Oenococcus oeni is the lactic acid bacteria species best adapted to wine. It proliferates during alcoholic fermentation and typically becomes the most abundant species during malolactic fermentation. A large diversity of O. oeni strains is present in all wine-producing regions. These strains group into several genetic lineages, which are more product-specific rather than linked to geography. I. e. strains of one genetic lineage are often detected in the same wine type produced in distant regions. This study aimed to investigate the biodiversity of O. oeni involved in malolactic fermentations of Cognac wines produced for distillation. These wines have several specific characteristics: they are low-pH white wines, low in alcohol, produced without added sulfites, and from a single grape variety (Ugni blanc). Nearly 3500 O. oeni isolates were obtained from 132 wines collected from 24 cellars across the main cognac-producing areas. 458 different strains were identified by genotyping, representing in between 1 and 249 isolates. A distribution analysis showed a large diversity of strains in each wine and cellar. Forty-nine strains were sequenced and a phylogenomic tree was reconstructed including 247 previously published genomes of strains from other regions and products. This tree revealed a new genetic lineage harboring exclusively Cognac strains whose predominance on Cognac wines was further confirmed by a specifically developed quantitative PCR test. The results highlight the existence of a new sub-lineage indicative of the domestication of O. oeni strains to a single type of wine, and across an entire wine-producing region.

The use of food preservatives is controversial these days, and sulphur dioxide (SO2) is no exception. Microbial communities have been studied particularly during the prefermentative and fermentative stages in the absence of added SO2. However, the microbial risks associated with the reduction or absence of SO2, particularly during the wine ageing process, have been little studied. The microbiological control of wine ageing is a key issue for winemakers wishing to produce high-quality wines without added SO2. The present study aims to evaluate the impact of different wine ageing strategies, depending on whether or not SO2 is added, on microbial population levels and diversity. Sulphite management had minimal impact on the population levels of yeast and acetic acid bacteria. Conversely, the population of lactic acid bacteria, whether from inoculation or spontaneous malolactic fermentation (MLF), was significantly impacted by SO2 management. High and stable population levels of around 105 CFU/mL were observed in the SO2-free modalities. Sulphiting after malolactic fermentation has a greater effect on lactic acid bacteria up to four months of ageing than standard SO2 addition at the time of vatting. Oenococcus oeni was the only species detected during ageing using a culturable approach. After five months of ageing, a high genetic diversity of Oenococcus oeni was highlighted in all modalities, even in the inoculated one with a commercial strain. The high population level of Oenococcus oeni was associated with acetic acid and D-lactic acid production, as well as mousy off-flavours. One sulfite treatment at the end of MLF inhibits the growth of Oenococcus oeni, but only temporarily. Winemakers should consider the stage and number of treatments to limit the development of lactic acid bacteria (LAB) during ageing and preserve the quality of red wine.

From the genus Oenococcus, the most studied species is O. oeni due to its role in malolactic fermentation (MLF) during the winemaking process. In 2014, a new member of the genus -alongside O. kitaharae and O. sicerae- was discovered: O. alcoholitolerans. Whereas O. oeni is found in oenological matrixes, from grapes to wine, O. alcoholitolerans is found in cachaça residues and bioethanol fermentation. Though these ecological niches are different, they share the presence of high ethanol concentrations, which could be related to their evolutionary adaptation. This study provides the first sequenced and circularized genome of O. alcoholitolerans (UFRJ-M7), which was used for a comparative genomic analysis with the species O. oeni. In addition, the physiological traits of both species were compared regarding malolactic fermentation performance and survival in conditions of increasing ethanol and acid. The results showed great genetic differences in genes related to defence mechanisms, transcriptional factors, and some aspects of metabolism, such as sugar catabolism, purine/pyrimidine, coenzyme and vitamin biosynthesis. All these variations can be attributed to the specific adaptations of O. oeni and O. alcoholitolerans to their respective ecological niches. Notably, O. alcoholitolerans has not been detected in analyzed wine samples. This observation aligns with laboratory-based physiological tests, which demonstrated that the combined stress of ethanol and low pH prevented the survival of O. alcoholitolerans, despite its ability to carry out the MLF.

La bactérie lactique Oenococcus oeni a accompagné depuis ses débuts la production du vin dont elle module le goût par diverses activités métaboliques. Les analyses génomiques récentes montrent une différenciation des souches du vin où pertes de gènes non essentiels, hypermutabilité, transfert horizontal de gènes et acquisition d’une résistance aux sulfites constituent des signes de domestication.

In the context of ecological transition, the use of wine by-products for industrial applications is a major challenge. Wine lees, the second wine by-product in terms of quantity, are a source of nutrients that can be used for stimulating the growth of microorganisms. Here, white wine lees were used as a stimulating agent for the growth of wine lactic acid bacteria (LAB) and to promote wine malolactic fermentation (MLF) in red wine.

Oenococcus oeni is the predominant lactic acid bacteria species in wine and cider, where it performs the malo-lactic fermentation (MLF). The O. oeni strains analyzed to date form four major genetic lineages named phy-logroups A, B, C and D. Most of the strains isolated from wine, cider, or kombucha belong to phylogroups A, B + C, and D, respectively, although B and C strains were also detected in wine. This study was performed to better understand the distribution of the phylogroups in wine and cider. Their population dynamics were determined by qPCR all through wine and cider productions, and the behavior of the strains was analyzed in synthetic wines and ciders. Phylogroups A, B and C were all represented in grape must and throughout the alcoholic fermentation, but on the transition to MLF, only phylogroup A remained at high levels in all wine productions. In the case of cider, phylogroups A, B and C were detected in stable levels during the process. When they were tested in synthetic wine and cider, all phylogroups performed MLF, but with different survival rates depending on the ethanol content. In this sense, ethanol and fermentation kinetics are the main agent that drives the selection of phylogroup A strains in wine, while B and C strains dominates in cider containing less ethanol.

La spectrométrie de masse de type MALDI-TOF a été adaptée afin d’être utilisée comme outil innovant d’identification au niveau de l’espèce des levures et bactéries isolées d’échantillons variés (moûts, vins, boissons). L’analyse d’un grand nombre de clones permet d’apprécier la diversité des espèces de levures, bactéries acétiques et lactiques présentes dès les phases pré-fermentaires, au cours des fermentations, pendant l’élevage ou après conditionnement. Dans le cas d’altération de produits, cet outil innovant participera à une meilleure maitrise des risques microbiologiques.

Oenococcus oeni is the most resistant lactic acid bacteria species to the environmental stresses encountered in wine, particularly the acidity, presence of ethanol and phenolic compounds. Indigenous strains develop spontaneously following the yeast-driven alcoholic fermentation and may perform the malolactic fermentation whereby improving taste, aroma, and the microbial stability of wine. However, spontaneous fermentation is sometimes delayed, prolonged or incomplete. In order to better control its timing and quality, O. oeni strains are selected and developed to be used as malolactic starters. They are prepared under proprietary manufacturing processes to survive direct inoculation and are predominantly provided as freeze-dried preparations. In this study, we have investigated the physiological and molecular alterations occurring in O. oeni cells prepared by an industrial process that consists of preconditioning protocols and freeze-drying, and compared them to the same strain grown in a grape juice medium. We found that compared to cultured cells, the industrial production process improved survival under extreme conditions, i. e. at low pH or high tannin concentrations. In contrast, cultured cells resumed active growth more quickly and strongly than freeze-dried preparations in standard pH wines. A proteomic analysis showed that during the industrial production most non-essential metabolic processes are shut down and components of the general and the stringent stress response are upregulated. The presence of major components of the stress response facilitates protein homeostasis and physiological changes that further ensure the integrity of cells.

There has been little exploration of how phages contribute to the diversity of the bacterial community associated with winemaking and may impact fermentations and product quality. Prophages of Oenococcus oeni , the most common species of lactic acid bacteria (LAB) associated with malolactic fermentation of wine, have been described, but no data is available regarding phages of O. oeni with true virulent lifestyles. The current study reports on the incidence and characterization of the first group of virulent oenophages named Vinitor, isolated from the enological environment. Vinitor phages are morphologically very similar to siphoviruses infecting other LAB. Although widespread during winemaking, they are more abundant in musts than temperate oenophages. We obtained the complete genomic sequences of phages Vinitor162 and Vinitor27, isolated from white and red wines, respectively. The assembled genomes shared 97.6% nucleotide identity and belong to the same species. Coupled with phylogenetic analysis, our study revealed that the genomes of Vinitor phages are architecturally mosaics and represent unique combinations of modules amongst LAB infecting-phages. Our data also provide some clues to possible evolutionary connections between Vinitor and (pro)phages associated to epiphytic and insect-related bacteria.

Oenococcus oeni is the most exploited lactic acid bacterium in the wine industry and drives the malolactic fermentation of wines. Although prophage-like sequences have been identified in the species, many are not characterized, and a global view of their integration and distribution amongst strains is currently lacking. In this work, we analyzed the complete genomes of 231 strains for the occurrence of prophages, and analyzed their size and positions of insertion. Our data show the limited variation in the number of prophages in O. oeni genomes, and that six sites of insertion within the bacterial genome are being used for site-specific recombination. Prophage diversity patterns varied significantly for different host lineages, and environmental niches. Overall, the findings highlight the pervasive presence of prophages in the O. oeni species, their role as a major source of within-species bacterial diversity and drivers of horizontal gene transfer. Our data also have implications for enhanced understanding of the prophage recombination events which occurred during evolution of O. oeni, as well as the potential of prophages in influencing the fitness of these bacteria in their distinct niches.

Winemakers are increasingly keen to limit the use of commercial yeasts in order to reduce oenological inputs. The preparation of an indigenous winery-made fermentation starter from grapes called ‘pied de cuve’ (PdC) is becoming popular, especially in organic farming systems. However, the implementation of the PdC method is still empirical and knowledge is lacking regarding the impact of PdC on S. cerevisiae diversity during alcoholic fermentation. In this study, the impact of PdC on S. cerevisiae genetic diversity and wine composition was evaluated at an industrial scale. Despite very low initial population level of S. cerevisiae before inoculation, the use of PdC was as efficient as Active Dry Yeast in terms of fermentation kinetics and chemical analyses on the resulting wines, except for one modality. At mid-fermentation, the diversity of S. cerevisiae strains was different depending on the PdC used, and was also different from that in the spontaneous fermentation with, in some cases, clonal expansion. Our results provide evidence that the use of PdC could secure the fermentation process more efficiently than spontaneous fermentation.

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.

Table S4 Public genome accession numbers (XLSX 12 kb)

The variation in descriptors for mousy off-flavor may be related to the different compounds involved in this spoilage, their concentrations, the matrix effect, individual detection abilities, the composition of a subject's saliva, and the pH of the tongue's surface. These different sources of variability partly explain the lack of consensus concerning the perception of this defect in wine. Several different sensory methods have been developed by wine professionals and scientists, based on the pH-dependency affecting the perception of some key mousy compounds. The objective of this study was to compare different sensory methods for detecting mousy character in red wine under standardized conditions, using alkaline paper strips and pH adjustment. Among the methods tested, adjusting pH to around 5 increased the consensus among tasters, and the detection and discrimination capacities of panelists.

Oenococcus oeni is the lactic acid bacteria species most commonly encountered in wine, where it develops after the alcoholic fermentation and achieves the malolactic fermentation that is needed to improve the quality of most wines. O. oeni is abundant in the oenological environment as well as in apple cider and kombucha, whereas it is a minor species in the natural environment. Numerous studies have shown that there is a great diversity of strains in each wine region and in each product or type of wine. Recently, genomic studies have shed new light on the species diversity, population structure, and environmental distribution. They revealed that O. oeni has unique genomic features that have contributed to its fast evolution and adaptation to the enological environment. They have also unveiled the phylogenetic diversity and genomic properties of strains that develop in different regions or different products. This review explores the distribution of O. oeni and the diversity of strains in natural habitats.

Oenococcus oeni is the lactic acid bacterium (LAB) that most commonly drives malolactic fermentation in wine. Although oenococcal prophages are highly prevalent, their implications on bacterial fitness have remained unexplored and more research is required in this field. An important step toward achieving this goal is the ability to produce isogenic pairs of strains that differ only by the lysogenic presence of a given prophage, allowing further comparisons of different phenotypic traits. A novel protocol for the rapid isolation of lysogens is presented. Bacteria were first picked from the center of turbid plaques produced by temperate oenophages on a sensitive nonlysogenic host. When streaked onto an agar medium containing red grape juice (RGJ), cells segregated into white and red colonies. PCR amplifications with phage-specific primers demonstrated that only lysogens underwent white-red morphotypic switching. The method proved successful for various oenophages irrespective of their genomic content and attachment site used for site-specific recombination in the bacterial chromosome. The color switch was also observed when a sensitive nonlysogenic strain was infected with an exogenously provided lytic phage, suggesting that intracolonial lysis triggers the change. Last, lysogens also produced red colonies on white grape juice agar supplemented with polyphenolic compounds. We posit that spontaneous prophage excision produces cell lysis events in lysogenic colonies growing on RGJ agar, which, in turn, foster interactions between lysed materials and polyphenolic compounds to yield colonies easily distinguishable by their red color. Furthermore, the technique was used successfully with other species of LAB.IMPORTANCE The presence of white and red colonies on red grape juice (RGJ) agar during enumeration of Oenococcus oeni in wine samples is frequently observed by stakeholders in the wine industry. Our study brings an explanation for this intriguing phenomenon and establishes a link between the white-red color switch and the lysogenic state of O. oeni It also provides a simple and inexpensive method to distinguish between lysogenic and nonlysogenic derivatives in O. oeni with a minimum of expended time and effort. Noteworthy, the protocol could be adapted to two other species of LAB, namely, Leuconostoc citreum and Lactobacillus plantarum It could be an effective tool to provide genetic, ecological, and functional insights into lysogeny and aid in improving biotechnological processes involving members of the lactic acid bacterium (LAB) family.

Avec le developpement des vins et cidres bio, on observe une vraie tendance a la realisation de fermentations spontanees, en laissant se developper les levures et bacteries indigenes. En effet, ces microorganismes sont parfois consideres comme des elements du terroir qui participent a la typicite des vins et des cidres. Pourtant, aucune connaissance scientifique ne permet d’affirmer une telle specificite, alors que la non-maitrise de ces microorganismes peut conduire a des difficultes de fermentation, des deviations aromatiques ou des alterations. Le projet Casdar Levains Bio s’est appuye sur un reseau de laboratoires, instituts techniques et associations de producteurs bio pour apporter les connaissances necessaires et des solutions pratiques pour realiser des fermentations indigenes avec un bon niveau de maitrise. Il a ete montre qu’il existe une grande diversite de souches de la levure Saccharomyces cerevisiae et de la bacterie lactique Oenococcus oeni, que des souches sont genetiquement adaptees a certains produits, mais pas a des regions ou a des sites de production. Des protocoles ont ete mis au point pour permettre de selectionner des souches issues des exploitations ou pour realiser des pieds de cuve de microorganismes indigenes. Certaines des solutions ont ete transferees avec succes aupres des producteurs.

The use of autochthonous strains of Oenococcus oeni for inducing malolactic fermentation (MLF) in wines is increasing because they are well adapted to the conditions of a specific area. The main aim of this work was to select O. oeni strains from Priorat (Catalonia, Spain) wines that would be able to carry out the MLF while maintaining the characteristics of the wine. Forty-five autochthonous strains were selected based on their degradation of L-malic acid, resistance to low pH and high ethanol, and the absence of biogenic amine genes. The three strains with the best characteristics were inoculated and MLF was carried out successfully with the final wines showing good characteristics. One of the inoculated O. oeni strains would be a good candidate to study as possible starter culture. It shows that autochthonous O. oeni strains, once selected, have the potential to be used as an inoculum for wines.