Study On The Fermentation Process And Biological Activity Of Lunasin Producted By Recombinant Pichia Pastoris

Lunasin is a polypeptide first isolated from soybean, contains 43 amino acids,molecular weight of 5 kDa, with antioxidant, anti-inflammatory, anti-cancer, lowering blood lipid functionIn this paper, the fermentation technology of high yield Lunasin of Pichia pastoris and the conditions of membrane separation and purification, as well as the activities of reducing blood lipid were studied. The main results are as follows(1)Study on growth characteristics of the strain. The growth curve of Pichia pastoris in YPG culture medium was determined by OD600, and the optimum time for the first stage seed solution and two stage seed solution culture was 28 h and 20 h,respectively. The expression for index determination of sorbitol concentration of 40g/L, adding 20 g/L mannitol.(2)High density fermentation process optimization.The dissolved oxygen feedback feeding strategiy and index- constant feeding two feeding strategies,dissolved oxygen feedback feeding strategy lunasin highest expression is 0.889 g / L index-constant feeding strategythe highest levels of expression of 1.805 g / L;The induction temperature was 24 C, the highest expression was 2.57 g/L;We compared the induction period of single carbon source and double carbon source feeding strategies(methanol- sorbitol and methanol- mannitol) supplement. It was found that the methanol-mannitol mixed fill materiafeeding strategy was more conducive to improve the lunasin expression and the highest expression level reached 3.02 g /L;The highest expression level of methanol compound nitrogen source was 3.67 g/L,which was 3.26 times of the original Lunasin expression.(3)Optimization of membrane separation process.The two stage membrane filtration of Vivaflow200 ultrafiltration membrane was studied to purify Lunasin and improve the recovery rate.Results showed that a membrane filtration optimal conditions feed pressure 0.2 MPa, pH 8.5, fermentation liquid temperature of 40 ℃,add 0.6 times the original volume of ultra pure water dilution and recovery rate reached 0.9412; secondary membrane filtering optimality conditions for feed pressureof 0.25 MPa, pH5.5, fermentation liquid temperature of 30 ℃.The dilution ratio was625 times and the recovery rate was 0.7895. The comprehensive recovery rate was0.7396 after two stage ultrafiltration separation.(4)Study on the activity of Lunasin in reducing blood lipid.High fat diet was fed to SD rats to build a high blood fat model, and to explore the effect of Lunasin on experimental hyperlipidemia in rats.Experimental results showed that lunasin can effectively reduce the hyperlipidemia rats serum alanine aminotransferase(ALT), total cholesterol(TC), low density lipoprotein cholesterol(LDL), the differences are significant level(P < 0.05), that lunasin can reduce serum alanine aminotransferase(ALT) 、cholesterol(TC) and low density lipoprotein(LDL)content.