Species diversity, community dynamics, and metabolite kinetics of the microbiota associated with traditional ecuadorian spontaneous cocoa bean fermentations

Traditional fermentations of the local Ecuadorian cocoa type Nacional, with its fine flavor, are carried out in boxes and on platforms for a short time. A multiphasic approach, encompassing culture-dependent and -independent microbiological analyses of fermenting cocoa pulp-bean samples, metabolite target analyses of both cocoa pulp and beans, and sensory analysis of chocolates produced from the respective fermented dry beans, was applied for the investigation of the influence of these fermentation practices on the yeast and bacterial species diversity and community dynamics during cocoa bean fermentation. A wide microbial species diversity was found during the first 3 days of all fermentations carried out. The prevailing ethanol-producing yeast species were Pichia kudriavzevii and Pichia manshurica, followed by Saccharomyces cerevisiae. Leuconostoc pseudomesenteroides (glucose and fructose fermenting), Fructobacillus tropaeoli-like (fructose fermenting), and Lactobacillus fermentum (citrate converting, mannitol producing) represented the main lactic acid bacterial species in the fermentations studied, resulting in intensive heterolactate metabolism of the pulp substrates. Tatumella saanichensis and Tatumella punctata were among the members of the family Enterobacteriaceae present during the initial phase of the cocoa bean fermentations and could be responsible for the production of gluconic acid in some cases. Also, a potential new yeast species was isolated, namely, Candida sorbosivorans-like. Acetic acid bacteria, whose main representative was Acetobacter pasteurianus, generally appeared later during fermentation and oxidized ethanol to acetic acid. However, acetic acid bacteria were not always present during the main course of the platform fermentations. All of the data taken together indicated that short box and platform fermentation methods caused incomplete fermentation, which had a serious impact on the quality of the fermented dry cocoa beans.     

Influence of Turning and Environmental Contamination on the Dynamics of Populations of Lactic Acid and Acetic Acid Bacteria Involved in Spontaneous Cocoa Bean Heap Fermentation in Ghana

The influence of turning and environmental contamination on six spontaneous cocoa bean heap fermentations performed in Ghana was studied through a multiphasic approach, encompassing both microbiological (culture-dependent and culture-independent techniques) and metabolite target analyses. A sensory analysis of chocolate made from the fermented, dried beans was performed as well. Only four clusters were found among the isolates of acetic acid bacteria (AAB) identified: Acetobacter pasteurianus, Acetobacter ghanensis, Acetobacter senegalensis, and a potential new Acetobacter lovaniensis-like species. Two main clusters were identified among the lactic acid bacteria (LAB) isolated, namely, Lactobacillus plantarum and Lactobacillus fermentum. No differences in biodiversity of LAB and AAB were seen for fermentations carried out at the farm and factory sites, indicating the cocoa pod surfaces and not the general environment as the main inoculum for spontaneous cocoa bean heap fermentation. Turning of the heaps enhanced aeration and increased the relative population size of AAB and the production of acetic acid. This in turn gave a more sour taste to chocolate made from these beans. Bitterness was reduced through losses of polyphenols and alkaloids upon fermentation and cocoa bean processing.     

THE MIGRATION OF PASSERINE NIGHT MIGRANTS ACROSS THE ENGLISH CHANNEL STUDIED BY RADAR

An account is given of the migration of British summer resident small passerine night migrants across the English Channel in spring and autumn, as detected by radar.
The main times of passage of each species have been determined from the trapping records of Dungeness Bird Observatory.
The tracks of the migrants are consistent with the view that they always head about NNW in spring and SSE in autumn but may be drifted by a cross-wind.
The wind-direction and speed had a paramount influence on the volume of migration in both spring and autumn. Temperature had no effect in autumn, and though there was more migration with warmer than cold weather in spring, this need not have been due to the influence of temperature as such.
Small reversed movements occurred, with southerly winds in autumn or, more doubtfully, with northerly winds in spring.     

Cocoa modulatory effect on rat faecal microbiota and colonic crosstalk

Previous studies have reported the effect of a cocoa-enriched diet on the intestinal immune system in rats. Cocoa contains fibre and polyphenols that can directly influence the intestinal ecosystem and its relationship with the immune system. The aim of this study was to evaluate the effects of a cocoa-enriched diet on gut microbiota, toll-like receptor (TLR) expression and immunoglobulin (Ig) A (IgA) intestinal secretion in rats. Four-week-old Wistar rats were fed a standard or cocoa diet for 6 weeks. Faecal samples were collected before the beginning of the diet and at the end of the study. After the nutritional intervention, colon samples were obtained to quantify TLR and IgA gene expression and IgA protein. Microbiota composition was characterized by fluorescent in situ hybridization (FISH) coupled to flow cytometry (FCM) analysis using specific probes directed to 16S rRNA of the main bacteria genus present in rat intestine. The cocoa dietary intervention resulted in a differential TLR pattern and a decrease in the intestinal IgA secretion and IgA-coating bacteria. Moreover there was a significant decrease in the proportion of Bacteroides, Clostridium and Staphylococcus genera in the faeces of cocoa-fed animals. In conclusion, cocoa intake affects the growth of certain species of gut microbiota in rats and is associated with changes in the TLR pattern which could be responsible for the changes observed in the intestinal immune system.     

Sensitive method for determination of DON in cocoa by means of HPLC-techniques

The mycotoxin deoxynivalenol (DON) is one of a group of mycotoxins known as type B trichothecenes and is particularly formed by the mould speciesFusarium graminearum andFusarium culmorum. The frequency of the occurrence of DON in certain raw materials and the concentrations found make it one of the world’s most significant mycotoxin contaminants. Positive findings of the toxin especially have been established in cereal-based foods, as well as in oilseeds. The main objective of this study was to set up a current situation assessment of the possible occurrence of deoxynivalenol in cocoa and cocoa products. As there was no analytical method for determining DON in cocoa and cocoa products, a special method was developed. The applicability and consistency of the method was confirmed by performing recovery assays on various cocoa products. A special post-column derivatisation procedure was developed to increase selectivity and raise sensitivity by a factor of 80. The method was used to test 230 samples for possible DON content, ranging from cocoa beans to cocoa bean shells, nibs, cocoa liquor and cocoa powders through to finished cocoa-based products. The results suggest that DON may occasionally occur in cocoa beans in very low concentrations. Presented at the 29^th Mykotoxin-Workshop, Fellbach, Germany, May 14–16, 2007     

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.     

Biodiversity conservation in cocoa production landscapes: an overview

Cocoa agroforests that retain a floristically diverse and structurally complex shade canopy have the potential to harbour significant levels of biodiversity, yet few studies have documented the plant and animal species occurring within these systems or within landscapes dominated by cocoa production. In this special issue, we bring together nine studies from Latin America, Africa and Asia that document the contribution of cocoa agroforestry systems to biodiversity conservation, and explore how the design, management and location of these systems within the broader landscape influence their value as habitats, resources and biological corridors. Tree diversity within the cocoa production systems is variable, depending on management, cultural differences, location and farm history, among other factors. Animal diversity is typically highest in those cocoa agroforests that have high plant diversity, structurally complex canopies, and abundant surrounding forest cover. In general, both plant and animal diversity within cocoa agroforests is greater than those of other agricultural land uses, but lower than in the original forest habitat. There are several emerging threats to biodiversity conservation within cocoa production landscapes, including the loss of remaining forest cover, the simplification of cocoa shade canopies and the conversion of cocoa agroforestry systems to other agricultural land uses with lower biodiversity value. To counter these threats and conserve biodiversity over the long-term, land management should focus on conserving native forest habitat within cocoa production landscapes, maintaining or restoring floristically diverse and structurally complex shade canopies within cocoa agroforests, and retaining other types of on-farm tree cover to enhance landscape connectivity and habitat availability.     

The geology of Kaldvelladalen

Langvatn lake is a large waterfilled kettle hole in glaciofluvial deposits developed during the last deglaciation. The main outflow from the lake occurs by percolation through the permeable sediments at the shoreline.
The sedimentary rocks and sediments (calcite bearing) within the watershed influence the quality of the lake waters.     

Intestinal immune system of young rats influenced by cocoa-enriched diet

Gut-associated lymphoid tissue (GALT) maintains mucosal homeostasis by counteracting pathogens and inducing a state of nonresponsiveness when it receives signals from food antigens and commensal bacteria. We report for the first time the influence of continuous cocoa consumption on GALT function in rats postweaning. Weaned Wistar rats were fed cocoa-enriched diets (4% or 10% food intake) for 3 weeks. The function of the primary inductive sites of GALT, such as Peyer's patches (PP) and mesenteric lymph nodes (MLN), was evaluated through an analysis of IgA-secretory ability and lymphocyte composition (T, B and natural killer cells), activation (IL-2 secretion and IL-2 receptor alpha expression) and proliferation. T-helper effector cell balance was also established based on cytokine profile (interferon gamma, IL-4 and IL-10) after mitogen activation. A 10% cocoa intake induced significant changes in PP and MLN lymphocyte composition and function, whereas a 4% cocoa diet did not cause significant modifications in either tissues. Cocoa diet strongly reduced secretory IgA (S-IgA) in the intestinal lumen, although IgA's secretory ability was only slightly decreased in PP. In addition, the 10% cocoa diet increased T-cell-antigen receptor gammadelta cell proportion in both lymphoid tissues. Thus, cocoa intake modulates intestinal immune responses in young rats, influencing gammadelta T-cells and S-IgA production.     

Cacao biotechnology: current status and future prospects

Theobroma cacao—The Food of the Gods, provides the raw material for the multibillion dollar chocolate industry and is also the main source of income for about 6 million smallholders around the world. Additionally, cocoa beans have a number of other nonfood uses in the pharmaceutical and cosmetic industries. Specifically, the potential health benefits of cocoa have received increasing attention as it is rich in polyphenols, particularly flavonoids. At present, the demand for cocoa and cocoa‐based products in Asia is growing particularly rapidly and chocolate manufacturers are increasing investment in this region. However, in many Asian countries, cocoa production is hampered due to many reasons including technological, political and socio‐economic issues. This review provides an overview of the present status of global cocoa production and recent advances in biotechnological applications for cacao improvement, with special emphasis on genetics/genomics, in vitro embryogenesis and genetic transformation. In addition, in order to obtain an insight into the latest innovations in the commercial sector, a survey was conducted on granted patents relating to T. cacao biotechnology.     

Ant assemblage structure on cocoa trees in smallholder farms in the Centre Region of Cameroon

We investigated the ant community structure in cocoa farms in the Centre Region of Cameroon. Ants were collected on the cocoa trees during the years 2006 and 2007 using chemical knock‐down. We tested the hypothesis of the existence of deterministic factor in the structuration of ant mosaic using C‐Score; we assessed the relationship between the numerical dominant and subdominant ant species using Spearman correlation test and discussed on the influence of vegetation structure and farm management on the ant community structure. A total of 53 ant species belonging to 20 genera and five subfamilies were identified from a set of 51,525 workers collected. C‐score analysis supported the hypothesis that ant community were structured by competition. Negative relationships were found between dominant ant species. Farming practices which were mainly pruning, chemical treatment and habitat structure appeared to influence the ecological status and distribution of dominant ant species.     

Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers

We evaluated the levels of (-)-epicatechin (EC) and its metabolites in plasma and urine after intake of chocolate or cocoa by male volunteers. EC metabolites were analyzed by HPLC and LC/MS after glucuronidase and/or sulfatase treatment. The maximum levels of total EC metabolites in plasma were reached 2 hours after either chocolate or cocoa intake. Sulfate, glucuronide, and sulfoglucuronide (mixture of sulfate and glucuronide) conjugates of nonmethylated EC were the main metabolites present in plasma rather than methylated forms. Urinary excretion of total EC metabolites within 24 hours after chocolate or cocoa intake was 29.8 ± 5.3% and 25.3 ± 8.1% of total EC intake. EC in chocolate and cocoa was partly absorbed and was found to be present as a component of various conjugates in plasma, and these were rapidly excreted in urine.     

Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers

We evaluated the levels of (-)-epicatechin (EC) and its metabolites in plasma and urine after intake of chocolate or cocoa by male volunteers. EC metabolites were analyzed by HPLC and LC/MS after glucuronidase and/or sulfatase treatment. The maximum levels of total EC metabolites in plasma were reached 2 hours after either chocolate or cocoa intake. Sulfate, glucuronide, and sulfoglucuronide (mixture of sulfate and glucuronide) conjugates of nonmethylated EC were the main metabolites present in plasma rather than methylated forms. Urinary excretion of total EC metabolites within 24 hours after chocolate or cocoa intake was 29.8 ± 5.3% and 25.3 ± 8.1% of total EC intake. EC in chocolate and cocoa was partly absorbed and was found to be present as a component of various conjugates in plasma, and these were rapidly excreted in urine.     

Landscape and farm scale management to enhance biodiversity conservation in the cocoa producing region of southern Bahia, Brazil

In southern Bahia, Brazil, large land areas are used for the production of cocoa (Theobroma cacao), which is predominantly grown under the shade of native trees in an agroforestry system locally known as cabruca. As a dominant forest-like landscape element of the cocoa region, the cabrucas play an important role in the conservation of the region’s biodiversity. The purpose of this review is to provide the scientific basis for an action plan to reconcile cocoa production and biodiversity conservation in southern Bahia. The available research collectively highlights the diversity of responses of different species and biological groups to both the habitat quality of the cabrucas themselves and to the general characteristics of the landscape, such as the relative extent and spatial configuration of different vegetation types within the landscape mosaic. We identify factors that influence directly or indirectly the occurrence of native species in the cabrucas and the wider landscape of the cocoa region and develop recommendations for their conservation management. We show that the current scientific knowledge already provides a good basis for a biodiversity friendly management of the cocoa region of southern Bahia, although more work is needed to refine some management recommendations, especially on shade canopy composition and density, and verify their economic viability. The implementation of our recommendations should be accompanied by appropriate biological and socioeconomic monitoring and the findings should inform a broad program of adaptive management of the cabrucas and the wider cocoa landscape.     

Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis

The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions.     

Cocoa biotechnology: status, constraints and future prospects

Current status and future prospects of cocoa biotechnology are reviewed. Potential for improving and modifying cocoa bean yield and quality are discussed. Prospects for producing cocoa components in vitro and cocoa butter substitutes in crops other than cocoa are examined. Application of complementary research tools is expected to allow significant enhancements in cocoa bean yield. Furthermore, cocoa varieties with modified characteristics are likely to become available, in particular varieties with increased cocoa butter content and modified fatty acid patterns. In vitro production of cocoa components is less likely.     

Evaluating Farmers’ Knowledge of Shade Trees in Different Cocoa Agro-Ecological Zones in Ghana

Shade trees are an integral part of most cocoa growing systems across the world. This study assesses farmers’ local knowledge of shade trees at two locations in Ghana with different climatic conditions and vulnerability to climate change. Akumadan is located in the northern dry part of the cocoa belt representing marginal conditions for cocoa cultivation, whereas Asankragua is located in the southern wet part, more ecologically favorable for cocoa cultivation. Inventories were conducted to assess the abundance of species, their structural characteristics and benefits to cocoa systems. Results show that cocoa farmers have detailed knowledge on the functions of shade trees in cocoa systems and prefer species that provide specific needs according to the location. However, abundance of species in a location does not necessary translate into beneficial impacts on cocoa productivity. In the drier part of the cocoa belt, income diversification through shade trees is an adaptation strategy to the increasingly marginal conditions for cocoa production, which has led to the high proportion of fruit trees among the most abundant species. In contrast, farmers in the southern part of the cocoa belt select shade trees for their high cocoa compatibility. Adaptation strategies for cocoa farmers should therefore promote species that best accommodate farmers’ economic, agronomic and environmental needs.     

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

Cocoa pulp fermentation is a spontaneous process during which the natural microbiota present at cocoa farms is allowed to ferment the pulp surrounding cocoa beans. Because such spontaneous fermentations are inconsistent and contribute to product variability, there is growing interest in a microbial starter culture that could be used to inoculate cocoa pulp fermentations. Previous studies have revealed that many different fungi are recovered from different batches of spontaneous cocoa pulp fermentations, whereas the variation in the prokaryotic microbiome is much more limited. In this study, therefore, we aimed to develop a suitable yeast starter culture that is able to outcompete wild contaminants and consistently produce high-quality chocolate. Starting from specifically selected Saccharomyces cerevisiae strains, we developed robust hybrids with characteristics that allow them to efficiently ferment cocoa pulp, including improved temperature tolerance and fermentation capacity. We conducted several laboratory and field trials to show that these new hybrids often outperform their parental strains and are able to dominate spontaneous pilot scale fermentations, which results in much more consistent microbial profiles. Moreover, analysis of the resulting chocolate showed that some of the cocoa batches that were fermented with specific starter cultures yielded superior chocolate. Taken together, these results describe the development of robust yeast starter cultures for cocoa pulp fermentations that can contribute to improving the consistency and quality of commercial chocolate production.     

Cocoa butter biosynthesis. Purification and characterization of a soluble sn-glycerol-3-phosphate acyltransferase from cocoa seeds

Glycerol-3-phosphate acyltransferase has been purified from the post-microsomal supernatant of cocoa seeds using differential ammonium sulfate solubility along with anion exchange and gel filtration chromatography. Chromatofocusing and isoelectric focusing revealed a series of proteins with acyltransferase activity having isoelectric points close to 5.2. Gel filtration on Sephacryl S-300 in 500 mM NaCl, along with polyacrylamide gel electrophoresis (denaturing and non-denaturing) and immunochemical analysis, gave evidence that the native enzyme has a molecular weight of 2 X 10(5) and consists of an aggregate of 10 Mr 20,000 subunits. The highly purified enzyme carries an acyl donor, probably acyl-CoA, although this is not firmly established. The hydrophobic nature of the purified enzyme was demonstrated by its firm binding to octyl-Sepharose. Mass spectrometric analysis of reaction products revealed the presence of both palmitic and stearic acids. Considering that 1) the fatty acids were derived from the purified enzyme; 2) they were found exclusively in the 1-position of glycerol 3-phosphate; 3) the fatty acid positioning and composition is consistent with that found in cocoa butter, the major storage product of cocoa seeds; and 4) the enzyme is found in the post-microsomal supernatant, it seems reasonable to conclude that the first step in cocoa butter biosynthesis is catalyzed by glycerol-3-phosphate acyltransferase in the cytoplasm of cocoa cotyledon cells.