From MicrobeWiki, the student-edited microbiology resource
Origin of Theobroma cacao
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. 
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 . 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 .
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 . Microbes arise from hands of workers, knives, unwashed baskets, and dried mucilage on fermentation boxes .
Containers wrapped in banana leaves are used to ferment up to 2000 kg of beans . 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 .
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 .
The bean pulp contains lots of fermentable sugars including glucose, fructose, and sucrose . 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 .
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 .
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 . 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 .
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 .
Lactic acid bacteria begin to grow when the pulp and “sweatings” are degraded and drained, and the yeast are dying . 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 .
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 . Most grew well between 40 to 45ºC, and at 7 to around 10% ethanol .
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 
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, Bacillus licheniformis, Bacillus subtilis, and Bacillus 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 .
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 fumigatus 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 .
Drying & Roasting
The drying and roasting process contains a very small portion of 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, in addition to that mold can begin to form if the drying conditions are not correct, but 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  although there are that 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 cocoa 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 . 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 . 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 .
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 .
Flavor and Aroma
There are several factors that appear to affect the flavor of chocolate, including the components that make up the bean pulp. Lipids comprise the majority of the cocoa bean (around 50%) of its overall weight. One study has claimed that the concentration of free fatty acids increases with the fungal degradation of the lipid matter in the cocoa bean, thus affecting the chocolate flavor .
During the fermentation of the bean pulp, bacteria produce various products such as alcohols, acetic acid, and organic acids, all of which can contribute to bean death. The chemical changes that arise from the bean death add to the initial aroma, coloring, and flavor of the cocoa; all of which are finalized in the drying and roasting stages . Acetic acids, such as A. aceti, A. pasteurianus and Gluconobacter oxydans, play a role in the aroma and odor during cocoa bean fermentation. They can produce various byproducts from metabolizing sugars and organic acids, and these byproducts can contribute to the aroma of cocoa beans during fermentation .
Yeast plays an important role in the fermentation of the cocoa bean by producing ethanol and ultimately acetic acid, which leads to bean death; thus setting off a series of biochemical changes that contribute to the flavor of the chocolate .Following the drying process, the water content of the bean pod is greatly reduced, at which point the beans are roasted in order to create the characteristic flavoring and odor of chocolate. Yeast plays a vital role in the final chocolate flavors of the roasted beans. It has also been found that the enzymes that the yeast releases are important for the chocolate precursor components. It is only when the cocoa beans have actually been roasted does the characteristic chocolate aroma occur .
Bacillus bacteria are also involved in the fermentation process of the cocoa beans, predominately in the end stages of fermentation when the environmental conditions of the process are more aerobic (oxygen containing) and less acidic. Some of these Bacillus bacteria that have been identified include Bacillus subtilis, Bacillus stearothermophilus, Bacillus cereus, Bacillus licheniformis, Bacillus coagulans, and Bacillus pumilus. The production of organic acids and pyrazines by Bacillus bacteria has been shown to affect the flavor of cocoa by allowing the enzymes produced to enter the cocoa beans and alter the chemical quantities inside .
Climate conditions, harvesting seasons, the ripeness of the cocoa pod (including the quality of the bean pulp) all affect the fermentation of the cocoa bean. In addition, the different flavors produced have been linked to the presence of esters in the chemical composition of the cocoa bean. These esters have been linked to the existence of yeasts and acetic acid bacteria such as Acetobacter and Gluconobacter. However, not all the chocolate odors are due to the presence of microbes. Some of the odors were due to the thermal conditions of the fermentation process. Some of these off-flavorings could have been the result of contamination in the drying process .
Health Benefits of Chocolate
It has been studied that the regular consumption of foods high in flavonoids can decrease the risk of cardiovascular disease . Specific to cardiovascular health, a specific sub-group of flavonoids known as flavan-3-ols (falvanols), are found in cocoa products such as chocolate . Various studies have shown improvements in blood pressure, and the functioning of endothelial cells and platelets due to the consumption of cocoa products that contain flavanols . The reason that dark chocolate is better for one’s health versus milk chocolate is that per serving, dark chocolate has almost 2.5 times that amount of flavonoids than milk chocolate; this may partly be due to the milk in milk chocolate inhibiting the absorption of those flavonoids by the intestine. Dark chocolate also has greater concentrations of phenols and catechins than milk chocolate .
Diseases affecting Theobroma Cacao
There are several various fungal diseases that have had detrimental effects on Theobroma cacao plant. The primary two diseases native to South America are caused by the fungi Crinipellis perniciosa (also known as Witches’ Broom disease) and Moniliophthora roreri (frosty pod rot or moniliasis disease). These fungi infect Theobroma L., and Herrania Goudot, in particular, T. cacao, which is the source of chocolate. This infection causes hypertrophy and hyperplasia in the cacao, ultimately resulting in tissue damage. C. perniciosa basidiospores infect cacao meristems, which leads to a newly disordered growth of shoots, “witches’ broom,” in the host. C. perniciosa also infects the early phases of cacao bean pod growth by breaking through the plant’s husks and destroying the cacao seeds. The M. roreri (frosty pod rot) pathogen is found only in the Theobroma and Herrania species. It results in outer and inner cacao bean pod damage, and results in the complete death and loss of the bean pods .
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. Studies are being conducted by observing the differences in mutans streptococci counts in placebo groups versus actual CBHE groups. The CBHE mouth wash is being tested as a means to lower mutans streptococci count and get rid of plaque build up on children’s teeth and studies have shown to have promising results .
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. 
C. Conjugation of the pUR400 gene into Escherichia coli HD701 from an Escherichia coli K12 strain gives them the ability to metabolize sucrose into hydrogen. Studies were done on Escherichia coli with and without the pUR400 that was conjugate into Escherichia coli, found that the parental strain was not able to metabolize sucrose into hydrogen although the recombinants were able to . The applications for this stems out into bioremediation of toxic wastes to wastes from production of food such as chocolate . Researchers have found that by taking chocolate waste and feeding it to Escherichia coli, the bacterium was able to produce hydrogen in return. Moreover the researchers were able to use this hydrogen to power a small fan through a fuel cell. 
D. Fermentation of Theobroma cacao is necessary in order to develop the flavor precursors of chocolate. This current study looks at a controlled study where the cacao seed, during fermentation, is inoculated with a hybrid yeast strain known as Kluyveromyces marxianus to see whether the increased pectinolytic activity would create a better quality of chocolate. The results showed that the hybrid yeast affected the overall degradation of the seed protein as well as reduced the titrable acidity; and overall, the beans inoculated with Kluyveromyces marxianus were preferred over the naturally fermented cacao beans. This preference in creating a better quality of the final product owes much of its success to improving the first 24 hours of fermentation by increasing the mass aeration of the fermenting seed. 
 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/>.
 Jespersen, L, Nielsen, DS, Hønholt, S, et al. (2005). Occurrence and diversity of yeasts involved in fermentation of West African cocoa beans. FEMS yeast research, 5(4-5), 441-53.
 Keen, CL, Holt, RR, Oteiza, PI, et al. (2005). Cocoa antioxidants and cardiovascular health. The American journal of clinical nutrition, 81(1 Suppl), 298-303S.
 Erdman, JW, Carson, L, Kwik-Uribe, C, et al. (2008). Effects of cocoa flavanols on risk factors for cardiovascular disease. Asia Pacific journal of clinical nutrition, 17 Suppl 1, 284-7.
 Ding, EL, Hutfless, SM, Ding, X, et al. (2006). Chocolate and prevention of cardiovascular disease: a systematic review. Nutrition & metabolism, 3, 2-. http://www.nutritionandmetabolism.com/content/3/1/2
 Lagunes Gálvez S., Loiseau G., Paredes J.L., Barel M., Guiraud J. P., Étude de la microflore et de la biochimie de la fermentation du cacao en République dominicaine, International Journal of Food Microbiology (in press). Barel M., Les arômes du chocolat, Chocolats et friandises, Académie française du chocolat et de la confiserie, 2001
 Jay, James Monroe, Martin J. Loessner, and David A. Golden. Modern Food Microbiology 7th ed. New York: Springer, 2005.
 Aime, MC. (2005). The causal agents of witches' broom and frosty pod rot of cacao (chocolate, Theobroma cacao) form a new lineage of Marasmiaceae. Mycologia, 97(5), 1012-.
 Macaskie, Lynne E. "Cr(VI) reduction by bio and bioinorganic catalysis via use of bio-H2: a sustainable approach for remediation of wastes." Journal of Chemical Technology & Biotechnology 82 (2007): 182182-89(8).
 Macaskie, Lynne E. "Production of H2 from sucrose by Escherichia coli strains carrying the pUR400 plasmid, which encodes invertase activity." Biotechnology Letters 26 (2004): 1879-883.
Edited by Rebecca Law, Brian Lew, Jason Ly, and Sahar Salek, students of Rachel Larsen