World J Microbiol Biotechnol (2013) 29:235–247
DOI 10.1007/s11274-012-1175-2
ORIGINAL PAPER
Evaluation of a potential starter culture for enhance quality
of coffee fermentation
Cristina Ferreira Silva • Danielle Marques Vilela •
Cecı́lia de Souza Cordeiro • Whasley Ferreira Duarte
Disney Ribeiro Dias • Rosane Freitas Schwan
Received: 12 June 2012 / Accepted: 18 September 2012 / Published online: 28 September 2012
Ó Springer Science+Business Media Dordrecht 2012
Abstract The coffee fermentation is characterized by the
presence of different microorganisms belonging to the
groups of bacteria, fungi and yeast. The objectives of this
work were to select pectinolytic microorganisms isolated
from coffee fermentations and evaluate their performance
on coffee pulp culture medium. The yeasts and bacteria
isolates were evaluated for their activity of polygalactu-
ronase (PG), pectin lyase (PL) and pectin methylesterase
(PME) and metabolites production. Among 127 yeasts
isolates and 189 bacterial isolates, 15 were pre-selected
based on their ability to produce PL and organic com-
pounds. These isolates were strains identified as Bacillus
cereus, Bacillus megaterium, Bacillus subtilis, Candida
parapsilosis, Pichia caribbica, Pichia guilliermondii and
Saccharomyces cerevisiae. When cultivated in Coffee peel
and pulp media in single culture or two by two mixed
inocula, different behavior concerning to PME, PL and PG
were found. The two principal components PC1 and PC2
accounted for 45.27 and 32.02 % of the total variance.
UFLA CN727 and UFLA CN731 strains were grouped in
the positive part of PC1 being characterized by 1,2-pro-
panediol, hexanoic acid, decanoic acid, nonanoic acid and
ethyl acetate. The UFLA CN448 and UFLA CN724 strains
were grouped in the negative part of PC1 and were mainly
characterized by guaiacol, butyric acid and citronellol.
S. cerevisiae UFLACN727, P. guilliermondii UFLACN731
C. F. Silva  C. de Souza Cordeiro  W. F. Duarte 
R. F. Schwan (&)
Department of Biology, Federal University of Lavras, Lavras,
MG CEP 37200-000, Brazil
e-mail: rschwan@dbi.ufla.br
D. M. Vilela  D. R. Dias
Department of Food Science, Federal University of Lavras,
Campus Universitário, Lavras, MG CEP 37200-000, Brazil
•
and C. parapsilosis UFLACN448 isolates are promising
candidates to be tested in future studies as coffee starter
cultures.
Keywords Pectinolytic enzymes  Pichia guilliermondii 
Saccharomyces cerevisiae  Organic acids 
Principal component analysis
Introduction
Coffee is one of the most appreciated non-alcoholic drinks,
and its consumption is distributed globally. It commands a
turnover close to US$10 billion per annum, which makes it
the second-most important commodity traded in world
markets, next to petroleum. Brazil is the leading producer
and exporter of Coffea arabica (ABIC 2010) followed
by Colombia, Paraguay, Venezuela, Indonesia, Ethiopia,
India, Mexico and 40 other countries. Coffee fruits are
processed after harvesting to allow spontaneous or indig-
enous fermentation to occur. This process may be either a
dry or wet process or a combination of both (a semi-dry
process). Arabica coffee (Coffea arabica) in Brazil and
Robusta coffee (Coffea canephora) in West Africa are
usually dry processed, which makes them ‘‘natural’’ cof-
fees. For dry processing, the fruits are harvested in several
stages of maturation (green, cherry, raisin and dry). The
coffee fruits remain intact after harvesting and are spread
out in thin layers (5–8 cm) on cement patios without being
washed to dry for up to 20 days. The pulp and mucilage
within the fruit ferment during this drying period (Silva
et al. 2000, 2008). Recent works point that coffee is sub-
strate favorable to the production of ochratoxin A (OTA)
especially when there is a microbiota already established
which can alter the substrate and favor the production of
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OTA by Aspergillus or Penicillium species (Mantle and
Chow 2000). Some yeast species isolated from coffee
fruits, can inhibit the growth of mycotoxigenic filamentous
fungi. Debaryomyces hansenii and Pichia anomala isolated
from dry processed coffee fruits were capable of inhibiting
or reducing sporulation and consequent toxin production
when co-cultivated in vitro with Aspergillus ochraceous,
Aspergillus parasiticus and Penicillium roqueforti (Ramos
et al. 2010).
In wet processing, the pulp is mechanically removed from
ripe fruits (called cherries). The fruits are then placed in fer-
mentation tanks and submerged in water for up to 48 h to
remove the mucilage. The fruits are then spread over patios or
dried mechanically. A large amount of water is used during the
pulp removal and in the fermentation tanks (Avallone et al.
2002; Masoud et al. 2004). Semi-dry processing is a variant
that combines a wet mechanical process to remove the pulp
and a dry process in which the depulped beans are spread in a
thin layer on cement patios to allow further aerobic degrada-
tion of the mucilage (Vilela et al. 2010).
The fermentation of coffee fruit is the processing step in
which the mucilage is degraded while the fruits are simul-
taneously dried to 11–12 % moisture. The length of time
required for fermentation differs among the three processing
methods. During fermentation, physicochemical changes
occur in grains, such as a reduction in water content and
simple sugars and the formation of aroma and flavour pre-
cursors (Vaast et al. 2006). The fermentation of coffee fruits
occurs naturally regardless of the processing method.
In all processing methods (dry, wet and semi-dry), the
objective of fermentation is to remove the mucilaginous
layer, which is rich in polysaccharides (pectins), and to
reduce the water content of the fruits. Coffee fruits contain
indigenous plant enzymes that degrade the mucilage layer,
but such activity is not sufficient for a complete and ade-
quate process (Agate and Bhat 1966). Fermentation allows
the growth of microorganisms that produce enzymes such
as polygalacturonases and pectin-lyases that are necessary
to depolymerise and hydrolyse the pectin present in the
mucilage (Agate and Bhat 1966; Masoud and Jespersen
2006). Such enzymes can be added during the fermentation
process, or they can be secreted by the fermentation
microorganisms (Kashyap et al. 2001; Jayani et al. 2005;
Uenojo and Pastore 2007). The removal of the mucilage by
microorganisms facilitates bean drying and produces
metabolites that diffuse into the interior of the coffee beans
and react with substances responsible for the flavour of the
final beverage. The microbiota present in coffee fruits is
dense and diverse and includes yeasts, filamentous fungi
and bacteria. The population of each microbial group
depends on the processing method and the extent of the
water loss on patios during the natural coffee drying pro-
cess (Silva et al. 2000, 2008; Sakiyama et al. 2001; Masoud
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World J Microbiol Biotechnol (2013) 29:235–247
et al. 2004; Masoud and Jespersen 2006; De Bruyne et al.
2007; Vilela et al. 2010).
During coffee fermentation, microorganisms are associ-
ated with the degradation of the pulp and mucilage of the
fruits, the production of pectinolytic enzymes, and the for-
mation of alcohols and acids, particularly acetic, lactic,
butyric and other long-chained carboxylic acids (Silva
2012). The main enzymes involved in the coffee fermenta-
tion is poligalacturonase (PG) which catalyses the hydrolysis
of a-1.4 glycosidic bonds into pectic acid (poligalacturonic
acid); pectin lyase (PL) which acts catalysing pectin break-
age by transelimination, releasing insaturated galacturonic
acids. The third enzyme is pectin methylesterase (PME)
responsible for the de-esterification of the methoxil group of
the pectin forming pectic acid and methanol.
Studies of the microbial diversity in coffee fruits are needed
to select microorganisms for starter cultures specific to dry and
wet fermentation processes. These microbial inocula could
enhance the organoleptic quality, reduce the processing time
and standardise the quality of aromatised coffee obtained from
particular coffee varieties. For coffee producers, the use of
starter cultures increases the beverage value without sub-
stantially increasing the cost. The selection of microorganisms
for coffee fermentation should be based on pectinase pro-
duction, and acidic and other metabolic compounds since
these factors interfere in the final beverage quality.
The principal component analysis (PCA) helps to identify
the important variables during the strain selection process.
PCA employs a mathematical procedure that transforms a set
of possibly correlated response variables into a new set of
non correlated variables called principal components (PCs).
Therefore the PCA can be used to reduce the number of
original variables to a few variables, by keeping only the
largest or most significant ones (Brereton 2000). This tool
has been used by different authors for a better understanding
of coffee characteristics as chemical composition, geo-
graphical origin and roasting methodology (Akiyama et al.
2003; Serra et al. 2005; Bicchi et al. 1997).
The objectives of this study were to isolate and to select
pectinolytic microorganisms from coffee fermentations and to
evaluate their performance in a coffee pulp culture medium.
Materials and methods
Microorganisms
Bacteria and yeast isolates from coffee fruit were obtained
from the Culture Collection of the Laboratory of Microbial
Physiology at DBI/UFLA, Lavras, Minas Gerais State,
Brazil. The microorganisms had been previously isolated
from coffee fruit (Coffea arabica L. var. Acaiá) during dry
and semi-dry fermentative processes (Silva et al. 2000,
World J Microbiol Biotechnol (2013) 29:235–247
2008; Vilela et al. 2010), and their potential for use as
culture starters in coffee fermentation was evaluated.
A total of 316 microorganisms including 127 yeast and 189
bacterial isolates were evaluated. Bacterial strains were
maintained on nutrient broth (3 % meat extract and 5 %
bacteriological peptone) and yeasts were maintained on
YPD (0.3 % yeast extract, 0.3 % malt extract, 0.5 % pep-
tone, 1 % glucose) at -80 °C.
Screening for pectinase activity
Pectinolytic activity was determined using the methods
described by Schwan et al. (1997) and Hanckin and Lacy
(1984). The yeasts and bacteria were grown on plates
containing mineral medium (The MP5 media contain glu-
cose 0.5 %; poligalacturonic acid 0.5; KH2PO4 0.6; yeast
extract 0.1; (NH4)2SO4 0.2; agar 1.5 and 0.1 mL of the
solutions FeSO4 0.0001 %; MgSO4 0.02; CaCl2 0.0001;
H3BO3 0.0002; MnSO4 0.0002; ZnSO47H2O 0.0014;
CuSO45H2O 0.001; MoO3 0.0002) plus polygalacturonic
acid (MP5) as substrate to determine the polygalacturonase
(PG) activity and mineral medium plus pectin as substrate
(MP7) to determine the pectin lyase activity (Hanckin and
Lacy 1984). The composition of the MP7 media is equal to
the MP5 media where the polygalacturonase acid was
replaced by pectin. Enzyme activity was indicated by the
formation of a clear halo around the colonies after pre-
cipitating polygalacturonic acid or pectin with 1 % cetyl
trimethyl ammonium bromide (CTAB). All reagents used
were purchased from Sigma–Aldrich and Merck.
Molecular identification of pectinolytic bacteria
and yeast
Bacteria and yeasts were identified by sequencing the 16S-
23S rDNA region for bacteria (16SF-R2: 50 -CGCGGGA
TCCTTGTACACACCGCCCGTC-30 ; 16S rDNA gene
9–25: 50 -GGCCGTCGACCCTTTCCCTCACGGTACTG-30 )
(Lechner et al. 1998) and the ITS1 -5.8S intergenic region of
the rDNA for yeasts (ITS1: 50 -CGTAGGTGAACCTG
CGG-30 ; ITS4: 50 -TCCTCCGCTTATTGATATGC-30 )
(Masoud et al. 2004). Purified PCR products were sequenced
using an ABI3730 XL automatic DNA sequencer, and
sequences were then compared to those available in the
GenBank database using the BLAST algorithm (National
Centre for Biotechnology Information, Maryland, USA).
Cultivation of strains
Fifteen isolates including bacteria and yeasts were culti-
vated in two different media (SPM and CPM) to analyse
the PG, PL and PME activity.
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Pectin medium (SPM)
Pectinase-producing bacteria and yeasts were grown in YPD
medium for 48 h at 28 °C. Flasks containing 300 mL of SPM
(synthetic pectin medium, containing 0.1 % glucose,
10 % citric pectin, 1.0 % KH2PO4, 0.5 % MgSO4.7H2O,
0.5 % CaCl2, 1.0 % (NH4)2SO4) were inoculated with
1.0 mg dry wt mL-1 equivalent of organisms from a 24 h
starter culture grown in YPD medium. The size of inoculum
was 104 cells mL-1. Cultures were incubated at 28 °C for 96 h
at 120 rpm and periodically sampled for PG, PL and PME
activity. Growth (dry wt mL-1) was determined from optical
density (OD600) measurements against an appropriate cali-
bration curve.
Coffee peel and pulp media (CPM)
The fifteen best enzyme producers were cultivated sepa-
rately and in pairs in CPM medium with modifications. The
preparation of inoculum was done as described before
(in SPM media).The pectinases were determined on the
following culture media: (a) CPM (4 % coffee
pulp ? mucilage) ? glucose (0.5 %), (b) CPM (4 %), and
(c) CPM ? minerals (K, P, Na, Mg, S, Ca). For preparation
of CPM medium, 40 g of the pulp and coffee peel was
boiled for 10 min and after filtered. The volume was
adjusted to 1L. When necessary, 0.13 % KH2PO4, 0.012 %
NA2HPO4, 0.05 % MgSO47H2O and 0.03 CaCl2 was
added. The same lot of coffee peel and pulp was used in all
experiments aiming to show no difference in the chemical
composition. Each culture was grown for 96 h at 28 °C and
120 rpm. Samples were taken every 12 h for microbio-
logical analysis; pH measurement; determination of PL, PG
and PME activity; and total protein and metabolites pro-
duction. After the samples were centrifuged at 4,800 rpm
for 10 min at 4 °C, the supernatant was collected to
determine the enzyme activity.
Enzyme assays
Pectin lyase (PL) activity (EC 4.2.2.10)
Pectin lyase activity in the cultures was determined spec-
trophotometrically using the method of Pitt (1988) as
modified by Kashyap et al. (2000). The reaction mixture
consisted of 5 mL of 1.0 % (w v-1) citrus pectin (85 %
esterified) in 0.5 M Tris–HCL, pH 6.8, and 1.0 mL of
culture supernatant. The reaction mixture was incubated for
2 h at 40 °C. The reaction was stopped by the addition of
0.6 mL of 9 % zinc sulphate and 0.6 mL of 0.5 M sodium
hydroxide. The mixture was centrifuged (6,000g, 5 min),
and 5 mL of supernatant was collected and mixed with
3 mL of 0.04 M thiobarbituric acid, 2.5 mL of 0.1 M HCl
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and 0.5 mL of distilled water. This mixture was boiled for
30 min then cooled to room temperature before reading the
absorbance at 550 nm. One unit of enzyme activity
(U mL-1) of pectin lyase was defined as the amount of
enzyme that increased the absorbance by 0.01 units.
Polygalacturonase activity (E.C. 3.2.1.67)
Polygalacturonase activity was assayed by measuring the
increase in reducing groups using the method described by
Schwan et al. (1997). The reaction mixture consisted of
1.0 mL of polygalacturonic acid 0.1 % (w v-1) in 0.1 M
citrate buffer, pH 5.0, and 1.5 mL of culture supernatant, and
it was incubated for 60 min at 40 °C. The reaction was
stopped by the addition of 1.5 mL of DNS (Miller 1959). The
mixture was boiled for 5 min and then cooled in an ice bath.
Absorbance was read at 600 nm, with the optical density
(OD600) determined using an appropriate calibration curve.
One unit of enzyme activity (U mL-1) was expressed as
1 lmol of galacturonic acid released mL-1 min-1.
Pectin methylesterase activity (E.C. 3.1.1.11)
Pectin methylesterase was assayed using the methodology
proposed by Baracat et al. (1989) by the continuous titrimetric
determination of the carboxyl groups liberated from methyl-
ester bonds. The reaction was carried out with three mL of the
enzymatic solution, to which 20 mL of 1 % Sigma citric pectin
in NaCl 0.1 M pH 7.5 solution was added and the mixture was
incubated for 30 min keeping the pH at 7.5 by the addition of
NaOH 0.02 M. PME activity was expressed as the micro-
equivalents of polygalacturonic acid produced mL-1 h-1.
Total protein
Total protein was determined using the method of Bradford
(1976), with bovine serum albumin (BSA) as the standard.
Chemical analysis
Coffee pulp and husks were physically and chemically
analysed by the determination of moisture, acidity, total
sugars, reducing and non-reducing sugars (AOAC 2000),
total and soluble pectin (Bitter and Muir 1962), acid
detergent fibre (ADF) (Mazzafera and Robinson 2000),
lignin and cellulose (van Soest and Wine 1968).
Alcohol, organic acids, carbohydrates and volatile
compounds
Ethanol, glycerol, organic acids (acetic acid, malic acid and
succinic acid) and carbohydrates (glucose, sucrose and
fructose) were quantified by high-performance liquid
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World J Microbiol Biotechnol (2013) 29:235–247
chromatography (HPLC) using a Shimadzu chromato-
graph, model LC-10Ai (Shimadzu Corp., Japan), equipped
with a dual detection system consisting of a UV detector
and a refractive index detector (RID—10A SPD-10Ai).
A Shimadzu ion exclusion column (Shim-pack SCR-101H,
7.9 mm 9 30 cm) was operated at a temperature of 30 °C
using 100 mM perchloric acid as the eluent at a flow rate of
0.6 mL-1. Sugars, glycerol and ethanol were detected by
RID. Acids were detected via UV absorbance (210 nm).
Compounds were identified by the comparison of their
retention times with the retention times of certified stan-
dards. Alcohols, sugars and acids were quantified using
calibration curves obtained from standard compounds.
Duplicate tests were run on all samples (Duarte et al.
2010).
Volatile compounds were analysed using a gas chro-
matograph (GC) Shimadzu model 17A, equipped with an
FID (flame ionisation detector) and a capillary column of
silica DB Wax (30 m 9 0.25 mm i.d. 9 0.25 lm) (J&W
Scientific). The oven temperature was maintained at 50 °C
for 5 min, raised to 190 °C by increments of 3 °C min-1
and then kept at 190 °C for 10 min. The injector and
detector temperatures were kept at 240 °C, and the carrier
gas (N2) was kept at a flow rate of 1.2 mL min-1. Injec-
tions of 1 lL were made in the split mode (1:10). The
volatile compounds were identified by comparing the
retention times of the samples with those of standard
compounds injected in the same conditions. The quantifi-
cation of volatile compounds was expressed as 4-nonanol
equivalent used at a final concentration of 126 mg L-1
(Duarte et al. 2010).
Statistical analysis
The data were analysed in a completely randomised design
with three replicates. A Tukey0 s test was performed using
SISVARÒ software version 5.0. Data of microbial metab-
olites were statistically analysed (principal component
analysis, PCA) using XLSTAT 7.5.2 (Addinsoft, New
York, NY, USA) software.
Results and discussion
Screening of pectinases production of bacteria
and yeast isolates
Bacteria and yeast isolates from dry and semi-dry coffee
processing were tested for their ability to secrete PG and
PL in semi-quantitative tests. After the sequencing, the
isolates were identified as Bacillus cereus UFLACN 445
and 446 (99 % similarity), B. megaterium UFLACN415
(99 % similarity), B. subtilis UFLACN406, 452 and 463
World J Microbiol Biotechnol (2013) 29:235–247
(99 % similarity), Candida parapsilosis UFLACN 426,
431, 448, 449 and 730 (99 % similarity), Pichia caribbica
UFLACN706, P. guilliermondii UFLACN731 (99 % sim-
ilarity) and S. cerevisiae UFLACN724 and 727 (99 %
similarity). The majority of positive strains were isolated
from dry processing, probably because in dry processing
the coffee fruit is fermented with the skin, pulp and
mucilage present, which favours microbial species that are
able to use pectin as a carbon and energy source.
Fifteen isolates were selected for enzyme production in
SPM and CPM (plus glucose) media because they had
halos wider than 30 mm diameter in the semi-quantitative
test. These species have been reported to be able to secrete
pectinases (Miyazaki 1991; Blanco et al. 1999; Corredig
et al. 2000; Singh et al. 1999; Takao et al. 2000; Kobayashi
et al. 2001). Pichia and Debaryomyces strains from coffee
fermentation have been reported to be pectinolytic and able
to inhibit ochratoxigenic filamentous fungi growth during
fermentative processes (Masoud and Kaltoft 2006; Masoud
and Jespersen 2006; Ramos et al. 2010). There is no
information in the literature about pectin lyase activity in
S. cerevisiae during coffee fermentation. This work is thus
the first to show pectin lyase activity in S. cerevisiae iso-
lated from coffee fermentation.
PL, PME and exo-PG secreted by yeasts and bacteria
in SPM and CPM culture media
The composition of coffee pulp and skin given in Table 1
was used to formulate a defined coffee culture medium
for pectinase production. Bacillus, Pichia, Debaryomyces,
Candida and Saccharomyces species were cultured in SPM
and CPM media for pectinase (PG, PL and PME) pro-
duction. Only 3 isolates (B. subtilis UFLACN406, B.
megaterium UFLACN415 and P. caribbica UFLACN706)
showed PG activity. Eleven isolates showed equal or
higher PL activity in SPM than in CPM medium and only 1
isolate (C. parapsilosis UFLACN449) showed higher PME
activity in CPM medium than SPM medium. This is
important because low PME activity does not release
methanol (Table 2).
Pectinase activity from yeast in single and mixed
culture
Yeasts isolates showed higher PL production (average of
60 and 53 % in SPM and CPM media, respectively)
(Table 2) compared to bacterial production in single cul-
ture. The monosaccharides in the pulp can be used to
induce yeast pectinase production (Masoud and Jespersen
2006), leading to greater activity. Higher production of
pectinases is usually associated with bacteria and fila-
mentous fungi because of the role of these enzymes in the
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Table 1 Physicochemical characteristics of pulp coffee (Coffea
arabica L.) for formulation media CPM in dry matter
Dry matter 20.25 %
Analysis
Protein 15.50
Ash 8.0
Acid detergent fiber 28.50
Fat 2.80
Total pectin 5.50
Lignin 12.0
Cellulose 16.20
Total sugars 11.50
infection process by plant pathogens (Blanco et al. 1999).
Of all isolates tested, four yeasts that showed higher pec-
tinase activity in CPM media were selected for fermenta-
tion on coffee pulp because this substrate is more similar to
coffee fruit composition. These isolates also produced
metabolites that are important for coffee quality.
The isolates S. cerevisiae UFLACN 724, S. cerevisiae
UFLACN 727, P. guilliermondii UFLACN 731 and
C. parapsilosis UFLACN 448 showed the highest PL
activity in CPM (2,637.00, 2,557.51, 2,344.86 and
2,636.76 U mL-1, respectively) (Table 2). S. cerevisiae
UFLACN 724 and UFLACN 727 showed PL activity in
SPM medium 50 % higher than the PL activity in CPM
culture medium. Several factors could explain the higher
activity in SPM: first, pectin may be inaccessible to
microorganisms because it is linked to cellulosic material
in the peel and pulp of the coffee fruit, and second, glucose
catabolism may be repressed.
Some isolates (UFLACN 727, UFLACN 730 and
UFLACN 731) showed increased enzyme production in the
presence of glucose compared to medium without glucose.
However, the increase in enzyme production did not result
in maximum enzyme activity. Schwan et al. (1997)
reported that pectinase from Kluyveromyces marxianus was
not repressed by carbohydrates, and Masoud and Jespersen
(2006) found that the synthesis of pectinase (PG) by yeast
requires the presence of monosaccharides. A third factor
that may have influenced the lower enzymatic activity in
CPM may have been the lack of a nitrogen source, which
was used to simulate the lack of nitrogenous compounds in
coffee fruit peel and pulp. Schwan and Rose (1994)
observed that an increase in inorganic ammonium sulphate
concentration did not influence the production of pectinase
in K. marxianus.
The PL activity from a mixed culture of yeast was, on
average, 20 times less than the activity found with a single
culture (Table 3). The maximum activity was obtained
with pure cultures of P. guilliermondii UFLACN 731 and
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Table 2 Production of pectin lyase (PL), pectin methylesterase (PME) and exo-polygalacturonase (Exo-PG) and pH value for different microorganisms of coffee (Coffea arabica L.) in
medium containing coffee pulp (CPM) and synthetic pectin (SPM) of single cell

123
Microorganisms pH Enzymatic activity (U mL-1)
PL PME Exo-PG
CPM SPM CPM SPM CPM SPM

Bacteria
B. cereus UFLACN445 4.3 766.24 ± 19.333 bE 1,582.32 ± 103.751 aF 789.33 ± 12.378 aE 830.83 ± 20.314 aF nd nd
4 3 4
B. cereus UFLACN446 4.4 1,405.25 ± 55.81 aC 1,485.83 ± 55.8 aF 809.13 ± 15.01 bE 2,547.68 – 102.628 aA nd nd
B. subtilis UFLACN406 4.1 1,313.38 ± 60.966 bD 1,733.62 ± 60.576 aF 2,202.63 ± 154.866 aA 2,284.06 ± 29.656 aB 84 ± 06 d 58 ± 03 d
B. megaterium UFLACN415 4.4 1,609.36 ± 90.611 aC 1,322.27 ± 23.486 bG 1,119.93 ± 27.202 bD 2,147.04 ± 25.554 aB 51 ± 01 d 37 ± 01 d
4 1 4 4
B. subtilis UFLACN452 4.0 1,177.65 ± 73.0 aD 1,221.53 ± 46.31 aG 1,940.07 ± 19.98 aA 2,034.39 ± 29.61 aB nd nd
B. subtilis UFLACN463 4 1,088.67 ± 22.794 aD 1,527.09 ± 101.513 aF 925.72 ± 22.733 bE 1,193.35 ± 38.011 aE nd nd
Yeast
C. parapsilosis UFLACN449 4.8 1,242.9 ± 28.708 aD 762.6 ± 11.253 bH 1,687.74 – 21.078 aC 690.38 ± 11.698 bF nd nd
6 3 7
C. parapsilosis UFLACN448 5.0 2,636.76 – 44.91 aA 2,580.68 – 50.36 aD 1,859.73 ± 23.71 bB 2,657.33 – 147.507 aA nd nd
C. parapsilosis UFLACN426 5.0 1,161.48 ± 16.572 bD 3,481.70 – 215.431 aC 1,129.11 ± 14.092 bD 1,739.93 ± 40.723 aC nd nd
1 1 8
C. parapsilosis UFLACN730 6.7 2,180.23 ± 161.25 aB 2,177.07 ± 71.74 aE 1,197.73 ± 10.66 aD 1,129.11 ± 14.092 aE nd nd
C. parapsilosis UFLACN431 5.0 1,711.76 ± 35.441 aC 1,399.47 ± 12.523 bG 1,118.25 ± 26.441 bD 1,755.68 ± 22.311 aC nd nd
1 1 1 1 1
P. caribbica UFLACN706 5.6 1,919.65 ± 20.74 aB 1,761.01 ± 23.23 aF 735.79 ± 21.87 bE 2,524.32 ± 76.0 aA 34 ± 0 d 60 ± 01 d
1 2 2 2
P. guilliermondii UFLACN731 6.1 2,344.86 – 51.23 bA 2,809.45 – 147.29 aD 1,208.83 ± 25.46 bD 1,508.54 ± 22.62 aD nd nd
S. cerevisiae UFLACN727 5.2 2,637.00 – 143.172 bA 4,961.07 – 175.301 aB 1,712.83 ± 40.631 bC 2,532.30 – 41.601 aA nd nd
S. cerevisiae UFLACN724 5.2 2,557.51 – 100.381 bA 5,256.12 – 161.611 aA 2,600.20 ± 96.171 aA 2,443.14 ± 89.281 aA nd nd
Combination of yeast isolates
UFLACN 448 and 724 5.1 89.80 ± 26.954 nD nD nD nD nD
UFLACN 448 and 727 5.2 111.69 ± 28.394 nD nD nD nD nD
UFLACN 448 and 731 5.2 136.03 ± 5.934 nD nD nD nD nD
UFLACN 727 and 724 5.0 107.48 ± 2.824 nD nD nD nD nD
UFLACN 727 and 731 5.1 119.21 ± 5.434 nD nD nD nD nD
UFLACN 731 and 724 5.1 139.83 ± 6.004 nD nD nD nD nD
Enzymatic activity (PL) from yeast isolates in mixed cultures using CPM media. Numbers in bold indicate the highest enzyme activity. Initial pH value was 5.6
Lower case letters indicate comparison of enzymatic activity (lines). Capital letters indicate comparison of different enzymes produced by isolates (columns)
nd not detected, nD not determined
Cultivation time: 112 h, 224 h, 336 h, 448 h, 560 h, 672 h, 784 h, 896 h
Means within different letters are statistically different at 95 % confidence level
World J Microbiol Biotechnol (2013) 29:235–247
World J Microbiol Biotechnol (2013) 29:235–247
S. cerevisiae UFLACN 724 (139.83 U mL-1). The low
enzyme activity in mixed culture could be due to compe-
tition for nutrients, preventing the isolates from reaching
the same activity levels as those found in a single culture.
Ciani et al. (2006) reported similar results in analysing
single and mixed cultures of Saccharomyces and non-
Saccharomyces strains for wine production. The advantage
of using single cultures is the possibility of greater control
and prediction of metabolic activities during cultivation
(Holzapfel 2002). The coffee fruits already present an
epiphytic natural microbiota of 104–107 UFC g-1. With
the inoculation of 2 or more species with populational
density higher than 107 UFC g-1 there would be a super-
position to the species naturally present. However, a
competition would be established between the inoculated
species compromising the control of the standardization of
the fermentative process.
Of the isolates, S. cerevisiae UFLACN 724, S. cerevi-
siae UFLACN 727, P. guilliermondii UFLACN 731 and
C. parapsilosis UFLACN 448 showed the highest PL
activity using CPM medium. Only the isolate S. cerevisiae
UFLACN724 presented higher PME activity in CPM than
in SPM medium (2,600.2 U mL-1) (Table 2). The metha-
nol released as a result of the PME activity was below the
detection level (6 mg L-1).
Pichia caribbica UFLACN 706 was the only yeast
isolate that produced exo PG in both CPM and SPM culture
media (34.0 and 60.0 U mL-1, respectively). Yeasts are
known to produce PG; however, it appears that coffee pulp
is not the ideal substrate for PG production. PG acts to
depolymerise pectin containing lower degrees of methyla-
tion than the pectin found in coffee pulp (over 70 %
esterified). The great majority of PG acts at low pH values
(5.0), but PG activity at neutral to basic pH (pH 7–11) has
been reported for Bacillus (Jayani et al. 2005). Thus, the
low yeasts PG activity in coffee pulp could be due to high
methylation of pulp, and not inactivation nor absence of
PG production. Most yeasts produce PG constitutively
(Blanco et al. 1999).
Pectinases activity from bacteria in single culture
Bacillus megaterium UFLACN 415 and B. cereus
UFLACN 446 showed the highest PL activity in CPM
medium after 12 h (1,405.25 U mL-1) and 48 h
(1,609.36 U mL-1) (Table 2). The PL activity was similar
in both culture media. B. subtilis UFLACN 406 and
B. megaterium UFLACN 415 were able to secrete PG;
however, the PG activity was 15 times lower than the PL
activity for these isolates. The high methylation of coffee
pectin seemed to inhibit the PG activity. In addition, the
glucose concentration in CPM medium could have inhib-
ited the enzyme activity, as reported by Henick-Kling et al.
241
(1998). The pectinase activity of all bacteria isolates was
an average of 1.7 times lower than the enzymes activity of
the yeast isolates.
Selection of starter culture in CPM medium
The criteria used to select microorganisms as a starter
culture for coffee fermentation were based on the pectin
lyase secretion, the physical and chemical changes, the
production of volatile compounds and the lack of produc-
tion of undesirable metabolites that could negatively affect
the sensory or health qualities of the final product.
The fifteen bacteria and yeasts that showed PL halo
production \30 mm diameter were evaluated for the con-
sumption of carbohydrates and the production of organic
acids during growth in CPM medium (Table 3).
Compared to the initial glucose concentration in CPM,
there was a remarkable increase in glucose concentration
(from 6.72 to 10.33 g L-1) (Table 3) after the cultivation
of B. cereus UFLACN 445, B. cereus UFLACN 446,
B. subtilis UFLACN 452 and B. subtilis UFLACN 463.
This increase may have been due to the breakage of the
pectin releasing glucose in the media. According to Garna
et al. (2004), the hydrolysis of pectin results in the release
of sugars, such as glucose, rhamnose and galactose.
Acetic and succinic acids were the most abundant acids
found in this study. The formation of alcohols (especially
ethanol) and acids, such as acetic, lactic, butyric and other
long-chained carboxylic acids, occurs during the natural
coffee fermentation process (Silva 2012). Acids that are
products of over-fermentation, such as butyric, acetic and
propionic acids, can damage the final quality of the bev-
erage by conferring an oniony flavour when present at
concentrations higher than 1 mg mL-1 (Lopez et al. 1989).
By contrast, malic and citric acids at concentrations
between 1 and 4 mg mL-1 confer a desirable acidity to the
product (Sivetz 1963; Jham et al. 2002). The microbial
activity might be detected in the final coffee aroma and
flavour if these acids diffused from pulp to bean and
remained after roasting. The highest concentrations of
acetic and succinic acids were produced by B. cereus
UFLACN 445 (0.29 and 0.65 g L-1, respectively) and
B. subtilis UFLACN 452 (0.43 and 0.45 g L-1, respec-
tively) (Table 3). Higher concentrations of acetic (0.05 and
1.5 g L-1) and succinic (0.06 and 0.9 g L-1) acids were
found by Rodrigues et al. (2007) in brewed coffee. Acetic
acid production is an aerobic metabolic process that can be
of bacterial origin or that can arise from the oxidation of
yeast-produced alcohol. The bacteria present on the surface
of the coffee cherries synthesise the acid that can then
migrate to the pulp and mucilage and interfere with the
organoleptic quality of the beans (Schwan et al. 2012).
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242 World J Microbiol Biotechnol (2013) 29:235–247

Table 3 Organic compounds obtained after fermentation from CPM (96 h of fermentation) with the fifteen different isolates selected
Microorganisms Compounds (g L-1)
Glucose Fructose Glycerol Ethanol Acetic acid Malic acid Succinic Methanol
acid

Bacteria
B. cereus UFLACN445 7.89 ± 0.31 0.48 ± 0.01 0.33 ± 0.0 0.30 ± 0.01 0.29 ± 0.01 ND 0.65 ± 0.05 ND
B. cereus UFLACN446 6.72 ± 0.47 0.06 ± 0.0 0.32 ± 0.0 0.26 ± 0.02 0.24 ± 0.0 ND 0.64 ± 0.01 ND
B. subtilis UFLACN406 1.24 ± 0.04 0.11 ± 0.0 0.27 ± 0.02 1.30 ± 0.1 0.05 ± 0.0 ND 0.50 ± 0.0 ND
B. megaterium 0.06 ± 0.0 ND 0.20 ± 0.01 1.55 ± 0.04 0.05 ± 0.0 ND 0.45 ± 0.0 ND
UFLACN415
B. subtilis UFLACN452 10.33 ± 0.74 0.50 ± 0.08 0.51 ± 0.02 0.44 ± 0.0 0.43 ± 0.02 ND 0.95 ± 0.02 ND
B. subtilis UFLACN463 7.53 ± 0.21 0.64 ± 0.1 0.38 ± 0.0 0.32 ± 0.0 0.24 ± 0.0 ND 0.69 ± 0.0 ND
Yeast
C. parapsilosis 0.04 ± 0.0 ND 0.11 ± 0.0 0.64 ± 0.05 ND ND 0.33 ± 0.0 ND
UFLACN449
C. parapsilosis ND ND 0.07 ± 0.0 0.93 ± 0..8 ND ND 0.24 ± 0.0 ND
UFLACN448
C. parapsilosis 0.02 ± 0.0 ND 0.10 ± 0.01 0.69 ± 0.0 ND ND 0.36 ± 0.0 ND
UFLACN426
C. parapsilosis ND ND ND 0.86 ± 0.1 ND ND 0.07 ± 0.0 ND
UFLACN730
C. parapsilosis ND ND 0.08 ± 0.0 1.19 ± 0.09 ND ND ND ND
UFLACN431
P. caribbica UFLACN706 ND ND 0.05 ± 0.0 0.88 ± 0.0 0.02 ± 0.0 ND 0.34 ± 0.0 ND
P. guilliermondii 0.06 ± 0.0 0.02 ± 0.0 0.05 ± 0.0 1.06 ± 0.03 ND 0.03± 0.33 ± 0.0 ND
UFLACN731
S. cerevisiae UFLACN727 0.05 ± 0.0 0.03 ± 0.0 0.08 ± 0.0 0.72 ± 0.0 ND 0.04 ± 0.0 0.22 ± 0.0 ND
S. cerevisiae UFLACN724 0.05 ± 0.0 0.05 ± 0.0 0.07 ± 0.0 0.90 ± 0.1 ND 0.04 ± 0.0 0.21 ± 0.0 ND
MCP media (Time 0) 5.1 ± 0.1 0.02 ± 0.0 ND ND 0.14± ND 0.18 ± 0.0 ND
ND non-detectable

Malic acid was only detected when P. guilliermondii
UFLACN731, S. cerevisiae UFLACN 724 and S. cerevisi-
ae UFLACN 727 were used to ferment CPM. Since malic
acid contains two carboxylic acid groups, it releases more
protons to the solution, thereby increasing the acidity
(Duarte et al. 2010). Ethanol was detected in all samples at
concentrations ranging from 0.64 to 1.19 g L-1. Ethanol
gives a sweet aroma to the final beverage; however, ethanol
and some organic acids that are not generated by thermal
reactions decrease after roasting (Gonzalez-Rios et al.
2007).
A principal component analysis was applied to the data
obtained from the HPLC analysis. The first and second
principal components (PC1 and PC2) accounted for 80.48 %
of the total variance (Fig. 1). All yeasts were grouped on the
negative part of the PC1 (62.90 % of the total variance).
Except for the isolates B. subtilis UFLACN 406 and
B. megaterium UFLACN 415, the bacteria isolates were
present in the positive part of the PC1, showing the different
fermentative behaviour profiles between yeast and bacterial
isolates. In the upper left quadrant (Fig. 1) of the PCA, the
isolates S. cerevisiae (UFLACN 724 and UFLACN 727),
P. guilliermondii UFLACN 731, C. parapsilosis (UFLACN
448, UFLACN 426) and P. caribbica UFLACN 706 were
characterised by the presence of malic acid. Isolates of
C. parapsilosis (UFLACN 730 and UFLACN 431) in the left
lower quadrant were characterised by the presence of etha-
nol. B. cereus (UFLACN 445 and UFLACN 446) and
B. subtilis (UFLACN 452A and UFLACN 463) were char-
acterised by the presence of residual sugars (glucose and
fructose), acids (succinic and acetic) and glycerol.
Forty-one volatile compounds were found in fermented
CPM medium (Table 4). Among them, only seven com-
pounds were found in all four fermented CPM media, namely,
acetaldehyde, acetoin, furfural, decyl aldehyde, isobutyric
acid, butyric acid and 2-phenylethanol (Table 4). The volatile
composition results showed that the evaluated yeasts can
affect the final product in different ways. The highest con-
centration of acetaldehyde, 0.82 mg L-1, was measured when
S. cerevisiae UFLA CN 727 was used to ferment CPM

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World J Microbiol Biotechnol (2013) 29:235–247
Fig. 1 Principal component
analysis (PCA) of organic
compounds found during
fermentation in CPM media
by the fifteen selected isolates
medium. The CPM medium fermented by C. parapsilosis
UFLA CN 448 showed the highest concentrations of acetoin
(4.18 mg L-1), furfural (4.85 mg L-1) and butyric acid
(4.20 mg L-1), while CPM media fermented by S. cerevisiae
UFLA CN 724 and by P. guilliermondii UFLA CN 731
showed the highest concentrations of isobutyric acid
(0.59 mg L-1) and 2-phenylethanol (1.81 mg L-1), respec-
tively. The volatile compounds identified in the fermented
CPM media belong to the classes of the most significant
contributors to coffee aroma listed by Buffo and Cardelli-
Freire (2004). As shown in Fig. 2, the principal component
analysis (PCA) allowed the differentiation between the four
fermented products using the different yeast strains. The two
principal components PC1 and PC2 accounted for 45.27 and
32.02 % of the total variance, respectively (Fig. 2a). In the
positive part of PC1, the UFLA CN 727 and UFLA CN 731
strains, which were characterised by 1,2-propanediol, hexa-
noic acid, decanoic acid, nonanoic acid and ethyl acetate, were
grouped. The UFLA CN448 and UFLA CN724 strains were
grouped in the negative part of PC1 and were mainly char-
acterised by guaiacol, butyric acid and citronellol (Fig. 2b).
On the basis of the higher PL activity in culture medium
containing coffee pulp and husks, the ability to hydrolyse the
pectin present in coffee and the presence of desirable meta-
bolic products, including malic acid, ethanol, 2-phenyletha-
nol, esters and terpene compounds, the isolates S. cerevisiae
243
UFLACN724, S. cerevisiae UFLACN727, P. guilliermondii
UFLACN731 and C. parapsilosis UFLACN448 were selec-
ted for fermentation in CPM medium.
The fermentation in CPM medium lasted 96 h. The
S. cerevisiae UFLACN724, S. cerevisiae UFLACN 727 and
P. guilliermondii UFLACN731 yeasts showed peaks in PL
activity after 12 and 24 h (Fig. 3). As expected, enzyme
activity was coincident with high cell viability (Fig. 3a–c).
During wet and semi-dry processes, pectin-rich mucilage
becomes more available to be attacked by pectinolytic
enzymes (Masoud et al. 2004; Masoud and Jespersen 2006;
Vilela et al. 2010). The decrease in enzyme activity after 24 h
might be due to inhibitory compounds produced during the
fermentation process.
Candida parapsilosis UFLACN448 showed increasing
enzyme activity during fermentation, reaching a peak after
72 h (2,636.76 U mL-1) (Fig. 3d). The PL secreted by this
isolate showed high stability in relation to the fermentation
time and variation of pH values (5.6–4.5). These findings are
important for both quality and rate of dry fermentation of
coffee, since the natural process might last up to 15 days
(Silva et al. 2000, 2008; Schwan and Wheals 2003).
Our data show that not all species of bacteria have high
PL activity, and thus, not all species would enhance pectin
hydrolysis at the beginning of the fermentation. The bac-
teria tested showed high residual sugar and acetic acid
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244 World J Microbiol Biotechnol (2013) 29:235–247

Table 4 Volatile compounds

Compounds (mg mL-1) Yeast
analysed by GC- FID of four
yeasts grown in CPM media UFLA CN727 UFLA CN724 UFLA CN731 UFLA CN448

Acetaldehyde 0.82 0.22 0.46 0.26

Butyraldehyde nd 0.17 nd nd
Ethyl acetate 0.33 nd nd nd
Methanol 0.45 nd 1.09 nd
Propyl acetate 169.37 nd nd nd
2-Methyl-1-butanol 0.26 nd 3.15 1.59
3-Methyl-1-butanol 0.58 nd nd nd
1-Pentanol nd nd nd 0.75
Acetoin 0.27 0.66 1.32 4.18
2-Heptanol nd nd nd 6.51
Ethyl lactate 1.32 0.36 2.25 nd
1-Hexanol 0.33 nd nd nd
Trans-3-hexen-1-ol 0.41 nd nd nd
2-Nonanone 0.36 nd 0.31 nd
Verbenone 0.38 nd nd nd
Furfural 3.18 2.93 2.88 4.85
Decyl aldehyde 1.20 0.77 0.69 1.19
Propionic acid 0.36 nd nd nd
Linalool 0.33 nd 0.38 nd
Isobutyric acid 0.45 0.59 0.57 0.39
1,2-Propanediol nd 0.26 0.59 nd
Butyric acid 1.39 1.65 1.09 4.20
Furfuryl alcohol 1.04 nd 0.63 1.06
Diethylsuccinate 0.27 nd 0.46 nd
a-Terpeniol 0.43 nd nd nd
1,3-Butanediol 0.63 nd 0.40 nd
b-Citronellol 0.91 1.35 nd 1.42
Phenylethyl acetate 0.82 nd nd nd
Hexanoic acid 068 0.44 1.03 nd
Geraniol 0,33 nd nd nd
Guaiacol nd 0.16 nd nd
2-Phenylethanol 0.24 0.24 1.81 0.52
2-Ethyl caproic acid 0.86 nd nd 2.95
Heptanoic acid 086 0.73 nd nd
Menthol nd nd nd 5.50
Diethyl malate nd nd 1.03 nd
Octanoic acid 0.34 1.03 nd 0.90
Nonanoic acid 5.60 0.49 nd 1.06
Decanoic acid 1.54 0.62 1.13 nd
Mono ethyl succinate 0.26 0.40 nd nd
Benzoic acid 0.36 nd 2.37 2.44

concentrations when cultivated in a coffee medium. More- fermentative ability of S. cerevisiae UFLACN727, P. guil-
over, specific strains of yeasts showed good potential to be used liermondii UFLACN731 and C. parapsilosis UFLACN448
as pure starter cultures to speed up and enhance the quality of a indicated that these strains should be tested in future studies as
dry fermentation process. In addition, the analysis of the coffee starter cultures.

123
World J Microbiol Biotechnol (2013) 29:235–247 245

Fig. 2 Principal component analysis (PCA) of volatile compounds identified by GC-FID in CPM fermented by four different isolates

Fig. 3 Pectin lyase activity, cellular viability and pH during the fermentation in CPM media for isolates S. cerevisiae UFLACN724
a, UFLACN727 b, P. guilliermondii UFLACN731 c and C. parapsilosis UFLACN448 d. Bars indicate standard deviation of the mean

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