Penicillium Viridicatum Research Articles

Conversion of the mycotoxin citrinin into dihydrocitrinone and ochratoxin A by Penicillium viridicatum

The mycotoxin citrinin, produced by Penicillium viridicatum, was found to be unstable in broth culture. This instability was investigated using the techniques of 14C-labelling, mass spectrometry and thin layer chromatography. It was concluded that intracellular factors released by P. viridicatum were responsible for the instability of the mycotoxin. The major products resulting from the breakdown of citrinin were identified as 3,4-dihydro-6,8-dihydroxy-3,4,5-trimethyl-isocoumarin-7-carboxylic acid (dihydrocitrinone) and ochratoxin A.

Penicillium viridicatum, Penicillium verrucosum, and production of ochratoxin A.

The taxonomy of the important mycotoxigenic species Penicillium viridicatum and P. verrucosum was reviewed to clarify disagreements relating to the three P. viridicatum groups erected by Ciegler and coworkers (A. Ciegler, D. I. Fennell, G. A. Sansing, R. W. Detroy, and G. A. Bennett, Appl. Microbiol. 26:271-278, 1973) and the mycotoxins produced by them. Cultures derived from the types of these two species and authentic cultures from each group and from many other sources were examined culturally, microscopically, and for mycotoxin production. It was concluded that P. viridicatum group II has affinities with P. verrucosum and not with P. viridicatum, as indicated by J. I. Pitt in the 1979 monograph (The Genus Penicillium and Its Teleomorphic States Eupenicillium and Talaromyces). As a result of this study it can now be unequivocally stated that the mycotoxins ochratoxin A and citrinin are not produced by P. viridicatum. Of species in subgenus Penicillium, only P. verrucosum is known to produce ochratoxin A.

Variation in nuclear DNA content in an ex-type isolate ofPenicilliummeasured by continuous flow microfluorimetry

The conidial DNA content of Penicillium viridicatum was examined using flow cytometry. Comparisons made between distinct cell lines derived from single conidial isolates showed reproducible differences in DNA content between 2 morphologically identifiable variants (red and white), These observations probably result from a stable aneuploid state in one of these variants.

Variation in nuclear DNA content in an ex-type isolate of Penicillium measured by continuous flow microfluorimetry

The conidial DNA content of Penicillium viridicatum was examined using flow cytometry. Comparisons made between distinct cell lines derived from single conidial isolates showed reproducible differences in DNA content between 2 morphologically identifiable variants (red and white), These observations probably result from a stable aneuploid state in one of these variants.

Morphological and biochemical variation in single isolates of Penicillium

Variations in morphological and biochemical characteristics in single strains were investigated in ex-type isolates of Penicillium viridicatum, P. aurantiocandidum and P. crustosum. Singleconidium isolates gave rise to two stable colony types (W-type and R-type) in all three species. A further unstable colony type (O-type) was seen only in P. viridicatum. Variations were seen between the different lines in both morphological and biochemical characters which are of taxonomic significance. The O-type colonies gave rise to significant numbers of abnormally large uninucleate conidia, the progeny of which showed properties in common with both stable lines. The variations observed may be present in nature or could have arisen in preservation. It is recommended that the extent of any variation present in single-conidium isolates should be considered in future descriptions of conidial fungi in order to clarify taxonomically significant characters. Similarly, the preservation and maintenance of reference cultures must be critically considered if they are to remain representative of their taxa.

Inhibition by antimicrobial food additives of ochratoxin A production by Aspergillus sulphureus and Penicillium viridicatum.

The effects of antimicrobial food additives on growth and ochratoxin A production by Aspergillus sulphureus NRRL 4077 and Penicillium viridicatum NRRL 3711 were investigated. At pH 4.5, growth and toxin production by both A. sulphureus and P. viridicatum were completely inhibited by 0.02% potassium sorbate, 0.067% methyl paraben, 0.0667% methyl paraben, and 0.2% sodium propionate. At pH 5.5, 0.134% potassium sorbate and 0.067% methyl paraben completely inhibited growth and ochratoxin A production by both fungi. Sodium bisulfite at 0.1%, the maximum level tested, was found to inhibit growth of A. sulphureus and P. viridicatum by 45 and 89%, respectively. Toxin production was inhibited by 97 and 99%, respectively. Sodium propionate (0.64%) at pH 5.5 inhibited growth of A. sulphureus and P. viridicatum by 76 and 90%, respectively. Toxin production was inhibited by greater than 99% for each fungus. Antimicrobial agents were ranked as to effectiveness by comparing the level required for complete inhibition of ochratoxin A production to the highest antimicrobial agent level normally used in food. At pH 4.5, the most effective inhibitor of growth and toxin production was potassium sorbate, followed by sodium propionate, methyl paraben, and sodium bisulfite, respectively, for both fungi. However, at pH 5.5, the most effective antimicrobial agents for inhibiting ochratoxin production were methyl paraben and potassium sorbate, followed by sodium propionate. Sodium bisulfite was not highly inhibitory to these toxigenic fungi at the higher pH value tested.

Postharvest production of ochratoxin A by Aspergillus ochraceus and Penicillium viridicatum in barley with different protein levels.

The production of ochratoxin A (OA) in barley by Aspergillus ochraceus and Penicillium viridicatum was measured at 12 and 25 degrees C. The grain had been fertilized with various amounts of nitrogen fertilizer (0, 90, or 240 kg/ha) and contained (at crop maturity) 9.1, 10.4, or 12.0% protein, respectively. The production of OA by both fungi increased as the protein concentration increased. Glutamic acid and proline were enriched relative to other amino acids as the protein concentration increased. The differences in OA production could not be explained by a differential effect of protein or amino acids on fungal growth in barley. However, glutamic acid and proline enhanced OA production in liquid cultures of both A. ochraceus and P. viridicatum.

Simultaneous isolation of xanthomegnin, viomellein, rubrosulphin, viopurpurin, and brevianamide A by preparative HPLC

A method for the isolation of xanthomegnin, viomellein, rubrosulphin, viopurpurin, and brevianamide A from Penicillium viridicatum (DSM 2447) is described. After extraction, HPLC was performed with a preparative silicagel column, eluted with toluene / ethyl acetate / formic acid (27/9/1, v/v/v) and dichloromethane / acetic acid (9/1, v/v). The toxins were detected with a UV-monitor. It was possible to isolate them in an absolutely pure state. The described method is operationally simple and very efficient.

The production of ochratoxin A and citrinin in barley

AbstractThe production of ochratoxin A by Aspergillus ochraceus and of ochratoxin A and citrinin by Penicillium viridicatum growing on previously sterilised barley for 200 days at 5, 10 and 20°C and a water activity of 0.85 is reported. A. ochraceus did not grow at 5°C, multiplied slowly at 10°C but did not produce toxin. At 20°C the organism multiplied more quickly and produced ochratoxin after 19 days, which slowly disappeared over the next 150 days. P. viridicatum grew slowly at 5°C but did not produce any toxin. It multiplied at 10°C and produced ochratoxin A which was only detectable during the period from 100 to 150 days. At 20°C both ochratoxin A and citrinin were produced. Ochratoxin A was detected after 10 days and was still present after 240 days, whereas citrinin was produced in large quantities between 118 and 129 days and then rapidly disappeared.

A selective and indicative medium for groups of Penicillium viridicatum producing different mycotoxins in cereals.

A medium, pentachloronitrobenzene-rose bengal-yeast extract-sucrose agar (PRYES), for the isolation of moulds occurring during storage of cereals has been developed and compared with other selective media. The basal medium is yeast extract agar containing 15% sucrose (w/v). In addition to the sucrose content further selective measures include the addition of antibacterial antibiotics chloramphenicol and chlortetracycline (50 mg/l), the fungicides rose bengal (25 mg/l each), and pentachloronitrobenzene (1 g/l) and a low incubation temperature (20 degrees C). Members of the Mucorales were completely inhibited, and fast-growing species of other moulds were slightly inhibited, allowing important storage moulds to develop. The important ochratoxin A and citrinin-producing Penicillium viridicatum group II was indicated by a typical violet brown reverse on PRYES. Producers of xanthomegnin and viomellein (P. viridicatum group I and P. aurantiogriseum) were indicated on PRYES by their yellow reverse and obverse colours. The medium was used for screening 40 samples of barley, and moulds with the characteristic colours were all identified as the species mentioned above.

Isolation of xanthomegnin from Penicillium viridicatum by preparative high-pressure liquid chromatography.

A method was developed for the production and purification of xanthomegnin from Penicillium viridicatum (NRRL 6430) cultured on rice at 15 degrees C for 29 days. Liquid-liquid extraction followed by high-pressure liquid chromatography afforded 440 mg of crystalline xanthomegnin per kg of rice.

Toxic effects of ochratoxin A and citrinin, alone and in combination, on chicken embryos.

The embryotoxic potential of ochratoxin A and citrinin was studied after administering, either subgerminally or intraamniotically, single mounting doses of the mycotoxins to chicken embryos on days 2, 3, and 4. The beginning of the embryotoxicity dose range was found to be between 0.01 to 0.05 microgram for ochratoxin A and 1 to 10 micrograms for citrinin. The maximum response to both mycotoxins occurred after administration on day 3. In addition to significant growth retardation of fetuses, exencephaly, microphthalmia, cleft beak, reduction deformities of the limbs, and abdominal wall and ventricular septal defects were encountered on day 8 of incubation. When 4 micrograms of citrinin was constantly added to ochratoxin A administered in the dose range of 0.03 to 0.5 microgram, a strictly additive effect was seen. It may be supposed that citrinin produced together with ochratoxin A in some strains of Penicillium viridicatum Westling does not potentiate the clear-cut embryotoxic action of the latter mycotoxin.

TOXICITY, MUTAGENICITY AND TERATOGENICITY OF BREVIANAMIDE, VIOMELLEIN AND XANTHOMEGNIN; SECONDARY METABOLITES OF PENICILLIUM VIRIDICATUM

The three major secondary metabolites of Penicillium viridicatum, brevianamide, xanthomegnin, and viomellein were tested for mutagenicity by the Salmonella/microsome test and for toxicity and teratogenicity by the chick embryo test. None were mutagenic. Viomellein was mildly toxic and teratogenic, xanthomegnin was the most toxic and was not a teratogen, and brevianamide was not toxic yet was the most teratogenic.

Production of ochratoxin A in barley by Aspergillus ochraceus and Penicillium viridicatum: effect of fungal growth, time, temperature, and inoculum size.

Moistened barley was inoculated with 1.4 x 10(3) and 1.4 x 10(5) spores, respectively, from ochratoxin A-producing strains of Aspergillus ochraceus and Penicillium varidicatum. To estimate fungal tissue in the barley, the amount of glucosamine was followed for 28 days at 10 and 25 degrees C. Ochratoxin A was also followed during the same period and under the same conditions. The data show that ochratoxin A could be detected 4 to 6 days after inoculation at 25 degrees C, and the maximal accumulation of ochratoxin A was observed 28 days after inoculation. After 28 days at 25 degrees C, the quantities of ochratoxin A were between 7 and 46 micrograms/g of grain. At 10 degrees C only P. viridicatum produced ochratoxin A. The results indicated that production of ochratoxin A is not associated with rapid increase of glucosamine in the barley.

Depletion of Sorbate from Different Media During Growth of Penicillium Species

Penicillium cyclopium, Penicillium roqueforti, Penicillium viridicatum, Penicillium puberulum, Penicillium cyclopium (atypical strain), Penicillium crustosum and Penicillium lanoso-viride were isolated from spoiled cheese. These molds grew and depleted sorbate from media when the chemical was present initially at a concentration of up to 3,000, 10,000, 6,000, 12,000, 12,000, 7,000 and 3,000 ppm, respectively. A combination of paper chromatography and spectrophotometry was used to determine amounts of residual sorbate. Seventy-one to 100% of sorbate present initially was depleted from media by the various molds during 4–20 days of incubation at 21°C and 22–48 days at 4°C. The substrate influenced growth of mold and depletion of sorbate, but uniform behavior was not observed for all the Penicillium species studied. For example, presence of 3,000 ppm of sorbate plus 1% casein in the medium inhibited P. cyclopium and P. lanoso-viride but not the other five species. Concentration of sorbate (3,000 – 9,000 ppm) plus temperature (4, 12, 21 °C) were important for inhibitory action of the preservative on P. cyclopium, P. viridicatum, P. crustosum and P. lanoso-viride but not P. puberulum, P. cyclopium (atypical strain) which grew at 4°C and depleted sorbate when the initial concentration was up to 9,000 ppm and P. roqueforti which grew at up to 6,000 ppm at the same temperature.

Ochratoxin A Production by Some Fungal Species in Relation to Water Activity and Temperature

The effects of water activity (aw) and temperature on growth of and ochratoxin A (OA) production by strains of Aspergillus ochraceus, Penicillium cyclopium, and Penicillium viridicatum were investigated. On agar media in which the aw had been adjusted by addition of sucrose or glycerol, the minimum aw values for OA production by A. ochraceus, P. cyclopium and P. viridicatum lay between 0.83–0.87, 0.87–0.90, and 0.83–0.86, respectively. At 24 C, optimum aw values for OA production by A. ochraceus and P. cyclopium were 0.99 and 0.95–0.99, respectively, whereas that of P. viridicatum varied and was 0.95 and 0.99 for the two strains tested. At optimum aw, the temperature range for OA production by A. ochraceus was 12–37 C, whereas that of P. cyclopium and P. viridicatum was 4–31 C. Optimum temperature for OA production by A. ochraceus was 31 C, whereas that of P. cyclopium and P. viridicatum was 24 C. On Edam cheese of 0.95 aw the minimum temperature for OA production by P. cyclopium was 20 C. On barley meal, P. viridicatum produced maximal quantities of OA at 0.97 aw and could produce OA at temperatures as low as 12 C.

Mycological survey of selected health foods

A survey was conducted to compare the total viable fungal content and the number of different mold species encountered in 10 types of health foods labeled organically grown and in the same foods without such a label. The foods were wheat flour, corn meal, brown rice, figs, split peas, pinto beans, soybeans, walnuts, pecans, and peanuts. Results showed no consistent difference in either the total viable fungal content or the number of different mold species encountered between the labeled and unlabeled foods. Two genera of yeasts (Rhodotorula and Saccharomyces) and 22 gener of molds, including more than 65 species, were encountered. The mold flora was dominated by Aspergillus glaucus, Aspergillus niger, Aspergillus flavus, Aspergillus candidus, Penicillium cyclopium, and Penicillium viridicatum. Isolates of the genera Alternaria, Cladosporium, Fusarium, and Helminthosporium also occurred in certain foods. At least 10 toxicogenic species of Aspergillus and Penicillium were encountered. A total of 87 cultures of these species, all isolated from health foods, were screened for laboratory production of their respective toxins. Toxin production potential of these 87 cultures did not differ from that of cultures of the same species isolated from conventional foods.

Structures of xanthoviridicatin D and xanthoviridicatin G, metabolites of penicillium viridicatum: application of proton and carbon-13 NMR spectroscopy

The structures of xanthoviridicatin D and xanthoviridicatin G, toxic metabolites of Penicillium viridicatum , have been shown to be 3 and 4a , respectively by spectroscopic studies.

Sensitivity to Natamycin (Pimaricin) of Fungi Isolated in Cheese Warehouses

The sensitivity to natamycin of molds and yeasts isolated in cheese warehouses where natamycin has been used for various periods was determined. After several years of continuous use of natamycin no natamycin-insensitive molds and yeasts were found. In 26 strains of molds isolated in cheese warehouses it was not possible under laboratory conditions to decrease the sensitivity for natamycin. Besides the sensitivity of fungi to natamycin, production of mycotoxins by the isolated molds was investigated. Strains of Penicillium viridicatum, Aspergillus versicolor, and Penicillium cyclopium isolated in the warehouses produced, under experimental conditions, ochratoxin A, sterigmatocystin, and penicillic acid, respectively.

Isolation and identification of xanthomegnin, viomellein, rubrosulphin, and viopurpurin as metabolites of penicillium viridicatum.

Four of the metabolites of Penicillium viridicatum 66-68-2 grown on rice cultures were isolated and identified as xanthomegnin, viomellein, rubrosulphin, and viopurpurin. Melting points, elemental analysis, and infrared, ultraviolet, and field desorption and electron impact mass spectra of the isolated compounds were consistent with values reported in the literature for these compounds. In addition, diacetate and triacetate derivatives were prepared, and the chemical and physical analyses of the derivatives were also consistent with literature data. Proton magnetic resonance spectroscopy and thin-layer chromatography were also used for the additional identification of selected compounds.