Preparation of monoclonal antibody against penicillic acid (PA) and its application in the immunological detection.

The high content of Penicillic acid (PA) in the feed pose threat to human health and cause serious losses to economic wealth through the enrichment effect of the food chain. The reliable and rapidly detection of PA is of significant importance to ensure food safety. In this study, indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and immunochromatographic test strips (ICTS) were established for PA determination based on anti-PA mAb secreted by 4H9 cell line. The linear range of ic-ELISA detection was 0.12-1.95 µg/mL, and the limit of detection (LOD) was 0.03 µg/mL. Then, conventional gold nanospheres (AuNS) with the average diameter of 20 nm were synthetized and AuNS-based strip was developed for rapidly detection of PA. The visual LOD (vLOD) of the AuNS-based strip was 3.9 µg/mL and the assay time of visual evaluation was less than 10 min without any instrument. To enhance the signal sensitivity of the ICTS, the larger size (about 85 nm) of gold nanoflowers (AuNFs) was prepared in our work, and was used as higher signal reporter to establish the AuNF-based strip for PA determination. Fortunately, the vLOD of AuNF-based strip was 0.97 µg/mL, which was approximately 4-fold lower than that of traditional AuNS-based strip. In summary, the rapid and sensitive immunoassays established in this study could be applied to detect and analyze the contamination of PA toxin in real food samples.

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

AIM: 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). METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

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 Vmax 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.

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 microg 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 microg kg(-1) and for the other compounds 5 microg 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.

Mycotoxin-forming ability of two Penicillium roqueforti strains in blue moldy tulum cheese ripened at various temperatures.

Isolated and identified toxigenic and nontoxigenic Penicillium roqueforti (PR) strains from moldy tulum cheeses were inoculated into tulum cheeses made in the laboratory and ripened at 5 and 12 degrees C. Mycotoxin (patulin, penicillic acid, PR toxin, and roquefortine) formation in the control and mold-inoculated cheeses were detected by thin-layer chromatography on the first through fourth months of ripening. Patulin, penicillic acid, and PR toxin were not detected in the experimental cheeses. Only roquefortine was detected in cheese inoculated with the toxigenic strain of the mold and ripened at 5 and 12 degrees C on the third and first months of ripening, respectively. Toxin in cheeses ripened at 5 and 12 degrees C was 2.1 to 2.4 and 2.1 to 3.8 mg/kg cheese, respectively.

Direct detection using the Drosophila DNA-repair test and isolation of a DNA-damaging mycotoxin, 5,6-dihydropenicillic acid, in fungal culture.

The Drosophila DNA-repair test was used in an attempt to detect fungal production of DNA-damaging mycotoxins without an extraction process. 29 species of fungi, 13 Aspergillus, 12 Penicillium and four Fusarium were inoculated directly to a Drosophila medium, and the larvae were then bred in the mouldy medium. Production of DNA-damaging mycotoxin was detected directly by counting the decrease in the survival of DNA-repair-deficient flies. With the direct detection method, Aspergillus ochraceus, A. parasiticus and A. versicolor produced DNA-damaging mycotoxins. The same results were obtained with the mouldy medium extract using the standard DNA-repair test. The direct detection method was convenient for surveying the fungal production of DNA-damaging mycotoxins. The extracts of A. parasiticus and A. versicolor contained aflatoxin B1 and sterigmatocystin, respectively. The DNA-damaging compound in the extract of A. ochraceus was isolated and purified to clear, colourless 'needles'. With nuclear magnetic resonance-mass spectroscopy spectra, the compound was confirmed to be 5,6-dihydropenicillic acid, the DNA-damaging potency of which has not been previously reported.

Contamination of dairy products by fungal metabolites.

The contamination of dairy products with various mycotoxins or other undesirable fungal metabolites can be attributed a.o. to ingestion of contaminated feed or the accidental development of molds and by consequence the excretion of fungal metabolites into the intermediate product. Different dairy products of commercial origin were examined: milk powder, reconstituted infant milk powder, and cheese. In addition to that, environmental factors contributing to the formation of the undesired fungal metabolites were studied. It was found that the presence of mycotoxins in dairy products is more related to the environmental factors causing mold growth on dairy products than to the ingestion of moldy feed by the cow.

High-performance liquid chromatographic determination of the mycotoxins patulin, penicillic acid, zearalenone and sterigmatocystin in artificially contaminated cocoa beans.

A high-performance liquid chromatographic (HPLC) method has been developed to allow the determination of patulin, penicillic acid, sterigmatocystin and zearalenone in samples of cocoa beans. When this method is combined with a method that was reported earlier for the determination of ochratoxin A [W. J. Hurst and R. A. Martin, Jr., J. Chromatogr., 265 (1983) 353], it allows for the determination of five mycotoxins. Samples were extracted with an acidic acetonitrile solution, partitioned with hexane to remove fat interferences and then partitioned with chloroform to remove the toxin containing fraction. Interferences were removed by the use of a bonded phase column followed by the final HPLC determination step, which uses a cyano column with a hexane-1-propanol-acetic acid mobile phase with dual channel UV detection at 245 and 280 nm. The method exhibits good linearity, accuracy and precision.

Mycotoxins in cereal grain. Part 12. Contamination with ochratoxin A and penicillic acid as indicator of improper storage of cereal grain.

Formation of ochratoxin A and penicillic acid in wheat kernels at 6 moisture levels: 15, 18, 21, 24, 27 and 30% at 15 degrees C has been examined during 4 months of storage. The minimum time for formation of significant amount (0.5-1 mg/kg) of ochratoxin A and penicillic acid (6-8 mg/kg) in stored grain has been found for the various water contents as follows: 18%-16 weeks, 21%-6 weeks, 24% and more - 2 weeks. At 15% of moisture content formation of ochratoxin A and penicillic acid was not observed until 4 months of storage.

Chemical confirmatory tests for ochratoxin A, citrinin, penicillic acid, sterigmatocystin, and zearalenone performed directly on thin layer chromatographic plates.

Published tests have been improved and a new procedure is described for chemical confirmation of mycotoxins directly on thin layer plates. After extraction and preliminary cleanup chromatography with n-hexane or chloroform, the mycotoxins ochratoxin A, citrinin, penicillic acid, sterigmatocystin, and zearalenone were easily separated by thin layer chromatography (TLC) using toluene-ethyl acetate-90% formic acid (6 + 3 + 1) developing solvent. In chemical confirmatory methods, the developed chromatogram was exposed to vapors of pyridine, acetic anhydride, or a mixture, or the mycotoxins were over-spotted. With this treatment, ochratoxin A, citrinin, penicillic acid, and zearalenone were converted to new fluorescent compounds, and observed under 365 nm light after re-chromatography with the same developing solvent. Sterigmatocystin was confirmed chemically using TLC plates impregnated with 0.6N H2SO4 or 10% oxalic acid in methanol. The described procedures are satisfactory for confirming mycotoxins present in standards, artificially contaminated grain samples (barley, corn, oat, rye, and wheat), and extracts from both fungal cultures and naturally contaminated grain samples.

High pressure liquid chromatographic determination of penicillic acid in chicken tissues.

Penicillic acid (PA) is a mycotoxin with reported cytotoxic, cardiotoxic, and carcinogenic activity and it can occur in high concentration in corn. The occurrence of PA in contaminated poultry feed represents a potential public health hazard. A reverse phase high pressure liquid chromatographic (HPLC) method is proposed for determining PA residues in chicken tissues. Optimization of chromatography was achieved for PA using a mobile phase consisting of acetonitrile: H2O. PA was detected by ultraviolet absorption at 254 nm, identified by retention time, and quantitated by peak area integration. Blood, parenchymal tissues, muscle, and alimentary tract contents were homogenized, sonicated, and acid treated followed by extraction with ethyl acetate and analysis by HPLC. Acute oral dosing of chickens with PA over a range of 50 to 550 mg/kg body weight resulted in detectable levels of the mycotoxin (confirmed by gas liquid chromatography) in gizzard muscle and contents, liver, kidney, heart, and intestinal contents. This method should prove useful both for the rapid and sensitive detection of PA residues in poultry and in further studies on the distribution and metabolism of this mycotoxin.

Mycotoxins in cereal grain. Part I. Ochratoxin, citrinin, sterigmatocystin, penicillic acid and toxigenic fungi in cereal grain.

Contamination with ochratoxin A mainly and also with citrinin, penicillic acid and sterigmatocystin was observed in moldy cereal grain samples (wheat, rye, and barley), during 1975-1978 years. The levels of cereal grain contamination in various years were very different. However usually during two months after harvest percentage of contaminated samples was 5-7% and ochratoxin A content not higher than 140 microgram/kg. During storage of grain with high moisture content slow increase of contamination level was observed-particularly during January and February - to level 1-3 mg/kg. Cereal grain from commercial channels was contaminated with fungi spores sometimes up to 10(9) spores per gram. Aspergillus and Penicillium were predominating species. Between 69 fungi isolates typical for barley kernels 13 were procedures of ochratoxin, sterigmatocystin, penicillic acid and F-2 toxin. Results for wheat and rye will be published later.

High-performance liquid chromatographic analysis of the mycotoxin penicillic acid and its application to biological fluids.

Penicillic acid (PA) is a carcinogenic food contaminant produced by several food-borne fungi. PA was resolved as a sharp peak by reversed-phase high-performance liquid chromatography on a small-particle (10 micrometer) column in 3-4 min by an elution system composed of acetonitrile (AN), water and glacial acetic acid (AC) with detection by ultraviolet (UV) absorbance at 254 nm. Peak height and peak area were related linearly to the amount of PA injected over a range of 5-500 ng. Reproducibility of retention time, peak height and peak area was demonstrated. The lower detection limit was 5 ng in two elution systems [AN-H2O-AC(45:55:2), flow-rate (F) 1 ml/min; AN-H2O-AC(40:60:2), F = 1.2 ml/min] and 10 ng in a third system [AN-H2O-AC(25:75:2), F = 1.6 ml/min]. Based on the sensitivity and separation of PA from (40:60:2), F = 12 ml/min; urine, AN-H2O-AC (25:75:2), F = 1.6 ml/min. Good recovery (89-98%) over a range of 1-50 microgram/ml of PA was obtained from PA-spiked plasma samples treated first with 25% HPO3 followed by extraction with chloroform. A single peak detected either by UV absorbance or by radioactivity was obtained when plasma samples spiked with [14C]PA were extracted. Good recovery (92-105%) of PA also was obtained from spiked urine and bile samples.

[A screening method for the determination of various mycotoxins in food (author's transl)]. / Verfahren zur Bestimmung verschiedener Mykotoxine in Lebensmitteln.

A simple analytical method for multiple mycotoxins was developed for detecting aflatoxin B1, B2, G1, G2, M1, and M2, sterigmatocystin, penicillin acid, ochratoxin A and patulin. These mycotoxins were extracted with ethyl acetate. The extract was cleaned up by chromatography on silica mini-column. Each fraction was separated by thin-layer chromatography. The final evaluation of the TLC-plates was carried out by fluorodensitometry. A considerable increase in fluorescence intensity was achieved by spraying the plates with paraffin after development. The detection limits of fluorescent mycotoxins and the fluorescent derivates of penicillic acid and sterigmatocystin were lowered to 1:10 up to 1:100. Spots of patulin were measured by fluorescence quenching.

Thin layer chromatographic determination of aflatoxins, ochratoxins, sterigmatocystin, zearalenone, citrinin, T-2 toxin, diacetoxyscirpenol, penicillic acid, patulin, and penitrem A.

A general method is described for determining 16 mycotoxins in mixed feeds and other food products used in the manufacture of these feedstuffs. The mycotoxins are extracted and cleaned up by extracting with solvents of different pH. Thin layer chromatography is used to separate the toxins; toxins are then quantitated by the limit detection method. The minimum detectable concentration of mycotoxins in various products is: aflatoxin B1 or G1, 4--5 micrograms/kg; ochratoxin A or ethyl ester A 140--145 micrograms/kg; citrinin 600--750 micrograms/kg; zearalenone, 410--500 micrograms/kg; sterigmatocystin, 140--145 micrograms/kg; diacetoxyscirpenol, 2400--2600 micrograms/kg; T-2 toxin, 800--950 micrograms/kg; patulin, 750--800 micrograms/kg; penitrem A 14,000--14,500 micrograms/kg; penicillic acid 3400--3650 micrograms/kg.

A study on the toxicity of spontaneously molded bread.

Molds of geni Penicillium, Aspergillus and Paecilomyces were found in spontaneously molded Finnish bread. Patulin was detected in 91% of 23 samples analysed in concentrations ranging from 27 to 138 microgram/kg. The toxin was found in dark bread in higher amounts than in white. Neither aflatoxins (12 samples) nor ochratoxin A (10 samples) were detected. Penicillic acid was found in one of five samples. No significant changes were found in the haemoglobin or leucocyte counts of rats kept on feed containing extracts of the molded bread. Extracts from bread contaminated with A niger were more toxic and less palatable than extracts from the other samples. The feeding test indicated a relatively low toxicity of molded bread.