Introduction

Alcoholic fermentation (AF) is the main microbiological process in winemaking, as it converts grape must into wine. This fermentation is carried out by the yeast Saccharomyces cerevisiae, although during the early stages, non-Saccharomyces yeasts are present in the fermenting must (Fleet et al., 1984). There is an increasing interest in these other yeasts (Padilla et al., 2016) due to the production of new aromas (Belda et al., 2017).

The yeast metabolism and the winemaking conditions greatly influence the final composition of the wine. Some compounds produced by yeasts—including non-Saccharomyces—during AF have a large impact on the subsequent malolactic fermentation (MLF), carried out mainly by the lactic acid bacterium Oenococcus oeni (Lonvaud‐Funel, 1999; Bartowsky, 2005; Balmaseda et al., 2018; Ferrando et al., 2020; Balmaseda et al., 2021). Some of these compounds are produced by the primary metabolism of yeast, such as ethanol and SO₂ (Arnink and Henick-Kling, 2005), certain organic acids (Lonvaud-Funel and Strasser de Saad, 1982), and medium-chain fatty acids (Guilloux-Benatier et al., 1998; Lonvaud‐Funel et al., 1988). Other compounds are delivered by the secondary metabolism as antimicrobial peptides (Branco et al., 2014) and proteinaceous compounds (Osborne and Edwards, 2007). Among the organic acids derived from the yeast metabolism, succinic acid seems to be the most related to MLF inhibition (Caridi and Corte, 1997; Son et al., 2009).

Succinic acid is one of the relevant organic acids in wine, whose main role is to confer microbial stability to wines in relation to acidity and pH. They also preserve the colour and sensory properties of wines. Tartaric and malic acids are generally the most prominent acids in wines, while others such as acetic, succinic, citric, lactic, and pyruvic can exist in minor concentrations (Mendes-Ferreira and Mendes-Faia, 2020). Tartaric and malic acids are already present in grape must, but no succinic or lactic acids are found in grapes. Instead, succinic acid is usually the predominant non-volatile organic acid formed by yeasts during AF (Thoukis et al., 1965).

Succinic acid is the 1,4-butanedioic acid (HOOC-CH₂-CH₂-COOH), with an m.w. of 118 g/mol and pK_a1 of 4.2 and pK_a2 of 5.6 (ChemIDplus, 2021). Its structure is like that of malic acid (HOOC-CHOH- CH₂-COOH), which has an m.w. of 134 g/mol, and which can also be called hydroxysuccinic acid. The organoleptic threshold of succinic acid in wine is 35 mg/L. It has an unusual bitter-salty taste and excess levels can have a negative impact on the mouthfeel of the wines (Coulter et al., 2004); thus, it may be beneficial to reduce it.

Succinic acid is an intermediate of the tricarboxylic acid cycle (TCA), and it, therefore, is produced by yeasts during the AF in the early fermentation stages (Conway and Brady, 1950), but also during the stationary phase (Lamikanra, 1997; Arikawa et al., 1999). However, it acts as an intermediary in other metabolic pathways such as γ-amino butyric acid (GABA) bypass, glyoxylic acid bypass and the methylcitric acid cycle. Therefore, due to mechanical stimuli and maceration of red grapes, the GABA concentration in must can increase and could encourage yeast to produce succinic acid (De Klerk, 2010).

S. cerevisiae strains are known to produce succinic acid, with values from 200 mg/L to more than 1 g/L (Heerde and Radler, 1978; Coulter et al., 2004; De Klerk, 2010; Zhu et al., 2020), thus explaining the increases of this acid in winemaking. This production of succinic acid can influence MLF, which has been shown with a cryotolerant S. cerevisiae strain that inhibits MLF (Caridi and Corte, 1997; Son et al., 2009). This organic acid has been described as a possible competitive inhibitor of MLF due to its similarity with L-malic acid (Lonvaud‐Funel et al., 1988; Caridi and Corte, 1997).

In addition, non-Saccharomyces yeasts can also significantly produce succinic acid. For example, Ciani and Maccarelli (1998) found that strains of Torulaspora delbrueckii produced it within the range of 0.3 to 0.8 g/L. Moreover, in sequential fermentation of S. cerevisiae – T. delbrueckii, succinic acid was produced up to 0.95 g/L (Zhu et al., 2020). Moreover, Contreras et al. (2014) found productions of 1 to 2 g/L of this acid by strains of Metschnikowia pulcherrima, Schizosaccharomyces malidevorans and Candida stellata.

Besides the above-mentioned studies of MLF inhibition by succinic-producing yeasts, the influence of succinic acid per se upon MLF and on O. oeni has been studied very little (Lonvaud-Funel and Strasser de Saad, 1982). Interestingly, it seems that succinic acid concentration can decrease during MLF (Yilmaz and Gökmen, 2021) and especially in induced simultaneous MLF with AF (Taniasuri et al., 2016). Consequently, its real influence on MLF remains unclear. Due to the lack of information and to clarify the impact of this compound in winemaking conditions, the main aim of this work was to evaluate for the first time the effect of different succinic acid concentrations on O. oeni strains in MLF. Keeping in mind the different dissociated or undissociated forms of succinic acid in the function of pH, assays at different pH—3.5 and 4.0—were included in the evaluation of the impact of succinic acid. To better understand the physiological response to succinic acid, the gene expressions related to stress and malolactic activity were also analysed.

Materials and methods

Results and discussion

2. Effect of succinic acid on malolactic fermentation

After the first experiments for comparing four different O. oeni strains, PSU-1 and CH11 were selected and used exclusively throughout the entire study since they were representative of the two behaviours observed in response to adding succinic acid. O. oeni CH11 showed a stronger inhibition of MLF in the presence of 2 g/L of succinic acid than strain PSU-1. This agrees with the above commented stronger growth inhibition of CH11 at 2 g/L of succinic acid added to the MRS medium. The other O. oeni strains VP41 and 1Pw13 showed similar behaviour to CH11 but with longer and slower MLF in all assays (Supplementary Figure 2). Control fermentations in WLM with strain PSU-1, at both pH 3.5 and 4.0, were completed in 120 h, and those with strain CH11 finished in 144 h (Figure 1). The bacterial population in the samples of these WLM fermentations was followed, but no increase was detected in any of them nor in controls (data not shown). It must be taken into account that growth is usually very difficult in the harsh conditions of WLM, similar to wine, with ethanol and low pH, as observed previously (Bordas et al., 2015; Jiang et al., 2018). Nonetheless, the inoculated population can survive and finish the MLF.

The results obtained in the presence of succinic acid varied depending on the pH and strains. Succinic acid inhibited the MLF of the two O. oeni strains at concentrations of 1 and 2 g/L at pH 3.5, whereas at pH 4 only the MLF with 2 g/L of succinic acid with O. oeni CH11 was inhibited in comparison to the control assay (0 g/L succinic acid). At pH 3.5 with 2 g/L of succinic acid, 43 % and 34 % of the L-malic acid was not consumed by CH11 and PSU-1, respectively, at the time that the control had exhausted all the L-malic acid. This shows that O. oeni CH11 was more sensitive to this concentration of succinic acid than PSU-1. The results with 1 and 2 g/L of succinic acid showed that the pH has a relevant influence on the MLF and the inhibitory effect of this organic acid. Meanwhile, at pH 3.5, succinic acid reduced the L-malic acid consumption rate in CH11 and PSU-1; however, at pH 4.0, both strains were able not only to carry out MLF but also to do so in less time than the control assay. Therefore, pH is an important factor because this physicochemical condition affects the dissociated and undissociated forms of succinic and malic acids, as observed by Augagneur et al. (2007).

Nevertheless, both strains CH11 and PSU-1 showed similar results in the 0.5 g/L succinic acid and in all cases, MLF finished about 24 h earlier than in the control assay. This best MLF performance in the presence of 0.5 g/L than without succinic acid was also found for the other two strains, 1Pw13 and VP41 (Supplementary Figure 1). Therefore, we have found that this low concentration of succinic acid could be slightly beneficial for O. oeni and the MLF, while levels of 1 g/L succinic acid or higher are clearly inhibitory. This probable benefit could be related to the above-commented decrease in succinic acid in some MLFs (Taniasuri et al., 2016; Yilmaz and Gökmen, 2021); however, we did not find significant variation in the succinic acid concentration in any case (data not shown).

Figure 1. L-malic consumption in WLM with 12 % ethanol (v/v) at pH 3.5 and 4.0 by O. oeni strains CH11 and PSU-1, in the presence of different concentrations of succinic acid: 0 g/L (control, blue), 0.5 g/L (red), 1 g/L (yellow), and 2 g/L (green). The values are the means of triplicates and the error bars represent the SD values.

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Anyway, it is clear that MLF is inhibited by a concentration of 1 g/L or higher of succinic acid for both strains and pHs. This inhibition is probably related to intracellular acidification by this acid, which would lead to a decrease in cell viability, affecting, in consequence, the MLF. Although, as commented above, we had not detected variation—neither decrease nor decrease—in biomass in these assays with WLM, the inhibition effect on cell growth was clear in the previous assays with MRS medium.

Regarding the important role that pH played in the performance of the MLF with the two strains, the results obtained with 1 and 2 g/L of succinic acid could be related to the dissociated and undissociated forms of succinic and L-malic acids. At pH 3.5, L-malic acid would be present at almost 50 % in dissociated monoanionic form (L-malic acid pK_a1 = 3.4) (Tourdot-Maréchal et al., 1993), whereas a large fraction of succinic acid (more than 70 %) is in its undissociated form (succinic acid pK_a1 = 4.2) (Jansen and van Gulik, 2014). However, at pH 4.0, only around 50 % of succinic acid would be undissociated (Jansen and van Gulik, 2014). At pH values above 4.5, the passive diffusion of L-malic acid into O. oeni cells would be negligible (Tourdot-Maréchal et al. (1993). However, at pH 3.2, the permeability of the cells to the undissociated acid by simple diffusion could represent more than 50 % of the total L-malic acid uptake. Considering that succinic acid and L-malic acid have a very similar chemical structure, it would be feasible that undissociated succinic acid could enter the O. oeni cell at pH 3.5, producing intracellular acidification due to the complete deprotonation of succinic acid at the cytosolic pH (O. oeni intracellular pH = 5.8–6.1; succinic acid pk_a1 = 4.2 pk_a2 = 5.6). Internal acidification has been associated with the inhibition of MLF by other acidic compounds at pH values close to 3, such as octanoic acid and some phenolic acids (Capucho and San Romao, 1994; Campos et al., 2003). The cytosolic acidification is known to inhibit the enzymatic activities of O. oeni in general and it affects the viability of the cells, as commented. However, moreover, the malolactic activity could be specifically affected due to the changes in the L-malate anionic state inside the cell. According to Acevedo et al. (2020), the optimal substrate for the malolactic enzyme in O. oeni is dianionic L-malate, which is the form present at the standard intracellular O. oeni pH (5.8–6.1). The internal acidification produced by succinic acid could convert dianionic L-malate into the monoanionic form, negatively affecting the malolactic enzyme efficiency. Altogether, the internal acidification of O. oeni, favoured at a low pH, could explain the stronger inhibition of succinic acid at pH 3.5 than at pH 4 observed in this work.

In regard to the composition of organic acids after MLF, there were no differences in L-lactic acid production (data not shown) because it was in accordance with the L-malic acid consumption. In addition, the amounts of acetic acid in each treatment were in accordance with the citric acid consumption and no statistical differences were found (data not shown). Neither one was above the acetic acid threshold (0.7 g/L) (Drysdale and Fleet, 1989).

3. Influence of succinic acid on MLF at different ratios of L-malic and succinic acid

As seen above, the increasing amounts of succinic acid exerted an inhibition effect on the L-malic acid consumption. To understand this effect in more depth, we carried out assays in the same WLM with different ratios of L-malic and succinic acids. In addition, we aimed to determine whether there was an inhibition threshold of succinic acid depending on the initial L-malic acid concentration. At the same time, these assays simulated the real situation of the diversity of initial L-malic acid levels (from 0.5 to 3 g/L in these assays) in different climatic zones of winemaking.

To determine this influence, L-malic acid consumption rates were calculated for the different assays, shown in Supplementary Table 1. As seen, the main significant inhibition of succinic acid was when L-malic acid was 0.5 or 1 g/L. To visualise these results better, they are summarised in Figure 2.

There is a significantly bigger inhibition of the consumption rate for most conditions when 2 g/L of succinic acid is present, in agreement with the inhibition of MLF kinetics we found and discussed above. In terms of the pH, a slightly greater L-malic acid consumption can be observed at pH 4.0 than at 3.5, as expected since the higher pH is less stressful for O. oeni. The consumption tendencies are similar for both the strains and the succinic concentrations for the two pHs (Figure 2).

Figure 2. L-malic acid consumption rate (mg/L·h) by O. oeni strains CH11 and PSU-1 in WLM with 12 % ethanol (v/v) at pH 3.5 and 4.0 with L-malic acid (0.5 g/L [black], and 1 g/L [grey]), at different concentrations of succinic acid. a–c Values of columns for each strain and pH are significantly different at p ≤ 0.05 according to the Tukey test.

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The most relevant result here is that there is generally more inhibition of the consumption rate at 0.5 g/L of L-malic acid than at 1 g/L, which is clearer for strain CH11 (Figure 2). It seems that succinic acid has an inhibiting effect on the consumption rate of L-malic acid when this is in a lower ratio with respect to succinic acid. That is, succinic acid has a clear inhibition effect on MLF when the concentration of this acid is higher than that of L-malic acid. In addition to the inhibition of MLF due to the already mentioned intracellular acidification caused by succinic acid, there could be competition between the succinic and L-malic acids for the malolactic enzyme due to their similar chemical structures, as suggested by Lonvaud-Funel and Strasser de Saad (1982).

It is particularly important to take this effect into account nowadays, when it is usual to have a low L-malic acid content in wine, mainly due to climate change that leads to less acidic grapes (Mira de Orduña, 2010).

5. Relative expression of genes related to stress and malolactic activity

To evaluate the possible inhibition of succinic acid in relation to the transcription of relevant genes associated with MLF development, we measured the relative expression of the following genes: i) mleA, encoding the malolactic enzyme, and mleP, encoding the L-malic acid transporter; ii) atpB, encoding the β-subunit of the F₁F₀ ATPase, associated with MLF activity and acid stress response (Cox and Henick-Kling, 1989; Fortier et al., 2003) and iii) hsp18, encoding for one of the most relevant stress proteins of O. oeni with a chaperone function, this gene is known to be activated in response to multiple stresses and has been proposed to be a stress adaptation indicator (Coucheney et al., 2005; Beltramo et al., 2006). The analysis was carried out with samples from the assay performed in WLM with the most inhibitory concentration of succinic acid (2 g/L). All samples were taken when the L-malic acid concentration was approximately 1 g/L in each case, at mid-MLF. Samples from cultures without added succinic acid were also analysed as control conditions.

In O. oeni CH11, the strain that showed the highest sensitivity to succinic acid, the gene hsp18 was clearly up-regulated at pH 3.5 and 4.0 (Figure 3). In contrast, the PSU-1 strain did not show overexpression of the hsp18 gene in any of the assayed conditions. These data suggest that the CH11 strain adopted this strategy to outcome the succinic acid inhibitory effect. However, O. oeni PSU-1, the strain least affected by succinic acid, might respond to stress differently, as seen in previous studies (Olguín et al. 2010).

No changes in expression were observed for atpB in any of the conditions studied. The transient activation of this gene has been described in response to acid shock (Fortier et al., 2003). In this study, there was no difference in the transcriptional levels of atpB at mid-MLF with or without succinic acid.

Surprisingly, O. oeni PSU-1 showed a 3.5 and 2.5-fold up-regulation of the genes mleP and mleA at pH 4 (Figure 3). This could be related to the faster MLF observed under these conditions (2 g/L succinic acid) with respect to the control condition at the same pH. However, the reason for the enhanced MLF due to the presence of succinic acid remains unclear and needs further research.

The main conclusion of the transcriptional study is that succinic acid does not inhibit the transcription of the mleA and mleP genes in any of the conditions. Therefore, the inhibition of MLF caused by this compound might be due to the intracellular acidification and possible substrate competition with L-malic acid at the enzymatic level, as previously discussed.

Figure 3. Relative expression (RE) of four genes related to stress (hsp18 and atpB) and malolactic activity (mleP and mleA) of strains CH11 and PSU-1 of O. oeni at middle MLF (approx. 1 g/L L-malic acid) in the presence of 2 g/L of succinic acid. The calibrator condition (RE = 1) was the absence of succinic acid. The data shown are mean values between both RE obtained with gene controls gyrA and gyrB with error bars representing SD values (n = 3).

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Conclusion

In this work, the effect of succinic acid on O. oeni and MLF has been evaluated for the first time. It shows that succinic acid can inhibit MLF at concentrations higher than 1 g/L, but it could be beneficial at a lower concentration, near 0.5 g/L. The inhibition is clear when L-malic acid is at a lower ratio with respect to succinic acid. As succinic acid is produced by yeasts, including non-Saccharomyces species, this variable effect is an example of an interaction between yeasts and LAB that can be negative or positive depending on the succinic concentration but also on the strains and other winemaking conditions. Therefore, one of the most important factors is the pH because this physicochemical condition influences the dissociated and undissociated forms of both acids. Consequently, the strain-dependent effect can lead to inhibition or promotion of MLF due to the cell response and adaptation. Further research is necessary to understand the molecular mechanisms of the influence of succinic acid on O. oeni and possibly the competence of succinic acid against L-malic acid at the enzymatic and transport levels.

Acknowledgements

This work was supported by grants AGL2015-70378-R and PGC2018-101852-B-I00, funded by Spanish MCIN/AEI/10.13039/501100011033 and, when appropriate, by ERDF "A way of making Europe", by the European Union or by the European Union Next Generation EU/PRTR. RT-G is grateful for the predoctoral fellowship from Fundación Carolina, which includes a partial stipend from the Mexican Government and University Rovira i Virgili.

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