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Ghanaian Cocoa Bean FermentationCharacterized by Spectroscopic andChromatographic Methods and ChemometricsPatrick C. Aculey, Pia Snitkjaer, Margaret Owusu, Marc Bassompiere, Jemmy Takrama, Lars Nørgaard, Mikael A. Petersen, andDennis S. Nielsen

Abstract: Export of cocoa beans is of great economic importance in Ghana and several other tropical countries. Raw cocoahas an astringent, unpleasant taste, and flavor, and has to be fermented, dried, and roasted to obtain the characteristic cocoaflavor and taste. In an attempt to obtain a deeper understanding of the changes in the cocoa beans during fermentationand investigate the possibility of future development of objective methods for assessing the degree of fermentation, anovel combination of methods including cut test, colorimetry, fluorescence spectroscopy, NIR spectroscopy, and GC-MSevaluated by chemometric methods was used to examine cocoa beans sampled at different durations of fermentation andsamples representing fully fermented and dried beans from all cocoa growing regions of Ghana. Using colorimetry itwas found that samples moved towards higher a∗ and b∗ values as fermentation progressed. Furthermore, the degree offermentation could, in general, be well described by the spectroscopic methods used. In addition, it was possible to linkanalysis of volatile compounds with predictions of fermentation time. Fermented and dried cocoa beans from the Voltaand the Western regions clustered separately in the score plots based on colorimetric, fluorescence, NIR, and GC-MSindicating regional differences in the composition of Ghanaian cocoa beans. The study demonstrates the potential ofcolorimetry and spectroscopic methods as valuable tools for determining the fermentation degree of cocoa beans. UsingGC-MS it was possible to demonstrate the formation of several important aroma compounds such 2-phenylethyl acetate,propionic acid, and acetoin and the breakdown of others like diacetyl during fermentation.

Keywords: cocoa, fluorescence, GC-MS, NIR, spectroscopy

Practical Application: The present study demonstrates the potential of using colorimetry and spectroscopic methods asobjective methods for determining cocoa bean quality along the processing chain. Development of objective methods fordetermining cocoa bean quality will be of great importance for quality insurance within the fields of cocoa processingand raw material control in chocolate producing companies.

IntroductionCocoa beans originate as seeds in the fruit pods of the tree

Theobroma cacao. They are the principal raw material of chocolate(Schwan and Wheals 2004). Approximately 2/3 of the world’scocoa is produced in West Africa. Ghana is the world’s 2nd largestproducer accounting for around 20% of the world production(Anon 2007). Being the largest export commodity cocoa is of greateconomical importance for Ghana as a country and of even biggersocio-economic importance in the cocoa growing communitiesand villages around the country.

Raw cocoa has an astringent, unpleasant taste, and flavor andhas to be fermented, dried, and roasted to obtain the characteristic

MS 20090994 Submitted 10/7/2009, Accepted 5/24/2010. Authors Aculey andTakrama are with Cocoa Research Inst. of Ghana (CRIG), Tafo-Akim, Ghana.Authors Snitkjaer, Owusu, Bassompiere, Nørgaard, Petersen, and Nielsen are withDept. of Food Science, Faculty of Life Sciences, Centre for Advanced Food Science(LMC), Univ. of Copenhagen, Denmark. Direct inquiries to author Nielsen (E-mail:[email protected]).

cocoa flavor and taste (Thompson and others 2001). Followingharvest the cocoa pods are opened and the pulp surrounding thebeans spontaneously inoculated with a range of microorganismsthat metabolise pulp sugars producing mainly ethanol and lacticacid. Some of the ethanol is further oxidised to acetic acid byacetic acid bacteria through an exothermal process. The ethanoland acetic acid penetrate the testa and in combination with theheat produced (reaching around 50 ◦C in the fermenting mass)kill the germ and break down cell walls in the bean. This initiatesthe processes leading to well fermented beans (Thompson andothers 2001; Schwan and Wheals 2004; Nielsen and others 2007).Following breakdown of the cell walls in the bean, numerous bio-chemical processes take place leading to the breakdown of proteinsto peptides and amino acids (Hashim and others 1998; Lerceteauand others 1999; Buyukpamukcu and others 2001); and break-down of anthocyanins to anthocyanidins and sugars (Pettipher1986; Wollgast and Anklam 2000). During fermentation and dry-ing the polyphenol content (including anthocyanidins) decreaseas some diffuse out of the beans while others are oxidised andpolymerise to insoluble high-molecular-weight compounds (tan-nins) during fermentation and drying (Pettipher 1986; Wollgast

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Cocoa bean fermentation characterization . . .

and Anklam 2000). This leads to changes in the internal color ofthe dried cocoa beans from the dark grey (slaty) color of the unfer-mented bean through the deep-purple color of under fermentedbeans to the brown color of the fully fermented bean (Pettipher1986; Wollgast and Anklam 2000).

The change in cocoa bean color during the fermentation isexploited in a simple method for determining the degree of fer-mentation called the cut test. In short, a number of beans arecut lengthwise and the internal color assessed. Brown beans areconsidered well fermented, violet beans partly fermented and grey(slaty) beans unfermented. It is generally believed that there is aninverse relationship between the flavor developed and the purplecolor retained in the beans. The method is widely used due to itssimplicity but is not without problems as color determination us-ing the cut test is considered subjective and difficult to standardise(Lopez 1984; Wood and Lass 1985).

Spectroscopic techniques such as NearInfraRed (NIR) spec-troscopy and fluorescence spectroscopy offer valuable alterna-tives to traditional methods for rapid determination of selectedchemical compounds as well as spectroscopic fingerprinting in awide range of food and food stuff matrices (Williams and Nor-ris 2001; Christensen and others 2006). Previously, NIR spec-troscopy has been used to determine quality parameters suchas fat, protein, and carbohydrates in cocoa and cocoa products(Kaffka and others 1982; Permanyer and Perez 1989). More re-cently, Whitacre and others (2003) successfully applied NIR topredict the content of proanthocyanidins in cocoa and Daviesand others (1991) attempted to predict the sensory quality ofthe finished chocolate with NIR measurements of ground rawcocoa beans with promising results. However, previously onlylittle attention has been given to the possibilities of investigat-ing changes during fermentation and none of the mentionedmethods have found commercial use in the cocoa/chocolateindustry.

In an attempt to obtain a deeper understanding of the changesin the cocoa beans during fermentation and investigate the possi-bility of future development of objective methods for assessing thedegree of fermentation, a novel combination of methods includ-ing NIR spectroscopy, fluorescence spectroscopy, colorimetry, andGC-MS evaluated by chemometric methods was used to examinecocoa beans sampled at different times of fermentation. Further-more, to investigate whether local differences in fermentation anddrying practices among farmers influence the quality of the fi-nal product, samples representing fully fermented and dried beansfrom all cocoa growing regions of Ghana were included in thestudy.

Materials and Methods

Cocoa fermentationsCocoa pods were harvested by traditional methods during Oc-

tober (average ambient temperature during the day 28 to 30 ◦C)in Mixed Hybrid Cocoa plantations in Ghana, near New Tafo(Eastern Region). The pods were harvested over 3 to 7 d andopened on the following day with a cutlass at the Cocoa ResearchInsti. of Ghana (CRIG) where the fermentation were carried outas well.

Following opening of the pods, the beans were placed inwooden trays (122 × 91 × 10 cm with a raffia mat at the bottomof each tray) with approximately 100 kg beans placed in each tray.Total of 8 trays were stacked on top of each other and the top tray

covered with plantain leaves. The whole stack was left to fermentfor 6 d.

At 24 h intervals approximately 300 g beans were sampled witha sterile plastic bag. After sampling the samples were divided into2 portions (12 samples), with one portion being sun-dried for 6to 10 d on raised raffia-mats and one portion being dried in afan-assisted air extraction oven at 50 ◦C for 3 d. After sun-dryingall samples were finally dried in a fan-assisted air extraction ovenat 50 ◦C for 48 h.

Furthermore, 26 samples representing fully fermented and dried(“ready-to-sell”) cocoa beans from farms representing all co-coa growing regions (Western, Brong-Ahafo, Ashianti, Central,Volta and Eastern region) of Ghana and 4 samples represent-ing fermentations carried out at CRIG were included in thestudy. The beans were bought directly from the farmers and sub-jected to same analyses as the beans sampled during the controlledfermentations.

The samples were airlifted to Denmark and stored at – 20 ◦Cuntil further analysis.

Cut testCut test was carried out basically as described by Wood and Lass

(1985) using a Magra Cutter (Model 12, Teserba, Switzerland).

Sample preparation for analysisTotal of 100 beans from each sample were ground in liquid

nitrogen using a coffee grinder (Braun). The grounded powderwas stored at – 20 ◦C until further analysis. To investigate thedifference between whole and de-shelled beans, samples of sun-dried beans from a tray fermentation (6 samples) carried out atCRIG were divided into 2 sub-samples. One sub-sample wascarefully de-shelled before grinding, and 1 sub-sample was groundas whole beans yielding a total of 12 samples.

Color measurement (lab system)The surface color of each sample was measured using a tris-

timulus color analyzer for measuring reflective colors of surfaceswith a Chroma Meter CR-300 (Minolta Camera Co. Ltd, Osaka,Japan). The instrument was standardised using a white calibrationplate (Calibration Plate CR-A43). Total of 20 g of ground co-coa beans was transferred into a white plastic dish and the surfacecolor determined. The colorimeter was set to perform 3 repeatedscans for each measurement and the measurements were done at4 different places on each sample giving 4 replicates per sample(Møller and others 2000).

Fluorescence spectroscopy—BioViewAuto-fluorescence was measured on a BioView spectrofluo-

rometer from Delta Lights & Optics (Lyngby, Denmark), with apulsating xenon lamp exciting at 20 nm broad bands from 260 to560 nm using rotating filters. The emission landscapes consistingof 20 nm broad bands from 300 to 600 nm were provided also bythe use of rotating filters and recollected in the BioView program(Svenstrup and others 2005). The samples were measured using afiber optic probe with direct contact to the sample analyzed. Onescan was performed per measurement. Each sample was measuredin duplicate.

Near infrared (NIR) spectroscopic analysisNear infrared spectra were acquired in reflectance mode, on

a spectrophotometer from Foss NIRSystems Inc. (Model 6500,

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Silver Springs, Md., U.S.A.) with a tungsten halogen lamp and aninternal ceramic lamp (Nielsen and others 2008). The instrumentwas calibrated and standardized with a pectin standard before themeasurements. The samples were measured in a special glass ringcap capable of holding approximately 5 g of ground powder. Thecup was carefully compacted with a back plate rubber seal andspun in a Spinning Module (NR6506). The spectrum recordedwas the average of 16 scans per sample. Each sample was measuredin duplicate. The results were recorded as log(1/R), where R isthe reflectance and a total of 850 variables were obtained in therange 800 nm to 2498 nm (with 2 nm intervals).

Dynamic headspace analysis–GC-MSVolatile aroma components from 5 g of ground, dried beans

were detected and identified basically following the procedure de-scribed by Juric and others (2003). Sampling was done on Tenax-TA traps at 30 ± 1◦C. The trapped volatiles were desorbed usingan automatic thermal desorption unit (ATD 400, Perkin Elmer,Norwalk, Conn., U.S.A.) and automatically transferred to a gaschromatograph-mass spectrometer (GC-MS, G1800A GCD Sys-tem, Hewlett-Packard, Palo Alto, Calif., U.S.A.). Separation ofvolatiles was carried out on a DB-Wax capillary column 30 mlong × 0.25 mm internal diameter, 0.25 μm film thickness. Themass spectrometric detector operated in the electron ionisationmode at 70 eV. Mass-to-charge ratios between 15 and 300 werescanned. Volatile compounds were identified by matching theirmass spectra with those of a commercial database (Wiley275.L,HP product nr G1035A). The software program, MSDChemsta-tion (Version E.01.00.237, Agilent Technologies, Palo Alto, Calif.,U.S.A.), was used for data analysis.

Data analysisCut test, colorimetric, fluorescence, NIR, and GC-MS data

were all subjected to Principal Component Analysis (PCA) forevaluating systematic variations. In short PCA provides an ap-proximation of a data matrix, X , expressed as the product of 2sets of vectors, T (scores) and P (loadings), that capture the latentfactors of X and are referred to as principal components (Woldand others 1987). The predictive performance of the GC-MS datawere evaluated using Partial Least Squares (PLS) regression (Haa-land and Thomas 1988). All models were validated using full crossvalidation (Wold 1978). The NIR spectra were Standard Nor-mal Variate (SNV) transformed and mean-centered (Barnes andothers 1989) and the GC-MS data were log10-transformed beforefurther analysis. All multivariate data analyses were performed us-ing MATLAB version 14 (MathWorks, Natick, Mass., U.S.A.)and LatentiX Version 1.00 (Latent5, Copenhagen, Denmark). Alldata were transferred to MATLAB and LatentiX using in-houseroutines written in MATLAB.

Results and DiscussionTwo different sample sets were investigated in the present study.

One set represented samples taken during controlled tray fermen-tations with 24 h intervals during 6 d of fermentation. The methodused for drying the fermented beans is known to influence thequality of the final product (Thompson and others 2001). Toinvestigate the effect of drying method, the beans were eithersun-dried or oven-dried following fermentation. A portion of thesun-dried beans was manually de-shelled following drying beforefurther analysis as it is known that the chemical composition ofthe shell is different from the cotyledon (Lopez and Dimick 1995).

Another sample set represented fully fermented and dried (com-mercial quality) cocoa beans obtained from farmers representingall cocoa growing regions in Ghana.

Cocoa bean changes during fermentation investigated bycut test, colorimetry, fluorescence, and NIR spectroscopy

Principal component analysis score plots describing the progressof fermentation using the traditional cut test and instrumen-tal methods based on colorimetry, fluorescence and NIR spec-troscopy are shown in Figure 1A to 1D.

As seen from Figure 1A, the drying method (sun comparedwith oven drying) heavily influences the cut test scores duringthe early stages of fermentation. From approximately 72 h of fer-mentation and onwards the drying method has only negligibleinfluence on the cut test scores. During the fermentation a clearprogress is observed, with the unfermented and shortly fermentedsamples being judged as “slaty” and “purple” followed by a movetowards “purple-brown” and “brown” by the end of fermentation(Figure 1A) which is in agreement with previously published re-sults (Lopez 1986). It is generally recognised that the drying rateand temperature influence the development of flavor and aromaprecursors during drying and that drying at a too high temperaturemay impair the development of these precursors (Thompson andothers 2001). In the present study, it was found that the oven-driedsamples appeared browner than the corresponding sun-dried beansand consequently more beans were judged as “brown” by the cuttest, probably due to more pronounced nonenzymatic browningby Maillard-type reactions (Kyi and others 2005).

In an attempt to determine the color of the fermented co-coa beans in a more objective manner than the visual assessmentof the cut test, colorimetry was used to determine developmentin the L∗a∗b∗-values of the cocoa beans during fermentation.In agreement with the cut test results, it was found that thedrying method also influences the colorimetry measurements asseen from Figure 1B. As fermentation time increase the sam-ples move towards higher a∗- and b∗-values as seen in the bi-plot(Figure 1B) corresponding well with the samples becoming in-creasingly brown with time. Ilangantileke and others (1991) alsoused color-measurements to estimate the degree of cocoa beanfermentation. Despite the experimental differences, comparableresults were obtained indicating the potential of colorimetry tobecome an objective and simple replacement for the cut test andmost probably grouping of samples in the categories slaty, purple,purple-brown, and brown is obtainable from a linear combinationof the L∗a∗b∗-values.

The samples are clearly separated on the basis of fermentationtime using fluorescence spectroscopy as seen from the score plotin Figure 1C (PC2 compared with PC3) indicating the potentialuse of this method for determining the degree of fermentation.Interestingly, whole beans grouped away from the de-shelled ovenand sun-dried beans indicating that the shell-content influencedthe fluorescence spectra more than the drying method. PCA ofthe NIR-spectra (Figure 1D) showed the same trend as the fluo-rescence spectra (Figure 1C) with a clear separation on the basisof fermentation time as well as shell content (whole beans com-pared with de-shelled beans). Recently, we have shown that NIR-spectra of cocoa beans sampled during fermentation are stronglycorrelated with microbiological changes (investigated using De-naturing Gradient Gel Electrophoresis) in the pulp surroundingthe beans (Nielsen and others 2008) underlining the potential of

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spectroscopic techniques as a valuable tool for investigating andcategorising cocoa beans.

Formation of volatile compounds duringfermentation investigated using GC-MS

Fermentation and drying are essential steps in the flavor forma-tion of cocoa beans and the flavor potential of the beans is an im-portant quality parameter (Schwan and Wheals 2004). Figure 2Ashows a clear change in the content and composition of volatilecomponents during fermentation. Samples fermented 0 to 24 hare placed to the left in the plot having negative values onPC1 while samples fermented for 72 to 120 h are placed tothe right having positive values on PC1. From Figure 2B itis seen that cocoa beans fermented for 24 h or less have rela-tively high concentrations of 2-methylpropanal, 2,3-butanedione(diacetyl)/2-pentanone, 2-pentanol, methyl acetate, 2-heptanone,2-pentyl propanoate, 1-pentanol, 2/3-methylbutanal, tetrahydro-2-methyl furan, 2-methyl-1-propanol, and ethyl acetate. Of thesecompounds, Frauendorfer and Schierberle (2006) reported 2/3-

methylbutanal to be among the most odour-active compoundsidentified in an extract from cocoa powder. During roasting a sig-nificant increase will occur due to Strecker degradation (Munchand Schierberle 1998; Schieberle 2005), and this is probably themost important source of 2/3-methylbutanal in cocoa. The co-coa beans in this study were analyzed unroasted, and the 2/3-methylbutanal found is most probably of microbiological origin. Itis well documented that lactic acid bacteria can convert isoleucineto 2-methylbutanal and leucine to 3-methylbutanal (Singh andothers 2003; Smit and others 2004). Yeasts have, however, beendemonstrated to reduce 2/3-methylbutanal to the correspondingalcohols rather efficiently (Perpete and Collin 2000), which wouldexplain the decline demonstrated during fermentation here. Mi-crobial activity is therefore not expected to be the main source of2/3-methylbutanal in cocoa.

Cocoa beans fermented for 72 h or more have higher lev-els of propanoic acid, linalool oxide, 3-hydroxy-2-butanone(acetoin), 2-methylpropanoic acid, 1-hydroxy-2-propanone, 3-methylbutanoic acid, acetic acid, 2-phenylethyl acetate, 2,3,5,

Figure 1–(A) Bi-plot (combining scores and loadings) of PC1 compared with PC2 from a PCA on oven-dried (OD, solid line) and sun-dried (SD, dashed)fermentation samples and cut test variables. (B) Bi-plot of PC1 compared with PC2 from a PCA on oven-dried (OD, solid line) and sun-dried (SD, dashed)fermentation samples and color (L∗a∗b∗) variables. (C) Score plot of PC2 compared with PC3 from a PCA on oven-dried (OD, solid line), sun-dried (SD,dashed), and sun-dried whole beans (SDW, dotted) fermentation samples and fluorescence data. (D) Score plot of PC1 compared with PC2 from a PCAon oven-dried (OD, solid line), sun-dried (SD, dashed), and sun-dried whole beans (SDW, dotted) fermentation samples and near infrared data (800 to2498 nm, SNV-transformed and mean-centered). Time is given in hours. P: purple, PB: purple brown, B: brown, S: slaty.


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6-tetramethylpyrazine, 2-pentyl acetate, benzaldehyde, trimethylpyrazine, benzene ethanol, 3-methylbutyl acetate, and linalool(Figure 2B and 2C). The increased levels of organic acids are a re-sult of the metabolisation of sugars from the pulp surrounding thecocoa beans (Bonvehi 2005). Propanoic acid, 2-methylpropanoicacid, 3-methylbutanoic acid, and acetic acid are all reported tobe important odor-active compounds in cocoa (Bonvehi 2005;Frauendorfer and Schierberle 2006; Krings and others 2006).

During fermentation diacetyl decreases and acetoin increases.This indicates activity of lactic acid bacteria and relatively anaer-obic conditions (reduction of diacetyl to acetoin; Kandler 1983).Furthermore, some acetate esters are formed during fermenta-tion (2-phenylethyl acetate, 2-pentyl acetate, and 3-methylbutylacetate) while others decrease (methyl acetate and ethyl acetate)(Figure 2B and 2C). Of these 2-phenylethyl acetate is reported tobe important for the aroma of cocoa (Bonvehi 2005; Frauendorferand Schierberle 2006; Krings and others 2006). The ester pro-duction is most likely a result of yeast metabolism. 2-Phenylethylacetate and 3-methylbutyl acetate are also important esters in beerand their production is reported to depend on the growth condi-tions of the yeast, among others, low levels of oxygen (Verstrepenmand others 2003).

The main part of the pyrazines in cocoa are formed during roast-ing. In fact, the level of tri- and tetramethylpyrazine has been sug-gested as an indicator of the degree of roasting (Ramli and others2006). The increasing levels of tri- and tetramethylpyrazine duringfermentation (Figure 2C) are, however, due to enzymatic activity.Gill and others (1984) demonstrated that tetramethylpyrazine is ametabolic product of Bacillus subtilis, and the formation indicatesB. subtilis activity during the fermentation. In general Bacillus spp.including B. subtilis reach high numbers during the later stagesof Ghanian cocoa fermentations (Carr and Davies 1980; Nielsenand others 2007). Ramli and others (2006) also found tetram-ethylpyrazine in unroasted beans, but not trimethylpyrazine.

Linalool (Figure 2B and C) is reported to be an odour-activecompound in cocoa (Bonvehi 2005; Frauendorfer and Schierberle2006; Krings and others 2006) and to contribute to the floweryand tea-like flavor of some cocoa varieties (Hansen and others1998). It has been suggested that glycosidases release glycoside-bound terpenes like linalool during fermentation, explaining theincrease observed (Hansen and others 1998).

Figure 3A and 3B support the close relationship between fer-mentation and the changes described previously. A PLS1 modelbased on all volatiles predicts the fermentation time reasonablewell (r = 0.86, RMSECV = 21.3, Figure 3B). In fact, acetic acidconcentration alone can reasonably predict fermentation time asseen from Figure 3A (r = 0.86, RMSECV = 21.3) and could pos-sibly be exploited as an objective and fairly simple measurementof the degree of fermentation. Overall, it is seen that the changesin levels of volatiles demonstrated here correspond well with thepresent knowledge on fermentation processes. Although roastinghas an immense impact on the odor of cocoa, for example byformation of pyrazines (Jinap and others 1998), some compoundsformed during fermentation such as organic acids, linalool, andesters persist and strongly influence taste and flavor of the finalproduct, chocolate.

Fermented and dried cocoa beans representing all cocoagrowing regions in Ghana investigated by cut test,colorimetry, fluorescence, and NIR spectroscopy

It is well known that different cocoa subspecies and varieties pro-duce cocoa beans with different aroma, flavor, and color potential


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(Wood and Lass 1985). Additionally, the influence of geographicorigin at the country level on aroma and flavor of cocoa beans hasbeen investigated to some extent (Hernandez and Rutledge 1994;Jinap and others 1995; Caligiani and others 2007). However, thevast majority of cocoa of commercial quality is traded as bulk ca-cao (for example, Ghana cocoa, grade I) where each lot representcocoa from numerous farmers and only little is known about theinfluence of geographic origin at the farm to farm level on co-coa beans. In the present study, samples of fermented and dried“ready-to-sell” cocoa beans representing all major cocoa grow-ing regions of Ghana have been investigated using the cut test,colorimetry, fluorescence, and NIR spectroscopy and GC-MS toelucidate differences from farm to farm and between the differentGhanaian cocoa growing regions.

As seen from Figure 4A, no major differences are seen betweenthe cut test values of the different Ghanaian cocoa growing regionsas the majority of the samples cluster together between the Brownand the Purple-Brown loading indicating that the cocoa is well-fermented (Wood and Lass 1985). Four farms (010, 015, 020, and229) including both farms representing the Ashanti Region clustercloser to the Purple and Slaty loadings indicating that the cocoa ispoorly fermented (Figure 4A).

The colorimetric analysis in general confirmed the cut test anal-ysis. However, of the 4 farm samples that clustered closer to theSlaty and Purple loadings (Figure 4A), only Farm 015 clusteredaway from the majority of samples in the PCA bi-plot based on thecolorimetric results (Figure 4B). The fluorescence and NIR spec-troscopic results confirmed this pattern with Farm 015 clusteringaway from the main cluster in Figure 4C as well as Figure 4D.

Interestingly, the samples representing farms in the Volta andthe Western regions in general (with the exception of Farm 019)clustered separately in the score plots based on colorimetric, fluo-rescence and NIR (Figure 4B to D) indicating regional differencesin the composition of Ghanaian cocoa beans.

Volatile compounds in cocoa beans representing allGhanaian cocoa growing regions investigated using GC-MS

The score plot in Figure 5A indicates that geographical dif-ferences in the composition of Ghanaian cocoa beans do exist

though distinct groupings are not evident. Cocoa beans fromthe Western farms do, however, differ from most of the otherfarms by having higher levels of benzaldehyde, 2-heptanone,2/3-methylbutanal, 2-nonanone, and 2,6-dimethyl-4-heptanoland lower levels of acetic acid, propanoic acid, 1-hydroxy-2-propanone, and 2-phenylethyl acetate (Figure 5B). This probablyreflect differences in fermentation practices as the production ofacetic acid during fermentation is dependent on oxygen avail-ability (Camu and others 2008) and esters such as 2-phenylethylacetate are most likely a result of yeast metabolism (Verstrepenmand others 2003).

Many of these compounds are—as mentioned previously—expected to be odor-active in the roasted cocoa and the re-gional differences are therefore important for the quality. Thedata could indicate that cocoa beans from Western farms areless fermented with lower levels of organic acids and higherlevels of 2/3-methylbutanal as mentioned previously. Corre-sponding to Figure 4B to 4D, the Volta farms are also heregenerally placed opposite to the Western farms in the PCAplot.

ConclusionsDegree of fermentation could, in general, be well described by

all the methods used. It was demonstrated that colorimetry hasthe potential to become an objective and simple replacement forthe cut test, but samples were also clearly separated on the basisof fermentation time using fluorescence spectroscopy and NIR-spectrometry. In addition, it was possible to link analysis of volatilecompounds and the formation of acetic acid with predictions offermentation time.

When cocoa beans from different regions in Ghana were com-pared no major differences were seen between the cut test values.On the contrary, the samples representing farms in the Volta andthe Western regions, in general, clustered separately in the scoreplots based on colorimetric, fluorescence, NIR, and GC-MS in-dicating regional differences in the composition of Ghanaian co-coa beans. Many of the detected compounds are expected to beodor-active in the roasted cocoa and the regional differences aretherefore important for the quality.

Figure 3–(A) Actual compared with Predicted plot (one PLS component) for time in hours for oven-dried (OD), sun-dried (SD), sun-dried whole beans(SDW) based on acetic acid as determined by GC-MS. (B) Actual compared with Predicted plot (one PLS component) for time in hours for oven-dried(OD), sun-dried (SD), sun-dried whole beans (SDW) based on all GC-MS data (log10 transformed).


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Figure 4–(A) Bi-plot of PC1 compared with PC2 from a PCA on farm samples and cut test variables. (B) Bi-plot of PC2 compared with PC3 from a PCAon farm samples and color data. (C) Score plot of PC2 compared with PC2 from a PCA on farm samples and autoscaled fluorescence data. (D) Score plotof PC1 compared with PC3 from a PCA on farm samples and near infrared data (800 to 2498 nm, SNV and mean-centered).

Figure 5–(A) Score plot of PC1 compared with PC2 from a PCA on farm samples and GC-MS data (log10 transformed). (B) Loading plot of PC1 comparedwith PC2 from a PCA on farm samples and GC-MS data (log10 transformed).


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AcknowledgmentThe research performed was partly financed through the EU

INCO project: “Developing biochemical and molecular markersfor determining quality assurance in the primary processing ofcocoa in West Africa—COCOQual” (ICA4-CT-2002–10040).This article is published with the kind permission of the Directorof CRIG. The authors are grateful for the technical assistanceof the staff of the Physiology/Biochemistry Div. of the CocoaResearch Inst. of Ghana.

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