International Journal of Food Microbiology 192 (2015) 7–12

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Glutaminase-producing Meyerozyma (Pichia) guilliermondii isolated from

Thai soy sauce fermentation
Phichayaphorn Aryuman a, Sittiwat Lertsiri a, Wonnop Visessanguan b, Nuttawee Niamsiri a,
Amaret Bhumiratana a, Apinya Assavanig a,⁎
a
Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand
b
National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Phaholyothin Rd., Khlong Nueng, Khlong Luang, Pathumthani 12120, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: In this study, 34 yeast isolates were obtained from koji and moromi samples of Thai soy sauce fermentation. How-
Received 25 March 2014 ever, the most interesting yeast strain was isolated from the enriched 2 month-old (M2) moromi sample and
Received in revised form 17 August 2014 identified as Meyerozyma (Pichia) guilliermondii EM2Y61. This strain is a salt-tolerant yeast that could tolerate
Accepted 15 September 2014
up to 20% (w/v) NaCl and produce extracellular and cell-bound glutaminases. Interestingly, its glutaminases
Available online 22 September 2014
were more active in 18% (w/v) NaCl which is a salt concentration in moromi. The extracellular glutaminase's
Keywords:
activity was found to be much higher than that of cell-bound glutaminase. The highest specific activity and
Soy sauce stability of the extracellular glutaminase were found in 18% (w/v) NaCl at pH 4.5 and 37 °C. A challenge test by
Salt-tolerant yeast adding partially-purified extracellular glutaminase from M. guilliermondii EM2Y61 into 1 month-old (M1)
Meyerozyma (Pichia) guilliermondii moromi sample showed an increased conversion of L-glutamine to L-glutamic acid. This is the first report of
Glutaminase glutaminase producing M. guilliermondii isolated from the moromi of Thai soy sauce fermentation. The results
Moromi suggested the potential application of M. guilliermondii EM2Y61 as starter yeast culture to increase L-glutamic
acid during soy sauce fermentation.
© 2014 Published by Elsevier B.V.

1. Introduction that converts L-glutamine originated from soy protein to L-glutamic

Meyerozyma (Pichia) guilliermondii belongs to the group of so-called
“flavogenic yeasts” (Tanner et al., 1945). Although, it is regarded as a
contaminant producing off-flavor compound, i.e. 4-ethylphenol, during
red wine production (Chatonnet et al., 1995; Dias et al., 2003), it is one
of the dominant yeasts found in koji and moromi of Japanese and
Chinese fermented soybeans (Kim et al., 2010). Wah et al. (2013)
reported that M. guilliermondii EM1Y52 produced volatile phenol
compounds and could also enhance the volatile flavor compound
production of Zygosaccharomyces rouxii in Thai soy sauce fermentation.
Soy sauce is made by fermentation of soybean combined with wheat
flour, rice flour, and brine (Fukushima, 2004; Mongkolwai et al., 1997).
Its production involves koji culture using Aspergillus oryzae and moromi
fermentation by adding brine solution into the koji. During koji culture,
A. oryzae mainly secretes protease and amylase to break down protein
and carbohydrate in soybean and flour into peptides, amino acids and
sugars, respectively (Mongkolwai et al., 1997). In addition to those en-
zymes, glutaminase is an important enzyme in soy sauce fermentation
⁎ Corresponding author at: Department of Biotechnology, Faculty of Science, Mahidol
University, 272 Rama VI Rd., Rachathevi, Bangkok 10400, Thailand. Tel.: + 66 2
2015300; fax: +66 2 3547160.
E-mail address: apinya.ass@mahidol.ac.th (A. Assavanig).
acid (Fukushima, 2004). Therefore, increasing production of glutaminase
during soy sauce fermentation could result in an increase of L-glutamic
acid which imparts so-called “umami” taste and improves the flavor in
soy sauce (Ohshita et al., 2000; Yano et al., 1988). L-Glutaminase in soy
sauce fermentation has been known to be produced by A. oryzae, howev-
er this fungal enzyme is markedly inhibited by high NaCl concentration
of 20–22% (w/v) which is the common condition in moromi fermenta-
tion (Yano et al., 1988). Meanwhile, salt-tolerant yeast involving in
moromi fermentation, Z. rouxii has been reported for its glutaminase
that can tolerate high salt concentration (Iyer and Singhal, 2008). Many
studies on the physiological roles, enzymatic properties, and application
of glutaminase from various microorganisms have been conducted,
including Micrococcus luteus (Moriguchi et al., 1994), Bacillus pasteurii
(Klein et al., 2002), Lactobacillus rhamnosus (Weingand-Ziade et al.,
2003), Lactobacillus reuteri KCTC3594 (Jeon et al., 2009), Saccharomyces
cerevisiae (Penninckx and Jaspers, 1985), Cryptococcus albidus (Iwasa
et al., 1987) and Debaryomyces sp. (Durá et al., 2002). In this study,
the production of glutaminase in salt-tolerant yeast M. guilliermondii
isolated from Thai soy sauce moromi is first reported. Since
M. guilliermondii is GRAS (Generally Recognized as Safe) organism
(Sibirny and Boretsky, 2009), understanding of its glutaminase produc-
tion would be beneficial in the development of the starter culture for
soy sauce fermentation.

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.019
0168-1605/© 2014 Published by Elsevier B.V.
8 P. Aryuman et al. / International Journal of Food Microbiology 192 (2015) 7–12
2. Materials and methods
2.1. Isolation of salt-tolerant yeasts from koji and moromi samples
Salt-tolerant yeasts were isolated from koji and moromi fermented
at 1 month (M1), 2 months (M2), and 3 months (M3) samples were
collected from a soy sauce factory in Samutprakan province, Thailand.
Isolation process was performed at 30 °C using G-medium (per L: 10 g
L-glutamine, 1 g K2HPO4, 1 g KH2PO4, 1 g NaCl, 0.5 g yeast extract,
0.1 g MgSO4·7H2O, 15 g agar, pH 7.0) (Wakayama et al., 2005) and
enrichment in YPD containing 10% (w/v) NaCl (10YPD) (Shumin et al.,
2012; Renouf and Lonvaud-Funel, 2007; Wah et al., 2013). All yeast col-
onies found were microscopically investigated. The isolated yeasts were
further determined for salt tolerance by measuring the growth
(OD600nm ≥ 0.5) in YPD broth supplemented with 0, 10, 15, 18, 20%
(w/v) NaCl, at 30 °C under shaking condition for 28 days. The yeast
isolates grown in NaCl concentration (≥15%) were determined for glu-
taminase production. Molecular identification of selected salt-tolerant
yeast producing the highest glutaminase was performed based on
sequence analysis of the D1/D2 domain on 26S rDNA (Ferreira et al.,
2010).
2.2. Investigation of extracellular, intracellular, and cell-bound glutaminase
activity in salt-tolerant yeast isolates
The selected salt-tolerant yeast isolates were cultured in 125 mL
Erlenmeyer flask containing 25 mL of YPD broth, incubated at 30 °C
with shaking (180 rpm) for 12 to 16 h. Cells were harvested by centrifu-
gation (3000 ×g, 4 °C, 15 min) and washed twice with 0.05 M phos-
phate buffer, pH 7.0. The cell concentration was adjusted to OD600 nm
1.5 before inoculation at 1% (v/v) inoculum into 100 mL of G-broth
(Wakayama et al., 2005) in 250 mL Erlenmeyer flask. After incubation
at 30 °C with shaking for 3 days, the supernatant was separated from
the yeast cells by centrifugation and determined for extracellular gluta-
minase activity. The yeast cells were washed once in sterile distilled
water. One gram of cell pellet was suspended in 1 mL of 50 mM Tris–
HCl buffer containing 5 mM EDTA, pH 7.5 and then disrupted by homog-
enization at 4 °C with equal weight of glass beads at 1-min burst with an
intermediary pause of 1 min. The disrupted cells were observed under
microscope (Durá et al., 2002). After centrifugation at 8000 × g, 4 °C
for 10 min, the liquid portion of cell lysate was subjected to determine
the intracellular glutaminase activity. Cell-bound glutaminase fraction
was prepared according to Soberón and González (1987). Briefly, cell
debris was separated from the cell lysate and then solubilized in the
Tris–HCl buffer containing 0.1% (v/v) Triton X-100 followed by centrifu-
gation at 8000 ×g, 4 °C for 10 min. The supernatant obtained was deter-
mined for cell-bound glutaminase activity.
2.3. Investigation of optimal pH, temperature, NaCl concentration, and
stability of crude extracellular glutaminase activity from M. guilliermondii
EM2Y61
The optimum pH for crude extracellular glutaminase activity was
assayed at 37 °C in Britton–Robinson's buffer (equi-volume of 0.04 M
boric acid, acetic acid, and phosphoric acid solution) containing
100 mM L-glutamine with and without 18% (w/v) NaCl in the pH
range of 3 to 9. For pH stability study, 10 mL of filter-sterilized extracel-
lular glutaminase fraction (Minisart Filters, 0.45 μm) were mixed with
40 mL of Britton–Robinson's buffer containing 18% (w/v) NaCl and incu-
bated at 37 °C for 72 h (Durá et al., 2004). The remaining glutaminase
activity was assayed under 18% (w/v) NaCl, at pH 4.5, 37 °C. The optimum
temperature and temperature stability were determined by incubating
the reaction mixture at 25, 30, 37, 40, 45 and 50 °C, pH 4.5, with and
without 18% (w/v) NaCl. The optimum NaCl concentration and stability
to NaCl concentration (i.e. 0, 5, 10, 15, 18 and 20% w/v) for extracellular
glutaminase activity were assayed at 37 °C, pH 4.5. Glutaminase
stability to NaCl concentration was monitored during 48 h of dialysis
against 100 volumes of Britton–Robinson's buffer with various NaCl
concentrations at 37 °C. The remaining glutaminase activity was
assayed at pH 4.5, 37 °C in the presence of 18% (w/v) NaCl.
2.4. Production and partial purification of extracellular glutaminase from
M. guilliermondii EM2Y61
Crude extracellular glutaminase was harvested from culture of the
selected yeast isolate in YPD broth containing 1% L-glutamine (w/v),
pH 4.5 by centrifugation. The enzyme was partially purified according
to the method of Kingcha et al. (2012). Briefly, 1000 mL of the superna-
tant was mixed with 20 g of Amberlite XAD-16 (Sigma-Aldrich, St. Louis,
USA) and stirred at 4 °C for 12 h. Amberlite was activated with 50% (v/v)
isopropanol and rinsed with deionized water prior to use. The slurry
was then loaded onto an Econo fast flow column (2.5 i.d. × 50 cm)
and washed with deionized water. Glutaminase fraction was eluted
with 5 mL 50% (v/v) ethanol and concentrated by a rotary evaporation.
2.5. Glutaminase activity assay
Glutaminase activity assay was based on the measurement of
L -glutamic acid produced under the defined conditions (Durá et al.,
2002). The reaction mixture containing 1.8 mL of 100 mM L-glutamine
in 50 mM sodium phosphate buffer (pH 7.0) with or without 20% (w/v)
NaCl was pre-incubated at 37 °C for 3 min before adding 0.2 mL of
crude enzyme sample. The reaction mixture was incubated at 37 °C for
30 min and stopped by adding 2 mL of 10% (w/v) trichloroacetic acid
(TCA) and incubating at 25 °C for 2 h (Sivaraman et al., 1997). After
centrifugation at 1000 ×g, 4 °C for 20 min, the supernatant was adjusted
to pH 8.6 with 10 N KOH prior to determination of L-glutamic acid using
L-glutamic acid assay kit (R-Biopharm AG, Germany). For the control
experiment, a sample was firstly added with 10% TCA to denature the
enzyme prior to adding L-glutamine substrate and measurement of
L -glutamic acid by the test kit. One unit (U) of glutaminase activity
was defined as the amount of enzyme that catalyzed the formation of
1 μmolL-glutamic acid per min at 37 °C, pH 7.0. Protein concentration
was determined by dyeing method using bovine serum albumin
(BSA) as a standard (Bradford, 1976).
2.6. Demonstration of glutaminase activity of M. guilliermondii EM2Y61 in
moromi model system
Fifty-mL portion of 1-month moromi (M1) was added with 100 mM
L-glutamine and partially-purified glutaminase to obtain the final con-
centration of 0.5 U, and incubated at 37 °C for 60 min. Glutaminase
was partially purified as mentioned above. Samples were collected at
0, 15, 30, 45 and 60 min of incubation and centrifuged at 3000 × g,
4 °C for 20 min (Kijima and Suzuki, 2007) prior to the determination
of L-glutamic acid by L-glutamic acid test kit.
2.7. Statistical analysis
All experiments were run in triplicate. A completely randomized de-
sign was used throughout this study. Data were subjected to one-way
analysis of variance and mean comparison with Duncan's test using
Statistic Package for Social Sciences (SPSS for Windows: SPSS Inc.,
Chicago, IL, USA).
3. Results and discussion
3.1. Isolation of M. guilliermondii from soy sauce fermentation process and
its production of glutaminase
Among 34 salt-tolerant yeasts (11 isolates from G-medium and 23
isolates from enrichment 10YPD medium), only 26 isolates (76.5%)
 P. Aryuman et al. / International Journal of Food Microbiology 192 (2015) 7–12 9

Fig. 1. Distribution of glutaminase activity in various cellular fractions of salt-tolerant yeast isolates. The activity was analyzed at pH 7.0, 37 °C for 30 min in 18% (w/v) NaCl. 1 U is defined as
the amount of glutaminase that catalyses the formation of 1 μmolL-glutamic acid per minute at 37 °C, pH 7.0. Total activity of extracellular glutaminase was defined as total units in
supernatant harvested. Total activity of intracellular glutaminase was defined as total units in total intracellular fraction obtained from cell lysis. Total activity of cell-bound glutaminase
was defined as total units in supernatant obtained after solubilization of cell debris.

Fig. 2. (a) pH profile; (b) pH stability of crude extracellular glutaminase activity of M. guilliermondii EM2Y61. Bars represent the standard deviation from triplicate experiments.
10 P. Aryuman et al. / International Journal of Food Microbiology 192 (2015) 7–12
produced glutaminase in G-medium. They were identified as
Zygosaccharomyces sp. (4 isolates, 15.4%), Debaryomyces sp. (15 isolates,
57.7%) and Pichia sp. (7 isolates, 26.9%) based on their cell morphology
and arrangement under light microscope and colonial appearance, ac-
cording to Kurtzman et al. (2011). All 26 yeast isolates grew best in
YPD broth, however they could slowly grow in YPD containing NaCl
up to 15% (w/v) and could survive in NaCl higher than 15%. Among
these isolates, only 3 isolates (EM2Y61, EM1Y52 and EKY62) could
grow in NaCl up to 20% and produced remarkably high glutaminase
activity.
From the results, glutaminase activity was variously distributed in
different cellular fractions as the extracellular, intracellular, and cell-
bound enzymes depending on the isolates (Fig. 1) when they were
cultivated in G-medium (Wakayama et al., 2005). Interestingly,
among 26 yeast isolates, 9 isolates produced 2 types and 17 isolates pro-
duced only one type of glutaminase. All of these glutaminase enzymes
were active in the presence of 18% (w/v) NaCl which is the NaCl concen-
tration employed in soy sauce moromi fermentation (Mongkolwai et al.,
1997). Moreover, the extracellular glutaminase activity was shown to
be the highest compared to intracellular and cell-bound glutaminase
activity. In yeasts, glutaminase was mostly found in intracellular frac-
tions, i.e. Debaryomyces spp. (Durá et al., 2002); C. albidus; S. cerevisiae
(Iwasa et al., 1987) and membrane-bound in S. cerevisiae (Iwasa et al.,
1987). Only halophilic yeast, i.e. Z. rouxii, which involves in moromi fer-
mentation, has been reported to produce extracellular glutaminase
(Iyer and Singhal, 2008). A. oryzae which takes part in koji fermentation
also produces extracellular glutaminase (Iyer and Singhal, 2008; Yano
et al., 1988). But A. oryzae glutaminase is markedly inhibited by
18% (w/v) NaCl whereas Z. rouxii glutaminase is partially inhibited by
18–20% (w/v) NaCl (Nandakumar et al., 2003). Among 3 isolates
(EM2Y61, EM1Y52 and EKY62), the isolate EM2Y61 that was obtained
Fig. 3. (a) Temperature profile; (b) temperature stability of crude extracellular glutaminase activity of M. guilliermondii (EM2Y61). Bars represent the standard deviation from triplicate
experiments.
from enriched culture of M2 moromi exhibited the highest extracellular
glutaminase activity (Fig. 1). It was noticed that these yeast isolates
were moderately halotolerant similar to Z. rouxii (Iyer and Singhal,
2008), S. cerevisiae (Silva-Graca et al., 2003), D. hansenii (Durá et al.,
2002) and P. guilliermondii (Butinar et al., 2005) which able to grow in
up to 18% (w/v) NaCl. Based on the partially amplified D1/D2 domain
of 26S rDNA sequence, these isolates of EM2Y61, EM1Y52 and EKY62
were positioned within the genus Meyerozyma (Pichia) and were
identical to U45709 of the type strain (NRRL Y-2075) of M.(Pichia)
guilliermondii. Hence, this study is the first report of salt-tolerant
M. guilliermondii producing the extracellular glutaminase.
To explore the possibility of using M.(Pichia) guilliermondii EM2Y61
(GenBank accession number: JX568888) for starter culture develop-
ment, optimal conditions and stability of crude extracellular glutamin-
ase of EM2Y61 were investigated. The optimum pH which was 4.5 of
this glutaminase was lower than those reported in other yeasts, i.e.
pH 6.0 of glutaminase from Candida albicans (Iwasa et al., 1987);
pH 7.5 of membrane-bound glutaminase A and pH 8.1 of intracellular
glutaminase B from S. cerevisiae (Soberón and González, 1987); and
pH 8.5 of glutaminase from Debaryomyces spp. (Durá et al., 2002). In
addition, the higher glutaminase activity of EM2Y61 was observed in
the reaction containing18% (w/v) NaCl at pH 4.5 (Fig. 2a). During
moromi fermentation, the pH of moromi liquid drops from 6.5–7.0 to
4.0–5.0 due to lactic acid bacteria such as Tetragenococcus halophilus,
converting sugars to acids. This acidic pH range is the suitable condition
for yeast growth during moromi fermentation (Kanbe and Uchida,
1982). The crude extracellular glutaminase of M. guilliermondii
EM2Y61was stable with the half-life longer than 12 h when incubated
at pH 3 to 5, at 37 °C. The activity lasted long for 24 h at pH 3 to 4.5
(Fig. 2b). The activity rapidly decreased at pH higher than 4.5. Since
the activity was relatively stable at the pH close to the optimum pH
 P. Aryuman et al. / International Journal of Food Microbiology 192 (2015) 7–12
(i.e. pH 4.5), this finding suggested that the native conformation and bi-
ological activity of the enzyme might be maintained in the acidic pH
(Frankenberger and Johnson, 1982). The glutaminase stability has
been reported: glutaminase from Debaryomyces spp. was isolated
from cure meat products showing the stability at pH 7.5–8.5 but it
was not stable at the acidic pH (Durá et al., 2004); glutaminase from
Stenotrophomonas maltophilia NYW-81 was isolated from soil samples
being active at pH 9 and having relative activities from 60–80% between
pH 5 and 10 (Wakayama et al., 2005). Such acid-stable property proba-
bly depended upon where the enzyme-producing microorganisms
were isolated. The pH of our moromi liquid at 4.5–5 was probably suit-
able for optimal activity and stability of EM2Y61 glutaminase.
The optimum temperature of crude extracellular glutaminase was
37 °C which was lower than those reported in other yeasts, such as
70 °C in C. albicans (Iwasa et al., 1987) and 40 °C in Debaryomyces spp.
(Durá et al., 2002). The optimal activity at 37 °C was observed in both
0 and 18% (w/v) NaCl but the latter was higher (Fig. 3a). The glutamin-
ase activity was gradually declined during 12 h of incubation at
25–37 °C with the residual activities of 90% at below 30 °C, and 85% at
37 °C. The half-life of the activity at 40–50 °C was longer than 12 h,
whereas it was more stable at below 37 °C (Fig. 3b). In contrast, gluta-
minase from Debaryomyces spp. was unstable at 37 °C with a half-life
as short as 7.4 h comparedto the highest stability at 25 °C with a half-
life of 135.8 h (Durá et al., 2004). C. albidus glutaminase has the activity
that remained higher than 90% after incubation at 60 °C (Sato et al.,
1999). On the other hand, S. cerevisiae glutaminase A (membrane-
bound glutaminase) was reported to be thermostable, but the B isozyme
(intracellular glutaminase) is thermolabile (Iwasa et al., 1987; Soberón
and González, 1987).
Fig. 4. (a) Salt concentration profile; (b) salt stability of extracellular glutaminase activity
of M. guilliermondii (EM2Y61). Bars represent the standard deviation from triplicate
experiments.
11
The EM2Y61 glutaminase in our study was relatively stable when
incubated in the presence of 15–20% (w/v) NaCl concentration for
12 h, with the residual activities of 85–90%, compared to 65–75% in 0–
10% (w/v) NaCl (Fig. 4b). The glutaminase activity remained more
than 50% after 24 h in the presence of NaCl concentration higher than
10% (w/v). The results showed that the specific glutaminase activity of
EM2Y61 was cultured in YPD medium, and could be increased from
0.36 to 0.43 U/mg protein when increasing the concentration of NaCl
from 0 to 18% (w/v) (Fig. 4a). However, the activity slightly declined
under 20% (w/v) NaCl (0.36 U/mg protein). The optimal NaCl concen-
tration for glutaminase activity during cultivation of M. guilliermondii
EM2Y61 was in agreement with those from C. albicans (Iwasa et al.,
1987), Cryptococcus nodaensis and Cryptococcus famata(Sato et al.,
1999). On the other hand, no activity of glutaminase A (membrane-
bound glutaminase) and B (intracellular glutaminase) from S. cerevisiae
(Soberón and González, 1987) and intracellular glutaminase from
Debaryomyces spp. were observed in 3% (w/v) NaCl (Durá et al., 2002).
3.2. Implication of M. guilliermondii glutaminase in moromi model system
Contents of L-glutamine in moromi samples determined by HPLC
were 2.10, 1.95, and 1.43 mM for M1, M2, and M3, respectively. A
large amount of L-glutamine in moromi was liberated from the soy pro-
tein, (Nakadai and Nasuno, 1989) into the mash through the actions of
proteinases and peptidases. Hence, we assumed that L-glutamine in the
moromi was a possible substrate for glutaminase that produced during
moromi fermentation. Since the highest concentration of L-glutamine
was observed in 1 month-moromi, it is possible to increase L-glutamic
acid by addition of extracellular glutaminase at this stage. Therefore,
the addition of partially-purified extracellular glutaminase from
EM2Y61 was challenged in the 1-month moromi model with a supple-
mentation of 100 mM L-glutamine. In the presence of 0.5 U of
M. guilliermondii EM2Y61 glutaminase, the concentration of L-glutamic
acid was significantly increased with the increasing time and reached
the maximum concentration at 60 min, which was 3 times higher than
the control with no addition of glutaminase (Fig. 5). The results demon-
strate the action of glutaminase to convert L-glutamine to L-glutamic acid
during moromi fermentation. However, the amount of L-glutamine
would be a limiting factor for the enhancement of L-glutamic acid
production. The results suggested that glutaminase activity could be
intensified by direct addition of glutaminase or inoculation of
glutaminase-producing starter culture. The application could be done
by using M. guilliermondii EM2Y61 as one of the yeasts in the starter
culture to increase L-glutamic acid during soy sauce fermentation.
Fig. 5. Effect of the addition of partially-purified extracellular glutaminase from
M. guilliermondii (EM2Y61) in 1-month moromi sample.
12 P. Aryuman et al. / International Journal of Food Microbiology 192 (2015) 7–12
4. Conclusions
Halotolerant yeast, M.(Pichia) guilliermondii EM2Y61, isolated from
Thai soy sauce fermentation could grow and produce extracellular glu-
taminase with high activity under high salt concentration. The highest
enzyme activity and stability were observed at pH 4.5, in 18% (w/v)
NaCl, at 37 °C. The results suggested that this enzyme could be active
during the moromi fermentation of Thai soy sauce production. There-
fore, M. guilliermondii EM2Y61 has potential to be used as a starter
culture at the early stage of moromi fermentation.
Acknowledgments
This work was supported by the Commission of Higher Education
in Thailand (CHE) and The Thailand Research Fund (grant no.
DBG5380022). Special thanks go to Dr. Thunyarat Pongtharangkul,
Department of Biotechnology, Faculty of Science, Mahidol University
for helpful suggestions on identification techniques, Ms. Supawan
Walaisri for co-operating in various microbiological techniques and
soy sauce factory in Thailand for kindly providing the koji and moromi
samples.
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