The Content of Polyphenolic Compounds in Cocoa Beans (Theobroma cacao L.), Depending on Variety,Growing Region,and Processing Operations: A Review

Polyphenols form the largest group of compounds among natural antioxidants, which largely affect the overall antioxidant and antifree radical activity of cocoa beans. The qualitative and quantitative composition of individual fractions of polyphenolic compounds, even within one species, is very diverse and depends on many factors, mainly on the area of cocoa trees cultivation, bean maturity, climatic conditions during growth, and the harvest season and storage time after harvest. Thermal processing of cocoa beans and cocoa derivative products at relatively high temperatures may in addition to favorable physicochemical, microbiological, and organoleptic changes result in a decrease of polyphenols concentration. Technological processing of cocoa beans negatively affects the content of polyphenolic compounds.     

Analysis of cocoa pulp and the formulation of a standardised artificial cocoa pulp medium

Results of analyses of cocoa pulp from the Ivory Coast, Nigeria and Malaysia are reported. These include pH, viscosity and a_w measurements and concentration of soluble sugars, vitamins and anions. Pulp from Malaysian cocoa pods had a higher water content, lower citrate, hemicellulose, lignin and pectin concentration and a higher pH than Ivorian or Nigerian cocoa pods. The composition of a defined cocoa pulp medium which has the same overall composition, pH, a_w and viscosity as cocoa pulp is described. The defined cocoa pulp medium supported the growth of yeasts, lactic acid bacteria and acetic acid bacteria.     

Aflatoxins and ochratoxin A in different cocoa clones (Theobroma cacao L.) developed in the southern region of Bahia,Brazil

Brazil is the sixth largest producer of cocoa beans in the world, after Côte d’Ivoire, Ghana, Indonesia, Nigeria and Cameroon. The southern region of Bahia stands out as the country’s largest producer, accounting for approximately 60% of production. Due to damage caused by infestation of the cocoa crop with the fungus Moniliophthora perniciosa, which causes ‘witch’s broom disease’, research in cocoa beans has led to the cloning of species that are resistant to the disease; however, there is little information about the development of other fungal genera in these clones, such as Aspergillus, which do not represent a phytopathogenicity problem but can grow during the pre-processing of cocoa beans and produce mycotoxins. Thus, the aim of this work was to determine the presence of aflatoxin (AF) and ochratoxin A (OTA) in cocoa clones developed in Brazil. Aflatoxin and ochratoxin A contamination were determined in 130 samples from 13 cocoa clones grown in the south of Bahia by ultra-performance liquid chromatography with a fluorescence detector. The method was evaluated for limit of detection (LOD) (0.05–0.90 μg kg^?1), limit of quantification (0.10–2.50 μg kg^?1) and recovery (RSD) (89.40–95.80%) for AFB₁, AFB₂, AFG₁, AFG₂ and OTA. Aflatoxin contamination was detected in 38% of the samples in the range of ?1, with AFB₁ in 25% of the total samples, whereas ochratoxin A was positive in 18% of the samples in the range of ?1.     

On-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavour of chocolates produced thereof

Cocoa bean fermentations controlled by means of starter cultures were introduced on several farms in two different cocoa-producing regions (West Africa and Southeast Asia). Two starter culture mixtures were tested, namely one composed of Saccharomyces cerevisiae H5S5K23, Lactobacillus fermentum 222, and Acetobacter pasteurianus 386B (three heaps and one box), and another composed of L. fermentum 222 and A. pasteurianus 386B (seven heaps and one box). In all starter culture-added cocoa bean fermentation processes, the inoculated starter culture species were able to outgrow the natural contamination of the cocoa pulp-bean mass and they prevailed during cocoa bean fermentation. The application of both added starter cultures resulted in fermented dry cocoa beans that gave concomitant milk and dark chocolates with a reliable flavour, independent of cocoa-producing region or fermentation method. The addition of the lactic acid bacterium (LAB)/acetic acid bacterium (AAB) starter culture to the fermenting cocoa pulp-bean mass accelerated the cocoa bean fermentation process regarding citric acid conversion and lactic acid production through carbohydrate fermentation. For the production of a standard bulk chocolate, the addition of a yeast/LAB/AAB starter culture was necessary. This enabled an enhanced and consistent ethanol production by yeasts for a successful starter culture-added cocoa bean fermentation process. This study showed possibilities for the use of starter cultures in cocoa bean fermentation processing to achieve a reliably improved fermentation of cocoa pulp-bean mass that can consistently produce high-quality fermented dry cocoa beans and flavourful chocolates produced thereof.     

Analytical evaluation of cocoa curing in the ivory coast

During an experimental cocoa curing on the Ivory Coast, samples of cocoa beans were collected every day, freeze-dried and analytically checked. Some of the analytical results are discussed in relation to the possibility of using chemical indices to assess the degree of fermentation of commercial cocoas.     

Dynamics and species diversity of communities of lactic acid bacteria and acetic acid bacteria during spontaneous cocoa bean fermentation in vessels

To speed up research on the usefulness and selection of bacterial starter cultures for cocoa bean fermentation, a benchmark cocoa bean fermentation process under natural fermentation conditions was developed successfully. Therefore, spontaneous fermentations of cocoa pulp-bean mass in vessels on a 20 kg scale were tried out in triplicate. The community dynamics and kinetics of these fermentations were studied through a multiphasic approach. Microbiological analysis revealed a limited bacterial species diversity and targeted community dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation, as was the case during cocoa bean fermentations processes carried out in the field. LAB isolates belonged to two main (GTG)₅-PCR clusters, namely Lactobacillus plantarum and Lactobacillus fermentum, with Fructobacillus pseudofilculneus occurring occasionally; one main (GTG)₅-PCR cluster, composed of Acetobacter pasteurianus, was found among the AAB isolates, besides minor clusters of Acetobacter ghanensis and Acetobacter senegalensis. 16S rRNA-PCR-DGGE revealed that L. plantarum and L. fermentum dominated the fermentations from day two until the end and Acetobacter was the only AAB species present at the end of the fermentations. Also, species of Tatumella and Pantoea were detected culture-independently at the beginning of the fermentations. Further, it was shown through metabolite target analyses that similar substrate consumption and metabolite production kinetics occurred in the vessels compared to spontaneous cocoa bean fermentation processes. Current drawbacks of the vessel fermentations encompassed an insufficient mixing of the cocoa pulp-bean mass and retarded yeast growth.     

Feasibility study on the use of Fourier transform near-infrared spectroscopy together with chemometrics to discriminate and quantify adulteration in cocoa beans

Fourier transform near-infrared (FT-NIR) spectroscopy combined with Support Vector Machine (SVM) and synergy interval partial least square (Si-PLS) was attempted in this study for cocoa bean authentication. SVM was used to develop an identification model to discriminate between fermented cocoa beans (FC), unfermented cocoa beans (UFC) and adulterated cocoa bean (5–40 wt/wt.% content of UFC). Si-PLS model was used to quantify the addition of UFC in FC. SVM model accurately discriminated the cocoa bean samples used. After cross-validation, the optimal identification rate was 100% in both the training set and prediction set at three principal components. For quantitative analysis, Si-PLS model was evaluated according to root mean square error of prediction (RMSEP) and coefficient of correlation in prediction (R_pred). The results revealed that Si-PLS model in this work was promising. The optimal performance of Si-PLS model showed an excellent predictive potential, RMSEP = 1.68 and R_pred = 0.98 in the prediction set. The overall results indicated that FT-NIR spectroscopy together with an appropriate multivariate algorithm could be employed for rapid identification of fermented and unfermented cocoa beans as well as the quantification of UFC down to 5% in FC for quality control management.     

Factors influencing quality variation in cocoa (Theobroma cacao) bean flavour profile — A review

This review examined the factors that influence flavour volatiles of cocoa beans and the volume of work that needs to be done on these factors and their impact on the flavour volatiles of commercial cocoa beans. Cocoa bean flavour is one of the most important quality attributes as flavour is central to acceptability of cocoa beans and cocoa products such as chocolate. The complex composition of cocoa bean flavour depends on bean genotype, postharvest treatments such as pulp pre-conditioning, fermentation and drying, industrial processes such as roasting as well as the type of soil and age of cocoa tree. The bean genotype determines the chemical composition of the bean, specifically the contents of bean storage proteins, polysaccharides, and polyphenols. This determines the quantities and type of precursors formed during fermentation and drying processes leading to flavour formation, hence, influencing both flavour type and intensity. Cocoa bean fermentation and drying result in the breakdown of the storage proteins by endogenous proteases into amino acids and short chain oligopeptides while the polysaccharides are also degraded by invertase to glucose and fructose. The amino acids, oligopeptides, glucose and fructose react with each other during the roasting process to produce the typical cocoa flavour volatiles. Polyphenols are also oxidized by polyphenol oxidase during fermentation and drying which reduce the astringency and bitterness of the beans, thus, enhancing the flavour of cocoa beans. However, the extent to which other factors such as age of the cocoa tree and soil chemical compositions influence the formation of flavour precursors and their relationships with final flavour quality remains unclear. With increasing demand for sustainable production of high quality cocoa beans, greater understanding of factors contributing to the variations in flavour character would have significant commercial implications.     

Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

Using a detailed HPLC–ESI-MS analysis including both high resolution and tandem MS analysis, a series of fourteen novel flavonoids were identified from alcoholic extracts of raw fermented cocoa beans from Cameroon. The novel compounds include a series of isomeric hexosides of flavan-3-ols and novel proanthocyanidine dimers and trimers and a sulfated flavan-3-ol. Additionally a jasmonic acid sulfate derivative was identified in the cocoa bean extracts as one of the major metabolites produced under fermentation stress. Therefore, the chosen HPLC–ESI-MS analysis method revealed a greater complexity of the cocoa bean polyphenolic extract than previously reported and allowed individual assignment of novel compounds.     

The effects of the organic acids in cocoa on the flavour of chocolate

Samples (54) of dried fermented cocoa beans from different world regions were analysed for levels of organic acids, pH and titratable acidity. The effects of the organic acids on the flavour characteristics of cocoa were examined by sensory evaluation of chocolate made from samples of cocoa beans. Concentrations (g kg^?1) of acids ranged from 1.3 to 11.8 for acetic, 1-6 to 9-9 for citric, 0.6 to 11.1 for lactic and 2.1 to 6.5 for oxalic. pH values ranged from 4.6 to 5.8, while titratable acidity ranged from 0.08 to 0.31 equivalents of sodium hydroxide per kg sample. Cocoas from South East Asia and the South Pacific tended to be more acidic than West African beans in terms of both chemical and sensory characteristics. Lactic and acetic acids were found to be in greater concentrations in cocoas from the former regions and were considered to be largely responsible for higher acid flavour scores. In contrast, citric and oxalic acids were generally lower in these beans. Flavour assessments of cocoas with and without added organic acids indicated that oxalic acid played an important role in chocolate flavour. These results suggest that a reduction in the levels of acetic and lactic acids only, may not be sufficient to produce a desirable flavour balance.     

Spontaneous organic cocoa bean box fermentations in Brazil are characterized by a restricted species diversity of lactic acid bacteria and acetic acid bacteria

Spontaneous organic cocoa bean box fermentations were carried out on two different farms in Brazil. Physical parameters, microbial growth, bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the fermented dry cocoa beans. The main end-products of the catabolism of the pulp substrates (glucose, fructose, and citric acid) by yeasts, LAB, and AAB were ethanol, lactic acid, mannitol, and/or acetic acid. Lactobacillus fermentum and Acetobacter pasteurianus were the predominating bacterial species of the fermentations as revealed through (GTG)₅-PCR fingerprinting of isolates and PCR-DGGE of 16S rRNA gene PCR amplicons of DNA directly extracted from fermentation samples. Fructobacillus pseudoficulneus, Lactobacillus plantarum, and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Also, three novel LAB species were found. This study emphasized the possible participation of Enterobacteriaceae in the cocoa bean fermentation process. Tatumella ptyseos and Tatumella citrea were the prevailing enterobacterial species in the beginning of the fermentations as revealed by 16S rRNA gene-PCR-DGGE. Finally, it turned out that control over a restricted bacterial species diversity during fermentation through an ideal post-harvest handling of the cocoa beans will allow the production of high-quality cocoa and chocolates produced thereof, independent of the fermentation method or farm.     

Analysis and occurrence of deoxynivalenol (DON) in cocoa

The primary objective of this study was to establish a current situation assessment of the possible occurrence of deoxynivalenol in cocoa and cocoa products. Since there was no analytic method for determining DON in cocoa and cocoa products, a special method was developed. The applicability and consistency of the method was confirmed by performing recovery assays on various cocoa products. A special post-column derivatisation procedure was used to increase selectivity and raise sensitivity by a factor of 80. The method’s limit of detection (LOD) was thereby reduced to 7 μg/kg; the limit of quantification (LOQ) was 14 μg/kg. The method was used to test 230 samples for possible DON content, ranging from cocoa beans to cocoa bean shells, nibs, cocoa liquor and cocoa powders through to finished cocoa-based products. In the case of cocoa beans and cocoa bean shells, DON content close to the detection limit was only determined in isolated cases. No DON content was detected in nibs, cocoa liquor, cocoa powders and finished cocoa-based products. Analogous to ochratoxin A and aflatoxins, the results show DON is more likely to be found in cocoa bean shells. Separation of shells during cocoa processing can reduce potential DON contents. Since no DON was determined in the fractions relevant for chocolate production, these assays show it does not represent a considerable issue for the cocoa and chocolate industry.     

Mycobiota of cocoa: from farm to chocolate

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

Temperature influences epimerization and composition of flavanol monomers,dimers and trimers during cocoa bean roasting

Cocoa consumption is suggested to promote many health benefits, since cocoa is a rich source of flavanols; but amounts and profiles of flavanols depend strongly on the bean type, origin and manufacturing process. Roasting is known as a crucial step in technical treatment of cocoa, which leads to flavanol losses and modifications, especially the epimerization of (−)-epicatechin to (−)-catechin. This study monitors the influence of cocoa bean roasting on the composition of flavanol monomers to trimers, with special focus on epimerization, which was quantified for procyanidin dimers, and also observed for trimers for the first time. Five dimeric and two trimeric potential epimerization products were detected and the extent of epimerization during cocoa roasting was shown to be a function of temperature. The data also showed remarkable variations in the change of flavanol content. The quantified flavanols decreased about 50% in Java beans and increased about 30% in Ivory Coast beans, despite being roasted under equal conditions.     

Free amino acid amounts in raw cocoas from different origins

Raw cocoa is the processed and traded form of the cocoa seed. Fresh seeds undergo fermentation and a drying process before they are prepared for transport and shipping. Depending on the local situation in the producer region the seeds are collected from big estates and are fermented and dried in big lots or they originate from small farmer's crop trees. In the subsequent transport and trading chain raw cocoas may be mixed and stored before they finally are sold and brought to the consumer countries. Local or regional variations in cocoa plant material, fermentation procedures and drying processes finally result in a typical traded good with respect to the amino acids, which form an important part of the flavour precursors. These free amino acids and their composition result from the fermentation procedure of fresh seeds. In the course of the fermentation specific cleavage of cocoa storage proteins delivers the amino acid patterns. In this study the variation of free amino acid amount and distribution of 108 commercially fermented and traded cocoa samples and two Theobroma grandiflorum (Willd. ex Spreng.) K. Schum samples were determined. This examination showed clearly, that content and distribution of free amino acids in raw cocoa from different origins vary greatly (5–25 mg g⁻¹ fatfree dry matter), in some cases country and even region-specific differences were apparent. It is important to notice typical, region-specific variations in the amounts and compositions of free amino acids.     

Occurrence of ochratoxin A in cocoa beans: Effect of shelling

The aim of this study was to investigate the influence of the shelling process on the presence of ochratoxin A (OTA) in cocoa samples. Twenty-two cocoa samples were analysed for the determination of OTA before (cocoa bean) and after undergoing manual shelling process (cocoa nib). In order to determine OTA contamination in cocoa samples, a validated high-performance liquid chromatography (HPLC) method with fluorescence detection was used for the quantitative analysis of ochratoxin A (OTA). In both types of samples, OTA was extracted with methanol-3% sodium hydrogen carbonate solution and then purified using immunoaffinity columns prior to HPLC analysis. Due to the fact that different recovery values were obtained for OTA from both types of samples, a revalidation of the method in the case of cocoa nibs was needed. Revalidation was based on the following criteria: Selectivity, limits of detection and quantification (0.03 and 0.1 µg kg^-1, respectively), precision (within-day and between-day variability) and recovery 84.2% (RSD = 7.1%), and uncertainty (30%). Fourteen of the twenty-two cocoa bean samples (64%) suffered a loss of OTA of more than 95% due to shelling, six samples suffered a loss of OTA in the range 65-95%, and only one sample presented a reduction of less than 50%. The principal conclusion derived from this study is that OTA contamination in cocoa beans is concentrated in the shell; therefore, improvements of the industrial shelling process could prevent OTA occurrence in cocoa final products.     

Improving chocolate flavor in poor-quality cocoa almonds by enzymatic treatment

Abstract: This paper proposes a method to enzymatically treat poor‐quality cocoa almonds (known as “slate”) to ensure the formation of chocolate flavor precursors. The production of flavor precursors improves the quality of these almonds, which are usually responsible for the low quality of the liquor produced. Proteases and carboxypeptidases from different sources were tested under various conditions. The different treatments were evaluated by chemical analysis (hydrolysis efficiency) and sensory analysis of the treated material compared to good‐quality cocoa almonds. The results show that it is possible, through the use of microbial enzymes, to generate the mixture of compounds that will release, after roasting, the characteristic chocolate flavor in poor‐quality almonds. However, it is necessary to optimize the conditions of enzymatic treatment to obtain better results and thus establish a process that can be used for industrial purposes for manufacturing cocoa and chocolate. Practical Application: The basidiomycete Moniliophtora perniciosa is the causative agent of witches’ broom disease (WBD) of the cocoa tree, whose seeds are the source of chocolate. It is the most important phytopathological problem of cocoa‐producing areas of the American continent, and has decimated the Brazilian cocoa industry. In Bahia (Brazil), M. perniciosa was identified in 1989 and, as a consequence of its spreading, the annual production of cocoa almonds dropped from 450000 to 90000 tons within 12 y, reducing export values from an all‐time high of about US$ 1 billion to 110 million. The high incidence of WBD incapacitates Brazil to produce enough cocoa almonds even for the internal market, leading the country to import low‐quality cocoa almonds mainly from African countries. Our work proposes an enzymatic treatment to increase the quality of that cocoa almonds and, consequently, to improve the quality of the chocolate produced and consumed in the country.     

Relationship between bioactive compounds and sensory properties of dark chocolate produced from Brazilian hybrid cocoa

This work evaluated the influence of bioactive compounds on the sensory profile and acceptability of dark chocolate produced from Brazilian hybrid cocoa. The results showed that higher contents of catechin, epicatechin, caffeine, total phenolic compounds and flavonoids contributed to higher intensity of bitterness, cocoa flavour, acid taste, and astringency, and negatively affected the acceptance of chocolate samples from the TSH1188, PH16, and CEPEC2002 varieties. Other varieties SR162 and BN34 showed lower contents of caffeine and phenolic compounds, and higher concentration of theobromine, revealed notes of sweet/caramel and fruity flavour, presented a higher acceptance. This research showed the great potential of the ‘Catongo’ (SR162) and BN34 hybrids from southern Bahia (Brazil) for the production of fine cocoa. The chemical and sensory evaluations may be a strategic tool to help cocoa producers in deciding which genetic varieties should be marketed as fine cocoa, adding value to the product and highlighting promising cocoa varieties.     

Effect of the types and concentrations of alkali on the color of cocoa liquor

BACKGROUND: The alkalization process is extensively used in the cocoa industry, but information is scarce and not easy to acquire. The goal of the study was to evaluate the effect of different types and concentrations of alkali on the color of cocoa liquor. Dried beans from Chuao (state Aragua, Venezuela) were used to produce cocoa liquors. Samples of liquors were alkalized with solutions of NaHCO₃, Na₂CO₃ and NaOH at concentrations of 10, 20 and 30 g kg^?1. RESULTS: The data showed that values of the coordinates L*, a* and b* decrease when liquors were treated with the three different types and concentrations of alkalis. Almost all samples had ΔE* values above 1. The ratios b*/a* and a*/b* and the proximate composition were also modified. Crude protein, crude fat and polyphenol concentrations were decreased and the ash content augmented as concentrations of the alkalis were increased. The fatty acid and sugar profiles were also affected. These ratios were most pronounced when NaOH was used. CONCLUSION: The selection of the type or concentration of alkali is a function of the type of product to be elaborated. Copyright © 2009 Society of Chemical Industry     

Fermentation of cocoa beans: influence of microbial activities and polyphenol concentrations on the flavour of chocolate

BACKGROUND: Spontaneous cocoa bean fermentation is characterised by a succession of microbial activities. Cocoa flavour precursors are developed during fermentation and drying of cocoa beans. Polyphenols and alkaloids contribute to astringency and bitterness of cocoa and chocolate. RESULTS: Population dynamics, metabolite target analyses, and chocolate production were performed for seven independent spontaneous cocoa bean heap fermentations in Ghana. Although the same micro‐organisms were involved in these heaps, carried out at different farms or in different seasons, heap temperatures and microbial metabolite concentrations were different. This could be due to heterogeneity and size of the heaps, but was mainly ascribed to microbial variability. Indeed, differences in microbial activity could be linked with the flavour of chocolates made from the corresponding dried, fermented cocoa beans. Whereas the polyphenol and alkaloid contents of cocoa beans were crop‐ and heap‐dependent, epicatechin and theobromine levels decreased during fermentation due to diffusion out of the bean cotyledons and polyphenol oxidation and condensation. Residual levels were responsible for the degree of bitterness of the final chocolates. CONCLUSION: Differences in microbial activities between different heap fermentations can result in dried fermented cocoa beans and chocolates with different flavour characteristics. Hence, fermentation control may direct the flavour of chocolate. Copyright © 2008 Society of Chemical Industry