| The heavy metal lead remains in food and feed through air,water,and soil pollution,and it is still a difficult problem to pose a serious threat to human andanimal health.There are few researches on heavy metals biosorbents that used in food,feed,human body and animal body,and most of them focus on lactic acidbacteria.Yeast and lactic acid bacteria can be mixed fermentation,supplementing many deficiencies in the application of single lactic acid bacteria fermentation.It can solve the harm caused by heavy metal pollution to food,human and animal to a great extent.In this study,6 yeast strains that isolated from heavy metal pollution areas in Inner Mongoliaas as the research objects.They have high adsorption and tolerance to heavy metal lead and can be used in food.In this project,firstly,the 6 yeast strains were screened for stress resistance and antioxidant capacity,and further explored adsorption characteristics and adsorption mechanism of the strains that were excellent selected.The mechanism of high concentration lead tolerance was analyzed by transcriptome technology.Finally,The animal model was used to verify the effect of alleviating lead poisoning.The research contents and results were listed as follows:Firstly,6 strains were screened by their beneficial properties with acid-base,NaCl tolerance,survivability under simulated gastrointestinal environment,and anti-oxidant capacity.It was found that all of 6 strains showed high resistance to acidand alkali,and could adapt to gastrointestinal tract of the acidic environment.W.anomalus QF11 grew best at pH 6;5 strains were resistant to NaCl with a co-ncentration of less than 6%,of which W.anomalus QF11 and W.anomalus QD28 showed the strongest NaCl tolerance.The survival rate of 5 strains in simulated gastrointestinal showed that W.anomalus QF11,M.chrysoperlae QK15,and M chrysoperlae QE112 contained higher survival rates.After 8 h,the survival rate of W.anomalus QF11 was 75.42%which showed the best activity;The result of the DPPH scavenging rate,hydroxyl free radical scavenging rate,superoxide anion scavenging rate and anti-lipid peroxidation ability of the three strains of bacteria showed that all the strains had certain abilities,especially inhibited lipid peroxidation which were much higher than Vc(100mg/mL).Two species of W.anomalus QF11 and M.chrysoperlae QK15 with different resistan(?)were chosen to conduct the following tests.Taking the adsorption capacity as the evaluation index,the ability of adsorbing ions in vitro of the two strains of W.anomalus QF11 and M.chrysoperlae QK15 was analyzed by investigating the influence of environmental factors.The resu-Its showed that pH value,mass concentration of Pb2+ Solution and the adsorption time possessed the same effect on the two strains,while the effect of mass con-centration was slightly different.Through the response surface optimization method,the optimal conditions for the two strains to adsorb Pb2+ were optimized.The optimal Pb2+ adsorption parameters of W.anomalus QF11 were when the bacterial cell concentration was 10.64 g/L,Pb2+concentration was 81.63 mg/L,pH value was 5.42,and the predicted adsorption capacity was 7.81mg/g,while the optimal Pb2+ adsorption parameters of M.chrysoperlae QK15 were when the cell concentra-tion was 12.46 g/L,the Pb2+concentration was 110.32 mg/L,and the pH value was 5.51.At this time,the Pb2+ predicted adsorption capacity was 5.80 mg/g.For clarifying the Pb2+ adsorption mechanism of two strains,we conducted the chemical reagent treatment test,adsorption stability test,scanning electron microscopy and transmission electron microscopy combined energy spectrum test,group masking,and Fourier infrared spectroscopy test.The results showed that most of the Pb2+ adsorbed by the two strains would be resolved,which proved that the Pb2+ adsorption mechanism of the two strains was mainly about the surface adsorption.The results of scanning electron microscope and transmission electron microscope could also prove that the Pb2+was adsorbed on the surface of the bacteria and accumulated a little in the cell.The main part of the bacteria that adsorbed heavy metals was the cell wall,and the amino,carboxyl,and phosphate groups on the surface of the bacteria played an important role in the Pb2+adsorption;The groups that participated in the Pb2+ adsorption were the protein amide on the cell wall band I,-CN-,-OH,-NH,phosphoric acid group(P=O,P-OH,)of the amine group,polysaccharide components on the cell wall,-CH2 group on the cell membrane,etc.The mechanism of resistance of W.anomalus QF11 to Pb2+was studied throughRNA-seq Transcriptomics.The results showed that there were 3638 differentially expressed genes identified between the Control group and the lead-treated group.Among them,1724 genes were up-regulated and 1914 genes were downregulated.3638 differentially expressed genes were significantly enriched in 29 GO terms(Padjust<0.05).These genes were mainly involved in cytoplasmic part;non-membrane-bounded;ribonucleoprotein complex;ncRNA metabolic process etc.The results ofsignificant enrichment analysis of KEGG pathway showed that 3638 differentially expressed genes were involved in two metabolic pathways:translation pathway andenergy metabolism pathway(Padjust<0.05).In addition the expression of a large number of genes in trehalose biosynthesis,heat shock proteins that repair cell damage,antioxidant enzymes,and glutathione metabolism pathways were up-regulated.Under the stress of Pb2+,the expression of some genes encoding transporters were up-regulated and some down-regulated.The mechanism of resistance of W.anomalus QF11 to lead involved many metabolic and biosynthetic proceses.For evaluating the effect of W.anomalus QF11 on lead relief effect of poisoning,we measured the lead content in feces,tissues,blood and oxidative stress related indicators in the liver of the lead exposure model group and W.anomalus QF11 intervention group,and observed the pathological of rat tissue sections.The results showed that compared with the blank group,rats in the lead model group had reached toxic lead poisoning and severe lead poisoning which meant the modeling was successful;Compared with the lead model group,the lead content in the blood,liver,kidney,and brain of the intervention group were all to a certain extent which could increase the lcontent in feces.The intervention effect on moderate lead poisoning was higher than that of severe lead poisoning;The liver,kidney and brain tissues of rats in the bacterial control group and the blank group were intact,and the rats in the model group Hepatic edema,Die's cavity appeared,renal tubular epithelial cells were lightly stained,granular degeneration,part of the epithelial cells fell off,cerebral hemorrhage was large and numerous,and cerebral edema appeared. |
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