Separation And Purification Of Bile Salt Hydrolase From Probiotics

The purpose of this study was to optimize fermentation conditions of bile salt hydrolase from Lactobacillus casei GL-B, thereby enhancing the activity of bile salt hydrolase. The effects of incubation conditions and the medium compoments were observed on the activity of bile salt hydrolase, mean when MRS broth is based medium. The optimal carbon, nitrogen, temperature, pH, culture time and stimulating factors of bile salt hydrolase fermentation were observed by a single factor experiment, and the optimum conditions were achieved. The fermentation condition of high yield bile salt hydrolase was determined by the five factors and four levers orthogonal test L16(45). The optimized fermentation condition was glucose2%, pancreas peptone1%, temperature37℃, pH7, inoculation of2%.In common MRS medium, the bile salt hydrolyse activity was0.543U, at optimum conditions, the bile salt hydrolyse activity was3.114U, increased about5times than before.In order to obtain higher activity of bile salt hydrolase, the induce experiment were carried out by the substrate induce to increase the activity of bile salt hydrolase. The induce matter was bile salt. The bile salt hydrolyse activity was3.46times than before.At the same time, the study of on the cholesterol-lowering mechanism by Lactobacillus casei GL-B bacteria was researched. Lactobacillus casei GL-B were cultured in MRS medium containing cholesterol plus taurocholic acid, and the other MRS which uninoculated sterile broth was the control. After cultured24h, the levers of cholesterol of MRS medium were measured. Results showed that the bacteria exhibited an ability to reduce cholesterol, and36.9%cholesterol can be removed. The process of cholesterol removing and cholesterol distributing in the precipitation were analyzed, the cholesterol-lowering reason may be due in part to the deconjugation of bile salts by the bacteria produce bile salts hydrolase, and cholesterol was co-precipitated with deconjugated bile. Another reason was bacterial assimilation, so that the lever of cholesterol was reduced.The bile salt hydrolase was purified by anion sulphate precipitation, followed by DEAE-Sepharose FF cation exchange chromatography and SEPHADEX G-100, and the bile salt hydrolse was washed with0.1mol/L-0.5mol/L NaCl at last. The molecular weight of BSH is about50kDa.The characteristics of the bile hvdrolase were observed. The optimum reaction pH was6, the optimum reaction temperature was37℃. The optimum effect of substrate concentration on BSH activity was6mmol/L, the BSH activity was highest. Different inhibitors had different effects, the effect of EDTA was the smallest, inhibited6.25%. The highest effect was carbamide, inhibited93.75%.