Cacao Procyanidins-Induced Lifespan Extension in Caenorhabditis elegans in a Nervous System and CaMKII-Dependent Manner.

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.

Integrating shotgun metagenomics and metabolomics to elucidate the dynamics of microbial communities and metabolites in fine flavor cocoa fermentation in Hainan.

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.

Influence of cocoa varieties on carbohydrate composition and enzymatic activity of cocoa pulp.

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.

The essential role of spontaneous and starter yeasts in cocoa and coffee fermentation.

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.

Metabolomics during the spontaneous fermentation in cocoa (Theobroma cacao L.): An exploraty review.

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.

Revealing the pathways of cadmium uptake and translocation in cacao trees (Theobroma cacao L.): A 108Cd pulse-chase experiment.

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 (108Cd) 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 108Cd via surface irrigation with a spiked solution. The increase in 108Cd isotopic abundance (IA) in the plant tissues was followed up for 548 days after spiking. The 108Cd 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 108Cd 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 108Cd 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.

An in-depth multiphasic analysis of the chocolate production chain, from bean to bar, demonstrates the superiority of Saccharomyces cerevisiae over Hanseniaspora opuntiae as functional starter culture during cocoa fermentation.

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.

Incubation tests mimicking fermentation reveal that phytate breakdown is key to lower the cadmium concentrations in cacao nibs.

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 (R2 = 0.56), suggesting hydrolysis of phytate and mobilisation of the associated Cd2+.

Molecular, biochemical and micromorphological responses of cacao seedlings of the Parinari series, carrying the lethal gene Luteus-Pa, in the presence and absence of cotyledons.

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.

Hazelnut peptide fractions preserve their bioactivities beyond industrial manufacture and simulated digestion of hazelnut cocoa cream.

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.

Diversity and determinants of bitterness, astringency, and fat content in cultivated Nacional and native Amazonian cocoa accessions from Ecuador.

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.

Single-base resolution methylomes of somatic embryogenesis in Theobroma cacao L. reveal epigenome modifications associated with somatic embryo abnormalities.

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.

Analysis of fruit ripening in Theobroma cacao pod husk based on untargeted metabolomics.

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.

Fatty acid tryptamide from cacao elongates Drosophila melanogaster lifespan with sirtuin-dependent heat shock protein expression.

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.

Unique metabolite profiles of Indonesian cocoa beans from different origins and their correlation with temperature.

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.

Drivers of cadmium accumulation in Theobroma cacao L. beans: A quantitative synthesis of soil-plant relationships across the Cacao Belt.

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).

Screening Wild Yeast Isolated from Cocoa Bean Fermentation Using Volatile Compounds Profile.

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.

TcTI, a Kunitz-type trypsin inhibitor from cocoa associated with defense against pathogens.

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 ß-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.

Improvement of endoglucanase production by Aspergillus brasiliensis in solid-state fermentation using cupuaçu (Theobroma grandiflorum) residue as substrate.

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.

Combination of Aerobic Training and Cocoa Flavanols as Effective Therapies to Reduce Metabolic and Inflammatory Disruptions in Insulin-Resistant Rats: The Exercise, Cocoa, and Diabetes Study.

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.