Development of superior yeast starter cultures for cocoa fermentations - improving chocolate quality and flavor

The fermentation of cocoa pulp, the first step in the production process of chocolate, is crucial for chocolate flavor development and pulp degradation. Despite the large annual production of cocoa beans, about 4 million tonnes per year, this fermentation process is one of the few remaining large-scale spontaneous fermentations in today’s food industry. Spontaneous cocoa pulp fermentations contain a wide variety of microorganisms, with lactic acid bacteria, acetic acid bacteria and yeasts being the main players. Previous studies revealed that many different yeasts are recovered from spontaneous fermentations, whereas the variation in the prokaryotic microbiome is much more limited. Because spontaneous fermentations lead to unwanted product variability and possibly off-flavor formation, there is a growing interest in microbial starter cultures that can be used to inoculate cocoa pulp fermentations, yet with limited success. Starter cultures have been successfully applied in the production process of many foods and beverages, where they greatly increase the efficiency and reproducibility of the fermentation process and result in augmented product consistency. We therefore aimed to develop optimal yeast-based cocoa starter cultures able to outcompete wild contaminants, consistently produce high-quality chocolate and tune chocolate flavor. We differentiated between starter cultures for the production of regular (bulk) chocolate, and specialty, aromatic chocolate with distinct fruity notes. We first characterized different spontaneous cocoa pulp fermentations in the two most prominent cocoa-exporting regions to investigate and characterize the microbiota involved. We established that the spontaneous fermentations were characterized by a core and a variable microbiota. The core microbiota consisted of a prokaryotic fraction, largely unaffected by geographical location, and a region-specific yeast fraction. A recurring multi-phase yeast profile was observed, characterized by a transient dominance of fast glucose-fermenting yeasts (Hanseniaspora spp.), which are quickly outcompeted by more thermotolerant yeasts (Saccharomyces and/or Pichia spp.). In a second part, we developed yeast hybrids with an improved thermotolerance that efficiently ferment cocoa pulp, starting from specifically selected industrial and indigenous cocoa Saccharomyces cerevisiae strains. The aromatic hybrids that were developed for a specialty, aromatic chocolate were additionally able to produce high concentrations of fruity esters. These esters, such as isoamyl acetate, are important flavor compounds in fermented beverages, lending a pleasant, fruity aroma to the product. Several laboratory and field trials showed that the new hybrids often outperformed their parental strains and were able to dominate pilot scale fermentations, resulting in much more consistent microbial profiles. Gas chromatography-mass spectrometry analysis of the cocoa liquor revealed a decrease in spoilage-related off-flavors in S. cerevisiae inoculated fermentations compared to spontaneous fermentations, and a specific increase in various acetate ester concentrations for fermentations inoculated with the aromatic yeast hybrids. Analysis of the resulting chocolate by an expert and a consumer panel showed that some cocoa batches fermented with specific starter cultures yielded superior chocolate, indicating that these starter cultures can be used for commercial production. In a last part, we characterized pectin and hemicellulose cell wall polysaccharides in cocoa pulp and their role in the degradation of the pulp. It is assumed that pulp degradation is the result of pectin breakdown caused by pectinolytic yeasts, while the contribution of the hemicellulose fraction has been neglected. Therefore, we first provided a comprehensive overview of the composition of pectin and hemicellulose cell wall polysaccharides. By consequently subjecting the cocoa pulp to different physicochemical, enzymatic and microbial treatments that mimicked the changing conditions during a fermentation process, we showed that increasing temperatures, endo-polygalacturonase and xyloglucanase reduce the viscosity of cocoa pulp, thereby contributing to the desired liquefaction. Taken together, the results describe the development of optimal S. cerevisiae starter cultures for cocoa pulp fermentations that improve the consistency and quality of commercial chocolate production and introduce desired product diversification. This work further provides a comprehensive overview of what contributes to changing the viscosity of cocoa pulp during fermentation.status: publishe