Investigating Key Volatile Compound Diffusion in Cocoa Beans during Yeast Fermentation-like Incubation.

An experimental setup was devised to investigate the permeability of cocoa bean seed coat and pulp to key volatile compounds during fermentation. Four labeled compounds (ethyl acetate-d3, ethyl octanoate-d15, 2-phenylethanol-d5, linalool-d5) and 2 unlabeled (beta-damascenone, delta-decalactone) were chosen for the investigation. The beans (cotyledons), depulped beans, or pulped beans were immersed separately in a concentrated solution of these volatile compounds at 36 or 46 °C for durations ranging from 3 to 120 h. The imbibed beans were dissected, and the cotyledons were analyzed by SPME-GC/MS. The diffusion of volatile compounds from the external solution to the seed was categorized into three groups: (1) not diffusible (ethyl octanoate-d15); (2) semidiffusible (ethyl acetate); and (3) totally diffusible (2-phenylethanol-d5, linalool-d5, beta-damascenone, delta-decalactone). The impact of the yeast on volatile compound diffusion was also investigated by immerging the pulped beans into the same concentrated solution with a yeast starter. Results highlighted the positive role of yeast in the diffusion of volatile compounds. The starter positively contributed to volatile compound diffusion after a transition phase occurring at approximately 48 h of fermentation, enriching the cocoa beans with key aromatic volatile compounds.

Thermo-adaptive evolution to generate improved Saccharomyces cerevisiae strains for cocoa pulp fermentations.

Cocoa pulp fermentation is a consequence of the succession of indigenous yeasts, lactic acid bacteria and acetic acid bacteria that not only produce a diversity of metabolites, but also cause the production of flavour precursors. However, as such spontaneous fermentations are less reproducible and contribute to produce variability, interest in a microbial starter culture is growing that could be used to inoculate cocoa pulp fermentations. This study aimed to generate robust S. cerevisiae strains by thermo-adaptive evolution that could be used in cocoa fermentation. We evolved a cocoa strain in a sugary defined medium at high temperature to improve both fermentation and growth capacity. Moreover, adaptive evolution at high temperature (40 °C) also enabled us to unveil the molecular basis underlying the improved phenotype by analysing the whole genome sequence of the evolved strain. Adaptation to high-temperature conditions occurred at different genomic levels, and promoted aneuploidies, segmental duplication, and SNVs in the evolved strain. The lipid profile analysis of the evolved strain also evidenced changes in the membrane composition that contribute to maintain an appropriate cell membrane state at high temperature. Our work demonstrates that experimental evolution is an effective approach to generate better-adapted yeast strains at high temperature for industrial processes.