Identification And High Cell Density Culture Of Lactic Acid Bacterium Lowwering Cholestrol

Lactobacillus are used for probiotic, the effects are generally related to inhibition of pathogenic species, reducing the risk of colon cancer, increasing the immune response, and decreasing serum cholesterol. The Coprecipitation suggested by Frand A.M. was the most popular theory to explain mechanism of Lactobacillus to reduce blood cholesterol level. In this theory, bile salt hydrolase(BSH) is the most important role to decrease blood cholecterol level. The bile salt was degraded to Amino acid and cholic acid, which was coprecipitated with cholesterol and excreted by metabolism. So the ability of cholesterol-lowering depend on the activity of bile salt hydrolase. The microbiological technology and molecular biological technology were utilized in the research to screen the Lactobacillus, which had ability of cholesterol-lowering and bile salt hydrolase gene.Lactic Acid Bacteria Lp-1was identified by experiments in vitro cholesterol-reducing and16S rRNA, and BSH gene was identified by PCR. The results indicated that Lactic Acid Bacteria Lp-1had the function of cholesterol-reducing in vitro. The strain Lp-1had bile salt hydrolase gene in genome, and sequence analysis showed that deoxyribose nucleotide of the bile salt hydrolase gene was98.9%identical to the published sequence. The homology of16S rRNA gene was99%between the strain Lp-1and published Lactobacillus plantarum. Thus, the Lactobacillus plantarum Lp-1had bile salt hydrolase gene, and produced bile salt hydrolase.It was varified in the pre-experiment that glucouse, yeast extract, tryptone lactoalbumin hydrolysate, temperature and inoculum size had strongly influence on cell density and bile salt hydrolase activity of Lactobacillins plantarum Lp-1. The interaction of these significant factors were studied in Box-Behnken Design experiment. The optimum condition of fermentation was estimated (per litre):glucose27.77g, yeast extract10.77g, lactoalbumin hydrolysate8.95g, tryptone6.93g, and temperature was35.62℃, pH6.5, inoculum size5.92%.pH control and glucose addtion were utilized in the treatment of high cell density culture. The effects of different neutralizing agent on cell growth and the comparison of decreasing volume of different neutralizing agent were studied,2M NaOH was selected as neutralizing agent in fermentation. The effects of different carbon and nitrogen ratio on cell growth and the change of reducing sugar concentration were studied. Finally, glucose was utilized in fed-batch culture, the concentation of glucose was50g/L and the added ratio was lmL/min. The cell density of Lactobacillius plantarum Lp-1incubated in optimum fermrntation conditions reached1.66×1010CFU/mL, which was1.8times to compare with batch culture. The bile salt hydrolase activity reached19.6IU/mL, which enhanced78.1%to compare with none-pH control.