| Aflatoxins widely exist in food, feed and agricultural products. It can not only make great harm to the health of human beings and animals, but also will bring huge economic losses at the same time. The present physical and chemical methods of aflatoxin’s removal have some defects such as long processing time, low grade product, easy producing by-products and having secondary pollution and so on. However, enzymatic degradation of aflatoxin has attracted much attention because of the advantages of high efficiency, strong specificity, no environmental pollution and so on. This research has used cumarin as the only carbon source to screen the microorganism that could degrade aflatoxin B1 efficiently, Also, the fermentation technology and enzyme preparation preparative technique have been optimized. The results confirmed that the fermentation technology in 50 L stirred tank reactor and enzyme preparation preparative technique are feasible effectively, which has provided a theoretical basis for industrial production aflatoxin B1-degrading enzyme and application. The details are as follows:1. 27 strains have been screened by using cumarin as the only carbon source that can grow well in the coumarin plant. After second screening, HSD8 strains is determined to be the best strain which can produce aflatoxin B1-degrading enzyme to degrade aflatoxin B1. It’s enzyme activity reach 433 U/m L. The strain was eventually identified as Sinomonas sp. through morphological, physiological and biochemical characteristics combined with phylogenetic analysis. There is no report of the microorganism production enzyme to degrade aflatoxin B1 at home and abroad.2. The growth curve of HSD8 strains has been draw and the logarithmic phase is determined at 4-16 h. Also, the fermentation conditions of HSD8 strains was optimized by single-factor test to improve its activity of AFB1-degradation enzyme. After the strain was cultured under the optimum condition(50 m L/250 m L, initial p H 5.0, inoculation amount 8%, 37°C and 160 r/min) for 48 h, the activity of AFB1-degradation enzyme in fermentation supernatant is elevated to 548 U/m L which is 23.7% higher than the result obtained under the initial fermentation condition.3. Using single-factor test to determine optimum carbon and nitrogen source and metal ions in the HSD8’s fermentation medium. Finally, carbon source is determined to be molasses, nitrogen source be dlammonium hydrogen citrate and metal ions be Mg2+ã€Mn2+ and Ca2+. Optimal culture medium obtained by Plackett-Burman design and Central Composite Design, and the composition is molasses 3 g/L, dlammonium hydrogen citrate 10 g/L, potassium dihydrogen phosphate 2 g/L, dipotassium hydrogen phosphate trihydrate 0.4 g/L, sodium chloride 4.5 g/L, manganese sulfate monohydrate 0.6 g/L, calcium chloride 0.6 g/L, magnesium sulfate 2.16 g/L, coumarin 0.17 g/L. The actual enzyme activity 736 U/m L is close to the predicted enzyme activity 741 U/m L through model validation test. The enzyme activity is 25.54% higher then that of pre-optimization4. HSD8 strains is used to ferment in a 50 L stirred tank reactor on the basis of shaking flask fermentation conditions. It has been determined through single-factor test that the aeration rate is 20 L/min, agitation speed is 200 rpm, inoculum size is 5% and fermentation period is 36 h., Taking three consecutive batch fermentation under the optimal conditions, the average enzyme activity reaches 794 U/m L. Compared with shaking flask fermentation, inoculation amount decreases 3%, fermentation period shortened 12 h, enzyme activity increases 7.9%. Using fermentation broth to prepare enzyme preparation and choosing 10% of maltodextrin as filler and 0.2% of Arabic gum as embedding medium, with the method of centrifugal spray drying to prepare enzyme preparation. The enzyme activity is 723 U/m L and the loss of enzyme activity is 8.9%. |
