Separation And Characteristics Of The Products Derived From Biodegraded Aflatoxin B₁

More than a quarter of grains are contaminated with mycotoxins in the world every year, which causes huge economic losses and arouses widespread concern in food safety. Therefore, aflatoxins are always been a major threat to the agriculture and food industry. In this thesis, the degradation of aflatoxin Bi with Pseudomonas stutzeri F4, the seperation, characteristics and the mutagenicity of the degradation products were investigated. The main results are as follows:1. AFB1(5μg/mL) was degraded by using F4cell suspension.86.6%of AFB1were degraded after72h cultivation, and no AFB1was detected at102h. Only10%degradation efficacy was kept when the cell suspension treated with SDS. In addition, the cell-free supernatant of the degraded solution taken from different time point also had the degradation ability to AFB1residue, and in which,60h sample showed the best degradation activity to84.26%after continuous cultivation for48h. When the60h supernatant treated with proteinase K and SDS, the degradation efficiency were decreased to45.42%and13.62%, respectively. This result showed that the active protein produced by F4was digested or inhibited by proteinase K and SDS. Meanwhile, Induction of1μg/mL of AFB1had no effect on the degradation activity of F4strain. It was indicated that a non-induced intracellular enzyme produced by F4was responsible for the degradation of AFB1.2. Separation and characteristics of the products derived from biodegraded AFB1. The possible maximum absorption wavelength (200nm～210nm and250nm～260nm) of biodegradation products from AFB1were determined by ultraviolet-visible wavelength scanning. HPLC gradient ultraviolet wavelength (every5nm) had detected that two new materials absorb at205nm produced, and were named product1and product2preliminarily. The two products had an obvious transformation to AFB1by detecting all time point samples. Product1could generate a faint yellow fluorescence by TLC detection. The first stage of mass (Q-TOF/MS) spectrum showed that the molecular weight of product2was206.0564, and the molecular formula was C11H10O6. Second stage of mass (Q-TOF/MS2) spectrums of AFB1and product2had many the same ion fragments, which indicated that product2had a high homology with AFB1parent structure, and further confirmed that product2was one of the AFB1biodegradation products. Also, the possible structural formula of product2was obtained by analyzing its polarity, the structures of its fragments and the pH change before and after degradation, named6-Methoxy-[4,9] benzo-2,10,11,13-tetrahydro-[11,13-oxirane] difuran. Thus, these results provide a basis for the pathway of AFB1biodegraded by F4.3. The mutagenicity of the degradation products was investigated by Ames test. The0h degradation solution (containing AFB1), the control of the0h degradation solution (no AFB1exist), the102h degradation solution (containing the products), and the control of the102h degradation solution (no products exist) were detected respectively by using a group of Ames test standard strains (TA97、TA98、TA100、 TA102) which had been qualified by the biological characteristics experiments. The number of revertant colonies on the four strains representing the102h degradation solution had a similar effect to the control of102h both in the terms of metabolic activation (S9+) and without metabolic activation (S9-), and had no concentration-related relationship existed over the four concentration gradients. Although the revertant colonies of the control of102h had a part of increase compared to the solvent control, but there was no concentration-related relationship existed. It is likely that the metabolites produced by F4had a micro-mutagenic effect. There was a sharp increase of the TA97(S9-)、TA98(S9-) and TA102(S9+) revertant colonies representing the0h degradation solution. Also, a distinct concentration-related relationship was existed. These phenomenons proved that AFB1had a high mutagenicity too. The condition of the revertant colonies representing the control of0h was consistent with the solvent control both in the terms of S9+and S9-. The results showed that the mutagenicity of AFB1was reduced greatly after102h biodegradation, and the total of the degradation products was classified as negative mutagenicity, that means low or no toxicity. It was implied that AFB1was degrded to low or no toxic products by Pseudomonas stutzeri F4.