Production of penicillic acid and patulin by an atypical Penicillium roqueforti isolate.

Simultaneous production of penicillic acid and patulin by an atypical strain of Penicillium roqueforti isolated from cheddar cheese is reported. Mycotoxin production was confirmed by thin-layer and gas-liquid chromatography and by ultraviolet, infrared, and mass spectral analyses. Culture extracts were toxic to Bacillus megaterium and chicken embryos. Commercial strains of P. roqueoforti used in production of blue-veined cheeses were shown not to produce penicillic acid and patulin.     

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.     

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.     

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.     

Isolation of Roquefortine C from Feed Grain

Roquefortine C was isolated from feed grain heavily infected by Penicillium roqueforti. The identity of the mycotoxin was confirmed by mass spectrometry. Other mycotoxins that are known to be produced by P. roqueforti such as PR toxin, patulin, and penicillic acid were not detected in the grain.     

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.     

Investigating the effect of patulin,penicillic acid and EDTA on biofilm formation of isolates from dental unit water lines

This study investigated the effect of patulin and penicillic acid, two known quorum-sensing inhibitors, and the common biocide ethylenediaminetetraacetic acid (EDTA) on the biofilm formation and auto-inducer (AI)-2 production of three isolates from dental unit water lines, Klebsiella sp., Bacillus subtilis and Bacillus cereus. Penicillic acid on its own had no effect on the biofilm formation of all isolates, whereas in combination with EDTA, it enhanced biofilm formation significantly in Klebsiella sp. and B. cereus. EDTA at concentrations greater than 10 μM promoted biofilm formation in B. cereus and B. subtilis. Patulin was found to promote biofilm formation in B. cereus up to 25 μM. A significant increase in biofilm formation was observed in B. cereus and B. subtilis at concentrations greater than 10 μM of patulin when combined with EDTA. The Vibrio harveyi BB170 AI-2 bioassay showed a positive response for Klebsiella sp. AI-2 production with a maximum fold induction at the late exponential growth phase. Addition of glucose prolonged the AI-2 production phase considerably. No significant effect of patulin, penicillic acid alone as well as in combination with EDTA was observed on AI-2 production by Klebsiella sp. The findings have important implications for the design of biofilm prevention and eradication strategies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of penicillic acid by Aspergillus sclerotiorum CGF

The production of penicillic acid by Aspergillus sclerotiorum CGF for the biocontrol of Phytophthora disease was investigated in submerged fermentation using media composed of different nutrients. Soluble starch was found to be the most effective substrate among the carbon sources used, and produced the highest penicillic acid concentration of 2.98 mg ml(-1). When organic nitrogen sources were used, pharmamedia, yeast extract, and polypeptone-S were found to be suitable organic nitrogen sources (2.46-2.71 mg ml(-1)). The production of penicillic acid was not detected in when inorganic nitrogen sources were used. Only Na2HPO4, among the metal ions and phosphate salts tested, increased the production of penicillic acid (approximately 20%). When A. sclerotiorum CGF was cultured in optimal medium [8.0% (w/v) soluble starch, 0.6% (w/v) yeast extract, and 0.3% (w/v) Na2HPO4], maximum penicillic acid concentration (approximately 9.40 mg ml(-1)) and cell mass (approximately 17.4 g l(-1)) were obtained after 12 days.     

Natural occurrence of mycotoxins in cereals

Besides peanuts and cottonseed, cereal grains are the most important feed and food source that occasionally are naturally contaminated with mycotoxins. The problem of mycotoxins occurring naturally in cereals, especially in corn, has become trouble-some because of changing agricultural technology. The mycotoxin problem in cereals is not restricted to any geographic or climatic region. Toxins are produced on cereals, both in the field and in storage; they involve both the grain and the whole plant. The genera of fungi most involved areAspergillus, Fusarium, Penicillium andClaviceps. Mycotoxins known to occur naturally in cereals include aflatoxins B₁, B₂, G₁ and G₂-as well as aflatoxins M₁ and M₂-ochratoxins A and B, penicillic acid, patulin, ergot, zearalenone, citrinin, T-2, tenuazonic acid, kojic acid and sterigmatocystin. Of these mycotoxins, aflatoxins, patulin, penicillic acid and sterigmatocystin are carcinogens.     

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.     

Mise en évidence de l'activité antibactérienne de quelques mycotoxines par utilisation de Bacillus thuringiensis (Berliner)

Antibacterial activity of pure preparations of mycotoxins, was studied against strains of Bacillus thuringiensis (Berliner). Most of fungal metabolites used provokes: 1) Growth bacterial inhibition similar to lethal effect of antibiotics 2) Enlargment of cell volume; this cell abnormality induction resembles those obtained with mitomycin C. The bioassay of fungal toxins with B. thuringiensis can be recommended as a sensitive technique to supplement chemical characterization. By using specific cell lines to such a mycotoxin and specific inactivators as coumarin for aflatoxin, or sulfhydryl compounds for penicillic acid and patulin.     

Physiological criteria and mycotoxin production as AIDS in identification of common asymmetric penicillia

The taxonomy of the asymmetric (predominantly terverticillate) penicillia is based on morphological differences that leave identification difficult. The application of physiological criteria facilitated the identification of the common asymmetric penicillia investigated. Changes in the placement of some strains of these penicillia made the connection to mycotoxin-producing ability clearer. The classical criterion of conidium color was deemphasized and replaced by the following criteria: (i) growth on nitrite-sucrose agar and (ii) growth and acid (and subsequent base) production on creatine-sucrose agar (containing bromocresol purple). Other criteria used or developed were: (iii) growth on sorbic acid plus benzoic acid agar (50 + 50 ppm, pH 3.8), (iv) growth on an agar containing 1,000 ppm propionic acid (pH 3.8), (v) growth on an agar containing 0.5% acetic acid, (vi) growth at 37 degrees C, (vii) growth rate on an agar containing 0.1% pentachloronitrobenzene, (viii) production of extracellular tricaproinase, and (ix) fasciculation on a medium containing 10 ppm botran (2,6-dichloro-4-nitroanilin). The pattern of extracellular metabolites after thin-layer chromatography was used as a chemotaxonomic criterion. The species investigated, the number of isolates investigated, and the toxins which some of these isolates produce were: Penicillium roqueforti (18) (patulin), P. citrinum (11) (citrinin), P. patulum (9) (patulin and griseofulvin), P. expansum (patulin and citrinin), P. hirsutum (13), P. brevicompactum (19), and P. chrysogenum (12). Widespread species of the P. cyclopium, P. viridicatum, and P. expansum series of Raper and Thom (A Manual of the Penicillia, 1949) were subdivided into four new groups: "P. crustosum pA" (29) (penitrem A), "P. melanochlorum" (29), "P. cyclopium p" (119) (penicillic acid and infrequently penitrem A), and "P. viridicatum o-c" (43) (ochratoxin A and citrinin). "P. viridicatum o-c" was separated from "P. cyclopium p" due to its ability to grow on nitrite as sole nitrogen source. The species and groups investigated were related to the new taxonomic classification of the genus Penicillium according to Pitt.     

Mycotoxin-Producing Potential of Mold Flora of Dried Beans

To evaluate the potential for mycotoxin production by molds in dried beans, the mold flora of 114 samples was determined both before and after surface disinfection of the beans with 5% NaOCl. Surface disinfection substantially reduced mold incidence, indicating that contamination was mainly on the surface. The flora, both before and after disinfection, was dominated by species of the Aspergillus glaucus group, the toxicogenic species A ochracues, Penicillium cyclopium, and P. viridicatum, and species of Alternaria, Cladosporium, and Fusarium. The toxicogenic species Aspergillus flavis, A. versicolor, Penicillium Citrinum, P. expansum, P. islandicum, and P. urticae were encountered less frequently. Of 209 species of Aspergillus and Penicillium screened for mycotoxin production on sterile rice substrate, 114 produced one or more of the following mycotoxins: A. flavus, aflatoxins; A. ochraceus, ochratoxins; A. nidulans, A. unguis, and A. versicolor, sterigmatocystin; P. cyclopium, penicillic acid; P. citrinum and P. viridicatum, citrinin; P. urticae, patulin and griseofulvin. Sterigmatocystin production by A. unguis is reported for the first time.     

Mode of Action of α, β Unsaturated Carbonyl Compounds

The toxicity of the α, β unsaturated carbonyl compounds (α, β UCC_s) (patulin, penicillic acid, parasorbic acid, tulipalin and plumbagin) towards Pythium sp. group F (Van Der Plaat -Niterinks 1981) was neutralized by the addition of an excess of cysteine. This suggests that the mode of action of these compounds could be due to a binding of the α, βi UCC_s to sulphydryl groups in enzymes or other macromolecules. Alcohol dehydrogenase (ADH), an enzyme with a sulphydryl group at the active site, was assayed spectrophotometrically and all the α, β UCC_s inhibited ADH.     

Mutagenicity to Salmonella typhimurium of some Aspergillus and Penicillium mycotoxins.

17 mycotoxins produced by various Aspergillus and Penicillium species were screened for their mutagenic activity to Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, both with and without metabolic activation. Austdiol, austocystins A and D, kojic acid and viridicatumtoxin were found to be mutagenic after metabolic activation, while austdiol was also mutagenic per se. Aflatoxin B1, sterigmatocystin and versicolorin A, which were used as positive controls were also mutagenic. No mutagenic activity was evident in the case of citrinin, cyclopiazonic acid, fumitremorgen B, griseofulvin, luteoskyrin, O-methylsterigmatocystin, mycophenolic acid, ochratoxin A, patulin, penicillic acid, secalonic acid D and TR2-toxin. A good relationship was found between the mutagenic activity, or lack of it, of most of the mycotoxins with existing data on carcinogenicity. Inadequate information on the carcinogenicity of austdiol, austocystins A and D, kojic acid and viridicatumtoxin precluded correlations with mutagenicity to S. typhimurium. The relationship between chemical structure and mutagenicity of the mycotoxins is discussed.     

Effect of preservatives on patulin production by Penicillium expansum.

Penicillium expansum has been grown on Capek-Dox medium using glucose and fructose as carbon source. Preservatives used in fruit processing and introduced in the medium were sorbic acid, formic acid, benzoic acid, SO2 and saccharose. Sulphur dioxide had a most inhibitory effect on mycelium growth and patulin production, formic acid concentration of 0.025% increased the amount of patulin by about 30% as compared to the culture with no preservatives. However its higher concentrations inhibited synthesis of this mycotoxin. Sorbic acid concentration of 0.1% stimulated the fungus strains examined in patulin synthesis but its highest amounts were detected using 0.0125% benzoic acid increased patulin secretion from 8 to 50% as compared to the control, depending on the strain examined. Saccharose concentration up to 50% clearly decreased patulin content in the medium until its total disappearance.     

Investigation of Penicillium chrysogenum Isolates for their Suitability as Starter Cultures

One hundred twenty three isolates ofP. chrysogenum were biologically tested in brine shrimp test and screened withStaphylococcus aureus for the detection of antibacterial activity. Furthermore, they were chemically examined (thin layer chromatographic method, TLC) for the synthesis of 8 mycotoxins (citrinin, cyclopiazonic acid, mycophenolic acid, patulin, penicillic acid, PR-toxin, ochratoxin A, and roquefortine). The results indicated that 85% of the tested isolates produce roquefortine and one isolate produces cyclopiazonic acid. Considering the results of the chemical assay for mycotoxins as well as the results of the brine shrimp test and the detection of antibacterial activity, 119 (97%) of the tested isolates are not suitable to be used as starter cultures for mould-fermented meats. The extracts of only 4 isolates were subjected to further biological tests in mice and the results indicated that only one isolate was non-toxinogenic.     

Type 2 Quorum Sensing Monitoring,Inhibition and Biofilm Formation in Marine Microrganisms

The quorum sensing (QS) dependent behaviour of micro-organisms, in particular expression of virulence genes, biofilm formation and dispersal, have provided impetus for investigating practical approaches to interfere with microbial QS. This study tests Halomonas pacifica and Marinobacter hydrocarbonoclasticus, two halophilic marine micro-organism, for their AI-2 dependent QS signalling and the effect of two well-known quorum-sensing inhibitors (QSIs), patulin and penicillic acid, on biofilm formation. We report, for the first time, the successful amplification of a putative luxS gene in H. pacifica using degenerated primers and AI-2 dependent QS as well as inhibition using QSIs. Penicillic acid had a strong inhibitory effect on AI-2 induction of H. pacifica at non-growth inhibitory concentrations, while patulin has an adverse effect only at the highest concentration (25 μM). QSIs effect on biofilm forming capability was isolate specific, with maximum inhibition at 25 μM of patulin in H. pacifica. In M. hydrocarbonoclasticus, no adverse effects were noted at any tested concentration of either QSIs. Detection of bioluminescence and the presence of a putative luxS gene provide biochemical and genetic evidence for the production of a signalling molecule(s) which is the essential first step in characterizing H. pacifica QS. This study highlights the importance of AI-2 dependent QS in a marine setting, not previously reported. It further suggests that QSI compounds must be selected in the specific system in which they are to function, and they cannot easily be transferred from one QS system to another.     

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.