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.

Starter Culture Development and Innovation for Novel Fermented Foods.

Interest in fermented foods is increasing because fermented foods are promising solutions for more secure food systems with an increased proportion of minimally processed plant foods and a smaller environmental footprint. These developments also pertain to novel fermented food for which no traditional template exists, raising the question of how to develop starter cultures for such fermentations. This review establishes a framework that integrates traditional and scientific knowledge systems for the selection of suitable cultures. Safety considerations, the use of organisms in traditional food fermentations, and the link of phylogeny to metabolic properties provide criteria for culture selection. Such approaches can also select for microbial strains that have health benefits. A science-based approach to the development of novel fermented foods can substantially advance their value through more secure food systems, food products that provide health-promoting microbes, and the provision of foods that improve human health. Expected final online publication date for the Annual Review of Food Science and Technology, Volume 15 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Domestication in dry-cured meat Penicillium fungi: Convergent specific phenotypes and horizontal gene transfers without strong genetic subdivision.

Some fungi have been domesticated for food production, with genetic differentiation between populations from food and wild environments, and food populations often acquiring beneficial traits through horizontal gene transfers (HGTs). Studying their adaptation to human-made substrates is of fundamental and applied importance for understanding adaptation processes and for further strain improvement. We studied here the population structures and phenotypes of two distantly related Penicillium species used for dry-cured meat production, P. nalgiovense, the most common species in the dry-cured meat food industry, and P. salamii, used locally by farms. Both species displayed low genetic diversity, lacking differentiation between strains isolated from dry-cured meat and those from other environments. Nevertheless, the strains collected from dry-cured meat within each species displayed slower proteolysis and lipolysis than their wild conspecifics, and those of P. nalgiovense were whiter. Phenotypically, the non-dry-cured meat strains were more similar to their sister species than to their conspecific dry-cured meat strains, indicating an evolution of specific phenotypes in dry-cured meat strains. A comparison of available Penicillium genomes from various environments revealed HGTs, particularly between P. nalgiovense and P. salamii (representing almost 1.5 Mb of cumulative length). HGTs additionally involved P. biforme, also found in dry-cured meat products. We further detected positive selection based on amino acid changes. Our findings suggest that selection by humans has shaped the P. salamii and P. nalgiovense populations used for dry-cured meat production, which constitutes domestication. Several genetic and phenotypic changes were similar in P. salamii, P. nalgiovense and P. biforme, indicating convergent adaptation to the same human-made environment. Our findings have implications for fundamental knowledge on adaptation and for the food industry: the discovery of different phenotypes and of two mating types paves the way for strain improvement by conventional breeding, to elucidate the genomic bases of beneficial phenotypes and to generate diversity.

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.

Autochthonous starter culture selection for Salame Piemonte PGI production.

Salame Piemonte is a dry-fermented meat product typical of the Piedmont region in Italy, manufactured using commercial starter cultures. This study aimed to select autochthonous starter cultures (ASCs) that could be used for sausage fermentation in order to strengthen the link with the geographical area of production and improve the sensory properties of the final product. A culture-dependent approach was adopted during three different spontaneous sausage fermentation processes to isolate and characterise the main bacterial resources involved. Dominant lactic acid bacteria (LAB) in each batch were Pediococcus pentosaceus, Latilactobacillus sakei, and Latilactobacillus curvatus; Staphylococcus xylosus was the most dominant coagulase-negative staphylococci (CNS) in all the studied batches. LAB and presumptive CNS isolates were further evaluated for their physiological properties and biotechnological potential. Thereafter, 11 strains were selected and evaluated for safety. Five selected strains (two P. pentosaceus, two L. sakei, and one S. xylosus strain) were used for pilot-scale Salame Piemonte production with seven different strain combinations. Based on the liking test, three ASC combinations led to the highest liking score compared to industrial products. These three ASCs were then used for the second pilot-scale sausage production confirming the high liking score. In summary, the use of P. pentosaceus and S. xylosus ASC significantly improved product sensory properties compared with that obtained using commercial starter cultures.

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.

Deciphering the Microbiota and Volatile Profiles of Algerian Smen, a Traditional Fermented Butter.

In Algeria, Smen is a fermented butter produced in households using empirical methods. Smen fermentation is driven by autochthonous microorganisms; it improves butter shelf-life and yields highly fragrant products used as ingredients in traditional dishes as well as in traditional medicine. The present study is aimed at investigating microbial diversity and dynamics during Algerian Smen fermentation using both culture-dependent and culture-independent approaches, as well as by monitoring volatile organic compound production. To reach this goal, fifteen Smen samples (final products) produced in households from different regions in Algeria were collected and analyzed. In addition, microbial and volatile compound dynamics at the different stages of Smen manufacturing were investigated for one Smen preparation. The results showed that Smen is a microbiologically safe product, as all hygiene and safety criteria were respected. The dominant microorganisms identified by both techniques were LAB and yeasts. Lactococcus spp. and Streptococcus thermophilus were the main bacterial species involved in spontaneous raw milk fermentation preceding butter-making, while lactobacilli and enterococci were the only bacteria found to be viable during Smen maturation. Regarding fungal diversity, yeast species were only recovered from two mature Smen samples by culturing, while different species (e.g., Geotrichum candidum, Moniliella sp.) were identified in all samples by the culture-independent approach. Using microbial analysis of a single batch, many of these were found viable during manufacturing. Concerning the volatile profiles, they were highly diverse and characterized by a high prevalence of short chain fatty acids, methylketones, and esters. Correlation analysis between microbial diversity and volatile profiles showed that several yeast (Moniliella sp., K. marxianus) and LAB (e.g., Lactococcus spp., S. thermophilus) species were strongly correlated with one or more volatile organic compound families, including several ethyl esters and methyl ketones that can be linked to pleasant, sweetly floral, fruity, buttery, and creamy odors. This study clearly identified key microorganisms involved in Smen fermentation and maturation that could be used in the future for better fermentation control and improvement of quality attributes.

Brine salt concentration reduction and inoculation with autochthonous consortia: Impact on Protected Designation of Origin Nyons black table olive fermentations.

Nyons table olives, named after the French city where they are processed, are naturally fermented black table olives. Their specificity relies on the use of the "Tanche" olive variety harvested at full maturity and their slow spontaneous fermentation in 10% salt brine driven by yeast populations. This study aimed at investigating the benefit of inoculating autochthonous consortia to produce Nyons table olives by fermentation in 10% salt brine and in reduced salt conditions (8%). Two strategies were evaluated: inoculation with a defined autochthonous consortium and inoculation by spent brine backslopping. To define the consortium, yeasts were selected among 48 autochthonous isolates and key features included high halotolerance, low pectinolytic and proteolytic activities, however none had ß-glucosidase activities. The consortium included eight yeast strains with distinct technological properties belonging to five dominant species, i.e. Citeromyces nyonsensis, Pichia membranifaciens, Wickerhamomyces anomalus, Zygotorulaspora mrakii and Candida atlantica. Fermentation trials were conducted over a year and compared by evaluating microbial community shifts (16S and ITS metagenetics) and volatile profiles (GC-MS). Regarding fermentations with the defined consortium, four out of five species implanted in early stages while one, Pichia membranifaciens, persisted and largely dominated by the end of the fermentation. Altogether, inoculation with the defined consortium did not disrupt microbial shifts compared to traditional fermentations although minor differences were observed in volatile profiles. The backslopping method yielded the highest impact on microbial populations and olive volatile profiles, with higher ester abundances at the end of fermentation. Finally, reduced salt in brine gave very promising results as no deleterious effects on microbial communities, volatile dynamics but also safety criteria of the olives were observed compared to traditional fermented olives.

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.

Use of metabarcoding and source tracking to identify desirable or spoilage autochthonous microorganism sources during black olive fermentations.

This study aimed at investigating the influence of the process environment and raw materials as sources of microorganisms during Nyons black table olive fermentations. Fermented olives and/or brine from spoiled fermentation tanks were analyzed and compared to good quality samples from fermentations collected during 3 consecutive harvest years. Fresh olives, salt and different process environment samples were also analyzed. Microbial diversity of all samples was analyzed using 16S and ITS2 amplicon sequencing and SourceTracker tool was used to investigate links between environment, raw materials and fermentation samples. First, comparison of microbial diversity in control and most spoiled fermentations revealed striking differences in bacterial composition with an overall higher diversity in spoiled fermentations especially for lactic acid bacteria with Lentilactobacillus buchneri, Lentilactobacillus parafarraginis dominating in brine and Pediococcus parvulus, Pediococcus ethanolidurans dominating in olive fruits. Fungal communities were similar in composition although higher abundances of Pichia membranifaciens and Penicillium carneum/roqueforti were observed in spoiled samples. Secondly, process environment samples were characterized by high bacterial and fungal diversity, especially compared to fresh olive fruits. Overall, dominant fungal species in control fermentations were also found in most environmental samples revealing a "house mycobiota". SourceTracker analysis further highlighted the contribution of brine and water from the optical sorter as a source of fungi. Most interestingly, spoilage fungi and most bacteria were retrieved in brine and environmental samples while others such as P. ethanolidurans were only found in environmental samples indicating that the studied spoilage originated from a fermentation deviation rather than a punctual contamination. Taken altogether, these results highlighted the positive and negative influence of the process environment and emphasized the relevance of studying it to better understand microbial vectors occurring during food fermentations, especially natural ones.

Linking Pélardon artisanal goat cheese microbial communities to aroma compounds during cheese-making and ripening.

Pélardon is an artisanal French raw goat's milk cheese, produced using natural whey as a backslop. The aim of this study was to identify key microbial players involved in the acidification and aroma production of this Protected Designation of Origin cheese. Microbial diversity of samples, collected from the raw milk to 3-month cheese ripening, was determined by culture-dependent (MALDI-TOF analysis of 2877 isolates) and -independent (ITS2 and 16S metabarcoding) approaches and linked to changes in biochemical profiles (volatile compounds and acids). In parallel, potential dominant autochthonous microorganism reservoirs were also investigated by sampling the cheese-factory environment. Complex and increasing microbial diversity was observed by both approaches during ripening although major discrepancies were observed regarding Lactococcus lactis and Lacticaseibacillus paracasei fate. By correlating microbial shifts to biochemical changes, Lactococcus lactis was identified as the main acidifying bacterium, while L. mesenteroides and Geotrichum candidum were prevalent and associated with amino acids catabolism after the acidification step. The three species were dominant in the whey (backslop). In contrast, L. paracasei, Enterococcus faecalis, Penicillium commune and Scopulariopsis brevicaulis, which dominated during ripening, likely originated from the cheese-making environment. All these four species were positively correlated to major volatile compounds responsible for the goaty and earthy Pélardon cheese aroma. Overall, this work highlighted the power of MALDI-TOF and molecular techniques combined with volatilome analyses to dynamically follow and identify microbial communities during cheese-making and successively identify the key-players involved in aroma production and contributing to the typicity of Pélardon cheese.

Deciphering Microbial Community Dynamics and Biochemical Changes During Nyons Black Olive Natural Fermentations.

French PDO Nyons black table olives are produced according to a traditional slow spontaneous fermentation in brine. The manufacture and unique sensorial properties of these olives thus only rely on the autochthonous complex microbiota. This study aimed at unraveling the microbial communities and dynamics of Nyons olives during a 1.5-year-long spontaneous fermentation to determine the main microbial drivers and link microbial species to key metabolites. Fermentations were monitored at a local producer plant at regular time intervals for two harvests and two olive types (organically and conventionally grown) using culture-dependent and metabarcoding (ITS2 for fungi, V3-V4 region for bacteria) approaches. Olives and brines were also sampled for volatiles, organic acids and phenolic compounds. No major differences in microbiota composition were observed according to olive type or harvest period. Throughout the fermentation, yeasts were clearly the most dominant. ITS2 sequencing data revealed complex fungal diversity dominated by Citeromyces nyonsensis, Wickerhamomyces anomalus, Zygotorulaspora mrakii, Candida boidinii and Pichia membranifaciens species. Bacterial communities were dominated by the Celerinatantimonas genus, while lactic acid bacteria remained scarce. Clear shifts in microbial communities and biochemical profiles were observed during fermentation and, by correlating metabolites and microbiota changes, four different phases were distinguished. During the first 7 days, phase I, a fast decrease of filamentous fungal and bacterial populations was observed. Between days 21 and 120, phase II, W. anomalus and C. nyonsensis for fungi and Celerinatantimonas diazotrophica for bacteria dominated the fermentation and were linked to the pH decrease and citric acid production. Phase III, between 120 and 183 days, was characterized by an increase in acids and esters and correlated to increased abundances of Z. mrakii, P. membranifaciens and C. boidinii. During the last months of fermentation, phase IV, microbial communities were dominated by P. membranifaciens and C. boidinii. Both species were strongly correlated to an increase in fruity esters and alcohol abundances. Overall, this study provides an in-depth understanding about microbial species succession and how the microbiota shapes the final distinct olive characteristics. It also constitutes a first step to identify key drivers of this fermentation.

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.

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.

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.