Effects of temperature and medium composition on inhibitory activities of gossypol‐related compounds against aflatoxigenic fungi

Aims

To investigate the effects of temperature and medium composition on growth/aflatoxin inhibitory activities of terpenoids gossypol, gossypolone and apogossypolone against Aspergillus flavus and A. parasiticus.

Methods and Results

The compounds were tested at a concentration of 100 μg ml^?1 in a Czapek Dox (Czapek) agar medium at 25, 31 and 37°C. Increased incubation temperature marginally increased growth inhibition caused by these compounds, but reduced the aflatoxin inhibition effected by gossypol. Gossypolone and apogossypolone retained good aflatoxin inhibitory activity against A. flavus and A. parasiticus at higher incubation temperatures. However, increased temperature also significantly reduced aflatoxin production in control cultures. The effects of the terpenoids on fungal growth and aflatoxin production against the same fungi were also determined in Czapek, Czapek with a protein/amino acid addendum and yeast extract sucrose (YES) media. Growth of these fungi in the protein‐supplemented Czapek medium or in the YES medium greatly reduced the growth inhibition effects of the terpenoids. Apogossypolone displayed strong anti‐aflatoxigenic activity in the Czapek medium, but this activity was significantly reduced in the protein‐amended Czapek and YES media. Gossypol, which displayed little to no aflatoxin inhibitory activity in the Czapek medium, did yield significant anti‐aflatoxigenic activity in the YES medium.

Conclusions

Incubation temperature and media composition are important parameters involved in the regulation of aflatoxin production in A. flavus and A. parasiticus. These parameters also affect the potency of growth and aflatoxin inhibitory activities of these gossypol‐related compounds against aflatoxigenic fungi.

Significance and Impact of the Study

Studies utilizing gossypol‐related compounds as inhibitory agents of biological activities should be interpreted with caution due to compound interaction with multiple components of the test system, especially serum proteins.     

Modelling the effect of water activity and temperature on growth rate and aflatoxin production by two isolates of Aspergillus flavus on paddy

Aims: This study was conducted to characterize the growth of and aflatoxin production by Aspergillus flavus on paddy and to develop kinetic models describing the growth rate as a function of water activity (a_w) and temperature. Methods and Results: The growth of A. flavus on paddy and aflatoxin production were studied following a full factorial design with seven a_w levels within the range of 0·82–0·99 and seven temperatures between 10 and 43°C. The growth of the fungi, expressed as colony diameter (mm), was measured daily, and the aflatoxins were analysed using HPLC with a fluorescence detector. The maximum colony growth rates of both isolates were estimated by fitting the primary model of Baranyi to growth data. Three potentially suitable secondary models, Rosso, polynomial and Davey, were assessed for their ability to describe the radial growth rate as a function of temperature and a_w. Both strains failed to grow at the marginal temperatures (10 and 43°C), regardless of the a_w studied, and at the a_w level of 0·82, regardless of temperature. Despite that the predictions of all studied models showed good agreement with the observed growth rates, Davey model proved to be the best predictor of the experimental data. The cardinal parameters as estimated by Rosso model were comparable to those reported in previous studies. Toxins were detected in the range of 0·86–0·99 a_w with optimal a_w of 0·98 and optimal temperature in the range of 25–30°C. Conclusions: The influences of a_w and temperature on the growth of A. flavus and aflatoxin production were successfully characterized, and the models developed were found to be capable of providing good, related estimates of the growth rates. Significance and Impact of the Study: The results of this study could be effectively implemented in minimizing the risk of aflatoxin contamination of the paddy at postharvest.     

Drought Stress and Preharvest Aflatoxin Contamination in Agricultural Commodity: Genetics, Genomics and Proteomics

Throughout the world, aflatoxin contamination is considered one of the most serious food safety issues concerning health. Chronic problems with preharvest aflatoxin contamination occur in the southern US, and are particularly troublesome in corn, peanut, cottonseed, and tree nuts. Drought stress is a major factor to contribute to preharvest afiatoxin contamination. Recent studies have demonstrated higher concentration of defense or stress-related proteins in corn kernels of resistant genotypes compared with susceptible genotypes, suggesting that preharvest field condition (drought or not drought) influences gene expression differently In different genotypes resulting in different levels of "end products": PR(pathogenesis-related) proteins in the mature kernels. Because of the complexity of Aspergillus-plant interactions, better understanding of the mechanisms of genetic resistance will be needed using genomics and proteomics for crop improvement. Genetic Improvement of crop resistance to drought stress is one component and will provide a good perspective on the efficacy of control strategy. Proteomic comparisons of corn kernel proteins between resistant or susceptible genotypes to Aspergillus flavus infection have identified stress-related proteins along with antifungal proteins as associated with kernel resistance. Gene expression studies in developing corn kernels are In agreement with the proteomic studies that defense-related genes could be upregulated or downregulated by abiotic stresses.     

Aflatoxin biosynthesis gene clusters and flanking regions

AIMS: To compare the biosynthetic gene cluster sequences of the main aflatoxin (AF)-producing Aspergillus species. METHODS AND RESULTS: Sequencing was on fosmid clones selected by homology to Aspergillus parasiticus sequence. Alignments revealed that gene order is conserved among AF gene clusters of Aspergillus nomius, A. parasiticus, two sclerotial morphotypes of Aspergillus flavus, and an unnamed Aspergillus sp. Phylogenetic relationships were established using the maximum likelihood method implemented in PAUP. Based on the Eurotiomycete/Sordariomycete divergence time, the A. flavus-type cluster has been maintained for at least 25 million years. Such conservation of the genes and gene order reflects strong selective constraints on rearrangement. Phylogenetic comparison of individual genes in the cluster indicated that ver-1, which has homology to a melanin biosynthesis gene, experienced selective forces distinct from the other pathway genes. Sequences upstream of the polyketide synthase-encoding gene vary among the species, but a four-gene sugar utilization cluster at the distal end is conserved, indicating a functional relationship between the two adjacent clusters. CONCLUSIONS: The high conservation of cluster components needed for AF production suggests there is an adaptive value for AFs in character-shaping niches important to those taxa. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first comparison of the complete nucleotide sequences of gene clusters harbouring the AF biosynthesis genes of the main AF-producing species. Such a comparison will aid in understanding how AF biosynthesis is regulated in experimental and natural environments.     

Electrochemical monitoring of aflatoxin M1 in milk samples using silver nanoparticles dispersed on α‐cyclodextrin‐GQDs nanocomposite

Aflatoxins are potential food pollutants produced by fungi. One of important toxins is aflatoxin M1 (AFM1). A great deal of concern is associated with AFM1 toxicity. In the present study, an innovative electrochemical interface for quantitation of AFM1 based on ternary signal amplification strategy was fabricated. In this work, silver nanoparticles was electrodeposited onto green and biocompatible nanocomposite containing α‐cyclodextrin as conductive matrix and graphene quantum dots as amplification element. Therefore, a multilayer film based on α‐cyclodextrin, graphene quantum dots, and silver nanoparticles was exploited to develop a highly sensitive electrochemical sensor for detection of AFM1. Fully electrochemical methodology was used to prepare a transducer on a glassy carbon electrode, which provided a high surface area toward sensitive detection of AFM1. The surface morphology of electrode surface was characterized by high‐resolution field emission scanning electron microscope. The proposed sensing platform provides a simple tool for AFM1 detection. Under optimized condition, the calibration curve for AFM1 concentration was linear in 0.015mM to 25mM with low limit of quantification of 2μM. The practical analytical utility of the modified electrode was illustrated by determination of AFM1 in unprocessed milk samples.     

黄曲霉菌aflR基因启动子序列变异与黄曲霉毒素产生相关联

为探讨黄曲霉菌aflR基因启动子序列变异与黄曲霉毒素产生的关系,收集黄曲霉菌、米曲霉菌和寄生曲霉菌若干株。在有利于黄曲霉毒素产生的条件下培养后,提取各菌株的总RNA,RT-PCR法检测aflR基因的mRNA表达水平;并应用ELISA法检测各菌株产生黄曲霉毒素B1的情况。提取各菌株的基因组DNA,PCR扩增aflR基因启动子序列并测序。应用基因分析软件将不产毒素的黄曲霉菌与产毒黄曲霉菌的aflR基因启动子序列进行比较,找出不产毒菌株aflR基因启动子序列的变异位点。ELISA法和RT-PCR法结果表明,产毒的黄曲霉菌菌株均有明显的aflR基因转录,而在2株不产毒的黄曲霉菌菌株中,一株aflR基因无转录,另一株仅有较低水平的转录。序列比较结果表明,不产毒黄曲霉菌菌株的aflR基因启动子序列存在如下共同变异位点:-90、-236、-253、-262、-282位。米曲霉菌产生黄曲霉毒素B1和aflR基因转录的检测均为阴性,并且其aflR基因启动子序列中存在与上述不产毒黄曲霉菌菌株相同的变异位点。寄生曲霉菌产生黄曲霉毒素B1和aflR基因转录的检测均呈阳性,并且其aflR基因启动子序列的上述5个位点与产毒黄曲霉菌完全一致。在不产毒素的黄曲霉菌aflR基因启动子序列中发现了5个共同变异位点,实验结果提示这些变异位点可能与黄曲霉毒素的产生有关。     

The effects of rumen fluid on the in vitro aflatoxin binding capacity of different sequestering agents and in vivo release of the sequestered toxin

In an in vitro experiment, aluminosilicates (Atox^® and Novasil™ Plus) and a yeast cell wall derivate (Mycosorb^®) were used as sequestering agents (SAs) to verify their capacity for binding aflatoxin B1 (AFB1) in vitro. SAs were individually mixed at three different ratios with AFB1 (1:5000, 1:50,000 and 1:500,000, w/w) in water (CTR), rumen fluid from a lactating cow with a low rumen pH (LRS) or rumen fluid from a dry cow with a high rumen pH (HRS), and then used in a 3 × 3 × 3 factorial arrangement of a completely randomized design. At the 1:500,000 AF:SA ratio Atox^® and Novasil™ Plus sequestered over 0.87 and 0.98 of the AFB1 in the CTR and rumen solutions (LRS and HRS), respectively. This efficacy decreased when the amount of clays was reduced, with higher values (P<0.001) for Atox^® compared with Novasil™ Plus (0.50 vs. 0.28 in CTR; 0.58 vs. 0.16 in LRS and 0.44 vs. 0.27 in HRS). Mycosorb^® had a lower sequestering efficacy (P<0.001) in all of tested experimental conditions, with 0.34 being the maximum value obtained in the CTR solution.     

Calcium uptake and efflux during the yeast to mycelium transition inSporothrix schenckii

A group of five children with kwashiorkor, seven with marasmic kwashiorkor and one underweight child were given an aflatoxin-free diet consisting of maize meal and milk powder. Blood specimens were collected on admission; on day 4 and 10, 24 hour urine and stool samples were collected for the first ten days. Serum, urine and stool samples were analysed for aflatoxins using high performance liquid chromatography with fluorescent detection, after various extraction and clean-up procedures. The children with kwashiorkor and marasmic kwashiorkor excreted aflatoxins in stools for up to 9 and 6 days after admission respectively. No aflatoxins were detected in the stools or urine of the underweight child. In kwashiorkor, urinary excretion ceased after 2 days, while in marasmic kwashiorkor urinary excretion persisted for 4 days. In stools, B₁ was the type of aflatoxin detected most frequently in kwashiorkor and least frequently in marasmic kwashiorkor. Aflatoxin M₂ was frequently detected in the stools of both groups of children.Estimates of the total amount of aflatoxin excreted by kwashiorkor and marasmic kwashiorkor indicate that these children were harbouring up to 4 g/kg body weight at the time of admission.These findings establish that aflatoxins accumulate in body fluids and tissues in kwashiorkor and marasmic kwashiorkor which is only slowly eliminated.     

Mycobiota in poultry feeds and natural occurrence of aflatoxins, fumonisins and zearalenone in the Rio de Janeiro State, Brazil

The intake of mycotoxin-contaminated feeds can lead to nutrient losses and may have adverse effects on animal health and on productivity. The aims of this study were (1) to determine the mycobiota present in poultry feed samples, and (2) to evaluate the natural occurrence of aflatoxin B₁, fumonisin B₁ and zearalenone. Fungal counts were similar between all culture media tested (10³ CFU g⁻¹). The most frequent genus isolated was Penicillium spp. (41.26%) followed by Aspergillus spp. (33.33%) and Fusarium spp. (20.63%). High precision liquid chromatography was applied to quantify aflatoxin B₁ and fumonisin B₁. Thin layer chromatography was used to determine zearalenone levels. Aflatoxin B₁ values ranged between 1.2 and 17.5 μg kg⁻¹. Fumonisin B₁ levels ranged between 1.5 and 5.5 μg g⁻¹. Zearalenone levels ranged between 0.1 and 7 μg g⁻¹. The present study shows the simultaneous occurrence of two carcinogenic mycotoxins, aflatoxin B₁ and fumonisin B₁, together with another Fusarium mycotoxin (zearalenone) in␣feed intended for poultry consumption. Many samples contained AFB₁ levels near the permissible maximum and it could affect young animals. A synergistic toxic response is possible in animals under simultaneous exposure.     

Fungi associated with post-harvest rot of black plum (Vitex doniana) in Nigeria

Groundnut samples from 21 selected markets in the 10 regions of Ghana yielded high levels of the aflatoxigenic fungus Aspergillus flavus on half-strength potato dextrose agar. The fungus was associated with 31.7 and 12.8%, respectively, of all damaged and undamaged kernels assayed. Only 0.24% of total kernels assayed yielded A. parasiticus. Other fungi detected from total kernels assayed were A. niger (34%), A. candidus (1.45%), A. tamarii (3.93%), A. ochraceous (5.26%), Fusarium spp. (1.7%) Penicillium spp. (5.19%), a Mucor sp. (2.3%), a Trichoderma sp. (0.2%), Rhizopus stolonifer (12%) and certain unidentifiable fungi (11.72%). Total aflatoxin levels ranging from 5.7 to 22, 168 ppb were identified with damaged kernel samples. The mycotoxin was not detected in 50% of undamaged kernel samples tested and very low levels mostly ranging from 0.1 to 12.2 ppb were associated with the undamaged samples that tested positive for aflatoxins. In a novel in vitro microbial assay to determine the effectiveness of certain plant extracts against aflatoxin synthesis, extracts from Xylopia aethiopica, Monodera myristica, Cinnamomum verum and Piper nigrum permitted fungal growth in 1.5% potato-dextrose broth while completely suppressing NOR formation. These extracts, however, could not suppress NOR formation in a yeast extract sucrose medium.