Hassan Gourama

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.

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.

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.

Anti-aflatoxigenic activity of Lactobacillus casei pseudoplantarum.

Lactobacillus casei pseudoplantarum 371 isolated from a silage inoculant was found to inhibit aflatoxins B1 and G1 biosynthesis by Aspergillus flavus subsp. parasiticus NRRI. 2999, in liquid medium. The inhibitory activity in the Lactobacillus cell-free supernatant was found to be sensitive to proteolytic enzymes such as trypsin and alpha-chymotrypsin, but resistant to pepsin. Lab-Lemco tryptone broth (LTB), supplemented with 20% of dialyzed protein concentrate of the supernatant, totally inhibited the production of aflatoxins B1 and G1. When the protein concentrate was digested with trypsin, the production of aflatoxins B1 and G1 was restored. The inhibitory activity of the supernatant was inactivated within 10 min at 100 degrees C. A. flavus grown in the Lactobacillus cell-free supernatant did not produce a mutagenic response in the Salmonella mutagenicity test. However, Lactobacillus casei pseudo plantarum 371 did not have an effect on aflatoxin production and mold growth as measured by ergosterol and plate count, when the organisms were inoculated together on sterile steamed rice.

Detection of Molds in Foods and Feeds: Potential Rapid and Selective Methods†

Most laboratories still rely on traditional microbiological methods to detect molds in foods and feed. These methods are modified bacteriological methods. Plate count techniques are time consuming and do not detect dead fungi, which are a sign of past contamination. Development of rapid methods to detect molds in foods is still in its embryonic stage. Recently mycologists have begun to develop media that are differential and selective for particular mold species. The use of these media is of great value for the detection of specific groups of fungi such as toxigenic fungi. Other potential rapid methods include chemical and biochemical assays for, e.g., chitin and ergosterol, and immunological and electrical impedance methods.

Relationship between aflatoxin production and mold growth as measured by ergosterol and plate count

Samples of sterile long grain white rice were inoculated with different inoculum levels of Aspergillus flavus subsp parasiticus spores and incubated at 25°C. Ergosterol content, mold count and aflatoxin production were determined. Ergosterol was determined using a method that consisted of methanol-hexane extraction, saponification and quantification using HPLC with UV-detection. At low inoculum levels mold spores and ergosterol were not detected until the third day of incubation. Generally aflatoxin B1 followed the same trend as ergosterol. At high inoculum levels ergosterol and mold spore counts were detectable before aflatoxin B1. There was no clear pattern between aflatoxin G1 production and ergosterol content. Ergosterol appears to be a sensitive early indicator of low levels of fungal activity and aflatoxin production in rice.

Antifungal Activity of 10-Oxo-trans-8-decenoic Acid and 1-Octen-3-ol against Penicillium expansum in Potato Dextrose Agar Medium

In mushrooms, 10-oxo-trans-8-decenoic acid (ODA) and 1-octen-3-ol are secondary metabolites produced naturally by the enzymatic breakdown of linoleic acid. Both compounds were determined to inhibit the mycelial growth of Penicillium expansum PP497A, a common food spoilage organism, when added to potato dextrose agar medium. ODA and 1-octen-3-ol were inhibitory at concentrations of > 1.25 mM (230 microg/g for ODA and 160 microg/g for 1-octen-3-ol). At pH 5.6, 1-octen-3-ol was more inhibitory than ODA. However, at pH 3.5, both compounds (especially ODA) were more inhibitory than they were at pH 5.6. This finding indicates that the undissociated carboxyl of ODA was important for inhibition. At a concentration of 2.5 mM and a pH of 3.5, ODA and 1-octen-3-ol inhibited growth by 43.1 and 41.9%, respectively. An additive effect was observed when both compounds were added at a combined concentration of > or = 1.25 mM; when both were added at a combined concentration of 2.5 mM, mycelial growth was inhibited by 48.8 and 72.8% at pHs of 5.6 and 3.5, respectively. Although the antifungal activity levels for these two compounds were lower than those observed for equal molar concentrations of sorbate, a common antifungal compound, these findings indicate that further investigation of the potential of ODA and 1-octen-3-ol for use as natural food preservatives is warranted.

Effects of modified atmosphere packaging and preservatives on the shelf-life of high moisture prunes and raisins

The growth of Aspergillus niger and Zygosaccharomyces rouxii on high moisture prunes and raisins in the presence of preservatives and packed under modified atmospheres was determined. Prunes and raisins adjusted to an aw of 0.84-0.87 in the presence of carbon dioxide atmospheres (40 and 80% CO2) did not support growth of A. niger. However, Z. rouxii spoiled the fruit samples, both in air and under CO2 conditions. Addition of low levels of K-sorbate (186 ppm in prunes and 153 ppm in raisins) or Na-benzoate (176ppm in prunes and 158ppm in raisins) delayed outgrowth of Z. rouxii. The inhibitory effect of preservatives was higher in raisins than in prunes. Modified atmospheres (40% CO2-60% N2 or 80% CO2-20% N2) combined with the addition of 417 and 343 ppm K-sorbate or 383 and 321 ppm Na-benzoate accomplished complete growth inhibition of Z. rouxii and extended the shelf-life of high moisture prunes and raisins at 30 degrees C for at least 6 months.

Effects of Potassium Sorbate and Natamycin on Growth and Penicillic Acid Production by Aspergillus ochraceus

Potassium sorbate at 500, 1000 and 1500 μg/ml delayed initiation of growth and sporulation by Aspergillus ochraceus 0L24 in yeast extract-sucrose (YES) broth at 15°C, 25°C and 35°C. At 25°C, sporulation and growth were more rapid. Potassium sorbate at 500 μg/ml resulted in an increase in mycelial weight, but at 1000 and 1500 μg/ml the mycelial mass was decreased. Potassium sorbate also reduced or prevented production of penicillic acid, especially at 15 and 35°C. Natamycin at 1, 10 and 20 μg/ml delayed initiation of growth and sporulation in YES broth. At 20 μg of natamycin/ml, mycelial growth was inhibited by 80 to 100% and penicillic acid production was completely inhibited. Growth and penicillic acid production on olive paste by A. ochraceus in the presence of potassium sorbate and natamycin showed that sorbate at 1500, 3000, and 6000 μg/g delayed growth and sporulation. Also, the extent of growth was greatly reduced by 3000 and 6000 μg of potassium sorbate/g. Penicillic acid production was reduced over the control at all the potassium sorbate levels. At 6000 μg of sorbate/g, no penicillic acid was detected after 21 d of incubation. Natamycin at 85, 175, and 350 μg/g delayed growth and sporulation by A. ochraceus on olive paste. Increasing levels of natamycin resulted in decreased growth. Production of penicillic acid was also decreased by natamycin, 350 μg of natamycin/g decreased penicillic acid production by 96%.

Effects of Oleuropein, Tyrosol, and Caffeic Acid on the Growth of Mold Isolated from Olives

The effects of oleuropein, tyrosol, and caffeic acid on the growth of mold species isolated from Moroccan olives were studied. Oleuropein at 0.2%, 0.4%, and 0.6% slightly stimulated fungal growth. Tyrosol at 0.2%, 0.4%, and 0.6% inhibited the growth of all mold species tested. Caffeic acid was less inhibitory than tyrosol.