Determination of aflatoxin risk components for in-shell Brazil nuts

A study was conducted on the risk from aflatoxins associated with the kernels and shells of Brazil nuts. Samples were collected from processing plants in Amazonia, Brazil. A total of 54 test samples (40 kg) were taken from 13 in-shell Brazil nut lots ready for market. Each in-shell sample was shelled and the kernels and shells were sorted in five fractions: good kernels, rotten kernels, good shells with kernel residue, good shells without kernel residue, and rotten shells, and analysed for aflatoxins. The kernel:shell ratio mass (w/w) was 50.2/49.8%. The Brazil nut shell was found to be contaminated with aflatoxin. Rotten nuts were found to be a high-risk fraction for aflatoxin in in-shell Brazil nut lots. Rotten nuts contributed only 4.2% of the sample mass (kg), but contributed 76.6% of the total aflatoxin mass (μg) in the in-shell test sample. The highest correlations were found between the aflatoxin concentration in in-shell Brazil nuts samples and the aflatoxin concentration in all defective fractions (R(2)=0.97). The aflatoxin mass of all defective fractions (R(2)=0.90) as well as that of the rotten nut (R(2)=0.88) were also strongly correlated with the aflatoxin concentration of the in-shell test samples. Process factors of 0.17, 0.16 and 0.24 were respectively calculated to estimate the aflatoxin concentration in the good kernels (edible) and good nuts by measuring the aflatoxin concentration in the in-shell test sample and in all kernels, respectively.     

Determination of aflatoxin risk components for in-shell Brazil nuts

A study was conducted on the risk from aflatoxins associated with the kernels and shells of Brazil nuts. Samples were collected from processing plants in Amazonia, Brazil. A total of 54 test samples (40?kg) were taken from 13 in-shell Brazil nut lots ready for market. Each in-shell sample was shelled and the kernels and shells were sorted in five fractions: good kernels, rotten kernels, good shells with kernel residue, good shells without kernel residue, and rotten shells, and analysed for aflatoxins. The kernel?:?shell ratio mass (w/w) was 50.2/49.8%. The Brazil nut shell was found to be contaminated with aflatoxin. Rotten nuts were found to be a high-risk fraction for aflatoxin in in-shell Brazil nut lots. Rotten nuts contributed only 4.2% of the sample mass (kg), but contributed 76.6% of the total aflatoxin mass (µg) in the in-shell test sample. The highest correlations were found between the aflatoxin concentration in in-shell Brazil nuts samples and the aflatoxin concentration in all defective fractions (R ^2?=?0.97). The aflatoxin mass of all defective fractions (R ^2?=?0.90) as well as that of the rotten nut (R ^2?=?0.88) were also strongly correlated with the aflatoxin concentration of the in-shell test samples. Process factors of 0.17, 0.16 and 0.24 were respectively calculated to estimate the aflatoxin concentration in the good kernels (edible) and good nuts by measuring the aflatoxin concentration in the in-shell test sample and in all kernels, respectively.     

Aflatoxin reduction in corn through field application of competitive fungi.

Soil in corn plots was inoculated with nonaflatoxigenic strains of Aspergillus flavus and A. parasiticus during crop years 1994 to 1997 to determine the effect of application of the nontoxigenic strains on preharvest aflatoxin contamination of corn. Corn plots in a separate part of the field were not inoculated and served as controls. Inoculation resulted in significant increases in the total A. flavus/parasiticus soil population in treated plots, and that population was dominated by the applied strain of A. parasiticus (NRRL 21369). In the years when weather conditions favored aflatoxin contamination (1996 and 1997), corn was predominately colonized by A. flavus as opposed to A. parasiticus. In 1996, colonization by wild-type A. flavus was significantly reduced in treated plots compared with control plots, but total A. flavus/parasiticus colonization was not different between the two groups. A change to a more aggressive strain of A. flavus (NRRL 21882) as part of the biocontrol inoculum in 1997 resulted in a significantly (P < 0.001) higher colonization of corn by the applied strain. Weather conditions did not favor aflatoxin contamination in 1994 and 1995. In 1996, the aflatoxin concentration in corn from treated plots averaged 24.0 ppb, a reduction of 87% compared with the aflatoxin in control plots that averaged 188.4 ppb. In 1997, aflatoxin was reduced by 66% in treated corn (29.8 ppb) compared with control corn (87.5 ppb). Together, the data indicated that although the applied strain of A. parasiticus dominated in the soil, the nonaflatoxigenic strains of A. flavus were more responsible for the observed reductions in aflatoxin contamination. Inclusion of a nonaflatoxigenic strain of A. parasiticus in a biological control formulation for aflatoxin contamination may not be as important for airborne crops, such as corn, as for soilborne crops, such as peanuts.     

Aflatoxin in betel nut and its control by use of food preservatives.

The occurrence of aflatoxins in market betel nut samples was studied. It was observed that several betel nut samples were infested with aflatoxin-producing fungus, Aspergillus flavus. Out of 32 samples collected from various places, 12 were positive for aflatoxin. Aflatoxin B1 was detected in all the positive samples. Other aflatoxins were also detected in some samples. Boric acid, propionic acid and potassium metabisulphite were used for the control of aflatoxin B1 on betel nuts. Propionic acid was most effective in inhibiting aflatoxin production on betel nut after intervals of 2 (62%) and 4 (85%) weeks. Controlling the occurrence of aflatoxin could safeguard the users from the health hazards of aflatoxins.     

Polymerase chain reaction-mediated characterization of molds belonging to the Aspergillus flavus group and detection of Aspergillus parasiticus in peanut kernels by a multiplex polymerase chain reaction

The Aspergillus flavus group covers species of A. flavus and Aspergillus parasiticus as aflatoxin producers and Aspergillus oryzae and Aspergillus sojae as koji molds. Genetic similarity among these species is high, and aflatoxin production of a culture may be affected by cultivation conditions and substrate composition. Therefore, a polymerase chain reaction (PCR)-mediated method of detecting the aflatoxin-synthesizing genes to indicate the degree of risk a genotype has of being a phenotypic producer was demonstrated. In this study, 19 strains of the A. flavus group, including A. flavus, A. parasiticus, A. oryzae, A. sojae, and one Aspergillus niger, were subjected to PCR testing in an attempt to detect four genes, encoding for norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-1), and a regulatory protein (apa-2), involved in aflatoxin biosynthesis. Concurrently, the strains were cultivated in yeast-malt (YM) broth for aflatoxin detection. Fifteen strains were shown to possess the four target DNA fragments. With regard to aflatoxigenicity, all seven aflatoxigenic strains possessed the four DNA fragments, and five strains bearing less than the four DNA fragments did not produce aflatoxin. When peanut kernels were artificially contaminated with A. parasiticus and A. niger for 7 days, the contaminant DNA was extractable from a piece of cotyledon (ca. 100 mg), and when subjected to multiplex PCR testing using the four pairs of primers coding for the above genes, they were successfully detected. The target DNA fragments were detected in the kernels infected with A. parasiticus, and none was detected in the sound (uninoculated) kernels or in the kernels infected with A. niger.     

Elimination of Aspergillus parasiticus from nut surface with low pressure cold plasma (LPCP) treatment

Low pressure cold plasma (LPCP) using air gases and sulfur hexafluoride (SF(6)) was developed and tested for anti-fungal efficacy against Aspergillus parasiticus on various nut samples. Artificially A. parasiticus contaminated hazelnuts, peanuts, and pistachio nuts were treated with air gases plasma and SF(6) plasma for up to 20 min duration. The sterilizing effect of LPCP on A. parasiticus was higher during the early treatment period than the later treatment period. Air gases plasma treatment for 5 min resulted in 1-log reduction of A. parasiticus and a further 5 min treatment resulted in additional 1-log reduction. SF(6) plasma application was more effective resulting in approximately a 5-log decrease in fungal population for the same duration. When effectiveness of plasma treatment against aflatoxins were tested, 20 min air gases plasma treatment resulted in a 50% reduction in total aflatoxins (AFB1, AFB2, AFG1, and AFG2), while only a 20% reduction in total aflatoxin was observed after 20 min SF(6) plasma treatment. In this study, a rapid, functional clean-up method for the elimination of aflatoxin producing fungus from shelled and unshelled nuts was investigated as a suitable fungal decontamination method.     

Distribution of aflatoxin-producing Aspergillus flavus and Aspergillus parasiticus in sugarcane fields in the southernmost islands of Japan

The distribution of Aspergillus flavus and Aspergillus parasiticus in sugarcane field soils and on harvested sugarcane stems was studied on seven islands of Okinawa and Kagoshima Prefectures, the southernmost prefectures in Japan. With the use of a combination of dilution plate and plant debris plate techniques, the fungi were detected on all seven islands studied and in 74% of 53 soil samples. The fungi were also found on the cut surfaces of sugarcane stems from one of the islands. A. parasiticus was the predominant fungus, although many atypical A. parasiticus isolates that produced metulated conidial heads were also obtained. The proportions of isolates testing positive for aflatoxin production were ca. 89% (146 of 164) of all isolates and ca. 69% of A. flavus isolates. More than 40% of A. flavus isolates also produced G aflatoxins. Scanning electron microscopic observation of conidial wall texture was useful in distinguishing A. parasiticus from A. flavus. Cyclopiazonic acid, an indole mycotoxin, was never synthesized by any of the A. parasiticus or G aflatoxin-producing A. flavus isolates tested.     

Brazil nuts: Benefits and risks associated with contamination by fungi and mycotoxins

The monotypic type genus Bertholletia produces commercially nutritionally harvested edible seeds, Brazil nuts. It is an important product from the Amazon forest in the food production chain, with a 2008 annual world production of 78,000 tonnes, being Brazil responsible for approximately 40% of it. Although there are beneficial nutritional properties, the prevailing mycobiota of Brazil nuts include fungi that are producers of aflatoxins, such as Aspergillus flavus, A. parasiticus and A. nomius. Aflatoxins have deleterious effects in consumption considering the global distribution chain, affecting major exporting countries. The present review is focused on the importance of Brazil nuts for the Amazon rainforest, emphasizing on the social and environmental impact of its production, on the mycobiota contamination of seeds, and on the presence of mycotoxins and related food safety aspects.     

INCIDENCE OF AFLATOXIGENIC ISOLATES OF ASPERGILLUS FLAVUS/PARASITICUS OBTAINED FROM GEORGIA CORN PROCESSING PLANTS

Two corn processing facilities within Georgia were evaluated in order to determine the incidence of Aspergillus flavus or A. parasiticus within the plant and in corn harvested and processed in 1984 and 1985. Conidia of A. flavus/parasiticus were found in all corn samples evaluated as well as in settled dust samples taken within these processing facilities. Isolates were obtained by using the differential/selective medium Aspergillus flavus/parasiticus agar. Upon subsequent culture only 55% of the selected isolates were confirmed as belonging to A. flavus/parasiticus group. Some of these isolates were randomly chosen and their ability to produce aflatoxins B₁, B₂, G₁, or G₂ evaluated. Thirty-two percent of the A. flavus/parasiticus isolates cultured for aflatoxin production were found to be aflatoxigenic.     

INFLUENCE OF OTHER FUNGI ON AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS IN MAIZE KERNELS

Experiments were designed to determine whether certain nontoxigenic fungi commonly isolated from maize kernels can affect aflatoxin B₁ development when inoculated with A. flavus onto individual unsterilized, and autoclaved maize kernels . Trichoderma viride and Aspergillus niger were found to be strongly antagonist inhibiting the growth of A. flavus by 87 and 66% respectively, whereas Aspergillus versicolor, Fusarium moniliforme, Paecilomyces variotii and Emericella quadrillineata inhibited the growth of A. flavus by less than 51%. Less aflatoxin B₁ was detected when A. flavus was paired with A. niger or T. viride than with the other test fungi. When A. niger or T. viride was introduced onto the kernels 72 h before inoculation with A. flavus, no aflatoxin B₁ was detected in unsterilized kernels and the levels of aflatoxin B₁ were greatly reduced from 700 ppb to 160 and 140 ppb in autoclaved kernels, respectively. When inoculation of A. flavus followed 72 h of incubation of either A. niger and T. viride, no aflatoxin B₁ was detected. However, when both A. niger and T. viride were introduced 72 h after inoculation with A. flavus, the levels of aflatoxin B₁ were reduced to 385 and 560 ppb, respectively in unsterilized and autoclaved maize kernels . Trichoderma viride and Aspergillus niger may be useful in biological control of aflatoxin contamination of maize kernels .; Accepted for Publication June 11, 1997     

Interaction of water activity and temperature on aflatoxin production by Aspergillus flavus and A. parasiticus in irradiated maize seeds.

The effects of aw (0.90, 0.95, 0.98) and temperature (25 degrees C, 30 degrees C, 35 degrees C) on aflatoxin production by Aspergillus flavus and Aspergillus parasiticus growing on irradiated maize seeds, were examined. Highest levels of aflatoxin were produced by A. parasitious at 25 degrees C and 0.98 aw and by A. flavus at 30 degrees C at 0.95 and 0.98 aw. At 0.90 aw toxin production was consistently low for both species at all temperatures. Temperature cycling of A. flavus between 25 degrees C and 35 degrees C each for 12 h resulted in higher aflatoxin synthesis than when incubated either at 25 degrees C or 35 degrees C.     

Detection of aflatoxin-producing molds in Korean fermented foods and grains by multiplex PCR

An assay based on multiplex PCR was applied for the detection of potential aflatoxin-producing molds in Korean fermented foods and grains. Three genes, avfA, omtA, and ver-1, coding for key enzymes in aflatoxin biosynthesis, were used as aflatoxin-detecting target genes in multiplex PCR. DNA extracted from Aspergillus flavus, Aspergillus parasiticus, Aspergillus oryzae, Aspergillus niger, Aspergillus terreus, Penicillium expansum, and Fusarium verticillioides was used as PCR template to test specificity of the multiplex PCR assay. Positive results were achieved only with DNA that was extracted from the aflatoxigenic molds A. flavus and A. parasiticus in all three primer pairs. This result was supported by aflatoxin detection with direct competitive enzyme-linked immunosorbent assay (DC-ELISA). The PCR assay required just a few hours, enabling rapid and simultaneous detection of many samples at a low cost. A total of 22 Meju samples, 24 Doenjang samples, and 10 barley samples commercially obtained in Korea were analyzed. The DC-ELISA assay for aflatoxin detection gave negative results for all samples, whereas the PCR-based method gave positive results for 1 of 22 Meju samples and 2 of 10 barley samples. After incubation of the positive samples with malt extract agar, DC-ELISA also gave positive results for aflatoxin detection. All Doenjang samples were negative by multiplex PCR and DC-ELISA assay, suggesting that aflatoxin contamination and the presence of aflatoxin-producing molds in Doenjang are probably low.     

黄曲霉毒素的检测及其降解方法进展

黄曲霉毒素主要是由真菌寄生曲霉和黄曲霉产生的次生代谢产物,具有极强的毒性、致癌性,在自然界中普遍存在。文中论述了不同物质中黄曲霉毒素的检测方法及应用,同时介绍了黄曲霉毒素的降解方法及成果,并对黄曲霉毒素检测及降解技术的发展趋势进行了展望。     

Identification and toxigenic potential of the industrially important fungi, Aspergillus oryzae and Aspergillus sojae

Mold strains belonging to the species Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu, and as protein production hosts in modern industrial processes. A. oryzae and A. sojae are relatives of the wild molds Aspergillus flavus and Aspergillus parasiticus. All four species are classified to the A. flavus group. Strains of the A. flavus group are characterized by a high degree of morphological similarity. Koji mold species are generally perceived of as being nontoxigenic, whereas wild molds are associated with the carcinogenic aflatoxins. Thus, reliable identification of individual strains is very important for application purposes. This review considers the pheno- and genotypic markers used in the classification of A. flavus group strains and specifically in the identification of A. oryzae and A. sojae strains. Separation of A. oryzae and A. sojae from A. flavus and A. parasiticus, respectively, is inconsistent, and both morphologic and molecular evidence support conspecificity. The high degree of identity is reflected by the divergent identification of reference cultures maintained in culture collections. As close relatives of aflatoxin-producing wild molds, koji molds possess an aflatoxin gene homolog cluster. Some strains identified as A. oryzae and A. sojae have been implicated in aflatoxin production. Identification of a strain as A. oryzae or A. sojae is no guarantee of its inability to produce aflatoxins or other toxic metabolites. Toxigenic potential must be determined specifically for individual strains. The species taxa, A. oryzae and A. sojae, are currently conserved by societal issues.     

黄曲霉素的毒害及防除

真菌在新陈代谢过程中会产生大量化学结构各异的活性物质，这些物质被称为真菌毒素，其中许多对人和动物有害。黄曲霉毒素，简称黄曲霉素，就是由黄曲霉和寄生曲霉两种真菌菌株产生的真菌毒素，具有强烈的毒性和致癌、致畸、致突变性。本文介绍了黄曲霉素的危害及防除措施。     

Aqueous extracts of Tulbaghia violacea inhibit germination of Aspergillus flavus and Aspergillus parasiticus conidia

Aspergillus flavus and Aspergillus parasiticus are important plant pathogens and causal agents of pre- and postharvest rots of corn, peanuts, and tree nuts. These fungal pathogens cause significant crop losses and produce aflatoxins, which contaminate many food products and contribute to liver cancer worldwide. Aqueous preparations of Tulbaghia violacea (wild garlic) were antifungal and at 10 mg/ml resulted in sustained growth inhibition of greater than 50% for both A. flavus and A. parasiticus. Light microscopy revealed that the plant extract inhibited conidial germination in a dose-dependent manner. When exposed to T. violacea extract concentrations of 10 mg/ml and above, A. parasiticus conidia began germinating earlier and germination was completed before that of A. flavus, indicating that A. parasiticus conidia were more resistant to the antifungal effects of T. violacea than were A. flavus conidia. At a subinhibitory extract dose of 15 mg/ml, hyphae of both fungal species exhibited increased granulation and vesicle formation, possibly due to increased reactivity between hyphal cellular components and T. violacea extract. These hyphal changes were not seen when hyphae were formed in the absence of the extract. Transmission electron microscopy revealed thickening of conidial cell walls in both fungal species when grown in the presence of the plant extract. Cell walls of A. flavus also became considerably thicker than those of A. parasiticus, indicating differential response to the extract. Aqueous preparations of T. violacea can be used as antifungal treatments for the control of A. flavus and A. parasiticus. Because the extract exhibited a more pronounced effect on A. flavus than on A. parasiticus, higher doses may be needed for control of A. parasiticus infections.     

Quality control of Aspergillus flavus and A. parasiticus agar and comparison with dichloran 18% glycerol agar: a collaborative study

AFPA culture medium, which is used for recognition of Aspergillus flavus and A. parasiticus, has been validated in a collaborative study including nine laboratories located in Australia, Brazil, Denmark, The Netherlands, Sweden and United Kingdom. Three freeze-dried fungal mixtures, containing A. flavus/A. parasiticus and background fungi, were produced and checked for homogeneity. The coefficients of variance were low, ranging from 0.81% to 1.09% for total fungal counts and between 2.50% and 2.72% for counts of A. flavus/A. parasiticus. The laboratories analysed the contents of two vials of each mixture on commercial A. flavus and A. parasiticus agar (AFPA), in-house-made AFPA, and on a standard media, dichloran 18% glycerol agar (DG18). Reproducibility values for counts of A. flavus/A. parasiticus indicated no differences between the commercial AFPA and the in-house-made AFPA. Variation between laboratories was low, indicating that the medium was effective in use. Reproducibility values for DG18 were higher. There were no differences in counts of A. flavus/A. parasiticus on AFPA and DG18. However, DG18 gave slightly higher total fungal counts compared to AFPA.     

Mycotoxins in edible tree nuts

Tree nuts (almonds, pistachios, and walnuts) are an exceptionally valuable crop, especially in California, with an aggregate value approaching $3.5 billion. Much of this economic value comes from overseas markets, with up to 60% of the crop being exported. The product can be contaminated with aflatoxins or ochratoxins, with the former being of special concern because of the strict regulatory levels (4 ppb total aflatoxins) applied by the European Community (EC). Natural, consumer-acceptable control methods are therefore required to conform to such limits. Research has shown that aflatoxin production is markedly decreased by the presence of natural antioxidants that occur in tree nuts, including hydrolysable tannins, flavonoids and phenolic acids. In vitro testing of individual compounds showed that the antiaflatoxigenic effect correlated with the structure and concentration of such compounds in individual nut varieties and species. This lead to the hypothesis that aflatoxin biosynthesis is stimulated by oxidative stress on the fungus and that compounds capable of relieving oxidative stress should therefore suppress or eliminate aflatoxin biosynthesis. Oxidative stress induced in A. flavus by addition of tert-butyl hydroperoxide to the media stimulated peak aflatoxin production and maintained high levels over time. However, aflatoxin formation was significantly inhibited by incorporation into the media of the antioxidant, tannic acid. Measures to increase natural products with antioxidant properties in tree nuts may thereby reduce or eliminate the ability of A. flavus to biosynthesize aflatoxins, thus ensuring levels at or below regulatory limits and maintaining export markets for U.S. tree nuts.     

AFLATOXIGENIC ASPERGILLI IN FOODS AND FEEDS IN UGANDA

Studies conducted during the sixties and the seventies on food crops in Uganda showed that the populace was exposed to consumption of aflatoxin-contaminated foods. These studies also linked the highest incidence of liver cancer in the world to the presence of high levels of aflatoxins in the food and beverages. After a lapse of a decade, it was of interest to investigate the occurrence of aflatoxins and aflatoxigenic fungi in staple Ugandan food crops and poultry feeds derived from these foodstuffs. A simple, rapid and reproducible procedure was used. The procedure consisted of growing or culturing feed grains on a selective medium, Aspergillus flavus/parasiticus agar (AFPA) followed by screening for aflatoxin producing fungi on a coconut agar medium (CAM) under UV light with a subsequent confirmatory screening method for aflatoxin production by the fungi in pure culture. Fifty-four samples consisting of corn and peanuts, soybean and poultry feed were analyzed for content of aflatoxigenic. A. flavus/parasiticus and 25 of the samples were also screened for aflatoxins B₁ and G₁, zearalenone, sterigmatocystin, ochratoxin A, citrinin, vomitoxin, and diacetoxyscirpenol (DAS). Aflatoxigenic A. flavus/parasiticus was detected from the majority of corn (77%), peanuts (36% human food and 83.3% animal feed) and poultry feed (66.6%). but not from soybean samples. Two samples out of 25 contained detectable levels of aJatosin B, (20 ppb). For the jirst time other mycotoxins, zearalenone (3 samples) and vomitoxin (2 samples) were detected in corn from Uganda.