Thursday, 30 March 2017 15:05

Chocolate: The Fermentation and Flavors of the Chocomicrobiome

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Published in Microbial Sciences

Cacao podFigure 1. Cacao pod of the Theobroma cacao tree. Source

Chocolate is one of the world's most loved and widely recognized flavors. People go nuts for the dark brown stuff, to the point where entire holidays revolve around the decadent sweet. From Halloween candy to Valentine's Day chocolates to Easter eggs, there is no doubt the world is crazy about this scrumptious treat. But how is chocolate made? What gives this simple seed its unique and irresistible flavor? For that, we can thank microbes and the metabolic process of fermentation.


From Bean to Bar

Whether you buy fancy truffles from the chocolatier or simply grab a bar of Hershey’s from the local supermarket, all chocolate begins as a seed from the Theobroma cacao tree, native to tropical regions of the Americas. Cocoa seeds (also commonly called beans, although they are not actually beans) grow in long ovoid pods and are harvested only when fully ripe to ensure enough cocoa butter and sugars have developed. The seeds and pulp are then removed from the pod and loaded into piles within wooden boxes lined with banana leaves, where the seeds undergo the essential flavor-producing process of fermentation. Only after this process, which can take up to a week to complete, are the seeds dried, ground, and processed into the chocolate we consume.


Fermentation for flavor

Cacao seeds showing the sticky white pulp that is broken down during cacao fermentation. Brown areas of the pulp have begun to ferment. Photo credit: Scott Chimileski Figure 2. Cacao seeds showing the sticky white pulp that is broken down during cacao fermentation. Brown areas of the pulp have begun to ferment. Photo credit: Scott Chimileski

Unlike most other commercially available fermented foods and drinks produced today, such as kombucha, beer, wine, or yogurt, cocoa seed fermentation does not rely on starter cultures. Cocoa seed fermentation occurs best when seeds are placed in a dark moist environment, and the internal temperature increases as fermentation progresses. Fermentation is a product of yeasts and bacteria that in combination produce ethanol, lactic acid, and acetic acid along with important aromatic flavor precursors that are responsible for chocolate’s memorable taste.


Microbial Cocoa Composition

Although there are some variations, the microbial species involved in cocoa fermentation are very consistent. Six species of microbes are the core components of cocoa seed fermentation. The group contains two bacterial species (Lactobacillus fermentum and Acetobacter pasteurianus) and four yeast species (Saccharomyces cerevisiae, Hanseniaspora opuntiaeHanguana thailandica, and Pichia kudriavzevii). Some variability in flavor arises from the presence or absence of bacterial species Lactobacillus plantarum and Lactobacillus pentosus, which produce lactic acid, and/or Gluconobacter frateurii, which produces acetic acid.


 dried cacao seeds Figure 3. dried cacao seeds. Source

These microbes eventually aid in the fermentation of the cacao seed as part of an ecological succession. Cocoa sugars found within the white pasty pulp that surrounds the seed, composed predominantly of glucose and fructose, attract S. cerevisiae and other yeasts first.  Other fungi such as Penicillium citrinum and Aspergillus fumigatus also contribute to seed and pulp fermentation. However, as fermentation progresses, the buildup of ethanol prevents further propagation of the yeast species allowing takeover by bacterial Lactobacillus and Acetobacter species.


Key to Cocoa

Saccharomyces cerevisiae SEMFigure 4. Scanning electron micrograph of Saccharomyces cerevisiae, one of the first microbes to colonize fermenting cacao seeds. Source

Cocoa seed fermentation produces the majority of flavor that we recognize as “chocolate.” In addition to the production of acetic and lactic acid, ethanol, and minor sugars such as glycerol and mannitol, a variety of volatile compounds are also produced. There are over 600 volatile compounds found in fermented cocoa seeds. In one study, a group of scientists led by Graham Fleet at the University of New South Wales reported concentrations of 40 volatile compounds found in cocoa seeds after fermentation that were not present before the process. Peter Schieberle, Ph.D. presented research at a meeting of the American Chemical Society regarding replicating the complex aroma of chocolate in the lab. Schieberle claims, “To make a very good cocoa aroma, you need only 25 of the nearly 600 volatile compounds present in the beans.” Interestingly, no individual compound was identified exhibiting the typical aroma of cocoa, instead the smell and flavor of cocoa is due to a combination of aromas.


Many studies have tried to elucidate the key odor-active compounds in chocolate. There is a general consensus that the aroma is a combination of pyrazines, aldehydes, esters, alcohols, acids, and hydrocarbons. Eleven particular compounds have been considered the major contributors of the overall flavor of chocolate in studies by Afoakwa et al and Liu et al. For a more comprehensive list of the aromatic components of chocolate, check out this review which lists both the pleasurable and not-so-pleasurable aromas we sense when consuming cocoa.


 Compound Perceived aroma
2-Methylpropanal malty, dark chocolate
3-methylbutanal malty, dark chocolate
phenylacetaldehyde rosy
2-ethyl-3,5-dimethylpyrazine popcorn-like, potato-like
tetramethylpyrazine milk coffee-mocha roasted, nutty
trimethylpyrazine earthy, nutty
2-acetyl-1-pyrroline popcorn-like
3-methylbutanoic acid cheesy, sweaty
3,5 (or 2)-diethyl-2 (or 5)-methylpyrazine cocoa, chocolate, rum, roasted
2-methylbutanal chocolate
furaneol caramel-like

Table 1: Compounds responsible for chocolate aroma. Adapted from review.


Imagine for a moment a world without chocolate. Without microbes, we would have no cocoa fermentation, and with no cocoa fermentation, we would have no cocoa flavor. The very essence of chocolate depends deeply on the metabolism of the tiny organisms that break down the simple sugars and fats of a simple seed. So next time you crave that bar of chocolate, brownie, hot cocoa or even sprinkle some cocoa powder on your latte, remember that chocolate isn’t just a powdered seed, but rather the delightful product of a complex micro-ecosystem.


Further Reading:

Cocoa Fermentation — Bringing Magic to the Cocoa

Dynamics and Biodiversity of Populations of Lactic Acid Bacteria and Acetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans in Ghana

A comparative study of aroma-active compounds between dark and milk chocolate: relationship to sensory perception.

Flavor formation and character in cocoa and chocolate: a critical review

Last modified on Friday, 31 March 2017 19:37
Monika Buczek

Senior Contributor Monika Buczek holds M.Phil. and Ph.D. Degrees in Molecular, Cellular and Developmental Biology from the City University of New York Graduate Center. She studies bacterial cell division and the proteolytic regulation of division proteins. Monika is a lecturer in Human Biology, Molecular Biology and Microbiology at the City College of New York as well as the Executive Director of the multidisciplinary CCNY Science Alliance. You can connect with Monika on LinkedIn and Twitter.