Effect of temperature and water activity on growth and ochratoxin A production by Australian Aspergillus carbonarius and A. niger isolates on a simulated grape juice medium

The effect of water activity (0.92, 0.95, 0.965 and 0.98) and temperature (15 °C, 25 °C, 30 °C and 35 °C) on growth rate and ochratoxin A (OA) production by five strains of Aspergillus carbonarius and two strains of A. niger isolated from Australian vineyards was characterised on a synthetic grape juice medium. Maximum growth for A. carbonarius occurred at ca 0.965 a_w and 30 °C, and for A. niger, at ca 0.98 a_w and 35 °C. The optimum temperature for OA production was 15 °C and little was produced above 25 °C. The optimum a_w for toxin production was 0.95–0.98 for A. carbonarius and 0.95 for A. niger. Toxin was produced in young colonies after and, typically, did not continue to accumulate the entire surface area of the plate was colonised. Rather, the amount decreased as colonies aged. Trends for growth and OA production were similar among Australian isolates and those from European grapes, as reported in the literature.     

Aflatoxin formation and gene expression in response to carbon source media shift in Aspergillus parasiticus

Aflatoxins are toxic and carcinogenic polyketide metabolites produced by fungal species, including Aspergillus flavus and A. parasiticus. The biosynthesis of aflatoxins is modulated by many environmental factors, including the availability of a carbon source. The gene expression profile of A. parasiticus was evaluated during a shift from a medium with low concentration of simple sugars, yeast extract (YE), to a similar medium with sucrose, yeast extract sucrose (YES). Gene expression and aflatoxins (B₁, B₂, G₁, and G₂) were quantified from fungal mycelia harvested pre- and post-shifting. When compared with YE media, YES caused temporary reduction of the aflatoxin levels detected at 3-h post-shifting and they remained low well past 12 h post-shift. Aflatoxin levels did not exceed the levels in YE until 24 h post-shift, at which time point a tenfold increase was observed over YE. Microarray analysis comparing the RNA samples from the 48-h YE culture to the YES samples identified a total of 2120 genes that were expressed across all experiments, including most of the aflatoxin biosynthesis genes. One-way analysis of variance (ANOVA) identified 56 genes that were expressed with significant variation across all time points. Three genes responsible for converting norsolorinic acid to averantin were identified among these significantly expressed genes. The potential involvement of these genes in the regulation of aflatoxin biosynthesis is discussed.     

Inhibitory effects of anethole and eugenol on the growth and toxin production of Aspergillus parasiticus

The antifungal and antiaflatoxigenic activity of anethole and eugenol which are active components of commonly used spices was studied against two strains of Aspergillus parasiticus. Anethole, up to concentration of 400 μg/ml where complete inhibition was observed, delayed growth and reduced mycelial weight but it showed a stimulative effect on the toxin production of both strains. At a concentration of 300 μg/ml, eugenol inhibited the growth of both strains; levels of eugenol below 200 μg/ml enhanced production of aflatoxin particularly by A. parasiticus NRRL 299.     

Spectral analyses of fluorescences in dent maize infected with Aspergillus flavus and Aspergillus parasiticus

Bright greenish yellow (BGYF) and blue white (BWF) fluorescences were associated with Aspergillus flavus and A. parasiticus infected maize. The fluorescences were studied spectrofluorometrically, the BGYF exhibiting a peak wave length between 480–485 nm and the BWF between 440–445 nm. Neither fluorescence varied in maize stored under different moistures and temperatures.

BWF was similar spectrally to the fluorescence of the endosperm of sound kernels but × 5 20 more intense. The spectrum of BWF was similar to Aflatoxin G₁ or a mixture of aflatoxin B₁, B₂, G₁ and G₂ when they were spotted on endosperm tissue. A color reference for BGYF was similar in peak wave length to BGYF. Amsoy soybeans without the seed coat fluoresced with a peak 470–475 nm and the intensity was low compared to BGYF in maize. A fluorescence of maize kernels visually similar to BGYF but not associated with Aspergillus infection or aflatoxin contamination was also investigated. This “false BGY” fluorescence was spectrally similar to the BGYF in infected kernels.     

Mycotoxin occurrence and Aspergillus flavus soil propagules in a corn and cotton glyphosate-resistant cropping systems

The effects of cotton-corn rotation and glyphosate use on levels of soil-borne Aspergillus flavus, aflatoxin and fumonisin contamination in corn and cotton seed were determined during 2002-2005 in Stoneville, Mississippi (USA). There were four rotation systems (continuous cotton, continuous corn, cotton-corn and corn-cotton) for both glyphosate-resistant (GR) and non-GR cultivars-herbicide system arranged in a randomized complete block design with four replications. Aspergillus flavus populations in surface (5-cm depth) soil, sampled before planting (March/April), mid-season (June) and after harvest (September), ranged from 1.47 to 2.99 log (10) cfu g^-1 soil in the four rotation systems. Propagules of A. flavus were higher in the continuous corn system compared to the continuous cotton system on three sample dates, and cotton rotated with corn decreased A. flavus propagules in three of nine sample dates. Propagules of A. flavus were significantly greater in plots with GR cultivars compared to non-GR cultivars in three samples. In cotton seed, aflatoxin and fumonisin levels were similar (≤4 µg kg^-1 and non-detectable, respectively) regardless of rotation and glyphosate. In corn grain, aflatoxin was above the regulatory level (≥20 µg kg^-1) only in GR cultivar in 2004 and 2005. Fumonisin was higher in non-GR cultivar (4 mg kg^-1) regardless of rotation in 2004; however, in 2002, 2003 and 2005, aflatoxin and fumonisin levels were similar regardless of rotation and glyphosate. These results indicate the potential for increased aflatoxin and fumonisin levels (1 of 4 years) in corn; however, climatic conditions encountered during this study did not allow for mycotoxin production. In laboratory incubation studies, fairly high concentrations of glyphosate were required to inhibit A. flavus growth; however no short-term effect of soil treatment with glyphosate on A. flavus populations were observed. These data suggest that altered populations of A. flavus or higher aflatoxin concentrations in corn grain were due to indirect effects of the GR cropping system.     

Impact of habitats on toxigenic potential of Aspergillus flavus

Toxigenic potentials of Aspergillus flavus strains isolated from different organic substrates, air and soils were evaluated. The frequency of occurrence of A. flavus ranged from 76% in maize to 87% (coconut and groundnut) and 92% in makhana (Euryale ferox indica.) Incidence was lowest in green gram (42%). Analysis of variance showed that the percentage incidence of A. flavus in the aerosphere of maize fields was significantly affected by season × location. In soil samples the frequency of occurrence of A. flavus was high during the monsoons (76% in non-diara land soils, 69% in diara land soil). Among 1706 isolates of A. flavus obtained from different sources, 826 (48.4%) were found to be toxigenic. The frequency of non-toxigenic strains of A. flavus was comparatively higher (ratio = 1.07:1) than the toxigenic strains. Percentage incidence of the toxigenic strains of A. flavus was the highest (73.3%; ratio = 0.36:1) in the soil samples. No attempt was made to differentiate A. flavus and A. parasiticus and therefore all references to A. flavus include A. parasiticus.     

Some biological and chemical characteristics of damaged corn

The predominant fungi isolated from damaged kernels of corn (maize) plated on agar media were species of Aspergillus (mainly A. glaucus) and Penicillium. Masses of spores and mycelium of Aspergillus, Penicillium, Cladosporium and Fusarium were present in the cavities of the embryo of many kernels from which no fungi grew. Some samples had a high percentage of blue-eye kernels, evidently caused mainly by A. restrictus and A. glaucus. The embryos of all damaged kernels were decayed to some extent by fungi. Weight averaged 51 · 7 lb/bu, not much below that of sound corn. Fat acidity values ranged from 90–324. No aflatoxin was found in any of the 24 samples tested.     

Biodiversity of Aspergillus section Flavi in the United States: A review

Fungi belonging to Aspergillus section Flavi are of great economic importance in the United States due to their ability to produce toxic and carcinogenic aflatoxins in agricultural commodities. Development of control strategies against A. flavus and A. parasiticus, the major aflatoxin-producing species, is dependent upon a basic understanding of their diversity in agricultural ecosystems. This review summarizes our current knowledge of species and population diversity in the United States in relation to morphology, mycotoxin production and genetic characters. The high genetic diversity in populations of aflatoxigenic fungi is a reflection of their versatile habits in nature, which include saprotrophic colonization of plant debris in soil and parasitism of seeds and grain. Genetic variation within populations may originate from a cryptic sexual state. The advent of intensive monoculture agriculture not only increases population size but also may introduce positive selective pressure for aflatoxin production due to its link with pathogenicity in crops. Important goals in population research are to determine how section Flavi diversity in agricultural ecosystems is changing and to measure the direction of this evolution.     

Aspergillus flavus link and its occurrence in relation to other mycoflora on stored spices

Spices are used sparingly to flavour foods; their mycoflora and its possible role in food infection has not been thoroughly investigated. In an examination of 6 spices, Aspergillus flavus, other Aspergillus species, and species of Rhizopus, Mucor, Penicillium, Tricothecium, and Fusarium were found on seed surfaces. A. flavus which is implicated in the production of carcinogenic aflatoxin, was mainly found on ginger, mustard, garlic, and pepper. The highest fungal counts (10.6 × 10⁴/g) occurred in stored pepper (Piper nigrum L.) and the lowest in curry leaves (Murraya koenigii L.). Contamination of pepper and mustard with A. flavus seems to be related to the processing and storage of these spices.     

Population ecology of Aspergillus flavus associated with Mississippi Delta soils

Understanding the source of Aspergillus flavus is required to manage aflatoxin contamination of maize (Zea mays L.). Studies assessed A. flavus propagules, Fusarium spp., and total fungi associated with Mississippi Delta soils. Soils from 12 and 15 sites were collected in 2000 and 2001, respectively. The propagule density of A. flavus ranged from log(10) 2.0 to 4.3 colony-forming units (cfu) g^-1 soil, while total fusaria ranged from log(10) 3.0 to 5.4 cfu g^-1 soil. The highest populations of A. flavus were associated with soils containing higher organic matter, especially in sites under a no-tillage management. The frequency of aflatoxin production in isolates ranged from 13 to 81% depending on soil. In 2001, there was a highly significant correlation between A. flavus and the history of maize cultivation. Soil fertility factors such as organic matter content, nitrate and extractable phosphorus correlated with the density of Aspergillus, Fusarium spp., and total fungi. The relationship between soil parameters and Aspergillus populations may be useful in predicting the contribution of soil microflora to aflatoxin contamination.     

Validation of an overall model describing the effect of three environmental factors on the apparent D-value of Bacillus cereus spores

Several factorial models extending the famous Bigelow model to describe the influence of the heating and recovery pH and a_w conditions on bacterial heat resistance have been developed. These models can be associated in an overall multifactorial model describing the influences of heating and recovery conditions on D values. For Bacillus cereus strain ADQP 407 the model parameters characterising the environmental factor influences (pH, Temperature, a_w) were evaluated. Determination of bacterial heat resistance in cream chocolate have been realised to validate these parameter values and to evaluate the level of the influence of food texture or different compounds not taken account of in the model.     

Development of a PCR protocol to detect aflatoxigenic molds in food products

Aflatoxins are secondary metabolites produced mainly by Aspergillus species growing in foodstuffs. Because aflatoxins have important health effects, the detection of early contamination of foods by aflatoxigenic molds should be useful. In the present work, a reliable conventional PCR method for detecting aflatoxigenic molds of various species was developed. Fifty-six aflatoxigenic and nonaflatoxigenic strains commonly reported in foodstuffs were tested. Aflatoxin production was first confirmed by micellar electrokinetic capillary electrophoresis or/and high-pressure liquid chromatography-mass spectrometry. Based on the conserved regions of the O-methyltransferase gene (omt-1) involved in the aflatoxin biosynthetic pathway, six primer pairs were designed. With only the designed primer pair AFF1-AFR3, the expected PCR product (381 bp) was obtained in all of the tested aflatoxigenic strains of various species and genera. Amplification products were not obtained with this primer pair for any of the nonaflatoxigenic reference molds. However, an amplicon of 453 bp was obtained for all aflatoxigenic and nonaflatoxigenic mold reference strains with a PCR protocol based on the constitutive fungal β-tubulin gene, which was used as a positive fungal control. The PCR protocol based on omt-1 detected as little as 15 pg of DNA from aflatoxigenic molds and 10(2) to 10(3) CFU/g in contaminated food samples. This PCR protocol should be used as a routine technique to detect aflatoxigenic molds in foods.     

Survey of mycoflora counts, aflatoxin production and induced biochemical changes in walnut kernels

Fungal infection and mycotoxin contamination in fresh and stored kernels of walnut (Juglans regia) collected from different localities of Uttaranchal (India) were investigated. Fresh samples carry a combination of field as well as storage fungi. Species of Alternaria, Aspergillus and Penicillium were predominant. Thirty-nine percent of Aspergillus flavus isolates were toxigenic and produced up to 2170 μg/l of aflatoxin B₁ in the liquid media. Aflatoxin B₁ was the most common mycotoxin encountered as a natural contaminant in the stored samples. Twenty-one percent of fresh samples contained aflatoxin B₁ in low concentrations. The concentration of aflatoxin B₁ in fresh as well as stored samples was in the range of 140–1220 μg/kg. Characteristic rotting was observed in fresh as well as stored samples. The walnut kernels exhibited significant reductions in the levels of oil, starch and protein content during fungal infection.     

The normal mycoflora of commodities from Thailand. 1. nuts and oilseeds

A comprehensive study was carried out of the fungi occuring in commodities normally traded in Thailand. Samples of major commodities were obtained from farmers' stocks and middlemen in major producing areas throughout the country. Retail samples were obtained from outlets in and around Bangkok. Samples were divided into two portions, one being examined in Bangkok, and the second in Sydney. After surface disinfection, fungi were enumerated by direct plating on dichloran rose bengal chloramphenicol agar, dichloran 18% glycerol agar, Aspergillus flavus and parasiticus agar and dichloran chloramphenicol peptone agar. Figures for percentage infection were calculated, and fungi were isolated and identified to species level. In all 602 samples were examined, and at North Ryde about 18 000 fungal isolates identified. Data obtained from 329 samples are reported here, comprising maize (154), peanuts (109), cashews (45) and copra (21). Major fungi in maize included Fusarium moniliforme (present in 97% of samples), Aspergillus flavus (85%), Penicillium citrinum (67%), Aspergillus niger (64%), Lasiodiplodia theobromae (58%) and Fusarium semitectum (45%). In peanuts, the major fungi were Aspergillus flavus (95% of samples), Aspergillus niger (86%), Rhizopus oryzae (60%), Eurotium rubrum (51%), Macrophomina phaseolina (49%), Penicillium citrinum (46%) and Eurotium chevalieri (46%). Invasion in cashews was lower, major fungi being Aspergillus flavus (60%), Nigrospora oryzae (58%), Aspergillus niger (53%), Chaetomium globosum (47%) and Eurotium chevalieri (40%). Aspergillus flavus (86% of samples) was again dominant in copra, with Rhizopus oryzae (52%), Aspergillus niger (43%), Eurotium chevalieri (43%) the only other species exceeding 40% infection. Aspergillus parasiticus was rarely seen, and Aspergillus nomius was reported from foods for the first time.     

THE AFLATOXIN CONTAMINATION OF FIG FRUITS IN AYDIN CITY (TURKEY)

The mold flora of 50 dried fig samples consumed in Turkey was examined and the aflatoxigenic ones were determined. Among 127 fungi isolated, 74 were Aspergillus, 24 were Trichoderma, 16 were Fusarium and 13 were Acremonium. Of the isolates, 17 were aflatoxigenic and four of them were capable to produce aflatoxin, three of which were characterized as A. flavus and one as A. parasiticus. Aflatoxin production of four strains was confirmed by high pressure liquid chromotography. The effect of UV irradiation on mold count and aflatoxin quantity was also tested. It was found that UV irradiation led to a decrease in the mold count and aflatoxin quantity.

PRACTICAL APPLICATIONS

Studies have shown that the concentration of aflatoxins may exceed the determined limits in dried figs. Its presence can be a potential threat to the health of consumers. Dried figs are one of the major agricultural export products of Turkey ( Senyuva et al. 2005 ). The effects of UV irradiation on mold flora of dried figs and aflatoxins have been examined. The Aspergillus flavus and parasiticus agar (AFPA) medium is used for detection of aflatoxigenic species, and coconut agar medium (CAM) is used to detect the aflatoxin-producing ability of aflatoxigenic strains. It was found that the reproduction of the molds in dried figs, consequently the aflatoxigenic mold strains, can be depressed by UV irradiation. It was found that increasing time of UV irradiation led to a decrease in the mold count in dried figs. In addition, a UV irradiation applied for 90 min, was found to decrease the aflatoxin quantity in dried figs in an amount of 25%. Because of inexpensiveness and easiness of the application it was concluded that the UV irradiation can be used as a practical application.     

The effect of storage time and agroecological zone on mould incidence and aflatoxin contamination of maize from traders in Uganda

A study to determine mould incidence and aflatoxin contamination of maize kernels was carried out among dealers (traders) in the three agroecological zones of Uganda. The maize kernels were categorized into those stored for two to six months or for more than six months to one year. Results indicate that the mean moisture content of the kernels was within the recommended safe storage levels of ≤ 15% but was significantly lower in the Highland maize kernels followed by the Mid-Altitude (dry) kernels while the Mid-Altitude (moist) kernels had the highest levels. Across the agroecological zones, Aspergillus, Fusarium, Penicillium and Rhizopus were the most predominant fungal genera identified and, among their species, A. niger had the highest incidence, followed by A. flavus, F. verticillioides, A. wentii, A. penicillioides and Rhizopus stolonifer. There were more aflatoxin positive samples from the Mid-Altitude (moist) zone (88%) followed by those samples from the Mid-Altitude (dry) zone (78%) while samples from the Highland zone (69%) were least contaminated. Aflatoxin levels increased with storage time such that maize samples from the Mid-Altitude (dry and moist) stored for more than six months had mean levels greater than the 20 ppb FDA/WHO regulatory limits. Aflatoxin B₁ was the most predominant type and was found to contaminate maize kernels from all the three agroecological zones. These results indicate that maize consumers in Uganda are exposed to the danger of aflatoxin poisoning. Thus, there is the need for policy makers to establish and enforce maize quality standards and regulations related to moulds and aflatoxins across the agroecological zones to minimize health hazards related to consumption of contaminated kernels.     

Factors influencing fungal and aflatoxin levels in Turkish hazelnuts (Corylus avellana L.) during growth, harvest, drying and storage: A 3-year study

The levels aflatoxins in Turkish hazelnuts have been monitored over a 3-years period (2002-2004). Periodical sampling was made in 72 different orchards at different locations representative of the hazelnut-growing areas and post-harvest applications. Various parameters (aflatoxins, water activity, moulds) were analysed and environmental conditions (temperature and relative humidity) recorded during growing and at different stages of harvest and post-harvest processing, involving three different harvesting methods (collection in nets, from the ground, etc.) and four drying techniques (traditional sun-drying, mechanical drying, etc.). Fungal and aflatoxin analyses (HPLC) showed no significant difference except between samples which had been in contact with the ground and those which had not (at 95% confidence level). Aflatoxins levels from the orchard recorded a maximum of 0.77 ± 0.08 ng g^-1 from a total of 1624 samples. Regarding harvesting and post-harvest processes, the only application where aflatoxins were detected was in samples which had been in direct contact with the ground (max. 3.18 ± 0.03 ng g^-1). Aflatoxin formation was low during storage (max. 0.34 ± 0.003 ng g^-1). As a result of mycological studies, a total of 5546 Aspergillus flavus (89%) and A. parasiticus (11%) species were isolated and identified from samples. The results indicated that harvesting hazelnuts into a canvas by shaking the trees, manual harvesting of mature hazelnuts where possible, use of jute instead of nylon sacks and mechanical drying technique would minimize aflatoxin levels in hazelnuts. These recommendations have been implemented and about 4000 people in the hazelnut industry have been trained in these practices.     

Effect of gas barrier characteristics of films on aflatoxin production by Aspergillus flavus in peanuts packaged under modified atmosphere packaging (MAP) conditions

The effects of the gas barrier characteristics of three films (ASI, ASII and ASIII) and storage temperature on the growth of, and aflatoxin production by, Aspergillus flavus in peanuts packaged in air and under a modified gas atmosphere of CO₂:N₂ (65:35) were investigated. Mold growth was barely visible in air packaged peanuts using high-medium barrier films (ASI and ASII) and stored at 20°C with more extensive growth occurring in air packaged peanuts stored at 25 and 30°C. Extensive mycelial growth and sporulation occurred in all air packaged peanuts in a low barrier film (ASIII), especially at 30°C. Gas packaging inhibited mold growth in peanuts packaged in a high gas barrier film at 20°C. However, mold growth occurred in gas packaged peanuts packaged in film ASII at higher storage temperatures while extensive mycelial growth was observed in all peanuts packaged in film ASIII irrespective of storage temperature. Levels of aflatoxin greater than the regulatory limit of 20 ng/g were detected in all air packaged peanuts with the highest level of aflatoxin (76 ng/g) being detected in peanuts packaged in a high gas barrier film ASI. Aflatoxin production was inhibited in gas packaged peanuts using a high barrier film. However, higher levels of aflatoxin were detected in all gas packaged peanuts in medium-low gas barrier films (ASII and ASIII), particularly at higher storage temperatures. This study has shown that MAP using a CO₂:N₂ (65:35) gas mixture was effective in controlling aflatoxin production by A. flavus in peanuts to levels less than the regulatory limit of 20 ng/g. However, the antimycotic effect of low O₂-high CO₂ atmospheres is dependent on the gas barrier characteristics of the packaging films, especially at higher storage temperatures.     

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.     

Comparison of surface sampling methods and cleanability assessment of stainless steel surfaces subjected or not to shot peening

The cleanability of AISI 304 stainless steel surfaces, indicated by the removal of Escherichia coli cells or Aspergillus niger spores was assessed by controlled inoculation and washing treatment of samples in standardised conditions. Two systems of recapture (Rodac plate technique and swabbing technique) were compared. Four industrial finishes, subjected or not to shot peening, contaminated at low concentration (1–10 cfu/cm²), and then washed with distilled water or alkaline detergent, were examined. The Rodac plate technique detected most of microorganisms inoculated (80% for E. coli cells and 67% for A. niger spores), whereas the swabbing technique recovered only 1% of the E. coli cells and 26% of the A. niger spores. Using the Rodac plate technique E. coli cells proved to be easily detachable from samples either with distilled water (98%) or alkaline detergent (>99%). For the surfaces contaminated with A. niger spores, the cleanability increased from 34% with distilled water to 77% with alkaline detergent. In these working conditions type of finish (shot treated or not) had no significant effect on cleanability of stainless steel.