Preparation of Goat β-Lactoglobulin

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scopic technique to the e x a m i n a t i o n of h e a t e d whey a n d ultrafiltrate systems. Acknowledgments

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Use of the electron microscope and special laboratory facilities of the Department of Yeterinary Anatomy (Dr. A. F. Weber, Ctmirman) is gratefully acknowledged. C. V. MORR R. V. JOSEPHSON E. L. THOMAS Department of Dairy Industries and S. P. FROMMES Department of Veterinary Anatomy University of Minnesota, St. Paul References

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(1) Ad_aehi, S. 1963. Electron Microscopic Observations of Alkaline E a r t h Metal-Caseinate Particles. J. Dairy Sci., 46: 743. (2) Bohren, H. U., and Wenner, V. R. 1961. Natural S¢ate of Milk Proteins. I. Composition of the Micellar and Soluble Casein of Milk After Ultracentrifugal Sedimentation. J. Dairy Sci., 44: 1213. (3) D'Agostino-Barbaro, A., and Calapaj, G. G. 1958. Electron Microscopy of Casein from the Milk of Several Species. Acta Med. Vet., 4: 9. (4) Hostettler, H., and Imhoff, K. 1951. Electronic-Optical Examinations on the Fine Structure of Milk. Milchwissensehaft, 6: 351. (5) Hostettler, If., and Imhoff, K. 1951. Electronic-Optical Examinations on the Fine Structure of Milk. Milchwissensehaft, 6" 4OO. (6) Hostettler, H., and hnhoff, K. 1952. Elektronenoptische Untersuehungen fiber die

(9)

(10)

(11)

(12)

(13)

(14) (15)

Submikroskopische Struktur yon Milch und Milcherzeugnissen. Landwirtsch. J ahrb. Schweiz, 66; 309. Hostettler, H., Imhoff, K., and Stein, J . 1965. Studies on the Effect of Heat Treatment and Lyophilization on the State of Distribution and Physiological Properties of Milk Proteins with Special Consideration of Heat Treatment Conditions Applied in Uperization. I. Effect on the State of Distribution of Milk Proteins. Milchwissenschaft, 20: 189. Kenkare, D. B., Morr, C. V., and Gould, I. A. 1964. Factors Affecting the H e a t Aggregation of Proteins in Selected Skimmilk Sera. J. Dairy Sci., 47:947. Knoop, E. 1961. Dairy Science as Influenced by the Results of Physical Fundamental Research. Kiel. Milchwirtsch. Forsch.-ber., 13 : 389. Knoop, E., and Wortman, A. 1960. Studies on the Size Distribution of Casein Particles in Cow's Milk, Goat's Milk and Hunmn Milk. Milchwissenschaft, 15: 273. Nitschmann, H. 1949. Determination of the Electron-Microscopic Size of Calcium Caseinate Particles in Cow Milk. Helv. Chim. Acta, 32: 1258. Saito, Z., and Hashimoto, Y. 1964. Electron Microscopic Studies on the Shape and Size of Calcium Caseinate-Phosphate Particles in Milk. J. Faculty Agr., Hokkaido Univ., 54 : 17. Shimmin, P. D., and Hill, R. D. 1964. An Electron Microscope Study of the Internal Structure of Casein Micelles. J. Dairy Research, 31: 121. Spinco Technic~tl Manual, Memo IX-B, 1.0, p. 4.0 (1959). Published by Beckman Instruments Co., Palo Alto, California. Tessier, H., and Rose, D. 1958. Calcium Ion Concentration in Milk. J. Dairy Sci., 41 : 351.

Preparation of Goat -Lactoglobulin Sen a n d C h a u d h u r i (11) r e p o r t e d the crystallization of g o a t fl-laetoglobulin f r o m a n isoelectric, salt-free solution by a modification of the method of A s c h a f f e n b u r g a n d D r e w r y (1). P h i l l i p s a n d J e n n e s s (8), however, were unsuccessful in t h e i r a t t e m p t s to crystallize g o a t fl-lactoglobulin in a similar fashion. A s k o n a s (2) also failed in a n earlier effort. I n all cases (2, 8, 11, 12), however, the p r o t e i n was crystallized f r o m solutions c o n t a i n i n g a m m o n i u m sulfate, as described by A s k o n a s (2). I n the course of' a n investigation c o m p a r i n g the a n t i g e n i c i t y of v a r i o u s fl-laetoglobulius, it became necessary to p r e p a r e g o a t fl-lactoglobulin f r o m large a m o u n t s of' pooled h e r d milk. The method employed is essentially t h a t of Sen a n d C h a u d h u r i (11), except f o r two modifications which enabled us to p r e p a r e a large q u a n t i t y o f crystalline m a t e r i a l in a relatively s h o r t

time. The modifications precede the a p p l i c a t i o n of the Sen a n d C h a u d h u r i p r o c e d u r e (11). The first modification requires removal of the cream f r o m g o a t ' s milk u s i n g a D e L a v a l cream separ a t o r ; 1 the second requires the acid p r e c i p i t a tion of casein at p H 4.2. 2 Experimental Procedure F o r t y - o n e liters of pooled h e r d goat milk were w a r m e d to 40-45 C, a n d the cream was i It is not implied t h a t the U.S. Department of Agriculture recommends produc~s of companies mentioned, to the possible exclusion of others in the same business. -"It was found that removal of casein by acid precipitation also facilitates the crystallization of cow fl-]actoglobulin C prepared according to the method previously described (5).

TECHNICAL

separated by twice passing the milk through a DeLaval cream separator. The skimmilk, p H 6.6, was then adjusted to p H 4.2 with 1.0 HC1, to precipitate the casein removed by filtration overnight. The p H of the filtrate (32.3 liters) was readjusted to 6.6 with cone NH~0H, and the procedure of Sen and Chaudhuri (11) followed. This procedure requires removal of contaminating caseins by addition of 20 g Na~SO~ per 100 ml filtrate. The casein-free filtrate is then adjusted to p H 3.0 to remove all whey proteins except fl-lactoglobulin. Crude fl-lactoglobulin is obtained by raising the p H of the filtrate to 6.6 and adding 20 g (NH,):SO~ per 100 ml filtrate. I t was found that the filtrate obtained after removal of the fl-lactoglobulin was not completely free of protein. Therefore, 15 g (NII,)~SO~ per 100 ml of filtrate were added, and the precipitated protein removed by filtration. This fraction had a gel-eleetrophoretic mobility similar to fl-lactoglobulin, and no further studies on this fraction have been undertaken. This fraction is discussed further below. The Sen and Chaudhuri procedure was carried out twice before an effort was made to crystallize the fl-lactoglobulin. This ensured removal of contaminating caseins and whey proteins which inhibit crystallization. The final concentrated solution of fl-lactoglobulin, p H 5.9, produced a heavy oil after dialysis. The volmne of the dialysate was reduced to one-half by pervaporation, and dialysis was then resumed, whereupon crystallization occurred almost immediately. This crystalline material was recrystallized four times from solution in 0.15 N NaC1 and subsequent dialysis. DEAE-eellulose column chromatography was carried out as previously described (3, 6, 9), except that the N'aC1 gradient extended to only 0.11 ~ and was contained in six chambers. Polyacrylamide-gel eleetrophoresis (10) was performed as described by Peterson (7), using approximately 1% protein solutions. A TrisEDTA-boric acid buffer (4) was employed with a 7-8% Cyanogum gel.

NOTES

407

Fro. 1. Po]yacrylamide-gel electrophoresis of various filtrates obtained during the preparation of goat fl-lactoglobulin. (For explanation, sue text.) (Figure 1, 3). This p H adjustment also removes the fast-moving substances (a-lactalburain) and leaves a filtrate containing fl-laetoglobulin (Figure 1, 3), which is in part removed

Results and Discussion

Figure 1 is a gel-eleetrophoretic pattern of the proteins in the various filtrates obtained during the preparative procedure, except for Figure 1, 4, a preparation of crystalline goat fl-lactoglobulin supplied by Miss N. Phillips, and Dr. R. Jenness, Department of Biochemistry, University of Minnesota. Figure 1, I is the .goat whey after acid precipitation of the caseins at p H 4.2. The whey contains slowmoving substances which tend to streak out from the origin and a substance, probably alactalbumin, which migrates faster than fllactoglobulin. The slower-moving substances are in part removed by precipitation with 20% Na~SO~ (Figure 1, 2) and completely removed by adjustment of the Na~SO~ filtrate to p H 3.0

FIG. 2. Four-times-crystallized goat ¢~-lactoglobulin obtained from isoelectric, salt-free solution.

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2500

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,

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FIG. 3. Polyac,'ylamide-gel electrophoresis of 1) 35% (iNH4)_~SO~ fraction; 2) crystalline goat ~-laetoglobulin (Phillips and Jenness); 3) fourtimes-reerystallized goat fl-lactog]obulin (present au±hors); 4) four-times-recrystallized cow fl-laetoglobulin B. by readjusting the p H 3.0 filtrate to p H 6.6 and adding (NH,)_~SO~ to 20%. The material remaining in the 20% (NH,)=SO, filtrate (Figure 1, 5) is completely removed by addition of (NH~)=SO, to 35% (Figure 1, 6). The 35% (NH~).~SO~ filtrate contained a small amount of material after dialysis which did not stain with Amido Black dye. I t is obvious from Figure 1 that the procedure gives sharp separations of the major proteins in goat whey, enabling their preparation in good yield. In the present instance, 26 g of four-timesrecrystallized fi-lactoglobulin (Figure 2) were obtained from 41 liters of pooled milk. In addition, 22.4 g of material, presmuably also fl-lactoglobulin, was in the mother liquors after c13-stallization. These yields compare favorably with those obtained in the preparation of cow fl-lactoglobulin. The crystals shown in Figure 2 are similar to those of Sen and Chaudhuri (11), and no effort was made to obtain the hexagonal bipyramids of Askonas (2). Figure 3, 3 shows the four-times-reerystallized goat fl-laetoglobulin compared with the preparation of Phillips and Jenness (Figure 3, 2) and four-timesrecrystallized cow fl-lactoglobulin B (Figure 3, 4) prepared in these laboratories. We have no satisfactory explanation for the fact that the preparation of Phillips and Jenness gives a narrower band after the electrophoresis represented by Figure 3. In agreement with pre-

80

I00

!20

140

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Fro. 4. OEAE-cellulose cohlnm chronm~ography of goat and cow fl-lactoglobulins. (For explanation, see text.) vious findings, goat fl-laetoglobulin has a slower mobility than cow fl-lactoglobulin B, presumably related to its higher isoelectric point (2, 8, 11, 12); the molecular weight of the two fllactoglobulins are virtually identical (2, 8, 11, 12). Figure 3, 1 shows that the material precipitating with 35% (NH~)fSO4 has the same mobility as the crystalline fl-lactoglobulin (Figure 3, 3). I f these two substances are indeed the same, the goat fl-lactoglobulin is nmch more soluble in (NH,)~SO4-Na~SO~ solutions at about p H 6.0 than cow fl-lactoglobulin. About 7.0 g of this material was obtained in the present instance, whereas a ma×imum of 3.0 g of material was obtained during preparation of cow fl-lactoglobulins. Figure 4 is the chromatographic separation of cow fl-laetoglobulin B and goat fl-laetoglobulin. The latter protein is eluted within the holdup volume of the column, probably a verification of its high isoeleetrie point of 5.9. The buffer employed in the chromatography is p H 5.8, so that the goat protein is probably not sufficiently anionic to exchange with the D E A E cellulose. Cow fl-lactoglobulin B, with a lower isoelectric point, exchanges with the D E A E cellulose and is eluted in normal fashion (3, 6, 9). The DEAE-cellulose chromatography provides a rapid method for distinguishing and separating cow and goat B-lactoglobulins. In summary, large quantities of crystalline goat fl-laetoglobulin have been prepared in relatively short time, utilizing a modification of the method of Sen and Chaudhuri (11).

TECt{NICAL NOTES Acknowledgments

We ttjank William C. Wagner of Pure Goat Products, Inc., Boyertown, Pennsylvania, for supplying the goat milk. We also thank Dr. Peter Hoagland for his advice and interest and Miss Ann Neistadt for excellent technical assistance. One of us (E. B. Kalan) wishes to express his gratitude to Dr. Robert Jenness, Department of Biochemistry, University of Minnesota, for a most stimulating experience at the latter's laboratories, where Ibis work was initiated. EDWIN B. KALAN and JAY J. BASCH Eastern Regional Research Laboratory Philadelphia, Pennsylvania 8 '~Eastern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. References

(1) Aschaffenburg, R., and Drewry, J. 1957. Improved Method for the Preparation of CrEstalline fl-Laetog]obulin and a-Lactalbumin from Cow's Milk. Biochem. J., 65: 273. (2) Askonas, B. A. 1954. Crystallization of Goat fi-Lactoglobulin. Biochem. J., 58: 332. (3) Basch, J. J., Kalan, E. B., and Thompson, M. P. 1965. Preparation of fl-Lactoglobulin C. J. Dairy Sci., 48: 604.

409

(4) Ferris, T. G., Easterling, R. E., a~ld Budd, R . E . 1964. Electrophoresis of Serum Proteins in Aerylamide Gel. V. Effect of Burfers on Separation and Mobility. Anal. Biochem., 8 : 477. (5) Kalan, E. B., Greeuberg, R., and Walter, M. 1965. Studies on fl-Lactoglobu]ins A, B, and C. I. Comp'~rison of Chemical Properties. Biochemistry, 4: 991. (6) Kalan, E. B., Greenberg, R., Walter, M., and Gordon, W. G. 1964. Chemical Properties of fi-Lactoglobulins A, B, and C. Bioehem. Biophys. Research Comm., 16: 199. (7) Peterson, R. F. 1963. High Resolution of Milk Proteins Obtained by Gel Electrophoresis. J. Dairy Sei., 46: 1136. (8) Phillips, N., and Jenness, R. 1965. Some Physical and Chemical Properties of Goat fi-Lactoglobulin. Biochem. Biophys. Research Comm., 21: 16. (9) Picz, K. A., Davie, E. W., Folk, J. E., and Gladner, J. A. 1961. fl-Laetoglobulins A and B. I. Chromatographic Separation and Anfiuo Acid Composition. J. Biol. Chem., 236 : 2912. (10) Raymond, S., and Nakamichi, M. 1962. Eleetrophoresis in Synthetic Gels. I. Relation of Gel Structure to Resolution. Anal. Bioehem., 3: 23. (11) Sen, A., and Chaudhuri, S. 1962. Non-casein Proteins of Goat's Milk. Nature, 195: 286. (12) Townend, R. 1965. Some Solution Properties of Capri4 fl-Lactoglobulln. Abstrs. 150th Meeting ACS, 76C: 158. Atlantic City, N.J., September, 1965.

Aroma Significance of Sulfur Compounds in Surface-Ripened Cheese Surface ripened cheeses of the T r a p p i s t or Limburger type are famous for a strong, putrid aroma suggestive of certain sulfur compounds. Indeed, methyl mereaptan and hydrogen sulfide have been identified in a few varieties of such cheese (4, 12, 13), but these two compounds are also known to occur in Cheddar (5-7), a nonp u t r i d type. To provide better understanding of the relationship of sulfur compounds to cheese aroma, we undertook an investigation of them in T r a p p i s t - t y p e cheese. The effects of reagents f o r sequestering sulfur-containing components on the aroma of some other cheese varieties (Cheddar, Blue, Brick, and Liederkranz) also were obsmwed. Experimental Procedures and Results

Treatment of cheese and panel evaluation. Details of the procedure used for treatment of the cheese with reagents have been previously ~Authorized for publication February 8, 1965, as paper no. 3098 in ihe Journal Series of the Pennsylvania Agricultural Experiment Station.

reported (10). Reagents f o r blocking functional groups were made up to 100 ml as follows: distilled water, as a control; 3 % ttgClz, to bind hydrogen sulfide (H._,S), mercaptans ( R S H ) , sulfides ( R S R ) and disulfides ( R S S R ) ; 4 % (Ha' (CN):, to bind H : S and R S H only; 1% Na._,CO.~, to bind strong and weak acids; 5 % N a H C O : , to bind strong acids only; 0.5% 2,4-dinitrophenylhydrazine (DNP) in 1 H:SO~, to bind bases and carbonyls. These solutions were mixed with 25 g of cheese in a W a r i n g Blendor and p r o m p t l y transferred to a foil-covered ground-glass stoppered flask for subsequent aroma evaluation. E i g h t members of the Dairy. D e p a r t m e n t staff evaluated differences in head-space aroma between treated and control samples. The investigation was conducted with 4-wk-old T r a p p i s t - t y p e cheese manufactured according to procedures (8) at the University Creamery. I n addition to the Trappist, Brick (manufactured at the University Creamery), Liederkranz, Cheddar, and Blue cheese (obtained f r o m retail stores in State College) were treated with the 4 % H g (CN)~