Screening Of Probiotic Lactic Acid Bacteria From Traditional Chinese Fermented Foods And Study Of Related Cholesterol-Lowering Mechanisms

In recent years,due to the improvement of people’s living standards,the rate of overweight and obesity has been rising.Cardiovascular and cerebrovascular diseases such as coronary heart disease,hypertension and atherosclerosis have become worldwide health problems.Hypercholesterolemia is one of the main factors that cause cardiovascular and cerebrovascular diseases.Statins are currently the most widely used cholesterol-lowering drugs with certain side effects.Therefore,finding new alternative or adjuvant therapies is an important goal in the treatment of hyperlipidemia.Some studies have reported the cholesterol-lowering effect of probiotic lactic acid bacteria,but the mechanism of their probiotic function is still unclear.In this study,lactic acid bacteria strains were isolated and purified from traditional Chinese fermented non-dairy foods(sauerkraut,pickle,cured beef,cured pork,and sausage),which were identified by 16 S r DNA sequencing and typed by Random Amplification of Polymorphic DNA.On the basis of isolation and identification,in vitro screening method was used to characterize and evaluate the cholesterol-lowering lactic acid bacteria with excellent probiotic,safety and industrial properties.Other probiotic function of the selected strain was also investigated.The cholesterol-lowering ability of selected lactic acid bacteria was investigated by in vitro and in vivo.The effects of cholesterol-lowering lactic acid bacteria on the expression levels of some key genes associated with cholesterol metabolism was evaluated to study the mechanisms for regulating cholesterol levels.The main results are as follows:(1)Isolation,identification and cluster of lactic acid bacteria.A total of 155 potential lactic acid bacteria strains were isolated from different kinds of traditional fermented foods using modified MRS medium and identified by 16 S r DNA sequencing as 60 Staphylococcus carnosus(38.7%),42 Lactobacillus plantarum(27.1%),34 Weissella cibaria(21.9%),12 Lactobacillus brevis(7.7%),5 Weissella confusa(3.2%)and 2 Lactobacillus sakei(1.3%).RAPD cluster analysis indicated that 155 strains showed diverse genotypes,which provides a genetic basis for the screening of lactic acid bacteria with probiotic properties and industrial potential.(2)In vitro probiotic properties of lactic acid bacteria.All 155 tested bacteria were screened for in vitro probiotic properties.Based on low p H and bile salt tolerance,10 strains were selected with the strongest tolerance,including 9 L.plantarum and 1 W.cibria.The results of cholesterol removal rate in high cholesterol medium showed that the same genus lactic acid bacteria from the same fermented food have diverse cholesterol removal ability.The tested lactic acid bacteria showed various bacteriostatic phenotypes against 4 pathogenic bacteria.The cholesterol-lowering ability,cell surface properties,antimicrobial ability and the adherence capacity to SW480 cells were strain-specific among tested strains.Using the comprehensive evaluation of in vitro probiotic properties,3 L.plantarum strians SK1,CB9 and CB10,which showed similar in vitro probiotic properties to the reference strain LGG,are the most potential probiotic lactic acid bacteria candidate strains.(3)Safety properties of selected lactic acid bacteria.10 tested strains were found negative to hemolytic toxicity,histamine and yramine producing,nitroreductase producing,and azoreductase producing.Seleted lactic acid bacteria showed extensive antibiotic resistance against 9 antibiotics.Antibiotic resistance genes str(A),str(B),ere(A)and tet(M)were detected in some strains by PCR.In particular,L.plantarum CB9 was sensitive to 7 of the 9 antibiotics and no corresponding resistance gene was detected.Based on in vitro probiotic properties and safety evaluation,the potential probiotic L.plantarum CB9 with cholesterol-lowering function was selected for further study.(4)Industrial properties of L.plantarum CB9.After passaging for 40 generations,CB9 maintained stable colony morphology and acid production capacity.The lactic acid yield was around 20 g/L.CB9 showed a high Na Cl tolerance and was able to grow at 6.5% salt concentration.High Na Cl concentration could inhibit the growth of L.plantarum CB9.CB9 exhibited rapid coagulation performance in litmus and 10% sterile skim milk.The acidification rate Δp H6 was 0.89±0.18,and then increased to 2.65±0.27 after 24 hours incubation.(5)Other probiotic function of L.plantarum CB9.L.plantarum CB9 significantly inhibited the adhesion of four strains of Helicobacter pylori in the AGS cell line;CB9significantly inhibited the adhesion of H.pylori HPAG1 on human gastric primary epithelial cells,maintained cell morphology and significantly down-regulated the secretion of IL-8 of cells;CB9 conditioned medium also showed the same inhibition effect on HPAG1 adhesion,which was not affected by heat-treatment;CB9 significantly down-regulated the relative m RNA expression of H.pylori colonization-related genes sab A and cag L,other relative genes alp A,alp B,bab A and lab A were not significantly affected.(6)Whole genome sequencing of L.plantarum CB9.The total length of CB9 genome is3380735bp;the GC content is 43.67%;the average length of 5S,16 S and 23 S r RNA are121 bp,1347bp and 2714 bp,respectively;the average length of t RNA is 75bp;and the total length of tm RNA is 368 bp.The KEGG metabolic pathway annotation showed that CB9 is enriched in the genetic information processing pathway and environmental information processing.Enrichment in other metabolic pathways such as carbohydrate metabolism,amino acid metabolism,cellular processes,and nucleotide metabolism were also found in CB9.The COG annotation classification showed that CB9 enriched the most genes in the Amino acid transport and metabolism pathway,followed by the Energy production and conversion pathway.In addition,significant enrichments in other functional categories such as Translation,ribosomal structure and biogenesis,Transcription,Replication,recombination and repair,Carbohydrate transport and metabolism were found.The results of subsystem classification showed that a total of 1099 genes of CB9 were divided into 194 subsystems that belong to 11 classes.One of the largest classes includes 64 subsystems and 413 genes that aggregate metabolic pathways associated with metabolism.The results of protein sequence similarity comparison showed that most of the protein sequence of CB9 was more than 90 % similar to the reference strain L.plantarum WCFS1.Finally,the circular genome map of L.plantarum CB9 was drawn.(7)In vitro and in vivo cholesterol-lowering effect of L.plantarum CB9.Results showed that the in vitro removal mechanism of CB9 in high cholesterol broth mainly relies on the synergy of assimilation and co-precipitation.Within the range of 0-10g/L bile salt concentration,cholesterol removal rate of CB9 increased along the bile salt concentrations,reached the peak value at 3 g/L bile salt contration and decreased with higher bile salt concentration.The cholesterol-lowering efficiency of heat-inactivated CB9 cells was significantly decreased compared to the viable cells.In the hypercholesterol rat model,L.plantarum CB9 inhibited the weight gain of rats significantly and alleviated the liver cell pathology.Reduction of the TC and LDL-C level in blood,TG level in liver,and TC level in feces were found significant in CB9 feeding group.Quantitative real-time PCR analysis revealed that the intake of CB9 in hypercholesterol rat significantly up-regulated the m RNA expression levels of FXR,CYP7A1,LDL-R and ABCA1 gene,and significantly down-regulated the m RNA expression levels of NPC1L1,HMGCR,FAS and SREBP gene.