Microbiota associated with commercial dry-aged beef in France.

Meat dry aging consists in storing unpackaged meat in a cold room, and at a specific and controlled relative humidity (RH), for a period of 1 to 5 weeks or more. This practice has become widespread in recent years due to its positive effect on the tenderness of the meat but also on other organoleptic characteristics and therefore its market value. The objective of this work was to study the bacterial and fungal microbiota of dry-aged beef at the commercial stage by both culture-dependent and -independent approaches. Fifty-eight samples of dry-aged meat from different producer types (meat processing plants, artisanal and supermarket butchers) were studied. The dry-aging conditions (temperature, RH) of the meats, as well as the surface pH and aw, were measured. The main microbial groups were enumerated by culture on various dedicated media. Concerning fungi, isolates of yeasts and molds (n = 257) were identified after dereplication by FTIR spectroscopy and/or sequencing of taxonomically relevant genes (26S rDNA, ITS, ß-tubulin, actin). Metagenetic analyzes targeting the V3-V4 regions of 16S rDNA and ITS2 were also performed. Overall, ripening practices were diversified with temperatures and RH between 0.5 and 2.8 °C (median = 2 °C) and 47 and 88 % (median = 70 %), respectively. The aerobic colony count varied between 1.97 and 10.91 log10 CFU/g (median = 8.32 log10 CFU/g) and was similar to that of Pseudomonas spp., indicating that this bacterial group was dominant. Yeast populations varied between <2 and 9.41 log10 CFU/g, while molds showed abundances between <2 and 7.7 log10 TFU/g, the highest values being found in meats matured with a high RH. Bacterial and mold counts were positively correlated with the dry-aging RH and, to a lesser extent, temperature. The main yeast species were Candida zeylanoides and Yarrowia alimentaria as well as Itersonilia pannonica (identified only in metagenetics). The dominant mold species were psychrophilic or psychrotrophic species, namely Mucor complex flavus and Helycostylum elegans/pulchrum that have already been shown to be associated with dry-aged beef meat. This study has identified the main microorganisms associated with dry-aged meat in France, which raises the question of their role in the organoleptic quality of these higher value products.

Discriminative power of DNA-based, volatilome, near infrared spectroscopy, elements and stable isotopes methods for the origin authentication of typical Italian mountain cheese using sPLS-DA modeling.

Origin authentication methods are pivotal in counteracting frauds and provide evidence for certification systems. For these reasons, geographical origin authentication methods are used to ensure product origin. This study focused on the origin authentication (i.e. at the producer level) of a typical mountain cheese origin using various approaches, including shotgun metagenomics, volatilome, near infrared spectroscopy, stable isotopes, and elemental analyses. DNA-based analysis revealed that viral communities achieved a higher classification accuracy rate (97.4 ± 2.6 %) than bacterial communities (96.1 ± 4.0 %). Non-starter lactic acid bacteria and phages specific to each origin were identified. Volatile organic compounds exhibited potential clusters according to cheese origin, with a classification accuracy rate of 90.0 ± 11.1 %. Near-infrared spectroscopy showed lower discriminative power for cheese authentication, yielding only a 76.0 ± 31.6 % classification accuracy rate. Model performances were influenced by specific regions of the infrared spectrum, possibly associated with fat content, lipid profile and protein characteristics. Furthermore, we analyzed the elemental composition of mountain Caciotta cheese and identified significant differences in elements related to dairy equipment, macronutrients, and rare earth elements among different origins. The combination of elements and isotopes showed a decrease in authentication performance (97.0 ± 3.1 %) compared to the original element models, which were found to achieve the best classification accuracy rate (99.0 ± 0.01 %). Overall, our findings emphasize the potential of multi-omics techniques in cheese origin authentication and highlight the complexity of factors influencing cheese composition and hence typicity.

Cytotoxic Effects of Major and Emerging Mycotoxins on HepaRG Cells and Transcriptomic Response after Exposure of Spheroids to Enniatins B and B1.

Mycotoxins, produced by fungi, frequently occur at different stages in the food supply chain between pre- and postharvest. Globally produced cereal crops are known to be highly susceptible to contamination, thus constituting a major public health concern. Among the encountered mycotoxigenic fungi in cereals, Fusarium spp. are the most frequent and produce both regulated (i.e., T-2 toxin, deoxynivalenol -DON-, zearalenone -ZEA-) and emerging (i.e., enniatins -ENNs-, beauvericin -BEA-) mycotoxins. In this study, we investigated the in vitro cytotoxic effects of regulated and emerging fusariotoxins on HepaRG cells in 2D and 3D models using undifferentiated and differentiated cells. We also studied the impact of ENN B1 and ENN B exposure on gene expression of HepaRG spheroids. Gene expression profiling pinpointed the differentially expressed genes (DEGs) and overall similar pathways were involved in responses to mycotoxin exposure. Complement cascades, metabolism, steroid hormones, bile secretion, and cholesterol pathways were all negatively impacted by both ENNs. For cholesterol biosynthesis, 23/27 genes were significantly down-regulated and could be correlated to a 30% reduction in cholesterol levels. Our results show the impact of ENNs on the cholesterol biosynthesis pathway for the first time. This finding suggests a potential negative effect on human health due to the essential role this pathway plays.

Ecological diversity and associated volatilome of typical mountain Caciotta cheese from Italy.

Traditional products are particularly appreciated by consumers and among these products, cheese is a major contributor to the Italian mountainous area economics. In this study, shotgun metagenomics and volatilomics were used to understand the biotic and abiotic factors contributing to mountain Caciotta cheese typicity and diversity. Results showed that the origin of cheese played a significant role; however, curd cooking temperature, pH, salt concentration and water activity also had an impact. Viral communities exhibited higher biodiversity and discriminated cheese origins in terms of production farms. Among the most dominant bacteria, Streptococcus thermophilus showed higher intraspecific diversity and closer relationship to production farm when compared to Lactobacillus delbrueckii. However, despite a few cases in which the starter culture was phylogenetically separated from the most dominant strains sequenced in the cheese, starter cultures and dominant cheese strains clustered together suggesting substantial starter colonization in mountain Caciotta cheese. The Caciotta cheese volatilome contained prominent levels of alcohols and ketones, accompanied by lower proportions of terpenes. Volatile profile not only demonstrated a noticeable association with production farm but also significant differences in the relative abundances of enzymes connected to flavor development. Moreover, correlations of different non-homologous isofunctional enzymes highlighted specific contributions to the typical flavor of mountain Caciotta cheese. Overall, this study provides a deeper understanding of the factors shaping typical mountain Caciotta cheese, and the potential of metagenomics for characterizing and potentially authenticating food products.

A new cheese population in Penicillium roqueforti and adaptation of the five populations to their ecological niche.

Domestication is an excellent case study for understanding adaptation and multiple fungal lineages have been domesticated for fermenting food products. Studying domestication in fungi has thus both fundamental and applied interest. Genomic studies have revealed the existence of four populations within the blue-cheese-making fungus Penicillium roqueforti. The two cheese populations show footprints of domestication, but the adaptation of the two non-cheese populations to their ecological niches (i.e., silage/spoiled food and lumber/spoiled food) has not been investigated yet. Here, we reveal the existence of a new P. roqueforti population, specific to French Termignon cheeses, produced using small-scale traditional practices, with spontaneous blue mould colonisation. This Termignon population is genetically differentiated from the four previously identified populations, providing a novel source of genetic diversity for cheese making. The Termignon population indeed displayed substantial genetic diversity, both mating types, horizontally transferred regions previously detected in the non-Roquefort population, and intermediate phenotypes between cheese and non-cheese populations. Phenotypically, the non-Roquefort cheese population was the most differentiated, with specific traits beneficial for cheese making, in particular higher tolerance to salt, to acidic pH and to lactic acid. Our results support the view that this clonal population, used for many cheese types in multiple countries, is a domesticated lineage on which humans exerted strong selection. The lumber/spoiled food and silage/spoiled food populations were not more tolerant to crop fungicides but showed faster growth in various carbon sources (e.g., dextrose, pectin, sucrose, xylose and/or lactose), which can be beneficial in their ecological niches. Such contrasted phenotypes between P. roqueforti populations, with beneficial traits for cheese-making in the cheese populations and enhanced ability to metabolise sugars in the lumber/spoiled food population, support the inference of domestication in cheese fungi and more generally of adaptation to anthropized environments.

Functional diversity of Bisifusarium domesticum and the newly described Nectriaceae cheese-associated species.

Bisifusarium domesticum is among the main molds used during cheese-making for its "anticollanti" property that prevents the sticky smear defect of some cheeses. Previously, numerous cheese rinds were sampled to create a working collection and not only did we isolate B. domesticum but we observed a completely unexpected diversity of "Fusarium-like" fungi belonging to the Nectriaceae family. Four novel cheese-associated species belonging to two genera were described: Bisifusarium allantoides, Bisifusarium penicilloides, Longinectria lagenoides, and Longinectria verticilliformis. In this study, we thus aimed at determining their potential functional impact during cheese-making by evaluating their lipolytic and proteolytic activities as well as their capacity to produce volatile (HS-Trap GC-MS) and non-volatile secondary metabolites (HPLC & LC-Q-TOF). While all isolates were proteolytic and lipolytic, higher activities were observed at 12 °C for several B. domesticum, B. penicilloides and L. lagenoides isolates, which is in agreement with typical cheese ripening conditions. Using volatilomics, we identified multiple cheese-related compounds, especially ketones and alcohols. B. domesticum and B. penicilloides isolates showed higher aromatic potential although compounds of interest were also produced by B. allantoides and L. lagenoides. These species were also lipid producers. Finally, an untargeted extrolite analysis suggested a safety status of these strains as no known mycotoxins were produced and revealed the production of potential novel secondary metabolites. Biopreservation tests performed with B. domesticum suggested that it may be an interesting candidate for biopreservation applications in the cheese industry in the future.

Authenticity and Typicity of Traditional Cheeses: A Review on Geographical Origin Authentication Methods.

Food fraud, corresponding to any intentional action to deceive purchasers and gain an undue economical advantage, is estimated to result in a 10 to 65 billion US dollars/year economical cost worldwide. Dairy products, such as cheese, in particular cheeses with protected land- and tradition-related labels, have been listed as among the most impacted as consumers are ready to pay a premium price for traditional and typical products. In this context, efficient food authentication methods are needed to counteract current and emerging frauds. This review reports the available authentication methods, either chemical, physical, or DNA-based methods, currently used for origin authentication, highlighting their principle, reported application to cheese geographical origin authentication, performance, and respective advantages and limits. Isotope and elemental fingerprinting showed consistent accuracy in origin authentication. Other chemical and physical methods, such as near-infrared spectroscopy and nuclear magnetic resonance, require more studies and larger sampling to assess their discriminative power. Emerging DNA-based methods, such as metabarcoding, showed good potential for origin authentication. However, metagenomics, providing a more in-depth view of the cheese microbiota (up to the strain level), but also the combination of methods relying on different targets, can be of interest for this field.

Impact of temperature application and concentration of commercial sanitizers on inactivation of food-plant fungal spores.

This study aimed at quantifying the impact of the concentration of four commercial sanitizers and temperature on mold spores inactivation. The sanitizers were based on the following fungicide molecules, ethanol (ARVO 21 SR), active chlorine (ARVO CLM 600), hydrogen peroxide (Nocolyse Food) and triamine (P3 Topax 960). Food plant spores were produced under a moderate water stress, 0.95 aw and dry-harvested to simulate airborne spores responsible for contamination in the food industry. First, Aspergillus flavus, Cladosporium cladosporioides, Mucor circinelloides, and two Penicillium commune isolates were tested against the sanitizers at 20 °C and at a concentration recommended by the manufacturers. Overall, A. flavus was the less resistant species. Second the effects of concentration and temperature were assessed on the most resistant species, i.e., P. commune UBOCC-A-116003 (ARVO 21 SR and P3 Topax 960), P. commune UBOCC-A-112059 (ARVO CLM 600), and M. circinelloides (Nocolyse Food). With the exception of ARVO 21 SR, the observed inactivation kinetics were downward concave. The time necessary to obtain 4 log reduction, t4D, was estimated by means of the Weibull model. At 20 °C and at the recommended concentration by the manufacturers, t4D (min) for the most resistant strains were equal to 2.14 (ARVO 21 SR), 7.35 (ARVO CLM 600), 39.3 (Nocolyse Food) and 82.8 (P3 Topax 960). T4D was increased at lower concentrations and temperatures. These effects were more pronounced for ARVO 21 SR, t4D were about 10 fold and 20 fold the above reported value, 2.14 min, at 8 °C and by diluting the sanitizer by a 10:8 factor, respectively. The least effect of temperature, 3 fold, was shown for ARVO CLM 600, while concentration of P3 Topax 960 had no significant effect on t4D within the recommended utilization range.

Effect of abiotic factors and culture media on the growth of cheese-associated Nectriaceae species.

Nectriaceae species have been described in various natural environments or as plant or human pathogens. Within this family, the Bisifusarium domesticum species is of particular interest for food mycologists as it is used for technological functions in various cheese productions. Moreover, it has only been isolated from the cheese environment so far and, until recently, was the only Nectriaceae species described in this food product. Recently, four novel cheese-associated Nectriaceae species have been described, including two associated to the Bisifusarium genus and two to a new genus, Longinectria gen. nov.. These observations raise questions concerning the potential adaptation of these species to the cheese environment. In this context, this study first focused on determining the impact of abiotic factors on the growth of isolates belonging to the five cheese-associated species (i.e. B. allantoides sp. nov., B. domesticum, B. penicilloides sp. nov., L. lagenoides gen. nov. sp. nov. and L. verticilliforme gen. nov. sp. nov.) but also included phylogenetically close species. To do so, fungal growth kinetics in liquid medium (Potato Dextrose Broth) were determined by laser nephelometry at different temperatures, pH and water activities using NaCl as a depressor. Growth modeling was then performed to estimate cardinal values for each abiotic factor. Secondly, fungal growth was also evaluated on Potato Dextrose Agar (synthetic medium), cheese agar (cheese-mimicking medium) and Raclette de Savoie cheese (actual cheese). Our results clearly highlighted physiological differences in growth characteristics between the studied cheese-associated Nectriaceae spp. and the "non-cheese" species which could suggest, for the former, an adaptation to this food matrix. Indeed, regarding the impact of the tested abiotic factors, statistical analyses confirmed this dichotomy, with for example the lowest optimal temperatures estimated for the cheese-associated species (Topt 19.1-23.1 °C) while the other Bisifusarium species exhibited the highest optimal temperatures (Topt 26.1-36.2 °C). As for the impact of growth media, radial growth measurements highlighted that B. domesticum was the least affected species for growth on Raclette de Savoie and even grew faster on cheese agar than on synthetic medium confirming its strong adaptation to the cheese environment.

Microbial Ecology of French Dry Fermented Sausages and Mycotoxin Risk Evaluation During Storage.

Dry fermented sausages are produced worldwide by well-controlled fermentation processes involving complex microbiota including many bacterial and fungal species with key technological roles. However, to date, fungal diversity on sausage casings during storage has not been fully described. In this context, we studied the microbial communities from dry fermented sausages naturally colonized or voluntarily surface inoculated with molds during storage using both culture-dependent and metabarcoding methods. Staphylococci and lactic acid bacteria largely dominated in samples, although some halotolerant genera (e.g., Halomonas, Tetragenococcus, and Celerinatantimonas spp.) were also frequently observed. Fungal populations varied from 7.2 to 9.8 log TFU/cm2 sausage casing during storage, suggesting relatively low count variability among products. Fungal diversity identified on voluntarily inoculated casings was lower (dominated by Penicillium nalgiovense and Debaryomyces hansenii) than naturally environment-inoculated fermented sausages (colonized by P. nalgiovense, Penicillium nordicum, and other Penicillium spp. and sporadically by Scopulariopsis sp., D. hansenii, and Candida zeylanoïdes). P. nalgiovense and D. hansenii were systematically identified, highlighting their key technological role. The mycotoxin risk was then evaluated, and in situ mycotoxin production of selected mold isolates was determined during pilot-scale sausage productions. Among the identified fungal species, P. nalgiovense was confirmed not to produce mycotoxins. However, some P. nordicum, Penicillium chrysogenum, Penicillium bialowienzense, Penicillium brevicompactum, and Penicillium citreonigrum isolates produced one or more mycotoxins in vitro. P. nordicum also produced ochratoxin A during pilot-scale sausage productions using "worst-case" conditions in the absence of biotic competition. These data provide new knowledge on fermented sausage microbiota and the potential mycotoxin risk during storage.

Tailor-made microbial consortium for Kombucha fermentation: Microbiota-induced biochemical changes and biofilm formation.

Kombucha is a very distinct naturally fermented sweetened tea that has been produced for thousands of years. Fermentation relies on metabolic activities of the complex autochthonous symbiotic microbiota embedded in a floating biofilm and used as a backslop for successive fermentations. Here, we designed a tailor-made microbial consortium representative of the core Kombucha microbiota to drive this fermentation. Microbial (counts, metagenetics), physico-chemical (pH, density) and biochemical (organic acids, volatile compounds) parameters were monitored as well as biofilm formation by confocal laser scanning microscopy and scanning electron microscopy. While nine species were co-inoculated, four (Dekkera bruxellensis, Hanseniaspora uvarum, Acetobacter okinawensis and Liquorilactobacillus nagelii) largely dominated. Microbial activities led to acetic, lactic, succinic and oxalic acids being produced right from the start of fermentation while gluconic and glucuronic acids progressively increased. A distinct shift in volatile profile was also observed with mainly aldehydes identified early on, then high abundances of fatty acids, ketones and esters at the end. Correlation analyses, combining metabolomic and microbial data also showed a shift in species abundances during fermentation. We also determined distinct bacteria-yeast co-occurence patterns in biofilms by microscopy. Our study provides clear evidence that a tailor-made consortium can be successfully used to drive Kombucha fermentations.

Impact of the physiological state of fungal spores on their inactivation by active chlorine and hydrogen peroxide.

This study aimed at assessing the impact of the physiological state of fungal spores on inactivation by sodium hypochlorite, 0.1% and 0.2% active chlorine, and 3% hydrogen peroxide. In this context, two physiological states were compared for 4 fungal species (5 strains). The first physiological state corresponded to fungal spores produced at 0.99 aw and harvested using an aqueous solution (laboratory conditions), while the second one corresponded to fungal spores produced under a moderate water stress (0.95 aw) and dry-harvested (mechanical harvesting without use of any water, mimicking food plant conditions). Aspergillus flavus "food plant" conidia were more resistant to all tested fungicide molecules than the "laboratory" ones. The same phenomenon was observed for Penicillium commune UBOCC-A-116003 conidia treated with hydrogen peroxide. However, this isolate did not exhibit any inactivation difference between "laboratory" and "food plant" conidia treated with sodium hypochlorite. Similarly, the physiological state of Cladosporium cladosporioides conidia did not impact the efficacy of the tested biocides. P. commune UBOCC-A-112059 "food plant" and "laboratory" conidia were more resistant to hydrogen peroxide and sodium hypochlorite, respectively. As for Mucor circinelloides, "laboratory" spores were more resistant to all disinfectant than the "food plant" ones. Noteworthy, regardless of the physiological state, all M. circinelloides and C. cladosporioides conidia were inactivated for 5 min treatment at 0.2% active chlorine and for 2.5 min treatment at 0.1% active chlorine, while the conidia of all the other species remained viable for these treatments. The obtained data indicate that the efficacy of disinfectant molecules depends not only on the encountered fungal species and its intraspecific diversity but also on the spore physiological state.

Domestication of the Emblematic White Cheese-Making Fungus Penicillium camemberti and Its Diversification into Two Varieties.

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, by divergence from the blue-green wild P. fuscoglaucum fungus. Another much more recent domestication event led to the generation of the P. camemberti clonal lineage as a sister group to P. biforme. Penicillium biforme displayed signs of phenotypic adaptation to cheese making relative to P. fuscoglaucum, in terms of whiter color, faster growth on cheese medium under cave conditions, lower amounts 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 varieties, 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 effects on cheese making.

Impact of intraspecific variability and physiological state on Penicillium commune inactivation by 70% ethanol.

The aim of this study was to assess the impact of the physiological state and intraspecific variability on the efficacy of 70% ethanol to inactivate conidia of Penicillium commune, used as a representative species of dairy product contaminants. Four physiological states were obtained by modifying the water activity during the production of conidia (0.995 and 0.950) and the harvesting conditions (hydrated and non-hydrated). These conditions were applied to four different P. commune strains isolated from contaminated dairy products. Five minutes exposure to 70% ethanol at ambient temperature allowed total inactivation of conidia (>4 log10) regardless of the physiological state or the strain. For 1 min exposure, regardless of the strains, only dry-harvested conidia produced at aw 0.950 exhibited survivors. Survival after 2 min exposure was observed for this physiological state for P. commune UBOCC-A-116003 only. For this strain, the impact of the physiological state was greater than 1.54 log10 between dry-harvested conidia produced at aw 0.950 that exhibited survivors after 1 min treatment and the 3 other kinds of conidia that were all inactivated. For 1 min exposure, by comparing the more resistant strain to the three other strains, the impact of the intraspecific variability was 2.35 log10. These results demonstrated that the physiological state of the conidia, the representativeness of the tested species and strains should be taken into account to assess the efficacy of disinfectants in dairies.

Antifungal activity of fermented dairy ingredients: Identification of antifungal compounds.

Fungi are commonly identified as the cause for dairy food spoilage. This can lead to substantial economic losses for the dairy industry as well as consumer dissatisfaction. In this context, biopreservation of fermented dairy products using lactic acid bacteria, propionibacteria and fungi capable of producing a large range of antifungal metabolites is of major interest. In a previous study, extensive screening was performed in vitro and in situ to select 3 dairy fermentates (derived from Acidipropionibacterium jensenii CIRM-BIA1774, Lactobacillus rhamnosus CIRM-BIA1952 and Mucor lanceolatus UBOCC-A-109193, respectively) with antifungal activity. The aim of the present study was to determine the main compounds responsible for this antifungal activity. Fifty-six known antifungal compounds as well as volatiles were targeted using different analytical methods (conventional LC and GC, GC-MS, LC-QToF). The most abundant antifungal compounds in P. jensenii-, L. rhamnosus- and M. lanceolatus-derived fermentates corresponded to propionic and acetic acids, lactic and acetic acids, and butyric acid, respectively. Many other antifungal compounds (organic acids, free fatty acids, volatile compounds) were identified but at lower levels. In addition, an untargeted approach using nano LC-MS/MS identified a 9-amino acid peptide derived from αs2-casein in the L. rhamnosus-derived fermentate. This peptide inhibited M. racemosus and R. mucilaginosa in vitro. This study provides new insights on the molecules involved in antifungal activities of food-grade microorganism fermentates which could be used as antifungal ingredients in the dairy industry.

Comparative genomics applied to Mucor species with different lifestyles.

BACKGROUND: Despite a growing number of investigations on early diverging fungi, the corresponding lineages have not been as extensively characterized as Ascomycota or Basidiomycota ones. The Mucor genus, pertaining to one of these lineages is not an exception. To this date, a restricted number of Mucor annotated genomes is publicly available and mainly correspond to the reference species, Mucor circinelloides, and to medically relevant species. However, the Mucor genus is composed of a large number of ubiquitous species as well as few species that have been reported to specifically occur in certain habitats. The present study aimed to expand the range of Mucor genomes available and identify potential genomic imprints of adaptation to different environments and lifestyles in the Mucor genus. RESULTS: In this study, we report four newly sequenced genomes of Mucor isolates collected from non-clinical environments pertaining to species with contrasted lifestyles, namely Mucor fuscus and Mucor lanceolatus, two species used in cheese production (during ripening), Mucor racemosus, a recurrent cheese spoiler sometimes described as an opportunistic animal and human pathogen, and Mucor endophyticus, a plant endophyte. Comparison of these new genomes with those previously available for six Mucor and two Rhizopus (formerly identified as M. racemosus) isolates allowed global structural and functional description such as their TE content, core and species-specific genes and specialized genes. We proposed gene candidates involved in iron metabolism; some of these genes being known to be involved in pathogenicity; and described patterns such as a reduced number of CAZymes in the species used for cheese ripening as well as in the endophytic isolate that might be related to adaptation to different environments and lifestyles within the Mucor genus. CONCLUSIONS: This study extended the descriptive data set for Mucor genomes, pointed out the complexity of obtaining a robust phylogeny even with multiple genes families and allowed identifying contrasting potentially lifestyle-associated gene repertoires. The obtained data will allow investigating further the link between genetic and its biological data, especially in terms of adaptation to a given habitat.

Independent domestication events in the blue-cheese fungus Penicillium roqueforti.

Domestication provides an excellent framework for studying adaptive divergence. Using population genomics and phenotypic assays, we reconstructed the domestication history of the blue cheese mould Penicillium roqueforti. We showed that this fungus was domesticated twice independently. The population used in Roquefort originated from an old domestication event associated with weak bottlenecks and exhibited traits beneficial for pre-industrial cheese production (slower growth in cheese and greater spore production on bread, the traditional multiplication medium). The other cheese population originated more recently from the selection of a single clonal lineage, was associated with all types of blue cheese worldwide except Roquefort, and displayed phenotypes more suited for industrial cheese production (high lipolytic activity, efficient cheese cavity colonization ability and salt tolerance). We detected genomic regions affected by recent positive selection and putative horizontal gene transfers. This study sheds light on the processes of rapid adaptation and raises questions about genetic resource conservation.

Production and migration of patulin in Penicillium expansum molded apples during cold and ambient storage.

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.

Identification and quantification of natural compounds produced by antifungal bioprotective cultures in dairy products.

In the context of growing consumer demand for clean label foods, antifungal cultures offer alternatives to chemical preservatives for the reduction of food fungal spoilage. Selected binary combinations of lactobacilli strains were recently successfully used to inhibit Penicillium commune and Mucor racemosus in four dairy products. Our aim was to identify the compounds most likely involved in their antifungal activity. Four chromatographic methods, targeting 56 antifungal compounds as well as volatiles, were combined. Overall, 53 antifungal compounds were detected, of which 33 were in significantly higher amounts in at least one product inoculated with an antifungal culture compared to the controls. They were present at concentrations below their MIC and thus could act in synergy. Among them, the most commonly identified were acetic, hydroxyphenyllactic, phenyllactic, 3-phenylpropanoic, 3-(4-hydroxyphenyl)propanoic and 5-oxopyrrolidine-2-carboxylic acids, diacetyl, acetoin, and an unidentified volatile. This extensive study contributes to improve the knowledge about the action mode of antifungal lactobacilli.

Expanding the biodiversity of Oenococcus oeni through comparative genomics of apple cider and kombucha strains.

BACKGROUND: Oenococcus oeni is a lactic acid bacteria species adapted to the low pH, ethanol-rich environments of wine and cider fermentation, where it performs the crucial role of malolactic fermentation. It has a small genome and has lost the mutS-mutL DNA mismatch repair genes, making it a hypermutable and highly specialized species. Two main lineages of strains, named groups A and B, have been described to date, as well as other subgroups correlated to different types of wines or regions. A third group "C" has also been hypothesized based on sequence analysis, but it remains controversial. In this study we have elucidated the species population structure by sequencing 14 genomes of new strains isolated from cider and kombucha and performing comparative genomics analyses. RESULTS: Sequence-based phylogenetic trees confirmed a population structure of 4 clades: The previously identified A and B, a third group "C" consisting of the new cider strains and a small subgroup of wine strains previously attributed to group B, and a fourth group "D" exclusively represented by kombucha strains. A pair of complete genomes from group C and D were compared to the circularized O. oeni PSU-1 strain reference genome and no genomic rearrangements were found. Phylogenetic trees, K-means clustering and pangenome gene clusters evidenced the existence of smaller, specialized subgroups of strains. Using the pangenome, genomic differences in stress resistance and biosynthetic pathways were found to uniquely distinguish the C and D clades. CONCLUSIONS: The obtained results, including the additional cider and kombucha strains, firmly established the O. oeni population structure. Group C does not appear as fully domesticated as group A to wine, but showed several unique patterns which may be due to ongoing specialization to the cider environment. Group D was shown to be the most divergent member of O. oeni to date, appearing as the closest to a pre-domestication state of the species.