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Previous Article | Next Article 
Applied and Environmental Microbiology, March 2007, p. 1809-1824, Vol. 73, No. 6
0099-2240/07/$08.00+0 doi:10.1128/AEM.02189-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Research Group of Industrial Microbiology and Food Biotechnology, Department of Applied Biological Sciences and Engineering, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium,1 BCCM/LMG Bacteria Collection and Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium,2 Cocoa Research Institute of Ghana, New Tafo, Akim, Ghana3
Received 18 September 2006/ Accepted 20 January 2007
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After removal of the beans from the pods, the first step in cocoa processing is a spontaneous 3- to 10-day fermentation of beans and pulp in heaps, boxes, baskets, or trays, of which spontaneous heap fermentation is the most widely used method in Ghana (4, 39). A microbial succession of yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) takes place during fermentation (3, 67, 73). The yeasts liquefy the pulp through depectinization, which reduces pulp viscosity, and produce ethanol from sugars (and citric acid) under anaerobic conditions in an acid (pH < 4.0), carbohydrate-rich environment. As pulp is drained away, ethanol formation proceeds and both temperature and pH increase, which creates ideal conditions for the growth of LAB and AAB. LAB convert sugars and organic acids mainly into lactic acid. As more air comes in, AAB start to grow that oxidize the ethanol, initially produced by the yeasts, to acetic acid. Ethanol and acetic acid diffuse into the beans, and this, in combination with the heat produced by this exothermic bioconversion, causes the death of the seed embryo as well as the end of fermentation. Also, this initiates biochemical changes in the beans, leading to the formation of precursor molecules for the development of a characteristic flavor and color of the beans (26, 73). These properties are further developed during drying, roasting, and final processing of well-fermented cocoa beans (10, 73). Besides the actions of yeasts, the activities of both LAB and AAB are essential for the production of high-quality cocoa. Bacillus spp. may develop at the end of fermentation (increased temperature and less acidic and more-aerobic conditions), but their role in the process is not clear (3, 51, 67).
Several studies have dealt with yeasts involved in cocoa bean fermentation (3, 30, 49, 63, 66). Few studies have focused on the population dynamics and biodiversity of LAB and AAB during cocoa bean fermentation in relation to the metabolomics of this ecosystem. This may be due to difficulties in accessing and sampling such fermentations and in isolating and growing AAB and to the limited biodiversity that has been seen among LAB and AAB from fermented cocoa beans (67). In general, AAB belonging to the genus Acetobacter have been found more frequently than those of Gluconobacter (8, 51, 53, 67, 72). In the case of LAB, lactobacilli dominate compared with Leuconostoc, Pediococcus, and Lactococcus (8, 54, 55, 72). As most of the microbiological studies on cocoa date many years back, classical culture-dependent methods were used and no molecular identification methods were included. Moreover, the taxonomy of both LAB and AAB has undergone several changes during the last decade (18). This requests an updated isolation, identification, and biodiversity study of LAB and AAB from fermenting cocoa beans, including both molecular identification and culture-independent methods.
Culture-independent methods such as denaturing gradient gel electrophoresis (DGGE) of PCR-derived gene amplicons have been developed to assist in the study of the microbial biodiversity and population dynamics of complex ecosystems over space and/or time, including food (19, 22, 47). Compared to traditional culturing, these methods, generally based on nucleic acids such as the 16S rRNA gene, aim at obtaining both a qualitative and a semiquantitative picture of a microbial community without the need to isolate and culture its single components. However, it has previously been demonstrated that while these techniques are valuable, there is a need to use a combined system to overcome the bias of the "culture-(in)dependent-only" approach. Therefore, culture-independent and cultivation methods should be applied in parallel for population dynamics and biodiversity studies. Recently, such an approach was applied to study the yeast microbiota during cocoa bean fermentation in Ghana (49). Concerning AAB, this technique has been applied to determine their diversity during wine and vinegar fermentations (16, 44). With respect to LAB, DGGE studies encompass several fermented foods, such as cheese, wine, sourdough, and fermented sausages(19, 58, 61).
The aim of the present study was to assess the population dynamics of LAB and AAB and the evolution of important fermentation parameters (temperature, pH, sugars, and metabolites) during spontaneous heap fermentations of cocoa beans in Ghana. Both culture-dependent and -independent methods were applied to monitor and identify LAB and AAB. Cluster analyses of the repetitive-sequence-based PCR (rep-PCR) and bacterial DGGE profiles were performed to reveal possible differences between fermentation processes. Through metabolite target analysis, a link was made between the substrates and metabolites found in pulp and beans and the microbes identified.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cocoa pods from mixed hybrid cocoa tree plantations (Criollo and Forastero) were harvested by traditional methods (such as manual harvest and transport in unwashed baskets) and used for fermentation within 2 to 3 days. Only matured pods were used for fermentation. Plantation workers cut the pods with unwashed machetes, beans plus surrounding pulp were scooped out manually, the placenta was not removed, and the husks were left to rot in the surroundings. At each farm, approximately 250 to 1,000 kg of wet beans and pulp was placed on banana and plantain leaves on the ground, resulting in heaps of 95- to 180-cm diameter and 40- to 64-cm height which were then covered with extra banana and plantain leaves and left to ferment. The beans were not mixed during fermentation. The entire fermentation lasted 6 days at both farms. The drainage of liquids produced during fermentation (sweatings) was allowed to penetrate into the ground. Drying of the fermented cocoa beans took around 10 to 14 days, depending on the weather, and resulted in amounts of 33 to 200 kg of dried beans. During fermentation, there was an on-line follow-up of temperature (outside and inside the heaps), pH (inside the heaps), and rainfall (pluviometer). Temperature and pH were measured by inserting a digital pH 340i sensor (WTW GmbH, Weilheim, Germany) in the middle of the fermenting cocoa bean mass.
Sampling.
Samples of the seven heaps were taken
according to a fixed time schedule, namely, at the start of the
fermentation (time zero, fresh cocoa beans) and after 6, 12, 18, 24,
30, 36, 42, 48, 54, 60, 66, 72, 84, 96, 120, and 144 h of
fermentation. Sampling was always done at the same depth of the bean
mass (approximately 30 cm from the upper surface) but in different
points of the heap. Each sample consisted of 600 g of beans
that was aseptically removed and transferred into sterile plastic bags.
Besides samples from the mucilaginous pulp of the opened pods, swab
samples corresponding to a surface of 25 cm2 were taken from
the environment (surfaces of cocoa pods, banana leaves, baskets,
machetes, and farmers' hands). For culture-independent and metabolite
analyses, 128 cocoa bean samples were cooled, frozen, and transported
on dry ice to Belgium.
Plating, enumeration, isolation, and maintenance.
In Ghana, the culture-dependent
approach was performed immediately after sampling (fresh samples were
transiently stored on ice and treated in the laboratory within
1 h). Therefore, 180 ml of 0.1% (wt/vol) peptone water
(Oxoid, Basingstoke, United Kingdom) was added to 20 g of
pulp and beans in a sterile stomacher bag that was vigorously shaken
for 3 min in a Stomacher 400 (Seward, Worthington, United Kingdom) to
obtain a uniform homogenate. Samples (1.0 ml) of the homogenate were
serially diluted 10-fold in 0.1% (wt/vol) peptone water, from which
aliquots (0.1 ml) were plated on different selective agar media that
were incubated at different temperatures for 1 to 4 days in a standard
incubator (Jouan, St. Herblain, France) for the monitoring, isolation,
and enumeration (by recording the number of CFU) of specific groups of
microorganisms responsible for fermentation: plate count agar (PCA;
Oxoid) for the total aerobic bacterial count (37°C), malt
extract agar (MEA; Oxoid) plus 100 mg liter–1 of
oxytetracycline for yeasts (37°C),
deoxycholate-mannitol-sorbitol (DMS) agar
(25) plus 400 mg
liter–1 of cycloheximide for AAB (42°C), de
Man-Rogosa-Sharpe (MRS
[15]) and medium 17 (M17)
agar (Oxoid) of Terzaghi and Sandine
(71) plus 400 mg
liter–1 of cycloheximide for LAB (37°C), and
kanamycin esculin azide (KAA, Oxoid) agar plus 400 mg
liter–1 of cycloheximide for enterococci
(37°C). Swabs were transferred to 10 ml of 0.1% (wt/vol)
peptone water and vortexed for 2 min; 0.1-ml aliquots were spread on
the agar media and incubated as described above. Morphologically
different colonies were picked up from a suitable dilution of each
sample on MRS, M17, KAA, and DMS agar media, grown in test tubes with
the appropriate medium, purified through subculturing and plating, and
stored at –80°C in the same medium supplemented with
25% (vol/vol) glycerol as a cryoprotectant. This culture-dependent
approach yielded 790 bacterial isolates (498 LAB isolates from MRS and
M17, 40 LAB isolates from KAA, and 252 AAB isolates) for identification
in Belgium. The numbering of the identified bacterial strains is listed
in Fig.
1 and
2. It turned out that approximately 15% of the LAB and
AAB isolates could not be revived after transport from Ghana to
Belgium.
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Direct extraction of DNA from fermented cocoa bean samples.
Twenty grams of frozen beans plus pulp samples was homogenized twice in a Stomacher 400 for 5 min, with
70 ml saline added each time. The combined fluid (±120 ml) was
removed by decanting and subsequently centrifuged at 170 x
g at 4°C for 5 min to remove large particles. The
supernatant was filtered through a 20-µm-pore-size filter
(Whatman, Brentford, United Kingdom). The filtrate was centrifuged at
8,000 x g at 4°C for 20 min to pellet the
cells, which were subsequently frozen at –20°C for at
least 1 h. The thawed pellet was washed in 1 ml TES buffer
(6.7%[wt/vol] sucrose, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA) and
resuspended in 300 µl STET buffer (8% [wt/vol] sucrose, 5%
[wt/vol] Triton X-100, 50 mM Tris-HCl, pH 8.0, 50 mM EDTA).
Seventy-five microliters of lysis buffer (TES containing 1,330 U
ml–1 mutanolysin and 100 mg ml–1
lysozyme; Sigma-Aldrich, St. Louis, MO) and 100 µl proteinase K
(TE containing 2.5 mg ml–1) were added, and the
suspension was incubated at 37°C for 1 h. After the
addition of 40 µl preheated (37°C) 20% (wt/vol) SDS in
TE buffer and a pinch of glass beads with a diameter of 150 to 212
µm (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 microliters 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 microcentrifugation (13,000 rpm for 5 min at 4°C)
using Phase Lock Gel tubes (Eppendorf AG, Hamburg, Germany). The
aqueous phase was further purified by using a NucleoSpin column
according to the manufacturer's instructions (Macherey Nagel GmbH,
Düren, Germany). This was done primarily to remove potentially
PCR-inhibiting compounds, as cocoa pulp contains polysaccharides,
proteins, enzymes, and polyphenols. Finally, the eluted phase was
carefully mixed with 70 µl 5 M NaCl and 1 ml isopropanol, and
the DNA was precipitated on ice for at least 15 min. The DNA was
collected by microcentrifugation (13,000 rpm for 30 min at
4°C), and the pellet was washed in ice-cold 70% (vol/vol)
ethanol. The DNA was dried in a vacuum excicator and resuspended in 100
µl TE. Three microliters of DNase-free RNase (10 mg
ml–1; Sigma-Aldrich) was added, and the whole
suspension was incubated at 37°C for 10 min. The
final samples were stored at –20°C until further
use.
PCR.
The primers used in this study were a
primer pair that amplifies DNA from species of LAB (LAC1-LAC2
[78]) and a primer pair
that amplifies DNA from species of AAB and LAB (WBAC1-WBAC2 [44]). LAC1
(5'-AGCAGTAGGAATCTTCCA-3') and
LAC2 (5'-ATTTCACCGCTACACATG-3')
target the V3-V4 region of the 16S rRNA gene, and WBAC1
(5'-GTCGTCAGCTCGTGTCGTGAGA-3')
and WBAC2
(5'-CCCGGGAACGTATTCACCGCG-3')
target the V7-V8 region of the 16S rRNA gene. To facilitate DGGE
separation, a GC-rich sequence
(5'-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC-3')
was attached to one of the primers in each primer pair. PCR
amplifications were performed using a DNA T3 thermocycler (Biometra,
Westburg, The Netherlands) in a final volume of 50 µl,
containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM
of each dATP, dCTP, dGTP, and dTTP, 0.2 µM of each primer, 1.25
IU of Taq DNA polymerase (Roche Diagnostics GmbH, Mannheim,
Germany), and 3 µl of extracted DNA (approximately 500 ng). One
single PCR core program was used for all primer pairs: initial
denaturation at 95°C for 5 min; 30 cycles of denaturation at
95°C for 20 s, annealing at a primer-specific
temperature (LAC, 61°C; WBAC, 67°C) for 45 s,
and extension at 72°C for 1 min; and a final extension at
72°C for 7 min, followed by cooling to 4°C. PCR
amplification products were stored at –20°C. Amplicons
(10 µl) were run in 1.5x TAE (40 mM Tris-acetate, 2 mM
Na2EDTA, pH 8.5) agarose (0.8%, wt/vol) gels at 100 V for 30
min, flanked by the EZ Load 100-bp molecular ruler (Bio-Rad, Hercules,
CA).
DGGE analysis. (i) Cocoa bean samples.
PCR products were
analyzed on DGGE polyacrylamide gels by using a protocol based on that
of Muyzer et al. (47).
The gels (160 by 160 by 1 mm) consisted of 8% (vol/vol) polyacrylamide
(National Diagnostics, Atlanta, GA) in 1x TAE buffer, using a
35 to 60% and a 50 to 70% denaturant gradient (100% denaturing
polyacrylamide solution corresponded with 7 M urea [National
Diagnostics] and 40% [vol/vol] formamide [Sigma]) for PCR products
obtained with the LAC1-LAC2 and WBAC1-WBAC2 primers, respectively.
Electrophoresis of PCR samples was carried out in 1.0x TAE
running buffer at 70 V for 16 h at a constant temperature of
60°C, using the DCode system apparatus (Bio-Rad). After
electrophoresis, all gels were stained with ethidium bromide (50
µl of ethidium bromide in 500 ml of 1.0x TAE buffer)
for 10 min, followed by visualization of the DGGE band profiles under
UV light. Digital capturing of images was performed with the Gel Doc EQ
system (Bio-Rad). The resulting fingerprint pictures were analyzed
using BioNumerics version 4.0 software (Applied Maths). DGGE analyses
were performed twice. Normalization of the gels was performed by using
band ladders of known bacterial DNA in three lanes in all gels.
Therefore, DNA originating from pure cultures of Lactobacillus
plantarum LMG 6907T, Lactobacillus fermentum
LMG 6902T, Leuconostoc mesenteroides subsp.
mesenteroides LMG 6893T, Lactobacillus
casei LMG 6904T, Pediococcus acidilactici LMG
11384T, and Lactobacillus acidophilus LMG
9433T and of L. plantarum LMG 6907T,
Enterococcus faecalis LMG 7937T, Acetobacter
pasteurianus LMG 1262T, Leuconostoc
mesenteroides subsp. mesenteroides LMG 6893T,
and Acetobacter syzygii LMG 21419T was mixed in
equal volumes of the same concentration and used as a reference ladder
for LAB and AAB, respectively, after the corresponding PCR amplicons
were positioned in a DGGE gel with the appropriate gradient. For
cluster analysis of DGGE profiles, the calculation of similarities in
the profiles of bands was based on the Dice coefficient to provide a
qualitative discrimination among the
patterns.
(ii) Colonies from agar plates.
For PCR-DGGE
analyses of bulk cells, colonies were washed off from an appropriate
dilution on MRS agar plates of the corresponding heap samples with
saline (0.85% [wt/vol] NaCl) in Ghana; this cell suspension was frozen
and transported on dry ice to Belgium. Total DNA from the cells was
extracted as described above for (GTG)5 PCR analysis.
PCR-DGGE was performed as described above for
LAB.
(iii) DNA sequencing.
For the sequencing of DGGE bands,
bands of interest were excised from the gels with a sterile blade,
mixed with 50 µl of sterile water, and incubated overnight at
4°C to allow the DNA of the bands to diffuse out of the
polyacrylamide gel blocks. Two microliters of this aqueous solution was
used to reamplify the PCR products with the same primers, including the
GC clamp. The amplicons were checked for purity by another DGGE run
under the conditions described above with amplified DNA of the original
sample as a control. Only reamplified PCR products migrating as a
single band and at the same position with respect to the control were
amplified with the primer without the GC clamp and sequenced in a
commercial facility using capillary sequencing technology (VIB,
Brussels, Belgium). Searches in the GenBank database were performed
with the BLAST program (1)
to determine the closest known relatives of the partial 16S rRNA gene
sequences obtained.
Metabolite target analysis. (i) Sample preparation.
Frozen samples of beans plus pulp
were used to prepare aqueous extracts for metabolite analysis. Beans
were physically separated from pulp by manual peeling. Samples (20 g)
of separate fractions of pulp and beans were mixed with 80 ml of
ultrapure water (MilliQ; Waters Corp., Milford, MA) with an Omnimixer
(Phillips, Brussels, Belgium) for 5 min. The homogenate was centrifuged
at 17,000 x g at 4°C for 15 min, and the
supernatant was retained. The sediment was washed with 20 ml of
ultrapure water and centrifuged, and the washing supernatants were
combined with the first ones to provide aqueous extracts for further
analyses. These extracts were clarified by filtration through
0.45-µm-pore-size filters (Whatman) before further
use.
(ii) Gas chromatography-mass spectrometry.
Short-chain fatty acids, branched short-chain fatty acids, and other
volatile compounds, as well as ethanol,methanol, acetaldehyde, diacetyl, and acetoin, in aqueous extracts were
determined with gas chromatography-mass spectrometry according to the
methods described by Van der Meulen et al.
(76), except that
2,6-dimethyl phenol was used as an internal standard in all
cases.
(iii) HPAEC with pulsed amperometric detection.
The amounts of glucose, fructose, sucrose, mannitol, and erythritol of
aqueous extracts were determined by high-pressure anion exchange
chromatography (HPAEC) with pulsed amperometric detection as described
by Van der Meulen et al. (76), except that
extracts (700 µl) were treated with acetonitrile (700
µl) to remove proteins.
(iv) HPAEC and conductivity with ion suppression.
Organic acids (citric acid, acetic
acid, lactic acid, gluconic acid, ketogluconic acids, formic acid,
oxalic acid, malic acid, and fumaric acid) in aqueous extracts were
determined by HPAEC and conductivity with ion suppression, using an
AS-19 column (Dionex, Sunnyvale, CA). The mobile phase, at a flow rate
of 1.0 ml min–1, consisted of ultrapure
water (0.015 µS cm–1; eluent A) and 100 mM
KOH (eluent B). The following gradient was applied: 0 min, 96% eluent A
and 4% eluent B; 20 min, 96% eluent A and 4% eluent B; and 60 min, 0%
eluent A and 100% eluent B. The aqueous extracts were treated with
acetonitrile as described above, appropriately diluted, filtered (0.2
µm, Minisart RC 4; Sartorius, Darmstadt, Germany) prior to
injection, and run together with the appropriate external
standards.
(v) Liquid chromatography-mass spectrometry.
Amino acids, amino acid metabolites,
and succinic acid in aqueous extracts were quantified through liquid
chromatography-mass spectrometry on a Waters 2695 chromatograph coupled
to a Quattro Micro mass spectrometer (Micromass; Waters Corp.).
Succinic acid and amino acid metabolites were determined according to
the method of Van der Meulen et al.
(76). In the case of
amino acids, a symmetry column (Waters Corp.) was used. The mobile
phase, at a flow rate of 0.2 ml min–1 and linearly
increasing to 0.5 ml min–1 over a period of 45 min
with a flow rate of 0.2 ml min–1 afterwards, was
composed of 0.1% (vol/vol) formic acid in ultrapure water (eluent A)
and 90% (vol/vol) acetonitrile in ultrapure water (eluent B). The
following gradient was used (vol/vol): 0 min, 90% eluent A and 10%
eluent B; 45 min, 10% eluent A and 90% eluent B; 46 min, 90% eluent A
and 10% eluent B; and 60 min, 90% eluent A and 10% eluent B. One
hundred microliters of internal standard (0.002% [wt/vol]
2-aminobutyric acid in ultrapure water) was added to 500 µl of
aqueous extract. The amino acids were derivatized using an AccQ Fluor
reagent kit according to the manufacturer's instructions (Waters
Corp.). The derivatized samples as well as the appropriate external
standards were injected.
All sample preparations and analyses were performed in triplicate, and the mean values ± standard deviations are represented as milligrams per gram of pulp or beans.
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RESULTS |
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The ambient temperature during the day and night was 24 to 39°C and 19 to 24°C, respectively, with maxima being slightly lower in the case of rainfall (Fig. 3B). The temperature inside the heaps went from on average 26.3°C (26.0 to 30.0°C) at the start of the fermentations to a maximum temperature of on average 43.5°C (42.2 to 47.7°C). Rainfall slightly influenced the temperature course of the heap, with ups and downs of 1 to 4°C (Fig. 3B).
In general, no pronounced differences were observed between the repetitions and the seasons for microbial counts, pH, and temperature during cocoa bean heap fermentation at both farms, indicating the validity of the sampling, isolation, and measurement procedures.
Identification of the isolates.
Phenotypic analyses
indicated that isolates from MEA were yeasts (large colonies and bigger
cells than those of LAB and AAB) and that 240 out of 382 isolates from
MRS, M17, and KAA belonged to the LAB group (gram positive; rods or
cocci; nonmotile; catalase negative; oxidase negative; production of
lactic acid, acetic acid, and/or ethanol), while 132 out of 170
isolates from DMS belonged to the AAB group (gram negative; rods;
motile or nonmotile; catalase positive; oxidase negative; production of
acetic acid, gluconic acid, and 2-keto-gluconic acid). Noteworthy was
the selective isolation of AAB on DMS and of L. plantarum on
KAA under the conditions used, representing 78% and 72% of the
isolates, respectively. Most L. plantarum strains were
isolated from MRS; almost no enterococci were found neither on KAA
(15%) nor on the other media (Fig.
4).
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DNA/DNA hybridizations between AAB isolate 444B and isolates 430A and 415, all from cluster II, revealed high DNA binding values of 99% and 88%, respectively, and intermediate DNA binding values with A. syzygii LMG 21419T (46%) and Acetobacter lovaniensis LMG 1617T (47%), the phylogenetically closest Acetobacter species, which are below species level (69). This novel species is further referred to as "Acetobacter ghanaensis" (I. Cleenwerck, N. Camu, K. Engelbeen, T. De Winter, K. Vandemeulebroecke, P. De Vos, and L. De Vuyst, unpublished results). DNA/DNA hybridizations between isolates 108B (cluster III) and 420A (cluster IV) revealed a DNA homology value of 75%. Hybridizations of both isolates against A. tropicalis LMG 21419T and the newly proposed species "Acetobacter senegalensis" LMG 1617T (B. Ndoye, I. Cleenwerck, K. Engelbeen, R. Dubois-Dauphin, A. T. Guiro, S. Van Trappen, A. Willems, and P. Thonart, unpublished results), their phylogenetically closest neighbors, revealed respective DNA hybridization values of 54 to 58%, which is below species level, and 79 to 81%, which is above the accepted limit (70%) for species delineation (69). This indicates that rep-PCR clusters III and IV represent two clusters of the newly proposed "A. senegalensis" isolates. The G+C contents of DNA from isolates 108B and 420A were 55.6 and 55.9 mol%, respectively. These values were similar to the G+C content of DNA obtained for the type strain of "A. senegalensis." Further, isolates 108B and 420A showed the same phenotypic features as the type strain of "A. senegalensis": growth on yeast extract and 30% (wt/vol) D-glucose, growth with ammonium as the sole nitrogen source and ethanol as the energy source, growth in the presence of 10% (vol/vol) ethanol, growth with glycerol as the sole energy source but not with maltose or methanol, and the ability to produce 2-keto-D-gluconic acid from D-glucose but not 5-keto-D-gluconic acid.
Cluster analyses of the rep-PCR profiles according to farm and season did not reveal significantly different results (data not shown).
Population dynamics of Ghanaian cocoa bean heap fermentations: culture-independent approach.
Figure
5 shows representative fermentation courses for all seven
spontaneous cocoa bean heap fermentations as obtained by a community
analysis of DNA samples through 16S rRNA PCR-DGGE with the LAC1-LAC2
primer pair (only heaps 2 and 5 are shown). The number and intensity of
visible bands that varied among samples with fermentation time and
among fermentations could be related to shifts of the bacterial
compositions and hence a reflection of the impact of certain strains on
fermentation (cf. infra) as well as heterogeneous
samplings. Concerning the microbial succession as determined by DGGE in
general, it turned out that L. fermentum (band i) was the most
dominant species throughout fermentation in all heaps (Fig.
5A and B), although
L. plantarum (band iii) was detected in heap fermentations 2,
6, and 7 mainly during the first part of the fermentation (Fig.
5A). In general, the
intensity of the band corresponding with L. fermentum
increased upon fermentation (Fig.
5A and B), in
some fermentations still visible after 66 h (Fig.
5B) but in
others already disappeared before that time (Fig.
5A). L.
pseudomesenteroides (band ii) was detected during the first
24 h of fermentation in heaps 2, 3, 4, 5, and 7 (Fig.
5A and B). Also, an
unidentified bacterium (band iv), of which the partial 16S rRNA gene
sequence was identical to that of the "W.
ghanaensis" isolates, appeared throughout heap
fermentations 2, 3, 4, 5, and 6 up to 48 h (Fig.
5B). Lanes without bands
as in heap 2 indicate time points below the detection limit of DGGE
(Fig. 5A), due to a
too-low number of LAB as a result of death or lysis of the cells (as it
was difficult to recover LAB and AAB towards the end of the
fermentations, too) (Table
1), and the increasing
possibility of interference by inhibiting compounds for DNA extraction
and PCR amplification present in the samples upon fermentation time.
Finally, PCR-DGGE analyses performed on DNA extracted from cell mass
recovered from MRS agar plates where colonies were picked from revealed
the same species identities (Fig.
5C). Using the WBAC
primers, PCR-DGGE analyses revealed again L. plantarum and
L. fermentum as the dominating species, while no AAB species
were detected, although PCR-DGGE analysis of AAB reference strains was
successful (data not shown). Taking into account the totality of
PCR-DGGE runs performed, no pronounced differences were observed
between the repetitions.
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In general, almost all free sucrose in the pulp was consumed at the start of fermentation (Fig. 3C). The citric acid of the pulp was rapidly consumed as well (within 30 to 48 h of fermentation) and paralleled LAB growth (Fig. 3F), causing the pH of the pulp to increase. The initial decrease in citric acid of the beans was less than that of the pulp (Fig. 3F). At the end of all fermentations, citric acid slightly increased or stabilized in both pulp and beans. Fructose and glucose disappeared simultaneously in the pulp and were almost exhausted after 36 to 48 h of fermentation, whereas mannitol increased from 20 h of fermentation and stabilized from 70 h of fermentation onward (Fig. 3C).Erythritol was not found. Sucrose was hydrolyzed into glucose and fructose in the beans (Fig. 3D). Ethanol increased upon fermentation, almost simultaneously in pulp and beans, and reached a maximum of 10 ± 0.40 to 25 ± 0.40 mg g–1 in the pulp after 30 to 36 h of fermentation, after which it declined (Fig. 3H). Acetic acid levels increased after 6 h of fermentation. Concentrations of 10 ± 0.11 to 15 ± 0.11 mg g–1 of acetic acid were found in the fermented cocoa bean pulp after 90 h of fermentation.
The total amount of organic acids differed from heap to heap (data not shown). Whereas the amounts of ethanol and acetic acid were always slightly higher in the pulp than in the beans, the amounts of lactic acid were considerably higher in the pulp than in the beans. Lactic acid reached a maximum concentration of 1.0 ± 0.06 to 9.0 ± 0.06 mg g–1 in the pulp after 48 h of fermentation. Succinic acid increased up to 0.10 ± 0.0025 to 0.25 ± 0.0025 mg g–1 and 0.23 ± 0.0035 to 0.65 ± 0.0035 mg g–1 in pulp and beans, respectively, after 40 to 60 h of fermentation. Ketogluconic acid, malic acid, and fumaric acid were not found; gluconic acid (2 ± 0.005 to 4 ± 0.005 mg g–1 in pulp and 0.2 ± 0.005 to 0.4 ± 0.005 mg g–1 in beans) and oxalic acid (1 ± 0.004 mg g–1 in pulp and 1 ± 0.004 to 2 ± 0.004 mg g–1 in beans) remained stable as a function of time. Fatty acids (except for acetic acid), methanol, acetaldehyde, diacetyl, and acetoin were not found under the analysis conditions.
There was an increase in all free amino acids upon fermentation from 1.1 ± 0.003 to 3.5 ± 0.003 mg g–1 to 4.4 ± 0.003 to 4.8 ± 0.003 mg g–1 in the pulp and from 1.7 ± 0.002 to 2.1 ±0.002 mg g–1 to 5.2 ± 0.002 to 5.9 ± 0.002 mg g–1 in the beans (depending on the amino acid), except for lysine and glutamine in the pulp, asparagine in pulp and beans, and aspartic acid in the beans, which all decreased. In general, hydrophobic amino acids increased and acid amino acids decreased upon fermentation time (data not shown). The glutamic acid content increased in the pulp and first decreased and then increased in the beans (data not shown). Arginine showed a decrease followed by an increase in the pulp, while histidine showed an increase followed by a decrease in the beans (data not shown). Aromatic amino acid metabolites such as phenyllactic acid and OH-phenyllactic acid were not found under the analysis conditions.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Population dynamics.
The studied Ghanaian cocoa bean heap
fermentations were characterized by a high initial level and rapid
growth of both yeasts and LAB, a simultaneous but slower growth of AAB,
and a lower maximum temperature compared with those of other cocoa bean
fermentations reported in the literature. In other reports, counts are
generally from an initial 3 to 7 and a maximum 7 to 9 to finally 2 to 3
log CFU g–1, from 4 to 5 and 8 to 9 to 1.5 log CFU
g–1, and from 3 to 4 and 6 to 7 to 0 log CFU
g–1 for yeast, LAB, and AAB, respectively, and the
maximum fermentation temperature is often up to 50°C
(3,
66). A clear three-phase
fermentation process of a well-ordered succession of microbial groups
and a timely production of metabolites was difficult to recognize. In
general, the succession of microorganisms during cocoa bean
fermentation actually reflects the environmental factors (temperature,
pH, and oxygen tension) and the metabolism of substrates of the cocoa
bean pulp or originating from the cocoa beans (depending on composition
and harvest conditions), thus resulting in the production of
significant amounts of ethanol, lactic acid, and acetic acid
(3,
66,
67). It is
likely to assume that pod ripeness and postharvest pod age (due to
transportation and transient storage) and hence the pH of the cocoa
bean pulp determine the initial amounts of yeasts and LAB. Moreover,
yeast metabolism favors the growth of aciduric LAB
(55). The speed of AAB
growth probably determines the time course within which a maximum
fermentation temperature is reached and hence of the whole
fermentation, as the heat generated through acetic acid formation will
be responsible for the death of almost all microorganisms involved in a
desirable fermentation. This depends mainly on the size of the heap and
the influence of turning, as both factors probably influence sweating
and aeration of the heap and hence full growth of yeasts, LAB, and AAB
(4,
28).
Biodiversity.
Although the microbial ecology might be
influenced by cocoa cultivar, pod age, fermentation method and site,
and sample collection, the present study revealed that the biodiversity
of both LAB and AAB in the fermentations analyzed was rather
restricted, in contrast with a rich and varied yeast microbiota
reported so far for (Ghanaian) cocoa bean fermentations
(3,
30,
49,
63,
66). Lactobacillus
fermentum, L. plantarum, and A. pasteurianus
were found to be the dominant species. Although earlier studies report
on a broad diversity of both LAB and AAB as well
(8,
53-55,
72), recent reports
mention the dominance of L. fermentum, L. plantarum,
L. mesenteroides, L. lactis, A.
pasteurianus, and Acetobacter aceti
(3,
67). This limited number
of taxa can be explained by an updated taxonomy of both LAB and AAB
during the last years
(18) as well as an
adaptation of only a few dominant species to the cocoa bean
fermentation conditions (cf. infra). For instance, Lactobacillus
cellobiosus was considered an important microorganism in cocoa
bean fermentation (3), but
this species has been transferred to L. fermentum
(14). Similarly,
Acetobacter rancens has been included in the species A.
pasteurianus (13).
Interestingly, new taxa of both LAB (e.g., species of
Weissella) and AAB (e.g., species of Acetobacter)
were found in the present study.
Dominant strains and microbial successions.
The present study showed that L.
plantarum and L. fermentum were the most dominant LAB
species in the Ghanaian cocoa bean heap fermentations performed.
Moreover, L. plantarum decreased and L. fermentum
increased upon fermentation time, the latter species often dominating
the whole fermentation course. These data were supported by both
PCR-DGGE and enumeration on MRS, M17, and KAA, the latter medium being
selective for L. plantarum under the conditions applied. Both
species have been found in cocoa bean fermentations before
(8,
54,
55,
72). To our knowledge,
this is the first paper to show their individual succession.
Culture-dependent microbiological analysis of AAB species further
indicated that A. pasteurianus fulfilled a key role in the
Ghanaian cocoa bean heap fermentations performed, given its isolation
and metabolic activity throughout fermentation. "A.
ghanaensis" and "A. senegalensis"
strains were not always isolated, and the latter strains disappeared
faster than the former strains. Although AAB isolated from fermenting
material are difficult to grow under laboratory conditions
(3,
48), they possibly grew
or remained viable under the anaerobic conditions of the heaps, as has
been found in wine fermentations
(23). DGGE analyses of
AAB did not produce satisfactory results; again, L. plantarum
and L. fermentum were detected with the WBAC primers used.
These primers were actually developed to monitor both LAB and AAB
during wine fermentation
(44). This underlines the
importance of the detection limit to carry out PCR-DGGE, which is
generally between 4 and 6 log CFU g–1 or higher,
depending on the bacteria investigated, and hence the importance of
detecting the >90% most numerous species of a community without
discriminating living from dead cells or cells in a viable but not
cultivable state (16,
19). The cultivable
amount of the total AAB during the fermentations performed was never
higher than 7 log CFU g–1, and this occurred only
temporarily in the middle of the fermentations. Also, it may be that
the detection limit of AAB in a mixed ecosystem is higher and/or that
differential or preferential PCR amplification of DNA of LAB instead of
AAB occurred. A possible solution for this monitoring problem could be
the use of different species-specific primers targeting other regions
of the 16S rRNA gene or other genes
(74) or increasing the
intensity of the PCR amplicons produced from DNA by applying a nested
PCR (23). Both the agar
medium used for isolation and the high temperature of the heaps might
be responsible for the selective isolation of A. pasteurianus,
as this species may prefer calcium lactate and mannitol and is more
heat resistant and ethanol tolerant
(3,
8). It has
been shown before that more-complex and more-differentiated media are
necessary for the isolation of non-Acetobacter species
(42). A.
pasteurianus has been found during cocoa bean fermentation before
(3,
53); A. syzygii
(41) and A.
tropicalis (40) have
not. As mentioned before, the clusters corresponding with
"A. ghanaensis" (cluster II) and
"A. senegalensis" (a newly proposed
heat-resistant AAB species isolated from mango fruit; clusters III and
IV) (B. Ndoye et al., unpublished results) represent new species of
Acetobacter.
In the beginning of the fermentations, L. pseudomesenteroides and E. casseliflavus were present, but they disappeared rather rapidly. These species are often associated with plant material (6, 20). As most species of Weissella and some other LAB were found only in one or a few samples in the beginning of the fermentations, they represent contaminants from the environment. They could be associated with pod surfaces, banana/plantain leaves, the materials used, and farmers' hands (51). However, they did not adapt to the matrix upon further fermentation (as revealed by their absence through isolation and analysis of PCR-DGGE profiles) and hence are not regarded as important for cocoa bean fermentation. In contrast, although "W. ghanaensis" could hardly be recovered by plating (in one sample only), it appeared throughout some of the fermentations, as revealed by PCR-DGGE. Therefore, it may be speculated that "W. ghanaensis" plays a role in the Ghanaian cocoa bean heap fermentation process.
All dominant microorganisms mentioned above come from the environment as well. L. plantarum and L. fermentum are indeed associated with plant material (50). A. pasteurianus, A. syzygii, and A. tropicalis are generally isolated from fermented foods, flowers and fruits, and fruits and fermented foods, respectively, almost always from tropical countries (40-42). Some of these heat-resistant strains are interesting for industrial fermentations at higher temperatures (48). Interestingly, only a few strains of the better-adapted populations of L. plantarum, L. fermentum, "W. ghanaensis," and A. pasteurianus outnumbered the rest of the microbiota and were responsible for spontaneous fermentation of the cocoa beans, as only a limited number of strains within a heap and among heaps were found. Indeed, isolates with highly similar or even identical (GTG)5 PCR fingerprints were frequently found within the set of LAB and AAB isolates recovered from the same fermentation heap, whereas the samples were always taken at the same depth but in different points of the heap, indicating clones of the same strain and a possible microbial succession during fermentation at the strain level. Moreover, as no differences were observed between the season and only slight differences could be detected between the farms (as revealed by cluster analysis of the PCR-DGGE profiles), it can be anticipated that the heap fermentations performed during this study were dominated by certain strains and were hence very reproducible, which may support the general high quality standard of Ghanaian fermented cocoa beans. This is in accordance with the results of Nielsen et al. (49) on yeast diversity. Furthermore, they showed grouping of the fermentations with respect to fermentation method, site, time, and season. Microbial succession at both species and strain level within a fermentation course is often reported for spontaneous food fermentations (30).
Metabolomics.
Metabolite target analysis during this
study revealed that sugars, in particular sucrose, were utilized by the
yeasts through invertase activity, being converted to ethanol and
carbon dioxide, while glucose, fructose, and citrate were used by LAB
to be converted into lactic acid, acetic acid, ethanol, and mannitol.
During fermentation, sucrose inversion took place in the beans due to
cotyledon invertase activity
(26) and/or induced acid
hydrolysis as a result of acetic acid penetration into the beans upon
fermentation. Glucose was preferentially fermented above fructose
following sucrose hydrolysis
(60). Although citrate
has been mentioned as an important carbon source for yeasts during
cocoa bean fermentation
(65), only a few yeasts
(e.g., Pichia fermentans) can assimilate citrate and of the
dominant species, such as Candida krusei, found in Ghanaian
cocoa bean heap fermentations, only some isolates are able to
assimilate citrate within a reasonable time
(30). This indicates that
citrate assimilation was due to LAB growth as hypothesized before
(3,
73), probably being
favored at low pH values
(52). Hence, it may be an
important selective parameter for LAB strains to be used in cocoa bean
fermentation. Leuconostoc spp., Enterococcus spp.,
and L. plantarum metabolize citrate
(52,
59,
70). This may explain
their survival during the first part of the fermentation course.
Although citrate consumption has been shown to enhance the growth of
Leuconostoc spp. and not that of L. plantarum
(52,
70), the latter species
is more acid and ethanol tolerant than the former one
(3,
45), possibly explaining
its dominance against L. pseudomesenteroides during the first
part of the fermentation course. However, citrate consumption caused
the pH of the pulp to increase from pH 3.5 to about pH 4.3, which is
slightly lower than reported elsewhere
(3,
67) but important for the
proteolysis stage of the fermentation
(5). The production of
succinic acid may be ascribed to citrate-fermenting LAB as well or to
the conversion of fumaric acid and malic acid
(36). The aciduric and
ethanol-tolerant character of L. fermentum
(3,
7,
68) may explain its
survival and dominance of the whole cocoa bean fermentation process. In
addition, strains of L. plantarum and L. fermentum
are able to produce antimicrobial substances that play a role in
bacterial ecology (17).
The increased population of L. fermentum upon fermentation
explains the simultaneous accumulation of mannitol, as L.
fermentum can use fructose as an alternative electron acceptor,
being completely converted to mannitol. The part of available fructose
that was used as an alternative electron acceptor by L.
fermentum to produce mannitol could hence not be converted to
nonvolatile lactic acid (undesired for cocoa beans) through glycolysis
by this or other LAB species. Reversely, the production of mannitol
enabled the production of extra acetic acid and ATP, contributing to
both volatile acidity (desired for cocoa beans) and enhanced growth
competitiveness, respectively
(80). Both physiological
characteristics likely influence the quality of fermented cocoa beans
(29,
34). Finally, AAB, in
particular A. pasteurianus, grew on ethanol, mannitol, and
lactate and converted ethanol into acetic acid. This oxidation process
was responsible for the increase in temperature inside the heap
(67), and hence, a
maximum population of AAB corresponded with a maximum fermentation
temperature. Part of the acetic acid volatilized and part penetrated
into the cotyledons of the beans and was, together with part of the
ethanol and the heat, responsible for the killing of the cocoa seed
embryo and changes in the subcellular structure of the beans
(10), being an important
end point of fermentation.
Free amino acids increased upon fermentation time, although they were present in smaller amounts than reported elsewhere (27, 38, 62). However, the use of aqueous extracts in the present study reflects the bioavailability of nutrients for microbial fermentation of the pulp instead of their total extractable concentrations, which is important for further processing of the beans. Different patterns have frequently been observed for different amino acids (10, 27, 38). While the increase of hydrophobic amino acids and hydrophilic oligopeptides is due to cocoa bean proteolytic activity (5, 26, 77), it is well known that LAB and AAB use free (acid) amino acids as a carbon/nitrogen source and a nitrogen source, respectively (24, 35, 37, 64). In general, proteolysis depends primarily on the fermentation conditions, namely, the duration and intensity of acidification, temperature, and aeration (10, 38). Also, oxidation, condensation, and complexation (with polypeptides) of polyphenols occur (10, 31). Consequently, the fermentation conditions determine the amount of free amino acids, oligopeptides, reducing sugars, and polyphenols of fermented, dried cocoa beans, which all play an important role in aroma precursor formation that is further developed during cocoa processing (10, 73). The metabolite results presented in this paper in combination with literature data indicate that a successful fermentation process is reached after about 72 h of spontaneous fermentation.
Conclusions.
The use of a multiphasic approach as
applied during this study increases our understanding of spontaneous
food fermentation processes. The combination of both microbiological
analyses, encompassing culture-dependent and -independent methods, and
metabolite analyses, encompassing pure culture and fermentation
samples, allows us to profile population dynamics and fermentation
courses. This approach permits the identification of specific
populations and metabolites useful in improving fermentation (with
respect to reproducible and standardized end products and fermentation
time) and organoleptic (with respect to reproducible consistency,
color, flavor, and taste) profiles. Although cocoa bean fermentation is
a heterogeneous process per se, the organoleptic quality of Ghanaian
fermented cocoa beans is frequently reported as excellent
(12,
81). Yet, fermentation of
cocoa beans depends on production methods, batch sizes, pod ripeness
and storage, and fermentation conditions. As shown in the present
paper, particular competitive strains of both LAB and AAB dominate the
Ghanaian cocoa bean heap fermentation process. With the purpose of
selecting starter cultures for controlled cocoa bean fermentations
(65,
67), we indicate that
these should be acid-tolerant, ethanol-tolerant, and citrate-utilizing
strains in the case of LAB (preferably a combination of L.
plantarum and L. fermentum) and acid-tolerant,
ethanol-tolerant, and heat-resistant strains in the case of AAB
(preferably A. pasteurianus). Nevertheless, further research
is needed to determine whether these species are indeed essential or
necessary to develop fermented, dried, and roasted cocoa beans for good
chocolate quality.
|
ACKNOWLEDGMENTS |
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The cooperation of the Ghanaian Cocoa Producers' Alliance (COCOBOD, Accra, Ghana) and the Cocoa Research Institute of Ghana is highly appreciated. An approval was obtained by the COCOBOD to cooperate with local farmers.
|
FOOTNOTES |
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Published
ahead of print on 2 February 2007.
|
REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-amylase production at pH 4.0. Int. J. Food
Microbiol.
80:77-87.[CrossRef][Medline]
) and/or 16S rRNA gene sequence analysis (indicated by
).
), LAB (MRS, •;
M17, 
; KAA, 
), AAB (DMS, 
), and total
aerobic bacteria (PCA, 
). (B) Temperature inside
(
, KAA; 
, M17; and 
, MRS. The dilutions from which the different species were picked are not
shown. Lb., Lactobacillus.
