# Cognac distillation wines host specific Oenococcus oeni strains representing a novel genetic lineage

Sayoko Matsumoto, Jana Rudolf, Cécile Miot-Sertier, Olivier Claisse,
Panagiotis Stamatopoulos, Xavier Poitou, Patrick Lucas

► To cite this version:

Sayoko Matsumoto, Jana Rudolf, Cécile Miot-Sertier, Olivier Claisse, Panagiotis Stamatopoulos, et al. Cognac distillation wines host specific Oenococcus oeni strains representing a novel genetic lineage. International Journal of Food Microbiology, 2026, 446, pp.111539. 10.1016/j.ijfoodmicro.2025.111539. hal-05448339

HAL Id: hal-05448339

https://hal.inrae.fr/hal-05448339v1

Submitted on 8 Jan 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
<figure><img src="image_2.png" /></figure>

# Cognac distillation wines host specific *Oenococcus oeni* strains representing a novel genetic lineage

Sayoko Matsumoto<sup>a</sup>, Jana Rudolf<sup>a,*</sup>, Cécile Miot-Sertier<sup>a</sup>, Olivier Claisse<sup>a</sup>, Panagiotis Stamatopoulos<sup>b</sup>, Xavier Poitou<sup>b</sup>, Patrick Lucas<sup>a</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

**Keywords:**

Lactic acid bacteria

Malolactic fermentation

Domestication

Genomics

Population structure

Phylogenomics

## ABSTRACT

*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.

## 1. Introduction

*Oenococcus oeni* is the principal lactic acid bacteria (LAB) species associated with the production of alcoholic beverages such as wine, apple cider and kombucha (Lorentzen and Lucas, 2019). It has a clonal population structure that fans out into currently four main genetic lineages (denominated A-D) with several hundred sequenced strains (Campbell-Sills et al., 2015; Lorentzen et al., 2019).

The majority of strains were isolated from malolactic fermentations (MLF) of wines and group into lineage A. These strains are tolerant to wine-typical characteristics such as low pH (~3.5) and high alcohol (~12%) content and are particularly prevalent after the alcoholic fermentation (AF) (Balmaseda et al., 2023). Commercially available malolactic starter strains all belong to lineage A (Campbell-Sills et al., 2015). They were selected to better control MLFs, i. e. to prevent stuck

fermentations and the development wine faults (Lonvaud-Funel, 1999; Torriani et al., 2011). Lineage B and C strains are well adapted to low pH, but are more sensitive to high alcohol concentrations, and generally do not survive the AF. They are hence abundant in grape must, but also in other low-alcohol fruit-based fermented products such as apple cider (Bilhère et al., 2009; Bridier et al., 2010). Lineage D is quite distinct from the other three genetic lineages and harbors a few strains isolated from fermented kombucha tea (Coton et al., 2017; Lorentzen et al., 2019) which is of low pH (~3.0) and contains very little alcohol (~1%). This adaptation of microorganisms to certain manufactured niche environments – also called domestication – is a well-known and wide-spread phenomenon (Campbell-Sills et al., 2015; Steensels et al., 2019).

Besides pH and ethanol, other particular physico-chemical constraints, such as the presence of phenolic compounds or sulfites, can result in the formation of sub-lineages (Campbell-Sills et al., 2017).

Abbreviations: AF, alcoholic fermentation; GJM, grape juice medium; LAB, lactic acid bacteria; MICFAM, MicroScope homologous gene family; MLF, malolactic fermentation; MLVA, multiple-locus variable number tandem repeat analysis; SNP, single nucleotide polymorphism; TR, tandem repeat.

* Corresponding author.

E-mail address: jana.rudolf@u-bordeaux.fr (J. Rudolf).

<footnote><sup>a</sup> Univ. Bordeaux, Bordeaux INP, Bordeaux Sciences Agro, INRAE, UMR 1366, OENO, ISVV, F-33140, Villenave d'Ornon, France</footnote>

<footnote><sup>b</sup> Hennessy, Rue de la Richonne-CS20020, 16101, Cognac, France</footnote>
Exemplary are two groups of A-strains isolated from either red (AR) or white wine (AW). Sub-lineage AR-strains are highly tolerant towards tannins but are sensitive to pH < 3.3 (Breniaux et al., 2018). Sub-lineage AW-strains, however, are sensitive to tannins, but are well adapted to high acidity. They also encode a particular glucan synthesis gene that promotes the production of a capsular dextran (Dimopoulou et al., 2014; Campbell-Sills et al., 2017). This gene is absent in all other A-strains and could explain the resistance to low-pH conditions (Dols-Lafargue et al., 2008).

This study was initiated with the aim of analyzing the biodiversity of LAB in wines produced for distillation in the Cognac region (referred to as: Cognac distillation wines). In the production of base wines for fruit-based spirits such as cognac or brandy, sulfur dioxide is not used for controlling microbial growth or oxidation due to an undesirable impact on the aroma composition of the distillate. LAB that survived the AF can hence develop freely and trigger spontaneous MLF during the storage period until distillation (Du Plessis et al., 2002). Metabolic activities of these bacteria can positively or negatively affect the quality of both base wines and distillates (Tsakiris et al., 2014). For example, L-lactic acid that is formed during MLF correlates with an increase in ethyl lactate, which may be detrimental to the product after distillation. Less commonly, MLF may be desired to reduce the ethanal content of the wines, and exceptionally it can be induced by the use of commercial O. oeni strains. The biodiversity of LAB in Cognac distillation wines remains poorly characterized, however, it is almost certain that O. oeni will be the dominant species as observed in the case of base wines

produced for South African brandy (Du Plessis et al., 2004). Apart from the already mentioned technical peculiarities (no added sulfites, a long storage period), cognac base wines have several other specific characteristics including the production i) in a single region, ii) almost exclusively from a unique grape variety (Ugni blanc), iii) from wines that have a very low pH (2.7-3.3) as well as a low ethanol content (<12 %).

The aim of this study was to investigate the biodiversity of LAB, and in particular *O. oeni*, in Cognac distillation wines following alcoholic fermentation. For this, we used well established methods based on MLVA-genotyping and profiling, followed by sequencing of a selection of strains and phylogenomic analyses (El Khoury et al., 2017). The results revealed an unexpected large diversity of *O. oeni* strains and the presence of a new cognac-specific sub-lineage, tallying well with the concept of domestication of microorganisms to certain practices and environmental conditions.

## 2. Materials and methods

### 2.1. Sampling and chemical analysis of wines from the Cognac region

Samples originated from 24 wineries located in the Cognac region (Fig. 1). The wineries were chosen according to their geographic location, in order to include multiple sites distributed throughout the main appellations of Cognac, namely Grande Champagne, Petite Champagne, Fins-Bois and Borderies.

All samples were processed as follows within 24-48 h after sampling:

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** The Cognac production area and the location of wineries. The production area of Cognac is represented with the six geographical appellation zones in different colors. The inset shows their location on a map of France relative to the cities of Paris and Bordeaux. The 24 wineries partaking in this study are indicated on the map and numbered as in Table S1.</figcaption></figure>
i) using a WineScan™ Auto (FOSS Analytical, Hilleroed, Denmark) interferometer the concentrations of L-malic acid and L-lactic acid were determined as well as the ethanol content and the pH, ii) bacterial colonies were counted and LAB isolated (Section 2.2), and iii) total microbial DNA was extracted for qPCR analysis (Section 2.4). The wine samples were initially collected with the aim to isolate LAB involved in MLF. However, chemical and microbiological analyses showed that not all wines were undergoing MLF at the time of sampling. Therefore, samples were grouped into four categories according to the MLF stage:

- Stage 0 (far before MLF): LAB populations >$10^5$/mL, L-malic acid >0.2 g/L, L-lactic acid <0.2 g/L

- Stage 1 (just before MLF): LAB populations >$10^5$/mL, L-malic acid >0.2 g/L, L-lactic acid <0.2 g/L

- Stage 2 (during MLF): LAB populations >$10^5$/mL, L-malic acid >0.2 g/L, L-lactic acid >0.2 g/L

- Stage 3 (after MLF): LAB populations >$10^5$/mL, L-malic acid <0.2 g/L, L-lactic acid >0.2 g/L.

Table S1 describes all samples, including their origin, MLF stage and main chemical and microbiological parameters.

## 2.2. Isolation of bacteria, culture conditions and storage

Wine samples were diluted in sterile normal saline solution. 100 µL of each dilution were plated onto a solid grape juice medium (solid GJM) containing 250 mL/L commercial red grape juice (Jaffaden, E.Leclerc®), 5 g/L yeast extract, 1 mL/L Tween®80 (Acros Organics/Thermo Scientific), 25 g/L agar and 100 mg/L pimaricin (Natacin, Caglificio Clerici S.p.A., Cadorago, Italy), adjusted to pH 4.8 (KOH) (El Khoury et al., 2017). Plates were incubated for seven days at 25 °C under anaerobic conditions using Oxoid AnaeroGen 2.5 L sachets (Thermo Fischer). Bacterial colonies were resuspended in 100 µL sterile MilliQ water. Ten µL of each suspension were inoculated into 90 µL of liquid GJM and incubated at 25 °C. After seven days, frozen stocks were prepared by addition of sterile glycerol to a final concentration of 20 % (v/v), and stored at -80 °C. The remaining 90 µL of colony suspension were frozen at -20 °C for subsequent molecular analyses.

All strains that were sequenced as part of this study were deposited in the collection of the Biological Resources Center OENO (CRBO, ISVV, Bordeaux University).

## 2.3. Genotyping of LAB isolates

From each sample, circa 30 bacterial colonies were directly genotyped by Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA, Claisse and Lonvaud-Funel, 2012) as we expected most of them to represent the species *O. oeni*. This number has been shown to be sufficient in order to detect the majority of *O. oeni* genotypes in wine (El Khoury et al., 2017, Romero et al., 2018). Per isolate, two multiplex PCR reactions (10 µL) were carried out with five different primer pairs (TR1-TR5) specific to *O. oeni* as described by Claisse and Lonvaud-Funel (2014) using 1 µL colony suspension (Section 2.2). After amplification, the two PCR reactions were diluted as described, pooled, and 2 µL of the mixture were supplemented with 9 µL of deionized formamide (Formamide Hi-Di™, Applied Biosystems). Fragment sizes were determined by using the Fragment Length Analysis service at Eurofins Genomics (Ebersberg, Germany). To obtain a digital MLVA-Profile for each isolate, the number of tandem repeats (TR) for each of the five TR regions was obtained from the respective chromatogram by using the GeneMarker software (Version 2.2, SoftGenetics, PA, USA). The new MLVA profiles (Table S2) were processed using BioNumerics Version 8.0 (Applied Maths NV, Sint-Martens-Latem, Belgium) and compared to our in-house database (El Khoury et al., 2017; Franquès et al., 2018; Lorentzen et al., 2019).

## 2.4. Extraction of genomic DNA

For genome sequencing, bacterial cells from 2 mL liquid cultures at stationary phase were collected by centrifugation for 10 min at 10000 xg. Genomic DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) following the manufacturer's protocol with the following modifications: i) bacterial cell pellets underwent a 3 h treatment with 600 µL of 10 mg/mL lysozyme in 50 mM EDTA, pH 8.0, at 37 °C previous to the cell lysis and ii) the resulting DNA pellets were rehydrated in 100 µL of 10 mM Tris, pH 8.0. The purity and the quantity of genomic DNA were determined using a BioSpec-nano photospectrometer (Shimadzu Biotech, Tokyo, Japan).

Cell pellets for qPCR assays were prepared directly from wine samples (biomass). For standard curve preparations, 10 mL bacterial cultures were used at the end of the exponential growth phase. Wine or cultures were centrifuged at 10000 xg for 15 min. Cell pellets were resuspended in 50 mM EDTA, pH 8.0, and divided in two equal parts. They were washed twice with 1 mL of 50 mM EDTA and centrifuged at 10000 xg for 5 min. The final cell pellets were kept at -20 °C until DNA extraction. All following work was carried out on ice or 4 °C: thawed cell pellets were resuspended in 300 µL 50 mM EDTA, pH 8.0, and added to 200 µL 0.1 mm diameter sterile Disruptor Beads (Scientific Industries, Inc., Bohemia, NY, USA). Cells were disrupted by using a FastPrep®-24 bead beating grinder and lysis system (MP Biomedicals, Santa Ana, CA, USA) for five cycles, with intermittent one minute cooling on ice, at 6.5 m/s for 45 s. Then, 300 µL of nuclei lysis solution and 200 µL of protein precipitation solution (Wizard Genomic DNA Purification Kit, Promega) were added to the cell lysate and kept on ice for five minutes. After centrifugation at 14000 xg for 5 min, 550 µL of supernatant were mixed with 55 µL of 10 % polyvinylpyrrolidone (PVP, Polyclar AT™, Himedia, Thane, India) in MilliQ water to bind polyphenolic compounds. They were precipitated by centrifugation at 14000 xg for 5 min. The supernatant was mixed with 500 µL isopropanol. After centrifugation at 14000 xg for 15 min, the supernatant was discarded, and the DNA pellet was washed with 300 µL of ice-cold 70 % ethanol. After a last centrifugation at 14000 xg for 5 min, the DNA pellet was air-dried for 15 min and rehydrated with 50 µL sterile water supplemented with 1.5 µL RNase A solution.

## 2.5. Genome sequencing, assembly, annotation and analyses

Genome sequencing was performed at the Institute of Microbiology and Genetics at the University of Göttingen (Germany). Briefly, Illumina shotgun paired-end (2 × 300 bp) libraries were generated from purified genomic DNA using the Nextera XT sample preparation kit as recommended by the manufacturer (Illumina, San Diego, CA, USA). The MiSeq system was used for sequencing as recommended by the manufacturer (Illumina). The quality control of raw sequence reads was carried out with FastQC v0.11.9 (Andrews, 2010) and the reads were quality-filtered using Trimmomatic v0.39 (Bolger et al., 2014). Reads were then de novo assembled using shovill 1.1.0 (Seemann, 2016) and the SKESA 2.4.0 assembler (Souvorov et al., 2018). Shovill was also used to estimate the sequencing depth, trimmed reads, assembling reads, contigs out, genome size, N50 and L50. Genome annotation was performed using the automatic annotation pipeline of the MicroScope platform (Vallenet et al., 2020). The genome browser and other tools implemented in MicroScope were used for comparative genomics analyses. For phylogenetic analyses, an ANIm (Average Nucleotide Identity using the MUMmer algorithm) pairwise distance matrix of newly produced and publicly available *O. oeni* genomes was calculated using Pyani 0.2.9 (Pritchard et al., 2015). Phylogenetic trees were reconstructed by the Neighbor Joining method using MEGA X (Kumar et al., 2018). Assembled genomes were deposited in GenBank under the accession numbers indicated in Table S3.
## 2.6. qPCR assay development to detect O. oeni AC-strains

In addition to the already described qPCR assays to identify lineage A-strains (Balmaseda et al., 2023), a new qPCR assay was developed for the detection of sub-lineage AC (lineage A-Cognac, this study) O. oeni strains by using the following primers: AC_Forward (5'-TTCAATTC-CATTAGGAGAACC-3') and AC_Reverse (5'-GACAGCTGTATCATGGCG-3'), and Taqman DNA probes named AC_Probe_FAM (5'-CATA-TATTTAGTTGGGCAAATGTGG-3'/5'-FAM, 3'-BHQ1) and Non-AC_Probe_TexasRed (5'-CATATATTTAGTTGCGGCAAATGTGG-3'/5'-TexasRed, 3'-BHQ2). The probes were designed to target a single nucleotide polymorphism (SNP) in a manganese transporter gene (PSU-1 OEOE_0827/OEOE_RS03975) that is specific to sub-lineage AC-strains (Fig. S1). PCR reactions (20 µL) consisted of 0.05 µM of each primer and probe, 10 µL 2× real-time PCR mix (iQ Supermix, Bio-Rad, CA, USA) and 1 µL of a 10× diluted DNA template extracted from a microbial culture or biomass. Samples were analyzed on a Bio-Rad CFX96 Real-Time PCR detection system in triplicate. After initial denaturation at 95 °C for 3 min, 45 cycles of a two-step PCR protocol were used consisting of 15 s denaturation at 95 °C and 1 min annealing/extension at 60 °C for the AC probe and 57 °C for the non-AC probe. The quantification of lineage A-strains was performed using the qPCR primers and probe described in Balmaseda et al. (2023) and the same protocol as for sub-lineage AC-strains.

Two qPCR standard curves were constructed to determine the populations of lineages A and AC-strains. For this, known quantities of template DNA isolated from the AC-strain O. oeni CRBO 2162 were used. First, the strain was grown in GJM until late exponential phase. A 10× dilution series was prepared. 10 µL of dilutions 10³-10⁶ were plated onto solid GJM in triplicate to determine cell numbers. In parallel, genomic DNA was extracted from 5 mL culture of each dilution (Section 2.4). To limit the loss of bacterial DNA during extraction, a carrier DNA originating from 10⁸ yeast cells was added. This carrier DNA was isolated from a culture of Saccharomyces cerevisiae Lalvin FC9 (Lallemand) grown overnight at 25 °C in YPG medium (10 g/L yeast extract, 10 g/L peptone and 20 g/L D-glucose, adjusted to pH 4.8). The qPCR reactions were performed as described above using primers and probes specific for the A- or AC-strains.

# 3. Results

## 3.1. Isolation and genotyping of O. oeni isolates performing MLF in Cognac distillation wines

The biodiversity of O. oeni in Cognac wines undergoing MLF was investigated by sampling 132 wine vats in 24 wineries located in the main production areas of the Cognac appellation in 2019 (Fig. 1). Most of these wineries are at least 10 km apart from each other, which should limit cross contamination of their indigenous microbiota. All wines were collected after the end of the AF. However, chemical and microbiological analyses showed that they were at different stages of MLF at the time of sampling (Table S1). Therefore, they were classified into four groups according to their state of MLF completion (Section 2.1 and Table S1): 23 samples displayed insufficiently low LAB populations and had not yet started the MLF (<10⁵ LAB cells/mL, "stage 0" in Table S1). LAB isolated from these samples were analyzed separately as it was not clear whether the corresponding bacteria were capable of performing the MLF or not. 109 samples contained sufficiently high LAB populations. Depending on the concentrations of malic and lactic acid, 15 samples were classed "stage 1" (just before MLF), 30 "stage 2" (during MLF) and 64 "stage 3" (shortly after MLF). Even though bacterial population can change following the MLF, stage 3 isolates were considered to have taken part in the MLFs as samples were collected just after completion.

No bacterial colonies were obtained from 11 of the 23 stage 0-samples. From all other 121 samples approximately 30 colonies were analyzed directly by genotyping. This was done to avoid a preliminary

species-specific PCR testing, given that O. oeni is the most abundant LAB species during MLF. Genotyping was performed by amplifying five O. oeni-specific genomic regions containing tandem repeats. PCR products were obtained for all bacterial isolates, indicating that O. oeni was indeed the predominant species in all 121 wine samples. In total, 3442 bacterial isolates were genotyped, i.e. 230, 443, 890 and 1879 from MLF stages 0 to 3, respectively. 458 different combinations of the five TR regions (MLVA profiles) were identified, suggesting the presence of at least 458 different O. oeni strains. The list of obtained MLVA profiles of all the isolates is shown in Table S2.

## 3.2. Diversity analysis of Cognac strains

From the MLF stages 1 to 3, 3212 isolates were genotyped and 431 strains identified, indicating that each strain was isolated on average about 7.5 times. However, this frequency varied greatly from one strain to another: 135 strains were detected only once, while the most abundant one was represented by 249 isolates (Fig. 2A).

Comparison of the obtained MLVA profiles with those reported in previous studies (Campbell-Sills et al., 2017; El Khoury et al., 2017) showed that eight were already attributed to commercial MLF starter strains (Fig. 2A and Table S2). They represent 326 bacterial isolates in total that originated from 12 wineries, among them was the most frequently detected strain (249 isolates). Almost all isolates of this strain (237 isolates) came from 10 wines of one winery. A second commercial strain was detected 61 times in two wines of another winery. The six other commercial strains were isolated only once (three strains) and 3, 4 and 6 times, respectively (Table S2).

The remaining 423 strains did not exhibit a commercial strain profile. Detection frequencies varied greatly the vast majority (354 strains, 83 %) were detected less than ten times, constituting 26 % of the isolates. Two strains were detected over 100 times (9 % of the 2887 isolates), nine between 50 and 100 times (24 % of isolates), and 58 between ten and 50 times (41 % of the isolates) (Fig. 2A).

In each of the wines collected at stages 1 to 3, the number of strains detected ranged from 1 to 21, with a majority of wines containing between 7 and 12 different strains (Fig. 2B). Therefore, it appears that MLFs in Cognac wines were rarely driven by a single dominant strain, but rather by a consortium of seven strains and more. Furthermore, it is likely that the number of strains was underestimated, as it was based on the analysis of only 32 random isolates per wine.

The stage of MLF appears to influence the number of detected strains (Fig. 2C). In the beginning (stage 1) and in the course (stage 2) of MLF an average of nine strains were detected. However, in reality their number was often lower with three to nine strains in half of the samples. In contrast, once MLF was completed (stage 3) more than nine strains were consistently detected in almost all wines.

The number of strains in each winery varied from 1 to 62 (Fig. 2D). This high variability correlated with the number of samples analyzed per winery (1 to 14). According to our observations, if at least six wines were sampled from one winery, more than 50 to 60 different strains could be easily isolated. Despite these variations, in all wineries at least one predominant strain was identified that represented more than 10 % of all isolates. Generally, less than four strains accounted for more than 50 % of the isolates per winery (Fig. 2D).

To determine if Cognac strains are present in other regions, the MLVA profiles of the 423 non-commercial O. oeni strains isolated from stages 1-3 were compared to those of 925 profiles previously reported (Campbell-Sills et al., 2017; El Khoury et al., 2017). The vast majority (328 strains, 77 %) had no counterparts in Nouvelle-Aquitaine, in other regions of France, or in wine-producing areas in other parts of the world (Fig. 2E). This suggests that most of the O. oeni strains detected in this study were specific to the region of Cognac.
<figure><img src="image_5.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_9.png" /></figure>

**Fig. 2.** Diversity of *O. oeni* strains in Cognac wines, wineries and regions. A. Number of isolates for each of the 431 *O. oeni* strains. Strains sharing the same genotype as commercial strains are indicated in blue and with an asterisk. B. Number of strains detected per wine collected at MLF stages 1–3 (excluding commercial strains and wines from which less than 25 isolates were genotyped for statistical analysis). C. Number of strains detected per wine at MLF stages 0 to 3 (6, 13, 28 and 56 wines of MLF stage 0, 1, 2 and 3 respectively). The box plots depict the number of strains per wine (excluding commercial strains and wines from which less than 25 isolates were genotyped at each stage). The median number of strains is indicated by a black line. D. Number and relative abundance of strains in 24 wineries at stages 1 to 3. Strains with the same genotype as commercial strains are shown in blue. E. Venn-diagram representing the number of Cognac strains sharing the same genotype as strains isolated from other regions (Nouvelle-Aquitaine, France, World). Only the 423 non-commercial strains isolated from wines at stages 1 to 3 were considered.

## 3.3. Genomic analysis of selected Cognac strains

### 3.3.1. Genome sequencing, assembly and annotation

The genetic diversity of Cognac strains involved in MLFs was further investigated by selecting and sequencing 49 strains. The selection criteria were as follows: i) isolates must have originated from wines at MLF stages 1 to 3, ii) all wineries from which strains were isolated had to be represented, and iii) the most abundant strains were chosen from each winery. The 49 selected strains accounted for 56 % of the isolates genotyped at MLF stages 1 to 3 (1797 out of 3212 isolates), and for over 40 % of the total number of isolates from 20 wineries (Fig. S2A). Most of these strains were detected in several wineries: 22 strains in 3 to 8 wineries, 10 strains in 2 wineries, and the remaining 17 in only one winery each (Fig. S2B).

Short-read sequencing and assembly produced draft genomes comprising 25 to 86 contigs (Table S3) that ranged from 1.74 to 1.92 Mb in size with an average GC content of 37.9 %. This is consistent with previously published data for the *O. oeni* species (Lorentzen and Lucas, 2019). The automatic annotation pipeline of MicroScope (Vallenet et al., 2020) detected an average of 1959 coding sequences (CDS) per genome, with extreme values of 1875 and 2163 CDS, suggesting a large genetic diversity among strains considering genome size (Table S3). The number of pseudogenes was quite small, ranging from 18 to 43.

### 3.3.2. Phylogenetic distribution

The phylogenetic distribution of the Cognac strains was examined by reconstructing phylogenomic trees using the 49 newly sequenced genomes in addition to 247 publicly available *O. oeni* genomes that were previously assigned to lineages A-D (Lorentzen et al., 2019). Fig. 3A shows an ANI-based neighbor joining phylogenetic tree. As expected for strains isolated from wine MLFs, the 49 Cognac strains were all located within lineage A. Interestingly, 34 of these strains clustered together in what appears to be a new sub-lineage exclusively composed of Cognac strains. The 15 other sequenced strains were dispersed throughout lineage A-strains. Six of them (CRBO 2180-2185) group together indicating very close relationship and suggesting a common ancestral strain. Two other strains are found next to each other on a long branch at the root of lineage A (CRBO 2168 and 2169). The phylogenetic distribution
<figure><img src="image_10.png" /><figcaption>**Fig. 3.** Phylogenetic clustering of Cognac strains. A. Neighbor Joining tree based on a distance matrix of 296 *O. oeni* genomes (49 from Cognac strains and 247 publicly available) calculated with ANIm. Strains from the four genetic lineages A, B, C, and D are depicted using different colors. The darker lines on the outside circle indicate the positions of the 49 Cognac strains. Sub-lineages AC, AC2 and AW are also highlighted. B. Unrooted tree produced using the iTOL web service (Letunic and Bork, 2021) showing the evolutionary distances between all accessible sequenced strains.</figcaption></figure>

of strains was also examined by producing an unrooted tree using the iTOL web server (Letunic and Bork, 2021) in order to visualize the actual evolutionary distances between strains (Fig. 3B). The tree confirmed the existence of a sub-lineage containing only the 34 mentioned Cognac strains. It was named "sub-lineage AC" (for lineage A, sub-lineage Cognac). The tree also showed that, although they clustered together,

AC-strains are genetically quite diverse. Indeed, the evolutionary dis-
tances within their sub-lineage are much greater than for other sub-
lineages such as AW (lineage A, White; Campbell-Sills et al., 2017).
Given their unusual position on the tree, the two Cognac strains that are
located relatively isolated on a long branch at the root of lineage A were
also assigned to a new sub-lineage, named AC2 (Fig. 3). However, more
strains are required to definitively confirm this second new sub-lineage.

### 3.3.3. Core and pan-genome analyses

The genetic characteristics of strains belonging to the new AC sub-lineage were explored by analyzing their core and pan-genomes, which were subsequently compared to those of strains from lineage A and sub-lineage AW. The latter were included in the comparison as both AW and Cognac strains develop better in acidic white wines (Breniaux et al., 2018). Core and pan-genomes were determined using the Pan/Core-Genome interface of MicroScope (Vallenet et al., 2020) (Fig. 4A, B).

As anticipated for strains belonging to the same sub-lineage, the core-genome of the 34 AC-strains (1519 MicroScope homologous gene families, MICFAMs) was significantly larger than that of the 171 A-strains (1200 MICFAMs), or, for a better comparison, the first 34 A-strains of the analysis (1420 MICFAMs) (Fig. 4A). However, it was considerably smaller than the core-genome of the 13 AW-strains (1816 MICFAMs). At the 13-strains level, the AC sub-lineage shared about 1580 MICFAMs, which still falls short of what is observed for AW-strains. This suggests that, although they formed a specific sub-lineage, AC-strains are more divergent than strains of the sub-lineage AW. This was confirmed by pan-genome analyses (Fig. 4B) which showed a far greater pan-genome for the 34 AC-strains (3190 MICFAMs) than compared to the AW-strains (2389 MICFAMs), even at the 13-genome stage (Fig. 4B). The curves of the pan-genomic evolution for both AC- and AW-strains seemed to reach a plateau, indicating that the current strains represent almost 100 % of the genetic diversity of their respected sub-lineage. By comparison, even at the 34-genome stage, the pan-genome of the whole lineage A excluding AC and AW was far larger (6561 MICFAMs), and the curve of the pan-genomic evolution was not yet leveling off.

Comparing the 1519 (MICFAMs) core-genes of AC-strains to the 6561 A and 2389 AW pan-genes, respectively, revealed that no gene was specific to AC-strains; all of them were present in at least one A or AW-strain (not shown). However, a comparison of core-genomes indicated that AC-strains shared 38 MICFAMs that were not present in the core-genomes of A- or AW-strains (Fig. 4C, Table S4). Many of the gene products might have functions linked to the modulation of cell wall properties (D-alanine:D-alanyl-carrier protein ligase, cell wall synthesis protein, methyl-accepting chemotaxis cell wall protein), transport across membranes (e.g. ammonium permease, ABC-type transporter components, divalent metal transporter, arabinose efflux permease), and gene regulatory functions (MerR family transcriptional regulator, LacI family DNA-binding transcriptional regulator). In addition, a putative immunity protein against plantaricin EF (metal-dependent membrane protease (immunity protein PlnI)) seems to be conserved in AC-strains and as well as a component of the vitamin B6 synthesis pathway (pyridoxine 4-dehydrogenase). Many of the hypothetical proteins are gene fragments and most likely not functional. The absence of these core-AC genes from the core-genome of other A-strains does not suggest that they are completely absent from this latter group. A nucleotide blast showed (data not shown) their presence in some, but not all other A-strains.

### 3.4. The distribution of AC-strains during MLF

Next, the aim was to develop a qPCR test in order to detect and quantify sub-lineage AC-strains during the MLF. Unfortunately, as mentioned before, no specific gene was detected in AC-strains thus preventing the design of PCR primers. However, a manual comparison of their genomes with publicly available genomes using the MicroScope software revealed a specific SNP that was then used as a PCR target. This SNP was a single nucleotide deletion discovered in the gene of a divalent metal cation transporter (PSU-1 OEOE_RS03975, Fig. S1A). This deletion has so far only been detected in the genomes of AC-strains and the two AC2-strains. To distinguish AC and non-AC-strains, two different

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**Fig. 4.** Core-genome and pan-genome analysis of Cognac strains. The evolution of the core-genomes (A) and pan-genomes (B) of strains from lineages AC, AW and A* was determined using the Pan/Core-Genome tool implemented in MicroScope (Vallenet et al., 2020). All genes were assigned to gene families defined by MicroScope (MICFAMs). Core- and pan-genome evolutions were calculated using the MICFAM parameters at a cutoff of 80 % amino acid identity/80 % alignment coverage. For clarity, core- and pan-genome evolutions are depicted only for the first 34 A-strains, although the analysis was performed using the 171 A*-strains. C. A Venn-diagram depicting the comparison of core-genes from strains belonging to lineages AC, AW and A* (A*-strains: 171 A-strains, excluding AC-, AC2- and AW-strains).</figcaption></figure>
probes were designed to the SNP-containing genomic region, one of which containing the deletion (Fig. S1B). qPCR trials were carried out using these probes; however, no PCR product could be detected using the non-AC probe on PSU-1, CRBO 2180 and CRBO 2188 (Fig. S3A). Instead, a published qPCR method (Balmaseda et al., 2023) was used to target lineage A-strains. Standard curves of the correspondence between Cq values and bacterial population were calculated for both qPCR assays since the qPCR condition for lineage A was modified (Fig. S3B).

These qPCRs were conducted to estimate of the percentage of AC-
strains during MLF by quantifying the population levels of both A- and
AC-strains in wine samples. From each winery one or two wine samples
from stages 1 or 2 were selected. In total, 23 wines from 13 wineries
were analyzed (Fig. 5A). All population levels determined by the qPCR
were consistent with plate counts (Table S1). AC-strains were predom-
inant in almost all wines except for wines no. 83, 84 and 94. However,
these were samples from wineries 5 and 11 where strains with com-
mercial profiles were highly abundant (Figs. 2, 5B). These results illus-
trate that by using this qPCR method, the proportion of AC-strains
within a population of *O. oeni* in wine can be estimated quite well.

4. Discussion

This study described the discovery of a new sub-lineage AC of the
*O. oeni* species that is specific to wines produced for distillation in the
Cognac region. These distillation wines are not only produced in a single
region, they exhibit a number of other particularities (i.e. low pH, low
ethanol concentration, no added sulfites, extended storage periods)
which would explain this product-specific adaptation, a process also
known as microbial domestication (Steensels et al., 2019). AC-strains
turned out to be genetically quite divergent when compared to other
sub-lineages (Fig. 3, Lorentzen et al., 2019).

The diversity of *O. oeni* has already been explored in numerous
studies and in different wine-growing regions. The number of identified
strains varies greatly depending on various factors such as the number of
participating wineries, the number of wines analyzed, and the number of
bacterial isolates genotyped, as well as the exact genotyping method
used. For example, Vigentini et al. (2016) analyzed 48 isolates from 13
cellars in the Aosta Valley in Italy by REA-PFGE (Restriction Enzyme
Analysis with Pulsed Field Gel Electrophoresis) and identified 28
different strains. However, REA-PFGE is a method difficult to implement

for a large number of samples. Cappello et al. (2022) used AFLP (Amplified Fragment Length Polymorphism) for genotyping 250 isolates from four cellars in the region of Apulia, Italy, which uncovered 192 different profiles. Larger scale studies were conducted by using MLVA-PCR for genotyping. For instance, Franquès et al. (2017) analyzed 538 isolates originating from nine cellars in the Catalan region of Priorat resulting in 164 different genotypes. In another study 1922 isolates from cellars in the Languedoc-Roussillon, Burgundy and New Aquitaine regions in France were genotyped by MLVA-PCR yielding 476 different profiles (El Khoury et al., 2017). Thus, it was not surprising to detect as many as 458 different genotypes from 132 wines of 24 cellars in the Cognac region by analyzing nearly 3500 isolates (Fig. 2, Table S2). This significant diversity of *O. oeni* strains appears to be the norm in most wine-growing regions. The total number of strains in the Cognac region is likely much higher. Indeed, quite a large number of strains were detected by analyzing just a few wine vats in a few cellars, thus it is likely that thousands of strains would be discovered by investigating more wines from this region.

Some strains were present in significantly higher proportions than
others but did not appear to be predominant (Fig. 2A). For example, the
most abundant strain (249 isolates) is a commercial one and is nearly the
unique strain present in the 10 tanks of one winery (237 isolates). The
abundance of this strain was at first puzzling, however, upon request the
winery confirmed that they had inoculated certain tanks with a com-
mercial product. The fact, that this strain was rarely detected in any of
the other wineries, indicates that it is not particularly well adapted to
Cognac wines. Most of the other commercial strains were detected
sporadically, suggesting that they were not intentionally inoculated and
might have been used in the past in these cellars or are contamination
from for example shared equipment. They were neither predominantly
detected in the analyzed wines, nor did they spread throughout the
entire region. For the distribution of indigenous (non-commercial)
strains the following was observed: in each cellar one or a small number
of strains were detected that represented more than 50 % of isolates
(Fig. 2D). Many of these strains were also identified in other cellars
(Fig. S2B), but none at highly abundant levels. Hence, this work is in
agreement with another previous study that showed a wide regional
variety of strains, without certain strains being significantly more
abundant than others (El Khoury et al., 2017).

Furthermore, it has been shown that the relative abundance of one

<figure><img src="image_14.png" /><figcaption>**Fig. 5.** The abundance of sub-lineage AC or lineage A *O. oeni* strains. A. The population estimates of AC (yellow) and A (pink) strains in wine samples during MLF stages 1 or 2 were determined by qPCR. Samples are numbered as in Table S1. The qPCR analysis was conducted in triplicate. Standard deviations are indicated. B. The figure depicts the proportion of AC (yellow) and commercial (blue) *O. oeni* strains in wine samples. The proportion is based on approximately 30 isolates genotyped by MLVA-PCR.</figcaption></figure>
strain within a population might change during the MLF (Reguant and Bordons, 2003; Reguant et al., 2005a, 2005b; González-Arenzana et al., 2012). This notion is particularly interesting for distillations wines as they often undergo long storage periods between the end of the MLF and distillation. It suggests that strains that are particularly abundant at a specific moment during the fermentation might almost completely disappear during subsequent winemaking stages (Reguant et al., 2005a; González-Arenzana et al., 2012).

It was anticipated that the *O. oeni* strains isolated from cognac base wines would belong to the genetic lineage A as they were isolated from wines post AF. Sequencing data and subsequent phylogenetic analyses confirmed this notion by placing all 49 Cognac strains in lineage A which was further confirmed by qPCR data (Figs. 3, 5). Among the four genetic lineages described to date (lineages A to D), lineage D has not been detected in wines, while lineages B and C are abundant in grape must but decline during AF. Generally, only strains of lineage A develop during or after AF, and perform MLF (Balmaseda et al., 2023). So far, only a few studies that focused on the ecology of *O. oeni* in wine-producing regions have also identified the genetic group of strains because the only methods currently available are MLST (Multi Locus Sequence Typing) and genome sequencing. Nevertheless, MLST studies have confirmed the presence of A-strains and rarely B-strains in wines from various regions in France (Bilhère et al., 2009; Bridier et al., 2010; El Khoury et al., 2017), Italy (Capozzi et al., 2015) and China (Wang et al., 2015). This was further confirmed by phylogenomic studies based on genome sequences (Campbell-Sills et al., 2015; Sternes et al., 2017; Lorentzen et al., 2019).

The relatively large genetic divergence of sub-lineage AC was rather unexpected as strains derived from the same region and the same type of wine. In fact, these strains are more diverse than for example sub-lineage AW-strains (Fig. 2). AC-strains share a small number of core genes that, although absent from the AW and A core-genomes, are not unique to this sub-lineage (Table S4). The main driver of diversity could lie in the long storage period of these wines before distillation. During "normal" wine production microorganisms can develop during the AF and MLF which last a few weeks. Once the AF (for most white wines) or the MLF (for most red wines) is completed, all microbial life is eliminated by racking, sulfiting, and other winemaking practices. In cognac base wines, however, no action is taken to remove microorganisms after the fermentations, and they can thus continue to proliferate up to six months before distillation. This and the circumstance that *O. oeni* lacks the DNA mismatch repair genes mutS and mutL (Marcobal et al., 2008) could account for different-rate genomic evolution and the larger diversity of these AC-sub-lineage strains.

## 5. Conclusion

This study provides the first comprehensive overview of the biodiversity of *O. oeni* strains performing MLF in Cognac distillation wines. The combined use of MLVA genotyping, DNA sequencing and phylogenomic analyses revealed an unexpectedly high diversity of *O. oeni* strains in these wines. A new sub-lineage was identified, designated AC (lineage A-Cognac), containing strains that are genetically distinct from all other *O. oeni* strains. They appear to have adapted to the unique physicochemical environment of Cognac base wines, characterized by low pH and low ethanol content, combined with particular practices such as the absence of added sulfites, and the use of a single grape variety (Ugni blanc).

The predominance and wide distribution of AC-strains across the main Cognac production areas suggest a long-term domestication process shaped by the local winemaking practices. While comparative genomic analyses identified several core genes potentially linked to stress response, transport, and cell wall functions, the molecular mechanisms underlying this adaptation remain to be elucidated.

Altogether, these findings highlight Cognac distillation wines as a distinct ecological niche for *O. oeni*, expanding our understanding of

microbial diversification in fermented beverages. Future work should focus on determining the physiological traits and metabolic capacities that distinguish AC-strains from other *O. oeni* of the A-lineage, as well as assessing their possible occurrence in other distillation wines such as armagnac or brandy. Such insights would contribute to a deeper understanding of microbial domestication and may open new perspectives for managing MLF in wines destined for distillation.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijfoodmicro.2025.111539.

## CRediT authorship contribution statement

**Sayoko Matsumoto:** Formal analysis, Writing – original draft, Methodology. **Jana Rudolf:** Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Data curation. **Cécile Miot-Sertier:** Formal analysis, Methodology. **Olivier Claisse:** Data curation, Methodology, Visualization. **Panagiotis Stamatopoulos:** Conceptualization, Investigation, Methodology, Validation, Writing – original draft. **Xavier Poitou:** Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Validation, Writing – original draft. **Patrick Lucas:** Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing.

## Funding

The work was supported by Jas Hennessy & Co, Cognac, France.

## Declaration of competing interest

The study was supported by Jas Hennessy & Co. This has not altered the authenticity of its experimental design, results and conclusions. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors are grateful to Judith Girard (Bordeaux University), Valentin Lebrec (Jas Hennessy & Co), Rebekah Hicks David (Jas Hennessy & Co), Adeline Barreau (Jas Hennessy & Co), and Microflora, for their contributions to the experiments. They also thank the 24 wineries that provided the wines used in this study.

## Data availability

Data will be made available on request.

## References

Andrews, S., 2010. FastQC: a quality control tool for high throughput sequence data. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/.

Balmaseda, A., Lorentzen, M., Dutilh, L., Bauduin, R., Guichard, H., Ollivier, S., Miot-Sertier, C., Lucas, P.M., 2023. Alcoholic fermentation drives the selection of *Oenococcus oeni* strains in wine but not in cider. Int. J. Food Microbiol. 400, 110276.

Bilhère, E., Lucas, P.M., Claisse, O., Lonvaud-Funel, A., 2009. Multilocus sequence typing of *Oenococcus oeni*: detection of two subpopulations shaped by intergenic recombination. Appl. Environ. Microbiol. 75, 1291–1300.

Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 30, 2114–2120.

Breniaux, M., Dutilh, L., Petrel, M., Gontier, E., Campbell-Sills, H., Deleris-Bou, M., Krieger, S., Teissedre, P.-L., Jourdes, M., Reguant, C., Lucas, P.M., 2018. Adaptation of two groups of *Oenococcus oeni* strains to red and white wines: the role of acidity and phenolic compounds. J. Appl. Microbiol. 125, 1117–1127.

Bridier, J., Claisse, O., Coton, M., Coton, E., Lonvaud-Funel, A., 2010. Evidence of distinct populations and specific subpopulations within the species *Oenococcus oeni*. Appl. Environ. Microbiol. 76, 7754–7764.

Campbell-Sills, H., El Khoury, M., Favier, M., Romano, A., Biasioli, F., Spano, G., Sherman, D.J., Bouchez, O., Coton, E., Coton, M., Okada, S., Tanaka, N., Dols-
Lafargue, M., Lucas, P.M., 2015. Phylogenomic analysis of *Oenococcus oeni* reveals specific domestication of strains to cider and wines. Genome Biol. Evol. 7, 1506–1518.

Campbell-Sills, H., Khoury, M.E., Gammacurta, M., Miot-Sertier, C., Dutilh, L., Vestner, J., Capozzi, V., Sherman, D., Hubert, C., Claisse, O., Spano, G., De Revel, G., Lucas, P.M., 2017. Two different *Oenococcus oeni* lineages are associated to either red or white wines in Burgundy: genomics and metabolomics insights. OENO One 51, 309.

Capozzi, V., Garofalo, C., Chiriatti, M.A., Grieco, F., Spano, G., 2015. Microbial terroir and food innovation: the case of yeast biodiversity in wine. Microbiol. Res. 181, 75–83.

Cappello, M.S., Falco, V., Curcio, R., Mita, G., Zapparoli, G., 2022. Molecular and physiological properties of indigenous strains of *Oenococcus oeni* selected from Nero di Troia wine (Apulia, Italy). Microorganisms 10, 795.

Claisse, O., Lonvaud-Funel, A., 2012. Development of a multilocus variable number of tandem repeat typing method for *Oenococcus oeni*. Food Microbiol. 30, 340–347.

Claisse, O., Lonvaud-Funel, A., 2014. Multiplex variable number of tandem repeats for *Oenococcus oeni* and applications. Food Microbiol. 38, 80–86.

Coton, M., Pawtowski, A., Taminiau, B., Burgaud, G., Deniel, F., Couloumme-Labarthe, L., Fall, A., Daube, G., Coton, E., 2017. Unraveling microbial ecology of industrial-scale Kombucha fermentations by metabarcoding and culture-based methods. FEMS Microbiol. Ecol. 93, fix048.

Dimopoulou, M., Vuillemin, M., Campbell-Sills, H., Lucas, P.M., Ballestra, P., Miot-Sertier, C., Favier, M., Coulon, J., Moine, V., Doco, T., Roques, M., Williams, P., Petrel, M., Gontier, E., Moulis, C., Remaud-Simeon, M., Dols-Lafargue, M., 2014. Exopolysaccharide (EPS) synthesis by *Oenococcus oeni*: from genes to phenotypes. PLoS One 9.

Dols-Lafargue, M., Lee, H.Y., Le Marrec, C., Heyraud, A., Chambat, G., Lonvaud-Funel, A., 2008. Characterization of *gtf*, a glucosyltransferase gene in the genomes of *Pediococcus parvulus* and *Oenococcus oeni*, two bacterial species commonly found in wine. Appl. Environ. Microbiol. 74, 4079–4090.

Du Plessis, H.W., Steger, C.L.C., Du Toit, M., Lambrechts, M.G., 2002. The occurrence of malolactic fermentation in brandy base wine and its influence on brandy quality. J. Appl. Microbiol. 92, 1005–1013.

Du Plessis, H.W., Dicks, L.M.T., Pretorius, I.S., Lambrechts, M.G., du Toit, M., 2004. Identification of lactic acid bacteria isolated from South African brandy base wines. Int. J. Food Microbiol. 91 (1), 19–29.

El Khoury, M., Campbell-Sills, H., Salin, F., Guichoux, E., Claisse, O., Lucas, P.M., 2017. Biogeography of *Oenococcus oeni* reveals distinctive but nonspecific populations in wine-producing regions. Appl. Environ. Microbiol. 83, e02322-16.

Franquès, J., Araque, I., Palahí, E., Portillo, M. del C., Reguant, C., Bordons, A., 2017. Presence of *Oenococcus oeni* and other lactic acid bacteria in grapes and wines from Priorat (Catalonia, Spain). LWT Food Sci. Technol. 81, 326–334.

Franquès, J., Araque, I., Khoury, M.E., Lucas, P.M., Reguant, C., Bordons, A., 2018. Selection and characterization of autochthonous strains of *Oenococcus oeni* for vinification in Priorat (Catalonia, Spain). OENO One 52.

González-Arenzana, L., Santamaría, P., Lopez, R., Tenorio, C., López-Alfaro, I., 2012. Ecology of indigenous lactic acid bacteria along different winemaking processes of Tempranillo red wine from La Rioja (Spain). Sci. World J. 2012, 796327.

Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K., 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549.

Letunic, I., Bork, P., 2021. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49, W293–W296.

Lonvaud-Funel, A., 1999. Lactic acid bacteria in the quality improvement and depreciation of wine. In: Konings, W.N., Kuipers, O.P., Huis Veld, J.H.J. (Eds.), Lactic Acid Bacteria: Genetics, Metabolism and Applications. Springer Netherlands, Dordrecht, pp. 317–331.

Lorentzen, M.P.G., Lucas, P.M., 2019. Distribution of *Oenococcus oeni* populations in natural habitats. Appl. Microbiol. Biotechnol. 103, 2937–2945.

Lorentzen, M.P., Campbell-Sills, H., Jorgensen, T.S., Nielsen, T.K., Coton, M., Coton, E., Hansen, L., Lucas, P.M., 2019. Expanding the biodiversity of *Oenococcus oeni* through comparative genomics of apple cider and kombucha strains. BMC Genomics 20, 330.

Marcobal, A.M., Sela, D.A., Wolf, Y.I., Makarova, K.S., Mills, D.A., 2008. Role of hypermutability in the evolution of the genus *Oenococcus*. J. Bacteriol. 190, 564–570.

Pritchard, L., Glover, R., Humphris, S., Elphinstone, J., Toth, I., 2015. Genomics and taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens. Anal. Methods 8.

Reguant, C., Bordons, A., 2003. Typification of *Oenococcus oeni* strains by multiplex RAPD-PCR and study of population dynamics during malolactic fermentation. J. Appl. Microbiol. 95, 344–353.

Reguant, C., Carreté, R., Constantí, M., Bordons, A., 2005a. Population dynamics of *Oenococcus oeni* strains in a new winery and the effect of SO₂ and yeast strain. FEMS Microbiol. Lett. 246, 111–117.

Reguant, C., Carreté, R., Ferrer, N., Bordons, A., 2005b. Molecular analysis of *Oenococcus oeni* population dynamics and the effect of aeration and temperature during alcoholic fermentation on malolactic fermentation. Int. J. Food Sci. Technol. 40, 451–459.

Romero, J., Ilabaca, C., Ruiz, M., Jara, C., 2018. *Oenococcus oeni* in Chilean red wines: technological and genomic characterization. Front. Microbiol. 9, 90.

Seemann, T., 2016. Shovill: Faster SPAdes Assembly of Illumina Reads.

Souvorov, A., Agarwala, A., Lipman, D.J., 2018. SKESA: strategic k-mer extension for scrupulous assemblies. Genome Biol. 19, 153.

Steensels, J., Gallone, B., Voordeckers, K., Verstrepen, K.J., 2019. Domestication of industrial microbes. Curr. Biol. 29, R381–R393.

Sternes, P.R., Costello, P.J., Chambers, P.J., Bartowsky, E.J., Borneman, A.R., 2017. Whole transcriptome RNAseq analysis of *Oenococcus oeni* reveals distinct intra-specific expression patterns during malolactic fermentation, including genes involved in diacetyl metabolism. Int. J. Food Microbiol. 257, 216–224.

Torriani, S., Felis, G.E., Fracchetti, F., 2011. Selection criteria and tools for malolactic starters development: an update. Ann. Microbiol. 61, 33–39.

Tsakiris, A., Kallithraka, S., Kourkoutas, Y., 2014. Grape brandy production, composition and sensory evaluation. J. Sci. Food Agric. 94 (3), 404–414.

Vallenet, D., Calteau, A., Dubois, M., Amours, P., Bazin, A., Beuvin, M., Burlot, L., Bussell, X., Fouteau, S., Gautreau, G., Lajus, A., Langlois, J., Planel, R., Roche, D., Rollin, J., Rouy, Z., Sabatet, V., Médigue, C., 2020. MicroScope: an integrated platform for the annotation and exploration of microbial gene functions through genomic, pangenomic and metabolic comparative analysis. Nucleic Acids Res. 48, D579–D589.

Vigentini, I., Praz, A., Domeneghetti, D., Zenato, S., Picozzi, C., Barmaz, A., Foschino, R., 2016. Characterization of malolactic bacteria isolated from Aosta Valley wines and evidence of psychrotrophy in some strains. J. Appl. Microbiol. 120, 934–945.

Wang, T., Li, H., Wang, H., Su, J., 2015. Multilocus sequence typing and pulsed-field gel electrophoresis analysis of *Oenococcus oeni* from different wine-producing regions of China. Int. J. Food Microbiol. 199, 47–53.