Preparation,Enzymatic Hydrolysis Of Black Bean 7S Globulin And Study On Slow Digestion Of Starch

Starches and glycogen in high-carbohydrate foods can be broken down into glucose and maltose byα-amylase andα-glucosidase,resulting in increased glucose levels in the blood and affecting human health.Inhibition ofα-amylase andα-glucosidase can effectively control the rise of blood glucose.At present,controlling the inhibitory activity of starch digestive enzymes to control human blood glucose has become a research focus.Black bean protein contains a complete variety of essential amino acids and a suitable proportion of essential amino acids.Black bean protein is a kind of high-quality plant protein with good biological activity and has certain inhibitory effect onα-amylase andα-glucosidase activities.In this paper,the inhibition rate ofα-amylase andα-glucosidase was used as the index to determine the optimal extraction process of black bean 7S globulin,and the influence of black bean 7S globulin and its enzymolysis peptide on the digestion characteristics of starch was further investigated,and the active component and its amino acid sequence were determined by further separation and purification of black bean 7S pellet protease hydrolysate.It provides a new idea for the exploitation and utilization of 7S globulin resources in black bean.First,the black bean 7S globulin was prepared by alkali extraction and acid precipitation method with the inhibition rates ofα-amylase andα-glucosidase as indexes.The extraction process was optimized by single factor and response surface experiments to determine the extraction conditions with the best inhibition effect on the activity of the two enzymes.Then,the inhibitory activity stability of black bean 7S globulin onα-amylase andα-glucosidase at different temperatures and p H was investigated,and the inhibitory types of black bean 7S globulin onα-amylase andα-glucosidase were deduced by hyperbolic reciprocal method.The results showed that black bean 7S globulin had the best inhibitory activity againstα-amylase when the ratio of solid to liquid was 1:13,the extraction p H was 4.8 and the concentration of sodium chloride was 0.16 mol/L,and the inhibitory activity againstα-amylase was 70.51%.Black bean 7S globulin can maintain good enzyme inhibition activity between 20 and 60℃,which is a heat-sensitive protein.When the temperature rises to 70℃,the inhibitory activity of the two enzymes almost drops to 0.In the range of p H 3 to 9,black bean 7S globulin showed good inhibitory activity againstα-amylase andα-glucosidase,and showed good acid-base tolerance.It was found that black bean 7S globulin had a non-competitive inhibition onα-amylase with a non-competitive inhibition constant of 12.89 mg/m L and a competitive inhibition onα-glucosidase with a competitive constant of 1.00 mg/m L.Secondly,the complex enzymolysis modification of black bean 7S globulin with alkaline protease and transglutaminase was carried out to explore the effect of black bean 7S globulin and its enzymolysis peptide on the slow digestion of starch.The results showed that theα-amylase andα-glucosidase of black bean 7S ball protease were stronger than that of black bean 7S globulin after simulated digestion in vitro.This may be because the gastrointestinal environment has a certain promotion effect on the bioactive peptide segment after enzymatic hydrolysis,so that it can further react in the gastrointestinal tract without being affected,so the inhibitory activity of the two enzymes gradually increases.After adding black bean 7S globulin and its hydrolyzed peptide to wheat starch,the hydrolysis rate of starch decreased,and protease hydrolyzed peptide had greater effect on the digestibility of starch.RDS,SDS and RS changed significantly with the increase of protease lysate.When the addition of protease hydrolysis peptide increased to 30%,RDS content decreased by 12.85%,RS content increased by 17.45%,and starch hydrolysis rate decreased by 14.77%compared with blank wheat starch.The equilibrium concentration C∞and k values of wheat starch hydrolysis showed a decreasing trend after the addition of black bean 7S globulin and its hydrolyzed peptide.The predicted blood glucose decreased to 82.90%and 72.55%,respectively,when the protein and peptide were added at 30%.In addition,black bean 7S protein and its enzymolysis peptide had good inhibition onα-amylase andα-glucosidase activities,and the inhibition effect of black bean 7S protein was stronger than that of enzymolysis peptide.When the concentration of 7S globulin was 20mg/m L,the inhibition rates ofα-amylase andα-glucosidase were 69.54±2.22%and 26.23±1.09%,and the IC₅₀values were 9.84 and 38.98mg/m L,respectively.The inhibition rates ofα-amylase andα-glucoside were 46.14±2.56%and 22.89±1.01%,respectively,and the IC₅₀values were 22.41 and 46.69 mg/m L,respectively,when the concentration of protease hydrolysate of 7S pellet was 20 mg/m L.Finally,the amino acid composition of black bean 7S globulin and its hydrolyzed peptide was analyzed.The results showed that both of them contained more hydrophobic amino acids,such as leucine,isoleucine and valine,and aromatic amino acids,including tyrosine.The hydrophobicity of amino acids was related to the inhibitory activity ofα-glucosidase,and aromatic amino acids were related to the inhibitory activity ofα-amylase.Three peptides with different molecular weights were isolated by ultrafiltration,among which the peptides smaller than 3Kda showed better inhibitory activity against the two enzymes.The molecular weight distribution analysis of the peptide showed that the molecular weight was mainly concentrated below 1000Da.Then Sephadex-15 dextran gel chromatography was used to further purify and collect two components.The inhibitory activity ofα-amylase andα-glucosidase was 24.17±0.83%and 9.06±0.57%,respectively,for componentⅡwith longer retention time in the gel chromatogram column.The structure of componentⅡwas characterized and five different amino acid sequences were obtained.They were respectively TLFLPH（Thr-Leu-Phe-Leu-Pro-His）,TFPYP（Thr-Phe-Pro-Tyr-Pro）,KLGLKF（Lys-leu-gl-Leu-Lys-phe）and FKSDRIFFA（Phe-Lys-Ser-Asp-Arg-Ile-Phe-Phe-Ala）and SFDYEPFY（Ser-Phe-Asp-Tyr-Glu-Pro-Phe-Tyr）.