Isolation And Purification Of α-glucosidase Inhibitory Peptides From Hot-pressed Peanut Meal Protein Hydrolysates

Type 2 diabetes is a common disease that endangers human health.At present,the commonly used strategy for the treatment of type 2 diabetes is to inhibit the activity ofα-glucosidase.However,the clinical inhibitors are generally chemical compounds,which are accompanied by a series of side effects,so the development of natural compounds with the activity ofα-glucosidase is of great significance.High temperature peanut meal is a by-product of deep processing of peanut oil.It is a cheap protein resource with rich protein content,and the content of arginine and hydrophobic amino acid in protein is high.The enzymatic hydrolysis product of peanut meal has potentialα-glucosidase inhibitory activity.Therefore,in this study,α-glucosidase inhibitory peptide was prepared by enzymolysis using high-temperature peanut meal protein as raw material.The influence of enzyme type on inhibitory activity was studied,and the optimal hydrolysis method was screened.Theα-glucosidase inhibition rate was used as tracking index to determine the method of separation and purification ofα-glucosidase inhibition peptide,and the primary structure ofα-glucosidase inhibition peptide was determined by nano liquid chromatography-tandem mass spectrometry.The interaction betweenα-glucosidase inhibitory peptides andα-glucosidase molecules was studied by molecular docking technique,and high activity peptides were further screened.The effects ofα-glucosidase inhibitory peptides on the structure ofα-glucosidase were studied by fluorescence spectroscopy to explore the inhibitory molecular mechanism.The main research results are as follows:（1）Different peptides were screened usingα-glucoside inhibition rate and hydrolysis degree as tracking indexes.The hydrolysis product（PMH-NA）obtained from the double enzyme hydrolysis of neutral protease and alkaline protease had the highestα-Glu inhibition rate（46.62±1.55%）,followed by PMH-Neu（43.61±1.25%）.PMH-Alc（35.41±2.00%）,PMH-Pro（34.77±1.80%）,PMH-Fla（26.48±1.56%）and PMH-Pap（24.01±2.6%）.The IC₅₀ value of PMH-NA was 5.63±0.19 mg/m L.The protein yield and TCA-NSI of the hydrolysate were also determined.PMH-NA had the highest protein yield of 89.20±0.66%.The highest TCA-NSI value was PMH-Alc（80.06±4.54%）.Followed by PMH-Pro（69.36±2.54%）,PMH-NA（60.22±1.25%）,PMH-Neu（56.53±2.56%）,PMH-Pap（43.48±1.54%）and PMH-Fla（43.41±1.57%）.It was found that PMH-NA had the best chemical properties.Therefore,neutral protease and alkaline protease were selected as the optimal method to prepareα-glucosidase inhibitory peptide.（3）A total of 643 peptides were detected from C-Ⅰcomponent,among which 23 peptides had potentialα-glucosidase inhibitory activity.After molecular docking,four peptides（FYNPAAGR,PGVLPVAS,FFVPPSQQ and FSYNPQAG）that can bind toα-glucosidase closely and have inhibitory effect were obtained.The binding energies were-8.1 kcal/mol,-8.6kcal/mol,-8.0 kcal/mol and-7.7 kcal/mol,respectively.The IC50 values of the four peptides were 235.5μM,1780.5μM,1307.3μM and 1603.1μM,respectively.FYNPAAGR exhibitedα-glucosidase inhibitory activity about 1.1 times that of acarbose.（4）FYNPAAGR can quench the endogenous fluorescence ofα-glucosidase but has no effect on the conformation of the enzyme.The K_q values of FYNPAAGR at different temperatures were 10¹² orders of magnitude,much larger than the maximum dynamic quenching constant(2×10¹⁰M^-1s^-1),indicating that static quenching was the main mechanism of the interaction between FYNPAAGR andα-glucosidase.TheΔH andΔS values of FYNPAAGR combined withα-glucosidase were higher than 0,indicating that the hydrophobic interaction was the main interaction force.FYNPAAGR was found to be a reversible competitive inhibitor of alpha-glucosidase by mapping the enzymatic reaction and the Lineweaver-Burk double reciprocal equation.The calculated inhibition constant K_i=0.16.