Food Microbiology 28 (2011) 1326e1338

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Spontaneous organic cocoa bean box fermentations in Brazil are characterized

by a restricted species diversity of lactic acid bacteria and acetic acid bacteriaq
Zoi Papalexandratou a, Gino Vrancken a, Katrien De Bruyne b, Peter Vandamme b, Luc De Vuyst a, *
a
Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2,
B-1050 Brussels, Belgium
b
Laboratory of Microbiology, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: Spontaneous organic cocoa bean box fermentations were carried out on two different farms in Brazil.
Received 1 February 2011 Physical parameters, microbial growth, bacterial species diversity [mainly lactic acid bacteria (LAB) and
Received in revised form acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from
19 May 2011
the fermented dry cocoa beans. The main end-products of the catabolism of the pulp substrates (glucose,
Accepted 1 June 2011
Available online 12 June 2011
fructose, and citric acid) by yeasts, LAB, and AAB were ethanol, lactic acid, mannitol, and/or acetic acid.
Lactobacillus fermentum and Acetobacter pasteurianus were the predominating bacterial species of the
fermentations as revealed through (GTG)5-PCR fingerprinting of isolates and PCR-DGGE of 16S rRNA gene
Keywords:
Organic cocoa
PCR amplicons of DNA directly extracted from fermentation samples. Fructobacillus pseudoficulneus,
Fermentation Lactobacillus plantarum, and Acetobacter senegalensis were among the prevailing species during the initial
Diversity phase of the fermentations. Also, three novel LAB species were found. This study emphasized the
(GTG)5-PCR possible participation of Enterobacteriaceae in the cocoa bean fermentation process. Tatumella ptyseos
16S rRNA gene-PCR-DGGE and Tatumella citrea were the prevailing enterobacterial species in the beginning of the fermentations as
Chocolate revealed by 16S rRNA gene-PCR-DGGE. Finally, it turned out that control over a restricted bacterial
species diversity during fermentation through an ideal post-harvest handling of the cocoa beans will
allow the production of high-quality cocoa and chocolates produced thereof, independent of the
fermentation method or farm.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction De Vuyst et al., 2010). The spontaneous cocoa bean fermentation

Nowadays, high-quality cocoa beans are of special interest, as
new consumer trends require chocolates with unique characteris-
tics and beneficial health effects (Ding et al., 2006; Nebesny et al.,
2007; Beckett, 2009). Therefore, current research focuses on the
development of new products by trying to understand and control
all factors that could affect the quality of chocolate, in particular the
cocoa bean fermentation process (Holm et al., 1993; Schwan, 1998;
Afoakwa et al., 2008; Hii et al., 2009; De Vuyst et al., 2010).
Development of the organoleptic properties of chocolate already
starts during post-harvest processing of the cocoa beans (Schwan
and Wheals, 2004; Afoakwa et al., 2008). Traditional fermentation
of cocoa beans followed by natural sun-drying represents the first
step of cocoa bean curing and hence of chocolate production
(Fowler et al., 1998; Wood and Lass, 2001; Thompson et al., 2007;
q All the authors are partners of the Flanders Research Consortium on Fermented
Foods and Beverages.
* Corresponding author. Tel.: þ32 2 6293245; fax: þ32 2 6292720.
E-mail address: ldvuyst@vub.ac.be (L. De Vuyst).
process differs from country to country, as it is often carried out
according to typical local ways, differing in fermentation method,
duration of the fermentation, and other operational practices on the
farm (pod/bean selection, periodical mixing, etc.), which may all
impact the quality of the final products (Ardhana and Fleet, 2003;
Lagunes-Gálvez et al., 2007; Nielsen et al., 2005, 2007b; Camu
et al., 2007, 2008a,b; Kostinek et al., 2008). In general, the seeds
of the cocoa tree, Theobroma cacao L., are manually removed out of
the freshly harvested cocoa pods, upon which fermentation starts,
as the carbohydrate-rich pulp that surrounds the beans is imme-
diately contaminated with the microbiota from the environment
(Ardhana and Fleet, 2003; Schwan and Wheals, 2004; Jespersen
et al., 2005; Thompson et al., 2007; De Vuyst et al., 2010).
Complex biochemical reactions outside and inside the beans are the
main consequence of microbial activities. Yeasts and lactic acid
bacteria (LAB) are the predominating microbial species at the initial
phases of the fermentation process (Schwan and Wheals, 2004;
Daniel et al., 2009; De Vuyst et al., 2010). The major constituents
of the fresh pulp, namely glucose, fructose, and citric acid, are fer-
mented into mainly ethanol and lactic acid. As a consequence, the

0740-0020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2011.06.003
 Z. Papalexandratou et al. / Food Microbiology 28 (2011) 1326e1338
microenvironment of the fermenting substrate changes, allowing
activities by obligate aerobic acetic acid bacteria (AAB) that are able
to grow through oxidation of the ethanol produced by the yeasts
into acetic acid (Schwan and Wheals, 2004; De Vuyst et al., 2010).
During fermentation, ethanol and acetic acid diffuse into the beans,
and this, in combination with the heat produced, causes the death
of the seed embryo. Consequently, the internal structure of the
cocoa beans breaks down, releasing all compounds of the storage
and pigment cells that can now interact through biochemical
reactions of activated endogenous hydrolases to develop flavor and
color precursors.
Today, Brazil represents the main cocoa producer of Latin
America, which corresponds to 4% of the global cocoa production
(www.worldcocoafoundation.org). In the early 1990s, a fungal
infection (i.e., witches’ broom disease) destroyed large cocoa
plantations (de Arruda et al., 2003; Griffith et al., 2003; Scarpari
et al., 2005). Today, cocoa manufacturers use witches’ broom-
resistant clones of the cocoa tree and cultivate organic cocoa
(Pereira et al., 1996; Anonymous, 2000; Faleiro et al., 2004;
Mondego et al., 2008). Unfortunately, Brazilian cocoa possesses
strongly acidic flavor, which is an undesirable characteristic for
chocolate making (Passos and Passos, 1985; Wood and Lass, 2001).
In the past, most of the microbiological studies of the cocoa bean
fermentation process worldwide were based on traditional plating,
isolation, and identification of the microbial colonies (Passos et al.,
1984a,b; Schwan et al., 1986; Ardhana and Fleet, 2003; Lagunes-
Gálvez et al., 2007). In particular, investigations of the microbiota
of Brazilian cocoa bean fermentations have been restricted mainly
to box fermentations and the isolation of yeasts and bacteria for
their phenotypic characterization (Passos et al., 1984a,b; Passos and
Passos, 1985; Schwan et al., 1986, 1995). Also, the impact of a starter
culture consisting of essential yeast and cocoa-specific LAB and AAB
species on the course of Brazilian cocoa bean box fermentations has
been reported (Schwan, 1998). All these studies have emphasized
the acidity of Brazilian cocoa. Further, they have revealed that the
initial phases of Brazilian cocoa bean box fermentations are
dominated by Lactobacillus plantarum, while Lactobacillus del-
brueckii, Leuconostoc mesenteroides, and Pediococcus acidilactici are
present at the end of the fermentations (Passos et al., 1984b).
Acetobacter aceti and Acetobacter pasteurianus are the main AAB
species (Passos and Passos, 1985). Additional studies have revealed
the involvement of Bacillus species in the later phases of Brazilian
cocoa bean fermentations, when the temperature in the boxes is
>50  C (Schwan et al., 1986). Recent sequencing data of 16S rRNA
gene clone libraries obtained from Brazilian cocoa bean fermenta-
tion samples have suggested a restricted bacterial species diversity,
including Lactobacillus fermentum and A. pasteurianus, during cocoa
bean box fermentations carried out with raw material of high
quality, clean equipment, and regular mixing (Garcia-Armisen
et al., 2010).
Recent studies of the LAB and AAB communities involved in
Ghanaian cocoa bean fermentations employed a multiphasic
approach, encompassing culture-dependent and -independent
methods as well as metabolite target analyses (Nielsen et al., 2005,
2007b; Camu et al., 2007, 2008b). Also, the study of Brazilian cocoa
bean fermentations requires further investigation as, in particular,
the role of LAB and AAB to get fermented dry cocoa beans of high-
quality needs to be clarified. Therefore, the aim of the present study
was to investigate microbial growth and bacterial species diversity
of traditional Brazilian organic cocoa bean box fermentations
culture-dependently and eindependently, as well as by analyzing
overall metabolism throughout fermentation and evaluating
impact on fermented dry cocoa bean and chocolate quality. Organic
cocoa was used as being representative for sustainable cocoa crop
cultivation.
1327
2. Materials and methods
2.1. Field experiments
During the main crop of 2007 (November 2007), two six-day
spontaneous cocoa bean box fermentations were performed in
Ilhéus (Bahia, Brazil) on two different farms, approximately 10 km
from each other. High-quality organic cocoa from the well-
maintained ‘Hawaii’ (Box 1 fermentation) and ‘Leão De Ouro’ (Box
2 fermentation) plantations was used. Cocoa pods of hybrids of
Criollo and Forastero were collected and opened manually at the
fields with clean machetes. A strict separation of healthy and
infected pods was made to avoid undesirable contamination of the
fermenting cocoa pulp-bean masses, and the placenta was removed
as well. All workers wore gloves. Fresh, selected cocoa beans were
placed into washed wooden containers with a capacity of 75 kg or
into baskets layered with clean sacks, before being transferred to
the final pre-washed wooden fermentation boxes. In the case of the
Box 1 fermentation, 1500 kg of cocoa beans were weighed and put
into a box of 1.2 m  1.2 m  1.0 m, which was placed under a metal
roof to protect the fermentation from bad weather conditions. The
fermenting cocoa pulp-bean mass was mixed after 54, 76, 96, and
120 h of fermentation by transfer of the contents from one box to
another identical box. In the case of the Box 2 fermentation, 1850 kg
of cocoa beans were placed into a box of 1.85 m  1.35 m  1.00 m,
which was standing inside a fermentary room. Mixing of the fer-
menting cocoa pulp-bean mass by transfer to another identical box
took place after 48, 72, 96, and 120 h. The sweatings produced
during both fermentations could drain away through a false bottom
of the boxes. After fermentation, the beans were sun-dried on
a wooden platform, which could be covered with a roof on wheels
in the case of rainfall. After two days of natural sun-drying, the
fermented cocoa beans of the Box 1 fermentation were artificially
dried at 60  C for 18 h, because of heavy rainfall and increased
humidity during sun-drying. Five days of sun-drying were suc-
ceeded by 10 h of controlled artificial drying at 60  C in the case of
the fermented cocoa beans of the Box 2 fermentation.
Samples of 500 g were collected in sterile bags after 0, 6, 12, 24,
30, 36, 48, 54, 60, 72, 84, 96, 120, and 144 h of fermentation and
stored at 20  C; an aliquot was used for immediate culture-
dependent microbiological analysis. Additional samples were
taken immediately after mixing the cocoa pulp-bean mass to
determine its influence on the microbiota. Sampling was always
done once per time point at the same depth of the fermenting
cocoa pulp-bean mass (approximately 40 cm from the upper
surface) but in different points of the boxes, as the periodical
mixing of the fermenting cocoa pulp-bean mass allowed its
homogenization. Also, swab samples corresponding to a surface of
25 cm2 were taken from the environment (pod surfaces; leafs;
workers’ hands; machetes; shovels used for mixing; transport and
fermentation boxes; and sacks). Environmental temperatures were
measured with online temperature recorders (DeltaTRAK, Pleas-
anton, CA, USA). Temperature and pH inside the fermenting cocoa
pulp-bean mass were monitored online with a digital pH 340i
sensor (WTW GmbH, Weilheim, Germany). Also, samples were
collected at the end of the drying process.
2.2. Culture-dependent microbiological analysis
Freshly taken samples, including swab samples, were trans-
ported to the laboratory for immediate plating on selective agar
media after appropriate dilution to enable cell count enumeration
[expressed as colony forming units (CFU) per gram], as described
previously (Camu et al., 2008b). The media used were the
following: malt extract agar (MEA, yeasts; Oxoid, Basingstoke,
1328 Z. Papalexandratou et al. / Food Microbiology 28 (2011) 1326e1338
Hamphsire, UK), plate count agar (PCA, total aerobic bacteria;
Oxoid), de Man-Rogosa-Sharpe agar (MRS, LAB; Oxoid), deoxy-
cholate mannitol sorbitol agar (DMS, AAB; Guiraud, 1998), and
acetic acid medium agar (AAM, AAB; Lisdiyanti et al., 2003). All agar
media were incubated at 37  C for 2e4 days. For a statistically
relevant investigation of the species diversity, 35% of the colonies
were picked up from MRS, DMS, and AAM agar of an appropriate
dilution, transferred into tubes containing 10 ml of the corre-
sponding medium, and grown at 37  C for another 2e4 days. This
resulted in a total of 1594 isolates: 632 from MRS agar, 452 from
DMS agar, and 510 from AAM agar. All isolates were stored in
cryovials containing the appropriate medium supplemented with
25% (vol/vol) of glycerol as a cryoprotectant. In addition, colonies
were washed off from MRS agar of a low sample dilution (100- to
1000-fold dilution) with 10 ml of saline (0.85%, wt/vol, NaCl) for
DNA extraction and analysis through denaturing gradient gel
electrophoresis (DGGE).
For classification and preliminary identification of these isolates,
their purity was checked, a catalase test was performed, and colony
morphology was examined (Camu et al., 2007). Total genomic DNA
was extracted from stock cultures that were propagated twice in
the appropriate medium and subjected to (GTG)5-PCR finger-
printing and numerical cluster analysis (with the Pearson correla-
tion coefficient), following the protocol described previously (Camu
et al., 2007; Papalexandratou et al., 2009). Representative isolates
of each (GTG)5-PCR cluster were subjected to further polyphasic
identification through partial 16S rRNA gene sequence analysis
(LAB and AAB) (Camu et al., 2007) and/or full 16S rRNA, pheS, atpA,
and rpoA gene sequence analysis (only LAB) (De Bruyne et al.,
2007).
2.3. Culture-independent microbiological analysis
2.3.1. Extraction of DNA from cocoa bean fermentation samples
Direct extraction of DNA from fermenting cocoa pulp-bean
samples was performed as follows. Collection and lysis of the
cells and phenol-chloroform-isoamyl alcohol extraction of their
DNA were performed according to the protocol of Camu et al.
(2007) with minor modifications (Lefeber et al., 2011). Briefly, to
get rid of cocoa pulp compounds potentially inhibiting polymerase
chain reactions (PCR), such as polysaccharides, proteins, enzymes,
and polyphenols, a NucleoSpin column (Macherey Nagel GmbH,
Düren, Germany) was used for further purification of the DNA-
containing aqueous phase, following the manufacturer’s instruc-
tions. All DNA samples were diluted to a final concentration of
50 ng/ml (Garcia-Armisen et al., 2010).
2.3.2. 16S rRNA gene-PCR-DGGE
A pair of primers that targets the V3-V4 region of the 16S rRNA
gene was used to amplify DNA from LAB species (LAC1-LAC2;
Walter et al., 2001) and a pair of universal primers (357f-518r;
Ercolini et al., 2001) was used to amplify DNA of the V3 region of the
16S rRNA gene of bacteria in general (Vasilopoulos et al., 2008). PCR
conditions, DGGE analyses, gel processing, DGGE band pattern
cluster analysis (with the band-based Dice coefficient), control of
the purity of DNA from excised bands, DNA band sequencing, and
BLAST (Basic Local Alignment Search Tool) analysis of the nucleotide
sequences in the GenBank database (http://www.ncbi.nlm.nih.gov/
BLAST) were performed as described previously (Camu et al., 2007).
2.4. Metabolite target analysis
Cocoa bean fermentation samples, both from pulp and beans, as
well as fermented dry cocoa beans were used to prepare aqueous
extracts for metabolite target analysis as described previously
(Camu et al., 2007). The aqueous extracts of the pulp samples were
centrifuged at 17,000  g at 4  C for 15 min to remove solids. During
the preparation of the bean samples, the centrifugation step was
omitted. To quantify metabolites, a standard addition protocol was
used; therefore, aqueous extracts were further prepared prior to
chromatographic analysis for removal of proteins and addition of
internal standards, as described by Vrancken et al. (2008), except
that proteins were removed with 600 ml of acetonitrile.
To measure the concentrations of acetic acid, citric acid, gluconic
acid, and lactic acid in the aqueous extracts, high performance
anion exchange chromatography (HPAEC) with conductivity under
ion suppression (CIS) was applied, using an ICS 3000 chromato-
graph (Dionex, Sunnyvale, CA, USA), equipped with an AS-19
column (Dionex). The concentrations of glucose, fructose, sucrose,
and mannitol in the aqueous extracts were determined by HPAEC
with pulsed amperometric detection (PAD; Dionex), using an ICS
3000 chromatograph equipped with a CarboPacÔ PA10 column
(Dionex). Aqueous extracts were run under the same conditions as
described previously (Camu et al., 2007).
The concentrations of ethanol present in the aqueous extracts
were measured using a gas chromatograph (Focus GC; Interscience,
Breda, The Netherlands), equipped with a Stabilwax-DA column
(Restek, Bellefonte, PA, USA) and a flame ionization detector (FID),
as described previously (Lefeber et al., 2011). Sample preparations
and ethanol analyses were performed in triplicate and the mean
values  standard deviations are presented.
2.5. Quality assessment of fermented dry cocoa beans
A cut test was performed on the fermented dry cocoa beans by
Barry Callebaut Brasil (Ilhéus, Brazil) to estimate their quality
(Camu et al., 2008a) and categorize them as grade I (maximum 3%
of moldy, slaty, insect damaged, germinated, or flat beans), grade II
(maximum 4% of moldy beans; maximum 8% of slaty beans;
maximum 6% of insect-damaged, germinated, and flat beans), or
sub-grade (if the percentage of defective cocoa beans was higher;
www.cocoafederation.com). Also, their moisture content was
determined (ISO 2291:1980).
2.6. Chocolate production and sensory analysis
Thirty kilograms of fermented dry cocoa beans of each
fermentation were converted into chocolate by Barry Callebaut
France (Louviers, France). The recipe and the procedure followed
for dark chocolate making have been described in detail previously
(Camu et al., 2008a). The chocolates produced were subjected to
a sensory analysis performed by a trained tasting panel of 17
members of Barry Callebaut France (Camu et al., 2008a). The flavor
descriptors were cocoa, sweet, fruity, aroma, roasted, sour, bitter,
and general appreciation, expressed as numerical values between
0 and 6. The chocolates were compared with a reference sample
with standard flavor descriptors to indicate flavor and taste
differences.
3. Results
3.1. Physical changes
The environmental temperature during the two Brazilian cocoa
bean box fermentations varied between 27e29  C and 22e23  C
during day and night, respectively (Fig. 1A and B). The temperature
inside the fermentary room of the Box 2 fermentation was always
2e3  C higher than the environmental temperature (Fig. 1B). The
temperature inside the fermenting cocoa pulp-bean mass increased
throughout the two fermentations, reaching a maximum value of
 Z. Papalexandratou et al. / Food Microbiology 28 (2011) 1326e1338 1329

Fig. 1. Course of the environmental temperature (B), the temperature inside the fermentary room (-), the temperature of the fermenting cocoa pulp-bean mass (continuous line,
left axis), and the pH (A, right axis) during the Box 1 (A) and Box 2 (B) organic cocoa bean fermentations carried out in Brazil. The arrows indicate mixing of the fermenting cocoa
pulp-bean mass during fermentation.

48.8  C (after 94 h) and 50.9  C (after 126 h) in the Box 1 and Box 2
fermentations, respectively. Mixing of the fermenting cocoa pulp-
bean mass had an immediate impact on its temperature, causing
a drop of 3e6  C (Fig. 1A and B). The pH of the fresh pulp was 3.5
and remained stable during the first 36 h of fermentation in the
case of Box 2 (Fig. 1B), whereas it reached a value of 4.0 after 24 h,
followed by a slight drop to pH 3.8, during the Box 1 fermentation
(Fig. 1A). Afterwards, the pH increased to a final value of 4.3 in both
box fermentations (Fig. 1A and B).
3.2. Culture-dependent microbiological analysis
3.2.1. Microbial growth
The application of a culture-dependent microbiological
approach by means of cultivation on selective agar media revealed
the succession of yeasts, LAB, and AAB during the two Brazilian
cocoa bean box fermentations (Fig. 2). The PCA counts increased
during the first 36 h and 54 h of the Box 1 and Box 2 fermentations
[initially 6.5 log (CFU/g) and 5.5 log (CFU/g), respectively] to
a maximum of 8.5 log (CFU/g). Yeasts (MEA counts) were prevailing
during the first 24 h of the fermentations. During the first 12 h of
the Box 2 fermentation, the MEA counts increased from an initial
population of 5.5 log (CFU/g) to 7.2 log (CFU/g). Their population
decreased from a maximum of 7.0 log (CFU/g) at the start and 7.2
log (CFU/g) after 12 h of fermentation in the Box 1 and Box 2
fermentations, respectively, to 2.0 log (CFU/g) and 2.8 log (CFU/g)
after 144 h of fermentation, respectively. LAB counts (MRS agar)
reached their peak [z 9.0 log (CFU/g)] 24 h and 54 h into
fermentation in the Box 1 and Box 2 fermentations, respectively.
The AAB counts (DMS agar) increased as a function of time and
reached approximately 8.0 log (CFU/g) at the later stage of both
fermentations (96e122 h). During both fermentations, colony
counts on AAM agar were higher [z2.0 log (CFU/g)] than those
found on DMS agar, although both media were used to count AAB.
AAM counts followed approximately those of MRS, indicating the
presence of both AAB and LAB.
The plating of swab samples showed that yeast and bacterial
counts were higher on the surface of the equipment used [boxes,
shovels, sacks; approximately 4.3e6.9 log (CFU/g)] than on the
surface of the plant materials [pods, leaves; approximately < 1.0e4.4
log (CFU/g)]. Low DMS [  2.0 log (CFU/g)], AAM [z3.0 log (CFU/g)],
MRS [z3.0 log (CFU/g)], and PCA [z4.0 log (CFU/g)] counts were
found on machetes and workers’ hands.
3.2.2. Identification of the bacterial isolates
Of the total of 1594 bacterial colonies isolated from fermenta-
tion and swab samples, 134 isolates could not be recovered after
transport from Brazil to Belgium. With the help of the catalase test,
in combination with visual examination of the colony morphology,
a fast exclusion from further analysis of non-target bacteria could
be achieved. Almost 50% of the MRS isolates from swab samples
were catalase-positive, while only 11% of the MRS isolates from
fermentation samples were catalase-positive; those catalase-
positive isolates represented non-LAB species that were not
further identified. Among the isolates picked up from the AAM agar,
mostly from fermentation samples, 50% were catalase-negative;
they were identified as Lb. plantarum and Lb. fermentum (see
below). This explains the higher total counts [z2.0 log (CFU/g)] on
AAM agar, compared to DMS agar, as mentioned above. Conse-
quently, purification and preliminary characterization of all
collected isolates resulted in a total of 1003 pure isolates: 473
potential LAB (MRS agar) and 530 potential AAB (287 isolates from
DMS agar and 243 isolates from AAM agar).
Cluster analysis of the (GTG)5-PCR fingerprints of the 473 LAB
isolates and sequence analysis of selected genes of representative
isolates of each cluster showed that Lb. fermentum was by far the

10 10
9 A 9 B
8 8
7 7
log (CFU/g)

log (CFU/g)

6 6
5 5
4 4
3 3
2 2
1 1
0 0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Time (h) Time (h)

Fig. 2. Microbial succession of yeasts (MEA counts, -), LAB (MRS counts, C), AAB (DMS counts, :; AAM counts, ,) and total aerobic bacteria (PCA counts, 6) during the Box 1 (A)
and Box 2 (B) organic cocoa bean fermentations carried out in Brazil. The arrows indicate mixing of the fermenting cocoa pulp-bean mass during fermentation.
1330 Z. Papalexandratou et al. / Food Microbiology 28 (2011) 1326e1338

most prevalent species during both box fermentations, represent-
ing 91.5% of all LAB isolates (Table 1). Most of the other LAB species
were only present at the beginning of the fermentations or were
isolated from swab samples. Few isolates were identified as Lb.
plantarum and Fructobacillus pseudoficulneus. Combination of 16S
rRNA, pheS, rpoA, and atpA gene sequencing data revealed the
isolation of three potentially novel LAB species, a Lactobacillus sp.
nov., a Weissella sp. nov., and a Fructobacillus sp. nov. These LAB
species probably did not play a key role during the fermentations,
as they represented only one (isolate M425), one (isolate M75), and
two (isolates M56 and M622) isolate(s), respectively. Isolate M425
was picked up during the fourth day of the Box 1 fermentation. It
showed low similarity (96.4%) in its 16S rRNA gene with that of
Lactobacillus jensenii ATCC 25258 (GenBank accession number
AF243176). Further multiple gene sequence analysis resulted in
82.3% pheS gene sequence similarity with Lactobacillus amylolyticus
LMG 18796T (GenBank accession number AM087724), 83.9% rpoA
gene sequence similarity with Lactobacillus crispatus LMG 18200,
and 86.5% atpA gene sequence similarity with Lactobacillus intesti-
nalis LMG 14196T. Isolate M75, present at the beginning of the Box 1
fermentation, showed 99.4% 16S rRNA gene sequence similarity
with Weissella ghanensis LMG 24287 (GenBank accession number
AM882998) as well as high similarity in the pheS gene (89.0%) with
W. ghanensis LMG 24286T (GenBank accession number FM202095)
and Weissella fabaria LMG 24289T (GenBank accession number
FM202097). However, the pheS gene sequence difference was too
large to attribute this isolate to one of the two former described
Weissella species. The M56 and M622 isolates, coming from initial
samples of the Box 2 fermentation, showed 99.2% 16S rRNA gene
sequence similarity and 94% atpA gene sequence similarity with
F. pseudoficulneus LC51 (GenBank accession number AY169967) and
LMG 23899 (GenBank accession number AM711274), respectively.
Classification and identification of the 287 DMS isolates through
(GTG)5-PCR fingerprinting and sequencing of the 16S rRNA gene of
representative isolates revealed two main groups, namely
A. pasteurianus (cluster I) and Acetobacter senegalensis (cluster II),
representing 73% and 27% of all DMS isolates, respectively (Fig. 3).
The latter species was mostly isolated from the initial phases of
both box fermentations. A wider AAB species diversity was found
among the isolates from AAM agar, but with A. pasteurianus as the
main representative (z73%) for the two cocoa bean box fermen-
tations. AAB species isolated during the first hours of both box
fermentations were A. senegalensis and Acetobacter fabarum.
Acetobacter peroxydans was only present in the Box 1 fermentation,
whereas Acetobacter indonesiensis and Acetobacter orleanensis
originated from the Box 2 fermentation. Besides good growth of
LAB on AAM agar, this medium allowed the occasional isolation of
non-Acetobacter species, including Gluconobacter oxydans and
Gluconacetobacter saccharivorans. Only few differences were
observed between the AAB species found on AAM agar of the Box 1
and Box 2 fermentations (Table 2).
3.3. Culture-independent microbiological analysis
16S rRNA gene-PCR-DGGE with LAC primers allowed a fast
monitoring of the prevailing LAB communities throughout the
entire Brazilian cocoa bean box fermentations. Lb. fermentum was
definitely the most prevalent LAB species during both box
fermentations, as revealed through sequencing of the very intense
DGGE band migrating approximately to the middle of the DGGE
gels (Fig. 4A and B). Some other LAB species could only be detected
during the first hours of the fermentations. No differences in LAB
species diversity were found between the two box fermentations,
although they were carried out at two different farms. L. plantarum
was detected during the first 30 h of Box 1, but it remained visible
for 92 h during the Box 2 fermentation. A DGGE band of low
intensity, corresponding with Weissella sp., was visible during the
first 12 h of the Box 1 and Box 2 fermentations, as well as a band of
stronger intensity of Leuconostoc pseudomesenteroides in the case of
Box 1. F. pseudoficulneus was mainly present at the beginning of the
Box 2 fermentation (first 30 h). A band at the bottom of the DGGE
gel of Box 2 at 96 h into fermentation corresponded with Lacto-
bacillus reuteri (Fig. 4B).
The DGGE pattern (LAC primers) of the DNAs extracted from the
bulk cells harvested from MRS agar confirmed the results of both
culture-dependent and culture-independent analyses described
above (data not shown). Lb. fermentum gave a strong band in all
DGGE fingerprints, underlining the prevalence of this species.
F. pseudoficulneus was detected in plate washes at 6 and 144 h into
the Box 2 fermentation and Lb. plantarum was more visible in plate
washes of the first 30 h of this fermentation.
The use of universal primers offered additional information
concerning the diversity of non-LAB species, in particular Enter-
obacteriaceae and AAB. Concerning LAB species, only the prevailing
Lb. fermentum could be retrieved. During the first 30 h of both box
fermentations, an intense band was seen in the middle of the DGGE

Table 1
Species diversity of lactic acid bacteria isolated from MRS agar and their isolation source during the Box 1 (231 isolates) and Box 2 (242 isolates) organic cocoa bean
fermentations carried out in Brazil, as revealed by (GTG)5-PCR fingerprinting classification and 16S rRNA, pheS, atpA, and/or rpoA gene sequencing of representative isolates of
each (GTG)5-PCR cluster.

LAB species Number of isolates

Box 1 Box 2

Fermentation samples Swab samples Fermentation samples Swab samples

Enterococcus casseliflavus e e e 1 (leaf)
Lactobacillus amylovorus 2 (end) e e e
Lactobacillus fermentum 188 (throughout) 16 (leaf, pod, hand, boxes, 187 (throughout) 38 (leaf, pod, hand, boxes,
sack, shovel) sack, shovel)
Lactobacillus pentosus e 2 (hand) e e
Lactobacillus plantarum 4 (throughout) 6 (pod, hand, box) 4 (throughout) 1 (leaf)
Lactobacillus sp. nov. 1 (end) e e e
Lactococcus lactis subsp. lactis e 2 (sack) e e
Leuconostoc pseudomesenteroides e 1 (sack) e 2 (machete)
Fructobacillus pseudoficulneus 5 (beginning) e e e
Fructobacillus sp. nov. e e 1 (beginning) 1 (shovel)
Pediococcus acidilactici e e 4 (end) 1 (leaf)
Streptococcus salivarius 2 (beginning) e 1 (beginning) 1 (pod)
Streptococcus sp. 1 (beginning) e e e
Weissella sp. nov. 1 (beginning) e e e
 Z. Papalexandratou et al. / Food Microbiology 28 (2011) 1326e1338 1331

Fig. 3. Cluster analysis of the generated and digitized (GTG)5-PCR fingerprints of the 287 potential acetic acid bacterial isolates from DMS agar. Banding patterns were clustered
together with reference strains by using the unweighted-pair group method with arithmetic averages (UPGMA) clustering algorithm, with correlation levels expressed as
percentage values of the Pearson correlation coefficient. Species validation of representative strains of each cluster was carried out by partial 16S rRNA gene sequence analysis
(indicated by *).

gels that corresponded with an enterobacterial species, Tatumella
ptyseos (Fig. 4C and D). Also, at the same time a band corresponding
with Tatumella citrea was present in the DGGE gel of the Box 2
fermentation. A band corresponding with chloroplast DNA that
could be amplified with these primers too was visible during the
first 30 h of both fermentations. Band (x) in Fig. 4C and D, visible
after 54 and 60 h of the Box 1 and Box 2 fermentations, respec-
tively, and migrating to the same height as the type strain of
A. pasteurianus, could only be revealed at genus level, as it showed
100% similarity with more than one AAB species. This band most
likely corresponded with A. pasteurianus, based on the results of the
culture-dependent analysis.
Mixing of the fermenting cocoa pulp-bean mass did not cause
any direct influence on the bacterial species diversity. Neither did
cluster analysis of the DGGE patterns with the LAC primers of the
samples of the Box 1 and Box 2 fermentations reveal an influence of
the farm on the LAB species diversity (Fig. 4E). Some differences
were only detected during the first hours of the fermentations
1332 Z. Papalexandratou et al. / Food Microbiology 28 (2011) 1326e1338

Table 2
Species diversity of acetic acid bacteria isolated from AAM agar and their isolation source during the Box 1 (108 isolates) and Box 2 (135 isolates) organic cocoa bean
fermentations carried out in Brazil, as revealed by (GTG)5-PCR fingerprinting classification and partial 16S rRNA gene sequencing of representative isolates of each (GTG)5-PCR
cluster.

AAB species Number of isolates

Box 1 Box 2

Fermentation samples Swab samples Fermentation samples Swab samples

Acetobacter fabarum 1 (beginning) e 1 (beginning) e
Acetobacter ghanensis e 1 (transport box) e e
Acetobacter indonesiensis e e 1 (beginning) e
Acetobacter indonesiensis/malorum/cerevisiae e 1 (sack) 2 (beginning) 1 (sack)
Acetobacter lovaniensis/fabarum e e e 1 (sack)
Acetobacter malorum/cerevisiae e 3 (box/machete) 4 (beginning) 9 (sack)
Acetobacter orientalis e e 1 (beginning) 1 (sack)
Acetobacter pasteurianus 39 (throughout) 51 (leaf, pod, hand, 63 (throughout) 24 (leaf, pod, hand,
boxes, sack, shovel) boxes, sack, shovel)
Acetobacter peroxydans 1 (beginning) e e e
Acetobacter pomorum e 1 (box) e e
Acetobacter senegalensis 4 (beginning) 5 (box) 19 (beginning) 5 (hand, leaf, pod, sack)
Gluconacetobacter saccharivorans 1 (beginning) e 1 (beginning) 1 (sack)
Gluconobacter oxydans e e 1 (beginning) e

(Fig. 4E: Box 1, Ia; Box 2, Ib), indicating that the LAB species diversity
stabilized after one day of fermentation, being represented by Lb.
fermentum (Fig. 4E, II). Cluster analysis of the DGGE patterns in the
case of the use of universal primers showed bacterial shifts during
the cocoa bean box fermentations, reflected in the presence of
three clusters representing the beginning (I), the middle (II), and
the end (III) of the box fermentations (Fig. 4F).
3.4. Metabolites
3.4.1. Cocoa bean fermentation samples
With respect to the microbial succession of yeasts, LAB, and AAB
during the two Brazilian cocoa bean box fermentations, microbial
growth resulted in consumption or conversion of the main
components of the fresh pulp. Figs. 5 and 6 include representative
graphs of the course of the main metabolites present in the pulp
and in the cocoa beans during the two box fermentations studied.
Pulp. The composition of the fresh pulp of the cocoa pods
collected at the two separate plantations differed. The fresh pulp
of the Box 1 fermentation consisted of 55.7  0.1 mg/g glucose,
50.5  0.1 mg/g fructose, and 8.1  0.1 mg/g citric acid (Fig. 5A and
E), while the fresh pulp of the Box 2 fermentation contained
42.8  0.1 mg/g glucose, 67.2  0.1 mg/g fructose, and
11.2  0.1 mg/g citric acid (Fig. 5B and F). No sucrose was found in
the pulps. A faster assimilation of both glucose and fructose was
seen in the case of the Box 2 fermentation compared to Box 1, as
these carbohydrates were almost exhausted after 30 h of
fermentation (Fig. 5B). Citric acid was consumed after two days of
both fermentations, but it increased again to final concentrations
of 2.3  0.1 mg/g and 3.5  0.1 mg/g at the end of the Box 1 and
Box 2 fermentations, respectively, because of drying effects
(Fig. 5E and F). A higher mannitol production was found during
the Box 1 fermentation, compared to the Box 2 fermentation,
which started after 24 h and reached a peak after 84 h of
fermentation (24.7  0.3 mg/g). Lactic acid was slightly produced
during the first 36 h of the Box 1 fermentation, followed by an
increase to 4.7  0.1 mg/g when glucose and fructose were
consumed completely (Fig. 5E). For the first 72 h of the Box 2
fermentation, the formation of lactic acid was slow and finally
increased to 4.1  0.1 mg/g (Fig. 5F). Gluconic acid was produced
after 48 h of the Box 1 fermentation, reaching a concentration of
3.1  0.1 mg/g, indicating oxidation of the remaining pulp glucose
(Fig. 5E). Fluctuations in the concentrations of gluconic acid in the
pulp of the Box 2 fermentation were found; it was produced at the
very beginning of that fermentation, then dropped to zero, and
finally formed again upon mixing of the fermenting cocoa pulp-
bean mass (Fig. 5F). Maximum ethanol concentrations were
reached after 54 h of fermentation (6.0  0.1 mg/g in Box 1;
7.1  0.1 mg/g in Box 2) (Fig. 5G and H). Acetic acid was present in
the pulp from the beginning of the fermentations and its
concentration increased to 12.0  0.4 mg/g (96 h) and
18.7  0.1 mg/g (120 h) in the Box 1 and Box 2 fermentations,
respectively (Fig. 5G and H). A decrease of the lactic acid and acetic
acid concentrations of the pulp later into the fermentations was
seen, indicating overoxidation of these organic acids into carbon
dioxide and water by AAB
Beans. Approximately 28.0 mg/g of sucrose was present in the
fresh cocoa beans of both box fermentations and it was converted
into glucose and fructose upon fermentation (Fig. 5C and D). Lactic
acid in the beans of both fermentations remained at low concen-
trations (approximately 1.0 mg/g). No gluconic acid was found in
the beans. Higher concentrations of ethanol were measured in the
beans in comparison with the pulp, indicating its immediate
diffusion into the beans or its direct evaporation from the pulp
during fermentation. The concentration of acetic acid in the beans
rose simultaneously with that produced in the pulp, because of
diffusion of acetic acid from the pulp into the beans.
3.4.2. Fermented dry cocoa beans
No sucrose was found in the beans (Fig. 6), indicating
a successful and complete fermentation. In addition, ethanol as the
main volatile present in the beans evaporated during drying. A
concentration effect of lactic acid and citric acid was found, because
of a decrease of the moisture content of the beans. The concen-
tration of acetic acid in the fermented dry beans was rather low
(approximately 7.5 mg/g), corresponding with a weak sour taste of
the beans (Fig. 6).
3.5. Quality assessment of fermented dry cocoa beans
The fermented dry cocoa beans of the Box 1 fermentation were
classified as grade I, indicating that the quality of these beans was
excellent and ideal for chocolate production. The low percentage of
violet beans (3%) showed that the fermentation process was
finished. The final moisture of the beans was 5.2% and 100 g of beans
corresponded with a total of 88 beans, indicating an individual mass
 Z. Papalexandratou et al. / Food Microbiology 28 (2011) 1326e1338
> 1 g. Unfortunately, the fermented cocoa beans of the Box 2
fermentation were smoke-contaminated during the artificial drying
step. The moisture content (4.4%), which was lower than required,
together with the overall characterization of the beans as smoked,
indicated a serious impact of an inappropriate treatment of the
beans during drying. The beans were obviously over-dried and were
irretrievably damaged by the poorly maintained equipment used for
artificial drying. Ten percent of the beans tested were still violet.
Therefore, they were classified as sub-grade.
3.6. Sensory analysis of chocolate
The trained tasting panel could observe a significant difference
between the dark chocolates made from the fermented dry cocoa
beans of the two Brazilian box fermentations (Fig. 7). None of the
chocolates were characterized as sour. The chocolate coming from
cocoa of the Box 1 fermentation was highly appreciated and was
characterized as a good example of high-quality dark chocolate.
Also, this product received high marks for fruitiness and aroma.
Although the dark chocolate made from fermented dry cocoa beans
of the Box 2 fermentation received positive values concerning
aroma and sweetness, this product was not appreciated because of
the strong smoky taste that dominated all other flavors.
4. Discussion
Up to now, only the Ghanaian cocoa bean fermentation process
has been studied with both culture-dependent and culture-
independent techniques, whether or not in combination with
a metabolite target analysis, in heaps (Jespersen et al., 2005; Nielsen
et al., 2005, 2007b; Camu et al., 2007, 2008a,b) and trays (Jespersen
et al., 2005; Nielsen et al., 2005, 2007b). The present study reports on
such a multiphasic approach applied on Brazilian organic cocoa bean
box fermentations. Unraveling the microbial species diversity of
cocoa bean fermentations worldwide is required to understand this
complex process and eventually allow its control by adapting agri-
cultural and operational practices on the farm and/or by adding
starter cultures (Schwan, 1998; Garcia-Armisen et al., 2010; Lefeber
et al., 2011; Papalexandratou et al., 2011).
In general, a succession of yeast, enterobacterial, LAB, and AAB
activities took place, which was accompanied with a temperature
and pH increase as a result of carbohydrate fermentation, citric acid
assimilation, and ethanol oxidation. These microorganisms origi-
nated from the environment, in particular boxes, shovels, and sacks,
which seemed to be contaminated, despite their washing after each
use. The potential involvement of Enterobacteriaceae in cocoa bean
fermentation has been suggested recently and was confirmed
during the present study (Garcia-Armisen et al., 2010; Lefeber et al.,
2011; Papalexandratou et al., 2011).
A restricted number of prevailing species of LAB and AAB was
found, as revealed through 16S rRNA gene-PCR-DGGE and (GTG)5-
PCR identification of 473 LAB and 530 AAB isolates, namely Lb.
fermentum (main strong DGGE band in the case of LAC and
universal primers; 91.5% of all cultivable LAB) and A. pasteurianus
(DGGE band in the case of universal primers corresponding with
AAB species; 73% of all cultivable AAB), respectively. Both species
have been isolated from cocoa bean fermentations, often as
dominating species, before (Nielsen et al., 2007b; Camu et al., 2007,
2008b; Kostinek et al., 2008). Despite their mesophilic character,
these species show close adaptation to the microenvironment of
the fermenting cocoa pulp-bean mass, allowing their isolation even
at high temperatures (>40  C) and low pH (3.9e4.5). This can be
ascribed to an adapted substrate catabolism and ethanol, acid, and
heat tolerance (Ardhana and Fleet, 2003; Ndoye et al., 2006; Camu
et al., 2007). In the past, Lb. fermentum has been isolated from
1333
maize-based fermented foods, vegetable fermentations, and cas-
sava fermentations, and A. pasteurianus from wine, vinegar, and rice
fermentations (Hammes and Vogel, 1995; Kersters et al., 2006).
Strictly heterofermentative Lb. fermentum (converting citric acid,
fermenting glucose, and using fructose as alternative external
electron acceptor to convert it into mannitol) and ethanol-oxidizing
A. pasteurianus are desirable during cocoa bean fermentation to
obtain volatile acetic acid out of carbohydrates and/or citric acid
and ethanol, respectively (De Vuyst et al., 2010).
During the initial stage of both fermentations of the present
study, a wider LAB and AAB species diversity was found than upon
further fermentation, encompassing mainly Lb. plantarum,
F. pseudoficulneus, and A. senegalensis. A. senegalensis has been found
for the first time during Ghanaian spontaneous cocoa bean heap
fermentations (Camu et al., 2007, 2008b), but was originally isolated
from mango fruit in Senegal (Ndoye et al., 2007). It is not as
competitive as A. pasteurianus (Camu et al., 2007). L. plantarum has
been reported in several studies of cocoa bean fermentations around
the world (Ardhana and Fleet, 2003; Schwan and Wheals, 2004;
Camu et al., 2007; Kostinek et al., 2008), but it seems to be less
adapted than Lb. fermentum with respect to ethanol and heat toler-
ance (Camu et al., 2008b). F. pseudoficulneus has been first isolated
from a ripe fig in the Alentejo region of Portugal (Chambel et al.,
2006). Although not considered a common cocoa-related LAB
species before, it has been detected by 16S rRNA gene-PCR-DGGE
with universal primers during cocoa bean fermentations per-
formed in Ghana (Nielsen et al., 2007b). It represents a later
synonym of Leuconostoc pseudoficulneum (Endo and Okada, 2008),
which is in turn phylogenetically related to Leuc. pseudomesenter-
oides and, therefore, it may have been misidentified before. The new
name is in accordance with its fructose-loving character (Endo et al.,
2009), a property explaining its occurrence in the beginning of the
cocoa bean fermentation process, which is characterized by the
formation of ethanol from glucose by yeasts and co-assimilation of
fructose and citric acid by LAB species as well as fructose catabolism
by fructose-loving LAB species. Although the heterofermentative
Leuc. pseudomesenteroides was not among the prevailing LAB
isolates at the beginning of the Brazilian box fermentations per-
formed during this study, it was occasionally represented as a clear
band in the DGGE gels of samples of both fermentations carried out,
indicating its presence in and hence accidental contamination from
the environment. However, it was not as competitive as other LAB
species, in particular Lb. fermentum. Random contaminants such as
Lb. reuteri do not play a role in the cocoa bean fermentation process.
Finally, three novel species could be isolated from the initial pool of
the LAB communities, namely a Lactobacillus sp., a Weissella sp., and
a Fructobacillus sp., which need further investigation with respect to
their identity. The finding of these new species confirms that fer-
menting cocoa beans are a chest of novel microbial species
(Cleenwerck et al., 2007, 2008; De Bruyne et al., 2008, 2009; Nielsen
et al., 2007a, 2010).
Enterobacteriaceae seemed to participate in the cocoa bean
fermentation process as well. Among them, Tatumella spp.
(T. ptyseos, T. citrea) seemed to be the main representatives during
the first 30 h of fermentation, as revealed by their intense DGGE
bands. Similarly, during the early stages of successful heap and
box fermentations carried out in Ivory Coast in 2006, Erwinia
spp. (Erwinia soli, Erwinia tasmaniensis) and Pantoea sp. were
detected culture-independently (Papalexandratou et al., 2011).
Also, Garcia-Armisen et al. (2010) have found that 2.8e3.3% of the
clones obtained from Ghanaian (60 h) and Brazilian (48 h)
fermentation samples represent enterobacterial species, showing
their decline upon fermentation. Whether they play a determining
role in the course of the cocoa bean fermentation is not known,
although they should be capable to perform pectin degradation
1334 Z. Papalexandratou et al. / Food Microbiology 28 (2011) 1326e1338

Fig. 4. PCR-DGGE patterns with the LAC1-LAC2 primers (35e60% denaturing gradients; A,B) and the universal 357f-518r primers (35e70% denaturing gradients; C,D) of the
amplified 16S rRNA gene fragments of DNA from the respective LAB and bacterial species present in the cocoa bean samples of the Box 1 (A,C) and Box 2 (B,D) organic cocoa bean
fermentations carried out in Brazil. The reference ladders (L) consisted of (a) Lactobacillus plantarum LMG 6907T; (b) Lactobacillus fermentum LMG 6902T; (c) Leuconostoc pseu-
domesenteroides 274 (Camu et al., 2007); (d) Pediococcus acidilactici LMG 11384T; (e) Lactobacillus casei LMG 6904T; (f) Acetobacter pasteurianus LMG 1262T; (g) Gluconacetobacter
europaeus LMG 18890T; and (h) Acetobacter senegalensis LMG 23690T. The lane numbers represent the time (h) of sampling during fermentation. The closest relatives of the
 80 80

70
A 70
B

Concentration (mg/g)
Concentration (mg/g)
60 60

50 50

40 40

30 30

20 20
10
10
0 0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Time (h) Time (h)
40 40
35
C 35
D

Concentration (mg/g)
Concentration (mg/g)

30 30
25 25
20 20
15 15
10 10
5 5
0 0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Time (h) Time (h)
12 12
E F
Concentration (mg/g)
Concentration (mg/g)

9 9

6 6

3 3

0 0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Time (h) Time (h)
20 20
G H
16 16
Concentration (mg/g)

Concentration (mg/g)

12 12

8 8

4 4

0 0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Time (h) Time (h)

Fig. 5. Course of sucrose (A), glucose (C), fructose (:), and mannitol (-) in the pulp (A,B) and beans (C,D) during the Box 1 (A,C) and Box 2 (B,D) organic cocoa bean fermentations
carried out in Brazil, respectively. Course of citric acid (C), lactic acid (:), gluconic acid (-), acetic acid (6), and ethanol (B) in the pulp during the Box 1 (E,G) and the Box 2 (F,H)
organic cocoa bean fermentations, respectively.

fragments sequenced (% of identical nucleotides compared to sequences retrieved from the GenBank database and accession numbers between brackets) were: (i) Lb. plantarum
(100%; EU637402.1); (ii) Weissella sp. (100%; AM882997.1); (iii) Lb. fermentum (100%; EU688978.1); (iv) Leuc. pseudomesenteroides (100%; DQ523483.1); (v) Fructobacillus pseudo-
ficulneus (100%; AY169967.1); (vi) Lactobacillus reuteri (100%; EU626022.1); (vii) Tatumella ptyseos (100%; EU344770.1); (viii) chloroplast DNA (100%; HQ244500.1); (ix) Tatumella
citrea (100%; EU344769.1); and () AAB species (100%; GQ246703.1, HM190252.1, AB485746.1). The arrows indicate points of mixing of the fermenting cocoa pulp-bean mass.
Dendrograms were derived from the cluster analysis of the PCR-DGGE patterns (LAC primers, E; universal primers, F) of the bacterial communities associated with the Box 1 and Box
2 organic cocoa bean fermentations carried out in Brazil, based on the Dice coefficient of similarity (weighted) and obtained with the UPGMA clustering algorithm. (E) Ia and Ib
represent the initial LAB communities during the Box 1 and Box 2 fermentations, respectively, and II the main LAB communities during both fermentations. (F) Shifts at the bacterial
communities during the (I) beginning, (II) middle, and (III) end of the Box 2 fermentation.
1336 Z. Papalexandratou et al. / Food Microbiology 28 (2011) 1326e1338
10
7
Concentration (mg/g)
6
0
Box 1 fermentation
glucose fructose mannitol
Fig. 6. Residual carbohydrates, mannitol, and organic acids in the fermented dry cocoa beans of the Box 1 and Box 2 organic cocoa bean fermentations carried out in Brazil.
(together with certain yeasts and perhaps Bacillus species although
the optimal pH and temperature for pectinolytic activity by the
latter are rather high), citric acid assimilation (together with Lb.
fermentum), and gluconic acid production from glucose during the
initial phase of the cocoa bean fermentation process (Pujol and
Kado, 2002; Garcia-Armisen et al., 2010; Ouattara et al., 2010).
Enterobacteriaceae are, for instance, involved in spontaneous sour
beer fermentation processes (Martens et al.,1991). T. ptyseos has first
been isolated from clinical sources, mainly sputum specimens
(Hollis et al., 1981), but recently it has been reported as a causal
agent of pink disease of pineapple fruit (Marín-Cevada et al., 2010),
a disease mostly associated with T. citrea (Pujol and Kado, 2002). The
latter species, also isolated during the initial stage of the cocoa bean
fermentations of the present study, represents a later synonym of
Pantoea citrea, a species that has been isolated from fruit and soil
samples in Japan (Kageyama et al., 1992; Brady et al., 2010).
Fig. 7. Flavor profiles of the chocolates made with fermented dry cocoa beans of the
Box 1 and Box 2 organic cocoa bean fermentations carried out in Brazil. The flavor
intensity increases from the center to the perimeter. The results were obtained through
a sensory analysis performed by a trained tasting panel of 17 members. The averages
are presented.
Box 2 fermentation
lactic acid acetic acid citric acid gluconic acid
The use of two different agar media for the isolation of AAB,
namely DMS (Guiraud, 1998) and AAM (Lisdiyanti et al., 2003),
enabled the isolation of Acetobacter as well as Gluconacetobacter
and Gluconobacter species. While only Acetobacter species have
been isolated during Ghanaian cocoa bean heap fermentations
(Camu et al., 2007, 2008b), former studies of cocoa bean box
fermentations have reported on the isolation of Gluconobacter
species as well, making use of non-DMS media (Passos and Passos,
1985; Schwan and Wheals, 2004). However, the latter species does
not seem to play a key role during the main fermentation stage, as it
was present only at the beginning of the cocoa bean fermentations
of this study, perhaps through environmental contamination of the
freshly harvested cocoa beans. In addition, it prefers glucose
instead of ethanol for oxidation and hence it is not competitive
enough toward yeasts and LAB species under the initial micro-
aerophilic conditions of the fermenting cocoa pulp-bean mass.
However, it may be responsible for gluconic acid production at the
beginning of the fermentation process as well. Finally, it was shown
that AAM agar was not suitable for AAB enumeration as acid-
tolerant LAB species, such as Lb. fermentum and Lb. plantarum,
could be isolated from this medium too. The presence of ethanol in
and the low pH of AAM agar, in contrast with MRS agar, did not
make the medium suitable for LAB enumeration either. Further-
more, as DMS agar seems to be selective for Acetobacter species
isolation, a combination of the main substrates of the two media
could possibly allow the formulation of an appropriate medium for
AAB enumeration and isolation.
The initial wide bacterial species diversity generally shifts into
the prevalence of a few species upon further fermentation, in
particular Lb. fermentum during the lactic acid fermentation phase
and A. pasteurianus during the acetic acid fermentation phase. Such
a restricted LAB and AAB species diversity during the major part of
the cocoa bean fermentation process seems to be necessary to
obtain well-fermented cocoa beans (Garcia-Armisen et al., 2010;
Lefeber et al., 2011; Papalexandratou et al., 2011), which is reflec-
ted in a homogeneously fermenting cocoa pulp-bean mass,
a predictable change of the microbial communities under the pre-
vailing conditions (substrates, pH, temperature, and oxygen
tension), low-acid fermented dry cocoa beans without residual
sucrose, and flavorsome chocolate, witnessed by the Brazilian
 Z. Papalexandratou et al. / Food Microbiology 28 (2011) 1326e1338
organic cocoa bean box fermentations of the present study. A wide
LAB species diversity and an onset of AAB growth too early during
cocoa bean fermentation results in fermented dry beans with a too
acidic flavor, the reason for which has to be sought for in the
fermentation practices, as has been shown for Brazilian cocoa bean
box fermentations in the past (Passos and Passos, 1985; Garcia-
Armisen et al., 2010; Papalexandratou et al., 2011). Neglecting
strict and careful separation of healthy and infected pods results in
poorly fermented cocoa, which was not the case during the present
study (Clapperton et al., 1994; Sukha, 2003). Further, frequent
mixing of the fermenting cocoa pulp-bean mass, without damaging
the cocoa beans, ensures its homogeneity and uniform fermentation
conditions, in particular in the case of large volumes of fermenting
beans (Wood and Lass, 2001; Camu et al., 2008b). However, the use
of boxes for the fermentation of cocoa beans causes increased
aeration of the fermenting cocoa-pulp-bean mass (Passos et al.,
1984b; Wood and Lass, 2001) and, therefore, enhanced growth of
AAB, in turn resulting in higher concentrations of acetic acid (Passos
et al., 1984b; Wood and Lass, 2001; Lagunes-Gálvez et al., 2007), in
comparison with heap fermentations unless turned frequently
(Camu et al., 2007, 2008b). Finally, the use of advanced equipment,
such as mechanical dryers, requires technical maintenance and
good operational practices too, as well-fermented cocoa beans can
easily be damaged by inferior post-fermentation handling, such as
poor artificial drying, in turn influencing chocolate quality produced
from concomitant damaged beans as experienced in the present
study (Wood and Lass, 2001; Thompson et al., 2007; Beckett, 2009).
5. Conclusions
Taken these and previous data together, it can be concluded that
apparently neither the farm nor the fermentation method influ-
ences the bacterial species diversity and metabolite course of cocoa
bean fermentation processes and hence the quality of fermented
dry cocoa beans and chocolates produced thereof, provided that
local agricultural and operational practices on the farm, encom-
passing crop cultivation, fermentation, and post-fermentation
handling, are carried out with care. A careful selection and pre-
processing of cocoa pods and beans, regular mixing of the fer-
menting cocoa pulp-bean mass, controlled duration of the
fermentation, all resulting in a restricted bacterial species diversity
during fermentation, and controlled drying with the appropriate
equipment will improve post-harvest curing of Brazilian cocoa and
hence make high-quality chocolate production possible, despite its
reputation as of inferior quality. As Lb. fermentum and
A. pasteurianus were the prevalent bacterial species during well-
performed cocoa bean heap and box fermentations in Ghana and
Brazil, they may be used as starter cultures for homogeneous, fast,
and successful controlled fermentation processes in the future.
Acknowledgments
This research was funded by the Research Council of the Vrije
Universiteit Brussel, the Federal Research Policy (Contract C3/00/
17), the Fund for Scientific Research-Flanders, the Flemish Institute
for the Encouragement of Scientific and Technological Research in
the Industry and Barry Callebaut N.V. In particular, we acknowledge
the help of Barry Callebaut Brazil (Flavio Fachinello and Anna-
Carolina Silva) and Barry Callebaut Belgium (Nicholas Camu and
Herwig Bernaert). The cooperation of the local farmers of the
‘Hawaii’ and ‘Leão De Ouro’ plantations (Rodovia Ilhéus/Uruçuca,
Bahia, Brazil) is highly appreciated.
1337
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