Non-volatile and volatile composition of West African bulk and Ecuadorian fine-flavor cocoa liquor and chocolate

https://doi.org/10.1016/j.foodres.2019.108943Get rights and content

Highlights

  • In-depth study of the composition of a bulk and a fine-flavor cocoa liquor and chocolate.

  • Investigation of volatile (HS-SPME GC–MS) and non-volatile metabolites (UPLC-HRMS).

  • A wide range of non-volatile compounds, both polyphenols and non-phenolic compounds, could be identified and quantified.

  • Principal component analysis revealed the main distinctive (non-)volatile constituents.

  • The suggested approach results in a thorough comparison of cocoa samples.

Abstract

Cocoa products are obtained from the seeds of Theobroma cacao L. In this research, cocoa liquor and chocolate produced from cocoa beans from West Africa (Forastero, “bulk” cacao) and Ecuador (Nacional variety, “fine-flavor” cacao), were investigated, using a novel approach in which various analytical techniques are combined in order to obtain in-depth knowledge of the studied cocoa samples. The levels of various classes of primary metabolites were determined and a wide range of secondary metabolites, including volatile organic acids, aldehydes, esters, pyrazines, polyphenols, methylxanthines and biogenic amines, were identified and/or quantified by HS-SPME GC–MS (headspace-solid phase microextraction gas chromatography - mass spectrometry). and UPLC-HRMS (ultra-performance liquid chromatography - high resolution mass spectrometry). Odor Activity Values (OAV) were calculated to assess the contribution of individual volatiles on the final aroma.

Various volatile aroma compounds were more abundant in the West African cocoa liquor and chocolate, while the Ecuadorian samples were richer in most quantified non-volatile metabolites. Principal component analysis (PCA) confirmed that the four samples can be clearly distinguished. Alcohols, pyrazines, amino acids and biogenic amines were found to be highly influential in causing this differentiation. The proposed approach can be useful in future studies on more extensive cocoa sample collections, in order to highlight similarities and pinpoint typical differences in chemical composition among these samples.

Introduction

Cocoa liquor is the product obtained from the fermented, dried, roasted and ground seeds of Theobroma cacao L. Together with cocoa butter (the fat pressed from cocoa liquor) and sugar, it is the major ingredient of dark chocolate. Cocoa is cultivated between 20° north and 20° south of the equator (Beckett, 2009), mainly in West Africa (representing over 70% of the world production), but also in Central and South America, and South-East Asia (ICCO, 2018). Generally, cocoa is classified into four main varieties based on geographic origin, pod morphology, bean yield, genotype, disease resistance and flavor characteristics. Forastero cocoa, the main variety cultivated in West Africa, is considered as bulk cocoa representing over 90% of the global cacao production. The Criollo, Trinitario and Nacional varieties form the fine-flavor cocoa group and represent 5–10% of the cocoa world production. The Nacional variety is exclusively grown in Ecuador and produces the typical Arriba beans with distinct floral and spicy flavor notes (Aprotosoaie, Luca, & Miron, 2016).

With regard to the chemical composition of cocoa samples (cocoa beans, cocoa liquors, chocolates, etc.), a rough division can be made between non-volatile and volatile components, each requiring different methods of analysis. Besides water and primary metabolites like fats, carbohydrates, proteins and dietary fibers, an important group of non-volatile components are the polyphenols, which can be further divided into three main subgroups: flavan-3-ols (mainly (+)-catechin and (−)-epicatechin), anthocyanins and proanthocyanidins (dimers, trimers and oligomers) (Wollgast & Anklam, 2000). Moreover, flavonoids like quercetin and luteolin have been reported, as were several phenolic acids, such as caffeic, ferulic, and coumaric acid (Aprotosoaie, Luca et al., 2016). Many reports were published with regard to the beneficial health effects of polyphenols, mainly because of their antioxidant potential and their protective effects on the cardiovascular system (Aprotosoaie et al., 2016, Crozier and Hurst, 2014).

Another important class of non-volatile compounds in cocoa are the alkaloids, the methylxanthines being the most abundant ones. Theobromine is the major alkaloid, followed by caffeine. Moreover, theophylline is present, but in much lower amounts. Just like the polyphenols, these methylxanthines contribute to the typical bitter taste of cocoa. Compared to coffee, cocoa contains significantly lower levels of caffeine. Theobromine levels are higher though, but its stimulating effects on the central nervous system are far less pronounced than those of caffeine (Aprotosoaie et al., 2016, Tuenter et al., 2018).

Amino acids and biogenic amines, like 2-phenylethylamine, dopamine and tyramine are also present. Especially during roasting, amino acids can be converted to biogenic amines via decarboxylation. Additional enzymatic reactions can occur, like the reaction of dopamine with acetaldehyde, which leads to the formation of salsolinol. Biogenic amines can exert important effects in the human organism. 2-Phenylethylamine, for example, has been referred to as the “love drug”, as it is associated with aphrodisiac effects. Moreover, mood lifting and heightened sensitivity were also associated to the presence of phenylethylamine and N-acylethanolamine. However, 2-phenylethylamine is only present in low amounts, and it does not reach the brain after oral intake. Nevertheless, some amines, at high levels, may cause adverse effects to human health. For example, high levels of tyramine, tryptamine and 2-phenylethylamine can cause migraine (do Carmo Brito et al., 2017, Tuenter et al., 2018).

Flavor is the most important criterion for chocolate quality. It is influenced by both the volatile and non-volatile fraction. During fermentation, the formation of flavor precursors like reducing sugars and free amino acids is initiated. Mainly during drying and roasting of the cocoa beans, these compounds can undergo further reactions, which are important for the final flavor and aroma of the cocoa liquors and chocolates. Reducing sugars can react with amino acids or oligopeptides via a Maillard reaction, leading to Amadori products. These can be further converted via multiple reactions into various compounds (aldehydes, ketones, furans, pyrazines, etc.) (Aprotosoaie, Luca et al., 2016). Finally, conching is of utmost importance as it eliminates undesirable volatiles and reduces the moisture content, thus enhancing the flavor of the final product (Afoakwa, 2011, Aprotosoaie et al., 2016). The specific cocoa aroma and final composition of cocoa liquors and chocolates are influenced by many factors, like the cocoa genotype, place of origin of the cocoa beans, season of harvesting, the practices of local farmers, and all subsequent processing steps (Afoakwa et al., 2008, Caligiani et al., 2014, Hinneh et al., 2019, Rottiers et al., 2018).

In the past decades, interest in “functional”, healthy, and high-quality food products has risen, and chocolate is one example of this. A lot of research has been carried out, mainly concerning the cardiovascular effects of (polyphenols in) chocolate. Nevertheless, it is crucial to perform these tests on well-characterized samples, and it is important to take into account also other (minor) constituents when assessing potential biological activities. However, in many studies the levels of flavan-3-ols, procyanidins, methylxanthines and the total phenolic content are determined, but a more detailed analytical characterization of the non-volatile phytochemicals in cocoa liquors and chocolates is relatively rare. Different techniques have been applied for the analysis of such samples, for example High Performance Liquid Chromatography – Ultraviolet detection (HPLC-UV), or Nuclear Magnetic Resonance (NMR) spectroscopy (Caligiani et al., 2014, Natsume et al., 2000). However, Ultra Performance Liquid Chromatography– High Resolution Mass Spectrometry (UPLC-HRMS) is the preferred technique when analyzing also minor non-volatile constituents, (like biogenic amines, as reported by Oracz and Nebesny (2014)) and is applied in this research. With regard to the volatile compounds, different extraction procedures combined with various analytical techniques were already tested (Counet et al., 2002, Frauendorfer and Schieberle, 2008, Owusu et al., 2011). Headspace Solid-Phase Microextraction (HS-SPME) is considered the preferred extraction technique in combination with GC–MS (Gas Chromatography – Mass Spectrometry) for profiling of the aroma of cocoa products and was used for analysis of the aroma compounds.

In this study, a comparison is made between cocoa liquors and chocolates, produced from a batch of “blended bulk cocoa beans”, harvested in West Africa, which is common practice in commercial mass production of chocolate, and cocoa beans of the Nacional variety, considered a fine-flavor cocoa, from Ecuador. The nutritional composition of the cocoa liquors was determined; the levels of various phytochemicals, volatile and non-volatile, were assessed by means of HS-SPME GC–MS and UPLC-HRMS. Additionally, the odor activity values (OAVs) of volatile compounds and the contribution of these compounds to overall aroma were calculated. Finally, principal component analysis (PCA) was carried out, based on the GC–MS and UPLC-HRMS data, in order to visualize whether the samples could be distinguished and if so, to look further into the exact compositional differences between the samples.

Section snippets

Raw materials

Two cocoa liquors were investigated: a commercial West African cocoa liquor (WA-CL) made from a blend of cocoa beans of different origins, supplied by Cacaolab bvba (Evergem, Belgium), and an Ecuadorian cocoa liquor (EC-CL) made from cocoa beans of the Nacional variety collected in the Sucumbíos province. The former is considered a bulk cocoa liquor, the latter a fine-flavor cocoa liquor. The cocoa liquor samples were obtained from well fermented and dried cocoa beans that were roasted and

Nutritional composition of cocoa liquors

Table 1 shows the nutritional composition of the cocoa liquors. Fat (>54%) was the major constituent for both origins, followed by total dietary fibers (14.1–19.8%) and proteins (14.7–15.6%). Ash and moisture content of the cocoa liquor samples were less than 3.5% and 1.5%, respectively. The nutritional composition of the cocoa liquors varied depending on their geographical origin. EC-CL showed a lower moisture content, a higher protein content and a lower total dietary fiber and ash content.

Conclusion

In this study, cocoa liquor and chocolate, produced with either a bulk cocoa blend of Theobroma cacao beans from West Africa or with fine-flavor cocoa beans of the Nacional variety, grown in Ecuador, were analyzed for a wide range of properties. The scope of this study was broader than most researches on cocoa, since a wide range of compounds and characteristics of the samples has been investigated. Volatile and non-volatile constituents were analyzed, both influencing the final flavor of

CRediT authorship contribution statement

Emmy Tuenter: Investigation, Methodology, Formal analysis, Writing - original draft, Writing - review & editing, Visualization. Claudia Delbaere: Investigation, Formal analysis, Writing - original draft, Writing - review & editing. Ann De Winne: Investigation, Formal analysis, Writing - original draft, Writing - review & editing. Sebastiaan Bijttebier: Investigation, Writing - original draft. Deborah Custers: Formal analysis, Writing - review & editing. Kenn Foubert: Conceptualization, Formal

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We would like to thank Professor Jenny Ruales from the Escuela Polytécnica Nacional in Ecuador for providing the Ecuadorian cocoa liquor. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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