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

Improving industrial yeast strains: exploiting natural and artificial diversity

Yeasts have been used for thousands of years to make fermented foods and beverages, such as beer, wine, sake, and bread. However, the choice for a particular yeast strain or species for a specific industrial application is often based on historical, rather than scientific grounds. Moreover, new biotechnological yeast applications, such as the production of second-generation biofuels, confront yeast with environments and challenges that differ from those encountered in traditional food fermentations. Together, this implies that there are interesting opportunities to isolate or generate yeast variants that perform better than the currently used strains. Here, we discuss the different strategies of strain selection and improvement available for both conventional and nonconventional yeasts. Exploiting the existing natural diversity and using techniques such as mutagenesis, protoplast fusion, breeding, genome shuffling and directed evolution to generate artificial diversity, or the use of genetic modification strategies to alter traits in a more targeted way, have led to the selection of superior industrial yeasts. Furthermore, recent technological advances allowed the development of high-throughput techniques, such as 'global transcription machinery engineering' (gTME), to induce genetic variation, providing a new source of yeast genetic diversity.status: publishe

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

Microbial starter cultures have extensively been used to enhance the consistency and efficiency of industrial fermentations. Despite the advantages of such controlled fermentations, the fermentation involved in the production of chocolate is still a spontaneous process that relies on the natural microbiota at cocoa farms. However, recent studies indicate that certain thermotolerant Saccharomyces cerevisiae cultures can be used as starter cultures for cocoa pulp fermentation. In this study, we investigate the potential of specifically developed starter cultures to modulate chocolate aroma. Specifically, we developed several new S. cerevisiae hybrids that combine thermotolerance and efficient cocoa pulp fermentation with a high production of volatile flavor-active esters. In addition, we investigated the potential of two strains of two non-Saccharomyces species that produce very large amounts of fruity esters (Pichia kluyveri and Cyberlindnera fabianii) to modulate chocolate aroma. Gas chromatography-mass spectrometry (GC-MS) analysis of the cocoa liquor revealed an increased concentration of various flavor-active esters and a decrease in spoilage-related off-flavors in batches inoculated with S. cerevisiae starter cultures and, to a lesser extent, in batches inoculated with P. kluyveri and Cyb. fabianii. Additionally, GC-MS analysis of chocolate samples revealed that while most short-chain esters evaporated during conching, longer and more-fat-soluble ethyl and acetate esters, such as ethyl octanoate, phenylethyl acetate, ethyl phenylacetate, ethyl decanoate, and ethyl dodecanoate, remained almost unaffected. Sensory analysis by an expert panel confirmed significant differences in the aromas of chocolates produced with different starter cultures. Together, these results show that the selection of different yeast cultures opens novel avenues for modulating chocolate flavor.status: publishe

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

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.status: publishe

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

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.status: publishe

Characterization and Degradation of Pectic Polysaccharides in Cocoa Pulp

Microbial fermentation of the viscous pulp surrounding cocoa beans is a crucial step in chocolate production. During this process, the pulp is degraded, after which the beans are dried and shipped to factories for further processing. Despite its central role in chocolate production, pulp degradation, which is assumed to be a result of pectin breakdown, has not been thoroughly investigated. Therefore, this study provides a comprehensive physicochemical analysis of cocoa pulp, focusing on pectic polysaccharides, and the factors influencing its degradation. Detailed analysis reveals that pectin in cocoa pulp largely consists of weakly bound substances, and that both temperature and enzyme activity play a role in its degradation. Furthermore, this study shows that pulp degradation by an indigenous yeast fully relies on the presence of a single gene (PGU1), encoding for an endopolygalacturonase. Apart from their basic scientific value, these new insights could propel the selection of microbial starter cultures for more efficient pulp degradation.status: publishe

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

<div><p>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, <i>Lactobacillus fermentum</i> and <i>Acetobacter pasteurianus</i>. 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 <i>Saccharomyces cerevisiae</i>. 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.</p></div

Temperature and pH of cocoa pulp in four Malaysian spontaneous cocoa pulp fermentations.

<p>*B1 and H1 (box and heap fermentation 1 respectively) were performed in the beginning of the harvest season (October 2011), while B2 and H2 (box and heap fermentation 2 respectively) were executed at the end of the harvest season (January 2012).</p><p>Different parameters are listed for each fermentation: pH_i = initial pH, pH_f = final pH, pH_min = minimal pH, pH_max = maximal pH, T_i = initial temperature, T_f = final temperature, T_min = minimal T, T_max =  maximal temperature.</p

Microbial cell counts during four Malaysian spontaneous cocoa pulp fermentations.

<p>A: yeast. B: lactic acid bacteria (LAB). C: acetic acid bacteria (AAB). Box fermentation 1 (October 2011, black), heap fermentation 1 (October 2011, grey), box fermentation 2 (January 2012, brown) and heap fermentation 2 (January 2012, orange). The dashed arrows indicate when the cell counts rise up to or drop below the detection limit [1.5 10⁴ colony forming units (CFU) g⁻¹]. Solid arrows indicate turning.</p