RESEARCH PAPERS

I N F L U E N C E O F K - C A S E I N A N D fl-LACTOGLOBULIN ON T H E H E A T S T A B I L I T Y O F S K I M M I L K 1

H. TESSIEI% AND DYSON ROSE Division of Bioscienees, National Research Council, Ottawa, Canada

SU~)/IARY

Milks from individual cows were classified according to their heat s tabi l i ty-pH relation as Type A (maximum and nfinimum heat stability) and Type B (no minimum heat stabil i ty). The heat stabili ty responses of these milks could be changed from Type A to B and from B to A by adding x-casein and fl-lacto- globulin, respectively. Addition of either no- or fl-casein did not affect the heat stability of milk. The heat s tab i l i ty -pH relation of milk heated at 90 C for 10 rain was no longer affected by the addition of K-casein.

Although much work has been done to deter- mine factors that influence stabili ty of milk, reactions responsible for aggregation and pre- cipitation of casein when milk is heated are poorly understood (2, 9-12). The heat stability of milk varies markedly with p H over a nar- row range (10), and this variation has been related to the fl-lactoglobulln content (11). Since fl-lactoglobulin is known to complex with K-casein at temperatures above 65C (6, 20), the influence of added K-casein and fl-lacto- globulin on the heat stability at 140 C of skim- milk, and milks modified by selection of coarse or fine caseinate particles, was studied.

~V[ATERIALS AND ~V[ETIIODS

Bulk milks, and milks from individual cows, obtained from the Central Experimental Farm, Ottawa, were used for preparat ions of casein fractions and for the heat stability tests, as-Ca- sein was prepared by the method of Zittte (19) ; fl-casein by urea fractionation (4). K-Casein was prepared according to Swaisgood and Brunner (13), or Zittle (18). All fractions were purified and freeze-dried as reported pre- viously (14). K-Caseins from individual milks were typed by starch gel electrophoresis (8). Sialic acid-depleted K-casein was prepared by the action of neuraminidase (15) i its sialic acid content was reduced by 80%. Weights of all caseins are reported on a freeze-dried basis without correction for the approximately 10% moisture present.

Commercial B-laetoglobulin three times erys- tallized (Nutrit ional Biochemical Corp.) and fl-lactoglobulins A and B, kindly supplied by Dr. R. Jenness, University of Minnesota, were

Received for publication July 14, 1964.

Issued as N.R.C. No. 8196.

used. Added proteins, expressed as milligrams per milliliter of milk, were dissolved by shak- ing mixtures on a wrist-action shaker for 15 rain.

fi-Lactoglobulin was estimated by the method of Asehaffenburg and Drewry (1). K-Casein was estimated by the method of Marier et al. (7), and the sum of • + K-casein by the method of Tessier et al. (14). Heat stabili ty was determined at 140 C over a p H range as previously described (10).

Based on the dissociation of a-casein com- plex by NaC1 (19), milk depleted in K-casein was prepared by adding 1.2 g of NaC1 per 100 ml of milk, centrifuging at 28,000 rpm 55 rain (Spinco Model L, No. 30 head) and suspending the sedimented casein phase in cent r i fugal se- rum prepared from untreated milk from the same milking. To determine the extent of K-ca- sein removal, 15 ml of the centrifugal sera from untreated and sodium chloride-treated samples were dialyzed against running tap water at 15 C overnight, made to p H 4.6, and the sialic acid content of the precipitated ca- seins determined.

Milks containing caseinate-phosphate micelles of a selected size range were prepared by sedi- menting the desired micelles (30,000 rpm, No. 30 head, Spinco L) and resuspending-them in t h e supernatant serum from a 60-rain een- trifugation. Coarse-particle milk contained the particles sedimented by a 7-rain centrifugation; fine-particle milk contained those sedimented by a 60-min but not by a 20-min eentrifuga- tion. Casein concentration in these prepara- tions was adjusted to that of the original milk.

RESULTS

Thirty-three milks were classified into Types A and B (Figure 1) o n t h e basis of their heat

1047

1048 H. TESSIER AND D. ROSE

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TYPE B MILK

I I I I ] I 6.4 6.5 6.6 6.7 6.8 6.9 7.0

pH

Fro. 1. p H - h e a t s t ab i l i ty curves of h ld iv idua] milks, e - - T y p e A mi lk ; X - - T y p e B milk.

stabil i ty-pH relation: Type A has both a maximum and a minimmn heat stability in the pH range 6.4 to 7.1, whereas type B has only a gradually increasing heat stability through- out this p i t range. Approximately two-thirds of the milks studied were of Type A. Milks from three cows varied between Types A and B during one lactation period, but most cows consistently produced one type of milk. All bulk milks studied were of Type A.

Addition of ~-casein to a Type A milk re- duced or eliminated the minimum in the heat stabil i ty-pH curve, but addition of similar amounts of fl- and as-caseins had only a slight effect (Figures 2, 3). The addition o£ 1 to 2 mg of K-casein per milliliter of milk eliminated the minimum in Type A milks. A plot of amounts of added K-casein against the increase in heat stability at the pH of minimum heat stability (pH 6.7-6.8) was essentially linear with all milks, but the slope of these plots varied between milks. The effect of added K-casein on the minimum heat stability was apparently independent of the initial K-casein content of the milk.

Addition of fl-laetoglobulin to Type B milks, or to Type A milks to which K-casein had been added (Figure 2), induced a minimum in the heat s tabi l i ty-pH plot. Attempts to determine the amount of fl-lactoglobnlia complexed by added K-casein, or vice versa, by observing the effect of various additions on the minimum heat

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FIG. 2. Effec t of var ious a m o u n t s o f added K-casein on the p H - h e a t s tab i l i ty of Type A milk. K-Casein added, m g / m l : O - - n o n e ; X- -0 .50 ; O - - 0.75; [ : ] - -1 .00; A - - 1 . 5 0 . No te : A - - 1 . 0 0 m g K-casein p lus 1.50 m g f l - lactoglobul in/ml.

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pH

FIG. 3. Effect of added K-, fl-, and a,-casein on the pH-heat stability of Type A milk. e - -none; X--2 mg K-casein; 0 - - 2 mg //-casein; U]--5 mg as-casein/ml.

HEAT STABILITY OF SKIMMILK 1049

stability, failed to give consistent results for different milks.

Type B milks, or Type A milks made to respond like Type B milks by added K-casein, were converted to Type A milks by removal of some K-casein by treatment with sodium chlo- ride (Figure 4A, B). NaC1 treatment of se-

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t~io. 4. (A) pH-heat stability curves of Type B milk. O--control ; X--NaCI treatment only ; O--NaC1 treatment plus 2 mg K-easein/ml. (B) pII-heat stability curves of Type A milk, O - - control; 0 - - 2 mg K-casein added/ml; X--2 mg K-casein/ml plus NaC1 treatment.

lected milks gave centrifuged sera significantly higher in K-casein content than the untreated control (Table 1). However, not all milks,

T A B L E 1

a-Casein content of c en t r i f uged sera

Type A Type A-B " Type B

- - - - ( % ) Unt r ea t ed 17.6 20.7 3] .6 NaCl - t rea ted 19.1 27.4 34.7

a This mi lk changed f r o m Type B to Type A du r ing the same l ac ta t ion period.

especially those of Type A, responded appre- ciably to this treatment. Addition of K-casein reversed the effect of sodium chloride treatment.

The heat stabili ty of coarse- and fine-particle milks usually differed from that of the original milk. In general, fine-particle milks had a higher heat stability throughout the pI-I range studied, and tended to be more of Type B than the original milks (Figures 5, 6). Occasional Type A milks, part ieular ly from cows giving milk that fluctuated between Type A and B during a lactation period, yieIded fine-particle milk that was distinctly Type B. Conversely, coarse-particle milks had a lower heat stability throughout the pI-I range, and were invariably Type A, even when the original milk was Type B (Figure 6). Addition of K-casein and fl-lacto-

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pH

FIo. 5. pI- I -heat s t ab i l i ty curves of Type A milk. ® - - o r i g i n a l sk immi lk ; X- -coarse -par t i c le mi lk ; O - - f i n e - p a r t i c l e milk•

globulin to coarse- and fine-particle milks pro- duced the same effects as did their addition to normal milks.

Fine-part icle milk contained from 25 to 50% more, and coarse-particle milk from 25 to 50% less, K-casein than the original milk. However,

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FIG. 6. p H - h e a t s t ab i l i ty curves o f Type B milk. O- - -o r ig ina l sk immi lk ; O- - f i ne -pa r t i c l e mi lk ; × - - c o a r s e - p a r t i c l e milk.

] 0 5 0 H. TESSIER AND D. ROSE

no relation between the K-casein content and heat stability response was apparent.

K-Casein, prepared by different methods (13, 18), from individual or bulk milks, K-caseins giving single a or b zones in starch gel electro- phoresis (8), and K-caseins treated with neur- aminidase to remove 80% of the siahc acid, did not differ detectably in their effect on heat stability when added to milk. Similarly, Types A and B fl-lactoglobulin did not differ in their effect on the heat stability.

When K-casein was added to a Type A milk that had been forewarmed at 90 C for 10 min, the heat stabil i ty-pH relation remained essen- tially unchanged (Figure 7). Moreover, even

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Fie. 7. Effect of the addition of K-casein on heated and nonheated Type A milk. @--not heated; O--not heated plus 2 mg K-casein/ml; ×--heated 90 C, 10 rain; A--heated 90 C, 10 min, plus 2 mg K-casein/ml.

if K-casein was added before forewarming, the forewarming treatment prevented conversion of the Type A to a Type B milk.

The fl-lactoglobulin, total K- and fl-casein contents of the 33 normal milks and several fine- and coarse-particle milks studied were not discernibly related to the maximum or minimum heat stabilities. Furthermore, Type A and Type B milks did not consistently differ in K-, fl-, and as-casein, or fl-lactoglobulin content.

DISCUSSIO~

Published data (11) indicate that the reac- tilon between heat-denatured fl-lactoglobulin and K-casein is a major factor responsible for the complex heat s tabil i ty-pH pattern of most milks, but a direct relation between fl-lacto- globulin or K-casein content and the heat sta- bi l i ty-pH pattern has not been established (12). The data in the present paper suggest that surface constituents of caleium-caseinate-phos-

phate micelles, rather than their gross compo- sition, must be considered in any explanation of heat stability. This same consideration may apply to the effect of fl-lactoglobulin-casein interactions on the rennetting of milk (5).

The addition of K-casein to milk having both a maximum and a minimum in its heat sta- b i l i ty-pH pattern (Type A milk) reduced or completely eliminated the minimum. Since the added K-casein would not penetrate the micelles, this suggests that the amount of surface K-ca- sein affects the heat stabil i ty-pH pattern, and that milks with no minimum in the heat sta- b i l i ty-pH pattern (Type B milks) contain an excess of K-casein on the micellar surfaces. In accord with this suggestion, removal of some of the K-casein from a Type B milk by treating the milk with sodium chloride converted it to a Type A milk. Similarly, Type B milk can be converted to Type A by the addition of fl-lac- toglobulin, suggesting that the heat stability- pH pattern is controlled by the proportions of surface K-casein and soluble fl-lactoglobulin present.

When milk is heated, the denaturing fi-lacto- globulin can presumably interact with itself, with soluble casein, and with casein exposed on the mieellar surface. I f so, the heat sta- b i l i ty-pH pattern will be affected by the pro- portions of these components and by those factors that influence the probability of a de- natured fl-lactoglobulin molecule interacting with another of the same type rather than with casein. Two of these factors are probably the rate and temperature of heating. Forewarming at 90 C may favor complexing of fl-lactoglobu- lin with K-casein on the micellar surface, thus assuring that the milk behaves as Type A, even though sufficient K-casein is present to give a Type B pattern when the milk is heated di- rectly at 140 C.

No relation between the K-casein content of acid casein from a given milk and the amount of K-casein on the micellar surfaces (as judged from the heat stabil i ty-pH pattern) was evi- dent. However, selection of fine micelles of high K-casein content tended to give milks with a higher surface K-casein (i.e., there was more probability of their being Type B) than did selection of coarse low K-casein particles. How- ever, fine and coarse micelles differ in their con- tent of inorganic calcium phosphate (3), and this may also influence their surface character- istics.

REFERENCES

(1 ) .A_SCHAFFENBURG, R., AND DREWRY, J . N e w Procedure for the Routine Determination of

HEAT STABILITY OF SKI3IMILK 1051

the Various Non-Casein Proteins of Milk. Prop. XVth Intern. Dairy Congr., 3: 1961. 1959.

(2) DAVIES, D. T., AND WHITE, J. C. D. Deter- minat ion of Beat Induced Changes in Pro- tein Stability and Chemical Composition of Milk. Proc. XVth Intern. Dairy Congr., 3: 1677. 1959.

(3) FORD, T. F., RAMSDELL, G. A., AND LAND- )~AN, S. G. Composition of the Casein Con- taining Particles of Milk. J. Dairy SCi. , 38: 843. 1955.

(4) HIPP, N. J., GROVES, M. L., CUSTER, ,]-. H., AND MCMEEKIN, T. L. Separation of a-, fl-, and 7-Casein. J. Dairy Sei., 35: 272. 1952.

(5) KANNAN, A., AND JENNESS, R. Relation of Milk Serum Proteins and Milk Salts to the Effects of Heat Treatment on Rennet Clot- ting. J. Dairy Sci., 44: 808. 1961.

(6) LONG, J. E., VAN WINKLE, Q., AND GOULD, I. A. Heat Induced Interaction Between Crude K-Casein and fl-Lactoglobulin. J. Dairy Sci., 46: 1329. 1963.

(7) MARIE/~ ,]-. R., TESSIEI~, H., AND ROSE, D. Sialic Acid as an Index of the K-Casein Content of Bovine Milk. J. Dairy Sci., 46: 373. 1963.

(8) NEELIN, J. M. Variants of K-Casein Re- vealed by Improved Starch Gel E]eetro- phoresis. J. Dairy Sei., 47:508. 1964.

(9) PYNE, G. T. Reviews of the Progress of Dairy Science. J. Dairy Research, 29:101. 1962.

(10) !~OSE, D. Variations in the Heat Stability and Composition of Milk from Individual Cows During Lactation. J. Dairy Sci., 44: 430. 1961.

(11) ROSE, D. Factors Affecting the pH-Sensi- tivity of the Heat Stability of Milk from Individual Cows. J. Dairy Sci., 44: 1405. 1961.

(12) ROSE, D. Heat Stability of Bovine Milk: A Review. Dairy Research Abstrs., 25(2): 45. 1963.

(13) S~VAISGOOD, H. E., AND BRUNblER, J . R. Characterization of K-Casein Obtained by Fractionation with Trlchloracetic Acid in a Concentrated lJrea Solution. J. Dairy Sci., 45: 1. 1962.

(14) TESSIER, H., ROSE, D., AND MARIER, J-. R. Turbidimetric Method for Estimating Sum of fl- and K-Casein in Whole Milk. J. Dairy Sci., 46: 651. 1963.

( ]5) THO~iPSON, M. P., AND PEPPER, L. Effect of Neuraminidase on K-Casein. J. Dairy Sci., 45: 794. 1962.

(]6) WAU~H, D. F. The Interaction of a,-, fi-, and K-Caseins in Mieelle Formations. Fara- day Society Discussions, No. 25. 1958.

(17) WAUGH, D. F., AND VOl~ HIPPEL, P. H. K-Casein and the Stabilization of Casein Micel]es. ~J. Am. Chem. Sot., 78: 4576. 1956.

(18) ZITTLE, C. A., AND CUSTER, J. H. Purifica- tion and Some of the Properties of as-Ca- sein and K-Casein. J. Dairy Sci., 46:1183. 1963.

(19) ZITTLE, C. A., CERBULIS , J., PEPPER, L., AND DELLA MONIOA, E. S. Preparation of Cal- cium-Sensitive a-Caseln. J. Dairy Sci., 42: 1897. 1959.

(20) ZITTLE, C. A., THOMPSON, M. P., CUSTER, J. H., AND CERBULIS, J. K-Casein-fl-Lacto- globulin Interaction in Solution when Heated. J. Dairy Sci., 45: 807. 1962.