Effect of spice essential oils on growth and aflatoxin B1 production by Aspergillus flavus

Aflatoxin B¹ production by Aspergillus flavus was studied in yeast extract sucrose broth in the presence of cinnamon, clove, almond and cardamom oils. Growth and aflatoxin B₁ production was inhibited by 0.5 μl cinnamon oil ml^-1 medium and by 1 μl clove oil ml_-1. Almond and cardamom oils only affected growth when their concentration exceeded 1.25 μl ml^-1 medium. Aflatoxin B₁ production was stimulated by 0.75 and 1 μl almond oil ml^-1 medium or by 0.25 and 0.5 μl cardamom oil ml^-1.     

Survey of fungal counts and natural occurrence of aflatoxins in Malaysian starch-based foods

In a survey of starch-based foods sampled from retail outlets in Malaysia, fungal colonies were mostly detected in wheat flour (100%), followed by rice flour (74%), glutinous rice grains (72%), ordinary rice grains (60%), glutinous rice flour (48%) and corn flour (26%). All positive samples of ordinary rice and glutinous rice grains had total fungal counts below 103 cfu/g sample, while among the positive rice flour, glutinous rice flour and corn flour samples, the highest total fungal count was more than 103 but less than 104 cfu/g sample respectively. However, in wheat flour samples total fungal count ranged from 102 cfu/g sample to slightly more than 104 cfu/g sample. Aflatoxigenic colonies were mostly detected in wheat flour (20%), followed by ordinary rice grains (4%), glutinous rice grains (4%) and glutinous rice flour (2%). No aflatoxigenic colonies were isolated from rice flour and corn flour samples. Screening of aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 using reversed-phase HPLC were carried out on 84 samples of ordinary rice grains and 83 samples of wheat flour. Two point four percent (2.4%) of ordinary rice grains were positive for aflatoxin G1 and 3.6% were positive for aflatoxin G2. All the positive samples were collected from private homes at concentrations ranging from 3.69–77.50 μg/kg. One point two percent (1.2%) of wheat flour samples were positive for aflatoxin B1 at a concentration of 25.62 μ};g/kg, 4.8% were positive for aflatoxin B2 at concentrations ranging from 11.25–252.50 μg/kg, 3.6% were positive for aflatoxin G1 at concentrations ranging from 25.00–289.38 μg/kg and 13.25% were positive for aflatoxin G2 at concentrations ranging from 16.25–436.25 μg/kg. Similarly, positive wheat flour samples were mostly collected from private homes. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of clove and cinnamon oils on growth of and aflatoxin production by Aspergillus flavus

The effect of different concentrations of clove and cinnamon oils was studied on the growth of and aflatoxin production by Aspergillus flavus in SMKY liquid medium. The effect of these compounds was also verified against aflatoxin production in maize. Significant reduction (P < 0.05) in the elaboration of aflatoxin in liquid culture after treatment with more than 100 μg ml^-1 of these compounds was recorded. Cinnamon oil exhibited maximum inhibitory action and reduced 78% aflatoxin formation on maize at 1000 mg kg^-1.     

Inhibition of fungal growth and infection in maize grains by spice oils

Essential oils of 12 spices were tested for inhibitory activity against fungal infection and mycelial growth in post-harvest maize grains during storage. It was observed that the oils of cassia, clove (30 μl/g grain and above), star-anise (40 μl/g grain and above, geranium (30 μl/g grain and above) and basil (50 μl/g grain) inhibit the in viuo mycelial growth of established seed-borne infections of Aspergillus flavus, Curvularia pallescens and Chaetomium indicum as well as preventing infection following inoculation with A. flavus, A. glaucus, A. niger and A. sydowi . These oils also preserved the grain from natural A. flavus infection during the experimental period. Nutmeg, ginger and cumin oil (all at 50 μ1/g grain) could check mould growth and grain infection for only a brief period.     

Aspergillus parasiticus growth and aflatoxin production on black and white pepper and the inhibitory action of their chemical constituents

Aspergillus parasiticus Speare NRRL 2999 growth and aflatoxin production in black and white pepper and the penetration of the fungus in black pepper corn over various incubation periods were studied. Also, the effects of piperine and pepper oil on growth and aflatoxin production were studied. Under laboratory conditions, black and white pepper supported aflatoxin production (62.5 and 44 ppb (ng/g), respectively) over 30 days of incubation. Fungal growth measured in terms of chitin was considerably less in white pepper than in black pepper. A histological study of black pepper corn showed the fungus penetrating up to the inner mesocarp and establishing itself in the middle mesocarp. Piperine and pepper oil were found to inhibit fungal growth and toxin production in a dose-dependent manner. Thus, both black and white pepper could be considered as poor substrates for fungal growth and aflatoxin production.     

Aspergillus parasiticus growth and aflatoxin production on black and white pepper and the inhibitory action of their chemical constituents.

Aspergillus parasiticus Speare NRRL 2999 growth and aflatoxin production in black and white pepper and the penetration of the fungus in black pepper corn over various incubation periods were studied. Also, the effects of piperine and pepper oil on growth and aflatoxin production were studied. Under laboratory conditions, black and white pepper supported aflatoxin production (62.5 and 44 ppb (ng/g), respectively) over 30 days of incubation. Fungal growth measured in terms of chitin was considerably less in white pepper than in black pepper. A histological study of black pepper corn showed the fungus penetrating up to the inner mesocarp and establishing itself in the middle mesocarp. Piperine and pepper oil were found to inhibit fungal growth and toxin production in a dose-dependent manner. Thus, both black and white pepper could be considered as poor substrates for fungal growth and aflatoxin production.     

Effect of selected inhibitors on growth,pigmentation, and aflatoxin production by Aspergillus parasiticus

We have treated a wild type strain of Aspergillus parasiticus with several known aflatoxin inhibitors in hopes of finding specific metabolic blocks in the aflatoxin biosynthetic pathway. In defined medium, benzole acid (2 and 3 mg/ml), cinnamon (1 mg/ml), and sodium acetate (5 mg/ml) were fungitoxic. Benzoic acid (0.5 and 1 mg/ml), chlorox (5 l/ml), and dimethyl sulfoxide (5 l/ml) did not affect dry weight or mycelial pigmentation. Sodium benzoate (1, 2, 4 and 8 mg/ml) added after 2 days growth inhibited aflatoxin production in two defined media. We were unable to confirm previously published reports that an uncharacterized yellow pigment accumulates with benzoate-inhibition of aflatoxin biosynthesis.     

Inhibitory Effect of Essential Oils on Aspergillus ochraceus Growth and Ochratoxin A Production

Ochratoxin A (OTA) is a mycotoxin which is a common contaminant in grains during storage. Aspergillus ochraceus is the most common producer of OTA. Essential oils play a crucial role as a biocontrol in the reduction of fungal contamination. Essential oils namely natural cinnamaldehyde, cinnamon oil, synthetic cinnamaldehyde, Litsea citrate oil, citral, eugenol, peppermint, eucalyptus, anise and camphor oils, were tested for their efficacy against A. ochraceus growth and OTA production by fumigation and contact assays. Natural cinnamaldehyde proved to be the most effective against A. ochraceus when compared to other oils. Complete fungal growth inhibition was obtained at 150–250 µL/L with fumigation and 250–500 µL/L with contact assays for cinnamon oil, natural and synthetic cinnamaldehyde, L. citrate oil and citral. Essential oils had an impact on the ergosterol biosynthesis and OTA production. Complete inhibition of ergosterol biosynthesis was observed at ≥100 µg/mL of natural cinnamaldehyde and at 200 µg/mL of citral, but total inhibition was not observed at 200 µg/mL of eugenol. But, citral and eugenol could inhibit the OTA production at ≥75 µg/mL and ≥150 µg/mL respectively, while natural cinnamaldehyde couldn’t fully inhibit OTA production at ≤200 µg/mL. The inhibition of OTA by natural cinnamaldehyde is mainly due to the reduction in fungal biomass. However, citral and eugenol could significant inhibit the OTA biosynthetic pathway. Also, we observed that cinnamaldehyde was converted to cinnamic alcohol by A. ochraceus, suggesting that the antimicrobial activity of cinnamaldehyde was mainly attributed to its carbonyl aldehyde group. The study concludes that natural cinnamaldehyde, citral and eugenol could be potential biocontrol agents against OTA contamination in storage grains.     

Production of aflatoxin in corn by a large-scale solid-substrate fermentation process

Culturing of Aspergillus flavus was conducted in static flask cultures and 4 in. × 5 ft columns (containing 7–8 kg corn) to measure the effects of moisture, temperature, and air flow upon growth and the production of aflatoxin. Aflatoxin levels as high as 6200 ppb (dry basis) in 10 days were observed. Conditions were selected (ca. 20% moisture, 0.008 liter air/kg corn/min air flow with 1.5 liter/kg/min recirculated) for production of aflatoxin in 1200 bushels of corn in a 18-ft diam corrugated steel Butler storage bin for preparation of contaminated corn for animal feeding trials and for testing of an ammoniation process for decontamination of aflatoxin in corn. A target level of 1000 ppb aflatoxin was attained.     

Effects of Moisture Content and Temperature on Aflatoxin Production in Corn

Samples of freshly harvested and remoistened corn, of various moisture contents, were stored at different temperatures; analyses for aflatoxin content were made periodically. At moisture levels above 17.5% and at temperatures of 24 C or warmer, aflatoxins were formed by Aspergillus flavus present in the original epiphytic mycoflora. Remoistened dried corn was subject to more rapid fungal deterioration and aflatoxin formation than freshly harvested corn. Screening of the fungi present in the corn revealed aflatoxin production only by A. flavus. The toxigenic strains produced only aflatoxins B(1) and B(2).     

Effect of climate and type of storage container on aflatoxin production in corn and its associated risks to wildlife species

The effects of grain storage containers on aflatoxin production, and the relationship between the level of aflatoxin and the number and weight of fluorescing kernels were determined in corn (Zea maize) stored in controlled climate regimes. Two hundred and forty 100-g samples were held up to 3 mos using four types of storage containers placed in four climates. Storage containers included corn placed in metal cans, paper bags, plastic bags, and paper bags placed in plastic bags. Climates were constant during the duration of the project and included a combination of temperatures and humidities. Temperatures were 29-32 C and 14-18 C; relative humidities were 85-88% and 35-40%. In addition, corn was exposed to environmental conditions conductive for aflatoxin production and 100 g samples were randomly collected, examined under ultraviolet light for fluorescence, and then quantified for aflatoxin levels. Corn samples tested negative for aflatoxin at the beginning of the project. Main (i.e., container, climate, and month) and interactive effects were not observed. Mean levels of aflatoxin ranged from 0 to 151 microg/kg. Aflatoxin was produced regardless of type of storage container, time of storage, and climatic conditions; however, only 8% of the samples produced aflatoxin levels that exceeded 50 microg/kg. Fluorescing corn ranged from 0 to 19 kernels per sample, while aflatoxin levels ranged from 0 to 1,375 microg/kg for the same samples. No relationships were found between the number and weight of fluorescing kernels of corn and aflatoxin levels. The black light test yielded a false negative rate of 23% when in fact the aflatoxin concentrations exceeded 50 microg/kg. Therefore, quantifying fluorescing grain under UV light should not be considered a feasible alternative for aflatoxin testing of grain intended for wildlife.     

Storage of pollen grains of Crotalaria retusa in oils

Summary Attempts were made to store pollen grains of Crotalaria retusa L. in a mineral oil (paraffin oil) and two vegetable oils (soybean oil and olive oil). Under laboratory conditions pollen grains not stored in oil lost in vitro germinability within 15–30 days, while those stored in oils maintained some degree of germinability even after 60 days. Pollen samples stored in oils at –20° C did not show any decline in germinability or pollen tube vigour even after 6 months of storage. The results amply demonstrate the feasibility of using oils for short- and long-term pollen storage.     

Occurrence of aflatoxin in some liver curative herbal medicines

Fifty herbal medicine samples of seven different taxa known to cure liver disorders were analysed for aflatoxin contamination. Twenty-three samples, out of 50, were contaminated with various levels of aflatoxins. Amongst the 23 contaminated samples the maximum level of aflatoxin B¹ recorded was 2.23 μg g^-1 in Asparagus racemosus and the minimum 0.28 μg g^-1 in Emblica officinalis . Aflatoxin G¹ was only found in one species, Terminalia belarica . Aflatoxin production of the isolates of Aspergillus flavus was also examined and the highest levels were produced by isolates from A. racemosus (1.07-2.47 μg ml^-1). Aflatoxin contamination of herbal drugs may be a risk for patients because the level of aflatoxins is much higher than the tolerance level fixed by the WHO for foods.     

Production of lactic acid and fungal biomass by Rhizopus fungi from food processing waste streams

This study proposed a novel waste utilization bioprocess for production of lactic acid and fungal biomass from waste streams by fungal species of Rhizopus arrhizus 36017 and R. oryzae 2062. The lactic acid and fungal biomass were produced in a single-stage simultaneous saccharification and fermentation process using potato, corn, wheat and pineapple waste streams as production media. R. arrhizus 36017 gave a high lactic acid yield up to 0.94–0.97 g/g of starch or sugars associated with 4–5 g/l of fungal biomass produced, while 17–19 g/l fungal biomass with a lactic acid yield of 0.65–0.76 g/g was produced by the R. oryzae 2062 in 36–48 h fermentation. Supplementation of 2 g/l of ammonium sulfate, yeast extract and peptone stimulated an increase in 8–15% lactic acid yield and 10–20% fungal biomass.

     

Comparative study on concentrations of deoxynivalenol and zearalenone in kernels of transgenic Bt maize hybrids and nontransgenic maize hybrids

A survey of aflatoxin contamination in selected Colombian foods was conducted over a 12-month period on a total of 248 samples. Samples were collected in supermarkets, retail stores and stock centres and were grouped into five categories: (1) corn and corn products, (2) cereal grains, (3) rice and rice products, (4) legume seeds; and (5) snacks and breakfast cereals. Aflatoxins were identified and quantitated using a liquid chromatographic technique with a limit of detection of 1 ng/g for each aflatoxin. Aflatoxins were detected in 14 of 109 samples of corn and corn products, 4 of 40 samples of rice and rice products, 2 of 30 samples of legume seeds, and 2 of 11 samples of snacks and breakfast cereals. None of the cereal grains samples analysed contained detectable levels of aflatoxins. Twelve of the total of 22 positive samples exceeded the maximum tolerable level of aflatoxin B₁ adopted in most countries (5 ng/g); 10 of these 12 samples corresponded to corn and corn products. The results of the present study indicate that aflatoxin B₁ contamination in certain foods in Colombia is a major public health concern. Continuous monitoring of aflatoxin B₁ levels in Colombian foods is advised.     

Determinatıon of ochratoxin A and total aflatoxin levels in corn samples from Turkey by enzyme-linked immunosorbent assay

Forty-seven samples of corn were collected from various street bazaars and market outlets in different regions of Turkey and total aflatoxin (AF) and ochratoxin A (OTA) levels were determined by enzyme-linked immunosorbent assay (ELISA) following sample preparation. Levels of AF and OTA in corn samples ranged between 1.75–120.3 μg/kg and 1.08–8.57 μg/kg, respectively. Although 53% of the samples analysed had no detectable levels of AF, 4% of similar samples were found to contain AFs above the acceptable limit of 10 μg/kg in Turkey. For OTA, 4% of the corn samples had levels above the acceptable limit (3 μg/kg) in Turkey, with over 43% samples not found to contain this mycotoxin. Although the levels of mycotoxins analysed in this study were not found to be high and the percentage of samples contaminated above permitted limits were low, the importance of overall daily dietary intake should not be underestimated and control of these fungal metabolites in corn must be explored to minimise the hazards they may cause in humans.     

Effect of phytate on aflatoxin formation by Aspergillus parasiticus grown on different grains

Aflatoxin production by Aspergillus parasiticus on corn, soybean, and cottonseed in the absence or presence of added sodium phytate was examined. No variation in aflatoxin concentrations was found in raw, chemically sterilized, or autoclaved soybeans whereas a five-fold reduction in total aflatoxins was found in cottonseed after addition of 330 g sodium phytate to 10 g of autoclaved material. However, phytate did not affect aflatoxin production on non-sterile cottonseeds, although in corn a slight inhibition was found. Extraction of raw soybeans with hexane allowed production of 20-fold more aflatoxins, but levels were still lower than those found on rice or corn. Part of this relative inhibition in soybeans may arise from a heat-unstable, polar solvent-soluble, dialyzable factor present in soybeans. Our results support the conclusion that phytate is not the factor in soy responsible for its relative resistance to aflatoxin formation.     

Aflatoxin content and number of fungi in poultry feedstuffs from Indonesia

The content of aflatoxin and associated fungi was determined in 56 samples, including 34 of corn, 10 of soybean meal, nine of rice bran and three of broken rice, collected from different poultry farms and poultry feedmills situated around Jakarta-Bogor, Indonesia.
Ninety-one per cent of the corn samples contained aflatoxins and the total concentration ranged from 22 to 6171 μ g/kg. With rice bran, 100% of the samples were positive for aflatoxin B₁, ranging from 36 to 71 μ g/kg. No aflatoxin was detected in samples of soybean meal or broken rice. All the samples were contaminated by several fungi (8 times 10³–5 times 10⁶cfu/g) and further identification was limited to Aspergillus flavus and A. parasiticus. The dominant species was A. flavus (2 times 10³–4 times 10⁶cfu/g in corn samples, 1·0 times 10³–1·0 times 10⁵cfu/g in soybean meal, 2 times 10⁴–4·4 times 10⁵cfu/g in rice bran and 2 times 10⁴–6 times 10⁴cfu/g in broken rice). Some of the corn samples also contained A. parasiticus (2 times 10³–9·5 times 10⁴cfu/g).     

Occurrence of aflatoxins and its management in diverse cropping systems of central Tanzania

The staple crops, maize, sorghum, bambara nut, groundnut, and sunflower common in semi-arid agro-pastoral farming systems of central Tanzania are prone to aflatoxin contamination. Consumption of such crop produce, contaminated with high levels of aflatoxin B₁ (AFB₁), affects growth and health. In this paper, aflatoxin contamination in freshly harvested and stored crop produce from central Tanzania was examined, including the efficacy of aflatoxin mitigation technologies on grain/kernal quality. A total of 312 farmers were recruited, trained on aflatoxin mitigation technologies, and allowed to deploy the technologies for 2 years. After 2 years, 188 of the 312 farmers were tracked to determine whether they had adopted and complied with the mitigation practices. Aflatoxigenic Aspergillus flavus and aflatoxin B1 contamination in freshly harvested and stored grains/kernels were assessed. A. flavus frequency and aflatoxin production by fungi were assayed by examining culture characteristics and thin-layer chromatography respectively. AFB₁ was assayed by enzyme-linked immunosorbent assay. The average aflatoxin contamination in freshly harvested samples was 18.8 μg/kg, which is above the acceptable standard of 10 μg/kg. Contamination increased during storage to an average of 57.2 μg/kg, indicating a high exposure risk. Grains and oilseeds from maize, sorghum, and sunflower produced in aboveground reproductive structures had relatively low aflatoxin contamination compared to those produced in geocarpic structures of groundnut and bambara nut. Farmers who adopted recommended post-harvest management practices had considerably lower aflatoxin contamination in their stored kernels/grains. Furthermore, the effects of these factors were quantified by multivariate statistical analyses. Training and behavioral changes by farmers in their post-harvest practice minimize aflatoxin contamination and improve food safety. Moreover, if non-trained farmers receive mitigation training, aflatoxin concentration is predicted to decrease by 28.9 μg/kg on average.