Effects of various acids and salts on growth and aflatoxin production by Aspergillus flavus NRRL 3145.

The effects of sodium chloride, sodium acetate, benzoic acid, sodium benzoate, malonic acid, and sodium malonate on growth and aflatoxin production by Aspergillus flavus were investigated in synthetic media. Sodium chloride at concentrations equivalent to or greater than 12 g/100 ml inhibited growth and aflatoxin production, while at 8 g or less/100 ml, growth and aflatoxin production were stimulated. At 2 g or less/100 ml, sodium acetate also stimulated growth and aflatoxin production, but reduction occurred with 4 g or more/100 ml. Malonic acid at 10, 20, 40, and 50 mM reduced growth and aflatoxin production (over 50%) while sodium malonate at similar concentrations but different pH values had the opposite effect. Benzoic acid (pH 3.9) and sodium benzoate (pH 5.0) at 0.4 g/100 ml completely inhibited growth and aflatoxin production. Examination of the effect of initial pH indicated that the extent of inhibitory action of malonic acid and sodium acetate was a function of initial pH. The inhibitory action of benzoic acid and sodium benzoate appeared to be a function of undissociated benzoic acid molecules. Aflatoxin reduction was usually accompanied by an unidentified orange pigment, while aflatoxin stimulation was accompanied by unidentified blue and green fluorescent spots but with lower Rf values that aflatoxins B1, G1, B2, and G2 standards.     

Inhibition of Aspergillus growth and aflatoxin release by derivatives of benzoic acid.

A study was conducted to determine the effects of o-nitrobenzoate, p-aminobenzoate, benzocaine (ethyl aminobenzoate), ethyl benzoate, methyl benzoate, salicylic acid (o-hydroxybenzoate), trans-cinnamic acid (beta-phenylacrylic acid), trans-cinnamaldehyde (3-phenylpropenal), ferulic acid (p-hydroxy-3-methoxycinnamic acid), aspirin (o-acetoxy benzoic acid), and anthranilic acid (o-aminobenzoic acid) upon growth and aflatoxin release in Aspergillus flavus NRRL 3145 and A. parasiticus NRRL 3240. A chemically defined medium was supplemented with various concentrations of these compounds and inoculated with spores, and the developing cultures were incubated for 4, 6, and 8 days at 27 degree C in a mechanical shaker. At the beginning of day 8 of incubation, aflatoxins were extracted from cell-free filtrates, separated by thin-layer chromatography, and quantitated by ultraviolet spectrophotometry. The structure of these aromatic compounds appeared to be critically related to their effects on mycelial growth and aflatoxin release. At concentrations of 2.5 and 5.0 mg per 25 ml of medium, methyl benzoate and ethyl benzoate were the most effective in reducing both mycelial growth and aflatoxin release by A. flavus and A. parasiticus. Inhibition of mycelial growth and aflatoxin release by various concentrations of the above-named aromatic compounds may indicate the possibility of their use as fungicides.     

Effect of selected inhibitors on growth,pigmentation, and aflatoxin production by Aspergillus parasiticus

We have treated a wild type strain of Aspergillus parasiticus with several known aflatoxin inhibitors in hopes of finding specific metabolic blocks in the aflatoxin biosynthetic pathway. In defined medium, benzole acid (2 and 3 mg/ml), cinnamon (1 mg/ml), and sodium acetate (5 mg/ml) were fungitoxic. Benzoic acid (0.5 and 1 mg/ml), chlorox (5 l/ml), and dimethyl sulfoxide (5 l/ml) did not affect dry weight or mycelial pigmentation. Sodium benzoate (1, 2, 4 and 8 mg/ml) added after 2 days growth inhibited aflatoxin production in two defined media. We were unable to confirm previously published reports that an uncharacterized yellow pigment accumulates with benzoate-inhibition of aflatoxin biosynthesis.     

Inhibition of Aflatoxin Production and Tentative Identification of an Aflatoxin Intermediate ?Versiconal Acetate” from Treatment with Dichlorvos

In general, aflatoxin production by Aspergillus flavus and A. parasiticus was greatly reduced in vitro in the presence of the insecticide dichlorvos. Reduction in yield of the toxins was accompanied by the apperance of a previously unidentified orange pigment. Spectral analyses of the pigment and of its methylated and acetylated derivatives indicated the compound to be versiconal acetate (IV). The data suggest that IV is an intermediate in the metabolic cycle that may terminate in the production of aflatoxin or of the versicolorins, or both. Dichlorvos apparently inhibits biosynthesis of the difurano ring structure common to the aflatoxins and the versicolorins.     

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.     

Inhibitory effect of molybdenum and vanadium salts on aflatoxin B1 synthesis by Aspergillus flavus

The effects of the elements zinc, manganese, iron, copper, molybdenum, and vanadium, added in various salt forms, on mycelial weights and aflatoxin B1 accumulation in the mycelium of Aspergillus flavus were investigated in liquid shake cultures. Ammonium heptamolybdate, when added to a complete medium at concentrations of 50-100 mg/L, appreciably reduced aflatoxin B1 accumulation without affecting growth of the fungus. Sodium molybdate and sodium monovanadate also reduced aflatoxin B1 yields without affecting mycelial growth but to a lesser extent. The addition of zinc sulphate stimulated aflatoxin B1 production in all media used. The influence of the other trace elements on aflatoxin production depended on the level of trace elements present in the basal medium. In general, manganese chloride had a stimulatory effect, whereas copper sulphate depressed yields. Mycelial levels of aflatoxin had peaked and then declined before mycelial dry weights had reached maximum. High yields of aflatoxin B1 were obtained in media having a final pH as low as pH 2.8.     

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.     

Enzymatic conversion of sterigmatocystin into aflatoxin B1 by cell-free extracts of Aspergillus parasiticus.

A cell-free extract, prepared from Aspergillus parasiticus ATCC 15517 grown in synthetic medium, was active in converting [14C]sterigmatocystin into aflatoxin B1 in the presence of reduced nicotinamide adenine dinucleotide phosphate. The activity was demonstrated by the time course of conversion and the linear dependence of the yield of product on enzyme concentrations. Optimum activity was obtained at pH 7.5 to 7.8 at 27 C. The results confirm sterigmatocystin as a biogenetic precursor of aflatoxin B1. Techniques were developed for enzymatic studies on aflatoxin biosynthesis.     

Enzymatic conversion of sterigmatocystin into aflatoxin B1 by cell-free extracts of Aspergillus parasiticus.

A cell-free extract, prepared from Aspergillus parasiticus ATCC 15517 grown in synthetic medium, was active in converting [14C]sterigmatocystin into aflatoxin B1 in the presence of reduced nicotinamide adenine dinucleotide phosphate. The activity was demonstrated by the time course of conversion and the linear dependence of the yield of product on enzyme concentrations. Optimum activity was obtained at pH 7.5 to 7.8 at 27 C. The results confirm sterigmatocystin as a biogenetic precursor of aflatoxin B1. Techniques were developed for enzymatic studies on aflatoxin biosynthesis.     

Effect of nutritional factors on aflatoxin B1 inhibition of growth inEscherichia coli andSalmonella typhi

The extent of growth inhibition by aflatoxin B1 in S. typhi and E. coli was greater in the presence of sodium citrate or sodium phosphate, palmitic and stearic acid than aflatoxin B1 alone. The addition of amino acids (glycine or glutamic acid) stimulated growth in E. coli and inhibited in S. typhi in the presence of aflatoxin B1. Other nutrients, such as yeast extract, lactose, or salt addition did not alter aflatoxin B1 antibacterial activity but decreased growth was observed in the presence of peptone.     

Yellow pigments used in rapid identification of aflatoxin-producing Aspergillus strains are anthraquinones associated with the aflatoxin biosynthetic pathway

Studies on biological control of aflatoxin production in crops by pre-infection with non-toxigenic Aspergillus flavus strains have created a need for improved methods to screen isolates for aflatoxigenicity. We have evaluated two empirical aflatoxigenicity tests: (i) yellow pigment production, and (ii) the appearance of a plum-red color in colonies exposed to ammonium hydroxide vapor. Yellow pigments from aflatoxigenic A. flavus were shown to function as pH indicator dyes. Seven pigments representing most of the pigmentation in extracts have been isolated using color changes when chromatography spots were exposed to ammonium hydroxide vapor to guide fractionation. Their structures have been shown to be norsolorinic acid, averantin, averufin, versicolorin C, versicolorin A, versicolorin A hemiacetal and nidurufin, all of which are known anthraquinone pigments on, or associated with, the aflatoxin biosynthetic pathway in Aspergillus spp. Thus, the basis of both empirical tests for aflatoxigenicity is detecting production of excess aflatoxin biosynthetic intermediates.     

Antimicrobial activity and inhibition of aflatoxin B1 formation by olive plant tissue constituents

Ethanolic extracts of olive callus tissues, added at 0.5 or 1.0% to media on which Aspergillus flavus was grown, inhibited aflatoxin production by 90% without inhibiting the fungal growth. The extract was found to contain mainly caffeic acid and, to a lesser extent, catechin and coumarins. The fungicidal and bactericidal activity of caffeic acid, catechin, coumarin and p-, o- or m-coumaric acid were tested and only caffeic acid and o-coumaric acid inhibited aflatoxin production. The inhibitory effect had no correlation with the growth of the fungus. Only coumarin at 10 mmol/1 totally inhibited fungal growth. Of the phenolic constituents of callus tissues tested, catechin and caffeic acid (10 mmol/1) showed bactericidal activity towards Pseudomonas aeruginosa and Staphylococcus aureus.     

Combined effects of solutes and food preservatives on rates of inactivation of and colony formation by heated spores and vegetative cells of molds.

The combined and independent effects of sucrose, sodium chloride, potassium sorbate, and sodium benzoate on heat inactivation of conidia of Aspergillus flavus and Penicillium puberulum, ascospores of Byssochlamys nivea, and vegetative cells of Geotrichum candidum were studied. In addition, the effects of solutes and preservatives on colony formation by unheated and heated conidia of A. flavus were evaluated. Increased concentrations of sucrose were accompanied by increased tolerance to heat by A. flavus, B. nivea, and G. candidum. Low concentrations (3 and 6%) of sodium chloride protected A. flavus and G. candidum, whereas up to 12% sodium chloride protected B. nivea, but had little effect on the heat stability of P. puberulum. Potassium sorbate and sodium benzoate acted synergistically with heat to inactivate all four molds. At the same concentration, the two preservatives had varied degrees of effectiveness on molds and were influenced by the type of solute in the heating menstrua. Heated conidia of A. flavus had increased sensitivity to preservatives and reduced water activity, whether achieved by the presence of sucrose or sodium chloride, thus demonstrating heat-induced injury. At the same concentration, potassium sorbate was clearly more inhibitory than was sodium benzoate to colony formation by A. flavus, and the presence of sucrose and sodium chloride enhanced this inhibition.     

山苍子油对霉菌抗菌性及其与黄曲霉产毒关系的研究

采用平板法比较天然增香剂山苍子油与合成食用防腐剂苯甲酸钠、山梨酸钾对8种霉菌的抗菌效力。结果表明,在培养基pH4．5时山苍子油对多数霉菌的最低抑菌浓度为1．77mg／ml,与山梨酸钾的抑菌强度相近,比苯甲酸钠强;但当培养基pH5．5以上时苯甲酸钠对霉菌几乎无效,山梨酸钾的抗菌效力也有减弱,而山苍子油受影响很小,其活性pH范围为4．5～8．5。山苍子油用脂肪酸蔗糖酯乳化,其抗菌效力增强一倍。同时,从山苍子油与黄曲霉产毒关系的实验中发现,山苍子油对黄曲霉产生黄曲霉毒素有较强的抑制作用。     

Antimicrobial activity and inhibition of aflatoxin B1 formation by olive plant tissue constituents

Ethanolic extracts of olive callus tissues, added at 0.5 or 1.0% to media on which Aspergillus flavus was grown, inhibited aflatoxin production by 90% without inhibiting the fungal growth. The extract was found to contain mainly caffeic acid and, to a lesser extent, catechin and coumarins. The fungicidal and bactericidal activity of caffeic acid, catechin, coumarin and p-, o- or m-coumaric acid were tested and only caffeic acid and o-coumaric acid inhibited aflatoxin production. The inhibitory effect had no correlation with the growth of the fungus. Only coumarin at 10 mmol/1 totally inhibited fungal growth. Of the phenolic constituents of callus tissues tested, catechin and caffeic acid (10 mmol/l) showed bactericidal activity towards Pseudomonas aeruginosa and Staphylococcus aureus.     

Inhibition of aflatoxin B production of Aspergillus flavus, isolated from soybean seeds by certain natural plant products

AIMS: The inhibitory effect of cowdung fumes, Captan, leaf powder of Withania somnifera, Hyptis suaveolens, Eucalyptus citriodora, peel powder of Citrus sinensis, Citrus medica and Punica granatum, neem cake and pongamia cake and spore suspension of Trichoderma harzianum and Aspergillus niger on aflatoxin B(1) production by toxigenic strain of Aspergillus flavus isolated from soybean seeds was investigated. METHODS AND RESULTS: Soybean seed was treated with different natural products and fungicide captan and was inoculated with toxigenic strain of A. flavus and incubated for different periods. The results showed that all the treatments were effective in controlling aflatoxin B(1) production. Captan, neem cake, spore suspension of T. harzianum, A. niger and combination of both reduced the level of aflatoxin B(1) to a great extent. Leaf powder of W. somnifera, H. suaveolens, peel powder of C. sinensis, C. medica and pongamia cake also controlled the aflatoxin B(1) production. CONCLUSIONS: All the natural product treatments applied were significantly effective in inhibiting aflatoxin B(1) production on soybean seeds by A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: These natural plant products may successfully replace chemical fungicides and provide an alternative method to protect soybean and other agricultural commodities from aflatoxin B(1) production by A. flavus.     

Inhibition of growth and aflatoxin biosynthesis of Aspergillus flavus by extracts of some Egyptian plants

The effect of five different concentrations (2, 4, 6, 8 and 10 mg ml-1) of an aqueous extracts of Lupinus albus, Ammi visnaga and Xanthium pungens were tested on growth and aflatoxin production by Aspergillus flavus in a chemically defined medium. All the plants inhibited mycelial growth and aflatoxin formation. The inhibitory effect was proportional with the applied concentration. Growth and aflatoxin production appeared to be correlated processes. The nature of the plant extract also affected the ratio of B1 to B2, and there was no correlation between the inhibition of aflatoxins or growth of the fungus and the resultant B1: B2 ratio.     

Influence of gamma irradiation of aflatoxin B1 production by Aspergillus flavus growing on some Nigerian foodstuffs

Spores of Aspergillus flavus (UI, 81) inoculated into some local food materials were irradiated at 62.5, 125.0, 250.0 and 500.0 krad, and the effect on aflatoxin B1 production on subsequent incubation was measured. The results show that aflatoxin B1 production decreased with increasing gamma irradiation dose in soya bean, groundnut, palm juice, while paw paw mash showed a relatively high yield of aflatoxin at 125.0 krad as compared to other irradiation levels tested except for the control. Irradiation of soya bean and groundnut inoculated with spores of Aspergillus flavus at 500.0 krad (pre-irradiation incubation period of 2 h) inhibited aflatoxin B1 production. Analysis of variance showed that media, pre-irradiation incubation periods and irradiation levels affected the total amounts of aflatoxin produced.     

Production of aflatoxin by an Aspergillus flavus isolate cultured under a limited oxygen supply

In a previous experiment on the preservation of hay of high moisture content with formic acid, among other agents, aflatoxin was formed in the hay, and aflatoxin-forming strains of Aspergillus flavus were isolated from this hay after incubation in air as well as in an anaerobic jar. One isolate from the anaerobic jar was cultivated in a chemostat (Bioflo model C 30; New Brunswick Scientific Co.) in a defined medium with added B vitamins, yeast extract, or formic acid, with or without gas flow (air or nitrogen). In all cases where spore germination occurred, aflatoxin was formed in the cultures with gas flow, and small quantities of aflatoxins B1 and B2 occurred even in an atmosphere of nitrogen. Addition of B vitamins and supply of traces of air gave an approximately 15-fold increase in the amount of aflatoxin in 2 days. Carbon dioxide enrichment hindered aflatoxin formation on the defined medium even in the presence of B vitamins, but when formic acid was added, small quantities (5 to 15 micrograms/liter) were formed, and this low level remained constant until the gas flow was started.     

Production of aflatoxin by an Aspergillus flavus isolate cultured under a limited oxygen supply.

In a previous experiment on the preservation of hay of high moisture content with formic acid, among other agents, aflatoxin was formed in the hay, and aflatoxin-forming strains of Aspergillus flavus were isolated from this hay after incubation in air as well as in an anaerobic jar. One isolate from the anaerobic jar was cultivated in a chemostat (Bioflo model C 30; New Brunswick Scientific Co.) in a defined medium with added B vitamins, yeast extract, or formic acid, with or without gas flow (air or nitrogen). In all cases where spore germination occurred, aflatoxin was formed in the cultures with gas flow, and small quantities of aflatoxins B1 and B2 occurred even in an atmosphere of nitrogen. Addition of B vitamins and supply of traces of air gave an approximately 15-fold increase in the amount of aflatoxin in 2 days. Carbon dioxide enrichment hindered aflatoxin formation on the defined medium even in the presence of B vitamins, but when formic acid was added, small quantities (5 to 15 micrograms/liter) were formed, and this low level remained constant until the gas flow was started.