| Aflatoxins are secondary metabolites produced by toxigenic strains of Aspergillus parasiticus and Aspergillus flavus and have caused worldwide concerns because of their teratogenic, mutagenic, hepatocarcinogenic and immunosuppressive properties. Among all types of aflatoxins, AFB1 is the most potent toxin and has been classified as a group 1 carcinogen by the International Agency for Research on Cancer(IARC). Aflatoxin contamination levels are variety in defferent natural feedstuff. It has the important theoretical basis and guiding significance to find out the reason and mechanism of difference of different raw material of aflatoxin contamination level, the effective prevention and control of aflatoxin contamination in the raw materials to ensure the quality and safety of feed products. Therefore, effect of the grain integrity on AFB1 production by Aspergillus parasiticus grown on the different grains and their by-products, on the basis of analysis of the influence of different raw materials may be components of the difference of aflatoxin contamination level, study the composition of the influence on AFB1 production by Aspergillus parasiticus, find out the reason and mechanism of difference of different raw material of aflatoxin contamination level. The main research contents and results are as follows: 1. Effect grain integrity of different grains and their by-products on AFB1production by Aspergillus parasiticus.The substrates(wheat, corn, soybean and peanut) and their by-products on AFB1 production by Aspergillus parasiticus were incubated at a constant temperature and humidity incubator for four time periods(0, 5, 10 and 15 days). AFB1 contents were measured by HPLC. The main results as follows:1) The results show that not sterilization and sterilization of the crushing feedstuff and their by-products with the extension of incubation time, the accumulation of AFB1 increased. AFB1 content of not sterilization samples and sterilization samples were significantly higher than their by-products in each training time point(P<0.05). AFB1 content of sterilization samples and their by-products were higher than not sterilization samples and in each training time point.2).The results show that the whole samples(wheat, corn, soybean and peanut) with the extension of incubation time, the accumulation of AFB1 increased. The maximum production of the AFB1 occurred on the 15 th day after inoculation, while that of corn appeared on the 5th day in surface sterilization treatment and high pressure sterilization treatment. The research show that they start growing part is different in different whole grains(wheat, corn, soybean and peanut). Corn and wheat start growthing in embryo first, but peanuts and soybean growth speed is faster than in soybean and wheat. The above results show that the grouth areas of different of the different materials(wheat, corn, soybean and peanut) associated with grain integrity and seed coat structure, finally produce the AFB1 content is different. This may be an important factor on AFB1 production in whole corn, wheat, peanuts, and soybeans by Aspergillus parasiticus. 2. Effect of oils and their levels on AFB1 production by Aspergillus parasiticus indifferent defatted samplesEffect of oils(wheat germ oil, corn germ oil, soybean oil, and peanut oil) on AFB1 production by Aspergillus parasiticus at the rates of 2g/100 g, 5g/100 g, 10g/100 g and 15g/100 g of substrate grown on defatted corn, defatted corn germ, defatted soybean, defatted wheat, defatted wheat germ, and defatted peanut meals, and incubated at a constant temperature and humidity incubator for four time periods(0, 5, 10 and 15 days). AFB1 contents were measured by HPLC. The main results are as follows:1) Effect of oils(wheat germ oil, corn germ oil, soybean oil, and peanut oil) on AFB1 production by Aspergillus parasiticus at the rates of 2, 5, 10 and 15g/100 g of substrate grown on the respective defatted wheat germ, defatted corn germ, defatted soybean, defatted corn, defatted wheat and defatted peanut meals. The results show that the samples showed a gradual increase in AFB1 production with an increase of incubation time from 0 to 10 days. The maximum production of the AFB1 occurred on the 15 th day after inoculation at oil doses of 2g/100 g, 5 g/100 g, 10g/100 g and 15g/100 g of substrates, while that of defatted corn, defatted soybean, defatted wheat, defatted wheat germ meals at oil doses of 15g/100 g of substrates and defatted wheat meals at oil doses of 2g/100 g and 10g/100 g of substrates appeared on the 10 th day. The results showed that the effect of oil levels on AFB1 production by Aspergillus parasiticus was time-and dose-dependent, but effect of different same added level of different oils in defatted wheat germ, defatted corn germ, defatted soybean, defatted corn, defatted wheat and defatted peanut meals on AFB1 production by Aspergillus parasiticus.2) Production of AFB1 by Aspergillus parasiticus with wheat germ oil, corn germ oil, soybean oil, and peanut oil at the rates of 2g/100 g, 5g/100 g, 10g/100 g and 15g/100 g of substrate grown on the six different defatted substrates(defatted wheat germ, defatted corn germ, defatted soybean, defatted corn, defatted wheat and defatted peanut meals), The research shows that oil level can strongly improve AFB1 production by Aspergillus parasiticus, and the results showed a significant difference between substrates in AFB1 production. When wheat germ oil, corn germ oil, soybean oil, peanut oil were added to the defatted soybean meal at the rate of 15g/100 g of substrate, the maximum production of the AFB1 is 1.4 times of the minimum value on the 10 th day after inoculation.When soybean oil was added separately to the defatted wheat germ, defatted corn germ, defatted soybean, defatted corn, defatted wheat and defatted peanut meals, the maximum production of the AFB1 is 31.3 times of the minimum value on the 10 th day after inoculation.The other three oils at a dose of 15g/100 g of substrate showed the similar tendency, a consistent pattern was found at the rate of 2g/100 g, 5g/100 g, 10g/100 g and 15g/100 g of substrate after 10 and15 days of incubation. These findings indicated that oil level can strongly improve AFB1 production by Aspergillus parasiticus, and the results showed a significant difference between substrates in AFB1 production, which may be caused by the properties of substrates or the interaction with oils. Our study provides more evidence that oil is only one of the factors for more AFB1 production, and the composition and content of the substrates may also play a role in the formation of AFB1. 3. Effects and mechanism of main composition of AFB1 by Aspergillus parasiticus inGMS liquid medium.Effect and mechanism of main compositions(amino acid, carbon, trace element, macro-nutrient and nitrogen) on AFB1 production in GMS liquid medium by Aspergillus parasiticus. The cultures were shaken at 150 cycles per min, 28℃ and harvested after 3 days. AFB1 contents were measured by HPLC, and relative expression levels were measured by quantitative Real-time PCR technique. The main results are as follows:1) The research shows that difference of content of Aspartic acid, Glutamic acid, Phenylalanine, Leucine, Lysine, Alanine Glycune and Methionine between several times more, and the difference of content of calcium and sodium is smaller, difference of content of others is bigger. The determination of carbon in raw material difference is small, low content and high content of less than 1.5 times. The differences of content of the nitrogen source in about 10 times in different materials.2) Aspartic acid, Glutamic acid, Phenylalanine, Leucine supported good toxin yield than Lysine, Alanine, Glycune. The optimal levels of iron, copper, manganese and zinc for aflatoxin production is 30mg/L, 24mg/L, 30mg/L and 10mg/L, respectively. The optimal level of magnesium and potassium for aflatoxin production is 0.4% and 0.1%, respectively. The optimal level of carbon(45%), nitrogen(4%) and carbon nitrogen ratio(4:1) stimulation of AFB1 production is most obvious. The above results show that different nutrients can promote AFB1 production by Aspergillus parasiticus in synthetic medium, and the optimal level of AFB1 production varies with nutrients.3) Compared with control group, aspartic acid of 1% could significantly increase afl R, afl O, afl D and afl U gene expression(P < 0.05). Phenylalanine and leucine added level at 2% and 0.5% respectively could significantly increase afl R, afl D, afl U, afl S and afl O gene expression(P < 0.05). Glutamic acid of 1% could markedly increase afl D, afl R, afl S and afl O gene expression(P < 0.05). Lysine could raise the trend of afl R, afl D, afl U, afl S and afl O gene expression, but no significant difference compared with control group. Iron(30 mg/L) significantly increase afl D, afl O and afl S gene expression(P < 0.05). Copper(24 mg/L) significantly increase afl R, afl D, afl S and afl O gene expression(P < 0.05). Zinc added level at 10 mg/kg significantly raised afl R and afl U gene expression(P < 0.05). Manganese(30mg/kg) significantly raise afl R, afl D, afl U and afl S gene expression(P < 0.05). Potassium(0.4%) and magnesium(0.1%) significantly increases afl R, afl S and afl U gene expression(P < 0.05) and afl R, afl D, afl U, afl S and afl O gene expression(P < 0.05), respectively. Carbon(45%) afl R, afl D, afl U and afl S gene expression was significantly increased(P < 0.05). Nitrogen(4%) compared with the control group significantly raised afl U, afl S and afl O gene expression(P < 0.05). The above results indicate that the appropriate level of different nutrients could increase AFB1 production, also could rise key genes(afl R, afl S, afl O, afl U and afl D) the relative expression in varying degrees in aflatoxin biosynthesis by Aspergillus parasiticus.In conclusion, this research mainly conclusions are as follows: 1) the integrity of seed and its coat of structure affect AFB1 production in different feedstuff by Aspergillus parasiticus. 2) The results showed that the effect of oil levels on AFB1 production by Aspergillus parasiticus was time-and dose-dependent. Oil is only one of the factors for more AFB1 production, and the composition and content of the substrates may also play a role in the formation of AFB1. 3) The results showed that the appropriate level of aspartic acid, glutamic acid, phenylalanine, leucine, iron, copper, manganese and magnesium could rise key genes(afl R, afl S, afl O, afl U and afl D) the relative expression in varying degrees in aflatoxin biosynthesis by Aspergillus parasiticus, and could increase AFB1 production. |
PDF Full Text Request