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Proanthocyanidins (OPACs) are the second largest class of plant metabolites after lignans. Although knowledge of their 3D conformations would add greatly to our understanding of their biological properties, very little has been published on the conformations of OPACs with a degree of polymerization (DP) above 4. We investigated the conformations of the linear epicatechin oligomers, prominent representatives of OPACs prevalent in apples and cocoa, where the epicatechin units are interconnected through the 4β-8 bonds. For DP-2 to DP-10 oligomers, conformational preferences reflected in the arrangement of consecutive flavan-3-ol units, are characterized by the φ torsion. For dimers, there are two energy wells corresponding to two preferred φ torsions, designated as compact and extended form. This behaviour is preserved in OPACs with higher DPs, but the most energetically favoured conformations are a combination of both, with compact-only or extended-only conformations being very unlikely. Thus, oligomers with DP ≥ 7 tend to assume an overall conformation approximating a spherical shape. This shape has a significant influence on the polarity of the OPAC oligomers expressed as 3D polar surface area, calculated using Spartan software for geometry-optimized 3D models, and possibly on other physicochemical properties. The results of polarity calculations provide a molecular-level rationale for the polarity-based chromatographic separation of the cocoa B-type procyanidins with DP range 4 to 10. In our experiments, using centrifugal partition chromatography (CPC) (a solvent system consisting of EtOAc-EtOH-water (6:1:5) v/v/v with aqueous phase stationary and upper phase mobile) we found that an enriched mixture of proanthocyanidins eluted first DP-1 (epicatechin) followed by consecutive elution of the DP-2 to DP-10 in the linear 4β-8 form. We demonstrated that such separation would not be possible if compact-only or extended-only conformations were present in solution. However, for the energy-favoured, spherically shaped conformations, the observed CPC elution order is fully justified., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cocoa (Theobroma cacao L.) is the basic raw material to produce chocolate and other derivatives such as cocoa butter, cocoa powder and cocoa liquor (cocoa paste), which requires a fermentation process that affects its chemical composition and sensory profile. The objective of this study was to monitor the biochemical, physical and sensory changes during fermentation of cocoa beans in cocoa bean processing plants in the department of Caquetá, Colombia. During fermentation, the temperature of the mass and the pH of the pulp and beans were monitored at the different cocoa bean processing plants (Sites ASOACASAN ASA, COMICACAO CMI, COMCAP COC). Also, at two points during fermentation (days 4 and 7), physical properties of the bean were determined, such as variables related to bromatological composition, polyphenolic compounds and antioxidant activity as sensory attributes at the different sites. An increase in dough temperature was found, however the pH of the cotyledon decreased during the fermentation process and the fat and moisture content varied with fermentation time. At the site level, total polyphenol content (TPC), total flavonoids (TF), 1,1-diphenyl-2-picrylhydrazil (DPPH) and ferric reducing antioxidant power (FRAP) contents were statistically different, with COC being different from the other sites. The TPC was higher at the COC site (507 mg gallic acid equivalent GAE/g Cocoa) with respect to the other sites (< 360 mg GAE/g Cocoa). The TF content followed a similar behavior to TPC, with significant differences between sites and differences between fermentation times for ASA. The TF was higher in COC (309.1 mg catechin/g cocoa) with respect to CMI (215.6 mg catechin/g cocoa) and ASA (185.7 mg catechin/g cocoa). Values in DPPH ranged from 5869.3 to 7781.8 μmol Trolox/g cocoa and for the FRAP assay ranged from 369.8 to 606.7 mg ascorbic acid AA/g cocoa among the sites. It was found that the time and management of the fermentation process has a significant impact on the parameters (biochemical, physical and sensory) of cocoa beans. Therefore, it is necessary to standardize the fermentation process to achieve a quality product that meets the needs of the market., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 González et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Advances in the understanding of bioavailability and metabolism of bioactive compounds have been achieved primarily through targeted or semi-targeted metabolomics approaches using the hypothesis of potential metabolized compounds. The recent development of untargeted metabolomics approaches can present great advantages in this field, such as in the discovery of new metabolized compounds or to study the metabolism of compounds from multiple matrices simultaneously. Thus, this study proposes the use of an untargeted metabolomics strategy based on HPLC-ESI-QTOF-MS for the study of bioavailability and metabolism of bioactive compounds from different vegetal sources. Specifically, this study has been applied to plasma samples collected in an acute human intervention study using three matrices ( Hibiscus sabdariffa , Silybum marianum and Theobroma cacao ). This approach allowed the selection of those significant variables associated with exogenous metabolites derived from the consumption of bioactive compounds for their subsequent identification. As a result, 14, 25 and 3 potential metabolites associated with supplement intake were significantly detected in the plasma samples from volunteers who ingested the H. sabdariffa (HS), S. marianum (SM) and T. cacao (TC) extracts. Furthermore, T max values have been computed for each detected compound. The results highlight the potential of untargeted metabolomics for rapid and comprehensive analysis when working with a wide range of exogenous metabolites from different plant sources in biological samples.

Some germination is known to occur during the process of fermentation in cocoa beans. The impact of this biological process on the course of cocoa fermentation is not known and was thus investigated. In order to determine the impact of germination at the molecular level as well as on flavor, an untargeted metabolomics approach using Ultra Performance Liquid Chromatography-Electrospray Ionization-Time of Flight-Mass Spectrometry (UPLC-ESI-ToF-MS) with simultaneous acquisition of low- and high-collision energy mass spectra (MS e ) was performed. Extracts of raw and germinated cocoa beans of the same origin were measured and compared for characteristic differences by unsupervised principal component analysis. OPLS-DA revealed 12-hydroxyjasmonic acid (HOJA) sulfate, (+)-catechin and (-)-epicatechin as most down-regulated compounds as well as two hydroxymethylglutaryl (HMG) glucosides A and B among others as decisive up-regulated compounds in the germinated material. Additionally, further HMG glucosides and 12-hydroxyjasmonic acid could be identified in cocoa for the first time by coelution with isolated and synthesized reference compounds. HOJA sulfate, which has been postulated in cocoa, and HOJA were revealed to impart bitter and astringent taste qualities.

A recently published untargeted metabolomics approach toward marker compounds of cocoa germination revealed and identified 12-hydroxyjasmonic acid sulfate, (+)-catechin, and (-)-epicatechin as the most downregulated compounds and two hydroxymethylglutaryl glucosides (HMG gluc) A and B, among others, as the decisive upregulated compounds in the germinated material. These findings were quantitatively evaluated using ultrahigh-performance liquid chromatography-tandem mass spectrometry not only in previously examined sample material but also in a vastly expanded array of cocoa samples of different provenience and process and in cocoa products such as cocoa liquor and chocolate. Hereby, yields of newly identified HMG gluc derivatives could be determined in raw, fermented, germinated, and alternatively processed cocoa, and isomers of HMG gluc A and B could be established as key process indicators. Based on unsupervised clustering and supervised classification, models could identify germinated samples in testing sets consisting of raw, fermented, and germinated samples.

An experimental setup was devised to investigate the permeability of cocoa bean seed coat and pulp to key volatile compounds during fermentation. Four labeled compounds (ethyl acetate- d 3, ethyl octanoate- d 15, 2-phenylethanol- d 5, linalool- d 5) and 2 unlabeled (beta-damascenone, delta-decalactone) were chosen for the investigation. The beans (cotyledons), depulped beans, or pulped beans were immersed separately in a concentrated solution of these volatile compounds at 36 or 46 °C for durations ranging from 3 to 120 h. The imbibed beans were dissected, and the cotyledons were analyzed by SPME-GC/MS. The diffusion of volatile compounds from the external solution to the seed was categorized into three groups: (1) not diffusible (ethyl octanoate- d 15); (2) semidiffusible (ethyl acetate); and (3) totally diffusible (2-phenylethanol- d 5, linalool- d 5, beta-damascenone, delta-decalactone). The impact of the yeast on volatile compound diffusion was also investigated by immerging the pulped beans into the same concentrated solution with a yeast starter. Results highlighted the positive role of yeast in the diffusion of volatile compounds. The starter positively contributed to volatile compound diffusion after a transition phase occurring at approximately 48 h of fermentation, enriching the cocoa beans with key aromatic volatile compounds.

Assessing the bacterial community composition across cacao crops is important to understand its potential role as a modulator of cadmium (Cd) translocation to plant tissues under field conditions; Cd mobility between soil and plants is a complex and multifactorial problem that cannot be captured only by experimentation. Although microbes have been shown to metabolize and drive the speciation of Cd under controlled conditions, regardless of the link between soil bacterial community (SBC) dynamics and Cd mobilization in the rhizosphere, only a few studies have addressed the relationship between soil bacterial community composition (SBCC) and Cd content in cacao seeds (Cd seed ). Therefore, this study aimed to explore the association between SBCC and different factors influencing the distribution of Cd across cacao crop systems. This study comprised 225 samples collected across five farms, where we used an amplicon sequencing approach to characterize the bacterial community composition. The soil Cd concentration alone (Cd soil ) was a poor predictor of Cd seed . Still, we found that this relationship was more apparent when the variation within farms was controlled, suggesting a role of heterogeneity within farms in modulating Cd translocation and, thus, seed Cd content. Our results provide evidence of the link between soil bacterial communities and the distribution of Cd across Colombian cacao crops, and highlight the importance of incorporating fine-spatial-scale studies to advance the understanding of factors driving Cd uptake and accumulation in cacao plants., Importance: Cadmium (Cd) content in cacao crops is an issue that generates interest due to the commercialization of chocolate for human consumption. Several studies provided evidence about the non-biological factors involved in its translocation into the cacao plant. However, factors related to this process, including soil bacterial community composition (SBCC), still need to be addressed. It is well known that soil microbiome could impact compounds' chemical transformation, including Cd, on the field. Here, we found the first evidence of the link between soil bacterial community composition and Cd concentration in cacao soils and seeds. It highlights the importance of including the variation of bacterial communities to assess the factors driving the Cd translocation into cacao seeds. Moreover, the results highlight the relevance of the spatial heterogeneity within and across cacao farms, influencing the variability of Cd concentrations., Competing Interests: The authors declare no conflict of interest.

Durian (Durio zibethinus) is a tropical fruit that has a unique flavor and aroma. It occupies a significant phylogenetic position within the Malvaceae family. Extant core-eudicot plants are reported to share seven ancestral karyotypes that have undergone reshuffling, resulting in an abundant genomic diversity. However, the ancestral karyotypes of the Malvaceae family, as well as the evolution trajectory leading to the 28 chromosomes in durian, remain poorly understood. Here, we report the high-quality assembly of the durian genome with comprehensive comparative genomic analyses. By analyzing the collinear blocks between cacao and durian, we inferred 11 Malvaceae ancestral karyotypes. These blocks were present in a single-copy form in cacao and mainly in triplicates in durian, possibly resulting from a recent whole genome triplication (WGT) event that led to hexaploidization of the durian genome around 20 (17-24) million years ago. A large proportion of the duplicated genes in durian, such as those involved in the lignin biosynthesis module for phenylpropane biosynthesis, are derived directly from whole genome duplication, which makes it an important force in reshaping its genomic architecture. Transcriptome studies have revealed that genes involved in feruloyl-CoA formations were highly preferentially expressed in fruit peels, indicating that the thorns produced on durian fruit may comprise guaiacyl and syringyl lignins. Among all the analyzed transcription factors (TFs), members of the heat shock factor family (HSF) were the most significantly upregulated under heat stress. All subfamilies of genes encoding heat shock proteins (HSPs) in the durian genome appear to have undergone expansion. The potential interactions between HSF Dzi05.397 and HSPs were examined and experimentally verified. Our study provides a high-quality durian genome and reveals the reshuffling mechanism of ancestral Malvaceae chromosomes to produce the durian genome. We also provide insights into the mechanism underlying lignin biosynthesis and heat stress tolerance., (© 2024. Science China Press.)

Introduction: The cacao tree (Theobroma cacao), a perennial crop that serves as a source of cacao beans, can suffer from drastic climate changes such as irregular rainfall and shorter rainy seasons. The search for hybrids which are capable of producing specific metabolites favoring adaptation in new climatic conditions is a challenge in cacao farming., Objectives: We aimed to (1) analyze the metabolic changes in calli of three cacao genotypes during water deficit induced by incubation with polyethylene glycol and (2) assess their response to water deficit stress with regard to somatic embryo differentiation., Methods: Metabolic profiling was carried out using 1 H-NMR spectroscopy and multivariate data analysis was applied to crude extracts of calli grown in non-stress or water deficit stress conditions., Results: Water deficit stress influences the capacity of calli to produce embryos. The SCA12 genotype exhibited the best conversion capacity under severe conditions and was considered as tolerant to drought, followed by the SCA6 genotype (mid-tolerant) and the MA12 genotype (sensitive). Fifty-four metabolites were identified in the three cacao genotypes and discriminant metabolites were identified. Metabolites involved in water stress tolerance such as fructose, trans-aconitic acid, leucine, and hydroxybenzene derivatives were observed in SCA12, the tolerant genotype., Conclusion: These results demonstrate the utility of 1 H-NMR metabolomics as an essential tool for the analysis of the drought tolerance characteristics of T. cacao., (© 2024 John Wiley & Sons Ltd.)

Migration of nib Cd to the testa during fermentation can be achieved with high temperatures (> 45 °C) and low nib pH values (< 5.0) using spontaneous fermentation. However, this low pH can lead to low flavor quality. This study used three controlled temperature fermentation treatments on three cacao genotypes (CCN 51, ICS 95, and TCS 01) to test its effects on the nib pH, the migration of nib Cd to the testa, and the liquor flavor quality. All treatments were effective in reducing the total nib Cd concentration. Nevertheless, the treatment with the higher mean temperature (44.25 °C) and acidification (pH 4.66) reached the highest mean nib Cd reductions throughout fermentation, a 1.37 factor in TCS 01, promoting the development of fine-flavor cocoa sensorial notes. In unfermented beans, the Cd concentration of nibs was higher than that of the testa, and the Cd migration proceeded down the total concentration gradient. However, Cd migration was observed against the concentration gradient (testa Cd > nib Cd) from the fourth day. Cd migration could increase by extensive fermentation until the sixth day in high temperatures and probably by the adsorbent capacity of the testa. Genotype-by-treatment interactions were present for the nib Cd reduction, and a universal percentage of decrease of Cd for each genotype with fermentation cannot be expected. Selecting genotypes with highly adsorbent testa combined with controlled temperatures would help reduce the Cd concentration in the cacao raw material, improving its safety and quality., (© 2024. The Author(s).)

The bioconversion of lignocellulosic biomass into novel bioproducts is crucial for sustainable biorefineries, providing an integrated solution for circular economy objectives. The current study investigated a novel microwave-assisted acidic deep eutectic solvent (DES) pretreatment of waste cocoa pod husk (CPH) biomass to extract xylooligosaccharides (XOS). The sequential DES (choline chloride/citric acid, molar ratio 1:1) and microwave (450W) pretreatment of CPH biomass was effective in 67.3% xylan removal with a 52% XOS yield from total xylan. Among different XOS of varying degrees of polymerization, a higher xylobiose content corresponding to 69.3% of the total XOS (68.22 mg/g CPH) from liquid fraction was observed. Enzymatic hydrolysis of residual xylan from pretreated CPH biomass with low commercial xylanase (10 IU/g) concentration yielded 24.2% XOS. The MW-ChCl/citric acid synergistic pretreatment approach holds great promise for developing a cost-effective and environmentally friendly method contributing to the sustainable production of XOS from agricultural waste streams., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Procyanidins are gaining attention due to their potential health benefits. We found that cacao liquor procyanidin (CLPr) from Theobroma cacao seeds increased the lifespan of Caenorhabditis elegans, a representative model organism for aging studies. The genetic dependence of the lifespan-extending effect of CLPr was consistent with that of blueberry procyanidin, which is dependent on unc-43, osr-1, sek-1, and mev-1, but not on daf-16, sir-2.1, or skn-1. The lifespan-extending effect of CLPr was inhibited by neuron-specific RNA interference (RNAi) targeting unc-43 and pmk-1, and in worms with loss-of-function mutations in the odr-3, odr-1, or tax-4 genes, which are essential in sensory neurons, including AWC neurons. It was also inhibited in worms in which AWC neurons or AIB interneurons had been eliminated, and in worms with loss-of-function mutations in eat-4 or glr-1, which are responsible for glutamatergic synaptic transmission. These results suggest that the lifespan-extending effect of CLPr is dependent on the nervous system. In addition, it also requires unc-43 and pmk-1 expression in nonneuronal cells, as demonstrated by the experiments with RNAi in wild-type worms, the neuronal cells of which are not affected by systemic RNAi. The osr-1 gene is expressed in hypodermal and intestinal cells and regulates the response to osmotic stress along with unc-43/calcium/calmodulin-dependent protein kinase II and the p38 mitogen-activated protein kinase pathway. Consistent with this, CLPr improved osmotic stress tolerance in an unc-43- and pmk-1-dependent manner, and it was also dependent on AWC neurons. The lifespan-extending and osmotic-tolerance-improving activities were attributed to procyanidins with a tetrameric or higher-order oligomeric structure., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The aim of this study was to investigate the dynamic profile of microorganisms and metabolites in Hainan Trinitario cocoa during a six-day spontaneous box fermentation process. Shotgun metagenomic and metabolomic approaches were employed for this investigation. The potential metabolic functions of microorganisms in cocoa fermentation were revealed through a joint analysis of microbes, functional genes, and metabolites. During the anaerobic fermentation phase, Hanseniaspora emerged as the most prevalent yeast genus, implicated in pectin decomposition and potentially involved in glycolysis and starch and sucrose metabolism. Tatumella, possessing potential for pyruvate kinase, and Fructobacillus with a preference for fructose, constituted the primary bacteria during the pre-turning fermentation stage. Upon the introduction of oxygen into the fermentation mass, acetic acid bacteria ascended to dominant within the microflora. The exponential proliferation of Acetobacter resulted in a decline in taxonomic richness and abundance. Moreover, the identification of novel species within the Komagataeibacter genus suggests that Hainan cocoa may serve as a valuable reservoir for the discovery of unique cocoa fermentation bacteria. The KEGG annotation of metabolites and enzymes also highlighted the significant involvement of phenylalanine metabolism in cocoa fermentation. This research will offer a new perspective for the selection of starter strains and the formulation of mixed starter cultures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A screening of 24 Chuncho cacao postharvest processes was carried out and three treatments were identified with the best levels of functional non-volatile metabolites and the best profile of volatile metabolites. The relationship between the sensory profile of flavor and aroma, volatile and non-volatile metabolites was investigated by multiblock analysis. The most prominent volatile metabolites were benzaldehyde (Bz), 2,3,5-trimethylpyrazine (TriMP), 2,3,5,6-tetramethylpyrazine (TetraMP), myrcene (Myr), limonene (LM), ethylphenyl acetate (EpHAc) and 2-phenylethyl acetate (pHEthAc), and functional nonvolatile metabolites were total flavonoids (Tflav), total polyphenols (TppH), catechins (Cat), epicatechin (EpCat), caffeine (Caf), and theobromine (TeoB). Sensory analysis by MFA (multiple factorial analysis) allowed checking the substrates of the metabolites and highlighting correlated flavor and aroma descriptors. Based on multiblock analysis, four new descriptors were identified for Bz, Myr, and LM, such as nut, astringent, bitter, and crude. For TetraMP the fruity flavor was identified and for TriMP it was identified as Malt flavor. For Cat, EpCat, Caf, TeoB, Tflav, and TppH, the descriptors bitter, astringent, floral, and fruity were identified. This study will allow Chuncho cocoa farmers and producers to have a cocoa paste production scheme with a high value of functional metabolites and sensory profile, and value this Peruvian accession., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In Brazil, after the witch's broom disease incidence, diverse cocoa hybrids were developed, and variations were reported on their composition and characteristics. Based on this, the present study aimed to evaluate the pulp composition of several cocoa hybrids in order to better understand these variations. Results show that cocoa pulp is composed, on average, of 76 % sugar, and a wide variation (20 %) was observed in sugar content between hybrids. Regarding the sugar profile, a prevalence of reducing sugars was observed. Pod origin also plays an important role in pulp composition, with variations between hybrids from Espírito Santo and Bahia states. In relation to the degree of ripeness, ripe pods showed higher fructose and glucose content, while unripe pods presented mainly sucrose. Similar to sugars, the cello-oligossacharides profile was influenced by the degree of pod ripeness and origin and most ripe samples presented mainly cellobiose, cellotriose and cellotetrose. In addition, the prebiotic potential of cocoa pulp was highlighted by cello-oligossacharides digestion assay which exhibited low rates of degradation. Varying enzymatic activity was observed amongst different pulp hybrids, with polyphenol oxidase showing a higher variation when compared to invertase and polygalacturonase ranging. This study shows that the pod hybrid, origin and ripening degree may change the cocoa pulp composition. Therefore, it is very important to understand and evaluate these variations, in order to obtain better results in pulp utilization either in cocoa fermentation or as a coproduct., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

The pathways through which cadmium (Cd) is taken up and loaded into cacao beans (nibs) are yet to be revealed. Previous work suggested that Cd loading into cacao nibs may occur via direct xylem uptake rather than phloem-mediated redistribution from the leaves. A stable isotope ( 108 Cd) pulse-chase experiment was set up to identify the pathways of Cd loading into cacao nibs. The topsoil beneath two mature cacao trees in the field was enriched in 108 Cd via surface irrigation with a spiked solution. The increase in 108 Cd isotopic abundance (IA) in the plant tissues was followed up for 548 days after spiking. The 108 Cd IA in the plant tissues increased from natural abundance (0.89 %) to 7.0 % (tree A) and 10.1 % (tree B) at equilibrium. The tracer was taken up in the plant tissues in the order immature leaves > mature leaves > nibs in both trees, while tracer uptake in flowers and cherelles was less consistent between the trees. Half of the equilibrium 108 Cd IA was reached in the nibs at 191 days after spiking, significantly later than corresponding values for mature (151 days) and immature leaves (117 days). Pod maturation from flower stage takes about 6 months with most Cd entering the nibs at the last stage of development. The rather slow rise in the 108 Cd IA in the nibs compared to the leaves hence suggests that Cd in cacao nibs likely originates from phloem-redistribution from the stem, branches or mature leaves and not from direct root-to-nib transport via the xylem., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Hanseniaspora opuntiae is a commonly found yeast species in naturally fermenting cocoa pulp-bean mass, which needed in-depth investigation. The present study aimed at examining effects of the cocoa isolate H. opuntiae IMDO 040108 as part of three different starter culture mixtures compared with spontaneous fermentation, regarding microbial community, substrate consumption, and metabolite production dynamics, including volatile organic compound (VOC) and phytochemical compositions, as well as compositions of the cocoa beans after fermentation, cocoa liquors, and chocolates. The inoculated H. opuntiae strain was unable to prevail over background yeasts present in the fermenting cocoa pulp-bean mass. It led to under-fermented cocoa beans after four days of fermentation, which was however reflected in higher levels of polyphenols. Cocoa fermentation processes inoculated with a Saccharomyces cerevisiae strain enhanced flavour production during the fermentation and drying steps, which was reflected in richer and more reproducible aroma profiles of the cocoa liquors and chocolates. Sensory analysis of the cocoa liquors and chocolates further demonstrated that S. cerevisiae led to more acidic notes compared to spontaneous fermentation, as a result of an advanced fermentation degree. Finally, different VOC profiles were found in the cocoa beans throughout the whole chocolate production chain, depending on the fermentation process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Belcolade NV and Cacaolab BV provided non-financial assistance (provision of infrastructure) to the work. Co-author Prof. Koen Dewettinck is founder and shareholder of the UGent spin-off company Cacaolab BV., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Yeasts are important microorganisms used in different fermentation processes. The cocoa beans must go through a correct fermentation process to obtain good-quality chocolate, which involves the action of yeasts and bacteria, and yeasts play a crucial role since they act in the first days of fermentation. In coffee, several studies have shown that the microbiota in the fruits is also a relevant factor. The fermentation process (regardless of the processing type) improves the beverage's quality. In this sense, studies using starter cultures in these two raw materials are important for better control of the process, and optimization of fermentation time, in addition to the improvement and diversification of volatile and non-volatile compounds produced by yeasts. Thus, this review discusses the importance and role of yeasts during fermentation, their metabolism, the produced compounds, and how yeast and the different chemical reactions help increase the quality of chocolate and coffee., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Investigations of the compatibility between cacao genotypes of the population of the Parinari series (Pa), resulting from the reciprocal crossing of Pa 30 × Pa 169 and Pa 121 × Pa 169, allowed the verification of the occurrence of the recessive lethal single character called Luteus-Pa. These genotypes have this gene in heterozygosity, which when intercross or self-fertilize, segregate in a 3:1 ratio. Normal (NS) and mutant (MS) seedlings grow normally and, after a period of approximately 30 days of age, MS leaves begin to show a metallic yellow color, followed by necrotic spots, and death of the entire seedling, approximately 40 days after the emergency. The work evaluate the molecular, biochemical and micromorphological responses in NS and MS, with and without cotyledons, resulting from the crossing of the Pa 30 × Pa 169 cacao genotypes, aiming to elucidate the possible lethal mechanisms of the homozygous recessive Luteus-Pa. The presence of the lethal gene Luteus-Pa in the seedlings of the cacao genotypes of the population of the Parinari (Pa), with and without cotyledons, resulting from the crossing of Pa 30 × Pa 169, in addition to regulating the synthesis of proteins related to the photosynthetic and stress defense processes, promoted an increase in the synthesis of proteins involved in the glycolic pathway, induced oxidative stress, altered the mobilization of cotyledonary reserves, the integrity of cell membranes, leaf micromorphology and induced the death of seedlings, soon after depletion of protein and carbohydrate reserves, especially in the absence of cotyledons., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Earlier studies revealed that cadmium (Cd) concentrations in cacao nibs can decrease by a factor up to 1.3 during fermentation. Here, fermentation was mimicked by incubating beans at different temperatures, and acetic acid and ethanol concentrations in the incubation media. Nib Cd concentrations decreased during incubation by mobilisation in the nibs and subsequent outward migration to the testa and the incubation solution. This was most pronounced when high concentrations of acetic acid were combined with high temperature, while ethanol had no statistically significant effect. Incubation under typical fermentation conditions (45 °C and 20.0 g acetic acid L -1 ) reduced the nib Cd concentration by a factor 1.3. This factor increased to 1.6 under more extreme conditions, i.e. 65 °C and 40 g acetic acid L -1 . The final nib Cd concentrations correlated well to nib phytate concentrations (R 2  = 0.56), suggesting hydrolysis of phytate and mobilisation of the associated Cd 2+ ., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ruth Vanderschueren and co-authors has patent #EP21174400.8 pending to KU Leuven. Ruth Vanderschueren and co-authors has patent #EP21164933.0 pending to KU Leuven., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Spontaneous fermentation is a process that depends on substrates' physical characteristics, crop variety, and postharvest practices; it induces variations in the metabolites that are responsible for the taste, aroma, and quality. Metabolomics makes it possible to detect key metabolites using chemometrics and makes it possible to establish patterns or identify biomarker behaviors under certain conditions at a given time. Therefore, sensitive and highly efficient analytical techniques allow for studying the metabolomic fingerprint changes during fermentation; which identify and quantify metabolites related to taste and aroma formation of an adequate processing time. This review shows that studying metabolomics in spontaneous fermentation permits the characterization of spontaneous fermentation in different stages. Also, it demonstrates the possibility of modulating the quality of cocoa by improving the spontaneous fermentation time (because of volatile aromatic compounds formation), thus standardizing the process to obtain attributes and quality that will later impact the chocolate quality., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cocoa (Theobroma cacao L.) is the only tree that can produce cocoa. Cocoa beans are highly sought after by chocolate makers to produce chocolate. Cocoa can be fine aromatic, characterized by floral and fruity notes, or it can be described as standard cocoa with a more pronounced cocoa aroma and bitterness. In this study, the genetic and biochemical determinants of sensorial notes and nonvolatile compounds related to bitterness, astringency, fat content, and protein content will be investigated in two populations: a cultivated modern Nacional population and a population of cocoa accessions collected recently in the Ecuadorian South Amazonia area of origin of the Nacional ancestral variety. For this purpose, a genome-wide association study (GWAS) was carried out on both populations, with results of biochemical compounds evaluated by near-infrared spectroscopy (NIRS) assays and with sensory evaluations. Twenty areas of associations were detected for sensorial data especially bitterness and astringency. Fifty-three areas of associations were detected linked to nonvolatile compounds. A total of 81 candidate genes could be identified in the areas of the association., (© 2022 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

The pod husk of Theobroma cacao (CPH) plays an important agronomical role, as its appearance is used as indicator of ripening, guiding the farmers in the harvest process. Cacao harvesting is not a standardized practice because farmers harvest between six up to eight months from flowering, guided by pod's color and shape. The mixture of cacao beans from different ripening stages (RS), negatively affecting the quality and price of grain. A way to help the farmers in the harvest standardization could be through the use of chemical markers and visual indicators of CPH ripening. This study analyses CPH's metabolic distribution of two cacao clones, ICS95 and CCN51 at six, seven, and eight months of ripening. Untargeted metabolomics was done using HPLC-MS/MS for biomarker discovery and association to cacao ripening. The results indicated a strong metabolic differentiation of the sixth month with the rest of the months independent of the variety. Also, metabolic differences were found between cacao clones for the seventh and eighth month. We annotated five potential biochemical markers including 3-caffeoylpelargodinin 5-glucoside, indoleacetaldehyde, procyanidin A dimer, procyanidin C1, and kaempferol. We further looked for correlation between patterns of progression of our markers against quantitative indicators of CPH appearance and texture, at the same ripening stages. We also performed a functional analysis and three possible metabolic pathways: flavone and flavonol biosynthesis, flavonoid biosynthesis, and tryptophan metabolism were identified associated with stress sensing, plant development and defense respectively. We found significant and positive correlations between green color density and all metabolites. For texture, the correlations were significantly negative with all metabolites. Our results suggest that about the sixth month is appropriate for harvesting cacao in the region of Caldas, Colombia in order to avoid all the metabolic variations occurring at later stages of ripening which impact the cacao bean quality. Therefore, studying the cacao ripening process can help in the estimation of the best harvest time and contribute to the standardization of harvest practices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cold press hazelnut cakes represent a concentrated source of proteins that can be industrially exploited. Previously, bioactive attributes of hazelnut protein hydrolysates including antihypertensive and antidiabetic activities were documented. Here, we made an attempt to utilize bioactive hazelnut protein hydrolysates (1 % w/w) in the manufacture of industrial hazelnut cocoa cream and investigate their stability through processing and simulated gastrointestinal digestion. The inclusion of bioactive peptide fractions was a safe practice in the microbiological sense. Proteolysis lowered the potential allergenicity of hazelnut proteins in the cocoa cream products up to about 20 %. In silico trypsinolysis predicted partial degradation for 51.8 % of the peptide sequences (i.e., 43/83) that were present in the hydrolysates. However, partial degradation and mixing of degraded vs non-degraded peptides preserved and/or further elevated bioactive attributes in the digested cocoa cream products in terms of Angiotensin converting enzyme (ACE)-inhibitory (up to about 92 %) and antidiabetic activities (between 7.5 and 44.4 %). In most cases, however, antioxidative activity was < 10 %. While simulated in vitro digestion potentially influenced the bioactive attributes of protein hydrolysates, the influence of cocoa cream processing and food matrix were relatively limited for hydrolysate fractions and more pronounced for protein isolates. Hazelnut press cakes represent a significant resource for the generation and industrial utilization of bioactive peptides, which could preserve their bioactivity beyond industrial manufacture and digestion and lead to slightly reduced allergenicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Propagation by somatic embryogenesis in Theobroma cacao has some issues to be solved, as many morphologically abnormal somatic embryos that do not germinate into plants are frequently observed, thus hampering plant production on a commercial scale. For the first time the methylome landscape of T. cacao somatic embryogenesis was examined, using whole-genome bisulfite sequencing technique, with the aim to understand the epigenetic basis of somatic embryo abnormalities. We identified 873 differentially methylated genes (DMGs) in the CpG context between zygotic embryos, normal and abnormal somatic embryos, with important roles in development, programmed cell death, oxidative stress, and hypoxia induction, which can help to explain the morphological abnormalities of somatic embryos. We also identified the role of ethylene and its precursor 1-aminocyclopropane-1-carboxylate in several biological processes, such as hypoxia induction, cell differentiation and cell polarity, that could be associated to the development of abnormal somatic embryos. The biological processes and the hypothesis of ethylene and its precursor involvement in the somatic embryo abnormalities in cacao are discussed., (© 2022. The Author(s).)

Chocolate flavors vary depending on the origin of the cocoa beans used. Differences in soil, microorganisms, and environmental factors contribute to the formation of flavor precursors in cocoa beans. During cocoa bean fermentation, environmental temperature has been shown to alter metabolite concentrations. However, the correlation between the metabolite profile of cocoa beans and the temperature of their region of origin has not been fully defined. In this study, the metabolite profiles of Indonesian cocoa beans of various origins were evaluated using gas chromatography/mass spectrometry-based analysis, and were found to differ depending on the origin of the bean. Subsequently, the correlation between metabolite profile and environmental temperature of the origin was assessed using orthogonal projection to latent structure regression (OPLS-R) analysis. The analysis revealed that seven metabolites were associated with temperature: γ-aminobutyric acid (GABA), ethanolamine, glycerol, isocitric acid + citric acid, succinic acid, malic acid, and saccharic acid. The findings of this study will be valuable to chocolate industries for the production of single-origin chocolates., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Life span is increasing in developed countries as Japan, and an aging society is becoming a problem. In fact, healthy lifespan is not extended, and it is desired to extend it by functional food. Cacao (Theobroma cacao) contains various active components and is considered a preventative agent against metabolic disease. In addition, it has long been thought that regular cacao intake extends a healthy lifespan. However, there is no direct evidence for this belief. The purpose of this study is to identify the cacao component that elongate the lifespan of D. melanogaster as a model organism and to elucidate its functional mechanism. The activation of sirtuins, a family of NAD+-dependent deacetylases, has been reported to extend the lifespans of various organisms. Heat shock factor 1 is known to be deacetylated by reaction with sirtuins, thereby inducing gene expression of various heat shock proteins by heat stress and effectively extending the lifespan of organisms. Therefore, we evaluated whether components in cacao activate sirtuins and extend the lifespan of D. melanogaster. In the process, we discovered the fatty acid tryptamide as a lifespan-elongating component of cacao. Therefore, we investigated whether the fatty acid tryptamide from cacao upregulates the genes of heat shock proteins. As a result, it was confirmed that the gene expression of multiple heat shock proteins was significantly increased. This suggests that fatty acid tryptamide may activate sirtuins, increase gene expression of heat shock proteins, and elongate the lifespan of D. melanogaster., (© 2022. The Author(s).)

Aims: Optimize the production of Aspergillus brasiliensis endoglucanase in a solid-phase bioprocess using cupuaçu shell as substrate., Methods and Results: The shells were supplemented with nitrogen and phosphorous and used as a substrate. The centesimal and inorganic composition of the residue was determined, and found to be rich in fibres, and possessed essential elements for fungal growth. In the initial cultivation of A. brasiliensis, endoglucanase activity of 7.35 U g -1 was obtained. A factorial experimental design was used to determine the most significant variables for the bioprocess. The interactions between moisture, temperature and nitrogen source were noteworthy (p < 0.05). From the rotational central composite design, the optimization of temperature and nitrogen supplementation was obtained, and this reached 40.50 U g -1 , which is an increase of more than five times the value obtained initially. The enzymatic extract was applied as the biocatalyst in the hydrolysis of cupuaçu shells and, after 48 h, it was possible to observe the production of reducing sugars., Conclusions: Cupuaçu shell can be used as a substrate for endoglucanase production by A. brasiliensis. The process was optimized for the cultivation temperature and the nitrogen source. The enzymatic extract can be applied in the hydrolysis of lignocellulosic biomass., Significance and Impact of the Study: Cupuaçu shells can be used to produce cellulases, a product of high added value that can generate economic and environmental benefits for communities and companies producing derivatives of the cupuaçu fruit., (© 2021 The Society for Applied Microbiology.)

We aimed to investigate the combined effects of aerobic exercise (EXE) and cocoa flavanol (COCOA) supplementation on performance, metabolic parameters, and inflammatory and lipid profiles in obese insulin-resistant rats. Therefore, 32 male Wistar rats (230-250 g) were fed a high-fat diet and a fructose-rich beverage for 30 days to induce insulin resistance. Next, the rats were randomized into four groups, orally administered placebo solution or COCOA supplementation (45 mg·kg-1), and either remained sedentary or were subjected to EXE on a treadmill at 60% peak velocity for 30 min, for 8 weeks. Blood samples and peripheral tissues were collected and processed to analyze metabolic and inflammatory parameters, lipid profiles, and morphological parameters. Supplementation with COCOA and EXE improved physical performance and attenuated body mass gain, adipose index, and adipocyte area. When analyzed as individual interventions, supplementation with COCOA and EXE improved glucose intolerance and the lipid profile reduced the concentrations of leptin, glucose, and insulin, and reduced homeostasis assessment index (all effects were p < .001 for both interventions), while ameliorated some inflammatory mediators in examined tissues. In skeletal muscles, both COCOA supplementation and EXE increased the expression of glucose transporter (p < .001 and p < .001), and combined intervention showed additive effects (p < .001 vs. COCOA alone or EXE alone). Thus, combining COCOA with EXE represents an effective nonpharmacological strategy to treat insulin resistance; it could prevent Type 2 diabetes mellitus by improving physical performance, glucose metabolism, neuroendocrine control, and lipid and inflammatory mediators in the liver, pancreas, adipose tissue, and skeletal muscle in obese male insulin-resistant rats.

Elevated cadmium (Cd) concentrations in cacao and cocoa-based products (e.g., chocolate) present a potentially serious human health risk. While recent regulatory changes have established a threshold of 0.8 mg kg-1 for Cd content of cocoa-based products, the biophysical factors (e.g., climatic or edaphic conditions) that determine the amount of soil-derived Cd in the cacao bean are poorly understood and have yet to be quantitatively assessed across diverse production contexts. To determine the primary drivers of cacao bean Cd, we used the scientific literature to systematically compile a database of climatic, edaphic, and plant data from across the Cacao Belt, which is approximately 20 degrees latitude on either side of the equator. From this compiled dataset, we then used boosted regression trees to quantitatively synthesize and evaluate these drivers of cacao bean Cd. Total soil Cd concentration, soil pH, and leaf Cd were the best predictors of bean Cd content. Notably, we found that both available soil Cd and soil organic carbon (SOC) content had negligible effects on bean Cd. However, soil pH and SOC decreased the degree of bioconcentration of total soil Cd in the bean Cd concentration. Thus, given the difficulty in remediating soil Cd enriched soils, our results suggest that Cd mitigation strategies targeting plant physiology-based approaches (e.g., breeding, rootstocks) have a higher probability of success than soil-based strategies (e.g., remediation)., Competing Interests: The authors have declared that no competing interests exist.

Yeasts are one of the main ingredients responsible for flavor precursors production associated with sensorial characteristics in chocolate. Using wild yeast isolated from cocoa beans fermentation is emerging as a strategy for developing starter cultures. However, the volatile compounds (VCs) produced by yeasts are not yet known. This study aimed to select wild yeasts with the potential to produce volatile compounds associated with desirable flavor attributes. A total of 150 wild yeasts strains were isolated from the spontaneous cocoa beans fermentation, of which 40 were identified by morphology and physiological features. VCs produced were identified and quantified using SPME-GC-MS and GC-FID and profiles were evaluated statistically by PCA and cluster analysis for the compounds that had a high odor threshold value. Thirty-six VCs produced by these yeasts were identified into six main families, namely esters, alcohols, acids, aldehydes, ketones, and pyrazines. PCA showed the separation of the yeasts into two main clusters. Strains, Y195 and Y246, belong to the first cluster and are the highest producers of alcohols related to floral perceptions. In the second cluster, thirty-three yeasts were grouped by their ability to produce esters. Of all of them, Y110MRS stood out for producing 2-phenyl ethyl acetate and isoamyl acetate associated with fruity perceptions. This screening allowed us to identify yeasts that produced VCs of technological interest and which could be used to develop a starter culture.

Protease inhibitors (PIs) are important biotechnological tools of interest in agriculture. Usually they are the first proteins to be activated in plant-induced resistance against pathogens. Therefore, the aim of this study was to characterize a Theobroma cacao trypsin inhibitor called TcTI. The ORF has 740 bp encoding a protein with 219 amino acids, molecular weight of approximately 23 kDa. rTcTI was expressed in the soluble fraction of Escherichia coli strain Rosetta [DE3]. The purified His-Tag rTcTI showed inhibitory activity against commercial porcine trypsin. The kinetic model demonstrated that rTcTI is a competitive inhibitor, with a Ki value of 4.08 × 10 -7  mol L -1 . The thermostability analysis of rTcTI showed that 100% inhibitory activity was retained up to 60 °C and that at 70-80 °C, inhibitory activity remained above 50%. Circular dichroism analysis indicated that the protein is rich in loop structures and β-conformations. Furthermore, in vivo assays against Helicoverpa armigera larvae were also performed with rTcTI in 0.1 mg mL -1 spray solutions on leaf surfaces, which reduced larval growth by 70% compared to the control treatment. Trials with cocoa plants infected with Mp showed a greater accumulation of TcTI in resistant varieties of T. cacao, so this regulation may be associated with different isoforms of TcTI. This inhibitor has biochemical characteristics suitable for biotechnological applications as well as in resistance studies of T. cacao and other crops., (© 2022. The Author(s).)

Background: Antioxidants are beneficial in myocardial infarction (MI). It is suggestive that Theobroma cacao (TC) with rich antioxidant properties can be of health benefits in myocardial injury., Aim: The study investigated the effect of Theobroma cacao on cardioprotection in isoproterenol-induced myocardial infarction in rats., Material and Methods: Male Wistar rats divided into four groups of 6 rats were used for the study. In group 1, 0.9% normal saline placebo was administered via oral gavage to the control. Group 2 was the MI induced group that was given 100 mg/kg body weight isoproterenol subcutaneously twice at an interval of 24 hours. Group 3 was administered TC for 2 weeks at 100 mg/kg bodyweight via the oral route. Group 4 was pretreated with TC (100 mg/kg) via oral route for 2 weeks, immediately followed by the administration of 100 mg/kg body weight isoproterenol subcutaneously twice at an interval of 24 hours. The rats were sacrificed using chloroform anesthesia, and blood samples collected via cardiac puncture. The serum was analyzed for troponin level, lactate dehydrogenase (LDH), and malondialdehyde (MDA) level., Results: The serum troponin, LDH, and MDA levels were found to be significantly (p<0.01) increased in the MI group compared with the control. Pretreatment with TC before MI induction significantly (p<0.01) prevented increased serum troponin, LDH, and MDA levels when compared with the MI group. There was also a significant (p<0.01) decrease in MDA in the TC group compared with the control., Conclusion: These results suggest that Theobroma cacao protects against isoproterenol-induced myocardial injury, possibly by preventing oxidative stress and consequent lipid peroxidation., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Dark chocolate has long been recognized for its mood-altering properties; however, the evidence regarding the emotional effects of daily dark chocolate intake is limited. Therefore, we aimed to investigate the effects of dark chocolate intake on mood in everyday life, with special emphasis on the gut-brain axis. Two different dark chocolates (85% and 70% cocoa content) were tested in this study. In a randomized controlled trial, healthy adults (20-30 y) consumed either 30 g/d of 85% cocoa chocolate (DC85, n=18); 70% cocoa chocolate (DC70, n=16); or no chocolate (control group, CON; n=14); for 3 weeks. Mood states were measured using the Positive and Negative Affect Schedule (PANAS). Daily consumption of dark chocolate significantly reduced negative affect in DC85, but not in DC70. To assess the association between the mood-altering effects of dark chocolate and the gut microbiota, we performed fecal 16S rRNA sequencing analysis for the DC85 and CON groups. Gut microbial diversity was significantly higher in DC85 than CON (P<.05). Blautia obeum levels were significantly elevated and Faecalibacterium prausnitzii levels were reduced in DC85 compared to CON (P<.05). Furthermore, we found that the observed changes in negative affect scores were negatively correlated with diversity and relative abundance of Blautia obeum (P<.05). These findings indicate that dark chocolate exerts prebiotic effects, as evidenced by its ability to restructure the diversity and abundance of intestinal bacteria; thus, it may improve negative emotional states via the gut-brain axis., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lipases (triacylglycerol hydrolases, EC 3.1.1.3) are a class of enzymes with high industrial importance. An option for the production of this enzyme is through fungal growth via solid-state fermentation (SSF). Thus, this research presents a study of lipase production by Penicillium roqueforti ATCC 10110 through SSF using cocoa bran residues ( Theobroma cacao ) as a substrate. To achieve maximum lipase production, fermentation time (0 to 120 h) and palm oil (PO) percentage (0 to 50%) were optimized through analysis of one factor at a time (OFAT), with lipase activity as the response. The amount of cocoa was fixed (5 g), the incubation temperature was maintained at 27 °C, and the moisture content was established at 70%. For a 72 h incubation, the highest enzyme activity achieved using SSF without adding PO was 14.67 ± 1.47 U g -1 , whereas with PO (30%), it was 33.33 ± 3.33 U g -1 , thus demonstrating a 44% increase in enzyme activity. Through the OFAT methodology, it was possible to confirm that supplementation with palm residue was efficient and maximized the lipase of P. roqueforti ATCC 10110.

Worldwide only 8% of the biomass from harvested cacao fruits is used, as cacao beans, in chocolate-based products. Cacao mucilage exudate (CME), a nutrient-rich fluid, is usually lost during cacao beans fermentation. CME's composition and availability suggest a potential carbon source for cellulose production. CME and the Hestrin and Schramm medium were used, and compared, as growth media for bacterial cellulose (BC) production with Gluconacetobacter xylinus. CME can be used to produce BC. However, the high sugar content, low pH, and limited nitrogen sources in CME hinder G. xylinus growth affecting cellulose yields. BC production increased from 0.55 ± 0.16 g L -1 up to 13.13 ± 1.09 g L -1 after CME dilution and addition of a nitrogen source. BC production was scaled up from 30 mL to 15 L, using lab-scale experiments conditions, with no significant changes in yields and production rates, suggesting a robust process with industrial possibilities., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The Theobroma cacao presents a wide diversity in pod color among different cultivars. Although flavonoid biosynthesis has been studied in many plants, molecular mechanisms governing the diversity of coloration in cacao pods are largely unknown. The flavonoid metabolite profiles and flavonoid biosynthetic gene expression in the pod exocarps of light green pod 'TAS 410' (GW), green pod 'TAS 166' (GF), and mauve pod 'TAS 168' (PF) were determined. Changes in flavonoid metabolites, particularly the anthocyanins (cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, and cyanidin O-syringic acid) were significantly up-accumulated in the mauve phenotype (PF) compared to the light green or green phenotypes, endowing the pod color change from light green or green to mauve. Consistently, the PF phenotype showed different expression patterns of flavonoid biosynthetic structural genes in comparison with GW/GF phenotypes. The expression level of LAR and ANR in GW/GF was significantly higher than PF, while the expression level of UFGT in GW/GF was lower than PF. These genes likely generated more anthocyanins in the exocarps samples of PF than that of GW/GF. Simultaneously, colorless flavan-3-ols (catechin, epicatechin and proanthocyanidin) content in the exocarp samples of PF was lower than GW/GF. Additionally, MYB (gene18079) and bHLH (gene5045 and gene21575) may participate in the regulation of the pod color. This study sheds light on the molecular basis of cacao pod color variation, which will contribute to breeding cacao varieties with enhanced flavonoid profiles for nutritional applications., (© 2021. The Botanical Society of Japan.)

Background: Cacao is important for the economy of many countries in the humid tropics. Its quality is affected when fruits are not collected at the optimal harvest point. The aim of this study was to obtain maturity indices for producers to facilitate the timely harvest and improve the development of fermentation and the sensorial quality of cacao. The growth and respiration processes, and the physicochemical changes during the maturation of three cacao genotypes were determined. Physiological follow-ups measuring fruit length and diameter were performed from 30 days after anthesis to fruit deterioration in the tree., Results: Growth equations were obtained, establishing four maturity stages based on days after anthesis, and fruit length and diameter. Nineteen descriptors were used for the characterization, and through Pearson's correlation and principal component analysis (PCA), five descriptors were identified as representative of the maturity stages of the cacao fruit. PCA results and respiration measurements established that stage 3 presented the highest substrate availability for obtaining good fermentation and quality cacao. This stage showed values between 124 and 197 days after anthesis with lengths between 167.7 and 249.73 mm, and diameters between 64.4 and 95.8 mm, according to the locality., Conclusion: Three growth phases of the cacao fruits were established considering days after anthesis. Both the edaphoclimatic conditions and the cacao genotype characteristics influenced this determination. Stage 3 of fruit maturation shows the best physicochemical conditions for good fermentation. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Background: Cadmium uptake by cacao plants can affect plant growth, consumer health and commercialisation. To develop mitigation strategies, it is essential to identify the soil characteristics that could influence this absorption. To determine the relationships between cadmium absorption and the soil characteristics of cacao areas, the responses at concentrations of 0, 2, 5, 10 and 20 μg g -1 of cadmium in three soils of these areas and an andisol were evaluated, using 120-day-old seedlings of four cultivars of Theobroma cacao L., Results: In the present study, several relationships were found between chemical and physical soil characteristics and available cadmium, such as real and bulk densities, as well as contents of iron, sand, magnesium, potassium, sodium and copper. Additionally, moderate to strong correlations between potassium (r 2  = -0.56) and real density (r 2  = 0.42), with foliar cadmium, were found. Moreover, a differential deleterious effect on cacao growth in variables such as biomass was corroborated in cadmium concentrations from 5 μg g -1 in soils. There were no statistical differences between cultivars with respect to cadmium uptake or plant growth. Finally, a multiple linear regression model is proposed to estimate the foliar cadmium content (r 2  = 0.878)., Conclusion: Some soil characteristics such as density, as well as sand, clay, aluminium, potassium and iron contents, should be considered before establishing cacao crops to avoid cadmium accumulation. The correlation between potassium with foliar cadmium indicated that potassium could be significant in cadmium uptake mitigation strategies. The high correlation between available cadmium and foliar cadmium indicates that the quantification methodology developed using ethylenediaminetetraacetic acid extractant may be a useful diagnostic tool. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

The variable quality of cocoa produced by farmers is still a problem in the value chain, strongly depending on microbial activities. We analyzed the variability of cocoa microbiota from all twelve producing regions in Cote d'Ivoire, and described the geographical distribution of isolated microbiota, using a mapping. Microbial species were identified by ribosomal genes sequencing, strains were typed by RFLP and their techno-functional capacities were further investigated. Results showed a restricted diversity of lactic acid bacteria (LAB) and acetic acid bacteria (AAB) with respectively 10 and 5 strains. The dominant LAB and AAB strains, notably Lactobacillus plantarum 1 A, Acetobacter pasteurianus 1 A, Acetobacter okinawensis 2 A, and Acetobacter tropicalis 3 A, were found in all regions assuming that the acid microbiota was weakly variable. In contrast, the distribution of their functional performance such as acidification capability was variable, stronger in strains from Nawa and Haut-Sassandra regions and weaker in Indenie-Djuablin and San Pedro; this distribution seemed to be random. Moreover, the study also revealed a complex yeasts population showing a wide genetic diversity with 22 species and 45 strains indicating an intraspecific heterogeneity. Strains were generally different from a region to another and the resulting yeasts microbiota was globally variable in the regions. Likewise, the functional capacities such as pectinolytic was weak in P. kudriazevii strain 2 K from Gboklè and strong in P. kudriazevii strain 2 A from Loh-Djiboua. Additionally, the quality of fermented beans was also variable in the regions. The great variation of yeasts strains in the different regions may be the main microbial factors responsible for variation of the fermented cocoa quality observed., (Copyright © 2021. Published by Elsevier Ltd.)

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., (© 2021 Society of Chemical Industry.)

Introduction: Cocoa flavanols (CF) may exert health benefits through their potent vasodilatory effects, which are perpetuated by elevations in nitric oxide (NO) bioavailability. These vasodilatory effects may contribute to improved delivery of blood and oxygen (O 2 ) to exercising muscle., Purpose: Therefore, the objective of this study was to examine how CF supplementation impacts pulmonary O 2 uptake ([Formula: see text]) kinetics and exercise tolerance in sedentary middle-aged adults., Methods: We employed a double-blind cross-over, placebo-controlled design whereby 17 participants (11 male, 6 female; mean ± SD, 45 ± 6 years) randomly received either 7 days of daily CF (400 mg) or placebo (PL) supplementation. On day 7, participants completed a series of 'step' moderate- and severe-intensity exercise tests for the determination of [Formula: see text] kinetics., Results: During moderate-intensity exercise, the time constant of the phase II [Formula: see text] kinetics ([Formula: see text]) was decreased by 15% in CF as compared to PL (mean ± SD; PL 40 ± 12 s vs. CF 34 ± 9 s, P = 0.019), with no differences in the amplitude of [Formula: see text] (A[Formula: see text]; PL 0.77 ± 0.32 l min -1 vs. CF 0.79 ± 0.34 l min -1 , P = 0.263). However, during severe-intensity exercise, [Formula: see text], the amplitude of the slow component ([Formula: see text]) and exercise tolerance (PL 435 ± 58 s vs. CF 424 ± 47 s, P = 0.480) were unchanged between conditions., Conclusion: Our data show that acute CF supplementation enhanced [Formula: see text] kinetics during moderate-, but not severe-intensity exercise in middle-aged participants. These novel effects of CFs, in this demographic, may contribute to improved tolerance of moderate-activity physical activities, which appear commonly present in daily life., Trial Registration: Registered under ClinicalTrials.gov Identifier no. NCT04370353, 30/04/20 retrospectively registered.

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.

In addition to the vast diversity of fauna and flora, the Brazilian Amazon has different climatic periods characterized by periods with greater and lesser rainfall. The main objective of this research was to verify the influence of climatic seasons in the Brazilian Amazon (northeast of Pará state) concerning the aromatic and bioactive profiles of fermented and dried cocoa seeds. About 200 kg of seeds was fermented using specific protocols of local producers. Physicochemical analyzes (total titratable acidity, pH, total phenolic compounds, quantification of monomeric phenolics and methylxanthines) and volatile compounds by GC-MS were carried out. We observed that: in the summer, the highest levels of aldehydes were identified, such as benzaldehyde (6.34%) and phenylacetaldehyde (36.73%), related to the fermented cocoa and honey aromas, respectively; and a total of 27.89% of this same class was identified during winter. There were significant differences ( p ≤ 0.05, Tukey test) in the profile of bioactive compounds (catechin, epicatechin, caffeine, and theobromine), being higher in fermented almonds in winter. This study indicates that the climatic seasons in the Amazon affect the aromatic and bioactive profiles and could produce a new identity standard (summer and winter Amazon) for the cocoa almonds and their products.

High-yield resistant hybrids are used in cocoa fermentation and result in chocolates with different sensorial profiles. This work aimed to characterize the fermentation microbiologically and physicochemically. Hybrids CEPEC 2004, FA13, PH15, and CEPEC 2002 were used for fermentation. The yeast, acetic acid bacteria, lactic acid bacteria, and mesophilic bacteria population were evaluated in their respective medium. Carbohydrates and acids were detected using a high-performance liquid chromatography system, and volatiles were analyzed using gas chromatography-mass spectrometry equipment. Finally, a consumer acceptance test followed by a check-all-that-apply question and a temporal dominance of sensations assessment was performed in chocolate. The fermentation resulted in a typical succession: yeast-dominated at first, followed by lactic acid, acetic acid, and mesophilic bacteria. In the pulp, carbohydrates and citric acid were consumed. Low concentrations of acetic acid (0.09-1.75 g/kg) were detected. Acids, esters, and alcohols were the most abundant groups. The chocolate profile resulted in sweet, acidic, and fruity, satisfying consumers' tastes. PRACTICAL APPLICATION:  The cocoa hybrid-mix fermentation can improve the fermentation process and chocolate quality.  The mixture generated a different sensory profile in comparison to other fermentations.  The fruity chocolate was accepted and liked by consumers., (© 2021 Institute of Food Technologists®.)

In this study, the antifungal potential of chemical constituents from Piper pesaresanum and some synthesized derivatives was determined against three phytopathogenic fungi associated with the cocoa crop. The methodology included the phytochemical study on the aerial part of P. pesaresanum , the synthesis of some derivatives and the evaluation of the antifungal activity against the fungi Moniliophthora roreri , Fusarium solani and Phytophthora sp. The chemical study allowed the isolation of three benzoic acid derivatives ( 1 - 3 ), one dihydrochalcone ( 4 ) and a mixture of sterols ( 5 - 7 ). Seven derivatives ( 8 - 14 ) were synthesized from the main constituents, of which compounds 9 , 10 , 12 and 14 are reported for the first time. Benzoic acid derivatives showed strong antifungal activity against M. roreri , of which 11 (3.0 ± 0.8 µM) was the most active compound with an IC 50 lower compared with positive control Mancozeb ® (4.9 ± 0.4 µM). Dihydrochalcones and acid derivatives were active against F. solani and Phytophthora sp., of which 3 (32.5 ± 3.3 µM) and 4 (26.7 ± 5.3 µM) were the most active compounds, respectively. The preliminary structure-activity relationship allowed us to establish that prenylated chains and the carboxyl group are important in the antifungal activity of benzoic acid derivatives. Likewise, a positive influence of the carbonyl group on the antifungal activity for dihydrochalcones was deduced.

Chocolate is an important source of free bioactive amines and amino acids which play important roles in human health. Considering the limited information on the bioaccessibility of these compounds from chocolate, the objective of this study was to characterize their profiles and bioaccessibility in 70% cocoa dark chocolate through in vitro simulation of oral, gastric and intestinal digestions. Seven amines were detected; polyamines were predominant before in vitro digestion, whereas tyramine, cadaverine and spermidine after digestion. All amines showed high bioaccessibility with slight influence of digestive enzymes. Amines increased after gastrointestinal digestion: tyramine (13-fold), tryptamine (9-fold), others (2.4-4.2-fold) and histamine appeared. All amino acids, GABA and ammonia were detected in chocolate, and their contents increased after in vitro digestion due to digestive enzymes (4.6, 2.8 and 2.1, respectively). Dark chocolate protein is a good source of tryptophan, phenylalanine + tyrosine, isoleucine, histidine, but limiting for lysine, leucine, and threonine., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Clovamide (trans-clovamide, (2S)-3-(3,4-dihydroxyphenyl)-2-[[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]amino]propanoic acid) is a naturally occurring caffeoyl conjugate and a potent antioxidant found in the phenolic fraction of Theobroma Cacao L. beans. This work quantified clovamide content in single-origin cocoa beans at different production stages (raw, roasted, and winnowed side and end products) by high-performance liquid chromatography with diode array detector (HPLC-DAD). We analyzed the antioxidant activities of clovamide and these extracts by measuring their superoxide radical scavenging capabilities in a Rotating Ring-Disk Electrode (RRDE) electrochemical system against in-situ generated superoxide radical. Our studies concluded a positive correlation between clovamide concentration and the overall antioxidant activities of beans, with the roasting step showing a reduction effect on both. The subsequent refining steps recover the clovamide concentration. Antioxidant studies on clovamide alone by RRDE and density functional theory (DFT) studies led to the conclusion that it is a powerful oxygen radical scavenger, partially contributed by its molecular catechol moieties., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The metabolic profile of Lavado cocoa was characterized for the first time by NMR spectroscopy, then compared with the profiles of fermented and processed varieties, Natural and commercial cocoa. The significant difference in the contents of theobromine and flavanols prompted us to examine the cocoa varieties to seek correlations between these metabolite concentrations and the anti-amyloidogenic activity reported for cocoa in the literature. We combined NMR spectroscopy, preparative reversed-phase (RP) chromatography, atomic force microscopy, in vitro biochemical and cell assays, to investigate and compare the anti-amyloidogenic properties of extracts and fractions enriched in different metabolite classes. Lavado variety was the most active and the catechins and theobromine were the chemical components of cocoa hindering Aβ peptide on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line., (Copyright © 2020 Elsevier Ltd. All rights reserved.)