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Physico-chemical changes in casein micelles of buffaloand cow milks as a function of alkalinisation

Sarfraz Ahmad, Michel Piot, Florence Rousseau, Jean François Grongnet,Frédéric Gaucheron

To cite this version:Sarfraz Ahmad, Michel Piot, Florence Rousseau, Jean François Grongnet, Frédéric Gaucheron.Physico-chemical changes in casein micelles of buffalo and cow milks as a function of alkalinisation.Dairy Science & Technology, EDP sciences/Springer, 2009, 89 (3-4), pp.387-403. �hal-00895811�

Original article

Physico-chemical changes in casein micellesof buffalo and cow milks as a function

of alkalinisation

Sarfraz AHMAD1, Michel PIOT1, Florence ROUSSEAU

1, Jean François GRONGNET2,

Frédéric GAUCHERON1*

1 INRA, UMR1253 Science et Technologie du Lait et de l’Œuf, 65 rue de Saint Brieuc,35042 Rennes Cedex, France

2 AGROCAMPUS OUEST, 65 rue de Saint Brieuc, 35042 Rennes Cedex, France

Received 14 November 2008 – Accepted 16 April 2009

Abstract – By modifying the forces (hydrophobic and electrostatic interactions, hydrogen bondingand presence of micellar calcium phosphate) responsible for the structure and the stability of caseinmicelles, alkalinisation induces a disruption of casein micelles in milk. The objective of this work wasto compare the alkalinisation-induced physico-chemical changes of caseinmicelles of buffalo and cowmilks with a special attention to the mineral fraction. The whiteness and viscosity were determined asglobal characteristics of milk. The aqueous and micellar phases of milks were ascertained for thedistributions of the concentrations of nitrogen, casein molecules, calcium, inorganic phosphate andwater as their supramolecular and molecular characteristics. These parameters were measured at sixpH values between pH 6.7 and 10.8. Between pH 6.7 and 10.8, the whiteness decreased from 73.5 to50.9 and from 71.3 to 50.9 units and the viscosity increased from 1.8 to 10.2 and from 1.5 to 4.8 mPa·sfor buffalo and cow milks, respectively. Simultaneously, > 90% of nitrogen contents were in thesupernatants of ultracentrifugation at pH 9.7 and 8.6 for buffalo and cow milks, respectively.Chromatographic analyses showed that caseins were totally solubilised at these pH values. Calciumand inorganic phosphate concentrations progressively increased in the supernatants of ultracentri-fugation and decreased in the ultrafiltrates.At alkaline pH, the negative charge of caseins increased andthe inorganic phosphate ion changed its ionisation state from HPO4

2− to PO43− form. This form has a

greater affinity for calcium and can demineralise casein micelles. The consequences weremodifications of protein-protein and protein-minerals interactions resulting in micellar disruption.The dissociations took place at pH 9.7 and 8.6 for buffalo and cow milks, respectively. Thesedifferences were due to higher concentrations of casein and minerals in buffalo than in cow milk,which were also our criteria of selection of the former as a model.

casein micelle / buffalo milk / cow milk / mineral / alkalinisation

摘要 – 碱化作用对水牛乳和牛乳酪蛋白胶束物理化学性质的影响○ 分子间作用力 (疏水和静电作用、氢键和胶束磷酸钙的存在) 的改变会影响酪蛋白胶束的结构和稳定性，碱化作用使得乳中酪蛋白胶束破坏○ 本研究比较了碱化作用对水牛乳和牛乳物理化学性质和酪蛋白胶束中矿物元素的影响○ 以白度和黏度作为综合指标来评价乳的表观特性○ 并测定了乳水相和胶束相中氮、酪蛋白分子、钙、无机磷酸盐和水分的分布○ 这些参数的测定是在pH 6.7–10.8 (6 个 pH 间隔)○ 在 pH 6.7–10.8, 水牛乳的白度从 73.5 单位 降到 50.9 单位，牛乳的白度从 71.3 单位降到 50.9 单位，而水牛乳的黏度从 1.8 mPaÆs 增加 10.2 mPaÆs，牛

*Corresponding author (通讯作者): frederic.gaucheron@rennes.inra.fr

Dairy Sci. Technol. 89 (2009) 387–403© INRA, EDP Sciences, 2009DOI: 10.1051/dst/2009020

Available online at:www.dairy-journal.org

Article published by EDP Sciences

乳的黏度从 1.5 mPaÆs 增加到 4.8 mPaÆs○ 同时，水牛在乳 pH 9.7 和牛乳在 pH 8.6 时，高于90% 的氮存在于水相中○ 色谱分析表明在这些 pH 值下，酪蛋白完全是可溶的○ 在水相中钙和无机磷酸盐的浓度增加而在胶体相中含量降低○ 在碱性 pH 下，酪蛋白的负电荷增加，无机磷酸盐从 HPO4

2− 转变成 PO43−

○ PO43−与钙形成不溶性的磷酸钙，导致了酪蛋白胶束

中钙的溶解○ 结果是蛋白-蛋白和蛋白-矿物元素之间的作用发生改变，进而导致胶束的破坏○ 水牛乳在 pH 9.7 和牛乳在 pH 8.6 时，酪蛋白胶束完全解聚，这种解聚 pH 的不同是由于水牛乳酪蛋白和钙的浓度高于牛乳○

酪蛋白胶束 / 水牛 / 奶牛 / 矿物元素 / 碱化作用

Résumé – Modifications physico-chimiques des micelles de caséines du lait de vache et du laitde bufflesse en fonction de l’alcalinisation. En modifiant les forces qui interviennent dans lastructure et la stabilité des micelles de caséines (interactions hydrophobes et électrostatiques,liaisons hydrogène et présence de phosphate de calcium micellaire), l’alcalinisation du lait induitune dissociation des micelles de caséines. L’objectif de ce travail était de comparer les changementsphysico-chimiques et particulièrement la fraction minérale des micelles de caséines induits parl’alcalinisation de laits de bufflesse et de vache. La blancheur et la viscosité étaient déterminéescomme caractéristiques globales du lait. Les distributions entre phases aqueuses et micellaires desconcentrations en azote, molécules de caséines, calcium, phosphate inorganique et eau étaientdéterminées comme caractéristiques supramoléculaires et moléculaires. Ces paramètres étaientmesurés à six valeurs de pH compris entre pH 6,7 et 10,8. Entre pH 6,7 et 10,8, la blancheurdiminuait de 73,5 à 50,9 et de 71,3 à 50,9 unités alors que la viscosité augmentait de 1,8 à 10,2 et de1,5 à 4,8 mPaÆs pour les laits de bufflesse et de vache, respectivement. Dans le même temps, plus de90 % des concentrations en azote étaient dans les surnageants d’ultracentrifugation des laitsde bufflesse et de vache à pH 9,7 et 8,6, respectivement. Les analyses chromatographiques ontmontré que les caséines étaient totalement solubilisées à ces valeurs de pH. Les concentrations decalcium et de phosphate inorganique augmentaient progressivement dans les surnageants d’ultra-centrifugation et diminuaient dans les ultrafiltrates. À pH alcalin, la charge négative des caséinesaugmente et l’ion phosphate change son état d’ionisation en passant de la forme HPO4

2− à la formePO4

3−. Cette forme a une forte affinité pour le calcium et peut déminéraliser les micelles decaséines. Les conséquences étaient des modifications dans les interactions protéines-protéines etprotéines-minéraux conduisant à une dissociation micellaire. Les dissociations étaient respective-ment atteintes à pH 9,7 et 8,6 pour les laits de bufflesse et de vache. Ces différences quantitativesétaient dues aux plus fortes concentrations de caséines et de minéraux dans le lait de bufflesse parrapport au lait de vache, ce qui correspondait à nos critères de choix de ce lait comme modèle.

micelles de caséine / lait de bufflesse / lait de vache / minéraux / alcalinisation

1. INTRODUCTION

Casein micelles consist of 94% of case-ins (αS1, αS2, β- and κ-CN) and 6% of inor-ganic constituents corresponding to micellarcalcium phosphate. Hydrophobic and elec-trostatic interactions, hydrogen bondingand presence of calcium phosphate linkedto casein molecules are the forces responsi-ble for the structure and the stability ofcasein micelle. Cleavage of these forcesby pH decrease, addition of calcium chela-tants or urea, cooling, high pressure treat-

ment and alkalinisation induce disruptionof micelles into smaller units [24, 25].

Some studies show that alkalinisationleads to a reduction in the turbidity of milk[21, 23] to become transparent as whey atpH close to 9 [22, 23]. Other authors indicatethat milk alkalinisation induces a gelation ofcasein dispersion [14, 18]. Rose [19] foundan increase in casein concentration in theserumabove pH7.Vaia et al. [20] determineddecreases in concentrations of calcium (Ca)(particularly ionic Ca) and inorganicphosphate (Pi) in the aqueous phase with

388 S. Ahmad et al.

increasing pH. As per these authors, suchdecreases in ionic concentrations improvethe solvent quality and facilitate the dissocia-tion of casein micelles. According to thesame authors, this dissociation is due todiminished cohesive interaction betweenthe hydrophobic regions of caseins resultingin disrupted casein micelles. In addition tomineral changes in the aqueous phase,Huppertz et al. [6] andVaia et al. [20] explainthat the changes in the ionisation of proteinscan also contribute to this disruption.

The objective of this work was to studythe evolution of physico-chemical propertiesof casein micelles and to propose a mecha-nism of the disruption of casein micellesinducedby alkalinisationwith a special atten-tion paid to the mineral fraction. In this case,alkalinisation is used as a way to identify thefactors that are involved in the structure andstability of caseinmicelles.Thechangeswerecompared between buffalo and cow milks.Buffalo milk was selected because of itshigher concentration of casein micelles thancow milk. Moreover, the casein micelles inbuffalo milk are more mineralised and lesshydrated than those in cow milk [1].

2. MATERIALS AND METHODS

2.1. Samples preparation

Partially skimmed milks of both specieswere prepared by centrifugation at 2000× gfor 10 min at 20 °C. Thimerosal 0.3 g·L−1

(Sigma-Aldrich, St Louis, USA) was addedin both milks as a preservative againstmicroorganisms. The global compositionof the partially skimmed milks of both spe-cies was with 8.4 and 7.7 g·kg−1 of fat, 45.4and 32.4 g·kg−1 of total proteins and 55.0and 50.0 g·kg−1 of lactose for buffalo andcow milks, respectively.

Samples were alkalinised at approximatepH 7, 8, 9, 10 and 11 by the addition of1 mol·L−1 NaOH under vigorous stirringat 20 °C (in addition to the control sample

that was unmodified). The dilution causedby the NaOH addition was kept constant byadding an appropriate volume of Milli-Qwater. Before analysis, the samples were leftovernight at room temperature.

2.2. Preparation of supernatantsof ultracentrifugation,ultrafiltrates and micellar pellets

Control and alkalinised samples wereultracentrifuged at 100 000× g during 1 h(Sorvall,Discovery90SE,Hitachi,Newtown,USA) at 20 °C with a T-865 rotor. One partof the supernatant was kept for compo-sitional measurements and the rest wasultrafiltered at 20 °C with a membraneVivaspin 20 (Vivascience – Sartorius group,Goettingen, Germany), (molecular weightcut-off: 10 000 g·mol−1) at 1800× g for 2 hin a centrifuge SV 11 TH (Firlabo, Lyon,France). Ultrafiltrates were used for composi-tional measurements and physico-chemicalcharacterisations. Micellar pellets weredrained to measure micellar hydration.

2.3. Physico-chemical characteristicsof milks

2.3.1. Evolution of pH as a functionof NaOH added

The pH values were measured as a func-tion of NaOH added. The volumes of1 mol·L−1 NaOH used to get the samedesired pH in both milks were noted. Theexperimental error of pH measurementwas ± 0.02.

2.3.2. Whiteness

A microcolor tristimulus colorimeter(Minolta chromameter CR-300, Carrières-sur-Seine, France) was used for measure-ment. Calibration was performed using theMinolta calibration plate (standard tristimu-lus values: Y = 92.4; x = 0.3161; y =0.3325). In this system, L value defines

Physico-chemical changes in casein micelles 389

the position of the samples on the light-darkaxis [11]. The experimental error was ± 0.1arbitrary units.

2.3.3. Viscosity

The equipment was composed of a vis-cometer with co-axial cylinders (Low shear30, Contraves, Switzerland) whose double-walled external cylinder served as a thermo-stat [12]. At the bottom of the externalcylinder, a refractometer Sopelem (Paris,France) had been fixed. The gradient ofthe speed of viscometer was kept as1267 s−1 for all experiments. The experi-mental error was ± 0.05 mPa·s.

2.4. Physico-chemical characteristicsof casein micelles

2.4.1. Size distribution of particles

Size distribution of particles was deter-mined by injecting milk (500 μL) directlyinto the Mastersizer 2000 granulometer(Malvern Instruments Ltd., Worcestershire,UK) that contained Milli-Q water(~ 100 mL) under a 1500 rpm stirring at20 °C. Measurements were performedthrough diffraction of light with He-Ne laserhaving 633 nm wavelength. Refractive indi-ces were 1.333 and 1.570 for water andcasein micelles, respectively [3].

2.4.2. Micellar hydration

The casein pellets obtained by ultracentri-fugation were weighed and then dried at103 °C for 7 h. The difference in weightbefore and after drying, expressed as gH2O·g

−1 dry pellet, was taken as the micellarhydration. The experimental error was± 0.05 g.

2.4.3. Protein contents in supernatantsof ultracentrifugation

Samples to determine total nitrogen con-tent in the supernatants of ultracentrifuga-

tion were prepared according to the IDFprotocol [7] and were determined usingKjeldahl method [8]. Nitrogen content wasconverted into protein content using 6.38as conversion factor [10]. The experimentalerror was ± 0.1 g.

2.4.4. Chromatographic profiles ofproteins in milks andsupernatants ofultracentrifugation

The proteins present in milks and super-natants of ultracentrifugation were analysedby reverse-phase high-performance liquidchromatography (RP-HPLC). Before theanalysis, samples were reduced by10 mmol·L−1 dithiothreitol in the presenceof 8 mol·L−1 urea, 44 mmol·L−1 trisodiumcitrate and 100 mmol·L−1 Tris-HCl (pH 8)for 1 h at 37 °C, then diluted with 0.3%(v/v) trifluoroacetic acid (TFA) (adequatedilution to reach pH 2) and filtered through0.45 μm Acrodisc filter (Labo Standa,Caen, France). The amount of the sampleinjected into RP-HPLC was 100 μL. Sepa-rations were carried out on a 15 cm VydacC4 column 214 TP 54 (Touzart etMatignon, Les Ulis, France). The chromato-graphic conditions were as described byJaubert and Martin [9] with slight modifica-tions. Solvents were composed of 0.106%(v/v) TFA in water (solvent A) and 0.1%(v/v) TFA in 80% (v/v) acetonitrile(solvent B). For protein separation, elutionwas achieved using a linear gradient ofsolvent B from 37% to 56% for 44 min, from56% to 100% for the next 4 min and thenfrom 100% to 37% for the next 11 min (thetotal analysis time for each sample was59 min) at a flow rate of 1 mL·min−1. Thewavelength for the detection was 214 nm.The protein fractions considered wereκ-CN + αS2-CN, αS1-CN, β-CN andβ-lactoglobulin (β-Lg). Results were alsoexpressed by the ratio of HPLC area ofprotein fractions in the supernatants of

390 S. Ahmad et al.

ultracentrifugation of alkalinised milk toHPLC area of protein fractions in the controlmilk. The experimental error on the chro-matographic area was ± 5%.

2.4.5. Mineral analysis of milks,supernatants ofultracentrifugation andultrafiltrates

Ca and Na concentrations were mea-sured by atomic absorption spectrometry(Varian 220FS Spectr AA, Les Ulis, France)[13]. Pi concentration was determined usingion chromatography coupled with sup-pressed conductivity detection (DionexDX 500, Dionex, Voisin-le-Bretonneux,France) [2]. Ion concentrations determinedin the ultrafiltrates were converted into dif-fusible concentrations by multiplying witha correction factor 0.96 [17]. This correctiontook into account the volume effect of pro-teins present in milk but excluded in the ul-trafiltrates of milk due to retention onmembranes. The experimental error for allminerals was ± 1%.

3. RESULTS

3.1. Physico-chemical characteristicsof milk

3.1.1. Evolution of pH as a functionof NaOH added

In comparison with cow milk, buffalomilk required more alkali to attain the samepH level (Fig. 1A). For example, to attainpH 9.7, 6 and 5 mL of 1 mol·L−1 NaOHwere added in buffalo and cow milks,respectively.

3.1.2. Whiteness

At pH 6.7, buffalo milk was slightlywhiter than cow milk. Then, alkalinisation

induced a decrease in the whiteness of bothmilks (Fig. 1B). The decrease was slower inbuffalo milk than in cow milk, but finally atpH 10.8, the values were similar for bothmilks.

3.1.3. Viscosity

At pH 6.7, buffalo milk was slightlymore viscous than cow milk. Then, the vis-cosities increased for both milks during theiralkalinisation (Fig. 1C). At pH 8.6 and 10.8,viscosities of buffalo milk were two timeshigher than those of cow milk.

3.2. Physico-chemical characteristicsof casein micelles

3.2.1. Size distribution of particles

At pH 6.7, two populations with respectto the size of particles were determined forboth milks. The first (between 0.03 and0.40 nm) corresponded to casein micellesand the second (between 0.7 and 6 μm) tofat globules (Fig. 2). The presence of fatglobules was confirmed through observa-tions using an optical microscope (resultsnot shown).

At alkaline pH, we again observed twopopulations but the first one was signifi-cantly reduced, indicating the disappearanceof casein micelles and the volume percent-age was shifted towards the second popula-tion as the laser granulometry measures thesize of the major particles on the basis of100% total volume. This second populationcould always correspond to the fat globulesand also to the aggregates constituted ofcaseins associated with fat globules or case-ins themselves. Indeed, the presence ofaggregates of caseins was observed in acomplementary experiment that was per-formed with native phosphocaseinate (fat-free purified casein micelles) and weobserved a similar result, i.e., appearanceof population with a size of about 6 μm(results not shown).

Physico-chemical changes in casein micelles 391

3.2.2. Micellar hydration

At pH 6.7, the amounts of water associ-ated to the casein pellet that was obtained

by ultracentrifugation were 2.0 and 2.3 gH2O·g

−1 dry pellet for buffalo and cowmilks, respectively (Fig. 3). During alkalin-isation of both milks, the amount of

35

45

55

65

75

85

Whi

tene

ss (a

rbitr

ary

unit)

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2

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L)

0

2

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6 7 8 9 10 11

pH

Visc

osity

(mPa

. s)

A

B

C

Figure 1. pH evolution (A), whiteness (B) and viscosity (C) of buffalo (♦) and cow (■) milks as afunction of alkalinisation.

392 S. Ahmad et al.

ultracentrifuged pellet decreased from12.87% to 0.77% and from 9.10% to 0.55%for buffalo and cow milks, respectively. Thewater content associated to these pelletsincreased between pH 6.7 and 8.6. Then, thecurves were not plotted for pH 9.7 and 10.8because the amounts of pellet were consid-ered too low to measure the water contents.

3.2.3. Protein contents insupernatants ofultracentrifugation

At pH 6.7, the protein contents inthe supernatants of ultracentrifugation of

buffalo and cow milks were 11.6 and9.5 g·kg−1, respectively (Fig. 4). Theseconcentrations corresponded to whey pro-teins, non-sedimentable caseins, proteose-peptones and small molecules containingnitrogen (free amino acids, urea and pep-tides). During alkalinisation, an increasein protein contents in the supernatantswas determined. At pH 8.6, about 89%and 59% of the total proteins were presentin the supernatant of cow and buffalomilks, respectively. At pH 10.8 and 9.7,the totality of the proteins was non-ultra-centrifugable for buffalo and cow milks,respectively.

0

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Figure 2. Size distribution profiles of particles of buffalo (A) and cow (B) milks as a function ofalkalinisation.

Physico-chemical changes in casein micelles 393

3.2.4. RP-HPLC profiles of proteinsin milks and supernatantsof ultracentrifugation

RP-HPLCanalysesof bothmilks revealeddifferences regarding their chromatographicprofiles. Proteinswere grouped into four frac-tions according to their retention times asκ-CN + αS2-CN, αS1-CN, β-CN and β-Lg(Fig. 5A andB). For bothmilks, thefirst frac-

tion was considered as a group of κ-CN andαS2-CN. As shown by Zicarelli [26], thesetwo caseins were more concentrated in buf-falo milk than in cow milk. The second frac-tion (αS1-CN) showed two variants in buffalomilk and one in cowmilk. αS1-CN was moreconcentrated in buffalomilk than in cowmilk[16, 26]. The third fraction (β-CN) of cowmilk presented two variants correspondingto two well-resolved chromatographic peaks

0

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4

5

6

6pH

Mic

ella

r hyd

ratio

n (g

H2O

. g–1

dry

pelle

t)

7 8 9 10 11

Figure 3. Micellar hydration of ultracentrifuged pellets of buffalo (♦) and cow (■) milks as afunction of alkalinisation. The curves were not plotted for pH 9.7 and 10.8 because the amounts ofpellet were considered as too low.

05

101520253035404550

pH

Tota

l pro

tein

s (g

. kg–1

)

6 7 8 9 10 11

Figure 4. Total protein content (nitrogen × 6.38) in the supernatants of ultracentrifugation ofbuffalo (♦) and cow (■) milks as a function of alkalinisation.

394 S. Ahmad et al.

while β-CN of buffalo milk presented onlyone chromatographic peak with two molecu-lar masses (results not shown). Out of thesetwo masses, one was well identified by thedata bank while the other remained unidenti-fied. So we could assume that there might betwo variants of the said casein in buffalomilktoo but co-eluted. By considering thisassumption, our results agreed with the find-ings of Zicarelli [26] who found two variantsof β-CN in both milks. The β-CN andαS1-CN covered up 70% of the chromato-graphic area of proteins of both milks. Thefourth fraction (β-Lg) showed only one

variant in the buffalo milk in comparison toits counterpart cow milk that showed twovariants (A and B). This result was in agree-ment with those obtained by Pellegrino et al.[15] who showed that buffalo milk did notcontain β-Lg A that was present in cowmilk.The α-lactalbumin must be present but wasnot detected owing to its low concentrationin milk. On the other hand, the chromato-graphic conditions were not optimised todetect this specific whey protein.

In parallel, the same chromatographicanalyses were performed on the superna-tants of ultracentrifugation of control and

κ-CN & αs2-CNαs1-CNβ-CN

β-Lg

0.00.51.01.52.02.5

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0Time (min)

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β0.51.01.52.02.5

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D

10 20 30 40 50 60 0 10 20 30 40 50 60

Figure 5. RP-HPLC profiles of proteins in control buffalo (A) and cow (B) milks and in thedifferent supernatants of ultracentrifugation obtained from alkalinised milks (C, D: pH 6.7; E, F:pH 9.7 and G, H: pH 10.8 for buffalo and cow milks, respectively). The wavelength for thedetection was 214 nm. The quantity of protein injected for milks in A and B was different fromthose injected for supernatants from C to H.

Physico-chemical changes in casein micelles 395

alkalinised milks of both species. At pH 6.7,β-Lg was the main protein detected in bothmilks (Figs. 5C and 5D). At pH 9.7, themajority of casein molecules were in thesupernatants of ultracentrifugation of bothmilks (Figs. 5E and 5F). At pH 10.8, theRP-HPLC profiles of both milks were qual-itatively altered with a less good resolutionof the chromatographic separation(Figs. 5G and 5H), suggesting biochemicalmodifications induced by this high pHvalue.

Calculations of the ratios (HPLC areas ofeach protein fraction present in the superna-tant of alkali added milks to HPLC areas ofeach protein fraction present in the controlmilk) showed the increase in casein contentsin the supernatants during alkalinisation.For buffalo milk (Fig. 6A), concentrationsof the different casein fractions increasedgradually and constantly till pH 9.7. Forcow milk (Fig. 6B), maximum increasewas observed at pH 8.6. No major changein the β-Lg content was observed for bothmilks as a function of alkalinisation.

3.2.5. Mineral analysis of milks,supernatantsof ultracentrifugationand ultrafiltrates

At pH 6.7, the milks contained 65.2and 33.1 mmol·L−1 of total Ca for buffaloand cow milks, respectively (of which 7.3and 8.6 mmol·L−1 were found in the aque-ous phases of both milks, respectively).Concentrations of 32.7 and 20.8 mmol·L−1

of total Pi were found in buffalo and cowmilks, respectively (ofwhich 10.9 mmol·L−1

were found in the aqueous phases of bothmilks).

In both milks, alkalinisation induced anincrease in the concentrations of Ca and Piin the supernatants of ultracentrifugation(Figs. 7A and 7C) and a decrease in theultrafiltrates (Figs. 7B and 7D). At pH 10.8,the totality of Ca was nondiffusible whileabout 1 mmol·L−1 of Pi remained in the

ultrafiltrates of both milks. In parallel, theconcentration of Na increased in the super-natants of ultracentrifugation and in theultrafiltrates of both milks (Figs. 8A and 8B).

4. DISCUSSION

Alkalinisation-induced modificationswere very similar for both milks, with slightdifferences depending on the pH values.Buffalo milk showed the maximum modifi-cations at higher pH (about 9.7) than cowmilk (about 8.6). This delay in the phys-ico-chemical changes can be related to theconcentrations of proteins especially caseins(about 35 and 25 g·kg−1 in cow and buffalomilks, respectively) and minerals especiallyCa (65.2 and 33.1 mmol·L−1 in buffalo andcow milks, respectively) and Pi (32.7 and20.8 mmol·L−1 in buffalo and cow milks,respectively) [1]. The first part of this inter-pretation concerning the dependence of al-kalinisation process on protein contents issupported by the results of Vaia et al. [20]who indicated that the disruption of caseinmicelles induced by alkalinisation wasslower when the concentration of proteinsincreased.

The physico-chemical changes inducedby alkalinisation can be discussed preciselyin terms of modifications in minerals equi-libria and changes in the state of ionisationof proteins along with other factors.

4.1. Alkalinisation-induced changesin the minerals equilibria and inthe state of ionisation of proteins

4.1.1. Changes in the mineralequilibria

At pH 6.7, macro-elements are distrib-uted in aqueous and micellar phases ofmilk. K, Na and Cl ions are mainly in theaqueous phase while Ca (CaCit−, Ca2+ andCaHPO4), Pi (H2PO4

− and HPO42−), citrate

and Mg2+ are partly bound to the casein

396 S. Ahmad et al.

0

50

100

6.7 7 7.8 8.6 9.7pH

Rat

io (H

PLC

are

a of

sup

erna

tant

s of

alk

ali

adde

d m

ilks

by H

PLC

are

a of

con

trol

milk

)

0

50

100

Rat

io (H

PLC

are

a of

sup

erna

tant

s of

alk

ali

adde

d m

ilks

by H

PLC

are

a of

con

trol

milk

)A

B

Figure 6. Increase in different protein fractions in the supernatants of ultracentrifugation(100 000× g for 1 h at 20 °C) of buffalo (A) and cow (B) milks as a function of alkalinisation.Increase in the contents was calculated as the ratio between HPLC area of each protein fractionpresent in the supernatants of alkali added milks to HPLC area of each corresponding proteinfraction present in the control milk at pH 6.7. The RP-HPLC profiles are shown in Figure 5. Blackhistograms denote κ-CN + αs2-CN; dark grey histograms denote αs1-CN; light grey histogramsdenote β-CN and white histograms denote β-Lg. The areas of HPLC profiles obtained at pH 10.8were not considered as the chromatographic peaks were not well separated.

Physico-chemical changes in casein micelles 397

micelles and partly present in the aqueousphase. About two-third of the Ca (four-fifthin buffalo milk), half of the Pi (two-third inbuffalo milk), one-third of the Mg and 10%of the citrate are in the micellar phase. Inthis phase, Ca is present in two forms:micellar calcium phosphate and Ca boundto phosphoseryl residues. All these formshave been shown by the symbol d1 inFigure 9.

During alkalinisation, the distribution ofthese ions between aqueous and micellarphases was quantitatively modified (step d2

in Fig. 9), showing that the milk systemevolved to a thermodynamically morefavourable state. Globally, Ca and Pidecreased in the aqueous phase andincreased in the supernatants of ultracentri-fugation (Fig. 7). During alkalinisation, thestate of ionisation of Pi changed fromHPO4

2− towards PO43−. The PO4

3− formhas a greater affinity for Ca(2.88 × 106 M−1) as compared to HPO4

2−

(642 M−1) [25] and this triply ionised formof Pi can be considered as an effective Ca

sequestrant (step d3 in Fig. 9). From theresults of Van Dijk which indicated that themicellar calcium phosphate was notdegraded during alkalinisation [21], it canbe suggested that the Ca exclusively interact-ing with phosphoseryl residues of caseinmicelles was trapped by the PO4

3− to formcalcium phosphate salt. As the calcium phos-phate in the aqueous phase of the milk wassaturated and the amounts of Ca and Pi inthe aqueous phase decreased during alkalini-sation (Figs. 7B and 7D), a precipitation ofthis salt can be suggested. It is noteworthythat the concentrations of these ionsincreased in the supernatants of ultracentrifu-gation (Figs. 7A and 7C), suggesting that thenewly formed calcium phosphate had a lowweight as it remained nonsedimentable byultracentrifugation. The analyses of alkalin-isedmilks performed to observe the potentialpresence of insoluble calcium phosphate saltcontent using optical and scanning electronmicroscopes (results not shown) remainedunsuccessful, indicating that this calciumphosphate was small enough in size.

05

1015202530

6 7 8 9 10 11

pH pH

Con

cent

ratio

n (m

mol

. L–1

)

0

10

20

30

40

50

Con

cent

ratio

n (m

mol

. L–1

)

02468

1012

C

A

02468

1012

6 7 8 9 10 11

B

D

CaPi

Ca

Ca

Ca

Pi

Pi

Pi

Figure 7. Ca (♦) and Pi (▲) concentrations in the supernatants of ultracentrifugation (A and C) andin the ultrafiltrates (B and D) as a function of alkalinisation. A and B correspond to buffalo milk andC and D to cow milks.

398 S. Ahmad et al.

As regards the location of this calcium phos-phate, it may be directly associated with pro-teins that probably contain nucleation pointsor may become part of micellar calciumphosphate (step d4 in Fig. 9).

In the interpretation of the changes in theminerals equilibria, it is also necessary tointegrate the presence of additional Na dueto addition of NaOH (Fig. 8 and step d5 inFig. 9). The consequences of the additionof Na were probably similar to thosedescribed after the addition of NaCl in theliterature [4, 5, 13]. It has been shown thataddition of NaCl induced mainly exchangesbetween added Na and Ca bound to casein

micelles via the phosphoseryl residues. Asimilar exchange between Ca and Na mayoccur after the addition of NaOH. In thiscase, Ca present in the micellar calciumphosphate remained unaffected and thisinterpretation is in accordance with theresults obtained by other authors [4, 5, 21].

4.1.2. Changes in the state ofionisation of proteins

The state of ionisation of proteins that isalso dependent on the pH value can also bediscussed to interpret the modificationsinduced by alkalinisation. By taking into

0

10

20

30

40

50

60

7080

90

100

[Sod

ium

] (m

mol

. L–1)

0

10

20

30

40

50

60

70

6 7 8 9 10 11

pH

[Sod

ium

] (m

mol

. L–1

)

pH

A

B

Figure 8. Sodium concentrations in the supernatants of ultracentrifugation (A) and in theultrafiltrates (B) of buffalo (♦) and cow (▲) milks as a function of alkalinisation.

Physico-chemical changes in casein micelles 399

account the pK values of carboxylic groupsof the differently charged amino groups(Fig. 10), the glutamic acid, aspartic acidand C-terminal remain completely dissoci-ated because their pKa values are ionisedat pH 6.7 and remain in the same state ofionisation during alkalinisation. For this rea-son, they probably do not contribute to thechanges in the proteins. In the opposite, areduction in the ionisation of the amino-terminus of proteins, ε-amino group oflysine and imidazole group of histidinealong with an increase in ionisation of thephenol group of tyrosine and sulphydrylgroup of cystein is strongly probable. It isalso possible that the glycosidic parts ofκ-casein may become more ionised(Fig. 9). These ionisation changes cumula-tively resulted in an increase in the negativecharge of casein molecules. Indeed, weobserved between pH 6.7 and 10.8, a

significant and similar increase in the negativecharge of the caseins from−20 to−24 mV forboth milks. This change of charge was alsoreported and discussed by Vaia et al. [20].The consequences of these charge changescould have resulted in some modifications inthe protein-protein interactions in the caseinmicelles leading towards disruption.

4.2. Disruption of casein micelleswith solubilisation of caseinmolecules, formation of smallparticles and aggregatesand changes in water associatedwith caseins

At pH 6.7, the majority (90–95%) of thecasein molecules are combined to formcasein micelles. A variety of molecularforces are involved in the structure andthe stability of casein micelles depends

Addition of NaOH

Aqueous phase

Micellar phase

Ca2+CaCit- CaHPO4

HPO42- PO4

3-H2PO4-

H+ H+

Na+

OH-

Micellar calcium phosphate (Ca, Po-Pi)

Calcium bound to phosphoseryl residues (Ca-Po)

+ H2O

2 Na+ replaces 1 Ca2+

which form a bridge

Calcium phosphate (Ca & Pi) associated with proteins

++

+

Addition of NaOH

Aqueous phase

Micellar phase

Ca2+

HPO42- PO4

3-H2PO4-

H+ H+

Na+

OH-

Micellar calcium phosphate (Ca, Po-Pi)

Calcium bound to phosphoseryl residues (Ca-Po)

+ H2O

2 Na+ replaces 1 Ca2+

which form a bridge

Calcium phosphate (Ca & Pi) associated with proteins

++

+

Figure 9. Proposed mechanism explaining the changes in the mineral equilibria duringalkalinisation. d1 : Ion distribution at pH 6.7. d2 : Modification of Pi ionisation during alkalinisation.d3 : Chelation of different calcium forms by triply ionised Pi. d4 : Formation of new calciumphosphate salts. d5 : Displacement of calcium by sodium (from NaOH).

400 S. Ahmad et al.

on a balance between electrostatic repul-sions and hydrophobic interactions. Inaddition, micellar calcium phosphate cross-links the casein molecules and neutralisesnegatively-charged phosphoseryl groups,allowing the formation of hydrophobicinteractions between caseins. In milk, themicellar calcium phosphate is in equilibriumwith the aqueous phase that is supersatu-rated with respect to calcium phosphatesalts [24, 25].

During alkalinisation, the minerals equi-libria and state of ionisation of proteins weremodified as previously discussed andinduced changes in the molecular organisa-tion of casein micelles. The decrease inwhiteness (Fig. 1B) (which is related to par-ticle concentration and structural organisa-tion such as size and contents in proteinand minerals) and the increase in viscosity(Fig. 1C) corresponded to a disruption of

casein micelles. Increase of nitrogen con-tents in the supernatant of ultracentrifuga-tion (Fig. 4) confirmed this disruption andidentification of caseins in these superna-tants by RP-HPLC showed that all caseinmolecules were solubilised without prefer-ence (Figs. 5 and 6). As a consequence ofthe demineralisation and disruption ofcasein micelles, the released casein mole-cules resulted in small particles and self-associated large aggregates (Fig. 2 andresults on fat-free native phosphocaseinatenot shown).

The modifications in the structural orga-nisation of casein molecules inducedchanges in the interactions between waterand casein molecules and might explainthe increase in viscosity determined in thisstudy (Fig. 1C) and were also describedby Plomley et al. [18]. These findings werealso in agreement with the increase in water

At pH ~6.7

Addition of NaOHNa+

OH-+

H2O

O H

N-terminal pK=8.0

N

+N

II III IH

H

C-O-IIO

C-O-IIO

asppK=3.6

glu pK=4.2

tyrpK=10.1

cyspK=8.2

hispK=6.0

lys pK=10.5

argpK=12.5 C-O-

IIO

H+N

H

H

H+N

HH

H+N

HH S H

C-terminal pK=2.2

At pH ~10.8O-

H

N-terminal

N

NII III I

C-O-IIO

C-O-IIO

asp

glu

tyr

cys

his

lys

argpK=12.5 C-O-

IIO

NH

H

H+N

HH

NH

HS-

C-terminal

SugarsOH

OHOH

OHOH

OH

SugarsO-

O-O-

O-

O-

O-

At pH ~6.7

Addition of NaOHNa+

OH-+

H2O

Addition of NaOHNa+

OH-+

Addition of NaOHNa+

OH-+

H2O

O H

N-terminal pK=8.0

N

+N

II III IH

HN

+N

II III IH

H

C-O-IIO

C-O-IIO

asppK=3.6

glu pK=4.2

tyrpK=10.1

cyspK=8.2

hispK=6.0

lys pK=10.5

argpK=12.5 C-O-

IIO

H+N

H

H

H+N

HH

H+N

HH S H

C-O-IIOC-O-IIO

C-O-IIOC-O-IIO

asppK=3.6

glu pK=4.2

tyrpK=10.1

cyspK=8.2

lys pK=10.5

argpK=12.5 C-O-

IIOC-O-IIO

H+N

H

H

H+N

H

H

H+N

HH

H+N

HH

H+N

HH

H+N

HH S HS H

C-terminal pK=2.2

At pH ~10.8O-

H

N-terminal

N

NII III I

C-O-IIO

C-O-IIO

asp

glu

tyr

cys

his

lys

argpK=12.5 C-O-

IIO

NH

H

H+N

HH

NH

HS-

C-terminal

O-H

N-terminal

N

NII III I

C-O-IIOC-O-IIO

C-O-IIOC-O-IIO

asp

glu

tyr

cys

his

lys

argpK=12.5 C-O-

IIOC-O-IIO

NH

HNH

H

H+N

HH

H+N

HH

NH

HS-

C-terminal

SugarsOH

OHOH

OHOH

OH

SugarsOH

OHOH

OHOH

OH

SugarsOH

OHOH

OHOH

OH

SugarsO-

O-O-

O-

O-

O-

SugarsO-

O-O-

O-

O-

O-

SugarsO-

O-O-

O-

O-

O-

Figure 10. Proposed mechanism explaining the changes in the ionisation of proteins between pH 6.7and 10.8. pK values are reported by Vaia et al. [20].

Physico-chemical changes in casein micelles 401

content associated with the pellets observedin Figure 3.

5. CONCLUSION

This study demonstrated that alkalinisa-tion of both milks induced changes in min-erals, caseins and water distributionsbetween aqueous and micellar phases.These changes were qualitatively similarbut some quantitative differences wereobserved in both milks. The richness of buf-falo milk as compared to that of cow milk incaseins (about 35 and 25 g·kg−1 in buffaloand cow milks, respectively), Ca (65.2 and33.1 mmol·L−1 in buffalo and cowmilks, respectively) and Pi (32.7 and20.8 mmol·L−1 in buffalo and cow milks,respectively) explained why this milkrequired more alkaline solution to increasethe pH. It also explained why buffalo milkshowed a delay in modifications as com-pared to its counterpart cow milk: majorand maximum modifications occurred atpH 9.7 and 8.6 in buffalo and cow milks,respectively. These modifications wererelated to changes in the minerals equilibriaand in the state of ionisation of proteins.During alkalinisation, the ionisation stateof Pi in the aqueous phase changed by los-ing one or two protons to be in the form ofPO4

3−. The ability of PO43− to chelate Ca

was more pronounced than the H2PO4−

and HPO42− forms. In parallel, ionisation

of proteins changed during alkalinisationand their negative charge increased. As aresult of this chelation of Ca linked to phos-phoseryl residues and changes in the pro-tein-protein interactions in the caseinmicelles, solubilisation of casein moleculestook place and a disruption of caseinmicelles was observed.

Acknowledgements: We thank J. Guilleminfrom G.I.E. Chataigneraie (Coopérative deBufflonnes, Zone Artisanale, Maurs, France) forfacilitating us with buffalo milk and Higher

Education Commission of Pakistan and INRAof France for providing funds to do this research.

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Physico-chemical changes in casein micelles 403