Study Of Biological Conditions Of Aflatoxin Production And Quality Assurance In Aflatoxin Analysis

Aflatoxins are widely distributed mycotoxins produced mainly by Aspergillus species. It can contaminate many kinds of crops products, including peanuts, corn, cottonseed, brazil nuts, pistachios, spices, figs, and copra etc. The International Agency for Research on Cancer (IARC) has classified aflatoxin B1 as a Group 1 human carcinogen. The objective of this project was to study biological control and quality assurance in aflatoxin analysis, which are two important elements of strategies to manage aflatoxins based on those foods with aflatoxin problems.Aflatoxin production was evaluated under different fermentation conditions including the temperature, pH and rotational speed. A.flavus, A.parasiticus standard strain and strains isolated from soil and peanut samples were used in the experiment. The optimal and worst conditions for aflatoxin production were tested by orthogonal experiment. This experiment simulated the temperature, pH and ventilation condition of grain storage. Aflatoxin production could be reduced by modifying storage conditions. The ventilation, temperature and pH of storage are key elements affecting aflatoxin production. Study showed that low temperature, well ventilation ,dry and alkaline store condition were good practice to avoid aflatoxin contamination.Using RT-PCR technique, Several structural genes in aflatoxin biosynthesis pathway were studied. Three genes including aflO, aflD and aflP, whose expression were found to be closely related to aflatoxin productivity, which could be used to distinguish aflatoxigenic strains from nontoxigenic strains. The results from RT-PCR were consistent with those from HPLC determination for aflatoxins.A nontoxigenic and competitive strains named A1025 was isolated by UV irradiation in this study. It could not produce aflatoxins and kept stability during 5 generation's study. Experiments were conducted to determine effect of A1025 to reduce aflatoxin production. When the spore concentration of A1025 was 10%, aflatoxins could not be detected in the fermentation broth. It was found that A1025 can effectively reduce aflatoxin production when it was inoculated in peanut and other agricultural products at different concentrations. When the inoculation concentration of A1025 is 10%, the aflatoxin in peanut is 1.26ppb, while the control group is 89.5ppb. When the inoculation concentration of A1025 reached to 15%, the aflatoxin could not be detected. The study showed that A1025 has effective inhibition for aflatoxin production in simulation experiment for peanut and other agricultural products.An HPLC method for the determination of aflatoxin B1 and total aflatoxins in peanut was validated, the routine qulity control measures were conducted and the measurement uncertainty were estimated. LOD was estimated to be 0.1μg/kg with signal to noise ratio at 3 to 1. LOQ was 0.3μg/kg(S/N=10). The correlation coefficient for B1 and G1 are 0.99982 and 0.99976 with concentrations ranging from 0 to 8μg/kg. And B2,G2 are 0.99934 and 0.99983 with concentrations from 0 to 2.4μg/kg. Positive quality control samples were analyzed to get the RSDr is 6.10% for aflatoxin B1 and 6.08% for Total aflatoxins. Using FAPAS surplus samples to get the RSDR is 7.0% for B1 and 4.8% for Total aflatoxins. Trueness were evaluated from FAPAS interlaboratory proficiency test. Z≤2 for both B1 and total aflatoxins. Blank samples were spiked and analyzed in routine quality control. The recovery for B1 and Total aflatoxins are 77.5%-86.3% and 70.4%-81.7%. An in-house positive quality control samples were prepared from a naturally contaminated whole peanut sample.The positive quality control samples were tested for homogeneity and quality control limits were estabilished.The approach in this method includes four main areas of the method which can affect the overall uncertainty: sample homogeneity, standard purity, accuracy and precision. The relative uncertainty which were calculated for each area were finally transferred to the Combined Relative Uncertainty and Expand Relative Uncertainty. Relative uncertainty from sample homogeneity (ru1) were 0.033 for aflatoxin B1 and 0.036 for total aflatoxins. Relative uncertainty from standard purity (ru2) were 0.0132 for B1 and 0.0647 for total aflatoxins. Relative uncertainty from accuracy (ru3) were 0.064 for B1 and 0.049 for total aflatoxins. Relative uncertainty from precision (ru4) were 0.070 for B1 and 0.048 for total aflatoxins. Based on above calculation, the combined relative uncertainty(ru) were 0.10 for both aflatoxin B1 and total aflatoxins. The expanded relative uncertainty(rU) is caculated using a coverage factor of 2 which give a level of confidence of 95%. The expanded uncertainty(U) were estimated as 0.20 multiply reported value of B1 or Total. The report for aflatoxin analysis is finally described as: B1( Total)±0.20×B1(total).A collaborative study was conducted to evaluate the method to determine aflatoxin B1,B2,G1,G2 and Ochratoxin A in ginseng and ginger. Coordinated by Dr. Mary Trucksess, Thirteen laboratories from 7 countries participated in this study. According to AOAC guidelines, participants validated the method and analyzed 30 blind samples including blank samples, aflatoxins spiked samples (with concentration range from 0.25 to 16.0μg/kg ), ochratoxin A spiked samples (with concentration range from 0.25 to 8.0μg/kg) and naturally contaminated samples. The test portion was extracted with methanol-0.5% aqueous sodium hydrogen carbonate solution(700+300,v/v). The sample extract was filtered, diluted with phosphate buffer and applied to immunoaffinity column containing antibodies specific for aflatoxins and ochratoxin A. The column was washed with water and the aflatoxins and ochratoxin A were eluted with methanol. Aflatoxins were quantified by reverse-phase liquid chromatography (HPLC) with electrochemical post column derivatization. The detection was achieved by fluorescence. In-house validation showed that the correlation coefficient for B1 and Ochratoxin A were 0.9999 and 0.99998 with concentrations ranging from 0 to 4.0ng/ml. B2 and G2 were 0.9997 and 0.9996 with concentrations 0 to 1.0 ng/ml. G1 was 0.9999 with concentration from 0 to 2.0 ng/ml. Recoveries by using spiked samples were 80% to 90% for total aflatoxins and 85% to 95% for Ochratoxin A. Collaborative study showed that RSDR was 5.7-28.6% for total aflatoxins and 5.5-10.7% for ochratoxin A. HorRat value was less than 2. The method showed acceptable accuracy and could satisfy analytical requirements to determine total aflatoxins and ochratoxin A simultaneously.The assessment was conducted according to ISO 13528: 2005(E) and the International Harmonized Protocol for Proficiency Testing. 95 laboratories participated in the study. The analytic samples for this testing scheme were prepared from naturally contaminated peanut butter. The Ss≤0.3σptest was used to evaluate the homogeneity of the test samples; Sample stability was confirmed with x ? y≤0.3σp. The performance of each laboratory was designated by a z-score that was calculated using robust statistics. The robust mean of the participants'results in this study was nearly coincident with the median. A modified Horwitz equation was used to determine the standard deviation. Laboratories whose performance ratings were questionable or unsatisfactory were re-evaluated in a second interlaboratory comparison. Of the 49 laboratories that reported results for total aflatoxins, 46 performed satisfactorily. 3 performed questionable. Of the 80 laboratories that reported results for aflatoxin B1, 73 performed satisfactorily. 4 performed questionable. 3 performed unsatisfactory; The analytic results were obtained by four methods: high performance liquid chromatography, enzyme-linked immunosorbent assay (ELISA), fluorometry and liquid chromatography with tandem mass spectrometry (LC/MS/MS).