Mycoflora,and mycotoxins production in Nigerian corn and corn-based snacks

Thirty-one fungal species, mostly toxigenic and belonging to 11 genera were isolated from corn, corn cake and corn roll snack samples.Aspergillus, Penicillum andFusarium accounted for 10, 6 and 3 of the species and altogether, they constituted 90, 94 and 88 percent of the total fungi in corn, corn cake and corn roll snack respectively. Mycotoxins (aflatoxins and ochratoxin A) were detected in 45, 80 and 12 percent while the means and ranges of the total aflatoxins recorded were: 200(25–770 ppb); 233(15–1070 ppb) and 55(10–160 ppb) for corn, corn cake and corn roll snack samples respectively. Ochratoxin A was detected at toxicologically significant levels in only 15 percent of the corn cake samples analyzed. All the strains ofAspergillus flavus andA. ochraceus tested produced aflatoxin B and ochratoxin A, respectively, when they were cultured on each of the three substrates. In each case, substantial quantities of the toxins were produced from 25 to 35°C with the peak level recorded at 30°C. Toxin production was detected only in substrates with 15 percent moisture content and above; reaching the maximum at 25 or 30 percent moisture level. No substantial differences in the amount of toxins were elaborated with further increase in substrates' moisture content. Of the three substrates, corn cake was the most suitable for aflatoxin B production while they were all equally suitable for the elaboration of ochratoxin A.     

Enzyme formation during solid-substrate fermentation in rotating vessels

Aspergillus awamori NRRL 4869 was cultured on the solid substrate, wheat bran, in a modified Rollacell apparatus to produce alpha-galactosidase and invertase. The swivel cap on the elongated bottle permits the introduction of air while the bottle rotates. Parameters of air flow rate (0.05-0.2 liter/kg/min), rpm (0.15-15 rpm), and weight of solids (150 and 300 g) were varied. At low air flow rates (0.05 liter/kg solids/min), alpha-galactosidase production was minimal independent of the rotation rate. At 0.15 rpm and 0.2 liter/kg solids/min air flow rate, invertase production ceased after five days; whereas alpha-galactosidase production continued. The modified Rollacell can be a useful apparatus for studying solid-substrate cultures.     

Production of aflatoxin by an Aspergillus flavus isolate cultured under a limited oxygen supply

In a previous experiment on the preservation of hay of high moisture content with formic acid, among other agents, aflatoxin was formed in the hay, and aflatoxin-forming strains of Aspergillus flavus were isolated from this hay after incubation in air as well as in an anaerobic jar. One isolate from the anaerobic jar was cultivated in a chemostat (Bioflo model C 30; New Brunswick Scientific Co.) in a defined medium with added B vitamins, yeast extract, or formic acid, with or without gas flow (air or nitrogen). In all cases where spore germination occurred, aflatoxin was formed in the cultures with gas flow, and small quantities of aflatoxins B1 and B2 occurred even in an atmosphere of nitrogen. Addition of B vitamins and supply of traces of air gave an approximately 15-fold increase in the amount of aflatoxin in 2 days. Carbon dioxide enrichment hindered aflatoxin formation on the defined medium even in the presence of B vitamins, but when formic acid was added, small quantities (5 to 15 micrograms/liter) were formed, and this low level remained constant until the gas flow was started.     

Production of aflatoxin by an Aspergillus flavus isolate cultured under a limited oxygen supply.

In a previous experiment on the preservation of hay of high moisture content with formic acid, among other agents, aflatoxin was formed in the hay, and aflatoxin-forming strains of Aspergillus flavus were isolated from this hay after incubation in air as well as in an anaerobic jar. One isolate from the anaerobic jar was cultivated in a chemostat (Bioflo model C 30; New Brunswick Scientific Co.) in a defined medium with added B vitamins, yeast extract, or formic acid, with or without gas flow (air or nitrogen). In all cases where spore germination occurred, aflatoxin was formed in the cultures with gas flow, and small quantities of aflatoxins B1 and B2 occurred even in an atmosphere of nitrogen. Addition of B vitamins and supply of traces of air gave an approximately 15-fold increase in the amount of aflatoxin in 2 days. Carbon dioxide enrichment hindered aflatoxin formation on the defined medium even in the presence of B vitamins, but when formic acid was added, small quantities (5 to 15 micrograms/liter) were formed, and this low level remained constant until the gas flow was started.     

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.     

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.     

Suppression of spore germination and aflatoxin biosynthesis in Aspergillus parasiticus during and after exposure to high levels of phosphine

Agar cultures of toxigenic Aspergillus parasiticus NRRL 2999 were exposed to phosphine (PH3), in levels ranging from 0 to 2000 ppm (vol/vol). It was found that with PH3 concentrations of 400 ppm or higher the growth of the fungus was totally arrested. When PH3 was vented and the agar plates were exposed to open air, 100% of the initial CFU developed into fully grown colonies after PH3 levels below 300 ppm, but at higher PH3 concentrations only 50% of the colonies developed. The same strain of A. parasiticus was inoculated into high moisture corn under conditions highly favorable for aflatoxin production, and it was exposed to a range of PH3 levels. After exposure to 500 ppm PH3, both fungal growth and aflatoxin synthesis resumed shortly after elimination of the toxic gas, but after exposure to PH3 levels of 1000 ppm and higher, the physical appearance of the contaminated corn was remarkably changed, showing reduced mycelial growth and almost complete absence of green pigmentation. In addition, aflatoxin synthesis was totally absent for the remainder of the experiment (20 days). These results strongly suggest that exposure to PH3 levels of 1000 ppm or higher could bring about persistent metabolic changes in surviving Aspergillus organisms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Natural occurrence of aflatoxins and ochratoxin a in corn and barley from mazandaran and golestan in north provinces of I. R. Iran

Fourteen barley and nine corn samples, destined for animal feed, collected from Golestan and Mazandaran provinces in the north of Islamic Republic of Iran (I. R. Iran) were analysed for aflatoxins (AF) and ochratoxin A (OA) by high performance liquid chromatography. In corn samples, aflatoxin B₁ (AFB₁) and aflatoxin B₂ (AFB₂) were detected in 8 (88.8%) and 6 (66.6%) samples at a mean level of 15.83 and 2.99 ppb (median 1.72 and 1 ppb), respectively. None of the corn samples contained detectable amounts of aflatoxin G₁ (AFG₁) and aflatoxin G₂ (AFG₂). Only one of the AF-contaminated samples was co-contaminated with OA at a concentration of 0.35 ppb. This is the first report concerning natural occurrence of OA and co-occurrence with AF in corn samples of north of I. R. Iran.     

Cyclopiazonic acid and aflatoxins production byAspergillus flavus isolated from Argentinian corn

Thirty-four isolates ofAspergillus flavus obtained from the main Argentinian corn production area were tested for their ability to produce both cyclopiazonic acid (CPA) on corn and on liquid media and aflatoxins on corn. Aflatoxins and CPA were quantified by comparison with standards. The last one was confirmed by mass spectrometry. All but one of the isolates produced CPA on liquid medium in a range between 3120 to 62500 μg/kg, 27/34 isolates produced CPA on corn at levels ranging from 833 to 10000 μg/kg and 5/34 isolates produced aflatoxin B1 in a range between 29 to 115 μg/kg. According to these findings, the percentage ofAspergillus flavus isolates with CPA production ability and their levels of CPA production were higher than the observed elsewhere. It was observed significant differences (p<0,01) between CPA production on corn (median: 1761 μg/Kg) and in liquid medium (median: 27950 μg/Kg). These data represent the first report of the co-production of CPA and aflatoxin B1 by isolates ofAspergillus flavus obtained from corn in Argentina.     

Simultaneous occurrence of fumonisin B1 and other mycotoxins in moldy corn collected from the People's Republic of China in regions with high incidences of esophageal cancer.

A total of 31 corn samples collected from households in the counties of Cixian and Linxian of the People's Republic of China, where high incidences of esophageal cancer have been reported, were analyzed for fumonisin B1 (FB1), aflatoxin, and total trichothecene mycotoxins. High levels of FB1 (18 to 155 ppm; mean, 74 ppm) were found in 16 of the samples that showed heavy mold contamination. FB1, at lower levels (20 to 60 ppm; mean, 35.3 ppm), was also found in 15 samples, collected from the same households, that did not show any visible mold contamination. The levels of aflatoxin in the samples were low (1 to 38.4 ppb; mean, 8.61 ppb). High levels of total type-A trichothecenes were also found in the moldy corn samples (139 to 2,030 ppb; mean, 627 ppb). Immunochromatography of selected samples revealed that these samples contained T-2 toxin, HT-2 toxin, iso-neosolaniol, monoacetoxyscirpenol, and several other type-A trichothecenes. The concentration of total type-B trichothecenes in 15 moldy corn samples was in the range of 470 to 5,826 ppb (mean, 2,359 ppb). High levels (3.7 to 5.0 mg/g) of FB1 were produced in corn in the laboratory by five Fusarium moniliforme strains isolated from the moldy corn. These fungi were also capable of forming various nitrosamines (5 to 16 micrograms per flask) in the presence of nitrate and precursor amines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental short time production of aflatoxins by Aspergillus parasiticus in mixed feeds as related to various moisture contents

Experimental short time production of aflatoxins in mixed feeds at 22, 28 and 37 °C as related to various moisture contents was studied. Growth of Aspergillus parasiticus was not observed in the meals with a moisture content ranging around 15% (22, 28 and 37 °C); the lowest quantifiable total aflatoxins at the fourth day was detected at 22 °C with 19.4% of moisture content; the higher total quantity of aflatoxins (113 mg/kg) was produced at 28 °C with 29.3% of moisture content. The ratio aflatoxin B₁/aflatoxin G₁ increased as the temperature raised.     

Survey of aflatoxins and ochratoxin A in stored tubers ofCyperus esculentus L.

The mold incidence, moisture contents, pH and levels of mycotoxins (aflatoxins B₁, G₁ and ochratoxin A) on/of/in rootstock snack (tubers ofCyperus esculentus L.) samples were monitored during a 150-day storage period. Whereas the mold incidence, moisture and mycotoxin levels increased with storage time, the pH declined during the same period. Altogether, 12 fungal species, mostly toxigenic, includingAspergillus flavus, A. parasiticus andA. ochraceus were isolated. At collection period only 3 of the 9 snack samples analysed contained trace amounts of aflatoxins. By 120th day, all the 9 samples were contaminated and the average levels were 454 and 80 ppb for aflatoxin B₁ and aflatoxin G₁ respectively on the 150th day. Ochratoxin A was not detected before 120th day and then only at low levels, occuring in a maximum of four samples and ranging between 10 and 80 ppb.     

Aflatoxin levels in corn available as wild turkey feed in Georgia

Samples of corn available as wildlife feed from retailers throughout Georgia (USA) were collected during April 1997 and analyzed for aflatoxin to determine if levels harmful to wild turkeys (Meleagris gallopavo) were present. Three of 31 (10%) samples collected from a 40-country area were positive. An enzyme-linked immunosorbent assay qualitatively determined that two samples contained from 0 to 20 ppb aflatoxin. A chromatography analysis of a third sample measured 380 ppb total aflatoxin. A small percentage of our sample of wildlife feed collected during one season contained levels of aflatoxin that may cause harm to turkeys, especially poults. However, because aflatoxin levels ranging from 100 to 400 ppb may cause liver dysfunction and immunosuppression in turkey poults and other wildlife, grains known to be contaminated with aflatoxin at levels unacceptable for domestic animal feeds (> or =100 ppb) should not be sold as wildlife feed. Further analyses of grains sold as wildlife feed should be conducted to address this potential problem.     

Correlation of Aflatoxin Contamination With Zinc Content of Chicken Feed

Feed samples from chicken houses in five commercial chicken operations were analyzed for Zn, Mn, Fe, Cu, Cd, and aflatoxin content. Mean aflatoxin content of these samples was 14 ppb (14 ng/g) as opposed to 1.2 ppb in samples taken when the feed was made. Aflatoxin content of the feed samples correlated (r = 0.325) significantly (P < 0.05) with Zn content but not with Mn, Fe, or Cu, all of which correlated significantly with Zn. Zn content of unamended feed (<20 ppm [20 μg/g]) is normally supplemented with a mineral premix containing Zn, Mn, Fe, and Cu to meet the nutrient requirements of chickens (40 ppm of Zn). The mean zinc concentration of the feed samples (117 ppm) was about threefold greater than the nutrient requirement and ranged from 58 to 162 ppm in individual samples. These field survey results parallel earlier reports of augmented production of aflatoxin in monocultures of aflatoxigenic fungi in corn and other ingredients supplemented with Zn. These results suggest that stricter control of Zn levels during manufacture could reduce aflatoxin contamination of feed consumed by chickens.     

Effect of Aeration on Growth and Aflatoxin Production by Aspergillus flavus in Submerged Culture

Aspergillus flavus ATCC 15517 produced up to 212 mg per liter of total aflatoxin in submerged culture in aerated (3,000, 6,000, 9,000, and 12,000 ml/min) and agitated medium in 14-liter fermentors with 10 liters of medium consisting of 2% yeast extract and 10% sucrose. Aflatoxin production increased with time. A maximum of 212 mg/liter was produced at 9,000 ml/min aeration, whereas the yield decreased substantially at the lower aeration rates. Two other strains of A. flavus synthesized aflatoxin in smaller quantities.     

Screening for resistance to Aspergillus flavus and aflatoxin production in groundnut

All the varieties, advanced breeding lines, germplasm lines, and wild species used in the experiments differed significantly for their ability to allow invasion and aflatoxin production by an aflatoxigenicAspergillus flavus strain. Infection and colonisation were strongly correlated (r = 0.82), while there was no relation between infection and aflatoxin content or colonisation and aflatoxin content (r = 0.15). The varieties ICGS11 and S 206 supported less infection and colonisation (range 35 to 40%). Lowest aflatoxin content was recorded in Chitra (3,200 ppb), while it was highest in Kaushal (38,250 ppb). A cross derivative of GAUG1 × NC Ac 17133 R F showed lowest infection and colonisation (86,3 and 25,28%, respectively), and also supported moderate aflatoxin production (4,000 ppb). Among germplasm lines spancross supported lowest aflatoxin production (2,026 ppb) while both the wild species vz. ICG 8127 and ICG 8128 were highly susceptible to infection, colonisation, and aflatoxin production.     

Incidence of aflatoxins and aflatoxin producing fungi in animal feedstuffs

Fifty four samples including 5 of broken rice, 8 of corn grains, 8 of corn gluten feed, 13 of cottonseed cake and 4 each of rice polish, corn gluten, sesame oil cake, guar meal and wheat bran were screened for the presence of aflatoxins. Among all the samples, 14 were damaged and 40 apparently undamaged. The incidende of aflatoxins was found to be 60, 25, 25, and 23 per cent in broken rice, corn grains, corn gluten feed and cottonseed cake. Aflatoxins were not detected from rice polish, corn gluten, sesame oil cake, guar meal and wheat bran. Damaged sample revealed a much higher incidence i.e. 50 per cent as compared to undamaged ones i.e. 7.5 per cent. Mean concentration of aflatoxin B and G was found to be 15.5 and 12.2 ppb respectively.Cultural examination of aflatoxin positive feedstuffs yielded 39 isolates of different fungi including 21 of Aspergillus, 7 of Mucor, 6 of Rhizopus, 4 of Fusarium and one of Penicillium. These strains when tested for aflatoxin producing ability, revealed this property in only one isolate, identified as Aspergillus parasiticus.     

Aflatoxin Production and Degradation by Aspergillus flavus in 20-Liter Fermentors

Yields of from 200 to 300 mg per liter of aflatoxins B(1) and G(1) were produced by two strains of Aspergillus flavus in 20-liter fermentors under proper conditions of inoculum (well-dispersed growth) and aeration (0.5 volume per volume per min of air, 300 rev/min, 30 psi back pressure, baffles). Peak yields were usually attained in 72 hr, after which the aflatoxin concentration declined rapidly. Degradation of aflatoxin depended primarily on mycelial lysis and high-aeration conditions. Cultures previously reported not to degrade aflatoxin could be induced to do so under these conditions. The percentage and rate of toxin degradation were independent of toxin concentration, and appeared to be nonenzymatic and nonspecific. Degradation simulating that occurring in the fermentor was achieved by reacting aflatoxin with peroxidized methyl esters of vegetable oil; initial degradation was rapid and appeared to involve a complex series of reactions.