Extraction And Isolation Of Antioxidant Peptides From Pueraria Lobata And Their Protective Effects On HepG2 Cells From Oxidative Damage

Objective:The preparation process of pueraria antioxidant peptide was improved,and its physicochemical properties and functional characteristics were analyzed using various modern analytical methods.The antioxidant activity of pueraria antioxidant peptide was studied from both chemical and cellular antioxidant aspects,laying a theoretical foundation for the research and development of pueraria polypeptide in health products and functional foods.Methods:1.Extraction and separation of antioxidant peptides from Pueraria lobata:Select multiple inspection indicators and screen 5 proteolytic enzymes to obtain the most suitable protease for protease hydrolysis of Pueraria lobata;Using the degree of hydrolysis（DH）and DPPH free radical scavenging rate as indicators,a single factor combined with response surface orthogonal experiment was used to optimize the protease hydrolysis process of Pueraria lobata;The obtained protease hydrolysate of Pueraria lobata was separated by ultrafiltration and components with a molecular weight of less than 5 k Da were collected.2.Functional characterisation of Pueraria lobata antioxidant peptides:functional characterisation of Pueraria lobata antioxidant peptides by measuring solubility,minimum gelling concentration,water holding capacity,oil holding capacity,emulsification and emulsion stability.3.Study on the structure and properties of antioxidant peptides from Pueraria lobata:The molecular weight of antioxidant peptides from Pueraria lobata was determined by polypropylene gel electrophoresis and size array chromatography,and the composition and content of amino acid components were analyzed;UV spectrophotometry,Fourier transform infrared spectroscopy,circular dichroism spectroscopy,fluorescence spectroscopy,and thermal stability analysis were used to study its spatial structure and properties.4.Study on the antioxidant activity of pueraria antioxidant peptides:The antioxidant activity of pueraria antioxidant peptides was studied using two evaluation methods:chemical antioxidant and cellular antioxidant;Chemical oxidation resistance mainly includes the determination of free radical scavenging ability and reducing ability;Cell antioxidation mainly involves the study of the repair effect of puerarin antioxidant peptides on H₂O₂induced oxidative damage models in Hep G2 cells,such as the measurement of cell apoptosis,mitochondrial membrane potential,lipid peroxidation,and related oxidation indicators.Results:1.By analyzing the results of various screening indicators,it was finally determined that alkaline protease was the most suitable protease for the hydrolysis of Pueraria lobata.According to the single factor combined response surface orthogonal experiment,the optimal enzymolysis process was determined as follows:substrate concentration 2%,enzymolysis ratio 2%,enzymolysis time 3 h,enzymolysis temperature 55℃,enzymolysis p H=9.The experimental verification showed that the process was stable and reliable.The molecular weight of pueraria antioxidant peptides obtained from the prepared pueraria protease hydrolysate after ultrafiltration separation significantly decreased,indicating that the separation and purification process is feasible.2.The results of the functional properties study showed that the functional properties of Pueraria Mirifica antioxidant peptides were improved compared to Pueraria Mirifica proteins,especially the solubility,which changed more significantly,significantly improving their application in functional foods.3.Structural properties showed that the molecular weight distribution of Pueraria lobata antioxidant peptides was mainly in the range of 1000Da-3000Da,with higher nutrient content;the spatial secondary structure was mainly irregularly curled;the relative fluorescence intensity increased after enzymatic digestion compared to protein,indicating higher hydrophobicity;the thermal transformation temperature decreased to 79.6℃.4.The results of chemical oxidation resistance showed that puerarin antioxidant peptides had a good ability to scavenge free radicals in vitro and a good reduction ability.Their scavenging rates and reduction abilities for DPPH,ABTS⁺,superoxide anion,and hydroxyl free radicals were 89.31%,93.14%,58.29%,85.74%,and 2.191,respectively;The results of cell antioxidation showed that puerarin antioxidant peptides had a certain repair effect on H₂O₂induced oxidative stress models,which could reduce the rate of apoptosis,reduce the production of ROS,inhibit lipid peroxidation,and improve the activity of antioxidant enzymes.Conclusion:This experiment used a single factor combined with response surface orthogonal experiment to determine the optimal process for protease hydrolysis of Pueraria lobata.The enzymatic hydrolysates obtained under this process were separated and purified to obtain Pueraria lobata antioxidant peptides,and their physicochemical properties,functional characteristics,and antioxidant activity were further studied.Through research,it has been found that kudzu root antioxidant peptides have higher solubility and smaller molecular weight distribution,which can improve the absorption and utilization rate of protein in the human body and have more nutritional value.In addition,they also have good antioxidant activity and have a certain development potential in preventing and treating diseases caused by oxidative stress,with good development prospects.