Journal of Aquatic Food Product Technology

ISSN: 1049-8850 (Print) 1547-0636 (Online) Journal homepage: http://www.tandfonline.com/loi/wafp20

Controlling Lipid Oxidation and Volatile

Compounds in Frozen Fried Fish Cake Prepared
with Rice Bran Hydrolysate

Supattra Supawong, Supawan Thawornchinsombut & Jae W. Park

To cite this article: Supattra Supawong, Supawan Thawornchinsombut & Jae W. Park
(2018): Controlling Lipid Oxidation and Volatile Compounds in Frozen Fried Fish Cake
Prepared with Rice Bran Hydrolysate, Journal of Aquatic Food Product Technology, DOI:
10.1080/10498850.2018.1508103

To link to this article: https://doi.org/10.1080/10498850.2018.1508103

Published online: 13 Aug 2018.

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JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY
https://doi.org/10.1080/10498850.2018.1508103

Controlling Lipid Oxidation and Volatile Compounds in Frozen

Fried Fish Cake Prepared with Rice Bran Hydrolysate
Supattra Supawonga,b, Supawan Thawornchinsombuta, and Jae W. Parkb
a
Department of Food Technology, Faculty of Technology, Khon Kaen University, Khon Kaen, Thailand; bOSU Seafood
Research & Education Center, Oregon State University, Astoria, Oregon, USA

ABSTRACT KEYWORDS
Fried fish cakes formulated with (1) no antioxidant (control), (2) 1% rice bran Rice bran hydrolysate; fried
hydrolysate (RBH), (3) 2% RBH, (4) 0.05% rosemary oil, and (5) 0.02% fish cake; freeze-thaw;
butylated hydroxyanisole/butylated hydroxytoluene (BHA/BHT) were inves- oxidative changes;
tigated for their oxidation values following 0–9 freeze-thaw cycles. Both headspace solid phase
lipid oxidation and protein oxidation were significantly obstructed when microextraction
RBH or BHA/BHT was used. RBH at 2% was equally effective as 0.02% BHA/
BHT. Headspace solid phase microextraction (HS-SPME), which measures
volatile compounds that determine lipid oxidation, demonstrated the effec-
tiveness of RBH. The development of rancid volatile compound (i.e., hex-
anal) levels in fried fish cakes decreased during 0–9 freeze-thaw cycles. This
study demonstrates that RBH is as an effective antioxidant comparable to
commercial antioxidants (BHA/BHT) in frozen fried fish cakes. Consequently,
RBH can be a consumer-friendly and natural antioxidant ingredient.

Introduction
Muscle food products are often stored at freezing temperatures to extend storage life. Freezing and
frozen storage, however, can cause chemical and structural changes in products, depending on their
characteristics (meat source, amount and type of lipids, presence of cryoprotectants, antioxidant
status, protective packaging used, etc.). Such changes alter the characteristics of proteins (denatura-
tion and aggregation) and lipids (oxidation) and lead to undesirable effects on many product
characteristics, resulting in reduced quality and shelf life of meat products (Serrano et al., 2006).
Several antioxidant compounds have been discovered in rice bran, such as tocopherol, tocotrienol,
gamma-oryzanol, polyphenolic compounds, flavonoids, and peptides. These bioactive compounds can
reduce the risks of developing several chronic diseases, such as cancer, cardiovascular disease, anti-
inflammatory, anti-diabetic, anti-hypertensive, and immunomodulatory properties (Sharma et al., 2011).
Polyphenol and proteins (peptides and amino acids) have both antioxidant activity and capabilities to
eliminate radicals and chelate metals (Cagdas and Kumcuoglu, 2015).
In addition, the antioxidant activity of rice bran extract compounds can retard lipid and protein
deterioration in food products. According to our preliminary study, rice bran hydrolysates (RBHs)
produced from hexane defatted rice bran using subcritical alkaline water (SAW) followed by
enzymatic hydrolysis demonstrated that 2% RBH was effective to minimize lipid oxidation in
Pacific whiting surimi gel. Thiobarbituric acid reactive substances (TBARS) value was reduced by
79.84% (Supawong et al., 2017a). Based on the results of that study, it was hypothesized that RBH
could also be used as an effective antioxidant in frozen fried products.

CONTACT Jae W. Park jae.park@oregonstate.edu OSU Seafood Research & Education Center, 2001 Marine Dr Ste 253,
Astoria, Oregon 97103, USA.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/wafp.
© 2018 Taylor & Francis
2 S. SUPAWONG ET AL.

Volatile compounds are usually produced during food frying through lipid oxidation, affecting
the characteristics of fried products, especially flavor. These compounds, which can be harmful to
human health, are hexanal (Pino et al., 2013), furan (Pérez-Palacios et al., 2012), trans, tran-2,4-
decadienal, tran-2-decenal, aldehyde, and its derivatives (Boskou et al., 2006). The prominent
aldehyde compound generated through oxidation is hexanal (Ross and Smith, 2006). Hexanal is
the only aldehyde that arises from both the 9 and 13 hydroperoxides of linoleate, and from other
unsaturated aldehydes formed during the oxidation of linoleate (Shahidi and Pegg, 1994). Trans,
trans-2,4-decadienal is also a harmful volatile compound of fried products. Boskou et al. (2006)
reported that these compounds have cytotoxic and genotoxic effects and support low density
lipoprotein (LDL) oxidation. The International Agency for Research on Cancer has indicated
furan as possibly carcinogenic to humans (Group 2B), and the US Department of Health and
Human Service has recorded furan in the human pathogen list (Pérez-Palacios et al., 2012).
Trans-2-decenal (oxygenated α,β-unsaturated aldehyde) also forms in fried meat when it is kept in
the refrigerator for too long and contributes to a stale smell (Christine and Grosch, 1991).
Recently, more attention has been placed on the evaluation of naturally occurring antioxidants.
RBHs prepared from rice bran, which is a co-product of rice processing, has become of our interest.
We hypothesized that incorporation of RBH can improve some quality attributes of frozen fried
products (i.e., fried fish cake), as its negative characteristic (brown color) could be overcome. In
addition, fried fish cake is very popular in Japan, China, South Korea, and Thailand because of its
crispy surface texture, good taste, high protein, and low fat content. Therefore, the aim of this study is
to evaluate the oxidative changes and volatile compounds of fried fish cakes subjected to extended
freeze-thaw cycles as affected by RBH, rosemary oil, and commercial antioxidants (BHA/BHT).

Materials and methods

Frozen threadfin bream (Nemipterus spp.) surimi was obtained from ManA Frozen Foods Co., LTD.
(Songkhla, Thailand). Industrial hexane-defatted rice bran (HDRB) was obtained from Kasisuri Co.
Ltd. (Ayudhaya, Thailand). Rosemary oil produced by steam distillation was purchased from Now
Foods (Bloomingdale, IL, USA). Butylated hydroxyanisole (BHA≥ 98.5% purity) and butylated
hydroxytoluene (BHT≥ 99.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Wheat
flour, corn flour, potato flour, pure canola oil, and plain salt (Signature kitchensTM) were purchased
from a local grocery store. Hexanal and trans,trans-2,4-decadienal were supplied from Alfa Aesar
(Haverhill, MA, USA). Furfuryl alcohol and trans-2-decenal were supplied from TCI America
(Portland, OR, USA) for headspace solid phase microextraction, and a standard solution of
100 mg L−1 were prepared. All of the chemicals and reagents were of analytical grade.

Rice bran hydrolysate preparation

RBH was produced from HDRB using SAW followed by enzymatic hydrolysis according to Supawong
et al. (2017a). HDRB was sifted through a 50 mesh screen and dissolved in distilled water at a 1:7 (w/v)
ratio with 1.5% (w/v) citric acid (soaking for 18 h). The suspension was adjusted to pH 8 using 2N
NaOH. The alkaline suspension was added to a glass bottle and heated under pressure at 130°C
(170 kPa) using an autoclave (Consolidated Stills & Sterilizers, Allston, MA, USA) for 2 h and allowed
to rest at room temperature for approximately 2 h. Autoclave treatment is often used to enhance the
extraction of proteins or other compounds from raw materials (Kaewjumpol, 2017). The suspension
was incubated with 2% Protease G6 (Siam Victory Chemical, Bangkok, Thailand) mixed with 98%
HDRB at 60°C, pH 8 for 6 h. The degree of hydrolysis was 26.6%. The protease G6, which is an alkaline
protein, was used to extract glutelin, the major protein in rice (Shih, 2003). Then, it was subsequently
heated to 95°C for 2 min to inactivate the enzyme. Next, the mixture was left at room temperature for
approximately 1 h before centrifuging at 10,000 x g for 15 min. The supernatant, namely RBH, was
adjusted to pH 7 using 1N HCl, then freeze-dried and kept at −18°C.
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 3

The composition and physicochemical properties of RBH

Protein content
The concentration of protein was measured using Kjeldahl analysis (with 5.95 conversion factor)
according to the AOAC Official Method (AOAC, 2000).

Total phenolic content

Total phenolic compound content was determined using the Folin-Ciocalteau method according to
Singleton and Rossi (1965) with some modifications. RBH solution (200 µL) was added to 800 µL of the
Folin–Ciocalteu solution and 4 mL deionized water. The mixture was vortexed for 1 min and left to stand
at room temperature for 10 min, followed by adding 2 mL of sodium carbonate solution (7.5%). The
reaction mixture was vortexed for 1 min after incubation at room temperature for 2 h. The absorbance of
phenolic content was measured spectrophotometrically (UV-2401PC Recording Spectrophotometer,
Shimadzu, Tokyo, Japan) at 765 nm. The quantification of phenolic compounds based on the standard
curve generated using gallic acid was expressed as mg gallic acid equivalent/g of sample (mg GAE/g RBH).

2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS•) radical scavenging activity

The ABTS radical scavenging activity was determined according to Yin et al. (2012) with slight
modification. Seven mM 2,2ʹ-azono-bis (3-ethylbenz-thiazoline-6-sulfonic acid) was prepared using
0.1 M phosphate buffer (pH 7.4) containing 0.818% NaCl and 0.0015% KCl. ABTS was mixed with an
equal volume of 2.45 mM K2S4O8 and incubated at refrigeration (4–5°C) temperature for 12 h in the dark
to form the ABTS radical. To 100 µL RBH solution, 3 mL of ABTS was added and uniformly mixed. The
resultant sample was incubated at room temperature for 6 min in the dark, and the absorbance at 734 nm
was measured. Trolox (6-hydroxy-2,5,7,8 tetramethyl-chroman-2-carboxylic acid) was used to construct
the standard curve and expressed as trolox equivalent antioxidant capacity (mg TEAC/g RBH).

Ferric reducing antioxidant power (FRAP)

Ferric reducing antioxidant power (FRAP) assay was carried out by the method of Benzie and Strain
(1996) with some modification. The method is based on the reduction of a ferric 2,4,6-tripyridyl-s-
triazine complex (Fe3+-TPTZ) by antioxidants to the ferrous form (Fe2+-TPTZ). RBH was added to
10 mM ferric-TPTZ reagent, and the increase in absorbance at 593 nm was measured at 8 min.
Deionized water was used as a control. Iron (II) sulfate (FeSO4) was used to construct the standard
curve and expressed as mg FeSO4/g RBH.

Color
CIE L* (lightness), a* (redness), and b* (yellowness) values of RBH powder were determined using a Minolta
colorimeter (CR-310; Minolta Camera Co. Ltd., Osaka, Japan). The instrument was calibrated using a
Minolta calibration plate and a Hunter Lab standard hitching tile according to the method of Park (1994).

Fourier transform infrared spectroscopy (FTIR) measurement of RBH

The conformation of freeze-dried RBH was obtained by an FTIR spectrometer (Bruker, Billerica, MA,
USA). FTIR spectra were obtained in the wave number ranging from 400 to 4,000 cm−1. Diffusive
reflectance of the IR was measured and recorded with an average of 32 scans at a resolution of 4 cm−1.

Fried fish cakes preparation

Five different fish cake paste samples were prepared using various antioxidants: No antioxidant (control),
1% RBH (1-RBH), 2% RBH (2-RBH), 0.05% rosemary essential oil (Rosemary), and 0.02% BHA/BHT
(BHA/BHT). Frozen surimi was tempered and cut into small pieces (~ 3 cm cubes). Surimi cubes were
chopped at 1,800 rpm for 1 min (UM 5 universal, Stephan Machinery Corp, Columbus, OH, USA). With
salt (2% w/w) addition, surimi was chopped for an additional 1 min at 1,800 rpm. Other dry ingredients
4 S. SUPAWONG ET AL.

(9% wheat flour, 5.6% mixed starch (corn: potato: wheat 1:2:1)) as well as different antioxidant additives
were added. Moisture content was adjusted to 68.3% using ice/water. After adding ice and other
ingredients, chopping resumed at 1,800 rpm for 1 min. The paste was further chopped at 3,600 rpm
under vacuum (40–60 kPa) for 3 min. Chopping was conducted below 25°C. Then, a 33 g sample of the
mixture from each formulation was put into a patty former to form a square shape (5 x 5 m x 1 cm)
before frying in canola oil at 177°C for 6 min using a deep fryer (Presto 05466 Dual ProFry Immersion
Element Deep Fryer, Eau Claire, WI, USA). The fried fish cakes were cooled by placing them on a rack at
room temperature for 30 s before freezing at −18°C for 2 h. The frozen fried samples were vacuum
packed and frozen at −18°C for 24 h. The oxidation analysis of the samples was conducted after 0, 1, 3, 6,
and 9 freeze-thaw cycles. Freeze-thaw was used to mimic long-term commercial cold storage. One freeze-
thaw cycle was defined as 2 days freezing at 18°C, followed by 1 day thawing at 5°C.

Oxidation properties of fried fish cakes

Lipid oxidation
Lipid oxidation was investigated by measuring TBARS, according to the method described by Buege and
Aust (1978). Each ground fried sample (0.5 g) was homogenized in 10 mL thiobarbituric acid (TBA)
solution containing 0.375%TBA, 15% trichloroacetic acid (TCA), and 0.25N HCl for 1 min at high speed
using a homogenizer (BioSpec, Bartlesville, OK, USA). The mixed solution was heated (95–100°C) for
10 min; then, the solution was cooled with running tap water and centrifuged at 3,600 × g for 20 min at 25°C.
The supernatant was obtained to measure absorbance at 532 nm (UV-2401PC Recording
Spectrophotometer, Shimadzu). The TBARS value results were reported as mg of malonaldehyde/kg of
sample.

Protein oxidation
Protein oxidation in fried samples was investigated by measuring the fluorescence emitted in buffer
extracts using fluorescence spectroscopy according to the method described by Armenteros et al.
(2009) with slight modifications. Fried samples were minced and homogenized at 1:10 (w/v) in
10 mM phosphate buffer (pH 6.0) containing 0.6 N NaCl using an ultraturax homogenizer (Biospec
Products) for 1 min and centrifuged at 20,000 x g for 20 min at 20°C. The emission spectra of the
supernatant in a quartz spectrofluorometer cell were examined at 350–550 nm for protein oxidation.
The excitation wavelength was set at 350 nm (LS 50B Perkin–Elmer Luminescence Spectrometer,
Beaconsfield, UK). The excitation and emission slit widths were set at 10 nm. The results were
reported as fluorescence intensity units emitted by protein oxidation products at 420 nm. These
values were corrected according to the protein content of each fried sample by multiplying by a
correction factor (Cf = Pt/Pp), where Pt is the total average of the protein contents from all samples,
and Pp is the mean of the protein content from each sample, respectively.

Headspace solid-phase microextraction (HS-SPME) coupled to gas chromatographic-flame

ionization detector (GC-FID) for analysis volatile compounds in fried fish cakes
HS-SPME procedure
The volatile compounds of fried samples after grinding were determined using HS-SPME with GC. A
ground sample (1 g) and deionized water (3 mL) were mixed in a 20 mL glass vial. The glass vial was
hermetically capped with a PTFE-faced silicone septum (both from Supelco) and vortexed for 3 min.
After homogenization, samples were vigorously stirred at 60°C for 5 min to provide an equilibrium
between the sample matrix and headspace. An SPME fiber coated with divinylbenzene-carboxen-
polydimethylsiloxane (DVB/CAR/PDMS, 30 µm thickness) (Supelco) assembled in an SPME Supelco
holder was used as a tool to extract volatile compounds from the samples. The SPME fiber was
extracted in the headspace for 30 min at 60°C. Then, the fiber was removed and immediately injected
into a GC injection port for thermal desorption at 250°C for 5 min.
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 5

A Shimadzu GC-2010 equipped with a FID and a Zb-Wax, #216604 Phenomenex capillary
column (30.0 m x 0.25 mm; 0.5 µm film thickness) (Zebron, Anaheim, CA, USA) was used. As
for the chromatographic conditions, helium (grade 5; Airgas, Grand Rapids, MI, USA) was used as a
carrier gas at a flow rate of 1.65 mL/min. The temperature of the oven was initially set at 45°C for
2 min and then was increased to 200°C at a rate of 5°C/min before holding at 200°C for 5 min. Total
run time was 38 min. The FID temperature was 280°C.
Volatile compounds were identified by comparing retention time with standard compounds and
quantified by external calibration curve method. For each individual volatile compound, a calibra-
tion curve (sample peak area/standard compound peak area versus sample amount/standard com-
pound amount) was constructed. The final results, expressed in µg/100 g sample, were counted for
the exact weight of the sample portion in the head space vial.

Experimental design and statistical analysis

The experimental design was a split plot design. The freeze-thaw cycle was allocated to a main plot,
while types of antioxidants were allocated to a sub plot. Statistical analysis was performed using the
analysis of variance on SPSS for Windows, version 19.0 (SPSS Inc., Chicago, IL, USA). Statistical
significance in differences among samples and between treatments was analyzed using Duncan’s
New Multiple Range Test. Differences were considered significant at p < 0.05.

Results and discussion

The composition and physicochemical properties of RBH
The composition and physicochemical properties of RBH are shown in Table 1. Its protein content,
total phenolic content, ABTS radical scavenging ability, and FRAP of RBH were 27.56%, 29.44 mg
GAE/g RBH, 13.35 mg TEAC/g RBH, and 8.35 mg FeSO4/g RBH, respectively.
According to the FTIR spectrum of RBH (Figure 1), a broad band at 3246 cm−1 belongs to
stretching vibration of phenolic hydroxyl group (-OH) representing hydrogen bonding (Cruz-
Espinoza et al., 2012). The regions of 1574 cm−1 referred to as C = O from amide I, 1389 cm−1
referred to C–N stretching from amide II, and 1266 cm−1 referred to as C–N stretching and N–H
deformation from amide III, similar to the results reported earlier (Yang et al., 2015). The most
distinctive spectral features for proteins are the strong amide I and II bands centered approximately
between 1600 cm−1 and 1400 cm−1, respectively (Oliver et al., 2009). The series of overlapping peaks
located in the region of 1140–1021 cm−1 resulted from vibration modes. Cael and Koenig (1974)
mentioned that, for carbohydrates, a series of overlapping peaks located in the region of
1180–953 cm−1 results from vibration modes, such as the stretching of C–C and C–O and the
bending mode of C–H bonds. These are often referred to as the “saccharide” bands and are the most
intense bands in the mid-infrared spectrum. A small shoulder at around 1650 cm−1 was formed.

Table 1. Physicochemical properties of RBH.

Measurement Value Unit
Color, protein content, yield and protein recovery of freeze dried RBH
Color (L*/a*/b*) 64.20/2.29/17.19
Protein content* 27.56 %
Protein recovery** 66.55 % (based on protein in defatted rice bran)
Total phenolic content and antioxidant activities of freeze dried RBH
Total phenolic content*** 29.44 mg GAE/g RBH
ABTS 13.35 mg TEAC/g RBH
FRAP 8.35 mg FeSO4/g RBH
*Protein content and protein recovery were determined by Kjeldahl analysis
** Protein content of defatted rice bran = 19.78%
*** Folin-Ciocalteau method
6 S. SUPAWONG ET AL.

Figure 1. FTIR spectra of rice bran hydrolysates.

These bands were referred to typical Maillard reaction products (MRPs), i.e., Schiff’s base imine
group (stretching) and enaminol group (stretching) (Yang et al., 2015). As Monajjemzadeh et al.
(2009) reported, the first step of the Maillard reaction (MR) leads to the formation of an imine
known as a Schiff’s base. The C = N stretching band appears at 1,630–1,650 cm−1 in the infrared
spectra of imine containing compounds. Wnorowski and Yaylayan (2003) monitored carbonyl-
amine reaction between pyruvic acid and α-amino alcohols by FTIR spectroscopy and reported
that the resultant Schiff’s base absorbs infrared light at about 1,647 cm−1. Namli and Turhan (2006)
followed the reaction process between benzaldehyde or salicylaldehyde or 2-pyridinecarboxaldehyde
with aniline and found that the resulting imine showed a band at about 1,630 cm−1 in FTIR spectra.
This observation confirmed the formation of covalent bonds between proteins and polysaccharides
through the MR in RBH, which was consistent with brown color (Table 1). FTIR results confirm that
RBH consisting of protein (amide I and II), saccharide, phenolic hydroxyl group, and MRPs is
responsible for antioxidant activity. Thamnarathip et al. (2016) reported that RBH contains various
chemicals demonstrating antioxidant effects, such as phenolic compounds. Sharma et al. (2011)
reported that rice bran protein hydrolysate is an excellent source of protein and provides bioactive
activities such as antioxidative, antimicrobial, antithrombotic, antihypertensive, opioid, and immu-
nomodulatory activities. The antioxidant activity of the RBH is inherent to the phenolic compounds,
protein content, characteristic amino acid sequences of peptides, and MRPs (Adebiyi et al., 2009).
Kaewjumpol (2017) reported that high content of lysine, glutamic acid, hydrophobic amino acids (i.e.,
glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), and
aromatic amino acids (i.e., phenylalanine and tryptophan) were found in the RBH, which was produced
under the same condition (SAW at 130°C for 2 h followed by 2% Protease G6 hydrolysis) and using the same
raw material (defatted rice bran) used in this study. These results confirmed that RBH obtained from SAW
extraction followed by enzymatic hydrolysis demonstrated high content of amino acids. The antioxidative
amino acids in rice bran protein hydrolysates, such as histidine, proline, tryptophan, leucine, valine, alanine,
and methionine, could contribute to the antioxidant activity (Thamnarathip et al., 2016). Some amino acids
(e.g., arginine, glycine, and histidine), small peptides, and nitrogenous metabolites directly scavenge oxygen
free radicals (Sarojnalini and Devi, 2014).
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 7

Kaewjumpol et al. (2017) reported six phenolic compounds (vanillic acid, syringic acid, vanillin,
p-coumaric acid, ferulic acid, and sinapic acid) identified in RBH were produced under the same
condition (SAW at 130°C for 2 h followed by 2% Protease G6 hydrolysis) and using the same raw
material (defatted rice bran) used in this study. RBH contained more p-coumaric acid (3.006 mg/g RBH)
than other phenolic compounds. Ferulic acid (1.056 mg/g RBH) was the second highest content followed
by vanillin (0.434 mg/g RBH) and sinapic acid (0.444 mg/g RBH), respectively. These results confirmed
that RBH obtained from SAW extraction followed by enzymatic hydrolysis demonstrated content of
phenolic compounds (vanillic acid, syringic acid, vanillin, p-coumaric acid, ferulic acid, and sinapic
acid). Polyphenol and proteins (peptides and amino acids) have both antioxidant activity and capabilities
to eliminate the radicals and chelate metals (Cagdas and Kumcuoglu, 2015). This was explained by the
role of the radical scavenging activity via H-atom donation of the hydroxyl group in phenolic com-
pounds. Another valuable property of phenolic compounds is their ability to chelate heavy metal ions.
Phenolic compounds chelate metal ions due to the presence of suitable functional groups: hydroxyl and
carboxyl (Kulbat, 2016). Phenolic compounds possessing hydroxyl and carboxyl groups are able to bind
particularly iron and copper. They may inactivate iron ions by chelating and additionally suppressing the
superoxide-driven Fenton reaction. This is believed to be the most important source of reactive oxygen
species (Michalak, 2006). Amino acids with a phenolic hydroxyl group in the peptides may also be
responsible for the radical scavenging activity (Thamnarathip et al., 2016). A relationship between
phenolic content and antioxidant activity has also been described by Velioglu et al. (1998). A statistically
significant relationship between total phenolic content and antioxidant activity was reported, with
R2 = 0.963 (p < 0.001) for flaxseed and R2 = 0.905 (p < 0.001) for cereal (Velioglu et al., 1998).
Another compound responsible for antioxidant activity in RBH is MRPs, which could be formed
during the thermolysis process of RBH. Yoshimura et al. (1997) investigated the antioxidative effect of
MRPs using a glucose-glycine model system and revealed that MRPs could function as electron donors.
Thus, the antioxidant effect of RBH is presumably linked to its proteins (peptide and amino acid),
phenolic compounds, and MRPs constituents in RBH. These chemical compositions strongly suggest
RBH has a high antioxidant potential in fried fish cake products.

Oxidation properties of fried fish cakes

Lipid oxidation in fried fish cakes
TBARS values indicate the secondary lipid oxidation products (carbonyls and aldehydes of hydrocar-
bons) affecting off-flavors in meat products (Hwang et al., 2013). Figure 2 shows the TBARS values (mg
malondialdehyde/kg sample) of various fried fish cakes as affected by antioxidants and freeze-thaw
cycles. A significant (p < 0.05) interaction on TBARS was found between various antioxidants and freeze-
thaw cycles. Control sample (no antioxidant) showed the highest TBARS content, which was signifi-
cantly (p ≤ 0.05) higher than the antioxidant treatments. Similarly, Ramos et al. (2012) reported TBARS
values increased with longer frozen storage of fried kamaboko. The samples formulated with RBH and
BHA/BHT showed little change in TBARS after 1 F/T cycle and maintained lower values (below 2.2) after
9 F/T cycles. However, as freeze-thaw cycles were extended, the TBARS increased significantly (p ≤ 0.05)
in the control, with the descending order of rosemary, 1% RBH, BHA/BHT, and 2% RBH.
Lipid oxidation was significantly reduced with RBH, as TBARS values were significantly lower compared
to the control at all freeze-thaw cycles (p ≤ 0.05). The addition of 2% RBH was slightly better than or as
effective as BHA/BHT in keeping TBARS values low, indicating RBH effectively inhibited lipid oxidation.
This is probably due to antioxidant compounds in RBH, such as proteins (peptide and amino acid), phenolic
compounds, and MRPs. According to our previous study (Supawong et al., 2017b), RBH produced from
hexane defatted rice bran using SAW followed by enzymatic hydrolysis showed high antioxidant activity in
both in vitro and in vivo tests. As related to the physicochemical properties of RBH, its protein content, total
phenolic content, and ABTS radical scavenging ability of RBH were 27.56%, 29.44 mg GAE/g RBH, and
13.35 mg TEAC/g RBH, respectively.
8 S. SUPAWONG ET AL.

Figure 2. Lipid oxidation of fried fish cakes formulated with various antioxidants during freeze-thaw cycles.

Our previous study demonstrated 2% RBH was effective to minimize lipid oxidation in Pacific
whiting surimi gel: TBARS value was reduced by 79.84% (Supawong et al., 2017a). Wongthahan and
Thawornchinsombut (2015) as well as Homehong (2015) revealed that fish patties made from tilapia
byproducts, when prepared with added rice bran protein hydrolysates, also showed lower lipid
oxidation during freezing and thawing. Additionally, whey protein prevented the formation of
hydroperoxide and TBARS, suggesting it can be used as an antioxidant in a model system
(Browdy and Harris, 1997) and in raw and cooked pork patties (McCarthy et al., 2001).
According to our previous study (unpublished data), fried fish cake treated with 2% RBH also had a
significantly higher (p ≤ 0.05) total phenolic content (63.93 mg GAE/100 g) than other treatments: 1% RBH
(47.20 mg GAE/100 g), BHA/BHT (26.68 mg GAE/100 g), rosemary (22.35 mg GAE/100 g), and control
(20.74 mg GAE/100 g) and showed the highest antioxidant activity (both DPPH and ABTS radical
scavenging activity).
The capacity of RBH to prevent lipid oxidation is probably due to the antioxidant compounds in
RBH, such as proteins (peptides and amino acids), phenolic compounds, and MRPs. According to
the physicochemical properties of RBH (Table 1), protein content, total phenolic content, ABTS
radical scavenging activity, and FRAP were 27.56%, 29.44 mg GAE/g RBH, 13.35 mg TEAC/g RBH,
and 8.35 mg FeSO4/g RBH, respectively. Amino acids are suggested to have antioxidant properties as
they react with oxidized lipids (Sarojnalini and Devi, 2014). In addition, phenolic compounds in
RBH are responsible for antioxidant activity. Hwang et al. (2013) reported that phenolic compounds
from Gwanghwayakssuk leaves showed high antioxidant functions in frozen fried chicken nuggets.
Thus, the capability of RBH to prevent lipid oxidation is presumably linked to its rich polyphenol, protein
(peptide and amino acid), and MRPs constituents that have capabilities to eliminate the radicals and chelate
metals (as described in section 3.1). This was explained by the role of the radical scavenging activity via
H-atom donation of the hydroxyl group in phenolic compounds. Peptide and amino acids directly scavenge
oxygen free radicals and MRPs could function as electron donor. Lipid oxidation is induced by oxy- and/or
lipid free radical generation and results in the generation of toxic compounds such as malondialdehyde and
cholesterol oxidation products. However, phenolic, proteins (peptides and amino acids), and MRPs in RBH
have multi-antioxidant activity. Thus, these compounds could eliminate the free radicals and reactive
oxygen species, resulting in prevention of lipid oxidation.
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 9

Protein oxidation in fried fish cakes

Protein oxidation was evaluated by measuring the loss of natural tryptophan fluorescence and the
gain of fluorescent protein oxidation products using fluorescence spectroscopy. The presence of the
aromatic ring in the amino acid tryptophan is responsible for the natural fluorescence emitted at
350 nm when it is excited at 280 nm. Changes in intrinsic fluorescence of tryptophan have been used
to monitor physicochemical changes in proteins (Estévez et al., 2008), including those derived from
oxidative stress (Giessauf et al., 1995). The decrease of tryptophan fluorescence may be due to the
oxidative degradation of tryptophan and its conversion into radicals (Giessauf et al., 1995). In
addition, the oxidation of sulfhydryl groups can lead to the loss of sulfhydryl groups, resulting in
the generation of disulfide bonds and protein carbonyl compounds. These processes are commonly
linked to a decrease in muscle protein functionality, leading to increased water loss, weaker protein
gels, or less stable emulsions (Giessauf et al., 1995). They are involved in cross-linking of damaged
proteins via Schiff base formation. Schiff bases are generated as a result of the reactions between lipid
oxidation products (aldehydes) and amino groups from the side chain of proteins. These compounds
are conjugated fluorophores with distinct spectral properties and might be detected by recording
fluorescence at around 450 nm when excited at around 350 nm. The measurement of their
fluorescence values has been used as a protein oxidation index (Giessauf et al., 1995). This technique
has been successfully used to investigate the protein oxidation of meat products (Armenteros et al.,
2009), bovine serum albumin (BSA) (Heinonen et al., 1998), and oil-in-water emulsion containing
different levels of myofibrillar proteins (Estévez et al., 2008).
The highest peak emission wavelength, indicating protein oxidation of fried fish cake, was 420 nm
(Figure 3A). This was in agreement with Estévez et al. (2008), who found that the highest peak
emission wavelength of emulsions containing different levels of myofibrillar proteins was 420 nm.
Armenteros et al. (2009) found that various meat samples (ground meat, dry-cured ham, sausage)
demonstrated that the highest peak of the fluorescence emitted by protein oxidation products was at
450 nm. Giessauf et al. (1995) and Estévez et al. (2008) speculated that the oxidative degradation of
tryptophan and its conversion into radicals might cause the loss of tryptophan fluorescence.
Furthermore, carbonyl compounds are formed through protein oxidation (Estévez et al., 2008).
Fluorescence intensity units at 420 nm ranged from 366 to 847 (Figure 3B), which was similar to
those reported by Armenteros et al. (2009) in meat products. Estévez et al. (2008) measured protein
oxidation in oil-in-water emulsions containing myofibrillar protein (0.5–2% based on lipid content).
Their fluorescence intensity unit emitted by protein oxidation products ranged from 200 to 400 nm.
In the current study, as freeze-thaw cycles extended, protein oxidation values significantly increased
(p ≤ 0.05) for the control and RBH-treated samples, respectively (Figure 3B). However, RBH or BHA/BHT-
treated samples controlled protein oxidation effectively. The addition of 2% RBH was as effective as or

Figure 3. Protein oxidation of fried fish cakes spectrum corresponding to fluorescent protein oxidation products (λ excita-
tion = 350 nm) in fried fish cakes at 9 freeze-thaw cycles (A), Protein oxidation of fried fish cakes formulated with various
antioxidants during freeze-thaw cycles (B).
10 S. SUPAWONG ET AL.

slightly better than BHA/BHT in maintaining constant fluorescence intensity units emitted by protein
oxidation products. It was clear that the addition of RBH in fried fish cakes markedly inhibited protein
oxidation, probably because RBH contained phenolic compounds that act as antioxidants by eliminating
free radical chain-type reactions (H-atom donation). As RBH also contained proteins (peptides and amino
acids), it acted as an antioxidant by eliminating the free radicals and reactive oxygen species.
The physicochemical properties of RBH (Table 1) revealed that the total phenolic content of RBH
was 29.44 mg GAE/g RBH. The 2% RBH-treated fried fish cakes had higher concentrations of total
phenolic content (63.93 mg GAE/100 g sample) than other treatments (BHA/BHT and rosemary) and
showed the highest antioxidant activity (both DPPH and ABTS radical scavenging activity) (unpub-
lished data). RBH produced using the same method and material used in this study demonstrated six
phenolic compounds (vanillic acid, syringic acid, vanillin, p-coumaric acid, ferulic acid, and sinapic
acid) (Kaewjumpol et al., 2017). Wang et al. (2015) reported that alkaline hydrolysis condition induced
RBHs to release their individually bound phenolic compounds, and ferulic acid was the major phenolic
compound in rice bran. They also identified a new compound, namely para-hydroxy methyl benzoate
glucoside, which showed high antioxidant in ABTS radical scavenging activity.
Heinonen et al. (1998) speculated that phenolic compounds have hydrogen donors and their
methoxyl substituents can form more stable phenoxyl radical intermediates, leading to high anti-
oxidant functions in preventing lipid oxidation and protein oxidation. Heinonen et al. (1998)
demonstrated that the lower polarity of phenolic compounds (ferulic acid and malvidin) may impart
a better affinity for the protein interface and better inhibit protein oxidation. The higher polarity of
phenolic compounds (gallic acid, delphinidin, and propyl gallate) may convert copper ions to a lower
valence state, causing them to have prooxidant activity. They may also bind strongly to protein and
contribute to the indicated loss of tryptophan fluorescence. This indicated that phenolic compounds
in RBH are the main compound that prevents protein oxidation in fried fish cake.
Lipid oxidation is induced by oxy- and/or lipid-free radical generation and results in the
generation of toxic compounds such as the malondialdehyde and cholesterol oxidation products.
Muscle cells contain high amounts of proteins, which can also be affected by oxidative reactions.
Free radicals react with side chains of proteins to produce protein free radicals, which react with
molecular oxygen to form peroxy radicals. The protein hydroperoxides may then be decomposed to
carbonyl derivatives. Oxidation of sulfhydryl groups may lead to the formation of either intra- or
inter-protein disulfide cross-links. The attack of reactive oxygen species on muscle proteins leads to
the loss of sulfhydryl groups and the generation of carbonyl compounds (Soyer et al., 2010).
Therefore, both lipid and protein oxidation are caused by reactive oxygen species and free radicals.
However, proteins (peptides and amino acids) and phenolic compounds in RBH have multi-
antioxidant activity. This was explained by the role of the radical scavenging activity via H-atom
donation of the hydroxyl group in phenolic compounds. Both peptides and amino acids directly
scavenge oxygen free radicals. Thus, these chemical compositions could eliminate the free radicals
and reactive oxygen species (Thamnarathip et al., 2016) and prevent lipid and protein oxidation.

Volatile compounds in fried fish cakes using HS-SPME-GC

Volatile compounds produced from lipid oxidation can cause rancid off-flavors and odors. Due to
the limitations of TBARS and the need to measure primary oxidation products, the measurement of
volatile compounds has become a popular indicator of lipid oxidation (Ross and Smith, 2006).
In the present study, we reported the quantification of hexanal, trans,trans-2,4-decadienal, furfuryl
alcohol, and trans-2-decenal from fried fish cakes with various antioxidants during freeze-thaw cycles. A
typical standard chromatogram of hexanal, trans,trans-2,4-decadienal, furfuryl alcohol, and trans-2-decenal
is shown in Figure 4A. A significant interaction between various antioxidants and freeze-thaw cycles on
hexanal levels of samples was found (p ≤ 0.05) (Figure 4B). There was no significant difference in trans,trans-
2,4-decadienal, furfuryl alcohol, and trans-2-decenal levels in samples with various antioxidants (p > 0.05).
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 11

Figure 4. Volatile compound results Standard HS-SPME-GC chromatogram of oxidatively derived aldehydes. [Peaks identified
corresponds to (a) hexanal, (b) tran-2-Decenal, (c) Furfuryl alcohol, and (d) tran-tran-2,4-Decadienal] (A). The quantification of
hexanal from fried fish cakes with various antioxidants during freeze-thaw cycles (B).

However, the lower values of trans,trans-2,4-decadienal, furfuryl alcohol, and trans-2-decenal content were
observed in samples formulated with RBH (Figure 5).
The evolution of the hexanal levels (Figure 4B) in the sample with 2% RBH was observed from
55.21 µg/100 g sample at 0 freeze-thaw cycle to 201.46 µg/100 g sample on 9 freeze-thaw cycle. The
control samples showed the highest hexanal levels throughout the freeze-thaw cycles (p ≤ 0.05),
ranging from 88.65 µg/100 g sample at 0 freeze-thaw cycle to 414.06 µg/100 g sample after 9 freeze-
thaw cycles. Similar to the effect on TBARS, RBH was found to be the most effective antioxidant,
followed by commercial antioxidants (BHA/BHT). These results agree with those reported by Pino
et al. (2013), who studied hexanal production using HS-SPME in precooked chicken meatballs
during frozen storage. They found that the concentration of hexanal was significantly higher in
the control samples, which ranged from 34 μg of hexanal/100 g of sample at the beginning and
280 µg/100 g sample after 144 h of refrigerated storage. They suggested that hexanal indicates lipid
oxidation of meat more effectively than any other volatile components.
The major primary products of lipid oxidation reaction, hydroperoxides, are relatively unstable and
essentially odorless and are decomposed into a wide range of secondary compounds, including alkanes,
alkenes, aldehydes, ketones, alcohols, esters, acids, and hydrocarbons. Of these compounds, aldehydes are
considered the most important breakdown products because they possess low threshold values and are the
major contributors to the development of volatile compounds, rancid off-flavors, and odors. However,
proteins (peptides and amino acids), phenolic compounds, and MRPs in RBH have multi-antioxidant
activity. Thus, these chemical compositions in RBH could eliminate the free radicals and reactive oxygen
species to prevent lipid oxidation to effectively control rancid volatile compounds in fried fish cake.

Conclusion
RBH demonstrated strong activity against lipid and protein oxidation in fried fish cakes. RBH was
also effective in controlling rancid volatile compounds as hexanal levels in fried fish cakes were
significantly (p < 0.05) low and increased slowly compared to other treatments during freeze-thaw
cycles. The reduction of oxidation and controlling rancid volatile compounds by RBH were attrib-
uted to polyphenol, proteins (peptides and amino acids), and MRPs. RBH also demonstrated multi-
antioxidant activity, as it minimized the free radicals, reactive oxygen species, and chelating metals.
Based on consumer concerns regarding safety and toxicity of synthetic antioxidants, RBH may prove
useful as a safe and health-friendly antioxidant for the food industry.
12 S. SUPAWONG ET AL.

Figure 5. The quantification of furfuryl alcohol (A), trans-2-decenal (B), and trans-trans-2,4-decadienal (C) from fried fish cakes with
various antioxidants during freeze-thaw cycles.
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 13

Acknowledgments
The authors greatly appreciate the financial support by the Thailand Research Fund through the Research and Researchers
for Industries (RRI program, Grant number-PHD57I0040). This research was also funded by Khon Kaen University through
the National Research Council of Thailand (Grant no. 592101/2016). Special thanks are due to Mr. Varapong Supachok
(Kasisuri Co., Ltd.) for supplying partial funds and raw materials (industrially defatted rice bran, IDRB).

Funding
This work was supported by the National Research Council of Thailand [grant number 592101/2016] and Thailand
Research Fund [grant number PHD57I0040].

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