Studies Of Cholesterol-lowering Mechanism On Exopolysaccharide And Bile Salt Hydro Lase Of Lactobacillus Plantarum

With the improvment of living standards,the number of people suffered from hyperlipidemia is rising.The high total cholesterol content in the body will adverse our health,although cholesterol has many positive functions,such as cell structure,fat digestion,endocrine balance and other important physiological functions.As a result,the treatment of cholesterol reduction by microorganism in the gut and the elucidation of the cholesterol-lowering mechanism will facilitate the development of cardiovascular disease drugs and health care products.A number of studies have shown that microbial exopolysaccharide(EPS)has the cholesterol-lowering ability.The productivity of microbial EPS is low,and different strains has its specificity.In addition,the pathways and mechanisms of cholesterol reduction by lactic acid bacteria are complex.There is co-precipitation mediated by bile salt hydrolase that have the ability to lower cholesterol,except adsorption by EPS.Therefore,this research will clarify the mechanisms and differences of cholesterol reduction by two different strains of Lactobacillus plantarum(La-10 and La-11)in function of EPS,activity of key enzyme,and expression levels of genes for bile salt hydro lase and key enzymes in EPS biosynthesis pathway.Firstly,the single factor optimization experiment was carried out on EPS extraction of La-10 and La-11.The optimum fermentation conditions were as follows: 4% inoculation amount of La-10 and La-11,30 g/L and 20 g/L sucrose content respectively,30 h fermentation time,30? fermentation temperature.For the qualitative experiment of EPS,the study obtained the best vitro cholesterol-lowering experimental conditions: 0.8 g/L of EPS concentration,15 h adsorption time,0? and 37? reaction temperature separately.In addition,this study also showed that the elimination rate of inactivated bacteria and fermentation broth of La-10 were 6.45% and 3.19% respectively,and those of La-11 were 9.99% and 5.79% respectively.Secondly,two key genes of synthetic gene cluster of EPS,named UDP-glucose-4 epimerase gene(galE3)and priming glycosyltransferase gene(cps4E),were analyzed in heterologous expression experiment.The molecular weight of protein encoded by galE3 was 36 kDa,and it performed stable.It was a hydrophilic and highly activated protein,which could be purified well by N ickel column affinity chromatography.But the protein encoded by cps4 E,an unstable product,was purified unsuccessfully by N ickel column affinity chromatography,which was 25 kDa.That should be further explorated for a better expression.The enzymatic reaction could produce 0.55 mg/mL UDP-galactose of La-10,and La-11 was 0.30 mg/mL by High Performance Liquid C hromatography(HPLC).Finally,the experiment analyzed the changes of expression levels of galE3 and cps4 E in synthetic pathway of EPS by RT-qPCR,and verified La-11's capacity of coprecipitation to lowering cholesterol,exploring its other mechanism.The relative expression levels of galE3 and cps4 E in La-10 increased by 3.06 and 3.34 times respectively,and in La-11 galE3 and cps4 E raised 3.69 and 4.22 times repectively.In addition,the relative expression levels of bsh1,bsh2,bsh3 in La-11 were raised 4.58,8.77,2.25 times respectively,but it showed no obvious changes with bsh4.Consequently,the mechanism of cholesterol reduction was investigated in this study.Results displayed that the mechanism of cholesterol reduction is not the same in different strains.This work will provide experimental foundation and theoretical basis for the development of high-yield EPS strain by genetic engineering techniques and the application of Lactobacillus plantarum in medicine and food siecnce.