Vol.4, No.5B, 136-140 (2013) Agricultural Sciences
Isolation of aromatic yeasts(non-Saccharomyces
cerevisiae) from Korean traditional nuruks and
identification of fermentation characteristics
Choi Ji Ho, Soo Hwan Yeo, Ji-Hye Park, Han Seok Choi, Ji-Eun Gang, Soo In Kim,
Seok Tae Jeong, So Ra Kim
Fermentation & Food Processing Division, Department of Agro-food Resource, NAAS, RDA, Suwon, Korea
Received 2013
Ethyl caproate and isoamyl alcohol are impor-
tant for the quality of Yakju, one of the Korean
traditional alcoholic beverages. From Korean
traditional fermentation agent, Nuruk, we have
isolated 8 yeasts which produced rich aromatic
compounds using YEPD agar plates (1% yeast
extract, 2% peptone, 2% dextrose, 2% agar)
containing 50 uL cerulenin at 30. The isolated
aromatic yeasts are identified as Pichia anomala
(4 strains), Pichia fabianii (2 strains), Pichia
farinose (1 strains), Geotrichum candidum (1
strains). We conducted alcohol fermentation
with each of the aromatic yeasts and the com-
pounds (ethyl caproate and isoamyl alcohol)
producing range were 59.5 - 193.2 ppm and 10.8
- 91.6 ppm respectively. As a control, Fermivin®,
famous aromatic wine yeast, was 89.4 ppm and
16.2 ppm respectively. We also find that the
isolated Pichia anomala could produce higher
level ethanol (4.2% - 5.0%, v/v) than other spe-
cies (0.4% - 2. 2%, v/v ). Using the arom atic yeas t s
in fermentation industries, w e expect to improve
the quality of traditional alcoholic beverages.
Keywords: Aromatic Yeast; Pichia sp.; Yakju;
Korean Traditional Alcoholic Beverage; Ethyl
Many Korean alcoholic beverages are made using nu-
ruk, a traditional Korean fermentation agent, which en-
ables the hydrolysis of the raw materials and is known to
produce richly flavored components in addition to alco-
hol, giving the final product a pleasant taste [7]. During
the fermentation process, the yeast cells create a number
of spectrums of volatile molecules, such as esters, or-
ganic acids, aldehydes, higher alcohols and carbonyl
compounds offering sensory characteristics for the bev-
erages [5]. The representative components of flavors in
traditional Korean alcoholic beverages are ethyl caproate,
producing an apple like flavor, isoamyl alcohol (i-AmOH),
exhibiting a banana like flavor and 2-phenyl alcohol,
which provides for a rose like essence; all compounds
which normally originate from alcohol fermentation yeasts
[2]. We aimed to screen for highly productive aromatic
compound strains with high levels of ethyl caproate
(primarily), i-AmOH and 2-phenylalcohol, amongst oth-
ers. Finding out the potential of the isolated yeast for use
in the brewing of yakju, a variety of traditional Korean
alcoholic beverage, was also an important objective.
Thus, we embarked on the isolation and identification of
76 wild yeast strains from traditional Korean nuruks. We
then screened 24 strongly cerulenin-resistant wild yeasts
using a cerulenin medium. As a second screening, the
cerulenin-resistant yeasts were subjected to an olfactory
evaluation to eliminate those which, despite being strongly
cerulenin-resistant, emitted an unpleasant odor. We fi-
nally isolated 8 strains of aromatic wild yeasts, which
also exhibited strong cerulenin resistance. The isolates
were 4 strains of Pichia anomala (AY1, AY4, AY41,
AY56), 2 strains of Pichia fabianii (AY42, AY47),
Pichia farinosa AY5 and Geotrichum candidum AY6.
Pichia fabianii AY47 was seen to produce a large
amount of ethyl caproate in this study. In research with
the traditional Japanese drink of sake, the highest ethyl
caproate producing curulenin-resistant mutant of Sac-
charomyces cerevisiae produced levels 5 times (7.4 ppm)
that of the parental strain [6]. By applying these non-
Saccharomyces yeasts to fermentation processes, we
expect the quality of traditional Korean alcoholic bever-
ages to be notably improved.
2.1. Application of Aromatic Y east s for
For the fermentation of yak ju , each 1 kg of non-glu-
Copyright © 2013 SciRes. Openly accessible at http://www.scirp.org/journal/as/
C. J. Ho et al. / Agricultural Sciences 4 (2013) 136-1 40 137
tinous rice was washed 10 times and soaked in clean,
drinkable tap water for 12 hours and then hourhalf-dried
for 2 hours. Subsequently, the rice was finely powdered
using a roll-mill (Dong-Gwang Inc., Daegu, Gyeongbuk,
Korea). 1.2 L of boiling water was added to the rice flour
(120% water to raw starch material, v/w) and gently
stirred until the mixture cooled down to approximately
30℃. Then 2 g of fermentation agent (sp 15,000,
Chungmubalhyo, Ulsan, Gyeongnam, Korea) was added
for the hydrolysis of the rice flour and the 8 isolated and
3 commercial yeasts, which were adjusted to 106/mL,
inoculated into the mixtures in quantities of 2 mL, re-
spectively. The mixture was fermented at 20℃ for 7
days. All fermentations were performed in triplicate.
2.2. Flavor Compound Analyses of
Fermented Alcoholic Beverages
The flavor compounds of each brewed sample were
analyzed by GC (GC 2010, Shimadzu Co., Kyoto, Japan).
The flavor compounds of each 1 μL cultured sample
were analyzed by GC (GC 2010, Shimadzu Co., Kyoto,
Japan). The column employed was a HP-INNOWAX
capillary column (60 m × 0.25 mm id × 0.25 μM film
thickness, J & W Scientific, Folsom, CA, USA). The
carrier gas was nitrogen and the flow rate was 22.0
cm/sec (linear velocity). The column’s oven temperature
was held at 45℃ for 5 min and then set to rise to 100℃
at a rate of 5℃ /min and sustained at 100℃ for 5 min.
Subsequently the column’s oven temperature was in-
creased to 200˚C at a rate of 10 /min and sustained for
10 min. Each sample injection volume was 1 μL and the
split ratio was 50 : 1. The injector temperature and detec-
tor (FID) temperature were sustained at 250℃.
2.3. Physico-Chemical Analyses of
Fermented Alcoholic Beverages
The pH was measured with a pH meter (Metrohm 691,
Metrohm, Harisau, Switzerland). Total acid content was
measured as lactic acid concentration and was deter-
mined by adding two to three drops of mixed indicator
(0.2 g of bromothymol blue and 0.1 g of neutral red in
300 mL of 95% ethanol) to each 10 mL sample and then
titrated with 0.1 N NaOH until the solution turned light
green. Phthalic acid (Sigma-Aldrich, St. Louis, Mo, USA)
was used as the standard [12]. Amino acidity was deter-
mined by adding two to three drops of phenolphthalein to
each 10 mL sample and then neutralized with 0.1 N
NaOH until the solution turned light red. Amino acidity
was measured by adding 5 mL of neutral formalin to the
sample and then measuring the amount of 0.1 N NaOH
consumed to turn the solution light red [12]. The concen-
tration of soluble solids was measured with a hand re-
fractometer (PR101; ATAGO Co. Ltd., Tokyo, Japan)
and expressed in Brix units (% sucrose). Reducing sugar
concentration was analyzed by the DNS (dinitrosalicylic
acid) method with a UV/VIS spectrophotometer (JP/U-
2000, Hitachi, Ltd., Tokyo, Japan), with the glucose
standard procured from Sigma-Aldrich (St. Louis, MO,
USA) [11]. Alcohol content was measured using a gra-
vimeter after the 100 mL of alcoholic beverage sample
was distilled and then adjusted to 15℃ [13].
2.4. Sensory Evaluation of Fermented
Alcoholic Beverages
7 experienced panelists from our Fermented Food
Science Division evaluated the 11 resulting alcoholic
beverages for taste on the basis of nine-point hedonic
scales (1 = dislike extremely, or extremely weak; 9 = like
extremely, or extremely strong). The experiment was
conducted in triplicate and the mean values and standard
deviation were calculated from the data obtained from
the three separate experiments. These data were then
compared using Duncan’s multiple range method.
3.1. Application of Aromatic Y east s for
The 8 isolated yeasts and 3 commercial yeasts, which
were adjusted to 106/ml and inoculated at 2 ml into the
yakju-making mixtures (approximately 2.2 kg respect-
ively). The adding ratio of water to this mixture was
120% of the quantity of rice (v/w). The mixture was
fermented at 20℃ for 7 days. The physicochemical
characteristics of the yakju after fermentation with the 8
isolates is shown in table 5. The overall pH range of the
yakju was between 3.1 - 3.4. This latter result is seen to
be quite low when compared to makg eo lli’s pH when fer-
mented using S. cerevisiae (the range was 3.5 - 4.2) and
similar to makgeolli fermented with P. anomala (the
range was 3.3 - 3.4) in Kim et al’s study [9]. In terms of
alcohol production capabilities, the commercial yeasts of
Saccharomyces cerevisiae (AY36, AY38 and AY39)
produced 14.97% - 15.87% alcohol (v/v), while Pichia
anomala (AY1 and AY4) produced 4.57% - 5.00% (v/v),
and other isolated yeasts produced between
(v/v). From Kim et al’s study [9] it does seem possible
that P. anomala, well as S. cerevisiae, can produce high
concen- trations of alcohol. In the latter study when us-
ing P.anomala Y197-13 and koji, the alcohol content of
the resulting makgeo lli was found to be 11.1% (the water
adding ratio was 186% of the raw starch material used).
Thus it may well be possible to raise the alcohol levels
produced by P. anomala (AY1 and AY4) by controlling
the fermentation environment. Alternatively, Pichia
anomala (AY1 and AY4) could be employed in the pro-
duction of alcoholic beverages with lower alcohol con-
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C. J. Ho et al. / Agricultural Sciences 4 (2013) 136-1 40
tents. In relation to levels of soluble solids, it was ob-
served that, for the most part, the isolated yeasts had
higher levels of soluble solids (26.43 - 21.17 brix) than
the commercial yeasts (14.30 - 13.13 brix). Due to the
fact that the isolated yeasts’ alcohol production ability is
lower than the commercial yeasts’, the remaining sugar
is likely to have brought about the higher levels of solu-
ble solid content. Likewise, in relation to reducing sugar,
the isolated yeasts were mostly seen to have higher levels
(2.22% - 9.98%, w/v) than the commercial yeasts (1.34%
- 1.93%, w/v). The probable reason for this is also likely
to be the same as with the case of soluble solids. It is
possible that the sugar-acid ratio will have a great effect
on the overall taste of the yakju. In champagne wine, the
sweetness contributes to a good perception of its taste.
This sweetness results from high concentrations of sugar
in the fermentation mixture used (20.5 g/l sugar - 4.22 g/l
tartaric acid) [20]. Further study is needed to discover an
appropriate sugar-acid ratio in order to provide for tastier
3.2. Flavor Compound Analyses of
Fermented Alcoholic Beverages
For decades, so-called non-Saccharomyces yeasts (such
as Pichia, Candida , Hanseniaspora, Hansenula etc.)
have been reported to be producers of high concentra-
tions of fermentation compounds, such as acetic acid,
esters and acetoin. The effects of these latter compounds
on the sensory qualities of wines have been similar to the
role of the AATase of S. cerevisiae in the production of
acetate esters, such as ethyl acetate. Ethyl acetate exhibits a
solvent like-odor and AY42 possessed the lowest amount
of this compound, 49.9 ± 8.6 mg/l – well below the 160
mg/l odor threshold in case of wine [1]. In the Japanese
drink sake, Saccharomyces cerevisiae produced ethyl
acetate in the range of 50 - 100 ppm (160% of water to
raw starch material v/w) as analyzed by quantitative
traits loci (QTL) [8]. In this study the yakju which used
AY4 was seen to be within the latter range even though
the water ratio differed (120% of water to raw starch
material v/w, Table 2). Isoamyl acetate (i-AmOAc),
which emits a banana-like aroma, is synthesized from
isoamyl alcohol and acetyl coenzyme A and is catalyzed
by alcohol acetyltransferase (AATase, EC [4].
i-AmOH and i-AmOAc are usually within the ranges of
190 - 350 ppm and 8.0 - 19.3 ppm, respectively, in sake
(160% of water to raw starch material v/w) [2,8]. Further
studies in relation to the E/A ratio of ya k ju are needed.
Ethyl caproate, with an apple like aroma, is synthesized
by the enzymes esterase and alcohol acyltransferase in
yeast, with the substrates being ethanol and caproic acid
or caproyl-CoA [6]. In this study, in most of the yakjus
ethyl caproate levels were higher than in sake (55.0 -
176.6 ppm) and wine. AY47 (176.6 ppm) and AY56
(170.1 ppm) were seen to be higher than those for the
other treatments, and the commercial yeasts AY36, AY38
and AY39 were significantly lower (11.4 -19.1 ppm).
There have been several reports where non-Saccharo-
myces yeasts have been described as producers of high
concentrations of some fermentation compounds such as
esters and acetic acid, which influence the sensory qual-
ity of wines [14]. In this study, the results further support
these reports. Generally in this study, the isolated yeasts
produced between 3.4 - 24.3 ppm of 2-phenylethyl alco-
hol. Of the commercial yeasts, AY38 produced the high-
est concentration of the compound at 39.4 ppm. In wines
2-phenylethyl alcohol’s concentration is generally be-
tween 53 - 82 ppm [1,14] and in Agave tequilana it is 3.3
- 26.6 ppm [3]. Pichia sp. produced more 2-phenyl alco-
hol (up to 18%) when sucrose was used as a carbon
source, and when phenylalanine (up to 0.1%) was added
to the medium. Hence, further study is required to see
how production levels are influenced by modifying the
compositions of the media.
3.3. Sensory Evaluation of the Brewed Yakju
The 8 isolated wild yeasts and 3 commercial yeasts
were used for the brewing of yakju. Following the com-
pletion of the brewing process the resulting concoctions
were subjected to sensory evaluations (Table 3). AY6
yakju was strongest among the strains in terms of sweet-
ness at 6.2 ± 0.9, and AY6 yakju’s soluble solid and re-
ducing sugar content also belonged to the highest group
as seen with Duncan’s multiple range test in Table 5.
AY42 yakju was rated to be the strongest in terms of
sourness among the strains, at 6.5 ± 0.8. In color, AY36
yakju received the highest score, at 7.4 ± 0.9, with a
clean green-yellow color. For flavor, AY6 and AY42
yakjus surpassed other treatments in terms of possessing
a fruity aroma. It is certain that the quantity of the aro-
matic compounds is not important. Instead what is im-
portant is having the right proportions of the compounds
(ethyl caproate, isoamyl alcohol, ethyl acetate, 2-phenyl
alcohol etc.) so as to produce a pleasant flavor [10].
AY42 yakj u , we surmise, had the best overall flavor
out of all the yakjus tested, when considering GC data
(Table 2). In terms of taste and overall preference, the
yakjus with the highest soluble solid and reducing sugar
content received the highest scores. However, AY42,
which scored the highest in overall preference, had a
high concentration of soluble solids, but a comparatively
low concentration of total acid content (Ta bl e 1 ). Com-
mercial yeasts. AY36, AY38 and AY39 received rela-
tively low scores in overall preference because of the
comparatively higher concentrations of ethanol, and the
panels gave their opinion that the high ethanol gave the
yakju a bitter taste. Meanwhile, AY4 has high potential
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C. J. Ho et al. / Agricultural Sciences 4 (2013) 136-1 40
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Table 1. Physicochemical characteristics of the low alcoholic beverages using isolated yeasts.
Yeasts pH Total acid
(lactic acid, %, w/v) Amino acidity
(mL) Alcohol
(%, v/v) Soluble solids
(% sucrose) Reducing sugar
(%, w/v)
AY1 3.12 ± 0.20f 1.20 ± 0.10ab 5.27 ± 0.84c 4.57 ± 1.97b 22.27 ± 2.31bc 7.99 ± 0.96b
AY4 3.32 ± 0.01abc 1.20 ± 0.05ab 5.34 ± 0.31c 5.00 ± 1.23b 21.17 ± 1.45c 8.41 ± 1.34b
AY5 3.28 ± 0.08bcd 1.17 ± 0.05abc 6.16 ± 0.46ab 2.17 ± 1.59c 24.37 ± 1.72ab 8.48 ± 0.22b
AY6 3.37 ± 0.03abc 1.09 ± 0.03bcde 6.21 ± 0.42ab 0.97 ± 0.74c 25.03 ± 0.72a 8.83 ± 0.27ab
AY36 3.40 ± 0.05ab 0.77 ± 0.03g 4.34 ± 0.07d 15.87 ± 0.06a 13.50 ± 0.26d 1.36 ± 0.11c
AY38 3.45 ± 0.02a 0.75 ± 0.04g 3.43 ± 0.09e 15.40 ± 0.20a 14.30 ± 0.40d 1.93 ± 0.27c
AY39 3.32 ± 0.04abc 0.91 ± 0.04f 3.63 ± 0.04e 14.97 ± 0.40a 13.13 ± 0.65d 1.34 ± 0.50c
AY41 3.23 ± 0.06cde 1.14 ± 0.08abcd 5.67 ± 0.69bc 1.00 ± 0.36c 24.40 ± 2.94ab 8.25 ± 0.45b
AY42 3.24 ± 0.13cde 1.06 ± 0.08cde 6.10 ± 0.13ab 0.47 ± 0.42c 25.53 ± 0.97a 8.11 ± 0.66b
AY47 3.30 ±0.08abcd 1.00 ± 0.11ef 6.41 ± 0.09a 0.43 ± 0.06c 26.43 ± 0.31a 7.85 ± 1.31b
AY56 3.28 ± 0.10bcd 1.04 ± 0.05de 6.40 ± 0.30a 0.77 ± 0.15c 25.30 ± 1.20a 9.98 ± 0.39a
Values are mean ±SD. a-d in a column by different superscripts are significantly different at the p < 0.05 by Duncan’s multiple range test (n = 3). Total acid was
converted to lactic acid (consumed ml of 0.1N NaoH 0.09). AY 36, AY38 and AY39 are commercial yeasts.
Table 2. Flavor compounds analysis of the fermented alcoholic beverages expressed as mg/l.
No. Acet aldehyde EtOAc Iso
butanol i-AmOH Ethyl
caprylate Acetic acid Furfral Linalool Butyrate Ethyl
AY1 1458.7±54.4abcd 293.6±32.6a - 8.5±1.8cd 74.7±38.6bc 4801.6±298.0a342.2±60.7a148.3±18.2ab11.7±4.0cde 55.0±5.8c6.6±2.1cde
AY4 1250.1±196.6cde 130.3±53.3b - 7.4±3.0cd 90.6±2.5b1213.2±114.2g79.2±23.3d131.2±22.9ab 8.8±1.7cde 66.0 ± 2.6c9.4±7.2cde
AY5 1567.0±157.2ab - - 12.2±2.1bcd 132.4±30.2a4028.4±161.5b68.9±5.6 148.4±23.8ab 15.7±11.7bcde 61.4 ±17.1c24.3±11.6b
AY6 1633.4±94.6a - - 6.97±1.3d 31.5±3.8de 3404.6±230.6bcd 232.3±79.3b143.7±49.0ab 6.1±0.8e 69.3±12.6c4.7±1.5de
AY36 1281.2±82.9bcde - - - 76.5±10.4bc 3180.4±124.8cd 40.5±4.6d30.7±2.4cd 7.3±0.9de 12.3±1.1d4.9±0.4de
AY38 1541.9±146.1abc - - 15.8±2.3ab 25.1±2.54de 2722.7±212.8de 37.2±3.8d22.5±2.2d7.2±0.8de 11.4±0.6d39.4±6.9a
AY39 1257.1±157.8cde 3.5±1.2d 5.3±0.3a 11.2±2.9bcd - 2351.5±132.0ef - 2.2±0.2a 24.3±2.2b 19.1±3.4d6.4±0.3cde
AY41 1444.2±83.7abcd 67.4±13.5c 3.3±0.9ab 14.2±6.2bc - 3718.7±478.7196.7±10.3bc146.8±22.8ab24.7±5.6b 59.0±7.6c14.2±3.0c
AY42 1504.8±182.1abc 49.9±8.6c - 7.7±1.7cd 65.3±9.7bc 3026.6±373.4cde 149.3±19.5c67.5±17.4c17.0±7.8bcd 99.4±16.5b5.6±0.7de
AY47 1386.6±97.4abcd 82.5 ± 6.6c - 6.7±1.7d 55.4±3.1cd 3452.1±192.8bcd 62.0 ± 19.4d152.6±25.1a7.6±2.4de 176.6±17.4a6.9±1.7cde
AY56 1398.7±38.4abcd - - 8.1±1.5cd 1.5±0.3e1851.1±173.2fg68.7 ± 12.0d125.1±17.8ab 53.4±5.1a 170.1±11.6a3.4±0.5e
Values are mean ±SD. a-e in a column by different superscripts are significantly different at the p < 0.05 by Duncan’s multiple range test (n = 3). AY36, AY38
and AY39 are commercial yeasts.
C. J. Ho et al. / Agricultural Sciences 4 (2013) 136-1 40
Table 3. Sensory evaluation of the brewed low alcoholic beverages.
Strain Sweetness Sourness Color Flavor Taste Overall preference
AY1 4.7±1.0cde 6.3±1.9ab 4.5±1.4de 4.0±2.3c 4.1±1.8cd 3.9±1.9de
AY4 5.0±1.1bcd 6.1±1.8ab 6.2±2.1abc 5.4±1.5ab 4.9±1.6abcd 5.2±1.6abc
AY5 5.0±1.6bcd 5.6±1.9ab 4.5±1.5de 4.0±2.5c 3.9±1.9d 3.4±2.1e
AY6 6.2±0.9a 6.3±1.3ab 5.3±2.0cde 6.0±1.9a 5.6±1.6ab 5.7±1.7ab
AY36 4.3±2.2de 5.2±2.1b 7.4±0.9a 5.3±1.9abc 4.5±2.1bcd 4.5±2.2bcde
AY38 4.3±2.0de 5.5±1.9ab 6.7±1.4ab 5.7±1.8ab 4.7±2.1abcd 4.8±2.2bcd
AY39 3.9±1.6e 5.4±1.9ab 6.7±1.1ab 5.1±1.8abc 4.0±1.9cd 4.0±1.8cde
AY41 5.4±1.4abc 6.2±1.4ab 5.9±2.2bc 5.4±1.4ab 5.3±1.6abc 5.4±1.6ab
AY42 5.9±1.1ab 6.5±0.8a 5.5±2.0bcd 6.4±1.4a 5.9±1.5a 6.1±1.5a
AY47 5.2±1.0bcd 5.7±1.6ab 4.2±1.3e 4.4±2.1bc 5.1±1.8abcd 4.7±1.9bcde
AY56 5.7±1.0abc 6.2±1.5ab 5.3±1.8cde 5.3±1.7abc 5.4±1.4ab 5.5±1.2ab
Values are mean ±SD. a-e in a column by different superscripts are significantly different at the p<0.05 by Duncan’s multiple range test (n = 3). AY36, AY38
and AY39 are commercial yeasts.
for use in a sweet-yakju because of its alcohol production
ability and AY6, AY41 and AY56 have also great poten-
tial for use to create fruity beverages. AY42, we suggest,
have the best flavor composition among the isolates for
the production of good quality of yak ju , when consider-
ing the GC analyses and sensory evaluations.
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