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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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Fructophilic lactic acid bacteria (FLAB) are a recently discovered group whose main characteristic is to prefer D-fructose over D-glucose. In this study, laboratory cocoa beans fermentation was analyzed by Illumina-based amplicon sequencing, indicating the presence of potential FLAB of the genera Fructobacillus and Lactobacillus. Eighty efficient fructose-fermenting isolates, obtained from fermenting cocoa pulp beans mass, were identified by 16S rRNA gene sequencing as Pediococcus acidilactici (n = 52), Lactobacillus plantarum (n = 10), Pediococcus pentosaceus (n = 10), Bacillus subtilis (n = 4), and Leuconostoc pseudomesenteroides (n = 4). The growth characteristics of all the 10 L. plantarum strains classified them as "facultatively" fructophilic bacteria, i.e., they grew on glucose without an external electron acceptor but the growth on fructose was faster. Among them, L. plantarum LPBF 35 was characterized by producing a range of aroma-impacting compounds (acetaldehyde, ethyl acetate, nonanal, and octanoic acid), being introduced into a cocoa fermentation process. Although the process started with approximately equal amounts of glucose and fructose, a concomitant, but faster utilization of fructose, was observed in cocoa fermentation conducted with L. plantarum LPBF 35 (with no residual fructose observed) when compared to control fermentation using a glucophilic strain (8.77 mg/g residual fructose) and a spontaneous process (8.38 mg/g residual fructose). L. plantarum LPBF 35 also showed an ideal profile of organic acid metabolism (citric acid consumption and lactic acid production) associated with cocoa fermentation. These results proved new insights on cocoa microbial activity and brings new perspectives on the use of lactic acid bacteria as starter culture.

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Cocoa fermentation is a spontaneous process shaped by a variable microbial ecosystem which is assembled due to cross-feeding relationship among yeasts and bacteria, resulting in a synchronized microbial succession started by yeasts, followed by lactic acid bacteria (LAB) and finalized by acetic acid bacteria (AAB). Several studies have indicated the effect of microbial interactions in food ecosystems highlighting the importance of quorum sensing (QS) in bacterial adaptation in harsh environments modulating several phenotypes such as biofilm formation, tolerance to acid stress, bacteriocin production, competence, morphological modifications, motility, among others. However, antagonic interactions also occur, and can be marked by Quorum Quenching (QQ) activity, negatively impacting QS regulated phenotypes. Our current knowledge regarding microbial cocoa composition and functioning is based on culture-based analysis and culture-independent PCR-based methods. Therefore, we set out to investigate the application of metagenomics analysis on a classical spontaneous cocoa fermentation in order to describe: (I) the microbial taxonomic composition; (II) the functional potential of the cocoa microbiome; (III) the microbiome putative QS potential; and (IV) the microbiome QQ potential. Both aims III and IV are related to the expression of effectors that may confer advantageous traits along fermentation which can explain their dominance in specific time zones during the entire process. We have observed a bacterial succession shaped by yeasts and filamentous fungi and then Enterobacteriaceales, LAB and AAB, as well as a diverse genetic metabolic potential related to proteins and carbohydrates metabolism associated to the yeast Saccharomyces cerevisiae and members of the Enterobacteriaceales order and LAB and AAB groups. In addition, in silico evidences of interspecific QS arsenal were found in members of the genera Enterobacter, Lactobacillus, Bacillus and Pantoea, while inferences of intraspecific QS potential were found in the members of the genera Bacillus, Enterobacter, Komagataeibacter, Lactobacillus and Pantoea. In addition, a QQ potential was detected in Lactobacillus and in AAB members. These findings indicate that QS and QQ may modulate bacterial dominance in different time points during fermentation, along with cross-feeding, being responsible for their maintenance in a large time range.

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