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The Microbiology of Cocoa Fermentation and its

Role in Chocolate Quality

Rosane F. Schwan & Alan E. Wheals

To cite this article: Rosane F. Schwan & Alan E. Wheals (2004) The Microbiology of Cocoa
Fermentation and its Role in Chocolate Quality, Critical Reviews in Food Science and Nutrition,
44:4, 205-221, DOI: 10.1080/10408690490464104

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Critical Reviews in Food Science and Nutrition, 44:205–221 (2004)
Copyright C Taylor and Francis Inc.
ISSN: 1040-8398
DOI: 10.1080/10408690490464104

The Microbiology of Cocoa

Fermentation and its Role
in Chocolate Quality

Rosane F. Schwan
Department of Biology, Federal University of Lavras, Lavras, Brazil

Alan E. Wheals
Department of Biology and Biochemistry, University of Bath, Bath, England

The first stage of chocolate production consists of a natural, seven-day microbial fermentation of the pectinaceous pulp
surrounding beans of the tree Theobroma cacao. There is a microbial succession of a wide range of yeasts, lactic-acid, and
acetic-acid bacteria during which high temperatures of up to 50◦ C and microbial products, such as ethanol, lactic acid,
and acetic acid, kill the beans and cause production of flavor precursors. Over-fermentation leads to a rise in bacilli and
filamentous fungi that can cause off-flavors. The physiological roles of the predominant micro-organisms are now reasonably
well understood and the crucial importance of a well-ordered microbial succession in cocoa aroma has been established.
It has been possible to use a synthetic microbial cocktail inoculum of just 5 species, including members of the 3 principal
groups, to mimic the natural fermentation process and yield good quality chocolate. Reduction of the amount of pectin by
physical or mechanical means can also lead to an improved fermentation in reduced time and the juice can be used as a
high-value byproduct. To improve the quality of the processed beans, more research is needed on pectinase production by
yeasts, better depulping, fermenter design, and the use of starter cultures.

Keywords Theobroma cacao, yeasts, lactic acid bacteria, acetic acid bacteria

1. INTRODUCTION Ecuador, but there are many other smaller producers, particu-
larly of “fine” cocoa, which constitutes about 5% of world trade.
1.1. Cocoa and Chocolate Firms that make chocolate almost exclusively are Mars, Hershey,
and Rowntree-Mackintosh, but other important companies are
Probably originating in Mesoamerica,1 chocolate or cacao the beverage conglomerate, Jacobs-Suchard, and several multi-
had already been used as a food, a beverage, and as medicine for nationals such as Nestlé, Cadbury-Schweppes, Philip Morris,
over 2,000 years before Hernando Cortés brought it to Europe Unilever, and Zareena.
in 1528.2,3 Its special status in human culture is reflected in its Trade in cocoa is complex: farmers produce fermented beans,
Latin name with genus Theobroma, meaning food of the gods. warehouses store beans, processors turn this into cocoa products,
The specific name, cacao, probably originated as an Olmec word traders ship to mainly North America and Europe, and manufac-
from Mexico.2 The principal varieties are Criollo, now rarely turers convert this into consumable products. The “first” world
grown because of its disease susceptibility, Forastero from the dominates the commodities market that determines the price of
Amazonas region, and a hybrid, Trinitario, the latter two form- cocoa for the “third” world farmers.
ing most of the “bulk” market. The Arriba type, with a “fine” After reaching a peak of well over US$3,000/tonne in 1977
flavor, is grown in Ecuador. World annual production is approxi- the price of roasted beans has fallen to an average about
mately 2.5M tonnes and the major producers are the Ivory Coast, US $1,000/tonne during the last decade. There has been a long
Ghana, Indonesia, Brazil, Nigeria, Cameroon, Malaysia, and battle in Europe to prevent chocolate products that contain
only approximately 20% (w/w) cocoa solids being called choco-
Address correspondence to Dr. Rosane F. Schwan, Department of Bio- late but a compromise has been reached with terms such as
logy, Federal University of Lavras, 37 200 000, Lavras, MG, Brazil. E-mail: “Family milk chocolate” being legally permitted (EU Direc-
rschwan@[Link] tive 2000/36/EC). This revision of the 1973 Council Directive
205
206 R. F. SCHWAN AND A. E. WHEALS

Figure 1 Schematic of a microbial succession during cocoa bean fermentations. The open boxes indicate the periods during the fermentations when a particular
microbial group is most abundant and/or important. The stars indicate the timing of peaks of metabolites and temperature.

(73/241/EEC) permits up to 5% non-cocoa vegetable fat to be cilaginous pulp produces ethanol and acids as well as liberating
used in the manufacture of chocolate throughout the EU. It heat. A schematic of a microbial succession (Figure 1) summa-
would probably result in a loss in demand for cocoa beans rizes the key events during the process that occurs during cocoa
exceeding 184,000 tonnes. If there were worldwide adoption, fermentations in Bahia, Brazil. Diffusion of these metabolites
the loss of revenue to cocoa producers could be more than triggers complex biochemical reactions to occur in the cotyle-
US$1.5bn. dons. The testa provides a barrier to acid penetration into the
bean and diffusion out of undesirable theobromine, caffeine,
and polyphenols. The seed embryo is killed and the fruit tissues
1.2. Fermentation degrade which makes it much easier to dry the beans. This can
be done in the sun (using movable roofs to protect from tropical
Mature fruits (pods) rise directly from the stem of the co- showers) with regular turning until the water content is less than
coa tree and are thick walled and contain 30–40 beans (seeds). 8%, which takes from one to four weeks. Alternatively, artifi-
Each bean consists of two cotyledons and an embryo (radicle) cial dryers are used but it is important to keep the temperature
surrounded by a seed coat (testa) and is enveloped in a sweet, not exceeding 60◦ C and to dry slowly (at least 48 hours) during
white, mucilaginous pulp that comprises approximately 40% of which time some excess acids may volatilize and some oxida-
seed fresh weight. A microbial fermentation and drying process tion will occur, both of which are beneficial. The beans can then
is required to initiate the formation of the precursors of cocoa be stored for up to a year but staling will eventually occur. At
flavor.4 Harvested seeds are immediately allowed to undergo a this stage the cut beans show a purple color due to the presence
natural fermentation during which microbial action on the mu- of anthocyanins.
 MICROBIOLOGY OF COCOA FERMENTATION 207

1.3. Processing Metabolic and epidemiological studies indicate that regular in-
take of such products increases the plasma level of antioxidants,
The next stage is to roast the beans from 5 to 120 minutes a desirable attribute as a defense against reactive oxygen species
and from 120◦ C to 150◦ C depending on nature of the beans and (ROS). The antioxidants in cocoa can prevent the oxidation of
the required product. There may also be a pre-roast and ther- LDL-cholesterol, related to the mechanism of protection in heart
mal shock (to loosen the husk). During this process the cracked disease. Likewise, a few studies show that ROS associated with
husks are air-separated (winnowing) from the entire separated carcinogenic processes is also inhibited.6 The fats from cocoa
cotyledons (nibs), which undergo a further series of chemical butter are mainly stearic triglycerides (C18:0) that are less well
reactions leading to the development of full chocolate flavor. absorbed than other fats and tend to be excreted in the feces.
The roasted beans are then processed into chocolate. The nibs Thus, cocoa butter is less bioavailable and has minimal effect
are ground several times at elevated temperatures to make a fluid on serum cholesterol.6
paste (cocoa liquor) that on cooling yields cocoa mass, a dark Since the starting material is sterile, the fermentation process
bitter material with astringent flavors from the polyphenols and creates hot, acid conditions, and the beans are roasted at over
tannins. Typically 2/3 of this material is then pressed to separate 100◦ C, it is not surprising that there has never been a single
cocoa butter, a pale yellow, fatty liquid without any cocoa flavor, report of Escherichia coli or Salmonella spp. contamination in
and cocoa (press) cake, a dark brown residue (58% of the total). cocoa mass although some bacilli may survive.7 Food poison-
The cocoa cake will then be ground to cocoa powder for use by ing organisms rarely have been reported in the final processed
the confectionery and other industries. Cocoa cake is a strongly chocolate, presumably arising from contamination at a late stage
flavored but inedible material that needs further processing to in the factory.8 The technology of chocolate production effec-
become palatable. To make finished chocolate products, includ- tively limits mycotoxin contamination by moulds that might
ing confectionery, most of the cocoa butter is mixed back with have occurred in the period at the end of fermentation, during
the cocoa mass (liquor) together with sugar, sweeteners, milk drying or if allowed to get wet during transport and storage.
products, emulsifiers, and cocoa butter substitutes depending on Mycotoxins have been found on shells but never in cotyledons,
the requirements of the final product. For the finest chocolate, perhaps because of the inhibitory presence of methylxanthines.
“conching” is performed in order to get fine crystallization. The
chocolate is typically heated to between 50 and 60◦ C for several
hours, although it can be up to 5 days for specialist chocolate, 1.5. Chemistry
while lecithin is added followed by repeated milder heating and
cooling cycles before filling moulds. Chocolate flavors and aromas have been the subject of exten-
Cocoa butter, like all fats, is composed of a mixture of fatty sive research. Unfermented cocoa seeds do not produce cocoa
acids and is typically the saturated fatty acids palmitic acid flavor on roasting so an understanding of the development of
(25%) and stearic acid (35%), the monounsaturated fatty acid cocoa flavor precursors during fermentation is required. Bitter
oleic acid (35%), and the polyunsaturated fatty acid linoleic acid and astringent flavors are due to polyhydroxyphenols such as
(3%) with some others (2%). The melting point of cocoa butter catechins, flavan-3-ols, anthocyanins, and proanthocyanadins.
is around 35◦ C with softening around 30–32◦ C and it becomes Polyphenols tend to diffuse out of the bean during the fermen-
brittle fracture below 20◦ C. tation and also are oxidized by polyphenol oxidazes to pro-
duce mostly insoluble tannins. There is also a loss during dry-
ing and roasting.9 Since there are abundant health claims for
1.4. Health and Nutrition polyphenols10−14 efforts are being made to maintain their levels
while avoiding taste problems.15 Theobromine and caffeine and
Most of the health problems associated with high chocolate their complexes are major components of cocoa’s bitter taste
consumption stem from the high concentration of carbohydrates but they also tend to diffuse out of the bean during fermentation.
in processed chocolate rather than the chocolate itself. The ba- Endogenous acids (malic, tartaric, oxalic, phosphoric, citric) are
sis of its “addictive” properties for chocoholics has not been probably less important because it is the diffusion of lactic and
identified although cannabinoids are found in chocolate at low acetic acids into the bean that dominate bean acidity. In turn they
levels.5 In Colombia the nutritional role of chocolate is empha- depend on the sugars in the pulp and availability of oxygen for
sized because natural cane sugar and chocolate are combined their production by bacteria. Some lactic acid is lost during dry-
into a nutritious beverage with an excellent balance of carbohy- ing but most of the acetic acid remains. Therefore it is important
drates, lipids, and proteins. Possible medicinal/health benefits to ensure that neither the initial conditions nor fermentation and
of chocolate have been reported for many years but it is only drying produce excess acid.
recently that some of these claims are being more clearly iden- The source of hundreds of volatiles found in roasted beans
tified and studied.3 Research shows that the cocoa bean and its (both fermented and unfermented) are the reducing sugars, free
derived products are rich in specific antioxidants, including cate- amino acids, and oligopeptides. The sugars come from sucrose
chins and epicatechin, and especially the polymers procyanidins and its hydrolysis products, glucose and fructose, in addition
and polyphenols similar to those found in vegetables and tea. to being released from glycosides. Most amino acids and
208 R. F. SCHWAN AND A. E. WHEALS

oligopeptides are produced during acid hydrolysis that occurs lined and covered with leaves, are also used. In larger farms
during fermentation. These compounds undergo non-enzymatic fermentations are performed in large, perforated wooden boxes
browning reactions during drying and roasting. These Mail- allowing pulp to drain away and air to enter. Although they can
lard reactions are condensations between the α-amino group hold up to 2000 kg of beans the depth does not exceed 50 cm to
of amino acids, proteins, or amines and the carbonyl group of ensure good aeration. The beans are covered with banana leaves
reducing sugars. They are quite distinct from caramelization of or sacking to conserve the heat generated during fermentation.
sugars, which does not involve amino acids. Typical “hammy” To ensure uniform fermentation and increase aeration, beans are
off-flavors are produced by over-fermentation when bacilli and manually turned up to once per day. Some are tiered on slopes
filamentous fungi grow on cocoa husks and nibs to produce that facilitates transfer of beans from one box to a lower one with
short chain fatty acids. Smoky off-flavors from wood fires used simultaneous aeration. Plantations usually ferment for a longer
for drying are now less of a problem. period than small-holders and 6 to 7 days is usual.
Chocolate shares with wine the distinction of being an ancient Changes in the local climatic conditions influence the se-
fermented product with a combination of nutritional, medicinal, quence of microorganisms involved in cocoa fermentation but
and mystical properties. The global improvement in wine quality a similar succession of groups of organisms has often been
over the last 25 years has been significantly due to better control reported.20,21,22 The microbial succession in the fermentation
of the fermentation process itself. The purpose of this review is process has been clearly established.16,20−24 Early on in the fer-
to describe research over the last 15 years into the fermentation mentation, several species of yeasts proliferate, leading to pro-
process and discoveries on how cocoa fermentation is involved duction of ethanol and secretion of pectinolytic enzymes. This
in production of chocolate flavor precursors. If implemented, is followed by a phase in which bacteria appear, principally
this knowledge will enable high quality natural chocolate to be lactic-acid bacteria and acetic-acid bacteria, which is followed
routinely produced and, perhaps yield better financial returns for by growth of aerobic spore-forming bacteria. Finally, some fila-
farmers. mentous fungi may appear on the surface. A comprehensive and
representative set of data from both the fermentation process
and subsequent sun-drying are shown in Figure 2 (previosuly
2. THE FERMENTATION PROCESS unpublished data).
The initial acidity of the pulp (pH 3.6), due to citric acid,
2.1. Cocoa Pulp: The Fermentation Substrate together with low oxygen levels, favor colonization by yeasts25
that are able to utilize pulp carbohydrates under both aerobic and
Cocoa pulp is a rich medium for microbial growth. It con- anaerobic conditions. The size of the yeast population increases
sists of 82–87% water, 10–15% sugar, 2–3% pentosans, 1–3% from 107 CFU/g of pulp to 108 CFU/g of pulp during the first 12 h
citric acid, and 1–1.5% pectin.16 Proteins, amino acids, vitamins (Figure 2), then remains almost constant for the next 12 h after
(mainly vitamin C), and minerals are also present. The concen- which there is a dramatic decline of four orders of magnitude
tration of glucose, sucrose, and fructose is a function of fruit over the next day followed by a slower decrease leading to a
age.17 More glucose and fructose and a slight increase in to- final population of only 10 viable cells per gram of pulp.22
tal sugar concentration were observed in samples 6 days after The amended conditions favor the development of lactic-acid
harvest than in freshly harvested (ripe) pods.18 In a comparative bacteria. The number of these organisms reaches a peak around
analysis of pulp from beans collected in the Ivory Coast, Nigeria, 36 hours after the fermentation process begins and the bacte-
and Malaysia, differences were found in the amounts of water, rial population reached 6.4 × 107 CFU/g of pulp (Figure 2).
citrate, hemicellulose, lignin, and pectin.19 Pectin content, ap- This period of time is coincident with the decline of the yeast
proximately 1% on a fresh weight basis, was found to 37.5 and population.22,24 The lactic acid bacteria exhibit the fastest
66.1 g kg−1 dry weight pulp. growth rate during the 16–48 h period of fermentation and are
Seeds within the ripe pod are microbiologically sterile. When present in greater numbers, but not necessarily in biomass, than
the pod is opened with a knife, the pulp becomes contaminated yeasts for a short period of time.22 As aeration of the ferment-
with a variety of microorganisms many of which contribute to ing mass increases and the temperature rises above 37◦ C, acetic
the subsequent fermentation. Organisms come mainly from the acid bacteria became the dominant organisms, and the popula-
hands of workers, knives, unwashed baskets used for transport tion reached a peak at 88 hours with 1.2 × 107 CFU/g of pulp.22
of seeds, and dried mucilage left on the walls of boxes from This stage in the microbial succession is reflected in a decline in
previous fermentations. the concentration of ethanol and lactic acid, and increase in acetic
acid. The exothermic reactions of acetic-acid bacteria raise the
temperature of the fermenting mass even further up to 50◦ C or
2.2. Microbial Fermentation more. The decrease in the number of acetic-acid bacteria from
three days onwards is probably due to their inhibition by the
On small-holdings, fermentations are often done in heaps high temperature in the cocoa mass. The strong odor of acetic
of beans from about 25 kg to 2000 kg enclosed by banana or acid, evident from 48 to 112 h, decreases progressively towards
plantain leaves with some turning to assist aeration. Baskets, the end of the fermentation. After 120 hours of fermentation
 MICROBIOLOGY OF COCOA FERMENTATION 209

Figure 2 Cell density in the fermentation box and during sun drying. The cocoa beans were taken to a sun drying platform after 156 hours. Counts are per ml
of pulp. Yeasts: open circles. Lactic-acid bacteria: open diamonds. Acetic acid bacteria: open squares. Spore-forming bacteria: closed circles. Filamentous fungi:
open triangles.

acetic acid bacteria were not found. There is a minor increase the end of the fermentation the beans are usually transferred
in the number of yeasts to 3.5 × 103 CFU/g of pulp22 around to platforms and sun-dried. During this process, commencing
132–160 hours. This is due to growth of thermotolerant yeasts after 156 hours, there is a sharp decrease in the total microbial
utilizing some of the acids coinciding with an increase in the population. During sun drying cocoa beans are often humidified
oxygen content in the fermenting mass22 as well as survivors in to help the workers remove the rest of the mucilage with their
the cooler external layers of the fermentation. feet but eventually only microorganisms that are able to form
Aerobic, spore-forming bacteria can be isolated during the spores, bacilli, and filamentous fungi can survive.
first three days of fermentation with populations around 104
CFU/g of pulp but their numbers remain virtually unchanged.
Thereafter they start to dominate the microbial population to 2.3. Yeasts
such an extent that they form over 80% of the microflora,22,25,26
reaching 5.5 × 107 CFU/g of pulp.22 This phase in the succession Yeasts have been isolated from cocoa fermentations by many
coincides with increases in oxygen tension, temperature, and pH groups23 but only four studies have simultaneously identified
of the fermenting mass. Filamentous fungi are found in small yeasts and bacteria (Table 1). To avoid confusion the names
numbers throughout the fermentation, most commonly in the used in the original literature have been retained but current
aerated and cooler, superficial areas of the fermenting mass. At nomenclature is given in the appendix (see page 221). Other

Table 1 Yeasts isolated from cocoa fermentations in four countries

Brazil22 Ghana32 Malaysia32 Belize100

Candida bombi, Candida pelliculosa, Candida Candida spp., Hansenula Candida spp., Debaryomyces Brettanomyces clausenii., Candida spp.,
rugopelliculosa, Candida rugosa, spp., Kloeckera spp., spp., Hanseniaspora spp., C. boidinii, C. cacoai, C. guilliermondii,
Kloeckera apiculata, Kluyveromyces Pichia spp., Hansenula spp., Kloeckera C. intermedia, C. krusei, C. reukaufii,
marxianus, Kluyveromyces thermotolerans, Saccharomyces spp., spp., Rhodotorula spp., Kloeckera apis, Pichia
Lodderomyces elongisporus, Pichia Saccharomycopsis spp., Saccharomyces spp., membranaefaciens, Saccharomyces
fermentans, S. cerevisiae var. chevalieri, Schizosaccharomyces spp., Torulopsis spp. cerevisiae, Saccharomyces chevalieri,
Saccharomyces cerevisiae, Torulaspora Torulopsis spp. Saccharomycopsis spp.,
pretoriensis Schizosaccharomyces malidevorans,
Schizosaccharomyces spp.
210 R. F. SCHWAN AND A. E. WHEALS

Table 2 Lactic acid bacteria isolated from cocoa fermentations in four countries
Brazil29 Ghana32 Malaysia32 Belize100

Lactobacillus. Acidophilus, Lb. brevis, Lb. casei, Lb. collinoides Lb. collinoides, Lb. brevis, Lb. buchneri, Lb. casei, Lb. Casei
Lb. Delbrueckii, Lb. fermentum Lb. fermentum Lb. plantarum pseudoplantarum, Lb. cellobiosus, Lb. delbrueckii,
Lb. Lactis, Lb. Plantarum Lb. mali Lb. fermentum, Lb. fructivorans, Lb. gasseri, Lb.
Lactococcus lactis, Leuconostoc mesenteroides, Lb. plantarum kandleri, Lb. plantarum, Leuconostoc
Pediococcus acidilactici, P. dextrinicus mesenteroides, Ln. oenos, Ln. paramesenteroides

studies have identified isolates of the genera Candida, Pichia, 2.4. Bacteria
Saccharomyces, Kloeckera, Trichosporon, and Schizosaccha-
romyces in Java;16 Kloeckera apis, Candida pelliculosa, Can- A. Lactic-Acid Bacteria
dida tropicalis, and Saccharomyces cerevisiae in Indonesia;27
Lactic-acid bacteria increased in numbers when part of the
and Pichia membranaefaciens, Saccharomyces cerevisiae, Can-
pulp and “sweatings” had largely drained away, and the yeast
dida zeylanoides, Torulopsis candida, T. castelli, and T. holmii
population was declining. Yeast metabolism favors the growth
in the Ivory Coast.28 It is not possible to determine whether
of acidoduric, lactic-acid bacteria. Of the lactic acid bacteria iso-
these differences in the yeast flora were due to geography or
lated from cocoa fermentations21 (Table 2), Lactobacillus fer-
to fermentation practices. In the most comprehensive study22
mentum, Lb. plantarum, Leuconostoc mesenteroides, and Lac-
(Table 1), frequency of species with time was also monitored in
tococcus (Streptococcus) lactis were the most abundant species
detail. Saccharomyces cerevisiae was the dominant yeast in the
in the first 24 h of fermentation. In Bahia (Brazil), six Lac-
cocoa beans taken from boxes immediately after filling. Kloeck-
tobacillus spp. and two species of the genus Pediococcus to-
era apiculata grew during the early phase of fermentation but
gether with Lactococcus lactis and Leuconostoc mesenteroides
declined rapidly such that it could not be isolated after 24 h
were isolated29 (Table 2). In general, the Lactobacillus spp. were
of fermentation which probably reflects its intolerance of ethanol
at concentrations above 4% (v/v).22 Kluyveromyces marxianus present at the early stages whereas Lactococcus spp. occurred
during the final stages of fermentation. Lactic acid bacteria were
grew slowly at the outset of fermentation and then declined
isolated in cocoa fermentation in Indonesia and Lactobacil-
gradually. Two different strains of S. cerevisiae dominated the
lus plantarum and Lactobacillus cellobiosus were the principal
alcoholic fermentation phase and survived throughout the fer-
species.27
mentation process. Small numbers of Pichia fermentans and
Lodderomyces ellongisporus were isolated but only during the
first few hours of fermentation. Candida spp. increased in num-
B. Acetic-Acid Bacteria
bers after 24 h. Candida rugosa was present up to the end of fer-
mentation when the temperature was approximately 50◦ C. Toru- After the decline in the populations of yeasts and lactic-acid
lospora pretoriensis and Kluyveromyces thermotolerans were bacteria, the fermenting mass becomes more aerated. This cre-
found also when the temperature of the fermenting mass was ates conditions suitable for the development of acetic-acid bacte-
approximately 50◦ C. The yeast flora was abundant and varied, ria. These bacteria are responsible for the oxidation of ethanol to
which is not surprising since cocoa bean pulp contains, on aver- acetic acid and further oxidation of the latter to carbon dioxide
age, 14% of sugars. Of these, 60% is sucrose and 39% a mixture and water. The acidulation of cocoa beans and the high tem-
of glucose and fructose.18 All these sugars are fermented by perature in the fermenting mass, which causes diffusion and
the above species, but even so, S. cerevisiae was the most com- hydrolysis of proteins in the cotyledons, has been attributed to
mon species of yeast identified in the study probably because the metabolism of these organisms. Thus the acetic acid bacteria
of its rapid growth and ethanol-tolerance. It was also found in play a key role in the formation of the precursors of chocolate
high numbers during the first 24 h of cocoa fermentation in flavor.30 In general, the members of genus Acetobacter were
Trinidad.21 Kluyveromyces marxianus, K. thermotolerans, Can- found more frequently than those of Gluconobacter (Table 3).31
dida spp, and Torulospora pretoriensis, which were present in Species of Acetobacter aceti and Acetobacter pasteurianus were
considerable numbers in the Brazilian study, have not been re- isolated in Indonesia but the populations were only approxi-
ported from cocoa bean fermentations in other countries.21,23 mately 105 to 106 CFU/g.27

Table 3 Acetic acid bacteria isolated from cocoa fermentations in four countries
Brazil31 Ghana32 Malaysia32 Belize100

Acetobacter aceti subsp. Acetobacter ascendens, A. Acetobacter lovaniensis, A. Acetobacter spp.,

liquefaciens, A. pasteurianus, A. rancens, A. xylinum, rancens, A. xylinum, Gluconobacter oxydans
peroxydans, Gluconobacter Glucononbacter oxydans Gluconobacter oxydans
oxydans subsp. suboxydans
 MICROBIOLOGY OF COCOA FERMENTATION 211

Table 4 Aerobic spore-forming bacteria isolated from cocoa fermentations temperature of the fermenting mass was around 50◦ C. It is not
in four countries uncommon for yeast species isolated from Brazil to show higher
Brazil26 Trinidad21 Ghana and Malaysia101 maximum growth temperatures than the corresponding species
Bacillus brevis, B. cereus, B. Bacillus cereus, B. Bacillus licheniformis,
isolated from temperate sources.38
circulans, B. coagulans, B. cereus var. B. subtilis
firmus, B. laterosporus, B. mycoides, B.
licheniformis, B. coagulans, B.
3. ROLES OF MICROORGANISMS DURING
macerans, B. megaterium, licheniformis, B.
B. pasteurii, B. polymyxa, megaterium, B. COCOA FERMENTATION
B. pumilus, B. pumilus, B.
stearothermophilus, B. stearothermophilus, The great majority of flavor compounds (ca. 400) are formed
subtilis B. subtilis due to biochemical and enzymatic reactions that occur within
the cotyledon. The major role of microorganisms is to produce
acids and alcohols that will penetrate the testa and start the chem-
C. Aerobic Spore-Forming Bacteria
ical reactions that will form the precursors of chocolate flavor.
Increased aeration, increased pH value (3.5 to 5.0) of co- There is no evidence that enzymes from the microorganisms
coa pulp, and a rise in temperature to about 45◦ C in the co- penetrate the testa and create flavor compounds but hydrolytic
coa mass in the later stages of fermentation are associated with enzymes inside the beans are activated by microbial metabolites
the development of aerobic spore-forming bacteria of the genus such as acetic acid.39,40,41 Many different species of microor-
Bacillus21,26,32 (Table 4). Many Bacillus spp. are thermotoler- ganisms have been characterized and the microbial succession
ant and others grow well at elevated temperatures. B. stearother- has been defined. So far the roles of all these microorganisms
mophilus, B. coagulans, and B. circulans were isolated from co- have not been explicitly described particularly in their relative
coa beans that had been subjected to drying and roasting (150◦ C) contribution to the overall quality of the final product. The first
temperatures.7 step in understanding this is to determine the physiology of the
Aerobic spore-forming bacteria produce a variety of chemi- microorganisms and what they contribute to the dynamics of the
cal compounds under fermentative conditions. These may con- fermentation process. Then it is possible to define the potential
tribute to the acidity and perhaps at times to the off-flavors of ecological roles of these microorganisms.
fermented cocoa beans. Indeed it has been suggested that C3 –C5
free fatty acids found during the aerobic phase of fermentation
and considered to be responsible for off-flavors of chocolate34 3.1. Roles of Yeasts
are produced by B. subtilis, B. cereus, and B. megaterium. Other
substances such as acetic and lactic acids, 2,3-butanediol, and A. Ethanol Production
tetramethylpyrazine, all of which are deleterious to the flavor of
chocolate, are also produced by Bacillus spp.34,35 The sugar-rich, acidic pulp presents ideal conditions for rapid
yeast growth. Conversion of sucrose, glucose, and fructose to
ethanol and CO2 is the primary activity of the fermentative
2.5. Filamentous Fungi yeasts. Measurements of ethanol show clearly how, after ris-
ing in concentration in the pulp, it penetrates the cotyledons of
the beans. However, it is reputedly the acetic acid that kills the
Filamentous fungi are not considered to be an important part
beans.30
of the microbial succession of cocoa fermentation.16 They have
been found quite often, however, in the well-aerated parts of the
fermenting mass and during the drying process.36,37 It is likely B. Breakdown of Citric Acid
that they may cause hydrolysis of some of the pulp and even
the testa of the seeds; they may also produce acids or impart Some of the yeasts, including Candida spp. and Pichia spp.,
off-flavors to the beans.37 Filamentous fungi isolated from fer- metabolize citric acid causing the pH value to increase in the
menting cocoa in Bahia were Aspergillus fumigatus, A. niger, pulp which allows growth of bacteria. The loss of citric acid
Fusarium moniliforme, F. oxysporum, Lasiodiplodia theobro- both in the “sweatings” and by microbial metabolism causes an
mae, Mucor racemosus, Mucor sp., Paecilomyces varioti, Peni- alkaline drift in pH. This, together with the increasing levels of
cillium citrinum, P. implicatus, P. spinosum, Thielaviopsis alcohol and aeration, inhibits the yeasts and their activity wanes.
ethaceticus, Trichoderma viridae, and three different isolates
of Mycelia sterilia.37 Although the numbers were small, a great
C. Production of Organic Acids
diversity of species was seen in the first 44 h of fermentation.
Thereafter Aspergillus fumigatus and Mucor racemous domi- Several of the yeast isolates produce organic acids including
nated the fungal population up to the end of fermentation. Most acetic, oxalic, phosphoric, succinic, and malic acids. These weak
of these fungi are reported to be unable to grow at tempera- organic acids will have a buffering capacity and will tend to
tures higher than 45◦ C, but they have been isolated when the reduce fluctuations in pH.
212 R. F. SCHWAN AND A. E. WHEALS

D. Production of Volatiles both necessary and sufficient for there to be representatives of

each physiological/ecological group to provide the appropriate
Yeasts produce a large array of aroma compounds, principally
transformations during the fermentation (see section 5).
fusel alcohols, fatty acids, and fatty acid esters42 and different
species produce different aromas.42,43 It is known that volatile
compounds are important in the development of full chocolate 3.2. Roles of Bacteria
flavor.44,45 The five major yeasts that produce these volatiles
(Kloeckera apiculata, S. cerevisiae, S. cerevisiae var. chevalieri, A. Lactic-Acid Bacteria
Candida sp., and Kluyveromyces marxianus) have been studied
individually. Kloeckera apiculata and S. cerevisiae var. cheva- • Production of lactic acid. The great majority of lactic-acid
lieri were the major producers of volatiles such as isopropyl bacteria isolated during cocoa fermentation utilize glucose
acetate, ethyl acetate, methanol, 1-propanol, isoamyl alcohol, via the Embden-Meyerhof pathway yielding more than 85%
2,3 butanediol, diethyl succinate, and 2-phenylethanol. Among lactic acid. However, some species utilize glucose via the hex-
the yeasts with high fermentative power S. cerevisiae var. cheva- ose monophosphate pathway producing 50% lactic acid, and
lieri produced large amounts of aroma compounds suggesting ethanol, acetic acid, glycerol, mannitol, and CO2 . Their rela-
that these strains might be collaborating in the elaboration of tive proportion will thus change the composition of the pulp
aroma and flavor characteristics (Schwan, R. F. unpublished ob- substrate and thus may consequently change the microbial
servations). Although this study was done in pure culture, it succession.
does give clues as to which species might be added. Studies on • Production of citric acid. Lactic-acid bacteria first contribute
wine fermentations have shown the importance of the range of to an increase in acidity by producing citric acid and then
volatiles that can be produced by different strains and different lower the pH by metabolizing it and liberating non-acid by-
species.43,46,47 products.51 All lactic acid bacteria isolated from cocoa fer-
mentations were able to metabolize malic and citric acids.29,51
Dissimilation of these acids leads to an overall drop in acidity
E. Production of Pectinolytic Enzymes and rise in pH value. Lactic acid bacteria are virtually non-
proteolytic and their ability to ferment amino acids is also
Some strains produce pectinolytic enzymes20,28,48−50 that restricted with only two, serine and arginine, that are exten-
break down the cement between the walls of the pulp cells and the sively attacked by some of these organisms.52
resultant juice (or “cacao honey”) drains away as “sweatings.”
The collapse of the parenchyma cells in the pulp between the
beans results in the formation of void spaces into which air perco- B. Acetic-Acid Bacteria
lates. Only 4 out of 12 yeast species showed pectinolytic activity • Production of acetic acid. These bacteria are responsible
(K. marxianus, S. cerevisiae var. chevalieri, Candida rugopel-
for the oxidation of ethanol to acetic acid and further oxi-
liculosa, and K. thermotolerans). Only the first two showed sub-
dation of the latter to carbon dioxide and water. The exother-
stantial activity and only K. marxianus produced large quantities
mic reactions of the acetic-acid bacteria raise the tempera-
of heat stable endopolygalacturonase (PG). It had strong macer-
ture of the fermenting mass, sometimes to 50◦ C or more.
ation activity that reduced cocoa pulp viscosity during the first
The acidity of cocoa beans, the high temperature in the fer-
36 hr of fermentation when K. marxianus was the most abun-
menting mass, and the diffusion and hydrolysis of protein in
dant pectinolytic yeast. PG of K. marxianus has been studied
the cotyledons has been attributed to the metabolism of these
in more detail.49,50 None of the bacterial species present in the
microorganisms. Concentrations of a maximum of 6 g/L of
early stages of the fermentation have been shown to have pecti-
pulp of acetic acid was found in cocoa pulp after 88 hours
nolytic activity. This enzyme activity is crucial during the first
of fermentation.24 However, it disappeared quickly from the
24 hours because it breaks down the pulp and allows penetra-
pulp when the mass temperature rose above 50◦ C.22,24 Part
tion of oxygen into the fermenting cocoa mass enabling aerobic
of this acid is volatilized and part penetrates the testa (ap-
acetic acid bacteria to grow.
proximately 2%) and is responsible for killing the embryo.53
Ethanol, acids, and water diffusing into the cotyledon act as
solvents so that cellular components are transported to sites of
F. Yeast Varieties
enzyme activity and vice versa. The detailed levels of chem-
It is likely that all of these biochemical transformations are ical reactions inside the bean are still unknown. It is clear
necessary for a normal fermentation and species that perform that excess acid will interfere with chocolate flavor22,54 even
some or all of them are probably essential but the other yeast though most of the acetic acid will eventually be volatilized.
species are probably unimportant. Indeed, some of them could
be defined as transients that only show spasmodic appearance
C. Aerobic Spore-Forming Bacteria
and it is possible that the different species found in different
countries or in different types of fermentation are not impor- The Bacilllus species that were isolated produced a variety of
tant in respect of the fermentation process per se. It may be chemical compounds under fermentative conditions such as 2,3
 MICROBIOLOGY OF COCOA FERMENTATION 213

butanediol, pyrazines, acetic, and lactic acid. These bacteria may 4.2. Mechanical Removal of Cocoa Pulp
contribute to the acidity and perhaps at times to off-flavors of
fermented cocoa beans. Oxygen is one of the factors that deter- Brazilian and Malaysian cocoas tend to be extremely acidic
mines the microbial succession. Facultatively anaerobic yeasts (cotyledon pH about 4.2) and this has adversely affected the de-
are metabolically active at the beginning of fermentation when velopment of their international markets. Removal of some of
oxygen is not available because of its occlusion by the mucilagi- the pulp before fermentation reduces acidity and this presents a
nous pulp surrounding the seed. Lactic acid bacteria are the next possible solution to the acidity problem. It was reported that at
group in the succession and are microaerophilic. When the pulp least 10% by weight can be removed by pressing the beans prior
has been degraded, oxygen becomes more plentiful and then the to fermentation without measurable consequences.59 A normal
strictly aerobic acetic acid bacteria develop. bean fermentation occurred when up to 20% of total fresh weight
of beans (including pulp) was removed.59 This produced a less
acidic cocoa in Brazil60 although the acidity of beans was not
3.3. Conclusions
reduced when some of the pulp was removed prior to fermen-
tation in Malaysia.61 A decrease in volume, water, and sugar
Growth in relation to sugar and oxygen are the key parame- content in cocoa pulp occurred when beans were spread out in a
ters that establish and change the microbial succession. Ethanol thin layer before fermentation in Malaysia and this method pro-
tolerant yeasts ferment the sugars at low pH (pH 3.5 and 4.2) duced cocoa with less acidity.62 Genetic differences in material
and pectinolytic enzymes open the structure of the pulp for the cultivated in Malaysia and Brazil may be responsible for these
ingress of air. Lactic acid bacteria are micro-aerophilic and mem- differences since cacao cultivars in Malaysia have about three
bers of the homolactic group are able to ferment sugars and tol- times more pulp sugars than the Brazilian comum cultivar.51,60
erate this acidity. The acetic acid bacteria are aerobic and can Using a modified domestic washing machine it was shown63
grow at high concentrations of ethanol and tolerate temperatures that partial (20%) removal of cocoa pulp gave an accelerated
around 45◦ C. They produce acetic acid from sugars and also can fermentation. There was a more rapid progression in the micro-
oxidize ethanol to acetic acid and then to CO2 and water. These bial succession, temperature increase, and rise in pH value of
conclusions about the physiological roles of the major groups the cotyledon from 4.8 (as in traditional cocoa fermentation) to
were experimentally tested (see section 5). 5.5. Unfortunately these results could not be reproduced using a
commercial depulping machine. This was probably due to dif-
ferences in the technology: centrifugation is the basis of separa-
4. COCOA PULP
tion in a washing machine while gentle scraping is the principle
of depulpers, but they also tend to remove the tightly adhering
Cocoa pulp is the raw material on which the fermentation mucilaginous layer immediately surrounding the bean.59 Pulp
proceeds and this section will describe how it seems to be a key extraction on a larger scale for the cacao juice industry has been
determinant of both quality and financial viability of the process. done with commercially available depulpers.64 Such depulpers
Not only is the quantity of pulp crucial in affecting the efficiency remove from 17 to 20% of pulp in terms of the fresh weight of
and nature of the fermentation, but excess pulp can also be sold the seed. Some depulpers leave loose mucilage, but little sugar
as a high value commodity. on the seeds. This mucilage blocks the void spaces in the cocoa
mass, impairs aeration, causes under-fermentation, and extends
4.1. Quantity of Pulp Surrounding the Cacao Seed the fermentation period. If this occurs, there is no reduction of
acidity of the cocoa compared to traditional box fermentations.
Not all pulp is necessary for a successful fermentation of The viscosity of the pulp still needs to be reduced in addition to
cocoa beans. Loss of pulp occurs naturally during a fermenta- reduction of pulp quantity.
tion because the ‘sweatings’ drain out through the holes in the Washing of the seeds has been used to produce a product
fermentation box. This liquid is almost transparent and is rich suitable for wine production.65 This also yielded a pulp-depleted
in fermentable sugars, pectin, and acids. In Brazil, it has been bean that when fermented gave rise to fermented cocoa beans
used traditionally to make jelly. Today the juice for commercial that were less acidic. Such a process does require a very high
jelly production is pressed from the seeds before fermentation. level of water quality and worker hygiene.
The economy of cocoa-producing areas in Brazil is very depen-
dent on the acceptance of its products in the market. Processing 4.3. Enzymatic Removal of Cocoa Pulp
of post-harvest residues and by-products of cacao (eg. cacao
juice, cacao jam, vinegar and liquor of cacao juice) may offer The addition of pectinolytic enzymes improves the efficacy
opportunities for diversification on farms, especially where co- of mechanical pulp extractors. One liter of a 0.2% (w/w) solution
coa production is the major enterprise.55 Revenue generated by of pectinase (Ultrazym 100G, Novo Nordisk Ferment) sprayed
these products exceeds that obtained from selling cocoa beans over the seeds and allowing a reaction time of 30 minutes, in-
to processors. Ghana and Malaysia are also developing these creases the quantity of pulp extracted to approximately 23%
industries.56,57,58 compared to the batch-type depulper.66,67 This value represents
214 R. F. SCHWAN AND A. E. WHEALS

an increase of about 5% of total weight over the machine. Based revealed a PG gene but not a PME gene suggesting either er-
on an assessment of both external and internal (“cut-test”) color roneous assays or mis-identification of species in at least one
of beans, total fermentation time was reduced from seven to four case. S. chevalieri, Candida norvegensis, and Torulopsis candida
days and the acidity of the final product was reduced.55 As the were the only pectinolytic yeasts isolated from cocoa fermenta-
pectin chains were broken by the added enzymes, the pulp had tions in another study.48,74 In trials with pure “starter” cultures
a lower viscosity. This change also helps pulp processing for of yeasts, including Kluyveromyces marxianus, among isolates
pasteurized juice as well as for cacao soft drink production that from cocoa fermentations, the pH value did not rise during the
is bottled and stored at ambient temperatures. Some laboratory early stages of fermentation.74 The researchers suggested that
experiments have suggested that the yield could be improved K. marxianus interfered with the development of the wild yeast
by inoculation with pectinolytic yeasts.67 However, the experi- flora. Among the other strains studied, C. norvegensis produced
ments are difficult to evaluate since only 1 kg samples of beans the greatest amount of extracellular enzyme. They found that
were used, no monitoring of the microbial population was done, the yeast enzymes had the same optimum pH value of activity
and the fermented beans were only analyzed by anthocyanin (5.0) but differed from each other in their optimum temperature
content. Since good color and flavor are also due to bacterial and thermal stability. The enzymes of T. candida and K. fragilis
activity, the results could have been partly due to other microbi- had the highest optimum temperature (60◦ C).
ological activity. Of the 12 yeast species isolated from cocoa fermentations
Addition of commercial enzymes is costly and prohibitive in Brazil, K. marxianus, S. cerevisiae var. chevalieri, C. ru-
on a large scale. Two alternative approaches are being explored gopelliculosa, and K. thermotolerans produced extracellular en-
to provide a better quality of fermented beans by speeding up dopolygalactoronase (endoPG).50 Neither PME nor PL was de-
this process; (1) to increase microbial pectinolytic activity at tected in culture filtrates. The amounts and properties of each PG
the onset of fermentation, and (2) making a source of enzyme differed but all were relatively unstable compared with that of K.
obtainable from yeast cultures themselves. marxianus, which was also found to be the most active producer
of PG. This strain fermented the major pulp sugars as well as
degrading pectin. High yields of PGs were obtained with self-
4.4. Pectinases Produced by Yeasts induced anaerobic batch fermentations of K. marxianus with
100 g l−1 glucose as the sole carbon source75 but production is
Pectins give pulp its sticky, viscous, and cohesive properties. inhibited by oxygen.76,77 Addition of pectin or polygalacturonic
Pectin and pectic acid, the natural substrates of pectic enzymes, acid to the growth medium did not increase enzyme secretion,
are branched heteropolysaccharides in which the backbone con- indicating that PG production is constitutive under these condi-
tains L-rhamnose residues and αa-D-(1,4)-linked residues of tions but it was unable to grow on pectin or galacturonic acid as
D-galactopyranosiduronic acid.68,69 The neutral sugars, D- the sole carbon source like Rhodotorula spp.78 PG secreted by
galactose and L-arabinose and sometimes D-xylose and K. marxianus could macerate potato and cucumber slices and
L-fucose, form the side-chains of the pectin molecule. The car- decrease the viscosity of cocoa pulp by 50% within 18 min.49,50
boxyl groups of the D-galactopyranosiduronic acid residues are These data suggest that the anaerobic conditions that rapidly
partially esterified with methanol. Some of the secondary alcohol predominate after initiation of natural cocoa fermentations are
groups at C-2 and C-3 are acetylated.69 The degree of esterifica- ideal for the appearance of the enzyme but that its production
tion, the proportion of neutral saccharides, and degree of poly- becomes self-limiting as the pulp drains away and air percolated
merization are the principal elements of heterogeneity in pectic through the fermenter.
compounds of diverse origins.69 Enzymes that attack pectin can The PG secreted from K. marxianus was characterized and
be assigned to two main groups: (1) de-esterifying enzymes showed activity from pH 4 to 6, with an optimum at pH 5
(pectinmethylesterases, PME) that remove the methoxyl groups typical of endoPG secreted by yeasts. Unlike some pectinases,
from the esterified acid, and (2) chain-splitting enzymes (de- K. marxianus endoPG activity was not affected by buffers used
polymerases) that split the βb-(1,4)-glycosidic bond, either by across the pH range studied. The effect of temperature on en-
hydrolysis (polygalacturonases, PG) or by trans-elimination doPG activity from K. marxianus was similar to that reported for
(pectin and pectate lyases, PL). An increasing number of yeast PGs from other yeasts. From concentrated culture supernatant of
species have been discovered to have pectinolytic activity.70 K. marxianus, gel filtration resolved four peaks containing PG
In Java pectinolytic yeasts belonging to the genera Candida, activities. The relative molecular masses were calculated and
Pichia, Saccharomyces, and Zygosaccharomyces were found.71 the four PG forms had apparent Mr of 47, 41, 35, and 33 kDa.
Yeasts from cocoa fermentations produced various pectinolytic According to analysis of all bands by densitometry, about 85%
enzymes that aided the maceration of cocoa pulp and the drainage of total protein secreted into culture medium by K. marxianus
of “sweatings.”28 They claimed that Saccharomyces chevalieri consisted of PG.50 A study of the kinetics of appearance of the
(now classified as S. cerevisiae72,73 ), Torulopsis candida, and T. enzyme using sub-cellular fractionation showed it was secreted
holmii produced PME and that S. chevalieri and Candida zey- by the classical yeast secretory pathway. Since high endoPG
lanoides secreted PG. Genome sequencing of Saccharomyces activity in early stages of fermentation speeds the fermentation
cerevisiae ([Link] has process and leads to better quality of chocolate, overproducer
 MICROBIOLOGY OF COCOA FERMENTATION 215

strains might be useful in improving quality. An attempt was would require satisfying the safety aspects for the regulatory au-
made to do this by conventional chemical mutagenesis using ni- thorities of the country concerned. This is a lengthy and costly
trosoguanidine. However, with a constitutive (deregulated) gene, process. Even more important is that GMOs are currently under
substantial improvements in productivity were never likely and public scrutiny and are often perceived as abnormal and unde-
in a screen of 18,000 mutagenized cells only a few strains pro- sirable. Chocolate produced with such a strain would never find
duced enhanced levels of the enzyme and the best was only 25% a market!
above wild type levels.50 It was concluded that a more directed Alternative strategies have been tried (increase in chromoso-
approach might be more profitable. mal copy number; site-directed mutagenesis of the active site
of endoPG) but without success (Jia and Wheals, unpublished
data). It therefore seems likely that the best approach would be
4.5. Genetics of EndoPG Production to screen additional strains for the desired enhanced activity.

The genes for endoPG have recently been cloned from strains
of Saccharomyces cerevisiae,79–82 Saccharomyces bayanus,83 5. CHOCOLATE QUALITY
and Kluyveromyces marxianus.82,84 One of the most interesting
observations with respect to Saccharomyces cerevisiae was that One of the reasons that chocolate quality has not been a pri-
the non-pectinolytic laboratory strains used in genetic studies ority for farmers is that there is no financial incentive to produce
contain a PG homologue.85 By isolating the structural gene and high quality fermented cocoa beans. Poor practices are widely
putting it on an expression vector it has been shown that it is reported and even led Ecuador, with special status for quality,
secreted and functional.79,82 Transcription analysis has shown to have its rating downgraded in 1994. However, as increasing
that the gene can be induced under special conditions of nitro- numbers of farmers and countries are attempting to take control
gen starvation with induction of the pseudohyphal development of all the processing that occurs in the tropical countries and the
pathway.86 Only one copy of the gene is present on the genome global market reaches saturation, quality will become even more
in both of these yeasts in contrast to filamentous fungi where important. This section is directed to pointing towards general
at least four are present in Aspergillus niger.87 Over-expression improvement in practice that can be achieved by using appro-
strains should facilitate the process of producing a pure enzyme. priate procedures.
In the absence of contaminating enzymes and undesirable by-
products, such as methanol from pectin methylesterase, PG from
K. marxianus could be used directly on cocoa beans to speed up 5.1. Starter Cultures
the process and enhance the quality of the final product.
As an alternative to over-producing strains, the possibility From knowledge of the microorganisms responsible for spon-
of local production of the enzyme has been investigated. This taneous cocoa fermentations and their physiological roles dur-
would have the additional benefit of being suitable for use on any ing the process, an attempt was made to manipulate the ferm-
other pectinaceous fruit. To this end the endoPG from a number entation.24 From 12 yeast species, and 30 bacterial species that
of these strains has been taken from the original cloning strain had been identified, a defined microbial cocktail was selected
(S. cerevisiae) and transferred in turn to both K. lactis and K. for use as an inoculum. It consisted of one pectinolytic yeast
marxianus to create new expression systems (Jia and Wheals, species, two lactic acid bacterial species, and two acetic acid
unpublished data). The advantage of these hosts is that (1) the bacterial species.
plasmid carrying the endoPG is stable and requires no selection The yeast Saccharomyces cerevisiae var. chevalieri produces
using conventional systems and (2) that the strains can be grown pectinase, can ferment all pulp sugars at pH 3.5–4.2, is ethanol
on either cheese whey (an industrial waste product) or on sugar tolerant, and was present at the beginning of natural fermen-
cane juice, a widely available and cheap commodity in tropical tation. The lactic-acid bacteria were selected after observing
countries where cacao is grown. The enzyme output from these production of lactic acid at acidic pH, oxygen requirement,
sources is at least 50% higher than wild strains and constitutes and temperature tolerance. Two species of Lactobacillus were
about 90% of secreted protein. The medium in which the cells selected—L. lactis and L. plantarum. The best producers of
are grown is thus suitable for direct use without purification or acetic acid that were also tolerant to temperatures of 45◦ C were
concentration. isolates of Acetobacter aceti. Gluconobacter oxydans was also
Although the addition of an enzyme would be useful, even added to oxidize the ethanol to acetic acid and to CO2 and water.
better would be the inoculation of fermentations with over- A cocktail of these microorganisms was inoculated on cocoa
producer strains, particularly if they were stable enough to con- beans immediately after the pod was broken open and left to
tinually re-infect fresh batches of cocoa beans. Indeed, K. marx- ferment in sterilized 200 kg wooden boxes for 7 days. The three
ianus has the status of an organism that is generally regarded as key metabolites in the pulp, ethanol, lactic acid, and acetic acid
safe (GRAS). However, over-expressing strains are constructed showed similar sequential rises and falls to that found in spon-
with heterologous DNA sequences and are therefore classified taneous fermentation. Contamination from extraneous microor-
as genetically modified organisms (GMOs). To use such a GMO ganisms was kept to a minimum. The beans were then dried and
216 R. F. SCHWAN AND A. E. WHEALS

roasted and chocolate was produced by the usual means. A taste to reduce contamination but at present farmers break open the
panel found the product as good as a “natural fermentation.” pods in the fields with contaminated machetés, transport them in
However, natural fermentations are subject to random fluctu- unwashed containers, and pour them straight into wooden fer-
ations in the inoculum and the fermentation does not always mentation boxes containing the residues of the previous batch.
proceed correctly. Defined cocktails should always be more re- Bringing unopened pods close to the boxes for washing would
liable if only because of the lack of spoilage organisms and the only help if there were a source of washing water that was free of
fermentation time was less than the normal fermentation. fecal contamination. It would also require disposal of the husks
These were encouraging results since the fermentation oc- that are currently left to rot in the fields. Knives, containers, and
curred normally and the product was more than acceptable. It fermentation boxes would all need decontaminating, perhaps
also suggests that the physiological analysis presented in section with a disinfectant. Further ahead we can envisage the need for
3 is correct and that the consortium of microorganisms was cor- proper pasteurization of the materials. This would certainly en-
rectly chosen with respect to their physiological roles. Clearly able control of the inoculum to be achieved but there could be
there is still room for improvement since there was no guarantee adverse effects on the chemistry of the pectin and the beans
that the best species had been selected or that they had been in- leading to deleterious changes in overall quality. Furthermore it
oculated at the most appropriate rates. For example, subsequent implies bringing the pods to a more centrally located “factory”
enzymological work suggested that Kluyveromyces marxianus with appropriate facilities and economies of scale. Field trials
alone or K. marxianus together with S. cerevisiae might be a are now in progress (RF Schwan, unpublished data).
better choice of yeast(s).
One way to explore the generality of this result is to examine
the species found in other countries (Tables 1–3). In the only 5.2. Manipulation of the Fermentation
other study where yeast identification went to species level, the
pectinase-producing variety of Saccharomyces cerevisiae (var Depulping is an approach that is advantageous to the course
chevalieri) was found. In the other studies both Saccharomyces of the fermentation because excess pulp can lead to an over-acid
and Candida genera were found, both of which include pecti- fermented bean but the precise design of the depulping machine
nolytic yeasts. It is likely therefore that all cocoa fermentations is important. Pulp removal by shearing alone in commercial
contained both strongly fermentative and pectinolytic yeasts. depulpers was not as effective as a combination of centrifugation
With respect to the bacteria, there is little change between coun- and shearing given by a domestic washing machine. New designs
tries and the ones used in the cocktail were always present. These may be needed and certainly the cost needs to be kept as low as
results show that representatives of the three major groups may possible.
be sufficient to complete the complex fermentation and that the The natural course of a fermentation takes about seven days
basic features of the microbiology of cocoa fermentation are and the microbial flora evolves in a more or less predictable
understood. manner. It has already shown that supplementing natural en-
Since there is no quick way in which raw materials and tree dopolygalacturonase (PG) with commercial pectinase led to a
varieties are going to change, inoculation with a defined starter faster fermentation and a higher quality product. If non-GMO
culture may be an important way in which reliable fermentations yeast strains can be produced that can secrete enhanced amounts
may be achieved relatively quickly. On a global scale, a large of endoPG, these should have the same effect as adding pecti-
number of companies already supply fresh pressed, dried, or nase. Aeration is known to be important in the fermentation but
“instant” yeast cultures for baking, brewing, or wine making. adequate supplies of oxygen are dependent on reduced viscos-
Some of these companies also have the capacity to produce ity of the pulp and regular turning of the bean mass. Mechanical
batches of special yeasts and other, non-yeast microorganisms turning is not a realistic alternative and forced aeration requires
to order. Once a suitable strain or, more likely, consortium of careful control.65 Redesign of fermenters could be possible and
strains has been defined it will be possible to create cultures for this is being investigated.
direct addition to initiate fermentations. Provided the product is It is likely that the events of the first 24 hours of the fer-
of high quality and the chocolate producers are prepared to pay mentation entrain the subsequent microbial succession (see
a premium for this enhanced quality then it will be economic for section 3.1.E) but a detailed study of the lactic acid and acetic
farmers to purchase and continue to use starter culture strains. acid bacteria may also be useful. Reducing the amount of free
Education and training for the farmers may also encourage them carbohydrate will undoubtedly affect the growth and biochem-
to let the fermentation take its full course. istry of the bacteria but it is unclear what effect this will have
A potential problem with using starter cultures is that the res- with respect to quality and flavor.
ident microbial flora will compete with, and may even outgrow, Bacterial spore-formers and filamentous fungi that appear
the starter culture inoculum. Clearly, starting with a high density during the latter stages of the fermentation are usually associated
will help the new population to establish itself but it will cer- with the appearance of off-flavors and spoilage. A more precise
tainly be necessary to reduce the host microbiota. This will not investigation needs to be done on this in order to establish a
be easy without a change in some traditional practices. For ex- clear endpoint to the fermentation when harvesting is optimal.
ample, washing the pods before breakage does help considerably It is rather difficult to assess this stage since the state of the beans
 MICROBIOLOGY OF COCOA FERMENTATION 217

throughout the box is not uniform but delay in termination could Although final acidity can be reduced by slow (sun) drying
lead to loss of quality. to allow the acids to volatilize94 and produce a less acid product,
it is the acidity during the fermentation that is more crucial for
flavor development.95 Although acetic and lactic acids produced
5.3. Flavor during fermentation are the key determinants, oxalic acid, one
of the endogenous acids, has been reported to be a significant
Under-fermented beans have an astringent and bitter taste contributor to flavor.96
due partly to the presence of high levels of polyphenols. At-
tempts have been made to stimulate polyphenol oxidase activity 5.4. Fermenter Design
that decreases during fermentation and drying, by acidic incu-
bation. This has had some success in ameliorating the effect Further work along these lines will produce a more precise
in some poorer quality Indonesian beans.88 Alternatively Mars understanding of the entire process and elucidate how the vari-
Inc. (Hackettstown, NJ) has patented a method to maintain high ous microbiological factors determine the final outcome of the
levels of putatively beneficial polyphenols.15 Chocolate flavor fermentation. Both the biology and the chemistry are complex
chemistry is very complex and is determined by both the co- and it will need a comprehensive, simultaneous, and dynamic
coa plant variety and the fermentation and roasting process89,90 analysis of all aspects in controlled conditions if a really good
and it would be premature to say that it is fully understood, understanding of the process is to be achieved such that de-
but work over the last thirty years, particularly by the Technical fined changes will have predictable and quantitative outcomes.
University of Braunschweig, has resulted in the determination To achieve some of these aims a sterilizable stainless steel ves-
of the more important chocolate flavor precursors.39,40,41,44 In sel of novel design capable of turning a 50 kg load of beans
essence, it is proposed to be the combination of two proteases, has been constructed. Inoculum, aeration, and turn rate can be
aspartic endopeptidase and serine carboxy-(exo)peptidase, on controlled, temperature monitored and samples taken at inter-
vicilin (7S)-class globulin (VCG) storage protein that produces vals. Early results show that it can mimic the natural conditions
the cocoa-specific precursors. Experiments with alternative, re- of fermentation boxes and produce fermented beans for making
lated storage proteins or alternative peptidases both fail to pro- good quality chocolate in five days (Freire, Schwan and Serodio,
duce appropriate flavor precursors. The aspartic endopeptidase unpublished data). Combined with defined inocula there is the
splits VCG at hydrophobic amino acid residues. The products prospect of producing the best quality chocolate reliably and in
of this hydrolysis are substrates for the serine exopeptidase that less time. Another approach has been to modify a rotary drier
removes the hydrophobic amino acid residues at the carboxyl enabling it to ferment up to 9 tons of beans.97 Initial results also
terminus of the hydrophobic oligopeptides. Roasting of these show that it performed well in comparison with traditional fer-
precursors in the presence of reducing sugars produced signif- mentations although the process was stopped after four days.
icant cocoa aroma. However, which of the hydrophobic amino Research with such fermenters will accelerate the number of
acid residues and hydrophilic oligopeptides are responsible for variables that can be studied and allow earlier use of this infor-
the cocoa aroma is not yet known.91 mation by farmers. These are the first reports of making large-
Both of these enzymes are very pH-dependent in their activ- scale, controllable, and mechanized fermenters in an otherwise
ity. When the pH during proteolysis approaches pH 3.8 (the op- very traditional industry.
timum for aspartic endopeptidase), more hydrophobic oligopep- It is clear that the research done by major manufacturers
tides and less free amino acids are produced. On the other hand on roasting and processing has enabled them to produce good
pH values close to 5.8 (the optimum for serine exopeptidase), quality products from inferior sources. However in a consumer-
lead to an increase in hydrophilic oligopeptides and hydropho- oriented world where less processing and additives are desirable,
bic amino acids. If the pH becomes too acid too soon (pH < attention to the primary aspects of good quality plant material
4.5) there will be both a final reduction in flavor precursors and and well controlled fermentations will lead to both improved
an over-acid product. Thus, with respect to the organic acids final products and the need for less processing.
that diffuse slowly into the cotyledons, timing of initial entry,
duration of the period of optimum pH and final pH are cru- 6. CONCLUSIONS
cial for optimum flavor. In other words, fermentation may still
be the key to cocoa quality.44 This is further emphasized by We propose that reliable fermentations giving consistently
an analysis of VCG proteins and their proteolytic degradation high quality products can be achieved by a combination of three
products amongst five widely used cacao genotypes (Forastero, procedures:
Criollo, Trinitario, SCA 12, and UIT1). Although they can give
rise to different quality chocolate, all had similar potential for • Control of the amount of free pulp at the start of the fermen-
producing raw cocoa with high aroma potential.92 Two aspartic tation such that the final pH is not too acid,
proteinase (EC 3.4.23) genes that are expressed during seed de- • Use of a defined starter culture so that there is a well ordered
velopment have been cloned from T. cacao93 and thus prospects succession and timely production of acids diffusing into the
for the identification of the flavor precursors are now good. cotyledons, and
218 R. F. SCHWAN AND A. E. WHEALS

• Improved fermenter design to optimize the physical aspects process and how it can pay dividends in improving the quality
of the process especially aeration of the final product.

Another approach to improving flavor would be to make choco-

late from Theobroma grandiflorum (cupuaçu). This has a dif-
ferent and more fruity flavor but can be processed in much the ACKNOWLEDGMENTS
same way98 to produce “cupulate.” Although there could be po-
Much of the work described in this paper was done in SETEA at CE-
tential competition with the chocolate market, evidence from
PLAC/CEPEC in Itabuna and RFS thanks her colleagues for their consistent
other commodities strongly suggests that the total market will support and help over many years, in particular the excellent technical staff who
increase. did much of the work. The authors thank CNPq, CAPES and the EC (INCO-DC
Relatively little attention has been paid to the microbiology IC18 CT97 0182) for financial support.
of cocoa fermentation over the last 20 years and yet it remains at
the heart of the question of quality. The clearest demonstration
that the basic physiology and ecology of cocoa fermentation is
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 MICROBIOLOGY OF COCOA FERMENTATION 221

Appendix: Revised names of genera and species

Former name Current name

Yeasts
Brettanomyces clausenii Dekkera anomala
Candida guilliermondii Candida guilliermondii var. guilliermondii and
Candida guilliermondii var. membranifaciens
Candida krusei Issatchenkia orientalis
Candida norvegensis Pichia norvegensis
Candida pelliculosa Pichia anomala
Candida reukaufii Metschnikowia reukaufii
Candida. Cacoai Pichia farinosa
Kloeckera apiculata Hanseniaspora uvarum
Kloeckera apis Hanseniaspora guilliermondii
Kluyveromyces fragilis Kluyveromyces marxianus
Saccharomyces chevalieri Saccharomyces cerevisiae
Saccharomyces fragilis Kluyveromyces marxianus
Schizosaccharomyces malidevorans Schizosaccharomyces pombe
Torulopsis candida Candida saitoana
Torulopsis castelli Candida castelli
Torulopsis holmii Saccharomyces exiguus
Bacteria
Gluconobacter oxydans subsp. suboxydans Gluconobacter oxydans
Lactobacillus acidophilus Thiobacillus acidophilus
Lactobacillus casei pseudoplantarum Lactobacillus paracasei subsp paracasei
Lactobacillus cellobiosus Lactobacillus fermentum
Lactobacillus kandleri Weissella kandleri
Lactobacillus plantarum Lactococcus plantarum