Screening And Functional Mechanism Of Cholesterol-lowering Probiotic Lactobacillus

Cardiovascular disease(CVD) is a leading cause of death in the world. Diet has been identified as a means of conrtolling serum cholesteorl concentrations,and many health conscious people are adopting measures to decrease risk by reducing blood serum cholesteorl.Several investigations have reported that a fermented milk product with lactic acid bacterria have a a action to decrease seurm cholesterol coneentration.1. 66 strains of bacteria were isolated from the traditional fermented foods. Cholesterol-enriched MRSO-CHOL medium was used to analyze their cholesterol-reducing ability. 5 strains were screened for their excellent cholesterol-reducing ability. Low pH and bile tolerance, deconjugation of sodium taurocholate were tested and compared with cholesterol-reducing ability of these 5 strains, respectively. Strain GUO was selected as the optimal strain for all its tested abilities were among the top three and its cholesterol-reducing ability was best. Strain GUO was identified as Lactobacilluss plantarum by using API identification system.2. The growth characteristics of strain GUO was studied. It grew at optimal temperature 30℃,and the minimum 12℃,maximum 43℃.As for initial pH value for cell growth, the optimal value was 5.7,as well the upper and lower limitations were 3.6 and 9.4, respectively. The growth of L. plantarum GUO culture was tested in MRS broth and 10 % SKM medium. It was found L. plantarum GUO began logarithmic stage and maximum growth at 2 h and 11h, respectively. But it grew slowly in 10 % SKM and produced little acid. Stored at 4℃in MRS (pH 3.8) and SKM (pH6.8),it was found the death stage of 90 % living cells of L. plantarum GUO,as directed to value S, was 15.2 d and 116.3 d, respectively.3. The results showed that the adhesion was bacterial cell concentration and incubation time dependent. When the bacterial cell concentration reached 108CFU/mL, after 2h's incubation, the mammal cell surface tended to be saturated by L. plantarum GUO. The adhesive ability of L. plantarum GUO to Caco-2 cells reached maximal when the bacteria cells were harvested in stationary phase. The pH value of the solution demonstrated significantly effect on the bacterial adhesive ability,of which acid pH was optimal for the adhesion among the serial pH values tested. Whereas no such phenomena was observed for addtion of Ca²⁺and Mg²⁺(100 mM).These findings indicated the adhesion of L. plantarum GUO to Caco-2 cells could be affected by various factors, among which the effect of adhesin and receptor was predominant.4. Two methods, incubation and Denaturing Gradient Gel Electrophoresis were applied to assess L. plantarum GUO's colonization in intestinal tract of Wistar rats. Results showed that L. plantarum GUO mostly colonized rats'colon and ileum, the amount reached separately 107cfu/g and 108cfu/g, only little L. plantarum GUO colonized the jejunum and the caecum, the amount was less than 105cfu/g, but it couldn't colonize the rectum and the duodenum. L. plantarum GUO colonized the colon for about 10 d, the ileum for 7 d and the caecum for only 3 d. 5. More studies on the mechanism of cholesterol removal in vitro was carried out. The results showed that L. plantarum GUO strain was able to precipitate and assimilate cholesterols accounting for 36.5 % and 63.5% respectively, which displayed assimilation as the predominant one. The specific activity of the enzyme was increased 2.5-fold, 20-fold, 39.6-fold, respectively. But SDS-PAGE showed that the purified sample was not homogeneous. Through PAGE preparation electrophoresis we got the electrophoretic pure enzyme, calculated the molecular weight was about 38,000Da. Characterization of the enzyme exhibited the optimal concentration of ATP and the concentration of the substrates was 2.5 mg/mL and150μg/mL respectively; the optimum pH and temperature was 7.0 and 55℃respectively and kept stable after 30 min maintenance below 60℃from pH 6.0 to 9.0.6. In accordance with methods of our country for toxicological assessment on food safety, antibiotic sensitivity test, Ames test, the acute toxicity test, 90 d feeding and the teratogenicity test were used to assay the safety of L. plantarum GUO. Antibiotic sensitivity test found that L. plantarum GUO showed no sensitivity to Norfloxacin and Gentamycin, but was significantly sensitive to Cefradine, Tetracycline, Chloramphenicol and Erythromycin. L. plantarum GUO in Ames test and the acute toxicity test showed to negative. The 90 d feeding test showed that L. plantarum GUO had no effect on the growth and development, tissue pathology, blood system, liver function and kidney function of the rat. The result of the teratogenicity test showed that L. plantarum GUO had no effect on the toxicity of the mothers body, the toxicity of the embryo and teratogenicity. In short, L. plantarum GUO was safe and has the potential for development into functional foods.7. Due to the functionality and biological activity of L. plantarum GUO, it will have a important meaning that is applied to milk industry. On the basis of the single factors experiment, we design and optimize the formula of fermented milk:15 % SKM,5 % glucose,3 % starter; the formula of fermented milk drink of L. plantarum GUO:the fermented base materials 30 %,sucrose 13 %,pectin 0.3 %,sodium citrate 0.1 %.8. The effects of L. plantarum GUO on serum lipids were estimated in SD rats fed cholesterol-enriched diets. Living strain L. plantarum GUO significantly reduced TC and LDL-C, the ability increased as the dose increased, and also increased HDL-C. Also, dead L. plantarum GUO culture could significantly reduce LDL-C and increase HDL-C.