Molecular identification of lactic acid bacteria involved in Ghanaian cocoa bean fermentation

Cocoa is a vital cash crop in Ghana, contributing approximately 15% of the country’s foreign exchange earnings and employing over 800,000 farmers. Fermentation significantly influences cocoa quality and flavour, yet controlling this process remains a challenge. This study investigated lactic acid bacteria (LAB) involved in Ghanaian cocoa fermentation by analyzing samples from the Eastern and Western North Regions. A combination of culture-dependent methods, 16S rRNA gene sequencing (Sanger platform), and phylogenetic analysis was used to identify and characterize LAB diversity. Bioinformatics tools, including BLASTN (NCBI) and R software (v4.4.0), were employed for taxonomy assignment and visualization, while phylogenetic analysis was performed using PhyML (v3.0) and iTOL. Three LAB species were identified: Limosilactobacillus fermentum, Lactiplantibacillus plantarum, and Lentilactobacillus hilgardii. L. fermentum dominated the fermentation process, forming two distinct phylogenetic clusters, suggesting genetic diversity. Temperature and pH analysis revealed similar trends across regions, with peak fermentation temperatures (47.1°C and 45.4°C) coinciding with the highest pH values (5.19 and 5.5) at 72 hours. Notably, L. plantarum was isolated at 72 hours, a stage characterized by a declining pulp pH (4.9) and a heap temperature of 45.4°C, suggesting its adaptation to intermediate fermentation conditions when acidification intensifies, and microbial succession progresses. Statistical analysis showed no significant regional differences in temperature (p > 0.05), but fermentation time significantly influenced both temperature and pH dynamics (p < 0.05). These findings enhance the understanding of LAB and physicochemical dynamics in cocoa fermentation, offering insights for optimizing fermentation processes to improve cocoa quality and chocolate flavour.