Influence of two anti-fungal Lactobacillus fermentum-Saccharomyces cerevisiae co-cultures on cocoa bean fermentation and final bean quality.
ABSTRACT: The growth of filamentous fungi during the spontaneous cocoa bean fermentation leads to inferior cocoa bean quality and poses a health risk for consumers due to the potential accumulation of mycotoxins. We recently developed anti-fungal cultures with the capacity to inhibit the growth of mycotoxigenic filamentous fungi on cocoa beans. However, it is not clear how these anti-fungal cultures affect the fermentation process and cocoa bean quality. For that, the anti-fungal co-cultures, Lactobacillus fermentum M017-Saccharomyces cerevisiae H290 (A) and Lb. fermentum 223-S. cerevisiae H290 (B), were applied to 180-kg box fermentations in Honduras in three time-independent replications each including a spontaneous control fermentation. The comparison of inoculated and spontaneous fermentation processes revealed that the co-cultures only marginally affected the fermentation process and cocoa bean quality. Microorganisms reached maximal levels of 6.2-7.6 log CFU/g of yeasts and acetic acid bacteria and 7.9-9.5 log CFU/g of lactic acid bacteria during all fermentations and led to maximal metabolite concentrations in bean cotyledons of 4-12 mg/g ethanol, 2-6 mg/g lactic acid and 6-14 mg/g acetic acid. The fermentation and drying processes resulted in 38-90 mg epicatechin equivalents/g in the cotyledons of dried beans. However, the co-cultures led to up to ten times higher mannitol levels in cotyledons of inoculated beans compared to beans during spontaneous fermentation, and caused a slower fermentation process, detectable as up to 8-12 °C lower temperatures in the centre of the fermenting pulp-bean mass and up to 22% lower proportions of well-fermented beans after drying. Co-culture B-with Lb. fermentum 223 -led to improved cocoa bean quality compared to co-culture A-with Lb. fermentum M017 -, i.e. cocoa beans with 0.5-1.9 mg/g less acetic acid, 4-17% higher shares of well-fermented beans and, on a scale from 0 to 10, to 0.2-0.6 units lower astringency, up to 1.1 units lower off-flavours, and 0.2-0.9 units higher cocoa notes. Therefore, the anti-fungal co-culture B is recommended for future applications and its capacity to limit fungal growth and mycotoxin production during industrial-scale cocoa bean fermentation should be investigated in further studies.
Project description:Traditional fermentations of the local Ecuadorian cocoa type Nacional, with its fine flavor, are carried out in boxes and on platforms for a short time. A multiphasic approach, encompassing culture-dependent and -independent microbiological analyses of fermenting cocoa pulp-bean samples, metabolite target analyses of both cocoa pulp and beans, and sensory analysis of chocolates produced from the respective fermented dry beans, was applied for the investigation of the influence of these fermentation practices on the yeast and bacterial species diversity and community dynamics during cocoa bean fermentation. A wide microbial species diversity was found during the first 3 days of all fermentations carried out. The prevailing ethanol-producing yeast species were Pichia kudriavzevii and Pichia manshurica, followed by Saccharomyces cerevisiae. Leuconostoc pseudomesenteroides (glucose and fructose fermenting), Fructobacillus tropaeoli-like (fructose fermenting), and Lactobacillus fermentum (citrate converting, mannitol producing) represented the main lactic acid bacterial species in the fermentations studied, resulting in intensive heterolactate metabolism of the pulp substrates. Tatumella saanichensis and Tatumella punctata were among the members of the family Enterobacteriaceae present during the initial phase of the cocoa bean fermentations and could be responsible for the production of gluconic acid in some cases. Also, a potential new yeast species was isolated, namely, Candida sorbosivorans-like. Acetic acid bacteria, whose main representative was Acetobacter pasteurianus, generally appeared later during fermentation and oxidized ethanol to acetic acid. However, acetic acid bacteria were not always present during the main course of the platform fermentations. All of the data taken together indicated that short box and platform fermentation methods caused incomplete fermentation, which had a serious impact on the quality of the fermented dry cocoa beans.
Project description:Spontaneous cocoa bean fermentations performed under bench- and pilot-scale conditions were studied using an integrated microbiological approach with culture-dependent and culture-independent techniques, as well as analyses of target metabolites from both cocoa pulp and cotyledons. Both fermentation ecosystems reached equilibrium through a two-phase process, starting with the simultaneous growth of the yeasts (with Saccharomyces cerevisiae as the dominant species) and lactic acid bacteria (LAB) (Lactobacillus fermentum and Lactobacillus plantarum were the dominant species), which were gradually replaced by the acetic acid bacteria (AAB) (Acetobacter tropicalis was the dominant species). In both processes, a sequence of substrate consumption (sucrose, glucose, fructose, and citric acid) and metabolite production kinetics (ethanol, lactic acid, and acetic acid) similar to that of previous, larger-scale fermentation experiments was observed. The technological potential of yeast, LAB, and AAB isolates was evaluated using a polyphasic study that included the measurement of stress-tolerant growth and fermentation kinetic parameters in cocoa pulp media. Overall, strains L. fermentum UFLA CHBE8.12 (citric acid fermenting, lactic acid producing, and tolerant to heat, acid, lactic acid, and ethanol), S. cerevisiae UFLA CHYC7.04 (ethanol producing and tolerant to acid, heat, and ethanol), and Acetobacter tropicalis UFLA CHBE16.01 (ethanol and lactic acid oxidizing, acetic acid producing, and tolerant to acid, heat, acetic acid, and ethanol) were selected to form a cocktail starter culture that should lead to better-controlled and more-reliable cocoa bean fermentation processes.
Project description:This survey reports for the first time the changed of quality of fermented cocoa (Theobroma cacao L.) beans. The quality evaluation and simultaneous detection of amino acids, flavor, procyanidin, color, fat, protein, antioxidant activity, and enthalpy were obtained for different fermentation stages of cocoa beans. The results showed that total essential amino acids contents ranged from 2.64 g/100 g to 3.68 g/100 g. A total of 88 compounds identified at the end of the fermentation belonged to alcohols, acids, esters, ketones, pyrazines, aldehydes, and terpenoids. One of the chemical groups that were present in highest abundance in the consummation treatments was acids, representing 56.04% of the total extracted area, followed by alcohols (22.95%) and ketones (9.40%). The colors of the beans in different fermentation stages were different, from deep purple to deep red-brown. Fermented cocoa beans were shown to be 53.45% and 13.51% bean butter and protein content, respectively. The value of denaturation enthalpy (?H) ranged from 30.4 (J/g) to 43.38 (J/g). The 3-day fermented sample had the highest ?H (43.38 J/g). When the fermentation process was complete, the procyanidin concentration of the beans decreased, with the final yield of procyanidin at 6.2%. During fermentation, the antioxidant capacity of beans gradually reduced. The fermenting of cocoa beans had a significant effect on the quality formation. The findings of this study constitute a basis for further investigations on the quality formation of cocoa during fermentation.
Project description:This is the first report on the phylogenetic analysis of the community diversity of a single spontaneous cocoa bean box fermentation sample through a metagenomic approach involving 454 pyrosequencing. Several sequence-based and composition-based taxonomic profiling tools were used and evaluated to avoid software-dependent results and their outcome was validated by comparison with previously obtained culture-dependent and culture-independent data. Overall, this approach revealed a wider bacterial (mainly ?-Proteobacteria) and fungal diversity than previously found. Further, the use of a combination of different classification methods, in a software-independent way, helped to understand the actual composition of the microbial ecosystem under study. In addition, bacteriophage-related sequences were found. The bacterial diversity depended partially on the methods used, as composition-based methods predicted a wider diversity than sequence-based methods, and as classification methods based solely on phylogenetic marker genes predicted a more restricted diversity compared with methods that took all reads into account. The metagenomic sequencing analysis identified Hanseniaspora uvarum, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus as the prevailing species. Also, the presence of occasional members of the cocoa bean fermentation process was revealed (such as Erwinia tasmaniensis, Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides, and Oenococcus oeni). Furthermore, the sequence reads associated with viral communities were of a restricted diversity, dominated by Myoviridae and Siphoviridae, and reflecting Lactobacillus as the dominant host. To conclude, an accurate overview of all members of a cocoa bean fermentation process sample was revealed, indicating the superiority of metagenomic sequencing over previously used techniques.
Project description:Chocolate production suffered a vast impact with the emergence of the "witches' broom" disease in cocoa plants. To recover cocoa production, many disease-resistant hybrid plants have been developed. However, some different cocoa hybrids produce cocoa beans that generate chocolate with variable quality. Fermentation of cocoa beans is a microbiological process that can be applied for the production of chocolate flavor precursors, leading to overcoming the problem of variable chocolate quality. The aim of this work was to use a cocktail of microorganisms as a starter culture on the fermentation of the ripe cocoa pods from PH15 cocoa hybrid, and evaluate its influence on the microbial communities present on the fermentative process on the compounds involved during the fermentation, and to perform the chocolate sensorial characterization. According to the results obtained, different volatile compounds were identified in fermented beans and in the chocolate produced. Bitterness was the dominant taste found in non-inoculated chocolate, while chocolate made with inoculated beans showed bitter, sweet, and cocoa tastes. 2,3-Butanediol and 2,3-dimethylpyrazine were considered as volatile compounds making the difference on the flavor of both chocolates. Saccharomyces cerevisiae UFLA CCMA 0200, Lactobacillus plantarum CCMA 0238, and Acetobacter pasteurianus CCMA 0241 are proposed as starter cultures for cocoa fermentation.
Project description:The aim of the current work was to use hyperspectral imaging (HSI) in the spectral range 1000-2500?nm to quantitatively predict fermentation index (FI), total polyphenols (TP) and antioxidant activity (AA) of individual dry fermented cocoa beans scanned on a single seed basis, in a non-destructive manner. Seventeen cocoa bean batches were obtained and 10 cocoa beans were used from each batch. PLS regression models were built on 170 samples. The developed HSI predictive models were able to quantify three quality-related parameters with sufficient performance for screening purposes, with external validation R2 of 0.50 (RMSEP?=?0.27, RPD?=?1.40), 0.70 (RMSEP?=?34.1?mg ferulic acid?g-1, RPD?=?1.77) and 0.74 (60.0?mmol Trolog?kg-1, RPD?=?1.91) for FI, TP and AA, respectively. The calibrations were subsequently applied at a single bean and pixel level, so that the distribution was visualised within and between single seeds (chemical images). HSI is thus suggested as a promising approach to estimate cocoa bean composition rapidly and non-destructively, thus offering a valid tool for food inspection and quality control.
Project description:Cocoa beans (Theobroma cacao L.) are the principal raw material for chocolate manufacture. Before cocoa beans are ready for the chocolate industry, farm-based fermentation and drying processes are key determinants of bean quality and hence the price. To improve its value, cocoa beans were dried in a modified greenhouse (MGHD), conventional greenhouse (CGHD), and open sun (OSD) dryers. The drying behavior, kinetics, and quality were evaluated. The MGHD was constructed by modifying a conventional greenhouse with a fleece of black polyester material. Evaluation of air properties of the dryers without and with cocoa beans showed that the MGHD had average temperatures of 2 and 8°C above, and relative humidity of 12.28% and 25.48% below the CGHD and OSD, respectively. The drying data were fitted to four thin layer mathematical models. The Page and Overhult models gave favorable ranges of R 2 (.976 to .987), chi-square (3.7 × 10-4 to 9.9 × 10-4), and root mean square (RMSE; 0.0188 to 0.0307) for the three dryers. The cocoa beans dried in the MGHD took a shorter time to reach the expected 5%-8% moisture content and were of grade one quality.
Project description: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.
Project description:In the present work, simulated cocoa fermentation was investigated at the level of metabolic pathway fluxes (fluxome) of lactic acid bacteria (LAB), which are typically found in the microbial consortium known to convert nutrients from the cocoa pulp into organic acids. A comprehensive (13)C labeling approach allowed to quantify carbon fluxes during simulated cocoa fermentation by (i) parallel (13)C studies with [(13)C6]glucose, [1,2-(13)C2]glucose, and [(13)C6]fructose, respectively, (ii) gas chromatography-mass spectrometry (GC/MS) analysis of secreted acetate and lactate, (iii) stoichiometric profiling, and (iv) isotopomer modeling for flux calculation. The study of several strains of L. fermentum and L. plantarum revealed major differences in their fluxes. The L. fermentum strains channeled only a small amount (4 to 6%) of fructose into central metabolism, i.e., the phosphoketolase pathway, whereas only L. fermentum NCC 575 used fructose to form mannitol. In contrast, L. plantarum strains exhibited a high glycolytic flux. All strains differed in acetate flux, which originated from fractions of citrate (25 to 80%) and corresponding amounts of glucose and fructose. Subsequent, metafluxome studies with consortia of different L. fermentum and L. plantarum strains indicated a dominant (96%) contribution of L. fermentum NCC 575 to the overall flux in the microbial community, a scenario that was not observed for the other strains. This highlights the idea that individual LAB strains vary in their metabolic contribution to the overall fermentation process and opens up new routes toward streamlined starter cultures. L. fermentum NCC 575 might be one candidate due to its superior performance in flux activity.
Project description:Cocoa bean fermentation is an important microbial process, where most metabolites that affect chocolate quality and aroma are generated. Production of reproducible high-quality beans is a major challenge because most fermentations occur in open containers with a lack of variable control. Here we present a study that aims to identify the effect of farm protocols, climate, and bean mass exposure, in the dynamics and composition of microbial communities. Using high-throughput sequencing of molecular markers for bacteria and yeasts, complemented with culture-based methods, we evaluated the microbial diversity and dynamics associated to spontaneous cocoa fermentation in two distinct agro-ecological zones in Colombia. The bacterial communities were classified at two levels of evolutionary relationship, at a coarse resolution (OTU-level) and at a finer resolution (oligotype-level). A total of six bacterial OTUs were present in both farms, following a microbial succession that starts with the Enterobacteraceae family (one OTU), transitioning to the Lactobacillaceae family (three OTUs), and finishing with Acetobacteraceae family (two OTUs). When undesirable practices were done, OTUs were observed at unexpected moments during the fermentation. At a finer taxonomic resolution, 48 oligotypes were identified, with 46 present in both farms. These oligotypes have different patterns of prevalence. In the case of Lactobacillaceae a high evenness was observed among oligotypes. In contrast, for Enterobacteraceae and Acetobacteraceae a high dominance of one or two oligotypes was observed, these oligotypes were the same for both farms, despite geographic location and season of sampling. When the overall fermentations were compared using correlations matrices of oligotypes abundance, they show a clear clustering by farm, suggesting that farm protocols generate a unique fingerprint in the dynamics and interactions of the microbial communities. The comparison between the upper and middle layers of the bean mass showed that environmental exposure affects the paces at which ecological successions occur, and therefore, is an important source of cocoa quality heterogeneity. In conclusion, the results presented here showed that the dynamics of microbial fermentation can be used to identify the sources of variability and evidence the need for better fermentation technologies that favor the production of reproducible high-quality cocoa beans.