Abbreviations

CFU colony forming unit; LAB lactic acid bacteria; MLF malolactic fermentation; SO₂ sulfur dioxide; SD standard deviation.

Introduction

Malolactic fermentation (MLF) is an important winemaking process performed by certain wine lactic acid bacteria (LAB), principally Oenococcus oeni. The onset of MLF, characterised by the conversion of L-malic acid to L-lactic acid and CO₂, commences after the malolactic bacteria population has grown to approximately 10⁶ cells/mL (Wibowo et al., 1985). Consequently, the success or failure of MLF is highly dependent on wine parameters that affect the growth and survival of malolactic bacteria, particularly wine pH, the concentrations of alcohol and sulfur dioxide, and temperature. In general, wine conditions conducive to MLF include < 13 % v/v alcohol, > pH 3.4, < 8 mg/L free SO₂, < 30 mg/L total SO₂, 18–22 °C, while conditions outside of this range can become more challenging for MLF (Krieger-Weber, 2017). Moreover, for any given wine, the ability of wine LAB to grow and conduct MLF is highly dependent on the species and strain of LAB involved and their respective tolerances to the latter parameters.

Another major factor affecting the growth and MLF capability of wine LAB is the potential for interaction with fermentation yeast, which can encompass a diverse range of Saccharomyces cerevisiae and also non-Saccharomyces strains. Such interactions are complex. MLF outcomes can be affected by metabolite production or physical cellular interactions (Bartle et al., 2019). Inhibitory yeast metabolites include the formation of SO₂ (Henick-Kling and Park, 1994; Larsen et al., 2003; Wells and Osborne, 2011), medium-chain fatty acids (Guilloux-Benatier et al., 1998), inhibitory peptides (Nehme et al., 2010) and succinic acid at concentrations greater than 1 g/L (Torres-Guardado et al., 2022). Further, in a study of the interactions between Leuconostoc oenos (O. oeni) PSU-1 and S. cerevisiae 522 during co-culture, King and Beelman (1986) reported that the greatest inhibition of early bacterial growth was caused not by yeast metabolites but by yeast growth itself.

Of the potential inhibitory yeast metabolites, the formation of yeast-derived SO₂ during fermentation is well recognised (Rauhut, 1993) and can create a significant obstacle for MLF. Although most S. cerevisiae strains produce low concentrations (10–30 mg/L) of SO₂, some can produce over 100 mg/L (Eschenbruch, 1974; Rauhut, 1993; Richter et al., 2013). Various compositional and vinification factors can also influence SO₂ production by yeast (Andorrà et al., 2018; Rauhut, 1993).

Whether intrinsically derived during fermentation or added during the winemaking process, SO₂ enters a pH-dependent equilibrium. In an aqueous solution at pH 3.5, only a minor proportion (2 %) of free SO₂ exists in the molecular, and primarily antimicrobial, form (Ribéreau-Gayon et al., 2006). At lower pH, such as the pH of white wine, this proportion increases. For example, at pH 3.2, the proportion of molecular SO₂ in an aqueous solution increases to 3.91 % (Ribéreau-Gayon et al., 2006).

The molecular SO₂ concentrations reported as inhibitory towards O. oeni and other wine LAB are varied. Fia et al. (2018) reported that molecular SO₂ concentrations between 0.2–0.6 mg/L impeded growth and MLF activity of an O. oeni strain in white wine, and Ribéreau-Gayon et al. (2006) indicated 0.15–0.4 mg/L molecular SO₂ had varying, wine-dependent, inhibitory effects on LAB survival. Carr et al. (1976), however, suggest that much lower molecular SO₂ concentrations (0.032 mg/L) completely suppressed the growth of heterofermentative cocci (isolated from cider) in a nutrient medium, whereas Lactobacillus plantarum (currently Lactiplantibacillus plantarum) could tolerate up to approximately 1.5 mg/L molecular SO₂.

Bisulfite can also readily form adducts with compounds in wine, particularly carbonyls such as acetaldehyde, pyruvic acid, and 2-ketoglutaric acid (respective K_d at pH 3.3: 1.5 × 10^-6, 5.55 × 10^-4 and 1.4 × 10^-4) (Burroughs and Sparks, 1973). Such bound forms of SO₂ may also exhibit inhibitory activity towards wine LAB, with inhibition in wine typically occurring at > 50 mg/L bound SO₂ (Fugelsang, 1997; Wibowo et al., 1985). However, this concentration also appears subject to variability; as little as 5 mg/L acetaldehyde- and pyruvic acid-bound SO₂ was inhibitory to the growth of wine LAB in a semi-defined culture medium (Wells and Osborne, 2012), and 10 mg/L acetaldehyde-bisulfite restricted the growth and MLF activity of some bacterial strains in red wine (Lafon-Lafourcade, 1975). Several authors (Larsen et al., 2003; Osborne and Edwards, 2006; Wells and Osborne, 2011) have demonstrated that wines fermented by yeast strains producing higher total SO₂ concentrations were generally inhibitory to O. oeni. However, Larsen et al. (2003) and Osborne and Edwards (2006) could not demonstrate a clear relationship between yeast-derived SO₂ production and bacterial inhibition and proposed other inhibitory mechanisms.

White and sparkling-based wines present unique and difficult challenges for MLF. Compared to red wines, white wines destined for MLF generally have higher SO₂ concentration (e.g., up to approximately 50 mg/L total SO₂) and lower pH (e.g., pH 3.1–3.3), which approaches the limits for O. oeni survival. In such cases, increases in SO₂ from the fermentation yeast strain can render MLF extremely challenging. Consequently, the tolerance and adaptive capability of the malolactic bacterial strain to these conditions becomes critical to the success or failure of MLF.

Another important factor that can affect MLF outcome is the timing of bacterial inoculation. Of the different inoculation timings, sequential and co-inoculation are considered two major options (Bartowsky et al., 2015; Sumby et al., 2019). Sequential inoculation, whereby the starter culture is inoculated after alcoholic fermentation (AF), reduces the risk of acetic acid accumulation from carbohydrate metabolism by heterofermentative LAB. However, exposure of the starter culture to a high alcohol concentration can be detrimental to bacterial survival and MLF capability. During co-inoculation, the malolactic starter culture is typically inoculated 24 hours or more after yeast, thus enabling the bacteria to acclimatise to increasing alcohol concentration and, further, allowing greater availability of grape-derived nutrients in the early stages of AF. Importantly, this strategy has been suggested to improve overall MLF efficiency and assist in overcoming difficult wine conditions (Bartowsky et al., 2015).

Some decades ago, the benefits of early inoculation of yeast and malolactic bacteria for rapid MLF induction were recognised (Beelman and Kunkee, 1985). A growing interest in co-inoculation (Bartowsky et al., 2015) has led to its successful application for MLF induction in white wine (Jussier et al., 2006; Sereni et al., 2020; Zapparoli et al., 2006), including white varieties with low pH (Guzzon et al., 2016; Knoll et al., 2012). However, the potential of co-inoculation for MLF induction in white musts/wines with challenging concentrations of SO₂ remains unexplored. Further, since the interactions between yeast and bacteria during co-inoculation are likely more complex than sequential inoculation (Zapparoli et al., 2006), a greater understanding of such interactions is required to improve MLF performance in harsh wine environments.

This study investigates the role of S. cerevisiae-derived SO₂ on MLF progress during the co-inoculated fermentation of Chardonnay. Representative yeast strains with varying SO₂ production potential were investigated for their interactions with O. oeni during co-fermentation in Chardonnay. The early, transient formation of acetaldehyde by yeast was observed to be an important factor modulating SO₂ resistance in malolactic starter cultures during co-fermentation. Finally, a pilot-scale study further explored the effect of different yeast-bacteria pairings, in which acetaldehyde production varied, on MLF progress and wine sensory properties.

Material and methods

Results

2. Effect of yeast strain and SO2 production on the survival of O. oeni during co-fermentation

The effects of two non-SO₂-producing yeast strains (AWRI796, AWRI1483) and two SO₂-producing yeast strains (AWRI1497 and AWRI2880) on the survival, growth and

MLF performance of the commercial O. oeni strain YV Select was investigated during co-fermentation in Chardonnay. The yeast strains were selected on their ability to modulate SO₂ concentration during fermentation in Chardonnay (Figure 1). The concentrations of total SO₂, L-malic acid, viable bacteria, and the duration of AF during co-fermentation of O. oeni YV Select with each yeast strain are shown in Figure 2.

Figure 1. Total SO2 concentration in finished Chardonnay wine following fermentation with different strains of S. cerevisiae.

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Yeast strains are identified by their AWMCC identification number on the y-axis. The dotted line indicates the total SO₂ concentration of the Chardonnay juice prior to fermentation. Strains marked with dark-coloured bars indicate those used in subsequent work. Bars show the mean total SO₂ concentration, with error bars showing the standard deviation (n = 3).

Bacterial viable cell concentrations in co-fermentations with each S. cerevisiae strain remained relatively constant (4.4 × 10⁶ (SD = 1.1 × 10⁶) CFU/mL) over the first 24 h following bacterial inoculation (days 1–2 of AF). However, bacterial viable cell concentrations in all co-fermentations declined over the subsequent 5 days (days 2–7 of AF). The extent of the decrease in bacterial viability varied greatly regardless of whether O. oeni was inoculated with SO₂-producing or non-SO₂-producing yeast strains.

For the non-SO₂-producing yeast strains, the bacterial viable cell population within 7 days decreased by >10²- and >10⁴-fold for AWRI796 and AWRI1483, respectively. The decrease in bacterial cell viability affected MLF duration, which increased from 36 days with AWRI796 to 64 days with AWRI1483. These co-fermentations were associated with decreases in total SO₂ concentration of 14 mg/L and 5 mg/L during AF.

Co-fermentations with the yeast strains AWRI1497 and AWRI2880 substantially increased total SO₂ concentration to 65 mg/L and 63 mg/L after 5 days (Figure 2). However, these yeast strains had different effects on bacterial survival and MLF activity despite producing similar concentrations of SO₂. For S. cerevisiae AWRI1497, the viable bacterial population diminished to non-detectable levels (< 20 CFU/mL) and failed to recover sufficiently to initiate MLF. Co-fermentations with S. cerevisiae AWRI2880 exhibited a comparatively small initial decline in bacterial viability to 8.1 × 10⁵ CFU/mL within the first 7 days, after which it increased to > 1.0 × 10⁶ CFU/mL, and ultimately completed MLF within 34 days. Moreover, although there was evidence (P < 0.05) for differences in the concentrations of some major final wine parameters (including alcohol, total acidity, acetic and succinic acids, and pH value) (Table 3), the magnitude of those were small and remained within a normal concentration range for wine.

Figure 2. Effects of S. cerevisiae (Sc) strains AWRI1483, AWRI796, AWRI1497, and AWRI2880 on MLF by O. oeni YV Select and bacterial survival following co-inoculation of Chardonnay juice.

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Figures show total SO₂, L-malic acid, and bacterial viable cell concentrations for respective yeast-bacteria combinations. O. oeni YV Select was inoculated 24 h after inoculation of each yeast strain. Blue shading indicates the time for completion of AF. Data are the average and standard deviation of 3 replicate fermentations. Data points for viable bacterial concentrations on days 7 and 14 in Sc AWRI1497 co-fermentations are below the limit of detection (1.3 log₁₀ CFU/mL).

3. Influence of early acetaldehyde production by SO2-producing yeast on O. oeni survival

Variations between yeast strains in the production of acetaldehyde may give rise to subtle variations in free SO₂ concentrations during the early stages of fermentation. Consequently, modulation of free SO₂ concentration during the crucial post-inoculation period may also affect bacterial survival. Whether variation in acetaldehyde formation by SO₂-producing yeast strains could explain the differential post-inoculation survival rates of O. oeni was investigated in the laboratory- and pilot-scale studies. We also sought to determine whether the effect of yeast on O. oeni survival reported in Figure 2 was specific to the bacterial strain used or whether that observation could be generalised to other bacterial strains.

Table 3. Chemical composition of Chardonnay wines after MLF following co-fermentation with different S. cerevisiae yeast strains and O. oeni YV Select.

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		AWRI796	AWRI1483	AWRI1497	AWRI2880
Alcohol	(%v/v)	13.4 (0.00) a	13.4 (0.00) a	13.4 (0.00) a	13.2 (0.06) b
pH		3.52 (0.01) c	3.57 (0.01) a	3.43 (0.01) d	3.54 (0.01) b
Total acidity	(g/L)	6.0 (0.0) b	5.6 (0.1) c	7.1 (0.1) a	5.7 (0.0) c
Malic acid	(g/L)	0.14 (0.18) b	0.04 (0.02) b	2.71 (0.02) a	0.15 (0.05) b
Acetic acid	(g/L)	0.40 (0.08) a	0.37 (0.09) a	0.27 (0.01) a	0.37 (0.01) a
Succinic acid	(g/L)	3.0 (0.0) a	2.8 (0.1) b	3.0 (0.1) a	2.8 (0.0) b
Total SO2*	(mg/L)	25.1 (5.6) b	33.7 (4.4) b	70.3 (1.1) a	64.1 (2.9) a
AF Ferm Time	(days)	10	12	11	8
MLF Ferm Time	(days)	37	64	na**	34

^* Total SO₂ determined after completion of AF.

^{* *} na; not applicable, wine treatment did not undergo MLF. Data are the mean (SD) of 3 replicate fermentations. Mean values in rows with different letters are significantly different (α = 0.05).

3.1 Laboratory scale studies

The effects of the two previously studied SO₂-producing yeast strains, S. cerevisiae strains AWRI1497 and AWRI2880, on the viability of O. oeni YV Select and two other commercial O. oeni strains (VP41 and CH35) following co-inoculation into Chardonnay was investigated (Figure 3). These three O. oeni strains are commercially available to the Australian wine industry. As with the previous experiment (Figure 2), AWRI1497 and AWRI2880 produced similar concentrations of total SO₂ (25–31 mg/L average maximum increase), but their effect on bacterial survival differed (Figure 3). The variation in bacterial survival was associated with the early transient formation of acetaldehyde and, consequently, MLF duration (Figure 3). Over the first 5 days of co-fermentations with S. cerevisiae AWRI1497, each O. oeni strain exhibited a similar, linear reduction in viability from approximately 5 × 10⁶ CFU/mL to 6 × 10² CFU/mL (Figure 3), coinciding with a slow increase in acetaldehyde concentration to a peak of 64 mg/L at day 4 (Figure 3). From day 5, recovery of YV Select and VP41 viability was observed, increasing to > 10⁶ CFU/mL at 44 days, with MLF completion at 58 days (Figure 3). In comparison, the recovery of O. oeni CH35 was slower, achieving > 10⁵ CFU/mL after 65 days (Figure 3), and although initiating L-malic acid degradation, MLF was still incomplete at 74 days (Figure 3).

Figure 3. Effects of acetaldehyde and SO2 production by S. cerevisiae strains AWRI1497 and AWRI2880 on bacterial survival and MLF duration by O. oeni strains YV Select, VP41, and CH35 following co-inoculation of Chardonnay.

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Figures show concentrations of total SO₂, L-malic aid, bacterial viable cell concentration, acetaldehyde and calculated molecular SO₂ for respective yeast-bacteria combinations. Shading indicates the time for completion of AF by respective yeast strains. Data are the average and standard deviation of 9 replicate fermentations. For the yeast/bacteria combination AWRI2880 and O. oeni CH35, data for 8 replicate fermentations are shown.

In contrast to S. cerevisiae AWRI1497, the impact of S. cerevisiae AWRI2880 on O. oeni strains YV Select and VP41 was relatively minor, their viability declining to approximately 2 × 10⁶ CFU/mL after 5 days and remaining at ≥ 1 × 10⁶ CFU/mL until 23 days (Figure 3). For both O. oeni strains, MLF was complete at 26 days (Figure 3). However, for co-fermentations of S. cerevisiae AWRI2880 and O. oeni CH35, a linear decrease in bacterial viability from 5 × 10⁶ CFU/mL to 3 × 10⁴ CFU/mL occurred during the first 5 days, followed by recovery to ≥ 1 × 10⁶ CFU/mL at 51 days and completion of MLF at 74 days (Figure 3). The average acetaldehyde concentration in co-fermentations with S. cerevisiae AWRI2880 increased rapidly, reaching 95 mg/L on day one and a maximum of 112 mg/L on day 3, ultimately decreasing to 50–53 mg/L on day 8 (Figure 3). Compared to S. cerevisiae AWRI1497, it is noteworthy that acetaldehyde production began within the first 24 h for AWRI2880, while SO₂ production was not observed until 2 days after inoculation.

Determination of the molar ratio of acetaldehyde to total SO₂ in the early stages of fermentation revealed these values were considerably higher for AWRI2880 than AWRI1497, particularly at, and immediately following, bacterial inoculation; day one (4.0 vs. 1.6), day two (2.7 vs. 1.9) and day three (2.7 vs. 1.6). At the end of AF, the average molar ratio of acetaldehyde to total SO₂ for each yeast-bacteria strain combination was similar (1.1–1.3). The concentrations of total SO₂ and acetaldehyde were used to calculate the concentration of molecular SO₂ over time (Figure 3). These calculations suggest that the variation in acetaldehyde concentrations produced by the two yeast strains resulted in differences in O. oeni molecular SO₂ exposure.

During co-fermentation with S. cerevisiae AWRI1497, the calculated molecular SO₂ concentration experienced by bacteria was 3.8–5.8 µg/L at days 1–3 and 7.1–15.6 µg/L at days 4–5. These molecular SO₂ concentrations were associated with a loss in bacterial viability. In contrast, the much lower calculated concentrations of molecular SO₂ during co-fermentation with S. cerevisiae AWRI2880 (i.e., approx. 0.9-1.7 µg/L at days 1–3 and 3.6–5.9 µg/L at days 4–5) had a negligible effect on the viability of O. oeni strains YV Select and VP41, although were associated with a reduction in viability of approximately 10² CFU/mL for O. oeni CH35. Interestingly, a subsequent increase in calculated molecular SO₂ concentration to 14.6 µg/L on day 8 in this co-fermentation did not impact bacterial viability further.

3.2 Direct acetaldehyde addition prior to O. oeni inoculation does not stimulate bacterial survival

If early rapid acetaldehyde accumulation contributes to O. oeni survival through quenching of molecular SO₂ concentration, adding exogenous acetaldehyde might stimulate a similar effect. This idea was tested in fermentations initiated with AWRI1497 by adding excess acetaldehyde (99 mg/L) four hours before inoculation with O. oeni YV Select. Bacterial viability was monitored and compared to fermentations without an acetaldehyde addition. The concentration of acetaldehyde in treated co-fermentations decreased rapidly (Figure S2). Within 24 h of acetaldehyde addition, the average concentration of acetaldehyde in treated and untreated ferments was indistinguishable (42–43 mg/L). The control and acetaldehydetreated co-fermentations also displayed similar bacterial viability declines to 87–100 CFU/mL at day 4 (Figure S2).

3.3 Pilot-scale study

The relevance of these observations to commercial wine production was demonstrated in a winery-based pilot-scale co-inoculation study. This study used commercial ADWY preparations of two non-SO₂-producing and two SO₂-producing yeast strains. Two of these were ADWY preparations of strains used in the first interaction experiment: AWRI796 (i.e., Maurivin AWRI796) and AWRI2880 (i.e., Anchor NT 50). The ADWY strain Actiflore Rosé was included as a second SO₂-producing strain because the commercial form of AWRI1497 (i.e., Lalvin T73) was unavailable in Australia. Lalvin CY3079 was used as the second non-SO₂-producing ADWY strain. The O. oeni strain VP41 was selected as a representative commercial malolactic bacterial strain.

Two distinct patterns of total SO₂ formation were evident in the early stages of co-fermentation (Figure 4). Two yeast strains, Actiflore Rosé and NT 50, yielded 56 and 50 mg/L total SO₂ on days 3 and 4, respectively. The other two yeast strains, AWRI796 and CY3079, yielded 18 and 21 mg/L total SO₂ on day 3 (Figure 4). Consistent with laboratory-scale experiments, the effects of the two high SO₂-producing yeast strains on O. oeni VP41 were notably different. The bacterial population in cofermentations with S. cerevisiae Actiflore Rosé declined approximately 10-fold to 1.9 × 10⁵ CFU/mL at day 9, recovering to 9.4 × 10⁵ CFU/mL at day 16, after which MLF was complete at day 37 (Figure 4, Table 4). In contrast, co-fermentations with S. cerevisiae NT 50 were stimulatory to bacterial growth, yielding a maximum bacterial cell population of 5.1 × 10⁶ CFU/mL at day 9 and rapid completion of MLF at day 13 (Figure 4). A similar pattern of bacterial growth and L-malic acid degradation occurred during co-fermentation with S. cerevisiae CY3079 and, to a lesser extent, AWRI796, which only produced maxima of 21 and 19 mg/L total SO₂ within the first five days of AF, respectively (Figure 4).

Consistent with the laboratory-scale interaction experiment 2, the peak acetaldehyde concentration observed during the early stages of co-fermentations with S. cerevisiae NT 50 was notably higher (183–182 mg/L maximum at days 2 and 3) compared to the other yeast strains, which produced 87–98 mg/L acetaldehyde at this time (Figure 4).

Figure 4. Effects of acetaldehyde and SO2 production by S. cerevisiae strains AWRI796, CY3079, Actiflore Rosé and NT 50 on bacterial survival and MLF duration by O. oeni VP41 in pilot-scale fermentations of Chardonnay.

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Figure panels show concentrations of total SO₂, L-malic acid, bacterial viable cell concentration, acetaldehyde and calculated molecular SO₂ for respective yeast-bacteria combinations. Blue shading indicates the duration of AF. Data are the average and standard deviation of 3 replicate fermentations (35 L).

Further, the calculated molecular SO₂ concentrations during the early stages of co-fermentation (Figure 4) are consistent with laboratory-based work. Higher molecular SO₂ concentrations were observed with S. cerevisiae Actiflore Rosé (i.e., approx. 0.2–2.5 µg/L at days 1–3, 7.5–13.4 µg/L at days 4–6 and 18.8–34.6 µg/L at days 7–9) and associated with bacterial inhibition. Lower concentrations were observed with S. cerevisiae NT 50 (i.e., approx. ≤ 1.0 µg/L at days 1–3, 1.6–4.4 µg/L at days 4–6 and 7.4–9.5 µg/L at days 7–9) which generally supported bacterial growth.

The concentrations of other chemical components of Chardonnay wines co-fermented with O. oeni VP41 and each yeast strain are shown in Table 4. There was evidence (P < 0.05) for differences in the concentrations of alcohol, total acidity, acetic acid and in pH value between wines produced by different yeast strains. Notably, acetic acid concentration increased by 0.16 g/L in NT 50 compared to CY3079 wines. Co-fermentations with the different yeast strains had a major effect on wine diacetyl concentration, which was only detected in wines coinoculated with the high SO₂-producing yeast strains. Wines produced with NT 50 (4.9 (SD = 0.6) mg/L) contained significantly (P < 0.05) greater diacetyl concentrations than wines produced with Actiflore Rosé (1.0 (SD = 0.1) mg/L). In contrast, diacetyl was not detected in wines produced from co-fermentations with either of the non-SO₂-producing yeast strains (CY3079 and AWRI796).

Sensory assessment of the Chardonnay wines by Napping combined with UltraFlash Profiling revealed that there were differences related to the coinoculation yeast strain (Figure 5). The AWRI796 co-inoculated wines were positioned to the left of Figure 5 and were described more often with the terms citrus, apple/pear, tropical fruit, floral, vanilla, flint, sourness, and sweetness. The CY3079 coinoculated wines were separated along Dimension 1 from the AWRI796 wines, with a lower citation frequency of apple/pear and floral terms. The Actiflore Rosé and NT 50 coinoculated wines were also positioned away from the AWRI796 wines, and stone fruit, woody, butter, and bitterness terms were used more frequently for these wines. The NT 50 coinoculated wines had the highest citation frequency for butter aroma and flavour.

Table 4. Chemical composition of Chardonnay wines after MLF following pilot-scale co-fermentation with different S. cerevisiae yeast strains and O. oeni VP41.

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		AWRI796	CY3079	Actiflore Rosé	NT 50
Alcohol	(%v/v)	11.9 (0.1) bc	12.0 (0.0) a	11.9 (0.0) b	11.8 (0.0) c
pH		3.41 (0.01) d	3.42 (0.00) c	3.48 (0.01) b	3.53 (0.01) a
Total acidity	(g/L)	7.0 (0.0) a	6.7 (0.0) b	6.6 (0.1) b	7.0 (0.1) a
Acetic acid	(g/L)	0.35 (0.00) b	0.27 (0.01) c	0.29 (0.01) c	0.42 (0.02) a
Succinic acid	(g/L)	4.8 (0.3) a	5.1 (0.1) a	4.8 (0.4) a	5.0 (0.1) a
Total SO2*	(mg/L)	20.2 (0.5) b	13.6 (0.8) c	40.8 (2.1) a	46.5 (3.7) a
Diacetyl**	(mg/L)	n.d.	n.d.	1.0 (0.1) b	4.9 (0.6) a
AF Ferm Time	(days)	21	9	7	6
MLF Ferm Time	(days)	13	9	37	13

*Total SO₂ determined after completion of AF.

**Diacetyl analysis was undertaken at the time of sensory analysis (approx. 7 months after bottling); n.d.: not detected, less than 0.1 mg/L. Data are the mean (SD) of 3 replicate fermentations. Mean values in rows with different letters are significantly different (α = 0.05).

Figure 5. Multiple factor analysis (MFA) map from Napping (N) assessment of replicate Chardonnay wines co-inoculated with O. oeni VP41 and different S. cerevisiae strains.

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Saccharomyces cerevisiae strains: AWRI796 (796, purple circles), CY3079 (CY, light brown circles), Actiflore Rosé (AR, pink circles) and NT 50 (NT, green circles). Sensory descriptors from Ultra Flash Profiling (UFP) are superimposed.

Discussion

As a prerequisite to examining the effects of yeast-derived SO₂ on O. oeni, the SO₂-producing potential of 94 wine yeast strains was investigated following fermentation in a commercially prepared, presulfited Chardonnay juice (2017). The SO₂-producing capability observed supports other phenotypic surveys (Richter et al., 2013; Suzzi et al., 1985) and reflects the categorisation that most strains exhibit low SO₂-producing ability (an increase of up to 22 mg/L SO₂ at the 90^th percentile), while some are high sulfite-producers (Eschenbruch, 1974). Although the fermentation conditions used here may not fully account for possible minor losses in SO₂ through gaseous exchange, the observation that approximately half of the yeast strains exhibited the ability to consume SO₂ in sulfited grape juice is of practical importance, yet such capability has received little attention. Wine yeasts utilise sulfite over sulfate as a source of sulfur, and can oxidise and reduce sulfite added to grape must (Eschenbruch, 1973). Recently, Ochando et al. (2020) showed that a winemaking S. cerevisiae strain (MC005) could produce or consume SO₂ in a synthetic grape juice medium and suggested that cellular uptake (diffusion) and production of SO₂ by yeast from the sulfate pool was in equilibrium with an initial threshold concentration of SO₂ in the juice. Similarly, for respective yeast strains in the current study, the final total SO₂ concentration of wines fermented using sulfited and non-sulfited juices was also in a similar range. For example, that of Chardonnay wine fermented with S. cerevisiae AWRI796 in sulfited (2017) and nonsulfited (2021 pilot-scale study) juices was 28 (SD = 1.5) mg/L and 20 (SD = 0.5) mg/L, respectively.

Representative yeast strains with differing SO₂ production exhibited wide-ranging impacts on the survival and MLF capability of O. oeni following co-inoculation in Chardonnay. This was highlighted in the first interaction experiment in which differential MLF duration was observed in ferments of non-SO₂-producing and SO₂-producing yeast strains. This observation supports the findings of others (Larsen et al., 2003; Osborne and Edwards, 2006) that yeast metabolites other than SO₂ are likely to affect bacterial survival. Of those yeast metabolites with the potential to cause inhibition of O. oeni, there were no major differences in the final concentrations of succinic acid (Torres-Guardado et al., 2022). This suggests that metabolites such as toxic fatty acids or bioactive peptides (Bartle et al., 2019) could be involved in bacterial inhibition in wines co-fermented with the non-SO₂-producing yeast strains. Further, a notable observation from this study was the highly divergent effects of the two SO₂producing yeast strains (AWRI1497 and AWRI2880) on the survival and growth of O. oeni. While the accumulation of yeast-derived SO₂ is generally associated with O. oeni inhibition (Larsen et al., 2003; Wells and Osborne, 2011), particularly in grape must already containing SO₂ (Henick-Kling and Park, 1994), the enhanced survival of O. oeni after co-inoculation with S. cerevisiae AWRI2880 compared to AWRI1497 appeared novel, and formed the basis of subsequent investigations.

Of the yeast metabolites capable of modulating yeastderived SO₂ toxicity towards O. oeni, acetaldehyde was considered a primary candidate as it binds strongly with SO₂, especially at low pH (K = 1.5 × 10^-6 at pH 3.3). Yeasts exhibit a strain-dependent ability to rapidly produce acetaldehyde during the early stages of AF, which is subsequently re-utilised as fermentation progresses (Cheraiti et al., 2010; Li and Mira de Orduña, 2017; Ochando et al., 2020). In the current study, peak acetaldehyde production was exceptionally high with the SO₂-producing S. cerevisiae strain AWRI2880. This phenotypic feature resulted in comparatively high molar ratios of acetaldehyde:SO₂ (up to approximately 4:1) in the first few days following coinoculation, and is proposed as a key factor contributing to the enhanced survival of O. oeni.

These observations support the findings of Carr et al. (1976), who reported that the addition of an excess quantity of acetaldehyde (or an equivalent quantity of H₂O₂) alleviated SO₂ inhibition of L. plantarum. Similarly, Larsen et al. (2003) observed improved growth and MLF rate of O. oeni in Chardonnay wine fermented by S. cerevisiae V-1116 after treatments with excess amounts (50 mg/L) of acetaldehyde (or H₂O₂) and concluded that inhibition of bacterial growth was due to inhibitory concentrations of free SO₂. Under the co-fermentation conditions of the current study, however, attempts to validate the link between bacterial survival and acetaldehyde:SO₂ ratio were prevented by the rapid utilisation of added acetaldehyde by S. cerevisiae AWRI1497 (99 mg/L consumed within ≤ 24 h), resulting in no noticeable impacts on bacterial survival. The utilisation of exogenous acetaldehyde by yeast has been previously described (Ochando et al., 2020) and reflects the tightly regulated and dynamic flux of this intermediate during fermentation.

Further insight into the impact of the relative proportions of acetaldehyde:SO₂ on O. oeni survival was obtained by calculating the concentrations of free and molecular SO₂ in fermentation samples using acetaldehyde-bisulfite equilibria. This enabled estimation of free and molecular SO₂ concentration well below the detection limit of most analytical methods (i.e., < 2–3 mg/L free SO₂), which can occur during and after white wine fermentation. Although becoming unreliable as acetaldehyde:SO₂ molar ratios approach unity (approximately < 1.1:1.0), these data revealed that the initial survival of O. oeni starter cultures following co-inoculation with SO₂-producing yeast strains could be linked to, and regulated by, a narrow threshold concentration of molecular SO₂ (approximately 1–35 µg/L). In particular, the second interaction experiment demonstrated that following co-inoculation with S. cerevisiae AWRI1497, initial exposure of malolactic starter cultures to only 4–6 µg/L molecular SO₂ was associated with considerable reduction in bacterial viability over the first few days of co-fermentation. On the other hand, following co-inoculation with S. cerevisiae AWRI2880, negligible loss in viability of O. oeni strains YV Select and VP41 occurred after exposure to an initial molecular SO₂ concentration of 1–2 µg/L. These findings suggest that molecular SO₂ concentrations almost 100-fold less than those previously reported by Ribéreau-Gayon et al. (2006) (0.15–0.4 mg/L) may cause inhibition of O. oeni in wine. Further, such inhibition appears to be alleviated, and malolactic starter performance optimised, in wine conditions where molecular SO₂ is almost completely absent in the first few days of co-fermentation.

The extremely low concentrations of molecular SO₂ associated with O. oeni inhibition observed here support other reports regarding the SO₂ sensitivity of this organism. For example, Carr et al. (1976) noted that 0.032 mg/L molecular SO₂ in a culture medium (pH 4) was sufficient to completely suppress and kill 3 of 4 wild heterofermentative cocci (isolated from cider). Similarly, Hood (1983) demonstrated that very low calculated levels of free SO₂ (0.05-0.8 mg/L), which can exist in equilibria with acetaldehyde-bisulfite, caused a dose-response inhibition in L. brevis and Leuc. oenos growth in a culture medium (pH 4) containing 10, 20, and 30 mg/L total SO₂. This author also reported that a calculated free SO₂ concentration of 0.8 mg/L (equivalent to 5 µg/L molecular SO₂) entirely suppressed the growth of Leuc. oenos LN6 in the same medium containing 30 mg/L total SO₂.

The current work has also provided further insight into the adaptive capability of O. oeni to SO₂. For example, in the pilot-scale experiment, exposure of O. oeni VP41 to an extremely low and incremental rise in molecular SO₂ concentration (up to approximately 10 µg/L) within the first 9 days of co-fermentation with S. cerevisiae NT 50 facilitated effective adaptation to molecular and total SO₂ concentrations of approximately 16 µg/L and 57 mg/L, respectively. This suggests that the adaptive capability of O. oeni towards SO₂ (Delfini and Morsiani, 1992; Guzzo et al., 1998) encompasses the molecular form of this compound. Overall, the evidence from this work suggests the survival and adaptation of O. oeni following co-inoculation with SO₂producing yeast strains requires that molecular SO₂ concentrations remain below a threshold inhibitory range in the early stages of co-fermentation, which exponentially increases from approximately 3–6 µg/L in the first three days of co-fermentation.

While evidence from the second interaction experiment indicates some strain-dependent reduction in O. oeni viability after exposure to ≥ 50–60 mg/L bound SO₂, it is unclear whether such inhibition was caused by bound SO₂ itself, or extremely low concentrations (1–6 µg/L) of molecular SO₂ accumulated over the first 5 days of co-fermentation, or a combination of both. Manufacturer recommendations for O. oeni CH35 indicate its suitability for MLF induction in wines of up to 45 mg/L SO₂, and those of O. oeni VP41 and YV Select indicate SO₂ tolerances of up to 60 mg/L and < 50 mg/L, respectively. Interestingly, Carr et al. (1976) report no indication of toxicity of acetaldehyde-bound SO₂ towards L. plantarum. However, further studies are needed to clearly define the tolerance and adaptive capabilities of O. oeni strains to molecular and bound forms of SO₂ under wine conditions.

Further insight into the potential effects of acetaldehyde-bound SO₂ on coinoculated O. oeni was indirectly gained from the concentrations of acetaldehyde observed during the latter stages of co-fermentation. The toxicity of acetaldehyde-bound SO₂, at least in part, requires the bacterial utilisation of acetaldehyde, and subsequent release of sufficient free SO₂ to affect growth and survival (Fornachon, 1963; Hood, 1983; Osborne et al., 2006; Wells and Osborne, 2012). Moreover, the ability of wine LAB to degrade acetaldehyde and SO₂-bound acetaldehyde (Jackowetz and Mira de Orduña, 2012; Jackowetz and Mira de Orduña, 2013; Osborne et al., 2000; Pan et al., 2011; Wells and Osborne, 2012) appears to coincide with bacterial growth (Fornachon, 1963; Hood, 1983; Osborne et al., 2006).

In white wine, acetaldehyde catabolism can initiate after the commencement of MLF and continue for up to several weeks after its completion (Jackowetz and Mira de Orduña, 2012). However, in the current pilot-scale study, such metabolism was not evident in post-MLF stages, nor during MLF in wines co-inoculated with S. cerevisiae Actiflore Rosé. An absence of acetaldehyde catabolism may reflect differences in wine parameters, such as pH, that are critical to the growth and MLF capability of O. oeni. Osborne et al. (2006) reported that acetaldehyde degradation by O. oeni in white wine containing acetaldehyde-bound SO₂ occurred at pH 3.6 but not at pH 3.3. Similarly, Flamini et al. (2002) indicated that following MLF with two different O. oeni strains, substantial acetaldehyde degradation occurred in Cabernet-Sauvignon wine (pH 3.55) but not in Chardonnay (pH 3.24). Hence, it appears unlikely that acetaldehyde metabolism is sufficiently active to account for the inhibition of O. oeni immediately following coinoculation in low pH Chardonnay wine observed in the current study.

In addition to affecting the survival and MLF capability of O. oeni during co-fermentation, the pilot-scale study demonstrated that the choice of coinoculation yeast strain also had a major impact on the production of diacetyl and on wine sensory properties. The diketone diacetyl is an important flavour compound imparting buttery aroma and flavour attributes, and can arise from LAB metabolism during MLF (Bartowsky and Henschke, 2004). Yeast can also synthesise and degrade diacetyl (Martineau and Henick-Kling, 1995; Mink et al., 2014) and, although the diacetyl concentration in newly fermented wine is typically low (Bartowsky and Henschke, 2004), some variation may occur depending on factors including yeast lees contact, SO₂ addition and grape variety (Martineau and Henick-Kling, 1995). For Chardonnay wines without MLF, Martineau et al. (1995b) reported an average diacetyl concentration of 0.22 (SD = 0.20) mg/L. The presence of yeast during and after MLF will also influence diacetyl content (Bartowsky and Henschke, 2004; Martineau and Henick-Kling, 1995).

Since Chardonnay wines in this study were co-fermented and left in contact with yeast lees for one week after MLF, the absence of diacetyl (< 0.1 mg/L) in wines co-inoculated with the low SO₂producing yeast strains AWRI796 and CY3079 is not unexpected. However, the higher concentrations of diacetyl in wines coinoculated with the high SO₂producing S. cerevisiae strains, particularly NT 50 (4.9 (SD = 0.6) mg/L), is surprising, and may relate to diacetyl binding and retention at elevated SO₂ concentration.

The observations regarding diacetyl were also reflected in wine sensory properties, whereby wines coinoculated with high SO₂producing yeast strains Actiflore Rosé and NT 50 had higher citation frequency for butter and woody attributes, and those co-inoculated with NT 50 had the highest citation frequency for butter. These results align with the sensory detection threshold for diacetyl in Chardonnay wine (0.2 mg/L, Martineau et al. (1995a)). However, while a bacterial origin of accumulated diacetyl is likely, a potential contribution from yeast (Martineau and Henick-Kling, 1995) cannot be discounted and requires investigation.

Conclusions

This study confirms that S. cerevisiae strain choice can affect the survival of O. oeni and the conduct of MLF during coinoculation. Immediately following bacterial inoculation, the early stages of co-fermentation provide a unique and metabolically dynamic environment, the composition of which is critical for bacterial survival and MLF efficiency.

Significantly, for coinoculation with SO₂-producing yeast strains, laboratory- and pilotscale studies have demonstrated a direct association between the survival of O. oeni and the molar ratio of acetaldehyde:SO₂ in the early stages of AF. Alleviation of SO₂inhibition of O. oeni and improved MLF efficiency was associated with an early, transient accumulation of a high concentration of acetaldehyde.

By applying relevant acetaldehyde-bisulfite equilibria, the survival and adaptation to SO₂ by O. oeni starter cultures following coinoculation appear to be regulated by an extremely low concentration range of molecular SO₂ (approximately 1–35 µg/L). Bacterial survival coincided with molecular SO₂ concentrations remaining below a minimum inhibitory range, which increased exponentially from 3–6 µg/L in the first 3 days of co-fermentation. Although some O. oeni strains may exhibit sensitivity to bound SO₂, the mechanism of such inhibition during co-fermentation remains unclear.

Moreover, the possible role of other mechanisms, such as medium chain fatty acid formation, should not be discounted to fully explain the complex interactions that may occur between yeast and malolactic bacteria during co-fermentation. Reliance on total/bound SO₂ concentration may only provide an indirect and potentially inaccurate measure of SO₂ toxicity towards O. oeni for co-inoculation. Equilibrium concentrations of molecular SO₂ should also be considered. Knowledge of both the SO₂- and acetaldehyde-producing potential of yeast strains are, therefore, important selection criteria for co-inoculation, particularly for white musts/wines in which elevated SO₂ concentrations are anticipated.

The choice of coinoculation yeast strain also influenced wine diacetyl concentration and sensory properties. Coinoculation with high SO₂-producing yeast strains produced wines with higher diacetyl concentration. These wines had a higher citation frequency for butter attributes.

Database

Access to all data from the study is provided in the following on-line repository file: https://datadryad.org/stash/share/a7q7umBAe6yKC_zHKzd5uJfrWwyUo6c1al0LwCLzakQ"

Acknowledgements

The authors gratefully acknowledge Pernod Ricard Winemakers for the generous supply of Chardonnay juice. We thank Dr Leigh Francis for helpful discussion with this manuscript, Dr Toni Garcia Cordente for undertaking HPLC analysis and quantification of organic acids, Charlotte Jordans and Mark Rullo for technical assistance, and AWRI sensory panellists for their time and assistance. Alchemy Agencies (Australia) is also thanked for providing NataP UltraPure used to prepare selective agar media. This work was supported by Wine Australia, with levies from Australia’s grapegrowers and winemakers and matching funds from the Australian Government. The AWRI is a member of the Wine Innovation Cluster in Adelaide.

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