Modulation of Aflatoxin B1 production by Aspergillus flavus
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Mycotoxins are toxic contaminants of foodstuffs produced by a wide range of fungal species. Aflatoxins are the only mycotoxins carcinogenic for humans. They are mainly produced by the Aspergillus genus and can be found at each step of the agrofood chain (e.g. field, storage, process). Due to climate changes, France is starting to be exposed to aflatoxins. In order to limit the consumer exposure, many prevention or decontamination techniques have been developed. To this aim, we started the development of a biocontrol against aflatoxins accumulation for maize field application. Actinomycetes, are soil-borne bacteria that has already been commercialized as biocontrol. In Petri dishes, we studied the in vitro interaction between some actinomycetes and Aspergillus flavus, the main aflatoxins producer. We revealed that the interaction reduced the aflatoxins content (monitored by HPLC). Moreover, some bacterial isolates were able to reduce pure-aflatoxin B1 added in the medium. To understand this mechanism, adsorption tests has been conducted. Otherwise, RT-qPCR methodology was used to study the impact of Streptomyces-Aspergillus sp. on aflatoxin gene expression. Finally, the current knowledge of the impact of environmental factors (temperature, water activity and incubation time) on aflatoxins production was supplemented.Keywords:
Aspergillus parasiticus
Aspergillus oryzae
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ycological examination of 500 samples of animal feeds and feed ingredients for Aspergillus flavus and Aspergillus parasiticus revealed that 180 (36%) samples yielded isolates of Aspergillus flavus and 65 (13%) samples gave isolates of Aspergillus parasiticus. Testing of the same samples for AFB1 contamination showed that 99 samples (19.8%) contained AFB1 at a rate of 125 ppb, 45 samples (9%) at a rate of 25-50 ppb, 32 samples (6.4%) at a rate of 101-200 ppb and 19 samples (3.8%) contained aflatoxin B1 at a rate of 201-2000 ppb. Screening of isolated strains of Aspergillus flavus and Aspergillus parasiticus for aflatoxin B1 production by culturing on YES medium supplemented with 0.019% P-cresol revealed that 81 (45%) out of 180 isolates of Aspergillus flavus and 16 (24.62%) out of 65 isolates of Aspergillus parasiticus produced aflatoxin B1. Testing the ability of 4 Lactobacillus strains for removal of aflatoxin B1 from liquid media after physical and chemical treatments revealed that the acidic and heat treatments of bacterial pellets significantly enhanced their ability to bind aflatoxin B1 but heat treatment was not as effective as acidic treatment. Screening the ability of either intact mycelium or fragmented mycelium or culture cell - free system of non - aflatoxin B1 producing Aspergillus flavus and Aspergillus parasiticus indicated that fragmentation increased the ability of tested strain to degrade aflatoxin B1. Culture cell free system showed the highest percent of aflatoxin B1 degradation. Aspergillus flavus showed higher percent of degradation than Aspergillus parasiticus.
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The influence of pyridazinone herbicides on aflatoxin production by Aspergillus flavus and A. parasiticus was studied in liquid media. Mycelia production was not affected by 20, 40, or 60 micrograms of herbicide per ml; however, aflatoxin production by A. parasiticus was higher in media with herbicide, whereas A. flavus produced lower aflatoxin levels.
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The amount of aflatoxin produced by Aspergillus flavus and Aspergillus parasiticus grown on various aged and non-aged seeds, kept at suitable conditions of temperature and moisture, is particularly related to the peroxide numbers of the seed oils. The addition of synthetic hydroperoxides to the cultures greatly increased aflatoxin production.
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Aflatoxins are polyketide secondary metabolites that are produced by certain fungal species in the Aspergillus section Flavi, particularly Aspergillus flavus and Aspergillus parasiticus which contaminate human food as well as animal feed. These are among the most carcinogenic substances known. Due to the toxic and carcinogenic properties of aflatoxins, there is a need to develop reliable methods to detect the presence of aflatoxigenic Aspergilli in contaminated food and feed. Not all Aspergillus strains are able to produce aflatoxins. It requires a detection methodology which can specifically distinguish between the aflatoxin producing and nonproducing strains of Aspergillus. Present communication reports validation of a PCR based detection system based on three genes viz., nor-1, apa-2 and omt-1 involved in aflatoxin biosynthesis, that can specifically distinguish the two aflatoxin producing species viz. Aspergillus flavus ,and Aspergillus parasiticus from non-producers i.e., A. niger, A. fumigates and A. oryzae.
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Nyjer seeds are oil rich (35-40% oil content) seeds of the plant Guizotia abyssinica , which is closely related to sunflower. They are pressed mechanically for cooking oil in Ethiopia and elsewhere. The remaining deoiled cake, which contains approximately 10% oil is commonly used as animal feed. This study investigated the effect of water activity and temperature on the growth and aflatoxin production of the four main forms of aflatoxin (B 1 , B 2 , G 1 and G 2 ) by Aspergillus flavus and Aspergillus parasiticus on ground nyjer seed with 10% oil. The ground nyjer seeds were adjusted to different water activity a w levels (0.82, 0.86, 0.90, 0.94 and 0.98 a w ) and incubated at 20, 27 and 35 °C, up to 30 days. Our results show that A. flavus and A. parasiticus had similar growth patterns in which the slowest fungal growth occurred on ground seeds with 0.86 a w at 20 °C. There was no fungal growth for either A. flavus or A. parasiticus at 0.82 a w . The most rapid growth conditions for A. flavus and A. parasiticus were 0.94 a w at 35 °C, and 0.94 a w at 20 °C, respectively. Aspergillus flavus produced aflatoxins (13 μg/kg aflatoxin B 1 ) only on seeds with 0.94 a w at 27 °C, while A. parasiticus produced high levels of aflatoxins under several conditions; the highest concentrations of aflatoxin B 1 (175 μg/kg) and AFG 1 (153 μg/kg) were produced on deoiled ground seeds with 0.94 a w at 27 °C. It is likely that storing ground deoiled nyjer seeds with a water activity up to 0.82 a w at 20 °C will reduce fungal growth aflatoxin production.
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Production of aflatoxins B1, B2, G1, and G2 in pure and mixed cultures of Aspergillus parasiticus and aspergillus flavus were compared. Differing percentages of A. parasiticus (NRRL 2999) and A. flavus (NRRL 5520) conidia were used as inoculum and allowed to grow in static liquid culture for 10 days. The pure and mixed cultures of A. parasiticus/A. flavus were extracted and the aflatoxins partially purified. High‐performance liquid chromatography was then used to determine aflatoxin concentrations. The maximal decreases of G1 and G2production occurred when the inocula went from 0 to 12.5% or 25% A. flavus. Aflatoxins G1and G2 comprised less than 10% of the total aflatoxins when the A. parasiticuslA. flavus inocula contained greater than 25% A. flavus. Production of aflatoxins B1 and B2 increased slightly as the percentage of A. flavus in the A. parasiticuslA. flavus mixed inoculum increased. A. flavus is apparently capable of suppressing accumulation of aflatoxins G1 and G2 by A. parasiticus when these fungi are grown in mixed culture.
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