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Monascus fermentation products can be consumed as food or feed supplements or used as food colouring or flavouring agents. In this study, qPCR and Q-TOF LC/MS methods were developed to monitor Monascus ruber and Monascus purpureus growth and extrolite (lovastatin, mevastatin, as well as the regulated mycotoxin, citrinin (CIT)) production, respectively. Wheat kernels were inoculated with one strain of each species during a solid-state fermentation followed over 63 days. Different growth and extrolite production patterns were clearly observed for the 2 tested strains. After 63 days, high lovastatin levels (up to 0.5 mg/g) were reached for M. ruber wheat fermented kernels while M. purpureus only yielded 0.1 mg/g of lovastatin at best, suggesting that M. ruber may be a better candidate for lovastatin production in a wheat-based model. Mevastatin levels were low and stable for both species. However, M. ruber fermented wheat kernels also contained the highest CIT content, up to 4.2 μg/g, i.e. at levels above the regulation threshold set by the European Union. CIT toxicity was then determined in vitro on bovine kidney cells, showing an IC10 of 6.10-4 M. At maximum concentrations encountered during solid-state fermentation, CIT toxicity was very low under chronic exposure.

Aims The complex mycobiota that colonizes traditional fermented sausages plays an important role in the organoleptic properties of such products. The aim of the present study was to investigate fungal diversity and mycotoxin production during maturation of PGI Salame Piemonte. Methods and results Casing and meat samples were collected at five sampling times from three different batches produced in the same factory and analysed using culture-dependent and independent approaches. Penicillium nalgiovense, which was deliberately inoculated, and Debaryomyces hansenii were the most dominant taxa in casings. Several other fungi mainly belonging to Penicillium crustosum, Penicillium glabrum, Penicillium nordicum, Cladosporium spp., Candida sake, Candida zeylanoides and Yarrowia divulgata were also identified. The casing mycobiota was compared to that of the meat using a metataxonomic approach and a higher fungal diversity was observed in meat as compared to casings. Mycotoxins and penicillin G were monitored using QTOF LC-MS and only trace amounts of roquefortine C were detected in two batches. Conclusions The present study highlighted the diversity of Salame Piemonte mycobiota and the important contribution of autochthonous fungi to its diversity. The absence of mycotoxins and penicillin G confirmed the high hygienic quality of the studied product regarding fungal and mycotoxin contamination. Significance and impact of the study For the first time, this study provides insights about Salame Piemonte mycobiota, which together with the bacterial microbiota and Salame Piemonte process specifications, are responsible for the product organoleptic properties.

Mycophenolic acid (MPA) is a secondary metabolite produced by various Penicillium species including Penicillium roqueforti. The MPA biosynthetic pathway was recently described in Penicillium brevicompactum. In this study, an in silico analysis of the P. roqueforti FM164 genome sequence localized a 23.5-kb putative MPA gene cluster. The cluster contains seven genes putatively coding seven proteins (MpaA, MpaB, MpaC, MpaDE, MpaF, MpaG, MpaH) and is highly similar (i.e. gene synteny, sequence homology) to the P. brevicompactum cluster. To confirm the involvement of this gene cluster in MPA biosynthesis, gene silencing using RNA interference targeting mpaC, encoding a putative polyketide synthase, was performed in a high MPA-producing P. roqueforti strain (F43-1). In the obtained transformants, decreased MPA production (measured by LC-Q-TOF/MS) was correlated to reduced mpaC gene expression by Q-RT-PCR. In parallel, mycotoxin quantification on multiple P. roqueforti strains suggested strain-dependent MPA-production. Thus, the entire MPA cluster was sequenced for P. roqueforti strains with contrasted MPA production and a 174bp deletion in mpaC was observed in low MPA-producers. PCRs directed towards the deleted region among 55 strains showed an excellent correlation with MPA quantification. Our results indicated the clear involvement of mpaC gene as well as surrounding cluster in P. roqueforti MPA biosynthesis.

SummaryDomestication involves recent adaptation under strong human selection and rapid diversification, and therefore constitutes a good model for studies of these processes. We studied the domestication of the emblematic white moldPenicillium camemberti, used for the maturation of soft cheeses, such as Camembert and Brie, about which surprisingly little was known, despite its economic and cultural importance. Whole genome-based analyses of genetic relationships and diversity revealed that an ancient domestication event led to the emergence of the gray-greenP. biformemold used in cheese-making, by divergence from the blue-green wildP. fuscoglaucumfungus. Another much more recent domestication event led to the generation of theP. camemberticlonal lineage as a sister group toP. biforme. Penicillium biformedisplayed signs of phenotypic adaptation to cheese-making relative toP. fuscoglaucum, in terms of whiter color, faster growth on cheese medium under cave conditions, lower levels of toxin production and greater ability to prevent the growth of other fungi. TheP. camembertilineage displayed even stronger signs of domestication for all these phenotypic features. We also identified two differentiatedP. camembertivarieties, apparently associated with different kinds of cheeses, and with contrasted phenotypic features in terms of color, growth, toxin production and competitive ability. We have, thus, identified footprints of domestication in these fungi, with genetic differentiation between cheese and wild populations, bottlenecks and specific phenotypic traits beneficial for cheese-making. This study has not only fundamental implications for our understanding of domestication but can also have important impacts on cheese-making.

Domestication involves recent adaptation under strong human selection and rapid diversification, and therefore constitutes a good model for studies of these processes. We studied the domestication of the emblematic white mold Penicillium camemberti, used for the maturation of soft cheeses, such as Camembert and Brie, about which surprisingly little was known, despite its economic and cultural importance. Whole genome-based analyses of genetic relationships and diversity revealed that an ancient domestication event led to the emergence of the gray-green P. biforme mold used in cheese-making, from the blue-green wild P. fuscoglaucum fungus. Another much more recent domestication event led to the generation of the P. camemberti clonal lineage from P. biforme. Both these domestication events were associated with strong bottlenecks. Penicillium biforme displayed signs of phenotypic adaptation to cheese-making relative to P. fuscoglaucum, in terms of its whiter color, faster growth on cheese medium under cave conditions, lower levels of toxin production and greater ability to prevent the growth of other fungi. The P. camemberti lineage displayed even stronger signs of domestication for all these phenotypic features. We also identified two differentiated P. camemberti lineages linked to different kinds of cheeses with contrasted phenotypic features in terms of color, growth, toxin production and competitive ability. We have, thus, identified footprints of domestication in these fungi, with genetic differentiation between cheese and wild strains and specific phenotypic traits beneficial for cheese-making. This study has not only fundamental implications for our understanding of domestication but can also have important impacts on cheese-making.

Moldy food products that are not subject to pathogenic bacterial contamination could be trimmed by consumers to remove fungal mycelium before consumption. However, prior to giving such recommendations to consumers, it is necessary to evaluate potential mycotoxin migration in these products. This study aimed at quantifying citrinin (CIT) and ochratoxin A (OTA) accumulation and migration in a French semi-hard Comte cheese after artificial inoculation with a CIT- and OTA-producing Penicillium verrucosum strain. At 8 °C, CIT and OTA production started after 14 days and 28 days incubation, respectively; while at 20 °C, both mycotoxins were produced from day 7. At 20 °C, maximum CIT concentration, about 50000 ng/g, was 20 fold that at 8 °C. Regardless of temperature, maximum OTA concentration was about 4000 ng/g cheese. Maximum concentrations were obtained in the upper part of the cheese, but depending on incubation time, mycotoxins were detected up to 1.6 cm in depth. As long as only white mycelium developed on the cheese surface, trimming can be acceptable, but a blue mold color (due to fungal sporulation) was associated with the accumulation of significant amounts of mycotoxins so the product should be discarded.

PR toxin is a well-known isoprenoid mycotoxin almost solely produced by Penicillium roqueforti after growth on food or animal feed. This mycotoxin has been described as the most toxic produced by this species. In this study, an in silico analysis allowed identifying for the first time a 22.4-kb biosynthetic gene cluster involved in PR toxin biosynthesis in P. roqueforti. The pathway contains 11 open reading frames encoding for ten putative proteins including the major fungal terpene cyclase, aristolochene synthase, involved in the first farnesyl-diphosphate cyclization step as well as an oxidoreductase, an oxidase, two P450 monooxygenases, a transferase, and two dehydrogenase enzymes. Gene silencing was used to study three genes (ORF5, ORF6, and ORF8 encoding for an acetyltransferase and two P450 monooxygenases, respectively) and resulted in 20 to 40% PR toxin production reductions in all transformants proving the involvement of these genes and the corresponding enzyme activities in PR toxin biosynthesis. According to the considered silenced gene target, eremofortin A and B productions were also affected suggesting their involvement as biosynthetic intermediates in this pathway. A PR toxin biosynthesis pathway is proposed based on the most recent and available data.

The ability of three Penicillium expansum isolates to produce patulin was first evaluated in YES medium after incubation at 25 °C to select a high patulin producer. Then, a spore suspension of the selected P. expansum 3.78 strain was inoculated onto the surface of Golden delicious apples and incubated at 8 or 20 °C until the mold lesion reached a diameter of 1, 2 or 3 cm. For each lesion size, patulin was quantified from apple samples cut into 1 cm depthwise fractions and widthwise sized cylinders. Maximum patulin concentration, about 80,000 ng/g apple, was obtained at 8 °C for the center and surface sample of the 3 cm diameter lesion. Patulin was systematically found at the highest concentration in the lesions, but still quantified up to one centimeter next to the lesion. Patulin concentrations were not significantly different between the 8 and 20 °C incubation temperature, except for the 3 cm large lesions. Based on these findings, and for lesions less than or equal to 3 cm in diameter, we recommend to consumers to cut off at least 1 cm around and below the mold spot to limit patulin exposure. Apples should also be stored at cool temperatures, below 8 °C, to delay lesion development.

International audience; This study is an attempt to go further in the comprehension of the effects of heavy fuel oil in the context of an accidental oil spill at sea. It focuses on the link between morphological and functional impacts of realistic doses of the dissolved fraction of a heavy fuel oil on fish gills. Juvenile turbot, Scophthalmus maximus were exposed to the dissolved fraction of a heavy fuel oil for 5 days and then placed 30 days in clean sea water for recovery. During the contamination period, the concentration of the 16 US EPA priority poly-aromatic hydrocarbons showed small variations around a mean value of 321.0 ± 9.1 ng l⁻¹ (mean ± SEM). The contamination induced a 64% increase in hepatic cytochrome P 450 1A (Western blot analysis). Osmolality, [Na⁺] and [Cl⁻] rapidly and significantly increased (by 14, 23 and 28% respectively) and slowly decreased to normal levels during the recovery period. At the same time, branchial histology showed decreases in the number of mucocytes (by 30%) and of chloride cells (by 95%) in the interlamellar epithelium. Therefore, it is suggested that the osmotic imbalance observed after the 5 days of exposure to the dissolved fraction of the heavy fuel oil is the consequence of the structural alteration of the gills i.e, the strong reduction of ionocyte numbers.

International audience; Flatfishes, turbots (Scophthalmus maximus), were injected intraperitoneally with two doses of fuel oil number 2. Biliary metabolites were evaluated by fixed fluorescence to verify the efficiency of intoxication. Ethoxyresorufin-O-deethylase (EROD) activity was compared with chromosomal damage measured by flow cytometry. The analysis of biliary metabolites showed a good dose-response relation and constitutes a clear reference for the subsequent measurements. Comparing flow cytometry and EROD results, a shorter delay of response for EROD activity was obtained, but chromosomal damage was significant only after one week. The persistence of the EROD response was shorter, while the genotoxic signal still persisted after one month. The measurement of chromosomal damage allowed a good differentiation between the two tested doses. In the case of EROD activity, the results were less clear. The results suggest that within a few weeks after exposure to fuel oil number 2, the measurements of chromosomal damage by flow cytometry can be used to detect a dose-dependent genotoxic response in fish.