Study On Purification Of Camellia Saponin By Mixed Microbial Fermentation

Camellia seed wastewater is the residual liquid after extracting oil from Camellia oleifera Abel seeds by way of aqueous-enzyme method. The camellia seed wastewater contains a lot of nutrients, especially Camellia Saponin. But by direct extraction, what Camellia Saponin we get are of darker color and lower purity. This article focuses on the purification technique by microbial fermentation of Camellia Saponin via using camellia seed wastewater by way of aqueous-enzyme method as raw materials and then bleached by macroporous resin. The main experimental procedures and results are as follows:1. Main components analysis of camellia seed wastewaterAfter testing the principle composition of camellia seed wastewater, the results show that camellia seed wastewater contains camellia saponin7%～8%,crude protein (desiccate)4%～6%, total water soluble sugars about0.7%and crude fat about0.7%.2. Microbial strain scelectionOne strain of Aspergillus oryzae, Aspergillus niger, Penicillium glaucum, Mucor mucedo, Rhizopus, Bacillus subtilis and Bacillus licheniformis respectively and some strains of Yeasts are selected when doing this camellia seed wastewater Plate Count Agar study. Through screening by comparing growth conditions on the Camellia seed wastewater Plate Count Agar and rescreening after single strain fermentation, the single stain is selected. The results tell that what cause little change in the content of camellia saponin as well as content of crude protein and the total water soluble sugars to decease in Camellia seed wastewater are Aspergillus oryzae, Aspergillus niger, Penicillium glaucum, Candida rugosa and candida tropicalis.The selected five strains:Aspergillus oryzae, Aspergillus niger, Penicillium glaucum,. Candida rugosa and candida tropicalis, are used’for various combination fermentation experiments. The experimental results show that the optimal fermentation combination is Candida rugosa and Aspergillus oryzae for it results in the nearly half reduction of crude protein and total water soluble sugars, which is more effective. Therefore, the best inoculation proportion of mixed bacteria is obvious, which is Candida rugosa and Aspergillus oryzae.3. Optimization of culture conditions of mixed strainsThe best culture condition of mixed bacteria is confirmed by camellia seed wastewater Plate Count Agar study. It is media amount27%, the total amount of inoculation of mixed strains8%, the best inoculation proportion2:3, the speed of constant temperature incubator150r/min, the fermentation temperature30℃and the fermentation time4d.The response surface analysis experiment at three levels of four factors is designed. Following are the conducted factors and levels:the amount of inoculation (6%,8/%,10%), the speed of constant temperature incubator (100r/min,150r/min,200r/min), the fermentation temperature(25℃,30℃,35℃), the fermentation time (3d,4d,5d). From the above study, we arrive at the conclusion that the optimal condition of the fermentation is: the total amount of inoculation of mixed stains8%, the speed of constant temperature incubator160r/min, the fermentation temperature33℃and the fermentation time4d.On this condition, the purity quotient of camellia Saponin gets improved from36.45%to55.87%and the color of cameiia saponin is changed from dark brown to pale brown by comparing with cameiia saponin by direct extraction.4. Purification and Bleaching experiments of the fermented liquidThe fermentation on Camellia seed wastewater is carried out under the optimal fermentation condition. After centrifugation and filtration, the fermented liquid is purified and bleached by macroporous resin. The experimental results show that the suitable resin to purify and bleach Camellia saponin is the density of cameiia saponin23mg/mL, the adsorption time110min; The optimal conditions for the purification and bleaching of macroporous resin is the ethanol concentration85%, the ratio of ethanol and pattern solution4:1and desorption time140min. Under such condition, the purity quotient of camellia Saponin gets improved to67.42%and the color of cameiia saponin is changed from pale brown to pale yellow.