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Chapter 17
The Functional Role of Lactic Acid Bacteria in Cocoa
Bean Fermentation
Luc De Vuyst, Timothy Lefeber, Zoe Papalexandratou, and Nicholas Camu
17.1. Introduction
17.1.1. History
The secrets of the cocoa tree, Theobroma cacao L.
(literally food of the gods), were first exploited
around 4000 years ago in the tropical rainforest of
Latin America. The fruits (cocoa pods) of this tree
contain seeds (cocoa beans) that can be processed
into cocoa powder, cocoa mass, and cocoa butter.
Cocoa (beans) is responsible for the unique flavor
and melt-in-the-mouth properties of chocolate. The
Biotechnology of Lactic Acid Bacteria Novel Applications
Edited by Fernanda Mozzi, Ral R. Raya and Graciela M. Vignolo
2010 Blackwell Publishing. ISBN: 978-0-813-81583-1
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303
304
Figure 17.1. Opened cocoa pod with beans embedded in a mucilaginous pulp.
phenols, alkaloids), whereas small volatile molecules, such as ethanol and acetic acid, can easily
penetrate it. Hence, this biological barrier controls
the kinetics of fermentation and concomitant diffusion processes (see below).
The cotyledons are basically made up of different
types of storage cells, namely white lipid/protein/
starch-containing cells and purple polyphenolcontaining cells, with parenchyma plasma forming
a grid between the two types of cells (Fig. 17.2B).
A
Cotyledons
Lipid/protein/starch
storage cells
Radicle
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Polyphenol
storage cells
Plasma
Fat vacuoles
Nucleus
Mitochondria
Pulp
Testa
Starch grains
Polyphenols
Protein granules
Whereas the storage cells of starch granules, aleurone grains, and fat droplets constitute the reserve
material for the embryo and lipids further form a
natural barrier around the water-soluble compounds
(enzymes, substrates, and inhibitors), the larger,
vacuolated, polyphenol-storage cells contain unique
cocoa bean components, including alkaloids (theobromine and caffeine) and polyphenols, both of
which are possibly involved in stress resistance
(light) responses of the plant.
The fat (cocoa butter) of the cocoa bean is important for chocolate production, as it will give chocolate its characteristic texture (Asep et al. 2008;
Beckett 2009). Fat makes up slightly more than 50%
of the mass of dry unfermented cocoa bean cotyledons; Forastero beans possess a higher fat content
than Criollo beans. The cocoa butter present in
the beans is a relatively simple fat, primarily composed of palmitic acid (C16 : 0, saturated, 25%),
stearic acid (C18 : 0, saturated, 35%), and oleic
acid (cis-C18 : 1, monounsaturated, 35%), with
small amounts of myristic, linoleic, linolic, and arachidic acids (5%). Stearic acid, the main fatty acid
in cocoa butter, has been suggested to be a nonatherogenic type of dietary saturated fat (non-cholesterolemic) because of its lower absorption (Ding
et al. 2006).
Cocoa bean quantities of alkaloids vary considerably in ripe seeds of different genetic origins, namely
about 0.7%3.0% of theobromine and 0.1%0.7%
of caffeine. Depending on the amount of anthocyanins, the polyphenol-storage or pigment cells, which
constitute 10%20% (dry mass) of the cotyledons
of cocoa beans, are white to deep purple. Three
groups of cocoa bean polyphenols (14%20% in
total) can be distinguished (Wollgast and Anklam
2000b; Ortega et al. 2008): monomeric catechins
or flavan 3-ols (ca. 37%, m/m), procyanidins or
(epi)catechin oligomers with a degree of polymerization higher than decamers (ca. 58%, m/m),
and anthocyanins or glycosylated derivatives of
anthocyanidins (ca. 4%, m/m). The main catechin is
()-epicatechin with up to 35% of the polyphenol
content. In smaller amounts, (+)-catechin, as well as
traces of (+)-gallocatechin and ()-epigallocatechin,
is found. Beans of the Forastero variety owe their
characteristic violet color to the anthocyanin content.
White beans of the Criollo type contain approximately two-thirds of the amount of polyphenols
found in Forastero beans and do not possess anthocyanins. Both alkaloids and polyphenols are linked
to cocoa bitterness. Besides alkaloids and polyphenols, unfermented cocoa beans mainly contain
alcohols, aldehydes, organic acids, and esters. It
is important to note that plant genetics (cultivar)
and environmental conditions of crop cultivation
(climate, soil quality and richness, water management, shade canopy management, pollination, etc.)
are the first determinants of the final cocoa flavor
(Sukha et al. 2008).
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307
Figure 17.3. The heap fermentation is the simplest and most commonly used method of fermentation on small farms. It
requires the simplest equipment at practically no cost, so that it can be run by a family. In a heap fermentation the wet beans
are piled on banana or plantain leaves, which are spread out in a circle on the ground, sometimes raised above soil level to
allow easy pulp drainage. When the heap is complete it is covered with more leaves, and these are often held in place by
small logs. The cover protects the fermenting mass against surface mold growth and keeps the heat inside. In general, sweatings are allowed to flow away and penetrate into the ground. The size of the heaps varies widely; heaps from about 25 to
2000 kg are common. This method is used throughout West Africa and almost exclusively in Ghana, where farmers are fermenting their beans in heaps of 200 to 500 kg for 6 days with care. The heap fermentation method has been used to produce
some of the worlds finest standard cocoa available, especially in Ghana.
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10
9
8
7
6
5
4
3
2
1
0
24
48
72
Time (h)
96
120
144
Figure 17.4. Population dynamics and succession of microbial activities during cocoa bean-pulp mass fermentation. LAB
(
); AAB (
); yeast (
); spore-forming bacteria (
); and filamentous fungi (
). The numbers indicate
maximal concentrations of ethanol (1), lactic acid (2), and acetic acid (3), and the maximum temperature (4).
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Table 17.1. Diversity of lactic acid bacteria associated with cocoa bean fermentations in different countries
Fermentation method
Microorganisms found
Reference
Trinidad
Box
Ghana
Box
Lact. collinoides/fermentum/mali/plantarum
Ghana
Ghana
Heap
Malaysia
Box
Lact. collinoides/plantarum
Brazil
Box
Belize
Box
Indonesia
Box
Lact. cellobiosus/hilgardii/plantarum
Dominican
Republic
Box
Nigeria
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data indicate that Lact. plantarum and Lact. fermentum seem to be indigenous to the fermentation of
cocoa beans worldwide. Moreover, Lact. fermentum
seems to play a significant role (see below).
Although a few yeasts assimilate citric acid
(Jespersen et al. 2005; Daniel et al. 2009), LAB
species are responsible for the breakdown of citric
acid during cocoa bean fermentation. Citric acid is
converted via oxaloacetate into acetate and pyruvic
acid, the latter, in turn, being converted into endproducts of pyruvate metabolism (lactic acid, acetic
acid, acetaldehyde, ethanol, diacetyl, etc.; Camu
et al. 2007, 2008a). Consumption of citrate by Leuc.
pseudomesenteroides (heterofermentative), Lact.
plantarum (homofermentative), and Lact. fermentum (heterofermentative) at the early stages of
Ghanaian cocoa bean heap fermentation, under low
pH conditions, explains the importance of these bacteria during this stage of the fermentation process,
thereby avoiding competition with the depectinizing
(citrate-negative) yeasts, which meanwhile degrade
sugars to ethanol anaerobically. Dominance of specific strains of Lact. fermentum throughout cocoa
bean fermentation, usually in succession of Lact.
plantarum that dominates at the beginning of the
fermentation, can be ascribed to their competitive
metabolism (efficient use of glucose and fructose as
energy source and external electron acceptor,
respectively), their acid and ethanol tolerance (lower
than for Lact. plantarum), and their higher oxygen
tolerance than Lact. plantarum (Camu et al. 2007,
2008a). Assimilation of citric acid causes the pH of
the cocoa pulp to increase from pH 3.5 to 4.25.0,
allowing the growth of other bacteria (Camu et al.
2007, 2008a). Another important feature of Lact.
fermentum growth is the production of mannitol out
of fructose, allowing more acetic acid to be produced, which takes place simultaneously with the
conversion of citric acid via pyruvate into acetic
acid, from a physiological point of view to regenerate the NAD+ cofactor and produce extra ATP,
explaining good growth of Lact. fermentum strains
at low pH (Camu et al. 2007, 2008a). Moreover,
metabolic connections exist between citrate fermentation and processes such as amino acid interconversion and mannitol production. As is the case for the
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Table 17.2. Functional role of lactic acid bacteria associated with cocoa bean fermentation.
Functional property
Causes a rapid start up of the fermentation under low pH conditions, avoiding competition
with sucrose- (glucose-)consuming, citrate-negative, depectinizing yeasts
Causes a pH increase (replacement of citric acid by lactic acid and acetic acid), in turn
controlling bacterial growth
Stimulates amino acid conversions
Production of mannitol
Favors production of acetic acid (volatile) instead of lactic acid (nonvolatile) out of fructose
Beneficial for the growth of AAB
Provides the necessary precursors for further flavor and color development during secondary
processing
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Carbohydrates
Pectins
Pulp
Hemicelluloses
Organic acids
Fermentable sugars
Microbial activity
Ethanol
Theobromine, Caffeine
Epicatechin, Catechin
Procyanidins
Complex tannins
Sugars
Anthocyanins, Cyanidins
Proteins, Peptides, Amino acids
Lactic
acid
Heat
Acetic
acid
Death of embryo
liberates enzymes
and substrates
Bean
Heat
CO2 + H2O
Figure 17.5. Biochemical changes and diffusion processes in pulp and beans during cocoa bean fermentation (after Lopez
1986).
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17.10.1. Rationale
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17.11. Conclusions
To conclude, it will be a challenge to introduce the
application of starter cultures in cocoa fermentation
practices, either to speed up and better control the
fermentation process or to target the fermentation
process toward predefined end-products. However,
to scale up the initiation of cocoa bean fermentation
with a defined inoculum to the farm level, whether
applied to small farm holdings or large estates, will
be an even greater challenge, especially as it relates
to starter culture production and maintenance,
inoculation and fermentation methods, and, last but
not least, extra costs.
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