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?