P R O T E O L Y T I C E N Z Y M E S I N HUMAN AND C O W ' S M I L K A. B. STORRS 1 A~D M. E. H U L L ttesearch Division, Armour and Company, Chicago Results obtained from feeding infants enzyme-treated milk suggest that the superiority of such milks may be associated with the added enzyme rather than with any physical or chemical changes in the milk proteins. Tests were made to determine the possible presence of the enzyme in treated cow's milk after pasteurization, and in human milk. The results provide a new concept in infant nutrition. Editor. The production of low curd-tension milk by modification with pancreatic enzymes was first proposed by Conquest et al. (4) in 1938. Later, a considerable amount of clinical investigation on such a product was reported by Blatt et al. (3) and Steigmann and Blatt (10). They showed that excellent results were obtained in infant nutrition with this modified milk and suggested its use in other situations where intolerance to cow's milk might be encountered. Blatt recommended the use of this milk unboiled, either diluted or undiluted, for the feeding of infants. Hess and Lundeen (6) at Michael Reese Hospital confirmed Blatt's work, obtaining good results when feeding unboiled Enzylac ~ to premature, as well as newborn, infants. These results suggested that the successful use of enzyme-treated milk might be due to something more than curd tension reduction. It was assumed that the additional effect was associated with the added pancreatic enzymes themselves, rather than with any physical or chemical change they might have had upon the proteins, such as reduction of curd tension or the liberation of proteoses, peptones, and amino acids. Enzyme-treated milk is produced commercially by adding pancreatic enzymes to raw milk, bringing the milk to pasteurization temperature (143 ° F.), holding it for 30 minutes, and then immediately cooling. While the milk is being heated, the enzymes act upon the milk proteins. The degree of hydrolysis has been estimated at only about 1% (12), which is insufficient to alter the appearance or flavor of the milk. It was assumed that the heat of pasteurization completely inactivated the added pancreatic enzymes, although occasionally low protein stability (11) is Received for publication September 16, 1955. 1 Present address: New 1Y[ilford, Conn. 2 Enzylac is the trade name for milk modified by a special pancreatic enzyme concentrate produced by the Armour Laboratories, a Division of Armour and Company, Chicago, Illinois. Enzylac is sold nationally by l~[edical Dairy Specialties of Chicago and its franchisees under license from the American Seal-Kap Corp., Long Island City, New York. 1097 10'98 A.B. STORIES AND :M. E. H U L L observed when treated milk is held for extended periods. From the commercial point of view protein instability has not been significant. From an academic viewpoint, however, it seemed desirable to determine the cause. Later experience seemed to indicate that a significant amount of enzyme activity might persist through pasteurization. The work reported in this paper was undertaken to study the effect of pasteurization upon proteolytic enzymes in milk, as well as to learn more about the enzyme activity of Enzylac milk and hmnan milk. E X P E R I M E N T A L PROCEDURE A N D R E S U L T S Tests for heat inactivation of enzymes in enzyme-treated milk. The enzyme used in this study was a special pancreatic concentrate used for the commercial production of Enzylac milk. It was added to whole milk at the level of 80 p.p.m., and the milk was then pasteurized in glass by heating to 143 ° F. and holding for 30 minutes. After pasteurization, the samples were cooled immediately in ice water and stored in a refrigerator. On the following day, after the addition of the necessary amount of antibiotic as indicated below, the samples were warmed to 100 ° F. and held at that temperature up to 6 hours, during which time the rate of enzymatic hydrolysis was followed by the determination of tyrosine by the Hull method (7). The method was modified slightly by using S & S No. 597 filter paper instead of No. 595 in order to remove the milk proteins more completely before measuring the tyrosine colorimetrically. Occasionally it was neces- TYROSINE LIBERATED FROM ENZYLAC, DURING IOO°F INCUBATION 0.8 o eg z 0.4 p- / ,.=, o.2 / o o ,.=, o. 06 2 4 6 HOURS, INCUBATED AT IO0°F FIG. 1. H e a t inactivation of proteolytie enzymes in Enzylac. PROTEOLYTIC ENZYMES IN MILK 1099 sary to either filter or centrifuge the final solution to remove a white g r a n u l a r precipitate, which i n t e r f e r e d with the colorimetric reading. F o r comparison, pancreatic enzyme concentrate was added to antibiotict r e a t e d r a w whole milk in the same quantities a n d the samples were incubated at 100 ° F. for 6 hours, followed b y a m e a s u r e m e n t of the tyrosine. The results of these tests, shown in F i g u r e 1, have been expressed in millig r a m s of tyrosine produced per liter of milk per m i l l i g r a m of pancreatic enzyme concentrate added. This method of expression makes possible a r e a d y comparison of rates of activity observed with v a r y i n g enzyme concentrations. The results show only the additional tyrosine produced by enzymatic action r a t h e r t h a n the total tyrosine in the samples. This was calculated b y s u b t r a c t i n g the tyrosine values of the a p p r o p r i a t e u n t r e a t e d control samples r u n concurrently. I t a p p e a r s t h a t about one-third of the added pancreatic enzymes are not inactivated b y pasteurization. I t was suggested t h a t this residual activity of enzyme-treated milk is a factor in the effectiveness of this milk as an i n f a n t food. I t was considered possible t h a t n a t u r e m i g h t provide enzymes in h u m a n milk to aid the i n f a n t d u r i n g the difficult period of a d j u s t m e n t f r o m p r e n a t a l n u t r i t i o n via the placenta to postn a t a l oral feeding. Consequently, a series of experiments was planned to determine the proteolytic enzyme activity of h u m a n milk as well as t h a t of cow's milk. Enzyme content of cow's milk and human milk. To demonstrate the presence of native enzyme activity in r a w h u m a n milk and r a w cow's milk, it was first necessary to eliminate any bacterial f e r m e n t a t i o n and consequent protein hydrolysis. Several anti-mierobials, including chlortetracycline ( A u r e o m y c i n ) , streptomycin, a n d h y d r o g e n peroxide, were tried without success ; finally, a m i x t u r e of bacitracin and p o l y m y x i n B was f o u n d to be satisfactory. The results obtained upon a sample of r a w cow's milk with a high bacteria content are shown b y samples A1 and A2 in Table 1. Total and proteolytic bacteria counts were made at the beginning and at the end of the 6-hour, 100 ° F. incubation period. 8 One-hundred-ml. samples o£ milk in 250-ml. glass-stoppered E r l e n m e y e r flasks were held at 100 ° F. Samples were removed periodically, and the tyrosine content was determined. The results obtained f r o m samples A1 and A2 show a large increase in the total bacteria count d u r i n g the 6-hour incubation period but a decreasing proteolytic count during this period. The addition of the antibiotics decreased the total and proteolytic counts appreciably. The effect of the bacteria u p o n the liberation is shown in the last group of figures. The milk sample without antibiotics liberated about f o u r times as m u c h tyrosine as the one containing antibiotics. E v i d e n t l y this high tyrosine liberation is caused by the effect of the bacteria on the milk protein or is indicative of a b r e a k d o w n of bacterial proteins. I n either case, it a p p e a r e d desirable to use milk samples containing as few bacTotal bacteria counts were made by Standard Plate Count (1), except that an incubation temperature of 98.6° F. was used. Proteo]ytic counts were made by counting the clear zones encircling the bacterial colonies after plating the colonies on Difco Plate Count Agar containing 1% skimmilk powder and incubating at a temperature of 98.6° 1~. TABLE 1 Effect of antibiotics on bacteria counts and tyrosine liberation in high and low bacteria count milks Antibiotics added (rag.) to 100 ml. milk Bacteria counts per ml. Enzylac added Proteolytie Total Tyrosine liberated (p.p.m.) Sample Bacitracin Polymyxin B (p.p.m.) 0 hr. 6 hr. 0 hr. 6 hr. 2 hr. 4 hr. 6 hr. A1 A2 B1 B2 C1 C2 C3 None 156 None 156 None None 156 None 15 None 15 None None 15 None None None None None 80 ~0 965,000 390,000 180 30 950 -- 208,000,000 65,000 28,800 ~10 299,500 107,500 1,300 17,650 13,150 30 20 20 --- 2,295 675 15,~00 ~10 78,500 24,600 1 12.0 3.0 0.0 1.8 1.5 33.0 30.2 16.0 4.0 1.5 1.8 3.0 48.5 42.7 20.4 6.0 1.5 3.2 11.7 63.5 58.3 P R O T E O L Y T I C ENZYMES I N MILK 1101 t e r i a as possible in order to have a m i n i m u m effect on the enzyme-protein system in the milk. The effect of a low bacteria count r a w milk u p o n tyrosine liberation was carried out in an experiment with r a w cow's milk taken as aseptically as possible f r o m a cow free f r o m mastitis. The results of this e x p e r i m e n t are shown in Table 1 b y samples B1 and B2 and indicate t h a t a v e r y small amount of tyrosine was liberated in either the sample with or t h a t without antibiotics. These results indicate t h a t the antibiotics used in this e x p e r i m e n t do not inhibit the liberation of tyrosine f r o m milk; however, to obtain a more complete answer to this question, the experiments conducted upon .samples C1, C2, and C3 were carried out with another sample of raw cow's milk having a low bacteria count. These results indicate a comparable liberation of tyrosine b y the enzymes in enzyme-treated milk with or without added antibiotics. The slight increase noted in the second sample was p r o b a b l y caused b y the bacteria t h a t developed because no antibiotic was present to inhibit them. TYROSINE LIBERATED DURING IOO°F INGUBATION I 1 I a 15 Q W I<~ n~ I.iJ m _~ IO (.9 hi Z ffl 0 ~- 5 Go~s I 2 I 4 HOURS I 6 :FIG. 2. P r o t e o l y t i c e n z y m e s i n v a r i o u s m i l k s . ( U p p e r o = r a w h u m a n , u p p e r ized E n z y l a c , lower o = r a w c o w ' s , a n d lower • = p a s t e u r i z e d eow~s) • = pasteur- 1102 A.B. S T O R R S A N D M. E. H U L L Samples of raw human milk, raw cow's milk, pasteurized cow's milk, and pasteurized enzyme-treated cow's milk were carried through a similar experiment. The bacteria counts on all these milks were below 1,000 per milliliter. The results indicate that raw human milk and enzyme-treated cow's milk contain substantially equal amounts of proteolytic enzyme as judged by the liberation of tyrosine at 100 ° F. Raw or pasteurized cow's milk contains about onefifth as much proteolytic enzymes as either raw human milk or pasteurized enzyme-treated cow's milk (Figure 2). DISCUSSION The finding that h u m a n milk contains appreciable quantities of proteolytic enzymes is of considerable interest nutritionally. The presence of proteolytie enzymes in human milk undoubtedly provides the i n f a n t with significant digestive assistance in its attempt to bridge the difficult period of adjustment from prenatal n u t r i t i o n via the placenta to postnatal oral feeding. The results reported here substantiate the experience gained through the commercial production of enzyme-treated milk, that there are appreciable quantities of proteolytic enzymes to be found in this pasteurized milk. The fact that the enzyme-treated milk has a comparable amount of proteolytic enzymes to that f o u n d in human milk m a y be one of the reasons that it is so well utilized by premature, as well as newborn, infants. Attempts to provide the infant with a milk similar in composition to human milk b y modifying cow's milk have not been entirely successful, as judged by the variety of infant feeding formulas that are in use and the number of prop r i e t a r y foods available. The fact that babies do best when fed breast milk supports the theory that human milk contains something not found in cow's milk. The presence of proteolytic enzymes in human milk may be responsible for the differences observed between infants fed human milk and those fed cow's milk. The intake of additional enzymes in the foods entering the intestinal tract, where optimum conditions for their activity ordinarily prevail, might conceivably provide a significant " b o o s t e r " effect which could be of assistance to individuals with lowered pancreatic activity. There is reason to suspect that such conditions do occur in young infants (2, 9), in some elderly people, and perhaps in some persons with allergies. The use of the amino acid tyrosine in the treatment of allergies (13) supplies a possible explanation for the value of enzyme-treated milk in such cases. The work reported here does not indicate the critical temperature at which proteolytic enzymes are inactivated. However, evaporated milk, which is exposed to a sterilization temperature of 240 ° F., contains no proteolytie activity (5). It would be logical to expect that the boiling of milk, a common practice in the feeding of infants, would also inactivate all proteolytic enzymes (8). According to the results shown in F i g u r e 2, pasteurization does not inactivate the proteolytic enzymes in cow's milk. I t is anticipated that some temperature between 143 ° and 212 ° F. would be the critical inactivation point for these enzymes. PROTEOLYTIC ENZYMES IN MILK 1103 P r o t e o l y t i c e n z y m e s t a k e n o r a l l y i n n o n p r o t e i n solutions w o u l d be d i g e s t e d b y the p e p s i n i n g a s t r i c juice. H o w e v e r , if these same e n z y m e s were a d d e d to a food c o n t a i n i n g c o n s i d e r a b l e p r o t e i n , such as milk, t h e e n z y m e s w o u l d be protected b y the a t t e n u a t i o n of the p e p s i n b y these p r o t e i n s . P a n c r e a t i c e n z y m e s are i n a c t i v e i n a n acid m e d i u m , such as the s t o m a c h p r o v i d e s ( 8 ) , b u t u p o n r e t u r n to a n e u t r a l or a l k a l i n e m e d i u m , t h e y r e g a i n t h e i r a c t i v i t y w i t h l i t t l e loss of potency. SUMMARY H u m a n m i l k c o n t a i n s p r o t e o l y t i c enzymes. A b o u t o n e - t h i r d of the p r o t e o l y t i c e n z y m e s a d d e d to cow's m i l k i n the p r o c e s s i n g of e n z y m e - t r e a t e d m i l k s u r v i v e p a s t e u r i z a t i o n . H u m a n m i l k a n d p a s t e u r i z e d e n z y m e - t r e a t e d c o w ' s m i l k cont a i n s u b s t a n t i a l l y e q u a l a m o u n t s of p r o t e o l y t i c enzymes. R a w or p a s t e u r i z e d cow's m i l k c o n t a i n s a b o u t one-fifth as m u c h p r o t e o l y t i c e n z y m e as e i t h e r r a w h u m a n m i l k or p a s t e u r i z e d e n z y m e - t r e a t e d milk. These r e s u l t s p r o v i d e a n e w c o n c e p t i n the n u t r i t i o n of i n f a n t s as well as others who do n o t t o l e r a t e cow's milk. REFERENCES (1) A.~ERIOANPUBLIC IIEALTH ASSOCIATION,INC. Standard Methods for the Examination of Dairy Products. 10th ed., 1953. (2) ANDERSEN,D. H. Pancreatic Enzymes in the Celiac Syndrome. Am. J. Diseases Children, 53: 643. 1942. (3) BLATT, M. L., HA~IS, E. H., 5ACOBS,tI. M., AND ZELDES, ~ . An Evaluation of Enzyme Treated Milk in Infant Feeding. J. Pediat., 17:435 (Oct.) 1940. (4) CONQUEST, V., TURNER, A. W., AND REYNOLDS, It. J. Soft Curd Milk Produced with Pancreatic Concentrate. J. Dairy Sci., 21: 361. 1938. (5) GRF~NE,H. T. Personal communication. (6) HESS, J. H., AND LUNDEEN, E. C. Premature Infant. J. B. Lippincott Co., Philadelphia. 1949. (7) HULL, M. E. Studies on Milk Proteins. II. Colorimetric Determinations of the Partial Itydro]ysis of the Proteins in milk. J. Dairy Sci., 30: 881. 1947. (8) NORTHROP, J. H., KUNITZ, M., AND HERRIOTT, R. Crystalline Enzymes. Columbia Univ. Press, New York. 1948. (9) RICHMOND, R. C., AND SHWACHMAN, ti. Studies of Fibrocystic Disease of the Pancreas (Mucoviscidosis): Chymotrypsin Activity of Duodenal Fluid. Pediatrics, 1 6 : 2 0 7 (Aug.) 1955. (10) ST~IC~ANN,F., ANn BLAir, M. L. Enzyme Treated Milk in the Dietary Management of Patients with Peptic Ulcer. Am. J. Digest. Diseases, 11 (9) : 276. 1944. (11) STORRS,A. B. A Test for the Protein Stability of Milk. J. Dairy Set., 25: 19. 1942. (12) STORI~S,A. B. Studies on Milk Proteins. III. The Modification of Milk by Pancreatic Enzymes. J. Dairy Sci., 30: 885. 1947. (13) WOODWARD, W. K. Response of Allergic Persons to Oral Administrations of an Epinephrine Precursor. U. S. Armed Eorces Med. J., 5: 1300. 1954.
