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Microbiological diversity associated with the spontaneous wet method of coffee fermentation
International Journal of Food Microbiology 210 (2015) 102–112
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Microbiological diversity associated with the spontaneous wet method of coffee fermentation Suzana Reis Evangelista a, Maria Gabriela da Cruz Pedroso Miguel a, Cristina Ferreira Silva a, Ana Carla Marques Pinheiro b, Rosane Freitas Schwan a,⁎ a b
Biology Department, Federal University of Lavras, CEP 37200-000, Lavras, MG, Brazil Food Sciences Department, Federal University of Lavras, CEP 37200-000, Lavras, MG, Brazil
a r t i c l e
i n f o
Article history: Received 17 March 2015 Received in revised form 5 June 2015 Accepted 7 June 2015 Available online 17 June 2015 Keywords: Arabica coffee Wet fermentation Microbiota PCR–DGGE Yeasts Bacteria
a b s t r a c t The evaluation of the microbiota present during coffee wet fermentation was done in two distinct regions of Minas Gerais, Brazil: one farm in the South of Minas Gerais (Lavras = L) and another farm in the savannah region (Monte Carmelo = MC). The yeast population ranged from 2.48 to 4.92 log CFU/g and from 2 to 4.81 log CFU/g, the mesophilic bacteria population ranged from 3.83 to 8.47 log CFU/g and from 5.37 to 7.36 log CFU/g, and the LAB population ranged from 2.57 to 5.66 log CFU/g and from 3.40 to 4.49 log CFU/g in the L and MC farms, respectively. Meyerozyma caribbica and Hanseniaspora uvarum were the dominant yeasts in coffee wet fermentation at L farm, and Torulaspora delbrueckii was the dominant yeast at MC farm. The species Staphylococcus warneri and Erwinia persicina were the predominant bacteria at L farm, and Enterobacter asburiae and Leuconostoc mesenteroides were the dominant species at MC farm. Lactic acid was the principal acid detected, reaching 2.33 g/kg at L farm and 1.40 g/kg at MC farm by the end of the process. The volatiles composition was similar for roasted coffee from the two different regions and furans, acids, and alcohol were the main groups detected. Temporal Dominance Sensation (TDS) analyses showed that the coffee beverage from L farm was dominated by citrus and herbaceous sensory characteristics, while the coffee from MC farm was dominated by citrus, herbaceous, and nuts sensory characteristics. Evaluating the microbiota in these two regions was important in improving the knowledge of the microbial species present during coffee wet fermentation in Brazil. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The coffee fruit usually consists of an outer layer of skin (peel), called the exocarp under which there is a layer of pulp, followed by mucilaginous mesocarp (mucilage) firmly adhered to the rigid layer called parchment (endocarp). The parchment protects two seeds surrounded by a thin membrane known as silver skin. The coffee fruit are subject to a fermentation process in order to remove the mucilage and pulp before drying. Three methods might be used to process coffee: the dry, semi-dry, and wet processes (Brando and Brando, 2014; Silva, 2014). In the dry method, the newly harvested whole fruit is fermented and dried on platforms after which the coffee beans are removed by hulling and polishing (removed the husk layer that cover the dry coffee beans). In the semi-dry method the coffee peel, pulp and part or all mucilage are removed mechanically and then the coffee is fermented and dried. The amount of mucilage removed depends on the characteristic of the machine used. In the wet method the peel and pulp are removed ⁎ Corresponding author. E-mail address: [email protected]fla.br (R.F. Schwan).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.06.008 0168-1605/© 2015 Elsevier B.V. All rights reserved.
mechanically, leaving the mucilage adhered to the beans. These pulped coffees are then transferred to water tanks where they are allowed to ferment for 6 to 72 h (depending on the environmental temperature), during which the remaining mucilage are degraded and solubilized. The beans are then removed from the tanks and sun dried. Brazil is the largest producer and exporter of coffee. The State of Minas Gerais, in the Southeast of Brazil, produces about 53% of all the coffee in Brazil, of which 85% is processed by dry method. In the last decade, coffee producers in the southern and central areas of Minas Gerais began to process coffee via wet fermentation (Sindcafé-MG, 2014). This processing produces a coffee with a different composition and sensory characteristics than coffee processed by the semi-dry or dry methods, which are alternatives used by the producer to suit different markets. Microorganisms are naturally present during coffee processing and use the various compounds in the pulp and mucilage as nutrients during the fermentation stages. They secrete organic acids and other metabolites that may affect the final sensory characteristics of the beverage (Silva, 2014). This microbiota may vary according to several factors: regional characteristics, coffee fruit composition and the method of fermentation. It is important to know the microbiota present during the processing of coffee, especially when selecting starter cultures that can
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be used in producing differentiated final products and inhibiting the growth of mycotoxigenic fungi (Masoud and Jespersen, 2006; Massawe and Lifa, 2010; Silva et al., 2012). Studies have been performed to identify the microbiota present in wet processing of coffee fruits in India, Hawaii, and Mexico (Agate and Bhat, 1966; Avallone et al., 2001; Frank et al., 1965). Classical morphological and biochemical characterization has been used to identify the microbiota, and species of Erwinia, Klebsiella, and Leuconostoc and the yeasts Kloeckera, Candida, Saccharomyces bayanus, Saccharomyces cerevisiae var. ellipsoideus, and Cryptococcus have been identified as contributing to these fermentations. Using a molecular approach, PCR–DGGE, Masoud et al. (2004) identified Pichia kluyveri, P. anomala, Hanseniaspora uvarum, Kluyveromyces marxianus, Candida pseudointermedia and Torulaspora delbrueckii, during wet coffee fermentation in Arusha, Tanzania (East Africa). Recently, yeasts species found in coffee processed by the wet method in Brazil were reported by Pereira et al. (2014). Pichia fermentans (YC5.2) and Saccharomyces sp. (YC9.15) were studied as having a potential for use as starter cultures for coffee wet fermentation. The bacteria population was not mentioned in their work (Pereira et al., 2014). Studies still are needed in Brazil to improve the knowledge of the microbiota present in coffee processing for each producing region due to the variation in climate and altitude. Our aim was to study the microbial diversity involved in wet coffee fermentation in two main producing regions of Brazil having distinct environmental characteristics using culture-dependent and culture-independent methods. The target metabolites present during the fermentation process were also analyzed using headspace solid-phase microextraction/gas chromatography (HS–SPME/GC) and high-performance liquid chromatography (HPLC). The sensory profile of the final coffee beverage was also evaluated. 2. Materials and methods 2.1. Processing of coffee fermentation The coffee fruits were fermented by the wet method. The experiments were performed in two geographically different regions of Minas Gerais, at a farm located in Monte Carmelo (MC), 870 m above sea level in the savannah (Cerrado) region, and at a farm located in Lavras (L), 919 m above sea level in the Atlantic Forest region. The experiments were done during two consecutive harvesting seasons (2012 and 2013). The fruit (60 kg) of Coffea arabica L. var. Acaiá was harvested at the mature stage (cherries) and was mechanically depulped in a horizontal machine (model BDSV-04; Pinhalense, São Paulo, Brazil), followed by for 48 h fermentation in a tank with 60 l of water to remove the mucilage. At the L farm, the temperature of fermentation was between 14 and 23 °C, and at the MC farm it was between 20 and 28 °C. Fermentations were done in duplicate at each farm. After fermentation, the coffees were placed on suspended platforms for sun drying until they reached approximately 11% moisture (224 h at L farm and 336 h at MC farm), measured using Moisture Meter G600i (GEHAKA, São Paulo, Brazil). The following samples were collected: cherries before fermentation, along the fermentation (0, 6, 12, 24, 36, and 48 h), and during drying (60, 112, 224, and 336 h). Samples (500 g) were placed aseptically in sterile plastic bags, in triplicate, and immediately transferred to the laboratory in iceboxes for microbiological analyses. For physicochemical analyses, coffee samples were frozen at −20 °C until analyzed. Sensory analysis and volatile compounds were evaluated in dried coffee beans. 2.2. Characterization and identification of microbiota 2.2.1. Quantification, isolation, and phenotypic characterization Coffee cherry sample (10 g) was added to 90 mL of sterile peptone water (in g/L: 1 bacteriological peptone [Himedia, Mumbai, India]), homogenized for 2 min in a Stomacher (Mayo Homogenius HG 400, São
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Paulo, Brazil), and used for decimal serial dilution. Bacteria and yeasts were enumerated by spread plating on MRS agar (Merck) for lactic acid bacteria, on plate count agar (PCA) (in g/L: 5 tryptone [Himedia], 2.5 yeast extract [Merck], 1 glucose [Merck], 15 agar Merck]), for total bacteria and on YPD agar (in g/L: 10 yeast extract [Merck], 10 peptone [Himedia], 20 dextrose [Merck], 20 agar [Merck]) for yeasts. MRS plates were incubated in an anaerobic jars at 30 °C for 3–4 days and PCA and YPD plates were incubated at 30 °C for 3–7 days and the morphological properties of the colonies (cell size, cell shape, edge, color, and brightness) were recorded and the square root of the number of colonies counted for each morphotype was purified by streaking on new agar plates (same culture media used for plating) (Senguna et al., 2009). The pure cultures were stored in an ultra freezer at −80 °C in the same broth culture media used for plating, containing 20% glycerol (w/w). The phenotypic characterization of the bacterial colonies was performed using Gram staining, catalase and oxidase activities, motility tests, growth in culture media with 1% and 5% (w/v) NaCl (salt tolerance), protease production, spore formation, and the ability to ferment glucose, sucrose, and xylose (Sigma, St. Louis, USA), as recommended in Bergey's Manual of Determinative Bacteriology (Holt et al., 1994). Yeast colonies were characterized for morphology and biochemical assessments as described by Kurtzman et al. (2011). A total of 435 isolates was obtained; 251 were from samples from L farm and 184 isolates were obtained from MC farm. The microbiota identified was the same in both years analysed. However, variation of less than 5% was found in the counting. 2.2.2. Genotypic identification The isolates (435) were grouped by phenotypic characteristics, as mentioned above, and representatives of each phenotypic group were subjected to molecular analyses (236 isolates). The genotypic characterization of the selected isolates was first performed by rep-PCR and subsequently by DNA sequencing. The bacterial and yeast cultures were grown under appropriate conditions, collected from agar plates with a sterile pipette tip, and resuspended in 50 μL of sterile Milli-Q water. The suspension was heated for 10 min at 95 °C, and three μL was used as a DNA template in the PCR experiments. The fingerprints of the genomic DNA were obtained via the PCR amplification of repetitive bacterial and yeast DNA elements (rep-PCR) using the (GTG)5 primer, as described by Nielsen et al. (2007). Amplified PCR products were separated via 2% (w/v) agarose gel electrophoresis at 70 V for 3 h, and the images were visualized and photographed using a transilluminator LPixImage (LTB 20 × 20 HE, LPix®, Brazil). The bacteria and yeasts representative of each group were subjected to 16S rRNA gene and internal transcribed spacer (ITS) region gene sequencing, respectively. The amplification of the 16S rRNA gene used the primers 27 F and 1512R (Devereux and Willis, 1995). The ITS region was amplified using the primers ITS1 and ITS4 (Nielsen et al., 2007). The amplified PCR products were sent for sequencing at the Advanced Genetics Technologies Center — AGTC (Kentucky, USA); the ABI3730 XL automatic DNA sequencer was utilized. The sequences were aligned using the BioEdit 7.7 sequence alignment editor and were compared to the GenBank database using the Basic Local Alignment Tool (BLAST) program (National Center for Biotechnology Information, Bethesda, MD) for the identification of isolates. 2.3. PCR–DGGE analysis Samples of coffee cherries (3 g) were mixed with 5 mL of Milli-Q water for 10 min then the coffee cherries were removed and the liquid phase was centrifuged at 100 ×g for 10 min at 4 °C. The pellet was used for DNA extraction. The total DNA was extracted from samples using the “DNA Purification from Tissues” protocol (QIAamp DNA Mini Kit [Qiagen, Hilden, Germany]) in accordance with the manufacturer's instructions. The DNA from the bacterial community was amplified with
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the primers 338fgc and 518r (Ovreas et al., 1997). A fragment of the D1region of the 26S rRNA gene was amplified with the eukaryotic universal primers NL1GC and LS2 (Cocolin et al., 2000). Reactions were performed according to Ramos et al. (2010). Aliquots (3 μL) of the amplification products were analyzed via electrophoresis on 1% agarose gels before they were used for DGGE. The PCR products were separated in polyacrylamide gels (8% (w/v) acrylamide:bisacrylamide [37.5:1]) in 1 × TAE buffer with a DCode system apparatus (BioRad Universal Dcode Mutation Detection System, Richmond, CA, USA). Denaturation gradients were used that varied from 15 to 55% for the bacterial products (100% corresponded to 7 M of urea and 40% [v/v] formamide) and from 20 to 60% for the yeast products. Electrophoresis was conducted at a constant voltage of 130 V for 6 h and at a constant temperature of 60 °C. Following electrophoresis, the gels were stained with SYBR-Green I (Molecular Probes, Eugene, UK) (1:10.000 v/v) for 30 min. The images were visualized and photographed using a transilluminator LPixImage (LTB 20 × 20 HE, LPix®, Brazil). Selected bands were excised and their DNA amplified using the primers 338fgc and 518r for bacteria and NL1 and LS2 for yeast. The PCR products were purified and sequenced at the Advanced Genetics Technologies Center — AGTC (Kentucky, USA); the ABI3730 XL automatic DNA sequencer was utilized. The sequences were compared to
the GenBank database using the BLAST algorithm (National Center for Biotechnology Information, Maryland, USA).
2.4. Analyses of organic acids The organic acids from the beans were analyzed using a highperformance liquid chromatography system (Shimadzu Corp., Japan) equipped with detection system consisting of a UV–Vis detector (SPD 10Ai). The extraction and operating conditions were performed according to the methodology described by Evangelista et al. (2014).
2.5. Analysis of volatile compounds Volatile compounds were extracted from the beans and analyzed according to Evangelista et al. (2014). The volatile compounds from each headspace analysis were defined by integrating the peak areas of all the identified compounds. Relative percent areas were used for semiquantitation of the target compounds. The relative percentage of the individual compounds was calculated from the total peak area of the volatile compounds on the chromatograms (Petisca et al., 2013).
Fig. 1. The populations of yeast (YEPG counts ), mesophilic bacteria (PCA counts ), and lactic acid bacteria (MRS counts ) in coffee wet processing at Lavras farm (A). The ), mesophilic bacteria (PCA counts ), and lactic acid bacteria (MRS counts ) in coffee wet processing at Monte Carmelo farm (B). populations of yeast (YEPG counts
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2.6. Analysis of sensory characteristics The samples of roasted coffee from each region were prepared according to the Specialty Coffee Association of America (SCAA, 2013). The coffee was roasted in a laboratory roaster (Probatino, Leogap model, Brazil) with a capacity of 150 g. The temperature used ranges from 180 to 200 °C and the time was around 10 min until the beans reach lightmedium roast. The coffee beans were ground in an electric mill (Pinhalense ML-1, Brazil). For sample uniformity evaluation, five cups of each sample were prepared. Clean and odor-free water was used. Water was brought to approximately 92–94 °C and poured directly on the ground coffee. The ratio was 8.25 g of coffee per 150 mL of water. Grounds were steeped for 3–5 min before the sample was evaluated. A panel of three trained coffee experts with Q-Grader Coffee Certificates evaluated the samples. The first sensorial evaluation was conducted to identify the most relevant of the coffee attributes, and the eight most cited terms were selected and retained for further Temporal Dominance of Sensations (TDS) evaluations (Pineau et al., 2009). The attributes selected by the panel were as follows: chocolate, bitter chocolate, caramel, citric, tobacco, butter, herbaceous, and nuts. Samples coded with three digits were submitted in a balanced order (Wakeling and MacFie, 1995) and evaluated in three replicates. The analysis was performed in 5 sequential sips, where each one last 15 s. For each sip, the panelist imbibed the coffee,
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moved it around in their mouths for 3 s, and swallowed it. The evaluation continued until no sensation was perceived or 15 s had passed. Data collection began upon tasting and consisted of selecting the most dominant attribute (among the eight attributes) perceived at that time using SENSOMAKER Software (Nunes and Pinheiro, 2012) and plotting as TDS curves showing the percentage of subjects which selected the attribute as dominant at a specific time — that is, the dominance rate (Pineau et al., 2009).
3. Result 3.1. Quantification and characterization of microbial population The mesophilic aerobic bacteria, LAB, and yeast populations found during coffee processing at the two farms, Lavras (L) and Monte Carmelo (MC), are shown in Fig. 1. The yeast population in coffee from L farm ranged from 2.5 to 4.9 log CFU/g and that for MC farm ranged from 2 to 4.8 log CFU/g. The population of mesophilic bacteria varied from 3.8 to 8.5 log CFU/g at L farm and from 5.4 to 7.4 log CFU/g at MC farm (Fig. 1). The LAB population varied from 2.6 to 5.7 log CFU/g at L farm and from 3.4 to 4.5 log CFU/g at MC farm.
Table 1 Yeasts and bacteria associated with fermentation and drying of coffee beans at Lavras farm (L) and Monte Carmelo farm (MC). Species identification
Population (log CFU/g) of the isolates from fruit coffee before and during processing Fermentation (hours)
Yeast L farm H. uvarum M. caribbica P. fermentans D. hansenii T. delbrueckii C. railenensis C. quercitrusa W. ciferrii Yeast MC Farm H. uvarum M. caribbica P. fermentans T. delbrueckii C. glabrata W. anomalus Bacteria L farm Enterococcus sp. O. pseudogrignonense C. taichungense C. bovis E. persicina S. warneri Pseudomonas sp. P. amylolyticus Curtobacterium sp. K. oxytoca B. amyloliquefaciens E. hermannii Bacteria MC farm E. asburiae E. ludwigii C. pallidum P. agglomerans P. dispersa K. oxytoca S. marcescens Microbacterium sp. M. laevaniformans W. cibaria Kocuria sp. L. mesenteroides
Drying (hours)
Coffee fruit
0
6
12
24
36
48
60
112
224
336
2.9 4.4 3.4 b2 b2 3.1 b2 b2
3.7 4.2 2.3 b2 b2 b2 2.3 b2
3.4 3.6 2.0 2.6 b2 b2 b2 b2
4.1 3.8 2.0 b2 b2 b2 b2 b2
3.6 3.3 b2 b2 b2 b2 b2 b2
3.8 4.9 b2 b2 b2 b2 b2 b2
b2 2.9 b2 b2 b2 b2 b2 b2
b2 2.5 b2 b2 b2 b2 b2 b2
2.8 3.5 b2 b2 b2 b2 b2 b2
3.0 3.9 3.0 b2 2.3 b2 b2 b2
3.6 4.0 b2 b2 b2 b2 b2 3.6
3.0 3.8 2.3 5.3 2.8 b2
2.5 b2 b2 b2 2.3 3.4
b2 b2 2.0 b2 b2 b2
b2 b2 2.5 b2 b2 b2
b2 4.0 b2 4.8 b2 b2
b2 b2 b2 4.5 b2 b2
b2 b2 b2 3.8 b2 b2
b2 b2 b2 4.3 b2 b2
b2 b2 b2 2.7 b2 b2
b2 b2 b2 2.9 b2 b2
⁎– – – – – –
b2 b2 4.9 b2 4.9 5.2 4.3 b2 4.0 4.3 4.8 4.0
b2 4.4 5.0 5.0 4.5 5.6 b2 b2 b2 b2 b2 b2
b2 b2 b2 4.0 4.0 6.1 b2 b2 b2 b2 b2 b2
b2 b2 b2 b2 5.5 6.7 b2 b2 6.2 b2 b2 b2
b2 b2 b2 b2 b2 7.1 b2 b2 b2 5.6 b2 b2
b2 b2 b2 b2 b2 8.5 b2 b2 b2 b2 b2 b2
b2 b2 b2 b2 6.1 7.2 b2 b2 b2 b2 b2 b2
b2 b2 b2 b2 6.1 6.6 b2 b2 b2 b2 b2 b2
b2 b2 b2 b2 b2 6.1 b2 b2 b2 b2 b2 b2
4.6 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2
b2 3.6 2.8 b2 b2 b2 b2 2.6 2.9 b2 b2 b2
4.7 5.7 4.6 b2 5.0 b2 4.7 b2 b2 b2 4.8 b2
5.3 4.3 b2 4.0 4.7 5.6 b2 4.8 4.8 b2 b2 b2
6.0 b2 b2 b2 b2 b2 b2 5.0 b2 6.1 b2 6.0
6.2 b2 b2 b2 b2 b2 b2 b2 b2 6.5 b2 b2
7.3 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2
7.4 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2
6.8 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2
5.9 b2 b2 b2 4.6 b2 b2 b2 b2 b2 b2 5.9
6.2 4.5 b2 b2 b2 b2 b2 b2 b2 b2 b2 4.2
b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 5.4
– – – – – – – – – – – –
⁎ Processing ends with 224 h in Monte Carmelo farm.
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A total of 251 microbial isolates were obtained from the samples of L farm: 59.4% yeasts and 40.6% bacteria (50.9% Gram-positive, and 49% Gram-negative). A total of 184 isolates were obtained from MC farm: 33.7% yeasts and 66.3% bacteria (33.6% Gram-positive, and 66.3% Gram-negative). The isolates were grouped by phenotypic characteristics (cell morphology and biochemical features) (data not shown), and 236 selected isolates were subjected to rep-PCR for genotypic characterization.
Representative isolates from each rep-PCR-profile were identified by sequencing. The identified species and the population during processing are shown in Table 1. The yeast species present in the coffee beans of L farm were Hanseniaspora uvarum (accession no KM402039–KM402045), Meyerozyma caribbica (accession no KM402046–KM402058), P. fermentans (accession no KM402059–KM402064), Debaryomyces hansenii (accession no KM402067), T. delbrueckii (accession
Fig. 2. PCR–DGGE patterns of the yeast and bacteria communities present during coffee wet processing at Lavras farm. Prokaryote (A) and Eukaryote (B). S = coffee cherries, 0 = 0 h, 1 = 4 h, 2 = 15 h, 3 = 22 h, 4 = 27 h, 5 = 40 h, 6 = 50 h, 7 = 140 h, 8 = 236 h, 9 = 360 h, H2O = water fermentation tank.
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no KM402068–KM402082), Candida railenensis (accession no KM402083), Candida quercitrusa (accession no KM402088), and Wickerhamomyces ciferrii (accession no KM402084) (Table 1). M. caribbica was the most prevalent yeast in the coffee fruit, during the fermentation and drying reached a maximum population of 4.9 log CFU/g at 36 h of fermentation. It was followed by
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H. uvarum with a population of 4.1 log CFU/g at 36 h of fermentation. The yeast species present in the coffee beans of MC farm were H. uvarum (accession no KM402039–KM402045), M. caribbica (accession no KM402046–KM402058), P. fermentans (accession no KM402059– KM402064), T. delbrueckii (accession no KM402068–KM402082),
Fig. 3. PCR–DGGE patterns of the yeast and bacteria communities present during coffee wet processing at Monte Carmelo farm. Prokaryote (A) and Eukaryote (B). S = coffee cherries, 0 = 0 h, 1 = 9 h, 2 = 20 h, 3 = 28 h, 6 = 54 h, 7 = 174 h, 8 = 202 h, H2O = water fermentation tank.
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Table 2 Species identified by PCR–DGGE using universal primers for yeast and bacteria. Bands
Acess number
Similarity (%)
Prokaryote
1 2,3, 27, 28, 4, 5 6 7, 8, 11, 12, 15, 16 9, 10 13,14 29, 30, 31, 32, 40, 41, 47, 48 33 34, 35 36, 37 38, 39 42, 43, 44 45, 46 49
AF515228 AB854267 HM756486 KF673524 KF031439 HQ683968 JX120129 FJ406528 JX315564 HG798481 KF453765 KF891342 KC430956 CP002272 JX242698
95 98 97 97 100 97 98 95 94 100 97 97 95 97 94
Leuconostoc pseudomesenteroides Weissella confusa Lysinibacillus fusiformis Lactobacillus fermentum Uncultured bacterium Klebsiella oxytoca Actinobacterium sp. Uncultured bacterium Acinetobacter schindleri Lactococcus lactis Enterobacteriaceae bacterium Enterobacter cloacae Enterobacter sp. Enterobacter lignolyticus Uncultured actinomycete
17, 18, 19, 20, 50, 51, 52 21, 22, 58, 59 23, 24, 60, 61, 62 25, 26, 63, 64 53 54, 55 56, 57
EU568995 JQ417238 KC510080 AY314792 EU650386 AY520372 HE799671
95 98 100 100 98 97 97
Eukaryote Meyerozyma caribbica Mitchella repens Pichia fermentuns Uncultured fungus Coffea arabica Candida sp. Torulaspora delbrueckii
C. glabrata (accession no KM402089–KM402091), and W. anomalus (accession no KM402085–KM402087) (Table 1). T. delbrueckii was the predominant yeast. It was detected in the coffee fruit with a high population of 5.3 log CFU/g and during the fermentation the maximum population was of 4.9 log CFU/g. M. caribbica was the second most significant yeast found in the coffee fruit (3.8 log CFU/g) and during fermentation with a population of 4 log CFU/g. The diversity of bacterial species was greater than that of the yeast: 23 bacteria and 10 yeast species were identified (Table 1). Klebsiella oxytoca (accession no KM402125–KM402126) was the only bacterial species found at both farms. Enterococcus sp. (accession no KM402100), Ochrobactrum pseudogrignonense (accession no KM402101–KM402102), Chryseobacterium taichungense (accession no KM402103–KM402104), C. bovis (accession no KM402105–KM402106), Erwinia persicina (accession no KM402108–KM402111), Staphylococcus warneri (accession no
KM402118–KM402119), Pseudomonas sp. (accession no KM402120), Paenibacillus amylolyticus (accession no KM402121), Curtobacterium sp. (accession no KM402123–KM402124), Bacillus amyloliquefaciens (accession no KM402133) and Escherichia hermannii (accession no KM402097), were found in samples from L farm (Table 1). S. warneri was the most prevalent bacteria, and was identified in the coffee fruit and during fermentation, reaching maximum population of 8.5 log CFU/g at 36 h of fermentation. E. persicina was the second most significant specie reaching the maximum value of 5.5 CFU/g at 12 h of fermentation. Enterobacter asburiae (accession no KM402092–KM402096), E. ludwigii (accession no KM402098–KM402099), C. pallidum (accession no KM402107), Pantoea agglomerans (accession noKM402112), P. dispersa (accession no KM402113–KM402117), Serratia marcescens (accession no KM402127), Microbacterium sp. (accession no
Table 3 Organic acids present in coffee during wet processing at Lavras (A) and Monte Carmelo. Time (hours)
L farm Fermentation tank 0 24 48 Drying 60 224 336 MC farm Fermentation tanks 0 24 48 Drying 60 112 224
Compounds (g/Kg) Citric
Malic
Succinic
Lactic
Acetic
0.08 ± 0.02 0.02 ± 0.01 0.07 ± 0.02
0.73 ± 0.35 0.16 ± 0.01 0.09 ± 0.01
0.48 ± 0.25 0.05 ± 0.01 nd
nd 0.62 ± 0.02 0.88 ± 0.07
Nd Nd 0.01 ± 0.00
0.10 ± 0.02 0.05 ± 0.00 0.20 ± 0.01
0.09 ± 0.03 0.08 ± 0.00 0.20 ± 0.01
0.02 ± 0.00 0.02 ± 0.00 0.08 ± 0.00
1.07 ± 0.02 1.09 ± 0.09 2.34 ± 0.45
Nd Nd Nd
0.18 ± 0.03 0.20 ± 0.01 0.08 ± 0.00
0.14 ± 0.00 0.06 ± 0.00 0.02 ± 0.00
0.40 ± 0.03 0.14 ± 0.02 0.05 ± 0.00
nd 0.34 ± 0.10 0.57 ± 0.05
Nd Nd 0.02 ± 0.00
0.03 ± 0.00 0.02 ± 0.00 0.04 ± 0.01
0.01 ± 0.00 nd nd
0.04 ± 0.00 0.12 ± 0.01 0.09 ± 0.00
1.03 ± 0.19 1.40 ± 0.12 1.08 ± 0.09
Nd Nd Nd
nd = not detected; ± = Standard deviation.
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KM402128–KM402129), M. laevaniformans (accession no KM402130), Weissella cibaria (accession no KM402131), Kocuria sp. (accession no KM402132), and Leuconostoc mesenteroides (accession noKM402134) were the bacteria isolated from MC farm (Table 1). E. asburiae was the most prevalent bacteria found in coffee fruit and during fermentation reaching maximum value of 7.4 log CFU/g at 36 h of fermentation. L. mesenteroides was the second most significant bacteria identified reaching the maximum value of 6 CFU/g at 12 h of fermentation. The microbiota identified was the same in both years analyzed. However, variation of less than 5% was found in the counting.
curves are presented in Fig. 4. In the coffee from L farm, sip 3 had dominant herbaceous and citric sensation characteristics, and sips 2 and 5 had a dominant citric sensation characteristic; in sips 1 and 4, no sensation characteristic was dominant (Fig. 4A). In the coffee from MC farm, sips 1 and 3 had a dominant citric sensation characteristic, sips 2 and 4 had a dominant herbaceous sensation characteristic, and sip 5 had a dominant nuts sensation characteristic (Fig. 4B). The sensations chocolate, bitter chocolate, caramel, tobacco, and butter did not trigger the most attention from the panelists. These results are represented in the graph below the line of significance (Fig. 4B).
3.2. Culture-independent microbiological analysis using PCR–DGGE
4. Discussion
Figs. 2 and 3 show the PCR–DGGE fingerprints of the bacteria and yeast communities. In general, species of bacteria present in the coffee before fermentation remained throughout the process and were also present in the water tank at the end of fermentation. The sequencing of the bands from L farm indicated the presence of Leuconostoc pseudomesenteroides (band 1), Weissella confusa (bands 2 and 3), Lysinibacillus fusiformis (bands 4 and 5), uncultured bacteria (bands 7, 8, 11, 12, 15, and 16), Lactobacillus fermentum (band 6), K. oxytoca (bands 9 and 10), and Actinobacterium (bands 13 and 14) (Fig. 2A and Table 2). MC farm samples presented W. confusa (bands 27 and 28), uncultured bacteria (bands 29, 30, 31, 32, 40, 41, 47, and 48), Acinetobacter schindleri (band 33), Lactococcus lactis (bands 34 and 35), Enterobacteriaceae bacterium (band 36 and 37), Enterobacter cloacae (bands 38 and 39), Enterobacter sp. (bands 42, 43, and 44), E. lignolyticus (bands 45 and 46), and uncultured actinomycete (band 49) (Fig. 3A and Table 2). The yeast species found in the coffee cherries before being placed into the tank remained throughout the fermentation process and some new species appear (Figs. 2B and 3B and Table 2). Both MC and L farms showed the presence of M. caribbica (bands 17, 18, 19, 20, 50, 51, and 52), Candida sp. (bands 54 and 55), T. delbrueckii (bands 56 and 57), P. fermentans (bands 23, 24, 60, 61, and 62), and uncultured fungi (bands 25, 26, 63, and 64). The plant species C. arabica (band 53) and Mitchella repens (bands 21, 22, 58 and 59) were also identified because the primers NL1 and LS2 are universal. Therefore, they can amplify the DNA of various eukaryotic organisms, including plants.
The mesophilic aerobic bacteria were dominant throughout the process and showed a high level of diversity (Figs. 1–3). A difference in the diversity of species of the prokaryotic and eukaryotic groups was observed among the regions studied, the main species of yeast and
3.3. Chemical analyses The main acid present in the coffee during fermentation and drying was lactic acid (Table 3), which showed increasing concentrations and reached a maximum value of 2.33 g/kg for L farm, while for MC farm, the maximum concentration was 1.07 g/kg. Acetic acid was detected in the coffee (0.02 g/kg) only at the end of fermentation in both experiments. Citric, malic and succinic acids were detected during fermentation tank. Propionic and butyric acids, which may impair the sensory characteristics of coffee were not detected. A total of 30 volatile compounds were detected via HS–SPME/GC (Table 4). Among these compounds, 18 were detected in green coffee and 25 were detected in roasted coffee. The composition of green coffee beans from the two farms was different: the green coffee from L farm showed alcohol (methanol), esters (ethyl butyrate and furfuryl acetate), and acids (hexanoic and nonanoic acid) as the main compounds, while the green coffee from MC farm showed alcohol (methanol), acids (isobutyric and hexanoic acid), and furan (furfuryl alcohol and furfural). The composition of the majority of volatile compounds in roasted coffee was similar. The main compounds were furans (furfuryl alcohol and furfural), acids (isobutyric acid), and alcohol (1-pentanol and 2-heptanol). 3.4. Sensorial analyses The most relevant attributes of the coffee were selected in the previous analysis and subsequently evaluated using TDS analysis. The TDS
Table 4 Relative percentage of volatile compounds identified in green and roasted coffee. Compounds
Green (%)
Roasted (%)
Coffee L farm
Coffee MC farm
Coffee L farm
Coffee MC farm
Ketones 2.3-butanedione 2-nonanone Total ketones
nd nd nd
nd nd nd
0.07 0.80 0.87
nd 1.28 1.28
Alcohols Methanol 1-propanol 1-pentanol 2-heptanol 3-methyl-1-pentanol 1-hexanol a-Terpineol 2-phenylethanol Total alcohols
16.10 nd nd nd 0.95 nd 1.03 1.41 19.49
12.24 1.17 nd nd nd 1.79 nd 0.65 15.85
0.10 nd 1.35 1.83 0.55 nd nd 0.08 4.91
0.09 nd 1.24 2.11 0.67 nd nd 0.06 4.18
Aldehydes Acetaldehyde Hexanal Octanal Nonanal Butyraldehyde Decyl aldehyde Total aldehydes
0.17 nd nd nd 0.91 nd 1.07
0.28 nd nd nd nd nd 0.28
0.10 0.01 0.44 0.55 0.66 0.34 2.10
0.14 nd 0.29 1.06 0.47 0.32 2.29
Acids Isobutyric acid Hexanoic acid Nonanoic acid Propanoic acid Total acids
nd 1.44 0.80 nd 2.23
3.11 2.36 0.95 nd 6.42
8.15 0.11 0.02 0.54 8.81
7.45 0.09 0.02 0.35 7.1
Esters Propyl acetate Ethyl butyrate Diethyl malonate Phenyl acetate Furfuryl acetate Diethyl succinate Total esters
0.52 3.27 nd nd 4.13 nd 7.92
nd 0.80 nd nd nd 1.14 1.95
0.61 nd 0.28 0.32 2.38 nd 3.59
0.76 nd 0.21 0.29 2.72 nd 3.98
Phenol Guaiacol
nd
nd
0.58
0.64
Burnt
Furans Furfuryl alcohol Furfural 5-methylfurfural Total furans Total GC area
nd nd 0.94 0.94 7163
2.72 2.70 0.58 5.99 9870
21.93 13.26 0.42 35.61 288,508
19.03 13.90 0.77 33.70 215,900
Burnt Almond/bitter Caramel
nd = not detected. a Czerny and Grosch (2000), Gonzalez-Rios et al. (2007).
Reference target compoundsa
Buttery
Green
Floral
Acrid/egg Green
Nutty
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bacteria were not the same between them (Table 1; Figs. 1 and 2). This difference could have been due to environmental temperature, humidity, composition of the remaining pulp and mucilage surrounding the coffee beans, and the altitude (Leong et al., 2014; Silva et al., 2008). The majority of the microbiota present throughout the wet processing was also detected in coffee fruit. The wet processing favored bacteria development that showed a high increase of the population in both regions. Studies of the microbiota from coffee undergoing different types
of processing also observed that some species already are naturally present in the fruit (Silva et al., 2000, 2008). Therefore, harvesting methods and fermentation process will directly interfere with microbiota (Avallone et al., 2001; Silva et al., 2000; Vilela et al., 2010). H. uvarum and T. delbrueckii were detected in higher numbers in coffee from L farm and MC farm, respectively, and were also reported in others studies that examined the wet and semi-dry processes (Masoud et al., 2004; Vilela et al., 2010). The presence of some yeasts,
Fig. 4. TDS curves of coffee from L farm (A) and coffee from MC farm (B). Chocolate ( ), Bitter chocolate ( ), Caramel ( ), Citric ( ), Tobacco ( ), Butter ( ), ), Nuts ( ). Chance level ( ) represents the dominance rate that an attribute can obtain by chance (one/number of attributes) Significance level ( ) expresses Herbaceous ( the smallest value of the proportion that is significantly (p = 0.05) higher than the chance level. Each slip duration of 15 s.
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such as Saccharomyces, Pichia, Candida and Meyerozyma provides benefits for coffee processing due to their important role in the degradation of mucilage rich in pectin and because they may inhibit the growth of mycotoxigenic fungi (Masoud and Jespersen, 2006; Massawe and Lifa, 2010; Silva et al., 2012; Evangelista et al., 2014). Therefore, these yeasts are interesting strains that could be used as starter cultures (Evangelista et al., 2014). P. fermentans was detected in both coffee regions. This specie produces volatile compounds that might interfere with the quality of coffee beverages (Pereira et al., 2014). Some bacteria identified in this work belong to genera commonly found during coffee processing, such as Erwinia, Klebsiella, Leuconostoc, Weissella, Enterococcus, Enterobacter, Serratia, and Bacillus. These genera have been identified during the natural, semi-dry, and wet fermentation of coffee (Avallone et al., 2001; Silva et al., 2000, 2008; Vilela et al., 2010). L. mesenteroides detected in coffee from MC farm is an important lactic acid bacteria present during the wet processing and species of Erwinia and Klebsiella are reported as important producers of pectinases (Avallone et al., 2001). Some species of yeast and bacteria found using the culturedependent method were not detected via PCR–DGGE (Figs. 2 and 3 and Table 2). Masoud et al. (2004) also reported that some microbial species were only detected using the culture-dependent method in the samples taken during wet processing. This fact could be explained by the difficulty of obtaining high-quality DNA suitable for PCR directly from the samples. The initial template DNA and template competition may affect the detection of rare microorganisms in the microbial population (Muyzer et al., 1993). All yeasts species detected in DGGE gels were also isolated from plating. Some bacterial species were detected only through PCR–DGGE. L. pseudomesenteroides, W. confuse, L. fusiformis, L. fermentum and Actinobacterium sp. were the species detected in L farm. A. schindleri, W. confusa, L. lactis, E. bacterium, E. cloacae and E. lignolyticus were the species detected in MC farm. This may have occurred because their population densities were lower than 2 log CFU/g, making them impossible to detect via the culture-dependent method (Figs. 2 and 3 and Table 2). Vilela et al. (2010) also reported that some microbial species were only detected using the DGGE method in the samples taken during semi-dry processing. The species L. pseudomesenteroides, W. confusa, L. lactis and Enterobacter sp. have been found in others studies (Leong et al., 2014; Vilela et al., 2010). The species L. pseudomesenteroides and W. confusa contribute in the fermentation process and might inhibit filamentous fungi growth (Leong et al., 2014). Therefore, molecular methods should always be used in conjunction with traditional methods to evaluate biodiversity. Acetic and lactic acids were produced throughout the fermentation. Lactic acid was the main acid found (Table 3). The presence of lactic acid bacteria, such as bacteria of the genera Weissella, Leuconostoc, and Lactobacillus identified throughout the fermentation process, may have contributed to this fact. The other acids detected were malic, citric, and succinic acids (Table 3). These acids are already naturally present in the coffee bean, and their concentrations decreased during fermentation, but succinic acid showed a slight increase at the end of the process. Succinic acid may be produced by Bacillus spp. (Silva et al., 2012) and by heterofermentative lactic acid bacteria (LAB) (Swiegers et al., 2005). The variation of acids during fermentation due to microbial metabolism changed the pH value and influences the microbiota present in the tank (Table 3). The diffusion of theses acids into the bean could influence the final beverage flavor and quality (Evangelista et al., 2014; Silva et al., 2012; Silva, 2014). The volatile compounds identified in the green coffee beans differed between the regions studied (Table 4) that may have influenced this difference is the microbiota involved during the processing of the coffee beans (Evangelista et al., 2014; Silva et al., 2012). Some compounds are known to play a role in aroma development during fermentation, such as the esters that were detected in the green coffee beans in both regions.
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However, after roasting, less difference in the volatile composition was observed (Table 4). The mechanisms of coffee aroma formation are extremely complex, and there is clearly a wide range of interactions between all the pathways involved. Maillard reactions occur between reducing carbohydrates and proteins and are responsible for the formation of many volatile compounds during roasting (López-Galilea et al., 2006). Each coffee sample was evaluated on the following attributes: aroma, flavor, aftertaste, acidity, body, balance, uniformity, clean cup, sweetness, and overall and was given a score for each of these attributes. The coffee of L farm received a score of 81 and MC farm of 80 (data not shown), being considered as very good special coffee according to SCAA (2013). Although both coffees were well accepted by the panelists, some distinguished flavors were found in the TDS analysis (Fig. 4). The coffee from L farm presented a nice citrus and herbaceous flavor, and the coffee from MC farm presented, addition to these flavors, nuts sensation characteristics. All these sensations are desirable in the final coffee beverage. The presence of volatile compounds differed between the regions and affected the beverages' final flavors. The presence of different microbial species in each region may have contributed to this difference. The microbial activity during fermentation produces volatile compounds that might influence the final taste of the beverage (Evangelista et al., 2014). Some volatile compounds detected in this study are described in the literature by providing aromatic notes such as caramel, nutty, burnt, buttery, floral, and almond (Table 4) (Czerny and Grosch, 2000; Gonzalez-Rios et al., 2007). The presence of furans and ketones were detected in both samples, which contributed to the citric and herbaceous flavors that were dominant in the sensory analysis (Fig. 4). The presence of furans can provide herbal or fruity notes, and ketones are described as providing buttery, caramel-like, musty, mushroom like, or fruity notes (López-Galilea et al., 2006). The evaluation of these two coffee-producing regions contributed to a better understanding of the microbiota present during coffee wet processing and some characteristics of this processing. In conclusion, this study evaluated coffee wet processing, which involved a variety of bacteria and yeasts, some of which prevailed during the fermentation process. The combination of two techniques — culture-dependent and independent methods — proved to be efficient for achieving an understanding of the microbial population responsible for wet coffee fermentation. Future work should be conducted to evaluate the production of enzymes by the main species identified and to select appropriate strains as starter cultures in coffee fermentation. Acknowledgments The authors thank the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico of Brasil (CNPQ), Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We also thank the Juliana farm, located in Monte Carmelo city, and the Resfriado farm, situated in Lavras in the state of Minas Gerais, Brazil, for collecting samples. References Agate, A.D., Bhat, J.V., 1966. Role of pectinolytic yeasts in the degradation of mucilage layer of Coffea robusta cherries. Appl. Microbiol. 14 (2), 256–260. Avallone, S., Guyot, B., Brillouet, J.-Marc, Olguin, E., Guiraud, J.-Pierre, 2001. Microbiological and biochemical study of coffee fermentation. Curr. Microbiol. 42, 252–256. Brando, C.H.J., Brando, M.F., 2014. Methods of coffee fermentation and drying. In: Schwan, R.F., Fleet, G.H. (Eds.), Cocoa and Coffee Fermentation. CRC Taylor & Francis, Boca Raton, FL, pp. 367–398. Cocolin, L., Bisson, L.F., Mills, D.A., 2000. Direct profiling of the yeast dynamics in wine fermentations. FEMS Microbiol. Lett. 189, 81–87. Czerny, M., Grosch, W., 2000. Potent odorants of raw Arabica coffee. Their changes during roasting. J. Agric. Food Chem. 48 (3), 868–872.
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Devereux, R., Willis, S.G., 1995. Amplification of ribosomal RNA sequences. In: Akkermans, A.D.L., Van Elsas, J.D., Bruijn, F.J. (Eds.), Molecular microbial ecology manual. Kluwer Academic, Dordrecht, pp. 1–11. Evangelista, S.R., Silva, C.F., Miguel, M.G.P.C., Cordeiro, C.S., Pinheiro, A.C.M., Duarte, W.F., Schwan, R.F., 2014. Improvement of coffee beverage quality by using selected yeasts strains during the fermentation in dry process. Food Res. Int. 61, 183–195. Frank, H.A., Lum, N.A., Cruz, A.S.D., 1965. Bacteria responsible for mucilage-layer decomposition in kona coffee cherries. Appl. Microbiol. 13, 201–207. Gonzalez-Rios, O., Suarez-Quiroza, M.L., Boulanger, R., Barel, M., Guyot, B., Guiraud, J.P., Schorr-Galindo, S., 2007. Impact of “ecological” post-harvest processing on coffee aroma: II. Roasted coffee. J. Food Compos. Anal. 20, 297–307. Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T., Williams, S.T., 1994. Bergey's manual of determinative bacteriology. ninth ed. W. & Wilkins, Baltimore. Kurtzman, C.P., Fell, J.W., Boekhout, T., 2011. The Yeasts, a Taxonomic Study. fourth ed. Elsevier, Amsterdan. Leong, K., Chen, Y., Pan, S., Chen, J., Wu, H., Chang, Y., Yanagida, F., 2014. Diversity of lactic acid bacteria associated with fresh coffee cherries in Taiwan. Curr. Microbiol. 68, 440–447. López-Galilea, I., Fournier, N., Cid, C., Guichard, E., 2006. Changes in headspace volatile concentrations of coffee brews caused by the roasting process and the brewing procedure. J. Agric. Food Chem. 54, 8560–8566. Masoud, W., Jespersen, L., 2006. Pectin degrading enzymes in yeasts involved in fermentation of Coffea arabica in East Africa. Int. J. Food Microbiol. 110, 291–296. Masoud, W., Cesar, L.B., Jespersen, L., Jakobsen, M., 2004. Yeast involved in fermentation of Coffea arabica in East Africa determined by genotyping and by direct denaturating gradient gel electrophoresis. Yeast 21, 549–556. Massawe, G.A., Lifa, S.J., 2010. Yeasts and lactic acid bacteria coffee fermentation starter cultures. Int. J. Postharvest Technol. Innov. 2, 41–80. Muyzer, G., Waal, E.C., Uitterlinden, A.G., 1993. Profile of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction: amplified genes coding for 16S rDNA. Appl. Environ. Microbiol. 59, 695–700. Nielsen, D.S., Teniola, O.D., Ban-Koffi, L., Owusu, M., Andersson, T.S., Holzapfel, W.H., 2007. The microbiology of Ghanaian cocoa fermentations analysed using culture dependent and culture independent methods. Int. J. Food Microbiol. 114, 168–186. Nunes, C.A., Pinheiro, A.C.M., 2012. Sensomaker Free user-friendly interface for sensory analysis Available from: www.ufla.br/sensomaker (Accessed at: Out. 2012). Ovreas, L., Forney, L., Daae, F.L., 1997. Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCRamplified gene fragments coding for 16S rRNA. Appl. Environ. Microbiol. 63, 3367–3373. Pereira, G.V.M., Soccol, V.T., Pandey, A., Medeiros, A.B.P., Lara, J.M.R.A., Gollo, A.L., Soccol, C.R., 2014. Isolation, selection and evaluation of yeasts for use in fermentation of coffee beans by the wet process. Int. J. Food Microbiol. 188, 60–66.
Petisca, C., Pérez-Palacios, T., Farah, A., Pinho, O., Ferreira, I.M.P.L.V.O., 2013. Furans and other volatile compounds in ground roasted and espresso coffee using headspace solid-phase microextraction: effect of roasting speed. Food Bioprod. Process. 91, 233–241. Pineau, N., Schlich, P., Cordelle, S., Mathonnière, C., Issanchou, S., Imbert, A., Rogeaux, M., Etiévant, P., Koster, E., 2009. Temporal dominance of sensations: construction of the TDS curves and comparison with time–intensity. Food Qual. Prefer. 20, 450–455. Ramos, C.L., de Almeida, E.G., Pereira, G.V.D.M., Cardoso, P.G., Dias, E.S., Schwan, R.F., 2010. Determination of dynamic characteristics of microbiota in a fermented beverage produced by Brazilian Amerindians using culture-dependent and culture-independent methods. Int. J. Food Microbiol. 140, 225–231. Senguna, I.Y., Nielsen, D.S., Karapinar, M., Jakobsen, M., 2009. Identification of lactic acid bacteria isolated from Tarhana, a traditional Turkish fermented food. Int. J. Food Microbiol. 135, 105–111. Silva, C.F., 2014. Microbial activity during coffee fermentation. In: Schwan, R.F., Fleet, G.H. (Eds.), Cocoa and Coffee Fermentation. CRC Taylor & Francis, Boca Raton, FL, pp. 398–423. Silva, C.F., Schwan, R.F., Dias, E.S., Wheals, A.E., 2000. Microbial diversity during maturation and natural processing of coffee cherries of Coffea arabica in Brazil. Int. J. Food Microbiol. 60, 251–260. Silva, C.F., Batista, L.R., Abreu, L.M., Dias, E.S., Schwan, R.F., 2008. Succession of bacterial and fungal communities during natural coffee (Coffea arabica) fermentation. Food Microbiol. 25, 951–957. Silva, C.F., Vilela, D.M., Souza, C.C., Duarte, W.F., Dias, D.R., Schwan, R.F., 2012. Evaluation of a potential starter culture for enhance quality of coffee fermentation. World J. Microbiol. Biotechnol. 29, 235–247. Sindicato da Indústria de Café do Estado de Minas Gerais (Sindcafé-MG), 2014. Available in: http://sindicafe-mg.com.br/plus/modulos/conteudo/?tac=tipos-de-cafe (Accessed at: Jan. 2014). Specialty Coffee Association of America (SCAA), 2013. Available in: http://www.scaa.org (Accessed at: April. 2013). Swiegers, J.H., Bartowsky, E.J., Henschke, P.A., Pretorius, I.S., 2005. Yeast and bacterial modulation of wine aroma and flavor. Aust. J. Grape Wine Res. 11, 139–173. Vilela, D.M., Pereira, G.V.D.M., Silva, C.F., Batista, L.R., Schwan, R.F., 2010. Molecular ecology and polyphasic characterization of the microbiota associated with semi-dry processed coffee (Coffea arabica L.). Food Microbiol. 27, 1128–1135. Wakeling, I.N., Macfie, H.J.H., 1995. Designing consumer trials balanced for first and higher orders of carry-over effect when only a subset of k samples from t may be tested. Food Qual. Prefer. 6, 299–308.
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Microbiological diversity associated with the spontaneous wet method of coffee fermentation Suzana Reis Evangelista a, Maria Gabriela da Cruz Pedroso Miguel a, Cristina Ferreira Silva a, Ana Carla Marques Pinheiro b, Rosane Freitas Schwan a,⁎ a b
Biology Department, Federal University of Lavras, CEP 37200-000, Lavras, MG, Brazil Food Sciences Department, Federal University of Lavras, CEP 37200-000, Lavras, MG, Brazil
a r t i c l e
i n f o
Article history: Received 17 March 2015 Received in revised form 5 June 2015 Accepted 7 June 2015 Available online 17 June 2015 Keywords: Arabica coffee Wet fermentation Microbiota PCR–DGGE Yeasts Bacteria
a b s t r a c t The evaluation of the microbiota present during coffee wet fermentation was done in two distinct regions of Minas Gerais, Brazil: one farm in the South of Minas Gerais (Lavras = L) and another farm in the savannah region (Monte Carmelo = MC). The yeast population ranged from 2.48 to 4.92 log CFU/g and from 2 to 4.81 log CFU/g, the mesophilic bacteria population ranged from 3.83 to 8.47 log CFU/g and from 5.37 to 7.36 log CFU/g, and the LAB population ranged from 2.57 to 5.66 log CFU/g and from 3.40 to 4.49 log CFU/g in the L and MC farms, respectively. Meyerozyma caribbica and Hanseniaspora uvarum were the dominant yeasts in coffee wet fermentation at L farm, and Torulaspora delbrueckii was the dominant yeast at MC farm. The species Staphylococcus warneri and Erwinia persicina were the predominant bacteria at L farm, and Enterobacter asburiae and Leuconostoc mesenteroides were the dominant species at MC farm. Lactic acid was the principal acid detected, reaching 2.33 g/kg at L farm and 1.40 g/kg at MC farm by the end of the process. The volatiles composition was similar for roasted coffee from the two different regions and furans, acids, and alcohol were the main groups detected. Temporal Dominance Sensation (TDS) analyses showed that the coffee beverage from L farm was dominated by citrus and herbaceous sensory characteristics, while the coffee from MC farm was dominated by citrus, herbaceous, and nuts sensory characteristics. Evaluating the microbiota in these two regions was important in improving the knowledge of the microbial species present during coffee wet fermentation in Brazil. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The coffee fruit usually consists of an outer layer of skin (peel), called the exocarp under which there is a layer of pulp, followed by mucilaginous mesocarp (mucilage) firmly adhered to the rigid layer called parchment (endocarp). The parchment protects two seeds surrounded by a thin membrane known as silver skin. The coffee fruit are subject to a fermentation process in order to remove the mucilage and pulp before drying. Three methods might be used to process coffee: the dry, semi-dry, and wet processes (Brando and Brando, 2014; Silva, 2014). In the dry method, the newly harvested whole fruit is fermented and dried on platforms after which the coffee beans are removed by hulling and polishing (removed the husk layer that cover the dry coffee beans). In the semi-dry method the coffee peel, pulp and part or all mucilage are removed mechanically and then the coffee is fermented and dried. The amount of mucilage removed depends on the characteristic of the machine used. In the wet method the peel and pulp are removed ⁎ Corresponding author. E-mail address: [email protected]fla.br (R.F. Schwan).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.06.008 0168-1605/© 2015 Elsevier B.V. All rights reserved.
mechanically, leaving the mucilage adhered to the beans. These pulped coffees are then transferred to water tanks where they are allowed to ferment for 6 to 72 h (depending on the environmental temperature), during which the remaining mucilage are degraded and solubilized. The beans are then removed from the tanks and sun dried. Brazil is the largest producer and exporter of coffee. The State of Minas Gerais, in the Southeast of Brazil, produces about 53% of all the coffee in Brazil, of which 85% is processed by dry method. In the last decade, coffee producers in the southern and central areas of Minas Gerais began to process coffee via wet fermentation (Sindcafé-MG, 2014). This processing produces a coffee with a different composition and sensory characteristics than coffee processed by the semi-dry or dry methods, which are alternatives used by the producer to suit different markets. Microorganisms are naturally present during coffee processing and use the various compounds in the pulp and mucilage as nutrients during the fermentation stages. They secrete organic acids and other metabolites that may affect the final sensory characteristics of the beverage (Silva, 2014). This microbiota may vary according to several factors: regional characteristics, coffee fruit composition and the method of fermentation. It is important to know the microbiota present during the processing of coffee, especially when selecting starter cultures that can
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be used in producing differentiated final products and inhibiting the growth of mycotoxigenic fungi (Masoud and Jespersen, 2006; Massawe and Lifa, 2010; Silva et al., 2012). Studies have been performed to identify the microbiota present in wet processing of coffee fruits in India, Hawaii, and Mexico (Agate and Bhat, 1966; Avallone et al., 2001; Frank et al., 1965). Classical morphological and biochemical characterization has been used to identify the microbiota, and species of Erwinia, Klebsiella, and Leuconostoc and the yeasts Kloeckera, Candida, Saccharomyces bayanus, Saccharomyces cerevisiae var. ellipsoideus, and Cryptococcus have been identified as contributing to these fermentations. Using a molecular approach, PCR–DGGE, Masoud et al. (2004) identified Pichia kluyveri, P. anomala, Hanseniaspora uvarum, Kluyveromyces marxianus, Candida pseudointermedia and Torulaspora delbrueckii, during wet coffee fermentation in Arusha, Tanzania (East Africa). Recently, yeasts species found in coffee processed by the wet method in Brazil were reported by Pereira et al. (2014). Pichia fermentans (YC5.2) and Saccharomyces sp. (YC9.15) were studied as having a potential for use as starter cultures for coffee wet fermentation. The bacteria population was not mentioned in their work (Pereira et al., 2014). Studies still are needed in Brazil to improve the knowledge of the microbiota present in coffee processing for each producing region due to the variation in climate and altitude. Our aim was to study the microbial diversity involved in wet coffee fermentation in two main producing regions of Brazil having distinct environmental characteristics using culture-dependent and culture-independent methods. The target metabolites present during the fermentation process were also analyzed using headspace solid-phase microextraction/gas chromatography (HS–SPME/GC) and high-performance liquid chromatography (HPLC). The sensory profile of the final coffee beverage was also evaluated. 2. Materials and methods 2.1. Processing of coffee fermentation The coffee fruits were fermented by the wet method. The experiments were performed in two geographically different regions of Minas Gerais, at a farm located in Monte Carmelo (MC), 870 m above sea level in the savannah (Cerrado) region, and at a farm located in Lavras (L), 919 m above sea level in the Atlantic Forest region. The experiments were done during two consecutive harvesting seasons (2012 and 2013). The fruit (60 kg) of Coffea arabica L. var. Acaiá was harvested at the mature stage (cherries) and was mechanically depulped in a horizontal machine (model BDSV-04; Pinhalense, São Paulo, Brazil), followed by for 48 h fermentation in a tank with 60 l of water to remove the mucilage. At the L farm, the temperature of fermentation was between 14 and 23 °C, and at the MC farm it was between 20 and 28 °C. Fermentations were done in duplicate at each farm. After fermentation, the coffees were placed on suspended platforms for sun drying until they reached approximately 11% moisture (224 h at L farm and 336 h at MC farm), measured using Moisture Meter G600i (GEHAKA, São Paulo, Brazil). The following samples were collected: cherries before fermentation, along the fermentation (0, 6, 12, 24, 36, and 48 h), and during drying (60, 112, 224, and 336 h). Samples (500 g) were placed aseptically in sterile plastic bags, in triplicate, and immediately transferred to the laboratory in iceboxes for microbiological analyses. For physicochemical analyses, coffee samples were frozen at −20 °C until analyzed. Sensory analysis and volatile compounds were evaluated in dried coffee beans. 2.2. Characterization and identification of microbiota 2.2.1. Quantification, isolation, and phenotypic characterization Coffee cherry sample (10 g) was added to 90 mL of sterile peptone water (in g/L: 1 bacteriological peptone [Himedia, Mumbai, India]), homogenized for 2 min in a Stomacher (Mayo Homogenius HG 400, São
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Paulo, Brazil), and used for decimal serial dilution. Bacteria and yeasts were enumerated by spread plating on MRS agar (Merck) for lactic acid bacteria, on plate count agar (PCA) (in g/L: 5 tryptone [Himedia], 2.5 yeast extract [Merck], 1 glucose [Merck], 15 agar Merck]), for total bacteria and on YPD agar (in g/L: 10 yeast extract [Merck], 10 peptone [Himedia], 20 dextrose [Merck], 20 agar [Merck]) for yeasts. MRS plates were incubated in an anaerobic jars at 30 °C for 3–4 days and PCA and YPD plates were incubated at 30 °C for 3–7 days and the morphological properties of the colonies (cell size, cell shape, edge, color, and brightness) were recorded and the square root of the number of colonies counted for each morphotype was purified by streaking on new agar plates (same culture media used for plating) (Senguna et al., 2009). The pure cultures were stored in an ultra freezer at −80 °C in the same broth culture media used for plating, containing 20% glycerol (w/w). The phenotypic characterization of the bacterial colonies was performed using Gram staining, catalase and oxidase activities, motility tests, growth in culture media with 1% and 5% (w/v) NaCl (salt tolerance), protease production, spore formation, and the ability to ferment glucose, sucrose, and xylose (Sigma, St. Louis, USA), as recommended in Bergey's Manual of Determinative Bacteriology (Holt et al., 1994). Yeast colonies were characterized for morphology and biochemical assessments as described by Kurtzman et al. (2011). A total of 435 isolates was obtained; 251 were from samples from L farm and 184 isolates were obtained from MC farm. The microbiota identified was the same in both years analysed. However, variation of less than 5% was found in the counting. 2.2.2. Genotypic identification The isolates (435) were grouped by phenotypic characteristics, as mentioned above, and representatives of each phenotypic group were subjected to molecular analyses (236 isolates). The genotypic characterization of the selected isolates was first performed by rep-PCR and subsequently by DNA sequencing. The bacterial and yeast cultures were grown under appropriate conditions, collected from agar plates with a sterile pipette tip, and resuspended in 50 μL of sterile Milli-Q water. The suspension was heated for 10 min at 95 °C, and three μL was used as a DNA template in the PCR experiments. The fingerprints of the genomic DNA were obtained via the PCR amplification of repetitive bacterial and yeast DNA elements (rep-PCR) using the (GTG)5 primer, as described by Nielsen et al. (2007). Amplified PCR products were separated via 2% (w/v) agarose gel electrophoresis at 70 V for 3 h, and the images were visualized and photographed using a transilluminator LPixImage (LTB 20 × 20 HE, LPix®, Brazil). The bacteria and yeasts representative of each group were subjected to 16S rRNA gene and internal transcribed spacer (ITS) region gene sequencing, respectively. The amplification of the 16S rRNA gene used the primers 27 F and 1512R (Devereux and Willis, 1995). The ITS region was amplified using the primers ITS1 and ITS4 (Nielsen et al., 2007). The amplified PCR products were sent for sequencing at the Advanced Genetics Technologies Center — AGTC (Kentucky, USA); the ABI3730 XL automatic DNA sequencer was utilized. The sequences were aligned using the BioEdit 7.7 sequence alignment editor and were compared to the GenBank database using the Basic Local Alignment Tool (BLAST) program (National Center for Biotechnology Information, Bethesda, MD) for the identification of isolates. 2.3. PCR–DGGE analysis Samples of coffee cherries (3 g) were mixed with 5 mL of Milli-Q water for 10 min then the coffee cherries were removed and the liquid phase was centrifuged at 100 ×g for 10 min at 4 °C. The pellet was used for DNA extraction. The total DNA was extracted from samples using the “DNA Purification from Tissues” protocol (QIAamp DNA Mini Kit [Qiagen, Hilden, Germany]) in accordance with the manufacturer's instructions. The DNA from the bacterial community was amplified with
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the primers 338fgc and 518r (Ovreas et al., 1997). A fragment of the D1region of the 26S rRNA gene was amplified with the eukaryotic universal primers NL1GC and LS2 (Cocolin et al., 2000). Reactions were performed according to Ramos et al. (2010). Aliquots (3 μL) of the amplification products were analyzed via electrophoresis on 1% agarose gels before they were used for DGGE. The PCR products were separated in polyacrylamide gels (8% (w/v) acrylamide:bisacrylamide [37.5:1]) in 1 × TAE buffer with a DCode system apparatus (BioRad Universal Dcode Mutation Detection System, Richmond, CA, USA). Denaturation gradients were used that varied from 15 to 55% for the bacterial products (100% corresponded to 7 M of urea and 40% [v/v] formamide) and from 20 to 60% for the yeast products. Electrophoresis was conducted at a constant voltage of 130 V for 6 h and at a constant temperature of 60 °C. Following electrophoresis, the gels were stained with SYBR-Green I (Molecular Probes, Eugene, UK) (1:10.000 v/v) for 30 min. The images were visualized and photographed using a transilluminator LPixImage (LTB 20 × 20 HE, LPix®, Brazil). Selected bands were excised and their DNA amplified using the primers 338fgc and 518r for bacteria and NL1 and LS2 for yeast. The PCR products were purified and sequenced at the Advanced Genetics Technologies Center — AGTC (Kentucky, USA); the ABI3730 XL automatic DNA sequencer was utilized. The sequences were compared to
the GenBank database using the BLAST algorithm (National Center for Biotechnology Information, Maryland, USA).
2.4. Analyses of organic acids The organic acids from the beans were analyzed using a highperformance liquid chromatography system (Shimadzu Corp., Japan) equipped with detection system consisting of a UV–Vis detector (SPD 10Ai). The extraction and operating conditions were performed according to the methodology described by Evangelista et al. (2014).
2.5. Analysis of volatile compounds Volatile compounds were extracted from the beans and analyzed according to Evangelista et al. (2014). The volatile compounds from each headspace analysis were defined by integrating the peak areas of all the identified compounds. Relative percent areas were used for semiquantitation of the target compounds. The relative percentage of the individual compounds was calculated from the total peak area of the volatile compounds on the chromatograms (Petisca et al., 2013).
Fig. 1. The populations of yeast (YEPG counts ), mesophilic bacteria (PCA counts ), and lactic acid bacteria (MRS counts ) in coffee wet processing at Lavras farm (A). The ), mesophilic bacteria (PCA counts ), and lactic acid bacteria (MRS counts ) in coffee wet processing at Monte Carmelo farm (B). populations of yeast (YEPG counts
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2.6. Analysis of sensory characteristics The samples of roasted coffee from each region were prepared according to the Specialty Coffee Association of America (SCAA, 2013). The coffee was roasted in a laboratory roaster (Probatino, Leogap model, Brazil) with a capacity of 150 g. The temperature used ranges from 180 to 200 °C and the time was around 10 min until the beans reach lightmedium roast. The coffee beans were ground in an electric mill (Pinhalense ML-1, Brazil). For sample uniformity evaluation, five cups of each sample were prepared. Clean and odor-free water was used. Water was brought to approximately 92–94 °C and poured directly on the ground coffee. The ratio was 8.25 g of coffee per 150 mL of water. Grounds were steeped for 3–5 min before the sample was evaluated. A panel of three trained coffee experts with Q-Grader Coffee Certificates evaluated the samples. The first sensorial evaluation was conducted to identify the most relevant of the coffee attributes, and the eight most cited terms were selected and retained for further Temporal Dominance of Sensations (TDS) evaluations (Pineau et al., 2009). The attributes selected by the panel were as follows: chocolate, bitter chocolate, caramel, citric, tobacco, butter, herbaceous, and nuts. Samples coded with three digits were submitted in a balanced order (Wakeling and MacFie, 1995) and evaluated in three replicates. The analysis was performed in 5 sequential sips, where each one last 15 s. For each sip, the panelist imbibed the coffee,
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moved it around in their mouths for 3 s, and swallowed it. The evaluation continued until no sensation was perceived or 15 s had passed. Data collection began upon tasting and consisted of selecting the most dominant attribute (among the eight attributes) perceived at that time using SENSOMAKER Software (Nunes and Pinheiro, 2012) and plotting as TDS curves showing the percentage of subjects which selected the attribute as dominant at a specific time — that is, the dominance rate (Pineau et al., 2009).
3. Result 3.1. Quantification and characterization of microbial population The mesophilic aerobic bacteria, LAB, and yeast populations found during coffee processing at the two farms, Lavras (L) and Monte Carmelo (MC), are shown in Fig. 1. The yeast population in coffee from L farm ranged from 2.5 to 4.9 log CFU/g and that for MC farm ranged from 2 to 4.8 log CFU/g. The population of mesophilic bacteria varied from 3.8 to 8.5 log CFU/g at L farm and from 5.4 to 7.4 log CFU/g at MC farm (Fig. 1). The LAB population varied from 2.6 to 5.7 log CFU/g at L farm and from 3.4 to 4.5 log CFU/g at MC farm.
Table 1 Yeasts and bacteria associated with fermentation and drying of coffee beans at Lavras farm (L) and Monte Carmelo farm (MC). Species identification
Population (log CFU/g) of the isolates from fruit coffee before and during processing Fermentation (hours)
Yeast L farm H. uvarum M. caribbica P. fermentans D. hansenii T. delbrueckii C. railenensis C. quercitrusa W. ciferrii Yeast MC Farm H. uvarum M. caribbica P. fermentans T. delbrueckii C. glabrata W. anomalus Bacteria L farm Enterococcus sp. O. pseudogrignonense C. taichungense C. bovis E. persicina S. warneri Pseudomonas sp. P. amylolyticus Curtobacterium sp. K. oxytoca B. amyloliquefaciens E. hermannii Bacteria MC farm E. asburiae E. ludwigii C. pallidum P. agglomerans P. dispersa K. oxytoca S. marcescens Microbacterium sp. M. laevaniformans W. cibaria Kocuria sp. L. mesenteroides
Drying (hours)
Coffee fruit
0
6
12
24
36
48
60
112
224
336
2.9 4.4 3.4 b2 b2 3.1 b2 b2
3.7 4.2 2.3 b2 b2 b2 2.3 b2
3.4 3.6 2.0 2.6 b2 b2 b2 b2
4.1 3.8 2.0 b2 b2 b2 b2 b2
3.6 3.3 b2 b2 b2 b2 b2 b2
3.8 4.9 b2 b2 b2 b2 b2 b2
b2 2.9 b2 b2 b2 b2 b2 b2
b2 2.5 b2 b2 b2 b2 b2 b2
2.8 3.5 b2 b2 b2 b2 b2 b2
3.0 3.9 3.0 b2 2.3 b2 b2 b2
3.6 4.0 b2 b2 b2 b2 b2 3.6
3.0 3.8 2.3 5.3 2.8 b2
2.5 b2 b2 b2 2.3 3.4
b2 b2 2.0 b2 b2 b2
b2 b2 2.5 b2 b2 b2
b2 4.0 b2 4.8 b2 b2
b2 b2 b2 4.5 b2 b2
b2 b2 b2 3.8 b2 b2
b2 b2 b2 4.3 b2 b2
b2 b2 b2 2.7 b2 b2
b2 b2 b2 2.9 b2 b2
⁎– – – – – –
b2 b2 4.9 b2 4.9 5.2 4.3 b2 4.0 4.3 4.8 4.0
b2 4.4 5.0 5.0 4.5 5.6 b2 b2 b2 b2 b2 b2
b2 b2 b2 4.0 4.0 6.1 b2 b2 b2 b2 b2 b2
b2 b2 b2 b2 5.5 6.7 b2 b2 6.2 b2 b2 b2
b2 b2 b2 b2 b2 7.1 b2 b2 b2 5.6 b2 b2
b2 b2 b2 b2 b2 8.5 b2 b2 b2 b2 b2 b2
b2 b2 b2 b2 6.1 7.2 b2 b2 b2 b2 b2 b2
b2 b2 b2 b2 6.1 6.6 b2 b2 b2 b2 b2 b2
b2 b2 b2 b2 b2 6.1 b2 b2 b2 b2 b2 b2
4.6 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2
b2 3.6 2.8 b2 b2 b2 b2 2.6 2.9 b2 b2 b2
4.7 5.7 4.6 b2 5.0 b2 4.7 b2 b2 b2 4.8 b2
5.3 4.3 b2 4.0 4.7 5.6 b2 4.8 4.8 b2 b2 b2
6.0 b2 b2 b2 b2 b2 b2 5.0 b2 6.1 b2 6.0
6.2 b2 b2 b2 b2 b2 b2 b2 b2 6.5 b2 b2
7.3 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2
7.4 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2
6.8 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2
5.9 b2 b2 b2 4.6 b2 b2 b2 b2 b2 b2 5.9
6.2 4.5 b2 b2 b2 b2 b2 b2 b2 b2 b2 4.2
b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 b2 5.4
– – – – – – – – – – – –
⁎ Processing ends with 224 h in Monte Carmelo farm.
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A total of 251 microbial isolates were obtained from the samples of L farm: 59.4% yeasts and 40.6% bacteria (50.9% Gram-positive, and 49% Gram-negative). A total of 184 isolates were obtained from MC farm: 33.7% yeasts and 66.3% bacteria (33.6% Gram-positive, and 66.3% Gram-negative). The isolates were grouped by phenotypic characteristics (cell morphology and biochemical features) (data not shown), and 236 selected isolates were subjected to rep-PCR for genotypic characterization.
Representative isolates from each rep-PCR-profile were identified by sequencing. The identified species and the population during processing are shown in Table 1. The yeast species present in the coffee beans of L farm were Hanseniaspora uvarum (accession no KM402039–KM402045), Meyerozyma caribbica (accession no KM402046–KM402058), P. fermentans (accession no KM402059–KM402064), Debaryomyces hansenii (accession no KM402067), T. delbrueckii (accession
Fig. 2. PCR–DGGE patterns of the yeast and bacteria communities present during coffee wet processing at Lavras farm. Prokaryote (A) and Eukaryote (B). S = coffee cherries, 0 = 0 h, 1 = 4 h, 2 = 15 h, 3 = 22 h, 4 = 27 h, 5 = 40 h, 6 = 50 h, 7 = 140 h, 8 = 236 h, 9 = 360 h, H2O = water fermentation tank.
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no KM402068–KM402082), Candida railenensis (accession no KM402083), Candida quercitrusa (accession no KM402088), and Wickerhamomyces ciferrii (accession no KM402084) (Table 1). M. caribbica was the most prevalent yeast in the coffee fruit, during the fermentation and drying reached a maximum population of 4.9 log CFU/g at 36 h of fermentation. It was followed by
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H. uvarum with a population of 4.1 log CFU/g at 36 h of fermentation. The yeast species present in the coffee beans of MC farm were H. uvarum (accession no KM402039–KM402045), M. caribbica (accession no KM402046–KM402058), P. fermentans (accession no KM402059– KM402064), T. delbrueckii (accession no KM402068–KM402082),
Fig. 3. PCR–DGGE patterns of the yeast and bacteria communities present during coffee wet processing at Monte Carmelo farm. Prokaryote (A) and Eukaryote (B). S = coffee cherries, 0 = 0 h, 1 = 9 h, 2 = 20 h, 3 = 28 h, 6 = 54 h, 7 = 174 h, 8 = 202 h, H2O = water fermentation tank.
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Table 2 Species identified by PCR–DGGE using universal primers for yeast and bacteria. Bands
Acess number
Similarity (%)
Prokaryote
1 2,3, 27, 28, 4, 5 6 7, 8, 11, 12, 15, 16 9, 10 13,14 29, 30, 31, 32, 40, 41, 47, 48 33 34, 35 36, 37 38, 39 42, 43, 44 45, 46 49
AF515228 AB854267 HM756486 KF673524 KF031439 HQ683968 JX120129 FJ406528 JX315564 HG798481 KF453765 KF891342 KC430956 CP002272 JX242698
95 98 97 97 100 97 98 95 94 100 97 97 95 97 94
Leuconostoc pseudomesenteroides Weissella confusa Lysinibacillus fusiformis Lactobacillus fermentum Uncultured bacterium Klebsiella oxytoca Actinobacterium sp. Uncultured bacterium Acinetobacter schindleri Lactococcus lactis Enterobacteriaceae bacterium Enterobacter cloacae Enterobacter sp. Enterobacter lignolyticus Uncultured actinomycete
17, 18, 19, 20, 50, 51, 52 21, 22, 58, 59 23, 24, 60, 61, 62 25, 26, 63, 64 53 54, 55 56, 57
EU568995 JQ417238 KC510080 AY314792 EU650386 AY520372 HE799671
95 98 100 100 98 97 97
Eukaryote Meyerozyma caribbica Mitchella repens Pichia fermentuns Uncultured fungus Coffea arabica Candida sp. Torulaspora delbrueckii
C. glabrata (accession no KM402089–KM402091), and W. anomalus (accession no KM402085–KM402087) (Table 1). T. delbrueckii was the predominant yeast. It was detected in the coffee fruit with a high population of 5.3 log CFU/g and during the fermentation the maximum population was of 4.9 log CFU/g. M. caribbica was the second most significant yeast found in the coffee fruit (3.8 log CFU/g) and during fermentation with a population of 4 log CFU/g. The diversity of bacterial species was greater than that of the yeast: 23 bacteria and 10 yeast species were identified (Table 1). Klebsiella oxytoca (accession no KM402125–KM402126) was the only bacterial species found at both farms. Enterococcus sp. (accession no KM402100), Ochrobactrum pseudogrignonense (accession no KM402101–KM402102), Chryseobacterium taichungense (accession no KM402103–KM402104), C. bovis (accession no KM402105–KM402106), Erwinia persicina (accession no KM402108–KM402111), Staphylococcus warneri (accession no
KM402118–KM402119), Pseudomonas sp. (accession no KM402120), Paenibacillus amylolyticus (accession no KM402121), Curtobacterium sp. (accession no KM402123–KM402124), Bacillus amyloliquefaciens (accession no KM402133) and Escherichia hermannii (accession no KM402097), were found in samples from L farm (Table 1). S. warneri was the most prevalent bacteria, and was identified in the coffee fruit and during fermentation, reaching maximum population of 8.5 log CFU/g at 36 h of fermentation. E. persicina was the second most significant specie reaching the maximum value of 5.5 CFU/g at 12 h of fermentation. Enterobacter asburiae (accession no KM402092–KM402096), E. ludwigii (accession no KM402098–KM402099), C. pallidum (accession no KM402107), Pantoea agglomerans (accession noKM402112), P. dispersa (accession no KM402113–KM402117), Serratia marcescens (accession no KM402127), Microbacterium sp. (accession no
Table 3 Organic acids present in coffee during wet processing at Lavras (A) and Monte Carmelo. Time (hours)
L farm Fermentation tank 0 24 48 Drying 60 224 336 MC farm Fermentation tanks 0 24 48 Drying 60 112 224
Compounds (g/Kg) Citric
Malic
Succinic
Lactic
Acetic
0.08 ± 0.02 0.02 ± 0.01 0.07 ± 0.02
0.73 ± 0.35 0.16 ± 0.01 0.09 ± 0.01
0.48 ± 0.25 0.05 ± 0.01 nd
nd 0.62 ± 0.02 0.88 ± 0.07
Nd Nd 0.01 ± 0.00
0.10 ± 0.02 0.05 ± 0.00 0.20 ± 0.01
0.09 ± 0.03 0.08 ± 0.00 0.20 ± 0.01
0.02 ± 0.00 0.02 ± 0.00 0.08 ± 0.00
1.07 ± 0.02 1.09 ± 0.09 2.34 ± 0.45
Nd Nd Nd
0.18 ± 0.03 0.20 ± 0.01 0.08 ± 0.00
0.14 ± 0.00 0.06 ± 0.00 0.02 ± 0.00
0.40 ± 0.03 0.14 ± 0.02 0.05 ± 0.00
nd 0.34 ± 0.10 0.57 ± 0.05
Nd Nd 0.02 ± 0.00
0.03 ± 0.00 0.02 ± 0.00 0.04 ± 0.01
0.01 ± 0.00 nd nd
0.04 ± 0.00 0.12 ± 0.01 0.09 ± 0.00
1.03 ± 0.19 1.40 ± 0.12 1.08 ± 0.09
Nd Nd Nd
nd = not detected; ± = Standard deviation.
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KM402128–KM402129), M. laevaniformans (accession no KM402130), Weissella cibaria (accession no KM402131), Kocuria sp. (accession no KM402132), and Leuconostoc mesenteroides (accession noKM402134) were the bacteria isolated from MC farm (Table 1). E. asburiae was the most prevalent bacteria found in coffee fruit and during fermentation reaching maximum value of 7.4 log CFU/g at 36 h of fermentation. L. mesenteroides was the second most significant bacteria identified reaching the maximum value of 6 CFU/g at 12 h of fermentation. The microbiota identified was the same in both years analyzed. However, variation of less than 5% was found in the counting.
curves are presented in Fig. 4. In the coffee from L farm, sip 3 had dominant herbaceous and citric sensation characteristics, and sips 2 and 5 had a dominant citric sensation characteristic; in sips 1 and 4, no sensation characteristic was dominant (Fig. 4A). In the coffee from MC farm, sips 1 and 3 had a dominant citric sensation characteristic, sips 2 and 4 had a dominant herbaceous sensation characteristic, and sip 5 had a dominant nuts sensation characteristic (Fig. 4B). The sensations chocolate, bitter chocolate, caramel, tobacco, and butter did not trigger the most attention from the panelists. These results are represented in the graph below the line of significance (Fig. 4B).
3.2. Culture-independent microbiological analysis using PCR–DGGE
4. Discussion
Figs. 2 and 3 show the PCR–DGGE fingerprints of the bacteria and yeast communities. In general, species of bacteria present in the coffee before fermentation remained throughout the process and were also present in the water tank at the end of fermentation. The sequencing of the bands from L farm indicated the presence of Leuconostoc pseudomesenteroides (band 1), Weissella confusa (bands 2 and 3), Lysinibacillus fusiformis (bands 4 and 5), uncultured bacteria (bands 7, 8, 11, 12, 15, and 16), Lactobacillus fermentum (band 6), K. oxytoca (bands 9 and 10), and Actinobacterium (bands 13 and 14) (Fig. 2A and Table 2). MC farm samples presented W. confusa (bands 27 and 28), uncultured bacteria (bands 29, 30, 31, 32, 40, 41, 47, and 48), Acinetobacter schindleri (band 33), Lactococcus lactis (bands 34 and 35), Enterobacteriaceae bacterium (band 36 and 37), Enterobacter cloacae (bands 38 and 39), Enterobacter sp. (bands 42, 43, and 44), E. lignolyticus (bands 45 and 46), and uncultured actinomycete (band 49) (Fig. 3A and Table 2). The yeast species found in the coffee cherries before being placed into the tank remained throughout the fermentation process and some new species appear (Figs. 2B and 3B and Table 2). Both MC and L farms showed the presence of M. caribbica (bands 17, 18, 19, 20, 50, 51, and 52), Candida sp. (bands 54 and 55), T. delbrueckii (bands 56 and 57), P. fermentans (bands 23, 24, 60, 61, and 62), and uncultured fungi (bands 25, 26, 63, and 64). The plant species C. arabica (band 53) and Mitchella repens (bands 21, 22, 58 and 59) were also identified because the primers NL1 and LS2 are universal. Therefore, they can amplify the DNA of various eukaryotic organisms, including plants.
The mesophilic aerobic bacteria were dominant throughout the process and showed a high level of diversity (Figs. 1–3). A difference in the diversity of species of the prokaryotic and eukaryotic groups was observed among the regions studied, the main species of yeast and
3.3. Chemical analyses The main acid present in the coffee during fermentation and drying was lactic acid (Table 3), which showed increasing concentrations and reached a maximum value of 2.33 g/kg for L farm, while for MC farm, the maximum concentration was 1.07 g/kg. Acetic acid was detected in the coffee (0.02 g/kg) only at the end of fermentation in both experiments. Citric, malic and succinic acids were detected during fermentation tank. Propionic and butyric acids, which may impair the sensory characteristics of coffee were not detected. A total of 30 volatile compounds were detected via HS–SPME/GC (Table 4). Among these compounds, 18 were detected in green coffee and 25 were detected in roasted coffee. The composition of green coffee beans from the two farms was different: the green coffee from L farm showed alcohol (methanol), esters (ethyl butyrate and furfuryl acetate), and acids (hexanoic and nonanoic acid) as the main compounds, while the green coffee from MC farm showed alcohol (methanol), acids (isobutyric and hexanoic acid), and furan (furfuryl alcohol and furfural). The composition of the majority of volatile compounds in roasted coffee was similar. The main compounds were furans (furfuryl alcohol and furfural), acids (isobutyric acid), and alcohol (1-pentanol and 2-heptanol). 3.4. Sensorial analyses The most relevant attributes of the coffee were selected in the previous analysis and subsequently evaluated using TDS analysis. The TDS
Table 4 Relative percentage of volatile compounds identified in green and roasted coffee. Compounds
Green (%)
Roasted (%)
Coffee L farm
Coffee MC farm
Coffee L farm
Coffee MC farm
Ketones 2.3-butanedione 2-nonanone Total ketones
nd nd nd
nd nd nd
0.07 0.80 0.87
nd 1.28 1.28
Alcohols Methanol 1-propanol 1-pentanol 2-heptanol 3-methyl-1-pentanol 1-hexanol a-Terpineol 2-phenylethanol Total alcohols
16.10 nd nd nd 0.95 nd 1.03 1.41 19.49
12.24 1.17 nd nd nd 1.79 nd 0.65 15.85
0.10 nd 1.35 1.83 0.55 nd nd 0.08 4.91
0.09 nd 1.24 2.11 0.67 nd nd 0.06 4.18
Aldehydes Acetaldehyde Hexanal Octanal Nonanal Butyraldehyde Decyl aldehyde Total aldehydes
0.17 nd nd nd 0.91 nd 1.07
0.28 nd nd nd nd nd 0.28
0.10 0.01 0.44 0.55 0.66 0.34 2.10
0.14 nd 0.29 1.06 0.47 0.32 2.29
Acids Isobutyric acid Hexanoic acid Nonanoic acid Propanoic acid Total acids
nd 1.44 0.80 nd 2.23
3.11 2.36 0.95 nd 6.42
8.15 0.11 0.02 0.54 8.81
7.45 0.09 0.02 0.35 7.1
Esters Propyl acetate Ethyl butyrate Diethyl malonate Phenyl acetate Furfuryl acetate Diethyl succinate Total esters
0.52 3.27 nd nd 4.13 nd 7.92
nd 0.80 nd nd nd 1.14 1.95
0.61 nd 0.28 0.32 2.38 nd 3.59
0.76 nd 0.21 0.29 2.72 nd 3.98
Phenol Guaiacol
nd
nd
0.58
0.64
Burnt
Furans Furfuryl alcohol Furfural 5-methylfurfural Total furans Total GC area
nd nd 0.94 0.94 7163
2.72 2.70 0.58 5.99 9870
21.93 13.26 0.42 35.61 288,508
19.03 13.90 0.77 33.70 215,900
Burnt Almond/bitter Caramel
nd = not detected. a Czerny and Grosch (2000), Gonzalez-Rios et al. (2007).
Reference target compoundsa
Buttery
Green
Floral
Acrid/egg Green
Nutty
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bacteria were not the same between them (Table 1; Figs. 1 and 2). This difference could have been due to environmental temperature, humidity, composition of the remaining pulp and mucilage surrounding the coffee beans, and the altitude (Leong et al., 2014; Silva et al., 2008). The majority of the microbiota present throughout the wet processing was also detected in coffee fruit. The wet processing favored bacteria development that showed a high increase of the population in both regions. Studies of the microbiota from coffee undergoing different types
of processing also observed that some species already are naturally present in the fruit (Silva et al., 2000, 2008). Therefore, harvesting methods and fermentation process will directly interfere with microbiota (Avallone et al., 2001; Silva et al., 2000; Vilela et al., 2010). H. uvarum and T. delbrueckii were detected in higher numbers in coffee from L farm and MC farm, respectively, and were also reported in others studies that examined the wet and semi-dry processes (Masoud et al., 2004; Vilela et al., 2010). The presence of some yeasts,
Fig. 4. TDS curves of coffee from L farm (A) and coffee from MC farm (B). Chocolate ( ), Bitter chocolate ( ), Caramel ( ), Citric ( ), Tobacco ( ), Butter ( ), ), Nuts ( ). Chance level ( ) represents the dominance rate that an attribute can obtain by chance (one/number of attributes) Significance level ( ) expresses Herbaceous ( the smallest value of the proportion that is significantly (p = 0.05) higher than the chance level. Each slip duration of 15 s.
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such as Saccharomyces, Pichia, Candida and Meyerozyma provides benefits for coffee processing due to their important role in the degradation of mucilage rich in pectin and because they may inhibit the growth of mycotoxigenic fungi (Masoud and Jespersen, 2006; Massawe and Lifa, 2010; Silva et al., 2012; Evangelista et al., 2014). Therefore, these yeasts are interesting strains that could be used as starter cultures (Evangelista et al., 2014). P. fermentans was detected in both coffee regions. This specie produces volatile compounds that might interfere with the quality of coffee beverages (Pereira et al., 2014). Some bacteria identified in this work belong to genera commonly found during coffee processing, such as Erwinia, Klebsiella, Leuconostoc, Weissella, Enterococcus, Enterobacter, Serratia, and Bacillus. These genera have been identified during the natural, semi-dry, and wet fermentation of coffee (Avallone et al., 2001; Silva et al., 2000, 2008; Vilela et al., 2010). L. mesenteroides detected in coffee from MC farm is an important lactic acid bacteria present during the wet processing and species of Erwinia and Klebsiella are reported as important producers of pectinases (Avallone et al., 2001). Some species of yeast and bacteria found using the culturedependent method were not detected via PCR–DGGE (Figs. 2 and 3 and Table 2). Masoud et al. (2004) also reported that some microbial species were only detected using the culture-dependent method in the samples taken during wet processing. This fact could be explained by the difficulty of obtaining high-quality DNA suitable for PCR directly from the samples. The initial template DNA and template competition may affect the detection of rare microorganisms in the microbial population (Muyzer et al., 1993). All yeasts species detected in DGGE gels were also isolated from plating. Some bacterial species were detected only through PCR–DGGE. L. pseudomesenteroides, W. confuse, L. fusiformis, L. fermentum and Actinobacterium sp. were the species detected in L farm. A. schindleri, W. confusa, L. lactis, E. bacterium, E. cloacae and E. lignolyticus were the species detected in MC farm. This may have occurred because their population densities were lower than 2 log CFU/g, making them impossible to detect via the culture-dependent method (Figs. 2 and 3 and Table 2). Vilela et al. (2010) also reported that some microbial species were only detected using the DGGE method in the samples taken during semi-dry processing. The species L. pseudomesenteroides, W. confusa, L. lactis and Enterobacter sp. have been found in others studies (Leong et al., 2014; Vilela et al., 2010). The species L. pseudomesenteroides and W. confusa contribute in the fermentation process and might inhibit filamentous fungi growth (Leong et al., 2014). Therefore, molecular methods should always be used in conjunction with traditional methods to evaluate biodiversity. Acetic and lactic acids were produced throughout the fermentation. Lactic acid was the main acid found (Table 3). The presence of lactic acid bacteria, such as bacteria of the genera Weissella, Leuconostoc, and Lactobacillus identified throughout the fermentation process, may have contributed to this fact. The other acids detected were malic, citric, and succinic acids (Table 3). These acids are already naturally present in the coffee bean, and their concentrations decreased during fermentation, but succinic acid showed a slight increase at the end of the process. Succinic acid may be produced by Bacillus spp. (Silva et al., 2012) and by heterofermentative lactic acid bacteria (LAB) (Swiegers et al., 2005). The variation of acids during fermentation due to microbial metabolism changed the pH value and influences the microbiota present in the tank (Table 3). The diffusion of theses acids into the bean could influence the final beverage flavor and quality (Evangelista et al., 2014; Silva et al., 2012; Silva, 2014). The volatile compounds identified in the green coffee beans differed between the regions studied (Table 4) that may have influenced this difference is the microbiota involved during the processing of the coffee beans (Evangelista et al., 2014; Silva et al., 2012). Some compounds are known to play a role in aroma development during fermentation, such as the esters that were detected in the green coffee beans in both regions.
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However, after roasting, less difference in the volatile composition was observed (Table 4). The mechanisms of coffee aroma formation are extremely complex, and there is clearly a wide range of interactions between all the pathways involved. Maillard reactions occur between reducing carbohydrates and proteins and are responsible for the formation of many volatile compounds during roasting (López-Galilea et al., 2006). Each coffee sample was evaluated on the following attributes: aroma, flavor, aftertaste, acidity, body, balance, uniformity, clean cup, sweetness, and overall and was given a score for each of these attributes. The coffee of L farm received a score of 81 and MC farm of 80 (data not shown), being considered as very good special coffee according to SCAA (2013). Although both coffees were well accepted by the panelists, some distinguished flavors were found in the TDS analysis (Fig. 4). The coffee from L farm presented a nice citrus and herbaceous flavor, and the coffee from MC farm presented, addition to these flavors, nuts sensation characteristics. All these sensations are desirable in the final coffee beverage. The presence of volatile compounds differed between the regions and affected the beverages' final flavors. The presence of different microbial species in each region may have contributed to this difference. The microbial activity during fermentation produces volatile compounds that might influence the final taste of the beverage (Evangelista et al., 2014). Some volatile compounds detected in this study are described in the literature by providing aromatic notes such as caramel, nutty, burnt, buttery, floral, and almond (Table 4) (Czerny and Grosch, 2000; Gonzalez-Rios et al., 2007). The presence of furans and ketones were detected in both samples, which contributed to the citric and herbaceous flavors that were dominant in the sensory analysis (Fig. 4). The presence of furans can provide herbal or fruity notes, and ketones are described as providing buttery, caramel-like, musty, mushroom like, or fruity notes (López-Galilea et al., 2006). The evaluation of these two coffee-producing regions contributed to a better understanding of the microbiota present during coffee wet processing and some characteristics of this processing. In conclusion, this study evaluated coffee wet processing, which involved a variety of bacteria and yeasts, some of which prevailed during the fermentation process. The combination of two techniques — culture-dependent and independent methods — proved to be efficient for achieving an understanding of the microbial population responsible for wet coffee fermentation. Future work should be conducted to evaluate the production of enzymes by the main species identified and to select appropriate strains as starter cultures in coffee fermentation. Acknowledgments The authors thank the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico of Brasil (CNPQ), Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We also thank the Juliana farm, located in Monte Carmelo city, and the Resfriado farm, situated in Lavras in the state of Minas Gerais, Brazil, for collecting samples. References Agate, A.D., Bhat, J.V., 1966. Role of pectinolytic yeasts in the degradation of mucilage layer of Coffea robusta cherries. Appl. Microbiol. 14 (2), 256–260. Avallone, S., Guyot, B., Brillouet, J.-Marc, Olguin, E., Guiraud, J.-Pierre, 2001. Microbiological and biochemical study of coffee fermentation. Curr. Microbiol. 42, 252–256. Brando, C.H.J., Brando, M.F., 2014. Methods of coffee fermentation and drying. In: Schwan, R.F., Fleet, G.H. (Eds.), Cocoa and Coffee Fermentation. CRC Taylor & Francis, Boca Raton, FL, pp. 367–398. Cocolin, L., Bisson, L.F., Mills, D.A., 2000. Direct profiling of the yeast dynamics in wine fermentations. FEMS Microbiol. Lett. 189, 81–87. Czerny, M., Grosch, W., 2000. Potent odorants of raw Arabica coffee. Their changes during roasting. J. Agric. Food Chem. 48 (3), 868–872.
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