对酪蛋白胶束结构影响的光谱学研究

第34卷,第1期            光谱学与光谱分析2014年1月            SpectroscopyandSpectralAnalysisVol畅34,No畅1,pp92‐97

January,2014 

CalciumSpectroscopicIononInvestigationtheStructuresofoftheCaseinInfluenceMicelles

ofWANGPeng‐jie1,LIUWUHongJian‐‐pnaing2,ZHANGHao1,GUOHui‐yuan1,

3,RENFa‐zheng1倡

1.BeijingLaboratoryforFoodQualityandSafety,CollegeofFoodScienceandNutritional2.UEngineeringniversityof,ChinaAlbertaAgricultural,EdmontonU,niversityCanada

,Beijing 100083,China

3.CollegeofFoodScienceandTechnology,GansuAgriculturalUniversity,Lanzhou 630070,China

Abstractment Theeffectsofcalciumextrinsicwerewithinfluorescencesynthetically(ANSexaminedionfluorescencebyonnonthestructuralpropertiesofcaseinmicellesinthecourseofheattreat)‐structure,waspositively‐invasivespectrometry.Thehydrophobicity,reflectedwithterthethegrowrangethofofcalcium0to12mmol・L-1.Meanwhile,theturbiditycorrelatedandwithstabilitytheconcentrationofcaseinmicellesofthealsocalciumincreasedeffectandmmolonpolydispersitythestructuralindexconcentrationscharacteristics.Comparedofwith.Howevercaseinthecalcium,oppositeion,resultsthecalciumwere‐observedchelator(forcitratehydrodynamic)hasanoppositediamethedexcalcium・L-1ion,the,neverthelesshydrophobicity,opposite,stabilitymicelles.Withinthecalciumconcentrationsrangeof0resultsandwereturbidityobservedwerefornegativelycorrelatedwiththeconcentrationingheat.Allheatmenttheresults.

indicatethatthecalciumioncouldbeusedtohydrodynamicmodifythestructuresdiameterofandcaseinpolydispersitymicellesKeywords Caseinmicelles;Calciumions;Structures;Spectroscopy

中图分类号:O657畅3  文献标识码:A   DOI:10畅3964/j畅issn畅1000‐0593(2014)01‐0092‐06

Introduction

sultintheofnoticeableuniquemilkeffects‐specificonmilkstability[2].groupto  Inthedairyindustry,variouskindsofmineralsareseinstotalcheeseimproveimportant,yoghurttheandqualityproteinofpowdersmilk‐basedproducts,suchusedasals,theproteinmostimportantinthemilkproteins,andrepresentpartsofcommercialdairycasein,existasmostmicellesmainly[3]

in.Thetheofmilk,togetherphysicochemicalformofcolloidalparticlessedimentationtheygreatlyroleintheprocessandthefinal.Thesequalitymineralsofproductsplayan,celles[4]allmilk‐basedproductsmainlydependcharacteristicsonexchangers[1]andinfluence.Ingeneralgelationthe,,allrennetsusceptibilitythemineralsaction,ofexistandthefoulingproductsindynamicofheattoe‐qareThe.

functionalandchemicalpropertiesofmilkuilibrium.Smallbetweenchangestheinthediffusibledistributionandtheofmineralsmicellarwouldphaserein‐thedeterminedbytheirstructures,whichcancaseinterationsmodificationinthestructuresofthemicroenvironmentcanbereflectedinofthemilk Received:2013‐04‐10,accepted:2013‐07‐11

 Foundationitem:InternationalScience&TechnologyCooperationProgramofChina(2011DFA32550),MinistryofScienceand Biography:WANGChinaPeng(2012‐jie,(1986BAD12—B)08)

,DoctoralCandidateofChinaAgriculturalUniversity  e‐mail:wpj1019@126.com

倡Correspondingauthor  e‐mail:renfazheng@263.net

by‐,‐12‐‐80%area.Caof‐,i.eminer‐caseinof.,the

mial‐‐.alteredmicellesThesebyal‐‐

echnologyofiontoofindurCaseinsspecieswiththebehydrophobicT

ity,sizeandconformationalstates.Fluorescentspectroscopycanbeusedtoquantifyproteinhydrophobicity[5].Mean‐while,dynamiclightscattering(DLS)andturbidity,whicharebothfastandnoninvasive,havebecomethemethodsofchoiceforevaluatingthehydrodynamicdiameter[6].

Inthedairyindustry,heattreatmentisanessentialpro‐cedure,aimingtoimprovethefinalqualityofraw‐milk‐prod‐uctsbyreducingthemicrobialrisksandtherefore,reducingtheriskofpoisoning.However,inothercases,wherecaseinmicellesareusedasfoodingredients,heattreatmentiswidelyappliedinimprovingthephysicochemicalpropertiesofsuchcitratebuffer(pH7畅0)toafinalconcentrationof1mg・

St.Louis,MO),accordingtoLecki[9],withslightmodifica‐tions.Caseindispersions(10mg・mL-1)weredilutedwithmL-1.Then,20mLofANS(8mmol・L-1inbuffersolu‐

tions)wasaddedto4mLofthedilutedsampleandmixed.RelativefluorescenceintensitywasmeasuredimmediatelywithaRF‐5301PCfluorescencespectrometer(Shimadzu,Ja‐pan)at390nm(excitationwavelength,slit5nm),400~650nm(emissionwavelength,slit5nm)andascanningspeedof10nm・s-1.

1畅3 Hydrodynamicdiameterandsizedistributionofcaseins

dairyseinmicellesformulationsbymanipulatingthefunctionalityoftheca‐micellesAsaal,heatuseful.

treatmenttoolforhasmodifyingbeenstudiedtheinstructures‐depth,andofcaseinsever‐ptherovenmechanismshavebeenpresented.Meanwhile,ithasbeenavailablemicellethatthecalciumionplaysacrucialroleinstabilizingponcaseintheeffectstructure[8].However,littleinformationisofropertiesmineralscalciumof.Thecaseinaimmicellesofheatofthisstudyinthetreatmentonthestructuralispresenceofvariouskindsofincaseinonmicellesthehydrophobicity.Agoodunderstanding,sizetoandinvestigateconformationaltheeffectofoftheseisimportantstatesseinthemicelleselucidation.

ofstructuresandfunctionalpropertiesofca‐1 MaterialsandMethods

1畅1 PreparationtheThecowmilkofincludedcaseinmicelles

inthisstudywascollectedmilkinstitution30waspreparedfarmofChinaAgriculturalUniversity.fromSkimTDwas3800minat25℃by,usingcentrifugationalab‐scaleofrawcentrifugemilkat4000(Xiangyigforweight℃.achieved,ChangshaThebyultracentrifugation,China).Separationat120000ofcaseingfor1micelleshat25ultrafiltrate.Thecaseinpelletsobtainedwerefreeze‐driedtoconstantazidewaterfreeze.‐driedcaseinmicelleswerere‐dissolvedinv)trypsinwasaddedtoinhibitSubsequentlybacterial,0grow畅02%th(,wand/v)0畅02%ofsodium(w/edtionstoDifferentinhibitorthecaseinconcentrationswasaddedtominimizeplasminactivity.

micellesolutionsofcalciumandcitratewereadd‐p.Aliquotsofthecasein-1

wereH6畅HClmicelleatdifferentfinalconcentra‐.Thesamplescaseinmicellewereadjustedsolutionsto

solutionsheated6by11畅2 wereatmolcooled65・℃L

for30min.Afterheattreatments,themgTheFluorescenceextrinsicstudyto25℃forfurtheranalysis.

fluorescenceofhydrophobicityofthecaseinofcaseinsolutionsmicelles

(10sulfonate・mL-1()ANSwas)measuredfluorescenceusingprobethe1‐(Sigmaanilino‐8‐ChemicalnaphthaleneCo.,

‐cellesTheNanoing,wereaverageBeckmandeterminedsizeandCoulterbypolydispersityindexofcaseinmi‐

Inclaser.,CAlight,USAscattering(DelsaTMdexexperimentalofofthesolventconditions1畅333and:temperature)viscosity1畅00225,under℃mPa,refractivethefollow‐・sin‐p100mLofrawskimmilkwasdispersedin10mL.ofAtotalmilk

(ermeatekDaMillipore.TheCorporationmilkpermeate,MA,wasobtainedbyultrafiltrationareanalysisindexcharacterizedcut‐offmembrane.Thedatawerebymean(MilliporeUSA)ofskimmilkusinga10‐exporteddiameterCorporationfromDthe[6,equipment5]and).polydispersityTheparticlesforfurther

quipment.directlyThepolydispersityandD[6,5]indexwaswas∑calculatedobtainedas

fromthee‐D[6,5]=

VjD3j∑whereVistherelativevolumefractionj

D2(1)

withjofallcaseinmicelles

dynamicdiametertriplicatediameterDj.Theresultswereexpressedasmeanhydro‐isanindex.Theofparticlehydrodynamic(nm).Eachpolydispersitydiameterexperimentwasconductedinlows

anddistributioniscalculatedofparticlesasfol‐

polydispersityindex=

50%

(2)

eterswhereatD90%90%,D10%andD50%representthehydrodynamicdiam‐

p1畅articles4 Quantification,respectively,10%andof.

50%cumulativevolumeofthecaseinαs1‐,αs2‐,β‐andSeparationspectrometryof‐HPLCκ‐caseinbyultraviolet

byαs1‐,αs2‐,β‐andκ‐caseinwascarriedout

natantreversed(100mmol(500‐pμhase・LL)-1wasHPLCTrisdiluted‐UVbasewithequipment,8mol3mL・Lof.Skim-1denaturingmilkorsolution

super‐2O,0畅3%v/vβ‐mercaptoethanolurea,13,adjustedg・L-1Na3citrate・2Htop20carriedH7畅0AoutwithusingHCl)reversed.Analysis‐phaseoftheHPLCmajor‐UVcasein(Shimadzucontents,wasLC‐

mm,Kyoto,Japan)withaKromasilC4column(4畅6×containing,300樻0畅1%,AkzoNobel(v/v)trifluoroacetic,Sweden).Solventacid(TFAAwas)andwater250sol‐

sensitivethanthoseusedinabsorptionspectroscopy.Further‐more,becauseemittedlightisdetectedatafixedwavelenguhatrightanglestotheexcitationradiation,fluorescentspectro‐scopicanalysisislessaffectedbyturbidmedia[7,9].Thefluo‐rescenceintensityofANSafterbindingtocaseinmicellesatdifferentcalciumconcentrationsisshowninFigure1.Itcanbeseenthatatthegivenheatingconditions(65℃for30min),thefluorescenceintensityofANSincreasedsignificant‐lywithincreasedcalciumconcentrations.Thisindicatedthatmorehydrophobicbindingsiteswereexposedathighercalci‐umconcentrations.However,whenthecaseinmicelleswereventBwasacetonitrilecontaining0畅1%(v/v)TFA.Theflowratewas0畅8mL・min-1.Alineargradientfrom30畅0%to50畅0%solventBover50minwasset.Afterfilteringthrougha0畅45μmfilter(Membrana,Wuppertal,Germa‐ny),a10μLsamplewasanalyzed.ThedissociationdegreeofindividualcaseinsfromthemicelleswasdeterminedbasedonthemethodofAnema[10].1畅5 Turbiditymeasurements

Theturbidityofcaseinsolutionswasdeterminedasde‐scribedbyPartschefeld,withslightmodifications.Thetrea‐tedsamplesweremeasuredatthewavelengthof633nmwithaUV‐visiblespectrophotometer(KeerdekeheatedcordingTurbiditywaswheretoindexPartschefeldwasused[11]to,expressasturbiditytheturbidity,Beijing=(w-values,China).wac‐0)/mmolhighestat90iswistheturbidityofthesampleaftertreatment,andw0w,found・L-1when℃for,andthethe30min,theANSfluorescencelowestcalciumANSconcentrationfluorescenceofcaseinsintensitysamplestheturbidity1畅6 weremeasuredofthesampleintriplicatebefore.heattreatment.Allthe0(asdatanotwhenshowntheconcentration).ofcalciumwas12mmolwithasesnaturationthosefunctionoftheThiscalciumtrendconcentrationofANSfluorescenceisnotgwereEachStatisticalperformedsampleAnalyses

wasusinganalyzedSPSSin13畅0triplicateforWindows.StatisticalXP(analyChica‐discussionoftreatedcaseinsatunder65℃ultrahigh,whichistemperaturesprobablyduecano,USA).TestsfordifferenceswereperformedusingDun‐‐heatingicant’s.

multiplerangemethod.P<0畅05wasconsideredsignif‐tensityconditionsincludedofin65this℃studyfor30wasmerelyfocused.rateofANSafterbindingtocaseinminmicelles.Thefluorescenceatdifferent2 Resultsanddiscussion

thatconcentrationswiththefluorescenceisintensityshowninofFigureANS1.Itcanbe2畅1 drophobicincreasedtrationsbindingcitratesitesconcentrationswereexposed.ItdecreasedindicatedsignificantlythatitsgboundaInHydrophobicityofcaseinmicelles

verywaterlow,quantumthehydrophobicyieldoffluorescentprobeANSexhib‐rateent..Citratecouldberegardedasatahighercalciumcitratechelatinghighlytofluorescentthehydrophobic.Fluorescentregionsfluorescenceprobesofproteins.Howeveraregenerally,itbecomes,oncemore

thecaseinreducedDependingmicellesthecalciumonthelevelwerecontentofdisruptedinchelatingagents,adding.

thecaseinmicellesFig畅1 FluorescenceintensityofANSincaseinmicellesasafunctionofcalciumionconcentrations

(theconcentrationsofcitratewere3and8mmol・L-1inAandB,respectively)

2畅2 developmentnoninvasiveDynamicSizecharacteristicslightscatteringofcasein(DLSmicelles

),forp,hasbecomethemethodofwhichchoiceisforbothevaluatingfastandthetheaccurateofdynamiclightscatteringholdsgreatsystemsarticlesize.Becauseandpolydispersitycaseinmicellesindexscatterinlightarangeveryofwellmicellar,the

responseinvestigationdeterminationofcaseinmicellesize,lightscatteringtoenvironmentalofitsinteractionswithothermoleculesarethatitfactors[12]avoidsdrastic.Thechangesmainadvantagesinwas6・wasL-1thedeon,thethe‐citin‐‐lesshy‐a‐‐.Thuscit‐,welloritsasenviof‐

intensityintensityaccordanttoThusobservedconcenpromiseasthe

ronmentofcaseinmicellesandthatthemeasurementsareper‐formedonalargenumberofparticles[13].AsshowninFigure2,thehydrodynamicdiameterofcaseinmicellesdecreaseswithincreasingcalciumconcentrations.Theioniccalciumcanshieldthenegativechargeonthesurfaceofcaseinmicelles.Atcalciumconcentrationsrangingfrom12畅0to0mmol・L-1,thesurfaceofcaseinmicellesbecomemorenegativelycharged.Meanwhile,duetothedecreasingcalciumconcen‐

trations,thestabilityofcaseinmicellesdecreasedandthere‐foretheybecamemoreinflatedstructures.Thisconsequentlyincreasedtheirhydrodynamicdiameter.Thehydrodynamicdi‐ameterofcaseinmicellesatdifferentcitrateconcentrationsisshowninFigure2.Thesizeofcaseinmicellesdecreasedwiththeincreasingcitrateconcentrations.Thus,comparedwiththecalcium,thecitratehasanoppositeeffectonthehydrody‐namicdiameterofcaseinmicelles.

Fig畅2 SizecharacteristicsofcaseinmicellesasafunctionofcalciumandBion,respectivelyconcentrations(theconcentrationsofcitratewere3and8mmol・L-1inA)

  TheparticlesizedistributionofasystemcanbeHoweverdexeffect2valueasindicatesthepolydispersityindex.Asmallpolydispersityexin‐pressed‐concentrationonthe,comparedstabilitywithofcaseincalciummicelles,thecitratehasanoppositedexalsodemonstratedaanarrownoticeableparticledecreasesizedistributioninpolydispersity.Figureinmicellarformofdecreasedcitrate,.

thepercentage.ofWithcaseinsthepresentincreasingmmolas・theL-1calciumconcentrationincreasedfrom0to12畅‐0creaseswhilewith.theThisincreasingmeansthattheheterogeneityofcaseinsin‐centration,thepolydispersityindexconcentrationdecreaseswithofcalciumincreasing.Meancon‐‐micellesofcitrate,suggestingtheheterogeneityofcasein2畅3 Stabilitydecreasedwithincreasingcitrateconcentration.

letspectrumofcasein(micellesdeterminedbyHPLC‐ultravio‐

Fig畅3 RP‐HPLCchromatogramsofcaseins

associationInthe2畅4 pableressthestabilitystatepresentUVofstudy)

,thedegreeofdissociationandtheofcaseincaseinfrommicellesthemicelles.wasusedtoex‐chromatographymethodforthecaseinanalysisisPresentlyhigh‐p,themostreli‐repeatableTurbidityTurbidity

hasbeenfoundtobeanextremelysensitivetrumcaseins(UV(HPLC)intandemwithanerformanceultravioletliquidspec‐calciumteractionsconcentrations.andThenoninvasiveturbidityisgivenofwaytheforincaseinmonitoringFiguremicellesthe5.Theatcaseinturbiditydifferent

rated.Theisshown)detectoreffectinofFigure.AcharacteristicRP‐HPLCprofileofthecalcium3on.Allthethecaseinswerewellsepa‐thecaseinsolutiondecreasedrapidlywithdecreasingcalciumisconcentrationcellarshowninFigure4.Thepercentagestabilityofcaseinsofcaseinpresentmicellesintheturbidityof.Howevercaseinmicelles,thecitrate.

hastheoppositeeffectcreasedformtrationfromincreased0to6mmolwhen・Lthe-1concentrations.However,whenofcalciummitheconcenin‐‐‐

3 Conclusion

-1

ageofcalciumwasstantof.caseinsThisindicatedpresenthigherthatinmicellarthan6mmol・L,thepercent‐thestabilityformremainedofcaseinlargelymicellesconin‐‐  ThestudysuggestedthatthemineralenvironmentcreasedL-1,andatcalciumitchangedconcentrationslittleathigherrangingcalciumfromconcentrations0to6mmol・.

calciummilkstronglyconcentrationsinfluencesrangingthestructurefrom0toof12caseinmmolmicelles・L-1,.hydrophobicityandturbidityofcaseinmicellesincreasedwith‐,inofoninintheAt

Fig畅4 Thestabilityofcaseinmicellesasafunctionofcalciumionconcentrations(theconcentrationsofcitratewere3and8mmol・L-1inAandB,

respectively)

Fig畅5 D[6,5]andturbidityofcaseinmicellesasafunctionofcalciumionconcentrations

(theconcentrationsofcitratewere3and8mmol・L-1inAandB,respectively)

increasingforcalciumconcentration,andconcentrationsthesizeandrangingpolydispersity0to12mmolindexbut・.theLMeanwhileopposite-1,the,wasatcitratefoundthepolydispersityindex.Thus,tyertiescitratehasanoppositeeffectoncomparedthephysicochemicalwiththecalciumcitrateandconcentrationturbidityofcaseinhydrophobici‐,butthemicellesoppositedecreasedwasfoundwithforincreasingthesize

heatstructuretreatmentofcaseinofcasein,micellescalcium.micellesionsTheseresultsindicatethatduring.

couldbeusedtomodifyReferences

[1] SalaonF,MiettonB,GaucheronF.Milchwissenschaft,2007,62(1):20.

[2] SinghH,RobertsMS,MunroPA,etal.JournalofDairyScience,1996,79(8):1340.[3] WangP,LiuH,WenP,etal.InternationalDairyJournal,2013,31(2):107.[4] FoxPF,BrodkorbA.InternationalDairyJournal,2008,18(7):677.[5] YükselZ,ErdemYK.JournalofFoodEngineering,2005,67(3):301.

[6] GebhardtR,DosterW,FriedrichJ,etal.EuropeanBiophysicsJournal,2006,35(6):503.

[7] ChakrabortyA,BasakS.JournalofPhotochemistryandPhotobiologyB:Biology,2007,87(3):191.[8] AnemaSG.FoodChemistry,2009,114(1):161.[9] LenckiRW.JournalofDairyScience,2007,90(1):75.

[10] AnemaSG,LiY.Lebensmittel‐Wissenschaftund‐Technologie,2000,33(5):335.[11] PartschefeldC,SchwarzenbolzU,RichterS,etal.Biotechnol.J.,2007,2(4):456.[12] TranLeT,SaveynP,HoaHD,etal.InternationalDairyJournal,2008,18(12):1090.[13] BeliciuCM,MoraruCI.JournalofDairyScience,2009,92(5):1829.

‐,propthe

钙离子对酪蛋白胶束结构影响的光谱学研究

王鹏杰1,吴建平2,张 昊1,郭慧媛1,刘洪娜3,任发政1倡

1.中国农业大学食品科学与营养工程学院,食品质量与安全北京实验室,北京 1000832.阿尔伯塔大学,加拿大埃德蒙顿市

3.甘肃农业大学食品科学与工程学院,甘肃兰州 630070

摘 要 综合运用动态光散射光谱、荧光光谱和高效液相‐紫外光谱法检测钙离子对酪蛋白胶束结构的影响。外源添加的钙离子的浓度从0增加至12mmol・L-1的过程中,酪蛋白胶束的外源ANS荧光强度和浊度一直增大,但是其体积平均直径和胶束的多分散指数是一直下降。同时,当外源添加的钙螯合剂(柠檬酸根)离子的浓度从0增加至12mmol・L-1的过程中,酪蛋白胶束的外源ANS荧光强度和浊度一直减小,但是酪蛋白胶束的体积平均直径、胶束的多分散指数和胶束的稳定性是增大的。因此,在对酪蛋白胶束的结构影响方面,钙离子和柠檬酸根离起到了相反的作用。研究证实,外源钙离子可以有效地调节酪蛋白胶束的结构,进而改善其功能特性。

关键词 光谱;钙离子;酪蛋白胶束;结构

(收稿日期:2013‐04‐10,修订日期:2013‐07‐11)  倡通讯联系人

钙离子对酪蛋白胶束结构影响的光谱学研究

作者:作者单位:

王鹏杰, 吴建平, 张昊, 郭慧媛, 刘洪娜, 任发政, WANG Peng-jie, WU Jian-ping, ZHANG Hao, GUO Hui-yuan, LIU Hong-na, REN Fa-zheng

王鹏杰,张昊,郭慧媛,任发政,WANG Peng-jie,ZHANG Hao,GUO Hui-yuan,REN Fa-zheng(中国农业大学食品科学与营养工程学院,食品质量与安全北京实验室,北京 100083), 吴建平,WU Jian-ping(阿尔伯塔大学,加拿大埃德蒙顿市), 刘洪娜,LIU Hong-na(甘肃农业大学食品科学与工程学院,甘肃 兰州,630070)光谱学与光谱分析

Spectroscopy and Spectral Analysis

2014(1)

刊名:

英文刊名:

年,卷(期):

本文链接:http://d.wanfangdata.com.cn/Periodical_gpxygpfx201401023.aspx

第34卷,第1期            光谱学与光谱分析2014年1月            SpectroscopyandSpectralAnalysisVol畅34,No畅1,pp92‐97

January,2014 

CalciumSpectroscopicIononInvestigationtheStructuresofoftheCaseinInfluenceMicelles

ofWANGPeng‐jie1,LIUWUHongJian‐‐pnaing2,ZHANGHao1,GUOHui‐yuan1,

3,RENFa‐zheng1倡

1.BeijingLaboratoryforFoodQualityandSafety,CollegeofFoodScienceandNutritional2.UEngineeringniversityof,ChinaAlbertaAgricultural,EdmontonU,niversityCanada

,Beijing 100083,China

3.CollegeofFoodScienceandTechnology,GansuAgriculturalUniversity,Lanzhou 630070,China

Abstractment Theeffectsofcalciumextrinsicwerewithinfluorescencesynthetically(ANSexaminedionfluorescencebyonnonthestructuralpropertiesofcaseinmicellesinthecourseofheattreat)‐structure,waspositively‐invasivespectrometry.Thehydrophobicity,reflectedwithterthethegrowrangethofofcalcium0to12mmol・L-1.Meanwhile,theturbiditycorrelatedandwithstabilitytheconcentrationofcaseinmicellesofthealsocalciumincreasedeffectandmmolonpolydispersitythestructuralindexconcentrationscharacteristics.Comparedofwith.Howevercaseinthecalcium,oppositeion,resultsthecalciumwere‐observedchelator(forcitratehydrodynamic)hasanoppositediamethedexcalcium・L-1ion,the,neverthelesshydrophobicity,opposite,stabilitymicelles.Withinthecalciumconcentrationsrangeof0resultsandwereturbidityobservedwerefornegativelycorrelatedwiththeconcentrationingheat.Allheatmenttheresults.

indicatethatthecalciumioncouldbeusedtohydrodynamicmodifythestructuresdiameterofandcaseinpolydispersitymicellesKeywords Caseinmicelles;Calciumions;Structures;Spectroscopy

中图分类号:O657畅3  文献标识码:A   DOI:10畅3964/j畅issn畅1000‐0593(2014)01‐0092‐06

Introduction

sultintheofnoticeableuniquemilkeffects‐specificonmilkstability[2].groupto  Inthedairyindustry,variouskindsofmineralsareseinstotalcheeseimproveimportant,yoghurttheandqualityproteinofpowdersmilk‐basedproducts,suchusedasals,theproteinmostimportantinthemilkproteins,andrepresentpartsofcommercialdairycasein,existasmostmicellesmainly[3]

in.Thetheofmilk,togetherphysicochemicalformofcolloidalparticlessedimentationtheygreatlyroleintheprocessandthefinal.Thesequalitymineralsofproductsplayan,celles[4]allmilk‐basedproductsmainlydependcharacteristicsonexchangers[1]andinfluence.Ingeneralgelationthe,,allrennetsusceptibilitythemineralsaction,ofexistandthefoulingproductsindynamicofheattoe‐qareThe.

functionalandchemicalpropertiesofmilkuilibrium.Smallbetweenchangestheinthediffusibledistributionandtheofmineralsmicellarwouldphaserein‐thedeterminedbytheirstructures,whichcancaseinterationsmodificationinthestructuresofthemicroenvironmentcanbereflectedinofthemilk Received:2013‐04‐10,accepted:2013‐07‐11

 Foundationitem:InternationalScience&TechnologyCooperationProgramofChina(2011DFA32550),MinistryofScienceand Biography:WANGChinaPeng(2012‐jie,(1986BAD12—B)08)

,DoctoralCandidateofChinaAgriculturalUniversity  e‐mail:wpj1019@126.com

倡Correspondingauthor  e‐mail:renfazheng@263.net

by‐,‐12‐‐80%area.Caof‐,i.eminer‐caseinof.,the

mial‐‐.alteredmicellesThesebyal‐‐

echnologyofiontoofindurCaseinsspecieswiththebehydrophobicT

ity,sizeandconformationalstates.Fluorescentspectroscopycanbeusedtoquantifyproteinhydrophobicity[5].Mean‐while,dynamiclightscattering(DLS)andturbidity,whicharebothfastandnoninvasive,havebecomethemethodsofchoiceforevaluatingthehydrodynamicdiameter[6].

Inthedairyindustry,heattreatmentisanessentialpro‐cedure,aimingtoimprovethefinalqualityofraw‐milk‐prod‐uctsbyreducingthemicrobialrisksandtherefore,reducingtheriskofpoisoning.However,inothercases,wherecaseinmicellesareusedasfoodingredients,heattreatmentiswidelyappliedinimprovingthephysicochemicalpropertiesofsuchcitratebuffer(pH7畅0)toafinalconcentrationof1mg・

St.Louis,MO),accordingtoLecki[9],withslightmodifica‐tions.Caseindispersions(10mg・mL-1)weredilutedwithmL-1.Then,20mLofANS(8mmol・L-1inbuffersolu‐

tions)wasaddedto4mLofthedilutedsampleandmixed.RelativefluorescenceintensitywasmeasuredimmediatelywithaRF‐5301PCfluorescencespectrometer(Shimadzu,Ja‐pan)at390nm(excitationwavelength,slit5nm),400~650nm(emissionwavelength,slit5nm)andascanningspeedof10nm・s-1.

1畅3 Hydrodynamicdiameterandsizedistributionofcaseins

dairyseinmicellesformulationsbymanipulatingthefunctionalityoftheca‐micellesAsaal,heatuseful.

treatmenttoolforhasmodifyingbeenstudiedtheinstructures‐depth,andofcaseinsever‐ptherovenmechanismshavebeenpresented.Meanwhile,ithasbeenavailablemicellethatthecalciumionplaysacrucialroleinstabilizingponcaseintheeffectstructure[8].However,littleinformationisofropertiesmineralscalciumof.Thecaseinaimmicellesofheatofthisstudyinthetreatmentonthestructuralispresenceofvariouskindsofincaseinonmicellesthehydrophobicity.Agoodunderstanding,sizetoandinvestigateconformationaltheeffectofoftheseisimportantstatesseinthemicelleselucidation.

ofstructuresandfunctionalpropertiesofca‐1 MaterialsandMethods

1畅1 PreparationtheThecowmilkofincludedcaseinmicelles

inthisstudywascollectedmilkinstitution30waspreparedfarmofChinaAgriculturalUniversity.fromSkimTDwas3800minat25℃by,usingcentrifugationalab‐scaleofrawcentrifugemilkat4000(Xiangyigforweight℃.achieved,ChangshaThebyultracentrifugation,China).Separationat120000ofcaseingfor1micelleshat25ultrafiltrate.Thecaseinpelletsobtainedwerefreeze‐driedtoconstantazidewaterfreeze.‐driedcaseinmicelleswerere‐dissolvedinv)trypsinwasaddedtoinhibitSubsequentlybacterial,0grow畅02%th(,wand/v)0畅02%ofsodium(w/edtionstoDifferentinhibitorthecaseinconcentrationswasaddedtominimizeplasminactivity.

micellesolutionsofcalciumandcitratewereadd‐p.Aliquotsofthecasein-1

wereH6畅HClmicelleatdifferentfinalconcentra‐.Thesamplescaseinmicellewereadjustedsolutionsto

solutionsheated6by11畅2 wereatmolcooled65・℃L

for30min.Afterheattreatments,themgTheFluorescenceextrinsicstudyto25℃forfurtheranalysis.

fluorescenceofhydrophobicityofthecaseinofcaseinsolutionsmicelles

(10sulfonate・mL-1()ANSwas)measuredfluorescenceusingprobethe1‐(Sigmaanilino‐8‐ChemicalnaphthaleneCo.,

‐cellesTheNanoing,wereaverageBeckmandeterminedsizeandCoulterbypolydispersityindexofcaseinmi‐

Inclaser.,CAlight,USAscattering(DelsaTMdexexperimentalofofthesolventconditions1畅333and:temperature)viscosity1畅00225,under℃mPa,refractivethefollow‐・sin‐p100mLofrawskimmilkwasdispersedin10mL.ofAtotalmilk

(ermeatekDaMillipore.TheCorporationmilkpermeate,MA,wasobtainedbyultrafiltrationareanalysisindexcharacterizedcut‐offmembrane.Thedatawerebymean(MilliporeUSA)ofskimmilkusinga10‐exporteddiameterCorporationfromDthe[6,equipment5]and).polydispersityTheparticlesforfurther

quipment.directlyThepolydispersityandD[6,5]indexwaswas∑calculatedobtainedas

fromthee‐D[6,5]=

VjD3j∑whereVistherelativevolumefractionj

D2(1)

withjofallcaseinmicelles

dynamicdiametertriplicatediameterDj.Theresultswereexpressedasmeanhydro‐isanindex.Theofparticlehydrodynamic(nm).Eachpolydispersitydiameterexperimentwasconductedinlows

anddistributioniscalculatedofparticlesasfol‐

polydispersityindex=

50%

(2)

eterswhereatD90%90%,D10%andD50%representthehydrodynamicdiam‐

p1畅articles4 Quantification,respectively,10%andof.

50%cumulativevolumeofthecaseinαs1‐,αs2‐,β‐andSeparationspectrometryof‐HPLCκ‐caseinbyultraviolet

byαs1‐,αs2‐,β‐andκ‐caseinwascarriedout

natantreversed(100mmol(500‐pμhase・LL)-1wasHPLCTrisdiluted‐UVbasewithequipment,8mol3mL・Lof.Skim-1denaturingmilkorsolution

super‐2O,0畅3%v/vβ‐mercaptoethanolurea,13,adjustedg・L-1Na3citrate・2Htop20carriedH7畅0AoutwithusingHCl)reversed.Analysis‐phaseoftheHPLCmajor‐UVcasein(Shimadzucontents,wasLC‐

mm,Kyoto,Japan)withaKromasilC4column(4畅6×containing,300樻0畅1%,AkzoNobel(v/v)trifluoroacetic,Sweden).Solventacid(TFAAwas)andwater250sol‐

sensitivethanthoseusedinabsorptionspectroscopy.Further‐more,becauseemittedlightisdetectedatafixedwavelenguhatrightanglestotheexcitationradiation,fluorescentspectro‐scopicanalysisislessaffectedbyturbidmedia[7,9].Thefluo‐rescenceintensityofANSafterbindingtocaseinmicellesatdifferentcalciumconcentrationsisshowninFigure1.Itcanbeseenthatatthegivenheatingconditions(65℃for30min),thefluorescenceintensityofANSincreasedsignificant‐lywithincreasedcalciumconcentrations.Thisindicatedthatmorehydrophobicbindingsiteswereexposedathighercalci‐umconcentrations.However,whenthecaseinmicelleswereventBwasacetonitrilecontaining0畅1%(v/v)TFA.Theflowratewas0畅8mL・min-1.Alineargradientfrom30畅0%to50畅0%solventBover50minwasset.Afterfilteringthrougha0畅45μmfilter(Membrana,Wuppertal,Germa‐ny),a10μLsamplewasanalyzed.ThedissociationdegreeofindividualcaseinsfromthemicelleswasdeterminedbasedonthemethodofAnema[10].1畅5 Turbiditymeasurements

Theturbidityofcaseinsolutionswasdeterminedasde‐scribedbyPartschefeld,withslightmodifications.Thetrea‐tedsamplesweremeasuredatthewavelengthof633nmwithaUV‐visiblespectrophotometer(KeerdekeheatedcordingTurbiditywaswheretoindexPartschefeldwasused[11]to,expressasturbiditytheturbidity,Beijing=(w-values,China).wac‐0)/mmolhighestat90iswistheturbidityofthesampleaftertreatment,andw0w,found・L-1when℃for,andthethe30min,theANSfluorescencelowestcalciumANSconcentrationfluorescenceofcaseinsintensitysamplestheturbidity1畅6 weremeasuredofthesampleintriplicatebefore.heattreatment.Allthe0(asdatanotwhenshowntheconcentration).ofcalciumwas12mmolwithasesnaturationthosefunctionoftheThiscalciumtrendconcentrationofANSfluorescenceisnotgwereEachStatisticalperformedsampleAnalyses

wasusinganalyzedSPSSin13畅0triplicateforWindows.StatisticalXP(analyChica‐discussionoftreatedcaseinsatunder65℃ultrahigh,whichistemperaturesprobablyduecano,USA).TestsfordifferenceswereperformedusingDun‐‐heatingicant’s.

multiplerangemethod.P<0畅05wasconsideredsignif‐tensityconditionsincludedofin65this℃studyfor30wasmerelyfocused.rateofANSafterbindingtocaseinminmicelles.Thefluorescenceatdifferent2 Resultsanddiscussion

thatconcentrationswiththefluorescenceisintensityshowninofFigureANS1.Itcanbe2畅1 drophobicincreasedtrationsbindingcitratesitesconcentrationswereexposed.ItdecreasedindicatedsignificantlythatitsgboundaInHydrophobicityofcaseinmicelles

verywaterlow,quantumthehydrophobicyieldoffluorescentprobeANSexhib‐rateent..Citratecouldberegardedasatahighercalciumcitratechelatinghighlytofluorescentthehydrophobic.Fluorescentregionsfluorescenceprobesofproteins.Howeveraregenerally,itbecomes,oncemore

thecaseinreducedDependingmicellesthecalciumonthelevelwerecontentofdisruptedinchelatingagents,adding.

thecaseinmicellesFig畅1 FluorescenceintensityofANSincaseinmicellesasafunctionofcalciumionconcentrations

(theconcentrationsofcitratewere3and8mmol・L-1inAandB,respectively)

2畅2 developmentnoninvasiveDynamicSizecharacteristicslightscatteringofcasein(DLSmicelles

),forp,hasbecomethemethodofwhichchoiceisforbothevaluatingfastandthetheaccurateofdynamiclightscatteringholdsgreatsystemsarticlesize.Becauseandpolydispersitycaseinmicellesindexscatterinlightarangeveryofwellmicellar,the

responseinvestigationdeterminationofcaseinmicellesize,lightscatteringtoenvironmentalofitsinteractionswithothermoleculesarethatitfactors[12]avoidsdrastic.Thechangesmainadvantagesinwas6・wasL-1thedeon,thethe‐citin‐‐lesshy‐a‐‐.Thuscit‐,welloritsasenviof‐

intensityintensityaccordanttoThusobservedconcenpromiseasthe

ronmentofcaseinmicellesandthatthemeasurementsareper‐formedonalargenumberofparticles[13].AsshowninFigure2,thehydrodynamicdiameterofcaseinmicellesdecreaseswithincreasingcalciumconcentrations.Theioniccalciumcanshieldthenegativechargeonthesurfaceofcaseinmicelles.Atcalciumconcentrationsrangingfrom12畅0to0mmol・L-1,thesurfaceofcaseinmicellesbecomemorenegativelycharged.Meanwhile,duetothedecreasingcalciumconcen‐

trations,thestabilityofcaseinmicellesdecreasedandthere‐foretheybecamemoreinflatedstructures.Thisconsequentlyincreasedtheirhydrodynamicdiameter.Thehydrodynamicdi‐ameterofcaseinmicellesatdifferentcitrateconcentrationsisshowninFigure2.Thesizeofcaseinmicellesdecreasedwiththeincreasingcitrateconcentrations.Thus,comparedwiththecalcium,thecitratehasanoppositeeffectonthehydrody‐namicdiameterofcaseinmicelles.

Fig畅2 SizecharacteristicsofcaseinmicellesasafunctionofcalciumandBion,respectivelyconcentrations(theconcentrationsofcitratewere3and8mmol・L-1inA)

  TheparticlesizedistributionofasystemcanbeHoweverdexeffect2valueasindicatesthepolydispersityindex.Asmallpolydispersityexin‐pressed‐concentrationonthe,comparedstabilitywithofcaseincalciummicelles,thecitratehasanoppositedexalsodemonstratedaanarrownoticeableparticledecreasesizedistributioninpolydispersity.Figureinmicellarformofdecreasedcitrate,.

thepercentage.ofWithcaseinsthepresentincreasingmmolas・theL-1calciumconcentrationincreasedfrom0to12畅‐0creaseswhilewith.theThisincreasingmeansthattheheterogeneityofcaseinsin‐centration,thepolydispersityindexconcentrationdecreaseswithofcalciumincreasing.Meancon‐‐micellesofcitrate,suggestingtheheterogeneityofcasein2畅3 Stabilitydecreasedwithincreasingcitrateconcentration.

letspectrumofcasein(micellesdeterminedbyHPLC‐ultravio‐

Fig畅3 RP‐HPLCchromatogramsofcaseins

associationInthe2畅4 pableressthestabilitystatepresentUVofstudy)

,thedegreeofdissociationandtheofcaseincaseinfrommicellesthemicelles.wasusedtoex‐chromatographymethodforthecaseinanalysisisPresentlyhigh‐p,themostreli‐repeatableTurbidityTurbidity

hasbeenfoundtobeanextremelysensitivetrumcaseins(UV(HPLC)intandemwithanerformanceultravioletliquidspec‐calciumteractionsconcentrations.andThenoninvasiveturbidityisgivenofwaytheforincaseinmonitoringFiguremicellesthe5.Theatcaseinturbiditydifferent

rated.Theisshown)detectoreffectinofFigure.AcharacteristicRP‐HPLCprofileofthecalcium3on.Allthethecaseinswerewellsepa‐thecaseinsolutiondecreasedrapidlywithdecreasingcalciumisconcentrationcellarshowninFigure4.Thepercentagestabilityofcaseinsofcaseinpresentmicellesintheturbidityof.Howevercaseinmicelles,thecitrate.

hastheoppositeeffectcreasedformtrationfromincreased0to6mmolwhen・Lthe-1concentrations.However,whenofcalciummitheconcenin‐‐‐

3 Conclusion

-1

ageofcalciumwasstantof.caseinsThisindicatedpresenthigherthatinmicellarthan6mmol・L,thepercent‐thestabilityformremainedofcaseinlargelymicellesconin‐‐  ThestudysuggestedthatthemineralenvironmentcreasedL-1,andatcalciumitchangedconcentrationslittleathigherrangingcalciumfromconcentrations0to6mmol・.

calciummilkstronglyconcentrationsinfluencesrangingthestructurefrom0toof12caseinmmolmicelles・L-1,.hydrophobicityandturbidityofcaseinmicellesincreasedwith‐,inofoninintheAt

Fig畅4 Thestabilityofcaseinmicellesasafunctionofcalciumionconcentrations(theconcentrationsofcitratewere3and8mmol・L-1inAandB,

respectively)

Fig畅5 D[6,5]andturbidityofcaseinmicellesasafunctionofcalciumionconcentrations

(theconcentrationsofcitratewere3and8mmol・L-1inAandB,respectively)

increasingforcalciumconcentration,andconcentrationsthesizeandrangingpolydispersity0to12mmolindexbut・.theLMeanwhileopposite-1,the,wasatcitratefoundthepolydispersityindex.Thus,tyertiescitratehasanoppositeeffectoncomparedthephysicochemicalwiththecalciumcitrateandconcentrationturbidityofcaseinhydrophobici‐,butthemicellesoppositedecreasedwasfoundwithforincreasingthesize

heatstructuretreatmentofcaseinofcasein,micellescalcium.micellesionsTheseresultsindicatethatduring.

couldbeusedtomodifyReferences

[1] SalaonF,MiettonB,GaucheronF.Milchwissenschaft,2007,62(1):20.

[2] SinghH,RobertsMS,MunroPA,etal.JournalofDairyScience,1996,79(8):1340.[3] WangP,LiuH,WenP,etal.InternationalDairyJournal,2013,31(2):107.[4] FoxPF,BrodkorbA.InternationalDairyJournal,2008,18(7):677.[5] YükselZ,ErdemYK.JournalofFoodEngineering,2005,67(3):301.

[6] GebhardtR,DosterW,FriedrichJ,etal.EuropeanBiophysicsJournal,2006,35(6):503.

[7] ChakrabortyA,BasakS.JournalofPhotochemistryandPhotobiologyB:Biology,2007,87(3):191.[8] AnemaSG.FoodChemistry,2009,114(1):161.[9] LenckiRW.JournalofDairyScience,2007,90(1):75.

[10] AnemaSG,LiY.Lebensmittel‐Wissenschaftund‐Technologie,2000,33(5):335.[11] PartschefeldC,SchwarzenbolzU,RichterS,etal.Biotechnol.J.,2007,2(4):456.[12] TranLeT,SaveynP,HoaHD,etal.InternationalDairyJournal,2008,18(12):1090.[13] BeliciuCM,MoraruCI.JournalofDairyScience,2009,92(5):1829.

‐,propthe

钙离子对酪蛋白胶束结构影响的光谱学研究

王鹏杰1,吴建平2,张 昊1,郭慧媛1,刘洪娜3,任发政1倡

1.中国农业大学食品科学与营养工程学院,食品质量与安全北京实验室,北京 1000832.阿尔伯塔大学,加拿大埃德蒙顿市

3.甘肃农业大学食品科学与工程学院,甘肃兰州 630070

摘 要 综合运用动态光散射光谱、荧光光谱和高效液相‐紫外光谱法检测钙离子对酪蛋白胶束结构的影响。外源添加的钙离子的浓度从0增加至12mmol・L-1的过程中,酪蛋白胶束的外源ANS荧光强度和浊度一直增大,但是其体积平均直径和胶束的多分散指数是一直下降。同时,当外源添加的钙螯合剂(柠檬酸根)离子的浓度从0增加至12mmol・L-1的过程中,酪蛋白胶束的外源ANS荧光强度和浊度一直减小,但是酪蛋白胶束的体积平均直径、胶束的多分散指数和胶束的稳定性是增大的。因此,在对酪蛋白胶束的结构影响方面,钙离子和柠檬酸根离起到了相反的作用。研究证实,外源钙离子可以有效地调节酪蛋白胶束的结构,进而改善其功能特性。

关键词 光谱;钙离子;酪蛋白胶束;结构

(收稿日期:2013‐04‐10,修订日期:2013‐07‐11)  倡通讯联系人

钙离子对酪蛋白胶束结构影响的光谱学研究

作者:作者单位:

王鹏杰, 吴建平, 张昊, 郭慧媛, 刘洪娜, 任发政, WANG Peng-jie, WU Jian-ping, ZHANG Hao, GUO Hui-yuan, LIU Hong-na, REN Fa-zheng

王鹏杰,张昊,郭慧媛,任发政,WANG Peng-jie,ZHANG Hao,GUO Hui-yuan,REN Fa-zheng(中国农业大学食品科学与营养工程学院,食品质量与安全北京实验室,北京 100083), 吴建平,WU Jian-ping(阿尔伯塔大学,加拿大埃德蒙顿市), 刘洪娜,LIU Hong-na(甘肃农业大学食品科学与工程学院,甘肃 兰州,630070)光谱学与光谱分析

Spectroscopy and Spectral Analysis

2014(1)

刊名:

英文刊名:

年,卷(期):

本文链接:http://d.wanfangdata.com.cn/Periodical_gpxygpfx201401023.aspx


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