The Isolation And Study Of Strain Which Effectively Remove ZEN And Positively Regulate The Intestinal Flora

Mycotoxin contamination has become an important factor leading to the global food safety problem and aroused wide concern.Zearalenone(ZEN),also known as F-2 toxin,is a non-steroidal toxin that is mainly produced by the fusarium.ZEN has strong estrogen effect,hepatotoxic,immunotoxic,genotoxic and reproductive toxicity,and can break the homeostasis of intestinal flora in body.Mycotoxins contaminate 25%of food and crops worldwide annually,causing economic losses in food production and livestock farming and harming the health of animals and humans.Therefore,it is imperative to remove the ZEN from contaminated grain and feed effectively.Compared with the traditional physical and chemical methods,biodegradation has the advantages of specificity,mild condition,non-destruction of nutrients and no secondary pollution,and has a good application prospect.Therefore,it has become the key method of ZEN detoxification research.The aim of this paper is to select a dual functional strain that can not only degrade ZEN effectively in vitro and vivo but also regulate the intestinal flora in vivo.The fifty strains of Bacillus were isolated from the contaminated feed by high temperature treatment and using ZEN as sole carbon and energy.Then preliminarily determined the degrading ability of all the strains using ELISA kit.Among them,the strain numbered 7showed the ability to degrade ZEN efficiently,and could degrade 90.4%of ZEN.The growth of the strain and the removal of ZEN were further detected by adding ZEN as the only carbon and energy source and strain 7 in minimal medium.The results showed that strain 7 could only grow in the presence of ZEN,while the concentration of ZEN decreased with the growth of strain 7,and ZEN was finally fully utilized by this strain.Therefore,strain 7 grows on the minimal medium by using ZEN and removes ZEN by degrading this toxin.ZEN degrading strain was identified as a Bacillus cereus by morphological identification,physiological and biochemical identification,16S rDNA sequence analysis and phylogenetic position analysis,which was temporarily named BC7strain.Degrading characteristics of BC7 strain in LB medium and simulated gastric fluid were detected by HPLC.The results showed that the BC7 strain could degrade 100%and89.31%of ZEN in LB medium and simulated gastric fluid in 24 hours,showing the effective degradation.Furthermore,the degradation of ZEN and the regulation of intestinal flora by BC7 strain in vivo were studied by constructing a mouse poisoning model.Forty female Balb/c of mice aged 3 weeks were randomly divided into 4 groups and treated with intragastric administration as follow:control group:200?L normal saline,BC7 group:200?L BC7culture(CFU=3.45×10~8/mL),ZEN group:200?L ZEN(10 mg/kg BW)and BC7+ZEN group:200?L BC7 culture(CFU=3.45×10~8/mL)+200?L ZEN(10 mg/kg BW).By measuring the weight of uterus,liver and other organs,observe the uterus,ovaries and liver tissue pathological slices and the detection of estradiol(E2),luteinizing hormone(LH),aspertate aminotransferase(AST)and third transaminase(ALT)levels to evaluate the efficacy of the degradation strains for the degradation of ZEN in the body.The results showed that:ZEN had severe damage to the uterus,ovary and liver,which significantly increased the relative weight of the uterus and liver(p<0.05).In addition,ZEN significantly increased the relative weight of kidney(p<0.05),decreased the relative weight of spleen(p<0.05),and decreased the relative weight of thymus significantly(p<0.01).The pathological results of the uterus showed that ZEN caused thinning myometrium,infiltrating eosinophils and fewer anomalous submucosal glands.The pathological results of the ovary showed that ZEN caused increased numbers of non-functional follicles,a thickened granular layer and uneven follicular antral fluid.The pathological results of liver showed that ZEN caused hepatocyte swelling,obliteration of blood sinusoids,degeneration of cytoplasmic contents,increases numbers of binucleated hepatocytes,cells with large nuclei and a disordered arrangement of hepatic cords.Moreover,ZEN significantly increased LH content(p<0.05),significantly decreased E2content(p<0.01),and significantly increased AST and ALT activity(p<0.05).The BC7strain had no significant effect on all the above detection indexes,showing certain safety.Adding the strain BC7,the relative weight of the uterus and liver was significant decreased(p<0.05),the relative weight of thymus gland was significantly increased(p<0.05),LH,AST and ALT were significantly reduced(p<0.05),E2 levels was increased significantly(p<0.05).Among them,E2,AST and ALT levels were close to normal levels.A healthy gut microbiome plays a vital role in maintaining health of the host organism.Mycotoxins can cause intestinal flora imbalance by removing beneficial bacteria and increasing intestinal pathogenic microorganisms.The experiment used the similarity of16S rDNA sequence to detect the microbial diversity of intestinal flora in mice to observe the effect of ZEN and its degradation strain on intestinal flora.Our results indicated that ZEN can destroy the balance of intestinal flora,decrease the abundance of Firmicutes,Blautia and Lactobacillus and increase the abundance of Bacteroidetes,Bacteroides and Desulfovibrio.The BC7 strain had no harmful effect on the composition and abundance of mice intestinal flora,and it also increased the abundance of Lactobacillus and Lachnospiraceae.Adding BC7 strain can restore the broken balance of intestinal flora by ZEN,decrease the abundance of Bacteroides and Desulfovibrio,increase the abundance of Blautia and Lactobacillus.Therefore,the following conclusions are drawn in this paper:BC7 strains can efficiently degrade ZEN in vitro,ease the poisoning effect caused by ZEN in mice effectively,regulate the disturbance of intestinal flora caused by ZEN in vivo,and also promote the growth of probiotics in the gut.