Detailed Analysis of the Microbial Population in Malaysian Spontaneous Cocoa Pulp Fermentations Reveals a Core and Variable Microbiota

Abstract

The fermentation of cocoa pulp is one of the few remaining large-scale spontaneous microbial processes in today's food industry. The microbiota involved in cocoa pulp fermentations is complex and variable, which leads to inconsistent production efficiency and cocoa quality. Despite intensive research in the field, a detailed and comprehensive analysis of the microbiota is still lacking, especially for the expanding Asian production region. Here, we report a large-scale, comprehensive analysis of four spontaneous Malaysian cocoa pulp fermentations across two time points in the harvest season and two fermentation methods. Our results show that the cocoa microbiota consists of a “core” and a “variable” part. The bacterial populations show a remarkable consistency, with only two dominant species, Lactobacillus fermentum and Acetobacter pasteurianus. The fungal diversity is much larger, with four dominant species occurring in all fermentations (“core” yeasts), and a large number of yeasts that only occur in lower numbers and specific fermentations (“variable” yeasts). Despite this diversity, a clear pattern emerges, with early dominance of apiculate yeasts and late dominance of Saccharomyces cerevisiae. Our results provide new insights into the microbial diversity in Malaysian cocoa pulp fermentations and pave the way for the selection of starter cultures to increase efficiency and consistency.

Tuning Chocolate Flavor through Development of Thermotolerant Saccharomyces cerevisiae Starter Cultures with Increased Acetate Ester Production

“…The suspension was powerfully shaken and manually kneaded for 5 min, and a 10-fold serial dilution in 0.1% peptone water was made of the homogeneous cocoa pulp solution. Next, 100 ml from each dilution was spread over three different selective agar plates, de Man-Rogosa-Sharpe medium (MRS) supplemented with 0.01% cycloheximide for LAB (30°C, 2 days), acetic acid medium (AAM) (42°C, 4 days) with 0.01% cycloheximide for AAB, and 2% YPD (30°C, 2 days) supplemented with 0.01% chloramphenicol for yeast growth (14).…”

“…For bacteria, six LAB and three AAB isolates were randomly picked from the countable plates for all time points. Sixteen (at the onset of the fermentation, when the largest yeast diversity was expected [14]) to 10 (at other time points) random yeast isolates per time point were taken to analyze the population dynamics. In total, 645 LAB, 274 AAB, and 1,148 yeast isolates were isolated.…”

“…In total, 645 LAB, 274 AAB, and 1,148 yeast isolates were isolated. All isolates were identified in Belgium to the species level by fingerprinting and rRNA sequencing, as described previously (14,29). Concisely, viable bacterial isolates were identified using a polyphasic approach, starting with a phenotypic characterization (catalase and oxidase activity).…”

“…These precursors will act as the substrate for Strecker degradation and Maillard reactions during roasting and conching (4). Yeasts are present throughout the fermentation process, with maximal population sizes reached in the middle of the process (12)(13)(14)(15). Apart from the primary metabolites ethanol and CO 2 , yeast cells produce numerous secondary flavor-active metabolites, including aldehydes, carbonyl compounds, esters, fatty acids, higher alcohols, organic acids, phenols, and sulfur-containing compounds (16).…”

Preliminary Analysis of Sugar Supplementation on Alcoholic Fermentation by <i>Meyerozyma guilliermondii</i>

“…Several studies geared towards their industrial applications, response to different stress conditions and genetic modification attempts via knowledge of its complete genome sequence, makes Saccharomyces cerevisiae the most prominent specie known to man [2], [3] [4]. S. cerevisiae has been widely employed in the brewing sector due to its ease of converting sugar-rich substrates into ethanol coupled with its unique tolerance to the end-product Fermentation by Meyerozyma guilliermondii and other inhibitory metabolic intermediates [5], [6].…”