Study On The Determination Of Aflatoxin-Producing Aspergillus Strains And The Conditions Of Aflatoxins Production

The aflatoxins are second fungal metabolites with highly toxic and carcinogenic properties produced by some strains of A.flavus and A.paraticus which are attented by all countries in world. However not all strains are able to produce aflatoxins, all strains of A.paraticus and parts strains of A.flavus can produce aflatoxins. It is reported by FAO that 25% crops are contaminated by aflatoxins and 2% crops lost nutritional and economical worth. It is pointed out that the research topics in this field are focusing on the determination technology of aflatoxins at present, however, the reports about the testing of aflatoxin prudoucing strains are very little. Traditional microbiologic detection methods are time consuming and easily appear false negitive results. For aflatoxin contamination grain, people only treat by chemical means to reduce the aflatoxins. This is because that we lack theoretical foundation of aflatoxin biosynthesis mechanism, condition and accumulation rule.This paper studies on three methods to distinguish aflatoxin-producing and non-producing Aspergillus strains and the accumulation rule of aflatoxin. The experiment simulates storage environment of grains to determine the optimal and minimum condition of aflatoxins production and the accumulation of aflatoxins. So we can determine the main environmental factors affecting the production of aflatoxins and predict that the strains produce aflatoxins in natural environment. This study provides the information for mechanism of aflatoxins production and given relevant prevention measures. The main results are as follows:1. The method of extracting aflatoxins from fermentation liquid is improved based on national standard using immunoaffinity columns to reduce interferences of other toxins. This method used enzyme loaded postcolumn and electrochemical detector to increase fluorescence intensity can qualitatively and quantitatively detect aflatoxin-producing strains.2. Cyclodextrin can enhance the natural fluorescence of aflatoxins. Strains are cultivated on common media used for testing mycotoxin production ability whichβ-cyclodextrin and methylatedβ-cyclodextrin is added. The aflatoxins-production strains are readily detectable by direct visualization of a bright blue or blue-green fluorescent area surrounding colonies when observed under long wave length (365-nm) UV light after 5 days of incubation at 28°C. We compare the effect and concentration ofβ-cyclodextrin and methylatedβ-cyclodextrin and pay attention to the specificity ofβ-cyclodextrin and methylatedβ-cyclodextrin to enhance fluorescence of aflatoxins. We found that methylatedβ-cyclodextrin can only enhance the fluorescence of aflatoxins and when the concentration is 6g/L the effect is best.3. The key genes aflO, aflD and aflP are selected from several structal genes in the biosynthesis pathway of aflatoxin whose concurrent expression can be used as marker of the aflatoxigenic potential of A.falvus and A.parasiticus. so the three key genes are the prerequisite for developing diagnostic RT-PCR based on gene expression by controlling up to which point the aflatoxin biosynthesis may occur or not.4. The optimal and lowest condition are selected by orthogonal experiment. This experiment simulates grain storage environment by temperature, PH and rotational speed. So we can see that changing storage condition to control aflatoxin producing is an efficient and easy method. The ventilation and temperature of storage condition and the PH of grain are efficient for aflatoxin production.5. This paper studies on the accumulation of aflatoxins which increases rapidly under the determinate optimal condition in a short time. So we can predict the model of aflatoxin production and determine the main factors influencing aflatoxin to control the aflatoxin contamination produced by Aspergillus strains during grain storage. Useful reference were provided by these results for the control of aflatoxin contamination.