Structural Characterization and Enzyme Inhibition Kinetics of Hypoglycemic Glycopeptides Derived from Buckwheat
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 prepare α-GIG.The method of β-elimination re-action was used to determine the type of glycopeptide bond and infrared spectroscopy was used to analyze the secondary structure.The inhibitory effects of α-GIG on α-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,the α-glucosi-dase inhibition rate was 70.83％.I1,I2,I3 were separated from Sephadex G-25,and I2 with the best enzyme inhibito-ry effect whose IC50 is 1.72 mg/mL.I2 was further separated and purified by RP-HPLC to obtain α-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 in α-GIG.The results of the enzyme inhibition kinetics study showed that α-GIG exhibited a mixed-type non-competitive inhibition of α-glucosidase.Therefore,α-GIG had the potential to be developed as a natural α-glucosidase inhibitor.
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