Effect of Citrus reticulata and Cymbopogon citratus Essential Oils on Aspergillus flavus Growth and Aflatoxin Production on Asparagus racemosus

Essential oils extracted from Citrus reticulata and Cymbopogon citratus were tested in vitro against the toxigenic strain of Aspergillus flavus, isolated from the tuberous roots of Asparagus racemosus, used in preparation of herbal drugs. The essential oils completely inhibited the growth of A. flavus at 750 ppm and also exhibited a broad fungitoxic spectrum against nine additional fungi isolated from the roots. Citrus reticulata and Cymbopogon citratus essential oils completely inhibited aflatoxin B₁ production at 750 and 500 ppm, respectively. During in vivo investigation, the incidence of fungi and aflatoxin B₁ production decreased considerably in essential oil-treated root samples. The findings thus indicate possible exploitation of the essential oils as effective inhibitor of aflatoxin B₁ production and as post-harvest fungitoxicant of traditionally used plant origin for the control of storage fungi. These essential oils may be recommended as plant-based antifungals as well as aflatoxin B₁ suppressors in post-harvest processing of herbal samples.     

The effect of <Emphasis Type="Italic">Baccharis glutinosa</Emphasis> extract on the growth of mycotoxigenic fungi and fumonisin B<Subscript>1</Subscript> and aflatoxin B<Subscript>1</Subscript> production

The aim of this study was to evaluate the effect of Baccharis glutinosa isolated extract on the growth of Aspergillus flavus and Aspergillus parasiticus, and their aflatoxin B₁ production; and growth of Fusarium verticillioides, and their fumonisin B₁ production. The three fungi were exposed to an antifungal fraction, designated as fraction F6-1, isolated from B. glutinosa by methanolic extraction followed by silica gel chromatography. The growth of the fungi was evaluated in kinetics of radial extension growth, kinetics of spores germination, length and diameter of hyphae, spores diameter, as well as in aflatoxin B₁ and fumonisin B₁ production. Fraction F6-1 caused radial growth inhibition of the three fungi mainly F. verticillioides. Spores germination of A. flavus and A. parasiticus was delayed in the early stage of the incubation time, although they completely germinated at 27 h. In contrast, spore germination of F. verticillioides was inhibited 87.7% up to 96 h. The lengths and diameters of hyphae, and spore diameters of the three fungi, were significantly smaller in comparison with those of the controls, and several morphological alterations were observed. Concerning aflatoxin B₁ and fumonisin B₁, fraction F6-1 did not show any inhibition effect at the concentration used. Fraction F6-1 was able to significantly inhibit the development of the three fungi, mainly F. verticillioides. The strong inhibitory effect of F6-1 on hyphae and spores suggests that it interacted with the fungi cell walls, which caused severe deformities. Nevertheless, this fraction was unable in inhibiting mycotoxin production from the three fungi at the concentration tested.     

Mycotoxins and mycotoxigenic moulds in nuts and sunflower seeds for human consumption

A survey was carried out to obtain data on the occurrence of mycotoxins and the mycotoxin-producing potential of fungi isolated from nuts (almonds, peanuts, hazelnuts, pistachio nuts) and sunflower seeds in Spain. Thin-layer chromatography was used to separate the toxins. Aflatoxins were detected in one sample of almonds (95 ppb aflatoxin B₁ and 15 ppb aflaxtoxin B₂) and in one sample of peanuts at a level below 10 ppb of aflatoxin B1. 100% of samples showed variable incidence of fungal contamination. The predominant fungi present in samples were Penicillium spp, Aspergillus niger, A. flavus, A. glaucus and Rhizopus spp. The results showed that isolates of different species were able to produce aflatoxins B₁, B₂, G₁, and G₂, sterigmatocystin, ochratoxin A, patulin, citrinin, penicillic acid, zearalenone, and griseofulvin.     

Aflatoxins in mutants of Aspergillus flavus

Mutants ofAspergillus flavus were recovered following the irradiation of conidia with ultraviolet light. Analysis of the mutants for aflatoxins B₁, B₂, G₁, and G₂ indicated a wide range of variability in aflatoxin levels. None of the isolates produced the G toxins, and four produced little or no aflatoxin B₂. Production of B₁ and B₂ by the mutants ranged from 1.3 µ;g/ml to 967 µg/ml and zero to 30 µg/ml, respectively. The correlation between production of B₁ and B₂ was statistically significant. There was no apparent correlation between nutritional requirement or conidial color and aflatoxin production.     

Evaluation of the mycological status of luncheon meat with special reference to aflatoxigenic moulds and aflatoxin residues

The luncheon meat samples analyzed, which were produced locally by the two main luncheon meat producing companies in Egypt were relatively highly contaminated either by moulds and yeasts in general, aflatoxigenic species and aflatoxin residues in particular. The most frequently encountered fungi from the samples were yeasts, Aspergillus niger, A. flavus, Penicillium chrysogenum, Rhizopus stolonifer, Mucor circinelloides. Less common were Cladosporium sphaerospermum, Alternaria alternata, Mycosphaerella tassiana, P. aurantiogriseum and P. oxalicum. The most important aflatoxigenic species, A. flavus, was isolated frequently. It was 10% of the total fungal isolates from both samples of the two companies. Seven luncheon meat samples out of 50 analyzed were positive for aflatoxin B₁ or B₁ and G₁, while all samples were negative for aflatoxins B₂, G₂, M₁ and M₂. Aflatoxin B₁ was detected only in 4 and 3 samples out of 25 analyzed from each of company A and B, respectively. The highest detectable level, 11.1 ppb, was recorded in a sample from company B and the least, 0.5 ppb, in a sample from company A. Aflatoxin G₁, at concentration of 3.2 ppb, was detected in only one sample of the aflatoxin B₁ – contaminated 3 samples of company B: this sample also had the highest level of aflatoxin B₁. Some luncheon meat samples had higher numbers of aflatoxigenic A. flavus than others, however these samples were negative for aflatoxins. The hazardous potential of such contamination will be discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of temperature on production of aflatoxin on rice by Aspergillus flavus

Summary The effect of temperature on formation of aflatoxin on solid substrate (rice) byAspergillus flavus NRRL 2999 has been studied in some detail. The optimum temperature for production of both aflatoxin B₁ and G₁ under the conditions employed is 28° C. Comparable yields of B₁ were obtained at 32° C, but considerably less G₁ was produced at this temperature. Both B₁ and G₁ were found in lesser amounts at temperatures above 32° C, and the aflatoxin content of rice incubated at 37° C was low (300–700 ppb) even though growth was good.Reducing the temperature from 28° to 15° C resulted in progressively less aflatoxin, but 100 ppb of B₁ was detected in cultures incubated 3 weeks at 11° C. No aflatoxin was produced at 8° C.The ratio of the four aflatoxins is affected by temperature. At the lower temperatures, essentially equal amounts of aflatoxin B₁ and G₁ were produced, whereas at 28° C, approximately four times as much B₁ was detected as G₁. At the higher temperatures, relatively less G was formed, until at 37° C, less than 10 ppb was detected.This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.     

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> NRRL 3357 and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> SRRC 143

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B₁ and B₂ biosynthesis, while A. parasiticus cultures had significantly increased B₁ and G₁ biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.     

In situ absorption of aflatoxins in rat small intestine

To evaluate the rate at which the four main aflatoxins (aflatoxins B₁, B₂, G₁ and G₂) are able to cross the luminal membrane of the rat small intestine, a study about intestinal absorption kinetics of these mycotoxins has been made. In situ results obtained showed that the absorption of aflatoxins in rat small intestine is a very fast process that follows first-order kinetics, with an absorption rate constant (k _a ) of 5.84±0.05 (aflatoxin B₁), 4.06±0.09 (aflatoxin B₂), 2.09±0.03 (aflatoxin G₁) and 1.58±0.04 (aflatoxin G₂) h^–1, respectively.     

Mycotoxigenic fungi in peanuts from different geographic regions of Egypt

To understand the importance of mycotoxigenic fungi in Egyptian peanuts, samples from five regions (Alexandria, El-Beheira, El-Sharqiya, El-Daqahelaya in northern Egypt and Asyut, southern Egypt) in two seasons (2007, 2008) were collected. Aspergillus was consistently the most frequent genus in seeds and in-shell peanuts and was the dominant mycotoxigenic component of the mycobiota. There was no direct correlation between the moisture content of the samples and the fungal populations on peanut seeds tested from different regions. The most common species were from Aspergillus section Flavi (4.7-78.3%), Aspergillus section Nigri (9.4–52.6%) and Aspergillus section Circumdati (5.1–30.9%). In the in-shell peanut samples, the lowest populations were recorded in El-Beheira and Asyut (3.7–4.0 log₁₀ CFU g^-1) and the highest in Alexandria and Elsharqiya (4.1–6.0 log₁₀ CFU g^-1). Aspergillus section Flavi and section Nigri were the most dominant, and Aspergillus section Circumdati were only found in samples in 2008. Both qualitative (coconut cream agar) and quantitative analyses (HPLC) were used to analyse the potential mycotoxin production by strains isolated from peanuts. Of a total of 88 Aspergillus section Flavi strains examined, 95% were A. flavus based on production of aflatoxin B₁ on yeast extract sucrose (YES) medium and confirmation using molecular analyses. Of 64 Aspergillus section Circumdati strains only 28% produced ochratoxin A (OTA), and were identified as A. westerdijkiae. No Aspergillus section Nigri strains produced OTA, and they were identified as A. niger (uniseriate). The presence of these toxigenic fungi indicates that there is a potential risk of mycotoxin contamination in Egyptian peanuts and suggests that problems can arise from contamination with both aflatoxins and perhaps also OTA.     

Aflatoxin is degraded at different temperatures and pH values by mycella of Aspergillus parasiticus

Summary Blended 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 were tested for their ability to degrade aflatoxins B₁ and G₁ at 7,19,28,36, and 45°C. Rates for degradation of aflatoxin B₁ and G₁ were maximum at 28°C. Intermediate rates of aflatoxin degradation were observed at 19 and 36°C while little aflatoxin was degraded at 7 and 45°C. Five different pH values (2.0, 3.0, 4.0, 5.0, and 6.5) were also tested to determine the effect of pH on ability of blended 9-day-old mycelia of A. parasiticus NRRL 2999 to degrade aflatoxins. The ability of mycelia to degrade aflatoxin was pH-dependent. Of the pH values tested, greatest rates of aflatoxin B₁ and G₁ degradation occurred when pH was in the range of 5 to 6.5. Little aflatoxin was degraded at pH 4.0 and essentially no aflatoxin was degraded by mycelia at pH 2.0 or 3.0 although some aflatoxin was degraded by acid conditions only at pH values of 4 or less.     

Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of Strawberry (Fragaria ananassa Duch.) fruits

Strawberry fungi were isolated from fresh fruits and juice on the two types of media (Sabouraud dextrose agar, SDA and potato-dextrose agar, PDA) at 28 °C. Nineteen fungal species belong to 12 genera were isolated from fruits and juice on both isolation media. The most common fungal genera and species were Aspergillus flavus, A. niger, Mucor racemosus, Neurospora crassa, Penicillium chrysogenum, Rhizopus stolonifer and Trichoderma harzianum. Twenty A. flavus and A. parasitics isolates were assayed for their abilities to produce aflatoxins. The concentration of aflatoxins ranged between 25.8–75.2 and 23.6–71.1 ng/ml at 350 and 365 nm, respectively. Among A. flavus and A. parasiticus strains tested, aflatoxin B contributed 30–60% of total isolates. However, G type contributed 85–90%. The R_f values of B₁, B₂, G₁ and G₂ were 0.79, 0.61, 0.44 and 0.32, respectively. High-performance liquid chromatography analysis of extracts revealed the presence of aflatoxins with variable levels.     

Experimental short time production of aflatoxins by Aspergillus parasiticus in mixed feeds as related to various moisture contents

Experimental short time production of aflatoxins in mixed feeds at 22, 28 and 37 °C as related to various moisture contents was studied. Growth of Aspergillus parasiticus was not observed in the meals with a moisture content ranging around 15% (22, 28 and 37 °C); the lowest quantifiable total aflatoxins at the fourth day was detected at 22 °C with 19.4% of moisture content; the higher total quantity of aflatoxins (113 mg/kg) was produced at 28 °C with 29.3% of moisture content. The ratio aflatoxin B₁/aflatoxin G₁ increased as the temperature raised.     

Incidence of seed-borne fungi and aflatoxins in Sudanese lentil seeds

Thirteen seed samples of lentil (Lens esculenta) were incubated on agar plate and moist filter papers (Moist Chambers) at 28 ± 2 °C for determination of the incidence of seed-borne fungi. Aflatoxins content of the seeds was measured using the bright greenish-yellow fluorescence test (BGYF) and thin-layer chromatography (TLC). Sixty-nine species and seven varieties, which belong to 24 genera of fungi, were isolated from this crop. Of these fungi, 51 species and two varieties are considered new for this crop, whereas seven genera and 13 species are new to the mycoflora of the Sudan. The genus Aspergillus (13 species and 6 varieties) which comprising 44% of the total colony count was the most prevalent genus followed by Rhizopus (2 species, 19%), Penicillium (6 species) and Fusarium (8 species) (12%), Chaetomium (3 species) and Cladosporium (5 species) (6%), where the 18 genera (1–4 species) showed very low level of incidence (19%). Of the possible pathogens of lentil plants, F. oxysporum the main cause of vascular wilt was recovered from seeds of this crop. Thin layer chromatographic analysis of chloroform extracts of 13 seed samples showed that only one samples was naturally contaminated with aflatoxins B₁, B₂, G₁ and G₂ (14.3 μg/kg). This revised version was published online in June 2006 with corrections to the Cover Date.     

The production of aflatoxin B1 or G1 by Aspergillus parasiticus at various combinations of temperature and water activity is related to the ratio of aflS to aflR expression

The influence of varying combinations of water activity (a_w) and temperature on growth, aflatoxin biosynthesis and aflR/aflS expression of Aspergillus parasiticus was analysed in the ranges 17–42°C and 0.90–0.99 a_w. Optimum growth was at 35°C. At each temperature studied, growth increased from 0.90 to 0.99 a_w. Temperatures of 17 and 42°C only supported marginal growth. The external conditions had a differential effect on aflatoxin B₁ or G₁ biosynthesis. The temperature optima of aflatoxin B₁ and G₁ were not at the temperature which supported optimal growth (35°C) but either below (aflatoxin G₁, 20–30°C) or above (aflatoxin B₁, 37°C). Interestingly, the expression of the two regulatory genes aflR and aflS showed an expression profile which corresponded to the biosynthesis profile of either B₁ (aflR) or G₁ (aflS). The ratios of the expression data between aflS:aflR were calculated. High ratios at a range between 17 and 30°C corresponded with the production profile of aflatoxin G₁ biosynthesis. A low ratio was observed at >30°C, which was related to aflatoxin B₁ biosynthesis. The results revealed that the temperature was the key parameter for aflatoxin B₁, whereas it was water activity for G₁ biosynthesis. These differences in regulation may be attributed to variable conditions of the ecological niche in which these species occur.     

Production of aflatoxin byAspergillus parasiticus and its control

The aim of the present work was to investigate the production of aflatoxin byAspergillus parasiticus and to find out the possible ways to control it. Of 40 food samples collected from Abha region, Saudi Arabia, only 25% were contaminated with aflatoxins. Oil-rich commodities had the highly contaminated commodities by fungi and aflatoxins while spices were free from aflatoxins.Bacillus megatertum andB cereus were suitable for microbiological assay of aflatoxins. Czapek’s-Dox medium was found a suitable medium for isolation of fungi from food samples. The optimal pH for the growth ofA. parasiticus and its productivity of aflatoxin B₁ was found at 6.0, while the best incubation conditions were found at 30°C for 10 days. D-glucose was the best carbon source for fungal growth, as well as aflatoxin production. Corn steep liquor, yeast extract and peptone were the best nitrogen sources for both fungal growth and toxin production (NH₄)₂HPO₄ (1.55 gL^-1) and NaNO₂ (1.6 gL^-1) reduced fungal growth and toxin production with 37.7% and 85%, respectively. Of ten amino acids tested, asparagine was the best for aflatoxin B₁ production. Zn²⁺ and Co²⁺ supported significantly both fungal growth, as well as, aflatoxin B₁ production at the different tested concentrations. Zn²⁺ was effective when added toA. parasiticus growth medium at the first two days of the culture age. The other tested metal ions expressed variable effects depending on the type of ion and its concentration. Water activity (a_w) was an important factor controlling the growth ofA. parasiticus and toxin production. The minimum a_w for the fungal growth was 0.8 on both coffee beans and rice grains, while a_w of 0.70 caused complete inhibition for the growth and aflatoxin B₁ production. H₂O₂ is a potent inhibitor for growth ofA. parasiticus and its productivity of toxins. NaHCO₃ and C₆H₅COONa converted aflatoxin B₁ to water-soluble form which returned to aflatoxin B₁ by acidity. Black pepper, ciliated heath, cuminum and curcuma were the most inhibitory spices on toxin production. Glutathione, quinine, EDTA, sodium azide, indole acetic acid, 2,4-dichlorophenoxy acetic acid, phenol and catechol were inhibitory for both growth, as well as, aflatoxin B₁ production. Stearic acid supported the fungal growth and decreased the productivity of AFB₁ gradually. Lauric acid is the most suppressive fatty acid for both fungal growth and aflatoxin production, but oleic acid was the most potent supporter. Vitamin A supported the growth but inhibited aflatoxin B₁ production. Vitamins C and D₂ were also repressive particularly for aflatoxin production The present study included studying the activities of some enzymes in relation to aflatoxin production during 20-days ofA. parasiticus age in 2-days intervals. Glycolytic enzymes and pyruvate-generating enzymes seems to be linked with aflatoxin B₁ production. Also, pentose-phosphate pathway enzymes may provide NADPH for aflatoxin B₁ synthesis. The decreased activities of TCA cycle enzymes particularly from 4th day of growth up to 10th day were associated with the increase of aflatoxin B₁ production. All the tested enzymes as well as aflatoxin B₁ production were inhibited by either catechol or phenol.     

Production and characterization of polyclonal and monoclonal antibodies against Aflatoxin B1 oxime-BSA in an enzyme-linked immunosorbent assay

From a single aflatoxin B₁ oxime — bovine serum albumin conjugate, polyclonal and monoclonal antibody preparations were produced. The four rabbit polyclonal antisera were specific for aflatoxin Bi in a microtitration plate enzyme — linked immunosorbent assay. The monoclonal antibodies showed a wide range of differing specificities, recognizing, for example, aflatoxins B₁, B₂, G₁ and G₂; B₁ and B₂; B₁ and G₁; and G₁ alone. No antibody preparations reacted with aflatoxin M₁. The significance of these results to the strategy of anti-aflatoxin antibody production for use in quantitative enzyme immunoassays is discussed.     

Mycoflora and mycotoxins of peanut (Arachis hypogaea L.) seeds in Egypt. III. Cellulose-decomposing and mycotoxin-producing fungi

From 40 peanut seed samples collected in Egypt, forty-three species and one variety of fungi, belonging to 16 genera, were collected. The most dominant genera were Aspergillus (11 species + one variety), Penicillium (11 species) and Fusarium (4 species). From the preceding genera A. fumigatus, A. flavus, A. niger, P. chrysogenum and F. oxysporum were the most frequent species.Forty-nine isolates belonging to 12 species and one variety were tested for production of mycotoxins, after growth on liquid medium containing two carbon sources (sucrose or cellulose). Thin layer chromatographic analysis revealed that the quality and quantity of mycotoxins was higher on sucrose than cellulose. Mycotoxins identified were aflatoxins B₁, B₂, G₁ & G₂, citrinin; fumagillin; diacetoxyscirpenol T-2 toxin; satratoxin H; and zearalenone.     

Dillapiol and Apiol as Specific Inhibitors of the Biosynthesis of Aflatoxin G1 in Aspergillus parasiticus

Dillapiol was isolated from the essential oil of dill as a specific inhibitor of aflatoxin G₁ production. It inhibited aflatoxin G₁ production by Aspergillus parasiticus with an IC₅₀ value of 0.15 μM without inhibiting aflatoxin B₁ production or fungal growth. Apiol and myristicin, congeners of dillapiol, showed similar activity with IC₅₀ values of 0.24 and 3.5 μM, respectively.     

Mutagenic activities of aflatoxin B 1 and G 1 in Neurospora crassa

Summary The mutagenicity and mutagenic specificity of aflatoxin B₁ and G₁ were studied with the adenine-3 (ad-3) test system of Neurospora crassa. Aflatoxin B₁ and G₁ failed to show mutagenicity in resting conidia, but both agents were mutagenic in growing vegetative cultures. The frequencies of ad-3 mutants induced by aflatoxin B₁ and G₁ (40g/ml) were 70.7x10^-6 survivors and 9.6x10^-6 survivors, respectively. Since aflatoxin B₁ gave a 177-fold increase over the spontaneous mutation frequency it is a rather potent mutagen, whereas aflatoxin G₁ gave only a 24-fold increase and so is only moderately mutagenic.Genetic analyses of ad-3 mutants induced by aflatoxin B₁ and G₁ indicate that both agents induce a low frequency of multilocus deletions. The spectra of point mutations at the ad-3A and ad-3B loci induced by aflatoxin B₁ and G₁ are not distinguishable from each other. Hence both agents probably induce the same relative frequencies of genetic alterations. The frequencies of leakiness, allelic complementation, and classes of complementation patterns among the ad-3 mutants induced by both agents are higher than the frequencies among ICR-170-induced mutants and somewhat lower than those among NA- and AP-induced mutants. The results of reversion tests with NA, MNNG, and ICR-170 indicate that in addition to multilocus deletion, aflatoxin B₁-induced ad-3 mutants consist of frameshifts, base-pair transitions, and possibly other types of intragenic alterations.     

Transformation of sterigmatocystin and O-methylsterigmatocystin by aflatoxigenic and nonaflatoxigenic field isolates of the Aspergillus flavus group

Transformation of sterigmatocystin and O-methylsterigmatocystin (two metabolic aflatoxin precursors) to aflatoxins by aflatoxigenic and nonaflatoxigenic field isolates of Aspergillus flavus was studied. The 24 nonaflatoxigenic isolates investigated failed to transform both precursors. Among the 8 aflatoxin-producing isolates used, 7 transformed both precursors whereas the remaining failed to transform both. According to these results, the usefulness of the measurement of enzymatic activities related to aflatoxin production in understanding the true status of conflictive field isolates is discussed.Abbreviations ST sterigmatocystin - OMST O-methylsterigmatocystin - AFB₁ aflatoxin B₁ - AFB₂ aflatoxin B₂ - AFG₁ aflatoxin G₁ - AFG₂ aflatoxin G₂ - GM growth medium of Adye and Mateles - RM replacement medium of Adye and Mateles