Journal of Functional Foods 16 (2015) 94–103

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Amino acid composition and in vitro antioxidant

and cytoprotective activity of abalone viscera
hydrolysate

Jae-Young Je a,1, Soo Yeon Park b,1, Joung-Youl Hwang c,

Chang-Bum Ahn b,d,*
a
Department of Marine-Bio Convergence Science, Pukyong National University, Busan 608-739, Republic of
Korea
b
School of Food Technology and Nutrition, Chonnam National University, Yeosu 550-749, Republic of Korea
c
Korea Abalone Laboratory, Naju 520-330, Republic of Korea
d
Division of Food and Nutrition, Chonnam National University, Gwangju 500-759, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Article history: Abalone viscera is one of the byproducts from abalone processing and is discarded as in-
Received 14 January 2015 dustrial waste. In this study, abalone viscera hydrolysates (AVH) were prepared using Alcalase,
Received in revised form 16 April Flavourzyme, Neutrase, and Protamex, and their oxygen radical absorbance capacity (ORAC)
2015 was evaluated. The AVH by Alcalase showed promising ORAC value, and further optimal
Accepted 17 April 2015 enzyme/substrate hydrolysis condition for production of the AVH was determined. The AVH
Available online also exerted strong hydrogen peroxide scavenging activity, Fe2+ chelating activity, and re-
ducing power. Furthermore, the AVH inhibited lipid peroxidation in a model system and
Keywords: protected hydroxyl radical-induced DNA damage. Amino acid profiles revealed that the AVH
Abalone viscera contained 57.1% antioxidant amino acids and 44.0% hydrophobic amino acids, which con-
Enzymatic hydrolysis tributed to its higher antioxidant activity. In addition, the AVH showed cytoprotective effects
Antioxidant activity against hydrogen peroxide-induced cytotoxicity of cultured hepatocytes, and significantly
Cytoprotection (p < 0.05) inhibited intracellular reactive oxygen species (ROS) and membrane lipid peroxidation.
Taken all together, these results suggest that the AVH might be useful as an ingredient for
functional foods.
© 2015 Elsevier Ltd. All rights reserved.

medicine and for tonics, and it also exhibits many physiologi-

1. Introduction
Abalone (Haliotis discus hannai) is one of the most valuable
marine gastropods and is widely cultured in east Asia, Aus-
tralia, America, and many other regions, and more than 90%
of the world abalone production is based on farming (Cook &
Roy Gordon, 2010). Abalone has been used in traditional
cal benefits such as fatigue recovery, and detoxification (Kim
& Pallela, 2012; Lee et al., 2008). Due to its nutritive and phar-
maceutical values, abalone mariculture has been increasing.
The total production from South Korea was estimated as 7580
metric tonnes in 2009, and various types of manufacturing prod-
ucts derived from abalone have also been significantly
increased.

* Corresponding author. School of Food Technology and Nutrition, Chonnam National University, Yeosu 550-749, Republic of Korea. Tel.:
+82 61 659 7411; fax: +82 61 659 7419.
E-mail address: a321@jnu.ac.kr (C.-B. Ahn).
1
These authors equally contributed to this work.
http://dx.doi.org/10.1016/j.jff.2015.04.023
1756-4646/© 2015 Elsevier Ltd. All rights reserved.
 Journal of Functional Foods 16 (2015) 94–103
In general, abalone viscera is considered as an inedible part
by producers as well as customers; thus, this visceral matter
is normally discarded as industrial waste and accounts for
15–25% of the total body weight of abalone. However, abalone
viscera contains rich organic contents, and in recent years, many
researchers have discussed specific health benefits such as an-
tioxidant, and immunomodulating activities of crude
polysaccharides from abalone viscera, thereby indicating
abalone viscera may be useful as an ingredient for functional
foods (Zhu et al., 2008, 2010, 2011). However, little informa-
tion for utilization of protein in abalone viscera exists despite
the high protein content (72%) in abalone viscera (dry basis)
(Zhu et al., 2011).
Bioactive peptides, specific protein fragments with the ability
to impact on body functions, have a high nutritional value and
health-related benefits such as antioxidant functions and blood
pressure reductions. The production of protein hydrolysates
by enzymatic hydrolysis with endopeptidases and exopepti-
dases is considered the most effective way to obtain protein
hydrolysates containing bioactive peptides with defined char-
acteristics (Clemente, 2000). Various food protein sources have
been exploited to produce protein hydrolysates with specific
bioactivities such as antioxidant and antihypertensive effects
(Bougatef et al., 2009; Chalamaiah, Dinesh Kumar, Hemalatha,
& Jyothirmayi, 2012); among them, seafood proteins have re-
ceived considerable attention for the production of bioactive
protein hydrolysates since they are produced in high amounts
as byproducts from food processing. An array of protein
hydrolysates exhibiting antioxidant, antihypertensive,
anticoagulant, and calcium binding activities has been ob-
tained from marine-derived proteins (Chalamaiah et al., 2012;
Jung & Kim, 2007, 2009; Lee, Qian, & Kim, 2010). Moreover, some
of these protein hydrolysates may exhibit multifunctional prop-
erties, thereby indicating that they may be useful as an ingredient
for functional foods.
Thus, in the present study, we investigated the produc-
tion of protein hydrolysates with antioxidant, hydrogen peroxide
scavenging and metal chelating activities. The cytoprotective
activity of the obtained hydrolysates against hydrogen per-
oxide-induced hepatic damage in hepatocytes was also
evaluated.
2. Materials and methods
2.1. Materials
All chemicals including fluorescein, Trolox (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid), 2,2-azo-bis(2-amidino-
propane) dihydrochloride (AAPH), Folin–Ciocalteu’s phenol
reagent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), hydrogen peroxide, thiobarbituric acid (TBA),
trichloroacetic acid (TCA), peroxidase, potassium ferricya-
nide, and ferrozine were purchased from Sigma Chemical Co.
(St. Louis, MO, USA). Diphenyl-1-pyrenylphosphine (DPPP) and
2′,7′-dichlorofluorescin diacetate (DCFH-DA) were obtained from
Molecular Probes Inc. (Eugene, OR, USA). Alcalase, Flavourzyme,
Neutrase, and Protamex were purchased from Novo Co.
(Novozyme Nordisk, Bagsvaerd, Denmark).
95
2.2. Preparation of abalone viscera hydrolysates (AVH)
Before digestion, the abalone viscera (AV) was washed using
tap water, and then freeze-dried. The protein content of AV was
29.90% by Kjeldahl method. The AV was digested with Alcalase,
Flavourzyme, Neutrase, and Protamex under optimal reac-
tion conditions (Table 1) to prepare AVH. Briefly, the enzyme
was mixed with a 12% substrate solution at a ratio of 1:25 (w/w).
Hydrolysis was performed with simple agitation for 8 h, and
then the mixture was boiled for 10 min to inactivate the pro-
tease. After selecting the optimal enzyme, new digestions using
E/S ratios of 1:50 and 1:100 were performed to determine the
optimal E/S ratio for this enzyme. Unhydrolyzed proteins were
removed using filter cloth, and the supernatant was lyophi-
lized and stored at −20 °C until use. Protein content of the
generated AVH was 19.30% by Kjeldahl method.
The degree of hydrolysis (%DH) was determined using a tri-
chloroacetic acid (TCA) method (Hoyle & Merrltt, 1994). DH is
calculated from the following equation:
%DH = (10% TCA soluble N in sample
Total N in sample) × 100
2.3. Determination of amino acid and free amino acid
compositions
Amino acid composition of AV and AVH was analyzed with an
amino acid analyzer (S433-H, Sykam GmbH, Germany). A 50 mg
of AV or AVH was hydrolyzed using 2 mL of 6.0 M HCl in a
sealed-vacuum ampoule at 110 °C for 24 h. Hydrochloric acid
was removed by rotary evaporator and brought to a final volume
of 10 mL with 0.2 M sodium citrate buffer (pH 2.2). Amino acids
were determined using a cation separation column (LCA K06/Na,
4.6 × 150 mm) with flow rates of 0.45 mL/min (buffer) and
0.25 mL/min (reagent) at wavelengths of 440 and 570 nm.
For determination of free amino acids, 2.0 g of AV or AVH
was homogenized at 12,000 rpm twice for 2 min with 75%
ethanol, followed by centrifuging at 2000× g for 30 min. The su-
pernatant was then removed by rotary evaporator, and to it was
then added 8 mL of distilled water with 5′-sulfosalicylic acid
(0.2 g) at 4 °C for 1 h. The mixture was centrifuged at 2000× g
for 30 min, and the 2-mL supernatant was transferred into a
tube containing 1 mL of 0.2 M lithium citrate buffer (pH 2.2)
added. Free amino acids were determined on the same amino
acid analyzer.
Table 1 – Enzymatic hydrolysis conditions, degree of
hydrolysis (DH) and yield of abalone viscera
hydrolysates (AVH).
Proteasea pH T (°C) DH (%) Yield (%)
Alcalase 7.0 50 60.04 ± 0.04 33.22 ± 0.20
Flavourzyme 7.0 50 58.64 ± 0.49 30.13 ± 0.32
Neutrase 7.0 50 50.89 ± 1.41 28.02 ± 0.26
Protamex 7.0 50 55.61 ± 0.60 34.64 ± 0.25
a
Enzymatic hydrolysis was conducted at E/S ratio of 1:25 for 8 h.
96 Journal of Functional Foods 16 (2015) 94–103
2.4. Measurement of antioxidant activity
2.4.1. ORAC assay
The ORAC values were determined according to the method
of Zulueta, Esteve, and Frigola (2009) with slight modifica-
tions. Briefly, 50 µL of AVH (1.0 mg/mL) or distilled water (control)
was mixed with 50 µL of fluorescein (78 nM) in a 96-well
microplate and incubated at 37 °C for 15 min, followed by the
addition of 25 µL of AAPH (221 mM). The fluorescence was re-
corded every 5 min for 60 min (λ excitation: 485 nm; λ emission;
535 nm) by microplate reader (SpectraMax M2/M2e; Molecu-
lar Devices, Sunnyvale, CA, USA). Trolox (1–20 µM) was used to
prepare a standard curve, and the ORAC values of AVH were
calculated by Trolox standard curve. The ORAC values were ex-
pressed as µM Trolox equivalent (TE)/mg AVH. The area under
curve (AUC) was calculated as:
AUC = ( 0.5 + f5 f0 + f10 f0 + … + fn +5 f0 ) ∗ 5
where f0 is the initial fluorescence and fn is the fluorescence
at time n.
2.4.2. Hydrogen peroxide scavenging activity
Hydrogen peroxide scavenging activity was determined ac-
cording to the method of Heo, Park, Lee, and Jeon (2005). A
100 µL of sodium phosphate buffer (0.1 M, pH 5.0) and the AVH
(0.3–2.4 mg/mL, final concentration) or buffer (control) were
mixed in a 96-well microplate, followed by the addition of 20 µL
of hydrogen peroxide (10 mM), and then it was incubated at
37 °C for 5 min. After the incubation, 30 µL of ABTS (1.25 mM)
and 30 µL of peroxidase (1 unit/mL) were added to the mixture,
and then the resulting mixture was incubated at 37 °C for
10 min. The absorbance was recorded at 405 nm by microplate
reader (SpectraMax M2/M2e). The hydrogen peroxide scaveng-
ing activity was calculated by the following equation.
Scavenging activity (% )
= [( A 405 of control − A 405 of sample) A 405 of control ] × 100
2.4.3. Ferrous ion chelating activity
The ferrous ion chelating activity was measured by Singh and
Rajini (2004), with modifications. The AVH (0.25–2.0 mg/mL, final
concentration) or distilled water (control) was mixed with
0.1 mM of FeCl2 for 30 s, followed by the addition of 0.25 mM
of ferrozine and then allowed to stand for 10 min. The absor-
bance was measured at 562 nm (SpectraMax M2/M2e). The
ferrous ion chelating activity was calculated by the following
equation.
Scavenging activity (% )
= [( A562 of control − A562 of sample) A562 of control ] × 100
2.4.4. Ferric reducing antioxidant power (FRAP)
FRAP of the AVH was determined according to the method of
Oyaizu (1986). The AVH (0.25–2.0 mg/mL, final concentration)
was mixed with 0.5 mL of sodium phosphate buffer (0.2 M, pH
6.6) and 0.5 mL of potassium ferricyanide (1%) followed by in-
cubation at 50 °C for 20 min, and then 0.5 mL of TCA (10%) was
added to the mixture and centrifuged at 1036× g for 10 min.
The supernatant (0.5 mL) was mixed with 0.5 mL of distilled
water and 0.1 mL of FeCl3 (0.1%), and the absorbance was mea-
sured at 700 nm (SpectraMax M2/M2e). The increase in
absorbance of the reaction mixture indicated the increase in
reducing power.
2.5. Inhibition of lipid peroxidation in mackerel lipid
model system
To evaluate lipid peroxidation inhibition ability, the total lipid
from mackerel was extracted by Bligh and Dyer’s (1959) method.
Then, 200 µL of 2.5% mackerel lipid (in ethanol), 200 µL of AVH,
and 50 mM sodium phosphate buffer (pH 7.0) were mixed to-
gether. The mixture was incubated in the dark at 40 °C for 4
days. After incubation, the mixture was tested for the forma-
tion of thiobarbituric acid reactive-substances (TBARS). TBARS
were determined by modifying the procedure of Stoilova,
Krastanov, Stoyanova, Denev, and Gargova (2007). The mixture
was mixed with TBA–TCA solution (20 mM TBA in 15% TCA)
and then heated in a boiling water bath for 15 min before
allowed to cool to room temperature. The mixture was then
mixed with chloroform and centrifuged at 1036× g for 15 min.
The chloroform layer was measured at an absorbance of 532 nm.
A malondialdehyde (MDA) standard curve was prepared, and
TBARS was expressed as µM of MDA. Butylated hydroxytolu-
ene (BHT, 0.2 mg/mL) was used as a positive control.
2.6. Protection against hydroxyl radical-induced DNA
damage
To study the protective effect of the AVH against hydroxyl
radical-induced DNA damage, the reaction was conducted in
an Eppendorf tube at a total volume of 12 µL containing 0.5 µg
of pBR322 plasmid DNA (Life Technologies, Seoul, Korea), 2 mM
FeSO4 and the AVH solution (in D.W.). The mixture was incu-
bated with 4 µL of H2O2 (10 mM) at 37 °C for 30 min (Yeung et al.,
2002). The mixture was subjected to 0.8% agarose gel electro-
phoresis. DNA bands (supercoiled and open circular) were
stained with ethidium bromide and quantified using Davinch-
Chemi™ imaging system (Core Bio, Seoul, Korea).
2.7. Cytoprotective activities of AVH against hydrogen
peroxide-induced hepatocyte damage
2.7.1. MTT assay
Hepatocytes (Chang liver cells, American Type Culture Collec-
tion, Rockville, MD, USA) were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 10% FBS, 100 U/mL
of penicillin, and 100 µg/mL of streptomycin at 37 °C in an at-
mosphere composed of 5% CO2 and 95% air.
Cell cytotoxicity of the AVH was evaluated using MTT assay.
The hepatocytes (1 × 104 per well) were incubated in a 96-
well plate for 24 h, followed by treatment with the AVH
(100–1000 µg/mL) for 24 h. The medium was removed, and a
100-µL MTT solution (1 mg/mL) was added, followed by incu-
bation for 4 h. The intracellular formazan product formed was
dissolved in DMSO, and the optical density at 540 nm was mea-
sured by means of a microplate reader.
To determine cytoprotective effect of the AVH against hy-
drogen peroxide-induced hepatocyte damage, the hepatocytes
 Journal of Functional Foods 16 (2015) 94–103 97

were pretreated with the AVH (50–250 µg/mL) and without 500
(control) for 2 h and then washed three times with phos- a A

(mM TE/mg sample)

phate buffered saline (PBS). The hepatocytes were then exposed 400 b
to 650 µM of H2O2 to give oxidative stress, followed by incu- c

ORAC value
d d
bation for 24 h. After 24-h incubation, MTT assay was performed 300
as described in the above method.
200
2.7.2. Determination of intracellular reactive oxygen species
(ROS) generation 100
Intracellular ROS in H2O2-stimulated hepatocytes was ana-
lyzed by staining using DCFH-DA. After treatment of the AVH 0
for 2 h, the hepatocytes were incubated with 20 µM of DCFH-

d
ou se

eu e

ot e
ex
ym

Pr ras
ze

la

am
ly
DA for 30 min. The hepatocytes were washed three times with

Fl lca

rz

t
ro
PBS, and 650 µM of H2O2 was added. Fluorescence was de-

A
yd

N
av
nh
tected on a fluorescence reader (SpectraMax M2/M2e) by

U
measuring at 485 nm and 535 nm for excitation and emis-
500
sion, respectively (Lee, Cho, & Je, 2013). The percentage of
a a a B

(mM TE/mg sample)

fluorescence intensity (ROS generation) was compared with that
400
of the control cells without the AVH, which were arbitrarily as-

ORAC value
signed a value of 100%.
300

2.7.3. Determination of cell membrane lipid peroxidation 200

Cell membrane lipid peroxidation was quantified by measur-
ing the amount of added DPPP oxidized to fluorescent DPPP
oxide. The hepatocytes were cultured to approximately 70–80%
confluence in a 10-cm culture dish and then washed three times
with PBS, followed by labeling with 13 µM DPPP in DMSO for
30 min. After washing three times with PBS, the hepatocytes
were seeded in a 96-well plate at 4 × 105 cells/mL using serum-
free DMEM followed by pretreatment with the AVH for 2 h. Then,
the mixture was incubated with 20 µL of AAPH (30 mM) to ini-
tiate cell membrane lipid peroxidation. After 6-h incubation,
the fluorescence due to the oxidation of DPPP was measured
at 361 nm and 380 nm for excitation and emission, respec-
tively (Lee et al., 2013). The percentage of fluorescence intensity
(membrane lipid peroxidation) was compared with that of the
control cells without the AVH, which were arbitrarily as-
signed a value of 100%.
100
0
1:25 1:50 1:100
E/S ratio
Fig. 1 – (A) Oxygen radical absorbance capacity (ORAC)
values of abalone viscera and its hydrolysates obtained
with different proteases. (B) Determination of optimal
enzyme/substrate (E/S) ratio for the production of abalone
viscera hydrolysates based on the ORAC assay. Bars with
different letters indicate means with significant differences
(p < 0.05). Values are expressed as means ± S.D. (n = 3).

of protein hydrolysates that ranged from 28.02 ± 0.26 to

2.8. Statistics
The data are presented as the mean ± standard deviation (SD)
of at least three independent experiments (n = 3). Differences
between means of each group were assessed by one-way analy-
sis of variance followed by Duncan’s test using PASW Statistics
19.0 software (SPSS, Chicago, IL, USA). A P-value < 0.05 was con-
sidered statistically significant.
3. Results and discussion
3.1. Preparation and ORAC value of AVH
Four different proteases (Alcalase, Flavourzyme, Neutrase, and
Protamex) were employed to produce bioactive protein hydro-
lysates from AV, and the hydrolysis reaction conditions, degree
of hydrolysis (DH) and protein hydrolysates yields are shown
in Table 1. The %DH ranged from 50.89 ± 1.41 to 60.04 ± 0.04,
and proteases used in this study produced similar amounts
34.64 ± 0.25%. Afterwards, we evaluated ORAC values of each
AVH and AV to determine the best protease to produce bioactive
AVH. As shown in Fig. 1A, the type of protease used during hy-
drolysis greatly affected the ORAC values of AVH. The AVH
produced by Alcalase showed the highest ORAC value (415 µM
TE/mg sample). These results indicated that Alcalase was the
more suitable protease for production of bioactive protein hy-
drolysates from AV. To determine the optimal E/S ratio,
enzymatic hydrolysis with Alcalase at various E/S ratios were
conducted and the ORAC values of the resultant AVH were de-
termined. As depicted in Fig. 1B, all AVHs produced using
different E/S ratios showed no significant difference for the
ORAC values; thus, we generated the AVH using Alcalase with
an E/S ratio of 1:100 for further study.
The ORAC assay measures the antioxidant capacity of pure
compounds and food-derived antioxidants, and this method
also evaluates the antioxidant activity of biological fluids in
subjects fed with antioxidant-rich foods (Prior et al., 2003). This
assay evaluates the ability to scavenge peroxyl radicals, a
common radical in human biology. Thus, the ORAC assay is
98 Journal of Functional Foods 16 (2015) 94–103

considered the most reliable method to measure the antioxi-

Table 3 – Free amino acid compositions of abalone
dant activity of food-derived antioxidants, and antioxidants with
viscera (AV) and abalone viscera hydrolysates (AVH).
high ORAC values may exhibit antioxidant effects on the human
Amino acid AV AVH
body (Park, Ahn, & Je, 2014). Several marine protein-derived hy-
drolysates were demonstrated to possess antioxidant activity. mg/100 g % mg/100 g %
For instance, ORAC values of 4.4 µM TE/mg protein, 3.5 µM TE/g amino amino
acid acid
protein, and 225 µM TE/g protein were reported for sea cu-
cumber hydrolysates, catfish hydrolysates, and pacific hake Phosphoserine 76.56 ± 2.31 2.5 447.84 ± 7.34 3.8
hydrolysates (Pérez-Vega, Olivera-Castillo, Gómez-Ruiz, & Taurine 1531.65 ± 15.82 49.5 5038.41 ± 21.55 42.5
Aspartic acid 121.12 ± 1.03 3.9 591.24 ± 5.71 5.0
Hernández-Ledesma, 2013; Samaranayaka & Li-Chan, 2008;
Threonine 69.49 ± 2.22 2.2 332.26 ± 3.44 2.8
Theodore, Raghavan, & Kristinsson, 2008). All of these values
Serine 79.50 ± 0.87 2.6 372.76 ± 4.07 3.1
are lower compared to the 415.8 µM TE/mg sample obtained Glutamic acid 209.10 ± 4.28 6.8 652.07 ± 8.23 5.5
for AVH in this work. Proline 61.56 ± 1.24 2.0 152.30 ± 2.30 1.3
Glycine 158.38 ± 3.03 5.1 392.54 ± 4.87 3.3
Alanine 153.04 ± 5.33 4.9 611.57 ± 6.33 5.2
3.2. Amino acid and free amino acid compositions
Valine 77.07 ± 1.87 2.5 336.39 ± 3.55 2.8
Methionine – 47.46 ± 0.83 0.4
Amino acid compositions of the AV and the AVH produced by Isoleucine 52.85 ± 0.98 1.7 203.63 ± 2.36 1.7
Alcalase hydrolysis are summarized in Table 2. In the AV, the Leucine 67.56 ± 1.26 2.2 472.06 ± 4.02 4.0
most abundant amino acids were found to be glutamic acid Tyrosine 60.24 ± 0.19 1.9 426.76 ± 3.88 3.6
(15%), aspartic acid (12.3%), and glycine (10.6%), followed by ar- phenylalanine 37.10 ± 1.02 1.2 250.20 ± 2.48 2.1
β-alanine – – 202.81 ± 1.99 1.7
ginine (7.2%), alanine (6.7%), leucine (5.8%), serine (5.8%), and
Histidine 8.75 ± 0.15 0.3 53.97 ± 0.83 0.5
threonine (5.7%). Essential amino acids in the AV were also
Ornithine 8.47 ± 0.56 0.3 31.61 ± 0.57 0.3
found in relatively high amounts, and the total percentage of Lysine 96.75 ± 2.57 3.1 464.34 ± 5.28 3.9
essential amino acids was 35.1%. In the case of the AVH, glu- Arginine 224.94 ± 5.63 7.3 765.93 ± 9.34 6.5
tamic acid (18.0%), aspartic acid (12.2%), and leucine (8.7%) were Total 3094.12 100.0 11,846.15 100.0
the major amino acids observed, but glycine (4.7%) was dra-
matically decreased compared to that of AV. The total percentage
was 42.3%, which is higher than that of the AV, thereby
indicating that the AVH can be used as good source of essen-
tial amino acids.
The contents of individual free amino acids of the AV and
Table 2 – Amino acid compositions of abalone viscera the AVH by Alcalase hydrolysis are shown in Table 3. The major
(AV) and abalone viscera hydrolysates (AVH). free amino acids found in the AV were taurine (49.5%), argi-
Amino acids AV AVH nine (7.3%), glutamic acid (6.8%), glycine (5.1%), and alanine
mg/100 g % mg/100 g % (4.9%). The amounts of total essential free amino acids and free
amino amino amino acids were found to be 409.5 and 3094.1 mg/100 g, re-
acid acid spectively. In the case of the AVH, the free amino acids found
Aspartic acid 3452.08 ± 30.35 12.3 3565.14 ± 32.45 12.2 in the highest concentration were found to be taurine (42.5%),
Threonine 1596.60 ± 21.46 5.7 1231.86 ± 20.49 4.2 arginine (6.5%), glutamic acid (5.5%), alanine (5.2%), and aspartic
Serine 1640.59 ± 13.24 5.8 1575.49 ± 12.57 5.4 acid (5.0%). The amounts of total essential free amino acids and
Glutamic acid 4224.92 ± 50.67 15.0 5230.58 ± 45.09 18.0 free amino acids were found to be 2160.3 and 11,846.2 mg/100 g,
Proline 887.52 ± 5.69 3.2 1001.37 ± 6.82 3.4 respectively, for the AVH. As expected, the amounts of total free
Glycine 2985.14 ± 8.91 10.6 1356.87 ± 6.89 4.7
amino acids were higher than that of AV, and this result may
Alanine 1893.85 ± 6.78 6.7 1509.65 ± 4.51 5.2
be due to the enzymatic hydrolysis of protein. The percent-
Cysteine 414.56 ± 3.24 1.5 1048.77 ± 10.86 3.6
Valine 1441.75 ± 7.36 5.1 1941.15 ± 7.89 6.7 age of essential free amino acids in the AV and the AVH was
Methionine 564.07 ± 3.88 2.0 498.094 ± 2.81 1.7 13.2 and 18.2%, respectively.
Isoleucine 1001.55 ± 10.22 3.6 1783.11 ± 13.92 6.1 Protein hydrolysates obtained after enzymatic hydrolysis are
Leucine 1639.24 ± 15.44 5.8 2532.77 ± 12.84 8.7 generally composed of free amino acids and short chain
Tyrosine 760.08 ± 3.45 2.7 956.62 ± 1.99 3.3 bioactive peptides, both of which exhibit many advantages as
Phenylalanine 780.98 ± 4.19 2.8 1542.00 ± 3.01 5.3
nutraceuticals or functional foods because of their amino acid
Histidine 1388.00 ± 7.45 4.9 1400.11 ± 6.29 4.8
Lysine 1466.05 ± 6.19 5.2 1404.30 ± 5.78 4.8
profile (Chalamaiah et al., 2012). These amino acids play an im-
Arginine 2013.79 ± 9.79 7.2 560.91 ± 2.46 1.9 portant role in various physiological activities of human body
Total 28,150.76 ± 67.24 100.0 29,138.79 ± 60.81 100.0 and affect either directly or indirectly in maintaining good
AAAa 49.6 57.1 health. Many marine protein-derived hydrolysates have been
HAA 33.4 44.0 reported to exhibit variation in their amino acid composition
a
Combined total of antioxidant amino acids (AAA) – aspartic acid, depending on the raw material, enzyme source, and hydroly-
glutamic acid, proline, cysteine, methionine, tyrosine, phenylala- sis conditions (Chalamaiah et al., 2012). Major amino acids were
nine, histidine, and lysine. Hydrophobic amino acids (HAA) – proline, also found to be glutamic acid, aspartic acid, and glycine in
alanine, cysteine, valine, methionine, isoleucine, leucine, tyro-
fish protein hydrolysates and shellfish hydrolysates (Ghassem
sine, and phenylalanine.
et al., 2014; Je, Park, Jung, & Kim, 2005; Yin et al., 2010). In the
 Journal of Functional Foods 16 (2015) 94–103 99

present study, AVH had higher essential amino acids (18.2%) at 2.0 mg/mL of the AVH. The calculated IC50 value was deter-
in total free amino acids than that of the AV (13.2%), thereby mined to be 0.65 ± 0.02 mg/mL.
supporting that the AVH, from the amino acid profile, may be Hydrogen peroxide was relatively unreactive as it is; however,
adequate as a potential ingredient of functional foods. it plays an important role in oxidative stress. It can be con-
The amino acid constituents and the sequence of the pep- verted to hydroxyl radicals by transition metals such as Fe2+
tides are very important for their antioxidant activity. The in Fenton reaction, and generated hydroxyl radicals cause oxi-
presence of hydrophobic amino acids and histidine, proline, dative damage to critical cellular biomolecules such as DNA,
methionine, tyrosine, lysine, phenylalanine, and cysteine has proteins, and membrane lipids (Seifried, Anderson, Fisher, &
been shown to enhance the potency of antioxidant peptides Milner, 2007). Therefore, scavenging and chelating of hydro-
through proton-donation ability, electron-donation ability, and/ gen peroxide and Fe2+ by antioxidants could prevent ROS
or direct lipid radical scavengers (Je, Qian, Byun, & Kim, 2007; generation, which initiated harmful reactions. Autolysis-
Samaranayaka & Li-Chan, 2011; Sarmadi & Ismail, 2010). It also assisted fish protein hydrolysates from Pacific hake possessed
has been demonstrated that acidic amino acids such as glu- 46.7% Fe2+ chelating activity at 5.0 mg/mL, which is lower than
tamic acid and aspartic acid exhibited strong antioxidant ability that obtained in this work (Samaranayaka & Li-Chan, 2008).
due to carboxyl and amino groups in the side chains as che- Rapeseed protein hydrolysates and their membrane ultrafil-
lator of metal ions (Sarmadi & Ismail, 2010; Udenigwe & Aluko, tration peptide fractions exhibited Fe2+ chelating activity and
2011). In the present study, we found 49.6% antioxidant amino their IC50 values were higher than that of the AVH (He, Girgih,
acids (AAA) and 33.4% hydrophobic amino acids (HAA) in the Malomo, Ju, & Aluko, 2013). In the present study, we demon-
AV whereas 57.1% AAA and 44.0% HAA were observed in the strated that the AVH had potent hydrogen peroxide scavenging
AVH. In view of these results, AVH was expected to exhibit and Fe2+ chelating hydrolysates.
strong antioxidant ability due to its higher content of AAA and The FRAP of the AVH was measured as the ability to reduce
HAA than AV (Table 2). In addition, free amino acid composi- Fe3+ to Fe2+, which indicated the capacity of antioxidants to
tions revealed that taurine content was dramatically increased donate an electron or hydrogen, and an increase in absor-
3.3-fold by enzymatic hydrolysis in the AVH compared to that bance at 700 nm indicated greater reducing power. As depicted
of the AV. It is well known that taurine has several beneficial in Fig. 2C, a dose-dependent increase of the FRAP was ob-
physiological actions such as antioxidation, protective effects served with an absorbance of 0.98 ± 0.01 at 2.0 mg/mL. Many
of renal damage, and anti-inflammation in brain (Chang et al., studies reported that protein hydrolysates from other sources
2014; Huxtable, 1992; Su et al., 2014), thus supporting the fact possessed strong FRAP. Fish protein hydrolysates from smooth
that the AVH may have health benefits. As expected, the ORAC hound muscle protein and yellow stripe trevally were re-
value of the AVH was higher than that of AV, and this result ported to have FRAP values of 0.60 at 2.0 mg/mL and 0.52 at
is attributed to high AAA (57.1%) and HAA (44.0%) contents in 3.6 mg/mL, respectively (Bougatef et al., 2009; Klompong,
the AVH (Fig. 1 and Table 2). Benjakul, Kantachote, & Shahidi, 2007). Additionally, only one
research for the FRAP of abalone viscera hydrolysates by alkali
protease, papain, neutral protease, pepsin, and trypsin was re-
3.3. Antioxidant activities
ported by Zhou et al. (2012) and abalone viscera hydrolysates
exhibited the FRAP value of below 0.90 at 10.0 mg/mL.
Next, we investigated hydrogen peroxide scavenging activity,
Fe2+ chelating activity, and reducing power of the AVH. As shown
in Fig. 2A, the AVH displayed effectively hydrogen peroxide scav- 3.4. Inhibition ability of lipid peroxidation in mackerel
enging activity in a dose-dependent manner, and 98.74 ± 0.25% lipid model system
scavenging activity was observed at 2.4 mg/mL of the AVH. The
calculated IC50 value, which is the concentration required to Lipid peroxidation proceeds via a free radical-mediated process
scavenge hydrogen peroxide by 50%, was determined to be in biological systems and thereby resulting in an oxidative al-
0.13 ± 0.01 mg/mL. The AVH also exhibited strong chelating ac- teration of polyunsaturated fatty acids in the cell membranes.
tivity toward Fe2+ (Fig. 2B), and it displayed 88% chelating activity In particular, lipid peroxidation in foods led to the development
H2O2 scavenging activity (%)

100
Fe2+ chelating activity (%)

a a
A C
Absorbance at 700 nm

100 b B a 1.0
80 b
c
80 c b
0.8 d

d 60
60 c
0.6

40 40 d 0.4

20 20 0.2

0 0 0.0
0.3 0.6 1.2 2.4 0.25 0.5 1 2 0.25 0.5 1 2
AVH (mg/mL) AVH (mg/mL) AVH (mg/mL)

Fig. 2 – (A) Hydrogen peroxide scavenging activity; (B) ferrous ion chelating activity; and (C) ferric reducing antioxidant
power of abalone viscera hydrolysates (AVH) obtained with Alcalase. Bars with different letters indicate means with
significant differences (p < 0.05). Values are expressed as means ± S.D. (n = 3).
100 Journal of Functional Foods 16 (2015) 94–103
80
a
60
MDA (µM)
40
20 c
d
e
0
0 0.1 0.5 1 BHT
AVH (mg/mL)
Fig. 3 – Lipid peroxidation inhibition ability of the abalone
viscera hydrolysates (AVH) in a model system. BHT was
used as a positive control. Bars with different letters
indicate means with significant differences (p < 0.05).
Values are expressed as means ± S.D. (n = 3).
of undesirable off-flavors, potentially toxic substances and de-
terioration (Park, Jung, Nam, Shahidi, & Kim, 2001). Therefore,
the measurement for inhibition of lipid peroxidation was an
important indicator for evaluating antioxidant ability with
regard to human health and food protection. In the present
study, total lipids were oxidized in a water/ethanol emulsion
at 40 °C in a dark room to evaluate lipid peroxidation inhibi-
tion ability of AVH. Peroxyl and alkoxyl radicals derived from
the preexisting lipid peroxide in emulsion initiated lipid
peroxidation (Cheng, Ren, Li, Chang, & Chen, 2003). Finally,
various reactive aldehydes are generated with MDA during lipid
peroxidation, which is one of the most frequently used indi-
cators of lipid peroxidation (Esterbauer & Cheeseman, 1990).
Thus, we determined TBARS in a water/ethanol emulsion after
4 days of incubation and TBARS value was expressed as MDA
concentration. As shown in Fig. 3, formation of MDA, which
is the final product of lipid peroxidation, without the AVH was
found to be 71.33 ± 0.58 µM, whereas this value dramatically
decreased (p < 0.05) in the presence of the AVH, in a dose-
dependent manner. The final concentration of MDA was
determined to be 9.94 ± 0.13 µM at 1.0 mg/mL. The lipid
peroxidation inhibition result suggested that AVH could inhibit
lipid peroxidation and this activity is attributed to its oxygen
radical (peroxyl and alkoxyl) scavenging activity.
3.5. Protection against hydroxyl radical-induced DNA
damage
DNA is another sensitive target for ROS-mediated oxidative
damage; thus, overproduction of ROS results in DNA damage,
which can initiate carcinogenesis and neurodegenerative dis-
eases, and the hydroxyl radical was considered to be a potent
DNA damage agent of physiological significance (You et al.,
2002). Thus, the potential protective effect of AVH against hy-
droxyl radical-induced DNA damage was evaluated in vitro.
Under oxidative conditions, the supercoiled (SC) DNA form can
be converted to open circular (OC) DNA form that in this system
Fig. 4 – Protective ability of the abalone viscera
hydrolysates (AVH) against hydroxyl radical-induced DNA
damage. Bars with different letters indicate means with
significant differences (p < 0.05). Values are expressed as
means ± S.D. (n = 3).
the conversion of SC to OC is provoked by strand breaks caused
by hydroxyl radicals, which can be visualized by electropho-
resis with ethidium bromide. As shown in Fig. 4, exposure of
hydroxyl radical derived from Fenton reaction resulted in DNA
damage with 16.83% SC DNA form. Co-treatment with the AVH
protected hydroxyl radical-induced DNA damage in a dose-
dependent manner, and the AVH protected SC DNA form with
68.31 ± 0.10% at 600 µg/mL. In the present study, the protec-
tive effect of the AVH against hydroxyl radical-induced DNA
damage was demonstrated. This activity may attribute to its
H2O2 scavenging and Fe2+ chelating activity (Fig. 2), although
we used Fenton reaction for generation of hydroxyl radical.
However, the AVH concentration used in this study was lower
compared to H2O2 scavenging and Fe2+ chelating assay, indi-
cating the AVH could be also quenched hydroxyl radical.
3.6. Cytoprotective activities of AVH against hydrogen
peroxide-induced hepatocyte damage
Hepatocytes were employed to evaluate cytoprotective effects
of the AVH against oxidative stress-induced damage. To de-
termine potential cytotoxic effects of the hydrolysates, cultured
hepatocytes were treated with various concentrations of AVH,
and then evaluations were performed using the MTT assay. As
shown in Fig. 5A, no significant cytotoxic effect up to 250 µg/
mL of the AVH occurred whereas the cell viability significantly
(p < 0.05) decreased at 500 and 1000 µg/mL of the AVH. Thus,
the non-cytotoxic concentration was applied to the next ex-
periments. Hepatocytes were exposed to 650 µM hydrogen
peroxide in order to induce oxidative damage. About 60.15%
cell viability compared to the non-treatment group (blank) was
observed by treatment with 650 µM hydrogen peroxide; however,
this cell viability was significantly (p < 0.05) increased by pre-
treatment with AVH in a dose-dependent manner, and the cell
viability reached up to 89.97% at 250 µg/mL of the AVH (Fig. 5B).
Hydrogen peroxide had been widely used to induce oxidative
 Journal of Functional Foods 16 (2015) 94–103 101

120 120
a a A a B
100 b c 100 b

Cell viability (%)

Cell viability (%)

bc
80 80 c
d
60 60
40 40
20 20
0 0
100 250 500 1000 Blank 0 50 100 250
AVH (µg/mL) H2O2 + AVH (µg/mL)
120 120
C
C D
D

lipid peroxidation (%)

a a
100 100
b

Membrane
80 c 80
ROS (%)

b c
d
60 60
40 40 d
20 20
0 0
0 50 100 250 0 50 100 250
H2O2 + AVH (µg/mL) AAPH + AVH (µg/mL)

Fig. 5 – (A) Cytotoxicity of the abalone viscera hydrolysates (AVH) toward cultured hepatocytes; (B) cytoprotective activity of
the AVH against hydrogen peroxide-induced cytotoxicity in cultured hepatocytes; (C) inhibition activity of the AVH on
intracellular ROS formation in cultured hepatocytes; and (D) inhibition activity of the AVH on membrane lipid peroxidation
in cultured hepatocytes. Bars with different letters indicate means with significant differences (p < 0.05). Values are
expressed as means ± S.D. (n = 3).

cell injury via generation of hydroxyl radical, and several reports
demonstrated that treatment of hydrogen peroxide induced
cell death and these cell deaths were inhibited by pretreat-
ment with antioxidants (He, Tao, & Liu, 2014; Linden, Gulden,
Martin, Maser, & Seibert, 2008). Wiriyaphan, Xiao, Decker, and
Yongsawatdigul (2015) also reported that threadfin bream
(Nemipterus spp.) surimi byproduct hydrolysates attenuated
t-butyl hydroperoxide-induced cytotoxicity. In the present study,
we also demonstrated that pretreatment with the AVH pro-
tected hydrogen peroxide-induced hepatocyte cell death, and
this observation was in accordance with literature data.
To evaluate cellular antioxidant activity of the AVH under
oxidative stress conditions, intracellular ROS formation was
assayed using DCFH-DA, which was widely used to measure
oxidative stress in cells. DCFH-DA was hydrolyzed to DCFH by
intracellular esterases, followed by oxidation of DCFH to form
fluorescent DCF in the presence of ROS. As shown in Fig. 5C,
intracellular ROS formation was significantly (p < 0.05) inhib-
ited by pretreatment with AVH in a dose-dependent manner,
and ROS generation decreased by 38.7% in the presence of
250 µg/mL of the AVH compared to control (with H2O2, but
without AVH). The ability of AVH to inhibit AAPH-induced mem-
brane lipid peroxidation was also evaluated in cultured
hepatocytes. AAPH treatment resulted in a significant in-
crease of membrane lipid peroxidation. However, pretreatment
with AVH significantly (p < 0.05) decreased membrane lipid
peroxidation in a dose-dependent manner in cultured hepa-
tocytes (Fig. 5D). The membrane lipid peroxidation activity of
the AVH was 69.6% at 250 µg/mL. It is generally believed that
overproduction of ROS typically induced cell death through oxi-
dative stress, which is strongly linked to a number of human
diseases and also is involved in the pathogenesis of hepatic
fibrosis (Poli, 2000). The AVH obtained in our study presented
comparable or even higher cellular ROS scavenging activity than
protein hydrolysates from silver carp, threadfin bream, and egg
membrane obtained by several authors (Malaypally et al., 2014;
Shi, Kovacs-Nolan, Jiang, Tsao, & Mine, 2014; Wiriyaphan et al.,
2015). Moreover, the AVH also inhibited membrane lipid
peroxidation induced by AAPH, which is an initiator of lipid
peroxidation, in cultured hepatocytes.
4. Conclusions
Abalone viscera hydrolysates (AVH) produced by enzymatic hy-
drolysis demonstrated that antioxidant activity was highly
dependent on the enzyme used during proteolysis. AVH ob-
tained by treatment with Alcalase displayed the highest ORAC
values. Hydrolysates also presented hydrogen peroxide scav-
enging activity, Fe2+ chelating activity, reducing power, protective
effects against hydroxyl radical-induced DNA damage, and lipid
102 Journal of Functional Foods 16 (2015) 94–103
peroxidation in a model system. The AVH was also found to
have high concentrations of antioxidant amino acids, which
may play an important role on its antioxidant activity. Fur-
thermore, the AVH protected cultured hepatocytes against
oxidative stresses through intracellular ROS scavenging and
membrane lipid peroxidation in cultured hepatocytes. Taken
all together, these results suggest that the AVH can be prom-
ising ingredients in functional foods.
Acknowledgement
This research was supported by the Marine product research
and development program funded by Jellanamdo.
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