Screening Of Exopolysaccharide-producing Lactic Acid Bacteria And The Functional Activities

Lactic acid bacteria(LAB)are commonly used probiotics.Exopolysaccharide(EPS)produced by LAB has a variety of biological activities,such as protecting bacteria,antioxidant,anti-tumor and regulating immune function.Moreover,the extracellular polysaccharides of LAB can be used as prebiotics which can be digested and absorbed by other probiotics in the intestine,promoting their growth and regulating intestinal flora.In this study,the EPS-producing LAB were screened from natural fermentation broth of fruits,and were isolated and identified.Their prebiotic characteristics such as the tolerance to simulated gastrointestinal environment,antimicrobial activity and antioxidant activity were investigated,and the fermentation conditions were optimized.The effects of the EPS on intestinal microbiota in mice were also studied by high throughput sequencing technology.This study would provide valuable reference for the research and application of EPS of LAB.The main results are showed as following:Four EPS-producing LAB strains named DNB1,DNC1,DNC6 and DNR3 were screened from orange and grapefruit natural fermented liquid by the methods of observation of ropy colony and analyzing polysaccharide production.The EPS yield of these four strains was 85.7?88.6?92.8 and 97.2 mg/L respectively.These four strains were identified as Lactobacillu plantarum,Enterococcus faecalis,Pedicoccus acidilacticii and Leuconostoc mesenteroide according to morphological characteristics,gram stain and 16 S r DNA sequencing.The prebiotic properties of four strains of EPS-producing LAB were further investigated.The results showed that four strains of LAB exhibited high tolerance to the culture medium of p H 3.0 and 4.0,and the survival rate was above 90%.Under the condition of p H 2.0,the survival rate of L.plantarum DNB1 is the highest(56.63%).Four strains presented high tolerance to artificial gastrointestinal fluid which showed more than 90% survival rate after incubated in the artificial gastric fluid for 2h and they could survive after incubated for 8h in artificial intestinal fluid.The fermentation broth of L.plantarum DNB1 exhibited antibacterial activities against Staphylococcus aureus,Bacillus subtilis,Escherichia coli.The fermentation conditions of L.plantarum DNB1 for the production of extracellular polysaccharides were studied.The fermentation conditions were obtained firstly by single factor test as followed: fermentation time of 20 h,fermentation temperature of 37 °C,inoculation rate of 3% and p H of 6.0.Three important factors affecting the synthesis of extracellular polysaccharides of L.plantarum DNB1 were selected: p H,fermentation temperature and inoculation rate.According to the method of response surface analysis,the optimal fermentation conditions were as follows: p H of 6.12,fermentation temperature of 37.5 °C and inoculation rate of 3.65%.Under the optimal fermentation conditions,the EPS yield can reach 95.8 mg/L,near the theoretical value(93.2 mg/L).The antioxidant capacities of the EPSs of LABs were studied.In vitro,the EPSs of the four strains displayed certain abilities to scavenge hydroxyl,1,1-diphenyl-2-picrylhydrazyl and 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)radicals,and EPS of L.plantarum DNB1 exhibited the highest antioxidant activity.In vivo,the EPS of L.plantarum DNB1 increased the activity of GSH-Px and total antioxidant capacity in the blood of mice,as a result it presented quite good antioxidant activity.The regulation effects of EPS on the intestinal flora of mice were investigated using high-throughput sequencing technology.The relative abundance of Lactobacillaceae was increased by 6.3%;and that of Staphylococcaceae was decreased by 6.32%;the relative abundance of Lactobacillus was increased from1.15% to 7.53% and that of Staphylococcus was decreased from 4.37% to 0.095%after the mice were fed with low dose of EPS for 28 days compared with pre-administration.Compared with the hd group(before fed with high dose of EPS),the relative abundance of the Lactobacillaceae of the Hd group(fed high dose of EPS for 28 days)was increased by 6.3% while the relative proportion of Staphylococcaceae decreased significantly from 3.78% to 0.34%;the relative abundance of Lactobacillus was increased significantly from 0.20% to 7.05% and that of Staphylococcus was decreased from 2.76% to 0.34%.Compared with the controlgroup C,the relative abundances of Lactobacillaceae in both Ld and Hd group were increased by 4.95% and 4.47% respectively.The UPGMA clustering tree results showed that,at the level of Phylum,the intestinal flora of the mice in the Ld group and the Hd group were closer,which was significantly different from that of the control group.