View of Optimization of centrifugal separation of α-lactalbumin and β-lactoglobulin

Vol.6(1997): 193-198.

Optimization ^of centrifugal separation of a-lactalbumin and (3-lactoglobulin

TuomoTupasela

AgriculturalResearch^Centreof^Finland,Food ResearchInstitute,FIN-31600Jokioinen, Finland, e-mail:tuomo.tupasela@mtt.fi

Petri Koskela

Departmentof^FoodTechnology,FIN-00014Universityof^{Helsinki,Finland} Eero^Pahkala,Veikko Kankare

AgriculturalResearch Centreof^Finland,Food ResearchInstitute,FIN-31600Jokioinen,Finland

Whey proteins, whichare mainly composedofP-lactoglobulin (P~lg) and a-lactalbumin(a-la),ac- countfor about 20%of theproteinsof^{bovine milk. Inthis}studyweinvestigatedthe effect ofpH, dry matter content,concentrationfactor, heat treatment and centrifugationon^{theseparation}of a-la from

P-lgusingclarifiedwheyas raw material,a-Laprecipitationwashighest, 23.3%,when thedrymat- ter contentranged from5.8% to25.7%.Theoptimum pH of a-laprecipitation dependedonthedry matter content.Theseparation efficiencyincreased when the concentration factor and heat treatment time at 55°C increased. Alonger centrifugation time and higher separation speed did not have a marked effect ontheseparation efficiency. Separationwas moreefficient with ahigher centrifuga- tionspeedatconcentration levels30 Xand 60 X.Theseparation efficiencydid notimprovewhen the temperaturewasraisedfrom 55°C to 65°C but itwasbetter ataconcentration level120 Xthan at60 Xand 30 X,and also at concentration level60 Xthan with30 X.

Key^words:a-lactalbumin,P-lactoglobulin, separationfractionation, filtration, concentration

ntroduction

Whey proteins, which aremainly composed of P-lactoglobulin (p-lg), ~3.3 g/kg whey, and a- lactalbumin(a-la),~1.0 g/kg whey,^accountfor about20% of the proteincontentofbovine milk (Walstra and Jenness 1984).Many efforts have been made to separate these major^components

withaview toproducing whey proteinconcen- trates with improved and specific functional properties(Amundson etal. 1982, Pearce 1983, Pearce 1987, Maubois etal. 1987, Rosenberg

1995).These efforts have involved gentle heat- ing of whey, followed by concentration of whey atacidic pH valuestoproduce selective precip-

itation of a-laorP-lg.

Pearce (1983) reported that maximum pre-

©Agricultural and Food ScienceinFinland Manuscriptreceived March 1997

cipitation of a-la in whey and optimum separa- tion of the proteinswereobtained^atabout 65°C when the wheywas kept_atapH range from 4.1 to4.3 for several minutes. No clarifyingpretreat- ment of whey was used. McDonough et al.

(1974) and de Wit (1984) showed that the pres- ence of fat impairs the whipping properties ofa whey protein concentrate (WPC).Lipidsarealso susceptible to oxidation and thus may contrib- ute toan off-flavour,which is best prevented by removal of lipids from whey (de Boer et al.

1977). In a clarification method introduced by Mauboisetal. (1987) for thepretreatment meth- od of whey, the residual fat and lipidcomponents wereremoved with microfiltration. Tupasela et al. (1994) studied the effect of clarification of whey on whey ultrafiltration. We discuss here the effect of clarification of wheyon the separa- tion of a-la from(i-lg. The effects of pH, con- centrationfactor,drymattercontent, heattreat- mentand centrifugationwere studied. All exper- imentswereconducted_on pilot-plantorlabora- tory scale.

Material and methods

Whey

Fresh edam cheesewhey wasobtained from the Food Research Institute’s dairy plant, and the cheese milk was pasteurized at74°C for 15s.

After the cheese had been made the whey was centrifuged and cooledto2°C inaprocess tank.

CaCI, solution (CaCl

2 x 2H,0 662.1 g/1 a.d.

H,O),720 ml per ¹⁰⁰I whey, was added^tothe whey and the pH of the whey was adjusted to 7.3 with 1 M NaOH solution using a process basedon that ofFauquant ^etal.(1985). Immedi-

ately after these treatments,the wheywasheat- edat 50°C for8 min in amixing tank provided witha heating/cooling jacket. After heat treat- mentthe whey wascooledto40°C with ice wa- ter.

Whey processing

Heat-treated wheywas microfiltered with an APV CL 3/40 microfiltration (MF) unit equipped with 0.2 pm ceramic membranes (Ceraver, France) in two modules. Each module had a membranearea of 1.4 m 2. In the MF unit the whey circulation speedwas 5 m/s and thetem- perature waskept atbetween 35 and 40°C with

aheat exchange section in the circulation loop.

The whey inlet pressure was 3 bar. The whey was microfiltered by recycling in abatch run.

The microfiltrationpermeate was collected and thetemperature was loweredto20°C in aproc- esstank by circulating icewaterin the agitator.

The MF unitwascleaned after eachrunwith 1^% NaOH solution.

Thepermeate wasconcentrated witha mem- brane ultrafiltration unit I ^(PCI Bro MK, UK;

cut-off9000) and unit II (Millipore, Pellicon Cassette, USA;cut-off 10 000^PTGC). The pH of the WPCwasadjustedtothe desired level with 2 MHCI, after which the WPC was heated ina waterbath to55°C or65°C for 5, 15or 30 min.

Thistreatmentcaused precipitation of a-la. The precipitate_wascollected by centrifugation^(Sor- vali RC-5B Superspeed Centrifuge using aSS- -34 rotor, Du Pont Instruments, Connecticut, USA), at5000 rpm and 10 000 rpm(3020g and

12 100 g, respectively).

Analysis

The total solids content of differently treated wheys was determined after drying the wheys for 16 h at 102°C using the modified method of IDF 4 (1958). The protein content was deter- mined by the Kjeldahl method with N conver- sion of 6.38 (TECATOR 1975). The pH was measured withaKnick Portamess752 pH meter (Berlin, Germany). a-La and (3-lg were identi- fied and quantified by aFPLC Mono Q HR 5/5 chromatography column(Pharmacia)connected toa UV detector(280 nm) using the modified method of Humbrey and Newsome(1984).

Vol. 6(1997): 193-198.

Results and discussion

Effect of whey dry matter content and pH on separation

Firstwestudied how ot-la precipitated when the pH range was from 3.2 to 4.5 at a dry matter content 12.5%. The a-la precipitation levelwas obtained when the(3-lg content in supernatant was determined. The results in Figure 1 show that the pH range from 3.4 to 3.8 is the _most favourable for the precipitation of a-la. When the drymatter contentof whey was 12.5% and the pH ranged from 3.2 to 4.5 the maximuma- la precipitation was achieved at apH value of around3.6. In thenexttrial _wehad^tendifferent whey drymatter contents(from 5.8%to25.7%) and a- la and (3-lg wereseparatedatseven dif- ferent pH values (from 3.4 to 4.0). Higher pH values, 3.9 and 4.0, _were still included in the experiment because the dry matter content of three samples were lower than earlier. Figure 2 shows thatatthe lowest drymattercontent,5.8%, a-la did notprecipitate well enough. When the drymatter ^contentwas raised^to 9.2%,more a- lawasprecipitated; maximum precipitation_was

achieved^at adrymatter content of23.3%. The pH optimum of a-la precipitation decreasedas the drymatter ^contentincreased. Figure 3 sug- gests that,in the separation of(3-lg froma-la, the drymatter content had _{a greater} effect than the proteincontent, because the drymattercon- tent line has a better positive correlation with the (3-lg content line than has the protein con- tent^(%,protein in drymatter) line.

Fig.

1.

Fig. 2.Effect ofdrymatter contentand pH ofwheyon(3-lgcontents(%)insupernatants.Drymattercontentsarepresented below the figure.

Effect of concentration ^{factor, heat}

treatment and centrifugation speed ^and

time on ot-la and (3-lg separation

In this trial we used three differentconcentra- tion factors, 30 X, 60 X and 120 X. The dry matter ^{contents (%)} were 11.6, 14.5 and 19.0, respectively. The whey was processed as de- scribed above. The pH before ^{treatments was} adjustedto3.6 with 2 M HCI. In trial 1 the^con- centration factorwas 30 X and thetemperature treatmentswere55°C/5min,55°C/15min,55°C/

30 min and 65°C/30 min. Each heat-treated^sam- ple was centrifuged ^at 5000 rpm/20 min and

10 000 rpm/20 min. In trial 2 the concentration factorwas60 X and thetreatmentwas asin trial 1. In trial 3 the concentration factorwas 120 X.

The sample was treated as in trial 1 and 2, but was centrifuged also at 5000 rpm/10 min and 10 000 rpm/10 min. The results are shown in Table 1.

From Table 1 it canbe seen that the ratios (cc-la/p-lg X 100)range from7 ^to 20. The lower thevalue, the purerwasthesupernatant withre- gard ^toP-lg. Note that when the concentration factor and heattreatmenttimeat55°Cincreased, the separation efficiency also increased. A long- er centrifugation time and greater separation

speed did not have a marked effect on separa- tion efficiency. Separation efficiency improved with the higher centrifugation speed atconcen- tration levels 30 X and 60 X but ^not when the temperature wasraised from 55°C^to65°C. Sep- aration efficiencywas betterat aconcentration level 120 X thanat60 X and 30X, and also at concentration level 60 X than^at30 X.

Conclusion

In this study oc-la precipitation was highest ^at 23.3%. The optimum pH of oc-la precipitation dependedon the drymatter content.Separation efficiency increased withanincrease in concen- tration factor and heat ^treatment time^at 55°C, and also witha higher centrifugation speed at concentration levels 30 X and 60 X. Therewas no improvement when the temperature was raised from 55°C ^to65°C. Separation efficiency was better atconcentration level 120 X thanat 60 X and 30 X, and concentration level 60 X than^at 30 X.

In the light of the aboveresults, and the in- dustrial potential for separating these major whey protein components from whey, in order^topro- Fig. 3. (3-lgcontents^(%)insupernatantversuswhey drymatter content^{( %)}andproteincontent^(%,protein in dry^matter).

Voi6(1997): 193-198.

Table I,Effect of concentration factor, heat treatment andcentrifugation speedand timeontx-la and(1-lg separation. Concentrationfactor⁼30 X, 60 Xand 120 X; heat treatment⁼55°C/5min,55°C/15min, 55°C/30 min and 65°C/30min; centrifugation speedand time ⁼5000 rpm/10 min, 5000 rpm/20 min,

10 000rpm/10min and 10 000 rpm/20 min.

Supernatant a-la/p-lg X 100ratio

Heat treatment Concentrationfactor Centrifugation speed and time

30 X 60 X 120 X

55°C/smin 11 5000 rpm/10 min

18 12 10 5000 rpm/20 min

10 10 000 rpm/10 min

20 12 10 10 000 rpm/20 min

55°C/15min 8 5000 rpm/10 min

16 11 8 5000 rpm/20 min

8 10 000 rpm/10 min

12 10 8 10 000 rpm/20 min

55°C/30min 7 5000 rpm/10 min

14 9 7 5000 rpm/20 min

7 10 000 rpm/10 min

13 8 7 10 000 rpm/20 min

65°C/30min 9 5000 rpm/10 min

13 11 8 5000 rpm/20 min

9 10 000 rpm/10 min

13 8 8 10 000 rpm/20 min

-=not determined

duce whey protein isolates with specific func- (dry matter content e. 13%)and temperature tional properties, themost suitable combination treatment at 55°C/30 min. This combination would be^aconcentration factor of30 X^to 60 X should be tested^onindustrial scale.

References

Amundson, C.H., Watanawanichakorn, S. ^& Hill, C.G.

1982.Production of enriched protein fractions of p-

lactoglobulinand ot-lactalbumin from cheese whey.

Journal of FoodProcessingand Preservation 6: 55- 71.

Boer, R., deWit,J.N. ^& Hiddink,J. 1977.Processingof whey by means of membranes and some applica- tions of whey protein concentrate. Journal of the Society^ofDairy Technology30: 112-120.

Fauquant,J., Vieco, E., Brule,G.^&Maubois,J.-L. 1985.

Clarification des lactosérums doux par agrégation thermocalciquede la matiére grasse résiduelle. Lait 65: 1-20.

IDF4.1958.Dry matterincheese and processed cheese.

Humbrey, R.S.^&Newsome,L.J. ^1984.High performance ion-exchange chromatographyof the major bovine

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Maubois, J.L., Pierre, A,, Fauquant, J.^&Piot, M. 1987.

Industrial fractionation of main whey proteins. IDF Bulletin212: 154-159.

McDonough,F.E.,Hargrove,R.E.,Mattingly,W.A.,^Posa- ti, L.R^&Alhford, J.A.^1974. Compositionand prop- erties ofwhey protein concentratesfrom ultrafiltra- tion. Journal of Dairy Science^57: 1438- 1443.

Pearce,R.J. 1983. Thermalseparationofp-lactoglobulin and a-lactalbumin in bovine Cheddar cheese whey.

Australian Journal of Dairy Technology38:144-148.

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150-153.

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SELOSTUS

oc-lactalbumiinin ja p-lactoglobuliinin sentrifugointierotuksen optimointi

Tuomo Tupasela, PetriKoskela,Eero Pahkala jaVeikko Kankare Maatalouden tutkimuskeskusja Helsingin yliopisto

Heraproteiinit käsittävät noin 20^% maidon proteii- neista. Heraproteiinit koostuvat pääasiassa (i-lakto- globuliinista(P-lg) jaa-laktalbumiinista (a-la). Teh- dyissä kokeissa tutkimmepH:n, kuiva-ainepitoisuu- den,väkevyyden, lämpökäsittelyn ja sentrifugoinnin vaikutusta a-la:n ja(3-lg:nerottumiseen kirkastetus- ta juustoherasta.

a-laktalbumiinin saostuminen onnistuiparhaiten, kun kuiva-ainepitoisuus oli23,3 ^%,kuiva-ainepitoi- suuden vaihdellessa 5,8 ja 25,7 %:n välillä. Kuiva- ainepitoisuus vaikutti a-la:n saostumiseen optimi pH- tasolla. Sentrifugointitulos parani,kun väkevyyttä ja

aikaa nostettiin 55°C:nlämpökäsittelyssä. Pidempi sentrifugointiaika ja suurempi sentrifugointinopeus eivät vaikuttaneet paljon sentrifugointitulokseen.

Konsentrointikertoimilla 30 X ja 60 Xsaatiinparempi sentrifugointituloskun sentrifugointinopeuttakasva- tettiin. Sentrifugointitulosei parantunut, kun käsit- telylämpötilaanostettiin 55°C:sta 60°C:een. Konsen-

trointikertoimella 120 X ^saatiin parempi sentrifu- gointituloskuin konsentrointikertoimilla 30 X ja 60 X, ja konsentrointikertoimella 60 X saatiiin parempi sentrifugointituloskuin konsentrointikertoimella 30 X.