INTRODUCTION
The Latin name for cocoa, Theobroma Cocao,
translates as “food for the gods”. The plant has its origin
in the upper Amazon, where archeologists discovered
theobromine alkaloid from the oldest organic cocoa
matter in history at the Santa Ana archaeological site
in La Florida, in the province of Zamora Chinchipe,
Ecuador. From this place, cocoa beans spread
throughout the rest of the continent. On the Yucatan
Peninsula, fragments of once-vast cocoa plantations
have been found on the territories that were occupied by
the Mayan civilization. The traces of cultivated cocoa
were also discovered in Central America on the territory
of the modern Mexico. Currently, cocoa is cultivated
in many tropical countries of the world. It grows in the
area between 20 degrees latitude north and south of the
equator [1, 2].
The cocoa beans come from the Theobroma cacao
tree. They grow in a pod that contains 30–40 beans
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wrapped in a jelly-like mucilaginous substance. Raw
cocoa has an unpleasant astringent taste, which means
that its volatile compounds have to be generated
artificially. During treatment, microorganisms modify
their state and components through various processes,
e.g. fermentation. After fermentation, beans are dried
and exposed to the sun. Only then do the typical sensory
properties develop, and the beans acquire the pleasant
characteristics we associate with chocolate [3, 4].
Fermentation is considered the most critical step
in the processing of cocoa, since the beans are the
main raw material in chocolate industry. It is at the
fermentation stage that they develop their sensory
properties. These properties come from aroma
precursors generated during chemical changes in
the phenolic content [5]. Fermentation occurs in the
pulp of the pod. The pulp is a white carbohydrate-rich
mucilaginous mass that surrounds and protects the
beans. The process lasts several days and depends on
several groups of microorganisms [6].
The microbial activity during cocoa fermentation
has complex biochemical implications. In fact, cocoa is
one of few foods where so many changes and processes
occur during the same process. It includes the successive
growth of several species of yeasts, lactic acid bacteria,
acetic acid bacteria, and, to greater or lesser extent,
species of Bacillus and filamentous fungi [7]. Yeasts
are active at the earliest stage of fermentation. The most
common strains include Saccharomyces spp, Candidda
spp, and Pichia spp. The yeast stage is followed by lactic
acid bacteria, which are represented by Lactobacillus
plantarum and Lactobacillus fermentum. Acid bacteria
acetics belong to the genus of Acetobacter, Acetobacter
pasteurianus being the most common.
What exactly determines the quality of the cocoa
beans still remains a mystery: groups of microorganisms
or individual species? Most likely, all the strains are
essential for the fermentation process, since new
genera and new species are constantly being discovered
[8, 9]. Therefore, the first objective of this review
was to specify the characteristics and reactions of
microorganisms during fermentation. The second
objective was to formulate some recommendations on
improving fermentation conditions or maintaining the
optimal ones to achieve the best transformation in the
chemical compounds.
STUDY OBJECTS AND METHODS
The bibliographic review was conducted according
to three databases, namely Science Direct, Scopus,
and Medline. The descriptors included the following
key words: cocoa fermentation, microbiology of cocoa
fermentation, and phenolic compounds in cocoa
fermentation. The articles were in English or Spanish
and indexed in high impact journals. They were
selected according to their relevance in terms of cocoa
fermentation. Of 350 initially selected articles, 149 were
excluded as irrelevant, and 101 did not fit the language
criterion. Out of 100 English and Spanish articles, only
50 were selected as corresponding with all the specified
requirements.
RESULTS AND DISCUSSION
Cocoa fermentation. Cocoa fermentation is a
post-harvest process which includes several stages.
The first step after cultivation is to open the pods and
remove the beans. They are covered with white pulp, or
mucilage, which is mainly sugars and water [10]. The
initial pH of the pulp is 3.6. It is a nutrient-rich medium
that encourages microbial growth. The pulp has the
following composition: about 85% of water, 10–15%
of sugars (the concentration of glucose, fructose, and
sucrose depending on the age and maturation), 2–3%
of pentoses, 1–3% of citric acid, and 1.5% of pectin,
proteins, amino acids, vitamins, and minerals. Vitamin
C and potassium are the most common representatives
of vitamins and minerals. They are minor but very
important components [10–12].
The microbiological changes during fermentation
are obvious. First, yeasts ferment pulp carbohydrates
and transform them into ethanol and carbon dioxide.
The secretions of their pectinolytic enzymes generate
anaerobic environment. The yeast stage takes
approximately 36 h. The next stage involves lactic
acid bacteria that appear between 16 and 48 h of
fermentation. They generate lactic and citric acid,
increase the acidity of the medium, and change the
composition of the pulp.
As fermentation continues, oxygen begins to come
in. As a result, the temperature rises above 37°C,
which boosts the growth of acetic acid bacteria. Their
population reaches its peak in 88 h. Between 48 and
112 h of fermentation, one can even feel the smell
emanating from acetic acid. After alcohol and lactic
acid turned to acetic acid, the temperature rises up to
50°C. The heat finally inhibits the microorganisms that
have a life span of 120 h. After fermentation, several
filamentous fungi have been registered in the surface
areas and in the excess fermentation mass [13–16].
Basically, the fermentation of cocoa beans begins
with the initial acidity of the mucilage and the low levels
of oxygen, which are the optimal conditions for yeasts.
As these factors decrease, the lactic acid bacteria reach
their maximum growth point. As their amount gradually
decreases, it is replaced by acetic bacteria, which
prefer ethanol, good aeration, and heat. Aerobic sporeforming
bacteria and filamentous fungi often appear at
the final stage of fermentation. They are responsible for
unpleasant flavors of fermented cocoa beans [17, 18].
On the other hand, prolonged fermentation leads to an
increase in bacilli and filamentous fungi, which can also
cause unpleasant flavors. The physiological functions of
the predominant microorganisms have been the subject
of countless studies, which established the crucial
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role of microbial succession in the development of the
characteristic cocoa aroma [16].
The sensory properties of cocoa beans can be
developed by basic conditions or by external factors,
especially those connected with fermentation. The cocoa
flavor increases as the fermentation time elapses, which
means a negative correlation with astringency. In other
words, the astringency of the beans decreases during
fermentation [19].
Stages and changes of cocoa beans. Fermentation
of raw cocoa beans occurs in two stages, which, in
turn, are divided into four steps. The first stage involves
microbial reactions that take place in the pulp and on the
surface of the beans. The second phase involves several
hydrolytic reactions that occur within cotyledons [20].
The system formed to ferment the mucilage that
covers the cocoa beans is metabolized by a succession
of microorganisms. When cocoa beans are harvested
and extracted from the pod, they are exposed to natural
biodiverse microflora that comes from the contact
with environment, crop handling personnel, transport
containers, tools, pod surfaces, etc. [21, 22].
Reactions initiated during fermentation continue at
the drying and roasting stage. Thus, oxidation reactions
reduce acidity and the amount of phenolic compounds
responsible for bitterness and astringency. Cocoa beans
can be dried in the sun or in special dryers. However,
the latter method often implies extra high temperatures,
which can harden the cotyledons and decrease the
quality of the finished product. The quality of cocoa
beans directly depends on the genotype, harvest,
fermentation, drying, and roasting. For instance, beans
of different cocoa genotypes should not be fermented
together, as it can spoil their sensory properties [23, 24].
Microorganisms present in cocoa fermentation.
Traditionally, cocoa fermentation is an uncontrolled
process initiated by microorganisms that naturally
appear in fermentation sites. These fermenting
organisms use pulp as the main substrate. At the onset
of fermentation, pulp reduces the diffusion of oxygen
within the mass of the fermented bean, thus creating
anaerobic conditions [25]. As it was already mentioned,
there are five main groups of microorganisms that
participate in cocoa fermentation: yeasts, lactic acid
bacteria, acetic acid bacteria, and various species
of bacilli and fungi. Unlike other fermented raw
materials, endogenous enzymes play a crucial role in
the development of the flavor of cocoa beans: without
fermentation, cocoa beans have no flavor. During
fermentation, microorganisms eliminate pulp and
produce indispensable metabolites [26, 27].
Yeasts. Yeasts are eukaryotic microorganisms with a
high biotechnological potential for food industry. Their
properties are completely different from prokaryotic
bacteria. Yeasts are resistant to antibiotics, sulfa drugs,
and other antibacterial agents. This resistance is genetic,
i.e. natural: it cannot be modified or transmitted to
other microorganisms [28]. Yeast particles are 5×10 μm
in size, which is significantly bigger than the size of
bacteria (0.5×5 μm) [29].
The yeast species that have been identified as
the main colonizers during cocoa fermentation are
the Saccharomyces cerevisiae, Candida pelliculosa,
Candida tropicalis, Candida zeylanoides, Torulopsis
candida, Torulopsis castelli, Torulopsis holmii,
Kloeckera apiculata, Kloeckera apis, Schizosaccharomyces,
Kluyveromyces marxianus, Pichia
membranifaciens, Pichia kudriavzevii, and Pichia
membranaefaciens. The Saccharomyces cerevisiae is the
most common strain reported in all cocoa plantations.
The exact reason why a certain strain of yeast enters
fermentation process still remains unknown. However,
the Kloeckera apiculata does not survive 24 h of
fermentation as it is inhibited by the concentration
of ethanol produced in the medium. As for the
Kluyveromyces marxianus, it grows slowly and degrades
gradually. Yeasts are active for approximately 48 h and
reach the peak of their activity in 24 h. By that time,
their activity has changed conditions of the medium, and
other microorganisms join in [16, 30, 31].
Yeasts play an important role in the pulp degradation
process. Cocoa pulp can be fermented to produce an
alcoholic beverage. Yeasts demonstrate pectinolytic
activity. The secondary products of yeast metabolism
involve organic acids, aldehydes, ketones, higher
alcohols, and esters. The production of glycosidases
enzymes is important and affects the quality of beans
and, subsequently, that of chocolate [32].
Lactic acid bacteria. Lactic acid bacteria comprise
a group of microorganisms linked by the formation of
lactic acid as the main metabolite. They are a product of
carbohydrate fermentation. Depending on the amount of
this product, they can be homo- or heterofermentative.
They share similar morphological, physiological,
and metabolic characteristics. They are Grampositive,
catalase and oxidase negative, not mobile,
and they do not form spores. They can be anaerobic,
microaerophilic, and airborne [33].
Lactic acid bacteria can appear at the onset of
fermentation. However, they increase their number
and become active only when the pulp with its sugars
begins to hydrolyze and leave the fermentative system,
which is boosts yeast metabolism. The main species
that have been isolated so far include Lactobacillus
plantarum, Lactobacillus fermentum, Lactobacillus
cellobiosus, Leuconostoc mesenteroides, Lactococcus
(Streptococcus) lactis, Pediococcus spp, and various
species of Bacillus.
As for heat-resistant flora, Lactobacillus curieae,
Enterococcus faecium, Fructobacillus pseudoficulneus,
Lactobacillus casei, Weissella paramesenteroides
and Weissella cibaria have also been registered,
but to a lesser extent. They exist during the first
72 h of fermentation and reach their peak in 36 h.
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Their maximum growth period is 16–48 h. The
prevailing species include Lactobacillus plantarum
and Lactobacillus fermentum. Lactic acid bacteria
mostly produce lactic acid, but they also generate small
amounts of alcohol and acetic acid from fructose and
glucose. In addition, they can use citric acid to produce
acetaldehyde, diacetyl, mannitol, acetic acid, and lactic
acid [34–36].
Acetic acid bacteria. Acetic bacteria are Gramnegative
and belong to the Acetobacteraceae family.
They are strict aerobics, non-spore-forming, ellipsoidal
or bacillus-shaped. They may occur in pairs or in chains.
Members of the Acetobacter genus are so common due
to their ability to grow in ethanol environment. Acetic
bacteria are known to partially oxidize a variety of
carbohydrates and to release various metabolites, e.g.
aldehydes, ketones, and organic acids, in different media.
For a long time, they have been used to perform specific
oxidation reactions via processes called “oxidative
fermentations” [37]. The first step in the production
of acetic acid is the conversion of ethanol from a
carbohydrate by yeasts. The second step is the oxidation
of ethanol to acetic acid by acetic acid bacteria [38].
During cocoa fermentation, the population of yeasts
and lactic acid bacteria decays, thus creating an aerobic
environment favorable for the growth of acetic bacteria.
The temperature reaches approximately 37°C. The
increase in temperature triggers protein hydrolysis and
acidification of the beans. As a result, ethanol dissolves
into acetic acid, carbon dioxide, and water. Some
strains appear at 24 h and reach their growth peak at
88 h. After 120 h, they can no longer be detected. They
begin to disappear when the mass reaches 50°C. Part of
the generated acid volatilizes, while the rest enters the
bean and is responsible for killing the germ [39]. These
bacteria play a fundamental role in the generation of
volatile compounds that affect the quality of chocolate.
The Acetobacter and Gluconobacter geni are usually
observed during fermentation, the most common being
Acetobacter aceti and Acetobacter pasteurianus, as
well as the recently discovered Acetobacter ascendens,
A. rancens, A. xylinum, A. lovaniensis, A. xylinum,
A. peroxydans, and Gluconobacter oxydans [16, 40, 41].
Fermentation stages.
Stage I. During the first stage of fermentation, the
volume of the pulp that surrounds the beans reduces
the diffusion of oxygen within the medium. This
is where the beans will be fermented in anaerobic
conditions. During this stage, first yeasts and then
lactic bacteria consume sugars and organic acids
from the pulp, thus producing ethanol, lactic acid,
etc. [25].
The yeast population starts with 107 CFU/g pulp
and reaches a maximum of 108 CFU/g pulp. After that,
it starts to decline until it reaches the bottom level
of 10 cells per gram of pulp. Yeasts are prevailing
microorganisms, and their depectinization activity
causes liquefaction of the pulp with its subsequent
drainage, or “sweating”. The pulp loses its viscosity
and lets in air [42]. As a result, the simple sugars of the
mucilage, namely sucrose, fructose, and glucose, turn
into ethanol. The pectin degrades, causing the texture of
the bean to change, and eliminates citric acid. The yeasts
which are generally responsible for metabolizing this
acid are Candida spp. and Pichia spp., which generate
an alkaline pH. This parameter, together with alcohol
and oxygen, coincidentally inhibits the yeasts and their
activity, but contributes to the development of lactic
bacteria. Yeasts also form such organic acids as acetic,
oxalic, phosphoric, and malic acids. They help reduce
pH fluctuations [43, 44].
Yeasts have become focus of numerous cocoa bean
fermentation studies since they release pulp degradation
enzymes. Moreover, they are also the main producers of
esters and higher alcohols, which can contribute to the
complex mix of aromatic volatile compounds that make
up the cocoa aroma. The main yeasts that generate these
volatile compounds are Candida sp., Kluyveromyces
marxianus, Kloeckera apiculata, S. cerevisiae, and
S. cerevisiae var. chevalieri [45].
The second phase of this stage involves several
hydrolytic reactions that occur within the cotyledons.
As the fermentation continues and the pulp drains, more
oxygen enters the system, thus creating the optimal
conditions for the growth of lactic bacteria [42]. They
colonize the cocoa mass, degrade the glucose of the pulp
into lactic acid, and assimilate the remaining citric acid.
Several studies on microbial fermentation indicate
that two most prevalent species in this process are
Lactobacillus plantarum and Lactobacillus fermentum.
They also produce acetate esters from acetic acid, which
give different tones to cocoa-based products [31]. Lactic
bacteria can reach a population of 6.4×107 CFU/g pulp.
At first, they increase the acidity by producing citric
acid, but then they lower the pH by releasing products
that are not acidic. Lactic acid bacteria are able to
metabolize malic acid. These bacteria have no major
proteolytic activity and can only ferment two types
of amino acids: serine and arginine. After all these
reactions, the environment is totally aerobic, which
allows for the growth of acetic bacteria [7, 34, 46].
Stage II. During the second stage, the environment
is oxygenated, and the pH has decreased due to the
removal of some components and variability of the
remaining compounds. At last, acetic acid bacteria can
convert the previously obtained ethanol into acetic acid
via the oxidation of alcohol. The optimal temperature
of the acetic fermentation process is between 28°C
and 30°C, and the optimum pH is 4.5. The oxidation
of ethanol is carried out in two stages. First, ethanol is
oxidized into acetaldehyde. Second, the acetaldehyde
becomes acetic acid. Other products include ethyl
acetate, butanol, isopropanol, intermediate acetaldehyde
compounds, and organic acids [47].
The formation of acetic acid is very important at this
stage of the process. It occurs due to the activity of acetic
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bacteria. The exothermic reactions of the bacteria raise
the temperature of the mass. The population reaches its
peak at 1.2×107 CFU/g pulp and falls down after three
days of activity precisely because of the high temperature
it generates. In some cases, the population can reach
3.5×103 CFU/g pulp [16, 48].
As the volume of oxygen increases, the pH reaches
3.5–5.0, and the temperature becomes 45–50°C. Under
these conditions, several aerobic spores of Bacillus
bacteria may appear in the fermentation. After the pile
of beans has been stirred, one can detect the presence
of Bacillus licheniformis, B. megaterium, B. pumilus,
B. pumilus. B. coagulans, B. circulans. B. subtilis.
B. cereus, and B. megaterium. Most of them are heattolerant
and can survive during drying and roasting.
They are capable of producing numerous enzymes,
both proteolytic and lipolytic, which catalyze reactions.
However, they give cocoa unpleasant taste and smell
since they degrade proteins and fats by producing
chemical substances that can distort the flavor [49, 50].
CONCLUSION
The fermentation stage is considered the most
important process in the transformation of cocoa to
chocolate. The changes that occur in its volatile and
aromatic compounds trigger structural changes in the
composition. The changes are due to the activity of
various microorganisms. Their main objective is to kill
the germ and thus stop the metabolism of the bean. The
resulting alcohol is broken down into acetic acid and
other acids, which produce desirable sensory properties
that will be accentuated during drying and roasting.
Yeasts, lactic bacteria, and acetic bacteria play
a fundamental role in the fermentation process. By
knowing the main strains, their action parameters, main
reactions, and products, food scientists can improve this
process to obtain cocoa beans with a better chemical
composition.
Each strain of microorganisms requires a separate
research with regard to the variety of cocoa beans.
The species vary from zone to zone, and plantations in
different parts of the world are unlikely to have similar
characteristics.
CONTRIBUTION
Roberto Ordoñez-Araque and Julio Urresto-Villegas
compiled the manuscript. Edgar Landines-Vera and
Carla Caicedo-Jaramillo collected the data, checked the
structure, and performed the final review.
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interest related to the publication of this article.
ACKNOWLEDGEMENTS
The present research is our homage to the indigenous
peoples of Ecuador, who started growing cocoa
5.000 years ago and introduced this delicious treat to the
world.

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