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.