Wine Innovation Advances As LEVEL2 SALVA Demonstrates Effective Bioprotection Against Brettanomyces

Naturally controlling Brettanomyces at the onset of wine production with LEVEL² SALVA™ - By Amandine Deroite, Marion Bastien, Ann Dumont, Anthony Silvano, José-Maria Heras & Anne Julien-Ortiz

LEVEL² SALVA™ is a new non-Saccharomyces yeast of the species Suhomyces pyralidae used as a specific bioprotection against Brettanomyces bruxellensis during the pre-fermentative steps of wine production. LEVEL² SALVA™ produces the specific Brettanomyces Inhibition Factor Spkt1, which disrupts the cell wall of Brettanomyces. Our study demonstrates its effectiveness in reducing Brettanomyces populations and volatile phenol production. It can be used at various stages of pre-fermentation, including on harvested grapes, all the way to tank filling. Additionally, LEVEL² SALVA™ enables a reduction in SO₂ usage without compromising wine stability and quality, offering a natural alternative for managing Brettanomyces contamination.

Introduction

Brettanomyces bruxellensis is a well-known spoilage microorganism in wine, as its development leads to the production of unpleasant aromas. The volatile phenols produced during Brettanomyces contamination are associated with faults in wine, as shown in Table 1. Even at low concentrations, below their perception threshold, the presence of those compounds can decrease the aromatic intensity of wines and hide the wine’s typicity.

Brettanomyces bruxellensis can be found at different stages of the winemaking process. It can also be present in vineyards, even on specific blocks within a vineyard. Previous studies have established the presence of Brettanomyces bruxellensis in several vineyards at various stages of grape development following veraison (Renouf et al., 2007).

1.   Classical control against Brettanomyces

The most common tool to avoid Brettanomyces bruxellensis contamination is SO₂ addition. However, according to Avramova and other authors (2018), adaptation phenomena led to a Brettanomyces bruxellensis subpopulation that is resistant to SO₂. With the tendency to decrease inputs, including sulphites, it is important to find alternative solutions to prevent and inhibit B. bruxellensis development during wine production and storage, in addition to considering this potential SO₂ resistance.

2.   The interest of controlling Brettanomyces in the pre-fermentative steps

As Brettanomyces is also found before the onset of fermentation, a preventive approach is needed to inhibit and control it by using bioprotection. They are found on grapes, even before the vinification process, as demonstrated by Renouf and Lonvaud-Funel (2007), who detected it in samples taken in four vineyards from the Bordeaux area at different development stages of the grape between veraison and harvest. Oro and other authors (2019) proved the strict relationship between Brettanomyces bruxellensis strains from the vineyard and from the winery with molecular analysis (26S-D1/D2 region of rDNA sequencing) of 62 samples from grapes, winery environments and fermenting musts. They showed that isolates from the vineyard and winery represented dominant and common biotypes, which indicated that vineyards are a contamination source of B. bruxellensis in the winery environment. Lately, Pigao and other authors (2021) also isolated Brettanomyces bruxellensis from 12 of 149 grape clusters (two years sampling) in a vineyard in Oregon.

Currently, bioprotection solutions with non-Saccharomyces yeasts in the pre-fermentative steps exist, but they are not really effective against Brettanomyces.

3.   A new non-Saccharomyces selection: LEVEL² SALVA™

Suhomyces pyralidae (formerly Candida pyralidae) IWBT Y1140 was isolated from South African Cabernet Sauvignon grape juice (Mehlomakulu et al., 2014) by the South African Grape and Wine Research Institute (University of Stellenbosch). It was shown to control and inhibit B. bruxellensis strains found in the wine environment. It is now available to wine producers under the name LEVEL² SALVA™.

This specific inhibiting activity is explained by the production of a Brettanomyces Inhibition Factor, named Spkt1. It was shown that its optimal activity conditions are compatible with winemaking conditions, mainly in terms of pH and temperature range. As it is sensitive to ethanol, it makes it ideal to be used in the pre-fermentative steps (Mehlomakulu et al., 2014).

As shown in Figure 1, LEVEL² SALVA™ inhibits Brettanomyces bruxellensis without impacting on Saccharomyces cerevisiae growth. This experiment confirms the efficiency of LEVEL² SALVA™ in the pre-fermentation steps in lab conditions. It also shows that Saccharomyces yeast alone is sometimes not sufficient to control the development of Brettanomyces.

3.1 LEVEL² SALVA™ characterisation

LEVEL² SALVA™ was characterised for its carbon metabolism, YAN consumption and fermentative capacity. With very low nitrogen needs and one of the lowest fermentative capacities of all non-Saccharomyces strains, combined with a good implantation and growth capacity, it is an excellent candidate for grape must bioprotection.

3.2 Brettanomyces Inhibition Factor Spkt1 mechanism

The Brettanomyces Inhibition Factor Spkt1 produced by LEVEL² SALVA™ is most probably an enzyme that disrupts specifically Brettanomyces bruxellensis cell wall. This hypothesis was confirmed by an experiment (Figure 2), inoculating LEVEL² SALVA™ and a Saccharomyces cerevisiae in a medium with no carbon source but containing laminarin. This laminarin molecule was used to mimic the cell wall of Brettanomyces. Saccharomyces cerevisiae, with no carbon source, cannot grow. With its Brettanomyces Inhibitor factor, LEVEL² SALVA™ was able to hydrolyse the laminarin to use a carbon source and grow on this medium. This shows that the Brettanomyces Inhibition Factor is solely produced by LEVEL² SALVA™.

Mehlomakulu and other authors (2017) performed scanning microscopy to validate the impact of the Brettanomyces Inhibition Factor Spkt1 produced by LEVEL² SALVA™ on Brettanomyces bruxellensis cell wall (Figure 3). After 24 hours of treatment, Brettanomyces strains presented wrinkles, indicating membrane degradation, while untreated cells remained smooth, with an intact membrane. 

4.   LEVEL² SALVA™ bioprotection efficiency against Brettanomyces: validation in grape must at lab scale

LEVEL² SALVA™ was fully characterised regarding several parameters (inoculation rate, ethanol tolerance, SO₂ tolerance, etc.) in our research laboratory in Blagnac, France, to determine the optimal conditions of use and of its Brettanomyces Inhibition Factor production.

Fermentation kinetics revealed that LEVEL² SALVA™ inoculation had no negative impact on fermentation duration.

Compared to a control, LEVEL² SALVA™ was also able to inhibit Brettanomyces bruxellensis and to significantly limit volatile phenol production from 66% to 91% depending on the volatile phenol (Figure 4).

The efficiency and specificity of LEVEL² SALVA™ to inhibit Brettanomyces bruxellensis were compared to two other non-Saccharomyces strains, NS2 and NS4, found on the market, with claims of their bioprotection properties. A negative control without any non-Saccharomyces was also tested, as well as with Brettanomyces bruxellensis alone. All the fermentations reached dryness with volatile acidity <0.3 g H2SO4/L (data not shown).

Brettanomyces bruxellensis was inoculated at 1 x 10³ CFU/mL, and its viability was monitored during fermentations by plating on selective medium (Figure 5). Saccharomyces cerevisiae, as well as the non-Saccharomyces NS2 and NS4, did not efficiently limit Brettanomyces’ growth. LEVEL² SALVA™ was the only yeast able to decrease the Brettanomyces population.

5.   Pilot and winery trials: scaling up

Many trials were conducted at pilot and winery scale on different red grape varieties (Merlot, Cabernet Sauvignon, Syrah, Tempranillo, etc.) in France, Spain, Italy and the United States. LEVEL² SALVA™ was inoculated at different prefermentative stages of the winemaking process, either on the grapes or at tank filling.

5.1 Recurrent grape block contamination

In a winery in Columbia Gorge AVA, WA, USA, recurrent Brettanomyces issues have been identified as coming from grapes grown in a specific block of their vineyard. A trial was done with 50% destemmed bunches of Syrah from this identified block and homogenously filled in 40 hL oak tanks (same provider, same age). As the elevator moved fruit into tank, 4 g/hL of SO₂ were added in both cases. Then, LEVEL² SALVA™ was inoculated in one tank compared to the control tank with no addition. Tanks were not mixed for 24 hours at 14°C, before the Saccharomyces cerevisiae inoculation. Both tanks followed the same winemaking process, and the wines were analysed at the end of the malolactic fermentation (MLF). Wines where LEVEL² SALVA™ was inoculated at early stages showed a very low level of Brettanomyces population was found (Figure 6). 

5.2 Addition of LEVEL² SALVA™ on harvested grapes

LEVEL² SALVA™ can also be applied in earlier stages of wine production, more specifically on the harvested grapes. A trial in Spain was conducted in which LEVEL² SALVA™ was applied by spraying on Tempranillo grapes that had been hand-harvested, compared to grapes without any addition. The same winemaking process was applied for both treatments. At the end of MLF, we observed that LEVEL² SALVA™ was able to efficiently limit the development of Brettanomyces (Figure 7), decreasing its population by a factor of 2.4.

 5.3 Early contamination of Brettanomyces: efficiency of LEVEL² SALVA™

An experiment was conducted in the laboratory of Microbiology of the Fondazione Edmund Mach (San Michele all’Adige, Italy) in a liquid red grape must (Rebo grape variety), inoculated with Brettanomyces at 1 x 10³ CFU/mL. After 12 hours, two modalities were performed (1 L bottle, three replicates for each). The first modality (“Control”) was inoculated with Saccharomyces cerevisiae and fermented at a temperature below 27°C. The second modality was inoculated with LEVEL² SALVA™ for 48 hours before Saccharomyces cerevisiae inoculation. At the end of alcoholic fermentation, both 4-ethylphenol and 4-ethylguaiacol were in lower concentration in the modalities with LEVEL² SALVA™ compared to the control (Figure 8).

5.4 Reduction in SO₂ usage with LEVEL² SALVA™

An experiment was conducted with the Università Degli Studi di Udine on Cabernet Franc. After crushing and destemming, the must with skins was homogeneously split into three 25-L tanks where Brettanomyces was inoculated at a level of 1 x 10³ CFU/mL. A control with no addition was compared to a SO₂ addition of 2.5 g/hL and an addition of LEVEL² SALVA™ (with no SO₂ addition). At the end of the alcoholic fermentation, volatile phenols were analysed. Levels of 4-ethylphenol and 4-ethylguaiacol were higher in the control with neither SO₂ nor LEVEL² SALVA™. In this experiment, LEVEL² SALVA™ was more effective than SO₂ as it reached the lowest level of volatile phenols (Figure 9). 

Another trial was conducted on a Merlot, in Bordeaux, France, comparing 5 g/hL of SO₂ addition to an addition of LEVEL² SALVA™ (with no SO₂ addition), both added at tank’s filling (80 hL stainless steel tanks) before a cold soak (four days at 10°C). A follow-up at different stages (end MLF and five months after) showed the presence of Brettanomyces at the end of MLF and significant growth five months after, during ageing, in the control treated with 5 g/hL of SO₂. No Brettanomyces were detected in the wines with LEVEL² SALVA™, neither after MLF nor five months after. This inhibition of Brettanomyces by LEVEL² SALVA™ was correlated with the absence of volatile phenols in the LEVEL² SALVA™ treatments (Figure 10).

 6.   An integral bioprotection approach to control Brettanomyces

Even at levels below their perception threshold, volatile phenols can negatively impact the sensory profile by reducing the fruit perception, masking the wine identity and its terroir signature. At higher concentrations, it clearly leads to a qualitative depreciation of the wine, with the presence of obvious defaults. Different natural solutions for grapes until the final wine during ageing can help to manage Brettanomyces contaminations. LEVEL² SALVA™ is a specific bioprotection solution that complements existing natural solutions.

In the vineyard, LALVIGNE BOTRYLESS™ (chitosan from Aspergillus niger) applied from post-flowering to veraison showed interesting results to reduce spoilage microorganisms, including Brettanomyces.

Then LEVEL² SALVA™, an innovative natural solution is able to prevent the Brettanomyces contamination from the earliest stages (on grapes, before transport, at reception, etc.) with its production of a specific Brettanomyces Inhibition Factor, disrupting the cell wall of Brettanomyces cells. An early inoculation of a selected Saccharomyces cerevisiae and a well-managed clean alcoholic fermentation will take over to occupy the microbiological space and avoid Brettanomyces development. Inoculation with selected bacteria is also key in the winemaking process. The use of Lactiplantibacilus plantarum ML Prime™ will result in an early stabilisation of the wines, while Oenococus oeni has a direct effect to control Brettanomyces population and prevents the production of volatile phenols. Depending on the feasibility of the winemaking process and the objectives of the winemaker, both co-inoculation and sequential inoculation can be interesting. NO BRETT INSIDE™ (chitosan-based solutions) is also an excellent curative tool in case of Brettanomyces development during ageing. The combination of those natural alternatives to sulfiting offers great natural opportunities to winemakers to manage Brettanomyces contaminations and mitigate their consequences on wine quality, preserving wine terroir signature and identity.

Conclusion

This paper demonstrates the efficacy of the new non-Saccharomyces LEVEL² SALVA™ as a specific bioprotection agent against Brettanomyces bruxellensis in the pre-fermentative stages of wine production. The inhibition mechanism, driven by the Brettanomyces Inhibition Factor Spkt1, effectively disrupts the cell wall of Brettanomyces, significantly decreasing their populations. Laboratory and pilot-scale trials confirm that LEVEL² SALVA™ significantly reduces volatile phenol production, which is crucial for maintaining wine quality. The trials also highlight the versatility of LEVEL² SALVA™, showing its effectiveness when applied at various stages of the winemaking process, including on harvested grapes and during tank filling. Additionally, the reduction in SO₂ usage without compromising wine stability underscores the potential of LEVEL² SALVA™ as a sustainable alternative in winemaking. Overall, LEVEL² SALVA™ offers a robust, natural solution for managing Brettanomyces contamination aligning with the industry’s move towards reduced chemical inputs and enhanced wine quality preservation.

References

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