domain: sde sdv

text:
Maria Dimopoulou
email: mdimopoulou@uniwa.gr

Marguerite Dols-Lafargue

Paloma López

Maria Teresa Dueñas

Exopolysaccharides Producing Lactic Acid Bacteria in Wine and Other Fermented Beverages: For Better or for Worse?

Keywords: lactic acid bacteria, wine, exopolysaccharides, fermented beverages

Lactic acid bacteria (LAB) from fermented beverages such as wine, cider and beer produce a wide range of exopolysaccharides (EPS) through multiple biosynthetic pathways. These extracellular polysaccharides constitute key elements for bacterial species adaptation to such anthropic processes. In the food industry, LAB polysaccharides have been widely studied for their rheological, functional and nutritional properties; however, these have been poorly studied in wine, beer and cider until recently. In this review, we have gathered the information available on these specific polysaccharide structure and, biosynthetic pathways, as well as the physiology of their production. The genes associated with EPS synthesis are also presented and compared. Finally, the possible role of EPS for bacterial survival and spread, as well as the risks or possible benefits for the winemaker and the wine lover, are discussed.

Introduction

Lactic acid bacteria (LAB) form a large group of bacteria, and are widely used throughout the world for the biotransformation of animal and plant resources. From very early on, they were valued to preserve and improve the organoleptic or nutritional properties of many foods. This is also the case in fermented beverages, where many distinct genera and species have been described [START_REF] König | Biology of Microorganisms on Grapes[END_REF]. However, not all species are found in all beverages. Species distribution seems rather specific to a particular type of fermented drink. Furthermore, LAB are sometimes hardly detectable in fruits and become dominant at certain steps in the production process. In fact, in wine and cider, LAB develops after the yeasts first drive alcoholic fermentation (AF). At the end of AF, upon lysis of the yeasts, the medium contains high levels of ethanol and very few residual sugars; it is acidic. Only certain species have been found to resist this process, including LAB of the genera Lactobacillus, Pediococcus, Oenococcus and Leuconostoc [START_REF] König | Maintenance of Wine-Associated Microorganisms[END_REF]. These bacteria multiply by consuming the elements left by the yeasts (sugars, acids). Depending on the metabolic pathways taken to break down the sugars, they produce either only lactic acid (homofermentative bacteria) or a mixture of lactic acid and acetate or ethanol (heterofermentative bacteria) [START_REF] König | Maintenance of Wine-Associated Microorganisms[END_REF]. However, the main visible transformation carried out by lactic acid bacteria in wine and cider is the conversion of the L-malic acid present into L-lactic acid and CO 2 . Although this is not fermentation sensu stricto, this reaction is called malolactic fermentation (MLF). MLF is important for three reasons [START_REF] Davis | Practical Implications of Malolactic Fermentation: A Review[END_REF]: (i) the transformation of malic acid (di-acid) into lactic acid (monoacid) softens the beverage, reducing its total acidity; (ii) the growth of bacteria is accompanied by the consumption of the residual substrates of the medium, which limits the risk of development of the spoilage flora during storage and aging and (iii) the release of aromas (from grapes, apples, yeasts or wood precursors) and the production of secondary metabolites by bacteria contribute to the final sensorial properties of the wine [START_REF] Lonvaud-Funel | Lactic Acid Bacteria in the Quality Improvement and Depreciation of Wine[END_REF][START_REF] Versari | Leuconostoc oenos and Malolactic Fermentation in Wine: A Review[END_REF][START_REF] Sumby | Microbial Modulation of Aromatic Esters in Wine: Current Knowledge and Future Prospects[END_REF] In the same way, LAB also modulate the ciders final quality [START_REF] Sánchez | Population Dynamics of Lactic Acid Bacteria during Spontaneous Malolactic Fermentation in Industrial Cider[END_REF][START_REF] Sánchez | Prevalent Lactic Acid Bacteria in Cider Cellars and Efficiency of Oenococcus oeni Strains[END_REF].

The success of MLF relies on the ability of indigenous LAB to survive and develop under particularly hostile physicochemical conditions. Only the most resistant LAB are selected. Though MLF can also be carried out by genera other than Oenococcus [START_REF] Lonvaud-Funel | Lactic Acid Bacteria in the Quality Improvement and Depreciation of Wine[END_REF][START_REF] Bartowsky | Oenococcus oeni and Malolactic Fermentation-Moving into the Molecular Arena[END_REF] most often, Oenococcus oeni becomes the dominant species and the main driver of MLF in temperate-zone wines or ciders, although it is hardly detected on the fruits [START_REF] Lonvaud-Funel | Lactic Acid Bacteria in the Quality Improvement and Depreciation of Wine[END_REF][START_REF] Sánchez | Prevalent Lactic Acid Bacteria in Cider Cellars and Efficiency of Oenococcus oeni Strains[END_REF][START_REF] Lorentzen | Distribution of Oenococcus oeni Populations in Natural Habitats[END_REF]. The bacterial strains found in ciders and wines are different, suggesting that a form of domestication has occurred within the species. Other species of the genus Oenococcus are found in fermented products made from sugar cane (O. alcolitolerans [START_REF] Badotti | Oenococcus alcoholitolerans Sp. Nov., a Lactic Acid Bacteria Isolated from Cachaça and Ethanol Fermentation Processes[END_REF]) or during the preparation of schochu (O. kitaharae [START_REF] Endo | a Non-Acidophilic and Non-Malolactic-Fermenting Oenococcus Isolated from a Composting Distilled Shochu Residue[END_REF]). O. sicerae is found specifically in ciders [START_REF] Cousin | Microorganisms in Fermented Apple Beverages: Current Knowledge and Future Directions[END_REF].

Certain particularly efficient LAB strains have been selected and are marketed as malolactic starters [START_REF] Bartowsky | Oenococcus oeni and Malolactic Fermentation-Moving into the Molecular Arena[END_REF]. Conversely, certain strains of LAB (including O. oeni) can cause deterioration before, during or after MLF, depending on their genome content and the associated metabolic abilities [START_REF] Bartowsky | Oenococcus oeni and the Genomic Era[END_REF]. Furthermore, the context (type of beverage or even type of wine considered) may also modulate the microbial metabolic activities and thus the risk associated with the presence of a specific strain. The machinery necessary for the biosynthesis of exopolysaccharides (EPSs) is one of the metabolic tools which differentiates the LAB strains. EPS can (i) contribute to bacterial survival in the specific context of the production of fermented drinks, (ii) improve the sensory properties of wines and other fermented drinks or (iii) on the contrary, lead to beverage spoilage [START_REF] Lonvaud-Funel | Lactic Acid Bacteria in the Quality Improvement and Depreciation of Wine[END_REF][START_REF] Dimopoulou | Exopolysaccharides Produced by Oenococcus oeni: From Genomic and Phenotypic Analysis to Technological Valorization[END_REF][START_REF] Dols Lafargue | Polysaccharide Production by Wine Lactic Acid Bacteria: Negative Trait or Potential Advantage? A Review[END_REF].

The aim of this review is to synopsize the current knowledge on the EPSs produced by the LAB of fermented drinks, namely, the nature and location of the EPSs, the genes involved and the active biosynthetic pathways, as well as the consequences of EPS production on the survival of the bacteria and on the quality of the beverages produced. Finally, research perspectives will be examined.

EPSs Produced and Biosynthetic Pathways

EPSs are extracellular glucidic polymers of variable size (a few tens of monosaccharides to several tens of thousands). They may consist of a single type of monomer (homopolysaccharides) or of several different monomers (heteropolysaccharides), they may be neutral or charged and may or may not contain non-carbohydrate substituents. Finally, they can be linear or branched. Among fermented drinks, the LAB EPSs O. oeni EPSs are the ones that have been the most studied-data on this species will therefore be presented first, and where possible, knowledge will be extended to other species (Table 1).

EPSs Produced by Wine and Cider LAB in Brief

The β-1,3 β-1,2 glucan was the first EPS produced by wine or cider LAB to be identified [START_REF] Llaubères | Structure of an Exocellular Beta-D-Glucan from Pediococcus sp., a Wine Lactic Bacteria[END_REF]. It comprises a backbone made of β-1,3 linked glucoses and branches made of a single β-1,2 linked glucose attached to each of the two or three residues of the main chain. This polymer will be referred to as "β-glucan" throughout this review. Its accumulation in beverages induces an increase in viscosity and even a ropy character: the wine, beer or cider can harbor an oily texture, even when β-glucan concentrations are as low as 12 mg/L [START_REF] Dimopoulou | Polysaccharide Production by Grapes Must and Wine Microorganisms[END_REF]. Many bacterial species are able to produce this specific glucan: O. oeni, Lactobacillus suebicus, Lactobacillus diolivorans, Pediococcus parvulus, Pediococcus damnosus, Lactobacillus collinoides, Lactobacillus brevis, Lactobacillus rossiae, Lactobacillus parabuchneri and Levilactobacillus brevis [START_REF] Werning | Pediococcus Parvulus Gtf Gene Encoding the GTF Glycosyltransferase and Its Application for Specific PCR Detection of β-D-glucan-producing Bacteria in Foods and Beverages[END_REF][START_REF] Dols-Lafargue | Characterization of Gtf, a Glucosyltransferase Gene in the Genomes of Pediococcus parvulus and Oenococcus oeni, Two Bacterial Species Commonly Found in Wine[END_REF][START_REF] Duenas | Heterofermentative Lactobacilli Causing Ropiness in Basque Country Ciders[END_REF][START_REF] Walling | A Putative Glucan Synthase Gene Dps Detected in Exopolysaccharide-Producing Pediococcus damnosus and Oenococcus oeni Strains Isolated from Wine and Cider[END_REF][START_REF] Fraunhofer | Characterization of β-Glucan Formation by Lactobacillus brevis TMW 1.2112 Isolated from Slimy Spoiled Beer[END_REF][START_REF] Jakob | β-Glucan Formation Is a Selective Advantage for Beer-Spoiling Levilactobacillus brevis TMW 1.2112 during Planktonic Growth[END_REF][START_REF] Garai-Ibabe | Naturally Occurring 2-Substituted (1,3)-Beta-D-Glucan Producing Lactobacillus suebicus and Pediococcus parvulus Strains with Potential Utility in the Production of Functional Foods[END_REF][START_REF] Dueñas-Chasco | Structural Analysis of the Exopolysaccharides Produced by Lactobacillus spp. G-77[END_REF][START_REF] Ibarburu | Production and Partial Characterization of Exopolysaccharides Produced by Two Lactobacillus suebicus Strains Isolated from Cider[END_REF][START_REF] Dueñas-Chasco | Structural Analysis of the Exopolysaccharide Produced by Pediococcus damnosus 2.6[END_REF][START_REF] Puertas | Disclosing Diversity of Exopolysaccharide-Producing Lactobacilli from Spanish Natural Ciders[END_REF]. Additionally, heteropolysaccharides, dextrans and fructans are also produced by O. oeni and some other species [START_REF] Dimopoulou | Polysaccharide Production by Grapes Must and Wine Microorganisms[END_REF].

Many wine or cider bacteria have been shown to produce several types of polymers simultaneously. For example, certain strains of O. oeni isolated from wine or cider produce both dextran (α-1,6-α-1,3 glucan), levan (β-2,6 fructan), β-glucan and heteropolysaccharides composed of galactose, glucose and rhamnose [START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF]. Pediococcus ethanolidurans isolated from Basque cider also produces both ropy β-glucan and a heteropolysaccharide composed of glucose, galactose, glucosamine and glycerol-3-phosphate [START_REF] Llamas-Arriba | Characterization of Pediococcus ethanolidurans CUPV141: A β-D-Glucanand Heteropolysaccharide-Producing Bacterium[END_REF].

However, depending on the conditions, one of the polymers formed is much more abundant than the others and explains the dominant phenotype of the strain: capsulated, mucous, ropy or sticky [START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF][START_REF] Dimopoulou | Exploration of Phenomena Contributing to the Diversity of Oenococcus oeni Exopolysaccharides[END_REF]. The production could be modulated by the presence of numerous phenolic compounds in these beverages [START_REF] Stivala | Effect of Phenolic Compounds on Viability of Wine Spoilage Lactic Acid Bacteria. A Structure-Activity Relationship Study[END_REF]. Furthermore, in wine or grape juice, the concentrations of the EPS produced are often hardly noticeable due to the presence of numerous other polysaccharides liberated from the grapes and yeasts [START_REF] Dols-Lafargue | Changes in Red Wine Soluble Polysaccharide Composition Induced by Malolactic Fermentation[END_REF][START_REF] Ciezack | Evidence for Exopolysaccharide Production by Oenococcus oeni Strains Isolated from Non-Ropy Wines[END_REF].

EPS Localization

EPS can be classified into three major groups, according to their exact external location [START_REF] Ryan | Sugar-Coated: Exopolysaccharide Producing Lactic Acid Bacteria for Food and Human Health Applications[END_REF]:

• WPS or wall polysaccharides, attached to the cell, covalently or not, but without forming a capsule.

•

CPS (or capsular polysaccharides), most of the time linked to peptidoglycan, forming either a thick and cohesive (capsule) or a thin and cohesive (film) outer layer.

•

Exocellular polysaccharides (or true EPSs), released into the environment surrounding the cell during planktonic growth. This kind of true EPS can also form a slime or a polymeric matrix during growth on solid media or biofilm formation.

The distinction between these polysaccharides is sometimes controversial. The capsules are observable in negative staining in classical microscopy, but some polymers can form a dense layer that is visible in electron microscopy but not thick enough to be visible in negative staining [START_REF] Weidenmaier | Teichoic Acids and Related Cell-Wall Glycopolymers in Gram-Positive Physiology and Host Interactions[END_REF][START_REF] Chapot-Chartier | Interactions of the Cell-Wall Glycopolymers of Lactic Acid Bacteria with Their Bacteriophages[END_REF]. In addition, CPS can be released depending on the growing conditions or due to unstable cell binding and can be mistaken for EPSs. Conversely, certain EPSs can be found linked to the cell and to the peptidoglycan, even non-covalently [START_REF] Weidenmaier | Teichoic Acids and Related Cell-Wall Glycopolymers in Gram-Positive Physiology and Host Interactions[END_REF].

For instance, most O. oeni strains studied are encapsulated by a heteropolysaccharidic layer and this kind of "capsule" seems to be present regardless of the culture conditions. However, some of these CPSs are released into the surrounding medium at the end of the stationary phase [START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF] or during growth on solid media [START_REF] Jakob | β-Glucan Formation Is a Selective Advantage for Beer-Spoiling Levilactobacillus brevis TMW 1.2112 during Planktonic Growth[END_REF]. On the other hand, the capsular β-glucan of P. parvulus 2.6, a ropy strain isolated from cider, can be released from the cell "capsule" into the medium by means of simple cell washes [START_REF] Fernández De Palencia | Probiotic Properties of the 2-Substituted (1,3)-β-d-Glucan-Producing Bacterium Pediococcus parvulus 2.6[END_REF].

Biosynthetic Pathways

The EPS biosynthetic pathways involving sugar nucleotides have been extensively studied in milk LAB (Streptococcus thermophilus, Lactobacillus rhamnosus and Lactococcus lactis) and in pathogenic streptococci (Streptococcus pneumonia and Streptococcus agalactiae) [START_REF] Kolkman | The Capsule Polysaccharide Synthesis Locus of Streptococcus pneumoniae Serotype 14: Identification of the Glycosyl Transferase Gene Cps14E[END_REF][START_REF] Kolkman | Functional Analysis of Glycosyltransferases Encoded by the Capsular Polysaccharide Biosynthesis Locus of Streptococcus pneumoniae Serotype 14[END_REF][START_REF] Stingele | Lactobacillus Helveticus Lh59 Secretes an Exopolysaccharide That Is Identical to the One Produced by Lactobacillus helveticus TN-4, a Presumed Spontaneous Mutant of Lactobacillus helveticus TY1-2[END_REF][START_REF] Stingele | Introduction of the Exopolysaccharide Gene Cluster from Streptococcus thermophilus Sfi6 into Lactococcus lactis MG1363: Production and Characterization of an Altered Polysaccharide[END_REF][START_REF] De Vuyst | Recent Developments in the Biosynthesis and Applications of Heteropolysaccharides from Lactic Acid Bacteria[END_REF][START_REF] Lebeer | Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase[END_REF]. Dextran and levan synthesis have been mainly studied in Lc. mesenteroides [START_REF] Monsan | Homopolysaccharides from Lactic Acid Bacteria[END_REF].

Similar pathways were found to be active in LAB of wine and fermented beverages. Actually, in these specific bacteria, three types of cellular machinery dedicated to the biosynthesis of EPSs have been described to date (Figure 1): (i) extracellular transglycosidases called glycansucrases, which use sucrose as a substrate and catalyze the synthesis of homopolysaccharides (α-glucans, β-fructans); (ii) isolated synthases that use nucleotide sugars as substrates and which alone catalyze the polymerization and export of homopolysaccharides; and (iii) complex systems involving nucleotide sugars as substrates and many enzymes that achieve the synthesis and export of complex heteropolysaccharides together [START_REF] Dimopoulou | Polysaccharide Production by Grapes Must and Wine Microorganisms[END_REF].

(i) Transglycosidases, which specifically use sucrose as a substrate (or glycansucrases), are classified into the CAZy GH-13, 68 and 70 families (www.cazy.org (accessed on 14 September 2021)) [START_REF] Lombard | The Carbohydrate-Active Enzymes Database (CAZy) in 2013[END_REF]. They catalyze the synthesis of homopolysaccharides made up of glucose or fructose, according to the following simplified reactions:

n sucrose → (fructose)n + n glucose (fructansucrase) n sucrose → (glucose)n + n fructose (glucansucrase)
These enzymes are exocellular (Figure 1). They catalyze the synthesis of polysaccharides via a succession of donor-acceptor type reactions (a processive mechanism [START_REF] Moulis | Understanding the Polymerization Mechanism of Glycoside-Hydrolase Family 70 Glucansucrases[END_REF]). They are produced by certain strains of different species found in fermented beverages (O. oeni, O. kitaharae, Lc. mesenteroides, Lc. pseudomesenteroides) [START_REF] Zhou | Characterization of a Dextran Produced by Leuconostoc pseudomesenteroides XG5 from Homemade Wine[END_REF][START_REF] Montersino | Evaluation of Exopolysaccharide Production by Leuconostoc mesenteroides Strains Isolated from Wine[END_REF][START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF][START_REF] Dimopoulou | Exploration of Phenomena Contributing to the Diversity of Oenococcus oeni Exopolysaccharides[END_REF][START_REF] Vuillemin | A Dextran with Unique Rheological Properties Produced by the Dextransucrase from Oenococcus kitaharae DSM 17330[END_REF]. The structure of the polymer produced varies depending on the producing enzyme, both in terms of the type of osidic bonds formed (α-1,6 and α-1,3 in varying proportions for glucans and mainly β-2,6 for fructans), and in terms of degree of polymerization. Interestingly, the dextran produced by the enzyme DsrOK from O. kitaharae is one of the largest ever studied [START_REF] Vuillemin | A Dextran with Unique Rheological Properties Produced by the Dextransucrase from Oenococcus kitaharae DSM 17330[END_REF]. The enzyme also displays a very high catalytic efficacy, whereas the enzyme DsrO of O. oeni is poorly efficient and quite unstable [START_REF] Dimopoulou | Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance[END_REF][START_REF] Vuillemin | A Dextran with Unique Rheological Properties Produced by the Dextransucrase from Oenococcus kitaharae DSM 17330[END_REF]. These enzymes are exocellular (Figure 1). They catalyze the synthesis of polysaccharides via a succession of donor-acceptor type reactions (a processive mechanism [START_REF] Moulis | Understanding the Polymerization Mechanism of Glycoside-Hydrolase Family 70 Glucansucrases[END_REF]). They are produced by certain strains of different species found in fermented beverages (O. oeni, O. kitaharae, Lc. mesenteroides, Lc. pseudomesenteroides) [START_REF] Zhou | Characterization of a Dextran Produced by Leuconostoc pseudomesenteroides XG5 from Homemade Wine[END_REF][START_REF] Montersino | Evaluation of Exopolysaccharide Production by Leuconostoc mesenteroides Strains Isolated from Wine[END_REF][START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF][START_REF] Dimopoulou | Exploration of Phenomena Contributing to the Diversity of Oenococcus oeni Exopolysaccharides[END_REF][START_REF] Vuillemin | A Dextran with Unique Rheological Properties Produced by the Dextransucrase from Oenococcus kitaharae DSM 17330[END_REF]. The structure of the polymer produced varies depending on the producing enzyme, both in terms of the type of osidic bonds formed (α-1,6 and α-1,3 in varying proportions for glucans and mainly β-2,6 for fructans), and in terms of degree of polymerization. Interestingly, the dextran produced by the enzyme DsrOK from O. kitaharae is one of the largest ever studied (ii) The synthase pathway involves a single membrane-spanning enzyme, which alone carries out the initiation of synthesis, the elongation of the polymer (processive enzyme) and its export through the membrane (Figure 1). The β-glucan causing wine or cider ropiness is produced by an enzyme of this type, called Gtf. The role of Gtf in the synthesis of P. parvulus and O. oeni β-glucan was demonstrated in 2006 and 2008 [START_REF] Werning | Pediococcus Parvulus Gtf Gene Encoding the GTF Glycosyltransferase and Its Application for Specific PCR Detection of β-D-glucan-producing Bacteria in Foods and Beverages[END_REF][START_REF] Dols-Lafargue | Characterization of Gtf, a Glucosyltransferase Gene in the Genomes of Pediococcus parvulus and Oenococcus oeni, Two Bacterial Species Commonly Found in Wine[END_REF]. Gtf is 32% identical to Tts, a synthase found in S. pneumoniae type 37, which produces a β-glucan with structure close to that produced by wine, beer and cider strains. This pneumococcal glucan is immunogenic in humans, as in mice, and was shown to be responsible for pneumococcal strain virulence [START_REF] Llull | A Single Gene (Tts) Located Outside the Cap Locus Directs the Formation of Streptococcus pneumoniae Type 37 Capsular Polysaccharide. Type 37 Pneumococci Are Natural, Genetically Binary Strains[END_REF]. (iii) The third pathway is the most complex (Figure 1). This pathway is sometimes called the Wzy-dependent pathway, based on the name given to the polymerase in E. coli [START_REF] Whitfield | Biosynthesis and Assembly of Capsular Polysaccharides in Escherichia coli[END_REF]. This pathway has been perfectly characterized in Gram-negative bacteria and partially in Gram-positive bacteria [START_REF] Kolkman | The Capsule Polysaccharide Synthesis Locus of Streptococcus pneumoniae Serotype 14: Identification of the Glycosyl Transferase Gene Cps14E[END_REF][START_REF] Kolkman | Functional Analysis of Glycosyltransferases Encoded by the Capsular Polysaccharide Biosynthesis Locus of Streptococcus pneumoniae Serotype 14[END_REF][START_REF] Stingele | Lactobacillus Helveticus Lh59 Secretes an Exopolysaccharide That Is Identical to the One Produced by Lactobacillus helveticus TN-4, a Presumed Spontaneous Mutant of Lactobacillus helveticus TY1-2[END_REF][START_REF] Stingele | Introduction of the Exopolysaccharide Gene Cluster from Streptococcus thermophilus Sfi6 into Lactococcus lactis MG1363: Production and Characterization of an Altered Polysaccharide[END_REF][START_REF] Whitfield | Biosynthesis and Assembly of Capsular Polysaccharides in Escherichia coli[END_REF]. The first step is the synthesis of a repeating oligosaccharidic unit through the transfer of monomers to a lipid transporter on the inner face of the cell membrane (Figure 1). This synthesis, carried out by a series of non-processive glycosyltransferases, is followed by the export of the repeating unit by a flippase (Wzx) and by the assembly of the exported repeating units by a polymerase attached to the external face of the cell membrane (Wzy). Regulating enzymes and factors modulate the chain length and the polymer release. The glycosyltransferase which initiates the synthesis of the repeat unit by transferring the first monomer to the lipid transporter is called the "priming glycosyltransferase". Several priming glycosyltransferases are found in O. oeni and complement each other [START_REF] Dimopoulou | Molecular Cloning, Expression and Characterization of Oenococcus oeni Priming Glycosyltransferases[END_REF], ensuring EPS formation even in cases in which mutations inactivate one of the enzymes.

The distinction between biosynthetic pathways using a transglycosidase and sucrose as a precursor or glycosyltransferase(s) and sugar nucleotides is very important from a physiological point of view-the biosynthetic pathways involving sugar nucleotides are very "expensive" for the bacteria from an energetic point of view. They compete for nucleotide sugars with the cell-wall synthetic pathways, which limits the production levels of liberated polymers to a few hundred mg/L, whereas the production levels can rise to a few tens of grams per liter, in the case of glucansucrase using sucrose [START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF][START_REF] Dimopoulou | Exploration of Phenomena Contributing to the Diversity of Oenococcus oeni Exopolysaccharides[END_REF][START_REF] Ciezack | Evidence for Exopolysaccharide Production by Oenococcus oeni Strains Isolated from Non-Ropy Wines[END_REF][START_REF] Gänzle | Novel Metabolites from Cereal-Associated Lactobacilli-Novel Functionalities for Cereal Products?[END_REF]. The O. oeni strains equipped with active glucansucrase or fructansucrases release from 0.2 to 8 g/L of polymers depending on the strains and growing conditions. Sucrose has to be added to the growth media [START_REF] Dimopoulou | Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance[END_REF][START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF]. In the presence of high sucrose concentrations, Lc. mesenteroides isolated from Spanish wines can produce 500 mg/L of dextran [START_REF] Montersino | Evaluation of Exopolysaccharide Production by Leuconostoc mesenteroides Strains Isolated from Wine[END_REF].

The Wzy-dependent pathway leads to the release of at most 250 mg/L of heteropolysaccharide in O. oeni and about 50 mg/L in P. ethanolidurans [START_REF] Llamas-Arriba | Characterization of Pediococcus ethanolidurans CUPV141: A β-D-Glucanand Heteropolysaccharide-Producing Bacterium[END_REF][START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF][START_REF] Ciezack | Evidence for Exopolysaccharide Production by Oenococcus oeni Strains Isolated from Non-Ropy Wines[END_REF]. Furthermore, depending on the bacterial strain and the growth conditions, the concentration of β-glucan can vary between 10 and 250 mg/L [START_REF] Dols-Lafargue | Characterization of Gtf, a Glucosyltransferase Gene in the Genomes of Pediococcus parvulus and Oenococcus oeni, Two Bacterial Species Commonly Found in Wine[END_REF][START_REF] Ibarburu | A Real-Time PCR Assay for Detection and Quantification of 2-Branched (1,3)-Beta-D-Glucan Producing Lactic Acid Bacteria in Cider[END_REF][START_REF] Ciezack | Evidence for Exopolysaccharide Production by Oenococcus oeni Strains Isolated from Non-Ropy Wines[END_REF].

Genes Associated with EPS Synthesis

Glycansucrases and autonomous synthase, which are enzymes capable of catalyzing the synthesis of polymers on their own, are generally encoded by isolated genes, whereas Wzy-dependant pathways are generally encoded by genes in large clusters or operons. The eps genes can be chromosomal or plasmidic. In wine and cider LAB, some of these genes seem extremely mobile, in particular the gtf gene, associated with the synthesis of the β-glucan causing wine or cider ropiness. In fact, it is carried by at least four different plasmids within the genus Pediococcus [START_REF] Werning | Pediococcus Parvulus Gtf Gene Encoding the GTF Glycosyltransferase and Its Application for Specific PCR Detection of β-D-glucan-producing Bacteria in Foods and Beverages[END_REF][START_REF] Walling | A Putative Glucan Synthase Gene Dps Detected in Exopolysaccharide-Producing Pediococcus damnosus and Oenococcus oeni Strains Isolated from Wine and Cider[END_REF][START_REF] Llamas-Arriba | Characterization of Pediococcus ethanolidurans CUPV141: A β-D-Glucanand Heteropolysaccharide-Producing Bacterium[END_REF][START_REF] Lonvaud-Funel | Antagonism between Lactic Acid Bacteria of Wines: Inhibition of Leuconostoc oenos by Lactobacillus plantarum and Pediococcus pentosaceus[END_REF], and it is chromosomic (but inserted in a phage remnant or even in a prophage) in O. oeni [START_REF] Dols-Lafargue | Characterization of Gtf, a Glucosyltransferase Gene in the Genomes of Pediococcus parvulus and Oenococcus oeni, Two Bacterial Species Commonly Found in Wine[END_REF][START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF]. Despite the high number of genetic locations, the gene is more than 95% conserved between the bacterial species capable of producing the ropy β-glucan: Lb. suebicus, Lb. diolivorans, P. parvulus, P. damnosus, Lb. collinoides and O. oeni [START_REF] Werning | Pediococcus Parvulus Gtf Gene Encoding the GTF Glycosyltransferase and Its Application for Specific PCR Detection of β-D-glucan-producing Bacteria in Foods and Beverages[END_REF][START_REF] Dols-Lafargue | Characterization of Gtf, a Glucosyltransferase Gene in the Genomes of Pediococcus parvulus and Oenococcus oeni, Two Bacterial Species Commonly Found in Wine[END_REF][START_REF] Walling | A Putative Glucan Synthase Gene Dps Detected in Exopolysaccharide-Producing Pediococcus damnosus and Oenococcus oeni Strains Isolated from Wine and Cider[END_REF][START_REF] Garai-Ibabe | Naturally Occurring 2-Substituted (1,3)-Beta-D-Glucan Producing Lactobacillus suebicus and Pediococcus parvulus Strains with Potential Utility in the Production of Functional Foods[END_REF][START_REF] Dueñas-Chasco | Structural Analysis of the Exopolysaccharides Produced by Lactobacillus spp. G-77[END_REF][START_REF] Ibarburu | Production and Partial Characterization of Exopolysaccharides Produced by Two Lactobacillus suebicus Strains Isolated from Cider[END_REF][START_REF] Dueñas-Chasco | Structural Analysis of the Exopolysaccharide Produced by Pediococcus damnosus 2.6[END_REF][START_REF] Puertas | Disclosing Diversity of Exopolysaccharide-Producing Lactobacilli from Spanish Natural Ciders[END_REF]. This suggests either a recent transfer between the bacteria or a high-level conserved amino acid sequence requirement for maintaining the activity.

Dextransucrase genes are generally chromosomic and highly conserved in O. oeni, even though the main habitat of the species, wine, does not contain any sucrose. Truncated forms are sometimes found but most of the time the dsrO gene is not truncated [START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF]. This nearly ubiquitous presence of a very large dextransucrase gene (>3000 bp) suggests that dextransucrase activity is important for the survival of the species, but this may be in a context other than wine. Other Oenococcus species also display dextransucrase genes, except in O. kitaharae these have not yet been characterized [START_REF] Vuillemin | A Dextran with Unique Rheological Properties Produced by the Dextransucrase from Oenococcus kitaharae DSM 17330[END_REF].

In LAB, the genes encoding the enzymes involved in the Wzy-dependent pathway are organized in clusters or operons, in which regulatory genes or genes for the synthesis of precursors are often found [START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF][START_REF] Pluvinet | Polymorphism of Eps Loci in Streptococcus thermophilus: Sequence Replacement by Putative Horizontal Transfer in S. thermophilus IP6757[END_REF][START_REF] Bentley | Genetic Analysis of the Capsular Biosynthetic Locus from All 90 Pneumococcal Serotypes[END_REF]. These clusters still exhibit a high density of coding zones and are chromosomic in the fermented-drink LAB strains studied to date. In O. oeni the eps gene cluster organization is often unidirectional (Figure 2). The 5 region is the most conserved one and it displays regulatory genes, the priming glycosyltransferase gene, then the genes encoding the other glycosyltransferases, together with genes involved in precursor biosynthesis, followed by wzy and wzx. Dimopoulou et al. found 14 distinct complete eps clusters out of 50 O. oeni genomes studied [START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF]. Recombination events are certainly at the origin of the final diversity observed. These recombination events may lean on the conserved 5 region of the eps gene cluster and on the 3 end, on the gene recP (Figure 2). The size of the chromosome region concerned in the recombination process can be as high as 50 kb. Such a diversity of eps gene clusters is often described as a component of adaptation to host defense mechanisms in pathogenic bacteria [START_REF] Milkman | Molecular Evolution of the Escherichia coli Chromosome. VI. Two Regions of High Effective Recombination[END_REF][START_REF] Cieslewicz | Structural and Genetic Diversity of Group B Streptococcus Capsular Polysaccharides[END_REF][START_REF] Mavroidi | Genetic Relatedness of the Streptococcus pneumoniae Capsular Biosynthetic Loci[END_REF]. Actually, more than 88 wzy-dependent gene clusters, causing as many serotypes, have been described in S. pneumoniae [START_REF] Bentley | Genetic Analysis of the Capsular Biosynthetic Locus from All 90 Pneumococcal Serotypes[END_REF]. This eps gene cluster is present in more than 85% of the O. oeni strains studied and it is associated with capsular polysaccharide synthesis. Only the last one presented in the bottom of this figure encodes an inactive pathway (no CPS produced; the gene transcription was not examined). The cluster is highly variable, especially in its 3′ end, whereas the 5′ end sequence is more conserved. The genes wzx and wzy are the most divergent ones and appear to be specific to each complete cluster. Several recombination events upstream and downstream of the eps locus may have led to the diversity observed. These events To sum up, in the O. oeni chromosome, regardless of the strain considered, there is at least one gene dedicated to EPS synthesis which encodes a functional pathway, and very often several genes encode several active pathways [START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF]. However, the selective advantage of such a high diversity of eps genes and clusters for O. oeni, a non-pathogenic bacterial species, remains unclear. This point will be further discussed in the following paragraphs.

This eps gene cluster is present in more than 85% of the O. oeni strains studied and it is associated with capsular polysaccharide synthesis. Only the last one presented in the bottom of this figure encodes an inactive pathway (no CPS produced; the gene transcription was not examined). The cluster is highly variable, especially in its 3 end, whereas the 5 end sequence is more conserved. The genes wzx and wzy are the most divergent ones and appear to be specific to each complete cluster. Several recombination events upstream and downstream of the eps locus may have led to the diversity observed. These events may lean on recP or the cluster of genes inserted between recP and the 3 end of the eps gene cluster, or even on the eps gene cluster itself. In parallel, recombination events may have occurred upstream of eps gene cluster 2, between the genes of amiO and wze or wzd.

What Could Be the Consequences for the Winemaker or the Wine Lover?

The exact role of polysaccharides in bacterial physiology is not perfectly understood. Nevertheless, the fact that the majority of the wine microorganisms have the ability to produce several EPS structures independently of the genus, species or strain could indicate the importance of these polymers for the survival of the individuals or for maintaining the species. The production of fermented beverages from fruits is a seasonal activity. Microorganisms must therefore survive not only during the beverage production process but also, once the beverages are produced, in cellars or factories and/or in orchards and vineyards for almost a year. LAB may therefore be able to survive in the gastrointestinal tract of insects and birds that gravitate to production areas and/or on production equipment or on trees, plants and soils, which would promote (1) their survival between seasons and ( 2) their natural spread or their human-driven spread (wine trade, exchange of winemaking materials, Figure 3). gene cluster, or even on the eps gene cluster itself. In parallel, recombination events may have occurred upstream of eps gene cluster 2, between the genes of amiO and wze or wzd.

What Could Be the Consequences for the Winemaker or the Wine Lover?

The exact role of polysaccharides in bacterial physiology is not perfectly understood. Nevertheless, the fact that the majority of the wine microorganisms have the ability to produce several EPS structures independently of the genus, species or strain could indicate the importance of these polymers for the survival of the individuals or for maintaining the species. The production of fermented beverages from fruits is a seasonal activity. Microorganisms must therefore survive not only during the beverage production process but also, once the beverages are produced, in cellars or factories and/or in orchards and vineyards for almost a year. LAB may therefore be able to survive in the gastrointestinal tract of insects and birds that gravitate to production areas and/or on production equipment or on trees, plants and soils, which would promote (1) their survival between seasons and ( 2) their natural spread or their human-driven spread (wine trade, exchange of winemaking materials, Figure 3).

EPSs for Bacteria Survival in Harsh Winemaking Conditions

The presence of an extracellular polymeric matrix could be implicated in the protection of the cell in various situations including wine or cider elaboration [START_REF] Kubota | Altered Gene Expression Levels of Matrix Metalloproteinases and Their Inhibitors in Periodontitis-Affected Gingival Tissue[END_REF][START_REF] Ramírez-Castrillón | GTG)5 MSP-PCR Fingerprinting as a Technique for Discrimination of Wine Associated Yeasts?[END_REF]. Indeed, many bacteria enhance EPS synthesis as a response to carbon dioxide release by yeasts during AF [START_REF] Cotter | Surviving the Acid Test: Responses of Gram-Positive Bacteria to Low PH[END_REF][START_REF] Chowdhury | Stress Response in Pathogenic Bacteria[END_REF] or in the case of various stresses such as low pH or high ethanol or phenolic compound concentrations [START_REF] Walling | A Putative Glucan Synthase Gene Dps Detected in Exopolysaccharide-Producing Pediococcus damnosus and Oenococcus oeni Strains Isolated from Wine and Cider[END_REF][START_REF] Dimopoulou | Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance[END_REF]. For example, β-glucan seems to increase the

EPSs for Bacteria Survival in Harsh Winemaking Conditions

The presence of an extracellular polymeric matrix could be implicated in the protection of the cell in various situations including wine or cider elaboration [START_REF] Kubota | Altered Gene Expression Levels of Matrix Metalloproteinases and Their Inhibitors in Periodontitis-Affected Gingival Tissue[END_REF][START_REF] Ramírez-Castrillón | GTG)5 MSP-PCR Fingerprinting as a Technique for Discrimination of Wine Associated Yeasts?[END_REF]. Indeed, many bacteria enhance EPS synthesis as a response to carbon dioxide release by yeasts during AF [START_REF] Cotter | Surviving the Acid Test: Responses of Gram-Positive Bacteria to Low PH[END_REF][START_REF] Chowdhury | Stress Response in Pathogenic Bacteria[END_REF] or in the case of various stresses such as low pH or high ethanol or phenolic compound concentrations [START_REF] Walling | A Putative Glucan Synthase Gene Dps Detected in Exopolysaccharide-Producing Pediococcus damnosus and Oenococcus oeni Strains Isolated from Wine and Cider[END_REF][START_REF] Dimopoulou | Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance[END_REF]. For example, β-glucan seems to increase the resistance of the producing bacteria to acidic pH [START_REF] Dols-Lafargue | Characterization of Gtf, a Glucosyltransferase Gene in the Genomes of Pediococcus parvulus and Oenococcus oeni, Two Bacterial Species Commonly Found in Wine[END_REF][START_REF] Jakob | β-Glucan Formation Is a Selective Advantage for Beer-Spoiling Levilactobacillus brevis TMW 1.2112 during Planktonic Growth[END_REF][START_REF] Fernández De Palencia | Probiotic Properties of the 2-Substituted (1,3)-β-d-Glucan-Producing Bacterium Pediococcus parvulus 2.6[END_REF]. In France, the prevalence of ropy O. oeni strains is particularly high in regions producing naturally more acidic wines (Bourgogne or Champagne) [START_REF] Dols-Lafargue | Characterization of Gtf, a Glucosyltransferase Gene in the Genomes of Pediococcus parvulus and Oenococcus oeni, Two Bacterial Species Commonly Found in Wine[END_REF][START_REF] Renouf | Interactions between Brettanomyces bruxellensis and Other Yeast Species during the Initial Stages of Winemaking[END_REF][START_REF] El Khoury | Biogeography of Oenococcus oeni Reveals Distinctive but Nonspecific Populations in Wine-Producing Regions[END_REF]. The increase in viscosity induced by β-glucan may slow down the diffusion of abiotic compounds, such as phenolic compounds, sulfites, and ethanol, and protect the bacteria. By constituting a protective layer, β-glucan is also involved in the resistance of LAB to lysozyme treatment [START_REF] Coulon | Lysozyme Resistance of the Ropy Strain Pediococcus parvulus IOEB 8801 Is Correlated with Beta-Glucan Accumulation around the Cell[END_REF]. LAB CPSs and dextrans can also offer protection during the freeze-drying step in malolactic starter production [START_REF] Dimopoulou | Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance[END_REF][START_REF] Caggianiello | Exopolysaccharides Produced by Lactic Acid Bacteria: From Health-Promoting Benefits to Stress Tolerance Mechanisms[END_REF]. On the other hand, the phage resistance associated with EPSs is controversial and EPSs could, depending on the case, serve as a target for attachment and recognition or, on the contrary, as a masking element for these targets [START_REF] Deveau | Effect of Exopolysaccharides on Phage-Host Interactions in Lactococcus lactis[END_REF][START_REF] Mccabe | The Targeted Recognition of Lactococcus lactis Phages to Their Polysaccharide Receptors[END_REF].

EPSs, Biotic Interactions and Species Survival

Beta-glucan was shown to promote the adhesion of bacteria to intestinal cells and to play a role in cross feeding in the intestinal tract, and this may promote microbial survival in the intestinal tracts of various animal and insects, and hence in bacterial dissemination and the colonization of new ecological niches [START_REF] Fernández De Palencia | Probiotic Properties of the 2-Substituted (1,3)-β-d-Glucan-Producing Bacterium Pediococcus parvulus 2.6[END_REF][START_REF] Russo | Beta-Glucans Improve Growth, Viability and Colonization of Probiotic Microorganisms[END_REF][START_REF] Stack | Association of Beta-Glucan Endogenous Production with Increased Stress Tolerance of Intestinal Lactobacilli[END_REF][START_REF] Arena | Beta-Glucans and Probiotics[END_REF]. In the same way, the high diversity of capsular heteropolysaccharides produced by O. oeni may be involved in bacterial survival in a biotic context, through interactions or adhesion to epithelial receptors in the gastrointestinal tracts of insects or animals. The capsule may also constitute a protection against host defense mechanisms and promote host colonization, as shown for many pathogenic Gram-positive and Gram-negative bacteria [START_REF] Milkman | Molecular Evolution of the Escherichia coli Chromosome. VI. Two Regions of High Effective Recombination[END_REF][START_REF] Llull | Genetic Bases and Medical Relevance of Capsular Polysaccharide Biosynthesis in Pathogenic Streptococci[END_REF][START_REF] Cooper | Biochemical, and Structural Analyses of Bacterial Surface Polysaccharides[END_REF]. Nevertheless, further work will be necessary to identify the hosts in which wine LAB are accommodated between two vintages, if any.

O. oeni glucansucrase genes are highly conserved, despite the fact that the encoded enzymes use a substrate, sucrose, that is rarely found in wine but is present in grapes. The production of dextran on grapes may assist the attachment of the bacteria to fruits or to vine woods. It may also promote their survival in the soil, as suggested a long time ago for Lc. mesenteroides [START_REF] Brooker | Ultrastructural Surface Changes Associated with Dextran Synthesis by Leuconostoc mesenteroides[END_REF]. Furthermore, EPS-producing LAB adheres better to S. cerevisiae cells, which efficiently consume lactic acid and consequently decrease the acidity of the medium, subsequently promoting bacterial growth [START_REF] Nguyen | Exopolysaccharide Production by Lactic Acid Bacteria: The Manipulation of Environmental Stresses for Industrial Applications[END_REF][START_REF] Tada | Fed-Batch Coculture of Lactobacillus kefiranofaciens with Saccharomyces cerevisiae for Effective Production of Kefiran[END_REF].

EPSs and Bacterial Colonization of the Production Cellar

EPSs may help the wine microorganism to invade during all the winemaking steps through promoting adhesion onto abiotic surfaces, i.e., stainless steel, plastic and glass surfaces, as well as the wood of oak barrels. Undoubtedly, the presence of EPSs modulates the physicochemical characteristics of the LAB cell surface [START_REF] Polak-Berecka | The Effect of Cell Surface Components on Adhesion Ability of Lactobacillus rhamnosus[END_REF]. In some cases, EPSs were shown to be implicated in the adhesion process [START_REF] Wang | The Interaction between Saccharomyces cerevisiae and Non-Saccharomyces Yeast during Alcoholic Fermentation Is Species and Strain Specific[END_REF][START_REF] Da Silva | Safety and Probiotic Functionality of Isolated Goat Milk Lactic Acid Bacteria[END_REF]. Adhered cells can, in the second phase, evolve into biofilms. Biofilms are complex structures composed mainly of an extracellular matrix, genetic material and microorganisms. Depending on the case, abundant EPSs can increase or decrease the adhesion capacity and biofilm formation [START_REF] Mah | Mechanisms of Biofilm Resistance to Antimicrobial Agents[END_REF].

The same could be assumed for the EPSs produced by wine LAB, i.e., dextran, βglucan and heteropolysaccharides. EPSs may be important for O. oeni biofilm settlement as the genes implicated in heteropolysaccharide and homopolysaccharide production are overexpressed under stressed biofilm conditions [START_REF] Bastard | Effect of Biofilm Formation by Oenococcus oeni on Malolactic Fermentation and the Release of Aromatic Compounds in Wine[END_REF]. More recently, it has been demonstrated through FT-IR spectra analysis that the metabolic fingerprint of the attached cells was significantly different from that of the planktonic ones, and the spectral zone associated with polysaccharides was discriminative. This suggests the importance of EPSs in biofilm formation on stainless steel surfaces [START_REF] Dimopoulou | Assessing the Biofilm Formation Capacity of the Wine Spoilage Yeast Brettanomyces bruxellensis through FTIR Spectroscopy[END_REF]. The β-glucan produced by O. oeni or recombinant L. lactis expressing gtf may be involved in the last steps of adhesion and biofilm consolidation, rather than primary adhesion [START_REF] Dols-Lafargue | Characterization of Gtf, a Glucosyltransferase Gene in the Genomes of Pediococcus parvulus and Oenococcus oeni, Two Bacterial Species Commonly Found in Wine[END_REF]. Regarding O. oeni dextran, the research carried out so far has not made it possible to determine whether it promotes or whether it prevents attachment to abiotic surfaces. This may depend on the strain and the context in which the cells develop. Nácher-Vázquez et al. [START_REF] Nácher-Vázquez | Dextran Production by Lactobacillus Sakei MN1 Coincides with Reduced Autoagglutination, Biofilm Formation and Epithelial Cell Adhesion[END_REF] compared the biofilm formation and aggregation ability of two different bacterial species, Lc. mesenteroides and Lb. sakei, both of which are great producers of dextrans. Interestingly, only Lb. sakei formed a biofilm on the tested abiotic surfaces, whereas dextran exerted an antiadhesive effect in the case of the strain of Lc. mesenteroides. Thus, the authors proposed that the role of dextran was involved in the aggregation process and biofilm consolidation, rather than in the primary adhesion process, which is rather linked to the surface proteins of the bacteria. Dextran, which is a neutral and weakly adhesive polymer, may aid in the attachment process by masking repulsion molecules. By contrast, it could prevent adhesion by masking motifs essential for primary adhesion [START_REF] Re | Autoaggregation and Hydrophobicity of 13 Strains of Bifidobacterium longum[END_REF][START_REF] Pérez | Surface Properties of Bifidobacterial Strains of Human Origin[END_REF].

The most important point is that biofilms are a strategy used to overcome stressful conditions [START_REF] Van De Guchte | Stress Responses in Lactic Acid Bacteria[END_REF]. O. oeni cells attached to stainless steel and oak wood surfaces can perform malolactic fermentation and display higher tolerance to wine stress than planktonic ones [START_REF] Bastard | Effect of Biofilm Formation by Oenococcus oeni on Malolactic Fermentation and the Release of Aromatic Compounds in Wine[END_REF]. In the same way, the biofilm cells of Lb. plantarum expressed higher resistance to acetic acid and low pH values than the planktonic cells [START_REF] Kubota | Altered Gene Expression Levels of Matrix Metalloproteinases and Their Inhibitors in Periodontitis-Affected Gingival Tissue[END_REF].

Beverage Spoilage and Possible Treatments

Some of the EPSs from wine bacteria are implicated in a specific form of wine spoilage, the disease known as "ropiness". The polymer produced increases the wine's viscosity and sometimes induces an oily sensation [START_REF] Lonvaud-Funel | Inhibition of Malolactic Fermentation of Wines by Products of Yeast Metabolism[END_REF]. This ropy character may be perceived with the naked eye and can occur in stainless-steel tanks, in barrels and even in the bottle. If the alteration remains at bottle opening, the consumer who discovers the defect sometimes definitively turns away from the wine from the area concerned and this may provoke great losses for the wine industry (Figure 3).

For the moment, the only molecule clearly implicated in ropiness is the β-glucan described in the previous paragraph, but other molecules could be implicated in other bacteria [START_REF] Walling | A Putative Glucan Synthase Gene Dps Detected in Exopolysaccharide-Producing Pediococcus damnosus and Oenococcus oeni Strains Isolated from Wine and Cider[END_REF]. Interestingly, wine LAB displaying the ropy phenotype in laboratory media may be isolated from non-ropy wines [START_REF] Ciezack | Evidence for Exopolysaccharide Production by Oenococcus oeni Strains Isolated from Non-Ropy Wines[END_REF]. This observation indicates that the effect of EPSs on wine depends on many factors and the presence of a "ropy" strain is not sufficient to provoke wine spoilage. In the first place, spoilage is not correlated with EPS quantity but mainly with EPS structure, as just 12 mg/L of β-glucan may provoke ropiness, whereas some g/L of dextran do not induce such a viscosity change. In addition, the role of the wine or cider matrix is essential as interactions of the polymer with the matrix may enhance the viscosity. Interestingly the Lactobacillus and Pediococcus strains producing β-glucan are also implicated in beer and cider spoilage [START_REF] Duenas | Heterofermentative Lactobacilli Causing Ropiness in Basque Country Ciders[END_REF][START_REF] Fraunhofer | Characterization of β-Glucan Formation by Lactobacillus brevis TMW 1.2112 Isolated from Slimy Spoiled Beer[END_REF][START_REF] Dueñas-Chasco | Structural Analysis of the Exopolysaccharide Produced by Pediococcus damnosus 2.6[END_REF][START_REF] Bockwoldt | Persistence and β-Glucan Formation of Beer-Spoiling Lactic Acid Bacteria in Wheat and Rye Sourdoughs[END_REF]. P. parvulus is the species that is most often involved in the ropiness of wine and ciders, as it develops after fermentation processes in the late stages of winemaking, even after bottling. O. oeni ropy strains are quite frequent but, as they develop in the early stages of winemaking, when wine supervision is easier, they are less dangerous for the final quality of the wine (Table 1). Indeed, the vigorous stirring of the wine is enough to make the ropy character disappear and a close supervision of the microbial flora during the following stages of winemaking may guarantee that the viscosity remains low. The well-developed qPCR protocols for the detection of ropy strains, through the detection of gtf genes, constitute an interesting tool for wine supervision, early reaction and curative or preventive treatments [START_REF] Werning | Pediococcus Parvulus Gtf Gene Encoding the GTF Glycosyltransferase and Its Application for Specific PCR Detection of β-D-glucan-producing Bacteria in Foods and Beverages[END_REF][START_REF] Ibarburu | A Real-Time PCR Assay for Detection and Quantification of 2-Branched (1,3)-Beta-D-Glucan Producing Lactic Acid Bacteria in Cider[END_REF][START_REF] Delaherche | Detection and Quantification of Brettanomyces bruxellensis and "ropy" Pediococcus damnosus Strains in Wine by Real-Time Polymerase Chain Reaction[END_REF]. However, LAB spoilage species could be resistant to SO 2 treatment, as well as to lysozyme [START_REF] Dols-Lafargue | Characterization of Gtf, a Glucosyltransferase Gene in the Genomes of Pediococcus parvulus and Oenococcus oeni, Two Bacterial Species Commonly Found in Wine[END_REF][START_REF] Coulon | Lysozyme Resistance of the Ropy Strain Pediococcus parvulus IOEB 8801 Is Correlated with Beta-Glucan Accumulation around the Cell[END_REF]. Nevertheless, the ropy strains could be remedied via the simultaneous enzymatic action of β-glucanase and lysozyme, as recommended by Coulon et al. [START_REF] Coulon | Lysozyme Resistance of the Ropy Strain Pediococcus parvulus IOEB 8801 Is Correlated with Beta-Glucan Accumulation around the Cell[END_REF]. A combined treatment of low concentrations of enterocin AS-48, along with high-intensity pulsed-electric field (HIPEF) treatment, was also shown to inactivate exopolysaccharide-producing LAB strains in apple juice, but these methods need to be investigated more and are not currently allowed [99-101].

Bacterial EPSs and Wine Sensorial Properties

For years, bacterial polysaccharides were considered to have no impact on wine, except for β-glucan accumulation and ropiness (see previous paragraph). However, bacterial polysaccharides accumulate in wine during MLF and later even when no viscosity increase is noticeable [START_REF] Dimopoulou | Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes[END_REF][START_REF] Dols-Lafargue | Changes in Red Wine Soluble Polysaccharide Composition Induced by Malolactic Fermentation[END_REF]. These biopolymers could directly or indirectly have an impact on the wine's sensory profile, as previously shown for yeast mannoproteins (MPs) [102-104]. In fact, MP enrichment of wines have been proven to modify wine astringency and sucrosity [105,106]. MPs have also been mentioned to play an important role in tartrate salt crystallization, although the MP concentration needs to be adjusted depending on the wine considered [107-109]. Additionally, recent studies have shown that MPs can interact with all anthocyanin families and modulate the bioaccessibility of polyphenolic compounds during digestion in the gastrointestinal tract [110]. In addition, pectin, a heteropolysaccharide mainly composed of partially methylated galacturonic acid, can inhibit proteins and tannin precipitation [111]. These interactions also depend on the hydrophobicity and molecular weight of the phenolic compounds [112]. In the same way, the EPSs liberated by LAB could interfere with tannins and salivary proteins and thus reduce the sense of astringency. This could be the case with O. oeni dextrans. Indeed, small commercial dextrans (>5000 da) were shown to increase the mouthfeel sensation and to decrease wine astringency (Dimopoulou et al., unpublished).

Even if polysaccharides are not volatile molecules, they may indirectly contribute to aroma perception. Olfactory perception is a complicated phenomenon in which molecular interactions play a crucial role. Dimopoulou et al. [START_REF] Dimopoulou | Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance[END_REF] suggested for the first time that the bacterial EPSs produced in wine could contribute to aromatic complexity and fruity aromas. These may affect the release of esters and alcohols from the liquid phase, affecting their concentration in the head space [113]. Indeed, the wine matrix plays an essential role in the in vivo aroma release [114]. When dextrans from Lc. mesenteroides were added to wine model medium, isoamyl acetate and ethyl hexanoate esters were salted out in the vapor phase as the presence of polysaccharides decreased their solubility [115]. Bastard et al. [START_REF] Bastard | Effect of Biofilm Formation by Oenococcus oeni on Malolactic Fermentation and the Release of Aromatic Compounds in Wine[END_REF] also suggest an interaction between the biofilm formed on the oak surface and wine, contributing to the expression of trans-whisky lactone complexity.

Conclusions

The ability of LAB to produce EPSs in wine and other fermented beverages has been known for a long time, but mainly in the context of ropy β-glucan accumulation and wine spoilage. Great genotypic and phenotypic diversity within LAB EPS producers, especially O. oeni, has been more recently elucidated. The more the research advances, the more the complexity of EPS biosynthesis in fermented beverages is unraveled, revealing the multiple biosynthetic pathways and the associated phenotypic diversity. The effects of the EPSs produced on bacterial survival and dissemination, as well as on wine quality, are complex and trigger many fascinating research perspectives for the future. An important question is whether the EPSs produced by LAB of fermented beverages ultimately have a positive or negative effect. No one-word answer to this question yet exists, and this will certainly require many years of work.

Figure 1 .

 1 Figure 1. Overview of the EPS biosynthetic pathways active in O. oeni and other LAB from fermented beverages. From top to bottom and right to left: (A) Dextran (α-glucan) and levan (β-fructan) synthesis from sucrose by external glycansucrases such as dextransucrase or levansucrase. (B) β-glucan synthesis involving UDP-glucose and the membrane spanning glycosyltransferase Gtf. (C) Heteropolysaccharide synthesis: the repeating unit is built via the involvement of priming glycosyltransferase and several other glycosyltransferases, a lipid carrier and activated substrates (UDP-oses). Then, the complete repeating unit is externalized by a flippase and is polymerized outside the cell before the release of the polymer. Gtase, glycosyltransferase. (i) Transglycosidases, which specifically use sucrose as a substrate (or glycansucrases), are classified into the CAZy GH-13, 68 and 70 families (www.cazy.org) [56]. They catalyze the synthesis of homopolysaccharides made up of glucose or fructose, according to the following simplified reactions: n sucrose  (fructose)n + n glucose (fructansucrase) n sucrose  (glucose)n + n fructose (glucansucrase)

Figure 1 .

 1 Figure 1. Overview of the EPS biosynthetic pathways active in O. oeni and other LAB from fermented beverages. From top to bottom and right to left: (A) Dextran (α-glucan) and levan (β-fructan) synthesis from sucrose by external glycansucrases such as dextransucrase or levansucrase. (B) β-glucan synthesis involving UDP-glucose and the membrane spanning glycosyltransferase Gtf. (C) Heteropolysaccharide synthesis: the repeating unit is built via the involvement of priming glycosyltransferase and several other glycosyltransferases, a lipid carrier and activated substrates (UDP-oses). Then, the complete repeating unit is externalized by a flippase and is polymerized outside the cell before the release of the polymer. Gtase, glycosyltransferase.

Foods 2021 , 17 Figure 2 .

 2021172 Figure 2. Consensus organization of eps gene cluster 2 in O. oeni (adapted from Dimopoulou et al. [35]).

Figure 2 .

 2 Figure 2. Consensus organization of eps gene cluster 2 in O. oeni (adapted from Dimopoulou et al. [35]).

Foods 2021 ,

 2021 10, x FOR PEER REVIEW 9 of 17

Figure 3 .

 3 Figure 3. Possible incidence of bacterial polysaccharides in the specific context of winemaking.

Figure 3 .

 3 Figure 3. Possible incidence of bacterial polysaccharides in the specific context of winemaking.

Table 1 .

 1 LAB EPSs isolated from fermented beverages.

	EPS Type	EPS Structure	Species	Niche	Implicated Genes	Consequences/Role of EPS	Reference
		β-glucan	Oenococcus oeni	wine, cider	gtf	ropy character, stress resistance	[17-19]
		β-glucan	Pediococcus damnosus	cider	gtf	ropy character	[17,20-23]
		β-glucan	Pediococcus parvulus	cider, wine	gtf	ropy character, stress resistance	[18,23]
		β-glucan	Pediococcus ethanolidurans	cider	-	ropy character	[24]
	homopolysaccharides	β-glucan	Pediococcus claussenii	beer	gtf	ropy character	[25,26]
		β-glucan	Lactobacillus brevis	beer	gtf2	ropy character, biofilm formation ethanol tolerance,	[26,27]
		β-glucan	Lactobacillus diolivorans	cider	gtf	-	[17]
		β-glucan	Lactobacillus suebicus	cider	gtf	ropy character	[28,29]

Table 1 .

 1 Cont.

	EPS Type	EPS Structure	Species	Niche	Implicated Genes	Consequences/Role of EPS	Reference
		β-glucan	Lactobacillus spp.	cider	-	ropy character	[30]
			Leuconostoc
		a-glucan	pseudomesen-teroides,	beer	dsr	increased viscosity	[31]
			Weissella confusa
		dextran	Leuconostoc pseudomesen-teroides	homemade wine	-	-	[32]
		glucan and fructan	Leuconostoc mesenteroides	grape must and wine	Glucosyltransferase gene	more or less mucoid strains	[33]
						lyoprotective ability
		dextran and levan	Oenococcus oeni	wine	dsrO and levO	to freeze-drying	[15,34,35]
						process
						aromatic
		glucose, galactose, rhamnose	Oenococcus oeni	wine	eps cluster	complexity, biofilm lyoprotective ability formation, capsule,	[15,34-36]
						to freeze-drying
	heteropolysaccharides	glucose, galactose, galactofuranose	Lactobacillus suebicus	cider	gtf	ropy character	[28,29]
		glucose, galactose,
		N-acetyl-glucosamine,	Lactobacillus suebicus	cider	eps cluster	ropy character	[37]
		phosphate
		glucose, galactose, glucosamine	Pediococcus ethanolidurans	cider	-	ropy character	[24]

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Conflicts of Interest:

The authors declare no conflict of interest.


Kévin Chighine

Estelle Léonce

Céline Boutin

Hervé Desvaux

Patrick Berthault

Xe Nmr-

29 Xe NMR-based biosensing on a benchtop spectrometer


PhD Valentin Flaudias
email: vflaudias@chu-clermontferrand.fr

Frederique Teisseidre

Ingrid De Chazeron

Maryline Chalmeton

Celian Bertin

Marie Izaute

Nadia Chakroun-Baggioni

Bruno Pereira

Georges Brousse

Pierre Maurage

A multi-dimensional evaluation of craving and impulsivity among people admitted for alcohol-related problems in emergency department

Keywords: Alcohol use disorders, craving, impulsivity, emergency department

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Craving and impulsivity are considered as key psychological dimensions involved in the emergence and development of alcohol-use disorders. Craving is an intense subjective desire and urge towards alcohol, promoting compulsive consumption [START_REF] Tiffany | A cognitive model of drug urges and drug-use behavior: role of automatic and nonautomatic processes[END_REF].

Impulsivity covers a wide-range of poorly planned and excessive behaviors often leading to negative consequences [START_REF] Moeller | Psychiatric aspects of impulsivity[END_REF], among which uncontrolled substance consumption [START_REF] Jentsch | Dissecting impulsivity and its relationships to drug addictions[END_REF]. These two factors, which have also been suggested as predictors of relapse following treatment [START_REF] Joos | The relationship between impulsivity and craving in alcohol dependent patients[END_REF], are conceptualized as multidimensional: First, [START_REF] Anton | What is craving?: Models and implications for treatment[END_REF] distinguished obsessive (i.e. intrusive thoughts or mental images) and compulsive (i.e. ritualized drinking behaviors) dimensions of craving, classically measured using the Obsessive Compulsive Drinking Scale [OCDS [START_REF] Anton | The Obsessive Compulsive Drinking Scale: a self-rated instrument for the quantification of thoughts about alcohol and drinking behavior[END_REF]].

Second, two main multidimensional impulsivity models are currently dominant in addictive states, namely (1) the UPPS [START_REF] Whiteside | The Five Factor Model and impulsivity: using a structural model of personality to understand impulsivity[END_REF], distinguishing four impulsivity factors: urgency (the tendency to produce rash behavioral responses when facing intense positive or negative emotional states), lack of perseverance (the difficulty to maintain one's attentional focus on a task which might be hard, long or boring), lack of premeditation (the difficulty to plan one's behaviors or to think about the consequences of an action before performing it), sensation seeking (the propensity to look for thrilling and unconventional life experiences, despite their riskiness); and (2) the Barratt Impulsiveness Scale (BIS, [START_REF] Patton | Factor structure of the Barratt impulsiveness scale[END_REF], considering three impulsivity factors: Attentional impulsivity (the inability to focus attentional resources on a task), Motor impulsivity (the production of behaviors without preliminary thinking), and non-planning impulsivity (the lack of consideration of future consequences of one's behavior).

While these two concepts have been widely explored in alcohol-use disorders, several questions remain, as craving and impulsivity have: (1) been mostly explored separately, their interactions not being fully understood; (2) centrally been studied in patients presenting severe alcohol-use disorders or among healthy social drinkers. Their respective roles on other types of population presenting alcohol-related disorders remain to be determined. In particular, patients admitted on emergency department for alcohol misuse constitute a population of interest, as they have specific characteristics: while they are most often seeking help in link with intense acute consumption [START_REF] Te Wildt | Alcohol related conditions represent a major psychiatric problem in emergency departments[END_REF], 75% of them will later be admitted in detoxification therapy for chronic alcohol-related disorders [START_REF] Djenati | Prévalence de l'abus d'alcool chez des patients admis aux Urgences[END_REF], highlighting the particularly stage of alcohol-use disorders they are confronted with (i.e. a problematic use of alcohol, but not yet intense enough to lead them towards detoxification therapy). However, craving and impulsivity have not yet been measured in this population.

We propose an integrative and joint evaluation of craving and impulsivity among patients admitted in emergency department, using a multimodal perspective including multiple impulsivity (UPPS, BIS) and craving [OCDS, craving visual analogue scale (VAS)] evaluations. We hypothesized that, beyond high impulsivity and craving, a strong link will be observed between the severity of alcohol-use disorders on the one hand, and compulsive craving, sensation seeking and non-planning impulsiveness on the other hand, these dimensions being the most related to the unplanned and dangerous behaviors observed in this population [START_REF] Cherpitel | Alcohol and Injury in the United States General Population: A Risk Function Analysis from the 2005 National Alcohol Survey[END_REF][START_REF] Zerhouni | Alcohol and Violence in the Emergency Room: A Review and Perspectives from Psychological and Social Sciences[END_REF].

Methods

Participants

The study was conducted in the University Hospital "Gabriel Montpied" (Clermont-Ferrand, France) between April and May 2012, and approved by the Ethical Committee of the Hospital. The inclusion criterion was to be admitted to the emergency department for alcohol misuse. 178 patients fulfilled this criterion, but 78 were excluded because they refused to take part in the study or presented psychiatric/neurological symptoms, and two were excluded because of their age (higher than 90 years old). The final sample thus comprised 98 patients.

Inclusion was conducted during the admission day, after the blood alcohol level was back to zero, when the mental state of patients allowed psychological evaluation, and when there was no severe withdrawal symptoms, as assessed by a senior medical doctor.

Clinical assessments

We used the AUDIT [START_REF] Gache | The Alcohol Use Disorders Identification Test (AUDIT) as a screening tool for excessive drinking in primary care: reliability and validity of a French version[END_REF][START_REF] Saunders | Development of the Alcohol Use Disorders Identification Test (AUDIT): WHO Collaborative Project on Early Detection of Persons with Harmful Alcohol Consumption--II[END_REF] to assess the severity of alcohol-use disorders, the UPPS [START_REF] Whiteside | The Five Factor Model and impulsivity: using a structural model of personality to understand impulsivity[END_REF][START_REF] Van Der Linden | A French adaptation of the UPPS Impulsive Behavior Scale -Confirmatory factor analysis in a sample of undergraduate students[END_REF] and the Barratt Impulsiveness Scale [BIS-11, (Patton et al., 1995)] to assess impulsivity, and the OCDS [START_REF] Anton | The Obsessive Compulsive Drinking Scale: a self-rated instrument for the quantification of thoughts about alcohol and drinking behavior[END_REF][START_REF] Chignon | Selfassessment questionnaire of alcoholic craving (ECCA Questionnaire: Behavior and Cognition in Relation to Alcohol: French translation and validation of the Obsessive-Compulsive Drinking Scale[END_REF]) and a single-item VAS to assess craving. The VAS instruction was "Indicate, on the following scale, the usual intensity of your desire for alcohol when it occurs, by putting a mark on the line going from "very weak desire" to "irresistible desire". The more your mark is on the right, the more intense is your desire". The VAS was thus a continuous measure as the mark could be placed at any level, and was then recoded as a 0 to 10 score (M = 5.01; SD = 3.05, 6 patients reporting a score of 0, 8 patients reporting a score of 10; one missing data;). All these assessments were self-reported questionnaires, and Cronbach's α are indicated in Table 1.

Procedure

Before starting the experiment, each patient was informed about the aims of the study.

Patients were free to participate and did not receive any compensation. The study was conducted individually, starting with the self-administered questionnaires followed by the AUDIT interview, carried out by two trained psychologists. The total procedure lasted one hour on average.

Results

Patients were between 18 and 65 years old (Mage=42.26; SD=12.89), and the sample consisted of 37 women (Mage=42.7; SD=14.85) and 61 men (Mag=41.98; SD=11.67). The mean AUDIT score was 23.66 (SD=8.29): A low dependence risk (score lower than 8) was present in 6% of the patients, 9% were hazardous drinkers (score between 9 and 15), 14% were harmful drinkers (score between 16 and 19), and 69 % presented a high dependence risk (score higher than 19) [START_REF] Babor | AUDIT: the alcohol use disorders identification test: guidelines for use in primary health care[END_REF]).

Spearman's correlations analyses showed that all sub-dimensions of craving were correlated with the severity of alcohol-use disorders. All impulsivity sub-dimensions were correlated with the severity of alcohol-use disorders, except the lack of premeditation (p=0.781) and the lack of perseverance (p=0.056).

Linear multiple hierarchical regression with all scores (normalized through log transformation) was conducted. The first step explored AUDIT scores as dependent variable and age and sex as predictive factors, and showed no significant effect. The second step added impulsivity (BIS/UPPS subscales) and craving (VAS/OCDS sub-scores) as predictive factors, showing that the only significant predictive factors for the AUDIT (R² =0.701, F (12,85) =16,627, p<0.001) were the OCDS compulsive subscale (p<0.001), the craving VAS (p=0.042) and the urgency dimension of the UPPS (Table 1).

<Insert Table 1 here > Receiver operating characteristic (ROC) curve analysis indicated which VAS score best predicted harmful consumption measured by the AUDIT (i.e., score higher than 16). A craving threshold score of three (on a VAS ranging from 0 to 10) optimized sensitivity (0.817) and specificity (0.800). The area under the curve was 0.876 and the asymptotic significance was lower than 9.001 [95% CI =0.790, 0.961].

Discussion

The primary aim of this study was to explore the role played by two major factors related to the severity of alcohol-use disorders (craving and impulsivity) in a population of patients admitted to emergency department for alcohol-related problems. Our results first confirmed that craving is an important factor linked with the severity of alcohol-use disorders in this population, particularly for its compulsive dimension which is more strongly involved than the obsessive one (more centrally involved in relapse and disease persistence, [START_REF] Anton | The Obsessive Compulsive Drinking Scale: a self-rated instrument for the quantification of thoughts about alcohol and drinking behavior[END_REF].

We also observed an important predictive effect of subjective craving, as assessed by the VAS, on alcohol-related disorder. This result complements previous ones, as this scale reflects the global subjective feeling regarding craving. Indeed, craving can be an unconscious process [START_REF] Sayette | The Role of Craving in Substance Use Disorders: Theoretical and Methodological Issues[END_REF] difficult to target in short medical care perspective, but the VAS offers a rapid evaluation of self-reported conscious craving. The ROC analysis moreover showed that a score above three on this scale, indicating moderate craving, constitutes an interesting threshold to detect patients presenting harmful drinking. Despite this potential clinical usefulness of the VAS scale to perform a quick-scan of patients regarding their craving intensity and alcohol-related disorders, it should be underlined that: (1) as the VAS is based on a one-item scale, it might lead to erroneous information and clinical decision when this item is misinterpreted by the patients, and it obviously do not replace an in-depth evaluation of the multidimensional concept of craving; (2) the threshold determined here could be strongly influenced by the population included in the study, and might thus not be generalizable to other populations (e.g., patients presenting more severe alcohol-use disorders) or contexts (e.g., other clinical settings or regions/countries).

Our results did not show any strong effect of impulsivity factors (UPPS, BIS) on alcohol-use severity when craving was included in the regression analysis. The only significant effect concerned the urgency dimension of the UPPS, which is in line with previous studies [START_REF] Lannoy | Binging at the campus: motivations and impulsivity influence binge drinking profiles in university students[END_REF]. As impulsivity is strongly involved in the emergence of excessive alcohol consumption (e.g., binge drinking, [START_REF] Cyders | The Role of Personality Dispositions to Risky Behavior in Predicting First Year College Drinking[END_REF], it could centrally be a predictor of problematic use. Our study suggests that subjective craving and compulsive behaviors are more linked with alcohol-related severity than impulsivity, which highlights the urgent need to take craving evolution into account when exploring the developmental aspects of addictive states and the specificities of each population. Moreover, the relationship between craving and urgency dimensions of impulsivity need more exploration: we exclusively used self-reported questionnaires and these results should be further explored through experimental tasks. Moreover, the correlational nature of this study, as well as the limits related to the AUDIT (e.g., grouping situational and long-term alcohol-consumption items in a global score) reduce the strengths of the conclusions that can be drawn.

As a whole, this study proposed to determine how impulsivity and craving measures can predict excessive alcohol consumption in an under-explored high-risk population admitted in emergency department. Results showed the important relationship between craving (particularly its subjective and compulsive aspects) and alcohol-use severity. While these results should be replicated on larger and multicentric samples, they suggest a potential interest of using a simple VAS craving assessment in clinical settings to obtain a fast while preliminary evaluation of this key psychological component.

TableTable 1 .

 1 Multiple hierarchical regression analyses determining the predicting value of craving (measured with OCDS and VAS) and impulsivity (measured with BIS and UPPS) on alcohol-use disorder score (measured with AUDIT).

	Dependent	Independent variables	Cronbach	Beta	t	p-value	95 % IC	R²	F	p-value
	variable		α					adjusted
	Step 1 AUDIT	Age	-	-0.019	-0.187	0.852	[-0.004_0.003]	0.014	0.675	0.511
	(α =0.776)	Gender	-	-0,116	-1.141	0.257	[-0.134_0.036]
	Step 2 AUDIT	Age	-	-0.079	-1.109	0.270
		Gender	-	-0.091	-1.467	0.146
		BIS Attentional Impulsivity	0.639	0.014	0.179	0.858
		BIS Motor Impulsivity	0.602	-0.023	-0.284	0.777
		BIS Non-planning Impulsivity	0.683	0.083	0.927	0.357
		OCDS Compulsive score	0.878	0.613	5.376	0.001
		OCDS Obsessive score	0.917	-0.045	-0.413	0.681
		UPPS Urgency	0.868	0.164	2.274	0.025
		UPPS Lack of Premeditation	0.867	0.109	1.383	0.170
		UPPS Lack of Perseverance	0.825	-0.092	-1.13	0.262
		UPPS Sensation Seeking	0.810	0.043	0.596	0.553
		Visual Analogue Scale of Craving	-	0.205	2.532	0.013

Financial support. This work was support by a grant from the Mission Interministérielle de Lutte Contre les Drogues Et les Conduites Addictives (MILDECA) and with a grant "Bourse de mobilité jeune chercheur » proposed by the Cancéropole Lyon Auvergne Rhône-Alpes (CLARA). PM (Senior Research Associate) is funded by the Belgian Fund for Scientific Research (F.R.S.-FNRS, Belgium)

Conflict of interest. None