Bandung, Indonesia
Bandung, Indonesia
Bandung, Indonesia
Bandung, Indonesia
Napa cabbage waste contains an organic component, cellulose, which can be utilised as an ingredient for cellulose-degrading enzyme production with the help of indigenous yeast. The aim of the research was to identify and characterise potential indigenous yeast isolated from napa cabbage waste, which has cellulose-degrading activity. Indigenous yeast were isolated and characterised using the RapID Yeast Plus System, then turbidity was used to determine the yeast total population. Indigenous yeast was grown at napa cabbage waste at 27, 37, and 40°C for three days, and cellulose-degrading activity was determined by the Dinitrosalicylic Acid (DNS) method. The potential yeast isolate with the highest cellulose-degrading activity was identified by a sequence analysis of the rRNA gene internal transcribed spacer (ITS) region with using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′- TCCTCCGCTTATTGATATGC-3′). The results were compared to the GenBank database using the Basic Local Alignment Search Tools/BLAST algorithm. Three species of indigenous yeast were isolated from napa cabbage waste (S2, S6, and S8). S8, incubated at 37ºC for three days, demonstrated the highest cellulose-degrading enzyme activity (1.188 U/mL), with the average activity of 0.684U/mL. Species identification results indicated that the S8 isolate had a 100% similarity to Pichia fermentans UniFGPF2 (KT029805.1).
Pichia fermentans, temperature, cellulase enzyme, internal transcribed spacer
INTRODUCTION
Napa cabbage (Brassica pekinesis L.) is one of the
most cultivated agricultural products in Indonesia. In
2014 its production reached 602 468 t. The local leader
in this field was the West Java province that yields
14.92 tons of napa cabbage per ha [1]. Since over 20% of
napa cabbage cannot be utilised [2], this waste amount
makes napa cabbage production inefficient.
Napa cabbage waste contains the same essential
component, namely, polysaccharides in the form of
cellulose, as napa cabbage itself. Cellulose is known
to be a constituent component of plant cell walls, and it
account for as much as 30–50% of total lignocellulose [3].
Currently, napa cabbage waste is used as animal
feed, while its value could be increased, e.g., through
production of cellulose-degrading enzymes.
Cellulose-degrading enzymes can be produced
from napa cabbage waste, which is high in cellulose
content, by yeast. Enzyme production by indigenous
cellulolytic yeast requires optimal conditions, however, it
is influenced by external factors, especially, temperature.
Thus, too low temperatures can inhibit enzyme
production because of the plasma membrane fluidity
decrease which leads to disturbed metabolic activity [4].
On the other hand, too high temperature can damage
cells and the structure of proteins, which are constituents
of enzymes. The fact that temperature is an easily
controlled parameter makes it possible to support yeast
growth during the fermentation of napa cabbage waste
for cellulose-degrading enzyme production. Therefore,
the aim of this research was to characterise and identify
indigenous yeast isolated from napa cabbage waste.
STUDY OBJECTS AND METHODS
The object of the research was napa cabbage waste
from the Gedebage Central Market in Bandung City,
Copyright © 2019, Utama et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International
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Foods and Raw Materials, 2019, vol. 7, no. 2
E-ISSN 2310-9599
ISSN 2308-4057
Research Article DOI: http://doi.org/10.21603/2308-4057-2019-2-321-328
Open Access Available online at http:jfrm.ru
Indigenous yeast with cellulose-degrading activity
in napa cabbage (Brassica pekinensis L.) waste:
Сharacterisation and species identification
Gemilang L. Utama* , Widia D. Lestari, Indira L. Kayaputri,
Roostita L. Balia
Universitas Padjadjaran, Bandung, Indonesia
* e-mail: g.l.utama@unpad.ac.id
Received July 31, 2019; Accepted in revised form August 27, 2019; Published October 21, 2019
Abstract: Napa cabbage waste contains an organic component, cellulose, which can be utilised as an ingredient for cellulose-degrading
enzyme production with the help of indigenous yeast. The aim of the research was to identify and characterise potential indigenous
yeast isolated from napa cabbage waste, which has cellulose-degrading activity. Indigenous yeast were isolated and characterised
using the RapID Yeast Plus System, then turbidity was used to determine the yeast total population. Indigenous yeast was grown
at napa cabbage waste at 27, 37, and 40°C for three days, and cellulose-degrading activity was determined by the Dinitrosalicylic
Acid (DNS) method. The potential yeast isolate with the highest cellulose-degrading activity was identified by a sequence analysis
of the rRNA gene internal transcribed spacer (ITS) region with using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4
(5′- TCCTCCGCTTATTGATATGC-3′). The results were compared to the GenBank database using the Basic Local Alignment Search
Tools/BLAST algorithm. Three species of indigenous yeast were isolated from napa cabbage waste (S2, S6, and S8). S8, incubated at 37ºC
for three days, demonstrated the highest cellulose-degrading enzyme activity (1.188 U/mL), with the average activity of 0.684U/mL.
Species identification results indicated that the S8 isolate had a 100% similarity to Pichia fermentans UniFGPF2 (KT029805.1).
Keywords: Pichia fermentans, temperature, cellulase enzyme, internal transcribed spacer
Please cite this article in press as: Utama GL, Lestari WD, Kayaputri IL, Balia RL. Indigenous yeast with cellulose-degrading
activity in napa cabbage (Brassica pekinensis L.) waste: Сharacterisation and species identification. Foods and Raw Materials.
2019;7(2):321–328. DOI: http://doi.org/10.21603/2308-4057-2019-2-321-328.
322
Utama G.L. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 321–328
Indonesia. We used the following materials: Potato
Dextrose Agar (PDA), Yeast and Mold Agar (YMA),
Nutrient Broth (NB), Thermo Scientific RapID
Yeast Plus System Kit, Carboxymethyle Cellulose
(CMC), distilled water, 0.85% NaCl, DNS reagent
(3.5-Dinitrosalicyclic acid), phosphate buffer solution
(pH 7), gelatin, antibiotics, KH2PO4, and MgSO4.
In our experiment we used nine treatments. Treatment
factors were the type of yeast isolate and incubation
temperature (Table 1). The isolation process of indigenous
cellulolytic yeast from each treatment lasted for three
days. The experiments were repeated three times.
The selection of the best treatment was performed
based on quantitative analysis by determining the
highest value of enzyme activity using Factorial
Randomized Block Design. According to the results of
isolation and identification of indigenous cellulolytic
yeast from napa cabbage waste, descriptive analysis on
the total population of yeast during the production of
cellulose-degrading enzymes was conducted.
Isolation and identification of indigenous yeast.
The isolation of indigenous cellulolytic yeast from napa
cabbage waste was carried out using the direct plating
method [5–6]. One gram of crushed napa cabbage
waste was added into 0.85% NaCl, inoculated into a
modified PDA (PDA with a 3% yeast extract and 10 ppm
antibiotics) and then incubated at 30°C for three days.
The biochemical activities of the selected isolates were
characterised with the help of RapID Yeast Plus System
Kit [7]. For species identification of potential indigenous
yeast with the highest cellulose-degrading enzyme
activity we used rRNA gene internal transcribed spacer
(ITS) region. Sequence analysis was carried out using
primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) as
forward and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)
as reverse. DNA amplification was performed by
Macrogen Inc. The results were compared with the
GenBank database using the BLAST algorithm [8].
Determination of total yeast population. Total
yeast population determination was carried out by
turbidimetry: 1 mL of the liquid culture was taken from
the enzyme production medium followed by absorbance
measurement [9]. This method is based on the
spectrophotometric measurement of the total population
at a wavelength of 600 nm [5].
Determination of cellulose-degrading enzyme
activity. Cellulose-degrading enzyme production was
carried out by the International Union’s recommended
method of Pure and Applied Chemistry (IUPAC)
with some modifications [10]. The salt media used
consisted of KH2PO4, Mg2SO4 and gelatin. The napa
cabbage waste was incubated in salt media at (1:2, w/v)
by adding 2% (v/v) of isolates [11]. The isolation was
carried out in an incubator at 27°C, 37°C and 45°C for
three days followed by stirring at 100 rpm for 60 min
at room temperature. Then every 24 h the fermented
solution was separated using a centrifuge to obtain crude
cellulose-degrading enzymes in supernatant, where
crude enzymes reacted with DNS (Dinitrosalicylic
Acid) reagent. Finally, spectrophotometric analysis was
carried out to obtain absorbance values which were used
to determine cellulose-degrading enzyme activity. The
control used was 3 mL of DNS reagent that was diluted
to 25 mL by distilled water.
RESULTS AND DISCUSSION
Characterisation of indigenous yeast. After
three days of incubation, eight isolates with different
characteristics were obtained (Table 2). S2, S6 and
S8 isolates displayed macroscopic morphological
characteristics similar to those of yeast.
Asliha and Alami state that macroscopically, yeast
are round, white, with membranous colony texture,
while microscopically, multilateral yeast bud and its
cell size ranges from 1 to 7 μm [12]. In addition, the
microscopic analysis allowed three isolates to be chosen
because they had cell size classified as that of yeast. An
average cell diameter of the S2, S6 and S8 was 3.87, 3.76
and 4.24 μm, respectively (Fig. 1). The selected isolates
Table 1 Treatment factors
Types of yeast isolates Incubation temperature
27°C 37°C 45°C
Isolate 1 (S2) A B C
Isolate 2 (S6) D E F
Isolate 3 (S8) G H I
Table 2 Characteristics of indigenous yeast isolates
Isolate Macroscopic characteristics
S1 Fungi, long white hyphae, aerobic, colonised
S2 Round, broken white coloured, wet, aerobic
S3 Round, broken white coloured, anaerobic
S4 Oval, broken white coloured, anaerobic
S5 Fungi, long white hyphae, aerobic, colonised
S6 Round, broken white coloured, aerobic
S7 Round, yellow, anaerobic
S8 Oval, yellow, anaerobic
Figure 1 Macroscopic and microscopic images of selected
indigenous yeast
9.35
5.11
4.34
0
2
4
6
8
10
0 0,5 1 1,5 2 2,5 3 3,5
× 109 CFU/mL
days
S2
11.31
12
S6
323
Utama G.L. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 321–328
were purified, and their biochemical activities were
tested using the RapID Yeast Plus System (Table 3).
The identification results are based on the
biochemical properties of the isolates tested against
the reacted compound. The glucose or glucoside
hydrolysis ability was only shown by S2 isolates against
β-Glucoside and β-Fucoside. Lopez et al. states that
several non-Saccharomyces yeast could be found in
soil, fruits, trees, and damaged food or drink that has
glycolytic β-glucosidase activity [13]. The biochemical
properties of indigenous yeast (Table 5) are also
supported by Mateo et al., who found the glycolytic
activity, especially β-glucosidase activity, on indigenous
yeas [14]. It implies that glucose can be hydrolysed into
acidic compounds which reduce pH until it changes the
colour of the resultants. Macroscopically, two isolates
identified as S6 and S8 had different characteristics.
They differed from the characteristics of Candida sp.
that has the anamorphous properties, does not have a
sexual reproduction phase, and has unstable phenotypic
characteristics [15]. Therefore, although the two isolates
were different in form, colour, and oxygen requirements,
they had the same biochemical activity.
Total population of indigenous yeasts. The results
of indigenous yeasts total population determination
during incubation are demonstrated in Fig. 2. During
day 1 of incubation, the total population in all
treatments decreased because the isolates still were
in the adaptation phase in the medium. This phase is
called the lag phase or the cell adaptation period of
new microorganisms to the environment [16]. Nguong
et al. states that it takes 16 h for yeast with biochemical
activity similar to that of the S2 isolate to adjust to a new
environment [17].
After the adaptation phase, the total population of
all treatments increased. The increase is the exponential
growth phase, where cells of microorganisms have
adapted to the environment and began to multiply so
that the number of mass cells or cell density increases
rapidly [16]. Spectrometric analysis preformed by
Kanti et al. revealed that the population of indigenous
yeast, such as Candida, Rhodothorula, Pichia, and
Debaryomyces, began to increase from 24th h and
reached a plateau by the 96th h (OD 600 nm) [5].
The highest total population was observed at the
incubation temperature of 27°C in all the treatments.
Mateo et al. state that the biochemical activity of the
indigenous yeast was maximal at the temperature of
30–40°C [14]. However, the isolate that belongs to the
Hanseniaspora genus that has biochemical activity
Table 3 Identification of indigenous yeast from napa cabbage
waste
Isolate S2 S6 S8
Glucose + + +
Maltose – – –
Sucrose – – –
Trehalose – – –
Raffinose – – –
Lipid – – –
NAGA – – –
αGlucoside – – –
βGlucoside + – –
ONPG – – –
αGalactoside – – –
βFucoside + – –
PHS – – –
PCHO – – –
Urea – – –
Prolyne – – –
Histidine + + +
Leucyl–Glycine – – –
(+) assimilates the substrate positively; and (-) assimilates the
substrate negatively
Figure 2 Total population of indigenous yeast: (1) – 27°C,
(2) – 37°C, (3) – 45°C
9.35
5.11
4.34
0
2
4
6
8
10
0 0,5 1 1,5 2 2,5 3 3,5
× 109 CFU/mL
days
S2
11.31
5.75
3.67
0
3
6
9
12
0 1 2 3
× 109 CFU/mL
days
S6
10.88
8.27
4.36
0
3
6
9
0 1 2 3
× 109 CFU/mL
days
S8
9.35
5.11
4.34
0
2
4
6
8
10
0 0,5 1 1,5 2 2,5 3 3,5
× 109 CFU/mL
days
S2
11.31
5.75
3.67
0
3
6
9
12
0 1 2 3
× 109 CFU/mL
days
S6
10.88
8.27
4.36
0
3
6
9
0 1 2 3
× 109 CFU/mL
days
S8 (1)
(2)
(3)
(1)
(2)
(3)
(1)
(2)
(3)
D
D
D
324
Utama G.L. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 321–328
similar to that of the S2 isolate had the maximum
biochemical activity at 28°C. Meanwhile, according to
Gänzle et al., a representative of the Candida genus with
biochemical activity similar to that of S6 and S8 isolates
grew rapidly at 27°C [18].
Cellulose-degrading enzyme activity. Cellulosedegrading
enzyme activity of indigenous yeast
is shown in Fig. 3. The highest enzyme activity
produced by S2 was 0.598 U/mL at an incubation
temperature of 45°C. The high temperature caused
an increase in the rate of biochemical reactions,
especially for indigenous yeast that has similar
biochemical activity with Hanseniaspora. Fennema
states that a high temperature affect various
reactions [19]. The enzyme belongs to the group of
mesozyme enzymes (in the range of 20–50°C) [20].
According to López et al., the glycolytic activity
(β-glucosidase) of H.guilliermondii at 28°C is about
0.064–2.887 U/mL [13, 21].
S6 isolates obtained at the incubation temperature
of 45°C also displayed a high enzyme activity. It is
because the growth of Candida-like organisms occurred
at the maximum temperature (40–45°C) [20]. As stated
by Shuler and Kargi, enzymes are Growth-associated
products, i.e. the growth of microorganisms is directly
proportional to the product concentration [16]. However,
the S8 isolate demonstrated the highest enzyme activity
when treated at 37°C: its value was 1.203 U/mL on day1
and 1.188 U/mL on day 2.
Table 4 shows analysis of variance. F-value was
greater than Pvalue probability (0.05), which indicated
the presence of at least one treatment that significantly
differed from the others. Hence, it required an additional
test, namely, the Duncan Test.
Table 5 demonstrates the Duncan Test results.
According to the data, the S8 treatment incubated at
37°C produced enzyme with an activity significantly
differing from the other treatments. This is in accordance
with the result of Sulman and Rehman, that Candidalike
organisms are able to produce cellulose-degrading
enzymes with the highest activity at 37°C [11]. The
growth of isolates at 27°C cannot produce enzymes
with high activity because energy supply from the
environment is low, while at 45°C the growth of isolates
is inhibited and the structure of the enzyme is denatured
so that the activity is not optimal. Therefore, incubation
at 37°C gives enough energy for isolates to grow without
damaging the structure of the enzyme produced.
Temperature greatly influences the enzymatic
activity and rigorous of yeast cell membranes, and
Figure 3 Cellulose-degrading enzyme activity: (1) – 27°C,
(2) – 37°C, (3) – 45°C
Table 4 Analysis of variance
Source df Sum of
squares
Mean
square
F-value P-value
Isolate (I) 8 0.779 0.097 89.722* 2.07
Temperature (T) 3 0.539 0.108 165.668* 2.74
I*T 24 0.837 0.035 32.118* 1.67
Replication 2 0.009 0.005 4.249 3.13
Error 70 0.076 0.001
Total: 107 2.240
*significant
0.259
0.115
0.598
0.0
0.2
0.4
0.6
0 1 2 3
U/ml
days
S2
0.125
0.142
0.879
0.9
2 3
days
S6
0.168
1.188
0.893
0.0
0.4
0.8
1.2
0 1 2 3
U/ml
days
S8
0.259
0.115
0.598
0.2
0.4
0.6
1 2 3
U/ml
days
S2
0.125
0.142
0.879
0.0
0.3
0.6
0 1 2 3
U/ml
days
S6
0.168
1.188
0.893
0.0
0.4
0.8
1.2
0 1 2 3
U/ml
days
S8
0.0
0 0.9
(1)
(2)
(3)
(1)
(2)
(3)
(1)
(2)
(3)
Table 5 Duncan test results
Yeast Temperature Average cellulose-degrading
enzyme activity (U/mL)
Significance
S8 37°C 0.684 a
S8 45°C 0.395 b
S6 45°C 0.384 b
S2 45°C 0.315 c
S2 27°C 0.196 d
S2 37°C 0.160 de
S6 37°C 0.146 e
S6 27°C 0.131 e
S8 27°C 0.129 e
The treatment marked with the same sign shows no significant
difference at the level of 5% according to the Duncan test
D
D
D
325
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S8 (Query 190511)
Figure 4 Phylogenetic tree of S8 isolate
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higher temperature can shorten the exponential phase
of yeast growth. In addition, higher temperature can
cause denaturation of ribosomes and membrane fluidity
problems. Thus, 30–35°C is the optimal temperature for
yeast metabolism, including the enzymatic activity [22].
The difference in S2 and S8 enzyme activities was
due to their different biochemical abilities. Lopez et al.
found that Hanseniaspora sp., which is similar to the
S2 isolate, was able to assimilate glycerol, galactose
and sucrose, unlike with Candida sp., which is similar
to S8 [21, 23].
The different activity of the enzyme produced by
S6 and S8 could be caused by different phylogenetics
between the two isolates. According to Birmeta et al.,
Candida sp. that was mentioned as C. krusei has close
proximity to P. fermentans having certainly different
biochemical ability than C. krusei [24]. P. fermentans
has an anamorphic form, Candida lambica, but it is
not uncommon to find C. lambica mis-identification as
C. krusei caused by similar biochemical abilities of the
yeast. Nevertheless, C. lambica is able to assimilate
xylose, compared to C. krusei cannot [25]. Meanwhile,
the ability of yeast to assimilate xylose has not been
determined by the RapID Yeast Plus System method, so
differences between C. krusei and C. lambica have not
been identified.
Species identification of potential indigenous
yeast with the highest cellulose-degrading activities.
The identification of the S8 isolate resulted in the
100% similarity to P. fermentans strain UniFGPF2
(KT029805.1). The phylogenetic tree (Fig. 4) shows
that the S8 isolate is also similar to P. kluyveri culture
CBS:188 (KY104555.1), P. fermentans strain UniFGPF1
(KT029804.1), P. fermentans strain UFLA CWFY24
(KM402062.1), and P. fermentans strain YF12b
(EU488722.1, DQ674358.1).
P. fermentans have the ability to ferment and
assimilate glucose, D-xylose, succinate, lactate, citrate,
and glycerol [24]. Candida lambica is an anamorphic
form of P. fermentans which can assimilate glucose
and xylose but cannot assimilate arabinose, galactose,
and selobiosa [26]. In addition, Issatchenkia orientalis,
a teleomorphic form of Candida krusei that usually
incorrectly identified as Candida lambica, can assimilate
glucose sufficiently but cannot assimilate galactose,
maltose, sucrose, lactose, raffinose, and trehalose [27].
According to Bengoa et al., despite P. fermentans
and C. lambica can growth at a temperature up to
37°C, the optimal temperature is 25–30°C [28]. Such
strain as I. orientalis has the unique properties, as this
microorganism can grow at a higher temperature level.
Miao et al. reported that I. orientalist strains optimally
grows and produces a high amount of ethanol at 41°C,
which indicates its thermostability [29].
CONCLUSION
Three species of indigenous yeast were isolated
from napa cabbage waste. The highest cellulosedegrading
enzyme activity (1.188U/mL) displayed the
S8 isolate incubated at 37°C for three days. Its average
cellulose-degrading activity was 0.684U/mL. According
To the species identification, the S8 isolate showed
a 100% similarity to Pichia fermentans UniFGPF2
(KT029805.1).
CONFLICT OF INTEREST
The authors declare no conflict of interests.
ACKNOWLEDGMENTS
The authors would like to thank the Student
Research Group, Vivi Fadila Sari, Isfari Dinika and
Syarah Virgina for their help with the experiments
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