采后杏果中果胶酶和螯合可溶果胶的交互作用的体外研究.pdf

Original articleExtraction and physicochemical properties of soya bean proteinand oil by a new reverse micelle system compared with otherextraction methodsGuanhao Bu,1Yingying Yang,1Fusheng Chen,1*Zhixiong Liao,2Yanxiu Gao,1Hongshun Yang,3,4Runjie Li,1Kunlun Liu1&Junting Zhao51 College of Food Science and Technology,Henan University of Technology,Zhengzhou 450001,China2 Xinke College,Henan Institute of Science and Technology,Xinxiang 453003,China3 Food Science and Technology Programme,Department of Chemistry,National University of Singapore,Science Drive 2,Singapore 117543,Singapore4 National University of Singapore(Suzhou)Research Institute,Suzhou Industrial Park,377 Lin Quan Street,Jiangsu 215123,China5 School of Chemistry and Chemical Engineering,Henan University of Technology,Zhengzhou 450001,China(Received 22 April 2013;Accepted in revised form 25 September 2013)SummaryReverse micelle extraction is a novel technology for the separation of plant components such as proteinsand oil.In this study,sodium bis(2-ethylhexyl)sulphosuccinate(AOT)reverse micelle system and AOT/Tween 85 reverse micelle system were used to extract soya bean protein and oil from soya bean flour.Thephysicochemical properties of the protein and oil extracted were investigated and compared with tradi-tional extraction methods.The results showed that the efficiency of forward extraction of soya bean pro-tein using an AOT/Tween 85 reverse micelle system was superior to that using an AOT reverse micellesystem at the optimal extraction conditions.In addition,soya bean proteins extracted using reversemicelle extraction had no unordered structure under Fourier transform infrared spectroscopy.The acidand peroxide values of oil products from two reverse micelle extractions were lower than that fromimmersion.The results indicated that AOT/Tween 85 reverse micelle system is effective in extracting soyabean protein and oil.KeywordsAOT,reverse micelle extraction,soya bean oil,soya bean protein,Tween 85.IntroductionReverse micelle extraction,a relatively new technologyfor liquidliquid extraction,especially for the isolationand purification of proteins,has recently received dueattention(Luisi et al.,1979;Harikrishna et al.,2002).Reverse micelles are formed by surfactants in nonpolarorganic solvents,while in their polar cores,nanometre-sized water pools are formed by the solubilisation inwater(Luisi et al.,1979;Naoea et al.,2011).Withself-assembly capacity,reverse micelle systems are notonly dynamic balancing systems but also stable andtransparent thermodynamic systems(Liu et al.,2006).The overall mechanism of reverse micelle extractionconsists of two fundamental steps:forward extraction(protein is transferred into polar water pools and oil istransferred into the organic phase)and backwardextraction(protein is released from polar water poolsand transferred into an aqueous phase to be recovered,resulting in separation of aqueous phase and oil phase)(Zhao et al.,2010a).The polar water pools inside reverse micelles cansolubilise hydrophilic biomolecules,such as proteins,enzymes,DNA,nucleic acids,short peptides andamino acids.The size of water pools plays a significantrole in the solubility of biomolecules in the micellecore.W0,the determining factor of the size of waterpools and the molar ratio,is the ratio of water andsurfactant,which can be adjusted by an aqueous buffercontaining an appropriate amount of salt(Ghazi et al.,2006).Reverse micelles form a three-phase systemincluding watersurfactantorganic solvent(i.e.theform of W/O(water/oil)with various shapes,such asspherical,oval and rod);thus in this system,the bio-molecules inside the polar water pools are protectedfrom denaturation by organic solvents.Reverse micelles are more suitable for separatingproteins than regular liquidliquid extraction or otherseparation methods because in those methods,the trans-*Correspondent:Fax:[phone];e-mail:International Journal of Food Science and Technology 2014,49,10791089doi:10.1111/ijfs.12403 2013 Institute of Food Science and Technology1079fer of proteins into solvents often results in irreversibledenaturation and loss of biological activity(Guo et al.,2008).Luisi et al.(1979)first pointed out the potentialof reverse micelle solubilisation of proteins for proteinseparation.Dekker et al.(1986)and Goklen&Hatton(1985)developed the process systematically for practicaluse of reverse micelle systems.Later on,many research-ers demonstrated the factors including molar ratio W0,aqueous phase pH and ionic strength(Rho&Kang,2004),surfactant type and concentration(Shin&Vera,2002),and co-surfactant(Lee et al.,2004)affecting pro-tein solubilisation based on the interactions betweenreverse micelles and proteins.Among these parameters,surfactant is well known as playing an important role instabilising protein solubilisation in reverse micelles.Most studies to date have focused on the extraction ofproteins by reverse micelle systems,whereas studies onthe extraction of oil have been rarely reported.With the development of protein extraction by ionicreverse micelles,protein sectional deactivation causedby the strong electrostatic interaction occasionallyoccurs in the extraction process(Harikrishna et al.,2002).Therefore,some nonionic surfactants are usedto form nonionic reverse micelles for protein extrac-tion to avoid protein deactivation.Tween 85 is usuallyused for reverse micelle extractions,and it does nothas detrimental effects on the structure,function andstability of proteins solubilised in reverse micelles(Sawada et al.,2004).However,the extraction effi-ciency by Tween 85 itself was very low.In this study,the efficiency of two reverse micellesystems,ananionicsurfactantnonionicsurfactantAOT/Tween 85 mixed system and an anionic surfac-tant AOT system in extracting protein and oil fromsoya bean flour,was investigated.Effects of variousfactors including soya bean flour concentration,W0,temperature,time,pH,ionic strength and ultrasonicpower on the efficiency of forward extraction of soyabean protein were examined.The physical and chemi-cal properties of oil extracted by reverse micelle werecompared with oil immersed,such as acid value(AV),peroxide value(POV)and fat acid(FA)composition.Materials and methodsMaterialsChemically pure AOT and Tween 85 were purchasedfrom Shanghai Haiqu Chemical Co.Ltd.(Shanghai,China)andShanghaiLichenLimitedCompany(Shanghai,China),respectively.All other reagentsused were of analytical grade.Full-fat soya bean floursieved through a 100-mesh screen and containing42.2%protein,4.3%moisture and 24.7%oil wasobtained from Anyang Mantianxue Food Manufactur-ing Co.Ltd(Anyang,Henan,China).Proximate composition analysisCrude protein of soya bean flour was determined usingthe micro-Kjeldahl method(Concon&Soltess,1973),whereas crude fat was determined by Soxhlet extrac-tion(AOAC,1984).The moisture content was mea-sured by drying in an oven at 105 C until constantweight was obtained.AV,POV and FA compositionwere determined according to standard methods of theInternational Union of Pure and Applied Chemistryfor analysis of oil and fats.Preparation of two reverse micelle systemsAn AOT reverse micelle was obtained by dissolvingAOT in isooctane and then injecting the mixture withKClphosphate buffer solution.A stock solution of0.08 g mL?1AOT reverse micelle system was obtainedby mixing AOT with isooctane and then stirring themixture using a magnetic stirrer at room temperature.When AOT was dissolved completely,KClphosphatebuffer solutions with certain pH and different concen-trations were added for creating water content.Thewater content in reverse micelles is expressed as W0,which is the molar ratio of water to surfactant.Thewater content was determined by the KarlFischermethod(Bu et al.,2012).AnAOT/Tween85reversemicellesystemwasachieved by dissolving AOT and Tween 85(mass ratio,4:1)in isooctane and n-octyl alcohol(volume ratio,4:1)and the same procedure was used for the AOTreverse micelle system(Hemavathi et al.,2010).Extraction of soya bean protein and oil by reverse micellesystemsSoya bean flour was added to the different reversemicelle systems and then extracted for a certain periodbyanultrasonic-assistedmethod.Theresultingmixture with protein dissolved in polar core and oilcontained in organic solvent phase was centrifuged at5000 9 g for 15 min,and clear supernatant was usedfor the next step of extraction.The above-describedprocedure is designated as the forward extraction.The same volume of clear supernatant and buffersolution was mixed and extracted in an ultrasoniccleaning machine.The resulting mixture was centri-fuged at 5000 9 g for 10 min.The oil and aqueousphases were then separated and stored.This step isdesignated as the backward extraction.The proteincontent in the aqueous phase was measured by a spec-trophotometer(UV-160A;Shimadzu,Kyoto,Japan)at280 nm.NaCl solution(0.1Min 70%ethanol solu-tion,with pH adjusted to 4 with 0.1MHCl and 0.1MNaOH)and the upper oil phase were mixed at theproportion of 1:2.5(v:v),stirred at 30 C for 1 h,left 2013 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2014Extraction of soya bean protein and oil G.Bu et al.1080to stand and stratified.Isooctane was subsequentlyremoved from the upper organic phase using a rotaryvacuum evaporator at 90 C.The oil product wasobtained following the above-described steps.Immersion of soya bean oil in petroleum etherFull-fat soya bean flour in petroleum ether was defat-ted for 12 h.Next,the petroleum ether was completelyrecovered,with the remaining material being the oilproduct(Ramadan et al.,2010).The preparation of protein samplesAround 1.0002.000 g protein of soya bean flour wasadded into the individual two reverse micelle solutions(AOT/Tween 85 reverse micelle system and AOTreverse micelle system),respectively.After forwardextraction and backward extraction,two kinds ofextraction solutions were added to the treated dialysisbag.Dialysis process lasted for 4872 h.During thisprocess,water was changed regularly about every 8 h.Finally,solutions were freeze-dried for protein samplecollection.Scanning electron microscopy analysisA low amount of each soya bean protein product wasglued on a double-sided adhesive tape,which was cov-ered by the glass slide.The sample was then coatedwith platinum of 10 nm thickness to make them con-ductive and examined with a JMS-5610 scanning elec-tronmicroscope(SEM)at20 kvtoobservethemicroscopicstructureofthesoyabeanprotein.Pictures were taken under a certain magnification(400 9).The morphological characteristics of proteinsamples were analysed on images acquired using aJMS-5610 scanning electron microscope at an acceler-ated voltage of 150 kv and a working distance of1015 mm(Zhao et al.,2010c).Infrared spectra measurementThe protein samples were prepared using the potas-sium bromide pellet method(Zhao et al.,2008c).Infrared spectra of soya bean protein were measuredwith a PerkinElmer Model GX Fourier transforminfrared spectrophotometer at 20 C.Reference spectrawere recorded under identical conditions,but withpotassium bromide containing no protein.Scanningrange of Fourier transform infrared spectroscopy was4000400 cm?1,andresolutionratiowas4 cm?1.Reactions inside the sample could be deducted basedon the changes in protein structure from infrared spec-tra.The second derivative and Fourier deconvolutiontechnique were used to improve the resolution ratio,and peak fit software was utilised to determine thelimit of amide belt I for gaining secondary deconvolu-tion spectra,which could analyse protein secondarystructure changes.Statistical analysisAll experimental data in this study were an average oftriplicate observations and subject to one-way analysisof variance(ANOVA)usingSAS9.0 software(SAS Insti-tute Inc.,Cary,NC,USA).Results and discussionThe yield and purity of soya bean protein productsUsing AOT/Tween 85 reverse micelle system,0.29 gprotein products was extracted from 1 g soya beanflour,while using AOT reverse micelle system,0.28 gprotein was extracted from 1 g soya bean flour.Thepurity of protein extracted by AOT reverse micelleextraction was 80.2%,and by AOT/Tween 85 reversemicelle extraction,it was 79.1%.Thus,the yield andpurity of protein products by these two systems weresimilar.The content of surfactant in soya bean proteinseparated by the reverse micelles is very low and mostof the surfactant retained in oil phase.If further purifi-cation is needed,soya bean protein could be washedwith 65%ethanol solution to remove any residualsurfactant(Zhu et al.,2010).Effects of various factors on the efficiency of forwardextraction of soya bean proteinAqueous phase pHThe aqueous phase pH in the reverse micelle systemsvaried from 6 to 10 as shown in Fig.1a.Soya beanprotein extraction efficiency increased with increasingaqueous phase pH,reaching a maximum at pH 7.0and decreased with a further increase in pH.At pH7.0,the efficiency of protein extraction in the AOT/Tween 85 system(92.5%)was higher than that in theAOT system(88.4%).Electrostatic interactions between the ionic surfac-tant molecules and the counter charge of protein mole-cules are considered the main driving force in theforward extraction process(Dekker et al.,1986).pHthat mainly affects the charge numbers of proteins is adominant factor for the extraction process.Proteins insolution exhibit an amphoteric ionisation phenome-non.When the net charge and electric field mobilityare equal to zero,the pH value stands for pI(proteinisoelectric point).When pH is higher than pI,the pro-teins are charged with negative electricity;otherwise,the proteins are charged with positive electricity.Inour two reverse micelle systems,surfactant molecules 2013 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2014Extraction of soya bean protein and oil G.Bu et al.1081304050607080901002030405060Temperature(C)Forward extractionefficiency(%)2040608010000.10.20.30.4Ionic strength(mol/L)Forward extraction efficiency(%)5060708090100150180210240270300Ultrasonic power(W)Forward extractionefficiency(%)708090100Volume ratio of isooctane and n-octylForward extractionefficiency(%)no n-octyl2:1 4:15:16:160708090100Mass ratio of AOT and Tween 85Forward extractionefficiency(%)1:12:13:14:15:120406080100678910pHForward extractionefficiency(%)AOTAOT/Tween 85AOTAOT/Tween 85AOTAOT/Tween 85AOTAOT/Tween 85AOTAOT/Tween 85AOT/Tween 85AOT/Tween 8540506070809010081012141618W0Forward extractionefficiency(%)(a)(b)(c)(d)(e)(g)(f)Figure 1 Effects of aqueous phase pH(a),W0(b),ionic strength(c),ultrasonic temperature(d),ultrasonic power(e),volume ratio of isooc-tane and n-octyl(f)and mass ratio of AOT and Tween 85(g)on the efficiency of forward extraction.2013 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2014Extraction of soya bean protein and oil G.Bu et al.1082were charged with negative electricity because AOT isan anion