Study On The Mechanism Of High Efficient Degradation Of Aflatoxin B1 By Intestinal Cobacteria Of Hermetia Illucens L

Aflatoxin is a class of stimulating metabolites produced by fungi such as Aspergillus flavus and Aspergillus parasiticus,and is widely found in various crops,with strong toxicity,carcinogenicity,teratogenicity and hepatotoxic effects.The physicochemical properties of aflatoxins are so stable that the processing of Park’s food is difficult to disrupt,posing a serious threat to humans and animals,and causing huge economic losses.Biodegradation of aflatoxins has received widespread attention due to its mild reaction conditions,non-polluting,no secondary contamination and loss of nutritional value of the product.The black water gadfly is able to tolerate high concentrations of aflatoxin and is widely used to biodegrade and utilize contaminated feed into insect protein and other resources.In this study,an ultra performance liquid chromatographic(UPLC)tandem four-stage time-of-flight mass spectrometry(TFS)method was established for the determination of aflatoxin B1 in blackwater gadfly larvae raised on contaminated peanut cakes.AFB1 was extracted from blackwater gadfly larval powder using methanol-aqueous solution(8:2,v/v)with the addition of 7%NaCl,purified by immunoaffinity column,gradient elution with methanol-ammonium acetate(10 mmol/L)aqueous solution as mobile phase,and detected by electrospray ionization(ESI)in positive ion mode with 7.0 min peak.The results showed that the linearity of AFB1 was good in the concentration range of 1 μg/kg-20 μg/kg with correlation coefficient>0.999,the recoveries of larval powder were 83.06%-118.36%and the relative standard deviations(RSDs)were 6%-10%,the recoveries of worm droppings were 76.6%-114.1%and the relative standard deviations(RSDs)were 0-3%,the limit of detection(LOD,S/N≥3)was 0.01μg/kg and the limit of quantification(LOQ,S/N≥10)was 0.05 μg/kg,and the method is simple and accurate.Screening strains that efficiently degraded aflatoxin B1 using the gut of black fly fly larvae as materials.In this experiment,coumarin was used as the only carbon source and energy source.After enrichment and screening,a strain that degraded AFB1 efficiently was obtained.According to the 16S rRNA sequence analysis,morphology,physiological and biochemical identification of the strain,the strain was identified as a microphila Stenotrophomonas acidophilus,named as Stenotrophomonas microalgae A-2.The degradation of AFB1 was studied by different components of A-2 bacteria cultured at 37℃ for 72h.The results showed that the degradation rate of A-2 bacteria supernatant on AFB1 was 95%,and the degradation rate of PBS suspension on AFB1 was 17.1%The degradation rate of AFB1 by cell disruption was 7.1%.Taking the fermentation supernatant of A-2 bacteria as the research object,the supernatant of A-2 fermentation liquid was filtered with 0.22 μm filter membrane,boiling water bath for 20 min,autoclaved,and concentrated 20 times,and 0.22 μm filter membrane was used to filter.The degradation rate of AFB1 was slightly degraded by the supernatant,and the degradation rate after treatment was 84.95%.Therefore,the degradation of AFB1 by A-2 bacteria may be degraded by extracellular secretions.After the remaining three treatment methods,the degradation efficiency of AFB1 has been improved,with significant differences.The degradation capacity of A-2 bacteria fermentation supernatant was studied under time,pH and different concentrations of AFB1,which showed that the degradation efficiency of A-2 bacteria supernatant was more than 90%after 24 h,and the optimal reaction pH=8.0,It can tolerate higher concentrations of AFB1,while the degradation efficiency is above 90%.The active substance that degrades AFB1 in the supernatant of A-2 bacterial fermentation broth was separated and purified.It was found that the active substance was more polar and the degradation activity was mainly present in the effluent.Mass spectrometry detection found that the peak time of the active material was about 2.0 min.Combining the first and second mass spectra,it was found that the active material has a molecular weight of 174 and a positive charge.It is speculated that its structure may be 1-methyltryptamine or 5-Methyl tryptamine.