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Influence of Protein Hydrolysis on the Susceptibility of Milk to Oxidized Flavor Development1
I N F L U E N C E OF P R O T E I N H Y D R O L Y S I S ON T H E S U S C E P T I B I L I T Y OF M I L K TO O X I D I Z E D F L A V O R D E V E L O P M E N T 1 T. L. FOICSTER, C. JENSEN, AND EMILY PLATtt North DaI~ota Agricultural Experi~e~tt Station, Fargo
Anderson (1, 2) cited a number of instances in which mild treatment of milk with a pancreatic concentrate, before pasteurization, gave protection against later development of oxidized flavor. Doan and Miller (5) and Corbett and T r a c y (4) confirmed these observations. The treatment has proved so successful that it has been adopted as commercial practice in many areas where Health D e p a r t m e n t approval could be obtained. Two pancreatic concentrates, at least, are presently being marketed for this purpose. Changes brought about by the mild treatment recommended were so slight that no definite differences in total protein or in formal titration could be d e tected by Conquest et al. (3), although the curd tension was lowered appreciably. Storrs (10) however, using a modified tyrosine test devised by Hull (8), found that the enzymatic treatment caused definite increases in tyrosine units. This evidence, with that of Doan and Miller (5) who obtained no protective action when trypsin solutions were denatured by heating, indicated that protein hydrolysis might be responsible for the added resistance to oxidized flavor in milk samples treated with pancreatic enzyme preparations. P r e l i m i n a r y observations b y F o r s t e r and Sommer (6), with crystalline trypsin, suggested that the protective action of commercial enzyme preparations could be due to hydrolytic cleavage of milk proteins. The work reported herein was undertaken with a view to obtaining additional information regarding tile influence of milk protein hydrolysis on the susceptibility of milk to oxidized flavor development. EXPERIMENTAL
The general procedure used in conducting each of the trials reported in Tables 2 and 3 was as follows: A sample of fresh, raw cow's milk was divided into twQ 500-ml. portions in amber glass, p y r e x bottles. One portion served as the control and the other received the enzyme treatment. This consisted of incubating the sample for 20 minutes at 40 ° C. in the presence of the required amount of enzyme, followed by in-bottle pasteurization at 62 ° to 63 ° C. for 30 minutes and cooling. Non-protein nitrogen determinations were made in quadruplicate by the method of Johnson (9) on 2 ml. portions of a trichloroacetic acid filtrate. This filtrate was p r e p a r e d by adding 1 ml. of milk to 25 ml. of 0.5 iV[ trichloroacetic acid, mixing well, and filtering after allowing 1 hour for precipitation. Susceptibility of each sample to oxidized flavor development was determined by setting up a series consisting of six 50-ml. portions. Copper, in the form of solutions of copper sulfate, then was added to give the desired Received f o r p u b l i c a t i o n A u g u s t 11, 1952. i P a r t i a l r e p o r t on P u r n e l l P r o j e c t 149 ~' R e l a t i o n of P r o t e i n s a n d N o n - P r o t e i n N i t r o g e n to D e v e l o p m e n t o f Oxidized F l a v o r in M i l k " p u b l i s h e d w i t h t h e a p p r o v a l of Director of t h e Station. 98
99
OXIDIZED FLAVOR IN MILK
a d d e d copper c o n c e n t r a t i o n s . P o r t i o n s 1, 2, 3, 4, 5, a n d 6, respectively, c o n t a i n e d 0, 10 -6, 10-5, 5 X 10 -~, 10 -4, a n d 5 X 10- ~ M/1. of a d d e d copper. The c o p p e r s u l f a t e s o l u t i o n s used were of such c o n c e n t r a t i o n t h a t n o more t h a n 0.5 ml. of a s o l u t i o n was r e q u i r e d to give the d e s i r e d a d d e d c o p p e r c o n c e n t r a t i o n . These six p o r t i o n s were e x a m i n e d f o r the p r e s e n c e of oxidized flavor a f t e r storage a t 0 ° to 4 ° C. for 48 hours. A n u n l e r i c a I v a l u e , r e f e r r e d to h e r e i n as the s u s c e p t i b i l i t y r a t i n g , t h e n was a s s i g n e d to the sample. This was b a s e d on its s u s c e p t i b i l i t y as det e r m i n e d b y the m i n i m u m c o n c e n t r a t i o n of a d d e d c o p p e r w h i c h p r o d u c e d the flavor. T a b l e I shows how the s u s c e p t i b i l i t y r a t i n g was assigned. I t will be TABLE 1 Relationship between susceptibility rating and minimum concentration of added copper required to produce oxidized flavor Susceptibility rating
Minimum cone. added copper to produce oxidized flavor. (M./1.)
6.0 5.0 4.0 3.5 3.0 2.5 2.0
none a 10.8 10-5 5 X 1 0 -5 10-~ 5 X 10-4 no oxidized flavor in 5 X 10-4b
A susceptibility rating of 6.0 was to be assigned if the flavor developed in that portion to which no copper was added. This never occurred. b A susceptibility rating of 2.0 was assigned when the oxidized flavor failed to appear even in the portion containing 5 X 10-4 M./1. of added copper. n o t e d f r o m t h i s t a b l e t h a t h i g h e r s u s c e p t i b i l i t y r a t i n g s were a s s i g n e d to s a m p l e s w h i c h developed the flavor w i t h low c o n c e n t r a t i o n s of a d d e d copper a n d lower r a t i n g s to those w h i c h r e q u i r e d g r e a t e r c o n c e n t r a t i o n s of a d d e d c o p p e r to produce the defect. D a t a s h o w i n g the i n f l u e n c e of p a n c r e a t i c e n z y m e t r e a t m e n t of m i l k o n n o n p r o t e i n n i t r o g e n a n d s u s c e p t i b i l i t y to oxidized flavor are p r e s e n t e d i n Tables 2 a n d 3, respectively. The d a t a i n T a b l e 2 show c l e a r l y t h a t t r e a t m e n t of m i l k w i t h a n y one of the t h r e e p a n c r e a t i c e n z y m e p r e p a r a t i o n s t e s t e d r e s u l t e d i n a s i g n i f i c a n t ( 5 % p o i n t F ) i n c r e a s e i n n o n - p r o t e i n n i t r o g e n . S u c h increases i n n o n - p r o t e i n n i t r o g e n c a n be i n t e r p r e t e d as i n d i c a t i n g p r o t e i n h y d r o l y s i s . T h e TABLE 2 Influence of pancreatic enzyme treatment of ~nilI¢ on non-protein nitrogen content Enzyme used Lactivase T-360 Trypsin Cryst. Trypsin
Rate used
No. of trials
(mg.ll.) 60 20 4
8 5 6
Mean non-protein nitrogen Control Treated Increase (mg.lL) 285.3 274.6 270.3
(reg.~L) 296.4 323.3 406.8
(reg.~L) 11.] 48.7 136.5
Analysis of variance F value a 5% point 7.43 51.76 17.78
5.59 7.71 6.61
The interaction of treatment X trials proved to be significant for each enzyme treatment, hence the variance for this interaction was used to test the significance of the enzyme treatment in each case.
100
T . L . F O R S T E R E T AL
TABLE 3 Influence of pancreatic enzyme treatment of milk on susceptibility to oxidized flavor
Enzyme used
Rate used
No. of trials
Mean Susceptibility rating Control Treated Decrease
t value ~
P
(rag.~1.)
Lactivase T-360 Trypsin Cryst. Trypsin
60 20 4
8 5 6
3.69 3.30 3.75
3.06. 2.00 2.92
0.63 1.30 0.83
7.696 10.612 7.912
< 0.01 < 0.01 ~ 0.01
Values paired. d a t a of T a b l e 3 show t h a t each e n z y m e t r e a t m e n t also r e s u l t e d in a s i g n i f i c a n t d e c r e a s e i n s u s c e p t i b i l i t y r a t i n g , i.e., r e d u c e d t h e t e n d e n c y t o w a r d o x i d i z e d flavor d e v e l o p m e n t . The d e c r e a s e s i n s u s c e p t i b i l i t y s h o w n in T a b l e 3 m i g h t h a v e b e e n due to p r o t e i n h y d r o l y s i s r e s u l t i n g f r o m t h e e n z y m e t r e a t m e n t s or m e r e l y to the a d d i t i o n of t h e e n z y m e p r e p a r a t i o n s . I n o r d e r to d e t e r m i n e w h i c h of the above m e n t i o n e d p o s s i b i l i t i e s was r e s p o n s i b l e , a n o t h e r s e r i e s of t r i a l s was c o n d u c t e d , u s i n g h e a t - d e n a t u r e d (10 m i n u t e s b o i l i n g ) enzymes. I n each of these t r i a l s a s a m p l e of f r e s h r a w m i l k was d i v i d e d i n t o f o u r 500-ml. p o r t i o n s i n a m b e r glass b o t t l e s a n d h e a t e d to t h e p a s t e u r i z i n g t e m p e r a t u r e of 62 ° to 63 ° C. The h e a t denatured enzymes, in aqueous solution, then were added and mixed and the m i l k h e l d f o r t h e r e q u i r e d 30 m i n u t e s p a s t e u r i z a t i o n e x p o s u r e , f o l l o w e d b y r a p i d cooling. One p o r t i o n , to w h i c h no e n z y m e was a d d e d , s e r v e d as the control, w h i l e L a c t i v a s e , T-360 t r y p s i n , a n d c r y s t a l l i n e t r y p s i n , r e s p e c t i v e l y , were a d d e d to t h e o t h e r t h r e e p o r t i o n s . N o n - p r o t e i n n i t r o g e n a n d s u s c e p t i b i l i t y t e s t s t h e n w e r e m a d e on these p o r t i o n s in t h e m a n n e r p r e v i o u s l y d e s c r i b e d . R e s u l t s of t h i s series of t r i a l s a r e s u m m a r i z e d i n T a b l e 4. T h e y show t h a t a d d i t i o n of t h e h e a t d e n a t u r e d e n z y m e p r e p a r a t i o n s h a d no a p p r e c i a b l e i n fluence on t h e n o n - p r o t e i n n i t r o g e n c o n t e n t , i.e., p r o t e i n h y d r o l y s i s d i d not t a k e TABLE 4 Influence o/ addition of heat denatured pancreatic enzymes to mil~ on non-protein nitrogen and s~.scep~ibility to oxidized flavor ~
Enzyme used None (control) Lactivase T-360 Trypsin Cryst. Trypsin
Rate used
Non-protein nitrogen
(mg./l.)
(mg./l.)
-60 20 4
236.5 b 239.9 240.6 237.8
Susceptibility rating 3.67 3.33 2.50 3.6]
t value ~
4.082 9.924 1.079
P °
~ 0.01 ~ 0.01 0.3 to 0.4
Av. of 9 trials. b Difference between mean non-protein nitrogen values for the four treatments were not significant at the 5% point. ( F ~ 1.22 and 5% pt. ---- 3.01) c Calculated for susceptibility rating data. Enzyme treatments compared individually with control. Values paired. place. Two of t h e enzymes, however, L a c t i v a s e a n d T-360 t r y p s i n , c a u s e d a p p r e c i a b l e a n d s i g n i f i c a n t ( P ~ < 0.01) l o w e r i n g of t h e s u s c e p t i b i l i t y r a t i n g . T h e
O X I D I Z E D FLAVOI% I N M I L K
101
small decrease in susceptibility rating resulting from the addition of heatdenatured crystalline trypsin proved not to be significant. DISCUSSION
Crystalline trypsin, when added at the rate of 4 rag. per liter, incubated for 20 minutes at 40° C. and then destroyed by pasteurization, caused considerable protein hydrolysis (Table 2) and markedly reduced the susceptibility of the treated milk to oxidized flavor development (Table 3). Heat-denatured crystalline trypsin added, at the same rate, to milk which was immediatelypasteurized caused no protein hydrolysis and no significant change in susceptibility to oxidized flavor (Table 4). The purity of a crystalline trypsin preparation preCludes the possibility of any enzyme activity except protein hydrolysis. Therefore, it may be concluded that the protein hydrolysis resulting from treatment of milk with crystalline trypsin was directly responsibfle for the observed decrease in susceptibility to oxidized flavor. Treatment of milk with Lactivase or T-360 trypsin, in a manner similar to that used when treating with crystalline trypsin but at higher rates, also caused protein hydrolysis (Table 2) and reduced susceptibility to development of oxidized flavor (Table 3). In these cases, however, the decreases in susceptibility cannot be ascribed entirely to protein hydrolysis, since addition of these enzyme preparations, after heat denaturation, resulted in decreases in susceptibility without causing any hydrolysis (Table 4). Furthermore, the decreases in susceptibility resulting from treatment of milk with the active enzyme preparations were not proportional to the degree of protein hydrolysis. Crystalline trypsin, for instance, showed ]8~ times the activity in causing increases in non-protein nitrogen as did Laetivase but was only 20 times more active in reducing susceptibility to oxidized flavor. Obviously, some other factor besides protein hydrolysis was involved in causing the decreased susceptibility observed when milk was treated with either Lactivase or T-360 trypsin. To explain the decreased susceptibility of milk treated with pancreatic enzymes, Forster and Sommer (6) advanced a theory based on the work of Hopkins (7). They suggested that such enzyme treatments cause a mild degree of protein hydrolysis, which in turn exposes readily oxidizable groups, such as -SH groups, which formerly were deep within the protein molecule. Oxidation of these groups then reduces the amounts of oxidizing agents and oxygen present in the milk to a point where lipid oxidation cannot proceed. Thus, the oxidized flavor resulting from lipid oxidation is prevented. The data presented in this paper support this theory. In addition, that part of the effect of Lactivase and T-360 trypsin which cannot be ascribed to their enzyme activity may still be explained on the basis of this theory. Neither Lactivase nor T-360 trypsin approaches the degree of purity to be found in crystalline trypsin. Unquestionably, both preparations contain appreciable amounts of protein hydrolytic products. Such products would, therefore, contain readily oxidizable groups which would function in the same manner as those resulting from enzyme hydrolysis of milk proteins. Both of these enzyme
102
T.L. FORSTER
ET AL
preparations were used in appreciable amounts, and it therefore is quite conceivable that sufficient readily oxidizable groups were added to impart the degree of protection from oxidized flavor noted in Table 4. Homogenization and high heat treatments (70 ° to 80 ° C.) are known to reduce the susceptibility of milk to development of oxidized flavor. It is possible that these factors, also, function by causing protein hydrolysis or denaturation. Further study is needed if the mechanism by which these factors function is to be clearly understood. SUMM&RY AND CONCLUSIONS
Treatment of fresh raw milk, before pasteurization, with active Lactivase (60 mg. per liter), T-360 Trypsin (20 mg. per liter) or crystalline trypsin (4 rag. per liter) resulted in appreciable protein hydrolysis as measured in terms of increase in non-protein nitrogen content. Such treatment always was accompanied by a decrease in susceptibility to oxidized flavor. Addition of the heat-denatured enzyme preparations caused no protein hydrolysis, but addition of heat-denatured Lactivase and T-360 trypsin did cause a decrease in susceptibility. Addition of heat-denatured crystalline trypsin, however, caused no appreciable decrease in susceptibility. It is concluded that (a) the protein hydrolysis induced by treating milk with crystalline trypsin is accompanied by, and responsible for, a decrease in the susceptibility of the mille to oxidized flavor development, and (b) the decrease in susceptibility to oxidized flavor development, caused by treating milk with Lactivase or T-360 trypsin, is partly due to hydrolysis of the milk protein and partly to some, as yet unknown, component in the enzyme preparations used. REFERENCES (1) ANDERSORI, E. O. Variations in Susceptibility of Milk as Secreted by the Cow. Proc. Intern. Assoc. Milk Dealers, Lab. See., 153. 1937. (2) ARIDERSORI, E. O. Preventing Development of Oxidized Flavor in Milk. Milk Dealer, 29(3) :32. 1939. (3) CONQUEST, V., TURNER, A. W., AND REYRIOLDS, I-~. J. Soft Curd Milk Produced with Pancreatic Concentrate. J. Dairy Sci., 21: 361. 1938. (4) CORBETT,W. J., AND TRACY, P. H. Experiments on the Use of Certain Antioxidants for Control of Oxidized Flavor in Dairy Products. Food Research, 6: 445. 194~1. (5) DOARI, F. J., AND MILLER, G. M. Trypsin An Anti-oxygen for Milk. Mi]k P l a n t Monthly, 29,9: 42. 1940. (6) FOaSTER, T. L., ARID SOI~MER, H. I~. Manganese, Trypsin, Milk Proteins and the Susceptibility of Milk to Oxidized Flavor Development. J. Dairy Sei., 34: 992. 1951. (7) HOPKIRIS, F. G. Glutathione. Its Influence in the Oxidation of F a t s and Proteins. Biochem. J., 19: 787. 1925. (8) HULL, M. E. Studies on Milk Proteins. II. Colorimetric Determination of the Partial Hydrolysis of the Proteins of Milk. J. Dairy Sci., 30: 881. 1947. (9) Jo~RIso~, M. J. Isolation and Properties of a Pure Yeast Polypeptidase. J. Biol. Chem., 137: 575. 1941. (10) STO•RS, A. B. Studies of Milk Proteins. I I I . The Modification of Milk by Pancreatic Enzymes. J. Dairy Sci., 30: 885. 1947.
Anderson (1, 2) cited a number of instances in which mild treatment of milk with a pancreatic concentrate, before pasteurization, gave protection against later development of oxidized flavor. Doan and Miller (5) and Corbett and T r a c y (4) confirmed these observations. The treatment has proved so successful that it has been adopted as commercial practice in many areas where Health D e p a r t m e n t approval could be obtained. Two pancreatic concentrates, at least, are presently being marketed for this purpose. Changes brought about by the mild treatment recommended were so slight that no definite differences in total protein or in formal titration could be d e tected by Conquest et al. (3), although the curd tension was lowered appreciably. Storrs (10) however, using a modified tyrosine test devised by Hull (8), found that the enzymatic treatment caused definite increases in tyrosine units. This evidence, with that of Doan and Miller (5) who obtained no protective action when trypsin solutions were denatured by heating, indicated that protein hydrolysis might be responsible for the added resistance to oxidized flavor in milk samples treated with pancreatic enzyme preparations. P r e l i m i n a r y observations b y F o r s t e r and Sommer (6), with crystalline trypsin, suggested that the protective action of commercial enzyme preparations could be due to hydrolytic cleavage of milk proteins. The work reported herein was undertaken with a view to obtaining additional information regarding tile influence of milk protein hydrolysis on the susceptibility of milk to oxidized flavor development. EXPERIMENTAL
The general procedure used in conducting each of the trials reported in Tables 2 and 3 was as follows: A sample of fresh, raw cow's milk was divided into twQ 500-ml. portions in amber glass, p y r e x bottles. One portion served as the control and the other received the enzyme treatment. This consisted of incubating the sample for 20 minutes at 40 ° C. in the presence of the required amount of enzyme, followed by in-bottle pasteurization at 62 ° to 63 ° C. for 30 minutes and cooling. Non-protein nitrogen determinations were made in quadruplicate by the method of Johnson (9) on 2 ml. portions of a trichloroacetic acid filtrate. This filtrate was p r e p a r e d by adding 1 ml. of milk to 25 ml. of 0.5 iV[ trichloroacetic acid, mixing well, and filtering after allowing 1 hour for precipitation. Susceptibility of each sample to oxidized flavor development was determined by setting up a series consisting of six 50-ml. portions. Copper, in the form of solutions of copper sulfate, then was added to give the desired Received f o r p u b l i c a t i o n A u g u s t 11, 1952. i P a r t i a l r e p o r t on P u r n e l l P r o j e c t 149 ~' R e l a t i o n of P r o t e i n s a n d N o n - P r o t e i n N i t r o g e n to D e v e l o p m e n t o f Oxidized F l a v o r in M i l k " p u b l i s h e d w i t h t h e a p p r o v a l of Director of t h e Station. 98
99
OXIDIZED FLAVOR IN MILK
a d d e d copper c o n c e n t r a t i o n s . P o r t i o n s 1, 2, 3, 4, 5, a n d 6, respectively, c o n t a i n e d 0, 10 -6, 10-5, 5 X 10 -~, 10 -4, a n d 5 X 10- ~ M/1. of a d d e d copper. The c o p p e r s u l f a t e s o l u t i o n s used were of such c o n c e n t r a t i o n t h a t n o more t h a n 0.5 ml. of a s o l u t i o n was r e q u i r e d to give the d e s i r e d a d d e d c o p p e r c o n c e n t r a t i o n . These six p o r t i o n s were e x a m i n e d f o r the p r e s e n c e of oxidized flavor a f t e r storage a t 0 ° to 4 ° C. for 48 hours. A n u n l e r i c a I v a l u e , r e f e r r e d to h e r e i n as the s u s c e p t i b i l i t y r a t i n g , t h e n was a s s i g n e d to the sample. This was b a s e d on its s u s c e p t i b i l i t y as det e r m i n e d b y the m i n i m u m c o n c e n t r a t i o n of a d d e d c o p p e r w h i c h p r o d u c e d the flavor. T a b l e I shows how the s u s c e p t i b i l i t y r a t i n g was assigned. I t will be TABLE 1 Relationship between susceptibility rating and minimum concentration of added copper required to produce oxidized flavor Susceptibility rating
Minimum cone. added copper to produce oxidized flavor. (M./1.)
6.0 5.0 4.0 3.5 3.0 2.5 2.0
none a 10.8 10-5 5 X 1 0 -5 10-~ 5 X 10-4 no oxidized flavor in 5 X 10-4b
A susceptibility rating of 6.0 was to be assigned if the flavor developed in that portion to which no copper was added. This never occurred. b A susceptibility rating of 2.0 was assigned when the oxidized flavor failed to appear even in the portion containing 5 X 10-4 M./1. of added copper. n o t e d f r o m t h i s t a b l e t h a t h i g h e r s u s c e p t i b i l i t y r a t i n g s were a s s i g n e d to s a m p l e s w h i c h developed the flavor w i t h low c o n c e n t r a t i o n s of a d d e d copper a n d lower r a t i n g s to those w h i c h r e q u i r e d g r e a t e r c o n c e n t r a t i o n s of a d d e d c o p p e r to produce the defect. D a t a s h o w i n g the i n f l u e n c e of p a n c r e a t i c e n z y m e t r e a t m e n t of m i l k o n n o n p r o t e i n n i t r o g e n a n d s u s c e p t i b i l i t y to oxidized flavor are p r e s e n t e d i n Tables 2 a n d 3, respectively. The d a t a i n T a b l e 2 show c l e a r l y t h a t t r e a t m e n t of m i l k w i t h a n y one of the t h r e e p a n c r e a t i c e n z y m e p r e p a r a t i o n s t e s t e d r e s u l t e d i n a s i g n i f i c a n t ( 5 % p o i n t F ) i n c r e a s e i n n o n - p r o t e i n n i t r o g e n . S u c h increases i n n o n - p r o t e i n n i t r o g e n c a n be i n t e r p r e t e d as i n d i c a t i n g p r o t e i n h y d r o l y s i s . T h e TABLE 2 Influence of pancreatic enzyme treatment of ~nilI¢ on non-protein nitrogen content Enzyme used Lactivase T-360 Trypsin Cryst. Trypsin
Rate used
No. of trials
(mg.ll.) 60 20 4
8 5 6
Mean non-protein nitrogen Control Treated Increase (mg.lL) 285.3 274.6 270.3
(reg.~L) 296.4 323.3 406.8
(reg.~L) 11.] 48.7 136.5
Analysis of variance F value a 5% point 7.43 51.76 17.78
5.59 7.71 6.61
The interaction of treatment X trials proved to be significant for each enzyme treatment, hence the variance for this interaction was used to test the significance of the enzyme treatment in each case.
100
T . L . F O R S T E R E T AL
TABLE 3 Influence of pancreatic enzyme treatment of milk on susceptibility to oxidized flavor
Enzyme used
Rate used
No. of trials
Mean Susceptibility rating Control Treated Decrease
t value ~
P
(rag.~1.)
Lactivase T-360 Trypsin Cryst. Trypsin
60 20 4
8 5 6
3.69 3.30 3.75
3.06. 2.00 2.92
0.63 1.30 0.83
7.696 10.612 7.912
< 0.01 < 0.01 ~ 0.01
Values paired. d a t a of T a b l e 3 show t h a t each e n z y m e t r e a t m e n t also r e s u l t e d in a s i g n i f i c a n t d e c r e a s e i n s u s c e p t i b i l i t y r a t i n g , i.e., r e d u c e d t h e t e n d e n c y t o w a r d o x i d i z e d flavor d e v e l o p m e n t . The d e c r e a s e s i n s u s c e p t i b i l i t y s h o w n in T a b l e 3 m i g h t h a v e b e e n due to p r o t e i n h y d r o l y s i s r e s u l t i n g f r o m t h e e n z y m e t r e a t m e n t s or m e r e l y to the a d d i t i o n of t h e e n z y m e p r e p a r a t i o n s . I n o r d e r to d e t e r m i n e w h i c h of the above m e n t i o n e d p o s s i b i l i t i e s was r e s p o n s i b l e , a n o t h e r s e r i e s of t r i a l s was c o n d u c t e d , u s i n g h e a t - d e n a t u r e d (10 m i n u t e s b o i l i n g ) enzymes. I n each of these t r i a l s a s a m p l e of f r e s h r a w m i l k was d i v i d e d i n t o f o u r 500-ml. p o r t i o n s i n a m b e r glass b o t t l e s a n d h e a t e d to t h e p a s t e u r i z i n g t e m p e r a t u r e of 62 ° to 63 ° C. The h e a t denatured enzymes, in aqueous solution, then were added and mixed and the m i l k h e l d f o r t h e r e q u i r e d 30 m i n u t e s p a s t e u r i z a t i o n e x p o s u r e , f o l l o w e d b y r a p i d cooling. One p o r t i o n , to w h i c h no e n z y m e was a d d e d , s e r v e d as the control, w h i l e L a c t i v a s e , T-360 t r y p s i n , a n d c r y s t a l l i n e t r y p s i n , r e s p e c t i v e l y , were a d d e d to t h e o t h e r t h r e e p o r t i o n s . N o n - p r o t e i n n i t r o g e n a n d s u s c e p t i b i l i t y t e s t s t h e n w e r e m a d e on these p o r t i o n s in t h e m a n n e r p r e v i o u s l y d e s c r i b e d . R e s u l t s of t h i s series of t r i a l s a r e s u m m a r i z e d i n T a b l e 4. T h e y show t h a t a d d i t i o n of t h e h e a t d e n a t u r e d e n z y m e p r e p a r a t i o n s h a d no a p p r e c i a b l e i n fluence on t h e n o n - p r o t e i n n i t r o g e n c o n t e n t , i.e., p r o t e i n h y d r o l y s i s d i d not t a k e TABLE 4 Influence o/ addition of heat denatured pancreatic enzymes to mil~ on non-protein nitrogen and s~.scep~ibility to oxidized flavor ~
Enzyme used None (control) Lactivase T-360 Trypsin Cryst. Trypsin
Rate used
Non-protein nitrogen
(mg./l.)
(mg./l.)
-60 20 4
236.5 b 239.9 240.6 237.8
Susceptibility rating 3.67 3.33 2.50 3.6]
t value ~
4.082 9.924 1.079
P °
~ 0.01 ~ 0.01 0.3 to 0.4
Av. of 9 trials. b Difference between mean non-protein nitrogen values for the four treatments were not significant at the 5% point. ( F ~ 1.22 and 5% pt. ---- 3.01) c Calculated for susceptibility rating data. Enzyme treatments compared individually with control. Values paired. place. Two of t h e enzymes, however, L a c t i v a s e a n d T-360 t r y p s i n , c a u s e d a p p r e c i a b l e a n d s i g n i f i c a n t ( P ~ < 0.01) l o w e r i n g of t h e s u s c e p t i b i l i t y r a t i n g . T h e
O X I D I Z E D FLAVOI% I N M I L K
101
small decrease in susceptibility rating resulting from the addition of heatdenatured crystalline trypsin proved not to be significant. DISCUSSION
Crystalline trypsin, when added at the rate of 4 rag. per liter, incubated for 20 minutes at 40° C. and then destroyed by pasteurization, caused considerable protein hydrolysis (Table 2) and markedly reduced the susceptibility of the treated milk to oxidized flavor development (Table 3). Heat-denatured crystalline trypsin added, at the same rate, to milk which was immediatelypasteurized caused no protein hydrolysis and no significant change in susceptibility to oxidized flavor (Table 4). The purity of a crystalline trypsin preparation preCludes the possibility of any enzyme activity except protein hydrolysis. Therefore, it may be concluded that the protein hydrolysis resulting from treatment of milk with crystalline trypsin was directly responsibfle for the observed decrease in susceptibility to oxidized flavor. Treatment of milk with Lactivase or T-360 trypsin, in a manner similar to that used when treating with crystalline trypsin but at higher rates, also caused protein hydrolysis (Table 2) and reduced susceptibility to development of oxidized flavor (Table 3). In these cases, however, the decreases in susceptibility cannot be ascribed entirely to protein hydrolysis, since addition of these enzyme preparations, after heat denaturation, resulted in decreases in susceptibility without causing any hydrolysis (Table 4). Furthermore, the decreases in susceptibility resulting from treatment of milk with the active enzyme preparations were not proportional to the degree of protein hydrolysis. Crystalline trypsin, for instance, showed ]8~ times the activity in causing increases in non-protein nitrogen as did Laetivase but was only 20 times more active in reducing susceptibility to oxidized flavor. Obviously, some other factor besides protein hydrolysis was involved in causing the decreased susceptibility observed when milk was treated with either Lactivase or T-360 trypsin. To explain the decreased susceptibility of milk treated with pancreatic enzymes, Forster and Sommer (6) advanced a theory based on the work of Hopkins (7). They suggested that such enzyme treatments cause a mild degree of protein hydrolysis, which in turn exposes readily oxidizable groups, such as -SH groups, which formerly were deep within the protein molecule. Oxidation of these groups then reduces the amounts of oxidizing agents and oxygen present in the milk to a point where lipid oxidation cannot proceed. Thus, the oxidized flavor resulting from lipid oxidation is prevented. The data presented in this paper support this theory. In addition, that part of the effect of Lactivase and T-360 trypsin which cannot be ascribed to their enzyme activity may still be explained on the basis of this theory. Neither Lactivase nor T-360 trypsin approaches the degree of purity to be found in crystalline trypsin. Unquestionably, both preparations contain appreciable amounts of protein hydrolytic products. Such products would, therefore, contain readily oxidizable groups which would function in the same manner as those resulting from enzyme hydrolysis of milk proteins. Both of these enzyme
102
T.L. FORSTER
ET AL
preparations were used in appreciable amounts, and it therefore is quite conceivable that sufficient readily oxidizable groups were added to impart the degree of protection from oxidized flavor noted in Table 4. Homogenization and high heat treatments (70 ° to 80 ° C.) are known to reduce the susceptibility of milk to development of oxidized flavor. It is possible that these factors, also, function by causing protein hydrolysis or denaturation. Further study is needed if the mechanism by which these factors function is to be clearly understood. SUMM&RY AND CONCLUSIONS
Treatment of fresh raw milk, before pasteurization, with active Lactivase (60 mg. per liter), T-360 Trypsin (20 mg. per liter) or crystalline trypsin (4 rag. per liter) resulted in appreciable protein hydrolysis as measured in terms of increase in non-protein nitrogen content. Such treatment always was accompanied by a decrease in susceptibility to oxidized flavor. Addition of the heat-denatured enzyme preparations caused no protein hydrolysis, but addition of heat-denatured Lactivase and T-360 trypsin did cause a decrease in susceptibility. Addition of heat-denatured crystalline trypsin, however, caused no appreciable decrease in susceptibility. It is concluded that (a) the protein hydrolysis induced by treating milk with crystalline trypsin is accompanied by, and responsible for, a decrease in the susceptibility of the mille to oxidized flavor development, and (b) the decrease in susceptibility to oxidized flavor development, caused by treating milk with Lactivase or T-360 trypsin, is partly due to hydrolysis of the milk protein and partly to some, as yet unknown, component in the enzyme preparations used. REFERENCES (1) ANDERSORI, E. O. Variations in Susceptibility of Milk as Secreted by the Cow. Proc. Intern. Assoc. Milk Dealers, Lab. See., 153. 1937. (2) ARIDERSORI, E. O. Preventing Development of Oxidized Flavor in Milk. Milk Dealer, 29(3) :32. 1939. (3) CONQUEST, V., TURNER, A. W., AND REYRIOLDS, I-~. J. Soft Curd Milk Produced with Pancreatic Concentrate. J. Dairy Sci., 21: 361. 1938. (4) CORBETT,W. J., AND TRACY, P. H. Experiments on the Use of Certain Antioxidants for Control of Oxidized Flavor in Dairy Products. Food Research, 6: 445. 194~1. (5) DOARI, F. J., AND MILLER, G. M. Trypsin An Anti-oxygen for Milk. Mi]k P l a n t Monthly, 29,9: 42. 1940. (6) FOaSTER, T. L., ARID SOI~MER, H. I~. Manganese, Trypsin, Milk Proteins and the Susceptibility of Milk to Oxidized Flavor Development. J. Dairy Sei., 34: 992. 1951. (7) HOPKIRIS, F. G. Glutathione. Its Influence in the Oxidation of F a t s and Proteins. Biochem. J., 19: 787. 1925. (8) HULL, M. E. Studies on Milk Proteins. II. Colorimetric Determination of the Partial Hydrolysis of the Proteins of Milk. J. Dairy Sci., 30: 881. 1947. (9) Jo~RIso~, M. J. Isolation and Properties of a Pure Yeast Polypeptidase. J. Biol. Chem., 137: 575. 1941. (10) STO•RS, A. B. Studies of Milk Proteins. I I I . The Modification of Milk by Pancreatic Enzymes. J. Dairy Sci., 30: 885. 1947.