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

Dark chocolate produced on equipment used to manufacture milk chocolate can contain milk due to cross-contact. This study evaluated the use of dry cleaning methods for removing milk chocolate residue from a butterfly or ball valve attached to a stainless steel pipe and from pilot-scale equipment used in chocolate manufacture. Milk-free dark chocolate (40 °C) was pumped through a milk chocolate-contaminated valve/pipe assembly after no cleaning, use of a pig purging treatment, or a 40 °C cocoa butter flush. Dark chocolate samples were collected at 7-sec intervals. Treatments investigated for the removal of residual milk chocolate from a conche and a ball mill included no cleaning, a 40 °C cocoa butter rinse, and wet cleaning. After cleaning, three batches of dark chocolate (40 °C) were processed in the ball mill and conche, and each batch was collected. Milk chocolate was processed on a 3-roll refiner, followed by push-through with dark chocolate (∼9 kg) with 0.3 kg samples collected at 5-min intervals. Dark chocolate samples were analyzed for milk concentrations by ELISA. Trials and analyses were completed in triplicate. Dark chocolate push-through alone resulted in milk concentrations ≥4,500 µg/g in samples obtained from the contaminated valve/pipe combinations within the first few seconds of collection, and ≥16.2 kg of dark chocolate was needed to obtain milk concentrations below the ELISA LOQ (2.5 µg/g). A pig purging treatment of the ball valve/pipe assembly resulted in milk concentrations below the ELISA LOQ. A cocoa butter flush of the butterfly valve/pipe decreased initial milk concentrations, but milk was detected until ≥18.7 kg dark chocolate purge. Milk concentrations in first batches of dark chocolate processed in a ball mill and conche without cleaning were ≥17,000 µg/g while the use of a cocoa butter rinse reduced milk levels in dark chocolate by ≥89%. Some dry cleaning treatments were effective at reducing levels of milk in dark chocolate due to cross-contact., (Published by Elsevier Inc.)

To reduce unwanted fat bloom in the manufacturing and storage of chocolates, detailed knowledge of the chemical composition and molecular mobility of the oils and fats contained is required. Although the formation of fat bloom on chocolate products has been studied for many decades with regard to its prevention and reduction, questions on the molecular level still remain to be answered. Chocolate products with nut-based fillings are especially prone to undesirable fat bloom. The chemical composition of fat bloom is thought to be dominated by the triacylglycerides of the chocolate matrix, which migrate to the chocolate's surface and recrystallize there. Migration of oils from the fillings into the chocolate as driving force for fat bloom formation is an additional factor in the discussion. In this work, the migration was studied and confirmed by MRI, while the chemical composition of the fat bloom was measured by NMR spectroscopy and HPLC-MS, revealing the most important triacylglycerides in the fat bloom. The combination of HPLC-MS with NMR spectroscopy at 800 MHz allows for detailed chemical structure determination. A rapid routine was developed combining the two modalities, which was then applied to investigate the aging, the impact of chocolate composition, and the influence of hazelnut fillings processing parameters, such as the degree of roasting and grinding of the nuts or the mixing time, on fat bloom formation.

In the present study, the effect of the addition of quince and collagen type I and III to dessert chocolate on its functional properties was determined. The study evaluated the antioxidant potential of the tested formulations using the FRAP method and the linoleic acid oxidation test and beta-carotene bleaching test. The tested samples were also evaluated for inhibitory activity against enzymes important in preventive health (inflammation and neurodegenerative disorders) namely: AChE, BChE, GR, GPx, COX, and SOD. The addition of quince and collagen to the chocolate samples resulted in higher activity compared to the control sample, as indicated by the FRAP test. The experiment highlighted the impact of including quince fruit on the antioxidant activity of the chocolate samples. Interestingly, merely increasing the quince fruit amount did not consistently enhance antioxidant potential. Specifically, chocolate samples with a lower proportion of quince fruit (2 g/100 g) exhibited greater antioxidant activity when supplemented with collagen I. Conversely, in samples with higher quince percentages (3 g and 4 g), those enriched with collagen III showed higher antioxidant activity. Similar correlations were observed in the linoleic acid oxidation test. Notably, samples containing 3 g and 4 g of quince and type III collagen demonstrated statistically similar highest antioxidant properties. Regardless of the collagen type used, there was no observed increase in activity towards the tested enzymes for samples with the lowest percentage of quince fruit. Both collagen types exhibited the highest activity in the inhibition assay against acetylcholinesterase and butyrylcholinesterase when combined with 3 g and 4 g of quince. Overall, the experimental incorporation of both fruit and collagen enhanced the chocolates' activity. Similarly to the antioxidant activity findings, chocolates with lower quince fruit quantities showed increased activity when supplemented with collagen III, while those with higher quince content (3 g and 4 g) displayed higher activity with collagen I. Bitter chocolate by itself is an attractive food product, rich in many bioactive compounds. However, enriching it with other attractive raw materials can make its properties and taste even more attractive.

Mass spectrometry has been widely accepted as a confirmatory tool for the sensitive detection of undeclared presence of allergenic ingredients. Multiple methods have been developed so far, achieving different levels of sensitivity and robustness, still lacking harmonization of the analytical validation and impairing comparability of results. In this investigation, a quantitative method has been validated in-house for the determination of six allergenic ingredients (cow's milk, hen's egg, peanut, soybean, hazelnut, and almond) in a chocolate-based matrix. The latter has been produced in a food pilot plant to provide a real and well-characterized matrix for proper assessment of method performance characteristics according to official guidelines. In particular, recent considerations issued by the European Committee for Standardization have been followed to guide a rigorous single-laboratory validation and to feature the main method performance, such as selectivity, linearity, and sensitivity. Synthetic surrogates of the peptide markers have been used both in native and labelled forms in matrix-matched calibration curves as external calibrants and internal standards, respectively. A two-order of magnitude range was investigated, focusing on the low concentration range for proper assessment of the detection and quantification limits (LOD and LOQ) by rigorous calibration approach. Conversion factors for all six allergenic ingredients have been determined for the first time to report the final quantitative information as fraction of total allergenic food protein (TAFP) per mass of food (µg TAFP /g food ), since such a reporting unit is exploitable in allergenic risk assessment plans. The method achieved good sensitivity with LOD values ranging between 0.08 and 0.2 µg TAFP /g food , for all ingredients besides egg and soybean, whose quantitative markers reported a slightly higher limit (1.1 and 1.2 µg TAFP /g food , respectively). Different samples of chocolate bar incurred at four defined concentration levels close to the currently available threshold doses have been analyzed to test the quantitative performance of the analytical method, with a proper estimate of the measurement uncertainty from different sources of variability. The sensitivity achieved resulted in compliance with the various threshold doses issued or recommended worldwide., (© 2023. The Author(s).)

The main component of chocolate, cocoa (Theobroma cacao), is a significant commercial agricultural plant that directly sustains the livelihoods of an estimated forty to fifty million people. The economies of many cocoa producing nations, particularly those in the developing world, are supported by cocoa export revenue. To ensure satisfactory yields, however, the plant is usually intensely treated with pesticides because it is vulnerable to disease and pest attacks. Even though pesticides help protect the cocoa plant, unintended environmental contamination is also likely. Honey, produced from nectar obtained by honeybees from flowers while foraging, can serve as a good indicator for the level of pesticide residues and environmental pesticide build-up in landscapes. Here, we use a systematic literature review to quantify the extent of research on residues of pesticides used in cocoa cultivation in honey. In 81% of the 104 studies examined for this analysis, 169 distinct compounds were detected. Imidacloprid was the most frequently detected pesticide, making neonicotinoids the most frequently found class of pesticides overall. However, in cocoa producing countries, organophosphates, organochlorines, and pyrethroids were the most frequently detected pesticides. Interestingly, only 19% of studies were carried out in cocoa producing countries. We recommend prioritizing more research in the countries that produce cocoa to help to understand the potential impact of pesticide residues linked with cocoa cultivation in honey and the environment more generally to inform better pesticide usage, human health, and environmental policies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Boakye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The chemical composition of dark chocolate has a significant impact on its complex flavor profile. This study aims to investigate the relationship between the volatile chemical composition and perceived flavor of 54 dark chocolate samples made from Trinitario cocoa beans from the Dominican Republic. The samples were evaluated by a trained panel and analyzed using gas chromatography-mass spectrometry (GC-MS) to identify and quantify the volatile compounds. Predictive models based on a partial least squares regression (PLS) allowed the identification of key compounds for predicting individual sensory attributes. The models were most successful in classifying samples based on the intensity of bitterness and astringency, even though these attributes are mostly linked to non-volatile compounds. Acetaldehyde, dimethyl sulfide, and 2,3-butanediol were found to be key predictors for various sensory attributes, while propylene glycol diacetate was identified as a possible marker for red fruit aroma. The study highlights the potential of using volatile compounds to accurately predict chocolate flavor potential.

The high incidence of aflatoxins (AFs) in chocolates suggests the necessity to create a practical and cost-effective processing strategy for eliminating mycotoxins. The present study aimed to assess the adsorption abilities of activated charcoal (A. charcoal), yeast ( Saccharomyces cerevisiae ), and the probiotic Lactobacillus rhamnosus as AFs adsorbents in three forms-sole, di- and tri-mix-in phosphate-buffered saline (PBS) through an in vitro approach, simulated to mimic the conditions present in the gastrointestinal tract (GIT) based on pH, time and AFs concentration. In addition, the novel fortification of chocolate with A. charcoal, probiotic, and yeast (tri-mix adsorbents) was evaluated for its effects on the sensory properties. Using HPLC, 60 samples of dark, milk, bitter, couverture, powder, and wafer chocolates were examined for the presence of AFs. Results showed that all the examined samples contained AFs, with maximum concentrations of 2.32, 1.81, and 1.66 µg/kg for powder, milk, and dark chocolates, respectively. The combined treatment demonstrated the highest adsorption efficiency (96.8%) among all tested compounds. Scanning electron microscope (SEM) analysis revealed the tested adsorbents to be effective AF-binding agents. Moreover, the novel combination of tri-mix fortified chocolate had a minor cytotoxicity impact on the adsorptive abilities, with the highest binding at pH 6.8 for 4 h, in addition to inducing an insignificant effect on the sensory attributes of dark chocolate. Tri-mix is thus recommended in the manufacturing of dark chocolate in order to enhance the safety of the newly developed product.

In this work, a rapid detection method using solid sampling electrothermal vaporization atomic absorption spectrometry (SS-ETV-AAS) was established for cadmium in chocolate. The instrumental system includes a solid sampling ETV unit, a catalytic pyrolysis furnace, an AAS detector, and a gas supply system with only an air pump and a hydrogen generator. Herein, MgO material with 1.0−1.5 mm particle size was first employed to replace the kaolin filler previously used to further shorten the peak width and to thereby improve the sensitivity. With 350 mL/min of air, a chocolate sample was heated for 25 s from 435 to 464 °C to remove water and organic matrices; then, after supplying 240 mL/min hydrogen and turning down air to 120 mL/min, a N2/H2 mixture gas was formed to accelerate Cd vaporization from chocolate residue under 465 to 765 °C. Under the optimized conditions, the detection limit (LOD) was obviously lowered to 70 pg/g (vs. previous 150 pg/g) with R2 > 0.999; the relative standard deviations (RSD) of repeated measurements for real chocolate samples ranged from 1.5% to 6.4%, indicating a favorable precision; and the Cd recoveries were in the range of 93−107%, proving a satisfied accuracy. Thus, the total analysis time is less than 3 min without the sample digestion process. Thereafter, 78 chocolate samples with different brands from 9 producing countries in China market were collected and measured by this proposed method. Based on the measured Cd concentrations, a dietary exposure assessment was performed for Chinese residents, and the target hazard quotient (THQ) values are all less than 1, proving no significant health risk from intaking chocolate cadmium for Chinese residents.

Chocolate is one of the most popular sweets in the world. In recent years, the bean-to-bar process for chocolate production has attracted global attention. Bean-to-bar is a method of managing the whole production process from cocoa beans to chocolate bars, including single-origin chocolate (SOC). Many manufacturers aim to produce high-quality chocolate to maximize the flavor of cocoa beans. However, chocolate compounds are very complex due to many processes, and there are a limited number of studies on the SOC produced from the bean-to-bar process. Therefore, understanding the effects of processing is important for the growth of the chocolate industry. The objective of this study was to investigate the processing effect on the component changes of SOC during the bean-to-bar process. In this study, the component changes during the bean-to-bar process were monitored using gas chromatography/mass spectrometry (GC/MS). Then, the characteristics of SOC from five regions in Indonesia were further investigated. Lastly, the component profiles were combined with the data obtained from sensory evaluation. Our results showed that the influence of the manufacturing process was greater than that of the difference in the cocoa production area. Moreover, 1-pentanol, raffinose, and heptanoic acid were correlated with sweetness and dairy flavor, whereas glutamic acid, tartaric acid, 3-methyl-2-butanone, mannitol, and ethyl cinnamate were correlated with bitterness, astringency, and cocoa flavor, which were shown to be affected by fermentation, roasting, and sugar addition. This information might provide a basis for improving the chocolate production process and its quality related to the component profiles., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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

The design and production of incurred test materials are critical for the development and validation of methods for food allergen analysis. This is because production and processing conditions, together with the food matrix, can modify allergens affecting their structure, extractability and detectability. For the ThRAll project, which aims to develop a mass spectrometry-based reference method for the simultaneous accurate quantification of six allergenic ingredients in two hard to analyse matrices. Two highly processed matrices, chocolate bars and broth powder, were selected to incur with six allergenic ingredients (egg, milk, peanut, soy, hazelnut and almond) at 2, 4, 10 and 40 mg total allergenic protein/kg food matrix using a pilot-scale food manufacturing plant. The allergenic activity of the ingredients incurred was verified using food-allergic patient serum/plasma IgE, the homogeneity of the incurred matrices verified and their stability at 4 °C assessed over at least 30-month storage using appropriate enzyme-linked immunosorbent assays (ELISA). Allergens were found at all levels from the chocolate bar and were homogenously distributed, apart from peanut and soy which could only be determined above 4 mg total allergenic ingredient protein/kg. The homogeneity assessment was restricted to analysis of soy, milk and peanut for the broth powder but nevertheless demonstrated that the allergens were homogeneously distributed. All the allergens tested were found to be stable in the incurred matrices for at least 30 months demonstrating they are suitable for method development., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

The consumption of food for pleasure is mainly associated with adverse health effects. This review was carried out to verify recent reports on the impact of chocolate and wine consumption on cardiovascular health, with a particular focus on atherosclerosis. On one side, these products have proven adverse effects on the cardiovascular system, but on the other hand, if consumed in optimal amounts, they have cardiovascular benefits. The submitted data suggest that the beneficial doses are 30-50 g and 130/250 mL for chocolate and wine, respectively, for women and men. The accumulated evidence indicates that the active ingredients in the products under consideration in this review are phenolic compounds, characterized by anti-inflammatory, antioxidant, and antiplatelet properties. However, there are also some reports of cardioprotective properties of other compounds such as esters, amines, biogenic amines, amino acids, fatty acids, mineral ingredients, and vitamins. Our narrative review has shown that in meta-analyses of intervention studies, consumption of chocolate and wine was positively associated with the beneficial outcomes associated with the cardiovascular system. In contrast, the assessment with the GRADE (Grading of Recommendations Assessment, Development and Evaluation) scale did not confirm this phenomenon. In addition, mechanisms of action of bioactive compounds present in chocolate and wine depend on some factors, such as age, sex, body weight, and the presence of additional medical conditions. Patients using cardiovascular drugs simultaneously with both products should be alert to the risk of pharmacologically relevant interactions during their use. Our narrative review leads to the conclusion that there is abundant evidence to prove the beneficial impact of consuming both products on cardiovascular health, however some evidence still remains controversial. Many authors of studies included in this review postulated that well-designed, longitudinal studies should be performed to determine the effects of these products and their components on atherosclerosis and other CVD (Cardiovascular Disease) disease.

Caffeine-related health incidents in New Zealand have escalated over the last two decades. In order to reduce the risk of substance-related harm, it is important to understand the consumers' motivations for its use. This is especially true for tertiary students who are presumed to be at a higher risk due to seeking out caffeine's well-known cognitive benefits as well as the targeted marketing of such products to young adults. This study examined the habits and motivations for caffeine consumption in tertiary students in New Zealand. A previously validated caffeine consumption-habits (CaffCo) questionnaire was administered online to 317 tertiary students ( n = 169 females), aged ≥16 years. Of the 99.1% of participants who regularly consumed caffeine, coffee (76.3%) tea (71.6%) and chocolate (81.7%) consumption were the most prevalent. Motivations for caffeinated-product consumption differed according to caffeine source. Tea was consumed for the warmth and taste, coffee was consumed to stay awake and for warmth, and chocolate, for the taste and as a treat. Marketing was not identified by participants as influencing their consumption of caffeinated products. Knowledge of motivations for caffeine consumption may assist in identifying strategies to reduce caffeine intake in those New Zealand tertiary students who regularly consume amounts of caffeine that exceed safe level.

Resistant starch (RS) is a part of insoluble dietary fiber, and it could be recognized as a functional food ingredient in some types of confectionery products that lack dietary fiber. Unlike dark and milk chocolate, white chocolate does not contain fat-free cocoa solids rich in dietary fiber. In the present study, 5%, 10%, and 15% of white chocolate were substituted with RS in order to improve the nutritional value of enriched white chocolate. The influence of RS on rheological, textural, and thermal properties of the chocolate fat phase was firstly investigated, and then further influence on physical properties, dietary fiber content, and sensory characteristics of enriched white chocolates were investigated. The obtained results showed that enriched chocolates had increased content of total dietary fiber and reduced total fats and protein content in accordance with the added amount of RS. At the same time, RS increased viscosity and reduced the hardness and volume mean diameter in enriched chocolates in accordance with the added amount. RS improved the nutritional composition of white chocolate by increasing the content of dietary fiber. At the same time, RS did not impair the color and sensory characteristics of enriched white chocolates.

In this study, dark chocolates (DCh) containing zinc lactate (ZnL) were enriched with extracts from elderberries (EFrE), elderflowers (EFlE), and chokeberries (ChFrE) to improve their functional properties. Both dried plant extracts and chocolates were analyzed for antioxidant capacity (AC) using four different analytical methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), cupric ion-reducing antioxidant capacity (CUPRAC), and ferric-reducing antioxidant power (FRAP), while total phenolic content (TPC) was determined by Folin-Ciocalteu (F-C) assay. An increase in antioxidant properties of fortified chocolates was found, and the bioaccessibility of their antioxidants was evaluated. The highest AC and TPC were found in ChFrE and chocolate with chokeberries (DCh + ChFrE) before and after simulated in vitro digestion. Bioaccessibility studies indicated that during the simulated digestion the AC of all chocolates reduced significantly, whereas insignificant differences in TPC results were observed between chemical and physiological extracts. Moreover, the influence of plant extracts on physicochemical parameters such as moisture content (MC), fat content (FC), and viscosity of chocolates was estimated. Furthermore, scanning electron microscopy with dispersive energy spectroscopy (SEM-EDS) was used to analyze surface properties and differences in the chemical composition of chocolates without and with additives.

Chocolate manufacture includes a complex tempering procedure to direct the crystallization of cocoa butter towards the formation of fat crystal networks with specific polymorphism, nano- and microstructure, melting behavior, surface gloss and mechanical properties. Here we investigate the effects of adding various minor non-triglyceride lipidic components to refined cocoa butter and chocolate on their physical properties. We discover that addition of saturated phosphatidylcholine and phosphatidylethanolamine to neutralized and bleached cocoa butter or molten and recrystallized commercial chocolate at 0.1% (w/w) levels, followed by rapid cooling to 20 °C in the absence of shear, accelerates crystallization, stabilizes the desirable Form V polymorph and induces the formation of chocolate with an optimal microstructure, surface gloss and mechanical strength. Final chocolate structure and properties are comparable to those of a commercial tempered chocolate. Minor lipidic component addition represents an effective way to engineer chocolate material properties at different length scales, thus simplifying the entire tempering process., (© 2021. The Author(s).)

Metagenomic studies about cocoa fermentation have mainly reported on the analysis of short reads for determination of operational taxonomic units. However, it is also important to determine metagenome-assembled genomes (MAGs), which are genomes deriving from the assembly of metagenomics. For this research, all the cocoa metagenomes from public databases were downloaded, resulting in five data sets: one from Ghana and four from Brazil. In addition, in silico approaches were used to describe putative phenotypes and the metabolic potential of MAGs. A total of 17 high-quality MAGs were recovered from these microbiomes, as follows: (i) for fungi, Yamadazyma tenuis ( n  = 1); (ii) lactic acid bacteria, Limosilactobacillus fermentum ( n  = 5), Liquorilactobacillus cacaonum ( n  = 1), Liquorilactobacillus nagelli ( n  = 1), Leuconostoc pseudomesenteroides ( n  = 1), and Lactiplantibacillus plantarum subsp. plantarum ( n  = 1); (iii) acetic acid bacteria, Acetobacter senegalensis ( n  = 2) and Kozakia baliensis ( n  = 1); and (iv) Bacillus subtilis ( n  = 1), Brevundimonas sp. ( n  = 2), and Pseudomonas sp. ( n  = 1). Medium-quality MAGs were also recovered from cocoa microbiomes, including some that, to our knowledge, were not previously detected in this environment (Liquorilactobacillus vini, Komagataeibacter saccharivorans, and Komagataeibacter maltaceti) and others previously described (Fructobacillus pseudoficulneus and Acetobacter pasteurianus). Taken together, the MAGs were useful for providing an additional description of the microbiome of cocoa fermentation, revealing previously overlooked microorganisms, with prediction of key phenotypes and biochemical pathways. IMPORTANCE The production of chocolate starts with the harvesting of cocoa fruits and the spontaneous fermentation of the seeds in a microbial succession that depends on yeasts, lactic acid bacteria, and acetic acid bacteria in order to eliminate bitter and astringent compounds present in the raw material, which will be further roasted and grinded to originate the cocoa powder that will enter the food processing industry. The microbiota of cocoa fermentation is not completely known, and yet it advanced from culture-based studies to the advent of next-generation DNA sequencing, with the generation of a myriad of data that need bioinformatic approaches to be properly analyzed. Although the majority of metagenomic studies have been based on short reads (operational taxonomic units), it is also important to analyze entire genomes to determine more precisely possible ecological roles of different species. Metagenome-assembled genomes (MAGs) are very useful for this purpose; here, MAGs from cocoa fermentation microbiomes are described, and the possible implications of their phenotypic and metabolic potentials are discussed.

Amino acids present ergogenic action, helping to increase, protect, and restore the muscular system of young athletes. Moreover, the encapsulation of five relevant amino acids in chocolate pellet form will appeal to them, facilitating their daily consumption. A reliable HPLC fluorimetric method was developed to detect and quantitatively determine L-Leucine, L-Isoleucine, L-Histidine, L-Valine, and β-Alanine in chocolate using aniline as an internal standard. Experimental design methodology was used to investigate and optimize the clean-up procedure of the samples. Therefore, three extraction techniques (solid-phase extraction (by two different SPE cartridges) and liquid-solid extraction (LSE)) were compared and evaluated. The LOQ values in chocolate varied from 24 to 118 ng/g (recovery 89.7-95.6%, %RSD < 2.5). Amino acids were pre-column derivatized with o -phthalaldehyde (OPA), while derivatization parameters were thoroughly investigated by experimental design methodology. The analysis was performed by HPLC-fluorescence (emission: λ = 455 nm, excitation: λ = 340 nm) method using a C 18 column and a mixture of phosphate buffer (pH = 2.8; 20 mM)-methanol as a mobile phase in gradient elution. The method was validated (r 2 > 0.999, %RSD < 2, LOD: 10 ng mL -1 for histidine and leucine, 2 ng mL -1 for alanine and valine, and 4 ng mL -1 for Isoleucine) according to the International Conference on Harmonization guidelines.

The content of polyphenols in chocolate depends on many factors related to the properties of raw material and manufacturing parameters. The trend toward developing chocolates made from unroasted cocoa beans encourages research in this area. In addition, modern customers attach great importance to how the food they consume benefits their bodies. One such benefit that consumers value is the preservation of natural antioxidant compounds in food products (e.g., polyphenols). Therefore, in our study we attempted to determine the relationship between variable parameters at the conching stage (i.e., temperature and time of) and the content of dominant polyphenols (i.e.,catechins, epicatechins, and procyanidin B2) in chocolate milk mass (CMM) obtained from unroasted cocoa beans. Increasing the conching temperature from 50 to 60 °C decreased the content of three basic flavan-3-ols. The highest number of these compounds was determined when the process was carried out at 50 °C. However, the time that caused the least degradation of these compounds differed. For catechin, it was 2 h; for epicatechin it was 1 h; and for procyanidin it was 3 h. The influence of both the temperature and conching time on the rheological properties of chocolate milk mass was demonstrated. At 50 °C, the viscosity and the yield stress of the conched mass showed its highest value.

Mental stress has been shown to induce cardiovascular events, likely due to its negative impact on vascular function. Flavanols, plant-derived polyphenolic compounds, improve endothelial function and blood pressure (BP) in humans, however their effects during stress are not known. This study examined the effects of acute intake of cocoa flavanols on stress-induced changes on vascular function. In a randomised, controlled, double-blind, cross-over intervention study, 30 healthy men ingested a cocoa flavanol beverage (high-flavanol: 150 mg vs. low-flavanol < 4 mg (-)-epicatechin) 1.5 h before an 8-min mental stress task). Forearm blood flow (FBF), BP, and cardiovascular activity were assessed pre- and post-intervention, both at rest and during stress. Endothelial function (brachial flow-mediated dilatation, FMD) and brachial BP were measured before the intervention and 30 and 90 min post-stress. FMD was impaired 30 min post-stress, yet high-flavanol cocoa attenuated this decline and remained significantly higher compared to low-flavanol cocoa at 90 min post-stress. High-flavanol cocoa increased FBF at rest and during stress. Stress-induced cardiovascular and BP responses were similar in both conditions. Flavanols are effective at counteracting mental stress-induced endothelial dysfunction and improving peripheral blood flow during stress. These findings suggest the use of flavanol-rich dietary strategies to protect vascular health during stress.

Cornelian cherry (Cornus mas L.) fruits are a valuable source of bioactive compounds that are responsible for the perception of bitter taste of chocolate products. The aim of the study was to validate the inhibitory effect of Cornus mas on the TAS2R3 and TAS2R13 bitter taste receptors and to assess the effect of masking the bitter taste of dark chocolate with the help of the sensory panel. Dark chocolate was prepared with an addition of 5% of freeze-dried cornelian cherry fruits and 108 CFU/g of Bacillus coagulans probiotic strains. Effect on the TAS2R receptors was evaluated in specially transfected HEK293T cells, and the inhibition ratio was measured using the calcium release test. Moreover, the total polyphenol content, antioxidant activity and simulated intestinal in vitro digestion were determined for the samples. The tested chocolate products were rich in chlorogenic, caffeic and sinapic acids. The addition of cornelian cherry positively affected the antioxidant activity. The phytochemicals of Cornus mas decreased the TAS2R13 activity by 132% after a 2-minute interaction and, % at the same time, inhibited the TAS2R3 activity by 11.5. Meanwhile, chocolate with the addition of fruit was less bitter according to the sensory panel., Competing Interests: The authors have declared that no competing interests exist.

This study aimed to identify the volatile compounds in the fermented and dried cocoa beans conducted with three distinct inoculants of yeast species due to their high fermentative capacity: Saccharomyces cerevisiae , Pichia kudriavzevii , the mixture in equal proportions 1:1 of both species, and a control fermentation (with no inoculum application). Three starter cultures of yeasts, previously isolated and identified in cocoa fermentation in the municipality of Tomé-Açu, Pará state, Brazil. The seeds with pulp were removed manually and placed in wooden boxes for the fermentation process that lasted from 6 to 7 days. On the last day of fermentation, the almonds were packaged properly and placed to dry (36 °C), followed by preparation for the analysis of volatile compounds by GC-MS technique. In addition to the control fermentation, a high capacity for the formation of desirable compounds in chocolate by the inoculants with P. kudriavzevii was observed, which was confirmed through multivariate analyses, classifying these almonds with the highest content of aldehydes, esters, ketones and alcohols and low concentration of off-flavours. We conclude that the addition of mixed culture starter can be an excellent alternative for cocoa producers, suggesting obtaining cocoa beans with desirable characteristics for chocolate production, as well as creating a product identity for the producing region.

Obesity remains a major public health problem due to its increasing prevalence. Natural products have become common as adjunct therapeutic agents for treating obesity and preventing metabolic diseases. Cocoa and its products are commonly consumed worldwide. Dark chocolate, a rich source of polyphenols, has received attention lately for its beneficial role in the management of obesity; however, conflicting results are still being reported. This scoping review aims to provide a comprehensive understanding of the existing literature on the relationship and effects of cocoa and dark chocolate intake among obese adults. We searched multiple databases for research investigating the consumption of cocoa and/or dark chocolate in managing obesity among adults. This review includes epidemiological and human studies that were published in English over the last 10 years. Our review of the current literature indicates that epidemiological and human trials with obese adults have shown inconsistent results, which may be due to the different populations of subjects, and different types of cocoa products and doses used for intervention. Studies among obese adults are mainly focusing on obese individuals with comorbidities, as such more studies are needed to elucidate the role of cocoa polyphenols in weight control and preventing the risk of chronic diseases among obese individuals without comorbidities as well as healthy individuals. Careful adjustment of confounding factors would be required. The effects of cocoa and dark chocolate intake on obese adults were discussed, and further research is warranted to identify the gaps.

Increasing evidence support a beneficial role of cocoa and cocoa products on human cognition, particularly in aging populations and patients at risk. However, thorough reviews on the efficacy of cocoa on brain processes in young adults do not exist precisely due to the limited number of studies in the matter. Thus, the aim of this study was to summarize the findings on the acute and chronic effects of cocoa administration on cognitive functions and brain health in young adults. Web of Science and PubMed databases were used to search for relevant trials. Human randomized controlled studies were selected according to PRISMA guidelines. Eleven intervention studies that involved a total of 366 participants investigating the role of cocoa on cognitive performance in children and young adults (average age ≤ 25 years old) were finally selected. Findings from individual studies confirm that acute and chronic cocoa intake have a positive effect on several cognitive outcomes. After acute consumption, these beneficial effects seem to be accompanied with an increase in cerebral blood flow or cerebral blood oxygenation. After chronic intake of cocoa flavanols in young adults, a better cognitive performance was found together with increased levels of neurotrophins. This systematic review further supports the beneficial effect of cocoa flavanols on cognitive function and neuroplasticity and indicates that such benefits are possible in early adulthood.

Chocolate is considered as both caloric and functional food. Its nutritional properties may be improved by addition of fiber; however, this may reduce polyphenols content. The aim of this research was to determine the influence of cocoa shell addition (as a source of fiber) and its combination with different ingredients (cocoa butter equivalents (CBE), emulsifiers, dairy ingredients) on polyphenols of dark and milk chocolates. Total polyphenol (TPC) and total flavonoid (TFC) contents were determined spectrophotometrically, identification and quantification of individual compounds by high pressure liquid chromatography and antioxidant capacity by ferric reducing antioxidant power (FRAP) assay. Results showed that even though addition of cocoa shell to chocolate results in reduced contents of TPC, TFC, and individual compounds, it is not significant compared to ones reported by other authors for commercial chocolates. Other ingredients influence determined values for all investigated parameters; however, additional research is needed to reveal exact mechanisms and implications.

Diabetic retinopathy (DR), one of the most common complications of diabetes, is the leading cause of legal blindness among adults of working age in developed countries. After 20 years of diabetes, almost all patients suffering from type I diabetes mellitus and about 60% of type II diabetics have DR. Several studies have tried to identify drugs and therapies to treat DR though little attention has been given to flavonoids, one type of polyphenols, which can be found in high levels mainly in fruits and vegetables, but also in other foods such as grains, cocoa, green tea or even in red wine. Flavonoids have anti-inflammatory, antioxidant and antiviral effects. Since it is known that diabetes induces oxidative stress and inflammation in the retina leading to neuronal death in the early stages of the disease, the use of these compounds can prove to be beneficial in the prevention or treatment of DR. In this review, we summarize the molecular and cellular effects of flavonoids in the diabetic retina.

The biological activity of chocolates gains more and more attention of consumers. Its antioxidant properties depend, among other factors, mainly on the origin of cocoa and the characteristics that this origin gives to the final product. Therefore, the aim of the study was to measure and compare the total content of polyphenols, antioxidant activity, and key odorants of commercial chocolates made from blend cocoa with single-origin ones. The highest content of polyphenols was found in 90% blend cocoa chocolate and single-origin samples, while the lowest content was exhibited by 100% chocolate from blend cocoa mass. The highest antioxidant activity measured by 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and ferric reducing antioxidant power (FRAP) assays was observed in the sample of chocolate with 90% cocoa solids from blend mass, followed by single-origin chocolates. A high positive correlation between ABTS assay and the total polyphenol and phenolic acids' content, as well as among the total content of polyphenols, flavonoids, and phenolic acids was found. Mineral composition analysis showed that dark chocolate is a valuable source of some elements, especially Mg, Fe, and Zn. Potentially toxic elements were not detected or below permitted limits. Moreover, it was noticed that the main volatile compound in all tested samples was acetic acid, but pyrazines were considered the most important group of chocolate odorants.

Since 1 January 2019 a maximum content of 0.6 mg kg -1 cadmium (Cd) in cocoa powder sold to the final consumer or as an ingredient in sweetened cocoa powder sold to the final consumer (drinking chocolate) is set by the Commission Regulation (EU) No. 488/2014. Monitoring compliance with the specified limit value requires analytical measuring methods and reference materials for quality control. However, suitable certified reference materials intended for quality assurance and quality control purposes are still lacking. Therefore, three cocoa reference materials (ERM®-BD513, ERM®-514 and ERM®-515) were developed according to the requirements of ISO 17034 and the recommendations of ISO Guide 35. The whole process of reference material development, including material preparation, assessment of homogeneity and stability, characterisation and value assignment is presented. The assignment of the certified mass fractions was based upon an interlaboratory comparison study involving 19 expert laboratories for Cd and 12 laboratories for acrylamide. The certified mass fractions and expanded uncertainties (k = 2) of the reference materials were (0.181 ± 0.009) mg kg -1 Cd (ERM®-BD513), (0.541 ± 0.024) mg kg -1 Cd (ERM®-BD514) and (0.690 ± 0.029) mg kg -1 Cd (ERM®-BD515). Acrylamide contents are given for information.

Dark chocolate samples were previously classified into four sensory categories. The classification was modelled based on volatile compounds analyzed by direct introduction mass spectrometry of the chocolates' headspace. The purpose of the study was to identify the most discriminant odor-active compounds that should characterize the four sensory categories. To address the problem, a gas chromatography-olfactometry (GC-O) study was conducted by 12 assessors using a comparative detection frequency analysis (cDFA) approach on 12 exemplary samples. A nasal impact frequency (NIF) difference threshold combined with a statistical approach (Khi² test on k proportions) revealed 38 discriminative key odorants able to differentiate the samples and to characterize the sensory categories. A heatmap emphasized the 19 most discriminant key odorants, among which heterocyclic molecules (furanones, pyranones, lactones, one pyrrole, and one pyrazine) played a prominent role with secondary alcohols, acids, and esters. The initial sensory classes were retrieved using the discriminant key volatiles in a correspondence analysis (CA) and a hierarchical cluster analysis (HCA). Among the 38 discriminant key odorants, although previously identified in cocoa products, 21 were formally described for the first time as key aroma compounds of dark chocolate. Moreover, 13 key odorants were described for the first time in a cocoa product.

We performed a comprehensive study encompassing chemical characterization and sensory evaluation of two types of dark chocolate, i.e., artisanal (Choco-A) and industrial (Choco-I), as well as an evaluation of onset of gastrointestinal symptoms and gastrointestinal motility in healthy subjects fed with dark chocolate. Proteomic, lipid and metabolite analysis were performed by LC-MS/MS analysis and the total phenol content and antioxidant activity were estimated in both types of chocolate. Fifty healthy volunteers joined the study of the sensory characteristics of both types of chocolate; another 16 subjects underwent the study of gallbladder and gastric emptying by functional ultrasonography and orocecal transit time by lactulose H 2 -breath test after ingestion of dark chocolate. Identification of polyphenols, amino acids and fatty acids was carried out in both types of chocolate analysed, and results confirmed their richness in polyphenols, amino acid derivatives and fatty acids (FAs) either saturated (stearic, myristic, palmitic, ecosanoic) or unsaturated (oleic and linolenic). For agreeability, Choco-A scored higher than Choco-I for smell, texture, and taste and they did not show significant differences in the gastrointestinal motility. In conclusion as for gastrointestinal motility studies, we report that the ingestion of a small amount of chocolate induced a mild gallbladder, gastric contraction and a fast transit time compared to the test meal in healthy subjects.

There is good evidence that cocoa flavonoids can acutely improve cognitive function in humans, possibly via mechanisms such as increased cerebral blood flow. To date, much of the evidence is based on measures of executive function with extracts and cocoa-based interventions with a high flavonoid content. The aim of the present study was to explore whether benefits to episodic verbal memory and mood are observed two hours post consumption of a commercially available dark chocolate (DC) bar relative to a 35 g white chocolate bar (WC). Ninety-eight healthy young adults ( n = 57 females) aged 18-24 years consumed either a 35 g DC bar or a calorie-matched low flavonoid WC bar. Verbal episodic memory and mood were assessed pre consumption and 2 h post consumption. An ANOVA analysis showed that the DC was associated with better verbal memory performance for several outcome measures of the Rey Auditory Verbal Learning Test relative to the WC, however, there were no effects on mood. These findings lend support to the notion that everyday available portions of dark chocolate can confer benefits to the brain in healthy consumers.

Background: While there has been an increasing interest in the health properties of chocolate, limited research has looked into the changes of antioxidants occurring in the time span from production to the best before date, which was a period of 18 months in this study., Methods: Humidity, ash, pH, acidity, fiber, carotenoids, retinols, tocopherols, sugars, proteins, theobromine, caffeine, polyphenols, fats, the peroxide value, organic acids, and volatile compounds, along with the sensory profile, were monitored at 18-week intervals for 18 months under conditions simulating a factory warehouse or a point of sale., Results: At the end of the storage period, more polyphenols were lost (64% and 87%) than vitamin E (5% and 14%) in cocoa mass and cocoa powder, respectively. Conversely, a greater loss in vitamin E (34% and 86%) than in polyphenols (19% and 47%) was shown in the hazelnut paste and gianduja chocolate, respectively. The sensory profiling of cocoa mass, cocoa powder, and hazelnut paste revealed increases in grittiness and astringency, as well as decreases in melting, bitterness, and toasted aroma. Moreover, in the hazelnut paste and gianduja chocolate, oiliness increased with a toasted and caramel aroma. Furthermore, dark chocolate was more gritty, acidic, and bitter. Milk chocolate lost its nutty aroma but maintained its sweetness and creaminess., Conclusions: These results should contribute an important reference for companies and consumers, in order to preserve the antioxidants and understand how antioxidants and sensory properties change from the date of production until the best before date.

Chocolate production is a complex process during which numerous chemical reactions occur. The most important processes, involving most of the reactions important for development of the proper chocolate flavor, are fermentation, drying and roasting of cocoa bean, and chocolate conching. During fermentation, formation of important precursors occurs, which are essential for further chemical reactions in the following processes of chocolate production. Roasting is one of the most important processes due to the occurrence of Maillard's reactions, during which aroma compounds are formed. In this paper, we have reviewed the most important chemical reactions that occur with proteins, carbohydrates, lipids, and polyphenols. Additionally, we present other components that may be naturally present or form during the production process, such as methylxanthines, aldehydes, esters, ketones, pyrazines, acids, and alcohols.

Acrylamide is formed during baking in some frequently consumed food products. It is proven to be carcinogenic in rodents and a probable human carcinogen. Thus, the food industry is working to find solutions to minimize its formation during processing. To better understand the sources of its formation, the present study is aimed at investigating how acrylamide concentration may be influenced by bakery-making parameters within a parallel strategy of mycotoxin mitigation (focusing specifically on deoxynivalenol-DON) related to wholegrain and cocoa biscuit production. Among Fusarium toxins, DON is considered the most important contaminant in wheat and related bakery products, such as biscuits, due to its widespread occurrence. Exploiting the power of a Design of Experiments (DoE), several conditions were varied as mycotoxin contamination levels of the raw materials, recipe formulation, pH value of dough, and baking time/temperature; each selected treatment was varied within a defined range according to the technological requirements to obtain an appreciable product for consumers. Experiments were performed in a pilot-plant scale in order to simulate an industrial production and samples were extracted and analysed by HPLC-MS/MS system. Applying a baking temperature of 200 °C at the highest sugar dose, acrylamide increased its concentration, and in particular, levels ranged from 306 ± 16 µg/Kg d.m. and 400 ± 27 µg/Kg d.m. in biscuits made without and with the addition of cocoa, respectively. Conversely, using a baking temperature of 180 °C in the same conditions (pH, baking time, and sugar concentrations), acrylamide values remained below 125 ± 14 µg/Kg d.m. and 156 ± 15 µg/Kg d.m. in the two final products. The developed predictive model suggested how some parameters can concretely contribute to limit acrylamide formation in the final product, highlighting a significant role of pH value (correlated also to sodium bicarbonate raising agent), followed by baking time/temperature parameters. In particular, the increasing range of baking conditions influenced in a limited way the final acrylamide content within the parallel effective range of DON reduction. The study represents a concrete example of how the control and optimization of selected operative parameters may lead to multiple mitigation of specific natural/process contaminants in the final food products, though still remaining in the sensorial satisfactory range., Competing Interests: The authors declare no conflict of interest.

The study aimed to compare the sugar (1992, 2017) and energy (2017) content of chocolate confectionery available in the UK between 1992 and 2017 using cross-sectional surveys. All major UK retailers operating at the time were included. Sugar content in 1992 was obtained from a booklet and sugar and energy content from 2017 were collected from product packaging in-store. In 1992, the average sugar content of chocolate confectionery was 46.6 ± 10.3 g/100 g and in 2017 it was 47.3 ± 12.1 g/100 g. Sugar content ranged from 0.5 to 75.2 g/100g, with large variations between different categories of chocolate and within the same category of chocolate. There were 23 products found in both 1992 and 2017. The average sugar content per 100 g for these products was 44.6 ± 9.4 g in 1992 and 54.7 ± 6.3 g in 2017, representing a 23% increase in sugar content ( p < 0.001). The results show that the sugar content of chocolate confectionery has increased since 1992, which is concerning. However, they also suggest sugar levels can be reduced because (a) lower sugar versions of the same products existed in 1992 and (b) there is a large variation in sugar and energy content between different categories of chocolate and within the same category in 2017.

In recent years, the consumption of chocolate and, in particular, dark chocolate has been "rehabilitated" due to its high content of cocoa antioxidant polyphenols. Although it is recognized that regular exercise improves energy metabolism and muscle performance, excessive or unaccustomed exercise may induce cell damage and impair muscle function by triggering oxidative stress and tissue inflammation. The aim of this review was to revise the available data from literature on the effects of cocoa polyphenols on exercise-associated tissue damage and impairment of exercise performance. To this aim, PubMed and Web of Science databases were searched with the following keywords: "intervention studies", "cocoa polyphenols", "exercise training", "inflammation", "oxidative stress", and "exercise performance". We selected thirteen randomized clinical trials on cocoa ingestion that involved a total of 200 well-trained athletes. The retrieved data indicate that acute, sub-chronic, and chronic cocoa polyphenol intake may reduce exercise-induced oxidative stress but not inflammation, while mixed results are observed in terms of exercise performance and recovery. The interpretation of available results on the anti-oxidative and anti-inflammatory activities of cocoa polyphenols remains questionable, likely due to the variety of physiological networks involved. Further experimental studies are mandatory to clarify the role of cocoa polyphenol supplementation in exercise-mediated inflammation.

Cocoa has beneficial health effects partly due to its high flavanol content. This study was aimed at assessing the absorption and metabolism of polyphenols in two soluble cocoa products: a conventional (CC) and a flavanol-rich product (CC-PP). A crossover, randomized, blind study was performed in 13 healthy men and women. On two different days, after an overnight fast, volunteers consumed one serving of CC (15 g) or CC-PP (25 g) in 200 mL of semi-skimmed milk containing 19.80 mg and 68.25 mg of flavanols, respectively. Blood and urine samples were taken, before and after CC and CC-PP consumption, and analyzed by high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QToF-MS). Up to 10 and 30 metabolites were identified in plasma and urine, respectively. Phase II derivatives of epicatechin were identified with kinetics compatible with small intestine absorption, although the most abundant groups of metabolites were phase II derivatives of phenyl-γ-valerolactone and phenylvaleric acid, formed at colonic level. 5-(4'-Hydroxyphenyl)-γ-valerolactone-sulfate could be a sensitive biomarker of cocoa flavanol intake. CC and CC-PP flavanols showed a dose-dependent absorption with a recovery of 35%. In conclusion, cocoa flavanols are moderately bioavailable and extensively metabolized, mainly by the colonic microbiota.

Cocoa is continuously drawing attention due to growing scientific evidence suggesting its effects on health. Flavanols and methylxanthines are some of the most important bioactive compounds present in cocoa. Other important bioactives, such as phenolic acids and lactones, are derived from microbial metabolism. The identification of the metabolites produced after cocoa intake is a first step to understand the overall effect on human health. In general, after cocoa intake, methylxanthines show high absorption and elimination efficiencies. Catechins are transformed mainly into sulfate and glucuronide conjugates. Metabolism of procyanidins is highly influenced by the polymerization degree, which hinders their absorption. The polymerization degree over three units leads to biotransformation by the colonic microbiota, resulting in valerolactones and phenolic acids, with higher excretion times. Long term intervention studies, as well as untargeted metabolomic approaches, are scarce. Contradictory results have been reported concerning matrix effects and health impact, and there are still scientific gaps that have to be addresed to understand the influence of cocoa intake on health. This review addresses different cocoa clinical studies, summarizes the different methodologies employed as well as the metabolites that have been identified in plasma and urine after cocoa intake.

Texture affects liking or rejection of many foods for clinically relevant populations and the general public. Phenotypic differences in chemosensation are well documented and influence food choices, but oral touch perception is less understood. Here, we used chocolate as a model food to explore texture perception, specifically grittiness perception. In Experiment 1, the Just Noticeable Difference (JND) for particle size in melted chocolate was ~5 μm in a particle size range commonly found in commercial chocolates; as expected, the JND increased with particle size, with a Weber Fraction of ~0.17. In Experiment 2, individual differences in touch perception were explored: detection and discrimination thresholds for oral point pressure were determined with Von Frey Hairs. Discrimination thresholds varied across individuals, allowing us to separate participants into high and low sensitivity groups. Across all participants, two solid commercial chocolates (with particle sizes of 19 and 26 μm; i.e., just above the JND) were successfully discriminated in a forced-choice task. However, this was driven entirely by individuals with better oral acuity: 17 of 20 of more acute individuals correctly identified the grittier chocolate versus 12 of 24 less acute individuals. This suggests phenotypic differences in oral somatosensation can influence texture perception of foods.

Chocolate elicits unique brain activity compared to other foods, activating similar brain regions and neurobiological substrates with potentially similar psychoactive effects as substances of abuse. We sought to determine the relationship between chocolate with varying combinations of its main constituents (sugar, cocoa, and fat) and its psychoactive effects. Participants consumed 5 g of a commercially available chocolate with increasing amounts of sugar (90% cocoa, 85% cocoa, 70% cocoa, and milk chocolates). After each chocolate sample, participants completed the Psychoactive Effects Questionnaire (PEQ). The PEQ consists of questions taken from the Morphine-Benzedrine Group (MBG), Morphine (M,) and Excitement (E) subscales of the Addiction Research Center Inventory. After all testing procedures, participants completed the Binge Eating Scale (BES) while left alone and allowed to eat as much as they wanted of each of the different chocolates. We found a measurable psychoactive dose⁻effect relationship with each incremental increase in the chocolate's sugar content. The total number of positive responses and the number of positive responses on the E subscale began increasing after tasting the 90% cocoa chocolate, whereas the number of positive responses on the MBG and M subscales began increasing after tasting the 85% cocoa chocolate sample. We did not find a correlation between BES scores and the total amount of chocolate consumed or self-reported scores on the PEQ. These results suggest that each incremental increase in chocolate's sugar content enhances its psychoactive effects. These results extend our understanding of chocolate's appeal and unique ability to prompt an addictive-like eating response.

Cocoa agroforestry systems have the potential to conserve biodiversity and provide environmental or ecological benefits at various nested scales ranging from the plot to ecoregion. While integrating organic practices into cocoa agroforestry may further enhance these potentials, empirical and robust data to support this claim is lacking, and mechanisms for biodiversity conservation and the provision of environmental and ecological benefits are poorly understood. A field study was conducted in the Eastern Region of Ghana to evaluate the potential of organic cocoa agroforests to conserve native floristic diversity in comparison with conventional cocoa agroforests. Shade tree species richness, Shannon, Simpson's reciprocal and Margalef diversity indices were estimated from 84 organic and conventional cocoa agroforestry plots. Species importance value index, a measure of how dominant a species is in a given ecosystem, and conservation status were used to evaluate the conservation potential of shade trees on studied cocoa farms. Organic farms recorded higher mean shade tree species richness (5.10 ± 0.38) compared to conventional farms (3.48 ± 0.39). Similarly, mean Shannon diversity index, Simpson's reciprocal diversity index and Margalef diversity index were significantly higher on organic farms compared to conventional farms. According to the importance value index, fruit and native shade tree species were the most important on both organic and conventional farms for all the cocoa age groups but more so on organic farms. Organic farms maintained 14 native tree species facing a conservation issue compared to 10 on conventional cocoa farms. The results suggest that diversified organic cocoa farms can serve as reservoirs of native tree species, including those currently facing conservation concerns thereby providing support and contributing to the conservation of tree species in the landscape., Competing Interests: The authors have declared that no competing interests exist.

The objective of this work is to determine the extent to which changes in the skim milk powder (SMP) manufacturing process alter the volatile profile of SMP, and whether these changes are carried through to a final product when the SMP is used as an ingredient and subjected to further processing. The manufacture of SMP is a multistage process involving a preliminary concentration step, heat treatment, and a drying stage. However, the methods and conditions used by the industry are not standardized, and the inherent variability in the production of SMP has consequences for the end-users, such as the confectionery industry, where the SMP is used as an ingredient during the production of milk chocolate, white chocolate, and caramel. This study investigates the effect of each stage of the manufacturing process on the concentration of reducing sugars and available amino groups (as precursors of the Maillard reaction) as well as on the volatile products of the Maillard reaction and lipid degradation. Eight types of SMP were produced using combinations of different processing conditions: concentration (by evaporation or reverse osmosis), heat treatment (low heat or high heat), and drying (spray-drying or freeze-drying). Maillard precursors were quantified after each processing stage and volatile compounds were extracted using solid-phase microextraction, and analyzed by gas chromatography-mass spectrometry. The resulting SMP were incorporated into a model white chocolate system, produced under varying conching conditions. We demonstrate not only that changes in the SMP manufacturing conditions affect the volatile profile of SMP, but also that these differences can be carried through to a final product when the SMP is used to prepare a model white chocolate. Understanding these differences is important to the industry for controlling the flavor of the end product., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The development of new methods for identifying a broad spectrum of analytes, as well as highly selective tools to provide the most accurate information regarding the processes and relationships in the world, has been an area of interest for researchers for many years. The information obtained with these tools provides valuable data to complement existing knowledge but, above all, to identify and determine previously unknown hazards. Recently, attention has been paid to the migration of xenobiotics from the surfaces of various everyday objects and the resulting impacts on human health. Since children are among those most vulnerable to health consequences, one of the main subjects of interest is the migration of low-molecular-weight compounds from toys and products intended for children. This migration has become a stimulus for research aimed at determining the degree of release of compounds from popular commercially available chocolate/toy sets. One of main objectives of this research was to determine the impact of time on the ecotoxicity (with Vibrio fischeri bioluminescent bacteria) of extracts of products intended for children and to assess the correlation with total volatile organic compound emissions using basic chemometric methods. The studies on endocrine potential (with XenoScreen YES/YAS) of the extracts and showed that compounds released from the studied objects (including packaging foils, plastic capsules storing toys, most of toys studied and all chocolate samples) exhibit mostly androgenic antagonistic behavior while using artificial saliva as extraction medium increased the impact observed. The impact of time in most cases was positive one and increased with prolonging extraction time. The small-scale stationary environmental test chambers - μ-CTE™ 250 system was employed to perform the studies aimed at determining the profile of total volatile organic compounds (TVOCs) emissions. Due to this it was possible to state that objects from which the greatest amounts of contaminants are released are plastic containers (with emission rate falling down from 3273 to 2280 ng/g of material at 6 h of conditioning in elevated temperature)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Jackfruit seeds are an under-utilized waste product in many tropical countries. In this work, we demonstrate the potential of roasted jackfruit seeds to substitute for cocoa powder in cappuccino formulations. Two different flours were produced from a hard variety jackfruit by drying or fermenting the seeds prior to roasting. Next, formulations were prepared with 50%, 75%, and 100% substitution of cocoa powder with jackfruit seed flours, totalizing seven with control formulation. The acceptance of cappuccinos by consumers (n = 126) and quantitative descriptive analysis (QDA®) were used to describe the preparations. Physicochemical properties were also evaluated. When 50% and 75% cocoa powder was replaced with dry jackfruit seed flour, there was no change in sensory acceptability or technological properties; however, it is possible to identify advantages tousing dry jackfruit seed flour, including moisture reduction and high wettability, solubility and sensory acceptation of the chocolate aroma. The principal component analysis of QDA explained90% variances; cluster analysis enabled the definition of four groups for six cappuccino preparations. In fact, dry jackfruit seed flour is an innovative cocoa powder substitute; it could be used in food preparations, consequently utilizing this tropical fruit waste by incorporating it as an ingredient in a common product of the human diet., Competing Interests: There is no conflict of interest with any of the authors, financial, personal or other relationships with other people or organizations of the paper being submitted.

Cocoa and dark chocolate have a wide variety of powerful antioxidants and other nutrients that can positively affect human health. Probiotic dark chocolate has the potential to be a new product in the growing number of functional foods. In this study, encapsulated potential probiotic Lactobacillus plantarum 564 and commercial probiotic Lactobacillus plantarum 299v were added in the production of dark chocolate. The results show very good survival of probiotic bacteria after production and during storage, reaching 10⁸cfu/g in the first 60 days and over 10⁶cfu/g up to 180 days. No statistically significant difference ( p > 0.05) in chemical composition and no major differences in the volatile profiles between control and experimental chocolate samples were observed, indicating no impact of probiotic bacteria on compositional and sensory characteristics of dark chocolate. The sensory evaluation of control and both probiotic dark chocolate samples showed excellent sensory quality after 60 and 180 days of storage, demonstrating that probiotics did not affect aroma, texture and appearance of chocolate. Due to a high viability of bacterial cells and acceptable sensory properties, it can be concluded that encapsulated probiotics Lb. plantarum 564 and Lb. plantarum 299v could be successfully used in the production of probiotic dark chocolate.

Scientific evidence of the association between free sugar consumption and several adverse health effects has led many public health institutions to take measures to limit the intake of added or free sugar. Monitoring the efficiency of such policies and the amount of free sugar consumed requires precise knowledge of free sugar content in different food products. To meet this need, our cross-sectional study aimed at assessing free sugar content for 10,674 pre-packaged food items available from major Slovenian food stores during data collection in 2015. Together, 52.6% of all analyzed products contained free sugar, which accounted for an average of 57.5% of the total sugar content. Food categories with the highest median free sugar content were: honey and syrups (78.0 g/100 g), jellies (62.9 g/100 g), chocolate and sweets (44.6 g/100 g), jam and spreads (35.9 g/100 g), and cereal bars (23.8 g/100 g). Using year-round sales data provided by the retailers, the data showed that chocolate, sweets, and soft drinks alone accounted for more than 50% of all free sugar sold on the Slovenian market. The results of this study can be used to prepare more targeted interventions and efficient dietary recommendations., Competing Interests: The authors declare no conflicts of interest.

Objective: To determine if ingestion of lycosome-formulated dark chocolate (DC) containing astaxanthin (ASTX) improves bioavailability of ASTX and affects markers of hypoxia and oxidative stress in aging individuals., Design: Randomized, blinded, four-arm, prospective study., Settings: Lycotec Ltd, Cambridge, United Kingdom and Institute of Cardiology, Saratov, Russian Federation., Participants: 32 healthy individuals aged 60-70 years with confirmed signs of oxidative stress (increased serum levels of oxidized LDL and malonic dialdehyde) randomized into four study groups (8 volunteers each)., Intervention: Volunteers of first group were given orally 10 gr of dark chocolate (DC). Individuals from the second group received 7 mg of astaxanthin (ASTX). Third group of volunteers was supplemented with 10 gr of DC and 7 mg of ASTX ingested simultaneously as two separate formulations. Last group of the individuals was given 10 gr of a lycosomal formulation of DC containing 7 mg of co-crystalized ASTX (L-DC-ASTX), a newly developed highly bioavailable nutraceutical composition of DC containing 2 groups of antioxidants (cocoa flavanols and ASTX). All formulations were given orally, once daily for a month., Measurements: Serum ASTX was measured by high-performance liquid chromatography. Nitric oxide, malonic dialdehyde and oxidized LDL were quantified spectrophotometrically. Oxygenation parameters were evaluated by near-infrared spectroscopy., Results: One month ingestion of singular formulation of ASTX lead to a 20 fold buildup in serum ASTX level whereas the 4 week ingestion of L-DC-ASTX formulation was accompanied by more prominent accumulation of ASTX in serum (a 40 fold increase over the basal values) at the same daily dose of ASTX. Both antioxidants taken separately decreased serum levels of oxidized LDL and malonic dialdehyde. However effect of L-DC-ASTX formulation was more prominent. ASTX ingested alone caused a borderline increase (p=0.054) in serum nitric oxide (NO) levels, whereas DC ingestion lead to small but statistically significant increase in serum NO concentration. Higher values of NO level were seen after co-ingestion of DC and ASTX, especially in case of L-DC-ASTX formulation suggesting additive/synergistic effects of DC and ASTX on nitric oxide production. These changes were in agreement with the increase in plasma oxygen transport and tissue oxygen saturation seen in the volunteers supplemented with L-DC-ASTX formulation., Conclusion: The nutraceutical formulation of DC and ASTX with an enhanced bioavailability of ASTX can be efficiently used for the correction of oxidative status in aging individuals., Competing Interests: All authors declare no conflict of interest involved

Background: Cocoa contains polyphenols that are thought to be beneficial for vascular health. Objective: We assessed the impact of chocolate containing distinct concentrations of cocoa on cerebrovascular function and cognition. Methods: Using a counterbalanced within-subject design, we compared the acute impact of consumption of energy-matched chocolate containing 80%, 35%, and 0% single-origin cacao on vascular endothelial function, cognition, and cerebrovascular function in 12 healthy postmenopausal women (mean ± SD age: 57.3 ± 5.3 y). Participants attended a familiarization session, followed by 3 experimental trials, each separated by 1 wk. Outcome measures included cerebral blood flow velocity (CBF v ) responses, recorded before and during completion of a computerized cognitive assessment battery (CogState); brachial artery flow-mediated dilation (FMD); and hemodynamic responses (heart rate and blood pressure). Results: When CBF v data before and after chocolate intake were compared between conditions through the use of 2-factor ANOVA, an interaction effect ( P = 0.003) and main effects for chocolate ( P = 0.043) and time ( P = 0.001) were evident. Post hoc analysis revealed that both milk chocolate (MC; 35% cocoa; P = 0.02) and dark chocolate (DC; 80% cocoa; P = 0.003) induced significantly lower cerebral blood flow responses during the cognitive tasks, after normalizing for changes in arterial pressure. DC consumption also increased brachial FMD compared with the baseline value before chocolate consumption ( P = 0.002), whereas MC and white chocolate (0% cocoa) caused no change ( P- interaction between conditions = 0.034). Conclusions: Consumption of chocolate containing high concentrations of cocoa enhanced vascular endothelial function, which was reflected by improvements in FMD. Cognitive function outcomes did not differ between conditions; however, cerebral blood flow responses during these cognitive tasks were lower in those consuming MC and DC. These findings suggest that chocolate containing high concentrations of cocoa may modify the relation between cerebral metabolism and blood flow responses in postmenopausal women. This trial was registered at www.ANZCTR.orgau as ACTRN12616000990426., Competing Interests: Author disclosures: CEM, HHC, KJG, KJS, KEP, LHN, and DJG, no conflicts of interest., (© 2017 American Society for Nutrition.)