The microbiology of Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods

Export of cocoa beans is of great economic importance in Ghana and several other tropical countries. Raw cocoa has an astringent unpleasant taste and a spontaneous fermentation is the first step in a process leading to cocoa beans with the characteristic cocoa flavour and taste. The microbiology of Ghanaian cocoa fermentations was investigated using culture-dependent and culture-independent methods. Samples were collected at 12 hour intervals during 96-144 hour tray and traditional heap fermentations. Yeast, Lactic Acid Bacteria (LAB), Acetic Acid Bacteria (AAB) and Bacillus spp. were enumerated on suitable substrates and identified using phenotypic and molecular methods. The yeast and bacterial micro-populations involved in the cocoa fermentation were further investigated using the culture-independent method Denaturing Gradient Gel Electrophopresis (DGGE). A microbiological succession was observed during the fermentations. At the onset of fermentation yeasts were the dominating microorganisms. Lactic Acid Bacteria became dominant after 12-24 h of fermentation and remained predominant throughout the fermentations with AAB reaching high counts in the mid phase of fermentation. Bacillus spp. were only detected during heap fermentations where they reached high numbers during the later stages of fermentation. Hanseniaspora guilliermondii was the predominant yeast during the initial phase and Pichia membranifaciens during the later phases of fermentation. A number of other yeast species including three putatively undescribed species were isolated during the fermentations. Lactobacillus fermentum was the dominant LAB in most samples. Several other LAB including Lactobacillus plantarum, Leuconostoc pseudomesenteroides, Leuconostoc pseudoficulneum, Pediocococcus acidilactici and a putatively undescribed LAB species were detected during the fermentations. Acetobacter syzygii, Acetobacter pasteurianus and Acetobacter tropicalis were the predominant AAB in all investigated fermentations. During the later stages of heap fermentation Bacillus licheniformis and occasionally other Bacillus spp. were detected in high numbers. In general the culture-based findings were confirmed using DGGE. However, DGGE indicated that Lc. pseudoficulneum plays a more important role during the fermentation of cocoa than expected from the culture-based findings as it yielded a strong band in most DGGE fingerprints. Cluster analysis of the DGGE fingerprints revealed that the DGGE fingerprints clustered according to fermentation site. Within each fermentation site the profiles clustered according to fermentation time. The DGGE method seems to offer a relatively fast and reliable tool for studying yeast and bacterial dynamics during cocoa fermentations.     

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

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

Yeast populations associated with Ghanaian cocoa fermentations analysed using denaturing gradient gel electrophoresis (DGGE)

The yeast populations associated with the fermentation of Ghanaian cocoa were investigated using denaturing gradient gel electrophoresis (DGGE). Samples were collected at 12-24 h intervals from heap and tray fermentations, at three different fermentation sites and different periods during the season. Eukaryotic universal primers were used to amplify a fragment of the 26S rRNA gene. The DGGE profiles were relatively complex, underlining that the fermentation of cocoa is a complex microbial process. The identities of selected fragments in the denaturing gels were revealed by sequencing. Hanseniaspora guilliermondii, Candida krusei and Pichia membranifaciens were detected from most fermentations, indicating their possible important role in the fermentation of Ghanaian cocoa. Saccharomyces cerevisiae and Candida zemplinina were almost exclusively detected during tray fermentations. The developed DGGE protocol was compared with traditional culture-based isolations. The results were comparable but slightly different, as one yeast species (C. zemplinina) was only detected using DGGE. On the other hand, Trichosporon asahii yielded only faint bands in the denaturing gels, despite the fact that it was detected using culture-based methods. Analysis of pure cultures showed that the targeted region of the 26S rRNA gene was poorly amplified in T. asahii, whereas all other investigated isolates were amplified efficiently using the chosen PCR approach. Cluster analysis revealed that the DGGE profiles clustered according to fermentation method and fermentation site. Furthermore, clustering according to progress in the fermentation was observed. The DGGE technique therefore seems to offer a relatively fast and reliable method for studying yeast population dynamics during cocoa fermentations.     

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

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

Theobroma cacao L., "The food of the Gods": quality determinants of commercial cocoa beans, with particular reference to the impact of fermentation

The quality of commercial cocoa beans, the principal raw material for chocolate production, relies on the combination of factors that include the type of planting material, the agricultural practices, and the post-harvest processing. Among these, the fermentation of the cocoa beans is still the most relevant since it is the process whereby the precursors of the cocoa flavor arise. The formation of these precursors depends on the activity of different microbial groups on the beans pulp. A comparison of fermentations in different countries showed that a well-defined microbial succession does not always take place and that the role of Bacillus spp. in this process remains unclear. Considering the overriding importance of the fermentation to achieve high quality commercial cocoa beans, we discuss the need of addressing the impact of the farming system, the ripeness state of the pods, and the role of microbial interactions on the fermentation in future research. In addition, the problem of high acidification cocoa beans, aspects dealing with the volatile fraction of the flavor, and the cocoa butter properties, all were identified as critical aspects that need further investigation. The standardization of the microbiological methods and the application of metagenomic approaches would magnify the knowledge in this domain.     

Post-harvest pod storage: A method for pulp preconditioning to impair strong nib acidification during cocoa fermentation in Malaysia

In Malaysia, pulp preconditioning by post-harvest storage of cocoa pods leads to the reduction of nib acidification during subsequent fermentation, reduction of the acid note and an increase in cocoa flavour in the resulting raw cocoa. Data from several shallow-box fermentations, with material from unstored and stored pods, are compared and interpreted, obtained in the years 1984 to 1987 in a cooperational investigation of the Malaysian Agricultural Research and Development Institute (MARDI), Malaysia, and the Botanical Institute, Technical University of Braunschweig (TUBS), FRG. Prior to and during fermentation, pulp volume and pulp sugars; pH value, acetic acid and lactic acid content in the pulp and nibs; and oxygen concentration and temperature in the mass were determined. Some flavour assessments from selected samples are given. The great reduction in pulp volume per seed rather than the decrease of pulp sugars per seed during pod storage was found to be of the most importance. Pulp-volume reduction enhances mass aeration and increases the ratio of respiration to ethanol fermentation and its subsequent oxidation to acetic acid. As a consequence, the acidification of the seeds during the formative stages of flavour precursors (after the death of the seeds) is strongly reduced. With effectively dry stored pods (pulp volume per gram of seed ≤0·6 ml) the anaerobic phase during the initial stages of fermentation which is common with unstored pods is suppressed. Under these conditions the nib pH value does not fall below 5·0 and no drastic acid (and flavour) degradation at the end of fermentation is necessary to reduce the acidity in the seeds.     

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

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

Enzyme activities in cocoa beans during fermentation

The activities of endoprotease, aminopeptidase, carboxypeptidase, invertase (cotyledon and pulp), polyphenol oxidase and glycosidases were studied during heap fermentation of ICS-95 cocoa beans. These enzymes are of key importance in flavour precursor formation and in pigment degradation during cocoa fermentation. Optimal extraction and assay conditions were established to characterise the enzymes reactions and to quantify and compare enzyme activities during cocoa fermentation. The enzymes exhibited large differences in pH optima and stability during fermentation. Aminopeptidase, invertase (cotyledon and pulp) and polyphenol oxidase were strongly inactivated, carboxypeptidase was partly inactivated, whereas endoprotease and glycosidases remained active throughout the fermentation. Since many enzymes are inactivated during fermentation, it is generally recognised that the actual period of enzyme action is short. Although our results confirmed total inactivation for some enzymes, we show that several key enzymes are not completely inactivated during fermentation. Therefore, some enzyme reactions can continue throughout the whole fermentation process. Only polyphenol oxidase was strongly inactivated during sun and artificial drying of the beans. The other enzymes were stable during the drying process. Enzymes like endoprotease and glycosidases are still active in properly fermented and dried beans. © 1998 SCI.     

Acidification,proteolysis and flavour potential in fermenting cocoa beans

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.     

Comparison of the bacterial species diversity of spontaneous cocoa bean fermentations carried out at selected farms in Ivory Coast and Brazil

To compare the spontaneous cocoa bean fermentation process carried out in different cocoa-producing regions, heap and box (one Ivorian farm) and box (two Brazilian farms) fermentations were carried out. All fermentations were studied through a multiphasic approach. In general, the temperature inside the fermenting mass increased throughout all fermentations and reached end-values of 42-48 °C. The main end-products of pulp carbohydrate catabolism were ethanol, lactic acid, acetic acid, and/or mannitol. In the case of the fermentations on the selected Ivorian farm, the species diversity of lactic acid bacteria (LAB) and acetic acid bacteria (AAB) was restricted. Lactobacillus fermentum and Leuconostoc pseudomesenteroides were the predominant LAB species, due to their ethanol and acid tolerance and citrate consumption. The levels of mannitol, ascribed to growth of L. fermentum, were fermentation-dependent. Also, enterobacterial species, such as Erwinia soli and Pantoea sp., were among the predominating microbiota during the early stages of both heap and box fermentations in Ivory Coast, which could be responsible for gluconic acid production. Consumption of gluconic acid at the initial phases of the Ivorian fermentations could be due to yeast growth. A wider microbial species diversity throughout the fermentation process was seen in the case of the box fermentations on the selected Brazilian farms, which differed, amongst other factors, regarding pod/bean selection on these farms as compared to fermentations on the selected Ivorian farm. This microbiota included Lactobacillus plantarum, Lactobacillus durianis, L. fermentum, Lactobacillus mali, Lactobacillus nagelii, L. pseudomesenteroides, and Pediococcus acidilactici, as well as Bacillus subtilis that was present at late fermentation, when the temperature inside the fermenting mass reached values higher than 50 °C. Moreover, AAB seemed to dominate the Brazilian box fermentations studied, explaining higher acetic acid concentrations in the pulp and the beans. To conclude, it turned out that the species diversity and community dynamics, influenced by local operational practices, in particular pod/bean selection, impact the quality of fermented cocoa beans.     

Comparison of enzyme activities involved in flavour precursor formation in unfermented beans of different cocoa genotypes

The activities of endoprotease, aminopeptidase, carboxypeptidase and invertase (cotyledon and pulp) were studied in unfermented beans of 10 genotype samples with different flavour characteristics (high and low cocoa flavour). Analysis of variance showed that significant differences in enzyme activities exist between certain genotypes. Aminopeptidase and endoprotease activities in beans of the PA7 genotype were higher than in all others. Principal component analysis (PCA) showed that the PA7 genotype (high cocoa flavour) was very different from the UIT1 genotype (low cocoa flavour). Although significant differences exist, no simple and general relationship is established between the flavour potential of a genotype and the level of key enzyme activities in unfermented beans. Carboxypeptidase is of key importance for peptide and free amino acid formation, but differences in enzyme activity could not be correlated to flavour potential of the genotype. It is suggested that the level of enzyme activities present in unfermented beans is not a limiting factor for optimal formation of flavour precursors during the fermentation process. © 2000 Society of Chemical Industry     

Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12–24 hour intervals during 120 h of fermentation. Yeast isolates were grouped by (GTG)₅-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12 h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12 h of fermentation whilst PFGE showed that ~ 88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed.     

Ghanaian Cocoa Bean Fermentation Characterized by Spectroscopic and Chromatographic Methods and Chemometrics

Abstract: Export of cocoa beans is of great economic importance in Ghana and several other tropical countries. Raw cocoa has an astringent, unpleasant taste, and flavor, and has to be fermented, dried, and roasted to obtain the characteristic cocoa flavor and taste. In an attempt to obtain a deeper understanding of the changes in the cocoa beans during fermentation and investigate the possibility of future development of objective methods for assessing the degree of fermentation, a novel combination of methods including cut test, colorimetry, fluorescence spectroscopy, NIR spectroscopy, and GC-MS evaluated by chemometric methods was used to examine cocoa beans sampled at different durations of fermentation and samples representing fully fermented and dried beans from all cocoa growing regions of Ghana. Using colorimetry it was found that samples moved towards higher a* and b* values as fermentation progressed. Furthermore, the degree of fermentation could, in general, be well described by the spectroscopic methods used. In addition, it was possible to link analysis of volatile compounds with predictions of fermentation time. Fermented and dried cocoa beans from the Volta and the Western regions clustered separately in the score plots based on colorimetric, fluorescence, NIR, and GC-MS indicating regional differences in the composition of Ghanaian cocoa beans. The study demonstrates the potential of colorimetry and spectroscopic methods as valuable tools for determining the fermentation degree of cocoa beans. Using GC-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. Practical Application: The present study demonstrates the potential of using colorimetry and spectroscopic methods as objective methods for determining cocoa bean quality along the processing chain. Development of objective methods for determining cocoa bean quality will be of great importance for quality insurance within the fields of cocoa processing and raw material control in chocolate producing companies.     

Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus predominate during well-performed Malaysian cocoa bean box fermentations,underlining the importance of these microbial species for a successful cocoa bean fermentation process

Two spontaneous Malaysian cocoa bean box fermentations (one farm, two plantation plots) were investigated. Physical parameters, microbial community dynamics, yeast and bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the respective fermented dry cocoa beans. Similar microbial growth and metabolite profiles were obtained for the two fermentations. Low concentrations of citric acid were found in the fresh pulp, revealing low acidity of the raw material. The main end-products of the catabolism of the pulp substrates glucose, fructose, and citric acid by yeasts, LAB, and AAB were ethanol, lactic acid, acetic acid, and/or mannitol. Hanseniaspora opuntiae, Lactobacillus fermentum, and Acetobacter pasteurianus were the prevalent species of the two fermentations. Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus pentosus, and Acetobacter ghanensis were also found during the mid-phase of the fermentation processes. Leuconostoc pseudomesenteroides and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Tatumella saanichensis and Enterobacter sp. were present in the beginning of the fermentations and they could be responsible for the degradation of citric acid and/or the production of gluconic acid and lactic acid, respectively. The presence of facultative heterofermentative LAB during the fermentations caused a high production of lactic acid. Finally, as these fermentations were carried out with high-quality raw material and were characterised by a restricted microbial species diversity, resulting in successfully fermented dry cocoa beans and good chocolates produced thereof, it is likely that the prevailing species H. opuntiae, S. cerevisiae, Lb. fermentum, and A. pasteurianus were responsible for it.     

Pod storage time and spontaneous fermentation treatments and their impact on the generation of cocoa flavour precursor compounds

Most of volatile flavour compounds occurred during the cocoa postharvest operations including pod storage and fermentation whom effects on these metabolites were aimed by the present study. Three different spontaneous fermentation treatments were performed on the farm, and the main isolated yeasts were identified using PCR-DGGE technique. Fermented cocoa beans were solar-dried, and their flavour compounds were identified using solid phase microextraction technique–gas chromatography–mass spectrometry. The main results showed that Pichia kudriavzevii was common yeast involved in the cocoa fermentation. Thirty flavour precursor compounds grouped into six chemical families including alcohols and pyrazines were identified in raw cocoa samples. ACP showed that short durations of pod storage and fermentation promote the formation of flavour precursors of cocoa. No spontaneous cocoa fermentation treatment influenced the occurrence of aroma compounds. The generation of flavour compounds depends only on the pod storage and the fermentative microorganisms of cocoa.     

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

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

The Precursors of Chocolate Aroma: Production of Free Amino Acids during Fermentation of Cocoa Beans

SUMMARY— Amino acids are produced during the fermentation of cocoa beans at a rate which is in agreement with the already established rapid rate of flavour and aroma development. The role of these compounds as aroma precursors receives further confirmation as a result of these studies, and an objective method of assessing the "degree of fermentation" of cocoa beans has been described and tested on a number of different commercial varieties of cocoa.     

Aroma precursors and methylpyrazines in underfermented cocoa beans induced by endogenous carboxypeptidase

Cocoa-specific aroma precursors and methylpyrazines in underfermented cocoa beans obtained from fermentation induced by indigenous carboxypeptidase have been investigated. Fermentation conditions and cocoa bean components were analyzed during 0 to 3 d of fermentation. Underfermented cocoa beans were characterized as having hydrophilic peptides and free hydrophobic amino acids much higher than unfermented ones. These 2 key components of cocoa aroma precursors may be produced from the breakdown of proteins and polypeptides by endogenous carboxypeptidase during the fermentation process. The enzyme was activated during fermentation. Polypeptides of 47, 31, and 19 kDa were observed in the samples throughout the 3-d fermentation period; however, only the first 2 polypeptides were remarkably reduced during fermentation. Since the 1st day of fermentation, underfermented cocoa beans contained methylpyrazines, a dominant group of cocoa-specific aroma. This might be due to microbial activities during fermentation, observed through a decrease of pH value and an increase of temperature of cocoa beans. The concentration of tetramethylpyrazines was significantly increased during the 3 d of fermentation. This may increase the cocoa-specific flavor to the beans.     

Non-conventional fermentation at laboratory scale of cocoa beans: Using probiotic microorganisms and substitution of mucilage by fruit pulps

In a non-conventional lab-scale fermentation of cocoa beans using probiotic microorganisms and substituting the cocoa pulp for fruit pulp, physicochemical, microbiological and quality parameters were investigated. Two hundred grams of beans were fermented in a controlled environmental chamber (temperature ramp rate of 25°C for 48 h; 35°C for 48 h and 45°C for 48 h; and 65% HR). pH, titratable acid, citric, lactic and acetic acids, as well as sugars and ethanol were measured. A cut test was also performed on the cocoa beans fermented 5 and 6 days. As the fermentation time progressed, citric acid concentration decreased until 0.53 g kg⁻¹, whereas both lactic and acetic acids increased until 0.44 and 16.58 g kg⁻¹, respectively. Sucrose content decreased from 12.26 g kg⁻¹ (in fresh) to 6.54 g kg⁻¹ on the 6th day. Fructose and glucose contents increased in the cotyledons from day five, reaching a maximum concentration of 1.14 and 1.01 g kg⁻¹, respectively, on day six. Yeasts were the main microorganisms during the first 24–48 h (8.4 log CFU g⁻¹), while bacterial counts reached its highest number (7.8 log CFU g⁻¹) on day four. Beans fermented 5 and 6 days resulted in more fermented beans (>81%) and less violet ones (<18.4%) than the control.     

Amyl alcohols as compounds indicative of raw cocoa bean quality

 Amyl alcohols were isolated from raw cocoa beans by steam distillation and quantified by means of gas chromatography. Concentration ratios between primary and secondary amyl alcohols and between alcohols, aldehydes and acetates were calculated. These ratios indicate the fermentation degree and possible staling of cocoa beans. Hence, flavour quality of raw cocoa beans may be measured and the method used for industrial quality control. Received: 27 October 1995/Revised version: 25 March 1996