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Food Sci Nutr. 2020;8:1874–1887.www.foodscience-nutrition.com
1 | INTRODUCTION
Skipjack tuna (Katsuwonus pelamis) is consumed worldwide because
of its abundance, nutritional value, and sensorial attributes (firm tex-
ture and flavorful flesh). This tuna is widely used as a raw material
for sashimi and sushi in Korea and Japan (Lee, Park, et al., 2016). In
2015, 51,201 metric tons of skipjack tuna were canned, account-
ing for 57% of all canned products in Korea (KOSIS, 2016). The an-
nual discard rate of fish processing byproducts has increased with
increasing fish production. The fish processing industry generates
large quantities of byproducts such as heads, skin, frames, roe, vis-
cera, and scales (Narsing Rao, Prabhakara Rao, Satyanarayana, &
Balaswamy, 2012), most of which are not used as sources of high
value-added products for human or animal consumption and are dis-
posed of as waste. However, these byproducts are excellent sources
of proteins (Lee, Lee, et al., 2016). Upcycling or reuse of byproducts
has gained wide attention in the seafood processing industr y owing
to its economic effectiveness (Lee, Lee, et al., 2016). In recent years,
Received: 16 December 2018
|
Revised: 16 January 2020
|
Accepted: 29 January 2020
DOI: 10.10 02/f sn3.1470
ORIGINAL RESEARCH
Food functionalities and bioactivities of protein isolates
recovered from skipjack tuna roe by isoelectric solubilization
and precipitation
Jang Woo Cha1 | In Seong Yoon1 | Gyoon-Woo Lee2 | Sang In Kang1 |
Sun Young Park1 | Jin-Soo Kim1,3 | Min Soo Heu2,3
This is an op en access article under t he terms of the Creat ive Commons Attributio n License, which permits use, dist ribution and reproduc tion in any medium,
provide d the orig inal work is proper ly cited .
© 2020 The Authors . Food Scie nce & Nutri tion published by Wiley Perio dicals, Inc.
Cha and Yoon e qually contri buted to this work .
1Depar tment of Seafood and Aquaculture
Science/Instit ute of Mari ne Indus try,
Gyeongsang National University,
Tongyeong, Korea
2Department of Food and Nutrition/
Instit ute of Marine Indus try, Gyeon gsang
Nationa l University, Jinju , Korea
3Research Center for Industrial
Develop ment of Seafood, Gye ongsang
National University, Tongyeong, Korea
Correspondence
Min Soo He u, Department of Food and
Nutrit ion/Ins titute of Marine Industr y,
Gyeongs ang National University, 501
Jinjuda ero, Jinju, Gyeong sangna m-do 52828,
Korea.
Email: minsheu@gnu.ac.kr
Funding information
Ministry of Oceans and Fisheries, Korea
Abstract
Four roe protein isolates (RPIs) from skipjack tuna were prepared using isoelectric
solubilization (pH 11 and 12) and precipitation (pH 4.5 and 5.5) (ISP) at different pH
points to evaluate their physicochemical and functional properties and in vitro bio-
activities. Moisture (<6.3%) and protein (71%–77%) content were maintained. Sulfur,
sodium, phosphorus, and potassium were the major elements, and glutamic acid and
leucine were the prevalent amino acids (12.2–12.8 and 9.6–9.8 g/100 g protein, re-
spectively) in RPIs. RPI-1 showed the highest buffering capacity at pH 7–12. RPIs and
casein showed similar water-holding capacities. At pH 12, RPI-1(pH 11/4.5) showed
the highest solubility, followed by RPI-3(pH 12/4.5), RPI-2(pH 11/5.5), and RPI-4(pH
12/5.5) (p < .05). Oil-in-water emulsifying activity indices of RPI-1 and RPI-3 signifi-
cantly differed. At pH 2 and 7–12, pH-shift treatment improved the food functional-
ity of RPIs, which was superior to positive controls (casein and hemoglobin). RPI-1
showed ABTS+ radical scavenging (102.7 μg/ml) and angiotensin-converting enzyme
inhibitory activities (44.0%).
KEYWORDS
food functionality, physicochemical characterization, roe protein isolates, skipjack tuna
|
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CHA et Al .
numerous studies have examined the use of protein-rich seafood
byproducts as nutraceuticals or protein and lipid supplements (Kim,
Yoon, Shim, & Lim, 2016).
Among fish byproducts, roes are nutritionally rich in essential
fatty acids, minerals, and amino acids (Heu et al., 2006; Park et al.,
2016). The production of caviar and roe products from other fish
species has been examined previously (Bledsoe, G., Bledsoe, C., &
Rasco, 2003).
Roe, a byproduct of fish processing (3.0%–30.0% depending on
species), contains 11% albumin, 75% ovoglobulin, and 13% collagen
(Sikorski, 1994) and is generally used as animal feed or in pet food
preparation. The underutilized skipjack tuna roe (STR) requires pro-
cessing methods for conversion to more marketable and acceptable
forms, such as protein concentrates, isolates, and extracts. Previous
studies characterized the ingredients, chemical composition (Heu
et al., 2006; Lee, Lee, et al., 2016), fractions, and characteristics of
the protease inhibitors (Kim et al., 2013), concentrates (Park et al.,
2016; Yoon, Lee, et al., 2018), and protein isolates (Lee, Park, et al.,
2016) in fish roes.
Isoelectric solubilization and precipitation (ISP) promotes the
selective pH-induced water solubility of muscle proteins, segre-
gates lipids, and removes materials unfit for human consumption,
such as bones, scales, and skin (Hultin & Kelleher, 1999). ISP is used
for protein recovery from inaccessible sources such as fish (Chen &
Jaczynski, 20 07), chicken (Tahergorabi, Beamer, Matak, & Jaczynski,
2011), and beef (Mireles, Gomez, & James, 2002) processing byprod-
ucts; additionally, the fat content in the recovered protein isolate
is significantly reduced. A major technology was developed for fea-
sible extraction of functional protein isolates from low-value raw
materials (dark muscle fish and fatty fish) and seafood processing
byproducts (fish cutoffs and fish frame) (Nolsoe & Undeland, 2009).
Proteins recovered from the ISP process exhibit desirable functional
proper ties and may have better gelling properties than proteins ob-
tained by conventional surimi processing (Kristinsson, Theodore,
Demir, & Ingadottir, 2005). Proteins are the basic functional compo-
nents of processed foods and determine the textural and nutritional
proper ties and contribute to the quality and sensorial at tributes of
foods (Mohamed, Xia, Issoufou, & Qixing, 20 09; Park et al., 2016).
No studies have examined the protein isolate preparation of skipjack
tuna roe or its food functional characteristics and bioactivity. The
purpose of this study was to investigate the physicochemical and
functional characteristics of protein isolates recovered from skipjack
tuna roe using the ISP process, as well as their antioxidative and an-
tihypertensive bioactivities.
2 | MATERIALS AND METHODS
2.1 | Chemicals
2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ammo-
nium salt (ABTS), casein, hemoglobin, hippuryl-his-leu acetate salt
(HHL), lung acetone powder from rabbit, mushroom tyrosinase,
potassium persulfate, and sodium L-tartrate were purchased from
Sigma-Aldrich. 3,4-Dihydroxy-L-phenylalanine (L-DOPA) was pur-
chased from Acros Organics. Hydrochloric acid (1 M) and sodium
hydroxide (1 M) were purchased from Yakuri Pure Chemicals Co.,
Ltd.. Folin–Ciocalteu reagent and acetic acid were purchased from
Junsei Chemical Co., Ltd. Sodium dodecyl sulfate (SDS) and glycine
were purchased from Bio Basic, Inc. Soybean oil was purchased from
Ottogi Co., Ltd. All other reagents were of analytical grade.
2.2 | Sample
Skipjack tuna roe was obtained from Dong won F & B Co., Ltd.
(Changwon, Korea), stored at −70°C in sealed polyethylene bags,
and transferred to the laboratory. Frozen roe was partially thawed
for 24 hr at 4°C and then cut into small pieces to 2.0 cm thickness
and minced with a food grinder. Minced roe was frozen at −20°C
until use.
2.3 | Preparation of roe protein isolates (RPIs)
Roe protein isolates were prepared as described by Lee, Lee, et al.
(2016). The frozen minced roe was homogenized with deionized dis-
tilled water (DDW) at a 1:6 (w/v) ratio using a homogenizer. The ho-
mogenate was adjusted to pH 11 and 12 with 2N Na OH. Solubilization
was allowed to occur at 4°C for 1 hr, followed by centrifugation at
12,00 0 × g and 4° C for 30 mi n. After cent rifugat io n, two alka li-so lu ble
(pH 11 and 12) supernatant fractions were collected.
To prepare the RPIs from the alkali-soluble frac tions by acid
precipitation, the pH of the fractions was readjusted to pH 4.5 and
5.5 (similar to the isoelectric point of fish proteins) by adding 2N
HCl. The suspensions were centrifuged at 12,000 × g and 4°C for
30 min. Precipitates obtained after ISP were washed with DDW and
centrifuged at 12,0 00 × g and 4°C for 30 min to remove the NaCl.
After centrifugation, the washed RPIs were lyophilized and labeled
as RPI-1 (pH 11/4.5), RPI-2 (pH 11/5.5), RPI-3 (pH 12/4.5), and RPI-4
(pH 12/5.5). These RPIs were stored at −20°C until further use.
2.4 | Physicochemical properties
2.4.1 | Proximate composition
The proximate composition was determined by the AOAC method
(AOAC, 2005). Moisture content (950.46) was determined by placing
a 0.5-g sample in an aluminum pan and drying in a forced air con-
vection oven at 105°C until a constant mass was reached. The ash
content (920.153) was determined by charring approximately 0.2 g
sample in a ceramic crucible over a hot plate and then heating in a
muffle furnace at 550°C until a constant mass was achieved. The
total crude protein content (928.08) was determined by the semi-
micro Kjeldahl method. Total lipid content (960.39) was determined
1876
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CHA et Al.
by the Soxhlet extraction method. Sample (5 g) was extracted with
dimethyl ether for 30 min at a drip rate of 10 ml/min. The total
lipid content was determined by gravimetry and expressed as a
percentage.
2.4.2 | Protein concentration
The protein concentration of the samples was determined as de-
scribed by Lowry, Rosebrough, Farr, and Randall (1951) using bovine
serum albumin as a standard.
2.4.3 | Total amino acid content
The samples were hydrolyzed by 6 N HCl at 110°C for 24 hr on a
heating block and filtered using a vacuum filtration device. Amino
acids were quantified with an amino acid analyzer (Biochrom 30,
Biochrom Ltd.) using sodium citrate buffers (pH 2.2) in step gradients.
The results are reported in milligrams of amino acid per 100 g protein.
2.4.4 | Mineral analysis
The mineral content of the samples was determined by inductively
coupled plasma optical emission spectrophotometry (Optima
4300 DV, PerkinElmer). The samples were dissolved in 10 ml of
70% (v/v) nitric acid and heated on a hot plate until digestion was
complete. The volume of the samples (in duplicates) was made up
to 100 ml with 2% nitric acid in a volumetric flask. The mineral
concentration was calculated and expressed as milligrams per
100 g sample.
2.4.5 | Color value
The sam ples were equili brate d to 20 ± 2°C for 2 hr prior to Hu nte r
color measurement. Color values were determined using a color
meter (ZE-2000 Nippon Denshoku Inc.). The colorimeter was
calibrated using a standard plate (L* [lightness] = 96.82, a* [red-
ness] = −0.35, b* [yellowness] = 0.59) supplied by the manufac-
turer. The values of the Commission Internationale d’Eclairage of
France (CIE) color system using tri-stimulus color values (L*, a*, and
b*) were determined. Whiteness was calculated using the follow-
ing equation:
2.5 | SDS–polyacrylamide gel electrophoresis
(SDS-PAGE)
The molecular weight distribution of proteins was investigated
by SDS-PAGE, which was performed according to the method of
Laemmli (1970). Briefly, 20 mg sample was solubilized in 5 ml of 5%
SDS. The protein solution was then mixed at a 4:1 (v/v) ratio with
SDS-PAGE sample treatment buffer (pH 6.8) and boiled at 100°C
for 3 min. Samples (20 µg protein) were loaded into a 10% Mini-
PROTEAN® TGX™ precast gel and electrophoresed at a constant
current of 10 mA using a Mini-PROTE AN® tetra cell (Bio-Rad).
2.6 | Protein functionalities
2.6.1 | Buffer capacity
Buffer capacit y was measured as described by Park et al. (2016).
The sample (300 mg) was dispersed in 30 ml distilled water, and
measured volumes of 0.5 M NaOH and HCl were added in small in-
crements. The corresponding changes in pH in alkaline and acidic
ranges were recorded. The amounts of base and acid added were
plotted against the pH, and the buffer capacity in each range was
expressed as the mean value of NaOH or HCl (in millimolar) required
per gram sample to induce a unit change in pH.
2.6.2 | Water-holding capacity (WHC)
The WHC of samples was measured as described by Park et al.
(2016). The sample (300 mg) was mixed with 30 ml DDW in a 50-ml
centrifuge tube. The mixture was thoroughly vortexed for 10 min at
20 ± 2°C and centrifuged at 12,000 × g for 20 min at 4°C. WHC was
determined from the difference in masses and expressed as grams of
water absorbed per gram protein.
where C is protein concentration (%).
2.6.3 | Protein solubility
The protein solubility of samples was determined according the
method of Park et al. (2016). First, 300 mg sample was dispersed in
30 ml DDW and the pH was adjusted to 2–12 with 2N HCl and 2N
NaOH. The mixture was stabilized at room temperature for 30 min
prior to centrifugation at 12,0 00 g for 20 min. Protein content in
the supernatant and total protein content in the sample were deter-
mined by Lowry's method after solubilizing the sample in 2N NaOH.
Protein solubility was calculated as follows:
Each measurement was replicated at least five times, and the re-
sults were expressed as the means ± SD.
Whiteness
=100−
√
(100−L*)2+a*2 +b
*2
WHC (g
∕g protein) =
Mass of pellet (g)
−
Mass of sample (g)
Mass of sample (g)×C
Solubility (%)
=
Protein content in supernatant
Total protein content in sample
×
100
|
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CHA et Al .
2.6.4 | Foaming capacity (FC) and foam stability (FS)
Foaming capacity (FC) and foam stability (FS) of the sample solu-
tion (1%, w/v) were determined as described by Park et al. (2016)
with slight modifications. Ten milliliters of 1% (w/v) sample solution
was transferred to a 25-ml volumetric cylinder. The solution was ho-
mogenized (Polytron® PT 1200E, Kinematica AG) at 12,500 rpm for
1 min at 20 ± 2°C. The sample was allowed to stand for 0, 15, 30, and
60 min, and FC and FS were calculated using the following equations:
where VT is the total volume after whipping, V0 is the initial volume,
and Ft and Vt are the total foam and total volume, respectively, after
standing at room temperature for different durations (t = 15, 30, and
60 min).
2.6.5 | Emulsifying properties
The emulsif ying activity index (E AI) and emulsion stability index (ESI)
were determined as described by Park et al. (2016). Soybean oil (Ottogi
Co., Ltd.) and 1% (w/v) sample at a 1:3 (v/v) ratio were homogenized at
a rate of 12,500 rpm for 1 min. An aliquot of the emulsion (50 μl) was
pipetted from the bottom of the volumetric cylinder 0 and 10 min after
homogenization and mixed with 5 ml of 0.1% SDS solution. The absorb-
ance of the mixture was measured at 500 nm (UV-2900, Hitachi).
The absorbances measured immediately (A0min) and at 10 min
(A10min) after emulsification were used to calculate EAI and ESI as
follows:
where A is the absorbance at 500 nm, DF is the dilution factor (100), l is
the path length of the cuvette (1 cm), φ is the oil volume fraction (0.25),
and C is the protein concentration in the aqueous phase (g/ml).
where ΔA = A0 min–A10min and Δt = 10 min. A0min and A10min are the
absorbance values measured at 0 and 10 min after emulsification,
respectively.
2.7 | Antioxidative and antihypertensive activity
2.7.1 | ABTS+ radical scavenging activity
ABTS+ radical scavenging activit y was determined using the ABTS
assay with slight modification based on the method of Binsan et al.
(20 08). Fresh ABT S+ solutions in ethanol were prepared for each assay.
Samples (1 ml) were mixed with 3 ml ABTS+ solution and incubated at
room temperatu re for 30 min in th e dark . Ab so rbance was mea su re d at
734 nm with a spectrophotometer. The IC50 value was defined as the
concentration required to scavenge 50% of ABTS+ radical.
Absorb an ce was mea su re d im me di at el y (A 734), and ABTS+ radical
scavenging activity was calculated as follows:
where control734 is the absorbance of the same reaction system with-
out the sample.
2.7. 2 | Tyrosinase inhibitory activity
The tyrosinase inhibitory activity was determined as described by Iida
et al. (1995) with some modifications. Briefly, 900 μl (50 U/ml reaction
mixture) mushroom tyrosinase was preincubated with the sample in
50 mM phosphate buffer (pH 6.8) for 30 min at room temperature.
Next, 300 μl 10 mM L-DOPA was added to the reaction mix ture, and
enzyme activity was monitored at room temperature by measuring the
change in abs or ba nce at 475 nm (UV-2900, Hi tachi) for 30 mi n at 1-min
intervals, which corresponded to the formation of dopachrome.
Samples without inhibitor were used as controls. The percentage
inhibition of enzyme activity by the active compounds was calcu-
lated as follows:
where control475 is the absorbance of a reac tion system without the
sample.
2.7. 3 | Angiotensin-converting enzyme (ACE)
inhibitory activity
Angiotensin-converting enzyme inhibitory activity was estimated
as described by Cushman and Cheung (1971) with slight modifica-
tions. ACE was extracted from 5 g lung acetone powder from rab-
bit with 100 ml 0.05 M sodium borate buffer (pH 8.3) containing
300 mM NaCl. A mixture of 100 μl sample, 50 μl ACE extract s and
50 μl 0.05 M sodium borate buffer (pH 8.3), was preincubated at
room temperature for 30 min before incubation with 50 μl substrate
(5 mM HHL in 0.05 M sodium borate buffer, pH 8.3) for 60 min at
37°C in a water bath. The reaction was terminated by adding 250 μl
1N HCl. The resulting hippuric acid was extracted with 1.5 ml ethyl
acetate. After centrifugation (1,890 × g, 10 min, 4°C), 1.0 ml of the
upper layer was transferred into a test tube and evaporated at 100°C
for 1 hr in a heating block. The hippuric acid was dissolved in 1.0 ml
distilled water, and absorbance was measured at 228 nm with a UV
Foaming capacity (%)
=
VT
V0
×
100
Foam stability (%)
=
(Ft∕Vt)
(FT
∕
VT) ×
100
EAI (m
2∕g) =
2×2.303×A×DF
l×𝜑×C
×
100
ESI ( min )
=
A0×Δt
ΔA
ABTS
+radical scavenging activity (%) =
(Control
734
−Sample
734
)
Control734
×
100
Tyrosinase inhibitory activity (%)
=
(Control
475 −
Sample
475)
Control
475
×
100
1878
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CHA et Al.
spectrophotometer. Absorbance was measured immediately (A2 28)
and used to calculate the ACE inhibitory activity as follows:
where the sample blank is the absorbance of the inactivated sample
before adding HHL and control blank is the absorbance of the inacti-
vated control before adding HHL.
2.8 | Statistical analysis
All analyses were performed in triplicate. Data were averaged, and
the SD was calculated. Data were analyzed by analysis of variance
using SPSS 12.0 K software (SPSS, Inc.) for Windows. Mean com-
parisons were performed using the multiple range Duncan's test
(p < .05).
3 | RESULTS AND DISCUSSION
3.1 | Physicochemical properties
3.1.1 | Proximate composition
Roe protein isolates were recovered from STR by ISP. The proxi-
mate composition, mineral content, and Hunter's color values of
the RPIs and positive controls (casein and hemoglobin) are shown in
Table 1. Freeze-dried STR (FDSTR) contained 6.2 ± 0.9% moisture,
71.1 ± 0.7% protein, 15. 2 ± 1.3% lipid, and 7.2 ± 0.1% ash. FDSTR
yield and protein yield were 26.7 and 19.0 g/100 g of STR , respec-
tively. The yields of RPIs produced from STR by ISP differed slightly
by 10.3%–13.0%. The recovery of RPIs from STR by ISP was 54.2%–
68.4%. This low protein recovery may be attributed to the presence
of alkaline insoluble proteins that did not dissolve during alkaline
solubilization (Lee, Lee, et al., 2016; Yoon, Kang, et al., 2018). The
RPI-2 showed the highest protein content (76.8 ± 0.3%), followed
by RPI-4 (75.4 ± 0.4%), RPI-1 (71.4 ± 0.8%), and RPI-3 (70.8 ± 0.7%).
However, these values were lower than those of the positive con-
trols (85.5 ± 0.0% for casein, 94.4 ± 0.4% for hemoglobin). The
protein content of RPIs was higher than those reported for fish pro-
tein powders from arrowtooth flounder and herring (Sathivel et al.,
2004). The protein content in fish eggs (Sathivel, Yin, Bechtel, &
King, 2009) and surimi (Huda, Abdullah, & Babji, 2001) was similar
to those in the RPIs. Lipid (13.8 ± 2.5–15.6 ± 1.0%) and ash content
(1.8 ± 0.6–3.6 ± 0.1%) in the RPIs were lower than those in FDSTR
(p < .05). This reduction in lipid and mineral content occurred be-
cause of mineral and fat migration into the processed water during
ISP (Lee et al., 2017; Lee, Lee, et al., 2016). The protein content of
RPI-2 and RPI-4 precipitated at pH 5.5 after alkaline solubilization
was significantly higher than that of RPI-1 and RPI-3 precipitated at
pH 4.5 (p < .05). In contrast, the total yield (10.3–10.9 g/100 g) and
protein yield (7.8–7.9 g/100 g) of RPI-1 and RPI-2 were significantly
lower than those of RPI-3 and RPI-4 (p < .05). Thus, alkali solubiliza-
tion at pH 12 was better than that at pH 11, while acid precipitation
did not affect protein yield (p < .05).
3.1.2 | Minerals
The mineral content of FDSTR, RPIs, and positive controls is
shown in Table 1. The major minerals in FDSTR were potassium
(1,097.4 ± 9.0 mg/100 g), sulfur (974.3 ± 57.7 mg/100 g), sodium
(615.2 ± 1.4 mg/100 g), and phosphorus (247.9 ± 1.5 mg/100 g). The
predominant minerals in casein were sulfur (1,984.1 ± 3.2 mg/100 g),
calcium (987.2 ± 17.3 mg/100 g), and sodium (706.3 ± 10.9 mg/100 g),
and their levels were higher than those in FDSTR. Sulfur
(442.0 ± 46.2 mg/100 g), iron (250.1 ± 1.8 mg/100 g), and sodium
(212.6 ± 1.8 mg/100 g) were the major minerals in hemoglobin, and
their levels were lower than those in FDSTR, except for iron. The sul-
fur and potassium content in RPIs were 482.5 ± 89.5–782.1 ± 47.2
and 18.4 ± 3.1–70.3 ± 0.6 mg/100 g, respectively, which were
significantly lower than those in FDSTR. The sodium (70.1 ± 0.6–
166.9 ± 1.1 mg/100 g) and phosphorus (482.5–782.1 mg/100 g)
levels in the RPIs were also significantly lower than those in
FDSTR (p < .05), while magnesium and calcium levels showed simi-
lar trends. However, the sodium content in the RPIs was lower
than those in crab (266.8 mg/100 g) (Gokoglu & Yerlikaya, 2003),
hoki (620.0 mg/100 g) (Gokoglu, Yerlikaya, & Cengiz, 2004), and
fish-based dishes (222.8 mg/100 g) (Martinez-Valverde, Periago,
Santaella, & Ros, 20 00). Phosphorus content in the RPIs was higher
than that in rainbow trout (337.8 mg/100 g) (Gokoglu et al., 20 04)
and European perch (215–230.0 mg /100 g) (Orban et al., 2007).
Overall, the total mineral content in RPI-1 (pH 11/4.5) and RPI-3 (pH
12/4.5) was significantly lower than those in RPI-2 (pH 11/5.5) and
RPI-4 (pH 12/5.5) (p < .05). Acid precipitation at pH 4.5 during ISP
was found to be effective for removing minerals (potassium, sodium,
sulfur, and phosphorus) except for iron. Therefore, most minerals
in FDSTR migrated into the processed water during ISP (Lee et al.,
2017; Lee, Lee, et al., 2016).
3.1.3 | Color values
The color properties of FDSTR and the RPIs are shown in
Table 1. The L* value of FDSTR (59.2) was higher than those of the
RPIs (51.9 ± 0.4–55.0 ± 0.2), and the overall brightness of the RPIs
obtained after ISP was decreased (p < .05). RPI-1 and RPI-3 showed
lower a* values (4.1 ± 0.0 and 4.3 ± 0.1, respectively) than the other
RPIs. Additionally, the b* value and color difference (ΔE) showed
similar trends, confirming that acid precipitation at pH 4.5 during ISP
caused color fading. The whiteness of RPI-1 (52.0 ± 0.1) and RPI-3
(49.6 ± 0.4) was significantly higher than those of the other RPIs
(p < .05), but lower than that of FDSTR (53.6 ± 0.3) (p < .05). The RPIs
obtained after ISP were also fainter than the dark brown FDSTR. The
ACE inhibitory activity (%)
=
[
1−
Sample
228
−Control Blank
228
Control228 −Control Blank228 ]
×
100
|
1879
CHA et Al .
TABLE 1 Proximate composition and mineral content of roe protein isolates (RPIs) recovered from skipjack tuna roe (STR) by isoelectric solubilization and precipitation process
FDSTR RPI −1 RP I−2 RPI−3 RPI−4 Casein Hb
Yield† (g) 26.7 10.9 10.3 13.0 12.2
Protein yield‡ (g) 19.0 7.8 7.9 9. 2 9.2
Moisture (%) 6.2 ± 0.9a5.7 ± 0. 3a4.5 ± 0.1bc 6.3 ± 0.1a4.9 ± 0.3b4.0 ± 0.2c2.0 ± 0.0d
Protein (%) 71.1 ± 0.7e71.4 ± 0.8e76.8 ± 0.3c70.8 ± 0.7e75.4 ± 0.4d85.5 ± 0.0b94.4 ± 0.4a
Lipid (%) 15.2 ± 1.3a15.6 ± 1.0a14.0 ± 2.7a14.7 ± 2.0a13.8 ± 2.5aND ND
Ash (%) 7.2 ± 0.1a3.6 ± 0.1b1.8 ± 0.6c3.3 ± 0.4b2.0 ± 0.1cND ND
Minerals (mg/100 g) K 1,097.4 ± 9.0a18.4 ± 3.1e70.3 ± 0.6c25.8 ± 2.9e45.2 ± 2.1d912.0 ± 12.3b71.4 ± 3.0c
Na 615.2 ± 1.4ab 70.1 ± 0.6b113.9 ± 3.2b147.0 ± 2.1b166.9 ± 1.1b706.3 ± 10.9a212.6 ± 1.8b
Mg 66.9 ± 0.1a1.7 ± 0.1e7.1 ± 0.1c2.3 ± 0.1d7.6 ± 0.2bND ND
Zn 39.3 ± 0.3c8.3 ± 0.0d46.5 ± 0.5b7.6 ± 0.1d51.4 ± 0.9aND ND
Ca 36.3 ± 0.2b4.8 ± 0.1d15.6 ± 0.3cd 7.8 ± 0.2d15.5 ± 0.2cd 987.2 ± 17.3a21.2 ± 0. 2c
Fe 9.2 ± 0.2de 10.8 ± 0.2d16.4 ± 0.3b12 .1 ± 0.2c16.4 ± 0.2b4.8 ± 0.0 f250.1 ± 1.8a
P247.9 ± 1.5a111.9 ± 0.6e115.5 ± 1.0 d120.3 ± 1.0c131.5 ± 1.4b34.7 ± 0.4f29.1 ± 0.3g
S974.3 ± 57.7b482.5 ± 89.5ef 683.7 ± 83.6cd 573.7 ± 72.1de 782.1 ± 47.2c1984.1 ± 3.2a442.0 ± 46.2f
Color values L* 59.2 ± 0.3a55.0 ± 0.2b52.2 ± 0.5c52.6 ± 0.5c51.9 ± 0.4c
a* 6.8 ± 0.1a4.1 ± 0.0 e5.3 ± 0.1b4.3 ± 0.1d5.1 ± 0.1c
b* 21.2 ± 0.0a16.2 ± 0.2d17.2 ± 0.2b16. 8 ± 0.2c17.3 ± 0.1b
ΔE 43.5 ± 0.3d44.7 ± 0.3c48.0 ± 0.4a47.3 ± 0.4b48.3 ± 0.3a
Whiteness 53.6 ± 0. 3a52.0 ± 0.1b48.9 ± 0.4d49.6 ± 0.4c48.6 ± 0.3d
Note: Value s are mean ± SD of triplicate determinations. Means with different letters within the s ame row are signific antly different at p < .05 by Duncan's multiple range test.
FDSTR, freeze-dried skipjack tuna roe; Hb, hemoglobin. ND: not determined; RPI-1 and RPI-2, roe protein isolate adjusted to pH 4.5 and 5.5, respectively, after alkaline solubilization at pH 11; RPI-3 and
RPI-4, roe protein isolate adjusted to pH 4.5 and 5.5, respectively, after alkaline solubilization at pH 12.
†Yield is weight (g) of each sample obtained from 100 g of raw STR .
‡Protein yield (g) = yield × protein (%).
1880
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CHA et Al.
differences in color values may have resulted from the fractionation
effect of the pigment induced by ISP. The relatively low L* and high
a* values indic ate that the product was brown. The color and white-
ness of the fish protein isolates may be partially affected by the pres-
ence of connective tissues, which increase brightness, whereas lipid
retention can affect yellowness. Additionally, the yellow-brownish
color of the product may be attributed to the deposition of heme
proteins, which may affect redness, or to the denaturation and oxi-
dation of hemoglobin (K ristinsson & Rasco, 2000).
3.1.4 | Total amino acids
The total amino acid composition (g/100 g protein, %) of FDSTR ,
RPIs, and positive controls is shown in Table 2. The protein content
of all samples ranged from 75.8% to 96.3% based on the dry weight.
The RPIs contained high levels of glutamic acid, aspartic acid, leu-
cine, and lysine, which accounted for 12.2%–12.8%, 8.8%–9.0%,
9.6%–9.8%, and 8.4%–8.8% of the total amino acids, respectively.
Intarasirisawat, Benjakul, and Visessanguan (2011) reported that the
major nonessential amino acids (NEAA) in defatted tuna roes were
glutamic acid and aspartic acid, while leucine and lysine were the
predominant essential amino acids (EAA), which agrees with our re-
sults. The lysine content in the RPIs was higher than that in Channa
striatus (6.94%) and Lates calcarifer (6.86%) (Narsing Rao et al., 2012).
The EAAs in the RPIs ranged from 52.9% to 53.8%, which was higher
than that in FDSTR (50.1%). The EA A/NEAA ratio in the RPIs ranged
from 1.12 to 1.17, which was higher than that in casein (0.92) and
slightly lower than that in hemoglobin (1.33). The protein nutritional
value of the RPIs was improved by 12%–17% compared with that
of FDSTR because of the decrease in NEAA content during ISP.
The amino acid compositions of FDSTR and the RPIs were similar
to those of mullet, cod, pollock, chinook salmon roe, and yellow fin
tuna roe isolates (Bledsoe et al., 2003; Lee, Park, et al., 2016). The
hydrophobic amino acid content in the RPIs and positive controls
was 45.0%–45.9% and 44.6%–49.5%, respectively. The significantly
lower content of proline and glycine in the RPIs compared with in
FDSTR may be because of the precipitation of collagenous mate-
rial (alkaline insolubles) during alkali solubilization (Lee, Lee, et al.,
2016; Yoon, Kim, & Heu, 2018). However, the hydrophobic amino
acid content , apart from glycine and proline, in the RPIs was signifi-
cantly higher than that in FDSTR ( p < .05). Overall, the EAA content
in FDSTR and the RPIs were higher than those in casein (47.9%) but
lower than those in hemoglobin (57.1%). Therefore, the concen-
trates and isolates from fish roe differed in composition based on
the habitat environment, although fish roe proteins showed excel-
lent nutritional at tributes (Lee, Lee, et al., 2016; Park et al., 2016).
Water-holding or fat-binding capacities are functional features that
are closely related to texture by interactions between water, oil, or
other food components. These functional properties are affected by
the degree of exposure of hydrophilic (Asp, Glu, Lys, and Arg) and
hydrophobic (Tyr, Ile, Val and Phe etc.) amino acid residues within
the protein (Jellouli et al., 2011).
3.2 | SDS–PAGE
The SDS-PAGE profiles of the STR and RPIs are shown in Figure 1.
The major protein fractions of STR were observed at 100–75, 50–37,
25, 15, and 15–10 kDa. A dense protein band at 103 kDa was de-
tected in the soluble and insoluble fractions of Alaska walleye pol-
lock roe (Bechtel, Chantarachoti, Oliveira, & Sathivel, 20 07), and
four dense protein bands of 40–100-kDa were observed in spray-
dried catfish protein powder (Sathivel et al., 2009). In the RPI protein
bands, the 42-kDa protein band of STR was absent and the 75–100-
kDa bands were weaker than those of STR. The disappearance of
the 42-kDa protein band indicates that this protein was not read-
ily soluble during alkaline solubilization. The protein bands of acid-
precipitated RPIs af ter alkaline solubilization were 75–100, 25, and
10 –15 kDa.
Al-Holy and Rasco (2006) predicted that three protein bands in
salmon caviar, with molecular weights of 96, 20, and 10 kDa, were vi-
tellin-like protein, lysozyme, and phosvitin, respectively. Additionally,
a protein with a molecular weight of 27 kDa in Sturgeon's caviar may
be an ovomucoid, which is generally a glycoprotein with a MW of
27–29 kDa (Al-Holy & Rasco, 2006). Intarasirisawat et al. (2011) re-
ported that protein bands of 32.5, 29, and 32.5 kDa were detected
in skipjack, tongol, and bonito roes, respectively, which were ovomu-
coid or phosvitin.
3.3 | Food functionalities
3.3.1 | Buffer capacity
Buffer capacit y is defined as the volume (ml) or amount (mmol) of
HCl or NaOH required to induce a unit change in pH. The buffer
capacities of the RPIs and positive controls are shown in Figure 2a.
Over a pH range of 2–6, RPI-4 required 23.2 mM HCl (average) to
cause unit pH change, which is higher than that required by the other
RPIs (19.9–22.8 mM HCl). In contrast, in the pH range of 6–12, RPI-1
required a higher alkali concentration (63.9 mM NaOH) to cause a
unit pH change compared with the other RPIs (39.9–51.8 mM NaOH).
In comparison, casein and hemoglobin required 18.1 and 26.3 mM
HCl (pH range 2–6) and 33.8 and 21.8 mM NaOH (pH range 6–12),
respectively, to cause a unit pH change. These values were signifi-
cantly lower than those required for the RPIs (p < .05). Therefore, the
buffer capacities of the RPIs and casein were higher under alkaline
conditions than under acidic conditions, showing a similar trend as
roe concentrates of yellowfin tuna (Park et al., 2016). Chalamaiah,
Balaswamy, Narsing Rao, Prabhakara Rao, and Jyothirmayi (2013)
reported that dehydrated egg protein concentrate required an aver-
age of 0.65 mmol HCl and 1.22 mmol NaOH/g to induce a unit pH
change under both acidic and alkaline conditions, which was higher
than that for defatted egg protein concentrate. The lower buffer ca-
pacity of the dehydrated egg protein concentrate may be related to
the presence of fatty components, which require more acid or alkali
to induce a unit pH change.
|
1881
CHA et Al .
Thus, RPIs recovered from STR were superior to those from
other species and may resist changes in external pH. These RPIs with
excellent buffering capacity can be used to develop protein-fortified
food components under different processing environments.
3.3.2 | WHC
Water-holding capacity depends on protein–water interactions that
affect protein function. Mohamed, Xia, Issoufou, and Qixing (2012)
reported that interactions between proteins and water or oil are
important in food systems, as they affect the flavor and texture of
food. WHC s (g per g protein) with or without pH-shift treatment (pH
2.0–12.0) of the RPIs and positive controls are shown in Figure 2b. In
the control samples, the WHCs of RPI-1 and RPI-3 (5.1 and 5.4 g/g
protein, respectively) were higher than those of RPI-2, RPI-4, and ca-
sein (3.3–3.8 g/g protein). After alkaline solubilization, the WHCs of
RPI-1 and RPI-3 (recovered at pH 4.5) were significantly higher than
those of RPI-2 and RPI-4 (recovered at pH 5.5). This may be because
of the fractionation effect between the recovered RPIs during ISP.
The WHC of solubilized hemoglobin (0.9 g/g protein) after pH-shift
treatment was significantly lower than those of the RPIs and casein
(p < .05).
The WHCs of the RPIs were 20.7 ± 0.0–36.7 ± 0.9 g/g protein
at pH 2 and 19.8 ± 8.8–40.6 ± 0.0 g/g protein at pH 10–12. Among
the RPIs, RPI-2 showed a relatively high WHC. In contrast, at pH
4–8, the WHC of the RPIs (3–6 g/g protein) was similar to that of the
control. Thus, pH-shift treatment significantly improved the WHC of
RPIs at pH values other than pH 4–8, at which WHC was minimized
because of increased electrostatic repulsion. Mohamed et al. (2012)
Amino acid FDSTR
RPIs
Casein HbRP I−1 R PI−2 RPI−3 RPI−4
Protein content
(%)†
75.8 75.7 80.4 75.6 79. 3 8 9.1 96.3
Thr 5.1a4.8bc 4.9b4.8bc 4.9b3 .9d4.7c
Val‡ 6.2d6.3cd 6.1e6.4c6.4c7. 3 b10.2a
Met‡ 2.8b3.1a3 .1a3.1 a3.2a1.3c0.0d
ILe‡ 5.1d6.3ab 6.2b6.4a6.4a5.7c0.8e
Leu‡ 8.3e9.8 b9.7bc 9. 6c9.8 b9.1d13.3a
Phe‡ 4.1d4.8bc 4.7c4.8bc 4.9b5.0b7. 6 a
His 3.5b2.9c3.0c2.9c3.0c2.9c6.4a
Lys 8.4d8.7b8.6c8.4d8.8b8.3d10. 3a
Arg 6.6a6.6a6.6a6.4b6.5ab 4.4c3.8d
EAAs (%) 50.1 53.3 52.9 52.9 53.8 47.9 57. 1
Asp 9.0b8.8c8.8c9. 0 b9.0b8.2d11.2a
Ser 6.0a5.9a5.7b5.6b5.4c4.0e4.4d
Glu 13.2b12.2e12.4d12.8c12.5d22 .1a9. 3f
Pro‡ 5.8b4.6c4.2d4.6c4.1de 10.0a4.0e
Gly‡ 4.7a3.6b3.6b3.7b3.7b2.4c4.6a
Ala‡ 6.8c7.4 b7. 4 b7. 2 bc 7. 2bc 3.8d9.0 a
Cys 1.1a0.9b1.2a0.9b0.8b0.5c0.1d
Tyr 3.4b3.3b3.8a3.4b3.4b1.2c0.3d
NEAAs (%) 49.9 4 6.7 47. 1 47. 1 46.2 52.1 42 .9
Tot al (%) 100.0 100.0 100.0 100.0 100.0 100.0 100.0
EAAs/NEAAs 1.00 1.14 1.12 1.12 1 .17 0.92 1.33
HAAs (%) 43.8 45.9 45.0 45.8 45.7 44.6 4 9.5
Note: Value s with dif ferent letters within the same row are significantly different at p < .05 by
Duncan's multiple range test. Data are means of duplicate determination.
Abbreviations: E AAs, essential amino acids; HA As, hydrophobic amino acids. FDSTR , freeze-dried
skipjack tuna roe; Hb, haemoglobin; NE AAs, nonessential amino acids; RPI-1 and RPI-2, roe protein
isolates adjusted to pH 4.5 and 5.5, respectively, after alkaline solubilization at pH 11; RPI-3 and
RPI-4, roe protein isolates adjusted to pH 4.5 and 5.5, respectively, after alkaline solubilization at
pH 12.
†Based on dry basis .
‡Hydrophobic amino acid.
TABLE 2 Amino acid composition
(g/100 g protein) of protein isolates
recovered from skipjack tuna roe (RPIs)
using isoelectric solubilization and
precipitation process
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CHA et Al.
reported that WHCs of protein isolates from tilapia were 2.63–
2.51 g/g protein, which are lower than those of the RPIs found in this
study. Azadian, Nasab, and Abedi (2012) reported that the WHC of
minced fish reached a minimum near the isoelectric point (pH 4–6).
Chalamaiah et al. (2013) reported that the lack of polar amino groups
on the surface of protein molecules reduced the WHC because
these polar groups are responsible for protein–water interactions.
The WHC of RPIs from STR was superior to that of yellow fin tuna
roe (Park et al., 2016), Labeo rohita fish egg (Balaswamy, Jyothirmayi,
& Rao, 2007), and mrigal egg protein concentrates (Chalamaiah et al.,
2013). Particularly, our results showed that structural changes in
proteins were actively induced at extreme pH (pH 2 and pH 10–12),
which increased the WHC by more than 5–8-fold compared with the
controls. This is because acidic and alkaline pH shif ts cause protein
conformational changes in the RPIs, allowing hydrophilic amino acids
to easily access the surrounding water and increase the WHC.
3.3.3 | Protein solubility
Solubility is an important functional propert y of proteins, as it af-
fects rheological, hydrodynamic, and sur face activity character-
istics. Protein solubility is also important in many protein-based
formulations (Park et al., 2016). The solubilities (%) of the RPIs with
and without pH-shift treatment are shown in Figure 2c. The solu-
bilities of the control RPIs (1.7 ± 0.0–6.7 ± 0.3%) were significantly
lower (p < .05) than that of hemoglobin (71.3 ± 16.8%). However,
the solubility of casein was the lowest (0.4%), and the protein was
nearly insoluble in a 1% dispersion. The solubility of pH-shifted RPIs
FIGURE 1 SDS-PAGE patterns of protein isolates isolated
from skipjack tuna roe (RPIs) using isoelectric solubilization and
precipitation. M, protein maker; STR, skipjack tuna roe; RPI-1, RPI-
2, RPI-3, and RPI-4, roe protein isolates adjusted to pH 4.5 and 5.5
after alkaline solubilization at pH 11 and 12
FIGURE 2 Buffer capacity (a), water-holding capacity (b), and
solubility (c) of protein isolates recovered from skipjack tuna roe
(RPIs) using isoelectric solubilization and precipitation without
(control) and with pH-shift treatment. Values are expressed as the
mean ± SD of triplicate determinations. Means with different letters
within the sample and pH are significantly different at p < .05 by
Duncan's multiple range test. Hb, hemoglobin
|
1883
CHA et Al .
increased significantly at pH 2 (17.8 ± 0.3–33.1 ± 12.4%) compared
with the controls. At pH 12, their solubilities ranged from 69.1 ± 25.8
to 92.9 ± 18.7%, indicating a higher rate of solubilit y increase at al-
kaline pH. After alkaline solubilization, the solubilities of RPI-1 and
RPI-3 after acid precipitation at pH 4.5 were significantly higher
than those of RPI-2 and RPI-4 recovered at pH 5.5. However, the
solubility of hemoglobin (82.3 ± 4.0–99.9 ± 17.9%) was not affected
by pH-shift treatment (pH 2–12). Casein at pH 2 (42.7 ± 4.9%) and
pH 7–12 (83.8 ± 7.2–90.3 ± 9.0%) showed significantly higher sol-
ubility than the controls. The RPIs and casein showed the lowest
solubilities near the isoelectric point of pH 4–6 because of acid- and
alkali-limited protein solubilization. These results indicate that ex-
treme pH changes (such as pH 2 and 12) affected protein solubil-
ity by exposing more charged and polar groups to the surrounding
water (Kristinsson & Rasco, 2000). The pH-dependent solubility of
proteins is important in food functional properties and food system
applications, particularly when the pH is below 4 or over 7, and is af-
fected by protein–protein and protein–solvent interactions and the
surface hydrophilic–hydrophobic balance (Gbogouri, Linder, Fanni, &
Parmentier, 2004). The high solubility of fish proteins is important
in food applications because it affects other functional properties
such as foam and emulsification characteristics (Kristinsson & Rasco,
2000).
3.3.4 | FC and FS
Dispersions (1%, w/v) of the RPIs and positive controls were pre-
pared in DDW to measure FC and FS. Before centrifugation, the FCs
of 1%-dispersed RPI-1 and RPI-3 (125.3 ± 2.2–128.6 ± 3.4%) were
lower than that of hemoglobin (138.9 ± 18.3%), although the dif-
ference was not significant (p > .05), while RPI-2, RPI-4, and casein
showed no FC. The FSs of RPI-1, RPI-3, and hemoglobin were rela-
tively st able at 81.1 ± 8.0–88.0 ± 4.0±% for 60 min (Table 3). The FC
and FS of the supernatant from dispersed samples af ter centrifuga-
tion with and without pH-shift treatment are shown in Table 3.
The FCs of RPI-1, RPI-3, and casein increased by 147.0 ± 4.6%,
135.8 ± 4.8%, and 109.4 ± 11.1%, respectively, compared with
the dispersed samples; however, RPI-2 and RPI-4 showed no
foaming. The FC of hemoglobin decreased (138.9 ± 18.3% to
120.9 ± 11.9%) after centrifugation. During pH-shift treatment
(pH 2–12), the FCs of RPI-1 and RPI-3 was significantly increased
at pH 2 (191.3 ± 5.8% and 159.3 ± 6.3% , respectively) and pH
10–12 (139.9 ± 0.9–208.7 ± 7.6%) compared with the controls
(147.0 ± 4.6% and 135.8 ± 4.8%, respectively) (p < .05); however,
FCs at pH 4–8 (128.8 ± 5.6–154.0 ± 2.8%) showed no significant
difference (p > .05). The FCs of RPI-2 and RPI-4 were significantly
increased at pH 2 (185.1 ± 10.8% and 168.7 ± 4.2%, respectively)
and pH 7–12 (118.6 ± 0.4–186.7 ± 13.0%) compared with the
controls, whereas no FC (100.0%) was observed at pH 4–6. The
maximum FC of the RPIs (183.4 ± 25.0–208.7 ± 7.6%) and casein
(184.8 ± 65.0%) were observed at pH 12 and 8, respectively. At
pH 2–12, hemoglobin showed 115.1 ± 8.8–140.0 ± 22.5% FC;
however, pH-shift treatment did not induce a significant differ-
ence (p > .05). Regardless of pH-shift treatment, the FCs of RPI-1
and RPI-3 were superior to those of RPI-2, RPI-4, casein, and he-
moglobin. Proteins must rapidly migrate to the air–water inter-
face, unfold, and rearrange at the interface to show good foam
performance. Mutilangi, Panyam, and Kilara (1996) suggested that
the foaming ability of proteins could be improved by increasing
flexibility, which exposes a larger number of hydrophobic residues
and reduces surface tension. The FS of hemoglobin (81.4 ± 3.4%)
was maintained for up to 60 min in control 2 (without pH-shift),
but the RPIs and casein showed no FS ( Table 3). The FSs of RPIs
(59.1 ± 4.3–77.2 ± 8.0%) were more stable at pH 7–12 than at pH
2–6 un til 60 min af ter wh ipp ing . Add iti o nal ly, RPI -1 and RP I-3 we re
more stable than RPI-2 and RPI-4, and the foam characteristics of
RPI-1 were superior to those of RPI-3. All samples showed a low
foaming capacity at pH 4–6 because of their low WHC and solu-
bility (Figure 2) near the isoelectric point, and FS depends on the
degree of protein–water and protein–protein interactions within
the matrix (Mutilangi et al., 1996).
3.3.5 | EAI and ESI
The oil-in-water EAI and ESI were determined to evaluate the ability
of the samples to emulsify in foods such as soups, sauces, confec-
tionery breads, and dairy product s. The EAI (m2/g protein) and ESI
(min) of the RPIs and positive controls are shown in Table 3. The
EAI of the dispersed RPIs (control 1) was 1.8 ± 0.1–3.9 ± 0.5 m2/g
protein, and RPI-3 showed the highest EAI (3.9 ± 0.5 m2/g protein).
However, the EAI of the RPIs was significantly lower than that of
hemoglobin (18.4 ± 2.1 m2/g protein) (p < .05). After centrifugation
(control 2), the EAI of the supernatant of the dispersed samples was
generally increased, except for that of RPI-4 (2.3 ± 0.5 m2/g protein).
The EAIs of RPI-1 (14.7 ± 1.1), RPI-3 (15.9 ± 2.1), and casein (7.0 ± 0.9)
were notably higher than those of the dispersions before centrifuga-
tion (control 1).
Th e ES I of he mo g l ob i n (c o n t r o l 1) wi t h an EAI of 18 . 4 ± 2.1 m2/g
protein was 20.0 ± 6.4 min, while those of the RPIs and casein
were undetectable. Af ter centrifugation (control 2), there was no
significant difference in the ESIs (30.7 ± 7.0–34.5 ± 4.6 min) of
RPI-1, RPI-3, and casein (p > .05), although their ESIs were signifi-
cantly higher than that of hemoglobin (20.1 ± 3.7 min) (p < .05).
The increase in EAI and ESI after centrifugation may be related
to the presence of insoluble par ticles in the dispersion that in-
terfere with emulsion layer formation. EAI assessment of RPIs
and positive controls after pH-shift (pH 2–12) treatment showed
that RPI-2, RPI-4, casein, and hemoglobin had the lowest EAIs
(2.3 ± 0.5–12.9 ± 1.7 m2/g protein) at pH 4 and RPI-1 and RPI-3
had the lowest EAIs (13.3 ± 0.8 and 9.2 ± 0.4 m2/g protein, re-
spectively) at pH 6 (Table 3). These results suggest that the FC
and emulsifying activity are closely related to protein solubility
in food, which was minimal in the RPIs and positive controls at
pH 4–6. At pH 2 and 7–12, the EAIs of all samples, except for
1884
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CHA et Al.
hemoglobin, were higher than that of control 2. At all pH ranges
except for pH 2, the EAIs of RPI-1 and RPI-3 were higher than
those of RPI-2 and RPI-4. The EAI of the RPIs showed no sig-
nificant differences (p > .05) at pH 2 and 7–12; however, the
emulsifying activity significantly increased with pH (p < .05). The
emulsion stability did not increase in proportion to the increase
in emulsif ying activity with pH. The large deviation in emulsion
stability after pH-shif t treatment may have occurred bec ause
of the nonuniformit y of emulsified particles. Mutilangi et al.
(1996) reported that a higher content of high-MW peptides or
TABLE 3 Foaming capacit y (FC, %), foam stability (FS, %, 60 min), emulsifying activity index (EAI, m2/g of protein), and emulsion stability
(ESI, min) with pH-shift of protein isolates recovered from skipjack tuna roe (RPIs)
Sample
RPI-1 (pH
11/4.5) RPI-2 (pH 11/5.5) RPI-3 (pH 12/4.5) RPI-4 (pH 12/5.5) Casein Hb
FC (%) Control 1 128.6 ± 3.4A100.0 ± 0.0B125.3 ± 2.2 A100.0 ± 0.0B100.0 ± 0.0B138.9 ± 18.3A
Control 2 147.0 ± 4.6Ac 100.0 ± 0.0Cd 135.8 ± 4.8Ac 100.0 ± 0.0Cd 109.4 ± 11.1BCb 120.9 ± 11.9Ba
pH 2 191.3 ± 5.8Ab 185.1 ± 10.8ABa 159.5 ± 6.3CDb 168.7 ± 4.2BCa 140.3 ± 23.6Dab 115.1 ± 8.8Ea
pH 4 148.0 ± 9.9Ac 100.0 ± 0.0Dd 137.8 ± 3.8ABc 100.0 ± 0.0Dd 109.1 ± 10.8CDb 125. 2 ± 18.7BCa
pH 6 144.0 ± 6.8Ac 100.0 ± 0.0Cd 139.8 ± 2.9Ac 100.0 ± 0.0Cd 107.0 ± 8.4BCb 126.1 ± 26.5ABa
pH 7 128.8 ± 5.6ABd 118.6 ± 0.4Bc 145.8 ± 8.4ABc 114.2 ± 5.6Bcd 158.6 ± 37.0Aab 132.0 ± 17.1ABa
pH 8 154.0 ± 2. 8ABc 131.8 ± 0.6ABbc 145.3 ± 3.9ABc 120.5 ± 5.8Bb 184.8 ± 65.0Aa 139.8 ± 21.7ABa
pH 10 197.6 ± 9.5Aab 128.2 ± 0.8Bbc 139.9 ± 0.9Bc 125.4 ± 2.2Bb 135.6 ± 12.8Bab 140.0 ± 22.5Ba
pH 12 208.7 ± 7.6Aa 186.7 ± 13.0Aa 20 0.3 ± 14.8Aa 183.4 ± 25.0Aa 130.5 ± 9.7Bab 129.7 ± 7.8Ba
FS (%,
60 min)
Control 1 88.0 ± 4.0A- 86.1 ± 7.3A- - 81.1 ± 8.0A
Control 2 - - - - - 81.4 ± 3.4a
pH 2 - 28.4 ± 1.8Bc -56.4 ± 4.9Ab 41.3 ± 9.8ABb 52.4 ± 12.2Ac
pH 4 - - - - 73.6 ± 3.1Ba 52.9 ± 7.5Ac
pH 6 43.3 ± 6.8Bb -- -76.9 ± 0.7Aa 76.0 ± 11.5Aab
pH 7 68.9 ± 8.0Aa - 71.8 ± 3.0Aa - 74.4 ± 4.5Aa 74.5 ± 6.6Aabc
pH 8 77.2 ± 8.0Aa - 73.7 ± 0.2Aa 65.4 ± 7.3Aab 43.1 ± 18.9Bb 63.0 ± 5.9Aabc
pH 10 73.6 ± 6.3ABa 78.4 ± 6.2Aa 75.0 ± 2.6Aa 70.4 ± 4.5ABa 62.4 ± 1.7ABa 56.1 ± 20.7Bbc
pH 12 69.2 ± 2.3Aa 59.1 ± 4.3Ab 60.5 ± 7.9Ab 61.7 ± 3.5Aab 69.1 ± 9.1Aa 57.1 ± 16.6Abc
EAI (m2/g
protein)
Control 1 3.1 ± 0.1BC 1.8 ± 0.1CD 3.9 ± 0.5B3.4 ± 0.2BC 0.4 ± 0.3D18.4 ± 2.1A
Control 2 14.7 ± 1.1Be 3.9 ± 0.9De 15.9 ± 2.1Be 2.3 ± 0.5Dd 7.0 ± 0.9Ce 19.1 ± 1.9Aab
pH 2 15.5 ± 1.2Be 20.3 ± 1.4Ac 14.2 ± 0.9Be 19.2 ± 3.9Ac 11.9 ± 1.1Bcd 20.1 ± 1 2.3Aab
pH 4 14.8 ± 0.9ABe 5.5 ± 0.1Ce 15.6 ± 2.9Ae 5.6 ± 0.3Cd 2.3 ± 0.5Df 12.9 ± 1.7Bc
pH 6 13.3 ± 0.8Ce 9.8 ± 2.5Dd 9.2 ± 0.4Df 23.8 ± 1.2Ac 9.1 ± 1.7Dde 18.1 ± 1.3Bab
pH 7 21.0 ± 0.2 ABd 18.8 ± 1.8Bc 20.5 ± 0.4Bd 23.7 ± 1.7Ac 13.8 ± 2.6Cc 18.4 ± 1.8Bab
pH 8 27.3 ± 0.9Ac 22.2 ± 1.0Bc 26.5 ± 1.7Ac 26.2 ± 1.3Ac 12. 8 ± 1.3Dc 18. 3 ± 0.5Cab
pH 10 33.6 ± 2.4Ab 36. 5 ± 0.7Ab 33.4 ± 2.7Ab 34.2 ± 5.4Ab 23.7 ± 2.7Bb 16.1 ± 4.4Cbc
pH 12 54.2 ± 4.8Aa 54.0 ± 4.1Aa 55.0 ± 0.6Aa 53.8 ± 9.2Aa 36.8 ± 2.2Ba 20.8 ± 1.5Ca
ESI (min) Control 1 - - - - 20.0 ± 6.4C
Control 2 30.7 ± 7.0Abc - 34.5 ± 4.6Ab - 31.8 ± 3.6Ab 20.1 ± 3.7B ab
pH 2 15.3 ± 1.5ABc 16.9 ± 1.7ABb 17.0 ± 1.1ABc 20.2 ± 7.9ABc 13.2 ± 2.3Bc 21.2 ± 2.0Aa
pH 4 16.7 ± 1.7Cc 56.2 ± 15.1Ab 17.2 ± 1.7Cc 29.1 ± 4.0BCc 37.6 ± 13.9Bb 18.5 ± 2.1Cab
pH 6 31.2 ± 3.7Bbc 61.0 ± 18.1Ab 55.3 ± 0.0Ab 53.1 ± 17.4Ab 23.5 ± 4.3Bbc 18.6 ± 1.2B ab
pH 7 56.2 ± 4.5Bab 58.2 ± 8.1ABb 37.3 ± 4.3Cab 67.6 ± 8.8Aab 14.8 ± 1.7Dc 18.4 ± 2.7Dab
pH 8 25.2 ± 2.9Bbc 70.5 ± 11.9Ab 23.6 ± 1.8Bab 63.4 ± 1.3Aab 16.0 ± 2.3Bc 18.3 ± 1.8Bab
pH 10 27.5 ± 6.3CDbc 38.2 ± 5.5Bb 29.8 ± 3.3BCb 53.5 ± 7.3Ab 26.1 ± 6.9CDbc 18.0 ± 1.2Dab
pH 12 83.4 ± 49.2ABa 127.2 ± 74.8Aa 114.3 ± 48.3Aa 83.8 ± 26.4ABa 62.7 ± 13.9ABa 16.6 ± 1.8Bb
Note: Controls 1 and 2 refer to the s amples before and after centrifugation of the 1% dispersions, respectively. Values represent the mean ± SD of
n = 3. Means with different capital letters within same row and small letters within the same column are significantly different at p < .05 by Duncan's
multiple range test.
-, not detected
|
1885
CHA et Al .
hydrophobic peptides contributes to the stabilit y of emulsions.
Additionally, low-MW peptides exhibit good emulsification prop-
erties, although they are not amphipathic. The emulsifying prop-
erties of proteins are largely af fected by solubility, molecular size,
surface hydrophobicity, net charge, steric hindrance, and molecu-
lar flexibility (Gbogouri et al., 200 4).
3.4 | Antioxidative and antihypertensive activity
Table 4 shows the ABTS+ radical scavenging activity (IC50, μg /ml ),
tyrosinase inhibitory activity (%), and ACE inhibitory activity (%)
of RPI-1. The ABTS+ radical scavenging assay can be performed
on both lipophilic and hydrophilic compounds and has been
widely used as an antioxidant activity assay. The ABTS+ radical
scavenging activity (IC50) of RPI-1 (1.5 ± 0.1 mg protein/ml) was
102.7 ± 1.0 μg/ml, which was better than those of alcalase and
Protamex hydrolysates from shrimp processing byproducts (160
and 170 µg/ml, respectively) (Kim et al., 2016). UV irradiation can
generate reactive oxygen species that influence skin pigmenta-
tion. Tyrosinase inhibitors have recently gained attention in the
medical and cosmetic industries because of their association with
hyper-pigmentation (Choi, Kim, & Lee, 2011). The tyrosinase in-
hibitory activity of RPI-1 was 13.5 ± 1.7%, and some whitening
effects were predicted. Choi et al. (2011) reported a tyrosinase
inhibitory activity of 31% for tuna cooking drip, but this value
increased with the dose of gamma irradiation. Choi et al. (2017)
reported that anchovy muscles not subjected to subcritical water
hydrolysis inhibited t yrosinase activity by 14.65%. In these ex-
perimental results and reports, tyrosinase inhibitory activity was
also observed in proteinous materials containing proteins or amino
acids, but its inhibitory activity was low. The inhibition of ACE, a
key enzyme that regulates blood pressure, has been recognized
as an effective therapy for treating hypertension. The ACE inhib-
itory activity of RPI-1 was 44.0 ± 4.9%. The current search for
natural ACE-inhibiting peptides has extended into seafood protein
sources, particularly seafood byproduct s. The hydrolysates of the
byproducts of skin (Ngo, Ryu, & Kim, 2014) and yellow sole frame
(Jung et al., 2006) showed 35%–86% ACE inhibitory activities,
which are consistent with our results. These results suggest that
RPIs from STR possess antioxidative and antihypertensive activi-
ties, which can be improved by enzymatic hydrolysis.
4 | CONCLUSION
The aims of this study were to investigate the physicochemical and
functional properties of protein isolates recovered from skipjack
tuna roe (STR) by ISP. The roe protein isolates (RPIs) were similar
or superior to the positive controls (casein and hemoglobin) and
other fish protein isolates in terms of buffering capacity, foaming
ability, and emulsifying ability. The RPIs were rich in proteins con-
taining essential amino acids and exhibited suitable functional char-
acteristics for supplementing surimi-based products and traditional
foods. They also showed in vitro antioxidant and antihypertensive
activities. This study suggests that high value-added produc ts can
be developed from fish roes, which are currently underutilized in the
seafood processing industry.
ACKNOWLEDGMENTS
This research was a par t of the projected titled “Development and
commercialization of traditional seafood products based on the
Korean coastal marine resources (2016),” funded by the Ministry of
Oceans and Fisheries, Korea.
CONFLICT OF INTEREST
The authors claim no conflict s of interest.
ETHICAL APPROVAL
This study did not involve human or animal testing.
ORCID
Min Soo Heu https://orcid.org/0000-0001-7609-0303
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How to cite this article: Cha JW, Yoon IS, Lee G-W, et al.
Food functionalities and bioactivities of protein isolates
recovered from skipjack tuna roe by isoelectric solubilization
and precipitation. Food Sci Nutr. 2020;8:1874–1887.
https ://doi.org/10.1002/fsn3.1470
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