microbiological and physicochemical characterization of small

Microbiological and Physicochemical Characterization of Small-ScaleCocoa Fermentations and Screening of Yeast and Bacterial Strains ToDevelop a Defined Starter Culture

Gilberto Vinícius de Melo Pereira, Maria Gabriela da Cruz Pedrozo Miguel, Cíntia Lacerda Ramos, and Rosane Freitas Schwan

Biology Department, Federal University of Lavras, Lavras, Minas Gerais, Brazil

Spontaneous cocoa bean fermentations performed under bench- and pilot-scale conditions were studied using an integratedmicrobiological approach with culture-dependent and culture-independent techniques, as well as analyses of target metabolitesfrom both cocoa pulp and cotyledons. Both fermentation ecosystems reached equilibrium through a two-phase process, startingwith the simultaneous growth of the yeasts (with Saccharomyces cerevisiae as the dominant species) and lactic acid bacteria(LAB) (Lactobacillus fermentum and Lactobacillus plantarum were the dominant species), which were gradually replaced by theacetic acid bacteria (AAB) (Acetobacter tropicalis was the dominant species). In both processes, a sequence of substrate con-sumption (sucrose, glucose, fructose, and citric acid) and metabolite production kinetics (ethanol, lactic acid, and acetic acid)similar to that of previous, larger-scale fermentation experiments was observed. The technological potential of yeast, LAB, andAAB isolates was evaluated using a polyphasic study that included the measurement of stress-tolerant growth and fermentationkinetic parameters in cocoa pulp media. Overall, strains L. fermentum UFLA CHBE8.12 (citric acid fermenting, lactic acid pro-ducing, and tolerant to heat, acid, lactic acid, and ethanol), S. cerevisiae UFLA CHYC7.04 (ethanol producing and tolerant toacid, heat, and ethanol), and Acetobacter tropicalis UFLA CHBE16.01 (ethanol and lactic acid oxidizing, acetic acid producing,and tolerant to acid, heat, acetic acid, and ethanol) were selected to form a cocktail starter culture that should lead to better-con-trolled and more-reliable cocoa bean fermentation processes.

Cocoa fermentation is a key step in the technological transfor-mation of cocoa into chocolate, because the highly bitter, as-

tringent unfermented cocoa beans lack the full chocolate flavor.The fermentation of cocoa beans is therefore the first step of thechocolate-making process, which consists of a natural, 5- to 7-daymicrobial fermentation of the pectinaceous pulp surrounding theseeds of the tree Theobroma cacao (37, 39). The cocoa pulp ishydrolyzed during fermentation; this aids the drying process byallowing the pulp to be drained. Most importantly, fermentationtriggers an array of chemical changes within the cocoa bean thatare vital to the development of the complex, beloved flavor of“chocolate.”

The fermentation of cocoa beans occurs at two levels. The firstinvolves reactions that take place in the pulp, in the outer part ofthe beans, and the second involves several hydrolytic reactionsthat occur within the cotyledons (39). The microbial activity in thecocoa pulp is a well-defined microbial succession led by yeasts,which dominate the microbial community during the first hours,followed by the lactic acid bacteria (LAB), which decline after 48 hof fermentation, and finally the acetic acid bacteria (AAB) (2, 39).The metabolic diversity of the yeast, LAB, and AAB strains duringcocoa fermentation can be interpreted as a natural consequence ofthe environmental conditions that influence their growth and se-lection. Changes in the pH, temperature, sugar content, and fer-mentation products exert selection pressure on the already exist-ing natural biotypes, favoring those strains that are better adaptedto this environment (41). An analysis of the bacterial and yeaststrains that survive under these stresses could provide useful in-formation concerning the ability of the yeasts and bacteria to ini-tiate growth and complete fermentation. To perform this process,the microbial cells must adapt their own physiology or behavior inresponse to changing environmental stresses (11).

Chocolate processors require a constant supply of cocoa beansthat must conform to an array of criteria. The industrialization ofthe cocoa fermentation process may allow greater control over thequality of the cocoa beans and the chocolate derived from them(36). The concept of industrializing traditional fermentation pro-cesses to enhance their performance and efficiency is not new. Forexample, wine, beer, cheese, distilled sugar cane beverages, andyogurt were all at one time produced using traditional processes(43). These fermentations have been developed into highly effi-cient, well-controlled processes in modern fermentor designs of-ten using defined starter cultures (12, 40). However, cocoa fer-mentation remains an empirical process that does not give rise tobeans of consistent quality, which obliges processors to alter theirformulations continually (18). The fermentation takes place un-der uncontrolled environmental conditions that often lead to un-successful fermentation, and the variable quality of the productmay reflect the vagaries of chance contamination. Although pre-liminary experiments using defined starter cultures demonstratesatisfying results (8, 19, 35, 36), only one particular study utilizedyeast, LAB, and AAB simultaneously as a defined microbial cock-tail (36), but without any prior study of the stress tolerance and/orthe fermentative kinetic parameters of the individual strains.

Received 9 April 2012 Accepted 20 May 2012

Published ahead of print 25 May 2012

Address correspondence to Rosane Freitas Schwan, [email protected].

Supplemental material for this article may be found at http://aem.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.01144-12

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The first objective of this study was to investigate the physico-chemical changes and the dynamics of the microbial communitystructure during bench- and pilot-scale spontaneous cocoa beanfermentations. Next, the technological potential of the yeast, LAB,and AAB isolates was evaluated using a polyphasic screening studythat measured the isolates’ stress tolerance and fermentation ki-netic parameters in a cocoa pulp-simulating medium.

MATERIALS AND METHODSBench- and pilot-scale cocoa bean fermentations. Freshly harvested co-coa pods, obtained from a cacao farm located in Itajuípe, Bahia State,Brazil, were broken open manually with a machete, and the beans wereimmediately transferred to the fermentation site. The beans were mixed ina clean vessel to obtain a homogeneous mixture of 20 kg of wet beans andwere transferred to the laboratory. A total of 500 g of cocoa beans wasdeposited in plastic containers (PCs) (dimensions, 15 cm by 10 cm by 7cm). The plastic containers were kept in incubators, and the temperaturewas adjusted every 12 h to simulate the temperature of large-scale fermen-tations: 28°C at 0 h, 30°C at 12 h, 32°C at 24 h, 35°C at 36 h, 38°C at 48 h,42°C at 60 h, 46°C at 72 h, and 48°C at 84 h, 96 h, 108 h, 120 h, 132 h, and144 h. To allow sampling every 12 h, 14 individual plastic containers wereprepared for each experiment. The pilot-scale fermentation was per-formed in a 0.015-m3 double-layer stainless steel conical tank (ST) (10 kgof cocoa beans; noncommercial bioreactors) with temperature control.The stainless steel tank and plastic containers contained holes at the bot-tom to allow drainage of the sweatings generated during the fermentation.The vessels were partially closed with a steel and plastic lid to ensureadequate insulation. The fermentations were turned every 24 h, and anatural fermentation proceeded for 168 h.

Every 12 h, 200-g samples were randomly collected in sterile bags. Thesamples for chemical and culture-independent analyses were sealed inplastic bags and were frozen (�20°C). Microbiological analyses were per-formed immediately after sampling.

Culture-dependent microbiological analysis. Twenty-five grams ofcocoa beans and adhering pulp was added to 225 ml saline-peptone water(0.1% [vol/vol] bacteriological peptone [Himedia], 0.8% [vol/vol] NaCl[Merck, Whitehouse Station, NJ]) and was homogenized in a stomacherat normal speed for 5 min, followed by serial dilutions. LAB were enumer-ated by pour plate inoculation on MRS agar (Merck) containing 0.2%(vol/vol) sorbic acid (Merck) and 0.1% (vol/vol) cycloheximide (Merck)to inhibit yeast growth and 0.1% (vol/vol) cysteine-HCl to produce an-aerobic conditions during incubation. AAB were enumerated by surfaceinoculation on GYC agar (50 g/liter glucose, 10 g/liter yeast extract, 30g/liter calcium carbonate, 20 g/liter agar [all from Merck] [pH 5.6]) con-taining 0.1% cycloheximide to inhibit yeast growth and 50 mg/liter pen-icillin (Sigma, St. Louis, MO) to inhibit LAB growth. Yeasts were enumer-ated by surface inoculation on YEPG agar (1% yeast extract [Merck], 2%peptone [Himedia], 2% glucose [Merck] [pH 5.6]) containing 100 mg/liter chloramphenicol (Sigma) and 50 mg/liter chlortetracycline (Sigma)to inhibit bacterial growth. Nutrient agar (NA) containing 0.1% cyclohex-imide (Merck) was used as a general medium for a viable mesophilicbacterial population and Bacillus spp. Plating was performed in triplicatewith 100 �l (surface spread technique) or 1,000 �l (pour plate technique)of each diluted sample. After the spreading, the plates were incubated at30°C for 3 to 4 days for growth of cultures on MRS agar, YEPG agar, andNA and at 25°C for 5 to 8 days for growth of cultures on GYC agar.Following incubation, the number of CFU was recorded, followed bymorphological characterization and counts of each colony type obtained.The square root of the number of colonies of each type was restreaked andpurified. The purified isolates originating from GYC agar, YEPG agar, andNA were stored at �80°C in YEPG broth containing 20% (wt/wt) glycerol,and the isolates originating from MRS agar were stored at �80°C in MRSbroth containing 20% (wt/wt) glycerol.

Phenotypic characterization of bacterial colonies originating fromMRS, GYC, and NA plates was performed by conventional microbiolog-

ical methods: Gram staining, microscopic examination, catalase and oxi-dase activity, a motility test, spore formation, acid and gas productionfrom glucose, and acid and gas production from lactate and acetate (onlyfor isolates originating from GYC agar plates showing a clear zone aroundthe colony [presumptive AAB]). Yeast colonies were physiologically char-acterized by determining their morphology, spore formation, and fer-mentation of different carbon sources according to the method of Kurtz-man et al. (17).

Molecular identification of representative microbial strains was per-formed by sequence analysis of the full-length 16S rRNA gene or theinternal transcribed spacer (ITS) region for bacteria or yeasts, respec-tively. Bacterial or yeast cultures were grown under appropriate condi-tions, collected from agar plates with a sterile pipette tip, and resuspendedin 40 �l of PCR buffer. The suspension was heated for 10 min at 95°C, and1 �l was used as a DNA template in PCR experiments. For bacterial iso-lates originating from MRS agar and NA, a 16S rRNA PCR was carried outusing primers 27-F and 1512-R (23). For bacterial isolates originatingfrom GYC agar plates showing a clear zone around the colony, primers16Sd and 16Sr were used for the amplification of the 16S rRNA generegion conserved among AAB (34). For yeast isolates, ITS-PCR was car-ried out using primers ITS1 and ITS4 (26). Isolates identified as Saccha-romyces cerevisiae had their identity confirmed through a species-specificPCR assay with HO gene-derived primers (32). The rRNA gene region wasamplified in a Thermo PCYL220 thermal cycler (Thermo Fisher ScientificInc., Waltham, MA). The PCR products were sequenced using anABI3730 XL automatic DNA sequencer. The sequences were aligned usingthe BioEdit 7.7 sequence alignment editor and were compared to theGenBank database using the BLAST algorithm (National Center for Bio-technology Information, Bethesda, MD).

For the differentiation of AAB species closely related by their rRNAgene sequences, some specific biochemical tests were performed in orderto validate the data obtained by 16S rRNA sequencing. Growth on 30%D-glucose, 0.3% maltose, 0.3% methanol, and 10% ethanol was examinedby using a basal medium (0.05% yeast extract, 0.3% [wt/vol] vitamin-freeCasamino Acids [Difco], and 2.5% agar) and appropriate concentrationsof carbon sources. The medium without the carbon source was used as acontrol. Growth was checked after 7 days of incubation at 28°C. Theutilization of ammonium as the sole nitrogen source in the presence ofethanol as a carbon source was tested using Frateur’s modified Hoyerethanol/vitamin medium containing 2.5% agar. The acid productionfrom D- and L-arabitol was tested in phenol red broth with the carbonsource added at a final concentration of 1%. The results were assessed withreference to the control after incubation at 28°C for 48 h. All the tests wereperformed as described previously (15, 34).

Total-community DNA isolation. Beans and pulp were physicallyseparated by adding 100 ml of sterile distilled water to 100 g of the cocoabeans and adhering pulp in a plastic bag and homogenizing in a stomacherat normal speed for 5 min. The pulp fraction was recovered by decanting.Forty milliliters of the pulp fraction was lyophilized, and the freeze-driedcocoa pulp was ground thoroughly with a sterile pestle. Thirty milligramsof freeze-dried pulp was mixed by homogenization twice in 1.5 ml ofphosphate-buffered saline. The combined fluids were mixed by vortexingfor a further 10 min and were subsequently centrifuged at 100 � g and 4°Cfor 10 min to remove large particles. The supernatant was further centri-fuged at 8,000 � g and 4°C for 20 min to pellet the yeast and bacterial cells,which were subsequently frozen at �20°C for at least 1 h. This procedurewas performed twice for the preparation of yeast and bacterial cells sepa-rately. Bacterial cells were lysed as described by Pereira et al. (31). For thelysis of yeast cells, the pellet was resuspended in 600 �l sorbitol buffer (1 Msorbitol, 100 mM EDTA, 14 mM �-mercaptoethanol) with 10 mg/ml oflysing enzymes and was incubated at 30°C for 1 h. A pellet of the total cellswas obtained by centrifugation for 5 min at 5,000 � g and were resus-pended in 180 �l of buffer ATL (supplied in the QiAamp DNA Mini Kit).After cell lysis, the DNA in the supernatant was further purified by usingstep 4 (bacterial procedure) and step 2 (yeast procedure) of the protocol

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for DNA purification from tissues (supplied in the QIAamp DNA MiniKit; Qiagen, Hilden, Germany). The final samples were stored at �20°Cuntil further use.

Nested PCR–DGGE strategy. In order to increase the sensitivity ofdenaturing gradient gel electrophoresis (DGGE) and to facilitate theDGGE by analyzing fragments of the same length, a two-step nested-PCRtechnique was applied. For analysis of bacterial diversity, primers 27F and1512R were used to amplify the nearly complete gene encoding 16S rRNAunder conventional PCR conditions in the first PCR step (23). Subse-quently, the product of the first PCR was used as a template for a nestedPCR targeting the V3 region of the 16S rRNA gene using primers GC-338fand 518r (29) to create a DNA fragment suitable for DGGE analysis. Foranalysis of yeast diversity, PCR amplification of the ITS regions was per-formed using primers ITS1-F and ITS4 in the first step, followed by nestedPCR using the DGGE primers GC-ITS1-F and ITS2 (42). Reactions wereperformed in a Mastercycler (Eppendorf, Hamburg, Germany). The PCRproducts were analyzed by DGGE using a Bio-Rad DCode Universal Mu-tation Detection system (Bio-Rad, Richmond, CA). The PCR products ofthe second step were loaded onto 8% (wt/vol) polyacrylamide gels in arunning buffer containing 1� TAE (20 mM Tris, 10 mM acetate, 0.5 mMEDTA [pH 8.0]). Optimal separation was achieved with a 30 to 55%urea-formamide denaturing gradient for the bacterial community and a12 to 60% gradient for the yeast community (100% corresponded to 7 Murea and 40% [vol/vol] formamide).

The DGGE bands of interest were excised from the gel with a sterilescalpel, disrupted in 60 �l of sterile Milli-Q water, and left overnight at4°C to let the DNA diffuse out of the bands. Ten microliters of the elutedDNA of each DGGE band was reamplified by using the appropriate prim-ers and the conditions described above. The PCR products for sequencingwere purified using the QIAquick PCR purification kit (Qiagen). Thesamples were analyzed with an automated DNA sequencer (Applied Bio-systems, Foster City, CA). Searches in GenBank with BLAST were per-formed to determine the closest known sequences obtained.

Physical-chemical analysis. The temperature of the fermenting cocoapulp-bean mass was determined every 12 h with a Delta Ohm portabledata logger, model HD 2105.2. For analysis of target metabolites, aqueousextracts from fermentation samples were obtained as described previously(26, 36). The amounts (mg g–1) of alcohols (ethanol and methanol), or-ganic acids (lactic, acetic, and citric acids), and carbohydrates (glucose,sucrose, and fructose) were determined from pulp and bean extracts witha high-performance liquid chromatography (HPLC) apparatus (HP series1200; Hewlett-Packard Company), equipped with an Aminex HPX-87Hcolumn (Bio-Rad Laboratories, Hercules, CA) connected to a refractiveindex (RI) detector (HPG1362A; Hewlett-Packard Company). All sam-ples were analyzed in triplicate, and the average values and standard de-viations are presented. The column was eluted with a degassed mobilephase containing 4 mM H2SO4 at 30°C at a flow rate of 0.6 ml/min.

Screening on agar plates for stress tolerance. For the analysis of tol-erance to ethanol, lactic acid, acetic acid, glucose, and fructose, approxi-mately 106 CFU ml�1 of the yeast or AAB isolates was plated on basalmedium (0.05% yeast extract, 0.3% [wt/vol] vitamin-free Casamino Ac-ids [Difco], and 2.5% agar), and the LAB isolates were plated on MRSagar. The plates were supplemented with either 6, 10, or 12% (vol/wt)ethanol; 1, 2, 3, or 5% (vol/wt) lactic acid; 1, 2, 3, or 5% (vol/wt) aceticacid; 5, 15, or 30% (wt/wt) glucose; or 5, 15, or 30% (wt/wt) fructose. Amedium without a carbon source was used as a control. Growth wasobserved after 7 days at 28°C. To evaluate heat tolerance, the isolates weregrown at 30, 37, and 45°C on YEPG agar (for yeast and AAB isolates) orMRS agar (for LAB isolates). To evaluate pH tolerance, each medium wasadjusted to pH 2, 3, or 5.

Evaluation of the fermentation performance of yeasts, LAB, andAAB. The LAB and AAB strain fermentations were performed in 2-literErlenmeyer flasks containing 500 ml of cocoa pulp simulation mediumfor LAB (PSM-LAB) (13 g/liter fructose, 25 g/liter glucose, 10 g/liter citricacid, 5 g/liter yeast extract, 5 g/liter soya peptone, 0.5 g/liter magnesium

sulfate-heptahydrate, 0.2 g/liter manganese sulfate-monohydrate, 1 ml/liter Tween 80) or cocoa pulp simulation medium for AAB (PSM-AAB)(10 g/liter calcium lactate-pentahydrate, 10 ml/liter ethanol, 10 g/literyeast extract, 5 g/liter soya peptone), as described by Lefeber et al. (20).PSM-LAB and PSM-AAB were supplemented with 20% (vol/vol) freshcocoa pulp. A cocoa pulp simulation medium for yeast (PSM-yeast) wasformulated containing 17 g/liter fructose, 25 g/liter glucose, 10 g/litercitric acid, 5 g/liter yeast extract, 5 g/liter soya peptone, and 20% (vol/vol)fresh cocoa pulp. The precultures were grown in the same substrate at30°C for 48 h and were then used to inoculate each of the fermentations(106 CFU ml�1). The fermentations were performed in triplicate for eachstrain at 30°C for 24 h under static conditions. The alcohol (ethanol andmethanol), organic acid (lactic, acetic, and citric acids), and carbohydrate(glucose, sucrose, and fructose) contents were quantified in triplicate at 0,12, and 24 h of fermentation using HPLC with a model HP series 1200system (Hewlett-Packard Company) equipped with an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA) connected to an RIdetector (HPG1362A; Hewlett-Packard Company). The column waseluted with a degassed mobile phase containing 4 mM H2SO4 at 30°C at aflow rate of 0.6 ml/min.

RESULTSMicrobial counts. The results of the culture-dependent microbi-ological approach demonstrated that the yeasts, LAB, and AABwere able to grow in bench- and pilot-scale cocoa fermentations(Fig. 1a). The LAB and yeasts developed simultaneously andreached a maximum population of 8 log CFU g�1 after 12 h of

FIG 1 (a) Evolution of LAB in MRS medium (hatched symbols), yeasts inYEPG medium (open symbols), and AAB in GYC medium (filled symbols).Squares, ST; triangles, PC. (b) Temperatures inside the ST Œ and the PC � andthe incubation temperature (�).

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fermentation. The LAB counts remained high throughout the fer-mentations, while the yeasts progressively decreased in numberand became undetectable after 72 and 108 h in the PC and STfermentations, respectively. The AAB population started to de-velop after 12 h (5.20 and 6.36 log CFU g�1 in the ST and PC,respectively) and was present up to 96 h after the start of fermen-tation. Although nutrient agar medium was used to monitor thegrowth of aerobic mesophilic bacteria and Bacillus spp., severalGram-negative and aerobic rods that produced catalase and werenon-spore forming (later identified as AAB) grew on this medium(data not shown).

Identification and distribution of the isolates. A total of 96MRS, 68 GYC, 98 YEPG, and 71 NA isolates were randomly pickedup at different times during the fermentation processes and wereidentified via biochemical and molecular methods (Tables 1 and2). The 16S rRNA gene sequence analysis identified all of the homo-fermentative or facultatively heterofermentative MRS isolates asLactobacillus plantarum (n � 39). The other 57 isolates were obli-gately heterofermentative organisms, and most of them wereidentified as Lactobacillus fermentum (n � 52); the remaining 5obligately heterofermentative isolates were closely related to Lac-tobacillus vaccinostercus. At the onset of fermentation, L. planta-rum was the dominant species of the LAB, with a population ofapproximately 4 log CFU g�1. After 12 to 24 h, L. fermentum wasthe dominant species isolated and reached a maximum popula-tion of 8.01 log CFU g�1 at 24 h and 8.09 log CFU g�1 at 60 h in thePC and ST, respectively.

All of the isolates from the GYC agar plates oxidized acetateand lactate to CO2 and H2O and were assigned to the genus Ace-

tobacter. The sequencing of the 16S rRNA genes and the specificbiochemical tests supported the identification of these isolates asfour different species of Acetobacter, namely, Acetobacter tropicalis(n � 39), A. malorum (n � 13), A. cerevisiae (n � 9), and A.ghanensis (n � 7). A. tropicalis was the dominant species andreached a maximum population after 36 h (5.74 log CFU g�1) and12 h (6.22 log CFU g�1) in the PC and ST, respectively.

The 16S rRNA gene sequence analysis demonstrated that arange of other bacterial species grew on NA, including members ofthe Gram-positive order Bacillales and the Gram-negative familiesEnterobacteriaceae and Acetobacteraceae. The members of the or-der Bacillales were subdivided into the endospore-forming bacte-ria Bacillus subtilis (n � 29) and Bacillus megaterium (n � 11) andthe non-spore-forming bacteria Staphylococcus pasteuri (n � 7),Staphylococcus aureus (n � 3), Staphylococcus saprophyticus (n �3), Staphylococcus equorum (n � 1), and Staphylococcus xylosus(n � 1). The family Enterobacteriaceae was represented by thespecies Tatumella saanichensis (n � 3), Pantoea agglomerans (n �3), and Pantoea terrea (n � 2), while the family Acetobacteraceaewas represented by the species A. malorum (n � 6), A. cerevisiae (n� 6), A. pomorum (n � 2), and A. pasteurianus (n � 2) (data notshown). In this medium, B. subtilis was the dominant species, witha maximum population of 6.98 log CFU g�1 at 24 h and 6.92 logCFU g�1 at 12 h in the PC and ST, respectively.

The yeast isolates from the YEPG agar plates were Saccharomy-ces cerevisiae (n � 63), Pichia kluyveri (n � 13), Hanseniasporauvarum (n � 9), Issatchenkia orientalis (n � 7), Debaryomycesetchellsii (n � 5), and Kodamaea ohmeri (n � 1). S. cerevisiae wasthe most prevalent yeast found at the start of fermentation and

TABLE 1 Phylogenetic affiliationsa and estimated average levels of yeast and bacterial isolates from PC fermentation

Isolate identificationGenBank accession no.of closest relative

Estimated average level (log CFU g�1)b of the isolate at the following fermentation time (h):

0 12 24 36 48 60 72 84 96 108 120 132 144

YeastsS. cerevisiae AM711362.1 5.32 7.08 8.29 6.17 5.87 �1 �1 �1 �1 �1 �1 �1 �1P. kluyveri FM199971.1 �1 �1 �1 6.09 6.01 �1 �1 �1 �1 �1 �1 �1 �1H. uvarum FJ515178.1 �1 �1 �1 �1 6.33 5.93 �1 �1 �1 �1 �1 �1 �1I. orientalis EU315767.1 �1 �1 �1 6.13 5.92 �1 �1 �1 �1 �1 �1 �1 �1

LABL. plantarum HQ293084.1 4.11 7.23 7.87 6.96 6.84 6.23 6.76 �10 �10 5.98 5.76 5.59 �10L. fermentum HQ293040.2 �10 7.01 8.01 7.02 6.92 6.77 6.08 6.60 6.52 6.17 6.02 5.91 6.01L. vaccinostercus AB218801.1 �10 �10 7.96 7.11 �10 �10 �10 �10 �10 �10 �10 �10 �10

AABA. malorum FJ831444.1 �1 �1 5.23 �1 �1 4.31 3.76 �1 3.49 �1 �1 �1 �1A. cerevisiae HM562995.1 �1 4.00 �1 �1 5.00 4.00 �1 �1 �1 �1 �1 �1 �1A. tropicalis DQ523494.1 �1 �1 4.00 5.74 5.52 4.74 3.84 3.12 �1 �1 �1 �1 �1A. ghanensis HM562984.1 �1 5.10 �1 5.00 �1 �1 3.87 �1 �1 �1 �1 �1 �1

Species isolated on NAB. subtilis HQ286641.1 5.91 6.73 6.98 5.76 4.00 �1 �1 �1 �1 �1 �1 �1 �1B. megaterium FJ174651.1 5.32 5.00 6.32 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1S. pasteuri HM130543.1 5.17 5.00 6.21 �1 6.00 �1 �1 �1 �1 �1 �1 �1 �1S. xylosus HM854231.1 �1 6.19 5.00 �1 6.00 �1 �1 �1 �1 �1 �1 �1 �1S. saprophyticus HM130543.1 �1 5.00 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1T. saanichensis EU215774.1 5.00 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1

a BLAST searches were based on sequences of type and cultured strains at GenBank (National Center for Biotechnology Information). An isolate was assumed to belong to a givenspecies if the similarity between the query rRNA gene sequence and the sequences in the databases was higher than 97%.b Obtained after morphological characterization and molecular identification of each colony type at the sampling time. A value of �1 log CFU g�1 means that no colonies werefound.

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grew to a maximum population of 8.29 log CFU g�1 and 8.07 logCFU g�1 at 12 h in the PC and ST, respectively. Occasionally,Pichia kudriavzevii, H. uvarum, and I. orientalis were found inboth fermentation processes, while D. etchellsii and K. ohmeri wereisolated only at 12 h and 24 h in the ST fermentation.

Culture-independent microbiological analysis using a nest-ed-PCR-DGGE strategy. As displayed in Fig. 2, the bacterial andyeast DGGE profiles revealed a stable microbial composition in-dependent of the fermentation time. The PCR-DGGE revealedthat Lactobacillus fermentum, L. plantarum, and Acetobacter spp.(A. tropicalis and Acetobacter senegalensis were the closest relativesfound using sequence comparisons) were the dominant bacterialspecies, as revealed through sequencing of the most intense bands(Fig. 2A). Members of the Enterobacteriaceae family (Tatumellaptyseos and Pantoea terrea) and species of Staphylococcus (S. sap-rophyticus and a Staphylococcus sp.) were identified at differenttimes in both fermentation processes, while a Bacillus sp. and B.subtilis were detected at the end of the ST fermentation.

The PCR-DGGE revealed that the yeast population was com-posed of a few species (Fig. 2B). The most intense bands corre-sponded to the Hanseniaspora species (�99% identity with H.opuntiae, H. uvarum, and H. guilliermondii). Saccharomycescerevisiae could also be detected in almost all of the samples, but itsband density was typically weak. Pichia kluyveri was detectedthroughout the ST fermentation, while a Debaryomyces sp. wasobserved at the beginning of the PC fermentation.

Temperature of the cocoa mass. The temperature inside the

PC and ST fermentations ranged from an initial 26.6°C to a max-imum 46 to 47°C obtained at 108 h of fermentation (Fig. 1b). After108 h of fermentation, a slight decrease to 45 to 44°C was ob-served.

Substrate consumption and fermentation products. Thesubstrate consumption and the metabolite production of thespontaneous cocoa bean fermentations are shown in Fig. 3. Dur-ing the first 12 h of fermentation, the hydrolysis of nearly 50% ofthe pulp sucrose increases the levels of glucose and fructose over90 mg/g up to 130 mg/g. Glucose and fructose were consumedsimultaneously and rapidly up to 36 to 48 h, and afterwards, nosignificant changes were observed. The major carbohydrate insidethe beans was sucrose (6.73 mg/g and 7.44 mg/g in the PC and ST,respectively), which was continuously hydrolyzed into fructoseand glucose.

Ethanol was produced and was subsequently consumed con-comitantly with the growth of the yeast and the AAB (maximumconcentrations, 65.37 mg/g at 36 h and 79 mg/g at 48 h in the PCand ST, respectively) (Fig. 3). The ethanol produced in the pulpdiffused into the beans and reached maximum concentrations of2.89 mg/g at 36 h and 4.51 mg/g at 84 h in the PC and ST, respec-tively, after which it began evaporating.

The ST fermentation exhibited an unexpected profile, with lit-tle acetic acid production; this acid was not oxidized during thefirst 48 h of fermentation, and, with a linear increase, peaked onlyat the end of the fermentation process (18.86 mg/g). During thePC fermentation, acetic acid quickly appeared within 48 h (from

TABLE 2 Phylogenetic affiliationsa and estimated average levels of yeast and bacterial isolates from ST fermentation

Isolate identificationGenBank accession no.of closest relative

Estimated average level (log CFU g�1)b of the isolate at the following fermentation time (h):

0 12 24 36 48 60 72 84 96 108 120 132 144

YeastsS. cerevisiae AM711362.1 5.30 7.14 8.07 6.27 6.45 5.71 5.93 4.45 3.95 �1 �1 �1 �1P. kluyveri FM199971.1 �1 6.00 �1 6.34 6.56 �1 �1 �1 �1 �1 �1 �1 �1H. uvarum FJ515178.1 �1 �1 �1 �1 5.00 5.65 �1 �1 �1 �1 �1 �1 �1I. orientalis EU315767.1 �1 �1 �1 6.56 5.00 �1 �1 �1 �1 �1 �1 �1 �1D. etchellsii AJ586528.1 �1 6.00 7.00 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1K. ohmeri EF196811.1 �1 6.00 7.00 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1

LABL. plantarum HQ293084.1 4.55 7.30 7.97 �10 �10 8.05 8.08 �10 �10 �10 �10 �10 �10L. fermentum HQ293040.2 �10 �10 8.01 7.16 7.72 8.09 �10 7.07 7.96 6.48 6.37 6.21 6.09

AABA. malorum FJ831444.1 �1 �1 6.08 5.51 �1 4.32 3.00 �1 �1 �1 �1 �1 �1A. cerevisiae HM562995.1 �1 5.00 �1 �1 5.00 4.61 �1 �1 �1 �1 �1 �1 �1A. tropicalis DQ523494.1 �1 6.22 5.00 5.17 5.10 4.37 3.84 3.61 3.50 �1 �1 �1 �1A. ghanensis HM562984.1 �1 �1 �1 �1 5.00 4.00 3.87 �1 �1 �1 �1 �1 �1

Species isolated on NAB. subtilis HQ286641.1 6.81 6.92 7.42 5.11 4.28 �1 �1 �1 �1 �1 �1 �1 �1B. megaterium FJ174651.1 5.00 6.31 7.00 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1T. saanichensis EU215774.1 �1 6.00 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1S. aureus FJ899095.1 5.00 6.00 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1S. equorum AM945662.1 �1 �1 �1 6.00 �1 �1 �1 �1 �1 �1 �1 �1 �1P. agglomerans EU596536.1 6.20 �1 7.00 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1P. terrea HM562993.1 �1 6.00 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1 �1

a BLAST searches were based on sequences of type and cultured strains at GenBank (National Center for Biotechnology Information). An isolate was assumed to belong to a givenspecies if the similarity between the query rRNA gene sequence and the sequences in the databases was higher than 97%.b Obtained after morphological characterization and molecular identification of each colony type at the sampling time. A value of �1 log CFU g�1 means that no colonies werefound.




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24 to 72 h). After 72 h of fermentation, the acetic acid was likelyoxidized to CO2 and H2O, dropping below 20 mg g�1 at the end ofthe fermentation. A portion of the acetic acid content produced inthe pulp diffused into the beans after 36 h of fermentation. Thefinal concentrations of acetic acid in the cotyledon were 1.58 mg/gand 3.82 mg/g in the PC and ST, respectively. The concentrationof lactic acid in the pulp increased in the first 36 to 48 h, followedby a drop coinciding with the appearance of the AAB; there wasagain an increase in the concentration during the last 3 days of theST fermentation. The final lactic acid content in the pulp was 6.2mg/g in the PC fermentation, and the concentration in the ST wasapproximately 3-fold higher (19.22 mg/g). The lactic acid contentin the cotyledons was nearly 2-fold higher at the end of the STfermentation process (1.89 mg/g and 0.97 mg/g in the ST and PC,respectively). Rapid decreases in the citric acid content in the pulpwere observed after 12 h and 48 h for the ST and PC fermentations,respectively, while inside the beans, the citric acid content wasstable during both fermentation processes, with final concentra-tions of 1.26 and 1.82 mg/g in the PC and ST, respectively.

Polyphasic selection study. To investigate the physiologicaladaptation of the yeasts (184 isolates), the LAB (156 isolates), andthe AAB (112 isolates) to the cocoa fermentation conditions, andto select for strains better adapted to this environment, severalgrowth parameters were evaluated. Initially, the isolates werescreened for their abilities to withstand the stressors imposed bycocoa fermentation, including variations in the pH, the tempera-ture, and the concentrations of ethanol, lactic acid, acetic acid,glucose, and fructose. Isolates from traditional cocoa fermenta-tions performed in wooden boxes in Brazil (G. V. M. Pereira et al.,submitted for publication) were also included in this step. Thebest-adapted strains were selected and were subjected to addi-tional kinetic analysis performed in cocoa pulp simulation media.Tables S3, S4, and S5 in the supplemental material summarize thekinetic analysis data.

Yeasts. All of the cocoa yeast isolates tolerated the pH range ofa typical cocoa fermentation process (from pH 2.0 to pH 5.0). Themaximum growth temperature was 30 to 37°C, except for four I.orientalis and three S. cerevisiae isolates, which were able to growup to 45°C. Although all of the isolates exhibited a remarkableability to grow in the presence of 15% glucose and fructose, thebest growth was observed in the presence of 5% carbohydrates.The majority of the yeasts identified as S. cerevisiae, P. kluyveri, andD. etchellsii were tolerant of media amended with as much as 12%ethanol, while the other yeast species tolerated only 6% ethanol(except for Pichia fermentans UFLA CHYA4.0 and UFLACHYD31.07, I. orientalis UFLA CHYB6.02 and UFLA CHYC6.02,and Candida humilis UFLA CHD14.32) (data not shown). Fromthese results, 15 well-adapted isolates of S. cerevisiae, P. fermen-tans, P. kluyveri, D. etchellsii, and I. orientalis were selected forsimulated cocoa fermentation in PSM-yeast. Isolates of K. ohmeri,Candida orthopsilosis, C. humilis, Candida intermedia, and Schizo-saccharomyces pombe, which were not resistant to the stressorsemployed in this study, were also included in this step. The fer-mentation kinetics of the pure cultures is reported in Table S3 inthe supplemental material. S. cerevisiae strains UFLA CHYC7.04and UFLA CHYB4.03 exhibited the highest conversion rate ofsubstrate to ethanol (49.33 and 49.81% of the theoretical value,respectively). Within the non-Saccharomyces species, D. etchellsiiUFLA CHYB5.56, C. humilis UFLA CHYD14.32, P. kluyveri UFLACHYC2.02, and I. orientalis UFLA CHYB6.02 and UFLACHYC6.02 exhibited the highest fermentation efficiencies (�35%conversion of the substrate into ethanol), while K. ohmeri BM2.01,P. fermentans UFLA CHYA4.01 and UFLA CHYD31.07, C.orthopsilosis UFLA CHYC5.02, C. intermedia UFLA CHYE21.01,and Schizosaccharomyces pombe UFLA CHYE5.39 exhibited thelowest values (�20% conversion). None of the organic acid con-tents changed significantly in PSM-yeast during 24 h of fermenta-tion.

FIG 2 16S rRNA gene PCR-DGGE profiles of the bacterial communities (A) and ITS PCR-DGGE profiles of the yeast communities (B) associated with the cocoabean fermentation samples from the PC and the ST. (A) The closest relatives of the fragments sequenced, based on a search of GenBank (�97% similarity), wereL. plantarum (bands 1, 2, and 3), L. fermentum (bands 4, 5, 6, 7, and 8), P. terrea (bands 9 and 10), a Staphylococcus sp. (band 11), B. subtilis (bands 12 and 13),an Acetobacter sp. (bands 14, 15, 16, 17, 18, 19, 20, and 21), S. saprophyticus (bands 22, 23, and 24), an uncultured Lactobacillus sp. (band 25), T. ptyseos (bands26 and 27), and a Bacillus sp. (bands 28 and 29). (B) The closest relatives of the fragments sequenced, based on a search of GenBank (�97% similarity), were aHanseniaspora sp. (bands 1, 2, 3, 4, 5, 6, 7, and 8), a Debaryomyces sp. (band 9), S. cerevisiae (bands 10 and 11), and P. kluyveri (bands 12 and 13).

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LAB. The results demonstrated the overall ability of the LABisolates to grow over a wide range of pH values and lactic acidconcentrations. In general, the majority of the L. plantarum and L.fermentum strains shared the ability to grow at 45°C and 12%ethanol, while Weissella sp. isolates did not (data not shown). Sixstrains of L. fermentum, five of L. plantarum, and one of Weissellaghanensis were selected on the basis of their growth properties forfurther kinetic analysis in PSM-LAB (see Table S4 in the supple-mental material). The strictly heterofermentative LAB strains (L.fermentum and the Weissella sp.) fermented glucose but not fruc-tose, converted citric acid, and accumulated lactic acid and aceticacid as the main products. Interestingly, the conversion of citricacid by the strictly heterofermentative LAB strains was more effi-cient than the conversion of carbohydrates (glucose or fructose)into these products. All the citric acid content was consumed in 12h of fermentation, while a residual content of carbohydrates was

observed at the end of fermentations. The production of lacticacid was higher for L. fermentum UFLA CHBE8.12 (7.72 g liter�1),while the production of acetic acid was higher for Weissellaghanensis UFLA CHBE8.23 and L. fermentum UFLA CHBE6.01(6.25 g liter�1). None of the strains of L. plantarum tested wereable to produce lactic or acetic acid when inoculated in the syn-thetic medium used in this study (see Table S4).

AAB. All of the cocoa AAB isolates grew in a basal mediumsupplemented with as much as 5% acetic acid and a pH adjusted to2.0, 3.0, or 5.0, while these isolates failed to grow at 15 and 30%glucose and fructose concentrations. All of the isolates grew at 30and 37°C, as well as in 6% ethanol. Only the A. tropicalis isolatesgrew at 45°C and in 12% ethanol (data not shown). Five stress-tolerant A. tropicalis strains and representative isolates of the otherAAB species were subjected to simulated cocoa fermentation inPSM-AAB (see Table S5 in the supplemental material). The AAB

FIG 3 (Top) Residual glucose, fructose, and sucrose levels. Samples were taken from the ST (triangles) and the PC (squares). Open symbols, sucrose; filledsymbols, glucose; hatched symbols, fructose. (Center and bottom) Production of ethanol, methanol, and organic acids during cocoa bean fermentation. Ethanoland organic acid contents in the pulp (open symbols) and in the beans (filled symbols) are shown. Error bars represent standard deviations.




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species oxidized almost all of the ethanol into acetic acid within 24h of fermentation. These species also oxidized lactic acid duringtheir growth. The production of acetic acid was higher for A. tropi-calis UFLA CHBE16.01, A. senegalensis UFLA CHBE6.16, A.ghanensis UFLA CHBA3.01, A. malorum UFLA CHBA1.01, andGluconobacter oxydans UFLA CHBD7.06, which produced 14.08,13.49, 13.25, 11.45, and 10.73 g/liter, respectively, than for theother isolates, which produced �10 g/liter.

DISCUSSIONMicrobiological and physicochemical performance of cocoabean fermentations. The microbiological communities in the co-coa bean fermentations performed under bench- and pilot-scaleconditions (Fig. 1a; Tables 1 and 2) were similar to those observedin previous spontaneous, larger-scale fermentations (2, 14, 16, 18,25, 27, 30, 39). The most common LAB (L. fermentum and L.plantarum), AAB (A. tropicalis), and yeast (S. cerevisiae and Han-seniaspora spp.) cocoa species appear to dominate the cocoa fer-mentations. The composition and metabolic activity of these well-adapted cocoa species remained unchanged from those in largercocoa fermentations once a stable ecosystem was achieved. Thesesimilarities suggested that the small-scale fermentations provideda suitable model system for larger-scale fermentations, at leastwith regard to microbial ecology. The use of a suitable modelsystem may allow the evaluation of a starter culture under con-trolled conditions prior to its use in the field.

Several studies have attempted to cure small quantities of cacaobeans (�20 kg) rapidly and effectively in order to research aspectsof the cocoa fermentations, such as the effects of the cultivar, podstorage, pulp removal, and acid production (5, 10, 33). However,in these studies, the temperature of the cocoa bean mass did notincrease normally, often reaching only 35 to 37°C. Therefore, inthe present study, the bench- and pilot-scale fermentations wereplaced in a temperature-controlled incubator. The program cho-sen was designed to mimic the increases observed in a traditionalfermentation, which allowed the successive growth of the yeastsand the LAB (25 to 32°C; 0 to 48 h), followed by the AAB (40 to48°C; 60 to 144 h). For both fermentation processes, the temper-ature of the cocoa bean mass closely followed the temperature ofthe incubator (Fig. 1b).

The culture-based approach demonstrated that the LAB spe-cies L. fermentum and L. plantarum and the yeast S. cerevisiaedominated the early stages of fermentation. The richness in fer-mentable carbohydrates and the low oxygen content of the cocoamass favored the growth of these microbial groups, which werehypothesized to rapidly metabolize reducing sugars and citric acidand to produce mainly ethanol and lactic acid (Fig. 3). In addition,the simultaneous growth of both the yeast and the LAB can beexplained by additional modes of LAB-yeast interaction. For ex-ample, the death and autolysis of the yeast cells releases vitaminsand other nutrients, and/or the CO2 produced by the yeast createsmicroaerophilic conditions, which favor LAB growth (9). More-over, in the beginning, L. plantarum (facultatively heterofermen-tative) dominated the LAB community, but after 36 h, L. fermen-tum (strictly heterofermentative) became the dominant LABspecies (Tables 1 and 2). A similar growth dynamic between thesetwo LAB species was observed during large-scale cocoa fermenta-tions (26). This result demonstrated that the yeasts were mostadaptable when they were associated with the facultatively hetero-fermentative LAB rather than the strictly heterofermentative LAB,

as observed previously during sourdough fermentations (13).Dircks (7) observed that the increased ethanol content during acontrolled inoculation of cacao bean fermentations using differ-ent yeast species inhibited the growth of L. fermentum. Despite thecomplex microbial ecology of cocoa bean fermentations, thesetypes of interactions have not been considered.

Higher concentrations of citric and lactic acid were recoveredduring PC fermentation, in which L. plantarum populations werehigher, and amounts of L. fermentum were smaller, than those forthe ST fermentation. The homolactic metabolism of L. plantarumallows this species to achieve a high cell density within a reasonablefermentation time and, in contrast to L. fermentum, to producelarge amounts of lactic acid. In contrast, the heterolactic metabo-lism of L. fermentum leads to the rapid conversion of citric acidand the production of almost equal amounts (on a mass basis) oflactic acid and acetic acid (3). This led to higher final concentra-tions of lactic acid (but not of citric acid) in the cocoa beans fromthe PC fermentation than in those from the ST fermentation (Fig.3). Although the diffusion of lactic acid into the cocoa beans con-tributes to the breakdown of the seed cell structure, because it isnonvolatile, such an excess is not reduced during drying, and highconcentrations of the residual lactic acid will impart a sour flavorto the chocolate (1).

The kinetics of ethanol production in the pulp of cocoa fer-mentations corresponded to yeast growth (specifically the growthof the yeast S. cerevisiae [Tables 1 and 2]) and the utilization of thesugars. However, over time, the ethanol concentration in eachfermentation varied with the lowest content recovered from thePC at 48 h of fermentation. This result may have been due to thehigher yeast population observed in the ST than in the PC fermen-tation. The metabolic activity of the yeasts and the LAB (temper-ature increase; ethanol and lactic acid accumulation [Fig. 1b and3]) favored AAB growth (Fig. 1a), which was accompanied by theproduction of acetic acid and a reduction in the concentration ofethanol (Fig. 3). This finding agreed with the classic description ofthese bacteria in the literature (22, 39). Both culture-independentand culture-based approaches suggested a major role for the genusAcetobacter in bench- and pilot-scale cocoa fermentations. Thepresence of Gluconobacter species, previously reported in largercocoa fermentations (22, 39), was not confirmed in this study. A.tropicalis was the dominant species in both fermentations. Thedominance of A. tropicalis was observed previously during largercocoa fermentations in Australia (7). The A. tropicalis species isassociated mainly with fruits and fermented foods and has beenselected to produce artisanal vinegar (24). The dominance of thisspecies during cocoa bean fermentation may be explained by itsacid and heat resistance (24). The levels of acetic acid produced inthe pulp from the PC fermentation were much lower than thoseproduced during ST fermentation. This difference might be due tothe low availability of oxygen inside the tank; consequently, lessethanol may be oxidized to acetic acid.

Bacillus, Staphylococcus, and Enterobacteria were isolated dur-ing the last 0 to 48 h. The PCR-DGGE profile confirmed the pres-ence of these three bacterial groups in both fermentation pro-cesses (Fig. 2). The main species isolated were Bacillus subtilis, B.megaterium, and Staphylococcus pasteuri. Their growth appearedto be inhibited by the high population of the LAB and/or yeast,and no growth was observed after 48 h of fermentation (Tables 1and 2). The appearance of Bacillus spp. during cocoa bean fermen-tations is less predictable than that of other microbial groups.

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Their role during cocoa fermentation is not well known, and thisbacterium has never been involved as a starter in attempts to con-trol the fermentation process. It is believed that the growth of alow population of certain Bacillus strains might have a beneficialaction, e.g., by acting as a complementary partner to the yeasts inthe pulp depectinization process during the advanced stage ofcocoa fermentation (28). However, a high population may con-tribute to the acidity and perhaps to the undesirable flavors offermented cocoa beans (38). In this sense, cocoa fermentationsconducted with a defined inoculum of LAB and yeasts may proveuseful for the control of Bacillus spp. during industrialized fer-mentations. Although the conditions in the cocoa mass do notfavor the development of Enterobacteria and Staphylococcus spe-cies (low pH and high temperatures), their presence was reportedusing culture-independent methods during Ghanaian, Brazilian,and Ivorian cocoa bean fermentations (30). The appearance ofthese bacterial species underlines the importance of monitoringthe hygiene of the fermentation process to ensure that these spe-cies do not become dominant and spoil the beans (2). Their pres-ence may indicate human contact with the beans or may also beassociated with pod surfaces and the material used.

The PCR-DGGE method has some potential limitations thatmade it necessary to use this technique in combination with aculture-dependent method. Species present in low concentrationscould occasionally be grown from agar plates but in many casesdid not generate a detectable DGGE band. Thus, it is possible thatthe DGGE fingerprint might mask the perturbations of low-abun-dance community members in our study. This finding illustratesthe intrinsic limitation of DGGE analysis in visualizing only thepredominant species of a microbial community (31). Interest-ingly, S. cerevisiae yielded only a relatively weak band in the dena-turing gels compared to Hanseniaspora spp. (Fig. 2b), even thoughS. cerevisiae dominated the yeast species isolated by using the plat-ing method from both fermentation processes. A possible expla-nation may be that the S. cerevisiae ITS fragment is less efficientlyamplified than that of the Hanseniaspora species present duringthe cocoa fermentation by using the protocol described here (25).An accurate assessment of the microbial ecologies using PCR-DGGE requires appropriately designed primers, and the use ofpoorly targeted primers will skew estimates of the microbial diver-sity present (4).

Polyphasic selection study. To our knowledge, this study rep-resents the first assessment of the growth and stress tolerance ofyeasts, LAB, and AAB under cocoa-fermenting conditions. Thecriteria used were based on the physical and chemical changes thateach microbial group faces in the cocoa pulp substrate when theirmetabolisms are most active. At the beginning of the process, theyeast and LAB cells are affected by osmotic stress due to the highsugar content in the cocoa pulp (39). As the fermentation pro-gresses, other stressors become relevant as ethanol and lactic acidaccumulate and the environment acidifies. After 2 days of fermen-tation, the AAB become the dominant group and are faced with anethanol-rich environment, followed by the accumulation of aceticacid and an increase in the temperature to a mean value of 45°C(39). In the present study, the results indicated that the stressors ofthe early fermentation phase influenced the prevalence of the best-adapted yeast, S. cerevisiae. In addition, some strains of the non-Saccharomyces yeasts, such as P. fermentans, P. kluyveri, D. etchell-sii, and I. orientalis, were also well adapted to the conditionsimposed. The limited number of yeasts capable of growth at 45°C

explains the decreasing yeast population (Fig. 1a) after AAB-facil-itated exothermic ethanol oxidation raises the temperature to48°C. Similarly, the abilities of the L. fermentum and L. plantarumstrains to grow over a wide range of pH values, temperatures, andethanol and lactic acid concentrations correlated with the stablepopulation of the LAB during the entire fermentation process(Fig. 1a). Finally, the ability of A. tropicalis isolates to tolerate thestressors imposed during the second stage of the fermentationmight be considered an advantage of this species, which led to itsdominance in the fermentations performed.

As expected, the fermenting capacity of S. cerevisiae strains wasgenerally higher than that of the non-Saccharomyces species inPSM-yeast (see Tables S3 to S5 in the supplemental material). Theproduction of ethanol by the yeasts could affect the course of thecocoa fermentation in several ways, including the inhibition ofcertain microbial species, the inability of the cocoa beans to ger-minate and their decompartmentalization, and the use of ethanolas a precursor for the formation of acetic acid. However, somenon-Saccharomyces strains (D. etchellsii UFLA CHYB5.56, C. hu-milis UFLA CHYD14.32, P. kluyveri UFLA CHYC2.02, and I. ori-entalis UFLA CHYB6.02 and UFLA CHYC6.02) also producedsignificant levels of ethanol. This result, together with the ability ofthese non-Saccharomyces strains to tolerate cocoa-fermentingconditions, indicated that these strains could be used in a con-trolled cocoa fermentation multiculture starter with S. cerevisiae.The secondary products of their metabolism (e.g., organic acids,aldehydes, ketones, higher alcohols, and esters) and their glycosi-dase production are likely to be significant and should impactbean and chocolate quality (2). These potentially important influ-ences have been overlooked in previous work on cocoa fermenta-tion.

The strictly heterofermentative L. fermentum strains werecharacterized by rapid citric acid conversion, and in the consump-tion of sugars, glucose was preferentially metabolized over fruc-tose. Citric acid is used for the oxidation of NADH plus H� tobypass the energy-limiting ethanol pathway and to maximize thegrowth rate on glucose, thereby producing mannitol and lacticacid plus acetic acid (21). The metabolism of citric acid by cocoa-specific L. fermentum has been observed previously (20). The con-sumption of citric acid resulted in the production of organic acidswith a higher pKa value, which increased the pH of the environ-ment and allowed better bacterial growth and microbiologicalcontrol of the environment (39).

All of the AAB strains oxidized lactic acid (through lactate dehy-drogenase, pyruvate decarboxylase, and acetaldehyde dehydrogenaseactivities) and ethanol (via the sequential action of pyrroquinoline-quinone-dependent alcohol dehydrogenase and acetaldehyde dehy-drogenase activities) into acetic acid in PSM-AAB. Thus, the simul-taneous metabolism of the LAB and yeasts stimulates the growth ofthe AAB during a natural cocoa fermentation. In agreement with theability to tolerate cocoa fermentation stress, the oxidation process ofA. tropicalis UFLA CBE16.01 was the most effective, followed by A.senegalensis UFLA CBE6.16 and A. ghanensis UFLA CBA3.01. Inter-estingly, the species A. senegalensis and A. ghanensis were recentlydescribed during Ghanaian cocoa bean fermentation processes (6),which likely explains their adaptation to the cocoa pulp habitat.

In conclusion, this study demonstrated that the bench- andpilot-scale cocoa fermentation ecosystems reach equilibriumstarting with the simultaneous growth of the yeasts and LAB,which are gradually replaced by the AAB. Overall, the dominant




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species largely overlapped with those commonly associated withlarger cocoa bean fermentations (S. cerevisiae and Hanseniasporaspp. in the yeast group, L. fermentum and L. plantarum in the LABgroup, and A. tropicalis in the AAB group), proving their adapta-tion to the cocoa environment under the conditions applied. Inboth processes, the course of substrate consumption and metab-olite production was similar to that in the larger, spontaneouscocoa bean fermentation processes. However, further studiesshould evaluate the impact of excessive production of these me-tabolites in the pulp on the technological and sensorial quality ofthe resultant chocolate.

The polyphasic selection study allowed us to construct a betterpicture of the physiology and ecology of the indigenous yeast,LAB, and AAB strains. As a result, some strains from these threemajor groups were selected as potential starter cultures. In partic-ular, L. fermentum UFLA CBE8.12 (citric acid fermenting, lacticacid producing, and tolerant to heat, acid, lactic acid, and etha-nol), S. cerevisiae UFLA CYC7.04 (ethanol producing and acid,heat, and ethanol tolerant), and A. tropicalis UFLA CBE16.01 (eth-anol and lactic acid oxidizing, acetic acid producing, and tolerantto acid, heat, acetic acid, and ethanol) were selected as candidatesfor a mixed-strain starter cocktail that should lead to better-con-trolled and more-reliable cocoa bean fermentation processes. Inaddition, analysis of the non-Saccharomyces strains (D. etchellsiiUFLA CYB5.56, C. humilis UFLA CYD14.32, P. kluyveri UFLACYC2.02, and I. orientalis UFLA CYB6.02 and UFLA CYC6.02)indicated that these should be tested in association with S. cerevi-siae for cocoa starter cultures in future studies.

ACKNOWLEDGMENTS

We thank the Brazilian agencies Conselho Nacional de DesenvolvimentoCientífico e Tecnológico of Brasil (CNPQ), Fundação de Amparo a Pes-quisa do Estado de Minas Gerais (FAPEMIG), and CAPES for scholar-ships, and Almirante Cacau Agrícola (Masterfoods) for financial support.

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