Mycotoxin formation in moist wheat under controlled temperatures

One-kilogram parcels of wheat with 20.5% moisture content were maintained at 15° and 22 °C for 10 weeks to study quality changes. Temperature, moisture, oxygen and carbon dioxide levels, microfloral incidence and abundance, seed germination, fat acidity values, aflatoxins, sterigmatocystin, ochratoxin A, penicillic acid, citrinin and zearalenone were monitored. By two weeks, trace levels of ochratoxin had formed at both temperatures. By 10 weeks, the wheat contained at least three times more ochratoxin A at 22 °C than at 15 °C. Strains of Penicillium verrucosum var. cyclopium were associated with ochratoxin A production. No other mycotoxins were detected. The effect of temperature was significant for all variables (greater effect at 22 °C) except A. glaucus gr. and Penicillium (P<.01). The effect of time was significant for all variables except bacteria (P<.01). The shape of the response was fully characterized by the linear and quadratic terms, except for % moisture which was linear only, and for bacteria for which time was not significant. The interaction between time and temperature was significant (P<.01) for total fungal propagule count, % moisture, and Aspergillus versicolor, indicative of the steeper rise in slope for 22 °C.     

Penicillic Acid Production by Blue-Eye Fungi on Various Agricultural Commodities

Of 10 Penicillium species reported to cause blue-eye disease of corn, four (P. martensii, P. palitans, P. cyclopium, P. puberulum) were found capable of producing the mycotoxin penicillic acid on various agricultural commodities. Commodities with high protein contents did not support toxin synthesis. The extent of toxin production varied with the strain of mold, the commodity, and the temperature; low temperatures (1 to 10 C) favored toxin accumulation.     

Isolation of secondary metabolites of Aspergillus ochraceus by HPLC

A method is described for the isolation and purification of ochratoxin A, ochratoxin B, ochratoxin ß mellein, 4-hydroxymellein and penicillic acid produced byAspergillus ochraceus in a synthetic liquid medium. Ochratoxin α, which was not found in the culture medium, was obtained by acid hydrolysis of ochratoxin A. A high pressure liquid Chromatograph equipped with Lichrosorb 100 and Lichrosorb RP-18 columns and UV and/or Refractive Index detectors was used.     

Quality changes in granary-stored wheat at 15 and 19% moisture content

Half-bushel parcels of wheat at 15 and 19% moisture content (MC) were implanted in bulks of dry stored oats in a farm granary for 60 weeks to monitor quality changes. Temperature, MC, fat acidity value (FAV), oxygen and carbon dioxide levels, germination, microfloral incidence and abundance, and major storage mycotoxins (aflatoxins, sterigmatocystin, ochratoxin A, citrinin, penicillic acid) were monitored at two-week or four-week intervals during storage. Wheat storage at 19% MC was characterized by higher temperature, MC, FAV, carbon dioxide level, and microfloral abundance, and lower levels of oxygen and germination as compared to 15% MC wheat; no mycotoxins were detected at either moisture. Approximately 19% MC may represent a critical moisture limit for mycotoxin formation in granary stored wheat, given the indigenous mycoflora.     

Production of Ochratoxins in Different Cereal Products by Aspergillus ochraceus

The effects of temperature and length of incubation on ochratoxin A production in various substrates were studied. The optimal temperature for toxin production by Aspergillus ochraceus NRRL-3174 was found to be around 28 C. Very low levels of ochratoxin A are produced in corn, rice, and wheat bran at 4 C. The optimal time for ochratoxin A production depends on the substrate, ranging from 7 to 14 days at 28 C. Ochratoxin B and dihydroisocoumaric acid, i.e., one of the hydrolysis products of ochratoxin A, were produced in rice but at levels considerably lower than ochratoxin A. No ochratoxin C was produced in rice at 28 C. When added to rice cereal or oatmeal, the toxin was found to be very stable over prolonged storage and even to autoclaving for 3 hr.     

Classification of terverticillate penicillia based on profiles of mycotoxins and other secondary metabolites

Strains of available terverticillate penicillium species and varieties were analyzed for profiles of known mycotoxins and other secondary metabolites produced on Czapek yeast autolysate agar (intracellular metabolites) and yeast extract-sucrose agar (extracellular metabolites) by using simple thin-layer chromatography screening techniques. These strains (2,473 in all) could be classified into 29 groups based on profiles of secondary metabolites. Most of these profiles of secondary metabolites were distinct, containing several biosynthetically different mycotoxins and unknown metabolites characterized by distinct colors and retardation factors on thin-layer chromatography plates. Some species (P. italicum and P. atramentosum) only produced one or two metabolites by the simple screening methods. The 29 groups based on profiles of secondary metabolites were known species or subgroups thereof. These species and subgroups were independently identifiable by using morphological and physiological criteria. The species accepted, the number of isolates in each species investigated, and the mycotoxins they produced were: P. atramentosum, 4; P. aurantiogriseum, 510 (group I: penicillic acid and S-toxin and group II: penicillic acid, penitrem A [low frequency], terrestric acid [low frequency], viomellein, and xanthomegnin); P. brevicompactum, 81 (brevianamid A and mycophenolic acid); P. camembertii group I, 38, and group II, 114 (cyclopiazonic acid); P. chrysogenum, 87 (penicillin, roquefortine C, and PR-toxin); P. claviforme, 4 (patulin and roquefortine C); P. clavigerum, 4 (penitrem A); P. concentricum group I, 10 (griseofulvin and roquefortine C), and group II, 3 (patulin and roquefortine C); P. crustosum, 123 (penitrem A, roquefortine C, and terrestric acid); P. echinulatum, 13; P. expansum, 91 (citrinin, patulin, and roquefortine C); P. granulatum, 6 (patulin, penitrem A, and roquefortine C [traces]); P. griseofulvum, 21 (cyclopiazonic acid, griseofulvin, patulin, and roquefortine C); P. hirsutum, 100 (group I: terrestric acid; group II: citrinin, penicillic acid , roquefortine C, and terrestric acid; and group III: roquefortine C and terrestric acid), P. hirsutum group IV, 2 (chaetoglobosin C); P. isariiforme, 1; P. italicum, 41; P. mali, 104; P. roquefortii, 78 (group I: mycophenolic acid, PR-toxin, and roquefortine C and group II: mycophenolic acid, patulin, penicillic acid [low frequency], and roquefortine C); P. viridicatum group I, 634 (brevianamid A [low frequency], penicillic acid, viomellein, and xanthomegnin), P. viridicatum group II and III, 494 (citrinin and ochratoxin A), P. viridicatum group IV, 12 (griseofulvin and viridicatumtoxin). It is proposed that profiles of secondary metabolites be strongly emphasized in any future revision of the penicillia.     

Management of environmental temperature and rations for poultry production in the hot and humid tropics

This paper reviews studies that have been conducted to determine how environmental temperature affects productivity of poultry and how these effects can be alleviated so that the bird will realise its full productive capacity. It is shown that temperature primarily affects production of poultry meat and eggs through food consumption. A key factor in efficient weight gain and/or egg production of poultry is optimum nutrient intake. High environmental temperatures cause food intake to decrease and often result in inadequate nutrient intake contributing to poor performance. When nutrient intake is shifted by the influence of temperature on food intake, the adverse effect on productivity (growth or egg output) may be alleviated through improved feed formulation by adjusting the nutrient density to compensate for the altered intake of food. Improved feed management and better housing systems can partially compensate for low feed intake caused by high environmental temperatures.     

Screening methods to detect toxigenic fungi in liquid medium

Rapid and sensitive methods for identifying toxin production by toxigenic fungi cultivated in liquid medium were developed. The production of aflatoxins B₁, B₂, G₁, G₂, sterigmatocystin, ochratoxin A, patulin, penicillic acid, citrinin and zearalenone was detected employing thin layer chromatography and high performance liquid chromatography detecion with or without extraction and purification procedures. No significant variation was found and toxins could be detected after 2–4 days incubation. The sensitivity of the methods and recovery after extraction have been estimated.     

Black soldier fly (Diptera: Stratiomyidae) larval heat generation and management

Mass production of black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), larvae results in massive heat generation, which impacts facility management, waste conversion, and larval production. We tested daily substrate temperatures with different population densities (i.e., 0, 500, 1000, 5000, and 10 000 larvae/pan), different population sizes (i.e., 166, 1000, and 10 000 larvae at a fixed feed ratio) and air temperatures (i.e., 20 and 30 °C) on various production parameters. Impacts of shifting larvae from 30 to 20 °C on either day 9 or 11 were also determined. Larval activity increased substrate temperatures significantly (i.e., at least 10 °C above air temperatures). Low air temperature favored growth with the higher population sizes while high temperature favored growth with low population sizes. The greatest average individual larval weights (e.g., 0.126 and 0.124 g) and feed conversion ratios (e.g., 1.92 and 2.08 g/g) were recorded for either 10 000 larvae reared at 20 °C or 100 larvae reared at 30 °C. Shifting temperatures from high (30 °C) to low (20 °C) in between (∼10-d-old larvae) impacted larval production weights (16% increases) and feed conversion ratios (increased 14%). Facilities should consider the impact of larval density, population size, and air temperature during black soldier fly mass production as these factors impact overall larval production.     

Wild rice as fermentation substrate for mycotoxin production

Many cereal grains have been studied for their suitability as substrates for the fermentative production of mycotoxins. However, except for aflatoxin, wild rice has not been investigated. Hence, five mold cultures known to produce the mycotoxins ochratoxin-A, penicillic acid, patulin, vomitoxin, and zearalenone were grown on wild rice under varying conditions of moisture and temperature to determine whether this grain would serve as a suitable substrate for toxin production. Under appropriate fermentation conditions, good yields of ochratoxin-A and moderate amounts of patulin were obtained, but only small amounts of penicillic acid, vomitoxin, and zearalenone were elaborated. An extract from a sample of naturally molded wild rice contained 0.8 microgram of patulin per g of rice. The predominating mold was identified as Aspergillus clavatus. Under identical cultural conditions, this isolate and a known patulin-producing strain of A. clavatus yielded approximately equivalent amounts of the mycotoxin.     

Wild rice as fermentation substrate for mycotoxin production.

Many cereal grains have been studied for their suitability as substrates for the fermentative production of mycotoxins. However, except for aflatoxin, wild rice has not been investigated. Hence, five mold cultures known to produce the mycotoxins ochratoxin-A, penicillic acid, patulin, vomitoxin, and zearalenone were grown on wild rice under varying conditions of moisture and temperature to determine whether this grain would serve as a suitable substrate for toxin production. Under appropriate fermentation conditions, good yields of ochratoxin-A and moderate amounts of patulin were obtained, but only small amounts of penicillic acid, vomitoxin, and zearalenone were elaborated. An extract from a sample of naturally molded wild rice contained 0.8 microgram of patulin per g of rice. The predominating mold was identified as Aspergillus clavatus. Under identical cultural conditions, this isolate and a known patulin-producing strain of A. clavatus yielded approximately equivalent amounts of the mycotoxin.     

Influence of water activity, temperature and time on mycotoxins production on barley rootlets

AIMS: The objective of this study was to determine the ochratoxin (OT) and aflatoxin (AF) production by three strains of Aspergillus spp. under different water activities, temperature and incubation time on barley rootlets (BR). METHODS AND RESULTS: Aspergillus ochraceus and Aspergillus flavus were able to produce mycotoxins on BR. Aspergillus ochraceus produced ochratoxin A (OTA) at 0.80 water activity (a(w)), at 25 and 30 degrees C as optimal environmental conditions. The OTA production varies at different incubation days depending on a(w). Aflatoxin B(1) (AFB1) accumulation was obtained at 25 degrees C, at 0.80 and 0.95 a(w), after 14 and 21 incubation days respectively. Temperature was a critical factor influencing OTA and AFB(1) production. CONCLUSIONS: This study demonstrates that BR support OTA and AFB(1) production at relatively low water activity (0.80 a(w)) and high temperatures (25-30 degrees C). SIGNIFICANCE AND IMPACT OF THE STUDY: The study of ecophysiological parameters and their interactions would determine the prevailing environmental factors, which enhance the mycotoxin production on BR used as animal feed.     

Simultaneous quanitative determination of secondary metabolites ofAspergillus ochraceus by TLC

A simple TLC method for the quanitative determination of mellein, 4-hydroxy-mellein, penicillic acid, ochratoxin A, B, α and β is described. Application of this technique permits metabolic studies of the influence of different factors on the formation ofAspergillus ochraceus metabolites.     

An enzyme-linked immunosorbent assay for monitoring of Aspergillus ochraceus growth in coffee powder, chilli powder and poultry feed

AIMS: The work was carried out to develop an immunoassay for estimation of Aspergillus ochraceus biomass on solid substrate. METHODS AND RESULTS: An indirect noncompetitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of fungal biomass in food commodities using antibody raised against A. ochraceus mycelial antigen. The sensitivity of the assay was linear in the range of 10-160 microg fungal biomass per millilitre extract of coffee (R(2)=0.989), poultry feed (R(2)=0.987) and chilli (R(2)=0.989). The growth of A. ochraceus in the food commodities like chilli, coffee beans and poultry feed, under the influence of two levels of moisture (20% and 30%) were monitored by the ELISA. The maximum fungal colonization was observed in poultry feed (9.8 and 11.8 mg g(-1)) followed by coffee beans (6.8 and 11.3 mg g(-1)) and chilli (5.1 and 6.3 mg g(-1)) at 20% and 30% moisture after 20 days of incubation. Similarly the fungus produced maximum ochratoxin A in poultry feed (25 and 120 microg g(-1)) followed by coffee beans (8 and 24 microg g(-1)) and chilli (0.2 and 0.45 microg g(-1)) at 20% and 30% moisture after 20 days of incubation. CONCLUSIONS: The method can be used for quantitative estimation of fungal biomass and comparison of fungal colonization in food substrates varying in composition. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be adapted for studying the fungal colonization in different solid substrates under different culture condition. The method is sensitive to mould colonization of >or=0.02% (w/w) and can be used for early detection of specific fungal infestation in food commodities.     

Production of ochratoxin a,citrinin, and ergosterol byPenicillium viridicatum in autoclaved and non-autoclaved wheat at low temperature

Wheat (moisture content: 26%) was autoclaved or left untreated, inoculated with conidia ofPenicillium viridicatum and stored at 10°C. The fungus grew on both substrates and was the dominant mould on the non-autoclaved grain. Autoclaving resulted in an earlier onset of ergosterol, ochratoxin A, and citrinin production due to accelerated mould growth. Yield of ochratoxin A increased while citrinin slightly decreased in autoclaved wheat.     

Bacterial bioluminescence as a bioassay for mycotoxins.

The use of bacterial bioluminescence as a toxicological assay for mycotoxins was tested with rubratoxin B, zearalenone, penicillic acid, citrinin, ochratoxin A, PR-toxin, aflatoxin B1, and patulin. The concentrations of mycotoxins causing 50% light reduction (EC50) in Photobacterium phosphoreum were determined immediately and at 5 h after reconstitution of the bacteria from a freeze-dried state. Generally, less toxins were required to obtain an EC50 at 5 h. The effects of the above mycotoxins on bioluminescence were determined after 5, 10, 15, and 20 min of incubation with the bacterial suspensions. The concentration of rubratoxin B necessary to elicit an EC50 increased with time, whereas the concentration of citrinin, penicillic acid, patulin, and PR-toxin necessary decreased with time. There was very little change in the concentration of zearalenone, aflatoxin B1, and ochratoxin A required to elicit an EC50 with time. The bacterial bioluminescence assay was most sensitive to patulin and least sensitive to rubratoxin B.     

Role of Penicillic Acid in the Phytotoxicity of Penicillium cyclopium and Penicillium canescens to the Germination of Corn Seeds

Penicillium cyclopium and Penicillium canescens cultures inhibited the germination of corn. The phytotoxic compound was isolated by solvent extraction and thin-layer chromatography on silica gel. The phytotoxin was identified as penicillic acid by mass spectrometry, nuclear magnetic resonance, and infrared spectroscopy. Gas-liquid chromatography on a capillary glass column separated the two epimeric forms of penicillic acid. The maximum production of penicillic acid was obtained with P. cyclopium cultures grown at 25°C. The phytotoxicity of penicillic acid was manifested by its ability to alter the germination of corn. The percent inhibition of germination was directly proportional to the logarithm of the penicillic acid concentration. Growth of the main root was reduced 50% by concentrations of 500 μg/ml.     

Mycotoxins and toxigenic fungi in sago starch from Papua New Guinea

AIMS: To assay sago starch from Papua New Guinea (PNG) for important mycotoxins and to test fungal isolates from sago for mycotoxin production in culture. METHODS AND RESULTS: Sago starch collected from Western and East Sepik Provinces was assayed for aflatoxins, ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin and zearalenone and all 51 samples were negative. Frequently isolated species of Penicillium (13), Aspergillus (five) and Fusarium (one) were cultured on wheat grain, and tested for the production of ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin, patulin and penicillic acid. All 12 isolates of P. citrinin and one of two A. flavipes isolates produced citrinin. A single isolate of A. versicolor produced sterigmatocystin. No other mycotoxins were detected in these cultures. CONCLUSIONS: No evidence was found of systemic mycotoxin contamination of sago starch. However, the isolation of several mycotoxigenic fungi shows the potential for citrinin and other mycotoxins to be produced in sago stored under special conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Sago starch is the staple carbohydrate in lowland PNG and the absence of mycotoxins in freshly prepared sago starch is a positive finding. However, the frequent isolation of citrinin-producing fungi indicates a potential health risk for sago consumers, and food safety is dependant on promoting good storage practices.     

Occurrence of trichothecenes, zearalenone and ochratoxin A in cereals and mixed feed from central Lithuania

A total of 92 samples — 23 winter wheat, 12 summer barley, 5 oats and 52 mixed feed — were collected from a state factory in Kaunas, Lithuania and were analysed for the presence of trichothecenes, zearalenone (ZEN) and ochratoxin A (OA) using gas chromatography with electron capture detection and immunoaffinity column/high performance liquid chromatography with fluorescence and UV detections. Deoxynivalenol (DON), nivalenol (NIV), T-2 toxin and HT-2 toxin were detected at concentrations above 10 μg/kg in 68%, 48%, 38% and 8% of cereal samples, respectively, and in 98%, 88%, 12% and 8% of samples of mixed feed for swine and poultry. More than 10 μg/kg of zearalenone and ochratoxin A were found in 58% and 92% of the mixed feed samples, respectively. The highest concentrations of all analysed trichothecenes in Lithuanian mixed feed and cereal grains, with an exception of T-2 toxin in one oat lot and one sample of mixed feed and OA in two mixed feed samples, were lower than those reported as Lithuanian advisory or tolerance limits.