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角蛋白废弃物转化为角蛋白水解物

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角蛋白废弃物转化为角蛋白水解物角蛋白废弃物转化为角蛋白水解物 第17卷第6期 2007年12月 皮革科学与工程 IEATHERSCIENCEANDENGINEERING Vo1.17,No.6 Decl2oo7 ArticalID:1OO4—7964(2OO7)O6一OOO6一O7 TreatmentofKeratinWasteintoKeratinHydrolysate P.Mokrejs1,D.Janacoza,FLangmaiera,MMladek,KK0lomaznik,Vasek . TomasBataUniversity,...
角蛋白废弃物转化为角蛋白水解物
角蛋白废弃物转化为角蛋白水解物 第17卷第6期 2007年12月 皮革科学与工程 IEATHERSCIENCEANDENGINEERING Vo1.17,No.6 Decl2oo7 ArticalID:1OO4—7964(2OO7)O6一OOO6一O7 TreatmentofKeratinWasteintoKeratinHydrolysate P.Mokrejs1,D.Janacoza,FLangmaiera,MMladek,KK0lomaznik,Vasek . TomasBataUniversity,FacultyofTechnology,DepartmentofProteinandLeather,haiti. TGM275,76272Zlin,TheCzechRepublic;2.TomasBataUniversity,Faculty0厂 AppliedInformatics,InstituteofProcessingControlandAppliedComputerScience, NdStranemi4511,76005Z/in,TheCzechRepublic) Abstract:Keratinwastearisingmainlyfrommeat-industryprocessingofpoultryrepresentsagreat quantityofwasteannuallywhichhasnotbeenutilisedproperly.Inaddition,intheproductionof hidesintoleathersandinwoolproductionthereiSanotherlargequantityofunder-utilisedkeratin waste.Keratin,duetoitshighproportionofvaluableprotein,couldbeusede.g.asanutrition infeed.Anotherhigh—valueaddedproductiSsolublekeratinhydrolysatethatmaybeprocessed intobiodegradablecoatingsorpackaging,foragriculturalfilms,forencapsulatingchemicals,in cosmeticsetc.Nevertheless,keratincontainslargeamountofdisulphidebondsandthereforeiS insolubleinmostcommonsolventsandresistanttoproteolyticenzymes.Tomakekeratinsolu— ble,disulphidebondshavetobecleavage.Presentedresearchpaperstudiesthepossibilitiesof extractionkeratinfromchickenfeathersandfromfleecetoproducesolublekeratinhydrolysate. Keratinmaterialwastreatedthroughtwophaseprocess.Inthefirstphase,therawkeratinmate— rialwasincubatedinwatersolutionof2-mercaptoethanoIand,inthesecondphase,incubatedin thesamesolutionwithenzymeatdefinedconditions(temperature,PHandtime).Experiments weredesignedby2一 levelfactorialschemewiththreeexperimentalfactorsandwithtworepetitions inthecentre.Thepercentageofinsolublekeratinmaterialwasdeterminedgravimetrically.The resultsares{atisticallyevaluated. Keywords:keratin;feathers;fleece;hydrolysate;extraction;enzyme CLCnumber!TS512Doclnnentcode:A 角蛋白废弃物转化为角蛋白水解物 摘要:角蛋白废弃物主要来源于家禽肉联加工厂生产时没有正确处 理掉的大量废物.而且,在革和毛织品的生 产过程中也存在大量的未加利用的角蛋白废弃物.角蛋白因其含有 较高的有利用价值的蛋白,可以增加食物中的 营养.另一种价值较高的角蛋白是角蛋白水解物,可以将其加工成可 生物降解的覆盖物或者包装袋,作为农业薄 膜,化妆用品的包装.然而由于角蛋白含有大量的双硫键而很难溶于 普通的溶剂中,并且抗蛋白酶作用.要想角 蛋白溶解就必须破坏双硫键.已有的研究报道指出用2步法处理角 蛋白材料,从小鸡羽毛和绒毛中萃取的角蛋白 可以转化成可溶解的角蛋白水解物.第一步,原角蛋白保温培养在巯 基乙醇溶液中,第二步在同第一步一样的溶 液(同样的温度,pH和时间)中加入酶.正交试验为2因素,3水平,重复 2次.定量测定不可溶解的角蛋白百分 数.根据统计学分析结果. 关键词:角蛋白;羽毛;绒毛;水解物;萃取;酶 lINIRODUCI1】_0N Invertebrates,thehornylayeroftheepider— misandepidermalappendages,suchashair,hairs, quoins,hoofs,feathers,scales,fleeceandnails, arealltheresultofanelaboratedifferentiation,or keratinization,ofspecializedepithelialeelltermed ReceivedDate:2007—12-03 *P.Mokrejs:Correspondingauthor.PhoneN.:-{-{-420576031230;FaxN.:- {-{-420576031563;e-mail:mokrejs@ft.utb.c2 第6期 P.M.krejs,etal:TreatmentofKeratinWasteintoKeratinHydrolysate7 keratinocytes.Duringthisprocess,living,grow— ing,epithelialtissueisconvertedintolifeless tough,insoluble,fibrousmaterial,thatshows verylittlespeciesspecificityandthatprovidesa Drotectiveoutercovering.Thismaterialismainly composedofthestructuralproteinkeratin.Adis— tinctivefeatureofkeratin,whencomparedtoother maiorfibrousproteins,suchascollagen,elastin, andmyofibrilalarproteins.istheoccurrenceo{a largeamountofcysteineresidues,mainlypresent asthedisulphidebondeddimericaminoacidcystine [1] Keratinismechanicallyresistantandchemical一 1vnoreactiveproteinoccurringamongstinallver— tebrates[21.Keratincannotbeconsiderasonepro— tein,butasbiologicalsystemcreatedfromthese— riesofmutuallydifferentproteins.Thecommon symboloftheseproteinsisinsolubilityinwater, highresistancetoproteolyticenzymesandoccur— renceofcross—bindingsofdisulphidetype.After cross—linkingtheseparateproteinscreatedimen— sionalnetworkinwhichtheirloosetheirowniden— tityandbecomeapartofmacromoleculewithvery highmolecularmass[引.Inmostkeratin-containing tissues,filamentsaresetinanon-fibrillarmatrix. Filament—matrixstructuresarebasicallycompos— ites,wherethefilamentiscomposedofamaterial withahighelasticitymodulusandthematrixdis— tributesthestressevenlyoverthefilaments,thus preventingthepropagationofcracks.Factors, suchasfilamentorientation,filamentpacking,na— tureandproportionofthematrixandcrystallinity alldeterminethemechanicalpropertiesofkeratins [4] Keratinsswellinwater.Waterdiffuseinto thefibrilstructureapartiallydisruptselectrovalent andhydrogenbondsresultinginlowercohesionof fibrils.Swellingofkeratinisimportantformany reactionsenablingchemicalstobetterdiffuseinto swollen”keratingel”.Theresistanceofkeratinsa- gainstacidsisquitegoodbecausedisulphidebonds areresistanttotheacidhydrolysis.Afteralong hydrolysisusingmineralacidsathighertempera— tureshydrolysisoccurswithoutrupturingo{cyste— ine.TotalhydrolysisofkeratinOccursonlyby heatingkeratinwith30ofsulphuricacidorwith 6Nofhydrochloricacidat11O?for24hours. Theinfluenceofalkalisismoreoutstandingand dependsmainlyonthepHoftheenvironment, temperatureandtime.Disulphidebondsofcyste— inesurrendersofhydrolyticalcleavage. Methodsforisolatingkeratinproteinsinvolve oxidation,sulphitolysisorreductionofthedisul— phidebonds.Keratinscanonlybeextractedfrom materialifthedisulphideandhydrogenbondsare broken.Treatmentofkeratinswithoxidativea— gentsmayleadalternativelybytwoprocesses. Firstly,oxidationofdisulphidebondswithout theircleavage,orsecondly,oxidativecleavageof disulphidebond.Disulphidebondsareoxidativeaf— fectedwithoutcleavagewiththesolutionsofhy— drogenperoxide,KMn04andbyphoto-oxidation. Oxidationproceedswithformationofsulphooxide tosulphone.Oxidationcleavageofkeratinsbythe mcansofperoxoacidsorsodiumchloriteleadsto thefcIrmationofkeratin-sulphoacids.Bytheopera, tionwithreductiveagentsreductivecleavageofdi— sulphidebondsoccurs,e.g.bythemeansofsul— phitesorthioglycolicacid.Afterreductivecleavage dissolutionofkeratinsbythemeanso{enzymeeas, Yoct.urs[引. Amildextractionprocedure,without significantpeptidebondhydrolysis,involvesthe useofthiols,like2一mercaptoethanol,inconcen, tratedureasolutionsatamoderatelyalkalinepH. Theamountofexcessthiolnecessaryforthere— ductionofdisulphidebondsinproteinsdependsup, ontheoxidation—reductionpotentialsofthiol—disul— phidepairsandonthereactivityofthedisulphide bonds.Whenfeathersweresolubilisedfollowing thisprocedureastablefeatherkeratinsolutionwas obtained.Schemeofreductionofdisulphidebonds bythiols[1]: 2R—S一+一CH2一S—S—CH2—2一CHz— S一+R—S—S—R 8皮革科学与工程第17卷 Toincreasethesolubilityoftheextractedker— atiFISintheabsenceofareducinganddisruptivea— gent,manydifferentapproacheshavebeenfol— lowed.mostofwhichinvolvechemicalmodifica— tionofthecysteineresiduesofthekeratin.A methodthatdoesnotinvolvechemicalmodification istheadditionofanionicsurfactanttothekeratin solution.Yamauchieta1.investigatedtheextrac— tionofwoolkeratinswithanaqueoussolutionofu— rea,2一mercaptoethanolandsodiumdodecylsul— phateassurfactant[引.Theyfoundthatthesurfac— tantacceleratedtheextractionandincreasedthe extractionyield.Italsostabilisedtheaqueouspro- teinsolutionafterremovalofureabydialysisa— gainstwatercontaining2一mercaptoethano1.The surfactantformsacomplexwiththekeratinandis removedmuchslowerbydialysisthanotherlow molecularweightcompounds. Severalproceduresaredescribedinthelitera— turetodissolvefeatherkeratins.Solubilisation methodswithconcomitantpeptidebondscission includeacidandalkalihydrolysis,reductionofdi— sulfidebondswithalkalinesodiumsulphidesolu— tions,acombinationofenzymaticandchemical treatment,andtheuseofammoniumcopperhy— droxide.Procedureswithoutsignificantpeptide bondscissioninwhichonlydisulfidebondsare splitincludesulphitolysisoroxidationofdisulfide bondswithperformicacid.Anothermildproce— dureinvolvestheuseofthiols.like2一mercaptoeth— anol.toreducethedisulfidebondsinconcentrated ureasolutionsatamoderatelyalkalinepH.When feathersaresolubilised,followingthisprocedure, astablefeatherkeratinsolutionisobtained.Re— movalof2一mercaDtoethanolandureafromthisso— lutionbydialysisresultsinaggregation.ofthekera— tinpolypeptidechainsandre-oxidationofthecys— teineresiduestoyieldawhite,opaquege1.Forthe developmentofbiodegradablematerialsfromfeath— erkeratins,likefilmsforcompostablepackaging orpapercoatings,water-solublederivativesare needed[71. Industrialresearchonkeratinsandkeratinhy— drolysatesisintensiveandnewclaimsaremadean- nually.Especiallythecosmeticsandtextileindus” tryareinterestedinproductsforstylingormodif- yinghairandwoo1.Shampoosandnailpolishre- moverwithhydrolysedkeratinsandeyelashmake- upbasedonwaxandkeratinhydrolysatesarejust examplesofthemanypatentedformulationsthat makeuseofkeratins[引.Applicationsarenumer— OUS,butarebasicallyproductformulationsusing keratinsasmaincomponents.Filmandcoating fromproteins,suchaswheatgluten,soyprotein, collagen,gelatine,milkproteinsandcornzein,for foodandnon-foofapplicationshavebeenextensive- lystudiedaswellasreviewed[9-113.Hydrolysates ofkeratinmayalsobeusedintheproductionofbi- odegradablefilmsandfoils.Inrecentyearsthere hasbeenanincreasinginterestobservedinthede— velopmentofmoreenvironmentfriendlycoatings [12] 2L1ERIALSANDMETHoDS Theaimofourexperimentswastoexamine thepossibilitiesofextractionkeratinfromfleece andchickenfeathersbyreductionofdisulphide bondswith2-mercaptoethanolfollowedbyenzy— matictreatment.F1eeceandfeathersweresupplied byanagriculturalfarminTheCzechRepublic. Apparatusandequipmentcomprised:drier WTBBinderE/B28(Germany).magneticstirrer IKARCTbasicwithexternalthermoregulator ETS-D4fuzzy(Germany),incubatorBinder BD23/RS422,23l(Germany),electronicbalance Kern770/GS/GJ(Germany),pHmeterPiccolo withAmplifiedElectrodeHI1295(Germany),fil— terpaperFilpapKA-1(TheCzechRepublic).2一 mercaptoethanolwassuppliedbySigma-Aldrich (St.Ix)uis,USA),proteinaseenzymeSavinase Ultra16IandlipaseenzymeGreasex50Iwas suppliedbyNovoNordiskDenmark.NaOHP.a. andNa2CO3P.a.weresuppliedbyPetrLukescom- pany(TheCzechRepublic). 第6期 P.Mokrejs,etal:TreatmentofKeratinWasteintoKeratinHydrolysate9 AgeneraIschemedescribingthewholeprocess ofstartingkeratinrawmaterialintokeratinhy— drolysateisshowninFig.1.Priortoexperiments ofreductivecleavage,rawkeratinmaterialwas preparedbycleaning,cutting,degreasinganddr— ying.150goffeathersandfleecerespectivelywas cleanedbyimmersingitinasolutionwhichwas composedof2litresof1ofNa2CO3at30?. Thesolutionwasthendrainedthroughasieveand keratinmaterialwassprayedwithastrongstream ofslightlyhotwater.Thedriedmaterialwascut byscissorsinsmallfilamentsuptothelengthof4 mm.Thismaterialwasthenextractedusing6gof enzymeGreasex50Iand2Iofwaterat40?for 8hoursatthepHof9.00.5(adjustedwith59/iof NaOH)undershortshakingevery8hours.After that,thesolutionwasdrainedthroughasieveand thedegreasedkeratinmaterialwassprayedwitha strongstreamofslightlyhotwater.Finallywas driedat103?andstoredataroomtemperaturein aclosedplasticbottle. Fig.1SchemeofproceduresoftreatmentkeratinrawmateriaIinto keratinhydrolysate 图1原角蛋白转化为角蛋白水解物处理过程 2.1Two-phasehydrolysisoffleece Experimentsweredonebythemeansoffacto— rialexperimentsontwolevels(min—max)with tworepetitionsinthecentre.Threeexperimental factorsweremonitored:timeof2州phaseofex— traction(2—8h),temperatureof2砌phaseofex— traction(50—70?)anddosageofenzyme(0.5 39/6,basedondrymatteroffleece).Inthefirst phaseofextraction,2goffleecewasweighintoa boilingflaskand100mIofpreheated(60?)wa, tersolutionof0.1mo1.I一2一mercaDtoethanolwas added.Theflaskwasincubatedat’(50?0.5)? for24hoursundershortshakingevery15minutes. Then,thepHwasadjustedto9.00.2byadding5 ofNaOH.Inthesecondphaseofextraction, thedefinedamountofenzynmwasaddedandthe mixturewasincubatedfordefinedtimeatdefined temperature(seeTab.1).Afterthat,themixture wasfilteredthroughafilterpaper.Theinsoluble fleecewaswashedwith400mlslightlyhotwater andthendriedonaPetridishat(103?2)?to constantweightandweighted.Theamountofex— tractedkeratinwascalculated. 2.2Two-phasehydrolysisofchickenfeathers Experimentsweredone,similarlytothosede— scribedatextractionofkeratinfromfleece,bythe meansofstatisticalschemeontwolevels(min max)withtworepetitionsinthecentre.Threeex— perimentalfactorsweremonitored:timeof2.a phaseofextraction(8—24h),temperatureof2州 phaseofextraction(50—70?)anddosageofen— zyme(26,basedondrymatteroffleece).In thefirstphaseofextraction,2gramsoffeathers wasweighintoaboilingflaskand100mlofpre— heated(70?)watersolutionof0.4mo1.I一12一 mercaptoethanolwasadded.Theflaskwasincuba— tedat(70士0.5)?for48hundershortshaking every1hourduringthefirst8hoursandthenever— Y10hours.Then,thepHwasadjustedto9.0j二 0.2byadding59/6ofNaOH.Inthesecondphase olextraction,thedefinedamountofenzymewas addedandthemixturewasincubatedfordefined timeatdefinedtemperature(seeTab.2).Fig.2 visualisesthedegreeofdecomposedfeathersduring thewholeprocess.Afterthat,themirXturewasfil— teredthroughafilterpaper. Theinsolublefeath’ers waswashedwith400mlslightlyhotwaterand thendriedonaPetridishat(103_4-2)?tocon— stantweightandweighted.Theamountofextrac— tedkeratinwascalculated. 10皮革科学与工程第17卷 Il’l”he(1曜rceofdecomposedfeathersduringtwophaseextrac— tionprocess.A.driedandcutfeathersbeforetreatment-B— thestateafterthe feathersimmersedin2-mereaptoethano!,C— 1”phaseofextraction.D=thestateafterthe2phaseofextrac— tion. 图22步法萃取后角蛋白分解程度 A:处理前干燥和剪掉的羽毛;B:羽毛浸入巯基乙醇溶液中;C: 第一步萃取后的状态;D;第二步萃取后的状态 3RESI?I’sANDDICION Experimentaldatawasevaluatedbycomputer— isedprogramStatisticalGraphicsSystem,Version 6.0suppliedbyManugistic,Inc.,Roekville, ? USA.’ 3.1Two-phasehydrolysisoffleece Resultsoftwophasehydrolysisoffleece basedonreductionofdisulphidebondbycombi— ning2一mercaptoethanolinthe1吼phaseofextrac— tior~andenzymeinthe2喇phaseofextractionare summarisedinTab.1.Contourdiagramsofex- tractedkeratinindependenceontimeofhydrolysis (2phase)anddosageofenzymeholdingtempera— tureat50,60and70?areshownonFig.3. Fromcontourdiagramsitisobviousthattheyield ofextractedkeratiniSinfluencednotonlybythe choiceoftimeofhydrolysisanddosageofenzyme butalsobytemperature.Prolongedhydrolysis(up to8h)with2.5(basedonstartingweightof fleece)ofenzymeleadsto69ofextractedkera— tinifholdingtemperatureat50?.AtthesalTle conditionsbutatincreasedtemperature60?the amountofextractedkeratinrisesupto75.5.If thetemperaturerisesto70?theamountofex— tractedkeratiniS82. Tab.1Resultsoftwophasehydrolysisoffleece 表12步法水解绒毛结果 Monitoredfactorsin2phaseoffleecetreatmentExtracted TJme/hTemperature/~2DosageofenzymeN’keratin/% 1)basedonstartingweightofdryfleece AB PerctntofextractedkeratinPerCentofextractedkeratin 234567B Timeofhydrolysi~Jh 3 25 2 】.5 】 0.5 0 234567B Pl且tof,1训Im Timeofhydroly出m 234567B Timeofhydrolysis/h Fig.3Contourdiagramofresultant.areaofextractedkeratinfrom fleeceindependenceOntimeofhydrolysis(2phase)anddosage ofenzyme Temperatureofhydrolysis:A=50?,B=60?andC一70? 图3第二步水解时间和酶用量萃取绒毛的等高线分布图 水解温度:A=50?,B=60?andC一70? 3.2Two-phasehydrolysisofchickenfeathers Resultsoftwophasehydrolysisoffeathers basedonreductionofdisulphidebondbycombi一 譬晕N80々口 连目蒈蜡Q %,?00?8口 第6期P.Mokrejs,etal:TreatmentofKeratinWa?
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