紫菜酶解产物锌螯合-α-葡萄糖苷酶抑制剂活性肽的研究
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摘要
研究紫菜酶解α-葡萄糖苷酶抑制活性肽与锌螯合反应的条件,并对锌-螯合-糖苷酶抑制剂活性肽的胃肠消化稳定性进行评价。对条斑紫菜在一定条件下进行内切与外切蛋白酶的复合酶解获得具备α-葡萄糖苷酶高抑制活性的多肽,采用超滤纳滤双膜组合分离后旋转蒸发浓缩冻干,获得的多肽质量浓度为1 mg/mL时对0.5 mg/mL的α-葡萄糖苷酶抑制率达68%;利用α-葡萄糖苷酶抑制活性肽与锌溶液反应进行螯合条件优化,螯合的最佳条件为:时间1.5 h,pH4.5,温度37℃,质量浓度6 mg/mL,得到的锌-螯合-糖苷酶抑制剂活性肽溶液螯合度为25.6%,螯合后对α-葡萄糖苷酶抑制活性提高到79.8%;建立体外胃肠消化模型,以α-葡萄糖苷酶抑制活性为指标,评价制备的锌-螯合-糖苷酶抑制剂活性肽的胃肠消化耐受性,结果表明:经过不同酶与底物比、时间胃消化后α-葡萄糖苷酶抑制活性下降均在7%左右,十二指肠消化后,抑制活性下降均在5%以内,具有良好的胃肠消化稳定活性。
In order to study the conditions of chelating Znic with α-glucosidase inhibitory active peptides from Porphyra yezoensis enzymatic hydrolysis and evaluate the stability of gastrointestinal digestion The polypeptide,inhibiting the α-glucosidase activity highly,would be gained by the compound enzymolysis technics with using protease of ex-and in-under various treatment factors.Thereafter,the hydrolysate was separated by filtering membrane technique combined ultrafiltration with nanofiltration firstly,and then concentration through rotary evaporator,which the inhibition ratio of α-glucosidase(0.5 mg/mL) could be reached 68% at 1 mg/mL of concentrated polypeptide solution;The optimization factors of chelating reaction involved polypeptide and zinc solution were1.5 h of reaction time,37 ℃ of temperature,6 mg/ml of polypeptide and pH 4.5 respectively,which the chelating degree of polypeptide and zinc had reached 25.6%, and the inhibition for α-glucosidase had increased to 79.8% after chelation;In addition,the model of gastrointestinal digestion in vitro would be built to evaluate the toleration of the chelate for gastrointestinal digestion by assessing their capacity to inhibit the α-glucosidase activity. The results indicated that the decrease of α-glucosidase activity only had 7% after gastrointestinal digestion under various reaction time and enzyme-substrate ratio;and then reaching 5% approximately after duodenal digestion. Therefore,the chelate of polypeptide and zinc solution was shown better stability of gastrointestinal digestion in this study.
In order to study the conditions of chelating Znic with α-glucosidase inhibitory active peptides from Porphyra yezoensis enzymatic hydrolysis and evaluate the stability of gastrointestinal digestion The polypeptide,inhibiting the α-glucosidase activity highly,would be gained by the compound enzymolysis technics with using protease of ex-and in-under various treatment factors.Thereafter,the hydrolysate was separated by filtering membrane technique combined ultrafiltration with nanofiltration firstly,and then concentration through rotary evaporator,which the inhibition ratio of α-glucosidase(0.5 mg/mL) could be reached 68% at 1 mg/mL of concentrated polypeptide solution;The optimization factors of chelating reaction involved polypeptide and zinc solution were1.5 h of reaction time,37 ℃ of temperature,6 mg/ml of polypeptide and pH 4.5 respectively,which the chelating degree of polypeptide and zinc had reached 25.6%, and the inhibition for α-glucosidase had increased to 79.8% after chelation;In addition,the model of gastrointestinal digestion in vitro would be built to evaluate the toleration of the chelate for gastrointestinal digestion by assessing their capacity to inhibit the α-glucosidase activity. The results indicated that the decrease of α-glucosidase activity only had 7% after gastrointestinal digestion under various reaction time and enzyme-substrate ratio;and then reaching 5% approximately after duodenal digestion. Therefore,the chelate of polypeptide and zinc solution was shown better stability of gastrointestinal digestion in this study.
引文
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[18]LIU Shanshan,AO Jing,XIE Ningning,et al.Study on the stability of casein antioxidant peptides during simulated gastrointestinal digestion[J].Journal of Chinese Institute of Food Science and Technology,2014,14(2):47-54.(in Chinese)
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[2]ZHOU Feng,LIU Dongdong,ZHOU Nandi,et al.Purification and characterization ofα-glucosidase inhibitor from proteolytic product of porphyra yezoensis[J].Natural Products Research and Development,2014,26(9):1463-1468.(in Chinese)
[3]WANG Liming,XIA Wenshui.Research on the mechanism of tea polysaccharide reducing blood glucose in vitro[J].Journal of Food Science and Biotechnology,2010,29(3):354-358.(in Chinese)
[4]尹尚军,斯越秀,钱国英.锌离子作为α-葡萄糖苷酶抑制剂的应用及其测定方法:中国,201210376435.7[P].2013-01-09.
[5]BUGLYO P,NAGY E M,FARKAS E,et al.New insights into the metal ion-peptide hydroxamate interactions:Metal complexes of primary hydroxamic acid derivatives of common dipeptides in aqueous solution[J].Polyhedron,2007,26(8):1625-1633.
[6]TORREGGIANI A,FINI G,BOTTURA G.Effect of transition metal binding on the tautomeric equilibrium of the carnosine imidazolic ring[J].Journal of Molecular Structure,2001,s 565-566(2):341-346.
[7]LIU Wen,LOU Qiaoming,YANG Wenge.Research progress of peptide-metal element chelate[J].Food and Fermentation Industries,2014,40(4):142-146.(in Chinese)
[8]YANG Jie,ZHAO Honglei,XU Shufen,et al.Antioxidant and zinc supplementation effect of zinc chelating small peptides from fish protein in mice[J].Journal of Huazhong Agricultural University,2011,30(4):516-520.(in Chinese)
[9]MEI Liping,SUN Xinda,QI Juanrei,et al.The significance of the trace elements in patients with diabetes and diadetic nephropathy[J].Chinese Journal of Healthy Laboratory Technology,2011,21(5):1198-1200.(in Chinese)
[10]PAULE S G,NIKOLOVSKI B,LUDEMAN J,et al.Ability of GHTD-amide and analogs to enhance insulin activity through zinc chelation and dispersal of insulin oligomers[J].Peptides,2009,30(6):1088-1097.
[11]GAO Suyun,PAN Sizhi,GUO Kangquan.The synthesis and preparation of Soybean hydrolysate separation protein chelating Zn(Ⅱ)-peptide[J].Food Science,2003,24(10):117-120.(in Chinese)
[12]XU Qingling,ZENG Qingzhu,YAN Lei,et al.Preparation and biological activities of Zn(II)-tilapia peptide complexes[J].Food Science,2003,24(10):117-120.(in Chinese)
[13]DU Fen,Hou Hu,ZHAO Yuran,et al.Digestibility of alaska pollock-derived metal chelating peptide in vitro gastrointestinal digestion[J].Modern Food Science and Technology,2016(7).(in Chinese)
[14]CRUZ-HUERTA E,GARCIA-NEBOT M J,MIRALLES B,et al.Caseinophosphopeptides released after tryptic hydrolysis versus simulated gastrointestinal digestion of a casein-derived by-product[J].Food Chemistry,2015,168:648-655.
[15]LIU Wenhan,WU Xiaoqiong,CHEN Dan,et al.Inderect study of zinc-cystine complex reaction by FAAS[J].Spectroscopy and Spectral Analysis,2008,28(1):209-213.(in Chinese)
[16]ZHANG Lixin,XUE Feng,GAI Ruiying.Screeningforα-glucosidase inhibitors from marine invertebrates extracts[J].Chinese Journal of Marine Drugs,2008,27(5):11-14.(in Chinese)
[17]杨杨.高剂量补锌导致海马锌含量减少,BDNF缺失和学习记忆障碍[D].武汉:华中科技大学,2013.
[18]LIU Shanshan,AO Jing,XIE Ningning,et al.Study on the stability of casein antioxidant peptides during simulated gastrointestinal digestion[J].Journal of Chinese Institute of Food Science and Technology,2014,14(2):47-54.(in Chinese)
[19]CHEN Chen,CHI Yujie,ZHAO Mingyang,et al.The inhibitory effect of egg white-derived peptides on the activity of angiotensin converting enzyme and their stability during simulated gastrointestinal digestion in vitro[J].Acta Nutrimenta Sinic,2012,34(3):274-277.(in Chinese)