One of the non-Saccharomyces yeasts studied is Metschnikowia pulcherrima. This article will focus on the antagonistic activity of a specific strain, M. pulcherrima LEVEL2 Guardia™, on other wine yeast species for bioprotection applications.
More and more winemakers are reducing their use of sulphites in wine in order to respond to consumer demands. Alternative biological solutions to control microbial contamination, while reducing the use of SO2, have recently been developed as bioprotection. One of the principles of bioprotection is based on the management of detrimental microbial populations more than their eradication. Moreover, having an alternative such as microbiological bioprotection can be an interesting option, especially in the context of global warming where the increase in pH renders SO2 less efficient.
With the continuous interest in the selection of new Saccharomyces cerevisiae and Oenoccocus oeni strains, particular attention has been on the selection of non-Saccharomyces species/strains for, amongst other things, the natural bioprotection abilities against spoilage yeasts or bacteria.
LEVEL2 Guardia™ – powerful antimicrobial action in red wines
LEVEL2 Guardia™ is the latest Metschnikowia pulcherrima yeast in the Lallemand portfolio. It was selected by the Institut Français de la Vigne et du Vin in Burgundy, France, for its suitable properties during the pre-fermentative steps in red winemaking, as well as its high ability to control other contaminating microorganisms.
In wine must, LEVEL2 Guardia™ can implement itself very efficiently and multiply, and by doing so, occupy the must environment to displace other species, even at low temperatures. As shown in Figure 1, a Pinot noir 2020 (IFV Beaune, Burgundy, France), LEVEL2 Guardia™ was able to multiply during a cold soak of five days at 10°C. Consequently, at the end of this pre-fermentative step a reduction of the spoilage yeast Hanseniaspora uvarum and other contaminating yeasts, in comparison with a control with SO2 addition, was seen.
FIGURE 1. Yeast count after a five day cold soak at 10°C in a Pinot noir (IFV Beaune, France, 2020). Trial comparing LEVEL2 Guardia™ added at 10 g/hL to a control with SO2 addition at 2.5 g/100 kg.
Another trial on a Grenache 2020 (INCAVI, Spain) also illustrates the good implantation of LEVEL2 Guardia™ at low temperature, as well as its high antimicrobial action against different microbial populations. As with the previous trial, LEVEL2 Guardia™ inoculation was measured against SO2 addition during cold soak of five days at 10°C. Results during the cold soak showed a good implantation of LEVEL2 Guardia™ and other contaminating species, such as Hanseniaspora numbers, were significantly reduced (Figure 2). Both tanks were then inoculated with the same Saccharomyces cerevisiae. Volatile acidity measured at the end of the alcoholic fermentation was significantly lower for the bioprotected wine (Figure 3).
FIGURE 2. Implantation control done during a five day cold soak at 10°C in a Grenache (INCAVI, Spain, 2020). Trial comparing LEVEL2 Guardia™ added at 10 g/hL to a control without bioprotection. No sulphites added in both cases.
FIGURE 3. Volatile acidity in Grenache wines (INCAVI, Spain, 2020). Trial comparing LEVEL2 Guardia™ added at 10 g/hL to a control without bioprotection. No sulphites added in both cases.
Why is LEVEL2 Guardia™ such a powerful bioprotection agent?
Metschnikowia pulcherrima is an interesting microorganism found in the must flora. As with Saccharomyces cerevisiae, within the specie, there are many different strains behaving differently from one another, hence the importance of selecting the right yeast for a specific application.
The mechanism of action, quite unique to this strain of M. pulcherrima, is its ability to secrete pulcherimmic acid. Pulcherimmic acid is a natural acid with no sensory impact, produced by some yeast species, especially M. pulcherrima who possesses the genes (PUL1, PUL2, PUL4, snf2) which enables its synthesis. When pulcherrimic acid is produced by the yeast, once excreted into the media, it will have a strong affinity for the free iron and subsequently chelate it (Figure 4).
Pulcherrimin is then formed. The iron present in the must is depleted and the growth of contaminating species (for example, Hanseniaspora, etc.) will be reduced as free iron is a necessary element for their growth. Figure 5 shows the different free and total iron concentration in a must where different M. pulcherrima strains, among which LEVEL2 Guardia™ and a selected Saccharomyces cerevisiae strain, were used.
FIGURE 4. Pulcherrimin biosynthesis of M. pulcherrima and its iron scavenging ability (Sipiczki, 2020).
FIGURE 5. Free and total iron concentration in must with different M. pulcherrima strains and a S. cerevisiae.
The positive association of LEVEL2 Guardia™ and Saccharomyces cerevisiae wine yeast
While LEVEL2 Guardia™ is exceedingly efficient at chelating free iron from the must environment and thus reduce the growth of other yeast species, it could be assumed that it can also affect the growth of the essential S. cerevisiae needed to complete the fermentation. However, the wine yeast S. cerevisiae has the ability to scavenge back the iron bound to pulcherrimic acid and use it for its metabolic functions. Thanks to the presence of the PUL3 and PUL4 genes within its genome (Krause et al., 2018), selected wine yeast S. cerevisiae can be inoculated following the use of LEVEL2 Guardia™.
Moreover, the implantation of the selected S. cerevisiae was shown to be even more efficient when LEVEL2 Guardia™ has been used prior to fermentation as shown in Figure 6, probably because of the strong limitation of contaminant flora.
FIGURE 6. Implantation control done halfway through alcoholic fermentation in a Grenache (INCAVI, Spain, 2020). Trial comparing LEVEL2 Guardia™ added at 10 g/hL to a control without bioprotection. No sulphites added in both cases.
During pre-fermentation, the must is susceptible to the development of undesirable microorganisms and protection of the must is necessary to avoid sensory deviation right at the onset of the winemaking process. The use of LEVEL2 Guardia™, for example during cold soak of red grapes, is an efficient alternative to SO2 to control a wide range of contaminants.
Contributing researchers: Anne Ortiz-Julien1, Anthony Silvano1, José Maria Heras2, Ann Dumont1, Marion Bastien1
[(1) Lallemand SAS – Blagnac, France, (2) Lallemand Bio, Spain.]
Note: Article previously published in Revue des Oenologues.
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