The following is a summary of the stated research mentioned above. The content summarized here, including the figures and tables, all belong to the researchers (unless otherwise indicated). The summary attempts to stay as close to the original paper as much as possible with some adjustments in regards to jargon, length, or to focus on bean to bar aspects.

Note From Geoseph

The tables and figures in this summary may seem daunting to most. Do not be overwhelmed. The tables compare data of the 6 cocoa hybrids analyzed in this study, and are great tools to compare and contrast various levels of volatile aromas (potential flavours) in unroasted (Table 2) and roasted (Table 3) cocoa beans grown in Cameroon. There is also some interesting information which suggests many of these aromas or aroma groups can be linked to specific cacao genotypes, suggesting aroma is also partially genetically linked, reinforcing the importance of cacao genetics when it comes to cacao and chocolate flavour. If you find the data too much, it’s worth skimming through the information as there are some great tidbits of information in this quite recent paper published in 2023.

Introduction

Theobroma Cacao is a perennial crop growing around the world in tropical and subtropical regions. In the 2021/2022 crop year, 5.4 million tons of cacao were produced worldwide. The top producers are Ivory Coast (number 1), Ghana, Ecuador, Cameroon, Nigeria, Brazil, and Indonesia among 58 other countries (ICCO, 2022). There are many genotypes of cacao, including hand-pollinated clones/hybrids created to be more disease tolerant or to somehow perform better for their intended purpose. There are three major groups of cocoa: Criollo (Fine), Forastero (Bulk), and Trinitario (hybrids of these two). Cameroon is the largest African producer of the Trinitario cocoa mainly from of SNK (Selection of Nkoemvone) and ICS (Imperial College Selection) stock. The SNK clones, known locally as LT (Local Trinitario) are the most representative in Cameroon. Pure Trinitario hybrid production is limited due to incompatibility cases between clones, but crossing Trinitario and Forastero shows promising results. Another cocoa found in Cameroon is Scavina (Forastero). It is known for floral aroma within the fruit pulp and fresh cotyledons (Kadow et al., 2013). On top of this, Scavina 12 pods appear to be higher in (-)-epicatechin (an antioxidant) (Akoa et al, 2021; Ondobo, 2014).

Overview of this study by Akoa et al. (2023).

Over the years, more than 600 volatile compounds have been identified in cocoa and cocoa by-products (Aprotosoaie et al., 2016). Cocoa is generally classified as either fine flavoured cocoa and bulk/ordinary cocoa. Fruity and floral volatiles are often associated with fine cocoa. These compounds were often classified as alcohols, esters, terpenoids, and ketones. These volatile compounds are impacted by many aspects including the genetics of the cacao, the environment it grows in, and steps of processing such as fermentation, drying, and roasting. Kongor et al. (2016) mentions genotypes are significantly related to their chemical composition. However, how the cocoa beans are treated post harvest also heavily influence their overall chemical composition including aroma volatiles.

One important step impacting aroma precursors (mainly sugars and amino acids) is the fermentation of the fresh cocoa beans. Essentially, the hydrolysis of sucrose creates fructose and glucose, which are the main two reducing sugars found in fermented cocoa beans. Roasting initiates biochemical reactions with these reducing sugars to produce the characteristic chocolate flavour and aromas found within the chocolate.

There is a lack of data on the biochemical and volatile aroma composition of controlled Trinitario and Scavina cocoa hybrids from Cameroon. The work here aims to characterize the sugars and volatile compounds of fermented dried and roasted nibs from six controlled cocoa hybrids. HPIC-ED (High Pressure Ion Chromatography) and HS-SPME-GC-MS (Headspace solid phase micro extraction, and gas chromatography/mass spectrometry) will be used to quantify and capture this.

Methods and Materials

Hand-pollination was used to produce six hybrids from 9 clones (see Table 1 for list of hybrids). Fermented dried roasted nibs were ground up and prepared. The HS-SPME was used to extract the volatiles, which were then analyzed using gas chromatography-mass spectrometry (GC-MS) to identify the compounds. Sugars were analyzed by High Pressure Ion Chromatography.

Results And Discussion

Reducing and non-reducing sugars

As seen in Figure 1, the sugars varied according to the genotype of cocoa as well as pre and post roasting. Figure 1A displays the amount of sucrose and reducing sugars for each of the six hybrids. Figure 1B displayed the amount of Maltose and non-reducing sugars for the same six hybrids.

Sugars post fermentation/drying

In Figure 1A, we see that in fermented & dried beans, fructose is the dominate sugar present. This is because sucrose is hydrolysed into fructose and glucose during fermentation, and microorganisms prefer to metabolize glucose, leaving behind higher levels of fructose (Reineccius et al., 1972).

In regards to fermented and dried cocoa beans, the hybrid with the highest level of fructose was SCA12 x ICS40 at about 3.9 g/100g dry weight (DW). The lowest concentration of fructose recorded was for SNK10 x IMC67 at 3.01 g/100g dry weight.

Fructose was 9-12 times more important than glucose and 5-7 times more important than sucrose. The ratio of fructose to glucose can be used as an indicator of when the fermentation is optimal. The ratio of these sugars can play a key role in the outcome of the Maillard reaction. Hinneh et al. (2018) (read my summary about it here) observed that a 7 day pre-conditional period increased the content of fructose in dried fermented beans. This appears to be dependant on the pulp, which is linked to genetics. Keep in mind some cocoa varietals require different fermentation practises to release more reducing sugars from sucrose (Niemenak et al., 2021). In relation to fructose, the levels of galactose were marginal in these hybrids.

Some studies of highlighted other supplementary sugars in cocoa (Niemenak et al., 2021; Reineccius et al., 1972). In this study, identified sugars included Maltose, Verbascose, Stachyose, and Raffinose (see Figure 1B).

Sugars post roasting

There was a great reduction in sugars observed during roasting. The most important sugar to point out is the level of fructose. It is well known that during roasting, the Maillard and Strecker degradations take place where they utilize fructose and glucose, combined with amino acids, to produce a range of volatile aromas. SNK hybrids (first three pairs in Figure 1A) saw the greatest reduction in reducing sugars during roasting. This reduction in sugar content may be dependant on the sugar type and genotype of cocoa.

In regards to the other sugars (Figure 1B), roasting resulted in a decrease in sugars of only 4 hybrids. In Hybrids SNK16 x T60/887 and ICS40 x UPA134 (First and last pair of data in Figure 1B) we see an increase in raffinose. There was a moderate increase in verbascose content in hybrid SNK16 x T60/887 post roasting.

Volatile compounds in fermented dried and roasted nibs

A Gas Chromatography-Mass Spectrometry analysis identified an array of 48 volatile compounds including acids, esters, alcohols, ketones, aldehydes, pyrazines, terpenes, lactones, pyrroles, and furans. Take a look at Table 2 to see the various levels of compounds associated with each of the six hybrids. Odor description gives you an idea of what we associated that volatile compound with in real life. In some cases the levels of various aromas are drastically different, even though each sample was treated the same way in regards to fermentation/drying/roasting/analyzing of the samples, showing the impact genetics has on compounds. These volatiles were classified as fruity (15), floral (11), chocolate/nutty (10), buttery/creamy (3), undesirable (4), and other (5) according to their odor characteristics.

Fruity Volatiles

There were 15 compounds classified as fruity in this study. These compounds differed from hybrid to hybrid, although some hybrids saw similar levels of various compounds. Esters are known as one of the most important type of volatile to fruity and floral flavours in foods, which are derived from the esterification (combining an organic acid with an alcohol) of alcohols. It is documented that esters are synthesized by yeasts from alcohols during anaerobic respiration (Rojas et al., 2002). They are the second most important group of volatile compounds after pyrazines. According to some studies, secondary alcohols and alkyl ketones which are identified as fruity volatiles are thought to be derived from fatty-acid volatiles (Kadow et al., 2013; Schwab et al., 2008).

Fermented dried beans had the highest level of the Ester, methyl acetate. Its concentration seemed highest in ICS and SCA12 hybrids more than in SNK hybrids. The hybrid SCA12 x ICS40 was dominated by ethyl acetate (fruity, aromatic) and 2-heptanol (fruity, lemon grass, floral, fresh, sweet, herbal, green). The fruity volatiles with high concentrations were made up mostly of esters and secondary alcohols. Many of these compounds were also identified in Ecuadorian genotypes found by Colonges et al. (2022). However, the results in this study differed from the results by Rottiers et al. (2019) who identified methyl ketones and their secondary alcohols as key odorants in fruity volatiles (as opposed to mostly esters). Esters and alcohols have been well documented as important compounds to cocoa aroma. Cocoa volatiles have also been suggested to be associated with cocoa genetics, and could be used as a tool capable of discriminating cocoa genotypes. For instance, Kadow et al. (2013) found that 2-pentanol (green, fruity, sweet, pungent) was specific to genotype SCA6, and 2-heptanol (fruity, lemon grass, floral, herb) was specific to EET62.

However, when cocoa is roasted, the total concentration of fruity volatiles dropped in all six hybrids analyzed. However, the differences appeared to be hybrid dependant. Hybrid ICS40 x SCA12 showed the highest level of fruity volatiles (~416 ug/g) and SNK10 x IMC67 showed the lowest level (~184 ug/g). Due to this drastic drop in these fruity compounds, they are often only detected as traces in cocoa products and by-products.

Floral Volatiles

Floral volatiles were represented by Terpenes, Esters, Alcohols, Aldehydes, and Ketones. The hybrid SNK109 x T79/467 fermented dried beans contained the highest concentration of floral volatiles (~97 ug/g). The lowest concentration of floral volatiles was found in the hybrid SNK16 x T60-887.

The floral compound 2-phenyl ethanol (Honey, floral rose, sweet, powdery, chocolate) found in the hybrid ICS40 x UPA134 was of importance. 2-phenylethanol and benzyl alcohol were also important in floral odor production. SNK hybrids were observed to contain higher levels of acetophenone (floral, fruity) even after roasting, which indicates even cocoa and chocolate products may contain higher detectable levels of floral notes (Bastos et al., 2019). Other authors have noted that high alcohol concentrations correlate to floral and sweet notes in fermented and dried cocoa beans (Aculey et al., 2010).

The hybrid SCA12 x ICS40 showed specific concentrations of terpenes (β-myrcene, trans-β-ocimene, linalool). Colonges et al. (2021) showed a correlation between these terpene compounds and genotype. Linalool is described as having a floral aroma and is a key aroma compound found in fine cocoa varietals. An increase in total floral volatiles was seen in roasted nibs vs the unroasted (fermented and dried) nibs made up mostly of terpenes and alcohols.

Chocolate/Nutty Volatiles

Chocolate and nutty volatiles were made up mostly of 5 pyrazines, 4 aldehydes, and 1 pyrrole. SCA12 x ICS40 had the highest concentration of chocolate and nutty aroma volatiles in unfermented samples, followed by other ICS40 hybrids. The compound benzaldehyde (almond) was the most dominate of nutty aldehydes identified. Benzaldehyde, 2-methylbutanal, and 3-methylbutanal are produced during the Maillard reaction while cocoa beans are roasting (Counet et al., 2002; Diab et al., 2014), but also from lactic acid bacteria during fermentation (formed from phenylalanine, isoleucine, and leucine respectively) (Bonnarme et al., 2004; Jinap et al., 1994).

Many studies have mentioned that many pyrazines originate from microbial origins, such as with fermented foods such as cocoa and soybeans (Besson et al., 1997; Selamat et al., 1994). Pyrazines in dried and fermented beans can be used as an indicator for a good fermentation and predict the quality of the beans (Jinap et al., 1994). Tetramethyl and trimethyl also represent nutty/chocolate flavour, but also associated with the more familiar or “basic” cocoa flavour. Hybrid ICS40 x UPA134 saw the highest percentage of tetramethyl at 72%, while SNK10 x IMC67 hybrid saw the lowest at 37%. In this study, hybrids could be discriminated according to levels of pyrazines present. Ziegleder (2009) found that Ghanian Forastero was richer in pyrazines (6.98 mg/kg) than Mexican criollo (1.42 mg/kg).

Roasting increased levels of pyrazines and aldehydes, with higher content of nutty/chocolate volatiles identified (except for in nibs of ICS40 hybrids). The ratio of tetramethyl pyrazine (TMP) to trimethyl pyrazine (TrMP) has been used as an indicator of well roasted cocoa beans, with a ratio value of 1.5-2.5 as optimal. Roasting parameters of beans depends on cocoa genotype, the material used (beans, nibs or liquor), the type of chocolate produced (milk or dark), and the desired attributes of the final product.

During roasting, two reactions (Strecker degradation and Maillard) which occur simultaneously are responsible for the rise in aldehyde concentrations. Aldehydes such as 3-methylbutanal (malty), 2-methylbutanal (chocolate), and benzaldehyde (floral) are developed both by fermentation and roasting, and are crucial aroma compounds contributing the overall flavour of chocolate. The pyrrole 2-acetylpyrrole (chocolate, hazelnut) is created during the Maillard reactions during roasting, and an important volatile in cocoa. Roasting should be adjusted in order to optimize increased levels of aldehydes and pyrazines as these are often important compounds identified in cocoa.

Creamy/Buttery Volatiles

This category of volatiles included 2 ketones (3-hydrooxybutanone and 2,3-butanedione) and one lactone (γ-butyrolactone). Acetoin and 2,3-butanedione were reported to be naturally present in cocoa beans (Ho et al., 2014). Acetoin (butter, milk) is produced from pyruvate via alcoholic fermentation (lactic acid bacteria) and from 2,3-butanediol from yeasts (Pretorius, 2000). High levels of 2,3-butanedione are associated with milky flavours, which was reported in Cameroonian Forastero.

Creamy and butter volatiles increased when fermented and dried nibs were roasted. The increase is dependent on acetoin content of the beans. The compound 2,3-butanedione decreased among all hybrid samples, but this was less important compared to the increase in acetoin. Acetoin has been classified as a technological marker for cocoa processing.

Undesirable Volatiles

These included acetic acid (sharp, pungent, sour), 3-methylbutanoic acid (sweaty), ethanol (alcohol), and 2-methoxy-phenol (smoky). The acetic acid was the most represented and gives off a sharp vinegar flavour. It is developed during fermentation during the aerobic phase via acetic acid bacteria, and by yeasts during the anaerobic phase of fermentation.

The 3-methylbutanoic acid was the second most represented volatile after acetic acid, and is derived from Leucine (an amino acid) putrefactive (decaying) bacteria during aerobic fermentation or from isobutyl acetate.

Roasting increased total undesirable volatiles including acetic acid and 3-methylbutanoic acids, which was also observed by Hinneh et al. (2020), but contrasted by Frauendorfer and Schieberle (2008). It is suggested that the heat during roasting unlocks the acids and makes them more available. Another hypothesis is that the heat causes methyl acetate to hydrolyse into methanol and acetic acid at high temperatures. A decrease in acetic acid occurred not during roasting by during grinding (data not shown).

Overview of volatiles

A Venn diagram was created to show an overview of the types of aromas (floral, fruity, nutty, buttery, etc.) with their associated chemical groups (esters, terpenes, alcohols, etc.). See Figure 2. For instance, fruity volatiles (shown in yellow) came from three chemical groups ketones, esters, and alcohols. Floral volatiles came from terpenes, ketones, alcohols, aldehydes, and esters. Nutty/chocolate flavours were made up of pyrazines, aldehydes, and pyrroles. And buttery/creamy volatiles were made up of ketones and lactones. Undesirable were mostly made up of acids.

As stated earlier, some findings show that specific chemical groups can describe the genotypic effect of a given cocoa. For example, terpenes were specific to SCA6 cocoa. Pyrazines are associated with Mexican Criollo. A genotype rich in terpenes will generally have very floral notes, and a genotype rich in pyrazines will have strong chocolate/nutty flavours. However, fruity notes did not have a strong genotype effect as they were not specific to one chemical group. It is suggested then that fruity flavours may be affected more by local growing environment and postharvest processes, but more investigation is required.

Correlation between reducing sugars and volatiles

Although hybrid SCA12 x ICS40 had the highest levels of reducing sugars, they did not have the highest concentration of total volatile compounds in roasted beans. Hybrid ICS40 x SCA12 with low levels of reducing sugars had high levels of total volatiles. It is well understood that aroma precursors (reducing sugars and amino acids) participate in the creation of volatile compounds. However, many authors point out there is no clear quantitative correlation between amounts of precursors to the aromatic compounds formed (Tran et al., 2015, Frauendorfer & Schieberle, 2008). On the contrary, Giacometti et al. (2015) claim Criollo cacao contains high levels of precursors that produce high levels of pyrazines. In this study though, the hybrid SCA12 x ICS40 which had the greatest level of reducing sugars did have the highest concentration of pyrazines specifically among the 6 hybrids.

Odor activity Values of Aroma Components

Although we see that there are many volatile compounds found at high concentrations in cacao, it’s important to note that this does not always directly correlate with the overall aroma/flavour of the cocoa beans. How much a volatile compound participates in the overall aroma depends on the odor-active value (OAV) of that particular compound. In other words, just because one aroma is found at higher concentrations, it does not mean that you would detect it compared to another aroma at lower concentrations. It is important to evaluate the OAV values in order to estimate if that particular aroma is present in concentrations above the threshold required in dark chocolate in order to play a key role in the overall flavour of the chocolate.

Since cocoa normally contains at least 50% fat, the odor threshold values were analyzed in an oil/lipidic media. The Odor Threshold Values (OTVs) of the main volatiles are shown in Table 4. An OAV greater or equal to 1 contributes to the aroma of the cocoa/chocolate (Frauendorfer & Schieberle, 2008).

Acetic acid concentrations and OAV’s increased during roasting, but decreased during conching and the “dilution of the cocoa liquors” when sugar and cocoa butter was added (Counet et al., 2002). The compounds 2-phenylethanol and 2-phenethyl acetate were recognized as odor-active compounds contributing floral and honey like flavours. The OAVs of aldehydes, pyrazines, and terpenes increased with roasting of the beans.

Principal component analysis (PCA) and Hierarchical cluster analysis heatmap (HCA heatmap)

Below are a couple of Figures that help offer a birds-eye-view of the data collected in this study. Figure 3 is a PCA (Principal Component Analysis) plots the 6 different hybrids and groups them with the volatiles most closely associated with them in this study. Figure 3A represents unroasted fermented/dried beans, and Figure 3B on the bottom represents the roasted nibs. The idea here is to study each graph to get an idea of 1) which aromas are associated or found in higher concentrations with which hybrids, and 2) how they shift from unroasted (A) to roasted samples (B).

Figure 4 is a Hierarchical Cluster Analysis Heatmap (HCA Heatmap) of the same data but only for unroasted fermented dried beans. On the bottom of the figure are the 6 hybrids, and on the right are the volatile aroma compounds identified in the study. Dark red represents an aroma compound in high abundance/concentration for that hybrid, pink being less so, and dark blue representing a very low abundance/concentration of that aroma compound. Scanning through this figure you can get a quick overview of which volatile compounds (aromas) were highest in which hybrids. The clusters of hybrids in Figure 4 correspond to the clusters of hybrids in Figure 3, displaying which hybrids are generally more closely associated according to their aroma concentrations.

In figure 4, the two columns on the left represent hybrids ICS40 x UPA134 and ICS40 x SCA12, both of which were high in 2-pentanol acetate, methyl acetate, 2-phenyl ethanol, linalool oxide, and 2-heptanone making them similar in this regard. What set these two hybrids apart were volatiles ethyl isopentanoate, 3-methyl butanal, and 3-methylbutanoic acid.

The second cluster in the middle were the two SNK hybrids (SNK10 x IMC67 and SNK16 x T60/887), and could be distinguished by high levels of isobutyl acetate, 2-nonanone, 2-nonanol, 2-heptanol and its esters. They differed in regards to their pyrazine content.

The third cluster was simply the SCA12 x ICS40 hybrid alone, which was a unique hybrid with high concentrations of 2,3-butanediol, linalool, β-myrcene, and cis/trans-β-ocimene. In this hybrid, the monterpene biosynthetic pathway appears to be more intense than in the other hybrids.

The fourth cluster was SNK109 x T79/467 hybrid alone, which was high in acetoin, 2-phenylethyl acetate, isoamyl acetate, acetic acid, 2-phenylethanal, ethyl phenyl acetate, 2-pentanol, benzyl alcohol and butyl acetate. The degradation pathway of L-phenylalanine in this hybrid seems to be very important.

Conclusion

Six hybrids of cocoa grown in Cameroon were differentiated according to biochemical and aromatic compounds. The compounds 2,3-butanediol, linalool, β-myrcene, cis/trans-β-ocimene, 2-nonanone, 2-nonanol, 2-heptanol, methyl acetate, acetophenone could be used to discriminate the cocoa hybrids into their native groups (Lower Amazonian, Upper Amazonian, Trinitario, and local Cameroonian Trinitario cocoa). Other volatile compounds (acetoin, 2-phenylethyl acetate, isoamyl acetate, and acetic acid) were specific to a given hybrid, irrespective of it’s native group. Some volatiles could be classified to specific chemical groups, such as floral (terpenes), chocolate/nutty (pyrazines), buttery/creaming (lactones), and undesirable aromas (acids/phenols).

Generally speaking, roasting reduced total amount of volatile compounds in all hybrids, with the greatest reduction seen with fruity volatiles, but other volatiles as mentioned above generally increased during roasting. The variation in concentration of volatile aroma compounds during roasting was unique to each hybrid, suggesting that cacao genotype should be considered when determining roasting parameters of cocoa.

There was no significant differentiation in regards to sugar content and volatile concentration, suggesting an absence of pollen effect on these compounds.

Sugar profile and volatile aroma composition in cocoa nibs from 6 Cameroonian fine-flavor cocoa hybrids