South Africa Wine Technical Yearbook 2025

bruxellensis cell wall. This hypothesis was confirmed by an experiment (Figure 2), inoculating LEVEL 2 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, LEVEL 2 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 LEVEL 2 SALVA™. Mehlomakulu and other authors (20170 performed scanning microscopy to validate the impact of the Brettanomyces Inhibition Factor Spkt1 produced by LEVEL 2 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. LEVEL 2 SALVA™ bioprotection efficiency against Brettanomyces : validation in grape must at lab scale LEVEL 2 SALVA™ was fully characterised regarding several parameters (inoculation rate, ethanol tolerance, SO 2 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 LEVEL 2 SALVA™ inoculation had no negative impact on fermentation duration. Compared to a control, LEVEL 2 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).

FIGURE 2. Saccharomyces cerevisiae A (blue) and LEVEL 2 SALVA™ (green) growth measured by OD600 in minimum media with laminarin.

FIGURE 3. Scanning microscopy of Brettanomyces bruxellensis (a) control; (b) 24 hours after exposure to the Brettanomyces Inhibition Factor produced by LEVEL 2 SALVA™. (Mehlomakulu et al. , 2017). (Images used with permission from Oxford Press.)

3.1 LEVEL 2 SALVA™ characterisation LEVEL 2 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 LEVEL 2 SALVA™ is most probably an enzyme that disrupts specifically Brettanomyces

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, LEVEL 2 SALVA™ inhibits Brettanomyces bruxellensis without impacting on Saccharomyces cerevisiae growth. This experiment confirms the efficiency of LEVEL 2 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 .

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TECHNICAL YEARBOOK 2025