RESEARCH ARTICLE
Assessment of the yeast species composition of cocoa bean
fermentations in different cocoa-producing regions using
denaturing gradient gel electrophoresis
Zoi Papalexandratou & Luc De Vuyst
Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit
Brussel, Brussels, Belgium
Correspondence: Luc De Vuyst, Research
Group of Industrial Microbiology and Food
Biotechnology (IMDO), Vrije Universiteit
Brussel (VUB), Pleinlaan 2, B-1050 Brussels,
Belgium. Tel.: + 32 2 6293245;
fax: + 32 2 6292720;
e-mail: ldvuyst@vub.ac.be
Received 10 January 2011; revised 8 July
2011; accepted 21 July 2011.
Final version published online 30 August
2011.
DOI: 10.1111/j.1567-1364.2011.00747.x
Editor: Cletus Kurtzman
Keywords
DGGE; yeast; cocoa bean fermentation.
Introduction
YEAST RESEARCH
Fermented, dry cocoa beans are the basic raw material for
chocolate production (Beckett, 2009). The raw cocoa
originates as seeds in fruit pods of the tree, Theobroma
cacao L., and has to undergo a natural fermentation and
drying process to develop essential colour and flavour
precursors (Schwan & Wheals, 2004; De Vuyst et al.,
2010). The latter compounds are transformed during
cocoa bean roasting and chocolate manufacturing into
the final characteristic chocolate flavour (Afoakwa et al.,
2008; Camu et al., 2008a; Beckett, 2009). The fermenta-
tion step plays an important role for the final quality of
the cocoa beans and, consequently, for chocolate pro-
duced thereof. As it remains a spontaneous process,
cocoa bean fermentation is affected by the surrounding
environment and local fermentation practices (Camu
et al., 2007; Thompson et al., 2007; De Vuyst et al., 2010;
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Abstract
The yeast species composition of 12 cocoa bean fermentations carried out in
Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-indepen-
dently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments,
obtained through polymerase chain reaction with universal eukaryotic primers,
was carried out with two different commercial apparatus (the DCode and CBS
systems). In general, this molecular method allowed a rapid monitoring of the
yeast species prevailing during fermentation. Under similar and optimal dena-
turing gradient gel electrophoresis conditions, the CBS system allowed a better
separated band pattern than the DCode system and an unambiguous detection
of the prevailing species present in the fermentation samples. The most fre-
quent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and
Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates
a restricted yeast species composition of the cocoa bean fermentation process.
Exceptionally, the Ivorian cocoa bean box fermentation samples showed a
wider yeast species composition, with Hyphopichia burtonii and Meyerozyma
caribbica among the main representatives. Yeasts were not detected in the sam-
ples when the temperature inside the fermenting cocoa pulp-bean mass reached
values higher than 45 °C or under early acetic acid production conditions.
Papalexandratou et al., 2011). In general, the sterile carbo-
hydrate-rich cocoa pulp that surrounds the cocoa beans in
the fruit pods is rapidly contaminated by microorganisms
present in the environment after opening of the pods
(Nielsen et al., 2005, 2007; Camu et al., 2007, 2008b). Fer-
mentation methods depend on the cocoa-producing region
and include heaps (Ghana, Ivory Coast), trays (Ghana,
Ivory Coast), boxes (Brazil, Malaysia), baskets (Ivory
Coast) and platforms (Ecuador) (Wood & Lass, 2001).
During fermentation, microbial successions occur as
the microenvironment (temperature, pH, oxygen avail-
ability) changes (Schwan & Wheals, 2004; De Vuyst et al.,
2010). The main microbial groups that occur during a
cocoa bean fermentation process are yeasts, lactic acid
bacteria (LAB) and acetic acid bacteria (AAB). Yeasts are
mainly responsible for ethanol production out of glucose
(from sucrose) during the early stages of the fermenta-
tion, when the cocoa pulp-bean mass is anaerobic with
FEMS Yeast Res 11 (2011) 564–574
26S rRNA gene-PCR-DGGE of cocoa bean fermentations
low pH and temperature (Ardhana & Fleet, 2003; Camu
et al., 2007, 2008b). Also, yeasts are the major contribu-
tors to pectinolysis for pulp removal, enabling air ingress
in the cocoa pulp-bean mass (Schwan et al., 1995;
Schwan & Wheals, 2004). LAB produce mainly lactic acid
and/or mannitol out of citric acid and/or fructose under
micro-aerophilic conditions, enabling a slight pH increase
of the fermenting cocoa pulp-bean mass (Camu et al.,
2007, 2008b). AAB appear at the later stage of fermenta-
tion, when more oxygen penetrates the fermenting cocoa
pulp-bean mass, and they mainly oxidize the ethanol pro-
duced by yeasts into acetic acid (Camu et al., 2007,
2008b).
The most frequently isolated yeast species are Saccharo-
myces cerevisiae, Hanseniaspora guilliermondii (anamorph
Kloeckera apis), Hanseniaspora opuntiae, Pichia fermen-
tans, Pichia kluyveri, Pichia kudriavzevii (formerly Issat-
chenkia orientalis, anamorph Candida krusei), Pichia
membranifaciens and Wickerhamomyces anomalus (for-
merly Pichia anomala) (Schwan et al., 1995; Ardhana &
Fleet, 2003; Schwan & Wheals, 2004; Jespersen et al.,
2005; Nielsen et al., 2005, 2007; Lagunes-Gálvez et al.,
2007; Leal et al., 2008; Daniel et al., 2009). Based on a
combination of molecular analyses and morphological
and physiological data, Daniel et al. (2009) have identi-
fied P. kudriavzevii, S. cerevisiae and H. opuntiae as the
main yeast species involved in Ghanaian cocoa bean heap
fermentations, confirming data from earlier studies
(Jespersen et al., 2005; Nielsen et al., 2005, 2007). Fur-
thermore, they have reported on several species that were
not found in cocoa bean fermentation before, such as
Candida carpophila, Candida orthopsilosis, Kodamaea
ohmeri, Meyerozyma caribbica and Saccharomycodes ludwi-
gii (Daniel et al., 2009). Also, the new species Candida
halmiae and Candida awuaii have been reported recently
(Nielsen et al., 2010).
Most of the studies dealing with yeast identification are
based on phenotypic characterization (Carr et al., 1979;
Ardhana & Fleet, 2003), followed by molecular identifica-
tion of the picked up isolates, often based on sequencing
of the D1/D2 domain of the 26S rRNA gene (Jespersen
et al., 2005; Nielsen et al., 2005; Daniel et al., 2009). PCR
denaturing gradient gel electrophoresis (PCR-DGGE) has
been proven to be an easy tool for monitoring the total
yeast species composition in, for instance, cocoa bean
(Nielsen et al., 2007), wine (Cocolin et al., 2000; Stringini
et al., 2009) and sourdough (Meroth et al., 2003) fermen-
tation samples. Using universal eukaryotic primers, the
D1/D2 domain of the 26S rRNA gene is usually targeted,
but other regions such as the 18S rRNA gene may be
used as well (Beh et al., 2006). It has been shown that
this kind of molecular approach overcomes the limita-
tions of culture methods and reveals species that might
FEMS Yeast Res 11 (2011) 564–574
565
fail to produce colonies on selected agar media. Hence, a
more accurate representation of the composition of yeast
species in food fermentation ecosystems may be obtained
(Giraffa, 2004). However, a few drawbacks of this tech-
nique have been reported. One drawback of several yeast
studies using DGGE is the multiple banding patterns dis-
played by single strains (Mills et al., 2002; Masoud et al.,
2004; Prakitchaiwattana et al., 2004; Garofalo et al., 2008;
Ongol & Asano, 2009). Also, some studies mention the
presence of single-stranded DNA (ssDNA) artefacts in the
DGGE patterns, although the actual reason of their
appearance has not been defined (Cocolin et al., 2000;
Masoud et al., 2004). One study has reported on the
influence of the DGGE apparatus on the bacterial and
yeast species composition of the ecosystem studied
(Ascher et al., 2010).
The aim of the present study was to rapidly monitor
the species composition of yeasts involved in traditional
spontaneous cocoa bean fermentations carried out in dif-
ferent cocoa-producing regions, namely Brazil, Ecuador,
Ivory Coast and Malaysia, through 26S rRNA gene-PCR-
DGGE, with a focus on the impact of the different origins
of the cocoa and methods of fermentation (in particular
heap, box and platform).
Materials and methods
Samples
Fermentation samples were available from 12 field experi-
ments set up in Ivory Coast (main crop of 2006; one
farm; heap and box), Brazil [main crop of 2006 (two
farms; box) and main crop of 2007 (two farms; box)],
Ecuador (main crop of 2008; two farms; box and plat-
form) and Malaysia (main crop of 2010; one farm, two
plantations; box). During fermentation, samples of 500 g
were taken every 6–12 h (and after mixing in the case of
box fermentations) from the same depth, but in different
points of the fermenting cocoa pulp-bean mass (c. 40 cm
from the upper surface). Freshly taken samples were
transported to the laboratory for immediate plating on
malt extract agar (MEA; Oxoid, Basingstoke, UK) supple-
mented with 100 mg g 1 oxytetracycline for yeast enu-
meration. Then, all fermentation samples were
temporarily stored at 20 °C before transport on dry ice
to Belgium for further culture-independent analysis. Yeast
counts were obtained after incubation of the agar media
at 37 °C for 1–3 days. This incubation temperature was
selected because of the high temperature inside the fer-
menting cocoa pulp-bean mass, where the samples were
withdrawn from.
Fermentation method, duration of fermentation, mix-
ing/spreading points during fermentation, initial and final
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
566 Z. Papalexandratou & L. De Vuyst

values of pH of and temperature inside the fermenting
cocoa pulp-bean mass and initial and maximum yeast
counts are summarized in Table 1.
DNA extraction
DNA was directly extracted from the fermentation sam-
ples, as described previously (Camu et al., 2007). The ori-
ginal protocol was as follows. Twenty grams of frozen
cocoa beans plus pulp samples were washed twice with
70 mL of saline solution [0.85% (w/v) NaCl]. The com-
bined fluid (±120 mL) was removed by decanting and
subsequently centrifuged at 170 g at 4 °C for 5 min to
remove large particles. The supernatant was filtered
through a coffee filter and the filtrate was centrifuged at
8000 g at 4 °C for 20 min to pellet the cells, which were
subsequently frozen at 20 °C overnight. The thawed pel-
let was washed in 1 mL of TES buffer [6.7% (w/v)
sucrose, 50 mM Tris–HCl, pH 8.0, 1 mM EDTA] and
resuspended in 300 lL of STET buffer [8% (w/v) sucrose,
5% (w/v) Triton X-100, 50 mM Tris–HCl, pH 8.0,
50 mM EDTA]. Seventy-five microlitres of lysis buffer
[TES containing 1330 U mL 1 of mutanolysin (Sigma-
Aldrich, St. Louis, MO) and 100 mg mL 1 of lysozyme
(Sigma-Aldrich)] and 100 lL of a solution of proteinase K
[2.5 mg mL 1 of TE buffer (10 mM Tris–HCl; 1 mM
EDTA, pH 8.0); VWR International, Darmstadt,
Germany] were added for cell lysis, and the suspension was
incubated at 37 °C for 1 h. After the addition of 40 lL
preheated (37 °C) 20% (w/v) sodium dodecyl sulphate in
TE buffer and a pinch of glass beads with a diameter of
150–212 lm (Sigma-Aldrich), cells were vortexed for 60 s
and incubated at 37 °C for 10 min, followed by a 10-min
incubation at 65 °C. One-hundred microlitres of TE buffer
were added, and the lysate was extracted with 1 volume of
phenol–chloroform–isoamyl alcohol (49 : 49 : 1) (Sigma-
Aldrich) for 30 s. Phases were separated by microcentrifu-
gation (18 900 g for 5 min at 4 °C) using Phase Lock Gel
tubes (Eppendorf AG, Hamburg, Germany). To get rid of
cocoa pulp compounds potentially inhibiting PCR, such as
polysaccharides, proteins, enzymes and polyphenols, a Nu-
cleoSpin column (Macherey Nagel GmbH, Düren,
Germany) was used for further purification of the DNA-
containing aqueous phase, following the manufacturer’s
instructions. The final DNA samples (50 ng lL 1) were
stored at 20 °C until further use.
For a few samples, an extra enzymatic treatment with
lyticase (20 mg g 1; Sigma-Aldrich) and lysing enzymes
from Trichoderma harzianum (40 mg g 1; Sigma-
Aldrich) was performed for cell lysis to optimize the
DNA extraction of yeasts (Meroth et al., 2003). How-
ever, as no different DGGE patterns were found, most

Table 1. Cocoa bean fermentations from which samples were available. The origin, fermentation practices and changes in physical parameters
and yeast counts during fermentation are given

Fermentation parameter T (°C) pH Yeast counts (CFU g 1)

Origin of the cocoa bean fermentation Duration (h) Mixing/spreading point (h) Ti Tf pHi pHf (CFU g 1)i (CFU g 1)max; h

Ivory Coast (main crop 2006; 1 farm)

Heap (H) 150 – 24.0 39.6 N/A N/A N/A N/A
Box (B) 150 24; 48 24.0 43.3 N/A N/A N/A N/A
Brazil (main crop 2006; 2 farms)*
Box (F1) 144 – 26.9 42.2 2.6 3.8 N/A N/A
Box (F2) 144 – 30.4 48.2 2.8 4.6 N/A N/A
Brazil (main crop 2007; 2 farms)†
Box (B1) 144 54; 76; 96; 120 25.6 48.5 3.5 4.3 7.09 7.16; 12
Box (B2) 144 48; 72; 96; 120 25.6 47.6 3.4 4.3 5.48 7.22; 12
Ecuador (main crop 2008; 2 farms)
Platform (P1; cocoa from farm 1) 96 50; 72 (spreading for 3 h) 25.9 46.3 3.9 4.5 4.85 7.88; 72
Box (I1; cocoa from farm 1) 96 24; 72 28.7 46.5 3.7 4.2 5.53 7.77; 36
Platform (P2; cocoa from farm 2) 96 54; 72 (spreading for 3 h) 26.6 49.0 3.4 3.7 3.70 7.77; 24
Box (I2; cocoa from farm 2) 96 24; 72 25.7 47.9 3.7 4.1 4.28 7.65; 42
Malaysia (main crop 2010; 2 plantations)
Box (M1) 120 48; 96 28.5 43.9 3.9 4.4 6.28 7.73; 12
Box (M2) 120 48; 96 31.8 42.7 3.9 4.2 5.29 7.24; 36

For each fermentation, the following parameters are listed: temperature (T) [initial (Ti) and final (Tf) temperature] and pH [initial (pHi) and final
(pHf) pH value] inside the fermenting cocoa pulp-bean mass. Yeast counts [colony forming units (CFU) g 1] were obtained by plating on malt
extract agar supplemented with 100 mg g 1 oxytetracycline (37 °C; 1–3 days): at the beginning (CFU g 1)i of the fermentation and at their max-
imum (CFU g 1)max during the fermentation. N/A, not available.
*Cocoa bean fermentations carried out without separation of healthy and infected pods and no regular mixing.
†
Cocoa bean fermentations carried out with organic cocoa.

ª 2011 Federation of European Microbiological Societies FEMS Yeast Res 11 (2011) 564–574
Published by Blackwell Publishing Ltd. All rights reserved
26S rRNA gene-PCR-DGGE of cocoa bean fermentations
experiments were carried out according to the original
protocol.
To extract DNA from yeast cultures to construct an
identification ladder, the protocol of Gevers et al. (2001)
was used with minor modifications (Camu et al., 2007).
Briefly, total genomic DNA was extracted from stock
cultures that were propagated twice in YG medium (glu-
cose, 20 g L 1; yeast extract, 5 g L 1) following the phe-
nol–chloroform–isoamyl alcohol method. The enzymatic
treatment for the DNA extraction consisted of a final
concentration of 5 U lL 1 of lysozyme (VWR Interna-
tional) and 0.8 U lL 1 of mutanolysine (Sigma-Aldrich),
suspended in TES buffer. The final DNA samples were
diluted in TE buffer to a concentration of 50 ng lL 1
and stored at 20 °C until further use.
26S rRNA gene-PCR-DGGE
The yeast 26S rRNA gene fragment (250 bp) was ampli-
fied using the eukaryotic universal primers NL1 (5′-GCA
TAT CAA TAA GCG GAG GAA AAG-3′), containing a
GC-clamp (5′-CGC CCG CCG CGC GCG GCG GGC
GGG GCG GGG-3′) at the 5′ end and LS2 (5′-ATT CCC
AAA CAA CTC GAC TC-3′) (Cocolin et al., 2000; Niel-
sen et al., 2007). The PCR mixture and conditions were
as reported by Vasilopoulos et al. (2008) and Nielsen
et al. (2007), respectively. To optimize the monitoring of
yeast species through 26S rRNA gene-PCR-DGGE, two
commercial DGGE apparatus were used: the DCode sys-
tem (gels of 16 cm 9 16 cm 9 0.1 cm; Bio-Rad, Hercu-
les, CA) and the CBS Scientific system (gels of
17.7 cm 9 22 cm 9 0.1 cm; CBS Scientific Company,
San Diego, CA). For both systems, electrophoresis of the
PCR amplicons was carried out in 1.09 TAE buffer
(40 mM Tris-acetate, 1 mM EDTA, pH 8.0) at 70 V for
16 h at a constant temperature of 60 °C. For the separa-
tion of the PCR amplicons, an optimization of the dena-
turing gradients (25–60%, 35–70% and 35–60%) was
performed. A 35–60% denaturing gradient was optimal
(data not shown). A reference ladder was included in all
gels for normalization of the gels for numerical analysis
with BIONUMERICS version 5.1 software (Applied Maths,
Sint-Martens-Latem, Belgium) as well as for preliminary
identification of DGGE bands in the lanes of the fermen-
tation samples. This ladder was constructed by running
PCR amplicons from DNA of several yeast strains on
DGGE gels of the DCode DGGE system. All these yeasts
were isolates from the Ecuadorian cocoa bean fermenta-
tion samples and were identified previously through a
multi-gene sequencing approach, including D1/D2 LSU,
ITS and ACT1 gene sequencing. The accession numbers
are given below. Using this ladder, in combination with
DGGE band sequencing, the yeast species composition
FEMS Yeast Res 11 (2011) 564–574
567
and community dynamics of all fermentation samples
were analysed. For numerical analysis of DGGE band pat-
terns, the band-based Dice coefficient was used to dis-
criminate among them concerning the influence of
mixing, duration of the fermentation or fermentation
method.
DGGE band sequencing
DGGE bands of interest were excised from the gels with a
sterile blade, mixed with 40 lL of sterile water and put at
4 °C for 24 h to let the DNA diffuse out of the bands.
Then, the bands were vigorously vortexed for 15–20 min
and microcentrifuged (1000 g) for 15 s to collect the
aqueous DNA solution. Three microlitres of this solution
were used for a PCR assay with the same primers as men-
tioned above, excluding the GC-clamp, under the same
PCR conditions. The PCR products were purified using
the Wizard Plus SV Minipreps DNA Purification System
(Promega, Madison, WI) and sequenced in a commercial
facility (VIB, Antwerp, Belgium). Searches in the GenBank
database were performed with the BLAST (Basic Local
Alignment Search Tool) program to determine the closest
known relatives of the partial 26S rRNA gene sequences
(http://www.ncbi.nlm.nih.gov/BLAST). Accession numbers
are indicated in parentheses at the appropriate places.
Results
Comparison of two commercial DGGE systems
for yeast species composition monitoring of
cocoa bean fermentation samples
Reference yeast strains
The PCR amplicons of DNA from the Ecuadorian cocoa
reference yeast strains migrated differently in the poly-
acrylamide gels of the two commercial systems and their
overall patterns differed at species level (Fig. 1). The
DCode system gave multiple banding patterns for almost
all strains tested (Fig. 1a). All S. cerevisiae strains gave the
same pattern, composed of three bands with the DCode
DGGE system, which showed 100% identity with S. cere-
visiae strain SS1-1 (HM123752) after BLAST analysis
(Fig. 1a). A better separation of the bands and a distinct
DGGE image were obtained with the CBS system; in
addition, no multiple banding patterns occurred, except
for P. kudriavzevii (Fig. 1b). Therefore, the final selection
of strains per species for the construction of an identifica-
tion ladder was based on their migration in gels of the
CBS DGGE system (Fig. 1b), which were (according to
the direction of electrophoresis): H. opuntiae Y120
(FR870033), Candida tropicalis Y49 (FR870028), Torulaspora
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
568
delbrueckii Y7 (FR870025), Kluyveromyces marxianus Y40
(FR870027), S. cerevisiae D652 (representative GenBank
accession number HM123752) and P. kudriavzevii D695
(representative GenBank accession number HM191632).
However, concerning Hanseniaspora, no distinction could
be made between the species H. opuntiae, H. guilliermon-
dii and Hanseniaspora uvarum, as these three species
yielded the same DGGE band because there are no differ-
ences in the nucleotide sequences of the corresponding
26S rRNA gene fragments. Although the two DNA bands
of P. kudriavzevii were close to each other, they did not
interfere with bands of other yeast strains in the ladder.
Also, an intense band was present in almost all DCode
DGGE patterns in the lower part of the gels, interfering
with the detection of species migrating almost at the same
height of this band, such as P. kudriavzevii (Fig. 1a). This
(a) 1 2 3 4 5 6 7 8 9 10 11 12 (b) 1 6 10 9 11 3
Fig. 1. DGGE patterns of identified cocoa yeast strains obtained with
the DCode DGGE system (a). Strains selected for the ladder (L)
construction, optimized using the CBS Scientific DGGE system (b).
The lanes represent: (1) Pichia kudriavzevii D695 (representative
GenBank accession number HM191632); (2) Pichia kudriavzevii D642
(representative GenBank accession number HM191632); (3) Saccharomyces
cerevisiae D652 (representative GenBank accession number HM123752);
(4) Saccharomyces cerevisiae D650 (representative GenBank accession
number HM123752); (5) Saccharomyces cerevisiae D534 (representative
GenBank accession number HM123752); (6) Torulaspora delbrueckii Y7
(FR870025); (7) Candida sorborivorans-like Y54 (FR870030); (8) Pichia
kluyveri Y57 (FR870031); (9) Kluyveromyces marxianus Y40
(FR870037); (10) Candida tropicalis Y49 (FR870028); (11)
Hanseniaspora opuntiae Y120 (FR870033); and (12) Rhodotorula
minuta Y50 (FR870029).
L 0 6 24 30 48 54
(a)
Fig. 2. Yeast DGGE patterns of the Ivorian cocoa bean box fermentation samples using the DCode DGGE system (a) and the CBS Scientific
DGGE system (b). The lane numbers represent the time (h) of sampling during fermentation. Lane L represents the ladder of the reference yeast
strains.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Z. Papalexandratou & L. De Vuyst
band was not visible in the gel of the CBS System, indi-
cating differences in gradient formation between the two
DGGE systems.
Fermentation samples
Both DGGE systems allowed fast monitoring of the yeast
species composition of the cocoa bean fermentation sam-
ples studied. However, an important difference in
detected yeast species between the two systems was found,
as illustrated for the Ivorian cocoa bean box fermentation
samples (Fig. 2). As mentioned above, a fuzzy band was
formed in the lower part of the DCode system gels in all
samples, making accurate detection and successful
sequencing of co-migrating yeast DNA bands difficult or
impossible, in particular, those corresponding to S. cerevi-
siae and P. kudriavzevii.
Yeast species composition of cocoa bean
fermentation samples
Comparing the semi-quantitative PCR-DGGE data with
culture-dependent data (Table 2), Hanseniaspora sp.
(100% identity with H. opuntiae, H. uvarum and H. guil-
liermondii) was prevailing in all fermentation samples,
independent of their origin and fermentation method
(Fig. 3). Based on the presence of DGGE bands corre-
sponding with P. kudriavzevii and/or S. cerevisiae, these
yeast species were the second most common ones found
in several fermentation samples of most fermentations
(Fig. 3). This indicated a restricted yeast species composi-
tion involved in the cocoa bean fermentation processes
(Fig. 3). An exception was the Ivorian box fermentation
that showed a wider yeast species composition, with
Hyphopichia burtonii and Meyerozyma caribbica among
the main representatives (Fig. 3a).
Pichia kudriavzevii was present at the beginning of the
Ivorian heap fermentation and was succeeded by Hanse-
niaspora sp. after 48 h of fermentation (Fig. 3a). In the
case of the two Brazilian cocoa bean box fermentations
72 78 L L 0 6 24 30 48 54 72 78 L
(b)
FEMS Yeast Res 11 (2011) 564–574
26S rRNA gene-PCR-DGGE of cocoa bean fermentations 569

Table 2. Overall results of the yeast species detected in each sample, including culture-dependent data from previous studies

Cocoa-producing region Culture-dependent analysis Culture-independent analysis Reference

Ivory Coast Not available Hanseniaspora sp. This study

Hyphopichia burtonii Meyerozyma caribbica
Pichia kudriavzevii
Pichia veronae/fabianni
Saccharomyces cerevisiae
Brazil Not available Candida jaroonii/friedrichii This study
Hanseniaspora sp.
Hanseniaspora vineae
Pichia kudriavzevii
Saccharomyces cerevisiae
Wickerhamomyces anomalus
Ecuador Candida sorbosivorans-like – This study; Z. Papalexandratou,
Candida tropicalis – G. Falony, E. Romanens, J.C. Jimenez,
– Debaryomyces sp./Candida sp. F. Amores, H.M. Daniel, L. De Vuyst,
Hanseniaspora opuntiae Hanseniaspora sp. unpublished results
– Issatchenkia tericola
Kluyveromyces marxianus –
Pichia kluyveri –
Pichia kudriavzevii Pichia kudriavzevii
Pichia manshurica –
Rhodotorula minuta –
Saccharomyces cerevisiae –
Torulaspora delbrueckii –
Malaysia Not available Hanseniaspora sp. This study
Pichia kudriavzevii
Saccharomyces cerevisiae
Torulaspora delbrueckii

(F1 and F2), Hanseniaspora sp. was present in parallel
with P. kudriavzevii during the first 36 and 54 h of
fermentations F1 and F2 respectively (Fig. 3b). However,
P. kudriavzevii was absent in the two Brazilian cocoa bean
box fermentations (B1 and B2) (Fig. 3c). In Ecuador, all
box and platform fermentations showed the same yeast
species composition; Hanseniaspora sp. and P. kudriavzevii
were both present, although the latter species appeared,
in some cases, as a band of low intensity (Fig. 3d and e).
During the Malaysian box fermentations, S. cerevisiae was
present during the whole fermentation process, together
with Hanseniaspora sp. (Fig. 3f).
A few single bands were detected during the first hours
of some fermentation, such as those corresponding with
Wickerhamomyces anomalus, Debaryomyces sp. (100%
identity with Debaryomyces hansenii, Debaryomyces nepal-
ensis and Debaryomyces mycophilus), Candida jaroonii/frie-
drichii and Hanseniaspora vineae, perhaps accidental
contaminants from the environment (Fig. 3c and d).
Although all fermentations lasted for 4–6 days, yeast
bands were not always found at the end of fermentation.
In contrast, in the case of the Malaysian box fermenta-
tions, yeasts were detected during the whole fermentation
process; in particular, Hanseniaspora sp. was still found
after 4 days of fermentation (Fig. 3f).
Based on numerical analysis of all DGGE band pat-
terns, no significant influence of fermentation practices,
such as mixing of the fermenting cocoa pulp-bean mass
or duration of the fermentation or fermentation methods
(heap, box, platform) on the yeast species composition
was seen (Fig. 4). However, yeasts were only detected for
a short time in the case of fermentations characterized by
practices such as no separation of healthy and infected
pods, no mixing of the fermenting cocoa pulp-bean mass
or spreading of the fermenting beans during fermentation
(Fig. 3b and d), indicating atypical processing.
Discussion
During the last decade, the culture-independent PCR-
DGGE method has been used frequently to monitor the
microbial species composition and community dynamics
during food fermentation processes; however, its detec-
tion limit is rather high (103–104 CFU g 1, depending on
the species and/or strain) and data are semi-quantitative
(Ercolini, 2004; Giraffa, 2004). In the present study, 26S
rRNA gene-PCR-DGGE was used for monitoring the
yeast community dynamics and species composition dur-
ing cocoa bean fermentations carried out in several
cocoa-producing regions. In general, DGGE allowed a fast

FEMS Yeast Res 11 (2011) 564–574 ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
570 Z. Papalexandratou & L. De Vuyst

(a) Ivorian box (B) fermentation Ivorian heap (H) fermentation Ecuadorian platform (P1) fermentation Ecuadorian platform (P2) fermentation
(d)
L 0 6 24 30 48 54 72 78 L 0 6 24 30 48 54 72 78 96 L 0 6 12 18 24 30 36 L 0 6 12 18 24 30 36
i i i i i i

iii vi
iv
v

ix x

xi xi xi xi

Brazilian box (F1) fermentation Brazilian box (F2) fermentation

(b) Ecuadorian box (I1) fermentation Ecuadorian box (I2) fermentation
L 0 6 12 24 30 36 L 0 6 12 24 30 36 42 48 54
i i
(e) L 0 6 12 24 24.5 30 36 48 L 0 6 12 24 24.5 30 36 48
i

xi xi
xi xi

(c) Brazilian box (B1) fermentation Brazilian box (B2) fermentation

L 0 6 12 24 30 36 48 54 60 72 L 0 6 12 24 30 36 48 54 60 72 (f) Malaysian box (M1) fermentation Malaysian box (M2) fermentation
i i L 0 6 12 24 30 36 L 48 48.5 54 60 72 84 84.5 L 96 120 L 0 6 12 24 30 36 L 48 48.5 54 60 72 84 84.5 L 96 120

i i i
ii

vii viii

ix ix ix ix

xi xi

Fig. 3. Yeast DGGE patterns of cocoa bean fermentation samples obtained with the CBS Scientific DGGE system. (a) Ivorian box and heap
fermentations carried out in 2006; (b) Brazilian box fermentations carried out in 2006; (c) Brazilian box fermentations carried out in 2007; (d)
Ecuadorian platform fermentations carried out in 2008; (e) Ecuadorian box fermentations carried out in 2008; (f) Malaysian box fermentations
carried out in 2010. The lane numbers represent the time (h) of sampling during fermentation. Lane L represents the ladder of the reference
yeast strains. The closest relatives of the fragments sequenced (% of identical nucleotides compared with sequences retrieved from the GenBank
database and representative Accession No. between parentheses) were: (i) Hanseniaspora opuntiae/uvarum/guilliermondii (100%; FM180549,
AB438210); (ii) Wickerhamomyces anomalus (100%; FJ515235); (iii) Hyphopichia burtonii (100%; GQ222349); (iv) Pichia veronae/fabianni
(100%; AB436465, EF550321); (v) Meyerozyma caribbica (100%; AB557838); (vi) Debaryomyces sp./Candida sp. (100%; FJ432624, AY520416);
(vii) Candida jaroonii/friedrichii (100%; AB292057); (viii) Hanseniaspora vineae (100%; HM191667); (ix) Saccharomyces cerevisiae (100%;
HM123752); (x) Issatchenkia terricola (100%; EF550233); and (xi) Pichia kudriavzevii (100%; HM191632).

detection of the main yeast species involved in the pro- could be eliminated by extension of the final elongation
cess, representing at least 90% of the total yeast commu- step of the PCR assays (Janse et al., 2004). Finally, the
nities, as has been seen for detailed studies on the presence of a fuzzy band in the lower part of the DCode
bacterial species composition revealed by plating, PCR- gels, which affects accurate sequencing of excised DGGE
DGGE and rDNA libraries (Garcia-Armisen et al., 2010). bands, has been ascribed to ssDNA artefacts that are not
However, the technique was not free of drawbacks, influenced differentially by the gradient (Heuer et al.,
mainly in the case of the DCode apparatus, as sometimes 1997; Cocolin et al., 2000). Most of these drawbacks were
multiple banding patterns were formed for a single spe- overcome when using the CBS apparatus, allowing a bet-
cies. The reasons for this appearance are not well under- ter separation of the yeast bands in the reference ladder
stood. In general, it may reflect artefacts of the PCR as well as distinct yeast DGGE patterns for all cocoa bean
assays using primers with GC clamps and DNA denatur- fermentations studied. Comparison of the two apparatus
ation kinetics during electrophoresis (Beh et al., 2006; revealed few differences in gradient formation, perhaps
Rettedal et al., 2010). Also, multiple bands could arise because of the different dimensions of the gels. The fact
from nucleotide variations among multiple rDNA copies that the DGGE apparatus can affect DGGE-based yeast
within a single strain or could indicate the presence of and bacterial community structure analysis has already
different strains within a species (Beh et al., 2006). Arte- been reported, in the case of soil samples (Ascher et al.,
facts of double bands of PCR amplicons of several species 2010).

ª 2011 Federation of European Microbiological Societies FEMS Yeast Res 11 (2011) 564–574
Published by Blackwell Publishing Ltd. All rights reserved
26S rRNA gene-PCR-DGGE of cocoa bean fermentations 571

100
20

30

40

50

60
10

70

80

90
Brazil B1 S3 (24 h)
IC Box S7 (78 h)
IC Box S5 (54 h)
IC Box S0 (0 h)
IC Box S3 (30 h)
IC Box S2 (24 h)
IC Box S1 (6 h)
IC Box S4 (48 h)
Ecuador I1 S1 (6 h)
Ecuador I1 S2 (12 h)
Ecuador P1 S1 (6 h)
Ecuador P1 S2 (12 h)
Ecuador P1 S3 (18 h)
Ecuador P1 S4 (24 h)
Ecuador P1 S5 (30 h)
Brazil F2 S0 (0 h)
Malaysia M1 S5 (36 h)
Brazil B2 S7 (54 h)
Ecuador I1 S5 (30 h)
Ecuador I1 S6 (36 h)
Ecuador I1 S7 (48h)
IC Heap S4 (48 h)
IC Box S6 (72 h)
IC Heap S6 (72 h)
Brazil B1 S5 (36 h)
Brazil B1 S7 (54 h)
Malaysia M1 S4 (30 h)
Malaysia M1 S6b (48.5 h)
Malaysia M1 S8 (60 h)
Malaysia M1 S9 (72 h)
Malaysia M1 S10a (84 h)
Malaysia M1 S10b (84.5 h)
Malaysia M1 S11 (96 h)
Malaysia M1 S12 (120 h)
Malaysia M2 S2 (12 h)
Malaysia M2 S3 (24 h)
Malaysia M2 S4 (30 h)
Malaysia M2 S5 (36 h)
Malaysia M2 S6 (48 h)
Malaysia M2 S8 (60 h)
Malaysia M2 S9 (72 h)
Malaysia M2 S1 (6 h)
Brazil B2 S6 (48 h)
Malaysia M1 S1 (6 h)
Malaysia M1 S3 (24 h)
Malaysia M2 S11 (96 h)
Malaysia M2 S12 (120 h)
Malaysia M1 S2 (12 h)
Malaysia M2 S10b (84.5 h)
Malaysia M1 S0 (0 h)
Malaysia M2 S6b (48.5 h)
Malaysia M2 S10 (84 h)
Malaysia M1 S7 (54 h)
Malaysia M2 S7 (54 h)
Malaysia M2 S0 (0 h)
Ecuador P2 S3 (18 h)
Ecuador P2 S4 (24 h)
IC Heap S5 (54 h)
Ecuador P1 S6 (36 h)
IC Heap S1 (6 h)
Brazil F1 S5 (36 h)
IC Heap S0 (0 h)
Brazil F2 S1 (6 h)
Brazil F2 S2 (12 h)
Brazil F2 S3 (24 h)
Brazil F2 S4 (30 h)
Ecuador I1 S0 (0 h)
Ecuador I1 S4 (24 h)
Ecuador I1 S4b (24.5 h)
Ecuador P2 S5 (30 h)
Ecuador P2 S6 (36 h)
IC Heap S3 (30 h)
IC Heap S2 (24 h)
Ecuador P2 S1 (6 h)
Ecuador P2 S2 (12 h)
Brazil F1 S0 (0 h)
Brazil F1 S2 (12 h)
Brazil F1 S3 (24 h)
Brazil F1 S4 (30 h)
Brazil F2 S5 (36 h)
Ecuador I2 S4 (24 h)
Ecuador I2 S4b (24.5 h)
Brazil F1 S1 (6 h)
Brazil B1 S1 (6 h)
Brazil B1 S2 (12 h)
Brazil B1 S4 (30 h)
Brazil B2 S3 (24 h)
Brazil B2 S4 (30 h)
Brazil B2 S5 (36 h)
Ecuador I2 S5 (30 h)
Ecuador I2 S6 (36 h)
Ecuador I2 S7 (48 h)
Brazil B1 S0 (0 h)
Brazil B2 S0 (0 h)
Brazil B2 S1 (6 h)
Brazil B2 S2 (12 h)
Brazil B1 S6 (48 h)
IC Heap S7 (78 h)
Ecuador P2 S0 (0 h)
Ecuador I2 S0 (0 h)
Ecuador I2 S1 (6 h)
Ecuador I2 S2 (12 h)

Fig. 4. Dendrogram derived from the cluster analysis of the 26S rRNA gene-PCR-DGGE patterns of the yeast communities associated with cocoa
bean fermentation samples from Ivory Coast, Brazil, Ecuador and Malaysia, based on the Dice coefficient of similarity (weighted) and obtained
with the UPGMA clustering algorithm.

FEMS Yeast Res 11 (2011) 564–574 ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
572
Concerning yeast species composition, Hanseniaspora
sp. was the predominating yeast species in all fermenta-
tions of the present study. For the Brazilian box fermen-
tations (B1 and B2), carried out with organic cocoa
under optimal fermentation practices such as selection of
healthy pods solely, use of washed equipment and regular
mixing during fermentation (Garcia-Armisen et al., 2010;
Papalexandratou et al., 2011), Hanseniaspora sp. was the
only species present throughout the fermentation process,
thus suggesting its possible importance for the success of
the fermentation. Distinction between Hanseniaspora
opuntiae, H. uvarum and H. guilliermondii is only possi-
ble by multi-gene sequence analysis (Cadez et al., 2003,
2006; Daniel et al., 2009). However, the results of the
culture-dependent study of Daniel et al. (2009) that used
accurate molecular identifications suggests that H. opunti-
ae is the predominant species during cocoa bean fermen-
tation (Daniel et al., 2009). In the study of Nielsen et al.
(2007), 247 yeast isolates were analysed revealing the
dominance of H. guilliermondii and P. membranifaciens.
In the study of Daniel et al. (2009), H. opuntiae prevails
during the initial phase of Ghanaian cocoa bean heap
fermentations, based on the analysis of 90 isolates from
seven fermentation heaps. The ethanol tolerance up to
5–10% (v/v) of H. opuntiae explains its dominance
during cocoa bean fermentation, wherein ethanol never
exceeds 2.5% (v/v) (Camu et al., 2007, 2008a, b; Daniel
et al., 2009). Originally, H. opuntiae was isolated from
Opuntia ficus-indica rot in Hawaii (Cadez et al., 2003).
During the present study and based on the samples
analysed, P. kudriavzevii and/or S. cerevisiae were the sec-
ond most common yeast species found for most fermen-
tations. The former species was less prevalent during the
Ecuadorian cocoa bean fermentations, whereas the latter
species prevailed during the Malaysian box fermentations.
In general, the most frequently isolated cocoa yeast spe-
cies is S. cerevisiae (Schwan & Wheals, 2004; De Vuyst
et al., 2010). Pichia kudriavzevii has been reported as an
acid- and ethanol-tolerant yeast species (Okuma et al.,
1986; Daniel et al., 2009). It ferments reducing carbohy-
drates at a slower rate than S. cerevisiae, but it is able to
use malic acid efficiently, in contrast to S. cerevisiae (Kim
et al., 2008). Although P. kudriavzevii partially reduces
citric acid, cocoa-originating H. opuntiae and S. cerevisiae
isolates do not assimilate citric acid, indicating a possible
(minor) role of P. kudriavzevii in citric acid conversion
besides LAB (Camu et al., 2007, 2008a, b; Daniel et al.,
2009). Meyerozyma caribbica and H. burtonii represented
the main yeast species involved in the Ivorian box fer-
mentations and the latter had not been yet documented
from cocoa bean fermentations before. Meyerozyma
caribbica was originally isolated from sugar cane in Cuba
(Vaughan-Martini et al., 2005). Hyphopichia burtonii was
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Z. Papalexandratou & L. De Vuyst
originally isolated from murcha, a natural starter culture
for the production of chunga/jand, a fermented liquor
from Nepal, and appears to possess amylolytic activity
(Takeuchi et al., 2006). It has been isolated from tempeh,
a fermented soy product as well (Aldsworth et al., 2009).
Pichia kudriavzevii, S. cerevisiae and H. opuntiae repre-
sent the main yeast species of seven Ghanaian cocoa bean
heap fermentations, as revealed through M13-PCR finger-
printing of yeast isolates and multiple locus gene sequenc-
ing in the study of Daniel et al. (2009). Application of 26S
rRNA gene-PCR-DGGE on Ghanaian heap and tray fer-
mentation samples with the DCode apparatus has revealed
similar results in the study of Nielsen et al. (2007),
whereby H. guilliermondii was the prevalent band followed
by P. membranifaciens and P. kudriavzevii. Although
H. guilliermondii and P. membranifaciens were not found
during the present study, the former species in the study
of Nielsen et al. (2007) should have been identified as
H. opuntiae (Daniel et al., 2009), based on the elongation
factor-1a and ACT1 gene sequences (Cadez et al., 2003,
2006). The absence of P. membranifaciens may be ascribed
to the dominance of H. opuntiae, as substrate competition
between these species has been reported (Jespersen et al.,
2005). Finally, high counts of the species P. kudriavzevii
have been found on the surface of pods infected with
black pod disease (Jespersen et al., 2005).
Concerning yeast community dynamics, no specific
temporal distribution was recognized for H. opuntiae,
P. kudriavzevii or S. cerevisiae, as all species could be found
at the beginning, in the middle or at the end of the cocoa
bean fermentation processes, reflecting their adaptation to
the cocoa pulp-bean mass ecosystem. Rarely encountered
species, such as Debaryomyces sp., H. vineae, C. jaroonii/
friedrichii and W. anomalus, only occurred during the ini-
tial phase of the fermentation, perhaps because of their
low ethanol tolerance. However, Hanseniaspora seems to
be more acid- and heat-tolerant, as it could still be found
after 4 days of fermentation, as seen in the Malaysian box
fermentations (Fig. 3f), when AAB have already produced
high amounts of acetic acid. In general, yeasts were not
detected in the case of a fermentation temperature above
45 °C or at high acidity. This was for instance the case
for the Brazilian box F1 fermentation, wherein AAB dom-
inated the fermentation and produced high amounts of
acetic acid early into the fermentation (Garcia-Armisen
et al., 2010; Papalexandratou et al., 2011).
In conclusion, a restricted yeast species composition
was found for different cocoa bean fermentations in dif-
ferent cocoa-producing regions, with H. opuntiae being
the prevailing species based on the presence of DGGE
bands, followed by P. kudriavzevii and S. cerevisiae. These
yeast communities could rapidly be monitored by 26S
rRNA gene-PCR-DGGE, not only with respect to their
FEMS Yeast Res 11 (2011) 564–574
26S rRNA gene-PCR-DGGE of cocoa bean fermentations
identities but also concerning their temporal dynamics.
The latter is much more difficult to perform culture-
dependently, unless hundreds of isolates are identified,
which is labour-intensive and time-consuming (usually
several weeks to months, depending on the number of
isolates). The culture-independent analysis reported in the
present study was performed on samples from different
origins, a comparison that was done for the first time
and contributed to the novelty of this study, and the
results could be obtained within 2 weeks based on frozen
samples. Moreover, they confirmed that all yeast species
involved in the fermentation process are cultivable. The
CBS Scientific DGGE system appeared to be more reliable
to monitor the yeast community dynamics and species
composition than the DCode apparatus, as a better band
separation occurred for PCR amplicons with a high GC
content. The detection of a few prevalent yeast species in
all fermentations worldwide will be useful for the devel-
opment of an ideal yeast starter culture in combination
with a bacterial cocktail to allow the control of cocoa
bean fermentation processes in the near future.
Acnkowledgements
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 Belgium (Nicholas Camu and
Herwig Bernaert). The cooperation of the local farmers of
the ‘Hawaii’ and ‘Leao De Ouro’ plantations (Rodovia
Ilhéus/Uruçuca, Bahia, Brazil) is highly appreciated. We
thank the Centre National de Recherche Agronomique
(Abidjan, Ivory Coast) for their cooperation. The experi-
ments in Ecuador were carried out in collaboration with
the Estación Experimental Tropical Pichilingue, Instituto
Nacional Autónomo de Investigaciones Agropecuarias
(INIAP) (Quevedo, Los Rios, Ecuador). The help of the
family Ong for the accomplishment of the Malaysian fer-
mentations (Triang, Pahang) is highly appreciated. Frédé-
ric Ravyts is thanked for the help to optimize the DGGE
gradients.
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