Exploration Of The Causes Of Abnormal Aflatoxin B₁ Contamination Events In Cottonseed Meal Of A Region In The Middle And Lower Reaches Of Yangtze River

Aflatoxin B₁（AFB₁） is extremely toxic, and potently harmful to human and animal health, which attracts widespread attention around of world. We found that AFB₁ contamination of cottonseed meal in a region of the middle and lower reaches of Yangtze River was very serious in 2013, which was abnormal and lead to seriously excessive AFB₁ in feedstuffs, caused tremendous economic losses to the local animal husbandry, especially to duck industry. So it is very important for us to find out the causes of abnormal aflatoxin contamination in cottonseed meal, so as to provide a reference for preventing recurrence of such incidents. In this study, we investigated cotton varieties and natural climate in that region during 2011 to 2015, isolated and identified the fungal species in cottonseed, screened aflatoxigenic strains and analyzed their aflatoxigenicity, compared the difference of AFB₁ production of aflatoxigenic Aspergillus flavus strains in different feed raw materials, as well as studied the effects of free gossypol on the AFB₁ production of high aflatoxigenic strain, aimed to clarify the reasons why aflatoxin B₁ contamination in cottonseed meal was so serious and abnormal. The main research contents and results are as follows:1. Investigation of cotton varieties and natural climate in a region of the middle and lower reaches of Yangtze River during 2011 to 2015 We carried out a survey of the main cotton varieties and natural climate of Cotton growing periods（which mainly includes the temperature and rainfall distribution） during 2011 to 2015 in a region of the middle and lower reaches of Yangtze River, in order to analyze whether the natural climate and cotton varieties are the reasons for the abnormal AFB₁ contamination level in cottonseed meal. The results show that, when compared with other years, the temperature of flowering and boll-forming stages（at July and August） in 2013 was significantly higher, while the rainfall had no significant difference, but was abundant, which could stimulate the growth of aflatoxigenic fungi. But in September, 2013 when the cotton boll opening most, the weather was warm, and the rainfall was significantly higher, far more than other years. This kind of weather was conducive to AFB₁ contamination. And the excessive rainfall probably lead to high moisture content of cottonseed during harvest and storage, and thus more easily contaminated by molds or mycotoxins. According to the survey of cotton varieties, it was found that the main cotton varieties in 2013 were roughly same as other years.2. Isolation and identification of the fungal species in cottonseed In this part, a cottonseed contaminated naturally with 862.25±67.02 μg/kg AFB₁ was used for isolation of fungal strains. Morphological characteristics and molecular analysis of the internal transcribed spacer（ITS） of r DNA were used for the identification of the isolates. The main results are as follows: we obtained 21 fungal isolates on Czapek Dox Medium from the cottonseed. The fungal isolates were divided into 6 genera: Aspergillus, Penicillium, Alternaria, Fusarium, Leptosphaeria and Arthrinium. Aspergillus was the most frequent genus, followed by Penicillium. The most common species were Aspergillus flavus, penicillum chrysogenum and Aspergillus versicolor.3. Screening of aflatoxigenic strains and analysis of their aflatoxigenicity in cottonseed Aflatoxigenic strains were screened with the AFPA medium and confirmatory aflatoxin production by high-performance liquid chromatography（HPLC）. The main results are as follows: five Aspergillus flavus strains were both found to be aflatoxigenic strains, and their aflatoxigenicity in the PDA medium were as follows: CS05> NRRL3357> CS17> CS12> CS08> CS07. Besides, the AFB₁ production of Aspergillus flavus CS05 was 16.91±1.79 μg/plate, Significantly higher than that of of Aspergillus flavus NRRL3357（10.24±0.86 μg/plate）（P<0.05）, which is usually used as a standard aflatoxigenic strain. Aspergillus CS05 was used as the test strain in the following experiment.4. The difference of AFB₁ production by aflatoxigenic Aspergillus flavus CS05 in different feed raw materials In this experiment, aflatoxigenic Aspergillus flavus CS05 and NRRL3357 were inoculated in different feed raw materials and by-products for 10 days, then determined aflatoxin production by HPLC, in order to compare the differences of AFB₁ production and the aflatoxigenicity of aflatoxigenic Aspergillus flavus NRRL3357 and CS05 in different feed raw materials and by-products. The main results are as follows: the AFB₁ productions of Aspergillus flavus CS05 in different feed materials and by-product had obvious differences. AFB₁ production in wheat, cottonseed meal and soybean meal were relatively low; while in maize the AFB₁ production was significantly higher, and after autoclaving the result was higher than maize; In the non-sterilized cottonseed and soybean, AFB₁ was relatively low, whereas the AFB₁ was significantly increased after autoclaving. In most of feed materials and by-products, aflatoxigenicity of Aspergillus Aflavus CS05 was stronger than that of Aspergillus Aflavus NRRL3357. In non-sterilization or chemical sterilization conditions, the AFB₁ production of granular cottonseed was higher than that of crushed cottonseed, while the AFB₁ production of granular cottonseed was higher than that of crushed cottonseed after autoclaving, which indicated that the microbial and free gossypol concentration affected the production of Aspergillus Aflavus CS05 in cottonseed. Free gossypol concentrations of local cottonseed meal samples were obviously low, which indirectly reflected that free gossypol concentrations of local cottonseed were lower than other regions.5. Effects of free gossypol on growth and aflatoxigenicity of Aspergillus flavus CS05 In this experiment, we added different concentrations of free gossypol in synthetic medium, and inoculated with Aspergillus flavus CS05 for 10 days, then measured colony diameter and determined aflatoxin production by HPLC. All of these were aimed to analysis the effects of different free gossypol levels on growth and aflatoxigenicity of Aspergillus flavus CS05. The results showed that, free gossypol with a concentration of 0³00 μg/m L did not affect the growth of Aspergillus flavus CS05 in PDA medium, but inhibited AFB₁ production of Aspergillus flavus CS05, and the highest inhibition rate reached 70.22%. In Czapek Dox Medium, free gossypol could inhibit the growth of Aspergillus flavus CS05, and the growth inhibition rates of different growth periods were also different. At the same time, free gossypol could also inhibit the AFB₁ production of Aspergillus flavus CS05, when free gossypol concentration was more than or equal to 75 μg/m L, the AFB₁ inhibition rate was over 90%. In a certain range of concentration, inhibitory effect increased with the increase of free gossypol concentration, when up to a certain extent, the inhibition effect subsequently tended to be stable.At last, the main Conclusions of this research are as follows: The abnormal Aflatoxin B₁ contamination events in cottonseed meal of a region in the middle and lower reaches of Yangtze River in 2013 were caused by several factors together. Firstly, free Gossypol could inhibit the growth and aflatoxigenicity of Aspergillus flavus, and the free gossypol concentrations of local cottonseeds were generally lower than other regions, which might be the basis for the sudden emergence of abnormal AFB₁ concentration in cottonseed meal. Secondly, there was a high aflatoxigenic strain in cottonseed, whose aflatoxigenicity is stronger than that of Aspergillus flavus NRRL3357, not only in the PDA medium, but also in different feed materials and by-products. This may be one of the root causes for the abnormal AFB₁ contamination of cottonseed meal in 2013. Thirdly, the significantly hot and humid weather in July and August, 2013, and the excessive rainfalls and warm weather in September, 2013 might be the reasons for the abnormal AFB₁ contamination in cottonseed meal.