Scientific research

An association has been identified between the extent of roasting and the amount of extractable protein from the cocoa bean, its nutritive value, and the sensorial quality of the liquor. Cocoa nibs from fermented seeds (Theobroma cacao L.) were precision-roasted at 150°C for 0, 30, 34, 38, 42, and 46 min and the protein fraction extracted. From the beginning of roasting, until minute 38, about 87% of the proteins were extractable, but the extractability substantially decreased to 72.7% at 42 min and to 65.3% at 46 min. Both total soluble protein determination and albumin concentration of the roasted nibs diminished slightly until minute 38, when acceptable sensory characteristics were obtained for the liquor. Both total nitrogen and capillary-electrophoretic separation and quantification of the albumin showed that the amounts of extractable protein in this fraction consistently followed a cyclic pattern until minute 42, irreversibly decreasing thereafter. Biological evaluation of the protein from the cocoa nibs roasted for the various times showed that at the point that the sensory rating approached that of a commercial liquor, the albumin content and nutritive value were still high. The findings suggest that with moderate, uniform roasting it may not be necessary to sacrifice the protein's biological value for the sensorial attributes of chocolate in a standard commercial roast.

Bean characteristics of Upper Amazon cacao (Theobroma cacao L.) progenies were compared with two control hybrids in a series of progeny trials in Ghana. Flavour was assessed using fermented and dried cacao beans. All but 8 of the 70 new hybrids being tested had bean weight >1.05 g which is acceptable to the chocolate manufacturers. Using Upper Amazon pollen parents led to increased percentage shell content of beans and a higher fat content of the nibs. A few of the inter-Upper Amazon hybrids were considered by the chocolate manufacturers to lack chocolate flavour. In general, the bean characteristics of the new hybrids did not differ significantly from the control crosses. Although the bean characters studied appeared to be largely genetically determined, they could also be influenced markedly by environmental factors. The chances of Ghanaian cacao beans falling outside the acceptable flavour range are not high provided that breeding is within the Amazonian Forastero group.

Among the methods of cocoa bean fermentation, heap method is the most common in small farms in Ghana and other West African countries. However, processing by this method requires a certain maiximum quantity of wet bean in order to generate heat in the mass sufficiently and thereby to ensure adequate fermentation. The size of heap often has a bearing on acidity of the processed beans, too large quanities often leading to higher acidity. The present experiment was taken up at the Cocoa Research Institute, Tafo, Ghana, to study the effect of heap sizes on temperature, pH and quality of beans.

Cocoa beans were fermented for 144 h using shallow wooden boxes at ambient temperature. Two major polypeptides were found to consist of the storage protein and an albumin fraction. The storage protein comprises two vicilin fractions with molecular weights of 47.1 and 39.2 kDa, and the albumin fraction has a molecular weight of 21.1 kDa. The degradation of vicilin fractions during the course of fermentation was visually detected by sodium docecyl sulphate-polyacrylamide gel electrophoresis. The albumin fraction was found to be the most resistant to proteolysis during fermentation. At the end of fermentation, the 39.2 kDa polypeptide was completely degraded but the 47.1 kDa polypeptide was still present at low intensity. The protein concentrations of 47.1 and 39.2 kDa polypeptides decreased from 1.74 to 0.03 µg and from 0.93 to 0.02 µg, respectively. The protein concentration of 46 and 46.5 kDa polypeptides increased from 0.06 to 0.34 µg and from 0.03 to 0.23 µg, respectively. This could be due to the result of the degradation products of the 47.1 kDa polypeptide.

Cocoa cotyledons contain vicilin (7S)-class globulin (VCG), a major storage protein. It is the native source of oligopeptides and free amino acids which have been identified as precursors of cocoa-specific aroma and are formed through proteolysis during fermentation. High-resolution electrophoresis of native proteins isolated from ripe, unfermented cocoa cotyledons harvested from different cultivars was used to determine genetic differences of the genotypes. Flavour differences have been reported to exist after standard fermentation in cocoa beans harvested from various genotypes. In this paper, SDS-PAGE and 2D IEF/SDS-PAGE polypeptide separation patterns are shown which were separately isolated from cotyledons of various genetic origins. Cotyledons from three cultivars belonging to genetically distant varieties and a hybrid, Criollo, Forastero and Trinitario, did not reveal any analytical identity differences of VCG subunit polypeptide bands. Additionally, proteins of cotyledons harvested from three of those clones which were reported to produce genotype-specific flavour differences in raw cocoa after standard fermentation, SCA 12, UIT1 and PBC 140, when analysed in the same way, did not indicate differences. Thus the cotyledon storage proteins from various genetically different cocoa trees are, within methodological limits, the same. We conclude that aroma differences in raw cocoa harvested from various genotypes are the result of other genetic, physiological or curing-related factors, but are not due to genetic differences of aroma precursors derived from storage proteins.

Acetone dry powder (AcDP) was prepared for six cocoa genotypes, namely Forastero (Amelonado type), Criollo, Trinitario, SCA 12, UIT1 and PBC 140. Hydrophobic oligopeptides were produced when autolysis of AcDP was carried out at pH 3.5. Comparative HPLC analysis showed that autolysis of AcDP from various genotypes revealed a similar pattern of oligopeptides. Most of the hydrophobic oligopeptides were not generated during autolysis of AcDP in the presence of protease inhibitor (Pepstatin A), indicating that the generation of these oligopeptides was due to the action of cocoa cotyledon aspartic endoprotease. This finding implies that the splitting action of aspartic endoprotease on vicilin (7S)-class globulin (VCG) from various genotypes was the same. The information from the study provides additional evidence that there are no obvious differences in VCG composition between various genotypes.

Cocoa beans are the principal raw material of chocolate manufacture. The beans are subject to a microbial fermentation as the first stage in chocolate production. The microbial ecology of bean fermentation (Forastero and Trinitario cultivars) was investigated at three commercial fermentaries in East Java, Indonesia by determining the populations of individual species at 12-h intervals throughout the process. The first 2-3 days of fermentation were characterised by the successional growth of various species of filamentous fungi, yeasts, lactic acid bacteria and acetic acid bacteria. The principal species found were Penicillium citrinum, an unidentified basidiomycete, Kloeckera apis, Saccharomyces cerevisiae, Candida tropicalis, Lactobacillus cellobiosus, Lactobacillus plantarum and Acetobacter pasteurianus. The later stages of fermentation were dominated by the presence of Bacillus species, mostly, Bacillus pumilus and Bacillus licheniformis. Glucose, fructose, sucrose and citric acid of the bean pulp were utilised during fermentation, with the production of ethanol, acetic acid and lactic acid that diffused into the beans. The filamentous fungi were notable for their production of polygalacturonase activity and probably contributed to the degradation of bean pulp.

Leucine and valine were reacted with fructose in a mixture of cocoa butter and water at different temperatures. The rate of the Strecker reaction is higher in cocoa butter-water than in water. In the presence of cocoa butter the two amino acids give some aldehyde also without sugar. The participation of cocoa butter in the production of flavor during the roasting of cocoa beans is therefore proposed.

Naturalized cocoa populations originating from the Oyapok and Tanpok basins in French Guiana were studied for their technological characters (bean count, fat content, purine content) and sensory characters (overall aroma intensity, cocoa flavour, acidity, bitterness, astringency, fruity or floral tastes, aftertaste, etc.), along with three controls (Amelonado and Ecuadorian varieties). The bean count in Guianan cocoa was higher than that of the controls, but it generally remained acceptable (below 100). Caffeine content was much higher than that of the Amelonado control. The overall aroma intensity and cocoa flavour of the chocolates made with the dry cocoa beans from Guianan trees were statistically superior to those of the industrial reference, the West African Amelonado. The other criteria studied, particularly the fat content, did not reveal any significant differences from the controls.

The relationship between perceived aroma and the volatile concentration measured in-nose was investigated during eating of a model food. Sensory ranking and time-intensity analysis (TI) were used to measure perceived aroma, while in-nose volatile concentration was monitored by atmospheric pressure ionization mass spectrometry, which produced time release data. A gelatine-sucrose gel with a range of gelatine concentrations (2-8% w/w) and flavoured with furfuryl acetate was used as the model food. Senosry scaling showed decreased flavour intensities and TI showed a decrease in the flavour perceived over time, as the gelatine concentration increased. Studies in model systems and in people demonstrated that the different rates of release observed for different gelatine concentrations were not due to binding of volatile to protein in the gel, nor to mucous membranes, but were due to different rates of gel breakdown in-mouth. There were no significant differences in the maximum in-nose volatile concentrations for the different gelatine concentrations, so the amount of volatile present did not correlate well with the sensory analysis. However, the rates of volatile release were different for the different gels and showed a good correlation with sensory data.

Volatile compounds in the aroma of five varieties of roasted and unroasted (raw) cocoa beans have been identified by mass spectral analysis and gas chromatography. The five common varieties selected for this study all contain the following compounds usually in this order of abundance: isovaleraldehyde, isobutyraldehyde, propionaldehyde, methyl alcohol, acetaldehyde, methyl acetate, n-butyraldehyde, and diacetyl. An additional eight compounds appear in smaller amounts. As evidenced by gas chromatographic analysis, the raw bean aroma contains the same components but in lower concentrations. The principal differences between varieties are shown to be due to the ratios of these compounds rather than new compounds. The effect of roasting period on the concentration of four aldehydes in the aroma of the ground bean is shown.

A survey of 56 cocoa farms in Ghana was carried out during the 1986 main crop to assess the influence of fermentation practices (post-harvest pod storage, cultivar, weight of ferments, number of turns and timing, drying) on quality. Variations in the frequency of turning of the ferments was noted with cocoa-producing region and cultivar. Sensory evaluation of chocolate samples made from the cocoa beans indicated that a short pod-storage and fermentation with a single turn after three days produced the most acceptable cocoa. The acceptability varied by region with the Eastern region producing the most acceptable cocoa. As with acceptability, chocolate flavour significantly improved with a short pod-storage time. A composite sample was average in terms of its sensory characteristics, supporting the concept that blending facilitates the balanced flavour characteristic of Ghana cocoa.

Conservation of cacao germplasm in the International Cocoa Genebank, Trinidad
The deliberate collection of exotic germplasm, begun in Trinidad by F.J. Pound, was for a very specific purpose. The main selection criterion was resistance to Witches' Broom disease of cacao. The collections, which were previously distributed over several sites in Trinidad, are now conserved at one site in Centeno, viz. the International Cocoa Genebank, Trinidad (ICG,T).
The genebank contains mainly collections obtained during Pound's expeditions to Peru (Upper Amazon Forastero) and Ecuador (Refractario). Smaller populations from Colombia (the Anglo-Colombian expedition of 1952) and from the Orienté of Ecuador are also represented. The original collections were later augmented by the LCT-EEN collections from Ecuador (1980-1985) and by those acquired by CRU's collecting expeditions to South America and Belize, Central America.

The collections presently conserved in the ICG,T include:
Imperial College Selections (1930-34)
Ecuadorian Refractario Collection (1937) - selected for apparent Witches' Broom disease resistance
Upper Amazon Collection (1937, 1942)
Anglo-Colombian expedition material (1952)
Ecuador Collection (1969-1973)
LCT-EEN Collection (1980-1985)
Cocoa Research Unit's Local Germplasm Collection (1991)
Caribbean Collections (Grenada - 1940's; Dominica, Martinique, Guadeloupe - 1986-1990)
Belize collection (1992, 1994, 1996)
French Guiana collection (1995)
Ecuadorian collection (2001)

The International Cocoa Gene bank, Trinidad, is an international cacao (Theobroma cacao L.) germplasm depository that conserves nearly 2500 accessions in its field collection. A portion of this germplasm was characterized for phenetic diversity with morphological descriptors from the International Board for Plant Genetic Resources cacao descriptor list. Data for 28 quantitative and 26 qualitative descriptors were obtained on 100 accessions representing 24 populations. Associations among the accessions were examined by hierarchical average linkage cluster analysis. Variances of the standardized values were computed for the quantitative descriptors. The diversity and evenness of the qualitative descriptors were assessed with the Shannon-Weaver diversity index (SWDI). The variances and the Shannon-Weaver diversity indices summarized the direct contributions of the quantitative and qualitative descriptors to the similarity measure. The variances of the standardized quantitative descriptors ranged from 0.03 for flower ligule length to 0.07 for fruit husk weight. About 75% of the fruit and bean descriptors had variances greater than 0.045, compared to 17% for the flower descriptors, indicating a relatively higher discriminative value of the former. Normalized SWDI values greater than 0.50 were obtained for 69% of the 26 qualitative descriptors. Eighty percent of the flower descriptors had SWDI values greater than 0.50, compared to 60% for those of the fruit and bean. Cluster analysis indicated rich phenetic diversity in this sample of germplasm. At the 75% level of similarity, the accessions were grouped in 11 clusters, each containing two or more accessions. Nine accessions were ungrouped. This diversity should prove useful for breeding programs. The observed link between geographic origin and accession grouping suggested that it is necessary to collect and conserve germplasm representing a broad geographic range.

Chemical reactions in cocoa seeds during fermentation and roasting may depend on post-mortem structural changes in the mesophyll cells. Aeration, temperature and acetic acid concentration vary considerably during commercial fermentation. Light and electron microscopic studies of seeds after artificial fermentations give evidence that the kind and the degree of subcellular structural changes depend on these variations. At 50°C in the presence of acetic acid (35 mM/litre, pH 4.0) water-containing compartments are destroyed shortly before the lipid vacuoles fuse. The hydrophilic particles of the plasm become dispersed within the lipid phase. These changes occur within 6-20 h independent of the presence of air. At 40°C in the absence of acetic acid (citric acid, pH 5.5) the seeds germinate and protein vacuoles in many cells of the mesophyll inflate considerably within 6h, which is before post-mortem structural changes, different from those following treatment in acetic acid at 50°C, become obvious. Incubation of cotyledon fragments instead of whole seeds submerged in buffer induced these structural changes as well and were even more pronounced. The significance of temperature differences in the range of 40-50°C and induction of germination during fermentation is discussed.

Characteristic post-mortem changes in subcellular structures are described which are caused by the penetration of acetic acid during incubation of cocoa seeds in aqueous media. In the storage cells lipid is agglomerated and separates from hydrophilic portions in proportion to the acetic acid concentration and pH. This effect is less pronounced at 50°C than at 40°C. In the absence of acetic acid or with very low concentration of undissociated acetic acid other substructural characteristics dominate in most of the cells. At 50°C Post-mortem changes do not induce lipid agglomeration. At 40°C the intact protein vacuole swells and its matrix structure becomes spongy as a response to in-vivo water absorption. Finally, it is shown that a concentration gradient persists in whole seeds for most of the time required for fermentation, because of the slow diffusion of acetic acid. The results are compared with temperature effects on subcellular structures and are discussed in relation to their significance for proteolysis in cocoa seeds during fermentation.

During anaerobic incubation in citric acid/NaOH (35 mmol; pH 5.5) at 40°C the fresh weight of ripe cocoa seeds (without testae) increases considerably (17-27% within 40 h) by water absorption. This is similar to water uptake during germination at 25-30°C. At 50°C in the same medium, weight increase (3.5-7.3% within 40 h) as well as net water uptake is low. Subcellular compartmentation is destroyed at 50 but not at 40°C. Since water uptake is greatly reduced or is impaired at 40°C in the presence of osmotically active substances like sucrose, it is concluded that intact vacuoles in the cotyledons are responsible for high water absorption. In the presence of acetic acid (200 mmol; 50°C) weight increase is low but loss of dry matter is increased by exudation. These findings are discussed with respect to protein vacuole swelling and proteolysis during fermentation and germination.

Investigations of proteolysis during anaerobic cocoa seed incubation have been extended by disc and SDS-gel electrophoretic protein analysis. Two protein bands (2.6 x 10⁴ and 4.4 x 10⁴ Dalton) were found to be vacuolar storage proteins, which accumulated during seed ripening (90 to 160 days after pollination) and which were specifically utilised during germination. Although the storage proteins are poorly soluble at pH 3.5-4.5, proteolysis during incubation of acetone dry powders is highest in this pH range. All proteins are digested at 50°C, pH 4.5. During seed incubation at 50°C, pH 4.5, however, the storage proteins are degraded preferentially although the cells are dead at 50°C. This degradation is increased by preincubation at 40°C instead of 50°C. The results are discussed in the light of structural peculiarities in the seed tissue and the possible role of specific endopeptidases and peptides in the formation of flavour precursors during fermentation.

Cocoa fermentations in Ghana and Trinidad as well as anaerobic fermentation-like incubations of fresh cocoa beans in Germany were carried out under controlled conditions. Samples of beans were taken during the course of these treatments and determinations were made as to acidification (pH, acetic acid content), proteolysis (free α-amino nitrogen, peptide nitrogen and SDS electrophoresis of the protein peptides) and flavour potential (gas chromatography of the highly volatile compounds, in particular isopentanal and organoleptic analysis after thin layer roasting). A positive correlation between acidification, proteolysis and the development of flavour potential during anaerobic fermentation can be demonstrated in principle. However, the flavour potential is increased if the temperature rise is comparatively slow in both normal fermentation and laboratory incubation. Strong acidification and high accumulation of amino acids and peptides were not essential for a good flavour potential. The isopentanal content proved to be a useful indicator of the progress of normal fermentation in the tropics. These findings can be interpreted on the basis of earlier results about germination-like processes in the protein vacuoles, pre- and post-mortem subcellular structures and the special characteristics of acetic acid diffusion. Conclusions which are relevant to the practice of cocoa fermentation are discussed in more detail.

In Malaysia, bean spreading was investigated as an alternative method to pod storage for preconditioning of pulp in order to reduce nib acidification during subsequent cocoa fermentation. Harvested pods were broken and the seeds were exposed to quick pulp surface drying either by spreading in full sunshine or by the use of a suitable drier. A decrease in the volume, water and sugar content was observed during spreading, which caused subsequent shallow box fermentation to run similarly to stored pod fermentation, showing a steep temperature increase, absence of an initial anaerobic phase, reduced acetic acid formation and a higher minimum pH value in the nibs. Irregularities in fermentations and practical problems are discussed.

Patent application lodged by Kraft Jacobs Suchard R&D, Inc.

The invention relates to a novel low-flavor cocoa, a method for its production and a use thereof. The novel low-flavor cocoa is obtainable from unfermented cocoa beans by a two step process. In the first step the unfermented beans are treated to destroy the cellular and subcellular structures and then in a second step they are subjected to an oxidation treatment. This method suppresses the formation of flavor and hence low-flavor cocoa is obtained which is e.g. useful as substitute for cocoa butter in the manufacture of chocolate and for the compensation of variations in the flavor intensity of untreated cocoa.

Excerpt from patent application:
It is the predominant view (cf. Belitz, Grosch, Lehrbuch der Lebensmittelchemie, 4th edition, 1992, p. 874, and Fincke, Handbuch der Kakaoerzeugnisse, 2nd edition, 1965, p. 321 et seq.) that most of the aroma compounds in cocoa are produced from so-called aroma precursors. These aroma precursors are produced from non aroma constituents of the unfermented cocoa beans by enzymatic reactions during the course of fermentation. Thus, fermentation is of particular importance for the flavor development. During the further treatment of cocoa, such as drying, roasting and conching, the precursors and aroma compounds undergo at least in part further quantitative and/or qualitative changes.

Various trials were made in the past to intensify the cocoa aroma and to reduce foreign aroma and taste notes by treating the cocoa nibs, cocoa beans and cocoa seeds. In this regard, attention should be drawn for example to W097-33484, EP-A-755632, EP-A-749694 and EP-A-102668.

The aim of this study was to determine the levels of nine phenols (phenol, guaiacol derivatives, xylenols, and cresols) by using a GC/MS technique in smoked samples under control, commercial samples taken from contaminated batches, and uncontaminated samples. The smoky taste of cocoa powder may have two origins: drying and storage. The sample was steam distilled, extracted with ethyl ether, concentrated, and chromatographed on a 50-m OV-351 fused silica column. The physicochemical data obtained were related to sensory evaluation and submitted to multivariate distribution (Mahalanobis distance) and Pearson Chi-squared test. The discriminatory phenols identified were phenol, 3-methylphenol (m-cresol), 2,3-dimethylphenol (2,3-xylenol), 3-ethylphenol, 4-ethylphenol, and total phenols. The results obtained imply that uncontaminated smoked cocoa samples should have the following maximum concentrations: phenol, 2 mg/kg; 3-methylphenol, 0.9 mg/kg; 2,3-dimethylphenol, 0.55 mg/kg; 3-ethylphenol, 0.90 mg/kg; 4-ethylphenol, 0.70 mg/kg; and total phenols, 9.6 mg/kg.

This paper describes the evaluation of purine alkaloids and diketopiperazines (DKPs) contents in 24 samples of processed non-alkalinized cocoa powder. The chemical data obtained were related to sensory evaluation and subjected to analysis of variance. During cocoa roasting, the content of DKPs can be increased, resulting in a negative influence on the sensorial quality of cocoa. A pronounced bitter metallic taste can originate from some of these compounds, and becomes stronger when these DKPs are combined with purine alkaloids. The cocoa roasting process at different temperatures (125, 130, 135, 140 and 145 °C) and durations (3, 10, 16 and 20 min) was evaluated by measuring the flavour indices, formol numbers and DKP contents in 24 samples of cocoa powder. The addition of reducing sugars was also evaluated in the same industrial processes in 27 other samples of cocoa powder. The higher the roasting temperature of the cocoa was, the more elevated the flavour index became, except at temperatures up to 140°C, where DKPs were generated in large proportions.

Most of the volatile compounds identified are highly significant in determining cocoa powder flavour, and this paper demonstrates that basic sensory perceptions (undesirable, bitter pungent, repulsive, fruity, nutty, floral, vegetal, and sweet chocolate) can be totally explained by aroma compounds with R2-adjusted values of 0.85 and greater.
Samples from five geographical origins of cocoa bean were characterized by chemical compounds and sensory attributes. The aroma extracts were obtained by a two-step procedure involving (1) preliminary steam distillation under reduced pressure to evaluate the methylpyrazines generated in roasted cocoa powder by spectrophotometry (flavour index), and (2) Likens-Nickerson's simultaneous steam distillation and solvent extraction method with added NaCl. The distilled compounds were separated by adsorption chromatography in six fractions depending on the polarity.
A combined total of 114 compounds were detected by gas chromatography/mass spectrometry, 110 of which were identified. About 15 components in the mean milligrams per kilogram range (1.09-4.67 mg kg^-1) and 95 components in the mean micrograms per kilogram range (12-980 µg kg^-1) were quantified.

The major components of cocoa aroma (mean greater than 1.30 mg kg^-1) were:
2,3,5,6-tetramethylpyrazine,
benzaldehyde,
2-phenylacetaldehyde,
acetophenone,
3-methylbutyric acid,
5-methyl-2-phenyl-2-hexenal,
ethyl phenylacetate, and
3-hydroxy-2-methyl-4-pyrone.

Cocoa sweatings, the pale yellowish liquid that drains off during cocoa fermentation, is the breakdown product of the mucilage surrounding the fresh cocoa bean, and constitutes about 10% of the weight of the cocoa fruit. On average, about 1.9 million l of sweatings are produced annually in Ghana during the cocoa harvesting season. It has been shown to be a suitable medium for the production of wines, alcohol, marmalade, jam and syrup. Its rapid collection in high yields and quality is the first step to its utilization on a commercial scale. Thus pure yeast culture fermentation of cocoa under controlled temperature conditions and its effect on yield of sweatings and final cocoa bean quality was investigated. Cocoa fermentations employing Saccharomyces chevalieri or Kluyveromyces fragilis alone gave significantly higher yields of sweatings (p 0.05) than controls. The initial rates of sweating by the two strains were also very high but dropped to a constant minimum value after 12h of fermentation. In contrast, fermentations employing Torulopsis candida or Candida norvengensis alone as well as different combinations of all the yeast strains did not give any significant difference in yield compared to controls (p 0.05). Fermentations using S. chevalieri alone or other combinations in which S. chevalieri was present gave beans with acceptable quality based on different quality indices used for grading cocoa beans commercially.

As previously reported the carboxypeptidase activity present in ripe, ungerminated Theobroma cacao seeds is involved in the proteolytic formation of the cocoa-specific aroma precursors. Therefore, we have investigated the specificity of this particular enzyme activity using crude homogenates of acetone dry powder prepared from unfermented, ripe cocoa seeds. Both oligopeptide mixtures derived from cocoa seed proteins and synthetic peptides have been found to be suitable substrates for this enzyme activity. However, peptides with carboxyterminal arginine, lysine or proline residues are resistant against degradation by the cocoa seed carboxypeptidase. The enzyme preferentially liberates hydrophobic amino acids, whereas acidic amino acids are released very slowly. The rate of hydrolysis is not only determined by the carboxyterminal, but also affected by the neighbouring amino acid residue. Furthermore, the specificity of the enzyme is influenced by the pH-value. The specificity of the cocoa seed carboxypeptidase activity is remarkably similar to the specificity of carboxypeptidase A from porcine pancreas which (in addition to the cocoa aspartic endoprotease) has recently been successfully used for the in vitro formation of the cocoa-specific aroma precursors. These results are discussed in the light of the pH-dependent generation of the cocoa-specific aroma precursors during fermentation of the cocoa seeds.

(PhD thesis for Pennsylvania State University)

Chocolate has recently gained attention for its high levels of flavanol antioxidants. These compounds, implicated in protection against cardiovascular disease, inflammation, and cancer, have become the object of a large quantity of research that seeks to fully appreciate the significance of flavanols for human health. Continuing in that vain, this thesis aims to recognize the bioactive role of flavanols in chocolate, but points out that flavanols are important not only in terms of human health, but also to other issues in chocolate production. Flavanols contribute to chocolate flavor and may also offer disease resistance to cacao seeds prior to harvest.
Both flavor and disease resistance are difficult to breed for and a better understanding of their association through flavanols could greatly assist in more efficient management of the crop. The goal of this thesis is to develop a marker for economically important traits through these central flavanol compounds. Since pigments in fresh cacao seeds are flavanols themselves, the thesis proposes that fresh seed color may be a ready indicator of native flavanol content in the fresh seed.
Two hundred seeds from 14 different varieties, chosen to cover the full range of color, were studied in order to develop a marker for flavanol content. Seeds were assessed in terms of light reflectance properties and mapped on scales of observable color based on a standard human observer. The measurements were compared to a chemical characterization of color based on anthocyanin concentrations from individual seed extracts. Through HPLC-MS, four anthocyanin pigments were identified, two of which were previously unknown in cacao. A significant correlation was found between total anthocyanin concentration and lightness of the seed. Additional variation in seed color was explained through in vivo effects such as copigmentation and pH of the cell environment.
In order to assess the ability of color to act as a marker for flavanols, concentrations of other flavanols (catechins, and procyanidins) were measured as well. Seeds were found to contain total extractable flavanol concentrations ranging from 1.25 to 26 µg/ml catechin equivalents per gram seed dry weight. A positive relationship was uncovered in the sample set between procyanidin and anthocyanin concentrations recorded for seeds. This trend was consistent for subsets of the data grouped by pod, with the exception of one pod whose seeds showed a negative relationship between anthocyanins and procyanidins. Apparently, all flavanol subspecies are coregulated in seeds throughout the species, although there is opportunity for exceptions or fine-tuning mechanisms.
In testing observable color as a predictor for flavanol content, a statistically significant relationship was established. Lightness of the outer surface of the seed was found to be the best measure of the flavanol content, with lighter seeds containing low levels of flavanols, and darker seeds containing higher concentrations. Colorimetric measurements of seed lightness can be used to estimate flavanol concentrations to within ± 4 µg/mL catechin equivalents per gram seed dry weight (on average).

Fermented cocoa beans (Theobroma cacao L., Sterculiaceae) from different countries of origin (Ecuador, Ghana, Trinidad) and cocoa beans roasted under defined conditions (industrial roasting; 150-220°C for 20 min, dry roasting in conventional oven) were analyzed for their contents of certain chiral hydroxy acids, catechins, and amino acids. Cocoa beans are fermented, dried, and industrially transformed by roasting for the production of chocolate, cocoa powders, and other cocoa-related products. Fermentation and roasting conditions influence the contents of chiral compounds such as hydroxy acids, amino acids, and polyphenols, depending on technological procedures as well as some technical parameters. The aim of this work was to check if the content and nature of the named chiral compounds present both in fermented and roasted cocoa beans could be related to the traditional parameters used to classify the variety of seeds and the degree of fermentation. The extent of racemization of amino acids in fermented cocoa beans was low while it slowly increased during roasting, depending on the temperature applied. L-lactic acid was always higher than the D-form while citric acid was generally the most abundant hydroxy acid detected in beans. A correlation was found between polyphenol content and degree of fermentation, while epimerization of (-)-epicatechin to (+)-catechin was observed during roasting. On the whole, results showed that several chiral compounds could be considered as good quality markers for cocoa seeds and cocoa-related products of different quality and geographic origin.

The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as "Weissella ghanaensis", was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named "Acetobacter senegalensis" (A. tropicalis-like) and "Acetobacter ghanaensis" (A. syzygii-like).

The influence of turning and environmental contamination on six spontaneous cocoa bean heap fermentations performed in Ghana was studied through a multiphasic approach, encompassing both microbiological (culture-dependent and culture-independent techniques) and metabolite target analyses. A sensory analysis of chocolate made from the fermented, dried beans was performed as well. Only four clusters were found among the isolates of acetic acid bacteria (AAB) identified: Acetobacter pasteurianus, Acetobacter ghanensis, Acetobacter senegalensis, and a potential new Acetobacter lovaniensis-like species. Two main clusters were identified among the lactic acid bacteria (LAB) isolated, namely, Lactobacillus plantarum and Lactobacillus fermentum. No differences in biodiversity of LAB and AAB were seen for fermentations carried out at the farm and factory sites, indicating the cocoa pod surfaces and not the general environment as the main inoculum for spontaneous cocoa bean heap fermentation. Turning of the heaps enhanced aeration and increased the relative population size of AAB and the production of acetic acid. This in turn gave a more sour taste to chocolate made from these beans. Bitterness was reduced through losses of polyphenols and alkaloids upon fermentation and cocoa bean processing.

(Abstract not publicly available)

Dietary polyphenols show a great diversity of structures, ranging from rather simple molecules (monomers and oligomers) to polymers. Higher-molecular-weight structures (with molecular weights of > 500) are usually designated as tannins, which refers to their ability to interact with proteins. Among them, condensed tannins (proanthocyanidins) are particularly important because of their wide distribution in plants and their contributions to major food qualities. All phenolic compounds are highly unstable and rapidly transformed into various reaction products when the plant cells are damaged (for instance, during food processing), thus adding to the complexity of dietary polyphenol composition. The polyphenol composition of plant-derived foods and beverages depends on that of the raw material used but also on the extraction process and subsequent biochemical and chemical reactions of plant polyphenols. The occurrence of specific tannin-like compounds (ie, thearubigins and theaflavins) arising from enzymatic oxidation is well documented in black tea. Various chemical reactions involving anthocyanins and/or flavanols have been demonstrated to occur during red wine aging. Current knowledge regarding the reaction mechanisms involved in some of these processes and the structures of the resulting products is reviewed. Their effects on organoleptic and nutritional quality are also discussed.

On astringency: [the] conversion of grape tannins to new products, particularly tannin-anthocyanin adducts, is generally reported to reduce wine astringency (Somers, 1971*). This was classically ascribed to an increase in molecular weight, because larger tannins were thought to be insoluble and thus non-astringent. However, recent studies showed that higher molecular-weight proanthocyanidins are both soluble and more astringent than the oligomeric proanthocyanidins (Vidal et al, 2003). Consequently, the decrease in astringency observed during wine aging is likely to involve acid-catalyzed processes leading to lowermolecular- weight species, rather than polymerization reactions. However, the taste of polyphenol reactions products and the effect on astringency of incorporating anthocyanin units into a tannin structure remain to be investigated.

* Somers, T. (1971). The polymeric nature of wine pigments. Phytochemistry, 10, 2175-2186 (abstract not publicly available)

From the book, on the subject of olfactory fatigue:
Chapter 28 - Otolaryngologic Principles - by William K. Chiang - p.420
Olfactory fatigue is the process of olfactory adaptation following exposure to a stimulus for a variable period of time. This leads to a temporal diminution of the smell. Unfortunately, this adaptation may lead to a false sense of security with continued exposure to a toxin. For example, hydrogen sulfide [...] is readily detectable as a distinct and offensive substance at the very low concentration of 0.025 ppm. At the higher and potentially toxic concentration of 50 ppm, the odor is less offensive, and recognition may disappear after 2-15 minutes of exposure. At an even higher concentration, when toxicity is likely, the onset of olfactory fatigue is even more rapid. The combination of rapid onset of olfactory fatigue and toxicity at high concentrations of hydrogen sulfide exposure has contributed to numerous fatalities.

The outstanding position of the Republic of Venezuela, as center of evolution of the best commercial cacaos, is emphasized. The taxonomy of the species of the genus Theobroma is briefly discussed, together with the systematic status of cultivated cacaos. The morphological characteristics of cacao pods and grading of fresh cacao beans are summarized and the regions of cacao culture in Venezuela listed. The evolution of cacao populations of Venezuela are discussed from the poorest (Amazonian Forastero), very near Calabacillo, to the highest (Red-shelled or Venezuelan Criollo and Green-shelled Criollo). In the past only Criollos were cultivated, at least in the Central and Western regions of Venezuela. The Amazonian Cacao has been introduced or diffused, and crossed with Criollos so that this speciation of cacao, marked by an increase of Criollo "blood" is now evident: Orinocan Forasteros, Trinitarian Forastero and Venezuelan Forastero. In addition, Porcelain [Porcelana] cacao (supposed to be an albino mutant of Amazonian Forastero or Calabacillo) and Porcelain x Criollos hybrids ("Criollain") are discussed. The actual evolution in the cacao population is deduced from the culture of the Central sector to the Andine regions.

The possibility of genetic effects on cocoa (Theobroma cacao L.) flavour was investigated. Consistent differences in flavour attributes, especially cocoa flavour intensity, acidity, sourness, bitterness, and astringency, were found among the West African Amelonado variety (AML), four Upper Amazon clones [Iquitos Mixed Calabacillo 67 (IMC67), Nanay 33 (NA33), Parinari 7 (PA7), and Scavina 12 (SCA12)], and a Nicaraguan Criollo (UIT1) grown in Sabah, Malaysia. The flavour of UIT1 was distinctly different from the West African standard, being characterized by intense bitterness and astringency associated with caffeine and polyphenols. These attributes were ameliorated by prolonged storage of the pods before processing the wet beans. The six genotypes differed also in bean size and butter fat content. The differences in flavour were independent of the differences in bean size. The results demonstrated a significant contribution of genotype to flavour in addition to effects of processing.

From Luna et al, 2002: "The investigations of Clapperton et al demonstrated a genotypic effect on sensory attributes such as astringency, bitterness, and cocoa flavor intensity. They showed a link between polyphenols, astringency, and cocoa flavor intensity and between alkaloids and bitterness intensity."

Cocoa and chocolate manufacturers buy beans for colour, fat and flavour. This paper deals with the contribution of planting material to these qualitative characteristics. All three could be tuned to manufacturers' requirements by the traditional processes of selection and plant breeding. Selection and breeding cannot realistically produce any more than a five point increase in fat content over the current average of about 55%. It is better to consider initially yields of cocoa butter per hectare, or better still, per unit labour cost, and select high yielding, disease resistant and easily managed planting material. Trials which have demonstrated clear and consistent effects of pollen donor on bean size and colour have shown no corresponding effect on flavour. Flavour characteristics are heritable but are expressed in fruit from the first generation of seedlings, not in the beans that result from the initial act of pollination. Flavour is the property of the mother tree alone, thereby facilitating selection. The use of a small scale flavour test which allows parents and individual progeny to be compared directly has shown that flavour characteristics may be segregated among progeny.

Notes on possible non-genetic factors affecting flavour outcomes:
"There was more variability in flavour development in certain genotypes, e.g. PA 13, KA 5-201 and ICS 95, than in others. Variable flavour development does not appear to be related to one or other of the fermentation media. [...] It is possible that an unrecognised, or unrecognisable, variation in the condition of the pods which were selected for the tests may account for these differences in flavour. The use of fewer pods for a flavour test makes selection for uniformity of ripeness and other conditions more critical."

"The intensity of the [floral/fruity] characteristic may differ between progeny although instances where the flavour was clearly present in two of the replicate preparations but absent in the third sounds a note of caution. Further testing and replication are required and are in progress."

Knight and Rogers (1953, 1955) postulated the existence of five incompatibility alleles, at a single locus, in the three self-incompatible but cross-compatible cacao genotypes studied by them. The five alleles differed in dominance, but two were equal; the same dominance sequence, S 1 > = S 2 > S 3 > S 4 > S 5 was exhibited in both male and female parts of the flower. Their criterion of compatibility and incompatibility was the retention or fall, respectively, of hand-pollinated flowers guarded from insect visits.

The flavor of eight cocoa liquors of different origins (Africa, America, and Asia) and different varieties (Fine grades: criollo, trinitario, and nacional. Bulk-basic grade: forastero.) was analyzed by headspace solid-phase microextraction mass spectrometry (HS-SPME-MS). Their procyanidin contents were quantified by HPLC-UV (280 nm). Fine varieties with short fermentation processes proved to contain more procyanidins, while criollo from New Guinea and forastero beans showed the highest aroma levels. The levels of cocoa aroma compounds formed during roasting are shown to vary directly with bean fermentation time and inversely with residual procyanidin content in cocoa liquor. Measurement of antioxidant activity in cocoa liquor proved to be a useful tool for assessing residual polyphenols.

BACKGROUND: The association between nasal allergy and loss or diminution of smell is frequently alluded to in the literature; however, neither the true prevalence of hyposmia in individuals with allergic rhinitis nor its bases have been established.
METHODS: We assessed olfactory threshold for phenylethyl alcohol in 91 patients with symptoms of allergic rhinitis and 80 nonatopic control subjects. To determine the degree to which nasal congestion contributes to hyposmia in allergic rhinitis, total nasal resistance was measured in 64 of the patients and 72 of the control subjects.
RESULTS: Olfactory thresholds were significantly higher in allergic patients than in control subjects (p < 0.001), with 23.1% of the patients demonstrating a clinically significant smell loss (defined as threshold at or above the 2.5th percentile of control values). Although nasal resistance was significantly higher among patients than among controls (p < 0.001), it was not related to olfactory threshold in either group. Clinical or radiographic evidence of sinusitis or nasal polyps or both in allergy patients was found to be significantly associated with hyposmia (p < 0.006).
CONCLUSIONS: The observed prevalence of hyposmia among patients with allergic rhinitis suggests that this is a major etiologic factor contributing to smell disorders. Sinusitis or nasal polyps or both may underlie many cases of allergy-related hyposmia.

It has been suggested in the literature that aldehydes are produced from the amino acids in roasting cocoa beans by the Strecker degradation. [U-14C]-L-Leucine [U-14C]-L-3-phenylalanine and [U-14C]-L-threonine-were used in an investigation to confirm this mechanism. The labelled amino acids were introduced into ripe cocoa beans which were then fermented, dried and roasted under conditions approaching those of normal practice. The carbonyl compounds were collected as 2,4-dinitrophenylhydrazones and identified by thin-layer chromatography (t.l.c.) and autoradiography. The major products from [14C]-L-leucine and [14C]-L-3-phenylalanine were isovaleraldehyde and phenylacetaldehyde respectively, which are the aldehydes expected from a Strecker degradation. Some condensation products and other radioactive carbonyl compounds were also noted. The degradation of threonine appeared to occur early in the roasting process and to be more complex. Acetaldehyde was identified and it is suggested that this was produced via the Strecker aldehyde.

Darwin, C. (1859)
The Origin of Species

Excerpt from CHAPTER II - VARIATION UNDER NATURE, regarding the differences between varieties, species, and genera:
"[...] what are varieties but groups of forms, unequally related to each other, and clustered round certain forms -- that is, round their parent-species. Undoubtedly there is one most important point of difference between varieties and species, namely, that the amount of difference between varieties, when compared with each other or with their parent-species, is much less than that between the species of the same genus. But when we come to discuss the principle, as I call it, of divergence of character, we shall see how this may be explained, and how the lesser differences between varieties tend to increase into the greater differences between species."

Cocoa seeds and pulp were fermented for 144 h, followed by natural drying. The tegument was removed and the cotyledons were broken into nibs which were roasted at 150°C for 30 min. Non-fermented material, material fermented for 24, 48 and 72 h, material fermented for 144 h and then dried, and also the roasted nibs, were all prepared for chemical and microscopic analyses. Light microscopy revealed the presence of anionic and cationic residues and of neutral sugars. During fermentation there was a reduction in the cytoplasmic content of phenolic compounds and in the number of protein bodies. The cell wall showed a reduction in anionic residues and a loss of crystallinity. These alterations were maximum after 72 h. Drying and roasting increased the number of damaged cells and reduced the amount of cytoplasmic material. The chemical analyses generally confirmed the microscopy results. The concentration of amino-terminal groups and total free amino acids increased during fermentation (up to 72 h), but returned to the initial values after roasting. The principal chemical changes were related to reducing sugars, free amino acids, proteins and phenols, and PCA was suggested as a useful tool to compare different samples. Microscopic analysis revealed the degradation of protein and phenolic bodies and cellular damage during roasting.

Protein hydrolysis using an exogenous protease on cocoa nibs was performed to verify the formation of precursors and the effect on cocoa flavour.
An experimental design was used to check the influence of temperature (30 to 70°C) and enzyme : substrate ratio [E/S] (97.5 to 1267.5 U g^-1 of protein). The % Degree of Hydrolysis (% DH) was affected mainly by [E/S] leading to a 4-fold increase (from 5 to 20 %) after 6 hours of treatment. During cocoa nibs roasting, there was a greater consumption of hydrolysis compounds in the sample treated with protease as compared to the control, indicating their participation in the Maillard reaction. An increased perception of chocolate flavour and bitter taste was observed in a product formulated with protease treated cocoa.

Nacional cocoa from Ecuador is a genetic group acknowledged for its Arriba floral flavour, which has partly been lost due to the introduction of other more productive varieties. Between 1996 and 2000, 115 Nacional cocoa trees were identified by high yield, resistance to diseases and intense floral flavour, and reproduced by grafting. Some of the progeny are now fruiting and show resistance to Crinipellis perniciosa with an intense floral flavour.

The objectives of this study were the selection of a strain of Saccharomyces cerevisiae, the elaboration of a fermentative process using cocoa (Theobroma cacao L.) fruit pulp, and the assessment of the acceptance of the elaborated beverage. Three S. cerevisiae strains (CA116, CA1162 and CA1183) were assessed while growing in a fruit pulp medium at different temperatures. The ethanol:biomass and glycerol:biomass ratios showed that there were no significant differences among the three strains at different temperatures. However, the strain CA1183 reached a higher ethanol production and yield and it was chosen as a starter to produce the cocoa beverage. The concentration of higher alcohols, methanol, esters and acetaldehyde found in the elaborated beverage was in accordance with the standards established for table wine. Sensory analysis revealed a high degree of acceptance amongst the great majority of tasters. It can be concluded that pulp processing into an alcoholic beverage is a realistic additional way of utilisation of the cocoa fruit.

Genetic distances among cacao cultivars were calculated through multivariate analysis, using the D2 statistic, to examine racial group classification and to assess heterotic hybrids. A 5 x 5 complete diallel was evaluated. Over a five-year period (1986-1990), five cultivars of the S1 generation, pertaining to the Lower Amazon Forastero and Trinitario racial groups and 20 crosses between the corresponding S0 parents were analyzed, based upon five yield components - number of healthy and collected fruits per plant (NHFP and NCFP), wet seed weight per plant and per fruit (WSWP and WSWF), and percentage of diseased fruits per plant (PDFP).
The diversity analysis suggested a close relationship between the Trinitario and Lower Amazon Forastero groups. A correlation coefficient (r) was calculated to determine the association between genetic diversity and heterosis. Genetic distance of parents by D2 was found to be linearly related to average performance of hybrids for WSWP and WSWF (r = 0.68, P < 0.05 and r = 0.76, P < 0.05, respectively). The heterotic performance for the same components was also correlated with D2, both with r = 0.66 (P < 0.05). A relationship between genetic divergence and combining ability effects was suggested because the most divergent cultivar exhibited a high general combining ability, generating the best performing hybrids. Results indicated that genetic diversity estimates can be useful in selecting parents for crosses and in assessing relationships among cacao racial groups.

Noted in results:
The cultivars studied included:
SIAL 169 - a Lower Amazon Forastero, originating from southern Bahia, and
CEPEC 1 - a spontaneous mutant with white seeds.

Over the 5 year period of this study, the mean yields of these cultivars were:
SIAL 169 - 3.76kg of wet seed per plant [or, 2.25 times the mean yield of the white-seeded cultivar]
CEPEC 1 - 1.67kg of wet seed per plant [or, 44.4% of the mean yield of the Lower Amazon cultivar SIAL 169]

We examined whether presenting an odor with a positive, neutral, or negative name would influence how people perceive it. In experiment 1, 40 participants rated 15 odors for their pleasantness, intensity, and arousal. In experiment 2, 30 participants passively smelled 10 odors while their skin conductance (SC), heart rate (HR), and sniffing were recorded. We found significant overall effects of odor names on perceived pleasantness, intensity, and arousal. Pleasantness showed the most robust effect of odor names: the same odors were perceived as more pleasant when presented with positive than with neutral and negative names and when presented with neutral than with negative names. In addition, odorants were rated as more intense when presented with negative than with neutral and positive names and as more arousing when presented with positive than with neutral names. Furthermore, SC and sniff volumes, but not HR, were modified by odor names, and the SC changes could not be accounted for by sniffing changes. Importantly, odor names presented with odorless water did not produce any effect on skin conductance and sniff volumes, ruling out the possibility that the naming-related findings were triggered by an emotional reaction to odor names. Taken together, these experiments show that there is a lot to a name, at least when it comes to olfactory perception.

Results are presented from a joint project between Sime Darby Plantations and Cadbury Ltd. to develop a raw cocoa processing method suitable for use in Malaysia, to produce cocoa beans equivalent to West African flavour. The results show that changing current Malaysian "industrial" practices so that they are closer to West African "individual grower" practices gives a significant flavour improvement. Pod storage, fermentation and drying were all identified as processing stages which influence flavour. Their optimum conditions for flavour improvement were established and practical ways of implementing these in the Malaysian estate situation were investigated and developed. The optimum processing method to produce Malaysian cocoa beans of flavour similar to West African beans involves storing pods for up to 12 days before splitting; fermenting beans for 5 full days with only a single turn after 48 hours; drying slowly by blowing ambient air through the beans for 80 hours to 20 per cent moisture and then blowing air at 60 centigrade grade until dry (8 per cent moisture). Practical considerations on the implementation of these novel processing methods are discussed and a number of operating advantages and opportunities highlighted

According to Hii et al, 2004: this study found that the dissipation of acetic acid is more efficient in natural drying as compared to the faster artificial drying where entrapment of acids causes high acidic flavour in cocoa beans.

The human eye color is a quantitative trait displaying multifactorial inheritance. Several studies have shown that the OCA2 locus is the major contributor to the human eye color variation. By linkage analysis of a large Danish family, we finemapped the blue eye color locus to a 166 Kbp region within the HERC2 gene. By association analyses, we identified two SNPs within this region that were perfectly associated with the blue and brown eye colors: rs12913832 and rs1129038. Of these, rs12913832 is located 21.152 bp upstream from the OCA2 promoter in a highly conserved sequence in intron 86 of HERC2. The brown eye color allele of rs12913832 is highly conserved throughout a number of species. As shown by a Luciferase assays in cell cultures, the element significantly reduces the activity of the OCA2 promoter and electrophoretic mobility shift assays demonstrate that the two alleles bind different subsets of nuclear extracts. One single haplotype, represented by six polymorphic SNPs covering half of the 3′ end of the HERC2 gene, was found in 155 blue-eyed individuals from Denmark, and in 5 and 2 blue-eyed individuals from Turkey and Jordan, respectively. Hence, our data suggest a common founder mutation in an OCA2 inhibiting regulatory element as the cause of blue eye color in humans. In addition, an LOD score of Z = 4.21 between hair color and D14S72 was obtained in the large family, indicating that RABGGTA is a candidate gene for hair color.

From the website of the University of Copenhagen's Faculty of Health Sciences:
http://healthsciences.ku.dk/focus/blueeyes/
New research shows that people with blue eyes have a single, common ancestor. Professor Eiberg and his team at the Faculty of Health Sciences have tracked down a genetic mutation which probably took place 6-10,000 years ago and is the cause of the eye colour of all blue-eyed humans alive on the planet today.
"Originally, we all had brown eyes", said Professor Eiberg from the Department of Cellular and Molecular Medicine. "But a genetic mutation affecting the OCA2 gene in our chromosomes resulted in the creation of a "switch", which literally "turned off" the ability to produce brown eyes". The OCA2 gene codes for the so-called P protein, which is involved in the production of melanin, the pigment that gives colour to our hair, eyes and skin. The "switch", which is located in the gene adjacent to OCA2 does not, however, turn off the gene entirely, but rather limits its action to reducing the production of melanin in the iris - effectively "diluting" brown eyes to blue. The switch's effect on OCA2 is very specific. If the OCA2 gene had been completely destroyed or turned off, human beings would be without melanin in their hair, eyes or skin colour - a condition known as albinism.

Raw cacao beans are heated in hot water or water vapor containing an acidic, alkaline or alcoholic substance without being subjected to fermentation or after being slightly fermented, whereby enzymes contained in the beans are inactivated, or microorganisms present on the beans are destroyed, nibs of the beans being thereby prevented from undergoing a color change. This method enables production of white cacao nibs, and it is thus possible to prepare white chocolates and other varieties of food having good flavor and taste by using such cacao nibs.

We studied the functional relationship between pollination intensity and fruit survival as well as the number of seeds per pod in the tropical tree Theobroma cacao L. on a Forastero Upper-Amazon clone (UPA 409) in Ivory Coast. Cutting the style 24 h after pollination allowed for counting the number of pollen grains deposited on a stigma without affecting fruit set and seed development. Forty-three pollen grains were necessary to reach 50% of maximum fruit set 28 days after pollination. Above 115 pollen grains, the proportion of developing ovaries reached a maximum of 88% 28 days after pollination and 75% at maturity. With fewer than 238 pollen grains per stigma, there was a close relationship between pollination intensity and number of seeds per pod; the pollen:seed ratio increased from 1.6:1 to 3.8:1 for PI increasing from 30 to 238 pollen grains. For higher pollination intensities, the average number of seeds per pod reached a maximum of 58. The relationship between pollination intensity and seed content was modelled. Results are consistent with the hypothesis that ovules attracted pollen tubes in a similar way regardless of whether or not they had already been reached by another pollen tube.

Excerpt from editorial foreword:
The subject of flavor and flavor ingredients, much like its close allies fragrance and perfumery, is still viewed by many through a veil of mystery. To contemporary practitioners of the "pure sciences", the subject of flavors is grossly misunderstood, suggestive of alchemy, and at times subject to condescending critiscism. No such indictments are further from realityw areas of research are more serious or embellished by more sound, scientific thought and sophisticated techniques.

Fermentation of Theobroma cacao L. seeds has been considered to be the most important factor influencing cocoa flavour and has accordingly received most attention. Development of a test procedure in which small amounts of wet cocoa seeds in net bags are inserted in fermentations of wet seeds from single or mixed genotypes has allowed the flavour potential of a wide range of genotypes to be examined. Flavour profiles of cocoa liquors from genotypes grown at BAL Plantations Sdn Bhd, Malaysia differed consistently for various replicate preparations indicating a strong genetic contribution to flavour. In the present study, similar flavour profiles were developed from cocoa from the same genotypes grown in Brazil and Malaysia and subjected to the same conditions of post-harvest processing. Genetic fingerprinting by Rapid Amplified Polymorphic DNA (RAPD) analysis confirmed that most of the genotypes tested for flavour in Brazil and Malaysia were the same, although there were also a few erroneous identifications. The results confirmed the contribution of genotype to cocoa flavour. There appeared to be a minor effect of environment on cocoa flavour development. Mis-identification of genotypes might be more common than is currently realized, and represent a problem for transferring results between various research programmes.

From the back cover:
For the best part of two centuries investigators have tried with varying degrees of success to identify the compounds which give roasted coffee its characteristic aroma and taste. The analytical methods and the state of progress in chemistry at the end of the 19th century did not allow for the separation, isolation and identification of the multitude of trace chemicals which are present in roasted coffee. By 1900, scarcely a dozen compounds had been identified. Since the beginning of the sixties, with the advent of gas chromatography and mass spectrometry, the number of identifications has increased tremendously. To date, 850 compounds have been identified in the flavor of roasted coffee and 300 in the smell of green coffee. In this work, the authors systematically review the non-volatile constituents of green coffee, including their structure, and discuss their important contribution as flavor precursors during the roasting process. They also trace the chronological discovery of the individual chemicals and critically examine the validity of their identification, highlighting the enormous progress, which has been realized during the 20th century and particularly in the last 40 years. For convenience, the constituents of green and roasted coffee have been distributed into chemical classes according to structure, systematic and empirical names, their CAS Registry Numbers and occasionally their FEMA classification. Comments are made on the origin or the formation during roasting of each individual compound.

A hierarchically structured vocabulary of mouth-feel sensations elicited by red wines has been produced. Represented as a wheel, this structured vocabulary should assist tasters in their interpretation and use of terminology relating to "in mouth" sensations produced by red wines. These terms and their definitions were generated by consulting the opinions of experienced wine tasters following exposure to an extensive range of commercial red wines. Logical relationships among the derived terms were formulated by analysis of "sorting data" provided by a combined group of experienced winemakers and wine-tasters.

Related study:
King et al (2003)
Effectiveness of the 'Mouth-feel Wheel' for the evaluation of astringent subqualities in British Columbia red wines

Red table wines of quality are characterized by pleasing and complex mouth-feel sensations, the most important of these being astringency. While a comprehensive set of terms has been developed over time to describe the flavor of red wines, an appropriate vocabulary describing the astringent sensations produced by these wines is not well defined. This paper presents a structured vocabulary derived by a panel of experienced wine tasters that can be used to describe the astringent sub-qualities of red wines. Multidimensional scaling of sorting data showed that an experienced panel and a group of skilled red wine-makers had similar interpretations of the relationships among the astringency terms. A tasting panel was successfully trained to identify and consistently rate the intensity of the astringent sub-qualities encountered in a set of one year old Shiraz wines. A novel approach of using finger touch standards to represent the astringent sensations experienced in the mouth was utilized.

As the largest class of natural products, terpenes [including linalool] have a variety of roles in mediating antagonistic and beneficial interactions among organisms. They defend many species of plants, animals and microorganisms against predators, pathogens and competitors, and they are involved in conveying messages to conspecifics and mutualists regarding the presence of food, mates and enemies. Despite the diversity of terpenes known, it is striking how phylogenetically distant organisms have come to use similar structures for common purposes. New natural roles undoubtedly remain to be discovered for this large class of compounds, given that such a small percentage of terpenes has been investigated so far.

We combined event-related functional magnetic resonance imaging (fMRI) with olfactory classical conditioning to differentiate the neural responses evoked during appetitive and aversive olfactory learning. Three neutral faces [the conditioned stimuli (CS+)] were repetitively paired with pleasant, neutral, or unpleasant odors [the unconditioned stimuli (UCS)] in a partial reinforcement schedule. A fourth face was never paired to odor [the nonconditioned stimulus (CS-)]. Learning-related neural activity, comparing unpaired (face only) CS+ stimuli with CS-, showed valence-independent activations in rostral and caudal orbitofrontal cortex (OFC). Medial OFC responded to the appetitive (app) CS+, whereas lateral OFC responded to the aversive (av) CS+. Within nucleus accumbens, neural responses showed divergent activation profiles that increased with time in response to the appCS+ but decreased in response to the avCS+. In posterior amygdala, responses were elicited by the appCS+, which habituated over time. In temporal piriform cortex, neural responses were evoked by the avCS+, which progressively increased with time.
These results highlight regional and temporal dissociations during olfactory learning and imply that emotionally salient odors can engender cross-modal associative learning. Moreover, the findings suggest that the role of human primary (piriform) and secondary olfactory cortices transcends their function as mere intermediaries of chemosensory information processing.

Episodic memory is often imbued with multisensory richness, such that the recall of an event can be endowed with the sights, sounds, and smells of its prior occurrence. While hippocampus and related medial temporal structures are implicated in episodic memory retrieval, the participation of sensory-specific cortex in representing the qualities of an episode is less well established. We combined functional magnetic resonance imaging (fMRI) with a cross-modal paradigm, where objects were presented with odors during memory encoding. We then examined the effect of odor context on neural responses at retrieval when these same objects were presented alone. Primary olfactory (piriform) cortex, as well as anterior hippocampus, was activated during the successful retrieval of old (compared to new) objects. Our findings indicate that sensory features of the original engram are preserved in unimodal olfactory cortex. We suggest that reactivation of memory traces distributed across modality-specific brain areas underpins the sensory qualities of episodic memories.

Background: Numerous studies indicate that flavanols may exert significant vascular protection because of their antioxidant properties and increased nitric oxide bioavailability. In turn, nitric oxide bioavailability deeply influences insulin-stimulated glucose uptake and vascular tone. Thus, flavanols may also exert positive metabolic and pressor effects.

Objective: The objective was to compare the effects of either dark or white chocolate bars on blood pressure and glucose and insulin responses to an oral-glucose-tolerance test in healthy subjects.

Design: After a 7-d cocoa-free run-in phase, 15 healthy subjects were randomly assigned to receive for 15 d either 100 g dark chocolate bars, which contained {approx}500 mg polyphenols, or 90 g white chocolate bars, which presumably contained no polyphenols. Successively, subjects entered a further cocoa-free washout phase of 7 d and then were crossed over to the other condition. Oral-glucose-tolerance tests were performed at the end of each period to calculate the homeostasis model assessment of insulin resistance (HOMA-IR) and the quantitative insulin sensitivity check index (QUICKI); blood pressure was measured daily.

Results: HOMA-IR was significantly lower after dark than after white chocolate ingestion (0.94 ± 0.42 compared with 1.72 ± 0.62; P < 0.001), and QUICKI was significantly higher after dark than after white chocolate ingestion (0.398 ± 0.039 compared with 0356 ± 0.023; P = 0.001). Although within normal values, systolic blood pressure was lower after dark than after white chocolate ingestion (107.5 ± 8.6 compared with 113.9 ± 8.4 mm Hg; P < 0.05).

Conclusion: Dark, but not white, chocolate decreases blood pressure and improves insulin sensitivity in healthy persons.

During the fermentation of cocoa (Theobroma cacao) mixtures, in proportions of 100/0; 75/25; 50/50 and 0/100 of criollo and forastero types cultivated in Cumboto, Venezuela, the effect of the blending on chemical properties was evaluated. Blends were fermented for 4 days in wooden boxes and cocoa mass was removed at 24 and 72 h after the initiation of the process. Analysis of humidity, tannins, proteins, pH, total acidity, reducing and total sugars were performed in the cotyledon and the pulp+testa at 0, 2 and 4 days of fermentation. The results revealed a similar behaviour of chemical parameters, during the fermentation of all blends. In cotyledons, humidity, tannins and total acidity increased while proteins, pH reducing and total sugars decreased. In the pulp+testa, tannins, proteins and pH, increased, humidity reducing and total sugars decreased, while total acidity decreased the second day and increased on the fourth day. In addition, variations of chemical characteristics of the blends were observed which were not related with the proportions of blending used and cannot therefore be attributed to the differences in the types of cocoas of the blends. They may be related to variations in the degree of maturity of the fruits as well as to the great diversity and heterogeneity of the materials which form the cocoa woods in the coastal region of Aragua State.

Cocoa seeds were collected from La Isleta, La Vega de Santa Cruz and El Paraiso plots of criollo, forastero amazonico and trinitario cocoas in Cumboto (Aragua, Venezuela), and the cotyledons and seed coat+pulp were analysed for chemical characteristics.

The cotyledons presented
49.52-52.24% fat,
35.8-36.87% humidity,
13.59-13.97% proteins,
7.62-8.07% total sugars,
3.59-3.67% ashes,
2.90-3.24% reducing sugars,
0.68-0.80% tannins,
0.31-0.35% total acidity and
pH of 6.35-6.39.

Seed coat+pulp had
78.00-80.02% humidity,
4.27-4.31% proteins,
35.99-46.04% total sugars,
17.30-19.14% reducing sugars,
12.47-14.15°Brix,
0.84% tannins
3.39-3.41% total acidity and
pH of 3.45-3.56.

The values obtained did not differ among the three cocoa types, except for the cotyledon's fat which presented the lowest value in the forastero and the highest value in the trinitario as well as humidity and total sugars of the seed coat+pulp which presented the lowest percentage in the trinitario. Similarities in characteristics among the plots were also observed; in the cotyledon the only differences were in fat (P>0.05) of the cocoa from La Isleta (49.34%), proteins from El Paraiso (12.78%) which were the lowest and reducing sugars from La Isleta (3.39%) which had the highest value. In the seed coat+pulp, differences occurred in the pH which was superior in El Paraiso (3.71) and total acidity of La Isleta (3.85%) which was higher than that of El Paraiso (2.91%). In conclusion, the cocoas of the three plots from Cumboto, showed similar chemical characteristics, indicating that materials from the area are not pure, but are hybridized and that most characteristics have not been significantly affected by plot handling and locality. Therefore, it may be concluded that the chemical characteristics evaluated, except for fat content, are not good indicators for detecting differences between cocoa types.

Representative condensed and hydrolyzable tannins and related simple phenolics were evaluated as biological antioxidants using cyclic voltammetry, the metmyoglobin assay, and the deoxyribose assay. The redox potentials of the tannins were similar to those of structurally related simple phenolics. However, the tannins were 15-30 times more effective at quenching peroxyl radicals than simple phenolics or Trolox. One of the tannins, polygalloyl glucose, reacted an order of magnitude more quickly with hydroxyl radical than mannitol. These results suggest that tannins, which are found in many plant-based foods and beverages, are potentially very important biological antioxidants.

The activities of endoprotease, aminopeptidase, carboxypeptidase, invertase (cotyledon and pulp), polyphenol oxidase and glycosidases were studied during heap fermentation of ICS-95 cocoa beans. These enzymes are of key importance in flavour precursor formation and in pigment degradation during cocoa fermentation. Optimal extraction and assay conditions were established to characterise the enzymes reactions and to quantify and compare enzyme activities during cocoa fermentation. The enzymes exhibited large differences in pH optima and stability during fermentation. Aminopeptidase, invertase (cotyledon and pulp) and polyphenol oxidase were strongly inactivated, carboxypeptidase was partly inactivated, whereas endoprotease and glycosidases remained active throughout the fermentation. Since many enzymes are inactivated during fermentation, it is generally recognised that the actual period of enzyme action is short. Although our results confirmed total inactivation for some enzymes, we show that several key enzymes are not completely inactivated during fermentation. Therefore, some enzyme reactions can continue throughout the whole fermentation process. Only polyphenol oxidase was strongly inactivated during sun and artificial drying of the beans. The other enzymes were stable during the drying process. Enzymes like endoprotease and glycosidases are still active in properly fermented and dried beans.

Using the penetration theory of interfacial mass transfer, mathematical models have been developed to describe flavor release from liquid foods in the mouth, which incorporates the effects of breathing and saliva dilution. It has been assumed that the rate-limiting step for flavor release is resistance to mass transfer across the liquid-gas interface. This model has been applied to two types of liquid foods: first, aqueous solutions containing aroma-binding macromolecules and, second, liquid oil-in-water emulsions. The initial rates of release were found to be independent of both the rate of saliva production and gas flow rates due to breathing. However, at slightly longer times the saliva flow rate greatly influenced the quantity of flavor available for perception in the headspace.

The free amino acids, peptide-N, reducing sugars (the flavour precursors in cocoa) and pyrazine profiles of mix hybrid cocoa beans fermented in a rotary drum reactor were monitored over a period of 6 days. As fermentation progressed, the acidic free amino acid concentration decreased significantly (P<0.05) by 15%, whereas total, hydrophobic and other free amino acids increased significantly by 148, 280 and 127%, respectively. In terms of hydrophobic/acidic/other free amino acids ratio, the unfermented cocoa beans contained 30%:18%: 52%, whereas those of fermented beans contained 46%: 6%: 48%. Concentrations of peptide-N and total reducing sugars were significantly (P<0.05) increased by 55 and 208%, respectively during fermentation; however, those of sucrose and total sugars decreased significantly (P<0.05) by 89% and 75%, respectively. The unfermented cocoa beans contained no pyrazine; however during fermentation, the 2-methyl-, 2,5-dimethyl-, 2,6-dimethyl-, 2,3-dimethyl-, trimethyl- and tetramethylpyrazine were formed. The two principle pyrazines were tetramethyl- (2099.30 g kg-1) and trimethylpyrazine (692.00 g kg-1).

A response surface methodology (RSM) was used to determine the optimum condition for mass and turning time during cocoa fermentation. Mass and turning time were used as independent variables; concentrations of free amino acids, peptide-N, sugars and pyrazines were the dependent variables. The R2 values for peptide-N, tetramethylpyrazine and total pyrazines were greater than 0·9. Both lower (10 kg) and higher (100 kg) cocoa mass together with lower (0 min) and higher (10 min) turning time gave products containing low concentrations of hydrophobic, total and other free amino acids, peptide-N, fructose, glucose and total reducing sugars; in contrast, those of acidic free amino acids gave higher concentrations. Trimethyl-, tetramethyl- and total pyrazines increased significantly (P<0.05) at higher mass (100 kg) and higher turning time (10 min). From the highest concentration of the important flavour precursors ie hydrophobic free amino acids, total reducing sugars and peptide-N, the recommended mass of cocoa beans and turning time for an optimum cocoa fermentation condition was 60 kg and 5 min, respectively.

The effect of drying time on free amino acid, peptide-N, sugar and pyrazine concentrations as well as the influence of bean depth and temperature on these compounds during cocoa drying was studied. Drying time, bean depth and temperature significantly decreased the concentration of free amino acids, peptide-N, total reducing sugars and sucrose and increased the concentration of trimethyl-, tetramethylpyrazine and total pyrazines in cocoa beans. The best drying treatment was obtained at the combination of bean depth/drying temperature of 8.3 cm/40°C. This was based on the fact that it produced significantly high concentrations of hydrophobic free amino acids, peptide-N and total reducing sugars and significantly low concentration of trimethyl-, tetramethylpyrazine and total pyrazines. Drying treatment of 1.5 cm/60°C significantly produced the lowest concentration of free amino acids, peptide-N and total reducing sugars and the highest concentration of pyrazines.

Excerpt from chapter 8.5 Cocoa
8.5.1 Fermentation
Acetic acid "then permeates the bean, lowering the internal pH to ~4.5 [...] when the death of the seed occurs the cells lose their structural integrity and with it the cell membranes lose their semi-permeability. The phenolic and purine metabolites are then able to slowly diffuse away from their sites of storage within the cell (often first noticed as the diffusion of pigments) [...] (i) Sucrose is converted to glucose and fructose by invertase [...] (ii) Polyphenol-oxidase converts the various phenolic metabolites [...]

8.5.2 Drying
[...] forced hot air drying may cause the shell of the bean to lose moisture preferentially. As a result the shells harden before all the water is lost and thereby the diffusion and loss of volatile acids from the beans is inhibited [...]

8.5.3 Roasting
Fermented beans "possess many of the flavour volatiles - such as alcohols and esters - but their aroma is not usually chocolate-like. [...] Almost all the reducing sugars and at least half of the amino acid content (either as free amino acids or small peptides) are lost in this roasting phase. [...]

'Good' cocoa liquors are not generally derived from cacao beans which possess too high a polyphenol content [...] Polyphenol-enzyme complexation invariably leads to a substantial decrease and eventually loss of enzymatic activity. It is therefore reasonable to suppose that at this point the rates of degradation of storage proteins and sucrose will steadily decrease [...] Depending on the availability of oxygen, the polyphenols themselves [will be] oxidised to quinones which will either form covalent bonds with the proteins (quinone tanning) or self-polymerise. With cacao beans which are rich in polyphenolic metabolites, these [enzymatic] reactions, essential to the devlopment of 'good'cocoa liquors, will therefore be arrested prematurely."

The southern border of Mesoamerica is traditionally drawn at the Ulua river of western Honduras, before dipping southward to include El Salvador, Pacific Nicaragua, and northwest Costa Rica. Recent work in the Department of Colon, Honduras, provides the earliest evidence of aboriginal occupation in the region and extends the established chronological sequence back more than a thousand years. A preliminary examination of the ceramics, and a comparison to other Preclassic sites, indicates that eastern Honduras, despite its later affiliation with Lower Central American cultural patterns, was probably participating in the cultural development and long-distance trade network of Early and Middle Preclassic Mesoamerican neighbors. Using ethno-historic analogy, the possibility of cacao as a Preclassic trade commodity is raised. Finally, it is suggested that the cultural frontier of Mesoamerica in the southeast be extended for the Preclassic time horizon.

The objective of this study is to assess the quality of cocoa beans produced by smallholders from different regions in Malaysia, such as Sabah, Sarawak, Perak, and Pahang. Most of the samples were obtained from beans fermented in plastic sacks, with a fermentation duration of 5 days and dried using natural technique in 4-5 days. Results showed no significant difference (p>0.05) among the bean samples for moisture content, pH, degree of fermentation (cut test score and fermentation index), and sensory evaluation (cocoa, bitterness, astringency, and sourness flavour attributes) based on the different locations. Cut tests showed that all the samples were well-fermented with percent brown beans of more than 60% which agreed well with the fermentation index. The quantity of slaty and purple beans was higher in the Sarawak and Pahang samples as compared to the Perak and Sabah samples. The presence of these beans in high amounts is not desired as unfermented and underfermented beans tend to introduce excessive bitterness and astringency in the finished products.
The pH values were mostly at the less acidic level (pH > 5.50) except for the Pahang samples which were at the medium level (pH 5.20-5.49). The pH values recorded were much less acidic than those reported by Jinap et al. (1995) between 4.64 and 4.85 for Malaysian cocoa beans. Highly acidic beans are associated with pH of less than 5.2.
Highly acidic beans are associated with low chocolate flavour, possibly due to over degradation of storage proteins.

Selected references:

Duncan et al, 1989
Improvement of Malaysian cocoa bean flavour by modification of harvesting, fermentation and drying method - found that dissipation of acetic acid is more efficient in natural drying as compared to the faster artificial drying where entrapment of acids causes high acidic flavour in cocoa beans.

Jinap, 1994
Organic acids in cocoa beans - A review - found that the best flavoured West African beans usually have pH values around 5.5.
Jinap, 1995 - found that naturally dried cocoa beans have been reported to have better flavour quality as compared to artificially dried beans due to the gentle drying process. Also, that chocolate made from medium pH beans received a higher response in strong chocolate flavour than those made from low or high pH beans.

Shahrir and Mamot, 1987
Determination of fermentation Index (FI) and its application to cocoa quality and grading - found that the colour of unfermented beans is generally slaty, while underfermented beans are purple, and fully fermented beans are brown in colour.

In this study fermented cocoa beans were dried in a direct solar dryer at three levels of loading (20, 30 and 60 kg). The drying platform was constructed with 1 cm thick plywood and measured 153 x 91.5cm. Surface mouldiness was found to be heavy in the 60 kg treatment, with beans appearing blackish. All the dried beans were reasonably acceptable in terms of vinegary odour and weak in alcohol odour. Weak odour was also detected for the faecal, rancid and cheesy odours. The 60 kg treatment was rated strong for wet sock odour due to poor drying condition. A significant difference (P < 0.05) was found between the 60 kg treatment and the lower loading treatments for pH and titratable acidity. A cut test showed that the lower loading treatments resulted in a higher percentage of brown beans. The 20 kg treatment showed the highest cut test score, which is significantly different (P < 0.05) from the 60 kg treatment. Fermentation index also showed a tendency for lower loading treatments to have a higher index. No significant difference (P > 0.05) was found among the treatments in terms of cocoa, astringency, bitterness and sourness flavour notes.However, better flavour was observed for beans from the 20 kg treatment. No mouldy off flavour was found in any of the dried beans. Overall quality assessment showed that the 20 kg treatment was able to produce reasonably good-quality beans as compared to other loadings and therefore is recommended for the direct solar dryer.

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) 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.

Handbooks for Genebanks: No. 4. International Plant Genetic Resources Institute, Rome

Excerpt:
Roberts (1973) introduced the terms "orthodox" and "recalcitrant" for the two categories of seed storage behaviour he identified. According to Roberts, orthodox seeds can be dried to low moisture contents (2-5%) without damage. In addition, their longevity increases with decreases in seed storage moisture content and temperature in a quantifiable and predictable way over a wide range of storage environments. In contrast, recalcitrant seeds cannot survive desiccation below a comparatively high (between 12 and 31%) moisture content. p.15

[Theobroma cacao has] "tropical-recalcitrant" seeds that are sensitive to damage from both desiccation and exposure to cool temperatures of 10-15°C or less. p.15

The "lowest safe moisture content" for cocoa (Theobroma cacao) is 23% (Mumford & Brett, 1982) p.28

Dark semi-sweet chocolate samples, obtained at various times during conching, were cooled and prepared for examination by scanning electron microscopy (SEM) in an attempt to observe structural changes during conching. In addition, ingredient mixtures containing known amounts of cocoa solids, microcrystalline cellulose, starch, sucrose and lecithin were studied to correctly identify the components of chocolate. Pulverized sucrose particles were easily characterized by their smooth surfaces and heterogeneous shapes. Defatted chocolate liquor was rougher in appearance and smaller in size than sucrose. Cellular detail of the cocoa bean was evident in liquor even after the disruptive effect of refining. Lecithin, when added to liquor-sugar and cocoa butter-sugar mixtures, had a remarkable and rapid effect in reducing three-dimensional structure. When processed chocolates from four different conches were examined by SEM, little or no differences were observed in the size or shape of the sugar or liquor particles as conching proceeded. Surface smoothening, however, was noted and attributed to the homogenization or spreading of cocoa butter during conching. When lecithin was added to chocolate during conching the surfaces were reduced and appeared to flatten. The effect of lecithin in reducing surface tension was also supported by viscometry. Viscometric measurements taken at this time showed that the increase in fluidity was accompanied by a sharp and rapid decrease in plastic viscosity.

The existing quality assessment methods for cocoa such as the cut-test and sensory test were evaluated. The results were compared with the fermentation index and color measurement as indirect quality assessment methods. Results of the cut-test score and sensory evaluation methods indicated variability in results and therefore were not adequate to assess bean quality. The fermentation index method was useful to distinguish different bean samples from 5 color categories. The fermentation index related well with other quality parameters, such as pH, reducing sugars, free amino acids, and cocoa color pigments. Direct color measurement had a good relationship with fermentation index, pH, reducing sugars, and free amino acids, as evidenced by high correlation values. Color measurement was appropriate to estimate cocoa bean quality.

Project carried out on behalf of ICCO (the International Cocoa Organization).

The definition of fine or flavour cocoa remains controversial as there is no single universally-accepted criterion that could be adopted as a basis for determining whether or not cocoa of a given origin is to be classified as fine or flavour cocoa. Relevant criteria include the genetic origin of planting material, morphological characteristics of the plant, flavour characteristics of cocoa beans produced, chemical characteristics of cocoa beans, colour of the cocoa beans and nibs, degree of fermentation, drying, acidity and off-flavours. Points are awarded or subtracted by the quality assessors, depending on the condition of the cocoa beans in relation to the above criteria. However, the measurement of these criteria does not reflect objectively the cocoa quality in terms of taste or flavour, causing chocolate manufacturers problems in standardizing their products. This difficulty is shared also by traders on the international market, who base their decisions mainly on the degree of fermentation and genetic origin of cocoa.

The main objective of this project was to develop the capacity of all involved in the cocoa sector to adequately differentiate between fine and bulk cocoa, thus improving the marketing position of fine or flavour cocoa. The specific objectives of the project were to establish physical, chemical and organoleptic parameters enabling the evaluation of cocoa quality in relation to genotype and environment, and to disseminate selected parameters, methodologies, standards and instruments to be used in the evaluation of cocoa quality.

Project implementation started in January 2001 and was carried out in Ecuador, Trinidad and Tobago, Papua New Guinea and Venezuela. The final project evaluation workshop was held in Guayaquil, Ecuador, in April 2006.

The results of the project clearly indicated that the physical parameters measured had proved to be inconclusive in differentiating fine from bulk cocoas. On the other hand, the theobromine/caffeine ratio had proved to be a clear indicator in differentiating fine cocoa from bulk cocoa. However, this ratio could not differentiate qualities of expected flavour profiles, although aromatic profiles were promising indicators in achieving both origin and flavour qualities differentiation. It was further demonstrated that all fine or flavour cocoa samples differed clearly in their organoleptic parameters from the Ghana bulk cocoa which was used as a reference. The project produced a spectrum of unique sensorial attributes for samples from each fine cocoa producing country that had participated in the project, concluding that the countries were not competing against each other but satisfied different flavour niche markets.

Related study:

Sukha, D.A.; Butler, D.R.; Umaharan, P.; Boult, E. (2007)
The use of an optimised organoleptic assessment protocol to describe and quantify different flavour attributes of cocoa liquors made from Ghana and Trinitario beans

The interaction between astringency and sweetness was investigated in red wine using time-intensity (T-I) methodology. Maximum intensity and total duration for astringency decreased significantly with increasing sucrose concentration, whereas no sweetness T-I parameter was significantly affected by astringency level. Although no differences in perception of astringency or sweetness were found as a function of PROP (6-n-propylthiouracil) taster status, there was a significant difference in intensity and persistence of astringency as a function of salivary flow status. Low -flow subjects rated astringency higher and recorded longer duration of astringent aftertaste than high -flow subjects, although there was no difference in sweetness responses as a function of salivary flow status.

The drying conditions to comply with are as follows: a temperature of 65-70°C (17-18 h), thickness 23-27 cm, and a moderate air flow of 0.4-0.5 m/s to avoid "caking".

As paraphrased by Bonvehi et al (1998):
"Artificial drying reduces the processing time and yields a more homogeneous product but increases acidity and incorporates a fruity taste to cocoa (Jacquet et al., 1980). It is convenient to dry at a moderate temperature (<80°C), because the more the temperature increases, the larger is the retention of acidity in the cotyledons (Jacquet et al., 1980)."

Samples of cocoa beans were taken on two separate occasions during heap and tray fermentations in Ghana, West Africa. In total 496 yeast isolates were identified by conventional microbiological analyses and by amplification of their ITS1-5.8S rDNA-ITS2 regions. For important species the identifications were confirmed by sequencing of the D1/D2 domain of the 5' end of the large subunit (26S) rDNA. Assimilations of organic acids and other carbon compounds were conducted. For dominant yeasts intraspecies variations were examined by determination of chromosome length polymorphism (CLP) using pulsed-field gel electrophoresis. For the heap fermentations maximum yeast cell counts of 9.1 x 10(7) were reached, whereas maximum yeast counts of 6.0 x 10(6) were reached for the tray fermentations. Candida krusei was found to be the dominant species during heap fermentation, followed by P. membranifaciens, P. kluyveri, Hanseniaspora guilliermondii and Trichosporon asahii, whereas Saccharomyces cerevisiae and P. membranifaciens were found to be the dominant species during tray fermentation followed by low numbers of C. krusei, P. kluyveri, H. guilliermondii and some yeast species of minor importance. For isolates within all dominant species CLP was evident, indicating that several different strains are involved in the fermentations. Isolates of C. krusei, P. membranifaciens, H. guilliermondii, T. asahii and Rhodotorula glutinis could be found on the surface of the cocoa pods and in some cases on the production equipment, whereas the origin of e.g. S. cerevisiae was not indicated by the results obtained. In conclusion, the results obtained show that fermentation of cocoa beans is a very inhomogeneous process with great variations in both yeast counts and species composition. The variations seem to depend especially on the processing procedure, but also the season and the post-harvest storage are likely to influence the yeast counts and the species composition.

Thirty-nine fermented and dried cocoa bean samples from 13 countries were evaluated for pH, titratable acidity and concentrations of volatile and nonvolatile acids. The correlation coefficient between pH and log10 titratable acidity was -0.94. Cocoa beans from Brazil and Far Eastern countries were highly acidic while those from Central American and South American countries were low in acidity. Samples from West African countries were intermediate with titratable acidity values from 0.12 to 0.15 meq NaOH/g sample and pH values from 5.20 to 5.49. Highly acidic beans were characterized by high concentrations of acetic and lactic acids. The high correlation between acetic acid and both pH (r=0.86) and titratable acidity (r = 0.91) indicated that this acid could be primarily responsible for high acidity in cocoa beans.

The objectives of the study were to evaluate the effect of drying on the pH, titratable acidity and volatile fatty acids (VTA), ie acetic, propionic, butyric, isobutyric and isovaleric acids content of cocoa beans, and on the flavour of the resultant chocolate. Freshly harvested cocoa pods from clones of PBC 123 and 128 (1:1 ratio) were stored for 9 days and fermented for 5 days with a single turning at 48 h. Four drying treatments, ie oven-drying at 60°C, air-blow drying, shade-drying and sun-drying were carried out. Samples were taken periodically for pH, titratable acidity, and VFA analysis. Physical characteristics were also recorded. The resultant beans were made into dark chocolate for flavour evaluation. The results showed that wet beans held in shady areas for 120 h will not dry quickly enough to inhibit mould and yeast and other putrefactive activities; rapid oven-drying of these still wet beans did not improve their flavour profile. There were no significant differences (P 0.05) between samples that have undergone sun-drying and air-blow drying in the acidity and C3-C5 VFA content; both were evaluated favourably by taste panellists. Oven-dried beans contain a high concentration of VFA and produced chocolates with a high intensity of off-flavour.

Flavour characteristics of the chocolate made from 14 dried, fermented cocoa bean samples from eight different countries of origin and the relationship with pH, titratable acidity and acetic and lactic acid were studied. The fermented dried cocoa beans were processed into semi-sweet dark chocolate and were evaluated for their flavour difference by the multiple comparison test using the Ghanaian sample as a reference. The descriptors and the intensities of the chocolate flavour perceived by the taste panel members were also obtained. There was no correlation between the flavour score and the pH, tritratable acidity, acetic and lactic acid concentrations. The study found that chocolate samples made from the low pH (4.75-5.19) and high pH (5.50-5.80) cocoa beans have low response in strong chocolate flavour. On the other hand, chocolate samples made from the Ghanaian and Nigerian beans which have medium pH values of 5.20-5.49 received a high response in strong chocolate flavour. More off-flavour descriptors were perceived from chocolate samples made from low-pH cocoa beans.

Incubation of unfermented and partly fermented cocoa beans in acetate buffer, pH5.5, at 45°C increased yellowness, total colour differences and fermentation index value of the cocoa bean powders and decreased cocoa procyanidins (monomers to pentamers), and their astringency. Fermentation index and (-)-epicatechin content, equivalent to those of fully fermented beans, were reached by unfermented beans after 4-8-h incubation, but not by partly fermented beans even after 16h. During incubation of partly fermented cocoa beans enriched with polyphenol oxidase, yellowness and fermentation index value were increased, whilst (-)-epicatechin was decreased. Tyrosinase had a less significant effect in yellow colour formation, but showed a significant reduction of (-)-epicatechin and increase in fermentation index compared with crude cocoa polyphenol oxidase. However, both enzymes have similar effects on procyanidin degradation and astringent taste reduction. Incubation of cocoa beans for 16h increased the cut test score of unfermented and partly fermented beans by 50 and 30%, respectively.

Changes in flavor release and aroma characteristics in a medium including food phenolics may be attributed to an intermolecular interaction between flavor compounds and phenolics. To investigate the interaction, one- and two-dimensional NMR studies have been carried out on the binding of two phenolics, gallic acid and naringin, with three aroma compounds, 2-methylpyrazine, vanillin, and ethyl benzoate. Evaluation of thermodynamic parameters and intermolecular nuclear Overhauser effects reveals that gallic acid can interact more strongly with aromatic flavors than naringin. The supramolecular complexation is also dependent on the structural nature of the flavors, with 2-methylpyrazine and vanillin interacting more strongly than ethyl benzoate. The interaction is principally pi-pi stacking between the galloyl ring and the aromatic ring of the aroma compounds, but secondary hydrogen-bonding effects help to stabilize the complex and enhance the specificity.

Research paper for Master of Science degree, University of Wisconsin-Stout, WI.

Chocolate liquor is the source of antioxidant flavanols (catechin and epicatechin) and methylxanthines (caffeine and theobromine) found in dark chocolate. Factors that can influence the flavanol and methylxanthine concentration of dark chocolate investigated in this study include the amount of chocolate liquor added, alkalization, and cacao bean genus. The purpose of this study was to quantify flavanols and methylxanthines in different dark chocolates from Legacy Chocolates with different weight percentages of chocolate liquor and different cacao bean genera, Criollo and Forastero. Chocolate samples were analyzed by reverse-phase high performance liquid chromatography (HPLC). Results indicated that the greater the percentage of chocolate liquor added to the final product, the more flavanol antioxidants present. When comparing chocolates with similar weight percentages, the Forastero genus had a significantly greater (p < 0.05) flavanol concentration than the Criollo genus. The Criollo genus resulted in a significantly greater (p < 0.05) caffeine content in dark chocolate when compared to a product prepared with similar weight percentages of chocolate liquor from the Forastero genus. Conversely, the Forastero genus produced a chocolate that was significantly greater (p < 0.05) in theobromine when compared to a Criollo product with similar weight percentages of chocolate liquor. Alkalization processing did not appear to affect catechin, epicatechin, caffeine, or theobromine concentrations in chocolates with similar weight percentages of chocolate liquor. Commercial brand chocolates were analyzed for comparison.

Note: The usage of the terms genus and genera in this abstract is technically incorrect. In fact, there is only one cocoa genus - Theobroma. And there is only one cocoa species - cacao. Forastero and Criollo are widely recognised cocoa cultivars.SM

The transport and uptake of inspired odorant molecules in the human nasal cavity were determined using an anatomically correct three-dimensional finite element model. The steady-state equations of motion and continuity were first solved to determine laminar flow patterns of odorous air at quiet breathing flow rates. The air stream entering the ventral tip of the naris travelled to the olfactory slit, and then passed through the slit in nearly a straight path without forming separated recirculating zones. The fraction of volumetric flow passing through the olfactory airway was about 10%, and remained nearly constant with variations in flow rate. The three-dimensional inspiratory velocity field was used in the solution of the uncoupled steady convective-diffusion equation to determine the concentration field in the airways and odorant mass flux at the nasal walls. The mass-transfer boundary condition used at the nasal cavity wall included the effects of solubility and diffusivity of odorants in the mucosal lining, and the thickness of the mucus layer. The total olfactory flux of odorants, that is highly correlated with perceived odor intensity, was determined as a function of all transport parameters in our model. Increase in nasal flow rate at a constant inlet concentration resulted in an increase in total olfactory uptake for all odorants. However, with increase in flow rate, the fractional uptake, i.e., total olfactory flux normalized by convective flux at the inlet, decreased for poorly soluble odorants, while it increased for highly soluble odorants. The pattern of flux (or imposed patterning) across the olfactory mucosa, that carries information concerning odor identity, was also determined as a function of transport parameters. There was an overall decrease in odorant flux as the location on the olfactory surface was varied from the anterior towards the posterior and from the inferior towards the superior ends. The flux pattern became more uniform, i.e., the steepness of the flux gradients across the olfactory surface decreased, as the mucus solubility of the odorants decreased. Different odorants generated discernibly different flux patterns across the olfactory mucosa that may contribute to the encoding of odor quality. Variation of total olfactory flux with time after cessation of airflow was determined by solving the unsteady diffusion equation in the air-phase. The flux decreased approximately exponentially with time. The rate of decay decreased as solubility and diffusivity decreased, but was very rapid over a wide range of the parameters, with time constants of less that 0.5 s for most odorants, implying a rapid decrease in perceived odor intensity with cessation of nasal airflow.

As determined by high performance liquid chromatography, (-)- epicatechin concentrations among freshly harvested beans of verified genetic origin ranged from 21.89-43.27 mg/g of dry defatted samples. Fermented beans showed much lower concentrations (2-10 mg). During fermentation, a trend towards decrease in (-)-epicatechin content was observed. Commercial beans from areas with reputations for shipping well-fermented products contained lower levels of (-)-epicatechin than beans from regions where fermentation is less extensive.

This research examined the effectiveness of the "mouth-feel wheel" for the assessment of astringent subqualities in 25 commercial British Columbia (BC) red wines. Twelve judges discussed, adapted and practised the definitions and reference standards (fabrics) to characterise the astringent subqualities. The astringent qualities were organised into six categories (surface smoothness, drying, dynamic, weight, complex ripe/integrated, unripe/unintegrated), each consisting of specific descriptors or subqualities (3≤n≤7), as outlined in the literature. The wines were evaluated in duplicate using a strict tasting protocol. Judges rated the magnitude of astringency and aftertaste and identified the astringent subqualities using a check-off system. The subquality frequencies were weakly correlated (r=0.40) with the astringent and aftertaste intensities. Judge consistency and overall performance were examined using chi-square and principal component analysis (PCA).
In general, judge consistency was poor, both within and between judges. The interrelationship of the astringent subqualities was explored using multidimensional scaling and PCA, employing distance and correlation matrices, respectively. Both techniques showed that the descriptors successfully identified different levels of astringency. However, the close location of some of the descriptors suggested that some judges may have been using different vocabularies to describe the same astringent subqualities.
It was believed that the lexicon describing the astringent subqualities would need simplification and clarification before it could be used effectively to describe BC red wines.

Related study:
Gawel et al (undated)
A "Mouth-feel Wheel": terminology for communicating the mouth-feel characteristics of red wine
http://www.winepros.com.au/pdf/mouthfeel.pdf

The production of the C3 to C5 volatile fatty acids during the processing of raw cacao has been studied by means of gas chromatography. These fatty acids are produced towards the end of fermentation and also during the drying phase, if conditions are conducive to this. The time of their appearances is related to the degree of aeration of the fermenting mass. In low concentrations, they may be regarded as contributing to normal chocolate flavour, but higher concentrations, such as are associated with poor conditions of processing, cause "dull", "insipid" off-flavours. Suggestions are made about the mechanism of their production and the microorganisms that may be involved.

The integration of olfactory and taste perception and the role of congruency and familiarity on perception have already been demonstrated in model solutions. The aim of the present study was to investigate the role of these factors in real food products. Therefore, we have investigated the impact of olfactory perception on perceived bitterness in a familiar (bitter cocoa beverage) and an unfamiliar (bitter milk) beverage. Sensory profilings with and without noseclip were conducted according to simultaneous product presentation. In a first experiment, an instant cocoa powder mixed with water was used to prepare a common base. Two types of flavourings were added: cocoa and vanilla, at three different levels (none, medium and high). Samples were compared within a flavouring type. In a second experiment a vanilla flavour was added at three levels to a milk base containing caffeine. The panellists scored bitterness, sourness, sweetness and body with noseclip. Without noseclip, overall aroma above the cup and in mouth were assessed in addition to the previous set of attributes. With noseclip, results showed that neither the cocoa nor the vanilla flavourings provided any additional taste to the beverages. Without noseclip, olfactory/taste interaction in the cocoa beverage led to an enhancement of bitterness induced by the cocoa flavouring and an increase in sweetness from the vanilla flavouring. On the contrary, in the caffeinated milk, the addition of vanilla flavouring did not significantly increase sweetness, but unexpectedly enhanced bitterness. This study is further evidence of the influence of olfaction on taste perception in complex matrices. In addition, our results suggest that taste–olfaction integration is product dependent and related to food experience, even when working with trained subjects. Furthermore, the unpleasantness due to the neophobia related to the consumption of a new product and to bitterness may enhance bitterness when the unfamiliarity of the product is increased by addition of vanilla flavouring to a bitter milk beverage.

Flavonoids are phenolic substances widely found in fruits and vegetables. Many epidemiological studies associate the ingestion of flavonoids with a reduced risk of cardiovascular disease and certain types of cancer. These effects are due to the physiological activity of flavonoids in the reduction of oxidative stress, inhibiting low-density lipoproteins (LDL) oxidation and platelet aggregation, acting as vasodilators in blood vessels, inhibiting the adherence of monocytes to the vascular endothelium, promoting fibrinolysis, acting as immunomodulators and anti-inflammatory agents and as inhibitors in the different phases of tumour process. Cocoa is an important source of polyphenols, which comprise 12-18% of its total weight on dry basis; the major phenolic compounds are epicatechin, proanthocyanidins and catechin. The levels of flavonoids contained are higher than the ones founds in apples, onions or wine, foods known for their high amount of phenolic compounds. Cocoa and cocoa products are important sources of flavonoids in our diet. In the Dutch population chocolate contributes up to 20% of the total flavonoid intake in adults, and in children the percentage is even higher. The bioavailability of these compounds depends on other food constituents, and their interaction with the food matrix. This article reviews current evidence on the health effects of cocoa flavonoids in our diet. The compiled data supports the premise that the consumption of cocoa flavonoids is beneficial to human health.

Among the most important volatile compounds in the aroma of strawberries are 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol) and its methoxy derivative (methoxyfuraneol, mesifuran). Three strawberry varieties, Malach, Tamar, and Yael, were assessed for total volatiles, Furaneol, and methoxyfuraneol. The content of these compounds sharply increased during fruit ripening, with maximum values at the ripe stage. An enzymatic activity that transfers a methyl group from S-adenosylmethionine (SAM) to Furaneol sharply increases during ripening of strawberry fruits. The in vitro generated methoxyfuraneol was identified by radio-TLC and GC-MS. The partially purified enzyme had a native molecular mass of ~80 kDa, with optimum activity at pH 8.5 and 37°C. A high apparent Km of 5 mM was calculated for Furaneol, whereas this enzyme preparation apparently accepted as substrates other o-dihydroxyphenol derivatives (such as catechol, caffeic acid, and protocatechuic aldehyde) with much higher affinities (Km ~ 105, 130, and 20 M, respectively). A Km for SAM was found to be ~5 M, regardless of the acceptor used. Substrates that contained a phenolic group with only one OH group, such as p-coumaric and trans-ferulic acid, as well as trans-anol and coniferyl alcohol, were apparently not accepted by this activity. It is suggested that Furaneol methylation is mediated by an O-methyltransferase activity and that this activity increases during fruit ripening.

Two-component mixtures of astringent materials were rated for perceived intensity of astringent and taste attributes over time. Components included alum (a complex salt), gallic acid (the monomeric component of hydrolyzable tannins), catechin (the monomeric component of condensed tannins) and citric acid. Mixtures of alum and gallic acid showed mixture suppression, in that the 50/50 mixture was less intense than either component in astringency, drying, roughing and puckery/drawing sensations. Suppression was seen at concentration levels producing moderate to strong astringency but was absent or less pronounced at lower concentration levels. A similar pattern held for citric acid, although the suppressive effects were less pronounced. Catechin and gallic acid mixtures were additive. Sensory interactions between astringent materials appears to depend on the substances involved and their concentrations (or intensity levels).

Qualitative and quantitative perceptual reactions to astringent materials were examined for three diverse chemical substances (alum, tannic acid and tartaric acid) at several concentrations producing moderate to strong levels of perceived sensation. Group discussions were held to determine language appropriate to describe the sensations arising from solutions of the three compounds and a composite ballot of six rating scales (astringency, mouth drying, puckery feeling, mouth roughing, bitterness and sourness) was developed. For both experiments, two concentrations of each compound were rated on the six attributes for five to six minutes, a discrete-point time-intensity scaling procedure. All ratings showed roughly exponential decays over time. The intensity ratings for each attribute were found to depend on both the particular astringent substance and concentration tested. The results from experiment 2 suggested that the four tactile attributes of drying, puckery feeling, roughing, and overall astringency may not be totally interchangeable and that there may be multiple sub-qualities in the sensory reactions grouped as astringency. It is recommended that future structure-activity studies make use of time-intensity procedures with multiple rated attributes, using 1 g/1 alum as a reference material, since it is relatively low in perceived bitterness and sourness, but produces pronounced drying, roughing, puckery/drawing sensations.

Black tea, green tea, red wine, and cocoa are high in phenolic phytochemicals, among which theaflavin, epigallocatechin gallate, resveratrol, and procyanidin, respectively, have been extensively investigated due to their possible role as chemopreventive agents based on their antioxidant capacities.
The present study compared the phenolic and flavonoid contents and total antioxidant capacities of cocoa, black tea, green tea, and red wine.
Cocoa contained much higher levels of total phenolics (611 mg of gallic acid equivalents, GAE) and flavonoids (564 mg of epicatechin equivalents, ECE) per serving than
black tea (124 mg of GAE and 34 mg of ECE, respectively),
green tea (165 mg of GAE and 47 mg of ECE), and
red wine (340 mg of GAE and 163 mg of ECE).
Total antioxidant activities were measured using the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays and are expressed as vitamin C equivalent antioxidant capacities (VCEACs). Cocoa exhibited the highest antioxidant activity among the samples in ABTS and DPPH assays, with VCEACs of 1128 and 836 mg/serving, respectively. The relative total antioxidant capacities of the samples in both assays were as follows in decreasing order: cocoa > red wine > green tea > black tea. The total antioxidant capacities from ABTS and DPPH assays were highly correlated with phenolic content (r2 = 0.981 and 0.967, respectively) and flavonoid content (r2 = 0.949 and 0.915). These results suggest that cocoa is more beneficial to health than teas and red wine in terms of its higher antioxidant capacity.

Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to evaluate genetic relationships within the Theobroma cacao species and to assess the organization of its genetic diversity. Genetic variability was estimated with 18 primers and 43 RFLP probes on 155 cocoa trees belonging to different morphological groups and coming from various geographic origins. The majority of the RFLP probes issued from low-copy DNA sequences. On the basis of on the genetic distance matrices, the two molecular methods gave related estimates of the genetic relationship between genotypes. Although an influence of cocoa morphological groups and geographical origins of trees was observed, a lack of gene differentiation characterized the T. cacao accessions studied. The continuous RFLP variability observed within the species may reflect the hybridization and introgressions between trees of different origins. Nevertheless, the Nacional type was detected to be genetically specific and different from well-known types such as Forastero, Criollo and Trinitario. Some of those genotypes were characterized by a low heterozygosity rate and may constitute the original Nacional pool. These results also provide information for the constitution of a cocoa tree core collection.

Recalcitrant seed axes were reported to survive to lower water contents under fast-drying conditions. The present study was to examine the hypothesis that drying rate and dehydration duration could interact to determine desiccation tolerance through different physico-chemical mechanisms. The effect of drying rate on desiccation tolerance of Theobroma cacao seed axes at 16°C was examined. Rapid-drying at low relative humidity (RH) and slow-drying at high RH were more harmful to cocoa axes, because electrolyte leakage began to increase and axis viability began to decrease at high water contents. Maximum desiccation tolerance was observed with intermediate drying rates at RH between 88% and 91%, indicating the existence of an optimal drying rate or optimal desiccation duration. This maximum level of desiccation tolerance for cocoa axes (corresponding to a critical water potential of -9 MPa) was also detected using the equilibration method, in which axes were dehydrated over a series of salt solutions or glycerol solutions until the equilibrium. These data confirmed that the physiological basis of the optimal drying rate is related to both mechanical stress during desiccation and the length of desiccation duration during which deleterious reactions may occur. The optimal drying rate represents a situation where combined damages from mechanical and metabolic stresses become minimal.

Results: Under the condition used in the present study, cocoa seeds can be easily stored for more than 2 months without significant loss of seed viability and vigour.

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.

The perceived dryness and bitterness associated with the astringent stimulus tannic acid were examined in a series of experiments. The method of magnitude estimation was used in conjunction with a sip and spit procedure to measure changes in these two oral sensations during repeated exposures to the test stimulus. Salivary volume was also measured. The results indicated (i) that mouth dryness increased significantly over time, whereas bitterness increased only slightly over time, and (ii) the addition of sweeteners to solutions of tannic acid reduced both perceived dryness and bitterness. It was concluded that the attenuation of dryness by sweeteners was probably related to increases in salivary volume produced by the sweeteners, although the lubricating characteristics of the viscous sucrose solutions may also have played a role.

From the back cover:
Covering a wide range of food and food product groups and including only key constituents and compounds contributing significantly to flavor, this reference presents knowledge of volatile compounds occurring in foods and beverages and describes their sensory properties and mechanisms of formation.
Drawing upon the expertise of more than 20 international authorities on flavor research from industry, research, and academia, Volatile Compounds in Foods and Beverages also provides information on recent developments in instrumental and sensory methodology ... traces the history of flavor research ... examines the occurance of off-flavor substances formed during processing or originating from the environment ... contains over 2,300 literature citations ... and much more.
This collection works as a vital reference for food scientists and technologists, sensory analysts, flavor chemists and analysts, flavorists, and food biochemists and biotechnologists; and serves as a helpful text for upper-level undergraduate and graduate students in flavor chemistry and food technology courses.

To facilitate the identification of Theobroma cacao L. that possess desirable traits to meet changing production and market conditions, there is a need to understand the genetic relationships among T. cacao germplasm. In addition, new cultivars are needed to provide more broadly based resistance to devastating diseases such as witches' broom disease [Crinipellis perniciosa (Stahel) Singer]. A subset of 270 T. cacao accessions based on (i) witches' broom disease resistance data, (ii) genetic characterization experiments, and (iii) a random sampling of recently acquired accessions was selected from the extensive germplasm collection at the Centro de Pesquisa do Cacau (CEPEC; Itabuna, Bahia, Brazil) in collaboration with Fazenda Almirante, a division of M&M Mars Incorporated in Itajuipe, Bahia, Brazil. Estimates of genetic distance based on random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic diversity among the selected accessions. Incorporation of recently acquired accessions (Accessions 181-270) did not increase the breadth of the distribution of genetic diversity already present within the "original" group sampled (Accessions 1-180) suggesting new accessions collected from already sampled geographic regions do not increase the existing genetic diversity in the germplasm collection. In addition, differences in RAPD marker frequencies were associated with accessions that had a high threshold of tolerance to witches' broom disease. Most accessions exhibiting tolerance to witches' broom disease were from the Upper Amazon region, with the exception of SGU 26, a hybrid from Guatemala. This suggests that the Upper Amazon is not the only region to have genes for resistance to witches' broom disease and stresses the need for further collection and examination of germplasm from other regions.

In Malaysia, pulp preconditioning by post-harvest storage of cocoa pods leads to the reduction of nib acidification during subsequent fermentation, reduction of the acid note and an increase in cocoa flavour in the resulting raw cocoa. Data from several shallow-box fermentations, with material from unstored and stored pods, are compared and interpreted, obtained in the years 1984 to 1987 in a cooperational investigation of the Malaysian Agricultural Research and Development Institute (MARDI), Malaysia, and the Botanical Institute, Technical University of Braunschweig (TUBS), FRG. Prior to and during fermentation, pulp volume and pulp sugars; pH value, acetic acid and lactic acid content in the pulp and nibs; and oxygen concentration and temperature in the mass were determined. Some flavour assessments from selected samples are given. The great reduction in pulp volume per seed rather than the decrease of pulp sugars per seed during pod storage was found to be of the most importance. Pulp-volume reduction enhances mass aeration and increases the ratio of respiration to ethanol fermentation and its subsequent oxidation to acetic acid. As a consequence, the acidification of the seeds during the formative stages of flavour precursors (after the death of the seeds) is strongly reduced. With effectively dry stored pods (pulp volume per gram of seed 0.6 ml) the anaerobic phase during the initial stages of fermentation which is common with unstored pods is suppressed. Under these conditions the nib pH value does not fall below 5.0 and no drastic acid (and flavour) degradation at the end of fermentation is necessary to reduce the acidity in the seeds.

In the United States, commercially available foods, including cocoa and chocolate, are being marketed with statements referring to the level of antioxidant activity and polyphenols. For cocoa-containing foods, there has been no comprehensive survey of the content of these and other chemistries. A survey of cocoa and chocolate-containing products marketed in the United States was conducted to determine antioxidant activity and polyphenol and procyanidin contents. Commercially available samples consisted of the top market share products in each of the following six categories: natural cocoa, unsweetened baking chocolate, dark chocolate, semisweet baking chips, milk chocolate, and chocolate syrup. Composite samples were characterized using four different methods: oxygen radical absorbance capacity (ORAC), vitamin C equivalence antioxidant capacity (VCEAC), total polyphenols, and procyanidins. All composite lots were further characterized for percent nonfat cocoa solids (NFCS) and percent fat. Natural cocoas had the highest levels of antioxidant activities, total polyphenols, and procyanidins followed by baking chocolates, dark chocolates and baking chips, and finally milk chocolate and syrups.
The results showed a strong linear correlation between NFCS and ORAC (R² = 0.9849), total polyphenols (R² = 0.9793), and procyanidins (R² = 0.946), respectively. On the basis of principal component analysis, 81.4% of the sample set was associated with NFCS, antioxidant activity, total polyphenols, and procyanidins. The results indicated that, regardless of the product category, NFCS were the primary factor contributing to the level of cocoa antioxidants in the products tested. Results further suggested that differences in cocoa bean blends and processing, with the possible exception of Dutching, are minor factors in determining the level of antioxidants in commercially available cocoa-containing products in the United States.

Increased 2-phenylethanol and glycerol formation in spontaneous grape must fermentation compared with fermentation with active dry yeast starters in wine can be observed. In extreme cases up to 280 mg/l 2-phenylethanol may be formed, whereas wines fermented by pure yeast starters display 20-40 mg/l. As 2-phenylethanol participated in wine bouquet and aroma formation, it is supposed by some winemakers that spontaneously fermented wines show larger scope to complete their character. Further research is however necessary to bring about more details in this aspect.

Incubation-activation of remaining key enzymes in dried under-fermented cocoa beans and its effect on aroma precursor formation has been studied using defatted unfermented and partly fermented cocoa bean powders. Results of the study showed that aspartic endoprotease, carboxypeptidase and invertase were significantly inactivated during fermentation and drying, and the effect of fermentation was significantly lower than that of drying. The enzyme activities remaining in these beans were still sufficient to carry out enzymatic reaction during incubation. Peptide patterns, resulting from incubation of unfermented and partly fermented beans powders, were quite similar to the well-fermented patterns. Meanwhile, free amino acid concentrations of the unfermented beans were significantly increased during the first 4 h of incubation and then remained constant; however, with partly fermented beans, the formation continued and the hydrophobic and total free amino acid concentrations reached the value of well-fermented beans after 24 h of incubation. Reducing sugar concentrations of both unfermented and partly fermented cocoa beans could reach the level of well-fermented beans by incubation.

A study on effect of polyphenol on pyrazine formation during cocoa liquor roasting has been carried out. Cocoa liquors, having polyphenol concentrations of 41, 58, 116, 143 and 170 g/kg, were roasted at 120°C for 15, 25, 35 and 45 min. Results of the study showed that, as polyphenol concentration increases, pyrazine formation decreases; this was due to the binding of polyphenol against pyrazine precursors, i.e. free amino acids and reducing sugars, and pyrazines formed during roasting. Reduction of the formations of 2,3,5-TrMP and 2,3,5,6-TMP occurred throughout roasting times, starting at 116 g polyphenol per kg and reaching maximum reduction at 116-170 g polyphenol per kg. The reduction against 2,5-DMP was not significant over roasting times, except at 35 min; however, formation of 2,3-DMP was reduced at roasting times of 25, 35 and 45 min. These results implied that the presence of polyphenol in cocoa has to be considered in many aspects, including its reduction of flavour formation during roasting, besides productions of astringency and bitterness of the product and its beneficial effect as an antioxidant.

Sensory properties of cocoa liquor roasted at 120°C for 15, 25, 35 and 45 min and containing different polyphenol concentrations (58, 116, 143 and 170 g/kg) were studied. Eight trained panellists carried out the sensory analysis using line scale with Ghanaian fermented cocoa liquor as a reference. The sensory attributes were cocoa flavour, astringency, bitterness, acidity/sourness, fruity/floral/bouquet, raw/green, smoky, mouldy/earthy and viscosity. Results of the study showed that as polyphenol concentration in cocoa liquor increased, cocoa flavour and viscosity decreased and astringency and bitterness increased; however, other sensory properties were not influenced by polyphenol concentration. An increase in roasting duration of cocoa liquors containing 58-143 g/kg polyphenol increased the flavour intensity; meanwhile that of contains 170 g/kg polyphenol, it was in vice versa. These findings indicated that cocoa polyphenol would cause negative effect on flavour properties, apart from its well-known benefit as preservative and antioxidant.

Research presented to: Conferencistas, Primer Congreso Venezolano del Cacao y su Industria (First Venezuelan Congress of the Cocoa Industry) AGETROP/CIRAD, France.

Stated agenda of conference: "to rescue the best cocoa in the world" (translated from notes by the Committee Organizer, March 2000)

Genetic diversity analysis of Venezuelan and some Mexican Criollo cacao trees, as well as individuals from the other morphogeographic groups, was performed using RFLP markers. Samples of Criollo trees were collected in ancient Venezuelan plantations without regard to agronomic traits. Also, some samples of Criollo could be taken in the Lacandón rainforest of Mexico, where wild Criollo cacao trees have been reported. In addition to those already mentioned samples of Criollo trees from several germplasm collections in Venezuela, Costa Rica and Mexico were also studied.
RFLP analyses revealed a high level of homozygosity in the Criollo clones collected in the ancient Venezuelan plantations and in the Lacandón rainforest, in contrast to "Criollo" clones sampled in germplasm collections. A significant molecular similarity between all the types of Criollo studied (Pentagona, Porcelana, Criollo Andino, Criollo de la Costa and the one from the Lacandón rainforest) was found, in spite of the dissimilar morphological traits that differentiate them. Criollo from germplasm collections appeared generally, to be more heterozygous and variable, perhaps due to selection for some agronomically important characteristics. This probably corresponds to partial introgressions of Forastero genes, which gave vigour and resistance to ancestral Criollo varieties.
From data obtained new hypotheses concerning evolution and domestication schemes could be proposed. The low genetic variability encountered among the Criollo varieties, suggests that the Criollo group originated from a reduced ancestral population and that man participated in the spread of individuals of this group in Central America.
In addition, consequences for breeding for quality traits can be derived since the poor yield and the pest susceptibility which has characterised the individuals of the Criollo group could be explained partly as a consanguinity depression phenomenon. Thus, for obtaining genetic gain but conserving quality, crosses between Criollo individuals must be avoided. Different breeding strategies for improvement of quality varieties are discussed.

Criollo cacao (Theobroma cacao ssp. cacao) was cultivated by the Mayas over 1500 years ago. It has been suggested that Criollo cacao originated in Central America and that it evolved independently from the cacao populations in the Amazon basin. Cacao populations from the Amazon basin are included in the second morphogeographic group: Forastero, and assigned to T. cacao ssp. sphaerocarpum. To gain further insight into the origin and genetic basis of Criollo cacao from Central America, RFLP and microsatellite analyses were performed on a sample that avoided mixing pure Criollo individuals with individuals classified as Criollo but which might have been introgressed with Forastero genes. We distinguished these two types of individuals as Ancient and Modern Criollo. In contrast to previous studies, Ancient Criollo individuals formerly classified as 'wild', were found to form a closely related group together with Ancient Criollo individuals from South America. The Ancient Criollo trees were also closer to Colombian-Ecuadorian Forastero individuals than these Colombian-Ecuadorian trees were to other South American Forastero individuals. RFLP and microsatellite analyses revealed a high level of homozygosity and significantly low genetic diversity within the Ancient Criollo group. The results suggest that the Ancient Criollo individuals represent the original Criollo group. The results also implies that this group does not represent a separate subspecies and that it probably originated from a few individuals in South America that may have been spread by man within Central America.

Cacao (Theobroma cacao L.) has been cultivated in Central America since pre-Columbian times. The type of cacao cultivated in this region was called Criollo; cacao populations from the Amazon basin were called Forastero. The type of Forastero most commonly cultivated until 1950 was named Amelonado. Historical data show Trinitario cacao to have originated in Trinidad, resulting from natural hybridisation between Criollo and Amelonado Forastero. Doubts persist on the source of the Amelonado Forastero involved in the origin of Trinitario; the Amelonado parent may have come from the Lower Amazon, the Orinoco or the Guyanas. Most of the cacao cultivated worldwide until 1950 consisted of Criollo, Trinitario and Amelonado. From the early 1950s, Forastero material collected in the Upper Amazon region during the 1930s and 1940s began to be employed in breeding programmes. To gain a better understanding of the origin and the genetic basis of the cacao cultivars exploited before the utilisation of germplasm collected in the Upper Amazon, a study was carried out using restriction fragment length polymorphism and microsatellite markers. Trinitario samples from 17 countries were analysed. With molecular markers, it was possible to clearly identify three main genotypes (represented by clones SP1, MAT1-6 and SIAL70) implicated in the origin of most Trinitario clones.

Polyphenol components were extracted from Malaysian mix-hybrid cocoa beans from different treatments of fermentation namely; post harvest pod storage, bean spreading and pressing. The polyphenol compounds were analyzed using high pressure liquid chromatography. The (-)-epicatechin, (+)-catechin, theobromine and caffeine of control treatment at 5 days pod storages were 11.87, 4.31, 21.08 and 3.85 mg/g, respectively. Pre-fermentation treatments were found to be significant in affecting the changes in acidity, degree of fermentation and the polyphenol content of cocoa bean. During fermentation, all pre-treated samples showed decreased levels of (-)-epicatechin and (+)-catechin but the rate of decrease were found to be different. It was also found that the (-)-epicatechin and (+)-catechin content before and after fermentation was affected by the type of pre-fermentation treatment used. Percentage lost for (-)-epicatechin and (+)-catechin during fermentation at different degree of fermentation ranged from 6 to 17% and 0.95 to 1.62%, respectively. It was observed that pod storage treatments until 15 days have significant effect in the reduction of acidity, fermentation index (FI) and polyphenol contents compared to spreading and pressing methods. Recoveries (Rv) ranged from 90.90 to 93.92% with % coefficient of variances (CVs) of 6.51, 8.57, 2.42 and 6.37 for (-)-epicatechin, (+)-catechin, theobromine and caffeine, respectively. This study indicates that 15 days pulp preconditioning is the optimum conditions for the degradation of (-)-epicatechin and (+)-catechin, theobromine and caffeine.

Nineteen cacao clones from Cameroon genebank were analysed. Fresh and fermented-like seeds were used. Two main polyphenols were present in our samples: catechin and epicatechin. Epicatechin represents 2-4% DM of defatted cocoa seed powder. Undefined substances called A, B and C were also found in cocoa seeds. Substance A is discussed as a derivative of caffeic acid and an ester-bound compound. Substances B and C are oligomeres of proanthocyanidins. Protocatechiuc acid and quercetin were not detected. Two anthocyanins were found in cocoa seeds: cyanidin-3-galactoside and cyanidin-3-arabinoside. They represent 0.02 0.4% DM of defatted cocoa seed powder. Total phenols, catechin, epicatechin and anthocyanin in fresh and fermented-like beans were genotype-dependent. Polyphenols from seeds of two different pods from the same clone showed a quantitative significant difference. Spearman's correlation test showed that there is no correlation between the number of seeds per pod, weight of pod and content of polyphenolic compounds. Nevertheless, a negative correlation was found between the number of seeds per pod and the catechin content (r=-0.463, P<0.01). Groupings of samples were observed using PCA and hierarchical cluster analysis. The separation between groups is related to their polyphenol and anthocyanin contents.

Novel mathematical models for flavour release during drinking are described, based on the physiology of breathing and swallowing. Surprisingly, we conclude that most flavour molecules arriving in the nose are extracted from liquid left in the throat, after swallowing. The models are fit to real time flavour release data obtained using APCI-mass spectrometry. Before modelling, raw data are corrected for the effects of varying airflow rate, using the signal from acetone in exhaled air. A simple equilibrium batch extraction model correctly describes flavour release during the first breaths after swallowing a flavoured liquid. It shows that for eight volatiles, whose in vitro air-water partition coefficients vary by a factor of 500, the apparent in vivo air-saliva partition coefficients vary only by a factor of five. To interpret the kinetics of flavour release longer after swallowing, diffusion of flavour into the throat lining is included. This is done using a three-layer model for mass transfer in the throat. An analytical solution of this model gives good fits to typical data. These models de-couple the physiological and physico chemical aspects of flavour release, clarifying the effect of behaviour on in-vivo flavour release.

Chapter 23: Domestication and Distribution of the Chocolate Tree (Theobroma cacao L.) in Mesoamerica
[Book edited by Arturo Gõmez-Pompa]

Excerpt:
The history of Theobroma cacao L. in Mesoamerica is especially important because historical and archaeological evidence place this area as the center of domestication for the species since at least 2,000 years ago. Mesoamerica represents the extreme northern distribution where T. cacao occurs naturally and is also the place where a unique segment of genetic diversity has already been recently documented (De la Cruz et al, 1995; Whitkus et al, 1998). [...] The objective of this chapter is to describe the diversity and distribution of cacao in ancient Mesoamerica and compare it with present times. Based on historical accounts and fieldwork, the occurence and distribution of putative wild and pre-Hispanic abandoned populations in present times in Mesoamerica will be described. New evidence is presented to propose that the domestication and use of cacao as chocolate in pre-Hispanic times was not exclusive to Mesoamerica, but to several indigenous groups from South America as well.

[...] Wolters (1999) suggests that Valdivia culture (3500-1600 B.C.), and all successive cultures in west Ecuador, conducted coastal shipping to Peru, Middle America, and southern Mexico from 2200 to 1450 B.C.

[...] The Maya developed a complex and sophisticated agroforestry system using shade trees, irrigation systems, nurseries, and transplantation techniques [...] One of these transplantation techniques involved wrapping the roots of seedlings in mats during the transplantation process (Alvarado-Tezozomoc [1598] 1944)

[...] Other sources not only take for granted that cacao was not cultivated in South America, but also even propose routes and possible actors (such as Spanish missionaries or settlers as responsible for bringing cacao and starting its cultivation in South America (Young 1994; Coe and Coe 1996). However, to paraphrase the astronomer Carl Sagan (1977), sometimes the absence of evidence is not evidence for absence.

The globulins of the fermented grain of the criollo, forastero and trinitario cocoa types in the Cumboto area, Aragua state, Venezuela were characterized by electrophoresis. Pods from two plants, three fruits per plant, were harvested for each type of cocoa in El Paraíso, La Vega de Santa Cruz and La Isleta parcels. Healthy pods corresponding to each type of cocoa were shelled by hand and underwent a fermentation process for a period of five days. The pulp was removed from the fermented grains, and the cotiledons were dried in an oven for 48h at 40°C. This was followed by a grinding process. The globulins were extracted using 0.5M NaCl and were analyzed by unidimensional PAGE-SDS electrophoresis in a 20% poliacrilamide plate gel. The results revealed significant variations (p<0.05) in the protein contents of the degreased powder of the fermented grains corresponding to the different cocoa types (17.6%-20.3%) and the different parcels (17.5%-20.3%). The amount of globulins also differed between parcels (15.3%-30.4%). However, the values did not show a statistical differentiation between the types (16.8%-24.9%). The electrophoretical analysis of the globulins showed patterns with a total of 49 soft bands with a relative mobility ranging from 0.214 to 0.971, and molecular weights in between 79 and 13.8kDa, which varied between plants, cocoa types, and parcels.

In order to isolate, identify and characterize the microflora of cacao beans before, during and after fermentation and locate possible sources contributing to microbial contamination, cacao beans from the Centeno and San Louis Estates in Trinidad were investigated. Prior to fermentation, the interior and exterior of the pods, hands of employees, utensils, dried pulp material of the sweatboxes and finally fruitflies (Drosophila melanogaster) were studied microbiologically. At Centeno Estate, beans were sampled at 5, 45 and 90 cm depths at 8-hr intervals for the first 72 hr and every 12 hr thereafter for 7 days. Sampling at San Louis Estate was carried out at 24 hr intervals for the same period. The changes in microbial population of the beans sampled at Centeno Estate ranged from 148,000/g at 0 hr to 410,000/g at the completion of the fermentation, whereas, at San Louis Estate they ranged from 680,000/g to 920,000/g during the same period. Taxonomical studies of isolates obtained during the fermentation period revealed the identification of 44 microorganisms at both Estates. Yeasts Zymomonas mobilis and several species of lactic acid organisms dominated the flora during the early stages of fermentation. As the fermentation progressed, these and other isolates were taken over by several species of genus Bacillus. Microbiological examination of dried and polished beans resulted in the identification of 22 organisms at Centeno Estate and 15 organisms at San Louis Estate

This study investigated the antioxidant capacity and total phenolic content of cocoa beans from different countries, namely Malaysia, Ghana, Ivory Coast and Sulawesi. The antioxidant capacity of water and ethanolic extracts prepared from cocoa beans was measured by three different assays. To estimate the total phenolic content, the assay using Folin-Ciocalteu reagent was used. The water extract showed the higher value of antioxidant activity based on β-carotene bleaching assay, while the ethanolic extract showed the highest scavenging and ferric reducing activities. Ghanaian cocoa beans showed the highest antioxidant and scavenging activities, followed by Ivory Coast, Malaysian and Sulawesian. However, Malaysian and Sulawesian beans exhibited the highest ferric reducing activity, compared to the other beans. The highest phenolic content was found in Malaysian beans, followed by Sulawesian, Ghanaian and Ivory Coast. A positive correlation existed for both ethanolic (r = 0.76) and water extracts (r = 0.78) between phenolic content and ferric reducing activity. Our results showed that antioxidant capacity and phenolic content of Malaysian cocoa beans were comparable to Ghanaian, Ivory Coast, and Sulawesian beans.

Immature harvest of cocoa to prevent losses due to pests and pilferage is common in Sri Lanka. The effect of maturity on the levels of some components of the Amelonado variety of cocoa grown in Sri Lanka is reported here. The major problem posed by immature harvest is the loss of fats which has been shown in this study to be of the order of 50% when the crop is harvested 1.5 months early, although the fatty acid composition does not vary significantly during this period. The pulp of immature cocoa pods contains less free sugars and produces less ethanol on yeast fermentation than pulp of mature pods but the methanol content of the distillate is higher in immature pulp. The pectin content of the pod endocarp declines as maturation proceeds.

Intensity of astringency and bitterness of seven flavonoid compounds was evaluated by a time-intensity (TI) procedure. Eighteen trained judges rated intensity continuously from ingestion, through expectoration at 10s until extinction of the sensation. The seven stimuli included two flavan-3- ol monomers, (+)-catechin and (-)-epicatechin, three dimers and two trimers synthesised from catechin or epicatechin by condensation with (+)-dihydroquercitin. As the degree of polymerisation increased, maximum bitterness intensity (I.max) and total duration (T.tot) decreased whereas astringency I.max increased. The monomers were significantly higher in bitterness at I.max than the dimers, which were significantly higher than the trimers. Astringency I.max of the monomers was lower than the dimers or trimers, although no significant difference was found in T.tot among the polymer classes. The bond linking the monomeric units had an influence on both sensory properties. The catechin-catechin dimer linked by a 4-6 bond was more bitter than both catechin-(4-8)-catechin and catechin-(4-8)-epicatechin. Astringency was affected by both the specific linkage and the identity of the monomeric units with the dimer, catechin-(4-8)-catechin, being lower in astringency than either catechin-(4-6)-catechin or catechin-(4-8)-epicatechin.

From the back cover:
This English translation of Emile Peynaud's classic work Le Goût du Vin represents the culmination of a lifetime's work and experience, combining scientific fact and professional expertise with a rich sense of the history, tradition and culture of winemaking and wine appreciation. For the modern wine professional, The Taste of Wine is a masterclass on the science, procedures and vocabulary of wine tasting as currently practised at the highest level; for the amateur oenologist, it is both an essential work of reference and the key to enjoyment of this infinitely various subject.

Emotions have a strong influence on the perception of visual and auditory stimuli. Only little is known about the relation between emotional stimulation and olfactory functions. The present study investigated the relationship between the presentation of affective pictures, olfactory functions, and sex. Olfactory performance was assessed in 32 subjects (16 male). Olfactory sensitivity was significantly reduced following unpleasant picture presentation for all subjects and following pleasant picture presentation for male subjects only. Pleasantness and intensity ratings of a neutral suprathreshold odor were related to the valence of the pictures: After unpleasant picture presentation, the odor was rated as less pleasant and more intense, whereas viewing positive pictures induced a significant increase in reported odor pleasantness. We conclude that inducing a negative emotional state reduces olfactory sensitivity. A relation to functional deviations within the primary olfactory cortices is discussed.

Fresh, unfermented cocoa beans (Theobroma cacao seeds) contain approximately 2% w/w epicatechin and almost exclusively epicatechin based procyanidin oligomers and polymers. These are concentrated largely in the flesh of the beans. Together with commonly encountered epicatechin oligomeric procyanidins, three new procyanidin natural products were isolated: epicatechin-(2 beta leads to 5,4 beta leads to 6)-epicatechin; 3T-O-beta-D-galactopyranosyl-ent-epicatechin-(2 alpha leads to 7,4 alpha leads to 8)-epicatechin; 3T-O-L-arabinopyranosyl-ent-epicatechin-(2 alpha leads to 7,4 alpha leads to 8)-epicatechin.

Communication from the Chocolate technology International ZDS Symposium, December 14-16, 2004, Köln

A general cocoa aroma development diagram was presented at ChocoTecnic'98. The diagram enabled characterization of the relative sizes of the volatile fractions of thermal or non-thermal origin. However, it was based on dried cocoas and could therefore not be used to distinguish between the respective roles played by fermentation and drying. To that end, a set of experiments was conducted under a cooperative project with Venezuela on Criollo cocoa aroma quality. As these are very recent results, we shall present them without going into reaction mechanism aspects. Cocoas were fermented in cubic (60 cm) wooden boxes for 4 days. The following fermentation conditions were studied. - 2 pod opening delays: 0 days and 5 days, - 3 turning rates: every 24 h, after 48 h, and after 24 and 72 h, - 3 harvest dates per pod opening delay. A sample was taken each day and divided into 2 parts; one was frozen at -80°C, the other was sun-dried. Volatile compounds were extracted by steam distillation and analysed by GC-MS (DB Wax column, 60 m x 0.32 mm). The fermentation time and harvest date were the only two factors that caused any significant differences in volatile compound contents between the experiments, the former being by far the more important. Fresh Criollo cocoa beans contained a qualitatively very large volatile fraction: 94 compounds were characterized. During cocoa drying, 26 new compounds appeared. This volatile fraction was much more developed than that of Forastero cocoas. During fermentation, it was primarily the alcohol and acid fractions that developed. The overall aldehyde, pyrazine, furan and phenol contents were in much smaller quantities and increased steadily during this operation. When compounds were considered individually, their contents evolved in several ways: 56 remained constant, 7 decreased, 19 increased and 10 followed a bell curve. The effect of drying was measured by the variation in volatile compound contents before and after drying. For instance, during this operation, overall alcohol contents (particularly ethanol) and acid contents (mainly acetic) decreased substantially. The aldehyde, ketone, pyrazine and pyrrole contents increased after one day's fermentation, whereas the phenol content and, to a lesser degree, the ester content decreased. Taken individually, the compound contents evolved in different ways: 43 appeared, 32 remained constant, 2 decreased, 73 increased and 11 followed a bell curve. The initial results of an analysis of the volatile fractions of cocoa sampled daily during 8 days of drying indicated that changes during drying were complex.

This study measured the ethanol and acetic acid in the cocoa pulp at intervals during fermentation. From these figures, the consumption of oxygen and the production of heat of the mass were calculated. The results show that there is a surprisingly high consumption of oxygen, and that the pulp should be aerobic in all except the first two days of the fermentation. Also, the fermentation and oxidation reactions in the pulp produce sufficient heat to explain the high temperatures of the fermentation; the reactions in the cotyledons do not need to be taken into account. The results underline the importance of ventilation and the need for a better comprehension of the methods by which ventilation can be controlled.

In this study, commercial Malaysian cocoa beans (SMC1A) were roasted in a forced airflow-drying oven for 20, 30, 40 and 50 min at 120, 130, 140, 150, 160 and 170°C. The products were evaluated for flavor compounds and sensory evaluation (as dark chocolate). The volatile fraction was isolated using the combined steam distillation-extraction procedure and was identified by gas chromatography-mass spectrometry. A quantitative descriptive analysis was used to evaluate the flavor intensity of the chocolates using a 9-point rating scale for selected flavor attributes, namely astringency, bitter taste, sour taste, cocoa and burnt. Panelists were asked to smell and taste the sample against a standard chocolate. It was found that there were significant differences in flavor compounds between the different conditions of roasting. The main flavoring compounds identified composed of aliphatic and alicyclic groups such as alcohol and ester, and heterocyclic groups such as pyrazine and aldehyde. A total of 19 volatile major components were identified: nine pyrazines (2,5-dimethyl-, 2,3-dimethyl-, 2-ethyl-6-methyl-, trimethyl-, 3-ethyl-2,5-dimethyl-, tetramethyl-, 2-ethenyl-6-methyl- and 3,5-dimethyl-2-methylpyrazine); five aldehydes (5-methyl-2-phenyl-2-hexenal, benzaldehyde, benzalacetaldehyde and α-ethyliden-benzenacetaldehyde); one methyl ketone (2-nonanone); two alcohols (linalool and 2-heptanol); and two esters (4-ethylphenyl acetate and 2-phenylethyl acetate). Based on the flavor profile of the compounds identified, an optimum production of the major flavoring compounds such as pyrazine, aldehyde, ketone, alcohol and ester occurred at 160°C for 30 min of roasting. Trimethylpyrazine and tetramethylpyrazine compounds together with 5-methyl-2-phenyl-2-hexanal were found to be good indicators for the evaluation of the roasting process. However, based on chocolate evaluation, the best roasting temperature was 150°C for 30 min, which gave the lowest astringency and at the same time gave the lowest bitter taste and low level of sour and burnt tastes. At 150°C roasting temperature, the desirable cocoa flavor was at its optimum. Correlation coefficients among certain volatile flavor and sensory characteristics of cocoa beans and dark chocolate were significant (P < 0.05).

The aroma of wine consists of 600 to 800 aroma compounds, many of which, especially those typical of the variety, are already present in the grapes. There are significant varietal differences between the aromagrams ('fingerprint patterns'). Thus the amount of some flavour compounds ('key substances') shows typical dependence on the variety. Especially monoterpene compounds play an important role in the differentiation of wine varieties. The German white wines can be differentiated into three groups only by quantitative determination of 12 monoterpenes ('terpene profile'). These groups are: 'Riesling type', 'Muscat type' and 'SilvanerWeißburgunder type'. Such 'terpene profiles' are also useful for the separation of real Riesling wines from others called Riesling (e.g. Welschriesling, Kap Riesling, Emerald Riesling) but not produced from grapes of the variety Riesling. Including further components and by means of statistical methods (discriminant analysis) even the different varieties within the mentioned groups for instance the 'Riesling' group (e.g. Riesling, Kerner, Ehrenfelser, Bacchus, Müller-Thurgau) can be separated from each other. An analytical characterization of the neutral ('Silvaner-type') grape varieties Silvaner, Rulánder (Pinot gris), Weißburgunder (Pinot blanc) is also possible with about 20 compounds (e. g. monoterpenes, alcohols). Computing at the same time free and glycosidically bound aroma components (monoterpenes, alcohols, norisoprenes) in discriminant analysis the characterization of the neutral grape varieties can be considerably improved. To identify compounds causing 'off-flavours' sniffing technique is the method of choice. The off-flavour is pinpointed during gas chromatographic separation of the complex aroma mixture by effluent sniffing. Once allocated, the chemical nature of the off-flavours is elucidated by spectroscopic methods. Substances contributing to the green pepper taint, the strawberry note, moussiness, corkiness, etc. in wine could be found in this way.

Roasting-induced change to carbohydrates and cell wall polysaccharides was investigated in three varieties of cocoa beans. The concentrations of glucose and fructose decreased after roasting but levels of the non-reducing sugars, sucrose, raffinose, stachyose and verbascose, were not markedly affected. Approximately 10% of the arabinose content of the polysaccharides was degraded but, overall, the pectic and hemicellulosic polymers remained intact after roasting. The degree of esterification and acetylation of the pectic polysaccharides were unaffected by roasting. Roasting did promote an interaction between polysaccharides, proteins, polyphenolics and Maillard products. This led to the formation of insoluble complexes which co-purified with, and augmented, the levels of cell wall material isolated from roasted compared to unroasted beans. The implications of the results are discussed in relation to the role that "Klason lignin" plays in the formation of these chemical amalgams during roasting.

Glc of the silylated sugars fraction of cocoa beans revealed fructose, sorbose, glucose, sucrose, inositol, mannitol, a pentitol, and traces of two unidentified sugars. The same sugars were present in all samples, irrespective of geographic origin. Relative concentrations were, however, quite different, even among lots of the same type of bean. Most of these differences were attributed to harvesting, fermenting, and drying variables. Unfermented Sanchez beans were 1% by wt sucrose, but fermented Bahia and Ghana beans averaged only 0.05 and 0.12% sucrose, respectively. Fermented varieties contained 2 to 16 times more fructose than glucose. The preferential consumption of the glucose moiety of sucrose during fermentation has implications important to the development of chocolate flavor during roasting. Results suggest that absorbed and occluded water-soluble constituents from the pulp contribute to the reducing sugars content of cocoa beans.

Glc of the pyrazine fraction from roasted cocoa beans yielded nine well resolved peaks which could be quantitated. When beans from several producing countries were roasted under identical conditions, pyrazines generated varied between 142 pg/ 100 g of beans and 698 pg/l00 g. The potential for generating pyrazines was greatest in samples from countries where beans are traditionally fermented. Tetramethylpyrazine, trimethylpyrazine, and pyrazines under a peak representing a mixture of 2-ethylpyrazine, 2,5-dimethylpyrazine, and 2,6-dimethylpyrazine were present in the highest concentrations. In fermented cocoa beans, pyrazine concentration increased rapidly during roasting to a near maximum value which did not change during extended roasting. Results indicate that fermentation influences the rate of formation and final concentration of pyrazines in roasted beans primarily through its effect on the free sugars. Ketoses dominated the sugars fraction (62x of total) in well fermented beans compared to 21 in nonfermented varieties. Tetramethylpyrazine was the only pyrazine detected in unroasted beans and then only in fermented samples.

The cocoa relatives T grandiflorum (cupuaçu) and T bicolor (macambo) are promising crop plants for sustainable agroforestry in the Amazon region of South America. The market for cupuaçu is expanding since the fruit flesh is utilised by the foodstuffs industry. Attempts to commercialise chocolate-like wares from the seeds have failed so far because of unreliable product quality. It is not known whether this is due to an insufficient aroma potential of cupuaçu seeds. We therefore investigated the proteolytic enzymes and the seed storage globulins which are both decisive for the formation of aroma precursors in cocoa. We found that the activities of the aspartic endopeptidase and the carboxypeptidase in T bicolor and T grandiflorum differed slightly from those in cocoa. The specificity of the carboxypeptidase for hydrophobic amino acids was quite similar across the three species, while the optimal pH of the T grandiflorum enzyme was lower than that of the other species. The qualitative and quantitative differences between the globulins indicate a lower maximum yield of aroma precursors in T grandiflorum and a higher maximum yield of aroma precursors in T bicolor, compared to cocoa. We conclude that the quality of chocolate-like products made from the studied cocoa relatives can be improved by adapting fermentation procedures to particular biochemical features of these seeds.

The destruction of free amino acids during a factory roast of Accra cocoa beans was studied. Different amino acids were destroyed at different rates, but none were destroyed completely. Of the total free amino acids present in the unroasted beans, some 50% were found in the roasted product.

The destruction of reducing sugars during the roasting of cocoa beans was investigated and found to be almost complete. The significance of this observation is discussed in relation to the possible role played by the reducing sugars in the deamination of the free amino acids of the cocoa bean, and to flavor development.

The sucrose in fresh cocoa beans is hydrolyzed to glucose and fructose during fermentation and the rate of the reaction confirms the possible inclusion of reducing sugars among the precursors of chocolate aroma. The optimal concentration of reducing sugars in the bean is reached at about the same time as maximal flavor development, and coincides approximately with the peak in amino acid concentration. An objective method for assessing the "Degree of Fermentation" of cocoa beans has been proposed and tested.

A preliminary study of the Strecker degradation of amino acids by reducing sugars in cocoa beans has revealed an unexpected temperature effect on the extent of the reaction which might influence the flavor of the product. Model amino acid/sugar systems were studied and the results support the hypothesis that there exists a relationship between the temperature of reaction, the extent of amino acid degradation and the production of flavor volatiles during the roasting of cocoa beans. A factory experiment, in which Accra cocoa beans were roasted at three different temperatures, provided supporting evidence of the influence of temperature on total flavor and on the strength of basic chocolate flavor.

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 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.

From the back cover:
The human organs of perception are continually being bombarded with chemicals from the environment. Our bodies have in turn developed complex processing systems that manifest themselves in our emotions, memory, and language. Yet the available data on the high-order cognitive implications of taste and smell are scattered among journals in many fields, with no single source synthesizing the large body of knowledge, much of which has appeared in the past decade.
This book presents the first multidisciplinary synthesis of the literature in olfactory and gustatory cognition. The book is conveniently divided into sections, including linguistic representations, emotion, memory, neural bases, and individual variation. Leading experts have written chapters on many facets of taste and smell, including odor memory, cortical representations, psychophysics and functional imaging studies, genetic variation in taste, and the hedonistic dimensions of odors. The approach is integrative, combining perspectives from neuroscience, psychology, anthropology, philosophy, and linguistics, and is appropriate for students and researchers in all of these areas who seek the authoritative reference on olfaction, taste, and cognition.

The maximum amounts of acetic acid produced by ripe and unripe cocoa beans were 157 mg and 110 mg/10 g wet wt of cotyledon, respectively. The unripe beans had a lower pH than the ripe beans after 6 days' fermentation. About 40% of ripe beans achieved a chocolate colour compared with 27% of unripe beans.

Cocoa fermentations were performed in wooden boxes under the following four experimental regimens: beans naturally fermented with wild microflora; aseptically prepared beans with no inoculum; and beans inoculated with a defined cocktail containing microorganisms at a suitable concentration either at zero time or by using phased additions at appropriate times. The cocktail used consisted of a yeast, Saccharomyces cerevisiae var. chevalieri, two lactic acid bacterial species, Lactobacillus lactis and Lactobacillus plantarum, and two acetic acid bacterial species, Acetobacter aceti and Gluconobacter oxydans subsp. suboxydans. The parameters measured were cell counts (for yeasts, filamentous fungi, lactic acid bacteria, acetic acid bacteria, and spore formers, including reisolation and identification of all residual cell types), sugar, ethanol, acetic acid, and lactic acid contents (and contents of other organic acids), pH, and temperature. A cut test for bean quality and a sensorial analysis of chocolate made from the beans were also performed. The natural fermentation mimicked exactly the conditions in 800-kg boxes on farms. The aseptic box remained largely free of microflora throughout the study, and no significant biochemical changes occurred. With the zero-time inoculum the fermentation was almost identical to the natural fermentation. The fermentation with the phased-addition inoculum was similar, but many changes in parameters were slower and less pronounced, which led to a slightly poorer end product. The data show that the nearly 50 common species of microorganisms found in natural fermentations can be replaced by a judicious selection and concentration of members of each physiological group. This is the first report of successful use of a defined, mixed starter culture in such a complex fermentation, and it should lead to chocolate of more reliable and better quality.

The first stage of chocolate production consists of a natural, seven-day microbial fermentation of the pectinaceous pulp surrounding beans of the tree Theobroma cacao. There is a microbial succession of a wide range of yeasts, lactic-acid, and acetic-acid bacteria during which high temperatures of up to 50°C and microbial products, such as ethanol, lactic acid, and acetic acid, kill the beans and cause production of flavor precursors. Over-fermentation leads to a rise in bacilli and filamentous fungi that can cause off-flavors. The physiological roles of the predominant micro-organisms are now reasonably well understood and the crucial importance of a well-ordered microbial succession in cocoa aroma has been established. It has been possible to use a synthetic microbial cocktail inoculum of just 5 species, including members of the 3 principal groups, to mimic the natural fermentation process and yield good quality chocolate. Reduction of the amount of pectin by physical or mechanical means can also lead to an improved fermentation in reduced time and the juice can be used as a high-value byproduct. To improve the quality of the processed beans, more research is needed on pectinase production by yeasts, better depulping, fermenter design, and the use of starter cultures.

3 cocoa varieties grown in low country intercrops and traditional mid-country plantations of Sri Lanka were compared. Significantly different temperature and pH changes occurred during fermentation of the varieties. The "sweat box" fermentation method, with mixing at 12 h intervals, could be used to obtain a satisfactory (85 per cent) level of fermentation. The optimum duration of fermentation for all varieties irrespective of location of growth was 6 days. These findings could help to upgrade the cottage scale industry in Sri Lanka.

Four mixing intervals (6, 12, 18 and 24 h) were compared with special reference to three selected cacao varieties from two locations of Sri Lanka. Cocoa beans were fermented for 6 days by using the "sweat box" fermentation method. Irrespective of location and variety, product prepared by using all mixing intervals were significantly different with variations in temperature as well as degree of fermentation. Frequent mixing (at 6 and 12 h) produced a higher number of well-fermented beans than other treatments. When practical aspects of fermentation in both small and large scale contexts are considered, mixing at 12 h intervals for 6 days can be recommended as the most suitable treatment for three varieties tested from two locations.

Notes from study regarding pH of pulp and cotyledon:
pH of fresh pulp: 3.5 - 4
pH of fresh cotyledons: 5.5 - 6.5
"Acid production is dependent on aeration (Quesnel 1968)"

A sample of 94 accessions of Theobroma cacao L. (cacao), representing four populations from the Brazilian Amazon (Acre, Rondônia, lower Amazon and upper Amazon) were analyzed using microsatellite markers to assess the genetic diversity and the natural population structure. From the 19 microsatellite loci tested, 11 amplified scorable products, revealing a total of 49 alleles, including two monomorphic loci.
The Brazilian upper Amazon population contained the largest genetic diversity, with the most polymorphic loci, the highest observed heterozygosity; and the majority of rare alleles, thereby this region might be considered part of the center of diversity of the species.
The observed heterozygosity for all the Brazilian populations (H_o = 0.347) was comparable with values reported for other similar upper Amazon Forastero cacao populations, with the Acre and Rondônia displaying the lowest values. The lower Amazon population, traditionally defined as highly homozygous, presented an unexpectedly high observed heterozygosity (H_o = 0.372), disclosing rare and distinct alleles, with large identity with the upper Amazon population. It was hypothesized that part of the lower Amazon population might derive from successive natural or intentional introduction of planting material from other provenances, mainly upper Amazon. Most of the loci exhibited a lower observed heterozygosity than expected, suggesting that self-pollination might be more common than usually assumed in cacao, but excess of homozygotes might also derive from sub-grouping (Wahlund effect) or from sampling related individuals. Most of the gene diversity was found to occur within groups, with small differentiation between the four Brazilian Amazon populations, typical of species with high gene flow.

Sensory-guided decomposition of roasted cocoa nibs revealed that, besides theobromine and caffeine, a series of bitter-tasting 2,5-diketopiperazines and flavan-3-ols were the key inducers of the bitter taste as well as the astringent mouthfeel imparted upon consumption of roasted cocoa. In addition, a number of polyphenol glycopyranosides as well as a series of N-phenylpropenoyl-L-amino acids have been identified as key astringent compounds of roasted cocoa. In the present investigation, a total of 84 putative taste compounds were quantified in roasted cocoa beans and then rated for the taste contribution on the basis of dose-over-threshold (DoT) factors to bridge the gap between pure structural chemistry and human taste perception. To verify these quantitative results, an aqueous taste reconstitute was prepared by blending aqueous solutions of the individual taste compounds in their "natural" concentrations. Sensory analyses revealed that the taste profile of this artificial cocktail was very close to the taste profile of an aqueous suspension of roasted cocoa nibs. To further narrow down the number of key taste compounds, finally, taste omission experiments and human dose/response functions were performed, demonstrating that the bitter-tasting alkaloids theobromine and caffeine, seven bitter-tasting diketopiperazines, seven bitter- and astringent-tasting flavan-3-ols, six puckering astringent N-phenylpropenoyl-L-amino acids, four velvety astringent flavonol glycosides, -aminobutyric acid, -aminoisobutyric acid, and six organic acids are the key organoleptics of the roasted cocoa nibs.

From the back cover:
Today's chemists need to know how hazardous the chemicals they work with are, and they want to understand the relationship between chemical and structural properties and toxicity. At the same time, modern society requires that chemists have this kowledge, as legislation makes the producers/users of chemicals responsible for any adverse effects. The book deals with the effects on both man and ecosystems. It stresses especially on the relationship between chemical structure and chemical properties/toxic effects and metabolic conversions. This is not just another book on toxicology. What makes this book special is that it is written from a chemical point of view. This textbook applies the basic principles of reactivity and reaction possibilities of organic compounds to metabolic reactions and living systems.

From p.37, on the subject of volatility:
[...] the principal route for exposure to chemicals in the workplace is via the lungs, and the chemicals inhaled are present in the air either as a vapour or as an aerosol. A compound's volatility will therefore to a large extent determine the risk for a chemical to be absorbed by persons exposed to it at workplaces, as well as influence its distribution in the environment. Many chemicals are liquid at room temperature but evaporate readily. A liquid stay liquid because the intermolecular forces hold the molecules together. However, by increasing the kinetic energy of the molecules, by for example increasing the temperature, molecules may become energetic enough to break free from the intermolecular forces and pass into the gaseous phase. Even at temperatures much lower than the boiling point, a small proportion of the molecules will have sufficient kinetic energy to leave the liquid. Therefore, all liquids have a vapour pressure at all temperatures, and wet clothes will dry at room temperature (and even at temperatures below 0°C when the water is frozen). When the temperature increases the vapour pressure does the same, and at temperatures when the vapour pressure is the same as the air pressure the liquid will boil. [...] the molecular weight of the compound in question may also be important.

An optimised protocol for organoleptic assessment of cocoa liquors was developed and used to determine what were the flavour attributes and sensory differences between samples of Ghana beans (normally classified as "bulk") and bean samples from local commercial clones and estates in Trinidad (normally classified as "fine or flavour" cocoa). The optimised protocol was validated by independent sensory assessment carried out at Masterfoods, UK. Trinitario samples from four local commercial clones and five commercial estates from Trinidad were investigated for their liquor quality, over three crop years using the optimised sensory panel and an appropriate sensory design with replications. The optimised protocol was not only able to consistently differentiate between the "bulk" and "fine or flavour" cocoa types but also able to consistently quantify the level of each attribute in genotypes, over replication and season. This method was used to delineate quantitative differences in flavour attributes among cocoa genotypes as well as to determine the influence of environment on cocoa flavour profiles. The optimised organoleptic protocol involves a detailed description of primary and secondary processing of beans, preparation of liquor, panellist selection, training, sensory design and data analysis methods. The optimised organoleptic protocol provides a robust methodology to improve cocoa quality by selection of genotype and environment.

See also:

INIAP (2007)
Project to Establish the Physical, Chemical and Organoleptic Parameters to Differentiate between Fine and Bulk Cocoa

Sukha and Butler (2005)
The CFC/ICCO/INIAP Cocoa Flavour Project - Investigating the Spectrum of Fine Flavour within Genotypes and between Origins
INGENIC Newsletter, Issue No. 10, Sep. 2005
Excerpt:
Individual flavour profiles of the different country clone samples provide interesting insight into the spectrum that exists under the designation "fine or flavour".
The Venezuelan Criollos (Guasare and Criollo Merideño) are organoleptically the most distinct set of samples with very nutty and some raw/beany flavour notes, however, they possess a unique caramel/malt/fudge-like flavour attribute that no other set of samples assessed under the purview of the CFC/ICCO/INIAP Cocoa Flavour Project possessed.
Ecuador Nacional types (CCAT clones) all contain elements of nutty flavour, but also a unique aromatic "floral" attribute that tends to be a combination of "herbal", "forest green" and moderate "fresh flower" aromas. This was quite distinct from the very sweet and pungent perfume and citrus-like floral attribute associated with CRU 2000. The other coded Trinidad clones, CRU 2001, 2002 and 2017 possessed a mixture of moderately acid "raisin" and "brown fruit" flavour notes, which were different from the fresh fruit, almost "banana"-like fruitiness, of the "KA" accession group from PNG.
The aromatic flavour attributes from the different country clones were, in all instances, found to contrast sharply to the dominant cocoa flavour and nutty attributes found in the Ghana reference sample.

Proteins were extracted from the seeds of Brazilian Comun cacao with a urea buffer and purified on a Sephadex G-25 column. The protein extract was then separated into eight groups using stepwise changes in pH on a Sephadex SP-25 cation exchange column. Each protein group was significantly different in amino acid composition from every other group. Based on this method, 13 amino acids should be considered when classifying various cacaos according to genetic origins. Glutamic acid, alanine, and arginine were most variable. Least likely to reflect differences among protein groups were lysine, histidine, cysteine, and methionine.

Boiling water extracts were cooled, centrifuged and injected into a high-pressure liquid chromatograph. Total alkaloid content among varieties ranged from 24-50 mg/g defatted cocoa with an average of 37 mg for 10 samples. Theobromine's share of the total ranged from 52-99%, averaging 87%. Among samples, Criollo had the lowest concentration of total alkaloids, but its caffeine content was highest. Alkaloid accumulation in seeds was greatest near the terminal stage of pod growth, and this extended into the ripening phase. One-fourth of the alkaloid content of cotyledons was lost during fermentation through migration into the testa and pulp sweatings.

Science at the Crossroads: Papers Presented to the International Congress of the History of Science and technology Held in London from June 29th to July 3rd, 1931 by the delegates of the U.S.S.R, Frank Cass and Co., 1931

Introduction:
Where are the beginnings of agriculture to be sought? Were they independent in different regions, in different continents? How is the geographical localisation of primitive agriculture to be explained? Which plants were first brought into cultivation? Which animals were first domesticated, and where? Where shall we find the primary sources of cultivated plants? How are modern domesticated held animals and cultivated plants connected with their wild related types? How did the evolution of cultivated plants and animals proceed? How are primary agricultural civilizations connected? Which implements were used by primitive agriculturists in different regions?

Viewed from the standpoint of concrete materialistic studies all these historical questions are very actual, and of great significance for modern agriculture. In contradistinction to past practice, the present-day investigator, faced with increasingly difficult economic conditions in the world, attempts to utilise the experience of the past in order to improve upon existing practice. In the Soviet Union, which is now building up socialism and socialistic agriculture, we are interested in the problem of the origin of agriculture, and of the origin of cultivated plants and animals chiefly from the dynamic viewpoint. By knowledge of the past, by studying the elements from which agriculture has developed, by collecting cultivated plants in the ancient centres of agriculture, we seek to master the historical process. We wish to know how to modify cultivated plants and domestic animals according to the requirements of the day. We are but slightly interested in the wheat and barley found in the graves of Pharaohs of the earliest dynasties. To us, constructive questions--problems which interest the engineer--are more urgent. It is much more important for us to know how Egyptian wheat differs from wheats of other countries, which characteristics in this Egyptian wheat are of importance in order to improve our wheat, to understand how this Egyptian wheat has originated. The investigator wishes to find the primary elements, "the bricks and mortar," from which the modern species and varieties were created. We need this knowledge in order to possess the initial material for practical plant and animal breeding. We study the construction of primitive agricultural implements in order to get indications for the construction of modern machinery.

In brief, the historical problems of this origin of agriculture, of the origin of cultivated plants and domesticated animals are especially interesting for us in the sense of mastering and controlling the breeding of cultivated plants and animals.

A range of structurally defined apple and grape proanthocyanidins was isolated in sufficient amount to carry out a formal sensory descriptive analysis study. Purified proanthocyanidin fractions differed in chain length, degree of galloylation and epigallocatechin content. Astringency attributes of the preparations in a model wine medium were rated while the fractions were held in the mouth and after expectoration. The degree of polymerization appeared to be the variable that discriminated among the fractions to the greatest extent. It affected both the overall astringency and the different individual astringency attributes, with increased drying, chalky, adhesive and pucker characters correlating with increasing chain length. A rougher sensation with increased coarseness, drying and chalkiness correlated with an increased degree of galloylation of the fractions. The presence of epigallocatechin units in the proanthocyanidin tended to lower the coarse perception.

The proteolytic formation of the cocoa-specific aroma precursors was investigated in vitro using protein substrates and proteases purified from ungerminated cocoa seeds. An aspartic endoprotease and a carboxypeptidase present in ungerminated cocoa seeds were found to be required for this process. Cocoaspecific aroma precursors were obtained by proteolytic digestion of the vicilin-like globulin but not by proteolysis of the albumin of cocoa seeds

Most of the thousands of grapevine cultivars (Vitis vinifera L.) can be divided into two groups, red and white, based on the presence or absence of anthocyanin in the berry skin, which has been found from genetic experiments to be controlled by a single locus. A regulatory gene, VvMYBA1, which could activate anthocyanin biosynthesis in a transient assay, was recently shown not to be transcribed in white berries due to the presence of a retrotransposon in the promoter. We have found that the berry colour locus comprises two very similar genes, VvMYBA1 and VvMYBA2, located on a single bacterial artificial chromosome. Either gene can regulate colour in the grape berry. The white berry allele of VvMYBA2 is inactivated by two non-conservative mutations, one leads to an amino acid substitution and the other to a frame shift resulting in a smaller protein. Transient assays showed that either mutation removed the ability of the regulator to switch on anthocyanin biosynthesis. VvMYBA2 sequence analyses, together with marker information, confirmed that 55 white cultivars all contain the white berry allele, but not red berry alleles. These results suggest that all extant white cultivars of grape vines have a common origin. We conclude that rare mutational events occurring in two adjacent genes were essential for the genesis of the white grapes used to produce the white wines and white table grapes we enjoy today.

The evolution of selfing vs. outbreeding has been of major interest to plant population biology. The independent historic introductions of self-compatible (Criollo) and self-incompatible (Forastero) genotypes of cacao in Trinidad have allowed us to study the selection acting upon an unnatural breeding system polymorphism. Field observations of an abandoned cacao plantation indicate that the self-incompatible phenotype has slightly increased in frequency within a single generation. The self-compatible (Criollo) trees produced significantly less flowers but still set more pods than did the self-incompatible (Forastero) trees, although compatibility types did not differ in tree size or mature seed production.
Greenhouse observations suggest that the apparent failure of self-compatibility to increase in the population is related to inbreeding depression resulting from selfing, expressed as reduced seedling establishment.

Criollo: self-compatible
Forastero: self-incompatible
Hybrids (Trinitario): either self-compatible or self-incompatible

Organic acids precipitated as lead salts from water extracts of cocoa beans were converted to trimethylsilyl ethers. TMS ethers were then separated and identified using a combined gas chromatograph-mass spectrometer. Citric, phosphoric, lactic, oxalic, malic, tartaric, succinic and gluconic acids were present in every sample examined, irrespective of geographic origin. Five of the acids were quantitated using a GLC procedure which involved the preparation of methyl esters from freeze-dehydrated, water extracts. Concentration ranges found, g/100g beans, were: lactic, 0.11-0.71; oxalic, 0.24-0.43; succinic, 0.02-0.07; malic, 0.02-0.10; citric, 0.56-1.32. Concentrations of lactic acid were lowest and citric acid levels tended to be highest in commercial samples from countries where cocoa beans usually are not subjected to a planned fermentation. These trends were confirmed in an experiment involving beans from Trinidad which had undergone a carefully controlled fermentation. Only minor differences were found in organic acid concentrations between roasted and unroasted cocoa beans.

[Zur Verbreitungsgeschichte und Ethnobotanik indianischer Kulturpflanzen, insbesondere des Kakaobaumes]

Amerindian coastal shipping along the Pacific coast of America has been neglected for the dispersal of pre-Columbus crop plants up to now. Indians of the Valdivia Culture and their successors travelled by boat or raft along the coasts from their west Ecuadorian home to Peru and Middle America since 2200 BC and to Southern Mexico since 1450 BC. The travelling ceased with the arrival of Spaniards in 1526. Presumably crop plant export occurred from western Ecuador to Peru and Middle America (sweet manioc, Annona cherimola, Carica papaya , early great-grained corn) and Mexico (tobacco). On the other hand the pre-Columbus crops (Persea americana, Capsicum annuum ) evolved in Mexico were brought to western Ecuador and neighbouring northern Peru. Shipment by sea would be faster and thus more successful than the time consuming transportation by land all the way through Colombia and Central America. For sweet manioc, only shipment of living cuttings appears as a feasible explanation. Similarly one may hypothesize that living plants of Theobroma cacao were shipped from South to Middle America on this Pacific sea route. Since neither wild cacao trees nor primitive cultigens are found in the area between Ecuador and Guatemala, thus the land route is highly unlikely. In the Mesoamerican Pre-classic Culture region the existence of cacao tree is proven since 500 BC, about thousand years later than the first contact of west-Ecuadorian Indians with Mexico. The use of T. cacao in indigenous medicine is reported. Surprisingly there is no evidence for pre-Columbus domestication of cacao in its natural habitat Amazonia. This may be explained by the great popularity of similar but much stronger acting drugs like the caffeine drug Guaraná and the cocaine drug Erythroxylum coca var. ipadu .

At the beginning of the 19th century cocoa production was confined to Central and South America and some of the Carribean islands plus some plantings of local significance in the East Indies. The total world production in the first five years of the century averaged only 135,000 tonnes.
Great changes were soon to come ...

Taxonomy
Common names
Botanic description
History of cultivation
Natural Habitat
Geographic distribution
Biophysical limits: Altitude: 100-300 m, Mean annual temperature: 26 deg. C, Mean annual rainfall: 1 000-3 000 mm
Reproductive Biology: Cacao is naturally out-breeding, and various insects are associated with its pollination, the main ones being thrips, midges, ants and aphids. It has a complex system of self-incompatibility. After successful pollination, fertilization takes place within 36 hours. The period between fertilization and pod maturation varies from 150 to 180 days.
Propagation methods: Cocoa seeds readily germinate when sown and do not pass through a dormancy period. They lose viability within 5-7 days of extraction from the pod unless specially treated, and germinate within 7-10 days. The plant can easily be propagated vegetatively by leaf-bud cutting, multiple-bud cutting, marcotting, budding, grafting and layering.
Tree Management: Farmers plant cocoa at high densities of 3000-4000 trees/ha because the resulting tall trees develop fewer lateral branches and more vertical suckers. This encourages flowering on the main stem at the expense of branches, particularly suitable for some lower Amazon Forastero varieties.
Germplasm Management: Seed storage behaviour is recalcitrant. Storage temperature between 4 and 15°C is damaging to seed viability and germination. Optimum storage temperature appears to be 17°C. Seeds tolerate desiccation to 25% mc when dried at 20°C, while only about 40-60% survive when dried at 10°C; seeds stored in pods at 5 or 10°C are killed within 2 days, and there is 100% survival when stored in pods at temperatures of 15-30°C for 3 weeks. Viability is reduced from 92% to 18% on desiccation from 45% to 36.7% mc; no seeds survive desiccation to 26% mc; 24% germination after 8 months subimbibed storage (41-42% mc) at 98% rh and 20°C with Thiram fungicide. Similarly, no seeds survive desiccation to below 20% mc, and no fresh seeds survive in storage at 4°C or 15°C.
Functional uses
Products
Pests and diseases
Bibliography

Protein was extracted from cocoa beans with a buffer solution and separated on Sephadex LH20. After dialysis and freeze dehydration protein preparations were characterized according to solubility in different aqueous media and electrophoretic behavior on disc gels. Compared to pigmented varieties, extracts from nonpigmented beans yielded only half as much protein off the Sephadex column, and nondialyzable solids were less pure with respect to protein (60% vs. 30% protein). Electrophoresis of pigmented varieties gave eight protein bands which were more intense and numerous than for nonpigmented types. Differences reflect protein-polyphenol interactions important to quality characteristics of chocolate, and methods used are suggested as adjuncts in breeding programs related to optimization of desirable quality characteristics.

During ripening of cocoa fruit the protein content of seeds decreased 25 percent, but consistent qualitative trends were not apparent. Most of the protein of unfermented beans could be solubilized and recovered. However, at the conclusion of fermentation only about one-third of the protein could be extracted from beans. The extractable protein fractions became progressively less pure during fermentation and at the end were only 40 percent protein. Fewer, more diffuse protein bands were evident on disc gels as fermentation advanced. Most of these changes are believed to involve protein interaction with polyphenols. Somewhat similar trends were noted during roasting and conching.

Background and Aims: Cocoa (Theobroma cacao) is indigenous to the Amazon region of South America, and it is well known that the Peruvian Amazon harbours a large number of diverse cocoa populations. A small fraction of the diversity has been collected and maintained as an ex-situ germplasm repository in Peru. However, incorrect labelling of accessions and lack of information on genetic diversity have hindered efficient conservation and use of this germplasm. This study targeted assessment of genetic diversity and population structure in a managed and a semi-natural population.
Key Results: Ten synonymous mislabelled groups were identified among the 105 accessions. The germplasm group in the Huallaga valley was clearly separated from the group in Ucayali valley by the Bayesian assignment test. The Huallaga group has lower genetic diversity, both in terms of allelic richness and of gene diversity, than the Ucayali group. Analysis of molecular variance suggested genetic substructure in the Ucayali group. Significant spatial correlation between genetic distance and geographical distances was detected in the Ucayali group by Mantel tests.
Conclusions: These results substantiate the hypothesis that the Peruvian Amazon hosts a high level of cocoa genetic diversity, and the diversity has a spatial structure. The introduction of exotic seed populations into the Peruvian Amazon is changing the cocoa germplasm spectrum in this region. The spatial structure of cocoa diversity recorded here highlights the need for additional collecting and conservation measures for natural and semi-natural cocoa populations.

The terpenes present in cocoa flavour were analysed by means of gas chromatography-mass spectrometry. The pyranoid linalool oxides, ocimene and nerolidol, were identified for the first time as cocoa volatiles. Linalool, which is the major terpene component in cocoa contributes markedly to the flowery and tea-like flavour of some cocoa varieties. Flavour grade beans contain relatively high concentrations of linalool and basic grade cocoas possess. The isolation of linalool by means of intensive (simultaneous distillation extraction) and more gentle methods (vacuum condensation) yields similar quantitative results. The estimation of the concentration ratio of linalool/benzaldehyde within steam distillates ist suitable for the industrial quality control of unroasted cocoas.

Quote from Counet et al (2004):
"According to Ziegleder, fine dried fermented cocoa beans (Ecuador, Trinidad, and Venezuela) contain up to 8 times more linalool than bulk-basic ones (Ghana, Ivory Coast, Brazil). Linalool might thus be responsible for the flowery tea note found in fine varieties"