Food Chemistry 206 (2016) 174–181

Contents lists available at ScienceDirect

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

Sequence, taste and umami-enhancing effect of the peptides separated

from soy sauce
Mingzhu Zhuang a, Lianzhu Lin a,b, Mouming Zhao a,b, Yi Dong a, Dongxiao Sun-Waterhouse a,b,
Huiping Chen a, Chaoying Qiu c, Guowan Su a,b,⇑
a
School of Food Science and Engineering, South China University of Technology, China
b
Guangdong Food Green Processing and Nutrition Regulation Technologies Research Center, Guangzhou 510650, China
c
Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China

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

Article history: Five tasty peptides were separated from soy sauce, by sensory-guided fractionation, using macroporous
Received 6 September 2015 resin, medium-pressure liquid chromatography and reverse phase-high performance liquid chromatog-
Received in revised form 7 March 2016 raphy, and identified by ultra-performance liquid chromatography tandem mass-spectrometry as
Accepted 17 March 2016
ALPEEV, LPEEV, AQALQAQA, EQQQQ and EAGIQ (which originated from glycinin A1bB2-445, glycinin
Available online 18 March 2016
A1bB2-445, cobyric acid synthase, leucine-tRNA ligase and glycoprotein glucosyltransferase, respec-
tively). LPEEV, AQALQAQA and EQQQQ tasted umami with threshold values of 0.43, 1.25 and
Keywords:
0.76 mmol/l, respectively. ALPEEV and EAGIQ had minimal umami taste, but ALPEEV, EAGIQ and LPEEV
Amino acid sequence
Peptide
showed umami-enhancement with a threshold estimated at 1.52, 1.94 and 3.41 mmol/l, respectively.
Separation In addition, the synthetic peptides showed much better sensory taste than mixtures of their constitutive
Soy sauce amino acids. It indicated that peptides might play an important role in the umami taste of soy sauce.
Umami taste Ó 2016 Elsevier Ltd. All rights reserved.
Umami-enhancement

1. Introduction
Soy sauce is one of the most commonly used seasoning prod-
ucts worldwide due to its strong and distinct umami taste
(Keshun, 1997). The pleasant umami taste of soy sauce is mainly
derived from peptides, amino acids and other taste-associated sub-
stances, such as nucleotides and salts (Li, Zhao, Zhao, & Cui, 2010).
Sodium salt, and some free amino acids, especially glutamic acid,
aspartic acid, non-charged medium-sized sweet taste-eliciting
amino acids (such as alanine, serine and glycine) and aromatic
amino acids (such as L-phenylalanine and L-tyrosine), are believed
to contribute significantly to the sensory characteristics including
umami taste of Japanese soy sauce (Lioe, Wada, Aoki, & Yasuda,
2007) or Indonesian soy sauce (Lioe, Selamat, & Yasuda, 2010).
Apriyantono, Setyaningsih, Hariyadi, and Nuraida (2004) proposed
that the most delicious fraction with a molecular weight of less
than 500 Da produced via ultrafiltration from Indonesian soy sauce
was responsible for the umami taste of the Indonesian soy sauce.
While there is a growing interest in the major constituents in soy
⇑ Corresponding author at: School of Food Science and Engineering, South China
University of Technology, China.
E-mail address: fegwsu@scut.edu.cn (G. Su).
sauce accounting for its umami taste, those in the Chinese soy
sauce have not yet been isolated and examined.
In non-soy sauce products, some peptides have already been
found to elicit umami taste, including those naturally occurring
in protein hydrolysates (such as the two novel umami-associated
peptides from peanut hydrolysate (Su et al., 2012)) and in fer-
mented products (such as the three sensory-active peptides from
Chinese rice wine (Han & Xu, 2011)), as well as those synthetic
peptides (such as Glu-Glu, Glu-Val, Ala-Asp-Glu, Ala-Glu-Asp,
Asp-Glu-Glu and Ser-Pro-Glu (Maehashi, Matsuzaki, Yamamoto,
& Udaka, 1999)). Following this logic, it is of high interest to inves-
tigate whether or not the peptides purified from the Chinese soy
sauce exhibit umami taste.
In the present study, the tasty peptides from Chinese soy sauce
were isolated and separated, via sensory-guided fractionation,
using macroporous resin, medium-pressure liquid chromatogra-
phy (MPLC), and reverse phase-high performance liquid chro-
matography (RP-HPLC). The amino acid sequences of the
resultant umami-tasting fractions were determined by ultra-
performance liquid chromatography tandem mass-spectrometry
(UPLC–MS/MS). These identified peptides were further subjected
to sensory evaluation to elucidate their taste, umami-enhancing
effects, as well as the structure–activity and dose–response
relationships.

http://dx.doi.org/10.1016/j.foodchem.2016.03.058
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
 M. Zhuang et al. / Food Chemistry 206 (2016) 174–181
2. Materials and methods
2.1. Soy sauce sample
Light Soy sauce was provided by the Lee Kum Kee CO. (Guangz-
hou, China), a portion of which was directly lyophilized using the
Alpha 1-4 LD plus freeze dryer (Marin Christ, Osterode, Germany),
and the dried soy sauce contained total nitrogen (TN) 4.29% (w/w),
ammonia nitrogen (AN) 2.45% (w/w) and sodium chloride 42.38%
(w/w). The other portion of original light soy sauce was concen-
trated by gentle rotary evaporation (55 °C), and then lyophilized,
which contained TN 6.45% (w/w), AN 3.71% (w/w) and sodium salt
17.97% (w/w). All the different dried soy sauces were stored at
20 °C until further purification.
2.2. Materials and chemicals
Five commercially available synthetic peptides (ALPEEV, LPEEV,
AQALQAQA, EQQQQ and EAGIQ) were of food grade and purchased
from GL Biochem. Comp., Shanghai, the purity of the synthetic pep-
tides was higher than 95%, and the solvent or chemical residues of
peptides were removed by GL Biochem Comp to ensure the safety of
the taste study. Food grade caffeine, citric acid, sodium chloride,
sucrose, monosodium glutamate (MSG) and food grade amino acids
(alanine (A), leucine (L), proline (P), glutamate (E), valine (V), glu-
tamine (Q), glycine (G), isoleucine (I)) were provided by Guangdong
Hui Xiang Source Biological Technology Co., Ltd. Macroporous
resins (XAD-16) was purchased from H&G Co., Ltd., Beijing, China.
All other chemicals for analyses were of analytical (AR) grade.
2.3. Separation and purification of soy sauce on XAD-16 macroporous
resin
The separation and purification of soy sauce on XAD-16 macro-
porous resin was carried out according to our previous research
(Zhuang et al., 2016). Soy sauce was sequentially eluted with
deionized water, 20% (v/v) ethanol and 40% (v/v) ethanol to sepa-
rate into three fractions (XAD-0%, XAD-20% and XAD-40%). Each
eluting fraction was concentrated by gentle rotary evaporation
(RE52AA, Yarong Equipment Co., Shanghai, China), then lyophilized
to remove analytical solvent in a freeze-dryer (Marin Christ, Oster-
ode, Germany) and stored at 20 °C until further analysis. Results
showed that XAD-0% fraction containing the highest peptides and
umami amino acids, displayed better sensory taste than XAD-20%
and XAD-40% fractions, especially the umami and salty taste. Thus,
the XAD-0% fraction was further purified in the present study.
2.4. MPLC separation and purification of the eluate from XAD-16
macroporous resin
The XAD-0% fraction was firstly reconstituted in Milli-Q water
to achieve a concentration of 10 mg/ml. An aliquot (20 ml), of such
a solution, was then carefully loaded onto the top of the C-18 gel
column (49  460 mm, Buchi, Swiss) at a constant temperature
(25 °C). The column was sequentially eluted with 1000 ml of 2%,
5%, 10% or 15% (v/v) ethanol, respectively, at a constant flow rate
of 10 ml/min and room temperature (25 °C). The elution process
was repeated, and each eluate was concentrated separately using
a rotary evaporator (RE52AA, Yarong Equipment Co., Shanghai,
China) at reduced pressure and 55 °C, lyophilized to remove
analytical solvent and stored at 20 °C until further use.
2.5. RP-HPLC fractionation of MPLC eluates
Fractionation of MPLC eluates by RP-HPLC was conducted fol-
lowing the method of Gu et al. (2014) with some modifications
175
(Gu et al., 2014). The freeze dried MPLC eluate fraction with the
most intense umami taste was redissolved in Milli-Q water and
the solution was loaded onto a XBridgeTM BEH130 semi-
preparative C18 RP-HPLC column (10  150 mm, 10 lm; Waters,
Milford, MA, USA). The column was eluted with a linear gradient
of acetonitrile (5–50%) and Milli-Q water (95–50%) at a flow rate
of 2 ml/min, for 25 min, at room temperature (25 °C). Elution of
the compounds of interest was monitored at 214 nm (Waters
2487 Detector), and the corresponding peak fractions were col-
lected, concentrated using the rotary evaporator, lyophilized to
remove analytical solvent and stored at 20 °C until further use.
2.6. Identification of tasty peptides by UPLC–MS/MS
The dried RP-HPLC fractions were reconstituted in Milli-Q
water. Among these solutions, those with the most intense umami
taste were subjected to UPLC–MS/MS analyses using a UPLC sys-
tem (Bruker, RSLC Dionex U3000) equipped with an electrospray
ionization-mass spectrometry (ESI-MS) (Bruker maxis impact)
coupled via tandem mass spectrometry (MS/MS) and a de-novo
sequencing software. Aliquots of test samples (1 ll) were injected
via an auto-sampler, and subsequently separated by reverse phase
C18 column (2.1  100 mm; 2.2 lm) at a flow rate of 0.3 ml/min.
Two injection replicates were run for each test sample. The
RP-HPLC fraction was purified using UPLC–MS/MS with prepara-
tive setting. Then the setting showed below: a gradient elution
was employed consisting of mobile phase A (H2O, 0.1% formic acid)
and B (acetonitrile): 0–5 min, A 98% v/v; 5–8 min, A 98% v/v to 95%
v/v; 8–15 min, A 95% v/v; 15–20 min, A 95% v/v to 85% v/v;
20–25 min, A 85% v/v to 80% v/v; 25–30 min, A 80% v/v to 70%
v/v; 30–40 min, A 70% v/v to 15% v/v; 40–45 min, A 15% v/v. All
the eluted peptides from the reverse phase column were analyzed
online by MS, and the peptides of interest were selected for MS/MS
sequencing. Spectra were recorded in positive ion mode over a
mass/charge (m/z) range of 50–1200. High purity N2 was used as
drying gas at a flow rate of 8.0 l/min and as nebulizing gas at a
pressure of 2.0 bar. The peptide sequencing was accomplished
through processing the MS/MS spectra.
2.7. Sensory evaluation
Analysis was carried out with a panel of 10 members (five men
and five women, aged from 25 to 35). Panellists were trained to
evaluate the taste of the aqueous solutions (2 ml each) of the fol-
lowing standard taste compounds through a triangle test: 1%
sucrose solution for sweet taste; 0.35% sodium chloride solution
for salty taste; 0.08% caffeine solution for bitter taste; 0.35% MSG
solution for umami taste; 0.08% citric acid solution for sour taste.
The samples (MPLC fractions, HPLC fractions and RP-HPLC frac-
tions) in deionized water were transferred into small glass cups,
respectively, and served in a rationalized order with an average
serving temperature at 23 ± 2 °C. Panellists were seated separately
in a sensory panel room, at 23 ± 2 °C, under normal lighting, in three
different sessions. They were asked to sip the sample and let it swirl
around in the mouth briefly and expectorated. To avoid fatigue and
carryover effect, the panelists were asked to wash their mouth with
50–60 ml drinkable water between testing two different samples.
2.7.1. Preliminary taste profiling
Taste profiles for a 1% solution of targeting peptide-containing
samples that have been passed through a sterile filter (medical-
grade) were analyzed using a 10-point intensity scale (0, no taste;
10, the strongest taste). Reference samples for sweet, bitter, sour,
salty and umami qualities were set up: sucrose solution (1%), caf-
feine solution (0.08%), citric acid solution (0.08%), sodium chloride
solution (0.35%), and MSG solution (0.35%). The threshold of
176 M. Zhuang et al. / Food Chemistry 206 (2016) 174–181
reference samples was defined as the scale of five points for
respective taste quality. The average rating points scored by differ-
ent assessors for the five taste qualities of each fraction derived
from soy sauce were recorded.
2.7.2. Descriptive analysis for qualitative evaluation of synthetic
peptides
A series of water solutions of five synthetic peptides were pre-
pared at different concentrations (i.e. 4, 2, 1, 0.5 and 0.25 g/l) for
descriptive evaluation. To understand the umami-enhancing effect
of synthetic peptides, the dose–response relationship of the five
synthetic peptides was also examined at the concentrations of 0,
0.25, 0.5, 1, 2 and 4 g/l, with/without MSG (0.03 mg/l). These sam-
ples were scored from 0 to 10, with zero score referring to water
medium (i.e. containing no synthetic peptides and no MSG), and
scores larger than zero representing either the threshold value of
a synthetic peptide solution (if the test sample in the absence of
MSG), or the umami-enhancing effect as a function of synthetic
peptide (if the test sample in the presence of MSG). (The threshold
value was the minimum identified concentration of sample, which
meant the taste intensity of this concentration was the lowest
intensity to be identified by people.)
2.7.3. Comparative evaluation between the synthetic peptides and
their amino acid components
This comparative study aimed to demonstrate the taste differ-
ence between a synthetic peptide and its constitutive amino acids
Fig. 1. Taste intensity of five taste reference samples and (A) four fractions separated by medium-pressure liquid chromatography (MPLC-1, MPLC-2, MPLC-3 and MPLC-4);
(B) four fractions derived from RP-HPLC (HPLC-1, HPLC-2, HPLC-3 and HPLC-4).
at the same molarity. Five solutions of the constitutive amino acids
were prepared, which corresponded to ALPEEV, LPEEV, AQALQAQA,
EQQQQ or EAGIQ synthetic peptides. The same reference samples
were used herein as those described in Section 2.7.1, and their tast-
ing score values were set as 10 points. The concentrations of the
synthetic peptides used herein were the same as their threshold
values determined in Section 2.7.2, while the type and concentra-
tion of the amino acid solution corresponded to those as synthetic
peptides’ amino acid components. The average score values of
different samples were obtained and plotted for comparison.
2.8. Statistical analysis
All tests were performed in triplicate for each test sample. All
the results were reported based on at least six measurements. Data
statistical analysis was performed with one-way ANOVA using
SPSS 11 (SPSS Inc., Chicago, USA). The mean values were consid-
ered significantly different at p < 0.05 (*represent p < 0.05), which
was assessed by the paired t test.
3. Results and discussion
3.1. Five taste qualities of the fractions from the XAD-0% separated by
MPLC and RP-HPLC
MPLC is widely used for the separation of natural compounds,
organic compounds, proteins and peptides due to its high safety,
 M. Zhuang et al. / Food Chemistry 206 (2016) 174–181 177

Fig. 2. MS/MS spectra of (A) ALPEEV peptide, (B) LPEEV peptide, (C) AQALQAQA peptide, (D) EQQQQ peptide, and (E) EAGIQ peptide. b and y represented the ions generated
from peptides.

repetition and reliability. Four fractions (MPLC-1, MPLC-2, MPLC-3
and MPLC-4) isolated by C-18 gel column displayed significant
(p < 0.05) differences in saltiness, bitterness and umami, although
having almost the same sweetness (50–60% sweetness of the 1%
sucrose solution) and sourness (40–50% sourness of the 0.08% citric
acid solution) (Fig. 1A).
The MPLC-1 fraction had the highest intensities of umami taste
(1.4 times as umami as the 0.35% MSG solution) and salty taste
(1.5 times as salty as the 0.35% sodium chloride solution), but
the least bitter taste (16% of the bitterness for the 0.08% caffeine
solution). The MPLC-2 tasted the least umami, while the MPLC-3
and MPLC-4 both had medium salty and umami taste intensities.
178 M. Zhuang et al. / Food Chemistry 206 (2016) 174–181
Fig. 2 (continued)
The bitter intensity of MPLC-2, MPLC-3 and MPLC-4 fractions was
quite close to that of the 0.08% caffeine solution.
HPLC is also a strong separation method to purify proteins and
peptides according to its high speed, efficiency, sensitivity and so
on. As the highest umami fraction, MPLC-1 was used for further
fractionation by RP-HPLC, and the resultant four sub-fractions
(HPLC-1, HPLC-2, HPLC-3 and HPLC-4) were collected and sub-
jected to taste evaluation (Fig. 1B). Among the HPLC derived frac-
tions, HPLC-3 had the highest umami intensity (1.8 times as
umami as the 0.35% MSG solution), and tasted the sweetest
(65% sweetness of that for the 1% sucrose solution). HPLC-4 had
the highest bitter intensity (1.5 times as bitter as the 0.08% caf-
feine solution), while HPLC-2 exhibited the highest sourness (the
same as that of the 0.08% citric acid solution). HPLC-2 and HPLC-
3 were the saltiest (both were 1.5 times as salty as the 0.35%
sodium chloride solution). HPLC-4 could be enriched with
hydrophobic amino acids or hydrophobic bitter peptides (Zhang,
Jiao, Liu, Wu, & Zhang, 2008). HPLC-3 was dominated by the
umami and salty tastes and was selected for further purification.
3.2. Identification of taste peptides in the HPLC-3 fraction by UPLC–
MS/MS
Fig. 2 depicts the UPLC-MS/MS traces for the HPLC-3 fraction.
The molecular mass of the peptides in HPLC-3 was determined as
657.3523 Da, 586.3144 Da, 401.7106 Da, 662.2846 Da and
517.2679 Da. The peptides identified in HPLC-3 were not found
compared with the sensory-active peptides from the protein data-
base. The major fragment products generated upon high-energy
collision-induced dissociation of the protonated peptides were
well characterized as b-type ions and y-type ions. Such b and y ions
are useful for the sequence determination indicating the genera-
tion of protonated molecules and subsequent charge-proximate
fragmentations; The b-type ions and y-type ions would have been
generated from the protonated peptides blocked on the N-terminal
amino via an intramolecular nucleophilic attack of the carbonyl
oxygen located at the N-terminal end of the molecule on the car-
bonyl carbon of the adjacent C-terminal residue through the proto-
nated peptide bond. Due to the extensive fractionation and
purification employed to generate HPLC-3 in this study, sample
matrix interference was largely reduced insufficient (which eased
the peptide sequencing and suggesting the feasibility for obtaining
reliable sequence matching and structural characterization on pep-
tides). As a result, five main peptides were identified in HPLC-3:
Ala-Leu-Pro-Glu-Glu-Val (ALPEEV) (Fig. 2A), Leu-Pro-Glu-Glu-Val
(LPEEV) (Fig. 2B), Ala-Gln-Ala-Leu-Gln-Ala-Gln-Ala (AQALQAQA)
(Fig. 2C), Glu-Gln-Gln-Gln-Gln (EQQQQ) (Fig. 2D) and Glu-Ala-
Gly-Ile-Gln (EAGIQ) (Fig. 2E), respectively.
As shown in Table 1, the MS/MS spectra of ALPEEV, LPEEV,
AQALQAQA, EQQQQ and EAGIQ peptides were well matched to
 M. Zhuang et al. / Food Chemistry 206 (2016) 174–181 179

Table 1
The spectrum matching between the synthetic peptides and commercially available proteins.

Protein Molecular mass of peptides (m/z) Fragment Sequence

Glycinin A1bB2-445 657.3523 f(440–445) N.ALPEEV.I
586.3144 f(441–445) A.LPEEV.I
Cobyric acid synthase 401.7106 f(59–66) R.AQALQAQA.C
Leucine-tRNA ligase 662.2846 f(158–162) E.EQQQQ.Q
L-Glycoprotein glucosyltransferase 517.2679 f(739–743) S.EAGIQ.R

Table 2
Descriptive sensory evaluation of five synthetic peptides and their monosodium glutamate (MSG)-enhanced solutions.

Synthetic peptides Sensory evaluation of peptide water solutions

In the absence of MSG In the presence of MSG
Basic taste Threshold value (mmol/l) Umami enhancement Threshold value (mmol/l)
ALPEEV Sour, astringent 0.76 Strong umami 1.52
LPEEV Sour, sweet, umami, astringent 0.43 Slight umami 3.41
AQALQAQA Sweet, umami, astringent 1.25 Undetected umami –
EQQQQ Sour, salty, umami, astringent 0.76 Undetected umami –
EAGIQ Sour, sweet, salty, astringent 0.97 Strong umami 1.94

those of glycinin A1bB2-445 f(440–445), glycinin A1bB2-445
f(441–445), cobyric acid synthase f(59–66), leucine-tRNA ligase
f(158–162) and L-glycoprotein glucosyltransferase f(739–743),
respectively. During soy sauce fermentation, most of the proteins
from the raw materials (including soybeans and wheat) were pri-
marily hydrolyzed into short peptides or free amino acids (Kijima
& Suzuki, 2007). Glycinin A1bB2-445 as a fraction of soybean pro-
tein, could be degraded into ALPEEV and LPEEV. Thus, it is under-
standable that these two peptides were identified from soy sauce
and its HPLC-3 fraction. in addition, the composition of soy sauce
was influenced not only by soybean and wheat, but also by
microorganisms such as Aspergillus oryzae and Aspergillus sojae.
Cobyric acid synthase, leucine-tRNA ligase and L-glycoprotein
glucosyltransferase are likely microbial proteins, from which
AQALQAQA, EQQQQ and EAGIQ peptides could be isolated. There-
fore, AQALQAQA, EQQQQ and EAGIQ could also occur in soy sauce.
3.3. Sensory evaluation of synthetic peptides
The five purified peptides (ALPEEV, LPEEV, AQALQAQA, EQQQQ
and EAGIQ) were reconstituted in deionized water and subjected to
preliminary sensory evaluation according to the five basic taste
qualities (Table 2). LPEEV, AQALQAQA and EQQQQ were perceived
umami with threshold values of about 0.43 mmol/l, 1.25 mmol/l
and 0.76 mmol/l, respectively. Astringency was detected in all
the synthetic peptide samples, which probably resulted from the
presence of hydrophobic amino acids along with the residual
sodium acetate introduced during the peptide synthesis process
(Brannan, Setser, & Kemp, 2001). The detected sour taste for the
five synthetic peptide samples likely came from the free amino
acids including those residual acidic amino acids left from the pep-
tide synthesis process (Kawai & Hayakawa, 2005). Some sweetness
was perceived for the LPEEV, AQALQAQA and EAGIQ samples,
while the EQQQQ and EAGIQ samples tasted salty. Further, strong
umami-enhancement in the presence of MSG seemed possible for
ALPEEV and EAGIQ exhibiting threshold values of 1.52 mmol/l
and 1.94 mmol/l, respectively. In comparison, the presence of
MSG only led to slight umami-enhancement for LPEEV (threshold
value about 3.41 mmol/l) and no umami-enhancement for
AQALQAQA and EQQQQ.
Fig. 3 further demonstrated the dose–response effect of the syn-
thetic peptides in the presence of MSG. For the solution containing
a constant concentration (0.03 mg/l) of MSG, adding EQQQQ and
AQALQAQA at either of all the concentrations used in this study
did not influence significantly (p > 0.05) the umami taste of the
MSG-containing solution. However, the umami intensity of the
MSG-containing solution exhibited a peptide dose-dependent
increase (from the peptide concentration of 0.25 g/l) for ALPEEV,
LPEEV and EAGIQ, until above 2 g/l, where decreasing umami taste
occurred probably due to the suppression by the inherent sourness
and astringency of peptides.
To further determine the possible contribution of constitutive
amino acids to the overall taste of peptides, the intensities of the
five basic tastes of each synthetic peptide as well as the mixture
of its constitutive, were evaluated (Fig. 4). From Fig. 4A, the ALPEEV
sample tasted dominantly sour (80% of the sourness of the 0.08%
citric acid solution), while its amino acid mixture was perceived
containing almost evenly sweetness, saltiness and sourness.
Overall, both the peptide sample and the amino acid mixture had
relatively low intensities of the five basic tastes with neither hav-
ing umami nor bitter tastes. For LPEEV, the peptide solution had
four times umami taste and 2-fold sweetness as much as for the
amino acid mixture. Overall, both the peptide sample and the
amino acid mixture had relatively low intensities of the five basic
tastes with the same sour intensity. The latter had the second
strongest taste as saltiness but the former tasted no saltiness
(Fig. 4B). In contrast, AQALQAQA (Fig. 4C), EQQQQ (Fig. 4D) and
Fig. 3. The dose–response relationship of the umami taste for the solutions of the
five synthetic peptides in the presence of monosodium glutamate (MSG).
180 M. Zhuang et al. / Food Chemistry 206 (2016) 174–181
Fig. 4. Taste intensity of synthetic peptide solutions and their corresponding
mixture of constitutive amino acids: (A) ALPEEV, (B) LPEEV, (C) AQALQAQA, (D)
EQQQQ and (E) EAGIQ peptides. The values in a column meant significantly
different between synthetic peptides and constitutive (p < 0.05).
EAGIQ (Fig. 4E) had a relatively stronger overall taste (which was
reflected by the magnitude of taste intensity), with no bitterness
detected in their peptide or amino acid samples. Their peptide
solutions significantly (p < 0.05) differed from corresponding
amino acids mixtures in taste profiles, indicating the critical role
of amino acid sequence in the taste of peptides. For AQALQAQA,
the peptide solution possessed greater umami and sweet tastes
but much less saltiness and sourness than the amino acid mixture;
For EQQQQ, the sweetless peptide solution tasted more umami but
less sour than the sweet amino acid mixture, although both having
the same saltiness as the 0.35% sodium chloride solution; For
EAGIQ, minimal umami taste and relatively high sweetness
(80% of that for 1% sucrose solution) were detected for both the
peptide solution and the amino acid mixture. The peptide solution
tasted saltier (70% of that for 0.35% sodium chloride) and more
sour (same as the 0.08% citric acid solution), compared to the
amino acid mixture. These results agreed with the finding that a
peptide (such as H-Glu-Pro-Ala-Asp-OH in chicken protein)
produced through umami-guided purification from the umami
proteins could taste umamiless (Temussi, 2012). While the amino
acids in native proteins adopt L-structure, the D-amino acids could
occur in synthetic peptides, hence, the difference between L- and
D-enantiomers could contribute to the different tastes of synthetic
peptides and native peptides (Su et al., 2012).
Umami peptides and umami-enhancing peptides are closely
involved in the taste response process for ingested foods
(Yamaguchi & Ninomiya, 2000). These peptides could be extracted
largely from animal protein hydrolysates (Behrens, Meyerhof,
Hellfritsch, & Hofmann, 2011) with some from plant proteins
(Dang, Gao, Ma, & Wu, 2015). Our previous study (Su et al.,
2012) had found two peptides from peanut hydrolysate possessing
umami taste and/or umami-enhancing effect. In order to develop
foods with desired umami taste, it is important to understand
the structure-taste relationship of amino acids and peptides.
Masatoshi, Soichi, Michiko, Hiromichi, and Masao (1975) identified
nine umami peptides (Glu-Glu, Glu-Asp, Thr-Glu, Glu-Ser,
Glu-Gly-Ser, Ser-Glu-Glu, Glu-Gln-Glu, Glu-Asp-Glu and
Asp-Glu-Ser) from fish protein (Masatoshi et al., 1975). Maehashi
et al. (1999) isolated some umami peptides from chicken including
Glu-Glu, Glu-Val, Ala-Asp-Glu, Ala-Glu-Asp, Pro-Glu-Glu and
Ser-Pro-Glu (Maehashi et al., 1999). Some previous studies corre-
lated the umami taste of peptides with the Asp, Asn, Gln or Glu
amino acid residues presented in the sequence (Temussi, 2012),
which was in agreement with our present findings on the umami
synthetic peptides. Controversial results were also reported by
Matoba (2006) and Tomoyuki, Ryoji, and Toshihide (2004), the for-
mer believed Phe-Ala-Leu-Pro-Glu-Tyr-Leu-Lys tasted bitter
(Masuda & Kitabatake, 2006), and the latter found that Ala-Pro-P
ro-Pro-Pro-Ala-Glu-Val-His-Glu-Val-Val-Glu mainly showed sour
(Tomoyuki et al., 2004). Thus, the peptides composed of umami
amino acids do not always elicit the umami taste of these umami
amino acids, due to other influencing factors including the interac-
tions between amino acids and the form in which peptides are
tasted (Sun-Waterhouse & Wadhwa, 2013).
This study has confirmed the important role of peptides in the
umami taste of soy sauce, and demonstrated the feasibility to pro-
duce the tasty peptides from soy sauce using sensory-guided sep-
aration techniques such as MARs, MPLC and RP-HPLC. UPLC–MS/
MS analysis revealed the amino acid sequences of the umami pep-
tides were ALPEEV, LPEEV, AQALQAQA, EQQQQ and EAGIQ (which
were derived from glycinin A1bB2-445, glycinin A1bB2-445, coby-
ric acid synthase, leucine-tRNA ligase and L-glycoprotein glucosyl-
transferase, respectively). LPEEV, AQALQAQA and EQQQQ tasted
 M. Zhuang et al. / Food Chemistry 206 (2016) 174–181
umami while ALPEEV and EAGIQ had minimal umami taste.
EQQQQ and AQALQAQA at either the concentrations used in this
study did not exert significant (p > 0.05) enhancement on the
umami taste of the MSG-containing solution, while ALPEEV, LPEEV
and EAGIQ displayed umami-enhancing effects in a peptide
dose-dependent manner at concentrations ranging 0.25–2.0 g/l.
Comparison of the five basic taste qualities of the peptide solutions
and the mixture of its constitutive amino acids further suggested
that the taste of peptides is governed by its amino acid sequence
rather than individual amino acids. Consequently, the peptides
might play an important role in the taste of soy sauce, and it needs
further research to study the concentration of the peptides in soy
sauce, respectively. In addition to the structure of peptides would
contribute to their sensory taste, the pH of peptides also would
impact the sensory taste. Thus further work on the relationship
between sensory taste and pH is worthy to be carried out.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
The authors gratefully acknowledge Natural Science Foundation
of China (No. 31301555), Natural Science Foundation of
Guangdong Province (No. S2013040015333) and University
Doctoral Fund Project of the Ministry of Education (No.
20130172120022) for their financial supports.
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