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Behaviour of calcium and phosphate in bovine casein micelles
Inf. Dairy Journal 6 (1996) 769-780 Copyright
0
PII:SO958-6946(96)00006-4
1996 Published by Elsevier Science Limited Printed in Ireland. All rights reserved 0958-6946/96/$15.00+0.00
ELSEVIER
Behaviour of Calcium and Phosphate in Bovine Casein Micelles
Zhu Ping Zhang” & Takayoshi Aok?* ‘United Chair of Applied Resource Chemistry, Course of Bioresource Science for Processing, United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890, Japan ‘Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Kagoshima 890, Japan (Received 26 April 1995; accepted 4 January 1996)
ABSTRACT The behaviour of calcium and phosphate in casein micelles was examined using 4’Ca and 32P by means of ultrafiltration and high performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea. Approximately 3040% of the phosphate present in the colloidal phase of a casein micelle dispersion (CMD) was estimated to be hard to exchange with the phosphate present in soluble phase. The distribution patterns of 45Ca and 32P between soluble and colloidal phases in CMD did not coincide with the protein elution patterns from high performance gel chromatography in the presence of urea. Only two peaks appeared in the distribution patterns of 45Ca and 32P in CMD. The proportions of 45Ca and “P in the fraction of casein aggregates cross-linked by micellar calcium phosphate (MCP) increased with the length of holding time after adding of 45Ca and 32P to CMD. The proportion of 45Ca in the fraction of casein aggregates cross-linked by MCP decreased slightly on cooling at 4°C. The proportion of 4’Ca and 32P distributed in casein aggregates crosslinked by MCP increased significantly on heating at 90°C. The results suggested that calcium andphosphate in CMD could be divided into three types. Copyright 0 1996 Published by Elsevier Science Limited
INTRODUCTION In bovine milk, calcium and phosphate are present in excess of their solubilities, but as a result of interaction with casein, do not precipitate (Holt, 1985). *Author to whom correspondence
should be addressed. 769
770
Z. P. Zhang, T. Aoki
Approximately one-third of the calcium in bovine milk is present in the serum phase as free calcium ions or complexed predominantly by citrate or phosphate (Holt et al., 1981). The remaining two-thirds are either directly bound to the caseins or are an integral part of the colloidal calcium phosphate complex in socalled casein micelles (Schmidt, 1982). The calcium phosphate associated with casein is called micellar calcium phosphate (MCP) or colloidal calcium phosphate. The MCP plays an important role in maintaining the integrity of casein micelles which disaggregate into submicelles when MCP is removed (McGann & Pyne, 1960; Schmidt & Buchheim, 1970; Morr et al., 1971). In some models of the structure of casein micelles (Schmidt, 1982; Walstra & van Vliet, 1986) MCP is considered to link submicelles. Using high-performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea, Aoki et al. (1987) showed that caseins are cross-linked through their ester phosphate groups by MCP. It has been also shown that at least three phosphate groups are needed for cross-linking of casein by MCP (Aoki et al., 1992). Although several models have been proposed (Holt et al., 1982; Schmidt, 1982; van Dijk, 1990), understanding of the structure of MCP is still limited. Yamauchi et al. (1969) and Yamauchi & Yoneda (1977) found that 3&40% of colloidal calcium was not exchanged even after 48 h at 1618 “C, and that the amount of exchangeable calcium depended on the pH and heat treatment of the milk. A better understanding of the behaviour of calcium and phosphate ions and their interaction with caseins is important, because these ions play an essential role in the formation of casein micelles and in milk stability (Schmidt, 1982; Holt, 1985). In the present study, the behaviour of calcium and phosphate in casein micelles was examined using 45Ca and 32P by means of ultrafiltration and high-performance gel chromatography in the presence of 6 M urea.
MATERIALS
AND METHODS
Preparation of casein micelle dispersion
Raw bovine skim milk was purchased from Kagoshima Milk Co. (Kagoshima, Japan). One mg of trypsin inhibitor (Sigma Chemical Co., St Louis, MO 63178 USA) was added to 500 mL of skim milk to stop the action of plasmin. Casein micelles were separated from the skim milk, which had been stored at 25°C for 2 h, by ultracentrifugation at 100,OOOxg for 1 h at 25°C and then dispersed in simulated milk ultrafiltrate (SMUF; Jenness & Koops, 1962), by ultrasonic treatment at 9 kHz for 18 min at about 20°C. The casein concentration in the casein micelle dispersion (CMD) was determined by the Kjeldahl method and adjusted to 2.5% by adding SMUF. Sodium azide was added of 0.05% to CMD as a preservative. Preparation of colloidal phosphate-free
CMD
Colloidal phosphate-free (CPF) CMD were prepared by the method of Morr et al. (1971). To 10 mL CMD in an ice water bath were added 50 mg EDTA Na2.2H20 and 80 mg EDTA Na4.4H20 and the solution dialysed for 2 days at 5°C against four changes of 50 times its volume of SMUF.
Calcium and phosphate in bovine casein micelles
171
Separation of soluble phase calcium 45CaC12 solution (Specific activity: 6.17 mCi mg-‘) was purchased from ICN Biomedicals, Inc. (Irvine, CA, U.S.A.). Soluble phase-calcium in CMD and CPF-CMD was separated by ultrafiltration at room temperature (22-25°C). To 25 mL of CMD or CPF-CMD, respectively, 0.25 mL of 45CaC12 solution (calcium concentration: 7.7 x lo-* mg mL_‘) were added, and mixed immediately. The mixture was ultrafiltered at appropriate intervals, usually, (1) immediately after addition of 45CaC12, (2) after standing at room temperature for 24 h, and (3) for 48 h. Each time, 6 mL of sample were ultrafiltered by using a Amicon Diaflo YM-10 ultrawas filtration membrane (Amicon Co., Danvers, MA, USA). Ultratiltrate obtained at a rate of 1 mL h-‘. The first 2 mL were discarded, and the next 1 mL retained for analysis. No precipitation was observed during holding the samples at room temperature for 48 h. Separation of soluble phase phosphate K2H32P04 solution (specific activity: 32.26 mCi mg-‘) was purchased from ICN Biomedicals Inc. (Irvine, CA, USA). Soluble phase-phosphate in CMD and CPF-CMD was separated by ultrafiltration at room temperature (22-25”(Z). To 25 mL of CMD or CPF-CMD, respectively, 0.25 mL of K2H3*P04 solution (phosphate concentration: 3 x 10e2 mg mL_‘) were added, and mixed immediately. The mixture was ultrafiltered at appropriate intervals, as described above. Each time, 2 mL samples were ultrafiltered through Centrisalt I (Sartorius AGW-3400 Goettingen, Germany, cut-off Mw 10,000). Cooling and heating of CMD After adding the 45CaC12 or K2H32P04 solution to the samples, the mixture was cooled in a 50 mL Pyrex flask at 4°C in a water bath for 6 h. Then, the flask was held at 25°C in a water bath for 6 h. The above operations were repeated for a total holding time of 48 h. After adding the 45CaC12 or K2H3*P04 solution to the samples, the mixture was heated at 90°C for 30 min and then held at 25°C for 24 h. This operation was repeated at 48 h. Highqperformance gel chromatography High-performance gel chromatography was performed at room temperature (2225°C) with a Shimadzu LC-5A chromatograph (Shimadzu Ltd, Kyoto, Japan) equipped with a Shimadzu SPD-2A spectrophotometric detector (Shimadzu Ltd) using a TSK-GEL G4000SW column (7.5 mm x 60 cm, Toso Ltd, Tokyo, Japan) attached to a TSK-GEL guard column (7.5 mm x 7.5 cm, Toso Ltd). Before analysis, 6 M urea-SMUF (USMUF), prepared by the method of Aoki et al. (1986), was passed through the system at a flow rate of 0.5 mL min-’ for more than 3 h. To 1 mL of CMD, 0.5 g of solid urea (specially prepared grade) and 2mercaptoethanol (to 10 IIIM) were added to reduce the caseins. The solution was allowed to stand overnight at 25°C and then filtered through a membrane filter
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(pore size, 0.45 pm; Japan Millipore Ltd, Yamagata, Japan) before injection. The injection volumes were 25 and 200 PL for analytical and preparative runs, respectively. Determination of calcium and phosphate Calcium was determined by using Calcium E-Test Wako (Wake, Ltd., Osaka, Japan) with minor modification. To 0.5 mL samples (which were diluted 100 fold with ion exchanged water), 2 mL of 3.3 M monoethanolamine buffer (pH 12.0) were added and mixed immediately. The mixture was allowed to stand for 10 min at room temperature (22-25’C), before addition of 1 mL of solution containing 0.25 mM methylxylenol blue and 31 IIIM 8-quinolinol. Inorganic phosphorus was determined in the TCA filtrate by the method of Allen (1940). Determination of radioactivity Samples (200 pL) were pipetted onto a crystalline Ready Cap (Beckman Instruments, Inc., Fullerton, CA, USA), and air-dried under a heat lamp at a temperature less than 70°C. When samples were dry, the Ready Cap was placed into a standard 20 mL liquid scintillation vial. Radioactivity was determined by a model LSC-3050 Aloka radiation counter (Aloka Co. Ltd, Tokyo, Japan). The proportion of radioactivity was calculated by subtracting the value of background. The radioactivity was also determined by using a /I-RAM radio-HPLC detector (IN/US Systems, Inc., Tampa, FL, USA). Calculation The proportions of the exchanged calcium and phosphate (% Caex or P,,) in the colloidal phase to total colloidal one were calculated by the following formula,
%Qx
or
P,, =
100 -45 Casol or 32P,01Cas01 or PsOi Casoi or PsOl x 100 ’ 100 - Casol or Psol 45Cas01or 32Ps01
where 45Ca,,i or 32Ps0iis the percentage of 45Ca or 32P activity remaining in the soluble phase; Casoi or Psol is the percentage of calcium or phosphate content in the soluble phase. This formula is essentially the same as that used by Neuman et al. (1949) or Falkenheim et al. (1951) for the radioisotopic study of bone minerals.
RESULTS Exchange of calcium in CMD Exchange of calcium between the soluble and colloidal phases of CMD was determined on four different lots of CMD by the ultrafiltration method. The results are given in Table 1. Approximately 45% of colloidal calcium was exchanged with soluble calcium within 1 h, while approximately a further IO-15% exchanged slowly. The approximate proportion of hard-to-exchange calcium estimated from lOO-% Ca,, 48 h after the addition of 45CaC12, was 45%. This was similar to the
Calcium and phosphate in bovine casein micelles
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Z. P. Zhang, T. Aoki
174
results of Yamauchi et al. (1969), who found that approximately 40% of colloidal calcium was hard to exchange with soluble calcium even after 48 h. In order to investigate the distribution of 45Ca in CMD, after adding 45CaC12 solution to CMD, high-performance gel chromatography was performed on a TSK-GEL G4000SW column in the presence of 6 M urea. The elution pattern of 45Ca obtained is shown in Fig. 1. Aoki et al. (1986) who separated CMD under similar conditions, concluded that fraction 1 was composed of casein aggregates cross-linked by MCP, while fraction 2 contained casein monomers that bound only calcium. Fraction 3 contained non-casein components present in sedimented casein micelles. The elution pattern of 45Ca in CMD did not completely coincide with the protein elution pattern, monitored by absorbance at 280 nm, from the TSK-GEL G4000SW column in the presence of urea (Fi!. 1). Only two peaks appeared in the distribution pattern of 45Ca in CMD. 4 Ca was eluted in the fraction containing casein aggregates (centered around 30 min) cross-linked by Fl
F2
F3
6
1 0 0
10
20
30
40
60
60
Retention time (min) Fig. 1. Elution
patterns of 45Ca and the proteins in casein micelles obtained by high performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea. Fl, fraction 1 consisted of casein aggregates cross-linked by MCP, F2, fraction 2 was composed of casein monomers; F3, fraction 3 was assigned to non-casein components contained in sedimented casein micelles. The dashed line indicates the proteins elution pattern. After holding for 0 (O), 24 (0) or 48 (A) h.
Calcium and phosphate
in bovine casein micelles
115
MCP and non-casein components contained in sedimented casein micelles. No 45Ca was eluted in the fraction containing monomeric caseins $centered around 40 min). To obtain further information on the distribution of 4 Ca in CMD, we enlarged the distribution patterns of 45Ca in casein aggregates cross-linked by MCP of CMD, as shown in the insert in Fig. 1. Table 2 shows the distribution of 45Ca in casein aggregates cross-linked by MCP and in fraction 3 of CMD. The proportion of 45Ca in casein aggregates cross-linked by MCP in CMD increased on standing. The distribution pattern of 45Ca in CPF-CMD was also examined; 45Ca appeared only in fraction 3 of CPF-CMD. Furthermore, the distribution pattern of 45Ca in USMUF showed that 45Ca was eluted only in fraction 3. Exchange of phosphate in CMD
Exchange of phosphate between the soluble and colloidal phases of CMD was determined on four different lots of CMD by the ultrafiltration method. The results in Table 1 showed that approximately 40% of colloidal phosphate was exchanged with soluble phosphate within 1 h, while approximately a further 20% exchanged slowly. The approximate proportion of hard-to-exchange phosphate, estimated from lOO-% P,, 48 h after the addition of K2H32P04, was 34%. In order to investigate the distribution of 32P in CMD, highperformance gel chromatography was performed. As shown in Fig. 2, the distribution pattern of 32P in CMD did not coincide with the total protein elution pattern from high-performance gel chromatography in the presence of urea. Only two peaks were observed in the distribution pattern of 32P in CMD. The insert in Fig. 2 shows the distribution patterns of 32P in casein aggregates cross-linked by MCP of CMD. The proportion of 32P in casein a gregates cross-linked by MCP of CMD is also given in Table 2; similar to ‘&a the proportion of 32P in casein aggregates cross-linked by MCP increased with the duration of holding. The distribution pattern of 32P in CPF-CMD was also examined; only one peak was observed in the distribution pattern of 32P in CPF-CMD, similar to 45Ca in CPF-CMD. Effect of cooling and heating
The effects of cooling on calcium and phosphate exchange and 45Ca and 32P distributions were examined. The proportion of hard-to-exchange calcium estimated at 48 h after the addition of 45CaC12was about 46% on cooling at 4°C; no significant change was observed. As shown in Table 3, the proportion of phosphate exchanged at 4°C 48 h after the addition of K2H32P04 was 54.5%, which was lower than that in untreated CMD after 48 h at room temperature (Table 1). The distribution of 45Ca and 32P in casein aggregates cross-linked by MCP was investigated. As shown in Table 4, the proportions of 45Ca and 32P in the fraction of casein aggregates cross-linked by MCP 48 h after the addition of 45CaC12or K2H32P04 on cooling at 4°C were 18.1%, which were somewhat lower than those in the fraction of casein aggregates cross-linked by MCP in unheated CMD (Table 2). The effects of heating on calcium and phosphate exchange and the distribution of 45Ca and 32P were also examined. As shown in Table 3, the proportion of exchanged phosphate 48 h after heating at 90°C was 69.8%, which was somewhat higher than that in unheated CMD (Table 1). The proportions of 45Ca and 32P in the fraction of casein aggregates cross-linked by MCP 48 h after heating at 90°C
Z. P. Z/rang. T. Aoki
776
TABLE 2 of 4’Ca and 32P in Chromatography Fractions dispersion.
Proportions
Time (h) after adding “Co or 32P
1 and 3 of a Casein Micelle
“Ca
0”
Fraction I
Fraction 3
Fraction I
Fraction 3
(%)
(%)
(%)
(%)
85.2fl.4 81.8fl.6 80.2f0.7
12.0 i2.6 16.5 ztO.7 18.5 ztl.6
88.0 f2.6 83.5 f0.7 81.5 fl.6
14.8fl.4 18.2fl.6 19.8xtO.7
24h 48h
32P
“Immediately after adding 45Ca or 32P to CMD. ‘Number of hours held at 25°C before sample’s preparation for chromatography. Each value is the means f standard deviation of four determinations.
I
Fl
F2
I
F3
I
I
35 30 A b
25
G E” 20 8 ;
15
: 10 5 0 0
10
20
30
40
50
60
Retention time (mln) Fig. 2. Distribution patterns of 32P in casein micelles obtained by high-performance gel chromatography on a TSK-GEL G4OOOSWcolumn in the presence of 6 M urea. Fl, fraction 1 consisted of casein aggregates cross-linked by MCP; F2, fraction 2 was composed of casein monomers; F3, fraction 3 was assigned to non-casein components contained in sedimented casein micelles. After holding for 0 (O), 24 (0) or 48 (A) h.
Calcium and phosphate in bovine casein micelles
717
TABLE 3
Effect of Cooling and Heating on the Exchange of Pi in Casein Micelle Dispersions as Measured by UF. Treatment
PSO/
Time (h) after adding 32P
32PSO/
O% P,.x
Cooling at 4°C
24 48
52.0 51.1
68.3 65.5
50.3 54.5
Heating at 90°C
24 48
49.3 47.9
59.6 56.8
66.0 69.8
The above values were mean values of two determinations.
TABLE 4
Effect of Cooling and Heating on the Distribution of 45Ca and 32P in Chromatography Fractions 1 and 3 of Casein Micelle Dispersion. Treatment
Time (h) after adding 4’Ca or 32P (%)
45Ca
32P
Fraction 1
Fraction 3
Fraction 1
Fraction 3
(%)
(%)
(%)
(%)
Cooling at 4°C
24 48
16.3 18.1
83.7 81.9
16.3 18.1
83.7 81.9
Heating at 90°C
24 48
26.4 31.2
73.6 68.8
25.6 30.1
74.4 69.9
The above values were mean values of two determinations.
were 3 1.2 and 30.1%, respectively, which were considerably higher than those in the fraction of casein aggregates cross-linked by MCP of unheated CMD (Table 2 and 4).
DISCUSSION Yamauchi et al. (1969) introduced the concept of hard-to-exchange calcium, which is part of the colloidal calcium phosphate. Pierre et al. (1983) found that approximately 3.5% of the colloidal calcium appeared to be hard to exchange. In the present study, we obtained similar results (Table 1). We also found that approximately 40% of colloidal phosphate was exchanged with soluble phosphate within 1 h, while approximatei$ a further 20% exchanged slowly (Table 1). However, the proportions of Ca and 32P in fraction 3 obtained by high-performance gel chromatography were higher than that in soluble phase obtained b ultrafiltration (Tables 1 and 2). This indicated that the exchanged 45Ca and K2 P in fraction 2 were re-exchanged during high performance gel chromatography, because 45Ca was eluted only in fraction 3 in USMUF. Disaggregation of casein micelles by urea, followed by gel permeation chroma-
778
Z. P. Zhang, T. Aoki
tography, resulted in a calcium phosphate-containing protein fraction and a fraction with a very low calcium phosphate content (Aoki et al., 1986). Probably, the exchanged 45Ca and 32P in a very low calcium phosphate-containing casein fraction were easily re-exchanged durin high-performance gel chromatography. On the other hand, the exchanged $5Ca and 32P in a high calcium phosphate-containing casein fraction were hardly re-exchanged during highperformance gel chromatography. Although the calcium and phosphate in the colloidal phase of CPF-CMD was exchanged rapidly, only one peak occurred in the distribution attern of 45Ca and 32P in CPF-CMD. This also suggested that the exchanged B Ca and 32P were re-exchanged during high-performance gel chromatography. van Dijk (1990) proposed an ion cluster model for MCP in which two phosphate groups, four inorganic phosphates and eight cations (mainly calcium) participate; he also suggested that two ‘unstable arms’ in C2SerP3 may act as complexes that are in fast equilibrium with the solution. According to his model, when 45Ca was exchanged into unstable arms, it became stable. The exchanged 45Ca in stable arms was hardly re-exchanged during high-performance gel chromatography. This may explain why the proportions of 45Ca and 32P in the fraction of casein aggregates cross-linked by MCP in CMD increased on standing after addition of 45Ca and 32P to CMD (Table 2). From above results, calcium and phosphate in CMD could be divided into the three types: i) exchanged and not re-exchanged; ii) exchanged and reexchanged; iii) hard to exchange. From Tables 1 and 3, it is noted that the proportion of exchanged phosphate at 4°C 48 h after the addition of K2H3*P04 was somewhat lower than that at 25°C 24 h after the addition of K2H3*P04. This may suggest that cooling to 4°C reduced the exchange of calcium and phosphate in CMD. Accordingly, the holding temperature is important for the exchange of calcium and phosphate in CMD. Yamauchi & Yoneda (1977) found that the exchangeability of colloidal calcium in milk with soluble calcium estimated at 4849 h after the addition of 45CaC12 was not changed by heating at 80 “C, but was reduced somewhat by heating at 100 “C. Pierre et al. (1983) found that the amount of hard-to-exchange calcium decreased with increasing temperature and holding time at the higher temperature. In the present study, the proportion of exchanged calcium and phosphate on heating at 90 “C were slightly higher than those in unheated CMD (Tables 1 and 3). Furthermore, the proportions of 45Ca and 32P in the fraction of casein aggregates cross-linked by MCP on heating at 90°C were considerably higher than those in the fraction of casein aggregates cross-linked by MCP in unheated CMD (Tables 2 and 4). In a 43Ca NMR study, Wahlgren et al. (1990) found that five different calcium environments existed in heated milk fractions. Pouliot et al. (1989) reported that the concentrations of calcium and phosphate in the soluble phase decreased within a very short time when the milk conditioned at 4°C was heated to 90°C. Approximately 60% of calcium and 40% of phosphate in the soluble phase were transferred to the colloidal phase on heating at 90°C for 40 min (Pouliot et al., 1989). Rose et al. (1959) stated that after cooling milk that had been heated at 93°C to 4°C for 20 h, 75-90% of the heat-precipitated calcium phosphate resolublized. It could be speculated that heating CMD at 90°C for 30 min promoted exchange of calcium and phosphate from the soluble to the colloidal phase to increase the proportion of 45Ca and 32P in the fraction of casein aggregates cross-linked by MCP.
Calcium and phosphate in bovine casein micelles
119
ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid for Scientific Research 07660362) from the Ministry of Education, Science, and Culture of Japan.
(No.
REFERENCES Allen, R.J.L. (1940). The estimation of phosphorus. Biochem. J., 34, 858-865. Aoki, T., Kako, Y. & Imamura, T. (1986). Separation of casein aggregates cross-linked by colloidal calcium phosphate from bovine casein micelles by high performance gel chromatography in the presence of urea. J. Dairy Res., 53, 53-59. Aoki, T., Umeda, T. & Kako, Y. (1992). The least number of phosphate groups for crosslinking of casein by colloidal calcium phosphate. J. Dairy Sci.. 75, 971 975.
Aoki, T., Yamada, N., Tomita, I., Kako, Y. & Imamura, T. (1987). Caseins are crosslinked through their ester phosphate groups by colloidal calcium phosphate. Biochim. Bz’ophys. Acra, 911, 238-243.
Falkenhein, M., Underwood, E.E. & Hodge, H.C. (1951). Calcium exchange: the mechanism of adsorption of calcium 45 by bone. J. Biol. Chem., 188,805-817. Holt, C. D. (1985). The milk salts: Their secretion, concentrations and physical chemistry. In Developments in Dairy Chemistry-3. Lactose and Minor Constituents, ed. P. F. Fox. Elsevier Applied Science Publisher, London, pp. 143- 18 1. Holt, C., Dalgleish, D.G. & Jenness, R. (1981). Calculation of the ion equilibria in milk diffusate and comparison with experiment. Anal Biocliern., 113, 154163. Halt, C., Hasnain, S.S. & Hukins, D.W.L. (1982). Structure of bovine milk calcium phosphate determined by X-ray absorption spectroscopy. Biochim. Biophys. Acta, 719, 299-303.
Jenness, R. & Koops, J. (1962). Preparation and properties of a salt solution which simulates milk ultrafiltrate. Neth. Milk Dairy J., 16, 153-164. Knoop, A.-M., Knoop, E. & Wiechen, A. (1979). Sub-structure of synthetic casein micelles. J. Dairy Res., 46, 347350.
McGann, T.C.A. & Pyne, G.T. (1960). The colloidal phosphate of milk. III. Nature of its association with casein. J. Dairy Res., 27, 403417. Morr, C.V., Josephson, R.V., Jenness, R. & Manning, P.B. (1971). Composition and properties of submicellar casein complexes in colloidal phosphate-free skim milk. J. Dairy Sci., 54, 1555-1563.
Neuman, W.F., Neuman, M.W., Main, E.R. & Mulryan, B.J. (1949). Deposition of uranium in bone. V. Ion-exchange studies. J. Biol. Chem., 179, 335-340. Pierre. A., Brule, G. & Fauquant. J. (1983). Study of Ca exchange in milk using 45Ca. Lait. 63, 473480. Pouliot, Y.. Boulet, M. & Paquin, P. (1989). Observations on the heated-induced salt balance changes in milk. I. Effect of heating time between 4 and 90°C. J. Dairy Res., 56, 185-192.
Rose, D. & Tessier, H. (1959). Composition of ultraliltrates from milk heated at 80 to 230°F. in relation to heated stability. J. Dairy Sci., 42, 9699980. Schmidt, D. G. (1982). Association of caseins and casein micelles structure. In Developments in Dairy Chemistry-l. Proteins, ed. P. F. Fox. Elsevier Applied Science Publisher, London, pp. 61-86. Schmidt, D.G. & Buchheim, W. (1970). An electron-microscopic investigation of the casein micelles in cow milk. Milchwissenschaft, 25, 596600. van Dijk, H.J.M. (1990). The properties of casein micelles. 1. The nature of the micellar calcium phosphate. Neth. Milk Dairy J., 44, 65-81.
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Wahlgren, N.M., Dejmek, P. 8z Drakenberg, T. (1990). A 43Ca and 31P NMR study of the calcium and phosphate equilibria in heated milk solutions. J. Dairy Res., 57, 355-364. Walstra, P. & van Vliet, T. (1986). The physical chemistry of curd making. Neth. Milk Dairy J., 40, 241-259.
Yamauchi, K. & Yoneda, Y. (1977). Effect of some treatments of milk on the exchangeability of colloidal calcium in milk with soluble calcium. Agric. Biol. Chem., 41,2395-2399.
Yamauchi, K., Yoneda, Y., Koga, Y. & Tsugo, T. (1969). Exchangeability calcium in milk with soluble calcium. Agric. Biol. Chem., 33, 907-914.
of colloidal
0
PII:SO958-6946(96)00006-4
1996 Published by Elsevier Science Limited Printed in Ireland. All rights reserved 0958-6946/96/$15.00+0.00
ELSEVIER
Behaviour of Calcium and Phosphate in Bovine Casein Micelles
Zhu Ping Zhang” & Takayoshi Aok?* ‘United Chair of Applied Resource Chemistry, Course of Bioresource Science for Processing, United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890, Japan ‘Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Kagoshima 890, Japan (Received 26 April 1995; accepted 4 January 1996)
ABSTRACT The behaviour of calcium and phosphate in casein micelles was examined using 4’Ca and 32P by means of ultrafiltration and high performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea. Approximately 3040% of the phosphate present in the colloidal phase of a casein micelle dispersion (CMD) was estimated to be hard to exchange with the phosphate present in soluble phase. The distribution patterns of 45Ca and 32P between soluble and colloidal phases in CMD did not coincide with the protein elution patterns from high performance gel chromatography in the presence of urea. Only two peaks appeared in the distribution patterns of 45Ca and 32P in CMD. The proportions of 45Ca and “P in the fraction of casein aggregates cross-linked by micellar calcium phosphate (MCP) increased with the length of holding time after adding of 45Ca and 32P to CMD. The proportion of 45Ca in the fraction of casein aggregates cross-linked by MCP decreased slightly on cooling at 4°C. The proportion of 4’Ca and 32P distributed in casein aggregates crosslinked by MCP increased significantly on heating at 90°C. The results suggested that calcium andphosphate in CMD could be divided into three types. Copyright 0 1996 Published by Elsevier Science Limited
INTRODUCTION In bovine milk, calcium and phosphate are present in excess of their solubilities, but as a result of interaction with casein, do not precipitate (Holt, 1985). *Author to whom correspondence
should be addressed. 769
770
Z. P. Zhang, T. Aoki
Approximately one-third of the calcium in bovine milk is present in the serum phase as free calcium ions or complexed predominantly by citrate or phosphate (Holt et al., 1981). The remaining two-thirds are either directly bound to the caseins or are an integral part of the colloidal calcium phosphate complex in socalled casein micelles (Schmidt, 1982). The calcium phosphate associated with casein is called micellar calcium phosphate (MCP) or colloidal calcium phosphate. The MCP plays an important role in maintaining the integrity of casein micelles which disaggregate into submicelles when MCP is removed (McGann & Pyne, 1960; Schmidt & Buchheim, 1970; Morr et al., 1971). In some models of the structure of casein micelles (Schmidt, 1982; Walstra & van Vliet, 1986) MCP is considered to link submicelles. Using high-performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea, Aoki et al. (1987) showed that caseins are cross-linked through their ester phosphate groups by MCP. It has been also shown that at least three phosphate groups are needed for cross-linking of casein by MCP (Aoki et al., 1992). Although several models have been proposed (Holt et al., 1982; Schmidt, 1982; van Dijk, 1990), understanding of the structure of MCP is still limited. Yamauchi et al. (1969) and Yamauchi & Yoneda (1977) found that 3&40% of colloidal calcium was not exchanged even after 48 h at 1618 “C, and that the amount of exchangeable calcium depended on the pH and heat treatment of the milk. A better understanding of the behaviour of calcium and phosphate ions and their interaction with caseins is important, because these ions play an essential role in the formation of casein micelles and in milk stability (Schmidt, 1982; Holt, 1985). In the present study, the behaviour of calcium and phosphate in casein micelles was examined using 45Ca and 32P by means of ultrafiltration and high-performance gel chromatography in the presence of 6 M urea.
MATERIALS
AND METHODS
Preparation of casein micelle dispersion
Raw bovine skim milk was purchased from Kagoshima Milk Co. (Kagoshima, Japan). One mg of trypsin inhibitor (Sigma Chemical Co., St Louis, MO 63178 USA) was added to 500 mL of skim milk to stop the action of plasmin. Casein micelles were separated from the skim milk, which had been stored at 25°C for 2 h, by ultracentrifugation at 100,OOOxg for 1 h at 25°C and then dispersed in simulated milk ultrafiltrate (SMUF; Jenness & Koops, 1962), by ultrasonic treatment at 9 kHz for 18 min at about 20°C. The casein concentration in the casein micelle dispersion (CMD) was determined by the Kjeldahl method and adjusted to 2.5% by adding SMUF. Sodium azide was added of 0.05% to CMD as a preservative. Preparation of colloidal phosphate-free
CMD
Colloidal phosphate-free (CPF) CMD were prepared by the method of Morr et al. (1971). To 10 mL CMD in an ice water bath were added 50 mg EDTA Na2.2H20 and 80 mg EDTA Na4.4H20 and the solution dialysed for 2 days at 5°C against four changes of 50 times its volume of SMUF.
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Separation of soluble phase calcium 45CaC12 solution (Specific activity: 6.17 mCi mg-‘) was purchased from ICN Biomedicals, Inc. (Irvine, CA, U.S.A.). Soluble phase-calcium in CMD and CPF-CMD was separated by ultrafiltration at room temperature (22-25°C). To 25 mL of CMD or CPF-CMD, respectively, 0.25 mL of 45CaC12 solution (calcium concentration: 7.7 x lo-* mg mL_‘) were added, and mixed immediately. The mixture was ultrafiltered at appropriate intervals, usually, (1) immediately after addition of 45CaC12, (2) after standing at room temperature for 24 h, and (3) for 48 h. Each time, 6 mL of sample were ultrafiltered by using a Amicon Diaflo YM-10 ultrawas filtration membrane (Amicon Co., Danvers, MA, USA). Ultratiltrate obtained at a rate of 1 mL h-‘. The first 2 mL were discarded, and the next 1 mL retained for analysis. No precipitation was observed during holding the samples at room temperature for 48 h. Separation of soluble phase phosphate K2H32P04 solution (specific activity: 32.26 mCi mg-‘) was purchased from ICN Biomedicals Inc. (Irvine, CA, USA). Soluble phase-phosphate in CMD and CPF-CMD was separated by ultrafiltration at room temperature (22-25”(Z). To 25 mL of CMD or CPF-CMD, respectively, 0.25 mL of K2H3*P04 solution (phosphate concentration: 3 x 10e2 mg mL_‘) were added, and mixed immediately. The mixture was ultrafiltered at appropriate intervals, as described above. Each time, 2 mL samples were ultrafiltered through Centrisalt I (Sartorius AGW-3400 Goettingen, Germany, cut-off Mw 10,000). Cooling and heating of CMD After adding the 45CaC12 or K2H32P04 solution to the samples, the mixture was cooled in a 50 mL Pyrex flask at 4°C in a water bath for 6 h. Then, the flask was held at 25°C in a water bath for 6 h. The above operations were repeated for a total holding time of 48 h. After adding the 45CaC12 or K2H3*P04 solution to the samples, the mixture was heated at 90°C for 30 min and then held at 25°C for 24 h. This operation was repeated at 48 h. Highqperformance gel chromatography High-performance gel chromatography was performed at room temperature (2225°C) with a Shimadzu LC-5A chromatograph (Shimadzu Ltd, Kyoto, Japan) equipped with a Shimadzu SPD-2A spectrophotometric detector (Shimadzu Ltd) using a TSK-GEL G4000SW column (7.5 mm x 60 cm, Toso Ltd, Tokyo, Japan) attached to a TSK-GEL guard column (7.5 mm x 7.5 cm, Toso Ltd). Before analysis, 6 M urea-SMUF (USMUF), prepared by the method of Aoki et al. (1986), was passed through the system at a flow rate of 0.5 mL min-’ for more than 3 h. To 1 mL of CMD, 0.5 g of solid urea (specially prepared grade) and 2mercaptoethanol (to 10 IIIM) were added to reduce the caseins. The solution was allowed to stand overnight at 25°C and then filtered through a membrane filter
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(pore size, 0.45 pm; Japan Millipore Ltd, Yamagata, Japan) before injection. The injection volumes were 25 and 200 PL for analytical and preparative runs, respectively. Determination of calcium and phosphate Calcium was determined by using Calcium E-Test Wako (Wake, Ltd., Osaka, Japan) with minor modification. To 0.5 mL samples (which were diluted 100 fold with ion exchanged water), 2 mL of 3.3 M monoethanolamine buffer (pH 12.0) were added and mixed immediately. The mixture was allowed to stand for 10 min at room temperature (22-25’C), before addition of 1 mL of solution containing 0.25 mM methylxylenol blue and 31 IIIM 8-quinolinol. Inorganic phosphorus was determined in the TCA filtrate by the method of Allen (1940). Determination of radioactivity Samples (200 pL) were pipetted onto a crystalline Ready Cap (Beckman Instruments, Inc., Fullerton, CA, USA), and air-dried under a heat lamp at a temperature less than 70°C. When samples were dry, the Ready Cap was placed into a standard 20 mL liquid scintillation vial. Radioactivity was determined by a model LSC-3050 Aloka radiation counter (Aloka Co. Ltd, Tokyo, Japan). The proportion of radioactivity was calculated by subtracting the value of background. The radioactivity was also determined by using a /I-RAM radio-HPLC detector (IN/US Systems, Inc., Tampa, FL, USA). Calculation The proportions of the exchanged calcium and phosphate (% Caex or P,,) in the colloidal phase to total colloidal one were calculated by the following formula,
%Qx
or
P,, =
100 -45 Casol or 32P,01Cas01 or PsOi Casoi or PsOl x 100 ’ 100 - Casol or Psol 45Cas01or 32Ps01
where 45Ca,,i or 32Ps0iis the percentage of 45Ca or 32P activity remaining in the soluble phase; Casoi or Psol is the percentage of calcium or phosphate content in the soluble phase. This formula is essentially the same as that used by Neuman et al. (1949) or Falkenheim et al. (1951) for the radioisotopic study of bone minerals.
RESULTS Exchange of calcium in CMD Exchange of calcium between the soluble and colloidal phases of CMD was determined on four different lots of CMD by the ultrafiltration method. The results are given in Table 1. Approximately 45% of colloidal calcium was exchanged with soluble calcium within 1 h, while approximately a further IO-15% exchanged slowly. The approximate proportion of hard-to-exchange calcium estimated from lOO-% Ca,, 48 h after the addition of 45CaC12, was 45%. This was similar to the
Calcium and phosphate in bovine casein micelles
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Z. P. Zhang, T. Aoki
174
results of Yamauchi et al. (1969), who found that approximately 40% of colloidal calcium was hard to exchange with soluble calcium even after 48 h. In order to investigate the distribution of 45Ca in CMD, after adding 45CaC12 solution to CMD, high-performance gel chromatography was performed on a TSK-GEL G4000SW column in the presence of 6 M urea. The elution pattern of 45Ca obtained is shown in Fig. 1. Aoki et al. (1986) who separated CMD under similar conditions, concluded that fraction 1 was composed of casein aggregates cross-linked by MCP, while fraction 2 contained casein monomers that bound only calcium. Fraction 3 contained non-casein components present in sedimented casein micelles. The elution pattern of 45Ca in CMD did not completely coincide with the protein elution pattern, monitored by absorbance at 280 nm, from the TSK-GEL G4000SW column in the presence of urea (Fi!. 1). Only two peaks appeared in the distribution pattern of 45Ca in CMD. 4 Ca was eluted in the fraction containing casein aggregates (centered around 30 min) cross-linked by Fl
F2
F3
6
1 0 0
10
20
30
40
60
60
Retention time (min) Fig. 1. Elution
patterns of 45Ca and the proteins in casein micelles obtained by high performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea. Fl, fraction 1 consisted of casein aggregates cross-linked by MCP, F2, fraction 2 was composed of casein monomers; F3, fraction 3 was assigned to non-casein components contained in sedimented casein micelles. The dashed line indicates the proteins elution pattern. After holding for 0 (O), 24 (0) or 48 (A) h.
Calcium and phosphate
in bovine casein micelles
115
MCP and non-casein components contained in sedimented casein micelles. No 45Ca was eluted in the fraction containing monomeric caseins $centered around 40 min). To obtain further information on the distribution of 4 Ca in CMD, we enlarged the distribution patterns of 45Ca in casein aggregates cross-linked by MCP of CMD, as shown in the insert in Fig. 1. Table 2 shows the distribution of 45Ca in casein aggregates cross-linked by MCP and in fraction 3 of CMD. The proportion of 45Ca in casein aggregates cross-linked by MCP in CMD increased on standing. The distribution pattern of 45Ca in CPF-CMD was also examined; 45Ca appeared only in fraction 3 of CPF-CMD. Furthermore, the distribution pattern of 45Ca in USMUF showed that 45Ca was eluted only in fraction 3. Exchange of phosphate in CMD
Exchange of phosphate between the soluble and colloidal phases of CMD was determined on four different lots of CMD by the ultrafiltration method. The results in Table 1 showed that approximately 40% of colloidal phosphate was exchanged with soluble phosphate within 1 h, while approximately a further 20% exchanged slowly. The approximate proportion of hard-to-exchange phosphate, estimated from lOO-% P,, 48 h after the addition of K2H32P04, was 34%. In order to investigate the distribution of 32P in CMD, highperformance gel chromatography was performed. As shown in Fig. 2, the distribution pattern of 32P in CMD did not coincide with the total protein elution pattern from high-performance gel chromatography in the presence of urea. Only two peaks were observed in the distribution pattern of 32P in CMD. The insert in Fig. 2 shows the distribution patterns of 32P in casein aggregates cross-linked by MCP of CMD. The proportion of 32P in casein a gregates cross-linked by MCP of CMD is also given in Table 2; similar to ‘&a the proportion of 32P in casein aggregates cross-linked by MCP increased with the duration of holding. The distribution pattern of 32P in CPF-CMD was also examined; only one peak was observed in the distribution pattern of 32P in CPF-CMD, similar to 45Ca in CPF-CMD. Effect of cooling and heating
The effects of cooling on calcium and phosphate exchange and 45Ca and 32P distributions were examined. The proportion of hard-to-exchange calcium estimated at 48 h after the addition of 45CaC12was about 46% on cooling at 4°C; no significant change was observed. As shown in Table 3, the proportion of phosphate exchanged at 4°C 48 h after the addition of K2H32P04 was 54.5%, which was lower than that in untreated CMD after 48 h at room temperature (Table 1). The distribution of 45Ca and 32P in casein aggregates cross-linked by MCP was investigated. As shown in Table 4, the proportions of 45Ca and 32P in the fraction of casein aggregates cross-linked by MCP 48 h after the addition of 45CaC12or K2H32P04 on cooling at 4°C were 18.1%, which were somewhat lower than those in the fraction of casein aggregates cross-linked by MCP in unheated CMD (Table 2). The effects of heating on calcium and phosphate exchange and the distribution of 45Ca and 32P were also examined. As shown in Table 3, the proportion of exchanged phosphate 48 h after heating at 90°C was 69.8%, which was somewhat higher than that in unheated CMD (Table 1). The proportions of 45Ca and 32P in the fraction of casein aggregates cross-linked by MCP 48 h after heating at 90°C
Z. P. Z/rang. T. Aoki
776
TABLE 2 of 4’Ca and 32P in Chromatography Fractions dispersion.
Proportions
Time (h) after adding “Co or 32P
1 and 3 of a Casein Micelle
“Ca
0”
Fraction I
Fraction 3
Fraction I
Fraction 3
(%)
(%)
(%)
(%)
85.2fl.4 81.8fl.6 80.2f0.7
12.0 i2.6 16.5 ztO.7 18.5 ztl.6
88.0 f2.6 83.5 f0.7 81.5 fl.6
14.8fl.4 18.2fl.6 19.8xtO.7
24h 48h
32P
“Immediately after adding 45Ca or 32P to CMD. ‘Number of hours held at 25°C before sample’s preparation for chromatography. Each value is the means f standard deviation of four determinations.
I
Fl
F2
I
F3
I
I
35 30 A b
25
G E” 20 8 ;
15
: 10 5 0 0
10
20
30
40
50
60
Retention time (mln) Fig. 2. Distribution patterns of 32P in casein micelles obtained by high-performance gel chromatography on a TSK-GEL G4OOOSWcolumn in the presence of 6 M urea. Fl, fraction 1 consisted of casein aggregates cross-linked by MCP; F2, fraction 2 was composed of casein monomers; F3, fraction 3 was assigned to non-casein components contained in sedimented casein micelles. After holding for 0 (O), 24 (0) or 48 (A) h.
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717
TABLE 3
Effect of Cooling and Heating on the Exchange of Pi in Casein Micelle Dispersions as Measured by UF. Treatment
PSO/
Time (h) after adding 32P
32PSO/
O% P,.x
Cooling at 4°C
24 48
52.0 51.1
68.3 65.5
50.3 54.5
Heating at 90°C
24 48
49.3 47.9
59.6 56.8
66.0 69.8
The above values were mean values of two determinations.
TABLE 4
Effect of Cooling and Heating on the Distribution of 45Ca and 32P in Chromatography Fractions 1 and 3 of Casein Micelle Dispersion. Treatment
Time (h) after adding 4’Ca or 32P (%)
45Ca
32P
Fraction 1
Fraction 3
Fraction 1
Fraction 3
(%)
(%)
(%)
(%)
Cooling at 4°C
24 48
16.3 18.1
83.7 81.9
16.3 18.1
83.7 81.9
Heating at 90°C
24 48
26.4 31.2
73.6 68.8
25.6 30.1
74.4 69.9
The above values were mean values of two determinations.
were 3 1.2 and 30.1%, respectively, which were considerably higher than those in the fraction of casein aggregates cross-linked by MCP of unheated CMD (Table 2 and 4).
DISCUSSION Yamauchi et al. (1969) introduced the concept of hard-to-exchange calcium, which is part of the colloidal calcium phosphate. Pierre et al. (1983) found that approximately 3.5% of the colloidal calcium appeared to be hard to exchange. In the present study, we obtained similar results (Table 1). We also found that approximately 40% of colloidal phosphate was exchanged with soluble phosphate within 1 h, while approximatei$ a further 20% exchanged slowly (Table 1). However, the proportions of Ca and 32P in fraction 3 obtained by high-performance gel chromatography were higher than that in soluble phase obtained b ultrafiltration (Tables 1 and 2). This indicated that the exchanged 45Ca and K2 P in fraction 2 were re-exchanged during high performance gel chromatography, because 45Ca was eluted only in fraction 3 in USMUF. Disaggregation of casein micelles by urea, followed by gel permeation chroma-
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Z. P. Zhang, T. Aoki
tography, resulted in a calcium phosphate-containing protein fraction and a fraction with a very low calcium phosphate content (Aoki et al., 1986). Probably, the exchanged 45Ca and 32P in a very low calcium phosphate-containing casein fraction were easily re-exchanged durin high-performance gel chromatography. On the other hand, the exchanged $5Ca and 32P in a high calcium phosphate-containing casein fraction were hardly re-exchanged during highperformance gel chromatography. Although the calcium and phosphate in the colloidal phase of CPF-CMD was exchanged rapidly, only one peak occurred in the distribution attern of 45Ca and 32P in CPF-CMD. This also suggested that the exchanged B Ca and 32P were re-exchanged during high-performance gel chromatography. van Dijk (1990) proposed an ion cluster model for MCP in which two phosphate groups, four inorganic phosphates and eight cations (mainly calcium) participate; he also suggested that two ‘unstable arms’ in C2SerP3 may act as complexes that are in fast equilibrium with the solution. According to his model, when 45Ca was exchanged into unstable arms, it became stable. The exchanged 45Ca in stable arms was hardly re-exchanged during high-performance gel chromatography. This may explain why the proportions of 45Ca and 32P in the fraction of casein aggregates cross-linked by MCP in CMD increased on standing after addition of 45Ca and 32P to CMD (Table 2). From above results, calcium and phosphate in CMD could be divided into the three types: i) exchanged and not re-exchanged; ii) exchanged and reexchanged; iii) hard to exchange. From Tables 1 and 3, it is noted that the proportion of exchanged phosphate at 4°C 48 h after the addition of K2H3*P04 was somewhat lower than that at 25°C 24 h after the addition of K2H3*P04. This may suggest that cooling to 4°C reduced the exchange of calcium and phosphate in CMD. Accordingly, the holding temperature is important for the exchange of calcium and phosphate in CMD. Yamauchi & Yoneda (1977) found that the exchangeability of colloidal calcium in milk with soluble calcium estimated at 4849 h after the addition of 45CaC12 was not changed by heating at 80 “C, but was reduced somewhat by heating at 100 “C. Pierre et al. (1983) found that the amount of hard-to-exchange calcium decreased with increasing temperature and holding time at the higher temperature. In the present study, the proportion of exchanged calcium and phosphate on heating at 90 “C were slightly higher than those in unheated CMD (Tables 1 and 3). Furthermore, the proportions of 45Ca and 32P in the fraction of casein aggregates cross-linked by MCP on heating at 90°C were considerably higher than those in the fraction of casein aggregates cross-linked by MCP in unheated CMD (Tables 2 and 4). In a 43Ca NMR study, Wahlgren et al. (1990) found that five different calcium environments existed in heated milk fractions. Pouliot et al. (1989) reported that the concentrations of calcium and phosphate in the soluble phase decreased within a very short time when the milk conditioned at 4°C was heated to 90°C. Approximately 60% of calcium and 40% of phosphate in the soluble phase were transferred to the colloidal phase on heating at 90°C for 40 min (Pouliot et al., 1989). Rose et al. (1959) stated that after cooling milk that had been heated at 93°C to 4°C for 20 h, 75-90% of the heat-precipitated calcium phosphate resolublized. It could be speculated that heating CMD at 90°C for 30 min promoted exchange of calcium and phosphate from the soluble to the colloidal phase to increase the proportion of 45Ca and 32P in the fraction of casein aggregates cross-linked by MCP.
Calcium and phosphate in bovine casein micelles
119
ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid for Scientific Research 07660362) from the Ministry of Education, Science, and Culture of Japan.
(No.
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of colloidal