Penicillic acid in fruits: method development, validation by liquid chromatography-tandem mass spectrometry and survey in southern China.

BACKGROUND: Penicillic acid (PA) is produced by Aspergillus spp. and Penicillium spp., which are common postharvest and storage fungi of fruits. PA can be of concern for human health because of its toxicity and high fruit consumption by the population. However, no data on PA occurrence in various fruits have yet been reported. A quick, easy, cheap, effective, rugged and safe (QuEChERS) approach for PA determination in various fruits was developed and applied to explore PA incidence in fruits. RESULTS: The modified QuEChERS procedure with extraction by ethyl acetate and purification by multi-walled carbon nanotubes (MWCNTs), primary secondary amine (PSA) and octadecyl silane (C18) was established to determine PA in various fruits by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The average recoveries were 72.9-102.2% and relative standard deviations (RSDs) were 1.3-7.9%. A total of 161 fruits samples, including kiwi, apple, peach, grape and mandarin/orange, were collected in southern China. The incidence of PA in fruits was 14.9% and the levels of PA contamination were 0.200-0.596 µg kg-1 . Our results suggested that orange/mandarin, grape and kiwi were favorable matrices for Aspergillus spp. and Penicillium spp. to produce PA, rather than peach and apple. CONCLUSION: To the best of our knowledge, this is the first report about PA contamination in various fruits in China. Our study emphasizes the necessity of the current established method, which could be used for continuous monitoring of PA and reducing the health risk to Chinese consumers. © 2020 Society of Chemical Industry.

Cryptic Secondary Metabolites from the Sponge-Associated Fungus Aspergillus ochraceus.

The fungus Aspergillus ochraceus was isolated from the Mediterranean sponge Agelas oroides. The initial fermentation of the fungus on solid rice medium yielded 16 known compounds (4⁻19). The addition of several inorganic salts to the rice medium mainly influenced the accumulation of these secondary metabolites. Fermentation of the fungus on white bean medium yielded the new waspergillamide B (1) featuring an unusual p-nitrobenzoic acid as partial structure. Moreover, two new compounds, ochraspergillic acids A and B (2 and 3), which are both adducts of dihydropenicillic acid and o- or p-aminobenzoic acid, were isolated from the co-culture of the fungus with Bacillus subtilis. Compound 2 was also detected in axenic fungal cultures following the addition of either anthranilic acid or tryptophan to the rice medium. The structures of the new compounds were established by 1D and 2DNMR experiments as well as from the HRMS data. The absolute configuration of 1 was elucidated following hydrolysis and derivatization of the amino acids using Marfey's reagent. Viomellein (9) and ochratoxin B (18) exhibited strong cytotoxicity against the A2780 human ovarian carcinoma cells with IC50 values of 5.0 and 3.0 µM, respectively.

Type 2 quorum sensing monitoring, inhibition and biofilm formation in marine microrganisms.

The quorum sensing (QS) dependent behaviour of micro-organisms, in particular expression of virulence genes, biofilm formation and dispersal, have provided impetus for investigating practical approaches to interfere with microbial QS. This study tests Halomonas pacifica and Marinobacter hydrocarbonoclasticus, two halophilic marine micro-organism, for their AI-2 dependent QS signalling and the effect of two well-known quorum-sensing inhibitors (QSIs), patulin and penicillic acid, on biofilm formation. We report, for the first time, the successful amplification of a putative luxS gene in H. pacifica using degenerated primers and AI-2 dependent QS as well as inhibition using QSIs. Penicillic acid had a strong inhibitory effect on AI-2 induction of H. pacifica at non-growth inhibitory concentrations, while patulin has an adverse effect only at the highest concentration (25 µM). QSIs effect on biofilm forming capability was isolate specific, with maximum inhibition at 25 µM of patulin in H. pacifica. In M. hydrocarbonoclasticus, no adverse effects were noted at any tested concentration of either QSIs. Detection of bioluminescence and the presence of a putative luxS gene provide biochemical and genetic evidence for the production of a signalling molecule(s) which is the essential first step in characterizing H. pacifica QS. This study highlights the importance of AI-2 dependent QS in a marine setting, not previously reported. It further suggests that 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.

First morphomolecular identification of Penicillium griseofulvum and Penicillium aurantiogriseum toxicogenic isolates associated with blue mold on apple.

Postharvest blue mold decay caused by Penicillium spp. is the most important disease of fresh apple fruit in the world, which extend from the field to the store. Two new Penicillium spp. responsible for apple fruit decay were recovered. The morphological and molecular features of Penicillium griseofulvum and Penicillium aurantiogriseum isolated from apple fruits were characterized morphologically and molecularly. Pathogenicity test exhibited that both P. griseofulvum and P. aurantiogriseum were responsible for blue mold decay in storage apple fruits. Lesion diameter indicated that P. aurantiogriseum was more aggressive than P. griseofulvum. All tested isolates were able to synthesize citrinin in addition to patulin. Not all of the isolates belonging to the same species showed the same profile of secondary metabolites. Microsatellite-primed polymerase chain reaction was able to differentiate these isolates at the species level and divided the analyzed isolates into two genetically different groups. Little intraspecific variability was evident. Microsatellite-primed polymerase chain reaction analysis proved to be an objective, rapid, and reliable tool to identify Penicillium spp. involved in blue mold of apple. This is the first report of occurrence of P. griseofulvum and P. aurantiogriseum on imported apple fruits in Saudi Arabia.

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 Na2HPO4, among the metal ions and phosphate salts tested, increased the production of penicillic acid (approximately 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) Na2HPO4], maximum penicillic acid concentration (approximately 9.40 mg ml(-1)) and cell mass (approximately 17.4 g l(-1)) were obtained after 12 days.

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.

New Penicillium species associated with bulbs and root vegetables.

Taxa of the Penicillium series Corymbifera are known for their strongly fasciculate growth and association with the rhizosphere of vegetables and flower bulbs. Using micromorphology, colony characteristics on various media and chemotaxonomic profiling, P. albocoremium sensu stricto and two new species, P. radicicola and P. tulipae, are redescribed during a taxonomic survey of P. albocoremium isolates contained within the IBT culture collection. Although these novel taxa are micromorphologically quite similar, their unique secondary metabolite profiles individually distinguish them from isolates of P. albocoremium. Moreover, the following metabolites produced by these species are known mycotoxins: citrinin, penicillic acid and terrestric acid by P. radicicola and terrestric acid and penitrem A by P. tulipae.

Mycotoxin production by molds isolated from 'Greek-style' black olives.

Seventeen mold strains were isolated from 'Greek-style' black olives produced in Morocco. Eight of these isolates were identified as Aspergillus flavus, seven as Aspergillus petrakii, and two as Aspergillus ochraceus Wilhelm. The A. flavus strains were tested for production of aflatoxins B1, B2, G1, and G2; and A. ochraceus and A. petrakii strains were tested for production of ochratoxin, penicillic acid, patulin, and citrinin. The organisms were tested for mycotoxin production on five different substrates, including rice powder-corn steep agar, autoclaved rice, yeast-extract sucrose broth (YES), potato dextrose agar (PDA), and fresh olive paste. All strains of A. flavus produced aflatoxins on all substrates except olive paste and PDA. In PDA, only two strains produced Aflatoxin B1. Five A. ochraceus group isolates produced penicillic acid on one or more of the substrates, but only two out of the five produced penicillic acid on olive paste. None produced ochratoxin, patulin or citrinin. Quantities of aflatoxin B1 produced in rice ranged from 5 to 14 micrograms/g of rice, and of penicillic acid 15-32 micrograms/g of rice. In olive paste, the concentrations of penicillic acid were 11.4 and 30.2 micrograms/g. Biological toxicity of extracts of mold cultures was confirmed using chicken embryos and a microbiological test. Crude extracts of cultures were also tested for mutagenicity using the Salmonella mutagenicity (Ames) Test, and some gave positive mutagenic responses.

In vitro metabolism of penicillic acid with mouse-liver homogenate fractions.

The metabolism of penicillic acid (PA), a carcinogenic mycotoxin, was studied in vitro using subcellular fractions of mouse-liver homogenates. PA reacted with glutathione (GSH) both enzymatically and non-enzymatically. Each reaction was of equal importance. The in vitro metabolism of PA using different hepatic subcellular fractions was essentially non-enzymatic when GSH was absent, but metabolism was strikingly increased when GSH was available. In the microsomal preparation in the presence of GSH 75% of the added PA was biotransformed within 30 min to metabolite(s) that were not extractable with organic solvents. HPLC analysis indicated that the metabolite(s) were more polar than the parent compound.

Three new chlorine containing antibiotics from a marine-derived fungus Aspergillus ostianus collected in Pohnpei.

A marine bacterium Ruegeria atlantica (designated as strain TUF-D) was isolated from a glass plate submerged in the coastal water. Three new chlorine containing compounds (1 to approximately 3), together with penicillic acid (4) were obtained from a marine-derived fungus Aspergillus ostianus strain TUF 01F313 isolated from a marine sponge at Pohnpei as antibacterial components against R. atlantica. The structures of three new antibiotics were determined based on their spectral data as 8-chloro-9-hydroxy-8,9-deoxyasperlactone (1), 9-chloro-8-hydroxy-8,9-deoxyasperlactone (2), and 9-chloro-8-hydroxy-8,9-deoxyaspyrone (3). Compound 1 inhibited the growth of R. atlantica at 5 microg/disc (inhibition zone: 12.7 mm), while 2 and 3 were active at 25 microg/disc (10.1 and 10.5 mm, respectively).

Preliminary report of the distribution of [14C]penicillic acid in rats: autoradiographic technique.

The distribution of [14C]penicillic acid in rats was determined by autoradiographic technique. Adult male rats were given [14C]penicillic acid orally and IV. According to autoradiographic analyses 24 hours later, high levels of radioactivity were in liver, heart, kidneys, lungs, and caudal part of the intestine. The significance of bile as an excretory route for penicillic acid was confirmed by the results from IV administration of the compound.

[Synthesis and identification of penicillic-acid antigens from Penicillium cyclopium].

To establish a new immune assay for Penicillic Acid (PA) from Penicillium cyclopium, we studied the synthesis of conjugated complete antigens for penicillic acid. PA was conjugated to bovine serum album (BSA) and ovalbumin (OVA) by 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC). The artificial antigens PA-BSA and PA-OVA were identified by ultraviolet spectrometric scanning, SDS-PAGE and immunization. Results showed that the absorption peak of conjugation were different from that of the carrier protein alone and of the PA. The conjugated ratio of PA and BSA was 23.2:1 and that of PA and OVA was 10.4:1. Balb/c mice were immunized by the artificial antigen of PA-BSA, with PA-OVA as coating antigen. The average titer of antiserums was more than 12 800 by indirect ELISA. The obtained antigens offered a basis for developing immunoassay method.

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.

Biliary excretion of [14C]penicillic acid by rats.

Adult male rats were given [14C]penicillic acid by oral intubation, and the biliary excretion of the compound was monitored. The significance of bile as an excretory route was confirmed and compared by using open and recycle cannulation of the bile duct. The biological half-retention life of [14C]penicillic acid in the bile was 3.63 h.

The absorption, distribution, and excretion of 14C-penicillic acid by rats.

Adult male and female rats were given 14C-penicillic acid (labeled in the 1, 3, and 5 positions by fermentation with acetate-1-14C) by oral intubation and were sacrificed at 1, 2, 4, 24, 48, 72, and 168 hours following administration of the labeled compound. Approximately 98 p. 100 of the 14C-radioactivity given was absorbed from the gastrointestinal tract of the rats during the first 48 hours. After 7 days, 82 p. 100 was recovered in the urine and 13 p. 100 in the feces. Less than 1 p. 100 of the total radioactivity was recovered as 14CO2 from the expired air; most of this amount was recovered within the first 72 hours after administration. Various tissues were, analyzed for 14C-radioactivity throughout the 7-day-period; no significant retention of radioactivity was apparent in any of the tissues examined. The level of 14C-penicillic acid reached a maximum in the blood and tissues 2-4 hours after administration. Red blood cells contained the highest concentration of 14C-radioactivity. Liver, bladder, kidneys, heart, and bile also showed a high level of 14C-radioactivity concentration. The estimated biological half-life of penicillic acid was 18.7 and 17.1 hours for the male and female, respectively. The estimated biological half-retention time from the point of administration by the red blood cells, liver, and kidney of the male and female rats was 24.2 and 14.0; 19.8 and 17.8; and 21.3 and 19.0 hours, respectively. Recovery studies indicated no unchanged penicillic acid in the urine.

In vivo and in vitro metabolism of [14C]penicillic acid.

In vivo and in vitro studies of the metabolism of [14C]penicillic acid showed a significant uptake of penicillic acid by red blood cells and a significant increase in radioactivity associated with the membrane fraction with time (1-4 hr); however, the majority of the red blood cell activity was intracellular. The 14C-radioactivity concentration in liver fractions (1.5 percent of the administered dose 2 hr following administration) was high in the RNA-DNA and protein fractions. The level of the 14C activity associated with these fractions increased with time (24 hr). Preliminary studies of the in vivo metabolism of [14C]penicillic acid by rats showed that a significant amount of radioactivity was excreted in the urine (82 percent of the administered dose after 7 days), of which strongly acidic urinary metabolites accounted for 23 percent; weakly to moderately acidic, 60 percent; strongly basic, 2 percent; and neutral, 15 percent. Isolation and identification studies of the neutral urinary fraction indicated that the major metabolites are non-ionic macromolecules (Mr < 5000). Biliary metabolites (10 percent of the administered dose 2 hr following administration) were 28 percent strongly acidic and 72 percent weakly to moderately acidic.

Pharmacokinetics of the mycotoxin penicillic acid in male mice: absorption, distribution, excretion, and kinetics.

The pharmacokinetics of penicillic acid (PA), a carcinogenic mycotoxin, was investigated in male mice. Absorption of PA after po administration of [14C]PA was rapid. Only a small percentage of the radioactivity in the plasma was unchanged PA. After ip or iv administration of [14C]PA (90 mg/kg), blood, liver, kidneys, intestine, lungs, heart, and spleen contained the largest amounts of radioactivity while brain tissue accumulated the least. Over 90% and approximately 60% of the administered radioactivity was excreted in the urine after iv and ip injection, respectively, but essentially no unchanged PA was detected in the urine. Over 25% of the administered radioactivity following an iv dose of [14C]PA (90 mg/kg) was excreted in the bile in 60 min; no unchanged PA was detected in the bile. The excretion of radioactivity in the bile was decreased in diethyl maleate-pretreated mice. Only a small amount of the administered radioactivity was recovered in the feces and as expired CO2. The unchanged PA concentration-time curve in plasma was best fit by three, two, and one compartment open models after iv, ip, and po administration, respectively. Based on these results, it was concluded that metabolism and not excretion of unchanged parent penicillic acid is the major process of elimination of PA from the blood. There are extensive route-dependent differences in the kinetic behavior of PA.