Aflatoxin formation in hay treated with formic acid and in isolated strains of Aspergillus flavus

The possibility of improving the keeping qualities of field-dried and hayloft-dried hay, with dry matter contents of 55 and 73% by adding sodium chloride, formic acid and propionic acid was investigated. Field-dried, sodium-chloride-treated hay was stored in containers, while controls and acid-treated hay were pressed into high-density bales. All hayloft-dried hay was stored in containers. Fungal attacks were very heavy, especially in the baled hay. The contaminating flora was examined at the temperature maximum and after 40 and 32 days storage for field-dried and hayloft-dried hay respectively. The genera Penicillium, Cladosporium and Trichothecium were predominant in hayloft-dried hay, and the differences between varying dry matter contents were insignificant. Mostly Aspergillus spp., but also Penicillium spp., occurred in the field-dried hay at 73% dry matter content but at 55% the flora consisted mostly of Aspergillus flavus, and the formic acid-treated hay contained almost a pure culture of this fungus. In addition, aflatoxin B₁ and G₁ (635–1000 μg/l) were demonstrated in this formic acid-treated hay.

Toxin formation in isolates of A. flavus from differently treated hay specimens was also investigated on various substrates.     

Multiplex real-time PCR for detection and quantification of mycotoxigenic Aspergillus, Penicillium and Fusarium

The most agriculturally and economically important classes of mycotoxins are produced by species of Aspergillus, Penicillium, and Fusarium. Rapid methods to detect mycotoxigenic fungi could help prevent mycotoxins from entering the food chain. The purpose of this research was to develop a multiplex real-time PCR assay to detect and quantify multiple species of mycotoxigenic fungi. A pair of broad-spectrum PCR primers was designed for amplification of the internal transcribed spacer (ITS) regions of rDNA from the mycotoxigenic species. An in silico analysis of the primers revealed the presence of amplification in more than 40 Aspergillus species, 23 Fusarium species, and 32 Penicillium species as well as 64 other fungal genera. Genus-specific Taqman probes were designed from the ITS sequences of the most important mycotoxigenic species of Fusarium, Penicillium, and Aspergillus. The specificity of the probes was established against a wide range of fungal species. As a multiplex assay, the linear range of detection was 1 pg to 10 ng of DNA. The assay was validated by analyzing fungal growth in distiller's grain (DG), an animal feedstock that is a by-product when ethanol is produced from corn. This assay could be used as an initial step to evaluate the mycotoxigenic potential of DG and various other agricultural commodities.     

Contamination with storage fungi of human food from Cameroon

In a mycological study, a total of 95 human food samples were investigated to evaluate the incidence of fungal contamination in Cameroon by conventional identification method and partly confirmed by DNA sequencing. The isolated fungal spp. were further studied to determine their toxigenic potentials. The investigation revealed the predominance of Aspergillus and Penicillium with 96% of samples contaminated with at least one species of these fungi, whereas the incidence of co-contamination of samples was 85%. Aspergillus flavus and Aspergillus parasiticus (Flavi section) were the most predominant species contaminating mainly maize and peanuts. In addition, P. crustosum and P. polonicum were the most common contaminants belonging to the genus Penicillium. On the other hand, A. ochraceus (Circumdati section) registered a low incidence rate of 5%, including other members of the Aspergillus group. Other members of the genera Rhizopus and Alternaria spp. were also registered in the study. A majority of fungal strains of A. ochraceus, A. parasiticus, P. crustosum and P. polonicum isolated were toxigenic, producing the mycotoxins tested for, while none was detected in cultures of A. fumigatus. The high incidence rate of fungi contamination coupled with their potentials in producing mycotoxins gives a strong indication that the samples tested may likely be contaminated with various mycotoxins. There is need for further study to assess the incidence of mycotoxins contamination in similar food samples.     

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.     

Fungal diversity in cow, goat and ewe milk

Knowledge of fungal diversity in the environment is poor compared with bacterial biodiversity. In this study, we applied the denaturing high-performance liquid chromatography (D-HPLC) technique, combined with the amplification of the ITS1 region from fungal rDNA, for the rapid identification of major fungal species in 9 raw milk samples from cow, ewe and goat, collected at different periods of the year. A total of 27 fungal species were identified. Yeast species belonged to Candida, Cryptococcus, Debaryomyces, Geotrichum, Kluyveromyces, Malassezia, Pichia, Rhodotorula and Trichosporon genera; and mold species belonged to Aspergillus, Chrysosporium, Cladosporium, Engyodontium, Fusarium, Penicillium and Torrubiella genera. Cow milk samples harbored the highest fungal diversity with a maximum of 15 species in a single sample, whereas a maximum of 4 and 6 different species were recovered in goat and ewe milk respectively. Commonly encountered genera in cow and goat milk were Geotrichum candidum, Kluyveromyces marxianus and Candida spp. (C. catenulata and C. inconspicua); whereas Candida parapsilosis was frequently found in ewe milk samples. Most of detected species were previously described in literature data. A few species were uncultured fungi and others (Torrubiella and Malassezia) were described for the first time in milk.     

A survey on distribution and toxigenicity of Aspergillus section Flavi in poultry feeds

Thirty-five samples of poultry feeds and corresponding raw materials (maize, soybean and meat meal) from a processing plant were analyzed to evaluate the distribution and toxigenicity of Aspergillus section Flavi isolates. Mycological analysis of the samples indicated the presence of five fungal genera (Aspergillus, Penicillium, Fusarium, Cladosporium, and Eurotium). Aspergillus flavus was the predominant species being present in 48.5% of the analyzed samples. Ninety-one isolates belonging to Aspergillus section Flavi were isolated; ninety were identified as A. flavus and only one as A. parasiticus. Fifty-seven isolates were capable of producing sclerotia, 41 were identified as L-type strains and 16 as type S. Fifty-seven percent of the isolates produced AFB₁ levels ranging from 0.05 μg/kg to 27.7 μg/kg whereas 86.8% produced CPA from 1.5 μg/kg to 137.8 μg/kg. L-strains produced from 0.05 to 14.8 μg/kg of aflatoxin and type S produced levels from 0.05 to 1.65 μg/kg. No significant differences in CPA production among S- and L-strains were observed. Sclerotial isolates produced AFB₁ levels ranging between 0.05 and 27.7 μg/kg and CPA levels from 3.8 to 47.3 μg/kg. More than half of the A. flavus isolates were able to produce AFB and CPA simultaneously. Twenty percent of the 35 samples were contaminated with aflatoxin B₁ whereas 34.3% were contaminated with CPA. The high rate of CPA producing isolates represents a potential risk of contamination with this toxin in poultry feeds.     

Application of Lactobacillus amylovorus as an antifungal adjunct to extend the shelf-life of Cheddar cheese

Lactobacillus amylovorus DSM 19280 is an antifungal strain that is inhibitory to a range of fungi including Penicillium expansum, Penicillium roqueforti, Aspergillus niger, Aspergillus fumigatus and Fusarium culmorum. In this study, the strain was used as an adjunct culture in a Cheddar cheese model system. During the ripening period, P. expansum spores were applied to the cheese surface to mimic fungal contamination. The presence of the antifungal L. amylovorus adjunct resulted in a four-day delay in appearance of Penicillium growth on the cheese in comparison to the adjunct-free control. When cheeses were exposed to natural airborne fungi, the presence of the adjunct resulted in a six-day delay in the appearance of mycelia on the cheese surface. Significantly, its presence had no detectable negative impact on cheese quality. The results indicate that the strain could have an application for extending the shelf-life of cheeses which are prone to fungal spoilage.     

A survey of pre-harvest ear rot diseases of maize and associated mycotoxins in south and central Zambia

Maize ear rots reduce grain yield and quality with implication on food security and health. Some of the pathogenic fungi produce mycotoxins in maize grain posing a health risk to humans and livestock. Unfortunately, the levels of ear rot and mycotoxin infection in grain produced by subsistence farmers in sub-Saharan countries are not known. A survey was thus conducted to determine the prevalence of the ear rot problem and levels of mycotoxins in maize grain. A total of 114 farmsteads were randomly sampled from 11 districts in Lusaka and southern provinces in Zambia during 2006. Ten randomly picked cobs were examined per farmstead and the ear rot disease incidence and severity were estimated on site. This was followed by the standard seed health testing procedures for fungal isolation in the laboratory. Results indicated that the dominant ear rots were caused by Fusarium and Stenocarpella. Incidence of Fusarium verticillioides ranged from 2 to 21%, whereas that of Stenocarpella maydis reached 37% on ear rot diseased maize grain. In addition, 2-7% F. verticillioides, and 3-18% Aspergillusflavus, respectively, were recovered from seemingly healthy maize grain. The mean rank of fungal species, from highest to lowest, was F. verticillioides, S. maydis, A.flavus, Fusarium graminearum, Aspergillus niger, Penicillium spp., Botrydiplodia spp., and Cladosporium spp. The direct competitive ELISA-test indicated higher levels of fumonisins than aflatoxins in pre-harvest maize grain samples. The concentration of fumonisins from six districts, and aflatoxin from two districts, was 10-fold higher than 2 ppm and far higher than 2 ppb maximum daily intake recommended by the FAO/WHO. The study therefore suggested that subsistence farmers and consumers in this part of Zambia, and maybe also in similar environments in sub-Saharan Africa, might be exposed to dangerous levels of mycotoxins due to the high levels of ear rot infections in maize grain.     

Production of fungal lipases using wheat bran and soybean bran and incorporation of sugarcane bagasse as a co-substrate in solid-state fermentation

Fungal strains were screened for lipase producing activities and 10 strains were classified as good producers. Aspergillus sp., Fusarium sp., and Penicillium sp. exhibited the highest activities when fermented in wheat bran (WB) and soybean bran (SB). No fungal growth was observed using sugarcane bagasse (CB). An experimental design was applied to incorporate CB into the fermentation process for lipase production by Aspergillus sp. and Penicillium sp., and to evaluate the best moisture content for the substrate. Strains studied achieved maximum lipase activities with 25% CB combined with 75% WB or SB at 40% moisture content. The highest lipase activities were observed for WB and SB, and for SB combined with CB using Aspergillus sp. Fermentation of 96 h was the optimum period for enzyme production.     

Effects of Tropical Citrus Essential Oils on Growth, Aflatoxin Production, and Ultrastructure Alterations of Aspergillus flavus and Aspergillus parasiticus

Ethyl acetate extracts and hydrodistillated essential oils from five cultivars of tropical citrus epicarps were evaluated for their inhibitory activities against Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus, and Penicillium sp. using disk diffusion and broth microdilution assays. Essential oils prepared from kaffir lime (Citrus hystrix DC) and acid lime (Citrus aurantifolia Swingle) epicarps exhibited stronger antifungal activity to all fungi than their ethyl acetate extracts with minimum inhibitory concentration and minimum fungicidal concentration values of 0.56 and 1.13 mg/ml (dry matter), respectively, against aflatoxin-producing A. flavus and A. parasiticus. The dominant components of the essential oil from kaffir lime were limonene, citronellol, linalool, o-cymene, and camphene, whereas limonene and p-cymene were major components of acid lime essential oil. Pure limonene, citronellal, and citronellol were five to six times less fungicidal than the natural essential oils, indicating the synergistic activity of many active compounds present in the oils. Kaffir and acid lime essential oils significantly reduced aflatoxin production of A. flavus and A. parasiticus, particularly lime essential oil, which completely inhibited growth and aflatoxin production of A. flavus at the concentration of 2.25 mg/ml. Target cell damage caused by acid lime essential oil was investigated under transmission electron microscopy. Destructive alterations of plasma and nucleus membrane, loss of cytoplasm, vacuole fusion, and detachment of fibrillar layer were clearly exhibited in essential-oil-treated cells.     

Aspergillus flavus infection and aflatoxin production in mixtures of high-moisture and dry maize

High-moisture (26·6–27·9% m.c.) and dry (9·8% m.c.) fractions of white and yellow maize were examined for fungal development and aflatoxin production during an 8-week incubation at 25°C. Treatment procedures included blending of either high-moisture white with dry yellow or high-moisture yellow with dry white maize fractions (average moisture in blend, 14%) and inoculation of some test maizes with A. flavus spores. At sampling time white and yellow components of maize blends were manually separated and all of the maize samples were analyzed for levels of moisture, fungal infection and aflatoxin. Moisture levels in maize blends equilibrated rapidly during the initial 2–4 days of incubation; neither dry yellow nor dry white exceeded 13% moisture during the trial period. Only a limited incidence of A. flavus was observed on uninoculated maize. but in samples treated with A. flavus spores a high infection rate developed; from 58 to 98% of the kernels in dry fractions of inoculated blends were infected with A. flavus during the trial. Aflatoxin was detected in high-moisture maize and in both high-moisture and dry fractions of inoculated maize blends. Up to 500 μg aflatoxin B₁/kg of corn was found after the 8-week incubation in a dry fraction of inoculated maize blends.     

Mycobiota and mycotoxin contamination of maize flours and popcorn kernels for human consumption commercialized in Spain

Mycobiota and co-occurrence of aflatoxins, citrinin, ochratoxin A and zearalenone in 30 samples of maize flours and 30 of popcorn kernels purchased in Spain for human consumption were determined. The mycotoxin-producing ability of Aspergillus, Fusarium and Penicillium spp. was also studied.     

Qualitative survey of fungi isolated from wine‐aging environment

The main topic of this paper is the study of the qualitative composition of fungal population in some cellar environments: superficial and inner layer of oak barrels, wall surfaces and aging wine. In addition, the influence of age of barrels and winery climatic conditions on fungal populations was evaluated. Two selected types of cellars were studied (a ground level and climate‐controlled cellar‐industrial winery and an unconditioned underground cellar‐family owned winery); 142 different strains were isolated and then identified through a morphology‐based method. Wood inner layer was characterised by the presence of few oak‐specialised endophytes (Trichoderma viride, Acremonium strictum and Cladosporium cladosporioides), whereas some strains of Aspergillus spp. and Penicillium spp. (principally Aspergillus nidulans, Aspergillus oryzae, Penicillium glabrum and Penicillium aurantiogriseum) connected with humidity and pollution were isolated. Aspergillus spp. and Penicillium spp. were isolated also in wall and barrel surfaces and in aging wine.     

The application of novel rotary plasma jets to inhibit the aflatoxin-producing Aspergillus flavus and the spoilage fungus,Aspergillus niger on peanuts

The major safety risk of peanuts is contamination with aflatoxin. Cold atmospheric plasma (CAP) has been demonstrated to inactivate fungi effectively. In this study, a novel CAP device with a rotary jet system was used to inactivate the existing A. flavus and A. niger on peanuts. The initial inoculation levels were 6.39 and 5.83 log CFU/g of A. flavus and A. niger, respectively. After treatments at 180 W for 7.5 min and 200 W for 5 min, A. flavus was not detected. For A. niger, the treatments at 180 W for 10 min and 200 W for 5 min resulted in undetected population. Observation under scanning electron microscope revealed the fungal spores were evidently damaged. The growth of A. flavus and aflatoxin concentrations were significantly lower (p < 0.05) on the group treated with 200 W for 5 min than other treatment groups stored for 29 d. Oil quality indexes of the CAP-treated peanuts were maintained in the range of excellent grades. This study demonstrated CAP effectively inhibited fungal growth and toxin production without adversely affected oil quality.     

Multiplex PCR assay for the detection of aflatoxigenic and non-aflatoxigenic fungi in meju, a Korean fermented soybean food starter

Aflatoxins are toxic secondary metabolites produced commonly by Aspergillus flavus and Aspergillus parasiticus. In this study, the possibility of using multiplex PCR was investigated to speed up and specify the detection of aflatoxigenic Aspergillus species in meju, a traditional Korean fermented soybean food starter. Two different sets of three primers were designed specifically for the omtB, ver-1, aflR, and omtA genes present in the aflatoxin biosynthesis cluster. The optimized multiplex PCR showed that only aflatoxigenic Aspergillus species gave three band patterns in both primer sets. The detection limits were determined as 125 pg/μl for genomic DNA from aflatoxigenic A. parasiticus KCCM 35078, and 10⁵ spores/g of meju sample for DNA extracted directly from meju. A total of 65 Aspergillus isolates from meju were tested for the presence of aflatoxigenic fungi by the application of multiplex PCR, and were analyzed by TLC and HPLC for the aflatoxin production in the culture filtrates. Results showed a good correlation between the presence of the aflatoxin biosynthesis genes analyzed by multiplex PCR and aflatoxin production by TLC and HPLC. This suggests that this multiplex PCR method may provide an accurate and specific detection of aflatoxigenic Aspergillus species in fermented soybean foods.     

Whole wheat and white wheat flour— the mycobiota and potential mycotoxins

Mold growth has detrimental effects on the quality of flour and may result in mycotoxin contamination. The search for potential mycotoxins—almost 400 are known—is time-consuming and expensive. However, detailed knowledge about the mycobiota and especially the toxin producing fungi enables the effective search for these toxic fungal metabolites. Therefore, a whole wheat flour and a white wheat flour (type 405) were investigated for their total qualitative as well as quantitative mycobiota. Overall, 51 species belonging to 14 different genera could be isolated. Total fungal counts of the whole wheat flour amounted to 1833 molds while the white wheat flour contained 1730 cfu 2 g⁻¹. The mycobiota of both flours was dominated by Aspergillus spp. accounting for 84% and 77·3% of the isolations, respectively. Fungi of the genus Penicillium spp. occurred only to a minor degree: 8% of the isolations in whole wheat flour and 15% in white wheat flour. Aspergillus candidus was the most frequently encountered mold.Penicillium aurantiogriseum , Cladosporium cladosporioides, A. flavus, Eurotium herbariorum,P. griseofulvum , P. brevicompactum and P. viridicatum were isolated to a lesser degree. From the 3563 identified fungi 93·3% (32 species) belong to the group of toxigenic molds.     

Lesser known sources of protein in some Nigerian peasant diets

The proximate mineral composition and indispensable amino acid pattern were determined for samples of Snail (Vivapara quadrata), periwinkle (Littorina littorea), crayfish (Palamonetes varians) and some locally smoked fresh water fish (Pisces spp.).The crude protein contents of smoked fish, crayfish, snail and periwinkle were 75·31, 74·84, 65·29 and 60·93% dry matter, respectively. These values were 56·9, 54·9, 35·2 and 26·1%, respectively higher than that of whole hen's egg. All the samples, except snail, also had higher ash values. Whole hen's egg, however, contained more crude fat (40·23%) and gross energy (34·74 Kj/g) than the test samples.The minerals calcium and phosphorus were higher in the test samples than in whole hen's egg. Samples analysed contained higher amounts of tryptophan than whole hen's egg and, with the exception of snail, the samples also contained more lysine. The test samples have high chemical scores ($\text{A}\text{E}$ ratios). Comparison has also been made of the indispensable amino acid patterns of these samples with those of cow's milk, human milk and the FAO (1957) provisional pattern.     

Aspergillus species from section Flavi isolated from soil at planting and harvest time in peanut‐growing regions of Argentina

Aspergillus species from section Flavi were isolated from soil samples in three peanut‐growing regions of Córdoba Province, Argentina. The samples were collected during the planting and harvest periods. Both total fungal population and Aspergillus species from section Flavi showed no significant differences between planting and harvest time in two of the regions evaluated. Only in one region were there significant differences in cfu g^?1 of total fungal population and Aspergillus species from section Flavi. A flavus was the dominant species isolated in all three localities during the planting and harvest periods. There were significant differences (p < 0.05) in the ratio of toxigenic and atoxigenic strains dependent on the period and the region evaluated. In one region, higher frequencies of toxigenic A flavus and A parasiticus in soil were found and a high contamination level of aflatoxins was detected in peanut seeds. Copyright © 2003 Society of Chemical Industry     

Multiplex Real‐Time PCR Method for Detection and Quantification of Mycotoxigenic Fungi Belonging to Three Different Genera

Abstract: Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real‐time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene‐producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON‐ and OTA‐producing fungi can be detected and quantified in a single reaction tube using this multiplex real‐time PCR method. Practical Application: This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal‐based foods.     

Isolation of Fungal Species from Fermentating Pearl Millet Gruel and Determination of their Antagonistic Activities against Indicator Bacterial Species

The mycological and physicochemical qualities (pH and titratable acidity) of fermenting pearl millet gruel were evaluated using routine methods. Several species of yeasts and moulds were isolated and the mould species identified based on their observable macroscopic and microscopic characteristics. The moulds identified included: Penicillium sp (FM1), Rhizopus spp (FM2, FM3 and FM6), Aspergillus flavus (FM7) and Aspergillus niger (FM8). These isolates were screened for production of antimicrobial compounds using agar well diffusion method. Escherichia coli, Staphylococcus aureus, Bacillus cereus, Bacillus lichieniformis, Salmonella spp., Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas syringae, Proteus sp. and Serratia sp. were utilized as indicator organisms. Secondary metabolites were also extracted from the respective moulds and the antimicrobial properties of these metabolites were tested against pure cultures of E. coli, S. aureus, P. flourescens and B. lichieniformis. All the moulds exhibited antimicrobial activity against P. flourescens while the metabolic extract of Aspergillus flavus (FM7) displayed the highest zone of inhibition (24 mm) against an overnight culture of P. flourescens.