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.

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.

Comparative analysis of five Mucor species transcriptomes.

Mucor species belong to the Mucorales order within the Mucoromycota phylum, an early diverging fungal lineage. Although Mucor species are often ubiquitous some species have been reported to specifically occur in certain ecological niches. In this study, similarities and differences of a representative set of Mucor species with contrasted lifestyles were investigated at the transcriptome level. Five strains pertaining to five different species were studied, namely M. fuscus and M. lanceolatus, two species used in cheese production (during ripening), M. racemosus, a recurrent cheese spoiler sometimes described as an opportunistic pathogen, M. circinelloides, often described as an opportunistic pathogen and M. endophyticus, a plant endophyte. A core transcriptome was delimited and a phylogenetic analysis led to an altered phylogenetic placement of M. endophyticus compared to previously published topologies. Interestingly, the core transcriptome comprising 5566 orthogroups included genes potentially involved in secondary metabolism. As expected, given the wide taxonomic range investigated, the five transcriptomes also displayed specificities that can be, for some of them, linked to the different lifestyles such as differences in the composition of transcripts identified as virulence factors or carbohydrate transporters.

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.

Production and migration of ochratoxin A and citrinin in Comté cheese by an isolate of Penicillium verrucosum selected among Penicillium spp. mycotoxin producers in YES medium.

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 Comté 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.

Selection of Algerian lactic acid bacteria for use as antifungal bioprotective cultures and application in dairy and bakery products.

In the context of a demand for "preservative-free" food products, biopreservation appears as a promising alternative to either replace or reduce the use of chemical preservatives. The purpose of this study was to evaluate the antifungal activity of a collection of lactic acid bacteria (n = 194), and then to evaluate the applicability and efficacy of selected ones used as bioprotective cultures against mold spoilers in dairy and bakery products. First, lactic acid bacteria were isolated from various Algerian raw milk samples and Amoredj, a traditional fermented product. Secondly, in vitro screening tests against Mucor racemosus UBOCC-A-109155, Penicillium commune UBOCC-A-116003, Yarrowia lipolytica UBOCC-A-216006, Aspergillus tubingensis AN, Aspergillus flavus T5 and Paecilomyces formosus AT allowed for the selection of 3 active strains, namely Lactobacillus plantarum CH1, Lactobacillus paracasei B20 and Leuconostoc mesenteroides L1. In situ tests were then performed to validate their activity in actual products (sour cream and sourdough bread) challenged with fungal spoilers. These tests showed that antifungal LAB could slow the fungal target growth and could be candidates of interest for industrial applications. Finally, organic acids and various antifungal compounds produced in sour cream and sourdough bread by the selected LAB, and thus potentially supporting the observed antifungal activity, were identified and quantified by HPLC and LC-QTOF.

Development of antifungal ingredients for dairy products: From in vitro screening to pilot scale application.

Biopreservation represents a complementary approach to traditional hurdle technologies for reducing microbial contaminants (pathogens and spoilers) in food. In the dairy industry that is concerned by fungal spoilage, biopreservation can also be an alternative to preservatives currently used (e.g. natamycin, potassium sorbate). The aim of this study was to develop antifungal fermentates derived from two dairy substrates using a sequential approach including an in vitro screening followed by an in situ validation. The in vitro screening of the antifungal activity of fermentates derivating from 430 lactic acid bacteria (LAB) (23 species), 70 propionibacteria (4 species) and 198 fungi (87 species) was performed against four major spoilage fungi (Penicillium commune, Mucor racemosus, Galactomyces geotrichum and Yarrowia lipolytica) using a cheese-mimicking model. The most active fermentates were obtained from Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei/paracasei and Lactobacillus plantarum among the tested LAB, Propionibacterium jensenii among propionibacteria, and Mucor lanceolatus among the tested fungi. Then, for the 11 most active fermentates, culture conditions were optimized by varying incubation time and temperature in order to enhance their antifungal activity. Finally, the antifungal activity of 3 fermentates of interest obtained from Lactobacillus rhamnosus CIRM-BIA1952, P. jensenii CIRM-BIA1774 and M. lanceolatus UBOCC-A-109193 were evaluated in real dairy products (sour cream and semi-hard cheese) at a pilot-scale using challenge and durability tests. In parallel, the impact of these ingredients on organoleptic properties of the obtained products was also assessed. In semi-hard cheese, application of the selected fermentates on the cheese surface delayed the growth of spoilage molds for up to 21 days, without any effect on organoleptic properties, P. jensenii CIRM-BIA1774 fermentate being the most active. In sour cream, incorporation of the latter fermentate at 2 or 5% yielded a high antifungal activity but was detrimental to the product organoleptic properties. Determination of the concentration limit, compatible with product acceptability, showed that incorporation of this fermentate at 0.4% prevented growth of fungal contaminants in durability tests but had a more limited effect against M. racemosus and P. commune in challenge tests. To our knowledge, this is the first time that the workflow followed in this study, from in vitro screening using dairy matrix to scale-up in cheese and sour cream, is applied for production of natural ingredients relying on a large microbial diversity in terms of species and strains. This approach allowed obtaining several antifungal fermentates which are promising candidates for dairy products biopreservation.

Effects of fusariotoxin co-exposure on THP-1 human immune cells.

Deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T2), fumonisin B1 (FB1), zearalenone (ZEA), and moniliformin (MON) mycotoxins are common food and feed contaminants produced by Fusarium spp. However, while they are usually found to co-occur in a large range of commodities, only few data are available on mycotoxin co-exposure effects and cellular response mechanisms. In this study, the individual and combined toxic effects of these fusariotoxins were evaluated on the THP-1 human immune cell line as major fusariotoxins are mostly potent immunomodulators. In particular, four relevant fusariotoxin mixtures, namely DON-MON, DON-FB1, DON-ZEA, and NIV-T2, were studied using several parameters including cell viability as well as the expression of cell surface markers and the main mitogen-activated protein kinases (MAPKs). After 48 h exposure, a reduction of cell viability in a dose-dependent manner was observed for T2, the most cytotoxic mycotoxin, followed by NIV, DON, MON, FB1, and ZEA. Regarding mycotoxin mixtures, they mainly showed antagonism on cell viability reduction. Interestingly, at concentrations inhibiting 50% of cell viability, most viable cells exhibited surface marker loss and thus became potentially non-functional. In addition, during the first 18 h of exposure, the effects of mycotoxin mixtures on early cell apoptosis and necrosis were found to be different from those induced by the toxins alone. At the molecular level, after 1 h exposure of individual and combined mycotoxins, the three main MAPK signaling pathways (p38, SAPK/JNK, and ERK1/2) were activated, highlighting a fast reaction of the exposed cells even at low cytotoxicity levels.

Technical note: High-throughput method for antifungal activity screening in a cheese-mimicking model.

In this study, we developed a high-throughput antifungal activity screening method using a cheese-mimicking matrix distributed in 24-well plates. This method allowed rapid screening of a large variety of antifungal agent candidates: bacterial fermented ingredients, bacterial isolates, and preservatives. Using the proposed method, we characterized the antifungal activity of 44 lactic acid bacteria (LAB) fermented milk-based ingredients and 23 LAB isolates used as protective cultures against 4 fungal targets (Mucor racemosus, Penicillium commune, Galactomyces geotrichum, and Yarrowia lipolytica). We also used this method to determine the minimum inhibitory concentration of a preservative, natamycin, against 9 fungal targets. The results underlined the strain-dependency of LAB antifungal activity, the strong effect of fermentation substrate on this activity, and the effect of the screening medium on natamycin minimum inhibitory concentration. Our method could achieved a screening rate of 1,600 assays per week and can be implemented to evaluate antifungal activity of microorganisms, fermentation products, or purified compounds compatible with dairy technology.

Penicillium roqueforti PR toxin gene cluster characterization.

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.

Genetic basis for mycophenolic acid production and strain-dependent production variability in Penicillium roqueforti.

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.

Microsatellite analysis of Saccharomyces uvarum diversity.

Considered as a sister species of Saccharomyces cerevisiae, S. uvarum is, to a lesser extent, an interesting species for fundamental and applied research studies. Despite its potential interest as a new gene pool for fermenting agents, the intraspecific molecular genetic diversity of this species is still poorly investigated. In this study, we report the use of nine microsatellite markers to describe S. uvarum genetic diversity and population structure among 108 isolates from various geographical and substrate origins (wine, cider and natural sources). Our combined microsatellite markers set allowed differentiating 89 genotypes. In contrast to S. cerevisiae genetic diversity, wild and human origin isolates were intertwined. A total of 75% of strains were proven to be homozygotes and estimated heterozygosity suggests a selfing rate above 0.95 for the different population tested here. From this point of view, the S. uvarum life cycle appears to be more closely related to S. paradoxus or S. cerevisiae of natural resources than S. cerevisiae wine isolates. Population structure could not be correlated to distinct geographic or technological origins, suggesting lower differentiation that may result from a large exchange between human and natural populations mediated by insects or human activities.

Effect of temperature, pH, and water activity on Mucor spp. growth on synthetic medium, cheese analog and cheese.

The Mucor genus includes a large number of ubiquitous fungal species. In the dairy environment, some of them play a technological role providing typical organoleptic qualities to some cheeses while others can cause spoilage. In this study, we compared the effect of relevant abiotic factors for cheese production on the growth of six strains representative of dairy technological and contaminant species as well as of a non cheese related strain (plant endophyte). Growth kinetics were determined for each strain in function of temperature, water activity and pH on synthetic Potato Dextrose Agar (PDA), and secondary models were fitted to calculate the corresponding specific cardinal values. Using these values and growth kinetics acquired at 15 °C on cheese agar medium (CA) along with three different cheese types, optimal growth rates (µopt) were estimated and consequently used to establish a predictive model. Contrarily to contaminant strains, technological strains showed higher µopt on cheese matrices than on PDA. Interestingly, lag times of the endophyte strain were strongly extended on cheese related matrices. This study offers a relevant predictive model of growth that may be used for better cheese production control but also raises the question of adaptation of some Mucor strains to the cheese.

1-Octanol, a self-inhibitor of spore germination in Penicillium camemberti.

Penicillium camemberti is a technologically relevant fungus used to manufacture mold-ripened cheeses. This fungal species produces many volatile organic compounds (VOCs) including ammonia, methyl-ketones, alcohols and esters. Although it is now well known that VOCs can act as signaling molecules, nothing is known about their involvement in P. camemberti lifecycle. In this study, spore germination was shown to be self-regulated by quorum sensing in P. camemberti. This phenomenon, also called "crowding effect", is population-dependent (i.e. observed at high population densities). After determining the volatile nature of the compounds involved in this process, 1-octanol was identified as the main compound produced at high-spore density using GC-MS. Its inhibitory effect was confirmed in vitro and 3 mM 1-octanol totally inhibited spore germination while 100 µM only transiently inhibited spore germination. This is the first time that self-inhibition of spore germination is demonstrated in P. camemberti. The obtained results provide interesting perspectives for better control of mold-ripened cheese processes.

Two novel Saccharomycopsis species isolated from black olive brines and a tropical plant. Description of Saccharomycopsis olivae f. a., sp. nov. and Saccharomycopsis guyanensis f. a., sp. nov. Reassignment of Candida amapae to Saccharomycopsis amapae f. a., comb. nov., Candida lassenensis to Saccharomycopsis lassenensis f. a., comb. nov. and Arthroascus babjevae to Saccharomycopsis babjevae f. a., comb. nov.

Three yeast strains related to members of the genus Saccharomycopsis were isolated. One strain (CLIB 1310) was isolated from olive brines of fermented black olives in France and two strains (CLIB 1454 and CLIB 1455) were isolated from a plant in French Guiana. Sequence analyses based on the D1/D2 domains of the nuclear large subunit rRNA gene, small-subunit rRNA gene and partial EF-1α gene revealed that the strains represented two novel taxa exhibiting extensive sequence divergence from the previously described species of the genus Saccharomycopsis. Two novel species are described to accommodate these newly isolated strains: Saccharomycopsis olivae sp. nov. (type strain CLIB 1310(T) = CBS 12701(T)) and Saccharomycopsis guyanensis sp. nov. (type strain CLIB 1455(T) = CBS 12914(T) and strain CLIB 1454). Both strains CLIB 1454 and CLIB 1455(T) displayed identical sequences but differed in their ability to metabolize sorbitol and in their morphology on agar medium. Candida amapae, Candida lassensensis and Arthroascus babjevae belonging to the Saccharomycopsis clade, are reassigned to Saccharomycopsis as novel combinations.

Filamentous Fungi and Mycotoxins in Cheese: A Review.

Important fungi growing on cheese include Penicillium, Aspergillus, Cladosporium, Geotrichum, Mucor, and Trichoderma. For some cheeses, such as Camembert, Roquefort, molds are intentionally added. However, some contaminating or technological fungal species have the potential to produce undesirable metabolites such as mycotoxins. The most hazardous mycotoxins found in cheese, ochratoxin A and aflatoxin M1, are produced by unwanted fungal species either via direct cheese contamination or indirect milk contamination (animal feed contamination), respectively. To date, no human food poisoning cases have been associated with contaminated cheese consumption. However, although some studies state that cheese is an unfavorable matrix for mycotoxin production; these metabolites are actually detected in cheeses at various concentrations. In this context, questions can be raised concerning mycotoxin production in cheese, the biotic and abiotic factors influencing their production, mycotoxin relative toxicity as well as the methods used for detection and quantification. This review emphasizes future challenges that need to be addressed by the scientific community, fungal culture manufacturers, and artisanal and industrial cheese producers.

Occurrence of the two major regulated mycotoxins, ochratoxin A and fumonisin B1, in cereal and cereal-based products in Europe and toxicological effects: A review.

Among cereal contaminants, mycotoxins are of concern due to their importance in terms of food and feed safety. The difficulty in establishing a diagnosis for mycotoxicosis relies on the fact that the effects are most often subclinical for chronic exposure and the most common scenario is multi-contamination by various toxins. Mycotoxin co-occurrence is a major food safety concern as additive or even synergic toxic impacts may occur, but also regarding current regulations as they mainly concern individual mycotoxin levels in specific foods and feed in the food chain. However, due to the large number of possible mycotoxin combinations, there is still limited knowledge on co-exposure toxicity data, which depends on several parameters. In this context, this systematic review aims to provide an overview of the toxic effects of two regulated mycotoxins, namely ochratoxin A and fumonisin B1. This review focused on the 2012-2022 period and analysed the occurrence in Europe of the selected mycotoxins in different food matrices (cereals and cereal-derived products), and their toxic impact, alone or in combination, on in vitro intestinal and hepatic human cells. To better understand and evaluate the associated risks, further research is needed using new approach methodologies (NAM), such as in vitro 3D models. KEY CONTRIBUTION: Cereals and their derived products are the most important food source for humans and feed for animals worldwide. This manuscript is a state of the art review of the literature over the last ten years on ochratoxin A and fumonisin B1 mycotoxins in these products in Europe as well as their toxicological effects, alone and in combination, on human cells. Future perspectives and some challenges regarding the assessment of toxicological effects of mycotoxins are also discussed.

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.

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.