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.

Notes From Geoseph

This is a summary of the paper “Effect of spontaneous fermentation location on the fingerprint of volatile compound precursors of cocoa and the sensory perceptions of the end-chocolate” by Kouassi et al. (2022). The results here demonstrate a few things which I find interesting. We have a batch of cocoa that was grown and harvested in the same are. However, instead of sending them all to the same fermentation facility the lot was divided and sent off to 5 different regions ranging from 150-550 km from the harvest area within Ivory Coast. The identical batch of beans were then fermented in exactly the same way. The result? The fermented cocoa beans were all of similar good quality no matter where they were fermented. This demonstrates fermentation method has a big impact on quality and consistency. However, what did differ, and in some ways quite greatly, was a shift in the concentrations of aroma compounds formed due to the local fermentation. Same beans genetically, same harvest time, same fermentation method. And what varied from region to region was the microbial (yeast, bacteria) population - which had a direct relationship to the formation of aromas and their concentrations. Same quality, yet different aroma profiles - resulting in chocolate with different aroma profiles. This study confirms what has already been suggested for years that microbial species and populations have a profound impact on the flavour of the cocoa bean, and as a result the flavour of the resulting chocolte.

Introduction

Chocolate is one of the most consumed foods worldwide due to its flavour and unique sensory characteristics (the way it feels and melts in the mouth). Chocolate is made from cocoa beans, and the flavour compounds of the cocoa bean are important indicators of how well or poorly the beans were fermented and dried. They are also very important to the sensory characteristics experienced by the consumer in its finished stage: chocolate. Many of these compounds are formed during the fermentation and drying process, with fermentation being crucial to the development of many precursor aromas that are formed during the later roasting stage.

Open fresh cocoa pod. Image by Science Of Chocolate.

Keep in mind there are many factors which influence aroma profile of the cocoa beans including cocoa bean genotype, soil conditions, age of the cocoa trees, postharvest treatments (such as pod storage), and the manufacturing of the beans into chocolate. There are many groups of flavour compounds involved in cacao and chocolate flavour including aldehydes, ketones, esters, alcohols, acids, and pyrazines.

The Ivory Coast leads in the world production of cocoa beans, and on-farm fermentation is still performed spontaneously, which often leads to cocoa beans with poor aromatic quality. However, some are abandoning spontaneous fermentation for controlled fermentation processes using starter cultures. Many studies have reported improvement of aromatic quality by using specific microorganism starter cultures. However, very few studies have identified the diversity of microbiota such as yeasts and acetic bacteria. This work will evaluate the effects of the fermentation location on the formation of flavour compounds in the beans, and how this translates to organoleptic (taste, flavour, sensory attributes) of the chocolate made from these beans.

Materials & Methods

Geographic characteristics of the 5 cocoa fermentation regions in the Ivory Coast.

Cocoa beans were harvested from mature pods of the Ivorian hybrid cultivar called “Mercedes” which is a cross between Amelonado and West African Trinitario. Cocoa pods were harvested on a 5 hecatare traditional farm in the AKoupe region during the main harvest season. The harvested pods were then divided into 5 batches and transported to 5 cocoa producing regions including Daloa, Akoupe, San Pedro, Soubre, and Djekanou (see Table 1). Once reaching their destination, the intact pods were soaked in a disinfecting agent for 15 minutes, and then opened with the seeds and mucilage removed. Beans were then sorted to discard any rotten, black, or germinated beans leaving only healthy beans to be fermented.

The fermentation process was carried out in duplicate, and each fermentation lasted for 5 days. They were started at the same time in their corresponding locations, and in the exact same method using banana leaves and plastic boxes. Cocoa beans fermented at Akoupe (where they were harvested) were considered the control for the fermentation location. After fermentation, a bean cut test was used using the Magra bean cutter, and the cut test score was calculated using the equation shown here:

The chocolate was produced from the 5 day fermented and dried cocoa beans from each of the 5 locations. The beans were roasted to 160°C/320°F for 30 minutes. The chocolate was judged by a panel of 12 people, using the Akoupe region chocolate as the control. The panel found significant differences in the organoleptic (sensory) attributes of the chocolates produced from the 5 different batches of beans.

Results & Discussion

Physical quality parameters of cocoa bean according to the fermentation location

Table 2 displays the results of the cut tests on the fermented dried beans. All batches contained over 80 good beans per 100g, indicating a good bean count, and no statistical difference found between the 5 regional batches. Low bean counts are often due to various factors such as genotype, age of the tree, growing conditions, and season of production. Of the unhealthy beans a very low percentage was due to slaty and moldy beans, with the rest being due to purple beans, defective (insect damage, germinated, flat) which were found in all 5 regional batches. The highest level of brown beans (optimal) were found in the batch fermented in the San Pedro region.

All batches had a very low percentage of mouldy beans. Low percentage of mouldy beans could be explained by the antifungal bacteria and yeast growth during fermentation inhibiting fungi growth. It’s believed the organic acids produced from lactic acid bacteria, proteinaceous compounds from yeasts, and high amounts of alcohol, and lower pH, organic acids, elevelated temperatures, and microaerophilic conditions in the mass also restrict mold growth.

Fingerprint of volatile compound precursors in the function of fermentation location of cocoa

You can see in the two images of Table 3 below, 34 volatile compound (aroma) precursors, which are categorized according to their chemical families: alcohols, aldehydes, ketones, acids, esters, pyrazines, and other compounds. It’s good to note that the compounds listed here are also in accordance with a study by Assi-Claire et al. (2019) who analyzed the aroma compounds identified in cocoa beans inoculated with Saccaromyces cervisiae (yeast). In comparison to another study by Valle-Epquin et al. (2020) who identified aroma compounds of Criollo cacao from Peru, it appears there are less volatile compounds detected here in this study compared to the Criollo cacao.

When looking at concentrations of aroma compounds, the alcohols Ethanol, 2-Pentanol, and 2-Heptanol stand out. In most cases, concentration of aroma compounds increased from very low or nothing in unfermented cocoa beans to much higher levels in 5 day fermented cocoa beans (regardless of location). However, it is interesting to note that Ethanol, 2-Pentanol, and 2-Heptanol were much higher in unfermented cocoa beans, and decreased substantially in the post-fermented beans. Higher levels of these three compounds appear to be characteristic of unfermented beans.

The overall increase in certain aromatic compounds in cocoa could be due to the microbial metabolism of yeasts and lactic acid and acetic acid bacteria during the fermentation. The yeasts specifically seem to directly impact levels of esters, which exhibit aromas such as flowery and honey. The results in this study suggest that not all aromatic compounds identified in the cocoa beans are due only to fermentation itself, but also to the location of the fermentation.

Alcohols

Alcohols tend to promote floral and sweet aroma notes. All the cocoa bean batches show high levels of alcohols by the end of fermentation, regardless of location. The high levels of alcohols can be explained by the post harvest storage of the pods before they were opened and fermented (Kone et al. 2016). The Soubre region batch exhibited higher concentrations of many of the alcohols, and this could be explained by the local endemic yeast strains in that fermentation. The most abundant alcohol was 2,3-butanediol (fruity, creamy, buttery) at 114.65 ug/g vs 6-61 ug/g identified in beans from the other fermentation locations. This compound is known to be a desirable compound in high quality cocoa products.

2-pentanol is also a key alcohol and was present in highest concentrations in both Soubre and Djekanou fermented batches. 2-heptanol is another key alcohol, and was highest in cocoa fermented in San-Pedro, again likely due to the metabolism of sugars in the fruit by local endemic yeast species.

Aldehydes

You can see in table 3 that nonanal (rose, orange peel) and benzaldehyde (sweet, almond, cherry) were the main aldehydes in all bean samples. Beans from San-Pedro and Djekanou had the highest levels of benzaldehyde. It is believed aldehydes can be formed from lipid oxidation or self-oxidation and the degradation of fatty acids during fermentation. However, Hamdouche et al. (2019) concluded aldehydes resulted from lactic fermentation from amino acids instead. Regardless of the pathway that leads to the production of these compounds, it is suggested that fermenting in these regions would allow for greater levels of these aromas (citrus, green, rose, fruity, almond, cherry).

Ketones

Cocoa beans fermented in Daloa region contained the highest concentrations of the ketones. Acetoin (buttery, sour milk, caramel) is a major ketone, and may be produced from pyruvate and butanediol during alcoholic fermentation due to the yeasts involved. However, Assi-Clair et al. (2019) concluded some ketones are not formed due to yeast metabolism. High levels of ketones are suggested to be key characteristics of higher quality cocoa beans. An ANOVA test (an analysis of variance statistical test) confirmed that fermentation location had a significant influence on production of ketones in the fermenting cocoa beans. Some ketones such as 2,3-butanedione, 2-acetoxy-3-butanone, and acetoin contribute buttery and creamy notes. Other ketones such as acetophenone, 2-nonanone, and 2-heptanone produce fruity, flowery, and sweet flavours.

Acids

Acetic acid and 2-methylpropanoic acid were the most abundant acids identified in all batches. Beans fermented in the Daloa region were highest in acetic acid (vinegar, peppers, green, sour), while beans from Soubre were highest in 2-methylpropanoic acid (rancid). It appears location also influences the generation of these aromatic acids. A great deal of acetic acid is removed via evaporation during roasting of the cocoa beans.

Esters

Table 3 shows that esters had a great presence in the aroma profile of the beans here. The results of esters identified were similar to those of Valle-Epquin et al. (2020) based on Criollo cocoa beans from Peru, but far lower than a study by Rodriquez-Campos et al. (2012) who identified 20 ester compounds.

Isoamyl acetate (sweet, fruity, banana) and 2-phenylethyl acetate (honey, flowery) had the highest concentrations in beans fermented in the Daloa region. Soubre region beans had the highest level of 2,3-butanediol, diacetate A (generally sweet, pleasant, fruity).

Esters are generally produced via the metabolism of yeasts from alcohols via esterification pathways. However, some esters are already found in the fresh cocoa bean mucilage (fruit). Esters are generally the second most important aromas to cocoa aroma profiles after pyrazines, and are siginficantly influenced by fermentation location.

Pyrazines

Cacao from the Daloa region contained the highest concentrations of pyrazines. Pyrazines are formed via microbial metabolism during fermentation, and then increase in concentration during roasting (Strecker degradation and Maillard reactions). Pyrazines are the most important group of aroma volatiles as they offer the most "chocolate” notes (Valle-Epquin et al. 2020). Pyrazines is also an indicator of beneficial yeasts and a good fermentation. Location of fermentation also significantly influences pyrazine levels.

Other volatile compound precursors

Table 3 shows other volatile compounds identified as well, and one important terpene, linalool. Linalool is formed from the breakdown of leucine from yeast activity during fermentation. Linalool greatly influences the aroma profile of chocolate and is a favorable aroma. Fermentation location had a significant influence on linalool and other volatile compounds.

Impact of fermentation location on the fingerprint of volatile compounds precursors of cocoa

A biplot shown in Figure 1 displays a visual of the relationship between fermentation region and the formation of flavour compounds in the cocoa beans. You can see the biplot is divided into 4 quadrants.

First thing to notice is that in the top left quadrant displays the aroma compounds associated with San-Pedro. And we can see that San-Pedro cacao the aromas associated with it is uniquely discriminated from the other four location batches. San-Pedro had high levels of the 8 compounds shown in the top left quadrant of the biplot, the batch with less numerous aromatic compound precursors.

Soubre and Djekanou are both in the negative axis shown in the bottom right quadrant, and their 12 corresponding aroma compounds associated with them.

On the positive axis there are 3 (San-Pedr, Akoupe, Daloa) with a combined number of 20 aroma compounds. The beans fermented at Daloa recorded the highest number of desirable aromatic compound precursors (esters, ketones, aldehydes), and this could be ascribe to the local endemic microbiota (yeasts in particular) as previously concluded by Maura et al. (2016). S. cerevisiae and P. kudriazevii yeasts produce a wide variety of desirable aromatic compounds in cocoa beans (Junior et al. 2021).

Sensory Attributes of Finished Chocolate

Chocolate was made from the 5 batches of cocoa beans fermented in different regions. The chocolate was analyzed by a panel of 12 people for various aspects such as odor, sweet, sour, bitter, astringent, cocoa aroma, fresh fruit, dried fruit, floral, vegetable, spicy, alcoholic aroma, animal, cocoa odor, and overall quality.

Figure 2 displays the results of this analysis visually for the 5 different chocolates made. All the chocolate samples scored the same for spicy and cocoa odor notes. The spicy flavour may be due to genotype and growing conditions of the tree (Bastos et al. 2019).

Chocolate made with cacao fermented at Daloa scored the highest for fresh fruit and floral. This could be due to highest concentrations of alcohols, ketones, and esters. Junior et al. (2021) demonstrated the ability of yeast to produce esters, alcohols, and aldehydes. This suggests that the Daloa region has endemic yeasts with the potential for aromatic compound production.

Chocolate made with cacao fermented at Soubre scored highest for sour, astringent, and bitter notes.

Chocolate made with cacao fermented in San Pedro had the highest score for overall quality, even though the the actual aroma compound content of the cocoa from San Pedro were low. This confirms that roasting has a big impact on improving the sensory perception of the cacao.

The highest scores for undesirable aromas (sour, bitter, astringent) were identified in both Soubre and Daloa region cocoa, possibly due to the high acid levels.

It’s interesting to point out that there was no direct relationship between the concentration of aromatic compounds in the cocoa beans, and the sensory attributes of the chocolate made from those beans. This is because the sensory attributes favoured in the chocolate are not only due to volatile profile, but also include non volatile factors. Kongor et al. (2016) concluded that concentrations and types of volatile compound precursors originating from microbial processes during fermentation are not a good indicator for cocoa aromatic quality. Valle-Epquin et al. (2020) confirmed that only after roasting the beans could the true fingerprint of the aroma of the cocoa beans.

Conclusion

The location of the fermentation process of the cocoa beans had no impact on the overall physical quality of cocoa beans. However, the region in which they were fermented did impact the aroma compounds in the cocoa beans likely due to the diversity of endemic microorganisms. A good fermentation will ensure good aromatic quality of the cocoa beans.

San-Pedro proved to be the region for the highest concentration of aroma compounds formed during fermentation, however, we know that sensory properties of the finished chocolate also involves roasting, not just what happens at fermentation.

There were some limitations to this study including as it lacked data on sensory perceptions of chocolate and the microbial ecology for each fermentation location. The data on the yeast itself will be published in a separate publication in the near future, and the analysis of the aromatic compounds in the finished chocolate will also be made known.

Fermentation location and local endemic microbes impacts aroma profile of cocoa beans.