| Lactic acid bacteria are widely found in fermented food and living environment,and are generally recognized as safe(GRAS).With the progress of human development,heavy metal lead pollution is also very extensive.More and more studies have found that lactic acid bacteria isolated from fermented food and soil environment have strong adsorption and removal effect on lead,but the tolerance and removal mechanism of lactic acid bacteria to lead remains to be clarified.Therefore,this paper intends to screen lactic acid bacteria with high tolerance to lead from fermented foods,and the mechanism of lactic acid bacteria was studied in vivo and in vitro.In vitro,the changes of cell structure and antioxidant properties of lactic acid bacteria were observed under different lead concentrations,and the adsorption and dissociation of lead by lactic acid bacteria under different conditions were studied to study the stress tolerance and adsorption mechanism of lactic acid bacteria to lead.In vivo,the lethal concentration of lead and the protective effect of lactic acid bacteria on nematodes exposed to lead were studied by establishing the model of Caenorhabditis elegans.The mechanism of lactic acid bacteria on host antagonism of lead poisoning was observed by the expression of nematode of stress-related genes.The main conclusions are as follows:(1)Screening of Lactic Acid Bacteria with High Lead ToleranceTen strains of lactic acid bacteria isolated from fermented vegetables were tested for lead tolerance,and a highly tolerant strain R was screened,which could grow at5000ppmmg/L lead concentration.The lead low tolerance strain P was selected as a control to study the growth ability,acid resistance,salt tolerance and bile salt resistance of the two strains of lactic acid bacteria.It was found that the characteristics of the R strain were better then that of P strain.In addition,both strains were found to survive in simulated gastric fluid by simulating the gastrointestinal environment in vitro,while only the R strain survived in the simulated intestinal fluid.Finally,16 S rDNA molecular identification showed that R strain was Lactobacillus plantarum and P strain was Lactobacillus fermenti.(2)Stress response of lactic acid bacteria to leadThe results showed that with the increase of lead concentration,the polysaccharide content of lactic acid bacteria cell wall decreased,the proportion of short-chain fatty acid and long-chain fatty acid changed.The membrane fluidity of lactic acid bacteria under lead exposure was significantly different.The positive region of PI-DNA could not be detected in the membrane fluidity of R cell of lactic acid bacteria under lead exposure,When the highest lead concentration was 500 mg/L,the cells were not damaged;while when the lead concentration was 50 mg/L,the P2 region reached the highest level of 13.74%.It may be that when the lead concentration was high,the bacterial body of P strain was damaged seriously,resulting in less DNA,so the P2 region was low.Lead exposure had an effect on the antioxidant properties ofLactobacillus stress response.With the increase of lead concentration,DPPH·scavenging rate and OH· scavenging rate increased at first and then decreased,while the anti-lipid peroxidation rate decreased.There was no significant effect on the antioxidant properties of iron ions.The DPPH· and OH· scavenging rates of strain R were significantly higher than those of strain P,but the anti-lipid peroxidation rate of strain R was lower than that of strain P.(3)Adsorption of lead by lactic acid bacteriaThe adsorption of lead by lactic acid bacteria was studied at different lead concentrations.The results showed that both lactic acid bacteria could adsorb lead,and the adsorption rate of R strain was higher than that of P strain at different lead concentration,and the highest adsorption rate of two strain were 95.78% and 87.9%respectively when the concentration of lead was 50mg/L.By observing the effects of various eluents on the adsorption stability of lactic acid bacteria to lead,it was found that the eluting effect of 1mmol/L EDTA was the best among the two strains,and the eluting effect of pure water was the worst.Among them,the content of lead adsorbed on R strain was more than that of P strain,so the adsorption of lead by R strain was more stable and not easy to be eluted.Among the cell components,the cell wall had the highest lead adsorption rate,followed by protoplasts.The cell wall and protoplast of strain R had the highest lead adsorption capacity of 70% and 24% respectively,which were higher than that of strain P(50% and 20%).Scanning electron microscopy and transmission electron microscopy(TEM)showed that the cells of strain R exposed to lead were intact and plump,and a large number of white particles were adsorbed on the surface,while strain P had damaged cells and fewer white particles were adsorbed on the surface.(4)Study on the mechanism of lactic acid bacteria alleviating lead toxicity by using C.elegans as a modelBoth lactic acid bacteria strains can be colonized in the worm,which provides a possibility forstudying the mechanism of lactic acid bacteria protecting nematodes exposed to lead.It was found that there was a linear correlation between lead concentration and nematode longevity in the selection of lead-lethal nematode concentration.400 mg/L and 800 mg/L were selected to study the protective effect of lactic acid bacteria on nematodes exposed to lead.The results showed that both strains could prolong the lifespan of nematodes exposure to lead,but the ratio of r strain to p strain could prolong the life span of nematodes by 4 days and 3 days,respectively,and the survival rate on the last day was 11.11% when the concentration of lead was400mg/L.When the lead concentration was 800 mg/L,strain R and strain P could prolong the life span of nematodes by 5 and 4 days respectively,and the survival rate on the last day was 15.66% and 13.16%,respectively.Therefore,strain R had a stronger effect in protecting nematodes.The gene expression of nematodes in different treatment groups was studied.It was found that the expression of hsp-16.2,mtl-1 and mtl-2 was up-regulated 14-fold,8-fold and 11-fold respectively in the lead exposure group,and6-fold in the lead exposure group.So these genes are designed to cope with environmental stimuli.However,SOD-3,a significantly changed antioxidant-relatedgene,was up-regulated 11 times in the R-strain protection group and 9 times in the P-protected group.Therefore,the presence of lactic acid bacteria can significantly up-regulate the antioxidant genes of nematodes,which may be used to alleviate nematode lead poisoning. |
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