Production of aflatoxin by Aspergillus parasiticus NRRL-2999 during growth in the presence of curing salts

Aspergillus parasiticus NRRL-2999 was inoculated into meat mixtures with curing salts and into yeast extractsucrose (YES) and sucrose-ammonium salts (SAS) broth with and without curing salts to determine if the presence of curing salts significantly affected growth and aflatoxin production by the mold. The effect of individual curing salts or curing salt mixtures on growth and toxin elaboration by the aspergillus was substrate dependent. When YES broth contained 100 ppm of NaNO₂, 2% NaCl, or 1 or 2% NaCl plus 200 ppm of NaNO₂ or 200 ppm of NaNO₃, growth and/or aflatoxin production was depressed. Biosynthesis of aflatoxin B₁ was enhanced by presence of 1 and 4% NaCl in YES broth. The SAS broth containing only NaCl or NaCl combined with nitrite or nitrate yielded less aflatoxin than did control broth or no aflatoxin at all. When compared to the control, an increase in growth and amount of aflatoxin occurred in SAS broth which contained 200 ppm of NaNO₃. Sausages containing 100 and 200 ppm NaNO₂ and no NaCl supported more mold growth and aflatoxin production than did control sausage with 3 % NaCl and 100 ppm of NaNO₂. Addition of 2 and 3 % NaCl and no nitrite to sausage resulted in less aflatoxin than in control sausage.     

Specific Features of Albumin Interactions with Hemiacetals of Aflatoxins and Sterigmatocystin

Aflatoxin B_2a (AB_2a), aflatoxin G_2a (AG_2a), and the hemiacetal of sterigmatocystin have been shown to form immunoreactive conjugates with albumin. The conjugates were formed following incubation of solution mixtures at room temperature for 1 h, as demonstrated by spectrophotometry and enzyme immunoassay. Anti-AB_2a antibodies reacted with AB_2a, aflatoxin B₁, and aflatoxin B₂ (100, 8.8, and 5.9%, respectively); a similar result was obtained for anti-AG_2a antibodies reacting with AG_2a, aflatoxin G₁, and aflatoxin G₂ (100, 2.5, and <1.0%, respectively). Binding of anti-AB_2a and anti-AG_2a antibodies to solid-phase conjugates of AB_2a or AG_2a exhibited similar analytical characteristics.     

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.     

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.     

The analysis of aflatoxins by high-performance liquid chromatography.

The potential of high-performance liquid chromatography as a technique for separating aflatoxins B₁ B₂, G₁, G₂, B_2a, Q₁, M₁, P₁, aflatoxicol, and a degradation product of aflatoxin B₁, 2,3-dihydrodiol, has been assessed. A microparticulate silica adsorption column used with a 1:1 chloroform -dichloromethane eluant provided good resolution of aflatoxins B₁, B₂, G₁, and G₂ but the addition of 1% propan-2-ol was necessary for the elution of aflatoxins M₁ and Q₁. By selecting appropriate solvent mixtures, good resolution of all of the aflatoxins studied was obtained using columns containing an octadecyl (C₁₈) reversed-phase bonded to a microparticulate support. Details are given for resolving: (1) aflatoxins B₁, B₂, G₁, and G₂ using a 5% tetrahydrofuran-15% dimethylformamide in water eluant and (2) aflatoxins B₁ B_2a, Q₁ M₁ P₁ aflatoxicol, and a product of aflatoxin B₁ 2,3-dihydrodiol treated with Tris-buffer, using either 15% dimethylformamide in water or 10% tetrahydrofuran in water as eluant.     

An enzyme immunoassay system for aflatoxin B1

Indirect enzyme immunoassay based on immobilized conjugate of aflatoxin B₁ carboxymethyloxime with bovine serum albumin and polyclonal rabbit antibodies allows determining aflatoxin B₁ with a low relative cross-reactivity against aflatoxin B₂, G₁, G₂, M₁ B_2a, and G_2a and sterigmatocystin (15.5, 15.5, 1.7, 1.0, 0.03, 0.03 and 0.01%, respectively) with a sensitivity of 0.04 ng per well or 4.0 ng per ml organic solvent.     

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.     

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.     

Reduction of aflatoxins by Rhizopus oryzae and Trichoderma reesei

This study evaluated the ability of the microorganisms Rhizopus oryzae (CCT7560) and Trichoderma reesei (QM9414), producers of generally recognized as safe (GRAS) enzymes, to reduce the level of aflatoxins B₁, B₂, G₁, G₂, and M₁. The variables considered to the screening were the initial number of spores in the inoculum and the culture time. The culture was conducted in contaminated 4 % potato dextrose agar (PDA) medium, and the residual mycotoxins were determined every 24 h by HPLC-FL. The fungus R. oryzae has reduced aflatoxins B₁, B₂, and G₁ in the 96 h and aflatoxins M₁ and G₂ in the range of 120 h of culture by approximately 100 %. The fungus T. reesei has reduced aflatoxins B₁, B₂, and M₁ in the 96 h and aflatoxin G₁ in the range of 120 h of culture by approximately 100 %. The highest reduction occurred in the middle of R. oryzae culture.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.     

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.     

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.     

Untersuchungen über den Einfluss von Aflatoxin B1 auf die Morphologie und die cytochemisch fassbare Aktivität einiger Enzyme von Mucor hiemalis (Mucorales)

Zusammenfassung Mit Hilfe cytochemischer Methoden wurde der Einfluß verschiedener Konzentrationen von Aflatoxin B₁ (100 ppm, 10 ppm, 0 ppm) auf die Enzyme Succinat-Dehydrogenase, L (+)-Lactat: NAD-Oxydoreduktase, Alkohol-Dehydrogenase, L-Isocitrat: NAD-Oxydoreduktase, Malat dehydrierendes Enzym (NAD als Akzeptor), NADPH₂-Cytochrom c-Reduktase, Cytochromoxydase und saure Phosphatase des PilzesMucor hiemalis untersucht. Succinat-Dehydrogenase, Alkohol-Dehydrogenase, L-Isocitrat: NAD-Oxydoreduktase und saure Phosphatase wurden bereits durch 10 ppm des Mycotoxins in ihren Aktivitäten vermindert. Atypische Sporenkeimung und verringerter Durchmesser der Hyphen waren die äußeren Kennzeichen der Aflatoxin B₁-Intoxikation. Die Ähnlichkeit des Eingriffs von Aflatoxin B₁ in den Stoffwechsel der Leber junger Enten und in denjenigen von Zellen vonMucor hiemalis wird diskutiert.

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Investigations of the influence of aflatoxin B₁ on the morphology and the cytochemically detectable activities of some enzymes ofMucor hiemalis (Mucorales). Using cytochemical methods the influence of various concentrations of aflatoxin B₁ (100 ppm 10 ppm, 0 ppm) on the enzymes succinic dehydrogenase, L(+)-lactate: NAD oxidoreductase, alcohol dehydrogenase, L-isocitrate: NAD oxidoreductase, malate dehydrogenating enzyme (NAD as acceptor), NADPH₂: cytochrome reductase, cytochrome oxidase and acid phosphatase of the fungusMucor hiemalis was investigated. The activity of succinic dehydrogenase, alcohol dehydrogenase, L-isocitrate: NAD oxidoreductase and acid phosphatase was reduced by the addition of only 10 ppm of the mycotoxin. The morphological symptoms of aflatoxin B₁ intoxication were atypical germination of the spores and reduced diameter of the hyphae. The similarity between the interference of aflatoxin B₁ with metabolism in the liver of ducklings and that of the cells ofMucor hiemalis is the subject of discussion.

     

Preparation, purification and characteristics of an aflatoxin degradation enzyme from Myxococcus fulvus ANSM068

Aims: To prepare, purify and characterize an extracellular enzyme from Myxococcus fulvus ANSM068, designated as myxobacteria aflatoxin degradation enzyme (MADE), which possesses degradation activity against aflatoxin B₁ (AFB₁), G₁ (AFG₁) and M₁ (AFM₁) in solution. Methods and Results: The culture supernatant of strain M. fulvus demonstrated high degradation ability against AFB₁ (71·89%), AFG₁ (68·13%) and AFM₁ (63·82%) after 48 h of incubation. An enzyme was purified from the supernatant of M. fulvus using ethanol precipitation and chromatography on DEAE‐Sepharose and Superdex 75. An overall 166‐fold purification of the enzyme with a recovery of 57% and a final specific activity of 569·44 × 10³ U mg^?1 was obtained using the present purification protocol. The apparent molecular mass of MADE was estimated to be 32 kDa by SDS‐PAGE. AFG₁ and AFM₁ were significantly degraded, by 96·96 and 95·80%, respectively, when treated with pure MADE (100 U ml^?1) produced by strain ANSM068. MADE exhibited the largest amount of activity at 35°C and pH 6·0, with Mg²⁺ ions greatly promoting and Zn²⁺ strongly inhibiting MADE activity. Conclusions: An aflatoxin degradation enzyme from bacterial isolates can effectively remove aflatoxin B₁, G₁ and M₁ in solution. Significance and Impact of the Study: The high activity and wide temperature and pH range of MADE for the degradation of aflatoxin have promising applications in control of mycotoxins during food and feed processing.     

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.     

Reduction of aflatoxin B1 levels by sheep saliva

This study determined the decrease of aflatoxin B₁ by sheep saliva at concentrations of 150 and 300 μg aflatoxin B-₁/L saliva. Analyses for aflatoxins B₁, M₁, and aflatoxicol (R₀) were performed after 2, 4, 6, 24, and 48 hours of incubation. Aflatoxin M₁ and R₀ were not detected and only residues of aflatoxin B₁ were found. 4 to 13% of aflatoxin B₁ were decomposed by sheep’s saliva within 2 hrs and 33 to 43% of aflatoxin B₁ after 24 hrs. Decomposition was affected by the aflatoxin concentration. Decrease of aflatoxin B₁ at 2, 4, 6 hrs was nearly three times higher at the low concentration (150 ppb) compared to the high concentration (300 ppb). After 48 hrs incubation more than 80% of the initial aflatoxin B₁ had been decomposed by the saliva.     

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.     

Degradation of Pure Aflatoxins by Tetrahymena pyriformis

Tetrahymena pyriformis W with nutrients, ca. 22 × 10⁶ cells, decreased the concentration of aflatoxin B₁ 58% in 24 hr and 67% in 48 hr. An unknown, bright-blue fluorescent substance was produced, with intensity about one-half that of the unchanged B₁, with an R_f of 0.52 compared with 0.59 for B₁ and 0.55 for B₂ on a thin-layer chromatography plate, and with an ultraviolet spectrum showing maxima of 253, 261, and 328 mμ. In a separate assay, the cells with nutrients did not degrade pure G₁. Starved, washed cells, ca. 11 × 10⁶, decreased the concentration of B₁ 50% in 10 hr, 70% in 22 hr, and 75% in 30 hr, producing the same unknown component. Ethyl alcohol, 1.96% (v/v), decreased cell populations and size, but the cells remained actively motile in broth plus the alcohol for 96 hr. In 72 hr, neither toxin (ca. 2 ppm) in combination with ethyl alcohol had more inhibitory effect on cell numbers, with or without nutrients, than was produced by alcohol alone. Aflatoxin B₁ had no observed effect on the viability of the starved cells for 30 hr or on the nourished cells for 72 hr. There was no noticeable effect of G₁ on the starved cells in 30 hr or on the nourished cells in 48 hr. After 72 hr with G₁ plus nutrients, many of the cells were round with blisters, nonmotile, and apparently dead or dying.     

Aflatoxin is degraded by heated and unheated mycelia,filtrates of homogenized mycelia and filtrates of broth cultures of Aspergillus parasiticus

Steaming one-half of a lot of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 for 6 min resulted in little or no subsequent degradation of aflatoxin B₁ or G₁ by these mycelia. The other half of these mycelia was not heat-treated and degraded aflatoxins B₁ and G₁ Filtrates of the growth substrate which remained after the mycelium was removed from 8- to 15-day old cultures of A. parasiticus NRRL 2999 did not degrade substantial amounts of aflatoxin B₁ or G₁, whereas mycelia originally produced on these filtrates degraded substantial amounts of both aflatoxins. The supernatant fluid from homogenates of 9-day-old mycelia of A. parasiticus NRRL 2999 degraded aflatoxins B₁ and G₁ when 0.1 M or 1.0 M phosphate buffer, pH 6.5, was used to suspend the homogenate. These data support the hypothesis that the aflatoxin degrading factor(s) present in the mycelium of A. purasiticus is/are enzyme(s) or at least influenced by enzyme(s).     

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.