Origin of Theobroma cacoa
Chocolate is derived from the seeds of the tree Theobroma cacao. It is normally found in humid, tropical regions of northern South and Central America. Major producers of chocolate are Ivory Coast, Ghana, Indonesia, Brazil, Nigeria, Cameroon, Malaysia, and Ecuador. Several types of the tree have been discovered including Criollo, Forastero, and Trinitario. Most chocolate producers currently use Forastero and Trinitario, however, because Criollo is highly susceptible to disease. (2)
Fermentation is the first process cocoa beans are subjected to in making chocolate. The process usually lasts up to seven days. Fermentation takes place in the pectinaceous pulp surrounding beans of this tree. Biochemical and enzymatic reactions that take place inside the cotyledon cause a variety of flavor compounds (5). The pulp surrounding the cocoa beans is fermented by various microbes including yeasts, lactic acid bacteria, and acetic acid bacteria. The resulting high temperature and products produced by these microbes, such as the ethanol from yeast, kill the beans and contribute to the flavoring of the chocolate (2).
Once the seeds are harvested, fermentation is usually begun immediately. The beans inside of the cocoa pods are in an environment such that no microbes can grow. However, upon cutting the cocoa pods open, the beans are exposed to microbes and the fermentation process is allowed to begin (2). Microbes arise from hands of workers, knives, unwashed baskets, and dried mucilage on fermentation boxes (5).
Containers wrapped in banana leaves are used to ferment up to 2000 kg of beans (2). The beans are covered in a white-cream, mucilaginous (protein/sugar coat) pulp that is solubilized, and the breakdown of the glue between the pulp cells walls and the cocoa honey (“sweatings”) are released through holes in the box containing the beans (1).
In the early stages of the fermenting process, yeasts produce ethanol and secrete enzymes that break down pectin. Bacteria (lactic acid and acetic acid bacteria) then appear, followed by aerobic spore-forming bacteria and filamentous fungi (2).
The bean pulp contains lots of fermentable sugars including glucose, fructose, and sucrose (1). It is an ideal medium for microbes to grow on because it is rich in nutrients. It is made up of 82-87% water, 10-15% sugar, 2-3% pentsans, 1-3% citric acid, and 1-1.5% pectin, along with various other proteins, amino acids, vitamins, and minerals (2).
Changes to Physical Conditions
Due to the presence of citric acid, the pulp is an acidic environment, with pH 3.0 to 3.5. As yeasts use up citric acid, pH increases to around 4.8 to 5.0. The yeast also convert sugars (glucose, sucrose, and fructose) into ethanol, increasing the concentration of ethanol for one or two days. The concentration then decreases gradually as it is oxidatively metabolized to acetic acid by acetic acid bacteria. Temperature rises throughout process due to the release of heat as a by-product of biochemical processes carried about by the microbes, from around 20 to 25ºC to 48 to 50ºC (1).
Yeast grow well in acidic environments and low oxygen levels, such as in the beginning stages of fermentation. In these early stages, yeast are very important in paving the way for further fermentation by bacteria. They convert sugars, such as sucrose, glucose, and fructose, into ethanol and CO2, decrease the acidity of the pulp by using up citric acid, and produce aromatic compounds, which contribute to the chocolate aroma and are important to development of flavor. In order to deal with fluctuations in bean conditions, some yeast produce weak organic acids to buffer fluctuations such as pH. Yeast are also responsible for degrading pulp and producing enzymes that break down pectin (1). This creates cavities in the cocoa where air can flow. However, this increased air flow, along with an increase in pH and concentration of alcohol, eventually kills off the yeast (2).
Prominent yeast in the first 24 to 36 hours of fermentation include Kloeckera apis (~70-90% of the total yeast grown), Kloeckera javanica and Kloeckera africana, Candida pelliculosa and Candida humicola (less than 5% of total yeast), Rhodotorula rubra and Rhodotorula glutinis. Saccharomyces cerevisiae and Candida tropicalis were also prominent during first 24-36 hours, but died off by the end of fermentation. Most grew only until about 37 to 40ºC, and up to around 5-10% ethanol (1).
Lactic acid bacteria begin to grow when the pulp and “sweatings” are degraded and drained, and the yeast are dying (2). The main function of lactic acid bacteria is to metabolize pulp sugars (glucose and fructose) and citrate to produce lactic acid, acetic acid, ethanol, and mannitol. The production of lactic and acetic acid contributes to the decrease in pH. Lactic acid bacteria have also been thought to contribute to yeast’s ability to use citrate as a carbon source. These products are good for acetic acid bacteria growth, and allow them to convert ethanol into acetic acid, releasing heat as a byproduct for the eventual cocoa bean death (3).
Predominant lactic acid bacteria in the first 36 to 48 hours of fermentation include Lactobacillus cellobiosus (60-85% of the total lactic acid bacteria grown), Lactobacillus plantarum, Lactobacillus hilgardii (only 2% of the total bacteria) (1), Lactobacillus fermentum, Leuconostoc mesenteroides, and Lactococcus lactis. (2) Most grew well between 40 to 45ºC, and at 7 to around 10% ethanol (1).
Towards the end of fermentation, the presence of yeast and lactic acid bacteria decline and the fermenting heap becomes more aerated. These conditions can therefore lead to the development of acetic-acid bacteria. This bacteria oxidizes ethanol to acetic acid, and also further oxidizes the acetic acid to carbon dioxide and water. These organisms are metabolized due to the acidulation of cocoa beans at high temperatures, which causes diffusion and hydrolysis of proteins in the cotyledons. Acetic acid bacteria primarily form the precursors of chocolate flavor. These include members of the genus Acetobacter as well as Gluconobacter. (2)
Aerobic Spore-Forming Bacteria
High temperatures and increase in pH along with increased aeration leads to the development of aeobic spore-forming bacteria of the genus Bacillus. This includes B. pumilus, B. licheniformis, B. subtilis, and B. cereus. The Bacillus spp. found during the aerobic phase of fermentation have been found to be responsible for the flavoring of chocolate. Aerobic spore-forming bacteria form chemical compounds that cause acidity and sometimes off-flavoring if fermentation continues for too long. (2)
Filamentous fungi are also found in the well-aerated parts of the fermented mass. They may cause hydrolysis of some of the pulp and produce acids, but are not considered important in microbial succession. Of the filamentous fungi, Aspergillus fumigates and Mucor racemous are the most present in the fungal population up to the end of fermentation. These fungi cannot grow at temperatures higher than 45°C, but can be isolated at a temperature of around 50°C. (2)
Drying & Roasting
The drying and roasting process contains a very small portion of the microbes such as, lactic acid bacteria, acetic-acid bacteria, aerobic spore forming bacteria, and others. The residual microbes will begin to form endospores as conditions such as levels of heat increase and the humidity decreases not much else is known. In the past bean drying involved the use of sunlight and spreading out the beans over a wide area; these days bean drying has evolved to using large machines that regulate temperature and humidity (7) although some may still dry their beans the traditional way. Drying begins when the beans are placed in a box with a piece of plastic covering it, the beans are then turned while being roasted at around 50°C to 60°C degrees and dried over the course of a few days until the water content drops to about 7 to 10%. The rate at which the beans dry is also an important factor involved in the study of chocolate making if conditions are not right then microbes such as fungi may begin to form. At this stage of chocolate production the maker would want to minimize the amount of microbes since they can alter the flavor of the chocolate. Research has found that rapid drying at low humidity and slow drying at high humidity were more harmful to cacao beans. They determined that this was due to a leakage of electrolytes and concluded that if one were to determine the optimal rate for drying, finding the point at which the least amount of damage done from mechanical and metabolic stresses would probably be the optimal point (10). Another has found that conditions for getting good results were brought up by drying at low temperature or occasional breaks from drying if dried at high temperature (11). A few years later, another group of researchers have specifically determined that the optimal conditions for drying cacao beans was at a temperature of 40°C and a depth of 8.3 cm this minimized the amount of free amino acids being produced however they did not mention how it affected the taste (12).
Once dried, the beans are cleaned to remove any remaining microbes and get rid of any excess material, they are then roasted at around 120°C, killing all remaining microbes, and bringing out the flavor and the scent. If should be known that some companies have been advertising the fact that bacteria and fungus that are involved in developing chocolate have been branded as dangerous when in fact they are not. After roasting the cacao beans are cleaned until there is just the nib portion of the bean left, and then they are grounded up and heated until the chocolate moves to a liquid state. The process ends in a step known as tempering where chocolate is given its shiny appearance.
A. Glucosyltransferases allow bacteria to stick to surfaces such as a child’s tooth. A new type of mouth wash containing cocoa bean husk extract (CBHE) has been developed and found to aid in anti-glucosyltransferase and antibacterial activity. It is being tested as a means to lower mutans streptococci count and get rid of plaque build up on children’s teeth (4).
B. To observe the interactions between microbial activities on the outside of beans and chemical processes inside, various tools can be used. Denaturing Gradient Gel Electrophoresis (DGGE) is used to monitor microbial changes during fermentation of cocoa. Near Infrared (NIR) spectroscopy is used to determine various components in cocoa beans. A number of cocoa fermentations bean samples are taken with 24 hour intervals to be dried and analyzed by NIR as well as simultaneously by DGGE. Using culture dependent and culture-independent methods, the microbiology of Ghanaian cocoa fermentations can be analyzed to determine whether fermentation determined using DGGE are correlated with that of NIR. (6)
C. Researchers have found that by taking chocolate waste and feeding it to Escherichia coli they were able to produce hydrogen, moreover the researchers were able to use this hydrogen to power a small fan through a fuel cell. (13)
 Ardhana, MM, & Fleet, GH. (2003). The microbial ecology of cocoa bean fermentations in Indonesia. International journal of food microbiology, 86(1-2), 87-99.
 Schwan, RF, & Wheals, AE. (2004). The microbiology of cocoa fermentation and its role in chocolate quality. Critical reviews in food science & nutrition, 44(4), 205-21.
 Camu, N, De Winter, T, Verbrugghe, K, et al. (2007). Dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in Ghana. Applied and environmental microbiology, 73(6), 1809-24.
 Srikanth, RK, Shashikiran, ND, & Subba Reddy, VV. (2008). Chocolate mouth rinse: Effect on plaque accumulation and mutans streptococci counts when used by children. Journal of the Indian Society of Pedodontics and Preventive Dentistry, 26(2), 67-70.
 Leal, GA, Gomes, LH, Efraim, P, et al. (2008). Fermentation of cacao (Theobroma cacao L.) seeds with a hybrid Kluyveromyces marxianus strain improved product quality attributes. FEMS yeast research, 8(5), 788-98.
 Nielsen, DS, Snitkjaer, P, & van den Berg, F. (2008). Investigating the fermentation of cocoa by correlating denaturing gradient gel electrophoresis profiles and near infrared spectra. International journal of food microbiology, 125(2), 133-40.
 Taylor, DA. (2005). Sweet deal for cocoa production?. Environmental health perspectives, 113(8), A516-.
 Rubini, MR, Silva-Ribeiro, RT, Pomella, AW, et al. (2005). Diversity of endophytic fungal community of cacao (Theobroma cacao L.) and biological control of Crinipellis perniciosa, causal agent of Witches' Broom Disease. International journal of biological sciences, 1(1), 24-33.
 Camu, N, González, A, De Winter, T, et al. (2008). Influence of turning and environmental contamination on the dynamics of populations of lactic acid and acetic acid bacteria involved in spontaneous cocoa bean heap fermentation in Ghana. Applied and environmental microbiology, 74(1), 86-98.
 Schwan, RF. (1998). Cocoa fermentations conducted with a defined microbial cocktail inoculum. Applied and environmental microbiology, 64(4), 1477-83.
 Faborode, MO. (1995). On the effects of forced air drying on cocoa quality. Journal of food engineering, 25(4), 455-.
 Hashim, P. (1999). Effect of drying time, bean depth and temperature on free amino acid, peptide-N, sugar and pyrazine concentrations of Malaysian cocoa beans. Journal of the science of food and agriculture, 79(7), 987-.
 Redwood, MD, Mikheenko, IP, Sargent, F, et al. (2008). Dissecting the roles of Escherichia coli hydrogenases in biohydrogen production. FEMS microbiology letters, 278(1), 48-55.
 "Chocolate: The Exhibition." Chocolate: The Exhibition. The Field Museum & The National Science Foundation. 27 Aug. 2008 <http://www.fieldmuseum.org/chocolate/>.
Edited by [Rebecca Law, Brian Lew, Jason Ly, and Sahar Salek], students of Rachel Larsen