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The Effect of Heat Treatment on the Sulfhydryl Groups in Skimmilk and Nonfat Dry Milk
T H E E F F E C T OF H E A T T R E A T M E N T ON T H E S U L F H Y D R Y L GROUPS IN SKIMMILK AND NONFAT DRY MILK GUNTER ZWEIG ~ND RICHARD J. BLOCK Bioche~deal Research Laboratories, Special Products Di¢,isio~, The Bo~'den Co., Yo~dcers, 25. Y.
At the present time, it is generally believed that cystine and cysteine are responsible for all the disulfide and sulfhydryl groups of proteins. Although a certain amount of evidence has appeared that this statement may not always be true (11), the experiments to be described on milk were carried out on the basis of this hypothesis. It is generally assumed also that the equilibrium between cystine and cysteine in a protein can be shifted with relative ease although in so doing the chemical, physical, and biological properties of the protein m a y be markedly affected. Thus, the protein, pre-keratin, is soluble and readily digestible and contains primarily cysteine. When the cysteine is oxidized in v i v o to cystine, the insoluble, indigestible protein, keratin, is formed. On the other hand, the hormone, insulin, is devoid of cysteine but contains a very sizable quantity of cystine. If only a very small proportion of the cystine is reduced to cysteine, the biological effects of insulin are lost (14). Milk proteins, specifically the fi-lactoglobulin fraction (3), also contain both cystine and cysteine. Numerous studies have shown that the presence of the sulfhydryl groups in milk, presumably all present in the proteins as cysteine, have a very marked effect on the flavor of fluid and dried milk, on the keeping qualities of evaporated and dried milk, and on other properties (1, 13, 15). The supposed deleterious effect of the -SH groups of fl-lactoglobulin on the baking qualities of bread dough made with improperly heated milk or milk products has been questioned by the observation that unheated fl-lactoglobulin does not cause poor loaf formation (7). The "cooked flavor" of heated milk has been associated with the decomposition of the -SH groups, possibly t o I~2S (5). The object of this study was to ascertain, if possible, the relationship between the appearance of the "cooked flavor" and the change in sulfhydryl groups on heating. Although considerable work had been carried out on this problem prior to our experiments, it was believed that the methods used for the estimation of the sulfhydryl groups were either nonspecific, very laborious, or required equipment not usually available in the laboratories of milk plants (4, 7, 8, 9, 12, 13). The introduction by Kolthoff (6) of a specific amperometric titration method for -SH groups and its adaptation to these groups in proteins by Benesch (2) appeared to offer a solution of these difficulties. Since our experiments were undertaken, Hutton and Patton (5) have published their results on milk, using the Kolthoff procedure. However, as the resutts of Itutton and Patton and ours do not agree in all respects, it was thought desirable to publish the present ret)ort. Received for publication ~ovember 25, ]952. 427
428
GUNTER
ZVv'EIG A N D RICH+4.RD J. B L O C K
EXPERIMENTAL
Theory. The amperometric titration of mercaptans (sulfhydryl groups) is based on the fact that when silver ions react with -SH compounds, au insoluble, v e r y slightly dissociated product results. As long as free -SH groups exist in solution or in fine suspension, there will be only a small amount of silver ions in solution, tIowever, when all the -SH groups have been converted to the silver mercaptides, the f u r t h e r addition of the soluble silver salt will result in the rapid increase of A g + ions in solution. This latter increase of A g + ions measured amperometrically, is the end-point of the reaction. Apparat~s. The equipment used is essentially that of Kolthoff (6). I t consists of a rotating platinum electrode, a reference electrode, a galvanometer and an automatic burette (Figure 1). The only change from the a p p a r a t u s described
FI~. 1. Apparatus for -SIt group analysis.
EFFECT
OF HEAT
ON SULFHYDRYL
GROUPS
429
by Kolthoff is that a connnercially available calomel electrode (Beckman No. 1170) is substituted for the external H g I 2 reference electrode. A n external potential of -0.15 V is applied by means of a d r y cell. Titration. One h u n d r e d ml. of 0.1 M sodium acetate and 1.0 ml. of ammonium hydroxide (sp. gr. 0.90) are mixed in a 250-ml. beaker.: The use of sodium acetate, in place of N H ~ N Q or NK.~NOs-C2H~OH (2, 5, 6), resulted in the titration of the -SH groups in milk proteins. A 25.0-ml. sample of skimmilk or 25.0 ml. of reconstituted skimmilk is added to the solution. A stream of nitrogen is then passed t h r o u g h the solution for 5 minutes at a rate to minimize foaming and surface denaturation. The gas is dispersed by a sintered glass bubbler. The contents of the beaker are titrated with 0.0'01 N A g N Q in the a p p a r a t u s described above. I n contrast to simple, soluble mercaptans such as cysteine, the milk protein titrations showed a sharp increase in diffusion c u r r e n t prior to the end-point, followed by a slow d r i f t towards zero diffusion current. ~ This phenomenon continued until the end-point was passed, after which the diffusion current (galvanometer readings) increased proportionally to the amount of A g + ions in solution. Because of the lack of a sharp end-point, the following procedure was employed. Silver nitrate was added r a p i d l y to the reaction vessel until a small excess was present, as indicated by the deflection of the galvanometer needle. Then 1.0-ml. aliquots of A g N Q solution were added, each spaced 60 seconds apart. The galvanometer responses were noted just before each successive addition. I n this way a series of points could be obtained. A straight line d r a w n through four or five of these points plotted on g r a p h paper and extrapolated to zero diffusion current gave accurate, reproducible end-points. A typical titration curve is shown in F i g u r e 2.
Milk samples. Fresh, raw whole milk was stored in the refrigerator for one day, and then the cream was removed. Nonfat d r y milk (vacuum roll or s p r a y dried) was furnished by D. E. Mook, New Products Research Laboratory, The Borden Company, N. Y. W h e y was prepared from raw skinmled milk by precipitating the casein with acetic acid at p i t 4.55. Protein-free whey was made by adding sufficient 50 per cent trichloracetic acid to the whey until the concentration of T.C.A. reached 10 per cent. The filtrate from this T.C.A. precipitation was called " p r o t e i n - f r e e w h e y . " The milk samples were heated without agitation in 500-ml. r o u n d bottom flasks immersed in a thermostatielly controlled water bath2 The heating was z The addition of a large amount of ammonium hyc~_roxi,l.emakes the titration feasible in the presence of chloride or bromide ions (6). The reaction in the presence of ammonia proceeds t~s follows : A g (NI-L)2 ÷ + I~Stt--~ l~SAg + NH, + + NH:~ 2 This drift in the diffusion current, which is encountered in the case of protein but not in the case of titrations of soluble mercaptans, m'ty be explained oa th~ bas:3 that an appreciable time is required for the Ag+ lolls to penetrate the protein m~]e?ule%
GUNTER
430 80
ZWEIG
AND
I
I
J. B L O C K
RICHARD
I
I
60
",'i,ro,io,'Wi,", , 0.00," A,~.% ~
/
/
Row skimmilk
u.l ¢Y p.
~,o ._J
ENDPOINT/ ENDPOINO~V o _~
c
4
~'~- °ENDP 8
I
12
I 20
t6
ML. A g N 0 3
Fro. 2. -SH group titration eurves for 1.75 mg. of cysteine.ItC1 and 25.0 m]. of raw ~klmmilk.
c a r r i e d o u t u n d e r a i r or n i t r o g e n . A f t e r t h e m i l k s a m p l e s h a d r e a c h e d t h e d e s i r e d t e m p e r a t u r e , a l i q u o t s w e r e r e m o v e d i m m e d i a t e l y f r o m t h e bath, cooled i n ice w a t e r , a n d t i t r a t e d w i t h A g N 0 3 . T h e above d e s c r i b e d p r o c e d u r e was m o d i f i e d as f o l l o w s f o r t h e studies of t h e r a t e of t h e r e a c t i o n . F o r t h e k i n e t i c r a t e studies, 10.0-ml. s a m p l e s of m i l k w e r e h e a t e d i n 25.0-ml. E r l e n m e y e r flasks i m m e r s e d in t h e w a t e r b a t h at 79.0 ° C. T h e flasks t h e n w e r e r e m o v e d f r o m th e b a t h a t t h e d e s i r e d i n t e r v a l s , a n d t h e c o n t e n t s w e r e t r a n s f e r r e d q u a n t i t a t i v e l y i n t o a 250-ml. b e a k e r c o n t a i n i n g t h e ice-cold s o d i u m a c e t a t e solution. T h e r e m a i n d e r of t h e p r o c e d u r e has b e e n described. TABLE 1 Sulfhydryl groups in milL, whey, etc. The values are calculated as milliequivalents of cysteine per liter of milk. -SH groups/1 Skimmilk ........................................................................................... Raw whey ......................................................................................... Boiled whey ..................................................................................... Protein-free whey ................................................................................ Casein .................................................................................................
0.264 0.316 0.022 0.000 0.000
Applying a thin layer of mineral oil serves as an excellent method of reducing the evaporation of water, especially at the higher temperatures. In order to minimize any protein denaturation other than that caused by the heat treatment, the milk samples were not agitated by stirring dmring the heat treatment.
EFFECT
OF HEAT
ON SULFHYDRYL
TABLE
The effect o f heat t r e a t m e n t
m. eq.
Temperature
GROUPS
431
2 on
-SILl g r o u p s in sl~immill¢
-Sit/1.
Sample I
Cooked flavor ~ score sample 2
Samole 2
(°C) untreated 0.264 0.308 28.5 ........ 0.300 33.8 0.328 38.0 0.296 0.320 43.5 0.328 48.2 0.29'2 50.0 0.292 58.5 0.284 0.280 69.0 0.224 0.272 78.2 0.180 80.0 0.196 100.0 (one hr.) 0.072 0.092 (All values are the average of four separate titrations.) *
-----+ + +
- - No ~~cooked ' ' flavor, + ~ Strong ~ccooked ' ' flavor. RESULTS AND DISCUSSION
Sulfhydryl grot~ps in milk at~d milk prod~ects. A l t h o u g h w e h a v e f o u n d t h a t the quantity
of -SIt groups
in milk and
its derivatives varies from batch
to
batch of milk, even when obtained from the same farm, the values given in Table 1 s h o w a t y p i c a l d i s t r i b u t i o n in milk, w h e y , casein, etc. It appears, in agreement with Hutton
(5) a n d L a r s o n
( 8 ) , t h a t all t h e - S H
groups of milk are in the whey proteins.
Effect of heating on -SH groups. T h e r e s u l t s o f h e a t i n g r a w s k i m m i l k i n a i r o n t h e - S H g r o u p s a r e p r e s e n t e d i n T a b l e 2 a n d F i g u r e 3. T h e v a l u e s o n u n h e a t e d 0.03
J v J .5
0
0.02
O
o
0,0 I
~---b
i
0
O O
I
l
I
I
20
40
60
80
TEMPERATURE
"*G
Fz(~. 3. The effect of temperature on -S]~ groqps in two samples of skimmilk. a Sample 1 b Sample 2
I00
G U N T E R Z W E I G AND RICHAI~D J. B L O C K
432
milk are numerically similar to those of Larson (8), who used an iodometrie method of analysis. I n contrast, I ~ l t t o n (5), employing a procedure similar to t h a t used in these studies, f o u n d only a p p r o x i m a t e l y one-half the q u a n t i t y of - S I t groups. I t a p p e a r s t h a t the substitution of sodium acetate for NH4NO 8 used by others (2, 5, 6) results in titration of the same n u m b e r of - S H groups as the more tedious iodometric procedure. The use of NH4NO~ b y Benesch (2) also m a y explain his inability to detect - S H groups in ovalbumin unless alcohol was added to the t i t r a t i o n mixture. Studies are u n d e r w a y in our l a b o r a t o r y on the s u l f h y d r y l groups of n a t u r a l and d e n a t u r e d ovalbumin. The values in Table 2 and F i g u r e 3 suggest t h a t heating causes a slight initial increase in titrable -SH groups a n d then with increased heating, a decrease. I t is seen in F i g u r e 3 that there is only a slight change in - S H concentration to a certain t e m p e r a t u r e . Above this t e m p e r a t u r e , the - S H concentration decreases v e r y r a p i d l y with increasing t e m p e r a t u r e . The b r e a k in the curve at which this r a p i d change occurs is called the " c r i t i c a l t e m p e r a t u r e " and, in our experience, lies between 58 and 69 ° C. The v a r i a b i l i t y of the " c r i t i c a l temperat u r e " f r o m batch to batch of milk m a y be related to the commercial experience that different samples of milk nmst be heated to different t e m p e r a t u r e s with variable holding times to achieve similar end-products. Table 2 also shows a p p r o x i m a t e cooked flavor scores. These were evaluated organoleptically and are consequently largely subjective. However, it is seen t h a t the a p p e a r a n c e of cooked flavor was observed at the " c r i t i c a l t e m p e r a t u r e . " This observation has been repeated m a n y times. T h e s u l f h y d r y l c o , t e n t of n o n f a t d r y m i l k . A l m m b e r of samples of nonfat d r y milk p r e p a r e d b y D. E. Moo~: were reconstituted and assayed for -SI{ groups. Some of the results are shown in Table 3 and F i g u r e 4. The two samples illustrated have " c r i t i c a l t e m p e r a t u r e s " at 71-77 ° C. I t a p p e a r s t h a t the time of heating (holding-time) p r i o r to drying at t e m p e r a t u r e s above t h a t of the " c r i t i c a l t e m p e r a t u r e , " has a pronounced effect on the loss of -SH groups, e.g., at 76.6 ° C. and holding-time of 5 minutes, the spray-dried milk contained 0.220 milliequivaleuts per 100 g. p o w d e r ; when the holding time was lengthened to 20 minutes, the value fell to 0.163 milliequivalents per 100 g. The results indicate that the loss of -SH groups in whey proteins is a function of TABLE
3
The effect of pre-heat treatment on - S H groups in nonfat dry milk
Temperature
Holding time
(° CJ
(~n.J
untre~.ted
63.0 71.4 71.4 76.6 76.6 82.2 93.4
in. eq. SH Groups/100 g.
roll-dried 0.219
30 5 10 5 20 10 10
0.186 0.170 0.160 0.125 0.103
spray-dried
0.240
0.243 0.243 0.240 0.220 0.163 0.156 0.143
E F F E C T OF H E A T ON S U L F H Y D R Y L G R O U P S
0.25
i
I
I
I
80
90
433
re o
0.20
0
_o
o~
_J o
035
o.~o 70
TEMPERATURE
FIG. 4. T h e e f f e c t o f p r e - h e a t t r e a t m e n t
on -SH
I00
-°C
groups in two samples of skimmilk powder.
O roll-dried powder • spray-dried powder time and temperature when the fatter is above the " c r i t i c a l t e m p e r a t u r e . " The effect of flash-heat treatment on the -SH, groups in reconstituted nonfat d r y milk is given in Table 4. The toss of -SH groups at the higher temperatures (120-150°'C.) is quite variable. This is a reflection of the fact that v e r y slight variations in the " h o l d i n g - t i m e " in this type of heat t r e a t m e n t may result in marked differences in the physical properties of final product. Kinetic studies. Larson and Jenness (10) reported that the loss of -SH groups in fl-lactoglobulin by heat is a first order reaction. The results given in Table 5 confirm their findings on skimmed milk. The data presented in Table 5, which are typical of m a n y experiments, show that a straight line is obtained when the log of the concentration of -SH groups is plotted against time. The results indicate also that the methods used by Larson (8) and b y us, the one based on o-iodosobenzoate and the other on the formation of silver mcrcaptide, are essentially equivalent. The effect of oxidizing agents. The widespread use of oxidizing agents for
GUNTER ZWEIG AND RICI~ARD g. BLOCK
434
TABLE 4 The effect of flash-heat treatment on - S H groups in nonfat dry ~nilk Temperature
m. eq. - S H g r o u p s / 1 0 0 g.
(°C) none 60.0 65.6 71.1 76.7 82.2 87.8 93.3 98.9 104.4 110.0 115.6 121.1 126.7 132.2 137.8 143.3 148.9
0.252 0.235 0.242 0,246 0.254 0.232 0,236 0.232 0.225 0.2Ol 0.208 0.190 0.164 0.135 0.155 0,171 0.124 0,134
TABLE 5 K i n e t i c study of reaction involving loss of sulfhydryl groups at 79.(?° C. Time 0 25.0 50.0 90.0 20.0
min. min. min. min. hr.
m. eq. - S H groups/1. 0.208 0.182 0.146 0.110 0.020
TABLE 6 The effect of oxidizing agents on - S H groups in skim~nilk at 25 ° C. Sample
m. eq. - S H / 1 .
:Raw skimmilk
0.206
R a w skimmilk -]0.03% IK,O~
0.214
R a w skimmilk ~0.03% IK~O, q8.2 X 10-4% Fe as YeCl,
0.190
R a w skimmilk --{0.03% H,_,O,,, -+8.2 X 1 0 4 % F e as FeSO~
0.190
R a w skimmilk -{0.03% H20~ q8.2 X 10 -~ Cu as CuSO~
0.218
R a w skimmilk -]4.2 X 10-s% KBrO8
0.220
E F F E C T OF H E A T ON S U L F H Y D R Y L G R O U P S
435
the " a g i n g " of flour, which is usually associated with the oxidization of -SH groups, p r o m p t e d us to a p p l y this procedure to milk. Unheated skimmed milk was treated with h y d r o g e n peroxide or KBrO~ with or without presence of small amounts of cupric, ferric, or ferrous salts (Table 6). The milk samples were analyzed for s u l f h y d r y l groups 20 hours after addition of the oxidizing agents. The results in Table 6 show that u n d e r the conditions used, these oxidants had no effect on the -SH groups in milk. Although K B r Q did not have a n y a p p a r e n t oxidizing effect on the -SH groups in milk at room temperature, it was thought that small quantities of KBrO~ m a y be able to lower the " c r i t i c a l t e m p e r a t u r e . " The results of one such experiment are summarized in Table 7. I t will be seen that within the experiTABLE 7 The effect of heat t r e a t m e n t on - S H g r o ~ p s in sIcim.mil~, p o t a s s i u m bromate a d d e d
m. eq.-SH/1. Temperature
Raw skimmilk
Raw skimmilk -{4.2 X 10-s% KBrO8
0.26 0.24 lost 0.18 0.16
0.28 0.23 0.22 0.20 0.16
(°c.)
38.0 48.0 59.0 67.0 77.0
mental error of the method employed, KBrO~ did not decrease the number of -SIt groups at the various temperatures tested. These findings m a y be explained on the basis that either the oxidizing agents were " u s e d u p " by other components of the milk before the -SH groups could be oxidized or that conversion of -SH to -SS- takes place, which then is reversed b y reducing substances in the milk. 4 SUMMARY
The s u l f h y d r y l groups in milk proteins have been determined by the amperometric-argentometrie method of Kolthoff. All or almost all the -SI{ groups in milk are present in the whey proteins. Heating milk causes an initial small rise in titrable -SH groups, followed by a marked decrease in -SH. The temperature at which the first sharp decrease in -SH groups is seen has been called the " c r i t i The disappearance of -SH groups observed when milk is heated may not necessarily be the result of oxidation to -SS-. Other reactions such as the formation of a sulfide (lanthionine) with the loss of II~S from 2 molecules of -StI; thiazolidine formation (-CH--Ctt2) ; djenkolie
/ acid type structure (CH~
S-ell..
1 \
NH ), etc. may account for the observations.
\
S-CIL-
I
/
CH2
S
436
GU:NTER ZWEIG AND RICHARD ;I. BLOCK
cal temperature." T h e r e is a n a p p a r e n t r e l a t i o n b e t w e e n t h e " c r i t i c a l t e m p e r a t u r e " a n d t h e a p p e a r a n c e o f c o o k e d f l a v o r . T h e loss o f - S H g r o u p s f o u n d o n heating milk above its "critical temperature" is a f i r s t o r d e r r e a c t i o n . H e a t i n g in the presence or absence of dissolved oxygen or in the presence of oxidizing a g e n t s d o e s n o t a f f e c t t h e v a l u e of t h e " c r i t i c a l t e m p e r a t u r e . " T h e m e t h o d o f K o l t h o f f is r e c o m m e n d e d a s a u s e f u l t o o l f o r t h e e v a l u a t i o n o f the effects of heat treatment on milk and milk products. ACKNOWLEDGMENT We wish to t h a n k D. E. Mook, Assistant Director, New Products Research Laboratory, and It. W. Howard, Research Director, Special Products Division, The Borden Company, New York, for their review of this manuscript and their many helpful suggestions.
REFERENCES
(1) ASHWORTH, V. S., AND KRUEGER, G. J-. Chemical Factors Affecting the Baking Quality of N o n f a t Milk Solids. Cereal Chem., 28: 145. 1951. (2) BENESCtt, R. E., AND BENESC~I, R. Amperometric Titration of Sulfhydryl Groups in Amino Acids and Proteins. Arch. Biochem., 19: 35-45. 1948. (3) BLOCK, R. J. et a~. Unpublished Experiments. (4) HARLAND, H. A., AND ASH~VORTI-I, U. S. Use of Thiamindisulflde for the Estimation of Reducing Substances in Processed Milk. J. Dairy Sci., 28: 15-23. 1945. (5) HUTTON, J. T., AND PATTON, S. The Origin of Sulfhydryl Groups in Milk Proteins and Their Contribution to " C o o k e d F l a v o r . " J. Dairy Sei., 35: 699-705. 1952. (6) KOLTHOFr, I. iV[., AND HARRIS, W . E . Amperomctric Titration of Mercaptans with Silver N i t r a t e Using the Rotating P l a t i n u m Electrode. Ind. Eng. Chem. Anal. ed., 18: 161-162. 1946. (7) LARSEN, R. A., ,]-ENNESS, R., AND GEDDES, W. F. Effect of Heat Treatment on the Sulfhydryl Groups of Milk Serum Proteins. ~crcal Clmm., 25: 287-297. 1949. The Effect of Various Milk Serum Proteins and Sulfhydryl Groups on Bread Quality. J. Dairy Sci., 35: 482. 1952. (8) LARSON, B. L., AND ,]-ENNESS, R. The Reducing Capacity of Milk as ~¢[easured by an Iodometric Titration. J. Dairy Sci., 33: 896-903. 1950. (9) LARSON, B. L., JENNESS, R., GEDDES, R. F., AND COULTER, S. T. An Evaluation of the Methods Used for Determining the B a k i n g Quality of N o n f a t Dry Milk Solids. Cereal Chem., 28: 351-370. 1951. (10) LARSON, B. L., AND JENNESS, R. Characterization of the Sulfhydryl Groups and Kinetics of H e a t Denaturation of Crystalline fl-Laetoglobulin. J. Am. Chem. Soc., 74: 3090. 1952. (11) LINDERSTROM-LANG, K., AND JACOBSEI~~, C. F. On the Properties of 2-Methylthiazoline and their Relation to the Protein Problem. J. Biol. Chem., 137: 443-455. 1941. (12) PATTON, S., AND JOSEPItSON, D . V . Observations on the Application of the Nitroprusside Test to }Ieated Milk. J. Dairy Sci., 32: 398. 1949. (13) STA~BERG, O. E., AND BAILEY, C. ]-I. The Effect of Heat Treatment of Milk in Relation to Baking Quality as Shown by Polarograph and F a r i n o g r a p h Studies. Cereal Chem., 1 9 : 507-517. :1942. (14) S~EgN, K. G., AND WttITE, A. Studies on the Constitution of Insulin. J. Biol. Chem., 1 1 7 : 95-110. 1937. (15) YAN DAM, B., ABMA, R. R., AND REVALLIER-WAI~EFEMIUS, J. G. The Use of Dried Skim Milk in Breadmaking. Netherlands Milk and Dairy J., 2: 148-161. 1948; also Chem. Abstracts, 43: 187. 1949.
At the present time, it is generally believed that cystine and cysteine are responsible for all the disulfide and sulfhydryl groups of proteins. Although a certain amount of evidence has appeared that this statement may not always be true (11), the experiments to be described on milk were carried out on the basis of this hypothesis. It is generally assumed also that the equilibrium between cystine and cysteine in a protein can be shifted with relative ease although in so doing the chemical, physical, and biological properties of the protein m a y be markedly affected. Thus, the protein, pre-keratin, is soluble and readily digestible and contains primarily cysteine. When the cysteine is oxidized in v i v o to cystine, the insoluble, indigestible protein, keratin, is formed. On the other hand, the hormone, insulin, is devoid of cysteine but contains a very sizable quantity of cystine. If only a very small proportion of the cystine is reduced to cysteine, the biological effects of insulin are lost (14). Milk proteins, specifically the fi-lactoglobulin fraction (3), also contain both cystine and cysteine. Numerous studies have shown that the presence of the sulfhydryl groups in milk, presumably all present in the proteins as cysteine, have a very marked effect on the flavor of fluid and dried milk, on the keeping qualities of evaporated and dried milk, and on other properties (1, 13, 15). The supposed deleterious effect of the -SH groups of fl-lactoglobulin on the baking qualities of bread dough made with improperly heated milk or milk products has been questioned by the observation that unheated fl-lactoglobulin does not cause poor loaf formation (7). The "cooked flavor" of heated milk has been associated with the decomposition of the -SH groups, possibly t o I~2S (5). The object of this study was to ascertain, if possible, the relationship between the appearance of the "cooked flavor" and the change in sulfhydryl groups on heating. Although considerable work had been carried out on this problem prior to our experiments, it was believed that the methods used for the estimation of the sulfhydryl groups were either nonspecific, very laborious, or required equipment not usually available in the laboratories of milk plants (4, 7, 8, 9, 12, 13). The introduction by Kolthoff (6) of a specific amperometric titration method for -SH groups and its adaptation to these groups in proteins by Benesch (2) appeared to offer a solution of these difficulties. Since our experiments were undertaken, Hutton and Patton (5) have published their results on milk, using the Kolthoff procedure. However, as the resutts of Itutton and Patton and ours do not agree in all respects, it was thought desirable to publish the present ret)ort. Received for publication ~ovember 25, ]952. 427
428
GUNTER
ZVv'EIG A N D RICH+4.RD J. B L O C K
EXPERIMENTAL
Theory. The amperometric titration of mercaptans (sulfhydryl groups) is based on the fact that when silver ions react with -SH compounds, au insoluble, v e r y slightly dissociated product results. As long as free -SH groups exist in solution or in fine suspension, there will be only a small amount of silver ions in solution, tIowever, when all the -SH groups have been converted to the silver mercaptides, the f u r t h e r addition of the soluble silver salt will result in the rapid increase of A g + ions in solution. This latter increase of A g + ions measured amperometrically, is the end-point of the reaction. Apparat~s. The equipment used is essentially that of Kolthoff (6). I t consists of a rotating platinum electrode, a reference electrode, a galvanometer and an automatic burette (Figure 1). The only change from the a p p a r a t u s described
FI~. 1. Apparatus for -SIt group analysis.
EFFECT
OF HEAT
ON SULFHYDRYL
GROUPS
429
by Kolthoff is that a connnercially available calomel electrode (Beckman No. 1170) is substituted for the external H g I 2 reference electrode. A n external potential of -0.15 V is applied by means of a d r y cell. Titration. One h u n d r e d ml. of 0.1 M sodium acetate and 1.0 ml. of ammonium hydroxide (sp. gr. 0.90) are mixed in a 250-ml. beaker.: The use of sodium acetate, in place of N H ~ N Q or NK.~NOs-C2H~OH (2, 5, 6), resulted in the titration of the -SH groups in milk proteins. A 25.0-ml. sample of skimmilk or 25.0 ml. of reconstituted skimmilk is added to the solution. A stream of nitrogen is then passed t h r o u g h the solution for 5 minutes at a rate to minimize foaming and surface denaturation. The gas is dispersed by a sintered glass bubbler. The contents of the beaker are titrated with 0.0'01 N A g N Q in the a p p a r a t u s described above. I n contrast to simple, soluble mercaptans such as cysteine, the milk protein titrations showed a sharp increase in diffusion c u r r e n t prior to the end-point, followed by a slow d r i f t towards zero diffusion current. ~ This phenomenon continued until the end-point was passed, after which the diffusion current (galvanometer readings) increased proportionally to the amount of A g + ions in solution. Because of the lack of a sharp end-point, the following procedure was employed. Silver nitrate was added r a p i d l y to the reaction vessel until a small excess was present, as indicated by the deflection of the galvanometer needle. Then 1.0-ml. aliquots of A g N Q solution were added, each spaced 60 seconds apart. The galvanometer responses were noted just before each successive addition. I n this way a series of points could be obtained. A straight line d r a w n through four or five of these points plotted on g r a p h paper and extrapolated to zero diffusion current gave accurate, reproducible end-points. A typical titration curve is shown in F i g u r e 2.
Milk samples. Fresh, raw whole milk was stored in the refrigerator for one day, and then the cream was removed. Nonfat d r y milk (vacuum roll or s p r a y dried) was furnished by D. E. Mook, New Products Research Laboratory, The Borden Company, N. Y. W h e y was prepared from raw skinmled milk by precipitating the casein with acetic acid at p i t 4.55. Protein-free whey was made by adding sufficient 50 per cent trichloracetic acid to the whey until the concentration of T.C.A. reached 10 per cent. The filtrate from this T.C.A. precipitation was called " p r o t e i n - f r e e w h e y . " The milk samples were heated without agitation in 500-ml. r o u n d bottom flasks immersed in a thermostatielly controlled water bath2 The heating was z The addition of a large amount of ammonium hyc~_roxi,l.emakes the titration feasible in the presence of chloride or bromide ions (6). The reaction in the presence of ammonia proceeds t~s follows : A g (NI-L)2 ÷ + I~Stt--~ l~SAg + NH, + + NH:~ 2 This drift in the diffusion current, which is encountered in the case of protein but not in the case of titrations of soluble mercaptans, m'ty be explained oa th~ bas:3 that an appreciable time is required for the Ag+ lolls to penetrate the protein m~]e?ule%
GUNTER
430 80
ZWEIG
AND
I
I
J. B L O C K
RICHARD
I
I
60
",'i,ro,io,'Wi,", , 0.00," A,~.% ~
/
/
Row skimmilk
u.l ¢Y p.
~,o ._J
ENDPOINT/ ENDPOINO~V o _~
c
4
~'~- °ENDP 8
I
12
I 20
t6
ML. A g N 0 3
Fro. 2. -SH group titration eurves for 1.75 mg. of cysteine.ItC1 and 25.0 m]. of raw ~klmmilk.
c a r r i e d o u t u n d e r a i r or n i t r o g e n . A f t e r t h e m i l k s a m p l e s h a d r e a c h e d t h e d e s i r e d t e m p e r a t u r e , a l i q u o t s w e r e r e m o v e d i m m e d i a t e l y f r o m t h e bath, cooled i n ice w a t e r , a n d t i t r a t e d w i t h A g N 0 3 . T h e above d e s c r i b e d p r o c e d u r e was m o d i f i e d as f o l l o w s f o r t h e studies of t h e r a t e of t h e r e a c t i o n . F o r t h e k i n e t i c r a t e studies, 10.0-ml. s a m p l e s of m i l k w e r e h e a t e d i n 25.0-ml. E r l e n m e y e r flasks i m m e r s e d in t h e w a t e r b a t h at 79.0 ° C. T h e flasks t h e n w e r e r e m o v e d f r o m th e b a t h a t t h e d e s i r e d i n t e r v a l s , a n d t h e c o n t e n t s w e r e t r a n s f e r r e d q u a n t i t a t i v e l y i n t o a 250-ml. b e a k e r c o n t a i n i n g t h e ice-cold s o d i u m a c e t a t e solution. T h e r e m a i n d e r of t h e p r o c e d u r e has b e e n described. TABLE 1 Sulfhydryl groups in milL, whey, etc. The values are calculated as milliequivalents of cysteine per liter of milk. -SH groups/1 Skimmilk ........................................................................................... Raw whey ......................................................................................... Boiled whey ..................................................................................... Protein-free whey ................................................................................ Casein .................................................................................................
0.264 0.316 0.022 0.000 0.000
Applying a thin layer of mineral oil serves as an excellent method of reducing the evaporation of water, especially at the higher temperatures. In order to minimize any protein denaturation other than that caused by the heat treatment, the milk samples were not agitated by stirring dmring the heat treatment.
EFFECT
OF HEAT
ON SULFHYDRYL
TABLE
The effect o f heat t r e a t m e n t
m. eq.
Temperature
GROUPS
431
2 on
-SILl g r o u p s in sl~immill¢
-Sit/1.
Sample I
Cooked flavor ~ score sample 2
Samole 2
(°C) untreated 0.264 0.308 28.5 ........ 0.300 33.8 0.328 38.0 0.296 0.320 43.5 0.328 48.2 0.29'2 50.0 0.292 58.5 0.284 0.280 69.0 0.224 0.272 78.2 0.180 80.0 0.196 100.0 (one hr.) 0.072 0.092 (All values are the average of four separate titrations.) *
-----+ + +
- - No ~~cooked ' ' flavor, + ~ Strong ~ccooked ' ' flavor. RESULTS AND DISCUSSION
Sulfhydryl grot~ps in milk at~d milk prod~ects. A l t h o u g h w e h a v e f o u n d t h a t the quantity
of -SIt groups
in milk and
its derivatives varies from batch
to
batch of milk, even when obtained from the same farm, the values given in Table 1 s h o w a t y p i c a l d i s t r i b u t i o n in milk, w h e y , casein, etc. It appears, in agreement with Hutton
(5) a n d L a r s o n
( 8 ) , t h a t all t h e - S H
groups of milk are in the whey proteins.
Effect of heating on -SH groups. T h e r e s u l t s o f h e a t i n g r a w s k i m m i l k i n a i r o n t h e - S H g r o u p s a r e p r e s e n t e d i n T a b l e 2 a n d F i g u r e 3. T h e v a l u e s o n u n h e a t e d 0.03
J v J .5
0
0.02
O
o
0,0 I
~---b
i
0
O O
I
l
I
I
20
40
60
80
TEMPERATURE
"*G
Fz(~. 3. The effect of temperature on -S]~ groqps in two samples of skimmilk. a Sample 1 b Sample 2
I00
G U N T E R Z W E I G AND RICHAI~D J. B L O C K
432
milk are numerically similar to those of Larson (8), who used an iodometrie method of analysis. I n contrast, I ~ l t t o n (5), employing a procedure similar to t h a t used in these studies, f o u n d only a p p r o x i m a t e l y one-half the q u a n t i t y of - S I t groups. I t a p p e a r s t h a t the substitution of sodium acetate for NH4NO 8 used by others (2, 5, 6) results in titration of the same n u m b e r of - S H groups as the more tedious iodometric procedure. The use of NH4NO~ b y Benesch (2) also m a y explain his inability to detect - S H groups in ovalbumin unless alcohol was added to the t i t r a t i o n mixture. Studies are u n d e r w a y in our l a b o r a t o r y on the s u l f h y d r y l groups of n a t u r a l and d e n a t u r e d ovalbumin. The values in Table 2 and F i g u r e 3 suggest t h a t heating causes a slight initial increase in titrable -SH groups a n d then with increased heating, a decrease. I t is seen in F i g u r e 3 that there is only a slight change in - S H concentration to a certain t e m p e r a t u r e . Above this t e m p e r a t u r e , the - S H concentration decreases v e r y r a p i d l y with increasing t e m p e r a t u r e . The b r e a k in the curve at which this r a p i d change occurs is called the " c r i t i c a l t e m p e r a t u r e " and, in our experience, lies between 58 and 69 ° C. The v a r i a b i l i t y of the " c r i t i c a l temperat u r e " f r o m batch to batch of milk m a y be related to the commercial experience that different samples of milk nmst be heated to different t e m p e r a t u r e s with variable holding times to achieve similar end-products. Table 2 also shows a p p r o x i m a t e cooked flavor scores. These were evaluated organoleptically and are consequently largely subjective. However, it is seen t h a t the a p p e a r a n c e of cooked flavor was observed at the " c r i t i c a l t e m p e r a t u r e . " This observation has been repeated m a n y times. T h e s u l f h y d r y l c o , t e n t of n o n f a t d r y m i l k . A l m m b e r of samples of nonfat d r y milk p r e p a r e d b y D. E. Moo~: were reconstituted and assayed for -SI{ groups. Some of the results are shown in Table 3 and F i g u r e 4. The two samples illustrated have " c r i t i c a l t e m p e r a t u r e s " at 71-77 ° C. I t a p p e a r s t h a t the time of heating (holding-time) p r i o r to drying at t e m p e r a t u r e s above t h a t of the " c r i t i c a l t e m p e r a t u r e , " has a pronounced effect on the loss of -SH groups, e.g., at 76.6 ° C. and holding-time of 5 minutes, the spray-dried milk contained 0.220 milliequivaleuts per 100 g. p o w d e r ; when the holding time was lengthened to 20 minutes, the value fell to 0.163 milliequivalents per 100 g. The results indicate that the loss of -SH groups in whey proteins is a function of TABLE
3
The effect of pre-heat treatment on - S H groups in nonfat dry milk
Temperature
Holding time
(° CJ
(~n.J
untre~.ted
63.0 71.4 71.4 76.6 76.6 82.2 93.4
in. eq. SH Groups/100 g.
roll-dried 0.219
30 5 10 5 20 10 10
0.186 0.170 0.160 0.125 0.103
spray-dried
0.240
0.243 0.243 0.240 0.220 0.163 0.156 0.143
E F F E C T OF H E A T ON S U L F H Y D R Y L G R O U P S
0.25
i
I
I
I
80
90
433
re o
0.20
0
_o
o~
_J o
035
o.~o 70
TEMPERATURE
FIG. 4. T h e e f f e c t o f p r e - h e a t t r e a t m e n t
on -SH
I00
-°C
groups in two samples of skimmilk powder.
O roll-dried powder • spray-dried powder time and temperature when the fatter is above the " c r i t i c a l t e m p e r a t u r e . " The effect of flash-heat treatment on the -SH, groups in reconstituted nonfat d r y milk is given in Table 4. The toss of -SH groups at the higher temperatures (120-150°'C.) is quite variable. This is a reflection of the fact that v e r y slight variations in the " h o l d i n g - t i m e " in this type of heat t r e a t m e n t may result in marked differences in the physical properties of final product. Kinetic studies. Larson and Jenness (10) reported that the loss of -SH groups in fl-lactoglobulin by heat is a first order reaction. The results given in Table 5 confirm their findings on skimmed milk. The data presented in Table 5, which are typical of m a n y experiments, show that a straight line is obtained when the log of the concentration of -SH groups is plotted against time. The results indicate also that the methods used by Larson (8) and b y us, the one based on o-iodosobenzoate and the other on the formation of silver mcrcaptide, are essentially equivalent. The effect of oxidizing agents. The widespread use of oxidizing agents for
GUNTER ZWEIG AND RICI~ARD g. BLOCK
434
TABLE 4 The effect of flash-heat treatment on - S H groups in nonfat dry ~nilk Temperature
m. eq. - S H g r o u p s / 1 0 0 g.
(°C) none 60.0 65.6 71.1 76.7 82.2 87.8 93.3 98.9 104.4 110.0 115.6 121.1 126.7 132.2 137.8 143.3 148.9
0.252 0.235 0.242 0,246 0.254 0.232 0,236 0.232 0.225 0.2Ol 0.208 0.190 0.164 0.135 0.155 0,171 0.124 0,134
TABLE 5 K i n e t i c study of reaction involving loss of sulfhydryl groups at 79.(?° C. Time 0 25.0 50.0 90.0 20.0
min. min. min. min. hr.
m. eq. - S H groups/1. 0.208 0.182 0.146 0.110 0.020
TABLE 6 The effect of oxidizing agents on - S H groups in skim~nilk at 25 ° C. Sample
m. eq. - S H / 1 .
:Raw skimmilk
0.206
R a w skimmilk -]0.03% IK,O~
0.214
R a w skimmilk ~0.03% IK~O, q8.2 X 10-4% Fe as YeCl,
0.190
R a w skimmilk --{0.03% H,_,O,,, -+8.2 X 1 0 4 % F e as FeSO~
0.190
R a w skimmilk -{0.03% H20~ q8.2 X 10 -~ Cu as CuSO~
0.218
R a w skimmilk -]4.2 X 10-s% KBrO8
0.220
E F F E C T OF H E A T ON S U L F H Y D R Y L G R O U P S
435
the " a g i n g " of flour, which is usually associated with the oxidization of -SH groups, p r o m p t e d us to a p p l y this procedure to milk. Unheated skimmed milk was treated with h y d r o g e n peroxide or KBrO~ with or without presence of small amounts of cupric, ferric, or ferrous salts (Table 6). The milk samples were analyzed for s u l f h y d r y l groups 20 hours after addition of the oxidizing agents. The results in Table 6 show that u n d e r the conditions used, these oxidants had no effect on the -SH groups in milk. Although K B r Q did not have a n y a p p a r e n t oxidizing effect on the -SH groups in milk at room temperature, it was thought that small quantities of KBrO~ m a y be able to lower the " c r i t i c a l t e m p e r a t u r e . " The results of one such experiment are summarized in Table 7. I t will be seen that within the experiTABLE 7 The effect of heat t r e a t m e n t on - S H g r o ~ p s in sIcim.mil~, p o t a s s i u m bromate a d d e d
m. eq.-SH/1. Temperature
Raw skimmilk
Raw skimmilk -{4.2 X 10-s% KBrO8
0.26 0.24 lost 0.18 0.16
0.28 0.23 0.22 0.20 0.16
(°c.)
38.0 48.0 59.0 67.0 77.0
mental error of the method employed, KBrO~ did not decrease the number of -SIt groups at the various temperatures tested. These findings m a y be explained on the basis that either the oxidizing agents were " u s e d u p " by other components of the milk before the -SH groups could be oxidized or that conversion of -SH to -SS- takes place, which then is reversed b y reducing substances in the milk. 4 SUMMARY
The s u l f h y d r y l groups in milk proteins have been determined by the amperometric-argentometrie method of Kolthoff. All or almost all the -SI{ groups in milk are present in the whey proteins. Heating milk causes an initial small rise in titrable -SH groups, followed by a marked decrease in -SH. The temperature at which the first sharp decrease in -SH groups is seen has been called the " c r i t i The disappearance of -SH groups observed when milk is heated may not necessarily be the result of oxidation to -SS-. Other reactions such as the formation of a sulfide (lanthionine) with the loss of II~S from 2 molecules of -StI; thiazolidine formation (-CH--Ctt2) ; djenkolie
/ acid type structure (CH~
S-ell..
1 \
NH ), etc. may account for the observations.
\
S-CIL-
I
/
CH2
S
436
GU:NTER ZWEIG AND RICHARD ;I. BLOCK
cal temperature." T h e r e is a n a p p a r e n t r e l a t i o n b e t w e e n t h e " c r i t i c a l t e m p e r a t u r e " a n d t h e a p p e a r a n c e o f c o o k e d f l a v o r . T h e loss o f - S H g r o u p s f o u n d o n heating milk above its "critical temperature" is a f i r s t o r d e r r e a c t i o n . H e a t i n g in the presence or absence of dissolved oxygen or in the presence of oxidizing a g e n t s d o e s n o t a f f e c t t h e v a l u e of t h e " c r i t i c a l t e m p e r a t u r e . " T h e m e t h o d o f K o l t h o f f is r e c o m m e n d e d a s a u s e f u l t o o l f o r t h e e v a l u a t i o n o f the effects of heat treatment on milk and milk products. ACKNOWLEDGMENT We wish to t h a n k D. E. Mook, Assistant Director, New Products Research Laboratory, and It. W. Howard, Research Director, Special Products Division, The Borden Company, New York, for their review of this manuscript and their many helpful suggestions.
REFERENCES
(1) ASHWORTH, V. S., AND KRUEGER, G. J-. Chemical Factors Affecting the Baking Quality of N o n f a t Milk Solids. Cereal Chem., 28: 145. 1951. (2) BENESCtt, R. E., AND BENESC~I, R. Amperometric Titration of Sulfhydryl Groups in Amino Acids and Proteins. Arch. Biochem., 19: 35-45. 1948. (3) BLOCK, R. J. et a~. Unpublished Experiments. (4) HARLAND, H. A., AND ASH~VORTI-I, U. S. Use of Thiamindisulflde for the Estimation of Reducing Substances in Processed Milk. J. Dairy Sci., 28: 15-23. 1945. (5) HUTTON, J. T., AND PATTON, S. The Origin of Sulfhydryl Groups in Milk Proteins and Their Contribution to " C o o k e d F l a v o r . " J. Dairy Sei., 35: 699-705. 1952. (6) KOLTHOFr, I. iV[., AND HARRIS, W . E . Amperomctric Titration of Mercaptans with Silver N i t r a t e Using the Rotating P l a t i n u m Electrode. Ind. Eng. Chem. Anal. ed., 18: 161-162. 1946. (7) LARSEN, R. A., ,]-ENNESS, R., AND GEDDES, W. F. Effect of Heat Treatment on the Sulfhydryl Groups of Milk Serum Proteins. ~crcal Clmm., 25: 287-297. 1949. The Effect of Various Milk Serum Proteins and Sulfhydryl Groups on Bread Quality. J. Dairy Sci., 35: 482. 1952. (8) LARSON, B. L., AND ,]-ENNESS, R. The Reducing Capacity of Milk as ~¢[easured by an Iodometric Titration. J. Dairy Sci., 33: 896-903. 1950. (9) LARSON, B. L., JENNESS, R., GEDDES, R. F., AND COULTER, S. T. An Evaluation of the Methods Used for Determining the B a k i n g Quality of N o n f a t Dry Milk Solids. Cereal Chem., 28: 351-370. 1951. (10) LARSON, B. L., AND JENNESS, R. Characterization of the Sulfhydryl Groups and Kinetics of H e a t Denaturation of Crystalline fl-Laetoglobulin. J. Am. Chem. Soc., 74: 3090. 1952. (11) LINDERSTROM-LANG, K., AND JACOBSEI~~, C. F. On the Properties of 2-Methylthiazoline and their Relation to the Protein Problem. J. Biol. Chem., 137: 443-455. 1941. (12) PATTON, S., AND JOSEPItSON, D . V . Observations on the Application of the Nitroprusside Test to }Ieated Milk. J. Dairy Sci., 32: 398. 1949. (13) STA~BERG, O. E., AND BAILEY, C. ]-I. The Effect of Heat Treatment of Milk in Relation to Baking Quality as Shown by Polarograph and F a r i n o g r a p h Studies. Cereal Chem., 1 9 : 507-517. :1942. (14) S~EgN, K. G., AND WttITE, A. Studies on the Constitution of Insulin. J. Biol. Chem., 1 1 7 : 95-110. 1937. (15) YAN DAM, B., ABMA, R. R., AND REVALLIER-WAI~EFEMIUS, J. G. The Use of Dried Skim Milk in Breadmaking. Netherlands Milk and Dairy J., 2: 148-161. 1948; also Chem. Abstracts, 43: 187. 1949.