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Quality of Tuna Fish Oils Extracted from Processing the By-Products of Three
Species of Neritic Tuna Using Supercritical Carbon Dioxide

Article  in  Journal of Food Processing and Preservation · May 2014

DOI: 10.1111/jfpp.12248

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 Journal of Food Processing and Preservation ISSN 1745-4549

QUALITY OF TUNA FISH OILS EXTRACTED FROM PROCESSING

THE BY-PRODUCTS OF THREE SPECIES OF NERITIC TUNA USING
SUPERCRITICAL CARBON DIOXIDE
SAHENA FERDOSH1, ZAIDUL ISLAM SARKER2,6, NIK NORULAINI3, ALEXANDRA OLIVEIRA4,
KAMARUZZAMAN YUNUS5, AHMED JALAL CHOWDURY5, JAHURUL AKANDA1 and MOHD OMAR1
1
School of Industrial Technology, Universiti Sains Malaysia, Penang, Malaysia
2
Faculty of Pharmacy, International Islamic University Malaysia, Kuantan Campus, Kuantan, Pahang 25200, Malaysia
3
School of Distant Education, Universiti Sains Malaysia, Penang, Malaysia
4
Kodiak Seafood and Marine Science Centre, University of Alaska Fairbanks, Kodiak, AK
5
Faculty of Science, International Islamic University Malaysia, Kuantan Campus, Kuantan, Pahang, Malaysia

6
Corresponding author. ABSTRACT
TEL: +6-046-585-435;
FAX: +6-046-585-435; Fatty acid constituents of total lipids extracted from the head, the skin and the
EMAIL: zaidul@iium.edu.my viscera of three neritic tuna species, namely Thunnus tonggol, Euthynnus affinis
and Auxis thazard, using supercritical carbon dioxide (SC-CO2) and Soxhlet
Received for Publication December 13, 2013
extraction method were determined and compared. Saturated fatty acid (39.7–
Accepted for Publication February 17, 2014
48.5%) was dominant in conjunction with monounsaturated fatty acid (21.9–
doi:10.1111/jfpp.12248 26.6%) and polyunsaturated fatty acid (PUFA) (24.1–27.9%) in all species, and
the difference between methods were nonsignificant. Docosahexaenoic acid was
the major PUFA, accounting for 17.0–19.9% in the head, 15.7–17.3% in the skin
and 14.3–16.1% in the viscera of total fatty acids. Total oil extracted by SC-CO2
had lower free fatty acid (FFA) and peroxide value (PV) content than that by
Soxhlet method. The ranges of FFA and PV were 1.8–5.0 and 1.2–2.4%, respec-
tively. It was concluded that SC-CO2 is an effective method to extract fish oil that
is rich in omega-3 fatty acids from tuna by-products.

PRACTICAL APPLICATIONS
Fish waste is a by-product of fish process and fish-based food industries. Fish oil
could be extracted from tuna fish waste that contains high amount of polyunsatu-
rated fatty acids, especially eicosapentaenoic acid and docosahexaenoic acid
(DHA). Supercritical fluid extraction could be applied to obtain refined, bleached
and deodorized fish oil from tuna waste without any toxic organic solvent. Tuna
waste oil contains long chain of omega-3,6 fatty acids, especially DHA, which have
potential health benefits in reducing the risk of various diseases.

(Thunnus tonggol), the eastern little tuna (Euthynnus affinis)

INTRODUCTION
Malaysia is surrounded by Andaman Sea, Straits of Malacca,
Sulu Sea and South China Sea. Its dynamic marine capture
fisheries plays a significant role in the national economy,
contributing to about 75% of the total yearly volume of
fishery products harvested. Wild tuna species, consisting
mainly of Oceanic tuna and neritic tuna, are an important
fishery resource in Malaysia, with growing interest by the
canning industry. The main neritic or coastal little tuna
species found in Malaysian waters are the longtail tuna
and the frigate tuna (Auxis thazard). More than 90% of the
tuna landings in Malaysia consist of neritic species, of which
49% are longtail tuna, 34% are eastern little tuna and 7%
are frigate tuna (Bakar and Hassan 2011). Neritic little tuna
are currently the main target tuna species in Malaysia and
the neighboring country, Thailand, because of their high
consumer demand and competitive prices offered by tuna
processors.
Fish processing may generate up to 50% of the whole fish
weight as by-products, depending on which components are

Journal of Food Processing and Preservation •• (2014) ••–•• © 2014 Wiley Periodicals, Inc. 1
TUNA FISH OILS EXTRACTED USING SC-CO2
further utilized for production of fishmeal, fish oils or other
specialty products such as fish gelatine and edible protein
powders (Babbit 1990). During processing, commonly pro-
duced by-products are heads, skin, viscera and frames,
which are generally discarded as processing leftovers or used
for production of animal feed or fertilizer. Proper utilization
of fishery by-products has many advantages; principally, it
increases the overall value of the catch, it reduces cost of
processing waste disposal or treatment and it ultimately
lowers environmental pollution.
The oil content of fish by-products is highly variable and
may range from 1.4 to 40.1% (Babbit 1990), depending on
the species and tissue type. It has been well established in
the current literature that marine oils are the most impor-
tant source of long-chain polyunsaturated fatty acid
(PUFA). In marine species, lipids are deposited beneath the
skin, in the muscle, head and in the viscera, mainly in livers
(Aryee and Simpson 2009). Not only lipid content is highly
variable, but also fatty acid composition of fish lipids is
diverse, being also distinguishable within a fish for its differ-
ent body parts (Saito et al. 1999; Aryee and Simpson 2009;
Sahena et al. 2010). Fatty acid composition of the fish may
differ due to feeding habits, environmental temperature,
fish age and sexual maturity, location of the catch and type
of species (Haliloğlu et al. 2004; Selmi and Sadok 2010).
Lohne (1976) found extensive lipid deposits in the head
and belly cavity of mackerel and capelin. Pink salmon
by-products have been reported to contain 10.9% oil in the
heads and 2% oil in the viscera (Bechtel 2003). Tuna heads
are known to be rich in omega-3 PUFA, mainly
docosahexaenoic acid (DHA) (Saito et al. 1999). Fish from
tropical climate were found to have lower amounts of total
lipids compared to fish from the Arctic region. Besides,
the lipids of freshwater feeds are characterized by
linoleic (C18:2ω-6) and linolenic (C18:3ω-3) acids and
eicosapentaenoic acid (EPA) (Haliloğlu et al. 2004). The
plankton of marine feeds presents low levels of ω-6 PUFA,
of which EPA and DHA are the predominant acids (Justi
et al. 2003). Thus, marine fish are distinguished by high
concentrations of ω-3 because they feed on plankton,
whereas freshwater fish mainly contains ω-6 fatty acids.
Many technologies have been developed for the extrac-
tion of fish oil in the food industry. Wet pressing is the most
common traditional process to obtain crude fish oil from
fresh fish at industrial scale, as described by the Food and
Agriculture Organization of the United Nations (FAO, FID
1986). The process involves the production of crude fish oil
through several steps, i.e., cooking of the raw material,
pressing of the cooked material and final filtration or cen-
trifugation to recover the oil from the extract (FAO, FID
1986). However, this is not feasible when the oil content
of the raw material is low (Rubio-Rodríguez et al. 2010).
New processes that use supercritical fluids are replacing
2 Journal of Food Processing and Preservation •• (2014) ••–•• © 2014 Wiley Periodicals, Inc.
S. FERDOSH ET AL.
conventional processes of lipid extraction. Supercritical
carbon dioxide (SC-CO2) is a low-temperature selective
extraction method that yields high-purity lipid extract
(Dron et al. 1997).
Tuna are economically important marine fish species in
Malaysia; however, there are no reports of total lipid
extracted by SC-CO2 and their fatty acid profile for the
by-products of T. tonggol, E. affinis and A. thazard from
Malaysian waters. Therefore, the objective of this research
was to determine the quality of tuna fish oils extracted from
the head, the skin and the viscera of three species of tropical
neritic tuna using SC-CO2. A secondary objective was to
compare these results with the data obtained by subjecting
the tissues to lipid extraction using the Soxhlet method.
EXPERIMENTAL PROCEDURE
Materials
Fresh tuna fish were collected from the fish landing center
of Batu Maung, Pulau Penang, Malaysia. The solvents used
for supercritical fluid extraction (SFE) were 99.9% pure
CO2 obtained from MOX-LINED gases Sdn Bhd (P.J., Selan-
gor, Malaysia) and absolute ethanol (99.7%) were provided
by QRëC (QREC [Asia] Sdn Bhd, Rawang, Malaysia). Puri-
fied helium as a carrier gas with a purity of 99.9% was also
purchased from MOX-LINED gases Sdn Bhd. Other sol-
vents used in this study were of analytical grade and were
obtained from QRëC (REC [Asia] Sdn Bhd).
Sample Preparation for the Experiments
Fresh fish samples were kept in plastic bags and transported
in an insulated icebox to the laboratory of Environmental
Technology, School of Industrial Technology, Universiti
Sains Malaysia, Malaysia. The samples were immediately
de-headed, gutted and washed with copious amounts of
cool water, and then the flesh and skin were separated using
a mechanical de-bonner. The head, skin and viscera were
stored at −20C separately, and then freeze-dried (model:
LABCONCO, Kansas City, MO) at a drying temperature of
−47C and under a vacuum of 0.133 bar. The dried samples
were kept at −80C until use. For experimental analysis, the
dried samples were ground with a dry mixer (Waring Labo-
ratory, Pompano Beach, FL) into particles ranging from 0.5
to 1.0 mm in size by being forced through a sieve.
Extraction of Total Oil Using the
Soxhlet Method
Five grams (dry weight) of head, skin and viscera powder
was weighed into a cellulose extraction thimble and covered
S. FERDOSH ET AL.
with glass wool and was extracted with 200 mL of n-hexane
for 6 h in a Soxhlet extractor. The experiment was carried
out in triplicate for each sample. The extracted oil was
evaporated under vacuum at 60C using a rotary evaporator
(Büchi-Rotavapor, Flawil, Switzerland) and then placed in
an oven at 45C for 1 h before being transferred into desicca-
tors before reweighing.
Supercritical Carbon Dioxide
(SC-CO2) Extraction
The experimental setup for the SC-CO2 extraction process
was followed according to Norulaini et al. (2009). The ISCO
SFE System (ISCO Inc., Lincoln, NE) consisting of a
supercritical fluid extractor (ISCO, SFX 220); a controller
(ISCO, SFX 200); a carbon dioxide cylinder; a chiller (Yih
Der BL-730); two syringe pumps (ISCO, model 100DX), a
CO2 pump and a co-solvent pump; and restrictor tempera-
ture controller associated with two coaxially heated capil-
lary restrictors (ISCO). The CO2 pump was fitted with a
cooling jacket to deliver CO2, and the co-solvent pump was
fitted to deliver co-solvents as modifiers/entrainers. To cool
up the pump’s head, an ethylene glycol–deionized water
mixture (50:50, v/v) was circulated through the cooling
jacket using a refrigerated bath circulator (model 631D,
Tech-Lab Manufacturing Sdn. Bhd., Selangor, Malaysia),
which can chill the coolant down to 0C. In each experiment,
2 g of ground sample (dry weight) was loaded into a 10-mL
sample cartridge and placed in the ISCO extraction
chamber and allowed to equilibrate at the desired tempera-
ture. The extractions were performed with CO2 and ethanol
(as a co-solvent) at 3 mL/min (2.4 mL CO2 and 0.6 mL
ethanol/ min, v/v), for 2 h continuously at 65C temperature
and 40 MPa pressure (Sahena et al. 2013). The restrictor was
maintained at 60C to avoid problems of restrictor plugging.
The extracts with ethanol were collected into a preweighed
blue cap bottle yield trap through a restrictor. The trap was
cooled with an ice–water mixture. The oil trap containing
the extracted oil with ethanol, as a residue of co-solvent, was
evaporated under vacuum at 40C using the rotary evapora-
tor (Büchi-Rotavapor), and then placed in the oven at 45C
for 30 min before being transferred into the desiccators.
The total oil yield and extractability were expressed as
percentage based on 100 g of ground dry sample, as
defined below:
Grams oil extracted
Yield (%) = × 100
Grams of sample
Total yield (g ) by SC-CO2 extaction
Extractibility (%) =
Total yield (g ) by solvent extraction
Journal of Food Processing and Preservation •• (2014) ••–•• © 2014 Wiley Periodicals, Inc.
TUNA FISH OILS EXTRACTED USING SC-CO2
Analysis of Fatty Acid
The fatty acid (FA) constituents of the total oil yield
extracted by the Soxhlet and SC-CO2 methods at 40 MPa,
3 mL/min and 65C was analyzed to determine the FA
profile. The FA constituents were analyzed by gas chroma-
tography (GC) with flame ionization detector (GC-2010
Plus AOC-5000, Shimadzu, Osaka, Japan). Fatty acid methyl
ester (FAME) was prepared by dissolving 50 mg of sample
into 0.95 mL of n-hexane and 0.05 mL of 1 M sodium
methoxide (30% methanol in sodium methoxide). The
mixture was then shaken vigorously using an auto-vortexer
(VF2, Janke and Kunkel, Staufen, Germany) for 30 s and
was allowed to stand for 5 min so that it formed a bilayer.
The clear upper layer containing the FAMEs (1 μL) was
pipette off and injected into the GC using the standard
PORIM (Palm Oil Research Institute of Malaysia 1995)
method. Supelco 37 component FAME mixture (Sigma-
Aldrich, Supelco, Bellefonte, PA) was used as the reference
standard fish oil (purity 99%). The BPX70 (70%
cyanopropyl polysilphenylene-siloxane [30 m × 0.32 mm ×
0.25 μm film thickness], SGE France) was purchased from
Sigma-Aldrich Co. (St. Louis, MO). The oven temperature
was set at 140C, held for 2 min, increased with a heating
rate of 5C/min up to a final temperature of 250C, and then
held for 10 min at 250C. Identification of chromatographic
peaks was performed using the retention time of the FAME
standard. The results of FA analysis were reported as the
average of three analyses for each sample investigated. The
FA components are expressed as a percentage by mass
of FAMEs.
Determination of Free Fatty Acid (FFA) and
Peroxide Value (PV)
Rancidity and oxidative stability analysis was conducted in
triplicate for each sample of tuna by-product oil extracted
by the Soxhlet and SC-CO2 methods. The percentage free
fatty acids (% FFA as oleic acid) and peroxide value (meq
O2/kg oil) were determined using the official method of
AOCS (2003).
Statistics
The experiments were performed in triplicate and each set
(1) of yields was averaged. The weighed means were derived
from an analysis of variance by Statistica version 10.0
(StatSoft Inc., Tulsa, OK). For tests of statistical significance
×100 between yield, FA and FFA from different species, the data
were subjected to unequal N Tukey’s HSD (honest signifi-
(2) cant difference) test for significant differences (P < 0.05).
3
TUNA FISH OILS EXTRACTED USING SC-CO2
RESULTS AND DISCUSSION
Total Oil Content
In this study, the total oil was extracted from the dissected
head, skin and viscera of the three species of tuna using
both Soxhlet as conventional method and SC-CO2 with
co-solvent method (Table 1). The total oil extracted using
Soxhlet was similar to the oil of SC-CO2 method and the
differences in the oil content among three species of tuna
extracted using both methods were nonsignificant. The oil
contents were significantly different among the dissected
body parts of the three species of tuna fish (Table 1). On the
contrary, the oil yield extracted from the same waste from
different species was found to be nonsignificant (Table 1).
In our study, the maximum lipid content was found in the
head of T. tonggol (36.2% d.b. [dry basis]) followed by the
skin of E. affinis (26.4% d.b.) and the viscera of A. thazard
(17.1% d.b.). This is in agreement with the reports of Vlieg
and Murray (1988). They extracted total oil by Folch
method from the head and viscera of albacore tuna, which
was 13.6 and 4.1% (w/w), respectively. Karunarathna and
Attygalle (2010) reported the oil content in the skin of
E. affinis and A. thazard to be 6.3 and 8.9% (w/w), respec-
tively. Orbital organ of T. tonggol contain 19.9% (w/w) total
lipid, as reported by Saito et al. (2005), which is in line with
the lipid content of the head of T. tonggol in our study. Lipid
content of fish can differ due to their size, age, season and
feeding habit (Saito et al. 2005; Selmi and Sadok 2010). The
oil yield was extracted at a lower temperature by SC-CO2
than the Soxhlet extraction, thus rendering a lower yield
than the Soxhlet method for all species and all dissected
parts (Table 1); the most possible reason was that at higher
temperature of Soxhlet extraction, the lipid cells ruptured to
a greater extent. In applying high temperature for the longer
extraction time of the Soxhlet method, some parts of fish
protein and other components may remain with the ren-
dered oil.
Fatty Acid Constituents in Tuna
By-Product Oil
The FA in terms of triglyceride constituents of head, skin
and viscera of T. tonggol are given in Table 2. The FA con-
Head Skin
Fish species Soxhlet SC-CO2 Soxhlet SC-CO2
Thunnus tonggol 36.2 ± 1.8 35.6 ± 2.4 22.4 ± 1.4 21.8 ± 1.8 13.6 ± 0.4 13.5c ± 0.6
a a b b
Euthynnus affinis 29.5a ± 0.9 28.4a ± 1.8 26.4b ± 1.7 24.8b ± 0.9 16.9c ± 0.9 16.1c ± 0.8
Auxis thazard 30.2a ± 1.3 29.5a ± 1.5 24.6b ± 0.8 23.8b ± 2.1 17.1c ± 0.6 16.8c ± 1.2
Note: Different superscript letters within rows indicate statistical difference at P < 0.05.
SC-CO2, supercritical carbon dioxide; SD, standard deviation.
4 Journal of Food Processing and Preservation •• (2014) ••–•• © 2014 Wiley Periodicals, Inc.
S. FERDOSH ET AL.
stituents in the oil extracted using both Soxhlet and SC-CO2
methods was found to be closer and the difference is non-
significant. Similarly, nonsignificant differences were found
in the FA composition of oil extracted from T. tonggol
(Table 2), E. affinis (Table 3) and A. thazard (Table 4).
Sahena et al. (2010) reported the FA composition of Indian
mackerel oil extracted by various techniques of SC-CO2 and
found that the differences were nonsignificant with the
Soxhlet extraction method.
The total amount of saturated fatty acid (SFA) constitu-
ents in the three dissected parts – head, skin and viscera – of
the three different species of tunas were found to be the
higher in percentage than either monounsaturated fatty
acid (MUFA) or PUFA. The viscera parts of all three species
contain significantly higher amount of total SFA than either
skin or head part. The viscera part of E. affinis contains the
highest amount of SFA (47.4–48.6%), followed by the skin
(41.4–41.9%) and head (41.3–41.4%) (Table 3). Similar
trend was found in T. tonggol, where SFA content was high
in the viscera (45.9–46.1%), followed by head (42.8–43.7%)
and skin (39.7–40.8%) (Table 2). In A. thazard, highest
(46.3–47.4%) SFA was found in the viscera part, followed
by head (42.6–43.2%) and skin (39.7–40.2%) (Table 4).
However, the SFA constituents of C16:0 (21.3–31.0%) was
the highest among all other SFA constituents in any dis-
sected parts of all three species of tuna, followed by C18:0
(5.8–9.2%) and C14:0 (4.3–6.1%). The differences among
the C16:0, C18:0 and C14:0 constituents in the head, skin
and viscera of all species of tuna were nonsignificant,
whereas significant differences were observed among the
total SFA between the by-products (Tables 2–4). Moreover,
the SFA content of C15:0 and C17:0 was claimed to be very
low (ranging from 0.7 to 1.8%) and the differences among
these FAs in three dissected parts – head, skin, viscera – of
all three species of tuna were nonsignificant.
Chantachum et al. (2000) reported that FA constituents
in the total oils extracted from both precooked and non-
precooked tuna head prepared under optimum conditions
(85C, 30 min) had high amounts of palmitic acid (C16:0),
oleic acid (C18:1), myristic acid (C14:0), stearic acid
(C18:0) and behenic acid (C22:0). Similarly, Intarasirisawat
et al. (2011) reported palmitic acid (C16:0) as the dominant
SFA constituent in the total lipids extracted from tuna roes.
Their findings are in line with our observations where
TABLE 1. COMPARISON OF TOTAL LIPID
Viscera
EXTRACTED BY SOXHLET AND SC-CO2 FROM
Soxhlet SC-CO2 NERITIC LITTLE TUNA WASTES (% DRY
c WEIGHT MEAN ± SD)
S. FERDOSH ET AL. TUNA FISH OILS EXTRACTED USING SC-CO2

TABLE 2. FATTY ACID COMPOSITION OF

Head Skin Viscera
THUNNUS TONGGOL WASTE OIL EXTRACTED Fatty acids
BY SOXHLET AND SC-CO2 (VALUES ARE (%) Soxhlet SC-CO2 Soxhlet SC-CO2 Soxhlet SC-CO2
MEAN ± SD) C14:0 5.6a ± 0.1 5.3a ± 0.1 4.5b ± 0.1 3.9b ± 0.1 4.4b ± 0.3 4.3b ± 0.1
C15:0 1.6a ± 0.1 1.6a ± 0.2 1.3a ± 0.1 1.7a ± 0.1 0.9b ± 0.1 1.2a ± 0.3
C16:0 27.8b ± 0.3 27.1b ± 0.2 25.7b ± 0.4 26.7b ± 0.3 31.0a ± 0.5 30.8a ± 0.7
C17:0 1.1a ± 0.1 1.1ab ± 0.1 0.7ab ± 0.1 1.1a ± 0.1 1.3a ± 0.3 1.1a ± 0.1
C18:0 6.2a ± 0.3 6.4a ± 0.2 6.2a ± 0.1 6.1a ± 0.1 6.9a ± 0.1 7.4a ± 0.07
C22:0 1.5 ± 0.1 1.4 ± 0.1 1.4 ± 0.0 1.4 ± 0.0 1.3 ± 0.0 1.4 ± 0.2
16:1n7 4.8b ± 0.3 5.1ab ± 0.2 5.9ab ± 0.2 6.5a ± 0.2 4.7b ± 0.2 5.4ab ± 0.1
18:1n9 14.0a ± 0.4 13.4a ± 0.3 13.8a ± 0.3 13.1a ± 0.2 12.8b ± 0.1 13.1b ± 0.2
18:1n7 2.1b ± 0.1 2.4b ± 0.1 2.3b ± 0.2 2.9ab ± 0.1 3.6a ± 0.3 2.8b ± 0.1
20:1n9 1.8a ± 0.1 1.5a ± 0.1 1.4a ± 0.1 1.2a ± 0.1 1.2a ± 0.1 0.9a ± 0.0
22:1n11 1.4a ± 0.2 1.4a ± 0.2 2.1a ± 0.2 1.9a ± 0.3 1.6a ± 0.2 1.1a ± 0.0
18:2n6 1.0a ± 0.1 1.2a ± 0.1 1.5a ± 0.1 1.4a ± 0.1 1.7a ± 0.1 1.4a ± 0.1
18:4n3 1.7b ± 0.1 1.1a ± 0.1 1.4a ± 0.1 1.0ab ± 0.1 0.5b ± 0.1 0.6b ± 0.0
20:4n6 3.6a ± 0.2 3.6a ± 0.2 3.2a ± 0.3 3.3a ± 0.2 3.8a ± 0.3 3.3a ± 0.2
20:5n3 1.6b ± 0.1 1.4b ± 0.1 1.4b ± 0.1 1.7b ± 0.1 2.5a ± 0.1 2.6a ± 0.3
22:5n3 0.7b ± 0.1 0.7b ± 0.1 1.5a ± 0.1 1.1a ± 0.1 0.8b ± 0.1 1.1a ± 0.1
22:6n3 19.0a ± 0.8 19.9a ± 0.6 17.0b ± 0.7 17.3b ± 0.8 16.1c ± 0.8 15.8c ± 0.6
ΣSFA 43.7b ± 0.9 42.8b ± 0.7 39.7c ± 0.9 40.8c ± 0.5 45.9a ± 0.7 46.1a ± 0.5
ΣMUFA 24.1bc ± 0.5 23.8bc ± 0.8 25.5a ± 0.6 25.5a ± 0.9 23.9c ± 0.6 23.2c ± 0.8
ΣPUFA 27.8a ± 0.6 27.9a ± 0.4 26.0a ± 0.3 25.8ab ± 0.5 25.3b ± 0.8 24.7b ± 0.4
Other FAs 4.3b ± 0.1 5.5b ± 0.1 8.8a ± 0.2 7.9a ± 0.2 4.9b ± 0.2 5.9b ± 0.2

Note: Different superscript letters within rows indicate statistical difference at P < 0.05.
FA, fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SC-CO2,
supercritical carbon dioxide; SD, standard deviation; SFA, saturated fatty acid.

TABLE 3. FATTY ACID COMPOSITION OF

Head Skin Viscera
EUTHYNNUS AFFINIS WASTE OIL EXTRACTED Fatty acids
BY SOXHLET AND SC-CO2 (VALUES ARE (%) Soxhlet SC-CO2 Soxhlet SC-CO2 Soxhlet SC-CO2
MEAN ± SD) C14:0 4.9 ± 0.2
a
5.0 ± 0.1
a
5.3 ± 0.1
a
5.2 ± 0.3
a
5.7 ± 0.1
a
5.3a ± 0.3
C15:0 1.8a ± 0.1 1.6a ± 0.1 1.2a ± 0.1 0.8a ± 0.1 1.4a ± 0.1 1.1a ± 0.1
C16:0 25.1b ± 1.0 26.1b ± 1.6 26.2b ± 1.0 26.1b ± 0.8 29.4a ± 0.6 29.7a ± 0.8
C17:0 1.3a ± 0.1 0.8ab ± 0.1 0.8b ± 0.0 1.5a ± 0.1 1.5a ± 0.1 1.2a ± 0.0
C18:0 6.9b ± 0.2 6.5b ± 0.2 7.1b ± 0.4 6.7b ± 0.1 9.2a ± 0.2 8.8a ± 0.6
C22:0 1.4 ± 0.1 1.4 ± 0.1 1.4 ± 0.1 1.3 ± 0.0 1.5 ± 0.1 1.4 ± 0.1
16:1n7 5.3a ± 0.2 5.9a ± 0.2 6.1a ± 0.3 6.5a ± 0.1 5.9a ± 0.3 5.3a ± 0.4
18:1n9 13.9a ± 0.5 14.8a ± 1.4 14.4a ± 0.3 14.1a ± 0.7 11.9b ± 0.9 12.6b ± 0.3
18:1n7 2.9a ± 0.4 2.5a ± 0.2 2.8a ± 0.1 2.6a ± 0.3 2.9a ± 0.1 2.2a ± 0.2
20:1n9 1.5a ± 0.1 1.5a ± 0.1 1.5a ± 0.2 1.4a ± 0.1 0.8a ± 0.0 1.2a ± 0.1
22:1n11 1.6a ± 0.3 1.5a ± 0.2 1.1a ± 0.1 2.0a ± 0.2 0.5b ± 0.0 0.7b ± 0.0
18:2n6 1.3a ± 0.2 1.4a ± 0.2 0.8b ± 0.1 1.5a ± 0.1 1.4a ± 0.1 1.5a ± 0.1
18:4n3 1.0ab ± 0.1 0.9b ± 0.1 2.0a ± 0.2 1.8a ± 0.3 0.5b ± 0.0 1.2ab ± 0.1
20:4n6 3.2b ± 0.2 3.6b ± 0.1 3.2ab ± 0.2 2.6b ± 0.1 3.2a ± 0.4 3.6a ± 0.2
20:5n3 1.3c ± 0.1 1.7c ± 0.2 1.7c ± 0.0 1.9c ± 0.2 3.7a ± 0.2 2.9bc ± 0.3
22:5n3 0.9b ± 0.1 0.6b ± 0.1 0.6b ± 0.1 1.4a ± 0.1 1.0a ± 0.3 1.3a ± 0.2
22:6n3 17.8a ± 1.0 18.0a ± 1.4 16.9b ± 0.6 15.7bc ± 0.8 14.3c ± 0.9 14.9c ± 0.6
ΣSFA 41.4b ± 0.4 41.3b ± 0.6 41.9b ± 1.0 41.4b ± 1.3 48.5a ± 0.6 47.4a ± 1.2
ΣMUFA 25.3a ± 0.7 26.2a ± 0.7 25.9a ± 1.1 26.6a ± 0.6 22.1b ± 0.9 21.9b ± 0.6
ΣPUFA 25.4a ± 0.9 26.2a ± 0.7 25.3b ± 0.6 24.8bc ± 1.1 24.1c ± 1.0 25.3b ± 0.7
Other FAs 7.9ab ± 0.1 6.2b ± 0.4 7.9a ± 0.2 8.5a ± 0.3 5.2b ± 0.2 5.4b ± 0.1

Note: Different superscript letters within rows indicate statistical difference at P < 0.05.
FA, fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SC-CO2,
supercritical carbon dioxide; SD, standard deviation; SFA, saturated fatty acid.

Journal of Food Processing and Preservation •• (2014) ••–•• © 2014 Wiley Periodicals, Inc. 5
TUNA FISH OILS EXTRACTED USING SC-CO2 S. FERDOSH ET AL.

TABLE 4. FATTY ACID COMPOSITION OF

Head Skin Viscera
Fatty acids AUXIS THAZARD WASTE OIL EXTRACTED BY
(%) Soxhlet SC-CO2 Soxhlet SC-CO2 Soxhlet SC-CO2 SOXHLET AND SC-CO2 (VALUES ARE
C14:0 5.3a ± 0.2 5.7a ± 0.2 6.1a ± 0.4 5.9a ± 0.2 4.7b ± 0.4 4.6b ± 0.3 MEAN ± SD)
C15:0 1.5a ± 0.1 0.9a ± 0.1 1.3a ± 0.2 1.7a ± 0.2 1.3a ± 0.1 1.3a ± 0.0
C16:0 27.4b ± 0.3 28.1b ± 0.4 21.6c ± 0.8 21.3c ± 1.2 29.7a ± 0.9 30.6a ± 0.9
C17:0 1.1a ± 0.1 1.3a ± 0.2 1.6a ± 0.1 1.6a ± 0.2 1.2a ± 0.2 1.4a ± 0.3
C18:0 6.2c ± 0.3 5.8c ± 0.3 8.1a ± 0.2 7.9ab ± 0.3 7.8ab ± 0.2 8.2a ± 0.3
C22:0 1.4 ± 0.0 1.4 ± 0.1 1.4 ± 0.1 1.4 ± 0.0 1.4 ± 0.0 1.4 ± 0.1
16:1n7 6.4ab ± 0.4 5.7b ± 0.3 7.7a ± 0.6 5.9ab ± 0.1 4.4c ± 0.2 4.6c ± 0.4
18:1n9 12.8b ± 0.6 13.6b ± 1.0 12.6b ± 0.6 12.7b ± 0.4 14.8a ± 0.7 13.8ab ± 0.9
18:1n7 2.2a ± 0.2 2.2a ± 0.1 2.4a ± 0.2 2.7a ± 0.1 2.8a ± 0.2 2.6a ± 0.2
20:1n9 1.1a ± 0.1 1.1a ± 0.1 1.3a ± 0.1 1.4a ± 0.3 0.9a ± 0.2 1.3a ± 0.2
22:1n11 0.9bc ± 0.1 1.0ab ± 0.1 1.9a ± 0.2 1.8a ± 0.1 0.5c ± 0.1 0.7c ± 0.1
18:2n6 1.6a ± 0.4 1.5a ± 0.2 0.9a ± 0.1 1.1a ± 0.2 1.0a ± 0.2 1.1a ± 0.0
18:4n3 1.4bc ± 0.1 0.9c ± 0.1 1.3ab ± 0.1 1.6a ± 0.2 0.9c ± 0.2 0.7c ± 0.1
20:4n6 3.1b ± 0.2 3.4ab ± 0.2 3.1a ± 0.3 2.9a ± 0.1 3.5a ± 0.2 3.7a ± 0.2
20:5n3 2.1b ± 0.1 2.3b ± 0.1 2.0a ± 0.2 2.7ab ± 0.2 3.0a ± 0.1 2.9a ± 0.2
22:5n3 1.5ab ± 0.3 0.9b ± 0.1 1.0b ± 0.2 1.1ab ± 0.1 1.6a ± 0.0 1.6a ± 0.1
22:6n3 17.1a ± 0.8 17.0a ± 1.3 15.9b ± 0.9 16.2b ± 0.9 15.9b ± 0.8 15.4b ± 0.6
ΣSFA 42.6bc ± 1.1 43.2b ± 1.2 40.2c ± 0.6 39.7cd ± 0.7 46.3a ± 0.9 47.4a ± 0.9
ΣMUFA 23.4bc ± 1.1 23.7bc ± 0.9 25.9a ± 0.9 24.4a ± 0.8 23.4c ± 0.4 22.9c ± 1.8
ΣPUFA 26.9a ± 0.8 25.9a ± 0.7 24.1b ± 0.6 25.6b ± 0.4 25.8b ± 1.0 25.4b ± 0.9
Other FAs 5.1bc ± 0.3 5.2bc ± 0.1 9.9 ± 0.1 9.7a ± 0.3 4.5c ± 0.2 4.3c ± 0.1

Note: Different superscript letters within rows indicate statistical difference at P < 0.05.
FA, fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SC-CO2,
supercritical carbon dioxide; SD, standard deviation; SFA, saturated fatty acid.

among the SFA, palmitic acid (C16:0) was found to be the
highest, which could be regarded the dominant fatty acid in
the total oil extracted using both Soxhlet and SC-CO2
extraction methods (Tables 2–4).
The total amount of MUFA constituents was high in the
skin, followed by head and viscera in all three species of
tuna fish (Tables 2–4). The highest amount of MUFA was
about 26.6% in the skin of E. affinis (Table 3), followed by
the skin (about 25.9%) of A. thazard (Table 4) and skin
(about 25.5%) of T. tonggol (Table 2). However, the differ-
ences in the MUFA in the skin of all three species were non-
significant. The percentage of C18:1n9 constituent was
higher (ranged from 11.9 to 14.8%) than any other MUFA
constituents among all dissected parts: head, skin and
viscera of all three species of tuna followed by C16:1n7
(ranging from 4.4 to 7.7%) and C18:1n7 (ranging from 2.1
to 3.6%). However, the differences in these three types of
MUFA constituents among the body parts of all three
species were nonsignificant, whereas the differences in the
percentages of other MUFA, e.g., C20:1n9 and C22:1n11
were nonsignificant in all the dissected parts in three species
of tuna fish, and the specific amount of those MUFA could
be considered as low (0.5–2.2%).
Other studies also reported the short-chain FA content to
be very low in the total oil extracted from fish by-products,
where the oleic acid (C18:1) is the dominant fatty acid, fol-
lowed by palmitoleic acid (C16:1) among the MUFA
(Stansby et al. 1990; Chantachum et al. 2000). On the con-
trary, Intarasirisawat et al. (2011) extracted total lipids from
tuna roes and reported oleic acid (C18:1n-9) constituent as
the main MUFA. In all the samples, the palmitoleic acid
(C16:1n-7) and cis-vaccenic acid (C18:1n-7) as the n-7 fatty
acids were found to be low. This is because the n-7 fatty
acids, especially the palmitoleic acid and cis-vaccenic acid,
are ubiquitous minor components in animal tissue
(Intarasirisawat et al. 2011).
The total amount of PUFA was significantly highest
(ranging from 25.4 to 27.9%) in the head part, followed by
the skin (ranging from 24.1 to 26.0%) and viscera (ranging
from 24.1 to 25.8%) parts. The C22:6n3 PUFA, defined as
DHA itself, was more than 80% among all other PUFA con-
stituents in all the individual dissected parts of head, skin
and viscera of all three species. The highest amount of DHA
(19.9%) was found in the head of T. tonggol (Table 2), fol-
lowed by 18.0% in the head of E. affinis (Table 3) and 17.1%
in the head of A. thazard (Table 4). The second highest
DHA content was observed in the skin of all three species of
tuna, where the highest amount (17.3%) of DHA was found
in T. tonggol (Table 2), followed by E. affinis (16.9%)
(Table 3) and A. thazard (16.2%) (Table 4). Similarly, the
last highest trend of DHA content was observed in the
viscera of all three species, where 16.1% DHA was obtained
in T. tonggol (Table 2), followed by 15.9% in A. thazard
(Table 4) and 14.9% in E. affinis (Table 3). Lipids from all

6 Journal of Food Processing and Preservation •• (2014) ••–•• © 2014 Wiley Periodicals, Inc.
S. FERDOSH ET AL.
tuna species by-products – head, skin and viscera – con-
tained a low level of EPA, ranging from 1.3 to 3.7%
(Tables 2–4). However, the viscera contains the highest
amount of EPA, followed by the skin and head in all three
species of tuna. The other PUFA, e.g., C18:2n6, C18:4n3,
C20:4n6 and C22:5n3, nonsignificantly exhibited at the
range of 0.5–3.8% within the dissected parts of all three
species of the tuna fish.
According to Stansby et al. (1990), who studied various
species of tuna, DHA is often considerably higher in the
total oils than in the total oils of most other species.
Shimada et al. (1997) also reported higher amount of
DHA (22.9%) than EPA (6.5%) in tuna oils. Similarly,
Chantachum et al. (2000) found that tuna oil from the head
exhibited a much higher content of DHA (18.8–25.5%)
than EPA (0.1%). Intarasirisawat et al. (2011) extracted total
lipids from tuna roes, which contained high amount of
DHA (20.5–26.2% of total lipids) along with the phospho-
lipids as the major component (51.2–54.9% of total lipids).
The authors claimed that DHA, an important n-3 fatty acid,
was the dominant PUFAs and attributed as much higher
than the EPA in all samples. Eyes in the tuna head could be
an important source of DHA, whereas the EPA content was
negligible (Chantachum et al. 2000). The authors also
reported that DHA contents in both crude oils from pre-
cooked and non-precooked tuna heads exhibited slightly
higher content of DHA in the former ones; thus, it could be
concluded that the high heat/temperature possibly resulted
in a higher release of phospholipids located in the cell mem-
brane. This could be the reason for the small differences in
the total oil content as well as in the fatty acid constituents
between the oils extracted from the different dissected parts
by Soxhlet and SFE methods in our study. The important
and essential characteristic of the tuna family is that the
DHA is still rich in the total oils of all tissues throughout
the dissected body parts (Saito et al. 2005).
Saito et al. (1999) analyzed the FA composition of the
lipid extracted from fish muscle and fish wastes: liver,
viscera, stomach of three highly migratory tuna species,
which belong to the tribes of tunas such as Euthynnus,
Thunnus and Auxis. The authors claimed that the DHA
content is comparatively high in the total lipid of all migra-
tory tuna species belonging to the tribes of tuna. Our obser-
vation is similar to that of other tuna species containing
comparatively high DHA among PUFA in the total oils of
other highly migratory tuna species belonging to the tribe
Thunnini (Medina et al. 1995; Saito et al. 1999, 2005). Total
SFA was always higher than total MUFA and total PUFA in
all dissected part in all species of tuna in our study. It might
be due to high seawater temperature due to tropical weather
in Malaysia. However, the visceral lipids contained lower
DHA level than the head and skin parts, which might be
dominated by prey lipids. The observation however is
Journal of Food Processing and Preservation •• (2014) ••–•• © 2014 Wiley Periodicals, Inc.
TUNA FISH OILS EXTRACTED USING SC-CO2
similar to the observation of SFA, MUFA and PUFA in the
total oils of T. tonggol (Saito et al. 2005). Saito et al. (1999)
also claimed relatively higher DHA in the total lipid content
of tissue of two tuna species, e.g., E. affinis and S. orientalis,
than their stomach contents. Thus, it may be concluded that
highly migratory marine fish such as tuna species, although
dominated mainly with SFA constituents in their total oil
but it is the incorporation of MUFA and PUFA where accu-
mulation of DHA is compulsory and relatively high among
all other PUFA, and this is due to SFA and MUFA in any
fatty fish. E. affinis, which belongs to the tribe Thunnini, is
expected to have a high level of DHA similar to that of other
Thunnini species such as bonito E. pelamis (Medina et al.
1995).
Saito et al. (2005) analyzed the lipid and fatty acid com-
positions of various dissected organs, including the stomach
contents of a highly migratory fish species T. tonggol. The
authors also reported that SFA, such as 16:0, 18:0 and
18:1n-9, and MUFA are major components in marine fish
rather than the DHA contents, which attributed less than
20% of the total fatty acids. The authors claimed high levels
of DHA in the neutral lipids a characteristic of tuna species,
such as the orbital fats and head oils. However, their obser-
vations are in agreement with the findings of this study
where only four fatty acid constituents were found as major
fatty acids in terms of tryglyceride compositions of all the
organs. These fatty acids could be listed as SFA, 16:0 and
18:0, and as MUFA, 18:1n-9, and n-3 PUFA: DHA as shown
in Tables 2–4. However, these reports strongly support our
observations, although there is a small difference in the FA
constituents compared with those reported by the above-
mentioned researchers. This was probably due to the differ-
ences in species, locations, nature of migration, food habit
of the tuna as well as the processing of the raw material
and the differences in the extraction methods of fish oil
extraction.
Whereas the head part contains the highest amount of
total PUFA followed by the skin and viscera part, and the
differences were significant. On the contrary, reverse trend
was found in the total MUFA, and it was high in the skin
part followed by viscera and head parts, and the differences
were not significant. The trend of neither PUFA nor MUFA
was found in the total amount of SFA content, which was
high in the viscera part followed by the head and skin parts,
and the differences were significant. These results are in line
with the studies of Saito et al. (2005) and Karunarathna and
Attygalle (2010).
Some unidentified FAs defined as other FAs were
observed in all dissected parts of all three species. These
other FAs could be SFA, MUFA and/or PUFA, and those FAs
were unidentified. The reason of this unidentification may
be due to the experimental errors or due to the limitation of
either using proper GC column, program settings especially
7
TUNA FISH OILS EXTRACTED USING SC-CO2 S. FERDOSH ET AL.

TABLE 5. FREE FATTY ACID AND PEROXIDE VALUE OF LITTLE TUNA WASTE OIL EXTRACTED BY SOXHLET AND SC-CO2 METHOD (VALUES ARE
MEAN ± SD)

Head Skin Viscera

Soxhlet SC-CO2 Soxhlet SC-CO2 Soxhlet SC-CO2
Free fatty acids (% as oleic acid)
Thunnus tonggol 2.3cd ± 0.2 1.8d ± 0.2 2.9c ± 0.1 1.8d ± 0.1 5.0a ± 0.4 3.8b ± 0.2
Euthynnus affinis 3.1c ± 0.1 2.6d ± 0.1 3.2c ± 0.1 2.7d ± 0.1 4.7a ± 0.1 3.9b ± 0.3
Auxis thazard 2.9cd ± 0.1 1.9e ± 0.1 3.2cd ± 0.2 2.5d ± 0.2 4.6a ± 0.3 3.6bc ± 0.3
Peroxide value (meq O2/kg oil)
Thunnus tonggol 1.7cd ± 0.2 1.2e ± 0.07 1.9b ± 0.10 1.4de ± 0.2 2.4a ± 0.2 1.9bc ± 0.1
Euthynnus affinis 1.6cd ± 0.1 1.2e ± 0.14 1.7b ± 0.16 1.4d ± 0.1 2.1a ± 0.2 1.8bc ± 0.1
Auxis thazard 1.8cd ± 0.1 1.3e ± 0.17 1.9b ± 0.08 1.6d ± 0.2 2.2a ± 0.1 2.0bc ± 0.2

Note: Different superscript letters within rows indicate statistical difference at P < 0.05.
SC-CO2, supercritical carbon dioxide; SD, standard deviation.

temperature setting and/or selecting GC standards.
However, the ranges of the other FAs were observed within
4.0–10% and they could be considered as low and
ignorable.
According to the report of Japan Aquatic Oil Association
(1989), marine fish species generally contain wide ranges of
FAs because they are on marine grazing food such as
phytoplanktones through zooplantone and micronektons
from where most of the lipids as well as PUFA are origi-
nated in fish. Fish oil is one of the main sources of marine
bioactive compound that plays a vital role in many biologi-
cal functions, which is potential for increasing the range of
functional seafood products (Kaur and Das 2011). Huynh
et al. (2007) reported that oleic acid (C18:1) plays a vital
role in energy metabolism during gonad development,
whereas palmitic acid (C16:0) is used as a source of poten-
tial metabolic energy during growth and roe formation in
fish. Goldberg et al. (2009) stated that palmitoleic acid is
involved in the regulation of insulin and systemic metabolic
homeostasis associated with type 2 diabetes, obesity, athero-
sclerosis and inflammatory disorders.
FFA and PV of Extracted Oil
The FFA content of oils extracted by Soxhlet and SC-CO2
method was studied to determine the effect of extraction
method on oil quality. According to the quality guideline for
edible crude fish oil, FFA and PV content should vary
usually between 2–5% and 3–20 meq/kg (Bimbo 1998).
Table 5 presents the levels of FFA and PV of oil extracted
from the head, skin and viscera of T. tonggol, E. affinis and
A. thazard. The FFA content of oil extracted by Soxhlet and
SC-CO2 method ranged from 2.3 to 5.0% and from 1.8 to
3.9%, respectively. Tuna viscera contains the highest PV
(1.9–2.4 meq/kg), followed by the skin (1.4–1.9 meq/kg)
and head (1.2–1.8 meq/kg). Generally, oils prepared at
higher temperature had higher FFA value (Chantachum
et al. 2000). Aryee and Simpson (2009) studied the effect of
extraction method on the FFA content of the oils. The
authors also reported that highest amount of FFA was
found in Soxhlet method than other methods reported,
which is in line with our results. A relatively lower FFA
content of SC-CO2 extracted oil in this study can be attrib-
uted due to low temperature and short extraction time. Fish
oil is highly susceptible to oxidative damage and the rate of
oxidation increases with increasing unsaturated fatty acid
content in oil. Therefore, mild extraction methods are pre-
ferred to minimize oxidative deterioration as well as forma-
tion of undesirable co-products (Aryee and Simpson 2009).
In all species of tuna by-products, viscera oil contains the
highest content of FFA and PV, followed by the skin and
head (Table 5). This higher percentage of FFA reflects the
degree of lipid hydrolysis in the raw material, mainly caused
by enzymatic and bacterial activity from microorganisms or
biological tissues (Ashie et al. 1996). The quality of SC-CO2
extracted fish by-product oil was superior to that of Soxhlet
extracted oil.
CONCLUSIONS
Among the individual body parts, the head is one of the
main waste parts of any species of tuna fish that is usually
discarded as major by-products during tuna processing in
fish industry. The total oil content was found higher
(36.2%) in the head part compared to any other individual
part of any species of tuna fish. The SC-CO2 extraction with
co-solvent could be applied as green processing method of
obtaining maximum and organic residue-free oil yield.
However, a part of total SFA and total MUFA, very high
amount of total PUFA constituents, ranging from 24.1 to
27.9%, was observed in all dissected parts such as the head,
skin and viscera individually in all three tuna species
analysed in this study. Among the PUFA constituents, DHA
alone was about 80% of the total PUFA in the tuna
by-products, where the head part was regarded to have the

8 Journal of Food Processing and Preservation •• (2014) ••–•• © 2014 Wiley Periodicals, Inc.
S. FERDOSH ET AL.
highest content of PUFA, especially DHA, among all other
by-product parts of all three species of tuna fish. Moreover,
the quality of the total oil extracted using both Soxhlet and
SC-CO2 methods were acceptable as relatively lower FFA
content was observed.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial support
under Research University Grant No. 1001/PTKIND/845032
provided by the Universiti Sains Malaysia, Malaysia, to carry
out this study.
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