Biosynthesis of ochratoxins by Aspergillus ochraceus.

Shaken liquid fermentation of an isolate of Aspergillus ochraceus showed growth-associated production of ochratoxins A and B, followed by production of a related polyketide diaporthin. Later, between 150 and 250 h, mellein accumulated transitorily. In contrast, shaken solid substrate (shredded wheat) fermentation over 14 days produced mainly ochratoxins A and B (ratio ca. 5:1) in very high yield (up to 10 mg/g). In these systems experiments with 14C-labelled precursors and putative intermediates revealed temporal separation of early and late stages of the ochratoxin biosynthetic pathway, but did not support an intermediary role for mellein. The pentaketide intermediate ochratoxin beta was biotransformed very efficiently into both ochratoxins A and B, 14 and 19%, respectively. The already chlorinated ochratoxin alpha was only biotransformed significantly (4.85%) into ochratoxin A, indicating that chlorination is mainly a penultimate biosynthetic step in the biosynthesis of ochratoxin A. This was supported by poor (1.5%) conversion of radiolabelled ochratoxin B into ochratoxin A. Experiments implied that some ochratoxin B may arise by dechlorination of ochratoxin A.     

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°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.     

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.     

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°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 μg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 μg/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.     

Biosynthesis of diaporthin and orthosporin by Aspergillus ochraceus

Diaporthin and orthosporin were characterised from the fungus Aspergillus ochraceus D2306. Diaporthin was identified by high-resolution electron impact mass spectrometry and 1H and 13C NMR spectroscopy, from which new spectroscopic assignments were made. Orthosporin was also identified by mass spectrometry and both fungal metabolites are reported for the first time as co-metabolites and also as products of A. ochraceus. The methylation inhibitor ethionine affected production of both diaporthin and orthosporin in spite of no obvious methylation step in the biosynthesis of orthosporin, implying that extracellular orthosporin may arise by de-O-methylation of diaporthin. The biosynthetic origin of diaporthin was demonstrated by incorporation of [1-14C]acetate and [methyl-14C]methionine administered in early idiophase.     

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.     

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.     

Mellein and 4-Hydroxymellein Production by Aspergillus ochraceus Wilhelm

Mellein and 4-hydroxymellein are isocoumarin compounds produced by Aspergillus ochraceus Wilhelm. They are structurally similar to the dihydroisocoumarin moiety of ochratoxin A, a toxic metabolite of the same fungus, and they possibly have similar biological properties. Production of mellein and 4-hydroxymellein on synthetic media and natural solid substrates was determined. Several carbon and nitrogen sources supported production of these metabolites in stationary culture. Additional zinc and molybdenum increased production of both metabolites in stationary culture, but were not required for maximum production in shaken culture. Mellein and 4-hydroxymellein were produced on yellow corn, but neither was produced on wheat, peanuts, or soybeans.     

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.     

Biodegradation of lignocellulosic waste by Aspergillus terreus

Biodegradation of lignocellulosic waste by Aspergillus terreus is reported for the first time. This isolate produced 250 CMCase (carboxymethyl cellulase or endoglucanase) U.ml(-1) and biodegraded hay and straw during 3 days and the biomass production on straw was 5g.L(-1) dry weight from 0.25 cm2 inoculated mycellium. This strain secreted endocellulases and exocellulases in the culture medium, but some of the enzymes produced, remained cell membrane bound. Cell bound enzymes were released by various treatments. The highest amount of endoglucanase and exoglucanase was released when the cells were treated with sonication. Aspergillus terreus was added to two tanks containing sugar wastewater and pulp manufacturing waste, as a seed for COD removal. This fungus reduced the COD by 40-80 percent, also, ammonia was reduced from 14.5 mM to 5.6 mM in sugar beet wastewater. The effects of crude enzyme of this fungus for COD removal was studied.     

Biodegradation of lignocellulosic waste by Aspergillus terreus

Biodegradation of lignocellulosic waste by Aspergillus terreus is reported for the first time. This isolate produced 250 CMCase (carboxymethyl cellulase or endoglucanase) U.ml^-1 and biodegraded hay and straw during 3 days and the biomass production on straw was 5g.L^-1dry weight from 0.25 cm² inoculated mycellium. This strain secreted endocellulases and exocellulases in the culture medium, but some of the enzymes produced, remained cell membrane bound. Cell bound enzymes were released by various treatments. The highest amount of endoglucanase and exoglucanase was released when the cells were treated with sonication. Aspergillus terreus was added to two tanks containing sugar wastewater and pulp manufacturing waste, as a seed for COD removal. This fungus reduced the COD by 40–80 percent, also, ammonia was reduced from 14.5 mM to 5.6 mM in sugar beet wastewater. The effects of crude enzyme of this fungus for COD removal was studied.     

Detection and quantification of Aspergillus ochraceus in green coffee by PCR

AIMS: The aim of this study was to detect and quantify DNA of the ochratoxinogenic fungus Aspergillus ochraceus in green coffee and to compare the results with the ochratoxin A content of naturally contaminated samples. METHODS AND RESULTS: A DNA extraction protocol based on a combination of ultrasonification and a commercial kit was tested for the recovery of fungal DNA. PCR and real-time PCR protocols were established for the detection of A. ochraceus. Sensitivity of the PCR was checked by the addition of inoculated green coffee and pure fungal DNA to uncontaminated green coffee samples. The A. ochraceus DNA content of 30 naturally contaminated green coffee samples was determined and compared with the ochratoxin A concentrations. CONCLUSIONS: Aspergillus ochraceus can be rapidly and specifically detected in green coffee by PCR. A positive correlation between the ochratoxin A content and the DNA quantity was established. Significance and Impact of the Study: This work offers a quick alternative to the conventional mycological detection and quantification of A. ochraceus in green coffee.     

An improved 11 alpha-hydroxylation of progesterone by Aspergillus ochraceus TS.

1. The stereospecific hydroxylation of progesterone exclusively to its 11 alpha-hydroxy derivative by Aspergillus ochraceus TS is reported. 2. There is no secondary metabolite (6beta, 11 alpha-dihydroxyprogesterone) formed even when the transformation was attempted with different concentrations of the substrate (200 microgram/ml-200 mg/ml) for prolonged with Zn2+ at a concentration of 50 microgram/ml identical results were obtained. 3. Similar results were also obtained at different pH values of the culture medium.     

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.     

利用赭曲霉NG1203羟基化齐墩果酸的研究

考察了利用赭曲霉Aspergillus ochraceusNG1203进行C11α-羟基化齐墩果酸生化反应的条件。得出了菌株摇瓶培养最佳培养基配方(g.L-1):葡萄糖20,玉米浆25,酵母膏3,K2HPO41.5,pH 6.0。菌株培养20 h,加入2 mg.L-1的齐墩果酸利于诱导羟基化酶的产生。菌株在28℃下以150 r.min-1振荡培养24 h,加入底物的乙醇溶液,使转化液中齐墩果酸的初始质量浓度达100 mg.L-1,转化液中乙醇体积分数最终达3%。经96 h转化,齐墩果酸转化率可达到10.12%。通过HPLC1、H NMR和13C NMR分析,结果表明产物为C11α-羟基齐墩果酸。     

Biochemical characterization and potential for textile dye degradation of blue laccase from Aspergillus ochraceus NCIM-1146

In our study, we produced intracellular blue laccase by growing the filamentous fungus Aspergillus ochraceus NCIM-1146 in potato dextrose broth. The enzyme was then purified 22-fold to a specific activity of 4.81 U/mg using anion-exchange and size exclusion chromatography. The molecular weight of purified laccase was estimated as 68 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme showed maximum substrate specificity toward 2,2′-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid than any other substrate. The optimum pH and temperature for laccase activity were 4.0 and 60°C, respectively. The purified enzyme was stable up to 50°C, and high laccase activity was maintained at pH 5.0 ∼ 7.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol, and L-cysteine. Purified laccase decolorized various textile dyes within 4 h in the absence of redox mediators. HPLC and FTIR analysis confirmed degradation of methyl orange. The metabolite formed after decolorization of methyl orange was characterized as p-N,N′-dimethylamine phenyldiazine using GCMS.     

Bioconversion of Progesterone by the Activated Immobilized Conidia of Aspergillus ochraceus TS

Progesterone was transformed to its 11α-hydroxy derivative (100% e.e) by the activated immobilized conidia of Aspergillus ochraceus TS. The immobilized preparation retained 79% of free conidial activity. The immobilized conidia, activated by nutrients, exhibited an increase in 11α-hydroxylation, and it was free of the side product 6β, 11α-dihydroxy progesterone. The half life and turnover of immobilized and activated immobilized conidia were 14 and 12 days and 187 and 416 μmoles of the product/g of conidia respectively. The pH and temperature profiles of the free conidia remained unaltered after immobilization and activation. Some germination of conidia inside the matrix owing to incubation with nutrients was detected by scanning electron microscopy. Received: 18 March 1999 / Accepted: 1 July 1999     

Two-Liquid phase reactor studies of 11alpha-hydroxylation of progesterone by Aspergillus ochraceus

The 11alpha-hydroxylation of progesterone by free cells of Aspergillus ochraceus has been examined in a reactor with an aqueous and a natural oil phase. As the proportion of oil is increased, the point of inversion from a continuous aqueous phase is affected by the cell concentration, as is the optimum ratio of the phases for conversion. High oil ratios are not productive because of poor mass transfer to the concentrated cells in the aqueous phase. The cells are stable in the presence of oleic acid for at least 24 h.     

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 pl values of the isozymes were different. The GSTs produced by Aspergillus ochraceus TS fairly share functional properties with mammalian cytosolic isozymes.