Investigating luxS gene expression in lactobacilli along lab-scale cocoa fermentations.

Previous metagenomic analyses have suggested that lactobacilli present potential for Quorum Sensing (QS) in cocoa fermentation, and in the present research, laboratory scale fermentations were carried out to monitor the expression of luxS, a universal marker of QS. For that, 96 h-fermentations were studied, as follows: F0 (non inoculated control), F1 (inoculated with yeasts, lactic acid bacteria, and acetic acid bacteria), F2 (inoculated with yeasts and acetic acid bacteria), F3 (inoculated with yeasts only). The parameters evaluated were: plate counting, quantification of key enzymes and analysis of volatile organic compounds associated with key sensory descriptors, using headspace gas chromatography-mass spectrometry (GC-MS). Furthermore, QS was estimated by the quantification of the expression of luxS genes by Reverse Transcriptase Real-Time PCR. The results demonstrated that microbial succession occurred in pilot scale fermentations, but no statistical differences for microbial enumeration and α-diversity index were observed among experiments and control. Moreover, it was not possible to make conclusive correlations of enzymatic profile and fermenting microbiota, likely due to the intrinsic activity of plant hydrolases. Regarding to the expression of luxS genes, in Lactiplantibacillus plantarum they were active along the fermentation, but for Limosilactobacillus fermentum, luxS was expressed only at early and middle phases. Correlation analysis of luxS expression and production of volatile metabolites evidenced a possible negative association of Lp. Plantarum with fermentation quality. In conclusion, these data corroborate former shotgun metagenomic analysis by demonstrating the expression of luxS by lactobacilli in pilot scale cocoa fermentation and evidence Lp. Plantarum is the main lactic acid bacteria related to its expression.

An in-depth multiphasic analysis of the chocolate production chain, from bean to bar, demonstrates the superiority of Saccharomyces cerevisiae over Hanseniaspora opuntiae as functional starter culture during cocoa fermentation.

Hanseniaspora opuntiae is a commonly found yeast species in naturally fermenting cocoa pulp-bean mass, which needed in-depth investigation. The present study aimed at examining effects of the cocoa isolate H. opuntiae IMDO 040108 as part of three different starter culture mixtures compared with spontaneous fermentation, regarding microbial community, substrate consumption, and metabolite production dynamics, including volatile organic compound (VOC) and phytochemical compositions, as well as compositions of the cocoa beans after fermentation, cocoa liquors, and chocolates. The inoculated H. opuntiae strain was unable to prevail over background yeasts present in the fermenting cocoa pulp-bean mass. It led to under-fermented cocoa beans after four days of fermentation, which was however reflected in higher levels of polyphenols. Cocoa fermentation processes inoculated with a Saccharomyces cerevisiae strain enhanced flavour production during the fermentation and drying steps, which was reflected in richer and more reproducible aroma profiles of the cocoa liquors and chocolates. Sensory analysis of the cocoa liquors and chocolates further demonstrated that S. cerevisiae led to more acidic notes compared to spontaneous fermentation, as a result of an advanced fermentation degree. Finally, different VOC profiles were found in the cocoa beans throughout the whole chocolate production chain, depending on the fermentation process.

Functional yeast starter cultures for cocoa fermentation.

The quest to develop a performant starter culture mixture to be applied in cocoa fermentation processes started in the 20th century, aiming at achieving high-quality, reproducible chocolates with improved organoleptic properties. Since then, different yeasts have been proposed as candidate starter cultures, as this microbial group plays a key role during fermentation of the cocoa pulp-bean mass. Yeast starter culture-initiated fermentation trials have been performed worldwide through the equatorial zone and the effects of yeast inoculation have been analysed as a function of the cocoa variety (Forastero, Trinitario and hybrids) and fermentation method (farm-, small- and micro-scale) through the application of physicochemical, microbiological and chemical techniques. A thorough screening of candidate yeast starter culture strains is sometimes done to obtain the best performing strains to steer the cocoa fermentation process and/or to enhance specific features, such as pectinolysis, ethanol production, citrate assimilation and flavour production. Besides their effects during cocoa fermentation, a significant influence of the starter culture mixture applied is often found on the cocoa liquors and/or chocolates produced thereof. Thus, starter culture-initiated cocoa fermentation processes constitute a suitable strategy to elaborate improved flavourful chocolate products.

Pangenome analyses of LuxS-coding genes and enzymatic repertoires in cocoa-related lactic acid bacteria.

Lactobacillaceae presents potential for interspecific Quorum Sensing (QS) in spontaneous cocoa fermentation, correlated with high abundance of luxS. Three Brazilian isolates from cocoa fermentation were characterized by Whole Genome Sequencing and luxS gene was surveyed in their genomes, in comparison with public databases. They were classified as Lactiplantibacillus plantarum, Limosilactobacillus fermentum and Pediococcus acidilactici. LuxS genes were conserved in core genomes of the novel isolates, but in some non-cocoa related Lactic Acid Bacteria (LAB) it was accessory and plasmid-borne. The conservation and horizontal acquisition of luxS reinforces that QS is determinant for bacterial adaptation in several environments, especially taking into account the luxS has been correlated with modulation of bacteriocin production, stress tolerance and biofilm formation. Therefore, in this paper, new clade and species-specific primers were designed for future application for screening of luxS gene in LAB to evaluate the adaptive potential to diverse food fermentations.

Metagenome-Assembled Genomes Contribute to Unraveling of the Microbiome of Cocoa Fermentation.

Metagenomic studies about cocoa fermentation have mainly reported on the analysis of short reads for determination of operational taxonomic units. However, it is also important to determine metagenome-assembled genomes (MAGs), which are genomes deriving from the assembly of metagenomics. For this research, all the cocoa metagenomes from public databases were downloaded, resulting in five data sets: one from Ghana and four from Brazil. In addition, in silico approaches were used to describe putative phenotypes and the metabolic potential of MAGs. A total of 17 high-quality MAGs were recovered from these microbiomes, as follows: (i) for fungi, Yamadazyma tenuis (n = 1); (ii) lactic acid bacteria, Limosilactobacillus fermentum (n = 5), Liquorilactobacillus cacaonum (n = 1), Liquorilactobacillus nagelli (n = 1), Leuconostoc pseudomesenteroides (n = 1), and Lactiplantibacillus plantarum subsp. plantarum (n = 1); (iii) acetic acid bacteria, Acetobacter senegalensis (n = 2) and Kozakia baliensis (n = 1); and (iv) Bacillus subtilis (n = 1), Brevundimonas sp. (n = 2), and Pseudomonas sp. (n = 1). Medium-quality MAGs were also recovered from cocoa microbiomes, including some that, to our knowledge, were not previously detected in this environment (Liquorilactobacillus vini, Komagataeibacter saccharivorans, and Komagataeibacter maltaceti) and others previously described (Fructobacillus pseudoficulneus and Acetobacter pasteurianus). Taken together, the MAGs were useful for providing an additional description of the microbiome of cocoa fermentation, revealing previously overlooked microorganisms, with prediction of key phenotypes and biochemical pathways. IMPORTANCE The production of chocolate starts with the harvesting of cocoa fruits and the spontaneous fermentation of the seeds in a microbial succession that depends on yeasts, lactic acid bacteria, and acetic acid bacteria in order to eliminate bitter and astringent compounds present in the raw material, which will be further roasted and grinded to originate the cocoa powder that will enter the food processing industry. The microbiota of cocoa fermentation is not completely known, and yet it advanced from culture-based studies to the advent of next-generation DNA sequencing, with the generation of a myriad of data that need bioinformatic approaches to be properly analyzed. Although the majority of metagenomic studies have been based on short reads (operational taxonomic units), it is also important to analyze entire genomes to determine more precisely possible ecological roles of different species. Metagenome-assembled genomes (MAGs) are very useful for this purpose; here, MAGs from cocoa fermentation microbiomes are described, and the possible implications of their phenotypic and metabolic potentials are discussed.

Integrating microbial metagenomics and physicochemical parameters and a new perspective on starter culture for fine cocoa fermentation.

Cocoa beans used for chocolate production are fermented seeds of Theobroma cacao obtained by a natural fermentation process. The flavors and chemical compounds produced during the fermentation process make this step one of the most important in fine chocolate production. Herein, an integrative analysis of the variation of microbial community structure, using a shotgun metagenomics approach and associated physicochemical features, was performed during fermentation of fine cocoa beans. Samples of Forastero variety (FOR) and a mixture of two hybrids (PS1319 and CCN51) (MIX) from Bahia, Brazil, were analyzed at 7 different times. In the beginning (0 h), the structures of microbial communities were very different between FOR and MIX, reflecting the original plant-associated microbiomes. The highest change in microbial community structures occurred at the first 24 h of fermentation, with a marked increase in temperature and acetic acid concentration, and pH decrease. At 24-48 h both microbial community structures were quite homogenous regarding temperature, acetic acid, succinic acid, pH, soluble proteins and total phenols. During 72-96 h, the community structure resembles an acidic and warmer environment, prevailing few acetic acid bacteria. Taxonomic richness and abundance at 72-144 h exhibited significant correlation with temperature, reducing sugars, succinic, and acetic acids. Finally, we recommend that dominant microbial species of spontaneous fine cocoa fermentations should be considered as inoculum in accordance with the farm/region and GMP to maintain a differential organoleptic feature for production of fine chocolate. In our study, a starter inoculum composed of Acetobacter pausterianus and Hanseniaspora opuntiae strains is indicated.

Mapping the functional and strain diversity of the main microbiota involved in cocoa fermentation from Cote d'Ivoire.

The variable quality of cocoa produced by farmers is still a problem in the value chain, strongly depending on microbial activities. We analyzed the variability of cocoa microbiota from all twelve producing regions in Cote d'Ivoire, and described the geographical distribution of isolated microbiota, using a mapping. Microbial species were identified by ribosomal genes sequencing, strains were typed by RFLP and their techno-functional capacities were further investigated. Results showed a restricted diversity of lactic acid bacteria (LAB) and acetic acid bacteria (AAB) with respectively 10 and 5 strains. The dominant LAB and AAB strains, notably Lactobacillus plantarum 1 A, Acetobacter pasteurianus 1 A, Acetobacter okinawensis 2 A, and Acetobacter tropicalis 3 A, were found in all regions assuming that the acid microbiota was weakly variable. In contrast, the distribution of their functional performance such as acidification capability was variable, stronger in strains from Nawa and Haut-Sassandra regions and weaker in Indenie-Djuablin and San Pedro; this distribution seemed to be random. Moreover, the study also revealed a complex yeasts population showing a wide genetic diversity with 22 species and 45 strains indicating an intraspecific heterogeneity. Strains were generally different from a region to another and the resulting yeasts microbiota was globally variable in the regions. Likewise, the functional capacities such as pectinolytic was weak in P. kudriazevii strain 2 K from Gboklè and strong in P. kudriazevii strain 2 A from Loh-Djiboua. Additionally, the quality of fermented beans was also variable in the regions. The great variation of yeasts strains in the different regions may be the main microbial factors responsible for variation of the fermented cocoa quality observed.

Influence of S. cerevisiae and P. kluyveri as starters on chocolate flavour.

BACKGROUND: Fermented cocoa beans (Theobroma cacao L.) are a pivotal raw material for chocolate production. A cocktail yeast applied in the cocoa fermentation process can promote the formation of pleasant metabolites. Saccharomyces, Pichia and Hanseniaspora have been widely used in fermentation to improve the final product organoleptic profile, highlighting that fermentation is a critical point for chocolate flavour precursor production. This study aims to evaluate the impact of Pichia kluyveri and Saccharomyces cerevisiae strains as starter cultures on the fermentation for two cocoa hybrids, FA13 and CEPEC2002. RESULTS: During fermentation processes, volatile organic compounds (VOCs) and protein profiles were assessed. Chocolates produced were also assessed regarding the presence of VOCs. Eighty VOCs were identified using gas chromatography coupled to mass spectrometry analysis. Mass spectrometry provided the protein profile evolution during fermentation and showed that the profiles changed with inoculation type (spontaneous versus inoculated fermentation). Chocolate obtained from FA13 inoculated with S. cerevisiae strain contained a greater amount of organics acids, being categorised as sourer than chocolate produced by spontaneous fermentation of FA13. CEPEC2002 inoculated with S. cerevisiae strain in co-culture with P. kluyveri strain generated less sour and sweeter chocolate than spontaneous fermentation only. CONCLUSIONS: Chocolates from inoculated assays with starter cultures were more accepted by evaluators, highlighting that P. kluyveri and S. cerevisiae influence the composition of VOCs. Besides, protein profiles also changed throughout fermentation. Further investigation should be conducted to clarify protein degradation dynamics during inoculated fermentations to define which of the microbial cultures positively affect the chocolate sensory characteristics. © 2021 Society of Chemical Industry.

Genome-scale metabolic modeling of Acetobacter pasteurianus 386B reveals its metabolic adaptation to cocoa fermentation conditions.

Acetobacter pasteurianus 386B has been selected as a candidate functional starter culture to better control the cocoa fermentation process. Previously, its genome has been sequenced and a genome-scale metabolic model (GEM) has been reconstructed. To understand its metabolic adaptation to cocoa fermentation conditions, different flux balance analysis (FBA) simulations were performed and compared with experimental data. In particular, metabolic flux distributions were simulated for two phases that characterize the growth of A. pasteurianus 386B under cocoa fermentation conditions, predicting a switch in respiratory chain usage in between these phases. The possible influence on the resulting energy production was shown using a reduced version of the GEM. FBA simulations revealed the importance of the compartmentalization of the ethanol oxidation reactions, namely in the periplasm or in the cytoplasm, and highlighted the potential role of ethanol as a source of carbon, energy, and NADPH. Regarding the latter, the physiological function of a proton-translocating NAD(P)+ transhydrogenase was further investigated in silico. This study revealed the potential of using a GEM to simulate the metabolism of A. pasteurianus 386B, and may provide a general framework toward a better physiological understanding of functional starter cultures in food fermentation processes.

Acetic acid bacteria (AAB) involved in cocoa fermentation from Ivory Coast: species diversity and performance in acetic acid production.

In this study, we investigated the diversity of AAB from fermenting cocoa and the production of acetic acid in response to various environmental conditions. Ribosomal 16S gene sequence analysis and PCR-RFLP showed a restricted microbiota mainly composed of Acetobacter pasteurianus, Acetobacter tropicalis and Acetobacter okinawensis sp., consistently found in all six regions studied. Meanwhile Acetobacter malorum, Acetobacter ghanensis and Gluconobacter oxydans were isolated as minor species in specific regions. The dominant species were mainly isolated in the first 72 h period of natural cocoa fermentation while the minor species were present toward the later stages. Acetobacter okinawensis, a newly isolated species, was able to yield an unusually high quantity, up to 62 g/L of acetic acid at 30 °C. However, a shift of temperature to 35 °C severely impaired acid production in most strains of this species. While acetic acid production increases for up to 6 days in Acetobacter okinawensis and Acetobacter pasteurianus, it decreases beyond 4 days in Acetobacter tropicalis strains. The production of acetic acid was strongly dependent on environmental conditions, with optimal production between pH 4 and 5, under ethanol concentration below 8% and temperatures above 35-40 °C, corresponding to conditions prevailing in the first half of fermentation process. Acetobacter tropicalis was more productive at higher ethanol concentration and Acetobacter okinawensis at low pH. Species diversity and different behavior of strains highlight the importance of valuable starter selection for well-controlled cocoa fermentation.

Effects of Maturity at Harvest and Fermentation Conditions on Bioactive Compounds of Cocoa Beans.

Cocoa beans and cocoa products contain considerable amounts of bioactive compounds. Harvesting cocoa fruit too early or too late may have effects on the phenolic and alkaloid concentrations of the cocoa powder. Fermentation, a primary processing used to transform cocoa beans to cocoa powder, may also influence the contents of bioactive compounds. In this study, proanthocyanidins, the major compounds in cocoa polyphenols, caffeine and theobromine of cocoa beans, were evaluated at different maturities at harvest, and with different fermentation durations, with and without the addition of a commercial enzyme, Pectinex® Ultra SP-L. The amounts of proanthocyanidins, caffeine and theobromine, and the antioxidant capacities of the unfermented cocoa beans increased as the fruits matured. The values ranged from 16.12-27.28 g catechin equivalents (CE)/100 g dry weight (DW); 99.66-173.61 mg/100 g DW; 556.39-948.84 mg/100 g DW; 23.23-26.32 mol Trolox equivalents (TE)/100 g DW, respectively. Prolonged fermentation with or without the addition of pectinase, from three to seven days, significantly reduced the amounts of these compounds present. Fermentation using the enzyme significantly reduced the proanthocyanidin content and antioxidant capacity of the cocoa powder, with the overall means decreasing from 8.93-4.93 g CE/100 g DW and from 15.81-12.95 g mol TE/100 g DW, respectively. Two-way ANOVA analyses showed that the proanthocyanidins, caffeine, theobromine contents and the antioxidant capacity of cocoa beans were strongly dependet to their stages of maturity, fermentation methods and fermentation duration.

Regulation of the synthesis of pulp degrading enzymes in Bacillus isolated from cocoa fermentation.

Pectin degrading enzymes are essential for quality of product from cocoa fermentation. Previously, we studied purified pectate lyases (Pel) produced by Bacillus strains from fermenting cocoa and characterized the cloned pel genes. This study aims to search for biological signals that modulates Pel production and regulators that influence pel gene expression. Strains were grown to the end of exponential phase in media containing various carbon sources. Pel enzymes production in Bacillus was unaffected by simple sugar content variation up to 2%. Additionally, it appeared that pel gene is not under the control of the most common carbon and pectin catabolism regulators ccpA and kdgR, which could explain the insensitivity of Pel production to carbon source variation. However, a 6-fold decrease in Pel production was observed when bacteria were grown in LB rich medium as opposed to basal mineral medium. Subsequently, bioinformatics analysis of cloned pel gene promoter region revealed the presence of DegU binding site. Furthermore, the deletion of degU gene dramatically reduces the pel gene expression, as revealed by real time quantitative PCR, showing an activation effect of DegU on Pel synthesis in Bacillus strains studied. We assumed that, during the latter stage of cocoa fermentation when simple sugars are depleted, production of Pel in Bacillus is stimulated by DegU to supply microbial cells with carbon source from polymeric pectic compounds.

The impact of yeast starter cultures on the microbial communities and volatile compounds in cocoa fermentation and the resulting sensory attributes of chocolate.

Theobroma cacao seeds are the main raw material for chocolate production. During their fermentation, a succession of microorganisms are responsible for the physicochemical changes occurring in the pulp and inside the beans. The aim of this study was to investigate the effects of yeast inoculation (Saccharomyces cerevisiae UFLA CA11, Pichia kluivery CCMA0237, and Hanseniaspora uvarum CCMA0236) on the profile of the volatile compounds and microbial communities in cocoa fermentation. The resulting chocolate was also evaluated by temporal dominance of sensations (TDS) analyses. The dominant microorganisms during spontaneous fermentation were S. cerevisiae, H. uvarum, H. guilliermondii, Lactobacillus fermentum, Pediococcus sp., and Acetobacter pasteurianus. Similarly, S. cerevisiae, P. kluyveri, Candida sp., Pediococcus sp., and A. pasteurianus were the predominant microorganisms assessed by Denaturing Gradient Gel Electrophoresis (DGGE) in inoculated fermentation. Sixty-seven volatile compounds were detected and quantified by gas chromatography/mass spectrometry (GC/MS) at the end of fermentation and chocolates. The main group of volatile compound found after the inoculated and spontaneous fermentations was esters (41 and 39 %, respectively). In the chocolates, the main group was acids (73 and 44 % from the inoculated and spontaneous fermentations, respectively). The TDS analyses showed a dominance of bitter and cocoa attributes in both chocolates. However, in the inoculated chocolate, lingering fruity notes were more intense, while the chocolate produced by spontaneous fermentation was more astringent. Thus, the inoculation of yeast influenced the microbial profile, which likely affected the volatile compounds that affect sensory characteristics, resulting in chocolate with dominant bitter, cocoa, and fruity attributes.

Electromagnetic fields effects on microbial growth in cocoa fermentation: A controlled experimental approach using established growth models.

Understanding the effects of electromagnetic fields is crucial in the fermentation of cocoa beans, since through precise control of fermentation conditions the sensory and nutritional properties of cocoa beans could be improved. This study aimed to evaluate the effect of oscillating magnetic fields (OMF) on the kinetic growth of the core microbial communities of the Collections Castro Naranjal (CCN 51) cocoa bean. The data was obtained by three different models: Gompertz, Baranyi, and Logistic. The cocoa beans were subjected to different OMF strengths ranging from 0 mT to 80 mT for 1 h using the Helmholtz coil electromagnetic device. The viable microbial populations of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeast (Y) were quantified using the colony-forming unit (CFU) counting method. The logistic model appropriately described the growth of LAB and Y under magnetic field exposure. Whereas the Baranyi model was suitable for describing AAB growth. The microbial populations in cocoa beans exposed to magnetic fields showed lower (maximum specific growth rate (µmax), values than untreated controls, with AAB exhibiting the highest average growth rate value at 5 mT and Y having the lowest average maximum growth rate value at 80 mT. The lower maximum specific growth rates and longer lag phases when exposed to magnetic fields compared to controls demonstrate the influence of magnetic fields on microbial growth kinetics.

Microbial composition and metabolic profiles during machine-controlled intra-factory fermentation of cocoa beans harvested in semitropical area of Japan.

Cocoa bean fermentation is typically performed in a spontaneous manner on farms in tropical countries or areas and involves several microbial groups. Metabolism by microbes markedly affects the quality of cocoa beans fermented and the chocolate produced thereof. The present study characterized the microbiota and their metabolic profiles in temperature- and humidity-controlled intra-factory cocoa fermentation in a semitropical area of Japan. Although environmental factors were uniform, the microbiota of cocoa beans subjected to intra-factory fermentation was not stable between tests, particularly in terms of the cell count levels and species observed. Fermentation was sometimes delayed, and fermenting microbes were present at very low levels after 24 hr of fermentation. Due to the unstable microbiota, the profiles of water-soluble compounds differed between tests, indicating the unstable qualities of the fermented cocoa beans. These results suggest the necessity of starter cultures not only in on-farm fermentation but also in machine-controlled intra-factory cocoa fermentation.

The Virome of Cocoa Fermentation-Associated Microorganisms.

Theobroma cacao plantations are of significant economic importance worldwide, primarily for chocolate production. During the harvest and processing of cocoa beans, they are subjected to fermentation either by microorganisms present in the environment (spontaneous fermentation) or the addition of starter cultures, with different strains directly contributing distinct flavor and color characteristics to the beans. In addition to fungi and bacteria, viruses are ubiquitous and can affect the quality of the fermentation process by infecting fermenting organisms, destabilizing microbial diversity, and consequently affecting fermentation quality. Therefore, in this study, we explored publicly available metatranscriptomic libraries of cocoa bean fermentation in Limon Province, Costa Rica, looking for viruses associated with fermenting microorganisms. Libraries were derived from the same sample at different time points: 7, 20, and 68 h of fermentation, corresponding to yeast- and lactic acid bacteria-driven phases. Using a comprehensive pipeline, we identified 68 viral sequences that could be assigned to 62 new viral species and 6 known viruses distributed among at least nine families, with particular abundance of elements from the Lenarviricota phylum. Interestingly, 44 of these sequences were specifically associated with ssRNA phages (Fiersviridae) and mostly fungi-infecting viral families (Botourmiaviridae, Narnaviridae, and Mitoviridae). Of note, viruses from those families show a complex evolutionary relationship, transitioning from infecting bacteria to infecting fungi. We also identified 10 and 3 viruses classified within the Totiviridae and Nodaviridae families, respectively. The quantification of the virus-derived RNAs shows a general pattern of decline, similar to the dynamic profile of some microorganism genera during the fermentation process. Unexpectedly, we identified narnavirus-related elements that showed similarity to segmented viral species. By exploring the molecular characteristics of these viral sequences and applying Hidden Markov Models, we were capable of associating these additional segments with a specific taxon. In summary, our study elucidates the complex virome associated with the microbial consortia engaged in cocoa bean fermentation that could contribute to organism/strain selection, altering metabolite production and, consequently, affecting the sensory characteristics of cocoa beans.

Integrating shotgun metagenomics and metabolomics to elucidate the dynamics of microbial communities and metabolites in fine flavor cocoa fermentation in Hainan.

The aim of this study was to investigate the dynamic profile of microorganisms and metabolites in Hainan Trinitario cocoa during a six-day spontaneous box fermentation process. Shotgun metagenomic and metabolomic approaches were employed for this investigation. The potential metabolic functions of microorganisms in cocoa fermentation were revealed through a joint analysis of microbes, functional genes, and metabolites. During the anaerobic fermentation phase, Hanseniaspora emerged as the most prevalent yeast genus, implicated in pectin decomposition and potentially involved in glycolysis and starch and sucrose metabolism. Tatumella, possessing potential for pyruvate kinase, and Fructobacillus with a preference for fructose, constituted the primary bacteria during the pre-turning fermentation stage. Upon the introduction of oxygen into the fermentation mass, acetic acid bacteria ascended to dominant within the microflora. The exponential proliferation of Acetobacter resulted in a decline in taxonomic richness and abundance. Moreover, the identification of novel species within the Komagataeibacter genus suggests that Hainan cocoa may serve as a valuable reservoir for the discovery of unique cocoa fermentation bacteria. The KEGG annotation of metabolites and enzymes also highlighted the significant involvement of phenylalanine metabolism in cocoa fermentation. This research will offer a new perspective for the selection of starter strains and the formulation of mixed starter cultures.

Optimization of cacao beans fermentation by native species and electromagnetic fields.

Acid and bitter notes of the cocoa clone Cacao Castro Naranjal 51 (CCN 51) negatively affect the final quality of the chocolate. Thence, the fermentative process of cocoa beans using native species and electromagnetic fields (EMF) was carried out to evaluate the effect on the yield and quality of CCN 51 cocoa beans. The variables magnetic field density (D), exposure time (T), and inoculum concentration (IC) were optimized through response surface methodology to obtain two statistically validated second-order models, explaining 88.39% and 92.51% of the variability in the yield and quality of the beans, respectively. In the coordinate: 5 mT(D), 22.5 min (T), and 1.6% (CI), yield and bean quality improved to 110% and 120% above the control (without magnetic field). The metagenomic analysis showed that the changes in the microbial communities favored the aroma profile at low and intermediate field densities (5-42 mT) with high yields and floral, fruity, and nutty notes. Conversely, field densities (80 mT) were evaluated with low yields and undesirable notes of acidity and bitterness. The findings revealed that EMF effectively improves the yield and quality of CCN 51 cocoa beans with future applications in the development and quality of chocolate products.

Bioaccessibility of bioactive amines in dark chocolates made with different proportions of under-fermented and fermented cocoa beans.

The influence of under-fermented (UF) cocoa (0 to 65 %) on bioactive amines in chocolate and their in vitro bioaccessibility was investigated. The same amines were found in all treatments; however, treatments were divided into two groups regarding total amines [0 & 20 % UF (34 mg/kg) and 35 to 65 % UF (17 mg/kg)] and phenolic levels [lower and higher, respectively]. Serotonin, tyramine, putrescine, cadaverine, agmatine and phenylethylamine were higher in chocolate with ≤ 20 % UF cocoa. Histamine and spermidine were not affected. Digestibility studies indicated that low levels of amines were present in the oral phase. Gastric digestion was effective in releasing tyramine, spermidine and phenylethylamine from conjugates. Serotonin and agmatine were not detected after in vitro digestion of chocolate with ≥ 35 % UF cocoa. Histamine was released during in vitro intestinal digestion. By adding different proportions of UF cocoa during chocolate production, the levels and bioaccessibility of amines can be modulated.

Yeast strains do have an impact on the production of cured cocoa beans, as assessed with Costa Rican Trinitario cocoa fermentation processes and chocolates thereof.

The microbiological and metabolic outcomes of good cocoa fermentation practices can be standardized and influenced through the addition of starter culture mixtures composed of yeast and bacterial strains. The present study performed two spontaneous and 10 starter culture-initiated (SCI) cocoa fermentation processes (CFPs) in Costa Rica with local Trinitario cocoa. The yeast strains Saccharomyces cerevisiae IMDO 050523, Hanseniaspora opuntiae IMDO 020003, and Pichia kudriavzevii IMDO 060005 were used to compose starter culture mixtures in combination with the lactic acid bacterium strain Limosilactobacillus fermentum IMDO 0611222 and the acetic acid bacterium strain Acetobacter pasteurianus IMDO 0506386. The microbial community and metabolite dynamics of the cocoa pulp-bean mass fermentation, the metabolite dynamics of the drying cocoa beans, and the volatile organic compound (VOC) profiles of the chocolate production were assessed. An amplicon sequence variant approach based on full-length 16S rRNA gene sequencing instead of targeting the V4 region led to a highly accurate monitoring of the starter culture strains added, in particular the Liml. fermentum IMDO 0611222 strain. The latter strain always prevailed over the background lactic acid bacteria. A similar approach, based on the internal transcribed spacer (ITS1) region of the fungal rRNA transcribed unit, was used for yeast strain monitoring. The SCI CFPs evolved faster when compared to the spontaneous ones. Moreover, the yeast strains applied did have an impact. The presence of S. cerevisiae IMDO 050523 was necessary for successful fermentation of the cocoa pulp-bean mass, which was characterized by the production of higher alcohols and esters. In contrast, the inoculation of H. opuntiae IMDO 020003 as the sole yeast strain led to underfermentation and a poor VOC profile, mainly due to its low competitiveness. The P. kudriavzevii IMDO 060005 strain tested in the present study did not contribute to a richer VOC profile. Although differences in VOCs could be revealed in the cocoa liquors, no significant effect on the final chocolates could be obtained, mainly due to a great impact of cocoa liquor processing during chocolate-making. Hence, optimization of the starter culture mixture and cocoa liquor processing seem to be of pivotal importance.