Preparation Of α-Glucosidase Inhibitory Peptide From Coix Seed And Study On Its Calcium-Zinc Chelate

The most common form of diabetes in China is type 2 diabetes mellitus(T2DM),which cannot be permanently cured.The probability of T2 DM patients suffering from complications such as osteoporosis increases,and the loss of trace elements is serious.However,clinical studies have shown that α-glucosidase(AG)inhibitors such as acarbose have obvious side effects.The α-glucosidase inhibitory peptide(AGIP),which is generated from plant protein,has drawn significant study interest from academics both domestically and internationally because of its diverse origins,natural safety,and long-term utility.Coix seed is a high-quality plant protein source that can be used to make AGIP.Then AGIP chelated with calcium and zinc to create new and functional trace element supplements,laying the theoretical groundwork for expanding the application scope and increasing the added value of coix seed peptides.Response surface methodology was used in this article to optimize the most effective technology for producing crude polypeptide fermentation broth from coix seed using mixed bacteria fermentation based on a single factor experiment.The high purity AGIP was obtained by separating and purifying the coix seed crude polypeptide fermentation broth,and its inhibitory effect on α-glucosidase and stability was investigated in vitro.The antioxidant and antibacterial properties of coix seed AGIP and calcium-zinc chelate were investigated,as well as their preparation technology and structure.The following were the main conclusions:1.Bacillus subtilis and Streptococcus thermophilus were selected from seven different bacteria to co-fermenting coix seed based on parameters such as the rate of AG inhibition,the degree of hydrolysis,and crude polypeptide production.The ratio of solid to liquid of 1:15g/m L,the ratio of Bacillus subtilis to Streptococcus thermophilus of 1:1,the inoculation amount of 3%,the fermentation temperature of 37°C,the initial pH of pH6.5,and the fermentation time of 3 days were demonstrated as the best conditions for each factor.Based on these,response surface technique was applied to determine the optimal preparation circumstances of coix seed crude polypeptide fermentation broth,which were as follows: the material to liquid ratio was1:12.9 g/m L,the inoculation amount was 3.2%,the initial pH was 6.23,and the fermentation time was 3.1 days.The rate of AG inhibition under the aforementioned circumstances was71.64%.2.AGIP was purified utilizing gel filtration chromatography followed by reversed-phase high performance liquid chromatography(RP-HPLC).AGIP had a purity of up to 93.91% and a molecular mass of around 7.8 k Da.The infrared and ultraviolet spectra revealed that AGIP had the typical structure of sugars and polypeptides,with the glycopeptide being of the O-link type.Its secondary structure was made up of the following elements: β-folding accounted for21.90%,random curling accounted for 23.92%,α-helix accounted for 28.12%,and β-rotation accounted for 26.06%.The examination of the kinetics of inhibition indicated that AGIP was reversible and mix-typed inhibitor of α-glucosidase.The quenching of AGIP on α-glucosidase was demonstrated by the fluorescence spectra to be a static mechanism.The conformation ofα-glucosidase may be changed during the formation of complex between AGIP and α-glucosidase,and AGIP and α-glucosidase shared only one binding site.Analysis of stability revealed that AGIP had excellent thermal and acid-base stability.High NaCl content had a significantly impact on AG inhibitory activity,and it was enhanced by simulated gastrointestinal digestion in vitro(P < 0.05).3.Four components were separated by Sephadex G-15.Among them,A3 showed the highest the chelating capacity of calcium and zinc,which were 30.42% and 47.54% respectively.When A3 was separated using RP-HPLC,the calcium and zinc chelation rates increased to39.45% and 56.3%,correspondingly,indicating that AGIP had the highest AG inhibitory action and chelation rates with calcium and zinc ions.Using calcium and zinc chelation rates as indicators,the optimum preparation conditions of AGIP-calcium-zinc(AGIP-Ca-Zn)chelate were determined to use single factor and response surface methodology.The following chelate conditions were found to be optimal: 4.4:1 mass ratio of AGIP to Ca-Zn,1:1 mass ratio of calcium to zinc,and pH 3.7.Zinc and calcium chelation rates were 52.63% and 63.79%,respectively,under these conditions.4.The structures of chelate and AGIP were compared and evaluated using ultraviolet,infrared,fluorescence,and Zeta potential characterisation.Research showed that the principal binding sites of AGIP with calcium and zinc were amino nitrogen and carboxylic acid group,and its spatial structure was also changed,and chelates was not as stable as AGIP.The AG inhibitory activity of chelates was significantly increased(P < 0.05).Positive antibacterial activity was detected in the AGIP-Ca-Zn chelate.The chelates hindered the growth of Escherichia coli、Staphylococcus aureus、Salmonella choleraescens、Vibrio parahemolyticus,but AGIP had no antibacterial activity.Moreover,AGIP-Ca-Zn chelate had modest antioxidant activity.The DPPH radical,OH radical,and iron reducing power of AGIP-Ca-Zn chelate were significantly raised in comparison to AGIP,while the ABTS radical scavenging rate was markedly reduced(P < 0.05).