Inoculation strategy-driven changes in volatiles and organoleptic properties of cider: Insights from GC-MS, GC-IMS, and multivariate statistical models

This study investigated the effects of co-fermentation with two non- Saccharomyces yeasts ( Pichia kluyveri X31-10 and Torulaspora delbrueckii X28-10) on cider flavor profile and aroma complexity using simultaneous and sequential inoculation strategies. Pure cultures of each strain were used as references. The two strains exhibited inoculation-order-dependent antagonistic interactions in growth dynamics, and mixed fermentations yielded higher final ethanol concentrations compared to the P. kluyveri pure culture. A total of 65 and 76 volatile compounds were identified by GC-MS and GC-IMS, respectively, with 12 compounds common to both methods. Esters and alcohols were the predominant classes. Based on odor activity values (OAVs) and partial least squares discriminant analysis (PLS-DA), isoamyl acetate and ethyl acetate were identified as key compounds discriminating between inoculation strategies. Partial least squares regression (PLSR) suggested a positive association between most volatile compound classes (excluding higher alcohols) and fruity and floral aroma attributes. These findings highlighted the potential of co-inoculation with P. kluyveri and T. delbrueckii as a strategy for tailoring cider aroma and enhancing sensory appeal, with implications for cider fermentation innovation. • Inoculation-order-dependent antagonistic interactions occurred between two yeasts • Early involvement of P. kluyveri contributed to enhanced fruity and floral aromas • Isoamyl acetate and ethyl acetate discriminated among inoculation strategies • Most volatile classes were positively associated with fruity and floral attributes