Access to this full-text is provided by Wiley.
Content available from Food Science & Nutrition
This content is subject to copyright. Terms and conditions apply.
412
|
Food Sci Nutr. 2019;7:41 2–4 24.
www.foodscience-nutrition.com
1 | INTRODUCTION
According to statistics from the Food and Agriculture Organization
of the United Nations (FAO), the total fishery production in 2015
was about 200 million tons (FAO, 2016). As the fish production is
increasing each year, the discarding rate of fish processing by-
products also increases. Fish processing industr y generates a wide
variety of by- products such as roe, visceral, heads, skin, frames,
and scales in large quantities (Klomklao & Benjakul, 2016). Most of
these by- products are disposed as waste, without processing into
Received: 30 January 2018
|
Revised: 31 July 2018
|
Accepted: 5 August 2018
DOI: 10.1002/fsn3.793
ORIGINAL RESEARCH
Food functionality of protein isolates extracted from Yellowfin
Tuna (Thunnus albacares) roe using alkaline solubilization and
acid precipitation process
In Seong Yoon1 | Hyun Ji Lee2 | Sang In Kang1,3 | Sun Young Park1,3 |
Young Mi Kang1 | Jin-Soo Kim1,3 | Min Soo Heu1,2
This is an op en acces s article unde r the terms of the Cre ative Commons At tribu tion License, which permits use, distr ibutio n and reproduc tion in any medium,
provide d the original wor k is prope rly cite d.
© 2019 The Auth ors. Foo d Science & Nutritio n published by Wiley Period icals , Inc.
1Research Center for Industrial Development
of Seafoo d, Gyeong sang National University,
Tongyeong, Korea
2Department of Food and Nutrition/Institute
of Marine I ndust ry, Gyeong sang National
University, Jinju, Korea
3Depar tment of S eafood and
Aquaculture Science/Institute of Marine
Industry, Gyeongsang National University,
Tongyeong, Korea
Correspondence
Min Soo He u, Depa rtme nt of Food an d
Nutrit ion/Ins titute of Marine Industry,
Gyeongs ang National Universit y, Jinju,
Korea.
Email: minsheu@gun.ac.kr
Funding information
This res earch was suppo rted by the Nati onal
Resear ch Foundation of Ko rea (NRF) fund ed
by the Min istry of Educ ation, S cience and
Technology (Grant/Award Number: NRF-
2014R1A1 A4A0100 8620). This stu dy was a
part of t he project tit led “Develop ment and
commer cialization of t raditional se afood produc ts
based on t he Korean coast al marine reso urces,”
funde d by the Ministr y of Oceans and Fis heries,
Korea
Abstract
Four types of roe protein isolates (RPIs) were prepared through the alkaline solubili-
zation and acid precipitation (ASAP) process, and their functional properties and in
vitro bioactivities were evaluated. Higher buffer capacity in pH- shift range of 8–12
was found in RPI- 1 (pH 11/4.5), required average 94.5 mM NaOH than that of other
RPIs to change the pH by 1 unit. All the samples of 1% dispersion (w/v) showed the
lowest buffering capacity near the initial pH. The water- holding capacities (WHC) of
RPIs and casein as controls without pH- shift were in range of 3.7–4.0 g/g protein, and
there were no significant differences (p > 0.05). At pH 2 and 8–12 with pH- shift,
WHC and protein solubility of RPIs were significantly improved compared to those of
controls. Foaming capacities of RPI- 1 and RPI- 3 were 141.9% and 128.1%, respec-
tively, but those of RPI- 2 and RPI- 4 were not detected. The oil- in- water emulsifying
activity index of RPI- 1 and RPI- 3 was 10.0 and 8.3 m2/g protein, which was not sta-
tistically different from casein (7.0 m2/g), but lower than that of hemoglobin
(19.1 m2/g). Overall, RPIs, casein, and hemoglobin exhibited lower food functionality
at pH 4–6 near isoelectric points. Through the pH- shift treatment, the food function-
alities of RPIs were improved over the controls, especially in the pH 2 and pH 8–12
ranges. RPI also showed in vitro antioxidant and antihypertensive activities.
Therefore, it has been confirmed that RPI extracted from yellowfin tuna roe has high
utility as a protein- or food- functional- enhancing material or protein substitute re-
source for noodles, confectioner y, baking, and surimi- based products.
KEYWORDS
acid precipitation process, alkaline solubilization, fish roes, food functionality, protein isolate
|
413
YOON et al.
value- added product s either for industrial applications or for animal
and human consumption. However, these processing by- products
can be good protein resources (Lee, Park et al., 2016). The global de-
mand for proteins is increasing, and more food proteins are needed
from the source of conventional proteins as well as the new source
of protein. If we accept that all proteins will have nutritional value,
the value in the food industr y for both conventional and new pro-
tein sources is required to have enough food functional properties
to allow the protein to be accepted as a food ingredient (Azadian,
Nasab, & Abedi, 2012; Horax, Hettiarachchy, Kannan, & Chen, 2011;
Lee et al., 2017). Among fish by- products, fish roes are highly nutri-
tious material rich in essential fatty acids, minerals, and amino acids
(Heu et al., 2006; Park et al., 2016).
Yellowfin tuna (Thunnus albacares) is an epipelagic fish that in-
habits the mixed surface layer of the ocean above the thermocline
(Kunal, Kumar, Menezes, & Meena, 2013) and is used in the canned
tuna industry. It is canned with total amount of 55,135 metric tons,
which accounted for 66% of total canned products in Korea (MOF,
2016). Tuna roe, a by- product generated from fish processing (1.5%–
3.0% of total weight), is generally used in animal feed or pet food
preparation (Heu et al., 2006; Klomklao & Benjakul, 2016; Lee, Park
et al., 2016; Lee, Lee et al., 2016). Thus, processing methods for con-
verting the underutilized yellowfin tuna roe into more marketable
and acceptable forms such as ex tracts, concentrates, isolates, and
hydrolysates are required.
Protein modification is mostly realized by enzymatic, physical,
and chemical treatment with resultant changes in structural, phys-
icochemical, and functional properties (Gehring, Gigliotti, Moritz,
Tou, & Jaczynski, 2011; Mohamed, Xia, Issoufou, & Qixing, 2012).
Alkaline solubilization and acid precipitation (ASAP) process consists
of isolating the protein components of fish tissue by acid or alkali and
then precipitating all soluble proteins near their isoelectric points
(Chaijan, Panpipat, & Benjakul, 2010; Yongsawatdigul & Park, 200 4).
This proce ss allows for sele ctive pH- ind uced water solubi lity of tissue
proteins with concurrent separation of lipids and removal of materi-
als not intended for human consumption, such as bones, scales, and
skin (Gehring et al., 2011; Tahergorabi, Beamer, Matak, & Jaczynski,
2011). The pH- shift causes structural changes in the protein, leading
to partial unfolding of proteins, thus resulting in more exposure of
the functional groups (A zadian et al., 2012). The major advantages
of this process include economic feasibility, high recovery yield, and
improved functionality ( Arfat & Benjakul, 2013). Various methods
of protein isolate preparation have been reported for different pro-
tein sources, including fish protein (A zadian et al., 2012; Mohamed
et al., 2012), chicken (Tahergorabi et al., 2011) and beef (Mireles
DeWitt, Gomez, & James, 20 02) processing by- products, oilseeds
(Horax et al., 2011), and cereals (Paraman, Hettiarachchy, Schaefer,
& Beck, 20 07), based on the solubility behavior of their proteins. The
proteins recovered by this process have good func tionality, and in
some cases, better gelation properties than proteins recovered with
conventional surimi processing (Chaijan et al., 2010; Kristinsson,
Theodore, Demir, & Ingadottir, 2005). Protein isolates are the basic
functional components of various high- protein processed food
products and thus determine the textural and nutritional properties
of the foods (Mohamed, Zhu, Issoufou, & Fatmata, 2009; Mustafa,
Al- Wessali, Al- Basha, & Al- Amir, 1986). These properties contribute
to the quality and sensory attributes of food systems.
In our earlier study, preparation of protein concentrate (Lee, Park
et al., 2016; Yoon et al., 2018) and isolates (Lee, Lee et al., 2016) from
tuna roe were conducted and their chemical and nutritional proper-
ties were evaluated. Also, functionalities of roe protein concentrate
from tuna were examined (Park et al., 2016; Yoon et al., 2018). The
aims of this study were to evaluate functional properties and in vitro
antioxidant and antihypertensive activities of extracted roe protein
isolates from yellowfin tuna by ASAP process for their industrial ap-
plication as functional protein ingredients and supplements.
FIGURE1 Flowchart of preparation for protein isolates from
yellowfin tuna roe by alkaline solubilization and acid precipitation
process
414
|
YOON et al .
2 | MATERIALS AND METHODS
2.1 | Material
Yellowfin tuna (Thunnus albacares) roe was obtained from Dongwon
F&B Co., Ltd. (Changwon, Korea). Frozen roe was partially thawed
for 24 hr at 4°C and then cut into small pieces with an approximate
thickness of 1.5–3 cm and minced with food grinder (SFM- 555SP,
Shinil Industrial Co., Ltd., Seoul Korea). The minced roes were stored
frozen at - 20°C until the protein isolates were prepared.
2.2 | Chemicals
Sodium dodecyl sulfate (SDS) and glycine were purchased from Bio
Basic Inc., (Ontario, Canada). 2,2′- azino- bis(3- ethylbenzothiazoline
- 6- sulfonic acid) diammouium salt (ABTS), hippurly- his- leu acetate
salt (HHL), lung acetone powder from rabbit, mushroom tyrosinase,
bovine serum albumin (BSA), casein, hemoglobin, sodium carbon-
ate, sodium hydroxide, sodium L- tartrate, and potassium hydroxide
were purchased from Sigma- Aldrich Co., LLC. (St. Louis, MO, USA).
3,4- Dihydroxy- L- phenylalanine (L- DOPA) was purchased from Acros
Organics (New Jersey, USA). Copper (II) sulfate pentahydrate was
purchased from Yakuri Pure Chemicals Co., Ltd. (Kyoto, Japan).
Folin- Ciocalteu’s reagent was purchased Junsei Chemical Co., Ltd.
(Tokyo, Japan). Soybean oil was purchased from Ottogi Co., Ltd.
(Seoul, Korea). All reagents used analytical grade.
2.3 | Preparation of roe protein isolates (RPIs)
Four types of RPIs were prepared by the method of our previous
report (Lee, Lee et al., 2016), and the process is shown in Figure 1.
Briefly, the frozen minced roe was par tially thawed and homog-
enized with deionized distilled water (DDW) at a ratio of 1:6 (w/v)
using a homogenizer (POLYTRON® PT 120 0E, KINEMATICA AG,
Luzern, Switzerland). The homogenate were divided into two por-
tions and then adjusted to pH 11 and 12 with 2 N NaOH, respec-
tively. Once the desired pH was reached, the alkaline solubilization
was allowed to take place at 4°C for 1 hr, followed by centrifuga-
tion at 12,000 g and 4°C for 30 min using a refrigerator centrifuge
(Supra 22K , Hanil Science Industrial Co., Ltd., Incheon, Korea).
After centrifugation, two alkaline solubles (pH 11 and 12) in the
supernatant fraction were collected. To prepare the isolates from
alkaline solubles through acid precipitation, those of pH were re-
adjusted by addition of 2 N HCl to pH 4.5 and 5.5, respectively, a
value near the isoelectric point (pH 4- 6) of fish proteins (Chaijan et
al., 2010; Pérez-Mateos, Boyd, & Lanier, 2004; Yongsawatdigul &
Park, 20 04). The suspensions were centrifuge d at 12,000 g and 4°C
for 30 min. The precipitates by alkaline solubilization and acid pre-
cipitation (ASAP) processing were additionally washed with DDW
by centrifugation at 12,00 0 g and 4°C for 30 min to remove the
NaCl. After centrifugation, the washed roe protein isolates (RPIs)
were lyophilized and referred to 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), respectively. All
samples were stored at - 20°C until further experiments. Freeze-
dried concentrate (FDC) from minced roe of yellowfin tuna as a
sample control was prepared using freeze dryer (PVTFD50A, il-
Shinbiobase Co., Ltd., Dongducheon, Korea), and casein and hemo-
globin, which isolated from bovine milk and blood, respectively,
as positive control were used. All experimental results were com-
pared with the sample and positive controls.
2.4 | Buffer capacity
Buffer capacity was estimated by the method of Park et al. (2016) with
slightly modified method of Narsing Rao and Govardhana Rao (2010).
Briefly, sample (300 mg) was dispersed in 30 ml of DDW and known
volumes of 0.5 M NaOH or 0.5 M HCl were added and corresponding
changes in pH in both alkali and acid ranges were noted. The quantity
of alkali and acid added was plotted against pH. Buffer capacity in each
range was expressed as the mean value of mM of NaOH or HCl per
gram of protein required to bring about a change in pH by 1 unit.
2.5 | Water- holding capacity
The water- holding capacity (WHC) of sample was measured follow-
ing the method of Park et al. (2016). Sample (300 mg) was dispersed
in 30 ml of DDW. The mixture was stirred using a magnetic stirrer
at room temperature for 1 hr and then centrifuged at 12,000 g for
20 min at 4°C. Then, the supernatant was removed, and the weight
of the pellet was determined.
where C is protein content (%).
2.6 | Protein solubility
The protein solubility was measured according to the method of Park
et al. (2016). Sample (300 mg) was taken in 30 ml of DDW and the pH
of the mixture was adjusted to pH 2, 4, 6, 7, 8, 10, and 12 with 0.5 N
HCl or 0.5 N NaOH. The mixture was stirred at room temperature
(25 ± 2°C) for 30 min and centrifuged at 12,00 0 g for 20 min at 4°C.
Protein content in the supernatant was determined using the Lowr y’s
method (Lowry, Rosebrough, Farr, & Randall, 1951), using bovine serum
albumin as a standard. Total protein content in the sample was deter-
mined using the Lowr y’s method after solubilization of the 20 mg sam-
ple in 0.5 N NaOH. Protein solubility was calculated as follows:
2.7 | Foaming capacity and foam stability
Foaming capacity (FC) and foam stability (FS) of sample solution
(1%, w/v) was determined according to the method of Park et al.
(2016). Briefly, 10 ml of 1% (w/v) sample solution was homogenized
WHC(g
∕g protein) =
(Weight of pellet (g) −Weight of sample (g)
Weight of sample (g)×C
Solubility(%)
=
Protein content in supernatant
Total protein content in sample
×
100
|
415
YOON et al.
in a 25- ml volumetric cylinder with a homogenizer at a speed of
12,500 rpm for 1 min at room temperature. The sample was allowed
to stand for 1, 15, 30, and 60 min, respectively. FC and FS were then
calculated by using the following equations:
Foaming capacity (%) = VT/V0 × 10 0
Foam stability (%) = (Ft/Vt)/(VT/V0) × 100
where VT is total volume after whipping; V0 is the original total
volume before whipping; and Ft and Vt are total foam and total vol-
ume after leaving at room temperature for different times (t = 15,
30 and 60 min).
2.8 | Oil- in- water emulsifying activity index and
emulsion stability index
The emulsifying activity index (EAI) and emulsion stability index
(ESI) were determined according to the method of Park et al. (2016).
Soybean oil (1 ml) and 3 ml of 1% (w/v) sample were mixed and ho-
mogenized at a speed of 12,500 rpm for 1 min. Aliquots of the emul-
sion (50 μl) were pipetted from the bottom of the container at 0 and
10 min after homogenization and diluted to 5 ml using 0.1% sodium
dodecyl sulfate (SDS) solution. The absorbance of the diluted solu-
tion was measured at 50 0 nm (UV- 290 0, Hitachi, Kyoto, Japan).
The absorbance measured at once (A0 min) and 10 min (A10 min)
after emulsion formations was used to calculate the emulsifying ac-
tivity index (EAI) and the emulsion stability index (ESI) as follows:
where A = absorbance (500 nm), DF = dilution factor (100), l = path
length of cuvette (1 cm), φ = oil volume fraction (0.25), and C is pro-
tein concentration in aqueous phase (g/ml)
where A0 and A10 are the absorbance measured at once and af ter
10 min, ΔA = A0–A10, and Δt = 10 min, respectively.
2.9 | ABTS+ radical scavenging activity
The ABTS+ radical scavenging activity was determined by the
method of Yoon et al. (2017) with slightly modified method of
Binsan et al. (2008). The stock solutions included 7.4 mM ABTS
and 2.6 mM potassium persulfate. The working solution was pre-
pared by mixing the two stock solutions in equal quantities and
allowing them to react for 12 hr at room temperature in the dark.
The solution was then diluted by mixing 2 ml ABTS solution with
50 ml ethanol, in order to obtain an absorbance of 1.0 ± 0.02 units
at 734 nm using a spec trophotometer. Fresh ABTS+ ethanolic so-
lution was prepared for each assay. Sample (1 ml) was mixed with
3 ml of ABTS+ solution, and the mixture was left at room tempera-
ture for 30 min in the dark. The absorbance was then measured
at 734 nm using a spectrophotometer (UV- 2900, Hitachi, Kyoto,
Japan). The IC50 value was defined as the concentration required
for scavenging 50% of ABTS+ radical.
The absorbance measured immediately A734 as follows:ABTS+
radical scavenging activity (%) =
where control734 is the absorbance of same reaction system without
sample.
2.10 | Tyrosinase inhibitory activity
The tyrosinase inhibitor y activity was measured by the procedure
described by Iida et al. (1995) with some modification. The reac-
tion mixture consist of 1.5 ml of 50 mM phosphate buffer (pH 6.8),
900 μl of mushroom tyrosinase (50 Unit/ml), 300 μl of sample, and
300 μl of 10 mM L- DOPA solution. Briefly, 900 μl (50 Unit/ml of
reaction mixture) of mushroom tyrosinase was preincubated with
the sample in 50 mM phosphate buffer (pH 6.8) for 30 min at room
temperature. Then, the 300 μl of 10 mM L- DOPA was added to the
reaction mixture and the enzyme reaction was monitored by meas-
uring the change in absorbance at 475 nm (UV- 2900, Hitachi, Kyoto,
Japan) and room temperature, corresponding to the formation of do-
pachrome, for 30 min at 1 min intervals.
Controls, without inhibitor, were routinely determined. The per-
cent inhibition of the enzyme by the active compounds was calcu-
lated as follows:Tyrosinase inhibitory activity (%) =
where control475 is the absorbance of same reaction system without
sample.
2.11 | Angiotensin- converting enzyme (ACE)
inhibitory activity
The ACE inhibitory activity was estimated using a modification of
the method of Cushman and Cheung (1971). The mixture of sample
(100 μl), 50 μl of ACE ex tracts from rabbit lung (Sigma- Aldrich Co.,
St. Louis, MO), and 50 μl of 0.05 M sodium borate buffer (pH 8.3)
were preincubated at room temperature for 30 min, after which
the mixture was reincubated with 50 μl of substrate (5 mM HHL in
0.05 M sodium borate buffer, pH 8.3) for 60 min at 37°C in water
bath. The reaction was terminated by adding 250 μl of 1 N HCl. The
resulting hippuric acid was ex trac ted with 1.5 ml of 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 dry bath. The hippuric acid was dissolved in 1.0 ml of
distilled water, and the absorbance was measured at 228 nm using
an UV- spectrophotometer (UV- 2900, Hitachi, Kyoto, Japan). The
IC50 value was defined as the concentration required for inhibiting
50% of ACE.
The absorbance measured immediately A228 as follows:
ACE inhibitory activity (%) =
EAI (m
2∕g) =
2×2.303×A×D
l×Φ×C
ESI(min)
=
A
0
×Δt
ΔA
(Control
734
−Sample
734
)
Control
734
×
100
(Control
475 −
Sample
475
)
Control
475
×
100
[
1−
Sample
228
−Control Blank
228
Control
228 −
Control Blank
228 ]
×
100
416
|
YOON et al .
where sample blank is the absorbance of inactivated sample, before
added HHL, and control blank is the absorbance of inactivated con-
trol, before added HHL.
2.12 | Statistical analysis
Each measurement was replicated at least triplicates, and the re-
sults were expressed as mean ± SD. The data were subjected to
analysis of variance (ANOVA), and the differences bet ween means
were evaluated by Duncan’s test (p < 0.05). SPSS statistic program
(SPSS 12.0 KO, SPSS Inc., Chicago, IL, USA) was used for data
analysis.
3 | RESULTS AND DISCUSSION
3.1 | Buffer capacity
Buffer capacity is defined as mL or mmol of HCl or NaOH needed to
change the pH one unit. Different nutrients in human food and ani-
mal feed increase the buffer capacity of food and feed, which is ver y
important in human and animals. The buffer c apacit y and pH val-
ues of FDC, RPIs, and the positive controls (casein and hemoglobin)
with pH- shift are presented in Table 1. The initial pH values of RPI- 1
(pH 3.6) and RPI- 3 (pH 3.5) in deionized distilled water (DDW) were
lower than those of RPI- 2 (pH 4.8) and RPI- 4 (pH 5.3). pH differ-
ences bet ween RPIs are caused by a difference between the target
value of pH (pH 4.5 and 5.5) in the acid precipitation of the ASAP
process. The initial pH values of FDC, casein, and hemoglobin (1%,
dispersion in DDW) were 5.8, 5.2, and 7.1, respectively. In the vicin-
ity of these initial pHs, the buffer capacit y of all samples (1% disper-
sion), including positive controls, was minimized and estimated to be
near the isoelectric point. The buffer capacity of FDC was an aver-
age of 26.2 mM HCl (pH 2- 6) and 68.7 mM NaOH (pH 8- 12) for the
change of one pH unit per gram of protein, respectively. For the RPIs
at acidic range (pH 2–6), averages ranging from 15.0 to 28.2 mM
HCl were needed per g protein for one pH unit change, whereas at
alkaline range (pH 8–12), averages ranging from 74.3 to 95.7 mM
NaOH were needed per g protein. Higher values for FDC may be
due to presence of fat components which need more acid or alkali
to bring pH change by one unit (Chalamaiah, Balaswamy, Rao, Rao,
& Jyothirmayi, 2013; Lee, Lee et al., 2016). Casein and hemoglobin
at pH range 2–6 required averages 18.1 and 25.0 mM HCl, respec-
tively, to change the pH by one unit. Averages 38.6 and 29.8 mM
NaOH were needed for casein and hemoglobin in the pH- shift range
8–12, respectively. Overall, higher buf fer capacities of all samples
were obser ved for FDC, RPIs, and positive controls in alkaline pH (8–
12) than in acidic pH (2–6) range. The buffer capacities of RPI- 1 and
RPI- 3 were significantly bet ter than those of other RPIs, FDCs, and
positive controls (p < 0.05). The buffer capacities of mrigal egg con-
centrate (Chalamaiah et al., 2013), gum karaya seed meal (Narsing
Rao & Govardhana Rao, 2010), yellowfin tuna roe concentrate (Park
et al., 2016), and skipjack tuna roe concentrate (Yoon et al., 2018)
were reported to be stronger in alkaline than acidity. In these results
and reports, RPIs extracted from yellow fin tuna roe were superior
to those of other species through comparison of buffer capacity and
were not expected to be affected by changes in external pH environ-
ment. Also, it will contribute to the design of procedures for scale-
up processing of protein isolates and hydrolysates (Narsing Rao,
Prabhakara Rao, Satyanarayana, & Balaswamy, 2012; Park et al.,
2016). Therefore, RPIs with excellent buffering capacity can be ap-
plied to the development of protein- fortified food components by
being applicable to various processing environments.
Sample FDC R P I - 1 R P I - 2 R P I - 3 R P I - 4 Casein Hemoglobin
Initial pH 5.8b3.6e4.8d3.5f5.3c5.2c7. 1a
pH 2 106. 5a66.6b1 07. 0 a48.6c70.5b70.4b100.56a
pH 3 41. 2b5.7de 15.9c4.4e13.7c8.8d47. 8 8 a
pH 4 19. 1b2.5ef 3.5 cd 3.1de 4.1c2.1f20.04a
pH 5 6.9c7.9b1.2d7.7 b0.7de 0.2e9.60 a
pH 6 1.4f13.3a5.6c11. 5b2.7e2.0ef 4.53d
pH 7 10.1d24.8ab 11. 5 cd 19.6 bc 6.8de 30.3a0.55e
pH 8 36.2b40.0b23.8d30.3c14 .2e46.2a6.99 f
pH 9 54.9ab 56.2a33.5 cd 43.5bc 24.2de 53.4ab 20 .52e
pH 10 84.6a7 7. 3 ab 53.4 cd 62. 5bc 37. 2d68.0abc 66.9 9abc
pH 11 150.1 a13 7. 3 b105.5c130.8b10 0. 3c102 .6c37. 61d
pH 12 311.1b41 8. 0a321.0b413.7a381.4a200.8c126.1d
Notes. Values represent the mean of n = 3.
Means with different small letters within same row are significantly diffe rent at p < 0.05 by Duncan’s
multiple range test.
FDC, freeze- dried concentrate; RPI- 1 and RPI- 2, roe protein isolate adjusting at pH 4.5 and 5.5, re-
spectively, after alkaline solubilization at pH 11; RPI- 3 and RPI- 4, roe protein isolate adjusting at pH
4.5 and 5. 5, respectively, after alkaline solubiliz ation at pH 12.
TABLE1 pH and buffer capacity
(accumulated mM of NaOH or HCl/g of
protein) of FDC , RPIs prepared by
pH- shift process
|
417
YOON et al.
3.2 | Water- holding capacity (WHC)
The WHCs ( g/g protein) of FDC , RPIs, and positive controls (ca-
sein and hemoglobin) without and with pH- shift from 2.0 to 12.0
are shown in Figure 2. Water- holding capacit y belongs to protein
functionality related to hydration by protein–water interactions,
and Mohamed et al. (2012) repor ted that protein interactions with
water or oil are important in food systems because they affect the
flavor and texture of the food. The WHCs of RPIs and casein with-
out pH- shift (controls) were in the range of 3.7–4.0 g/g protein
with no significant differences (p > 0.05). The WHC of hemoglobin
(0.9 g/g protein) was significantly lower than those of FDC, RPIs,
and casein (p < 0.05). In case of pH 2, the WHC of RPIs exhibited
a 20- 23 g/g of protein range, and at pH 12, showed a range of
20- 34 g/g of protein. Among the RPIs, RPI- 1 and RPI- 2 showed a
relatively high water- holding capacit y. On the other hand, in the
range of pH 4- 8, WHCs (3- 8 g/g protein) of RPIs were similar to
the controls without pH- shift treatment. The pH- shift treatment
significantly improved the WHC of RPIs at pH values except for
the pH range of 4- 8, which minimized the water- holding capacity
due to the increased electrostatic repulsion (Azadian et al., 2012).
Mohamed et al. (2012) reported that WHCs of protein isolates
from tilapia were 2.63–2.51 ml/g, and lower than those of the
RPIs in this study. Azadian et al. (2012) repor ted that the lowest
WHC was observed in minced fish (pH 6.3) near the isoelectric
point compared with protein isolates of silver carp. The WHC of
the mrigal defatted egg protein concentrate is higher than that of
the Labeo rohita fish egg protein concentrate, and the high WHC
of the mrigal defatted egg protein concentrate may be due to the
presence of polar groups such as COOH and NH2 (Chalamaiah
et al., 2013). Tan, Ngoh, and Gan (2014) reported that the lack of
polar amino groups on the surface of protein molecules causes
WHC to be lowered because the polar groups in the protein are re-
sponsible for protein–water interactions. This is due to the acidic
and alkaline pH- shift which leads to conformational changes in the
protein within the RPIs, allowing hydrophilic amino acids to easily
access the surrounding water, and increase WHC.
3.3 | Protein solubility
The protein solubilities (%) of FDC and the RPIs without and with
pH- shift (pH 2–12) are shown in Figure 3. Protein solubility is an
important parameter influencing other functionalities of proteins,
such as foaming, emulsifying, and gel properties (A zadian et al.,
FIGURE2 Water- holding capacity of
protein isolates recovered from yellowfin
tuna roe by alkaline solublilization and
acid precipitation process without
and with pH- shift. FDC, freeze- dried
concentrate; RPI- 1 and RPI- 2, roe protein
isolate adjusting at pH 4.5 and 5. 5,
respectively, after alkaline solubilization at
pH 11; RPI- 3 and RPI- 4, roe protein isolate
adjusting at pH 4.5 and 5.5, respectively,
after alkaline solubilization at pH 12.
Values are means ± standard deviation
of triplicate determinations. Means with
different small letters within the same pH
and capital letters within same sample
are significantly different at p < 0.05 by
Duncan’s multiple range test
418
|
YOON et al .
2012; Kinsella, 1976; Mohan, Ramachandran, & Sankar, 2006). The
solubilities (2.3–3.2%) of RPIs without pH- shift (controls) were
significantly lower than that (71.3%) of the hemoglobin as a posi-
tive control (p < 0.05). However, the protein solubility (0.4%) of
other positive control casein was found to be almost insoluble in
1% dispersion. Protein solubilities of pH-shifted RPIs were signifi-
cantly increased 12.6–24.5% at pH 2 compared to those without
pH-shift treatment (controls). Also, at pH 12, their protein solu-
bilities ranged from 20.4 to 41.6%, indicating a higher solubility
increase rate at alkaline pH- shift. Among the RPIs at pH 12, RPI- 2
had the highest solubility (41.6%), followed by RPI- 1 (35.8%), RPI- 4
(21.3%), and RPI- 3 (20.4%; p < 0.05). Also FDC (53.8%), as a sample
control, showed significantly higher solubility than RPIs. Around
the isoelectric point at pH 4–6, the RPIs exhibited the lowest
solubility because of acid and alkali limiting protein solubilization.
However, the solubility (82.3%–99.9%) of hemoglobin was not af-
fected by pH variation, and casein showed about 90% solubility
in the range of pH 7- 12. After alkaline solubilization of the ASAP
process, the solubilities of protein isolates (RPI- 2 and 4) recovered
in acid precipitation at pH 5.5 were significantly higher than those
(RPI- 1 and 3) recovered at pH 4.5. These results indicate that ex-
treme pH variations, such as pH 2 and 12, were able to improve
protein solubility due to the exposure of more charged and polar
groups to the surrounding water (Kristinsson et al., 20 05). The pH-
dependent protein solubility is important in functional properties
and applications related to food systems, especially at pH < 4 or
> 7 (Kinsella, 1976), and is influenced by protein–protein, protein–
solvent interactions, and surface hydrophobic–hydrophilic balance
of the protein (Horax et al., 2011). The high solubility of fish pro-
teins is an important feature in many food applications and af fect s
other functional proper ties such as foam and emulsification prop-
erties (Kristinsson & Rasco, 2000). The protein solubilities of the
RPIs extracted from yellowfin tuna roe and positive controls at
various pH values may provide useful pointers on how well pro-
tein isolates will per form when incorporated into food systems
(Mohamed et al., 2012).
3.4 | Foaming capacity and foam stability
RPIs (1%, w/v) were dispersed in DDW and their foaming proper-
ties, such as foaming capacity (FC) and foam stability (FS), were
analyzed (Table 2). To compare the foaming properties of solubilized
protein, dispersed RPIs were centrifuged and the supernatant was
analyzed for FC and FS. Before centrifugation (control 1s), the FCs
FIGURE3 Protein solubility of
protein isolates recovered from yellowfin
tuna roe by alkaline solublilization and
acid precipitation process at initial pH
and various pH. FDC , freeze- dried
concentrate; RPI- 1 and RPI- 2, roe protein
isolate adjusting at pH 4.5 and 5. 5,
respectively, after alkaline solubilization at
pH 11; RPI- 3 and RPI- 4, roe protein isolate
adjusting at pH 4.5 and 5.5, respectively,
after alkaline solubilization at pH
12.Values are means ± standard deviation
of triplicate determinations. Means with
different small letters within the same pH
and capital letters within same sample
are significantly different at p < 0.05 by
Duncan’s multiple range test
|
419
YOON et al.
TABLE2 Foaming capacity (FC, %) and foam stability (FS, %) of protein isolates recovered from yellowfin tuna roe by alkaline solublilization and acid precipitation process with pH- shift
FDC R P I - 1 R P I - 2 R P I - 3 R P I - 4 Casein Hemoglobin
Control 1 FC (%) 121.4 ± 6.3b109.0 ± 3.1bc 100.0 ± 0.0c114.2 ± 1.2bc 100.0 ± 0.0c100.0 ± 0.0c138.9 ± 18.3a
15 min 88.6 ± 8.1 - - - - - 88.6 ± 8.9
60 min 81.5 ± 7.0 - - - - - 81.1 ± 8.0
Control 2 FC (%) 178 .2 ± 19.7aBCD 141.9 ± 3.7bBC 100.0 ± 0.0eE 128.1 ± 4.9bcBCD 100.0 ± 0.0eC 109.4 ± 11.1deB 120.9 ± 11.9cdA
15 min 79.8 ± 6.9 73.1 ± 3.9 - 53.4 ± 8.3 - 69.3 ± 22.4 94.6 ± 5.7
60 min 58.8 ± 4.1 - - - - - 81.4 ± 3.4
pH 2 FC (%) 212.5 ± 13.4aA 132.4 ± 6.5cdBC 176.4 ± 6.9bB 131.7 ± 7.3cdBC 124.4 ± 6.7cdB 140.3 ± 23.6cAB 115.1 ± 8.8dA
15 min 67.6 ± 6.4 68.2 ± 6.4 64.1 ± 2.5 42.6 ± 3.2 - 73.6 ± 7.0 87.4 ± 9.7
60 min 54.9 ± 7.4 0.0 ± 0.0 40.5 ± 2.8 - - 41.3 ± 9.8 52.4 ± 12.2
pH 4 FC (%) 170.7 ± 11.6aD 133.6 ± 2.2bBC 122.4 ± 0.8bcD 134.3 ± 1. 2bB 123.3 ± 0.1bcB 109.1 ± 10.8 cB 125.2 ± 18.7bcA
15 min 36.2 ± 1.3 70.5 ± 4.4 - 69.7 ± .8 - 83.4 ± 1.2 75.8 ± 8.0
60 min - - - - - 73.6 ± 3.1 52.9 ± 7.5
pH 6 FC (%) 175.3 ± 0.2aCD 126.1 ± 2.0bC 127.3 ± 3.6bCD 124.7 ± 0.0bCD 100.0 ± 0.0cC 107.0 ± 8.4bcB 126.1 ± 26 .5bA
15 min 77.0 ± 0.8 66.7 ± 2.9 57.4 ± 1.2 - - 90.3 ± 4.7 82.5 ± 8.1
60 min 63.6 ± 0. 8 - - - - 76.9 ± 0.7 76.0 ± 11.5
pH 7 FC (%) 191.7 ± 2.3aBC 127.5 ± 3.9cC 121.0 ± 2.1cD 123.2 ± 2.1cD 127.2 ± 0.8cB 158 .6 ± 37.0bAB 132.0 ± 17.1bcA
15 min 82 .9 ± 2.9 84.4 ± 0.7 82.6 ± 1.6 78.8 ± 3.8 69.6 ± 1.8 88.4 ± 3.2 82.7 ± 11.3
60 min 69.3 ± 0.6 69.5 ± 2.2 69.3 ± 2.9 65.5 ± 0.7 55.2 ± 0.9 74.4 ± 4.5 74.5 ± 6.6
pH 8 FC (%) 180.9 ± 5.9abBCD 129.8 ± 1.5cC 133.5 ± 2. 3bcCD 127.7 ± 4.1cBCD 123.7 ± 1.5cB 184. 8 ± 65.0aA 139.8 ± 21.7abcA
15 min 82.6 ± 3.4 84.3 ± 4.2 80.7 ± 0.5 78.5 ± 3.6 82.2 ± 3.2 65.9 ± 22.3 79.4 ± 8.4
60 min 69.8 ± 0.2 71.2 ± 1.7 70.4 ± 0.9 67.4 ± 4.4 70.9 ± 0.1 43.1 ± 18.9 63.0 ± 5.9
pH 10 FC (%) 197.1 ± 4.7aAB 133.7 ± 6.7bcBC 129.6 ± 5.7bcCD 125.1 ± 1.6bcCD 118.9 ± 0.8cB 135.6 ± .8bcAB 140.0 ± 22.5bA
15 min 71.4 ± 14.4 59.8 ± 5.3 84.7 ± 4.5 85.2 ± 3.1 84.2 ± 7.6 85.5 ± 2.6 82.2 ± 12.2
60 min 57.1 ± 14.3 41.8 ± 5.9 65.3 ± 3.7 63.9 ± 11.9 65.3 ± 13.1 62.4 ± 1.7 56.1 ± 20.7
pH 12 FC (%) 196.4 ± 7. 5aAB 182.9 ± 11.0abA 199.6 ± 10.2aA 173.1 ± 4.6bA 166. 8 ± 16. 3bA 130.5 ± 9.7cAB 129.7 ± 7.8cA
15 min 75.1 ± 7.9 76.7 ± 9.9 84.8 ± 1.3 77.5 ± 8.8 81.1 ± 4.8 81.3 ± 11.6 84.1 ± 5.5
60 min 63.5 ± 8.1 55.3 ± 6.1 69.7 ± 7.3 56.4 ± 11.0 61.4 ± 3.8 69.1 ± 9.1 57.1 ± 16.6
Notes. Controls 1 and 2 refer to samples b efore and after centrifugation, respec tively.
Values represent the mean of triplicate determinations.
Means with different small letters within same row and capital let ters within same column are significantly different at p < 0.05 by Duncan’s multiple range test.
FDC, freeze- dried concentrate; RPI- 1 and RPI- 2, roe protein isolate adjusting at pH 4.5 and 5.5, respectively, after alkaline solubilization at pH 11; RPI- 3 and RPI- 4, roe protein isolate adjus ting at pH 4.5
and 5.5, respectively, after alkaline solubilization at pH 12; - , Not detected.
420
|
YOON et al .
TABLE3 Emulsifying activity index (EAI) and emulsion stability index (ESI) of FDC, RPIs prepared by pH- shift process at various pH
Sample FDC R P I - 1 R P I - 2 R P I - 3 R P I - 4 Casein Hemoglobin
EAI (m2/g of protein) Control 1 8.3b3.0c1.4c2.3 cd 2.0 cd 0.4d18.4 a
Control 2 12.8bDEF 10.0bcDE 4.2dF 8.7cE 6.6cdE 7.0 cdE 19.1 aAB
pH 2 16.1bC 14.2bcD 14. 8bcE 16.5bD 16.4bD 11.9cCD 2 0.1aAB
pH 4 9.7 bF 6.4cdE 5.7dF 5.4dF 7. 7cE 2.3eF 12.9aC
pH 6 15.9aCD 10.8 bD 15.9aE 8.2cE 9.8 bcE 9.1 bcDE 18.1aAB
pH 7 11.1eEF 24.5 aC 21.7abD 20.0bcC 16.6cdD 13.8deC 18.4bcAB
pH 8 13.0dCDE 25.5aBC 25.8aC 23.6abB 21.6 bC 12.8dC 18.3cAB
pH 10 22.2bB 31. 2aA 32.5aB 31.7aA 31.1aA 23.7bB 16.1cBC
pH 12 38.1aA 2 9. 5 bAB 39. 2 aA 23.7cB 26.4bcB 36.8aA 20.8cA
ESI (min) Control 1 19.5b40.2a25.3b41. 2a32.0ab 19. 3 b20.0b
Control 2 17. 3 cCD 23.2bcB 26.1abCD 22.9bcD 16.5 cC 31.8aB 20.1bcAB
pH 2 14.3bcD 1 7.9ab cB 19.3aD 1 7.1 abcD 18 .3abC 13 .2cC 21. 2aA
pH 4 14.2cD 32. 8abB 45.1aA 29. 1abc CD 44.6aAB 3 7.6aB 18.5bcAB
pH 6 21.0dABC 63.6aA 45.7abcA 48 .1abAB 31.7bcdBC 23.5cdBC 18 .6dAB
pH 7 19.5 cABC 34.0bB 4 4.5bA 41. 0bBC 61.4aA 14.8cC 18.4cAB
pH 8 22.6cAB 24.9cB 40.6bAB 56.4aA 4 9. 5abAB 16 .0cC 18.3cAB
pH 10 18.0cBCD 28 .1bcB 33.6bBC 25.5bcD 4 9.4aAB 26.1bcBC 18.0cAB
pH 12 24.1bcA 24.0bcB 33.3bBC 18 .3cD 22.8bcC 62.7aA 16.6 cB
Notes. Controls 1 and 2 refer to samples b efore and after centrifugation, respec tively.
Values represent the mean of triplicate determinations.
Means with different small letters within same row and capital let ters within same column are significantly different at p < 0.05 by Duncan’s multiple range test.
FDC, freeze- dried concentrate; RPI- 1 and RPI- 2, roe protein isolate adjusting at pH 4.5 and 5.5, respectively, after alkaline solubilization at pH 11; RPI- 3 and RPI- 4, roe protein isolate adjus ting at pH 4.5
and 5.5, respectively, after alkaline solubilization at pH 12; - , Not detected.
|
421
YOON et al.
(100–114.2%) of the RPIs were lower than that (121.4%) of FDC
(p < 0.05). RPI- 3 had the highest FC (114.2%) among the RPIs, fol-
lowed by RPI- 1 (109.0%). The FCs of RPI- 2, RPI- 4, and casein were
not detected. However, the FC (138.9%) of hemoglobin was signifi-
cantly higher than those of FDC and the RPIs (p < 0.05), because
of its high solubilit y in DDW (Figure 3). After centrifugation (con-
trol 2s), the FCs of FDC, RPI- 1, RPI- 3, and casein were increased to
178.2%, 141.9%, 128.1%, and 109.4%, respectively, but the FCs of
RPI- 2 and RPI- 4 were not still detected. The FCs of the RPIs upon
pH- shift were higher at pH 12 (166.8% for RPI- 4, 173.1% for RPI- 3,
182.9% for RPI- 1, and 199.6% for RPI- 2) than at other pH- shif t val-
ues (p < 0.05). In all pH ranges, the FC of FDC ranged from 170.7%
to 212.5% and was higher than those of the RPIs and positive con-
trols (casein and hemoglobin). To show good foaming, a protein must
migrate rapidly to the air–water interface, unfolding, and rearrang-
ing at the interface (Halling & Walstra, 1981; Klompong, Benjakul,
Kantachote, & Shahidi, 2007). Mutilangi, Panyam, and Kilara (1996)
suggested that the foaming capacity of a protein was improved by
making it more flexible, exposing more hydrophobic residues, and
increasing its capacity to decrease surface tension.
Prior to centrifugation (control 1s), the FSs of the RPIs were not
detected because their FC was too low to keep the foam layer after
whipping. After centrifugation (control 2s), FS tended to decrease
with increasing time. The foams of RPI- 1 (73.1%) and RPI- 3 (53.4%)
were kept for the first 15 min and then completely disappeared after
15 min. However, the foams of RPI- 2 and RPI- 4 disappeared within
15 min. The FS of RPIs with pH- shift was more stable in the range of
pH 7–12 than in the acidic range of pH 2- 6. The FSs of RPIs were un-
stable near the isoelectric point in the range of pH 4–6. In the range
of pH 7–12, the FSs of FDC, RPIs, and positive controls were stable
for 60 min. The lowest foaming capacity of all samples at pH 4- 6 is
due to low water- holding capacity (Figure 2) and solubility (Figure 3)
at pH near the isoelectric point, and foam stability depends on the
degree of protein–water and protein–protein interactions within
foam layer (Mutilangi et al., 1996; Naqash & Nazeer, 2013). Our re-
sults also revealed that foaming properties were pH- dependent and
that the protein isolation conditions according to the A SAP process
influenced foam ability (Mohamed et al., 2012).
3.5 | Oil- in- water emulsifying activity index
(EAI) and emulsion stability index (ESI)
The EAI and ESI were performed to assess the ability to act as emul-
sifiers in a variety of foods, such as soups, sauces, confectioner y
breads, and dairy products (Can Karaca, Low, & Nickerson, 2011).
The EAI (m2/g of protein) estimates the ability of the protein to aid
in the formation and stability of a newly created emulsion by con-
tributing units of area of inter face stabilized per unit weight of pro-
tein, which is determined by the turbidit y (Park et al., 2016). The E AI
(m2/g of protein) and ESI (min) values of FDC, RPIs, and the positive
controls are shown in Tables 3. Before centrifugation (control 1s),
there were no significant differences among the EAI values (2.0-
3.0 m2/g of protein) of the RPIs (p > 0.05) except for RPI- 2 (1.4 m2/g
of protein). The RPIs had significantly lower E AI values than that
of FDC (8.3 m2/g of protein; p < 0.05). Compared with casein and
hemoglobin as the positive controls, the RPIs were higher that of
casein (0.4 m2/g of protein), but lower than that of hemoglobin
(18.4 m2/g of protein; p < 0.05). After centrifugation (control 2s), the
EAI values (4.2- 19.1 m2/g of protein) of the supernatants of all sam-
ples were improved compared to the dispersions before centrifuga-
tion (control 1s). The lowest EAI values of the RPIs were at pH 4
(5.4–7.7 m2/g of protein) with a coincidental decrease in solubility
(Figure 3). Since the lowest solubility occurred at pH 4, peptides
could not migrate rapidly to the interface (Klompong et al., 2007),
but the EAI increased as pH moved away from pH 4. The EAI values
of the RPIs were highest at pH 10, in the range of 31.1–32.5 m2/g
of protein, except for RPI- 2. Compared to FDC, the RPIs had higher
EAI values from pH 7 to 10. The EAI of casein was higher as pH
increased. However, the EAI of hemoglobin (16.1–20.8 m2/g of pro-
tein) was similar at all pH- shift values, except for pH 4.
After centrifugation (control 2s), the ESI values of the RPIs and
FDC slightl y decreased, exce pt for RPI- 2, which in creased to 26.1 m in
compared to control 1s. The ESI values of casein and hemoglobin
increased to 31.8 and 20.1 min, respectively. The ESI values of the
RPIs were decreased at extreme pH- shift of pH 2 (17.1–19.3 min) and
pH 12 (18.3–33.3 min). However, the ESI values of FDC and hemo-
globin (14.2–24.1 min) were similar at all pH ranges. In the case of
casein, the ESI was the highest at pH 12 (62.7 min). The emulsifica-
tion capacity is an oil- in- water surface active phenomenon, which
depends on the hydrophilic or hydrophobic nature of the peptides
and ionic charges on particles (Chalamaiah et al., 2013; Gbogouri,
Linder, Fanni, & Parmentier, 2004). The improvement in emulsifica-
tion activity and emulsion stability by centrifugation is presumably
due to the presence of insoluble particles in the dispersion which
interferes with the formation of the emulsion layer. However, the
emulsion stability did not increase in proportion to the increase in
emulsifying activity according to pH rise. The large deviation of the
emulsion stability by the pH- shift treatment was presumed to be
caused by the nonuniformit y of emulsified par ticles. These results
indicate that RPI- 1 and RPI- 3 are somewhat superior to RPI- 2 and
RPI- 4 over the full range of pH, although there is no significant dif-
ference in the emulsifying activity and stabilit y of RPIs by pH- shift
treatment.
3.6 | Antioxidant and antihypertensive activity
In the above experimental results, RPI- 1 was found to be relatively
superior in buffer capacity, WHC, solubilit y, foaming, and emulsify-
ing abilit y of RPIs extracted from yellowfin tuna roe through ASAP
process, and its antioxidant and antihypertensive activity were in-
vestigated. Table 4 showed the ABTS+ radical scavenging activity
(IC50, μg/mL), tyrosinase inhibitory activity (%), and ACE inhibitory
activity (%) of RPI- 1 (1% dispersion). Measurement of ABTS+ radical
scavenging activity can be applied to both oleophilic and hydrophilic
compoun ds and has been wide ly used as an antioxida nt activity ass ay
(You, Zhao, Cui, Zhao, & Yang, 20 09). The ABTS+ radical scavenging
422
|
YOON et al .
activity (IC50) of the supernatant (1.3 mg protein/ml) of 1% RPI- 1
dispersion was 82.9 μg/ml and showed better scavenging activity
than that (160- 170 μg/ml) of enzyme hydrolysates from shrimp pro-
cessing by- products (Kim, Yoon, Shim, & Lim, 2016). It also exhibited
similar or slightly weaker scavenging activity than those of isolate
processed water (33- 97 μg/ml, Lee et al., 2017), extracts (28- 45 μg/
ml), and cooking drips (55- 110 μg/ml, Yoon et al., 2017) of fish roe.
Recently, tyrosinase inhibitors have become increasingly import-
ant in pharmaceutical and cosmetic products in relation to hyperpig-
mentation (Choi, Kim, & Lee, 2011; Schurink, van Berkel, Wichers,
& Boeriu, 2007). The tyrosinase inhibitory activity of RPI- 1 was
14.0%, and some whitening effects could be expected. Tyrosinase
inhibitory activities of isolate processed waters of fish roes ranged
from 14.6 to 20.8% (Lee et al., 2017). Yoon et al. (2017) reported
that water extrac ts from fish roes showed tyrosinase inhibitory ac-
tivities (14.6%- 20.8%) relatively higher than those (0.4%- 2.5%) of
heat- treated cooking drips, but they were not expected to have a
whitening effect. Choi et al. (2011) reported that the tyrosinase in-
hibitory activity of tuna cooking drip was 31%, but the activities in-
creased in accordance with the absorbed dose of gamma irradiation.
Choi et al. (2017) reported that anchov y muscle hydrolysate with
subcritical water hydrolysis showed about 14.7% of tyrosinase inhib-
itory activit y. In these experimental results and reports, tyrosinase
inhibitory activity was also found in proteinous materials containing
protein or amino acid, but its inhibitor y activity was not strong.
The inhibition of ACE, a key enzyme regulating the blood pres-
sure, has been recognized as the most effective therapy for the
treatment of hypertension. ACE inhibitory activity of RPI- 1 (1.3 mg
protein/ml) was 35.7%. Current research on natural ACE inhibition
peptides has extended to seafood protein sources, particularly sea-
food by- product s. Lee et al. (2017) and Yoon et al. (2017) showed
that the 50% ACE inhibitory activity concentration of processed
waters recovered from fish roes ranged from 1.2 to 2.0 mg/ml,
and these processed waters recovered through heat or alkali/acid
treatment showed no difference according to treatment method
in ACE inhibitory activity. On the other hand, the enzyme hydroly-
sates of skate skin gelatin (Ngo, Ryu, & Kim, 2014), yellow sole frame
(Jung et al., 2006), skipjack roe (Intarasirisawat, Benjakul, Wu, &
Visessanguan, 2013), and Pacific cod skin (Himaya, Ngo, Ryu, & Kim,
2012) showed 35%- 86% ACE inhibitory ac tivit y and similar or supe-
rior to the results of this experiment. According to these results and
reports, RPIs extracted from yellowfin tuna roe showed antioxidant
and antihypertensive activities and could improve these bioactivities
through enzymatic hydrolysis.
4 | CONCLUSION
The roe protein isolates recovered from yellow fin tuna contained es-
sential amino acids- rich proteins in our previous study (Lee, Lee et al.,
2016) and had food components suitable as surimi- based products
and as protein substitutes or enhancers in traditional foods. In this
study, protein isolates of yellowfin tuna roe as a processing by- product
were extracted using the ASAP process and were determined to their
food functionalities and bioactivity. Yellowfin tuna roe protein isolates
were similar or superior to those of the positive controls and many
other fish protein isolates in terms of buffering capacity, foaming, and
emulsifying ability, except for the solubility. The overall func tionality
of the protein isolate measured in this experiment was low at pH 4- 6
near the isoelectric point, where buffer capacity, WHC, and solubility
are minimal. In addition, RPIs were also confirmed to have in vitro an-
tioxidative and antihypertensive activities, and thus, it could be used
as a health functional material. These RPIs can be used as an egg white
substitute for surimi- based products and as a development material
for animal protein fortification or new agricultural and marine fusion
products in snacks, noodles, confectionery, and baking. However,
these protein isolates require modification to enhance their functional
proper ties and to serve as better functional ingredients in food ap-
plications. Therefore, it is necessary to improve the solubility of RPIs
through enzymatic hydrolysis for the enhancement of food and health
functionalities of RPIs. This study also suggests that it would be an
opportunity for the development of high value- added products from
tuna roes that are in the seafood processing industry.
ACKNOWLEDGMENTS
This research was supported by the National Research Foundation
of Korea (NRF) funded by the Ministry of Education, Science and
Technology (NRF- 2014R1A1A4A0100 8620). This study was a part
of the project titled “Development and commercialization of tra-
ditional seafood product s based on the Korean coastal marine re-
sources,” funded by the Ministr y of Oceans and Fisheries, Korea.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
DECLARATION
This study has nothing to do with human and animal testing.
Sample Proteina(mg/ml) ABTS+(IC50, μg /ml)
Tyrosinase
inhibitory
activity (%)
ACE inhibitory
activity (%)
RP I - 1b1.3 ± 0.1 82.9 ± 0.9 1.4 ± 0.0 35.7 ± 2.2
Notes. IC 50, the half maximal inhibitory concentration.
Values represent the mean ± SD of n = 3.
aBase on the Lowry ’s et al. (1951) methods; bSupernatant of 1% dispersion after centrifugation.
TABLE4 ABTS+ radical scavenging
activity, tyrosinase inhibitory activity, and
angiotensin- converting enzyme (ACE)
inhibitory activity of RPI- 1 of protein
isolate recovered from yellowfin tuna roe
|
423
YOON et al.
ORCID
Min Soo Heu http://orcid.org/0000-0001-7609-0303
REFERENCES
Arfat, Y. A., & Benjakul, S. (2013). Effect of zinc sulphate on gelling
proper ties of phosphorylated protein isolate from yellow stripe
trevally. Food Chemistry, 141, 2848–2857. ht tps://doi.org/10.1016/j.
foodchem.2013.05.112
Azadian, M., Nasab, M. M ., & Abedi, E. (2012). Comparison of functional
proper ties and SDS- PAGE patterns between fish protein isolate
and surimi produced from silver carp. European Food Research and
Technology, 235, 83–90. https://doi.org/10.1007/s00217-012-1721-z
Binsan, W., Benjakul, S., Visessanguan, W., Roy trakul, S., Tanaka, M., &
Kishimura, H. (2008). Antioxidative ac tivit y of Mungoong, an ex-
tract paste, from the cephalothorax of white shrimp (Litopenaeus
vannamei). Food Chemistry, 106, 185–193. https://doi.org/10.1016/j.
foodchem.2007.05.065
Can Kar aca, A ., Low, N., & Nickerson, M. (2011). Emulsifying properties
of chickpea, faba bean, lentil and p ea proteins produced by isoelec-
tric precipitation and salt extraction. Food Research International, 44,
2742–2750. https://doi.org/10.1016/j.foodres.2011.06.012
Chaijan, M., Panpipat, W., & Benjakul, S. (2010). Physicochemical and
gelling properties of short- bodied mackerel (Rastrelliger brachysoma)
protein isolate prepared using alkaline- aided process. Food and
Bioproducts Processing, 88, 174–180. https://doi.org/10.1016/j.
fbp.2009.11.003
Chalamaiah, M., Balaswamy, K., Rao, G . N., Rao, P. G. P., & Jyothirmayi,
T. (2013). Chemical composition and f unctional properties of mri-
gal (Cirrhinus mrigala) egg protein concentrates and their applica-
tion in pas ta. Journal of Food Technology, 50, 514–520. https://doi.
or g /10 .1007/s1319 7-011- 0 35 7-5
Choi, J. S., Jang, D. B., Moon, H . E., Roh, M. K., Kim, Y. D., Cho, K. K .,
& Choi, I. S . (2017). Physiological properties of Engraulis japoni-
cus muscle protein hydrolysates prepared by subcritical water hy-
drolysis. Journal of Environmental Biology, 38, 283 –28 9. ht t p s: //d oi .
org/10.22438/jeb/
Choi, J. I., Kim, J. H., & Lee, J. W. (2011). Physiologic al properties of
tuna cooking drip hydrolysate prepared with gamma irradiation.
Process Biochemistry, 46, 1875–1878. ht tps://doi.org/10.1016/j.
procbio.2011.06.005
Cushman, D. W., & Cheung, H. S. (1971). Spectrophotometric assay
and properties of the angiotensin- converting enzyme of rab-
bit lung. Biochemical Pharmacology, 20, 16 37–1648. ht tps://doi.
org /10.1016/0 006-2952(71) 90292-9
FAO. (2016). Global aquaculture production (fish stat). URL ht tp://www.
fao. org /3/a-i5555e .pd f. Accessed 29/08/2 017.
Gbogouri, G. A ., Linder, M., Fanni, J., & Parmentier, M. (2004). Influence
of hydrolysis degree on the functional properties of salmon by prod-
uct hydrolysates. Journal of Food Science, 69, 615–622. https://doi.
org /10.1111/j .1365-2621.2004.tb 09 90 9.x
Gehring, C. K ., Gigliotti, J. C ., Moritz, J. S., Tou, J. C., & Jac zynski, J.
(2011). Functional and nutritional char acteristics of proteins and
lipids recovered by isoelectric processing of fish by- product s and
low- value fish e a review. Food Chemistry, 124, 422–431. https://doi.
org/10.1016/j.foodchem.2010.06.078
Halling, P. J., & Walstra, P. (1981). Protein stabilized foams and emulsions.
Critical Reviews in Food Science and Nutrition, 15, 155–203. https://
doi.org /10.10 80/10 40 839810952731 5
Heu, M. S., Kim, H. S., Jung, S. C., Park, C . H., Park, H. J., Yeum, D. M.,
… Kim, J. S. (2006). Food component characteristic s of skipjack
(Katsuwonus pelamis) and yellowfin tuna (Thunnus albacares) roes.
Journal of the Korean Fisheries Society, 39, 1–8 .
Himaya, S. W. A., Ngo, D. H., Ryu, B., & Kim, S. K. (2012). A n active pep-
tide purified from gastrointestinal enzyme hydrolysate of Pacific cod
skin gelatin attenuates angiotensin- 1 converting enzyme (ACE) ac-
tivity and cellular oxidative stress. Food Chemistry, 132, 1872–1882.
https://doi.org/10.1016/j.foodchem.2011.12.020
Horax, R., Hettiarachchy, N., Kannan, A ., & Chen, P. (2011). Protein
extraction optimisation, characterisation, and functionalities
of protein isolate from bitter melon (Momordica charantia)
seed. Food Chemistry, 1 24, 545–550. https://doi.org/10.1016/j.
foodchem.2010.06.068
Iida, K., Hase, K ., Shimomura, K ., Sudo, S., Kadot a, S., & Namba, T. (1995).
Potent inhibitors of t yrosinase activity and melanin biosynthesis
from Rheum officinale. Planta Medica, 61, 425–428. https://doi.
org /10.1055/s-20 06 -95812 9
Intarasirisawat, R., Benjakul, S., Wu, J., & Visessanguan, W. (2013).
Isolation of antioxidative and ACE inhibitory peptides from pro-
tein hydrolysate of skipjack (Katsuwana pelamis) roe. Journal
of Functional Foods, 5, 1854–1862. https://doi.org/10.1016/j.
jff.2013.09.006
Jung, W., Mendis, E., Je, J., Park, P., Son, B. W., Kim, H. C., … K im, S.
(2006). Angiotensin I- converting enzyme inhibitory peptide from
yellowfin sole (L imanda aspera) frame protein and its antihyperten-
sive effect in spontaneously hypertensive rat s. Food Chemistry, 94 ,
26–32. https://doi.org/10.1016/j.foodchem.2004.09.048
Kim, S. B., Yoon, N. Y., Shim, K. B., & Lim, C. W. (2016). Antioxidant and
angiotensin I- converting enzyme inhibitory activities of northern
shrimp (Pandalus borealis) by- products hydrolysate by enzymatic hy-
drolysis. Fisheries and Aquatic Sciences, 19, 1–6.
Kinsella, J. E. (1976). Functional proper ties of proteins in food. Critical
Reviews in Food Science and Nutrition, 7, 219–225. https://doi.
org /10.1080/1040839760 952720 8
Klomklao, S., & Benjakul, S. (2016). Utilization of tuna processing by-
products: Protein hydrolysate from skipjack tuna (Katsuwonus pela-
mis) viscera. Journal of Food Processing an d Preser vation, 41, e12970.
https ://doi.or g/10 .1111/ jfpp.1 2970
Klompong, V., Benjakul, S., K antachote, D., & Shahidi, F. (2007).
Antioxidative activity and functional properties of protein hydro-
lysate of yellow stripe trevally (Selaroides leptolepis) as influenced
by the degree of hydrolysis and enzyme t ype. Food Chemistry, 102,
1317–1327. https://doi.org/10.1016/j.foodchem.2006.07.016
Kristinsson, H. G., & Rasco, B. A . (200 0). Biochemic al and functional
proper ties of Atlantic salmon (Salmo salar) muscle hydrolyzed with
various alkaline proteases. Journal of Agricultural and Food Chemistry,
48, 657–666. https://doi.org/10.1021/jf990447v
Kristinsson, H. G., Theodore, A. E., Demir, N., & Ingadottir, B. (2005).
A comparative study between acid- and alkali- aided processing and
surimi processing for the recovery of proteins from channel catfish
muscle. Journal of Food Science, 70, C298–C3 06.
Kunal, S. P., Kumar, G., Menezes, M. R., & Meena, R . M. (2013).
Mitochondrial DNA analysis reveals three stocks of yellow-
fin tuna Thunnus albacares (Bonnaterre, 1788) in Indian waters.
Conservation Genetics, 14, 205–213. https://doi.org/10.1007/
s10592-013-04 45-3
Lee, H. J., Lee, G. W., Yoon, I. S., Park, S. H., Park, S. Y., Kim, J. S., & Heu,
M. S. (2016). Preparation and characterization of protein isolate from
yellowfin tuna Thunnus albacares roe by isoelectric solubilization/
precipitation process. Fisheries and Aquatic Sciences, 19, 14. https://
doi.org/10.1186/s41240-016-0014-z
Lee, H. J., Park, S. H., Yoon, I. S ., Lee, G. W., Kim, J. S., & Heu, M. S. (2016).
Chemical composition of protein concentrate prepared from yellow-
fin tuna Thunnus albacores roe by cook- dried process. Korean Journal
of Fisheries and Aquatic Sciences, 19, 12. https://doi.org/10.1186/
s41240-016-0012-1
Lee, G. W., Yoon, I. S., Kang, S. I., Lee, S. G., Kim, J. I ., Kim, J. S., & Heu, M.
S. (2017). Func tionality and biological activity of isolate processed
424
|
YOON et al .
water generated during protein isolate prepar ation of fish roes using
an isoelectric solubilization and precipitation process. Korean Journal
of Fisheries and Aquatic Sciences, 50, 694 –70 6 .
Lowry, O. H., Rosebrough, N. J., Farr, A. L., & R andall, R. J. (1951). Protein
measurement with the Folin phenol reagent. Journal of Biological
Chemistry, 193, 265–275.
Mireles DeWitt, C. A., Gomez, G., & James, J. M. (2002). Protein ex-
traction from b eef heart using acid solubilization. Journal of Food
Science, 67, 3335–3341. https://doi.org/10.1111/j.1365-2621.2002.
tb0 9588 .x
MOF (2016). Ministry of Ocean and Fisheries. Yearbook of ma-
rine resource. URL http://www.mof.go.kr/article/view.do?ar-
ticleKey=13940&boardKey=32¤tPageNo=1 Accessed
29/0 8 /201 7.
Mohamed, B. K. F., Xia, W., Issoufou , A., & Qixing, J. (2012). Influence
of pH Shift on func tional proper ties of protein isolated of tilapia
(Oreochromis niloticus) muscles and of soy protein isolate. Food and
Bioprocess Technology, 5, 2192–2 200 .
Mohamed, T. K., Zhu, K ., Issoufou, A., & Fatmata, T. (2009). Func tionality,
in vitro digestibility and physicochemical properties of two varieties
of defatted foxtail millet protein concentrates. International Journal
of Molecular Sciences, 10, 5224–523 8. h ttps://doi.org/10. 3390/
ijms101252 24
Mohan, M., Ramachandran, D., & Sankar, T. V. (2006). Functional prop-
erties of Rohu (Labeo rohita) proteins during iced storage. Fo od
Research International, 39, 847–854. htt ps://doi.org/10.1016/j.
foodres.2006.04.003
Mustaf a, A. L ., Al-Wessali, M. S., A l-Basha, O. M., & Al-Amir, R. H. (1986).
Utilization of cowpea flour and protein isolate in bakery products.
Cereal Foods World, 31, 756–758.
Mutilangi, W. A. M., Panyam, D., & Kilara, A. (1996). Functional prop-
erties of hydrolysates from proteolysis of heat- denatured whey
protein isolate. Journal of Food Science, 61, 27 0–274. ht t ps ://doi .
org /10.1111/j .1365-2621.1996 .tb14174.x
Naqash, S. Y., & Nazeer, R. A . (2013). Antioxidant and functional proper-
ties of protein hydrolysates from pink perch (Nemipterus japonicus)
muscle. Journal of Food Science and Technolog y, 50, 972–978. https://
doi.org /10.10 07/s13197-011-0416-y
Narsing Rao, G., & Govardhana Rao, D. (2010). Chemical and functional
charac terization of Gum karaya (Sterculia urens L.) seed meal. Food
Hydrocolloids, 24, 479–485.
Narsing Rao, G., Prabhakara Rao, P., Satyanar ayana, A ., & Balaswamy,
K. (2012). Functional properties and in vitro antioxidant activity of
roe protein hydrolys ates of Channa striatus and L abeo rohita. Fo od
Chemistry, 135, 1479–148 4.
Ngo, D. H., Ryu, B., & Kim, S. K. (2014). Active peptides from skate
(Okamejei kenojei) skin gelatin diminish angiotensin- I convert-
ing enzyme activity and intracellular free radical- mediated oxi-
dation. Food Chemistry, 143 , 246–255. https://doi.org/10.1016/j.
foodchem.2013.07.067
Paraman, I., Hettiarachchy, N. S., Schaefer, C., & Beck , M. I. (20 07).
Hydrophobicity, solubilit y, and emulsifying properties of
enzyme- modified rice endosperm protein. Cereal Chemistry, 84,
343–349. https://doi.org/10.1094/CCHEM-84-4-0343
Park, S. H., Lee, H. J., Yoon, I. S., Lee, G. W., Kim, J. S., & Heu, M. S.
(2016). Protein functionalit y of concent rates prepared from yel-
lowfin tuna (Thunnus albacares) roe by cook- dried process. Foo d
Science and Biotechnology, 25, 1569–1575. https: //doi.or g/10.1007/
s10068-016-0242-0
Pérez-Mateos, M., Boyd, L., & L anier, T. (200 4). Stability of omega- 3
fatty acids in fortified Sumi seafood during refrigerated storage.
Journal of Agricultural and Food Chemistry, 52, 7944–7949. https://
doi.org/10.1021/jf049656s
Schurink, M., van Berkel, W. J., Wicher s, H. J., & Boeriu, C. G. (2007).
Novel peptides with tyrosinase inhibitory activity. Peptides, 28, 485–
495. https://doi.org/10.1016/j.peptides.2006.11.023
Tahergorabi, R., Beamer, S. K., Matak, K. E., & Jaczynski, J. (2011). Effect
of isoelectric solubilisation/precipitation and titanium dioxide on
whitening and texture of proteins recovered from dark chicken- meat
processing by- products. LWT- Food Science and Technology, 44, 896–
903. https://doi.org/10.1016/j.lwt.2010.10.018
Tan, E. S., Ngoh, Y. Y., & Gan, C. Y. (2014). A comparative study of physi-
cochemical characteristics and functionalities of pinto bean protein
isolate (PBPI) against the soybean protein isolate (SPI) after the ex-
traction optimisation. Food Chemistry, 152, 4 47–455. https://doi.
org/10.1016/j.foodchem.2013.12.008
Yongsawatdig ul, J., & Park, J. W. (200 4). Effects of alkali and acid solubili-
zation on gelation characteristics of rockfish muscle proteins. Journal
of Food Science, 69, 499–505.
Yoon, I. S., Le e, G. W., Kang, S. I., Park, S . Y., K im, J. S., & Heu, M. S. (2017).
Food functionality and biological activit y of processed waters pro-
duced during the preparation of fish roe concentrates by cook- dried
process. Korean Journal of Fi sheries and Aqua tic Sciences, 50, 5 06–519.
Yoon, I. S., Lee, G. W., Kang, S. I., Park, S. Y., Lee, J. S., Kim, J. S., & Heu,
M. S. (2018). Chemical composition and f unctional properties of
roe concentrates from skipjack tuna (Katsuwonus pelamis) by cook-
dried process. Food Sciences an d Nutrition, 6, 1276–1286. https://doi.
org /10.1002/f sn3 .676
You, L., Zhao, M., Cui, C ., Zhao, H., & Yang, B. (2009). Effect of de-
gree of hydrolysis on the antioxidant activity of loach (Misgurnus
anguillicaudatus) protein hydrolysates . Innovative Fo od Science &
Emerging Technologies, 10, 235–240. https://doi.org/10.1016/j.
ifset.2008.08.007
How to cite this article: Yoon IS, Lee HJ, Kang SI, et al. Food
functionality of protein isolates extracted from Yellowfin Tuna
(Thunnus albacares) roe using alkaline solubilization and acid
precipitation process. Food Sci Nutr. 2019;7:412–424. ht t p s : //
doi.org/10.1002/fsn3.793
Available via license: CC BY
Content may be subject to copyright.
