Determination of Aflatoxin B1 in Feedstuffs without Clean-Up Step by High-Performance Liquid Chromatography.
A reliable and rapid method has been developed for the determination of aflatoxin B1 (AFB1) in four kinds of feedstuffs comprising broken rice, peanuts, corn, and fishmeal. A sample preparation was carried out based on the QuEChERS method with the exclusion of the clean-up step. In this study, AFB1 was extracted using acetonitrile/methanol (40/60 v/v), followed by partitioning with sodium chloride and magnesium sulfate. High-performance liquid chromatography with precolumn derivatization and fluorescence detection was performed. The coefficients of determination were greater than 0.9800. Throughout the developed method, the recovery of all feedstuffs achieved a range of 82.50-109.85% with relative standard deviation lower than 11% for all analytes at a concentration of 20-100 ng/g. The limit of detection (LOD) ranged from 0.2 to 1.2 ng/g and limit of quantitation (LOQ) ranged from 0.3 to 1.5 ng/g. The validated method was successfully applied to a total of 120 samples. The occurrence of AFB1 contamination was found at the following concentrations: in broken rice (0.44-2.33ng/g), peanut (3.97-106.26ng/g), corn (0.88-50.29 ng/g), and fishmeal (1.06-10.35 ng/g). These results indicate that the proposed method may be useful for regularly monitoring AFB1 contamination in feedstuffs.
Biocontrol of Aspergillus flavus on peanut kernels using Streptomyces yanglinensis 3-10.
The bacterium, Streptomyces yanglinensis 3-10, shows promise in the control of many phytopathogenic fungi. In this study, S. yanglinensisand its antifungal substances, culture filtrate (CF3-10) and crude extracts (CE3-10), were evaluated for their activity in reducing growth and aflatoxin AFB1 production by Aspergillus flavus, both in vitro and in vivo on peanut kernels. The results showed that in dual culture conditions, S. yanglinensis reduced the mycelial growth of A. flavus about 41% as compared to control. The mycelial growth of A. flavus was completely inhibited on potato dextrose agar amended with CF3-10 at 3% (v/v) or CE3-10 at 2.5 μg/ml. In liquid culture experiments, growth inhibition ranged from 32.3 to 91.9% with reduction in AFB1 production ranging from 46.4 to 93.4% using different concentrations of CF3-10 or CE3-10. For in vivo assays, CF3-10 at 0.133 ml/g (v/w) or CE3-10 at 13.3 μg/g (w/w) reduced the postharvest decay of peanut kernels by inhibiting visible growth of A. flavus leading to an 89.4 or 88.1% reduction in AFB1 detected, respectively. Compared with the controls, CF3-10 and CE3-10 in A. flavus shake culture significantly reduced expression levels of two AFB1 biosynthesis genes, aflR and aflS. Furthermore, electron microscopy observation showed that CF3-10 (2%, v/v) caused hyphae growth to be abnormal and shriveled, cell organelles to degenerate and collapse, large vacuoles to appear. These results suggest that S. yanglinensis 3-10 has potential as an alternative to chemical fungicides in protecting peanut kernels and other agricultural commodities against postharvest decay from A. flavus.
Evaluation of aflatoxins and Aspergillus sp. contamination in raw peanuts and peanut-based products along the supply chain in Malaysia.
The peanut supply chain in Malaysia is dominated by three main stakeholders (importers, manufacturers, retailers). The present study, aimed to determine the levels and critical points of aflatoxins and fungal contamination in peanuts along the supply chain. Specifically, two types of raw peanuts and six types of peanut-based products were collected (N = 178). Samples were analysed for aflatoxins by using High Performance Liquid Chromatography. Results revealed that the aflatoxins contamination was significantly higher (P≤0.05) in raw peanuts and peanut-based products from the retailers. However, there was no significant different (P≥0.05) in fungal contamination for both types of peanuts except for the total fungal count in raw peanuts from the retailers. Furthermore, raw peanut kernels from the retailers were the most contaminated ones ranged from <LOD - 1021.4 µg/kg (mean: 120.7 µg/kg, median: 1.4 µg/kg) followed by the samples collected from the manufacturers which was ranged from <LOD - 181.9 µg/kg (mean: 20.5 µg/kg, median: 0.0 µg/kg). About 38% and 22% of the samples from the retailers and manaufacturers were found to have exceeded the Malaysian Regulation limit (raw peanuts:15 µg/kg; peanut-based products:10 µg/kg) respectively. In contrast, no aflatoxins were detected in samples from the importers. On the other hand, 15.0% and 5.9% of peanut-based products from retailers and manufacturers respectively were found to have exceeded the limit. Fungal contamination (0.3 - 3.6 log CFU/g) was relatively higher in raw peanuts compared to that of peanut-based products (0.6 - 2.7 log CFU/g). In conclusion, the manufacturers and retailers were the critical points for aflatoxins contamination in peanuts. However, fungal contamination was more critical in the raw peanuts compared to peanut-based products. The study was limited by a minimal number of samples from the importer. Therefore, further investigations on a larger sample size should be conducted to confirm the findings in this present study.
Effect of potassium sorbate (E‐202) and the antifungal PgAFP protein on Aspergillus carbonarius growth and ochratoxin A production in raisin simulating media.
BACKGROUND: Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium fungi. The presence of OTA in raisins is mainly related to the black Aspergillus spp. contamination. This toxin poses risks to human and animal health due to their high toxicity and carcinogenicity. Therefore, new strategies to avoid the risk associated to OTA are necessary. RESULTS: In the present study, a comparison between the effects of the antifungal protein PgAFP and potassium sorbate (E-202), on the growth of Aspergillus carbonarius, biosynthetic- and stress-related gene expression and its OTA production at two water activity (aw ) levels 0.95 and 0.93 aw , was carried out. The results showed that PgAFP successfully controlled OTA production, whereas E-202, although was able to decrease Aspergillus carbonarius growth, caused a significant increase in OTA production by the fungus. CONCLUSION: PgAFP protein, a biological compound with an antifungal activity, is safer to use compared to E-202 and may be proposed as a food preservative and a useful biocontrol strategy to control ochratoxigenic A. carbonarius in raisins.
Polyphasic approach to the identification and characterization of aflatoxigenic strains of Aspergillus section Flavi isolated from peanuts and peanut-based products marketed in Malaysia.
Peanuts are widely consumed as the main ingredient in many local dishes in Malaysia. However, the tropical climate in Malaysia (high temperature and humidity) favours the growth of fungi from Aspergillus section Flavi, especially during storage. Most of the species from this section, such as A. flavus, A. parasiticus and A. nomius, are natural producers of aflatoxins. Precise identification of local isolates and information regarding their ability to produce aflatoxins are very important to evaluate the safety of food marketed in Malaysia. Therefore, this study aimed to identify and characterize the aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi in peanuts and peanut-based products. A polyphasic approach, consisting of morphological and chemical characterizations was applied to 128 isolates originating from raw peanuts and peanut-based products. On the basis of morphological characters, 127 positively identified as Aspergillus flavus, and the other as A. nomius. Chemical characterization revealed six chemotype profiles which indicates diversity of toxigenic potential. About 58.6%, 68.5%, and 100% of the isolates are positive for aflatoxins, cyclopiazonic acid and aspergillic acid productions respectively. The majority of the isolates originating from raw peanut samples (64.8%) were aflatoxigenic, while those from peanut-based products were less toxigenic (39.1%). The precise identification of these species may help in developing control strategies for aflatoxigenic fungi and aflatoxin contamination in peanuts, especially during storage. These findings also highlight the possibility of the co-occurrence of other toxins, which could increase the potential toxic effects of peanuts.
Cultural and Genetic Approaches to Manage Aflatoxin Contamination: Recent Insights Provide Opportunities for Improved Control.
Aspergillus flavus is a morphologically complex species that can produce the group of polyketide derived carcinogenic and mutagenic secondary metabolites, aflatoxins, as well as other secondary metabolites such as cyclopiazonic acid and aflatrem. Aflatoxin causes aflatoxicosis when aflatoxins are ingested through contaminated food and feed. In addition, aflatoxin contamination is a major problem, from both an economic and health aspect, in developing countries, especially Asia and Africa, where cereals and peanuts are an important food crops. Early measures for control of A. flavus infection and consequent aflatoxin contamination centered on creating unfavorable environments for the pathogen and destroying contaminated products. While development of atoxigenic (non-aflatoxin producing) strains of A. flavus as viable commercial biocontrol agents has marked a unique advance for control of aflatoxin contamination, particularly in Africa, new insights into the biology and sexuality of A. flavus are now providing opportunities to design improved atoxigenic strains for sustainable biocontrol of aflatoxin. Further, progress in the use of molecular technologies such as incorporation of antifungal genes in the host and host-induced gene silencing, is providing knowledge that could be harnessed to develop germplasm that is resistant to infection by A. flavus and aflatoxin contamination. This review summarizes the substantial progress that has been made to understand the biology of A. flavus and mitigate aflatoxin contamination with emphasis on maize. Concepts developed to date can provide a basis for future research efforts on the sustainable management of aflatoxin contamination.
Risk assessment of exposure to aflatoxin B1 and ochratoxin A through consumption of different Pistachio (Pistacia Vera L.) cultivars collected from four geographical regions of Iran.
Iran is one of the main suppliers of pistachio for the European market accounting for over 90% of its demands; hence, efficient analytical methods are required for detection of mycotoxins contamination in pistachio kernels before exporting them. In this study, aflatoxin B1 (AFB1) and ochratoxin A (OTA) levels in five pistachio cultivars collected from four sites of Iran, were measured by HPLC. Based on the results, risk assessment for AFB1 and OTA residues was done. The highest mean concentrations of AFB1 and OTA were found in Ahmad-aghaei (4.33 and 2.19 ng/g, respectively) and Akbari (4.08 and 1.943 ng/g, respectively) cultivars from Rafsanjan, Iran. Even the highest concentrations of AFB1 and OTA in analyzed samples were lower than the corresponding maximum limits set by EU authorities. The hazard index (HI) value for consumers of Iranian pistachio is below one. It could be concluded that consumption of pistachio cultivated in these regions poses no health risk of mycotoxins exposure.
Effects of packaging and pre-storage treatments on aflatoxin production in peanut storage under controlled conditions.
This study reports on aflatoxin production and peanut (Bailey's variety) quality, for four peanutpre-storage treatments; [(Raw clean (Raw-Cl), Raw inoculated with Aspergillus flavus NRRL 3357 (Raw-Inf), inoculated partially roasted but not-blanched (PRN-blanch); and inoculated partially roasted, blanched with discolored nuts sorted out (PR-blanched)]. All four treated samples were packaged in four different packaging systems [polypropylene woven sacks (PS), hermetic packs (HP), hermetic packs with oxygen absorbers (HPO), and vacuumed hermetic packs (HPV)] and stored under controlled conditions at a temperature of 30 ± 1 °C and water activity of 0.85 ± 0.02, for 14 weeks. Raw-Inf samples in PS had a higher fungal growth with a mean value of 8.01 × 104 CFU/g, compared to the mean values of samples in hermetic packs: 1.07 × 103 CFU/g for HP, 14.55 CFU/g for HPO, and 57.82 CFU/g for HPV. Similarly, the hermetic bags were able to reduce aflatoxin level of the Raw-Inf samples by 50.6% (HP), 63.0% (HPV), and 66.8% (HPO). Partial roasting and blanching in PS also reduced aflatoxin level by about 74.6%. Quality maintenance was the best for peanuts in HPO, recording peroxide value (PV) of 10.16 meq/kg and p-Anisidine (p-Av) of 3.95 meq/kg compared to samples in polypropylene woven sacks which had PV of 19.25 meq/kg and p-Av of 6.48 meq/kg. These results indicate that using zero-oxygen hermetic packaging, instead of the conventional polypropylene woven sacks, helped to suppress aflatoxin production and quality deterioration. Also, partially roasted, blanched and sorted peanuts showed a potential for reducing aflatoxin presence during storage.
Insight into Genes Regulating Post-harvest Aflatoxin Contamination of Tetraploid Peanut from Transcriptional Profiling.
Post-harvest aflatoxin contamination is a challenging issue that affects peanut quality. Aflatoxin is produced by fungi belonging to the Aspergilli group, and is known as an acutely toxic, carcinogenic and immune-suppressing class of mycotoxins. Evidence for several host genetic factors that may impact aflatoxin contamination has been reported, e.g., genes for lipoxygenase (PnLOX1 and PnLOX2/PnLOX3 that showed either positive or negative regulation with Aspergillus infection), ROS, and WRKY (highly associated with or differentially expressed upon infection of maize with A. flavusAspergillus flavus between resistant (ICG 1471) and susceptible (Florida-07) cultivated peanut genotypes. The gene expression profiling analysis was designed to reveal differentially expressed genes in response to the infection (infected vs mock-treated seeds). In addition, the differential expression of the fungal genes was profiled. The study revealed the complexity of the interaction between the fungus and peanut seeds as expression of a large number of genes was altered including some in the process of plant defense to aflatoxin accumulation. Analysis of the experimental data with 'keggseq', a novel designed tool for KEGG enrichment analysis, showed the importance of alpha-linolenic acid metabolism, protein processing in endoplasmic reticulum, spliceosome, and carbon fixation and metabolism pathways in conditioning resistance to aflatoxin accumulation. In addition, co-expression network analysis was carried out to reveal the correlation of gene expression among peanut and fungal genes. The results showed the importance of WRKY, TIR-NBS-LRR, ethylene, and heat shock proteins in the resistance mechanism.
Aspergillus section Flavi and aflatoxins in dried figs and nuts in Algeria.
The presence of Aspergillus section Flavi and aflatoxin (AF) contamination was investigated in 112 samples of peanuts, almonds and dried figs collected in Algeria. The occurrence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) in different commodities has been determined with a sensitive method based on high performance liquid chromatography (HPLC) coupled with fluorescence detection with post-column photochemical derivatisation. Analytical results indicated that 28 samples of peanuts, 16 samples of almonds and 26 samples of dried figs contained detectable levels of AFs. A total of 69 samples (61.6%) were contaminated with AFB1 ranging from the limit of quantification to 174 µg kg-1. AFB2 was found in 12 samples (10.7%) and varied from 0.18 to 193 µg kg-1. Seven samples revealed AF concentrations lower than the limit of quantification. Eleven peanut and fourteen dried fig samples exceeded the European maximum limits for AFB1.