemic agent.
hyperglycemia (Amiot et al., 2016). Another study showed that PPs from cranberry and strawberries improve the insulin
sensitivity as well (Paquette et al., 2017). Beneficial effects of some of the dietary PPs as an antihyperglycemic agent are
summarized in Fig. 3.
Polyphenols as Antioxidant
Polyphenols and their bioactive compounds have the potential to act as an antioxidant or prooxidant because of the particular structure of PPs and the context of cellular redox that may increase or decrease the levels of oxidant scavenging
proteins, oxidized lipids and proteins (Kim et al., 2014; Peng et al., 2016). They have the capacity to hunt free radicals,
elevate the reduction of hydroperoxide formation and quench the production of reactive oxygen species (ROS), including
peroxyl radical, nitric oxide, hydroxyl radical, superoxide radical, singlet oxygen, nitrogen dioxide, and peroxynitrite, via
modulation of various enzymes activities for development of ROS, that is cyclooxygenase, microsomal monooxygenase,
xanthine oxidase, mitochondrial succinoxidase, and NADH oxidase (Lamoral-Theys et al., 2010; Schepetkin et al., 2016).
Clinical and epidemiological studies suggested that extended intake of PPs increases antioxidant capacity of plasma, enhancing the circulating inflammatory markers and postprandial glycemic response (Forbes-Hernandez et al., 2016). It is
established that novel pyranoanthocyanins and other PPs from staghorn sumac (Rhus hirta L.) exerted strong antiinflammatory activity in chemical based assays, but activated in cell based assays (Peng et al., 2016). PPs also have direct interactions with vital cellular receptors or chief signaling pathways beyond antioxidant capacities (Bahadoran et al., 2013).
They also stabilize the oxidant-antioxidant balance, improve the endogenous antioxidant system, and prevent the oxidative
damage. The role of green tea Catechinsincluding Epicatechin (EC), Epigallocatechin (EGC), Epicatechin-Gallate (ECG),
and Epigallocatechin-Gallate (EGCG), has also been described to increase the plasma total antioxidant capacity, reduce
the stress-sensitive signaling pathways, prooxidant enzymes, and stimulate the antioxidant enzymes, including glutathione
peroxidase, catalase, and superoxide dismutase (Bahadoran et al., 2013). However, Kim et al. (2014) demonstrated that
214 SECTION | B Therapeutic Foods and Ingredients
they have little to no effect on the following biological actions: inhibition of histamine release, inhibition of leukotriene B4
release, Angiogenesis, RyR1 activation, Cytotoxicity in oral cavity, FAS inhibition, SIRT1 activation, and Na/H exchanger
inhibition. The most abundant EGCG is responsible for many beneficial effects in animal, clinical, and cell structure
studies, as compared to other green tea Catechins. It acts as an antioxidant as it improves the mitochondrial function and
upgrades the lipid infusion-mediated insulin resistance; it is associated with increased activity of antioxidant enzymes
including glutathione peroxidase and superoxide dismutase (SOD) experiment done in vivo (Li et al., 2011). Besides the
action of antioxidant, some studies suggested that EGCG acts as a prooxidant, as it autooxidizes and produces hydrogen
peroxide in cell culture media. By the addition of catalase and SOD, some cellular activity is abolished by inhibiting the
effect of EGCG of autooxidation (Mooi et al., 2010; Forester and Lambert, 2011).
Polyphenols and Cardiovascular Rehabilitation
Literature shows that polyphenolic compounds have the potential to slow down the digestion and absorption process of dietary lipids, lipoprotein metabolism, and improve the dyslipidemia levels. Weakening of cardiac pump functioning, chronic
increase in cAMP, mitochondrial dysfunctioning, and protein posttranslational alterations, are considered contributing factors (Oudot et al., 2016). Procyanidins present in apples induce the intestinal lipoprotein production, decrease the apolipoprotein B synthesis and secretion, and inhibit the esterification of cholesterol. The oligomeric procyanidins present in
apples enhances the inhibition of triglycerides absorption and pancreatic lipase. Catechins contained in green tea interact
with proteins of cholesterol translocation from the enterocyte brush border (multidrug resistance P-glycoprotein 1, ATPbinding cassette proteins, Niemann-Pick C-1 like 1 protein B type1-scavenger receptors), altering their functional capacity,
attenuating cholesterol absorption effectively and also reducing the blood pressure (BP) (De Pascual-Teresa et al., 2010;
Grassi et al., 2008). Tart cherries rich in anthocyanins are also used as a medicinal food for reduction of hyperlipidemia,
fatty liver, and hepatic steatosis, via enhancement of hepatic Peroxisome proliferator-activated receptor alpha (PPARα)
(Seymour et al., 2008). Consumption of Concord grape juice has also been associated with cardiovascular health. Cocoa
beverages or drinks containing flavanols have also been linked with a reduction in blood pressure, and act as antihypertensive and have vasculature-associated effects (Galleano et al., 2010; Davison et al., 2010; Berry et al., 2010). Purified
compounds of PPs, such as Quercetin capsules and EGCG tablets, help to decrease the BP (Egert et al., 2010). Soy and
soy-based products rich in phytoestrogens and isoflavones have also been associated with reduction of systolic and diastolic
levels (Galleano et al., 2010). Immunology has a fundamental function in the development of cardiovascular pathologies.
Oleuropein, polyphenols in olive oil, enhances the functional activity of immune-competent cells (macrophages) and also
scavenges the free radicals thus surrogate the markers of cardio-protection (Visioli and Bernardini, 2011).
Polyphenols as Brain Modulator
Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most prevalent disorders of the geriatric population which
have profound economical and social implications on any society. The exact cause of these disorders is not yet known, but it
has been proposed that neuronal and physiological degradation is associated with the age-related neurodegenerative changes
triggered by multifactorial actions, including glutamatergicexcito toxicity, neuroinflammation, increases in iron, and/or depletion of endogenous antioxidants (Chalopin et al., 2010; Queen and Tollefsbol, 2010). Therefore, with the advent of neuroprotective drugs, organic nutritional agents such as polyphenols, were also give due importance in the light of their potential
beneficial effects (Casamenti and Stefani, 2017). Polyphenol-rich foods such as Gingko Biloba, blueberry, cocoa, olive
leaves, extra virgin olive oil, and tea, were tested on different models and beneficial effects have been demonstrated with
respect to learning capabilities and memory (Vauzour, 2012; Field et al., 2011; Ebrahimi and Schluesener, 2012; Casamenti
and Stefani, 2017). Another review depicted that intake of PPs exerted positive effects in many neurodegenerative disorders
(Sarubbo et al., 2017). The citrus flavanone of tangerines has also been associated to serve as a neuroprotective agent to
maintain the nigrostriatal integrity and functionality following lesioning with 6-hydroxydopamine occurring in Parkinson’s
disease (Calabrese et al., 2012). Vauzour (2012) and Lamoral-Theys et al. (2010) demonstrated that polyphenols and their
metabolites do not act as conventional hydrogen-donating antioxidants in vivo, but modulate the cells through actions at
lipid kinase and protein kinase signaling pathways. However, little has been achieved in bringing them into routine clinical
applications (Ebrahimi and Schluesener, 2012). Beneficial effects of some of the dietary polyphenols are shown in Fig. 4.
Polyphenols and Cancer
Prevalence and motility of cancer are increasing daily, in spite of extensive research on chemoprevention in the last decade. Chemoprevention is the focus of attention as an alternate approach in control and prevention of any type of cancer (Paluszczak et al., 2010; Scoditti et al., 2012). Various studies suggest that curcumin, genistein, resveratrol, and
Novel Nutraceutical Compounds Chapter | 12 215
FIG. 4 Role of polyphenols as brain modulator.
e­ pigallocatechin-3-gallate, regulate different micro RNA in different types of cancers (Devi et al., 2017). Studies indicate
that consumption of fruits and vegetables, five times a day, reduce 50% risk of developing different types of cancer, as
compared to those who have fewer than two servings a day. Consumption of whole grains has also been investigated in
reduction of development of colorectal cancer (Fresco et al., 2010; Ghanemi et al., 2017). In addition, various studies have
been conducted on specific edible plant parts, and their effect on cancer cell structures. Human and animal models resulted
showed positive response, which is illustrated in Table 3. Green tea polyphenols (GTPs) given in the form of Polyphenon E
(Mitsui Norin, Japan) causes cell cycle arrest and apoptosis in prostate cancer cells, by suppressing class I histone deacetylases (HDAC) (Thakur et al., 2012). In another study, it was determined that GTPs had double roles, to alter chromatin
modeling and DNA methylation (epigenetic mechanisms of gene regulation) which makes them an excellent candidate for
chemoprevention of prostate cancer (Pandey et al., 2010; Cimino et al., 2016), and was also termed a chemo-preventive
agent (Chen et al., 2011). Phenolic compounds present in extra virgin olive oil or any other food confirmed in in vivo that
they decrease the DNA damage, and subsequently lessen the risk of developing cancer, most notably breast cancer (Visioli
and Bernardini, 2011; Paluszczak et al., 2010). Phenolic acids, cinnamic acids, and derivatives are responsible for Caspase
activation, calcium influx, ROS production, PARP cleavage, and calmodulin activation marked as apoptotic markers. The
curcuminoids phenolic derivative present in turmeric, Curry Zingiberaceae, and curry spice, is associated with activation of
Caspase and reducing regulation of Bcl-2, Bcl-XL, thus having proapoptotic properties (Fresco et al., 2010).
TABLE 3 Polyphenols as Anticancer Agent
Polyphenols
Source
Type of Cancer
Cell Type
Dosage
Duration
Effect
References
Green tea polyphenols
(GTPs)
Polyphenon E (Mitsui Norin, Japan)
Prostate cancer
LNCaP cells
(harboring wild-type
p53) and PC-3 cells
10–80 mg/
mL of GTP
24 h
Cell cycle arrest and
apoptosis by suppressing
class I histone deacetylases
Thakur et al.
(2012)
Green tea polyphenols
(GTPs)
5 or 10 μM 5-aza-2′-deoxycytidine
(Sigma, St. Louis, MO), 10 nM
Trichostatin A (Sigma), 5–20 μM
EGCG and 1–10 μg/mL
Polyphenon E (Mitsui Norin, Japan)
Prostate cancer
LNCaP cells
1–10 μg/mL
1–7 days
Dual potential to alter DNA
methylation and chromatin
modeling.
Excellent candidate for the
chemoprevention
Pandey
et al. (2010)
Baicalein (BA), Decitabine
(DAC), Myricetin (MYR),
Protocatechuic acid (PCA),
Phloretin (PHR), Sinapic
acid (SIA), Syringic acid
(SRA), Resveratrol (RES),
Rosmarinic acid (RA), and
Ellagic acid (EA)
Sigma (St. Louis, MO, USA).
Betanin (BET), cyanidin (CYA) and
galangin (GAL) ABCR (Karl- sruhe,
Germany)
Breast cancer
MCF7 cell line
10–40 μM
72 h
All inhibits the DNA methyl
transferase activity
Paluszczak
et al. (2010)
Oleuropein and
hydroxytyrosol resveratrol
and quercetin
Virgin olive oil and red wine
–
Endothelial cell
(angiogenic)
0.1–
50 μmol/L
1–4 h
Reduce inflammatory
angiogenesis through MMP9 and COX-2 inhibition
Scoditti
et al. (2012)
Epigallocatechin-3-gallate
(EGCG) and Theaflavin (TF)
Tea polyphenols poly (lactide-coglycolide) nanoparticles
Various human
cancer cell lines
Namely A549 (lung
carcinoma), HeLa
(cervical carcinoma)
and THP-1 (acute
monocytic leukemia)
–
Cell
proliferation
assay and
cell cycle
analysis
Inhibiting cell proliferation,
metastasis, angiogenesis
and apoptosis biomarkers.
First mode nanoparticlemediated delivery of
phytochemicals
Singh et al.
(2011)
Flavonols
Tea extracts of 6 Ardisia species
[A. japonica (AJ), A. escallonioides
(AES), A. mamillata (AM), A.
compressa (AC), A. crenata (ACR),
and A. elliptica (AE)]
Liver cancer
Cell line (HepG2)
0.0123
ng/mL to
389.05
mg/mL
24 h
Greatest anticancer
potential in vitro
Newell
et al. (2010)
Polyphenols
Novel nutrient mixture (NM)
containing ascorbic acid, lysine,
Proline, and green tea extract
Liver cancer
HCC cell line SkHep-1
0.5%
04 weeks
Inhibits tumor weight
and burden of SK-Hep-1
xenografts by 42% and 33%
Roomi et al.
(2010)
Polyphenon-B
Black tea
Hepatocar­cino­
genesis
HepG2 cells in vitro
20–
100 μg/mL
MTT assay
Effectively inhibits
proliferation and induces
apoptosis
Murugan
et al. (2010)
Polyphenol
Curcumin
Fibroblast-like
synoviocytes (FLS)
MH7A cells and
RA-FLS
–
ELISAs
Antiinflammatory properties
Induces apoptosis in FLS
Kloesch
et al. (2013)
Novel Nutraceutical Compounds Chapter | 12 217
3.4
Omega 3-Fatty Acids
Eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) are the most important omega 3-fatty
acids, but now n-3 docosapentaenoic acid (DPA; 22:5n-3) is also under consideration for being beneficial to human health.
Omega 3-fatty acids are categorized by the double bond position nearest the methyl end of the hydrocarbon chain being on
carbon number three (counting the methyl carbon as number one). Due to the long hydrocarbon chain, these three are all
termed as “very long chain n-3 fatty acids”.
3.4.1 Absorption and Mechanism of Action
Metabolically DPA, DHA, and EPA are inter-related through a single pathway, by which synthesis of EPA occurs from the
simpler plant-derived n-3 fatty acids. Linolenic acid (18:3n-3) fatty acid, which is an essential fatty acid in animals, is the
initial substrate required for this pathway. Three steps are involved for conversion of the a-linolenic acid to EPA which is
catalyzed in turn by following enzymes: delta-6 desaturase, elongase, and delta-5 desaturase (Calder, 2014). These enzymes
are then mutually united with the corresponding omega-6 (n-6) fatty acid biosynthetic pathway of conversion of linoleic
acid (18:2n-6) to arachidonic acid (20:4n-6). Consumption of linoleic acid in a higher amount in diet favors greater conversion to arachidonic acid, as compared to a-linolenic acid (Kaur et al., 2011). Conversion at low rates may be affected by age,
gender (Blasbalg et al., 2011), genetics (Koletzko et al., 2011), hormonal, and disease status. Where the biological effects
of a-linolenic acid are demonstrated, they seem to be related to conversion to EPA; therefore the limited conversion of the
a-linolenic acid to EPA and further to DHA occurs, which limits its functionality and health impact. One-step b-oxidation
of DHA can be used to produce EPA, a process sometimes referred to as retro conversion (Calder, 2014; Baker et al., 2016).
Biosynthesis of very long chain n-3 fatty acids is shown in Fig. 5.
3.4.2
Omega-3 Fatty Acids and Beneficial Health Effects
Different experimental approaches have been adopted in research, to see the effect of n-3 fatty acids on cell culture, through
human and animal models. The first line of approach in studies is investigating the association between the n-3 fatty acids
intake from diet, or in specific body pools, such as in blood serum or plasma or in erythrocytes, and clinical biomarkers of
certain diseases risks or manifestations. Such studies are termed ecological studies (comparison between populations and
subpopulation), case-control studies (comparison between diseased and nondiseased individuals) and prospective cohort
studies (follow-up studies). The second line of approach adopted in research, is the trial or intervention of omega-3 fatty
acids among animal or human models, and usually termed single blind or double blind randomized controlled trials. In both
types of approach, a number of beneficial effects has been reported, however in this section, a limited number of the most
important benefits are emphasized.
Omega-3 Fatty Acids and Cancer
A wide spectrum of biological actions are performed by EPA and DHA that may affect the tumor cell proliferation and
variability, such as DHA promoting tumor cell apoptosis by inducing oxidative stress. EPA and DHA also have the ability to substitute arachidonic acid in cell membranes, which result in decreased production of prostaglandin E2 mediators
which initiate tumor growth and cell proliferation (Merendino et al., 2013; Wang and Dubois, 2010; Vaughan et al., 2013).
Through these potential activities, EPA, and DHA directly scavenge the cancer cells and effect the environment of the
tumor. Recent evidence provides in-depth detailed critical analysis of the mechanism by which very long chain n-3 fatty
acids affect the tumor cell invasion, metastasis, and proliferation (Merendino et al., 2013; Gleissman et al., 2010), and the
efficiency of these n-3 fatty acids on human subjects (Vaughan et al., 2013). However, the ratio of n-6/n-3 matters significantly in initiation and progression of cancers. Joshi et al. (2016) stated that low n-6/n-3 ratio has been found beneficial for
management of cancer cell hallmarks. Some case-control and prospective studies recommended that omega-3 fatty acids
reduce the risk of prostate, breast, and colorectal cancer, thought some findings are inconsistent. A recent systematic review
established that these n-3 fatty acids have a protective role against breast cancer (Gerber, 2012), however, consumption of
either EPA or DHA revealed lower risk towards the development of breast cancer in another recent prospective cohort study
among postmenopausal women (Sczaniecka et al., 2012). The variation in concentration of n-3 fatty acids is also stated to
be low in blood lipids and cells in some cancer patients, as compared to controls, due to changes in dietary habits and an
altered metabolism (Murphy et al., 2012).
Along with the effect of lowering the risk of developing cancer, n-3 fatty acids also seem to be effective in the treatment
of cancer. Patients with lung cancer receiving EPA and DHA supplementation had improved energy, appetite, body weight,
and quality of life (Calder, 2014). Similarly, physical weakness and inflammation were seen among breast cancer patients
218 SECTION | B Therapeutic Foods and Ingredients
FIG. 5 Biosynthesis of very long chain n-3 fatty acids.
who had higher levels of n-3 fatty acids in their bloodstreams, as compared patients who had lower blood n-3 fatty acids
levels (Alfano et al., 2012). In another study, patients with lung cancer who were given supplementation of EPA and DHA
of 1.8 g per day, showed improved appetite, as compared to another group without supplementation. Similarly, the intervention of 2.9 g of EPA and DHA per day showed improvement in cognitive functioning, physical functioning, overall health
status, and quality of life among small cell lung cancer patients (Van Der Meij et al., 2012). Supplementation of 2.2 g per
Novel Nutraceutical Compounds Chapter | 12 219
day of n-3 fatty acids (EPA and DHA) also enhances the bodyweight and muscle mass during chemotherapy. Therefore, it
is proven, mostly through clinical trials on humans, that supplementation of EPA and DHA of around 2 g per day may have
the adjuvant potential to chemotherapy in different types of cancers.
Omega-3 Fatty Acids and Cardiovascular Diseases
Literature shows that incidence of CVDs is much lower in the population of Northern Canada, Alaska, and Greenland. This
is due to the regular consumption of their traditional diet which is based on whole meat and fatty fish, despite of high dietary
fat intake. A similar trend had been depicted in Japanese population who also continue to consume traditional foods. A very
recent meta-analysis (16 studies with over 422,000 individuals) studied the relationship of circulating or dietary fatty acids,
including n-3 fatty acids, with the outcome of coronary risk. It was found that individuals who had n-3 fatty acids intake
were at the top third position, with a relative risk of 0.87 [95% confidence interval (CI 0.78–0.97)], compared to another
group. Furthermore, the aggregate of 13 more studies with over 20,000 individuals showed relative risk of 0.78 (95% CI
0.65–0.94), 0.79 (95% CI 0.67–0.93), and 0.75 (95% CI 0.62–0.89) who were in the top third of circulating EPA, DHA
and both EPA and DHA (Chowdhury et al., 2014). It was therefore concluded that very long chain n-3 fatty acids have a
protective effect, and reduce the risk of development of CVDs. Contrary to this, another study indicated that this fatty acid
had no effect after the intervention of this fatty acid for more than 6 years of postintervention on cardiovascular outcomes
(Investigators, 2016). They basically lower the blood pressure, plasma triacylglycerol concentrations, and inflammation in
the body (Saravanan et al., 2010; De Caterina, 2011), so the healthier the profile of these risks, the lower the chances of
occurrence of developing CVDs.
Research also indicates that death rate is reduced in patients who were receiving EPA and DHA with a dose ranging
from 500 mg to 1.8 g per day for duration of 1–5 years (Calder, 2014). It is reported that n-3 fatty acids possess the beneficial effects by three mechanisms: alter the cardiac electrophysiology resulted in lower heart rate, increase in heart rate
variability, and the arrhythmias (Xin et al., 2013); act as antithrombotic resulted from the release of eicosanoid mediators
which control the platelet aggregation from arachidonic acid and EPA, so at the end reduces clot formation in the body;
exert antiinflammatory action to stabilize the atherosclerotic plaques preventing their rupture so reduce the onset of stroke
and myocardial infarction (Cawood et al., 2010).
Omega-3 Fatty Acids and the Brain
The human brain is made up of lipids (>50% of the dry weight of the brain), phospholipids in particular. It contains a high
amount of DHA as compared to other body tissues, and contributes in neuroplasticity, stabilizes the neuronal membrane,
neurotransmission and signal transduction (Parletta et al., 2013; Smith et al., 2016). DHA amounts dramatically increase
during the brain growth spurt as the weight of the human brain increases, the amount of DHA increases by 3–4-fold and
35-fold of the total brain. A metaanalysis of 17 clinical trials showed that supplementation of DHA to preterm infants exerted minimum support to neurodevelopment (Schulzke et al., 2011). One more meta-analysis of 11 randomized controlled
trials among >5000 pregnant mothers did not conclude that intake of n-3 fatty acids improved the cognitive development of
their offspring (Gould et al., 2013). It is now assumed that consumption of n-3 fatty acids possess beneficial effects beyond
infancy, and is possible more important a little later on in life. Children who have low serum EPA and DHA levels may
face autistic spectrum disorders or hyperactivity disorders than healthier ones. Dyspraxia and dyslexia might be associated
with the deficiency of some sort of fatty acids; it has been proved that increasing the EPA and DHA intake for a longer
duration, reflected positive effects on learning, attention and behavioral disorders (Yui et al., 2012; Milte et al., 2012).
Supplementation from 0.2 to 2.2 g per day of EPA (60% more EPA than DHA) was effective in primary depression concluded from 15 RCTs (Sublette et al., 2011). Likewise, intake of 2 g per day of EPA improves unipolar and bipolar depressive disorder after a 4-week intervention (Calder, 2014; Borsini et al., 2017). One gram of EPA gave substantial benefits to
subjects with borderline personality disorders, and DHA also produces antiaggressive effect. Postmortem findings showed
that subjects with Alzheimer’s disease contained less DHA than those not afflicted with the disease (Cunnane et al., 2012).
Intervention of 1.5 g per day of EPA and DHA for 6 months showed improvement in memory performance among subjects
with mild Alzheimer’s disease, however, other studies with varying doses and ratios of n-3 fatty acids reported no beneficial
contribution to cognitive abilities among people with Alzheimer’s disease (Quinn et al., 2010; Calder, 2014).
Omega 3 Fatty Acids and Inflammation
Normal host defense mechanisms of the body use inflammation as a basic component to initiate immune response, and
later function in tissue repair. Normally this inflammatory response is self-resolving and serves to protect the host from
further damages. For the self-defense of the immune system and inflammatory components, EPA and DHA exerted the
most vital/beneficial effect on the system (Calder, 2013a,b; Da Silva et al., 2016). When an immune system continuously
220 SECTION | B Therapeutic Foods and Ingredients
experiences inflammatory triggers, the body’s normal mechanism is disturbed, resolving factors induced tolerance and
allow inflammation to become chronic. This state then tortures host tissues and becomes pathological. Such adverse inflammatory responses can be seen in asthma, atopic dermatitis, inflammatory bowel diseases (IBD), rheumatoid arthritis
(RA), and psoriasis conditions (Calder et al., 2013; Gan et al., 2017). It is reported that n-6 fatty arachidonic acid is the key
precursor to the inflammatory process and fatty acids, for the release of eicosanoids-intimately involved in inflammation.
Contrary to the effect of arachidonic acid, it is reported that n-3 fatty acids give rise to mediators (protectins, resolvins,
prostaglandins, and resolvins) that are less antiinflammatory, inflammation resolving or proinflammatory (Calder, 2013a).
They also produce inflammatory cytokines and leukocyte migration. The incorporation of EPA and DHA into inflammatory
cell membranes influences the transcription factor activation, gene expression, and cell signaling, and therefore act as an
antiinflammatory. This has been proven in many clinical trials (Calder, 2013a) of rheumatoid arthritis (Miles and Calder,
2012), and inflammatory bowel diseases (Calder, 2009). Consumption of n-3 fatty acids by lactating and pregnant mothers will decrease the allergic risk in their neonates, and also boost the immune system of their babies to reduce the allergic
response later on in their lives (Noakes et al., 2012). Consumption of very long chain n-3 fatty acids in high amounts can
also be used to treat frank inflammatory conditions, while lower doses protect the low-grade inflammatory conditions.
4.
FUTURE PROSPECTS
Nutraceuticals are intended to play a significant role in future therapeutic developments, however, and an important precursor to their successful application should be standardization of their safety, purity, and efficacy by regulatory authorities.
This administrative and regulatory work should be done without impeding innovation or progress. The current knowledge
based on research via various models is undoubtedly a major challenge for physicians, nutritionists, food chemists, pharmacologists, and the food technologists. Nutraceuticals are considered important as powerful tools by the public health authorities, for prevention and treatment against nutritionally induced acute and chronic diseases. They also have significant
economic potential. However, in clinical practice the use of nutraceuticals is emergent, and there remains a dire need to
research important clinical and pharmaceutical issues.
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