Study On The Cholesterol Reducing Activity In A Cell-Free Supernatant Of Lactobacillus Reuterin JC-I

At present, people's living standard becomes better. But the unreasonable diet leads to the rising incidence of cerebrovascular, hypertension and arteriosclerosis disease year by year. Several studies have reported a high correlation between hypercholesterolemia and the incidence of these diseases. In the biological field, the research about the lactobacillus removing cholesterol has been just to start, and it has become the focal point. However, the precise mechanisms underlying lactic acid bacteria (LAB)-associated cholesterol reduction have yet to be determined. The study continues to explore the mechanism underlying cholesterol reduction by LAB in vitro.The lactobacillus reuteri exists generally in human and animal's intestinal tract. They can survive in the environment which is acid and has bile. It has been reported as one of the LAB with the cholesterol reduction ability. This study discovered that L.reuteri strain JC-I had the cholesterol reduction ability in the Cell-free supernatant (CFS).The removal rate may achieve 68.57%, and it declines along with the concentration of CFS. Precipitation phenomenon showed that the CFS of L.reuteri strain JC-I contain bile salt hydrolyase (BSH).In order to elucite the correlation between BSH activity and the cholesterol-reducing ability of L.reuteri strain JC-I, BSH roles in cholesterol removal were measured by heat treatment and pH adjustment. The remaining BSH activities after various treatments were measured by HPLC. The cholesterol removing ability and BSH activity of CFS both reduced significantly after heat treatment. While the co-precipitation caused by BSH cannot occurred in pH7.0, the CFS still present the cholesterol-removing ability. Beside the BSH, it was speculated there may be another effective component in the CFS with the ability to remove cholesterol.The cholesterol reduction activity of the components extracted by ethanol, ethyl acetate, and 70% AS was determined by using procedures identical to those just described. The majority of activity was found in the precipitate by a 70% AS solution. After TCA and the SDS processing, CFS no longer displayed the removal ability. After treatment with a variety of enzymes, CFS did not present the cholesterol reduction ability. It can be concluded that this effective components are proteins.In the process of purification of cholesterol-reducing components in CFS, CFS with cholesterol-reducing activity was partially purified by precipitation with 50%-70% AS, this being followed by DEAE Sepharose F.F column. Using a utilizing 25mM PBS (pH 7.0) including the 0-0.5mol/L NaCl as the eluent, there is one peak exhibit cholesterol-reducing activity. The specific activity was 61.6U/mg, and the protein is purified by about 11.7 folds. The molecular weight is determined by SDS-PAGE, and the estimated molecular weight is about 60kDa.