Skip to main content
SpringerLink
Log in

A quantitative model of the bovine casein micelle: ion equilibria and calcium phosphate sequestration by individual caseins in bovine milk

  • Original Article
  • Published:
European Biophysics Journal Aims and scope Submit manuscript

Abstract

The white appearance of skim milk is due to strong light scattering by colloidal particles called casein micelles. Bovine casein micelles comprise expressed proteins from four casein genes together with significant fractions of the total calcium, inorganic phosphate, magnesium and citrate ions in the milk. Thus, the milk salts are partitioned between the casein micelles, where they are mostly in the form of nanoclusters of an amorphous calcium phosphate sequestered by caseins through their phosphorylated residues, with the remainder in the continuous phase. Previously, a salt partition calculation was made assuming that the nanoclusters are sequestered only by short, highly phosphorylated casein sequences, sometimes called phosphate centres. Three of the four caseins have a proportion of their phosphorylated residues in either one or two phosphate centres and these were proposed to react with the nanoclusters equally and independently. An improved model of the partition of caseins and salts in milk is described in which all the phosphorylated residues in competent caseins act together to bind to and sequester the nanoclusters. The new model has been applied to results from a recent study of variation in salt and casein composition in the milk of individual cows. Compared to the previous model, it provides better agreement with experiment of the partition of caseins between free and bound states and equally good results for the partition of milk salts. In addition, new calculations are presented for the charge on individual caseins in their bound and free states.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • 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

    Article  CAS  Google Scholar 

  • Aoki T, Yamada N, Tomita I, Kako Y, Imamura T (1987) Caseins are cross-linked through their ester phosphate groups by colloidal calcium-phosphate. Biochem Biophys Acta 911:238–243

    CAS  PubMed  Google Scholar 

  • Aoki T, Yamada N, Kako Y (1990) Relation between the colloidal calcium-phosphate cross-linkage and release of beta casein from bovine casein micelles on cooling. Agric Biol Chem 54:2287–2292

    CAS  Google Scholar 

  • Aoki T, Sakamoto H, Kako Y (1991) Cross-linking of caseins by colloidal calcium phosphate in the presence of urea. Int Dairy J 1:67–75

    Article  CAS  Google Scholar 

  • Aoki T, Umeda T, Kako Y (1992) The least number of phosphate groups for cross-linking of casein by colloidal calcium phosphate. J Dairy Sci 75:971–975

    Article  CAS  PubMed  Google Scholar 

  • Baumy JJ, Guenot P, Sinbandhit S, Brulé G (1989) Study of calcium binding to phosphoserine residues of β-casein and its phosphopeptide (1–25) by 31P NMR. J Dairy Res 56:403–409

    Article  Google Scholar 

  • Belton PS, Lyster RLJ, Richards CP (1985) The P-31 nuclear magnetic resonance spectrum of cows milk. J Dairy Res 52:47–54

    Article  CAS  PubMed  Google Scholar 

  • Bhattacharyya J, Das KP (1999) Molecular chaperone-like properties of an unfolded protein, alpha(s)-casein. J Biol Chem 274:15505–15509

    Article  CAS  PubMed  Google Scholar 

  • Bijl E, van Valenberg HJF, Huppertz T, van Hooijdonk ACM (2013) Protein, casein, and micellar salts in milk: current content and historical perspectives. J Dairy Sci 96:5455–5464

    Article  CAS  PubMed  Google Scholar 

  • Bijl E, de Vries R, van Valenberg H, Huppertz T, Van Hooijdonk T (2014) Factors influencing casein micelle size in milk of individual cows: genetic variants and glycosylation of kappa-casein. Int Dairy J 34:135–141

    Article  CAS  Google Scholar 

  • Borgia A, Borgia MB, Bugge K, Kissling VM, Heidarsson PO, Fernandes CB, Sottini A, Soranno A, Buholzer KJ, Nettels D, Kragelund BB, Best RB, Schuler B (2018) Extreme disorder in an ultrahigh-affinity protein complex. Nature 555:61

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Clegg RA, Holt C (2009) An E. coli over-expression system for multiply-phosphorylated proteins and its use in a study of calcium phosphate sequestration by novel recombinant phosphopeptides. Protein Expr Purif 67:23–34

    Article  CAS  PubMed  Google Scholar 

  • Creamer LK, Berry GP, Mills OE (1977) A study of the dissociation of beta -casein from the bovine casein micelle at low temperature. N Z J Dairy Sci Technol 12:58–66

    CAS  Google Scholar 

  • Cross KJ, Huq NL, Reynolds EC (2016) Casein phosphopeptide-amorphous calcium phosphate nanocomplexes: a structural model. Biochemistry 55:4316–4325

    Article  CAS  PubMed  Google Scholar 

  • Dalgleish DG (2011) On the structural models of bovine casein micelles-review and possible improvements. Soft Matter 7:2265–2272

    Article  CAS  Google Scholar 

  • Dalgleish DG, Parker TG (1980) Binding of calcium ions to bovine alpha-S1-casein and precipitability of the protein-calcium ion complexes. J Dairy Res 47:113–122

    Article  CAS  Google Scholar 

  • Davies DT, Law AJR (1977) Improved method for quantitative fractionation of casein mixtures using ion-exchange chromatography. J Dairy Res 44:213–221

    Article  CAS  Google Scholar 

  • Davies DT, Law AJR (1980) Content and composition of protein in creamery milks in southwest scotland. J Dairy Res 47:83–90

    Article  CAS  Google Scholar 

  • Davies DT, Law AJR (1983) Variation in the protein-composition of bovine casein micelles and serum casein in relation to micellar size and milk temperature. J Dairy Res 50:67–75

    Article  CAS  Google Scholar 

  • Davies DT, White JCD (1960) The use of ultrafiltration and dialysis in isolating the aqueous phase of milk and in determining the partition of milk constituents between the aqueous and disperse phases. J Dairy Res 27:171–190

    Article  CAS  Google Scholar 

  • de Kruif CG (2014) The structure of casein micelles: a review of small-angle scattering data. J Appl Crystallogr 47:1479–1489

    Article  CAS  Google Scholar 

  • de Kruif CG, Holt C (2003) Casein micelle structure, functions and interactions. In: Fox PF, McSweeney PLH (eds) Advanced dairy chemistry, vol 1A. Proteins. Kluwer Academic/Plenum, New York, pp 675–698

    Google Scholar 

  • De Sa Peixoto P, Silva JVC, Laurent GP, Schmutz M, Thomas D, Bouchoux A, Gesan-Guiziou G (2017) How high concentrations of proteins stabilize the amorphous state of calcium orthophosphate: a solid-state NMR study of the casein case. Langmuir

  • Fang ZH, Bovenhuis H, Delacroix-Buchet A, Miranda G, Boichard D, Visker MHPW, Martin P (2017) Genetic and nongenetic factors contributing to differences in alpha-S-casein phosphorylation isoforms and other major milk proteins. J Dairy Sci 100(7):5564–5577

    Article  CAS  PubMed  Google Scholar 

  • Farrell H Jr, Cooke P, Wickham E, Piotrowski E, Hoagland P (2003) Environmental influences on bovine κ-casein: reduction and conversion to fibrillar (amyloid) structures. J Protein Chem 22:259–273

    Article  CAS  PubMed  Google Scholar 

  • Froehlich JW, Chu CS, Tang N, Waddell K, Grimm R, Lebrilla CB (2011) Label-free liquid chromatography-tandem mass spectrometry analysis with automated phosphopeptide enrichment reveals dynamic human milk protein phosphorylation during lactation. Anal Biochem 408:136–146

    Article  CAS  PubMed  Google Scholar 

  • Froloff N, Windemuth A, Honig B (1997) On the calculation of binding free energies using continuum methods: application to MHC class I protein-peptide interactions. Protein Sci 6:1293–1301

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gagnaire V, Pierre A, Molle D, Léonil J (1996) Phosphopeptides interacting with colloidal calcium phosphate isolated by tryptic hydrolysis of bovine casein micelles. J Dairy Res 63:405–422

    Article  CAS  PubMed  Google Scholar 

  • Gebauer D, Coelfen H (2011) Prenucleation clusters and non-classical nucleation. Nano Today 6:564–584

    Article  CAS  Google Scholar 

  • Gonzalez-Jordan A, Thomar P, Nicolai T, Dittmer J (2015) The effect of pH on the structure and phosphate mobility of casein micelles in aqueous solution. Food Hydrocolloids 51:88–94

    Article  CAS  Google Scholar 

  • Gower LB (2008) Biomimetic model systems for investigating the amorphous precursor pathway and its role in biomineralization. Chem Rev 108:4551–4627

    Article  CAS  PubMed  Google Scholar 

  • Heck JML, Schennink A, van Valenberg HJF, Bovenhuis H, Visker M, van Arendonk JAM, van Hooijdonk ACM (2009) Effects of milk protein variants on the protein composition of bovine milk. J Dairy Sci 92:1192–1202

    Article  CAS  PubMed  Google Scholar 

  • Hill AV (1910) Proceedings of the physiological society: January 22, 1910. J Physiol 40:i–vii

  • Holt C (1985) The milk salts: their secretion, concentrations and physical chemistry. In: Fox PF (ed) Developments in dairy chemistry: lactose and minor constituents, vol 3. Elsevier, London, pp 143–181

    Chapter  Google Scholar 

  • Holt C (1997) The milk salts and their interaction with casein. In: Fox PF (ed) Advanced dairy chemistry, vol 3. Lactose salts and vitamins. Water, Chapman and Hall, London, pp 233–254

    Google Scholar 

  • Holt C (2004) An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein micelles and its application to the calculation of the partition of salts in milk. Eur Biophys J Biophys Lett 33:421–434

    Article  CAS  Google Scholar 

  • Holt C (2013) Unfolded phosphopolypeptides enable soft and hard tissues to coexist in the same organism with relative ease. Curr Opin Struct Biol 23:420–425

    Article  CAS  PubMed  Google Scholar 

  • Holt C, Dalgleish DG, Jenness R (1981) Inorganic constituents of milk.2. Calculation of the ion equilibria in milk diffusate and comparison with experiment. Anal Biochem 113:154–163

    Article  CAS  PubMed  Google Scholar 

  • Holt C, Davies DT, Law AJR (1986) Effects of colloidal calcium phosphate content and free calcium ion concentration in the milk serum on the dissociation of bovine casein micelles. J Dairy Res 53:557–572

    Article  CAS  Google Scholar 

  • Holt C, Wahlgren NM, Drakenberg T (1996) Ability of a beta-casein phosphopeptide to modulate the precipitation of calcium phosphate by forming amorphous dicalcium phosphate nanoclusters. Biochem J 314:1035–1039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Holt C, Timmins PA, Errington N, Leaver J (1998) A core-shell model of calcium phosphate nanoclusters stabilized by beta-casein phosphopeptides, derived from sedimentation equilibrium and small-angle X-ray and neutron-scattering measurements. Eur J Biochem 252:73–78

    Article  CAS  PubMed  Google Scholar 

  • Holt C, de Kruif CG, Tuinier R, Timmins PA (2003) Substructure of bovine casein micelles by small-angle X-ray and neutron scattering. Colloids Surf A 213:275–284

    Article  CAS  Google Scholar 

  • Holt C, Sorensen ES, Clegg RA (2009) Role of calcium phosphate nanoclusters in the control of calcification. FEBS J 276:2308–2323

    Article  CAS  PubMed  Google Scholar 

  • Holt C, Lenton S, Nylander T, Sorensen ES, Teixeira SCM (2014) Mineralisation of soft and hard tissues and the stability of biofluids. J Struct Biol 185:383–396

    Article  CAS  PubMed  Google Scholar 

  • Horne DS (1998) Casein interactions: casting light on the black boxes, the structure in dairy products. Int Dairy J 8:171–177

    Article  CAS  Google Scholar 

  • Huppertz T, Gazi I, Luyten H, Nieuwenhuijse H, Alting A, Schokker E (2017) Hydration of casein micelles and caseinates: implications for casein micelle structure. Int Dairy J 74:1–11

    Article  CAS  Google Scholar 

  • Ibsen CJS, Gebauer D, Birkedal H (2016) Osteopontin stabilizes metastable states prior to nucleation during apatite formation. Chem Mater 28(23):8550–8555

    Article  CAS  Google Scholar 

  • Ingham B, Erlangga GD, Smialowska A, Kirby NM, Wang C, Matia-Merino L, Haverkamp RG, Carr AJ (2015) Solving the mystery of the internal structure of casein micelles. Soft Matter 11:2723–2725

    Article  CAS  PubMed  Google Scholar 

  • Jahnen-Dechent W, Heiss A, Schaefer C, Ketteler M (2011) Fetuin-A regulation of calcified matrix metabolism. Circ Res 108:1494–1509

    Article  CAS  PubMed  Google Scholar 

  • Ketto IA, Knutsen TM, Øyaas J, Heringstad B, Ådnøy T, Devold TG, Skeie SB (2017) Effects of milk protein polymorphism and composition, casein micelle size and salt distribution on the milk coagulation properties in Norwegian Red cattle. Int Dairy J 70:55–64

    Article  CAS  Google Scholar 

  • Lam E, Holt C, Edwards P, McKinnon I, Otter D, Li N, Hemar Y (2017) The effect of transglutaminase treatment on the physico-chemical properties of skim milk with added ethylenediaminetetraacetic acid. Food Hydrocolloids 69:329–340

    Article  CAS  Google Scholar 

  • Lenton S, Nylander T, Teixeira SCM, Holt C (2015a) A review of the biology of calcium phosphate sequestration with special reference to milk. Dairy Sci Technol 95:3–14

    Article  CAS  PubMed  Google Scholar 

  • Lenton S, Seydel T, Nylander T, Holt C, Hartlein M, Teixeira S, Zaccai G (2015b) Dynamic footprint of sequestration in the molecular fluctuations of osteopontin. J R Soc Interface 12(110):20150506

    Article  PubMed  PubMed Central  Google Scholar 

  • Lenton S, Nylander T, Holt C, Sawyer L, Härtlein M, Müller H, Teixeira SCM (2016) Structural studies of hydrated samples of amorphous calcium phosphate and phosphoprotein nanoclusters. Eur Biophys J Biophys Lett 45:405–412

    Article  CAS  Google Scholar 

  • Liao Y, Weber D, Xu W, Durbin-Johnson BP, Phinney BS, Lonnerdal B (2017) Absolute quantification of human milk caseins and the whey/casein ratio during the first year of lactation. J Proteome Res 16:4113–4121

    Article  CAS  PubMed  Google Scholar 

  • Little EM, Holt C (2004) An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein phosphopeptides. Eur Biophys J Biophys Lett 33:435–447

    Article  CAS  Google Scholar 

  • Liu DZ, Weeks MG, Dunstan DE, Martin GJO (2013) Temperature-dependent dynamics of bovine casein micelles in the range 10–40 degrees C. Food Chem 141:4081–4086

    Article  CAS  PubMed  Google Scholar 

  • Lyster RLJ (1981) Calculation by computer of individual concentrations in a simulated milk salt solution.2. An extension to the previous model. J Dairy Res 48:85–89

    Article  CAS  Google Scholar 

  • Marchin S, Putaux J-L, Pignon F, Léonil J (2007) Effects of the environmental factors on the casein micelle structure studied by cryo transmission electron microscopy and small-angle X-ray scattering/ultrasmall-angle X-ray scattering. J Chem Phys 126:045101

    Article  CAS  PubMed  Google Scholar 

  • Mazzali M, Kipari T, Ophascharoensuk V, Wesson JA, Johnson R, Hughes J (2002) Osteopontin—a molecule for all seasons. QJM 95:3–13

    Article  CAS  PubMed  Google Scholar 

  • McMahon DJ, McManus WR (1998) Rethinking casein micelle structure using electron microscopy. J Dairy Sci 81:2985–2993

    Article  CAS  Google Scholar 

  • McMahon DJ, Oommen BS (2012) Casein micelle structure, functions and interactions. In: Fox PF, McSweeney PLH (eds) Advanced dairy chemistry, vol 1A. Proteins: basic aspects. Springer, New York, pp 185–210

    Google Scholar 

  • Mekmene O, Gaucheron F (2011) Determination of calcium-binding constants of caseins, phosphoserine, citrate and pyrophosphate: a modelling approach using free calcium measurement. Food Chem 127:676–682

    Article  CAS  PubMed  Google Scholar 

  • Mekmene O, Le Graet Y, Gaucheron F (2009) A model for predicting salt equilibria in milk and mineral-enriched milks. Food Chem 116:233–239

    Article  CAS  Google Scholar 

  • Ono T, Murayama T, Kaketa S, Odagiri S (1990) Changes in the protein-composition and size distribution of bovine casein micelles induced by cooling. Agric Biol Chem 54:1385–1392

    CAS  Google Scholar 

  • Ono T, Ohotawa T, Takagi Y (1994) Complexes of casein phosphopetides and calcium phosphate prepared from casein micelles by tryptic digestion. Biosci Biotechnol Biochem 58:1376–1380

    Article  CAS  Google Scholar 

  • Parker TG, Dalgleish DG (1981) Binding of calcium ions to bovine beta-casein. J Dairy Res 48:71–76

    Article  CAS  PubMed  Google Scholar 

  • Poth AG, Deeth HC, Alewood PF, Holland JW (2008) Analysis of the human casein phosphoproteome by 2-D electrophoresis and MALDI-TOF/TOF MS reveals new phosphoforms. J Proteome Res 7:5017–5027

    Article  CAS  PubMed  Google Scholar 

  • Pouget EM, Bomans PHH, Goos JACM, Frederik PM, de With G, Sommerdijk NAJM (2009) The initial stages of template-controlled CaCO3 formation revealed by cryo-TEM. Science 323:1455–1458

    Article  CAS  PubMed  Google Scholar 

  • Rollema HS (1992) Casein association and micelle formation. In: Fox PF (ed) Advanced dairy chemistry, vol 1. Elsevier Science Publishers, Barking, pp 111–140

    Google Scholar 

  • Schmidt DG (1982) Association of caseins and casein micelle structure. In: Fox PF (ed) Developments in dairy chemistry, vol 1. Elsevier, London, pp 61–86

    Google Scholar 

  • Sleigh RW, Mackinlay AG, Pope JM (1983) NMR-studies of the phosphoserine regions of bovine alpha-S1-casein and beta-casein—assignment of P-31 resonances to specific phosphoserines and cation binding studied by measurement of enhancement of H-1 relaxation rate. Biochem Biophys Acta 742:175–183

    CAS  PubMed  Google Scholar 

  • Thomsen JK, Jakobsen HJ, Nielsen NC, Petersen TE, Rasmussen LK (1995) Solid-state magic-angle spinning 31P-NMR studies of native casein micelles. Eur J Biochem 230:454–459

    Article  CAS  PubMed  Google Scholar 

  • Thorn DC, Meehan S, Sunde M, Rekas A, Gras SL, MacPhee CE, Dobson CM, Wilson MR, Carver JA (2005) Amyloid fibril formation by bovine milk kappa-casein and its inhibition by the molecular chaperones alpha(s-) and beta-casein. Biochemistry 44:17027–17036

    Article  CAS  PubMed  Google Scholar 

  • Thorn DC, Ecroyd H, Sunde M, Poon S, Carver JA (2008) Amyloid fibril formation by bovine milk alpha(s2)-casein occurs under physiological conditions yet is prevented by its natural counterpart, alpha(s1)-casein. Biochemistry 47:3926–3936

    Article  CAS  PubMed  Google Scholar 

  • Thorn DC, Ecroyd H, Carver JA, Holt C (2015) Casein structures in the context of unfolded proteins. Int Dairy J 46:2–11

    Article  CAS  Google Scholar 

  • Treweek TM, Thorn DC, Price WE, Carver JA (2011) The chaperone action of bovine milk alpha(S1)- and alpha(S2)-caseins and their associated form alpha(S)-casein. Arch Biochem Biophys 510:42–52

    Article  CAS  PubMed  Google Scholar 

  • Vekilov PG (2010) Nucleation. Cryst Growth Des 10:5007–5019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wahlgren NM, Dejmek P, Drakenberg T (1993) Binding of Mg2+ and Ca2+ to beta casein A(1)- a multinuclear magnetic resonance study. J Dairy Res 60:65–78

    Article  CAS  Google Scholar 

  • Waugh DF (1971) Formation and structure of casein micelles. In: McKenzie HA (ed) Milk proteins chemistry and biology, vol II. Academic Press, New york and London, pp 3–85

    Chapter  Google Scholar 

  • White JCD, Davies DT (1958) The relation between the chemical composition of milk and the stability of the caseinate complex.1. General introduction, description of samples, methods and chemical composition of samples. J Dairy Res 25:236–255

    Article  CAS  Google Scholar 

  • White JCD, Davies DT (1963) The determination of citric acid in milk and milk sera. J Dairy Res 30:171–189

    Article  CAS  Google Scholar 

  • Yong YH, Foegeding EA (2010) Caseins: utilizing molecular chaperone properties to control protein aggregation in foods. J Agric Food Chem 58:685–693

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Liu D, Liu X, Hang F, Zhou P, Zhao J, Zhang H, Chen W (2018) Effect of temperature on casein micelle composition and gelation of bovine milk. Int Dairy J 78:20–27

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Dairy Science and Technology Group, Wageningen University, Bornse Weilanden 9, Wageningen, 6708 WG, The Netherlands

    Etske Bijl & Hein van Valenberg

  2. FrieslandCampina, Stationsplein 4, Amersfoort, 3818 LE, The Netherlands

    Thom Huppertz

  3. Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow, G12 8QQ, UK

    Carl Holt

Authors
  1. Etske Bijl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thom Huppertz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hein van Valenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Carl Holt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carl Holt.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 887 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bijl, E., Huppertz, T., van Valenberg, H. et al. A quantitative model of the bovine casein micelle: ion equilibria and calcium phosphate sequestration by individual caseins in bovine milk. Eur Biophys J 48, 45–59 (2019). https://doi.org/10.1007/s00249-018-1330-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00249-018-1330-2

Keywords

Search

Navigation