Effect of Zinc, Copper, and Iron on Ochratoxin A Production

The effect of zinc, copper, and iron levels on production of ochratoxin A by Aspergillus ochraceus Wilhelm in a synthetic medium in a shake culture was investigated. Optimal concentrations of ZnSO₄, CuSO₄, and FeCl₃ for ochratoxin A production were 0.055 to 2.2 mg/liter, 0.004 to 0.04 mg/liter, and 1.2 to 24 mg/liter, respectively. Zinc and copper levels greater than optimum reduced the rate of ochratoxin accumulation without altering either glutamate or sucrose utilization. Ochratoxin A production was correlated with rapid utilization of sucrose by the fungus and decreasing pH of the medium. Most of the glutamic acid was removed from the medium prior to ochratoxin production. There was no correlation between mycelial dry weight and ochratoxin A production.     

Biosynthesis of Ochratoxin A

Biosynthesis of ochratoxin A by Aspergillus ochraceus Wilh. was investigated by radiolabeling experiments in which phenylalanine-1-¹⁴C and sodium acetate-2-¹⁴C were supplied to the fungus in sucrose-yeast extract medium. Results showed that phenylalanine was incorporated unaltered into the phenylalanine moiety of ochratoxin A, whereas the isocoumarin moiety of ochratoxin A was mostly derived via acetate condensation.     

Kinetics of ochratoxin A production in different Aspergillus species

Kinetics of ochratoxin A production was examined in a number of ochratoxin producing isolates representing different sections of the Aspergillus genus. Both weak and high ochratoxin producers were tested using immunochemical or high-performance liquid chromatograhic methods. All isolates were found to produce the highest amounts of ochratoxin A after 7-10 days of incubation. Ochratoxin production varied between 30 - 5 x l0(5) ng ml(-1) among the Aspergillus isolates tested. The A. albertensis and A. melleus isolates examined were found to produce ochratoxin A constitutively. A. albertensis produced the highest amounts of ochratoxin A at 30 degrees C after 7 days' incubation in YES liquid medium. Ergosterol content and ochratoxin production of A. albertensis cultures were in good correlation.     

Assessment of ochratoxin A and tenuazonic acid in Canadian ice-wines

Twenty-six samples of commercial ice-wine made from late-harvested grapes were assayed for the mycotoxins ochratoxin A and tenuazonic acid. Canadian wines originated in British Columbia (18) and Ontario (8). For comparison two German wines from Hesse were also studied. Four additional samples of research ice-wine originating in were also studied. In all wine samples, assays using immuno-affinity chromatography and fluorescence liquid chromatography indicated ochratoxin A below 0.15 μg/L, the limit of determination of the method. Tenuazonic acid was determined by solidphase micro-extraction and liquid chromatography and was below the limit of determination (70 μg/L) in all samples. The European Union food tolerance limit for ochratoxin A in wine is 2 μg/L. A tolerance for tenuazonic acid has not yet been established.     

Detoxification of ochratoxin A,a food contaminant: Prevention of growth of Aspergillus ochraceus and its production of ochratoxin A

The toxicity of ochratoxin A (OTA), a mycotoxin produced by fungi ofAspergillus orPenicillium genera is now well documented. Its nephrotoxicity, immunosuppression, teratogenicity, and carcinogenicity have been widely studied. Physical and biochemical methods have been studied to prevent these toxinogenicAspergillus andPenicillium from producing OTA, and/or to destroy the mycotoxin when already produced in a liquid or a solid medium. Repeated freezing at ? 20?C and thawing at + 26?C aleatory reduce OTA production in a liquid medium. Exposure to UV B for different periods of time is efficient in preventing OTA production in a liquid medium. Gamma-irradiation from 2 to 5 kGy gives good results in preventing the production of OTA or destroying it when already produced. Carboxypeptidase is very efficient at 5 units/50 ml in a liquid medium for cleaving the OTA already produced.     

Investigation of Ochratoxin A biosynthetic genes in<Emphasis Type="Italic">Penicillium verrucosum</Emphasis> by DDRT-PCR experiments: differential expression of OTA genes

A defined minimal medium has been developed which is either restrictive or permissive for the production of ochratoxin A byPenicillium verrucosum simply by changing the nitrogen and carbon source. The combination of ammonium/glycerin promoted, whereas the combination nitrate/glucose repressed ochratoxin production.In parallel mutants ofP verrucosum have been constructed by restriction endonuclease mediated integration, which were not able to produce ochratoxin. Different types of transformants, which either produce no detectable amounts of ochratoxin A but ochratoxin α, strains which produce only ochratoxin B and strains which produces none of these secondary metabolites, have been observed.     

Biochemical Characterization of Ochratoxin A-Producing Strains of the Genus Penicillium

In order to explore the biochemical scope of ochratoxin A-producing penicillia, we screened 48 Penicillium verrucosum isolates for the production of secondary metabolites. Fungal metabolites were analyzed by high-pressure liquid or gas chromatography coupled to diode array detection or mass spectrometry. The following metabolites were identified: ochratoxins A and B, citrinin, verrucolones, verrucines, anacines, sclerotigenin, lumpidin, fumiquinazolines, alantrypinones, daldinin D, dipodazine, penigequinolines A and B, 2-pentanone, and 2-methyl-isoborneol. By use of average linking clustering based on binary (nonvolatile) metabolite data, the 48 isolates could be grouped into two large and clearly separated groups and a small outlying group of four non-ochratoxin-producing isolates. The largest group, containing 24 isolates, mainly originating from plant sources, included the type culture of P. verrucosum. These isolates produced ochratoxin A, verrucolones, citrinin, and verrucines and had a characteristic dark brown reverse color on yeast extract-sucrose agar medium. Almost all of a group of 20 isolates mainly originating from cheese and meat products had a pale cream reverse color on yeast extract-sucrose agar medium and produced ochratoxin A, verrucolones, anacines, and sclerotigenin. This group included the former type culture of P. nordicum. We also found that P. verrucosum isolates and three P. nordicum isolates incorporated phenylalanine into verrucine and lumpidin metabolites, a finding which could explain why those isolates produced relatively lower levels of ochratoxins than did most isolates of P. nordicum.     

Brine Shrimp (Artemia salina L.) Larvae as a Screening System for Fungal Toxins

Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (mug/ml): aflatoxin G(1), 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid gamma-lactone gave no mortality at 10 mug/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B(1), diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 mug/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 mug/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and beta-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B(1) and B(2), kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and beta-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates.     

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.     

Fast determination of ochratoxin A in serum by liquid chromatography: comparison with enzymic spectrofluorimetric method

A rapid high-performance liquid chromatographic method for the determination of low concentrations of ochratoxin A in serum is described. The extraction procedure was simple and short, and liquid chromatographic analysis was carried out isocratically on a reversed-phase C₁₈ column, with methanol—water—acetic acid (30:70:1) as mobile phase and fluorescence detection (excitation at 336 nm, emission at 465 nm). The examined concentration range, 5–50 ng/ml ochratoxin, the recovery method was 87–94%, compared with 62–67% for the enzymic spectrofluorimetric method. The high-performance liquid chromatographic method was faster because the extraction procedure was shorter, and more sensitive so that small sample volumes could be used.     

New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated in Penicillium species. In Aspergillus species, only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producing A. carbonarius ITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in an Aspergillus species. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.     

In-vitro screening ofSaccharomyces strains for ochratoxin A removal from liquid medium

The aptitude of twentySaccharomyces sensu stricto strains to remove ochratoxin A from a synthetic medium containing 1.1 ng/mL, about half of the European Community limit, was evaluated using four to six mg of biomass (wet weight)/mL. Seven satins show high levels of ochratoxin A removal, 0.72-1.10 ng/mL, equivalent to 66–100% of the available toxin, and unitary removing ativity of 14.31–27.24 pg/mg of biomass. Further research will be carried out to study the mechanism of OTA removal and to confirm the ability of the most efficacious strains ofSaccharomyces sensu stricto to remove OTA from contaminated wort and grape must during alcoholic fermentation.     

Development of a Defined Medium for Growth of Cercospora rosicola Passerini

A simple synthetic liquid medium containing a single amino acid, glucose, salts, trace metals, and thiamine was developed for cultivation of Cercospora rosicola Passerini. Thiamine was shown to be important to growth. Culture of C. rosicola Passerini in a chemically defined medium makes possible studies of (+)-abscisic acid biosynthesis and regulation.     

Preparative high-performance liquid partition chromatography of proteins.

Methods for preparative high-performance liquid chromatography (hplc) of proteins are described. Both normal and reverse-phase chromatography were studied and adapted to the fractionation of proteins in quantities of up to 50 mg. Lichrosorb Diol was used as a “normal phase” for chromatography of hydrophobic proteins. Lichrosorb RP-8 was used for reversephase chromatography of proteins.     

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.     

Metabolism of ochratoxin A by primary cultures of rat hepatocytes.

Association of ochratoxin A with cultured rat hepatocytes occurs at 4 degrees C, and the saturation level in the medium is 0.3 mM ochratoxin A, with maximal binding after 60 min. At 37 degrees C the level of cell-associated ochratoxin A increased up to 6 h and remained at 2 nmol of toxin per mg of cell protein for 30 h. With increasing concentrations of ochratoxin A, increasing amounts of the toxin accumulated in the cells; saturation occurred at a concentration of 0.3 mM. Ochratoxin A was metabolized by hepatocytes at 37 degrees. (4R)-4-Hydroxyochratoxin A appeared in the medium at a maximal level (about 30 nmol/mg of cell protein) at an ochratoxin A concentration of 0.25 mM after 48 h of incubation. Small amounts of (4S)-4-hydroxyochratoxin A were detected only after incubation for 22 h or longer.     

Determination of ochratoxin A in urine and faeces of swine by high-performance liquid chromatography

Sensitive methods for the determination of ochratoxin A in urine and faeces of swine are described. The samples were extracted with chloroform at pH <2, and the extracts were cleaned up by a combination of solid-phase extraction and liquid—liquid partition. High-performance liquid chromatography with fluorescence detection was used for detection and determination. The detection limits were 0.3 ng/ml for urine and 1.5 ng/g for faeces. Recoveries of ochratoxin A from spiked samples of urine and faeces were 93% and 60%, respectively. Because of the low detection limit and the fast and relatively easy performance, the method for the determination of ochratoxin A in urine proved suitable for the estimation of possible contamination of live animals.     

Occurrence of ochratoxin A in herbal drugs of Indian origin — a report

This paper contains a report of occurrence of ochratoxin A in some common herbal medicines collected from different store-houses and shop-keepers of Bihar, India. Of 129 samples of 9 plants, 55 were found to be contaminated with various levels of ochratoxin A. The level of ochratoxin A was found maximal in barks ofHolarrhena antidysenterica (1.14 – 2.34 μg/g) whereas it was minimal in rhizomes ofTacca aspera (0.3 – 0.74 μg/g).Aspergillus ochraceus, A sulphureus and Penicillium viridicatum isolates obtained from drug samples were also examined for their toxigenic potentials. 19 isolates ofA ochraceus, 13 ofA sulphureus and 37 isolates ofP viridicatum were found to be toxigenic out of 67, 33, and 107 isolates, respectively. The ochratoxin A produced by Aochraceus was in the range of 0.09 to 2.44 μg/mL, byA sulphureus 0.1 to 1.76 μg/mL, and byP viridicatum 0.14 to 2.78 μg/mL of the culture filtrate.     

Metabolism of ochratoxin B and its possible effects upon the metabolism and toxicity of ochratoxin A in rats

A metabolic product was formed from ochratoxin B by rat liver microsomal fractions in the presence of NADPH. It was isolated from the incubation mixture by extraction, thin-layer chromatography, high-pressure liquid chromatography, and crystallization. On the basis of mass and nuclear magnetic resonance spectroscopy, the structure is suggested to be 4-hydroxyochratoxin B. The Km for the formation of 4-hydroxyochratoxin B was determined, and the hydroxylation of ochratoxin A was not altered by the presence of ochratoxin B. Rats were given ochratoxin A or B, or a mixture of both intraperitoneally. The ratios of the three metabolites, ochratoxin A, (4R)-4-hydroxyochratoxin A, and ochratoxin alpha, excreted in the urine did not change in the presence of ochratoxin B. Ochratoxin B was metabolized to 4-hydroxyochratoxin B and ochratoxin beta, but in a different ratio than for the ochratoxin A metabolites. When given intraperitoneally, ochratoxin beta was excreted within 24 h. In rats treated with ochratoxin A alone, the food intake was reduced by 50%, and histologically severe lesions, degeneration, and necrosis were observed in the proximal tubules. When ochratoxin A and B given in combination, the animals were clinically unaffected and histologically there was only slight damage of proximal tubules. These observations indicate that ochratoxin B considerably reduces the toxic effects of ochratoxin A.     

A liquid culture system for shoot proliferation and analysis of pharmaceutically active constituents of Catharanthus roseus (L.) G. Don

An efficient and cost effective micropropagation protocol using liquid medium was developed for Catharanthus roseus, a commercially important medicinal plant. Comparative analysis of shoot growth and proliferation in liquid Murashige and Skoog (MS) medium supplemented with different concentrations of cytokinins [6-Benzyladenine (BA), Kinetin (KN) and Thidiazuron (TDZ)] was conducted. Better response in terms of shoot proliferation, shoot diameter, number of leaves/shoot, number of branches/shoot, fresh weight and dry weight was observed in a liquid medium vis-à-vis solid medium. A sample of 20 ml of liquid medium supplemented with 5 ??M of BA was optimized for propagation of C. roseus by a liquid culture system. Among various concentrations of auxins tried, 1-Naphthaleneacetic acid (NAA) 5 ??M was found to be the best for root induction. Quantification of pharmaceutically important constituents (vincristine and vinblastine) and total alkaloid content of microshoots grown in solid and liquid medium as well as in vitro raised plants and mother plant was also conducted, hitherto unreported in this high-value medicinal plant. This work further lays the foundations for the shifting of plant production from small to commercial scale.