Harnessing Pectinase-Producing Microorganisms from Cocoa (Theobroma cacao L.) Fermentation: Isolation, Characterization, and Prospects for Starter Culture Development

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Cocoa bean quality is strongly influenced by microbial fermentation, which drives flavour development through enzymatic activity. Despite its importance, the specific microorganisms, particularly those producing pectinase, remain poorly characterized. This study aimed to identify and evaluate pectinase-producing microbes from fermenting cocoa mass and assess their impact on fermentation performance. In the first experiment, 9 bacterial and 14 yeast strains were isolated and screened on pectinase screening agar. Three yeast strains (FF1D3, FF2D1, and NA) showed high pectinolytic activity. FF1D3 and FF2D1 were identified as Pichia kudriavzevii, whereas NA showed only a low-confidence closest BLAST match to Candida orthopsilosis and was therefore not advanced as a starter culture candidate. These strains had the highest polygalacturonase activity at 24–48 h and pectin lyase activity at 48–72 h. In the second experiment, inoculating cocoa beans with these strains significantly enhanced fermentation kinetics, with yeast populations reaching log109, indicating enhanced fermentation via accelerated sugar utilization. Brix values (1.97–3.11) and pH reduction (p < 0.01) confirmed active microbial metabolism and effective acidification. Phenolic content varied significantly (p < 0.01), with FF2D1 and combined treatments showing elevated levels due to strain-dependent enzymatic hydrolysis. The combined starter culture showed potential to improve fermentation consistency and accelerate sugar utilization. These findings highlight the potential of targeted microbial inoculation to optimise cocoa fermentation and quality.