Extraction,Antioxidant And Antifatigue Activities Of Pueraria Lobata Protein

Objective:In this study,Pueraria lobate was extracted by alkaline acid precipitation method,and then its structure,function and biological activity were investigated,so as to provide a basic theoretical basis for the development and utilization of plant protein resources.Method:1.Extraction of Pueraria lobate protein:Based on the protein extraction yield as an investigation index,the extraction process of different extraction methods of Pueraria lobate protein was determined,a single factor examination and response surface design experiments were used to determine the optimal extraction process,and the crude protein of Pueraria lobate was refined by ultrafiltration membrane separation.2.Structural properties of Pueraria lobate protein:the structural properties of Puerarin were investigated by SDS-PAGE electrophoresis,amino acid analysis,ultraviolet spectroscopy,infrared spectroscopy,and thermal stability determination.3.Functional properties of Pueraria lobate protein:the functional properties of Pueraria lobate protein including solubility,gelling properties,water holding(oil)property,emulsifying and emulsifying stability,blistering and foaming stability were investigated;4.Activity study of Pueraria lobate protein:the in vitro antioxidant activity of Pueraria lobate protein was determined by reducing power assay with hydroxyl radical scavenging,DPPH radical scavenging and ABTS~+radical scavenging assays using V_C as positive control and HepG2 cells,the antioxidant activity of Pueraria lobate protein was investigated by measuring cell viability and the contents of factors such as knot and MDA by CCK-8 assay,in vivo antioxidant activity of Pueraria lobate protein was investigated using a mouse aging model,and the relative biochemical indexes were determined and the anti fatigue activity of Pueraria lobate protein was investigated using a mouse force depletion swimming test.Result:1.Pueraria lobate protein was extracted by alkaline acid precipitation method and the optimal extraction process parameters were obtained through single factor investigation and response surface design experiments as follows:extraction temperature of 45°C,extraction time of 2h,feed to liquid ratio of 1:20(g/m L),alkaline extraction pH of 10,Pueraria lobate protein extraction yield of 11.73%and puerarin crude protein content of 60.18%;the refinement process was studied as follows:Pueraria lobate protein was dissolved by the graded water-soluble method twice.The white content was 68.09%;the membrane separation process parameters were as follows:the sample concentration was 10 mg/ml,a 10 kDa ultrafiltration membrane was used,and the protein content was 77.20%after separation by centrifugation at 4000 r/min for 8 min.2.The results of structural property study showed that Pueraria lobate protein has isoelectric point of pH=3.5 and solubility of 84.96%,and puerarin contains 17 amino acids,and amino acids composition is balanced.The results of SDS polyacrylamide gel electrophoresis showed that the subunits of Pueraria lobate protein were mainly distributed in the 10.0-15.0 kDa,15.0-25.0 kDa,35-40 kDa,55.0-70.0 kDa intervals,and the 15.0-25.0.The minimum gelation concentration(LGC)of Pueraria lobate protein was 12%;denaturation temperature was 82.5°C,△H=30.45 J/g;More total sulfhydryl group in Pueraria lobate protein was(81.37±1.83)μM/g,and disulfide bond content was(13.36±0.16)μM/g.3.Functional characterization the results of this study showed that pH,temperature and ionic strength significantly affected the functional properties of Pueraria lobate protein such as water holding(oil)property,emulsifying and emulsifying stability,blistering and foaming stability.4.Bioactivity studies showed that the free radical scavenging and total reducing capacity of Pueraria lobate protein became stronger with the increasing concentration,and exhibited an obvious dose-effect relationship.Compared with the hydrogen peroxide injury model group,Pueraria lobate protein was able to enhance the viability of HepG2 cells,improve the viability of T-AOC,and reduce the content of MDA.Compared with the D-galactose-induced senescence mouse model,Pueraria lobate protein administration group could have Effectively reduced the total carbonyl and MDA levels in serum and liver tissue of mice,while it could elevate the GSH-PX viability and SOD viability in serum and liver tissue of mice.Anti fatigue assay results showed that Pueraria lobate protein could prolong the swimming time of mice,enhance the viability of lactate dehydrogenase,reduce the serum urea nitrogen content,improve the liver glycogen content,and have obvious anti fatigue activity.Conclusions:Pueraria lobate protein obtained by alkaline precipitation method combined with membrane separation technique showed better structural and functional properties.In addition,Pueraria lobate protein has obvious in vivo,in vitro antioxidant activities and anti fatigue activities,and the above findings may provide a basis for the study of Pueraria lobate protein and its further product development.