Isolation,Identification,Optimization And Characteristics Of An Aflatoxin B1-degrading Strain

Aspergillus flavus has tremendous harmfulness to domestic animals’health and production, endangering the food terminal and human health,so degrading toxin research is urgently needed. Biodegradation doesn’t cause secondary pollution for raw material, has higher specificity, and bold toxin recombination back. It’s an efficient and safety degradation method. This research filtrate AFB1 bacterial strain of highly efficient degradation from sample that combine the phenotype and molecule authenticate bacterial strain species aimed to research the inhibiting effect for germs, the toxic effect for experimental animals, evaluate its security, optimize the bacterial strains’growth condition, optimize the fermentation mediums’nutritional ingredient and fermoentation conditions by single factor, use response surface experiment to notable factors, make sure the optimal submerged culture to reach the best degradation ability, the preliminary study of the degradation mechanism of AFB1 high efficiency degradation bacterium, confirming the presence of degradation component parts; research the influence of the temperature and proteinase K ability, ammonium sulfate to crude extraction of active ingredients, to detect the degradation of crude enzyme activity. The main results showed as follows:l.From all samples, chosen preliminarily 33 strains of which plants grow well. The degradation rate of AFB 1 standard compound sieve combines with HPLC to detect the strain F6 was up to 83.2%. The analysis results of Bacteria, bacterial colonies properties, dyeing and biochemical identification of judgment for the Bacillus, bacterial DNA sequence amplification, 16S rRNA sequence alignment were homology to egetables international standard strains of Bacillus ATCC700005 nucleotide was 99.3%, deciding the F6 bacterial strain as Bacillus oleronius, named Bacillus oleronius GX01 (GenBank KP297896).2.Bacillus oleronius GX01 had good inhibitory to three kinds of intestinal pathogenic bacteria, especially for salmonell,the grade was++. It was resistance the penicillin, cephalosporin and forests amine drug, the sulfa and quinolones were most sensitive, the other class of antibiotics with high sensitivity. There was no significant influence for Lavage 1 x 1010CFU/(mL·kg)Bacillus oleronius GX01 for weight gain, the dirty body ratio, blood indicators index, biochemical index, liver and kidney structure for mice. Above the three results demonstrate that Bacillus oleronius GX01 has high safety, has the safety basis of applying to the product.3.Bacillus oleronius GX01 were grown well in 25-40℃ and PH 5-8 nutrient broth medium. When arrived to 34℃ and the pH of 7, fertility is the most strong. Bacterial strain GX01 was needed oxygen. The greater amount of the triangle bottle,the smaller bottles of strains in the ventilation, which was disadvantage to Bacterial strains’breeding, Strain GX01 inoculation concentration within 10%, the strain of fertility increased with the growth of inoculation concentration, more than 10%is suppressed in fertility.4. Bacillus oleronius GX01 attains the best degradating effect in the single factor experimental conditions of glucose carbon sources, nitrogen sources. pH 8.0 beef paste,6% quantity of 48 h,100/250 mL bottle liquid quantity, fermentation,37℃.10 mmol/L Mg2+ has promoted Bacillus oleronius GX01 degradation to AFB1,10 mmol/L Cu2+、Ca2+、Fe2+、 Mn2+、Zn2+、A13+、Cd2+have different degree for restrain bacterial strains’degradation property.5. On the basis of single factor optimization, the response values for AFB1 degradation rate, select the factors which has significant influence to AFB1 degradation rates’initial pH, inoculation quantity and fluid volume, building the model of quadratic response surface to optimize. Through the response surface we draw the conclusion of the highest degradation rate of AFBl,they were initial pH 7.61, quantity of 6.29%, fluid volume 93.3 mL. Experimental verify that on optimizing condition that fermentation will improve the degradation rate of AFB1 to 88.81%.6.The clear liquid on the Fermented liquid is the main degradation part of Bacillus oleronius GX01 to AFB1, degradation activity was 83.0%. The degrade activity which after heating the supernatant fluid and plus protease K were significantly decreased only 16.2%and 23.9%, which explained the substance by Bacillus oleronius GX01 degradation degrade AFB1 was secretion of the cell body to the activity of extracellular enzymes.60%saturation of ammonium sulfate precipitation supernatant of crude enzyme degradation of AFB1 performance of up to 65.6%.Is obtained by separation identification Bacillus oleronius GX01 efficient degradation of AFB1. Its security, optimization of fermentation conditions and the degradation of active site of research provide for the future safety production and application of theoretical reference basis and fermentation, lay the foundation of further studies the strains of AFB1 biodegradable.