Breeding Strategy To Generate Robust Yeast Starter Cultures for Cocoa Pulp Fermentations

Abstract

Cocoa pulp fermentation is a spontaneous process during which the natural microbiota present at cocoa farms is allowed to ferment the pulp surrounding cocoa beans. Because such spontaneous fermentations are inconsistent and contribute to product variability, there is growing interest in a microbial starter culture that could be used to inoculate cocoa pulp fermentations. Previous studies have revealed that many different fungi are recovered from different batches of spontaneous cocoa pulp fermentations, whereas the variation in the prokaryotic microbiome is much more limited. In this study, therefore, we aimed to develop a suitable yeast starter culture that is able to outcompete wild contaminants and consistently produce high-quality chocolate. Starting from specifically selected Saccharomyces cerevisiae strains, we developed robust hybrids with characteristics that allow them to efficiently ferment cocoa pulp, including improved temperature tolerance and fermentation capacity. We conducted several laboratory and field trials to show that these new hybrids often outperform their parental strains and are able to dominate spontaneous pilot scale fermentations, which results in much more consistent microbial profiles. Moreover, analysis of the resulting chocolate showed that some of the cocoa batches that were fermented with specific starter cultures yielded superior chocolate. Taken together, these results describe the development of robust yeast starter cultures for cocoa pulp fermentations that can contribute to improving the consistency and quality of commercial chocolate production. Microbial fermentation is a crucial step in the production process for many foods and beverages, including chocolate, beer, wine, bread, and cheese. The quality of these products strongly depends on the microbes present, with even slight deviations in the microbial population yielding marked differences in product characteristics. For thousands of years, these fermentation processes were conducted spontaneously, relying on the inoculation of a complex mixture of microbes present in the environment. However, since the development of techniques to isolate and maintain pure microbial cultures in the late 19th century, an increasing number of producers have adopted the idea of using a defined starter culture (1). This practice greatly increased the reproducibility and efficiency of the fermentation process and resulted in augmented product consistency. However, starter cultures were not adopted in all fermentation industries. One of the most striking examples is the cocoa industry, where the production (about 4 ϫ 10 6 tons of beans per year) largely depends on the complex and highly variable microbial population present at cocoa farms, where the cocoa beans are exposed to the natural environment to start the fermentation process (2).The species diversity of spontaneous cocoa pulp fermentations has received much scientific attention, with several studies describing the microbiome across the world (3-9). Interestingly, the variable envi...

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“…Robust S. cerevisiae strains (i.e., strains that thrive in the highly selective cocoa pulp environment) were previously selected and developed to increase the efficiency and consistency of cocoa pulp fermentations (29). In this study, three such robust strains were selected for further breeding: Y927, an indigenous cocoa strain dominant in spontaneous fermentations; Y115, a bioethanol strain that outcompetes Y927 in a cocoa pulp environment; and their hybrid H28, which shows even better growth in cocoa pulp (Table 2).…”

“…Volatiles of the cocoa liquors and chocolates were extracted and quantified using GC-MS, according to a protocol described previously (29). In short, 5 g of each sample was liquefied at 50°C, spiked with 4 l of a 4-heptanone internal standard stock solution (0.32296 g l Ϫ1 ), and stirred.…”

“…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).…”

“…Given the economic importance of increased efficiency and consistency of cocoa pulp fermentation, several research teams have investigated the possibility of developing starter cultures for controlled fermentation (20,24,(26)(27)(28)(29)(30). An important finding of these studies is that the success of the starter culture depends largely on yeasts, since they have been shown to be indispensable in the development of the chocolate flavor, while other microbes, such as lactic acid bacteria, probably play a much less prominent role (10,26,29,31).…”

“…An important finding of these studies is that the success of the starter culture depends largely on yeasts, since they have been shown to be indispensable in the development of the chocolate flavor, while other microbes, such as lactic acid bacteria, probably play a much less prominent role (10,26,29,31). Moreover, our research group recently showed that yeast strains that show tolerance to high temperatures are especially suited as starter cultures, because they can outcompete and suppress the growth of the native cocoa microbiota, which increases the consistency and quality of the end product (29). Moreover, our trials also suggested that at least part of the yeast-related flavor(s) (or flavor precursors) produced during fermentation is retained in the final chocolate, despite the high temperatures used during roasting and conching.…”