Penicillic Acid Research Articles

Complex etiology, prophylaxis and hygiene control in mycotoxic nephropathies in farm animals and humans.

Various etiological factors contributing to the development of mycotoxic nephropathy in farm animals and humans are reviewed. The possible synergistic effect between ochratoxin A (OTA) and other mycotoxins, as penicillic acid (PA) and fumonisin B(1) (FB(1)), contributing to this nephropathy is also considered and discussed. The most convenient ways of prophylaxis and various preventive measures against OTA contamination of feeds or foods are reviewed. A reference is made concerning the most successful methods of veterinary hygiene control in the slaughterhouses in order to prevent the entering of OTA in commercial channels with a view to human health. The economic efficacy of these prophylactic procedures is also considered. An evaluation of human exposure to OTA is made.

LC–MS/MS multi-method for mycotoxins after single extraction, with validation data for peanut, pistachio, wheat, maize, cornflakes, raisins and figs

Mycotoxin analysis is usually carried out by high performance liquid chromatography after immunoaffinity column cleanup or in enzyme-linked immunosorbent assay tests. These methods normally involve determination of single compounds only. EU legislation already exists for the aflatoxins, ochratoxin A and patulin in food, and legislation will come into force for deoxynivalenol, zearalenone and the fumonisins in 2007. To enforce the various legal limits, it would be preferable to determine all mycotoxins by routine analysis in different types of matrices in one single extract. This would also be advantageous for HACCP control purposes. For this reason, a multi-method was developed with which 33 mycotoxins in various products could be analysed simultaneously. The mycotoxins were extracted with an acetonitrile/water mixture, diluted with water and then directly injected into a LC–MS/MS system. The mycotoxins were separated by reversed-phase HPLC and detected using an electrospray ionisation interface (ESI) and tandem MS, using MRM in the positive ion mode, to increase specificity for quality control. The following mycotoxins could be analysed in a single 30-min run: Aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 toxin, HT-2 toxin, α-zearalenol, α-zearalanol, β-zearalanol, sterigmatocystin, cyclopiazonic acid, penicillic acid, fumonisins B1, B2 and B3, diacetoxyscirpenol, 3- and 15-acetyl-deoxynivalenol, zearalanone, ergotamin, ergocornin, ergocristin, α-ergocryptin, citrinin, roquefortin C, fusarenone X, nivalenol, mycophenolic acid, alternariol and alternariol monomethyl ether. The limit of quantification for the aflatoxins and ochratoxin A was 1.0 µg kg−1 and for deoxynivalenol 50 µg kg−1. The quantification limits for the other mycotoxins were in the range 10–200 µg kg−1. The matrix effect and validation data are presented for between 13 and 24 mycotoxins in peanuts, pistachios, wheat, maize, cornflakes, raisins and figs. The method has been compared with the official EU method for the determination of aflatoxins in food and relevant FAPAS rounds. The multi-mycotoxin method has been proven by the detection of more than one mycotoxin in maize, buckwheat, figs and nuts. The LC–MS/MS technique has also been applied to baby food, which is subject to lower limits for aflatoxin B1 and ochratoxin A, ergot alkaloids in naturally contaminated rye and freeze-dried silage samples.

Rapid analysis of fungal cultures and dried figs for secondary metabolites by LC/TOF-MS

A liquid chromatography–time-of-flight mass spectrometry (LC/TOF-MS) method has been developed for profiling fungal metabolites. The performance of the procedure in terms of mass accuracy, selectivity (specificity) and repeatability was established by spiking aflatoxins, ochratoxins, trichothecenes and other metabolites into blank growth media. After extracting, and carrying out LC/TOF-MS analysis, the standards were correctly identified by searching a specially constructed database of 465 secondary metabolites. To demonstrate the viability of this approach 11 toxigenic and four non-toxigenic fungi from reference collections were grown on various media, for 7–14 days. The method was also applied to two toxigenic fungi, A. flavus (200–138) and A. parasiticus (2999–465) grown on gamma radiation sterilised dried figs, for 7–14 days. The fungal hyphae plus a portion of growth media or portions of dried figs were solvent extracted and analysed by LC/TOF-MS using a rapid resolution microbore LC column. Data processing based on cluster analysis, showed that electrospray ionization (ESI)–TOF-MS could be used to unequivocally identify metabolites in crude extracts. Using the elemental metabolite database, it was demonstrated that from culture collection isolates, anticipated metabolites. The speed and simplicity of the method has meant that levels of these metabolites could be monitored daily in sterilised figs. Over a 14-day period, levels of aflatoxins and kojic acid maximised at 5–6 days, whilst levels of 5-methoxysterigmatocystin remained relatively constant. In addition to the known metabolites expected to be produced by these fungi, roquefortine A, fumagillin, fumigaclavine B, malformins (peptides), aspergillic acid, nigragillin, terrein, terrestric acid and penicillic acid were also identified.

Inhibition of NF-Kappa B Activation by Penicillic Acid and Dihydropenicillic Acid Isolated from Fungi

In the course of our screening of NF-KB inhibitors from microbial secondary metabolites, we isolated two 2(5H)-furanones, penicillic acid and dihydropenicillic acid, from the culture filtrate of Penicillium. Both penicillic acids inhibited LPS-induced NO production and NF-KB activation. They did not inhibit protein synthesis. Interestingly, penicillic acid inhibited advanced glycation end product (AGE)-induced NO production.

Interactions of Organic Anion Transporters and Organic Cation Transporters With Mycotoxins

Mycotoxins are secondary metabolites of moulds that which exert adverse effects in humans and animals. It is known that direct cellular toxicity is often associated with increased cellular accumulation of toxic compounds, and membrane transport may be the first fundamental stage in the development of the cytotoxicity. To elucidate the entry pathway for mycotoxins into cells, we have investigated the interactions of human and rat organic anion transporters (hOATs/rOats) and human organic cation transporters (hOCTs) with mycotoxins using cells stably expressing hOATs/rOats/hOCTs. The mycotoxins tested were aflatoxin B1, α-zearalenol, citrinin, citrioveridine, cyclopiazonic acid, fumonisin B1, gliotoxin, patulin, penicillic acid, rubratoxin B, and zearalenone. These mycotoxins inhibited organic anion uptake mediated by hOAT1-4, and organic cation uptake mediated by hOCT1-2. By comparing the IC50values of mycotoxins for hOATs, it was found that hOAT1 and hOAT3 exhibited higher affinity interactions with mycotoxins than hOAT2 and hOAT4. There was no interspecies difference between humans and rats for the interactions of OATs with mycotoxins except that of OAT3 with rubratoxin B. Finally, we observed that hOAT1-4 and hOCT1-2 mediated the uptake of aflatoxin B1. In conclusion, hOATs and hOCTs interacted with various mycotoxins. Considering the localization of hOATs/rOats and hOCTs, it was suggested that these transporters were the possible entrance pathway for mycotoxins in kidney and liver, leading to the induction of adverse effects in humans and rats.

English

The toxigenesis of one Penicillium aurantiogriseum and one Penicillium viridicatumisolates was investigated. Sterile culture filtrates of both fungi had a clear antibacterial effect only against Bacillus subtilis. The effect on B. subtilis varied with amount of filtrate used and temperature. The antibacterial activity of chloroform extracts varied with the nature of media used to grow the fungi. Different mycotoxins were identified in the fungal cultures using thin-layer chromatography. P. aurantiogriseum was found to produce penicillic acid, terrestric acid and aurantiamine, while penicillic acid, terrestric acid, brevianamide A and xanthomegnin were produced by P. viridicatum.   Key words: P. aurantiogriseum, P. viridicatum, mycotoxins, antibacterial activity.

Morphological and Ultrastructural Studies for the Biological Action of Penicillic Acid on Some Bacterial Species

Morphological and Ultrastructural Studies for the Biological Action of Penicillic Acid on Some Bacterial Species

Ergosterol and mycotoxins in grain dusts from fourteen Belgian cereal storages: A preliminary screening survey

AbstractGrain dusts from farms and storage companies are generally used in animal feeding. They can give rise to airborne dust in the environment of workers or accidentally contaminate the following stored grains. Because dusts are potential mycotoxin‐rich materials, this preliminary screening survey was undertaken on citreoviridin, citrinin, cyclopiazonic acid, deoxynivalenol, gliotoxin, helvolic acid, mycophenolic acid, nivalenol, ochratoxin A, patulin, penicillic acid, secalonic acid D, sterigmatocystin, zearalenol and zearalenone. Furthermore, ergosterol was determined as a fungal growth marker. Fourteen grain dusts collected from farms and storage companies in Belgium were assayed and toxins co‐occurred at uneven distributions with wide ranges of concentrations. Median concentrations exceeded 1 µg g−1 for penicillic acid, gliotoxin, deoxynivalenol, helvolic acid, mycophenolic acid, patulin, sterigmatocystin and zearalenol. Using the median values, an assessment of worker exposure indicated that mycotoxin uptake through dust inhalation may simultaneously contribute to 0.5, 0.5, 0.7 and 0.1% of the respective tolerable daily intake of ochratoxin A, patulin, deoxynivalenol and zearalenone. In spite of these low values, the question of multi‐contamination of grain dusts and, consequently, exposure to several mycotoxins should not be underestimated. Moreover, inhalation of contaminated airborne aerosols can represent an additional route of exposure which has not been exhaustively investigated so far. Copyright © 2007 Society of Chemical Industry

Well-known quorum sensing inhibitors do not affect bacterial quorum sensing-regulated bean sprout spoilage

To investigate the potential of quorum sensing inhibitors (QSI) as food preservative agents in a food product, where bacterial spoilage is controlled by quorum sensing (QS). The effects of well-known QSI were tested on spoilage phenotypes and on QS-regulated genes of a bean sprout spoiling bacterial isolate (Pectobacterium A2JM) in laboratory substrates and in a bean sprout model system. The acylated homoserine lactones (AHL) analogues PenS-AHL and HepS-AHL decreased the specific protease activity of Pectobacterium A2JM in broth but did not reduce the expression of a QS-regulated secretion protein, and were without effect on soft rot of bean sprouts. The QSI ProS-AHL, furanone C-30, patulin, penicillic acid and 4-nitropyridine-N-oxide did not have any effect on protease activity, on gene expression or bean sprout appearance at nongrowth inhibitory concentrations. Extracts from garlic and bean sprouts induced the QS system of Pectobacterium in bean sprouts and a broth system, respectively. Among the several well-known QSI compounds, only PenS-AHL and HepS-AHL, inhibited QS-regulated protease activity of Pectobacterium A2JM in broth cultures, but had no effect on bean sprout spoilage. The 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.

Four psychrotolerant species with high chemical diversity consistently producing cycloaspeptide A, Penicillium jamesonlandense sp. nov., Penicillium ribium sp. nov., Penicillium soppii and Penicillium lanosum

Penicillium jamesonlandense is a novel species from Greenland that grows exceptionally slowly at 25 degrees C and has an optimum temperature for growth of 17-18 degrees C. The novel species is more psychrotolerant than any other Penicillium species described to date. Isolates of this novel species produce a range of secondary metabolites with a high chemical diversity, represented by kojic acid, penicillic acid, griseofulvin, pseurotin, chrysogine, tryptoquivalins and cycloaspeptide. Penicillium ribium, another novel psychrotolerant species from the Rocky Mountains, Wyoming, USA, produces asperfuran, kojic acid and cycloaspeptide. Originally reported from an unidentified Aspergillus species isolated from Nepal, cycloaspeptide A is reported here for the first time from the two novel Penicillium species and two known psychrotolerant species with high chemical diversity, Penicillium soppii and Penicillium lanosum. All species, except P. ribium, produce a combination of cycloaspeptide and griseofulvin. However, P. ribium (3/5 strains) produced the precursor to griseofulvin, norlichexanthone. The type strain of Penicillium jamesonlandense sp. nov. is DAOM 234087(T) (=IBT 21984(T) = IBT 24411(T) = CBS 102888(T)) and the type strain of Penicillium ribium sp. nov. is DAOM 234091(T) (=IBT 16537(T) = IBT 24431(T)).

Cytotoxicity assessment of gliotoxin and penicillic acid in Tetrahymena pyriformis

Various studies have documented the associations between mold exposure and effects on health. Mycotoxins, which occur in spores and mold fragments, can be involved in processes that have pathological effects, such as adynamia of the immune system, recurrent infections of the respiratory tract, or asthma. Using Tetrahymena pyriformis, a single-cell organism well established as a suitable model for human respiratory epithelium-cell functionalities, we investigated dose-response relationships of the mycotoxins gliotoxin and penicillic acid. Our study focused on the viability (cell count, MTT assay), energy levels (adenosine-5'-triphosphate content), energy-providing processes (MTT reduction per cell), and cell respiration (oxygen consumption). Both mycotoxins acted as cytotoxins in a dose-dependent manner. Gliotoxin had a stronger inhibitory effect (EC50 0.38 microM) than did penicillic acid (EC50 343.19 microM). The energy-providing processes were not inhibited or were only weakly inhibited under the influence of gliotoxin, whereas penicillic acid caused stimulation of the physiological parameters. Summarizing the results, it is clear that the two investigated mycotoxins must have different modes of action. They are not only different in the strength of their toxic effects but also in a variety of physiological aspects. In addition, T. pyriformis showed differences in its ability to overcome the negative effects of particular mycotoxin exposures.

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 Na 2HPO 4, among the metal ions and phosphate salts tested, increased the production of penicillic acid (≈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) Na 2HPO 4], maximum penicillic acid concentration (≈9.40 mg ml −1) and cell mass (≈17.4 g l −1) were obtained after 12 days.

Inactivation of GDP-mannose dehydrogenase from Pseudomonas aeruginosa by penicillic acid identifies a critical active site loop

The pathogenic bacterium Pseudomonas aeruginosa synthesizes alginate as one of a group of virulence factors that are produced during infections. The enzyme GDP-mannose dehydrogenase catalyzes the committed step in alginate biosynthesis. We show here that penicillic acid is an irreversible inactivator of GDP-mannose dehydrogenase. Inactivation occurs with a rate constant of 0.39 ± 0.01 mM −1 min −1 at pH 8.0, and does not exhibit saturation behavior. Partial protection from inactivation is afforded by GDP-mannose, but not by the other substrate, NAD +. GMP and NAD + together provide complete protection against inactivation. Analysis by mass spectrometry confirmed that the enzyme is alkylated at multiple cysteine residues by penicillic acid, including Cys 213, Cys 246, and the active site cysteine, Cys 268. However, the pH dependence of the inactivation rate suggested that alkylation of a single cysteine residue is sufficient to inactivate the enzyme. The C268A mutant protein was also susceptible to inactivation by penicillic acid. The presence of NAD + and GMP provided partial protection of Cys 246 and Cys 268, and almost complete protection of Cys 213. Cys 213 is located on a helix that forms part of the binding pocket for GDP-mannose, and forms a hydrogen bond with Asn 252. Asn 252 is located on a loop that surrounds GDP-mannose. The C213A mutant enzyme exhibits a V max that is 1.8-fold greater than the wild-type enzyme, suggesting that the interaction between Cys 213 and Asn 252 helps to hold the loop in place during catalysis, and that opening the loop to release product is partially rate-limiting. Cys 246 is adjacent to the GDP-mannose binding loop, and its alkylation may also interfere with loop movement.

Identity and effects of quorum-sensing inhibitors produced by Penicillium species

Quorum sensing (QS) communication systems are thought to afford bacteria with a mechanism to strategically cause disease. One example is Pseudomonas aeruginosa, which infects immunocompromised individuals such as cystic fibrosis patients. The authors have previously documented that blockage of the QS systems not only attenuates Ps. aeruginosa but also renders biofilms highly susceptible to treatment with conventional antibiotics. Filamentous fungi produce a battery of secondary metabolites, some of which are already in clinical use as antimicrobial drugs. Fungi coexist with bacteria but lack active immune systems, so instead rely on chemical defence mechanisms. It was speculated that some of these secondary metabolites could interfere with bacterial QS communication. During a screening of 100 extracts from 50 Penicillium species, 33 were found to produce QS inhibitory (QSI) compounds. In two cases, patulin and penicillic acid were identified as being biologically active QSI compounds. Their effect on QS-controlled gene expression in Ps. aeruginosa was verified by DNA microarray transcriptomics. Similar to previously investigated QSI compounds, patulin was found to enhance biofilm susceptibility to tobramycin treatment. Ps. aeruginosa has developed QS-dependent mechanisms that block development of the oxidative burst in PMN neutrophils. Accordingly, when the bacteria were treated with either patulin or penicillic acid, the neutrophils became activated. In a mouse pulmonary infection model, Ps. aeruginosa was more rapidly cleared from the mice that were treated with patulin compared with the placebo group.

Determination of selected mycotoxins in mould cheeses with liquid chromatography coupled to tandem with mass spectrometry

A simple and feasible method is described for analysing nine mycotoxins in cheese matrix. The method involves liquid extraction followed by high performance liquid chromatographic separation and mass spectrometric detection of the analytes, and allows the determination of aflatoxins B1, B2, G1, G2 and M1, ochratoxin A, mycophenolic acid, penicillic acid and roquefortine C simultaneously. Average recoveries of the mycotoxins from spiked samples at concentration levels of 5–200 µg kg−1 ranged from 96–143%. Within-day relative standard deviations at these concentration levels varied from 2.3–12.1%. The limit of quantification for aflatoxin M1 was 0.6 µg kg−1 and for the other compounds 5 µg kg−1. The method developed was applied for analysing these mycotoxins in blue and white mould cheeses purchased from Finnish supermarkets. Roquefortine C was detected in all of the blue mould cheese samples in concentrations of 0.8–12 mg kg−1. One blue cheese contained also 0.3 mg kg−1 mycophenolic acid. The other investigated mycotoxins were absent in the samples.

Phytotoxins from the fungus Malbranchea aurantiaca

Bioassay-directed fractionation of an ethyl acetate extract from cultures of the fungus Malbranchea aurantiaca led to the isolation of two phytotoxic compounds, namely, 1-hydroxy-2-oxoeremophil-1(10),7(11),8(9)-trien-12(8)-olide ( 1) and penicillic acid ( 2). The structure of 1 was established by spectroscopic and X-ray crystallographic analyses. Metabolites 1 and 2 caused significant inhibition of radicle growth of Amaranthus hypochondriacus with IC 50 values 6.57 and 3.86 μM, respectively. In addition, 1 inhibited activation of the calmodulin-dependent enzyme cAMP phosphodiesterase (IC 50 = 10.2 μM).

Fungal populations and mycotoxins in silage in Assiut and Sohag governorates in Egypt, with a special reference to characteristic Aspergilli toxins

Forty silage samples were collected from Assiut and Sohag governorates in Egypt to measure the presence of fungal population in silage. Forty-three species and 2 species varieties belonging to 17 genera were isolated using glucose Czapek's and Sabouraud's dextrose agar media at 28 degrees C. The most prevalent genera were Aspergillus (57.5 and 100 of the samples), Penicillium (100 and 55%) on the two mentioned media, respectively. Also, Fusarium oxysporum and Gibberella fujikurori were recovered in moderate incidences. Mycotoxin profiles were also determined in these samples: Aflatoxins showed the highest incidence rates of occurrence, it occurred in 22.5% of all samples analyzed. Other mycotoxins were detected from all samples (T2 toxins and sterigmatocystin at incidence of 7.5 and 5%, respectively). The screening of the characteristics mycotoxins of different isolates of Aspergillus isolated from silage samples was tested. The results clarified that some mycotoxins (aflatoxins-aspergillic acid-beta nitro propionic acid-cyclopiazonic acid-kojic acid and sterigmatocystin) were produced by some isolates of A. flavus. Some isolates of A.fumigatus could produce gliotoxin and verrucologen. All of A. niger isolates tested were able to produce kojic acid. One isolate of A. ochraceous formed ochratoxin A and other isolate produced penicillic acid. Concerning A. terreus isolates, the results showed that 5 isolates were able to produce citrinin and 4 isolates had ability to produce patulin. A. versicolor isolates showed the ability to produce ochratoxin A.

Characterization and regulation of new secondary metabolites fromAspergillus ochraceusM18 obtained by UV mutagenesis

UV irradiation of Aspergillus ochraceus NRRL 3174 conidia led to stable mutations in ochratoxin and penicillic-acid pathways. These mutants, especially M18, produced an unexpectedly large number of new metabolites. Two new compounds were purified by TLC and HPLC and their chemical structures were determined. They are 2,10-dimethyl 4-hydroxy-6-oxo-4-undecen-7-yne (1) and 4-(3-methyl-2- butenyl) oxy 1-phenyl acetic acid (2). Compound 1 is very active against Gram-positive bacteria, such as Staphylococcus aureus and Bacillus subtilis, but inactive against Gram-negative bacteria, fungi, and yeasts. However, compound 2 has no antibiotic activity. The production of 1 was generally associated with growth, whereas that of compound 2 was dissociated from growth. The biosynthesis of these 2 metabolites was influenced by the sources of carbon and nitrogen.

Penicillium frequentans isolated from Picea glehnii seedling roots as a possible biological control agent against damping‐off

AbstractPicea glehnii seedlings are affected by damping‐off fungi in nurseries. The aims of this study were (1) to isolate fungi grown in the seedling rhizosphere in forest soil of P. glehnii, (2) to select fungi that produce antifungal compounds against Pythium vexans, and (3) to examine whether or not selected fungi can protect seedlings from P. vexans. Penicillium frequentans from Picea glehnii seedling roots produced antibiotic penicillic acid. Penicillic acid did not cause significant phytotoxicity to the seedlings. Penicillium frequentans increased the average percentage of surviving seedlings when inoculated together with Pythium vexans, but the increase was not significant. Vigorous mycelial growth of P. frequentans around seedling roots seems to be one of the mechanisms for protection, but the amount of penicillic acid was too low to show antifungal activity in the seedling rhizosphere.

Combined effects of selected Penicillium mycotoxins on in vitro proliferation of porcine lymphocytes

The in vitro effect of combinations of the Penicillium mycotoxins citrinin (CIT), cyclopiazonic acid (CPA), ochratoxin A (OTA), patulin (PAT), penicillic acid (PIA) and roquefortine C (RQC) on mitogen induced lymphocyte proliferation was determined using purified lymphocytes from six piglets. Dose-response curves for each mycotoxin and mycotoxin combinations were generated. The combined effects of toxin pairs based on IC20 were illustrated in isobole diagrams and statistically calculated. OTA and CIT elicited a synergistic effect. Four toxin pairs elicited additive effects, four pairs less-than-additive effects and six pairs independent effects. Thus, the majority of toxin pairs tested produced lower combined effects than an additive effect. The results indicate that the sum effect of all toxins is less than that from the summation of concentrations of the individual compounds, adjusted for differences in potencies.