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. 2018 Jun 12:9:1276.
doi: 10.3389/fmicb.2018.01276. eCollection 2018.

Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance

Maria Dimopoulou  1 , Jerôme Raffenne  1 , Olivier Claisse  1 , Cécile Miot-Sertier  1 , Nerea Iturmendi  2 , Virginie Moine  2 , Joana Coulon  2 , Marguerite Dols-Lafargue  1
Affiliations

Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance

Maria Dimopoulou et al. Front Microbiol. .

Abstract

Oenococcus oeni is the lactic acid bacterium that most commonly drives malolactic fermentation (MLF) in wine. Though the importance of MLF in terms of wine microbial stability and sensory improvement is well established, it remains a winemaking step not so easy to control. O. oeni displays many adaptation tools to resist the harsh wine conditions which explain its natural dominance at this stage of winemaking. Previous findings showed that capsular polysaccharides and endogenous produced dextran increased the survival rate and the conservation time of malolactic starters. In this paper, we showed that exopolysaccharides specific production rates were increased in the presence of single stressors relevant to wine (pH, ethanol). The transcription of the associated genes was investigated in distinct O. oeni strains. The conditions in which eps genes and EPS synthesis were most stimulated were then evaluated for the production of freeze dried malolactic starters, for acclimation procedures and for MLF efficiency. Sensory analysis tests on the resulting wines were finally performed.

Keywords: Oenococcus oeni; exopolysaccharides; malolactic fermentation; stress; wine.

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Figures

Figure 1
Figure 1
Organization of the eps2 gene cluster in O. oeni IOEB_S277 and AWRI_B429. Putative rho-independent terminators (hairpin structures with polyT) are indicated. A promoter (−10 and −35 consensus sequences) was found upstream wzd in both strains. The stars indicate the genes whose expression level was studied by RT-qPCR.
Figure 2
Figure 2
Bacterial growth, EPS synthesis and relative gene expression in SMD medium supplemented or not with sucrose for strains O. oeni AWRI_B429 and IOEB_S277. (A) Bacterial growth on glucose medium (formula image) and on glucose+sucrose medium (formula image). (B) Polysaccharide concentration (n = 3) on glucose medium (formula image) and on glucose+sucrose medium (formula image). (C,D), Expression level relative to gyrA (2ΔCT) at the indicated time: the genes are described in the following order: pgm, ldh, wobA, wzx and dsrO. The blue point represents the expression ratio between wzx and wobA: relative gene expression on glucose medium (C) and on glucose+sucrose medium (D).
Figure 3
Figure 3
Expression of eps genes in response to medium change in O. oeni AWRI_B429.(A) Method: the bacterial cells were initially grown in Grape Juice medium until absorbance reached 0.6. After centrifugation, they were re-suspended in 8 different fresh growth media. After 3 and 24 h of incubation in the new medium, the cell cultivability (CFU counts) and the cell viability (fluorescent labeling and microscopy) were measured, and RNA extraction and RT-qPCR were realized. (B) Relative gene expression level of target genes after 24 h of incubation in different culture media. The reference gene was gyrA. The target gene expression level relative to gyr A (2ΔCT) was then compared to the relative expression in the control condition (2ΔΔCT). The relative expression in control condition was set to 1 for each gene. The results after 3 h of incubation are shown in Supplemental figure 2. (C) Expression level ratio of wzx/wobA after 24 h (see Supplemental figure 2 for expression level after 3 h).
Figure 4
Figure 4
Influence of the acclimation medium (standard or supplemented with sucrose) on survival (A) and malolactic fermentation kinetics (B). Seven O. oeni strains, belonging to 3 distinct families regarding their eps genes, were produced at laboratory scale in a freeze dried form, then acclimated and inoculated in wine (Pessac Leognan, 2012). The survival rate was calculated by comparing the initial cell cultivability (inoculum) with the cultivability after 48 h in wine; the Vmax of the malolactic fermentation was the ratio of the initial concentration of malic acid to the days needed to complete its consumption. Error bars represent the standard deviation of two biological replicates. formula image acclimation to wine performed in standard conditions. formula image acclimation to wine performed in the presence of 5 g/l sucrose.
Figure 5
Figure 5
Mean-centered intensities of sensory characteristics descriptors of wines inoculated with two different O. oeni strains (IOEB_S450 and S11) after acclimation without or with sucrose. The control is the wine that didn't realize the malolactic fermentation in which sucrose was added (5 mg/l) or not to mimic sucrose addition via the acclimated inoculum. The wine used for the experiment was a Merlot from 2012 (pH 3.5, ethanol 12% v/v and malic acid 3.5 g/l, no added sulfites). Error bars represent the standard deviation of two biological replicates.
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