size of native and heated casein micelles, content of protein and minerals in milk from norwegian...

*Corresponding author. Tel.: #47-64-94-85-50; fax: #47-64-94-37-89.

E-mail address: [email protected] (T.G. Devold).

International Dairy Journal 10 (2000) 313}323

Size of native and heated casein micelles, content of proteinand minerals in milk from Norwegian Red Cattle*e!ect of milk

protein polymorphism and di!erent feeding regimes

Tove Gulbrandsen Devold*, Margreet Jansen Brovold, Thor Langsrud,Gerd Elisabeth Vegarud

Department of Food Science, Agricultural University of Norway, P.O. Box 5036, N-1432 Aas, Norway

Received 28 September 1999; accepted 18 June 2000

Abstract

Milk samples of 59 cows of the Norwegian Red Cattle breed receiving three di!erent supplementary concentrates, were analysed forgenotypes of caseins and whey proteins, the content of di!erent milk salts (Ca2`, Ca, Mg and citrate), the content of total protein,casein and whey protein and the mean micellar size of native and heated casein micelles. The genotype of a

41-casein had a statistically

signi"cant e!ect on the content of protein and casein, and the content of whey protein and the casein number were signi"cantlyin#uenced by di!erent feeding regimes, and the content of citrate. The mean size of native and heated casein micelles was signi"-cantly in#uenced by the feeding regimes, genotype of a

41-casein (native mean size only) and i-casein, pH and the content of casein,

whey protein and casein number. The heat-induced changes in mean micellar size were signi"cantly a!ected by the calcium ionactivity which accounted for approximately 40% of the total variation. ( 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Milk protein polymorphism; Feeding; Protein content; Mineral content; Size of casein micelles; Size of heated casein micelles

1. Introduction

Numerous studies highlight the in#uence of milk pro-tein polymorphism on milk composition and technolo-gical properties of the milk. As recently reviewed indetail, genetic variants of all major milk proteins havebeen demonstrated to in#uence the composition of milkproteins including total protein, casein, casein numberand whey proteins (Bovenhuis, Van Arendonk, & Kor-ver, 1992; Jakob & Puhan, 1992; Ng-Kwai-Hang& Grosclaude, 1992; Delacroix-Buchet, Le"er, & Nuyts-Petit, 1993; Jakob, 1994; Ng-Kwai-Hang, 1997; Puhan,1997; Lodes, Buchberger, Krause, Aumann, & Kloster-meyer, 1997). In a few publications the impact of geneticpolymorphism of some or all milk proteins on meanmicellar size is discussed (Delacroix-Buchet et al., 1993;Lodes, Krause, Buchberger, Aumann, & Klostermeyer,1996; Walsh et al., 1998). In spite of the vast amount of

research in this "eld, the signi"cance of genotype e!ects isstill controversial and depends on the breed, the statist-ical model used for the evaluation of data and theauthor's interpretation.

In addition to inheritable factors of each animal, thecontent of protein and of other milk constituents arein#uenced by feeding. Only a few short-term studies havereported on the relationship between feeding and milkcomposition (Mackle & Bryant, 1996). Nutritional vari-ations a!ect proportions and concentrations of di!erentmilk proteins. In Western countries there is an increasingdemand/interest for dairy products made from milk fromecologically fed cows. An ecological feeding regime im-plies the use of ecologically grown feeding materials, andthis change in diet may in#uence the composition of theprotein fraction of the milk and thereby its functionalproperties and the quality of the products made from it.

Di!erent temperature treatments (cooling or heating)are used in the production of all dairy products in orderto improve the technological properties and to prolongthe shelf life. The e!ect of heat treatment on milk proteinsand the milk salt system have been extensively reviewed(Singh & Creamer, 1992; Holt, 1995; Jelen & Rattray,

0958-6946/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.PII: S 0 9 5 8 - 6 9 4 6 ( 0 0 ) 0 0 0 7 3 - X

1995; Singh, 1995), primarily with the objective of ex-plaining the heat stability of milk expressed as the heatcoagulation time at 1403C (HCT). The e!ect of milkcomposition, additives and possible pretreatments onHCT is mainly of interest in the production of traditionalsterilised milk and UHT products. It is not obviouswhether the results obtained at this temperature can beextrapolated directly to lower temperatures, e.g. 903Cwhich is commonly used in the production of fermentedmilk products.

Heating milk above 703C causes denaturation of wheyproteins (Singh & Creamer, 1992; Singh, 1995). The mainheat-induced complex is formed between denaturedb-lactoglobulin and i-casein at the micellar surface viasulphydryl}disulphide interchange reactions and hydro-phobic interactions (Elfagm & Wheelock, 1978; Haque& Kinsella, 1988). However the fate of the denaturedwhey proteins, i. e. whether they associate with the caseinmicelles or form aggregates consisting only of whey pro-teins, depends on several factors: heating temperature,time, pH, ionic strength and the concentrations of solublecalcium and phosphate and dissociation of i-casein fromcasein micelles (Singh & Fox, 1985, 1986, 1987a, b).

Allmere, AndreH n, and BjoK rck (1997) found that afterheating the milk samples at 903C for 10min and sub-sequent cooling to 223C, almost all a-lactalbumin wasrecovered in the whey fraction after ultracentrifugation.These results con#ict with the "ndings of Parnell-Clunies, Kakuda, and Irvine (1988) who separated thecasein/denatured whey protein complex by isoelectricprecipitation at pH 4.6. These "ndings indicate that thedenaturation of a-lactalbumin may be sometimes revers-ible, as suggested by RuK egg, Moor, and Blanc (1977).

When milk is heated at temperatures up to 903C,changes in the size of casein micelles are reported; how-ever, the magnitude of the changes is dependent on theanalytical method used (Singh & Creamer, 1992). Dal-gleish, Pouliot, and Paqiun (1987) studied the e!ect ofheat treatment on the mean micellar size by the use ofphoton-correlation spectroscopy (PCS) and observedonly minor changes. More pronounced changes havebeen revealed by the use of gel permeation chromatogra-phy on porous glass and electron microscopy (Moham-mad & Fox, 1987).

In addition to the interactions between heat-denaturedwhey proteins and i-casein, heat-induced changes at thistemperature are primarily due to the precipitation ofcalcium phosphate as reviewed by Singh and Fox (1989).This insoluble material is protected against sedimenta-tion through the association with casein micelles (Even-huis & de Vries, 1956), and on subsequent cooling, someor all of this material is redissolved, if the heating temper-ature had not exceeded 1103C (Kannan & Jenness, 1961).

The e!ect of milk protein polymorphism on the heat-induced interactions between whey proteins and caseinmicelles has been the subject for only a few investigations

(Dannenberg & Kessler, 1988a, b; Parnell-Clunies et al.,1988; Allmere et al., 1997; Allmere, AndreH n, LundeH n,& BjoK rck, 1998). These authors have concentrated main-ly on the di!erent polymorphs of i-casein and b-lacto-globulin and their impact on denaturation rate and lossof native b-lactoglobulin from the whey fraction.

The objectives of this study were as follows:

f screening of genetic polymorphism of the major milkproteins in milk from the university herd of the Norwe-gian Red Cattle breed,

f e!ects of genetic milk protein variants on the contentof protein and minerals,

f e!ects of genetic milk protein variants on mean size ofnative and heated casein micelles,

f e!ects of di!erent feeding regimes on content of pro-tein and minerals,

f e!ects of di!erent feeding regimes on mean size ofnative and heated casein micelles.

2. Materials and methods

2.1. Milk samples

Milk samples were obtained from 58 heifers in theuniversity herd of the national Norwegian Red Cattlebreed. The animals, being out on pasture, were all in midlactation. According to the feeding regimes the animalswere divided into three groups. Group 1 (30 animals) andGroup 2 (11 animals) were fed commercial supple-mentary concentrates and the third group (Group 3E,17 animals) received ecologically rolled barley as asupplement.

Milk samples of 40mL from each cow were collected(from morning milking), preserved, defatted and kept atroom temperature for measurements of pH, Ca2`-ionactivity and mean micellar size and separation of thewhey fraction. The milk samples were then kept at!203C for further use.

Sodium azide (0.02w/v%) was used as an anti-micro-bial agent and was added from a 8.0w/v% stock solutionimmediately after collection of the milk samples.

The milk fat was removed by low-speed centrifugationat 2000 g for 30 min at 303C. The samples were rapidlycooled at 43C for 3min to solidify the fat, and the skim-med milk was removed through a folded "lter.

2.2. Protein content

The total protein content of the skimmed milk (TN)and the whey fraction (WPN including whey proteinsand non-protein nitrogen) was determined by the semi-micro Kjeldahl method (IDF standard 20B, 1993) usingmodi"ed amounts of sample and reagents. To samples ofapproximately 0.5mL of milk or 2.0mL of whey, 3mL of

314 T.G. Devold et al. / International Dairy Journal 10 (2000) 313}323

https://www.researchgate.net/publication/237656178_Relation_of_Milk_Serum_Proteins_and_Milk_Salts_to_the_Effects_of_Heat_Treatment_on_Rennet_Clotting1?el=1_x_8&enrichId=rgreq-a63a8071-c7a3-4e61-9871-3d1960ede051&enrichSource=Y292ZXJQYWdlOzIyMjA2Nzg3OTtBUzoyMDExMTk1NzQ4MjcwMDhAMTQyNDk2MjA1MTczNA==

concentrated sulphuric acid and one Kjeltab containing1.5 g K

2SO

4and 7.5mg Selenium were added.

After complete digestion and prior to destillation,30mL water and 25mL NaOH (33w/v%) were added tothe samples. The ammonia liberated during destillationwas collected in 4% boric acid, and the distillate wastitrated against 0.01 N HCl. The protein content of thesamples was calculated from the nitrogen content usinga conversion factor of 6.38.

The whey fraction was obtained from the skimmedmilk by isoelectric precipitation of the caseins at pH 4.6,according to a modi"ed method of Ascha!enburg andDrewry (1959) and Langsrud and Hadland (1971).

These results were used to calculate the protein con-tent of the casein fraction as CN"(TN!WPN)]6.38and also the casein number.

2.3. Genetic variants of caseins and whey proteins

The genetic variants of the di!erent whey proteinsfrom individual animals were determined by isoelectricfocusing according to the method of Vegarud et al. (1989)and the casein polymorphs were determined according tothe method of Erhardt (1989) using a modi"ed mixture ofcarrier ampholytes, Ampholine pH 3.5}5.0, PharmalytepH 4.2}4.9, Pharmalyte pH 4.5}5.4, Ampholine pH 4}6and Pharmalyte pH 5}6 in the ratio 3 : 2 : 1.25 : 1.25 : 1.

2.4. Mean size of native and heated casein micelles

Mean size of casein micelles was determined by photoncorrelation spectroscopy (PCS) on skimmed milks. Themeasurements were performed using a Coulter ModelN4MD apparatus (Coultronics, Hialeah, FL, USA)equipped with a He}Ne laser (632.8 nm).

The instrument was operated in the unimodal modeand the measurements were performed in glass cuvettesat a scattering angle of 903 at 203C. Viscosity and refrac-tive index of the solvent}simulated milk ultra"ltrate(SMUF, Jennes, & Koops, 1962)*were 1.002 cP and1.333, respectively. After a delay of 90 s, each sample wasrun in triplicate, the length of each run was 90 s, sampletime was set to auto.

Prior to analysis the samples of skimmed milk werediluted 1 : 1000 in simulated milk ultra"ltrate (SMUF) toobtain a concentration satisfying the required count rate(photons per second) speci"ed by the manufacturer.

To avoid the interference of dust during the lightscattering measurements, the SMUF was "lteredthrough a 0.22lm "lter, the cuvettes were carefully rinsedin SMUF between each run, and the diluted sampleswere "ltered through a 0.8lm "lter.

Due to practical reasons the milk samples were heatedimmediately after collection and skimming, and the sizedetermination experiments were performed the followingday. To avoid changes in size distribution of heated

casein micelles due to whey protein renaturation andreversible changes in milk salt balance, glutardialdehyde(GA) was added to the heated milk samples immediatelyafter cooling to room temperature.

In order to investigate the e!ect of the concentration ofGA, "nal concentrations of 0.5, 1.0 and 2.0% (v/v) weretried out.

1.0, 2.0 and 4.0% (v/v) GA was added to SMUF, andthe pH was adjusted to 6.7 with 1 M KOH in order toavoid the pH e!ect of the acidic glutaraldehyde. Thissolution was added to the heated milk samples to obtain"nal concentrations of GA of 0.5, 1.0 and 2.0% (v/v). Themean size of the casein micelles was measured after 2, 5,10, 15 and 30 min and after 24 and 48 h to observepossible changes.

Skimmed milk samples were heated to 70, 80 and 903Cfor 5, 10, 15 and 30 min prior to cooling to room temper-ature.

2.5. Content of milk salts

The calcium ion activity was measured using a pH andion meter (Radiometer, DK) equipped with an ion selec-tive electrode and a calomel reference electrode. Theapparatus was calibrated using Ca-containing standardsolutions prepared according to Holt, Dalgleish, andJennes (1981).

The total contents of calcium and magnesium weredetermined by atomic absorption (GBC Atomic absorp-tion spectrophotometer GBC 906, Scienti"c EquipmentPFY, Ltd., Melbourne, Victoria, Australia).

The content of citrate was analysed using high-pressure liquid chromatography as previously des-cribed (Narvhus,"steraas, Mutukumira, & Abrahamsen,1998).

2.6. Statistical analyses

The general linear models procedure of MINITAB(version 12.1, 1998) was adopted to examine the experi-mental data. Statistical signi"cance was examined by themethod of least squares by use of a linear model whichincluded stage of lactation, group of cows (to evaluate thee!ect of di!erent feeding regimes), genetic variants of a

41,

b-, i-casein and b-lactoglobulin as "xed e!ects in allmodels. In addition, the content of casein and wheyprotein, casein number, pH and minerals examined(Ca2`, Ca, Mg and citrate) were included as covariablesin the models for mean size of native and heated caseinmicelles. In the models for content of protein, casein andnon-casein proteins, pH and minerals examined (Ca2`,Ca, Mg and citrate) were included as covariables.

The least-frequent genetic variants; a41

-casein CC,b-casein A1B/A2B, i-casein BB were omitted from thestatistical analysis due to the low number of cows (onlyone or two animals).

T.G. Devold et al. / International Dairy Journal 10 (2000) 313}323 315

tovede

Highlight

Fig. 1. Frequency distribution of genetic variants of a41

-, b- and i-casein and b-lactoglobulin in milk from the university herd of Norwe-gian Red Cattle.

3. Results and discussion

3.1. Frequency distribution of genetic variants

Fig. 1 shows the frequency distribution of genetic vari-ants of caseins and whey proteins. The most dominantpolymorphs from the animals included in this study werea41

-casein BB (83%), b-casein A1A1, A1A2 and A2A2(21, 52 and 24%, respectively) i-casein AA, AB and AE(65, 14 and 18%, respectively) and b-lactoglobulin ABand BB (34 and 52%, respectively). a

41-Casein CC, b-

casein A1B and A2B and i-casein BB occurred at verylow frequencies. This fact complicates the calculations ofaverage results and properties related to di!erent poly-morphs. For the two remaining major milk proteins,a42

- casein and a-lactalbumin, only one genetic variantwas observed for each protein: a

42-casein AA and a-

lactalbumin BB.These results are in good agreement with preliminary

results from a study including 800 animals of the samebreed (Devold, unpublished results); however, consider-able variation was observed when compared with otherwestern breeds concerning frequencies and genotype ofb-casein and i-casein (McLean, Graham, Ponzoni,& McKenzie, 1984; Bovenhuis et al., 1992; Delacroix-Buchet et al., 1993; Buchberger, 1995; Ikonen, Ojala,& Ruottinen, 1997; Winkelman & Wickham, 1997).

3.2. Milk composition

Table 1 shows the average milk composition of thecows with di!erent feeding regimes and the di!erentgenetic variants.

Table 2 displays the e!ects of di!erent variables on thecontent of protein, casein, whey protein and casein num-ber. The genotype of a

41-casein had a statistically signi"-

cant e!ect on the content of protein and casein, and thecontent of whey protein and the casein numberwas signi"cantly in#uenced by group of cows (di!erent

feeding regime). Among the components in the milk saltfraction examined, the content of citrate was signi"cantlycorrelated with the content of whey protein and thecasein number.

3.2.1. Ewect of feeding regimeAs shown in Tables 1 and 2, the content of whey

protein and the casein number was signi"cantly in-#uenced by the feeding regime.

The highest content of protein and casein (3.50 and2.72%) was observed in milk from cows in feeding regime2 (Group 2), while the content in milk from cows infeeding regime 1 (Group 1) and feeding regime 3E (Group3E) were 3.19/2.39% and 3.23/2.48%, respectively, how-ever, these di!erences did not prove to be statisticallysigni"cant.

The total whey protein content (0.79%) in milk fromcows in feeding regime 1 (Group 1) was signi"cantlyhigher than the whey protein content (0.75%) in milkfrom the cows fed ecologically (Group 3E).

The casein number of Group 2 and Group 3E (77.7and 76.9, respectively) was signi"cantly higher than thecasein number in Group 1 (75.3).

3.2.2. Ewect of genetic variants of as1-caseinThe average content of protein, casein and whey pro-

tein derived from our data was signi"cantly higher for theBB genotype than for the BC genotype of a

41-casein

(3.28/2.50/0.79 and 3.11/2.37/0.74% respectively) asshown in Tables 1 and 2. This was not in agreement withmost previous results as reviewed by Puhan (1997) andJakob (1994) or with the "ndings of Lodes et al. (1997)who found the BC genotype to have a signi"cantly highercontent of protein and casein. This e!ect is explained bymany investigators by the higher content of casein asso-ciated with the a

41-CN C as reviewed by Jakob (1994).

Other workers found no e!ect of polymorphism ofa41

-casein on milk protein content (McLean et al., 1984;Gonyon et al., 1987; Ng-Kwai-Hang, Monardes, & Hay-es, 1990).

3.2.3. Ewect of genetic variants of b-caseinAs shown in Tables 1 and 2, no signi"cant e!ect of

di!erent polymorphs of b-casein was observed in thecontent of protein and casein; the trend was as follows:A2A2'A1A2, A1A1 (3.39/2.61'3.23/2.44, 3.17/2.45%).

The lowest content of whey protein was observed forthe A1A1 variant (0.73% vs. 0.79 and 0.78% for the A1A2and A2A2 variants, respectively). Regarding casein num-ber, the lowest value was observed for the A1A2 variant(75.5 vs. 77.1 and 77.0 for the A1A1 and A2A2 variants,respectively). These "ndings indicate that the proportionsof the di!erent protein fractions in individual milk sam-ples may vary according to genotypes of b-casein.

McLean et al. (1984) found no statistically signi"cantrelationship between di!erent polymorphs of this protein


Table 1Average milk composition for di!erent feeding regimes and genotypes of milk proteins

N pH Milk content Mean micellar size

Ca2` Ca Mg Citrate Protein Casein W. prot. Casein Native Heated Incr.(mM) (mM) (mM) (ppm) (%) (%) (%) no. (nm) (nm) (nm)

Fed. Reg. GR. 1 30 6.70 2.49 25.49 4.04 1987 3.19 2.39 0.79 75.3 175 183 11GR. 2 11 6.74 2.25 28.92 4.49 1864 3.50 2.72 0.78 77.7 180 190 10GR. 3E 17 6.68 1.97 25.09 3.98 1831 3.23 2.48 0.75 76.9 191 201 9

Geneticvariants

a41

-CN BB 48 6.70 2.31 26.04 4.13 1918 3.28 2.50 0.79 76.0 182 191 11a41

-CN BC 8 6.73 2.12 25.03 3.90 1822 3.11 2.37 0.74 76.2 178 187 10a41

-CN CC 2 6.73 2.56 29.64 4.46 2316 3.25 2.66 0.59 81.7 161 171 10b-CN A1A1 12 6.69 2.18 25.34 4.24 2088 3.17 2.45 0.73 77.0 184 190 8b-CN A1A2 30 6.70 2.37 25.98 4.05 1899 3.23 2.44 0.79 75.5 181 191 12b-CN A1B 1 6.67 2.57 28.00 3.40 1871 3.24 2.42 0.82 74.7 189 194 14b-CN A2A2 14 6.72 2.15 26.86 4.23 1807 3.39 2.61 0.78 77.1 177 186 10b-CN A2B 1 6.68 3.00 21.90 3.20 2053 3.25 2.48 0.77 76.3 181 192 11i-CN AA 38 6.70 2.30 25.82 4.07 1903 3.23 2.46 0.77 76.0 181 191 11i-CN AB 8 6.72 2.15 26.66 4.09 1940 3.39 2.59 0.80 76.4 175 183 10i-CN AE 11 6.69 2.32 26.48 4.29 1983 3.27 2.51 0.76 76.6 186 192 8i-CN BB 1 6.63 2.88 23.6 3.60 1607 3.06 2.35 0.71 76.8 151 155 8b-lg AA 5 6.63 2.35 24.51 4.05 1817 3.26 2.45 0.80 75.4 190 207 20b-lg AB 23 6.72 2.33 26.59 4.16 1931 3.35 2.54 0.81 75.9 180 192 13b-lg BB 30 6.70 2.25 25.84 4.07 1924 3.19 2.45 0.75 76.6 179 185 7

Table 2Source of variation for content of protein, casein, whey protein and casein number

Variable Protein Casein Whey protein CN. no.

E!ect of variable

a41

-CN (BB'BC)! (BB'BC)! " "

b-CN " " " "

i-CN " " " "

b-LG " " " "

Fed. reg. " " (Gr.1'Gr.3E)! (Gr.2, Gr.3E'Gr.1)!W.O.Lact. " " " "

Casein (%) " " " "

WP (%) " " " "

Casein no. " " " "

pH " " " "

Ca2` " " " "

Ca " " " "

Mg " " " "

Citrate " " ! !

!p(0.05."N.S."non-signi"cant.

and the content of protein and casein; however theseresults con#ict with the "ndings of Lodes et al. (1997),who found the relationship (A1A1'A1A2'A2A2) tobe signi"cant by similar to the trends observed by othersas reviewed by Puhan (1997).

3.2.4. Ewect of genetic variants of i-caseinThe polymorphism of i-casein has received consider-

able interest, and especially much attention has been paid

to the BB variant due to its reported association withhigher protein content and superior cheese making prop-erties (van den Berg, Escher, de Konig, & Bovenhuis,1992; Puhan, 1997; Walsh et al., 1998). As previouslymentioned the i-casein BB variant occurs at a very lowfrequency in milk from Norwegian Red Cattle. Most inves-tigators seem to agree that the protein content in milk isrelated to the di!erent polymorphs of i-casein in thefollowing way: BB'AB'AA (van den Berg et al., 1992;


Table 3Mean size of the casein micelles in unheated and heated (903C in 5 and30min) skimmed milk samples from a selection of individual cows with(2%) and without (native) addition of 2% glutardialdehyde (GA)

Cow no. Mean size of casein micelles (nm)

Native 2% GA Di!

2235 2235, room temp 165 162 3903C, 5min 177 170 7903C, 30min 185 185 0

2315 2315, room temp 154 155 !1903C, 5min 162 154 8903C, 30min 165 161 4







Aleandri, Buttazoni, Schneider, Caroli, & Davoli, 1990;review by Jakob, 1994) As can be derived from Table 1,our results show a similar trend concerning protein andcasein content: AB'AE'AA, although the di!erenceswere not statistically signi"cant (Table 2). Very few pub-lished results are available concerning the variant AE ofi-casein. This may be due to inadequate resolution in theseparation techniques used to identify the genotypes ofthis protein. In the study by Lodes et al. (1997) nosigni"cant e!ect of i-casein polymorphism was observedin the content of protein and casein, with a trend(AA'AE'AB) opposite to our "ndings.

3.2.5. Ewect of genetic variants of b-lactoglobulinTables 1 and 2 display the e!ects of b-lactoglobulin

polymorphs on the content of protein, casein, non-caseinprotein and casein number. No statistically signi"cantrelationships were observed. The highest content of pro-tein and casein was observed for the AB variant(3.35/2.54% vs. 3.26/2.45 and 3.19/2.45% for the AA andBB variants, respectively). As pointed out by Jakob(1994) and Puhan (1997) the e!ect of this protein on theprotein content of milk is still controversial; however,several workers observed trends opposite to our "ndingsregarding the content of protein and casein. Concerningthe content of whey protein (AA, AB'BB) and caseinnumber (BB'AB'AA), the trends of our results agreewith relationships mentioned by Jakob (1994) and Puhan(1997), but in their reviews they reported only on thehomozygote variants.

3.3. Mean size of native and heated casein micelles

3.3.1. Ewect of the addition of glutardialdehydeGlutardialdehyde (GA) was added to the heated milk

samples to avoid possible disaggregation and restruc-turation of casein micelles/casein aggregates. Table 3 dis-plays the mean size of the casein micelles in unheated andheated (903C, 5 or 30min) skimmed milk samples froma selection of individual cows with and without the addi-tion of 2% glutardialdehyde (GA). Fixation of caseinmicelles with GA stabilised the casein micelles against thepossible salt balance induced changes, and caused onlya small decline in size, 3}6 nm, in most samples.

Table 4 shows the e!ect of di!erent concentrations ofGA (0.5, 1.0 and 2.0%) on the mean size of casein micellesin unheated and heated milk samples. Only minor di!er-ences within the experimental error were observed be-tween the di!erent GA concentrations. GA is widely usedto stabilise aggregated states of proteins and preventdissociation induced by changes in temperature, pH ionicstrength, etc.

When milk is heated, changes in the milk salt balancetake place, as reviewed by Singh and Creamer (1992). Theconcentrations of soluble phosphate and of soluble andionic calcium are reduced, the extent being dependent on

the severity of the heat treatment. The newly formedinsoluble material is protected against sedimentationthrough the association with casein micelles. On sub-sequent cooling some or all of this material are redissol-ved, if the heating temperature did not exceed 1103C. Itremains uncertain whether or not this will lead to cha-nges in the micellar size distribution, but to avoid pos-sible disaggregation and restructuration of caseinmicelles/casein aggregates due to reversible changes inthe milk salt balance prior to determination of mean size,glutardialdehyde (GA) was added to the heated milksamples.

Addition of GA leads to a stabilisation of proteinaggregates by the formation of intra- and intermolecularcovalent crosslinks. The reaction between GA and pept-ide chains is complex and involves the e-amino group oflysine (Lundblad & Noyes, 1984).

The remaining experiments on mean size of heatedcasein micelles were performed with 2.0% GA.

3.3.2. Ewect of various temperatures and holding timesWhen milk is heated, several changes in the protein

fraction take place. The degree of changes is dependent


Fig. 2. Mean size of casein micelles in unheated milk, native and GA-"xed (GA, UNH) and milk heated, GA-"xed, at di!erent temperatures andholding times from two di!erent cows (nos. 2235 and 2391).

Table 4E!ect of di!erent concentrations of GA (0.5, 1.0 and 2.0%) on the meansize of casein micelles in unheated and heated milk samples from twoanimals (cow nos. 2235 and 2262)

Cow no. Temp (3C) Mean size of casein micelles

Native 0.5% GA 1.0% GA 2.0% GA

2235 199 189 192 194803C, 5min 191 192 193803C, 30min 196 199 201903C, 5min 199 203 205903C, 30min 284 283 290

2262 160 156 153 154803C, 5min 153 155 156803C, 30min 161 160 159903C, 5min 160 159 157903C, 30min 185 183 180

on the heating temperatures and times employed. Di!er-ences in the size distribution of the casein micelles occurpartly because of dissociation/aggregation phenomenataking place in the casein fraction and partly because ofassociation of denatured whey proteins.

Individual milk samples from a selection of cows wereheated at 70, 80, and 903C for 5, 10, 15 and 30min. Fig. 2shows the mean micellar size in milk from two animals(cows nos. 2235 and 2391) responding quite di!erently toheat treatments. At 70 and 803C both showed a slightdecrease in mean size, and at 903C the mean micellar sizeof milk from cow no. 2391 remained constant, while themean size of the casein micelles of milk from cow no. 2235showed a marked increase.

The most pronounced e!ect on mean micellar size, ifany, was observed at 903C for 30min in all heated sam-ples; when this e!ect was large, size increments wereobserved at 903C in 5 min as well.

The mean sizes of native and heated (903C, 30min, 2%GA) casein micelles from individual animals (58 cows) of

the university herd are displayed in Fig. 3. The nativemean sizes are within the 149}222nm range, which indi-cates large variations between individual animals. Thechanges in the mean size of the casein micelles due to heattreatment (903C, 30min, 2% GA) varied between di!er-ent animals from!7 to 52nm. These heat-induced cha-nges were independent of native size as con"rmed bya linear regression coe$cient of R2"70.

Dalgleish et al. (1987b) analysed the mean micellar sizeof pooled milk samples heated at temperatures up to903C by means of photon correlation spectroscopy andobserved only minor changes, however, our results indi-cate large individual di!erences.

3.3.3. Ewect of milk compositionThe mean size of casein micelles has been reported to

be in#uenced by the milk composition, genetic variantsof milk proteins, content of protein, casein, and wheyprotein (Delacroix-Buchet et al., 1993; Lodes et al., 1996;Walsh et al., 1998). A similar relationship may be ex-pected for the relationship between the mean size ofheated micelles and milk composition. In order to high-light the impact of the di!erent compositional factors onthe mean size of native and heated micelles and heat-induced changes in size, analysis of variance was appliedto the experimental data (Table 5).

The average results for all compositional factors fordi!erent feeding regimes and di!erent polymorphs ofcaseins and b-lactoglobulin are included in Table 1.

As can be derived from Table 5, mean size of nativeand heated casein micelles was signi"cantly in#uenced bythe following parameters: group of cows (di!erent feedingregime), genotype of a

41-casein (native mean size only)

and i-casein, pH and the content of casein, whey proteinand casein number. None of the components in the milksalt fraction examined did signi"cantly in#uence themean size of native and heated micelles. The heat-in-duced changes in mean micellar size were signi"cantly


Fig. 3. Scatter plot of mean size of native and heated (903C, 30min,2%GA) casein micelles for all animals in the experiment.

Table 5Source of variation for mean size of native and heated casein micellesand for heat-induced change in size

Variable Mean size of casein micelles

Native Heated Change

E!ect of variable

a41

-CN (BB'BC)! # #

b-CN # # #

i-CN (AA, AE'AB)" (AA, AE'AB)! #

b-lg # # #

Fed. reg. (Gr.3E'Gr.1)! (Gr.3E'Gr.1)! #

W.O.Lact. # # #

Casein (%) " " #

WP (%) " " #

Casein no. " ! #

pH ! ! #

Ca2` # # !

Ca # # #

Mg # # #

Citrate # # #

!p(0.05."p(0.01.#N.S."non-signi"cant.

a!ected by the calcium ion activity. Analysis of regres-sion revealed that calcium ion activity accounted forapproximately 40% of the variability in heat-inducedchanges in micellar size (R2"42%) regardless of thegenetic variants of milk proteins.

3.3.4. Ewect of feeding regimeThe mean size of native casein micelles was a!ected by

feeding regime; the mean size of micelles (191 nm) in milkfrom the ecologically fed cows (Group 3E) was signi"-cantly larger than the mean micellar size (175 nm) in milkfrom cows in feeding regime 1 (Group 1). Size of caseinmicelles has been reported to be inversely related to thecontent of protein/casein in the milk, although in thisstudy no such simple relationship was observed regard-ing the di!erent feeding regimes; the average pro-

tein/casein contents in group 3E and group 1 were3.23/2.48 and 3.19/2.39%, respectively. The highest pro-tein content (3.50%) was observed in the milk from cowsin feeding regime 2 (Group 2), having medium sizedcasein micelles (180 nm).

A similar relationship was observed between the meansize of heated micelles and feeding regimes; the averagemean micellar size (201 nm) in milk from cows fed eco-logically (Group 3E) remained signi"cantly larger thanthe micelles (183 nm) in milk from cows in feeding regime1 (Group 1) after heating. The di!erent feeding regimesdid not in#uence the degree of heat-induced changes inmicellar size.

3.3.5. Ewect of genetic variants of as1-caseinAccording to Table 5, a

41-casein signi"cantly a!ected

the average mean size of native casein micelles; a41

-caseinBB gave signi"cantly larger micelles than the BC variant(182 and 178 nm, respectively). Lodes et al. (1996) ob-served a similar trend; however, in their study the meansizes of the two genotypes were not signi"cantly di!erent.

The di!erences in the average mean size of heatedmicelles between a

41-casein polymorphs show a similar

but not signi"cant trend.As previously discussed, the average content of pro-

tein, casein and whey protein derived from our data wassigni"cantly higher for the BB genotype than the BCgenotype. This is in contrast with the "ndings of Lodeset al. (1997), who showed the content of protein andcasein of the BC genotype being signi"cantly higher.

3.3.6. Ewect of genetic variants of b-caseinNo signi"cant relationship was observed between the

genetic variants of b-casein and average mean size ofnative micelles, as displayed in Table 5. Our results(A2A2(A1A2(A1A1) were similar to the statisticallysigni"cant trend observed by Lodes et al. (1996).

The di!erent b-casein polymorphs had no e!ect onchanges observed during heating.

3.3.7. Ewect of genetic variants of i-caseinConsistent with earlier "ndings (Delacroix-Buchet

et al., 1993; Lodes et al., 1996; Walsh et al., 1998) geno-types of i-casein signi"cantly in#uenced the averagemean micellar size. As can be derived from Tables 1 and3, micelles containing i-casein AB were signi"cantlysmaller than those with the AA and AE polymorphs.A similar di!erence was observed after heating.

3.3.8. Ewect of genetic variants of b-lactoglobulinOur results in Table 5 showed no signi"cant e!ect of

di!erent b-lactoglobulin variants, however, on an aver-age, native micelles in milk with the AA polymorph werelarger (190 nm) than with the AB and BB variants (180and 179nm, respectively, Table 1). In the report by Lodeset al. (1996) the same trend was statistically signi"cant.


These di!erences became more pronounced after heat-ing, AA'AB'BB ( 207, 192 and 185 nm, respectively);however, they did not prove to be statistically di!erent.

These results cannot be explained by di!erent reactionrates of the loss of b-lactoglobulin genotypes duringheating at 903C. The BB variant has been reported to bemore heat sensitive than the AA variant (Dannenberg& Kessler, 1988a; Parnell-Clunies et al., 1988; Allmereet al., 1997) at this temperature.

The di!erences in heat-induced changes in mean micel-lar size for various polymorphs of b-lactoglobulin mightbe related to the di!erences in the micellar surface area.The micelles having the largest mean size have thesmallest total surface area to be covered with denaturedwhey proteins during heat treatment leading to an in-crease in the thickness of the whey protein layer.

During heating, pH of milk is reported to in#uencethe fate of whey proteins and i-casein, the latter attemperatures above 903C. In our study, pH of the milkproved to statistically a!ect the mean size of native andheated casein micelles, and the most pronounced di!er-ence in pH related to milk protein polymorphs wasassociated with b-lactoglobulin. The lowest pH value wasfound in the milk containing the AA genotype. This milkhad the largest mean size of native and heated caseinmicelles.

Our results show a trend similar to the e!ects on meanmicellar size observed when pH was increased by addi-tion of alkali. An increase in pH from 6.7 to 6.9 led to anincrease in micellar diameter (Dalgleish et al., 1987).

After heating above their respective denaturation tem-perature, the two major whey proteins, a-lactalbuminand b-lactoglobulin are present in the micelle fraction(Manji & Kakuda, 1986; Law, Banks, Horne, Leaver,& West, 1994). The main complex formed is probablybetween b-lactoglobulin and i-casein (Hill, 1989; Jang& Swaisgood, 1990) through the formation of disul"debonds and hydrophobic interactions (Smits & vanBrouwershaven, 1980; Noh and Richardson, 1989; Noh,Richardson, & Creamer, 1989). The above investigationscannot fully explain the fate of the whey proteins afterheat treatment; either forming a complex with micellari-casein or producing very large aggregates of self-asso-ciated denatured whey proteins precipitated togetherwith casein micelles during the separation step (ultra-centrifugation or isoelectric precipitation) as suggestedby Old"eld, Singh, and Taylor (1998).

4. Conclusion

It was not within the scope of this study to perform anin-depth investigation of the e!ects of nutrient supply onmilk composition. However, our results indicate that thefeeding regime a!ects content of proteins and minerals,that the di!erent genotypes of a

41-casein signi"cantly

a!ected the composition of the protein fraction in themilk, and that the di!erent genotypes of a

41-casein

and i-casein a!ected the mean size of native andheated casein micelles. In spite of the many publishedreports on the topic, the e!ects of milk protein genotypeson the protein composition are con#icting, and furtherinvestigations on the milk from Norwegian Red Cattlewould be important to con"rm the trends observed inthis study.

Acknowledgements

This paper was presented at the workshop on &MilkProteins*Structure and Functional Properties' held inNaantali, Finland, 28}30 November 1998. The workshopwas organized as part of activities of the Nordic networkon Milk Proteins, which was funded by the Nordic Acad-emy for Advanced Study (NorFA).

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