Evaluation of gamma-irradiation in cocoa husk

gamma-Irradiation was investigated as a technique to improve the hygienic quality of cocoa husk. Cocoa husk is a byproduct of cocoa bean processing industry. It contains approximately 57.5% (w/w) dietary fiber (nonstarch polysaccharides plus lignin), 15% (w/w) crude protein, 10.7% (w/w) mineral elements, 2.32% (w/w) cocoa butter, and 2.8% (w/w) carbohydrates (free sugars plus starch). The effect of irradiation on the growth rates of microorganisms are reported. Total counts, enterobacteriaceae, coliforms, Staphylococcus aureus, Streptococcus "D" of Lancefield, and yeast and mold counts before and after irradiation at 5, 8, and 10 kGy were determined. Cocoa husk was irradiated in open containers. An irradiation dose of 5 kGy was already sufficient to decrease the microbial counts to a very low level. No alteration in dietary fiber was measured in the irradiated product and no significant differences were detected between irradiated and nonirradiated cocoa husk.     

Improved high-performance liquid chromatography method to determine theobromine and caffeine in cocoa and cocoa products

At present, the commonly used HPLC method for the analysis of caffeine and theobromine contents in aqueous cocoa extracts employs direct application of the extracts on the column. This practice gradually reduces the efficiency of the column and shortens its life. Also, this method gives inflated values due to interfering substances and difficulty in achieving baseline resolution. In the improved method, the interfering cocoa pigments are effectively removed by passing the aqueous extract through a Sep-pak C(18) cartridge. Subsequent injection on a C(18) reverse-phase column employing acetonitrile and water (20:80) as the mobile phase reduces the analysis time without affecting either resolution of the peak or the accuracy of caffeine and theobromine determination or achieving baseline resolution. Therefore, this method is ideally suited for rapid routine analysis of cocoa and its products.     

Cluster analysis for the systematic grouping of genuine cocoa butter and cocoa butter equivalent samples based on triglyceride patterns

The triglyceride profile of cocoa butters (CBs) from different geographical origins, varieties, growing seasons, and a number of cocoa butter equivalents (CBEs) was determined by capillary gas liquid chromatography. Hierarchical cluster analysis was applied to the five main triglycerides of the samples for the ability to find natural groupings among (a) CBs of various provenance and (b) CBE samples of different types. The samples were clustered using Ward's method, and the similarity values of the linkages were represented by dendrograms. The five triglycerides contained adequate information to obtain a meaningful sample differentiation. This information can be used to assess the purity and the origin of the CB sample examined.     

Reclamation of degraded cocoa lands using Albizia Zygia

The structure of the population of Albizia zygia and the regeneration potential of the species from seed were studied in three site conditions (mature cocoa stand, fallow and intact natural forest) in the moist semi-deciduous forest zone in Ghana. The potential of the species to regenerate vegetatively was also assessed. Different population structures and different natural regeneration status were observed for each site. Regeneration from seed appeared to be of a little importance in the fallow site, whereas vegetative regeneration was found to be a major mechanism of secondary succession in abandoned cocoa farms. The population of Albizia zygia in the forest appeared to be stable, while in the mature cocoa fields it was in decline. Efficient seed pretreatment techniques and vegetative propagation methods using juvenile cuttings, which are useful for enhanced management of the species, are also presented. Management strategies using the species for the rehabilitation of degraded cocoa farms are discussed.     

Chemical composition and flavor of ecuadorian cocoa liquor

The contribution of the chemical composition to the flavor of cocoa liquor from an Ecuadorian selfed population of clone EET 95 was investigated. Polyphenols, purine alkaloids, organic acids, and sugars were quantified, and the key sensory characteristics of cocoa were scored by a trained panel. Despite the short bean fermentation (2 days) commonly used for Arriba cocoa, acetic acid content was closely correlated to liquor pH, demonstrating its essential role in cocoa liquor acidification. Polyphenols were positively correlated to astringency, bitterness, and the green note and negatively correlated to the fruity character. Alkaloid and polyphenol levels fluctuated significantly within the selfed progeny and tended to be lower than those of the heterozygous clone EET 95 (inbreeding effect). These results support the idea that polyphenols might be essential to the overall perception of cocoa liquor characteristics and indicate that the composition and the sensory quality of cocoa liquor are the result of both a genotypic contribution and the conditions of fermentation and roasting.     

Occurrence of ochratoxin A in cocoa products and chocolate

In this work, the occurrence of ochratoxin A (OTA) in 170 samples of cocoa products of different geographical origins was studied. An immunoaffinity column with HPLC separation was developed to quantify low levels of OTA in cocoa bean, cocoa cake, cocoa mass, cocoa nib, cocoa powder, cocoa shell, cocoa butter, chocolate, and chocolate cream with >80% recoveries. The method was validated by performing replicate analyses of uncontaminated cocoa material spiked at three different levels of OTA (1, 2, and 5 microg/kg). The data obtained were related on the acceptable safe daily exposure for OTA. The highest levels of OTA were detected in roasted cocoa shell and cocoa cake (0.1-23.1 microg/kg) and only at minor levels in the other cocoa products. Twenty-six cocoa and chocolate samples were free from detectable OTA (<0.10 microg/kg). In roasted cocoa powder 38.7% of the samples analyzed contained OTA at levels ranging from 0.1 to 2 microg/kg, and 54.8% was contaminated at >2 microg/kg (and 12 samples at >3 microg/kg). Ochratoxin A was detected in cocoa bean at levels from 0.1 to 3.5 microg/kg, the mean concentration being 0.45 microg/kg; only one sample exceeded 2 microg/kg (4.7%). In contrast, 51.2% of cocoa cake samples contained OTA at levels > or =2 microg/kg, among which 16 exceeded 5 microg/kg (range of 5-9 microg/kg). These results indicate that roasted cocoa powder is not a major source of OTA in the diet.     

Characterization of peptides formed during fermentation of cocoa bean.

Analysis by SDS-PAGE and GPC-MS of fermented cocoa extracts shows changes in the amount and composition of the major proteins, accompanied by formation of complex distributions of peptides. MS/MS studies and application of SEQUEST sequencing software have allowed identification of two related peptides, a hexapeptide and a nonapeptide, formed from vicilin, one of the cocoa storage proteins. Time course studies of the two peptides show different abundance profiles and indicate, in part, production of the hexapeptide from the nonapeptide.     

Obtention and characterization of phenolic extracts from different cocoa sources

The aim of this study was to evaluate several cocoa sources to obtain a rich phenol extract for use as an ingredient in the food industry. Two types of phenolic extracts, complete and purified, from different cocoa sources (beans, nibs, liquor, and cocoa powder) were investigated. UPLC-MS/MS was used to identify and quantify the phenolic composition of the extracts, and the Folin-Ciocalteu and vanillin assays were used to determine the total phenolic and flavan-3-ol contents, respectively. The DPPH and ORAC assays were used to measure their antioxidant activity. The results of the analysis of the composition of the extracts revealed that the major fraction was procyanidins, followed by flavones and phenolic acids. From the obtained results, the nib could be considered the most interesting source for obtaining a rich phenolic cocoa extract because of its rich phenolic profile content and high antioxidant activity in comparison with the other cocoa sources.     

Physicochemical properties of acidified skim milk gels containing cocoa flavanols

The physicochemical properties of acidified milk gels after the addition of cocoa flavanols were studied. As the flavanol level increased (from 0 to 2.5 mg/g), syneresis and gel elasticity (tan δ) were found to significantly increase and decrease, respectively. Flavanol addition reduced the stress at fracture, with no changes in fracture strain, suggesting that the bond type (i.e., covalent vs noncovalent) was the underlying factor explaining the ease of fracture. Gels made from recombined milks containing the casein fraction of heated milk and the serum of heated flavanol/milk mixtures showed the lowest values of G' and fracture stress. It was concluded that whey proteins/flavanol interactions were responsible for the poor mechanical properties of flavanol-added acidified milk gels. High-performance liquid chromatography analysis of milk sera showed that 60% of the total available monomeric flavanols was found in the serum phase from which 75% was non-associated to whey proteins. Concomitantly, >70% of flavanols with degree of polymerization >3 were found to be associated with the casein fraction.     

Inhibition of key digestive enzymes by cocoa extracts and procyanidins

This study determined the in vitro inhibitory effects of cocoa extracts and procyanidins against pancreatic α-amylase (PA), pancreatic lipase (PL), and secreted phospholipase A(2) (PLA(2)) and characterized the kinetics of such inhibition. Lavado, regular, and Dutch-processed cocoa extracts as well as cocoa procyanidins (degree of polymerization (DP) = 2-10) were examined. Cocoa extracts and procyanidins dose-dependently inhibited PA, PL, and PLA(2). Lavado cocoa extract was the most potent inhibitor (IC(50) = 8.5-47 μg/mL). An inverse correlation between log IC(50) and DP (R(2) > 0.93) was observed. Kinetic analysis suggested that regular cocoa extract, the pentamer, and decamer inhibited PL activity in a mixed mode. The pentamer and decamer noncompetitively inhibited PLA(2) activity, whereas regular cocoa extract inhibited PLA(2) competitively. This study demonstrates that cocoa polyphenols can inhibit digestive enzymes in vitro and may, in conjunction with a low-calorie diet, play a role in body weight management.     

Characterization of cocoa butter and cocoa butter equivalents by bulk and molecular carbon isotope analyses: implications for vegetable fat quantification in chocolate

The fatty acids from cocoa butters of different origins, varieties, and suppliers and a number of cocoa butter equivalents (Illexao 30-61, Illexao 30-71, Illexao 30-96, Choclin, Coberine, Chocosine-Illipé, Chocosine-Shea, Shokao, Akomax, Akonord, and Ertina) were investigated by bulk stable carbon isotope analysis and compound specific isotope analysis. The interpretation is based on principal component analysis combining the fatty acid concentrations and the bulk and molecular isotopic data. The scatterplot of the two first principal components allowed detection of the addition of vegetable fats to cocoa butters. Enrichment in heavy carbon isotope ((13)C) of the bulk cocoa butter and of the individual fatty acids is related to mixing with other vegetable fats and possibly to thermally or oxidatively induced degradation during processing (e.g., drying and roasting of the cocoa beans or deodorization of the pressed fat) or storage. The feasibility of the analytical approach for authenticity assessment is discussed.     

Phosphatase activity of soil as affected by cocoa pod ash

Results of the effect of cocoa pod ash and KHCO₃ on phosphatase activity of some cocoa soils of Ghana are reported. Phosphatase activity of control soils generally decreased with duration of incubation. Higher rates of the ash application, regardless of time, resulted in a lower phosphatase activity than the control. This inhibition, among other factors, may be due to the presence of high available P and certain cations such as Mn and Zn. The KHCO₃ increased the activity of the phosphatase in all soils when compared with the control.     

Tetrahydro-beta-carbolines, potential neuroactive alkaloids, in chocolate and cocoa

Tetrahydro-beta-carbolines (THbetaCs), potential neuroactive alkaloids, were found in chocolate and cocoa. 6-Hydroxy-1-methyl-1,2, 3,4-tetrahydro-beta-carboline (6OHMTHbetaC), 1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid (THCA), 1-methyl-1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA) in both diastereoisomers (1S,3S and 1R,3S), and 1-methyl-1,2,3, 4-tetrahydro-beta-carboline (MTHbetaC), besides serotonin and tryptamine biogenic amines, were identified and quantified in dark chocolate, milk chocolate, cocoa, and chocolate-containing cereals by RP-HPLC-fluorescence and HPLC-MS. For each THbetaC, the concentration ranges were determined: 6OHMTHbetaC (0.16-3.92 microg/g), THCA (0.01-0.85 microg/g), 1S,3S-MTCA (0.35-2 microg/g), 1R,3S-MTCA (0.14-0.88 microg/g), and MTHbetaC (nd-0.21 microg/g). The highest content was generally found in chocolates and cocoas, but cereals containing chocolate also showed an appreciable amount of THbetaCs. The possible biological implications of this novel group of alkaloids in chocolate are discussed.     

Inositol penta- and hexaphosphate content of some cocoa soils in Ghana

The inositol penta- and hexaphosphate content of some typical cocoa soils in Ghana was measured by anion exchange chromatography with HCOONH₄ as eluant. The values which ranged from 6 to 35 ppm P (average 18 ± 8 ppm P) and accounted for 6% to 40% (average 14%) of the organic phosphorus are within those ranges reported for Canadian, Nigerian, but much lower than those for Scottish soils. The penta- and hexaphosphate content of the soil significantly correlated with total P, organic P, organic C, total N, Fe and P retention capacity. Incubation of soil for 11 weeks did not alter the inositol penta- and hexaphosphate content of the soil. Inorganic P adsorption could be decreased by adding inositol hexaphosphate. These data support the suggestion that a role of the esters in nutrition of crops is probably through the indirect effect of blocking of retention sites and agents which would otherwise be free to bind inorganic phosphorus.     

Determination of cocoa butter equivalents in milk chocolate by triacylglycerol profiling

An analytical approach for the detection and quantification of cocoa butter equivalents (CBEs) in milk chocolate is presented. It is based on (i) a comprehensive standardized database covering the triacylglycerol composition of a wide range of authentic milk fat (n=310), cocoa butter (n=75), and CBE (n=74) samples and 947 gravimetrically prepared mixtures thereof, (ii) the availability of a certified cocoa butter reference material (IRMM-801) for calibration, (iii) an evaluation algorithm, which allows a reliable quantification of the milk fat content in chocolate fats using a simple linear regression model, (iv) a subsequent correction of triacylglycerols deriving from milk fat, (v) mathematical expressions to detect the presence of CBEs in milk chocolate, and (vi) a multivariate statistical formula to quantify the amount of CBEs in milk chocolate. The detection limit was 1% CBE in chocolate fat (0.3% CBE in milk chocolate, having a fat content of 30%). For quantification, the average error for prediction was 1.2% CBE in chocolate fat, corresponding to 0.4% in milk chocolate (fat content, 30%).     

Predictive relationship between polyphenol and nonfat cocoa solids content of chocolate

Chocolate is often labeled with percent cocoa solids content. It is assumed that higher cocoa solids contents are indicative of higher polyphenol concentrations, which have potential health benefits. However, cocoa solids include polyphenol-free cocoa butter and polyphenol-rich nonfat cocoa solids (NFCS). In this study the strength of the relationship between NFCS content (estimated by theobromine as a proxy) and polyphenol content was tested in chocolate samples with labeled cocoa solids contents in the range of 20-100%, grouped as dark (n = 46), milk (n = 8), and those chocolates containing inclusions such as wafers or nuts (n = 15). The relationship was calculated with regard to both total polyphenol content and individual polyphenols. In dark chocolates, NFCS is linearly related to total polyphenols (r2 = 0.73). Total polyphenol content appears to be systematically slightly higher for milk chocolates than estimated by the dark chocolate model, whereas for chocolates containing other ingredients, the estimates fall close to or slightly below the model results. This shows that extra components such as milk, wafers, or nuts might influence the measurements of both theobromine and polyphenol contents. For each of the six main polyphenols (as well as their sum), the relationship with the estimated NFCS was much lower than for total polyphenols (r2 < 0.40), but these relationships were independent of the nature of the chocolate type, indicating that they might still have some predictive capabilities.     

Changes in key aroma compounds of Criollo cocoa beans during roasting

Application of a comparative aroma extraction dilution analysis on unroasted and roasted Criollo cocoa beans revealed 42 aroma compounds in the flavor dilution (FD) factor range of 1-4096 for the unroasted and 4-8192 for the roasted cocoa beans. While the same compounds were present in the unroasted and roasted cocoa beans, respectively, these clearly differed in their intensity. For example, 2- and 3-methylbutanoic acid (rancid) and acetic acid (sour) showed the highest FD factors in the unroasted beans, while 3-methylbutanal (malty), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (caramel-like), and 2- and 3-methylbutanoic acid (sweaty) were detected with the highest FD factors in the roasted seeds. Quantitation of 30 odorants by means of stable isotope dilution assays followed by a calculation of odor activity values (ratio of the concentration/odor threshold) revealed concentrations above the odor threshold for 22 compounds in the unroasted and 27 compounds in the roasted cocoa beans, respectively. In particular, a strong increase in the concentrations of the Strecker aldehydes 3-methylbutanal and phenylacetaldehyde as well as 4-hydroxy-2,5-dimethyl-3(2H)-furanone was measured, suggesting that these odorants should contribute most to the changes in the overall aroma after roasting. Various compounds contributing to the aroma of roasted cocoa beans, such as 3-methylbutanoic acid, ethyl 2-methylbutanoate, and 2-phenylethanol, were already present in unroasted, fermented cocoa beans and were not increased during roasting.     

Assessment of cocoa input needs using soil types and soil spectral analysis

In this study, diffuse reflectance spectroscopy (DRS) approach was examined for making input recommendations in the smallholder cocoa farms of Papua New Guinea (PNG). Soil samples were collected from four provinces of PNG. Soil samples from four different depths (0–10, 10–30, 30–60 and 60–90 cm) of 32 profiles in each of these site were used to create a database of soil chemical and physical properties. Spectral reflectance values at 1 nm interval covering visible to shortwave‐infrared (350–2,500 nm) were collected for each of these soil samples to develop partial least squares regression models. Soil textural fractions, soil organic carbon contents and available N were well predicted by the DRS approach with R² values larger than 0.75. Moderate to poor estimation efficiencies were observed for remaining parameters. Nevertheless, the estimated soil attributes and their corresponding measured soil parameters were used as inputs to an input recommendation model of soil diagnosis to create input recommendation for a targeted cocoa yield of 1,000 kg dry cocoa beans ha^‐1 Resulting input recommendations were similar for both of these input sources (measured and DRS‐estimated) suggesting that the DRS approach may provide an easy way to create input recommendations.