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Pilot scale isolation of proteins from field peas (Pisum sativum L.) for use as food ingredients

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Pilot scale isolation of proteins from field peas (Pisum sativum L.) for use as food ingredients
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  *Correspondent: Fax: +613 921 68284. e-mail:s9340176@cougar.vut.edu.au Pilotscale isolationofproteins fromfieldpeas( Pisumsativum    L.) foruse as foodingredients Shaojun Tian*, William S. A. Kyle & Darryl M. Small School of Life Sciences and Technology, Victoria University of Technology, Melbourne, Victoria 8001, Australia Summary The protein and starch fractions of field pea ( Pisum sativum L.) are potentially a sourceof novel ingredients for food processing. As part of research to elucidate the functionalproperties of field pea proteins, the pilot scale extraction of proteins has been undertaken.Two different approaches have been used and the recoveries of protein compared. Thefirst involved extraction with a salt solution followed by decanting and clarification toremove solids. The solution was then further concentrated and salt removed by ultrafil-tration and diafiltration. The second procedure was based upon alkaline extractionfollowed by decanting and recovery by isoelectric precipitation and neutralisation. Bothspray drying and freeze drying methods were employed. The protein isolates have beencompared and characterised with respect to solubility, chemical composition and toelectrophoretic patterns. Overall, the proteins extracted with salt exhibited better physicalproperties than proteins extracted with alkali, in terms of colour and particle size. Thesolubilities showed little variation and the electrophoretic patterns were similar. Freezedrying resulted in isolates with darker colour compared with spray drying. Keywords Isolation, particle size, pea proteins, spray and freeze drying, ultrafiltration. Introduction Field pea ( Pisum sativum L.) is a crop of increas-ing importance in the world market. Like otherlegume seeds, field pea is characteristically rich inprotein and contains high levels of lysine which isimportant in balancing the deficiencies of thisessential amino acid in cereal-based diets(Gueguen, 1991). Hence the protein fractions of field pea offer potential as a source of novelingredients for food processing. However, thetechnological uses of legume proteins dependlargely on the physico-chemical properties whichare necessary for their successful incorporationinto food systems. Processing conditions such asthe method of isolation, the effect of pH andextracting agent play an important role(Damodaran, 1996). Particle size and the dryingmethods may also be significant (Sathe etal  .,1982).Field pea proteins and starches have been frac-tionated by dry milling and air classification tech-niques (Vose, 1980; Sumner etal  ., 1981; Sosulski& McCurdy, 1987). Whole or dehulled field peaseeds are pin milled to yield flours with a specificparticle size which can be further separated intoprotein and starch fractions using an air classifi-er. This physical separation of the protein-richfraction is efficient and convenient because iteliminates the handling of large volumes of slur-ries. However, the functional properties of theprotein concentrate obtained by physical process-es are poorer than for the product obtained bywet processes (Gueguen, 1991).In order to prepare grain legume protein iso-lates, wet processes are most widely used. Oneapproach involves using alkali solution to solu-bilise the proteins and the insoluble material isremoved by centrifugation. By adding acid to the International Journal of Food Science and Technology 1999, 34 , 33–39 ©1999 Blackwell Science Ltd 33  supernatant, the protein isolate is precipitated iso-electrically. The product is then dried using eithera spray, drum or freeze drying method (Klein &Raidl, 1985; Sumner etal  ., 1981). Ultrafiltrationand reverse osmosis can also be used instead of acidification to recover the extracted proteins(Bramsnaes & Olsen, 1979; Deeslie & Cheryan,1991). Another wet process involves proteinextraction with sodium chloride solution and pre-cipitation of the isolate by water dilution (Murray etal  ., 1981; Abdel-Aal etal  ., 1986). However,there is relatively little documentation on thelarge scale fractionation of field pea proteinsbased on wet extraction which might relate tocommercial production.Accordingly, the purpose of the current studywas to: 1 Use salt (NaCl) solution to extract pea pro-teins in a pilot factory with the combination of membrane processes to desalt and concentratethe proteins; 2 Compare the physico-chemical properties of the product with the field pea protein isolateproduced by traditional alkaline extractionand precipitation at isoelectric pH; 3 Evaluate any differences in the protein isolatesresulting from the application of spray dryingand freeze drying. Materials andmethods Materials Dehulled, split field peas (Dunn type –  250 kg)were obtained in Victoria, Australia and a flourwas produced by grinding the seeds in a localcommercial plant. Laboratory preparation of protein isolates For comparative purposes, Osborne fractions(Osborne, 1924) and protein isolates were pre-pared from field pea flour. For the extraction of protein isolate, field pea flour was suspended indistilled water (1:5 w/v), adjusted to pH 9.0 andstirred for 1 h. After centrifugation and filtration,the pH was adjusted to 4.5 and the protein recov-ered by centrifugation at 8000   g  for 20 min.The protein extracts were then neutralised andfreeze dried. In order to extract globulin andalbumin fractions, field pea flour was suspended1:10 (w/v) in salt solutions (0.5M NaCl) for 1 hat room temperature, and then centrifuged at10000   g  for 20 min. The supernatant was col-lected and dialysed at 4°C against deionisedwater for 5 days; dialysate was changed everyday. The contents of dialysis tubes were cen-trifuged at 10000   g  for 20 min and the pellet(globulin fraction) was freeze-dried. The super-natant (albumin fraction) was concentrated bypassing through the ultrafiltration membranes (4plates, MW cut-off at 5000, Millipore Crop.,Bedford, USA) followed by freeze drying. Afteralbumins and globulins were extracted, the sub-sample from the centrifuge was extracted withbutan-1-ol  0.05% DTT (1:10 w/v) under mag-netic stirring for 1 h for prolamin fractions. Themixture was centrifuged at 12000   g  for 20 minand the supernatants were dialysed againstdeionised water for 5 days at 4°C and thenfreeze-dried. For glutelin extraction, the residuefrom the prolamin-alcohol solutions was re-extracted twice with 0.1M NaOH. The combinedsupernatants following centrifugation wereadjusted to pH 4.5. The resulting micellar systemwas centrifuged at 12 000   g  for 20 min and thepellet obtained was neutralised and freeze-driedto give the glutelin fraction. Pilot plant preparation of protein isolates The pilot scale isolation of field pea proteins wasconducted in the pilot scale facilities of theAustralian Food Industry Science Centre,Werribee, Victoria. The outlines of the processesto prepare the protein isolates extracted withalkaline solution and salt solution are sum-marised in Fig.1 and Fig.2, respectively.Field pea flour (190 kg) was mixed with 950 Lfiltered water at 40°C in a 1000 L vat with vig-orous agitation. The pH was adjusted to 9.0 using4M NaOH. After 1 h, the mixture was pumped toa decanter centrifuge (Westfalia Separator AG,Model D-59302 Oelde, F.R. Germany) wheremost of the carbohydrate solid was separatedfrom the protein solutions. In order to furtherpurify the proteins, the solution was then passedthrough a clarifier centrifuge (Westfalia SeparatorAG, Model D-4740 Oelde, F.R. Germany) to Pilotscale isolation of field pea proteins Shaojun Tian et al. International Journal of Food Science and Technology 1999, 34 , 33–39 34 ©1999 Blackwell Science Ltd  remove the fine particles of the carbohydrates.The extract of the proteins was adjusted to pH4.5 by using 4M HCl and mixed for 0.5 h. Thecurd precipitated by acid was concentrated bypassing the mixture to the clarifier and the wheydiscarded. The curd was washed with 200 L fil-tered water and reclarified. The product was thenneutralised to pH 7 by 4M NaOH with mild agi-tation and dried either by spray drier (NIROFSD-4) or by freeze drier.For protein isolation using a salt solution, 60 kgfield pea flour was mixed with 600 L 0.5M sodi-um chloride solution and the pH was adjusted topH 7. After 1 h of agitation, the slurry was passedthrough the decanter to separate the proteinextracts from the carbohydrate. The protein solu-tion was purified by clarification and then passedthrough the ultrafiltration and continuous diafil-tration system (DDS Model 37, 6.6 m 2 , Pasilac-Danish Turnkey Dairies Ultrafiltration Plants,Denmark) to remove salt and concentrate the pro-teins. The operation was conducted at 48°C. Themembrane used was plate and frame, polysulfoneGR 61 PP with a molecular weight cut-off at 20kDa. The product was either spray dried or freezedried. Before use of the freeze dried extract, theprotein isolates were reground in a hammer mill(Cereal Mill 6000, Newport Scientific Pty Ltd,Sydney, Australia) to pass a 400 micron screen. Analysis procedures All procedures were in triplicate and mean resultsare presented.Proximate analyses for chemical compositionwere as described by AACC (1983), these includ-ed procedures for moisture (air-oven method44–15A), crude protein (method 46–13), crude fat(method 30–25), crude fibre (method 32–10), totalash (method 08–03).Colour was measured with a MinoltaChromameter (Model CR-300, Minolta CameraCo., Osaka, Japan) on the dried samples. Particlesize measurement was performed on a MalvernMastersizer-X particle size analyser (MalvernInstruments Ltd, Worcester, UK.) Pilotscale isolation of field pea proteins Shaojun Tian et al. International Journal of Food Science and Technology 1999, 34 , 33–39 35 ©1999 Blackwell Science Ltd Figure 1 Flowchart of the process to prepare field peaprotein isolate with alkaline solution. Figure 2 Flowchart of the process to prepare field peaprotein isolate with salt solution.  Electrophoresis of proteins SDS–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out with samples reduced byuse of 2-mercaptoethanol and 2 min of heating at100°C. Polyacrylamide slab gels (12.5%) wereprepared as described by Laemmli (1970). Thegels were stained with 0.5% Coomassie Blue R250for 30 min and destained with ethanol/aceticacid/water (3.5/1/5.5 v/v/v). The standard proteinsused (Sigma Chemical Co., St. Louis, Mo) were:phosphorylase b (Mr 94 000), bovine serum albu-min (67 000), ovalbumin (43 000), carbonic anhy-drase (30 000), soybean trypsin inhibitor (20 100)and  -lactalbumin (14 400). Determination of the solubilities of theproteins extracted Protein isolates (0.4%, w/v) were suspended underpH 2, 3, 4, 5, 6, 7, 8, 9, 10 at room temperatureand stirred for 30 min. The respective slurrieswere then centrifuged at 4000   g  for 20 min. Thesoluble protein contents were determined by themethod of Lowry etal  . (1951) following appro-priate dilution. Standards containing 0–100mg of protein were prepared from 0.1% bovine serumalbumin solution. Results anddiscussion Composition and protein recovery Results of the proximate analyses of the seedflour and the protein isolates are listed in Table1.Crude fat was not removed in these processesbecause the fat content in field pea is relativelylow (2.68% on dry basis) and the additional stepto extract the oil would increase the productioncost. The protein recovery rates for alkalineextraction and salt extraction were 59% and 40%,respectively. Isolates extracted with salt containedmore protein than those extracted with alkali andboth had higher ash content than the srcinalflour. This reflects the small amount of saltremaining after processing as well as the salt pro-duced due to the food-grade acid and alkali usedfor protein precipitation and neutralisation(Sosulski & McCurdy, 1987).It is likely that the further application of ultra-filtration and diafiltration would probablyincrease the protein content and decrease the saltcontent of the dried product. However, the result-ing recovery of the protein would be lower.Another consideration is that continuous diafil-tration system adds water to the retentate as per-meate is removed (Nichols & Cheryan, 1981).Accordingly the addition of steps to remove saltwould result in problems being encountered dur-ing subsequent drying of the isolate. If morewater were used to wash the protein extracts, theconcentration of the proteins would not be highenough (less than 8–10%) for spray drying.Similarly during freeze drying, more time andenergy would be needed to remove the excesswater. However, the primary advantage of con-tinuous diafiltration is to keep protein concentra-tion low during processing which reduces yieldlosses due to membrane adsorption.For the other process where alkali was theextracting agent, use of water to wash the iso-electric precipitated proteins could increase theprotein content by 6%, but the recovery rate Pilotscale isolation of field pea proteins Shaojun Tian et al. International Journal of Food Science and Technology 1999, 34 , 33–39 36 ©1999 Blackwell Science Ltd ComponentFlourSPI b API c Residued Moisture (%)8.63.93.758.3Protein (Nx6.25) (%)28.881.177.11.84Crude fat (%)2.682.542.430.65Crude Fibre (%)1.100.070.021.23Ash (%)2.675.565.971.29Carbohydrate (%)64.810.714.595.0Salt (NaCl) (%)—5.365.23—a: Moisture valuesexpressed ‘as is’, others on a dry weight basis.b: Pilot scale salt (0.5MNaCl) extracted protein isolate.c: Pilot scale alkaline (pH9) extracted protein isolate.d: Residue fromalkaline extraction.e: Carbohydrate calculated by difference. Table 1 Proximate composition of field pea flour (dehulled) andprotein isolates   would be decreased by 7%. In addition, the clar-ification process is time consuming and henceonly one washing process was used in the pro-cessing of the protein isolate. Colour and particle size The colour parameters of the flour and the pro-tein isolates are presented in Table2. Proteinsextracted using salt had a lighter colour than theisoelectric precipitated proteins. On the otherhand, freeze-dried samples had a much darkercolour than the spray-dried products. This resultconfirmed the observation of Sumner etal  . (1981)who claimed that the freeze-dried products mayhave been darkened by oxidation of componentssuch polyphenols. The results of particle sizeanalyses are shown in Table3. The productextracted with salt gave, after spray-drying, a rel-atively even size with most particles between 10and 100 mm diameter. On the other hand, forspray dried isolate that was extracted with alkali,there was a wide range of fine particles. Freeze-dried products showed a larger particle sizebetween 100  m and 400  m and this could bereduced by regrinding. However this is time con-suming and the heat generated in the grindingprocedure may cause partial denaturation of theprotein molecules. Solubility and SDS–PAGE The structural characteristics of the proteins havebeen investigated using SDS–PAGE. The elec-trophoretic separation patterns of the pilot scaleisolates have been compared with those of frac-tions isolated in the laboratory by the traditionalOsborne procedures (Fig.3). For further com-parison, a laboratory extraction using the samealkaline solution was also prepared and subjectedto electrophoresis (lane 10, Fig.3). This extractshowed a similar pattern to the pilot scale iso-lates.The four different laboratory fractions (albu-min, globulin, prolamin and glutelin) extractedfrom field peas have distinct patterns of subunits. Pilotscale isolation of field pea proteins Shaojun Tian et al. International Journal of Food Science and Technology 1999, 34 , 33–39 37 ©1999 Blackwell Science Ltd Table 2 Colour parameters of field pea flour and protein isolates Table 3 A comparison of theparticle sizes of field pea flourand protein isolates ColourFlourSPI.1 a SPI.2 b API.1 c API.2 d L*88.9574.4283.6665.8679.29a*  5.55  2.35  4.35  1.07  3.31 b*  22.97  25.69  21.12  22.90  23.96a: Pilot scale salt (0.5M NaCl) extracted protein isolate by freeze drying.  b: Pilot scale salt (0.5M NaCl) extracted protein isolate by spray drying.c: Pilot scale alkaline (pH 9) extracted protein isolate by freeze drying.d: Pilot scale alkaline (pH 9) extracted protein isolate by spray drying . FlourSPI a API.1 b API.2 c Mean particle diameter(m)21.437.421.783.1Proportion of particles (%) with diameterless than  d 1  m2.30.15.10.15  m17.32.19.11.710  m27.97.616.15.050  m81.464.392.831.3100  m93.388.399.158.2400  m99.999.8100.098.0600  m100.0100.0100.0100.0a: Pilot scale salt (0.5MNaCl) extracted protein isolate by spray drying.b: Pilot scale alkaline (pH9) extracted protein isolate by spray drying.c: Pilot scale alkaline (pH9) extracted protein isolate by freeze drying.d: expressed as percentage of particles having a diameterless than that indicated.
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