Introduction
With the increase in alcohol levels and loss of acidity and freshness due to climate change, acidifying wines with different acids of chemical origin is common. This acidification has some limitations in terms of legislation (even banned in some appellations) and also in terms of impact (from an acidification point of view and also from a sensory one). A new natural way to increase freshness and acidity is now available for winemakers with the use of Level2 Laktia™ Lachancea thermotolerans. This non-fermenting non-Saccharomyces yeast found in the must environment naturally acidifies the wines when converting sugars into lactic acid. Lowering the wine pH will also help with the efficiency of SO2 and help reduce its use.
Why Lachancea thermotolerans?
Lachancea thermotolerans (L. thermotolerans) is a ubiquitous yeast species found worldwide, in anthropic and wild habitats (Hranilovic et al., 2017), including the grape and wine ecosystem. It is found in the must and wine environment, but cannot complete wine fermentation alone, mainly because of high oxygen needs (Holm Hansen et al., 2001; Petitgonnet et al., 2019) and limited resistance towards ethanol. A sequential inoculation with a fermenting yeast, Saccharomyces cerevisiae, is essential to complete fermentation.
The main technological interest of L. thermotolerans inoculation during wine fermentation is its ability to lower the pH in wine (Comitini et al., 2011; Morata et al., 2019; Vaquero et al., 2020). This acidification is due to lactic acid production from pyruvate through a reaction catalysed by three lactic acid dehydrogenases LDH1, 2 and 3 (Figure 1). This carbon metabolism is “competing” with ethanol production, slightly decreasing the final alcohol content and is dependent on the L. thermotolerans strain and the fermentation conditions. In addition to freshness improvement, L. thermotolerans inoculation during wine fermentation can lead to higher glycerol production, impacting the mouthfeel (Comitini et al., 2011; Gobbi et al., 2013), with significant heterogeneity between strains among this species.
FIGURE 1. Fermentative metabolism scheme of sugar in L. thermotolerans. Scheme from Sgouros et al. (2020) based on KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway Database.
L. thermotolerans inoculation also generally leads to higher ester production (ethyl lactate and ethyl acetate) and 2-phenyl ethanol (Comitini et al., 2011; Gobbi et al., 2013; Morata et al., 2019; Sgouros et al., 2020; Hranilovic et al., 2021) with some differences between strains both in terms of ester compounds and concentration. Hranilovic et al. (2021) have also shown an increase in linalool liberation, consistent with L. thermotolerans β-glucosidase activity as described by Comitini et al. (2011).
There is high variability among L. thermotolerans strains. For example, in the same matrix (wine conditions), lactic acid production can vary from strain to strain (Hranilovic et al., 2018). Sgouros et al. (2020) and Gatto et al. (2020) have shown that high lactic acid producers present a higher LDH2 expression than low lactic acid producers.
Selection and characterisation of Lachancea thermotolerans Level2 Laktia™
Lachancea thermotolerans Level2 Laktia™ was selected in the Rioja region in Spain from Tempranillo. It became available to winemakers in 2018, mainly for use in warm to hot climate regions to provide a blending component to increase the acidity and freshness of wines naturally.
Lab scale characterisation in different red wine conditions
At our research centre in Blagnac (France), a Box Behnken experimental design was used to predict Level2 Laktia™ lactic acid production behaviour in red wine, depending on different conditions of temperature (from 20 – 28°C), inoculation rate (from 10 – 30 g/hL) and initial pH (from 3.3 – 4). Fermentations were performed in a Merlot (239 g/L of sugar, 193 mg/L of YAN) in 200 mL fermenters. After 48 hours, levels of lactic acid were analysed.
The impact of environmental factors is represented through response surface plots (Figure 2). Lactic acid production increases with a higher initial pH, a higher Level2 Laktia™ inoculation rate and temperatures between 18 – 22°C.
FIGURE 2. Response surface plots showing Level2 Laktia™ lactic acid production variation against pH, inoculation rate and temperature after 48 hours in Merlot.
These results were also confirmed in a Syrah (239.6 g/L of sugar and 144 mg/L of YAN) in 200 mL fermenters. Lactic acid production is impacted the same way by environmental conditions, even though resulting concentrations might be different from one must to another due to other environmental factors (maceration, SO2 addition, nitrogen, turbidity, etcetera).
These experiments help identify the best conditions to support lactic acid production with Level2 Laktia™. For the next step of our characterisation trials, the inoculation rate has been fixed to the optimal dosage (25 g/hL).
Pilot and winery trials in white and red winemaking
As indicated in the introduction, a remarkable phenotypic diversity is found within L. thermotolerans strains. A characterisation was conducted at pilot and winery scales to confirm its impact on red and white wines. Since 2018, nearly 70 trials have been conducted on 16 different grape varieties in five countries. The objectives were to have a better understanding of the environmental factors impacting lactic acid production from Level2 Laktia™ and its positive sensory impact. A regular nutrition strategy with organic yeast nutrient addition after Level2 Laktia™ inoculation was followed for most trials.
Red winemaking
In contrast to laboratory trials, temperatures used for Level2 Laktia™ inoculation were between 18 and 20°C, which is typical during this winemaking step. Most of the time, there was no SO2 addition or minimal (from 2 – 4 g/hL). The main parameters studied were the contact time of Level2 Laktia™, as well as different processes, such as traditional maceration, carbonic maceration or flash pasteurisation (liquid phase). The control was, in some instances, chemical acidification at 1.5 g/L of tartaric acid addition to the must.
Among the 29 trials in red winemaking conditions, the highest levels of lactic acid (>5 g/L) were obtained with the longest contact time (72 hours) with Level2 Laktia™ and/or flash pasteurisation. Carbonic maceration does not seem to positively or negatively impact Level2 Laktia™ metabolism. A contact time of 24 hours generally led to the production of lactic acid between 2 and 3 g/L, while 48 hours gave higher concentrations, between 3 and 4 g/L. As at laboratory scale, a higher pH (>3.6) favoured lactic acid production. A summary of the impact of the environmental factors in red winemaking is presented in Figure 3.
FIGURE 3. Lactic acid production and key environmental factors in red winemaking at 25 g/hL of inoculation rate of Level2 Laktia.
* These levels can inhibit lactic acid bacteria (malolactic fermentation).
We observed, in some cases, a decrease in final ethanol content (from 0.2 – 0.7% v/v reduction in final wines). A maximum decrease was observed when high lactic acid was produced (>5 g/L).
Lactic acid is known to have an inhibitory impact on lactic acid bacteria and, therefore, malolactic fermentation. To ensure a complete malolactic fermentation, co-inoculation with a selected MLF strain is recommended. Lactobacillus plantarum (ML Prime™) can be inoculated simultaneously as Level2 Laktia™ or at the same time as selected Saccharomyces cerevisiae. With Oenococcus oeni starter culture, it has to be inoculated 24 hours after S. cerevisiae. For a sequential inoculation, if the lactic acid level is higher than 3 g/L, wine should be blended to decrease the level of lactic acid.
Regarding the sensory impact, the acidity was better integrated and balanced with Level2 Laktia™ compared to tartaric acid addition. The wines were generally more complex, fruitier and less bitter. Figure 4 shows the results of a sensory evaluation conducted in Merlot (south of France), where the freshness of the wine was perceived as more integrated compared to the control with no acidification. A summary of the comments to describe the wines from winemakers from different countries after using Level2 Laktia™ is also shown below.
FIGURE 4. Merlot (south of France) fermented with Level2 Laktia™ and selected S. cerevisiae compared to S. cerevisiae alone.
The figure below shows some tasting comments from wine trials in Italy and Spain on the impact of Level2 Laktia™ on the sensory profile of red wines.
White and rosé winemaking
Among the 38 trials conducted on white and rosé, Level2 Laktia™ produced from 0.18 – 8.7 g/L of lactic acid. This variation can be explained by environmental factors. Initial SO2, temperature at Level2 Laktia™ inoculation and contact time before Saccharomyces cerevisiae inoculation are key parameters impacting the lactic acid production in white and rosé wines.
A trial conducted at INRAE Pech Rouge (France) in 2019 illustrates differences in lactic acid production. A must of Maccabeu with a high initial pH (3.74) was homogenously distributed in 50 L tanks. Different levels of initial SO2 (0 or 15 mg/L), temperatures (14 or 18°C) and contact time of Level2 Laktia™ (24 or 72 hours) were applied in comparison with a control with the Saccharomyces cerevisiae only. The same inoculation rate of Level2 Laktia™ was used (25 g/hL). After the targeted contact time and for the control, Lalvin QA23 was inoculated, and the lactic acid production was evaluated at the end of the alcoholic fermentation (Figure 5). SO2 addition was the most impacting factor, as the lowest lactic acid production was obtained with 15 mg/L of SO2: on average, 0.8 g/L of lactic acid compared with 3.8 g/L without SO2. A longer contact time (72 hours) and a higher temperature (18°C) resulted in higher lactic acid production (respectively 5.4 and 3.9 g/L). We also observed a decrease of 0.5% v/v of alcohol.
FIGURE 5. Lactic acid production at the end of alcoholic fermentation. Maccabeu 2019, INRAE Pech Rouge, France with and without Level2 Laktia™. (Sugars = 215 g/L – pH = 3.74 – total acidity = 2.97 g/L TH2 – YAN = 119 mg/L – malic acid = 1.02 g/L.)
Lactic acid production was impacted similarly in all the winery/pilot trials. The lowest levels of lactic acid were obtained with higher levels of initial SO2, a lower initial temperature and/or a shorter contact time, as well as a low initial pH (<3.2). At the laboratory scale, we observed an interaction between these environmental factors with a positive or negative cumulative effect.
An added benefit of the use of Level2 Laktia™ is as the final pH is lowered, the SO2 is more effective at this low pH than it would be at a higher one. This impact is very useful in a strategy to reduce the use of SO2. In terms of sensory impact, the figure below shows winemaker comments after using Level2 Laktia™ in French, Italian and Spanish wines.
Conclusions
It has become common practice to acidify musts, often with tartaric acid, but now there is a biological/non-chemical alternative with Level2 Laktia™. This non-Saccharomyces yeast of the specie Lachancea thermotolerans converts a few grams of sugar into lactic acid at the start of alcoholic fermentation. It is natural acidification, and depending on the must conditions (pH, temperature and SO2), it can produce varied lactic acid concentrations. Moderate temperature and higher pH at inoculation appear to increase lactic acid production, while higher SO2 seems to decrease its production in white and rosé wines. When used before the juice is inoculated with a selected Saccharomyces cerevisiae wine yeast, Level2 Laktia™ will help the wine have more freshness and balance and accentuate the fruit aromas.
Bibliography
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– For more information, contact Piet Loubser at ploubser@lallemand.com.
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