This paper explores the broad probiotic and functional properties of two non-Saccharomyces strains (MI120 and MI125) and one Saccharomyces cerevisiae BB06 strain (as a reference probiotic). Torulaspora delbrueckii MI120 and Starmerella bacillaris MI125 were identified via 5.8S rDNA sequencing. All the strains survived well in simulated gastrointestinal conditions and had strong antioxidant activity (>68%). S. bacillaris MI125 excelled in antimicrobial activity against Gram-positive and Gram-negative bacteria. S. bacillaris MI125 and Sacch. cerevisiae BB06 resisted all the tested antibiotics. No strain displayed hemolytic behavior. The freeze-dried yeast strains achieved survival rates between 76.62% and 93.38%. Based on our physiological characterization analysis (carbon assimilation, ethanol tolerance, acetic acid and H₂S production, temperature and low pH tolerance, enzymatic pattern, and killer phenotype), all the strains showed interesting attributes, with Sacch. cerevisiae BB06 fermenting vigorously in malt extract medium. Beer fermented with T. delbrueckii MI120 had the highest phenolic content (96.02 μg GAE·mL⁻¹) and antioxidant activity (90.43%), matching commercial Sacch. cerevisiae US-05 in sensory traits such as taste and aroma. However, after two months in bottled beer, the yeast viability decreased to 2–3 log CFU·mL⁻¹. The pilot brewing and the assessments of the strains’ technological, physico-chemical, and sensorial properties confirmed their suitability for industrial brewing. Overall, T. delbrueckii MI120 emerges as a promising brewing strain, and S. bacillaris MI125 is a potential probiotic.

The industrial use of non-Saccharomyces yeasts offers a robust bioprocess strategy for modulating volatile biochemical profiles and reducing climate-driven ethanol production in fermented beverages. This study explores the metabolic diversity of indigenous non-Saccharomyces, with a particular focus on Starmerella bacillaris MI151, selected for sequential inoculation with Saccharomyces cerevisiae MI118. Pilot-scale bioreactor fermentations (25 L) of two Romanian matrix-specific aromatic grape cultivars, Busuioacă de Bohotin and Tămâioasă Românească, exhibited clear strain-dependent carbon flux changes. The sequential culture actively triggered the glycerol-pyruvic pathway, which consistently reduced the final ethanol concentration to up to 0.94% (v/v) and maintained a balanced profile of volatile compounds. GC-MS-based metabolomic profiling revealed substantial changes in the esterification kinetics and glycosidic precursor cleavage. The sequential fermentation resulted in a synergistic increase in n-hexyl acetate (up to 2310.01 µg/L) and bypassing of the standard enzymatic repression, freeing highly volatile monoterpenes (β-citronellol and β-geraniol) in the analyzed matrices. This targeted microbial system successfully modulated the organic acid–phenolic balance, neutralizing harsh structural finishes, as confirmed by quantitative sensory mapping. Finally, these specific local yeast strains exhibit strong bioprocess scalability, offering a predictable, non-engineered strategy to lower the ethanol yield while driving targeted flavor enhancement in climate-vulnerable viticultural regions.