Selection Of Cholesterol-lowering Lactic Acid Bacteria And Its Effects On Rats Fed With High-cholesterol Diet

In recent years,coronary heart disease(CHD)has attacked more and more people and became the major cause of morbidity and mortality in many countries.One of the important risk factors for CHD is hypercholesterolemia.The cholesterol lowering ability of lactic acid bacteria(LAB)without side effects makes them more and more attractive.Seventy-nine strains of LAB isolated from fermented food were screened in vitro for their ability to assimilate cholesterol.Then,ten strains which exhibited higher ability of cholesterol assimilation were investigated with the characteristics of acidic resistance,bile salt tolerance,and cell adhesion.According to the results,the best strain LP96 was picked out,and used to evaluate its effects on the high-cholesterol diet-fed rats.(1)The strains were separated using the spread-plate method on MRS agar with 2%(m/v)CaCO3 for their lactic acid productivity.Then,the colonies with lactic acid productivity were picked randomly from the agar medium for Gram stain and catalase test.The Gram-positive and catalase-negative with lactic acid productivity colonies were considered to be lactic acid bacteria.(2)The MRS-CHO broth was prepared by supplementing MRS broth with 0.1% sodium thioglycolate,0.2% sodium taurocholate,and 100?g/mL cholesterol.Each freshly prepared strain culture was inoculated 1m L in 9m L MRS-CHO broth and maintained anaerobically at 37? for 24 h,and the MRS-CHO broth without inoculation was used as the control.The remaining cholesterol contents in the supernatant and control broth were determined using the o-phthalaldehyde method.Based on the result,we selected ten strains(LPN5,LP130,LP96,LPZ,LPB,LP37,LP157,LP183,LPN21 and LP192B)that were better capable in lowering cholesterol and identified those strains through 16 S rRNA gene sequences.According to the result of 16 S rRNA gene sequences,the five strains LPN5,LP130,LP96,LPZ and LPB belong to Lactobacillus plantarum,and the other five strains LP37,LP157,LP183,LPN21 and LP192 B belong to Pediococcus pentosaceus.(3)These ten strains were selected for characteristic tests of acid and bile salt tolerances,cell adhesion ability.It was found that the strains LP96 and LP157 showed high tolerance to artificial acidic conditions(above 80%)and had strong resistance to bile salt(25.07%,25.36%),and assimilated nearly same cholesterol level and the strain LP37,LPN21,LP96 were excellent at adsorbability(145.67,140.26,73.21 strains adhering to one cell).Base on the results,LP96 was selected for further research.(4)Identification of LP96 was carried out by physiology and biochemistry property,colony morphology,and 16 S rRNA gene sequences.The strain LP96 was identified as Lactobacillus plantarum.(5)The possible mechanisms of cholesterol reduction in vitro by strain LP96.Cholesterol removal possible mechanisms by growing,resting and dead cells has been investigated.The resistances of the bacterias incubated in MRS-THO or MRS to the ultrasound also been studied.The result shows that different state cells have different mechanisms and the cholesterol in the medium increases the resistance of the bacteria to ultrasound.(6)The rats were randomly divided into five groups of five members each.According to the grouping of rats the different diets and different solution gastric gavage were received.This study lasted for 5 weeks,and the food and water were available ad libitum for all rats.Body weight was recorded at the beginning,ending of the study,and at the same time of the every week during the study.At the end of the experiment,rats were fasted overnight(12h)and euthanized by guillotine.Blood samples and liver were collected to examine the concentrations of serum triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C)in the serums and TC,TG in the liver.The concentrations of superoxide dismutase(SOD),catalase(CAT)and methane dicarboxylic aldehyde(MDA)in the blood were measured.One gram of digesta samples was used for enumeration of LAB,Coliforms,and Bifidobacteria by means of selective agar media.The results demonstrated that the levels of TG,TC,LDL-C and liver TC and TG were reduced significantly in the groups that received the strain LP96 solution compared with the model group,and that the HDL-C serum levels were increased without any significant difference.Furthermore,LP96 also showed good antioxidative activity and improvement of intestinal microbial balance in the rats.(7)Fermentation ability of strain LP96.The changement of acidity,pH,the number of living bacterium and protein degradation ability in the milk during the fermentation of LP96 were measured and the result shows that LP96 have the potential ability of fermentation.