Effects of β-Lactoglobulin on the Rheological Properties of Casein Micelle Rennet Gels

Effects of @-Lactoglobulinon the Rheological Properties of Casein Micelle Rennet Gels S.-Y. PARK," K. NAKAMURA,t and R. NIKI* *Department of Bioscience and Chemistry, Faculty of Agriculture, tDepartment of Polymer Science, Faculty of Science, University of Hokkaido, Sapporo 060, Japan

ABSTRACT

volves the aggregation of para-casein by a nonenzymatic reaction ( 3 , 8). The properties of casein The effects of heat treatment on the gelation micelle ( C M ) gels induced by rennet are affected by properties of casein micelle suspensions in the many factors, especially by heat treatment of raw presence or absence of P-LG were studied using a milk ( 2 8 , 29). In general, heat treatment greatly rheolograph-sol apparatus, scanning electron influences the quality of dairy products and is one of microscopy, and transmission electron microscopy. In the most critical operations during their manufacture. the absence of 0-LG, the characteristic parameters of Heat treatment significantly changes milk proteins, the gelation curves (gel modulus, gelation time, and through denaturation, particularly whey proteins, gelation rate) changed slightly as temperature in- resulting in adsorption on CM and possible intercreased from 65 to 80°C. In the presence of 0-LG, the action between /3-LG and K-CNvia hydrophobic interthree parameters were strongly dependent on the action ( 7 ), disulfide and sulfhydryl interchange reactemperature of the treatment; the gel modulus and tion (13, 26, 321, and crosslinking between (3-LG and the gelation rate at 80°C were about 50 and 12.5%, K-CN on the micellar surface (11, 16). respectively, of the values found a t 65°C. After heat In addition, complex formation between 0-LG and treatment at various temperatures, the amounts of 0- K-CNcaused by the heat treatment markedly impairs LG in the ultracentrifugal supernatant of the samples the rennetability of heated milk (20, 2 7) . That is, were estimated; the amounts of 0-LG decreased as interactions between thermally denatured 0-LG and temperature of heat treatment increased. This result K-CNoccur via the formation of intermolecular disulmay mean that complex formation by interaction be- fide bonds, which in turn impairs the formation of the tween 0-LG and K-CN is occurring. The microstruc- rennet casein gels. Although many of the mechanisms tures showed many fine grains on the surface of of gelation and the structure of the resulting gelation casein micelles when the samples were heated with 0- and network have been examined, the mechanism for LG. formation of rennet casein gels is not completely un( Key words: casein micelles, /%lactoglobulin, rheol- derstood. In this study, we investigated the effects of ogy, rennet gels) heat on the gelation properties of CM suspensions Abbreviation key: CM = casein micelles, CMP = with or without /3-LG.The CM suspension was heated caseomacropeptide, G = storage modulus, G ' = loss with /3-LG to investigate its rennetability and the modulus; SEM = scanning electron microscopy, SH = formation of rennet casein micelle gels. Then, viscosulfhydryl, TEM = transmission electron microscopy. elastic properties of rennet casein gels and the microstructure of the rennet casein gels were examined by scanning electron microscopy ( SEM) and INTRODUCTION transmission electron microscopy ( TEM) . The gelation of milk by rennet (chymosin) is an essential step in the production of cheese. Gelation is MATERIALS AND METHODS a two-stage process; the first stage involves enzymatic production of para-casein micelles and caseomacro- Preparation of CM Suspension peptide ( C M P ) from K-CN on the surface of the Fresh, raw milk was obtained from Holstein cows micelles by the chymosin, and the second stage inof the university farm. The raw milk was skimmed by centrifugation at 2000 x g for 20 niin at 20°C. Sodium azide (0.02%) and potassium penicillin G (50,000 Received August 31, 1995 units/L) were added to the skim milk to prevent Accepted July 1, 1996. 1996 J Dairy Sci 79:2137-2145

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bacterial growth, and E-amino-caproic acid (0.2 mlM) was added to prevent the action of plasmin. The CM were fractionated as a pellet from the skimmed milk by ultracentrifugation at 70,000 x g for 70 min at 20°C. A 3% CM suspension (wt/vol) was prepared by dispersing CM pellets in the simulated milk serum ultrafiltrate without lactose ( 141. Preparation of Rennet Solution

Commercial calf rennet from Chr. Hansen Biosystems, Inc. (Copenhagen, Denmark) was dissolved in 3 M NaCl solution. The activity of rennet solution was measured by the method of Berridge (1). Heat Treatment of Samples

The CM suspension or CM suspension containing 0-LG was heated in rubber-stoppered tubes at various temperatures for 30 min in a thermostatically controlled ethyleneglycol bath. After heat treatment, the samples were immediately cooled to 30°C and used for viscoelastic measurements and preparation of samples for SEM and TEM. Analysis of CMP

The amount of CMP that was liberated from CM by rennet was estimated by analyzing the elution curve from HPLC ( 3 0 ) . The CM suspension ( 2 ml) was treated by rennet at 30°C, and then the reaction was stopped at an appropriate time by the addition of 4 ml of 12% TCA solution. The precipitate in the mixture was filtered, and the filtrate was analyzed for CMP using an HPLC system (Nihon Bunko Co., Tokyo) equipped with a TSK 2000 SW column (39 cm x 0.75 cm; Tosoh Co., Tokyo, Japan). Determination of @-LG in Ultracentrifugal Supernatant

The concentration of free P-LG in the ultracentrifugal supernatant was determined t o estimate the amount of P-LG binding with CM. The CM solutions containing 0.32% 0-LG were heated at 65, 70, 75, and 80°C for 30 min and ultracentrifuged at 70,000 x g and 20°C for 70 min. After the ultracentrifugation, the supernatant ( 5 pl) was subjected to PAGE ( 181, followed by staining with Coomassie brilliant blue. The signal intensity was measured by densitometric scanning; the amount of free 6-LG was estimated from a standard curve that had a linear range of 0.2 to 3.2 mg/ml. The amount of 6-LG binding with CM Journal of Dairy Science Vol. 79, No. 12, 1996

was calculated as the difference between total and free B-LG. Determination of Sulfhydryl Groups

The sulfhydryl ( S H ) groups in the 8-LG and CM suspensions were determined by the Grasseti method ( 9 ) using DTNP [2,2'-dithiobis-(5nitropyridine)l, as modified by Obata et al. (24); 1.0 ml of 8-LG and CM suspension was mixed with 1.0 ml of a 0.1 M phosphate buffer solution (0.1 M, pH 6.8) and then added to 0.5 ml of a 5.0 x M DTNP ethanol solution. After incubation for 20 min at 20"C, 2.5 ml of 10% perchloric acid were added to this reaction mixture, which was then clarified by centrifugation at 1000 x g for 10 min. The supernatant was passed through a 0.20-pm membrane filter (Dismic-13cp; Advantec, Tokyo, Japan), and the absorbance of the filtrate was measured at 386 nm. Viscoelastic Measurements

The storage modulus ( G') and loss modulus ( G") were measured (Rheolograph-Sol apparatus; Toyoseiki Seisakusho, Tokyo, Japan); 4-ml samples of 3% CM suspension and of 3% CM suspension heated with 0.32% 0-LG were held for 5 min at 30"C, and the rennet solution ( 1 rennet unit) was added in a ratio of 1:250 (vol/vol); then the mixture was stirred for 30 s. Some of the reaction mixture (1.68 ml was put into the cell of a rheological apparatus. The measuring blade was inserted into the cell, and the surface of the sample was covered with silicone oil to prevent evaporation. The sample solution was subjected to 2 Hz of sinusoidal shear oscillations with an amplitude of 0.25 mm. SEM

The rennet solution (1 rennet unit) was added to the CM suspension ( 8 ml) in a ratio of 1:250 (voll vol) and stirred for 30 s.Some of the reaction mixture (0.5 ml) for SEM was put into a small test tube and held in a water bath of 30°C. The residual reaction mixture was used for rheological measurements to monitor the gelation process and to decide the time of sampling for SEM. The time of sampling is shown by the arrows in Figure 1. To the sample, 3 ml of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 6.0) were added, fixed at room temperature, and stored for 1 d at 4°C. The fixed samples were cut into blocks ( c a . 2 x 2 x 3 mm) and fixed in 3 ml of new 2.5% glutaraldehyde solution. The samples were washed three times with 0.1 M sodium cacodylate

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buffer solution ( p H 6.0). The samples were first placed in a 25% dimethylsulfoxide solution, then transferred to a 50% solution, and freeze-fractured (perpendicular cryofractured) using 50% dimethylsulfoxide solution in liquid nitrogen. To avoid ice crystal artifacts and phase separation during freezing, dimethylsulfoxide was essential. The cryofractured pieces were thawed in the 50% dimethysulfoxide solution. Cryofractured samples were washed three times with 0.1 M sodium cacodylate buffer (pH 6.0). The samples were postfixed in a 2% osmium tetroxide solution in 0.1 M sodium cacodylate buffer, pH 6.0. The samples were dehydrated in a graded series of ethanol solutions (50, 70, 90, and 99.5%) and transferred to t-butyl alcohol ( 1 to 2 ml). Butyl alcohol was changed three times. The samples in tbutyl alcohol were placed in a refrigerator ( 2 to 4°C ). The t-butyl alcohol was then frozen within a few minutes. The container was transferred into the bell jar of a vacuum evaporator and was evaporated with a rotary pump. The frozen t-butyl alcohol was completely sublimated in 2 to 3 h. After sublimation, the samples were left for additional 20 min until warmed to room temperature ( 1 2 ) . The dried specimens were coated with 18 nm of Au-Pd and Pt-Pd in an ion beam sputtering system (E-101 and E-1030, respectively; Hitachi Co., Tokyo, J a p a n ) using argon gas, and samples were examined using a scanning electron microscope (model S-800; Hitachi Co.). TEM

The samples for SEM were also used as the sample for TEM; a portion of the samples that were dried in t-butyl alcohol for SEM was embedded in Epon 812 resin mixture for 24 h at 60°C. Thin sections were cut on an Ultratome I11 (Ultracut N; Reichert Nissei, Tokyo, Japan), mounted on copper grids, and stained with solutions of uranyl acetate and lead citrate. The samples were examined in a transmission electron microscope (model JEM S-100s; JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV. RESULTS AND DISCUSSION

Rheological Properties

The effects of heat treatment of CM suspension in the presence or absence of 0-LG on the gelation of CM by rennet were investigated (Figure 1 ) . The gelation experiments were carried out at 30°C. The gelation process was examined by continuous measurements of the G of CM suspension after the addition of rennet to the samples that had been heated at differ-

TABLE 1. Three gelation parameters of casein micelles treated a t different temperatures with and without 0-LG. Temperature Control4 Heated5 65°C 70°C 75°C 80°C Heated with p-LG6 65°C 70°C 75°C 80°C

%at1

(Pa) 133

to.? (min) 20

t93 (min) 17

119 110 118 114

30 32 32 32

21 24 21 21

124 105 90 53

31 47 92 113

24 36 51 75

'Gel modulus measured a t the longest time. ZHalf gelation time. 3Time of gelation. 4The unheated 3% casein micelle suspension. 53% Casein micelle suspension. 63% Casein micelle suspension with 0.32% P-LG.

ent temperatures between 65 and 80°C for 30 min. The gelation curve 1 in Figure 1 is typical. The G value increased with time after a latent period and approached an asymptotic value. The time development of the G ' is quite similar to that of G , although data are not shown in Figure 1. Here, we used only G data t o analyze the kinetics of gelation. To characterize the kinetics of the gelation for the rennet gel, we defined the following parameters by analyzing the gelation curve: the gelation time was the time at which G deviated from the baseline; the gel modulus Gsat was measured at the longest time, and the half-gelation time (t0.5) was when G was equal to Gsa,/2 after the gelation time. Values for the three parameters are shown in Table 1. The gelation curves of heated CM and of heated CM containing 0-LG (Figure 1) show a decrease in the gel modulus and in the gelation rate and an increase in the gelation time compared with the gelation curve of the unheated control sample (curve 1 ) . The gel modulus, the gelation time, and the gelation rate of samples heated without 0-LG were changed by heat treatment ( a t 65 to 80°C for 30 min) compared with the native CM suspension. The CM may not be changed markedly by heating at 65 to 80°C for 30 min ( 28 1. However, Jenness and Patton ( 151 stated that the level of the dissolved calcium decreased when milk was heated at 67 to 98°C. Thus, the decrease in Ca2+concentration might have been a factor resulting in lower G values of the samples heated without 0LG ( 2 3 ) than those of unheated CM. The three gel parameters of the samples heated with 0-LG were influenced markedly by the heat Journal of Dairy Science Vol. 79, No 12, 1996

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treatment (Table 1) compared with gel parameters of the samples without 0-LG. The changes in the parameters were dependent on the temperature of the heat treatment. The gel modulus and the rate of gelation of the sample heated at 80°C were about 2 times smaller and 3.6 times greater, respectively, than those of samples heated at 65°C. Gelation time of the samples heated with P-LG at 80°C were 3 times longer than those of samples heated at 65°C. Wilson and Wheelock ( 3 1) reported that the clotting time of milk by rennet increased as heating temperature and time increased. Rennet clotting time depends on the intensity of the heat treatment, which could be

related to the extent of denaturation of P-LG and complex formation ( 4 1. Liberation of CMP After Renneting

The amount of CMP that was liberated from intact CM suspensions and from CM suspensions heated at various temperatures (65, 70, 75, and 80°C for 30 min) is shown as a function of time after the addition of rennet (Figure 2). The amount of liberated CMP increased rapidly for about 20 min and reached a constant value about 30 min after rennet addition. This tendency did not vary much among the four

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Figure 1. The storage modulus (G’), as a function of time after renneting, of samples heated for 30 min at 65°C ( a i , 70°C i b ) , 75°C ( c ) , and 80°C ( d ) : 1 ) Unheated control ( 3 % casein micelle suspensioni, 2 ) heated casein micelle suspension, and 3 ) heated casein micelle

suspension with 0-LG. Journal of Dairy Science Vol. 79, No. 12, 1996

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gregate or form a gel until about 85% of its K-CNhas reacted after the addition of rennet ( 2 ) . Changes in @-LG During Heating

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Time (min) Figure 2. The amount of caseomacropeptide (CMPI liberated from casein micelles ( a 1 and casein micelles with 6-LG ( b ), heated a t various temperatures as a function of time after renneting a t 30°C. A 3% casein suspension in simulated milk serum ultrafiltrate: unheated control ( 01, heated for 30 min a t 65°C ( O ) , 70°C (o),75°C ( A), or 80°C ( 0 ) .

samples, indicating that the heating of CM by themselves in this experiment did not influence the enzymatic reaction (primary phase of gelation by chymosin) (Figure 2a). Nevertheless, the liberation of CMP from the samples heated with 0-LG was influenced by the heat treatment (Figure 2b). During the initial phase of the reaction, the amount of liberated CMP increased rapidly but reached a plateau after 40 min. The heat treatment of the CM suspension with 0-LG resulted in a lower liberation rate of CMP and a lower amount of CMP. Chymosin has high substrate specificity as a digestive enzyme. Chymosin attacks only the Phelo5Met106 bond of K-CNand, consequently, produces only one CMP per K-CN molecule. The decrease in the quantity of CMP liberated from samples heated with P-LG suggested that the state of K-CN in CM was altered by heating with 0-LG to inhibit the enzymatic action of chymosin. Damicz and Dziuba ( 6 ) reported that, when the solution containing K-CN and P-LG was heated, the amount of NPN released, corresponding to CMP in this paper, decreased as the heating temperature increased. Furthermore, the lower liberation rate of CMP and the lower amount of CMP could have caused the increase in the gelation time (Table 1) because CM in milk do not start to ag-

Changes in 8-LG binding with CM for various heat treatments are shown in Figure 3. The CM suspensions containing 0.32% 0-LG were heated (65, 70, 75, and 80°C for 30 min) and ultracentrifuged. The free 0-LG in the ultracentrifugal supernatant was determined electrophoretically to estimate the amount of 0-LG binding with CM. The quantity of free 0-LG in the samples heated at 65°C was equal t o that of the control sample, which consisted of CM and unheated 0-LG. The percentage of free 0-LG content was defined as (C/CLG) x 100, where CLG and c = total and free concentrations of P-LG, respectively. The quantity of free 0-LG decreased abruptly as the temperature of heat treatment increased above 70°C. Free 0-LG content of the sample heated at 80°C was 10%; thus, about 90% of total added 0-LG was sedimented with CM, indicating that P-LG formed a complex with CM upon heating. Changes in Free SH Groups During Heating

For CM, the concentration of K-CNwas about 0.15 M,cysteine content was 0.30 d. Added 6-LG

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( S H ) groups D-LG.

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produced another 0.17 mM of free cysteine. The total cysteine concentration was 0.47 mM. The measured SH content for the 3% CM suspension with @-LG heated at 30°C (control) was 0.24 mM. This value was about one-half of the total cysteine concentration. The SH content was defined as (CSHIo.24 mM ) x 100, where CSH = concentration of free SH. The temperature dependence of SH content was similar to that of @-LG, which suggested that the heat treatment at 70°C brought about the formation of the intermolecular disulfide bonds (S-S bonds) between SH groups of @-LGand K-CN.Dalgleish ( 5 ) , in a study with urea chromatography, reported that the increase in temperature of heat treatment caused the increase in the aggregated ( @-LGand K-CN)material.

the results obtained by Patoka et al. (25), who observed “spikes” and “hairs” or filamentous attachments on the surfaces of CM, when skim milk was heated with whey ultrafiltration retentate, in which the main protein is 0-LG. Therefore, it could be assumed that the appendages observed on the heated CM consisting of @-LGwere the denatured @-LG, probably bound with K-CN on CM. From the observations of SEM and TEM, it was concluded that the appendages formed between @-LG and K-CNduring heat treatment may directly affect the viscoelastic characteristics of casein micelle rennet gels. Effects of 8-LG on Curd Formation

The process of curd formation can be explained as a two-stage reaction of renneting: the enzymatic reacThe SEM ( 1 7 ) in Figure 4 show the microstruc- tion and the subsequent coagulation stage. It has not tures of rennet gels of intact CM and CM with 0-LG. been established, however, whether the effects of The samples were obtained 3 h after renneting for heating slow down either stage. Van Hooydonk et al. ( 3 0 1 reported that the enzymatic attack of chymosin intact CM and 10 h after renneting for CM with @-LG. upon the micellar K-CNis slowed, but Marshall ( 1 9 ) The micrographs under low magnification illustrate has suggested that the enzymatic stage is unaffected the typical open spongy structures of casein rennet and that the major effect of heating is to hinder the gels. However, the CM gels in the samples with @-LG coagulation. In this paper, the heating process afhad coarser structures than did gels with only CM. fected the primary phase of gelation of CM by chymoUnder high magnification, CM only (Figure 4, a 2 ) sin (Figure 2). As was described previously, the forhad smooth surface particles; CM were clustered and mation of complexes between denatured @-LGand Kfused together to form the open structure. In micro- CN increased as temperature of heat treatment ingraphs of gels of CM heated with P-LG, many fine creased (Figure 3). The electron microscope showed grains can be clearly observed on the surface of CM that the surface of CM and the microstructure of (Figure 4, b2). In Figure 4 ( b 3 ) , the grains can be rennet gel were changed by heat treatment (Figure seen protruding from the surface of the CM gels. The 4);that is, many fine grains were found on the surfine grains observed on CM are thought to be the @- face of CM particles of samples heated with @-LG. LG and K-CNcomplexes produced by the heat treat- These results may imply that heating CM with 0-LG ment of the samples. Mohammad and Fox ( 2 2 ) affects not only the enzymatic stage of gelation but reported that @-LG, which forms complexes with also the coagulation stage. micellar K-CN during heating, appeared to be atThe reduction of the gel modulus and the prolongatached to the micellar surfaces when milk was heated tion of gelation time may be caused by the increase in at pH I 6.7. The micellar surfaces changed from a electrostatic repulsion and by the stearic hindrance smooth appearance to a ragged appearance with that arose from the adsorption of P-LG on the surface many appendages. Other authors (10, 2 11, observing of CM. In principle, the elastic modulus of gel is CM in milk by TEM, have reported a ragged appear- considered to be proportional to the number of elastiance with many appendages. cally active network chains in the gel per unit The microstructure of rennet gels of intact CM and volume. On the basis of this assumption, the decrease of CM heated with 0-LG was observed by TEM. The in the gel modulus indicates that the gels of heated electron micrographs of gels under lower magnifica- samples contain a less elastically active network tion (Figure 5, A1 and B1) show the thread-like chain than that of the control sample. structure of CM gels. Under higher magnification In conclusion, three factors apparently contribute (Figure 5 , A2 and B2), the CM heated without P-LG to the changes in the formation of CM rennet gels by had smooth contours, and CM in the samples heated heat treatment. First, the reduction in the rate of with P-LG had coarser contours ( B 2 ) with many enzymatic reaction may relate to the interaction beappendages on them. This observation agrees with tween denaturated 8-LG and CM and to an increase Microstructure of Rennet Gel

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EFFECTS OF 8-LACTOGLOBULIN ON RENNET GELS

214 3

Figure 4. Scanning electron micrographs of renneted gels of casein micelles ( a ) and casein micelles with 0-LG ( b 1. heated previously a t 80°C for 30 min. Scale bars: 10 pm ( a 1 and b l ) , 0.5 pm ( a 2 and b2), and 0.2 pm ( a 3 and b 3 ) .

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Figure 5. Transmission electron micrographs of renneted gels of casein micelles ( A ) and casein micelles with P-LG ( B ) , heated previously a t 80°C for 30 min. The magnifications of micrographs of A1 and B1 are same as those of A2 and B2, respectively. Scale bars: 0.5 pm ( A 2 ) and 0.25 pm ( B 2 ) .

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EFFECTS OF P-LACTOGLOBULIN ON RENNET GELS

in electrostatic repulsion between chymosin and its substrate. Second, stearic hindrance of the 0-LG associated with K-CNmay retard the enzymatic reaction rate. Third, coagulation reaction between para-casein micelles results from the subtle balance between the hydrophobic interactions and hydrophilic interactions. The P-LG may be bound on the surface of CM by the disulfide reaction, which makes new environments on the surface of CM and results in the weaker curd formation. ACKNOWLEDGMENT

The authors thank T. Ito of the Electron Microscope Laboratory, Faculty of Agriculture, Hokkaido University, for skillful technical assistance in providing electron micrographs. REFERENCES 1Berridge, N. J . 1945. The purification and crystallization of rennet. Biochem. J. 39:179. 2 Dalgleish, D. G. 1979. Proteolysis and aggregation of casein micelles treated with immobilized or soluble chymosin. J. Dairy Res. 46553. 3 Dalgleish, D. G. 1986. Page 579 in Advanced Dairy Chemistry. Vol. 1. Proteins: The Enzymatic Coagulation of Milk. P. F. Fox, ed. Elsevier Appl. Sci., London, England. 4Dalgleish, D. G. 1990. The effect of denaturation of 0lactoglobulin on renneting: a quantitative study. Milchwissenschaft 45491. 5 Dalgleish, D. G. 1990. Denaturation and aggregation of serum proteins and caseins in heated milk. J . Agric. Food Chem. 38: 1995. 6 Damicz, W., and J. Dziuba. 1975. Studies on casein proteolysis. I. Enzymatic phase of casein coagulation as influenced by heat treatment of milk proteins. Milchwissenschaft 30:399. 7Doi, H., S. Ideno, F. Ibuki, and M. Kanamori. 1983. Participation of the hydrophobic bond in complex formation between Kcasein and (3-lactoglobulin. Agric. Biol. Chem. 47:407. 8Fox, P. F., and D. M. Mulvihill. 1990. Page 121 in Food Gels: Casein. P. Harris, ed. Elsevier Appl. Sci., London, England. 9Grassetti, D. R., and J. F. Murray. 1969. The use of 2,2'-dithiobis-(5-nitropyridine) as a selective reagent for the detection of thiols. J. Chromatogr. 41:121. lOHarwalkar, V. R., and H. J. Vreeman. 1978. Effect of added phosphate and storage on changes in ultra-high temperature short-time sterilized concentrated skimmilk. 2. Micelle structure. Neth. Milk Dairy J. 32:204. 11Heertje, I. J . Visser, and P. Smits. 1985. Structure formation in acid milk gels. Food Microstruct. 4:267. 12Inoue, T., and H. Osatake. 1988. A new drying method of biological specimens for scanning electron microscopy: the tbutyl alcohol freeze drying method. Arch. Histol. Cytol. 51:53.

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13 Jang, H. D., and H. E. Swaisgood. 1990. Disulfide bond formation between thermally denatured P-lactoglobulin and *-casein in casein micelles. J. Dairy Sci. 73:900. 14 Jenness, R., and J. Koops. 1962. Preparation and properties of a salt solution which simulates milk ultrafiltrates. Neth. Milk Dairy J . 16:153. 15 Jenness, R., and S . Patton. 1959. Page 329 in Principles of Dairy Chemistry. John Wiley & Sons, Inc., New York, NY. 16Kalab, M., P. Allan-Wojtas, and B. E. Phipps-Todd. 1983. Development of microstructure in set-style nonfat yoghurt-a review. 1983. Food Microstruct. 2:51. 17Kalab, M., and V. R. Harwalkar. 1973. Milk gel structure. 1. Application of scanning electron microscopy to milk and other food gels. J. Dairy Sci. 56:835. 18 Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head bacteriophage Ta. Nature (Lond.) 227: 680. 19Marshal1, R. J. 1986. Increasing cheese yields by high heat treatment of milk. J . Dairy Res. 53:313. 20McKenzie, H. A. 1971. Page 257 in Milk Proteins, Chemistry and Biology. Vol. 2. H. A. McKenzie, ed. Academic Press, London, England. 21 McMahon, D. J., and B. H. Tousif. 1993. Effect of whey protein denaturation on structure of casein micelles and their rennetability after ultra-high temperature processing of milk with or without ultrafiltration. Int. Dairy J. 3:239. 22Mohammad. K. S., and P. F. Fox. 1987. Heat-induced microstructural changes in casein micelles before and after heat coagulation. N. Z. J . Sci. Technol. 22:191. 23Nakamura, K., and R. Niki. 1993. Rheological properties of casein micelle gels: the influence of calcium concentration on gelation induced by rennet. Biorheology 30:207. 24 Obata, A., and M. Matsuura. 1993. Decrease in the gel strength of tofu caused by an enzyme reaction during soybean grinding and its control. Biosci. Biotechnol. Biochem. 57542. 25 Patocka, G., P. Jelen, and M. Kalab. 1993. Thermostability of skimmilk with modified caseidwhey protein content. Int. Dairy J . 3:35. 26 Purkayastha, R., H. Tessier, and D. Rose. 1967. Thiol-disulfide interchange in formation of 0-lactoglobulin-K-casein complex. J. Dairy Sci. 50:764. 27Sawyer, W. H. 1968. Heat denaturation of bovine /3 lactoglobulins and relevance of disulfide aggregation. J. Dairy Sci. 51:323. 28Singh, H., and L. K. Creamer. 1986. Page 621 in Advanced Dairy Chemistry. Vol. 1. Proteins: Heat Stability of Milk. P. F. Fox, ed. Elsevier Appl. Sci., London, England. 29Van Hooydonk, A.C.M., P. G. de Koster, and I. J. Boerrigter. 1987. The renneting properties of heated milk. Neth. Milk Dairy J. 41:3. 30Van Hooydonk, A.C.M., and C. Olieman. 1982. A rapid and sensitive high-performance liquid chromatography method of following the action of chymosin in milk. Neth. Milk Dairy J. 36: 153. 31 Wilson, G. A., and J. V. Wheelock. 1972. Factors affecting the action of rennin in heated milk. J. Dairy Res. 39:413. 32Zahuru1, H., M. M. Kristjansson, and J. E. Kinsella. 1987. Interaction between K-casein and &lactoglobulin: possible mechanism. J . Agric. Food Chem. 35644.

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