Cloning, Expression And Cholesterol-lowering Function Of Bile Salt Hydrolase BSH Gene Derived From Intestinal Lactobacillus

With the continuous improvement of people's living standard,the diet structure with high fat and high calorie makes the patients with dyslipidemia increase significantly.Dyslipidemia cause a series of chronic cardiovascular diseases such as hyperlipidemia,hypertension,atherosclerosis and coronary heart disease.As an important probiotics in the human gut,lactic acid bacteria have been proven to have the ability to improve blood lipid levels.Many domestic and foreign literatures have reported that lactic acid bacteria with high bile salt hydrolase(BSH)activity can significantly reduce serum cholesterol in humans and animals.However,all these studies were based on the strain level.In this paper,molecular biological methods were used to construct recombinant BSH lactic acid bacteria to study its cholesterol-lowering function.The results could effectively eliminate the differences between strains and improve the accuracy.Firstly,403 strains of lactic acid bacteria were isolated from the intestinal contents of chickens,ducks and pigs by plate method.Then,sixteen strains of high BSH active lactic acid bacteria were obtained through preliminary screening by ninhydrin method and species identification.Subsequently,substrate specificity of sixteen strains of BSH active lactic acid bacteria was quantitatively determined by high performance liquid chromatography(HPLC).The results showed that one strain of Enterococcus durans specific hydrolyzed taurocholic acid(TCA)and taurochenodesoxychollic acid(TDCA).Four strains of Lactiplantibacillus plantarum and three strains of Enterococcus specific hydrolyzed glycocholic acid(GCA)and glycodeoxycholic acid(GDCA).Two strains of Lactobacillus amylovorus and six strains of Lactobacillus johnsonii hydrolyzed glycocholic acid(GCA),glycodeoxycholic acid(GDCA),taurocholic acid(TCA)and taurochenodesoxychollic acid(TDCA).The above BSH active lactic acid bacteria with different substrate specificity provide important research materials for this study.Next,using bsh-deficient strain Lactiplantibacillus plantarum NB5462 as the host and p SIP411 vector,sixteen bsh recombinant intracellular expression strains were constructed.HPLC results showed that all the 16 recombinant strains of BSH had different substrate specificity and BSH activity,but it was significantly lower than the corresponding wild bacteria.In order to improve the BSH activity,a recombinant surface display strain,named L.plantarum YB334 S,was constructed using NB5462 as host and p SIPmc S vector.Immunofluorescence results indicated that the recombinant BSH was successfully expressed on the cell wall,but HPLC results showed that the recombinant BSH had no activity.Based on the above results,three strains of BSH recombinant lactic acid bacteria with high activity were constructed and obtained: 1.YB32,which specifically hydrolyzed taurine conjugated bile acid;2.YB81,which specifically hydrolyzed glycine conjugated bile acid;3.YB334,which hydrolyzed taurine and glycine conjugated bile acid.The growth curves and enzyme activities of the three strains showed that the enzyme activities of the recombinant strains were stable after 7 h of induction culture.The results of simulated gastric and intestinal fluid test showed that the three recombinant strains all showed good tolerance to gastric and intestinal fluid.Finally,in this study,three strains of BSH recombinant lactic acid bacteria with different substrate specificity were applied to high-fat mouse model to study the cholesterollowering function of BSH.The results showed that the intervention of recombinant lactic acid bacteria YB32,YB81 and YB334 could significantly inhibit the weight gain of high-fat mice.YB334 inhibited the weight gain of mice most obviously.The intervention of three recombinant lactic acid bacteria could significantly reduce the serum levels of total cholesterol,triglyceride and low-density lipoprotein of high-fat mice.The serum total cholesterol concentration decreased by YB334 was significantly higher than that by YB32,and the serum total cholesterol concentration decreased by YB32 was significantly higher than that by YB81.All of them could significantly reduce the levels of total cholesterol and triglyceride in liver of high-fat mice,and reduce the accumulation of cholesterol in liver.YB334 significantly reduced the triglyceride concentration in liver of mice than YB81.All of them could improved liver damage.The results of this study indicated that BSH recombinant lactic acid bacteria had cholesterol-lowering function,and the cholesterollowering effect of different substrate specific BSH lactic acid bacteria was different.In conclusion,through in vitro screening of BSH active lactic acid bacteria and recombinant expression of BSH,the cholesterol-lowering function of lactic acid bacteria BSH enzyme which hydrolyzed taurine and glycine conjugated bile acid was confirmed in vivo in this paper,and the effects were found to be different.These results provide a research basis for clarifying the mechanism of regulating and lowering cholesterol by lactic acid bacteria BSH in the future,and also provide ideas for developing probiotics in preventing and treating hypercholesterolemia and maintaining body health.