Potential of Neurospora Sitophila As a Starter Culture in Cocoa Fermentation: Effects on Fermentative Dynamics, Bioactive Compounds, and Structural Properties of the Cocoa Bean and Cocoa Liquor

Cocoa fermentation is a critical determinant of the physicochemical and functional quality of cocoa derivatives, yet filamentous fungi remain an underexplored option as starter cultures. This study evaluated the potential of Neurospora sitophila as a fungal starter culture for cocoa fermentation, assessing its effects on fermentative dynamics, bioactive compounds, and structural properties of cocoa beans and liquor across three inoculum concentrations (5 × 103, 5 × 104, and 5 × 105 spores/mL) and a non-inoculated control. Physicochemical, colorimetric, chromatographic (HPLC), and spectroscopic (FTIR) analyses were performed throughout fermentation, drying, roasting, and liquor production, while thermal properties (DSC) were assessed in the resulting liquor. Fermentation time, rather than inoculum concentration, was the principal factor associated with pH decline (∼6.0 to ∼4.9–5.0), fermentation index progression, colorimetric changes, and bioactive compound dynamics, as indicated by multivariate (PCA, HCA) and FTIR analyses. N. sitophila inoculation produced compound-, parameter-, and stage-specific effects without a uniform dose-dependent pattern. NS5 showed the most pronounced spectral changes during fermentation, greater retention of epicatechin, catechin, theobromine, and caffeine in the liquor, and thermal behavior within the range commonly associated with the β(V) cocoa butter polymorph. Treatment effects on color were limited relative to processing stage, and no treatment performed consistently better across all parameters. Overall, N. sitophila shows potential as a fungal starter culture, but further studies incorporating microbiological monitoring, shorter fermentation times, sensory evaluation, and formal optimization are needed before establishing technological application.