S. cerevisiae wine fermentation is linked to specific esters enhancement

    Increase of fruity aroma during mixed T. delbrueckii / S. cerevisiae wine fermentation is linked to specific esters enhancement Philippe Renault, Joana Coulon, Gilles de Revel, Jean-Christophe Barbe, Marina Bely PII: DOI: Reference:

S0168-1605(15)00240-8 doi: 10.1016/j.ijfoodmicro.2015.04.037 FOOD 6902

To appear in:

International Journal of Food Microbiology

Received date: Revised date: Accepted date:

23 February 2015 17 April 2015 24 April 2015

Please cite this article as: Renault, Philippe, Coulon, Joana, de Revel, Gilles, Barbe, Jean-Christophe, Bely, Marina, Increase of fruity aroma during mixed T. delbrueckii / S. cerevisiae wine fermentation is linked to specific esters enhancement, International Journal of Food Microbiology (2015), doi: 10.1016/j.ijfoodmicro.2015.04.037

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ACCEPTED MANUSCRIPT Title Increase of fruity aroma during mixed T. delbrueckii / S. cerevisiae wine fermentation is

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linked to specific esters enhancement

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Author names

Philippe Renault1,2, Joana Coulon2, Gilles de Revel1,3, Jean-Christophe Barbe1,3, Marina

(1)

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Bely1*

Univ. Bordeaux, ISVV, EA 4577, Unité de recherche Œnologie, 33140 Villenave d'Ornon,

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France

Biolaffort, 33100 Bordeaux, France

(3)

INRA, ISVV, USC Oenologie, 33140 Villenave d'Ornon, France

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(2)

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*corresponding author. Tel.: +33-5-5757-5866; Fax: +33-5-5757-5813.

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Email address: [email protected]

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ACCEPTED MANUSCRIPT Abstract The aim of this work was to study ester formation and the aromatic impact of T. delbrueckii when used in association with S. cerevisiae during the alcoholic fermentation of must. In

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order to evaluate the influence of the inoculation procedure, sequential and simultaneous

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mixed cultures were carried out and compared to pure cultures of T. delbrueckii and S. cerevisiae.

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Our results showed that mixed inoculations allowed the increase, in comparison to S. cerevisiae pure culture, of some esters specifically produced by T. delbrueckii and significantly correlated to the maximal T. delbrueckii population reached in mixed cultures.

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Thus, ethyl propanoate, ethyl isobutanoate and ethyl dihydrocinnamate were considered as activity markers of T. delbrueckii. On the other hand, isobutyl acetate and isoamyl acetate

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concentrations were systematically increased during mixed inoculations although not correlated with the development of either species but were rather due to positive interactions between these species.

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Favoring T. delbrueckii development when performing sequential inoculation enhanced the

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concentration of esters linked to T. delbrueckii activity. On the contrary, simultaneous inoculation restricted the growth of T. delbrueckii, limiting the production of its activity

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markers, but involved a very important production of numerous esters due to more important positive interactions between species. These results suggest that the esters concentrations enhancement via interactions during mixed modalities was due to S. cerevisiae production in response to the presence of T. delbrueckii.

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Finally, sensory analyses showed that mixed inoculations between T. delbrueckii and S. cerevisiae allowed to enhance the complexity and fruity notes of wine in comparison to S. cerevisiae pure culture. Furthermore, the higher levels of ethyl propanoate, ethyl isobutanoate, ethyl dihydrocinnamate and isobutyl acetate in mixed wines were found responsible for the increase of fruitiness and complexity.

Keywords: Non-Saccharomyces, Torulaspora delbrueckii, wine, fermentation, mixed inoculation, esters.

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ACCEPTED MANUSCRIPT 1. Introduction

The fermentation of grape must is a complex microbial process, involving sequential

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development of various yeast communities including Saccharomyces and non-Saccharomyces

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species. The nutrient competition and the increasing ethanol content gradually eliminate the less tolerant species, thus favouring the development of S. cerevisiae which then completes

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the fermentation (Heard and Fleet 1985, 1986). The dominance of non-Saccharomyces yeasts during the early stages of the reaction has a major impact on the aromatic composition and sensory properties of wine (Ciani et al., 2010; Domizio et al., 2007; Fleet 2008; Jolly et al.,

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2014; Renouf et al., 2007; Romano et al., 2003; Swiegers et al., 2005). Consequently, many researchers have investigated the specific metabolisms of the various non-Saccharomyces

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yeast species and their potential applications in the wine industry (Andorra et al., 2011; Capece et al., 2011; Comitini et al., 2011; Domizio et al., 2011; Jolly et al., 2003; Moreira et

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al., 2011; Tofalo et al., 2012).

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In this context, Torulaspora delbrueckii, one of the few non-Saccharomyces yeast species currently commercialized, has been described as having a positive impact on the organoleptic

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quality of wines. Firstly, its low production of compounds like acetic acid, ethyl acetate, acetaldehyde, acetoin, hydrogen sulphide, and volatile phenols minimises off-flavours (Cabrera et al., 1988; Ciani and Maccarelli 1998; Ciani and Picciotti 1995; Herraiz et al., 1990; Martinez et al., 1990; Plata et al., 2003; Renault et al., 2009; Shinohara et al., 2000).

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Secondly, numerous authors (Azzolini et al., 2012; Comitini et al., 2011; King and Dickinson, 2000; Hernandez-Orte et al., 2008) showed that the strong β-glucosidase activity of this species enhanced wine aroma by modulating the levels of nor-isoprenoids, terpenols, and lactones by hydrolysing their respective precursors. This species also releases 2-phenylethanol at higher levels than S. cerevisiae (Herraiz et al., 1990 ; Moreno et al., 1991 ; Renault et al., 2009). However, concerning esters, a report from Viana et al. (2008) indicates that in synthetic media, T. delbrueckii produces less ester acetates and ethyl hexanoate than S. cerevisiae. In similar conditions, other authors confirmed the low production capacities of T. delbrueckii towards isoamyl acetate, and as well as ethyl butanoate, hexanoate and octanoate (Hernandez-Orte et al., 2008; Plata et al., 2003 ; Renault et al., 2009). Sadoudi et al. (2012) showed a lower production than S. cerevisiae for isoamyl, hexyl and 2-phenylethyl acetate, but a higher production of ethyl 4-hydroxybutanoate, ethyl 3-hexanoate and diethyl succinate. It is important to note that esters production by this species 3

ACCEPTED MANUSCRIPT is strain dependent (Renault et al., 2009) and that results are different when T. delbrueckii is associated to S. cerevisiae in mixed cultures.

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Despite a good ethanol production for a non-Saccharomyces yeast, between 7 and 12 % (v/v)

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(Cabrera et al., 1988, Ciani and Maccarelli 1998; Ciani and Piccioti 1995, Herraiz et al., 1990, Renault et al., 2009), the use of T. delbrueckii in pure culture leads to stuck fermentations.

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Mixed inoculations of selected strains of this species with S. cerevisiae have thus been proposed to modulate wine flavour and ensure complete alcoholic fermentation. In these conditions, authors have demonstrated that besides the reduction of off-flavours compounds

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like volatile acidity, acetaldehyde and acetoin (Bely et al., 2008; Ciani et al., 2006, Herraiz et al., 1990), the mixed inoculation of these two yeast species gives the systematic increase of 2-

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phenylethanol, terpenols and lactones (Azzolini et al., 2012; Comitini et al., 2011; Herraiz et al., 1990; Sadoudi et al., 2012). Results concerning esters production by T. delbrueckii / S. cerevisiae mixed inoculations remain confusing. According to Herraiz et al. (1990), mixed

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inoculations allow an increase, in comparison to pure cultures, of the total ester concentration

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and in particular the levels of isoamyl acetate and ethyl methanoate, octanoate, hexanoate and 3-hydroxybutanoate. On the contrary, Sadoudi et al. (2012) and Comitini et al. (2011) showed

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that total esters concentration of mixed inoculations was lower than that of a pure S. cerevisiae culture with a significant reduction in acetate esters and in particular isoamyl acetate. Azzolini et al. (2012) found no difference in the overall esters concentration between mixed T. delbrueckii / S. cerevisiae and pure S. cerevisiae cultures, but the level of some of

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them in mixed culture was higher (such as ethyl 3-hydroxybutanoate) while others were lower (such as isoamyl acetate). These overall contradicting results concerning esters concentrations in mixed inoculations may be due to the fact that their production depends on the development of each species during fermentation which has so far not been taken into account by authors in their discussion. The growth of the 2 yeasts may be different as a result of difference in must composition, fermentation temperature and initial concentration of each species. Furthermore, different interactions between yeasts (Albergaria et al., 2010; Fleet, 2003; Nissen et al., 2003, Renault et al., 2013) may be involved and modify the metabolic behavior of the 2 species.

Hence, the aim of this work was to study ester formation and the aromatic impact of T. delbrueckii when used in association with S. cerevisiae during the alcoholic fermentation of must. In these conditions, esters were evaluated at 40% of fermentations and at the end of 4

ACCEPTED MANUSCRIPT fermentations conducted with pure and mixed cultures of the two species. To gain more insights on aroma release, the growth of the 2 species as well as the fermentation kinetics

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were also monitored throughout the fermentation.

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ACCEPTED MANUSCRIPT 2. Materials and methods

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2.1 Microorganisms

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In this study, 3 commercial strains from Laffort company (France) were used. The 2 S. cerevisiae strains were Zymaflore® X5 and Zymaflore® FX10 for laboratory and winery scale TD N. SACCH.

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experiments respectively. T. delbrueckii Zymaflore® Alpha

was used in both

types of experiment. Yeasts were grown at 24°C on complete YPDA medium (1% yeast

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extract, 1% peptone, 2% dextrose) solidified with 2% agar and adjusted to pH 4.8.

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2.2 Fermentation medium

2.2.1 Laboratory scale

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The medium used in this study was a Sauvignon blanc must from Bordeaux area, pH: 3.15,

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with a sugar concentration of 203 g/L and an available nitrogen concentration adjusted to 210 mg/L (i.e. amino acids: 114 mg/L and ammonia: 96 mg/L). The total and free sulfur dioxide

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concentrations were respectively 60 and 19 mg/L. Before yeast inoculation, the must was sterilised by filtration (0.45 µm nitrate cellulose membrane, Millipore, Molsheim, France)

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2.2.2 Winery scale

The medium used in winery scale was a Merlot must from Bordeaux area, pH: 3.54, with a sugar concentration of 258 g/L and an available nitrogen concentration adjusted to 210 mg/L (i.e. amino acids: 119 mg/L and ammonia: 91 mg/L). The total and free sulfur dioxide concentrations were respectively 29 and 17 mg/L.

2.3 Fermentation conditions

2.3.1 Laboratory scale

Fermentation kinetics were monitored by CO2 release (Bely et al., 1990 a, b). The amount of CO2 release (g/L) was determined by automatic measurement of fermentor weight loss every 6

ACCEPTED MANUSCRIPT 20 minutes. The CO2 production rate (g/L/h) was obtained by polynomial smoothing of the last 11 CO2 measurements. Weight loss due to evaporation was under 2%.

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Yeasts were pre-cultured in Erlenmeyer flasks filled with must at 24°C for 24 or 48h for S.

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cerevisiae and T. delbrueckii, respectively. Fermentations were carried out at 24°C with agitation in 1.2 L fermentors locked to maintain anaerobiosis throughout alcoholic

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fermentation (CO2 was released through a sterile air outlet condenser). Four different fermentations were conducted: two with pure cultures and two with mixed cultures. Two types of mixed cultures were carried out: simultaneous mixed modality where T. delbrueckii

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and S. cerevisiae were inoculated at the same time and sequential mixed modality where T. delbrueckii was inoculated 24h before S. cerevisiae yeast. Single and mixed cultures were

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inoculated with 1 x 107 viable cells/mL for T. delbrueckii and 2 x 106 viable cells/mL for S. cerevisiae. All experiments were performed in triplicate.

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2.3.2 Winery scale

Fermentations were carried out in 200L tanks at temperatures between 18 and 22°C.

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Fermentation kinetics were evaluated by monitoring the density. Two different fermentations were carried out: a pure culture of S. cerevisiae and a sequential mixed culture where T. delbrueckii was inoculated 24h before S. cerevisiae yeast. Single and mixed cultures were inoculated with 1 x 107 viable cells/mL for T. delbrueckii and 2 x 106 viable cells/mL for S.

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cerevisiae. All experiments were performed in duplicate.

2.4 Population dynamics

In mixed cultures, yeast growth was determined by plate counting on 2 different agar media. Samples were withdrawn throughout fermentation and diluted appropriately. NonSaccharomyces cells were counted using a specific agar medium (NS): YPDA (1% yeast extract, 1% peptone, 2% dextrose, 2% agar; pH 4.8) supplemented with 1 µg/mL cycloheximide to promote the growth of T. delbrueckii Alpha and inhibit that of S. cerevisiae X5. The number of S. cerevisiae was given as the difference between the total plate count using YPDA medium and the plate count using NS medium. Yeast growth in single cultures was determined using only the YPDA medium. Plates were incubated at 24 °C for 4 days before counting. 7

ACCEPTED MANUSCRIPT The level of yeast population was only measured for experiments carried out in laboratory.

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2.5 Fermentation product analysis

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Ethanol concentration (% vol.) was measured by infrared refractance (SpectraAnalyser, Axflow, Plaisir, France) and sugar (g/L) was determined chemically by colorimetry (460 nm)

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in continuous flux (Sanimat, Montauban, France). These analyses were carried out by Sarco laboratory (Floirac, France).

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2.6 Esters analysis

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Concentration of esters was determined using a head space solid phase microextraction (HSSPME) followed by gas chromatography-mass spectrometry (GC-MS) as described by Antalick et al. (2010).

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The fiber (Supelco, Bellefonte, PA) was coated with 100 μm stationary phase

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polydimethylsiloxane film (PDMS-100). Twenty µL of a stock solution of internal standards, ethyl-d5 butyrate, ethyl-d5 hexanoate, ethyl-d5 octanoate, and ethyl-d5 cinnamate at 200

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mg/L each in absolute ethanol (analytical grade, 99.97%, Scharlau Chemie S.A, Barcelona, Spain), was added to 25 mL of the samples. A 10 mL sample was placed in a 20 mL headspace vial, 3.5 g of sodium chloride was added, and the vial was tightly sealed with a PTFE-lined cap. The solution was homogenized in a vortex shaker and then loaded onto a lheim an der Ruhr, Germany) autosampling device.

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Gerstel (

The program consisted of swirling the vial at 500 rpm at 40 °C for 2 min, then inserting the fiber into the headspace at 40 °C for 30 min as the solution was swirled again, then transferring the fiber to the injector for desorption at 250 °C for 15 min. Gas chromatography analyses were carried out on an HP 5890 GC system coupled to an HP 5972 quadrupole mass spectrometer (Hewlett-Packard), equipped with a Gerstel MPS2 autosampler. Injections were in splitless mode for 0.75 min, using a 2 mm I.D. non-deactivated direct linear transfer (injector temperature, 250 °C; interface temperature, 280 °C) and a BP21 capillary column (50 m × 0.32 mm, film thickness, 0.25 μm, SGE). The oven temperature was programmed at 40 °C for 5 min, then raised to 220 °C at 3 °C/min, and held at that temperature for 30 min. The carrier gas was Helium N55 (Air Liquide, France) with a column-head pressure of 8 psi. The mass spectrometer was operated in electron ionization mode at 70 eV with selected-ion-

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ACCEPTED MANUSCRIPT monitoring (SIM) mode. Esters were characterized by comparing their linear retention indices and mass spectra with those of standards.

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2.7 Sensory Analyses

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2.7.1 General Conditions

Sensory analyses were performed as described by Martin and de Revel (1999). Samples were evaluated in individual booths, using covered, black ISO glasses (NF V09-110, 1971)

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containing about 50 mL of liquid, coded with three-digit random numbers. Sessions lasted approximately 5 min.

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The panel was composed of 33 professional tasters (17 women and 16 men) aged between 24 and 60 years and distributed between researchers (ISVV, Bordeaux University) selected for

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their experience in assessing fruity aromas in red wine and winemakers of Bordeaux area.

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2.7.2 Discriminative testing methods

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Triangular tests were performed in a three-alternative choice. For each triangular test, three numbered samples were presented in random order: two identical and one different. Each judge used direct olfaction to identify the sample perceived as different in each test and gave an answer, even if he was not sure. The results of all of the triangular tests were statistically

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analyzed, according to the tables given in the literature (Martin and de Revel, 1999) and based on the binomial law corresponding to the distribution of answers in this type of test.

2.7.3 Descriptive testing methods

Sensory profiles of wines were evaluated for overall aromatic complexity, fruit aroma intensity and vegetal intensity. For each sample, the subject rated the intensity of these descriptors on a 0 to 7 scale where 0 corresponds to “no odour perceived” and 7 to “very intense”. All descriptors were mean-centered per panelist and scaled to unit variance. Data were then analyzed by single-factor variance analysis (ANOVA, p < 0.05).

2.7.4 Esters adjustment (winery scale)

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ACCEPTED MANUSCRIPT In order to study the impact of some esters on the sensory characteristics of wine, pure chemicals were directly added into the wine before tasting. Grade purity compounds were obtained from commercial sources as follows: ethyl propanoate

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from Sigma−Aldrich (Saint-Quentin-Fallavier, France) and ethyl isobutanoate, ethyl

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dihydrocinnamate and isobutyl acetate from VWR Prolabo (Fontenay-sous-bois, France).

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2.8 Statistical analysis

In order to compare modalities, data were analyzed by single-factor variance analysis

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(ANOVA, p < 0.05 ) after the verification of variance homogeneity (Levene test, p > 0.05). Thereafter a multiple comparison test (Duncan) was applied to classify the different

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modalities (p < 0.05). Correlations between variables were studied using Spearman’s rank correlations (ρ, p < 0.05). All these tests were carried out with R program as the principal

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component analysis (PCA).

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ACCEPTED MANUSCRIPT 3. Results

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3.1 Laboratory scale

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3.1.1 Growth and fermentation kinetics

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Single and mixed cultures were inoculated with 1 x 107 viable cells/mL for T. delbrueckii and 2 x 106 viable cells/mL for S. cerevisiae. Four different fermentations were carried out: two pure cultures and two mixed cultures. For simultaneous mixed culture, T. delbrueckii and S.

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cerevisiae were inoculated at the same time while T. delbrueckii was inoculated 24h before S.

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cerevisiae in sequential mixed culture.

As shown in Table 1, all the cultures completed the fermentation (residual sugar < 2 g/L) except for the pure T. delbrueckii culture which expectedly presented a residual sugar

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concentration above 100 g/L and an ethanol content of 6.2 % vol. All other modalities showed

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an ethanol content around 12 %/vol.

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Fermentation kinetics varied markedly from one culture to another. Indeed, the fermentation involving the inoculation with T. delbrueckii alone presented a low maximum CO2 production rate (Vmax) (Table 1). By contrast, the S. cerevisiae fermentation harbored a high Vmax resulting in the shortest fermentation duration (FD).

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Maximal populations (Xmax) were reached for all modalities during the first 20% of alcoholic fermentation. Xmax of T. delbrueckii and S. cerevisiae were significantly higher when inoculated alone than in the presence of the other species (Table 1). Both species hence had an influence on each other’s development involving intermediate fermentation kinetics for mixed modalities.

In the sequentially inoculated fermentation, the addition of S. cerevisiae 24h after the inoculation with T. delbrueckii allowed the latter to develop from 1 x 107 viable cells/mL to 4.1 x 107 viable cells/mL within 24h and permitted this species to start the fermentation. The inoculation ratio after 24h was then largely in favor of T. delbrueckii (about 20/1) but allowed a sufficient development of S. cerevisiae (from 2 x 106 to 2.4 x 107 viable cells/mL) to achieve fermentation. This modality presented, thus, a Vmax close to that of T. delbrueckii pure

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ACCEPTED MANUSCRIPT culture resulting in the longest fermentation duration of modalities with complete fermentation (Table 1).

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In the simultaneous mixed modality, where both species were added at the same time, the

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initial inoculation ratio (5/1: 1 x 107 viable cells/mL for T. delbrueckii and 2 x 106 viable cells/mL for S. cerevisiae) was less favorable to T. delbrueckii than in the sequential mixed

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modality. The growth of T. delbrueckii was thus limited as showed by its Xmax which was significantly lower in simultaneous fermentation (4.3 x 107 viable cells/mL) than in sequential mixed culture (6.1 x 107 viable cells/mL) (Table 1). On the other hand, S. cerevisiae presented

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that of pure S. cerevisiae culture (Table 1).

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a more important growth in simultaneous culture involving fermentation kinetics closed to

3.1.2 Ester production

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In this study, we chose to classify the different esters in function of their abundance in S.

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cerevisiae pure culture rather than in function of their chemical family (ethyl fatty acid esters, higher alcohol acetates…). Thus, the seven esters present at highest concentrations (ethyl

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butanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, isoamyl acetate, hexyl acetate and phenylethyl acetate) were named “major esters”, the others being considered as “minor esters”.

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3.1.2.1 Final ester composition

When comparing single species inoculations, the sum of all measured esters at the end of fermentation was 5 fold higher with S. cerevisiae (6289 µg/L) than with T. delbrueckii (1181 µg/L) as shown in Table 2. Hence, most esters, and in particular the 7 major esters, were found at a much lower concentration in the T. delbrueckii fermentation (915 µg/L) than compared to the S. cerevisiae (6107 µg/L). However, one interesting thing to consider is that if these esters represent 97% of the total ester concentration in S. cerevisiae, they only participate in 77% to the overall esters in T. delbrueckii, meaning that other esters have a much higher importance in that species. Indeed, « minor » esters can be considered as produced preferentially by T. delbrueckii (266 µg/L) in contrast to S. cerevisiae (182 µg/L). This is particularly true for three of them, ethyl propanoate, ethyl isobutanoate and ethyl

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ACCEPTED MANUSCRIPT dihydrocinnamate which are respectively found at 5, 3 and 67 fold higher concentrations in T. delbrueckii pure fermentation than S. cerevisiae pure fermentation.

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In the sequentially inoculated fermentation, overall ester production (6926 µg/L) was higher

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than in both pure cultures. The differences were significant with T. delbrueckii alone (6 fold higher) and non-significant with S. cerevisiae alone (Table 2). The 7 major esters were found

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at much higher concentrations than in the T. delbrueckii pure culture, and close to that involving S. cerevisiae alone, even if large variations could be seen among the different esters, such as isoamyl acetate which is significantly higher in the sequentially inoculated

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culture than in the S. cerevisiae fermentation. The other esters (except phenylethyl acetate) were at lower concentrations which made up for the difference. The most important feature

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however concerned the minor esters. They indeed showed a total concentration of 454 µg/L, which is much more than what measured in the pure cultures fermentations. This increase was essentially due to isobutyl acetate and to 2 esters previously described as preferentially

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produced by T. delbrueckii: ethyl propanoate and ethyl isobutanoate. Ethyl dihydrocinnamate

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(third ester preferentially produced by T. delbrueckii) concentration was lower than with T.

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delbrueckii alone, but higher (6 fold more) than with S. cerevisiae.

The simultaneous mixed modality allowed the most important total ester concentration (9836 µg/L) to develop, and was significantly higher than that involving pure S. cerevisiae culture (+ 36 %). This difference was mainly due to the increase of total major esters concentration

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and especially isoamyl acetate and to a lesser extent ethyl butanoate, ethyl decanoate and phenylethyl acetate. The concentration of minor esters (340 µg/L) was lower than that involving the sequentially inoculated fermentation (454 µg/L) but significantly higher than that involving S. cerevisiae pure culture. Hence, the ethyl propanoate, ethyl isobutanoate and ethyl dihydrocinnamate concentrations measured in that fermentation were intermediate to that of the sequentially inoculated culture and the pure S. cerevisiae culture.

It's interesting to note that for modalities which enabled a complete alcoholic fermentation (i.e., 3 modalities inoculated with S. cerevisiae), the more T. delbrueckii grew (Table 1) the more ethyl propanoate, ethyl isobutanoate and ethyl dihydrocinnamate concentrations were important (Table 2). In these conditions, the concentrations of ethyl propanoate, ethyl isobutanoate and ethyl dihydrocinnamate were significantly correlated to the maximal T. delbrueckii population (Spearman test, p < 0.05). Although not correlated with the 13

ACCEPTED MANUSCRIPT development of either species, mixed inoculations allowed, in comparison to pure cultures, a systematic increase of isobutyl and isoamyl acetate.

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3.1.2.2 Intermediate ester composition

In addition to ester concentrations at the end of the fermentation, esters were also measured at

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40 % of alcoholic fermentation completion in order to better understand the formation of "key" esters during mixed inoculations.

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As shown in Fig. 1, esters compositions at 40% of alcoholic fermentation were different among all 4 fermentations even though the sequentially inoculated culture was closer to the T.

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delbrueckii pure culture and the simultaneously inoculated culture closer to the S. cerevisiae pure culture. Furthermore, ester composition at 40 % of alcoholic fermentation in mixed modalities presented different profiles to that at the end of the fermentation process, while in

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pure fermentations the profiles were quite similar. The discrimination of modalities carried

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out by principal components analysis based on the 20 ester concentrations represented about

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80% of variance for PC1 and PC2 axes (Fig. 1).

Pure cultures of T. delbrueckii or S. cerevisiae showed at 40 % of alcoholic fermentation similar concentrations to that at the end of alcoholic fermentation (Fig. 2). This is mainly explained by a very slight decrease, during the fermentation, of the concentrations of major

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(Fig. 2) and minor (Fig. 3) esters. The situation was much different for mixed inoculations where contrarily to pure cultures, total esters concentration increased from mid to the end of the fermentation (Fig. 2).

Thus, at 40 % of alcoholic fermentation achievement, the sequentially mixed fermentation showed a total ester concentration 3 fold higher than that of T. delbrueckii and 2 times lower than the fermentation involving S. cerevisiae alone, whereas its final concentration was respectively 6 fold higher and equivalent. For simultaneous mixed modality, the intermediate concentration was similar to that of S. cerevisiae pure culture whereas its final concentration was higher (+36%). This is mainly explained by an increase in mixed modalities of almost all esters and notably isoamyl acetate which doubled its concentration in the second part of the fermentation process (Fig. 2). After 40 % of alcoholic fermentation the minor esters concentrations in mixed cultures were similar to that in T. delbrueckii pure culture, but 14

ACCEPTED MANUSCRIPT already higher than with S. cerevisiae pure culture. The synthesis during the second part of alcoholic fermentation leads to a minor esters concentration higher in mixed cultures than in the pure cultures. This is especially true for ethyl propanoate (Fig. 3) and ethyl isobutanoate

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(data not shown), but not for isobutyl acetate (Fig. 3) which presence is not linked, as already

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mentioned, to T. delbrueckii growth.

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In conclusion, our results showed that wine produced from mixed cultures resulted in an increase of several minor esters (ethyl propanoate, ethyl isobutanoate, ethyl dihydrocinnamate and isobutyl acetate) than compared to wine produced with S .cerevisiae. This experiment was

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performed in both white (Semillon and Sauvignon Blanc) and red wines (Merlot and Cabernet Sauvignon) (data not shown). Furthermore, it was found that in white and red wines the

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concentration of these minor esters were always increased in fermentations performed with mixed cultures (data not shown). Thus, because of laboratory constraints it was decided to

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continue larger scale experimentation.

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3.2 Winery scale: sensory impact of minor esters generated by mixed inoculations

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In order to confirm laboratory scale fermentations, larger experiments with either S. cerevisae alone or a sequentially mixed inoculation of T. delbrueckii / S. cerevisiae were performed in duplicate in 200L tanks. Wines were produced from a red must (Merlot) in a winery from the Bordeaux area. Inoculation levels and procedures were similar to the ones performed

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previously in the laboratory.

Large scale results confirmed the ones obtained with laboratory fermentations. Total esters concentration in sequentially mixed wines was higher than in the S. cerevisiae fermented wine with concentrations respectively of 2075 and 1774 µg/L (Table 3). As in the laboratory scale fermentations, this difference is due in part to major esters (especially isoamyl acetate), but mostly to minor esters and in particular ethyl propanoate, ethyl isobutanoate and to a lesser extend ethyl dihydrocinnamate and isobutyl acetate.

In order to evaluate the aromatic impact involved by the increase of these four minor esters during mixed inoculation, pure chemicals compounds of ethyl propanoate, ethyl isobutanoate ethyl dihydrocinnamate and isobutyl acetate were added to S. cerevisiae pure treatment to reach concentrations found in sequential mixed treatment. This new modality named "S. 15

ACCEPTED MANUSCRIPT cerevisiae + esters" or "S. cerevisiae adjusted wine" presented the same major esters concentration as S. cerevisiae pure culture but higher minor ester concentration (+ 208 µg/L) (Table 3). This resulted in a wine with an overall esters concentration intermediate to that of

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the S. cerevisiae pure fermentation and the sequentially mixed one.

First, sequentially mixed and pure S. cerevisiae wines were compared by triangular sensory

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analysis. They were perceived as different by the sensory panel. Then the S. cerevisiae adjusted wine was introduced and it was perceived as significantly different from the control S. cerevisiae wine but not from the sequentially mixed one. Hence, the addition of the 4 minor

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esters impacted the sensory profile of the wine.

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The panel was then asked to rate the 3 wines according to the following descriptors : aromatic complexity, fruitiness and vegetal notes. The sequentially inoculated wine and the S. cerevisiae adjusted wine were perceived significantly more fruity and complex than the pure

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S. cerevisiae (Fig. 4). Furthermore, no significant differences were found for these characters

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between the sequential modality and the S. cerevisiae adjusted wine. Although the vegetal note was perceived more intensively for the pure S. cerevisiae wine, the difference with the 2

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others modalities were not significant.

These results hence show that the higher levels of ethyl propanoate, ethyl isobutanoate, ethyl dihydrocinnamate and isobutyl acetate found in mixed T. delbrueckii / S. cerevisiae wines

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were responsible for the increase of fruitiness and complexity in comparison to pure S. cerevisiae culture. In other words, these 4 esters can be considered as aromatic markers of T. delbrueckii during mixed inoculations.

16

ACCEPTED MANUSCRIPT 4. Discussion

From this study, it appears that T. delbrueckii produced, in pure fermentation, less overall

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esters than S. cerevisiae and in particular less major esters (ethyl butanoate, ethyl hexanoate,

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ethyl octanoate, ethyl decanoate, isoamyl acetate, hexyl acetate and phenylethyl acetate). However, a few minor esters were produced in more important concentrations by T.

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delbrueckii. These results are in agreement with those from numerous authors (HernandezOrte et al., 2008; Herraiz et al., 1990 ; Moreno et al., 1991 ; Plata et al., 2003; Renault et al., 2009; Sadoudi et al., 2012; Viana et al., 2008). Our study shows that, ethyl propanoate, ethyl

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isobutanoate and ethyl dihydrocinnamate are esters specifically produced by T. delbrueckii. These results clearly point out that the biosynthesis of these esters is different, or at least not

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regulated in the same way in T. delbrueckii as in S. cerevisiae species. According to Sumby et al. (2010), the enzymatic accumulation of esters in wine during S. cerevisiae fermentation is known to be the result of the balance of the enzymatic synthesis

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and hydrolysis reactions involving esterases (Saerens et al., 2006) and synthesis reactions

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involving acetyltransferases (Lilly et al., 2000; Mason and Dufour, 2000; Verstrepen et al., 2003). Different enzyme activities, or even presence/absence of these enzymes among the two

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species could explain the different esters concentrations measured. In this study, principal component analyses of 20 ester concentrations showed that the fermentations in which both species were inoculated present quite different profiles than the pure cultures. Although carried out from an identical yeast couple, the 2 mixed modalities had

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very different esters compositions. Thus, the inoculation protocol is a key factor in the achievement of mixed inoculations because it generates different growth kinetics of the two yeast species modifying final ester compositions. Sequentially inoculated fermentation are therefore largely dominated by T. delbrueckii hence explaining the low fermentation kinetics between. When simultaneous mixed culture was performed, the growth of T. delbrueckii was limited resulting in kinetics close to those of pure S. cerevisiae culture. The limited development of T. delbrueckii in simultaneously inoculated fermentation in comparison to sequentially inoculated culture is similar to results found by Bely et al. (2008) and Ciani et al. (2006).

Our results show that the production of some minor esters in mixed modalities are directly linked to the growth of T. delbrueckii. Thus, for modalities that enabled complete alcoholic fermentation (i.e., 3 modalities inoculated with S. cerevisiae), the more T. delbrueckii grew 17

ACCEPTED MANUSCRIPT the more the ethyl propanoate, ethyl isobutanoate and ethyl dihydrocinnamate concentrations were high. In these conditions, the final concentrations of these 3 esters were notably linked to the maximal T. delbrueckii population. Moreover, the extended viability of T. delbrueckii in

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the sequentially inoculated fermentation compared to those involving T. delbrueckii alone

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(populations at 40% of alcoholic fermentation were significantly different and respectively 5.9 x 107 and 4.3 x 107 viable cells/mL) seemed to be linked to an ester concentration

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enhancement. Hence these esters can be considered as growth and/or activity markers of T. delbrueckii in mixed inoculations.

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Some esters, such as isobutyl acetate and isoamyl acetate, seem to be more linked to yeast interactions than simply to T. delbrueckii activity. They are indeed systematically more

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abundant in mixed inoculated cultures than in pure fermentations although T. delbrueckii is itself a weak producer and S. cerevisiae population was less important than in its pure culture. Furthermore, in pure fermentations the maximal isoamyl acetate concentration is reached at

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40 % of alcoholic fermentation achievement. These results confirm those of Lee et al. (2004)

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and Vianna and Ebeler (2001) with fermentations performed in Chardonnay wines. On the contrary, the isoamyl acetate concentration doubled in the second part of alcoholic

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fermentation in mixed modalities, involving more important final concentrations than in pure modalities. This is probably due to positive interaction between both species. It is important to note that isoamyl acetate concentration and maximal S. cerevisiae population were higher in simultaneously inoculated fermentations than in the sequentially inoculated ones. These

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results suggest that the enhancement of isoamyl acetate production during mixed inoculation is due to S. cerevisiae in response to the presence of T. delbrueckii. The same remarks can be made concerning phenylethyl acetate, ethyl butanoate and ethyl decanoate, which are also produced at highest concentrations in simultaneous mixed modality.

Finally, in the sequential culture, where T. delbrueckii leads the alcoholic fermentation, the overall esters production is close to that of S. cerevisiae, but with a minor/major esters balance far different. This modality involved the highest minor esters concentration thanks to an important production of T. delbrueckii activity markers. When the mixed inoculation was performed simultaneously, T. delbrueckii was less present during the alcoholic fermentation and the production of minor esters was thus less important while that of major esters is enhanced in comparison to the S. cerevisiae pure culture. This modality allowed the production of the most important final esters concentration. 18

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Large scale fermentations on Merlot confirmed the laboratory results and also showed that the sequentially inoculated wine was more fruity and complex than the one fermented with S.

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cerevisiae alone. These results are also in accordance with those from Azzolini et al. (2012).

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In order to precisely study the impact of the activity markers of T. delbrueckii (ethyl propanoate, ethyl isobutanoate and ethyl dihydrocinnamate) as well as isobutyl acetate

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(which systematically increases in mixed inoculations) their concentrations were artificially adjusted in S. cerevisiae fermented wine to the level which they were found in sequentially inoculated wine. These additions restored the more fruity and complex intensities perceived in

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the sequentially inoculated wine. These molecules are hence responsible for the enhanced fruitiness and complexity of mixed T. delbrueckii / S. cerevisiae wine and can be considered

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as aromatic T. delbrueckii markers.

Among these specific esters, only ethyl isobutanoate is present above its perception threshold

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(15 µg/L, Ferreira et al., 2000). There is hence no doubt about its fruity contribution

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(strawberry, kiwi, fruity) to wines fermented partially with T. delbrueckii. However, the three other esters: ethyl propanoate (ripe strawberry ; perception threshold : 2100 µg/L, Pineau,

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2007), ethyl dihydroxycinnamate (fruity, pineapple, almond ; perception threshold : 1,1 µg/L, Ferreira et al., 2000) and isobutyl acetate (solvent, fruity; perception threshold : 1600 µg/L, Aznar et al., 2003) presented concentrations below their perception threshold, questioning the effective contribution of these molecules to the wine aroma. However, it has been recently

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shown that through particular perceptive interactions certain esters can have an impact on wine aroma, even if present far below their perception threshold (Pineau, 2007; Atanasova et al., 2004). Furthermore, according to Pineau et al. (2009), slight single ester variations among complex mixtures can lead to aroma modulation. This is the case for ethyl propanoate, clearly involved in black fruit/jammy notes in red wines in spite of its concentration being below its perception threshold. Furthermore, according to Lytra et al. (2013), isobutyl acetate and ethyl propanoate are particularly implicated in this perceptive interaction phenomena and enhance the intensity of fresh fruit and black fruit notes. As a consequence, their omission among a mix of different esters can significantly impact the aroma perception of the matrix, and their presence (even if below their perception thresholds) in this esters mix, lowers the perception threshold of this aromatic pool, suggesting that isobutyl acetate and ethyl propanoate can be considered as aroma enhancers. In our experiences, the systematic increase of ethyl propanoate and isobutyl acetate during mixed inoculation allowed the enhancement of fruity 19

ACCEPTED MANUSCRIPT notes in comparison to S. cerevisiae pure fermentation. Concerning ethyl dihydrocinnamate, no information on its aroma impact can be found in the literature.

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Besides these esters, others might also be involved in the fruitiness perception of the wines.

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Although they were not statistical differences among the sequentially inoculated fermentations and the S. cerevisiae wine in which esters were adjusted, the complexity and

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fruity note were slightly more important in mixed inoculation. This could be linked to the isoamyl acetate concentration (perception threshold 30 µg/L, banana aroma, Ferreira et al., 2000), which was not artificially adjusted, present in concentrations higher than its

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perceptions threshold for the 2 modalities but in more important concentration for the T. delbrueckii / S. cerevisiae wine. Furthermore, another compound also to be considered, ethyl-

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3-hydroxybutanoate, has been described as an aroma enhancer (Lytra et al. 2013) but not measured in our study. According to Herraiz et al. (1990) and Azzolini et al. (2012), this compound concentration was systematically increased in mixed T. delbrueckii/S. cerevisiae

Acknowledgements

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inoculation in comparison to pure S. cerevisiae culture.

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The authors thank Aquitaine Regional Council for its financial support and Virginie Moine,

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Warren Albertin, Sophie Tempere, Georgia Lytra and Laurent Riquier for their precious help.

20

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ACCEPTED MANUSCRIPT Figure captions

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Fig. 1. Principal Component Analysis (PCA) based on the 20 esters concentrations for T. delbrueckii pure culture (Td), S. cerevisiae pure culture (Sc), sequential mixed culture (Seq) and simultaneous mixed culture (Sim) after 40% of the alcoholic fermentation (- 40% AF) and at the end of fermentation (- end AF). Modalities were carried out in triplicate.

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Fig. 2. Total, main esters and isoamyl acetate concentrations for T. delbrueckii pure culture (T. delbrueckii), S. cerevisiae pure culture (S. cerevisiae), sequential mixed culture (Sequential) and simultaneous mixed culture (Simultaneous) after 40% of the alcoholic fermentation (40% AF) and at the end of fermentation (end AF). Average values of three experiments.

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Fig. 3. Minor esters, propanoate ethyl and isobutyl acetate concentrations for T. delbrueckii pure culture (T. delbrueckii), S. cerevisiae pure culture (S. cerevisiae), sequential mixed culture (Sequential) and simultaneous mixed culture (Simultaneous) after 40% of the alcoholic fermentation (40% AF) and at the end of fermentation (end AF). Average values of three experiments.

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Fig. 4. Descriptive sensorial analysis of red wines after blending of duplicates. Grades ranked from 0 (poorly intense) to 7 (very intense).

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Figure 1

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29

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D

Figure 2

30

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Figure 3

31

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Figure 4

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Sequential mixed culture

Simultaneous mixed culture

S. cerevisiae pure culture

6.2 ± 0.3 a

11.9 ± 0.8 b

12.4 ± 0.4 b

11.8 ± 0.4 b

107 ± 4 b

Sugar (g/L) Vmax (g/L/h)

0.39 ± 0.01

a

0.60 ± 0.10

a

0.56 ± 0.03

b

473 ± 3 d

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350 ± 7 b

FD (h)

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T. delbrueckii pure culture

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Ethanol (% vol.)

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Table 1: Ethanol and residual sugar concentrations, kinetic parameters and maximal cell populations in pure and mixed T. delbrueckii and S. cerevisiae cultures (laboratory scale conditions)

0.60 ± 0.10

a

0.90 ± 0.20

a

0.84 ± 0.01

c

1.00 ± 0.01

d

390 ± 2 c

334 ± 3 a

Maximal population (viable cells/mL) 8.1 x 107 ± 2.8 x 106 /

6.1 x 107 ± 7.1 x 106

b

4.3 x 107 ± 3.5 x 106

a

2.4 x 107 ± 7.1 x 105

a

4.4 x 107 ± 2.3 x 106

b

/ 7.6 x 107 ± 1.8 x 106

D

S. cerevisiae

c

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T. delbrueckii

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Average values of three experiments ± standard deviation a, b, c, d represents significantly different statistical groups (p < 0.05) Vmax: maximum CO2 production rate; FD: fermentation duration

33

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ACCEPTED MANUSCRIPT Table 2: Final esters concentrations in pure and mixed T. delbrueckii and S. cerevisiae cultures (laboratory scale conditions)

Simultaneous mixed culture

29 ± 4

a

220 ± 14

b

Ethyl hexanoate

120 ± 10

a

671 ± 27

Ethyl octanoate

187 ± 13

a

Ethyl decanoate

458 ± 69 64 ± 1

Sum

c

1301 ± 96

c

580 ± 41

b

1364 ± 91

c

1407 ± 130

c

a

533 ± 17

a

878 ± 38

c

614 ± 41

b

a

3834 ± 101

c

4957 ± 84

d

1828 ± 63

b

179 ± 17

b

219 ± 16

c

236 ± 16

c

455 ± 69

b

540 ± 31

c

435 ± 3

b

6472 ± 287

b

9496 ± 336

c

53 ± 10

a

a

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Ethyl isobutanoate *

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Ethyl dihydrocinnamate

b

c

233 ± 8

d

110 ± 14

b

37 ± 10

a

41 ± 3

c

24 ± 2

b

8,8 ± 1,1

a

b

0.49 ± 0,08

a

1,4 ± 0,2

c

0.87 ± 0,04

b

0.87 ± 0,08

b

0.34 ± 0,05

a

1,3 ± 0,1

b

1,1 ± 0,1

b

2,0 ± 0,1

c

0.25 ± 0,04

a

1,7 ± 0,1

d

1,2 ± 0,1

c

0.71 ± 0,02

b

9,3 ± 0,9

a

0.10 ± 0,00

a

0.27 ± 0,05

b

0.20 ± 0,00

b

0.43 ± 0,06

c

c

1,3 ± 0,2

b

0,34 ± 0,04

a

0.20 ± 0,01

a

a

1,1 ± 0,0

a

1,0 ± 0,0

a

1,1 ± 0,0

a

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Ethyl trans- 2-hexenoate

6107 ± 369

195 ± 12 24 ± 2

Ethyl 2-methylbutanoate

Ethyl cinnamate

1195 ± 60

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Ethyl propanoate

Ethyl phenylacetate

b

a

Minor esters (µg/L)

Ethyl dodecanoate

c

3,9 ± 1,0

915 ± 104

Ethyl isovalerate

286 ± 20

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Phenylethyl acetate

d

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Ethyl butanoate

Hexyl acetate

343 ± 16

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Major esters (µg/L)

Isoamyl acetate *

S. cerevisiae pure culture

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Sequential mixed culture

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T. delbrueckii pure culture

Compounds

14 ± 1

1,1 ± 0,0

15 ± 3

b

41 ± 3

c

c

43 ± 3

Propyl acetate

11 ± 2

a

56 ± 11

c

53 ± 3

c

36 ± 4

b

Isobutyl acetate *

12 ± 1

a

99 ± 9

c

103 ± 5

c

51 ± 3

b

Butyl acetate

0.12 ± 0,01

a

2,7 ± 0,2

b

5,1 ± 0,6

c

2,8 ± 0,1

b

Octyl acetate

0.05 ± 0,04

a

0.21 ± 0,03

a

0.71 ± 0,06

b

0.81 ± 0,16

b

Sum

266 ± 3

Total sum (µg/L)

1181 ± 107

b

454 ± 35

d

340 ± 30

c

182 ± 22

a

a

6926 ± 322

b

9836 ± 366

c

6289 ± 392

b

Average values of three experiments ± standard deviation a, b, c, d represents significantly different statistical groups (p < 0.05) * Isoamyl acetate, ethyl isobutanoate and isobutyl acetate are respectively also called 3-methylbutyl acetate, ethyl 2-methylpropanoate and 2-methylpropyl acetate

34

ACCEPTED MANUSCRIPT Table 3: Final esters concentrations in pure S. cerevisiae (adjusted or not with esters) and sequential T. delbrueckii and S. cerevisiae cultures (winery scale conditions)

S. cerevisiae + esters

T

Sequential mixed culture

IP

S. cerevisiae pure culture

Compounds

146 ± 6

169 ± 1

146 ± 6

Ethyl hexanoate

338 ± 13

355 ± 4

338 ± 13

Ethyl octanoate

409 ± 25

377 ± 7

409 ± 25

Ethyl decanoate *

273 ± 14

126 ± 6

273 ± 14

Isoamyl acetate *

318 ± 13

536 ± 10

318 ± 13

Hexyl acetate *

1,0 ± 0,1

1,6 ± 0,1

0,98 ± 0,10

29 ± 2

36 ± 1

29 ± 2

1514 ± 112

1601 ± 88

1514 ± 112

143 ± 11

267 ± 8

267 ± nd

49 ± 5

110 ± 6

110 ± nd

NU

Ethyl butanoate

MA

SC R

Major esters (µg/L)

Phenylethyl acetate *

TE

D

Sum Minor esters (µg/L)

CE P

Ethyl propanoate *

Ethyl isobutanoate *

Ethyl dihydrocinnamate

AC

Isobutyl acetate *

9 other minor esters Sum *

Total sum (µg/L)

0,39 ± 0,10

0,53 ± 0,10

0,53 ± nd

16 ± 1

38 ± 2

38 ± nd

52 ± 20

58 ± 14

52 ± 20

260 ± 24

474 ± 19

468 ± nd

1774 ± 142

2075 ± 115

1981 ± nd

Average values of three experiments ± standard deviation * indicates significant differences (p < 0.05) between S. cerevisiae pure culture and sequential mixed culture nd means no determined

35

ACCEPTED MANUSCRIPT Highlights T. delbrueckii in association with S. cerevisiae impacts the aromatic profile of wines

•

The inoculation procedure has a major impact when performing mixed cultures

•

Increased concentrations of isobutyl and isoamyl acetate in mixed cultures

•

Several minor esters are markers of T. delbrueckii metabolism

AC

CE P

TE

D

MA

NU

SC R

IP

T

•

36