Corn as a source of mycotoxins in Indonesian poultry feeds and the effectiveness of visual examination methods for detecting contamination

Every truck load of corn (n=52) entering and every batch of poultry feed (n=290) leaving a Bogor feedmill over one year was analysed for aflatoxins, zearalenone, ochratoxin A and sterigmatocystin. Fifty loads of corn and 274 of the batches of chicken feed contained aflatoxins. Zearalenone was detected in 11 corn samples but was not found in the formulated feed. Ochratoxin A was detected in one corn sample, but not in feed. Corn can account for all of the aflatoxin in the feed since levels were always lower in the finished product. There was no quantitative association between the proportion of bright green-yellow fluorescent, purple or mouldy kernels and the mycotoxin contents of the composite samples. Nevertheless, the absence of abnormal kernels indicates higher quality corn since the highest levels of mycotoxins occurred in the abnormal kernels.     

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.     

A new report of pre-harvest ear rot of corn caused by Geotrichum candidum from Iran

In summer of 2004, samples of husk looseness ear of corn (Zea mays) (cv. 700-Karaj) were collected from corn fields in Ali-Abad (Jiroft region), Kerman province, Southeastern Iran, for diagnosis of an unusual ear decay. A fungus was isolated from the rotting kernels and subsequently identified as Geotrichum candidum. The fungal pathogen was found to be closely related to G. citri-aurantii (citrus race) based on morphological, physiological and pathogenicity properties. The fungal pathogenicity test was demonstrated by fulfilling Koch's postulates. The pathogen caused rot disease on husk looseness corn kernels in soft-dough stage of ripening. The fungus was also pathogenic on ripe lemon and green and ripe tomato fruits. Fungal isolates of corn were compared to isolates from soft-rotten potato tubers. These two groups of isolates were highly similar on the basis of their morphological, biochemical and pathogenicity characteristics. To our knowledge, this is the first known report of corn ear rot caused by G. candidum in the world.     

Preharvest aflatoxin contamination: effect of moisture and substrate variation in developing cottonseed and corn kernels.

Variations in moisture and substrate in preharvest corn kernels and cottonseed were linked with the ability of Aspergillus parasiticus to infect the seed and produce aflatoxin. Osmotic pressures and moisture content (MC) levels of developing starch-rich corn kernels and lipid-rich cottonseed were determined. For in vivo studies, corn kernels and cottonseed were inoculated with A. parasiticus conidia and retained on plants through maturation. For in vitro studies, samples of corn kernels and cottonseed were collected at various stages, sterilized, inoculated, incubated for 2 weeks, and assayed for toxin. Aflatoxin levels were highest in corn kernels inoculated at 28 days postflowering (52% MC) in both the in vivo and in vitro tests. Toxin concentrations in cottonseed were greatest with inoculation at 35 days postflowering (70% MC) in seed retained on the plant, but toxin accumulation continued to increase with the maturity of the seed inoculated in cottonseed used in the in vitro trials. Moisture and substrate conditions in the midrange of seed development provided optimum conditions for fungal development and toxin production in seed retained on the plant.     

Preharvest aflatoxin contamination: effect of moisture and substrate variation in developing cottonseed and corn kernels

Variations in moisture and substrate in preharvest corn kernels and cottonseed were linked with the ability of Aspergillus parasiticus to infect the seed and produce aflatoxin. Osmotic pressures and moisture content (MC) levels of developing starch-rich corn kernels and lipid-rich cottonseed were determined. For in vivo studies, corn kernels and cottonseed were inoculated with A. parasiticus conidia and retained on plants through maturation. For in vitro studies, samples of corn kernels and cottonseed were collected at various stages, sterilized, inoculated, incubated for 2 weeks, and assayed for toxin. Aflatoxin levels were highest in corn kernels inoculated at 28 days postflowering (52% MC) in both the in vivo and in vitro tests. Toxin concentrations in cottonseed were greatest with inoculation at 35 days postflowering (70% MC) in seed retained on the plant, but toxin accumulation continued to increase with the maturity of the seed inoculated in cottonseed used in the in vitro trials. Moisture and substrate conditions in the midrange of seed development provided optimum conditions for fungal development and toxin production in seed retained on the plant.     

Production of fumonisin B1 and B2 byFusarium moniliforme isolated from Korean corn kerneis for feed

The natural occurrence of fumonisin B₁ (FB₁) and B₂ (FB₂), a promoter for hepatocarcinogenesis, was investigated in Korean corn kernels for feed by HPLC with fluorescence detection. From the 12 corn kernel samples, FB₁ was detected in 5 samples at levels ranging from 53 to 1327ng/g, while FB₂ was found in 4 samples from 69 to 680ng/g. In the positive samples, the average concentrations of FB₁ and FB₂ were 506 and 288ng/g, respectively. One sample (No. K3) showed the highest FB₁ and FB₂ contents as 1327 and 680ng/g, respectively. In the micrological survey on 5 positive samples for FB₁ and FB₂, 6 strains ofFusarium monlliforme were isolated, and all these isolates had a producibility of FB₁ and FB₁, with maximum levels of 80.7 to 180.9 g/g.Thisis the first report on the natural co-contamination of FB₁ and FB₂ in Korean corn kernels for feed, and on the ability ofF. moniliforme isolated from corn kernels for feed in Korea to produce FB₁ and FB₂.     

Processing of transgenic corn seed and its effect on the recovery of recombinant beta-glucuronidase

The tools of plant biotechnology that have been developed to improve agronomic traits are now being applied to generate recombinant protein products for the food, feed, and pharmaceutical industry. This study addresses several processing and protein recovery issues that are relevant to utilizing transgenic corn as a protein production system. The gus gene coding for beta-glucuronidase (rGUS) was stably integrated and expressed over four generations. The accumulation level of rGUS reached 0.4% of total extractable protein. Within the kernel, rGUS was preferentially accumulated in the germ even though a constitutive ubiquitin promoter was used to direct gus expression. Fourth-generation transgenic seed was used to investigate the effect of seed processing on the activity and the recovery of rGUS. Transgenic seed containing rGUS could be stored at an ambient temperature for up to two weeks and for at least three months at 10 degrees C without a significant loss of enzyme activity. rGUS exposed to dry heat was more stable in ground than in whole kernels. The enzyme stability was correlated with the moisture loss of the samples during the heating. Transgenic seed was dry-milled, fractionated, and hexane extracted to produce full-fat and defatted germ fractions. The results of the aqueous extraction of rGUS from ground kernels, full-fat germ, and defatted-germ samples revealed that approximately 10 times more rGUS per gram of solids could be extracted from the ground full-fat germ and defatted-germ than from the kernel samples. The extraction of corn oil from ground germ with hot hexane (60 degrees C) did not affect the extractable rGUS activity. rGUS was purified from ground kernels and full-fat germ extracts by ion exchange, hydrophobic interaction, and size exclusion chromatography. Similar purity and yield of rGUS were obtained from both extracts. Biochemical properties of rGUS purified from transgenic corn seed were similar to those of E. coli GUS.     

Imaging and automated detection of Sitophilus oryzae (Coleoptera: Curculionidae) pupae in hard red winter wheat

Computed tomography, an imaging technique commonly used for diagnosing internal human health ailments, uses multiple x-rays and sophisticated software to recreate a cross-sectional representation of a subject. The use of this technique to image hard red winter wheat, Triticum aestivm L., samples infested with pupae of Sitophilus oryzae (L.) was investigated. A software program was developed to rapidly recognize and quantify the infested kernels. Samples were imaged in a 7.6-cm (o.d.) plastic tube containing 0, 50, or 100 infested kernels per kg of wheat. Interkernel spaces were filled with corn oil so as to increase the contrast between voids inside kernels and voids among kernels. Automated image processing, using a custom C language software program, was conducted separately on each 100 g portion of the prepared samples. The average detection accuracy in the five infested kernels per 100-g samples was 94.4 +/- 7.3% (mean +/- SD, n = 10), whereas the average detection accuracy in the 10 infested kernels per 100-g sample was 87.3 +/- 7.9% (n = 10). Detection accuracy in the 10 infested kernels per 100-g samples was slightly less than the five infested kernels per 100-g samples because of some infested kernels overlapping with each other or air bubbles in the oil. A mean of 1.2 +/- 0.9 (n = 10) bubbles (per tube) was incorrectly classed as infested kernels in replicates containing no infested kernels. In light of these positive results, future studies should be conducted using additional grains, insect species, and life stages.     

Morphological Observations of Diplodia maydis on Synthetic and Natural Substrates as Revealed by Scanning Electron Microscopy

Mycelial and spore morphology of Diplodia maydis were investigated by using scanning electron microscopy after growth on various media and natural substrates (oat and corn kernels, and corn husks). Of several specimen preparation methods studied, Parducz fixation followed by critical-point or freeze-drying gave adequate preservation for pycnidia, mycelia, and spores. Morphological characteristics were similar in rotary and reciprocal shaker cultures and differed from that found in stationary cultures in the amount of slime-like material produced and precipitated matter on the mycelial surfaces. In general, mycelial surfaces were smooth. Large areas of coalesced material were present in all samples examined. Slime-like material produced in liquid media appeared as a finely laced net, randomly appearing throughout the mycelia with bead-like structures present along the net. A fine netting also was observed interspersed among the spores inside the pycnidia obtained from oats. Slime-like material was observed to cover the pycnidia produced on oat and corn kernels. In the latter case, the spores were less protected by the outer slime-like covering. Thickened node-like structures were observed in mycelial mats produced in modified Fries 2 medium, on potato dextrose agar plates, and on infected oats. Round and ovate thickened node-like structures were observed in mycelium produced on corn kernels. In general, node-like structures were less abundant in mycelia from naturally infected substrates. Conidia were commonly rounded to tapered and two celled, with a distinctive ridged septum at the middle. Dried spores were collapsed in a characteristic flask-like fashion.     

Occurrence of Ochratoxin A-Producing Fungi in Commercial Corn Kernels in Argentina

Potentially ochratoxigenic Aspergillus and Penicillium species were identified and the natural occurrence of ochratoxin A (OTA) in corn kernels was evaluated. Likewise, the capacity to produce OTA by Aspergillus section Nigri and Circumdati was investigated. A total of 50 corn samples for human consumption was collected in the south of Córdoba Province. The surface-disinfected method for mycobiota determination was used. The OTA detection was performed by HPLC. OTA production was tested in strains belonging to section Nigri and Circumdati. Statistical analysis demonstrated that the specie A. flavus was isolated in higher frequency (p<0.01) from corn kernels in DRBC and DG18 media. The percentage of corn kernels contaminated by A. niger var. niger was similar in DRBC and DG18 media. The frequency of grains contaminated by A. flavus and A. niger var. awamori was higher than A. niger var. niger and A. japonicus var. japonicus (p<0.01) in DG18 media. The other potentially ochratoxigenic species, A. ochraceus, was isolated between 5% and 10% of the corn kernels in DG18 and DRBC media, respectively. The OTA producing species P. verrucosum was not isolated. All samples of corn were OTA negative (<1 ng g⁻¹). Thirty strains (25%) of the black Aspergillus were OTA producers. From four strains of A. ochraceus isolated, only one produced OTA. Due to the storage variable conditions could not be adequate in this substrate, the presence of ochratoxigenic strains of section Nigri and OTA needs to be evaluated for a longer time to establish the toxicological risk for human beings. The contamination of stored corn kernels with A. flavus and Aspergillus section Nigri was significant.     

Extraction, characterization and potential applications of cellulose in corn kernels and Distillers’ dried grains with solubles (DDGS)

Cellulose with properties suitable for films and absorbents has been extracted from corn kernels and DDGS. Although DDGS is an inexpensive and abundant co-product that contains valuable components, it is currently not being used for industrial applications. DDGS contains about 9–16% cellulose by weight but the properties of cellulose in DDGS or even in corn kernels such as degree of polymerization (DP), morphology and crystallinity of cellulose have not been studied. In this study, cellulose was extracted from corn kernels and DDGS using alkali and enzymes. A minimum crude cellulose yield of 1.7% and 7.2% with cellulose content of 72% and 81% was obtained from corn kernels and DDGS, respectively. The solids obtained after extraction with cellulose contents ranging from 35% to 81% were made into films with tensile strength and elongation up to 42.5 MPa and 3.3%, respectively, using water and without any additional chemicals. The cellulose obtained holds water up to 9 times its weight and could therefore be used as an absorbent. The cellulose could also be used as paper, composites, lubricant and nutritional supplement.     

Mycoflora of Argentinian corn harvested in the main production area in 1990

Corn (Zea mays) is the main cereal produced in and exported from Argentina. The risk of contamination by mycotoxins is related to the mycoflora associated with the corn kernels. This paper reports on the identification of internal and external mycoflora of corn kernels harvested in the main production area in Argentina in 1990. A mycological survey was carried out on 178 corn samples, from five locations in that area and the isolation frequency and relative density of the prevalent fungal genera compared. GenusFusarium was the most prevalent component of the internal seedborne mycoflora in the five locations.Penicillium was prevalent in all locations, taking into account the frequency. However, this genus was predominant only in two locations, when the relative density was considered. The predominantFusarium wasF. moniliforme and the most frequently isolated species ofAlternaria, Aspergillus andPenicillium wereA. alternata, A. flavus andP. decumbens, respectively.Diplodia species were not isolated from any of the samples.     

Penicillium-species fungi--producers of ochratoxin A in grain]

The occurrence of ochratoxin A and ochratoxigenic fungi in the commercial batches of various domestic grains (wheat, rye, barley, corn and rice) has been studied. Penicillium cyclopium, P. viridicatum and P. chrysogenum isolated from grain synthesized on a sucrose-yeast medium predominantly patulin, penicillic and kojic acids. Only 4.4% of the fungal isolates were able to synthesize ochratoxin A. The concentration of the mycotoxin accumulated by the fungi was less than 500 micrograms/kg. 230 samples of wheat and 502 samples of corn were examined. The analysis showed that ochratoxin A was present in 0.9% and 0.1% of samples tested, respectively. The mycotoxin accumulated in grain mainly during its spontaneous heating and was concentrated in mold-damaged kernels.     

Factors influencing the inhibition of aflatoxin production in corn by Aspergillus niger

Aspergillus niger, a mold commonly associated with Aspergillus flavus in damaged corn, interferes with the production of aflatoxin when grown with A. flavus on autoclaved corn. The pH of corn-meal disks was adjusted using NaOH-HCl, citric acid-sodium citrate, or a water extract of A. niger fermented corn. Aflatoxin formation was completely inhibited below pH 2.8-3.0, irrespective of the system used for pH adjustment. When grown in association with A. flavus NRRL 6432 on autoclaved corn kernels, A. niger NRRL 6411 lowered substrate pH sufficiently to suppress aflatoxin production. The biodegradation of aflatoxin B1 or its conversion to aflatoxin B2a were eliminated as potential mechanisms by which A. niger reduces aflatoxin contamination. A water extract of corn kernels fermented with A. niger caused an additional inhibition of aflatoxin formation apart from the effects of pH.     

Aspergillus flavus Infection and Aflatoxin Production in Corn: Influence of Trace Elements

Distribution of trace element levels in corn germ fractions from kernels naturally infected with Aspergillus flavus and from kernels free of the fungus demonstrated an association between the presence of A. flavus and higher levels of metals. A. flavus production of aflatoxin on various autoclaved corn media showed that ground, whole corn was an excellent substrate; similar high levels of toxin were observed on full-fat corn germ but endosperm and defatted corn germ supported reduced yields. The influence of trace elements and their availability in defatted corn germ to A. flavus-mediated aflatoxin biosynthesis were measured. Enrichment of the substrate with 5 to 10 mug of manganese, copper, cadmium, or chromium per g of germ increased toxin yields. Addition of lead or zinc (50 to 250 mug/g) also enhanced toxin accumulation. Aflatoxin elaboration was reduced by the addition of 25 mug of cadmium per g or 500 mug of copper per g of germ.     

Binomial sequential sampling plans for late instars of European corn borer (Lepidoptera: Crambidae), corn earworm (Lepidoptera: Noctuidae), and damaged kernels in sweet corn ears

Late-season infestations of European corn borer, Ostrinia nubilalis (Hübner), and corn earworm, Helicoverpa zea (Boddie), were sampled to develop binomial sequential sampling plans for larval infestations and damaged kernels in sweet corn, Zea mays L., ears, near harvest. Fields were sampled to obtain a range of larval densities likely to be encountered over a range of infestation levels and field conditions. Binomial sampling plans were developed for O. nubilalis larvae, H. zea larvae, O. nubilalis, and H. zea larvae combined, and for damaged sweet corn kernels. Observed densities ranged from 0.01 to 4.40 larvae per ear for O. nubilalis, 0.005-1.62 larvae per ear for H. zea, and 0.004-36.12 damaged kernels per ear. Results of resampling analyses, based on the proportion of ears infested with one or more larvae, or damaged kernels, indicated an average sample size of 34-37 ears was necessary to classify whether larval infestations, or the incidence of damaged kernels, exceeded 5%. Two operating characteristic curves are presented for each of the four sampling plans. Initial results, with upper bounds of 0.10, and alpha (type I) and beta (type II) error rates at 0.10 and 0.05, respectively, resulted in a 90% probability of making the correct management decision at infestation levels >10%. To improve performance of the sampling plans, we modified the binomial plans by reducing the upper bound to 0.075, while maintaining the same error rates. This plan resulted in a higher probability (>95%) of making the correct management decision to reject a sweet corn load when infestation levels are >10%.     

Emetic and refusal activity of deoxynivalenol to swine.

The minimum emetic dose of deoxynivalenol to swine weighing 9 to 10 kg was 0.05 mg/kg of body weight intraperitoneally and 0.1 to 0.2 mg/kg orally. There was no emesis by undosed pigs consuming vomitus from pigs orally dosed with deoxynivalenol or penned with such pigs without access to vomitus. Analysis by gas-liquid chromatography of a sample of Gibberella zeae-infected corn containing about 25% visually damaged kernels indicated 12 ppm of deoxynivalenol. Deoxynivalenol added to feed reduced feed consumption of 20- to 45-kg pigs, ranging from a 20% decrease with 3.6 ppm to 90% reduction with 40 ppm. Loss in weight was associated with feed refusal. Feed refusal, however, was much greater for naturally infected corn samples than for feeds with equal concentrations of the pure compound added, indicating the involvement of an additional factor(s) in the swine refusal response.     

Aflatoxin contamination caused by natural fungal infection of preharvest corn

Summary Aflatoxin contamination of developing corn (Zea mays L.) kernels caused by natural infection byAspergillus flavus Link ex Fries was studied in hybrids developed for the U.S. corn belt and for the southern U.S. and grown at diverse locations in 1977. Planting dates were staggered to examine the effect of crop maturity on infection by the toxin-producing fungus. A broad range of toxin values was observed at harvest; some levels exceeded the highest that had been previously recorded in corn. The highest concentration of aflatoxin B₁ detected was 8030 ppb. Levels of toxin differed significantly among planting dates in Florida and Georgia; the second planting date at these locations contained the highest toxin levels. Elevated concentrations of toxin were characteristic of kernel samples from southern locations and southeast Missouri; at these locations samples from hybrids developed for the south had significantly lower levels of toxin than hybrids developed for the corn belt. Ears with heavy insect damage had higher toxin levels than ears with less evidence of insect attack.Mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

OCCURRENCE OF A STELAR LESION DURING IMBIBITIONAL CHILLING OF ZEA MAYS L.

Radicle growth of the corn inbred Oh51A was reduced by imbibitional chilling at 5 C. The effect was mediated by the initial moisture of the kernel prior to hydration. Five percent initial moisture kernels were injured while 13% initial moisture kernels were not. The formation of a structural lesion in the radicle during the first 24 hr of hydration at 5 C of 5% initial moisture kernels was correlated with subsequent radicle growth reduction at 25 C. The lesion did not form during hydration of 13% initial moisture kernels at 5 C, in kernels hydrated at 25 C, in unimbibed kernels, or in heat-killed, cold-imbibed kernels. The lesion also did not appear during the imbibitional chilling of the corn inbred B8 which did not exhibit radicle growth reduction. Two sources of Oh51A with similar lesion frequencies were different in the severity of growth reduction. While the lesion became sealed at 25 C subsequent to cold hydration, growth reduction differences were not fully explained by this phenomenon. In one source more seedlings exhibited reduced growth than expected from the frequency of observable lesions. Therefore, other types of damage contributed to the reduction of radicle growth.