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

This research paper may seem a bit daunting and too technical to those of you who don’t read research papers very much. However, I encourage you to at least skim through it, and at the very least look at the graphs and figures. Try and recognize some patterns and get an idea of how much aroma compounds and other compounds can change (increase, decrease, maybe increase again). Fermentation is not only about “improving flavour” or “building up the aroma precursors”. It will help you appreciate how complex curing cocoa beans is as well as how complex tracking and building up favorable aromas can be. This research paper looked at one type of bean from one area in Mexico. Beans from all sorts of regions and various genotypes require slightly different fermentation methods.

As well, this research paper is from 2010, and refers to 3 cacao varieties (Forastero, Trinitario, and Criollo) which is a bit outdated today in regards to known varietals. However, the content in this paper in regards to compounds in cocoa beans during fermentation and drying still stands.

Introduction

The cocoa bean has been used in Mexico since pre-Hispanic times to create traditional beverages. Cocoa beans are classified by aromatic quality according to the compounds present. Factors which influence quality include cacao genotype, agroclimatic conditions, fermentation, drying, and the manufacturing process. All of these factors impact the composition of volatile and non-volatile compounds which define the quality of the final cocoa product (Brito et al., 2000).

During fermentation, the mucilaginous pulp surrounding the seeds is eliminated, heat is generated, and the pH drops (acidity increases) which inhibits the seed from germinating (Thompson, Miller, & Lopez, 2001). The pulp is rich in fermentable sugars including glucose, fructose, and sucrose along with inorganic salts and has a pH from about 3 to 3.5 due to high levels of citric acid. This substrate is ideal to allow yeast and lactic acid bacteria to grow.

Yeast

Yeasts ferment the carbohydrate-rich pulp and produce mostly ethanol and carbon dioxide. Schwan and Wheals (2004) found the yeasts Kloeckera apiculata and Saccharomyces cerevisiae var. chevalieri produced desirable compounds such as isopropyl acetate, ethyl acetate, 1-propanol, isoamyl alcohol, 2,3-butanediol, diethyl succinate, and phenylethanol. They also found that at the end of alcoholic fermentation, lactic acid bacteria and acetic acid bacteria began to grow.

Lactic Acid Bacteria

The lactic acid bacteria grows from consuming glucose and citric acid from the pulp, and in turn produced lactic acid. Lagunes-Galvez et al. (2007) reported that lactic acid was at its highest concentration in the fermenting mass at day 5 of fermentation. Lactic acid is not a favorable component of cocoa bean quality, and if a chocolate product contains lactic acid, it’s considered an undesirable flavour.

Acetic Acid Bacteria

Acetic acid bacteria oxidize the ethanol produced by alcoholic fermentation, and produces acetic acid and ethyl acetate. If acetic acid is too high, this may be detrimental to the quality of the cocoa products. During fermentation, acetic acid enters the bean and causes a decrease in the pH from 6.5 to 4.5 (Thompson et al., 2001). If cocoa beans have too high a pH after fermentation (5.5-5.8) they are considered poorly fermented. Cocoa beans with a lower pH of 4.75-5.19 are considered well fermented (Afoakwa et al., 2008). Chocolate made with cocoa beans with either a very high pH or very low pH had less chocolate flavour and more off flavours, while cacao with an intermediate pH of 5.20-5.49 produced chocolate with higher notes of chocolate flavour (Jinap, Dimick, & Hollender, 1995).

Drying

Drying reduces the moisture content of the cocoa beans to under 8% moisture. Beans are usually dried in the sun, but sometimes other drying methods are used. Drying also reduces the acidity and astringency of cocoa, and decreases the volatile compounds content (Afoakwa et al., 2009).

The volatile and non-volatile compound data was analyzed using PCA (principal component analysis). This is used to gather large amounts of data and organize them in a way to observe trends, clusters, and compare/contrast the data. There will be some PCA graphs later to visually show you the various trends and clusters.

The aim of this research was to identify the different volatiles (alcohols, aldehydes, ketones, esters, carboxylic acids, and pyrazines) as well as non-volatile compounds (sugars and carboxylic acids) during both fermentation and drying processes.

Materials & Methods overview

Cocoa beans of the Forastero variety from Cunduacan, Tabasco, Mexico were harvested by traditional methods and transported to the laboratory. Natural fermentation was carried out with 1000 kg of raw cocoa with pulp, in wooden boxes about 1m cubed at ambient temperature from 8 days. Cocoa beans were manually turned and moved from one box to the other once per day to ensure aeration and uniform fermentation.

The fermented beans were then placed on a concrete floor and spread out about 5-10 cm thick and sun dried. These beans were mixed manually each day for 5 days. Sub-samples of 2kg were obtained each day of fermentation and drying and labelled as the following:

Beans at zero time (before fermentation) labelled RC0
Fermentation samples labelled FC1-FC8
Drying samples labelled D1-D5

Samples were frozen and sent to the laboratory, then analyzed with the testa (husk around the bean) removed. Each sample was divided into two, one analyzed for volatiles and the other analyzed for non-volatiles. 5 grams of cocoa beans were also analyzed for pH and titratable (total) acidity, indicating the high values of acidity in cocoa beans can be indicated by these values.

Changes of pH during fermentation and drying processes

During fermentation, pH values of cocoa beans decrease (beans come more acidic). The pH decreases from 6.4 at RC0 (before fermentation) to 4.5 after 8 days of fermentation. It is suggested this decrease in pH is due to the acids produced from lactic and acetic acid bacteria during the fermentation process (Afoakwa et al., 2008; Thompson et al., 2001). However, too low a pH can indicate low quality cocoa beans. Jinap et al. (1995) reported that roasted beans with a lower pH (4.75-5.19) scored lower for notes of chocolate. Portillo et al. (2007) found that if pH was lower than 4.5, that overall aromatic potential was decreased as well.

As the cocoa beans dry, there is a loss of both water and volatile acids when drying occurs at a slower/more moderate length of time. Garcia-Alamilla et al. (2007) reported that if the drying temperature was around 60°C the beans had greater concentrations of acids including acetic, propionic, isobutyric, and isovaleric acids. In this experiement, drying temperature averaged 47°C.

In this experiment, cocoa bean acidity increased from 0.0062% to 0.106% after 8 days of fermentation. There is a high correlation between acetic acid and lactic acid with pH and titratable acidity

Sugar and non-volatile acids

Before fermentation occurs, cocoa beans have higher concentration of sucrose and lower concentrations of reducing sugars (glucose and fructose). This was also observed and reported here (Figure 1a). Reducing sugars are reducing agents which will help to form aroma compounds as the beans are dried and roasted. During fermentation, sucrose was depleted as it was converted to glucose and fructose (whose concentrations increased nearly three fold).

Organic acids were determined before fermentation, where citric acid concentration was higher than malic, lactic, oxalic, and succinic non-volatile acids (Figure 1b). During fermentation, Oxalic and malic acid levels did not change significantly. However, malic and lactic acids significantly increased during both fermentation and drying. Lactic acid was highest at day 3 of fermentation (Fig 1b). The presence of lactic acid is not favorable for cocoa quality as it will produce excessive sourness and can mask chocolate flavour (Lagunes-Galvez et al., 2007; Thompson et al., 2001).

volatile compounds produced during fermentation

There were 39 compounds identified during both the fermentation and drying stages in this research (Table 1). Some are responsible for favorable flavour notes, and some for less desirable off-flavours (ex/ propanoic and butanoic acid).

Six principal alcohols were identified during fermentation, two of which were amyl alcohols (3-methyl-1-butanol, and 3-methyl-2-butanol). See Figure 2a. Amyl alcohols are common compounds found in food flavour, and some used to evaluate cocoa flavour and fermentation degree. The alcohols here are produced during fermentation of the sugars present in the cocoa beans. Some such as 3-methyl-1-butanol, 2, 3-butanediol, and phenylethyl alcohol (honey, spice, floral, caramel) have also been reported in cacao fermented with K. apiculata and S. verevisiae var. chevalieri yeasts. These compounds are considered desirable in high-quality cocoa products. These two compounds have also been reported to be derived from amino acid catabolism (breakdown) during fermentation (Afoakwa et al., 2008). Phenylethyl alcohol is a precursor to the aldehyde phenylacetaldehyde, which is then oxidized to the ester phenylethyl acetate.

Acetoin (associated with butter and cream flavour) is one of the aldehydes with the highest concentration, and may be produced by alcohol fermentation from pyruvate and butanodiol (Pretorious, 2000). There were 7 esters identified during fermentation, of which ethyl acetate had the greatest concentration at day 3 (Figure 2c). Ethyl Acetate is produced during the esterification from acetic acid and ethanol (Pretorious, 2000).

Oberparleiter & Ziegleder, (1997) proposed using ratios of aldehydes-amyl alcohols and acetates-amyl alcohols as a way to evaluate fermentation degree. They suggest a ratio of methyl-1-butyl acetate:methyl-1-butanol that is higher than 1.5 means the beans are over fermented and produces a hammy flavour defect. This hammy flavour comes from the isobutyric and isovaleric acids formed enzymatically during fermenting for a prolonged duration. In this study there was a ratio of 0.99 at day 6 of fermentation and 1.68 at day 8, which means the mass was over fermented according to their method. There was an increase of isobutyric and isovaleric acids after four days (Figure 2d). In this example, it is suggested to have stopped the fermentation at day 6 when the ratio was lower to avoid esterification of amyl alcohols to amyl acetates.

Other acids identified included propionic, hexanoic, octanoic, nananoic, and dodecanoic acids. Propionic and butyric acid would have a negative effect on the cocoa aroma quality (Serra-Bonvehi, 2005).

volatile compounds produced during the drying process

Alcohol concentrations of 2,3-butanediol (buttery, creamy, fruity) and 1,3-butanediol increased during the drying process (Figure 3). Phenylethyl alcohol (honey, spice, floral, caramel) and benzyl alcohol (sweet, floral) decreased. The compound 3-methyl-2-butanol (fruity) was identified during fermentation, but not during the drying process (Figure 3a). This suggests that fermentation did not stop after fermentation, and that volatile alcohol was produced as a precursor to other compounds (i.e. 2,3-butanediol to produce 2,3-butanedione; Figure 3a and 3b). The concentration of many aldehydes decreased as well during the drying process. Tetramethylpyrazine significantly increased in concentration during drying (Figure 3b), and is associated with characteristic cocoa and roasted coffee flavours.

Drying reduces volatile acids as well as total polyphenols, and converts the flavour precursors into pyrazines and aldehydes (both of which contain an array of compounds important to overall flavour and quality). Flavour development within the bean continues during drying. It was also found that 3-methyl-1-butanol acetate and isobutyl acetate (fruit, apple, banana) increased in concentration during drying. Isobutyl acetate is a precursor of isobutyric acid (off-notes such as rancid, butter, cheese, hammy). Ethyl acetate concentration also increased, decreased, and then increased again during drying (Figure 3c). This paralleled the trend of acetic acid levels during drying in Figure 3d. Ethyl acetate is a product of esterification from acetic acid and ethanol (Pretorious, 2000). This suggests that acid bacteria were still present after fermentation and throughout the drying process. Acetic acid concentration increased during the drying process and was higher than isobutyric, isovaleric, hexanoic, octanoic, and nonanoic acid which all diminished during the drying processes (Figure 3d). An increase in temperature and aeration favored compound volatilization of short chain volatile acids (Nogales et al., 2006).

Principal Component Analysis (PCA)

Note: A PCA can be a bit complicated for those who have never looked at one. Check out this brief video which better explains how to interpret these PCA graphs.

A PCA was performed to determine the most important volatile and non-volatile compounds present during fermentation and drying. The PCA is a variable-reduction technique which organizes data visually based on similarities and differences to display trends, clusters, and other observations and define the number of “principal components”. To see which data points are correlated and which are not. Figures 4 and 5 display the PCA results for fermentation and drying respectively.

Each PCA requires a PC1 for the x-axis on the bottom (horizontal), and a PC2 for the y-axis on the left (vertical). In this case, PC1 represents 38.8% of data variance and PC2 represents 22.2% of data variance. Keep in mind PC1 is more important than PC2. The analysis includes pH, titratable acidity, sugars, non-volatile acids, and all volatile compounds.

Fermentation PCA

PC1 on the positive x-axis is highly influenced by volatile compounds such as methyl acetate , acetoin (butter, cream), 2,3-butanedione (butter), phenylethyl acetate (sweet, fruit, honey), and 3-methyl-1-butanol acetate, as well as by succinic acid, lactic acid, malic acid, and titratable acidity. You can see all of these compounds clustered towards the very left of the graph about mid way up in Figure 4a. These compounds increased in concentration significantly at mid-fermentation (FC3, FC4, FC5). Compounds with the highest concentrations were methyl acetate, acetoin, lactic acid, and 2,3-butanedione.

The PC1 negative axis (on the very left of the graph) grouped compounds identified before and on day 1 of fermentation (RC0 and FC1). These include sucrose, citric acid, oxalic acid, and dodecanoic acid, 2-pentanol, phenylacetaldehyde, and pH parameters. These compounds decreased during fermentation.

The PC2 on the positive y-axis (Top right quadrant of Figure 4a) was influenced by isobutyric, isovaleric, and propanoic acid, all of which increased in the last days of fermentation (FC6-FC8; Figure 4b).

PC2 on the negative y-axis included ethyl lactate, nananoic acid, ethyl acetate, 3-methyl-1-butanol and 2,3-butanediol (bottom right, negative quadrant). These compounds were most important at day 2 of fermentation (FC2).

Drying PCA

The drying process could be grouped into 3 groups (Figure 5b). The PC1 for drying on the positive axis (top right of Figure 5) was strongly influenced by phenyl acetaldehyde (honey, flower, sweet), glucose, octanoic acid (cheese, fatty), phenylethyl alcohol (honey, spice, floral, caramel), and hexanoic acid (pungent, rancid). These compounds decreased during the drying process.

The negative axis of PC1 we see volatile compounds including 3-methyl-1-butanol acetate, 3-methyl-2-butanol acetate, 2-methyl-1-propanol (wine), acetic acid (sour, astringent, vinegar), and tetramethylpyrazine (milk-coffee, roasted, chocolate). These compounds increased in concentration during the drying process, and are associated with the last two days of drying (DC4 and DC5).

PC2 on the positive axis we see titratable acidity, 2,3-butanedione, fructose, and methyl acetate. These compounds decreased in concentration during day 2 and 3 of drying. The negative axis of PC2 was mostly influenced by acetoin.

Conclusion

This research proposes various compounds which can be used as indicators for evaluating cacao during the fermentation process.

The PCA depicted the fermentation stage into four main groups, and into three main groups for the drying process. These identifying groups may be able to help search for indicators of off-flavours such as isobutyric (rancid, cheese, hammy), isovaleric (sweat, rancid), and propionic (pungent, rancid) acids.

The oxidation of 3-methyl-1-butanol (malty, bitter, chocolate) to 3-methyl-1-butanol acetate could be used to evaluate the degree of fermentation. The critical point in traditional fermentation of cocoa beans appear to be at day three, where the formation of acetic and lactic acid increased.

Identifying the main compounds produced during fermentation and drying may help in deciding when to stop a fermentation and prevent off-flavours.

Dynamics of volatile and non volatile compounds in cocoa during fermentation and drying processes