Effects of Mineral Salts and Calcium Chelating Agents on the Gelation of Renneted Skim Milk

Effects of Mineral Salts and Calcium Chelating Agents on the Gelation of Renneted Skim Milk

J. Dairy Sci. 84:1569–1575  American Dairy Science Association, 2001. Effects of Mineral Salts and Calcium Chelating Agents on the Gelation of Renne...

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J. Dairy Sci. 84:1569–1575  American Dairy Science Association, 2001.

Effects of Mineral Salts and Calcium Chelating Agents on the Gelation of Renneted Skim Milk P. Udabage* I. R. McKinnon* and M. A. Augustin† *Chemistry Department, Monash University, Clayton, Victoria 3168, Australia †Food Science Australia, Werribee, Victoria 3030, Australia

ABSTRACT The effects of adding CaCl2, orthophosphate, citrate, EDTA, or a mixture of these, to reconstituted skim milk (90 g of solids/kg solution) on the gelation of renneted milk were mediated by changes in Ca2+ activity and the casein micelle. At pH 6.65, the addition of citrate or EDTA, which removed more than 33% of the original colloidal calcium phosphate with the accompanying release of 20% casein from the micelle, completely inhibited gelation. Reformation of the depleted colloidal calcium phosphate and casein in the micelle, by the addition of CaCl2, removed this inhibition. When the minimum requirements for colloidal calcium phosphate and casein in the micelle were met, the coagulation time decreased with increasing Ca2+ activity, leveling off at high Ca2+ activity. The storage modulus of renneted gels, measured at 3 h, increased with increasing colloidal calcium phosphate content of micelles up to a level at which it was ∼130% of the original colloidal calcium phosphate in the micelles. Further increases in colloidal calcium phosphate by the addition of CaCl2, orthophosphate, or mixtures of these, which did not change the proportion of casein in the micelle, decreased the storage modulus. The gelation of the renneted milk was influenced by Ca2+ activity, the amounts of colloidal calcium phosphate, and casein within the micelle, with the effects of colloidal calcium phosphate and casein within the micelle clearly dominating the storage modulus. These results are consistent with the model of Horne (Int. Dairy J. 8:171–177, 1998) which postulates that, following cleavage of the stabilizing κcasein hairs by rennet, the properties of the rennet gel are determined by the balance between the electrostatic and hydrophobic forces between casein micelles. (Key words: minerals, caseins, rennet, gelation) Abbreviation key: CCP = colloidal calcium phosphate, CT = coagulation time, FOQELS = fiber optic

Received August 3, 2000. Accepted February 5, 2001. Corresponding author: P. Udabage; e-mail: sandani.udabage@ foodscience.afisc.csiro.au.

quasi elastic light scattering, IMCU = international milk clotting units, SMP = skim milk powder, NPN = nonprotein nitrogen, NCN = noncasein nitrogen, TN = total nitrogen, TCA = trichloroacetic acid. INTRODUCTION Rennet cleaves the κ-CN hairs that provide steric stabilization to casein micelles. Dalgleish (1979) suggested that a minimal cleavage of 86 to 90% of κ-CN was required for the aggregation of renneted casein micelles at pH 7. However, others (Fox and Mulvihill, 1990; van Hooydonk et al., 1986) found that aggregation occurred at a lower cleavage when calcium concentration or temperature was increased at a fixed pH. Shalabi and Fox (1982) found that renneted milk did not gel when the colloidal calcium phosphate (CCP) content of micelles was decreased by approximately 30% at constant Ca2+ activity. Dalgleish (1992) suggested that the increase in the coagulation rate of renneted micelles arises from the neutralization of negative charge within the micelles, with the decrease in repulsion allowing the close approach, thereby promoting hydrophobic interactions, which are necessary for gel formation, to occur. Although renneted micelles do not gel at 15°C (Brinkhuis and Payens, 1984), gels formed at 30°C do not dissolve on cooling to 5°C, suggesting that salt bridges and hydrophobic interactions play a role in formation and maintenance of the gel network (Walstra and van Vliet, 1986). Recently Horne (1998) hypothesized that the structure of rennet gels could be rationalized in terms of a model that treated caseins as a hydrophobic colloid, in which the interaction energy between the caseins is considered to be the sum of electrostatic repulsion and hydrophobic interactions. The addition of calcium and orthophosphate salts affects the properties of rennet gels (Zoon et al., 1988). The addition of salt additives affects Ca2+ activity, CCP, the proportion of casein in the micelle, and the ionic strength of the milk, but the relative contributions of each of these factors to the formation of rennet gels is not clear. In this work, the rennet gels were made at constant pH and temperature

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so that the effects of changes in the mineral and casein equilibria of milk on the aggregation of renneted casein micelles and the gel development could be investigated. Two independent methods, light scattering and rheological measurements, were used to study the renneting process. Renneting behavior was rationalized in terms of the available information on partitioning of caseins and minerals in milk and their effects on the interactions between caseins. MATERIALS AND METHODS Materials Skim milk powder (SMP) was a sample manufactured at the pilot plant of Food Science Australia, Melbourne Laboratory. The skim milk had been pasteurized at 72°C for 30 s before concentration and drying. Unless otherwise stated, all chemicals were analytical grade obtained from BDH Chemicals Australia (Kilsyth, Victoria 3137, Australia). Preparation of Reconstituted Skim Milk SMP was reconstituted in ultra-pure water to obtain 150 g/kg of milk solids suspension. Milk suspensions contained sodium azide (0.42 g/kg) as a bacteriocide. The suspension was stirred for 1 h and left to equilibrate at ∼20°C for 2 h. This milk was diluted with ultra-pure water, salt solutions or a mixture of these to obtain 90 g/kg milk solids suspensions. The salt solutions were added dropwise with continuous stirring. The pH was then adjusted by dropwise addition of 1 M NaOH or 1 M HCl, and the suspension was finally diluted to 90 g/ kg of milk solids. When both an orthophosphate salt and CaCl2 or a citrate salt and CaCl2 were added, the orthophosphate or citrate salt was added about 5 min before the addition of CaCl2. The salt solutions used were 500 mmol of CaCl2/kg, 100 mmol of orthophosphate (mixture of Na2HPO4 and NaH2PO4)/kg, 1 mol of NaCl/kg, 200 mmol of Na2H2EDTA/kg, and 100 mmol of trisodium citrate/kg. The mixtures were stirred and equilibrated overnight at 25°C. All milks had a final pH of 6.65 ± 0.05 at 25°C. All amounts were measured by mass, and concentrations are quoted as mass of solute per mass of solution. For renneting experiments, milk suspensions with slightly higher milk solids concentration and the additive were prepared so that when rennet was added, 90 g/kg of milk solids suspensions with the required amount of additive was obtained. Calcium Activity Ca2+ activity was measured at 25.0 ± 0.1 and 31.0 ± 0.1°C with a Ca-ion selective electrode with a reference Journal of Dairy Science Vol. 84, No. 7, 2001

Calomel electrode (Ion 85 analyzer from Radiometer A/ S, DK-2400, Copenhagen, Denmark). Calibrations were carried out with CaCl2 solutions, with an ionic strength (I) of 0.08 M, adjusted with KCl. The activity coefficient of Ca2+ in the calibration solutions at 25 and 31°C was calculated to be 0.425 and 0.422, respectively, from the extended Debye-Huckel approximation (MacInnes, 1961). Values of 0.5115 mol−1/2 L1/2 and 0.3291 × 1010 mol–1/2 L1/2 m−1 at 25°C and 0.5161 mol−1/2 L1/2 and 0.3301 × 1010 mol−1/2 L1/2 m−1 at 30°C (Robinson and Stokes, 1959) were used for the constants A and B, respectively, in the calculation at 25 and 31°C. The value of 6 × 10−10 m tabulated by Kielland (1937) for Ca2+ was used at both temperatures for the ion size parameter, assuming no change with temperature. Ultracentrifugation of Milk Samples were centrifuged at 78,000 × g at 25°C for 90 min with a Beckman L8-80M ultracentrifuge with a type 55.2 Ti rotor [Beckman Instruments Australia (Pty) Ltd., Gladesville, NSW 2111, Australia]. The supernatant, including the opalescent layer, was carefully removed, leaving a firm pellet. The opalescent layer was included in the supernatant for all analyses of nonpelleted components. In all analyses, the composition of the pelleted material, which was taken as the colloidal components of the micelle, was determined as the difference between the amount of the component present in the total milk (i.e., before centrifugation), and the supernatant obtained as above. Nitrogen Analyses The nonprotein nitrogen (NPN) and noncasein nitrogen (NCN) fractions of skim milk and the supernatant were obtained by a procedure based on the Standards Association of Australia (1988). The N analysis of these fractions and the total nitrogen (TN) were determined by the Kjeldahl method based on the Standards Association of Australia (1991), but using Kjeltabs Cu/3.5 as the catalyst, 10 ml of concentrated sulphuric acid, and 4 ml of 30% hydrogen peroxide. No correction for the volume of the precipitate was made, as the amount of precipitate and all of the subsequent analyses were determined on a mass basis. To convert nitrogen to protein concentration, a factor of 6.38 was used (International Dairy Federation, 1993). Analyses were carried out on the unadjusted milk and the supernatant fractions from all milks. For samples to which EDTA had been added, we corrected for the contribution of nitrogen from EDTA, assuming that EDTA nitrogen partitioned in the same way as milk NPN. A similar correction was made for azide nitrogen in all samples.

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Mineral Analyses Minerals (Ca, Mg, and Pi) were separated from the proteins by diluting milk or supernatant (5 g) with ultra-pure water (20 g) and 240 g of trichloroacetic acid per liter (TCA; 25 g) and filtration of the precipitated protein. Calcium and Mg in diluted TCA filtrates were determined by atomic absorption spectroscopy (Perkin Elmer Atomic Absorption Spectrophotometer-AA 3110) using an air/acetylene flame and wavelengths of 422.7 and 285.2 nm, respectively (Schmidt et al., 1977). Lanthanum chloride at a concentration of 1 g/kg was used as an ionization suppressant. For the control milk and samples with 10 mmol added Ca or 30 mmol added Pi/ kg milk, both the milks and the supernatants from centrifugation were analyzed. For other samples only the supernatant from centrifugation was analyzed. The Pi in diluted TCA filtrates was determined by the method of Allen (1940) with a Hitachi U-2000 spectrophotometer at 695 nm. In the control milk and milk with added Pi, both the milk and the supernatant after centrifugation were analyzed. In other samples, only the supernatants after centrifugation were analyzed. Renneting The stock rennet solution (single-strength 145-Halal calf rennet with 90% chymosin and 10% bovine pepsin from Chr. Hansen, Bayswater, Victoria 3153, Australia) was diluted with ultra-pure water just before renneting. Diluted rennet was added to milk suspensions that had been equilibrated at 31.0 ± 0.1°C for 45 min. The final strength of the rennet was 0.035 International Milk Clotting Units (IMCU)/g. Dynamic Rheological Measurements The rheological characteristics of the milk during renneting were measured at 31.0 ± 0.1°C (pH 6.65 ± 0.05) with a Bohlin VOR Rheometer (Bohlin Reologi AB, Sweden) in oscillation mode, operated at a frequency of 1 Hz and a strain of 0.0103 (amplitude 5%). A Bohlin C25 concentric measuring cylinder was used with a 1.257 mNm torsion bar. Measurements were started 5 min after mixing of rennet to allow time for mixing the milk and rennet, and placing the sample in the cup. The time between rennet addition and a sharp decrease in the measured phase angle was taken as the coagulation time (CT). The change in the phase angle by rheometry reflects the stage when the suspensions change from a purely viscous state to a primarily visco-elastic state. Measuring Light Scattering by Fiber Optic Quasi Elastic Light Scattering Fiber optic quasi elastic light scattering (FOQELS) measurements were performed at 31.0 ± 0.1°C on undi-

luted renneted samples in a Brookhaven Zeta-Plus fitted with a BI 90 correlator board (Brookhaven Instrument Corporation, Holtsville, New York, NY), using a laser of 671 nm and a scattering angle of 155°. The measurements were analyzed using the method of cumulants (Brown and Pusey, 1975). Diluted rennet was added at a final rennet concentration of 0.035 IMCU/g to samples that had been equilibrated for 45 min at 31.0 ± 0.1°C. A small quantity of the mixture (4 ml) was placed in a heated cuvette to measure light scattering. The correlation functions were averaged over 2 min. Measurements were started 2 min after mixing of rennet for all samples. Correlation Between Renneting Measurements at 31°C and Partitioning of Milk Components at 25°C Partitioning of minerals and caseins in milks at 25°C had been previously determined with the same batch of milk (Udabage, 1999), but the specific partition of the milk components at 31°C was not determined. It was of interest to correlate the available information on partitioning at 25°C to the properties of renneted milks at 31°C. The absolute amounts of minerals and protein in the colloidal phase of milk vary with temperature. It is well documented (Dalgleish and Law, 1988; Pouliot et al., 1989a, 1989b; Law, 1996) that increasing the temperature affects the distribution of minerals and casein, with the serum concentrations decreasing with increasing temperature. The effects of increasing temperature from 25 to 31°C were small. A good correlation was obtained between the Ca2+ activity at the two temperatures [Ca2+ activity at 25°C = Ca2+ activity at 31°C * 1.08 + 0.07; r2 = 0.999, n = 6]; this suggested that similar trends might be expected at 25 and 31°C at constant pH for pelleted Ca and Pi and therefore also for CCP and casein with added salts and calcium chelating agents. RESULTS The changes in dynamic moduli obtained during gelation of renneted milk had features similar to those reported previously (Walstra and van Vliet, 1986; Zoon et al., 1988). The addition of CaCl2, orthophosphate, citrate, EDTA, or a mixture of these, to milk influenced CT and storage modulus at 3 h but did not affect the phase angle (Table 1). The addition of CaCl2 reduced CT, whereas salts that complexed calcium and reduced Ca2+ activity increased CT. The addition of CaCl2 or orthophosphate increased the storage modulus at 3 h, as did the addition of mixtures of these salts. Depending on the level of chelating agent, added citrate or EDTA Journal of Dairy Science Vol. 84, No. 7, 2001

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UDABAGE ET AL. Table 1. Effects of additives on renneting of milk at 31°C and the composition of colloidal material upon ultracentrifugation of milk at 25°C.1

Additive (mmol/kg) Control (none) CaCl2 5 10 20 30

2+

Casein in micelle

Storage modulus2 (Pa)

Phase angle (°)

Ca activity c° = 1 mM

(g/kg)

CCP3

26.0

169

13.0

0.81

25.72

2.00

19.0 15.5 14.0 15.0

197 226 179 188

12.8 12.4 13.3 11.6

1.19 1.75 3.28 5.54

NDa 27.21 27.11 26.99

ND 2.63 3.15 3.44

29.0 29.5 28.5 34.0 39.5

223 202 159 185 240

14.0 13.4 12.8 13.2 14.0

0.69 0.63 0.54 0.42 0.35

ND ND 26.97 26.65 26.11

ND ND 2.14 2.32 2.53

17.5 27.5

216 188

11.6 11.7

0.89 0.43

27.11 27.04

2.99 3.46

30.5 179.0 No gelb

145 >1 No gelb

13.7 14.6c No gelb

0.72 0.66 0.56

ND 24.41 18.07

ND 1.59 1.10

34.5 84.5 No gelb

127 8 No gelb

13.5 14.7 No gelb

0.72 0.67 0.55

ND 24.27 20.50

ND 1.70 1.34

237 183

12.9 12.4

1.31 0.97

27.07 26.81

2.40 2.15

CT (min)

Pi 2.5 5 10 20 30 Pi + CaCl2 10 + 10 30 + 10 EDTA 2.5 5 10 Citrate 2.5 5 10 Citrate + CaCl2 5 + 10 10 + 10

19.3 27.0

ND = Not determined. no gel = No gel for 5 h. c 14.6 = Phase at 5 h all others at 3.10 h from mixing of rennet. 1 Mean values of duplicate or more analysis except coagulation time (CT), phase angle and storage modulus of milk with 5 mmol of added Ca/kg of milk, and 5 mmol of added EDTA/kg of milk, which were that of a single analysis. SD: CT, 1.9; phase angle, 0.5; storage modulus, 20; activity, 0.01; casein in micelle, 0.25; and CCP, 0.04. 2 Storage modulus at 3.10 h from mixing of rennet. 3 CCP = [Ca+Pi (as PO43−)]. a b

reduced the storage modulus or completely inhibited gelation. Increasing the concentration of Ca of milk suspensions with added citrate restored the ability of these milks to coagulate (Table 1). The CT was not affected by the addition of 50 mmol of NaCl/kg (26.5 min), but adding 100 mmol of NaCl/kg of milk increased the CT to 34 min. Adding NaCl increased the storage modulus. Values of 198 and 206 Pa were obtained with 50 and 100 mmol added NaCl/kg of milk, respectively, at 3 h. Thus, the primary cause of the differences in renneted gels with the added sodium orthophosphate was not the increase in the amount of Na, but the effect of orthophosphates. Many other workers have also found similar effects with the addition of calcium salts (Bohlin et al., 1984; Jen and Ashworth, 1970; McMahon et al., 1984; Patel and Reuter, 1986; Zoon et al., 1988), orthophosphates Journal of Dairy Science Vol. 84, No. 7, 2001

(McMahon et al., 1984), EDTA or citrate (Jenkins and Emmons, 1983), and NaCl (Zoon et al., 1989). The effects of adding CaCl2, orthophosphate, citrate, EDTA, or a mixture of these on the gelation properties of renneted milk could be related to changes in Ca2+ activity, CCP, and proportion of casein in the micelle at 25°C (Table 1). Removal of more than 33% of the original CCP with the accompanying release of 20% casein from the micelle, by addition of citrate or EDTA (10 mmol/kg of milk) completely inhibited gelation. Reformation of the depleted CCP and casein in the micelle by the addition of CaCl2, removed this inhibition. From these experiments, it was not possible to separate the roles of CCP and casein in the micelle, as the decrease in CCP was accompanied by release of casein from the micelle. The importance of CCP in rennet gel formation has been observed in other work. Shalabi and Fox

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Figure 1. The relationship between the storage modulus of renneted milk gels at 3.10 h and the amount of colloidal calcium phosphate (as % of control milk colloidal calcium phosphate) in micelles.

(1982) also observed superior renneting properties when CCP was reformed in milks. When Lucey et al. (1996) acidified milk to pH 4.6, neutralized to pH 6.6, and dialyzed against the control milk, they observed inferior renneting properties in milks where the original Ca2+ activity was restored but not that the original concentration of CCP. Figure 1 shows that the storage modulus of renneted gels increased with increasing CCP content of micelles up to a to a level to which it was ∼130% of the original CCP in the micelles. Further increases in CCP by addition of CaCl2, orthophosphate, or mixtures of these, which did not change the proportion of casein in the micelle, decreased the storage modulus. The effect of casein in micelles was similar. When the minimum requirements for CCP and casein in the micelle for gel formation were met, CT decreased with increasing Ca2+ activity, leveling off at high Ca2+ activity (Figure 2). Normalization of the storage modulus, using the method of Horne (1995), resulted in a single master

Figure 2. The dependence of rennet coagulation time on the Ca2+ activity. The filled symbols represent milk suspensions containing 5 mmol of EDTA or citrate/kg of milk with low content of colloidal calcium phosphate and casein in micelles.

Figure 3. A typical analysis of the renneting process using fibre optic quasi elastic light scattering, illustrating the onset of aggregation. (Duplicates were similar except that of the milk suspension with 10 mmol of added EDTA/kg of milk only one analysis was performed) (—䊐—) Control (none), (—䊏—) 10 EDTA, (—䊊—) 10 Citrate − mmol additive/kg of milk.

curve (not shown), suggesting the storage modulus and its kinetic development was a function of the final modulus and CT. Assessing Renneting Properties by FOQELS The apparent particle diameters obtained upon renneting are plotted in Figure 3. The aim of these experiments was to obtain qualitative information about particle aggregation. In these milk suspensions, the particle concentrations were such that particle-particle interactions and multiple scattering of light before it reaches the detector were significant. Thus, there was no simple relationship between the actual diameter of the casein micelle and apparent diameters as calculated by the software. However, in any one milk suspension, changes in the actual particle diameter would be reflected qualitatively by changes in the apparent diameter as measured here. Thus, the decrease in apparent diameter of a respective milk suspension reflected the cleavage of κ-CN. As particles were attracted to each other and gelation occurred, the particles lost their identity and the concept of particle diameter became meaningless. Nonetheless, the changes in viscosity and the restriction of motion that accompanied the aggregation and gelation processes were directly indicated by increases in the apparent diameter. When the rennet was added, the apparent micellar diameter decreased in all suspensions. The time to reach the minimum diameter, at which the rate of decrease in size was balanced by the aggregate growth rate, was defined as the aggregation time. The aggregation time depended on the composition of the milk. The aggregation time obtained by light scattering occurred before CT obtained by rheometry, but followed the same Journal of Dairy Science Vol. 84, No. 7, 2001

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trends. This is in agreement with the observations of McMahon et al., (1984), in which aggregation by turbidity measurements were observed well before visual observation of coagulation. In milk suspensions with 10 mmol of added citrate or EDTA per kilogram of milk, there were increases in particle diameter (Figure 3) but no gel formation for up to 5 h after rennet addition. Thus, renneted particles depleted in CCP with reduced proportions of casein in the micelle were attracted to each other, but a gel network was not formed. This was presumably because there was insufficient material (CCP and casein in the micelle), hence inadequate electrostatic and hydrophobic forces between the particles, to sustain a network. DISCUSSION From these experiments, different stages of the renneting process could be identified. In light scattering experiments, the enzymic cleavage of κ-CN by chymosin and the aggregation time were identified. By rheometry, the onset of coagulation was identified by the change in the phase angle, and the development of the gel was observed by the increase in the storage modulus. The aggegation times and CT and gel development of renneted milk were influenced by Ca2+ activity, CCP, and the amounts of casein within the micelle, with the effects of CCP and casein within the micelle clearly dominating gel formation. There were minimum requirements for CCP and the proportion of casein in the micelle for gel formation and an optimum ratio for CCP:casein in the micelle for gel development. When the minimum requirements for CCP and the proportion of casein in the micelle were met, the aggregation times and CT were controlled by the Ca2+ activity. The decreased electrostatic repulsion between renneted micelles due to increasing Ca2+ activity and CCP, coupled with the decrease in steric stabilization and electorstatic repulsion with the removal of the stabilizing layer by chymosin, promoted Van der Waals attractions that led to aggregation. In the presence of adequate CCP and casein in the micelles, hydrophobic interactions between casein molecules and salt bridging through CCP resulted in gelation. The results further suggested that a proper balance of electrostatic and hydrophobic interactions was needed for gel development, with excessive CCP having a destabilizing effect on gelation of renneted micelles. The effects that we have observed were consistent with Horne’s model of casein gels (1998), which states that the properties of the gel are determined by the balance electrostatic and hydrophobic interactions. In addition, our results suggested an even greater involveJournal of Dairy Science Vol. 84, No. 7, 2001

ment of CCP in the linking of casein network, promoting gelation, and at high enough concentrations affecting the development of the gel. ACKNOWLEDGMENT P. Udabage was supported by a joint postgraduate scholarship from Monash University and the Dairy Research and Development Corporation of Australia. REFERENCES Allen, R.J.L. 1940. The estimation of phosphorus. Biochem. J. 34:858–865. Bohlin, L., P. Hegg, and H. Ljusberg-Wahren. 1984. Viscoelastic properties of coagulating milk. J. Dairy Sci. 67:729–734. Brinkhuis, J., and T. A. Payens. 1984. The influence of temperature on the flocculation rate of renneted casein micelles. Biophys. Chem. 19:75–81. Brown, J. C., and P. N. Pusey. 1975. Photon correlation study of polydisperse samples of polystyrene in cyclohexane. J. Chem. Phys. 62:1136–1144. Dalgleish, D. G. 1979. Proteolysis and aggregation of casein micelles treated with immobilized or soluble chymosin. J. Dairy Res. 46:653–661. Dalgleish, D. G. 1992. The enzymic coagulation of milk. Pages 579– 619 in Advanced Dairy Chemistry. Vol. 1. P.F. Fox, ed. Elsevier Applied Science, London, UK. Dalgleish, D. G., and A.J.R. Law. 1988. pH-Induced dissociation of bovine casein micelles. I. Analysis of liberated caseins. J. Dairy Res. 55:529–538. Fox, P. F., and D. M. Mulvihill. 1990. Casein. Pages 121–171 in Food Gels. P. Harris, ed. Elsevier Applied Science, London, UK. Horne, D. S. 1998. Casein interactions: Casting light on the black boxes, the structure in dairy products. Int. Dairy J. 8:171–177. International Dairy Federation. 1993. Milk: Determination of Nitrogen Content. Brussels: IDF (FIL-IDF Standard no. 20B). Jen, J. J., and U. S. Ashworth. 1970. Factors influencing the curd tension of rennet coagulated milk. Salt balance. J. Dairy Sci. 53:1201–1206. Jenkins, K. J., and D. B. Emmons. 1983. Inhibitory effects of fatty acids, chelating agents, and other substances on rennet clotting of skim milk. J. Dairy Sci. 66:719–726. Kielland, J. 1937. Individual activity coefficients of ions in aqueous solutions. J. Am. Chem. Soc. 59:1675–1678. Law, A.J.R. 1996. Effects of heat treatment and acidification on the dissociation of bovine casein micelles. J. Dairy Res. 63:35–48. Lucey, J. A., C. Gorry, B. O. Kennedy, M. Kalab, R. Tan-Kinita, and P. F. Fox. 1996. Effect of acidification and neutralization of milk on some physico-chemical properties of casein micelles. Int. Dairy J. 6:257–272. MacInnes, D. A. 1961. Chapter 7 in The Principles of Electrochemistry. Dover Publication, Inc., New York. Debye-Hu¨ckel method for the theoretical calculation of activity coefficients. pp 137–151. McMahon, D. J., R. J. Brown, G. H. Richardson, and C. A. Ernstrom. 1984. Effects of Ca, Pi, and bulk culture media on milk coagulation properties. J. Dairy Sci. 67:930–938. Patel, R. S., and H. Reuter. 1986. Effect of sodium, calcium and inorganic phosphate on properties of rennet coagulated milk. Lebensmittel-Wissenschaft-und-Technologie 19:288–291. Pouliot, Y., M. Boulet, and P. Paquin. 1989a. Observations on heatinduced salt balance changes in milk. I. Effect of heating time between 4 and 90°C. J. Dairy Res. 56:185–192. Pouliot, Y., M. Boulet, and P. Paquin. 1989b. Observations on heatinduced salt balance changes in milk. ii. Reversibility on cooling. J. Dairy Res. 56:193–199. Robinson, R. A., and R. H. Stokes. 1959. Electrolyte solutions. Butterworths Publications Ltd., London.

GELATION OF RENNETED MILK Schmidt, D. G., J. Koops, and D. Westerbeek. 1977. Properties of artificial casein micelles. 1. Preparation, size distribution and composition. Neth. Milk and Dairy J. 31:328–341. Shalabi, S. I., and P. F. Fox. 1982. Influence of pH on the rennet coagulation of milk. J. Dairy Res. 49:153–157. Standards Association of Australia. 1988. Methods of chemical and physical testing for the dairying industry. General methods and principles—Determination of nitrogen—Nitrogen fractions from milk. (SAA AS 2300.1.2.2). Standards Association of Australia. 1991. Methods of chemical and physical testing for the dairying industry. General methods and principles—Determination of nitrogen—Reference Kjeldahl method. (SAA AS 2300.1.2.1).

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Udabage, P. 1999. The composition and the renneting behaviour of skim milk. Ph.D. thesis. Monash University, Australia. van Hooydonk, A.C.M., H. G. Hagedoorn, and I. J. Boerrigter. 1986. The effect of various cations on the renneting of milk. Neth. Milk Dairy J. 40:369–390. Walstra, P., and T. van Vliet. 1986. The physical chemistry of curd making. Neth. Milk Dairy J. 40:241–259. Zoon, P., T. van Vliet, and P. Walstra. 1988. Rheological properties of rennet-induced skim milk gels. 3. The effect of calcium and phosphate. Neth. Milk Dairy J. 42:295–312. Zoon, P., T. van Vliet, and P. Walstra. 1989. Rheological properties of rennet-induced skim milk gels. 4. The effect of pH and NaCl. Neth. Milk Dairy J. 43:17–34.

Journal of Dairy Science Vol. 84, No. 7, 2001