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The changing rule of enzyme-induced release of bitter peptides in

wheat gluten hydrolysates

RSC Advances Accepted Manuscript

Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Bo-Ye Liu,a Ke-Xue Zhu,a Xiao-Na Guo,a Wei Peng,a and Hui-Ming Zhoua,*
Extraction of wheat gluten hydrolysates prepared by Proteax with isobutyl alcohol opened a new avenue for exploring the
release characteristics of bitter peptides. This study was first to search for the suitable extraction conditions of bitter
peptides and sum up the changing rule of enzyme-induced release of bitter peptides. Effects of amino acid sequence,
amino acid composition and molecule weight distribution on bitterness intensity of isobutyl alcohol extracts were
discussed. The results showed amino acid composition and molecular weight distribution had a more significant impact on
bitterness intensity. Bitter peptides that had a higher proportion of bitter amino acids were gradually released within 2 h
of hydrolysis reaction, then the content of long-chain bitter peptides gradually reduced, so bitterness intensity increased
first and then decreased. It was found that the peptide fraction ranging 500-1000 Da had the strongest bitter taste, which
should be further hydrolyzed for the scientific production of small peptide powder.

Introduction which provided a new thinking and orientation for the deep
1
In the current study, more than half of bioactive peptides exploitation of small peptide powder. Although sequential

described in the literature and found in on-line databases were hydrolysis could provide many targeted desirable

small peptides (2-6 amino acids). Small peptides had been characteristics, bitter taste couldn't be removed completely and
2
reported to be more rapidly and evenly absorbed than large was still the main limitation factor for various applications of

peptides. Therefore, there was a pragmatic explanation to that protein hydrolysates. It is significant to investigate the

phenomenon as many industrial processes using enzymatic changing rule of enzyme-induced release of bitter peptides in

hydrolysis liberated small peptides. Wheat gluten (WG) is an the process of sequential hydrolysis.

abundant, relatively inexpensive and safely edible source of

Proteax is a proteolytic enzyme preparation manufactured
pure natural plant protein.3 Enzymatic hydrolysis of WG to
by fermenting process with a selected strain of Aspergillus
produce small peptides was a promising area and had a far-
oryzae. Proteax yielded high endo- and exo-activities of protein
reaching significance in theory and practice. Sequential
and was suitable for preparing low-bitterness small peptide
hydrolysis of WG using endo- and exo-peptidase not only could
powder.7 Considering its exploitation value, it was very
improve the utilization rate of protein and the yield of small
necessary to optimize the hydrolysis reaction system of
peptides4 but also could reduce bitter taste of peptide powder,5, 6
Proteax. The hydrolysis reaction system was a complex

enzymatic hydrolysis process. The primary task should be to

State Key Laboratory of Food Science and Technology, Collaborative Innovation summarize the changing rule of enzyme-induced release of
Center for Food Safety and Quality Control, School of Food Science and
Technology, Jiangnan University, Wuxi 214122, Jiangsu Province, People’s
Republic of China. E-mail: hmzhou@jiangnan.edu.cn; Fax: +(86) 510 85809610.
bitter peptides and expatiate debittering effect of the
†Electronic Supplementary Informa)on (ESI) available: See DOI:
10.1039/x0xx00000x exopeptidase based on the enzymatic properties and the

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knowledge on the structure of bitter peptides. Finally, some Enzymatic Hydrolysis of Wheat Gluten
useful suggestions were proposed on the preparation of small WG was dispersed as a 5% (w/v) solution in deionised water

peptide powder using sequential hydrolysis. and stirred for 1 h at room temperature. The suspension was

adjusted to pH 7.0 with 1 mol/L sodium hydroxide and kept at

Previous research mainly focused on the forming
50 °C in a water bath. The reaction was then initiated by the
mechanism,8, 9 isolation and purification10 and removal methods
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addition of Proteax to give an enzyme-to-substrate (E:S) ratio

of biter peptides.11 Until recently, there were relatively few
of 1:50 (w/w) and continued up to 6 h. The proteolysis reaction
studies on the release characteristics of biter peptides in protein

RSC Advances Accepted Manuscript

was stopped at intervals of 5, 10, 15, 30, 60, 120, 180, 240, 300
hydrolysates. Due to the higher hydrophobicity of biter
and 360 min, with the enzyme immediately inactivated by heat
peptides, isobutyl alcohol12 could be a useful solvent for the
treatment in boiling water for 15 min. Then the resulting
extraction of bitter peptides. A better understanding of the
hydrolysates were centrifuged at 10000 g for 15 min at 4 °C in
structural properties of bitter peptides was essential for the
a ZOPR-52D refrigerated centrifuge (Hitachi Koki Co. Ltd.,
control of their properties during the processing and application
Tokyo, Japan). The supernatants were freeze-dried and stored at
of small peptide powder. For above purpose, the main objective
‒20 °C.
of the current research was to determine amino acid

composition, molecule weight distribution and amino acid

Sensory Analyses
sequence of bitter peptides in the hydrolysis reaction system of
The sensory evaluation panelists (5 male and 5 female, ages
Proteax. The combination of sensory analysis and electronic
between 20 and 35 years) were trained three times a week with
tongue measurements was employed to elucidate the changing
quinine standards for a period of 1 month. Ten trained panelists
trend of bitter intensity. The effect of amino acid composition,
were chosen to evaluate the bitter taste of wheat gluten
molecule weight distribution and amino acid sequence on
hydrolysates (WGHs) and isobutyl alcohol extracts. The
bitterness intensity was emphatically discussed. Further
standard solutions were presented in concentrations of 0,
apprehension of this knowledge will be very meaningful for the
8.0×10-6, 1.6×10-5, 2.4×10-5, 3.2×10-5 and 4.0×10-5 g/mL that
application of sequential hydrolysis and the scientific
were scored 0, 1, 2, 3, 4 and 5, respectively. The standard
production of small peptide powder.
condition for sensory evaluation was maintained at room

temperature of 25 °C for WGHs at the concentration of 1%

Experimental (g/mL) at pH 6.5. The final values given by the panelists were
Materials and Reagents averaged.
WG (75% protein, wet base) was provided by Nan Tong Lian

Hai Wei Jing Co., Ltd. (Jiangsu, China). ProteAX (1400 U/g,
Taste Dilution Analysis
combined proteases) was donated by Amano Enzyme Inc
The bitterness of WGHs at different times (0−6 h) of enzymatic
(Nagoya, Japan). All other reagents and chemicals were of
hydrolysis was evaluated by the TDA method,13 with some
analytical grade.
minor changes. Briefly, freeze-dried enzymatic hydrolysates of

1 g were dispersed in 100 mL of deionised water at room

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temperature. WGHs were diluted stepwise at 1:1 ratio with volumetric flasks with trichloroacetic acid (10%, v/v). After

deionized water, which were then presented to a trained sensory incubation for 1 h at room temperature, the solution was filtered

panel (5 male and 5 female, ages between 20 and 35 years) in through Whatman filter paper No 4. The filtrate was

the order of increasing solute concentrations, and judged in a centrifuged at 7,000×g for 10 min, and the supernatant was

triangle test. Taste dilution (TD) factor was defined as the stored at 4 °C before injection. For the determination of the
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dilution at which a taste difference between the diluted sample total amino acids, isobutyl alcohol extracts (0.12 g) were

and two blanks (1% WG solution) could just be detected. The hydrolyzed at 110 °C for 24 h with 6 mol/L HCl in evacuated

RSC Advances Accepted Manuscript

TD value was the average value of the evaluation results sealed tubes. Then the sample (25 mL) was transferred to

provided by the inspectors. The differences among the results volumetric flasks and filtered. The filtrate was then transferred

should not be more than one of the dilution levels. to a 10 mL beaker and placed in a vacuum desiccator to remove

hydrochloric acid. The dried sample was then dissolved in 1

Isobutyl Alcohol Extraction mL 0.02 mol/L HCl and kept at 4 °C before injection. External
The 10 g of freeze-dried WGHs at different times (10 min, 15 standards were used for quantification. The content of different

min, 30 min, 120 min and 300 min) of enzymatic hydrolysis amino acids including free amino acids or the total amino acids
was dispersed in deionised water to the concentration (5%, were expressed as mg/g isobutyl alcohol extracts.
10%, 15%, 20% and 25% (w/v)) required. Then the solution

was mixed with equal volume isobutyl alcohol to give a two-

Determination of Molecular Weight Distribution
phase system. The mixture was shaken for about 2 min. When The MW distribution profiles of isobutyl alcohol extracts

the liquid was divided into two distinct layers after 15 min, were determined by high-performance gel-filtration

isobutyl alcohol phase and aqueous phase were separated, chromatography (HPGFC) on a TSK gel G2000 SWXL

freeze-dried and stored at ‒20 °C for further analysis. The 7.8×300 mm column (Tosoh Co., Tokyo, Japan) with Waters

nitrogen recovery of the two phases was measured separately 1525 liquid chromatography system (Waters Co., Milford, MA,

by the micro-Kjeldahl procedure (N×5.7). USA) according to the method of Liu et al,15 with some minor

changes. HPGFC was carried out with the mobile phase

(acetonitrile/water/trifluoroacetic acid, 10/90/0.1, v/v/v) at a

Analysis of Amino Acid
The amino acid composition of isobutyl alcohol extracts was flow rate of 0.5 mL/min and monitored at 220 nm and 30 °C.

determined according to the method reported by Fekkes et al.14 The standards used from Sigma were cytochrome C (12.5 kDa),

The amino acids in the sample were analyzed using an Agilent aprotinin (6.5 kDa), bacitracin (1450 Da), tetrapeptide GGYR

liquid chromatograph 1,100 with a UV detector operated at 338 (451 Da), and tripeptide GGG (189 Da).

nm. The ODS Hypersil (250×4.6 mm) column was applied at

40 °C with the mobile phase delivered at a flow rate of 1

Electronic Tongue Measurements
mL/min. For the determination of free amino acids, isobutyl Electronic tongue measurements were carried out with the taste

alcohol extracts (1.0 g) were filled up to 50 mL in sensing system TS-5000Z (Insent Inc., Japan). This electronic

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tongue was equipped with five lipid membrane sensors mean before applying PLSR analyses. By applying PLSR

indicating different taste qualities and corresponding reference analysis to standardized data, importance of peaks for each

electrodes. These sensors represent the gustatory stimuli umami attribute could be compared quantitatively based on regression

(SB2AAE), bitterness (SB2C00), saltiness (SB2CT0), sourness coefficients and loading weights for each predictor or X

(SB2CA0), and the nociceptive sensation astringency variable used in PLSR models.
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(SB2AE1). The sensor outputs were obtained as values

consequently. Each sample was measured four times with the Results and discussion

RSC Advances Accepted Manuscript

sensor of bitterness. One measurement cycle measured a Sensory Characterization of Wheat Gluten
reference solution (Vr) followed by the sample solution (Vs), a
Hydrolysates

short (2×3 s) cleaning procedure, the aftertaste (Vr') of

reference solution, and a cleaning procedure for 330 s. The

aftertaste was measured by determining the change of

membrane potential caused by the substance adsorption to the

lipid membrane after the short cleaning procedure. The

calculation formula of sensor output for taste and aftertaste

was:

Sensor output for taste = Vr − Vs (1)

Sensor output for aftertaste = Vr' − Vs ( 2)

Statistical Analysis Fig. 1 Bitterness intensity scores and taste dilution factor of wheat gluten

All of the tests of WGHs and isobutyl alcohol extracts were hydrolysates.

performed in triplicate and values were expressed as mean±

Sensory characterization of wheat gluten hydrolysates was
standard deviation (SD). The analysis of variance (ANOVA) of
evaluated from the bitterness intensity score and taste dilution
data was carried out using SPSS software (version 16.0, SPSS
factor respectively. As shown in Fig. 1, bitterness intensity of
Inc., Chicago, IL, USA). The differences between means were
WGHs increased continuously within 120 min of the enzymatic
evaluated by Duncan's multiple range test with a confidence
hydrolysis reaction. Bitterness intensity of WGHs reached
interval of 95%.
the highest level at 120 min, bitterness intensity score and taste

Partial least squares regression (PLSR) analysis was used dilution factor was 3.72 and 32 respectively. Bitterness

to explore the relationship between isobutyl alcohol extraction, intensity of WGHs decreased continuously during 120-300 min,

sensory data, the amino acid composition and molecular weight and bitterness intensity score and taste dilution factor was 1.33

distribution through UNSCRAMBLER ver. 9.7 (CAMO ASA, and 16 at 300 min respectively. Bitterness intensity score and

Oslo, Norway). All variables were centered and standardized taste dilution factor no longer reduced and began to stabilize

(1/Sdev) so that each variable has a unit variance and zero after 300 min. Bitterness intensity of WGHs increased at first

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and then decreased within 300 min, which indicated that the extract free amino acid with isobutyl alcohol because

release characteristics of bitter peptides obviously changed and of its low molecular weight. A part of bitter-tasting free amino

the resulting WGHs should be extracted with isobutyl alcohol acids remained in aqueous phase, bitter taste could not be

within the above-mentioned period of time. Bitterness intensity completely removed. Bitter-tasting free amino acid had the

scores and taste dilution factor were so low that the release higher bitter taste threshold17, 18
than bitter peptide under
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characteristics of bitter peptides couldn't be obtained at 5 min. normal circumstances and would not generate strong bitter

Along with the prolonging of reaction time, taste dilution taste. When bitterness intensity score of aqueous extracts was

RSC Advances Accepted Manuscript

factors increased at 10 min, 15 min, 30 min and 120 min. reduced to about 0.2, all the bitter peptides could be considered

Bitterness intensity scores decreased significantly at 300 min. to have been extracted with isobutyl alcohol. It was worth

Therefore, in order to study the release characteristics of bitter noting that the excessive increase in the concentration of

peptides, it was recommended to extract bitter peptides from WGHs would cause the bitterless substances of aqueous phase

WGHs at the above time point. to be extracted to isobutanol phase, which would confuse

structural characteristics of bitter peptides.

Extraction of Bitter Peptides with Isobutyl

In order to extract bitter peptides efficiently from WGHs,
Alcohol
When isobutyl alcohol was used to extract strong hydrophobic the concentration of WGHs at 10 min, 15 min, 30 min, 120 min

bitterness substances from WGHs, these differences in the and 300 min should be 10%, 15%, 15%, 20% and 15%

concentration of WGHs could affect nitrogen recovery of respectively. When the above conditions were met, bitterness

isobutyl alcohol phase and aqueous phase. As shown in Fig. 2, intensity score of isobutyl alcohol extracts (abbreviated

nitrogen recovery of isobutyl alcohol phase increased and respectively as Pro-10M, Pro-15M, Pro-30M, Pro-120M and

nitrogen recovery of aqueous phase decreased as the Pro-300M) was 2.74, 3.21, 3.60, 4.18 and 3.39 respectively.

concentration of WGHs increased, which was consistent with The results revealed that bitterness intensity of bitter peptides

previous studies.16 When the volume of the aqueous solution increased first and then decreased during the releasing process

was constant, the dissolving capacity was limited, so the of bitter peptides.

increase in the concentration of WGHs was beneficial to

transfer hydrophobic bitterness substances from aqueous phase

to isobutyl alcohol phase. In the process of transfer, bitterness

A a
intensity of isobutyl alcohol extracts increased gradually and

bitterness intensity of aqueous extracts decreased gradually.

When the concentration of WGHs reached a certain

extent, the further increase in the concentration could not

reduce bitterness intensity of aqueous extracts. The

most reasonable interpretation was that it was difficult to

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Fig. 2 Effect of the concentration of wheat gluten hydrolysates on nitrogen

recovery (A 10 Min; B 15 Min; C 30 Min; D 120 Min; E 300 Min) and bitterness

intensity (a 10 Min; b 15 Min; c 30 Min; d 120 Min; e 300 Min).

B b

Amino Acid Sequence of Bitter Peptides

The release characteristics of bitter peptides changed
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in terms of amino acid sequence, the proportion of bittter amino

acids and the relative molecular weight distribution. In the

RSC Advances Accepted Manuscript

following, the changes were discussed in detail. For proteax of

WG, peptide bonds were preferentially cleaved at the carboxyl-

C c terminal of glutamine (Gln), serine (Ser), threonine (Thr) and

methionine (Met) followed by cleavage on the carboxyl-

terminal of arginine (Arg) and finally on the carboxyl-terminal

of lysine (Lys), alanine (Ala), phenylalanine (Phe) and leucine

(Leu). According to the primary structure of wheat glutenin and

wheat gliadin provided by the National Center for

Biotechnology information, amino acid sequence of bitter

peptide (the proportion of bitter amino acid19 was more than

D d
50%) was identified by the cleavage of the primary structure

and was listed in Table 1.

A part of hydrolysis sites could not be cleaved in practice

under the influence of complex molecular configurations.

Amino acid sequence of bitter peptide in Table 1 might be real,

or it might be an important part of the real amino acid sequence

of bitter peptide from WGHs. In either case, amino acid

E e
sequence of bitter peptide was an important expression means

of bitter taste characteristics, which would affect the change of

bitterness intensity. In the initial reaction period, bitter peptides

with a high proportion of bitter amino acid were released from

WG, and a part of them had the particular amino acid sequence

(the adjacent basic amino acid and hydrophobic amino acid,20

basic amino acids located at the N-terminus ) which elicited

strong bitter taste. Then the particular amino acid sequences

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(HIPGLERPS, RYEAIRAIVYS, IALRT, AVRVPVPQ, VPF, RL, VL, QL, FL, VPL, PF, PPPF, DVVL, VQPL) with the

RVPFGVGT, AKRLVLFGIVVIALVALT, LIPCRDVVLQ, Phe and Leu located at the C-terminus had a small bitter taste

LPRMCNVYIPPYCS) were further hydrolyzed by cleavage of threshold. Therefore, the effect of amino acid sequence on bitterness

Arg. Proteax finally cleaved peptide bonds on the carboxyl- intensity was to decrease first and then increase.

terminal of Lys, Ala, Phe and Leu. The newly generated

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peptides (HIPGL, PL, PPL, PPF, PVL, PIL, IPF, IL, VF, IIL,

RSC Advances Accepted Manuscript

Table 1 Amino acid sequence of bitter peptide from wheat gluten hydrolysates

Protein composition Amino acid sequence of bitter peptide

Peptide bond hydrolysis (the carboxyl-
Peptide bond hydrolysis (the carboxyl-
terminal of glutamine, serine, threonine
terminal of arginine)
and methionine)
Peptide bond hydrolysis (the carboxyl-
terminal of lysine, alanine, phenylalanine
and leucine)

HIPGLERPS, PLPPQ, HHHQ, PIQ, FPQ,
PPLS, PPFS, PVLPQ, LPPFS, PPFS,
Low molecular weight PILPQ, PVLLQ, IPFVHPS, ILQ,
glutenin subunit LNPCKVFLQ, PVAM, LPQ, IPQ,
RYEAIRAIVYS, IILQ, FLQ, PHQ,
LELM, IALRT, LPT, RVPFGVGT
HIPGLER, PLPPQ, HHHQ, PIQ, FPQ,
PPLS, PPFS, PVLPQ, LPPFS, PPFS,
HIPGL, PL, PPQ, HHHQ, PIQ, PPL,
PILPQ, PVLLQ, IPFVHPS, ILQ,
PPF, PVL, PIL, IPF, VHPS, IL, VF, PVA,
LNPCKVFLQ, PVAM, LPQ, IPQ,
IR, IVYS, IIL, PHQ, VPF
YEAIR, AIVYS, IILQ, FLQ, PHQ,
LELM, IALR, LPT, VPFGVGT

AKRLVLFGIVVIALVALT, LPWS, LVLFGIVVIALVALT, LPWS, PLQ, AVPPK, RL, VL, GIVVIA, PWS, PL,
High molecular weight PLQ, GYYPS, YYPGQ, GYYPT, GYYPS, YYPGQ, GYYPT, GHYPAS, GYYPS, YYPGQ, GYYPT, GHYPA,
glutenin subunit GHYPAS, HPQ, PHYPAS, PGHYPAS, HPQ, PHYPAS, PGHYPAS, GYYIT, HPQ, PHYPA, PGHYPA, GYYIT,
GYYIT, PCHVS, PVAQLPT PCHVS, PVAQLPT PCHVS, PVA, QL

FLILALLAIVAT, AVRVPVPQ, FLILALLAIVAT, AVR, VPVPQ,

VPLVQ, FPPQ, PYPQ, PFPS, PYLQ,
PFPQ, PFPPQ, LPYPQ, PPPFS, PIS,
Wheat gliadin
ILPQ, LIPCRDVVLQ, VQPLQ,
AIHNVVHAIILHQ, YPS, LPQ,
LPRMCNVYIPPYCS
VPLVQ, FPPQ, PYPQ, PFPS, PYLQ,
PFPQ, PFPPQ, LPYPQ, PPPFS, PIS,
ILPQ, LIPCR, DVVLQ, VQPLQ, YPS,
LPQ, AIHNVVHAIILHQ, LPR,
MCNVYIPPYCS
FL,IL,VR,VPVPQ, VPL, PF, PPQ,
PYPQ, PPPF, PIS, IL, IPCR, DVVL,
VQPL, YPS, IHNVVHA, IIL, PR,
MCNVYIPPYCS

were relatively high, which was consistent with amino acid

Amino Acid Composition of Peptides composition of WG. The results indicated that hydrophilic
WGHs at different times (0−5h) of enzymatic hydrolysis were amino acids of hydrophobic peptides were also extracted with
extracted with isobutyl alcohol. These extracts showed the isobutyl alcohol, and the content of hydrophobic amino acids

different amino acid composition of peptides in Table 2. The could only vary in a certain range.
content of glutamine (gln), proline (Pro), glycine (Gly) and Leu

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Hydrophobic peptides gradually had a higher proportion of

hydrophobic amino acids within 0-120 min of enzymatic

hydrolysis. Two reasons could explain this trend. Firstly, the

degradation of high molecular weight peptides generated low

molecular weight peptides, some of which had a higher

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proportion of hydrophobic amino acids. Secondly, WG had a

large number of hydrophobic amino acids and

RSC Advances Accepted Manuscript

exhibited poor solubility. For proteax of WG, peptide bonds

were preferentially cleaved at the carboxyl-terminal of Gln, Ser,

Thr and Met. The hydrophilic area of WG was preferred to

participate in hydrolysis reaction. The macromolecular proteins

which had strong hydrophobic interaction were removed from

WGHs after centrifugation. These macromolecular proteins

gradually participated in the enzyme reaction and constantly

released bitter peptides which contained higher proportion of

hydrophobic amino acids with increscent DH. Therefore, it was

reasonable for the increase in the content of hydrophobic amino

acids (Phe、Ile、Leu、Pro) and the decrease in the content of

hydrophilic amino acids (Asp、Glu、Ser、Thr、Cys).

The endo-activity of Proteax was very low within 120-

300 min of enzymatic hydrolysis. Hydrophobic peptides which

had the high proportion of hydrophobic amino acids

no longer generated. Proteax had high exo-activity to cut free

amino acid from the C-terminal of hydrophobic peptides, so the

content of peptide-bound amino acids decreased from 317.49

mmol/g to 286.15 mmol/g. Arg and Lys had the stimulating

unit of bitter taste determinant sites,21, 22 Tyr, Val, Met, Phe, Ile

and Leu had the binding unit of bitter taste determinant sites.

The decrease in the content of the above bitter amino acids

would produce a debittering effect. Therefore, the effect of

amino acid composition on bitterness intensity was to increase

first and then decrease.

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Table 2 Amino acid composition of peptides (mmol/100 g isobutyl alcohol extracts)

Concentration (mmol/100 g) (mean±SD)

Peptide-bound amino acida
Pro-10M Pro-15M Pro-30M Pro-2H Pro-5H
b a b b c
Asp 20.66±0.82 19.28±0.43 18.31±0.21 17.08±0.58 15.30±0.65d

Gluc 209.64±6.97a 198.38±6.31ab 187.41±7.11bc 181.25±7.14c 178.51±6.71c

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Ser 27.05±0.65a 25.72±0.72ab 24.36±1.32bc 23.53±0.70cd 22.67±0.31d

abc a c bc
His 3.80±0.16 4.02±0.08 3.32±0.50 3.46±0.20 3.93±0.04ab

RSC Advances Accepted Manuscript

Gly 41.58±1.89a 41.03±2.07a 40.94±1.34a 39.83±1.53a 40.72±2.01a

Thr 5.63±0.28a 5.34±0.27a 5.26±0.30a 3.72±0.18b 2.78±0.17c

Arg 15.34±0.19ab 15.95±0.89a 14.35±1.05b 10.92±0.42c 8.54±0.79d

ab ab a bc
Ala 23.02±0.49 23.29±0.39 24.30±1.17 22.21±1.37 20.71±0.78c

Tyr 11.81±0.29b 12.34±0.45ab 12.97±0.72a 12.01±0.34b 10.84±0.31c

Cys 1.34±0.12a 1.22±0.11ab 1.11±0.16b 0.70±0.10c 0.65±0.09c

Val 24.56±1.02a 24.81±0.76a 25.51±1.49a 23.73±0.95a 19.60±1.58b

a a a a
Met 7.25±0.15 7.48±0.35 7.43±0.70 6.86±0.69 4.34±0.57b

Phe 24.2±1.30cd 25.86±1.62bc 27.80±1.43ab 28.80±0.96a 22.42±1.68d

Ile 24.86±1.17c 26.14±0.86bc 27.61±0.80ab 29.54±0.71a 26.24±1.56bc

Leu 46.02±2.58b 46.55±2.93b 49.72±1.88ab 51.63±3.36a 34.60±1.91c

c b b a
Lys 4.69±0.27 5.60±0.20 6.13±0.39 7.10±0.60 5.59±0.16b

Pro 95.95±4.90c 104.14±7.51bc 109.86±5.63ab 114.90±5.27ab 117.52±6.74a

Trp n.dd n.dd n.dd n.dd n.dd

Total 587.41±5.25a 587.16±5.70a 586.38±8.44a 577.26±5.82a 534.98±6.78b

Total hydrophobic amino acide 287.46±10.03bc 299.30±7.08b 313.16±3.83a 317.49±6.41a 286.15±4.21c

Total bitter amino acidf 258.49±10.11c 272.88±7.48b 284.69±3.36ab 288.94±5.50a 253.63±5.32c

The values in the same row followed by different letters are significantly different (p< 0.05).

a
Peptide-bound amino acids were calculated by subtracting free amino acids from total amino acids.

b
Aspartic acid + asparagine.

c
Glutamic acid + glutamine.

d
Not determined.

e
Total hydrophobic amino acids contained Gly, Ala, Val, Leu, Pro, Met, Phe, Trp and Ile.

f Total bitter baste amino acids contained Pro, Tyr, Lys, His, Arg, Val, Met, Phe, Ile, Leu, Trp.

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Molecule Weight Distribution of Isobutyl Alcohol should be discussed separately. The content of the long-chain
Extracts peptide of isobutyl alcohol extracts gradually decreased within
The fraction below 180 Da was mainly free amino acids, which
0-120 min of enzymatic hydrolysis, bitterness intensity of
wasn't the main bitter substances of isobutyl alcohol extracts.
isobutyl alcohol extracts must have been decreased. However
Table 3 showed that Pro-10M extract had the highest content of
the hydrolysis of WG generated hydrophobic peptides, which
the peptide fraction ranging 3000-6000 Da, which was only
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gradually had a higher proportion of hydrophobic amino acids.

4.63%. Moreover, peptide chain of the peptide fraction was so
The main bitter substances had the smaller bitter taste threshold
long that it was difficult to contain a high proportion of bitter

RSC Advances Accepted Manuscript

as a whole, so bitterness intensity of isobutyl alcohol extracts
amino acids, so the peptide fraction wasn't also the main bitter
gradually increased. The endo-activity of Proteax was very low
substances. The peptide fraction above 6000 Da23 didn't have a
within 120-300 min of enzymatic hydrolysis. The proportion of
bitter taste. The main bitter substances of isobutyl alcohol
hydrophobic amino acids of hydrophobic peptides no longer
extracts focused on the peptide fractions ranging 180-500 Da,
increased significantly. The long-chain peptide showed the
500-1000 Da and 1000-3000 Da. Bitter peptide had the smaller
strongest bitter taste, the decrease of its content caused
bitter taste threshold as bitter taste determinant sites of peptide
a reduction of bitterness intensity of Pro-2 H extract. Therefore,
distributed densely within a certain spatial range of taste bud. In
the effect of molecular weight distribution on bitterness
other words, when bitter peptide had a higher proportion of
intensity was to increase first and then decrease.
bitter amino acids or a larger amount of bitter amino acids, it

would have the smaller bitter taste threshold. At the beginning

of the hydrolysis reaction, the proportion of bitter amino acids Table 3 Molecule weight distribution of isobutyl alcohol extracts

in the long-chain peptide (500-1000 Da and 1000-3000 Da) The values in the same row followed by different letters are significantly different (p < 0.05).

remained at low level. Based on randomness appeared in Molecul

ar
Pro-10M Pro-15M Pro-30M Pro-2H Pro-5H
hydrolysis reaction, a part of the short-chain peptide (180-500 weight
(kDa)
Da) that contained more bitter taste of amino acids had the ≥10 4.05±0.22a 2.66±0.19b 1.84±0.06c 1.11±0.05d 0.75±0.07e
6-10 9.78±0.52a 8.79±0.30b 6.96±0.17c 3.86±0.19d 2.14±0.16e
stronger bitter taste. In the later stages of the hydrolysis
a b c d
3-6 4.63±0.18 3.73±0.12 3.03±0.19 1.94±0.03 1.58±0.14e
reaction, the long-chain peptide that had a high proportion of 1-3 17.91±1.43a 16.23±1.40ab 14.61±0.52b 11.44±0.70c 7.32±0.49d
a a a a
0.5-1 25.21±1.52 25.15±1.59 24.76±1.56 23.28±1.40 14.92±0.96b
bitter amino acids showed the strongest bitter taste.
d d c b
0.18-0.5 32.03±2.11 35.36±2.24 39.37±1.82 44.52±0.9 55.62±2.07a
≤0.18 6.39±0.38d 8.08±0.17c 9.43±0.37c 13.85±0.93b 17.67±1.40a
The content of peptide fractions ranging 500-1000 Da

and 1000-3000 Da decreased respectively from 25.21% and

17.91% in Pro-10M extract to 14.92% and 7.32% in Pro-5H
extract. The content of peptide fractions ranging 180-500 Da
increased from 32.03% in Pro-10M extract to 55.62% in Pro-
5H extract. The decrease of relative average molecular weight
caused different release characteristics of bitter peptides, which
Representation of Sensor Response of the
Electronic Tongue
To compare the intensity of the sense, the initial sensor output
of Pro-10M extract was defined as zero, being the frame of
reference. The initial sensor outputs of other treatments were
converted into new voltage values by TS-5000Z software

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system. A greater number represented the higher taste intensity.

The intensities of taste sense was a measure of the size of taste

sense produced by isobutyl alcohol extracts in a certain period

of time. The sensor outputs of bitterness and bitter aftertaste

(aftertaste-B) represented the instantaneous maximum intensity

Published on 10 October 2016. Downloaded by Northern Illinois University on 24/10/2016 14:59:31.

and sustained intensity of bitter taste, respectively. As shown in

Fig. 3, The sensor outputs of bitterness and aftertaste-B for

RSC Advances Accepted Manuscript

isobutyl alcohol extracts both increased first and then

decreased, Pro-2H extract had the highest instantaneous

Fig. 3 The sensor outputs of isobutyl alcohol extracts obtained by the electronic
maximum intensities (3.727) and sustained intensities (1.583).
tongue.

These results indicated that the proportion of hydrophobic

amino acids gradually increased, so bitter peptides had a longer

duration of action with taste bud, and bitter taste determinant

sites of bitter peptides became denser. This phenomenon was
stopped after 2 h of hydrolysis reaction. The content of long-
chain bitter peptides began to gradually reduce. Amino acid
composition and molecular weight distribution had a more
significant impact on bitterness intensity than amino acid
sequence. In order to find a better way for the reduction of
bitter taste, the internal connection between amino
composition, molecular weight distribution and bitterness
intensity should be further discussed.
Relationship between Isobutyl Alcohol Extracts,
Sensory Attributes ， Amino Acid Composition
and Molecular Weight Distribution
ANOVA-PLSR was used to process the mean data accumulated
from HPLC and the sensor outputs obtained by the electronic
tongue. Seventeen kinds of amino acids and seven kinds of the
peptide fraction were used as variables in the subsequent PLSR
analysis. The X-matrix was designed as amino acid
acid
composition and molecular weight distribution; the Y-matrix
was designed as isobutyl alcohol extracts and sensory variables.
The calibrated explained variance for this model was PC1 =

81% and PC2 = 18%. PC1 versus 2 (Fig. 2) and PC2 versus 3

were explored. PC2 versus 3 results was not presented here, as

the additional information was not gained through their

examination. Further, PCs didn't provide any predictive

improvement in the Y-matrix obtained. Fig. 4 was presented as

correlation loadings plot. The big circles indicated 50% and

100% explained variances, respectively. Seven Y variables (Y1

(the sensor outputs of bitterness), Y2 (the sensor outputs of

aftertaste-B), Pro-10M, Pro-15M, Pro-30M, Pro-2H, Pro-5H)

and twenty-four X variables (The peptide fraction above 10000

Da, the peptide fraction ranging 6000-10000 Da, the peptide

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fraction ranging 3000-60000 Da, the peptide fraction ranging

1000-3000 Da, the peptide fraction ranging 500-1000 Da, the

peptide fraction ranging 180-500 Da, the peptide fraction below

180 Da, aspartic acid, glutamic acid, serine, histidine, glycine,

threonine, arginine, alanine, tyrosine, cysteine, valine,

Published on 10 October 2016. Downloaded by Northern Illinois University on 24/10/2016 14:59:31.

methionine, phenylalanine, isoleucine, leucine, lysine, praline

were arranged in the order listed above and

RSC Advances Accepted Manuscript

numbered separately in Arabic numerals. ) were placed

between the inner and outer ellipses respectively, indicating

they were well explained by the PLSR model.

Fig. 4 An overview of the variation found in the mean data from the partial least

Pro-10M and Pro-5H were located in the left side of Y squares regression (PLSR) correlation loadings plot for isobutyl alcohol extracts.

The model was derived from amino acid composition and molecular weight
axis, Pro-30M and Pro-2H were located in the right side of Y
distribution as the X-matrix and extracts and sensory attributes as Y-matrix.
axis, so X axis represented bitter taste which increased in
Elipses represent r2 = 0.5 and 1.0, respectively.
intensity from left to right. Pro-10M and Pro-15M were located

in the lower side of X axis, Pro-5H and Pro-2H were located in

the upper side of X axis, so Y axis represented the average

relative molecular weight which decreased in size from bottom

Conclusions
The release of bitter peptides was a dynamic evolution process,
to top. According to the distribution of the independent
which was an existing phenomenon and regulated by the degree
variables, two groups of the independent variables were found
of hydrolysis, the enzyme active sites and amino acid sequence
to have a strong impact on bitter taste of isobutyl alcohol
of protein. The characteristic changes of bitter peptides gave
extracts. The most influential group was phenylalanine, leucine
rise to different bitterness intensity in the process. To
and tyrosine. The second-most influential group was valine,
investigate the characteristic changes and explain scientific
methionine, isoleucine, alanine, lysine and the peptide fraction
principles of bitter taste changes, then the changing rule of
ranging 500-1000 Da. The proportion of bitter taste amino acid
enzyme-induced release of bitter peptides was proposed. Amino
had the most important influence on bitterness intensity of
acid composition and molecular weight distribution had a more
bitter peptides. The peptide fraction ranging 500-1000 Da
significant impact on bitterness intensity than amino acid
showed the stronger bitter taste than the other peptide fractions.
sequence. The peptide fraction ranging 180-500 Da, 500-1000
The most likely explanation was that the peptide fraction
Da and 1000-3000 Da had gradually a higher proportion of
ranging 500-1000 Da had a larger amount of bitter amino acids
bitter taste amino acid, which showed stronger bitter taste. This
on the premise of containing a high proportion of bitter amino
phenomenon was stopped after 2 h of hydrolysis reaction. The
acids.
content of long-chain bitter peptides began to gradually reduce,

especially for the peptide fraction ranging 500-1000 Da having

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the strongest bitter taste. Bitterness intensity of bitter peptides 5. L. Li, Z.-Y. Yang, X.-Q. Yang, G.-H. Zhang, S.-Z.

increased first and then decreased in the hydrolysis Tang and F. Chen, Journal of Industrial
reaction system of Proteax.
Microbiology & Biotechnology, 2008, 35, 41-47.

According to above finds, the optimization methods 6. F. Liu and M. Yasuda, Journal of Industrial

would be further explored. For instance, it was very meaningful Microbiology & Biotechnology, 2005, 32, 487-489.
Published on 10 October 2016. Downloaded by Northern Illinois University on 24/10/2016 14:59:31.

to design complex enzymatic hydrolysis. Alcalase or 7. B.-Y. Liu, K.-X. Zhu, W. Peng, X.-N. Guo and H.-
Flavourzyme might be useful to further reduce the content of M. Zhou, RSC Advances, 2016, 6, 27659-27668.

RSC Advances Accepted Manuscript

long-chain bitter peptides. In the future, more attention should
8. N. Ishibashi, T. Kubo, M. Chino, H. Fukui, I.
be focused on the scientific production of small peptide powder
Shinoda, E. Kikuchi and H. Okai, Agricultural and
when preparing low-bitterness peptide powder by sequential
Biological Chemistry, 1988, 52, 95-98.
hydrolysis with endo- and exo-peptidase.
9. N. Ishibashi, K. Sadamori, O. Yamamoto, H.

Kanehisa, K. Kouge, E. Kikuchi, H. Okai and S.

Acknowledgements
The study was supported by National High Technology Fukui, Agricultural and Biological Chemistry, 1987,

Research and Development Program of China (863 Program, 51, 3309-3313.

NO. 2013AA102201), the Key R & D Programs of Jiangsu
10. X. Liu, D. Jiang and D. G. Peterson, J Agric Food
Province (BE2015327) and the Jiangsu province "Collaborative
Chem, 2014, 62, 5719-5725.
Innovation Center for Modern Grain Circulation and Safety"
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12. G. Lalasidis, Annales De La Nutrition Et De

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Page 15 of 15
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Graphical abstract
DOI: 10.1039/C6RA22155F
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