Wine spoilage yeasts are one of the main issues in the winemaking industry, and the control of the Brettanomyces and Pichia genus is an important goal to reduce economic loses from undesired aromatic profiles. Previous studies have demonstrated that Candida intermedia LAMAP1790 produces antimicrobial peptides of molecular mass under 10 kDa with fungicide activity against Brettanomyces bruxellensis, without affecting the yeast Saccharomyces cerevisiae. So far, it has not been determined whether these peptides show biocontroller effect in this yeast or other spoilage yeasts, such as Pichia guilliermondii. In this work, we determined that the exposure of B. bruxellensis to the low-mass peptides contained in the culture supernatant of C. intermedia LAMAP1790 produces a continuous rise of reactive oxygen species (ROS) in this yeast, without presenting a significant effect on membrane damage. These observations can give an approach to the antifungal mechanism. In addition, we described a fungicide activity of these peptides fraction against two strains of P. guilliermondii in a laboratory medium. However, carrying out assays on synthetic must, peptides must show an effect on the growth of B. bruxellensis. Moreover, these results can be considered as a start to develop new strategies for the biocontrol of spoilage yeast.

Brettanomyces bruxellensis is regarded as the main spoilage microorganism in the wine industry, owing to its production of off-flavours. It is difficult to eradicate owing to its high tolerance of adverse environmental conditions, such as low nutrient availability, low pH, and high levels of ethanol and SO₂. In this study, the production of volatile phenols and the growth kinetics of isolates from various regions of Chile were evaluated under stressful conditions. Through randomly amplified polymorphic DNA (RAPD) analysis, 15 strains were identified. These were grown in the presence of p-coumaric acid, a natural antimicrobial and the main precursor of off-flavours, and molecular sulfur dioxide (mSO₂), an antimicrobial synthetic used in the wine industry. When both compounds were used simultaneously, there were clear signs of an improvement in the fitness of most of the isolates, which showed an antagonistic interaction in which p-coumaric acid mitigates the effects of SO₂. Fourteen strains were able to produce 4-vinylphenol, which showed signs of phenylacrylic acid decarboxylase activity, and most of them produced 4-ethylphenol as a result of active vinylphenol reductase. These results demonstrate for the first time the serious implications of using p-coumaric acid, not only for the production of off-flavours, but also for its protective action against the toxic effects of SO₂.

Fungal nitrogen metabolism is a central determinant of yeast growth, fermentative performance, and secondary metabolism in fungi associated with fermented environments. For Saccharomyces cerevisiae, the assimilation of nitrogen sources found in grape must, including ammonium, free amino acids, and peptides, has been widely characterized, establishing a consumption hierarchy in which proline is considered a non-preferential nitrogenous source. However, the regulation and hierarchy of nitrogen-source utilization in non-conventional fungi remain poorly characterized, particularly in yeasts adapted to anthropized fermentative niches. In this context, Brettanomyces bruxellensis represents a useful fungal model to study the different nitrogen sources that are used, strain-dependent physiology, and survival under nutrient-limited fermentative conditions. We hypothesized that apparent depletion of amino acid and the expression of selected amino acid permease genes are strain-dependent traits linked to the survival of B. bruxellensis. In this study, we evaluated the strain-dependent physiology and amino acid depletion patterns of two B. bruxellensis strains from wine (LAMAP2480 and LAMAP1359). In both cases, the highest apparent depletion values under the 20 amino acid condition were observed for arginine, glutamine, tryptophan and proline, indicating that these compounds contributed substantially to the organic nitrogen pool under the assayed conditions. Only LAMAP2480 isolate was able to grow in the condition without added amino acids, indicating strain and nitrogen source dependence under limited organic nitrogen availability. Transcriptional analysis of GAP1, GNP1, and TAT1 permeases showed expression patterns dependent on both the strain and the available nitrogen source. This is the first study to combine the evaluation of physiological performance, amino acid consumption, apparent nitrogen depletion derived from amino acids normalized to OD₆₀₀ (OD-AA-N depletion), and the transcriptional responses of selected amino acid permease genes. This knowledge may contribute to understanding the survival of B. bruxellensis in fermentation environments with limited nutritional resources.

Brettanomyces bruxellensis has been described as the main spoilage microorganism in wines due to its ability to produce volatile phenols, which negatively impact the final product’s organoleptic properties. This yeast can grow and survive in environments that are too nutritionally poor and stressful for other microorganisms, and one of the stressful conditions it can endure is the high alcohol content in wine. In this study, cell wall morphology and the expression of some genes related to its composition were characterized under increasing ethanol concentrations to establish a possible ethanol resistance mechanism. B. bruxellensis LAMAP2480 showed greater resistance to β-1,3-glucanase activity when grown in media supplemented with 5% or 10% ethanol compared with the control assay (without ethanol). Transmission electron microscopy showed no significant differences in cell wall thickness during the different adaptation stages. However, the amount of wall polysaccharides and chitin briefly increased at 1% ethanol but returned to baseline at 5% and 10%. The amount of wall-associated protein increased progressively with each increment in ethanol concentration. In addition, overexpression of the ROM2 and KNR4/SMI1 genes was observed at 10% ethanol. These results suggest that the integrity of the cell wall might play an important role in the adaptation of B. bruxellensis to an ethanol-containing medium.