The development of non-alcoholic beer (NAB) with health benefits, using non-conventional potential probiotic yeasts, offers an interesting alternative to standard NAB brewing strategies. In this study, potential probiotic non-Saccharomyces yeasts Pichia manshurica, Kluyveromyces lactis, and Kluyveromyces marxianus, along with commercial probiotic yeast Saccharomyces boulardii, were characterised and tested for functional NAB production, whereas P. manshurica was used in NAB production for the first time. Growth and viability were assessed across a range of temperatures, pH, and iso-α-bitter acids. The tested yeasts withstood conditions typical of the beer matrix and human digestive tract and had a positive phenolic off-flavour phenotype. Two strains, K. lactis and K. marxianus, showed strong β-glucosidase activity, which may enhance beverage aroma complexity. Ethanol levels in beers fermented with non-Saccharomyces yeasts remained below the NAB limit (≤0.5% v/v). An analysis of volatile organic compound profiles revealed the potential of these yeasts to produce higher alcohols and esters valuable from a brewer’s perspective. This study provides valuable insight into novel probiotic fermentations and the potential application of unconventional yeasts in functional, aromatic, and health-oriented non-alcoholic beverages.

The development of innovative non-alcoholic beer (NAB) with health benefits, with the use of non-conventional potential probiotic yeasts offers an interesting alternative to standard NAB brewing strategies. In this study, potential probiotic non-Saccharomyces yeasts Pichia manshurica, Kluyveromyces lactis, and Kluyveromyces marxianus, along with commercial probiotic yeast Saccharomyces boulardii, were characterized and tested for functional NAB production, whereas P. manshurica was used in NAB production for the first time. Growth and viability were assessed across a range of temperatures, pH, and iso-α-bitter acids. The tested yeasts withstood conditions typical of the beer matrix and human digestive tract and had positive phenolic off-flavour phenotype. Two strains, K. lactis and K. marxianus, showed strong ß-glucosidase activity, which may enhance beverage aroma complexity. Ethanol levels in beers fermented with non-Saccharomyces yeasts remained below the NAB limit (≤0.5% v/v). Analysis of volatile organic compound profiles revealed the potential of these yeasts to produce higher alcohols and esters valuable from a brewer’s perspective. Sensorial evaluation of prepared NAB produced with potential probiotic non-Saccharomyces yeasts revealed floral and clove-like aroma traces. This study provides valuable insight into novel probiotic fermentations and the potential application of unconventional yeasts in functional, aromatic, and health-oriented non-alcoholic beverages.