Studies On The Probiotic Properties Of Lactobacillus

In recent years, probiotics potential probiotic function has attracted more and more attention.Probiotics are defined as live micro-organisms which when administered in adequate amounts confer health benefit on the host. The theoretical basis for the selection of probiotic micro-organisms contains safety functional and technological aspects.As the following aspects:no pathogenicity; stable survival ability in the acid and bile salt; adhesion and colonization on the digestive tract surface; not carrying transferable antibiotic genes; having antagonistic effect on pathogenic bacteria; can promote immune but will not lead to inflammation; antimutagenic and anticarcinogenic effects; good organoleptic properties; phage resistance; processing and storage stability. Lactic acid bacteria is a kind of probiotics,It can enhance human immunity, prevent or improve the treatment of diarrhea, prevent or treat other gastrointestinal diseases, prevent infection of the reproductive system, reduce serum cholesterol, promote the absorption of nutrients and so on. The source of the lactic acid bacteria are widely, according to the standard to screen probiotic lactic acid bacteria, and exploring the method of high density fermentation to improve the bacteria probiotic effects. In addition,in the function of lowering serum cholesterol, bile salt hydrolase plays an important role. Bile salt hydrolase can hydrolyze conjugated bile salts generated free bile salts and taurine or glycine. This promotes the synthesis of bile salts by cholesterol from scratch, thereby reducing the serum cholesterol level. Therefore, the research of bile salt hydrolase of the reduction of serum cholesterol in clinical efficacy has great application prospect.Probiotic properties of multi Lactobacillus strains preserved in the lab were evaluated in my study.We screened a strain of L. plantarum with excellent performance, named as L. plantarum SDMCC050209. We optimised culture conditions using raw materials of industrialization, improved the fermentation cell density and decreased the cost of production Finally, Bshl gene from L. plantarum SDMCC050209 were cloned and expressed in Escherichia coli. The main results are as follows:1. The vitro evaluation of probiotic properties of LactobacillusThrough screening to the laboratory 6 strains for their resistance to bile salts, gastrointestinal fluid resistance, degradation of cholesterol and other probiotic properties,we screened a L. plantarum strain with good probiotic properties, and named as L. plantarum SDMCC050209. The probiotic characteristics performance of the strain are as follows:In the pH2.5 environment the viable counts of strain decreased only one magnitude after 6h cultured; In the 0.3% bile salt cultured after 6h, there are still about 104 survival cell; It has strong tolerance to the artificial gastric juice and artificial intestinal juice, under the both of the conditions,it was cultured after 6h the viable count of strain decreased about two orders of magnitude; It has the good cholesterol removal ability, cholesterol removal rate reached more than 60%.2. Optimization of culture conditions of L. plantarumThe strain of L. plantarum SDMCC050209 high density fermentation conditions were optimized. Through the orthogonal test and other optimization method we obtained the optimum formula and culture conditions:0.5% soybean meal,1% yeast powder,4% brown sugar and inorganic salt,37℃ 60r/min shaking culture by 24h, viable count can reach 109 orders of magnitude. The experiments showed that in the culture medium added 0.5% fructooligosaccharides or 0.5% of oligo isomaltose sugar,L. plantarum SDMCC050209 in optimizing cultivation of 37℃ cultured after 24 hours, the maximum cell density increased 1.5.3. Cloning of bile salt hydrolase gene from L. plantarum SDMCC050209 and its expression in Escherichia coliUsing polymerase chain reaction (PCR),we cloned bile salt hydrolase gene enzyme bshl encoded on chromosome from L. plantarum SDMCC050209.The size of the gene is 975 bp, it consists of 324 amino acid residues.The catalytic reaction center of bshl is Cysteine2, the other four strictly conserved amino acid sites (Asp18, Asn 21, Asn 175. Arginine 228) also play an important role in the enzymatic catalytic reaction. In order to study the enzyme characterization, bshl gene was cloned into Escherichia coli expression vector, to construct the recombinant plasmid pET28b-bshl, and then we imported the E. coli BL21 (DE3) to construct the recombinant bacteria E. coli/pET28b-bshl. The determination of recombinant strain E. coli/pET28b-bshl of bile salt hydrolase activity was 1.15U/mg-1. The main reason of low activity is the E. coli/BL21 expression production in the BSH most formed inclusion body. Through the Ni column we purified the bile salt hydrolase formed inclusion body,At the expected size appeared the target band. The inclusion bodies were stepwise dialysis refolding. We measured the inclusion bodies enzyme activity by the method of free amino acid,the enzyme activity was undetectable,so under these conditions, the inclusion body renaturation was not successful.