Sequential co-fermentation of Pichia kluyveri and Saccharomyces cerevisiae enhances volatile complexity while retaining fermentation efficiency in wort

Non- Saccharomyces yeasts offer a natural route to enhance beer aroma diversity. We evaluated the fermentation performance and volatile profiles of a novel wild isolate, Pichia kluyveri TP1, and Saccharomyces cerevisiae US-05 in brewer’s wort. Molecular identification (ITS and 26S rDNA) confirmed the isolate as P. kluyveri , while its inability to grow at 37°C support its biological safety for food application. In monoculture, P. kluyveri TP1 exhibited rapid initial growth but failed to utilize maltose and sucrose, limiting ethanol yield to ∼0.42% alcohol by volume (ABV). Conversely, S. cerevisiae US-05 reached ∼3.83% ABV. A sequential strategy, inoculating P. kluyveri 48 h prior to US-05, harnessed early-stage ester production while retaining fermentation efficiency. This efficiency was evidenced by a final alcohol content (3.75% ABV) and residual sugar level (14.60 g/L) statistically comparable to the US-05 monoculture. HS-SPME–GC-MS identified 15 key volatiles. Sequential fermentation significantly increased acetate esters, specifically isoamyl acetate and phenylethyl acetate, with relative odor activity values (rVOA) of 4.61 and 2.45, respectively. Principal component analysis confirmed that co-fermented beers occupied a distinct, synergistic aroma space. These findings suggest P. kluyveri TP1 serves as an effective, flavor adjunct, enhancing aromatic complexity without sacrificing attenuation or production stability.