Screening And Functional Study Of Cholesterol-Lowering Lactic Acid Bacteria From Marine Resources

At present,coronary heart disease,hypertension,atherosclerosis and other cardiovascular diseases are increasingly high and seriously threaten human health and life,high serum cholesterol content is the main factor inducing these diseases.Drug therapy is the main method,but it will bring many side effects and the effect is unstable.Therefore,it is urgent to find a safe and effective microecological agent to assist drug therapy to reduce serum cholesterol level.In this study,multiple strains of lactobacillus were isolated from the viscera of Mesoplodon densirostris,and a lactobacillus with high cholesterol lowering function was further selected as the target strain through in vitro cholesterol lowering experiment.From growth characteristics,probiotics characteristics,security characteristics three aspects evaluate the target strains,optimizing the fermentation conditions,and discussed its role in vitro cholesterol mechanism,and finally study the effects of target strains on high-fat mice,so as to provide theoretical reference for the development of new cholesterol-lowering microecological products,and the results are as follows:(1)Study of screening,separation and characteristics.A strain of lactobacillus with cholesterol degradation rate of 48.82% was obtained from Beaked Whale viscera by traditional lactobacillus screening method and phthalaldehyde method,and it had the activity of bile saline hydrolysate.The strain was identified as Lactobacillus plantarum and labled as HJ-S2(NCBI registration number: MK248728;CGMCC No:17720),construct its evolutionary tree.In this paper,the growth characteristics of HJ-S2 strain were firstly studied,and the growth curve of the strain was determined.The target strain had a certain acid production capacity and the degradation rate reached the highest of 45% at 42 h in the decay period.Secondly,the probiotic characteristics of HJ-S2 strain were studied.In Vitro,Under being cultured for 3 h at pH3 or insolvent containing 0.3% bile salt,the tolerance rate reached 82.73% and 80.62% respectively.The release amount of free cholic acid of HJ-S2 strain was 3.35 ?mol/mL,showing a certain capacity ofdebinding bile salts.Analysis of adhesion to human colon cancer cell HT-29 of132.52±5.42,which has a certain colonization ability.The Lactobacillus plantarum HJ-S2 can providing a theoretical basis for subsequent strains to colonize the intestine and play a probiotic role.Finally,the safety characteristics of HJ-S2 were studied.The result of antibacterial suggested metabolites of the strain HJ-S2 have certain inhibitory effect on E.coli,S.areaus and Salmonella.The strain HJ-S2 was sensitive to tetracycline,streptomycin,ampicillin and rifampicin,and resistant to penicillin,gentamicin,erythromycin and kanamycin,and the strain was also not hemolytic.Preliminary study on cholesterol lowering mechanism of strain HJ-S2 in vitro.The results showed that the cholesterol lowering mechanism of the bacteria in different states was different.The growth cell bacterial suspension had the highest cholesterol lowering ability and the absorption of cholesterol was mainly assimilation.Cholesterol in MRS-CHOL medium can change the toughness of cell wall and improve the it's resistance to ultrasound significantly.(2)Optimization of fermentation conditions.Single factor and orthogonal experiment was used to optimize the fermentation conditions from two following aspects of internal and external factors.Result: The medium conditions were peptone5 g/L,glucose 25 g/L,cholesterol 1.0 g/L and bile salt 2.0 g/L.The fermentadion conditions were temperature 37?,pH 6,incubation time 24 h and inoculation amout1%.Under the optimized condition,the cholesterol-lowering rate reached to69.81%,significantly increased than before,which provided the production process and reference basis for the development and application of functional probiotics.(4)Experimental study on mice.The mouse high-fat model was constructed and the strain HJ-S2 was applied to the six-week male mice fed with high-fat diet.The results showed gastric perfusion of HJ-S2 could decrease the mice serum contents of total cholesterol(TC),total triglyceride(TG)and low density cholesterol(LDLC)and atherosclerosis index(AI)significantly,increase the content of high density lipoprotein(HDLC),fecal water content and feed utilization.Meanwhile,strain HJ-S2 could also improve the fat accumulation in the abdomen and kidney of the high-fat mice,and repair the damaged cells in the liver and pancreas of the high-fat mice.Theresults showed that HJ-S2 could effectively improve the health status of high-fat mice and reduce the risk of disease.