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The influence of magnetic fields on biological systems, including fermentation processes and cocoa bean fermentation, is an area of study that is under development. Mechanisms, such as magnetosensitivity, protein conformational changes, changes to cellular biophysical properties, ROS production, regulation of gene expression, and epigenetic modifications, have been identified to explain how magnetic fields affect microorganisms and cellular processes. These mechanisms can alter enzyme activity, protein stability, cell signaling, intercellular communication, and oxidative stress. In cacao fermentation, electromagnetic fields offer a potential means to enhance the sensory attributes of chocolate by modulating microbial metabolism and optimizing flavor and aroma development. This area of study offers possibilities for innovation and the creation of premium food products. In this review, these aspects will be explored systematically and illustratively. [ABSTRACT FROM AUTHOR]

In Ecuador, various processes are applied during cocoa post-harvesting. This study, therefore, explored fermentation parameters across two locations with 2–7 independent runs, focusing on temperature, microbial counts, pH during fermentation and drying, and their impact on cocoa bean quality. Factors including fermentation devices (jute bags, plastic bags, and wooden boxes), pre-drying, turning during fermentation, fermentation duration, and drying temperature were investigated. Fermenting in plastic bags without pre-drying or turning and fermenting in jute bags for only 40 ± 2.0 h yielded low maximal fermentation temperatures Tmax (31.1 ± 0.4 °C and 37.6 ± 1.8 °C), leading to bitter, astringent, woody, and earthy cocoa liquor. Longer fermentation (63 ± 6 h) in wooden boxes with turning (Wt) and in jute bags with pre-drying and turning (Jpt) achieved the highest Tmax of 46.5 ± 2.0 °C, and a more acidic cocoa liquor, particularly in Wt (both locations) and Jpt (location E). Therefore, it is recommended to ferment for a minimum duration from day 1 to 4 (63 ± 6 h), whether using plastic bags (with mandatory pre-drying) or jute bags (with or without pre-drying or turning). Furthermore, this study underscores the risks associated with excessively high drying temperatures (up to 95.2 ± 13.7 °C) and specific dryer types, which can falsify cut-tests and introduce unwanted burnt-roasted off-flavors in the cocoa liquor. [ABSTRACT FROM AUTHOR]

The influence of magnetic fields on biological systems, including fermentation processes and cocoa bean fermentation, is an area of study that is under development. Mechanisms, such as magnetosensitivity, protein conformational changes, changes to cellular biophysical properties, ROS production, regulation of gene expression, and epigenetic modifications, have been identified to explain how magnetic fields affect microorganisms and cellular processes. These mechanisms can alter enzyme activity, protein stability, cell signaling, intercellular communication, and oxidative stress. In cacao fermentation, electromagnetic fields offer a potential means to enhance the sensory attributes of chocolate by modulating microbial metabolism and optimizing flavor and aroma development. This area of study offers possibilities for innovation and the creation of premium food products. In this review, these aspects will be explored systematically and illustratively.

Fine-aroma cocoa (Theobroma cacao) is one of Ecuador's most iconic export products and comprises 63% of world production. Nevertheless, few advances have been made to improve fermentation processes that might benefit the development of chocolate's organoleptic characteristics. The study of starter cultures, which seek to improve organoleptic properties or decrease fermentation time, has been investigated in other countries. The aim of this study was to analyze the effect of a native microbial cocktail based on two yeasts (Torulaspora delbrueckii and Hanseniaspora uvarum), a lactic acid bacterium (LAB) (Limosilactobacillus plantarum), and an acetic acid bacterium (AAB) (Acetobacter ghanensis) inoculated at the beginning of the fermentative process while tracking physical and biochemical variables, microbial population dynamics, and bean fermentation time. The starter culture caused changes in sugar and acid content and increased polyphenols, which in turn generated temperature and pH changes in the dough. The dynamics of yeast, AAB, and mesophilic microorganisms remain higher than the controls throughout the process. A decrease in filamentous fungi that affect the flavor and quality of beans was observed due to the production of acetic acid or secondary metabolites from yeasts and LAB, and resulted in 24% greater fermentation than spontaneous fermentation in only 96 h. [ABSTRACT FROM AUTHOR]

In Ecuador, various processes are applied during cocoa post-harvesting. This study, therefore, explored fermentation parameters across two locations with 2–7 independent runs, focusing on temperature, microbial counts, pH during fermentation and drying, and their impact on cocoa bean quality. Factors including fermentation devices (jute bags, plastic bags, and wooden boxes), pre-drying, turning during fermentation, fermentation duration, and drying temperature were investigated. Fermenting in plastic bags without pre-drying or turning and fermenting in jute bags for only 40 ± 2.0 h yielded low maximal fermentation temperatures Tmax (31.1 ± 0.4 °C and 37.6 ± 1.8 °C), leading to bitter, astringent, woody, and earthy cocoa liquor. Longer fermentation (63 ± 6 h) in wooden boxes with turning (Wt) and in jute bags with pre-drying and turning (Jpt) achieved the highest Tmax of 46.5 ± 2.0 °C, and a more acidic cocoa liquor, particularly in Wt (both locations) and Jpt (location E). Therefore, it is recommended to ferment for a minimum duration from day 1 to 4 (63 ± 6 h), whether using plastic bags (with mandatory pre-drying) or jute bags (with or without pre-drying or turning). Furthermore, this study underscores the risks associated with excessively high drying temperatures (up to 95.2 ± 13.7 °C) and specific dryer types, which can falsify cut-tests and introduce unwanted burnt-roasted off-flavors in the cocoa liquor.

[Display omitted] • Cocoa fermentation time was reduced by 20% using starter culture. • Kluyveromyces marxianu s from mezcal fermentation prevailed during cocoa fermentation. • Starter culture reduced acetic acid and increased 2-Phenylethyl acetate in cocoa fermented. • Starter cultures lead to the formation of aromatic esters that produce floral notes. Traditional cocoa bean fermentation is a spontaneous process and can result in heterogeneous sensory quality. For this reason, yeast-integrated starter cultures may be an option for creating consistent organoleptic profiles. This study proposes the mixture of Hanseniaspora opuntiae and Kluyveromyces marxianus (from non-cocoa fermentation) as starter culture candidates. The microorganisms and volatile compounds were analyzed during the cocoa fermentation process, and the most abundant were correlated with predominant microorganisms. Results showed that Kluyveromyces marxianus, isolated from mezcal fermentation, was identified as the dominant yeast by high-throughput DNA sequencing. A total of 63 volatile compounds identified by HS-SPME-GC–MS were correlated with the more abundant bacteria and yeast using Principal Component Analysis and Agglomerative Hierarchical Clustering. This study demonstrates that yeasts from other fermentative processes can be used as starter cultures in cocoa fermentation and lead to the formation of more aromatic esters, decrease the acetic acid content. [ABSTRACT FROM AUTHOR]

Compared with other fermentation processes in food industry, cocoa bean fermentation is uncontrolled and not standardized. A detailed mechanistic understanding can therefore be relevant for cocoa bean quality control. Starting from an existing mathematical model of cocoa bean fermentation we analyse five additional biochemical mechanisms derived from the literature. These mechanisms, when added to the baseline model either in isolation or in combination, were evaluated in terms of their capacity to describe experimental data. In total, we evaluated 32 model variants on 23 fermentation datasets. We interpret the results from two perspectives: (1) success of the potential mechanism, (2) discrimination of fermentation protocols based on estimated parameters. The former provides insight in the fermentation process itself. The latter opens an avenue towards reverse-engineering empirical conditions from model parameters. We find support for two mechanisms debated in the literature: consumption of fructose by lactic acid bacteria and production of acetic acid by yeast. Furthermore, we provide evidence that model parameters are sensitive to differences in the cultivar, temperature control and usage of steel tanks compared with wooden boxes. Our results show that mathematical modelling can provide an alternative to standard chemical fingerprinting in the interpretation of fermentation data.

Cocoa bean fermentation is an important process in the manufacturing of cocoa products. It involves microbes, such as lactic acid bacteria, yeast, and acetic acid bacteria. The presence of mold in cocoa bean fermentation is undesired, as it reduces the quality and may produce mycotoxins, which can cause poisoning and death. Aspergillus niger is a fungus that produces ochratoxin A, which is often found in dried agricultural products such as seeds and cereals. In this study, we applied indigenous Candida famata HY-37, Lactobacillus plantarum HL-15, and Acetobacter spp. HA37 as starter cultures for cocoa bean fermentation. We found that the use of L. plantarum HL-15 individually or in combination Candida famata HY-37, Lactobacillus plantarum HL-15, and Acetobacter spp. HA-37 as a starter for cocoa bean fermentation can inhibit the growth of A. niger YAC-9 and the synthesis of ochratoxin A during fermentation and drying. With biological methods that use indigenous Lactobacillus plantarum HL-15 individually or in combination with Candida famata HY-37 and Acetobacter spp. HA-37, we successfully inhibited contamination by ochratoxin-A-producing fungi. Thus, the three indigenous microbes should be used in cocoa bean fermentation to inhibit the growth of fungi that produce mycotoxins and thus improve the quality. [ABSTRACT FROM AUTHOR]

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.

Acetic acid bacteria are involved in many food and beverage fermentation processes. They play an important role in cocoa bean fermentation through their acetic acid production. They initiate the development of some of the flavor precursors that are necessary for the organoleptic quality of cocoa, and for the beans' color. The development of starter cultures with local strains would enable the preservation of the microbial biodiversity of each country in cocoa-producing areas, and would also control the fermentation. This approach could avoid the standardization of cocoa bean fermentation in the producing countries. One hundred and thirty acetic acid bacteria were isolated from three different cocoa-producing countries, and were identified based on their 16S rRNA gene sequence. The predominate strains were grown in a cocoa pulp simulation medium (CPSMAAB) in order to compare their physiological traits regarding their specific growth rate, ethanol and lactic acid consumption, acetic acid production, and relative preferences of carbon sources. Finally, the intraspecific diversity of the strains was then assessed through the analysis of their genomic polymorphism by (GTG)5-PCR fingerprinting. Our results showed that Acetobacter pasteurianus was the most recovered species in all of the origins, with 86 isolates out of 130 cultures. A great similarity was observed between the strains according to their physiological characterization and genomic polymorphisms. However, the multi-parametric clustering results in the different groups highlighted some differences in their basic metabolism, such as their efficiency in converting carbon substrates to acetate, and their relative affinity to lactic acid and ethanol. The A. pasteurianus strains showed different behaviors regarding their ability to oxidize ethanol and lactic acid into acetic acid, and in their relative preference for each substrate. The impact of these behaviors on the cocoa quality should be investigated, and should be considered as a criterion for the selection of acetic acid bacteria starters. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cocoa (Theobroma cacao L) is a tropical plant that is widely used as a basic ingredient in chocolate production. The quality and taste of chocolate are greatly influenced by the stages and processes of post-harvest handling. Some post-harvest processes that are recognized for improving cocoa quality are pod storage, fermentation, and drying. The quality of cocoa beans can be known based on several physical and chemical parameters, including the color and pH of cocoa beans. This study aimed to analyze the effect of pod storage before fermentation (X1), aeration rate (X2) and aeration mode (X3) during the fermentation process on pH (Y1), chroma (Y2), and hue (Y3) of cocoa beans after fermentation and drying. Response surface methodology (RSM) was used to observe the relationships among the X1, X2, X3 variables and the Y1, Y2, and Y3 of dried cocoa beans, with the following treatments: X1 = 1; 5; 9 days, X2 = 0.2; 0.3; 0.4 liters/minute, X3 = intermittent 1; intermittent 2; and continue. The pH, chroma and hue of dried cocoa beans were: 4.873-6.517, 9.99-32.20, and 28.85-71.53, respectively. Furthermore, analysis of variance showed that pod storage before fermentation process significantly affected the hue but did not significantly affect the pH and chroma. The aeration rate had a significant effect on the pH and hue but did not have a significant effect on the chroma, while the aeration mode had no significant effect on the pH, chroma, and hue of dried cocoa beans. [ABSTRACT FROM AUTHOR]

Cocoa bean fermentation is an important process in the manufacturing of cocoa products. It involves microbes, such as lactic acid bacteria, yeast, and acetic acid bacteria. The presence of mold in cocoa bean fermentation is undesired, as it reduces the quality and may produce mycotoxins, which can cause poisoning and death. Aspergillus niger is a fungus that produces ochratoxin A, which is often found in dried agricultural products such as seeds and cereals. In this study, we applied indigenous Candida famata HY-37, Lactobacillus plantarum HL-15, and Acetobacter spp. HA-37 as starter cultures for cocoa bean fermentation. We found that the use of L. plantarum HL-15 individually or in combination Candida famata HY-37, Lactobacillus plantarum HL-15, and Acetobacter spp. HA-37 as a starter for cocoa bean fermentation can inhibit the growth of A. niger YAC-9 and the synthesis of ochratoxin A during fermentation and drying. With biological methods that use indigenous Lactobacillus plantarum HL-15 individually or in combination with Candida famata HY-37 and Acetobacter spp. HA-37, we successfully inhibited contamination by ochratoxin-A-producing fungi. Thus, the three indigenous microbes should be used in cocoa bean fermentation to inhibit the growth of fungi that produce mycotoxins and thus improve the quality.

Fine flavor cocoa is worldwide renowned to produce origin chocolates with special aromas, e.g. fruity or floral, in addition to its chocolate aroma. This research aims to elucidate fine flavor dynamics during fermentation by analyzing the sugar, free amino acid (FAA) and volatile profile. Ecuadorian Trinitario beans (Sacha Gold) were sampled after 0, 18, 24, 48 and 66 h of spontaneous fermentation. The unfermented beans contained significant sucrose, glutamic acid and asparagine amounts while the fermented beans (66 h) contained more flavor precursors, e.g. glucose, fructose, hydrophobic and other FAA. Forty-one volatiles were identified, including 13 fruity- and 12 floral-like, derived from various metabolic pathways. Whereas the level of fatty acid-derived fruity volatiles decreased, the amount of amino acid-derived fruity and floral volatiles increased and floral terpenes remained stable. Some fine volatiles were assumed to be pulp-derived (e.g. linalool, β-myrcene, 2-heptyl acetate) or intrinsic to the bean (e.g. 2-heptanol, 2-heptanone, 2-pentanol), while others were generated during fermentation by microbial synthesis (e.g. 2-phenylethanol, isoamyl alcohol). Multivariate analysis clustered samples according to fermentation time and quality. These findings demonstrate that cocoa fermentation is essential for the formation of flavor precursors and the development or preservation of important fine aroma compounds. Trinitario (or hybrids), one of the cocoa varieties with fine flavor potential, is cultivated all over the world and hence, care should be taken during post-harvest to fully exploit this fine flavor character and deliver high-quality cocoa beans with fine sensory characteristics. [ABSTRACT FROM AUTHOR]

Acetic acid bacteria are involved in many food and beverage fermentation processes. They play an important role in cocoa bean fermentation through their acetic acid production. They initiate the development of some of the flavor precursors that are necessary for the organoleptic quality of cocoa, and for the beans’ color. The development of starter cultures with local strains would enable the preservation of the microbial biodiversity of each country in cocoa-producing areas, and would also control the fermentation. This approach could avoid the standardization of cocoa bean fermentation in the producing countries. One hundred and thirty acetic acid bacteria were isolated from three different cocoa-producing countries, and were identified based on their 16S rRNA gene sequence. The predominate strains were grown in a cocoa pulp simulation medium (CPSM-AAB) in order to compare their physiological traits regarding their specific growth rate, ethanol and lactic acid consumption, acetic acid production, and relative preferences of carbon sources. Finally, the intraspecific diversity of the strains was then assessed through the analysis of their genomic polymorphism by (GTG)5-PCR fingerprinting. Our results showed that Acetobacter pasteurianus was the most recovered species in all of the origins, with 86 isolates out of 130 cultures. A great similarity was observed between the strains according to their physiological characterization and genomic polymorphisms. However, the multi-parametric clustering results in the different groups highlighted some differences in their basic metabolism, such as their efficiency in converting carbon substrates to acetate, and their relative affinity to lactic acid and ethanol. The A. pasteurianus strains showed different behaviors regarding their ability to oxidize ethanol and lactic acid into acetic acid, and in their relative preference for each substrate. The impact of these behaviors on the cocoa quality should be investigated, and should be considered as a criterion for the selection of acetic acid bacteria starters.

Abstract Contamination with filamentous fungi during cocoa bean fermentation and drying reduces the quality of cocoa beans and poses a health risk for consumers due to the potential accumulation of mycotoxins. The aim of this study was to develop anti-fungal lactic acid bacteria (LAB)-yeast co-cultures by selecting anti-fungal strains best adapted to the cocoa bean fermentation process from 362 LAB and 384 yeast strains isolated from cocoa bean post-harvest processes. The applied multiphasic screening approach included anti-fungal activity tests in vitro and in vivo and assessment of the carbon metabolism and stress tolerance of the anti-fungal strains in a cocoa pulp simulation medium. The anti-fungal strains, Lactobacillus fermentum M017, Lb. fermentum 223, Hanseniaspora opuntiae H17, and Saccharomyces cerevisiae H290, were selected based on their high fungal growth inhibition capacity and their well-adapted metabolism. Up to seven filamentous fungal strains of the genera Aspergillus , Penicillium , and Gibberella were inhibited on average by 63 and 75% of the maximal inhibition zone by M017 and 223, respectively, and by 25 and 31% by the strains H17 and H290, respectively. Both Lb. fermentum strains converted the medium's glucose, fructose, and citric acid into 20.4–23.0 g/l of mannitol, 3.9–6.2 g/l acetic acid, and 8.6–10.3 g/l lactic acid, whereas the two yeast strains metabolized glucose and fructose to produce 7.4–18.4 g/l of ethanol. The Lb. fermentum strains were further characterized as particularly tolerant towards ethanol, acetic acid, and heat stress and both yeast strains tolerated high amounts of ethanol and lactic acid in the medium. Finally, the anti-fungal in vivo assays revealed that the two Lb. fermentum strains completely inhibited growth of the citrinin-producing strain, P. citrinum S005, and the potentially fumonisin-producing strain, G. moniliformis S003, on the surface of cocoa beans. Furthermore, growth of the aflatoxin-producer A. flavus S075 was inhibited after 10-14 days by all four selected anti-fungal strains, i.e. Lb. fermentum M017, Lb. fermentum 223, H. opuntiae H17, and Sacc. cerevisiae H290, at 51–95% when applied as single cultures and at 100% when the strains were combined into four co-cultures, each composed of a Lb. fermentum and one of the two yeast strains. As a conclusion, these four LAB-yeast co-cultures are recommended for future applications to limit the growth of filamentous fungi and the concomitant mycotoxin production during the fermentation of cocoa beans. Highlights • Four anti-fungal LAB-yeast co-cultures selected from 362 LAB and 384 yeasts • Broadband anti-fungal activity against up to 7 filamentous fungi in vitro • 100% growth inhibition of aflatoxin-producing strain A. flavus S075 on cocoa beans • Carbon metabolism adapted to substrates typically occurring in cocoa pulp • High tolerance towards ethanol, lactic acid, acetic acid, and/or heat stress [ABSTRACT FROM AUTHOR]

Cocoa beans ( Theobroma cacao L.) are the raw material for chocolate production. Fermentation of the bean pulp by microorganisms is essential for developing the precursors of chocolate flavour. Currently, the cocoa fermentation is still conducted by an uncontrolled traditional process via a consortium of indigenous species of yeasts, lactic acid bacteria and acetic acid bacteria. Although the essential contribution of yeasts to the production of good quality beans and, typical chocolate character is generally agreed, the roles of lactic acid bacteria and acetic acid bacteria are less certain. The objective of this study was to investigate the contribution of LAB and AAB in cocoa bean fermentation by conducting small scale laboratory fermentations under aseptic conditions, inoculated with different groups of microorganisms previously isolated from spontaneous cocoa fermentations. The inoculation protocols were: (1) four yeasts Hanseniaspora guilliermondii , Pichia kudriavzevii , Kluyveromyces marxianus and Saccharomyces cerevisiae ; (2) four yeasts plus the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum ; (3) four yeasts plus the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateuri and (4) four yeasts plus two lactic acid bacteria and two acetic acid bacteria. Only the inoculated species were detected in the microbiota of their respective fermentations. Beans from the inoculated fermentations showed no significant differences in colour, shell weights and concentrations of residual sugars, alcohols and esters (p>0.05), but they were slightly different in contents of lactic acid and acetic acid (p<0.05). All beans were fully brown and free of mould. Residual sugar levels were less than 2.6 mg/g while the shell contents and ethanol were in the range of 11–13.4% and 4.8–7 mg/g, respectively. Beans fermented in the presence of LAB contained higher levels of lactic acid (0.6-1.2 mg/g) whereas higher concentrations of acetic acid (1.8–2.2 mg/g) were detected in beans inoculated with AAB. Triangle and hedonic sensory evaluations of chocolates prepared from beans taken from the three fermentations showed no significant differences (p > 0.05). It was concluded that the growth of lactic acid bacteria and acetic acid bacteria may not be essential for the fermentation of cocoa beans. [ABSTRACT FROM AUTHOR]

Cocoa bean fermentation relies on the sequential activation of several microbial populations, triggering a temporal pattern of biochemical transformations. Understanding this complex process is of tremendous importance as it is known to form the precursors of the resulting chocolate’s flavour and taste. At the same time, cocoa bean fermentation is one of the least controlled processes in the food industry. Here, a quantitative model of cocoa bean fermentation is constructed based on available microbiological and biochemical knowledge. The model is formulated as a system of coupled ordinary differential equations with two distinct types of state variables: (i) metabolite concentrations of glucose, fructose, ethanol, lactic acid and acetic acid and (ii) population sizes of yeast, lactic acid bacteria and acetic acid bacteria. We demonstrate that the model can quantitatively describe existing fermentation time series and that the estimated parameters, obtained by a Bayesian framework, can be used to extract and interpret differences in environmental conditions. The proposed model is a valuable tool towards a mechanistic understanding of this complex biochemical process, and can serve as a starting point for hypothesis testing of new systemic adjustments. In addition to providing the first quantitative mathematical model of cocoa bean fermentation, the purpose of our investigation is to show how differences in estimated parameter values for two experiments allow us to deduce differences in experimental conditions.

Cocoa is part of the cultural heritage in many areas of South and Central America and has played an important role in the history of human culture there. The modern methods of cocoa bean production for the purpose of the manufacture of modern chocolate are tied to the origin and development of cocoa bean fermentation and processing methods and the science of microbiology. To date, however, there has not been a study that discusses the impacts of both science and culture on the evolution of cocoa beans and cocoa bean processing. This work provides both a detailed overview of the evolution and historical development of cocoa, from its earliest forms to modern chocolate manufacturing, an in-depth discussion of the biochemistry of cocoa bean fermentation, as well as a compilation of primary research studies with details on fermentation methods, the scientific bases of interactions in microbial fermentations, and methods for their investigation, as well as metabolites that are produced. As a result, we present here the major microorganisms among all the ones that have been identified in previous studies. This database will aid researchers seeking standardized inoculants to drive cocoa bean fermentation, as well as serve as a guide for inventorying and assessing other food evolution-related studies regarding traditional and artisanal-based food systems. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry ( MALDI- TOF MS) is a powerful biotyping tool increasingly used for high-throughput identification of clinical microbial isolates, however, in food fermentation research this approach is still not well established. This study examines the microbial biodiversity of cocoa bean fermentation based on the isolation of micro-organisms in cocoa-producing regions, followed by MALDI- TOF MS in Switzerland. A preceding 6-week storage test to mimic lengthy transport of microbial samples from cocoa-producing regions to Switzerland was performed with strains of Lactobacillus plantarum, Acetobacter pasteurianus and Saccharomyces cerevisiae. Weekly MALDI- TOF MS analysis was able to successfully identify microbiota to the species level after storing live cultures on slant agar at mild temperatures (7°C) and/or in 75% aqueous ethanol at differing temperatures (−20, 7 and 30°C). The efficacy of this method was confirmed by on-site recording of the microbial biodiversity in cocoa bean fermentation in Bolivia and Brazil, with a total of 1126 randomly selected isolates. MALDI- TOF MS analyses revealed known dominant cocoa bean fermentation species with Lact. plantarum and Lactobacillus fermentum in the lactic acid bacteria taxon, Hanseniaspora opuntiae and S. cerevisiae in the yeast taxon, and Acet. pasteurianus, Acetobacter fabarum, Acetobacter ghanensis and Acetobacter senegalensis in the acetic acid bacteria taxon. Significance and Impact of the Study Microbial identification with MALDI-TOF MS has increased the number of samples that can be analysed in a given time, a prerequisite for high-throughput methods. This method is already widely used for the identification of clinical microbial isolates, whereas in food fermentation research, including cocoa bean fermentation, microbiota is mostly identified by time-consuming, biochemical-based phenotyping and molecular approaches. This study presents the use of MALDI-TOF MS for characterizing the microbial biodiversity of cocoa bean fermentation. The feasibility of MALDI-TOF MS identification of cocoa-specific microbiota has been shown with samples collected during on-site studies in two countries of origin, Bolivia and Brazil. [ABSTRACT FROM AUTHOR]

Acetobacter ghanensis LMG 23848 T and Acetobacter senegalensis 108B are acetic acid bacteria that originate from a spontaneous cocoa bean heap fermentation process and that have been characterised as strains with interesting functionalities through metabolic and kinetic studies. As there is currently little genetic information available for these species, whole-genome sequencing of A. ghanensis LMG 23848 T and A. senegalensis 108B and subsequent data analysis was performed. This approach not only revealed characteristics such as the metabolic potential and genomic architecture, but also allowed to indicate the genetic adaptations related to the cocoa bean fermentation process. Indeed, evidence was found that both species possessed the genetic ability to be involved in citrate assimilation and displayed adaptations in their respiratory chain that might improve their competitiveness during the cocoa bean fermentation process. In contrast, other properties such as the dependence on glycerol or mannitol and lactate as energy sources or a less efficient acid stress response may explain their low competitiveness. The presence of a gene coding for a proton-translocating transhydrogenase in A. ghanensis LMG 23848 T and the genes involved in two aromatic compound degradation pathways in A. senegalensis 108B indicate that these strains have an extended functionality compared to Acetobacter species isolated from other ecosystems. [ABSTRACT FROM AUTHOR]

Cocoa bean fermentation is still a spontaneous curing process to facilitate drying of nongerminating cocoa beans by pulp removal as well as to stimulate colour and flavour development of fermented dry cocoa beans. As it is carried out on farm, cocoa bean fermentation is subjected to various agricultural and operational practices and hence fermented dry cocoa beans of variable quality are obtained. Spontaneous cocoa bean fermentations carried out with care for approximate four days are characterized by a succession of particular microbial activities of three groups of micro-organisms, namely yeasts, lactic acid bacteria ( LAB) and acetic acid bacteria ( AAB), which results in well-fermented fully brown cocoa beans. This has been shown through a plethora of studies, often using a multiphasic experimental approach. Selected strains of several of the prevailing microbial species have been tested in appropriate cocoa pulp simulation media to unravel their functional roles and interactions as well as in small plastic vessels containing fresh cocoa pulp-bean mass to evaluate their capacity to dominate the cocoa bean fermentation process. Various starter cultures have been proposed for successful fermentation, encompassing both cocoa-derived and cocoa nonspecific strains of (hybrid) yeasts, LAB and AAB, some of which have been implemented on farms successfully. [ABSTRACT FROM AUTHOR]

Background: Lactobacillus fermentum 222 and Lactobacillus plantarum 80, isolates from a spontaneous Ghanaian cocoa bean fermentation process, proved to be interesting functional starter culture strains for cocoa bean fermentations. Lactobacillus fermentum 222 is a thermotolerant strain, able to dominate the fermentation process, thereby converting citrate and producing mannitol. Lactobacillus plantarum 80 is an acid-tolerant and facultative heterofermentative strain that is competitive during cocoa bean fermentation processes. In this study, whole-genome sequencing and comparative genome analysis was used to investigate the mechanisms of these strains to dominate the cocoa bean fermentation process. Results: Through functional annotation and analysis of the high-coverage contigs obtained through 454 pyrosequencing, plantaricin production was predicted for L. plantarum 80. For L. fermentum 222, genes encoding a complete arginine deiminase pathway were attributed. Further, in-depth functional analysis revealed the capacities of these strains associated with carbohydrate and amino acid metabolism, such as the ability to use alternative external electron acceptors, the presence of an extended pyruvate metabolism, and the occurrence of several amino acid conversion pathways. A comparative genome sequence analysis using publicly available genome sequences of strains of the species L. plantarum and L. fermentum revealed unique features of both strains studied. Indeed, L. fermentum 222 possessed genes encoding additional citrate transporters and enzymes involved in amino acid conversions, whereas L. plantarum 80 is the only member of this species that harboured a gene cluster involved in uptake and consumption of fructose and/or sorbose. Conclusions: In-depth genome sequence analysis of the candidate functional starter culture strains L. fermentum 222 and L. plantarum 80 revealed their metabolic capacities, niche adaptations and functionalities that enable them to dominate the cocoa bean fermentation process. Further, these results offered insights into the cocoa bean fermentation ecosystem as a whole and will facilitate the selection of appropriate starter culture strains for controlled cocoa bean fermentation processes. [ABSTRACT FROM AUTHOR]

A high-resolution functional metagenomic analysis of a representative single sample of a Brazilian spontaneous cocoa bean fermentation process was carried out to gain insight into its bacterial community functioning. By reconstruction of microbial meta-pathways based on metagenomic data, the current knowledge about the metabolic capabilities of bacterial members involved in the cocoa bean fermentation ecosystem was extended. Functional meta-pathway analysis revealed the distribution of the metabolic pathways between the bacterial members involved. The metabolic capabilities of the lactic acid bacteria present were most associated with the heterolactic fermentation and citrate assimilation pathways. The role of Enterobacteriaceae in the conversion of substrates was shown through the use of the mixed-acid fermentation and methylglyoxal detoxification pathways. Furthermore, several other potential functional roles for Enterobacteriaceae were indicated, such as pectinolysis and citrate assimilation. Concerning acetic acid bacteria, metabolic pathways were partially reconstructed, in particular those related to responses toward stress, explaining their metabolic activities during cocoa bean fermentation processes. Further, the in-depth metagenomic analysis unveiled functionalities involved in bacterial competitiveness, such as the occurrence of CRISPRs and potential bacteriocin production. Finally, comparative analysis of the metagenomic data with bacterial genomes of cocoa bean fermentation isolates revealed the applicability of the selected strains as functional starter cultures. [ABSTRACT FROM AUTHOR]

To monitor dominant species of lactic acid bacteria during cocoa bean fermentation, i.e. Lactobacillus plantarum and Lactobacillus fermentum , a fast and reliable culture-independent qPCR assay was developed. A modified DNA isolation procedure using a commercial kit followed by two species-specific qPCR assays resulted in 100% sensitivity for L. plantarum and L. fermentum . Kruskal–Wallis and post-hoc analyses of data obtained from experiments with cocoa beans that were artificially spiked with decimal concentrations of L. plantarum and L. fermentum strains allowed the calculation of a regression line suitable for the estimation of both species with a detection limit of 3 to 4 Log cells/g cocoa beans. This process was successfully tested for efficacy through the analyses of samples from laboratory-scale cocoa bean fermentations with both the qPCR assay and a culture-dependent method which resulted in comparable results. [ABSTRACT FROM AUTHOR]

Cocoa beans ( Theobroma cacao L.) are the raw material for chocolate production. Fermentation of cocoa pulp by microorganisms is crucial for developing chocolate flavor precursors. Yeasts conduct an alcoholic fermentation within the bean pulp that is essential for the production of good quality beans, giving typical chocolate characters. However, the roles of bacteria such as lactic acid bacteria and acetic acid bacteria in contributing to the quality of cocoa bean and chocolate are not fully understood. Using controlled laboratory fermentations, this study investigated the contribution of lactic acid bacteria to cocoa bean fermentation. Cocoa beans were fermented under conditions where the growth of lactic acid bacteria was restricted by the use of nisin and lysozyme. The resultant microbial ecology, chemistry and chocolate quality of beans from these fermentations were compared with those of indigenous (control) fermentations. The yeasts Hanseniaspora guilliermondii , Pichia kudriavzevii , Kluyveromyces marxianus and Saccharomyces cerevisiae , the lactic acid bacteria Lactobacillus plantarum , Lactobacillus pentosus and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in control fermentations. In fermentations with the presence of nisin and lysozyme, the same species of yeasts and acetic acid bacteria grew but the growth of lactic acid bacteria was prevented or restricted. These beans underwent characteristic alcoholic fermentation where the utilization of sugars and the production of ethanol, organic acids and volatile compounds in the bean pulp and nibs were similar for beans fermented in the presence of lactic acid bacteria. Lactic acid was produced during both fermentations but more so when lactic acid bacteria grew. Beans fermented in the presence or absence of lactic acid bacteria were fully fermented, had similar shell weights and gave acceptable chocolates with no differences in sensory rankings. It was concluded that lactic acid bacteria may not be necessary for successful cocoa fermentation. [ABSTRACT FROM AUTHOR]

Abstract: Cocoa beans (Theobroma cacao) are the major raw material for chocolate production and fermentation of the beans is essential for the development of chocolate flavor precursors. In this study, a novel approach was used to determine the role of yeasts in cocoa fermentation and their contribution to chocolate quality. Cocoa bean fermentations were conducted with the addition of 200ppm Natamycin to inhibit the growth of yeasts, and the resultant microbial ecology and metabolism, bean chemistry and chocolate quality were compared with those of normal (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii and Kluyveromyces marxianus, the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in the control fermentation. In fermentations with the presence of Natamycin, the same bacterial species grew but yeast growth was inhibited. Physical and chemical analyses showed that beans fermented without yeasts had increased shell content, lower production of ethanol, higher alcohols and esters throughout fermentation and lesser presence of pyrazines in the roasted product. Quality tests revealed that beans fermented without yeasts were purplish-violet in color and not fully brown, and chocolate prepared from these beans tasted more acid and lacked characteristic chocolate flavor. Beans fermented with yeast growth were fully brown in color and gave chocolate with typical characters which were clearly preferred by sensory panels. Our findings demonstrate that yeast growth and activity were essential for cocoa bean fermentation and the development of chocolate characteristics. [Copyright &y& Elsevier]

Background: Acetobacter pasteurianus 386B, an acetic acid bacterium originating from a spontaneous cocoa bean heap fermentation, proved to be an ideal functional starter culture for coca bean fermentations. It is able to dominate the fermentation process, thereby resisting high acetic acid concentrations and temperatures. However, the molecular mechanisms underlying its metabolic capabilities and niche adaptations are unknown. In this study, whole-genome sequencing and comparative genome analysis was used to investigate this strain's mechanisms to dominate the cocoa bean fermentation process. Results: The genome sequence of A. pasteurianus 386B is composed of a 2.8-Mb chromosome and seven plasmids. The annotation of 2875 protein-coding sequences revealed important characteristics, including several metabolic pathways, the occurrence of strain-specific genes such as an endopolygalacturonase, and the presence of mechanisms involved in tolerance towards various stress conditions. Furthermore, the low number of transposases in the genome and the absence of complete phage genomes indicate that this strain might be more genetically stable compared with other A. pasteurianus strains, which is an important advantage for the use of this strain as a functional starter culture. Comparative genome analysis with other members of the Acetobacteraceae confirmed the functional properties of A. pasteurianus 386B, such as its thermotolerant nature and unique genetic composition. Conclusions: Genome analysis of A. pasteurianus 386B provided detailed insights into the underlying mechanisms of its metabolic features, niche adaptations, and tolerance towards stress conditions. Combination of these data with previous experimental knowledge enabled an integrated, global overview of the functional characteristics of this strain. This knowledge will enable improved fermentation strategies and selection of appropriate acetic acid bacteria strains as functional starter culture for cocoa bean fermentation processes. [ABSTRACT FROM AUTHOR]

Abstract: Two spontaneous Malaysian cocoa bean box fermentations (one farm, two plantation plots) were investigated. Physical parameters, microbial community dynamics, yeast and bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the respective fermented dry cocoa beans. Similar microbial growth and metabolite profiles were obtained for the two fermentations. Low concentrations of citric acid were found in the fresh pulp, revealing low acidity of the raw material. The main end-products of the catabolism of the pulp substrates glucose, fructose, and citric acid by yeasts, LAB, and AAB were ethanol, lactic acid, acetic acid, and/or mannitol. Hanseniaspora opuntiae, Lactobacillus fermentum, and Acetobacter pasteurianus were the prevalent species of the two fermentations. Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus pentosus, and Acetobacter ghanensis were also found during the mid-phase of the fermentation processes. Leuconostoc pseudomesenteroides and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Tatumella saanichensis and Enterobacter sp. were present in the beginning of the fermentations and they could be responsible for the degradation of citric acid and/or the production of gluconic acid and lactic acid, respectively. The presence of facultative heterofermentative LAB during the fermentations caused a high production of lactic acid. Finally, as these fermentations were carried out with high-quality raw material and were characterised by a restricted microbial species diversity, resulting in successfully fermented dry cocoa beans and good chocolates produced thereof, it is likely that the prevailing species H. opuntiae, S. cerevisiae, Lb. fermentum, and A. pasteurianus were responsible for it. [Copyright &y& Elsevier]

Abstract: To speed up research on the usefulness and selection of bacterial starter cultures for cocoa bean fermentation, a benchmark cocoa bean fermentation process under natural fermentation conditions was developed successfully. Therefore, spontaneous fermentations of cocoa pulp-bean mass in vessels on a 20 kg scale were tried out in triplicate. The community dynamics and kinetics of these fermentations were studied through a multiphasic approach. Microbiological analysis revealed a limited bacterial species diversity and targeted community dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation, as was the case during cocoa bean fermentations processes carried out in the field. LAB isolates belonged to two main (GTG)5-PCR clusters, namely Lactobacillus plantarum and Lactobacillus fermentum, with Fructobacillus pseudofilculneus occurring occasionally; one main (GTG)5-PCR cluster, composed of Acetobacter pasteurianus, was found among the AAB isolates, besides minor clusters of Acetobacter ghanensis and Acetobacter senegalensis. 16S rRNA-PCR-DGGE revealed that L. plantarum and L. fermentum dominated the fermentations from day two until the end and Acetobacter was the only AAB species present at the end of the fermentations. Also, species of Tatumella and Pantoea were detected culture-independently at the beginning of the fermentations. Further, it was shown through metabolite target analyses that similar substrate consumption and metabolite production kinetics occurred in the vessels compared to spontaneous cocoa bean fermentation processes. Current drawbacks of the vessel fermentations encompassed an insufficient mixing of the cocoa pulp-bean mass and retarded yeast growth. [Copyright &y& Elsevier]

Abstract: Samples of cocoa beans were taken on two separate occasions during heap and tray fermentations in Ghana, West Africa. In total 496 yeast isolates were identified by conventional microbiological analyses and by amplification of their ITS1-5.8S rDNA-ITS2 regions. For important species the identifications were confirmed by sequencing of the D1/D2 domain of the 5′ end of the large subunit (26S) rDNA. Assimilations of organic acids and other carbon compounds were conducted. For dominant yeasts intraspecies variations were examined by determination of chromosome length polymorphism (CLP) using pulsed-field gel electrophoresis. For the heap fermentations maximum yeast cell counts of 9.1×107 were reached, whereas maximum yeast counts of 6.0×106 were reached for the tray fermentations. Candida krusei was found to be the dominant species during heap fermentation, followed by P. membranifaciens, P. kluyveri, Hanseniaspora guilliermondii and Trichosporon asahii, whereas Saccharomyces cerevisiae and P. membranifaciens were found to be the dominant species during tray fermentation followed by low numbers of C. krusei, P. kluyveri, H. guilliermondii and some yeast species of minor importance. For isolates within all dominant species CLP was evident, indicating that several different strains are involved in the fermentations. Isolates of C. krusei, P. membranifaciens, H. guilliermondii, T. asahii and Rhodotorula glutinis could be found on the surface of the cocoa pods and in some cases on the production equipment, whereas the origin of e.g. S. cerevisiae was not indicated by the results obtained. In conclusion, the results obtained show that fermentation of cocoa beans is a very inhomogeneous process with great variations in both yeast counts and species composition. The variations seem to depend especially on the processing procedure, but also the season and the post-harvest storage are likely to influence the yeast counts and the species composition. [Copyright &y& Elsevier]

• Temperature and acetic acid are the greatest influencers of cocoa fermentation. • Factors involved in cocoa fermentation show high degree of interaction. • Outcome of cocoa fermentation can be predicted using controlled incubation systems. Cocoa bean fermentation still remains a rather empirical process. The research presented here employed an artificial system of fermentation, using controlled incubations, in order to achieve greater control over the external influences that cocoa beans are exposed to, with the aim of experimentally modelling changes to bean components (responses). Experimental design was used, in a first-ever attempt, to study the effects of five factors and their interactions on the profiles of pH, peptides, and flavanols in the bean during the incubations. Temperature, incubation time and the concentration of acetic acid were the main factors influencing the three responses. Moreover, there was a significant amount of factor interaction, revealing the process to be more complex than initially thought, especially with respect to the role of ethanol. Using the model, one was also able to accurately predict the response of the bean to the exposure to specific factors. [ABSTRACT FROM AUTHOR]