在加热的脱脂脱脂牛奶中,PH值、钙络合剂和乳固体浓度对水溶性蛋白质聚集体形成的影响

Effects of pH,calcium-complexing agents and milk solids concentrationon formation of soluble protein aggregates in heated reconstituted skim milkJayani Chandrapala a ,b ,*,Mary Ann Augustin b ,Ian McKinnon a ,Punsandani Udabage ba School of Chemistry,Monash University,VIC 3800,AustraliabCSIRO Division of Food and Nutritional Sciences,671Sneydes Road,Werribee,VIC 3030,Australiaa r t i c l e i n f oArticle history:Received 31August 2009Received in revised form 30April 2010Accepted 17May 2010a b s t r a c tThe proportion of protein present in the supernatant after centrifugation and the formation of soluble protein aggregates in heated (90 C for 10min)reconstituted skim milk were investigated as a function of (a)pH,(b)milk concentration 9e 21%,w/w (milk solids non-fat,MSNF )and (c)addition of calcium-complexing agents (orthophosphate or EDTA).Compositional changes in milk,resulting from pH adjustment or salt addition,altered the distribution of caseins between the serum and micellar phases of milk prior to heating,and in fluenced the amount and composition of soluble aggregates formed during heat treatment.Although milk pH was a good predictor of the aggregation behaviour of the proteins during heating of milk of different solids concentrations,neither the proportion of protein in the supernatant prior to heating nor the pH alone could predict the aggregation behaviour when the composition was adjusted with mineral salts.Crown Copyright Ó2010Published by Elsevier Ltd.All rights reserved.1.IntroductionThermal processing of milk changes the composition and properties of milk proteins and alters the physical properties of the milk.Whilst some changes,such as those used to improve the texture of yoghurt products,are desirable,others,such as gel formation during the manufacture of Ultra-High Temperature milk are highly undesirable.These heat-induced changes are dependent on heating conditions (temperature e time pro file)as well as on pH,milk concentration and other aspects of milk composition (Anema,2000;Law &Leaver,1997).The pH,temperature of heating,duration of heat treatment and the composition of milk play dominant roles in determining the extent of casein solubilization upon heating.Low levels of k -casein were dissociated from the casein micelles on heating at pH 6.6,whereas about 50%was soluble on heat treatment at pH 7.1at 120 C;most dissociation of casein processes occur rapidly,with the majority occurring during the initial stages of heating,with little further changes on prolonged heating (Anema &Klostermeyer,1997).The in fluence of milk concentration on the dissociation behaviour of casein micelles during heating is depen-dent on temperature and pH (Anema,1998).Changes in the parti-tioning of caseins between the two phases occur on the addition ofsalts;the addition of phosphates and 10mmol kg À1of EDTA to milk did not alter the effective diameter of the micelles,whereas the addition of higher levels of EDTA resulted in complete disintegra-tion of the micelles (Udabage,McKinnon,&Augustin,2000).During thermal processing,sulphydryl e disul fide interchange reactions between the cysteine residues of k -casein (and some-times a S2-casein)and the whey proteins are denatured,resulting in the formation of aggregates (Law &Leaver,1997).Aggregates can also be formed between the whey proteins themselves (Guyomarc ’h,Law,&Dalgleish,2003;Old field,Singh,&Taylor,2005).Non-covalent interactions such as hydrophobic,ionic and van der Waals forces are also involved in aggregation of proteins (Law &Leaver,1997;Old field et al.,2005)with non-covalently bonded complexes being formed prior to intermolecular disulphide formation (Guyomarc ’h et al.,2003).Vasbinder and de Kruif (2003)suggested that heating at pH >6.6leads to a partial coverage of the casein micelles,whereas heating at pH <6.6leads to an attachment of almost all of the denatured whey proteins to casein micelles.At pH 6.55,the coverage is homogeneous,but decreasing or increasing the pH further leads to inhomogeneous coverage of the casein micelles.The dissociation of k -casein from the casein micelle upon heating has been linked to the formation of aggregates in the serum (Anema,2007),but whether the formation of serum aggregates is a cause or a consequence of the dissociation of k -casein is unclear.The formation of soluble aggregates as affected by changes in (a)pH at 25 C (del Angel &Dalgleish,2006;Donato &Dalgleish,2006;Jean,Renan,Famelart,&Guyomarc ’h,2006;Menard,Camier,&*Corresponding author.Tel.:þ61383446488;fax:þ61393475180.E-mail address:jayanic@.au (J.Chandrapala).Contents lists available at ScienceDirectInternational Dairy Journaljou rn al homepage :/locate/idairyj0958-6946/$e see front matter Crown Copyright Ó2010Published by Elsevier Ltd.All rights reserved.doi:10.1016/j.idairyj.2010.05.008International Dairy Journal 20(2010)777e 784Guyomarc’h,2005;Renan et al.,2006;Singh&Latham,1993),(b) casein micelle structure(del Angel&Dalgleish,2006;Renan, Guyomarc’h,Chatriot,Gamerre,&Famelart,2007),(c)genetic variants(Donato,Guyomarc’h,Amiot,&Dalgleish,2007)and(d) protein composition(Donato et al.,2007;Guyomarc’h et al.,2003) have been extensively examined.A molar ratio of1e5whey proteins to1k-casein was found for the soluble complexes heated at90 C(Anema,2007).The size of these complexes was found to be w20e50nm(del Angel&Dalgleish,2006).The soluble aggre-gate formation on heating increases with increase in pH prior to heating(del Angel&Dalgleish,2006;Donato&Dalgleish,2006; Jean et al.,2006;Menard et al.,2005;Renan et al.,2006).It is also known that pH affects the distribution of the caseins between the serum and micellar phase of milk(del Angel&Dalgleish,2006; Donato&Dalgleish,2006).The concentration and speciation of proteins in milk are affected by concentration(Anema,1998)and the addition of calcium-complexing agents(Udabage et al.,2000). In addition,concentration of milk reduces pH.The manner in which these factors interact and influence aggregate formation when milk is heated is not yet known.This study examined the formation and the composition of the soluble aggregates in heated(90 C for10min)reconstituted skim milk as a function of milk solids concentration,in the range9e21%, w/w,milk solids non-fat(MSNF),over a range of pH.The effect of addition of calcium-complexing agents(orthophosphate or EDTA; 10e30mmol kgÀ1)to9%(w/w)MSNF milk on formation of soluble aggregate was also examined.The aim was to determine if the observed effect of pH on the formation of soluble aggregates is due to changes in the concentration of protein and protein distribution rather than pH per se.2.Materials and methods2.1.MaterialsA commercial low-heat(72 C for20s)skim milk powder(SMP) was obtained from Tatura Milk Industries(PO Box213,Tatura,VIC 3616,Australia).The composition of the SMP(w/w)was36.5% protein,48.45%lactose,3.5%moisture,8.8%ash and0.525%fat.The extent of whey protein denaturation of the SMP was measured using the method reported by Parris and Baginski(1991),and showed that5.4%of the total whey protein present in the SMP was denatured.Ultra-pure(MilliQ)water was used at all times.2.2.Preparation of reconstituted skim milk solutionsReconstituted skim milk(either180g kgÀ1MSNF or300g kgÀ1 MSNF)were prepared as described by Chandrapala(2009).The reconstituted skim milk samples were kept overnight at4 C and then equilibrated at25 C for1h.Minor pH adjustments were carried out as necessary and small amounts(w0.5g)of MilliQ water were added to obtain milk solutions at the desiredfinal concentration9e21%,w/w,(MSNF)and pH(6.2e8.5).For the preparation of milk solutions with added salts(ortho-phosphate or EDTA),the salt solutions were added drop-wise with continuous stirring.The salt solutions used were100mmol kgÀ1of orthophosphate(equimolar mixture of Na2HPO4and NaH2PO4)and 200mmol kgÀ1of Na2H2EDTA.Calculated amounts of MilliQ water were added before the addition of the respective salt solution,such that,at the end of the addition andfinal pH adjustment,only a small amount of MilliQ water was required to obtain the9%(w/w) MSNF solution with the required amounts of the additive (10e30mmol kgÀ1orthophosphate or EDTA).The milk solutions were kept overnight at4 C.All amounts were measured by mass.All reconstituted milk samples were prepared in duplicate unless otherwise stated.2.3.Heat treatment and pH measurements of reconstitutedskim milk solutionsAliquots(50g)of milk were transferred to stainless steel tubes and heated in a water bath at90 C.The time to reach90 C was 8min,and the milk samples were held for a further10min at90 C. After heat treatment,the samples were cooled by immersion for 5min in a water bath held at25 C.All heat treatments were replicated at least in duplicate.The pH of the milk samples at25 C and at90 C was measured by an InPro2000liquid electrolyte pH electrode with an integrated temperature sensor(Mettler Toledo, PO Box173,Port Melbourne,VIC3207,Australia)connected to a Metrohm pH meter(Metrohm AG,Oberdorfstrasse68,9101 Herisau,Switzerland).2.4.Ultracentrifugation of skim milk samples and measurementof nitrogen contentThe heated and unheated milk solutions were ultracentrifuged at33,000Âg for60min at25 C using a Beckman LK90M ultra-centrifuge with a type55.2Ti rotor(Beckman Instruments Australia (Pty)Ltd,Gladesville,NSW2111,Australia).Approximately30min prior to ultracentrifugation,the concentrated milk solutions (12e21%,w/w,MSNF)were diluted to9%by weight with MilliQ water.In some samples(i.e.,21%(w/w)MSNF,pH at25 C<6.4), where soft gels were formed on heating,these were broken by vigorous shaking prior to dilution,using the method adopted by Anema(2000),to ensure redispersion.After centrifugation,the clear supernatant(excluding the opalescent layer)was carefully removed andfiltered using a0.45-m mfilter and stored at4 C until further analysis.These were generally used within1e2days after separation.Separate SEC(size-exclusion chromatography)experi-ments performed on fresh(0days)and stored(2days)superna-tants showed no change in the amounts and sizes of the soluble aggregates.A supernatant sample obtained from21%(w/w)MSNF undiluted skim milk was used to determine if there was an effect of dilution prior to centrifugation.The results indicated that dilution did not alter either the amount or the size of the soluble aggregates. In calculating the proportion of the total amount of a component in the supernatant,the mass of the opalescent layer was included as part of the total mass of supernatant.The total nitrogen content of selected supernatants of recon-stituted milk solutions before and after heating was determined using a LECO FP-2000Nitrogen Analyser(LECO Australia Pty Ltd, Castle Hill,NSW,Australia).The protein nitrogen content of the supernatant was calculated by subtracting the contribution from non protein nitrogen of the total milk.A factor of6.38was used to convert nitrogen to protein concentration(IDF,1993).2.5.Size-exclusion chromatographySize-exclusion chromatography(SEC)of the supernatants from different skim milk samples was conducted using a Shimadzu LC20 HPLC system equipped with a UV e vis detector operating at280nm (Shimadzu scientific instruments(Oceania)Pty Ltd,Mount Wav-erly,VIC3149,Australia).Samples(20m L)of thefiltered superna-tants were injected into a Phenomenex TSK GEL17u G5000PW 1000A column(Phenomenex Australia Pty LTD,PO box4084, Lane Cove,NSW2066,Australia)and run at0.5mL minÀ1in 100mmol LÀ1ammonium bicarbonate buffer at pH7.0;the total run time was35min.J.Chandrapala et al./International Dairy Journal20(2010)777e784 778SEC was also carried out on a larger scale to enable collection of sufficient sample for the determination of the composition of the aggregates.For this purpose,a40mL aliquot of supernatant was introduced to the column(5cm diameter and90cm long packed with Sephacryl S500HR,Amersham Bioscience Baulkham Hills, NSW,Australia)which had a nominal fractionation range of 40e100,000kDa.Ammonium bicarbonate(100mmol LÀ1,pH7)at aflow rate of3mL minÀ1was used as the running buffer.The column was run on a Pharmacia FPLC system(PO Box57,West Ryde,NSW2114,Australia)as described earlier(Chandrapala, 2009)The total elution time for each sample was1000min.Frac-tions were collected between270and470min(at5min intervals) using a LKB Superac2211Fraction Collector.Fractions were pooled in groups of three and freeze-dried before further analysis by SDS polyacrylamide gel electrophoresis.The elution profiles obtained from SEC carried out on the larger scale were similar to those obtained on the smaller scale(data not shown).All SEC experiments were performed at least in duplicate. Repeat measurements resulted in very similar elution profiles.2.6.Sodium Dodecyl Sulfate(SDS)polyacrylamide gelelectrophoresisMono-dimensional SDS-PAGE was used to determine the proportion of casein of the fraction collected from SEC corre-sponding to the soluble aggregates.The fractions were dissolved in ammonium bicarbonate buffer(100mmol LÀ1,pH7)to a crude total protein content of2.5mg mLÀ1.Precast4e12%acrylamide gels (NuPage Novex Bis Tris gels,Invitrogen Australia Pty Ltd,Mt.Wa-verley,Victoria,Australia)were used and electrophoresis was per-formed under both reducing and non-reducing conditions as described earlier(Chandrapala,2009).Commercial samples of sodium caseinate and whey protein isolate obtained from Murray Goulburn Co-operative Ltd(PO Box4307,Melbourne,VIC3001, Australia)were run as standards.The gels were stained using an Invitrogen Coomassie Blue staining kit following the manufac-turer’s instructions(Invitrogen Australia Pty Ltd,Mt.Waverley, Victoria,Australia).The stained gels were then photographed and the photographs were analysed using Scion Image software(Scion Corporation,82,Worman’s Mill Court,Suite H Frederick,Maryland 21701,USA).For reduced gels,the integrated intensities of each band were analysed to determine the relative proportions of the major proteins in the soluble aggregate fraction.All experiments were carried out at least twice as full replicates. All data were expressed as means with standard deviations.3.Results and discussion3.1.Protein content of supernatants of centrifuged milkk(9e21%,w/w,milk solids non-fat)at different pH valuesThe proportions of protein present in supernatants(expressed as%of total protein)as a function of initial pH at25 C for heated and unheated9%(w/w)MSNF milk are shown in Fig.1.For unheated milk at pH6.67and25 C,21.0Æ0.3%of the total protein was found in the supernatant.This value corresponds to1.6%of the caseins being present in the serum phase,somewhat lower than the value of6.5%found by Udabage et al.(2000)or the value of10% found by Griffin,Lyster,and Price(1988).This difference may be due to the inclusion(in both the previous studies)of the opalescent layer as part of the supernatant,use of different centrifugation speeds or to innate differences in the milk.As the milk pH was increased,the proportion of total protein in the supernatant of unheated milk increased linearly,to37.9%at pH8.52.The increased proportion of total protein present in supernatant with increasing pH implies an increase in the net dissociation of caseins (predominately k-casein)from the micelles,as reported by others (Anema,1998,2007;Anema&Klostermeyer,1997).In the present study,the changes in the proportion of total protein present in supernatant of heated milk were found to be dependent on pH(Fig.1).At pH<6.65,there was less protein in supernatant after heating than prior to heating,due to the removal of the large aggregates during centrifugation,which were formed on heating.At pH w6.65,heating caused little change in the proportion of protein remaining in the supernatant,suggesting that any removal of whey proteins by centrifugation was balanced by dissociation of caseins from the micelles.However,at pH>6.65, the proportion of protein in supernatant was higher after heating than prior to heating,which may be due to the dissociation of caseins from the micelles and/or to the formation of soluble aggregates.These results are in agreement with the model proposed by Vasbinder and de Kruif(2003).The trends towards increasing proportion of protein in super-natant of heated concentrated milk with increase in pH prior to heating are similar to those observed for that of9%(w/w)MSNF milk.The results demonstrate that,for a particular milk sample,the pH prior to heating is a good predictor of the proportion of protein in the supernatant,when milk is concentrated prior to heat treat-ment(Fig.2)and that the proportion of total protein in the supernatant is almost independent of the milk concentration. Anema(1998)observed considerable pH-and temperature-dependent dissociation behaviour of caseins from casein micelles of 10%and25%TS milk over the entire temperature range (20e120 C),irrespective of milk solids concentrations.These results are in line with those obtained by Anema(1998).3.1.2.Effect of addition of orthophosphate or EDTA to milk(9%,w/w,milk solids non-fat)The proportions of protein in supernatants(expressed as%of total protein)at different initial pH values at25 C for heated and unheated9%(w/w)MSNF milk containing different amounts of orthophosphate or EDTA are given in Table1.On addition of EDTA, the sequestration of calcium by EDTA resulted in the transfer of caseins from the micelles to the supernatant,and hence increased the proportion of total protein present in the supernatant,which also increased with increased pH,as reported by Udabage et al.(2000).In the present study,there was a small increase in the proportion of proteins in supernatant on addition of10mmol kgÀ1ortho-phosphate,with the effect decreasing as the level oforthophosphatepH at 25°CProteininsupernatantofmilk(%)Fig.1.Proportions of total protein present in supernatants(expressed as%of total protein in milk)of unheated(>)and heated(A)reconstituted9%(w/w)MSNF milk as a function of pH at25 C prior to heating.J.Chandrapala et al./International Dairy Journal20(2010)777e784779increased from 10to 30mmol kg À1,except at pH 7.2.Udabage et al.(2000)reported a decrease in level of supernatant casein on addi-tion of orthophosphate at pH 6.65,with the effect decreasing as the level of orthophosphate added increased from 10e 30mmol kg À1,while Gaucher,Piot,Beaucher,and Gaucheron (2007)found an increase in protein content of the supernatant on addition of milks with added orthophosphate at pH 6.8.Gaucher et al.(2007)demonstrated that the addition of high levels (>50mmol L À1)of orthophosphate result in the precipitation of calcium hydrogen phosphate with consequent dissociation of the casein micelles.EDTA chelates calcium without the precipitation of calcium phosphate,as EDTA is a strong chelating agent with 6co-ordination sites.On the other hand,the addition of phosphate results in calcium phosphate precipitation concurrently with the chelation of calcium.This effects a change in the dissociation of casein from the casein micelles.However,the exact mechanism for this increase in the amount of protein present in the supernatant on addition of orthophosphate is not yet known.In all samples containing EDTA,the proportion of proteins present in supernatants obtained after heating was considerably less than in supernatants obtained prior to heat treatment.For the example,with 20mmol L À1added EDTA at pH 7.11prior to heat treatment,w 96%of the protein,was present in the supernatant.Heat treatment resulted in aggregation of the proteins such that almost 40%was removed by the subsequent centrifugation after heating.It appears that the dissociated caseins tend to reassociate during heating and are then precipitated with the pelleted phase during centrifugation.3.2.Examination of the soluble aggregates in the supernatants of centrifuged milk by size-exclusion chromatography3.2.1.Supernatants of unheated and heated 9%(w/w)milk solids non-fat milk at pH 6.65To elucidate the nature of the soluble aggregates produced by the proteins on heating,the supernatants from unheated and heated reconstituted skim milk (9%(w/w)MSNF pH 6.65)prepared by centrifugation were analysed by SEC e HPLC (Fig.3).The chro-matographic pro files obtained were similar to those obtained previously (del Angel &Dalgleish,2006;Donato &Dalgleish,2006;Donato et al.,2007;Guyomarc ’h et al.,2003;Menard et al.,2005;Renan et al.,2006,2007).The earliest eluting peak (Peak 1)was the excluded volume of the column.This peak does not contain proteinaceous material (Guyomarc ’h et al.,2003)and may be composed of very small fat globules (Donato &Dalgleish,2006).Peak 2has been attributed to soluble aggregates (whey proteins and k -casein)formed on heating (Guyomarc ’h et al.,2003).Previous studies have estimated that the sizes of these aggregates are 20e 50nm (del Angel &Dalgleish,2006).Peak 3was assigned to native whey proteins and mono-meric caseins,and decreased on heating due to the involvement of the whey proteins and monomeric caseins in the formationofpH at 25CP r o t e i n i n s u p e r n a t a n t s o f h e a t e d m i l k s (%)Fig.2.Proportions of total protein present in supernatants (expressed as %of total protein)of heated milk as a function of the pH at 25 C prior to heating:-,9%(w/w)MSNF;:,12%(w/w)MSNF;,,15%(w/w)MSNF;6,18%(w/w)MSNF;C ,21%(w/w)MSNF.Table 1The proportion of total protein present in supernatant and the casein component of the soluble aggregates (expressed as %of total protein in the soluble aggregates)for heated reconstituted 9%(w/w)MSNF solutions with/without added (a)ortho-Mean values and standard deviation of (n !2)replicates.Pooled standard deviation for pH ¼Æ0.02.Nd ¼Not determined.bThe amount of caseins present in soluble aggregates expressed as %of the total protein content in the soluble aggregate fraction collected through SEC.5101520253035Elution time/minA b s o r b a n c e /a r b i t o r y u n i t sPeak 1Peak 3Peak 4Peak 4Peak 3Peak 2abFig.3.SEC e HPLC pro files of supernatants obtained from (a)heated and (b)unheated 9%(w/w)MSNF milk at pH 6.65.The peaks were assigned as follows:Peak 1(w 9min)is the excluded volume of the column;Peak 2(w 11min)contains soluble aggregate peak;Peak 3(w 18min)contains native whey proteins and monomeric caseins;Peak 4(w 21min)includes orotic acid and other dialyzable solutes.J.Chandrapala et al./International Dairy Journal 20(2010)777e 784780aggregates.Peak4has been identified as being made up of small molecules such as orotic acid and other dialyzable solutes and has been used as an internal standard(Guyomarc’h et al.,2003).3.2.2.Supernatants of heated milk(9e21%,w/w,milksolids non-fat)The SEC e HPLC elution profiles of supernatants of heated milk (9e21%(w/w)MSNF)at their unadjusted pH values and at a constant pH at25 C of 6.65are given in Fig.4(a)and(b), respectively.Increasing milk concentration(9e21%(w/w)MSNF), which decreased the natural pH from pH6.65to6.43,decreased the level of soluble aggregates formed on heating,as reflected by the reduced area of the soluble aggregate peak.A higher proportion of micelle-bound denatured whey protein aggregates is expected at lower pH(Vasbinder&de Kruif,2003),but it is not possible to distinguish between the contributions,due to the lower pH and the effect of milk concentration,as the unadjusted pH of the milk is reduced with increasing total solids.The decrease in pH before heating as a result of increasing solids concentration alters the distribution of proteins within the phases and,in addition,the pH decreases on heating.The decrease in pH with increasing temper-ature is mainly due to the decreased solubility of calcium phos-phate resulting re-equilibration of phosphate species within the serum phase and increased production of Hþions.Both these factors will contribute to a change in dissociation of casein from the casein micelles,and hence the distribution of the proteins within the phases.Further experiments were carried out with milk at varying concentrations,but adjusted to the same pH prior to heating in order to discriminate between effects of pH and total solids.The profiles for milk with9e21%(w/w)MSNF at pH 6.65prior to heating(Fig.4(b))were very similar to each other.The formation of soluble aggregates was almost similar in size and amounts,although slight changes were observed at higher concentrations. For18and21%(w/w)MSNF milk,there were increased amounts of soluble aggregates formed during heating and slight shifts towards shorter elution times as milk concentration increased,indicating the formation of larger aggregates at higher milk solids concen-tration.Similar trends were obtained for other pH values in the range of6.2e7.2(data not shown).Overall,it can be concluded that pH plays a predominant role in determining the soluble aggregate formation behaviour,whereas milk concentration plays a very minor role.3.2.3.Supernatants of unheated and heated milk(9%,w/w,milk solids non-fat)with added orthophosphate or ethylene diamine tetraacetic acid(EDTA)Fig.5shows the SEC e HPLC profiles of supernatants obtained from unheated and heated9%(w/w)MSNF milk with or without various amounts of added EDTA at pH7.2at25 C.In supernatants from unheated milk with added calcium-complexing agents,there was a peak eluting at w13e14min(denoted as Peak A in Fig.5(a)). SDS-PAGE analysis of this region in milk without additives has shown that this peak contains small quantities of caseins,a signif-icant amount of b-lactoglobulin and traces of a-lactalbumin (Donato&Dalgleish,2006).However,the area of Peak A increased with addition of orthophosphate or EDTA,and with increasing amounts of added calcium-complexing agents,in the present study. Hence,Peak A can be attributed to caseins,which dissociate from the micelle on addition of calcium-complexing agents.The increase in the area of Peak A was more pronounced with the addition of EDTA than with orthophosphate,because EDTA is the more powerful complexing agent and chelates calcium more strongly than orthophosphate.Total protein level in the supernatantof 05101520253035Absorbance/arbitoryunits05101520253035Elution time/minElution time/minAbsorbance/arbitoryunitsbaFig.4.SEC e HPLC profiles of supernatants obtained from heated milk(a)at theirunadjusted pH values at25 C(1,21%(w/w)MSNF,pH6.43;2,18%(w/w)MSNF,pH6.46;3,15%(w/w)MSNF,pH6.50;4,12%(w/w)MSNF,pH6.55;5,9%(w/w)MSNF,pH6.65)and(b)at pH6.65at25 C prior to heating(1,21%(w/w)MSNF;2,18%(w/w)MSNF;3,15%(w/w)MSNF;4,12%(w/w)MSNF;5,9%(w/w)MSNF).05101520253035Elution time/minAbsorbance/arbitoryunits05101520253035Elution time/minAbsorbance/arbitoryunitsControl10mmol kg-120mmol kg-1Control10mmol kg-120mmol kg-1Peak B**Peak AabFig.5.SEC e HPLC profiles of unheated and heated supernatants obtained from9%(w/w)MSNF milk with or without added EDTA(10or20mmol kgÀ1)at pH7.2at25 Cprior to heating.J.Chandrapala et al./International Dairy Journal20(2010)777e784781。