2017 7 Vol.33,No.7 Jul.2017 DOI:10.13652/j.issn.1003-5788.2017.07.002 Analysisofglycosylatedcaseininwholemilkpowder SUN Jing-li LI Lin-qiang ZHU Li-li TANG Lu WU Yuan (, 710119) (Colegeof Food Engineering and NutritionalScience,Shaanxi Normal University,Xi'an,Shaanxi710119,China) :, groups.thepercentage(46.13±0.46)% ofcaseinextracted ph bytannicacid wassignificantlyhigherthanthatofother methods, SDS-PAGE,.TheresultsofcaseinSDS-PAGEelectrophoresisboth, showedthesame3bandseitherdiferentacidsordiferentmethods, oncaseinextraction,andtheirmolecularweightswere34,24,and,ph4.6 60ku,respectively,andthe34and24ku wereglycosylatedcasein bythemethodofperiodate-schifalkalidyeing.theresultsofmorse 2% (ph3.58) (40.45± experimentshowedthatmilkpowdercaseinexistglycosylatedcasein. 0.66)%, Thecontentoffreeaminoacidfrom wholemilkpowderwassignifi- (46.13±0.46)%, cantlyhigherthanthatfromfreshmilkafterpapainhydrolysis(p< SDS-PAGE, 0.05).Wholemilkpowderhasglycosylatedcasein,theirmolecular 3, 34,24,60ku weightsare34and24kurespectively,whichareeasytohydrolysis., 34,24ku Keywords:wholemilkpowderreconstitutedmilkcaseinglycosy-, lation,, 34,24ku, : Abstract:Thisstudyinvestigatedglycosylatedcaseinin whole milk powderandexploreditsenzymaticproperty.thecaseinextraction weightpercentageweredeterminedbydiferentph,diferentacids, anddiferentmethods.thecaseinwasanalyzedbysds-pageelec- trophoresis,and the glycosylated casein wereidentified via the methodofperiodate-schifalkalidyeingandtheexperimentofmorse.the caseinwashydrolyzedbypapainandtheaminoacidcontentwasdeter- minedaccordingly.results:thepercentageofcaseinextractedunderph 4.6wassignificantlyhigherthanthatofothergroups.The weightpercentageofcaseinextractedfrom aceticacid (ph 3.58)was (40.45±0.66)%,which wassignificantlyhigherthanthatofother [1],, 50%, [2-3] [4], [5] α-1- (AGP) [6] [7-8] [9-11],,, : ( :2016NY-212) 170 :,,,, : (1971 ),,,, E-mail:lilinq@snnu.edu.cn :2017 03 15, 6
2017 7, 1.2.1 (1),, 1.3 [12~14], 1 40,,, 3,200 g 1.1 5min 2mg 1 ml, 1.1.1 10min,, : 10mL15%, 40 min Mark:18~94ku, Thermo, 4 ml3%, : 800U/mg, Bioreagent, 1h,, :, 10μL, 80V 40min,120V : (Tris) N,N,N,N- (TEMED) (SDS) G-250 α- :, 1.1.2 [15-17] ( ) :800B, : :PowerPacTM Universal, 10% Bio-rad :KQ-250DB, 1h, 2 ( 0.5h), Schif 1h(Schif : 1g 400 ml, :HY-2, 12mol/L HCl4.2 ml 3.2g,4, :722, 2g, 1min ), (0.5% 1.2 7% 1 1 ),4, 1.2.1 1.4.2 [18], : 2 1 7 (g/ml), 5 ml,, 1 ml 1% 1% ph(4.6,4.8,5.0,5.4,5.6,5.8) ( 5mL 0.1% NaCl ), (5% 200 g 10min,,, (1) α-, 95%, )2, W = m1 m 100%, (1) : W,% m1,g m,g 1.2.2 1 7 (g/ml) ( 0.1% NaCl ), 10 ml,, 5 ml, 1mL 0.1% ( 0.1% NaCl, (0.5%,1%,2%,3%,4%,5%), ), 37 6h [19-20] ph,200 g 10min,, 1.5.2, 1.2.1 (1) (0.0,1.0,2.0,5.0,10,15,20μg/mL) 1.0 ml 1.2.3 7 25mL, 2 mlph5.5 5mL, 10 mlph4.6 ( ) ph4.6 ( 2.5h, G-250 2h,, 1.4 1.4.1 ( ) SDS-PAGE 1h, 2 ( 0.5h), 1%, 1.5mL 2, 1.5 1.5.1 ph4.6, 0.1%,, 2% (2g 100 ml, )2 ml 15 min,, ) 0.1% ( ) ( 570nm, (y =0.0319x + ) 5% ( ), 0.0025,R 2 =0.9995), 40,,, 7
: 1.6 3, SPSS19.0, ±, LSD 5% 2 2.1 ph 4.6, (28.38±2.49)%,, [21-22] ph4.6 3 ph, Figure3 Influenceoflacticaciddiferentadditionsonex-, tractionrateofproteinandphvalueofsolution 1 Figure1 Influenceofaceticacid-sodiumacetatebufer solutiononproteinextractionrate 2.2 2~5, 2%, (40.45±0.66)%, 2%, (39.31±2.76)%, 3%, (37.44±1.04)%, 4%, (31.74±4.20)%,, > > > ph 5 ph > > >, ph Figure5 Influenceofphosphateaciddiferentadditionson,,, extractionrateofproteinandphvalueofsolution [23], 4 ph Figure4 Influenceofhdrochloricaciddiferentadditionson extractionrateofproteinandphvalueofsolution, 2.3 6, (46.13± 0.36)%,, (P< 0.05)[ 3 (P>0.05)], [24~25], 2 ph,, Figure2 Influenceofaceticaciddiferentadditionsonex- tractionrateofproteinandphvalueofsolution,, 8
2017 7 2.5 9 3 ( 9a), a. G-250 b. 1~3. 9 6 Figure9 AnalysisofSDS-PAGEoncaseinfromreconstitu- Figure6 Influenceofdiferentmethodsonprotein tedmilktreatedbydiferentdyeingmethods extractionrate 24,34,60ku 2 2.4 SDS-PAGE ( 9b), 24,34ku,, 7, 3 9a, 60ku Mark, 24,34,60ku,, [29~32] [26~28] 2.6, 10 8,, 3, Mark,, 24,34,60ku,, 2.5,, Figure10 1~4., 7 ph4.6, 2 [33~36] Figure7 AnalysisofSDS-PAGEoncaseinfromreconstituted milktreatedbydiferentacidsatph4.6, [37-38],, 10 Morseexperimentofcasein 2.7, ( ) 1~5. - 8 Figure8 AnalysisofSDS-PAGEoncaseinfromreconstitu- tedmilktreatedbydiferentextractionmethods 11 Figure11 Influenceofglycosylationonhydrolysisdegree ofcasein 9
:,,2006,28(6):178-179. [18]. [M]. :,2003:1-2. [19],,,. 3 [J]. :,2007,28(10S):, 29-31.,, [20],,,., [J].,2008(2):162-165. 3, 2, [21],,,., 34,24ku [J].,2008,33(12):, 262-267. [22],,,., [J]. :,2016(1):109-111., [23]. [D]. :,2015:23-36. [24],,. [J]. [1],,,.,2016,57(8):1335-1337. [J]. :,2013,29(1):1-3. [25],. [J]., [2],,. [J].,2002,28(12):57-61. [3],. [J]. : [J].,2014,35(20):101-103.,2012(12):1746-1759. [27]PORTUGALC A M.MilkProtein,UF Application[M].Ber- [4]. / [D]. :, lin:springerberlin Heidelberg,2016:1-20. 2009:1-4. [28]LEJARSA,NOGRE M,TELLIER M,etal.Methodforthe [5]. [J].,1996(1): extractionofoneorseveralproteinspresentinmilk:ep,wo/ 22-23. 2007/138198[P].2007-02-25. [6]ORCZYKP,OWICZ M,BERGHAUSEN M,etal.O -Glycosy- [29]BASCHJJ,GREENBERG R,FARRELL H MJr,etal.Iden- lationofα-1-acidgly-coproteinofhumanmilkislactationstage tificationofthemilkfatglobulemembraneproteins(i):isola- related[j].breastfeeding Medicine,2015,10(5):270-276. tionofmajorproteinsfromelectrophoreticgelsandcomparison [7]PICARIELLO G,FERRANTIP,MAMONE G,etal.Identifi- oftheiraminoacidcompositions[j].biochimicaetbiophysica cationofn-linkedglycoproteinsinhumanmilkbyhydrophilicin- Acta,1985,830(2):127. teractionliquidchromatographyandmassspectrometry[j].pro- [30]RIORDAN O N.Targetingtheglycomeofmajormilkproteins teomics,2008,8(18):3833-3847. [8]JOLANTA L K,IWONA K P,MARTA B M,etal.Lectin- basedanalysisoffucosylatedglycoproteinsofhumanskim milk during47daysoflactation[j].glycoconjugatejournal,2015,32 (9):665. from milk glycoproteins by novel endo-beta-n-acetyglu- cosaminidase[j].dissertations& Theses-Gradworks,2015,15 (10):15-16. [9]. [D]. : [32]KARAVS,LEP A,KIRMIZ N,etal.Oligosaccharidesre-,2007:1-5. leasedfrom milkglycoprote-insareselectivegrowthsubstrates [10]. α- β- for infant-associated bifidobacteria [J ]. Applied & [D]. :,2013:3-4. EnvironmentalMi-crobiology,2016,82(12):3622. [11],,,. [J]. [33],,,. α- β-,2015,36(12):385-389. [C]// [12],,,. :,2011:26-32. [J]. :,2016, 44(11):149-154. [13],,,. [35] (CPP) [J].,2010,26(6):51-53. [14]. SDS-PAGE [D]. :,2015:28-46. [15]. [M]. : 137-140.,2005:145-151. [37]. [16],. [J]. [D]. :,2003:2-6.,2006(s1):534-535. 2004(3):61-62. [26],,,. [J].FebsJournal,2016,278(19):3769-3781. [31]KARAV S.SelectiveprebioticactivityofN-glycansreleased [34],,,. [J].,2005,33(10):4-8. [J].,2000(6):15-16. [36],,,. [J].,2008,39(5): [38],,,. α- β- [17],.PAS [J]. [J].,2010,26(3):94-96. 10