Separation, Purification And Functional Activities Of Active Proteins From The Fermentation Products Of Armillaria Mellea

Corn gluten meal is a by-product of the starch processing industry.It mainly contains more than 60%zein and about 20%gluten and microglobulins.Due to its lack of tryptophan and other essential human,it is limited in the field of food processing.application.Therefore,many scholars have done a lot of research on the deep processing of corn protein powder,including the extraction of corn yellow pigment,enzymatic hydrolysis of corn active peptides and so on.This research group has been devoted to the systematic research on the modification of corn gluten by large fungi for many years.The results of the previous research indicate that the zein protein powder is subjected to fungal fermentation,and the protein in the fermentation product has certain functional activity.Therefore,based on the results of previous studies in this study,we selected Me-01,He-02,and Am-07,three large fungi,for solid-state fermentation.The strain Am-07,which has high protein conversion ability,was selected as the index of total protease activity and protein conversion rate,and a series of research work on protein composition analysis,separation and purification and bioactivity evaluation in Am-07 transformation products was carried out.The experimental data and the corresponding conclusions have laid the necessary theoretical basis for the in-depth study of the biotransformation,functional activity and functional food development of zein.1.Screening of different strains and fermentation methods using protease activity and protein conversion rate as indicatorsThrough the determination of the total protease activity of different fermentation modes of Me-01,He-02,and Am-07,it was determined that the fermentation mode of the three strains was solid state fermentation;and by screening the strains based on the protein conversion rate,it was determined Am-07 had better protein conversion,so the follow-up trial selected Am-07 for solid state fermentation tests.2.Analysis of protein components in Am-07 transformed zein flourThrough continuous culture of Am-07 for 20 days,the protein components in the fermentation product were determined every 24 hours.It was found that as the fermentation time increased,the protease activity and protein content of the fermentation product first increased and then decreased.Am-07 mainly produced acidic protease during the solid-state fermentation,and its total protease activity was highest at 313.13U/g on the 15th day,and its protein content reached the maximum at213.67 mg/g on the 15th day.By using different protease activities and protein content as variables and using spss19 for correlation and multiple regression analysis,a model for the relationship between protein content in different proteases and fermentation products during solid state fermentation was established.The correlation analysis found that acidic proteases,alkaline proteases,and neutral proteases have a certain correlation with the proteins in the fermentation products.The higher the protease activity,the more protein content in the fermentation products.The relationship between the protein content in the fermentation product and the protease activity was modeled by spss regression analysis:Protein content=-0.184+0.185×acid protease+0.227×neutral protease+0.089×alkaline proteaseBy comparative analysis of amino acid composition and content before and after fermentation of Am-07,it was found that the amino acid composition of the fermentation product maintained its original mode through solid state fermentation.Through comparative analysis of hydrophilic and hydrophobic amino acids,it was found that the proportion of hydrophobic aqueous amino acids after Am-07fermentation was reduced by 11.36%before fermentation.3.Separation and purification of protein in Am-07 solid state fermentation product.Separation and purification of the protein in the Am-07 solid-state fermentation zein protein powder by ammonium sulfate fractionation precipitation,Sephadex G-50chromatography,and preparative high-performance liquids,and simultaneous DPPH·and O₂^-·clearance rates.The determination of the final determination of B8components has a good antioxidant capacity.The determination of the molecular weight of the B8 fraction was found to be a single band with a molecular weight of10.2 kDa.As determined by an automatic amino acid analyzer,the B8 component protein consists of 9 amino acids.Its components are:threonine,serine,cysteine,tyrosine,phenylalanine,lysine,and histidine.,arginine,proline,these amino acids have better antioxidant effect.4.Evaluation of Antioxidant Activity of Protein in Am-07 Solid Fermentation Product.The H2O2 induced HepG2 cells were used to establish an oxidative injury model,and the protective effect of B8 component proteins on oxidative damage was verified.The MTT assay showed that the B8 component protein in the range of 12.5-50?g/mL had no significant effect on HepG2 cell viability.This concentration range was selected for subsequent experiments.Different concentrations of proteins can reduce the activity of T-SOD?total superoxide dismutase?,GSH?reduced glutathione?,and T-AOC?total antioxidant capacity?in HepG2 cells and reduce The content of LDH?lactate dehydrogenase?protects H2O2 from oxidative damage of HepG2 cells.