Possible synergistic effect of nisin and propionic acid on the growth of the mycotoxigenic fungi Aspergillus parasiticus, Aspergillus ochraceus, and Fusarium moniliforme.

The effects of nisin and propionic acid (PA) on aflatoxin production and on mycelial growth and spore germination of the mycotoxigenic fungi Aspergillus parasiticus, A. ochraceus, and Fusarium moniliforme were investigated. The growth of A. ochraceus was completely inhibited on media containing PA with nisin in concentrations of 0.05% PA with 1,000 ppm nisin, and 0.1% PA with 500 or 1,000 ppm nisin. The growth of both F. moniliforme and A. parasiticus was completely inhibited by PA with nisin at a concentration of 0.1% PA with 1,000 ppm nisin. Nisin alone caused a significant increase in mycelial growth when applied to A. ochraceus at 500 or 1,000 ppm and when applied to A. parasiticus at 1,000 ppm. Spore germination of A. ochraceus was completely inhibited on media containing 0.1% PA with 500 or 1,000 ppm nisin. Spores of F. moniliforme failed to germinate in 0.05% PA with 500 or 1,000 ppm nisin, whereas spores of A. parasiticus did not germinate on media containing 0.1% PA with 1,000 ppm nisin. For all three fungi tested, the inhibitory effect on mycelial growth was found to be fungistatic rather than fungicidal. The combined treatment of PA with nisin produced better fungistatic activity than treatment involving either material alone. Nisin, applied alone, did not stimulate aflatoxin production (expressed by microg toxin/mg mycelium), but the combined treatment at certain concentrations was inhibitory to aflatoxin B1 or G1. The production of aflatoxin G1, but not of B1, was stimulated in 0.05% PA with 1,000 ppm nisin and on media containing 0.1% PA with 100 ppm nisin. Nisin is currently applied in foods to prevent spoilage induced by bacteria but not by mold. The results of the present study indicate that a combined treatment of nisin in small concentrations of PA might be useful in preventing mold damage in certain foods and stored grain.

Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Escherichia coli.

Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, and each was cloned into the E. coli expression vector pGEX2T. Upon induction, the bacteria overexpressed 38- and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively. The chimeric proteins were isolated and affinity purified for use as antigens. The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn gain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) against A. parasiticus and A. flavus than against the other fungi tested and the corn grain. The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible. Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods.

Detection of aflatoxigenic molds in grains by PCR.

Aflatoxins are carcinogenic metabolites produced by several members of the Aspergillus flavus group in grains and floods. Three genes, ver-1, omt-1, and apa-2, coding for key enzymes and a regulatory factor in aflatoxin biosynthesis, respectively, have been identified, and their DNA sequences have been published. In the present study, three primer pairs, each complementing the coding portion of one of the genes, were generated. DNA extracted from mycelia of five Aspergillus species, four Penicillium species, and two Fusarium species was used as PCR template for each of the primer pairs. DNA extracted from peanut, corn, and three insect species commonly found in stored grains was also tested. Positive results (DNA amplification) were achieved only with DNA of the aflatoxigenic molds Aspergillus parasiticus and A. flavus in all three primer pairs. The detection limit of the PCR was determined by using the primer pairs complementing the omt-1 and ver-1 genes. Sterile corn flour was inoculated separately with six different molds, each at several spore concentrations. Positive results were obtained only after a 24-h incubation in enriched media, with extracts of corn inoculated with A. parasiticus or A. flavus, even at the lowest spore concentration applied (10(2) spores per g). No DNA spores per g). It is concluded that genes involved in the aflatoxin biosynthetic pathway may form the basis for an accurate, sensitive, and specific detection system, using PCR, for aflatoxigenic strains in grains and foods.

Production of patulin by different strains of Penicillium expansum in pear and apple cultivars stored at different temperatures and modified atmospheres.

It was shown that the ability of three different strains of Penicillium expansum (NRRL 2034, NRRL 6069, and CBS 481.84) to grow and to produce patulin in pears (cv. Spadona) and apples (cv. Starking) varied under the different temperatures tested (0, 3, 6, 17 and 25 degrees C). Strain NRRL 2034 did not produce patulin at 0 or 25 degrees C while the two other strains produced the toxin at all temperatures, the maximum production occurring at 25 degrees C (for pears) and 17 degrees C (for apples). No significant differences in pathogenicity, as determined by lesion diameter, was recorded among the strains. Patulin production was totally inhibited when the fungi were grown in apples stored under 3% CO2/2% O2 (25 degrees C). The weight of infected tissue in apples contaminated with any of the strains and stored at that modified atmosphere was 70% that of the control. Under 3% CO2/10% O2 or 3% CO2/20% O2, strain NRRL 2034 did not produce patulin while the two other strains produced the toxin in different amounts.

Production of zearalenone in vitro and in corn grains stored under modified atmospheres.

The production of zearalenone by an isolate of Fusarium equiseti was studied in chemically defined medium and in corn grains stored under modified atmospheres. An increase in the concentrations of sucrose or xylose in Czapek's medium resulted in increased toxin production, while no toxin was produced when lactose was present in the medium. Methionine (10(-2) and 10(-3) M) and cystine (10(-3) M) added to Czapek's medium inhibited zearalenone production. When amino acids or nitrogen salts were added as the sole nitrogen source, only alanine, tryptophan and NH4Cl totally inhibited zearalenone production. Zearalenone production was inhibited almost completely in high-moisture corn grains (27%) kept under atmospheres enriched with high CO2 levels (60%, 40% or 20%) with either 20% or 5% O2. However, a lower amount of CO2 was needed to inhibit fungal development and toxin formation when a reduced O2 level was applied.

Fumigant toxicity of essential oils against four major stored-product insects.

The fumigant toxicity of 28 essential oils extracted from various spice and herb plants and some of their major constituents were assessed for adult coleopteransRhyzopertha dominica, Oryzaephilus surinamensis, Tribolium castaneum, andSitophilus oryzae. Three groups of active materials were distinguished: (1) The compounds terpinen 4-ol, 1,8-cineole, and the essential oils of three-lobed sage, sage, bay laurel, rosemary, and lavender were most active againstR. dominica; (2) The compounds linalool,α-terpineol, and carvacrol and the essential oils of oregano, basil, Syrian marjoram, and thyme were most active againstO. surinamensis; and (3) the compound 1,8-cineole and the essential oils anise and peppermint were active againstT. castaneum.

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.

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 T-2 toxin production on high-moisture corn kernels by modified atmospheres.

The fungus Fusarium sporotrichioides, capable of producing T-2 toxin (T-2), was grown on irradiated corn kernels remoistened to 22% and kept in atmospheres of different CO2-O2 combinations. The production of T-2 was totally inhibited under 60% CO2-20% O2, whereas only trace amounts were detected when the gas combination was 40% CO2-5% O2. Under all other combinations tested, the amount of T-2 produced was reduced by 25 to 50% as compared with the control. Fungal growth was not inhibited by any of the gas mixtures examined, and the growth rate (measured by direct plating, dilution method, and CO2 production) was almost identical to that in grains kept under air. It is concluded that although F. sporotrichioides is tolerant to high CO2 levels, T-2 formation on corn can be inhibited by CO2 concentrations less than that required to inhibit fungal growth.

Effect of early stages of fungal development on the nutritional value of diets for broiler chicks.

During 65 d of storage a gradual increase in fungal activity (evaluated by CO2 production) was observed in a diet with its moisture content elevated to 136 g/kg. This activity was inhibited by supplementation of the wetted diets with either calcium propionate (3 g/kg) or Agrosil (2 g/kg). The fat content of the wetted untreated diet decreased between the 18th and the 45th d of storage from 38 to 29 g/kg. This change was prevented by the addition of either of the two fungistats. The weight gains of 7-d-old female broiler chicks fed on the wetted diets with or without the fungistats from the 18th d after their preparation for 27 d, did not differ significantly (P greater than 0.05) from those of birds fed on the unwetted diet. However, the food:gain ratio of chicks fed on the unwetted diet was significantly (P less than 0.05) better than that of chicks fed on the fungistat-free wetted diet. The results from birds fed on the fungistat-supplemented wetted diets were intermediate. It is concluded that the early stages of fungal activity (characterised by increased CO2 production, without changes or with only a slight decrease in fat content) have only a minor effect on the nutritional value of diets.

Effects of cysteine and structurally related compounds on ochratoxin production by Aspergillus ochraceus.

Addition of 10(-2) M L-cysteine, L-cystine, or S-ethyl-L-cysteine to a synthetic medium containing xylose as the sole carbon source did not decrease ochratoxin production by Aspergillus ochraceus. At that concentration, DL-homocysteine thiolactone HC1, DL-cysteine HC1, L-ethionine, S-methyl-L-cysteine, and glutathione (reduced) strongly inhibited ochratoxin production. DL-Homocysteine thiolactone HC1, DL-cysteine HC1, and L-ethionine also strongly inhibited fungal growth. At lower concentrations (10(-3) and 10(-4) M) only L-ethionine decreased the toxin production. Ochratoxin inhibition caused by DL-homocysteine thiolactone HC1, DL-cysteine HC1, and glutathione was observed only in cases where the pH of the media was below 5.0. The inhibition caused by 10(-3) M ethionine was partially prevented by the addition of 10(-3) M methionine but this was not the case after the addition of S-methyl-L-cysteine to the medium.

Studies of the fungistatic activity of antifungal compounds in mash and pelleted feeds.

The effect of calcium propionate (CP) and Agrosil (AG) as mold inhibitors in wetted mash and pelleted feed was studied using both commercial cattle and poultry rations. Number of fungal colonies isolated after pelleting was markedly reduced; however, wetting the pellets accelerated the build-up of inoculum and resulted in spoilage. The addition of CP (.3%) to the cattle ration before pelleting prevented mold proliferation during one month of storage while the number of fungal colonies counted in pellets treated with AG (.15%) markedly increased over that period. However, AG had a longer fungistatic effect than CP in preserving the mash diet. Both materials, applied at .2%, were ineffective in preserving wet pelleted poultry feed. After 17 days of storage, feed treated with either of the agents was visibly moldy. In all cases, an increase in mold population was concomitant with elevated carbon dioxide concentrations, which indicated the sensitivity of this parameter for measuring fungal activity. Fat content of the diets remained unchanged during the storage period in spite of increased fungal activity.

Ochratoxin production by Aspergillus ochraceus as affected by methionine and structurally related compounds.

When 10(-2) M of L- or D-methionine was added to a synthetic medium containing xylose as the sole carbon source, ochratoxin production by Aspergillus ochraceus was strongly inhibited. At that concentration methionine derivatives, e.g., alpha-methyl-DL-methionine, DL-methionine sulfoxide, and L-methionine sulfone, did not inhibit ochratoxin production, whereas DL-methionine S-methyl sulfonium chloride (MMSC) inhibited ochratoxin production to a large extent. L-Methionine, as well as MMSC, also completely inhibited sclerotia formation, while D-methionine and DL-methionine sulfoxide caused only a partial inhibition. At lower concentrations (10(-3) and 10(-4) M), none of the compounds exhibited inhibitory effects. In cases where strong ochratoxin inhibition was detected, fungal radial growth or mycelial dry weight was inhibited by only 10-25%, while the initial pH of the medium dropped from approximately 6.5 to approximately 4.4-5.0. Adjustment of the initial pH of media supplemented with 10(-2) M L-methionine, D-methionine, or MMSC to a pH of approximately 7.8 did not change the inhibitory effects on ochratoxin production in media containing L-methionine. On the other hand, sclerotia formation was restored in all three treatments.

Ochratoxin A production by Aspergillus ochraceus Wilhelm grown under controlled atmospheres.

When Aspergillus ochraceus NRRL 3174 was grown under controlled atmospheres with 1 and 5% O2 and without CO2, the amount of ochratoxin produced was the same as that produced by the control colonies. Increasing the O2 level up to 40% reduced ochratoxin production by 75%, whereas at 60% O2, ochratoxin production was enhanced. In atmospheres enriched with 10 or 20% CO2, ochratoxin production was reduced when O2 concentrations were below 20% and enhanced when the O2 concentration was 40 or 60% O2. Ochratoxin production was completely inhibited at 30% CO2 and above, regardless of the O2 level. Colony growth was partially inhibited at 60% CO2, and no growth occurred at 80% CO2 or above. However, when colonies inhibited by 60% CO2 or above were subsequently exposed to air, radial growth, number of sclerotia formed, and the amount of ochratoxin produced were the same as in the control colonies. The results indicate that A. ochraceus is tolerant to CO2 concentrations higher than those required to control storage insects.

The nutritional value of moldy grains for broiler chicks.

The effect was determined of mold development in corn and sorghum grains on their lipid content and nutritional value for broiler chicks. The grains, whole or ground, with their original moisture content (12.1 to 13.0%) or increased moisture content (15.0% moisture), were stored for 63 to 96 days prior to their incorporation into the diets fed to the chicks. Increasing the moisture content caused the development of the naturally occurring fungi (mainly Penicillium and Aspergillus spp.). The moldy grains did not contain aflatoxin B1, ochratoxin A, patulin, sterigmatocystin or zearalenone. Storage of whole or ground grains or of moistened whole corn did not result in differences in their fat content, in the metabolizable energy (ME) of the diets containing these grains, or in the performance of chicks fed these diets, but moistened whole sorghum affected performance adversely. Fat content in moistened ground grains decreased markedly during storage, but fatty acid ratios, vitamin E, carotene, xanthophyll, and protein levels were not markedly affected. These ground moldy grains reduced the dietary fat level during the 3 weeks of the feeding period in two out of three experiments and significantly (P less than .05) lowered ME values and depressed performance. Soybean oil supplementations to diets containing these grains increased dietary ME values and partially or completely restored performance. It is concluded, therefore, that the decreased energy level in diets containing ground moldy grains (not containing mycotoxins) is an important factor for their reduced nutritional value.