Analysis Of Intervention Effect Of Pichia Guilliermondii On Toxity Of AFB₁ And T-2 Toxin In Dried Red Fish Based On Intestinal Bacteria

Dried fish is a kind of flavor salted aquatic product in coastal areas,which is easily polluted by fungi and its mycotoxins in the environment during processing and storage.According to incomplete statistics,dried fish are generally seriously polluted by Fusarium spp.and Aspergillus spp.,and also contained high levels of aflatoxin B1(AFB1)and T-2 toxin(T-2).AFB1 and T-2 are highly toxic secondary metabolites of fungi,which will cause serious damage to the liver and immune system,and can also induce structural disorder of intestinal bacteria.Therefore,the ingestion of AFB1 and T-2 contaminated dried fish will bring potential safety risks to public health.So,the reduction toxicity of AFB1 and T-2 in dried fish products is one of the key scientific issues to prevent and control its harm.The research was showed that Saccharomyce and its metabolites can protect animal from mycotoxin.It can not only significantly alleviate the symptoms of mycotoxin poisoning,but also improve the gastrointestinal function and digestive capacity of animals.However,there are few reports on the mechanism of Saccharomyce in eliminating mycotoxin toxicity in animals.At the same time,the results of macrogenomics research have shown that intestinal bacteria is very important to the immune and liver function of the host.The changes of its quantity and composition may in turn affect the health of the host,indicating that intestinal flora may be a potential target for yeast to eliminate mycotoxin toxicity.Therefore,yeast fermentation can be used to intervene mice which exposed to AFB1 and T-2 toxin of dried fish,and study the mechanism of damage intervention of yeast fermentation on the body exposed to AFB1 and T-2 toxin in the dried fish,so as to provide a theoretical basis for reducing the damage of AFB1 and T-2 toxin of dried fish in the future by adding yeast fermentation.In this study,two kinds of mycotoxins,AFB1 and T-2,which were the most polluted in dried red fish,and Pichia guilliermondii MH 211588.1(PG),which was isolated by CAMT team at the early stage were selected,respectively administration dried red fish+AFB1,dried red fish+T-2,PG fermentation+dried red fish+AFB1 and PG fermentation+dried red fish+T-2 to SPF mice and pseudo-germfree mice(PM).After 7 days,the changes of peripheral blood cells were measured by blood analyzer,the activities of liver function enzymes were measured by microplate spectrophotometer,and the number and composition of intestinal bacteria were measured by high-throughput sequencing.In orger to explore the intervention effect of PG fermentation on the immune damage,coagulation disorder,liver damage and intestinal bacteria disorder of AFB1 and T-2 in dried fish fed with mice,pearson correlation coefficient analysis method was used to screen the related bacteria,and the self-established PM mice model was used to verify whether the intestinal flora is the target of PG fermentation.Conclusion:(1)Immunotoxicity and hepatotoxicity of AFB1 and T-2 toxins in dried red fish to SPF mice and intervention of PG fermentation:AFB1 in the dried red fish can significantly reduce the number of white blood cells(WBC)and lymphocyte(LYM)in the blood of mice(P<0.05);the number of platelet(PLT),the activity of acid phosphatase(ACP)and alkaline phosphatase(AKP),and the ratio of aspartate aminotransferase alanine aminotransferaseand(AST/ALT)increased(P<0.05),indicating that AFB1 toxin in dried red fish has obvious immunotoxi city and hepatotoxicity.At the same time,T-2 toxin in the dried red fish can significantly reduce the blood index of mice and significantly increase the activity of functional enzymes in the liver,indicating that T-2 toxin in the dried red fish has obvious immunotoxi city,hematotoxicity and hepatotoxicity.After adding PG fermentation,there was no difference in every index between PG intervention group and control group,indicating that PG fermentation had a good intervention effect on the immunotoxicity and hepatotoxicity of AFB1 and T-2 toxin in the dried red fish.(2)Effects on intestinal bacteria disorder of AFB1 and T-2 toxins in dried red fish and intervention of PG fermentation:The results showed that AFB1 and T-2 toxin in the dried red fish could significantly reduce bacterial alpha and beta diversity of intestine,while AFB1 and T-2 toxin in the dried red fish could significantly change the bacterial composition of intestine,mainly affecting the relative abundance of Prevotellaceae,Bacteroidales S24-7,Clostridiaceae,Alcaligenaceae.These bacteria were significantly correlated with blood index and liver function enzyme activity(P<0.05).After adding PG fermentation,the bacterial diversity and abundance of intestine were no different from those in the control group,and the correlation between intestinal bacteria and blood index and liver function enzyme activity was significantly reduced,which indicated that PG fermentation had a better intervention effect on the changes of bacterial structure and composition in intestine induced by AFB1 and T-2 toxin,and might intervene the toxicity of AFB1 and T-2 toxin by regulating the abundance of Prevotellaceae,Bacteroidales S24-7,Clostridiaceae,Alcaligenaceae.(3)Immunotoxicity and hepatotoxicity of AFB1 and T-2 toxins in dried red fish to pseudo-germfree mice and intervention of PG fermentation:After 3 days of administration of vancomycin,neomycin and metronidazole,most of the predominant intestinal bacteria of normal SPF mice were killed,and the number and bacterial species in intestine was decreased significantly(P<0.05).The PM mice model constructed was met the experimental requirements.Compared with SPF mice,WBC,LYM,PLT and other blood indexes in peripheral blood of PM mice fed AFB1 and T-2 toxin in dried red fish was increased significantly(P<0.05),ACP,AKP and AST/ALT enzyme activities was decreased significantly(P<0.05).The results showed that immunotoxi city,hematotoxicity and hepatotoxi city of AFB1 and T-2 toxin were related to the number and species of intestinal bacteria.At the same time,PG fermentation was significantly decreased WBC,LYM,PLT and other blood indexes in PM mice fed AFB1 and T-2 toxin(P<0.05),ACP,AKP and AST/ALT enzyme activities was significantly increased(P<0.05),indicating that the intervention effect of PG fermentation on toxicity of AFB1 and T-2 toxin in dried red fish is weakened,indicating that the number and species of intestinal bacteria is the target of PG fermentation to exert its intervention effect on the toxicity of AFB1 and T-2 toxin in dried red fish.