Antibacterial Properties Of Foodborne Lactobacillus Casei NA-2 And Activities Of Its Exopolysaccharide

Many Lactobacillus spp.are probiotics,showing activities of inhibiting pathogenic bacteria,regulating intestinal flora,immunoregulation and anti-tumor.The exopolysaccharide(EPS)secreted by Lactobacillus spp.is a natural component with a variety of functional activities,such as anti-biofilm,antioxidant and immunomodulatory.Fermented vegetable is a kind of acidic fermented food,which is rich in Lactobacillus spp.In this thesis,we choose the traditional fermented vegetable,Suan Cai,from Northeast China as the raw material and isolated an EPS-producing Lactobacillus from Suan Cai.After evaluated the physiological activities of the Lactobacillus,we extracted the EPS and studied its physico-chemical property,structural characteristics and physiological functions.The EPS-producing Lactobacillus spp.generally exhibit“mucoid”and“ropy”phenotpyes.In this study,a“mucoid”EPS-producing Lactobacillus was found from Suan Cai.After 16S r DNA gene sequence,phylogenetic analysis and the whole genome sequencing of the“mucoid”strain indicated that it was Lactobacillus casei,named as L.casei NA-2.The resistances to acid and bile of L.casei NA-2were analyzed.The results showed the survival rate of L.casei NA-2 can reach 70%after growing for 2h during the condition of p H 2-4,and the survival rate was still higher than 50%after 4 h.In the culture medium supplemented with 0.03%?0.3%bile salt,L.casei NA-2 can survived more than 70%after 4 h.Above all,L.casei NA-2 exhibited good resistances to acid and bile salt.After L.casei NA-2co-cultured with Bacillus cereus,Salmonella typhimurium,Escherichia coli O157:H7 and Staphylococcus aureus for 24 hours,pathogens were only survived less than 0.5%,and even no viable strains could be detected after 48 h.However,the growth of L.casei NA-2 was not obviously affected by the four pathogens.The co-culture systems were then observed by scanning electron microscope,showing the phenomenon of adhension between L.casei NA-2 and pathogenic bacteria.Furture,the mixture of L.casei NA-2 and each pathogen at the ratio of 1:1 was co-cultured with Caco-2 cells,suggesting the inhibition ability of L.casei NA-2 on the adhesion of the four pathogens to Caco-2 cells,with the highest inhibition rate of 50%.Hot water extraction and ethanol precipitation were used to extract the EPS on the surface of L.casei NA-2.After removing the proteins with TCA,the primary analysis of the EPS was analyzed.The result of fourier transform infrared spectroscopy(FT-IR)demonstrated the characteristic peaks in EPS,like hydroxyl and C-H.The total sugar and protein were 88%and 0.28%,respectively,determined by phenol-sulfuric acid method and coomassie brilliant blue(CBB).After hydrolysis with sulfate,the monosaccharide compositions in EPS were measured by TLC and HPLC.The result showed that the EPS were heteropolysaccharides contained rhamnose,mannose and glucose residues with the molar ratio of 24.3:1.0:42.9.The EPS was a mixture of three polysaccharides with molecular weights of 6.4×10~5 Da,2.0×10~5 Da and 1.4×10~4Da,determined by HPLC.The anti-biofilm activity of the EPS from L.casei NA-2 was analyzed by fluorescence spectrophotometry.The results suggested that the EPS could inhibit biofilm formation and disperse the biofilm of B.cereus,S.typhimurium,E.coli O157:H7 and S.aureus,which is consistent with the antibacterial activity.L.casei NA-2 EPS had the ability to scavenge hydroxyl radicals,superoxide radicals and DPPH.The scavenging activity of the EPS on superoxide radicals was significantly higher than ascorbic acid.It was found that the EPS can up-regulate the production of ROS and TNF-?in RAW 264.7 mancrophages through NF-?B and JNK signaling pathways,activating cellular immunity.After LPS-induced macrophages were treated with the EPS,expression of i NOS was reduced in cells,contributing the reduction of NO,showing a potential anti-inflammatory function of the EPS.