Molecular Characterization of Microorganisms, Pectinolytic Potential and Volatile Compound Formation During Spontaneous Fermentation of Criollo Cacao (Theobroma cacao L.)

Spontaneous fermentation of Criollo cacao involves temporal changes in culturable microorganisms and in the volatile compound profile, with potential implications for the development of aroma-related characteristics. This study aimed to characterize culturable microorganisms, assess their apparent pectinolytic potential, and evaluate the temporal dynamics of volatile compounds during spontaneous fermentation of Criollo cacao from the Lagunas rural community, Amazonas, Peru. Microorganisms were isolated at different fermentation stages, and pectinolytic potential was evaluated by screening for pectin-degradation halos on a mineral medium supplemented with pectin. Selected isolates were molecularly identified by sequencing the ITS region for yeasts and the 16S rRNA gene for bacteria. Volatile compounds were analyzed by headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME/GC-MS). All isolates evaluated in the pectin assay produced visible degradation halos, although halo diameter and area varied among isolates, indicating differences in apparent pectinolytic potential. Molecular characterization showed frequent recovery of Pichia kudriavzevii, Saccharomyces cerevisiae, and Hanseniaspora opuntiae among yeasts, whereas Acetobacter species were frequently recovered among bacteria. Volatile profiling revealed temporal changes in the relative abundance of alcohols, esters, aldehydes, ketones, terpenes, acids, and other compound families. Exploratory Spearman correlation analysis identified several strong temporal associations between microbial recovery frequency and VOC families, particularly involving P. kudriavzevii, Acetobacter pasteurianus, A. tropicalis, and Bacillus subtilis. However, these associations did not remain significant after correction for multiple comparisons. Overall, the integrated culture-dependent, molecular, pectinolytic screening, and volatile profiling approach provides preliminary evidence of coordinated temporal patterns between culturable microorganisms and volatile compounds during spontaneous Criollo cacao fermentation and identifies candidate isolates for further functional evaluation.