The Microbiology of Chocolate
Christine L. Case, Ed.D.
Biology Professor
Skyline College


BIOL 240



Chocolate comes from the seeds of the cacao or chocolate tree, Theobroma cacao. Linnaeus ascribed the genus name meaning "food of the gods" to the Greek words, theos, (god) and broma (food) and the specific epithet from the Native American word for the plant. The chocolate tree is native to Latin America and was transplanted to West Africa in 1824 by the Portuguese. Presently, Cote d'Ivoire and Ghana produce nearly half of the world's chocolate.

The chocolate tree produces small flowers along its trunk that are pollinated by midges (a type of fly). Each pollinated flower produces a large pod containing 30 to 40 bitter seeds embedded in a sweet, sticky pulp. This sweet pulp was a staple food of the Mayans 2000 years ago. The Mayans and Aztecs also used the seeds to make a chocolate drink. While Columbus brought the chocolate seeds to Europe in 1502, the Spanish in the 17th century added sugar to the bitter beverage to produce the "food of the gods" that Linnaeus and many others have enjoyed.
cocoa pod

Cacao seeds must be fermented, dried, and roasted to produce the chocolate flavor. Fermentation and drying are done at the farm that grows the chocolate trees. After harvesting, pods are split with a hammer or machete to reveal the seeds covered with pulp. Split seeds are piled in a heap covered with banana leaves or placed in a covered box. While in this heap, the sticky pulp becomes a turbid broth and the cacao seeds absorb flavors from the surrounding broth. The chemical composition of the pulp (below) changes after being kept in this heap for five to seven days.

Pulp Composition
  Before fermentation After fermentation
Sucrose 12% 0%
Citric acid 1-3% 0.5%
Pectin 1-1.5% _
pH 3.7 6.5
Ethyl alcohol _ 0.5%
Acetic acid _ 1.6%

These changes are the result of microbial growth. The sterile pulp gets inoculated with a variety of microorganisms from the machete, workers' hands, carrying-baskets, and fermentation boxes. During the first 24 hours, the seeds germinate and plant enzymes hydrolyze the sucrose to glucose and fructose. As microbes grow on the sugars, they produce heat which is trapped by the cover on the heap. The temperature in the fermentation heap reaches 40-50°C which kills the plant embryo.

Chocolate acquires its color and flavor during fermentation. A variety of microorganisms grow in the fermentation heap, but they do not all grow at the same time (see graph).


When one organism starts growing it alters the environment and inhibits its own growth but the new conditions are favorable for another species. This is called microbial or ecological succession.

The pulp contains mostly water with 10-15% sugars. The high sugar content in the pulp favors the growth of yeasts which ferment sugars to ethyl alcohol in the anaerobic heap. Eleven different species of yeasts have been isolated with Saccharomyces cerevisiae, and of that group, Candida rugosa, and Kluyveromyces marxianus are the most abundant. In addition to producing ethyl alcohol, the yeasts hydrolyze the pectin that covers the seeds. Experimental fermentations indicate that S. cerevisiae decreases the bitterness of the final product. Without pectin, the bitter alkaloids may leach out of the seed or be altered by alcohol that can now enter the seeds.

The yeast are killed by the alcohol they produce and, as the temperature rises, lactic acid bacteria such as Lactobacillus and Streptococcus grow. The pulp is stirred and drained to aerate it. The presence of oxygen and the lower pH now favor the growth of acetic acid bacteria, Acetobacter and Gluconobacter. After five days, the fermented mass contains up to 108 microbes per gram. The beans are then dried and as they dry, molds including Geotrichium grow. Geotrichium oxidizes the lactic acid to acetic acid and succinic acid.

If fermentation is allowed to continue beyond five days, microbes may start growing on the beans instead of on the pulp. Off-tastes result when Bacillus and filamentous fungi, including Aspergillus, Penicillium, and Mucor, hydrolyze lipids in the beans to produce short-chain fatty acids. As the pH approaches 7, Pseudomonas, Enterobacter, or Escherichia may grow and produce off-tastes and odors.

Dried beans are bagged for sale to chocolate manufacturing companies. Chocolate beans are sold at the Coffee, Sugar, and Cocoa Exchange in New York City. The first step in manufacturing is to roast the beans at 121°C. Roasting kills most of these microbes although some species of Bacillus may survive the roasting process.

In continuing efforts to produce consistent and high quality flavor, researchers are studying how many pods to put in the heap, how long to ferment, how to cover the heap, and when to stir the pile. Additionally, microbiologists are examining the roles of each microbe in the fermentation process to determine whether direct inoculation with one or more species is better than the present method of fermenting with wild-type microorganisms. Biochemists and food scientists are also investigating the chemicals responsible for chocolate flavor.

Chocolate Products
Microorganisms are also used in the production of finished chocolate products. Alpha amylase obtained from Aspergillus is used to hydrolyze starch for chocolate syrup and invertase from Saccharomyces is used to hydrolyze sucrose in filling mixtures to make soft-centered chocolate-covered candies.



Fungal diseases of cacao trees result in economic loss in Latin America and Africa. Phytophthora spp. are the most important of the cacao pathogens; these fungi cause black pod disease. The fungus grows on the ripening pod and seed coats. As the mycelia develop the pod blackens and is unusable.

Witches' broom, Crinipellis perniciosa, is the other serious fungal pathogen of cacao trees in Latin America. The fungus grows in the new buds, flowers, and seeds. Seeds will not develop on infected plants. The disease can cause yield losses of 75 percent in susceptible varieties

Biological Controls
In 2011, USDA researchers reported fungi in disease-free Peruvian cacao trees. The fungi may provide protection against diseases such as witches' broom disease either by stimulating the immune system of the plants or through direct parasitism or antibiotic effects against pathogens.

New varieties
In 2011, U. S. Agricultural Research Service scientists released nine new, high-yielding selections of Theobroma cacao. The new selections could offset some disease losses because they yield more beans than current varieties. About 30 to 40 percent of the world's cacao production is lost each year, mostly through fungal diseases and pests such as black pod, witches' broom, frosty pod rot and the cocoa pod borer.

Quality Control
Microbiologists play a role in the quality control lab of chocolate manufacturers. They test materials and finished products for spoilage organisms and pathogens. Standard plate counts and total yeast and mold counts are performed regularly. Molds can spoil chocolate by hydrolyzing lipids. The resulting fatty acids cause rancid tastes. Additionally, chocolate is tested for the following specific bacteria: Staphylococcus aureus, coliforms, and Salmonella. Several outbreaks of salmonellosis have been traced to chocolate that was contaminated after roasting. Current research is aimed at developing rapid methods such as ELISA tests and DNA probes to detect Salmonella in chocolate.