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Non-specificity of nutritional substrate for ochratoxin A production by isolates of Aspergillus ochraceus.

Aspergillus ochraceus is an important contaminant of diverse substrates, such as cereals, coffee, grapes and derivates. This fungus produce a nephrotoxic metabolite, ochratoxin A (OTA), whose presence on food and feeds may be an important risk for animal and human health. The aim of this work was to evaluate the significance of the origin of A. ochraceus isolates on their OTA production patterns on different substrates (yeast extract sucrose (YES) broth, irradiated barley grains, irradiated green coffee beans and sterilized grapes) and under different environmental conditions. Results did not show a significant influence of the isolation source on OTA-production profiles by A. ochraceus isolates on several substrates, since the isolates which produced the highest OTA amounts in vitro (YES medium) were also the isolates with the highest OTA yields on the other substrates. Abiotic factors assayed (water activity, temperature and substrate) affected significantly OTA productions by A. ochraceus. Maximum OTA amounts were detected at 25 degrees C and 0.98 a(w) on all substrates tested. The highest OTA accumulations found on the different substrates were: green coffee beans (> 2 mg g(-1)), barley grains (approximately 1 mg g(-1)), YES medium (13.9 microg ml(-1)) and grape (approximately 3 ng g(-1)).

Aspergillus ochraceus↗

Immunochemical identification of fungi using polyclonal antibodies raised in rabbits to exoantigens from Aspergillus ochraceus.

Aspergillus ochraceus Wilhelm is a producer of highly toxic mycotoxin, ochratoxin A, and is known to contribute to the damage of grain on storage. Rabbit antibodies were produced against the exoantigens of A. ochraceus, and ELISA and immunoblotting procedures were used to characterize their reactivity towards fungal exoantigens of four Aspergillus, seven Penicillium, three Fusarium, one Mucor and one Alternaria species. The antibodies were specific for A. ochraceus and essentially cross-reacted neither with other fungi nor with the water-soluble grain components. These antibodies, therefore, have the potential of being utilized for the discriminative monitoring of A. ochraceus in foodstuffs and feeds.

Animals↗

Amplification and diversity analysis of ketosynthase domains of putative polyketide synthase genes in Aspergillus ochraceus and Aspergillus carbonarius producers of ochratoxin A.

The diversity of polyketide synthase (PKS) genes in Aspergillus ochraceus NRRL 3174 and Aspergillus carbonarius 2Mu134 has been investigated using different primer pairs previously developed for the ketosynthase (KS) domain of fungal PKSs. Nine different KS domain sequences in A. ochraceus NRRL 3174 as well as five different KS domain sequences in A. carbonarius 2Mu134 have been identified. The identified KS fragments were distributed in five different clusters on the phylogenetic tree, indicating that they most probably represent PKSs responsible for different functions.

Amino Acid Sequence↗

Ochratoxin production by the Aspergillus ochraceus group and Aspergillus alliaceus.

Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30 degrees C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 microg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 microg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.

Aspergillus ochraceus↗

[Preparations of extracellular proteinases from Aspergillus ochraceus 513 and Aspergillus alliaceus 7dN1].

Preparations of extracellular proteolytic enzymes with high anticoagulant activity resembling protein C activators were isolated from the culture liquids of Aspergillus ochraceus 513 and Aspergillus alliaceus 7 dN1 by precipitation with ammonium sulfate and subsequent purification from ammonium ions by gel filtration on a column with Sephadex G-25. The pH and temperature activity optima and stability of the proteolytic enzymes from A. ochraceus 513 and A. alliaceus 7 dN1 were determined.

Ammonium Sulfate↗

ZrO₂@C-based colorimetric/photothermal dual-mode immunosensor coupled with a novel monoclonal antibody for quantification of Aspergillus ochraceus biomass.

Aspergillus ochraceus contaminates agricultural products and produces nephrotoxic, carcinogenic ochratoxin A (OTA), posing severe food safety hazards. A dual-signal lateral flow immunochromatographic assay (dLFIA) based on ZrO₂@C nanoprobes was established for quantitative detection of A. ochraceus biomass. A novel monoclonal antibody (mAb 4B4) was prepared as the capture antibody to immobilize A. ochraceus mycelial lysate antigen on the test line, and a rabbit polyclonal antibody (pAb G2801) as the detection antibody to modify ZrO₂@C composites (synthesized via UiO-66 pyrolysis) into 200 nm colorimetric/photothermal nanoprobes. This dLFIA achieved limits of detection of 0.164 μg/mL (colorimetric) and 0.517 μg/mL (photothermal). This efficient and reliable method allows quantitative analysis of A. ochraceus biomass, which is suitable for routine monitoring of fungal contamination in agro-food matrices.

Antibodies, Monoclonal↗

Transformation of steroids by spores of microorganisms. I. Hydroxylation of progesterone by conidia of Aspergillus ochraceus.

Conidia of Aspergillus ochraceus convert progesterone into 11alpha-hydroxyprogesterone and 6beta, 11alpha-dihydroxyprogesterone. The conversion ability does not depend on the sporulation medium. Transformation depends on the strain and on the conidia concentration. Adaptation has never been observed. Age and storage of conidia, pH, aeration-agitation, nitrogen source, metal ions, chelating agents, and metabolic activators showed no great influence within wide limits. Mercuric chloride, p-chloromercuribenzoate, NaN(3), and KCN inhibit conversion. Glucose is necessary, but can be replaced completely by d(+)-xylose, and partially by some other carbon sources. The ratio mono-/di-hydroxyprogesterone is influenced by progesterone concentration and period of incubation; also, a mutant that accumulates only monohydroxyprogesterone has been produced. Conidia of A. ochraceus also hydroxylate a variety of steroids. Spores of certain streptomycetes, phycomycetes (mucors), ascomycetes, and deuteromycetes are active. Most reactions already observed with vegetative cells have been repeated with spores. In general, spores of a particular organism effect fewer reactions than its mycelium, and fewer products accumulate.

Aspergillus↗

Molecular analysis of variability within the toxigenic Aspergillus ochraceus species.

Genetic variability of Aspergillus ochraceus was examined at the DNA level. Based on the HaeIII-Bg/II generated mitochondrial DNA restriction profiles, most isolates could be classified into two distinct groups. These two groups could also be distinguished by the random amplified polymorphic DNA technique, and with telomeric PCR amplifications. Phylogenetic analysis of sequences of the intergenic transcribed spacer region of some of the strains resulted in a dendrogram with the same topology as that based on mitochondrial DNA and amplified DNA data. None of the isolates with type 2 mtDNA profiles produce ochratoxins. Some strains (e.g., A. ochraceus ICMP 939) displayed strain-specific mitochondrial DNA patterns, and their amplified DNA profiles were also different from all other A. ochraceus strains examined.

Aspergillus↗

Mixed cultivation of Trichoderma reesei and Aspergillus ochraceus for improved cellulase production.

Aspergillus ochraceus IMI 317911 was screened to be a high cellobiase producing strain (2.40 IU/ml). The mixed batch cultivation of Trichoderma reesei QM 9414/Rut C-30 and A. ochraceus IMI 317911 resulted in a balanced enzyme as compared to singly grown cultures. The milled rice straw (6%, w/v) as carbon source was found suitable for production of cellulases resulting in a Filter Paper Activity (FPA) of 1.83 IU/ml and a cellobiase production of 1.63 IU/ml after 7 days of stirred cultivation. An overall cellulase productivity of 10.89 IU/l/h and an enzyme ratio to cellobiase of 1.12 was achieved.

Aspergillus ochraceus↗

Transformation in Aspergillus ochraceus.

Mutants (lysine requiring) of Aspergillus ochraceus were kept under starvation conditions for 15 days and finally were treated with DNA of a 40-h-old culture of the wild strain. The donor DNA-treated mutant conidia were then grown on plates containing minimal medium at 28 degrees C for 4 days. The number of transformed cells was estimated by colony counting and hence percentage transformants. The transforming activity of the donor DNA was found to be inhibited by the action of heat and variation of pH, and also varied with the period of starvation and with the concentration of donor DNA.

Aspergillus ochraceus↗

Characterization of a novel microsomal glutathione S-transferase produced by Aspergillus ochraceus TS.

Purification and characterization of microsomal glutathione S-transferase produced by Aspergillus ochraceus TS are reported. The isozymes are located in microsomes and were active against 1-chloro-2,4-dinitrobenzene, ethacrynic acid, 1,2-dichloro-4-nitrobenzene, trans-4-phenyl-3-buten-2-one,p-nitrobenzyl chloride and bromosulphophthalein. They were inhibited by N-ethylmaleimide and bromosulphophthalein. The GST isozymes produced by Aspergillus ochraceus TS are indistinguishable in respect of their molecular mass both in native and denatured state. The subunit of the purified protein had an apparent M(r) of 11 kDa while molecular mass of the native protein is around 56 kDa. The substrate specificity and pI values of the isozymes were different. The GSTs produced by Aspergillus ochraceus TS fairly share functional properties with mammalian cytosolic isozymes.

Aspergillus ochraceus↗

Effect of water activity and temperature on mycelial growth and ochratoxin A production by isolates of Aspergillus ochraceus on irradiated green coffee beans.

Aspergillus ochraceus as a fungal contaminant and ochratoxin A (OTA) producer plays an important role in coffee quality. Temperature and water activity (a(w)) significantly influence mycelial growth and OTA production by isolates of A. ochraceus on green coffee beans. Maximum mycelial growth was found at 30 degrees C and 0.95 to 0.99 a(w). A marked decrease in growth rate was observed when temperature and a(w) were reduced. At 0.80 a(w), mycelial growth occurred only at 30 and 20 degrees C for one isolate. Maximum OTA production was found at 20 degrees C and 0.99 a(w). At 10 degrees C, OTA was not produced, regardless of a(w). Similarly, no OTA was detected at 0.80 a(w). OTA production ranged from the limit of detection (40 ng g(-1) of green coffee) to 17,000 ng g(-1) of green coffee. Significant intraspecific differences in mycelial growth and OTA production were found. Primary data for lag phases prior to mycelial growth under the influence of temperature and a(w) were modelled by multiple linear regression, and the response surface plots were obtained.

Aspergillus ochraceus↗

Production of ochratoxin A by Aspergillus ochraceus in a semisynthetic medium.

Aspergillus ochraceus NRRL 3174 produced 29 mg of ochratoxin A per 100 ml of nutrient medium consisting of 4% sucrose and 2% yeast extract. Ochratoxin A was the sole metabolite present in the chloroform extracts of the growth medium. Trace amounts of ochratoxin B were produced in a 1% yeast medium, and a comparatively large amount of ochratoxin B was produced in media containing 16 and 32% sucrose.

Aspergillus↗

[Proteolytic enzymes produced by Aspergillus ochraceus in relation to their plasma coagulating and fibrinolytic activities].

The paper describes some properties of coagulases and fibrinolytic enzymes isolated from a proteolytic complex produced by Aspergillus ochraceus HP-19 during submerged cultivation on a synthetic medium. Proteolytic enzymes with the plasmocoagulating activity hydrolyzed casein at the maximum rate at 45 degrees C and pH 8.0--10.0. The coagulases were stable at pH 5.0--7.0 and were rather resistant to low pH values. The enzymes were entirely inactivated at 55 degrees C within 20--30 min. The activity of the coagulases was inhibited with the ions of Cu, Co, Ag, Pb, Mn, Zn and Hg (1.10(-3) M) by 100, 91, 85, 50, 50, 38 and 25%, respectively. The coagulases were entirely inhibited with EDTA whereas PCMB and PMSF inhibited their activity only to a small extent. The mechanism for blood clotting with the coagulases of Aspergillus ochraceus HP-19 is presumed to consist in the activation of protrombin via its limited specific proteolysis. The fibrinolytic enzymes of Aspergillus ochraceus HP-19 had the optimal pH 8.5 for casein, were stable at pH 6.0, and entirely inactivated at 55 degrees C within 5 min. In contrast to coagulases, they were resistant to the action of heavy metal ions. The enzymes were stabilized by the ions of Ca. The activity of the fibrinolytic enzymes of Aspergillus ochraceus HP-19 was completely inhibited with PMSF. Therefore, they belong to the class of serine proteases.

Aspergillus↗

Characterization of progesterone 11 alpha-hydroxylase of Aspergillus ochraceus TS: a cytochrome P-450 linked monooxygenase.

The monooxygenase of Aspergillus ochraceus TS capable of 11 alpha-hydroxylation of progesterone has been resolved into three components and characterized as (i) cytochrome P450, (ii) NADPH-cytochrome P450-reductase and (iii) phosphatidyl choline. The 11 alpha-hydroxylase was observed to be NADPH dependent, and hydroxylation was enhanced by a NADPH regenerating system. This fungal monooxygenase has many features in common with that of mammalian liver microsomes. The role of mammalian cytochrome P450 inducers were tested for induction of 11 alpha-hydroxylase in Aspergillus ochraceus TS. The reductase has been partially purified.

Aspergillus↗

Molecular typing of Aspergillus ochraceus and construction of species specific SCAR-primers based on AFLP.

70 strains of Aspergillus ochraceus mainly isolated from Brazilian coffee related sources were investigated for genetic relatedness using automated laser fluorescence analysis of AFLP fragments. Cluster analysis of fingerprints revealed a very close relationship among most of the strains. Based on these results, a sub-set of characteristic A. ochraceus strains was chosen for the detection of marker sequences. These sequences were obtained from silver stained AFLPs separated on polyacrylamide gels. A number of bands characteristic for A. ochraceus were detected and cut out from the gels. DNA was reamplified, cloned and fragments were sequenced. Based on these sequences a set of SCAR PCR-primers was constructed. PCRs were optimised for specificity and subsequently tested against a panel of Aspergillus species. Using this approach a PCR specific for Aspergillus ochraceus was developed.

Aspergillus ochraceus↗

[Thrombolytic and pharmacodynamic properties of Aspergillus ochraceus protease].

The protease isolated from Aspergillus ochraceus exerts a strong thrombolytic action. In in vitro experiments and in tests with animals (mice, rats, dogs) it caused lysis of fibrinous clots. The thrombolytic effect of the protease is conditioned by its direct influence on the fibrin. Besides the thrombolytic action the protease, used within a definite ranges of doses, displays antithrombotic properties. The paper cites data on the acute toxicity (in mice, rats and rabbits) and on the influence of the protease on isolated organs and on the organism of the animals.

Animals↗

Mycotoxicity of Aspergillus ochraceus to chicks.

Five isolates of Aspergillus ochraceus, obtained from peanuts, were grown separately on sterile, moist corn for 14 days and fed to 1-day-old Babcock B-300 cockerels to evaluate their toxic effects. Two isolates were highly toxic, causing death of all birds during the 1st week of the experiment. Two isolates were moderately toxic, causing severe growth suppression with some deaths occurring throughout the 3-week test period. One isolate had no apparent effect. When the two most toxic isolates (diets) were diluted, survival time increased but severe growth suppression was evident. Postmortem examinations revealed a few small hemorrhages in the proventriculi of birds which died between the 2nd and 5th days. Emaciation, dehydration, and dry, firm gizzard linings were observed throughout the experiment. Extensive hepatic injury consisting of either fatty changes or necrotic foci was the principal microscopic finding. Suppression of bone marrow activity and depletion of lymphoid elements in the spleen and bursa of Fabricius were also found. The severity of the histopathological changes was directly related to the concentration of ochratoxin A in the diets.

Animals↗