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On the Structure of Casein Micelles
On the Structure of Casein Micelles By Hein J. M. van Dijk’ CAMPINA MELKUNIE B. V., PO BOX 222,3440 AE WOERDEN, THE NETHERLANDS
We have examined the partition of calcium (Ca), magnesium (Mg), and phosphate (P) between the casein micelles and various aqueous phases. After addition of > 5% NaC1, we have (Ca, + Mg,)/Pi, = 2.0, and, after addition of NaOH, notable changes in the partition were found to occur at = 2.0 (subscripts: c = colloidal, i = inorganic, o = orapproximately Pic/€’, ganic colloidal). We have also examined the disintegration of the casein micelles after addition of NaOH. Above pH 9, skim milk slowly becomes as transparent as whey. The speed of this process is increased if the protein amino groups have been succinylated or it is carried out at a higher pH, and is decreased due to addition of NaCl or amidation of carboxyl groups. Even low concentrations of added NaCl (i.e. 10-20 mM) strongly retard the disintegration but hardly influence the reaggregation. The following model is proposed for the casein micelles, and especially for the native (-like) micellar calcium phosphate (nMCP) .
Proposition 1 Most nMCP bridges in raw milk contain 2 phosphoserine residues (Po), 4 Pi, ions, and 8 divalent cations. As the value of (Ca, Mgc)/Pic can be lowered from -2.3 to only 2.0
+
by addition of NaC1, while roughly half of the Ca, can the dissolved Ca in the same period,3 it appears that nMCP may be 2.0. In addition, our results suggest that with nMCP) is also 2.0. Finally, it is most likely that cluster interconnects two P, groups. 2*4
be exchanged by (Ca + Mg)/PiC in Pic/(Po associated every nMCP ion
Proposition 2 Casein molecules of the same kind are bound together in pairs by a number of nMCP bridges (nMCP dimers). After addition of 6 M urea5 or NaOH (pHlo)* to milk, the casein molecules associated with the nMCP are in units of, say, 2-20 molecules. Present affiliation: Tastemaker, PO Box 414, 3770 AK Barneveld, The Netherlands
166
O n the Structure of Casein Micelles
All Po groups in these units are associated with the nMCP.236Since most of these Po groups are present in clusters (which are not all identical), and since below pH 6.7 in most sub-micelles it is predominantly the asl-casein which associates with the nMCP,* it is barely conceivable that the Po groups find each other at random. Steric hindrance will not allow this in most cases. Both before and after the formation of nMCP, most caseins are in the form of ‘sub-micelles’ of diameter between about 15 and 20nm.7 Hydrophobic bonding is most likely involved in their f ~ r m a t i o n . ~
Proposition 3 Mainly on account of the fact that nMCP and casein-(Ca + M g ) considerably reduce the charge on the sub-micelles, they aggregate into ‘casein micelles ’. Since the amount of nMCP grows with increasing pH,’ and since the nMCP-links between casein molecules have a relatively long lifetime ,2*6the above results suggest that the casein sub-micelles are not linked together by such ‘permanent’ nMCP bridges. As the casein molecules will carry on average a net charge of about -17 at pH 6.7, and as 17 Ca, 1 Mg, 5.6 P043-, and 2.4 P 0 4 2 - ions are on average associated per casein m o l e ~ u l e these , ~ ~ ~ions compensate for ca. 90% of the net charge of the casein molecules in the casein micelle. The above results2 suggest that this is the main cause for the aggregation of the sub-micelles.
References 1. H.J.M. van Dijk, Neth. Milk Dairy J . , 1991, 45, 241. 2. H.J.M. van Dijk, Neth. Milk Dairy J . , 1992, 46, 101. 3. K. Yamauchi, Y . Yoneda, Y. Koga, and T. Tsugo, Agrzc. Biol. Chem., 1969, 33, 907. 4. H.J.M. van Dijk, Neth. Milk Dairy J . , 1990, 44, 65. 5. T. Aoki, Y. Kako, and T. Imamura, J . Dairy Res., 1986, 53, 53. 6. H.J.M. van Dijk, Neth. Milk Dairy J . , 1990, 44, 111. 7. D . G. Schmidt, Neth Milk Dairy J . , 1980, 34, 42.
We have examined the partition of calcium (Ca), magnesium (Mg), and phosphate (P) between the casein micelles and various aqueous phases. After addition of > 5% NaC1, we have (Ca, + Mg,)/Pi, = 2.0, and, after addition of NaOH, notable changes in the partition were found to occur at = 2.0 (subscripts: c = colloidal, i = inorganic, o = orapproximately Pic/€’, ganic colloidal). We have also examined the disintegration of the casein micelles after addition of NaOH. Above pH 9, skim milk slowly becomes as transparent as whey. The speed of this process is increased if the protein amino groups have been succinylated or it is carried out at a higher pH, and is decreased due to addition of NaCl or amidation of carboxyl groups. Even low concentrations of added NaCl (i.e. 10-20 mM) strongly retard the disintegration but hardly influence the reaggregation. The following model is proposed for the casein micelles, and especially for the native (-like) micellar calcium phosphate (nMCP) .
Proposition 1 Most nMCP bridges in raw milk contain 2 phosphoserine residues (Po), 4 Pi, ions, and 8 divalent cations. As the value of (Ca, Mgc)/Pic can be lowered from -2.3 to only 2.0
+
by addition of NaC1, while roughly half of the Ca, can the dissolved Ca in the same period,3 it appears that nMCP may be 2.0. In addition, our results suggest that with nMCP) is also 2.0. Finally, it is most likely that cluster interconnects two P, groups. 2*4
be exchanged by (Ca + Mg)/PiC in Pic/(Po associated every nMCP ion
Proposition 2 Casein molecules of the same kind are bound together in pairs by a number of nMCP bridges (nMCP dimers). After addition of 6 M urea5 or NaOH (pHlo)* to milk, the casein molecules associated with the nMCP are in units of, say, 2-20 molecules. Present affiliation: Tastemaker, PO Box 414, 3770 AK Barneveld, The Netherlands
166
O n the Structure of Casein Micelles
All Po groups in these units are associated with the nMCP.236Since most of these Po groups are present in clusters (which are not all identical), and since below pH 6.7 in most sub-micelles it is predominantly the asl-casein which associates with the nMCP,* it is barely conceivable that the Po groups find each other at random. Steric hindrance will not allow this in most cases. Both before and after the formation of nMCP, most caseins are in the form of ‘sub-micelles’ of diameter between about 15 and 20nm.7 Hydrophobic bonding is most likely involved in their f ~ r m a t i o n . ~
Proposition 3 Mainly on account of the fact that nMCP and casein-(Ca + M g ) considerably reduce the charge on the sub-micelles, they aggregate into ‘casein micelles ’. Since the amount of nMCP grows with increasing pH,’ and since the nMCP-links between casein molecules have a relatively long lifetime ,2*6the above results suggest that the casein sub-micelles are not linked together by such ‘permanent’ nMCP bridges. As the casein molecules will carry on average a net charge of about -17 at pH 6.7, and as 17 Ca, 1 Mg, 5.6 P043-, and 2.4 P 0 4 2 - ions are on average associated per casein m o l e ~ u l e these , ~ ~ ~ions compensate for ca. 90% of the net charge of the casein molecules in the casein micelle. The above results2 suggest that this is the main cause for the aggregation of the sub-micelles.
References 1. H.J.M. van Dijk, Neth. Milk Dairy J . , 1991, 45, 241. 2. H.J.M. van Dijk, Neth. Milk Dairy J . , 1992, 46, 101. 3. K. Yamauchi, Y . Yoneda, Y. Koga, and T. Tsugo, Agrzc. Biol. Chem., 1969, 33, 907. 4. H.J.M. van Dijk, Neth. Milk Dairy J . , 1990, 44, 65. 5. T. Aoki, Y. Kako, and T. Imamura, J . Dairy Res., 1986, 53, 53. 6. H.J.M. van Dijk, Neth. Milk Dairy J . , 1990, 44, 111. 7. D . G. Schmidt, Neth Milk Dairy J . , 1980, 34, 42.