Quality differentiation of cocoa beans: implications for geographical indications.

Geographical indications may stimulate collective actions of governance for quality control, trade and marketing as well as innovation based on the use of local resources and regional biodiversity. Cocoa production, however, dominated by small family agriculture in tropical regions, has rarely made use of such strategies. This review is aimed at understanding major research interests and emerging technologies helpful for the origin differentiation of cocoa quality. Results from literature search and cited references of publications on cocoa research were imported into VOSviewer for data analysis, which aided in visualizing major research hotpots. Co-occurrence analysis yielded major research clusters which guided the discussion of this review. Observed was a consensus recognizing cocoa quality resulting from the interaction of genotype, fermentation variables and geographical origin. A classic view of cocoa genetics based on the dichotomy of 'fine versus bulk' has been reexamined by a broader perspective of human selection and cocoa genotype evolution. This new approach to cocoa genetic diversity, together with the understanding of complex microbiome interactions through fermentation, as well as quality reproducibility challenged by geographical conditions, have demonstrated the importance of terroir in the production of special attributes. Cocoa growing communities around the tropics have been clearly enabled by new omics and chemometrics to systematize producing conditions and practices in the designation of specifications for the differentiation of origin quality. © 2021 Society of Chemical Industry.

Genetic diversity and ancestry of cacao (Theobroma cacao L.) in Dominica revealed by single nucleotide polymorphism markers.

Cacao (Theobroma cacao L.), an introduced tree crop in Dominica, is important for foreign exchange earnings from fine or flavour cocoa. The genetic structure of farmed cacao in Dominica was examined to identify varieties for conservation, breeding, and propagation to improve their cocoa industry. Cacao trees (156) from 73 sites over seven geographical regions were genotyped at 192 single nucleotide polymorphism (SNP) markers. Identity, regional differentiation, phylogenetic, multi-variate, ancestry, and core collection analyses were performed. Farmed cacao germplasm had moderate gene diversity (He = 0.320 ± 0.005) from generally unique trees, but cocoa growing regions were genetically similar. Synonymous matching (16.3%) showed that some clonal material was supplied to farmers. Cacao trees were mainly mixed from Amelonado, Criollo, Iquitos, Contamana, and Marañon ancestries, with predominantly Amelonado-Criollo hybrids. Criollo ancestry, linked to fine or flavour cocoa, was found at more than 30% in 28 unique trees. Forty-five trees, containing the SNP diversity of cacao in Dominica, are recommended as a core germplasm collection. This study identifies promising trees for improving cocoa quality; provides genetic evidence that community, regional, or country-wide pooling would not compromise the exclusive fine or flavour cocoa industry; and discusses other implications towards improving the Dominican cocoa industry.

Diversity in Composition of Bioactive Compounds Among 26 Cocoa Genotypes.

Composition of bioactive compounds in cocoa beans is critical to the sensory and nutritional quality of cocoa based products. Twenty-six cocoa bean genotypes were freshly collected from the same plantation location in Indonesia. The bioactive compounds in these raw cocoa genotypes were identified and quantified. The results showed a great diversity in the composition of bioactive compounds among the 26 cocoa samples. The concentrations of methylxanthines, epicatechin, proanthocyanidin (PA) B-type oligomers, clovamide, and anthocyanins were important variables that differentiated these genotypes. MCC 01, SUL 3, ICCRI 03, and ICS 60 genotypes had the highest contents of flavan-3-ols including PAs and have the potential to be developed for "healthy" product formulations. Some genotypes such as DR 1, DR 2, DR 38, ICS 13, KPC 1, KW 617, RCC 71, and TSH 858 could be favored by industries due to the potential to be made into end-products with brighter appearance.

Study of the physical and chemical changes during the maturation of three cocoa clones, EET8, CCN51, and ICS60.

BACKGROUND: Colombia is known for its production of fine and aromatic cocoa; however, the lack of homogeneity in the ripeness stage of cocoa fruit affects the final quality of cocoa beans. Therefore, the aim of this work was to identify parameters that can be use as indicators of ripeness in cocoa fruit in order to homogenize the characteristics of raw cocoa used in the production of cocoa-products industry. The parameters evaluated were fruit, seed and pod weight, firmness, color, polar and equatorial diameters of the fruit, seed moisture content, total titratable acidity, pH, and total soluble solids of pulp. RESULTS: Factors such as seed weight, firmness, diameters, total soluble solids, pH, and acidity were affected by the clone factor, whereas seed weight, pH, and total titratable acidity were affected by ripeness stage. CONCLUSION: Identification of indicators of ripeness for cocoa fruit is a complex task due to the influence of the clone on the evolution of the physicochemical characteristics of cocoa fruit during its maturation process. Thus, indicators must be developed for each clone, and at least two parameters among color, pH, and total titratable acidity should be used to determine the ripeness stage of cocoa fruit. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Differentiation of Ecuadorian National and CCN-51 cocoa beans and their mixtures by computer vision.

BACKGROUND: Ecuador exports two major types of cocoa beans, the highly regarded and lucrative National, known for its fine aroma, and the CCN-51 clone type, used in bulk for mass chocolate products. In order to discourage exportation of National cocoa adulterated with CCN-51, a fast and objective methodology for distinguishing between the two types of cocoa beans is needed. RESULTS: This study reports a methodology based on computer vision, which makes it possible to recognize these beans and determine the percentage of their mixture. The methodology was challenged with 336 samples of National cocoa and 127 of CCN-51. By excluding the samples with a low fermentation level and white beans, the model discriminated with a precision higher than 98%. The model was also able to identify and quantify adulterations in 75 export batches of National cocoa and separate out poorly fermented beans. CONCLUSION: A scientifically reliable methodology able to discriminate between Ecuadorian National and CCN-51 cocoa beans and their mixtures was successfully developed. © 2017 Society of Chemical Industry.

Origin-based polyphenolic fingerprinting of Theobroma cacao in unfermented and fermented beans.

A comprehensive analysis of cocoa polyphenols from unfermented and fermented cocoa beans from a wide range of geographic origins was carried out to catalogue systematic differences based on their origin as well as fermentation status. This study identifies previously unknown compounds with the goal to ascertain, which of these are responsible for the largest differences between bean types. UHPLC coupled with ultra-high resolution time-of-flight mass spectrometry was employed to identify and relatively quantify various oligomeric proanthocyanidins and their glycosides amongst several other unreported compounds. A series of biomarkers allowing a clear distinction between unfermented and fermented cocoa beans and for beans of different origins were identified. The large sample set employed allowed comparison of statistically significant variations of key cocoa constituents.

Comprehensive Chemical Fingerprinting of High-Quality Cocoa at Early Stages of Processing: Effectiveness of Combined Untargeted and Targeted Approaches for Classification and Discrimination.

This study investigates chemical information of volatile fractions of high-quality cocoa (Theobroma cacao L. Malvaceae) from different origins (Mexico, Ecuador, Venezuela, Columbia, Java, Trinidad, and Sao Tomè) produced for fine chocolate. This study explores the evolution of the entire pattern of volatiles in relation to cocoa processing (raw, roasted, steamed, and ground beans). Advanced chemical fingerprinting (e.g., combined untargeted and targeted fingerprinting) with comprehensive two-dimensional gas chromatography coupled with mass spectrometry allows advanced pattern recognition for classification, discrimination, and sensory-quality characterization. The entire data set is analyzed for 595 reliable two-dimensional peak regions, including 130 known analytes and 13 potent odorants. Multivariate analysis with unsupervised exploration (principal component analysis) and simple supervised discrimination methods (Fisher ratios and linear regression trees) reveal informative patterns of similarities and differences and identify characteristic compounds related to sample origin and manufacturing step.

Roasting conditions for preserving cocoa flavan-3-ol monomers and oligomers: interesting behaviour of Criollo clones.

BACKGROUND: Cocoa bean roasting is important for creating the typical chocolate aroma through Maillard reactions, but it is also a key step deleterious to the polyphenol content and profile. RESULTS: Compared with usual roasting at 150 °C, keeping the beans for 30 min at 120 °C or for 1 h at 90 °C proved much better for preventing strong degradation of native P1, P2 and P3 flavan-3-ols in cocoa (shown for Forastero, Trinitatio and Criollo cultivars). Surprisingly, Cuban, Mexican and Malagasy white-seeded beans behaved atypically when roasted for 30 min at 150 °C, releasing a pool of catechin. Enantiomeric chromatographic separation proved that this pool contained mainly (-)-catechin issued from (-)-epicatechin by epimerisation. As the (-)-epicatechin content remained relatively constant through Criollo bean roasting, flavan-3-ol monomers must have been regenerated from oligomers. This emergence of (-)-catechin in Criollo beans only, reported here for the first time, could be due to increased flavan-3-ol monomer stability in the absence of anthocyanidin-derived products. CONCLUSION: The degradation rate of flavan-3-ols through roasting is higher in cocoa beans containing anthocyani(di)ns. The liberation of a pool of (-)-catechin when submitted to roasting at 150 °C allows to distinguish white-seeded cultivars. © 2017 Society of Chemical Industry.

Food Fingerprinting: Characterization of the Ecuadorean Type CCN-51 of Theobroma cacao L. Using Microsatellite Markers.

The cocoa type "Colección Castro Naranjal 51" (CCN-51) is known for its resistance to specific climate conditions and its high yield, but it shows a weaker flavor profile and therefore is marketed as bulk cocoa. In a previous study, the two cocoa types Arriba and CCN-51 could easily be distinguished, but differences among the CCN-51 samples were observed. This was unexpected, as CCN-51 is reported to be a clone. To confirm whether CCN-51 is a pure clone, 10 simple sequence repeats (SSR) located on the nuclear genome were used to analyze various CCN-51 samples in comparison to the cocoa varieties Arriba and Criollo. As expected, there are differences in the SSR pattern among CCN-51, Arriba, and Criollo, but a variability within the CCN-51 sample set was detected as well. The previously described sequence variation in the chloroplast genome was confirmed by a variability in the microsatellite loci of the nuclear genome for a comprehensive cultivar collection of CCN-51 of both bean and leaf samples. In summary, beneath somaclonal variation, misidentification of plant collections and also sexual reproduction of CCN-51 can be suggested.

Cocoa content influences chocolate molecular profile investigated by MALDI-TOF mass spectrometry.

BACKGROUND: Chocolate authentication is a key aspect of quality control and safety. Matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) mass spectrometry (MS) has been demonstrated to be useful for molecular profiling of cells, tissues, and even food. The present study evaluated if MALDI-TOF MS analysis on low molecular mass profile may classify chocolate samples according to the cocoa content. RESULTS: The molecular profiles of seven processed commercial chocolate samples were compared by using MALDI-TOF MS. Some ions detected exclusively in chocolate samples corresponded to the metabolites of cocoa or other constituents. This method showed the presence of three distinct clusters according to confectionery and sensorial features of the chocolates and was used to establish a mass spectra database. Also, novel chocolate samples were evaluated in order to check the validity of the method and to challenge the database created with the mass spectra of the primary samples. Thus, the method was shown to be reliable for clustering unknown samples into the main chocolate categories. CONCLUSION: Simple sample preparation of the MALDI-TOF MS approach described will allow the surveillance and monitoring of constituents during the molecular profiling of chocolates.

DNA-based differentiation of the Ecuadorian cocoa types CCN-51 and Arriba based on sequence differences in the chloroplast genome.

Two cocoa types, Arriba and CCN-51, are being cultivated in Ecuador. With regard to the unique aroma, Arriba is considered a fine cocoa type, while CCN-51 is a bulk cocoa because of its weaker aroma. Because it is being assumed that Arriba is mixed with CCN-51, there is an interest in the analytical differentiation of the two types. Two methods to identify CCN-51 adulterations in Arriba cocoa were developed on the basis of differences in the chloroplast DNA. On the one hand, a different repeat of the sequence TAAAG in the inverted repeat region results in a different length of amplicons for the two cocoa types, which can be detected by agarose gel electrophoresis, capillary gel electrophoresis, and denaturing high-performance liquid chromatography. On the other hand, single nucleotide polymorphisms (SNPs) between the CCN-51 and Arriba sequences represent restriction sites, which can be used for restriction fragment length polymorphism analysis. A semi-quantitative analysis based on these SNPs is feasible. A method for an exact quantitation based on these results is not realizable. These sequence variations were confirmed for a comprehensive cultivar collection of Arriba and CCN-51, for both bean and leaf samples.

Accurate determination of genetic identity for a single cacao bean, using molecular markers with a nanofluidic system, ensures cocoa authentication.

Cacao (Theobroma cacao L.), the source of cocoa, is an economically important tropical crop. One problem with the premium cacao market is contamination with off-types adulterating raw premium material. Accurate determination of the genetic identity of single cacao beans is essential for ensuring cocoa authentication. Using nanofluidic single nucleotide polymorphism (SNP) genotyping with 48 SNP markers, we generated SNP fingerprints for small quantities of DNA extracted from the seed coat of single cacao beans. On the basis of the SNP profiles, we identified an assumed adulterant variety, which was unambiguously distinguished from the authentic beans by multilocus matching. Assignment tests based on both Bayesian clustering analysis and allele frequency clearly separated all 30 authentic samples from the non-authentic samples. Distance-based principle coordinate analysis further supported these results. The nanofluidic SNP protocol, together with forensic statistical tools, is sufficiently robust to establish authentication and to verify gourmet cacao varieties. This method shows significant potential for practical application.

Merits of random forests emerge in evaluation of chemometric classifiers by external validation.

Real-world applications will inevitably entail divergence between samples on which chemometric classifiers are trained and the unknowns requiring classification. This has long been recognized, but there is a shortage of empirical studies on which classifiers perform best in 'external validation' (EV), where the unknown samples are subject to sources of variation relative to the population used to train the classifier. Survey of 286 classification studies in analytical chemistry found only 6.6% that stated elements of variance between training and test samples. Instead, most tested classifiers using hold-outs or resampling (usually cross-validation) from the same population used in training. The present study evaluated a wide range of classifiers on NMR and mass spectra of plant and food materials, from four projects with different data properties (e.g., different numbers and prevalence of classes) and classification objectives. Use of cross-validation was found to be optimistic relative to EV on samples of different provenance to the training set (e.g., different genotypes, different growth conditions, different seasons of crop harvest). For classifier evaluations across the diverse tasks, we used ranks-based non-parametric comparisons, and permutation-based significance tests. Although latent variable methods (e.g., PLSDA) were used in 64% of the surveyed papers, they were among the less successful classifiers in EV, and orthogonal signal correction was counterproductive. Instead, the best EV performances were obtained with machine learning schemes that coped with the high dimensionality (914-1898 features). Random forests confirmed their resilience to high dimensionality, as best overall performers on the full data, despite being used in only 4.5% of the surveyed papers. Most other machine learning classifiers were improved by a feature selection filter (ReliefF), but still did not out-perform random forests.

Ultra-barcoding in cacao (Theobroma spp.; Malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA.

PREMISE OF STUDY: To reliably identify lineages below the species level such as subspecies or varieties, we propose an extension to DNA-barcoding using next-generation sequencing to produce whole organellar genomes and substantial nuclear ribosomal sequence. Because this method uses much longer versions of the traditional DNA-barcoding loci in the plastid and ribosomal DNA, we call our approach ultra-barcoding (UBC). METHODS: We used high-throughput next-generation sequencing to scan the genome and generate reliable sequence of high copy number regions. Using this method, we examined whole plastid genomes as well as nearly 6000 bases of nuclear ribosomal DNA sequences for nine genotypes of Theobroma cacao and an individual of the related species T. grandiflorum, as well as an additional publicly available whole plastid genome of T. cacao. KEY RESULTS: All individuals of T. cacao examined were uniquely distinguished, and evidence of reticulation and gene flow was observed. Sequence variation was observed in some of the canonical barcoding regions between species, but other regions of the chloroplast were more variable both within species and between species, as were ribosomal spacers. Furthermore, no single region provides the level of data available using the complete plastid genome and rDNA. CONCLUSIONS: Our data demonstrate that UBC is a viable, increasingly cost-effective approach for reliably distinguishing varieties and even individual genotypes of T. cacao. This approach shows great promise for applications where very closely related or interbreeding taxa must be distinguished.

[Fat and fatty acids content in chocolate products]. / Zawartosc tluszczu i kwasów tluszczowych w wyrobach czekoladowych.

The aim of this study was the comparison of fat and fatty acids content in chocolate products. Fifteen chocolate products divided into 3 groups--truffles, chocolates candy and chocolates cream were used in the investigations. Crude fat content in the chocolates products was determined on Soxhlet automatic apparatus. The saturated and unsaturated fatty acids were determined using gas chromatographic method. The highest content of fat, average 25.1%, was found in candy and cream chocolates. Saturated fatty acids in fat of investigated groups of chocolate products comprised above 52%, except truffles and chocolates candy with nuts. PUFA content was similar in the all chocolate product groups. Palmitic, stearic, oleic and linoleic acids dominated in the examined chocolate products. Oleic and linoleic acids content was higher in chocolate products with nuts.

Adding value to cocoa (Theobroma cacao L.) germplasm information with domestication history and admixture mapping.

A sound understanding of crop history can provide the basis for deriving novel genetic information through admixture mapping. We confirmed this, by using characterization data from an international collection of cocoa, collected 25 years ago, and from a contemporary plantation. We focus on the trees derived from three centuries of admixture between Meso-American Criollo and South American Forastero genomes. In both cacao sets of individuals, linkage disequilibrium extended over long genetic distances along chromosome regions, as expected in populations derived from recent admixture. Based on loose genome scans, genomic regions involved in useful traits were identified. Fifteen genomic regions involved in seed and fruit weight variation were highlighted. They correspond to ten previously identified QTLs and five novel ones. Admixture mapping can help to add value to genetic resources and thus, help to encourage investment in their conservation.