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     

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.     

Drying kinetics of the individual layer of cocoa beans during heat pump drying

Investigation was carried out to study the drying kinetics of the individual layer of cocoa beans, namely the testa and cotyledon, during heat pump drying. The cocoa beans were dried in thin layer using dehumidified air at 28.2 °C, 40.4 °C and 56 °C. Moisture contents of the bean, testa and cotyledon were recorded during drying. Reduction in moisture content was relatively fast at the testa as compared to the cotyledon in the initial two hours of drying. Subsequent drying showed a crossover region where the moisture content of the testa was higher than the cotyledon. The final moisture content of the testa was found to be higher than the inner cotyledon at the end of drying. This showed that moisture migrated from the cotyledon (lower moisture content) to the testa (higher moisture content) during drying. Although such transfer mechanism seems contradict but this can be further explained by the mass transfer potential concept as postulated by Luikov where the transfer of moisture is due to moisture potential difference instead of moisture content difference. Product quality analyses showed percent retention of cocoa polyphenols ranged from 44% to 73% as compared to freeze dried sample. Bean hardness was found reasonably comparable to the commercial sample and increased with decreasing moisture content.     

Quality of cocoa beans dried using a direct solar dryer at different loadings

In this study fermented cocoa beans were dried in a direct solar dryer at three levels of loading (20, 30 and 60 kg). Surface mouldiness was found to be heavy in the 60 kg treatment, with beans appearing blackish. All the dried beans were reasonably acceptable in terms of vinegary odour and weak in alcohol odour. Weak odour was also detected for the faecal, rancid and cheesy odours. The 60 kg treatment was rated strong for wet sock odour due to poor drying condition. A significant difference (P < 0.05) was found between the 60 kg treatment and the lower loading treatments for pH and titratable acidity. A cut test showed that the lower loading treatments resulted in a higher percentage of brown beans. The 20 kg treatment showed the highest cut test score, which is significantly different (P < 0.05) from the 60 kg treatment. Fermentation index also showed a tendency for lower loading treatments to have a higher index. No significant difference (P > 0.05) was found among the treatments in terms of cocoa, astringency, bitterness and sourness flavour notes. However, better flavour was observed for beans from the 20 kg treatment. No mouldy off flavour was found in any of the dried beans. Overall quality assessment showed that the 20 kg treatment was able to produce reasonably good‐quality beans as compared to other loadings and therefore is recommended for the direct solar dryer. Copyright © 2006 Society of Chemical Industry     

Performance of different drying methods and their effects on the chemical quality attributes of raw cocoa material

Studies were carried out to investigate the impact of different drying processes on the chemical quality traits of raw cocoa beans. The pH of less fermented cocoa is higher than the well‐fermented cocoa’s. The sun‐dried beans pH ranged from 4.5 to 5.5, while the pH of both oven‐ and mixed‐dried beans was between 3.8 and 5.2. The sun‐dried beans contained lower volatile acidity than oven‐dried beans. Artificially dried beans resulted in higher free acidity content when compared to both sun‐ and mixed‐dried beans. Ammonium nitrogen content in raw cocoa beans is not influenced by the drying methods. Free fatty acid content increases slowly but remains below the critical value of 1.75% whatever the drying processes. While oven‐dried beans show the FFA content above 0.70% both of sun‐ and mixed‐dried beans are associated with FFA content below 0.70%.     

Effects of incubation and polyphenol oxidase enrichment on colour, fermentation index, procyanidins and astringency of unfermented and partly fermented cocoa beans

Incubation of unfermented and partly fermented cocoa beans in acetate buffer, pH 5.5, at 45 °C increased yellowness, total colour differences and fermentation index value of the cocoa bean powders and decreased cocoa procyanidins (monomers to pentamers), and their astringency. Fermentation index and (–)‐epicatechin content, equivalent to those of fully fermented beans, were reached by unfermented beans after 4–8‐h incubation, but not by partly fermented beans even after 16 h. During incubation of partly fermented cocoa beans enriched with polyphenol oxidase, yellowness and fermentation index value were increased, whilst (–)‐epicatechin was decreased. Tyrosinase had a less significant effect in yellow colour formation, but showed a significant reduction of (–)‐epicatechin and increase in fermentation index compared with crude cocoa polyphenol oxidase. However, both enzymes have similar effects on procyanidin degradation and astringent taste reduction. Incubation of cocoa beans for 16 h increased the cut test score of unfermented and partly fermented beans by 50 and 30%, respectively.     

Drying kinetics,physico‐chemical properties,antioxidant activity and phenolic composition of foam‐mat dried germinated rice bean (Vigna umbellata) hydrolysate

This research was aimed to study physico‐chemical properties and antioxidant activities of foam‐mat dried germinated rice bean (Vigna umbellata) hydrolysate. Germination led to an increase in released phenolic content and antioxidant activity (DPPH radical scavenging activity and FRAP) of rice bean hydrolysate. The hydrolysate obtained from germinated rice bean (GRB) and non‐germinated rice beans (NGRBs) was foam‐mat dried at 60 and 70 °C. Semi‐theoretical and empirical model could suitably describe the drying characteristic of foamed bean hydrolysate. Total phenolic content and antioxidant activities of foam‐mat dried samples decreased with increasing air‐drying temperature (P ≤ 0.05). Gallic acid, catechol and epicatechin were major phenolic compounds in foam‐mat dried samples prepared from both GRB and NGRB. The higher phenolic content and antioxidant activities were found in foam‐mat dried hydrolysate of GRB. Electron spin resonance spectrometry revealed that foam‐mat dried rice bean hydrolysate showed a strong ability to scavenge free radicals, especially carbon‐centred radicals.     

Effect of drying time,bean depth and temperature on free amino acid,peptide‐N,sugar and pyrazine concentrations of Malaysian cocoa beans

The effect of drying time on free amino acid, peptide‐N, sugar and pyrazine concentrations as well as the influence of bean depth and temperature on these compounds during cocoa drying was studied. Drying time, bean depth and temperature significantly decreased the concentration of free amino acids, peptide‐N, total reducing sugars and sucrose and increased the concentration of trimethyl‐, tetramethylpyrazine and total pyrazines in cocoa beans. The best drying treatment was obtained at the combination of bean depth/drying temperature of 8.3 cm/40 °C. This was based on the fact that it produced significantly high concentrations of hydrophobic free amino acids, peptide‐N and total reducing sugars and significantly low concentration of trimethyl‐, tetramethylpyrazine and total pyrazines. Drying treatment of 1.5 cm/60 °C significantly produced the lowest concentration of free amino acids, peptide‐N and total reducing sugars and the highest concentration of pyrazines. © 1999 Society of Chemical Industry     

Batch drying of banana pieces — effect of stepwise change in drying air temperature on drying kinetics and product colour

Slices of banana were dried in a two-stage heat pump dryer capable of producing stepwise control of the inlet drying air temperature while keeping absolute humidity constant. Two stepwise air temperature profiles were tested. The incremental temperature step change in temperature of the drying air about the mean air temperature of 30°C was 5°C. The total drying time for each temperature-time profile was 240 min. The drying kinetics and temporal colour change of the products dried under these stepwise variation of the inlet air temperature were measured and compared with constant air temperature drying. The effects of the profile starting temperature and cycle time on both drying kinetics and product quality were also studied. It was observed that by employing stepwise-varying drying air temperature with appropriate starting temperature and cycle time, it was possible to reduce significantly the drying time to reach the desired moisture content with improved product colour.     

Oxidation of polyphenols in unfermented and partly fermented cocoa beans by cocoa polyphenol oxidase and tyrosinase

Unfermented and partly fermented dried cocoa beans have an excessively astringent and bitter flavour owing to their high polyphenol content. Studies on the remaining polyphenol oxidase activity and the oxidation of polyphenols in these beans have been conducted in relation to two factors, ie incubation time (0, 1, 2, 4, 8 and 16 h) and pH of incubation (3.5, 4.5, 5.5 and 6.5). Owing to unsuccessful polyphenol oxidation during incubation of partly fermented beans, incubation–enrichment treatments were carried out by combining incubation time (0, 8, 16, 24 and 32 h) and addition of crude cocoa polyphenol oxidase and purified tyrosinase from mushroom at 88 and 8800 units g^?1 and pH 5.5. Results showed that the remaining polyphenol oxidase activities of unfermented and partly fermented dried beans were 1 and 0.08% with specific activities of 9 and 1% respectively. The polyphenols of unfermented beans were effectively oxidised by incubation at 45 °C and pH 3.5–6.5 without enzyme enrichment; while those of partly fermented beans required enzyme enrichment. Both crude cocoa polyphenol oxidase and tyrosinase could be used for enzyme enrichment, but tyrosinase seemed to be more effective. © 2002 Society of Chemical Industry     

Identification and characterisation of the major aspartic proteinase activity in Theobroma cacao seeds

Theobroma cacao seeds contain an unusually high level of aspartic proteinase activity. Although this activity is central to the development of high‐quality cocoa flavour, the T cacao polypeptide responsible has not yet been definitively identified. Here we report the identification and characterisation of an active protein complex from T cacao seeds with an apparent molecular weight of approximately 50 kDa. This active complex contains at least two polypeptides: an approximately 30.5 kDa aspartic proteinase, the product of the TcAP2 gene, and an associated polypeptide, the 20.5 kDa trypsin inhibitor protein. The active complex co‐eluted off a size exclusion column with another complex containing the trypsin inhibitor and a putative acid chitinase. The 30.5 kDa TcAP2 proteinase is apparently a monomeric aspartic proteinase with optimal activity between 42 and 47 °C and an optimal pH of 3.0. Significant inactivation of the TcAP2 activity occurs at acid pH around 47–52 °C, a temperature potentially obtained during cocoa bean fermentation. SDS‐PAGE analysis showed that the purified TcAP2 complex efficiently degrades the cacao seed storage protein vicilin into peptides smaller than 10 kDa. In addition, high‐resolution size exclusion chromatography showed that this proteinase is capable of degrading proteins into peptides as small as di‐ and tripeptides, indicating for the first time that the main T cacao seed aspartic proteinase can produce very small peptide products. Our results demonstrate that the aspartic proteinase encoded by the TcAP2 gene plays a critical role in the production of cocoa flavour precursor peptides during cocoa bean fermentation. Copyright © 2005 Society of Chemical Industry     

Effects of drying conditions in hybrid dryer on the GABA rice properties

In this study, pre-germinated rough rice was dried under various conditions using a hybrid dryer that incorporated heat pump dryer with fluidized bed drying (FBD) system in order to compare with the single-stage drying by hot air dryer. The main objective was to investigate the effect of drying conditions on the cooking and physical properties, nutrients and sensorial qualities of GABA rice products. It appeared that the FBD at 100–140 °C led to noticeable increase of head rice yield. The germination process caused the decline of gelatinization enthalpy of brown rice flour, the change of pasting properties, the reduction of cooking time and escalation of the total phenolic content, antioxidant activity and GABA content. The FBD at higher temperature significantly raised the degree of starch gelatinization and caused longer cooking time, higher hardness and adhesiveness of cooked GABA rice. Three-stage drying comprising FBD at 60 °C for 10 min in the first stage followed by FBD at 100 °C in the second stage and then heat pump drying at 45 °C together with tempering step between drying stages was recommended due to its superior GABA rice product quality.     

Antioxidant Properties of Cocoa Beans (Theobroma cacao L.): Influence of Cultivar and Roasting Conditions

Effects of various roasting conditions on antioxidant properties of five Theobroma cacao L. varieties were investigated. The cocoa beans were roasted at four different temperatures (110–150°C) and three different air humidities (0.3–5.0%). The raw cocoa beans were characterized by high antioxidant activities. The antioxidant properties of the roasted cocoa beans varied markedly among the analyzed cultivars and geographical regions and were affected by roasting conditions. Generally, cocoa beans of the cv. Forastero from Brazil exhibited higher total phenolic content, free radical scavenging activity, and metal chelating ability than samples of the other analyzed cocoa varieties. Roasting at 110°C caused negligible changes in total phenolics content and antioxidant activity of cocoa beans, while almost all samples tended to have lower antioxidant potential when roasting temperature increased. The air humidity used in roasting did not affect the total phenolics content and antioxidant activity for lowest roasting temperature (110°C). Moreover, the obtained results revealed that thermal processing at the higher temperatures and elevated air humidity resulted in the higher antioxidant capacities. It was also found that the ferrous ion chelating activity of cocoa beans increased with the roasting temperature (in the range from 110 to 150°C), with the exception of cv. Trinitario from Papua New Guinea. The data showed that roasting at lower temperatures with humid air are more favorable in terms of preserving the bioactivity of roasted cocoa beans.     

Amadori-Verbindungen als Aromavorstufen in Kakao

Amadori-compounds as cocoa aroma precursors Several Amadori-compounds (fructose-amino acids), important intermediates of Maillard reaction, have been found in unroasted cocoa beans some years ago. Now, in cocoa additional fructose-phenylalanine (fru-phe) could be detected by means of a modified extraction procedure. For studying the contribution of Amadori-compounds fru-phe and fru-leu (fructose-leucine) were roasted at 145°C. The roasted flavours contained a lot of key compounds of cocoa flavour. If cocoa was moist-thermally treated and dried the level of Amadori-compounds could be raised and the roasted aroma intensified. Amadori-compounds obviously contribute to flavour formation in cocoa. This effect may by used technologically to increase flavour yield.     

Tocopherols in cocoa butter obtained from cocoa bean roasted in different forms and under various process parameters

Obtaining good quality chocolate strongly depends on raw material, i.e. cocoa beans. The processing of cocoa beans consists of some important steps, including fermentation, drying fermented beans and roasting. Traditionally roasting is performed on whole beans but currently, roasting crushed cocoa beans or cocoa liquor becomes more and more popular. Many biologically active compounds may be found in the cocoa beans, including tocopherols. This work investigates the influence of the constant or variable roasting process parameters (temperature, velocity and relative humidity of roasting air) on the tocopherol concentration in cocoa butter (CB) extracted from cocoa beans originating from Togo and roasted in two different forms, namely as whole and crushed beans. Whole cocoa beans were roasted to a 2% moisture content and crushed cocoa beans were firstly partially dried which further enables easier dehusking, then ground, dehusked and roasted until their humidity decreased to around 2%. Roasting resulted in lowering the content of individual tocopherols in analyzed material. The degree of degradation of tocopherols in CBs was different, depending on the form of roasted beans from which these CBs were extracted. Higher concentrations were determined in CBs extracted from beans roasted in the form of crushed samples comparing to CBs obtained from beans roasted as a whole. The study investigates different roasting parameters of crushed beans, none of which drastically lowered the concentration of tocopherols in extracted CBs. Their concentration in CBs extracted from whole beans was, on the other hand, influenced by roasting air parameters. In case of whole beans roasted under constant parameters, application of 150 °C proved to be more favorable than roasting at 135 °C, as well as application of “dry” air and 1 m/s roasting air velocity. Discussing the variable roasting process parameters, in case of applied roasting temperature it is more favorable to change it from 150 °C to 135 °C, than the other way round. Changing the relative humidity of roasting air from 5 to 0.3%, lower degradation of tocopherols in CB occurs when the process is conducted at 150 °C. It may be further concluded that a direct dependence between the velocity of roasting air varied during the process and the concentration of tocopherols in extracted CB may not be indicated. In conclusion, it is stated that the temperature of the air applied during the roasting process has the decisive influence on the tocopherol content in CBs extracted from cocoa beans subjected to the process.     

Activation of remaining key enzymes in dried under-fermented cocoa beans and its effect on aroma precursor formation

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

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.     

Effects of ultrasound on the physicochemical properties and microstructure of salted‐dried grass carp (Ctenopharyngodon idella)

Ultrasound treatment was used for accelerating drying process and shortening drying time of salted‐dried grass carp, in parallel with evaluation of physicochemical properties and microstructure. The grass carp blocks were salted with ultrasound‐assisted treatment at 0–480 W, 40 kHz for 120 min, then put into heat pump dryer for drying at 50% humidity, 30 °C for 50 hr. The moisture content results showed that ultrasound treatment could significantly promote water loss rate. As the ultrasound power increased, the hardness, the relative content of monounsaturated fatty acid and polyunsaturated fatty acid and total content of free amino acid decreased while the saturated fatty acid and sensory scores increased, respectively. Furthermore, microstructure of the dried‐salted grass carp was studied using paraffin sections and the scanning electron microscope to understand the change of microstructure. In general, ultrasound‐assisted salting significantly increased drying rate, and improved the quality of salted‐dried grass carp. This study shows the potential application of ultrasound in food drying process.

Practical applications

The ultrasound was applied in order to facilitate the traditional process to produce salted‐dried grass carp. And physicochemical properties and microstructure of salted‐dried fish were measured. The results showed that the application of ultrasound could significantly promote the mass transfer of water and improved the product's appeal for customers. The outcome of this study demonstrated a method for producing better quality dried‐salted fish products with a fast speed in the future and showed a great potential of ultrasound in food drying process.     

The effect of solar drying and heating on the hardness of Phaseolus beans during storage

Small white beans (Phaseolus vulgaris) were dried and heat processed before storage. Water removal in a solar drier was four times faster than conventional field drying in the pods. Heat treatments included particle-to-particle roasting using hot sand, and microwave heating. All samples stored for 212 days at low moisture contents (4–5%) showed a slow rate of hardening at a low temperature of 18°C and a high temperature of 34°C. However, heat treated and control beans became much harder when stored at a humidity of 9% and a temperature of 34°C. Heating and solar drying are effective treatments prior to bean storage, as they greatly reduce moisture to safer levels.