HAL Id: hal-00929619https://hal.archives-ouvertes.fr/hal-00929619

Submitted on 1 Jan 1998

HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.

Casein micelle size in relation with casein compositionand αs1, αs2, β and casein contents in goat milkAlice Pierre, Françoise Michel, Yvon Le Graët, Laurence Zahoute

To cite this version:Alice Pierre, Françoise Michel, Yvon Le Graët, Laurence Zahoute. Casein micelle size in relation withcasein composition and αs1, αs2, β and casein contents in goat milk. Le Lait, INRA Editions, 1998,78 (6), pp.591-605. <hal-00929619>

Lait (1998) 78, 591-605© InralElsevier, Paris

Original article

Casein micelle size in relation with casein compositionand asl' as2' ~ and K casein contents in goat milk

Alice Pierre*, Françoise Michel, Yvon Le Graët, Laurence Zahoute

Laboratoire de recherches de technologie laitière, Inra, 65, rue de Saint-Brieuc,35042 Rennes cedex, France

(Received 4 July 1997; accepted 22 July 1998)

Abstract - The relationship between micelle size and casein micelle composition was studied on21 individual goat milks from animais homozygous for as1casein variants A, B2, C, E, F and O. A largevariation in milk composition was obtained, as asl secretion levels varied from 7.2 g-kg"! in A milksto none in 0 milks. Mean micelle size (MMS), determined by photon correlation spectroscopy, var-ied between samples from 192 nm to 287 nm. This was explained by the different aspects of thehistograms of casein distribution according to the size, determined from transmission electronmicroscopy data, which showed a maximum either in the low diameter range (20-130 nm) or in thelarge diameter region (130-260 nm), or even intermediary figures with a bimodal distribution. Thecaseins, as1CN, as2CN, ~CN and KCN were determined in milks from nitrogen malter determinations(N x 6.38) and RP-HPLC analysis of casein. Polynomial relations were calculated between micellesize and milk composition al parameters. MMS was correlated on one side to the as1CN and KCN lev-els in milks (g-kg'") and, on the other side, to the proportions of as1CN %, as2CN % and ~CN % intotal casein. These polynomial relations allowed the prediction of the mean micelle size in milksfrom the casein levels, with aiS % accuracy. Monofactorial correlations also showed a significanteffect of as1CN (r = -0.77), but not any of KCN. Mineral composition of milks was determined,calcium by atomic absorption spectrophotometry and phosphorus, by a colorimetrie method. Goat milkswere characterized by a constant colloidal inorganic P level (12.4 (SO = 1.7) mmol-kg "). In contrast,colloidal Ca (Cac), SerineP and total colloidal P (Pc) were correlated to the total casein content. Theratio CaclPc was the most constant parameter in goat milks, amounting 1.22 (SO = 0.05), presumablycharacterizing an unique mode of association of caseins in milk. No significant correlation wasobtained between the colloidal Ca and P levels in milks and the size of micelles. © InralElsevier, Paris.

/1

1

1

1

1

goat milk 1 casein 1 micelle size 1 asJ caseinl colloidal calcium and phosphorus

Résumé - Taille des micelles du lait de chèvre en relation avec la composition de la caséine etles teneurs en caséines asJ' as2' ~ et Kdans le lait. La relation entre la taille des micelles de caséineet la composition de la fraction caséine des laits a été étudiée sur 21 laits individuels provenant de

* Correspondence and reprints. E-mail: apierre@labtechno.roazhon.inra.fr

591

592 A. Pierre et al.

chèvres homozygotes pour les variants de la caséine as!, A, B2, C, E, Fou 0, de manière à obtenirune grande variation de composition des laits. En effet, à ces variants correspondent des niveaux desécrétion différents de la caséine as!' de 7,2 g·kg-I pour le variant A à une absence totale pour levariant O. La taille moyenne des micelles a été mesurée par spectrophotométrie de corrélation de pho-tons; la distribution de la caséine selon le diamètre micellaire a été déterminée par microscopieélectronique à transmission. La teneur en caséine totale et la composition de la caséine ont été obte-nues à partir du dosage de la matière azotée totale (N x 6,38) et de l'analyse de la caséine par chro-matographie liquide à haute pression en phase inverse. Les minéraux colloïdaux, calcium et phosphore,ont été dosés respectivement par spectrophotométrie d'absorption atomique et par une méthodecolorimétrique. La taille moyenne des micelles dans les laits variait de 192 à 287 nm. La variation aété expliquée par l'aspect des histogrammes de distribution de la caséine en fonction de la taille,qui montrait, selon le lait, soit un maximum pour des valeurs faibles (20-130 nm), soit un maxi-mum pour des valeurs élevées (130-260 nm), soit encore une distribution bimodale. Une relation poly-nomiale a été calculée, reliant la taille moyenne des micelles (MMS) et les paramètres de composi-tion des laits (as1CN, as2CN, pCN et KCN). MMS était significativement corrélée d'une part à la teneuren aslCN et KCN des laits, d'autre part à la proportion de aslCN %, as2CN % et pCN % dans la caséinetotale. Ces relations ont permis de déterminer la taille micellaire moyenne des laits de chèvre à par-tir de leurs teneurs en caséines asl' as2' pet K, avec une incertitude de 15 %. Les corrélations mono-factorielles ont aussi été calculées, montrant un effet significatif de la teneur en as 1CN (r = - 0,77),mais non de la KCN. La composition minérale des laits de chèvre était caractérisée par une teneurconstante en phosphate inorganique colloïdal (12,4 [SO = 1,7] mmol-kg-'), quelle que soit la teneuren caséine totale, et par des teneurs variables en calcium colloïdal (Cac), en P lié aux sérines et en Pcolloïdal (Pc) qui étaient corrélées à la teneur en caséine totale du lait. Le rapport CaclPc est apparucomme un paramètre invariant, ayant une valeur de 1,22 (SO = 0,05) dans les laits de chèvre, iden-tique à la valeur déjà déterminée dans le lait de vache. Ce rapport semble caractériser un moded'association unique des caséines dans les micelles des laits. Aucune relation significative entreminéraux et taille micellaire n'a été mise en évidence. © InralElsevier, Paris.

lait de chèvre/ caséine / taille de micelle / caséine asl / calcium colloïdal/phosphore colloïdal

1. INTRODUCTION

Casein in milk is structured in micelles,the size of which is dependent on marn-malian species [1]. Casein micelle compo-sition includes, in addition to minerais, dif-ferent caseins, as\' as2' ~ and K casein forthe most part, the relative proportions ofwhich are fairly constant within speciesexcept in colustrum, but variable betweenspecies. Many investigations have been doneon cow milk to determine the relation be-tween micelle size and casein composition.KeN content was found to be in inverse rela-tion to mean micelle size, in cow milks fromvarious breeds [17] and in cow milks con-taining different KeN variants [12]. In addi-tion, the KCN proportion in artificialmicelles made from bovine caseins was

found related to micelle size in the sameway [23]. Among goat milks, a difference inmean micelle size was reported in relation toas!CN variants [20, 24]; consequently,as! CN cou Id also be a factor of variation ofmicelle size.

Goat milk is characterized by a highgenetic variability of nature and quantity ofcaseins, stemming mainly from as!CNcasein with about 14 variants already iden-tified [13]. The as1CN variability resultsnot only in the substitution or deletion ofone or two amino acids (A, B2 and C vari-ants), but also in large deletions of thesequences. In the F variant, for ex ample,the segment 59 to 95, containing a SerPcluster, is deleted. The as1CN variabilityalso induces variation in the secretion levels[6]. In goat milks its content varies from

Micelle size and case in composition 593

7.2 g-kg! milk for the A variant, to nonefor the 0 variants. From the comparison ofA and 0 goat milks, Pierre et al. [18] haveobserved differences in casein micelle sizedistribution, which explained differences inmean micelle size: the A milks were char-acterized by a narrow distribution of micellesizes centered on a low diameter range whileo milks had a broad distribution up to largediameters. Micelle size variation may there-fore be related to the Œs1CNlevel in milk.

Such goat milks, having a casein com-position varying to a larger extent than cowmilks, were expected to be a good model tostudy the relation between casein composi-tion and micelle size. It is the purpose ofthis paper to study the size characteristicsof micelles (mean micelle size and size dis-tribution of casein), and their chernical com-position (casein proportions and colloidalCa and P) in individual goat milks, fromanimais being homozygous for the variousŒs1CN variants, and to search for the rela-tions between size and composition.

2. MATERIALS AND METHODS

Goat milks were provided by Stationd'Amélioration Génétique des Animaux deToulouse (Inra). They were from 21 goats,homozygous for the A, B2, C, E, F, 0 alleles ofus1CN. Lactation numbers were the same (2nd)as weil as lactation stages (180-215 d).

Determination of total nitrogen (TN) and ofpH 4.2 soluble nitrogen (SN), extracted by theRowland procedure [22], were made by Kjel-dahl analysis (N x 6.38). Casein (CN) yield wascalculated as: CN = TN - SN. The pH was cho-sen at 4.2 for the separation of SN, because itcorresponds to the isoelectric pH of goat case inin milk, as determined in preliminary experi-ments. Phosphorus was determined by a photo-metric method [5] and calcium by atomic absorp-tion spectrophotometry (Spectra A 300, Varian,Palo Alto, Ca, USA). Analyses of Ca and P wereon total milk (T) and on ultrafiltrates, obtainedeither at the natural pH of milk (UF), or after aIN-HCl addition up to pH 5.0 (UF 5.0). Ultra-filtration was carried on through CF25 Centri-con membranes (Amicon, Paris, France) at 20 "C

and it involved a centrifugation step (750 g,45 min). The following fractions were then cal-culated: colloidal P (Pc) and colloidal Ca (Cac),as the difference [T-UF]; inorganic colloidal P(Pic), as [UF 5.0 - UF]; Serine P (SerP), as[Pc - Pic].

HPLC chromatography (Varian 5 000, PaloAlto, Ca, USA) allowed the separation of indi-vidual components in total casein. Reverse phaseseparation on a C4 column - 4.6 mm diameter,150 mm length (Vydac Interchim, Montluçon,France) was achieved using as eluents, (A) 0.1 %trifluoroacetic acid (TFA) in water, and (B)0.096 % TFA in acetonitrile/water (80/20, v/v).Separation was obtained in 30 min on the col-umn at 40 "C, with a flow rate of 1 mlvmirr 'and eluent B increasing from 37 to 53 % [18].Sam pie preparation involved separation of caseinfrom milk at 30 "C by precipitation at pH 4.2.Complete description of experimentals could befound elsewere [18]. Proportions of the variouscaseins in total casein were obtained from thepeak areas on the HPLC profiles, corrected bythe specifie relative absorbance of each caseincalculated from the results of Jaubert [II]: KeN,0.934; us2CN, 0.877; uslCN, 0.810; ~CN, 1.0.The contents of the caseins in milk were calcu-lated from the measured proportions combinedwith the total casein content of milk estimatedby N analysis.

Average case in micelle size was determinedby photon correlation spectroscopy (PCS) onskimmed milks. Measurements were performedwith a Coulter N4MD apparatus (Coultronics,Hialeah, FL, USA), equiped with a He Ne laserlight (632.8 nm), with the following experimen-tal conditions: scattering angle 90°, temperature20 "C, viscosity 1.002 cP and refractive indexof the solvent 1.333 (water), measure time 600 s,sample time 4.5 us. From the measured auto-correlation function of the scattered light wascalculated an intensity-weighted average diffu-sion coefficient. It allowed the calculation of anaverage diameter corresponding to the size of aspherical particle with an equivalent diffusioncoefficient. Experimental methods were the sameas already reported [18].

Histograms of micelle size distribution weredetermined on Il of the milks only, chosenamongst those having the highest and lowestindividual casein content relative to us1CN,us2CN or KeN in order to increase any differ-ences. Observations of milk micelles were madeon a Philips CM 12 apparatus (Philips Industrie,Bobigny, France), on a transmission mode, witha 80 kV voltage. Milk samples were skimmed

594 A. Pierre et al.

as already described, then diluted 11250 in0.2 mol-L'! sodium cacodylate buffer at pH 7.4.An aliquot was dropped on a collodion-treatedcarbon grid (300 mesh, 3 mm diameter). After30 s, excess sample was removed on a filter paperand the grid was immersed in a 2 % (w/v) uranylacetate solution for 2 min. The grid was thendrained and air dried. Histograms were calcu-lated from transmission electron micrographs(TEM) according to Schmidt et al. [23]. Fromthe plates obtained, the number fractions ofmicelles in 14 size classes of 20 nm width weredetermined by counting random fields. About1100 individual diameters of particles were mea-sured for each milk. Ali the micelles present onthe plates were measured. Results were treated asdescribed by Schmidt et al. [23] to obtain thevolume moment average diameter. Micelles ofdiameter ~ 130 nm are referred to here as "largemicelles". The relative volume fraction of largemicelles was calculated from TEM data. Assum-ing a constant micelle density, the volume dis-tribution is the same as the casein mass distri-bution.

Polynomial analysis was a multivariate anal-ysis of the least square type.

3.RESULTS

3.1. Size of casein micelles in milks

3.1.1. Mean micelle size

Mean micelle sizes in milks were deter-mined by PCS. The results are grouped intable 1 according to the us1CN variants inmilks. Three groups of milks, A, (H2 + C),(E +F + 0) can be distinguished, differingsignificantly in mean micelle size (P = 0.05), .in spite of the small number of milks in eachgroup. Such results are consistent with thoseof Remeuf [20].

3.1.2. Casein micelle size distribution

Histograms of casein micelle size distri-bution were obtained from TEM. Typicalhistograms of milks of each variant groupare presented in figure 1. Comparing Amilks and 0 milks, a large difference incasein micelle size distribution wasobserved. In two of the A milks (out ofthree) the distribution was centered on small

Table I. Mean micelle size (MMS), obtained by PCS, in goat milks according to their asl casein vari-ants. Minimum and maximum values are the lowest and highest values obtained for individu al milksinside a same variant group.Tableau I. Taille micellaire moyenne (MMS), obtenue par PCS, dans les laits de chèvre contenantdifférents variants de la caséine asl et valeurs minimale et maximale obtenues pour des laits indivi-duels à l'intérieur d'un même groupe de variants.

MMS: Mean micelle size(nm)

as1CN Number Mean Minimum Maximumvariant ofsamples value value

A 6 221 192 236

82 1 247C 2 248 248 249

E 6 264 241 287F 3 266 255 2710 3 262 255 283

Figure 1. Casein distri-bution according tomicelle size (fromTEM) in individual goatmilks with differentas1CN variants: 3 dif-ferent A variant milks;one milk only for eachof the other variants.Figure 1. Histo-grammes de distributionde la caséine selon lataille micellaire (obte-nue par TEM) dans deslaits individuels dechèvre contenant desvariants différents de lacaséine asl'

Micelle size and casein composition

35Amilks

30~~ 25c0

20t5jg 15CDE 10:::3(5> 5

0

35

~ 30;f.~ 25o~ 20

CD 15

§ 10(5> 5

o

DB milk.Cmilk

35,--------------------,CE milk

~ 30;,g .Fmilk~c 250t5 20jgCD15E:::3 10(5> 5

0

35.Omilk

~ 30;,g~c 250t5 20jg

15CDE

10:::3(5> 5

0<20 50 90 130 170 210 250

Diameter(nm)

595

diameter values (20-130 nm), as alreadyreported [18], while in 0 milk.most of thecasein was in large micelles (:2 130 nm).Milks with B2, C, E and F variants showedintermediate profiles, sorne.of the milks hav-

ing a bimodal distribution. Thus, the profilesprogressively changed from A to 0 milks;the small micelle peak decreased in E and Fmilks,and disappeared in 0 milk. The largemicelle peak increased up to a maximum in

596 A. Pierre et al.

o milk in which it accounted for about 80 %of the casein. The proportion of casein inlarge micelles (TEM) was found correlatedwith the mean micelle size in the milks,determined by PCS, with a correlation coef-ficient r = 0.76. An individu al variability ofmicelle distribution was also observed insidea same variant group, as shown by the com-parison of histograms of the three differentA milks reported infigure J. A bimodal dis-tribution was observed fore one of the Amilks (looting similar to those of the E orF milks, which was confirmed by the sta-tistical comparison of these distributions(rCA, F) = 0.91).

3.2. Amount of colloidal mate rialin milks

3.2.1. Caseins

Total casein level in milks varied from17.1 to 33.1 g·kg-1 (figure 2a), due to theirdifferent us1CN and ~CN contents. Con-

182a

16

14

0;-12Cl...S10III 0C 0'iii 8IIIcao 6

4

2

o /3CN

O-l-~----"',Lj"~--~---+-----I15 20 25 30 35

Total casein (g.kg '1)

1 J3CN = 0.3x+4.4 ; as1 CN = 0.7X-13.41

trarily, no variation ofKCN and us2CN con-tents was related to the total casein content(figure 2b). The average amounts of us2CNand KCN in milks were respectively 4.4(SD = 0.8) g-kg! and 4.5 (SD = 0.5) g-kg'.

3.2.2. Colloidal Ca and P

Colloidal phosphorus (Pc) levels arereported infigure 3a as a function of totalcasein, as weil as the levels of its two maincomponents, SerP and Pic. An increase in Pcwas observed in relation with the amountof SerP, while Pic remained at a quite con-stant level, 12.4 (SD = 1.7) mmol-kg ",whatever be the total casein content in milk.Colloidal calcium (Cac) also increased withthe total casein content (figure 3b) and washighly correlated with Pc (r = 0.97). Cac/Pcratio had quite the same value in ail themilks and amounted 1.22 (SO = 0.05). Con-trarily, Cac/Pic increased from 1.69 to 2.24,as a result of the Cac variation, with a meanvalue Cac/Pic = 1.94 (SD = 0.14).

2b8

.... ..~ & ....... ..6

4

2

o ..1..- __ --'- --'----__ --' -'

KCN8

6

4

2

o -1----+----+-----+----115 20 25 30 35

Total case in (g.kg .1)

• • •

las2CN = 4.4 (SO = 0.8); KCN = 4.5 (SO = 0.5) 1

Figure 2. Levels of caseins in individual goat milks having different total casein contents. g·kg-1

(n = 21). 2a: us1CN and ~CN. 2b: us2CN and KeN.Figure 2. Teneurs en caséines des laits individuels de chèvre ayant différentes teneurs en caséine totale.g-kg-' (n = 21). 2a: us1CN et ~CN. 2b: us2CN et KCN.

Micelle size and casein composition

3a30 -,--------------,

25 • Pc•20

à à àà4....0.- ·.I:J.-lï --t:>-trm;- -- ----

~ à à à •

~

.... SerP•• •••••5

o +----t------t---+-----i15 20 25 30 35

Total case in (g.kg-1)

Pc = 0.37x+10.4; Pic = 12.4 (50 = 1.7)SerP = 0.29x

597

3b

•

3 uii:ÙCIl(J

ù't

2(J

• Cac30 •25

"Cl.if 20oE.§. 15uCIl(J

Cac/Pic:c :c

1----:c~ • .or-&-

_x-x" :c "X' :c:c

10

Cac/Pc. .- .. .. .., '.~..o 1

15 20 25 30 35Total casein (g.kg -1)

5

Cac - 0.47x+12.3 ; Cac/Pic = 0.025x+1.3Cac/Pc = 1.22 (50 = 0.05)

Figure 3. Colloidal mineraIs in individual goat milks (mmol-kg"). 3a. Colloidal P (Pc), inorganic col-loidal P (Pic), serine P (SerP). 3b. Colloidal Ca (Cac) and molar ratios, CaclPc and CaclPic (n = 20).Figure 3. Minéraux colloïdaux dans les laits individu~ls de chèvre (rnmol-kg "). 3a. P colloïdal (Pc),P inorganique colloïdal (Pic), P des phosphosérines (SerP). 3b. Ca colloïdal (Cac) et rapports molaires,CaclPc et CaclPic (n = 20).

3.3. Composition of casein micelles

3.3.1. Proportions of caseinsand minerais

As a result of the variation in the amountsof the caseins in milks, a wide variation intheir relative proportions in total casein wasobserved: KeN (12 % to 26 %), as2CN (Il% to 29 %), as1CN (l % to 30 %), ~CN(39 % to 58 %). Also varied the colloidalCa and P fractions (expressed as mmol-g ' oftotal casein): Cac/CN (0.75 to 1.48), Pc/CN(0.65 to 1.23), Pic/CN (0.36 to 0.76).

3.3.2. Correlations between caseinsproportions and colloïdalCaandP r

Correlation coefficients between micellarconstituents were calculated and are report-

ed in table II. The large variation of asl CN %induced negative correlations with othercaseins, as the variables were linked, leadingto a correlation coefficient in the range r =-0.80. In the mineral fraction, Cac/CN andPic/CN were highly correlated (r = 0.95),according to the relation: Cac/CN = 1.49Pic/CN + 0.22. In this relation, the value ofthe slope, 1.49, corresponded to the meanvalue of the Ca/P ratio in the colloidal inor-ganic calcium phosphate of the milks andthe constant, 0.22 represented the averageamount of Ca directly bound tocasein inthe milks, 0.22 mmolg:' CN [8]. Betweencaseins and minerals, the highest correla-tion coefficients were obtained betweenas2CN % and Pic/CN (r = 0.86) and betweenas2CN % and Cac/CN (r = 0.82). KCN %was positively correlated with Pic/CN(r = 0.78). The negative correlation coeffi-cient obtained between aslC~ % and Pic/CN

598 A. Pierre et al.

Table II. Correlation coefficients between casein proportions (% of total casein) and colloidal Ca andP (mmol-g " CN) in the micelles of individual goat milks having different aslCN variants (n = 20).Tableau II. Coefficients de corrélation entre les proportions des caséines (pourcentage de la caséinetotale) et les teneurs en calcium et phosphore colloïdaux (mmol-g! CN) dans les micelles de caséinedes laits de chèvre contenant différents variants de la caséine asl (Il = 20).

aslCN % aszCN % I3CN % KCN% Pic/CN Pc/CN

aszCN % -0.80I3-CN % -0.79 nsKCN% -0.85 0.67 0.49Pc/CN -0.58 0.80 ns 0.63Pic/CN -0.76 0.86 ns 0.78Cac/CN -0.61 0.82 ns 0.65 0.95 0.97Cac/Pic 0.79 -0.58 -0.64 -0.70 -0.63

ns: not significant (P = 0.05).ns : non significatif (P = 0.05).

(r = -0.76) was in agreement with the resultsof Schmidt et al. [23].

3.4. Relation between micelle sizeand composition of casein

3.4.1. Correlations

Correlation coefficients between sizeparameters of casein micelles (mean micellesize, casein in large micelles) and casein orminerallevels in milks (g-kg'") or caseinsproportions in micelles (% total casein), arereported in table Ill. ln relation to caseinlevels in milks (gkg'), the higher correla-tions were observed with total casein andas1CN. No correlation was observed withKCN. Most of the coefficients related tocasein in large micelles are higher than thoseof mean micelle size. Considering nowcasein proportions (% of total casein), thehigher correlations with size parameterswere obtained with aslCN % (r = -0.73 and-0.86), and also with as2CN % (r = 0.61and 0.86). KCN % showed a positive coef-ficient with MMS (r = 0.46). When the par-tial correlation, meaning the correlation at aconstant as 1CN proportion, was calculatedbetween size and KCN %, the coefficient

obtained was r = -0.35, not significant atP = 0.05, but only at P = 0.20. Infigure 4 ispresented the relation between mean micellesize and casein proportions. ln the hypoth-esis of a Iinear relation, regression coeffi-cients were calculated which explained 53 %of the variance when related to as1CN, andrespectively 37 % for as2CN, 44 % for ~CNand 21 % for KCN. The amount of caseinin large micelles was presented infigure 5 asa function of the aslCN proportion and ofthe Pic/CN ratio. as1CN % explained 74 %of the total variance (figure 5a). In relationwith the Pic/CN variation (figure 5b), thecurve showed a biphasic course, casein inlarge micelles increased for Pic/CN valuesfrom 0.42 up to about 0.55 and, for thehigher values of the ratio, a plateau wasreached corresponding to 80 % of casein inlarge micelles. Other colloidal mineraI frac-tions in milks were not highly correlatedwith micelle size parameters, neither con-sidering their proportions nor their contentsin milks (table Ill).

3.4.2. Polynomial regressions

Polynomial relations were also calculatedbetween micelle size parameters and the

Micelle size and casein composition

Table III. Correlation coefficients between size parameters (Mean micelle size, MMS, obtained byPCS; casein in large micelles, :::::130 nm, from TEM) and caseins and colloidal minerais (g-kg! andpercentage of casein) in individual goat milks with different asl CN variants; n : number of samples.Tableau III. Coefficients de corrélation entre les paramètres de taille micellaire, taille micellairemoyenne (MMS), obtenue par PCS, et proportion de la caséine dans les micelles de taille z 130 nm(TEM) et la composition (caséines et minéraux colloïdaux) dans les laits individuels de chèvresayant des variants de la caséine asl différents; n : nombre d'échantillons.

MMS: Mean Casein in Total caseinmicelle size large micelles (g-kg! milk)

(nm) (%)

g-kg! milk n=21 n= 11 n=21Total casein -0.72 -0.69as1CN -0.77 -0.86 0.85aszCN ns ns nspCN ns ns 0.70KCN ns ns ns

Percentage of total caseinas1CN% -0.73 -0.86 0.82aszCN % 0.61 0.86 -0.71PCN% 0.66 ns -0.52KCN % 0.46 0.73 -0.82Pic/CN 0.43 0.77 -0.72

mmol-kg :' milk n =20 n =20Pc -0.61 ns 0.53Pic ns ns nsCac -0.54 ns 0.57Cac/Pic -0.64 -0.75 0.75

ns: not significant CP = 0.05).ns : non significatif CP = 0,05).

asl' as2' ~, K casein levels in milks. Theircharacteristics are reported in table IV. Twodifferent relations were calculated, one fromthe caseins contents (g-kg ") of the milks,the other from the casein proportions ( %).The results show that the aslCN and KCNcontents of milks were significantly in rela-tion with micelle size. The two coefficientswere negative, the higher being ascribed toKCN (-20.4). Considering now casein rela-tive proportions, as1CN %, as2CN % and~CN % had significant coefficients, ail threepositive. KCN % showed no significant rela-tion.

4. DISCUSSION

4.1. Micelle size

Profiles of TEM histograms of caseindistribution versus micelle diameter variedaccording to the milks. Casein in goat milksseemed to be preferentially distributed eitherin small micelles, or in large micelles, oreven, in various relative proportions amongthese two populations according to the milk,leading to bimodal or widely varying dis-tributions. The prevalence of one on theother explained the variations observed in

599

600 A. Pierre et al.

45 50 55 60 10 15 20~CN(%) KCN(%)

Figure 4. Relation between mean micelle size (MMS), obtained by PCS, and the proportion of thedifferent caseins in the total casein of individual goat milks (n = 21). 4a. Cls1CN. 4b. Cls2CN. 4c.pCN. 4d. KCN.Figure 4. Relation entre la taille micellaire moyenne (MMS), obtenue par PCS, et la proportion desdifférentes caséines dans la caséine totale de laits individuels de chèvre (n = 21). 4a. Cls1CN.4b. Cls2CN.4c. pCN. 4d. KCN.

300,--------------,

250

Ê.5. 200

.~:E

150

• •y - -1.6x + 271

R2·O.53100-t---+---+---t---f---+---jf---J

o 5 10 15 20 25 30lXs1CN(%)

300,----------------,

250Êe;;;200:E:E

150 y. 2.9x + 109R2·O.44

4c

~

..•• • •

. .••

100+-----j---t----j----jf----j

35 40

the mean micelle size (MMS). These varia-tions of MMS were in fact better correlatedto the levels of aslCN and KeN in the rnilks,than to the as1CN genetic variant itself.

Mean micelle size (MMS) was found inrelation with casein micelle compositionaccording to the following relations:

(a) MMS 1 (nm) = -6.4 as1CN - 20.4 KeN+364 ;

(b) MMS2 (nm) = 0.95 aslCN % +3.9 aszCN % + 3.5 ~CN %.

These equations allowed the determina-tion of MMS with about 15 % accuracy.MMS were calculated for the 21 goat milksusing these equations, and the results plottedagainst the experimental data obtained by

4a

• •y = 3.3x + 189

R2 = 0.37

10 25 3015

4d •

~- ".~~_ ..

••• •

y = 2.9x + 195R2 = 0.21

25 30

PCS measurements (figure 6). The relationsobtained explained respectively 78 and 62 %of the variance. Consequently, the equationsallow one to calculate an estimated valuefor mean micelle size in goat milk from itscasein content or its casein composition.

In equation (a), relative to casein con-tents, two caseins intervenes, as1CN andKeN. The as1CN content was found nega-tively correlated to micelle size, which hasnever been reported up to now. This wasconfirmed by the value of the monofacto-rial correlation coefficient between micellesize and as1CN (table Ill) which was alsonegative (r = -0.77). It might be a specifiefeature of goat milks, but, more likely, thelarge variation of as1CN content in the goat

Micelle size and casein composition 601

Table IV. Polynomialrelationsbetweenmean micellesize (MMS),measuredby PCS, andcaseincom-position in goat milks (number of samples: 21).Tableau IV. Relations polynomiales entre la taille micellaire moyenne (MMS), obtenue par PCS etla composition de la caséine des laits de chèvre (nombre d'échantillons: 21).

x coefficients relating to:

y Caseins/g-kg " milk

asl as2 ~ K Constant F r2

(a) Mean micelle size -6.4 ns ns -20.4 364 29 0.76(MMSl)SD 6 27

Casein % of total casein

as! as2 ~ K

(b) Mean micelle size 0.95 3.9 3.5 ns 0 1282 0.99(MMS2)SD 0.3 1.1 0.5

F: F-value; r2: regression coefficient; SD: standard deviation; ns: not significant (P = 0.05).F : valeur de F ; r2 : coefficient de régression; SD : écart type; ns : non significatif (P = 0,05).

milks studied allowed to highlight and quan-tifYthe relation, which did not appeared incow milk, as the variation of its as! CN levelis lower. The coefficient ascribed to KCNin equation (a), is also negative and has thehigher value. This result agrees weil withthe data already obtained on cow milk: KCNcontent of individual milks was reported tobe inversely related to micelle size [12, 17].The coefficient relating to KCN, -20.4,seems valid for milks others than goat milk.As an example, the results obtained byGutiérrez-Adan et al. [7] on the milks oftransgenic mices bearing the bovine KCNgene can be quantitatively explained by thevariation in their bovine KCN contents. lnfigure 7 is reported, as a function of thebovine KCN contents of the mice milks, onone side the experimental mean micelle sizes(MMS) measured by these authors, and, onthe other side, the mean micelle sizes(MMS-calc) calculated from the experi-mental size determined in normal mice milk,then modified as a function of the bovineKCN content, according to the -20.4 KCN

relation. The difference betweèn the tworelations is not significant (P = 0.05), mean-ing that the calculation is a good predictionof the sizes.

In another respects, it is noteworthy thatthe monofactorial correlation between theKCN level and micelle size was not signif-icant (table /If), contrary to equation (a).

In equation (b), in which intervene therelative proportions of the caseins (%) ingoat milks, micelle size was significantlyrelated to the proportions of caseins otherthan KCN, the most to ~CN %, due to itshigh coefficient and to its large proportion inmilk, and, to a lesser extent, to as1CN %and as2CN %. No significant effect of KCNon the size was observed, when KCN wasexpressed as a percentage. More, the mono-factorial correlation between size and KCN %gave a positive hardly significant coeffi-cient (r = 0.46). Only after calculation ofthe corresponding partial correlation of KCN %at a constant as1CN %, a negative coeffi-cient for KCN % became apparent. So, it

602 A. Pierre et al.

100UI

5a 5bal~ 80 • •(J • •'E •al 60 ,f:l •.!!! •.: 40c • • •.~ 20 Y = -1.5x + 79III

R2 = 0.74U~ 0 ,0

0 10 20 30 0.30 0.40 0.50 0.60 0.70 0.80us1CN (%) Pic/CN

Figure S. Relation between the proportion of casein contained in large micelles (~ 130 nm, TEM) andthe proportion of as1CN (a) and the Pic/CN ratio (b), in the total casein of individual goat milks(n = Il).Figure S. Relation entre la proportion de caséine incluse dans les grosses micelles (~ 130 nm, TEM)et la proportion de caséine asl (a) et le rapport Pic/CN (b) dans la caséine totale de laits individuelsde chèvre (n = Il).

300 .,.------------------,

Ê.sNtJ):E:E 260....tJ):E:EuralN

'iii 220'C

~:lUëüo

r·· ..w .

: MM52 = 0.64x + 93 :

R2 = 0.62 o o

Figure 6. Relation between themean micelle sizes experimentallymeasured by PCS (MMS) and thecalculated sizes obtained eitherfrom equation (a) involving caseinIevels in milk, (MMS 1), or fromequation (b) involving casein pro-portions, (MMS2). Diameters innm (n = 21).Figure 6. Relation entre les taillesmicellaires obtenues par PCS(MMS) et les tailles calculées àpartir de l'équation (a) faisantintervenir les teneurs en caséine(MMS 1), et de l'équation (b) fai-sant intervenir les proportions descaséines (MMS2). Diamètres ennm (n = 21).

................. ' -

1

MM51 = 0.78x + 55R2 = 0.78

180 +---'----+--~-_+_------'--------j

180 220 260Experimental sizes, MM5 (nm)

1_ Eqn (a) 0 Eqn (b) 1

seems that the weight of the cxs1CN level,having the greater range of variation in goatmilks matched with a passably high coeffi-cient, totally masked or modified the weightof the KCN variation, in spite of its highercoefficient. A negative coefficient for KCN %

300

would be in agreement with the resultsobtained either in cow milk or in goat milkcomparing the composition of the small andlarge micelles of individu al milks [2-4, 14,15, 19,24]. Schmidt et al. [23] also demon-strated that the size of micelles (artificial

Micelle size and casein composition 603

Figure 7. Mean micelle sizes in themilks of transgenic mice, as a functionof their bovine KCN content. MMS,experimental results from Gutiérrez-Adan et al. [7]; MMS-calc., micellesizes calculated from the experimen-tal size determined in normal micemilk, then modified as a function ofthe bovine KCN content of the micemilk, using the -20.4 KCN coefficient.Figure 7. Taille moyenne des micellesde caséine en fonction de la teneur enKCN dans des laits de souris transgé-niques. MMS exp., résultats expéri-mentaux de Gutiérrez-Adan et al. [7];MMS-calc., tailles calculées à partir dela valeur expérimentale obtenue dansle lait de souris normale et corrigée enfonction de la teneur en caséine Kbovine des laits, en utilisant le coeffi-cient -20.4 KCN.

micelles) was inversely related to KCN pro-portions. However, conditions of their exper-imentation were such (variation of KCN pro-portions at a constant casein level in thesuspension) that there was an 'one-to-one'relation between KCN concentrations andKCN proportions. Thus, from their results, itwas not possible to conclude which of thetwo was the efficient parameter. In contrast,when individual milks were studied, notonly KCN concentration varied, but also,and independently, their total casein con-centration.

Considering micelle size in relation withcolloidal minerais, no main correlation wasobtained, as already reported for cow milkcasein micelles by Schmidt et al. [23], apartthe relation reported between casein in largemicelles and Pic/CN.

4.2. Micelle composition

The wide range of variation observed inthe casein levels and in their proportionswas however compatible with the edifica-

300-,------------------,

250Ê..sCI):E:E200

..r----------------: MMS = -17.6x + 263 :, 1

1 R2 = 0.85 :~- - - - - - - - - - - - - - --'•

• •'.•MMS-calc= -20.3x + 263

R2 = 0.99

150-j----+---+-----+------1o 1 234

KeN bovine (g-l<g-1)

1. MMS 0 MMS-calè 1

tion of micelles in ail the goat milks, asobserved by TEM. Micelles appeared, inevery case, as constituted of granular aggre-gates of various sizes, similar to thosealready described by Richardson et al. [21]as weil in goat milk, as in cow milk and ewemilk.

The levels of colloidal minerai in goatmilks showed many similarities with thatof cow milk at a same total casein content(table V). Sorne of the parameters deter-mined were found varying according to thetotal casein content in goat milks, as SerP,Pc and Cac, while others remained constantin ail the milks, as Pi (12.4 (SD = 1.7)mrnol-kg "), or Cac/Pc, 1.22 (SD = 0.05).The constant Cac/Pc ratio observed in eithercow or goat milk, confirmed the main role ofcalcium phosphate during casein aggregationinto micelles [8,9, 16].

4.3. Conclusion

The unique structural appearance ofmicelles assorted of a same minerai com-position could mean that a same.mineral

604 A. Pierre et al.

Table V. Composition of micellar calcium phos-phate in cow milk and in a goat milk having thesame total casein content (25.8 g·kg-').Tableau V. Composition du phosphate de cal-cium micellaire dans le lait de vache et dans unlait de chèvre ayant la même teneur en caséinetotale (25,8 g-kg:").

Cow milk* Goat milk**

Cac 21.2 24.0Pc 17.3 19.6Pic la 12.4ScrP 7.3 7.2CaclPic 2.13 1.94CaclPc 1.23 1.22

* From [10] and [25]; ** fromfigure 3.* Voir [ID) et [25]; ** voirfigure 3.

structure could be involved in the aggrega-tion of caseins into micelles in ail the milks,whatever the species and whatever the pro-portions of the caseins.

Contrarily, the preferential edification oflow size or large size micelles, leading todifferent mean micelle sizes, seemed to bedependent on the aslCN and KCN levels inmilks. It is suggested that the aslCN andKCN levels in milks could be the influentfactors on micelle size regulation, or at least,be in close relation with them, although ourexperiments have not demonstrated theywere causal. However, it is not yet possibleto explain the results observed from the pre-sent knowledge on goat milk.

ACKNOWLEDGMENTS

We are grateful to J.L. Maubois for facilitiesand for his critical reading of the manuscript.Thanks to J. Léonil for helpful advice and toC. Holt for useful criticisms during the prepara-tion of the manuscript. We thank E. Manfredi(Inra, Station d'amélioration génétique des ani-maux, Toulouse, France) for providing us withthe goal milks and also P. Chastin, J. Martin and

M. Bouvier, who sorted out the goats and orga-nized collection and expedition of milks.

Electron microscopy measurernents were per-formed at the Centre de microscopie électron-ique à transmission de l'université de Rennes-I(METUR), département de Métabio. The workwas parti y supported by a grant of the EEC AIRProject CT 94-1441.

REFERENCES

[1] Buchheim W., Lund S., Scholtissek J., Vergle-ichende Untersuchungen zur Struktur und Grol3evon Casein micellen in der milch verschiedenerSpecies, Kiel Milch. Forsch. 41 (1989) 253-265.

[2] Dalgleish D.G., Home D.S., Law AJ.R., Size-related differences in bovine casein micelles,Biochirn. Biophys. Acta 991 (1989) 383-387.

[3] Davies D.T., Law AJ.R., Variation in the pro-tein composition of bovine case in micelles andserum casein in relation to micellar size andmilk temperature, 1. Dairy Res. 50 (1983) 67-75.

[4] Donelly WJ .. Mc Neil G.P., Buchheirn W., McGann T.C.A., A comprehensive study of therelationship between size and protein composi-tion in natural bovine casein micelles, Biochern.Biophys. Acta 789 (1984) 136-143.

[51 FIL-lOF. Determination of total phosphoruscontent in cheese. Standard n? 33B (1982).

[61 Grosclaude F., Mahé M.F., Brignon G., Di Sta-sio L., Jeunet R., A mendelian polymorphismunderlying quantitative variations in goatu" casein, Genet. Sel. Evol. 19 (1987) 399-411.

[71 Gutiérrez-Adan A., Maga E.A., Meade H., Shoe-maker C.F., Medrano J.F., Anderson G.B., Mur-ray J.D., Alterations of the physical character-istics of rnilk from transgenic mice producingbovine x-casein, J. Dairy Sei. 79 (1996) 791-799.

[8] Holt C; Inorganic constituents of milk III. Thecolloidal calcium phosphate of cow milk,J. Dairy Res. 49 (1982) 29-38.

[9] Holt c, Hasnain S.S., Hukins D.W.L., Struc-ture of bovine milk calcium phosphate deter-mined by X-ray absorption spectroscopy,Biochern. Biophys. Acta 719 (1982) 299-303.

[ 10] Holt c., The rnilk salts: their secretion, concen-trations and physical chemistry, in: Fox P.F.(ed), Developments in Dairy Chemistry 3. Lac-tose and minor constituents, Elsevier AppliedScience Publishers, London, 1985, pp. 143-181.

[III Jaubert A., Influence of sorne physicochemicalparameters (pH, temperature, ionie strength) oncomposition and structure of the goat caseinmicelle, 1992, Thesis ENSA Rennes.

Micelle size and casein composition

[12] Lodes A, Krause 1., Buch Berger J., AumannJ.,Klostermeyer H., The influence of genetic vari-ants of milk proteins on the compositional andtechnological properties of milk. 1 Case inmicelle size and the content of non-glycosylatedx-casein, Milchwissenshaft 51 (1996) 368-373.

[13] Martin P., Addeo F., Genetic polymorphism ofcase in in the milk of goats and sheep, in: lOFSpecial Issue 9603. Production and utilization ofewe and goat milk, 1996, pp. 45-58.

[14] Mc Gann T.C.A, Keamey RD., Donelly W.1.,Developments in column chromatography forthe separation and characterization of case inmicelles, J. Dairy Res. 46 (1979) 307-311.

[15] Mc Gann T.C.A., Donelly W.1., Keamey RD.,Buchheim W., Composition and size distributionof bovine casein micelles, Biochem. Biophys.Acta 630 (1980) 261-270.

[16] Mc Gann T.C.A, Buchheim W., Keamey RD.,Richardson, T., Composition and ultrastructureof calcium phosphate-citrate complexes inbovine milk systems, Biochem. Biophys. Acta760 (1983) 415-420.

[17] Nuyts-Petit V., Delacroix-Buchet A, Vassal L.,Influence de trois haplotypes des caséines as l'

p et K fréquents en race bovine normande surla composition du lait et l'aptitude à la fabrica-tion fromagère, Lait 77 (1997) 625--639.

[18] Pierre A., Michel F., Le Graët Y., Variation insize in goat milk casein micelles related to caseingenotype, Lait 75 (1995) 489-502.

605

[19] Pierre A, Michel F., Zahoute L., Composition ofcasein micelles related to size in goat milks withA and a asl casein variants, in: Home S.D.,Muir D. (eds), Symposium Proceedings 'Caseinsand caseinates, Structures, interactions, network,Ayr, Scotland, 1997.

[20] Remeuf F., Influence du polymorphisme géné-tique de la caséine a$l caprine sur les carac-téristiques physico-chimiques et technologiquesdu lait, Lait 73 (1993) 549-557.

[21] Richardson B.C., Creamer L.K., Pearce K.N.,Munford RE., Comparative micelle structureII. Structure and composition of casein micellesin ovine and caprine milk as compared withthose in bovine milk, J. Dairy Res. 41 (1974)239-247.

[22] Rowland S.1., The determination of the nitro-gen distribution in milk, J. Dairy Res. 9 (1938)42-46.

[23] Schmidt D.G., Koops J., Westerbeek D., Prop-erties of artificial casein micelles. 1 Prepara-tion, size distribution and composition, Neth.Milk Dairy J. 31 (1977) 328-341.

[24] Tziboula A., Horne D.S., Influence of caseingenotype on the physicochemical properties ofcaprine milk, in: IDF Special issue 9603. Pro-duction and utilization of ewe and goat milk,1996, pp. 282.

[25] White J.C.D., Davies D.T., The relation betweenthe chemical composition of milk and the sta-bility of the caseinate complex 1. General intro-duction, description of samples, methods andchemical composition of samples, J. Dairy Res.25 (1958) 236-255.