Study On The Strain And Activity Of Cholesterol-lowering Probiotics

Probiotics are beneficial to human health.Some strains have been found to have cholesterol-lowering functions,but there are still insufficient studies on cholesterol-lowering functions.First,optimization of cholesterol-MRS medium and cholesterol determination method for screening cholesterol-lowering probiotics.Then,the probiotics with strong cholesterol-lowering ability were screened out from the five probiotics with initial screened,and the fermentation characteristics of the five strains were analyzed simultaneously.Through acid and bile salt resistance experiments,high-quality strains resistant to high acidity and high bile salt environment were screened out.Furthermore,the strain was subjected to an animal experiment to confirm the cholesterol-lowering effect in vivo.Finally,the genetic characteristics of probiotics'cholesterol-lowering function were discussed at the molecular level.The main results were as follows:(1)The cholesterol-MRS medium was optimized.The crude cholesterol solution is subjected to a hot water bath at 90°C,vortexed,and sonicated to help prepare a clear and transparent cholesterol micelle solution.Then magnetic stirring of the MRS medium after adding the cholesterol micelles accelerates the uniform and stable existence of cholesterol in the MRS,which is beneficial to the long-term fermentation and cholesterol reduction of the probiotic strains in vitro.(2)The method for determining cholesterol is optimized.It was determined that the optimized cholesterol extraction step,saponification,removal of Tween-80 in the MRS medium,and the addition of the cholesterol solution were all beneficial to increase the accuracy of the modified OPA method for determining cholesterol in the cholesterol-MRS fermentation broth.(3)Using the optimized cholesterol-MRS medium and cholesterol determination method,three probiotics with a cholesterol removal rate of about 20%in vitro were screened from 5probiotics for the next acid and bile salt resistance experiments.All the 5 probiotics had the ability to lower cholesterol.When cultured for 72 h,the cholesterol-lowering effect of the strains was ranked from large to small:Lactobacillus casei(LCA-1)>Lactobacillus rhamnosus(LR-D)>Lactobacillus plantarum(LP-C)>Lactobacillus acidophilus(LAC-D)>L.reuteri(LSY-1),the cholesterol removal rates were 25.26%,22.18%,22.03%,14.06%,13.66%,respectively.When cultured for 24 h,the viable counts of the five probiotics in the cholesterol-containing medium were all above 1.0×10~8 CFU/m L,which was able to maintain a high viable count.And the cholesterol removal rate of the five probiotics is related to its live bacteria.(4)The strains of LCA-1,LR-D and LP-C were tested for acid and bile resistance.The strain LCA-1 had the worst tolerance,followed by LR-D and LP-C.The strain LP-C can survived for more than 24 hours in a highly acidic environment of p H 2.5 and can grow normally in a p H3.0 environment.Secondly,LP-C can tolerate 0.5%high bile salt environment,and the number of live bacteria cultured for 8 h can be maintained above 1.0×10~6 CFU/m L.(5)Lactobacillus plantarum LP-C had the function of lowering cholesterol in vivo,and the optimal dosage of Lactobacillus plantarum LP-C for daily gavage is 1.0×10~9 CFU/m L,and the dose of LP-C liquid was intragastrically administered to rats.After 45 days,serum TC,TG,and LDL-C of high-fat rats decreased by 12.73%,44.33%,and 15.79%,respectively,while HDL-C concentration was not affected.(6)High-throughput sequencing analysis of LP-C.The Lactobacillus plantarum LP-C genomic information contains a piece of chromosome information and a plasmid information,wherein the chromosome size is 3246747 bp,the GC content is 44.51%;the plasmid size is19592 bp,and the GC content is 40.49%.The KEGG analysis showed that the Bile salt hydrolase gene was present in the genome of the strain.Finally,the genomic information mining of LP-C was carried out.In addition to obtaining cholesterol-related genes,LP-C has also acquired other genes that are beneficial to the host,such as gene clusters that synthesize terpenes and bacteriocins,and genes that are resistant to some antibiotics and drugs.