In recent years, the frequency of occurrence of mousy off-flavours in wines has increased. This could be caused by the significant decrease in sulphur dioxide addition during processing, the increase in pH or even the trend for spontaneous fermentation in wine. This off-flavour was associated with Brettanomyces bruxellensis or lactic acid bacteria metabolisms. Three N-heterocyclic compounds (APY, ETHP, ATHP) were described as involved in mousiness perception. Thus far, no study addressed the variability in that N-heterocycles production according to microorganism strains from different species. Twenty-five wines presenting mousy off-flavour were analysed. In total, 252 bacteria with 90.5 % of Oenococcus oeni and 101 yeast strains with 53.5 % of Saccharomyces cerevisiae were isolated and identified. Their capacity to produce mousy compounds was investigated using Stir Bar Sorptive Extraction-Gas Chromatography-Mass Spectrometry (SBSE-GC-MS) and a standardised N-heterocycle assay medium. While four and three species of yeast and bacteria, respectively, were isolated from mousy wines, only three species of microorganisms were associated with N-heterocycles production: B. bruxellensis, Lentilactobacillus hilgardii and Oenococcus oeni. The screening was then extended to collection strains for these three species to improve their genetic representativity. Our results show that the levels and the ratios of the three N-heterocycles present huge variations according to the species. In addition, it has been shown that in most mousy wines, B. bruxellensis was not found. Finally, an interesting correlation between ATHP and ETHP was identified.

The use of food preservatives is controversial these days, and sulphur dioxide (SO2) is no exception. Microbial communities have been studied particularly during the prefermentative and fermentative stages in the absence of added SO2. However, the microbial risks associated with the reduction or absence of SO2, particularly during the wine ageing process, have been little studied. The microbiological control of wine ageing is a key issue for winemakers wishing to produce high-quality wines without added SO2. The present study aims to evaluate the impact of different wine ageing strategies, depending on whether or not SO2 is added, on microbial population levels and diversity. Sulphite management had minimal impact on the population levels of yeast and acetic acid bacteria. Conversely, the population of lactic acid bacteria, whether from inoculation or spontaneous malolactic fermentation (MLF), was significantly impacted by SO2 management. High and stable population levels of around 105 CFU/mL were observed in the SO2-free modalities. Sulphiting after malolactic fermentation has a greater effect on lactic acid bacteria up to four months of ageing than standard SO2 addition at the time of vatting. Oenococcus oeni was the only species detected during ageing using a culturable approach. After five months of ageing, a high genetic diversity of Oenococcus oeni was highlighted in all modalities, even in the inoculated one with a commercial strain. The high population level of Oenococcus oeni was associated with acetic acid and D-lactic acid production, as well as mousy off-flavours. One sulfite treatment at the end of MLF inhibits the growth of Oenococcus oeni, but only temporarily. Winemakers should consider the stage and number of treatments to limit the development of lactic acid bacteria (LAB) during ageing and preserve the quality of red wine.

The presence of mousy off-flavours in wine, attributed to the production of N-heterocycles by lactic acid bacteria or Brettanomyces bruxellensis, poses a significant challenge in the winemaking industry. This study is the first one to investigate the impact of co-cultures focusing on B. bruxellensis with two common wine microorganisms, Pediococcus parvulus and Saccharomyces cerevisiae on the formation of mousy compounds. The screening was conducted in a synthetic medium under controlled conditions. It reveals the nuanced effects of the microorganism combinations. Depending on the P. parvulus or S. cerevisiae strain co-inoculated with B. bruxellensis a synergistic effect is observed on the 2-acetyltetrahydropyridine (ATHP) production, while an inhibitory effect on 2-acetylpyrroline (APY) production by P. parvulus is highlighted when co-inoculated with B. bruxellensis. These findings shed light on the potential impact of the alcoholic fermentation yeast (S. cerevisiae) on the production of mousy off-flavours. Finally, a correlation between ATHP and 2-ethyltetrahydropyridine (ETHP) appears to be confirmed with a ratio of 1:10 between ETHP and ATHP produced in the N-heterocycles assay medium.

Brettanomyces bruxellensis is a yeast that causes spoilage in red wines due to its ability to produce volatile phenols, compounds associated with major sensory defects. Specific monitoring of low populations of this species in complex ecosystems such as wine during fermentation or aging often relies on plating onto selective media supplemented with cycloheximide. However, the variability of B. bruxellensis sensitivity to this antibiotic needs to be better characterized. A collection of 175 B. bruxellensis strains was, thus, grown on YPD medium supplemented with increasing concentrations of cycloheximide (0 to 0.5 g.L⁻¹), and yeast development was monitored for 20 days by image analysis. This study revealed significant inter-strain variability, with some strains showing very late or even no growth at high cycloheximide concentrations. The cycloheximide inhibitory effect was also dose- and population-dependent. In addition, colony size was frequently reduced at high doses. Additional tests were conducted on a subset of strains grown in wines with either low pH or high alcohol content or containing sulfur dioxide and then plated in the presence of increasing concentrations of cycloheximide. This revealed a cumulative effect of wine and cycloheximide stresses that resulted in an even higher delay in yeast detection. The results confirm the huge phenotypic diversity of the species and highlight the need to adapt the plates’ incubation time, particularly when the selectivity and the doses of cycloheximide needed are high (samples taken in pre-fermentation phases or during fermentation) or in case of stressful wine analysis, to minimize the risk of false negatives.