Alleviative Effects Of Bacillus Coagulans XY2 On Copper-induced Toxicity And Its Mechanism Research

Copper（Cu）is an essential trace element that plays an important role in human metabolism,but it is toxic to organisms at a high concentration level.As an environmental pollutant,copper can be enriched into the human body through the food chain,and its overload is associated with various human disease states.The traditional treatment strategy is using metal chelating agents to promote the excretion of copper salts with certain side effects and limitations.Probiotics,regarded as beneficial microorganisms for promoting human health,have functions of antioxidant capacity,immune-enhancing properties,intestinal barrier protection and intestinal microbiota modulation.Several studies have reported that probiotics show positive effects on alleviating and intervening heavy metals toxicity.However,evidence for relieving copper-induced toxicity by probiotics is still limited.Therefore,research on screening probiotics with the potential ability to resist copper toxicity and finding the mechanism is of great significance to reduce the harm of copper overload.In this thesis,we used zebrafish larvae to construct a in vivo screening model and one novel strain named Bacillus coagulans XY2 was discovered with the best protective activity,and its possible mechanism was explored.The main results are as follows:（1）A zebrafish in vivo screening model was constructed.Using the mortality rate as the evaluation index,strain XY2 was screened out with a good alleviation effect on the toxicity induced by Cu SO₄.This strain was identified as B.coagulans by 16S r RNA gene sequencing.Physical and chemical tests results indicated that B.coagulans XY2could utilize 36 kinds of carbon sources and was tolerant to the acidic environment.The in vitro and in vivo safety were confirmed by hemolysis tests and acute oral toxicity tests in mice.（2）The zebrafish model was further used to study the mechanism of B.coagulans XY2 alleviating the toxicity of Cu SO₄.The results indicated that B.coagulans XY2exhibited a high in vitro antioxidant activity,and alleviated Cu SO₄-induced oxidative stress in vivo by regulating enzymatic activities of SOD,GSH-Px and gene transcriptional levels of sod1,gstp1 and cat.The intervention of B.coagulans XY2reduced the neutrophils infiltration level in the larvae lateral line under Cu SO₄ exposure and inhibited the expression level changes of inflammatory cytokine genes il-1βand il-10 induced by Cu SO4,and alleviated the inflammatory damage of zebrafish.（3）The mice model was used to study the effects of Cu SO₄ and B.coagulans XY2on gut microbiota.The results indicated that Cu SO₄ exposure reduced the diversity and richness of the microbiota,and the intervention of B.coagulans XY2 could restore the Chao index,Ace index,Shannon index and Simpson index.There were significant differences in the microbiota structure among the groups.The intervention of B.coagulans XY2 significantly inhibited the down-regulation of the abundances of Coriobacteriaceae＿UCG-002 and Intestinimonas caused by Cu SO₄ treatment,and also caused changes in the abundances of Desulfovibrio,Alistipes and norank＿f＿Oscillospiraceae,suggesting that B.coagulans XY2 might alleviate copper toxicity by modulating the intestinal microbiota.（4）The whole genome of B.coagulans XY2 was sequenced by third-generation sequencing technology Oxford Nanopore Technologies（ONT）.The predicted genome length was 3299321 bp,and the GC content was 46.88%.The predicted coding gene length was 2719974 bp and the number of coding genes was 3274.93.03%of the genes were annotated to B.coagulans in the Nr database annotation results.16 antioxidant activity-related genes and 2 immune system-related genes were found in the GO database annotation results.These results provided a reference and basis for genomic information for future research on B.coagulans XY2.