33 6 2017 6 Vol.33,No.6 Jun.2017 DOI:10.13652/j.issn.1003-5788.2017.06.030 Preparation,separationandaminoacidsequenceanalysisofcorn peptideswithhypoglycemicactivity 1,2 1 1 1 3 3 HU Yu-hang 1,2 DAI Jun 1 CHEN Shang-wei 1 ZHU Song 1 ZHANG Xue-jun 3 ZHANG Ping 3 (1., 2141222., 214122 3., 274108) (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. 87.27% Keywords:Cornpeptidehydrolysishypoglycemicactivityamino 3,CP1 HepG2 acidsequence, α- (34.64%)CP1 RP HPLC, 2012 14, CP1-13 α-,, 39.50% UPLC Q TOF MS/MS, [1],2016 2.5 10 8 t, 2016 A-P-A-L-L-P-F 2.18 10 8 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 ),,, E-mail:daihplc@163.com :2017 04 26,,, [3],,,, :1, 2,, Luis Mojica [4] 147
2017 6 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], 1.2.1 (glucagon), MCP-1 α- ( -1), PYY( ), (0.22μm) Taisuke Mochida [8] GLP-1 1.2.2 GPL-1, (1) :GB5009.3 2010 NoriyukiHiguchi [9] (2) :GB5009.4 2010 GK, (3) :GB5009.5 2010 GLP-1GIP,, (5) :100g,80, 1000mL,90 1h, 65,pH 5.5,, 0.5g/100g 65,, 2h,4000r/min 10 min,, 2,, 1 1.1 1.1.1 (CGM): 68.27%, ( ) α- :4000U/g, 60 3 (Alcalase):2.4L(2.4 AU/g),, 1 [10] [11]16 (QB/T4707 2014) 767 P-2Gel:, HepG2: CtccbioScience FBS DMEM:, Hyclone DMSO:, α- :100UN, ( ) TFA:, 1 2.5 2.0 1 15 NaOH HCl NaCO3 :, 2 3 3.0 3.5 2.5 3.0 1 20 1 25 1.1.2 :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 6.5 148 (4) :GB/T14772 2008 1.2.3 CGM, 80 20 min,,, 90 20 min,, : ph [E]/[S], (DH ) [10] ph 8.5, 1 Table1 Designfactorsoftheorthogonaltestabout enzymaticprocess A /% B /h C (g/ml) 1.2.5 180D
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 1 3.0 1.0 30 2 3.5 1.5 45 3 4.0 2.0 60 1.2.6 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 3 3 200~400 nm :BEH C18 (2.1 mm 150 mm, 1.7μm) :45, :0.3 ml/min : 1.2.7 1mg/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 7.80 1.35 67.27 5.52 1000r/min 5min,, 4 3.41 10 5 cels/ml,100μl/ 96, 37,5% 0.74 81.25 3.28 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 1.2.9 RP HPLC,37 TOF MS ESI+ 3.5kV 30 V 100 : 400, 700L/h, 50L/h 6/20V 20~ 2 2.1 3, α- 81.25%( ),, 1.2.8 (RP-HPLC) 2.3 (RP 5, : [15] HPLC) Waters2545 >ph> Z, 149
2017 6 4, Table4 Resultsandanalysisoforthogonaltestabout 37.10% enzymaticprocess 2.4 A B C DH/% /%, 1 3, 1 1 1 3 23.86 1.57 CP1,CP2,CP3 3 2 1 2 1 25.44 1.86, 3 1 3 2 25.26 1.71 4 2 1 1 25.44 2.20 5 2 2 2 26.14 1.92 6 2 3 3 25.97 2.08 7 3 1 2 27.02 1.87 8 3 2 3 26.32 1.98 9 3 3 1 27.19 2.08 k1 24.85 25.44 26.02 DH k2 25.85 25.97 26.14 k3 26.84 26.14 25.38 R 1.99 0.70 0.76 k1 1.71 1.88 2.05 k2 2.06 1.92 1.84 k3 1.98 1.96 1.87 R 0.35 0.08 0.21 5, CP1 Table5 Resultsandanalysisoforthogonaltest aboutenzymaticprocess, MTT, HepG2 ( A B C X /% Y /% Z 1 1 1 1 86.78 71.36 0.79 2 2 2 2 77.86 87.27 0.83 3 3 3 3 68.84 91.55 0.80 4 1 2 3 78.51 83.45 0.81 5 2 3 1 69.54 90.36 0.80 6 3 1 2 75.79 78.82 0.77 7 1 3 2 72.23 78.27 0.75 8 2 1 3 78.92 78.82 0.79 9 3 2 1 71.68 82.50 0.77 k1 2.38 2.42 2.28 k2 2.26 2.28 2.26 k3 2.16 2.11 2.26 R 0.21 0.31 0.02 k1 2.33 2.29 2.44 k2 2.56 2.53 2.44 k3 2.53 2.60 2.54 R 0.23 0.31 0.10 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] 150 1 Figure1 SeparationofcornpeptidesbyPolyacrylamide gelcolumn 2.5 6, MTT GC/OD, ), LIJiang-tao [16] α-,3 CP1 34.59% 2.6 (Bio-GelP-2Gel) CP1 RP HPLC, 2 14,α- 3, CP1-13 39.5%, 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]
33 6 : 6 Table6 Thehypoglycemicactivityinvitroofthecornpeptides(X±S,n=3) / GC/ (mg ml -1 ) (mmol L -1 ) OD (A490) GC/OD/ (mmol L -1 ) α- /% 1.00 3.79±0.53 0.46±0.02 * * 8.25±1.15 0.10 3.59±0.05 0.49±0.04 * * 7.41±0.12 28.75±1.63 0.01 3.43±0.09 0.52±0.04 6.56±0.17 1.00 4.60±0.70 0.46±0.03 * * 9.33±1.42 CP1 0.10 3.90±0.32 0.47±0.01 * * 7.73±0.62 34.59±1.68 0.01 3.48±0.11 0.49±0.02 * * 6.84±0.21 1.00 2.55±0.34 0.49±0.01 * * 5.53±0.74 CP2 0.10 3.32±0.33 * 0.49±0.04 * 6.97±0.69 22.80±1.60 0.01 4.24±0.38 0.50±0.01 8.30±0.74 1.00 2.88±0.82 0.46±0.02 * * 6.25±0.82 CP3 0.10 2.71±0.17 0.48±0.03 * * 5.73±0.35 17.64±1.47 0.01 2.44±0.14 0.51±0.03 5.03±0.28 6.30±0.99 * 0.52±0.02 11.84±1.47 CK 3.75±0.59 0.54±0.02 6.87±1.08 GC,OD MTT OD,GC/OD *,P< 0.05**,P<0.01 2 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
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