| Cadmium（Cd）is a common heavy metal in the environment.It is characterized by high toxicity,low degradation,and strong accumulation.The human body can be exposed to Cd through food,water,air and other media.Among them,food intake is considered to be the main pathway of Cd exposure in non-occupational population.Cd can enter the human body through the food chain and accumulates,causing Cd poisoning symptoms.In human health problems caused by exposure to heavy metals in food,intestinal microorganisms play an important role in metabolizing and excreting heavy metals.In vitro bionic digestion model was built,fecal bacteria from healthy hosts were collected for intestinal microbial colonization,and the PBET-SHIME simulation digestion device was built from the perspective of multi-phase continuous dynamics to analyze the bioaccessibility of Cd in four common vegetables（pepper,mustard,pakchoi and cabbage）in different digestion phase（gastric,small intestine and colon）in a lead-zinc mining area in Guizhou province.To clarify the exposure dose of Cd in the process of human digestion,and study the effect of intestinal microorganisms on the bioaccessibility of Cd in vegetables,and its necessity in health risks assessment.By means of high-throughput sequencing,microbial cultography and other technologies,the important bacterial genera affecting the bioaccessibility of Cd were identified from the perspective of intestinal flora species composition,intestinal colony level and intestinal microflora,and the core strains with obvious Cd accumulation ability were preferentially screened.Through multiple characterization methods,whole genomics and comparative genomics,the binding characteristics and action mechanism of the target strain on Cd were clarified,and the correlation analysis of its probiotics and safety characteristics was conducted,and the influence of different prebiotics on the proliferation of the target strain was explored.The results of this study will promote the health risk assessment of dietary heavy metal exposure in a more comprehensive and scientific way,and also provide reference for future studies on human health effects based on the regulation of intestinal microorganisms to promote metabolism of heavy metal pollutants in the host body.The main research results are as follows:（1）In vitro condition,the optimized SHIME model can be used to culture human intestinal flora,so that the community composition and activity reach a stable state.The 16S r RNA results showed that Proteobacteria,Bacteroidetes and Firmicutes accounted for 57.46%,11.69%and 12.21%of the total bacterial communities in the two volunteers.It accounted for 57.16%,20.13%and 14.91%of the total bacteria in the children volunteers.Acetic acid was the main component of short-chain fatty acids（SCAFS）produced by intestinal microorganisms of two volunteers,accounting for 47.49～51.26%of total organic acids.（2）The bioaccessibility and risk quotient（HQ）of Cd in vegetables in colon phase were found to be low by PBET-SHIME model analysis.The results showed that the bioaccessibility of Cd in the colon phase was 6.98±5.96%～23.82±9.92%in adults,and in children:7.61±5.32%～24.82±10.87%,respectively,decreased about36.71～49.12%and 38.87～46.99%.Compared with the small intestine phase,the gut microbiota reduced the bioaccessibility of Cd in vegetables,and was effected by the differences in host metabolism.In the health risk assessment,the HQ calculated based on the bioaccessibility of Cd in the colon phase was the lowest,with the average HQ value of adults and children ranging from 1.25×10-3±1.53×10-6～3.33×10-1±2.15×10-3.The results indicates that the health risk assessment process considering the bioaccessibility of heavy metals in colon phase will be more scientific and reliable.（3）An gut strain ZY-2 with excellent tolerance and binding ability to Cd was selected from human gut microbiota,belonging to Enterococcus and Enterococcus faecalis strain ATCC19433.The removal rate of Cd in solution reached 49.11%within 2 hour.In vitro characterization showed that Cd binding of the strain mainly occurred in the cell wall,and a small part of Cd binding occurred in protoplasts,indicating that the mechanism of Cd binding was bioadsorption and bioaccumulation.Through FTIR and Raman,phosphoric acid group,amidoⅰgroup（N-H）and hydrocarbon group（C-H）are all involved in the binding of Cd in this strain,which involves complex reaction,ion exchange,extracellular adsorption and intracellular diffusion.（4）The coding genes associated with Cd tolerance and binding in the intestinal strain ZY-2 genome were identified.Annotated as bivalent metal ion binding genes such as znu A,znu B,znu C,mnt A,mnt B and mnt C in COG database;Annotated as divalent ion transport genes such as cop A,znt A,mnt H and czr A.The genes related to metal ion binding and transport were annotated in the GO database,such as gyr B,gdp P,rad A,isp F,sda A,hpr T,fts H,rha D,nrd B,feo A,cat E,man B,tly C,pyr C,adh E and lig A.Several two-component regulated metabolic pathways（ko02020）in the KEGG database are also involved in binding metal ions.（5）The comparative genomics found that the strain ZY-2 genome carried a large number of functional genes annotated for the beneficial effects of antibacterial activity,antioxidant activity,amino acid and vitamin synthesis,which indicated that the strain ZY-2 had probiotic properties to the host.Moreover,the analysis of mobile elements found that the genome of the strain did not have the basic conditions to cause host infection and spread drug resistance through mobile elements,such as gene island and plasmid.Therefore,the gut strain ZY-2 could be safely used as probiotics in the gene level.In conclusion,the bionic digestion model was used to confirm that gut microbiota can reduce the bioaccessibility of cadmium in vegetables,and also reduce the risk of human health caused by cadmium through diet.In vitro experiments,a variety of characterization methods and biological sequencing technology were also used to confirm the probiotics and safe strains of human intestinal flora with reduction effect on heavy metal cadmium at the gene level,which provided feasible ideas for the subsequent improvement of human health risk assessment and the use of human gut microbiota to metabolize heavy metals through dietary intake. |
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