Extraction, Separation And Enzymatic Hydrolysis Of Soyasaponins

In order to increase physiological acticity of soybean saponin, the extraction, separation and enzyme hydrolysis of soybean saponin were studied. The enzyme used to hydrolyze soybean saponin was produced from microbial strains. The purification and characteriazation of saponin enzyme was studied also. And the behavior of soybean saponin in the traditional soybean food fermentation was discussed.AB-8 resin column was used to extract soybean saponin. The optimum condition of extraction was that the concentration of ethanol used for soaking saponin and eluting saponin from column was 40% and 50% respectively, and the flow speed was 6.3 x 10~3nrmin"1. The content of protein and sugar in eluant was determined through Folin-Phenol and 3,5-dinitro-salicylic acid methods respectively. The adsorption capacity of AB-8 resin was 103.2mg-g", and the extract coefficient of soybean saponin was 90%. The method was convenient and useful.The soybean saponin was separated through silica column chromatography. Vacuum pump was used to raise pressure on the column, so the separation was fast and efficacious. In this method, the silica was used as station phase, and the solution of chloroform: methanol=9:l, 8.5:1.5, 8:2 and 7.5:2.5 was used as eluant in proper order. In the result, the soybean saponin that Rf was 0.46 was obtained (Developing solvent was chloroform: methanol: water=36:25:6), which was determined by HPLC-MS. It was proved that the soybean saponin was group B saponin, that consists of soyasaponin I , II ,111, IV and V.The Aspergillus sp.48s, sp.848s and sp.39s strains of 8 type strains highly produced the enzyme which hydrolyzed soybean saponin to lower sugar saponin. The optimal time and pH was 12h and 5 respectively in enzyme hydrolysis. The new saponins that molecular weight was 668 and 780 were obtained, they were the product of group B saponin hydrolyzed by enzyme form sp.48s. Their structure is3-O-[p-D-glucuronopyranosyl] soyasapogenol B with 2 molecular water and 3-O-[a-L-rhamnopyranosyl ( 1 -2 ) -p-D-glucuronopyranosyl]soyasapogenol B respectively. The optimal enzyme production was obtained after fermented 48h for sp.48s and sp.848s and 36h for sp.39s. The enzyme was purified by the method of gradient protein chromatography of middle pressure with the Biologic Chromatography System. The molecular weight was 34,000 for the sp.48s strain, 31,000 for sp.848s and 50,000 for sp.39s strain. As for the p-nitrophenyl- -glucosidase produced from sp.48s, sp.848s and sp.39s, the optimal pH was 5 and the optimal temperature was 40 C.The component and content of soybean saponin was changed in the process of miso fermentation. On the one hand, the content of saponin was increased firstly, and decreased at regular time, which could help people control the ferment time and obtain miso production that consists of more soybean saponin. On the other hand, the enzyme in Koji could hydrolyze soybean saponin to lower sugar saponin that had highly physiological activity. And the new saponin in miso Koji was the same as the lower sugar saponin produced from hydrolysis of saponin. So the mechanism was the same in the two processes.