microorganisms

Article
Isolation, Identification, and Characterization of
Pectinolytic Yeasts for Starter Culture in
Coffee Fermentation
Mesfin Haile and Won Hee Kang *
Department of Horticulture, Kangwon National University, Chuncheon 24341, Korea; mesfinhaile97@gmail.com
* Correspondence: whkang@kangwon.ac.kr




Received: 26 August 2019; Accepted: 27 September 2019; Published: 28 September 2019


Abstract: This experiment was carried out to identify and select pectinolytic yeasts that have potential
use as a starter culture for coffee fermentation during wet processing. The coffee fruit was fermented
for 48 h at 28 ◦ C and a sample was taken from the fermented solution and spread onto yeast
extract-peptone-dextrose agar (YPDA) media and incubated at 28 ◦ C. A total of 28 yeasts were
isolated, eight of which had the ability to produce pectinase enzymes. The species of those eight
yeasts were molecularly identified and confirmed. These yeasts are Wickerhamomyces anomalus (strain
KNU18Y3), Saccharomycopsis fibuligera (strain KNU18Y4), Papiliotrema flavescens (strain KNU18Y5
and KNU18Y6), Pichia kudriavzevii (strain KNU18Y7 and KNU18Y8), and Saccharomyces cerevisiae
(strain KNU18Y12 and KNU18Y13). The pectin degradation index of S. fibuligera (strain KNU18Y4),
W. anomalus (strain KNU18Y3), and P. flavescens (strain KNU18Y6) were higher compared to the others,
at 178%, 160%, and 152%, respectively. The pectinase enzyme assays were made on two growth
media: coffee pulp media (CPM) and synthetic pectin media (SPM). S. fibuligera (strain KNU18Y4)
and W. anomalus (strain KNU18Y3) had great potential in producing polygalacturonase (PG) and
pectin lyase (PL) compared to others in both media. However, S. cerevisiae strains (KNU18Y12 and
KNU18Y13) produced higher pectin methylesterase (PME). Using MEGA 6 software, the phylogenetic
trees were constructed to determine the evolutionary relationship of newly identified yeasts from our
experiment and previously published yeast species. The sequences of the yeasts were deposited in
the National Center for Biotechnology Information (NCBI) database.

Keywords: coffee; enzymes; polygalacturonase; pectin lyase; pectin methylesterase; starter culture

1. Introduction
Coffee is one of the essential worldwide commodities, next to crude oil. Coffee is the major export
commodity of around 60 tropical and subtropical countries [1,2]. Ethiopia is the origin of arabica
coffee, and as such, has a long tradition of roasting and drinking this deeply cultural non-alcoholic
beverage. Brazil ranks as the leading producer and exporter of arabica coffee, followed by Indonesia,
Ethiopia, Philippines, Mexico, Vietnam, and 40 other countries [3]. After harvesting, the coffee fruit is
processed using various methods: wet processing, dry processing, and semi-dry processing. During
the wet-processing method, the pulp is mechanically separated from fully ripe coffee cherries. Then
the fruit is collected and naturally fermented following the process of removal of mucilage by placing
it in a fermentation tank and submerging in water for at least 6–24 h [4]
The ultimate goal of fermentation is to remove the mucilage layer, which is made up of complex
compounds. Mucilage is a sticky substance and adheres to the coffee pulp, and it comprised around
5% of the coffee cherries in dry matter basis [5]. At the time of maturity of the coffee cherry, calcium
pectates found in the middle lamella and protopectin transformed into pectin from the cellular wall [6].

Microorganisms 2019, 7, 401; doi:10.3390/microorganisms7100401 www.mdpi.com/journal/microorganisms

Microorganisms 2019, 7, 401 2 of 16

The mesocarp of the coffee cherry is a translucent and gelatinous sweet substance which comprised
sugar, minerals and pectin [5]. Silva [7] explained that the microorganisms are responsible for the
degradation of the pulp and mucilage (polysaccharides) of the coffee fruit during fermentation. In
addition, the production of pectinolytic enzymes and formation of alcohols and acids (butyric acetic,
lactic, and other long-chained carboxylic acids) are associated with the microbes [7]. The fermentation
processes depend on the growth and metabolic activities of various groups of microorganisms such
as yeasts, Gram-negative bacteria, bacilli, filamentous fungi and, lactic acid bacteria and acetic acid
bacteria [8]. However, the presence of the microorganism during the fermentation of coffee cherries is
relying on the plant variety, processing method, moisture content, the colonizing species enzymatic
capacity, the competition of substrates, environmental factors and the microorganism’s antimicrobial
activity [9].
The microbial enzymes (pectinases) are responsible for the breaking down of pectin substances.
Pectinases are enzymes, which degrade pectin substances and have great importance in the food
industry. Reports indicate that microbial pectinases comprised the global food enzymes market by
25% [10]. They hold the first position among the commercially produced industrial enzymes. These
enzymes are environmentally friendly and sustainable with potentially wide applications in many
industrial activities, such as tea and coffee fermentation, oil extraction, clarification of juice and wines
etc. [11]. The main enzymes involved in coffee fermentation are polygalacturonase (PG), pectin lyase
(PL), and pectin methylesterase (PME) [12]. These three enzymes have a potential of the complete
digestion of pectin to produce galacturonic acid and its oligomers [13,14]. The selection of potential
starter culture for coffee fermentation includes production of these enzymes by the microorganisms.
The microorganisms used in the production of fermented foods and beverages partly determine their
quality. Some of the yeasts that isolated and identified in our study were evaluated for their effect on
the coffee quality parameters after fermenting the green coffee beans and found positive results [15].
The search for microbial diversity during coffee processing is required to select microorganisms
for starter cultures for the fermentation processes. The isolation and selection of microorganisms for
coffee fermentation have significant scope in identifying additional source organisms. The ultimate
objectives of this experiment were to isolate, identify, and characterize pectinolytic yeasts. We also
evaluated their efficiencies in producing PG, PL, and PME using the synthetic pectin media (SPM) and
the coffee pulp-culture media (CPM).

2. Materials and Methods

2.1. Spontaneous Fermentation and Yeast Isolation

Ripened coffee cherries (20 kg) were harvested from well-managed coffee (Coffea arabica L.)
trees that were grown in Chuncheon, Republic of Korea. The coffee cherries were depulped and
naturally fermented in 50 mL plastic container at 28 ◦ C without additional starter cultures for 48 h.
Various wild yeasts responsible for enhancing spontaneous fermentation activity were expected on
the coffee fruit. A sample solution was taken from the fermentation container and serially diluted
(10 −2 , 10 −3 , 10 −4 , and 10 −5 ). Small droplets (30 µL) of the serially diluted solutions were plated
onto YPDA (yeast extract-peptone-dextrose agar) media and spread using a glass rod. The cultures
in Petri dishes were sealed with Parafilm to prevent contamination of undesired microorganisms.
Then the cultures were incubated at 28 ◦ C for four days. The pure culture was used to screen yeast
isolates that have distinctive colonial morphologies. They were sub-cultured onto other new YPDA
media. The morphological features of the yeast colony that were recorded were color (white and
yellow), shape (circular, ovoid), surface (smooth, rough, dry, and powdery), and elevation (flat, raised,
convex, pulvinate, and umbonate). The YPDA media was used to culture the yeast from all samples.
The media compositions were: dextrose (20 gL −1 ), yeast extract (5 gL −1 ), peptone (10 gL −1 ), and agar
(15 gL −1 ). To suppress the growth of bacteria, 0.1 gL −1 ampicillin powder was added into the media
Microorganisms 2019, 7, 401 3 of 16

after autoclaving when the solution cooled to 50 ◦ C. All chemicals for this experiment were purchased
from Sigma Aldrich LLC (St. Louis, MO, USA).

2.2. Pectinolytic Yeast Screening

Pectinolytic activity was assessed using the protocol described by Schwan et al. [16]. The yeasts
were grown on plates comprising mineral media: MnSO4 (0.05 gL −1 ), KH2 PO4 (0.2 gL −1 ), (NH4 )2 SO4
(1 gL −1 ), CaCl2 (0.05 gL −1 ), MgSO4 (0.8 gL −1 ); yeast extract (1.0 gL −1 ); citric pectin (2.0 gL −1 ); and
agar (15 gL−1 ). Enzyme activity was shown by the development of a clear color change (yellow color)
around the colonies after flooding the plate with 50 mM potassium iodide–iodine solution.

2.3. Pectin Degradation Index (PDI) %

The diameter of the yeast colonies and the pectin degradation zones were measured using a ruler
after growing the yeasts on a Petri dish (YPDA + pectin) for five days. The degraded area showed
a clear zone after flooding with iodine solution. The clear zone and colony diameters were used to
compute the pectin degradation index (PDI), according to the following formula:

Pectin degradation index (PDI) % = (CD + CZ)/CD × 100

where CD is the colony diameter and CZ is the clearing zone diameter.

2.4. Molecular Identification and Phylogenetic Tree Analysis of Yeasts

The yeasts were grown on yeast extract-peptone-dextrose (YPD) broth media for 48 h and the
yeast cells were collected via centrifugation (10× rpm, 6 min). The yeasts DNA that suitable for use
as a template for polymerase chain reaction (PCR) amplification was extracted from approximately
1 × 107 cells. The yeasts were then identified by sequencing the 26S ITS rRNA region of the gene. The 26s
rRNA universal primers were used (Table 1). Purified PCR products were sequenced using ABI3730
XL automatic DNA sequencers that were then compared to those available in the GenBank database
using the Basic Local Alignment Search Tool (BLAST) algorithm, National Center for Biotechnology
Information, MD, USA (NCBI). The phylogenetic tree was made by the neighbor-joining method for
this alignment using the Molecular Evolutionary Genetics Analysis (MEGA 6, PSU, USA) software
with 1000 bootstrap replications. The final sequences were submitted to the GenBank.

Table 1. Details of the primers used in the present study.

Primer Name Sequence Type

Forward 5’-ACCCGCTGAACTTAAGC -3’ Universal
Reverse 5’ -TACTACCACCAAGATCT -3’ Universal

2.5. Synthetic Pectin Media and Coffee Pulp Media Preparation for Enzymatic Assays
The SPM was prepared with the following formulation: 10% citric pectin, 0.1% glucose, 0.5%
MgSO4 , 1.0% KH2 PO4 , 1.0% (NH4 )2 SO4 , and 0.5% CaCl2 . The CPM was prepared by measuring 400 g
of coffee pulp, including mucilage (Coffea arabica L.), and 0.5% glucose. The pulp was boiled using
water for 10 min and adjusted to 1 L [17]. Active yeast cells that were grown for 48 h were added with
1.0 × 10 4 cells mL −1 (4.0 Log colony forming unit (CFU) mL −1 ). The cultures were incubated at 28 ◦ C
for 96 h at 120 rpm and periodically sampled at 24, 48, 72, and 96 h. The supernatants were collected
for PG, PL, and PME activity from both SPM and CPM growing conditions.
In addition, the fermentation characteristics of the CPM culture, such as yeast cells, pH, and
◦ Brix, were regularly measured at 24-h intervals. Serial dilutions were prepared from the fermented

solution and plated onto YPDA media. The living yeast colonies were counted using the Neubauer
hemocytometer slide (Electron Microscopy Sciences, PA, USA) and expressed in Log CFU value.
Microorganisms 2019, 7, 401 4 of 16

A pH meter was used to measure the pH of the fermented solution. A refractometer (ATAGO Pocket
Refractometer, Tokyo, Japan) was used to estimate the ◦ Brix of the media. All the above measurements
were made in three replications regularly at 24-h intervals.
The protein content of the coffee pulp used to make the CPM was analyzed using the protocol
mentioned by Bradford [18], and bovine serum albumin (BSA) was used as a standard. The total
sugars [19] and soluble pectin [20] were determined.

2.6. Enzyme Assays

2.6.1. Pectin Lyase Activity

The pectin lyase activity was estimated in the culture using the method described by Pitt [21] as
modified by Kashyap et al. [22]. 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. Then, 0.6 mL of 9% zinc sulfate and 0.6 mL of 0.5 M sodium hydroxide
were added to discontinue the reaction. After that, using a centrifuge (6000 g for 5 min.), 5 mL of
supernatant was collected from the mixture and combined with 3 mL of 0.04 M thiobarbituric acid,
2.5 mL of 0.1 M HCl, 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 using a spectrophotometer. One unit of
enzyme activity (U mL −1 ) of pectin lyase increased the absorbance by 0.01 units.

2.6.2. Polygalacturonase Activity

The polygalacturonase activity was examined according to the protocol defined by
Schwan et al. [16]. A measurement of 0.1% polygalacturonic acid (w v −1 ) was added in 0.1 M
citrate buffer, and the pH was adjusted to 5.0. Then, the reaction mixture was prepared using 1 mL of
PG from the first buffer mixed with 1.5 mL of sample supernatant; this was followed by incubation
for 1 h at 40 ◦ C. The reaction was stopped by the addition of 1.5 mL of DNS [23]. The mixture was
boiled for 5 min and then cooled in an ice bath. The absorbance reading was made at 600 nm using
a spectrophotometer and measured with a proper calibration curve. One unit of enzyme activity
(U mL −1 ) was expressed as 1 mol of galacturonic acid liberated mL −1 min.

2.6.3. Pectin Methylesterase

Pectin methylesterase was measured using the protocol introduced by Baracat et al. [24] with
the continuous titrimetric determination of the carboxyl groups liberated from methyl ester bonds.
The reaction was carried out with 3 mL of the enzymatic micro solution, to which 20 mL of 1% Sigma
citric pectin in NaCl 0.1 M pH 7.5 solution was added. The mixture was incubated for 30 min keeping
the pH at 7.5 by the addition of NaOH 0.02 M. The absorbance reading was made at 550 nm using
a spectrophotometer. PME activity was expressed as the micro equivalents of polygalacturonic acid
produced mL −1 h −1 .

2.7. Statistical Analysis

Analysis of variance (ANOVA) was computed for testing the significances of the experiment.
Data were summarized in an Excel program and analyzed using SAS statistical software (SAS Institute,
Cary, NC, USA). Mean separation was done using Fisher’s Least Significant Difference.

3. Results

3.1. Isolation, Selection, and Morphological Characterization of Yeasts

The fermentation of coffee cherries was done for 48 h with yeasts and other microorganisms that
naturally existed on the coffee fruit. The samples were taken and spread on YPDA media to isolate the
yeasts. A total of 28 yeasts were isolated during coffee fermentation using the wet-processing method.
Microorganisms 2019, 7, 401 5 of 16

Among the isolated yeasts, eight of them showed pectinolytic activity after testing them on YPDA
media enriched with citrus pectin. The isolated yeasts colonies were morphologically characterized
according to their shape, color, elevation surface, and margin (Table 2).

Table 2. Morphological features of the eight yeast colonies.

Yeasts Shape Color Elevation Surface Margin

W. anomalus KNU18Y3 Circular White Raised Smooth Entire
S. fibuligera KNU18Y4 Circular White Convex Rough Entire
P. flavescens KNU18Y5 Circular Yellow Pulvinate Rough Entire
P. flavescens KNU18Y6 Circular Yellow Pulvinate Smooth Entire
P. kudriavzevii KNU18Y7 Circular White Umbonate Smooth Entire
P. kudriavzevii KNU18Y9 Circular White Pulvinate Smooth Entire
S. cerevisiae KNU18Y12 Circular White Convex Smooth Entire
S. cerevisiae KNU18Y13 Circular White Pulvinate Smooth Entire

3.2. Pectin Degradation Index (PDI) %

As shown in Table 3, the maximum pectin degradation index was derived after five days of
incubation on YPDA media that has a citrus pectin. The PDI% of yeasts was ranged from 110 to 178%.
The highest PDI was obtained from S. fibuligera (strain KNU18Y4) at 178%. The PDIs of W. anomalus
(strain KNU18Y3) and P. flavescens (strain KNU18Y6) were 160% and 152%, respectively (Table 3).
However, relatively, the lowest PDI % was obtained from P. kudriavzevii KNU18Y7, and it was 110%.
The PDI of the S. cerevisiae KNU18Y12 and S. cerevisiae KNU18Y13 was 121% and 118%, respectively.

Table 3. Pectin degradation index after 5 days incubation on YPDA media that supplemented a
citrus pectin.

Yeasts PDI %
W. anomalus KNU18Y3 160b ± 1.73
S. fibuligera KNU18Y4 178a ± 4.04
P. flavescens KNU18Y5 129c ± 4.62
P. flavescens KNU18Y6 152b ± 4.04
P. kudriavzevii KNU18Y7 110d ± 2.89
P. kudriavzevii KNU18Y9 125c ± 4.04
S. cerevisiae KNU18Y12 121cd ± 2.31
S. cerevisiae KNU18Y13 118cd ± 4.62
Results are presented as mean ± standard deviation (n = 5). Means denoted with different letters within column are
significantly different.

Molecular Identification and Phylogenetic Trees

Based on the preliminary pectinolytic yeasts screening results, eight isolates were selected and
identified molecularly by sequencing the 26s rRNA gene (D1/D2 region) using universal primers
(Table 1). The phylogenetic trees were constructed using MEGA 6 software to reveal the evolutionary
distance between the yeasts, which were identified from our study and previously reported yeast species
in the National Center for Biotechnology Information (NCBI) database (Figure 1A–E). The identified
yeasts were submitted to the GenBank with the accession numbers mentioned in Table 4.
Microorganisms
Microorganisms 2019,7,7,401
2019, 401 6 of 619of 16

Table 4. The accession number of the identified yeasts species and strain code.
Table 4. The accession number of the identified yeasts species and strain code.
Accession Number Yeast Species Name Strain Code
Accession Number
MH483547 Yeast Species Name
Wickerhamomyces anomalus Strain Code
KNU18Y3
MH483929
MH483547 Saccharomycopsis fibuligera
Wickerhamomyces anomalus KNU18Y4
KNU18Y3
MH483929
MH484046 Saccharomycopsis fibuligera
Papiliotrema flavescens KNU18Y4
KNU18Y5
MH484046
MH485393 Papiliotrema flavescens
Papiliotrema flavescens KNU18Y5
KNU18Y6
MH485393 Papiliotrema flavescens KNU18Y6
MH488970 Pichia kudriavzevii KNU18Y7
MH488970 Pichia kudriavzevii KNU18Y7
MH487468
MH487468
Pichia kudriavzevii
Pichia kudriavzevii
KNU18Y9
KNU18Y9
MH491423
MH491423 Saccharomyces cerevisiae
Saccharomyces cerevisiae KNU18Y12
KNU18Y12
MH488975
MH488975 Saccharomyces cerevisiae
Saccharomyces cerevisiae KNU18Y13
KNU18Y13

Figure 1. Cont.
Microorganisms 2019, 7, 401 7 of 16
Microorganisms 2019, 7, 401 7 of 19

Figure
Figure 1. Phylogeneticrelationship
1. Phylogenetic relationship between
between thethe identified
identified yeasts
yeasts and other
and other 26S rRNA
26S rRNA sequences
sequences of
ofpublished
published strains.
strains. A: (A) Wickerhamomyces
Wickerhamomyces anomalus
anomalus strainstrain KNU18Y3,
KNU18Y3, (B) Saccharomycopsis
Saccharomycopsis fibuligera
fibuligera strain
strain KNU18Y4,
KNU18Y4, (C) Papiliotrema
Papiliotrema flavescens
flavescens (strain (strain KNU18Y5,
KNU18Y5, KNU18Y6), KNU18Y6), (D) Pichia
Pichia kudriavzevii kudriavzevii
(strain KNU18Y7 (strain
KNU18Y7 and KNU18Y9)
and KNU18Y9) and Saccharomyces Saccharomyces
and (E)cerevisiae (straincerevisiae
KNU18Y12 (strain KNU18Y12 and KNU18Y13).
and KNU18Y13).
Microorganisms 2019, 7, 401 8 of 16

3.3. Fermentation Characteristics

The living yeast cells were counted at every 24-h interval. A significant difference (p < 0.05)
in living yeast cells were observed among the evaluated yeasts at different fermentation hours
(Table 5). An increasing trend in living yeasts cell was observed from the initial inoculation up to 48 h of
fermentation. Then, decreasing growth trends were observed in all yeasts at 72 and 96 h of fermentation.
The maximum living yeast cell growth was found from all yeasts at 48-h fermentation. The number of
living yeast cells of S. cerevisiae KNU18Y13 were significantly higher (8.23 Log CFU mL−1 ) compared
to other yeasts at 48 h fermentation. Among the tested yeasts, S. cerevisiae strain KNU18Y13 had a
unique characteristic in terms of growth habit. It is a highly reproducible yeast compared to other
yeasts and can ferment very quickly, with a noticeably high production of carbon dioxide. From
the experimented yeasts, S. fibuligera showed low reproduction rates (at each fermentation period
compared to others (Table 5)). There was no significant difference (p > 0.05) in living yeast cells at 72 and
96 h of fermentation among W. anomalus KNU18Y3, P. flavescens KNU18Y6, and S. cerevisiae KNU18Y13.

Table 5. The living cells of yeast species at different fermentation hours (24, 48, 72, and 96) in CPM.

Log CFU mL−1

Yeasts
24 h 48 h 72 h 96 h
W. anomalus KNU18Y3 7.08f ± 0.23 8.21b± 0.32 7.22a ± 0.52 7.23a ± 0.10
S. fibuligera KNU18Y4 6.80g ± 0.11 8.04e ± 0.52 7.11d ± 0.41 7.12c ± 0.08
P. flavescens KNU18Y5 7.10e ± 0.21 8.17d ± 0.46 7.16b ± 0.36 7.15b ± 0.15
P. flavescens KNU18Y6 7.13d ±0.14 8.20b ± 0.26 7.23a ± 0.21 7.22a ± 0.17
P. kudriavzevii KNU18Y7 7.17c ±0.12 8.19c ± 0.13 7.15c ± 0.25 7.15bc ± 0.20
P. kudriavzevii KNU18Y9 7.13d ± 0.15 8.18cd ± 0.26 7.16b ±0.14 7.17b ± 0.16
S. cerevisiae KNU18Y12 7.19b ± 0.09 8.18cd ± 0.18 7.17b ± 0.11 7.16b ± 0.19
S. cerevisiae KNU18Y13 7.22a ± 0.18 8.23a ± 0.12 7.22a ±0.32 7.21a ± 0.15
Results are presented as mean ± standard error (n = 5). Means denoted with different letters in each column are
significantly different.

The protein content, the total sugar, and total pectin in the coffee pulp that was used to make the
CPM were measured as dry matter, accounting for 14.30%, 12.45%, and 6.70%, respectively. The pH
of fermented solutions was regularly monitored and recorded. The initial (0 h) pH of the CPM
was adjusted at 5.72. The pH of the CPM continuously dropped starting from the initial hours of
fermentation until 72 h and began increasing at 96 h in all yeasts except the S. fibuligera KNU18Y4
inoculated media. Comparatively, the pH of the CPM with the S. fibuligera KNU18Y4 continuously
declined from the 24-h period to 96 h at a slow rate. In the P. flavescens KNU18Y5 inoculated treatment,
the pH decreased from the initial fermentation until 48 h and began increasing at 72 and 96 h. During
the first 24-h fermentation, the pH decreased at a higher rate compared to other fermentation hours.
The ◦ Brix of the CPM was measured from beginning to end of the fermentation process. The initial
◦ Brix of the CPM was 5.4. There were significant differences among yeasts in reducing the ◦ Brix

contents during each fermentation period. The ◦ Brix was continuously dropped in all fermentation
treatments (Table 6). During the first 24 h of fermentation, the ◦ Brix decreased at a higher rate compared
to the other fermentation periods. The W. anomalus KNU18Y3, S. fibuligera KNU18Y4, P. flavescens
KNU18Y6, and S. cerevisiae (KNU18Y12 and KNU18Y13) strains inoculated CPM ◦ Brix was declined
below 1% at the end of 96 h fermentation. After 96 h fermentation, the ◦ Brix of CPM was changed
from 5.4 to nearly 1.0% in P. flavescens KNU18Y5 and P. kudriavzevii strains (KNU18Y7 and KNU18Y9).
The lowest ◦ Brix was recorded from W. anomalus KNU18Y3 inoculated CPM (0.40%) at the same time
the pH was lower (4.75) compared to other treatments after 96 h fermentation.
Microorganisms 2019, 7, 401 9 of 16

Table 6. The pH and Brix of fermented coffee pulp media by different yeast species.

pH Brix◦
Strains Code
24 h 48 h 72 h 96 h 24 h 48 h 72 h 96 h
KNU18Y3 4.93b ± 0.21 4.62g ± 0.14 4.60e ± 0.23 4.75h ± 0.41 2.13b ± 0.11 0.9d ± 0.04 0.46d ± 0.02 0.40b ± 0.01
KNU18Y4 5.23a ± 0.42 5.01a ± 0.11 4.86c ± 0.27 4.83f ± 0.35 2.93a ± 0.23 1.77a ± 0.12 1.75a ± 0.04 0.96a ± 0.02
KNU18Y5 5.04ab ± 0.12 4.97b ± 0.13 5.28b ± 0.17 5.86c ± 0.23 2.10b ± 0.14 1.66ab ± 0.08 1.15b ± 0.03 1.03a ± 0.05
KNU18Y6 4.94b ± 0.13 4.77e ± 0.21 4.75cd ± 0.31 4.80g ± 0.24 2.06b ± 0.19 1.16cd ± 0.01 0.55c ± 0.06 0.46b ± 0.04
KNU18Y7 4.95b ± 0.14 4.73f ± 0.23 5.68a ± 0.30 5.91b ± 0.41 1.80c ± 0.09 1.70ab ± 0.10 1.15b ± 0.07 1.01a ± 0.03
KNU18Y9 4.99b ± 0.15 4.95c ±0.25 5.65a ± 0.24 5.96a ± 0.21 1.90c ± 0.18 1.77a ± 0.08 1.10b ± 0.07 1.01a ± 0.04
KNU18Y12 4.86b ± 0.15 4.85d ± 0.15 4.82c ± 0.18 5.13d ± 0.17 1.30d ± 0.17 1.03d ± 0.06 0.65c ± 0.04 0.60b ± 0.05
KNU18Y13 4.90b ± 0.17 4.85d ± 0.14 4.64de ± 0.19 5.08e ± 0.12 1.20d ± 0.13 1.40bc ± 0.10 0.60c ± 0.08 0.36b ± 0.04
Results are presented as mean ± standard error (n = 5). Means denoted with different letters in each column are significantly different.
Microorganisms 2019, 7, 401 10 of 16

3.4. Enzyme Assays

3.4.1. Pectin Lyase

The PL activity of different yeasts was determined at different fermentation hours in both CPM
and SPM growth conditions. The PL activity of S. fibuligera KNUY18Y4 and W. anomalus KNU18Y3
were significantly (p < 0.05) higher compared to other yeasts during all fermentation periods in both
CPM and SPM growth conditions (Table 7). The maximum PL activity was found during the 24-h
fermentation from all yeasts in both media. Regarding the fermentation hours, the highest PL activity
of S. fibuligera KNUY18Y4 obtained at 24 h fermentation and it was 17.66 and 117.55 U mL −1 . Similarly,
the highest PL activity of W. anomalus KNU18Y3 was 16.96 U mL−1 in CPM and 16.83 U mL −1 in
SPM condition. The PL activity showed a decreasing trend as the fermentation hours progressed
from 24 to 96. The PL activity of the yeasts showed similar patterns in both media, and the results
were approximate. In both media conditions, the lowest PL activity was obtained from S. cerevisiae
KNU18Y12, P. kudriavzevii KNU18Y9, S. cerevisiae KNU18Y13, and S. cerevisiae KNU18Y12 during the
24, 48, 72, and 96-h fermentation periods, respectively. The PL activity between P. flavescens strains
(KNU18Y5 and KNU18Y6), did not show a remarkable difference at 24 h in the CPM condition and
it was 16.16 and 16.42 U mL −1 , respectively (Table 7). However, at 48, 72, and 96 h, a significant
difference between P. flavescens strains was observed in CPM and at 72 h in SPM growth condition.
The P. kudriavzevii KNU18Y7 produce significantly higher at 48 and 96 h compared to P. kudriavzevii
KNU18Y9 in both media types. In CPM condition, the PL activity of P. kudriavzevii KNU18Y7 at 48
and 96 h was 14.57 and 7.81 U mL −1 , respectively. However, in SPM condition, the PL activity of
P. kudriavzevii KNU18Y7 was 14.51 and 7.76 U mL −1 at 48, and 96 h. Generally, the PL activity of all
yeasts was approximately increased by double at 24 and 48 h compared to 72 and 96 h durations in
both media conditions.

3.4.2. Polygalacturonase Activity

The PG activity of eight yeasts was evaluated at different fermentation hours by growing them
in CPM and SPM conditions. Like PL, the PG activity of S. fibuligera KNUY18Y4 and W. anomalus
KNU18Y3 were significantly (p < 0.05) higher compared to the other yeasts at 24, 48, and 72 h
fermentation, respectively, in both CPM and SPM (Table 8). The highest PG activity of S. fibuligera
KNUY18Y4 was found at 48 h fermentation and it was 8.28 and 8.21 U mL −1 in CPM and SPM
condition, respectively. However, relatively, the maximum PG activity of W. anomalus KNU18Y3 was
obtained at 24 h fermentation and it accounts 8.01 U mL −1 in CPM and 7.98 U mL −1 in SPM. Regarding
the fermentation time, the highest PG activity was found at 24 h from all yeasts except S. fibuligera
KNUY18Y4 in both media. At 24-h fermentation, the lowest PG was secreted by S. cerevisiae strain
KNU18Y13 and it was 6.06 U mL −1 in CPM and 6.03 U mL−1 in SPM conditions. We found a significant
difference between the two strains of P. flavescens (KNU18Y5 andKNU18Y6) in producing PG at 48 and
96 h, respectively, in both media, and at 24 h in the SPM condition. However, at 72 h fermentation in
both media and 24 h fermentation in the CPM condition, these two strains of P. flavescens did not show
significant variation in producing the PG enzyme (Table 8). Like P. flavescens strains, a similar result
trend was observed between the P. kudriavzevii strains (KNU18Y7 and KNU18Y9). Regardless of yeasts,
the highest activity of P. kudriavzevii KNU18Y9 (6.98 and 6.67 U mL −1 ) and P. kudriavzevii KNU18Y7
(6.54 and 6.41 U mL −1 ) was found at 24-h fermentation in both media types (Table 8). Generally, apart
from S. fibuligera KNUY18Y4 at 48 h, the PG activity showed a decreasing trend as the fermentation
hours exceeded from 24 to 96.
Microorganisms 2019, 7, 401 11 of 16

Table 7. Pectin lyase (PL) activity of the yeasts after fermentation in CPM and SPM (24, 48, 72, and 96 h).

Enzymatic Activity (U mL−1 )

Strains Code CPM SPM
24 h 48 h 72 h 96 h 24 h 48 h 72 h 96 h
KNU18Y3 16.91b ± 0.16 15.44b± 0.06 8.56ab ± 0.23 8.23a ± 0.10 16.83ab ± 0.12 15.32b ± 0.40 8.41b ± 0.69 7.12a ± 0.23
KNU18Y4 a
17.66 ± 0.13 16.25a± 0.03 8.75a ± 0.04 8.24a ± 0.12 17.55a ± 0.29 16.13a ± 0.21 8.65a± 0.23 7.14a ± 0.40
KNU18Y5 16.16 cd ± 0.12 14.68c ± 0.06 8.11d ± 0.08 7.75b ± 0.05 16.12abc ± 0.46 14.6cd ± 0.12 8.98d ± 0.35 7.69bc ± 0.23
KNU18Y6 16.42bcd ± 0.39 14.34de ± 0.07 8.47abc ± 0.06 7.32c ± 0.08 16.31abc ± 0.12 14.21de ± 0.29 8.34b ± 0.29 7.28cde ± 0.40
KNU18Y7 16.45bc ± 0.21 14.57cd ± 0.12 8.21cd ± 0.04 7.81b ± 0.09 16.23abc ± 0.10 14.51cd ± 0.06 8.11cd ± 0.49 7.76b ± 0.20
KNU18Y9 16.20cd ± 0.19 14.13e ± 0.07 8.38bcd ± 0.09 7.41c ± 0.14 16.13abc ± 0.29 14.09e ± 0.29 8.26bc ± 0.15 7.31d ± 0.40
KNU18Y12 14.93e ± 0.01 14.78c ± 0.10 8.39bcd ± 0.04 7.31c ± 0.18 14.86c ± 1.59 14.59cd ± 0.06 8.21bc ± 0.71 7.21e ± 0.12
KNU18Y13 15.80d ± 0.23 14.78c ± 0.06 7.67 e ± 0.03 7.78b ± 0.07 15.62bc ± 0.23 14.66c ± 0.17 7.59e ± 0.23 7.64c ± 0.13
Results are presented as mean ± standard error (n = 3). Means denoted with different letters in each column are significantly different.

Table 8. Polygalactrunase (PG) activity of the yeasts after fermentation in CPM and SPM (24, 48, 72, and 96 h).

Enzymatic Activity (U mL−1 )

Strains Code CPM SPM
24 h 48 h 72 h 96 h 24 h 48 h 72 h 96 h
KNU18Y3 8.01ab ± 0.47 7.76b ± 0.12 6.08b ± 0.96 6.01c ± 0.05 7.98b ± 0.35 7.71b ± 0.23 6.02bcd ± 0.17 5.91d ± 0.23
KNU18Y4 8.21a ± 0.50 8.28a ± 0.07 8.01a ± 0.11 7.75a ± 0.04 8.15a ± 0.29 8.21a ± 0.29 7.96a ± 0.29 7.71a ± 0.35
KNU18Y5 7.61abc ± 0.41 7.39c ± 0.06 6.07b ± 0.08 5.33d ± 0.09 7.51d ± 0.23 7.32d ± 0.23 6.01bcd ± 0.40 5.23f ± 0.52
KNU18Y6 7.98ab ± 0.47 7.68b ± 0.05 5.97b ± 0.18 5.88c ± 0.02 7.82c ± 0.46 7.61c ± 0.12 5.89d ± 0.52 5.7e ± 0.10
KNU18Y7 6.54cd ± 0.25 6.42e ± 0.09 6.03b ± 0.19 6.16c ± 0.21 6.41g ± 0.35 6.36g ± 0.20 5.94cd ± 0.21 6.02c ± 0.23
KNU18Y9 6.98bcd ± 0.32 6.77d ± 0.10 6.22b ± 0.05 6.59b ± 0.20 6.82e ± 0.29 6.67e ± 0.10 6.17bc ± 0.13 6.48b ± 0.40
KNU18Y12 6.71cd ± 0.28 6.58de ± 0.09 6.02b ± 0.05 6.15c ± 0.15 6.68f ± 0.23 6.49f ± 0.40 6.21b ± 0.06 6.05c ± 0.23
KNU18Y13 6.06d ± 0.13 5.94f ± 0.10 5.75c ± 0.12 5.22d ± 0.14 6.03h ± 0.18 5.88h ± 0.35 5.75d ± 0.27 5.32f ± 0.32
Results are presented as mean ± standard error (n = 3). Means denoted with different letters within each column are significantly different.
Microorganisms 2019, 7, 401 12 of 16

Table 9. Pectin methylesterase (PME) activity of the yeasts after fermentation in CPM and SPM (24, 48, 72, and 96 h).

Enzymatic Activity (U mL−1 )

Strains CODE CPM SPM
24 h 48 h 72 h 96 h 24 h 48 h 72 h 96 h
KNU18Y3 15.11c ± 0.47 15.68c± 0.14 13.61bcd ± 0.81 12.25cd ± 0.92 15.05b ± 0.35 15.56f ± 0.09 13.49bc± 0.25 12.15bc ± 0.13
KNU18Y4 15.23c ± 0.21 15.96bc ± 0.12 13.86bc ± 0.92 12.56bc ± 0.13 15.07b ± 0.15 15.72ef ± 0.12 13.71abc ± 0.15 12.41b ± 0.10
KNU18Y5 15.21c ± 0.29 16.34b ± 0.16 13.31d ± 0.12 12.01d ± 0.18 14.98b ± 0.18 16.25c ± 0.11 13.26c ± 0.12 11.98c ± 0.06
KNU18Y6 15.32bc ± 0.24 16.07bc ± 0.18 13.57cd ± 0.10 12.38bcd ± 0.16 15.17b ± 0.19 16.02cde ±0.11 13.33c ± 0.07 12.19bc ± 0.22
KNU18Y7 15.15c ± 0.17 16.19bc ± 0.20 13.56cd ± 0.11 12.26bcd ± 0.06 15.02b ± 0.12 15.94de ± 0.17 13.36c ± 0.16 12.12bc ± 0.12
KNU18Y9 15.23c ± 0.08 16.3b ± 0.19 13.83bc ± 0.15 12.43bc ± 0.08 14.98b ± 0.14 16.15cd ± 0.08 13.68abc ± 0.09 12.27bc ± 0.11
KNU18Y12 16.53a ± 0.13 18.25a ± 0.12 14.20a ± 0.09 13.00a ± 0.15 16.32a ± 0.07 18.01a ± 0.08 14.06a ± 0.15 12.85a ± 0.14
KNU18Y13 16.02ab ± 0.11 17.86a ± 0.23 13.90ab ± 0.08 12.64ab ± 0.06 15.89a ± 0.16 17.63b ± 0.06 13.81ab ± 0.13 12.42b ± 0.09
Results are presented as mean ± standard error (n = 3). Means denoted with different letters in each column are significantly different.
Microorganisms 2019, 7, 401 13 of 16

3.4.3. Pectin Methylesterase

There were significant (p < 0.05) differences among the PME activity of yeasts at different
fermentation hours in both the CPM and SPM growth conditions (Table 9). During 24 h fermentation,
the PME activity was not significantly different among the tested yeasts, except for the S. cerevisiae
strains, in both media types. PME activity of all yeasts showed an increasing trend as the fermentation
proceeded from 24 to 48 h. However, a decreasing trend observed at 72 and 96 h fermentation in both
media conditions. The highest PME activity throughout the fermentation period was obtained from
all yeasts at 48 h fermentation in both media types. Comparatively, a significant PME activity found
from S. cerevisiae strains (KNU18Y12 and KNU18Y13) in both media. The maximum PME activity of
S. cerevisiae strains (KNU18Y12 and KNU18Y13) was 18.25 and 16.02 U mL−1 in CPM and it was 18.01
and 17.63 U mL−1 in SPM condition, respectively (Table 9). Unlike PL and PG, the PME activity of
S. fibuligera KNU18Y4 and W. anomalus KNU18Y3 yeasts was lower in both media types compared
to other yeasts. At 48 h, the PME activity of W. anomalus KNU18Y3 was 15.68 and 15.56 U mL −1 in
CPM and SPM condition, respectively. However, the S. fibuligera KNU18Y4 PME activity was higher at
48 h fermentation compared to other fermentation hour treatments and it was 15.96 U mL −1 in the
CPM condition, and 15.72 U mL −1 in the SPM condition. The PME activity did not show a significant
difference between P. flavescens strains KNU18Y6 and KNU18Y5 at each fermentation period in both
media (Table 9). Likewise, the PME activity was not significantly different between the P. kudriavzevii
strains (KNU18Y7 and KNU18Y9) in both media at 24, 48, 72, and 96 h of fermentation.

4. Discussion
Fermentation is an important step during coffee processing to remove the mucilage (sticky
polysaccharide substance) form the parchment coffee. However, fermentation in wet processing is
critical because it creates a diverse aroma and flavor beyond its natural flavor and taste. Pectinase
enzyme accelerates tea fermentation by breaking down the pectin substance which presents in the
tea leaves [25]. Pectinase enzyme-producing yeasts are responsible for speeding up the fermentation
process and removing the pectin substances during coffee processing. For these reasons, isolation,
identification, and characterization of yeasts that can produce pectinase enzymes (pectin lyase,
polygalacturonase, and pectin methyl esterase) are essential. This process needs frequent study to
develop starter cultures for coffee fermentation. A microbiological culture that facilitates fermentation
activities is called starter culture. Starter cultures are widely used in the food industry to prepare such
products as yogurt, wine, and beer [26]. In controlled coffee fermentation, starter culture may improve
the quality of coffee and increase its economic value, thereby enhancing the incomes of farmers [27].
Yeast populations increased as the fermentation duration increased from 0 to 96 h. However, the rate
of yeast growth showed a sharper increase during the first 48 h, a result supported by Kamassah et
al. [28]. The growth of the yeasts decreased and approximately leveled out to a steady growth at 72 and
96 h compared to the first 48 h. The decrease after 48 h might be associated with pH changes (Table 6)
and depletion of the substrates, such as the total soluble solid content (Table 6). The change in pH
occurred during fermentation of CPM with different yeasts. The decreases in pH during fermentation
are associated with the production of acids. It was found that the production of organic acid and
absorption of amino acids have significant effects. Pectinolysis enabled reduction in demucilization
time which was evident with reduction in pH value [29]. As the yeasts grew in the CPM for 96 h, the
◦ Brix was substantially reduced. However, the ability to decrease the ◦ Brix content was significantly

varied among species as well as within strains (Table 6).

Pectinase enzyme is produced by several microorganisms. It was reported in bacteria such as
Xanthomonas sp., Bacillus sp. and in very few yeasts such as S. cerevisiae and Candida boidinii [30].
Pectinase activity was found in yeasts and yeast-like microorganism such as Dioszegia sp., Phenoliferia
glacialis and Tetracladium sp. isolates and the enzyme was identified as polygalacturonase [31]. In our
experiment, the PL, PG, and PME activities of yeasts decreased as the fermentation hours increased from
all yeasts. A similar result reported by Oumar et al., [32] the PL activity of Bacillus subtilis strain Btk 27
Microorganisms 2019, 7, 401 14 of 16

was reduced as the incubation time extended more than 48 h. The pectinase enzyme production among
yeasts was different. This indicated that the ability of the microorganism producing the pectinase
enzyme varied among yeasts species and strains. We have identified a pectinase enzyme-producing
S. cerevisiae (KNU18Y12 and KNU18Y13) during coffee fermentation by wet processing. Furthermore,
we have identified two pectinolytic P. kudriavzevii yeast strains. Koffi et al. [33] isolated and identified
S. cerevisiae YB14 and P. kudriavzevii YP13 yeasts that were able to produce the pectinase enzyme during
cacao fermentation. However, pectinolytic P. kudriavzevii yeast was examined and showed a potential
ability in producing PG enzymes during cacao mucilage fermentation [34]. In this experiment, a
potential pectinase enzyme-producing yeast, W. anomalus, was identified during a wet-processing
method. The PL and PG activity W. anomalus in our experiment was significantly higher than other
examined yeasts following S. fibuligera KNU18Y4. Martos et al. [35] reported a potential pectinase
enzyme-producing W. anomalus from citrus peel. They also mentioned its maximal PG activity was at
pH 4.5. However, in our experiment, we did not adjust the pH of the medium during fermentation
activity, rather the pH and the enzyme activity was measured at 24 h interval. The pH was 4.93 after
24-h fermentation in W. anomalus inoculated media and the PG activity was higher compared to the
rest other fermentation durations (48, 72, and 96). The PG activity of yeasts produced in an optimum
pH in the acidic region between 3.5 and 5.5 [35]. The W. anomalus is grouped as a biosafety level 1
organism that can grow under severe environmental stress conditions, such as high and low pH, high
osmotic pressure, anaerobic conditions, and low water activity [36]. A study showed that as the pH
increased from 3 to 4, the Aspergilus niger pectinase enzyme production also increased [37].
To the best of our knowledge, the other three yeasts S. fibuligera KNU18Y4 and two P. flavescens
(KNU18Y5 and KNU18Y6) are being reported for the first time as pectinase enzyme-producing yeasts
that are identified during coffee fermentation in the wet-processing method. The production of
pectinase enzymes varies among the identified yeasts in our experiment. These variations might be
associated with the difference of microorganism population (Table 5) and pH condition (Table 6) during
the fermentation period. Pectinase production by fungi varies according to the type of strain and
cultivation conditions (initial pH, inoculum size, and incubation period) [38]. Regarding the pectin
lyase activity, the first 24-h fermentation period produced a greater amount in all tested yeasts in
both media types. Besides the yeasts’ ability to produce the pectinase enzyme, we have evaluated the
impact of some of these yeasts by fermenting green coffee beans to improve the antioxidant activity,
total polyphenol, flavonoid, and tannin contents [15]. They showed positive results in improving the
flavonoid, polyphenol, and antioxidant activity [15].

5. Conclusions
In this study, we have isolated, identified, and characterized eight pectinase enzyme-producing
yeasts during wet-processing methods of fermenting coffee. Out of the identified yeast, S. fibuligera
KNU18Y4 and W. anomalus KNU18Y3 have the prominent capability of producing the PG and PL
enzymes in both the coffee pulp media and synthetic pectin media. The two S. cerevisiae strains
(KNU18Y12 and KNU18Y13) produce a higher PME. We have also identified two strains of P. flavescens
(KNU18Y5 and KNU18Y6) and S. fibuligera KNU18Y4 that have not previously been reported as
pectinase enzyme-producing yeast. Regarding the fermentation duration, the 24 and 48 h cultivation
of the tested yeasts produced a higher PL enzyme in both media types. Comparatively, the PG and
PME activity of all yeasts was high at 24 and 48 compared to 72 and 96 h fermentation. Generally, these
yeasts have the potential to be used as a starter culture during fermentation of coffee. Additionally,
these yeasts can also be used in the food industry for pectinase enzyme production. Isolation and
identification of yeasts to develop starter cultures for coffee fermentation are relevant and should be
continued in the future.

Author Contributions: Conceptualization, W.H.K., M.H.; Data curation, M.H.; formal analysis, M.H.;
investigation, W.H.K.; methodology, M.H.; resources, W.H.K.; software, M.H.; supervision, W.H.K.; validation,
Microorganisms 2019, 7, 401 15 of 16

W.H.K.; writing—original draft preparation, M.H.; writing—review and editing, W.H.K., M.H.; project
administration, W.H.K.
Conflicts of Interest: The authors declare no conflict of interest.

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