Penicillium Metabolites Research Articles

First Report on Mycotoxin Contamination of Hops (Humulus lupulus L.).

The presence of mycotoxins and other toxic metabolites in hops (Humulus lupulus L.) was assessed for the first time. In total, 62 hop samples were sampled in craft breweries, and analyzed by a multi-toxin LS-MS/MS method. The study collected samples from craft breweries in all of the Croatian counties and statistically compared the results. Based on previous reports on Alternaria spp. and Fusarium spp. contamination of hops, the study confirmed the contamination of hops with these toxins. Alternaria toxins, particularly tenuazonic acid, were found in all tested samples, while Fusarium toxins, including deoxynivalenol, were present in 98% of samples. However, no Aspergillus or Penicillium metabolites were detected, indicating proper storage conditions. In addition to the Alternaria and Fusarium toxins, abscisic acid, a drought stress indicator in hops, was also detected, as well as several unspecific metabolites. The findings suggest the need for monitoring, risk assessment, and potential regulation of Alternaria and Fusarium toxins in hops to ensure the safety of hop usage in the brewing and pharmaceutical industries. Also, four local wild varieties were tested, with similar results to the commercial varieties for toxin contamination, but the statistically significant regional differences in toxin occurrence highlight the importance and need for targeted monitoring.

Multiple mycotoxins associated with maize (Zea mays L.) grains harvested from subsistence farmers’ fields in southwestern Ethiopia

Fifty-four maize grain samples freshly harvested from subsistence farmers’ fields in southwestern Ethiopia were analyzed for multiple mycotoxins using liquid chromatography-tandem mass spectrometric (LC-MS/MS) method following extraction by acetonitrile/water/acetic acid on a rotary shaker. The grain samples were contaminated with a total of 164 metabolites, of which Fusarium and Penicillium metabolites were the most prevalent accounting for 27 and 30%, respectively. All the major mycotoxins and derivatives except one (citrinin) were of Fusarium origin. Zearalenone was the most frequent major mycotoxin occurring in 74% of the samples at concentrations of 0.32–1310 µg/kg. It was followed by nivalenol (63%), zearalenone-sulfate (44%), and fumonisin B1 (41%). Nivalenol, nivalenol glucoside, and fusarenon-X were detected at unusually high levels of 8–1700 µg/kg, 21–184 µg/kg, and 33–149 µg/kg, respectively. Deoxynivalenol and DON-3 glucoside contaminated 32% of the samples, each at levels of 15.9–5140 µg/kg and 10–583 µg/kg, respectively. Moniliformin and W493B occurred in 96 and 22% samples at levels of 3.27–4410 µg/kg and 3–652 µg/kg, respectively. Fumonisins were also detected in the samples at levels of 9–6770 µg/kg (B1), 16–1830 µg/kg (B2), 9.5–808 µg/kg (B3), and 1.3–128 µg/kg (A1). This study confirmed the presence of an array of mycotoxins contaminating maize grains right from the field. The effect of the co-occurring mycotoxins on consumers’ health should be investigated along with that of the newly emerging ones. Results of the current study call for application of pre-harvest mycotoxin mitigation strategies to safeguard maize-based food and feed.

Ochratoxin A: Overview of Prevention, Removal, and Detoxification Methods.

Ochratoxins are the secondary metabolites of Penicillium and Aspergillus, among which ochratoxin A (OTA) is the most toxic molecule. OTA is widely found in food and agricultural products. Due to its severe nephrotoxicity, immunotoxicity, neurotoxicity, and teratogenic mutagenesis, it is essential to develop effective, economical, and environmentally friendly methods for OTA decontamination and detoxification. This review mainly summarizes the application of technology in OTA prevention, removal, and detoxification from physical, chemical, and biological aspects, depending on the properties of OTA, and describes the advantages and disadvantages of each method from an objective perspective. Overall, biological methods have the greatest potential to degrade OTA. This review provides some ideas for searching for new strains and degrading enzymes.

Penicillium molds impact the transcriptome and evolution of the cheese bacterium Staphylococcus equorum.

The observation that Penicillium molds can inhibit the growth of Staphylococcus was a catalyst for the antibiotic revolution. Considerable attention has been paid to purified Penicillium metabolites that inhibit bacteria, but little is known about how Penicillium species impact the ecology and evolution of bacteria in multispecies microbial communities. Here, we investigated how four different species of Penicillium can impact global transcription and evolution of a widespread Staphylococcus species (S. equorum) using the cheese rind model microbiome. Through RNA sequencing, we identified a core transcriptional response of S. equorum against all five tested Penicillium strains, including upregulation of thiamine biosynthesis, fatty acid degradation, and amino acid metabolism as well as downregulation of genes involved in the transport of siderophores. In a 12-week evolution experiment where we co-cultured S. equorum with the same Penicillium strains, we observed surprisingly few non-synonymous mutations across S. equorum populations evolved with the Penicillium species. A mutation in a putative DHH family phosphoesterase gene only occurred in populations evolved without Penicillium and decreased the fitness of S. equorum when co-cultured with an antagonistic Penicillium strain. Our results highlight the potential for conserved mechanisms of Staphylococcus-Penicillium interactions and demonstrate how fungal biotic environments may constrain the evolution of bacterial species.IMPORTANCEFungi and bacteria are commonly found co-occurring both in natural and synthetic microbiomes, but our understanding of fungal-bacterial interactions is limited to a handful of species. Conserved mechanisms of interactions and evolutionary consequences of fungal-bacterial interactions are largely unknown. Our RNA sequencing and experimental evolution data with Penicillium species and the bacterium S. equorum demonstrate that divergent fungal species can elicit conserved transcriptional and genomic responses in co-occurring bacteria. Penicillium molds are integral to the discovery of novel antibiotics and production of certain foods. By understanding how Penicillium species affect bacteria, our work can further efforts to design and manage Penicillium-dominated microbial communities in industry and food production.

Mixtures of mycotoxins, phytoestrogens and pesticides co-occurring in wet spent brewery grains (BSG) intended for dairy cattle feeding in Austria

Spent brewery grains (BSG) are the main by-product of beer production and are incorporated in rations of food-delivering animals, mainly dairy cows. Like other agricultural commodities, BSG can be contaminated by a broad spectrum of natural and synthetic undesirable substances, which can be hazardous to animal and human health as well as to the environment. The co-occurrence of mycotoxins, phytoestrogens, other fungal and plant secondary metabolites, along with pesticides, was investigated in 21 BSG samples collected in dairy farms in Austria. For this purpose, a validated multi-metabolite liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI–MS/MS) was employed. Metabolites derived from Fusarium, Aspergillus, Alternaria and pesticide residues, were ubiquitous in the samples. Zearalenone (ZEN), T-2 and HT-2 toxins were the only regulated mycotoxin detected, albeit at concentrations below the European guidance values for animal feeds. Ergot alkaloids, Penicillium-derived metabolites, and phytoestrogens had occurrence rates of 90, 48 and 29%, respectively. Penicillium metabolites presented the highest levels among the fungal compounds, indicating contamination during storage. Aflatoxins (AFs), ochratoxins and deoxynivalenol (DON) were not detected. Out of the 16 detected pesticides, two fungicides, ametoctradin (9.5%) and mandipropamid (14.3%) revealed concentrations exceeding their respective maximum residue level (MRL) (0.01 mg kg−1) for barley in two samples. Although based on European guidance and MRL values the levels of the detected compounds probably do not pose acute risks for cattle, the impact of the long-time exposure to such mixtures of natural and synthetic toxicants on animal health and food safety are unknown and must be elucidated.

Rasprostranjenost Penicillium metabolita u kukuruzu gajenim u Srbiji

Maize can be contaminated with a wide range of fungal secondary metabolites that decrease the quality and safety of maize and maize-derived products. The increase of natural fungal metabolites occurrence in maize, influenced by climate changes, is recognized as a significant issue in recent years. Therefore, the main aim of this study was to investigate the influence of weather conditions on the natural occurrence of Penicillium spp. metabolites in maize kernel samples. The survey was conducted for two maize vegetation seasons 2016 and 2017. In total 458 maize samples were analyzed by liquid chromatography-tandem mass spectrometry method. The samples originated from the Autonomous Province of Vojvodina, and regions of Bačka, Srem, and Banat. Among 45 Penicillium metabolites investigated 16 and 18 were detected in samples from 2016 and 2017, respectively. The most commonly occurring Penicillium metabolite in both years was oxaline, which was detected in more than 90% of analyzed samples. Questiomycin A, 7hydroxypestalotin, pestalotin, and mycophenolic acid were also very frequently detected Penicillium metabolites. This is one of the unique studies in the Republic of Serbia, as well as in this part of Europe, investigating the occurrence of a great number of Penicillium metabolites in maize samples.

Rasprostranjenost Penicillium metabolita u kukuruzu gajenim u Srbiji

Maize can be contaminated with a wide range of fungal secondary metabolites that decrease the quality and safety of maize and maize-derived products. The increase of natural fungal metabolites occurrence in maize, influenced by climate changes, is recognized as a significant issue in recent years. Therefore, the main aim of this study was to investigate the influence of weather conditions on the natural occurrence of Penicillium spp. metabolites in maize kernel samples. The survey was conducted for two maize vegetation seasons 2016 and 2017. In total 458 maize samples were analyzed by liquid chromatography-tandem mass spectrometry method. The samples originated from the Autonomous Province of Vojvodina, and regions of Bačka, Srem, and Banat. Among 45 Penicillium metabolites investigated 16 and 18 were detected in samples from 2016 and 2017, respectively. The most commonly occurring Penicillium metabolite in both years was oxaline, which was detected in more than 90% of analyzed samples. Questiomycin A, 7hydroxypestalotin, pestalotin, and mycophenolic acid were also very frequently detected Penicillium metabolites. This is one of the unique studies in the Republic of Serbia, as well as in this part of Europe, investigating the occurrence of a great number of Penicillium metabolites in maize samples.

Tea: Transfer of Mycotoxins from the Spiked Matrix into an Infusion.

Recent surveys report the occurrence of Aspergillus and Penicillium metabolites (aflatoxins (AFLs), ochratoxin A (OTA), cyclopiazonic and mycophenolic acids (MPA), sterigmatocystin (STC), citrinin), Fusarium (trichothecenes, zearalenone (ZEA), fumonisins (FBs), enniatins (ENNs)) and Alternaria (alternariol (AOH), its methyl ether (AME), tentoxin (TE), and tenuazonic acid (TNZ)) toxins in dry Camellia sinensis and herbal tea samples. Since tea is consumed in the form of infusion, correct risk assessment needs evaluation of mycotoxins’ transfer rates. We have studied the transfer of AFLs, OTA, STC, deoxynivalenol (DON), ZEA, FBs, T-2, and HT-2 toxins, AOH, AME, TE, ENN A and B, beauvericin (BEA), and MPA from the spiked green tea matrix into an infusion under variation of preparation time and water characteristics (total dissolved solids (TDS) and pH). Analytes were detected by HPLC-MS/MS. The main factors affecting transfer rate proved to be mycotoxins’ polarity, pH of the resulting infusion (for OTA, FB2, and MPA) and matrix-infusion contact period. The concentration of mycotoxins increased by 20–50% within the first ten minutes of infusing, after that kinetic curve changed slowly. The concentration of DON and FB2 increased by about 10%, for ZEA, MPA, and STC it stayed constant, while for T-2, TE, AOH, and AFLs G1 and G2 it went down. Maximum transfer correlated well with analytes polarity. Maximum transfer of ENNs, BEA, STC, ZEA, and AOH into infusion was below 25%; AFLs—25–45%; DON, TE, and T-2 toxins 60–90%, FB1—80–100%. The concentration of OTA, MPA, and FB2 in the infusion depended on its pH. At pH about four, 20%, 40%, and 60% of these toxins transferred into an infusion, at pH about seven, their concentrations doubled. Water TDS did not affect transfer significantly.

Co-occurrence and toxicological relevance of secondary metabolites in dairy cow feed from Thailand

ABSTRACT The occurrence of secondary metabolites and co-contaminants in dairy cow feed samples (n = 115), concentrate, roughage, and mixed feed, collected from Ratchaburi and Kanjanaburi provinces, Thailand, between August 2018 and March 2019 were investigated using LC-MS/MS based multi-toxin method. A total of 113 metabolites were found in the samples. Fungal metabolites were the predominant compounds, followed by plant metabolites. Among major mycotoxins, zearalenone and fumonisins were most frequently detected in concentrate and mixed feed samples, while deoxynivalenol and aflatoxin B1 were found at the frequency lower than 50%. Other metabolites, produced by Aspergillus, Fusarium, Penicillium, and Alternaria species, occurred in the samples. Flavoglaucin, 3-nitropropionic acid, averufin, and sterigmatocystin were the most prevalent Aspergillus metabolites. Common Fusarium metabolites occurring in the samples included moniliformin, beauvericin, and enniatins. For Penicillium metabolites, mycophenolic acid, questiomycin A, quinolactacin A, oxaline, citrinin, and dihydrocitrinone were frequently detected. The toxic Alternaria metabolites, alternariol, and alternariol monomethyl ether showed the high incidence in the samples. Plant metabolites were commonly found, mainly cyanogenic compounds and isoflavones, from cassava and soybean meal used as feed ingredients. Overall, 96.6% of feed samples contained at least two metabolites, in a range from 2 to 69. According to co-contamination of mycotoxins found in feed samples, zearalenone were mostly found in combination with fumonisin B1, deoxynivalenol, and aflatoxin B1. Fumonisin B1 co-occurred with aflatoxin B1 and deoxynivalenol. The mixtures of deoxynivalenol and aflatoxin B1, and of zearalenone, fumonisin B1 and deoxynivalenol were also found. Due to known individual toxicity of fungal and plant metabolites and possible additive or synergistic toxic effects of multi-mycotoxins, the occurrence of these metabolites and co-contaminants should be monitored continuously to ensure food safety through the dairy supply chain.

Fungi and their metabolites in grain from individual households in Croatia

ABSTRACT A total of 117 fungal metabolites were detected in grains collected in Gunja-G (flooded village) and Gornji Stupnik-GS (control village), located in the Zagreb County, Croatia. Major mycotoxins and derivatives (17), ergot alkaloids (14), Fusarium (23), Aspergillus (18), Penicillium (18), Alternaria (7) and other fungal and unspecific metabolites (20) were found. A higher number of metabolites co-occurred per sample in grains from G (115) than in GS (91). Regulated mycotoxins were below maximum limits except fumonisins B1,2 in 15–20% of grains and aflatoxin B1. Fusarium metabolites contaminated more than 50% of grains at both locations. Besides FB1,2, bikaverin, aurofusarin, culmorin and 15-hidroxyculmorin were detected at relatively high concentrations. Ergot alkaloids were detected at 2–18 times higher concentrations in grains from G as compared to GS. Majority of Aspergillus mycotoxins were present at a low frequency (5–15%). Penicillium metabolites recovered with higher frequency in GS (55–70%) than in G (20–55%). Alteranaria metabolites prevailed in grains from G (60–80%).

Fungal and chemical diversity in hay and wrapped haylage for equine feed

The presence of fungi and mycotoxins in silage (fermented maize) for cattle and other ruminants have been studied extensively compared to wrapped haylage (fermented grass) for horses and other monogastric animals. The purpose of this work was to examine the fungal diversity of wrapped haylage and conventional hay and to analyse the forage sample for fungal metabolites. Faeces samples were also analysed to study the fate of fungi and metabolites. Fungal diversity of the samples was determined by direct plating on DG18, V8 and MEA and chemical analyses were done using LC-MS/MS. The results show that Sordaria fimicola was common in both hay and haylage, while Penicillium spp. was prevalent in haylage and Aspergillus spp. in hay. Communiols were found in all types of samples together with gliocladic acid. Roquefortines and fumigaclavines were found in haylage with no visible fungal growth, but not in hay. In haylage hot spot samples, a series of Penicillium metabolites were detected: Andrastins, fumigaclavines, isofumigaclavines, marcfortines, mycophenolic acid, PR toxins, and roquefortines. Penicillium solitum was found repeatedly in haylage and haylage hot spot samples and viridicatols were detected in a hot spot sample, which has not been reported before. Even haylage with no visible fungal growth contained more metabolites than hay. Individually, the metabolites detected in haylage may, in high doses, be mutagenic, neurotoxic or immunosuppressive; but the synergistic effect of small doses may also have other or greater negative health effects on equines than on ruminants.

(3R, 7R)-7-Acetoxyl-9-Oxo-de-O-Methyllasiodiplodin, a Secondary Metabolite of Penicillium Sp., Inhibits LPS-Mediated Inflammation in RAW 264.7 Macrophages through Blocking ERK/MAPKs and NF-κB Signaling Pathways.

Twelve polyketones were isolated from the fermentation broth of Penicillium sp., including six new compounds (supplementary material). Penicillium sp. is widely used in clinic as a highly effective and low toxic antibiotic. Among these compounds, (3R, 7R)-7-acetoxyl-9-oxo-de-O-methyllasiodiplodin named PS-2 showed significant anti-inflammatory activity. So, the anti-inflammatory mechanism of PS-2 was investigated by using lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. The results showed that PS-2 can significantly inhibit the overproduction of nitric oxide (NO), prostaglandin E2 (PGE2), and interleukin-6 (IL-6), whereas it showed no inhibition on the release of pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α). Cell-free colorimetric method demonstrated that PS-2 could obviously inhibit the enzymatic activity of cyclooxygenase-2 (COX-2). Western blot results indicated that PS-2 could significantly inhibit high expression of iNOS and COX-2 proteins. Further investigations on the anti-inflammatory mechanism showed that PS-2 could suppress the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), but did not exhibit obvious inhibition on the phosphorylation of c-JunN-terminal kinase (JNK) and phosphorylated 38 (p38). In addition, PS-2 inhibited the degradation of inhibitor of kappa-B alpha (IκB-α) and translocation to nucleus of nuclear factor kappa-B (NF-κB) p65 in RAW 264.7 macrophages. These results suggested that PS-2 might be an effective intervention against inflammatory diseases.

Effect of agronomic programmes with different susceptibility to deoxynivalenol risk on emerging contamination in winter wheat

Deoxynivalenol (DON) is the most prevalent mycotoxin in small cereal crops throughout the world, and its occurrence is closely linked to the presence of Fusarium Head Blight (FHB) disease. In order to minimize the sanitary risk, wheat cropping systems are commonly designed to control DON contamination, as this represents the main target contaminant. However, several other mycotoxins and secondary metabolites produced by Fusarium and other fungal species have been detected in wheat. The objective of this study was to evaluate whether the application of agronomic programmes with different susceptibility to DON contamination could also affect the occurrence of emerging mycotoxins in wheat kernels. Field experiments have been conducted in North Italy, under naturally-infected conditions, over a period of 3 growing seasons, by comparing 4 field programmes, which were constituted by the combination of wheat cultivars (a durum wheat variety that is susceptible to DON contamination and a common moderately resistant one) and 2 fungicide applications at heading (untreated control compared to an azole application at heading). Grain samples have been analyzed by means of a dilute-and-shoot multi-mycotoxin LC–MS/MS method, and 43 fungal metabolites were detected. In addition to DON, the most abundant compounds were aurofusarin, culmorin and deoxynivalenol-3-glucoside, which were detected in all the growing seasons and agronomic strategies. Other trichothecenes and zearalenone derivatives were also found, but in clearly lower concentrations. Contamination by enniatins and moniliformin, produced by other Fusarium species e.g. Fusarium avenaceum, alternariol, alternariol methyl ether and tentoxin, produced by Alternaria species, has been observed for all the compared growing seasons. The presence of other mycotoxins and secondary metabolites was clearly affected by the climatic conditions: fumonisins, beauvaricin, bikaverin, fusaric acid and butenolid were detected in the warmer growing seasons, while chrysogine, infectopyrone, secalonic acid and ergot alkaloids (sum of 13 toxins) were only found in the more rainy and cool seasons. Equisetin, decalonectrin, toxin T-2 and HT-2 were only found in traces. The application of the field programmes clearly affected DON contamination in each growing season: a significant increase in this toxin has been observed moving from the lowest risk agronomic strategy to the highest one. The application of the most favourable DON control field programme (a moderately resistant variety combined with fungicide application at heading) reduced the content of this mycotoxin by 89%, compared to the worst programme (untreated susceptible variety). The application of the less risky agronomic strategy for DON contamination led to a significant reduction (>84%) of all the other mycotoxins produced by the DON producing fungal species. Moreover, although the considered agronomic factors (variety susceptibility and fungicide application) resulted in a control efficacy that varied in function of the environmental conditions and the type of mycotoxin, the results show a clear reduction trend, after the application of agronomic strategies that are able to minimize the DON content, for almost all the other Fusarium, Alternaria, Claviceps and Penicillium metabolites. The results summarized in this work, which have been obtained under different environmental and agronomical conditions, allow a first assessment to be made of the agronomic strategies that could be applied to control emerging mycotoxins in wheat.

Synthesis, stereochemical assignment, and bioactivity of the Penicillium metabolites penicillenols B1 and B2

The Penicillium metabolites penicillenols B1 and B2 were synthesised for the first time by elimination of a mesylated penicillenol A precursor as obtained from an l-threonine derived tetramic acid. The (Z,S)- and (E,S)-configured diastereomers were identical to the natural compounds as to NMR spectra and optical rotations. Both isomers showed antiproliferative effects against cancer and endothelial cell lines and penicillenol B1 was also notably antibiotic against Staphylococcus aureus.

A Conserved Dedicated Olfactory Circuit for Detecting Harmful Microbes in Drosophila

Flies, like all animals, need to find suitable and safe food. Because the principal food source for Drosophila melanogaster is yeast growing on fermenting fruit, flies need to distinguish fruit with safe yeast from yeast covered with toxic microbes. We identify a functionally segregated olfactory circuit in flies that is activated exclusively by geosmin. This microbial odorant constitutes an ecologically relevant stimulus that alerts flies to the presence of harmful microbes. Geosmin activates only a single class of sensory neurons expressing the olfactory receptor Or56a. These neurons target the DA2 glomerulus and connect to projection neurons that respond exclusively to geosmin. Activation of DA2 is sufficient and necessary for aversion, overrides input from other olfactory pathways, and inhibits positive chemotaxis, oviposition, and feeding. The geosmin detection system is a conserved feature in the genus Drosophila that provides flies with a sensitive, specific means of identifying unsuitable feeding and breeding sites.

Antifungal effect of Penicillium metabolites against some fungi

Microorganisms are increasingly exploited as a source of new biological control agents. Genus Penicillium is a source of novel bioactive molecules which can be used as antifungal agents. The objective of this study was to evaluate the antifungal potential of Penicillium strains. Culture filtrates of two Penicillium species were tested for their antifungal potential by well diffusion assays. Filtrate of Penicillium isolates showed high antifungal effects on mycelial growth of Fusarium oxysporum, Fusarium solani, Macrophomina phaseolina, Aspergillus japonicus var aculeatus and Cladosporium cladosporioides. But Penicillium italicum inhibit the fungal growth from 45 to 68% as compared to Penicillium simplissimum (25–68%). However in case of A. japonicus var aculeatus, Penicillium spp. extracts were equally effective and reduce the colony growth up to 68%. However, P. simplissimum extract was least effective in case of M. phaseolina, where it decreased the colony growth only 25%.

ChemInform Abstract: Hydrogenation of Vermistatin.

Hydrogenation of vermistatin, a metabolite ofPenicillium vermiculatum, affordedcis- andtrans-3-(2′-carboxy-4′,6′-dimethoxybenzyl)-6-propyltetrahydro-4H-pyran-4-one and 4,6-dimethoxy-3-(4′-propylcyclopentanon-2′-yl)phthalide. The latter compound was found to be the strongest inhibitor of RNA synthesis in P 388 leukemia cells of all compounds tested.

Citromycetins and Bilains A–C: New Aromatic Polyketides and Diketopiperazines from Australian Marine-Derived and Terrestrial Penicillium spp.

Chemical analysis of an Australian marine-derived isolate of Penicillium bilaii, collected from the Huon estuary, Port Huon, Tasmania, yielded the known fungal aromatic polyketides citromycetin (1) and citromycin (2) together with two dihydro analogues, (-)-2,3-dihydrocitromycetin (3) and (-)-2,3-dihydrocitromycin (4). An Australian terrestrial isolate of Penicillium striatisporum collected near Shalvey, New South Wales, also yielded citromycetin (1), citromycin (2), and the new dihydro analogue (-)-2,3-dihydrocitromycetin (3), together with fulvic acid (5), anhydrofulvic acid (6), and a selection of new methoxylated analogues, 12-methoxycitromycetin (7), 12-methoxycitromycin (8), (-)-12-methoxy-2,3-dihydrocitromycetin (9), and 12-methoxyanhydrofulvic acid (10). P. bilaii also yielded the rare siderophore pistillarin (11), the known diketopiperazines cyclo-(L-Phe -L-Pro) (12), cyclo-(L-Pro-L-Tyr) (13), cyclo-(L-Pro-L-Val) (14), and cis-bis(methylthio)silvatin (15), and three new diketopiperazines, bilains A-C (16-18). The structures for the Penicillium metabolites 1- 18 were assigned by a combination of detailed spectroscopic analysis, including correlation with relevant literature data, chemical derivatization, degradation, and biosynthetic considerations. The citromycin polyketides 2 and 4 and the diketopiperazine 15 were weakly cytotoxic.

Natural and directed biosynthesis of communesin alkaloids

A role for tryptophan, acetate, mevalonate and methionine in the biosynthesis of communesins A and B, novel structurally-related and biologically-active Penicillium metabolites, has been established by isotopic labelling techniques. The incorporation of 14C-tryptamine has also been demonstrated. dl-2- 13C-tryptophan specifically enriched two carbon atoms in the 13C NMR spectrum, thereby defining the intra-molecular arrangement of the two tryptophan-derived moieties. Feeding differentially labelled precursors during communesin production showed that tryptophan and methionine are involved early in the biosynthesis and that mevalonate provides an isoprene which is added later. A biosynthetic pathway involving an early precursor based on tryptophan is proposed. Indole- N-( 13C-methyl) tryptophan was not incorporated into communesins implying that N-methylation of tryptophan is not the first step of the communesin biosynthetic pathway. During deamination of indole- N-( 13C-methyl) tryptophan to 1- 13C-methylindole-3-carboxylic acid communesin biosynthesis was inhibited. Of several halogenated indoles tested for directed biosynthesis, only dl-6-fluoro-tryptophan and 6-fluoro-tryptamine caused accumulation of the corresponding monofluoro-analogues of communesins A and B.

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Due to infections with moulds already in the vineyards, the formation of mycotoxins is possible under certain circumstances during the process of red wine making. At this, metabolites ofPenicillium spp. are of major importance, as this species are to be found frequently on grapes. Beside the nephrotoxic citrinin, which is often co-occurring with ochratoxin A, the occurrence of mycophenolic acid (MPA), a substance of immunosuppressive action, was investigated since it is formed by a great number of Penicillium-species. The detection of these compounds was carried out by means of ELISA and LC-MS. As testing material 44 red wine samples of different provenience and vintages were used. Mycophenolic acid could be detected in 91 % of the samples. The maximum content amounted to 130 ng/ml, yet most of the samples resulted in much lower concentrations of between 3 and 20 ng/ml. The extent of contamination seems to depend rather on the origin of the wine than on the vintage. In particular samples from Southern Europe were most contaminated. This could be due to different practises in wine-making. Citrinin was not detectable in any sample (< 0,2 ng/ml). Regarding the detected concentrations of MPA and citrinin, there is probably no concern for consumers' health. However, the degree of contamination of wine with MPA may well serve as an indicator for hygiene in production.