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     

Natural maize phenolic acids for control of aflatoxigenic fungi on maize

ABSTRACT:  Natural phytochemicals may be an alternative to synthetic chemicals for controlling fungal growth and mycotoxin production in stored maize. A key to progress in this field is to select the best natural maize phytochemicals to be applied in a storage maize ecosystem. This research was undertaken to evaluate the effects of the natural phytochemicals trans-cinnamic acid (CA) and ferulic acid (FA) alone at concentrations of 20 to 30 mM and in 5 combinations on Aspergillus flavus Link and A. parasiticus Speare populations and aflatoxin B₁ production. Studies on Aspergillus population and aflatoxin B₁ production were carried out in maize grain in relation to a water activity a_w of 0.99, 0.97, 0.95, and 0.93. CA and FA at concentrations of 25 to 30 mM, respectively, and CA-FA mixture T9 (25 + 30 mM) were the treatments most effective at inhibiting A. flavus and A. parasiticus population at all a_w assayed after 11 d of incubation. At all a_w values, the mixture CA-FA T9 (25 + 30 mM) completely inhibited (100%) aflatoxin B₁ production by both strains at a_w= 0.99, 0.97, 0.95, and 0.93. Decreased aflatoxin B₁ levels in comparison with the control were observed with mixtures CA-FA T6 (10 + 25 mM), T7 (20 + 20 mM), and T8 (20 + 30 mM) of both strains in the majority of a_w assayed. The data show that CA and FA could be considered as effective fungitoxicants for A. flavus and A. parasiticus in maize in the a_w range 0.99 to 0.93. The information obtained shows promise for controlling aflatoxigenic fungi in stored maize.     

INFLUENCE OF MOISTURE CONTENT AND STORAGE TEMPERATURE ON THE PRODUCTION OF AFLATOXIN BY ASPERGILLUS FLAVUS EA-81 IN MAIZE AFTER EXPOSURE TO GAMMA RADIATION

The effect of gamma radiation on aflatoxin production by Aspergillus flavus EA-81 in maize with different initial moisture levels was determined over a 15-day period. The viability of A. flavus on maize decreased over time with increasing moisture contents and storage at 8C. After 45 days at 28C, levels of viable conidiospores of A. flavus increased from 4.5 × 10⁷ to about 3.0 × 10⁸ per gram of maize. Levels of aflatoxin B₁ produced by A. flavus were 10 μg kg^-1 in the maize stored at 8C after 45 days. Production of aflatoxin was highest at 40% moisture and 28C. Irradiation of 1.0 or 2.0 kGy greatly reduced the level of mold growth relative to unirradiated controls. A dose of 4.0 kGy eliminated all viable fungi. Aflatoxin B₁ production decreased with increased levels of irradiation and was negligible at 4.0 kGy. When maize was inoculated after irradiation and stored, the spore counts and aflatoxin levels were higher than in unirradiated and inoculated controls after 30 days. Apparently, the natural competitive microflora prevented growth and thus limited higher concentrations of aflatoxin in maize.     

AFLATOXIN PRODUCTION ON TWO MUSHROOM SUBSTRATES

Two fungi, Boletus edulis and Agaricus bisporus, were tested as substrates for two known aflatoxigenic fungi, Aspergillus flavus ATCC 15548 and A. parasiticus NRRL 2999. Both autoclaved substrates supported mycelial growth, sporulation, and aflatoxin production; however, the B. edulis substrate allowed more rapid mold growth and greater toxin production than did the A. bisporus substrate under laboratory conditions. Both aflatoxins B₁ and AFG₁ were produced with AFG₁ being the predominant toxin. Aflatoxins B₂ and AFG₂ were not detected. Although toxin was produced at low levels, the highest mean being 0.55 μg/g substrate for AFB₁ and AFG₁, both mushrooms apparently contained minimal nutrients for toxigenic mold growth and failed to cause antimycotic or antiaflatoxigenic responses. Routinely used aflatoxin extraction and analytical procedures appear applicable for such testing of mushrooms.     

REDUCTION OF FUNGI AND MYCOTOXINS FORMATION IN SEEDS BY GAMMA-RADIATION

Ninety samples of maize, chick-peas and groundnut seeds collected from the Egyptian market were found to be heavily contaminated by molds. Alternaria, Aspergillus, Cladosporium, Eurotium, Fusarium, Mucor, Penicillium and Rhizopus were the most common fungal genera isolated from nondisinfected seeds . Aspergillus alutaceus, A. flavus, Fusarium verticillioides and F. oxysporum were isolated from all surface-disinfected seeds and were reported to produce ochratoxin A, aflatoxin B₁ and zearalenone, respectively. Irradiation at a dose 4.0 kGy reduced the mold growth greatly relative to unirradiated controls. There was no growth at dose 5.0 kGy. On the basis of the radiation survival data, the decimal reduction values D₁₀ for A. alutaceus, A. flavus and F. verticilliodies were 0.70. 2.10 and 0.93 kGy in maize. A dose of 5 kGy inhibited the toxigenic molds and mycotoxin formation in seeds. Aflatoxin B₁ and ochratoxin A were detected in maize and chick-peas, whereas zearalenone was detected in maize samples. Application of radiation at a dose of 6.0 kGy detoxified aflatoxin B₁ by 74.3–76.7%, ochratoxin A by 51.3–96.2% and zearalenone by about 78%.     

Efficacy of extract and essential oil of Lantana indica Roxb. against food contaminating moulds and aflatoxin B1 production

The study investigates the antifungal and antiaflatoxigenic efficacy of Lantana indica against Aspergillus flavus , a key storage fungus. The leaf essential oil of L. indica was found more active than leaf extracts. The oil absolutely inhibited the growth of A. flavus at 1.5 mg mL⁻¹ while ethanolic and chloroform extracts of leaf show MIC at 7.5 and 10.0 mg mL⁻¹ concentrations respectively. The oil also showed pronounced antiaflatoxigenic efficacy and completely inhibited the aflatoxin B₁ production at 0.75 mg mL⁻¹. The ethanolic and chloroformic extracts inhibited the aflatoxin B₁ production at 5.0 and 7.5 mg mL⁻¹, respectively while other extracts exhibited poor efficacy. The L. indica essential oil exhibited broad fungitoxic spectrum against twelve different storage moulds. The present findings may recommend the L. indica essential oil and its bioactive leaf extracts as natural preservative would of immense significance in view of the environmental and toxicological implications by indiscriminate use of synthetic pesticides .     

Efficacy of Artabotrys odoratissimus oil as a plant based antimicrobial against storage fungi and aflatoxin B1 secretion

The essential oil of Artabotrys odoratissimus R.Br. was evaluated for antifungal activity against some storage fungi causing contamination of food stuffs. Minimum inhibitory concentration of the oil was found to be 750 μL L⁻¹ against Aspergillus flavus Link. It was found superior over different prevalent synthetic fungicides which inhibited the growth of A. flavus between 1000–5000 μL L⁻¹. The oil exhibited broad fungitoxic spectrum against fourteen different storage fungi. Aspergillus fumigatus was inhibited at 1000 μL L⁻¹ whereas Cladosporium cladosporioides , Curvularia lunata , Fusarium oxysporum , Helminthosporium oryzae , Macrophomina phaseolina , Microsporum gypseum , Mucor racemosus , Penicillium italicum , Pythium debaryanum , Rhizoctonia solani , Sclerotium rolfsii and Trichoderma viride at 500 μL L⁻¹. Aspergillus niger was found to be inhibited only 84.9% at 1000 μL L⁻¹. In addition, the oil showed significant efficacy in arresting aflatoxin B₁ secretion by the toxigenic strain (Navjot 4NSt) of A. flavus at 750 μL L⁻¹. The efficacy of A. odoratissimus oil as aflatoxin suppressor is being reported for the first time.     

PHYTIC ACID INHIBITS the PRODUCTION of AFLATOXIN B1

Phytic acid inhibition of Aspergillus flavus aflatoxin B₁ production is well observed. Although this fungus grew well in Czapak-Dox medium, mycotoxin production was eliminated by adding a small amount of phytic acid. Possible reasons are discussed, and the importance of some metallic ions is observed. Results suggested that phytate may be an effective anti-AFB₁ agent for preventing the contamination of the fungus.     

Essential Oil of Aegle marmelos as a Safe Plant-Based Antimicrobial Against Postharvest Microbial Infestations and Aflatoxin Contamination of Food Commodities

ABSTRACT:  The essential oil of  Aegle marmelos  L. Correa (Rutaceae) showed strong fungitoxicity against some storage fungi-causing contamination of foodstuffs. The oil also showed efficacy as aflatoxin suppressor at 500 μL/L as it completely arrested the aflatoxin B₁ production by the toxigenic strains (Navjot 4NSt and Saktiman 3NSt) of  Aspergillus flavus  Link. Keeping in view the side effects of synthetic fungicides,  A. marmelos  oil may be recommended as an antimicrobial of plant origin to enhance the shelf life of stored food commodities by controlling the fungal growth as well as aflatoxin secretion. This is the 1st report on aflatoxin B₁ inhibitory nature of this oil.  A. marmelos  oil may be recommended as a novel plant-based antimicrobial in food protection over synthetic preservatives, most of which are reported to incite environmental problems because of their nonbiodegradable nature and side effects on mammals. The LD₅₀ of  Aegle  oil was found to be 23659.93 mg/kg body weight in mice ( Mus musculus  L.) when administered for acute oral toxicity showing nonmammalian toxicity of the oil. GC-MS analysis of the oil found DL-Limonene to be major component.     

A SIMPLE SOLID-PHASE RADIOIMMUNOASSAY FOR AFLATOXIN B1

A solid-phase radioimmunoassay (RIA) for aflatoxin B₁ (afla B₁) was developed. This method involved the incubation of afla B₁, both labelled and unlabelled, with immunoglobulin (IgG)-sepharose gel which was prepared by conjugation of the IgG highly specific to afla B₁ with CNBr-activated sepharose gel, followed by a filtration step. The binding capacity was determined by counting the radioactivity in the filtrate. Studies with different afla B₁ analogues revealed that the IgG-gel bound most effectively with B₁. Binding of afla B₂, G₁, G₂, and aflatoxicol to the IgG-gel was less effective in comparison with the IgG before coupling. Between 0.5–5.0 ng per assay, the displacement of radioactivity from the gel was directly proportional to the amount of afla B₁ present. Using a simple extraction procedure without clean-up step, the recovery yields for afla B₁ in the contaminated corn or wheat at levels of 5 ppb or above were above 60%.     

INHIBITION OF ASPERGILLUS PARASITICUS GROWTH AND TOXIN PRODUCTION BY GARLIC

Mycelial growth and toxin production by Asperigillus parasiticus were inhibited by garlic concentrations of 0.3–0.4%. When the fungus was grown in broth, aflatoxin B₁ production was inhibited at a garlic concentration of 0.3% while aflatoxin G₁ production was inhibited by 0.25% garlic. When growth on rice, aflatoxin B₁ was detected at garlic concentrations of up to 2.5%. Aflatoxin G₁ was detected at 1.25% garlic concentration but not above this level.     

PREPARATION AND CHARACTERIZATION OF AFLATOXIN B2a-HEMIGLUTARATE AND ITS USE FOR THE PRODUCTION OF ANTIBODY AGAINST AFLATOXIN B1

Hemisuccinate (HS) and hemiglutarate (HG) of aflatoxin B_2a (afla B_2a) were prepared by refluxing afla B_2a with the corresponding anhydride and 4-N, N-dimethylaminopyridine in tetrahydrofuran. Two epimers of the respective HS or HG which show different chromatographic behavior and physiochemical properties were isolated and characterized. Afla B_2a-HS hydrolyzes very rapidly in aqueous solution and was not used for further study. Afla B_2a-HG hydrolyzes at a much slower rate and was selected for the coupling to protein. Using the mixed anhydride method, as much as 12 moles of afla B_2a-HG were conjugated to each mole of bovine serum albumin (BSA). The antibody obtained from rabbits immunized with afla B_2a-HG BSA is most specific to afla B₁ and shows little cross reaction with afla G₁ and aflatoxicol. The lower limit for detection of afla B₁ by radioimmunoassay using this antibody is in the range of 30–50 pg per assay.     

Aflatoxin Removal from Pistachio Nuts by Natural Natrolite

ABSTRACT: Natrolite was used to explore a physical and safe method to decontaminate aflatoxin-containing pistachio lots. Pistachio nuts with low, medium, and high levels of aflatoxin were subjected to a washing process with a slurry of 5% natrolite. Using thin-layer chromatography and scanning, the aflatoxin contents of the samples were measured. Aflatoxins B₁ and B₂ were found in pistachio nuts. The majority of total aflatoxins was aflatoxin B₁ Natrolite treatment resulted in a 38% to 100% reduction in aflatoxin B₁, depending on the initial aflatoxin level. Although natrolite was demonstrated to be an effective candidate to reduce aflatoxin B₁, its efficacy against aflatoxin B₂ was limited.     

Aflatoxin levels in dried figs, nuts and paprika for export from Turkey

Three hundred and thirteen of 2643 dried fig, two of eighty hazelnut, sixteen of twenty-eight pistachio, five of ten peanut and nineteen of twenty-three paprika samples for export from Turkey were contaminated with total aflatoxins in the range of 0.2–162.76, 5.46–6.55, 2.31–63.11, 0.75–26.36 and 1.79–6.55 μg kg⁻¹, respectively. Samples were collected from January to August 2007 and tested for aflatoxins (B₁, B₂, G₁ and G₂) by immunoaffinity column extraction using RP-HPLC. Fifty-six of the 313 dried fig, all of the contaminated hazelnut and pistachio, two of the sixteen peanut and three of the nineteen paprika samples exceeded the regulatory limits of the European Union. The ratio of the different types of aflatoxin present in each sample exhibited great variability. For example, of 313 contaminated fig samples, 159 contained only aflatoxin B₁, eighty-five contained B₁ (49.7%) + G₁ (50.3%), twenty-two contained only G₁, twenty contained B₁ (89.4%) + B₂ (10.6%), thirteen contained B₁ (73.7%) + B₂ (10.8%) + G₁ (15.5%) and fourteen contained all four types, B₁ (26%) + B₂ (2.5%) + G₁ (66.5%) + G₂ (5%).     

COMBINED EFFECT OF pH AND HEAT TREATMENT ON DEGRADATION OF AFLATOXINS IN DRIED FIGS

Dried figs are sensitive commodities to aflatoxin contamination. Although preventive methods are the logical solution to aflatoxin problems, once the product is contaminated, decontamination procedures are inevitable. In this study, the effectiveness of a procedure consisted of acidification/alkalization, and heat treatment in degradation of aflatoxins was evaluated. The pH of dried fig extracts was adjusted to 3.1, 3.5, 6, 8 or 10 by adding acid or base. Extracts were heated at 50, 75 or 98C for 1 or 2 h, and then the residual aflatoxin B₁, B₂, G₁ and G₂ were determined. The highest level of degradation for aflatoxin B₁ (97  ±  1%) and B₂ (87  ±  1%) were observed at pH 10 in samples heated at 98 and 50C, respectively. Some treatments resulted in 100% degradation of aflatoxin G₁ and G₂ so that they could not be detected.

PRACTICAL APPLICATIONS

Aflatoxin contamination is a serious problem for a number of processed and non-processed foods, including dried figs. This not only presents severe risks to human and animal health but also causes economic problems for countries such as Turkey, U.S.A., Greece and Spain, which produce and export dried figs. It is clear that detoxifying studies are unavoidable when the amount of crop contaminated by toxins is considered. Therefore, the food industry is in search of applications that are effective in mycotoxin detoxification and adaptable to food processes. This is the first report on degradation of aflatoxins in naturally contaminated dried figs by such a promising method.     

Effect of Soybean Volatile Compounds on Aspergillus flavus Growth and Aflatoxin Production

ABSTRACT:  Soybean homogenates produced volatile compounds upon exposure to lipase. These induced volatiles were identified by SPME. Seventeen volatile compounds identified by SPME were chosen for determination of their ability to inhibit Aspergillus flavus growth and aflatoxin B₁ (AFB1) production in a solid media assay. These volatiles included aldehydes, alcohols, ketones, and furans. Of the tested compounds, the aldehydes showed the greatest inhibition of fungal growth and AFB1 production. These compounds inhibited up to 100% of the observed growth and AFB1 production as compared to the controls. The greatest activity by the aldehydes to disrupt growth was ranked as follows: 2,4 hexadienal > benzaldehyde > 2-octenal > ( E )-2-heptenal > octanal > ( E )-2-hexenal > nonanal > hexanal. The greatest activity by the aldehydes to reduce AFB1 was ranked as follows: ( E )-2-hexenal > 2,4 hexadienal > ( E )-2-heptenal > hexanal > nonanal. ( E )-2-hexenal and ( E )-2-heptenal were tested further in an A. flavus -inoculated corn kernel assay. Both compounds prevented colonization by A. flavus and eliminated AFB1 production when exposed to compound volumes < 10 μL as also shown in the solid media assay. The results suggest that soybeans react to lipase by production of potent antifungal volatiles.     

RESEARCH NOTE: MYCOTOXINS IN FENNEL SEED

Ten samples of fennel seed from fields in India were examined for the presence ofaflatoxins B₁, B₂, G₁ and G₂. Five of the samples were fluorescent or became fluorescent during a 12 month storage period. Only aflatoxins B₁, B₂ and G₁ were identified. One nonfluorescent sample contained detectable aflatoxin B₁ after 12 months of storage.     

贵州秋季栽培不同荞麦品种成熟期果实中黄曲霉及其毒素的分离与鉴定

本研究以贵阳秋季栽培的2个米苦荞(F.tataricum,贵米苦荞18-1号和贵黑米苦荞12号)、2个多苦荞(F.tatari-cymosum,贵多苦荞003C和贵多苦荞60)、2个甜荞(F.esculentum,贵红花甜荞2号和1412-1)、2个常规苦荞(F.tataricum,定苦荞1号和六苦2017)为材料。对其成熟期种子果壳和籽粒进行了黄曲霉分离鉴定,并采用高效液相色谱法对所有品种果壳和籽粒中分离出的黄曲霉菌株进行AFB₁、AFB₂、AFG₁和AFG₂毒素的检测。结果表明,所有品种果壳中均没有分离出黄曲霉菌落;4类荞麦籽粒中仅米苦荞分离出了黄曲霉菌落,共分离出4株黄曲霉菌株。其中贵米苦荞18-1号黄曲霉带菌率为1.56%,贵黑米苦荞12号黄曲霉带菌率为0.78%。分离菌株形态学和ITS序列扩增产物测序结果与已知黄曲霉菌序列完全一致。毒素检测结果表明不同品种之间产毒素差异显著,所有品种籽粒中只有米苦荞中检出4种毒素,贵米苦荞18-1号产AFB₁最高为(5.861±0.055) μg/kg、AFB₂最少为(1.605±0.052) μg/kg,贵黑米苦荞12号产AFB₁最高为(14.475±0.533) μg/kg、AFG₂最少为(3.393±0.151) μg/kg;籽粒产毒量远大于分离菌株产毒量;各分离出菌株之间产毒素能力差异显著,最大产AFT为(11.102±0.095) μg/kg、最小产AFT为(1.794±0.024) μg/kg。上述结果显示供试米苦荞籽粒带菌来源可能是由于果壳开裂籽粒外露后部分籽粒被直接侵染所致。所得结果可为米苦荞中黄曲霉抗性育种研究及荞麦种子的保存、运输、储藏等研究奠定基础。     

DETOXIFICATION OF GROUNDNUT SEEDS BY UREA AND SUNLIGHT

A fourteen hour exposure to sunlight destroys about 90 and 77% aflatoxin B₁ added to groundnut flakes with and without fat whereas only about 50% of the toxin is destroyed when present as a natural contaminant. Treating the groundnut flakes with 20% urea and 2% soybean flour (a source of urease) at 50% moisture would bring about 70% destruction of aflatoxin B₁; In large scale trials, destruction was about 85%. Treatment with urea does not bring down the PER value of the material, which is 1.5 after treatment as against 1.6 in the untreated groundnuts.     

Inhibition of Fungal Growth on Wheat and Rye Bread by Modified Atmosphere Packaging and Active Packaging Using Volatile Mustard Essential Oil

ABSTRACT: Wheat and rye bread artificially inoculated with molds were packed in modified atmospheres of 0%, 50%, 75%, or 100% CO₂ balanced with N₂, and 3 levels of residual O₂, 1%, 0.03%, or <0.01%/O₂-absorber, and stored for 30 to 35 d. Modified atmosphere packaging (MAP) was quantitatively more effective for rye bread because fewer mold species grew at elevated CO₂. However, the major rye bread contaminant, Penicillium roqueforti , was the overall most CO₂-resistant mold and only the use of O₂-absorber could prevent growth of this species. On wheat bread, the most CO₂-tolerant mold was Penicillium commune , growing in 99% CO₂ (with high residual O₂), and Aspergillus flavus was the mold species that grew at lowest O₂ in 75% CO₂ treatment. The spoilage yeast/"chalk mold" Endomyces fibuliger was less affected by the different O₂ levels than the true filamentous molds, and none of the tested MAP treatments could prevent growth, but lag-phase was increased with O₂-absorber on wheat bread and decreased with 1% residual O₂ on rye bread. Experiments with volatile mustard oil showed that A. flavus and Eurotium repens were the most mustard oil-resistant species on wheat and rye bread, respectively. A combination strategy with MAP and mustard oil proved most optimal, and total inhibition was achieved with 2 μL mustard oil/rye bread slice and between 2 and 3 μL/wheat bread. Results indicated that the nature and surface area of the product influences effectiveness of active packaging with mustard oil.