Cloning And Expression Of Bile Salt Hydrolse Gene Of Bifidobacterium Longum NCC2705and Discription Of Its Charactistics

Bile salt hydrolase of Bifidobacterium may be related to Bifidobacterium bile toleranceand cholesterol-lowering ability, so Bifidobacterium which can overexpress bile salt hydrolasegene by means of genetic engineering and fermentation engineering may be used ascholesterol-lowering drug or healthy food and has a very significant application value.Bifidobacterium longum is usually isolated from feces of adult and infant, because they canadapt to high concentrations of bile salts (concentration is usually in5mM below), and theycan make use of oligosaccharides, they particularly suitable for the growth in the colon.Bifidobacterium longum is a kind of Bifidobacterium which is available for the healthy foodcurrently. In this study, we used Bifidobacterium longum NCC2705as a template, cloned bilesalt hydrolase gene, and built three strains,they are pGEX-4T-1-bsh/BL21(DE3),pDG7-bsh/NCC2705and pMgap-bsh/NCC2705. In order to make a contribution to thedevelopment of high efficiency cholesterol-lowering Bifidobacterium,we studied theinducible expression conditions of bsh in E. coli., and we studied the bile salt tolerance,cholesterol degradation, and characteristics of fermented milk of Bifidobacterium by theoverexpression of bsh.Using conventional prokaryotic expression method, we constructed plasmidpGEX-4T-1-bsh and transformed it into E. coli BL21(DE3).After induced expression of bshwith IPTG in E.coli pGEX-4T-1-bsh/BL21(DE3), according to the results of SDS-PAGE gelelectrophoresis of purified GST-bsh fusion protein and GST empty tag of bacteria expressionbroth supernatant, we analysed that bile salt hydrolase has a molecular weight of about35Kda.We also built plasmids of pMgap-bsh and pDG7-bsh,and transformed NCC2705, sothat bsh can be overexpressed in NCC2705.We made studies on growth curve, bile tolerance and cholesterol-lowering ability ofNCC2705, pDG7-bsh/NCC2705and pMgap-bsh/NCC2705, results show that their growthcurves show little different; bile tolerance of three strains are diffrent, pMgap-bsh/NCC2705showed higher bile tolerance than NCC2705or pDG7-bsh/NCC2705; three strains haveabilities of lowering cholesterol, and the way is not a single,when we added bile salts in the medium, although growth of strains was significantly suppressed, cholesterol degradation rateof the strains was significantly increased, and the cholesterol degradation rate can reach morethan35%, and pMgap-bsh/NCC2705has higher cholesterol degradation rate than NCC2705and pDG7-bsh/NCC2705NCC2705and pMgap-bsh/NCC2705were fermented mixed with yogurt bacteriarespectively, optimization of process parameters were inoculated with6%fermentation agent,inoculation ratio of Bifidobacterium and mixed fermentation agent is1:2, fermentation5h in39℃. After fermentation, lactic acid degree, sensory indicators and number of viable cells infermented milk can conform commodity yogurt. During cold storage at4℃,the acidity offermented milk is120oT below, the number of viable cells is108cfu/mL above, and both oftheir acidity increased with the extension of the cold storage time, viable number are reducedwith the extension of the cold storage time,and the yogurt which has NCC2705changedfaster.