Screening, fermentation performance and biotransformation potential of maltose-negative yeasts for aroma enhancement in non-alcoholic beer production.

The global non-alcoholic beer (NAB) market is experiencing unprecedented growth due to a general trend in reduced alcohol consumption. Despite this growth, NABs often do not match their alcoholic counterparts in sensory quality, particularly in aroma complexity and "worty" notes. Yeasts can contribute to aroma not only by producing ethanol, esters, and higher alcohols, but also through biotransformation of bound hop- and malt-derived precursors, releasing low-threshold volatile thiols such as 3-mercaptohexan-1-ol (3MH), 3-sulfanyl-4-methylpentan-1-ol (3S4MP) and 4-mercapto-4-methylpentan-2-one (4MMP), which can impart tropical, guava, grapefruit and blackcurrant notes. Here, we screened 86 yeasts, including non-Saccharomyces species, via a multi-step screening strategy combining micro fermentation-based assessment of maltose utilisation, inability to produce volatile phenolic off-flavours via decarboxylation, biotransformation potential (assessed qualitatively via growth on cysteine as sole nitrogen source, indicative of β-lyase activity), and aromatic potential. Nine strains meeting these criteria were applied in wort fermentation trials. Strain selection to pilot-scale trials was determined by sensory analysis and chemical analysis, including direct quantification of thiol release via GC-MS/QQQ. Of these, Pichia membranifaciens and Saccharomycodes ludwigii were identified as most suitable for non-alcoholic IPA-style beer production, exhibiting enhanced fruity and floral aromas relative to a commercial maltose-negative Saccharomyces cerevisiae strain. In two pilot-scale trials using different hop varieties, and in one trial with the addition of thiol precursors, GC-MS/QQQ analyses combined with sensory evaluation showed that yeast strain selection and precursor supplementation both enhanced hop-derived aroma expression, particularly when dry hopping during active fermentation. Direct quantification confirmed that precursor supplementation increased the wort bound-thiol precursor concentration up to 2.3-fold, supporting precursor availability as a key driver of thiol release. These findings demonstrate that targeted, low-cost micro fermentation-based yeast screening, combined with hop and precursor management, provides a practical route to producing aromatic, hop-forward non-alcoholic beers with enhanced tropical and fruity character.