Study On The Aflatoxin M1Degrading Bacterium

Aflatoxin (Aflatoxin, AF) is a secondary metabolite produced by aspergillusparasiticus, aflatoxin, ochratoxin and other fungi, with high carcinogenicity, highteratogenicity and high mutagenicity. It was designated as carcinogen I by World HealthOrganization. In2011, a overproof of aflatoxin M1(AFM1) content was detected in abatch of one dairy, which is much more harmful than melamine. So AFM1became to drawattention of consumers and researchers. The present method of controlling aflatoxin M1isjust by controlling the content of its precursors aflatoxin B1in the feed, reducing theconversion of aflatoxin M1to prevent contamination of dairy products. However, AFM1has emerged in dairy products, there was no an effective way to remove to date. In recentyears, the methods of biodegradation attracted more and more attention, which has theadvantages of effectiveness, less side effect, little loss of food nutrition and even increasingthe nutritional content and so on.. According to this method, we selected a bacterial straincapable of degrading AFM1; optimized the fermentation conditions of bacterial strain;studied the mechanism of how bacterial strain degrades aflatoxin; preliminary separationand extraction of the active substance of degradation. The results were as follows:(1) Screening for strain which can degradate AFM1: In the experiment,38kinds ofanimal manure, a kinds of activated sludge samples from the sewage plant and3kinds ofsoil samples of peanut, we used coumarin as the sole carbon and energy sources topreliminary screen the objective strain, finaly we get50bacterium in total. Then we usedthe rate of degradation rate as the index to screen again, and we got a strain which candegredate the AFM1, the rate of degredation could reached89.55%. We put it as numberE-1-1-1.(2) Bacterial taxonomic identification of strain E-1-1-1: Through the partition linesand Gram staining we could see that the strain was small, round, milk white, and moist, themiddle colony was convex, the edge smooth and tidy. The bacteria was gram positive,rod-shaped and blunt ends. The identification results of Biolog display that the similarity ofthe physiological and biochemical test of E-1-1-1and bacillus pumilus was greater than0.6,the similarity of E-1-1-1and other strains was less than0.05, then finaly the result foridentification was Bacillus pumilus. Molecular identification of16SrDNA results showedthat the similar rate of E-1-1-1and Bacillus pumilus was99%Combining the colony morphology, Biolog physiological and biochemical characteristics,16SrDNA moleculesequencing as well as phylogenetic tree results of the bacteria strain, the E-1-1-1wasinitially identified as Bacillus pumilus (Bacillus pumilus) in bacterial taxonomy.(3) Optimization of conditions of degradating AFM1by Bacillus pumilus E-1-1-1:Through the single factor experiment, for E-1-1-1strain, the optimal initial pH value is6;the optimal temperature is37℃; the best inoculation is3%; Orthogonal designs wereperformed for PH, temperature and inoculums size and identified the best cultivationconditions: in the case of the temperature42℃, initial pH5.5, and inoculums size3%,degradation rate reached the peak of92.5%.(4) Study on the degradation mechanism of AFM1by Bacillus pumilus E-1-1-1: Thisstudy primarily explored the mechanism of how E-1-1-1bacteria strain removing aflatoxinM1and proposed the removing approach is degradation rather than adsorption. The activesubstances of degradation are mainly from bacterial extracellular metabolites, anddegradation rate is76.9%. The treatment of ammonium sulfate precipitation of75%forfermentation supernatant could obtain the highest protein degradation rate of54.33%. Thebest reaction temperature of42℃could obtain the highest protein degradation rate of67.5%. The ANOVA analysis through SPSS software was performed. The P value <0.05indicated the significant difference among temperature gradients and revealed thesignificant effect of temperature on activated proteins. In addition, proteins are not verystrict to the PH condition, which has a wide range from5to7. This is a essentialcharacteristic of activated protein. Precipitated proteins by75%ammonium sulfate werefurther separated by ultrafiltration, which showed the degradation of proteins withmolecular mass less than1KD was22.6%, those between1KD~3KD degradation was12.7%, between3KD~5KD was21.2%, between5KD~10KD degradation rate was7.1%,and more than10KD was30.7%. The sum degradation rate of every component is94.3%,which is close to the previously measured degradation rate (92.5%) of bacterial suspension.The potential reason is that the activated protein decomposing aflatoxin may be a complexprotein.