Lloyd B. Bullerman

Aspergillus flavus and Aspergillus parasiticus: aflatoxigenic fungi of concern in foods and feeds: a review

Aspergillus flavus and the closely related subspecies parasiticus have long been recognized as major contaminants of organic and nonorganic items. A. flavus , a common soil fungus, can infest a wide range of agricultural products. Some A. flavus varieties produce aflatoxins, which are carcinogenic toxins that induce liver cancer in laboratory animals. A. flavus var. flavus , A. flavus subsp. parasiticus , and A. nomius share the ability to produce aflatoxins. Identification of the A. flavus species group is mainly based on the color and macroscopic and microscopic characteristics of the fungus. A. flavus growth and aflatoxin biosynthesis depend on substrate, moisture, temperature, pH, aeration, and competing microflora. The growth of A. flavus and aflatoxin production are sometimes unavoidable. Aflatoxins are considered natural contaminants; the ideal control approach is prevention of mold growth and aflatoxin production. The detection of members of the A. flavus species group in foods and feed is generally carried out by using plate techniques such as surface spread or direct plating. Research on alternative fungal detection methods is still in its infancy. Few immunoassay techniques have been investigated in this regard. Aflatoxins are generally analyzed by chemical methods, although immunochemical methods which use antibodies are becoming common analytical tools for aflatoxins.

Comparative antimycotic effects of selected herbs, spices, plant components and commercial antifungal agents

The antifungal effects of 16 ground herbs and spices, 4 other plant materials, 3 commercial antifungal agents, tannic acid and 2 experimental mold inhibitors were tested against seven mycotoxin- producing molds. Of the 26 substances tested, cloves, cinnamon, mustard, allspice, garlic, and oregano at the 2% level in potato dextrose agar, completely inhibited growth of all seven mycotoxigenic molds for various times up to 21 d. The remaining compounds either caused little or no inhibition. Powdered pomegranate peel was a good inhibitor against four Penicillium species. Potassium sorbate at 0.3% was highly effective against all seven mold strains. The antifungal antibiotic, natamycin (pimaricin), was also highly effective. Combinations of different levels of potassium sorbate and cloves showed an enhanced or possible synergistic inhibitory effect on growth of all seven molds tested, indicating the possibility of using spices and commercial antifungal agents together in small amounts to obtain antimycotic activity.

Fumonisins in food

Occurrence of Fumonisins in Foods and Feeds: Fumonisins: History, Worldwide Occurrence and Impact W.F.O. Marasas Occurrence of Fumonisins in the US Food Supply A.E. Pohland Occurrence of Fusarium and Fumonisins on Food Grains and in Foods L.B. Bullerman Occurrence and Fate of Fumonisins in Beef J.S. Smith, R.A. Thakur Analytical Aspects of Fumonisins: Analytical Determination of Fumonisins and Other Metabolites Produced by Fusarium moniliforme and Related Species on Corn R.D. Plattner, et al. Quantitation and Identification of Fumonisins by Liquid Chromatography/Mass Spectrometry S.M. Musser NMR Structural Studies of Fumonisin B1 and Related Compounds from Fusarium moniliforme B.A. Blackwell, et al. Determination of Underivatized Fumonisin B1 and Related Compounds by HPLC J.G. Wilkes et al. Analysis of Fumonisin B1 in Corn by Capillary Electrophoresis C.M. Maragos, et al. Isolation and Purification of Fumonisin B1 and B2 from Rice Culture F.I. Meredith, et al. Immunochemical Methods for Fumonisins F.S. Chu Microbiological Aspects of Fumonisins: Introductory Biology of Fusarium moniliforme J.F. Leslie Genetic and Biochemical Aspects of Fumonisin Production A.E. Desjardins, et al. Fusaric Acid and Pathogenic Interactions of Corn and Noncorn Isolates of Fusarium moniliforme: A Nonobligate Pathogen of Corn C.W. Bacon, D.M. Hinton Fumonisins in Maize Genotypes Grown in Various Geographic Areas A. Visconti Liquid Culture Methods for the Production of Fumonisin S.E. Keller, T.M. Sullivan Biosynthesis of Fumonisin and AAL Derivatives by Alternaria and Fusarium inLaboratory Culture C.J. Mirocha, et al. Metabolism and Toxicity of Fumonisins: Fumonisin Toxicity and Metabolism Studies at the USDA W.P. Norred, et al. The Mycotoxin Fumonisin Induces Apoptosis in Cultured Human Cells and in Livers and Kidneys of Rats W.H. Tolleson, et al. Fumonisin B1 Toxicity in Male SpragueDawley Rats G. Bondy, et al. Biological Fate of Fumonisin B1 in Foodproducing Animals D.B. Prelusky, et al. Hepatotoxicity and Carcinogenicity of the Fumonisins in Rats: A Review Regarding Mechanistic Implications for Establishing Risk in Humans W.C.A. Gelderblom, et al. Fumonisin Toxicity and Sphingolipid Biosynthesis A.H. Merrill Jr, et al. Effects of Processing on Fumonisins: Distribution of Fumonisins in Food and Feed Products Prepared from Contaminated Corn G.A. Bennett, et al. Effect of Processing on Fumonisin Content of Corn P.A. Murphy, et al. Reduction of Risks Associated with Fumonisin Contamination in Corn D.L. Park, et al. Effect of Thermal Processing on the Stability of Fumonisin L.S. Jackson, et al. Regulatory Aspects of Fumonisins: Regulatory Aspects of Fumonisins in the United States T.C. Troxell 4 additional articles. Index.

Prevention of mold growth and toxin production through control of environmental conditions

Environmental conditions influence mold growth and mycotoxin production. Such things as water activity (aw), temperature, pH and atmosphere can strongly affect and profoundly alter patterns of growth and mycotoxin production. Generally, maintenance of low temperatures will prevent aflatoxin production in stored products, whereas other toxins such as penicillic acid, patulin, zearolenone and T-2 toxin may be produced at low temperatures. Toxic Penicillium and Fusarium species are generally more capable of growth at low temperatures than are toxic species of Aspergillus . Temperature interacts with aw to influence mold growth and mycotoxin production. Aflatoxin B1 can be produced at conditions of aw and temperature which are close to the minimum aw and temperature for growth. On the other hand, patulin, penicillic acid and ochratoxin A are produced within a narrower range of aw and temperature, compared with those for growth. In fact, production of patulin and penicillic acid by Penicillium species appears to be confined to high aw values only. In optimal substrates, the minima of aw and temperature for growth and toxin production may be lower than in other substrates. It appears that pH and substrate composition have no great effect on growth of toxic molds, but may have a great influence on toxin production. Presence of CO2 and O2 influences mold growth and mycotoxin production. A 20% level of CO2 in air depresses aflatoxin production and markedly depresses mold growth. Decreasing the O2 concentration of air to 10% depresses aflatoxin production, but only at O2 levels of less than 1% are growth and aflatoxin production completely inhibited. With patulin- and sterigmatocystin-producing molds, concentrations of 40% CO2 depress growth and toxin production, but a level of 90% CO2 is needed to completely inhibit production of these toxins. Decreasing O2 concentration to 2% depresses production of patulin and sterigmatocystin but does not affect fungal growth. Only at levels down to 0.2% are growth and toxin production completely inhibited. Controlled atmospheres with increased CO2 (above 10%) and decreased O2 (2%) can be used to retard mold growth. Exclusion of O2 by vacuum packaging in materials with low O2 permeability will depress or even prevent aflatoxin production. Presence of other microorganisms may also restrict fungal growth and mycotoxin production. Aflatoxin production by Aspergillus flavus in mixed cultures with Aspergillus niger is less than in pure culture. Mixtures of fungi growing in grains and nuts in competition with A. flavus seem to prevent aflatoxin production. Other organisms including Rhizopus nigricans , Saccharomyces cerevisiae , Brevibacterium linens and some lactic acid bacteria have been shown to reduce growth and aflatoxin production by Aspergillus parasiticus . In general, mold growth and mycotoxin production can be prevented by employing various measures based on knowledge of the factors involved. Choice of the measures depends upon the type of product, storage period and available techniques.

Mould spoilage and mycotoxin formation in grains as controlled by physical means

Low oxygen concentrations (less than 1%) and/or increased concentrations of CO2 or N2 have been found to be highly effective in preventing the development of mould on grain and in inhibiting selected mycotoxins, e.g. aflatoxins, ochratoxin, patulin, penicillic acid and T-2. However, the levels of CO2 needed to inhibit mould growth are much higher than those required for the inhibition of mycotoxin production. The degree of inhibition achieved by elevated CO2 concentrations is dependent on other environmental factors, such as relative humidity (RH) and temperature. Nevertheless, the biosynthetic pathways for mycotoxin production are merely blocked, but not damaged by high CO2 levels. Irradiation has been shown to destroy the conidia of moulds but the information concerning the effect of irradiation on mycotoxin formation seems to be contradictory. Aflatoxin production was increased in irradiated wheat grain, but decreased in barley and maize when the grain was irradiated prior to inoculation. The number of spores in the inoculum, grain condition, relative humidity and other environmental factors could all affect the results obtained. However, ochratoxin formation by Aspergillus ochraceus was consistently enhanced by irradiation of spores or mycelium.

Inhibition of Growth and Aflatoxin Production of Aspergillus flavus by Lactobacillus Species

A mixture of Lactobacillus species from a commercial silage inoculum reduced mold growth and inhibited aflatoxin production by Aspergillus flavus subsp. parasiticus . Actively growing Lactobacillus spp. cells totally inhibited germination of mold spores. Culture supernatant broth from the mixture of strains inhibited mold growth but did not destroy mold spore viability. Some mold spores were observed microscopically to have germinated and produced short nonbranching germ tubes; then growth ceased. While the pH of the culture broth and supernatant were about 4.0, acidification of nonfermented broth to pH 4.0 with HCl and lactic acid did not cause a similar inhibition of spore germination. The mixture of Lactobacillus species growing in a dialysis sack inhibited aflatoxin production by the A. flavus culture growing outside of the sack in broth, whereas mold growth was not affected. The pH values outside of the dialysis sack in the control and the treatments were similar (6 to 7) throughout the incubation period. When a dialysis sack with a molecular weight cutoff (MWCO) of 1,000 was used, there was little inhibition of aflatoxin B1 production, but with MWCOs of 6,000 to 8,000 and 12,000 to 14,000 aflatoxin production was greatly inhibited. In mixed culture experiments, levels of aflatoxin B1 and G1 were depressed compared to the control (monoculture). Mold growth in this case was also reduced compared to the monoculture system. Purified isolates of Lactobacillus from the commercial mixture had a slight effect on mold growth and aflatoxin production, but supernatant liquid of one isolate was quite inhibitory to production of aflatoxins B1 and G1, without affecting mold growth.

Stability of Fumonisins in Thermally Processed Corn Products

Little is known about the stability of fumonisins in corn-based foods during heating. This study investigated the effects of canning, baking, and roasting (dry heating) processes on the stability of fumonisins in artificially contaminated and naturally contaminated corn-based foods. All samples were analyzed for fumonisin levels by both a commercial enzyme-linked immunosorbent assay (ELISA) and a high-performance liquid chromatographic (HPLC) method. Canned whole-kernel corn showed a significant (P < or = 0.05) decrease in fumonisins by both ELISA (15%) and HPLC (11%) analyses. Canned cream-style corn and baked corn bread showed significant (P < or = 0.05) decreases in fumonisin levels at an average rate of 9% and 48%, respectively, as analyzed by ELISA. Corn-muffin mix artificially contaminated with 5 micrograms of fumonisin B1 (FB1) per g and naturally contaminated corn-muffin mix showed no significant (P < or = 0.05) losses of fumonisins upon baking. Roasting cornmeal samples artificially contaminated with 5 micrograms of FB1 per g and naturally contaminated cornmeal samples at 218 degrees C for 15 min resulted in almost complete loss of fumonisins.

Antimycotic and antiaflatoxigenic effect of lactic acid bacteria: a review

Lactic acid bacteria are extensively used in the fermentation of a wide variety of food products and are known for their preservative and therapeutic effects. Many lactic acid bacteria species have been reported to inactivate bacterial pathogens, and numerous antibacterial substances have been isolated. However, the antimycotic and antimycotoxigenic potential of lactic acid bacteria has still not been fully investigated. Fermented foods such as cheese can be contaminated by molds and mycotoxins. Mold causes spoilage and renders the product unusable for consumption, and the presence of mycotoxins presents a potential health hazard. A limited number of reports have shown that lactic acid bacteria affect mold growth and aflatoxin production. Although numerous lactic acid bacteria such as Lactobacillus spp. were found to inhibit aflatoxin biosynthesis, other lactic bacteria such as Lactococcus lactis were found to stimulate aflatoxin production. The morphology of lactic acid bacteria cells has also been found to be affected by the presence of fungal mycelia and aflatoxin. Lactococcus lactis cells became larger and formed long chains in the presence of Aspergillus flavus and aflatoxins. Numerous investigations reported that low pH, depletion of nutrients, and microbial competition do not explain the reason for aflatoxin inhibition. Some investigators suggested that the inhibition of aflatoxin is due to lactic acid and/or lactic acid bacteria metabolites. These metabolites have been reported to be heat-stable low-molecular-weight compounds.

Inhibition of mold growth and aflatoxin production by Lactobacillus spp.

The effect of three individual species of lactobacilli ( Lactobacillus acidophilus , L. bulgaricus , and L. plantarum ) and a commercial silage inoculant, containing three different strains of the same species, on growth and aflatoxin production of A. flavus subsp. parasiticus NRRL 2999 was determined. The study was done in three substrates; a liquid semi-synthetic broth, rice, and corn. The effect of the growing cell masses of the lactobacilli as well as the effect of metabolic products contained in cell free filtrates were determined in the liquid medium. The cells were effective in preventing growth of the mold, and bacterial metabolites were effective in reducing the amount of aflatoxin produced, although growth was not affected. The prevention of growth that was observed was determined to be relative to a pH effect and microbial competition; however, the lower levels of aflatoxin obtained in the presence of cell free supernatant culture fluids could not be explained on the basis of pH or competition. Mold growth was not affected by the presence of the silage inoculant on the rice and corn. However, increased levels of aflatoxin B1 were observed in the presence of the silage inoculant on rice, and decreased levels of aflatoxin G1 were observed on the presence of the silage inoculant on corn.

Effect of Processing on Fusarium Mycotoxins

Mycotoxins are secondary metabolites produced by a wide variety of fungal species that contaminate food or feed. Fumonisins (FUM), deoxynivalenol (DON) and zearalenone (ZEN) are examples of common mycotoxins in grains that have been shown to affect human and/or animal health. Physical, chemical and biological methods have been used for decontaminating grains containing these toxins. Some treatments reduce the concentration of mycotoxins while others are ineffective. For example, removal of damaged grain by density segregation can reduce DON and ZEN concentrations in corn and wheat. In contrast, thermal processing is usually ineffective for reducing the FUM and ZEN content of foods. More work is needed to identify effective methods for detoxifying mycotoxin contaminated food.