Factors influencing the inhibition of aflatoxin production in corn by Aspergillus niger

Aspergillus niger, a mold commonly associated with Aspergillus flavus in damaged corn, interferes with the production of aflatoxin when grown with A. flavus on autoclaved corn. The pH of corn-meal disks was adjusted using NaOH-HCl, citric acid-sodium citrate, or a water extract of A. niger fermented corn. Aflatoxin formation was completely inhibited below pH 2.8-3.0, irrespective of the system used for pH adjustment. When grown in association with A. flavus NRRL 6432 on autoclaved corn kernels, A. niger NRRL 6411 lowered substrate pH sufficiently to suppress aflatoxin production. The biodegradation of aflatoxin B1 or its conversion to aflatoxin B2a were eliminated as potential mechanisms by which A. niger reduces aflatoxin contamination. A water extract of corn kernels fermented with A. niger caused an additional inhibition of aflatoxin formation apart from the effects of pH.     

Fungal Contamination of Raw Materials of Some Herbal Drugs and Recommendation of <Emphasis Type="Italic">Cinnamomum camphora</Emphasis> Oil as Herbal Fungitoxicant

The paper explores fungal infection and aflatoxin B(1) contamination of six medicinal plant samples viz. Adhatoda vasica Nees, Asparagus racemosus Linn., Evolvulus alsinoides Linn., Glycyrrhiza glabra Linn., Plumbago zeylanica Linn. and Terminalia chebula Retz. A total of 858 fungal isolates were detected from the raw materials. Maximum number of fungal isolates was detected from A. racemosus (228). The genus Aspergillus was found to be the most dominant genus causing infection to most of the raw materials. Among the 32 isolates of A. flavus tested, 13 isolates were found to be toxigenic elaborating aflatoxin B(1). The highest elaboration of aflatoxin B(1) was 394.95 ppb by the isolates of A. flavus from G. glabra. The essential oil of Cinnamomum camphora (L.) Presl showed efficacy in arresting aflatoxin B(1) by the toxigenic strain. The growth of a toxigenic strain of A. flavus decreased progressively with increasing concentration of essential oil from leaves of C. camphora. The oil completely inhibited aflatoxin B(1) production even at 750 ppm. Hence, the oil of C. camphora is recommended as herbal fungitoxicant against the fungal contamination of the raw materials.     

Aflatoxin B1 and fumosin B1 in mixed cultures of Aspergillus flavus and Fusarium proliferatum on maize

Production of aflatoxin B1 and fumonisin B1 in pure and mixed cultures of Aspergillus flavus and Fusarium proliferatum were determined on irradiated maize seeds inoculated with different spore concentrations at 0.97 water activity (a(w)) and a temperature of 25 degrees C. The highest levels of aflatoxin B1 were produced by A. flavus at the lowest levels of inoculum (10(3) spore ml(-1)). There was no spore concentration influence on fumonisin B1 production after 10, 20 and 35 days of incubation. When A. flavus was co-inoculated with F. proliferatum, aflatoxin B1 production was inhibited. The higher the inocula levels of Fusarium produced, the higher the inhibition and this inhibition increased during the incubation period. Total inhibition was reached at 35 days of incubation. There was no interaction influence on fumonisin B1 production at all inoculum levels assayed. These results suggest that under optimal environmental conditions of substrate, water activity and temperature, the interaction between A. flavus and F proliferatum could produce inhibition of aflatoxin B1 and stimulation of fumonisin B1.     

Effects of Moisture Content and Temperature on Aflatoxin Production in Corn

Samples of freshly harvested and remoistened corn, of various moisture contents, were stored at different temperatures; analyses for aflatoxin content were made periodically. At moisture levels above 17.5% and at temperatures of 24 C or warmer, aflatoxins were formed by Aspergillus flavus present in the original epiphytic mycoflora. Remoistened dried corn was subject to more rapid fungal deterioration and aflatoxin formation than freshly harvested corn. Screening of the fungi present in the corn revealed aflatoxin production only by A. flavus. The toxigenic strains produced only aflatoxins B(1) and B(2).     

Partial characterization of the mode of action of benzoic acid on aflatoxin biosynthesis.

Aflatoxin production by a toxigenic strain of Aspergillus flavus was greatly reduced by benzoic acid and sodium benzoate in synthetic media. The reduction was accompanied by the appearance of a yellow pigment. Spectral analyses partially characterized this pigment as closely related to an acetyl derivative of a versiconal-type compound. A cell-free extract prepared from A. flavus grown in synthetic media was active in converting this yellow compound into aflatoxin B1 in the presence of reduced nicotinamide adenine dinucleotide phosphate at 25 degrees C (pH 7.4). In the presence of benzoic acid and its salt or autoclaved cell-free extract, conversion of yellow compound to aflatoxin B1 was prevented. These results suggest that the yellow compound is an intermediate in the secondary metabolic cycle involved in aflatoxin B1 production. Benzoic acid, sodium benzoate, or autoclaving the cell-free extract appear to have respectively blocked or denatured an enzymatic step late in the biosynthetic pathway of aflatoxin B1.     

Variability among atoxigenic Aspergillus flavus strains in ability to prevent aflatoxin contamination and production of aflatoxin biosynthetic pathway enzymes.

Five strains of Aspergillus flavus lacking the ability to produce aflatoxins were examined in greenhouse tests for the ability to prevent a toxigenic strain from contaminating developing cottonseed with aflatoxins. All atoxigenic strains reduced contamination when inoculated into developing bolls 24 h prior to the toxigenic strain. However, only one strain, AF36, was highly effective when inoculated simultaneously with the toxigenic strain. All five strains were able to inhibit aflatoxin production by the toxigenic strain in liquid fermentation. Thus, in vitro activity did not predict the ability of an atoxigenic strain to prevent contamination of developing bolls. Therefore, strain selection for competitive exclusion to prevent aflatoxin contamination should include evaluation of efficacy in developing crops prior to field release. Atoxigenic strains were also characterized by the ability to convert several aflatoxin precursors into aflatoxin B1. Four atoxigenic strains failed to convert any of the aflatoxin biosynthetic precursors to aflatoxins. However, the strain (AF36) most effective in preventing aflatoxin contamination in developing bolls converted all tested precursors into aflatoxin B1, indicating that this strain made enzymes in the aflatoxin biosynthetic pathway.     

Influence of microbial co-inhabitants on aflatoxin synthesis of Aspergillus flavus on maize kernels

The co-inhabiting mycoflora with Aspergillus flavus observed on individual maize kernels was evaluated for its influence on aflatoxin synthesis. All 13 types of associations of different fungal species inhibited aflatoxin B₁ and G₁ production at different levels (34·3–100%). Inhibition of radial growth of A. flavus by Fusarium moniliforme (59·8%), Trichoderma viride (72·5%) and Rhizopus nigricans (42%) could be directly correlated to the per cent inhibition of aflatoxin production. High levels of inhibition of aflatoxin elaboration were noted in competition of A. flavus with other toxigenic moulds.     

Incidence of aflatoxin producing strains and aflatoxin contamination in dry fruit slices of quinces (Cydonia oblonga Mill.) from the Indian state of Jammu and Kashmir

An investigation was undertaken to obtain data on the occurrence of aflatoxins and the aflatoxin producing potential of Aspergillus flavus strains isolated from dry fruit slices of quinces produced in jammu and Kashmir, India. A total of 147 A. flavus isolates recovered from dr fruit slices were grown in liquid rice flour medium and screened for the production of various aflatoxins by thin layer chromatography. The results showed that 23.14% of the tested isolates were aflatoxigenic, producing aflatoxins B1 and B2 in varying amounts. Aflatoxins G1 and G2 were not detected. All 25 of the investigated market samples were also found to be aflatoxin B1 positive and the level of contamination ranged from 96 to 8164 micrograms/kg of the dry fruit which is quite high in comparison to the permissible level of 30 ppb. As per these results biochemical composition of dry fruit slices of quinces, along with climatic conditions seem to be very favourable for aflatoxin production by the toxigenic A. flavus strains. Therefore, monitoring of aflatoxins in dry fruit slices of quinces is recommended for this region.     

Intrafungal distribution of aflatoxins among conidia and sclerotia of Aspergillus flavus and Aspergillus parasiticus

This research examines the distribution of aflatoxins among conidia and sclerotia of toxigenic strains of Aspergillus flavus Link and Aspergillus parasiticus Speare cultured on Czapek agar (21 days, 28 degrees C). Total aflatoxin levels in conidia and sclerotia varied considerably both within (intrafungal) and among strains. Aspergillus flavus NRRL 6554 accumulated the highest levels of aflatoxin (conidia: B1, 84000 ppb; G1, 566000 ppb; sclerotia: B1, 135000 ppb; G1, 968000 ppb). Substantial aflatoxin levels in conidia could place at risk those agricultural workers exposed to dust containing large numbers of A. flavus conidia. Cellular ratios of aflatoxin B1 to aflatoxin G1 were nearly identical in conidia and sclerotia even though levels of total aflatoxins in these propagule types may have differed greatly. Aflatoxin G1 was detected in sclerotia of all A. flavus strains but in the conidia of only one strain. Each of the A. parasiticus strains examined accumulated aflatoxin G1 in both sclerotia and conidia. These results are examined in the context of current evolutionary theory predicting an increase in the chemical defense systems of fungal sclerotia, propagules critical to the survival of these organisms.     

Positive correlation exists between glutathione S-transferase activity and aflatoxin formation in Aspergillus flavus.

The presence of glutathione (GSH) S-transferase activity, using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate, has been established in the cytosolic fraction of the toxigenic (aflatoxin producing) and nontoxigenic strains of Aspergillus flavus. Significant differences in the GSH S-transferase activity were observed between the toxigenic and non-toxigenic strains. A positive correlation has been demonstrated for the first time between aflatoxin formation and a biochemical parameter, namely GSH S-transferase activity. The evidence in support of A. flavus GSH S-transferase induction by endogenous aflatoxins is as follows: (i) the age-related production of aflatoxin follows the same pattern as the cytosolic GSH S-transferase activity profile; (ii) significantly higher enzyme activity was associated with mycelia of a toxigenic strain grown in medium supporting high aflatoxin production (sucrose-low-salts medium) while the enzyme activity was low in medium producing less aflatoxin (glucose-ammonium nitrate medium). The GSH S-transferase activity of the non-toxigenic strain was hardly affected by a change in the medium as it produces no aflatoxins; and (iii) the toxigenic strain demonstrated significantly higher apparent Vmax. with no change in Km as compared with the non-toxigenic strain. This indicates that the enzyme induction by endogenous aflatoxins is similar to the action of phenobarbitol and other inducing drugs (Kaplowitz et al., 1975).     

Effects of neem leaf extract on production of aflatoxins and activities of fatty acid synthetase,isocitrate dehydrogenase and glutathione S-transferase in Aspergillus parasiticus

The relationship between the activities of 3 cytosolic enzymes with aflatoxin biosynthesis in Aspergillus parasiticus cultured under different conditions has been investigated in order to find out the role of each enzyme in aflatoxin biosynthesis. Basically the activity of isocitrate dehydrogenase (IDH) was higher in non-toxigenic strains as compared to its counterpart toxigenic fungi (p < 0.05). In contrast, the activities of fatty acid synthase (FAS) as well as glutathione S-transferase (GST) were higher (P < 0.05) in toxigenic strains than that of the non-toxigenic fungi. Aflatoxin production was inhibited in fungi grown in presence of various concentrations of neem leaf extract. Aflatoxin was at its lowest level (>90% inhibition) when the concentration of neem extract was adjusted to 50% (v/v). No significant changes in FAS and IDH activities were observed when aflatoxin synthesis was under restraints by neem (Azadirachta indica) leaf extract. During a certain period of time of culture growth, when aflatoxin production reached to its maximum level, the activity of FAS was slightly induced in the toxigenic strains fed with a low concentration (1.56% v/v) of the neem leaf extract. At the time (96 h) when aflatoxin concentration reached to its maximum levels, the activity of GST in the toxigenic fungi was significantly higher (i.e., 7–11 folds) than that of non-toxigenic strains. The difference was highest in mycelial samples collected after 120 h. However unlike FAS and IDH, GST was readily inhibited (67%) in mycelia fed with 1.56% v/v of the neem extract. The inhibition reached to maximum of 80% in samples exposed to 6.25–12.5% of the extract. These results further substantiate previous finding that there is a positive correlation between GST activity and aflatoxin production in fungi.This revised version was published online in October 2005 with corrections to the Cover Date.     

Mold flora and aflatoxin contamination of stored and cooked samples of pearl millet in the Paharia tribal belt of Santhal paragana, Bihar, India

Stored and cooked samples of pearl millet (Pennesetum typhoides), which is regularly consumed as food by the Paharia tribe in the hilly regions of Santhal Pargana, Bihar State, India, that were harvested in January 1989 were analyzed for mold flora, natural occurrence of Aspergillus flavus and A. parasiticus, and incidence and levels of aflatoxin B1. Of the 22 fungal species isolated, A. flavus and A. parasiticus were the predominant species (63.8%) during the rainy season, followed by other species of Aspergillus, Penicillium, Fusarium, Rhizopus, Helminthosporium, and Curvularia. Screening of 169 A. flavus and A. parasiticus strains showed that 59 of them were toxigenic, producing various combinations of aflatoxins B1, B2, G1, and G2. The amounts of aflatoxin B1 ranged between 4 and 30 mg/100 ml of liquid medium. Analysis of stored and cooked samples also revealed a high incidence and alarming levels of naturally produced aflatoxin B1. Forty-nine of 75 stored and 16 of 38 cooked samples contained various combinations of aflatoxins. The levels of aflatoxin B1 ranged between 17 and 2,110 ppb in stored samples and 18 and 549 ppb in cooked samples. The correlation of insect damage with A. flavus and A. parasiticus incidence and quantity of aflatoxin B1 was found to be insignificant.     

Mold flora and aflatoxin contamination of stored and cooked samples of pearl millet in the Paharia tribal belt of Santhal paragana, Bihar, India.

Stored and cooked samples of pearl millet (Pennesetum typhoides), which is regularly consumed as food by the Paharia tribe in the hilly regions of Santhal Pargana, Bihar State, India, that were harvested in January 1989 were analyzed for mold flora, natural occurrence of Aspergillus flavus and A. parasiticus, and incidence and levels of aflatoxin B1. Of the 22 fungal species isolated, A. flavus and A. parasiticus were the predominant species (63.8%) during the rainy season, followed by other species of Aspergillus, Penicillium, Fusarium, Rhizopus, Helminthosporium, and Curvularia. Screening of 169 A. flavus and A. parasiticus strains showed that 59 of them were toxigenic, producing various combinations of aflatoxins B1, B2, G1, and G2. The amounts of aflatoxin B1 ranged between 4 and 30 mg/100 ml of liquid medium. Analysis of stored and cooked samples also revealed a high incidence and alarming levels of naturally produced aflatoxin B1. Forty-nine of 75 stored and 16 of 38 cooked samples contained various combinations of aflatoxins. The levels of aflatoxin B1 ranged between 17 and 2,110 ppb in stored samples and 18 and 549 ppb in cooked samples. The correlation of insect damage with A. flavus and A. parasiticus incidence and quantity of aflatoxin B1 was found to be insignificant.     

Survey of the mycoflora and mycotoxins of cotton seeds and cotton seed products in Egypt

Thirty-nine species and 16 fungal genera were isolated from Egyptian cotton seeds, cotton seed meal and cotton seed cake on 1% glucose-Czapek's agar medium incubated at 28 °C. Aspergillus was the most frequent genus and it emerged in 87–100% of the samples contributing 70–98% of total fungi in the three substrates tested. The most common species were A. niger, A. flavus, A. fumigatus, A. terreus and Rhizopus stolonifer; A. niger, A. fumigatus and Penicillium corylophilum; and A. niger, A. flavus, A. terreus, A. nidulans and Rhizopus stolonifer, respectively. Cotton seeds and cotton seed products were naturally contaminated by aflatoxin B₁ and B₂. About 16% of the different substrates tested were positive for aflatoxin contamination. No citrinin, ochratoxin A, patulin, sterigmatocystin, diacetoxyscirpenol, T-2 toxin or zearalenone were detected in the samples assayed.     

Interactions of saprophytic yeasts with a nor mutant of Aspergillus flavus.

The nor mutant of Aspergillus flavus has a defective norsolorinic acid reductase, and thus the aflatoxin biosynthetic pathway is blocked, resulting in the accumulation of norsolorinic acid, a bright red-orange pigment. We developed a visual agar plate assay to monitor yeast strains for their ability to inhibit aflatoxin production by visually scoring the accumulation of this pigment of the nor mutant. We identified yeast strains that reduced the red-orange pigment accumulation in the nor mutant. These yeasts also reduced aflatoxin accumulation by a toxigenic strain of A. flavus. These yeasts may be useful for reducing aflatoxin contamination of food commodities.     

Effects of soil moisture and temperature on preharvest invasion of peanuts by the Aspergillus flavus group and subsequent aflatoxin development

Four soil temperature and moisture treatment regimens were imposed on Florunner peanuts 94 days after planting in experimental plots in 1980. At harvest (145 days after planting), the incidence of the Aspergillus flavus group and the aflatoxin concentration were greatest in damaged kernels. Extensive colonization of sound mature kernels (SMK) by the A. flavus group occurred with the drought stress treatment (56% kernels colonized); colonization was less in the irrigated plot (7%) and the drought stress plot with cooled soil (11%) and was intermediate in the irrigated plot with heated soil (26%). Aflatoxin was virtually absent from SMK with the last three treatments, but it was found at an average concentration of 244 ppb (ng/g) in drought-stressed SMK. Colonization of SMK by the A. flavus group and aflatoxin production were greater with hot dry conditions. Neither elevated temperature alone nor drought stress alone caused aflatoxin contamination in SMK. When the ratio of SMK colonized by A. flavus compared with A. niger was greater than 19:1, there was aflatoxin contamination, but there was none if this ratio was less than 9:1. Irrigation caused a higher incidence of A. niger than drought did. This may have prevented the aflatoxin contamination of undamaged peanuts.     

Effects of soil moisture and temperature on preharvest invasion of peanuts by the Aspergillus flavus group and subsequent aflatoxin development.

Four soil temperature and moisture treatment regimens were imposed on Florunner peanuts 94 days after planting in experimental plots in 1980. At harvest (145 days after planting), the incidence of the Aspergillus flavus group and the aflatoxin concentration were greatest in damaged kernels. Extensive colonization of sound mature kernels (SMK) by the A. flavus group occurred with the drought stress treatment (56% kernels colonized); colonization was less in the irrigated plot (7%) and the drought stress plot with cooled soil (11%) and was intermediate in the irrigated plot with heated soil (26%). Aflatoxin was virtually absent from SMK with the last three treatments, but it was found at an average concentration of 244 ppb (ng/g) in drought-stressed SMK. Colonization of SMK by the A. flavus group and aflatoxin production were greater with hot dry conditions. Neither elevated temperature alone nor drought stress alone caused aflatoxin contamination in SMK. When the ratio of SMK colonized by A. flavus compared with A. niger was greater than 19:1, there was aflatoxin contamination, but there was none if this ratio was less than 9:1. Irrigation caused a higher incidence of A. niger than drought did. This may have prevented the aflatoxin contamination of undamaged peanuts.     

Aflatoxin contamination of some common drug plants

This paper deals with the natural occurrence of aflatoxins in some common drug plants collected from storehouses in Bihar, India. Of 15 samples analyzed, 14 were aflatoxin positive. The highest level of aflatoxin contamination was detected in the seeds of Piper nigrum (1.20 micrograms/g), followed by the level detected in the seeds of Mucuna prurita (1.16 micrograms/g), and the lowest level was detected in the bark of Acacia catechu (0.09 micrograms/g). Of 158 isolates of Aspergillus flavus obtained from as many samples of drug plants, 49 were found to be toxigenic. The amount of aflatoxin B1 elaborated by the toxigenic isolates was in the range of 0.86 to 5.24 micrograms/ml of culture filtrate.     

Aflatoxin contamination of some common drug plants.

This paper deals with the natural occurrence of aflatoxins in some common drug plants collected from storehouses in Bihar, India. Of 15 samples analyzed, 14 were aflatoxin positive. The highest level of aflatoxin contamination was detected in the seeds of Piper nigrum (1.20 micrograms/g), followed by the level detected in the seeds of Mucuna prurita (1.16 micrograms/g), and the lowest level was detected in the bark of Acacia catechu (0.09 micrograms/g). Of 158 isolates of Aspergillus flavus obtained from as many samples of drug plants, 49 were found to be toxigenic. The amount of aflatoxin B1 elaborated by the toxigenic isolates was in the range of 0.86 to 5.24 micrograms/ml of culture filtrate.     

传统发酵豆瓣中产毒黄曲霉高效拮抗菌的筛选

从自然发酵的豆瓣中筛选出对产毒黄曲霉菌的生长及其毒素合成均有抑制作用的细菌, 在蚕豆天然培养基(BAM)上利用菌落对峙实验初筛和滤纸片复筛得到1株有较高抑制产毒黄曲霉活性的菌株L4。对L4进行形态学、生理生化特征及16S rRNA序列同源性分析, 鉴定此菌株为枯草芽孢杆菌(Bacillus subtilis)。在抑制黄曲霉生长和黄曲霉毒素B1 (AFB1)合成的研究中表明, 在L4与黄曲霉菌共同培养15 d后, 黄曲霉菌丝产量和黄曲霉毒素B1 产量均比黄曲霉单独培养时显著降低(P < 0.01), AFB1合成受到明显抑制, 抑制率达93.7%。当黄曲霉孢子液与L4发酵上清液1: 1 (V/V)混合后接种在玉米粒上时, 黄曲霉在玉米上的生长和孢子萌发均得到完全抑制。