Cacao Proceso1

Food Evolution: The Impact of Society and Science
on the Fermentation of Cocoa Beans

Gulustan Ozturk and Glenn M. Young
Abstract: Cocoa is part of the cultural heritage in many areas of South and Central America and has played an
important role in the history of human culture there. The modern methods of cocoa bean production for the purpose of
the manufacture of modern chocolate are tied to the origin and development of cocoa bean fermentation and processing
methods and the science of microbiology. To date, however, there has not been a study that discusses the impacts of
both science and culture on the evolution of cocoa beans and cocoa bean processing. This work provides both a detailed
overview of the evolution and historical development of cocoa, from its earliest forms to modern chocolate manufacturing,
an in-depth discussion of the biochemistry of cocoa bean fermentation, as well as a compilation of primary research studies
with details on fermentation methods, the scientific bases of interactions in microbial fermentations, and methods for
their investigation, as well as metabolites that are produced. As a result, we present here the major microorganisms among
all the ones that have been identified in previous studies. This database will aid researchers seeking standardized inoculants
to drive cocoa bean fermentation, as well as serve as a guide for inventorying and assessing other food evolution–related
studies regarding traditional and artisanal-based food systems.
Keywords: chocolate, cocoa bean fermentation, food, science, society
Introduction As a building block for future work, our review is comprised of 2

Cocoa beans, unlike other fermented foods, require fermen- major parts. Part 1 considers evolution and historical development
tation to develop essential color and flavor to make them con- of cocoa from its earliest forms as an alcoholic beverage, the more
sumable (Martelli and Dittmar 1961; Ostovar and Keeney 1973; recent nonalcoholic beverage that was introduced to Europe after
Wood 1984; Schwan and Wheals 2004; Afoakwa and others 2008). the Spanish conquest of the New World, and, finally, tracing its
Fermentation is one of the oldest food-processing technologies, ultimate development into modern chocolate products.
predating human history, and it plays an important role by im- Cocoa beans for the manufacture of chocolate are one of the
proving the shelf-life and safety of various foods (Van Hijum and only remaining fermented products in which the microorganisms
others 2013). Cocoa bean fermentation is one of the few remain- that drive the fermentation come naturally from the environment
ing spontaneous microbial processes that occur at the farm through rather than being inoculated with a starter cocktail. This adherence
traditional practices, and subsequent processing happens at other to tradition is because cocoa bean growing and processing (that
facilities downstream in the food chain (Schwan and Wheals 2004; is, fermentation and drying) are performed at the level of individ-
Schwan and others 2015). The importance of cocoa bean fermen- ual farms, which is the result of the unique history/evolution of
tation for chocolate quality has been recognized for over 100 y and cocoa bean production and the subsequent chocolate manufactur-
has been reviewed by others from various different perspectives, ing industry and a lack of a clear demonstration that fermentation
such as chemistry and microbiology of fermentation (Schwan and is both important to final product value and controllable through
Wheals 2004; Afoakwa and others 2008; Lima and others 2011). explicit intervention.
Recently, Schwan and Fleet (2015) published a book with the Because of these factors underlying cocoa fermentation prac-
experts of this field about cocoa processing. To date, however, tices, there is a diversity and variety in the microbiota that drive
there has not been a study that discusses the impacts of both sci- the fermentation process, as well as the risk of inconsistent quality.
ence and culture on the evolution of cocoa beans and cocoa bean Fermentation is thought to be important because the chemical
processing. precursors from “bad” to “good” chocolate flavor are developed
at this stage, and it is therefore important to understand these
complex processes, how the microbial interactions are important
in the development of flavor precursors that determine the overall
CRF3-2017-0009 Submitted 1/12/2017, Accepted 3/7/2017. Authors are with quality of the final product.
Dept. of Food Science and Technology, Univ. of California, Davis, CA, U.S.A. Direct
inquiries to author Ozturk (E-mail: gozturk@ucdavis.edu).
In Part 2, we explore the ecology of cocoa fermentation.
This is intriguing because of the historical and geographic

C 2017 Institute of Food Technologists®
doi: 10.1111/1541-4337.12264 Vol. 16, 2017 r Comprehensive Reviews in Food Science and Food Safety 431
The impact of society and science on cocoa beans . . .
Figure 1–Schematic diagram of the Mesoamerican timeline. The large orange-shaded regions and the small blue-shaded regions represent the older
cocoa-based drink and chocolate, respectively (Adjusted from according to Pohl 2003).
expansion of cocoa production from its biological origin. Our Gulf Coast area, Pre-Olmec people were consuming cocoa-based
goal was to explore common themes and differences in the mi- beverages by 1750 B.C.E. (Powis and others 2007). Later, the
crobial communities that ultimately provide essential chemical Olmec civilization, centered around San Loranzo, shows evidence
changes leading to good chocolate flavors regardless of the origin of of the consumption of cocoa-based beverages between 1800 and
production. 400 B.C.E. (Powis and others 2007, 2011). The Olmec culture
spread cocoa among the indigenous and traditional communities
Origin and Early Uses of Cocoa throughout Mesoamerica––the word for cocoa itself (kakawa)
Amazonian origins comes from the Olmec language (Coe and Coe 1996; Kaufman
The question of where cocoa originated is complex. All wild and Justeson 2007). The residues of cocoa beverages on serving
relatives of domesticated cocoa trees are native to South America, vessels suggest that the Mayans were consuming cocoa-based
the Oronico and Amazon River basins (Motamayor and oth- beverages by at least 600 B.C.E. (Hurst and others 2002; Powis
ers 2002; Hurst and McGovern 2007; Henderson and others and others 2002). Mayan paintings dating from 700 A.D. provide
2007). Other studies, however, contended that the origin of the strong evidence that the peoples of this region already had
cocoa bean was in Central America (Schwan and Wheals 2004). switched to consuming cocoa drinks made from the roasted
Scholars in the field of genetics investigating the origins of and ground cocoa beans, with these beverages being consumed
cocoa beans have suggested that the origins of wild cocoa trees are especially by the elite, as well as being used as a key element
found to be at multiple locations, probably within the Oronico and in ceremonies (Vail 2009; Powis and others 2011). Cocoa beans
Amazon River basins (present-day Colombia and Venezuela) prior were used for everyday exchange and as a form of currency
to 1900 B.C.E. (Motamayor and others 2002). Domestications (Million 1955; Christopher 2013). In addition, beans were used
would have occurred in these areas subsequently, where they then to pay taxes in post-Classic times (900 to 1590 A.D; Million 1955;
spread throughout the region, most likely through trade (Hurst Rosemary and Henderson 2010). Cocoa was a part of the Mayan
and McGovern 2007; Young 2007; Vail 2009; Clement and oth- creation myth (Dillinger and others 2000; Vail 2009) and a Mayan’s
ers 2010; Chaves-López and others 2014). What is not known, relationship with cocoa started at birth, with a naming ritual in
however, is the earliest domestication of cocoa trees in South which the priest and mother presented cocoa to the newly-named
America. baby (Vail 2006). After death, an individual was buried with
Genetic evidence also clarifies the larger dispersal of cocoa personalized cocoa cups (Seawright 2012). From the 14th to the
beans, showing that cocoa beans gradually spread throughout the 16th centuries, as the Aztecs (Nahua people) dominated large
Amazon Valley and were introduced into Central America and parts of Mesoamerica, cocoa became, consequently, integral to all
Mexico by humans (Motamayor and others 2002). In the Amazo- of their social and ritual occasions. Because the climate was not
nian region, it was common to use the fruits of the cocoa bean for conducive to the cultivation of cocoa trees, the Aztecs traded co-
their sweet pulp to produce alcoholic beverages (Henderson and coa beans with the Mayas. There is also an evidence for cocoa use
others 2007). This product was probably the form in which cocoa north of the Mexican border in a time frame of about A.D. 1000
from Amazonian Ecuador reached Mesoamerica (Brown 2009; to 1125. This indicates exchange with cocoa in Mesoamerica and
Vail 2009; Clement and others 2010; Rosemary and Henderson cylinder jars and cocoa beverages suggest that the Chacoan ritual
2010). involved the drinking of cocoa (Crown and Hurst 2009). In the
16th century, the Spaniard Cortez was exposed to cocoa beans
Spread to mesoamerica upon his arrival in Mexico, and he brought the seeds to Europe.
The Mesoamerican phase of the cocoa story starts along the Europeans used ground cocoa beans as a drink like the Aztecs,
Pacific coast, with the earliest evidence of cocoa consumption and cocoa became a popular drink in Spanish courts and soon
among the Mokaya, one of the earliest Mesoamerican cultural spread throughout Europe (Wood 1984; Verna 2013).
groups, from 1900 to 1500 B.C.E. (Powis and others 2007; In the Amazonian region, it was common to use the fruits of
Figure 1). Residues from the preparation of cocoa-based bever- the cocoa bean for their sweet pulp to produce alcoholic beverages
ages in ceramic vessels have been found in the present-day state of (Henderson and others 2007). These beverages may have been the
Pasa de la Amada on the Pacific Coast of Chiapas, Mexico. In the product form people consumed that provided the incentive for
432 Comprehensive Reviews in Food Science and Food Safety r Vol. 16, 2017
cocoa distribution from the Amazonian region to Mesoamerica The Cultivation of Cocoa
(Brown 2009; Vail 2009; Clement and others 2010; Rosemary and Cocoa is a native to South American tropical regions (Mota-
Henderson 2010). However, one cannot rule out other uses of co- mayor and others 2002; Hurst and McGovern 2007). The tree
coa that may have also contributed to its spread to Mesoamerica. Theobroma cacao L. is the only species among the 22 Theobroma
Evidence in support of the beverage theory derives from the dis- species to produce the beans used in chocolate production (Wood
covery of theobromine, a compound unique to cocoa among 1984). Theobroma cacao L. is a diploid perennial tree, ranging from
Mesoamerican plants, in the residue inside spouted drinking ves- 8 to 15 m in height (Wood 1984; Fowler 1999). Although there
sels that the indigenous inhabitants of Mesoamerica used about is not much diversity, diploids often are allowed to be developed
1900 B.C.E. (Hall and others 1990; Hurst and others 2002; as part of breeding programs to get new varieties. Traditionally,
Powis and others 2007). This alcoholic beverage was replaced (by there are 2 main cultivars, Forastero and Criollo, based on the fla-
700 A.D.) by nonalcoholic, frothed cocoa made up of ground co- vor/sensory attributes of the cocoa beans and geographical ori-
coa beans, water, and chili. We speculate that if the beans and pulp gins (Morris 1882; Wood 1984; Cheesman 1944). However, a
were separated, the beans were then subsequently roasted to extend new genetics-based classification of cocoa beans has been pro-
the beans’ shelf-life. It is likely that the cocoa beans were roasted posed (Motamayor and others 2008), one that includes 10 groups:
initially for preservation, but later for the resulting unique aroma. Amelonado, Contamana, Criollo, Curaray, Guiana, Iquitos, Maraňón,
Roasting thus allows the beans to keep longer and gain proper- Nanay, Nacional, and Purús. The names of these groups are de-
ties that become more valuable. This change in product form is termined according to the geographical location (South America:
marked by a completely new serving vessel technology developed Fitzcarrald, Marañon, Serra do Moa, Iquitos, Vaupés, Carauari,
to support the new frothed cocoa (Henderson and others 2007). Purús, Monte Alegre, Gurupa, and Upper and Middle Solimðes)
Linguistic evidence suggests that cocoa had become familiar and or traditional cultivar most represented in that specific cluster
important to highland peoples of Mexico by around 1350 B.C.E. (Amelonado, Criollo, and Nacional; Motamayor and others 2008).
(Coe and Coe 2007; Brown 2009). This new classification system more accurately represents the ge-
netic diversity available to breeders (Motamayor and others 2013).
The Invention of Chocolate Although it is difficult to determine the timeline and spread
Even after this Mesoamerican innovation became a food of of cocoa trees to Mesoamerica, it is easy to identify particular
interest to Europeans in the 17th century, it was still 200 y before cultivars through genetic analysis. The type of cultivar that de-
the advent of the modern chocolate industry, when cocoa beans veloped through cultivation by local people in Mesoamerica was
were processed into cocoa powder and cocoa butter was used to Criollo (Motamayor and others 2002). The high demand and pop-
make chocolate candy and eventually chocolate bars. ularity of cocoa beans during the Spanish colonial period led to
From the time “chocolate” was introduced to Europe in the the spread of the cocoa tree rootstocks around the world, aided
17th century via the Spanish court until the 19th century, the by the establishment and spread of European colonies around the
cocoa beverage remained a spicy-bitter drink (Wood 1984; Coe globe. Cocoa cultivation spread to parts of South America and the
and Coe 2007; Verna 2013). Later, Europeans learned to use sugar Caribbean islands. The cultivation of cocoa was started in the 16th
(Old World) and vanilla (another import from the New World) century in Venezuela, and around the year 1600, Criollo cocoa was
to create a “softened” cocoa version, and soon this version of taken across the Pacific to the Philippines. The Criollo type was
“chocolate” spread throughout Europe (Wood 1984; McNeil introduced to Trinidad in 1678 from Venezuela. The majority of
2006; Verna 2013). By the mid-1600s, chocolate had gained cocoa bean production in the 16th and 17th centuries was Criollo
widespread popularity among the elite in France. The 1st hot cocoa, and during the 18th century Forastero, what was called
chocolate shop was opened in London in 1657, and by the 1700s the Amelonado type until 1950, began to be grown and used for
these chocolate houses were very common in England. In the manufacture (Wood 1984). Beginning in the 17th century, the
18th century, with the invention of the steam engine, cocoa beans Nacional type was planted in Ecuador (Wood 1984). During the
were ground by machine. In 1828, the Dutch chemist Coenraad 18th century, the Lower Amazonian Forastero cultivar was intro-
Van Houten invented the cocoa press and separated the cocoa duced to Trinidad, where the already established Criollo trees and
powder from the cocoa butter. This separation of the cocoa butter newly introduced Forastero trees gave rise to a new hybrid, called
allowed the making of modern chocolate. In 1847, the 1st com- Trinitario, that is both disease-resistant and productive (Cheesman
mercially available solid chocolate bar made from cocoa beans, 1944; Bartley 2005). Most of the cocoa trees introduced into
sugar, and cocoa butter was produced by Joseph Fry. In 1875, Africa and Asia originated from Criollo, Amelonado (Forastero), and
the 1st milk chocolate was made in Switzerland by Daniel Pe- Trinitario cultivars (Wood 1984; Motamayor and others 2013). In
ter with condensed milk powder obtained from Henry Nestle. In the early 19h century, the Forastero variety was introduced to West
1879, Rudolphe Lindt invented a process called conching: mixing and Central Africa (Wood 1984; Aikpokpodion 2012). Today,
cocoa with milk, vanilla, and extra cocoa butter for 12 to 48 h Forastero and its varietal crosses are the most commonly grown and
at a controlled temperature for the smoothest form of chocolate used beans. These beans make up about 95% of the world’s pro-
possible (Wood 1984; Verna 2013). This process was adopted as a duction and are grown primarily in West Africa. They make up
standard part of the chocolate-making process that marks the birth most of the “bulk/ordinary” beans grown (ICCO 2014a). Cocoa
of chocolate as we know it today. is now grown in more than 50 countries (ICCO 2014a).
The 20th century saw the proliferation of chocolates, includ-
ing Kisses from Hershey in America, Milky Way from Frank Worldwide Culture of Cocoa
Mars, Godiva Belgian chocolates, Nutella, and Kinder Eggs The major cocoa-producing countries are located in the cocoa
(Verna 2013). A present-day annual production of 4.3 million belt 23.5°N to 23.5°S of the equator and include Côte d’Ivoire,
tons of cocoa beans (in 2014) attests to the fact that people Ghana, Nigeria, Cameroon, Uganda, Togo, Sierra Leone, Mada-
around the world enjoy chocolate in its numerous different forms gascar (Africa); Indonesia, Malaysia, Papua New Guinea, India
(ICCO 2014a). (Asia); and Brazil, Ecuador, Mexico, Peru, Dominican Republic,

C 2017 Institute of Food Technologists® Vol. 16, 2017 r Comprehensive Reviews in Food Science and Food Safety 433
Figure 2–Countries producing cocoa beans in the cocoa belt, located at 23.5°N to 23.5°S of the equator. The red solid line represents the equator, and
the dashed lines in each hemisphere represent the locations of 23.5°S and 23.5°N of the equator. The region between red-dashed lines indicates the
cocoa belt. Each color represents the amount of cocoa production in million tons (MT).
Figure 3–Countries processing the most cocoa beans are outside the cocoa belt. The red solid line represents the equator, the dashed lines in each
hemisphere represent the locations of 23.5°S and 23.5°N of the equator. The region between red-dashed lines indicates the cocoa belt. Most of the
processing is outside the cocoa belt. We are defining processing of cocoa as any procedure that starts with grinding of cocoa beans. Each color
represents the amount of cocoa production in million tons (MT).
Colombia, Venezuela, Guatemala, Haiti, (South America) Africa are imported to the European Union (54%), then North
(Figure 2). The worldwide annual production of cocoa beans America (13%), and Asia (12.4%). Latin America is the 2nd
was approximately 4.3 million tons in 2013/2014 (ICCO 2014a). largest source of cocoa beans for processing regions such as the
Among these regions, the largest producers are Côte d’Ivoire, European Union and North America (ICCO 2014a). Manufac-
Ghana, and Indonesia (ICCO 2014b). Worldwide, there are ap- turers convert cocoa beans into consumable chocolate products
proximately 5 to 6 million cocoa farmers, and 95% of the cocoa (Saltini and others 2013). Most of the beans produced globally are
beans come from small family farms (ICCO 2014c). These farm- processed in countries that are outside the cocoa belt (Schwan and
ers produce the dried fermented beans, which then are sold to Wheals 2004; Lopes and Pires 2014). The countries that process
distributers (Schwan and Wheals 2004; Saltini and others 2013; the most cocoa beans are The Netherlands, Germany, the U.S.A.,
Lopes and Pires 2014). The majority of cocoa beans from West Brazil, Côte d’Ivoire, Ghana, Indonesia, and Malaysia (Figure 3;
Figure 4–Schematic representation of the cocoa value chain from farm to consumer. Cocoa beans go through a variety of processes that start at
harvesting of the fruit at the farm up to the end of industrial processing by food companies.
ICCO 2014a). Among these countries, The Netherlands processes that this fermentation provides precursors that contribute to the
the largest quantity of cocoa beans, amounting to 13% of world- desired characteristics of cocoa beans used for producing chocolate
wide production (ICCO 2014c). Côte d’Ivoire, the top producing (Ostovar and Keeney 1973; Wood 1984; Schwan and Wheals
country is also the 3rd-highest processor of cocoa beans (ICCO 2004).
2014b). The 1st cocoa pods are produced 2 to 3 y after planting. The oval
Fine-flavor cocoa beans are often considered to originate from pods are around 12 to 30 cm in length, and contain 30 to 40 beans
specific regions/cultivars (Wood 1984; Fowler 1994; Amoa-Awua (Figure 5). After the pods are harvested (4 to 7 mo after pollina-
2015), with the designation based on the quality and flavor of tion), they are stored for a number of days before they are opened,
the cocoa beans produced, specifically, the sensory properties that and the beans are removed by hand or machine. Natural fermen-
these cocoa beans might contribute to the final chocolate. How- tation of the mucilaginous pulp that surrounds the fresh raw cocoa
ever, in practice that is not how the industry operates. Rather, it beans is required for the development of the unique aroma and taste
operates through collective agreements requiring that only a cer- of chocolate (Wood 1984; Lopes and Pires 2014). This fermenta-
tain amount of beans be produced and sold with the fine-flavor co- tion process is accompanied by drying. After fermented beans are
coa designation (ICCO 2010, 2015). Because of these production dried, distributors buy them directly from farmers. Cocoa beans
restrictions, other beans that may have the same sensory properties are stored in warehouses until purchased by and transported to food
end up being labeled as bulk/ordinary beans (ICCO 2015). From companies for processing. There, the beans are roasted (via various
the Int. Cocoa Agreement, countries producing the fine/flavor methods and for various lengths of time, depending on the partic-
cocoa are Colombia, Costa Rica, Dominican Republic, Peru, ular needs/specifications of processor/manufacturer) and then fur-
Ecuador, Grenada, Indonesia, Jamaica, Madagascar, Papua New ther processed into cocoa liquor, cocoa butter, and cocoa powder.
Guinea, Saint Lucia, São Tomé & Prı́ncipe, Trinidad and Tobago, Processing continues to chocolate and beyond, with products run-
Bolivia, and Venezuela, and they all export exclusively or par- ning the gamut from chocolate to pates, powders, cakes, soaps, and
tially fine/flavor cocoa (ICCO 2010). Compared to the inter- cosmetics.
national market for bulk cocoa, fine/flavor cocoa production is
estimated to be less than 5% of the world cocoa production
(ICCO 2015).
Cocoa Fermentation
Fermentation is a spontaneous and complex process driven by a
large number of fungi and bacteria within particular growing and
How Cocoa is Processed processing environments (Ostovar and Keeney 1973; Wood 1984;
Cocoa beans go through a variety of processes that begin with Schwan and Wheals 2004; Schwan and others 2015). The pods are
the harvesting of the fruit at the farm and end with the indus- naturally inoculated with these various fungi and bacteria when
trial manufacturing of products by food companies (Figure 4). they are cut and handled during harvest (Ostovar and Keeney
The fermentation of the cocoa beans, which occurs within a di- 1973; Wood 1984; Schwan and Wheals 2004), and they are sub-
versified system of small-scale cocoa farms where the fermen- sequently exposed to these various fungi and bacteria during the
tation is a spontaneous process in the cocoa pulp, driven by fermentation/drying process from a variety of additional sources
the naturally occurring local microbiota. It has been proposed and forces such as wind. The drying/fermenting beans also come

Figure 5–Traditional process of fermenting cocoa beans in Vietnam. These pictures provide an overview of the primary operations used to produce
fermented cocoa beans. (A) Cocoa tree in Vietnam. (B) Harvested cocoa pods. (C) Pod breaking. (D) Broken pod and cocoa beans covered with pulp. (E)
Removing of cocoa beans covered with pulp by hand. (F) Mixing the beans and pulp. (G) Beans covered with mucilaginous pulp. (H) Box fermentation.
(I) Basket fermentation. (J) Late stage of fermentation. (K) Beans are covered with banana leaves. (L) Drying process. (M) Dried cocoa beans. (N), (O),
Fermented and dried cocoa beans are bagged for distribution. (P) Cocoa beans in storage.
into contact with a variety of inoculating surfaces with their own Flavor and Aroma Compounds in Cocoa Beans
varied local microorganisms. These inocula include tool surfaces Fermentation is required for developing the microbial metabo-
and various containers where the pods are held for fermentation lites essential for chocolate flavor and aroma (Martelli and Dittmar
and drying, the pod surfaces themselves, knives, laborers’ hands, 1961; Ostovar and Keeney 1973; Wood 1984; Thompson and
banana or plantain leaves (which are used to cover the piles of beans others 2007). The uniqueness of chocolate flavor is driven by the
during fermentation), and residual microorganisms from previous genetic constitution of the cocoa variety, although the fermenta-
fermentations. tion process releases and develops this flavor potential (Lopes and
Fermentation proceeds continuously during the stacking of co- Dimick 1995; Afoakwa and others 2008). The beans themselves
coa beans in heaps (Ghana, Ivory Coast), trays (Ghana, Ivory consist of 2 cotyledons (nibs) and an embryo covered by a seed
Coast), boxes (Brazil, Malaysia), baskets (Ivory Coast), and on coat (testa), all of which are the source of characteristic flavor and
platforms (Ecuador), with successive rotations of the beans during aroma compounds (Rohan and Connell 1964; Rohan and Stew-
the fermentation duration. The microorganism proliferation varies art 1967; Figure 6). The beans contain approximately 32% to 39%
during the fermentation, and the contribution of each group of water, 30% to 32% fat, 8% to 10% proteins (an albumin and a
microorganisms in the fermentation develops particular character- vicilin-class (7S) globulin), 2 % to 3% cellulose, 4% to 6% starch,
istics in the cocoa beans (Ostovar and Keeney 1973; Schwan and 4% to 6% pentosans, 2% to 3% sucrose, 1% acids (mainly citric,
Wheals 2004). oxalic, and malic acids), 1% to 3% theobromine, and 0.2% to 1%
The duration of fermentation depends on the type of cocoa and caffeine. Early in the fermentation process, pulp sugars, which pro-
the region where it is grown. For instance, Criollo, with a nutty and vide the substrates for the subsequent fermentation, are converted
mild chocolate flavor, needs only a short period of fermentation, to ethanol and lactic acid through the action of yeast and lactic
although Forastero, with a stronger chocolate flavor, requires 5 to 8 acid bacteria; later on ethanol is oxidized to acetic acid by acetic
d in order to achieve full flavor development. Trinitario also requires acid bacteria (Ostovar and Keeney 1973). Changes in pH, rise of
5 to 8 d of fermentation (Wood 1984). the temperature of the stack, and penetration of pulp fermentation
seeds do not produce cocoa flavor on roasting, the development

of cocoa flavor precursors during fermentation is required (Rohan
1964; Voigt and Biehl 1995).
The Study of Fermentation

The flavor and aroma characteristics of chocolate require fer-
mentation, and it is this phase that is the least controlled and the
least understood aspect of the overall cocoa production process.
To begin to understand the complexity of the fermentation pro-
cess and the microbiota involved, we review here what has been
published in the literature. We refer to Table 1 for describing the
overview of the approaches and major findings from these studies
to understand the microbiota of cocoa fermentation.
Over the past decade, as molecular profiling methods have been
Figure 6–Diagram of a cocoa bean. The bean is made up of 3 primary
adopted to better explore the microbial systems involved in co-
components (1) The testa (seed coat) is the outer membrane. (2) Kernel
(cotyledon). (3) Embryo is covered by seed coat. coa bean production, the microbial ecology of the cocoa bean
fermentation processes has received increased attention.
Several studies have been devoted to the microbial diversity of
products into cotyledons ultimately kills the embryo. This process spontaneous cocoa pulp fermentations in different places through-
creates the environmental conditions that induce changes both in out the world, and the results showed that both the initial and final
the structure of the seed at the subcellular level and in the metabo- microbial populations vary by location. Early research was lim-
lites present in the beans. These biochemical reactions result in the ited to microbial isolation and characterization through culture-
production of chocolate flavor precursors such of fructose and glu- based detection methods (Martelli and Dittmar 1961; Ostovar and
cose and peptides and hydrophobic free amino acids, specifically Keeney 1973; Passos and others 1984; Samah and others 1993;
leucine, alanine, phenylalanine, and tyrosine, all released during Ardhana and Fleet 2003; Lagunes and others 2007). These early
fermentation by aspartic proteinase and carboxypeptidase activi- culturing methods have proven to be unreliable for the com-
ties in the fermented beans (Rohan and Stewart 1967; Lopez and plete characterization of microbial ecosystems (for example, new
others 1978; Biehl and Passern 1982; Biehl and others 1985). This techniques have revealed that these older methods have resulted
characteristic flavor and color are further developed during bean in the misidentification of some microorganisms; Giraffa and
drying and the subsequent roasting and conching processes used Neviani 2001; Mayo and others 2014). To overcome this limi-
for chocolate manufacture (Schwan and Wheals 2004; Afoakwa tation, the application of culture-independent analysis methods,
and others 2008; Fowler 1999; Owusu and others 2011). based on DNA or RNA extracted directly from the samples, have
allowed for the detailed monitoring and description of fermenta-
High-Quality Traits of Cocoa Beans tion ecosystems (Mayo and others 2014; Table 1.)
The degree and time course of acidification of the cotyledons, For example, although for bacteria the 16sRNA gene is ampli-
the length of the fermentation process, and the final pH are the fied, the 26sRNA gene is amplified for yeast and mold (Schwan
most important elements affecting the raw cocoa quality (Voigt and and others 2015). The denaturing gradient gel electrophoresis
Biehl 1995; Schwan and Wheals 2004; Afoakwa and others 2008). (DGGE) approach, which provides sequence-specific separation
During the fermentation, metabolites––the ultimate source from of polymerase chain reaction (PCR) amplicons, and pyrosequenc-
fermentation––react with sugars and amino acids to contribute ing analysis have been used to study the microbial composition
to the flavor and color of the cocoa beans and also react with of cocoa beans (Camu and others 2007; Kostinek and others
alkaloids to contribute to the bitterness. Theobromine, caffeine, 2008; Daniel and others 2009; Garcia-Armisen and others 2010;
and polyphenols are the key compounds contributing to the bitter Papalexandratou and De Vuyst 2011a; Papalexandratou and others
taste as well as the astringent mouth-feel after consumption of 2011b). High-throughput DNA sequencing was also recently uti-
roasted cocoa (Stark and others 2005). A complexity of aromatic lized to gain a more comprehensive understanding of the bacterial
terpene and lipid metabolites also contribute greatly to the flavor population of cocoa beans (Illeghems and others 2012; Table 1).
of cocoa. What is interesting about these molecular approaches, compared
There is a strong influence of both the environment and the to the traditional approaches, is that new groups of strains have
genetic origins of cocoa beans on flavor development. For in- been reported. It is clear from the molecular approaches that the
stance, the Criolla cultivar contains additional flavors from the bacterial diversity of cocoa beans in the population is far higher
pulp, because pulp content and composition, as well as the cotyle- than previously realized. Because the fermenting cocoa bean is a
don composition (phenols and alkaloid contents) vary depending very complex ecology, combining community composition, and
upon the different cultivars (Kim and Keeney 1984; Afoakwa and metabolism of microbiota will provide deeper insight into the
others 2008). The essential precursors of the specific aroma com- complexity of cocoa fermentation systems.
ponents are generated during the fermentation process. The flavor The development of metagenomics, based on identification
precursors are further transformed, by Maillard reactions, into without cultivation, is opening new possibilities for the identi-
cocoa flavor compounds during roasting (Quesnel 1965; Rohan fication of previously non-isolated and non-identified microbial
and Stewart 1967; Biehl and others 1985; Voigt and others 1994; species from fermented foods and improving their performance to
Thompson and others 2001; Kratzer and others 2009; Ziegleder achieve more efficient fermentation (Handelsman 2004; Smid and
2009). Roasting takes between 5 and 120 min at a temperature Hugenholtz 2010). Such metagenomic approaches have been used
of 120 to 150 °C , depending on the nature of the beans and the recently to reveal the complexity of cocoa fermentation systems
requirements of the final product. Because unfermented cocoa (Illeghems and others 2012, 2015).

Table 1–An overview of the approaches and major findings from various studies to understand the microbiota of cocoa fermentation. The variety, geographical origin, fermentation type, and processing.
conditions all have an impact on the fermentation of cocoa
Variety Country Fermentation Metabolites Metabolites Cocoa

References method, duration Techniques Major findings Cocoa pulp bean
Forastero Brazil Wooden Standard plating Saccharomyces rosei, Hansenula anomala, Pichia fermentans, Pichia
Martelli and tanks:168ha methods membranaefaciens and Trichosporon cutaneum were identified only
Dittmar 1961 Saccharomyces rosei, Hansenula anomala, Pichia fermentans showed
fermenting capacity on the sugars of cocoa pulp. Saccharomyces species
were responsible for the alcoholic phase of the cocoa fermentation
Forastero B1, B2:168ha Standard plating Major aim is to isolate identify and characterize the microorganisms and
Trinadad Samples from methods their sources before, during, and after cocoa fermentation.
Ostovar and surface of Lactobacillaceae, Bacillaceae, Pseudomonadaceae, Micrococcaceae,
Keeney 1973 various sources Corynebacteriacea, Enterobacteriaceae, Propionibacteriaceae,
Actinomycetaceae, Azotabacteraceae Brevibacteriaceae were identified.
Bacillus cereus was the only common organism from the surfaces of
various sources
Brazil Passos and B:160ha mixed Standard plating Eight homolactic species: Lactobacillus plantarum, Lactobacillus casei,
others 1984 methods Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus lactis,
Pediococcus cerevisiae, Pediococcus acidilactici, Streptococcus lactis. Two
heterolactic species: Leuconostoc mesenteroides and Lactobacillus brevis.
Among the species of LAB, Lactobacillus plantarum was the most
common
Bahia Schwan and B:164ha Standard plating Aerobic spore forming bacteria isolated from traditional cacao
others 1986 methods fermentations in Bahia in the genus Bacillus: B. subtilis, B. licheniformis,
B. firmus, B. coagulans, B. pumilus, B. macerans, B. polymyxa, B.
laterosporus, B. stearothermophilus, B. circulans, B. pasteurii, B.
megaterium, B. brevis, and B. cereus.
Samah and others Ripe cocoa pods Standard plating The population sizes of acetic acid and lactic acid bacteria appeared to be AAM :14 LM :4.5
1993 were stored for methods directly correlated with the levels of acetic and lactic acids, respectively.
7 days after The tartaric, propionic, succinic, citric and oxalic acids were also detected.
harvesting. B, Regularly turned beans provided higher percentage of brown beans
turned (fermented)
daily:144ha
Brazil Schwan and Kluyveromyces Enzyme assays The effects of polysaccharides, sugar, and ammonium sulphate
Rose 1994 marxianus (CCT concentrations on endopolygalacturonase (PG) secretion were
3172) grown investigated. Replacing glucose in the medium with sucrose or varying
under different the concentration of ammonium sulphate in media had no effect on PG
medium secretion. Growth in fructose-containing medium or xylose-containing
medium retarded secretion of PG.
Brazil Schwan and Kluyveromyces Screening for During early cocoa pulp fermentations, Kluyveromyces marxianus is the
others 1996 marxianus (CCT pectinase activity most pectinolytic yeast. Endo-polygalacturonase (PG) is the 85% to 90%
3172) was and of total secreted protein. Purified PG comprises 4 proteins of Mr 45, 42,

isolated from characterization of 39, and 36 kDa.
cocoa polygalacturonase
fermentations (PG)
Brazil Schwan and 12 cocoa pulp Screening for Among 12 yeast strains, only Kluyveromyces marxianus (CCT 3172),
others 1997 degrading yeast pectinase activity Kluyveromyces thermotolerans (CCT 1701), Saccharomyces cerevisiae var.
strains and chevalier (CCT 1698), and Candida rugopelliculosa (CCT 1702) produced
characterization of extracellular, constitutive PG. Kluyveromyces marxianus was the most
polygalacturonase pectinolytic with 85% of total secreted protein consisting of a
(PG) constitutive endopolygalacturonase
(Continued)

Table 1–Continued.

b Brazil Schwan BA: natural ferm. Standard plating This is the1st report of successful use of a defined, mixed starter culture in BA: LAM :1.6,
1998 with wild methods Sensory such a complex fermentation: Saccharomyces cerevisiae var. chevalieri, AAM :5,5 EM :8
microflora; BB: analysis Lactobacillus lactis, Lactobacillus plantarum, Acetobacter aceti and BC: LAM :2,
aseptically Gluconobacter oxydans subsp. suboxydans were used. With the zero-time AAM :6 EM :7.1
prepared beans inoculum the fermentation was almost identical to the natural BD: LAM :1.8,
with no fermentation AAM :5.9 EM :7.5
inoculum; BC:
with the zero
time, defined
cocktail
inoculated BD:

a defined
cocktail at by
using phased
additions at
appropriate
times:168ha
Forastero Estate A: Standard plating The principal species found were Penicillium citrinum, an unidentified Estate A:B Estate A:B
Trinitario Forastero,B1:144ha methods basidiomycete, Kloeckera apis, Saccharomyces cerevisiae, Candida F0-F :62-11,42-9, F0-F :1-0.4,0.8-0.3,
Indonesia Estate B: tropicalis, Lactobacillus cellobiosus, Lactobacillus plantarum and G0-F :41-7,24-5, G0-F :0.7-0.1,0.6-
Ardhana and Trinitario,B2:96ha Acetobacter pasteurianus. The later stages of fermentation were S0-F :32-0,18-0, 0.1, S0-F :19-0,18-0,
others 2003 Estate C: dominated by the presence of Bacillus species, mostly, Bacillus pumilus E0-F :0.5-0.1,0.3- E0-F :0.2-0.4,0.2-1.6,
Trinitario,B3:96ha and Bacillus licheniformis. 1.6, CA0-F :9-4,7.4-3.5,

CA0-F :24-11,21- LA0-F :0.1-2,0.1-1.8
9, AA0-F :1-25,0.7-15
LA0-F :0.3-6,0.3-
5
AA0-F :0.4-
12,0.4-10
Ghanan Jespersen H:72ha T:72ha Amplification of their Heap ferm.:Candida krusei was the dominant species followed by Pichia
and others Swab samples ITS1-5.8S rDNA- membranifaciens, Pichia kluyveri, Hanseniaspora guilliermondii, and
2005 from the various ITS2 regions 26S Trichosporon asahii. Tray ferm.:Saccharomyces cerevisiae and Pichia
surfaces rRNA gene membranifaciens were the dominant species followed by low numbers of
amplicon Candida krusei, Pichia kluyveri, Hanseniaspora guilliermondii. Isolates of
sequencing CLP, Candida krusei, Pichia membranifaciens, Hanseniaspora guilliermondii,
PFGE Trichosporon asahii, and Rhodotorula glutinis could be found on the
surface of the cocoa pods or equipment production equipment
Ghana Nielsen and H:144ha -turning 26S rRNA gene DGGE protocol was compared with culture-based methods. Candida
others 2005 H2:72ha no amplicon zemplinina was only detected using DGGE but Trichosporon asahii
turning T:96ha sequencing (detected using culture-based methods) yielded only faint bands in the
PCR-DGGE denaturing gels. Hanseniaspora guilliermondii, Candida krusei, and
Pichia membranifaciens were detected from most ferm. Saccharomyces
cerevisiae and Candida zemplinina were almost exclusively detected in
tray ferm.
Ghana Nielsen and Traditional ferm. 16S rRNA gene A novel species, Lactobacillus ghanensis is proposed. The type strain is
others 2007a Ghanaian cocoa amplicon L489T ( = DSM 18630T = CCUG 53453T)
beans sequencing
Ghana Nielsen and H1(no 16S rRNA and 26S Hanseniaspora guilliermondii, was dominant during the initial phase, and
others 2007b turning):96a rRNA gene Pichia membranifaciens the later phases of ferm. Lactobacillus
H2(turning):144ha amplicon fermentum was the dominant Lactobacillus plantarum, Pediocococcus
T1,2:96ha sequencing acidilactici Leuconostoc pseudomesenteroides, and Leuconostoc
PCR-DGGE pseudoficulneum were detected. Acetobacter syzygii, Acetobacter
pasteurianus and Acetobacter tropicalis were the predominant. Bacillus
licheniformis and occasionally other Bacillus spp. were detected in high
numbers during later phases of heap ferm.
(Continued)
Vol. 16, 2017 r Comprehensive Reviews in Food Science and Food Safety 439

Trinitario B:144ha Standard plating The most abundant Candida insconspicua, Lactobacillus plantarum, and G0-F :55-2,
Dominican methods Acetobacter lovaniensis S0-F :87-11,
Republic Biochemical CA0-F :24-2.5,
Lagunes and characteristics AA0-M-F :0-22.5-
others 2007 5, LA0-M-F :0-2-0,
E0-M-F :0-9.5-0
Mixed Criollo and H1,2,3:144ha Population dynamics Four main LAB: Lactobacillus plantarum, Lactobacillus fermentum, Representative Representative H5:
Forastero H4,5,6,7:144ha of both LAB and Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other H5: G0-F :1-5, F0-F :0.5-5,
Ghana Camu AAB during taxa,Weissella. Only four AAB: Acetobacter pasteurianus, Acetobacter G0-F :52.5-2.5, S0-F :11.9-0.2 MM :0
and others fermentation 16S syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like F0-F :57-4, LA0-F :0-3.7
2007 rRNA gene bacteria. L. plantarum, L. fermentum, and A. pasteurianus, were S0-F :0-0 MM :14 CA0-F :0-3,
amplicon originated from the environment. A newly proposed Weissella sp., LA0-F :0-8 SA0-F :0.13-0.225
sequencing referred to as “Weissella ghanaensis, Two new species of Acetobacter CA0-F :9.2-2.2, AA0-F :0-6.9 EM :11
PCR-DGGE were found as well, namely,“Acetobacter senegalensis” (A.tropicalis like) SA0-F :0.29-0.1
and “Acetobacter ghanaensis” (A. syzygii-like) AA0-F :0-5.5
EM :22.5
Gnaha Cleenwerck Traditional heap 16S rRNA gene A novel species, Acetobacter ghanensis sp. nov. is proposed. The type strain
and others ferm. of amplicon is R-29337T ( = 430AT = LMG 23848T = DSM 18895T)
2007 Ghanaian cocoa sequencing
beans Phylogenetic
analysis
Gnaha Cleenwerck Traditional heap (GTG)5-PCR A novel species, Acetobacter fabarum sp. nov. is proposed. The type strain is
and others fermentations fingerprinting strain 985(T) ( = R-36330(T) = LMG 24244(T) = DSM 19596(T))
2008 of Ghanaian
cocoa beans
Ghana De Vuyst Ghanaian (GTG)5-PCR Acetobacter pasteurianus (cluster I, 100 isolates), Acetobacter syzygii- or
and others fermented fingerprinting 16S Acetobacter lovaniensis-like (cluster II, 23 isolates), and Acetobacter
2008 cocoa beans rRNA gene tropicalis-like (clusters III and IV containing 4 and 5 isolates, respectively)
amplicon were differentiated. A. syzygii-like and A. tropicalis-like strains reported
sequencing for the first time. Reclassifications: Acetobacter aceti LMG 1531,
DNA:DNA Gluconacetobacter xylinus LMG 1518, and Gluconacetobacter xylinus
hybridization subsp. sucrofermentans LMG 18788T
Ghana Camu and Farm site: 16S rRNA gene Acetobacter pasteurianus, Acetobacter ghanensis, Acetobacter H10:H11:H12:H13
others 2008a H8,H12:turning amplicon senegalensis, and a potential new Acetobacter lovaniensis-like species, H10:H11:H12:H13 AA0-M-F :0-10-4,0-5-
H9,13 no sequencing Lactobacillus plantarum and Lactobacillus fermentum were identified. G0-F :48-1,40- 4,0-6-6,0-7-7,
turning Factory PCR-DGGE Sensory Turning of the heaps increased the production of acetic acid and this turn 10,41-4,49-12, EM :7,11,5.5,11

site: analysis gave a more sour taste to chocolate. During fermentation of cocoa bean F0-F :51-2,50-
H10:turning processing, bitterness was reduced through losses of polyphenols and 9,44-8,51-15,
H11:no turning alkaloids MM :13,20,3,10
AA0-M-F :0-15-
5,0-4-3,0-12-
10,0-11-10,
EM :10,13.5,17,2
(Continued)


Ghana Camu and Farm 16S rRNA gene Differences in microbial activities between different heap fermentations H1:2:3:4:5:6:7 H1:2:3:4:5:6:7
others 2008b A:H1,H3,H4,H6 amplicon can result in different flavor characteristics in dried fermented cocoa CA0-F :6.17- CA0-F :5.32-3.23,
Farm sequencing beans and chocolates. Therefore, fermentation may affect the flavor of 0.12,0.07- 6.50-3.48,3.18-
B:H2,H5,H7 PCR-DGGE chocolate 0.31,6.33- 3.27,
0.18,5.87- 3.98-2.7,5.48-3.3,
0.65,7.98- 4.17-2.78,6-2.02,
1.26,9- E0-F :0.33-
1.47,9.18-1.91, 4.02,0.07-

E0-F :0.25- 8.01,0.23-
5.6,0.03- 1.90,0.74-2.65,0.5-
5.5,0.185.5, 7.09,0.23-
0.18-4.45,0.05- 5.79,0.95-6.57,
8.15, LA0-F :0-0,0.03-
0–6.4,0-3.53, 0.03,
LA0-F :0.25- 0-0,0.63-0.63,0.08-
5.6,0.03- 0.08,0.3-0.3,0.22-
5.5,0.18- 0.22,AA0-F :4.75-
4.45,0.05- 4.75,6.73-6.73,
8.15,0-6.4,0- 11.15-11.15,6.65-
3.53, 6.65,6.42-
AA0-F :0.11- 6.42,9.20-
3.60,0.12- 9.20,5.20-5.20 Dry
7.25,0.18- cb
12.50, CAF :5.80,3.95,4.80,
0.43-9.40,0.12- 4.93,5.53,5.82,7.58
5.95, AAF :4.12,5.07,3.32,
0.07-9.40,0.52- 6.95,3.55,7.87,7.73,
483 LAF :2.77,2.30,3.67,
3.90,3.42,2.45,1.92
GF :1.61,1.46,1.20,
1.13,1.19,2.21,3.09
FF :4.72,3.74,3.92,
4.68,3.40,5.65,6.53,
EF :0.27,0.38,0.20,
0.47,0.49,0.5,0.51
MF :1.05,1.33,0.48,
2.70,0.77,0.53,0.46
Quattara and Bacilli were This study is the first report on Bacillus pectinolytic strains involved in cocoa
others 2008 collected from fermentation. It indicates that Bacillus sp. is able to produce pectinolytic
different ferm. enzymes under cocoa fermentation conditions. So the degradation of the
Tarpaulin, pulp during cocoa fermentation might not be only due to pectinolytic
wooden box enzymes produced by yeasts but rather by a combined action of enzymes
and banana produced by both yeast and Bacillus strains.
leaves:144ha
Nigeria Kostinek T:120ha ,H:144ha (rep)-PCR-based A total of 193 LAB strains were isolated. 40 (20.7%) heterofermentative:
and others typing Lactobacillus brevis or Lactobacillus fermentum. 110 (57%)
2008 homofermentative rods: Lactobacillus plantarum. 35 (18.1%)
homofermentative cocci: Pediococcus acidilactici
(Continued)

b Brazil Leal and Plastic laundry 26S rRNA, nested Inoculation with Kluyveromyces marxianus strain caused changes in yeast
others 2008 baskets:144ha PCR–DGGE population dynamics, leading to an overall improvement of quality
covered with attributes of the final product in comparison with natural fermentation.
banana leaves. Sensorial evaluation of the chocolate obtained from beans fermented
Two batches with the K. marxianus inoculation was more accepted by analysts in
natural comparison with the one from cocoa obtained through natural
fermentation, fermentation
two with
Kluyveromyces
marxianus
hybrid strain
MMIII-41
De Bruyne and Ghanaian 16S rRNA gene Weissella ghanensis LMG 24286T is proposed. The type strain is 215T
others 2008 fermented amplicon (5LMG 24286T 5DSM 19935T )
cocoa beans sequencing
Ghana Daniel and A:H1,2,3:72ha M13-based Pichia kudriavzevii (Issatchenkia orientalis), Saccharomyces cerevisiae, and
others 2009 B:H4,5,6,7:72ha PCR-fingerprint Hanseniaspora opuntiae formed the major components. Hanseniaspora
profiles Swab opuntiae was identified conclusively for the first time from cocoa
samples fermentations
Ghana De Bruyne Ghanaian 16S rRNA gene Two novel Lactobacillus fabifermentans sp. nov. (type strain LMG 24284T
and others fermented amplicon 5DSM 21115T ) and Lactobacillus cacaonum sp. nov. (type strain LMG
2009 cocoa beans sequencing 24285T 5DSM 21116T ) are proposed
Ghana De Bruyne Ghanaian 16S rRNA gene A novel Weissella fabaria sp. nov., with strain 257T (5LMG 24289T 5DSM
and others fermented amplicon 21416T ) is proposed
2010 cocoa beans sequencing
Ghana, Brazil H1-60hsampling 16S rRNA gene Lactobacillus fermentum is by far the dominant species, followed by
Garcia- Armisen time B-B1,B2- amplicon Acetobacter pasteurianus. The presence of Gluconacetobacter species,
and others 48hsampling sequencing Enterobacteriaceae related to Erwinia/Pantoea/Tatumelle by 16 S rDNA
2010 time F-B3,B4- PCR-DGGE library sequencing
84hsampling
time
Quattara and Bacillus fusiformis Pectate lyases from They showed original features in regard to their high activity on a large
others 2010 (BS90), Bacillus the three main range of substrates, including highly methylated pectin
subtilis (BS66), pectinolytic Bacillus
and Bacillus strains isolated
pumilus (BS22) from fermenting
were isolated cocoa beans were
from purified and
fermenting characterized
cocoa beans
Lefeber and others Lactobacillus Investigate growth A mixture of Lactobacillus plantarum 80, Lactobacilllus fermentum 222, Cocult. L.
2010 plantarum, and metabolite and Acetobacter pasteurianus 386B can now be considered a plantarum 80,L.

Lactobacillus production in PSM mixed-strain starter culture fermentum 222:
fermentum, and in laboratory ferm. G0-F :152-
Acetobacter 42,F0-F :152-
pasteurianus 0,CA0-F :47-
0,LA0-F :0
95,AA0-F :0 120,
MM :136
Monocult.A.
pasteurianus
386B LA0 -F :73-
0,M0-F :225- 19,
AA0-M-F :0-120-
15
(Continued)


Brazil Santos and B:120ha PCR-DGGE: using Lac1-Lac2:Lactobacillus, Pediococcus, Leuconostoc, and Weisella.
others 2011 specific primers: Lac3-Lac2:Lactococcus, Streptococcus, Enterococcus, Tetragenococcus,
Lac1-Lac2 and and Vagococcus
Lac3-Lac2.
Ghana Nielsen and cocoa bean heap 26S rRNA gene Three novel species: Candida halmiae (group A, type strain G3T 5CBS
others 2011 fermentations amplicon 11009T 5CCUG 56721T ); Geotrichum ghanense (group B, type strain
in Ghana sequencing G6T 5CBS 11010T 5CCUG 56722T ) and Candida awuaii (group C, type
Rep-PCR
strain G15T 5CBS 11011T 5CCUG 56723T ) are proposed
Côte d’Ivoire plastic vessels with (GTG)5-PCR Lactobacillus plantarum, Lactobacillus fermentum, with Fructobacillus G0-144 :96.5-0 S0-144 :19.8-1.61
Lefeber and temperature- fingerprinting 16S pseudofilculneus occasionally, mainly Acetobacter pasteurianus, besides F0-144 :182-0 F0-144 :3.60-2.11

others 2011a controlled rRNA gene minor clusters of Acetobacter ghanensis and Acetobacter senegalensis M0-144 :0-5.6 G0-144 :2.32-2.16
amplicon were found. Tatumella and Pantoea were detected culture-independently E0-:144 :0-3.68 M0-144 :0-14
sequencing at the beginning of the fermentations CA0-144 :11.3- E0-:144 :1-2.65
PCR-DGGE Sensory 0.63 CA0-144 :7.39-5.41
analysis LA0-144 :0-3.74 LA0-144 :0-0.76
AA0-144 :0-10.6 AA0-144 :0-14.1
GA0-M-144 :0-
8.52-1.42
Lefeber and others Cocoa-specific and Kinetic investigation The cocoa-specific Lactobacillus fermentum strains can be categorized as L. fermentum
2011b cocoa- of carbohydrate the ones best adapted to the cocoa pulp ecosystem and show interesting 222:M103:M158:M332,G0-F :142-
nonspecific LAB fermentation and functional roles 85,140-80,142-
strains from citric acid 78,145-65,

Ghanaian and conversion by F0-F :142-
Brazilian cocoa various LAB strains 38,140-0,142-
bean heap/box 0,140-10,
fermentation as CA0-F :55-0,60-
a starter culture 0,48-0,57-
a cocoa pulp 0,LAM :0-75,0-
simulation 94,0-84,0-92,
medium:48h AA0-F :0-100,0-
130,0-125,0-
130,
MF :95,138,137,130
Brazil, Ecuador, 26S rRNA gene The most frequent yeast species were Hanseniaspora sp, Pichia kudriavzevii,
Ivory Coast Brazil:B(F1,F2,B1,B2): amplicon and Saccharomyces cerevisiae, independent of the origin of the cocoa.
Malaysia Pa- 144ha sequencing PCR- New species Meyerozyma caribbica and Hyphopichia burtonii
palexandratou Ecuador:B(I1,I2),P DGGE, represented the main yeast species in the Ivorian ferm.
and De Vuyst (P1,P2):96ha ,
2011a IvoryCoast:H,B:150ha
Malaysia:B(M1,M2):
120ha
Nacional (GTG)5-PCR Pichia kudriavzevii, Pichia manshurica, Saccharomyces cerevisiae, P1:B1:P2:B2 P1:B1:P2:B2 Dry cb:
×Trinitario B1:120ha ,B2:120ha fingerprinting 16S Leuconostoc pseudomesenteroides Fructobacillus tropaeoli-like, and G0-F :46-2,41- GF :2.6,6,1.6,1.8,
Ecuador Pa- mixed and rRNA gene Lactobacillus fermentum were prevailing species. Tatumella saanichensis 2,35-0,37- FF :5.8,3.1,5.3,7,
palexandratou P1:96ha , amplicon and Tatumella punctata (Enterobacteriaceae) present during the initial 3,F0-F :55-2,65- SF :1.1-1.5-1.2,1.8,
and others P2:120ha Swab sequencing phase, Acetobacter pasteurianus, generally appeared later during 1,35-0,34-2 S:0 MF :2.4,1.5,0.7,3,
2011b samples from PCR-DGGE Sensory fermentation. A potential new yeast species was isolated, namely, MF :35,15,0,9 LAF :2.1, 1.9,0.2,0.6,
surfaces analysis Candida sorbosivorans-like. CA0-F :6-2,6- CAF :5,6, 6,5,
2,10-2,8-3 AAF :6,13,6,7,
AA0-F :0-4,0-5,3- GAF :4,0,2,0
7,3-11
GAM :11.5,21.3.1,
8.9,14.8,
LAM :5,6.1,1.7,4.5
(Continued)

Hybrids of Criollo B1:144ha , (GTG)5-PCR Lactobacillus fermentum and Acetobacter pasteurianus were the B1:B2 S:0 B1:B2 S:28-1,27-2.5,
and Forastero B2:144ha fingerprinting 16S predominating species. Fructobacillus pseudoficulneus, Lactobacillus G0-F :55.5-9,43- G0-F :2-16,1-14,
Brazil Pa- mixed rRNA gene plantarum, and Acetobacter senegalensis (initial phase), Tatumella 8, F0-F :3-12.5,4-12,
palexandratou spontaneous amplicon ptyseos and Tatumella citrea (in the beginning) were the prevailing F0-F :50-2,67-9, MM :3.5,3.5, Dry cb:
and others organic cocoa sequencing species. This study emphasized the possible participation of MM :24-9, GF :1.4,3.7,FF :8.5,5.7,
2011c beans Swab PCR-DGGE Sensory Enterobacteriaceae in the cocoa bean fermentation process. Good CA0-F :8- MF :2.2,0.7,LAF :2.1,
samples from analysis post-harvest process of the cocoa beans will allow the production of 2.5,11.1-3.2, 2.1,AAF :8.5,7,
surfaces high-quality cocoa and chocolates independent of the fermentation LAM : 4.6,4, CAF :2.3,5,GAF :4.4,
method or farm GAM :3,11, 3.3
AAM :11,8,18
EM :6,7
Criollo and H1,B1:150ha 16S rRNA gene Ivory Coast: Lactobacillus fermentum, Leuconostoc pseudomesenteroides, Ivory coast: Ivory coast Fresh
Forastero hybrid B1,B2:144ha amplicon Erwinia soli and Pantoea sp.were predominant. Brazil: Lactobacillus G0-F :50- cb:EM :12.9-15.2
Ivory Coast, sequencing plantarum, Lactobacillus durianis, L. fermentum, Lactobacillus mali, 0,F0-F :55-0 SM :3-2.5 Dry
Brazil Pa- PCR-DGGE Lactobacillus nagelii, Leuconostoc pseudomesenteroides, and Pediococcus S:0,MM :1.7, cb:CA:4.6-7.8
palexandratou acidilactici, as well as Bacillus subtilis. Local operational practices, in AAM :24.1-15.1, AA:10.2-12.9
and others particular pod/bean selection influenced species diversity and EM :12- Brazil:Fresh
2011d community dynamics and impacted the quality of fermented cocoa beans 20,GA0-F :2-0 cb:GM :4FM :4,AA:
Brazil: G0-F :60- 22.4-19,EM :7.7-
0,F0-F :75-0 5.2dry
S:0,MM :7,CA:1, cb:GA:3.1-4.8
AAM :28,EM :20,
GA0-M-F :2.5-19-
4
Brazil Copetti and The cocoa samples Samples: 25 before Filamentous fungi were found in lower levels during fermentation. During
others 2011 came from three ferm. 51 different the six days of fermentation the fungi most commonly isolated were:
major farms and times of ferm. (1-6 Monascus ruber, Penicillium paneum, Geotrichum candidum and Absidia
represented the days), 85 different corymbifera
different stages times of sun drying
of production (1-14 days) and 65
the storage period
(up to one year)
Ivory Coast Pectinolytic ARDRA 16S rRNA Six Bacillus species were identified: B. subtilis, B. pumilus, B. sphaericus, B.
Quattara and Bacillus strains gene partial cereus, B. thuringiensis, together with B. fusiformis (for the first time). The

others 2011 isolated from sequencing best pectate lyase (PL) producers, yielding at least 9 U/mg of bacterial
fermenting dry weight, belonged to B. fusiformis, B. subtilis, and B. pumilus species
cocoa beans while those belonging to B. sphaericus, B. cereus and B. thuringiensis
generally showed a low level of activity.
(Continued)


b West Africa, Starter culture A:L. 16S and 26S rRNA In all starter culture-added cocoa bean fermentation processes, the A:I(H with, A:I(H with, without
Malasia Lefeber fermentum 222 PCR-DGGE Sensory inoculated starter culture species were able to outgrow the natural without turning),
and others and A. analysis contamination of the cocoa pulp-bean mass and they prevailed during turning), M(B),B:M(B),G(H)
2012 pasteurianus cocoa bean fermentation. For the production of a standard bulk M(B),B:M(B),G(H) Dry cb: S:0
386B chocolate, the addition of a yeast/LAB/AAB starter culture was G0-F :37-10,65- G:1.6,2,1.2,1.6,1.9,
H1,H2,H3:no necessary. 6,30-0,45-0,51- F:6.5,8.8,4.5,5.3,7.8,

mixing,:120ha H4,H5,H6,H7:mixing 0,F0-F :45-10,72- M:1.7,1.9,0.7,0.4,0.5,
B1:96ha Starter 16,30-0,56- LA:1.6,1.2,3,1.3,1.1,
culture B: 0,60-15,M0-F :0- AA:3.1,2.9,5.2,4.1,
S.cerevisiae 25,0-25,0-15,0- 3.9
H5S5K23, L. 7,0-
fermentum 222, 5,EM :11,6,13,17,18
and A. CA0-M-F :7.2-0,6-
pasteurianus 0,4-0,5.5-0,3-
386B,H1,H2,H3:mixing, 0,LA0-M-F :0-8-
and B1:mixing, 8,0-5-0,0-10.5-
10,0-5.5-5.5,0-
3-3,AA0-M-F :0-
11-7,0-10-6,0-
7-4,0-6-6,0-8-
8,GA0-M-F :0,0-
5,0,0,0
Brazil Pereira and Spontaneous 16S rRNA gene Saccharomyces cerevisiae, Lactobacillus fermentum and Lactobacillus ST:PC S0-F :119- ST:PC S0-F :7.2-0,6.8-0,
others 2012 cocoa bean amplicon plantarum, Acetobacter tropicalis were the dominant species. 37,120-60, G0-M-F :2-4-2.6,1.5-
fermentations sequencing nested Lactobacillus fermentum UFLA CHBE8.12, Saccharomyces cerevisiae G0-M-F : 4-2.75,F0-M-F :2-2.9-
performed PCR–DGGE UFLA CHYC7.04, and Acetobacter tropicalis UFLA CHBE16.01 were 37-130-10,40- 2.5,1-3.5-2.5,
ST:168ha , selected to form a cocktail starter culture 100-3,F0-M-F :30- CA0-M-F :2.3-3-1.9,
turned 122-3,37-95- 1.3-3-1.2,
PC:144ha were 3,CA0-M-F :20- EM :5-3,AA0-M-F :0-
kept in 21-4,21-22.5- 4.9-4,0-9-1.9,
incubators, 17.5, LA0-M-F :1-2.25-
EM :79- 2,0.55-1-1
65,AA0-M-F :0-
19-19,0-69-20,
LA0-M-F :2-22.5-
20,2-31.5-6
Brazil llleghems B:3-6d sampling taxonomic analysis Hanseniaspora uvarum, Hanseniaspora opuntiae, Saccharomyces cerevisiae,
and others at 30h i.e., 454 Lactobacillus fermentum, and Acetobacter pasteurianus as the prevailing
2012 pyrosequencing species. Fungal and bacterial species that were not yet associated with
(Metagenomic cocoa bean fermentations were identified: Vanderwaltozyma polyspora,
sequencing Zygosaccharomyces rouxii Moniliophthora perniciosa, Eremothecium
analysis) gossypii, Candida albicans, Erwinia tasmaniensis, Erwinia amylovora,
Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus,
Lactococcus lactis, Leuconostoc mesenteroides, Oenococcus oeni, and
Pectobacterium carotovorum
(Continued)

b Mixed hybrids SST:144ha , 16S rRNA gene The dominant species of major physiological roles were the same for SST:B1:B2 S0-F :10- SST:B1:B2
Brazil Pereira B1,B2:144ha ,both amplicon fermentations in SST, relative to boxes. Saccharomyces cerevisiae, 5,12.3-4.3,8- S0-M-F :5.7-7.5-0,6.8-
and others turned sequencing nested Hanseniaspora sp. Lactobacillus fermentum, Lactobacillus plantarum, 7,G0-M-F :32- 7.5-0,6.8-0,
2013 PCR–DGGE Acetobacter tropicalis, and Bacillus subtilis. A greater diversity of 125-10, G0-M-F :1.25-2.75-
bacteria and non-Saccharomyces yeasts was observed in box 145-17,132-19, 1.75
fermentations. The SST system markedly altered the pro-portion of yeasts F0-M-F :40-139- F0-M-F :3.8-5.5-5,2.4-
relative to box fermentations by decreasing their population size and 10, 160-35, 5.1-2.5,2.4-4.7-4.5,
diversity. A potentially novel acetic acid bacteria belonging to the genus 150-50, EM :3.25,3.6,3.6
Asaia was isolated during fermentation in B1. EM :71,64,48, LA0-M-F :0-1.8-1.2,0-
LA0-M-F :0-24- 1.6-1,0-1.5-1.1,
11,0-17-7,0-23- AA0-M-F :0-13-5.5,0-
10, 8.5-5.5,0-4.5-1.7,
AA0-M-F :0-57- CA0-M-F :1.6-5.5-
58,0-35-35,0- 2.25,
21-21, 1.4-2-0,2.5-2.4-0.2
CA0-M-F :15-19-
4.5,15-7.5-
2,14.5-14.5-0
Mixed hybrids B1, (GTG)5-PCR Hanseniaspora opuntiae, Lactobacillus fermentum, and Acetobacter B2: B2: S0-M-120 :8-12-2
unknown origin B2:120ha mixed fingerprinting pasteurianus were the prevalent species of the two fermentations. S0-120 :0,G0-120 :40- G0-120 :1.8-4,
Malaysia Pa- Swab samples M13-PCR DGGE During the initial phase: Leuconostoc pseudomesenteroides and 2, F0-120 :1.8-2.8,
palexandratou from surfaces 16S rDNA, 26S Acetobacter senegalensis. The mid-phase:Saccharomyces cerevisiae, F0-120 :51- LA0-120 :1-1.9
and others rDNA gene Lactobacillus plantarum, Lactobacillus pentosus, and Acetobacter 3,M0-120 :0-8, CA0-120 :0-2.1
2013 amplicon ghanensis.Tatumella saanichensis and Enterobacter sp. were in the LA0-120 :5.8-0, AA0-120 :0-
sequencing beginning and a Bacillus sp. were late fermentation. The presence of CA0-120 :0-11.8, 7.1,EM:0-8 Dry cb:
PCR–DGGE Sensory Enterobacter and Bacillus species on the surfaces of pods and shovels AA0-120 :0- S:0,G:1.1,F:2.6,
analysis 9, EM :0-17 LA:2.3,AA:7.5
Adler and others Lactobacillus 13C labeling based L.actobacillus fermentum NCC 575 might be one candidate due to its
2013 plantarum, metabolic flux superior performance in flux activity. This study results suggest that a
Lactobacillus analysis PSM-LAB single strain of L. fermentum is able to dominate the whole community
fermentum and drive the fermentation
b Forastero Ghana T1-6:120ha Two (GTG)5-PCR Inoculation:Pichia kluyveri CH, Lactobacillus fermentum L18, and G0-120 :50-10 G0-120 :2-6 F0-120 :1,6-5
Crafack and defined mixed fingerprinting 16S Acetobacter pasteurianus A149 seemed to have a positive influence on F0-120 :58-11 S0-120 :11-0
others 2013 starter cultures rDNA 26S rDNA the flavor S0-120 :0 M0-120 :0-1
used: gene amplicon M0-120 :0-14 E0-:24 :0-12
sequencing PFGE E0-:24 :0-24 CA0-120 :6-0
PCR-DGGE Sensory CA0-120 :6-0 LA0-120 :0-2
analysis LA0-120 :0-2.5 AA0-120 :0-9
AA0-120 :0-12
Brazil Ecuador Spontaneous 16S rRNA gene A novel species Weissella fabalis sp. nov. classified as M75T (5LMG 26217T
Malaysia cocoa bean amplicon 5CCUG 61472T )
Snauwaert and fermentations sequencing

others 2013 DNA–DNA
hybridization
Ghana Afoakwa H:144h turning Chemical composition The effects of pod storage (as a means of pulp preconditioning) and
and others Pod storage for Mineral analysis fermentation on the chemical composition and physical characteristics of
2013 7,14,21d Physical qualities Ghanaian cocoa beans were investigated. Proportion of cocoa nibs
increased and no appreciable changes were noted with the proportion of
germs with fermentation and increasing pod storage
Malaysia early and late rep-PCR,16S rDNA “core’’ bacterial and yeast microbiota; Lactobacillus fermentum, Acetobacter
Meersman and harvest, 26S rDNA gene pasteurianus, Saccharomyces cerevisiae, Hanseniaspora thailandica,
others 2013 B1,H1:144ha amplicon Hanseniaspora opuntiae, and Pichia kudriavzevii. New species been
B2, sequencing associated with spontaneous cocoa pulp fermentations, namely Candida
H2,:120ha turned, asiatica, Candida fructus, Candida metapsilosis, Rhodotorula
mucilaginosa, and Torulaspora globosa
(Continued)


Adler and others Acetobacter 13C labeling based The importance of a well-balanced microbial consortium for a successful Cocultivation of
2014 pasteurianus metabolic flux fermentation process was underlined. AAB performed the best and both microbes
NCC 316, analysis PSM-AAB produced the largest amounts of acetate in mixed culture experiments AA:
Lactobacillus when lactic acid bacteria and yeasts were both present. The pathway 117.2±3.5,121.5±15,6,Only
fermentum NCC activities of LAB, AAB, and yeast are important for the successful L. fermentum:
575,Saccha- fermentation of cocoa pulp AA:106.6±2.0,76.0
romyces ±2.2 Only
cerevisiae NYSC, S.cerevisia:
Acetobac- AA:7.5±1.3,

terghanensis 21.0±5.7
DSM18895
Trinitario, Papua Contact with pods rDNA sequencing Hanseniaspora guilliermondii, Pichia kudriavzevii, Kluyveromyces Control ferm.: Control ferm.:
New Guinea Ho and transfer RFLP analysis marxianus, Lactobacillus plantarum, Lactobacillus fermentum, S0-F :0,G0-F :48- S0-F :17-2,G0-F :0.5-4,
and others into P, 144ha Acetobacter pasteurianus, and Gluconobacter frateurii were the major 17.5, F0-F :1-4,EM :5.2
2014 Natamycin was species in the control ferm. In the presence of Natamycin, the same F0-F :71-0,EM :6.5 CA0-F :17-
used to inhibit bacterial species grew but yeast growth was inhibited. Quality tests CA0-F :45- 11,SA0-F :13-
the growth of showed that beans fermented without yeasts were purplish-violet in color 50,SA0-F :5- 22.5,LA0-F :3-
yeasts and not fully brown, and chocolate prepared from these beans tasted 20,LA0-M-F :0- 12.5,AAM :19,MA0-F :
more acid and lacked characteristic chocolate flavor. Therefore, yeasts 25-25 2-1,OA0-F :6.5-6.5
are essential for successful cocoa fermentation AAM :15,MA0-F :8- Natamycin treated:
0 OA0-F :0 S0-F :17-0,G0-F :1-4,

Natamycin F0-F :1-4,EM :0.2,
treated: CA0-F :17-
S0-F :0,G0 :48-35 18.5,SA0-F :14-
F0-F :71- 16.5,LA0-F :2.5-
16,EM :0.3 8,AAM :15,MA0-F :2-
CA0-F :45- 1,OA0-F :7.5-7.5
60,SA0-F :5-
15,LA0-M-F :0-
25-
20,AAM :14,MA0-F :9-
9,OA0-F :0
Ghana Moens and Four cocoa-specific AAB strains were Acetobacter pasteurianus 386B displayed beneficial functional roles:a fast
others 2014 AAB strains, A analyzed kinetically oxidation of ethanol and lactic acid
cocoa pulp and metabolically
simulation during laboratory
medium in fermentations
15-liter
fermenters: 48h
Trinidad and Fresh cocoa seeds Biochemical quality All samples from fermentation-like incubation were within the range of aa1,2.3 : 25.0 ±
Tobago Kadow were objected parameter analysis biochemical quality attributes. In terms of flavor precursors, raw cocoa 5.2, 16.2 ±0.4,
and others to three types of Free amino acids, from fermentation-like incubation has a particularly high chocolate 15.4 ± 1.7
2015 fermentation- reducing sugars, flavor potential. rs1,2,3 : 28.0 ±
like incubation phenolic 6.7, 21.1 ± 4.0
without compounds and 16.4 ± 1.0
microorganisms organic acids pc1,2,3 : 3.4 ±
for 5d 1.9, 5.1 ± 2.4,
2.2 ± 1.6
AA1,2,3 : 14.6 ±
3.0, 8.5 ± 0.8
8.1 ± 0.2
(Continued)

Forastero B1:168ha , 26S rRNA gene The detected predominant yeast varied from one technique to another. In
Trinitario B2:192ha amplicon total, 20 different yeasts could be identified. Important differences
Criollo Mexico mixed sequencing between the species detected in the two sampling. The predominant
Arana-Sánchez PCR–DGGE yeasts were Pichia kudriavzevii, Saccharomyces cerevisiae, and
and others PCR–RFLP Hanseniaspora sp.
2015
Trinitario Contact with pods rDNA sequencing Hanseniaspora guilliermondii, Pichia kudriavzevii, Kluyveromyces Lysozyme treated: Lysozyme treated
Australia Ho and transfer RFLP analysis marxianus, Saccharomyces cerevisiae, Lactobacillus plantarum, S0-F :0,G0-F :50- S0-F :17-1,G0-F :0-4,
and others into P1, P2. Lactobacillus pentosus, Lactobacillus fermentum, Acetobacter 20, F0-F :0-3,EM :5
2015 Nisin and pasteurianus and Gluconobacter frateurii were the major species in F0-F :68-0,EM :10 CA0-F :16-
lysozyme were control fermentations. Beans fermented in the presence or absence of CA0-F :40- 11,LA0-F :0-
used to restrict lactic acid bacteria were fully fermented, had similar shell weights and 42,LA0-F :0-10, 4,AAM :20 Control
the growth of gave acceptable chocolates with no differences in sensory rankings. Lactic AAM :17 Control ferm.:
lactic acid acid bacteria may not be necessary for successful cocoa fermentation. ferm.:S0-F :0, S0-F :17-1,G0-F :0-5,
bacteria G0 :48-20,F0 :72- F0-F :0-
0, 4,EM :6,CA0-F :16-
EM :10,CA0-F :42- 11,LA0-F :0-
50 14,AAM :19
LA0-F :0-
25, AAM :15

Brazil Illeghems B:3-6d sampling a comparative The role of lactic acid bacteria: the heterolactic ferm. and citrate
and others at 30h metagenomic assimilation pathways. The role of Enterobacteriaceae: mixed-acid ferm.,
2015 analysis MG-RAST methylglyoxal detoxification pathways, and other potential roles,
pectinolysis and citrate assimilation
Malaysia Ba. rep-PCR,16S rDNA New hybrid yeast strains were developed to improving the consistency and
Meersman and SA,SB:spontaneous 26S rDNA gene quality of commercial chocolate production
others 2015a ferm.:96ha amplicon
turning 12 sequencing GC-MS
inoculated ferm. analysis of cocoa
liquor
b Ivory Coast Lab-scale ferm. in 16S rDNA, 26S rDNA Control chocolate flavor by inoculating highly aromatic yeasts. New
Malaysia nutrient-rich gene amplicon Saccharomyces cerevisiae hybrids that are both thermotolerance and
Meersman and growth medium sequencing GC-MS efficient cocoa pulp fermentation with a high production of volatile
others 2015b and cocoa pulp analysis of cocoa flavor-active esters were developed. The potential of 2 species (Pichia
(Ivory Coast). liquor and kluyveri and Cyberlindnera fabianii) that produce very large amounts of
Pilot-scale ferm. chocolate fruity esters were investigated
in cocoa
pulp(Malasia)
Ivory Coast Koné B, P, and H(using 26S rRNA gene S. cerevisiae, C. tropicalis, P. kudriazevii, P. galeiforms, G. geotrichum, and W.

and others plantain amplicon anomalus were the major species. Common aroma compounds between
2016 leaves):168ha sequencing each yeast and raw cocoa samples were identified. P. kudriavzevii, S.
PCR-DGGE cerevisiae, G. geotrichum and W. anomalus could be thought as the most
SPME–GC/MS important contributors to the formation of aroma compounds in cocoa in
Ivory Coast.
Abbreviations: B, Wooden box; BA, BB, BA, BD, all wooden box-A,B,C,D, respectively; Ba., basket; H, heap; P, plastic boxes; PC, plastic containers; SST/ST, stainless steel tank; d, days; Cb, dry cocoa beans; G, glucose; S, sucrose; F, fructose; M, ,mannitol; E, ethanol; AA, acetic acid; CA, citric
acid; LA, lactic acid; SA, succinic acid; OA, oxalic acid; Aa, amino acids; Rs, reducing sugars; Pc, phenolic compounds; S0 , complete fermentation; X0 , 0 hour; XM , maximum; XF , final; Cocult., coculture; Monocult., monoculture; Ferm., fermentation; I, Côte d’Ivoire; M, Malaysia; G, Ghana;
AAB, acetic acid bacteria; LAB, lactic acid bacteria; PSM-AAB, cocoa pulp simulation medium for acetic acid bacteria; PSM-LAB, cocoa pulp simulation medium for lactic acid bacteria; 16S rRNA/ 26S rDNA gene amplicon sequencing, sequencing of PCR amplicons in the bacterial 16S
species gene or 26S fungal species gene; DGGE, denaturing gradient gel electrophoresis; rDNA sequencing, ribosomal DNA sequencing; RFLP analysis, restriction fragment length polymorphism; rep-PCR fingerprinting, repetitive DNA sequence-based polymerase chain reaction; GC-MS,
gas chromatography mass spectrometry; MG-RAST, metagenome rapid annotation using subsystems technology; CLP, chromosome length polymorphism; PFGE, pulsed-field gel electrophoresis; (GTG)5-PCR fingerprinting, amplification of repetitive bacterial DNA elements through the
polymerase chain reaction using the (GTG)5 primer; ARDRA, amplified ribosomal DNA restriction analysis; SPME-GC/MS, solid-phase micro-extraction and gas chromatography coupled with mass spectrometry.
a Different time points.
b Starters were used.

Figure 7–Microbes found during cocoa fermentation. Core and subcore groups. Acetic acid bacteria; A.: Acetobacter G.: Gluconobacter; Ga.:
Gluconacetobacter; M.: Microbacterium mold; A.: Aspergillus; Ab.: Absidia; As.: Ashbya; E.: Eurotium; G.: Geotrichum; M.: Moniliophthora; P.:
Penicillium Pa.: Paecilomyces; W.: Wallemia. Lactic acid bacteria; E.: Enterococcus; F.: Fructobacillus; L.: Lactobacillus; Leuc.: Leuconostoc;Lc.:
Lactococcus; O.: Oenococcus; P.: Pediococcus; St.: Streptococcus; W.: Weissella. V.: Vagococcus yeast; C.: Candida; Cr.: Cryptococcus; D.: Debaryomyces;
De.: Dekkera; E.: Eremothecium; I.: Issatchenkia; H.: Hanseniaspora; Ha.: Hansenula; Hy.: Hyphopichia; K.: Kluyveromyces; Ko.: Kodamaea; Kl.:
Kloeckera; L.: Lachancea; Lo.: Lodderomyces; M.: Meyerozyma; Mo.: Moniliophthora; P.: Pichia; R.: Rhodotorula S.: Saccharomyces; Sa.:
Saccharomycopsis; Sc.: Saccharomycodes; Sch.: Schizosaccharomyces; St.: Starmerella; T.: Torulaspora; Tr.: Trichosporon; W.: Wickerhamomyces; V.:
Vanderwaltozyma Y.: Yamadazyma;Ya.: Yarrowia; Z.: Zygosaccharomyces Other Gram-negatif bacteria; A.: Aerobacter; Az.: Azotomonas; Ch.:
Chryseobacterium; E.: Escherichia; En.: Enterobacter; Er.: Erwinia; F.: Frateuria; K.: Klebsiella; M.: Meiothermus; P.: Pseudomonas; Pe.: Pectobacterium;
Pr.:Providencia; S.: Salmonella; T.: Tatumella; Z.: Zymomonas Other Gram-pozitif bacteria; Ar.: Arthrobacter; B.: Bacillus; Br.: Brevibacterium; C.:
Cellomonosa, Co.: Corynebacterium; M.: Micrococcus; Pr.: Propionibacterium; S.: Staphylococcus
∗ :Basionym: Cr. glabrata (Cr. glabratus), G. oxydans (A. oxydans), Lc. lactis (St. lactis), L. fermentum (L. cellobiosus; L. fermenti), M. tetragenus (Gaffkya
tetragena), P. dextrinicus (Lc. fermenti), P. kudriavzevii (I. orientalis), P. damnosus (P. cerevisiae), R. rubra (R. mucilaginosa), W. anomalus (P. anomala),
Y. mexicana (P. mexicana).
Microbial Ecology of Cocoa Bean Fermentation variety of approaches and a variety of different results (that is,
The advent of such molecular ecology approaches have im- different methods yielded different sets of microorganisms), and
pacted our view of the complexity of the microbial communities are summarized in Table 1. As the table shows, the fermentation
within the cocoa bean fermentation. The microbial composition resulting in unique flavor and aroma is related to the cocoa variety,
profile of the fermented cocoa beans is theorized to be one of the the fermentation procedure, and its geographical origin.
factors influencing taste and flavor of the cocoa products. There Although spontaneous cocoa pulp fermentations have been
are many factors––including cultivar, location, fermentation type, investigated in different geographical locations using culture-
and the length of fermentation––that influence the collection of dependent and/or culture-independent approaches (Kostinek and
microbes involved in the fermentation (Ostovar and Keeney 1973; others 2008; Camu and others 2007, 2008a; Daniel and others
Ardhana and Fleet 2003; Schwan and Wheals 2004; Afoakwa and 2009; Papalexandratou and De Vuyst 2011a; Papalexandratou and
others 2008). Therefore, in our review, we find that there are a others 2011b,c; Jespersen and others 2005; Nielsen and others

2005, 2007b; Lagunes-Galvez and others 2007), common micro- (Illeghems and others 2012) through metabolomic and metaflux-
bial groups consistently occur during the cocoa bean fermentation ome studies. In addition, metagenomics analysis evidence provides
process: indigenous species of yeasts, lactic acid bacteria (LAB), and a hypothetical role for Enterobacteriaceae species during the cocoa
acetic acid bacteria (AAB; Ostovar and Keeney 1973; Ardhana and bean fermentation processes (Illeghems and others 2015). This
Fleet 2003; Schwan and Wheals 2004). These microbes are the type of study does not prove any contribution but adds to the
source for generating (through fermentation) the ethanol, lactic intrigue that microbial diversity during natural fermentations is a
acid, and acetic acid that are considered to be essential precursors consequence of complex inter-species interactions.
for driving the conversion of raw cocoa beans to the state needed There are 2 studies by Ho and others (2014, 2015) in which
prior to roasting (Forsyth and Quesnel 1963; Quesnel 1965; De they attempted to directly test the role of specific microorgan-
Brito and others 2001; Schwan and Wheals 2004). Sugar-rich and isms in cocoa bean fermentation. In the 1st study they asked the
acidic environmental conditions are ideal for rapid yeast growth. question, “How important are the yeasts in the fermentation?” In
The yeasts are responsible for pectinolysis of the cocoa pulp and this study, controlled cocoa bean fermentations were conducted
the production of ethanol from glucose. These modified condi- in the presence and absence of natamycin, an antibiotic that in-
tions favor the development of lactic acid bacteria, and LAB then hibits the growth of fungi. They surveyed the microbial population
consume glucose, fructose, and citric acid and produce mannitol, to define how inhibition of fungal growth affected the sequence
lactic acid, and acetic acid. When aeration of the fermenting mass of selected yeast species outgrowth and decline. Strikingly, they
increases and the temperature rises above 37 °C, the dominant reported that natamycin blocked Pichia guilliermondii, Hansenias-
organisms that are present include acetic acid bacteria. Acetic acid pora guilliermondii, Pichia kudriavzevii, Cryptococcus flavescens, and
bacteria oxidize ethanol and lactic acid into acetic acid (Ostovar Kluyveromyces marxianus growth without dramatically affecting the
and Keeney 1973). growth of the microbes, most notably the acetic acid bacteria and
Figure 7 shows the cumulative list of microbes that are present in lactic acid bacteria. This is interesting because it has been pro-
cocoa bean fermentation. Among each consortium member, there posed that, at least in part, growth of acetic acid bacteria depend
are many varieties and differences in each study. This difference and on yeast providing an essential metabolic substrate (Ostovar and
variety among the microbial population account for specialized Keeney 1973; Schwan and others 1995; Camu and others 2007;
species that are associated with cocoa beans originating from a Nielsen and others 2007; Thompson and others 2013). Likewise,
specific region. it has been considered that yeast metabolism favors the growth of
lactic acid bacteria (Schwan and Wheals 2004). It was found that
Studying Microbes the peak of populations of acetic acid bacteria and LAB did not
How the fermentation process is carried out has an impact significantly change and that the sequenced growth occurring was
on the product downstream because of the different complex- not affected. This suggests that the diversity of microbes in the
ity of microbes growing and the metabolites that are produced fermentation results in a robust pattern of microbial outgrowth
(Table 1). Given the importance of the microbiota of the pro- that ultimately generates desirable metabolites.
cessing environment in natural cocoa fermentations, farmhouse When metabolites were quantified from fermentations where
fermentations, and their surrounding environments are analyzed fungal growth was inhibited, there was no significant change in
together, with the goal of describing microbial transactions within the amount of acetic acid and lactic acid that accumulated in
the entire fermentation ecosystem. As expected, environmental beans. Although acetic acid present in cocoa bean fermentations
organisms detected in processing environments dominated the sur- is theorized to be largely due to acetic acid bacteria which oxidize
face microbiota of cocoa beans, demonstrating the importance of the ethanol produced by yeast, there are clearly alternative routes
the processing environment for populating cocoa bean microbial at play. Acetic acid bacteria may generate acetic acid by direct
communities (Ostovar and Keeney 1973; Papalexandratou and metabolism of sugars (Swiegers and others 2005). It is also known
others 2011b,c, 2013; Table 1). that acidic acid bacteria can generate acetic acid from lactic acid
Much of the research focuses on microbial diversity, identifi- produced by the LAB that consume the sugars (Camu and others
cation, and quantity of microbial communities in cocoa (Martelli 2007; Nielsen and others 2007). This may also explain why overall
and Dittmar 1961; Ostovar and Keeney 1973; Samah and others sugar consumption remains similar when comparing fermentations
1993; Ardhana and Fleet 2003; Jespersen and others 2005; Nielsen with and without natamycin treatment.
and others 2005, 2007b; Camu and others 2007, 2008; Lagunes- Yeasts do, nonetheless, play a critical role in producing cer-
Galvez and others 2007; Cleenwerck and others 2008; Kostinek tain volatile compounds, such as higher alcohols and esters since
and others 2008; Daniel and others 2009; Papalexandratou and natamycin treatment correlated with a significant reduction of
De Vuyst 2011a). Knowledge of the diversity of these consortium these metabolites in beans. This is important because these com-
members raises many questions about the molecular interactions pounds have an important impact on the estery, fruity, and floral
that occur between the consortium members. Cocoa fermenta- characteristics of chocolate flavor (Roelofsen 1958). When shell
tions have a complex microbial ecology consisting of interactions content, cut test, and certain sensory assessments such as color,
between a wide range of lactic acid bacteria, acetic acid bacteria, flavor, and overall acceptability from the chocolates prepared from
yeast cells, possibly molds, species of Bacillus, and members of the fermented beans were evaluated, the results were clearly different
Enterobacteriaceae (Ostovar and Keeney 1973; Schwan and others in cocoa bean fermentations in the absence of yeasts. Beans fer-
1986; Ouattara and others 2008). mented in the absence of yeasts were heavier, had a higher content
The roles of each particular group in the complex consortium of shell and moister, sticky material leading to the conclusion that
have been investigated. For example, a recent study identified the pulp had not been fully degraded. This result is consistent with
the functionalities of LAB, AAB, and Enterobacteriaceae, and their previous observations implicating yeast in degrading pulp pectins
possible interactions at a fixed time point during a spontaneous (Leal and others 2008). Furthermore, beans fermented in the ab-
Brazilian cocoa bean box fermentation process (Illeghems and oth- sence of yeast retained a purplish color, a property that led to
ers 2015). This study confirmed the activities of LAB and AAB chocolate, made from these beans, to have a lighter brown color
and was rated lower in sensory evaluations. Another characteristic Absidia corymbifera represented the highest occurrence among sam-
that changed was the content of pyrazines, which are essential pre- ples during the 6 d of fermentation (Copetti and others 2011). In
cursors for specific earthy, roasty aroma, and flavor (Schnermann addition, pectinolytic Bacillus species have been isolated from fer-
and Schieberle 1997). The formation of pyrazines depends on the menting cocoa beans via their ability to produce pectate lyase
Maillard reaction between sugars (fructose and glucose) and pep- (PL). The best PL producers belonged to Bacillus fusiformis, Bacillus
tides and also hydrophobic free amino acids, specifically leucine, subtilis, and Bacilus pumilus (Ouattara and others 2011).
alanine, phenylalanine, and tyrosine released during fermentation As illustrated by Table 1, the scientific evidence to date shows
by aspartic proteinase and carboxypeptidase activities (Rohan and that the diversity and complexity of the microbial population
Stewart 1967; Lopez and others 1978; Biehl and Passern 1982; within the cocoa bean fermentation process is important for the
Biehl and others 1985). The change in pyrazine content remains flavor outcomes in many different final products.
puzzling because there were no apparent differences in the con-
tent of sugars or amino acids in beans with or without natamycin Traditional Fermentation Versus Industrial Production
treatment. These results may suggest there are, as yet, unidentified Although traditional natural fermentation is perhaps the oldest
roles for fungi in the formation of pyrazines. food-preserving process, with its knowledge derived from the past
In a follow-up study nisin and lysozyme were used to restrict to the present within local communities (Caplice and Fitzgerald
growth of sensitive LAB (Ho and others 2015). It was reported 1999; Sieuwerts and others 2008), most modern fermented food
that the growth of LAB during fermentation of these beans was products rely on scientifically formulated starter cultures to in-
inhibited or restricted without dramatically affecting the growth of oculate and to control a fermentation. The unique and diverse
other microbes, most notably the acetic acid bacteria and yeasts. characteristics of traditional fermentation products are strongly re-
This suggests growth of other species that do not require LAB. lated to the diversity of their microbiota and microbial activities,
Their observations further support the idea that microbial diver- and detailed knowledge of the microbial consortium and char-
sity accounts for the robustness of cocoa fermentations. Further acterization of these traditional products was necessary for the
extending this investigation, a survey of metabolites in beans pro- development of industrial production (Sieuwerts and others 2008;
duced in the presence and absence of nisin and lysozyme showed Van Hijum and others 2013). Together with the advent of micro-
no change in acetic acid levels that accumulated. This is not sur- biology, technological innovation has allowed the manufacturing
prising because populations of acetic acid bacteria were main- of many fermentation products on a large scale, such as dairy prod-
tained. The metabolite analysis also revealed an unexpected result; ucts, bread, and beer (Caplice and Fitzgerald 1999; Sieuwerts and
levels of citric acid decreased similarly during fermentations with others 2008). However, a byproduct of this industrial approach has
and without nisin and lysozyme. This is surprising because previ- been that only a small part of the microbial wealth of traditional
ous reports theorized LAB were essential for citric acid metabolism fermentation methods is now used (Van Hijum and others 2013).
(De Vuyst and others 2010). Clearly, citric acid degradation in Therefore, this often diminishes the unique characteristics of the
beans needs further investigation to define alternative routes for original product that originally made the product popular (Smid
its metabolism. It was found that lactic acid levels were reduced and Hugenholtz 2010; Van Hijum and others 2013).
2.8-fold when LAB were completely removed from the fermenta- The origin, spread, and historical development of cocoa out-
tions. The remaining lactic acid could be attributed to the growth lined earlier has led to a system of production for cocoa beans
of yeast cells (Plessas and others 2008). Collectively, these different unique among modern fermented food products in that it is still
studies highlight the robust nature of natural fermentations with a produced within a diversified system of small-scale cocoa farms
diversity of microbes and their metabolites. where the fermentation is a spontaneous process driven by the
Other parameters such as volatiles, sensory characteristics, and naturally occurring local microbiota, rather than via standardized
physical appearance have been examined. These analyses revealed inoculants on an industrial scale, with subsequent processing oc-
no apparent changes between beans fermented in the presence curring at other facilities downstream in the food chain (Schwan
or absence of lactic acid bacteria. Furthermore, there was no dif- and Wheals 2004; Schwan and others 2015). This approach has
ference for sensory properties of chocolate made from fermented led to the remarkably diverse microbiota involved in cocoa bean
beans regardless of whether LAB growth was inhibited or restricted fermentation, as described earlier, even as the results of fermenta-
(Ho and others 2015). We speculated LAB are not essential, at least, tion can be variable in terms of quality (Schwan and Wheals 2004;
when in a community of diverse microbes as is found in natural Lefeber and others 2012; Crafack and others 2013; Schwan and
cocoa bean fermentations. others 2015).
In another study, 4 cocoa-specific AAB species were ana- To better control fermentation and the overall quality of the
lyzed kinetically and metabolically during laboratory fermenta- product, it is proposed as desirable to replace this traditional natural
tions (Moens and others 2014). Among these, Acetobacter pasteuri- cocoa bean fermentation process with starter cultures like those
anus 386B displayed beneficial functional roles, including a fast used in other fermented food products, and there have been some
oxidation of ethanol and lactic acid. Adler and others (2014) in- promising attempts made to accomplish this goal, as described in
vestigated metabolic fluxes of acetic acid bacteria under laboratory the literature (Schwan 1998; Leal and others 2008; Lefeber and
conditions, and they concluded that the largest amounts of acetate, others 2012; Crafack and others 2013), but none has yet to be put
the key compound for desirable cocoa flavors, were produced by into practice at the farm level.
acetic acid bacteria when lactic acid bacteria and yeasts were both One of the earliest reports of the successful use of a defined,
present. mixed-starter culture was by Schwan (1998). Here, the natural
When the mycobiota of cocoa beans was studied, with co- fermentation conditions were almost identical to those found in
coa samples collected from the different stages of production, Brazilian farms, and 1 yeast species, 2 lactic acid bacteria, and 2
the largest numbers and diversity of fungi were observed in the acetic acid bacteria were used as a defined microbial inoculum
samples collected at the farm, especially during drying and stor- cocktail in wooden boxes with aseptically prepared cocoa beans.
age. Monascus ruber, Penicillium paneum, Geotrichum candidum, and Beans inoculated with a defined cocktail at time zero were almost

identical to those processed through natural fermentation. Later, https://www.intechopen.com/books/genetic-diversity-in-plants/defining-

an attempt was made to exclude the influence of the natural mi- genetic-diversity-in-the-chocolate-tree-theobroma-cacao-l-grown-in-
west-and-central-africa
crobiota through the use of a specially designed stainless steel tank
Amoa-Awua W. 2015. Methods of cocoa fermentation and drying. In:
(SST) system. The SST system markedly altered the proportion Schwan RF, Fleet GH, editors. Cocoa and coffee fermentation. Boca
of yeasts relative to standard box fermentations by decreasing their
Raton FL: CRC Taylor & Francis. p 71–128.
population size and diversity (Pereira and others 2013). Arana-Sánchez A, Segura-Garcı́a LE, Kirchmayr M, Orozco-Ávila I,
In another study, Lefeber and others (2012) investigated a farmLugo-Cervantes E, Gschaedler-Mathis A. 2015. Identification of
implementation of starter culture for the fermentation of co- predominant yeasts associated with artisan Mexican cocoa fermentations
using culture-dependent and culture-independent approaches. World J
coa beans that resulted in improved fermentation. The inocu- Micro Biotechno 31:359–69.
lated starter culture species were able to outgrow the natural Ardhana MM, Fleet GH. 2003. The microbial ecology of cocoa bean
contamination of the cocoa pulp bean mass and they prevailed fermentations in Indonesia. Int J Food Microbiol 86(1-2):87–99.
during cocoa bean fermentation. The sensory analysis demon- Bartley BGD. 2005. The genetic diversity of cacao and its utilization. CABI
International, Wallingford, Oxfordshire: CABI Publishing.
strated that the application of both added starter cultures resulted
in fermented dry cocoa beans that gave a reliable flavor, inde- Biehl B, Passern D. 1982. Proteolysis during fermentation-like incubation of
cocoa seeds. J Sci Food Agric 33:1280–90.
pendent of the cocoa-producing region or fermentation method.
Biehl B, Brunner E, Passern D, Quesnel VC, Adomako D. 1985.
In another recent study, Meersman and others (2015b) investi- Acidification, proteolysis, and flavor potential in fermenting cocoa beans. J
gated the possibility of generating highly aromatic yeast hybrids Sci Food and Agric 36:583–98.
to control the chocolate flavor. They concluded that selection ofBrown CH. 2009. Development of agriculture in prehistoric Mesoamerica:
the linguistic evidence. Staller JE, Carrasco M, editors. Pre-Columbian
different yeast cultures modulates the final flavor of chocolate and
foodways. Berlin, Germany: Springer. p 71–107.
therefore provides different chocolate types to consumers. Only
Camu N, De Winter T, Verbrugghe K, Cleenwerck I, Vandamme P,
this study showed significant differences between inoculated and Takrama JS, Vancanneyt M, De Vuyst L. 2007. Dynamics and biodiversity of
spontaneous fermentations. However, with respect to these earlier populations of lactic acid bacteria and acetic acid bacteria involved in
studies on starter cultures, the overview of the data in Figure 7 spontaneous heap fermentation of cocoa beans in Ghana. Appl Environ
reveals that many common species of microorganisms found in Microbiol 73(6):1809–24.
natural cocoa bean fermentations might be replaced by this core Camu N, De Winter T, Addo SK, Takrama JS, Bernaert H, De Vuyst L.
2008a. Fermentation of cocoa beans: influence of microbial activities and
microbiota, which is the group of microorganisms among all the polyphenol concentrations on the flavor of chocolate. J Sci Food Agric
consortium members that has been frequently found in all studies. 88:2288–97.
While moving cocoa bean fermentation towards a more modern Camu N, González A, De Winter T, Van Schoor A, De Bruyne K,
model of employing a starter culture and streamlining the process Vandamme P, Takrama JS, Addo SK, De Vuyst L. 2008b. Influence of
turning and environmental contamination on the dynamics of populations
has some advantages (consistent quality, more product, more profitof lactic acid and acetic acid bacteria involved in spontaneous cocoa bean
for farmers), such a strategy also risks losing the microbiotic di-
heap fermentation in Ghana. Appl Environ Microbiol 74(1):86–98.
versity and resultant variation in flavor that makes chocolate soCaplice E, Fitzgerald GF. 1999. Food fermentations: role of microorganisms
unique (and popular). in food production and preservation. Int J Food Microbiol 50:131–49.
Chaves-López C, Serio A, Grande-Tovar CD, Cuervo-Mulet R,
Delgado-Ospina J, Paparella A. 2014. Traditional fermented foods and
Acknowledgments beverages from a microbiological and nutritional perspective: the
G. Ozturk was supported by the Turkish Ministry of National Colombian heritage. Comp Rev Food Sci Food Safety 13:1031–1048.
Education. The authors declare no conflicts of interest, nor any Cheesman EE. 1944. Notes on the nomenclature, classification and possible
competing financial interests. relationships of cocoa populations. Trop Agric 21:144–59.
Christopher H. 2013. Cacao’s relationship with Mesoamerican society. Spect
Authors’ Contributions 3:48–60.
G.O. wrote the manuscript, prepared the literature overview, Cleenwerck I, Camu N, Engelbeen K, De Winter T, Vandemeulebroecke K,
De Vos P, De Vuyst L. 2007. Acetobacter ghanensis sp. nov., a novel acetic acid
and interpreted data. G.M.Y. conceived the study, participated in bacterium isolated from traditional heap fermentations of Ghanaian cocoa
data interpretation, participated in coordination of the study, and beans. Int J Syst Evol Microbiol 57:1647–52.
performed critical revisions of the manuscript. Cleenwerck I, Gonzalez A, Camu N, Engelbeen K, De Vos P, De Vuyst L.
2008. Acetobacter fabarum sp. nov., an acetic acid bacterium from a Ghanaian
cocoa bean heap fermentation. Int J Syst Evol Microbiol 58(9):2180–5.
References Clement CR, De Cristo-Araújo M, Coppens D’Eeckenbrugge G, Alves
Pereira A, Picanço-Rodrigues D. 2010. Origin and domestication of native
Adler P, Bolten CJ, Dohnt K, Hansen CE, Wittmann C. 2013. Core Amazonian crops. Diver 2(1):72–106.
fluxome and metafluxume of lactic acid bacteria under simulated cocoa pulp
fermentation conditions. Appl Environ Microbiol 79(18):5670–81. Crafack M, Mikkelsen MB, Saerens S, Knudsen M, Blennow A, Lowor S,
Takrama J, Swiegers JH, Petersen GB, Heimdal H, Nielsen DS. 2013.
Adler P, Frey LJ, Berger A, Bolten CJ, Hansen CE, Wittmann C. 2014. The
Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus
key to acetate: metabolic fluxes of acetic acid bacteria under cocoa pulp
in a defined mixed starter culture for cocoa fermentation. Int J Food
fermentation simulating conditions. Appl Environ Microbiol 80:4702–16.
Microbiol 167(1):103–16.
Afoakwa EO, Paterson A, Fowler M, Ryan A. 2008. Flavor formation and
Crown PL, Hurst WJ. 2009. Evidence of cacao use in the Prehispanic
character in cocoa and chocolate: a critical review. Crit Rev Food Sci Nutr
48(9):840–57. American Southwest. PNAS 106(7):2110–13.
Afoakwa EO, Quao J, Takrama J, Budu AS, Saalia FK. 2013. Chemical Coe SD, Coe MD. 1996. The true history of chocolate. London: Thames
composition and physical quality characteristics of Ghanian cocoa beans as and Hudson.
affected by pulp pre-conditioning and fermentation. J Food Sci Technol Coe SD, Coe MD. 2007. The true history of chocolate. 2nd ed. New York:
50(6):1097–105. Thames and Hudson.
Aikpokpodion PO. 2012. Defining genetic diversity in the chocolate tree, Copetti MV, Lamanaka BT, Frisvad JC, Pereira JL, Taniwaki MH. 2011.
Theobroma cacao L. grown in West and Central Africa. In: Caliskan M, Mycobiota of cocoa: from farm to chocolate. Food Microbiol
editor. Genetic diversity in plants. InTech Publishing. Available from: 28(8):1499–504.
Daniel HM, Vrancken G, Takrama JF, Camu N, De Vos P, De Vuyst L. International Cocoa Organization (ICCO). 2014c. Cocoa market update.
2009. Yeast diversity of Ghanaian cocoa bean heap fermentations. FEMS London: (Online).
Yeast Res 9(5):774–83. International Cocoa Organization (ICCO). 2015. Available from:
De Brito ES, Garcı́a NHP, Gallão MI, Cortelazzo AL, Fevereiro PS, Braga http://www.icco.org/about-cocoa/fine-or-flavour-cocoa.html. Accessed
MR. 2001. Structural and chemical changes in cocoa (Theobroma cacao L) 2015 May 15.(Online).
during fermentation, drying, and roasting. J Sci Food Agric 81:281–8. Jespersen L, Nielsen DS, Hønholt S, Jakobsen M. 2005. Occurrence and
De Bruyne K, Camu N, Lefebvre K, De Vuyst L, Vandamme P. 2008. diversity of yeasts involved in fermentation of West African cocoa beans.
Weissella ghanensis sp. nov., isolated from a Ghanaian cocoa fermentation. Int FEMS Yeast Res 5(4-5):441–53.
J Syst Evol Microbiol 58:2721–5. Kaufman T, Justeson J. 2007. The history of the word for cacao in ancient
De Bruyne K, Camu N, De Vuyst L, Vandamme P. 2009. Lactobacillus Mesoamerica. Anci Meso 18:193–237.
fabifermentans sp. nov. and Lactobacillus cacaonum sp. nov., isolated from Kadow D, Niemenak N, Rohn S, Lieberei R. 2015. Fermentation-like
Ghanaian cocoa fermentations. Int J Syst Evol Microbiol 59:7–12 incubation of cocoa seeds (Theobroma cacao L.) reconstruction and guidance
De Bruyne K, Camu N, De Vuyst L, Vandamme P. 2010. Weissella fabaria sp. of the fermentation process. Food Sci Technol 62:357–61.
nov. from Ghanaian cocoa fermentation. Int J Syst Evol Microbiol Koné KM, Guéhi TS, Durand N, Ban-Koffi L, Berthiot L, Tachon FA, Brou
60(9):1999–2005. K, Boulanger R, Montet D. 2016. Contribution of predominant yeasts to
De Vuyst L, Camu N, De Winter T, Vandemeulebroecke K, Van de Perre V, the occurrence of aroma compounds during cocoa bean fermentation. Food
Vancanneyt M, De Vos P, Cleenwerck I. 2008. Validation of the Res Int 89:910–17.
(GTG)(5)-rep-PCR fingerprinting technique for rapid classification and Kim H, Keeney PG. 1984. Epicatechin content in fermented and
identification of acetic acid bacteria, with a focus on isolates from Ghanaian unfermented cocoa beans. J Food Sci 49:1090–2.
fermented cocoa beans. Int J Food Microbiol 125(1):79–90.
Kratzer U, Frank R, Kalbacher H, Biehl B, Wöstemeyer J, Voigt J. 2009.
De Vuyst L, Lefeber T, Papalexandratou Z, Camu N. 2010. The functional Subunit structure of the vicilin-like globular storage protein of cocoa seeds
role of lactic acid bacteria in cocoa bean fermentation. In: Mozzi F, Raya and the origin of cocoa- and chocolate-specific aroma precursors. Food
RR, Vignolo GM, editors. Biotechnology of lactic acid bacteria: novel Chem 113:903–13.
applications. Oxford UK: Wiley-Blackwell. p 301–25.
Kostinek M, Ban-Koffi L, Ottah-Atikpo M, Teniola D, Schillinger U,
Dillinger TL, Barriga P, Escárcega S, Jimenez M, Lowe DS, Grivetti LE. Holzapfel WH, Franz CM. 2008. Diversity of predominant lactic acid
2000. Food of the gods: cure for humanity? A cultural history of the bacteria associated with cocoa fermentation in Nigeria. Curr Microbiol
medicinal and ritual use of chocolate. J Nutri 130(8):2057–72. 56(4):306–14.
Forsyth WGC, Quesnel VC. 1963. The mechanism of cacao curing. Adv Lagunes Gálvez S, Loiseau G, Paredes JL, Barel M, Guiraud JP. 2007. Study
Enzymol Relat Areas Mol Biol 25:457–92. on the microflora and biochemistry of cocoa fermentation in the
Fowler MS. 1994. Fine or flavor cocoas: current position and prospects. Dominican Republic. Int J Food Microbiol 114(1):124–30.
Cocoa growers’ bulletin. Bournville: Cadbury Ltd. p 17–23. Leal GA, Gomes LH, Efraim P, De Almeida Tavares FC, Figueira A. 2008.
Fowler MS. 1999. Cocoa beans: from tree to factory. In: Beckett ST, editor. Fermentation of cacao (Theobroma cacao L.) seeds with a hybrid
Industrial chocolate manufacture and use. 3rd ed. Oxford: Blackwell Kluyveromyces marxianus strain improved product quality attributes. FEMS
Science. p 8–35. Yeast Res 8:788–98.
Garcia-Armisen T, Papalexandratou Z, Hendryckx H, Camu N, Vrancken Lefeber T, Janssens M, Camu N, De Vuyst L. 2010. Kinetic analysis of strains
G, De Vuyst L, Cornelis P. 2010. Diversity of the total bacterial community of lactic acid bacteria and acetic acid bacteria in cocoa pulp simulation
associated with Ghanaian and Brazilian cocoa bean fermentation samples as media toward development of a starter culture for cocoa bean fermentation.
revealed by a 16 S rRNA gene clone library. Appl Microbiol Biotechnol Appl Environ Microbiol 76(23):7708–16.
87(6):2281–92. Lefeber T, Gobert W, Vrancken G, Camu N, De Vuyst L. 2011a. Dynamics
Giraffa G, Neviani E. 2001. DNA-based, culture-independent strategies for and species diversity of communities of lactic acid bacteria and acetic acid
evaluating microbial communities in food-associated ecosystems. Int J Food bacteria during spontaneous cocoa bean fermentation in vessels. Food
Microbiol 67:19–34. Microbiol 28(3):457–64.
Hall, GD, Tarka SM, Hurst WJ, Stuart D, Adams REW. 1990. Cacao Lefeber T, Janssens M, Moens F, Gobert W, De Vuyst L. 2011b. Interesting
residues in ancient Maya vessels from Rı́o Azul, Guatemala. Ameri Antiq starter culture strains for controlled cocoa bean fermentation revealed by
55(1):138–43. simulated cocoa pulp fermentations of cocoa-specific lactic acid bacteria.
Handelsman J. 2004. Metagenomics: application of genomics to uncultured Appl Environ Microbiol 77(18):6694–8.
microorganisms. Microbiol Mol Biol Rev 68:669–85. Lefeber T, Papalexandratou Z, Gobert W, Camu N, De Vuyst L. 2012.
Henderson JS, Joyce RA, Hall GR, Hurst WJ, McGovern PE. 2007. On-farm implementation of a starter culture for improved cocoa bean
Chemical and archaeological evidence for the earliest cacao beverages. Proc fermentation and its influence on the flavor of chocolates produced thereof.
Natl Amer Sci 104(48):18937–40. Food Microbiol 30(2):379–92.
Ho VT, Zhao J, Fleet G. 2014. Yeasts are essential for cocoa bean Lima LJ, Almeida MH, Nout MJ, Zwietering MH. 2011. Theobroma cacao L.,
fermentation. Int J Food Microbiol 174:72–87. “The food of the Gods”: quality determinants of commercial cocoa beans,
with particular reference to the impact of fermentation. Crit Rev Food Sci
Ho VT, Zhao J, Fleet G. 2015. The effect of lactic acid bacteria on cocoa Nutr 51(8):731–61.
bean fermentation. Int J Food Microbiol 205:54–67.
Lopes AS, Dimick PS. 1995. Cocoa fermentation. In: Reed G,
Hurst WJ, Tarka SM, Powis TG, Valdez F, Hester TR. 2002. Archaeology: Nagodawithana TW, editors. Enzymes, biomass, food, and feed. Weinheim:
cacao usage by the earliest Maya civilization. Nature 418(6895):289–90. VCH. p 561–77.
Hurst WJ, McGovern PE. 2007. Chemical and archaeological evidence for Lopez AS, Dimick PS, Walsh RM. 1987. Scanning electron microscopy
the earliest cacao beverages. PNAS 104(48):18937–40. studies of the cellular changes in raw, fermented and dried cocoa beans.
Illeghems K, De Vuyst L, Papalexandratou Z, Weckx S. 2012. Phylogenetic Food Struc 6(1):9–16.
analysis of a spontaneous cocoa bean fermentation metagenome reveals new Lopez UV, Pires JL. 2014. Botany and production of cocoa. In: Schwan RF,
insights into its bacterial and fungal community diversity. PLoS One Fleet GH, editors. Cocoa and coffee fermentation. Boca Raton: CRC
7(5):38040. Taylor & Francis. P 43–71.
Illeghems K, Weckx S, De Vuyst L. 2015. Applying meta-pathway analyses Martelli HL, Dittmar HF. 1961. Cacao fermentation. V. Yeasts isolated from
through metagenomics to identify the functional properties of the major cacao beans during the curing process. Appl Microbiol 9:370–1.
bacterial communities of a single spontaneous cocoa bean fermentation Mayo B, Rachid CTCC, Alegrı́a Á, Leite AMO, Peixoto RS, Delgado S.
process sample. Food Microbiol 50:54–63. 2014. Impact of next generation sequencing techniques in food
microbiology. Curr Geno 15(4):293–309.
International Cocoa Organization (ICCO). 2010. International cocoa
agreement. London: (Online). McNeil CL. 2006. Traditional cacao use in modern Mesoamerica. In:
McNeil CL, editor. Chocolate in Mesoamerica: a cultural history of cacao.
International Cocoa Organization (ICCO). 2014a. Quarterly bulletin of Gainesville: University Press of Florida. p 341–66.
cocoa statistics. 2014/15. Vol. XLI, No. 1. (Online).
Meersman E, Steensels J, Mathawan M, Wittocx PJ, Saels V, Struyf N,
International Cocoa Organization (ICCO). 2014b. Cocoa market situation. Bernaert H, Vrancken G, Verstrepen KJ. 2013. Detailed analysis of the
2013/2014. London. (Online).

microbial population in Malaysian spontaneous cocoa pulp fermentations Papalexandratou Z, Camu N, Falony G, De Vuyst L. 2011d. Comparison of
reveals a core and variable microbiota. PLoS One 8(12):81559. the bacterial species diversity of spontaneous cocoa bean fermentations
Meersman E, Steensels J, Paulus T, Struyf N, Saels V, Mathawan M, Koffi J, carried out at selected farms in Ivory Coast and Brazil. Food Microbiol
Vrancken G, Verstrepen KJ. 2015a. Breeding strategy to generate robust 28(5):964–73.
yeast starter cultures for cocoa pulp fermentations. Appl Environ Microbiol Papalexandratou Z, Lefeber T, Bahrim B, Lee OS, Daniel HM, De Vuyst L.
81:6166–76. 2013. Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum,
Meersman E, Steensels J, Struyf N, Paulus T, Saels V, Mathawan M, Allegaert and Acetobacter pasteurianus predominate during well-performed Malaysian
L, Vrancken G, Verstrepen KJ. 2015b. Tuning chocolate flavor through cocoa bean box fermentations, underlining the importance of these
development of thermotolerant Saccharomyces cerevisiae starter cultures with microbial species for a successful cocoa bean fermentation process. Food
increased acetate ester production. Appl Environ Microbiol. (online). Microbiol 35(2):73–85.
Millon RF. 1955. When money grew on trees: a study of cacao in ancient Pereira GVM, Miguel MG, Ramos CL, Schwan RF. 2012. Microbiological
Mesoamerica [PhD thesis]. New York: Columbia University. p 1–604. and physicochemical characterization of small-scale cocoa fermentations and
screening of yeast and bacterial strains to develop a defined starter culture.
Moens F, Lefeber T, De Vuyst L. 2014. Oxidation of metabolites highlights Appl Environ Microbiol 78(15):5395–405.
the microbial interactions and role of Acetobacter pasteurianus during cocoa
bean fermentation. Appl Environ Microbiol 80(6):1848–57. Pereira GVM, Magalhães KT, de Almeida EG, da Silva Coelho I, Schwan
RF. 2013. Spontaneous cocoa bean fermentation carried out in a
Morris D. 1882. Cacao: how to grow and how to cure it. Jamaica: novel-design stainless steel tank: influence on the dynamics of microbial
Government Printing Establishment. p 45. populations and physical-chemical properties. Int J Food Microbiol
Motamayor JC, Risterucci AM, Lopez PA, Lanaud C. 2002. Cacao 161(2):121–33.
domestication I: the origin of the cacao cultivated by the Mayas. Heredity Plessas S, Fisher A, Koureta K, Psarianos C, Nigam P, Koutinas, A. 2008.
89:380–6. Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus,
Motamayor JC, Lachenaud P, Silva e Mota JW, Loor R, Kuhn DN, Brown and L. helveticus for sourdough bread making. Food Chem 106:985–
JS, Schnell RJ. 2008. Geographic and genetic population differentiation of 90.
the Amazonian chocolate tree (Theobroma cacao L.). PLoS One 3:10:e3311. Powis TG, Valdez FJR, Hester TR, Hurst WJ, Tarka SM. 2002. Spouted
Motamayor JC, Mockaitis K, Schmutz J. 2013. The genome sequence of the vessels and cacao use among the Preclassic Maya. Latin Ame Antiq
most widely cultivated cacao type and its use to identify candidate genes 13(1):85–106.
regulating pod color. Geno Bio 14(6):53. Powis TG, Hurst WJ, Rodriguez MC, Ortiz PC, Blake M, Cheetham D,
Nielsen DS, Hønholt S, Tano-Debrah K, Jespersen L. 2005. Yeast Coe MD, Hodgson G. 2007. Oldest chocolate in the New World. Antiq
populations associated with Ghanaian cocoa fermentations analyzed using 81(314):302–5.
denaturing gradient gel electrophoresis (DGGE). Yeast 22(4):271–84. Powis TG, Cyphers A, Gaikwad NW, Grivetti L, Cheong K. 2011. Cacao
Nielsen DA, Teniola OD, Ban-Koffi L, Owusu M, Andersson TS, Holzapfel use and the San Lorenzo Olmec. PNAS 108(21):8595–600.
WH. 2007. The microbiology of Ghanaian cocoa fermentations analyzed Pohl J. 2003. Timeline of major cultures of Mesoamerica. Foundation for the
using culture-dependent and culture independent methods. Int J Food advancement of Mesoamerican studies Inc. (FAMSI). Viewed 12 May 2015.
Micro 114:168–86.
Quesnel VC. 1965. Agents inducing the death of cacao seeds during
Nielsen DS, Schillinger U, Franz MAPC, Bresciani J, Amoa-Awua W, fermentation. J Sci Food Agric 16:441–7.
Holzapfel WH, Jakobsen M. 2007a. Lactobacillus ghanensis sp. nov., a motile
lactic acid bacterium isolated from Ghanaian cocoa fermentations. Int J Sys Roelofsen PA. 1958. Fermentation, drying, and storage of cacao beans. Adv
Evo Microbiol 57:1468–72. Food Res Int 8:225–96.
Nielsen DS, Teniola OD, Ban-Koffi L, Owusu M, Andersson TS, Holzapfel Rohan TA. 1964. The precursors of chocolate aroma: a comparative study of
WH. 2007b. The microbiology of Ghanaian cocoa fermentations analyzed fermented and unfermented cocoa beans. J Sci Food Agric 29:456–9.
using culture-dependent and culture-independent methods. Int J Food Rohan TA, Connell M. 1964. The precursors of chocolate aroma: a study of
Microbiol 114(2):168–86. the flavonoids and phenolic acids. J Food Sci 29:460–3.
Nielsen DS, Jakobsen M, Jespersen L. 2011. Candida halmiae sp. nov., Rohan TA, Stewart T. 1967. The precursors of chocolate aroma: production
Geotrichum ghanense sp. nov. and Candida awuaii sp. nov., isolated from of free amino acids during fermentation of cocoa beans. J Food Sci
Ghanaian cocoa fermentations. Int J Sys Evo Microbiol 60:1460–5. 32:395–8.
Ostovar K, Keeney PG. 1973. Isolation and characterization of Rosemary AJ, Henderson JS. 2010. Forming Mesoamerican taste: cacao
microorganisms involved in the fermentation of Trinidad’s cacao beans. J consumption in formative period contexts. In: Staller J, Carrasco M,
Food Sci 38:611–7. editors. Pre-colombian foodways. New York: Springer. p 157–73.
Ouattara HG, Koffi BL, Karou GT, Sangare A, Niamke SL, Diopoh JK. Samah AO, Ibrahim N, Alimon H, Karim AMI. 1993. Fermentation studies
2008. Implication of Bacillus sp. in the production of pectinolytic enzymes of stored cocoa beans. W J Microbiol Biotechnol 9:603–4.
during cocoa fermentation. World J Microbiol Biotechnol 24:1753–60.
Saltini R, Akkerman R, Frosch S. 2013. Optimizing chocolate production
Ouattara HGL, Reverchon S, Niamke SL, Nasser W. 2011. Molecular through traceability: a review of the influence of farming practices on cocoa
identification and pectate lyase production by Bacillus strains involved in bean quality. Food Control 29:167–87.
cocoa fermentation. Food Microbiol 28(1):1–8.
Santos TF, Santana LK, Santos AC, Silva GS, Romano CC, Dias JC,
Owusu M, Petersen MA, Heimdal H. 2011. Effect of fermentation method, Rezende RP. 2011. Lactic acid bacteria dynamics during spontaneous
roasting, and conching conditions on the aroma volatiles of dark chocolate. J fermentation of cocoa beans verified by culture-independent denaturing
Food Process Preserv 36:446–56. gradient gel electrophoresis. Genet Mol Res 10(4):2702–9.
Passos FML, Silva DO, Lopez A, Ferreira CLLF, Guimaraes WV. 1984. Schnermann P, Schieberle P. 1997. Evaluation of key odorants in milk
Characterization and distribution of lactic acid bacteria from traditional chocolate and cocoa mass by aroma extract dilution analysis. J Agric Food
cocoa bean fermentations in Bahia. J Food Sci 49:205–8. Chem 45:867–72.
Papalexandratou Z, De Vuyst L. 2011a. Assessment of the yeast species Schwan RF, Vanetti MCD, Silva DO, Lopez A, De Moraes CA. 1986.
composition of cocoa bean fermentations in different cocoa-producing Characterization and distribution of aerobic, spore-forming bacteria from
regions using denaturing gradient gel electrophoresis. FEMS Yeast Res cacao fermentations in Bahia. J Food Sci 51:1583–4.
11(7):564–74.
Schwan RF, Rose AH. 1994. Polygalacturonase production byKluyveromyces
Papalexandratou Z, Falony G, Romanens E, Jimenez JC, Amores F, Daniel marxianus: effect of medium composition. J Appl Bacteriol 76:62–7.
HM, De Vuyst L. 2011b. Species diversity, community dynamics, and
metabolite kinetics of the microbiota associated with traditional Ecuadorian Schwan RF, Rose AH, Board RG. 1995. Microbial fermentation of cocoa
spontaneous cocoa bean fermentations. Appl Environ Microbiol beans, with emphasis on enzymatic degradation of the pulp. J App
77(21):7698–14. Bact-Symp Supp 79:96–107.
Papalexandratou Z, Vrancken G, De Bruyne K, Vandamme P, De Vuyst L. Schwan RF, Cooper RM, Wheals AE. 1996. Endopolygalacturonase of the
2011c. Spontaneous organic cocoa bean box fermentations in Brazil are yeast Kluyveromyces marxianus is constitutive, highly active on native pectin
characterized by a restricted species diversity of lactic acid bacteria and and is the main extracellular protein. In: Visser J, Voragen AGJ, editors.
acetic acid bacteria. Food Microbiol 28(7):1326–38. Pectins and pectinases. Amsterdam: Elsevier. p 861–8.
Schwan RF, Cooper RM, Wheals AE. 1997. Endopolygalacturonase Thompson SS, Miller KB, Lopez AS. 2001. Cocoa and coffee. In: Doyle MP,
secretion by Kluyveromyces marxianus and other cocoa pulp-degrading yeasts. Beuchat LR, editors. Food microbiology: fundamentals and frontiers. 2nd
Enzyme Microb Technol 21:234–44. ed. Washington DC: ASM Press p 721–33.
Schwan RF. 1998. Cocoa fermentation conducted with a defined microbial Thompson SS, Miller KB, Lopez AS. 2007. Cocoa and coffee. In: Doyle
cocktail inoculum. Appl Environ Microbiol 64:1477–83. MP, Beuchat LR, editors. Food microbiology: fundamentals and frontiers.
3rd ed. Washington DC: ASM Press. p 837–49.
Schwan RF, Wheals AE. 2004. The microbiology of cocoa fermentation and
its role in chocolate quality. Crit Rev Food Sci Nutr 44(4):205–21. Thompson SS, Miller KB, Lopez A, Camu N. 2013. Cocoa and coffee. In:
Doyle MP, Beuchat LR, Montville TJ, editors. Food microbiology:
Schwan RF, Pereira GVM, Fleet GH. 2015. Microbial activities during fundamentals and frontiers, 4th ed. Washington DC: ASM Press. p 881–
cocoa fermentation. In: Schwan RF, Fleet GH, editors. Cocoa and coffee 99.
fermentations. Florida: CRC Press. p 129–92.
Vail G. 2009. Cacao use in Yucatan among the Pre-hispanic Maya. In:
Schwan RF, Fleet GH. 2015. Cocoa and coffee fermentations. Florida: CRC Grivetti LE, Shapiro HY, editors. Chocolate: history, culture, and heritage.
Press. p 129–92. New Jersey: John Wiley & Sons Inc. p 3–15.
Seawright C. 2012. Life, death, and chocolate in Mesoamerica: The Aztecs Van Hijum SA, Vaughan EE, Vogel RF. 2013. Application of state-of-art
and the Maya; where did the ritual use of cacao originate? Essay for sequencing technologies to indigenous food fermentations. Curr Opin
Archaeology of Ancient Mexico, culminating with the Aztec Empire at Biotechnol 24(2):178–86.
LaTrobe University. Verna R. 2013. The history and science of chocolate. 2013. Malaysian J
Sieuwerts S, de Bok FAM, Hugenholtz J, van Hylckama Vlieg JET. 2008. Pathol 35(2):111–21.
Unraveling microbial interactions in food fermentations: from classical to Voigt J, Heinrichs H, Voigt G, Biehl B. 1994. Cocoa-specific aroma
genomics approaches. Appl Envi Microbiol 74(16):4997–5007. precursors are generated by proteolytic digestion of the vicilin-like globulin
Smid EJ, Hugenholtz J. 2010. Functional genomics for food fermentation of cocoa seeds. Food Chem 50:177–84.
processes. An Rev Food Sci Technol 1:497–519. Voigt J, Biehl B. 1995. Precursors of the cocoa specific aroma components
are derived from the vicilin-class (7S) globulin of the cocoa seeds by
Snauwaert I, Papalexandratou Z, De Vuyst L, Vandamme P. 2013.
proteolytic processing. Botanica Acta 108:283–9.
Characterization of strains of Weissella fabalis sp. nov. and Fructobacillus
tropaeoli from spontaneous cocoa bean fermentations. Int J Syst Evol Wood GAR. 1984. History and development. In: Wood GAR, Lass RA,
Microbiol 63:1709–16. editors. Cocoa. 4th ed. Oxford, United Kingdom: Blackwell Science. p
1–10.
Stark T, Bareuther S, Hofmann T. 2005. Sensory-guided decomposition of
roasted cocoa nibs (Theobroma cacao) and structure determination of taste Young AM. 2007. The chocolate tree: a natural history of cacao. Gainesville,
active polyphenols. J Agric Food Chem 53:5407–18. FL: University Press of Florida.
Ziegleder G. 2009. Flavor development in cocoa and chocolate. In: Beckett
Swiegers JH, Bartowsky EJ, Henschke PA, Pretorius IS. 2005. Yeast and ST, editor. Industrial chocolate manufacture and use. York, UK: Blackwell
bacterial modulation of wine aroma and flavor. Aust J Grape Wine Res Publishing. p 169–91.
11:139–73.


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Food Evolution: The Impact of Society and Science

on the Fermentation of Cocoa Beans

Introduction As a building block for future work, our review is comprised of 2

seeds do not produce cocoa flavor on roasting, the development

The Study of Fermentation

Variety Country Fermentation Metabolites Metabolites Cocoa

C 2017 Institute of Food Technologists®

Variety Country Fermentation Metabolites Metabolites Cocoa

C 2017 Institute of Food Technologists®

Trinitario,B3:96ha and Bacillus licheniformis. 1.6, CA0-F :9-4,7.4-3.5,

Variety Country Fermentation Metabolites Metabolites Cocoa

C 2017 Institute of Food Technologists®

Variety Country Fermentation Metabolites Metabolites Cocoa

C 2017 Institute of Food Technologists®

Variety Country Fermentation Metabolites Metabolites Cocoa

C 2017 Institute of Food Technologists®

Variety Country Fermentation Metabolites Metabolites Cocoa

C 2017 Institute of Food Technologists®

strains from citric acid 78,145-65,

Variety Country Fermentation Metabolites Metabolites Cocoa

C 2017 Institute of Food Technologists®

Variety Country Fermentation Metabolites Metabolites Cocoa

C 2017 Institute of Food Technologists®

Variety Country Fermentation Metabolites Metabolites Cocoa

C 2017 Institute of Food Technologists®

Variety Country Fermentation Metabolites Metabolites Cocoa

C 2017 Institute of Food Technologists®

0 OA0-F :0 S0-F :17-0,G0-F :1-4,

Variety Country Fermentation Metabolites Metabolites Cocoa

25, AAM :15

C 2017 Institute of Food Technologists®

identical to those processed through natural fermentation. Later, https://www.intechopen.com/books/genetic-diversity-in-plants/defining-

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