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33 6 2017 6 Vol.33,No.6 Jun.2017 DOI:10.13652/j.issn.1003-5788.2017.06.

1 Vol.33,No.6 Jun.2017 DOI: /j.issn Preparation,separationandaminoacidsequenceanalysisofcorn peptideswithhypoglycemicactivity 1, HU Yu-hang 1,2 DAI Jun 1 CHEN Shang-wei 1 ZHU Song 1 ZHANG Xue-jun 3 ZHANG Ping 3 (1., , , ) (1.TheSchoolof Food Scienceand Technologyof Jiangnan University,Wuxi,Jiangsu214122,China 2.State Key Laboratoryof Food Scienceand Technologyof Jiangnan University,Wuxi,Jiangsu214122,China 3.Shangdong Tianjiu Biotechnology Co,Ltd.,Heze,Shandong274108,China) experimenttoobtaintheoptimalcombinationfor:amountofenz- : (DH ) chromatographicseparation.theresultofhypoglycemicactivityin,,ph 8.5,60 vitroshowedthatcp1 hadthebeterefectonpromotingnormal : 3.5g/100g, HepG2cels glucoseconsumption,andtheα-glycosidicenzymein- 2h, 1 20 (g/ml), DH 27.02%, hibitoryactivityreached34.64%,afterrp-hplccolumnpurifica- tion,14componentsweregotenthethirteenth (CP1-13)hadthe 85.23%, bestα-glycosidicenzymeinhibitoryactivity,andreached39.50%. : 50,pH3.5, 1.5g/100mL, DeterminedbyUPLC-Q-TOF-MS/MS,theaminoacidsequence 45min, 77.86%, wasa-p-a-l-l-p-f % Keywords:Cornpeptidehydrolysishypoglycemicactivityamino 3,CP1 HepG2 acidsequence, α- (34.64%)CP1 RP HPLC, , CP1-13 α-,, 39.50% UPLC Q TOF MS/MS, [1], t, 2016 A-P-A-L-L-P-F t :, Abstract:Inthisstudy,thedegreeofhydrolysis(DH )andpolypep-, tide content were index optimization for corn gluten meal of,, hydrolysisprocess.undertheconditionofph8.5,60,orthogonal [2], ymeenzyme3.5g/100g,hydrolysistime2h,solid-liquidratio1 20(g/mL)onthiscondition,theDHis27.02%,peptidecontentis 85.23%.Meanwhile,optimizedthedecolorizationtechnology,the bestconditions:temperature 50,pH 3.5,activated carbon dosage:1.5g/100ml,decoloringtime45minunderthecondition, cornpeptideyieldwas77.86%,thedecolorizationratewas87.27%. Afterdecoloringanddesalination,threecomponentswereisolatedby :,, : (1957 ),,, daihplc@163.com : ,,, [3],,,, :1, 2,, Luis Mojica [4] 147

2 Caco-2, :WatersAcquityUPLC, Wa- 24.5% Rim ters Nasri [5] :Waters2545, Waters α- :WatersAcquity [6] Dybowskin-2CDYa UPLC-Waters MALDI Synapt Q-TOF, Waters, 1.2 [7], (glucagon), MCP-1 α- ( -1), PYY( ), (0.22μm) Taisuke Mochida [8] GLP GPL-1, (1) :GB NoriyukiHiguchi [9] (2) :GB GK, (3) :GB GLP-1GIP,, (5) :100g,80, 1000mL,90 1h, 65,pH 5.5,, 0.5g/100g 65,, 2h,4000r/min 10 min,, 2,, (CGM): 68.27%, ( ) α- :4000U/g, 60 3 (Alcalase):2.4L(2.4 AU/g),, 1 [10] [11]16 (QB/T ) 767 P-2Gel:, HepG2: CtccbioScience FBS DMEM:, Hyclone DMSO:, α- :100UN, ( ) TFA:, NaOH HCl NaCO3 :, :SCIENTZ-10N, 1.2.4, :BeckmanJ-26xp, [12], :RE-52A,,:Z=0.5X +0.5Y, :HL-2D, X (%),Y (%) :HD-3, 50,, 2, :ThermoScientific8000, :XDS-1A,, ph (4) :GB/T CGM, min,,, min,, : ph [E]/[S], (DH ) [10] ph 8.5, 1 Table1 Designfactorsoftheorthogonaltestabout enzymaticprocess A /% B /h C (g/ml) D

3 33 6 : 2, Waters xbridge PrepC18 (250 mm Table2 Designfactorsoftheorthogonaltestabout decolorationprocess 19mm,5μm) 220nm, 20, 50 mg/ml, 500μL, 10 ml/min ApH B / C / :A,B 0.05% TFA, : (10-2 g ml -1 ) min 0~40min,95% A~65% A40~45min,65% A~20% A α-,, (Bio-GelP-2Gel,4.5cm 90cm), - - (UPLC 1g 30 ml Q TOF MS/MS),, (1) : WATERS ACQUITY 2.5mL/min,220nm,, UPLC WATERS ACQUITY PDA ~400 nm :BEH C18 (2.1 mm 150 mm, 1.7μm) :45, :0.3 ml/min : mg/mL, 5μL :A0.1%,B (1)α- : α-,0~40 min,100% A~70% A40~45 min,, [13],, 70%~20% A45~50min,20%~0% A50~55min,0%~ 300μL, 0.2U α- 50μL 100% A 2mg/mL 100 μl,, 37 (2) : WATERS MALDISYNAPT Q- 10min, 5 mmolpnpg100μl 20min, 0.5 molnaco3800μl,405nm A1,, A2, A0 (1) : 2000m/z 1800V A0 - ( A1 -A2) I = 100%, (1) : A0 I α-,% A0 A1 A2 3 CGM (2) : Table3 CompositionofCGMandthesample HepG2,,PBS 1, afterpretreatment % EDTA,37 1~3 min, 10% DMEM r/min 5min,, cels/ml,100μl/ 96, 37,5% CO2,95% 24h, 2.2 DMEM 4, DH : >, HepG2 >, A3B3C2 (0.01mg/mL), 3, 24h,,, [14] : > MTT, 10μL MTT, >, A1B1C2 3~4h, 200μL DMSO,, A3B1C2,DH 10min492nm OD, 27.02%, 1.87% [14] 45~50 min,20% A~20% A50~55 min,20% A~95% A55~60min,95% A~95% A RP HPLC,37 TOF MS ESI+ 3.5kV 30 V 100 : 400, 700L/h, 50L/h 6/20V 20~ , α %( ),, (RP-HPLC) 2.3 (RP 5, : [15] HPLC) Waters2545 >ph> Z, 149

4 , Table4 Resultsandanalysisoforthogonaltestabout 37.10% enzymaticprocess 2.4 A B C DH/% /%, 1 3, CP1,CP2,CP , k DH k k R k k k R , CP1 Table5 Resultsandanalysisoforthogonaltest aboutenzymaticprocess, MTT, HepG2 ( A B C X /% Y /% Z k k k R k k k R Pro-Ile,Val-Pro-Leu) (Gly-Pro-X) : 50,, (Gly-Pro-X), ph3.5, :1.5g/100 ml, DPP-IV X-Pro-Phe Pro, 45min, 77.86%, 87.27%, ( )90.91% L-L-P-F, ( )88.85%, 80% 75%, [19] Figure1 SeparationofcornpeptidesbyPolyacrylamide gelcolumn 2.5 6, MTT GC/OD, ), LIJiang-tao [16] α-,3 CP % 2.6 (Bio-GelP-2Gel) CP1 RP HPLC, 2 14,α- 3, CP %, 2.7 CP1-13 UPLC Q TOF MS/MS, 4,Biolynx A-P-A-L-L-P-F (DPP-Ⅳ) X-AlaX-ProN [17], (GLP-1) Tadashi Hatanaka [16] DPP-Ⅳ, Pro, (Ile- DPP-IV, Zhuang Hong [18]

5 33 6 : 6 Table6 Thehypoglycemicactivityinvitroofthecornpeptides(X±S,n=3) / GC/ (mg ml -1 ) (mmol L -1 ) OD (A490) GC/OD/ (mmol L -1 ) α- /% ± ±0.02 * * 8.25± ± ±0.04 * * 7.41± ± ± ± ± ± ±0.03 * * 9.33±1.42 CP ± ±0.01 * * 7.73± ± ± ±0.02 * * 6.84± ± ±0.01 * * 5.53±0.74 CP ±0.33 * 0.49±0.04 * 6.97± ± ± ± ± ± ±0.02 * * 6.25±0.82 CP ± ±0.03 * * 5.73± ± ± ± ± ±0.99 * 0.52± ±1.47 CK 3.75± ± ±1.08 GC,OD MTT OD,GC/OD *,P< 0.05**,P< CP1 Figure2 ThechromatogramofCP1separatedbyPreparativeRP-HPLC 3 CP1 α- Figure3 Theinhibitionactivityofα-glycosidaseofthe fractionsseparatedbyrp-hplc 3 Q-Q-L-L-P-FMaZhi-li [20] 4 Figure4 3 CP1-13 MS/MSsepctrumoftheaminoacidsequence ofcp1-13, R-L-L-P-F 3 (1), 4, X-L-L-, :ph8.5, 60, P-F 3.5g/100g, 2h, 1 20 (g/ml) 151

6 ,pH3.5, 1.5g/100 ml, anddipeptidylpeptidase-iv ActivityinRats[J].Endocrinology, 45min, 2010,151(7): CP1-13, [9]HIGUCHIN,HIRA T,YAMADA N,HARA H.OralAdmin- A-P-A-L-L-P-F, istrationofcornzein HydrolysateStimulatesGLP-1and GIP, [11]40-41 SecretionandImprovesGlucoseTolerancein MaleNormalRats α- andgoto-kakizakirats[j].endocrinology,2013,154(9): A-E-P-N-L-S-G-D-A-P, [10],. [J].,2010,36(7): (2), [11]. [D]. :,2015., CP1-13, [12],,. [J].,2012(4): [1].2016/17 : [EB/OL].( )[ ].htp://www. [14]YAMASHITA R,SAITO T,SATOH S,etal.Efectsofde- chinabgao.com/stat/stats/81417.html. hydroepiandrosteroneon gluconeogenicenzymesand glucose [2],,. uptakein human hepatomacelline,hepg2[j].endocrine [J].,2013,4(3): Journal,2005,52(6): [3]. [D]. :, [15],,,. 2016:2-4. [J].,2015,31(5): ,221. ches[j].journaloffunctionalfoods,2017,31: [5]NASRIR,ABDELHEDIO,JEMILI,etal.Amelioratingefectsof gobyfishproteinhydrolysatesonhigh-fat-high-fructosediet-induced hyperglycemia,oxidativestressanddeteriorationofkidneyfunction inrats[j].chem BiolInteract,2015,242: [6]. Dybowskin-2CDYa [18]ZHUANG Hong,TANG Ning,YUAN Yuan.Purificationand [D]. :,2015: identificationofantioxidantpeptidesfromcornglutenmeal[j]. [4]MOJICA L,MEJIA E G D,MENJIVAR M.Evaluationofthe hypoglycemicpotentialofablackbeanhydrolyzedproteinisolate anditspurepeptidesusinginsilico,invitroandinvivoapproa- [7],,. Glu- JournalofFunctionalFoods,2013,5(4): cagon MCP-1 PPPYY [J]. [19],,,. ACE,2014,34(3): [J].,2011,26(4): [8]MOCHIDA T,HIRA T,HARA H.TheCornProtein,Zein Hydrolysate,AdministeredintotheIleum AtenuatesHypergly- cemiaviaitsdualactiononglucagon-likepeptide-1secretion [13],,,. α- [J].,2016(6): [16]LIJiang-tao,HE Hui,MA Zhi-li,etal.Apoptosisinhuman hepatoma HepG2celsinducedbycornpeptidesanditsanti- tumoreficacyin H22tumorbearingmice[J].FoodandChemi- caltoxicology,2013,51: istry,2014,29(6): [17] HATANAKA T,KAWAKAMI K, URAJI M.Inhibitory efectofcolagen-derivedtripeptidesondipeptidylpeptidase-iv activity[j].journalofenzymeinhibitionand MedicinalChem- [20]MA Zhi-li,ZHANG Wen-jun,YU Guo-cai,etal.Theprimary structureidentificationofacornpeptidefacilitatingalcoholmetabo- lismbyhplc-ms/ms[j].peptides,2012,37(1): ( 109) (23):48-50., [6]. [J]. :,2007(29):299. [7],. [J].,2017(1): [8],. [J].,2015 (3): [9]. : [J].,2015(3): [1]. [J].,2015 [10],. [J]., (12): (9): [2]. [J].,2007(5): [3]. [J].,2014(9): [4],. : [J]. [12]. [J].,2014(11):21. :,1988(4):71. [13]. [D]. :, [5]. : [J]., [11],. [J].,2012(2): :10-18.

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; ; [3] 3 [4] ( ) 3.1 ; ( ) ; ; [2] [5] 106 33 6 2017 6 Vol.33No.6 Jun.2017 DOI:10.13652/j.issn.1003-5788.2017.06.021 Theapplicationoffolkartemotionalelementsin traditionalfoodpackagingdesign LI Li ( 410114) (Schoolof Artand DesignChangsha Universityof