Effect of Cycling Temperatures on Aflatoxin Production by Aspergillus parasiticus and Aspergillus flavus in Rice and Cheddar Cheese

Initiation of growth, sporulation and aflatoxin production at cycling temperatures took less time than at 15°C but more than at 18°C and 25°C. A. parasiticus produced more aflatoxins on rice under cycling temperatures than at 25°C, 18°C or 15°C, while A. flavus produced less aflatoxin under cycling temperatures. A. parasiticus produced more aflatoxins on cheese under cycling temperatures than at 18°C or 15°C, but much less than at 25°C. A. flavus produced less aflatoxins on cheese under cycling temperatures than at 18°C and 25°C. Both organisms produced trace amounts of toxins at 15°C on cheese. Preincubation at 25°C for 2 days before temperature cycling did not increase aflatoxin production on rice but increased production on cheese. The rate of aflatoxin production on cheese decreased as the temperature decreased. No growth, sporulation or aflatoxin production was observed at 5°C on either rice or cheese.     

Aspergillus Parasiticus Growth and Aflatoxin Production on Dehydrated Taro

A study was made of Aspergillus parasiticus growth and aflatoxin production on four taro media. The critical equilibrium relative humidity (ERH) for natural mold growth on unsterilized dehydrated taro was 88% at 20°C. However, nontoxigenic A. parasiticus NRRL 1957 did not grow at this ERH on dehydrated raw taro incubated at 20°, 30°, or 40°C. Instead, the growth of A. parasiticus NRRL 1957 on dehydrated taro was optimum at 30°C and an ERH of 96%. Aflatoxin production by toxigenic A. parasiticus NRRL 2999 was investigated on four taro media under optimal growth conditions. Only moderate quantities of aflatoxins were produced by A. parasiticus NRRL 2999 on uncooked dehydrated taro, but cooking or supplementation with peptone stimualted mycelial growth and aflatoxin production slightly. Nevertheless, growth and aflatoxin production on cooked or peptone-supplemented taro media was low.     

Selected plant essential oils and their main active components,a promising approach to inhibit aflatoxigenic fungi and aflatoxin production in food

Recent research has showed that Aspergillus flavus and Aspergillus parasiticus are aflatoxigenic species that can become very competitive in the framework of climate change. Aflatoxins show carcinogenic, mutagenic, immunotoxic and teratogenic effects on human and animals. Effective and sustainable measures to inhibit these species and aflatoxins in food are required. Origanum vulgare and Cinnamomum zeylanicum essential oils (EOs) and their major active constituents, carvacrol and cinnamaldehyde, respectively, were assayed for inhibiting these species and aflatoxin production in maize extract medium under different environmental conditions. Doses of 10–1000 mg l^?1 were assayed and the effective doses for 50 (ED₅₀) and 90% (ED₉₀) growth inhibition were determined. The ED₅₀ of cinnamaldehyde, carvacrol, oregano EO, and cinnamon EO against A. flavus were in the ranges 49–52.6, 98–145, 152–505, 295–560 mg l^?1 and against A. parasiticus in the ranges 46–55.5, 101–175, 260–425 and 490–675 mg l^?1, respectively, depending on environmental conditions. In A. flavus treatments ED₉₀ were in the ranges 89.7–90.5, 770–860 and 820–>1000 mg l^?1 for cinnamaldehyde, carvacrol and cinnamon EO, and in A. parasiticus treatments in the ranges 89–91, 855–>1000 and 900–>1000 mg l^?1, respectively. ED₉₀ values for oregano EO against both species were >1000 mg l^?1. Growth rates of both species were higher at 37 than at 25°C and at 0.99 than at 0.96 a_w. Aflatoxin production was higher at 25 than at 37°C. Stimulation of aflatoxin production was observed at low doses except for cinnamaldehyde treatments. The effectiveness of EOs and their main constituents to inhibit fungal growth and aflatoxin production in contact assays was lower than in vapour phase assays using bioactive EVOH-EO films previously reported.     

Effect of synthetic antioxidants on stored maize grain mycoflora in situ

BACKGROUND: The influence of a mixture of butylated hydroxyanisole (BHA) and propyl paraben (PP) (each at a concentration of 20 mmol L^?1) on mycoflora and Aspergillus section Flavi populations in stored maize grain was evaluated. A survey of 120 maize samples was carried out from June to November 2005. RESULTS: The predominant populations in non‐treated (control) maize between the first and sixth sampling periods were Aspergillus section Flavi and Penicillium. Aspergillus flavus was the fungus most frequently isolated from both control and antioxidant‐treated kernels. All samples of control and antioxidant‐treated maize kernels were negative for aflatoxins during the 6 month storage period. Aspergillus flavus and A. parasiticus strains showed a variable ability to produce aflatoxins. The contribution of the strains to silo community toxigenicity was higher for A. flavus L (large) and S (small) strains in the fourth sampling period. CONCLUSION: Antioxidant treatment negatively affected natural maize mycoflora and Aspergillus section Flavi populations between the second and sixth months of storage. Copyright © 2007 Society of Chemical Industry     

Mould contamination and the influence of water activity and temperature on mycotoxin production by two aspergilli in melon seed

Both the moisture levels and the incidence of mould contamination recorded for shelled water melon seed samples (22) obtained from 9 markets were generally higher as those recorded for the unshelled seed samples. 16 fungi, mostly toxigenic, were isolated from the surface-disinfected mouldy seeds. Of these isolates, 7, 3 and 2 species belonged to Aspergillus, Penicillum and Fusarium genera, respectively, while Botryodiplodia, Rhizopus, Sclerotium and Syncephalastrum had one respresentative each. Production of aflatoxins (B₁, B₂) by 5 toxigenic strains of Aspergillus flavus and of ochratoxin A by 4 toxigenic strains of A. ochraceus in melon seed at varying water activity (a_w) and temperature levels were investigated. Of the a_w levels (0.65, 0.70, 0.80, 0.90 and 0.98) provided, toxins were detected only at and above 0.80 with the peak production recorded at either 0.90 or 0.98 a_w level. Whereas aflatoxins were produced and detected under all the test temperatures (15, 20, 25, 30, 35, and 40 °C), the elaboration of ochratoxin A was detected only as from 25 to 40 °C. Optimum temperature for toxin production by all the strains of the two fungi used was 30 °C.     

Aflatoxin-producing potential of Aspergillus flavus and Aspergillus parasiticus isolated from samples of smoked-dried meat

Over a period of three years 420 samples of various smoke-dried meat products, collected from individual households in different region of Croatia were analysed for the presence of aflatoxigenic strains of the Aspergillus flavus group. Strains of A. flavus and A. parasiticus were present in 17,8% of the samples, and aflatoxin-producing ability was tested in 75 strains. In relation to sequential method of aflatoxin detection, 5 of 8 isolates were found in the first step (fluorescence in aflatoxin-producing ability medium - APA) and all of them in the second step (extraction method from syntheses on moist shredded wheat - SW). A. flavus strains produced mainly aflatoxin B₁, and had various levels of toxigenicity (1.4–3.12 mg/kg). Some strains of A. parasiticus produced all four aflatoxins B₁ B₂ G₁ G₂, while the other ones produced AF B₁ + G₁ only, with concentrations of aflatoxins from 0.1 to 450 mg/kg.     

Modeling Growth Rate and Assessing Aflatoxins Production by Aspergillus flavus as a Function of Water Activity and Temperature on Polished and Brown Rice

Abstract: The aim of this study was to model the radial growth rate and to assess aflatoxin production by Aspergillus flavus as a function of water activity (a_w 0.82 to 0.92) and temperature (12 to 42 °C) on polished and brown rice. The growth of the fungi, expressed as colony diameter (mm) was measured daily, and the aflatoxins were analyzed using HPLC with a fluorescence detector. The growth rates were estimated using the primary model of Baranyi, which describes the change in colony radius as a function of time. Total of 2 secondary models were used to describe the combined effects of a_w and temperature on the growth rates. The models were validated using independent experimental data. Linear Arrhenius–Davey model proved to be the best predictor of A. flavus growth rates on polished and brown rice followed by polynomial model. The estimated optimal growth temperature was around 30 °C. A. flavus growth and aflatoxins were not detected at 0.82 a_w on polished rice while growth and aflatoxins were detected at this a_w between 25 and 35 °C on brown rice. The highest amounts of toxins were formed at the highest a_w values (0.90 to 0.92) at a temperature of 20 °C after 21 d of incubation on both types of rice. Nevertheless, the consistencies of toxin production within a wider range of a_w values occurred between 25 to 30 °C. Brown rice seems to support A. flavus growth and aflatoxin production more than the polished rice. Practical Application: The developed models can be used to estimate to what extent the change in grain ecosystem conditions affect the storage stability and safety of grains without the need for running long‐standing storage study. By monitoring the intergranular relative humidity and temperature at different locations in the storage facility and inputting these data into the models, it is directly possible to assess either the conditions are conductive for the growth of A. flavus or aflatoxin production.     

Some Factors Influencing Aflatoxin Production in Fermented Sausages

Aspergillus flavus NRRL-6549 and NRRL-6550 were tested for their ability to produce aflatoxins, but only NRRL-6549 produced detectable amounts of B₁. Summer sausages prepared and inoculated with NRRL-6549 were smoked and held at 10 or 30°C for 3 or 6 wk at low or high humidities. Mold growth occurred on both inoculated and uninoculated sausages, the amount increasing with time, temperature and humidity. Smoking delayed but did not prevent mold growth. At 3 wk, aflatoxins were detected only in unsmoked inoculated sausages held at 30°C and high relative humidity. After 6 wk, B₁ was found only in unsmoked inoculated sausages held at 10°C and high humidity, but at 30°C was present at 2.6–6.6 μg/kg in all inoculated samples at both low and high humidities.     

Polyphasic characterization of Aspergillus section Flavi isolated from animal feeds in Algeria

In Algeria, little information is available on the population structure of Aspergillus section Flavi in raw materials and resultant animal feeds. A total of 172 isolates belonging to Aspergillus section Flavi were recovered from 57 animal feeds and identified on the basis of macro and micro-morphological characters, mycotoxin production and genetic relatedness. For the molecular analysis, sequencing of the calmodulin gene (CaM) and the internal transcribed spacer (ITS) regions were performed for representative isolates. Four distinct morphotypes were distinguished: Aspergillus flavus (78.5%), Aspergillus tamarii (19.2%), Aspergillus parasiticus (1.7%), and Aspergillus alliaceus (0.6%). All A. flavus isolates were of the L type and no correlation between sclerotia production and aflatoxigenicity was observed. Our results showed that 68% of the A. flavus strains produced aflatoxins B (AFB), and 72.7% were cyclopiazonic acid (CPA) producers. The three isolates of A. parasiticus were able to produce AFB and aflatoxins G but not CPA whereas, all the strains of A. tamarii produced only CPA. The obtained results revealed the presence of different species of Aspergillus section Flavi, among which were aflatoxin producers. This study provides evidence useful for considerations in aflatoxin control strategies.     

Isolation, enumeration and PCR characterization of aflatoxigenic fungi from food and feed samples in India

A total of 54 market samples comprising nine different food and feed commodities from Mysore city were examined in order to isolate aflatoxin-producing fungi as well as to assess aflatoxins in the commodities. Thirty-two samples were contaminated with aflatoxigenic fungi and the total mycoflora and aflatoxigenic fungi in different food and feed commodities were in the range of 0.2–260 and 0–100 cfu×10³/g, respectively. In total, 136 fungi were isolated, of which 32 were Aspergillus flavus strains and 26 of them were found to produce aflatoxins. A. flavus group of fungi comprising A. flavus, A. parasiticus, A. oryzae, A. sojae were characterized by using Aspergillus differential medium and PCR. The PCR was performed using two different sets of primers specifically targeted to aflR and omt genes of aflatoxin biosynthesis pathway. Most of the fungi belonging to A. flavus group reacted positively with the primers resulting in expected size amplicons of 796 bp for aflR and 404 bp for omt. Among the nine commodities screened for aflatoxin only, groundnut and groundnut cake were contaminated with aflatoxins B₁ and B₂. The aflatoxin contamination in these commodities exceeded the Indian regulatory limit of 30 μg/kg.     

Influence of Temperature, pH, Water Activity and Antifungal Agents on Growth of Aspergillus flavus and A. parasiticus

Effect of temperature, pH, water activity, and nine antifungal agents on growth of Aspergillus flavus and A. parasiticus was determined on Sabouraud-Dextrose Agar and on corn. Maximal growth of the two molds occurred at 33°C, the highest temperature used, pH of 5.0 and a_w of 0.99. At 15°C, growth was observed at a_w of 0.95 but not 0.90. Slight growth was observed at an a_w, of 0.85 at 27°C and 33°C. Nine antifungal agents (Botran, Orthocide, Poly-ram 80, Topsin-M, Thiram, Imazalil, sodium propionate, sodium sulfite and DDVP) were tested for inhibition of growth. Activity of the antifungals increased as the a_w was decreased. All antifungals showed inhibitory activity, but Imazalil and DDVP were the most effective agents at the lowest concentrations.     

Influence of temperature on growth rate and lag phase of fungi isolated from Argentine corn

The influence of temperature on the growth of nine strains of fungi belonging to the genera Eurotium, Aspergillus, Penicillium and Fusarium has been investigated for the temperature range 15–35° C. The lag phase and the growth rate were evaluated by using a laboratory medium. The maximum growth rate for E. repens, A. wentii and P. chrysogenum was observed at about 25° C, for P. citrinum near 30° C and for F. semitectum and F. moniliforme between 20 and 25° C. The growth rate of A. niger, A flavus and A. parasiticus increased with increasing temperatures in the range studied. For all strains studied it appeared that the higher the growth rate the lower the lag phase was.     

Aflatoxigenic fungi and aflatoxin in cocoa

This paper reports the occurrence of aflatoxigenic fungi and the presence of aflatoxins in 226 cocoa samples collected on Brazilian farms. The samples were taken at various stages of fermentation, drying and storage. A total of 819 potentially aflatoxigenic fungi were isolated using Dichloran 18% Glycerol agar after surface disinfection, and identified by standard techniques. The ability of the fungi to produce aflatoxins was determined using the agar plug technique and TLC. The presence of aflatoxins in cocoa samples was determined by HPLC using post-column derivatization with bromide after immunoaffinity column clean up. The aflatoxigenic fungi isolated were Aspergillus flavus, A. parasiticus and A. nomius. A considerable increase in numbers of these species was observed during drying and storage. In spite of the high prevalence of aflatoxigenic fungi, only low levels of aflatoxin were found in the cocoa samples, suggesting the existence of limiting factors to the accumulation of aflatoxins in the beans.     

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.     

Production of cyclopiazonic acid by aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus

96 strains of Aspergillus flavus isolated from samples of stored grain and smoke-dried meat products were examined for ability to produce cyclopiazonic acid and aflatoxins, grown on mycological broth medium and malt extract agar. Five strains produced cyclopiazonic acid in the range of 0.5 — 30 mg/kg and 9 produced aflatoxin B1 (0.1 — 14.8 mg/kg) but none of them produced both cyclopiazonic acid and aflatoxins.     

Occurrence of aflatoxins in milk thistle herbal supplements

Milk thistle (MT) dietary supplements are widely consumed due to their possible liver-health-promoting properties. As botanicals they can be contaminated with a variety of fungi and their secondary metabolites, mycotoxins. The aflatoxigenic fungus Aspergillus flavus has been previously isolated from these commodities. Currently, there is no published method for determining aflatoxins (AFs) in MT. Therefore, a liquid chromatography (LC) method validated for aflatoxin analysis in botanicals was evaluated and applied to MT. The method consisted of acetonitrile/water extraction, immunoaffinity column clean-up, LC separation, post-column photochemical reaction derivatisation and fluorescence detection. The average recoveries for AFs added to MT seeds, herb, oil-based liquid extract and alcohol-based liquid extract were 76% or higher. The mean relative standard deviation was <10%. The limit of detection (LOD) was 0.01?µg kg^?1 and the limit of quantification (LOQ) was 0.03?µg kg^–1. The method was used to conduct a small survey. A total of 83 MT samples from the US market were analysed. AFs were detected in 19% of the samples with levels ranging from 0.04 to 2.0?µg kg^–1. Additionally, an aflatoxigenic A. flavus strain from ATTC and an A. parasiticus strain isolated from MT herb powder were found to produce high amounts of aflatoxins (11,200 and 49,100?µg kg^–1, respectively) when cultured in MT seed powder. This is the first study reporting on aflatoxin contamination of MT botanical supplements and identifying methodology for AF analysis of these commodities.     

The production of aflatoxins in fresh date fruits and under simulated storage conditions

Sixteen varieties of date fruit (Phoenix dactylifera) at three stages of maturation (Kimri, Rutab and Tamr) were examined for the presence of fungi and analysed for aflatoxins B₁, B₂, G₁ and G₂ and sterigmatocystin. Single samples of each variety were used in the study. Samples as received were initially examined for mycoflora and toxin levels and then stored at 98% relative humidity and 30 °C for 14 days to investigate the effects of possible adverse storage conditions on mycoflora and, in particular, aflatoxin formation. All samples showed an absence of aflatoxins and their precusor, sterigmatocystin, after adverse storage for 14 days, although aflatoxin‐producing Aspergillus flavus isolates were identified in 10 varieties at the first stage of maturation (Kimri). High fungal counts were associated with the Rutab stage and low counts with the Tamr stage. The counts of A flavus ranged from 5.00 to 8.16 log₁₀(cfu g^?1) under simulated storage conditions, and three varieties contained significant levels of aflatoxin B₁ or B₂ ranging from 35 to 11 610 µg kg^?1. Sterigmatocystin was not detected in any of the samples as received or under simulated storage conditions. © 2002 Society of Chemical Industry     

Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR

Aflatoxins are important mycotoxins that represent a serious risk for human and animal health. These mycotoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus, two closely related species with different array of aflatoxins. In this work, two specific quantitative PCR (qPCR) assays were developed to detect and quantify both species in wheat flour using primers based on the multicopy ITS2 rDNA target sequence. The species specificity of the assays was tested in a wide range of strains of these species and others colonizing the same commodities. The sensitivity of the assay was estimated in 2.5 pg/reaction in both species. Discrimination capacity for detection and relative quantification of A. flavus and A. parasiticus DNA were analyzed using samples with DNA mixtures containing also other fungal species at different ratios. Both qPCR assays could detect spore concentrations equal or higher than 10⁶ spores/g in flour samples without prior incubation. These assays are valuable tools to improve diagnosis at an early stage and in all critical control points of food chain integrated in HACCP strategies.     

Aspergillus Section Flavi and aflatoxins in dusts generated by agricultural processing facilities in the Philippines

Relatively few data exist regarding concentrations of aflatoxins and their causative organisms in dusts within occupational environments. Here, we examined Aspergillus Section Flavi populations and aflatoxin levels in 54 samples of dusts generated by agricultural processing facilities as possible indicators of aflatoxin exposure in the Philippines. The average incidence of Aspergillus Section Flavi expressed as a percentage of total mould populations in rice dust, corn dust, feed dust and copra dust were 8, 4, 31 and 10%, respectively. Predominant aflatoxigenic fungi isolated were Aspergillus flavus and A. parasiticus with ratios of 31:1, 40:5, 16:4 and 1:1 in rice dust, corn dust, feed dust and copra dust, respectively. Aflatoxins produced by selected isolates in in vitro rice culture ranged from 100 µg kg^?1 to 100.5 mg kg^?1. Toxigenicity of isolates based on the average aflatoxin concentrations produced by positive isolates were in the order of copra dust > corn dust > rice dust > feed dust. Average natural concentrations of aflatoxins in rice dust, corn dust, feed dust, and copra dust were 25, 6, 15 and 10 µg kg^?1, respectively. Estimates of the amount of inhaled aflatoxins by workers in an 8‐h work shift ranged from 0.06 to 114 ng, the average of which is higher than the amount of aflatoxins ingested by Filipinos due to the consumption of polished rice. The presence of highly toxigenic Aspergillus Section Flavi and aflatoxins in agricultural dust is a critical health risk for workers, considering the frequency of exposure and the possibility of inhalation and subsequent absorption of aflatoxins in the respiratory tract. Copyright © 2006 Society of Chemical Industry     

Spoilage moulds and aflatoxins from poultry feeds

Aflatoxin producing strains of Aspergillus flayus Link (IMI 280819) and A. oryzae (Ahlb.) Cohn (IMI 280831) were among the eleven spoilage moulds isolated from five types of poultry feeds. The recorded pH and moisture content values of the various feeds are conducive to mould deterioration. All the four principal aflatoxins (B₁, B₂, G₁, G₂) were detected in the analysed feeds though at toxicologically ‘safe’ levels for most farm animals. Significant quantities of aflatoxin B₁ were produced by the two fungal isolates in all the five classes of poultry feeds with A. flavus yielding the larger amounts. Optimum aflatoxin B₁ production and mycelial growth in chick mash infusion medium were recorded for both species at 30 and 35 °C, respectively and similarly on the 8th and 6th day respectively when cultures were incubated at 30 °C.