Enzymatic function of the nor-1 protein in aflatoxin biosynthesis in Aspergillus parasiticus

The nor-1 gene is involved in aflatoxin biosynthesis in Aspergillus parasiticus and was predicted to encode a norsolorinic acid ketoreductase. Recombinant Nor-1 expressed in Escherichia coli converted the 1' keto group of norsolorinic acid to the 1' hydroxyl group of averantin in crude E. coli cell extracts in the presence of NADPH. The results confirm that Nor-1 functions as a ketoreductase in vitro.     

Occurrence of aflatoxin B1 and ochratoxin A in Lebanese cultivated wheat

An extensive survey of filamentous fungi isolated from wheat grown and consumed in Lebanon and their capacity to produce aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) was conducted to assess fungi potential for producing these toxins in wheat. From the 468 samples of wheat kernel, collected at preharvest stage from different locations during 2008 and 2009 cultivation seasons, 3,260 fungi strains were isolated with 49.4% belonging to Penicillium spp. and 31.2% belonging to Aspergillus spp. Penicillium spp. was detected on wheat samples with a high amount of P. verrucosum (37.0%). Among the different Aspergillus spp. isolated, A. niger aggregate was predominant and constituted 37.3%. whereas the isolation rate of A. flavus and A. ochraceus was 32.2 and 25.6%, respectively. The ability to produce OTA and AFB₁ by isolates belonging to Aspergillus spp. and Penicillium spp. was analyzed by high performance liquid chromatography with fluorescence detector (HPLC-FLD). It was found that 57.0% of Penicillium spp. and 80% of A. ochraceus isolates tested produced OTA, respectively, at maximum concentrations of 53 and 65 μg/g CYA. As for the aflatoxinogenic ability, 45.3% of A. flavus produced AFB₁, with maximum concentration of 40 μg/g CYA. A total of 156 wheat samples were analyzed for the levels of OTA and AFB₁ by HPLC-FLD. The results showed that 23.7% were contaminated with OTA, at a concentration higher than 3 μg/kg and 35.2% of these samples were contaminated with AFB₁ at concentration higher than 2 μg/kg. The risks originating from toxin levels in wheat produced in Lebanon should be monitored to prevent their harmful effects on public health.     

Stimulation of aflatoxin B1 and T-2 toxin production by sorbic acid

Aspergillus flavus grown on yeast extract-sucrose medium produced higher amounts of aflatoxin B1 in the presence of 0.025% sorbic acid than without this chemical with a maximum at 17 days of incubation. Addition of 0.05 to 0.0125% sorbic acid stimulated T-2 toxin production of Fusarium acuminatum cultures grown on maize meal. The highest amounts of the mycotoxin were detected in 14-day-old cultures containing 0.025% sorbic acid. It is assumed that certain amounts of sorbic acid near the minimal inhibitory concentration reduce the activity of the tricarboxylic acid cycle; this may lead to an accumulation of acetyl coenzyme A, which is an essential intermediate in the biosynthesis of aflatoxin B1 and T-2 toxin.     

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.     

Study of aflatoxin B1 production by Aspergillus parasiticus in bee pollen of Greek origin

Aflatoxin B₁ (AFB₁) is a carcinogenic metabolite produced by certain Aspergillus species such as A. parasiticus and A. flavus. The beneficial properties of bee pollen have transformed this commodity into an increasingly frequent component of the human diet. As bee pollen is a substrate on which aflatoxigenic fungi can grow, AFB₁ production is likely. In the present study, we describe a method for aflatoxin B₁ determination in bee pollen utilising high pressure liquid chromatography (HPLC) with a fluorescence detector (FD). The recovery factor of the method was found to be 111% (RSD% 1.61), while the detection limit (LOD) was 0.08 ng AFB₁/g. An additional aim of this study was to investigate the growth of A. parasiticus and AFB₁ production in bee pollen. Results indicated that no mycelial growth was observed and no AFB₁ was detected in bee pollen samples containing natural microbiota throughout the entire observation period (20 days). In contrast, AFB₁ production in treated bee pollen samples (15 g pollen/flask) inoculated with A. parasiticus was significantly higher (p?≤?0.05) compared to control samples (treated but not inoculated) throughout the entire incubation period, while no mycelial growth was apparent. Maximum production was observed on the 12th day (79.29 ng AFB₁/flask and 32.44 ng AFB₁/flask for inoculated and non-inoculated bee pollen, respectively). As a result, AFB₁ production in bee pollen is likely even following a minor contamination, which could occur randomly.     

Stimulation of aflatoxin B1 and T-2 toxin production by sorbic acid.

Aspergillus flavus grown on yeast extract-sucrose medium produced higher amounts of aflatoxin B1 in the presence of 0.025% sorbic acid than without this chemical with a maximum at 17 days of incubation. Addition of 0.05 to 0.0125% sorbic acid stimulated T-2 toxin production of Fusarium acuminatum cultures grown on maize meal. The highest amounts of the mycotoxin were detected in 14-day-old cultures containing 0.025% sorbic acid. It is assumed that certain amounts of sorbic acid near the minimal inhibitory concentration reduce the activity of the tricarboxylic acid cycle; this may lead to an accumulation of acetyl coenzyme A, which is an essential intermediate in the biosynthesis of aflatoxin B1 and T-2 toxin.     

Effect of aflatoxin B1 on germination index and seedling growth in wheat varieties

The effect of different concentrations of aflatoxin B₁ (100, 250, 500, 1000, 2000 µg/l) was studied on germination index and seedling growth in three varieties of wheat seeds. Inhibition in the above process was directly influenced by the concentration of toxin. Concentration of toxin had highly significant effect (p<0.001) for seed germination rate and radicle and plumule development. Inhibition dose for 50% reduction in germination rate (ID₅₀) determined by probit analysis was maximum for the variety HP-129 (895 µg/l).     

Detoxification of aflatoxin B1 by acidogenous yoghurt

Serial concentrations of aflatoxin B₁ ranged from 200 to 1000 p.p.b. were assayed for detoxification by acidogenous yoghurt. Thin-layer chromatography analysis revealed a complete transformation of 800 p.p.b. of aflatoxin B₁ to a new fluorescing compound corresponding to aflatoxin B_2a which is referred as hydroxydihydroaflatoxin B₁. Partial conversion was present in yoghurt sample containing 1000 p.p.b. Toxicity test on chickens, confirmed Ciegler findings.     

食用菌对黄曲霉毒素B1的脱毒作用研究

目的 研究平菇、金针菇、黑木耳等七种食用菌对黄曲霉毒素B1（AFB1）的脱毒效果。方法 首先通过降解圈直径与菌落直径之比（Da/Dm）筛选出具有脱毒作用的株菌;然后通过AFB1残留量筛选出脱毒能力最强的菌株;最后将该菌株接种于含AFB1的玉米粉和大米中,考查其对粮食中AFB1的脱毒效果。结果 初筛发现平菇、黑木耳、金针菇对AFB1有较强脱毒作用,其Da/Dm分别为1.6±0.02、1.5±0.01、1.4±0.02;复筛发现黑木耳的脱毒能力最强,与AFB1共培养10 d后能清除88.16%的AFB1;进一步发现黑木耳对玉米粉和大米中AFB1有一定去除作用,清除率分别为62.4%和15.73%。结论 黑木耳对受AFB1污染的粮食作物有较好的脱毒作用,可用其控制食品与饲料中的AFB1。     

Potential for aflatoxin B1 and B2 production by Aspergillus flavus strains isolated from rice samples

In this study, we investigated the potential for aflatoxin B₁ (AFB₁) and B₂ (AFB₂) production in rice grain by 127 strains of Aspergillus flavus isolated from rice grains collected from China. These strains were inoculated onto rice grains and incubated at 28 °C for 21 days. AFB₁ and AFB₂ were extracted and quantified by high-performance liquid chromatography coupled with fluorescence detection. Among the tested strains, 37% produced AFB₁ and AFB₂ with levels ranging from 175 to 124 101 μg kg⁻¹ for AFB₁ and from not detected to 10 329 μg kg⁻¹ for AFB₂. The mean yields of these isolates were 5884 μg kg⁻¹ for AFB₁ and 1968 μg kg⁻¹ for AFB₂. Overall, most of the aflatoxigenic strains produced higher levels of AFB₁ than AFB₂ in rice. The obtained information is useful for assessing the risk of aflatoxin contamination in rice samples.     

Comparison of antibody production against aflatoxin B1 in goats and rabbits.

Bacteria were found that are capable of producing good yields of β-amylase in unrefined media. The culture filtrates are free of α-amylase and isoamylase.     

Reduction of aflatoxin B1 levels by sheep saliva

This study determined the decrease of aflatoxin B₁ by sheep saliva at concentrations of 150 and 300 μg aflatoxin B-₁/L saliva. Analyses for aflatoxins B₁, M₁, and aflatoxicol (R₀) were performed after 2, 4, 6, 24, and 48 hours of incubation. Aflatoxin M₁ and R₀ were not detected and only residues of aflatoxin B₁ were found. 4 to 13% of aflatoxin B₁ were decomposed by sheep’s saliva within 2 hrs and 33 to 43% of aflatoxin B₁ after 24 hrs. Decomposition was affected by the aflatoxin concentration. Decrease of aflatoxin B₁ at 2, 4, 6 hrs was nearly three times higher at the low concentration (150 ppb) compared to the high concentration (300 ppb). After 48 hrs incubation more than 80% of the initial aflatoxin B₁ had been decomposed by the saliva.