Food Chemistry 192 (2016) 1068–1077

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Evaluation of umami taste in mushroom extracts by chemical analysis,

sensory evaluation, and an electronic tongue system
Chanvorleak Phat a, BoKyung Moon b, Chan Lee a,⇑
a
Department of Food Science and Technology, Chung-Ang University, Anseong-Si, Gyeonggi-Do 456-756, Republic of Korea
b
Department of Food and Nutrition, Chung-Ang University, Anseong-Si, Gyeonggi-Do 456-756, Republic of Korea

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

Article history: Seventeen edible mushrooms commercially available in Korea were analysed for their umami taste
Received 20 April 2015 compounds (50 -nucleotides: AMP, GMP, IMP, UMP, XMP; free amino acids: aspartic, glutamic acid) and
Received in revised form 29 June 2015 subjected to human sensory evaluation and electronic tongue measurements. Amanita virgineoides
Accepted 22 July 2015
featured the highest total 50 -nucleotide content (36.9 ± 1.50 mg/g), while monosodium glutamate-like
Available online 23 July 2015
components (42.4 ± 6.90 mg/g) were highest in Agaricus bisporus. The equivalent umami concentration
(EUC) ranged from 1.51 ± 0.42 to 3890 ± 833 mg MSG/g dry weight; most mushrooms exhibited a high
Keywords:
umami taste. Pleurotus ostreatus scored the highest in the human sensory evaluation, while Flammulina
Mushrooms
Umami taste
velutipes obtained the maximum score in the electronic tongue measurement. The EUC and the sensory
Equivalent umami concentration score from the electronic tongue test were highly correlated, and also showed significant correlation with
Sensory evaluation the human sensory evaluation score. These results suggest that the electronic tongue is suitable to
Electronic tongue determine the characteristic umami taste of mushrooms.
Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction
Mushrooms have been used as food and traditional medicines
in Asia for centuries (Kalač, 2012). Generally, mushrooms contain
about 57% carbohydrates, 25% protein, 5.7% fat, and 12.5% ash
(Kalač, 2009). Moreover, mushroom proteins include all the
essential amino acids that cannot be synthesised by our body
and therefore must be supplied with the diet. The total fat content
of mushroom is low and features a high proportion of polyunsatu-
rated fatty acids, ranging from 72% to 85% (Kalač, 2012). In
addition, the strong flavour and taste of mushroom contribute to
their extensive consumption as raw food, functional food, and
seasoning (Khan, Khan, Hossain, Tania, & Uddin, 2009).
The characteristic flavour substances or umami tastes in mush-
rooms, which represent the basic tastes enhanced by free amino
acids and 50 -nucleotides, can be analysed as volatile and
non-volatile components (Li et al., 2014) by high-performance
liquid chromatography (HPLC) and other methods. ‘‘Umami’’ was
coined as a term for savoury and delicious taste, and was recog-
nised as a basic taste typified by the amino acid glutamic acid
and its salt monosodium glutamate (MSG), which yield a savoury,
brothy, rich, or meaty taste sensation (Yamaguchi, 1991). The free
⇑ Corresponding author at: Department of Food Science and Technology, Chung-
Ang University, Anseong-Si, Gyeonggi-Do 456-756, Republic of Korea.
E-mail address: chanlee@cau.ac.kr (C. Lee).
amino acids, glutamic acid and aspartic acid, and the
50 -nucleotides, inosine 50 -monophosphate and guanosine
50 -monophosphate, were later identified as the main umami sub-
stances. Umami substances are naturally found in a variety of
foods, including meat, cheese, seafood and vegetables, and they
are the predominant flavour substances of mushrooms
(Yamaguchi, 1991). Water-soluble taste components, such as free
amino acids and 50 -nucleotides, make an important contribution
to the typical mushroom flavour (Dermiki, Phanphensophon,
Mottram, & Methven, 2013) and the combination of free amino
acids gives rise to a unique natural flavour (Mau, 2005).
Human sensory evaluation has been employed for the determi-
nation of the umami tastes in mushrooms. Chemical analysis by
HPLC offers quantitative data that cannot be explained in terms
of overall taste, because this method detects each taste substance
separately and cannot reveal taste–substance interactions such as
synergistic and suppression effects (Kobayashi et al., 2010). On
the other hand, sensory evaluation provides integrated, direct
measurements of perceived intensities of target attributes, such
as appearance, colour, aroma, taste, and texture (Bleibaum et al.,
2002). Nevertheless, sensory evaluation is time-consuming, expen-
sive, and might vary depending on daily conditions. Thus, a new
method has been developed for the evaluation of many tastes at
the same time, using only a taste sensor itself (Tran, Suzuki,
Okadome, Homma, & Ohtsubo, 2004). These methods can integrate

http://dx.doi.org/10.1016/j.foodchem.2015.07.113
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
 C. Phat et al. / Food Chemistry 192 (2016) 1068–1077
predictive relationships between sensory and instrumental mea-
surements. The electronic tongue measurement offers satisfactory
taste results that are close to a human sensory evaluation
(Kobayashi et al., 2010). There has been limited information to date
regarding the taste characteristics of mushrooms using the
electronic tongue.
In the present study, we aimed to evaluate the umami taste prop-
erties of various mushroom types available in Korean domestic mar-
kets in order to provide a complete range of information, which will
be very useful for both consumers and industrial applications in the
development of natural seasonings or food additives from
mushrooms. For this purpose eight umami components were anal-
ysed in mushroom extracts through chemical analysis using a
high-performance liquid chromatography (HPLC) and liquid chro-
matography–tandem mass spectrometry (LC–MS/MS) analysis,
and their taste was compared by sensory evaluation. Furthermore,
the correlations between the human sensory evaluation scores,
the electronic tongue test (taste sensing system TS-5000Z), the
levels of umami equivalent concentration, and the level of each
umami component were investigated by statistical analysis.
2. Materials and methods
2.1. Samples
Seventeen commercially available mushroom samples (Agaricus
bisporus, Amanita virgineoides, Auricularia auricula-judae,
Flammulina velutipes, Grifola frondosa, Hericium erinaceus,
Hypsizigus marmoreus, Lentinus edodes, Pleurotus cornucopiae var.
citrinopileatus, Pleurotus eryngii, Pleurotus ferulae, Pleurotus ostrea-
tus, Pleurotus salmoneostramineus, Polyozellus multiplex, Ramaria
botrytis (Pers.) Ricken, Sparassis crispa, and Tremella fuciformis) were
collected from the market or artificially cultivated using strains
from Rural Development Administration, Republic of Korea. Fresh
mushrooms were immediately freeze-dried, milled using a food
blender (HR 2860, Ya Horng Ele. Co., Ltd., Guan Cuangdong,
China), and stored at 20 °C until further analysis.
2.2. Standards and reagents
50 -Nucleotide standards [adenosine 50 -monophosphate sodium
salt (AMP), cytidine 50 -monophosphate disodium salt (CMP), gua-
nosine 50 -monophosphate disodium salt (GMP), inosine
50 -monophosphate disodium salt (IMP), and uridine
50 -monophosphate disodium salt (UMP)] were purchased from
Sigma–Aldrich, Yongin, Korea. Xanthosine 50 -monophosphate
(XMP) was supplied by Santa Cruz Biotechnology (Santa Cruz,
CA). Phosphoric acid 85% was obtained from Sam-Cheon chemicals,
Jeollanam, Korea. Methanol and water (HPLC gradient grade) were
purchased from Burdick & Jackson, Morristown, NJ. Aspartic acid
and glutamic acid were obtained from Junsei Chemical Co., Ltd.,
Tokyo, Japan and Sigma–Aldrich, Yongin, Korea, respectively.
2.3. Equipment
A Gilson HPLC system (Middleton, UK) equipped with a binary
pump, vacuum degasser, column oven, UV detector (254 nm), and
fluorescence detector (335 and 440 nm excitation and emission
wavelengths, respectively) was employed for umami taste mea-
surements. Gemini-NX C18 (5 lm, 4.60  250 mm, Phenomenex,
Torrance, CA) and Eclipse XDB C18 (5 lm, 4.60  150 mm;
Agilent, Santa Clara, CA) columns were used for this analysis.
The electronic tongue measurement was conducted with the
taste sensing system TS-5000Z (Intelligent Sensor Technology,
Inc., Kanagawa, Japan).
1069
2.4. 50 -Nucleotide assay
50 -Nucleotides were extracted and analysed as described by Pei
et al. (2014). Freeze-dried mushroom powder (500 mg) was
extracted with 50 mL of deionised water. This suspension was
heated to boiling for 1 min, cooled, and then centrifuged at
4000 rpm for 30 min. The extraction was filtered using a 0.22-lm
nylon filter, prior to HPLC analysis.
50 -Nucleotides were separated with a Gemini-NX 5 lm C18
(250  4.60 mm) column using an isocratic mobile phase of 5% A
and 95% B for 40 min (A: methanol and B: 0.05% phosphoric acid)
at a flow rate of 0.7 mL/min and UV detection at 254 nm. Each
50 -nucleotide was identified by matching its retention time with
that of an authentic standard, and quantified using its respective
calibration curve.
2.5. Free amino acid assay
Aspartic acid and glutamic acid were analysed and identified
using HPLC according to Pereira, Pontes, Câmara, and Marques
(2008). Free amino acids were extracted by suspending 500 mg of
homogenised sample powder in 50 mL of 0.1 M HCl and shaking
for 45 min at ambient temperature, followed by filtration through
a Whatman No. 4 filter paper. This sample did not exhibit any fluo-
rescence; it was derivatised using o-phthalaldehyde (OPA). OPA
derivatisation solution was prepared in a 10-mL flask by dissolving
250 mg of reagent in 1.5 mL ethanol and bringing the volume to
10 mL with 0.4 M borate buffer (pH 10.5). Finally, 200 lL of
2-mercaptoethanol were added. The reagent solution was left to
settle for 90 min and then stored in dark glass vials at 4 °C; it was
freshly prepared every 9 days. The derivatisation procedure was
performed in the sample injection loop according to the following
sequence: 10 lL of buffered sample mixture were transferred to
the injection loop followed by 10 lL of OPA solution and main-
tained for 3 min to promote the derivatisation reaction. The flow
rate was set to 1 mL/min and the column temperature was main-
tained at 35 °C. Mobile phase A contained 1% of tetrahydrofuran,
8% methanol, and 91% phosphate buffer (10 mM), and mobile phase
B consisted of 80% methanol and 20% phosphate buffer (10 mM).
The gradient program used is shown in the Supplemented material.
2.6. LC–MS/MS
LC–MS/MS (LTQ velos, Accela HPLC; Thermo, Waltham, MA)
equipped with an electrospray ionisation interface (ESI) was
applied to confirm the 50 -nucleotides. A C18 analytical column
(4.60  250 m, Phenomenex, CA, USA) was used for the analysis.
The liquid chromatography was performed under isocratic condi-
tions as described in the 50 -nucletide assay. A volume of 20 lL
was injected for LC–MS/MS, and the molecular weight of each sam-
ple was compared with that of the standards. The mass spectrom-
eter was operated in negative electrospray ionisation (ESI) mode.
Mass spectrometry was carried out in scan mode from m/z 50 to
m/z 2000. ESI-MS conditions were as follows: capillary voltage of
3 kV, pressure of nebuliser 40 psi, gas (nitrogen) temperature of
350 °C, cone gas and desolvation gas flows of 50 and 600 L/h,
respectively.
2.7. Equivalent umami concentration (EUC)
The EUC value (mg MSG/g) reflects the concentration of MSG
equivalent to the umami intensity given by a mixture of MSG
and 50 -nucleotides and is represented by the following equation
(Yamaguchi, 1991)
X X X
Y¼ ai bi þ 12:18ð ai bi Þð aj bj Þ;
1070 C. Phat et al. / Food Chemistry 192 (2016) 1068–1077

where Y is the EUC of the mixture in mg MSG/g; ai is the concentra-
tion (mg/g) of each umami amino acid [aspartic acid (Asp) or
glutamic acid (Glu)]; aj is the concentration (mg/g) of each umami
50 -nucleotide [50 -inosine monophosphate (50 -IMP), 50 -guanosine
monophosphate (50 -GMP), 50 -xanthosine monophosphate
(50 -XMP), or 50 -adenoshine monophosphate (50 -AMP)]; bi is the
relative umami concentration (RUC) for each umami amino acid
to MSG (Glu, 1; Asp, 0.077); bj is the RUC for umami 50 -nucelotide
to 50 -IMP (50 -IMP, 1; 50 -GMP, 2.3; 50 -XMP, 0.61; 50 -AMP, 0.18);
and 12.18 is a synergistic constant based on the concentration
(mg/g) used.
2.9. Statistical analysis
All assays were carried out in triplicate. The results are
expressed as mean ± standard deviation (SD). The experimental
data were subjected to analysis of variance for a completely ran-
domised design using Statistical Analysis System software (SAS
Institute., Cary, NC, USA, 2002). Spearman’s Rank Correlation
statistical treatment was conducted using IBM SPSS Statistics
version 21 (SPSS Inc., Chicago, IL). A p-value <0.05 was considered
statistically significant.

3. Results and discussion

2.8. Sensory evaluations

3.1. 50 -Nucleotides
2.8.1. Human sensory evaluation
Ten trained panellists (8 females and 2 males) aged between 24
The levels of six 50 -nucleotides were analysed in the mushroom
and 37 years participated in the sensory evaluation. Based on a
extracts. As shown in Table 1, AMP was detected in 15 out of 17
screening test, the panellists were selected from graduate students
samples. The highest AMP concentration was measured in an
at the Department of Food Science and Nutrition at Chung-Ang
extract from P. salmoneostramineus at 30.9 ± 0.01 mg/g. All
University, South Korea. All participants were familiar with umami
seventeen samples contained CMP at a concentration ranging
taste and had previous experience with sensory evaluation. During
from 0.03 ± 0.03 to 21.1 ± 0.76 mg/g with the extract from
five 1-h training sessions, the panellists were trained with different
Ama. virgineoides exhibiting the highest CMP concentration. IMP
concentrations of MSG solutions (0.03, 0.09, 0.15, 0.21, 0.27, or
was detected in sixteen mushroom samples, with the lowest con-
0.30 g/100 mL) to accustom them to the evaluation scales and
centration measured in the extract from Auri. auricular-judae, while
the intensity of umami taste of the standard solutions.
P. ostreatus contained the highest IMP level. G. frondosa appeared to
The sensory evaluation of the samples was performed in tripli-
have the highest UMP concentration among the tested mushrooms.
cate on different days. Sample solutions were prepared by extrac-
Only one mushroom, P. cornucopiae var. citrinopileatus, did not con-
tion of mushroom powders with boiling water (1% w/v) for 5 min.
tain any UMP in its extract. XMP was detected in all samples except
The panellists were presented with 30 mL of each sample together
for Aga. bisporus, with concentration ranges of 0.01–
with a glass of warm water, a spit cup for expectoration, a paper
4.74 ± 0.15 mg/g. Interestingly, GMP could not be detected in any
napkin, and palate cleansers (white bread) in random order in
of the mushroom samples.
three different sessions. Five to six samples were served in each
Yang, Lin, and Mau (2001) reported that L. edodes contains
session and panellists were given a break between each session.
9.51–24.2 mg/g of total 50 -nucleotides, and the total 50 -nucleotide
In order to avoid temperature differences, all samples were kept
content in common mushrooms was around 11.35 mg/g; this value
and served at 45 °C. Panellists evaluated the intensity of umami
was similar to that observed in this study. The total 50 -nucleotide
taste using an 11-point scale, where 1 means very weak, 6 means
content of P. ostreatus extract has previously been reported in dif-
medium, and 11 means very strong umami taste (He et al., 2009).
ferent studies worldwide, but the measured values were very low
compared to those in our study; this difference might be related to
2.8.2. Electronic tongue measurement
differences in the cultivation conditions between the samples
The electronic tongue system (taste sensing system TS-5000Z,
(Beluhan & Ranogajec, 2011; Tsai et al., 2009; Yang et al., 2001).
Japan) consists of reference electrodes, multichannel lipid/polymer
Lee, Jian, and Mau (2009) reported an XMP concentration of
membrane electrodes, an auto-sampler, an electronic unit for data
0.56 mg/g in Hyp. marmoreus extract, which is very similar to the
acquisition, and a personal computer with an advanced chemo-
results in our study.
metric software package (Intelligent Sensor Technology, Inc.,
Tsai et al. (2009) identified GMP, IMP, and XMP as the flavour
Kanagawa, Japan) (Tran et al., 2004). The response intensity of each
50 -nucleotides. Mushroom flavour 50 -nucleotide levels have been
sensor was measured with an Ag/AgCl reference electrode, which is
reported in several studies, ranging from 0.54 to 9.00 mg/g (Tsai
the most commonly used in this field (Kobayashi et al., 2010). The
et al., 2009; Yang et al., 2001; and Beluhan & Ranogajec, 2011).
potentiometric differences between each coated sensor and the
According to Yang et al. (2001), flavour 50 -nucleotides can be clas-
reference electrode contribute to the intensity value of the mea-
sified as low (<1 mg/g), medium (1–5 mg/g) and high (>5 mg/g).
sured samples (Chen, Zhao, & Vittayapadung, 2008).
Among the 17 mushroom samples tested here, P. ostreatus, H. eri-
Each sample was measured after the electric potentials of all
naceus, and Aga. bisporus possessed the highest levels of flavour
membranes had been stabilised in standard solutions. These stan-
50 -nucleotides of 14.8 ± 0.05, 10.3 ± 0.28, and 7.54 ± 0.33 mg/g,
dard solutions were prepared by dissolving the respective com-
respectively. Nine mushroom samples were classified in the
pound in 1 L of distilled water, and included a salty solution
medium range and five (Auri. auricular-judae, P. ferulae,
(0.045 g tartaric acid and 22.37 g potassium chloride), sour solu-
P. salmoneostramineus, S. crispa, T. fuciformis) were in the low range.
tion (0.45 g tartaric acid and 2.24 g potassium chloride dissolved
As the umami taste of mushrooms is elevated by the level of
in 1 L of distilled water), umami solution (0.045 g tartaric acid,
flavour 50 -nucleotides, the results suggest that our samples pro-
2.24 g potassium chloride, and 1.87 g monosodium glutamate), a
mise a good potential for these mushrooms to be employed as food
bitter (+) solution (0.045 g tartaric acid, 2.24 g potassium chloride,
seasonings or food additives.
and 0.04 g quinine hydrochloride), a bitter () solution (0.045 g
tartaric acid, 2.24 g potassium chloride, and 100 lL iso-a-acid),
and an astringent solution (0.045 g tartaric acid, 2.24 g potassium 3.2. Free amino acids
chloride, and 0.05 g tannic acid). Sample solutions were prepared
by extraction of mushroom powders with boiling water (1% w/v) According to Yamaguchi (1991), among all free amino acids,
for 5 min and centrifugation for 10 min at 3000 rpm before only aspartic acid and glutamic acid contribute to the characteris-
analysis. tic umami taste. Therefore, we focused on these two amino acids
 C. Phat et al. / Food Chemistry 192 (2016) 1068–1077 1071

Table 1
Individual 50 -nucleotides content (mg/g) of mushroom samples.

Samples AMP CMP GMP IMP UMP XMP Flavour 50 - Total 50 -

nucleotides nucleotides
Agaricus bisporus 1.77 ± 0.60f 3.46 ± 0.30f ND 7.54 ± 0.34b 0.26 ± 0.03jk ND 7.54 ± 0.33c 13.03 ± 0.56ef
Amanita virgineoides 8.67 ± 0.04c 21.13 ± 0.76a ND 1.08 ± 0.14f 5.29 ± 0.31d 0.76 ± 0.14ef 1.84 ± 0.20f 36.93 ± 1.50a
Auricularia auricula-judae 0.22 ± 0.18h 0.03 ± 0.03k ND 0.03 ± 0.03h 0.05 ± 0.01k 0.07 ± 0.09j 0.10 ± 0.08i 0.40 ± 0.27l
Flammulina velutipes 9.79 ± 1.39b 20.51 ± 0.95ab ND 4.51 ± 0.19d 0.32 ± 0.03j 0.43 ± 0.12gh 4.94 ± 0.24d 35.55 ± 2.62a
Grifola frondosa 0.76 ± 0.10gh 1.88 ± 0.02gh ND 1.02 ± 0.34f 10.40 ± 0.14a 0.44 ± 0.06gh 1.46 ± 0.34fg 14.50 ± 0.44e
Hericium erinaceus 2.27 ± 1.07f 3.47 ± 1.40f ND 5.57 ± 0.14c 8.48 ± 0.18b 4.74 ± 0.15a 10.31 ± 0.28b 24.53 ± 1.38d
Hypsizigus marmoreus 3.71 ± 0.62e 2.87 ± 0.14f ND 4.18 ± 0.17d 0.25 ± 0.13jk 0.59 ± 0.14fgh 4.76 ± 0.20d 11.81 ± 0.83f
Lentinus edodes 1.70 ± 0.52fg 4.98 ± 0.14e ND 0.35 ± 0.01g 2.38 ± 0.08e 2.17 ± 0.05b 2.52 ± 0.27e 11.58 ± 0.82f
Pleurotus cornucopiae var. ND 1.15 ± 0.10hij ND 0.40 ± 0.14g ND 1.09 ± 0.49d 1.49 ± 0.62fg 2.64 ± 0.53j
citrinopileatus
Pleurotus eryngii 0.67 ± 0.16h 19.70 ± 0.16b ND 3.28 ± 0.04e 5.50 ± 0.06c 1.49 ± 0.07c 4.77 ± 0.04d 30.65 ± 0.06c
Pleurotus ferulae ND 2.69 ± 0.07fg ND 0.09 ± 0.01h 0.12 ± 0.00jk 0.65 ± 0.05efg 0.74 ± 0.06h 3.55 ± 0.13hi
Pleurotus ostreatus 5.44 ± 0.55d 9.33 ± 0.69c ND 13.93 ± 0.05a 0.51 ± 0.02i 0.88 ± 0.10de 14.81 ± 0.05a 30.08 ± 0.37c
Pleurotus salmoneostramineus 30.86 ± 0.01a 0.98 ± 0.03ij ND 0.11 ± 0.01h 0.87 ± 0.11h 0.03 ± 0.00j 0.14 ± 0.01i 32.84 ± 0.46b
Polyozellus multiplex 0.04 ± 0.02h 0.26 ± 0.04jk ND 1.05 ± 0.02f 1.19 ± 0.02g 0.34 ± 0.05hi 1.39 ± 0.07g 2.88 ± 0.14i
Ramaria botrytis (Pers.) Ricken 0.64 ± 0.36h 1.27 ± 0.09hi ND 0.96 ± 0.01f 1.44 ± 0.08f 0.12 ± 0.01ij 1.08 ± 0.04gh 4.44 ± 0.39h
Sparassis crispa 0.59 ± 0.61h 0.37 ± 0.25jk ND 0.08 ± 0.04h 0.07 ± 0.03k 0.01 ± 0.00j 0.09 ± 0.03i 1.12 ± 0.62k
Tremella fuciformis 0.13 ± 0.04h 8.15 ± 0.18d ND 0.06 ± 0.00h 0.05 ± 0.01k 0.06 ± 0.01j 0.12 ± 0.01i 8.44 ± 0.14g

Values are expressed as mean ± standard deviation of triplicate analysis.

ND: not detected.
Flavour 50 -nucleotides = GMP + IMP + XMP.
a–l
Values bearing different superscript lowercase letters within the same column are significantly different (Duncan, p < 0.05).

instead of profiling all free amino acids in the mushroom samples.
Aspartic and glutamic acid were detected in all samples. The
highest aspartic acid concentration was found in Aga. bisporus
(18.1 ± 2.57 mg/g), while the highest glutamic acid concentration
was detected in Ama. virgineoides (35.0 ± 3.66 mg/g; Table 2).
Beluhan and Ranogajec (2011) reported that the combination of
aspartic acid and glutamic acid contributed to the MSG-like taste
or palatable taste. MSG-like components also affect the EUC levels
of mushrooms; those with high concentration of MSG-like
compounds tend to have high EUC values as well. All 17
mushrooms tested here exhibited wide ranges of MSG-like
levels from 0.94 ± 0.17 to 42. 4 ± 6.90 mg/g. Aga. bisporus,
P. salmoneostramineus, and Ama. virgineoides featured the highest
levels of MSG-like compounds at 42.4 ± 6.90, 41.9 ± 3.57, and
41.8 ± 4.45 mg/g, respectively. Various other studies have reported
a high content of MSG-like compounds in other edible mushroom
types, such as Craterellus cornucopioides (45.85 mg/g), Phellinus
linteus (42.43 mg/g), and P. ostreatus (41.26 mg/g) (Beluhan &
Ranogajec, 2011; Liang, Tsai, Huang, Liang, & Mau, 2010). The
MSG-like components in common mushrooms were previously
measured at 11.44 mg/g of dry weight (Zhang, Venkitasamy, Pan,
& Wang, 2013). Tsai, Weng, Huang, Chen, and Mau (2006)
determined the level of MSG-like compounds in G. frondosa as
6.51 mg/g, which was lower than the results in our study.
3.3. LC–MS/MS
The LC–MS/MS full-scan negative electrospray ion (ESI) mass
spectra for 50 -nucleotides are shown Fig. 1. The mass spectra for
CMP, AMP, UMP, IMP, XMP, and GMP showed the protonated
molecular ion at m/z 322, 346, 323, 347, 363, and 344, respectively.
50 -Nucleotides detected by HPLC were analysed by additional
LC–MS/MS and compared with protonated molecular ions of the
standards (Supplemented material). According to Lorenzetti, Lilla,
Donato, and Nucci (2007), the negative-ion mode of ESI-MS seems
to be a logical starting point for nucleotide analysis because of the
presence of one or more negatively charged phosphate groups in
the molecules. In their study, the m/z values of AMP and GMP were
348.10 and 364.10, respectively. Mateos-Vivas et al. (2015) also
reported that the mass-to-charge ratio for AMP was m/z 361, and

Table 2
Content of aspartic acid, glutamic acid and equivalent umami concentration (EUC) in mushroom samples.

Samples Aspartic acid (mg/g) Glutamic acid (mg/g) MSG-like (mg/g) EUC (mg MSG/g)
Agaricus bisporus 18.1 ± 2.57a 24.3 ± 4.38bc 42.4 ± 6.90a 2,480 ± 358b
Amanita virgineoides 6.80 ± 0.08cd 35.0 ± 3.66a 41.8 ± 4.45a 1380 ± 55.0c
Auricularia auricula-judae 0.33 ± 0.05g 0.61 ± 0.13h 0.94 ± 0.17f 1.51 ± 0.42d
Flammulina velutipes 1.60 ± 0.36fg 5.83 ± 1.44gh 7.43 ± 1.80f 480 ± 152d
Grifola frondosa 2.48 ± 0.37f 12.2 ± 1.87ef 14.6 ± 2.23de 226 ± 83.2d
Hericium erinaceus 4.76 ± 1.32e 10.3 ± 2.81fg 15.0 ± 4.13cde 1160 ± 395c
Hypsizigus marmoreus 3.09 ± 0.59f 19.7 ± 3.90cd 22.8 ± 4.49bcd 1280 ± 183c
Lentinus edodes 1.95 ± 0.03fg 9.54 ± 2.43fg 11.5 ± 0.51e 243 ± 20.6d
Pleurotus cornucopiae var. citrinopileatus 5.56 ± 1.38de 17.6 ± 4.22de 23.1 ± 5.61bc 252 ± 153d
Pleurotus eryngii 5.74 ± 0.81de 9.50 ± 1.23fg 15.2 ± 2.03cde 532 ± 65.5d
Pleurotus ferulae 2.91 ± 0.36f 12.3 ± 1.93ef 15.2 ± 2.29cde 86.8 ± 11.9d
Pleurotus ostreatus 7.66 ± 1.56c 20.0 ± 4.27cd 27.6 ± 10.38b 3890 ± 833a
Pleurotus salmoneostramineus 13.9 ± 1.19b 28.0 ± 2.38b 41.9 ± 3.57a 2040 ± 180b
Polyozellus multiplex 2.13 ± 0.15fg 3.38 ± 0.43h 5.51 ± 0.36f 58.3 ± 8.59d
Ramaria botrytis (Pers.) Ricken 1.29 ± 0.16cfg 2.63 ± 0.37h 3.92 ± 0.52f 41.1 ± 7.04d
Sparassis crispa 2.42 ± 0.68f 11.8 ± 8.12efg 14.2 ± 7.68e 40.4 ± 17.4d
Tremella fuciformis 1.36 ± 0.18fg 3.60 ± 0.44h 4.96 ± 0.69f 8.88 ± 4.32d

Values are expressed as mean ± standard deviation of triplicate analysis.

MSG-like = aspartic acid + glutamic acid.
a–h
Values bearing different superscript lowercase letters within the same column are significantly different (Duncan, p < 0.05).
1072 C. Phat et al. / Food Chemistry 192 (2016) 1068–1077

Fig. 1. LC–MS and LC–MS/MS chromatogram for samples (A) P. salmoneostramineus (AMP), (B) Ama. virgineoides (CMP), (C) P. ostreatus (IMP), (D) H. erinaceus (UMP), (E) H.
erinaceus (XMP).
 C. Phat et al. / Food Chemistry 192 (2016) 1068–1077 1073

Fig. 1 (continued)

that for UMP was m/z 334. In a study by Yang, Li, Feng, Hu, and Li findings were similar to the results of our present study.
(2010), it was reported that AMP, GMP, and UMP are correspond- Comparable results were also reported by Wang et al. (2014) and
ing to mass-to-charge m/z 348, 364, and 325, respectively. These Yamaoka et al. (2010).
1074 C. Phat et al. / Food Chemistry 192 (2016) 1068–1077

Fig. 1 (continued)

3.4. Equivalent umami concentration (EUC)
According to Mau (2005), the EUC values can be grouped into
four levels as: (1) >10,000 mg/g dry weight, (2) 1000–
10,000 mg/g dry weight, (3) 100–1000 mg/g dry weight, and (4)
<100 mg/g dry weight, corresponding to >10, 1–10, 0.1–1 and
<0.1 g MSG/g, respectively. As demonstrated in Table 2, the EUC
values of the mushrooms determined here ranged from
1.51 ± 0.42 mg MSG/g dry weight in Auri. auricula-judae to
3890 ± 833 mg MSG/g dry weight in P. ostreatus. Six mushroom
samples were classified as level (2), and five and six mushrooms
samples as levels (3) and (4), respectively. In the study by
Beluhan and Ranogajec (2011), the EUC levels of P. ostreatus and
auricular-judae (2.46 ± 1.33), which also showed the lowest EUC
levels (Table 2). These results suggest that the human sensory eval-
uation yields results very similar to those of the umami intensity
determination using HPLC.
Human sensory test was performed to evaluate the taste of food
samples in various studies, including juices, breads, meat, tea, and
many more (Bleibaum et al., 2002; He et al., 2009). A study on the
quality of bread supplemented with mushroom mycelia by
Table 3
Umami taste intensity based on sensory evaluations by human and electronic tongue
test.

F. velutipes were measured at 1505.5 ± 21.9 and 737.8 ± 9.1 mg Samples Human sensory Electronic tongue
MSG/g, respectively. These results were comparable to those in test test
the present study. Although previous reports have evaluated the Agaricus bisporus 8.60 ± 1.67a 13.81 ± 0.13b
EUC levels of mushrooms, only a few mushrooms were evaluated Amanita virgineoides 6.00 ± 1.22b 12.11 ± 0.17f
in each study (Cho, Choi, & Kim, 2010; Lee et al., 2009). In contrast, Auricularia auricula-judae 2.46 ± 1.33d 8.45 ± 0.33j
Flammulina velutipes 4.00 ± 2.68bcd 14.35 ± 0.20a
our study provided complete information related to the umami
Grifola frondosa 5.60 ± 2.41bc 12.81 ± 0.05de
taste from a wide range of mushroom samples. Thus, the calculated Hericium erinaceus 4.83 ± 1.94bcd 13.58 ± 1.61c
EUC values in this study will prove helpful for the consideration of Hypsizigus marmoreus 4.80 ± 2.28bcd 14.16 ± 0.03a
these mushrooms as food additives or food seasoning components. Lentinus edodes 4.00 ± 1.22bcd 11.83 ± 0.00g
Pleurotus cornucopiae var. 4.67 ± 1.03bcd 8.96 ± 0.11i
citrinopileatus
3.5. Sensory evaluations Pleurotus eryngii 4.40 ± 2.30bcd 12.98 ± 0.12d
Pleurotus ferulae 5.00 ± 2.10bcd 13.41 ± 0.07c
3.5.1. Human sensory evaluation Pleurotus ostreatus 9.33 ± 1.51a 13.81 ± 0.21b
Pleurotus salmoneostramineus 6.00 ± 1.41b 12.61 ± 0.02e
A sensory evaluation was also performed to measure umami
Polyozellus multiplex 3.00 ± 1.22cd 11.75 ± 0.00g
intensity in all mushroom samples. Significant differences in the Ramaria botrytis (Pers.) Ricken 3.00 ± 1.87cd 10.96 ± 0.09h
umami taste intensity among the samples were observed Sparassis crispa 3.60 ± 1.52bcd 12.58 ± 0.12e
(p < 0.05). Table 3 shows that the umami taste intensity was high- Tremella fuciformis 2.42 ± 0.62d 12.74 ± 0.00e
est in the mushroom extract of P. ostreatus (9.33 ± 1.51), which also Values are expressed as mean ± standard deviation of triplicate analysis.
exhibited the highest EUC level. The lowest intensity level was a–h
Values bearing different superscript lowercase letters within the same row are
observed in the extract from T. fuciformis (2.42 ± 0.62) and Auri. significantly different (Duncan, p < 0.05).
 C. Phat et al. / Food Chemistry 192 (2016) 1068–1077 1075

(A)
Sourness
20
10
0 Flammulina velutipes
Umami -10 Bitterness
-20 Hypsizigus marmoreus
-30
-40 Pleurotus ostreatus
-50
Agaricus bisporus
Hericium erinaceum
Astringency Saltiness
Pleurotus ferulae

Richness

(B)
Sourness
20
10 Pleurotus eryngii
0
Umami -10 Bitterness Grifola frondosa
-20
Tremella fuciformis
-30
-40 Pleurotus
salmoneostramineus
Sparassis crispa
Astringency Saltiness
Amanita virgineoides

Richness

(C)
Sourness
30
20 Lentinus edodes
10
Umami 0 Bitterness
-10 Polyozellus multiplex
-20
-30 Ramaria botrytis (Pers.)
-40
Ricken
Pleurotus cornucopiae
Astringency Saltiness var. citrinopileatus
Auricularia auricula-
judae

Richness
Fig. 2. Spider plot for the sensory score based on the taste sensing system of mushroom samples (A) F. velutipes, Hyp. marmoreus, P. ostreatus, Aga. bisporus, H. erinaceus, P.
ferulae; (B) P. eryngii, G. frondosa, T. fuciformis, P. salmoneostramineus, S. crispa, Ama. virgineoides; and (C) L. edodes, Pol. multiplex, R. botrytis (Pers.) Ricken, P. cornucopiae var.
citrinopileatus, Auri. auricula-judae.

Ulziijargal, Yang, Lin, Chen, and Mau (2013) revealed that the
umami intensity of all mycelium-supplemented breads was higher
than that of white bread, and the sensory profiles of these breads
were moderately acceptable in flavour and overall scores. In
another study, Dermiki et al. (2013) mentioned that the sensory
analysis of meat samples containing shiitake mushroom extract
scored higher in umami compared to the control. This result sug-
gests that shiitake mushroom extract could be used to enhance
the umami taste of food without significantly changing the flavour
attributes of the final products. Bai, Guo, Ma, Guo, and Lin (2013)
also performed sensory evaluation of fermented tea with medicinal
mushrooms and the results revealed that the low-grade tea leaves
1076 C. Phat et al. / Food Chemistry 192 (2016) 1068–1077

were significantly upgraded and the flavour of the fermented tea Table 4
was also improved by fermentation with the medicinal mushroom. Correlation coefficients between sensory evaluations, EUC and each of umami
components.

3.5.2. Electronic tongue measurement Variables Correlation p value

coefficients
The sensory evaluation of mushroom extracts was performed
using the electronic tongue test. Umami taste intensity based on EUC and human sensory evaluation*** 0.86 <0.00001
EUC and electronic tongue measurement* 0.57 0.02
the electronic tongue measurement ranged from 8.45 ± 0.33 to
Human sensory evaluation and electronic 0.51 0.04
14.35 ± 0.20. All mushroom samples showed negative scores in tongue measurement*
saltiness and sourness (Fig. 2). The bitterness of all samples was Human sensory evaluation and AMP* 0.58 0.01
relatively high. Mushrooms with low umami taste score appeared Human sensory evaluation and IMP* 0.57 0.02
to have very high bitterness score, whereas mushrooms with high Human sensory evaluation and XMP 0.19 0.47
Human sensory evaluation and flavour 5’- 0.54 0.02
umami taste levels also featured high levels of saltiness. The
nucleotides*
umami intensity from this evaluation is presented in Table 3. Human sensory evaluation and aspartic 0.87 <0.00001
Umami taste intensity significantly differed among the samples acid***
of mushroom extracts (p < 0.05). F. velutipes exhibited the highest Human sensory evaluation and glutamic 0.88 <0.00001
acid***
intensity with a score of 14.35 ± 0.20 and the lowest intensity
Human sensory evaluation and MSG-like*** 0.89 <0.00001
was observed in Auri. auricula-judae (8.45 ± 0.33). Electronic tongue measurement and AMP* 0.53 0.03
The electronic tongue or taste sensing system was also success- Electronic tongue measurement and IMP** 0.65 0.004
fully employed in various studies by Chen et al. (2008) and He et al. Electronic tongue measurement and XMP 0.08 0.76
(2009). Bleibaum et al. (2002) conducted a study in which apple Electronic tongue measurement and flavour 0.64 0.005
5’-nucleotides**
juice quality was compared by both human sensory evaluation
Electronic tongue measurement and aspartic 0.38 0.13
and electronic tongue sensors. Results from those studies implied acid
that the electronic tongue system is capable of evaluating taste Electronic tongue measurement and glutamic 0.37 0.15
characteristics of a variety of samples. Tran et al. (2004) applied acid
Electronic tongue measurement and MSG-like 0.40 0.11
the electronic tongue system for the taste analysis of brown rice.
In their study, the sensory scores for umami and sweetness of Correlation is significant at p value <0.05.
*
cooked brown rice with different milling yields were compared, p value <0.05.
**
and it was concluded that the electronic tongue could successfully p value <0.01.
***
p value <0.001.
be employed for the evaluation of rice taste. It is possible to predict
rice umami taste using the taste sensing system. Taste differences another and that these methods provide comparable results and
of brown rice with different milling yields can be determined not can be used equivalently.
only by physicochemical methods but also using the taste sensor. The correlation between the human sensory evaluation and the
Another application of the electronic tongue test in the food electronic tongue measurement was identified by He et al. (2009)
science field was performed by Qiu, Wang, and Gao (2015) for eval- in a study assessing Chinese tea. The results showed that electronic
uation of processed strawberry juice, in which basic tastes such as tongue sensors were correlated best with human sensory evalua-
sour, salty, sweet, bitter, and savory were compared between sam- tion, which is in agreement with the results of our study.
ples. The results from that study proposed that utilisation of the Kobayashi et al. (2010) also reported that taste sensors yield
electronic tongue represents a fast and cheap tool for qualitative results closer to human sensory scores for samples with similar
discrimination between processed strawberry juices. taste but different taste intensity, and the correlation between
these two methods was very high, suggesting that the electronic
3.6. Correlation between sensory evaluations and umami intensity tongue can function as a taste sensor for an objective taste evalu-
ation. In a study by Tran et al. (2004), it was stated that significant
The Spearman’s rank correlation is a non-parametric measure of differences of umami taste were observed between samples with
statistical dependence between two variables. The Spearman’s different milling yields when evaluated by electronic tongue sys-
rank correlation coefficient rs is computed by using rank scores Ri tems, suggesting that differences of brown rice samples can be
for Xi and Ci for Yj. These rank scores are defined as follows: determined by both physiochemical methods and by electronic
X tongue systems. Correlation between human sensory evaluation,
Ri ¼ r
k<i k
þ ðr i þ 1Þ=2 for i ¼ 1; 2; . . . ; R
chemical analysis, and electronic tongue system was also reported
X in other studies by Bleibaum et al. (2002), Dermiki et al. (2013),
Cj ¼ c
h<j h
þ ðcj þ 1Þ=2 for j ¼ 1; 2; . . . ; C Jiang, Luo, and Ying (2015).

The formulas for rs and its asymptotic variance can be obtained

from the Pearson formulas by substituting Ri and Cj for Xi and Yj,
respectively.
Correlation is considered significant at a p-value <0.05. The EUC
values of mushroom samples showed a correlation with both the
score from the human sensory evaluation and the electronic ton-
gue measurement score (Table 4). Moreover, umami taste intensity
based on the human sensory evaluation also significantly corre-
lated with that of the electronic tongue measurement. The human
sensory evaluation had a tendency to correlate well with all com-
ponents of umami taste except for XMP. In the same way, umami
taste scores from the electronic tongue measurement exhibited
strong correlation with IMP and flavour 50 -nucleotides, and moder-
ate correlation with the AMP level. It can be concluded from these
results that all the three analysis methods correlated well with one
4. Conclusion
In this study, the umami taste of mushroom extracts was anal-
ysed by HPLC, human sensory evaluation, and electronic tongue
measurement. In addition, the correlations between sensory eval-
uations (human and electronic tongue), EUC, and each umami taste
component were measured. The EUC results showed that the levels
of umami taste compounds in some mushroom extracts were very
high. Based on these findings, mushrooms should be considered as
a good raw material and natural source for industrial seasoning
manufacture.
Correlations between EUC levels and umami taste scores of the
electronic tongue measurement, as well as between the score from
the human sensory evaluation and the electronic tongue
 C. Phat et al. / Food Chemistry 192 (2016) 1068–1077
measurement were observed. EUC value was strongly correlated
with umami intensity obtained from the electronic tongue mea-
surement suggesting that the electronic tongue is capable of iden-
tifying the umami intensity in samples. These results are promising
in terms of the application of the electronic tongue as an objective
measurement for conventional sensory evaluations or chemical
analyses of the umami taste of mushrooms. This methodology
could therefore potentially play a key role in food processing
applications.
Acknowledgements
This work was supported by the GRRC program of the
Gyeonggi province (GRRC-CAU-2012-B01), Republic of Korea
and by the development of mushroom products and related
functional resources of the Rural Development Administration
(PJ907021102012), Republic of Korea.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2015.
07.113.
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