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Food Microbiology 25 (2008) 951957

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Food Microbiology
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Succession of bacterial and fungal communities during natural coffee (Coffea arabica) fermentation
Cristina Ferreira Silva a, Luis Roberto Batista b, Lucas Magalhaes Abreu a, Eustaquio Souza Dias a, Rosane Freitas Schwan a,
a b

Departamento de Biologia, Universidade Federal de Lavras, UFLA, 37.200-000 Lavras, MG, Brazil Departamento de Ciencia dos Alimentos, Universidade Federal de Lavras, UFLA, Lavras, MG, Brazil

a r t i c l e in fo
Article history: Received 4 April 2008 Received in revised form 8 July 2008 Accepted 9 July 2008 Available online 11 July 2008 Keywords: Bacteria Yeasts Filamentous fungi Coffee processing Microbial succession

abstract
Bacteria, yeasts and lamentous fungi were isolated during natural coffee processing. Bacteria were isolated in greater numbers at the beginning of the fermentation, when the moisture of the coffee beans was around 68%. Gram-positive bacteria represented 85.5% of all bacteria isolated, and Bacillus was the predominant genus (51%). Gram-negative species of the genera Serratia, Enterobacter and Acinetobacter were also found. Approximately 22% of 940 randomly chosen isolates of microorganisms were yeasts. Debaryomyces (27%), Pichia (18.9%) and Candida (8.0%) were the most commonly found genera, and these three genera tended to appear more often as the fruit was fermented and dried. Aspergillus was the most abundant genus besides Penicillium, Fusarium and Cladosporium, with 42.6% of the total fungi isolates. The genera and species identied included members known to have pectinase and cellulase activities. Of the 10 organic acids analyzed and quantied in coffee beans, acetic and lactic acids may have been generated by microbial activity. Butyric acid was not detected in any sample. & 2008 Elsevier Ltd. All rights reserved.

1. Introduction Brazil is the largest producer of coffee (4.2 M tons), followed by Paraguay, Venezuela, Colombia, Indonesia, Ethiopia, India, Mexico and another 40 countries. Two coffee species dominate the world market: Coffea arabica (arabica) and C. canephora (robusta). Arabica and Robusta coffees account for 76.4% and 23.6% of world production, respectively (Coltro et al., 2006). Coffee cherries are processed by one of the two methods (Schwan and Wheals, 2003). In Colombia, Central America and Hawaii, the wet method is used for Arabica coffee. In the wet method the hand-picking mature cherries are mechanical depulping and then fermented for approximately 2448 h to remove the mucilage layer. The dry process, which results in so-called unwashed or natural coffee, is the oldest and simplest method of processing coffee. The dry process is often used in countries where rainfall is scarce and long periods of sunshine are available to dry the coffee properly. The dry method is used for about 95% of Arabica coffee produced in Brazil, most coffee produced in Ethiopia, Haiti, Indonesia and Paraguay, and some Arabica produced in India and Ecuador. This method involves fermentation of whole fruit and usually produces coffee that is heavy in body, sweet, smooth and complex. The

Corresponding author. Tel.: +55 35 38291614; fax: +55 35 38291100.

E-mail address: rschwan@ua.br (R.F. Schwan). 0740-0020/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2008.07.003

coffee fruits are spread on the ground (earth, platforms, concrete or asphalt) in layers approximately 10 cm thick, heaped at night and respread each day (Schwan and Wheals, 2003). Over the course of 1025 days of sun drying, natural microbial fermentation occurs that can inuence in the nal quality of the product (Schwan and Wheals, 2003; Silva et al., 2000). Fermentation of pectinaceous sugars produces ethanol and acetic, lactic, butyric and higher carboxylic acids. The formation of butyric and propionic acids from bacterial fermentation causes a loss of quality due to diffusion of the acids into the beans (Amorim and Amorim, 1977). Bacteria, yeasts and lamentous fungi have already been reported during fermentation by the wet method (Masoud and Kaltoft, 2006; Avallone et al., 2001), but only one comprehensive study of dry processing has been published (Silva et al., 2000). The microbiota involved in dry processing are much more varied and complex than those found during wet fermentation, but the actual role of each group of microorganisms during coffee fermentation by natural processing is still unknown. The microbial succession and consortium of bacteria, yeast and lamentous fungi and their metabolites during natural coffee fermentation remain to be studied. An understanding of microbial dynamics during natural fermentation should enable more rapid fermentation and better quality of the nal product. Therefore, the objectives of this work were to investigate the natural microbial fermentation of coffee cherries, to isolate and

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characterize the microorganisms involved, and to evaluate the biochemical characteristics of coffee during fermentation, drying and storage.

2.2.2. Identication of yeasts All yeast isolates were characterized by determining their morphology, spore formation, assimilation and fermentation of different carbon sources according to Kurztman and Fell (1998) and Barnett et al. (2000). 2.2.3. Identication of lamentous fungi Filamentous fungi were initially cultured on PDA medium (Merck) and observed with an optical microscope for preliminary identication. This was done by morphotypic analysis of the colony, especially color and appearance, using the method of Pitt and Hocking (1997). Initial identication of the genera Penicillium, Fusarium, Cladosporium and Aspergillus was made with microscopic slide examination of spores and mycelium. Further support for fungi identication was found in Christensen (1982), Nelson et al. (1983) and Pitt and Hocking (1997). 2.3. Chemical analysis Samples of coffee fruits and beans were taken and characterized with respect to total soluble solids, starch, total sugars, reducing sugars, total titratable acidity, phenolics, pH and moisture (A.O.A.C., 2000), pectins (Bitter and Muir, 1962), polyphenol oxidase (PPO) (Mazzafera and Robinson, 2000), peroxidase (Matsuno and Uritani, 1972), lignin and cellulose (Van Soest and Wine, 1968). 2.4. Organic acids Organic acids were analyzed using a high-performance liquid chromatography system (Shimadzu, model LC-10Ai (Shimadzu Corp., Japan) with a UV detector at 210 nm. A Shimadzu ion exclusion column (Shim-pack SCR-101H) operated at a temperature of 40 1C was used to achieve chromatographic separation. Two gram of sample was mixed with 18 ml of water for 30 min. Water-soluble acids were eluted with 0.01 M potassium dihydrogen phosphate buffer solution at a ow rate of 0.6 ml/min. Samples were microltered through a 0.2 mm cellulose acetate lter and directly injected (20 ml) onto the chromatographic column. Standard solutions (malic acid, lactic acid, acetic acid, butyric acid, propionic acid, citric acid, oxalic acid, succinic acid, tartaric acid) were prepared by dilution of the individual compounds in ultra pure water. All samples were examined in triplicate. The coefcient of variation was less than 5% in each case.

2. Materials and methods 2.1. Sampling One hundred and eight kg of coffee cherries from C. arabica var. Acaia were hand-picked at the mature stage (red cherries) from a farm at 750800 m above sea level situated in Lavras in the state of Minas Gerais, Brazil. The beans were fermented and dried by the natural method; they remained on a concrete platform for about 25 days until reaching a moisture level of 1112%. The environmental temperature was 2528 1C during the day and 1820 1C during the night. The beans were then packed in either polystyrene bags or jute sacks and stored in a cold chamber at 3 1C with 59% relative humidity for 140 days.

2.2. Microbiological analyses Samples were aseptically taken every 24 h throughout coffee processing. Each sample included 200 fruits that were added to a bottle containing 1800 ml of saline-peptone water (0.1% bacteriological peptone, Himedia, Mumbai, India and 0.8% NaCl, Merck, Darmstadt, Germany). After mixing for 15 min at 150 rpm in an orbital shaker, ten-fold dilutions were prepared. Enumeration of microorganisms was carried out using ve different culture media. Plate count agar (PCA, Merck) and WL differential medium (Cat. no. 242510 Difco) were used as general media, Eosyn Methylene Blue Agar (Oxoid, Basingtstoke, UK) was used to enumerate Enterobacteriaceae, MRS medium (Oxoid) with 0.25% (v/v) of nystatin (Sigma, St. Louis, USA) was used for the enumeration of lactic acid bacteria (Almeida et al., 2007) and DG18 (Dicloran Glycerol 18%) agar (Oxoid) containing 100 mg chloramphenicol (Sigma) and 50 mg chlortetracycline (Sigma) per liter was used to enumerate yeasts and lamentous fungi (Hocking and Pitt, 1980). After spreading, plates were incubated at 28 1C for 48 h for bacteria and 5 days for lamentous fungi and yeast. For each type of medium containing isolated colonies, the square root of the number of colonies was taken at random for identication (Holt et al., 1994). Isolates were puried and stored at 80 1C in 20% glycerol.

2.2.1. Identication of bacteria The size, shape, color, height and edge of each colony were noted and Gram staining, presence of catalase and motility were assayed before replicating the bacteria onto slants of PCA and preserving them at 80 1C in 20% glycerol for subsequent identication. Gram-negative bacteria were identied using Bac-Tray Kits I, II and III (Difco), following the manufacturers instructions. Gram-positive bacteria were subdivided into spore-formers and non-spore-formers by heating at 80 1C for 10 min to kills the vegetative cells (Almeida et al., 2007). Subsequent identication using biochemical and motility tests proceeded as recommended in Bergeys Manual of Determinative Bacteriology (Holt et al., 1994) and The Prokaryotes (Hammes and Hertel, 2003) and conrmation was made using API 50 CHB galleries (BioMerieux). Presumptive lactobacilli were counted on MRS agar. Biochemical characterization of strains was performed with API ID 32 for lactococci and enterococci, and API 50 CHL (BioMerieux) for lactobacilli and leuconostocci.

3. Results 3.1. Microbiological analyses A total of 940 microbial colonies isolated from the experimental samples were analyzed. Eighty ve percent of the isolates could be identied to species level. The incidence of bacteria, yeast and lamentous fungi and the variation in water activity (aw) during coffee cherry and bean fermentation and drying are shown in Fig. 1. Ripe coffee cherries presented 69% moisture at the beginning of fermentation (time 0) that represented 0.85 aw (Fig. 1). From the 12th to the 16th day of fermentation the decrease in the moisture values was not 40.23%. The moisture reached 15%, 13% and 11%, on the 18th, 20th and 22nd fermentation/drying days, respectively. On the 22nd day, the coffee beans had 11% moisture and 0.52 aw. This aw value was considered safe for bean storage because it does not favor microorganism development (Bytof et al., 2005). After fermentation and

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100

Fungi

Yeast

Bacteria

aw

1 0.9

80 % of isolates number

0.8 0.7

60

0.6 0.5 aw

40

0.4 0.3

20

0.2 0.1

0 0 2 4 6 8 10 Time (days) 12 14 16 18 20 22 A B Storage C D E F

Fig. 1. Frequency of lamentous fungi, yeasts and bacteria isolated from fruits and grains of natural coffee during fermentation, drying and storage. () Water activity of coffee fruits. (A, B, C) Microorganisms isolated from coffee beans stored in jute bags, (D, E, F) microorganisms isolated from coffee beans stored in polystyrene bags from 40, 90 and 140 days at 3 1C and 59% relative humidity.

Table 1 Chemical and physical characteristics of coffee beans after 140 days storage in jute bags (J) and polystyrene bags (P) at 3 1C and 59% relative humidity Characteristics Coffee beans stored in jute bags 7Standard deviation 19.0571.25 20074.88 68.6972.65 133.5576.99 6.90170.96 0.36070.06 1.5470.34 1.1270.09 899.0979.01 881.2277.99 9.670.98 19.4071.76 Coffee beans stored in polystyrene bags 7Standard deviation

Water content (%) Acidity (ml NaOH 0,1 N) Polyfenol oxidase (U/g/min) Peroxidase (U/g/min) Phenolic compounds (%) Reducing sugars (%) Total sugars (%) Sucrose (%) Total Pectin (mg/10 g) Soluble Pectin (mg/100 g) Lignin (%) Cellulose (%)

13.7371.09 25076.77 89.3673.44 123.3374.33 7.68570.34 0.40870.08 1.6870.45 1.2170.06 73777.56 640.0978.94 7.470.78 19.8071.54

drying, the coffee beans were stored at 3 1C and 59% relative moisture in jute bags and polystyrene packaging for 140 days. Coffee bean re-hydration was observed during this period. Coffee beans in polystyrene packaging presented a 2% moisture increase as compared to the value observed on the 20th day of coffee cherry fermentation. An 8% moisture increase was detected in beans stored in jute sacks. The chemical and physical characterization of green coffee beans stored in the two different types of packaging at the end of storage is shown in Table 1. Except for cellulose concentrations, all chemical characteristics were different, indicating that the type of coffee bean storage and packaging inuenced the chemical composition. Coffee beans stored in polystyrene bags presented higher acidity values than those stored in jute sacks (Table 1). PPO and peroxidase enzyme levels are parameters that can be related to coffee beverage quality; high PPO activities indicate better quality of coffee (Amorim and Amorim, 1977). Beans stored in jute sacks presented a color change (whitening) that was conrmed by high peroxidase activity (133.5 U/g/min). The reducing sugar and sucrose contents were 0.408 and 1.21%, respectively, for beans stored in polystyrene bags; these values were higher than those observed for beans stored in jute sacks. Sucrose content is a desirable characteristic because it is a

precursor of the aromatic compounds that develop during bean roasting (Farah et al., 2006). Bacterial populations were predominant in coffee cherries sampled on the bush (time 0); they represented 96.3% of the total isolated microorganisms (bacteria, lamentous fungi and yeasts) as shown in Fig. 1. The bacterial population was X50% of the microorganisms isolated until the 8th day of fermentation. After this time the bacterial population decreased to o10% of total isolates by the 14th day of fermentation. An increase in the yeast population was observed as compared to the bacterial population at alternate times during fermentation and drying. The yeast count was greater compared than the bacteria count on fermentation days when aw was between 0.82 and 0.85. During storage, yeasts were detected in small numbers in beans stored in polystyrene packaging for 40 days (Fig. 1, letter D) and in jute packaging at 140 days. Filamentous fungi were detected in small numbers in coffee cherries on the bush and in greater populations at the end of the fermentation and drying period. The greater variability in fungi species did not result in numerical predominance of isolates. The microbial population present in the coffee cherries and beans varied in number and species. This variation seems to have been inuenced by the available aw as well as coffee cherry and bean moisture. Four different phases were observed during the processing: from 0 to 4, 6 to 12, 14 to 18 and 20 to 22 days of fermentation and drying. After the 4th day, three different Gramnegative bacteria, six Gram-positive bacteria, eight yeasts and fteen lamentous fungi species were identied. Yeasts and lamentous fungi showed higher diversity of species than bacteria, although the bacterial population was always higher. Species of the Enterobacteriaceae family predominated among the Gram-negative bacteria, and species of the Bacillus genus predominated among the Gram-positive isolates. A decrease in the bacterial population was observed from the 6th to the 12th day of processing, but there was simultaneously a signicant increase in yeasts. Most lamentous fungi species were present at approximately 102 CFU/ml until the 8th fermentation and drying day, while the yeast population ranged from 106 to 103 CFU/ml depending on the species. Among the identied fungi, a reduction in number of species was observed for the Penicillium and Fusarium genera when compared to initial time point (Table 2).

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Table 2 Dominant microbial species isolated from coffee fruits during 22 days of fermentation and drying Time (days) 0 2 4 6 8 12 14 16 18 20 22 Bacteria (X104 UFC/g) Yeast (X104 UFC/g) Filamentous fungi (X104 UFC/g)

Bacillus cereus, B. subtilis (6), B. macerans Acinetobacter sp, B. cereus, B. polymyxa (2), B. subtilis (5) Arthrobacter sp. (1), B. polymyxa (2), B. subtilis (8) B. cereus, B. subtilis (9) Acinetobacter sp. B. Cereus (2), B. macerans (1), Enterobacter agglomerans (1), Yersinia sp (1) B. cereus (2), B. subtilis (16), Arthrobacter (1) B. megaterium (1), B. subtilis (11) B. subtilis (5) B. megaterium (3), B. polymyxa (2), B. subtilis (8) B. subtilis (7), B. megaterium (1) B. subtilis (1), B. megaterium (3)

Candida saitoana (1) Candida fermentati (1) Stephanoascus smithiae (1) Debaryomyces polymorphus (2) Pichia guilliermondii (1) P. guilliermondii (3) P. guilliermondii (7) C. membranifaciens (1) D. hansenii(1), P. guilliermondii (6), P. burtonii, P. anomala (2) D. polymorphus (1), P. guilliermondii (4) S. smithiae (1), P. guilliermondii (8), D. polymorphus (2), D. hansenii (10), P. Burtonii (1), P. anomala (2) Arxula adeninivorans (1), D. hansenii (36), P. anomala (3), P. guilliermondii (5), P. sydowiorum (1) P. anomala (1), P. sydowiorum (2), P. subpelliculosa (1) Saccharomyces cerevisiae (1), D. hansenii (6), Arxula adeninivorans (1) Pichia anomala (1)

Cladosporium cladosporioides (7) C. cladosporioides (3) C. cladosporioides (2) C. cladosporioides C. cladosporioides (5) C. cladosporioides (3), Penicillium corylophilum (2) P. chrysogenum (2), P. brevicompactum (9) Fusarium solani (3) C. cladosporioides (2), P. roqueforti (6) P. solitum, P. brevicompactum (12), Aspergillus avus (3), A. ochraceus (12) A. avus (5), A. tamarii, A. niger (20), A. sydowii

( )numbers between brackets indicated number of isolates.

Table 3 Species of bacteria, yeast and lamentous fungi isolated from green coffee storage in jute bags (J) and polystyrene bags (P) during 140 days at 3 1C and 59% relative humidity Time (days) 40 P Bacteria Yeast Fungi

Bacillus subtilis (6), B. cereus* (3) B. megaterium (3)

Pichia anomala (2), Debaryomyces. hansenii (2)

40 J 90 P 90 J 140 P 140 J

B. subtilis (2), B. megateriun (2) B. cereus B. subtilis (3), B. megateriun (4) B. cereus B. subtilis B. subtilis (6), B. cereus (10), B. megaterium (6), B. macerans, Kurthia sp Kurthia sp (2), Tatumela ptyseos, Serratia rubidea, S. plymutica, Acinetobacter (2), Providencia mirabilis, B. subtilis (4), B. cereus (2), B. megaterium, B. macerans

Pichia anomala (4), Debaryomyces. hansenii (2)

Aspergillus avus (6), Penicillium citrinum (2), P. corylophilum, P. chrysogenum, P. roqueforti A. avus A. avus (2), P. brevicompactum, P. viridicatum, P. citrinum F. concolor, P. roqueforti, P. citrinum, P. solitum A. avus (21), A. niger (13), A. foetidius, A. dimorphicus (2), P. crustosum A. avus (11), A. niger (9), F. lateritium, P. citrinum (2), C. cladosporioides

P polystyrene. J jute bags. *B. cereus group (B. cereus, B. thuringiensis, B. anthracis (Castanha et al., 2007)). ( )numbers between brackets indicated number of isolates.

During the fermentation and drying period between the 14th and 18th days, ve different Gram-positive species, eleven yeast species and seventeen lamentous fungi species were identied (Table 2). Of the yeast isolates, 59% belonged to the Debaryomyces genus (Table 2). Penicillium brevicompactum and A. niger predominated in this nal period of the fermentation and drying process. The presence of three microbial groups in populations smaller than 102 CFU/ml was observed during green bean storage in a cold chamber. Spore-forming bacteria of the Bacillus genus, Pichia anomala and Debaryomyces hansenii isolates and species of Fusarium, Aspergillus and Penicillium were identied in coffee beans packed in jute sacks or polystyrene bags. Water reabsorption was observed during storage and consequently, increased microbial presence was detected in the green beans. Moisture in the beans stored in jute sacks sampled on the 140th day was 19%, which corresponds to 0.83 aw. The presence of several Gram-negative and Gram-positive bacteria and lamentous fungi species was detected in this period. The greatest variability in species occurred between the 6th and 18th days of

fermentation when the moisture conditions in the coffee cherries were between approximately 30% and 17%.

3.2. Bacterial identication Of all the bacterial isolates (375 isolates) identied, Grampositive bacteria belonging to the Bacillus genus were predominant (80.4%); this genus was found in the coffee beans throughout fermentation, drying and storage (Tables 2 and 3). Of the spore forming Gram-positive species, members of B. subtilis and B. cereus (B. cereus, B. thuringiensis, B. anthracis (Castanha et al., 2007) were dominant. The B. subtilis species was always present in greater numbers than the other species of this genus (134 identied isolates), and it was detected during the whole processing period. The other species identied from the Bacillus genus were B. cereus, B. megaterium, B. macerans and B. polymyxa. Non-sporulating Gram-positive bacteria were represented by ve isolates, of which three belonged to the Kurthia genus and two

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belonged to the Arthrobacter genus. Isolates from Kurthia spp. were only encountered on the 140th day of storage both in the jute and polystyrene packaging (Table 3). Gram-negative bacteria represented 19% of the total of bacterial isolates and were identied at fermentation days 0, 2 and 8. Gram-negative bacteria belonged predominantly to the Enterobacteriaceae family, and the most frequent genera were Enterobacter and Serratia. These bacteria presented greater species diversity as compared to the Gram-positive bacteria, but with lower incidence (o104 UFC/g). The following species were identied: Enterobacter agglomerans, E. cloacae, E. aerogenes, E. sakasaki, Acinetobacter sp, Serratia plymutica, Tatumella ptyseos, Pseudomonas paucimobilis, P. putrefaciens, P. alcalifaciens, Providencia mirabilis, Klebsiella oxytora, K. ozanae, Shigella dysenteriae and Yersinia sp. 3.3. Yeast identication The second microbial group in the succession isolated and identied from coffee cherries and beans was yeasts. These microorganisms presented a lower incidence with 202 isolates and 20 different species. D. hansenii represented 42.6% of the total yeast isolates and was widely distributed during coffee processing. During storage this species was isolated once at the 40th day in polystyrene packaging. The second most frequent genus was Pichia guilliermondii, which represented 20.6% of the total yeast and was isolated and identied at almost all sample times during fermentation and drying, but was not detected during storage. 3.4. Identication of lamentous fungi A total of 363 lamentous fungi isolates were identied, namely 132 Aspergillus, 101 Penicillium, 58 Cladosporium, 44 Fusarium, 15 Pestalotia and 13 Paecelomyces. Cladosporium cladosporioides was the fungi species most commonly found in coffee cherries (time 0) and was also isolated during the rst 18 days of coffee cherry fermentation. Fusarium species were isolated in coffee cherries on the bush and during fermentation, drying and storage in jute packaging (90th and 140th days) and in polystyrene packaging (140th day). Of the most frequent species of this genus, Fusarium lateritium and F. solani were found most in coffee cherries on the bush after C. cladosporioides. F. illudens was isolated in fruits on the bush (time 0) as well as on the 8th and 16th days of drying on the ground (Table 2).

Isolates of the Penicillium genus were identied during the whole process, except for at the 4th and 8th days. They were also found throughout storage in both types of packaging (Tables 2 and 3). P. brevicompactum was the most abundant species throughout the coffee fermentation and drying process (Tables 2 and 3). The second most abundant Penicillium species was P. roquefortii, representing 14.8% of the total isolates of this genus (Table 3). P. citrinum was isolated and identied in coffee cherries with 1814% moisture (14, 18 and 20 fermentation and drying days). During storage, P. roquefortii was found in beans in jute packaging (90th day) and in polystyrene packaging on the 40th, 90th and 140th storage days (Table 3). A total of 132 isolates of the Aspergillus genus were identied as belonging to the following species in decreasing order of number of isolates: A. avus, A. niger, A. ochraceus, A. tamarii, A. sydowii, A. foetidius and A. dimorphicus. These isolates were detected starting on the 8th fermentation day for coffee cherries on the ground, but were more abundant during storage, where they represented 59.6% of the total isolates (Tables 2 and 3). A. avus, the most abundant species, was more common during storage at 140 days in both types of packaging, although a greater number of isolates was observed in jute bags. A. niger represented 37.5% of the total of Aspergillus isolates and was only found on the last day of coffee processing (22nd day) with 30 isolates, and during storage in jute and polystyrene packaging (140 days) (Tables 2 and 3). A. ochraceus was found only on the 20th drying day, and was not detected at all during storage. Several lamentous fungi species were encountered during the fermentation and drying period of the coffee cherries on the ground.

3.5. Chemical analyses Organic acid analysis of the coffee cherries permitted correlation of the presence of microorganisms that produce these acids with the quality and fermentation of the natural coffee cherries. Of the 10 organic acids analyzed and quantied, four are microbial fermentation products (acetic, lactic, butyric and propionic acids). Butyric acid was not detected in any of the samples analyzed. The presence of this acid in coffee beans generally gives an unpleasant avor to the beverage and thus results in quality loss. Acetic acid was detected on the 2nd and 12th fermentation days, mainly in the pulp and mucilage fractions (Fig. 2). Acetic acid production is an aerobic metabolic process that can be of bacterial origin or the product of the oxidation of yeastproduced ethanol. The bacteria present on the surface of coffee

16 14 12 10 mg/g 8 6 4 2 0 0 2 4 6 8 12 Days of fermentation 14 16 mg/g

16 14 12 10 8 6 4 2 0 0 2 4 6 8 12 14 16 Days of fermentation 18 20 22

Fig. 2. Organic acids present in pulp+mucilage (A) and beans (B) of coffee fruits during fermentation and drying process. () fumaric acid, (+) oxalic acid, (m) citric acid, (B) acetic acid, (E) succinic acid, (&) propionic acid and (J) malic acid.

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cherries produce this acid, which can then migrate to the pulp and mucilage, where it can interfere with the organoleptic quality of the beans. Data on Fig. 2 shows that on the 8th fermentation day, the pulp and mucilage sample presented the greatest quantied value of lactic acid; this was the last day when malic acid was detected in that sample.

4. Discussion In this study, the sample was taken from the same batch of coffee cherries stripped manually from the bush and containing only red cherries; a microbial ecological succession was observed. This microbial succession was diversied and the number of individuals and species were inuenced principally by the moisture content of the coffee cherries and beans (Fig. 1). Differences in moisture content and chemical composition of the coffee also inuenced competition for the substrate and, therefore, affected colonization by these microorganisms. Thus, it is important to establish an epiphytic population of coffee cherries and beans because compounds secreted by microorganisms during their metabolism may migrate to the seed used to infuse the coffee beverage. Coffee bean moisture can be correlated with aw and is a factor that affects the degree of colonization and the colonizing species (Magan and Lacey, 1984). Differences in the microbial populations detected in studies on natural coffee can be explained by varying exposure times and surfaces (fermentation and drying ground) where coffee cherries are submitted to atmospheric conditions during the processing. The exposure time varies because climatic conditions during fermentation and drying can be different in each coffee-producing area. The Bacillus species predominated in our bacteria samples. This genus is characterized by its frequency in soils and sporulation that promotes cell survival in adverse conditions. Some Bacillus species can produce a series of extracellular enzymes that degrade complex compounds such as cellulose and pectin (Coughlan and Mayer, 1991). These two polymers are found in the skin, pulp and mucilage of coffee cherries, which therefore can be attacked by microbial enzymes. The cellulolytic ability of bacillus may contribute to the depolymerization of cellulose-containing complexes during fermentation of coffee cherries. Isolates of the B. cereus group (B. polymyxa and B. subtilis) were the most frequently encountered cellulolytic Bacillus species. Species such as E. aerogenes, E. cloacae and isolates of the Klebsiella genus found in this study on natural coffee were also identied in wet-processed Robusta coffee from the Congo (Van Pee and Castelein, 1972) and also in fresh C. arabica pulp in Colombia; Serratia and Pseudomonas were also found (Blandon et al., 1998). In this study, the capacity of the bacteria isolates to use polygalacturonic acid and pectin was tested (data not shown). None of the bacteria secreted polygalacturonase, but T. ptyseos, P. putrefaciens, E. aerogenes, Acinetobacter sp and P. mirabilis produced pectin lyase. As the pectin in coffee mucilage has a high methylation content (6070%), these bacteria might participate in the degradation of the mucilage of natural coffee cherries, thereby accelerating the fermentation process. There was a high diversity of species of lamentous fungi in fresh-harvested red cherries. In this study several lamentous fungi species were identied, but in reduced numbers, during the fermentation and drying period of coffee berries on the ground. However, during the storage period, fungal diversity decreased. Aspergillus and Penicillium dominated and were constantly present in stored coffee. The fungal incidence can be inuenced by different growth requirements and also by the anti-fungal activity of bacteria and yeasts. The ratio of fungi to yeasts in coffee cherries was always

inversely proportional, i.e., increases in the D. hansenii population promoted a decrease in the fungal population. D. hansenii has been reported as a possible biological control for prevention of degradation of stored fruit and grains (Payne and Bruce, 2001). The other yeasts identied have not presented anti-microbial potential to date in the literature, but may be important for natural coffee fermentation because they have pectin lyase activity. These species include Pichia burtonii, Debaryomyces polymorphus, Arxula adeninivorans, P. holstii and P. anomala. D. hansenii and P. guilliermondii were the two most frequently occurring species in the yeast group and were capable of growing on pectin in synthetic medium (data not shown). Therefore, these yeasts might aid pectin depolymerization in coffee mucilage and pulp. Pichia kluyveri and P. anomala isolated during coffee fermentation by the wet method showed high pectinolytic activity (Masoud and Jespersen, 2006). It can thus be concluded that both methods of coffee processing utilize pectinase activity to hydrolyze the pulp and mucilage. In addition to abiotic factors such as temperature and aw, microbial interactions can be governed by the microorganisms that comprise the communities during fruit ripening, fermentation and drying. At the start of natural coffee fermentation, simple sugars such as glucose, sucrose and fructose may be used, thus decreasing the availability of these sugars for other microorganisms. Complex polymers such as cellulose and pectin may become primary carbon sources, but their use depends on the depolymerization process. The isolation of microorganisms from the surface of coffee cherries and beans allowed the identication of microbial succession in these substrates during the fermentation, drying and storage period of natural coffee. This succession of bacteria, yeasts and lamentous fungi is most likely inuenced by coffee berry and bean moisture, temperature, competition for substrates, the enzymatic capacity of the colonizing species and antimicrobial activity. The constant demand of the consumer market for high-quality coffees has led to the need to understand the total microbiotic environment of dry or naturally processed coffee, and to determine the role of these microorganisms in the organoleptic characteristics of the nal beverage. Studies on natural coffee microbes always emphasize lamentous fungi isolation and identication, but the predominant microorganisms during the fermentation and drying period are bacteria and yeasts. Traditionally, naturally processed coffees originate from stripping the berries at various stages of ripeness. In this study, the coffee berries were harvested at a single stage of ripeness (red cherry) and placed on the fermentation and drying platform. This methodology was different from that used in other studies already published and allowed us to verify the existence of the succession of microbial groups. Microbial succession was established with the predominance of bacteria at the initial fermentation stages and with the presence of lamentous fungi and yeasts throughout the entire process on fermentation days when the aw was around 0.8. Gram-positive sporulating bacteria were isolated throughout the fermentation and storage period, perhaps because of spore formation under adverse conditions. Filamentous fungi were also detected throughout the process, possibly due to isolation of spores present on the surface of cherries because there was no visible mycelia colonization. Gram-negative bacteria predominated in the initial fermentation phases (up to the 12th day19% moisture) because they are less resistant to low moisture content than Gram-positive bacteria. D. hansenii and Pichia were the most frequent among the yeast isolates, but were still present in smaller populations than bacteria and fungi species. However, the yeasts identied in this study have been reported to inhibit of lamentous fungi mycelial growth and could

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thus be potentially used for biocontrol of lamentous fungi growth. This inhibition of fungal development may be of particular importance in coffee regions where the atmospheric conditions are adverse during natural coffee fermentation (high humidity, no sunshine and high rainfall).

Acknowledgements The authors thank Fundac ao de Amparo a Pesquisa - Estado de Minas Gerais (Fapemig) and Conselho Nacional Desenvolvimento Cientco e Tecnologico do Brasil (CNPq) nancial support. CFS and LMA thank CAPES (Coordenac ao - Aperfeic oamento de Pessoal de Nvel Superior) and CNPq scholarships. References
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