荞麦源降血糖糖肽结构表征及酶抑制动力学  

Structural Characterization and Enzyme Inhibition Kinetics of Hypoglycemic Glycopeptides Derived from Buckwheat

作　　者：周柳莎 郦萍[1] 顾双[1] 俞瑜媛 胡香莲 施永清[1] Zhou Liusha;Li Ping;Gu Shuang;Yu Yuyuan;Hu Xianglian;Shi Yongqing(College of Food and Biological Engineering,Zhejiang Gongshang University,Hangzhou 310018)

机构地区：[1]浙江工商大学食品与生物工程学院,杭州310018

出　　处：《中国食品学报》2024年第3期86-98,共13页Journal of Chinese Institute Of Food Science and Technology

摘　　要：为实现荞麦蛋白资源高值化利用,从6个菌株中筛选出植物乳杆菌和嗜热链球菌作为发酵菌株,制备α-葡萄糖苷酶抑制糖肽(α-GIG)。采用单因素实验确定优化中心点,然后做响应面试验优化发酵工艺。通过Sephadex G-25和反向高效液相色谱法(RP-HPLC)对发酵液进行分离纯化,制备α-GIG。采用β-消除反应方法确定α-GIG中糖肽键类型,红外光谱扫描法测定二级结构。采用Lineweaver-Burk作图法探究酶抑制动力学,确定α-GIG对α-葡萄糖苷酶的抑制作用类型。结果表明:在料液比1∶14.8、pH 7.5、接种量2%、发酵2.8 d条件下,发酵产物对α-葡萄糖苷酶抑制率达70.83%。发酵液经Sephadex G-25分离得到I1,I2,I33个组分,其中I2对α-葡萄糖苷酶抑制作用较强,IC50达1.72 mg/mL。经RP-HPLC进一步分离纯化I2,得到α-GIG,其纯度为94.17%。红外光谱扫描确定α-GIG的二级结构为:β-折叠占70.51%,α-螺旋占18.95%,β-转角占10.54%。α-GIG中糖肽键类型为O-糖肽键。酶抑制动力学研究表明,α-GIG对α-葡萄糖苷酶的抑制作用类型为混合型非竞争性抑制。α-GIG具有开发为天然α-葡萄糖苷酶抑制剂的潜力。To realize the high-value utilization of buckwheat protein resources,α-glucosidase-inhibiting glycopeptide(α-GIG)was prepared by fermentation of Lactobacillus plantarum and Streptococcus thermophilus which were selected from 6 strains.One-factor-at-a-time method aimed to determine the level of experimental factors and a further response surface experiment was used to optimize fermentation.The fermentation broth was separated and purified by Sephadex G-25 and reversed phase-high performance liquid chromatography(RP-HPLC)to prepareα-GIG.The method ofβ-elimination reaction was used to determine the type of glycopeptide bond and infrared spectroscopy was used to analyze the secondary structure.The inhibitory effects ofα-GIG onα-glucosidase was investigated by enzyme kinetics.The results showed that when the inoculum was 2%,pH was 7.5,solid-liquid ratio was 1∶14.8 and fermentation time was 2.8 d,theα-glucosidase inhibition rate was 70.83%.I1,I2,I3 were separated from Sephadex G-25,and I2 with the best enzyme inhibitory effect whose IC50 is 1.72 mg/mL.I2 was further separated and purified by RP-HPLC to obtainα-GIG with a purity of 94.17%.Fourier transform infrared spectroscopy suggest that the secondary structure composition was estimated as 70.51%β-folding,18.95%α-helices and 10.54%β-corner.β-elimination reaction demonstrated the existence of O-glycosidic linkage inα-GIG.The results of the enzyme inhibition kinetics study showed thatα-GIG exhibited a mixed-type noncompetitive inhibition ofα-glucosidase.Therefore,α-GIG had the potential to be developed as a naturalα-glucosidase inhibitor.

关 键 词：荞麦 Α-葡萄糖苷酶 响应面 结构表征 抑制动力学 

分 类 号：TS210.1[轻工技术与工程—粮食、油脂及植物蛋白工程]