Research On Soy Isoflavones Producted By Lactococcus Lactis Fermentation

Soybean isoflavone is a biological active substance, which is extracted from the soybean.In recent years, research shows that, soybean isoflavones have health care function such as the antitumor activity, resistance to disease of heart head blood-vessel, resistance of osteoporosis and prevent the decline of immunity and so on. With the improvement of people’s living standard, soybean isoflavone as medicine or health food raw material or additives, the market demand increased year by year. People pay an attention to soybean isoflavones more and more. And soybean isoflavones became the focus of research, the development and market foreground of soybean isoflavones product is very wide. The traditional soybean isoflavone industrial was limited by low yield, the complicated methods of extraction and purification and the ripe period of soybean raw material itself. So, soybean isoflavone was obtained by the other way becoming the new direction of the research.Firstly we studied the biological characteristics of Lactococcus lactis with the soybean isoflavone synthase (IFS) gene, confirmed the growth curve of engineering strain and the change of the acidity in the fermentation process. Then we screened and optimized the culture medium, fermentating conditions and the substrate by single factor experiment and the orthogonal experiment. Finally, the genetic stability of engineering strains was preliminary studied. The main research results were as follows:The growth curve of engineering strains Lactococcus Lactis NZ3900/pNZ8149-IFS1was tested by monitoring OD600of LM17broth, the last phase of logarithm growing of strain was8-10hours.The acidity curve of engineering strains was tested, it demonstrates because of producing lactic acid in the fermentating process, lead to the medium pH value fall, dropped from the initial pH value around7to4.8or so.Using buffer solution adjust the initial pH value of medium ot ferment,which make the logarithm of bacteria growth period be prolonged and reduced the decline of the pH value due to accumulation of lactic acid, and finally only reduced0.4pH value unit.By single factor experiment and orthogonal test we optimized the culture medium components:3%lactose,2%soybean peptone,1%yeast extract powder,2%disodium phosphate,0.4%magnesium sulfate,0.1%ascorbic acid; the optimal fermentation conditions:With0.2mol/L citric acid adjusting the pH value of fermentation medium for6.5, inoculum size3%, at30℃still cultruing for4hours, then added substrate for the end concentration0.2mg/ml and10ng/ml inducers nisin, continue to cultivate with150revolutions per minute at24℃for20hours. In this condition, the output of soybean isoflavones was up to12.5mg/L.We screened the chrysanthemum as raw material which was extracted in a boiling water bath for2hours with the ratio material to liquid1:30by the single factor experiment. Extracts as substrates were used for the fermentation of engineering strains to produce soybean isoflavones. And finally the yield was up to6.6mg/L.The genetic stability of engineering strain was identified throuth the method of the PCR of purpose gene, restriction enzyme digested of the plasmid and SDS-PAGE detection. The plasmid loss rate of strain was calculated by the copy plate counting method. The results showed that the restructuring plasmid of engineering strain has good genetic stability in50generation and purpose gene was expressed stably. The ability of0,10,20,30,40and50generation strains to produce soybean isoflavones by fermented has no significant difference. The concentration of lactose in fermentation has a certain effection on the genetic stability of engineering strains. Moreover, when used lactose above2%, is beneficial to the stability of the restructuring plasmid.