515Lait 85 (2005) 515526 INRA, EDP Sciences, 2005DOI: 10.1051/lait:2005037

Original article

Effect of heat treatment at alkaline pH on the rennet coagulation properties of skim milk

Olivia MNARD, Bndicte CAMIER, Fanny GUYOMARCH*

UMR 1253, Science et Technologie du Lait et de luf, Inra-Agrocampus Rennes, 65 rue de Saint-Brieuc, 35042 Rennes Cedex, France

Received 24 February 2005 Accepted 19 May 2005

Published online 1 October 2005

Abstract Reconstituted skim milk was heated at 90 C for 30 s at pH values ranging from 6.6 to8.1, stored overnight at 5 C then renneted at pH values ranging from 6.2 to 6.6. The heat-induceddisimprovement of the rennet coagulation properties of the milk (longer rennet coagulation time andlower gel firmness) were partially reduced as heat-treatment pH increased, except at pH 6.6. Theseproperties were related to the increased heat-induced dissociation of micellar -casein and subse-quent formation of serum, rather than micelle-bound, heat-induced whey protein/-casein aggrega-tes. It was postulated that such distribution of the denatured whey protein and -casein slightlyreduced the detrimental effects of heating on the enzymatic coagulation, as well as helped earlierdestabilisation of the micelles. These results are discussed with respect to the amplitude of the effectof the heat-treatment pH, as well as the side effects of alkalinisation and heat treatment on caseindissociation.

whey protein / heat treatment / rennet coagulation / pH

pH pH6.6~8.1 90 C 30s, 5 C pH6.2~6.6 pH pH6.6 - - - pH / / / pH

Rsum Effets dun traitement thermique pH alcalin sur laptitude du lait crm la coa-gulation prsure. Du lait crm reconstitu est trait thermiquement 90 C pendant 30 s despH variant de 6,6 8,1, stock 5 C pendant une nuit puis emprsur des pH variant de 6,2 6,6.La perte daptitude la coagulation prsure des laits chauffs (temps de coagulation plus long etmoindre fermet) a t rduite pour des pH de traitement thermique croissants, sauf pH 6,6. Cesproprits ont t mises en relation avec un taux de dissociation accru de la casine quand le pHde traitement thermique augmente, et donc une proportion accrue dagrgats thermo-induits de pro-tines sriques dnatures et de casine dans la phase soluble du lait. Il est postul que cette dis-tribution en protines rduit lgrement les effets rdhibitoires du traitement thermique sur lacoagulation enzymatique, et acclre la dstabilisation des micelles de casines. Ces rsultats sont

* Corresponding author ( ): fanny.guyomarch@rennes.inra.fr

Article published by EDP Sciences and available at http://www.edpsciences.org/lait or http://dx.doi.org/10.1051/lait:2005037

516 O. Mnard et al.

discuts relativement lamplitude de leffet du pH de traitement thermique ainsi qu la dissocia-tion alcaline ou thermo-induite des autres casines.

protine srique / traitement thermique / coagulation prsure / pH

1. INTRODUCTION

Incorporation of the whey proteins intocheese has long been extensively investi-gated as a means of adding value to dairyproducts, for economic reasons as well as toincrease nutritive or textural properties ofcheeses. Ultrafiltration of cheese milk,addition of particulated whey proteins (byheating and homogenisation of whey) and/or heat treatment of the milk are the threemethods generally used to meet these goals.Of the three methods, heating is the leastsatisfactory, as heated milk exhibits extendedrennet coagulation time (RCT) and reducedgel firming rate, eventually leading to theformation of soft, grainy and humid curds[7, 19, 29, 31]. These heat-induced techno-logical changes have been correlated to thedenaturation of whey proteins and forma-tion of both micelle-bound and soluble pro-tein aggregates from the interactionbetween denatured whey proteins and -casein. The enzymatic phase of renneting isslowed due to steric hindrance of thePhe105-Met106 bond of the -casein mole-cule by the attached whey proteins. Curdfusion is also inhibited as whey protein-coated micelles are better at retaining theirintegrity [10, 23, 31, 33].

Upon heating at pH > 6.7, heat-inducedmilk protein aggregates are reported to besmaller, more numerous [24] and with ahigher proportion of disulphide bonds com-pared with hydrophobic interactions [12,13]. Upon heating milk at pH > 6.9, the pro-portion of heat-induced whey protein/-casein aggregates found in the serum phaseis increased, while casein micelles aredepleted in -casein as a result of their inter-action with denatured whey protein and dis-sociation [25, 2628, 32]. The objective ofthis study was to investigate whether therennet coagulation properties of skim milkheated at various pH values > 6.5 would beimproved by the fact that the heat-induced

whey protein/-casein aggregates would bepresent in the serum phase rather than on themicelle surface. The related depletion of themicelles in -casein could also help reduceflocculation time since less stabilising -casein is present from the start of the enzy-matic reaction.

Information on the effect of alkalineheat-treatment of milk prior to renneting isscarce and results are contradictory due tothe various experimental conditions used,especially with respect to post-heatingtreatments, e.g. pH changes or storage. VanHooydonk et al. [31] reported poor rennet-ing properties of milk heated at pH 7.5,while Leaver et al. [18] observed that thechymosin reaction rate decreased withincreasing pH > 6.7, but not beyond 7.8.Conversely, Imafidon and Farkye [14, 15]found that milk heated at pH 7.5 then acid-ified or pH-cycled has a lower RCT thanmilk heated at pH 6.7, as later agreed bySingh and Waungana [29]. Cheeses obtainedwere not different with respect to composi-tion, and Banks et al. [8] noticed that heat-induced defects of cheese curds werereduced when heating of the cheese milkwas performed at pH 7.1 or 7.5. Vasbinderand de Kruif [32] reported better rennetingproperties of skim milk heated at pH 6.9rather than 6.7.

2. MATERIALS AND METHODS

2.1. Reconstituted skim milk

Milk was reconstituted as 100 gL1ultra-low heat skim milk powder (wheyprotein nitrogen index = 9.5 [25]) and0.5 gL1 sodium azide in stirred deionisedwater at 40 C. The milk was stirred for atleast one hour following complete dissolu-tion, then left overnight at 5 C to completethe process.

Rennet coagulation properties of heated milk 517

2.2. Milk ultrafiltration permeate

Milk ultrafiltrate (MUF) was preparedfrom fresh pasteurised milk on a 8 kgmol1TAMI membrane (Tami Industries, Nyons,France) and stored at 5 C after addition of0.5 gL1 sodium azide.

2.3. Other materials

Other chemicals were from Sigma (St-Quentin-Fallavier, France), Panreac (Bar-celone, Spain), Merck (Fontenay-sous-bois,France), Prolabo (Fontenay-sous-bois, France)and Carlo Erba (Val-de-Reuil, France) andwere of analytical grade. Solvents wereobtained from Carlo Erba and were ofHPLC grade.

2.4. pH adjustmentThe adjustments of the pH of heat treat-

ment or renneting were, respectively, per-formed ~2 h prior to heat treatment, and ~2 hprior to Formagraph analysis. The reconsti-tuted skim milk samples were first equili-brated at room temperature for at least 1 h,then pH was adjusted using HCl and NaOH5 molL1 or 0.5 molL1 at the followingvalues: heat-treatment pH 6.6, 7.1, 7.6 and8.1; renneting pH: 6.2, 6.3, 6.4, 6.5 and 6.6.Much care was taken so that the volume ofHCl or NaOH added to the milk samples toreach the heat treatment or the renneting pHwas minimum ( 3 mLL1) to avoid signif-icant dilution.

Samples were left with agitation for afurther ~30 min at room temperature toensure equilibration prior to final pHadjustment. Adjusted pH values were allwithin 0.05 pH units.

2.5. Heat treatment

Six hundred mL of reconstituted skimmilk were heat-treated at 90 C for 30 s ona recirculating tubular heat-exchanger com-posed of an inox tubular coil (8 mm diam-eter, 7 m length) plunged in a thermostatedwater-bath and connected to a recirculatingcentrifuge pump (Micropump Inc., Vancou-ver, Canada). The flow rate was 135 Lh1and the flow was turbulent to ensure proper

heat transfer (Re ~3200). The heating-upperiod was < 4 min. The milk was thencooled down to room temperature in agi-tated ice water. After 1 h at room tempera-ture, samples were taken for separation ofthe micelle and serum phases, then the milkwas stored overnight at 5 C.

2.6. Rennet coagulation properties

Rennet coagulation properties of heat-treated or control pH-adjusted milks wereevaluated on day 1 after heat treatmentusing a Formagraph rheometer (Foss Elec-tric, Nanterre, France) through the meas-urement of the rennet coagulation time(RCT) and of gel firmness taken at 1 RCTafter coagulation (aR).

Ten-mL milk samples were equilibratedat 35 C prior to inoculation with 30 L oftraditional rennet solution (Berthelot 530,Laboratoires Abia, Meursault, France) dilutedto 10% w/w in deionised water. Forma-graph measurement was monitored at 35 Cfor 2 h.

2.7. Separation of the serum and colloidal phases of skim milk

Separation of the serum and colloidalphases of heat-treated and control milksamples was performed after heat treat-ment, cooling and equilibration of the milkat room temperature for 1 h. Separation wasperformed on 15-mL aliquots of milk usingultracentrifugation on a Sorvall Discovery90 SE centrifuge (Kendro Laboratory Prod-ucts, Courtabuf, France) equipped with a50.2 Ti rotor (Beckman Coulter, Fullerton,CA, USA). The samples were spun at19 400 rpm (~33 000 average g) for 65 minat 20 C. The supernatants were collectedby simply pouring them into a containerwithout draining or washing of the pellets.This fraction was designated as the serumphase or serum. The pellets were resus-pended in a volume of milk ultrafiltrateequivalent to the removed supernatant(~12.5 mL), at 5 C for at least 48 h underconstant agitation. This fraction was desig-nated as the colloidal or micellar phaseof the milk sample.

518 O. Mnard et al.

The materials found in the serum andmicellar phases of heated and unheatedskim milk separated by the above methodhave been extensively described elsewhere[11]. The low centrifugation speed used inthis method prevented sedimentation of theprotein materials present in the serum phaseof the milk, at the expense of small quanti-ties of residual micellar material thatremained in the supernatant [11].

2.8. Quantification of heat-induced protein transfers by reverse-phase high performance liquid chromatography (RP-HPLC)

The protein composition of the initialmilk samples and of their serum and colloi-dal phases separated by ultracentrifugationwere determined by RP-HPLC as describedin [16]. Briefly, the samples were diluted5 times with denaturing buffer (7 molL1urea, 20 mmolL1 Bis-Tris Propane, pH7.5, + 5 LmL1 of fresh -mercaptoetha-nol) then incubated for 1 h at 37 C. The col-umn was an Apex wide-pore C18 columnof 25 cm length, 0.46 cm inner diameter and7 m bead diameter (Jones Chromatogra-phy, Hengoed, UK). Buffer A was 0.106%v/v trifluoroacetic acid (TFA) in Milli-Qwater (Waters, Molsheim, France). BufferB was 0.1% v/v TFA in 80% v/v acetonitrilein Milli-Q water. Loop size was 30 L, tem-perature was 46 C, flow rate was1 mLmin1 and detection was at 214 nm.

The proportions of -casein and wheyproteins in both the micellar and the serumphases of the milk were calculated by divid-ing the peak area of the protein (-casein orwhey proteins) in each fraction (micellar orserum phase) by the average peak area ofthe same protein in the total reconstitutedskim milk. This average peak area was theaverage of the peak areas of the total con-sidered protein found in 16 reconstitutedskim milk samples, at the various heat-treatment pH values, heated or not. The var-iation coefficients for the peak area of total-casein or total whey protein were < 10%,as expected, since pH change or heat treat-ment should not affect the overall proteincomposition of the milk. A correction factorof 12.5/15 was taken into account in the cal-

culation of the proportions of -casein orwhey proteins in the serum phase, as theremoval of the centrifugal micellar pellet(~2.5 mL) from the milk samples (15 mL)mathematically increases the concentrationof the serum species.

2.9. Analysis of the milk serums by size exclusion fast protein liquid chromatography (SE-FPLC)

Size exclusion FPLC analysis of theserums of heat-treated and control pH-adjustedmilks was performed within 1 week afterheat treatment on a Sephacryl S-500 Hi-Prep16/90 column (Amersham Biosciences,Orsay, France). The samples were filteredthrough 5-m filters (Pall Life Science, St-Germain-en-Laye, France). The separationwas performed at room temperature underisocratic conditions using 0.1 molL1 Tris,0.5 molL1 NaCl and 10 mmolL1 NaN3,pH 7, as the mobile phase. Loop size was0.5 mL, flow rate was 0.5 mLmin1, andabsorbance was monitored at 280 nm.Five mL eluate fractions were collectedevery 10 min, dialysed against deionisedwater, concentrated by freeze-drying andanalysed by RP-HPLC as described above.

2.10. Micelle size measurement by dynamic light scattering (DLS)

The particle size in the resuspended pel-let samples was measured within 1 weekafter heat treatment using dynamic lightscattering (DLS) at a set angle of 90 on aZetasizer Malvern 3000 HS (MalvernInstruments, Orsay, France). The laser wasa He-Ne laser, with 633 nm wavelength.The solution was brought to 25 C in a ther-mostated water-bath, diluted in milk ultra-filtration permeate (MUF) to meet theZetasizer operating range, and allowed tostand at 25 C for 20 min to ensure properequilibrium of the diluted system prior toanalysis. The solution was then transferredto 2-mL disposable cuvettes for measure-ment. The refractive index of the MUF was1.3416, the refractive index of the proteinparticles was 1.5 and the viscosity of theMUF was 0.99 mPas at 25 C. The results

Rennet coagulation properties of heated milk 519

given are the average of 10 readings, andeach sample was analysed 2 or 3 times. Thedata was visualised using a Contin model,and the mode(s) of the weight-averagedparticle size distribution were considered.

3. RESULTS

3.1. Rennet coagulation properties of milk heat-treated at alkaline pH

Figure 1 shows the average rennet coag-ulation time (RCT) values of the skim milksamples adjusted to pH values between 6.6and 8.1, with or without heat treatment,stored overnight at the adjusted pH, thenrenneted at pH values ranging from 6.2 to6.6. Variation of the renneting pH ensuredthat the optimum observation pH was visi-ble, i.e. where gelation occurs and the effectof heating pH, if any, is not dominated bythe effect of acidification on chymosinactivity.

The effects of heating and of the rennet-ing pH on the RCT were very significant(P0 < 0.001). The heat treatment of milkinduced considerable increase in the RCT,while acidification prior to rennetinginduced faster destabilisation of the milk

system, as shown by reduction of the RCT.Both these effects are well documented, asstated in the introduction. Milk acidifica-tion prior to renneting is known to enhancechymosin activity, as well as the release ofcolloidal calcium into soluble form,decrease in repulsive electrostatic interac-tion between micelles, and subsequentdestabilisation of micelles and increasedgel firming [14, 20, 22, 32, 34].

Heating skim milk at pH values rangingfrom 6.6 to 8.1 also showed a significanteffect on the RCT, albeit to a lower extentthan either the heating pH or renneting pHfactors (P0 < 0.01). In heated samples, theRCT of the milk increased from pH 6.6 to7.1, then decreased with increasing pH ofheat treatment in the alkaline range 7.1 to8.1 (P0 < 0.001). The effect was strongestat renneting pH values of 6.4 to 6.6, wherethe positive effect of acidification on chy-mosin action did not totally screen the effectof heat-treatment pH.

Figure 2 shows the average gel firmness,aR, of the same skim milk samples adjustedto pH values between 6.6 and 8.1, with orwithout heat treatment, stored overnight atthe adjusted pH, then renneted at pH valuesranging from 6.2 to 6.6.

Figure 1. Rennet coagulation time (RCT) at 35 C of reconstituted skim milk samples as a functionof heat treatment (ctl = unheated control milk, htd = milk heated at 90 C for 30 s), heat-treatmentpH (6.6, 7.1, 7.6 or 8.1) and renneting pH (6.2, 6.3, 6.4, 6.5 or 6.6). The presented data were theaverage of 2 repeated experiments analysed twice each. Data is missing for control milk at pH 7.1.See Section 2.6 for experimental details.

520 O. Mnard et al.

Again, the effects of both heat treatmentand renneting pH were very significant(P0 < 0.001). Heat treatment induced areduction in the gel firming rate (not shown)and gel firmness, and acidification counter-acted these effects due to a higher level ofionic calcium and decreased repulsive elec-trostatic interaction between micelles, aspreviously well reported in the literature.The effect of heat-treatment pH was not sig-nificant (P0 > 0.05) when the aR responseat all levels of the 3 factors (heat treatment,renneting pH and heat-treatment pH) areconsidered. It was, however, significantwhen heated samples were only considered(P0 < 0.01). Gel firmness, aR, tended toincrease upon increasing pH of heat treat-ment, except between pH 6.6 and 7.1. Thepositive effect of the heat-treatment pH onaR values was therefore strongest in thealkaline pH range 7.1 to 8.1 (P0 < 0.001).This increase was more visible at rennetingpH values of 6.5 or 6.6 where gels did notalways form after heat treatment at pH 6.6or 7.1 but reached significant aR valuesafter heat treatment at pH 7.6 or 8.1.

These results showed a slight but signif-icant recovery of the rennet coagulationproperties of skim milk after heat treatment

at alkaline pH values ranging from 7.1 to8.1. The RCT responses obtained at pH 6.6were often smaller than at higher pH, andaR values obtained at pH 6.6 were also oftenlarger than those found at higher pH. Theseresults distorted the otherwise rather linearand positive effect of alkaline pH of heattreatment on rennet coagulation propertiesof heated skim milk. These differencescould be explained by the strong effect ofacidification prior to rennet coagulation, asshown in Figures 1 and 2. Natural milk pHis usually 6.7 and adjusting the milk pH toeven pH 6.6 introduces an acidificationeffect on skim milk rennet coagulationproperties, as commonly exploited in thecheesemaking process [9, 21]. It is regret-table that the natural pH value of skim milkwas not initially chosen as the start of theheating pH range explored in the presentstudy.

3.2. Heat-induced protein transfers between the serum and colloidal phases of milk

Figure 3 shows the changes in the distri-bution of the -casein and of the whey pro-teins between the serum and the colloidal

Figure 2. Gel firmness (aR) of reconstituted skim milk rennet gels obtained at 35 C as a functionof heat treatment (ctl = unheated control milk, htd = milk heated at 90 C for 30 s), heat-treatmentpH (6.6, 7.1, 7.6 or 8.1) and renneting pH (6.2, 6.3, 6.4, 6.5 or 6.6). The presented data were theaverage of 2 repeated experiments analysed twice each. The symbol (*) indicates 0 mm, i.e., no gelwas formed. Data is missing for control milk at pH 7.1. See Section 2.6 for experimental details.

Rennet coagulation properties of heated milk 521

phases of reconstituted skim milk due to pHchanges towards alkaline values or to heat-ing at various heat-treatment pH valuesranging from 6.6 to 8.1.

Figure 3 shows the pH-dependent pro-tein exchanges between the colloidal andthe serum phases during heating. The pro-portion of dissociated -casein significantlyincreased with increasing pH, reaching upto almost 60% of the total protein after heat-ing at pH 7.6 and 8.1 (Figs. 3a and 3b). Con-versely, little -casein was dissociated atpH 6.6 (~10%). The proportion of wheyproteins found in the micellar phase signif-icantly decreased with increasing pH, from~60% of the total whey proteins at pH 6.6to ~20% at pH 7.6 and 8.1 (Figs. 3c and 3d).No significant heat-induced change couldbe found at pH 7.6 and 8.1, which indicatedthat no (heat-denatured) whey proteinswere bound to the micelles as a result of heattreatment at these alkaline pH values. Theproportion of about 20% of the total wheyprotein found in the micellar pellet of

unheated milk samples seemed too high tobe accounted for only by the serum trappedwithin the casein micelles pellet upon ultra-centrifugation. Another possible explana-tion may be based on the locally higherbackground noise of the RP-HPLC profilesin the whey protein region (not shown).Overall mass balance for the whey proteinsconsistently exceeded 100% by about 10%,which supports this explanation.

The results shown in Figure 3 thereforesuggest that the heat-induced transfers ofthe two protein species were somehowrelated. More whey protein was attached tothe micelles when less -casein was disso-ciated (at acidic pH values) or the whey proteinremained in the serum phase when -caseinextensively dissociated (at alkaline pHvalues).

Figures 3a and 3b also show a slight dis-sociation of the -casein from the micelleas the pH of the unheated control milkincreased. About 10% and 20% of the total-casein was found in the serum phase of

Figure 3. (Left) Proportions of -casein in the micellar phase (Fig. 3A) and in the serum phase(Fig. 3B) of reconstituted skim milk as a function of heat treatment (grey = unheated control; black =heated at 90 C for 30 s) and heat-treatment pH. (Right) Proportions of whey proteins in the micellarphase (Fig. 3C) and in the serum phase (Fig. 3D) of reconstituted skim milk as a function of heattreatment (grey = unheated control; black = heated at 90 C for 30 s) and heat-treatment pH. Theresults are given in percent of the total considered protein (total RP-HPLC area) found in the initialreconstituted skim milk. The presented data were the average of 2 repeated experiments analysedtwice each. See Sections 2.7. and 2.8. for experimental details.

522 O. Mnard et al.

the milk at pH 7.6 and 8.1, respectively,compared with less than 5% at pH 6.6 and7.1 (Fig. 3b). Similar alkaline-induced dis-sociation rates were also calculated for - ands-caseins, which indicated a non-specificphenomenon (not shown). The heat-induceddissociation rates were conversely muchlower in the case of - and s-caseins thanfor -casein (not shown). This indicated asignificantly higher sensitivity of this caseinto heat-induced dissociation from themicelles, in agreement with previous reports[1, 17, 30].

3.3. Concentration and size of the heat-induced serum protein aggregates

Concentration and size of the heat-induced serum whey protein/-casein aggre-

gates were estimated by FPLC separation ofthe milk supernatants. Figure 4 shows typ-ical elution profiles obtained for serums ofthe unheated skim milk samples at pH 6.6to 8.1 (top) and serums of the same samplesafter heat treatment of the milk (bottom).Peak identification was performed after RP-HPLC analysis of the collected eluate fractions(and in comparison with earlier work [11]).

Figure 4a shows that the serums ofunheated reconstituted skim milk containedindividual proteins (mostly native wheyproteins) as well as small non-protein com-ponents. The serums also contained two dis-tinct protein materials eluting at ~270 and300 min that were interpreted as two groupsof submicelles or other specific caseinmaterials (105 gmol1-range) with consist-ent composition [11]. Figure 4a also shows

Figure 4. Typical elution profiles of the serum phase of control (Fig. 4a) and heated (90 C for 30 s Fig. 4b) skim milk samples at various heat-treatment pH values and analysed by size-exclusionfast protein liquid chromatography (SE-FPLC) on a Sephacryl S-500 HR Hi-Prep 16/90 columnunder isocratic conditions in 0.1 molL1 Tris and 0.5 molL1 NaCl. All samples were preparedand injected at least twice with satisfactory repeatability. See Sections 2.7 and 2.9 for experimentaldetails.

Rennet coagulation properties of heated milk 523

a significant increase in the two serumcasein materials as the pH value of skimmilk increased, in agreement with the alka-line-induced dissociation reported earlier(see Sect. 3.2.).

The heating of the milk induced a strongdecrease in individual whey protein, as wellas a large increase in the two peaks at 270and 300 min (Fig. 4b). The latter resultsstrongly suggest an increased casein disso-ciation upon heating, as reported earlier.The early eluting 270-min peak, however,became very rich in whey protein and -casein, and it is highly probable that a newco-eluting peak was generated during heatingthat corresponded to heat-induced aggre-gates composed of denatured whey proteinsand -casein [11]. This aggregate peak elutedlater, therefore the present aggregates wereprobably smaller than in previous reports[11, 16]. This was accounted for by the lowerheat-treatment regime used in this studycompared with previous studies which alsoused this FPLC method. The aggregatepeak eluted at later elution times and its areatended to increase as heat-treatment pHincreased (Fig. 4b). However, the concom-itant increase and the slight shift toward ear-lier elution times of later eluting caseinmaterial showed less resolution at pH 8.1.These results indicate that a larger amountof smaller serum whey protein/-caseinaggregates were formed upon heating milkat alkaline pH values.

3.4. Micelle size

The estimation of the amount and/or thesize of micelle-bound whey protein/-caseinaggregates was measured through micellesize measurement by dynamic light scatter-ing (DLS) analysis of the centrifugal pelletsof the milk samples at pH 6.6 to 8.1, withor without heating, resuspended in milkultrafiltrate. Figure 5 shows the averagevalues of micelle size as a function of heat-treatment pH and heating.

No significant differences were foundbetween micelle sizes in control and heatedmilk samples, or across the pH values tested.There was a tendency for larger size disper-sion as well as increased micelle diameterwith increasing pH, possibly as a combined

result of increased dissociation of caseinblocks (as seen in Fig. 4) and swelling of thecasein micelle due to the increase in theelectrostatic repulsion between negatively-charged casein molecules. Earlier studieshave reported in detail the effect of heat-treatment pH values ranging from 6.5 to 6.7and heating regime [3, 4]. In those studies,it was clearly demonstrated that increasingheating time or temperature, as well asdecreasing the heat-treatment pH led to anincrease in average micelle size. Theseresults correlated well with the amount ofdenatured whey protein found to be associ-ated with the casein micelles as a functionof heating time, heating temperature andheat-treatment pH [3, 6]. The effect of theheat-treatment pH was, however, negligi-ble and the effect of the heat-treatment itselfseemed barely significant, at as low a heat-ing regime as 90 C for 30 s [3, 4], as usedin the present study.

4. DISCUSSION

The results obtained in this study showthat more -casein dissociated from themicelles and less denatured whey proteinswere bound to the micelles as heat-treat-ment pH increased from 6.6 to 8.1. As aconsequence, the two protein species werein the same phase at the end of the heating

Figure 5. Average particle size as measured bydynamic light scattering (DLS) in the resus-pended pellet fraction of control (open sym-bols) and heated (closed symbols) skim milk atvarious heat-treatment pH values. The pre-sented data were the average of 2 repeatedexperiments analysed 3 to 5 times each. SeeSection 2.10 for experimental details.

524 O. Mnard et al.

process. This heat-induced distribution ofthe two protein species was accounted forby their interaction through hydrophobicbonding and thiol/disulfide interchanges toform heat-induced serum whey protein/-casein aggregates, in agreement with previ-ous reports [36, 26, 32]. The analysis of theserum phase of reconstituted skim milkheated at pH values ranging from 6.6 to 8.1suggested that smaller but more numerousheat-induced aggregates were present in theserum phase as heat-treatment pH increased,in agreement with previous studies [12, 24,32]. The analysis of the micellar phase ofthe same milk samples did not, however,indicate that less whey protein/-caseinaggregates could be bound to the caseinmicelles as heat-treatment pH increased.Earlier studies using the same method, how-ever, reported a clear correlation between alower heat-treatment pH and a higher pro-portion of micelle-bound whey protein/-casein aggregates [35]. The results of thepresent study were accounted for by themild heating regime. They could also beexplained due to the effect of increasing pHvalues in the alkaline range on the swellingof the micelle and on the proportion of wheyprotein transferred to the micellar phase uponheating (i.e. very little or none, see Fig. 3).

The increased proportion of heat-inducedaggregates in the serum phase of milk, aspH increases, appears to be related to theincreased dissociation rate of the caseinmolecules, and especially, of -casein, inalkaline pH and/or high temperature condi-tions [1]. The interaction between -caseinand denatured whey proteins, and formationof aggregates upon heating, further enhancethe dissociation rate of -casein, but thequestion of whether this interaction pre-cedes, or follows, the dissociation of -casein is still debated [2, 4]. The smallersize of the heat-induced (serum) aggregatesof whey proteins and -casein, as pHincreases, may also be related to the higherdissociation rate of -casein ([24], Fig. 3),leading to a higher proportion of -caseinin the aggregates [11]. The poor resolutionof the heat-induced aggregates present inthe serum samples by SE-FPLC (Fig. 4) didnot, unfortunately, allow satisfactory quan-titative analysis of their composition.

Furthermore, original relationships weredrawn between this pH-dependent distribu-tion of the heat-induced whey protein/-casein aggregates across the serum and col-loidal phases, and the rennet coagulationproperties of the heated skim milk. Goodcorrelations (R2 > 85%) were found betweenthe average values of both RCT and aR(taken for a renneting pH of 6.5) and theheat-induced protein exchanges betweenthe serum and the micelle phases, at pH val-ues > 6.7. The RCT decreased and gel firm-ness, aR, increased as heat-induced -caseindissociation increased and whey proteinassociation decreased, as postulated. TheseR2 values were, however, probably affectedby the distribution of the data for the disso-ciation of -casein and the association ofthe whey protein, where the values found atpH 7.6 and 8.1 were very close and bothquite different from those found at pH 7.1(Fig. 3). Further research with a better con-trolled and wider spread of dissociation/association proportion values would beneeded to confirm these relationships.

In an earlier study, Imafidon and Farkye[14] heated milk at pH 7.5 and at 75 C for16 s, followed by acidification to pH 6.4prior to renneting. The RCT of the alkalineheat-treated milk was 6.07 min, versus21.91 min for the control sample (not heat-treated, not acidified). The RCT was evenshorter when the alkaline heat-treated milksample was pH-cycled (i.e. stored over-night at pH 5.5 then adjusted to pH 6.4)prior to renneting. Singh and Waungana[29] confirmed that alkaline heat-treatmentfollowed by pH-cycling gave the highestrennet coagulation properties for heated milk.These results indicated a positive effect ofalkaline heat-treatment on rennet coagula-tion properties of heated milk; however, thiseffect was often also dependent on otherexperimental conditions such as storage con-ditions or pH changes that made it difficultto study independently. The present studyused very standardised storage conditionswith respect to temperature and duration,and kept the samples at their heat-treatmentpH up to the last couple of hours before ren-neting in order to limit possible effects ofpH changes other than that under study.

Rennet coagulation properties of heated milk 525

The present results show slightly improvedrennet coagulation properties of heatedskim milk, when the heat treatment is per-formed at alkaline pH up to 8.1. Theseresults could be of interest to partiallyrecover the renneting properties of a milk,heated to improve cheese yield through therecovery of the heat-denatured whey pro-teins. Further research, however, needs tobe undertaken to ensure that the heat-induced whey protein/-casein aggregatesare or can be retained in the curd upon cut-ting and drainage. It is suggested thatcycling heat-treatment pH values couldhelp further aggregate the serum whey pro-tein/-casein aggregates up to sizes that wouldensure proper trapping in the rennet curd.Optimisation of the alkaline heat-treatmentmethod would also be needed, e.g. by con-trolled neutralisation of the milk after heattreatment, to ensure that minimum caseinloss is suffered from alkalinisation of themilk and further technological operationsup to moulding.

Acknowledgements: The authors wish to thankM.-H. Famelart and G. Brul for advice and dis-cussion during this work, as well as S. Delpineand J. Hannon for careful reading of the manu-script.

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