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• Most research on cultured-food acceptance examines cultured meat. • Cultured foods span from cultured animal-flesh (meat) to cultured plants (cacao). • Two studies assess people's reactions to cultured foods of different cell sources. • Foods with higher animal likeness are seen as particularly unnatural and disgusting. • Perceived unnaturalness and disgust significantly predict cultured food acceptance. [ABSTRACT FROM AUTHOR]

To reach the sustainable development goals on waste recycling, cacao pod husk (CPH), produced as an agricultural waste byproduct during the cacao bean processing was applied to manufacture CPH pectin for developing the potential for diverse application in aquaculture, minimizing CPH impact to the environment and bringing benefits to the agriculture and aquaculture industries. In this study, CPH pectin (5 g/kg diet) and Lactobacillus plantarum (LP; 1010 cfu/kg diet) were separately introduced to the diets of Litopenaeus vannamei for a 56-day feeding trial, and two synbiotic combinations of CPH pectin and LP (CPH pectin at 5 g/kg diet + LP at 107 cfu/kg diet or at 1010 cfu/kg diet) were also conducted. After the 56-day feeding trial, significantly elevated percent weight gain, percent length gains and feeding efficiency in L. vannamei were only observed in synbiotic combination of CPH pectin at 5 g/kg diet and LP at 107 cfu/kg diet treatment, and the remainder of the treatments remained consistently similar to the control. Significantly increases in total haemocyte count, granular cells, phenoloxidase activity, and respiratory bursts were observed in L. vannamei fed with synbiotics at 7–28 days of feeding, accompanied by significant promotion of phagocytic activity and clearance efficiency in response to V. alginolyticus challenge during 56 days of feeding trial. Furthermore, at the end of the 56 days of feeding trial, shrimp receiving CPH pectin and/or LP treatments showed a significantly higher survival ratio against V. alginolyticus infection and hypothermal stress. It was therefore concluded that CPH pectin or LP was confirmed as an immunostimulant for L. vannamei to trigger immunocompetence through oral administration without negative effects within 56 days of feeding trial, and the synbiotic combination of CPH pectin and LP exhibited complementary and synergistic effects on growth performance and immunocompetence in L. vannamei. ● Both CPH pectin and LP promoted the immunocompetence and resistance. ● Synbiotic combination of CPH pectin and LP further improve the growth performance. ● The sustainable application of cacao industrial waste benefit shrimp cultivation. [ABSTRACT FROM AUTHOR]

The cacao crop spread throughout the world from its center of origin in South America. However, one of its main diseases cacao swollen shoot disease (CSSD), which is caused by the complex of cacao-infecting badnaviruses, was first detected in Africa. Here we investigate the relationship between the genetic diversity of the viruses that cause CSSD and their geographical distribution, and the possible relationship between the genetic diversity of the virus and the symptoms they trigger. We also sought to determine the evolutionary models that could explain this relationship and evaluate the evolutionary history of the virus through ancestral state reconstructions and temporality analyses. A high mutation rate was observed along with a clear phylogenetic signal in the geographic distribution and in the symptoms associated with each viral species. The molecular clock provided new evidence on the emergence and speciation of cacao-infecting badnaviruses, suggesting a possible American origin of these viruses. • A high mutation rate was observed of the CSSV species. • Geographical region and Symptoms traits showed a clear phylogenetic signal. • In Africa, Ghana was found as the center of virus dispersal. • CSSV possibly originated from America. • The evolutionary history of CSSD together with cacao spread was reconstructed. [ABSTRACT FROM AUTHOR]

The disposal of cacao pod husk, a byproduct of cacao bean processing, can cause serious adverse environmental impacts, motivating scientist to explore and develop potential beneficial applications of this resource. Dried cacao pod husk was extracted with ethanol to obtain a 10.6% pectin of cacao pod husks (pCPH), and its effects on the immunocompetence of Litopenaeus vannamei were estimated. Measured variables included total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, as well as phagocytic activity and clearance efficiency against Vibrio alginolyticus after receiving pCPH at 0, 1.5, 3, and 6 μg shrimp−1 for 0, 1, 3 and 7 days via injection, and their resistance to thermal stress and V. alginolyticus infection were further evaluated. No significant differences were observed in total haemocyte count, differential haemocyte count, and respiratory bursts in shrimp receiving pCPH at 1.5 μg shrimp−1 for 1 day; however, these variables were significantly elevated after 3 days of injection, compared to the control group. The significantly increased phenoloxidase activity was assessed in shrimp receiving pCPH at 1.5, 3 and 6 μg shrimp−1 within 3 days, and activity returned to the baseline after 7 days. Furthermore, the reduced phenoloxidase activity per granulocytes or respiratory bursts per haemocytes maintained homeostasis following the variation of haemogram. For gene expression assessments in haemocytes, the immune-related genes of the lipopolysaccharide and β-1,3-glucan binding protein, prophenoloxidase II and anti-lipopolysaccharide factor as well as innate immune signaling pathway-related genes of toll-like receptors 1 and 3 significantly increased after shrimp received pCPH for 1 day. The increases in phagocytic activity and clearance efficiency were only detected in shrimp receiving pCPH at 6 μg shrimp−1 within 7 days, compared to the control. There was no significant difference in the mortality ratio of shrimp against hyperthermal stress when they received pCPH for 1 day, and the significant higher resistance to hypothermal stress and V. alginolyticus infection were found in shrimp received pCPH at 6 μg shrimp−1 for 1 days than those in the other treatments. It is therefore found that pCPH triggers immune responses serving as an immunostimulant capable of enhancing resistance against V. alginolyticus and hypothermal stress. • Pectin of cacao pod husk (pCPH) enhance immunocompetence of shrimp. • The survival ratio against hypothermal stress and pathogen infection are promoted by pCPH. • pCPH induce LGBP, proPOII, TLR1, TLR3, and ALF gene expression in haemocytes. [ABSTRACT FROM AUTHOR]

Immunostimulation is a novel method and a promising development in aquaculture. Products derived from plants exhibit various biological activities. In this study, the hot-water extract isolated from fresh cacao (Theobroma cacao) pod husks (CPHs) was administered by injection to evaluate cellular signaling pathways of innate immunity, the immunostimulating potential, disease resistance, and hypothermal tolerance of white shrimp, Litopenaeus vannamei. Results showed significant increases in the total hemocyte count, semigranular cells, granular cells, phenoloxidase activity, and respiratory bursts (RBs) of hemocytes per unit of hemolymph at 1 day, and in phagocytic activity toward and the clearance efficiency of Vibrio alginolyticus at 1–3 days after shrimp were injected with fresh CPH extract at 40 μg shrimp−1. However, only RBs per hemocyte had significantly decreased at 1 day after the injection. All immune parameters had returned to control levels by 3 days after receiving fresh CPH extract except for RBs, phagocytic activity, and the clearance efficiency, which had returned to control values by 7 days. Furthermore, at 1 day after the injection, the peroxinectin, prophenoloxidase (proPO) II, toll-like receptors (TLR) 3, signal transducer and activator of transcription (STAT), and crustin in shrimp receiving fresh CPH extract at 20 μg shrimp−1, and the lipopolysaccharide and β-1,3-glucan-binding protein, proPO II, TLR1, and STAT in shrimp receiving fresh CPH extract at 40 μg shrimp−1 were significantly higher than those of shrimp receiving saline. After injecting fresh CPH extract at 10–40 μg shrimp−1 and 40 μg shrimp−1, the mortality of shrimp challenged with V. alginolyticus and exposed to 14 °C for 96 h significantly decreased, respectively. These results suggest that fresh CPH extract can be used as an immunostimulant and a physiological regulator for shrimp through injection administration to enhance immunological and physiological responses, which can elevate the resistance against V. alginolyticus and tolerance against hypothermal stress in L. vannamei. • The resistance to pathogen or hypothermal stress increased in shrimp receiving CPH extract. • CPH extract promoted the immune responses of shrimp through injection. • Innate immunity- and proPO-related genes expressions were induced by injecting CPH extract. [ABSTRACT FROM AUTHOR]

A nanobiosensor was developed for the electrochemical detection of Phytophthora palmivora , a notorious pathogen of cacao causing severe crop loss. Sandwich hybrids between two oligonucleotide probes and the genomic DNA of P. palmivora were prepared and electrochemically detected. The oligonucleotide probes were designed based on the ITS sequence of the P. palmivora field isolates. The detection of sandwich hybrids with P. palmivora genomic DNA and the selectivity of the nanobiosensor towards it compared to other cacao-associated fungal pathogens were demonstrated. The detection limit was determined to be at 0.30 ng DNA μL−1. Detection of P. palmivora DNA on cacao samples was also demonstrated. The result of the preliminary analysis on cacao pod samples shows the potential of the developed nanobiosensor for reliable and more cost-effective analysis of field samples. • Oligonucleotide probes were designed based on the ITS sequence of P. palmivora. • The electrochemical detection scheme was confirmed to work in the presence of P. palmivora genomic DNA. • The selectivity of detection assay was demonstrated, and the detection was found to be sensitive down to 0.30 ng DNA μL-1. • Detection trials in pods showed successful results and discrimination of symptoms caused by P. palmivora from other pathogens. [ABSTRACT FROM AUTHOR]

Highlights • Mp-AOX diminishes hydrogen peroxide generation by mitochondria. • Mp-AOX has antioxidant function. • Mp-AOX expression decreases growth ratio and biomass of S. cerevisiae. • S. cerevisiae expressing Mp-AOX as high-throughput platform to inhibitor screening. Abstract The fungus Moniliophthora perniciosa is the causal agent of witches' broom disease (WBD), one of the most devastating diseases of cacao, the chocolate tree. Many strategies to control WBD have been tested so far, including the use of agrochemicals such as the strobilurins. Strobilurins are fungicides of the QoI family, and they are used in the control of a wide array of fungal diseases in many different crops, including cereals, field crops, fruits, tree nuts, and vegetables. These drugs act by specifically inhibiting fungal respiration at the Qo site of complex III, which is a component of the main mitochondrial respiratory chain. However, M. perniciosa is resistant to this family of chemicals. It has been postulated that this resistant phenotype is, at least in part, a result of the strong ability of this fungus to counteract the oxidative stress generated by the impairment of the main mitochondrial respiratory chain, through the activation of an alternative oxidase (Mp-AOX). To test this hypothesis, we expressed functional mitochondria-localized Mp-AOX in the model yeast Saccharomyces cerevisiae. We demonstrated that heterologous expression of Mp-AOX strongly inhibits hydrogen peroxide production by mitochondria. It also diminishes the total cell amount of oxidized glutathione (GSSG), resulting in a fifty-fold higher GSH/GSSG ratio in cells expressing Mp-AOX than in wild type cells. In addition, Mp-AOX activity decreases yeast growth rate and leads to low biomass production. Therefore, we propose the use of this heterologous expression system to direct the development of new inhibitors of fungal AOX by comparing the differences in optical density of Mp-AOX-expressing cells in the presence and absence of potential AOX inhibitors. Together, our results confirm the antioxidant role of Mp-AOX and provide an in vivo platform to be used in the screening of new fungicides based on Mp-AOX inhibition. [ABSTRACT FROM AUTHOR]

Black pod rot, caused by Phytophthora palmivora (Butler) is one of the most important diseases affecting the Theobroma cacao L. crop. The objective of the present work is to describe a methodology for the isolation and identification of fluorescent Pseudomonas antagonists of P. palmivora. Methods for collecting samples and obtaining bacterial isolates from the rhizosphere of T. cacao are described. The selection of antagonistic isolates is first carried out in a dual culture assay against a reference strain of P. palmivora , previously isolated from the same ecosystem. The strains selected for exhibiting plathogen's growth inhibition are submitted to a foliar test to determine in vivo antagonistic activity by treating detached leaves with the bacterial isolates and the pathogen. The bioprotection effect of bacteria against P. palmivora is also evaluated in planta under controlled conditions, by pre-treating roots with bacterial suspension and further inoculating the pathogen onto the leaves. The identification of bacterial isolates is performed using polyphasic taxonomy including physiological, biochemical and molecular methods. The methodology described here is a practical guide for selecting Pseudomonas with biocontrol potential against P. palmivora in the T. cacao agroecosystem. • The isolation of fluorescent Pseudomonas from T. cacao rhizosphere is described. • Antagonistic isolates against Phytophthora palmivora are selected in vitro and in vivo. • The bioprotection effect is evaluated in plants pre-treated with the selected strains. • Identification of selected antagonistic isolates by polyphasic taxonomy is described. [ABSTRACT FROM AUTHOR]

Abstract The PR-1 proteins (pathogenesis-related protein 1) are involved in plant defense mechanisms against various pathogens. The genome of cacao ( Theobroma cacao ) encodes 14 PR-1 proteins, named TcPR-1a to TcPR-1n. Two of them, TcPR-1f and TcPR-1g, have a C-terminal expansion with high similarity to protein kinase domains, suggesting a receptor-like kinase (RLK) protein architecture. Moreover, TcPR-1g is highly expressed during cacao response to Witches' Broom Disease, caused by the fungus Moniliopthora perniciosa. Here we describe a structural genomics approach to clone, express and purify the kinase domains of TcPR-1f and TcPR-1g. Escherichia coli BL21(DE3)-R3 cells were used for protein expression and co-expression of Lambda Protein Phosphatase was critical for successfully obtaining soluble recombinant protein. We expect that the ability to express and purify the kinase domains of TcPR-1f and TcPR-1g will further our understanding of the role these proteins play during cacao defense response. Highlights • We report the cloning, expression, and purification of kinase domains of two PR-1 Receptor-Like Kinases (PR-1Rk). • Recombinant PR-1RK were expressed in E. coli BL21(DE3)-R3-pRARE2 and BL21(DE3)-R3-lambda-PPase. • Co-expression of lambda-PPase were critical for the production of the recombinant kinase domains in soluble form. [ABSTRACT FROM AUTHOR]

Phylloplanins are potential antimicrobial compounds that can be found on the surface of some plants. These compounds were initially identified in Nicotiana tabacum as proteins secreted by short glandular trichomes (STG), which play an important role in controlling infection caused by Peronospora tabacina , especially by acting on the phylloplane, the first site of plant-pathogen interaction. A protein similar to phylloplanins, containing an Ole e 1 domain, was identified in the Theobroma cacao genome database. In this study, we analyzed the Open Read Frame (ORF) and the promoter of T. Cacao phylloplanin ( Tcphyll ) using bioinformatics tools, the expression pattern of its transcript by RT-qPCR and the promoter activity driving the β-glucuronidase (GUS) expression in transgenic tobacco. The results indicate that Tcphyll encodes a protein of 158 amino acid residues, with signal peptide and potential glycosylation and phosphorylation sites, which are grouped with phylloplanins instead of allergenic pollen grain proteins. Lower levels of Tcphyll transcripts were found in flowers, and higher levels were found in shoot apical meristem, 2–3 cm segment with leaf primordia and, rich in glandular trichomes. Accumulation of transcripts in meristems increased on the first day after inoculation with Moniliophthora perniciosa , and it was reduced after the second day. Promoter analysis identified 40 cis- acting regulatory elements; the GUS expression, under the control of 731 base pairs of the Tcphyll promoter, was observed in tall and short glandular trichomes. The results indicate that Tc PHYLL is the first characterized phylloplanin from a perennial tree. [ABSTRACT FROM AUTHOR]

In recent years several studies have attempted to understand the use of caffeinated beverages in North America before the coming of Europeans using absorbed residues. These studies have focused on the two key plant sources of caffeine in North America: Theobroma cacao (cacao) and Ilex vomitoria (yaupon holly). The authors initiated a study to explore the possibility that one or both plants were used at the Mississippian period (900–1600 CE) center of Etowah in northern Georgia. In the process, a series of problems with methodologies in use were revealed. Key among those were limitations on the methods used to identify ancient caffeinated beverage residues, distinguish them from modern contamination, and differentiate residues made by each plant. In this paper we explore what our data from the Etowah site reveal about methodologies currently in use and make suggestions for future studies of residues created by caffeinated beverages in North America. [ABSTRACT FROM AUTHOR]

Extracts from plant products can promote growth, can act as immunostimulants, and have antibacterial and antiparasitic properties. These extracts can be used as alternatives to the chemical treatments commonly used to prevent and control disease in aquatic species. Research on the subject has focused on identifying invasive plants or agricultural waste products that can be used as immunostimulants. The present study further identified an optimal means of extracting pectin from cacao pod husks to promote growth performance and immunocompetence in Litopenaeus vannamei that would both reduce production costs and enable waste recycling. The byproducts of pectin extraction from cacao pod husks, that is, dried cacao pod husk powder (DCP), steamed DCP (sDCP), hot water–treated cacao pod husk powder (HCP), hot water–treated cacao pod husk supernatant (HCS), and cacao pod husk pectin (CPH pectin), were used to create five experimental diets, which were administered to five groups. The control group was fed a basal diet. The growth and immunocompetence of the shrimp were determined after 30, 60, 90 and 120 days of feeding. To identify the most cost-effective means of obtaining dried cacao pod husks, this study firstly determined the costs and effectiveness of the sun-drying, dehumidification, and heated-wind drying techniques. According to the results of growth performance, the CPH pectin group had higher survival but lower weight gain than the DCP, sDCP, HCP, and HCS groups did. At 30, 60, and 90 days, the clearance efficiency of the experimental groups was higher than that of the control group. At 60 days, the experimental groups had significantly higher phagocytic activity than the control group did. However, at 30 and 90 days the HCP, HCS and CPH pectin group had higher phagocytic activity. The total hemocyte count, differential hemocyte count, phenoloxidase activity, and respiratory bursts of the CPH pectin group were higher at 30 days but the same as those of the control group at 60 and 90 days. After 120 days of feeding trial, the resistance of L. vannamei fed with diets containing byproducts of pectin extraction from cacao pod husks significantly enhanced compared to that in BD group when they were infected with Vibrio aliginolyticus for 168 h, and the related higher survival rate can be observed in HCP, HCS and CPH pectin groups. The study findings suggest that diet-administered HCP and HCS have long-term immunostimulant potential and that CPH pectin has potential in the early stages of feeding. In addition, when heated air drying was employed, a moisture level of below 10% was obtained within 12 h. The results of this study indicate that adding HCP obtained from heated air–dried cacao pod husks to the feed of L. vannamei is the most cost-effective and sustainable means of promoting long-term growth performance and immunocompetence in the species. • HCP promotes long-term growth performance and immunocompetence in shrimp. • The costs and effectiveness of the heated-wind drying techniques were proper for extraction. • The sustainable application of cacao industrial waste benefit shrimp cultivation. [ABSTRACT FROM AUTHOR]

Necrosis- and ethylene-inducing proteins are effector molecules of microorganisms able to induce cell death in plant tissues and/or ethylene biosynthesis. The fungus Moniliophthora perniciosa , which causes the witches' broom disease in Theobroma cacao , contains five genes that encode these proteins (MpNep1-5) in its genome. Among these, MpNep2 is the most expressed Nep during disease's development, especially in the necrotic phase. Although widely studied, little is known about the mechanisms by which these proteins induce cell death. In this perspective, the present study aimed to identify potential Mp NEP2 target proteins in protein extracts of Theobroma cacao (genotype Catongo) and propose, from the results achieved, mechanisms by which Mp NEP2 can induce the process of cell death. Molecular targets captured in vitro by r Mp NEP2 immobilized on CNBr-Sepharose were identified by ms/ms. Candidate targets were identified as an Auxin Response Factor, Sphingosine Kinase and a Formin like protein. These proteins are known to participate in important processes in primary metabolism, molecular function and regulation of the plant's response. The targets: Mp NEP2 interactions were validated in silico. We discussed the different signaling pathways, membrane modulation and cell cytoskeleton, by which Mp NEP2 can act and induce responses in the plant that leads to necrosis. [Display omitted] • For the first time the possible molecular mechanisms behind cell death induced by NEP2, an elicitor secreted by M. perniciosa , are described. • NEP2 interacts with an Auxin Response Factor, a Sphingosine Kinase and a Formin-like protein during the infection process. • NEP2 interaction with target proteins modulates signaling pathways and trigger cell modifications that leads to cell death. [ABSTRACT FROM AUTHOR]

The extract from Theobroma cacao L, pod husk served as the immunostimulant to enhance the immunity and resistance against Lactococcus garvieae of Macrobrachium rosenbergii. In this study, we employed the injection method and dietary administration method to determine the effect of cocoa pod husk (CPH) on M. rosenbergii. The non-specific immune parameters and disease resistance were evaluated after the prawn injected with 1 μg prawn−1 CPH extract (C1), 3 μg prawn−1 CPH extract (C3), and 5 μg prawn−1 CPH extract (C5) for 1, 3, and 7 days. The results showed a significant increase of total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), and phagocytic activity and clearance efficiency to L. garvieae. The non-specific immune parameters, physiological parameters, and disease resistance and growth performance were evaluated after the prawn fed with 1 g kg−1 CPH extract diet (CD1), 3 g kg−1 CPH extract diet (CD3) and 5 g kg−1 CPH extract diet (CD5). The results showed a significant increase in all immune parameters and showed a significant decrease in physiological parameters. No significant difference was observed in growth performance of prawn fed with the CPH containing diet. Both injection and dietary method showed a significant increase in disease resistance against to L. garvieae. We therefore recommend that CPH extract can be used as a immunostimulant for prawn by dietary administration to regulate immune responses, and carbohydrate metabolism lead to enhance resistance against pathogen. • CPH extract is a potential immunostimulant for M. rosenbergii. • Both injection and dietary administration of CPH extract promoted the immunocompetence and resistance of M. rosenbergii. • Dietary administration of CPH extract enhance the anti-stress ability of M. rosenbergii. [ABSTRACT FROM AUTHOR]

Analysis of the complex composition of cacao beans during the fermentation stages provided critical information for evaluating its quality and nutritional attributes. In this work, the effect of fermentation on cacaos' nutritional, mineral, and bioavailability was established from day 0 (d0) to day 8 (d8). Standard AOAC method was used to determine the proximate composition. Total mineral content was determined using the cost-effective MP-AES. Simulation of the human digestive process from the mouth to the small intestine was used to establish the mineral bioavailability. As a result, increasing fermentation days reduces the amount of nutritional content and increases its fat content. Fermentation causes an outward migration, which reduces total mineral content while having a positive effect on reducing potential heavy metals such as Pb and Ni at about 60 % and 50 %, respectively, in d6 at pH 4.84. Mobility of the minerals increases as the pH level decreases due to the penetration of organic acid in the beans. The high bioavailability of Mn, Ca, Fe, and Zn was noted in d5 and d6. Aside from flavor precursor development, results suggest that fermenting cacao beans to its optimum day reduces the risk of heavy metals and positively enhances the bioavailability of minerals necessary for human nutrition. • The total compositions reduce as fermentation progresses, except for fat. • Appropriate fermentation resulted in pH and heavy metals' reduction such as Pb and Ni. • MP-AES successfully determined the elemental composition of fermented beans. • Fermentation enhances the mineral bioavailability on particular days. [ABSTRACT FROM AUTHOR]

Hypothesis Silver nanoparticles (AgNPs) have emerged as a powerful weapon against antibiotic resistant microorganisms. However, most conventional AgNPs syntheses require the use of hazardous chemicals and generate toxic organic waste. Hence, in recent year’s, plant derived and biomolecule based synthetics have has gained much attention. Cacao has been used for years for its medicinal benefits and contains a powerful reducing agent - oxalic acid. We hypothesized that, due to the presence of oxalic acid, cacao extract is capable of reducing silver nitrate (AgNO 3 ) to produce AgNPs. Experiments In this study, AgNPs were synthesized by using natural cacao extract as a reducing and stabilizing agent. The reaction temperature, time and reactant molarity were varied to optimize the synthesis yield. Findings UV–visible spectroscopy (UV–vis), dynamic light scattering (DLS) and transmission electron microscopy (TEM) characterization demonstrated that the synthesized AgNPs were spherical particles ranging in size from 35 to 42.5 nm. The synthesized AgNPs showed significant antibacterial activity against clinically relevant pathogens such as Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus and Staphylococcus epidermidis . Importantly, these green AgNPs are not cytotoxic to human dermal fibroblasts (HDFs) at concentrations below 32 μg/ml. We conclude that cacao-based synthesis is a reproducible and sustainable method for the generation of stable antimicrobial silver nanoparticles with low cytotoxicity to human cells. The AgNPs synthesized in this work have promising properties for applications in the biomedical field. [ABSTRACT FROM AUTHOR]

Fermentation of cocoa ( Theobroma cacao ) is the pivotal in the post-harvest process. Solid state fermentation of the cocoa with inoculum of 10–60% of defined starter consortia of Saccharomyces cerevisiae , Lactobacillus plantarum and Acetobacter aceti in cocoa fermentation boxes was carried out. The microbial ecology, metabolism, bean chemistry and chocolate quality along with natural (control) fermentation were evaluated. An inoculum of 10% resulted to temperature of 42 °C and pH 5.5. The variation in microbial metabolites was indicative with reference to alcohol (2.3 ± 0.2 mg/g), lactic acid (0.1 ± 0.3 mg/g), acetic acid (1.6 mg/g), anthocyanin (8.5 ± 0.5 mg/kg) and total polyphenols (34 ± 0.2 mg ECE/g). The HPLC profiles revealed key alkaloids such as theobromine (2.2 ± 0.08 mg/g), caffeine (1.92 ± 0.06 mg/g), theophylline (0.42 ± 0.03 mg/g). The sensory profile with 10% inoculum scored 8.5 on hedonic scale and was significant ( p < 0.05). High density cell culture (30–60%) resulted in undesirable fermentation leading to low-quality cocoa beans. Use of 10% inoculum of starter consortia regulated microbial succession, consistent fermentation and development of desirable characteristics of the cocoa beans and potentially reduced the fermentation time to 3 days, as against that of 5–7 days in case of natural fermentation. [ABSTRACT FROM AUTHOR]

UV Raman spectroscopy with excitation wavelengths below 270 nm is a very promising technique for obtaining fluorescence-free Raman spectra, since fluorescence usually can be found only at wavelengths longer than 300 nm. Moreover, using an excitation source in the UV range leads to the resonance enhancement of the Raman signals for many molecules. Here, we introduce UV Raman spectroscopy as a tool for investigating various colored food samples, which exhibit strong fluorescence using the VIS or NIR Raman excitation. In this context, brewed coffee, green and black tea, cacao and cola beverage and dark edible oil (pumpkin seed oil) have been investigated. Using an excitation source at 266 nm yielded well-resolved, fluorescence-free spectra of all investigated samples. [ABSTRACT FROM AUTHOR]

Thaumatin-like proteins (TLPs) are found in diverse eukaryotes. Plant TLPs, known as Pathogenicity Related Protein (PR-5), are considered fungal inhibitors. However, genes encoding TLPs are frequently found in fungal genomes. In this work, we have identified that Moniliophthora perniciosa , a basidiomycete pathogen that causes the Witches' Broom Disease (WBD) of cacao, presents thirteen putative TLPs from which four are expressed during WBD progression. One of them is similar to small TLPs, which are present in phytopathogenic basidiomycete, such as wheat stem rust fungus Puccinia graminis . Fungi genomes annotation and phylogenetic data revealed a larger number of TLPs in basidiomycetes when comparing with ascomycetes, suggesting that these proteins could be involved in specific traits of mushroom-forming species. Based on the present data, we discuss the contribution of TLPs in the combat against fungal competitors and hypothesize a role of these proteins in M. perniciosa pathogenicity. [ABSTRACT FROM AUTHOR]

We have provided evidence that the stimulatory effects of (−)-epicatechin ((−)-EPI) on endothelial cell nitric oxide (NO) production may involve the participation of a cell-surface receptor. Thus far, such entity(ies) has not been fully elucidated. The G protein-coupled estrogen receptor (GPER) is a cell-surface receptor that has been linked to protective effects on the cardiovascular system and activation of intracellular signaling pathways (including NO production) similar to those reported with (−)-EPI. In bovine coronary artery endothelial cells (BCAEC) by the use of confocal imaging, we evidence the presence of GPER at the cell-surface and on F-actin filaments. Using in silico studies we document the favorable binding mode between (−)-EPI and GPER. Such binding is comparable to that of the GPER agonist, G1. By the use of selective blockers, we demonstrate that the activation of ERK 1/2 and CaMKII by (−)-EPI is dependent on the GPER/c-SRC/EGFR axis mimicking those effects noted with G1. We also evidence by the use of siRNA the role that GPER has on mediating ERK1/2 activation by (−)-EPI. GPER appears to be coupled to a non Gα i/o or Gα s , protein subtype. To extrapolate our findings to an ex vivo model, we employed phenylephrine pre-contracted aortic rings evidencing that (−)-EPI can mediate vasodilation through GPER activation. In conclusion, we provide evidence that suggests the GPER as a potential mediator of (−)-EPI effects and highlights the important role that GPER may have on cardiovascular system protection. [ABSTRACT FROM AUTHOR]

The cocoa tree, Theobroma cacao L., suffers large yield losses in Aceh Indonesia due to the disease black pod rot, caused by Phytophthora spp. Despite having the largest area under cacao production in Sumatra, farmers in the Aceh region have low overall production because of losses to insect pests and black pod rot. Trichoderma spp. were isolated from the roots and leaves of cacao trees and screened as potential biological control agents. Isolates used in the study were Trichoderma asperellum isolates T2 and T4, Trichoderma longibrachiatum isolates T15 and T16, and Trichoderma virens isolates T1 and Tv. T1, T2, T4, and Tv completely colonized and destroyed Phytophthora tropicalis and Phytophthora palmivora mycelium in precolonized plate assays. All six isolates reduced P . tropicalis , but none reduced the growth of P. palmivora in dual plate assays. Phytophthora growth was suppressed on MIN media amended with sterile heat inactivated Trichoderma culture filtrates, with Tv best suppressing growth of both Phytophthora spp . T. virens isolate Tv was the only isolate observed coiling around P . tropicalis mycelium and disrupted the formation of P. palmivora sporangia. Of all six isolates, only Tv reduced P . palmivora lesion expansion in a detached pod assay, reducing severity by 71%. Tv also reduced P. palmivora infection on seedlings when applied aerially at 1 × 10 6 and 1 × 10 8 conidia/ml, by 19% and 59%, respectively. T. virens isolate Tv is a mycoparasite, antagonizes Phytophthora in a dual plate assay, and shows antibiosis against Phytophthora spp., suggesting that multiple modes of action contribute to its ability to limit Phytophthora lesion expansion on cacao pods and seedlings. [ABSTRACT FROM AUTHOR]

• 44 yeast strains were tested against Moniliophthora roreri ; Frosty pod rot of cacao. • Six strains totally control M. roreri in vitro. • Seven yeasts were identified by molecular tools and found to belong to two genera. • Antifungal properties involved a combination of mechanisms. • Co-culture and VOCs reduced mycelial growth and spore numbers of M. roreri. Frosty pod rot is the most significant disease of cacao (Theobroma cacao L.) in Tropical America, enormously decreasing cacao production in these regions. The introduction of the disease in Cruzeiro do Sul, Brazil, increased cacao growers' concern. Preventive control measurements are already being studied and established. Therefore, this study aimed to isolate and identify yeasts from cacao tissues with the potential to control Moniliophthora roreri , the causal agent of frosty pod rot disease, and further evaluate some of their possible antagonistic mechanisms of action. The initial screening was conducted through a dual-culture with two M. roreri isolates from Ecuador and forty-four yeast isolates (38 from Ecuador, six from Brazil) from cacao tissues, cultivated separately. Yeast antagonistic activity was verified by co-cultivation, volatile organic compounds, cell-free suspensions, and thermostable compounds. Moreover, the biofilm-forming capacity of yeast isolates was examined. Seven yeast isolates were selected and showed an inhibitory effect of up to 71% in dual-cultures. None of them grew in body temperature. The isolates were identified by primer pairs of the D1/D2 region of the large subunit ribosomal ribonucleic acid (LSU rRNA) of Hannaella pagnoccae (INIAP-YYL 111, INIAP-YYL 112 and INIAP-YML 324), H. luteola (INIAP-YYL 521), Vishniacozyma carnescens (INIAP-YYL 612), Hannaella spp. (INIAP-YYL 123) and Rhodotorula paludigena (LEV 169), the last one already studied against Moniliophthora perniciosa in Brazil. Seven out of the forty-four yeast studied showed high inhibitory potential against M. roreri by different antagonistic mechanisms: co-cultivation in culture medium, volatile organic compounds, cell-free and thermostable compounds, and capacity for biofilm formation, some of these characteristics are promising for further use of yeasts as a biocontrol agent of M. roreri. [ABSTRACT FROM AUTHOR]

[Display omitted] • Native strain of Metarhizium anisopliae isolated from naturally infected adult of Helopeltis theivora. • In cage and field trials, M. anisopliae reduced H. theivora incidence in cocoa. • A M. anisopliae proved more effective in field condition by protecting the cocoa pods against H. theivora infestation. The tea mosquito bug, Helopeltis spp. is a destructive pest of cocoa across the globe. In the climate change scenario, the tea mosquito bug species complex, viz. Helopeltis theivora Waterhouse , 1886 , H. bradyi Waterhouse , 1886, and H. antonii V. Signoret, 1858, poses a serious threat to cocoa cultivation in India. Presently, systemic and/or broad-spectrum insecticides are used for mirid management by cocoa growers. However, concerns about their adverse effects on human health and the environment prompted us to search for alternative eco-friendly options for their management. Entomopathogenic fungi (EPF) are an effective and eco-friendly alternative to chemical insecticides for the management of cocoa mirids. The use of native strains of biological control agents improves their success rate. Hence, the present investigation was conducted to isolate, characterize and evaluate an indigenous insect-pathogenic isolate of EPF Metarhizium anisopliae against the mirid H. theivora. An extensive survey was conducted in the Dakshina Kannada district of Karnataka, India, to collect the adults of H. theivora naturally infected with entomopathogenic fungi. A pure culture of the EPF named as TMBMA1 was isolated from the infected adults. Based on the detailed morphological and molecular characterization, the isolate TMBMA1 was identified as Metarhizium anisopliae (Metsch.) Sorokin. The isolate TMBMA1 was found to be highly effective against H. theivora in an in-vitro bioassay and caused 100% insect mortality at 1 × 108 and 1 × 109 conidial suspensions 6 days post inoculation. Similarly, TMBMA1 was found effective in significantly reducing the incidence of mirids in field evaluations conducted for two consecutive years. TMBMA1 was found better than other commercial biopesticides and was comparable with the recommended chemical insecticide Lambda-cyhalothrin. In conclusion, our results reveal that the native EPF, M. anisopliae isolate TMBMA1 has great potential against the mirid H. theivora and can serve as an eco-friendly and sustainable alternative for mirid management in cocoa and other plantations in India. [ABSTRACT FROM AUTHOR]

We report the green synthesis of palladium/CuO nanoparticles (Pd/CuO NPs) using Theobroma cacao L. seeds extract and their catalytic activity for the reduction of 4-nitrophenol and Heck coupling reaction under aerobic conditions. The catalyst was characterized using the powder XRD, TEM, EDS, UV–vis and FT-IR. This method has the advantages of high yields, elimination of surfactant, ligand and homogeneous catalysts, simple methodology and easy work up. The catalyst can be recovered from the reaction mixture and reused several times without any significant loss of catalytic activity. [ABSTRACT FROM AUTHOR]

Seeds from Theobroma cacao progenies derived from the self-pollination of ‘ Catongo ’ב Catongo ’ and the crossing between CCN-10×SCA-6 were immersed for 24 h in different Cd solutions (2; 4; 8; 16 and 32 mg L −1 ) along with the control treatment (without Cd). Shortly after, the seeds were sown in plastic tubes containing organic substrate and were grown in a greenhouse for 60 days. The treatment with Cd was observed to cause morphological, biochemical, molecular and ultrastructural changes in both progenies of T. cacao. There has been deformation in chloroplasts, nuclear chromatin condensation, and reduction in thickness of the mesophyll. As for ‘ Catongo ’ב Catongo ’, a decrease in thickness of the epidermis was noted on the abaxial face. There has been increased guaiacol peroxidase activity in the roots of CCN-10×SCA-6, as well as in the ’ ‘ Catongo ’ב Catongo ’ leaves. In the presence of Cd, CCN-10×SCA-6 showed increased expression of the genes associated with the biosynthesis of phytochelatin (PCS-1) and class III peroxidases (PER-1) in leaves, and metallothionein (MT2b), in roots. In ‘ Catongo ’ב Catongo ’, there has been an increase in the expression of genes associated with the biosynthesis of PER-1 and cytosolic superoxide dismutase dependent on copper and zinc (Cu–Zn SOD Cyt ) in leaves and from MT2b and PCS-1 and roots. There was higher accumulation of Cd in the aerial parts of seedlings from both progenies, whereas the most pronounced accumulation was seen in ’ ‘ Catongo ’ב Catongo ’. The increase in Cd concentration has led to lower Zn and Fe levels in both progenies. Hence, one may conclude that the different survival strategies used by CCN-10×SCA-6 made such progeny more tolerant to Cd stress when compared to ’ ‘ Catongo ’ב Catongo ’ . [ABSTRACT FROM AUTHOR]

Despite remarkable advances in combination antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) infection remains incurable due to the incomplete elimination of the replication-competent virus, which persists in latent reservoirs. Strategies for targeting HIV reservoirs for eradication that involves reactivation of latent proviruses while protecting uninfected cells by cART are urgently needed for cure of HIV infection. We screened medicinal plant extracts for compounds that could reactivate the latent HIV-1 provirus and identified a procyanidin trimer C1 derived from Theobroma cacao as a potent activator of the provirus in human T cells latently infected with HIV-1. This reactivation largely depends on the NF-κB and MAPK signaling pathways because either overexpression of a super-repressor form of IκBα or pretreatment with a MEK inhibitor U0126 diminished provirus reactivation by C1. A pan-PKC inhibitor significantly blocked the phorbol ester-induced but not the C1-induced HIV-1 reactivation. Although C1-induced viral gene expression persisted for as long as 48 h post-stimulation, NF-κB-dependent transcription peaked at 12 h post-stimulation and then quickly declined, suggesting Tat-mediated self-sustainment of HIV-1 expression. These results suggest that procyanidin C1 trimer is a potential compound for reactivation of latent HIV-1 reservoirs. [ABSTRACT FROM AUTHOR]

We address the issue of contamination in sampling and storage procedures and the requirements of sample size and controls necessary to assay ceramic vessels for absorbed chemical residues. We focus our discussion on the detection and quantification of the three methylxanthines – caffeine, theobromine and theophylline as a means to infer whether Mississippian and Southwestern vessels had been used for the consumption of a stimulating drink made from the seeds of Theobroma cacao , a tree that grows in the Mesoamerican tropics. Our research detected two statistically differentiated concentration levels of methylxanthines on objects in museum storage: vessels with low levels of methylxanthines from airborne particulates that we attribute to environmental contamination, and vessels with significant higher levels of the methylxanthines that we attribute to the archaeological record reflecting prehistoric cacao consumption. We propose that cacao was imported into the American Midwest/Southeast during the Mississippian platform mound tradition AD 1000–1300 and into the American Southwest during the Chaco Great House tradition AD 900–1200 and the Hohokam platform mound tradition AD 1300–1400. [ABSTRACT FROM AUTHOR]

In this work, we determined the degree of filamentous fungi contamination in cocoa fermented seeds and the fungal potential to produce enzymes that could contribute to the cocoa quality. Internal transcribed spacer amplicon sequencing (ITS) showed 11 different species with Byssochlamys spectabilis, Aspergillus niger, Aspergillus flavus, Aspergillus carbonarius, Circinella muscae and Penicillium citrinum as the most abundant species. Our results evidenced intra and inter-species differences in the enzymes production. Cellulases, β-glucosidase, ornithine decarboxylase and phenylalanine decarboxylase were the most diffused enzymes expressed in the 53 strains here studied. Moreover, A. niger (6/12), A. carbonarius (2/3), and P. citrinum (3/3) showed high pectinolytic activity. Remarkable was the amino decarboxylase activity of P. citrinum , and A. flavus strains. For the first time we reported the presence of B. spectabilis in cocoa fermented beans, which could play an important role in the biogenic amines formation. In addition, we explored the capability of the Aspergillus section Nigri strains, to produce ochratoxin A (OTA) in a cacao model system (CPMS) and in malt extract medium (MEL). We observed that CPMS, but not MEL, stimulated the OTA production in 6 out 15 strains of Aspergillus section Nigri, reaching values ranging between 1.70 and 4995 μg OTA kg−1 dry mycelium. [Display omitted] • 53 strains of 11 different mould species were recognized in Colombian cocoa fermented seeds. • High intraspecific variability was found in extracellular enzymes of moulds. • B. spectabilis was first encountered in cocoa fermented beans. • Aspergillus section Nigri isolated from cacao showed OTA production ability in cocoa model system. [ABSTRACT FROM AUTHOR]

Highlights: [•] Ni content was determined in foods which importantly contribute to Ni dietary intake. [•] The highest concentration of Ni was found in soya and cacao based food products. [•] Cooking of acidic food in stainless steel pot leached Ni in very low concentrations. [•] Ni in cacao drinks made with water or milk is present in low molecular mass species. [•] The results considerably contribute to the Slovenian database regarding food safety. [Copyright &y& Elsevier]

Abstract: Young Theobroma cacao pods, known as cherelles, are commonly lost to physiological thinning known as cherelle wilt. Cherelles are susceptible to frosty pod rot caused by Moniliophthora roreri. We studied the cherelle wilt process and its impact on M. roreri infection using microscopic, metabolite, and gene expression analyses. Wilt was associated with increased levels of tricarboxylic acid cycle intermediaries and decreased levels of major metabolites. Expression changes of cacao ESTs in response to wilt suggest induction of the polyamine, ethylene, and jasmonic acid biosynthetic pathways and regulation of abscisic acid and cytokinin levels. M. roreriinfection caused little alteration of cherelle physiology. M. roreri responded to the late stage of wilt by altering the expression of M. roreri ESTs associated with metabolite detoxification and host tissue degradation. The environment of the wilting cherelles may truncate the disease cycle of frosty pod rot, by limiting M. roreri sporulation and stopping the lifecycle. [Copyright &y& Elsevier]

Previous work has shown that cacao nib cadmium (Cd) concentrations decrease during fermentation, but only when reaching sufficiently low nib pH. In this work, lab-scale experiments (5 kg units) with lactic and acetic acid amendments were ineffective at reducing the total nib Cd concentration. In contrast, the water-extractable fraction of the nib Cd concentration clearly increased when the pH was decreased. When single pod derived beans were embedded inside a full-scale fermentation box to monitor the effect of the fermentation effect with high precision, nib Cd concentrations decreased by a factor 1.25 (P-value <0.05) after four days of fermentation. Visualisation of the mobile Cd gradient within beans with LA-ICP-MS (using imprints of transversal cuts exposed to a metal binding gel) revealed that fermentation enhances the Cd mobility in the nibs. • Fermentation induces outward migration of Cd from cacao nib to mucilage and testa. • Water extractable Cd in cacao nibs increases with decreasing pH. • Organic acid treatments during lab scale fermentation did not lower nib Cd content. • Mobile Cd gradients within cacao beans were visualized with LA-ICP-MS. [ABSTRACT FROM AUTHOR]

Twenty-two cocoa bean samples were collected from different geographic origins of Indonesia. The composition of methylxanthines, polyphenols, key odorant volatiles, and minerals in the samples were quantified to better understand the quality variation of the beans. The results revealed great diversity in chemical composition of these beans. Unfermented cocoa beans were rich in methylxanthines and polyphenols but had low amounts of key odorant volatiles. Fermented samples shared similar characteristics (less bioactive compounds and abundant key odorant volatiles) regardless of their geographic origins. The origins of the samples could not be determined using the results of bioactive compounds and key odorant volatiles probably due to the influence of post-harvest processing. Separation of samples based on their fermentation degree (under-fermented and fermented samples) during principal component analysis (PCA) improved the accuracy of the determination of geographic origins. The origin of cocoa beans could be delineated by PCA using mineral composition data of the fermented samples. • Chemical profiles of 22 cocoa bean samples are diverse. • Fermentation degree affected chemical composition in beans more than origins. • Beans with similar fermentation degrees had similar bioactive composition. • Principal component analysis using minerals data delineated the origin of the beans. [ABSTRACT FROM AUTHOR]

Abstract: We report the first analysis of polygalacturonase regulation in the basidiomycete Moniliophthora perniciosa. Non-secreted and secreted polygalacturonase activity was obtained from M. perniciosa cultivated on bran-based solid medium or liquid media containing additional carbon sources or cacao extracts (infected or not by the fungus). Polygalacturonase activity assays were carried out under different temperatures and incubation periods. The best secreted polygalacturonase activity was obtained when the enzymatic assay was made at 50 °C for 10 min. Moreover, the polygalacturonase activity was enhanced when the fungus was cultivated on potato dextrose medium, in the presence of additional fermentable carbon sources, in the presence of cacao pulp or non infected cacao extracts. [Copyright &y& Elsevier]

Abstract: Theobroma cacao was detected in the ceramic assemblage at the 8th century Site 13, Alkali Ridge, southeastern Utah. The presence of this Mesoamerican beverage during the Pueblo I period is the earliest reported use of cacao in the northern American Southwest, coming centuries earlier than the recently documented Pueblo II consumption of cacao in cylinder jars, sharp-shouldered pitchers and shallow bowls at Pueblo Bonito, Chaco Canyon. Analogous to the situation at Chaco, cacao was found at Site 13 in a new vessel form decorated with a distinctive design system that contrasted markedly with designs and vessel forms in the local black-on-white ceramic assemblage. We postulate that Abajo R/O at Site 13 represents a ceramic tradition brought by one of the many groups moving into the northern Southwest. The detection of cacao in their ceramic vessels represents new evidence for the migration model that for centuries brought people with Mesoamerican beliefs, ritual practices and a new subsistence lifeway into the American Southwest. [Copyright &y& Elsevier]

Abstract: Theobroma cacao pods were inoculated with meiospores of Moniliophthora roreri (Mr), a hemibiotrophic basidiomycete causing frosty pod rot. Pods were malformed 30 days after inoculation (DAI) and sporulation was observed 60 DAI. Glucose and asparagine concentrations decreased and mannitol and malonate increased in infected pods 30 DAI. By 60 DAI, most carbohydrates, amino acids, and organic acids were drastically reduced by infection. Mannitol and succinic acid levels increased 60 DAI and likely originated from Mr. RT-qPCR analysis of cacao ESTs indicated a strong response to infection 30 DAI in malformed pod. Evidence indicated that biotrophic hyphae colonized pods and a shift to necrotrophic growth occurred later (during the end stages of infection). Expression of cacao ESTs associated with plant hormone biosynthesis and action was altered. Changes in the expression of Mr ESTs in response to nutrient deficiency in pure culture were small. Changes in Mr gene expression patterns and levels of specific metabolites in necrotic sporulating pods 60 DAI compared to malformed pods 30 DAI indicated that the glyoxylate cycle of Mr was up regulated during the shift from biotrophic to necrotrophic phases of the disease cycle. [Copyright &y& Elsevier]

Abstract: In this study, we investigated whether cacao liquor procyanidin (CLPr) extract, which consists of 4.3% catechin, 6.1% epicatechin, 39.4% procyanidins and others, ameliorated hyperglycemia and obesity in C57BL/6 mice fed a control or high-fat diet for 13weeks. CLPr suppressed high-fat diet-induced hyperglycemia, glucose intolerance and fat accumulation in white adipose tissue. CLPr also promoted translocation of glucose transporter 4 (GLUT4) and phosphorylation of AMP-activated protein kinase α (AMPKα) in the plasma membrane of skeletal muscle and brown adipose tissue. Phosphorylation of AMPKα was also enhanced in the liver and white adipose tissue. CLPr up-regulated the gene and protein expression levels of uncoupling protein (UCP)-1 in brown adipose tissue and UCP-3 in skeletal muscle. These results indicate that CLPr is a beneficial food material for the prevention of hyperglycemia and obesity. Activation of AMPKα, translocation of GLUT4 and up-regulation of UCP expression in skeletal muscle and adipose tissue are involved in the molecular mechanisms by which CLPr prevents hyperglycemia and obesity. [Copyright &y& Elsevier]

Abstract: Variables that influence the citric-acid extraction of pectins from cacao pod husk were examined. A screening study tested the main parameters influencing pectin yield and uronic acid content by a factorial fractional 33−1 design. Further, response surface methodology was applied using a central composite design to examine the effect of a greater region of variable values on pectin yield and uronic acid content. The yield was optimized by increasing the temperature and time. None of the variables had a significant effect on the uronic acid content, and there was lack of fit of the model to the uronic acid content. From the fitted model, extraction conditions with aqueous citric acid at pH 3.0 for 95 min at 95 °C provided a predicted yield of approximately 9.0 g/100 g dry cacao pod husks. The obtained experimental value for the yield was 10.1 ± 0.3 g/100 g dry cacao pod husks, with the pectins containing 65.1 ± 0.8 g uronic acid/100 g fraction, DE 40.3% and DA 15.9%. At 5 g/100 g aqueous solution, the fraction behaved as a concentrated solution and presented a non-Newtonian shear-thinning behavior, well described by Cross Model. Additionally, the fraction formed gels at acidic pH and high sucrose content. [Copyright &y& Elsevier]

Abstract: The hemibiotrophic basidiomycete fungus Moniliophthora perniciosa, the causal agent of Witches’ broom disease (WBD) in cacao, is able to grow on methanol as the sole carbon source. In plants, one of the main sources of methanol is the pectin present in the structure of cell walls. Pectin is composed of highly methylesterified chains of galacturonic acid. The hydrolysis between the methyl radicals and galacturonic acid in esterified pectin, mediated by a pectin methylesterase (PME), releases methanol, which may be decomposed by a methanol oxidase (MOX). The analysis of the M. pernciosa genome revealed putative mox and pme genes. Real-time quantitative RT-PCR performed with RNA from mycelia grown in the presence of methanol or pectin as the sole carbon source and with RNA from infected cacao seedlings in different stages of the progression of WBD indicate that the two genes are coregulated, suggesting that the fungus may be metabolizing the methanol released from pectin. Moreover, immunolocalization of homogalacturonan, the main pectic domain that constitutes the primary cell wall matrix, shows a reduction in the level of pectin methyl esterification in infected cacao seedlings. Although MOX has been classically classified as a peroxisomal enzyme, M. perniciosa presents an extracellular methanol oxidase. Its activity was detected in the fungus culture supernatants, and mass spectrometry analysis indicated the presence of this enzyme in the fungus secretome. Because M. pernciosa possesses all genes classically related to methanol metabolism, we propose a peroxisome-independent model for the utilization of methanol by this fungus, which begins with the extracellular oxidation of methanol derived from the demethylation of pectin and finishes in the cytosol. [Copyright &y& Elsevier]

Abstract: The present work was carried out to study the mycobiota of cocoa beans from farm to chocolate. Four hundred and ninety-four samples were analyzed at various stages of cocoa processing: (i) primary stage at the farm (fermentation, drying, and storage), (ii) secondary stage at processing (testa, nibs, liquor, butter, cake and powder) and (iii) the final chocolate product (dark, milk, white and powdered) collected from retail outlets. Direct plating or dilution plating on Dichloran 18% Glycerol agar were used for cocoa beans and processed product analyses, respectively. Fungi were isolated and identified using different keys of identification. The largest numbers and diversity of fungi were observed in the samples collected at the farm, especially during drying and storage. The species with the highest occurrence among samples were: Absidia corymbifera, Aspergillus sp. nov., A. flavus, Penicillium paneum and yeasts. A total of 1132 potentially toxigenic fungi were isolated from the following species or species groups: A. flavus, Aspergillus parasiticus, Aspergillus nomius, Aspergillus niger group, Aspergillus carbonarius and Aspergillus ochraceus group. The highest percentage of toxigenic fungi was found at the drying and storage stages. The industrial processing reduced the fungal contamination in all fractions and no fungi were found in the final chocolate products. The knowledge of which fungi are dominant at each processing stage of cocoa provides important data about their ecology. This understanding leads to a reduction in fungal spoilage and mycotoxin production in this product. [Copyright &y& Elsevier]

Abstract: We propose the existence of extensive trade and interaction among the peoples of the American Southwest and Mesoamerica. We base this inference on our detection of the widespread presence of theobromine, the biomarker for cacao, in 50 of 75 vessels used by Ancestral Puebloans (previously referred to as Anasazi) elites and non-elites from the Four Corners area and Hohokam elites of the Gila and Salt river valleys in the prehistoric American Southwest. We used a non-invasive, non-destructive aqueous sampling procedure that provides conservation advantages over the current methods that require crushing and boiling sherds or removing residues from vessel surfaces. Analysis of these samples by sensitive LC-MS instrumentation capable of detecting nanogram quantities of material revealed theobromine in non-local vessel forms found in elite burials in great house and platform mound sites as well as in local vessel forms used by non-elites living in small unit-pueblos. After elimination of plants native to the Southwest as the source of theobromine, we conclude that either Theobroma cacao or Theobroma Bicolor was imported from its homeland in the Mesoamerican tropical coastal lowlands. Our results are at odds with the current consensus that there was little systematic commerce between Mesoamerican and Southwestern polities. We suggest that cacao was exchanged for high quality turquoise such as that mined in the Cerrillos, New Mexico mining district. We conclude that, far from being isolated developmentally, this trade integrally tied populations in the American Southwest to the socio-political and economic activities of Mesoamerican states. [Copyright &y& Elsevier]

Abstract: Sixty-nine endospore-forming bacterial endophytes consisting of 15 different species from five genera were isolated from leaves, pods, branches, and flower cushions of Theobroma cacao as potential biological control agents. Sixteen isolates had in vitro chitinase production. In antagonism studies against cacao pathogens, 42% inhibited Moniliophthora roreri, 33% inhibited Moniliophthora perniciosa, and 49% inhibited Phytophthora capsici. Twenty-five percent of isolates inhibited the growth of both Moniliophthora spp., while 22% of isolates inhibited the growth of all three pathogens. Isolates that were chitinolytic and tested negative on Bacillus cereus agar were tested with in planta studies. All 14 isolates colonized the phyllosphere and internal leaf tissue when introduced with Silwet L-77, regardless of the tissue of origin of the isolate. Eight isolates significantly inhibited P. capsici lesion formation (p =0.05) in detached leaf assays when compared to untreated control leaves. ARISA with bacilli specific primers amplified 21 OTUs in field grown cacao leaves, while eubacteria specific primers amplified 58 OTUs. ARISA analysis of treated leaves demonstrated that inundative application of a single bacterial species did not cause a long-term shift of native bacterial communities. This research illustrates the presence of endospore-forming bacterial endophytes in cacao trees, their potential as antagonists of cacao pathogens, and that cacao harbors a range of bacterial endophytes. [Copyright &y& Elsevier]

Abstract: The main aim of this work was to produce fruit wines from pulp of gabiroba, cacao, umbu, cupuassu and jaboticaba and characterize them using gas chromatography–mass spectrometry for determination of minor compounds and gas chromatography-flame ionization detection for major compounds. Ninety-nine compounds (C6 compounds, alcohols, monoterpenic alcohols, monoterpenic oxides, ethyl esters, acetates, volatile phenols, acids, carbonyl compounds, sulfur compounds and sugars) were identified in fruit wines. The typical composition for each fruit wine was evidenced by principal component analysis and Tukey test. The yeast UFLA CA 1162 was efficient in the fermentation of the fruit pulp used in this work. The identification and quantification of the compounds allowed a good characterization of the fruit wines. With our results, we conclude that the use of tropical fruits in the production of fruit wines is a viable alternative that allows the use of harvest surpluses and other underused fruits, resulting in the introduction of new products into the market. [ABSTRACT FROM AUTHOR]

Abstract: Cocoa beans are rich source of polyphenols, contributing about 10% of the dry weight of the whole bean and its derivative chocolate, particularly dark chocolate, is considered one of the major contributors of antioxidants to the American diet after fruits and vegetables. At present the wide variation in cocoa processing and in the content and profile of polyphenols make it difficult to determine to what extent the findings about positive effects expressed in different studies, translate into tangible clinical benefits. Moreover, before claiming any healthy properties to a plant, natural product or food item on human subject, a basic research project approved by scientific and ethical commissions has to be performed. Until now the definition, composition, manufacturing specifications, packaging and labelling of cocoa and chocolate products in Europe, are regulated by “Directive 2000/36/EC of the European parliament and of the council”. The definitions take changes in consumer tastes, chocolate composition and labelling into account, but do not consider the real potential of healthy, beneficial and nutraceutical effects. In fact, they fail to establish an official analytical methodology for the quantification of phenolic compounds in cocoa and chocolate. Moreover quantification of these compounds is not used in product classification. This article reviews many qualitative differences of cocoa and chocolate, in particular dark chocolate, aiming to establish the different implications for public health through the use of the analyzed concentration of polyphenols in cocoa products. [Copyright &y& Elsevier]

• This is the first proteomics study of M. perniciosa. • We identified an 82-key protein set associated to fungus development. • Proteins crucial for primordium and basidiocarp formation were identified. • Main proteins are related to cell proliferation, stress response, and morphogenesis. Moniliophthora perniciosa is a hemibiotrophic fungus that causes witches' broom disease in cacao (Theobroma cacao L.). The biotrophic fungal phase initiates the disease and is characterized by a monokaryotic mycelium, while the necrotrophic phase is characterized by a dikaryotic mycelium and leads to necrosis of infected tissues. A study of the necrotrophic phase was conducted on bran-based solid medium, which is the only medium that enables basidiocarp and basidiospore production. Six different fungal developmental phases were observed according to the mycelium colour or the organ produced: white, yellow, pink, dark pink, primordium and basidiocarp. In this study, we identified notable proteins in each phase, particularly those accumulated prior to basidiocarp formation. Proteins were analysed by proteomics; 2-D gels showed 300–550 spots. Statistically differentially accumulated spots were sequenced by mass spectrometry and 259 proteins were identified and categorized into nine functional classes. Proteins related to energy metabolism, protein folding and morphogenesis that were potentially involved in primordium and basidiocarp formation were identified; these proteins may represent useful candidates for further analysis related to the spread and pathogenesis of this fungus. To the best of our knowledge, this report describes the first proteomic analysis of the developmental phases of Moniliophthora perniciosa. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cocoa is derived from the seeds of Theobroma cacao. • It has numerous biological properties, i.e. antioxidant, antiproliferative, antinflammatory. • Cocoa shows noticeable value in diet and health. Cocoa is derived from the seeds of Theobroma cacao L., an evergreen tree typical of tropical regions. It contains numerous phytochemicals, with polyphenols representing the largest groups of compounds inside the seed, and has been implicated in numerous biological properties, such as antioxidant, antiproliferative, antiapoptotic, anti-inflammatory, anti-cancer. Moreover, cocoa has been investigated in different health conditions, including heart diseases, dyspepsia, nervous system diseases, circulation problems, and many others. Given its high consumption in many countries all over the world, it is important to know and understand its effects on human health. In addition, the cocoa bean shell, a by-product of the process of cocoa preparation, has been gaining remarkable interest due to its high content of phytochemicals. This review summarizes the available literature and works on the health benefits of cocoa and cocoa bean shells. Moreover, the current review focuses on studies investigating their possible therapeutic roles in cancer and the underlining potential mechanisms of action. [ABSTRACT FROM AUTHOR]

Abstract: Alternative measures to chemical fungicides are needed to control Phytophthora megakarya, the main causal agent of black pod disease in Central and West Africa. Precolonized plate and detached cacao pod assays were used to screen fungal isolates for mycoparasitism on P. megakarya. Of over 200 isolates screened, only Trichoderma asperellum isolates 659-7, PR10, PR11, and PR12 were capable of necrotrophic mycoparasitism in both assays. Additional in vitro mycoparasitism assays demonstrated that T. asperellum 659-7, PR10, PR11, and PR12 were mycoparasitic on Phytophthora capsici, Phytophthora citrophthora, and Phytophthora palmivora; other causal agents of black pod worldwide. Culture filtrates from these T. asperellum isolates contained substantial laminarinase activity and lesser amounts of caboxymethylcellulase activity which could function in degrading cell walls of Phytophthora during mycoparasitism. Sequence analysis of the gene for translation elongation factor 1 (tef1) confirmed the identification of these isolates as T. asperellum. Molecular fingerprinting using RAPD and UP-PCR demonstrated high genetic similarity between isolates 659-7, PR11, and PR12 and high dissimilarity between PR10 and the other three isolates. Cacao trees sprayed with T. asperellum 659-7, PR10, PR11, or PR12 had a significantly lower percentage of diseased pods than the nontreated control in both short-term and long-term field screening experiments, but not lower than that for the chemical fungicide control treatment. Data presented here demonstrate for the first time the potential of mycoparasitic isolates of T. asperellum for suppression of black pod of cacao in Cameroon. [Copyright &y& Elsevier]

Abstract: This study asks whether exposure to images of chocolate induces cravings and guilty feelings in females. A further aim was to examine whether these effects are heightened in the case of dieters. The participants, 85 females, saw a series of enticing media images, either of chocolate or of non-food products. Two thirds of the sample were dieting or had dieted in the past; 15% had been on seven or more diets. After viewing the images all participants completed the Attitudes to Chocolate Questionnaire (ACQ) [Benton, Greenfield, & Morgan (1998). The development of the attitudes to chocolate questionnaire. Personality and Individual Differences, 24(4), 513–520]. The different conditions affected only those who dieted. Dieters had significantly higher ACQ scores after viewing the chocolate images than the non-dieters. It is suggested that dietary restriction increases desire for forbidden foods, in the form of craving, and may induce negative affect such as guilt, anxiety and depression. [Copyright &y& Elsevier]

Abstract: Moniliophthora perniciosa infects young meristematic tissue of Theobroma cacao and induces abnormal shoot development and ultimately necrosis. We analyzed culture filtrates of M. perniciosa to identify metabolites with possible effects on witches’ broom disease symptom development. All isolates produced salicylic acid (SA), phenyllactic acid and mandelic acid. When SA levels were measured in plant tissues, infected brooms had 5 times more SA compared to healthy shoots. In contrast, leaf inoculation with basidiospores and application of chemical elicitors did not alter leaf SA content. These findings suggest that the necrotrophic phase of M. perniciosa causes SA accumulation in diseased tissues. [Copyright &y& Elsevier]

The infection process of Moniliophthora perniciosa on cacao pods of different resistance sources to witches' broom disease of cacao; i.e., TSH1188 (resistant) and Catongo (susceptible), was evaluated at the histological and enzymatic levels. Cacao pods were inoculated with a basidiospore suspension and were assessed weekly for witches' broom symptoms. Histological evaluations revealed differences between genotypes in the pre and post penetration events of M. perniciosa. Compared with catongo, the onset and development of fungal colonization were delayed and less intense in pods of TSH1188. Also, peroxidase activities were higher during the early stages of the infection (0–48 HAI) in the incompatible interaction. These results suggested that TSH1188 cacao pods have structural and biochemical mechanisms which may hinder/delay the infection of M. perniciosa. [Display omitted] • Moniliophthora perniciosa infect cacao pods regardless of pod age. • The rate of the infection process differed between cacao genotypes. • The resistance mechanism was specific to a genotype. • Early and high peroxidase activity inhibited the advanced stages of infection. [ABSTRACT FROM AUTHOR]