β-glycosidase activity associated with the formation of aroma compounds in native non-Saccharomyces yeasts isolated from cocoa bean fermentation

Description of the subject. The use of non-Saccharomyces yeast for improving fine cocoa flavor perception has been poorly explored. Objectives. In this study, we investigated the contribution of the non-Saccharomyces yeasts producing β-glycosidase isolated from the spontaneous fermentation of cocoa to the quality and production of aromas during the alcoholic fermentation of a medium based on cocoa pulp. Method. The screening for β-glycosidase activity and molecular identification of non-Saccharomyces yeast isolates from Amazonian cocoa bean fermentations were performed on agar plates with esculin as a substrate and by 5.8S ITS rDNA sequence analysis, respectively. The yeasts producing β-glycosidases were used as a starter culture for alcoholic micro-fermentations of a medium based on cocoa pulp, incubated at 30 °C for 48 h. The quantification of organic acids, ethanol, and sugars was obtained by HPLC, and aroma compounds after cocoa pulp fermentation were identified by GC-FID and GC-MS. Results. Twenty-six non-Saccharomyces yeast isolates were selected by β-glycosidase activity, comprising species such as Pichia kudriavzevii (n = 15), Pichia sp. (n = 2), Pichia sporocuriosa (n = 1), Candida orthopsilosis (n = 3), Issatchenkia sp. (n = 1), I. orientalis (n = 1), and P. kudriavzevii/I. orientalis (n = 3). All yeast strains exhibited rapid growth in cocoa pulp medium, low ethanol production, high organic acid production, and higher glucose consume than fructose. These yeasts produced thirty different volatile compounds. The main groups included alcohols (3), esters (19), terpenes (4), phenols (2), organic acids (2), and aldehydes (3), with 67% of these having fruity and floral aromas. Pichia sp., P. kudriavzevii/I. orientalis and Issatchenckia sp. were the largest producers of volatile compounds. Notably, this study identified volatile compounds with fruity aromas previously unassociated with cocoa fermentations for the first time. Conclusions. These data demonstrate the potential of β-glycosidase-producing non-Saccharomyces yeasts to improve floral and fruity aromas, suggesting their promising use as a starter culture for cocoa fermentation.