# Impact of *Oenococcus oeni* esterase EstA7 on wine aromatic compounds

M. DARSONVAL*, H. ALEXANDRE, C. GRANDVALET

UMR Procédés Alimentaires et Microbiologiques (UMR PAM),
Equipe Vin, Aliment, Microbiologie, Stress(VALMiS), AgroSup Dijon - Université de Bourgogne,
Institut Universitaire de la Vigne et du Vin, Dijon, France

## *Oenococcus oeni* – esters – EstA7 – wine aromatic profile

## Introduction

Nowadays, malolactic fermentation (MLF) is sought in most red wines and some white wines. Despite a large microbial diversity in grape must, *O. oeni* predominates to provide the MLF and represents the majority of malolactic ferments commercialized. Besides the total acidity decrease of wine, numerous studies report that *Oenococcus oeni* can have a potential impact on the chemical composition of wine by releasing esters, attractive aromatic compounds, by the action of their own esterases [1]. The characterization of *O. oeni* esterases with potential aromatic interest is necessary for fundamental perspectives and opens the way to oenological applications using lactic acid bacteria (LAB). Esters form one of the most important families of aromatic compounds in wine and are responsible for the fruity aroma of young wines. The main wine esters are derived from grape berry or produced by yeasts during the alcoholic fermentation (AF) by enzymatic catalysis [2]. While extensive research has focused on esterase activity of yeasts, the impact of *O. oeni* esterases during MLF remains relatively little-explored [5]. Current knowledge is mainly based on ester profile study of oenological yeast strains or LAB strains in wine-like conditions. Previous studies have already characterized four genes encoding esterases from *O. oeni* (*estA2*, *estA7*, *estC* and *estB*) under conditions relevant to winemaking [3-6]. The esterases EstB28 and EstC of the *O. oeni* Ooeni28 strain were shown to have a dual activity of hydrolysis and synthesis on natural esters [6]. We have focused our study on EstA7 and established a link between the flavor profile and this esterase. We have successfully cloned and *in vivo* expressed the *estA7* gene in *O. oeni* ATCC BAA-1163. The *O. oeni* recombinant strains were used to inoculate Aligoté wine and performed MLF. The flavor profile of each wine was established by SPME-GCMS. This study is the first report of the characterization of EstA7 from *O. oeni* and shows that EstA7 is able to synthesize esters in wine.

## Materials and methods

### Cloning and expression of the estA7 gene

Based on the published sequences of *estA7* (JX215244) esterase gene from strain *O. oeni* Ooeni28 (Lallemand, nuovi Ceppi OO2), the *estA7* gene encoding putative esterase was identified and successfully amplified from *O. oeni* ATCC BAA-1163 strain. The coding sequence of *estA7* from ATCC BAA-1163 (accession number KT716344) was then cloned into an *E. coli*-LAB shuttle vector developed in our laboratory [7]. Resulting plasmid (pSYNestA7) was transferred in *E. coli* MG2163 strain (ECestA7 strain) and *O. oeni* ATCC BAA-1163 (OoestA7 strain). Enzymatic activity of EstA7 was confirmed by enzymatic assay first in *E. coli* (EcestA7 strain) then in *O. oeni* (OoestA7 strain) using pNP-butanoate as synthetic substrate at 410 nm at 37°C. *O. oeni* strain with native plasmid (Oosyn strain) was used as a control.

### MLF and ester analysis

The two recombinant *O. oeni* strains were used to inoculate sterile white wine from Aligoté grape variety to perform MLF. Ester concentrations were managed before and after MLF by SPME GC-MS method [6]. Analyses were performed on three independent samples in triplicate. One-way ANOVA was used to compare the concentrations of each wine ester concentration from each recombinant strain of *O. oeni* ($p \le 0.05$).
# Results and discussion

## Characterization of esterase activity

The esterase specific activity of EstA7 assayed in *E. coli* and *O. oeni* recombinant strains was normalized relative to the esterase specific activity of the strain carrying native plasmid (ECSyn and Oosyn). In the *E. coli* recombinant strain, the relative esterase activity observed was 5-fold higher than in the ECSyn strain (Figure 1). In the *O. oeni* recombinant strain, relative esterase activity was 12-fold higher than in Oosyn. These findings validated our cloning and expression system in *O. oeni* using the *estA7* gene and suggested that high enzymatic activity observed in recombinant strain could have the potential to affect the ester profile of wine during the MLF (Figure 1). Moreover, we noticed that the relative esterase activity level was 2-fold higher in *O. oeni* recombinant strains than in *E. coli*. This result could be explained by the use of an expression system designed for LAB.

<figure><img src="image_121.png" /><figcaption>**Fig. 1** - Relative esterase activity level of *E. coli* and *O. oeni* recombinant strains towards the synthetic substrate pNP-butanoate.</figcaption></figure>

## Modification of ester profiles after MLF

Based on previous results, we have established the ester profile of the Aligoté wine undergoing MLF by the recombinant strains of *O. oeni*. The concentration of eight main esters by GC-MS-SPME after and before the MLF (ethyl acetate, isobutyl acetate, ethyl butanoate, isoamyl acetate, ethyl hexanoate, hexyl acetate, ethyl lactate and ethyl octanoate) has been monitored (Table 1 and Figure 2). In wine having undergone MLF by the *O. oeni* strain with native plasmid (Oosyn), significant changes in ester composition consisted in the increase of ethyl lactate and ethyl octanoate and the decrease of isoamyl acetate and isobutyl acetate, and suggested a reduction of the banana aroma and an intensification of the ripe fruit flavor (Figure 2). The quantification of these esters confirmed the fermentative abilities of our laboratory strain *O. oeni* ATCC BAA-1163 and was essential to discern its ester profile on Aligoté variety. Concentration of ethyl lactate and ethyl acetate increased significantly in the two wines with MLF performed by Oosyn and OoestA7 strains relative to the non-inoculated wine, meaning that EstA7 has no detectable hydrolyzing or synthesizing activity towards these esters. Knowing that ethyl lactate formation is correlated with the degradation of malic acid, these results were not surprising [1]. The significant increase of hexyl acetate concentration during MLF performed by OoestA7 strain suggests that EstA7 is capable to synthesize this ester. Moreover, the concentration of ethyl octanoate and isoamyl acetate seemed to be increased in lower degree. EstA7 could play a more important role in ester synthesis rather than hydrolysis.

<table><caption>**Table 1** - Concentrations of selected esters prior and post MLF in Aligoté wine</caption><thead><tr><th>Ester (µg.L<sup>-1</sup>)</th><th>No inoculation</th><th>Oosyn</th><th>OoestA7</th><th>Aroma characteristics[1]</th></tr></thead><tbody><tr><td>Ethyl acetate</td><td>50 164±5 616</td><td>43 749±8 725</td><td>45 769±4 128</td><td>Fruity, Solvent(at > 100 mg.mL<sup>-1</sup>)</td></tr><tr><td>Isobutyl acetate</td><td>52±7</td><td>33±13</td><td>32±6</td><td>Fruity, banana</td></tr><tr><td>Ethyl butanoate</td><td>56±7</td><td>46±9</td><td>47±4</td><td>Floral, strawberry</td></tr><tr><td>Isoamyl acetate</td><td>4 800±623</td><td>3 307±666</td><td>3 895±423</td><td>Fruity, banana</td></tr><tr><td>Ethyl hexanoate</td><td>836±119</td><td>899±11</td><td>913±132</td><td>Fruity, strawberry</td></tr><tr><td>Hexyl acetate</td><td>197±24</td><td>145±3</td><td>178±24</td><td>Pear, banana</td></tr><tr><td>Ethyl lactate</td><td>14 410±830</td><td>33 682±5 804</td><td>27 695±4 762</td><td>Milk, buttery</td></tr><tr><td>Ethyl octanoate</td><td>1 731±130</td><td>2 611±135</td><td>3 329±649</td><td>Fruity, ripe fruit</td></tr></tbody></table>
<figure><img src="image_122.png" /><figcaption>**Fig. 2** - Ester concentrations of Aligoté wine not inoculated (white) or inoculated with *O. oeni* ATCC BAA-1163 recombinant strains: Oosyn strain (grey) or OoestA7 strain (black). Significant differences are based on one-way analysis of variance followed by a Tukey's multiple test (p < 0.05).</figcaption></figure>

## Conclusion

Little is known about EstA7 from *O. oeni* because of its instability and its difficulty to purify *in vitro* [8]. Our findings report for the first time that EstA7 of *O. oeni* is able to synthesize ester in wine. Indeed, EstA7 has an impact on hexyl acetate, an ester associated with the pleasant aroma descriptor of pear and banana sought in wine according its specificity. Our work underlines that EstA7 might be an interesting oenological selection criterion. Indeed, the expression level of *estA7* could be used as a direct application to screen future commercial malolactic ferments. For the first time in *O. oeni*, we have successfully expressed a gene encoding a functional protein, we can now consider the investigation of the role of the other esterases in *O. oeni* [5, 6].

## Acknowledgements

This work was supported by the Ministère de la Recherche et de l'Enseignement Supérieur and the Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement (AgroSup Dijon).

## References

1. Sumby KM, Grbin PR, Jiranek V (2010) Microbial modulation of aromatic esters in wine: current knowledge and future prospects. *Food Chem* **121**: 1-16.

2. Swiegers JH, Bartowsky EJ, Henschke PA et al (2005) Yeast and bacterial modulation of wine aroma and flavour. *Aust J Grape Wine Res* **11**: 139-173.

3. Matthews A, Grbin PR, Jiranek V (2007) Biochemical characterisation of the esterase activities of wine lactic acid bacteria. *Appl Microbiol Biotechnol* **77**: 329-337.

4. Sumby KM, Matthews AH, Grbin PR et al (2009) Cloning and characterization of an intracellular esterase from the wine-associated lactic acid bacterium *Oenococcus oeni*. *Appl Environ Microbiol* **75**: 6729-6735.

5. Sumby KM, Grbin PR, Jiranek V (2013) Characterization of EstCOo8 and EstC34, intracellular esterases, from the wine-associated lactic acid bacteria *Oenococcus oeni* and *Lactobacillus hilgardii*. *J Appl Microbiol* **114**: 413-422.

6. Sumby KM, Jiranek V, Grbin PR (2013) Ester synthesis and hydrolysis in an aqueous environment, and strain specific changes during malolactic fermentation in wine with *Oenococcus oeni*. *Food Chem* **141**: 1673-1680.

7. Darsonval M, Alexandre H, Msadek T et al (2015) The antisense RNA approach: a new application for *in vivo* investigation of the stress response of *Oenococcus oeni*, a wine-associated lactic acid bacteria. *Appl Environ Microbiol* **82**: 18-26.

8. Sumby KM, Grbin PR, Jiranek V (2012) Validation of the use of multiple internal control genes, and the application of real-time quantitative PCR, to study esterase gene expression in *Oenococcus oeni*. *Appl Microbiol Biotechnol* **96**: 1039-1047.
# The exopolysaccharides (EPS) of *Oenococcus oeni* and their implication in Champagne wine

M. DIMOPOULOU<fnref n="1" />*, Y. VASSEROT<fnref n="2" />, R. MARCHAL<fnref n="2" />, M. DOLS-LAFARGUE<fnref n="1" />,<fnref n="3" />

Champagne wine – polysaccharides – *Oenococcus oeni*

## Introduction

Champagne is a sparkling wine elaborated exclusively from grapes grown in the area of Champagne in the northeast of France, under strict regulation conditions. The wine is characterized by a great acidity, freshness and crispy taste. The malolactic fermentation (MLF), realized by the lactic acid bacteria, is taking place in most cases to ensure the microbial stability, especially during the second fermentation in the bottle. *Oenococcus oeni* is the selected species to drive the MLF. It is naturally present on grapes, in the cellar or can be added in the form of malolactic starters. Recently, the analysis of 50 genome sequences of *O. oeni* revealed the presence of multiple biosynthetic pathways for exopolysaccharide (EPS) production [1]. Interestingly all the strains isolated from Champagne wines formed a subgroup sharing the same *eps* gene profile. The Champagne strains were singular as they did not display a functional *eps2* cluster and they all present a glycosyltransferase gene (*gtf*) implicated in the production of β-glucan and responsible for a ropy character observed in laboratory culture conditions. The prevalence of the *gtf* gene in a collection of more than 200 strains was previously shown to be less than 7.5%, while more than 85% of the *O. oeni* strains studied displayed a functional *eps2* cluster.

The bacterial polysaccharides have never been considered to be of special interest in Champagne wine. However, β-glucan was previously shown to protect the bacteria from acid stress [2]. The purpose of our study was to analyze a greater collection of bacterial strains isolated from Champagne wines, regarding their EPS production profile and to answer the two following questions: (i) Is the presence of the *gtf* gene a common feature of the *O. oeni* strains isolated from Champagne? (ii) Do all the *O. oeni* strains isolated from Champagne display the same *eps* gene profile? The strains were characterized at the genotypic level by specific PCR reactions and VNTR analysis, and then at the phenotypic level through the quantification of the EPS produced.

## Materials and methods

*Strains and culture conditions*

The 55 *O. oeni* strains studied were either commercial malolactic starters (3) recommended for Champagne or acidic white wines, or isolates from white (49) or red (3) wines in various regions of France including Champagne (Table 1). They were stored in glycerol (50%) at -80°C.

The bacteria were routinely grown on grape juice medium made of commercial red grape juice 250 mL.L⁻<fnref n="1" />, yeast extract 5 g.L⁻<fnref n="1" /> and Tween 80 1 mL.L⁻<fnref n="1" />, pH 4.8 and sterilised for 20 min at 121°C. When stated in the text, 10 g.L⁻<fnref n="1" /> of sucrose were added to grape juice medium before sterilisation. Alternately, a semi defined (SMD) was used [1]. It contained either glucose 20 g.L⁻<fnref n="1" /> or glucose and sucrose, 10 g.L⁻<fnref n="1" /> each, as growth substrates. Growth was carried out at 25°C, for 7 to 15 days, without any agitation, except before sampling. Growth was monitored through OD₆₀₀ measurements. The ropy phenotype was estimated by observation of the cell deposit behavior during vortex agitation before sampling.

<footnote n="1">¹Univ. Bordeaux, ISVV, EA4577 Œnologie, F-33140 Villenave d'Ornon, France</footnote>

<footnote n="2">²Université de Reims, B.P. 1039, 51687 Reims, Laboratoire d'Œnologie, UPRES EA 2069, URVVC, Faculté des Sciences, Cedex 02, France</footnote>

<footnote n="3">³Ecole Nationale Supérieure de Chimie, de Biologie et de Physique, Institut Polytechnique de Bordeaux, Avenue Pey Berland, 33607 Pessac, France</footnote>
<table><caption>**Table 1** - The 55 *Oenococcus oeni* isolates with their corresponding origin</caption><thead><tr><th>Isolates</th><th>Origin</th><th>Isolates</th><th>Origin</th></tr></thead><tbody><tr><td>1</td><td>Champagne White wine</td><td>29</td><td>Chablis White wine</td></tr><tr><td>2</td><td>Champagne White wine</td><td>30</td><td>Bourgogne White wine</td></tr><tr><td>3</td><td>Champagne White wine</td><td>31</td><td>Bourgogne White wine</td></tr><tr><td>4</td><td>Champagne White wine</td><td>32</td><td>Bourgogne White wine</td></tr><tr><td>5</td><td>Champagne White wine</td><td>33</td><td>Champagne White wine</td></tr><tr><td>6</td><td>Champagne White wine</td><td>34</td><td>Champagne White wine</td></tr><tr><td>7</td><td>Champagne White wine</td><td>35</td><td>Commercial strain</td></tr><tr><td>8</td><td>Champagne White wine</td><td>36</td><td>Commercial strain</td></tr><tr><td>9</td><td>Champagne White wine</td><td>37</td><td>Commercial strain</td></tr><tr><td>10</td><td>Champagne White wine</td><td>38</td><td>Champagne White wine</td></tr><tr><td>11</td><td>Champagne White wine</td><td>39</td><td>Bourgogne White wine</td></tr><tr><td>12</td><td>Champagne White wine</td><td>40</td><td>Bourgogne White wine</td></tr><tr><td>13</td><td>Champagne White wine</td><td>41</td><td>Bourgogne White wine</td></tr><tr><td>14</td><td>Champagne White wine</td><td>42</td><td>Bourgogne White wine</td></tr><tr><td>15</td><td>Champagne White wine</td><td>43</td><td>Bourgogne White wine</td></tr><tr><td>16</td><td>Champagne White wine</td><td>44</td><td>Bourgogne White wine</td></tr><tr><td>17</td><td>Champagne White wine</td><td>45</td><td>Bourgogne White wine</td></tr><tr><td>18</td><td>Champagne White wine</td><td>46</td><td>Bourgogne White wine</td></tr><tr><td>19</td><td>Champagne White wine</td><td>47</td><td>Bourgogne White wine</td></tr><tr><td>20</td><td>Champagne White wine</td><td>48</td><td>Bourgogne White wine</td></tr><tr><td>21</td><td>Champagne White wine</td><td>49</td><td>Bourgogne White wine</td></tr><tr><td>22</td><td>Champagne White wine</td><td>50</td><td>Bourgogne White wine</td></tr><tr><td>23</td><td>Champagne White wine</td><td>51</td><td>Bourgogne White wine</td></tr><tr><td>24</td><td>Champagne White wine</td><td>52</td><td>Bourgogne White wine</td></tr><tr><td>25</td><td>Jura White wine</td><td>53</td><td>Bordeaux Red wine</td></tr><tr><td>26</td><td>Jura White wine</td><td>54</td><td>Bordeaux Red wine</td></tr><tr><td>27</td><td>Jura White wine</td><td>55</td><td>Bordeaux Red wine</td></tr><tr><td>28</td><td>Chablis White wine</td><td></td><td></td></tr></tbody></table>

## EPS analysis

For EPS concentration measurements, the whole culture medium was centrifuged (10,000 x g, 15 min, 4°C). Three volumes of ethanol-HCl 1N (95-5) were added to the supernatant to precipitate the polysaccharides. The tubes were let to stand for 24 hours at 4°C. Then, they were centrifuged (10,000 x g, 15 min, 4°C), and the pellet was washed with ethanol (80% vol), centrifuged again, dried for 20 min at 65°C and dissolved in distilled water. The amount of neutral polysaccharides was determined by the anthrone-sulfuric acid method [4], using glucose as the standard. For each sample, the polymer precipitation and assays were done in triplicate.

## PCR and VNTR analysis

Total genomic DNA was purified using the Wizard Genomic DNA purification kit (Promega, Madison, WI, USA). For PCR analysis, primers were designed according to the *eps* gene sequences published by Dimopoulou *et al.* [1]. The *gtf* gene was screened using PF1 (GATTGTAATAAAATAAAAAGACCC) and PF8 (CATATGATAACACGCAGGGC) and its chromosomal localization by the primers AGAGAA TTCATAGTTCACTGGGCTACCA and TTAGCTAAAATAGGGAGGATGCTATCTGTG. The operons *eps1* and *eps2* were screened using op1dir and op1rev (GTTGAGTTGGATGATCGCACCGAACCTG) and (GAATCTAATTATCAAGTCAGAATCGACCGA) or op2dir (CAAAGAAATACGAATGTTACGA GACGGAA) and op2rev TCATTTCGGTTCCTTGATTCCTTTTT). The *dsrO* gene was analyzed using the primers ATGAGAGATGGGGATAAAAATCGTCCG and AATTGTGATCCTGTAACTGCAATT CCG. Thirty PCR cycles (30 s at 95°C, 30 s at 55°C, 2 min at 72°C) were performed before agarose gel electrophoresis followed by ethidium bromide staining. VNTR analysis was carried out as described by Claisse and Lonvaud-Funel [3].
Results and discussion

In order to determine if the *O. oeni* isolates from Champagne all displayed the same *eps* gene profile, a large collection of isolates (30) of this viticulture region was analyzed. VNTR analysis was used to examine their distribution (Figure 1): 17 distinct VNTR profiles were obtained. Indeed some of the studied isolates displayed exactly the same VNTR profile and may correspond to the same strain. These isolates thus formed a bigger circle (Figure 1). A color distinction was made according to the presence (in black) or absence (in gray) of the *gtf* gene in the bacterial chromosome (PCR check). Eleven isolates, corresponding to 10 distinct strains, harbored the *gtf* gene in their chromosome. Conversely, the 19 isolates which were *gtf* negative corresponded to 7 strains according to the VNTR analysis. More than half of the Champagne strains examined (10/17 or 59%) were thus *gtf* positive.

<figure><img src="image_123.png" /><figcaption>Fig. 1 - Representation in tree cover minimal by BioNumerics, after VNTR analysis of the 30 *O. oeni* isolates from the Champagne region (including the malolactic starters). The number of isolates represented by every circle is indicated when they are more than one. The isolates colored in black are *gtf* positive (11) while the gray ones are *gtf* negative (19) according to PCR results.</figcaption></figure>

The same analysis was carried out with the 55 isolates mentioned in table 1. Forty-four distinct VNTR profiles were obtained. Among these, 41 originated from white and Champagne wines and 3 from red wines (added as controls). Among the 41 distinct strains from white and Champagne wines, 27 were found *gtf* positive, namely 66% (Figure 2). All the malolactic starters recommended for Champagne or white wines were also *gtf* positive, while all the red wine strains introduced as controls were *gtf* negative.

Compared to the percentage of the *gtf* positive strains previously observed among isolates from all over the world (7.5%), these results confirmed the high prevalence of the *gtf* gene in Champagne and French white wine *O. oeni* strains. However, they also indicated that Champagne and white wine strains were not necessarily *gtf* positive.

The 44 strains were further characterized for *eps* gene and EPS phenotypes (Figure 2). The Champagne strain *O. oeni* IOEB 0205 previously characterized [2] and displaying the *eps* profile specific for Champagne strains according to genome analysis [1] was used as control. When the *gtf* gene was present, it was most of the time (60%) located in the same chromosomal locus as that described for *O. oeni* IOEB 0205 [2]. This may be due to either (i) a similar mode of acquisition for all the *gtf* positive strains studied or (ii) most probably a strong relationship between all the *gtf* positive strains that may descend from the same ancestor having acquired the *gtf* gene before this strain divergence.
The 44 strains were then characterized for the other *eps* genes (Figure 2). Regarding the type of *eps1* and *eps2* gene cluster present, the 44 tested isolates were less similar. Actually, 19/44 strains had the same *eps1* model and 20/44 the same *eps2* model as the reference strain (IOEB 0205). However, only 10 isolates displayed exactly the same *eps* profile as the reference strain (*gtf*, *eps1*, *eps2* and *dsrO*) (Figure 2).

<figure><img src="image_124.png" /><figcaption>**Fig. 2** - Dendrogram based on VNTR sequences for 44 strains (isolates from wine with distinct VNTR profiles) of *O. oeni*, with their corresponding exopolysaccharide genotypic and phenotypic profile.</figcaption></figure>

The target locus tested by PCR is: A: glycosyltransferase gene *gtf*, B: glycosyltransferase gene localization: positive result if identical to *O. oeni* 0205, C: *eps1* type *O. oeni* 0205, D: *eps2* type *O. oeni* 0205, E: dextransucrase gene. The black box shows the presence of the target locus and the gray box its absence, according to the PCR results. The culture medium used for the EPS concentration measurements was supplemented with either glucose (G) or glucose and sucrose (G/S). The color indicates the different EPS production level as follows: (■): 0-50 mg.L⁻¹, (■): 50-150 mg.L⁻¹, (■): 150-1000 mg.L⁻¹, (■): >1 g.L⁻¹. The bacterial strains are indicated in numbers.
The polysaccharide concentration produced was determined after growth into two different culture media: SMD Glucose and SMD Glucose/Sucrose (Figure 2). The EPS concentration produced was always higher than 50 mg.L⁻¹ when the *gtf* gene was present in the genome of the bacteria. In this case, the culture medium was always ropy. Some of the strains studied were also able to produce more than 1 g.L⁻¹ of EPS, when the sucrose was added in the medium. However, as already observed, the presence of the *dsrO* gene in the chromosome was not always correlated with an over production of EPS in the presence of sucrose [2] (Figure 2).

Conclusion

The aim of this study was to investigate the *eps* gene profile of *O. oeni* strains isolated from Champagne and other French white wines. We first confirmed the high prevalence of the *gtf* gene among these strain (66%). Our results suggest that this is probably due to the acquisition of the gene by a common ancestor, putatively associated with increased fitness to white wines [2] but this fitness remains to be demonstrated in *O. oeni*. However, we also found that Champagne strains do not obligatory display exactly the same *eps* profile. In the future it will be interesting to study oenological consequences of the presence of *O. oeni* EPS in white sparkling and non-sparkling wines.

References

1. Dimopoulou M, Vuillemin M, Campbell-Sills H et al (2014) Exopolysaccharide (EPS) synthesis by *Oenococcus oeni*: from genes to phenotypes. *PLoS One* 9: e98898.

2. Dols-Lafargue M, Lee HY, le Marrec C et al (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.

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

4. Ludwig TG, Goldberg JV (1956) The anthrone method for the determination of carbohydrates in foods and in oral rinsing. *J Dent Res* **35**: 90-94.
# Wine bacteria to control volatile phenols and *Brettanomyces*

M. DELERIS-BOU<fnref n="1" />*, S. KRIEGER-WEBER<fnref n="1" />, J.P. OSBORNE<fnref n="2" />, V. GERBAUX<fnref n="3" />, C. BRIFFOX<fnref n="3" />, A. DUMONT<fnref n="1" />

Selected wine bacteria – malolactic fermentation – hydroxycinnamic acids – volatile phenols

## Introduction

This article explores a natural way to control the spoilage yeast *Brettanomyces* and the volatile phenols it produces by using selected wine bacteria to conduct malolactic fermentation (MLF).

The yeast *Brettanomyces/Dekkera* is considered a contaminant in wine and its growth must be limited. The major problem associated with *Brettanomyces* spoilage is the production of volatile phenols: 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG). The volatile phenols 4-EP and 4-EG are produced from the hydroxycinnamic acids p-coumaric acid and ferulic acid respectively through a two-step enzymatic process. The hydroxycinnamic acid is first converted to a vinylphenol via a cinnamate decarboxylase before being reduced to an ethylphenol via a vinylphenol reductase (figure 1).

<figure><img src="image_125.png" /><figcaption>**Fig. 1** - Biochemical pathways of volatile phenol production. Production potential of volatile phenols is linked with the levels of hydroxycinnamic acids present.</figcaption></figure>

p-Coumaric acid and ferulic acid are naturally present in grapes and are commonly present as esters of tartaric acid known respectively as coutaric and ferulic acid. As it was recently shown that *Brettanomyces bruxellensis* was unable to metabolize these tartaric acid ester bound hydroxycinnamic acids [1], any conversion from tartaric acid bound hydroxycinnamic acid into its free form needs to be taken into consideration. Because of this, the capacity of selected wine lactic acid bacteria (LAB) to perform this conversion was investigated.

Although *Brettanomyces* can be detected at any stage of the winemaking process, it is typically detected after alcoholic fermentation (AF) and before spontaneous malolactic fermentation or during barrel aging.

<footnote n="1">¹Lallemand SAS France, 19 rue des Briquetiers, 31702 Blagnac, France</footnote>

<footnote n="2">²Oregon State University, Dept of Food Science and Technology, 100 Wiegand Hall, Corvallis, OR 97331, USA</footnote>

<footnote n="3">³IFV, 6 rue du 16ᵉme Chasseurs, 21200 Beaune, France</footnote>
In addition to control the precursors of volatile phenols, it is also important to limit the growth of *Brettanomyces* and consequently reduce the production of volatile phenols. One way to achieve this is by avoiding lag phase after AF by applying an early inoculation of selected wine bacteria. This process was investigated in Pinot noir [2] and in practical cases [3].

# Materials and methods

*Screening of wine LAB for the ability to degrade tartaric esterified hydroxycinnamic acids*

The study was conducted in the laboratory of J. Osborne at Oregon State University (USA). 10 strains of Lallemand collection (Alpha™, VP41™, PN4™, O-MEGA™, Beta™, V22™, MT01™, Elios Alto™, Elios 1™, MT01™) were tested for their ability to degrade tartaric esterified hydroxycinnamic acids. Trials were conducted in a Pinot noir wine produced following standard procedures (yeast strain RC212, Fermaid K addition, 27°C fermentation temperature). Basic wine parameters after pressing were pH 3.53, TA 0.67 g/100mL, and 13.2% alcohol. After settling, the wine was pad filtered (2-3 µm) prior to being sterile filtered (0.45-µm PES cartridge). Pinot noir wine was inoculated with bacteria at approximately 1 x 10⁶ CFU/mL. Treatments were performed in triplicate and a non-inoculated control was also prepared. After the completion of MLF (< 30 mg/L malic acid), samples were taken and assessed for hydroxycinnamic acid content by HPLC-DAD according to Burns and Osborne [4].

In an additional trial [5], Pinot noir wines where MLF was conducted with *Oenococcus oeni* strain Alpha™, VP41™ and LAB1 were inoculated with *Brettanomyces bruxellensis* UCD-2049 at a rate of approx. 1 x 10⁴ CFU/mL. After 40 days, samples were taken and assessed for free and tartaric esterified hydroxycinnamic acid (HPLC-DAD) and 4-EP and 4-EG (GC-MS). Treatments were performed in triplicate and a non-inoculated control was also prepared.

*Influence of inoculation with selected wine bacteria on the growth of *Brettanomyces* and the subsequent production of volatile phenols*

- Practical case 1: Pinot noir experiment – Burgundy

The study was conducted by IFV (France – V. Gerbaux). Trial was run in a Pinot noir wine made from grapes of the Bourgogne Hautes Côtes de Beaune appellation. Basic wine parameters were pH 3.36, alcohol 12.8%v/v, TA 7.6 g/L tartaric acid and malic acid 1.8 g/L. 2 commercial LAB were used for inoculation of the wine at 2 x 10⁶ CFU/mL right after the end of AF. Concentrations of 4-EP and 4-EG were determined by gas chromatographic analysis.

- Practical case 2: Cabernet Franc experiment – Bordeaux area

The grapes were separated in 2 homogeneous lots. Must parameters were potential alcohol 13.2%v/v, pH 3.49, TA 4.55 g/L H₂SO₄ and malic acid 2.4 g/L. Both lots were inoculated with a selected yeast at 20 g/hL. The first lot was inoculated 72h after yeast addition (co-inoculation) with the bacteria Beta™. The other lot was not inoculated in bacteria (spontaneous MLF). Levels of ethylphenols were determined in both cases at the end of MLF.

- Practical case 3: Cabernet Sauvignon – Languedoc-Roussillon

The grapes were separated in 2 homogeneous lots. Must parameters were potential alcohol 12.8%v/v, pH 3.48, TA 4.6 g/L H₂SO₄ and malic acid 2.1 g/L. Both lots were inoculated with a selected yeast at 20 g/hL. The first lot was inoculated 48h after yeast addition (co-inoculation) with the bacteria Beta™. The other lot was not inoculated. Levels of 4-EG and 4-EP were determined in both cases at the end of MLF.

# Results and discussion

The first step to controlling *Brettanomyces* is respecting winemaking best practices. It is important to have an integrated strategy that takes into account the interdependence of diverse wine parameters, such as grape quality, SO₂, pH, wine temperature, nutrients, oxygen, barrel condition and oenological practices. Good cellar hygiene, reducing the lag phase between the end of AF and the beginning of MLF, and early stabilization, along with proper SO₂ dosage, greatly minimize the risk of microbial spoilage. The winemaker's strategy to limit the risk of developing *Brettanomyces* has three key factors: the presence of precursors for volatile phenols, the growth phases of *Brettanomyces* and the wine conditions. Selected wine bacteria can prevent *Brettanomyces* development by taking into consideration these three aspects.
### Limit the levels of precursors for volatile phenols

The objective is to investigate the capacity of selected wine bacteria to degrade hydroxycinnamic acids bound to tartaric esters present in wine into the free form which are precursors for volatile phenol production by *Brettanomyces*. Results for 4 selected strains are presented in table 1.

<table><caption>**Table 1** - Concentration (mg/L) of hydroxycinnamic acids in Pinot noir wine at the completion of MLF after inoculation with 4 commercial wine LAB</caption><thead><tr><th></th><th>Caftaric acid</th><th>Coutaric acid</th><th>Caffeic acid</th><th>p-coumaric acid</th><th>Ferulic acid</th></tr></thead><tbody><tr><td>PN4™</td><td>23.2 ± 0.4</td><td>6.6 ± 0.1</td><td>2.4 ± 0.2</td><td>0.9 ± 0.1</td><td>3.5 ± 0.2</td></tr><tr><td>OMEGA™</td><td>24.1 ± 1.3</td><td>6.9 ± 0.4</td><td>3.0 ± 0.1</td><td>1.0 ± 0.1</td><td>3.3 ± 0.3</td></tr><tr><td>Beta™</td><td>25.0 ± 2.2</td><td>7.0 ± 0.6</td><td>2.6 ± 0.5</td><td>0.8 ± 0.3</td><td>4.2 ± 0.5</td></tr><tr><td>V22™</td><td>25.8 ± 1.3</td><td>7.1 ± 0.3</td><td>2.4 ± 0.1</td><td>0.6 ± 0.1</td><td>3.8 ± 0.1</td></tr><tr><td>Control (no MLF)</td><td>25.1 ± 1.1</td><td>6.8 ± 0.5</td><td>2.2 ± 0.2</td><td>0.9 ± 0.3</td><td>4.1 ± 0.3</td></tr></tbody></table>

At the completion of MLF, no change was noted in the concentration of the tartaric esterified hydroxycinnamic acids (caftaric and coutaric acid) or the corresponding free hydroxycinnamic acids (p-coumaric and ferulic acid) for any of the ten strains tested.

In an additional trial the impact of a wine LAB strain known to degrade tartaric esterified hydroxycinnamic acids on volatile phenol production by *Brettanomyces* was investigated. The results are shown in table 2.

<table><caption>**Table 2** - Concentration (mg/L) of hydroxycinnamic acids in Pinot noir wine at the completion of MLF after inoculation with 3 commercial wine LAB and after 40 days of growth of *B. bruxellensis* UCD-2049 including final 4-EP and 4-EG concentrations (µg/L) in the wine (adapted from [5])</caption><thead><tr><th rowspan="2"></th><th colspan="4">At the completion of MLF (initial)</th><th colspan="6">After 40 days of growth of B. bruxellensis UCD-2049 (final)</th></tr><tr><th>Caftaric acid</th><th>Caffeic acid</th><th>Coutaric acid</th><th>p-coumaric acid</th><th>Caftaric acid</th><th>Caffeic acid</th><th>Coutaric acid</th><th>p-coumaric acid</th><th>4-EP</th><th>4-EG</th></tr></thead><tbody><tr><td>LAB1</td><td>11.1 ± 2.2</td><td>16.5 ± 4.1</td><td>3.5 ± 0.7</td><td>6.9 ± 1.7</td><td>9.9 ± 0.7</td><td>17.1 ± 1.6</td><td>2.5 ± 0.3</td><td>0.1 ± 0.0</td><td>1580.3 ± 154.1</td><td>270.7 ± 131.5</td></tr><tr><td>Alpha™</td><td>31.6 ± 0.2</td><td>3.3 ± 0.1</td><td>10.2 ± 0.2</td><td>1.4 ± 0.4</td><td>30.1 ± 0.2</td><td>3.2 ± 0.2</td><td>9.6 ± 0.1</td><td>0.1 ± 0.0</td><td>180.1 ± 12.8</td><td>70.7 ± 1.5</td></tr><tr><td>VP41™</td><td>31.3 ± 0.2</td><td>2.8 ± 0.1</td><td>9.9 ± 0.2</td><td>1.2 ± 0.6</td><td>29.5 ± 0.2</td><td>2.7 ± 0.2</td><td>9.6 ± 0.1</td><td>0.1 ± 0.0</td><td>216.2 ± 13.8</td><td>80.8 ± 3.8</td></tr><tr><td>Control (no MLF)</td><td>29.7 ± 0.2</td><td>2.5 ± 0.4</td><td>9.2 ± 0.7</td><td>1.6 ± 0.2</td><td>28.5 ± 2.9</td><td>2.2 ± 0.6</td><td>9.4 ± 0.7</td><td>0.1 ± 0.0</td><td>260.1 ± 42.1</td><td>80.2 ± 6.4</td></tr></tbody></table>

No significant changes in free or tartaric acid ester bound hydroxycinnamic acids were observed for wines that underwent MLF with Alpha™ or VP41™ when compared to the control without MLF. On the contrary, wine inoculated with LAB1 had significantly lower concentrations of caftaric and coutaric acid and higher concentrations of caffeic and p-coumaric acid. The increase in the concentration of volatile phenol precursors led to elevated production of 4-EP and 4-EG in the wine by *Brettanomyces* compared to the other three lots.

### Control of MLF to prevent the growth of *Brettanomyces* bruxellensis and avoid production of volatile phenols

The period from the end of AF to the start of MLF is particularly conducive to the development of *Brettanomyces*: the wine is not protected by SO₂, there are still some nutrients available to the spoilage yeast, and competition from other wine micro-organisms is hardly a threat, as the yeast has finished and is dying off and the indigenous lactic bacteria are not yet established. The use of selected wine bacteria is a solution to shorten the time lapse between AF and MLF and thereby prevent the development of *Brettanomyces*. The three practical cases summarized in table 3 show how MLF control can permit to limit the content of volatile phenols in the final wines when compared to non-inoculated wines.
<table><caption>**Table 3** - Volatile phenol production in Pinot noir, Cabernet-Franc and Cabernet-Sauvignon wines after MLF induced by inoculation with selected wine bacteria versus a control which underwent spontaneous fermentation</caption><thead><tr><th></th><th>Bacteria</th><th>Timing of addition</th><th>4-EP (µg/L)</th><th>4-EG (µg/L)</th></tr></thead><tbody><tr><td rowspan="3">Pinot noir</td><td>Lalvin 31</td><td>End of AF</td><td>46</td><td>20</td></tr><tr><td>FML Expertise S</td><td>End of AF</td><td>32</td><td>15</td></tr><tr><td>Spontaneous</td><td>-</td><td>1119</td><td>551</td></tr><tr><td rowspan="2">Cabernet-Franc</td><td>Beta<sup>TM</sup></td><td>Co-inoculation</td><td colspan="2">Total = 5</td></tr><tr><td>Spontaneous</td><td>-</td><td colspan="2">Total = 680</td></tr><tr><td rowspan="2">Cabernet-Sauvignon</td><td>Beta<sup>TM</sup></td><td>Co-inoculation</td><td>205</td><td>78</td></tr><tr><td>Spontaneous</td><td>-</td><td>1667</td><td>349</td></tr></tbody></table>

## Conclusion

Managing the winemaking process through secure AF and MLF is a good starting point to prevent the development of undesirable indigenous flora. It is also important to utilize wine LAB strains for MLF that cannot degrade tartaric esterified hydroxycinnamic acids as this process will lead to increased volatile phenol precursors in the wine. Moreover, appropriate inoculation strategies (co-inoculation, and early or sequential inoculation right after AF) have been shown to be effective tools to prevent the development of *Brettanomyces*.

## Abbreviations

AF, alcoholic fermentation; MLF, malolactic fermentation; 4-EP, 4-ethylphenol; 4-EG, 4-ethylguaiacol; LAB, lactic acid bacteria.

## References

1. Schopp LM, Lee J, Osborne JP et al (2013) Metabolism of nonesterified and esterified hydroxycinnamic acids in red wines by *Brettanomyces bruxellensis*. *J Agric Food Chem* **61**: 11610-11617.

2. Gerbaux V, Briffox C, Dumont A et al (2009) Influence of inoculation with malolactic bacteria on volatile phenols in wines. *Am J Enol Vitic* **60**: 233-235.

3. Pillet O, Dessup E, Rémond E (2007) Préserver les arômes fruités du vin : nouveaux enjeux dans la gestion de la fermentation malolactique. Partie 2/2 : La co-inoculation en levures et bactéries lactiques : préserver les arômes fruités et lutter contre les contaminations de type *Brettanomyces*. *Rev Oenol* **124**: 1-3.

4. Burns TR, Osborne JP (2013) Impact of malolactic fermentation on the color and color stability of Pinot noir and Merlot wine. *Am J Enol Vitic* **64**: 370-377.

5. Chescheir S, Philbin D, Osborne JP (2015) Impact of *Oenococcus oeni* on wine hydroxycinnamic acids and volatile phenol production by *Brettanomyces bruxellensis*. *Am J Enol Vitic* **66**: 357-362.
# Caractérisation des phénomènes de bio-adhésion à l'origine des altérations des vins

P. POUPAULT*

Institut Français de la Vigne et du Vin, Pôle Val de Loire Centre, F-37400 Amboise, France

Bio-adhésion – micro-organismes – matériaux – vin

## Introduction

L'hygiène est un des facteurs les plus importants pour l'obtention de vins répondant qualitativement au marché. L'incidence du niveau d'hygiène et des conditions de conservation en bouteilles sur l'apparition des phénols volatils d'un vin rouge a pu être mise en évidence [1]. La mise en place de procédures d'hygiène plus strictes et plus adaptées réduit significativement les populations résiduelles sur le matériel de transfert ou d'élevage [2], mais les phénomènes de bio-adhésion restent récurrents. Une meilleure connaissance des caractéristiques bio-adhésives des micro-organismes d'altération a été entreprise en conditions maîtrisées, dans un environnement plus ou moins favorable. L'utilisation de la microscopie confocale laser à balayage et à épifluorescence permet de caractériser la bio-adhésion de différentes levures du genre *Brettanomyces*.

A l'échelle du circuit-test, en conditions dynamiques maîtrisées, les résultats précédents sont confirmés. Le positionnement d'un manchon équipé de coupons extractibles dans le circuit-test permet de modéliser des cinétiques d'encrassement [3] et l'aptitude des micro-organismes à former des biofilms, à résister à une procédure de nettoyage-désinfection, voir même à recontaminer un vin par les surfaces en contact.

## Matériel et méthodes

### Essais d'adhésion

Les caractéristiques microbiologiques et physico-chimiques de plusieurs souches de levures du genre *Brettanomyces* sont évaluées par les tests MATH et MATS (Microbial Adhesion To Hydrocarbon / Solvent) et les mesures de mobilité électrophorétique. Les essais d'adhésion en conditions statiques, après sédimentation, sont observés par microscopie à épifluorescence après coloration par une solution d'orange acridine. Pour les essais de biofilms, l'observation des structures en trois dimensions obtenues après mise en suspension et remplissage des puits est réalisée à l'aide d'un Microscope Confocale Laser à Balayage (après coloration au Syto 9). L'ensemble des images est analysé avec le logiciel IMARIS pour une reconstruction en 3D, par l'équipe UMR-INRA d'AgroParisTech.

### Essais à l'échelle du circuit-test

Sur circuit-test, le CETIM (Centre technique des industries mécaniques) propose une configuration de tuyauterie présentant une cartographie hydrodynamique homogène pour l'implantation d'un manchon porte-coupons, grâce à l'utilisation de la simulation numérique d'écoulement. Les coupons extractibles permettent de suivre l'adhésion des micro-organismes dans le temps et de caractériser leur cinétique d'encrassement. Grâce à cette même configuration – manchon avec coupons extractibles – l'aptitude des cellules viables cultivables survivant à une procédure de nettoyage-désinfection est mesurée pour trois souches différenciées par leur comportement bio-adhésif au laboratoire.

## Résultats et discussion

### Comportement bio-adhésif

Les tests MATH et MATS montrent des différences dans les caractéristiques morphologiques et physico-chimiques de 24 levures de la filière viti-vinicole. Les souches se distinguent pour leur croissance, hydrophilie, mobilité électrophorétique (figure 1). Il en est de même pour les tests réalisés sur un panel de 12 souches de levures du genre *Brettanomyces*.
<figure><img src="image_126.png" /><figcaption>**Fig. 1** - Mobilité électrophorétique des souches (UMR INRA AgroParisTech).</figcaption></figure>

Pour les tests d'adhésion, quelle que soit la souche, l'adhésion la plus élevée est obtenue sur l'acier inoxydable 316 (photo 1). Sur les trois matériaux (acier 316, PET, verre), les niveaux de biocontamination se sont révélés souche-dépendants. Les observations microscopiques ont mis en évidence une distribution relativement homogène des cellules adhérentes sur l'acier inoxydable et le verre et plus hétérogènes sur le PET.

<figure><img src="image_127.png" /><figcaption>**Photo 1** - Adhésion sur l'acier des souches 23A, 8 et 20.</figcaption></figure>

Enfin, concernant les biofilms, toutes les souches ont montré une capacité à former des structures 3D, l'architecture de ces structures est souche-dépendante. On observe des structures éparses, plus denses ou continues (photo 2).

<figure><img src="image_128.png" /><figcaption>**Photo 2** - Biofilms formés par les souches à la surface des microplaques.</figcaption></figure>
La biocontamination des surfaces dépend de la nature des matériaux et de celle des souches contaminantes ; ces données ont sûrement un impact sur l'efficacité des procédures d'hygiène.

## Cinétique d'encrassement

En conditions dynamiques, sur circuit-test, l'aptitude de 3 souches de levures du genre *Brettanomyces* à la bio-adhésion est confirmée sur acier inoxydable 316. La simulation numérique utilisée par le CETIM a permis la conception puis la réalisation du manchon en acier inoxydable ; incorporé (photo 3) au circuit-test et muni de 16 coupons extractibles, il a permis de suivre dans le temps la cinétique de bio-adhésion. Les résultats confirment les observations faites au laboratoire.

<figure><img src="image_129.png" /><figcaption>**Photo 3** - Manchon incorporé dans le circuit-test et coupon extractible incorporé à chacune des faces du manchon.</figcaption></figure>

Des informations complémentaires apparaissent. Des différences de comportement bio-adhésif sont mises en évidence sur le niveau d'encrassement (nombre de cellules vivantes adhérentes sur les coupons), pic d'adhésion (dans le temps). Les niveaux d'adhésion (exemple de la souche Br8 sur le graphique 1) sont importants. L'aptitude des souches à « survivre » à une procédure de nettoyage-désinfection est évaluée, par leur capacité à recontaminer les surfaces (coupons extractibles du circuit-test) après passage d'un vin synthétique stérile. Des différences entre les trois souches apparaissent également.

<figure><img src="image_130.png" /><figcaption>**Graphique 1** - Nombre de cellules vivantes adhérentes sur les coupons en fonction du temps de circulation pour la souche Br8.</figcaption></figure>
Conclusion

En conditions maîtrisées au laboratoire (statique) puis sur circuit-test (dynamique), l'aptitude des trois souches de levures du genre *Brettanomyces* est confirmée. La plate-forme microscopique de l'équipe de l'UMR INRA d'AgroParisTech l'a mis en évidence. Il semble clairement que l'aptitude à l'adhésion soit très sensiblement liée à la nature de la souche de levure et au type de matériau.

Les caractéristiques morphologiques et physiologiques de la souche semblent avoir une incidence directe
sur le pouvoir d'adhésion mais également sur la capacité de la souche à résister à une procédure de
nettoyage-désinfection, puis à recontaminer un autre vin par l'intermédiaire des surfaces. Les résultats
obtenus grâce à la conception – par le CETIM – du manchon équipé de coupons extractibles confirment des
niveaux et pics d'adhésion différents entre les souches. Il en est de même, apparemment, pour la stratégie
de résistance de la souche. Ces données sont à prendre en compte aujourd'hui pour une meilleure gestion
des paramètres hydrodynamiques de la procédure.

Remerciements

Ces travaux ont bénéficié de l'appui financier de FranceAgriMer.

Références

1. Poupault P (2011) Niveau d'hygiène et risque d'altération : modélisation sur circuit-test et validation sur site. 9ème Symposium International d'œnologie de Bordeaux.

2. Poupault P (2012) Altérations microbiennes : origine et aspects préventifs pour l'élaboration des vins. Salon Vinitech, Bordeaux.

3. Poupault P, Pierrat D, Hermon C (2014) Utilisation du circuit-test pour la modélisation des cinétiques d'encrassement lors du transfert du vin. Journée de Restitution RMT Chlean, Paris, Maison du Lait.
# Effect of wine phenolic compounds on the lytic activity of oenophages

P. BALLESTRA<fnref n="1" />*, F. JAOMANJAKA<fnref n="1" />, M. CAYSSIALS<fnref n="1" />, C. PHILIPPE<fnref n="1" />, A. LAGARDE<fnref n="2" />, M. BLANCHE-BARBAT<fnref n="1" />, A. DECENDIT<fnref n="2" />, E. RENOUF<fnref n="2" />, C. LE MARREC<fnref n="2" />, S. CLUZET<fnref n="2" />

*Oenococcus oeni* – lytic phage – phenolic compounds – red wine

## Introduction

Bacteriophages infecting the lactic acid bacterium *Oenococcus oeni*, responsible of driving the malolactic fermentation (MLF) in wine, were first reported in the late of 1970's [1, 2]. Since then, many oenophages were isolated directly from distinct geographical origin wines, especially with sluggish or delayed MLF, or after induction from *O. oeni* lysogenic strains [3-10]. Some of these bacteriophages were characterized in terms of morphology, structural protein composition, lytic spectra, genome size, restriction enzyme analysis and/or DNA homology.

More recently, several prophage complete sequences were provided through the whole-genome sequencing of *O. oeni* lysogenic strains [11, 12]. Their analysis revealed an expanded genetic diversity of oenophages. Genome sequences of *O. oeni* prophages were clustered into four integrase groups (A to D) which were related to the chromosomal integration site [12]. PCR-based methods were developed for detection of *O. oeni* lysogenic strains and for detection and classification of temperate oenophages [12, 13]. The high prevalence of lysogeny in the *O. oeni* species was confirmed [11-13] and some prophages were annotated [12].

Despite the advances in the *O. oeni* bacteriophage molecular characterization, the role of these viruses in the wine ecosystem is still not clearly defined. Some oenophages would be able to induce *O. oeni* growth inhibition and MLF failures in red wines, even at low pH [2, 4], while others on the contrary would not affect MLF, probably due to their potential inactivation, total or partial, by wine conditions, such as low pH (<3.5), ethanol above 5% (v/v) and/or sulfur dioxide content [3, 5]. It was also reported that monomeric phenols of wine, especially caffeic acid, would affect the oenophages infective capacity [5]. In the same way, other wine phenolic compounds might act against *O. oeni* phages since they are inhibitory to animal or bacterial viruses [14, 15].

Actually, the risk of MLF failure associated to phage attack is difficult to assess and may be wrongly neglected. A better understanding of the interaction of the phage with its host and with its environment is needed to point out the factors which govern the activity of oenophages in wine. In this context, the aim of this work was to investigate the effect of wine phenolic compounds on the lytic activity of *O. oeni* bacteriophages.

## Materials and methods

### Isolation of phenolic fractions

A phenolic rich extract obtained from a Cabernet Sauvignon red wine was fractionated with centrifugal partition chromatography (CPC) according to the protocol previously described by Pawlus et al. [16]. A total of 104 phenolic fractions were obtained. Fractions displaying similar phenolic profiles on thin layer chromatogram (TLC) were pooled.

<footnote n="1">¹Univ. de Bordeaux, ISVV, EA 4577, USC 1366, INRA, IPB, Unité ŒNOLOGIE, chemin de Leysotte, F-33882 Villenave d'Ornon, France</footnote>

<footnote n="2">²Univ. de Bordeaux, ISVV, EA 3675, GESVAB, chemin de Leysotte, F-33882 Villenave d'Ornon, France</footnote>
## Quantification of oenophages lytic activity

The resulting 32 fractions were added at a rate of 2 or 10% to Ca-MRS culture medium, and cultures of *Oenococcus oeni* IOEB-S 277, infected or not with the lytic phage OE33PA (=Φ19-277), were conducted in microplate incubator at 30°C. Bacterial growth was assessed by OD600. Effective infection by OE33PA with a multiplicity of infection (MOI) of 0.1 resulted in the inhibition of bacterial growth and then in the lysis of cells. Phenolic fractions were considered as active when their presence in the culture medium induced a significant reduction of the inhibitory activity of the phage on the bacterial growth.

## Analysis of phenolic compounds

The phenolic compounds present in major quantities in active fractions were tentatively identified by liquid chromatography-mass spectrometry (LC-MS) and their impact on OE33PA stability and lytic activity on its bacteria host was studied. They were added to Ca-MRS culture medium for *Oenococcus oeni* growth at concentration levels found in wine. The cultures infected or not with OE33PA at a MOI of 0.01 were conducted in 10-mL vessel at 25°C in triplicate. Bacterial growth was assessed by OD600 and by counting on grape juice (GJ) agar plate. Phage counts were carried out using the classical double-layer plating technique, using Ca-MRS agar.

## Results and discussion

On the 32 phenolic fractions obtained from the Cabernet Sauvignon red wine, only two fractions, designated F9 and F11, were shown to influence the inhibitory activity of OE33PA on *O. oeni* bacterial growth (Table 1). Added at 10% (v/v) to culture medium, F9 and F11 induced a reduction in the inhibition rate of bacterial growth from 100% to 8.44% and 36.68%, respectively.

<table><caption>**Table 1** - Influence of two phenolic fractions from a red wine (Cabernet Sauvignon) on the inhibitory activity of OE33PA on *O. oeni* bacterial growth. As phenolic compounds of the fractions F9 and F11 were dissolved in ethanol, this solvent was added to reference cultures. Cultures, done in duplicate, were stopped in stationary phase after 40h incubation at 30°C. OD measurements were done at 600 nm.</caption><thead><tr><th rowspan="3">Culture medium<br>Ca-MRS with:</th><th colspan="4">Bacterial growth of <i>Oenococcus oeni</i> strain IOEB-S 277</th></tr><tr><th colspan="2">Control cultures</th><th colspan="2">Cultures infected by phage OE33PA (MOI=0.1)</th></tr><tr><th>Final OD value</th><th>Initial OD value</th><th>Final OD value</th><th>Inhibition rate (%)</th></tr></thead><tbody><tr><td>2% ethanol</td><td>0.542 ± 0.002</td><td>0.293 ± 0.003</td><td>0.278 ± 0.000</td><td>100</td></tr><tr><td>10% ethanol</td><td>0.513 ± 0.003</td><td>0.292 ± 0.005</td><td>0.260 ± 0.001</td><td>100</td></tr><tr><td>2% F9</td><td>0.485 ± 0.011</td><td>0.321 ± 0.003</td><td>0.314 ± 0.004</td><td>100</td></tr><tr><td>10% F9</td><td>0.735 ± 0.017</td><td>0.498 ± 0.013</td><td>0.715 ± 0.070</td><td>8.44</td></tr><tr><td>2% F11</td><td>0.552 ± 0.010</td><td>0.293 ± 0.001</td><td>0.295 ± 0.002</td><td>99.23</td></tr><tr><td>10% F11</td><td>0.506 ± 0,000</td><td>0.307 ± 0.006</td><td>0.433 ± 0.009</td><td>36.68</td></tr></tbody></table>

$$ \text{Inhibition rate} = \frac{\text{final OD value of control culture} - \text{final OD value of infected culture}}{\text{final OD value of control culture} - \text{initial OD value of infected culture}} \times 100 $$

LC chromatograms recorded for the fractions F9 and F11 at 306 nm are shown in Figure 1 (A and B, respectively). In fraction F9 (Figure 1A), 9 peaks were detected. Among the compounds present in higher amount, those at t<sub>R</sub> of 26.1 and 30.9 have been identified as *trans*-resveratrol and quercetin, respectively. The peaks eluted at 24.1 min and 30.1 min have been identified as myricetin and *cis*-resveratrol. Fraction F11 (Figure 1B) presented one prominent peak (t<sub>R</sub> 13.7) and 3 major other ones (t<sub>R</sub> 23.6, 25.7 and 30.9) among 14 detected peaks. The main phenolic compound has been shown to correspond to *p*-coumaric acid. Two of the other three major compounds have been identified as myricetin and quercetin.
<figure><img src="image_131.png" /><figcaption>**Fig. 1** - LC chromatograms at 306 nm of the F9 (A) and F11 (B) fractions. Peak identification was based on (i) analysis of the retention times ($t_R$), wavelengths of maximum absorbance ($\lambda_{max}$), pronated molecules ($[M+H]^+$) and major fragment ions (MS/MS in positive mode) and (ii) comparison to published literature [17-19].</figcaption></figure>

Within the compounds identified in active fractions, quercetin was the first to be tested for its ability to reduce the lytic activity of phage OEPA33. Preliminary experiments were performed to investigate the stability of the phage at 25°C in presence of quercetin. No decrease in phage count was observed during the five days of incubation in the Ca-MRS medium supplemented with quercetin at 50 or 100 mg/L (data not shown). Efficiency of infection by OE33PA was then investigated in presence of quercetin, as shown in Figure 2 (A and B). The addition of quercetin at 50 mg/L to the culture medium slowed the bacterial growth but limited the effects of phage attack. The inhibition rate of bacterial growth due to OE33PA reached 97.35% when infection was done in Ca-MRS and dropped to 58.39% when the medium was supplemented with quercetin (after 58h incubation at 30°C, Figure 2A). In the same way, the viral population was significantly lower when quercetin was included in the culture medium.

<figure><img src="image_132.png" /><figcaption>**Fig. 2** - Bacterial growth of *Oenococcus oeni* IOEB-S 277 at 25°C in cultures infected or not with phage OE33PA. The phage was added at t=0 at MOI 0.01. Values represent the absorbance at 600 nm (A) and the bacterial counts (B) of one of three independent experiments.</figcaption></figure>
The negative effect of quercetin on the growth of *O. oeni* has already been reported by Garcia Ruiz et al. [20]. Our results suggest that quercetin may modify *O. oeni* physiology so that the lytic development of OE33PA is reduced. Changes induced by quercetin may affect the cell wall and limit the adsorption of the phage to the bacterium. Same experiments were done with *trans*-resveratrol at 58 mg/L. The results showed no reduction of the lytic activity of OE33PA. The presence of *trans*-resveratrol, at a concentration level found in wines, induced only a decrease in growth of *O. oeni*. Such inhibiting effect has already been reported but at higher concentrations [20].

Our study should now be extended to *p*-coumaric acid and other phenolic compounds identified in the fractions F9 and F11.

## References

1. Sozzi T, Maret R, Poulin JM (1976) Mise en évidence de bactériophages dans le vin. *Experientia* **32**: 568-569.

2. Sozzi T, Gnaegi F, D'Amico N et al (1982) Difficultés de fermentation malolactique du vin dues à des bactériophages de *Leuconostoc oenos*. *Rev Suisse Vitic Arboric Hortic* **14**: 17-23.

3. Davis C, Neliane F, Silveira A et al (1985) Occurrence and properties of bacteriophages of *Leuconostoc oenos* in Australian wines. *Appl Environ Microbiol* **50**: 872-876.

4. Henick-Kling T, Lee TH, Nicholas DJD (1986) Inhibition of bacterial growth and malolactic fermentation in wine by bacteriophage. *J Appl Bacteriol* **61**: 287-293.

5. Henick-Kling T, Lee TH, Nicholas DJD (1986) Characterization of the lytic activity of bacteriophages of *Leuconostoc oenos* isolated from wine. *J Appl Bacteriol* **61**: 525-534.

6. Nel L, Wingfield BD, Van der Meer LJ et al (1987) Isolation and characterization of *Leuconostoc oenos* bacteriophages from wine and sugarcane. *FEMS Microbiol Lett* **44**: 63-67.

7. Arendt EK, Lonvaud A, Hammes WP (1991) Lysogeny in *Leuconostoc oenos*. *J Gen Microbiol* **137**: 2135-2139.

8. Arendt EK, Hammes WP (1992) Isolation and characterization of *Leuconostoc oenos* phages from German wines. *Appl Microbiol Biotechnol* **37**: 643-646.

9. Tenreiro R, Santos R, Brito L et al (1993) Bacteriophages induced by mitomycin C treatment of *Leuconostoc oenos* strains from Portuguese wines. *Lett Appl Microbiol* **16**: 207-209.

10. Poblet-Icart M, Bordons A, Lonvaud-Funel A (1998) Lysogeny of *Oenococcus oeni* (syn. *Leuconostoc oenos*) and study of their induced bacteriophages. *Curr Microbiol* **36**: 365-369.

11. Borneman AR, McCarthy JM, Chambers PJ et al (2012) Comparative analysis of the *Oenococcus oeni* pan genome reveals genetic diversity in industrially-relevant pathways. *BMC Genomics* **13**: 373.

12. Jaomanjaka F, Ballestra P, Dols-Lafargue M et al (2013) Expanding the diversity of oenococcal bacteriophages: insights into a novel group based on the integrase sequence. *Int J Food Microbiol* **166**: 331-340.

13. Doria F, Napoli C, Costantini A et al (2013) Development of a new method for detection and identification of *Oenococcus oeni* bacteriophages based on endolysin gene sequence and randomly amplified polymorphic DNA. *Appl Environ Microbiol* **79**: 4799-4805.

14. Konowalchuk J, Speirs JI (1976) Virus inactivation by grapes and wines. *Appl Environ Microbiol* **32**: 757-763.

15. Lee A, Eschenbruch R, Waller J (1985) Effect of phenolic compounds, ethyl alcohol, and sodium metabisulphite on the lytic activity of phage PL-1 on a *Lactobacillus casei* S. strain. *Can J Microbiol* **31**: 873-874.

16. Pawlus AD, Cantos-Villar E, Richard T et al (2013) Chemical dereplication of wine stilbenoids using high performance liquid chromatography-nuclear magnetic resonance spectroscopy. *J Chromatogr A* **1289**: 19-26.

17. Montoro P, Piacente S, Oleszek W et al (2004) Liquid chromatography/tandem mass spectrometry of unusual phenols from *Yucca schidigera* bark: comparison with other analytical techniques. *J Mass Spectrom* **39**: 1131-1138.

18. Pellati F, Orlandini G, Pinetti D et al (2011) HPLC-DAD and HPLC-ESI-MS/MS methods for metabolite profiling of propolis extracts. *J Pharm Biomed Anal* **55**: 934-948.

19. Ma YL, Li QM, Van den Heuvel H et al (1997) Characterization of flavone and flavonol aglycones by collision-induced dissociation tandem mass spectrometry. *Rapid Commun Mass Spectrom* **11**: 1357-1364.

20. Garcia-Ruiz A, Moreno-Arribas V, Martin-Alvarez PJ et al (2011) Comparative study of the inhibitory effects of wine polyphenols on the growth of enological lactic acid bacteria. *Int J Food Microbiol* **145**: 426-431.
# La bio-protection du moût par l'utilisation de levures non-Saccharomyces

A. DELAHERCHE<fnref n="1" />*, A. IMMELE<fnref n="2" />, C. PAGAGNI<fnref n="1" />, D. MAUREL<fnref n="1" />, W. MULINAZZI<fnref n="1" />

Levure non-Saccharomyces – bio-protection – sulfitage

## Introduction

Le millésime 2014 confirme le réchauffement climatique et pose une situation microbiologique inédite. Nous observons, non seulement une infection acétique hors normes, mais aussi une énorme quantité de germes par baie, qui pose un problème de fermentation prématurée et compromet le débourbage des jus blancs. Température, blessure des grains par *Drosophila suzukii*, maturité et pH favorisent la multiplication de la microflore sur le raisin, mais au détriment de l'implantation du genre *Saccharomyces*, et au profit de nombreux autres, dont *Brettanomyces bruxellensis* et les bactéries. Les piqûres fulgurantes en début de vinification ou les odeurs de cheval après la fermentation malolactique peuvent être spectaculaires, mais le plus souvent les microorganismes impactent la qualité des vins de façon plus insidieuse. Les vinificateurs se soucient beaucoup de l'extraction du potentiel aromatique du raisin, mais pas toujours de l'effet « masqueurs d'arômes » dont est responsable une partie de la microflore. Il est vrai que dans des vignobles septentrionaux ou avant le réchauffement climatique, l'objectif majeur était d'extraire les arômes : par vendange fraîche et de moindre maturité la microflore était facilement tenue à l'écart avec 5g/hl de SO₂. Par une maturité et une température plus élevée, les priorités s'inversent : le potentiel aromatique et phénolique s'extraient facilement mais la microflore devient envahissante et le sulfitage inefficace. Et la menace microbiologique s'aggrave si des pourritures s'installent : les millésimes 2006 et 2014 étaient une illustration de ces phénomènes. Tout porte à croire qu'ils seront de plus en plus fréquent et que nous devrons adapter nos techniques de vinification.

## Les levures sélectionnées ne standardisent pas les vins

C'est une question fréquente dans les vignobles : « le levurage systématique, par quelques souches majeures, n'est-il pas une source d'uniformisation ou de perte de typicité locale » ? Il faut se rappeler que le genre *Saccharomyces* ne produit aucune extravagance, c'est une levure relativement neutre qu'elle soit sélectionnée ou d'origine indigène. Sa principale qualité est de protéger le jus contre l'invasion d'autres genres. Elle évite la production de masqueurs d'arômes et aide ainsi à l'expression de la typicité. L'élaboration de la complexité et de la personnalité d'un vin ne se joue pas sur l'une ou l'autre variété de *Saccharomyces* fermentaire. Certes elle peut favoriser des thiols volatils ou des esters dominants comme l'isoacétate d'amyle, si elle est mise dans des conditions précises de fermentation, mais ce ne sont pas des arômes durables. Ils laissent facilement la place à une expression locale, s'il y en a une !

## Le sulfitage a plus d'effet sur la standardisation des vins que le levurage

La personnalité d'une cuvée se forme dès la phase pré-fermentaire. A la récolte ou au pressurage des centaines d'espèces différentes sont dispersées dans le jus. Il faut rappeler que le jus de raisin, tant qu'il est emprisonné dans le grain, est stérile. L'originalité d'une cuvée n'est pas nécessairement due à une souche spécifique de levure. Elle est liée à un agencement de genres de micro-organismes très variés en nombre et proportion. La configuration particulière, en genre, nombre, proportion et succession de la flore, participe à la complexité typique d'un cru. Or cette configuration, reposant sur des centaines d'espèces, est détruite par

<footnote n="1">¹AEB France, 10 rue du stade, 68240 Sigolsheim, France</footnote>

<footnote n="2">²VINEDIA, 2 rue Root, 68150 Osteheim, France</footnote>
le sulfitage. Bien sûr, tous les micro-organismes ne sont pas éliminés. Le SO₂ opère une sélection et laisse subsister quelques souches résistantes. Mais le sulfitage est-il un sélectionneur pertinent ? Oui, dans certaines conditions, pour assurer la sécurité et vinifier un vin loyal et marchand, mais au prix de la perte d'éléments caractéristiques de la cuvée.

D'autre part, la sélection par le SO₂, favorise l'émergence de souches résistantes aux sulfites, productrices d'H₂S ou d'éthanal. Le vin final pourrait être moins expressif et combiner plus de sulfites.

## Comment bénéficier de la flore indigène en évitant ses dérives ?

L'expression de la flore indigène et des levures sélectionnées ne sont pas incompatibles. La microflore pré-fermentaire indigène non-saccharomyces peut se révéler positive dans des conditions particulières dont les ordres de grandeur peuvent se résumer par une vendange saine, un pH inférieur à 3.5 et une maturité inférieure à 14% d'alcool potentiel.

Dans ces conditions, il est possible de laisser s'exprimer la flore indigène avant d'implanter une levure de l'espèce saccharomyces. Bien sûr, pour bénéficier du travail de cette flore, il faut proscrire tout sulfitage. Pour laisser de la place à la flore spontanée, il faudra retarder le levurage de 24 à 72 heures après le débourbage ou après l'encuvage. Mais le non-levurage ou le levurage retardé présente des risques importants que le vinificateur devra savoir gérer. On ne le répétera jamais assez : ce n'est pas l'abandon du sulfitage qui présente des risques, mais l'abandon du levurage.

## La protection biologique pour une meilleure typicité

Même dans nos régions septentrionales de Bourgogne, d'Alsace ou de Champagne, la climatologie actuelle demande une protection biologique et autorise rarement la fermentation spontanée sous peine de montée en acidité volatile et autres odeurs acétates. Le millésime 2014 s'est caractérisé par un inoculum de population indigène très important, qui dépassait parfois 2 millions de germes/mL, dès la récolte, et avec une proportion de bactéries acétiques inédites. En vinification en blanc, nous avons observé des démarrages de fermentation spontanée dans les 2 heures qui suivaient le pressurage. Avec de tels inoculums le sulfitage est inopérant et nous l'avons confirmé sur ce millésime.

## Vinification en blanc

La vinification en blanc pourrait s'orientée vers l'abandon de l'utilisation du SO₂ et du débourbage statique, au profit de la bio-protection et du débourbage dynamique.

<figure><img src="image_133.png" /><figcaption>Fig. 1 - Incidence de l'apport de Primaflora sur de déclenchement de la FA.</figcaption></figure>
La figure 1 ci-dessus montre que la bio-protection ne provoque pas de fermentation prématurée. Dans ces conditions climatiques, la stratégie sera, à l'avenir, d'abandonner le débourbage statique au profit de la flottation ou de la filtration des moûts. La bio-protection par l'addition de 3g/hl de Primaflora s'est montrée efficace contre les formations d'acétates. Le débourbage statique peut encore s'envisager quand on travaille en raisin entier jusqu'au pressurage et que l'on sépare les premiers jus d'écoulage, les plus chargés en micro-organismes indigènes et acétiques. Ces jus devraient être filtrés. Cette pratique de « fractionnement microbiologique » au moment du pressurage, proposée par Nicolas Secondé, a permis de limiter les départs en fermentation prématurés et d'autoriser un temps de débourbage statique.

## Vinification en rouge

La bio-protection par 5g/hl de Primaflora VR dès la machine à vendanger en remplacement du sulfitage a été appliquée pour la première fois à grande échelle dans divers vignobles, alors que le millésime présentait des risques microbiologiques particulièrement élevés. Cette démarche s'est montrée payante et a rassuré des œnologues très inquiets à l'idée d'abandonner le sulfitage des moûts. La réussite des vinifications en rouge, aussi bien vis-à-vis des « *Brettanomyces* » que des bactéries acétiques, repose sur ces deux opérations, sans sulfitage :

- Utilisation de Primaflora VR le plus tôt possible, à la vigne.

- Levurage très homogène appliqué en cours d'encuvage en goutte à goutte ou par pompe doseuse.

## Conclusion

Dans l'œnologie actuelle, il n'y a pas de gestion microbiologique pré-fermentaire. Cette étape étant neutralisée par le SO₂. Le sulfitage des moûts est un facteur important de réduction de la typicité. La lutte biologique, encore appelée bio-protection, permet de limiter des déviations aromatiques liées à un développement de la levure d'altération *Brettanomyces bruxellensis* et de favoriser la typicité. Le millésime 2014, très touché par *Drosophila Suzuki* a montré, la pertinence de cette stratégie. La gestion pré-fermentaire des microorganismes des moûts devrait faire partie de l'art de vinifier et ne plus se résumer au sulfitage.
# Selection characterization and evaluation of autochthonous *Saccharomyces cerevisiae* strains as wine starters for industrial fermentations of Agiorgitiko and Moschofilero grape musts

D. KONTOGIANNATOS<fnref n="1" />, A. BELKA<fnref n="1" />, V. TROIANOU<fnref n="1" />, Y. KOTSERIDIS<fnref n="1" />,<fnref n="2" />*

*Saccharomyces cerevisiae* – autochthonous strain – wine starters – Greek cultivars

## Introduction

The purpose of this study was to standardize a methodology for the isolation of indigenous *S. cerevisiae* yeast strains from two protected designations of origin located in Greece. The isolated yeast strains were assessed as potential fermentation starters in order to enhance the expression of the regional characteristics through the produced wines and to improve the quality. Indigenous *S. cerevisiae* yeast strains were isolated from spontaneously fermented grapes of cv. Agiorgitiko (Nemea region) and cv. Moschofilero (Mantinia region). 28 and 33 yeasts were isolated from Agiorgitiko and Moschofilero spontaneously fermented wines and two of them (one from each fermentation) were selected according to their unique random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) patterns. These strains, L2M (Moschofilero) and Soi2 (Agiorgitiko) will be assessed in industrial fermentations of Agiorgitiko and Moschofilero grape musts.

## Materials and methods

### Fermentation conditions

Two different spontaneous fermentations were performed in 5000-l stainless-steel fermenters, which contained 2000 l and 3000 l of Agiorgitiko and Moschofilero musts during the 2013 vintage. Fermentations were carried out until Bé was equal to 0. After the completion of the fermentations, 50-ml samples from the middle zone of each fermenter were taken in sterile falcon conical tubes. The samples were placed at -20°C for temporary storage. Serial dilutions of wine samples (10⁻<fnref n="1" />-10⁻¹⁰) were made up in 2% w/v peptone solutions. 100 µl of each dilution was inoculated on YPD agar plates and incubated for 2 days at 28°C. 10⁻⁴ and 10⁻⁵ dilutions of Agiorgitiko and Moschofilero plates, respectively, allowed us to clearly isolate 28 and 33 yeasts, respectively.

### PCR conditions

RAPD-PCR was used to analyze 28 and 33 yeasts from Agiorgitiko and Moschofilero wines, respectively. For this analysis, yeasts were screened with the RAPD-12 (5'-ACGGCGATGA-3'), OPA-12 (5'-TCGGCGATAG-3') and OPC-06 (5'-GAACGGACTC-3') primers. RAPD-PCR profiles were analyzed by 1% w/v agarose gel electrophoresis. Images of DNA patterns obtained after agarose gel electrophoresis were analyzed using the PyElph software [1] in order to prepare the phylogenetic dendrograms using the neighbor joining method. Reproducibility of banding patterns for all isolates was also evaluated.

## Results and discussion

Since the aim of the study was to isolate the most phylogenetically scarce *S. cerevisiae* yeast strains, from both Agiorgitiko and Moschofilero spontaneously fermented wines, research for RAPD-PCR primers was

<footnote n="1">¹Oenolysis, 16 Marathonos Avenue, 15351, Pallini, Greece</footnote>

<footnote n="2">²Department of Food Science and Technology, School of Food, Biotechnology and Development, Agricultural University of Athens, Iera Odos 75, 11855, Athens, Greece</footnote>

<footnote>*Corresponding author: ykotseridis@aua.gr</footnote>
conducted. This should produce few but distinct PCR bands instead of a large number of indiscernible ones. Moreover, in the first RAPD-PCR screen a less time-consuming protocol was adopted which, on the one hand, resulted in the isolation of low quality genomic DNA but, on the other hand, allowed to screen a large number of colonies.

28 yeasts from Agiorgitiko and 33 yeasts from Moschofilero were DNA extracted and analyzed in RAPD-PCR analysis using the RAPD12 primer. RAPD12 primer (5'-ACGGCGATGA-3') resulted in three major types of RAPD-PCR patterns (A, B and C) (Fig. 1). And the three kinds of patterns were observed in the isolated yeasts of both Agiorgitiko and Moschofilero varieties (Fig. 1). Two strains/28 yeasts (7.1%) from Agiorgitiko and two strains/33 yeasts (3%) from Moschofilero spontaneously fermented musts presented unique RAPD-PCR patterns compared with the dominant ones. In the case of Moschofilero, the unique pattern consisted of three bands of ~1200 bp, ~1000 bp and ~800 bp (pattern D), and the strain was named L2M, while in the case of Agiorgitiko the unique pattern consisted of four major bands of ~1250 bp, ~800 bp, ~450 bp and ~350 bp (pattern E), and the strain was named Soi2 (Fig. 1). Pattern A was observed in 28.6% of the total analyzed yeasts (8/28) in the Agiorgitiko variety and 30.3% (10/33) in the Moschofilero variety. Pattern B was observed in 35.7% of the total analyzed yeasts (10/28) in the Agiorgitiko variety and 36.4% (12/33) in the Moschofilero variety. Finally, pattern C was observed in 28.6% of the total analyzed yeasts (8/28) in the Agiorgitiko variety and 30.3% (10/33) in the Moschofilero variety.

<figure><img src="image_134.png" /><figcaption>**Fig. 1** - RAPD-PCR analysis of Agiorgitiko and Moschofilero indigenous yeast strains primed-off with the RAPD-12 primer.</figcaption></figure>

One strain from each RAPD-PCR pattern was selected in order to proceed to further molecular analyses. The L2M (pattern D), M9 (pattern C) and L26A (pattern B) strains were selected from Moschofilero grapes, while the Soi2 (pattern E) and Soi103 (pattern A) strains were selected from Agiorgitiko grapes (Fig. 1).

Further RAPD-PCR analyses were performed with DNA isolated from the 5 indigenous *S. cerevisiae* yeast strains and 3 commercial ones that have been used extensively from the two wineries in the past. The commercial yeast strains were (a) SC22 (Fermentis), which is particularly adapted to cultivars like Cabernet Sauvignon, Cabernet Franc and Merlot, (b) Saint Georges S101 (Fermentis), which is adapted to cultivars like Pinot Noir and Gamay as well as to all other light-body red varieties intended to produce wines with a rapid turnover (young Tempranillo, Sangiovese, Cinsault, Pinotage, Grenache) and (c) Martin Viallate's 58W3 strain. RAPD-PCR analysis with OPA-12 and OPC-06 primers showed a clear phylogenetic relationship between all strains tested (Fig. 2). OPC-06 resulted in bands of low polymorphism so it was discarded from further analyses (Fig. 2). The RAPD fragments which were generated with OPA-12 primer were further analyzed using the PyElph 1.4 software (see M&M). Reproducibility of banding patterns for 8 strains was also evaluated. Phylogenetic analysis revealed that the two Moschofilero isolates (L2M and M9) grouped together in a separate clade far away from the other yeast strains (L26A, Soi2, Soi103, 58W3 and Sc22), while the Soi2 strain which was isolated from the Agiorgitiko spontaneous fermentation was grouped in a second clade separated from the L26A, Soi103, 58W3 and Sc22 ones (Fig. 3). 58W3, Soi103, S101, Sc22 and L26A seemed to be the most phylogenetically related compared with the other strains (Fig. 3).
<figure><img src="image_135.png" /><figcaption>**Fig. 2** - RAPD-PCR analysis of Agiorgitiko and Moschofilero indigenous yeast strains primed-off with the OPA-12 and OPC-06 primers.</figcaption></figure>

<figure><img src="image_136.png" /><figcaption>**Fig. 3** - Phylogenetic analysis of RAPD-PCR patterns of Agiorgitiko and Moschofilero indigenous yeast strains primed-off with the OPA-12 primer.</figcaption></figure>

The combination of phylogenetic, biochemical and oenological analyses revealed that Soi2 (Agiorgitiko) and L2M (Moschofilero) yeast strains seem to be the best candidates for proceeding into industrial fermentations of Agiorgitiko and Moschofilero musts. As a result, during the 2014 vintage these strains were used in order to perform industrial vinifications. Three industrial vinifications per yeast strain were performed in 5000-l capacity stainless-steel wine tanks. Both Soi2 and L2M yeast strains were used either for the fermentation of Agiorgitiko and Moschofilero musts. For Moschofilero fermentation, a commercial yeast strain frequently used for Moschofilero wine production was used (CYS2), while for Agiorgitiko fermentation a common commercial yeast strain for this variety was used (CYS3). Fermentation's behaviour was checked daily by temperature and sugar content measurements. Average sugar content consumption slopes were calculated during fermentations while wine analytical parameters were calculated after the completion of the fermentations.

Our results showed similar fermentation behaviour for L2M and Soi2 strains in both Moschofilero and Agiorgitiko fermentations when compared with the CYS2 and CYS3 reference yeasts (Fig. 4). For Moschofilero fermentations, similar analytical characteristics were observed for both indigenous strains compared with the control CYS2 strain (data not shown). Ethanol content, free and total SO₂, pH, total acidity and density were similar in all strains tested (data not shown.). CYS2 resulted in the lower concentration levels of reducing sugars in Moschofilero fermentations, while both indigenous yeast strains resulted in higher but not prohibitive volatile acidity levels (data not shown). For Agiorgitiko fermentations, similar analytical characteristics were observed between all strains tested regarding the ethanol content, free and total SO₂, pH, total acidity and density (data not shown). And in this case the L2M resulted in higher volatile acidity and reduced sugar levels, while Soi2 and CYS3 resulted into wines with similar characteristics regarding these two parameters. Soi2 resulted in higher colour density and hue levels (data not shown). Moreover, sensory evaluation tests resulted in almost similar scores. According to the global sensory quality assay these wines presented similar sensory profiles when compared to the control (fermented with the reference CYS2 and CYS3 strains for Moschofilero and Agiorgitiko, respectively) after comparative evaluation of the appearance, aroma, flavour and texture of the wines (data not shown).
<figure><img src="image_137.png" /><figcaption>**Fig. 4** - Sugar consumption slopes (°Bé at 20°C) of industrial vinifications of Moschofilero (A) and Agiorgitiko (B) grape musts fermented with the L2M and Soi2 S. cerevisiae strains. CYS2 and CYS3 were used as reference strains for Moschofilero and Agiorgitiko, respectively.
Data are expressed as mean ± SD of three experimental replicates.</figcaption></figure>

**Conclusion**

We have developed an easy-to-use laboratory methodology which can be directly applied in oenological practices in order to phylogenetically distinguish S. cerevisiae autochthonous yeast strains isolated from Greek terroirs. Our methodology clearly distinguished S. cerevisiae yeast strains from spontaneously fermented musts of Nemea (Agiorgitiko) and Mantinia (Moschofilero) regions (Greece), in order to assess their oenological, molecular and biochemical characteristics as potential autochthonous yeast starters for industrial fermentations. Our results demonstrate the feasibility of using this methodology for the isolation and characterization of S. cerevisiae indigenous yeast strains in a commercial context. Moreover, we found that L2M and Soi2 strains could be easily used as wine starters for alcoholic fermentations of Moschofilero and Agiorgitiko musts; surprisingly Soi2, even if it was isolated from Agiorgitiko spontaneous fermentations, could be also exploited for the fermentation of Moschofilero or more generally for the industrial fermentations of white grape musts.

**Acknowledgements**

We would like to thank Ms. Z. Konstantina (Domaine Zafeiri, Nemea, Greece) and Mr. G. Papadopoulos (Domaine Papadopoulos, Mantinia, Greece) for providing us with the Agiorgitiko and Moschofilero grape musts, respectively. This program is funded by the Operational Program "Human Resources Development" of the National Strategic Framework (NSRF) 2007-2013.

**References**

1. Pavel AB, Vasile CI (2012) PyElph - a software tool for gel images analysis and phylogenetics.
*BMC Bioinform* **13**: 9.
# Acetic acid bacteria detection and quantification in red wine by qPCR

C. LONGIN*, M. GUILLOUX-BENATIER, H. ALEXANDRE

UMR Procédés Alimentaires et Microbiologiques, Equipe VAIMiS (Vin, Aliment, Microbiologie, Stress),
AgroSup Dijon – Université de Bourgogne, IUVV, rue Claude LADREY, BP 27877, 21000 Dijon, France

AAB – microbiological internal control – DNA extraction – qPCR

## Introduction

Acetic acid bacteria (AAB) (*Acetobacter* spp., *Gluconobacter* spp., *Gluconacetobacter* spp.) are wine spoilage microorganisms. They metabolize ethanol to acetaldehyde which leads to an increase of acetic acid during winemaking, aging or wine storage.

Many techniques allow the detection of AAB such as nested PCR or by detection of *adhA* gene by PCR. Other techniques allow the interspecies detection of the presence of AAB [1] such as DGGE, TGGE, PCR-RFLP, MALDI-TOF-MS [2] AFLP, RAPD, ERIC-PCR and REP-PCR. However, today quantification of total AAB or species specific AAB could only be done using qPCR [1].

Detection and quantification of AAB is useful during winemaking and wine storage in order to prevent AAB development and increase in volatile acidity. However, the reported qPCR protocols are always based on wine artificially enriched in AAB without growth in wine medium. Moreover, enumeration by common laboratory media is uncertain and not reproducible, especially the samples coming from extreme environments such as wine [3] Therefore, the aim of our study was to develop a qPCR protocol that allows the detection-quantification of AAB naturally present in wine.

## Materials and methods

### Bacterial strains and growth conditions in red wine

Four AAB were used: *A. aceti* DSM 3508; *A. pasteurianus* (isolated from vinegar); *G. oxydans* DSM 7145; and *Ga. liquefaciens* CIP 103109. Cells were pre-adapted to alcohol using a synthetic wine medium (5%v/v) and then inoculated in filtrated (0.2-µm) Pinot noir red wine (pH: 3.5; TAV: 12%v/v).

### Internal control for DNA isolation and amplification

An internal control was included in the assay by adding 10⁴ cells/mL of *Escherichia coli* in order to have a control for DNA preparation as well as PCR amplification. 4 log cells/mL of internal control gave a Cq value of 25.5 ± 0.4.

### DNA isolation

Two DNA extraction protocols were tested to compare and highlight the most discriminating:

- CTAB (cetyltrimethylammonium bromide) [4], the most used method in microbiology laboratories for AAB DNA extraction (Ausubel method),

- A technique developed for the extraction and the purification of plant DNA [5], which also uses CTAB (Doyle method).

At the beginning of the DNA extractions, the AAB pellets were treated or not with PVPP in order to eliminate phenolic compounds: No treatment (NT): pellets were suspended in lysis buffer; PVPP: pellets were suspended in lysis buffer supplemented with PVPP to obtain a final level of 1% (w/v).

Each bacterium species was analyzed in red wine in three independent experiments and for each individual experiment, three replicates were performed. Moreover, three wines naturally contaminated were analyzed.

<footnote>*Corresponding author: cedric.longin@u-bourgogne.fr</footnote>
## Real-time PCR amplification

Acetic acid bacteria primers amplifying the 16S rRNA gene were selected [6]: AAB-F (5' TGAGAGGATGA TCAGCCACACT 3'), the reverse primer AAB-R (5' TCACACACGCGGCATTG 3'). BSA (400 ng/µL) or PVP (0.5% (w/v)) addition in the qPCR mix was assessed in order to improve PCR amplification quality.

# Results and discussion

## Specificity

The specificity of the PCR assays was tested *in silico* and *in vitro* against *Oenococcus oeni*, *Pediococcus* sp. and *Lactobacillus* sp. The specificity of the qPCR assay was also tested against a panel of yeast strains encountered in wine: *Zygosaccharomyces bailii*, *Candida vini*, *Pichia membranifaciens*, *P. fermentans* and *Saccharomyces cerevisiae*. C_q value of *O. oeni* is 25.9 ± 0.4 whatever the cell concentration tested. Moreover, for the yeasts mentioned earlier, C_q value is 30.3 ± 0.3. So, no interferences with the AAB detection and quantification were shown.

## DNA isolation methods comparison and PCR inhibition assay

DNA extraction of AAB grown in red wine was performed after addition of 10⁴ cells/mL *E. coli* (internal control) using two different methods (Doyle and Ausubel methods) with or without PVPP treatment during cell lysis. Then, qPCR was performed either with BSA or PVP or without (control). For *A. pasteurianus* (Figure 1) and the three other AAB (data not shown), the Doyle method with or without PVPP lead to a C_q much lower (more efficient) than the Ausubel method. Moreover, the figure shows that PVPP improves the qPCR efficiency. However, using BSA and PVP in the qPCR mix does not enhance qPCR efficiency for the Doyle method. From this, the Doyle DNA extraction method was used for all AAB DNA extraction in red wine.

<figure><img src="image_138.png" /><figcaption>**Fig. 1** - Results of qPCR (C_q) after Doyle and Ausubel DNA extraction with or without PVPP during cell lysis of a sample of *A. pasteurianus* in red wine. BSA or PVP were added to the qPCR mix and qPCR efficiency was compared to Control.</figcaption></figure>

## Linearity, repeatability and reproducibility in red wine

After growth of each AAB in red wine and enumeration by flow cytometry, Doyle DNA extraction with PVPP was realized (three independent experiments, three replicates). An example of a representative standard curves in red wine for the four AAB is presented in Figure 2. From this, efficiency, r² and y-intercept were determined. The four AAB have an efficiency, r² and y-intercept statistically identical following an analysis by a Tukey test (data not shown). These results highlight that whatever the bacteria species present in red wines the C_q for a given population is similar, hence, our method allows a reliable quantification of various AAB specie in wines.

## Evaluation of the method with naturally spoiled red wines

AAB were quantified in three red wine samples enriched with our internal control (Figure 2). Wine dilutions were performed to assess and validate the linearity of the method. No statistic differences between red wines artificially and naturally contaminated were measured (data not shown). The trend curve equation for detection and quantification of AAB in red wines is: -0.45*C_q* + 14.11.
<figure><img src="image_139.png" /><figcaption>**Fig. 2** - Example of standard curves Cq as a function of log cells/mL obtained from Doyle DNA extraction from AAB artificially inoculated in red wine and 3 red wines naturally contaminated obtained by dilution of initial AAB sample before extraction. Standard deviation mean for Cq is 1.7.</figcaption></figure>

### Limits of detection (LOD) and quantification (LOQ)

The method for LOD and LOQ determination uses slope, residue standard deviation and standard deviation of the intercept obtained from linearity validation experiments [7]. With this method, LOD and LOQ values of 2 and 5.2·10³ cells/mL, respectively, were obtained.

## Conclusion

In this study, we have developed a qPCR method that allows reliable detection and quantification of acetic acid bacteria in red wine. We have shown that the previously reported DNA extraction method was not efficient enough for a precise quantification (high AAB LOD & LOQ). Finally, as far as we know, we have for the first time validated a qPCR protocol for AAB quantification in wines artificially or naturally contaminated. Specific AAB species quantification is not possible with our method, however, our main goal is to quantify AAB in wines whatever the species present in order to evaluate the risk of spoilage.

## Acknowledgements

The authors gratefully thank the Regional Council of Burgundy and the Interprofessional Office of Burgundy Wines for providing financial support.

## References

1. Sengun IY, Karabiyikli S (2011) Importance of acetic acid bacteria in food industry. *Food Control* **22**: 647-656.

2. Andrés-Barrao C, Benagli C, Chappuis M et al (2013) Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting. *Syst Appl Microbiol* **36**: 75-81.

3. Valera MJ, Torija MJ, Mas A et al (2013) *Acetobacter malorum* and *Acetobacter cerevisiae* identification and quantification by Real-Time PCR with TaqMan-MGB probes. *Food Microbiol* **36**: 30-39.

4. Ausubel FM, Brent R, Kingston RE et al (1992) *Short Protocols in Molecular Biology*. John Wiley & Sons Inc, London.

5. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. *Focus* **12**: 13-15.

6. Valera MJ, Torija MJ, Mas A et al (2015) Acetic acid bacteria from biofilm of strawberry vinegar visualized by microscopy and detected by complementing culture-dependent and culture-independent techniques. *Food Microbiol* **46**: 452-462.

7. OIV (2005) Practical guide for the validation, quality control, and uncertainty assessment of an alternative oenological analysis method. *Compendium of International Analysis of Methods - OIV, Guide for the Validation – Quality Control*.
# Carbohydrate metabolism
## of *Brettanomyces bruxellensis*

A. CIBRARIO<fnref n="1" />*, E. CANAC<fnref n="1" />, M. DOLS-LAFARGUE<fnref n="1" />,<fnref n="2" />, P. BALLESTRA<fnref n="1" />

*Brettanomyces bruxellensis* – carbohydrate requirements – diversity – adaptation

## Introduction

Among wine spoilage yeasts, *Brettanomyces bruxellensis* is the most dreaded. It can be detected at almost every stage of vinification, from alcoholic fermentation to bottling. It produces volatile phenolic compounds which cause off-flavors described as spicy, sweaty animal, leather, medicinal, smoked and clove-like aromas [1, 2]. This wine alteration is an important issue in winemaking as it causes the wine rejection by the consumers and devalues the image of the producer. All actors in the sector are looking forward to solutions to detect and prevent the development of these yeasts.

The nutrient requirements of *B. bruxellensis* remain unclear probably because the species displays a high level of genomic and phenotypic diversity [3]. Defining the carbohydrate requirements of various *B. bruxellensis* strains would contribute to a better knowledge of the species phenotypic diversity. It would also enlighten us about the *B. bruxellensis* adaptation to microbial competition in wine.

## Materials and methods

### Yeasts

Ten *B. bruxellensis* strains named A to J were analyzed. These strains were isolated from either red wine, white wine, grapes or wine deposit and stored in glycerol (50%) at -80°C.

### Growth conditions

Initially, the yeast strains were grown on YPD plates with the following composition (20 g.L⁻<fnref n="1" /> glucose, 20 g.L⁻<fnref n="1" /> agar, 10 g.L⁻<fnref n="1" /> yeast extract, 10 g.L⁻<fnref n="1" /> peptone, pH 4.8). For culture in liquid medium, YNB broth was used. It contained 6.7 g.L⁻<fnref n="1" /> of yeast nitrogen base with ammonium sulphate (Difco™) and 10 g.L⁻<fnref n="1" /> of a single carbohydrate (name stated in the text). Sixteen sugars were chosen because they are naturally present in wine or must [4, 5]: monosaccharides (D-glucose, D-fructose, D-ribose, D-xylose, L-arabinose, D-mannose, D-galactose, L-sorbose), disaccharides (saccharose, trehalose, cellobiose, melibiose, lactose and maltose) and polyols (D-mannitol and D-sorbitol). The pH was adjusted to 3.5 and the medium was sterilized by filtration (0.2-µm). Cultures were carried out at 25°C, under agitation (250 RPM, aerobic conditions).

The liquid cultures were started by propagating a colony in 2 mL of liquid glucose YNB medium. This tube was then used to inoculate subsequent cultures and the inoculum rate was adapted to ensure an initial OD₆₀₀ equal to 0.05. Growth was monitored for 15 to 20 days through OD₆₀₀ measurements confirmed with cellular numeration on Malassez cells. A control culture in the absence of sugar enabled to check the absence of growth in the absence of sugar (final OD₆₀₀< 0.3).

### HPLC analysis

The concentration of residual sugar and metabolic products (such as acetic acid and ethanol) was analyzed in the culture supernatants by HPLC using a Biorad Aminex HPX-87H column maintained at room temperature, and a Waters (Milford, USA) system, consisting of a pump (Waters 600), an injector (Waters 717) and a refractometer (Waters 2414). The mobile phase was H₂SO₄ 5mM, and had a constant flow rate of 0.5 mL/min.

<footnote n="1">¹Univ. Bordeaux, ISVV, EA4577 Œnologie, F-33140 Villenave d'Ornon, France</footnote>

<footnote n="2">²Ecole Nationale Supérieure de Chimie, de Biologie et de Physique, Institut Polytechnique de Bordeaux, Avenue Pey Berland, 33607 Pessac, France</footnote>
Results and discussion

Growth was monitored through OD measurements for 15 to 21 days and acidification of the media was detected by pH measurement at the end of the culture. Results for strain A are represented in figure 1. The control experiment confirmed the absence of growth without carbon source (OD<0.1).

<figure><img src="image_140.png" /><figcaption>Fig. 1 - Growth monitoring of *Brettanomyces bruxellensis* strain A on YNB medium and different carbohydrates over a period of two weeks.</figcaption></figure>

For strain A, at stationary phase, when the sugar was metabolized, OD was usually included between 1.8 and 2: this was the case for growth in media containing D-glucose, D-fructose, D-galactose, D-mannose, cellobiose, maltose, trehalose or sucrose. A pH decrease of about 1 unit was observed due to the formation of acetic and lactic acid (aerobic conditions) and these tests were considered positive.

Strain A did not grow on L-sorbose, D-xylose, L-arabinose, D-melibiose, and D-mannitol: the OD remained low (<0.15), the pH was stable and the analysis of the culture supernatant indicated no sugar consumption nor product formation (ethanol, acetic acid or other products of metabolism). These sugars were thus considered as not metabolized by strain A.

In the case of D-ribose, lactose and D-sorbitol, strain A grew to a maximum OD of 0.5, and this growth was associated with a slight decrease of the pH and metabolite production indicating that the sugar was indeed metabolized. This was considered as an intermediate result. Longer experiments (more than 15 days) or pre-acclimatization tests would be necessary to understand whether it is a true limited growth or if the result is due to a delay in the establishment of the metabolic arsenal.

The carbon profile consumption of strains A to J is summarized in Table 1. As previously observed for strain A, when the carbon source was metabolized (positive test, +), the final OD was higher than 1.5 and growth was associated with production of acetic acid and ethanol. The negative (-) and intermediate (+*) tests were also defined as those described above for strain A.

None of the strains studied was able to metabolize all the 16 sugars of the test. Three sugars, D-xylose, L-arabinose (pentose) and L-sorbose, were not metabolized (OD<0.1, no acidification and no production of secondary metabolites) by any of the strain studied. On the other hand, all the strains were able to ferment nine out of the 16 sugars including D-glucose, D-fructose, D-galactose, D-mannose (4 out of the 5 hexoses tested), sucrose, maltose, trehalose and cellobiose.

In addition, five strains were able to grow in an intermediate way on D-ribose, lactose or D-sorbitol, to a maximum OD around 0.5 associated with a pH decrease of 0.3. Strain A was able to metabolize all three but also with intermediate efficiency.
<table><caption>**Table 1** - Overview of the results of the growth tests on sole carbon source obtained with the ten *B. bruxellensis* strains studied</caption><thead><tr><th>Substrate</th><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th><th>F</th><th>G</th><th>H</th><th>I</th><th>J</th></tr></thead><tbody><tr><td>D-Glucose</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D-Fructose</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D-Galactose</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D-Mannose</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>L-Sorbose</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D-Xylose</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D-Ribose</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td></tr><tr><td>L-Arabinose</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Melibiose</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Cellobiose</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Maltose</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Lactose</td><td>+</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Trehalose</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Saccharose</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D-Mannitol</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D-Sorbitol</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td></tr></tbody></table>

Therefore, the *B. bruxellensis* strains studied did not present a high level of phenotypic diversity regarding their growth substrate: the strain selection grew efficiently with most of the hexoses and disaccharides analyzed but disliked pentoses and polyols. However, this result should be confirmed with a larger strain collection and the experiment should be extended to non wine strains. Indeed, recent studies using whole genome sequencing and genetic analysis such as DNA fingerprinting [6] or microsatellites analysis [7] have shed light on the genetic diversity among *Brettanomyces* strains. The genetic traits enabled to group the strains and the genetic groups matched with the group made based on the substrate of origin of the strains (such as wine, beer, cider, kumbucha and bioethanol fermentation). It would be interesting to investigate whether this genetic diversity is associated with diversity in term of carbohydrate consumption to see if this low level of diversity (i) is confirmed for wine strains, (ii) if it differs for non wine strains, and (iii) if it is the result of an adaptation to wine or a general trait in the species.

The low phenotypic diversity observed here is quite surprising as *Oenococcus oeni*, a bacterial species which shares the same ecological niche as *B. bruxellensis* (wine after alcoholic fermentation) and which has been reported to compete with *B. bruxellensis* [8], displays a very high level of diversity regarding the accepted growth substrate. Actually, among the substrates analyzed here, *O. oeni* was reported to metabolize from 3 to 13 sugars depending on the strain [9]. As a result, *O. oeni* and *B. bruxellensis* should compete for D-glucose, D-mannose, cellobiose and trehalose which are accepted as growth substrate by a high proportion of strains in both species, and which are all present at significant concentration in wine after alcoholic fermentation. On the other hand, *B. bruxellensis* may take advantage of the presence of D-fructose, D-galactose, maltose or sucrose which are rarely accepted as growth substrate by *O. oeni* strains. Fructose is the most abundant of these 4 carbohydrates in wine. *O. oeni* does not use it for growth but all strains studied reduce it into mannitol. A slightly different form of competition for fructose may thus exist since mannitol is not used by any of the species. Sucrose is only encountered in musts and may benefit to *B. bruxellensis* in the early stages of winemaking, while maltose is very rare in wine. Galactose may be slowly liberated from grape polysaccharides and may advantage *B. bruxellensis* later in the winemaking process.

Conversely, *O. oeni* may take advantage of the presence of pentoses or melibiose which are encountered in most wine either free or associated into grape polysaccharides. Indeed, *O. oeni* efficiently grows on meliobiose (73% of the strain studied), ribose (90% of the strains studied) and arabinose (82% of the strains studied). A lower proportion also grows on xylose (24%). Lactose, L-sorbose and polyols should be regarded as marginal substrate for both species. The two former being rare in wine, this result is not surprising.

<footnote>*: OD between 0.4 and 1.</footnote>
Conclusion

For the ten *B. bruxellensis* strains studied, coming from wine or grapes, carbohydrate consumption is quite similar even though ribose, lactose and sorbitol differentiate 4 out of the 10 strains. This result should be confirmed on a larger number of wine strains and compared with that observed for strains isolated from other beverages, and the links with genetic diversity (microsatellite analysis) should be analyzed. Moreover, genetic diversity can also be studied regarding the level of ploidy [3, 6-7]. Carbohydrate profile may rely on the level of ploidy of a strain, as additional genetic material increases the potent metabolic equipment.

Moreover, it should be confirmed that the carbohydrate consumption profiles of *B. bruxellensis* is different from that of its main competitor in wine *O. oeni* and that carbohydrate availability is a key element for the establishment of one or the other species.

References

1. Chatonnet P, Viala C, Dubourdieu D (1997) Influence of polyphenolic components of red wines on the microbial synthesis of volatile phenols. *Am J Enol Vitic* **48**: 443-448

2. Gerbaux V, Jeudy S, Monamy C (2000) Etude des phénols volatils dans les vins de Pinot noir en Bourgogne. *Bull OIV* **835/836**: 581-599.

3. Borneman AR, Zeppel R, Chambers PJ et al (2014) Insights into the *Dekkera bruxellensis* genomic landscape: comparative genomics reveals variations in ploidy and nutrient utilisation potential amongst wine isolates. *PLoS Genet* **10**: e1004161.

4. Pellerin P, Cabanis C (1998) Les glucides. In: Flanzy C (ed.) *Elements d'Oenologie*, Tec&Doc Lavoisier.

5. Ribéreau-Gayon P., Dubourdieu D, Donèche B et al (2004) *Traité d'Œnologie Volume 1: Microbiologie du vin, Vinifications*. Dunod.

6. Crauwels S, Zhu B, Steensels J et al (2014) Assessing genetic diversity among *Brettanomyces* yeasts by DNA fingerprinting and whole-genome sequencing. *Appl Environ Microbiol* **80**: 4398-4413.

7. Albertin W, Panfili A, Miot-Sertier C et al (2014) Development of microsatellite markers for the rapid and reliable genotyping of *Brettanomyces bruxellensis* at strain level. *Food Microbiol* **42**: 188-195.

8. Renouf V (2006) Description et caractérisation de la diversité microbienne durant l'élaboration du vin: interactions et équilibres – relation avec la qualité du vin. *PhD thesis*, Bordeaux University, ISVV.

9. Gammacurta M, Le Marrec C, Lonvaud-Funel A et al (2011) Diversity of carbohydrate catabolic pathways in *Oenococcus oeni*. In: *Oeno 2011*. Dunod.
# Comparative metabolomics in yeast cells after dehydration stress

G. LÓPEZ-MARTÍNEZ, A. BORRULL, M. POBLET, N. ROZÈS, R. CORDERO-OTERO*

*University Rovira i Virgili, Department of Biochemistry and Biotechnology, 1 Marcel·lí Domingo Str., 43007, Tarragona, Spain*

Viability – dehydration stress – metabolite extraction – wild yeast

## Introduction

Identifying changes in metabolites resulting from desiccation stress would permit optimisation of the drying process. During the application of stress, desiccation and rehydration, the yeast's metabolic processes are in a suspended state induced by cell water removal [1]. However, as soon as water becomes available, these organisms rapidly swell and return to active life.

At present, Villas-Bôas et al. [2] made a number of advances in yeast sample preparation procedures, with a focus on the following steps: cold methanol quenching, metabolic extraction by chemical treatment and sample concentration through solvent evaporation. Other well-known and widely used extraction methods in yeast research include methanol-chloroform, pH and boiling ethanol; however, problems have been identified in each case, including the recovery of non-phosphorylated compounds, compound stability at extreme pH levels and compound thermolability, respectively [3]. In the case of metabolite extraction from *E. coli* and *S. cerevisiae*, these effects can be mitigated by extracting in 50% (V·V⁻¹) methanol after a -80°C cycle [2, 4].

For the present study, *Saccharomyces cerevisiae*, *Naumovia castellii*, and *Saccharomyces mikatae*, which each possess different dehydration tolerance capabilities, were selected. Through metabolomic characterisation, the aim of this study was to identify the putative compounds involved in overcoming such stress. Our analysis included *S. mikatae* from the sensu stricto group, which includes yeast species that are closely related to *S. cerevisiae*. In addition, we selected *N. castellii*, belonging to the sensu lato group, which includes heterogeneous yeast species that diverge more significantly from *S. cerevisiae* [5]. In tandem with these two strains, the metabolism of *S. cerevisiae* was analysed. These species differ from each other in a very limited number of physiological characteristics, some of which may be controlled by single mutated genes [6].

## Materials and methods

### Strains, growth conditions and the desiccation-rehydration process

The yeast species *Saccharomyces cerevisiae* (CECT-1477: Burdeos sparkling wine), *Naumovia castellii* (CECT-11356: Finland soil) and *Saccharomyces mikatae* (CECT-11823: Japan monosporic culture) were grown in shake flasks (170 rpm) over 24 hours in YPD (1% (W·V⁻¹) yeast extract, 2% (W·V⁻¹) bacto-peptone and 2% (W·V⁻¹) glucose at 28°C by inoculating with overnight liquid culture at an initial OD₆₀₀ of 0.5. The effect of different compounds during rehydration was studied by adding each compound individually to the pure water basal condition. The desiccation-rehydration process and cell viability determination by flow cytometry were performed as previously described by Rodríguez-Porrata et al. [6].

### Metabolite extraction

Frozen cells were pelleted and resuspended in methanol-water (1:1, V·V⁻¹) to 400 µL, as described by Villas-Bôas et al. [2], and 10 µL of ribitol at 2 mg·mL⁻¹ was added as an internal standard (IS). To optimise this step, samples were placed for 10 min at 90°C, for 15 min in the presence of 0.1 mm glass beads and were then disrupted in a multitube bead-beater using 3 cycles of 10 s/beat, followed by 30 s rest for cooling. The 4 h dried residue was redissolved and derivatised for 1 h at 40°C in 50 µL of 20 mg mL⁻¹ methoxyamine hydrochloride in pyridine, followed by a 90-min treatment at 40°C with 70 µL MSTFA.
### GC-MS analysis

Analytical gas chromatography was performed using an Agilent Technologies Network CG System 6890N. Detection of compounds was carried out with an inert Mass Selective Detector 5975. A 2-µL cellular extract was injected with a split ratio of 20:1 into a DB-5HT column (30 m x 0.25 mm x 0.1 µm) with an automatic injector 7683B. Helium was used as the carrier gas at a constant flow of 1.0 mL·min⁻¹. The injector temperature was 200°C. The column oven temperature was initially held at 80°C for 4 min and then increased first to 200°C at the rate of 5°C·min⁻¹ and subsequently to 300°C at the rate of 25°C·min⁻¹, where it was held for 7 min. The MSD transfer temperature was 300°C. The MS quadrapole and source temperatures were maintained at 180°C and 280°C, respectively. The MS data were acquired in electronic ionisation (EI) scan mode at 70 eV for the range of 35-650 amu after a solvent delay of 4 min. Post-run analysis was performed with the Agilent MSD Chemstation. The relative abundance was corrected by the internal standard peak. These values were related to the 1x10⁹ colony formation units (cfu) according to the cells plated and counted after 24 h of culturing and desiccation-rehydration treatment.

### Flow cytometry analysis

Flow cytometry was carried out using a CYFlow® space instrument. The two-colour fluorescent probe LIVE/DEAD® Yeast Viability Kit was used. An overnight YPD culture of each *Saccharomyces* sp. strain was taken as a control of full viability (99% by FUN1 red-orange/Calcofluor white stain).

## Results and discussion

### *Yeast cell viability after dehydration stress*

The mean value of viable cells after rehydration was calculated after taking into account viability before drying. The survival rates for *S. mikatae* and *N. castellii* were lower than that for *S. cerevisiae*, with viabilities recorded at 20%, 40% and 84%, respectively.

### *Metabolomic profiling of yeast cells during the desiccation process*

A total of 44 peaks were identified and quantified for each strain before dehydration (BD) and after rehydration (AR). Multivariate data analysis was performed using principal component analysis (PCA), including normalisation according to the IS and cfu correction (see m&m), to examine the variations among metabolites between BD and AR steps in *S. cerevisiae*, *N. castellii* and *S. mikatae* (data no shown). As summarised in Fig. 1, these results may suggest that the viability of *N. castellii* is negatively affected during stress synergy due to inefficient detoxification and/or a poor capacity for metabolic esterification of phosphoric acid into nucleic acids, proteins, lipids, and sugars [8]. No significant differences among the yeast species were recorded BD in terms of phosphate content. However, between the BD and AR steps, *S. mikatae* and *S. cerevisiae* reduced their phosphate content by 70% and 30%, respectively, while *N. castellii* showed a 100% increase. The fact that *N. castellii* accumulated double the amount of phosphate AR compared to *S. cerevisiae* did not enhance its dehydration tolerance, with viability of 60% and 95%, respectively. However, *S. mikatae* showed a significantly lower content of phosphate after the drying and rehydration process, which was in line with the low cell viability level of 36%. A 50% increase in cellular glycerol accumulation was observed during stress imposition for *N. castellii* and *S. cerevisiae*, while *S. mikatae* showed 50% less and the lowest viability level of 36% (Fig. 1). Glycerol biosynthesis is an important side-reaction of the glycolytic pathway, as it provides NAD⁺ that is necessary for cellular glycolysis activity [7]. Our results suggest that cells undergoing glycerol synthesis during the dehydration process possess a backup of NAD⁺ for swift glycolytic activation when cell activity resumes. The NAD⁺ generated is oxidised to maintain redox balance by the conversion of glyceraldehyde-3-phosphate into 1,3- bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At the GAPDH glycolytic pathway step, under stress conditions, glycolytic flux is strongly prone to glycerol accumulation depending on the kinetic characteristics of GAPDH [9]. Pyruvate acts as a precursor for a side-reaction in α-alanine synthesis. Increases of 3-fold and 2-fold were observed for intracellular α-alanine in *N. castellii* and *S. cerevisiae*, respectively, while *S. mikatae* showed a 2-fold decrease between the BD and AR steps (Fig. 1).

The acetyl-Co-A branch point metabolite could be transformed into citric acid or lipids and fatty acids, such as palmitoleic acid, which were increased in *N. castellii* and *S. cerevisiae* during the dehydration process, while a 30% reduction was observed in *S. mikatae*. Meanwhile, the only intermediate in the TCA that showed significant variations during stress imposition was succinic acid, the levels of which were increased by 9-fold and 2-fold in *N. castellii* and *S. cerevisiae*, respectively, while no changes were
recorded in *S. mikatae*. *S. cerevisiae* does not accumulate succinic acid intracellularly [10], which may tentatively explain the insignificant differences between BD and AR values for *S. mikatae*, while *N. castellii* showed the highest accumulation, which suggests a metabolic difference with respect to the other two yeast species. In the TCA cycle, succinic acid is converted to oxaloacetate, which can be transaminated to form aspartate. All of the yeasts showed a very similar abundance of aspartic acid BD. However, this content in *N. castellii* and *S. cerevisiae* increased by 13-fold after rehydration, while in the case of *S. mikatae*, this level was only increased by 3.5-fold (Fig. 1). Taking into collective consideration the changes in both aspartic and succinic acid abundance, it may be suggested that *S. mikatae* cells do not present TCA activity during the dehydration and rehydration process, in contrast to *N. castellii* and *S. cerevisiae*. The TCA intermediate 2-oxoglutarate is also a branch point metabolite, and through transamination, it may form lysine and glutamate that could be used to synthesise other amino acids, such as glutamine, proline, and arginine. Our results indicated that the three yeast species showed similar contents of lysine and glutamine BD, whereas during stress imposition, the changes in abundance of these amino acids did not correlate with the putative changes in their TCA activity. *S. mikatae* showed 3-fold less lysine after stress induction, while *N. castellii* and *S. cerevisiae* showed increases of 3-fold and 2-fold, respectively. The glutamine abundance was significantly reduced in *S. mikatae* and *S. cerevisiae* by 6-fold and 3-fold, respectively. In fact, one of the two anaplerotic pathways responsible for replenishing the TCA cycle with intermediates, which maintains its function, is the glyoxylate cycle, in which isocitrate dehydrogenase can form 2-oxoglutarate from isocitrate. The participation of both the TCA and glyoxylate cycles for providing 2-oxoglutarate to the cell makes it very difficult to understand the glutamine and lysine abundance variations during stress induction. In addition, these variations were not correlated with the dehydration tolerance profiles of the different yeast species.

<figure><img src="image_141.png" /><figcaption>**Fig. 1** - Relative abundance of significant and stress-related compounds BD (white bars) and R (grey bars) in *S. cerevisiae*, *S. castellii*, and *S. mikatae*. The values represent the means of three independent experiments. \*Results for each compound with statistically significant differences (*p*-value<0.05) compared to the BD condition.</figcaption></figure>

*Effect of compound supplementation on yeast cell viability*

We next sought to ascertain whether the low viability of *S. mikatae* after the rehydration process could be
due to the low levels of lysine, Na₂HPO₄, or glutamine. A statistically significant increase of 20% in cell
survival driven by 1, 2 and 5% lysine or 10% glutamine supplementation was observed only in *S. mikatae*
(Fig. 2); *S. cerevisiae* did not show beneficial effects when submitted to the same rehydration conditions.
Unexpectedly, phosphate supplementation was detrimental and increased cell death to 30% compared to
the pure water reference condition (data not shown).
<figure><img src="image_142.png" /><figcaption>**Fig. 2** - Effect of rehydration treatments on cell viability. *S. mikatae* yeast cells were incubated at 37°C for 30 min in pure water or in the presence of lysine (Lys), phosphate (Pho), or glutamine (Gln). The scale of relative viability (%) indicates the percentage of experimental values for the different yeasts relative to the non-complemented condition (H₂O). The values represent the means of three independent experiments. \*Results with statistically significant differences (*p*-value<0.05) compared to the H₂O condition.</figcaption></figure>

## Conclusion

The high mortality rate showed by *S. mikatae* and *N. castellii* was not due to a single metabolite; rather, the accumulation of both succinic and phosphoric acid by *N. castellii* during stress imposition may have led to the reduced cell viability rate of 60%. In addition, the metabolite profiles suggested the activity of the glycolytic pathway and TCA cycle in *N. castellii* and *S. cerevisiae*. However, *S. mikatae* displayed very low activity with respect to these pathways, which could have led to a reduction in secondary metabolites required for viability AR. Furthermore, our results demonstrated that the presence of lysine or glutamine during the rehydration process had a positive effect on the recovery of *S. mikatae* cell activity; on the other hand, supplementation with these compounds might be yeast dependent.

## Acknowledgements

This work was financially supported by grants from the Spanish government (Projects AGL2010-22001-C02-02 and AGL2012-40018-C02-01, awarded to NR and RC, respectively). GL and AB would also like to thank the Rovira i Virgili University for the doctoral fellowships FI-DBR 2012, AGAUR and 2009BRDI/12/12, respectively.

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# Distribution of *Oenococcus oeni* strains during malolactic fermentation over regions and products in France and Lebanon

M. EL KHOURY<fnref n="1" />*, O. CLAISSE<fnref n="1" />, H. CAMPBELL-SILLS<fnref n="1" />,<fnref n="2" />, P.M. LUCAS<fnref n="1" />

*Oenococcus oeni* – diversity – indigenous – SNP

## Introduction

Microbial biogeography intends to document the spatial diversity of microorganisms at different environmental scales. Its goal is to propose hypotheses that explain the processes responsible for the creation of diversity patterns and the factors influencing their evolution [1]. This field proved to be of particular interest in enology when questions about the diversity of indigenous microorganisms - associated with grapes and wine - and terroir were being brought up [2-4].

An important microbial consortium interacts from the vineyard until the end of the fermentation process [5-7]. Among them, *Oenococcus oeni* is the main species of lactic acid bacteria of enological interest. While this species is barely identified on the grape and in the must, it develops right after the alcoholic fermentation and quickly becomes the predominant species [8, 9]. It is known to perform most of the malolactic fermentation (MLF) not only in red wine, but also in champagne, white wine and cider. The diversity of *O. oeni* has often been studied with the aim of selecting performant strains to produce malolactic starters [10, 11]. The use of new genotyping methods and phylogenetic approaches has allowed a more precise and easy identification of strains in the case of spatial or temporal inventory. Numerous studies have reported the high genetic and phenotypic diversity of this species in wines [12-14]. Population structure analyses also revealed that the strains of this species form two major genetic groups, named A and B, which are subdivided into subgroups eventually specific to a region or a product [15-17].

The aim of this study was to determine the biogeography of this species based on the analysis of a large collection of strains isolated from several wine and cider-producing regions. Predominant bacteria present in wines and cider during MLF were isolated by plating on specific media then identified at the species and strain levels by 16S rDNA and VNTR analysis. *O. oeni* strains were further analyzed phylogenetically by using a new method based on SNP analysis which was developed specially for this purpose.

## Materials and methods

### Bacteria isolation and preservation

The strains of *O. oeni* were isolated from 235 malolactic fermentations during a sampling campaign of wines and cider from historic wine producing regions and cider in France; few additional samples were taken from wineries in Lebanon. Details of the number of samples per region are given in Table 1. For 3 vintages, 2011, 2012, 2013, a total of 189 red wines, 36 white wines and 9 cider were analyzed. Strains were isolated on solid grape juice medium (GJM) with pimaricine (100 mg/L) (Sigma), after incubation for 7 days under anaerobic conditions at 25°C. For each sample, 15 colonies were cultivated in liquid GJM under the same conditions. An aliquot of the culture was preserved at -80°C with 30% glycerol (Sigma). The other part was centrifuged 5 min at 10000 g, washed with distilled water and kept frozen at -20°C until genotyping. 110 strains isolated from red and white wine, cider, liquor wine and old vintages from 1989 until 2008 were added to this study. They were obtained from the Centre de Ressources Biologiques Oenologique (CRB OENO) from Bordeaux University at ISVV (http://www.crboeno.univ-bordeauxsegalen.fr/).

<footnote n="1">¹Univ. Bordeaux, ISVV, EA 4577 Œnologie, F-33882 Villenave d'Ornon, France</footnote>

<footnote n="2">²Research and Innovation Centre, Fondazione Edmund Mach, San Michele all'Adige, Italy</footnote>

<footnote>*Corresponding author: mariette.elkhoury@gmail.com</footnote>
## Species identification and strain genotyping

Colonies of *O. oeni* were identified and genotyped by multiplex VNTR analysis [18]. Other bacterial species were identified by 16s rDNA sequence analysis.

## Phylogenetic analysis

A genotyping method based on single nucleotide polymorphism (SNP) analysis was developed to classify strains in genetic groups. Based on the whole-genome alignments of the sequences of 50 *O. oeni* strains, 10 phylogenetic clusters were defined, which allowed the selection of 40 SNP characterizing each cluster. The iPLEX Gold kit (Sequenom Inc., San Diego, CA) was employed to amplify all of the 40 SNP for each strain. The multiplex PCR product was then spotted on a SpectroCHIP and analyzed by MALDI-TOF mass spectrometry. Raw data were instantly treated using SpectroCALLER to define the SNP then the SpectroACQUIRE software was used to extract SNP data that were imported into MEGA 6.0.5 (Tamura, Stecher, Peterson, Filipski, and Kumar 2013) and BioNumerics v5.1. (Applied Maths, Belgium) for phylogenetic analysis.

## Results and discussion

A total of 2997 colonies of *O. oeni* were isolated from 226 wines and 9 cider (Table 1). This represents 93.3% of all the bacterial colonies analyzed and confirms that this species is predominant during MLF. Shannon-Weaver diversity index values were close to 4.5 in regions where more than 500 colonies were isolated. In agreement with previous studies, this indicates that the diversity of *O. oeni* strains is very high in each region. This also confirmed that the sampling was representative of the regional diversity of the strains during MLF. VNTR analysis showed that the 2997 colonies represented at least 514 strains. 87% of these strains were detected only in one region, indicating that each region holds a unique *O. oeni* population. All the strains were specific for either cider or wine since no strain was detected in both products.

<table><caption>**Table 1** - Number of *O. oeni* strains isolated in each region and values of the diversity and evenness indexes</caption><thead><tr><th>Region</th><th>Number of samples</th><th>Number of O. oeni clones</th><th>Number of O. oeni strains</th><th>Shannon diversity index</th><th>Pielou evenness index</th><th>Number of unique strains</th></tr></thead><tbody><tr><td>Aquitaine</td><td>80</td><td>1072</td><td>200</td><td>4.57</td><td>0.86</td><td>150</td></tr><tr><td>Burgundy</td><td>70</td><td>837</td><td>142</td><td>4.19</td><td>0.84</td><td>124</td></tr><tr><td>Languedoc</td><td>36</td><td>514</td><td>134</td><td>4.28</td><td>0.87</td><td>93</td></tr><tr><td>Roussillon</td><td>8</td><td>117</td><td>29</td><td>2.96</td><td>0.88</td><td>22</td></tr><tr><td>Val de Loire</td><td>9</td><td>104</td><td>25</td><td>2.7</td><td>0.86</td><td>25</td></tr><tr><td>Bretagne (cider)</td><td>32</td><td>353</td><td>57</td><td>3.17</td><td>0.82</td><td>46</td></tr><tr><td>Lebanon</td><td>235</td><td>2997</td><td>514</td><td></td><td></td><td></td></tr></tbody></table>

The total number of strains represents the number of different VNTR profiles when comparing all the regions together.

The phylogenetic relationships among the strains were determined by multiplex SNP genotyping. Figure 1a shows that most of the strains (84.2%) belong to the genetic group A and only 12.6% belong to the group B. Previous studies have also reported the predominance of group-A strains in wine [15, 16]. Interestingly, 18 strains (3.2%) were not classified in any of these groups and form supposedly a third group “C” as previously suggested [16]. Cider strains belong exclusively to subgroups B and C (Figure 1b). Wine strains were spread throughout the 3 genetic groups independently of their region of origin. However, some subgroups hold strains isolated from the same type of wine: A5 seems specific to strains from white wine and champagne, A2-8 to strains from red wine from Burgundy and subgroups B and C for cider strains.
<figure><img src="image_143.png" /><figcaption>**Fig. 1** - Minimum spanning trees presenting the 514 strains of *O. oeni* based on the phylogenetic cluster and the region of origin. a) The proportion of each phylogenetic cluster. Cluster A: green, cluster B: blue, cluster C: yellow. b) The proportion of strains of each region in each cluster. Aquitaine: red, Burgundy: turquoise blue, Languedoc-Roussillon: green, Val de Loire: pink, Lebanon: yellow, Cider Brittany, Normandy and Asturias: brown.</figcaption></figure>

Unlike the current idea that strains selected from a regional wine are specific to this particular region or terroir, our results show that these unique strains are not so far specific to their region of origin. Indeed, it appears that the indigenous population of *O. oeni* is actually a patchwork of strains belonging to different genetic groups. The phylogenetic groups of this species are very widely spread over all the regions that were analyzed. However, a separation in the population is observed between strains isolated from cider, red wines and white wines. The detection of genetically related strains in these products reveals that some strains have evolved to develop in specific types of wines.

## Conclusion

Taken together, these results emphasize the importance of the genetic diversity of the species *O. oeni* and support the idea that local winemaking processes and specific wine characteristics lead to the local development or selection of specific strains.

## Acknowledgements

This work was supported in parts by the French Ministry of Agriculture Agri-Food and Forestry (AAP 2012, CASDAR project, Levains Bio) and the European commission (FP7-SME project Wildwine, grant agreement no. 315065). Mariette El Khoury was the recipient of a fellowship from the French Ministry of Higher Education and Research.
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11. Solieri L, Genova F, De Paola M et al (2010) Characterization and technological properties of *Oenococcus oeni* strains from wine spontaneous malolactic fermentations: a framework for selection of new starter cultures. *J Appl Microbiol* **108**: 285-298.

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# Genomics of *Oenococcus oeni*: a new strategy for the selection of efficient malolactic starters

M. FAVIER<fnref n="1" />,<fnref n="2" />*, H. CAMPBELL-SILLS<fnref n="2" />, J. COULON<fnref n="1" />, V. MOINE<fnref n="1" />, P. LUCAS<fnref n="2" />

*Oenococcus oeni* – genome – plasmid – selection

## Introduction

*Oenococcus oeni* plays an essential role in winemaking as the main lactic acid bacteria involved in malolactic fermentation (MLF), which brings deacidification, aromatic complexity and microbial stability to wines. Nowadays, MLF is more and more performed by the addition of industrial starters, *O. oeni* being the main species selected because of its high tolerance to acidity and alcohol related stresses. However, strains can exhibit large phenotypic variations such as tolerance to freeze-drying, abilities to survive in wine and to lead a complete and rapid MLF. Many *O. oeni* genes involved in wine adaptation have been identified but have not always been truly efficient for the selection of enological strains which remains essentially based on time-consuming phenotypic techniques. This study aims at identifying specific genes involved in strain performances, through a systematic analysis and comparison of different *O. oeni* genomes.

## Materials and methods

*Strains and phenotypic determinations*

A collection of 44 *O. oeni* strains from diverse geographical and product origins was studied. All strains were produced in a freeze-dried form before testing their survival and fermentative capacities in model wine. Briefly, strains were cultivated by successive batches in the production media incubated at 25°C and concentrated by centrifugation. The pellet was prepared by adding a cryoprotectant before the freeze-drying cycle which consists in an overnight freezing at -80°C followed by a 24h drying step in a lyophilizer. Then the lyophilisates were inoculated at 10⁷ cell/mL in a model wine and MLF was carried out at 20°C. Thus the freeze-drying yield, the 24h survival rate and the MLF kinetic phenotypes were determined for all 44 strains.

*Genomic analysis*

For each phenotype, the genome of 4 strains with low and 4 strains with high performances were compared to construct a list of candidate genes only present in the efficient strains. In parallel, genetic analysis of pOENI-1 plasmids was performed to detect other potential candidate genes. The distribution of selected genes was studied by PCR detection in the 44 *O. oeni* strain collection. The ROC (Receiver Operating Curves) statistical analysis was used to validate genes as genetic markers of technological interest in *O. oeni* strains.

## Results and discussion

*Technological properties of 44 O. oeni strains*

First, the phenotypes representing the technological and enological skills of the strains were determined. Each of them was freeze-dried and then inoculated into a model wine to induce MLF. This experiment allowed the ranking of the strains according to their freeze-drying yield, survival in wine and MLF kinetics (Figure 1). As expected, *O. oeni* strains showed a large phenotypic diversity. The strains were more or less adapted to the production process and encountered stresses, which led to variable freeze-drying yields from

<footnote n="1">¹BIOLAFFORT, Bordeaux, France</footnote>

<footnote n="2">²Univ. Bordeaux, ISVV, Unité de recherche Œnologie, EA4577, USC 1366 INRA, Villenave d'Ornon, France</footnote>

<footnote>*Corresponding author: marion.favier@laffort.com</footnote>
1 to 40 g.L⁻¹. Some of them were not able to survive in wine 24h after inoculation, whereas others stayed viable and even developed. Among the collection, 36 strains induced a MLF more or less rapid and complete (from 14 to >100 days) and 7 of them were not able to start MLF after 42 days, probably because of population decline in the days after inoculation. Thanks to this phenotypic characterization, we could easily rank the strains and create distinct groups based on their technological skills.

<figure><img src="image_144.png" /><figcaption>**Fig. 1** - Technological properties of 44 *O. oeni* strains. (A) Freeze-drying yields. Values correspond to the mass of standardized freeze-dried bacteria obtained from 1 liter of culture. (B) Survival rates in wine. The viable cells were determined by epifluorescence after 24h in wine. (C) MLF kinetics of 18 indigenous strains. Green, commercial strains; orange, indigenous strains.</figcaption></figure>

*Identification of potential technological genes by genome comparison*

Secondly, a genomic comparative study was performed to define a list of candidate genes specific to efficient strains for each technological property (freeze-drying yield, survival rate, MLF kinetics). The core-genome shared by all strains was excluded and only the shell-genome, shared by some strains, was analyzed. The genome comparisons based on the detection of present/absent genes highlighted 22 genes discriminating strains according to their phenotypes. These genes are present only in strains with the best technological skills and are located in three regions of genomic plasticity, two of them being already described in previous genomic studies (Figure 2) [1, 2]. The region 1 contains 30 genes which are certainly involved in sugar transport, metabolism and carotenoid biosynthesis, the region 2 is composed of 12 genes related to sugar and amino acid metabolisms, and the region 3 encodes 4 genes closely related to
the pentose phosphate pathway. According to genetic analysis, the efficient strains possess the partial or
complete regions whereas they were completely absent in the strains with low skills.

In parallel, the study of the plasmid "pOENI-1" isolated from a commercial strain also revealed two accessory genes, *oye* and *tauE* (respectively encoding an oxidoreductase of the "Old Yellow Enzyme" family and a permease of the "sulfite export TauE/SafE" family), frequently associated with malolactic starters and indigenous strains causing spontaneous MLF (Figure 3) [3, 4].

<figure><img src="image_145.png" /><figcaption>Fig. 2 - Regions of technological interest on the strain PSU-1 chromosome.</figcaption></figure>

<figure><img src="image_146.png" /><figcaption>Fig. 3 - pOENI-01 genetic map.</figcaption></figure>

Validation of selected genes by statistical analysis

Finally, the statistical distribution of these genes was studied among the 44 phenotyped *O. oeni* strains. Five genes representative of genomic regions 1 (nqr), 2 (araC), 3 (xyl) and pOENI-01 plasmids (*oye*, *tauE*) were analyzed by PCR. We can observe the absence of these genes in 2 strains characterized by low technological properties whereas they were all present in 4 performant strains carrying also the plasmid. The construction of ROC (Receiver Operating Characteristic) curves allowed us to assess the quality of these candidate genes as markers of technological interest traits in all *O. oeni* strains. The ROC tool is a statistical measure of the binary classifier performance which allows (i) to identify and describe an interesting gene and its discrimination power and (ii) to predict the technological properties of a strain carrying this gene (Table 1, Figure 4). For example, the significant ROC analysis obtained for the *tauE* gene showed a performance test with technological relevance, characterized by a threshold value of ≤ 38 days, a sensitivity of 64% and a specificity of 73%. In other words, 64% of *tauE* positive strains were able to lead MLF in less than 38 days and 73% of *tauE* negative strains performed MLF in more than 38 days.
Only the gene *xyl* representative of the region 4 did not allow to discriminate strains according to their phenotypes. It hence cannot be considered as reliable genetic marker. Besides, the simultaneous presence of the 4 genes *nqr*, *araC*, *oye* and *tauE* statistically highlighted efficient strains, able to be produced in a freeze-dried form, survive to wine inoculation and lead to a complete and rapid MLF (Table 1).

<figure><img src="image_147.png" /><figcaption>Fig. 4 - Example of a significant ROC analysis.</figcaption></figure>

<table><caption>Table 1 - Significant ROC analysis</caption><thead><tr><th rowspan="2"></th><th colspan="3">Number of strains</th><th colspan="2">ROC curve</th></tr><tr><th>Absent gene</th><th>Present gene</th><th>Threshold value</th><th>Sensitivity (%)</th><th>Specificity (%)</th></tr></thead><tbody><tr><td><strong>Production yield</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>nqr*araC</td><td>15</td><td>27</td><td>&gt; 30.6 g/l</td><td>63</td><td>73</td></tr><tr><td><strong>Survival</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>arac</td><td>12</td><td>30</td><td>&gt; 21 %</td><td>77</td><td>70</td></tr><tr><td><strong>MLF</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>tauE</td><td>33</td><td>11</td><td>&le; 38 days</td><td>64</td><td>73</td></tr><tr><td>oye*tauE</td><td>36</td><td>8</td><td>&le; 30 days</td><td>75</td><td>86</td></tr></tbody></table>

## Conclusion

In enology, the use of selected *Oenococcus oeni* strains as malolactic starters is more and more recommended to perform MLF. Their selection protocol is mainly based on long and tedious empirical approaches. In this context, our study aimed at identifying technological interest genes specific to efficient strains, by combining the phenotypic characterization of 44 *O. oeni* strains and some genome comparisons. Twenty-four genes associated to four regions of genome plasticity located on the chromosome or a plasmid were identified as potential candidates to sign the technological superiority of some *O. oeni* strains. The use of an original statistical tool, the ROC analysis, allowed to define which genes had a high discriminant power and also to predict phenotypic traits in strains which possess these genes. Thanks to this approach, we can consider to preconize a first selection step of efficient *O. oeni* strains by an easy and quick PCR detection of these genes. Moreover, a more comprehensive study of these genomic regions could allow us to gain insight on wine adaptation of *O. oeni*.

## References

1. Bon E, Delaherche A, Bilhere E et al (2009) *Oenococcus oeni* genome plasticity associated with fitness. *Appl Environ Microbiol* **75**: 2079-2090.

2. Borneman A, McCarthy J, Chambers P et al (2012) Comparative analysis of the *Oenococcus oeni* pan genome reveals genetic diversity in industrially-relevant pathways. *BMC Genomics* **13**: 373.

3. Bilhère E (2009) Etude de la diversité génétique intraspécifique chez la bactérie lactique *Oenococcus oeni*. *Thèse de doctorat*, Université de Bordeaux II.

4. Favier M, Bilhère E, Lonvaud-Funel A et al (2012) Identification of pOENI-1 and related plasmids in *Oenococcus oeni* strains performing the malolactic fermentation in wine. *PloS One* **7**: e49082.
# Influence of selected wine lactic acid bacteria on the sensory profile of wines

M. DELERIS-BOU*, A. SILVANO, S. KRIEGER-WEBER

Lallemand SAS France, 19, rue des Briquetiers, 31702, Blagnac, France

Selected wine bacteria – malolactic fermentation – wine aroma – bacteria timing of inoculation

## Introduction

Malolactic fermentation (MLF) is the decarboxylation of L-malic acid to L-lactic acid and CO2 by wine lactic acid bacteria (LAB). Aside from L-malic acid consumption, wine LAB can metabolise other wine components during malolactic fermentation and contribute to the wine sensory profile. Through the citric acid metabolism, LAB produce diacetyl which contributes to wine complexity or give a buttery character depending on its concentration. Wine selected bacteria have a considerable number of other metabolic pathways and enzymes that lead to hydrolysis and synthesis of volatile secondary compounds at concentrations above their perception threshold. Secondary compounds that have been shown to change during MLF include ethyl and acetate esters, higher alcohols, carbonyls, volatile fatty acids and sulfur compounds. It was reported that these changes depend on the strain used to conduct MLF [1-3]. Practical MLF trials with several selected bacteria were conducted in different wines. Aroma analysis and wine tasting were done in order to check bacterial strain sensory contribution.

## Materials and methods

### Selected wine LAB strains

The *Oenococcus oeni* strains under freeze-dried form (MBR®) were directly inoculated into must/wine either 24h after yeast addition (co-inoculation) or after alcoholic fermentation (AF) (sequential inoculation).

### Wines/Regions

MLF trials were conducted in different grape varieties and countries: Chardonnay (Val de Loire, France), Merlot (Bordeaux, France), Barbera (Piemonte, Italy) and Riesling (Baden, Germany).

### Aroma analysis and wines tasting

SPME-GC/MS was used for aroma analysis. Wine tasting sessions were done with an international jury of 60 experts. Statistical result analysis was done with Fizz software (Biosystemes, France).

## Results and discussion

Diacetyl is associated with the buttery character of wine. It is formed as an intermediate metabolite in the reductive decarboxylation of pyruvic acid to 2,3-butanediol and mainly comes from the citric acid metabolism. This metabolism is highly LAB strain dependant [4]. The sensory threshold of diacetyl in wine has been shown to strongly depend upon the style and the type of wine and has been determined to vary from 0.2 mg/L for Chardonnay wine, 0.9 mg/L for Pinot Noir to 2.8 mg/L for Cabernet Sauvignon wine. In a study done at the AWRI by Bartowsky et al. [5] in Cabernet Sauvignon wines in Southern Australia, the results show a variation of diacetyl content in the final wines from 4 to 14 mg/L depending on the 7 wine LAB added in sequential inoculation. Another comparative practical trial with 7 selected LAB added in sequential inoculation in a Barbera wine also clearly shows the bacteria strain impact on diacetyl content (figure 1). While strains like S4 and S6 produce less than the sensory threshold thanks to a late citric acid metabolism, some others as S2 and S5 are able to produce more than 9 mg/L. In this trial, the spontaneous malolactic fermentation leads to a very high level of diacetyl (18.8 mg/L).
<figure><img src="image_148.png" /><figcaption>**Fig. 1** - Results of diacetyl analyses in a comparative trial of MLF with 6 different selected bacteria (sequential inoculation) and a control (spontaneous) in a Barbera wine. Italy, 2013.</figcaption></figure>

Another trial was conducted in a Chardonnay wine in France. MLF was induced with 2 different selected bacteria inoculated after AF. In correlation with the low diacetyl produced by the S4 bacteria in Barbera, the Chardonnay wine inoculated with this strain was found distinct from the one inoculated with the S7 strain, especially on the buttery notes (figure 2). The S7 strain is known for having an early metabolism of citric acid which means for having a high potential to produce diacetyl (results not shown). MLB strains produce significant different concentrations of diacetyl when inoculated in sequential inoculation. It is not the case in co-inoculation, when the bacteria are added to the must 24h after yeast. During AF, under the reductive conditions generated by active yeast cells which consume part of the oxygen available, diacetyl is immediately reduced to acetoin then 2,3-butanediol which results in a wine with very little lactic/buttery flavours. Other factors play a role on diacetyl final content in wine such as citric acid level, temperature, bacteria inoculation rate, timing of SO₂ addition after MLF, and contact with lees [1].

<figure><img src="image_149.png" /><figcaption>**Fig. 2** - Results of a tasting in a Chardonnay comparative trial of MLF with 2 selected wine bacteria (sequential inoculation).</figcaption></figure>

Another comparative MLF trial with 4 selected wine bacteria was conducted in the Bordeaux area. Durations of MLF range from 14 to 36 days depending on the strain inoculated. Some aroma compounds were analysed in the 4 final wines: 3 ethyl-esters, 2-phenylethanol, isoamyl acetate and β-ionone (figure 3). The concentrations of the 6 compounds were affected by the strain used for MLF and were all above their odour threshold. As previously studied [5], *O. oeni* strains can have varying impact on the profiles of esters that can affect berry fruit properties of wines. Variations of glycosylated bound flavour compounds, such as C₁₃-norisoprenoids, could be also of importance as they can contribute to the fruity notes of red wines.
<figure><img src="image_150.png" /><figcaption>**Fig. 3** - Analysis of 4 ethyl-esters, isoamyl acetate, 2-phenylethanol and β-ionone in a Merlot comparative trial with 4 selected wine LAB.</figcaption></figure>

Wine tastings were organized after large scale trials of MLF on Barbera and Riesling wines, which respectively compared MLF results obtained with 2 and 3 bacteria strains added in co-inoculation. The results of descriptive sensory analyses (figures 4 and 5) confirm that the bacteria strain has a significant impact on the wine profile. Indeed, in Barbera wines, fresh fruit and spicy note descriptors were different depending on the bacteria strain used for MLF. In Riesling, olfactive aromatic intensity and fruitiness of wines were statistically found different, and in a less extent floral notes, aromatic intensity in mouth, balance and mouthfeel in function of the bacteria strain.

<figure><img src="image_151.png" /><figcaption>**Fig. 4** - Results of a tasting in a Barbera comparative trial of MLF with 2 selected wine bacteria (sequential inoculation).</figcaption></figure>
<figure><img src="image_152.png" /><figcaption>Fig. 5 - Results of a tasting in a Riesling comparative trial of MLF with 3 selected wine bacteria (co-inoculation).</figcaption></figure>

Conclusion

Selected wine LAB are involved in modification of aroma compounds such as buttery and fruity-berry aromas. In relation with recent and ongoing research on this contribution, practical MLF trials highlight how wine aroma composition and sensory perception can be modulated by the choice of the bacteria strain.

References

1. Bartowsky E, Henschke P (2004) The ‘buttery’ attribute of wine—diacetyl—desirability, spoilage and beyond. *Int J Food Microbiol* **96**: 235-252.

2. Knoll C, Fritsch S, Schnell S et al (2012) Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma. *World J Microbiol Biotechnol* **28**: 1143-1153.

3. Sumby K, Jiranek V, Grbin PR (2013) Ester synthesis and hydrolysis in an aqueous environment, and strain specific changes during malolactic fermentation in wine with *Oenococcus oeni*. *Food Chem* **141**: 1673-1680.

4. Krieger S, Déléris-Bou M, Dumont A et al (2012) Sculpting the aromatic profile of wines through diacetyl management. *Winemaking Update No. 1*.

5. Bartowsky E, Costello P, Krieger-Weber S et al (2011) Influence of MLF on the fruity characters of red wine. Bringing chemistry and sensory science together. *Wine Vitic J* **6**: 27-33.
# Inter-laboratory comparison of available
## *Brettanomyces* quantification kits

C. LONGIN¹*, C. PETITGONNET¹, V. SERPAGGI², J. MAUPEU³, A. VALLET- COURBIN³,
S. ROUSSEAUX¹, M. GUILLOUX-BENNATIER¹, H. ALEXANDRE¹

¹UMR PAM, équipe VAIMiS (Vin, Aliment, Microbiologie, Stress), IUVV,
rue Claude LADREY, 21000 Dijon, France

²Inter-Rhône, service technique, 2260 route de grès, 84100 Orange, France

³Microflora - ADERA, ISVV, Unité de Recherche Œnologique, 33882 Villenave d'Ornon cedex, France

*Brettanomyces* – quantification – DNA extraction – q-PCR

## Introduction

*Brettanomyces bruxellensis* is well adapted to winemaking conditions, i.e. high ethanol concentration [1] and low pH [2], which allows it to develop in difficult environment such as wine.

Due to the production of volatile phenols and other compounds responsible for organoleptic defects, *Brettanomyces* is regarded as spoilage yeast encountered mainly in red wine. It is able to transform *p*-coumaric, ferulic and caffeic acids, respectively, to 4-ethyl-phenol, 4-ethyl-gaïcol and ethyl-4-catechol via two enzymes [3, 4]. These volatile and odorous molecules are the source of phenol and animal character recalling "horse stable", "horse sweat" sometimes called "Brett character". Other molecules are responsible for organoleptic deviations described as "mouse taste" [3]: the tetrahydropyridines. However, despite previous knowledge on this yeast, *Brettanomyces* contamination is still hard to manage and is considered as the main microbiological problem in red wines. Therefore, the detection and quantification of this yeast is essential in order to prevent wine spoilage. Several specific detection and quantification kits based on q-PCR assay are commercially available. Although these kits are frequently used not only by private enological laboratories but also by research laboratories, neither inter-laboratory nor inter-assay comparisons have been published so far. The aim of this work is to highlight advantages and disadvantages of each kit to help professionals choose the most suitable kit according to their needs.

## Materials and methods

*Brettanomyces bruxellensis* strains and growth conditions

*Brettanomyces bruxellensis* strains from Rhône Valley and Bordeaux were used in this study. Cells were pre-adapted to alcohol using a synthetic wine medium (5%v/v) and then inoculated in filtrated (0.2-µm) red wine from three vineyards (Bordeaux, Burgundy and Rhône Valley).

Conditions

Wines of Bordeaux and Rhône Valley were inoculated at 10³ cells.mL⁻¹ and then analyzed by three different q-PCR kits. Moreover, *Brettanomyces* cells (about 10⁶ cells.mL⁻¹) from two different wines (Burgundy and Bordeaux wines) were treated using either bisulfate (molecular form > 2 mg.L⁻¹) or chitosan (0.1 g.L⁻¹). Wine samples were collected at different time (0, 7, 14 days) after sulfite or chitosan addition (KiOfine-B, ICV). For the latter treatment, sampling was either done on wine with or without racking. The assays were conducted over time. For each sample, DNA was extracted and amplified (three different kits tested) in order to assess if DNA from dead cells could be amplified and thus give false positives.

Measurement of the *Brettanomyces* level

*Brettanomyces* wine concentration was checked using a selective medium, ITV medium (SM) (glucose 2%, yeast extract 1%, bacto-peptone 2%, *p*-coumaric acid 0.1%, ferulic acid 0.1%, bromocresol green 0.003% and chloramphenicol 0.02%). In parallel, the level of total cells was quantified by flow cytometry (FCM) or
DEFT (Direct Epifluorescent Filter Technique) (DEFT). Fluorescein diacetate (FDA) was used to quantify yeast cell viability by FCM and CFDA associated to propidium iodide (PI) was used for DEFT.

### DNA extraction and q-PCR kits

The present study compares the performances of three different *Brettanomyces* DNA extractions and q-PCR kits. Three kits were used in this study and named K1, K2 and K3. These kits are used routinely in different laboratories. Each of the three partners: Inter-Rhône (IR), Microflora (MF) and University Institute of Vine and Wine (IUVV) tested two extraction kits (one kit common for two laboratories) with four repetitions.

## Results and discussion

### *Brettanomyces* quantification in red wines

Results of *Brettanomyces* quantification on specific agar medium (SM) or by flow cytometry (FCM) or direct epifluorescent filter technique (DEFT) were compared to quantification results obtained using the three kits (Table 1). The results show that SM and FCM or DEFT led to statistically acceptable results for the two wines tested by the three labs. The mean levels of *Brettanomyces* cells measured with these techniques for Bordeaux and Rhône wines are 3.53 ± 0.28 and 3.88 ± 0.27 log cells.mL⁻¹, respectively. For all quantifications obtained with the three different kits, repeatability is satisfactory independent of the lab. For the Bordeaux wine, quantification performed by SM and FCM/DEFT are homogenous whatever the lab. Moreover, whatever the kit used, q-PCR results are not significantly different between labs, except for kit K3 (Table 1). For the kits K1 and K2, the difference between two labs is less than 1 log cells.mL⁻¹ (Table 1). For Rhône wine, quantification performed by SM and FCM/DEFT are homogenous whatever the lab. At statistical level, only kit K1 has a satisfactory reproducibility. However, the variation between labs for kits K2 and K3 is lower than 1 log cells.mL⁻¹. Results obtained with kit K3 show the most important heterogeneity for the two wines analyzed whatever the lab (Table 1). These differences probably come from matrix variability and/or manipulations and need to be further investigated. Moreover, it seems that the population obtained by specific q-PCR is underestimated by about 1 log cells.mL⁻¹ for the two wines studied.

<table><caption>**Table 1** - Concentrations of *Brettanomyces* cells (log cells.mL⁻¹) determined in Bordeaux and Rhône Valley red wines quantified by MF, IR and IUVV labs, means of four trials per kit (mean of standard deviation between kit repeatability is 6.0 ± 5.9%). Anova with Tukey test was performed to enumeration results for each wine.</caption><thead><tr><th rowspan="2">Labs</th><th colspan="5">Bordeaux wine</th><th colspan="5">Rhône wine</th></tr><tr><th>SM</th><th>FCM or EM</th><th>K1</th><th>K2</th><th>K3</th><th>SM</th><th>FCM or EM</th><th>K1</th><th>K2</th><th>K3</th></tr></thead><tbody><tr><td>IR</td><td>3.7<sup>b</sup></td><td>3.8<sup>b</sup></td><td>/</td><td>1.5<sup>ef</sup></td><td>6.3<sup>a</sup></td><td>4.2<sup>ab</sup></td><td>3.7<sup>b</sup></td><td>/</td><td>3.9<sup>b</sup></td><td>2.5<sup>d</sup></td></tr><tr><td>MF</td><td>3.7<sup>b</sup></td><td>3.3<sup>bc</sup></td><td>2.1<sup>cdef</sup></td><td>/</td><td>1.9<sup>def</sup></td><td>3.7<sup>bc</sup></td><td>4.0<sup>ab</sup></td><td>2.7<sup>d</sup></td><td>/</td><td>1.4<sup>e</sup></td></tr><tr><td>IUVV</td><td>3.6<sup>b</sup></td><td>3.1<sup>bcd</sup></td><td>3.0<sup>bcde</sup></td><td>2.4<sup>cdef</sup></td><td>/</td><td>4.2<sup>ab</sup></td><td>3.7<sup>b</sup></td><td>3.0<sup>cd</sup></td><td>4.6<sup>a</sup></td><td>/</td></tr></tbody></table>

/: not analyzed

### *Brettanomyces* quantification in red wines after microbiological treatments (SO₂ / Chitosan)

In order to assess if q-PCR could give false positives, *Brettanomyces* cells were treated as described above.

### Bordeaux wine

- After SO₂ treatment, the mean total population obtained by DEFT after 2 weeks is 5.5 ± 0.2 log cells.mL⁻¹. However, no viable cells could be detected by DEFT and culture medium (SM). Quantification by both kits (K1 and K3) led to 6.3 ± 0.2 and 1.6 ± 0.2 log cells.mL⁻¹, respectively.
These results demonstrate that even 2 weeks after sulfite treatment, dead cells still give a positive signal by q-PCR.

- After chitosan treatment and racking, no viable cells could be detected using classical microbiological methods and q-PCR with K3. However, using q-PCR K1, 1.3 ± 0.7 log cells.mL⁻¹ were detected after 2 weeks. This residual population could be due to false positives.

*Burgundy wine*

- After SO₂ treatment, the mean total population obtained by FCM after 2 weeks is 5.2 ± 0.2 log cells.mL⁻¹. However, no viable cells could be detected by FCM and SM both. Quantification by both kits (K2 and K1) led to 5.1 ± 0.1 and 5.1 ± 0.3 log cells.mL⁻¹, respectively. These results demonstrate that even 2 weeks after sulfite treatment, dead cells still give a positive signal by q-PCR as Bordeaux wine.

- After chitosan treatment without racking, the mean total population obtained by FCM after 2 weeks is 5.2 ± 0.2 log cells.mL⁻¹, whereas viability measuring by SM and FCM decreases to the limit of detection after treatment. No *Brettanomyces* cell is detected by K1, whereas K2 gives about 1.8 ± 0.1 log cells.mL⁻¹ after 2 weeks.

**Conclusion**

In this study, we have validated that the classical enumerations (SM, FCM or DEFT) led to statically identical results, whereas molecular techniques led to different results probably due to matrix and manipulations. The different kits do not have the same effectiveness depending on laboratories and wine matrix tested. Compared to conventional methods of microbiological counts, most of the time, the kits seem to underestimate the viable population. However, the presence of dead cells could lead to an overestimation of the viable *Brettanomyces* population. These variations could be controlled by the use of an internal standard. This study must be continued on more wine samples.

**Acknowledgements**

The authors gratefully thank FranceAgriMer for providing financial support for this national project called GNBrett.

**References**

1. Conterno L, Joseph CML, Arvik TJ et al (2006) Genetic and physiological characterization of *Brettanomyces bruxellensis* strains isolated from wines. *Am J Enol Vitic* **57**: 139-147.

2. Gilis J-F, Strehaiano P (1999) Study of industrial alcoholic fermentation contaminations by *Brettanomyces* yeast. *Institut national polytechnique de Toulouse*.

3. Suárez R, Suárez-Lepe JA, Morata A et al (2007) The production of ethylphenols in wine by yeasts of the genera *Brettanomyces* and *Dekkera*: a review. *Food Chem* **102**: 10-21.

4. Tchobanov I, Gal L, Guilloux-Benatier M et al (2008) Partial vinylphenol reductase purification and characterization from *Brettanomyces bruxellensis*. *FEMS Microbiol Lett* **284**: 213-217.
# Low environmental impact sanitization treatments: effect of electrolyzed water and ozone treatments on grape and wine microbiota

F. CRAVERO<fnref n="1" />, K. RANTSIOU<fnref n="1" />, V. ENGLEZOS<fnref n="1" />, F. TORCHIO<fnref n="2" />, S. GIACOSA<fnref n="1" />, S. RÍO SEGADE<fnref n="1" />, L. ROLLE<fnref n="1" />, L. COCOLIN<fnref n="1" />*

Grape ecology – wine ecology – ozone – electrolyzed water

## Introduction

The microbial community present on the grape surface has been well documented to have a significant impact on wine quality and composition. The main microorganisms present on the surface of grapes are yeast, bacteria and moulds. Since they are the first to colonize the must, the sterilization of the grape surface is fundamental, in order to avoid the unwanted spoilage microorganisms to colonize the must and produce chemical compounds of unpleasant origin, such as acetic acid [1], in vinification without sulfite additions.

Several studies have proposed the use of Ozone (either in water or in gaseous form) as well as Electrolyzed Water as eco-friendly approaches to control the growth of various microorganisms, mainly due to their high oxidizing potential and their low environmental impact [2, 3].

The aim of this work was to evaluate the impact of these treatments on the yeast ecology in post-harvest grapes as well as their impact during the fermentation process, by using culture-dependent (traditional plate counts) and independent techniques. After each treatment, the grapes were crushed and then divided in two fermentation series: spontaneous and inoculated. Samples were collected before and after each sanitizing treatment and during the fermentation and subsequently subjected to DGGE analysis in order to understand the effectiveness of each treatment on the grape berry microbiota and the fermentation ecology.

## Materials and methods

### Grapes and fermentations

Barbera (*Vitis vinifera* L.) grapes were used in this study. The grapes were harvested in the Piedmont wine region (Asti province, northwest Italy) with a good phytosanitary condition, when the maturity was optimal for the production of Barbera wines. The grapes without the main stalk were divided in batches of about 2 kg each, and then placed in a single layer into perforated boxes. The batches were treated in duplicate with: 1. Electrolyzed Water (a. 100 mL at 40 mg/L free chlorine, b. 100 mL at 400 mg/L free chlorine); 2. Ozone Water (a. 5 ppm for 6 minutes, b. 5 ppm for 12 minutes); and 3. Ozone Gas (a. 30 ppm for 12 hours, b. 30 ppm for 24 hours). After treatments, the crushed grapes were divided in two fermentation series: spontaneous and inoculated. The fermentations were monitored with microbiological and chemical analyses.

### Microbiological analysis

The microbial community present on the grape surfaces as well as during the fermentations process was determined by using a multi-phasic approach: culture-dependent and independent techniques. For the culture-dependent analysis, samples were serially diluted in Ringer solution and then plated in duplicate WL Nutrient medium (Biogenetics, Milano, Italy), followed by an incubation for 3-4 days at 25°C.

<footnote n="1">¹University of Torino, Dipartimento di Scienze Agrarie, Forestali e Alimentari, Agricultural Microbiology and Food Technology Sector, Largo Paolo Braccini, 2 -10095 Grugliasco, Torino, Italy</footnote>

<footnote n="2">²Istituto di Enologia e Ingegneria Agro-Alimentare, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29122 Piacenza, Italy</footnote>
After the incubation period, the yeast total population was counted. The DGGE was used for the independent microbiology approaches. The DGGE analyses were performed by using the protocol described by Alessandria et al. [4].

### Chemical analysis

The concentrations of organic acid (citric acid, tartaric acid and malic acid), reducing sugars (glucose and fructose) and ethanol product were determined by using the protocols described by Giordano et al. [5].

## Results and discussion

The yeast total population present on the grape berry surface, before and after the treatments with Ozone Water, Ozone Gas and Electrolyzed Water, showed a reduction of their population. In fact, the yeast population present on the control trial was in the order of about $10^5$ CFU/mL, while in the case of the Electrolyzed Water (400 mg/L), Ozone Water (12 minutes at 30 ppm) and Ozone Gas (24 hours at 30 ppm), the yeast population was decreased by one logarithm to about $10^4$ CFU/mL. In another study, the Electrolyzed Water was already proposed as possible post-harvest control (250 ppm from 10 min) of *Botrytis cinerea* on grape table surface, since it was able to reduce the surface infection for 7 days and the disease severity at 2% after 10 days of storage at 25°C [6]. In this context, Ozone treatments in water and in gaseous form have been applied to ensure the storage of table grapes, the Ozone was able to reduce the contamination by *Botrytis cinerea* during storage [7, 8].

As well as decreased yeast populations, all the treatments demonstrated a selective effect against the yeast species present on the grape surface. In fact, these results were in accordance with the results obtained by the DGGE analysis.

The DGGE analysis during all fermentations confirmed the different microbiota ecology found on the grape surface between the treatments, particularly in the first fermentation step.

The counts on WL Nutrient medium during the spontaneous and inoculated fermentations highlighted the effectiveness of the treatments, with respect to the control; particularly, a decrease of the apiculate yeasts in the first part of the fermentation was observed. These results are very interesting, since apiculate yeasts were considered as high producers of acetic acid [9, 10]. The chemical analysis confirmed also this trend since control wines were characterized by a higher quantity of acetic acid, while the samples treated and inoculated showed low quantity of acetic acid.

## Conclusion

The results of this work highlight the possibility of using the Ozone (in water and gas form) and the Electrolyzed Water as possible alternatives to sulfur dioxide ($SO_2$), in order to control fermentations. The treatments reduced and modified the microbial ecology on the grapes, particularly the Electrolyzed Water with a concentration of about 400 mg/L and the Ozone Water (12 minutes). The results appear promising, since the use of Ozone and Electrolyzed Water in combination with the inoculation of *S. cerevisiae* in the must have resulted in optimum fermentation kinetics.

## Acknowledgements

The authors would like to thank Cristian Carboni and DenoraNEXT for the Ozone and Electrolyzed Water generators used in this work.

## References

1. Romano P, Suzzi G, Comi G et al (1993) Higher alcohol and acetic acid production by apiculate wine yeasts. *J Appl Bacteriol* **73**: 126-130.

2. Khadre M, Yousef A, Kim J (2001) Microbiological aspects of ozone applications in food: a review. *J Food Sci* **66**: 1242-1252.
3. Huang Y, Hung YC, Hsu SY et al (2008) Application of electrolyzed water in the food industry. *Food Control* **19**: 329-345.

4. Alessandria V, Marengo F, Englezos V et al (2015) Mycobiota of Barbera grapes from the Piedmont region from a single vintage year. *Am J Enol Vitic* **66**: 244-250.

5. Giordano M, Rolle L, Zeppa G et al (2009) Chemical and volatile composition of three Italian sweet white Passito wines. *J Int Sci Vigne Vin* **43**: 159-170.

6. Guentzel J, Lam KL, Callan MA et al (2010) Postharvest management of gray mold and brown rot on surfaces of peaches and grapes using electrolyzed oxidizing water. *Int J Food Microbiol* **144**: 54-60.

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# Managing wild yeast potential as an added terroir layer for wines' regional distinctiveness

N. FONTES<fnref n="1" />*, F. CENTENO<fnref n="2" />, M.F. TEIXEIRA<fnref n="2" />, R. TENREIRO<fnref n="3" />, B. CABRAL ALMEIDA<fnref n="1" />, A. GRAÇA<fnref n="1" />

Wine yeasts – starter strains – autochthonous strains – terroir – uniqueness – regional

## Introduction

Wine flavor is the culminating result of a vast number of factors such as grape variety, geographical and cultural grape-growing conditions, grapes' microbiome and winemaking itineraries. Thus, this complex character of wine has implicated the use of selected commercial yeast, for a more controlled fermentation process and for the production of specific aromatic profiles and the reduction of sensory defects. However, the widespread use of these starter cultures can lead to a loss of typicality and complexity of the wines and their overall standardization, as well as ecological imbalances that promote the growth of undesirable species such as *Brettanomyces*. Moreover, available commercial «strains» are numerous and one same yeast may often have different names or acronyms, a consequence of this practice being the standardization of sensory characters resulting from the fermentation process, affecting quality wines.

The use of “so called” autochthonous yeast strains selected with characteristics of a starter culture could be an important tool in improving the organoleptic and sensory characteristics of typical wines of a given region, as well as providing for valorization and preservation of the microbial biodiversity [1]. In fact, it has been suggested that the land from which the grapes are grown imparts a unique quality to the wine, especially when spontaneous fermentations are carried out [2, 3].

Indeed, many studies of wine fermentations in various regions of the world have demonstrated the important contribution of indigenous wine yeasts to the sensory properties and overall appeal of the final product [4-7]. The present work aims to study the oenological potential of using two autochthonous wild yeasts belonging to a specific *terroir* as starter cultures.

## Materials and methods

### *Yeast strains and fermentation conditions*

The autochthonous yeast strains – QC386 and QC496 – have been selected over a collection of autochthonous wild yeasts of 768 isolates owned by Sogrape Vinhos S.A, originated from four wine regions in Portugal, including the Dão Region. Rather than the typical pre-selection of any particular taxon (namely *S. cerevisiae*), a universal approach was applied to all isolates allowing for an individual evaluation of each isolate's potential, regardless of species. On the other hand, a more practice-focused adaptation of the OIV protocol for yeast selection was used [8]. Industrial fermentations were conducted to evaluate the fermentation efficiency of the locally identified yeast strains – QC386 and QC496 – against commercial yeast preparations. For this purpose, a total volume of around 500 000 L, over two vintages (2013 and 2014), of white and red must (both a blend of grape varieties) was separately inoculated with commercial yeasts LALVIN QA23 (for white must) and LALVIN D254 (for red must) and the chosen wild strains from the collection.

### *PCR analysis*

Genome PCR-fingerprinting techniques [9] were applied to identify and distinguish the inoculated strain from the remaining yeast flora present in the grape must.

<footnote n="1">¹Sogrape Vinhos S.A., Aldeia Nova, 4430-809 Avintes, Portugal</footnote>

<footnote n="2">²Proenol - Indústria Biotecnológica Lda, Travessa das Lages 267, Apto 547, 4405-194 Canelas, Vila Nova de Gaia, Portugal</footnote>

<footnote n="3">³Universidade de Lisboa, Faculdade de Ciências, Instituto de Biossistemas e Ciências Integrativas (BioISI), Edifício TecLabs, Campus da FCUL, Campo Grande, 1749-016 Lisboa, Portugal</footnote>

<footnote>*Corresponding author: natacha fontes@sogrape.pt</footnote>
## Results and discussion

General technological characteristics influencing vinification were evaluated. The effects of different sugars, glucose and fructose, which are the main sugars found in grape must, on the kinetic performance of yeast fermentation are represented in Figures 1 and 2. Results showed similar kinetics profile between autochthonous selected yeasts and commercial strains, both for sugar and nitrogen consumption (Figures 1 and 2). However, QC496 showed a higher consumption rate in white wines, at a lower fermentation temperature (16°C), resulting in shorter fermentation time (Figure 1). In red wines, the difference in kinetics properties between yeast strains was not as evident (Figure 2). It should be stressed that genomic analysis by PCR-fingerprinting, performed in all the 32 fermentation vats (10 of white and 22 of red), confirmed the inoculated starters as the dominant yeasts during the fermentation process (data not shown).

<figure><img src="image_153.png" /><figcaption>**Fig. 1** - Glucose (A), Fructose (B) consumption profiles and alcohol production - AbV (C) observed at 16°C for QC386 (■) QC496 (●) and QA23 (▲) in white wine fermentations. (D) Glucose, Fructose and YAN consumption in the 1st alcoholic fermentation phase ($t_{50}$) and the 2nd phase ($t_{end}$). Data represent values of 2014 vintage.</figcaption></figure>

<table><caption>**Table 1** - Fermentation performance of autochthonous (QC386 and QC496) and commercial (QA23) yeast strains in white wine (values represent the average between 10 different replicates over 2013 and 2014 vintages). Vm: average consumption velocity of sugars.</caption><thead><tr><th rowspan="2">Yeast strain</th><th colspan="3">Fermentation duration (days)</th><th rowspan="2">Sugar consumption (%)</th><th rowspan="2">Vm (g/L day-1)</th><th rowspan="2">ΔYAN (%)</th></tr><tr><th>Average</th><th>Max</th><th>Min</th></tr></thead><tbody><tr><td>QA23</td><td>17</td><td>23</td><td>14</td><td>99</td><td>12.6</td><td>68</td></tr><tr><td>QC386</td><td>27</td><td>41</td><td>15</td><td>97</td><td>7.8</td><td>63</td></tr><tr><td>QC496</td><td>17</td><td>22</td><td>12</td><td>99</td><td>12.5</td><td>76</td></tr></tbody></table>

Concerning the yeast nitrogen demand during alcoholic fermentation (AF), globally there are no important differences between yeast strains (yeast assimilable nitrogen consumption during AF – ΔYAN – Tables 1 and 2). However, when analyzing the 1st AF phase ($t_{50}$ - midpoint of the AF – time-duration in days, for AbV > 8% vol.) separately from de 2nd phase ($t_{end}$ - days from the AF midpoint day until end of
fermentation), where fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation, a different pattern of substrate consumption is observed among yeast strains, more pronounced in red musts (Figures 1 and 2).

A

<figure><img src="image_154.png" /></figure>

B

<figure><img src="image_155.png" /></figure>

C

<figure><img src="image_156.png" /></figure>

D

<figure><img src="image_157.png" /><figcaption>**Fig. 2** - Glucose (A), Fructose (B) consumption profiles and alcohol production - AbV (C) observed at 25°C for QC386 (■) QC496 (●) and D254 (▲) in red wine fermentations. (D) Glucose, Fructose and YAN consumption in the 1st alcoholic fermentation phase ($t_{50}$) and the 2nd phase ($t_{end}$). Data represent mean values of 2014 vintage replicates.</figcaption></figure>

<table><caption>**Table 2** - Fermentation performance of autochthonous (QC386 and QC496) and commercial (QA23) yeast strains in red wine (values represent the average between 22 different replicates over 2013 and 2014 vintages). Vm: average consumption velocity of sugars.</caption><thead><tr><th rowspan="2">Yeast strain</th><th colspan="3">Fermentation duration (days)</th><th rowspan="2">Sugar consumption (%)</th><th rowspan="2">Vm (g/L day-1)</th><th rowspan="2">ΔYAN (%)</th></tr><tr><th>Average</th><th>Max</th><th>Min</th></tr></thead><tbody><tr><td>D254</td><td>6</td><td>7</td><td>5</td><td>98</td><td>33.3</td><td>59</td></tr><tr><td>QC386</td><td>6</td><td>8</td><td>5</td><td>98</td><td>31.6</td><td>57</td></tr><tr><td>QC496</td><td>5</td><td>6</td><td>4</td><td>99</td><td>37.7</td><td>42</td></tr></tbody></table>

Strain QC496 showed a higher sugar consumption rate, both for glucose and fructose, as well as YAN consumption during the 1st AF phase, consuming the same fructose at a higher rate with much less YAN demand during the 2nd AF phase. In addition, this autochthonous strain showed a greater organoleptic potential as judging by higher alcohol production (data not shown) and the evaluated sensory attributes assessed by a blind tasting with a professional panel of 15 oenologists (Figure 3).

Judging from the obtained fermentation kinetics and sensory properties, wild yeasts are presumed to be more competitive than commercial ones since they seem better adapted to the ecological and technological features of their own wine-growing area. Indeed, genome PCR-fingerprints have shown them as the dominant yeast strains during industrial fermentations, while competing with other autochthonous yeasts present in the must.
<figure><img src="image_158.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_159.png" /><figcaption>**Fig. 3** - Ranking box-plots for the evaluated sensory attributes of the white (A) and red (B) wines produced with autochthonous (QC386 and QC496) and commercial yeasts (QA23 and D254).</figcaption></figure>

**Conclusion**

The recognition and preservation of microorganisms present in vineyard and their preservation through cellar operations is a strategic activity for the oenologist who wants to express his own *terroir* in his wines.

This study gave the final confirmation on the existence of autochthonous yeasts from the Dão Region suitable for selection as starter cultures. They allow for features to potentially ensure preservation of regional distinctiveness while allowing for the production of premium quality wines, thus adding an extra layer of *terroir* to Dão wines.

**Acknowledgements**

This work was supported by Sogrape Vinhos SA. The authors thank Proenol-Indústria Biotecnológica Lda. and Universidade de Lisboa, Faculdade de Ciências, Instituto de Biossistemas e Ciências Integrativas (BioISI) for all the support provided during the course of this study.
Abbreviations

AbV, alcohol by volume; AF, alcoholic fermentation; YAN, yeast assimilable nitrogen; PCR, polymerase chain reaction; OIV, International Organization of Vine and Wine.

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8. McGuire D, Lemsaddek A, Tenreiro R et al (2011) Selection and characterization of autochthonous yeast starter strains isolated from Portuguese musts, pp. 439-444. *In 9e Symposium International d'Oenologie de Bordeaux (Oeno 2011)*.

9. Capece A, Salzano G, Romano P (2003) Molecular typing techniques as a tool to differentiate non-*Saccharomyces* wine species. *Int J Food Microbiol* **84**: 33-39.
# Monitoring extracellular proteolytic activity of wine yeasts during alcoholic fermentation

L. CHASSERIAUD<fnref n="1" />,<fnref n="2" />*, C. MIOT-SERTIER<fnref n="1" />,<fnref n="3" />, J. COULON<fnref n="2" />, W. ALBERTIN<fnref n="1" />,<fnref n="2" />, M. BELY<fnref n="1" />

Proteolytic activity – wine yeasts – white grape must – proteases

## Introduction

Alcoholic fermentation is a complex process that involves sequential development of different yeasts species. The first hours of fermentation are dominated by the growth of non-*Saccharomyces* yeasts [1-3]. These yeasts initiate alcoholic fermentation thus leading to environmental modifications (increasing ethanol concentration, presence of inhibitors, competition for nutrients...), then *Saccharomyces cerevisiae* becomes predominant and achieves alcoholic fermentation.

Among non-*Saccharomyces* yeasts, the genera *Hanseniaspora*, *Candida*, *Metschnikowia*, *Pichia*, *Torulaspora* and *Issatchenkia* are frequently found in fermenting grape must [4-7]. It is now well known that non-*Saccharomyces* species produce secondary compounds (volatile thiols, esters, higher alcohols, acetic acids...) and can impact positively or negatively on wine characteristics [8, 9]. They can also secrete extracellular enzymes that can have an interest in winemaking. Indeed, several enzymatic activities can improve wine quality (glycosidases, β-lyases...) or the winemaking process.

Proteases could have an impact on wine aroma or liberate peptides and amino acids from proteins and so increase assimilable nitrogen for yeasts in must.

If *Saccharomyces cerevisiae* is not known as a great producer of extracellular proteases, several studies have shown that some non-*Saccharomyces* species and particularly *Metschnikowia pulcherrima* have an interesting potential [10-13]. Nevertheless, most of the existing methods to reveal proteolytic activity require the use of laboratory medium, with conditions very different from grape must. Here we propose a method to assess proteolytic activity of yeasts directly in fermenting white grape must in order to gain insight on this issue in winemaking conditions.

## Materials and methods

### *Yeast strains*

7 yeast strains were used in this study (4 *Metschnikowia pulcherrima*, 1 *Metschnikowia* spp. (99% of identity with *M. andauensis*), 1 *Saccharomyces cerevisiae* and 1 *Torulaspora delbrueckii*) (table 1).

<table><caption>**Table 1 - Strains used in alcoholic fermentations**</caption><thead><tr><th>Species</th><th>Strain</th><th>Collection</th></tr></thead><tbody><tr><td rowspan="4">Metschnikowia pulcherrima</td><td>L1329</td><td>Centre de Ressources Biologiques Oenologiques, Bordeaux, France</td></tr><tr><td>Y6259</td><td>Northern Regional Research Laboratory</td></tr><tr><td>NZ366</td><td>Centre de recherche Pernod-Ricard, Créteil, France</td></tr><tr><td>IWBT Y1123</td><td>Institute for Wine Biotechnology, Stellenbosch, South Africa</td></tr><tr><td>Metschnikowia spp.</td><td>L0563</td><td>Centre de Ressources Biologiques Oenologiques, Bordeaux, France</td></tr><tr><td>Saccharomyces cerevisiae</td><td>X5</td><td rowspan="2">Laffort, Bordeaux, France</td></tr><tr><td>Torulaspora delbrueckii</td><td>Alpha</td></tr></tbody></table>

<footnote n="1">¹Univ. Bordeaux, ISVV, EA 4577, Œnologie, F-33140 Villenave d'Ornon, France</footnote>

<footnote n="2">²BioLaffort, 33100 Bordeaux, France</footnote>

<footnote n="3">³INRA, ISVV, Unité de recherche Œnologie, 210 Chemin de Leysotte, 33140 Villenave d'Ornon, France</footnote>
### Preculture conditions

Except for *S. cerevisiae* and *T. delbrueckii*, two preculture steps were necessary before fermentation. First, strains were grown at 24°C for 24h in YPD-based medium containing 1% yeast extract (w/v, Difco Laboratories, Detroit, MI), 1% Bacto peptone (w/v, Difco), and 2% glucose (w/v). Then, cultures were transferred in half diluted grape must and grown at 24°C for 24h. For *S. cerevisiae* and *T. delbrueckii* only growth in half diluted grape must is necessary.

### Alcoholic fermentations

Alcoholic fermentations were carried out in Sauvignon Blanc grape must from the Bordeaux region (reducing sugars 203 g/L, assimilable nitrogen 150 mg/LN, free SO₂ 18 mg/L, total acidity 4.19 g/L H₂SO₄) at 18°C with agitation and in 125-mL bioreactors. The different strains were inoculated at 5.10⁶ viable cells/mL in pure cultures. Fermentations kinetics were monitored by CO₂ release with regular weight loss measurement.

### Cell enumeration

Yeast growth during fermentation was monitored by plate counting on a YPD-based medium (1% yeast extract (w/v), 1% Bacto peptone (w/v), 2% glucose (w/v), 2% agar (w/v)).

### Proteolytic activity

In order to monitor the proteolytic activity directly in fermenting grape must, a chromogenic protease substrate was used: azocasein (Sigma). Azocasein proteolysis liberates a free dye into the supernatant, which can be quantified by optical density measurement at 440 nm. A solution of azocasein at 20 mg/mL was prepared in NaOH 0.1 M and added in bioreactors at 1.5 mg/mL final concentration. To verify that azocasein does impact yeast growth and fermentations, assays were carried out with and without azocasein. Samples were taken all along alcoholic fermentation. Reaction was stopped with trichloroacetic acid (10% final concentration v/v, Sigma). Samples were then centrifuged at 14 000 rpm for 10 minutes. Optical density of the supernatant was measured to evaluate the proteolytic activity of the different species.

### Data analysis

All fermentations were performed in triplicate. Several growth and fermentation parameters were calculated for each species: growth rate (h⁻¹), maximum population size (CFU.mL⁻¹), fermentation lag phase (time between the inoculation and the beginning of alcoholic fermentation, h), maximum fermentation rate (g.L⁻¹.h⁻¹) and maximum CO₂ release (g.L⁻¹). R software was used for statistical analysis (R Development Core Team, 2008). Variance heteroscedasticity led us to apply Kruskal-Wallis test to determine the difference between fermentation with and without azocasein. The statistical level of significance was set at p≤0.05.

## Results and discussion

### Effect of azocasein on growth and alcoholic fermentation

Kruskal-Wallis test on the different parameters revealed that azocasein had no significant impact on growth and fermentation for the different strains tested (*S. cerevisiae* X5, *T. delbrueckii* Alpha, *Metschnikowia* spp. L0563, *M. pulcherrima* IWBT Y1123). This substrate will be use to measure proteolytic activity of several yeast strains in oenological conditions.

### Proteolytic activity of yeasts during alcoholic fermentation

Evolution of optical density of the different yeasts due to the degradation of azocasein is given in figure 1 and growth kinetics in figure 2. No variation was detected for *T. delbrueckii* and *S. cerevisiae*, showing no protease activity, in accordance with previous studies [10]. In contrast, a strong increase in the optical density, reflecting a high proteolytic activity was observed for all *Metschnikowia* strains tested. The proteolytic activity of *M. pulcherrima* was already highlighted in laboratory medium [10]. Differences between *Metschnikowia* strains were observed in terms of proteolytic activity. In our conditions, *Metschnikowia* spp. L0563 has the strongest activity, leading to an increase of OD440 nm up to 0.21, unlike *M. pulcherrima* Y6259 which reaches only 0.17.

For all strains except *M. pulcherrima* Y6259, proteases are active from the start of growth, the cumulated activity being maximal at the end of growth. For *M. pulcherrima* Y6259, the proteolytic activity is detected only at the end of growth.
<figure><img src="image_160.png" /><figcaption>**Fig. 1** - Proteolytic activity (evolution OD440 nm) of different yeast strains during alcoholic fermentation.</figcaption></figure>

<figure><img src="image_161.png" /><figcaption>**Fig. 2** - Growth kinetics of different yeast strains during alcoholic fermentation.</figcaption></figure>

## Conclusion

In this work, we propose for the first time a test to monitor easily yeast proteolytic activity all along alcoholic fermentation, directly in the fermenting grape must. *Metschnikowia* species seem particularly active but differences could be observed between strains.

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