Increased Aflatoxin Production by Aspergillus parasiticus Under Conditions of Cycling Temperatures

The effects of cycling temperatures (5°C for 12 hr and 25°C for 12 hr) on aflatoxin production by Aspergillus parasiticus NRRL 2999 in yeast extract sucrose (YES) medium were studied. Cycling temperatures, after preincubation at 25°C for various times, resulted in more aflatoxin B₁, G₁, and total aflatoxin production than did constant incubation at either 25°C, which is generally considered to be the optimum for aflatoxin production, or 15°C, which is the same total thermal input as the 5-25°C temperature cycling. With increased preincubation time at 25°C, toxin production increased and the lag phase of growth was shortened or not evident. Cultures that were preincubated at 25°C for 1, 2, and 3 days prior to onset of temperature cycling showed the greatest increase in maximum aflatoxin production over the 25°C and 15°C constant temperatures. Cultures that were not preincubated at 25°C but subjected to constantly fluctuating temperatures produced maximum amounts of aflatoxin equivalent to cultures incubated at a constant 25°C. The maximum aflatoxin production at all temperatures studied occurred during the late log phase of growth and at pH minimums. Aflatoxins were found in higher concentrations in the broth than the mycelia under temperature cycling conditions, at 15°C, and at 25°C during the first 21 days of incubation, whereas greater amounts of toxin were retained in mycelium at 25°C in the later incubation period (28-42 days).     

Aflatoxin production in three selected samples of triticale,wheat and rye grown in Argentina

Little is known about mycotoxin contamination of triticale, a hybrid resulting from crossing wheat and rye. The purpose of the present work was to evaluate triticale as a substrate for aflatoxin accumulation in comparison with its parents. Aflatoxin (B₁, B₂, G₁ and G₂) accumulation curves were obtained for the three substrates inoculated with Aspergillus parasiticus NRRL 2999 and incubated at 25 °C and water activity 0.925 for 10 weeks. Wheat and triticale were poor substrates for aflatoxin production. Rye was more prone than the other substrates to fast colonisation by A parasiticus and accumulated larger aflatoxin quantities over the whole incubation period. The maximum aflatoxin concentration in rye (11 840 µg kg⁻¹) was significantly larger (p < 0.05) than those obtained in wheat (2150 µg kg⁻¹) and triticale (2850 µg kg⁻¹). © 2000 Society of Chemical Industry     

Effect of gamma irradiation on aflatoxin production in barley

Barley was inoculated with conidia of Aspergillus parasiticus NRRL 2999, and at 0°C was left to equilibrate at three moisture levels: 17, 20 and 25%. Gamma irradiation at five doses, 0, 50, 100, 200 and 400 krad, was applied to the grain either soon after moisture equilibration (3 days after inoculation) or 10 days later (13 days after inoculation). Increasing the radiation dose resulted in decreasing aflatoxin formation, with one exception: 200 krad applied 13 days after inoculation on barley stored at a moisture level of 25% (100% relative humidity) and 25°C led to higher aflatoxin production than at 100 or 50 krad. For the control of aflatoxin contamination in barley, storage at low moisture content appears to be more practical and effective than irradiation.     

Induction of chitinase in response to Aspergillus infection in sorghum (Sorghum bicolor (L) Moench)

Chitinase is an antifungal protein which is induced in higher plants during infection and stress. In this paper the induction of chitinase activity in response to infection by Aspergillus parasiticus (NRRL 2999) in six sorghum (Sorghum bicolor (L) Moench) genotypes and its association with aflatoxin production were investigated. Chitinase was induced in all six genotypes (two each of red, yellow and white sorghum) when infected by A parasiticus (NRRL 2999). The induction of chitinase activity was highest in the white cultivars, followed by the yellow and red genotypes, compared with healthy grains. In the white cultivars the chitinase activity increased on the 6th and 9th days after infection and was four‐ to fivefold higher than in healthy grains. The total aflatoxin produced was lower in the red genotypes than in the yellow and white genotypes. The white genotypes showed maximum total aflatoxin production at 6 days after infection. The aflatoxins produced in the white genotypes were comparable to those in the red genotypes. There was a significant positive correlation between chitinase activity and aflatoxin production in red sorghum (r² = 0.600, p ≤ 0.001) and white sorghum (r² = 0.411, p ≤ 0.001). Maximum chitinase activity was observed on day 12 in all genotypes under healthy conditions. Copyright © 2004 Society of Chemical Industry     

Effect of Cycling Temperatures on Aflatoxin Production by Aspergillus parasiticus and Aspergillus flavus in Rice and Cheddar Cheese

Initiation of growth, sporulation and aflatoxin production at cycling temperatures took less time than at 15°C but more than at 18°C and 25°C. A. parasiticus produced more aflatoxins on rice under cycling temperatures than at 25°C, 18°C or 15°C, while A. flavus produced less aflatoxin under cycling temperatures. A. parasiticus produced more aflatoxins on cheese under cycling temperatures than at 18°C or 15°C, but much less than at 25°C. A. flavus produced less aflatoxins on cheese under cycling temperatures than at 18°C and 25°C. Both organisms produced trace amounts of toxins at 15°C on cheese. Preincubation at 25°C for 2 days before temperature cycling did not increase aflatoxin production on rice but increased production on cheese. The rate of aflatoxin production on cheese decreased as the temperature decreased. No growth, sporulation or aflatoxin production was observed at 5°C on either rice or cheese.     

Effect of Caffeine on Aflatoxin Production on Cocoa Beans

The effects of caffeine on aflatoxin production by Aspergillus parasiticus NRRL 2999 were studied on 13 cocoa bean types. Caffeine levels from bean types ranged from 0.30-3.6 mg/g and only very low levels of aflatoxin were produced on bean types having > 1.80 mg caffeine/g. These data provide additional evidence that caffeine is an effective inhibitor of aflatoxin production and help explain why aflatoxin does not accumulate in cocoa beans under natural storage conditions.     

Isolation of a Caffeine-Resistant Mutant of Aspergillus parasiticus

A caffeine-resistant mutant of Aspergillus parasiticus NRRL 2999 was isolated and subsequently designated strain BCR1. The mutant strain grew in the presence of > 8 mg/mL caffeine, while growth of the parent strain was delayed by 1 mg/mL and inhibited by 2 mg/mL. Strain BCR1 produced abundant amounts of aflatoxin only when cultured in media containing caffeine. Residual caffeine analyses indicated that caffeine-resistance in BCR1 was not due to the metabolic elimination of caffeine.     

Inhibitory effect of phenolics extracted from sorghum genotypes on Aspergillus parasiticus (NRRL 2999) growth and aflatoxin production

The inhibitory activity of bioactive polyphenols present in six sorghum genotypes—two red (AON 486 and IS 620), two yellow (LPJ and IS 17779) and two white (SPV 86 and SPV 462) varieties—on Aspergillus parasiticus (NRRL 2999) growth and aflatoxin production was evaluated. In the first experiment the production of aflatoxins in the six sorghum genotypes after removal of surface phenolics by acidic methanol treatment was studied and compared with that in untreated grains. Aflatoxin production was found to be fourfold higher in treated grains. The total phenols and bioactive polyphenols extracted by acidic methanol were quantified using the Folin–Denis method and the bovine serum albumin–benzidine conjugate procedure respectively. In the second experiment the effect of extracted sorghum phenolics under in vitro conditions on fungal growth and aflatoxin production was studied at two concentrations (0.01% and 0.1%) of phenolics. Extracted phenolics added to yeast extract sucrose (YES) medium at 0.1% concentration showed an inhibitory effect on aflatoxin production. At 0.01% phenolic concentration, aflatoxin production was minimal on day 3 after infection. At other time points the aflatoxin content was similar to that in the control. At 9 days after infection the fungal biomass in IS 620 was significantly lower than that in the control. At 0.1% phenolic concentration, aflatoxin production was minimal and the red genotype IS 620 showed maximum resistance. Fungal biomass was lowest at all growth stages in IS 620 as compared with the control. Polyphenol oxidase (PPO) activity was not detected in A. parasiticus grown on YES medium (control). PPO activity was not induced in A. parasiticus by the addition of phenolics to the liquid culture medium (no PPO activity was detected in the culture medium). The inhibitory activity of bioactive polyphenols could be attributed to the lack of PPO enzyme in this fungus. Copyright © 2007 Society of Chemical Industry     

A New Method for Aflatoxin-Free Storage of Agricultural Commodities

Aflatoxin production was observed in the 2 kg lots of peanuts and corn that were stored for 90 days at ambient temperature (28–30°C) and a relative humidity of 100% after infecting with the spores of aflatoxin producing strain Aspergillus parasiticus NRRL 3145. Treatment of the samples with an aqueous solution of 2-chloroethylphosphonic acid prevented aflatoxin formation in both the commodities, whereas, the untreated lots supported aflatoxin formation.     

Evaluating,the inhibitory action of honey on fungal growth, sporulation, and aflatoxin production

Summary Unprocessed honey was inoculated with toxigenic strains ofAspergillus flavus NRRL 5862 andA. parasiticus NRRL 2999. The fungi grew and sporulated in varying amounts of honey diluted with water, but none of the cultures produced detectable levels of aflatoxin. Growth and subsequent sporulation were seen only in media containing up to and including 60% of honey. Media having 40% of honey showed growth and sporulation by day two. Neither species ofAspergillus produced toxins even in 10% honey. These results confirm our earlier observations that pure honey inhibited fungal growth and now even diluted honey seems capable of inhibiting toxin production or possibly neutralizing it. The general procedures recommended by the AOAC for extraction and thin layer chromatography were applied successfully in analyzing the honey substrate for aflatoxin.

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Abschätzung der Hemmwirkung von Honig auf Pilzwachstum, Sporulierung und Aflatoxin-Produktion

Zusammenfassung Unbehandelter Honig wurde mit toxigenen Stämmen vonAspergillus flavus NRRL 5862 undAspergillus parasiticus NRRL 2999 beimpft. Der Pilz wuchs und sporulierte in mit verschiedenen Wasser-Mengen verdünntem Honig, aber keine der Kulturen produzierte nachweisbare Mengen von Aflatoxin. Wachstum und nachfolgende Sporulation konnte nur in Medien mit nicht mehr als 60% Honig entdeckt werden. Medien mit 40% Honig zeigten innerhalb von 2 Tagen Wachstum und Sporulation. Selbst in 10% wäßriger Honiglösung produzierte keine derAspergillus-Arten Toxine. Diese Ergebnisse erhärten unsere früheren Beobachtungen, daß reiner Honig das Pilzwachstum hemmt und daß sogar verdünnte Honig-Lösungen fähig sind, die Toxin-Produktion zu hemmen. Die von AOAC empfohlenen Arbeitsanweisungen für Extraktion und Dünnschichtchromatographie waren auch bei der Honig-Analyse auf Aflatoxin erfolgreich.

     

Aflatoxins in sunflower seeds. Influence of fungicides and insecticides

The influence of ten commercial fungicides and insecticides on growth and formation of aflatoxin B₁ by Aspergillus parasiticus NRLL 2999 and A. parasiticus RC 12 was investigated in laboratory media and for sunflower seeds. Four of the five fungicides investigated in concentrations corresponding to commercial practice inhibited growth and toxin production in the laboratory media. The fifth compound appeared to stimulate growth but formation of aflatoxin B₁ could not be detected. Among the five insecticides investigated, one inhibited toxin formation but not growth and one inhibited growth as well as toxin formation. The results obtained in laboratory media could not be confirmed for sunflower seeds. Generally inhibition was much less and after prolonged incubation of sterile seeds inoculated with A. parasiticus spores, treated seeds even tended to develop higher levels of aflatoxin B₁ than the controls.     

Production of Blue-Fluorescent Pyrazines by A. parasiticus

Aspergillus parasiticus NRRL 2999 did not produce aflatoxins on peptone-mineral salts medium, but did accumulate blue-fluorescent material having R_f's on thin-layer chromatographic plates similar to aflatoxins B₁ and B₂. The blue-fluorescent material was ultimately resolved into nine compounds. The four major compounds were tentatively identified by mass spectrometry as deoxyaspergillic acid, flavocol, deoxydehydroxymutaaspergillic acid, and 2-hydroxy-3,6-di-sec-butylpyrazine, blue-fluorescent pyrazines that have previously been isolated from other aspergilli. Evaluation of AOAC- recommended solvent systems for aflatoxin analyses indicated that diethyl ether/methanol/water (98:1:1) was the best TLC solvent system for separating aflatoxins from interferring fluorescent material. Pyrazine production was inversely related to carbohydrate content of the medium. Care should be taken in evaluating aflatoxin analyses of material with high protein and low carbohydrate levels.     

Moisture-equilibrium relative humidity relationships in pistachio nuts with particular regard to control of aflatoxin formation

Adsorption and desorption isotherms have been determined both manometrically and by weight equilibration for Turkish pistachio nut kernel, shell and hull. Comparison of the calculated and experimentally determined adsorption isotherms for whole nuts showed good correlation. Nuts inoculated with Aspergillus flavus conidia were equilibrated to various ERH levels and stored in controlled environment cabinets at 28°C. Competitive growth of xerophilic strains of A. amstelodami prevented growth and aflatoxin production by the A. flavus at ERHs of 86% and below. At 88% ERH marked aflatoxin production occurred but competition was observed between the A. flavus and A. niger. In sealed containers metabolic moisture from growth of A. amstelodami raised the ERH from the initial 85% and permitted toxin production by A. flavus. The results are discussed in relation to post-harvest handling and storage of pistachio nuts.     

Production of aflatoxins in sausage,salami, sucuk and kavurma

Forty nine meat product samples were examined for the fungal genera. Penicillium sp. was detected in 74.8% of samples. No sample contained Aspergillus parasiticus or Aspergillus flavus. Production of aflatoxins in sausage, salami, sucuk and kavurma by A. parasiticus and A. flavus was studied at different temperatures. A. parasiticus and A. flavus produced no aflatoxins on meat products samples at 15°C. Sucuk was a poor substrate for A. parasiticus and A. flavus at 25°C. Sausage, salami and kavurma were favorable substrates for aflatoxin production by A. parasiticus at 25°C.     

Further Characterization of a Caffeine-Resistant Mutant of Aspergillus parasiticus

Studies were performed to characterize further Aspergillus parasiticus BCR1, a caffeine-resistant mutant of A. parasiticus NRRL 2999, particularly in regard to its caffeine-dependent production of aflatoxins. The enhanced synthesis of aflatoxins by caffeine was highly specific since neither dimethylxanthines nor purines could substitute for the trimethylxanthine. Caffeine's effects were phase dependent and only increased toxin formation if added early in the microorganism's life cycle. The ability of BCR1 to exclude caffeine appeared dependent on the initial levels of caffeine in the growth medium. Respiration and glucose utilization in the wild type strain were inhibited strongly by caffeine, but BCR1 was resistant to these effects. Comparison of glucose uptake kinetics in the wild type and mutant strains indicated that caffeine inhibition of aflatoxin synthesis in the wild type was not due to a disruption of glucose transport.     

Kultur vonAspergillus parasiticus im Apfelsaft II. Einflu? von Butylhydroxyanisol (BHA) and Butylhydroxytoluol (BHT) auf Pilzwachstum and Aflatoxin-Biosynthese

Zusammenfassung Aspergillus parasiticus NRRL 2999 wurde im Apfelsaft (aus Sirup) in Gegenwart von BHA bzw. BHT (100, 200, 300 oder 400 mg/1) bis zu 15 Tage bei 25 °C ruhend bebrütet. Myceltrockengewicht, pH and Konzentrationen der Aflatoxine B₁ und G₁ wurden in dreitägigen Abständen bestimmt. Der pH-Anfangswert von 2,5 blieb durchwegs unverändert. BHA unterdrückte das Wachstum and die Toxinanhäufung in der beobachteten Zeitspanne. Es kam jedoch zu konzentrationsabhängigen Wachstumsverzögerungen und zu früheren Toxin-Höchst-werten als in der Kontrollreihe, was auf eine Anpassung ans Milieu hinweist. BHT führte erst ab 200 mg/l zu einer ca. 25%igen Wachstumshemmung (Löslich-keitsgrenze von BHT zwischen 200 and 300 mg/1) und bei 200 mg/l zur ca. 45%igen Hemmung der Toxinanhäufung. Bei 100 mg/l wirkte BHT fördernd auf die Toxinsynthese (1,90 mal mehr G1 and 6,65 mal mehr B₁ als in der Kontrollreihe). Bei allen getesteten Konzentrationen von BHA bzw. BHT sowie in der Kontrollreihe wurde Aflatoxin G₁ starker als B₁ angehauft.

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The cultivation ofAspergillus parasiticus on apple juice II. Influence of butylated hydroxyanisole and butylated hydroxytoluene on fungal growth and aflatoxin biosynthesis

Summary Aspergillus parasiticus (NRRL 2999) was incubated in apple juice (from syrup), quiescently at 25 °C for up to 15 days in the presence of butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) at concentrations of 100, 200, 300, or 400 mg/l. Mycelial dry weight, pH and concentration of aflatoxin B₁ and G₁ were measured every 3 days with the initial pH of 2.5 remaining unchanged in all samples. BHA suppressed fungal growth and toxin accumulation during the observed incubation period. However, a concentration-dependent growth delay and earlier peaks of toxin accumulation suggested environmental adaptation of the mould. BHT (from 200 mg/l onwards), led to growth inhibition by about 25% (solubility limit of BHT lies between 200 and 300 mg/1), and at a concentration of 200 mg/1, it led to a reduction of toxin accumulation by approximately 45%. At 100 mg/l, however, BHT stimulated aflatoxin production (1.90 times more G, and 6.65 times more B₁ than in the controls). At all tested concentrations of BHA or BHT, as well as in the controls, the accumulation of aflatoxin G₁, without exception, surpassed that of B₁.

     

Kultur vonAspergillus parasiticus im Apfelsaft

Zusammenfassung Kulturen vonAspergillus parasiticus NRRL 2999 in Apfelsaft (aus Sirup) wurden Natriumbenzoat bzw. Kaliumsorbat (100, 200, 300 und 400 mg/l zugesetzt, danach wurden diese bei 25 °C, 3, 6, 9, 12, oder 15 Tage bebrütet und Myceltrokkengewicht, pH und Konzentration der Aflatoxine B₁ und G₁ bestimmt. Der pH-Anfangswert von 2,5 blieb in allen Fällen während der ganzen Inkubationsdauer unverändert. Natriumbenzoat unterdrückte bei allen getesteten Konzentrationen das Wachstum und förderte die Biosynthese von Aflatoxin G₁, während es die Anhäufung von B₁ wenig beeinflußte. Kaliumsorbat förderte das Wachstum bei 100 mg/kg, doch alle getesteten Konzentrationen inhibierten die Produktion der Toxine erheblich (keine nachweisbaren Mengen von B₁ und 3- bis 5 mal weniger G₁ als in der Kontrollreihe). Ausnahmslos wurde Aflatoxin G₁ stärker als B₁ angehäuft.

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The cultivation ofAspergillus parasiticus on apple juiceI. Influence of sodium benzoate and potassium sorbate on fungal growth and aflatoxin biosynthesis

Summary Sodium benzoate or potassium sorbate (100, 200, 300 and 400 mg/l) were added to cultures ofAspergillus parasiticus NRRL 2999 on apple juice (from syrup) and incubated quiescently at 25 °C for 3, 6, 9, 12 or 15 days. The cultures were analyzed for pH, mycelial dry weight and accumulation of aflatoxin B₁ and G₁. The initial pH of 2.5 remained constant in all instances throughout the incubation period. Sodium benzoate, at all concentrations, supressed fungal growth and stimulated the biosynthesis of G₁, whereas little influence was exerted upon the accumulation of B₁. Potassium sorbate stimulated fungal growth at 100 mg/l, while at all concentrations it considerably inhibited toxin production (no detectable amounts of B₁ and 3 to 5 times less G₁ than in controls). The concentration of G₁ surpassed that of B₁ without exception.

     

Stimulation of Aflatoxin Production in Media Supplemented with Taro

The effect of taro supplementation on aflatoxin production by Aspergillus parasiticus was studied on wheat and in the liquid synthetic glucose-low salts (GLS) medium of Adye and Mateles (Biochem. Biophys. Acta. 86: 418, 1964). Wheat containing added taro extract showed increased growth and sporulation and had 30% more aflatoxins. Adding 5% uncooked or cooked taro shreds to the liquid GLS medium increased toxin production two- to three-fold.     

Selected plant essential oils and their main active components,a promising approach to inhibit aflatoxigenic fungi and aflatoxin production in food

Recent research has showed that Aspergillus flavus and Aspergillus parasiticus are aflatoxigenic species that can become very competitive in the framework of climate change. Aflatoxins show carcinogenic, mutagenic, immunotoxic and teratogenic effects on human and animals. Effective and sustainable measures to inhibit these species and aflatoxins in food are required. Origanum vulgare and Cinnamomum zeylanicum essential oils (EOs) and their major active constituents, carvacrol and cinnamaldehyde, respectively, were assayed for inhibiting these species and aflatoxin production in maize extract medium under different environmental conditions. Doses of 10–1000 mg l^?1 were assayed and the effective doses for 50 (ED₅₀) and 90% (ED₉₀) growth inhibition were determined. The ED₅₀ of cinnamaldehyde, carvacrol, oregano EO, and cinnamon EO against A. flavus were in the ranges 49–52.6, 98–145, 152–505, 295–560 mg l^?1 and against A. parasiticus in the ranges 46–55.5, 101–175, 260–425 and 490–675 mg l^?1, respectively, depending on environmental conditions. In A. flavus treatments ED₉₀ were in the ranges 89.7–90.5, 770–860 and 820–>1000 mg l^?1 for cinnamaldehyde, carvacrol and cinnamon EO, and in A. parasiticus treatments in the ranges 89–91, 855–>1000 and 900–>1000 mg l^?1, respectively. ED₉₀ values for oregano EO against both species were >1000 mg l^?1. Growth rates of both species were higher at 37 than at 25°C and at 0.99 than at 0.96 a_w. Aflatoxin production was higher at 25 than at 37°C. Stimulation of aflatoxin production was observed at low doses except for cinnamaldehyde treatments. The effectiveness of EOs and their main constituents to inhibit fungal growth and aflatoxin production in contact assays was lower than in vapour phase assays using bioactive EVOH-EO films previously reported.     

Effects of Sorbate and Propionate on Growth and Aflatoxin Production of Sublethally injured Aspergillus parasiticus

Growth and aflatoxin production by injured spores (heat, 55°C for 15 min, γ-irradiation, 50 Krad) of Aspergillus parasiticus in the presence of sorbate (0.05 and 0.1%) and propionate (0.2 and 0.4%) were studied in YES broth at pH 4.5, 5.5 and 6.5, and 25°C for 21 days. Aflatoxin production was accelerated in the early stages of growth by γ-irradiation, but not heat. Growth and aflatoxin were delayed in cultures from low numbers of uninjured spores. Aflatoxin increased and was produced sooner by low numbers of uninjured spores in 0.05% sorbate. Both inhibitors slowed growth of injured spores more than non-injured. Inhibition of aflatoxin was dependent on the concentration of inhibitor, pH and injury.