Can. Inst. Food Sci. Technol. J. Vol. 17. No. 3. pp. 137-142. 1984

RESEARCH

Effluent Proteins from Rapeseed-CheeseWhey Protein Coprecipitation Process

Lilian U. Thompson, Patrick Chu, Maria Lo,Debbie Markovitch and Maria Siu

Department of Nutritional SciencesUniversity of Toronto

Toronto, OntarioM5S lA8

AbstractSodium hexametaphosphate, polyacrylic acid (PAA) and fer­

ripolyphosphate were used in recovering the effluent proteins froma rapeseed-cheese whey protein coprecipitation process. PAA wasthe most effective precipitant with %070 recovery of the effluent pro­tein. The PAA protein isolates had the highest protein (75070) andlowest ash (8070) content, lightest colour, highest nitrogen solubili­ty at acidic pH (74070), and best fat absorption (323070), emulsifying(74.5 mL oil/g sample) and whipping capacities (324070). Its pro­tein quality, measured as NPR and PER, was good but lower thanthat of the rapeseed-cheese whey protein coprecipitate and casein.As suggested by gel chromatograms, the purified PAA protein isolatewas uncomplexed with the PAA. The PAA protein isolate has poten­tial use as an eggwhite substitute or as nutritional supplement tobeverages.

ResumePour la recuperation des proteines d'effluent d'un procede de

coprecipitation des proteines de colza et de lactoserum, on a faitappel a I'hexametaphosphate de sodium, a l'acide polyacrylique(PAA) et au ferripolyphosphate. Le PAA fut I'agent coagulant leplus efficace avec une recuperation de 96070 des proteines de I'ef­fluent. Les isolats proteiques PAA furent les plus riches en protei­nes (75070) et les plus faibles en cendres (8070). De plus, la couleurfut la plus pale, la solubilite azotee en milieu acide fut la plus ele­vee (74070), et les proprietes emulsifiantes (74.5 mL huile/g d'echan­tillon) et de fouettement (324070) furent les meilleures. Leur qualiteproteique mesuree en termes de CPN et CEP (NPR et PER) futbonne, mais inferieure acelle de la caseine et du coprecipite colza­lactoserum. Tel qu'indique par la chromatographie sur gel, I'isolatproteique PAA purine n'a pas forme de complexe avec la PAA.L'isolat proteique PAA offr~ des possibilites comme substitut aublanc d'oeuf ou cornme supplement nutritionnel dans les breuvages.

IntroductionPreparation of oilseed protein concentrates often in­

volves dispersion of the meal or flour in water, adjust­ment to the isoelectric pH with HC1, and stirring withor without the presence of heat. Since many oilseedshave an isoelectric pH at 4-5 and since the natural pHof cottage cheese whey is 4.6, whey is an ideal dispers­ing medium for protein concentrate preparation.Cheese whey can reduce the acidulant requirement andincrease the protein yield since it has approximately1010 protein which can coprecipitate with oilseed pro­teins under the same conditions. When cottage cheese

whey is used as a dispersing agent, the protein con­centration is referred to as coprecipitation process(Thompson, 1977, 1978; Thompson et al., 1979).

Coprecipitation of rapeseed protein with cottagecheese whey protein by heat treatment (95°C, 15 min)at pH 4.6 resulted in complete sparing of acidulant anda higher nitrogen yield than separate precipitation orconcentration of the two proteins (Thompson, 1977).The nitrogen yield, however, was only 47% with therest lost in the effluent. If the coprecipitation is to bean economical venture, it is important to recover alsothese wasted proteins. The effluent proteins are acidsoluble and non-heat coagulable and may be suitablefor beverage supplementation.

Precipitation of cheese whey (Hidalgo, et al., 1973)and rapeseed proteins (Thompson et al., 1976, 1982;Liu et al., 1982; Gilberg and Tornell, 1976) by sodiumhexametaphosphate (SHMP) and of cheese whey pro­teins by ferripolyphosphate (FIP) (lones et al., 1972)or polyacrylic acid (PAA) (Sternberg et al., 1976) havebeen investigated. This paper describes the recoveryof the effluent proteins by SHMP, FIP and PAA andthe evaluation of the products.

Materials and MethodsDehulled, hexane extracted rapeseed flour was ob­

tained from the Food Research Institute, CanadaDepartment of Agriculture, Ottawa, Ontario and cot­tage cheese whey obtained from Gay Lea Foods,Weston, Ontario. SHMP (Fisher Scientific Co.) wasused as a I % solution. PAA (Good Rite K702, B.F.Goodrich Co., Kitchener, Ontario), supplied as a 25%solution, was diluted to I % prior to use. FIP, withFe to P ratio of 1:6 and aged 8 weeks at 4°C, wasprepared according to lones et al. (1972).

Rapeseed-cheese whey protein coprecipitate wasprepared according to the procedures of Thompson(1977). Rapeseed flour (50 g) and cheese whey (3000mL) mixture was adjusted to pH 4.6 with 1 N HCI,heated at 95°C for 15 min and then cooled to roomtemperature. After centrifugation (1000 X g, 20 min),

Copyright e 1984 Canadian Institute of Food Science and Technology

137

the precipitate was washed twice with distilled water,neutralized and freeze dried. All supernatants afterprecipitation and washing were combined to producethe effluent sample (ES). The ES contained 0.103070total nitrogen, 49.3070 of which was protein nitrogenand 50.7% was nonprotein nitrogen.

The effect of pH and volume of precipitant solu­tions (PAA, SHMP or FIP) on the precipitation ofeffluent protein was determined initially by the rapidscreening method of Hansen et al. (1971). To 20 mLof ES, varied amounts of precipitant solutions wereadded to obtain a precipitant to ES volume ratios of0.025 to 0.5. After adjustment to various pH valueswith 0.1 N HCI or NaOH and centrifugation (1000 xg, 20 min), 5 mL of supernatant were mixed with anequal volume of mercuric chloride solution (4% in ab­solute alcohol) and immediately examined for floc­culated proteins. Low degree of flocculation suggestedlow protein concentration in the supernatant and hencemore protein precipitation by the polyanion. Thisscreening method could be used since no interactionwas observed between the precipitants and mercuricchloride. The actual nitrogen precipitation yields weredetermined only under conditions (pH and precipitantconcentration) which gave the least flocculation. Thisinvolved nitrogen analysis (AOAC, 1980) of the ESbefore and after treatment by the precipitants andcalculation of the yield as:

Y = 100 (NI - N2)/NIWhere: Y = 010 yieldNI = g nitrogen in ES before precipitationN2 = g nitrogen in ES after precipitation

Protein isolates were prepared from the ES asfollows: 1% SHMP was added to ES at a volume ratioof 0.20: 1 and then adjusted to pH 3 with 1 N HCl.After centrifugation (1000 x g, 20 min), theprecipitate was washed once with distilled water, ad­justed to pH 7.0 with 1 N NaOH and freeze dried. Thesame procedure was followed with FIP as precipitantexcept that the FIP to ES volume ratio was 0.30:1 andthe pH was 2.5.

With PAA, the procedure was also similar exceptfor the following: 0.5 g filter aid (Celite, Fisher Scien­tific Co.) was added per liter ES, 1% PAA to ESvolume ratio was 0.25 and the pH was 4.0. Afterwashing, the precipitate was dispersed in distilled water(1/5 of the initial volume of ES), MgC03 was added(0.8 gig PAA used), and stirred for 1 h to raise thepH to 7 prior to centrifugation (1000 x g, 20 min).The precipitate containing the filter aid andmagnesium polyacrylate was removed and the super­natant with the dissolved protein isolate was freezedried. The purification method was similar to that ofSternberg et al. (1976).

The preparation of SHMP protein isolates couldalso be achieved without significant change in yield bydirect addition of equivalent amount of SHMP crystalsto the ES. Similarly, an equivalent amount of 25%PAA may be added directly to the ES in the prepara­tion of PAA protein isolates.

Moisture, protein (N x 6.25, microkjeldahl meth­od) and ash were determined by standard procedures

138 / Thompson et al.

(AOAC, 1980). Non-protein nitrogen was determinedas soluble nitrogen in 12% TCA. Phosphorous wasdetermined according to Fiske and Subbarow (1925)and iron by atomic absorption spectroscopy. Thesamples were hydrolyzed by 6 N HCI for 24 h at 110°Cunder vacuum and analyzed for amino acids using aBeckman amino acid analyzer Model 120C. Cystinewas measured as cysteic acid by performic acid oxida­tion of the samples followed by acid hydrolysis asabove.

Functional properties were determined as follows:colour lightness (% YClE) by reflectance spec­trophotometry (Clydesdale and Francis, 1969)fat ab­sorption by the method of Lin et al. (1974), nitrogensolubility by AACC (1969) method, emulsifying capa­city by titration of 1% sample dispersion with colouredcorn oil as described by Marchall et af. (1975) andwhipping capacity as % volume increase of 50 mL 3%sample dispersion after whipping in a high speedblender for 6 min (Lawhon and Cater, 1971). Fat ab­sorption, emulsifying and whipping capacities were ex­pressed both on per gram sample and per gram pro­tein basis.

The following were analyzed by gel chromato­graphy: ES proteins, effluent protein isolates obtain­ed by SHMP, FIP or PAA precipitation under op­timum conditions. Chromatography was carried outusing Sephadex G-loo (Pharmacia Fine Chemicals) in2.6 x 40 cm column with 0.05 M borate buffer (pH8) as eluent flowing at 60 mL/h. Absorbance wasmonitored at 280 nm. The protein isolates weredispersed in the borate buffer and centrifuged. Thesupernatants and ES were dialyzed against the bufferfor 48 h prior to introduction of 2.5 mg nitrogen sam­ple to the column.

The net protein (NPR) and protein efficiency ratios(PER) were determined by a 10 d rat feeding experi­ment with 30 male weanling rats (70-80 g initial bodyweight; Woodlyn Farms, Guelph, Ontario) randomlydistributed into 5 diet groups. The diets were preparedaccording to the formulation specified by AOAC(1980). Four groups were fed basal diets containing10% proten provided by either casein, casein + PAA,rapeseed-cheese whey protein coprecipitate, or PAAprotein isolate. PAA was added to the casein at thesame level used to precipitate the effluent protein(0.075 g PAA/loo g protein). The fifth group was feda non-protein diet. The rats were individually housedin hanging, galvanized, wire meshed cages at 23°C andon a 12 h light dark cycle. All diets and water weregiven ad libitum. The rats were weighed and food in­takes were measured every 2 days. PER was equal tothe ratio of weight gain to protein intake while NPRwas the ratio of weight gain of test animal plus weightloss of animals fed the non-protein diet to protein con­sumed by the test animal.

Results and DiscussionPolyanions can interact with the positively charged

amino acid side chains of the proteins yielding solu­ble or insoluble protein complexes. These reactions arepH dependent. With decreasing pH, the number of

J. Inst. Can. Set. Teehnol. Aliment. Vcl. 17. No. 3, 1984

>Zo

ca~....

....e:....A.

U....Cll:A. 20z

SHMP :ES (¥Jv) FIP:ES (v/vI- 0.20 60- 0.15 -0.30-- 0.10 -0.25-'0.20

20 50

....e:....a. 10u....Cll:a.z S#.

>z 15o

4

0L-....J~ .L.. ......_ ......

2 3

pH

10

oFig. 1. Effect of pH and ratio (v/v) of 1010 sodium hex­

ametaphosphate (SHMP) to effluent sample (ES) on thenitrogen precipitation yield.

2 3

pH

5

pH

Fig. 3. Effect of pH and ratio (v/v) of 1010 polyacrylic acid (PAA)to effluent sample (ES) on the nitrogen precipitation yield.

.

PAl.: ES ('Iv)

-0.25-o.a-0.150-00.10

..

o •~1--~2~-""'"':!'3---4~--....!5~---16-.J

Fig. 2. Effect of pH and ratio (v/v) of ferripolyphosphate (FIP)to effluent sample (ES) on the nitrogen precipitation yield.FIP was aged 8 weeks at 4°C according to Jones et al. (1972).

washed, adjusted to pH 7 with 1 N NaOH and freezedried without further purification. The PAA proteinprecipitate, on the other hand, was upgraded orpurified by dispersing the sample in water, addingMgC03 and centrifugation. The PAA was separatedfrom the protein as magnesium polyacrylate and thesupernatant which contained the effluent proteins was

free positive charges on the protein increases andthereby also the capacity of the proteins to interactwith the polyanions. Excess amount of polyanionsadded will result in a net charge on the aggregates giv­ing a soluble, charged complex. Inadequate additionof polyanion will cause incomplete protein precipita­tion. Hence there exists an optimum amount of poly­anion and pH for maximum protein precipitation(Hidalgo and Hansen, 1969, 1971; Gillberg andTornell, 1976).

The effect of precipitant to ES volume ratio and ofpH on the 070 precipitation of nitrogen in the ES areshown in Figures 1-3. When PAA was used as precipi­tant, the highest % nitrogen yield was 47% at pH 4and a 1% PAA:ES volume ratio of 0.25. With SHMP,the highest yield was 12% at pH 3 and 1% SHMP:ESvolume ratio of 0.20, while with FIP, the highest %yield was 41.2% at pH 2.5 and FIP:ES volume ratioof 0.30. PAA gave the best nitrogen yield, however,the value was still low probably due to the high con­centration of unprecipitable non protein nitrogen inthe effluent (50.7% of total nitrogen). This might beexpected since both rapeseed and cheese whey containconsiderable amounts of non protein nitrogen (Bhat­ty, 1972; Rizvi and Josephson, 1975). The proteinnitrogen yields by PAA, FIP and SHMP precipitationunder the optimum conditions were 95.7, 82.9 and23.9% respectively. With 47% protein nitrogenrecovery from the coprecipitation process (Thompson,1977), precipitation of the effluent proteins by PAAwould give a 98% total recovery of protein from therapeseed and cheese whey.

The SHMP and FIP protein precipitates were

Can. Inst. Food Sci. Technol. J. VO!. 17. No. 3. 1984 Thompson et al. / 139

Table I.Chemical composition of effluent protein isolaters.

EFFLlUT SNIPt.E

Fig. 4. Gel patterns of effluent proteins, and polyacrylic acid(PAA), sodium hexametaphosphate (SHMP), and fer­ripolyphosphate (FIP) protein isolates.

FIP

8.017.3 (18.8)58.6 (63.7)1I.3 (12.3)9.6 (10.4)

2.55 ± 0.212.ll ± 0.192.50 ± 0.092.58 ± 0.30

Proteinefficiency ratio

SHMP

9.053.9 (59.3)21.4 (23.5)6.4 (7.0)

na

precipitant I

Net proteinratio

4.83 ± 0.291

4.58 ± 0.245.32 ± 0.235.35 ± 0.41

PAA

11.765.8 (74.5)26.8 (7.7)

na3

na

IMean ± standard error.

Protein source

Moisture (%)Protein (N x 6.25) (%)Ash(%)P(%)Fe(%)Essential amino acids

(g116 g N)Iys 7.2 9.1 8.1thre 2.8 2.6 3.2cys + met 6.3 5.6 5.7val 3.2 3.0 3.4ileu 3.6 4.3 3.5leu 6.7 7.6 6.8phen + tyr 8.4 7.8 7.3

Rapeseed - cheese wheyprotein coprecipitate

PAA proteinCaseinCasein + PAA

IPAA = polyacrylic acid; SHMP = sodium hexametaphosphate;IFIP = ferripolyphosphate.2dry basis3not analyzed

Table 3.Protein quality of polyacrylic acid (PAA) protein isolate.

teins as compared with ES proteins suggests greaterrecovery of the high molecular weight protein fractionsby the PAA. The elution volumes were the same forboth the ES and PAA proteins suggesting that thePAA is not complexed with the protein and that theES protein was not significantly changed by theprecipitation process.

The SHMP protein isolate showed only a single peaksuggesting precipitation of primarily the highmolecular weight proteins by the SHMP. Since SHMPdid not precipitate the low molecular weight proteinfraction, it gave a lower protein recovery.

With the FIP protein isolate, the appearance of 2peaks suggests recovery of both the high and lowmolecular weight protein fractions of the effluent.However, the second fraction was eluted at an earlierstage probably due to the FIP which remained com­plexed to the protein.

The chemical composition of the three proteinisolates are given in Table 1. As might be expected,the PAA protein isolates had the most protein(74.5070). The FIP protein isolate had the least protein(18.8%) because of the considerable amount of ash(63.7%) which could then be attributed to elevated Feprobably (10.4%) and P (12.3%) levels. The FIPisolate could be used for iron supplementation as hasbeen suggested by other workers (Imado et al., 1962;Jones, et al., 1972, 1975; Amantea et al., 1974). Theessential amino acid composition of the three isolates

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then freeze dried. Magnesium polyacrylate may betreated with H2S04 to regenerate the PAA which canthen be recycled (Sternberg et al., 1977). PAA pro­tein precipitate was chosen for purification since PAAgave the highest protein precipitation yield and can berecovered and recycled. PAA also does not yet havefederal blanket approval for all food areas althoughit is approved for use in some food related products.

Gel filtration of the ES proteins, PAA, SHMP, andFIP protein isolates was done to determine which pro­tein fractions were precipitated by the three poly­anions. The PAA recovered both the high and lowmolecular weight protein fractions of the ES (Figure4). However, a higher ratio of the high molecularweight to low molecular weight proteins for PAA pro-

140 / Thompson et af. J, Inst, Can, Sci. Technol. Aliment. Vot. 17, No. 3, 1984

Table 2. Some functional properties of effluent protein isolates.

Emulsifying Whippingcapacity capacity

mL oillg mLoil/gB2sample protein AI (% YCIE)

74.5 113.2 323.5 327.8 71.761.5 114.1 88.5 109.5 46.349.5 286.1 63.5 244.7 63.6

130.0 141.6 na na 88.5na na 1003 na na

87.5 148.3 35.6 40.2 58.8

na

215

491.3401.7

1068.2198.3

mLllOOgprotein

323.3216.5184.8182.0

na

FatAbsorption

127

inL/IOOgsample

na

66.388.071.768.6

pH 7

Nitrogensolubility

(%)

73.56.06.0

50.0na4

pH 3

Product

PAA proteinSHMP proteinFIP proteinPromine DEgg whiteRapeseed - cheese whey

protein coprecipitate 2.3 10.7

1% volume increase of 50 mL 3% sample dispersion2% volume increase per g protein3% volume increase of 50 mL fresh egg white4not analysed

differed only slightly. Of interest, however, is the veryhigh lysine, cystine and methionine concentrations inall three. Since these amino acids are limiting in manyplant protein sources, the supplementary value of theeffluent protein isolate to other plant proteins may beconsidered as good.

The functional properties of the effluent proteinisolates are given in Table 2. The nitrogen solubilityof the proteins at pH 7 ranged from 66-880/0, was closeto that of Promine D (a commercial soy isolate) andwas much better than the coprecipitates. At acidic pH,the SHMP and FIP proteins did not dissolve well sincethe isoelectric pH's of the complexes were 2.5-3.0(Figures 1 and 2). On the other hand, the solubilityof PAA protein at acidic pH remained high (73.5%)since this protein is not complexed with the polyanion.The excellent nitrogen solubility of the PAA proteinat acidic and neutral pHs suggests uses in milk andacidic type beverages.

The fat absorption capacities of the three isolateswere better than Promine D and the coprecipitate. ThePAA protein had the highest value of the three isolateswhen expressed on per sample weight basis but FIPprotein had the highest value on per weight proteinbasis.

The emulsifying capacity of the three isolates on persample weight basis was poorer than the coprecipitateand Promine D. On protein basis, the FIP protein ex­celled the rest. The whipping capacity of the PAA pro­tein surpassed that of egg white, the coprecipitate andthe other protein isolates. With egg white equal to100%, the PAA protein gave 323% whipping capaci­ty. The utilization of the PAA protein as an egg whitesubstitute remains to be explored.

The PAA protein had a yellow tinge and had thelightest colour which remained light and acceptableeven after dispersion in water. By contrast, the FIPand SHMP proteins were light tan.

It appears from the foregoing that PAA is an ef­fective precipitant and may be considered the best forthe recovery of proteins from the effluent of rapeseed­whey protein coprecipitation process. The NPR andPER of the PAA protein isolate were therefore deter­mined and compared with that of the rapeseed-whey

protein coprecipitate, casein, and casein with addedPAA. PAA was added to casein to determine whetherany residual PAA in the protein isolate has any effecton protein quality. To maximize the PAA concentra­tion in the diet, a concentration equivalent to the totalamount of PAA that was used in the preparation ofthe dietary effluent protein isolate was added to thecasein.

The PAA had no effect on the protein quality asreflected by similar values for casein and casein +PAA (Table 3). The PAA protein had high PER andNPR although lower than that of the coprecipitate andcasein.

In conclusion, PAA is an effective precipitant, bet­ter than SHMP or FIP, for recovery of effluent pro­teins. Recovery of cheese whey and rapeseed proteinsby coprecipitation with acid and heat followed byprecipitation of the effluent proteins by PAA gave a98% total protein yield. The PAA-protein isolates hadhigh protein and essential amino acid contents andgood functional properties. The protein quality washigh but lower than that of rapeseed-cheese whey pro­tein coprecipitate and casein. The good whippabilityand nitrogen solubility at acidic pH suggest potentialapplication of PAA protein isolates as egg whitesubstitute or as nutritional supplement to beverages.

AcknowledgementThe authors thank E. Reyes for technical assistance,

J.D. Jones for providing the rapeseed flour andAgriculture Canada and Natural Sciences andEngineering Research Council of Canada for finan­cial support.

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Cereal Chemists, St. Paul, MN.Amantea, a.F., Kason, C.M., Nakai, 5., Bragg, D.B. and Emmons,

D.B. 1974. Preparation of ferric whey proteins by heating.Can. Inst. Food Sci. Tech. J. 7:199.

AOAC. 1980. "Official Methods of Analysis." Association of Of·ficial Analytical Chemists. Washington, DC.

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Bhatty, R.S. 1972. A note on trichloroacetic acid precipitation ofoil seed proteins. Cereal Chem. 49:729.

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Fiske, C.H. and Subbarow, Y. 1925. The colorimetric determina­tion of phosphorous. J. BioI. Chem. 66:325.

GilIberg, L. and Tornell, B. 1976. Preparation of rapeseed proteinisolates. Precipitation of rapeseed proteins in the presenceof polyacids. J. Food Sci. 41:1070.

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lones, S.B., Kalan, E.B., Jones, T.C., Hazel, J.F., Edmonston,L.F., Booth, A.N. and Fritz, J.C. 1975. FIP - whey pro­tein powders. Their potential as nutritional iron sup­plements. J. Agric. Food Chem. 23:981.

Lawhon, J. and Cater, C. 1971. Effect of processing methods andpH of precipitation on the yield and functional proper­ties of protein isolates from glandless cottonseed. J. FoodSci. 36:372.

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Thompson, L.V., Allum-Poon, P. and Procope, C. 1976. Isola­tion of rapeseed protein using sodium hex­ametaphosphate. Can. Inst. Food Sci. Tech. J. 9:15.

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Accepted January 18, 1984

J. Inst. Can. Sci. Technol. Aliment. Vo!. 17, No. 3. 1984