PUFAs in Fish:

Extraction,
Fractionation,
Importance
in Health
F. Sahena, I.S.M. Zaidul, S. Jinap, N. Saari, H.A. Jahurul,
K.A. Abbas, and N.A. Norulaini

ABSTRACT: Polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA), are currently in demand in the pure form and actively being studied to understand their potential roles in
human health. Arachidonic acid, 20:4 (n-6), and DHA, 22:6 (n-3), are important in normal neurodevelopment and
visual function. Infants fed formula often have low blood lipid 20:4 (n-6) and 22:6 (n-3). Consumption of fish oils may
increase the 20:5 (n-3) (EPA) and 22:6 (n-3) (DHA) in human blood. Some marine fish oils contain higher amounts
of arachidonic acid, EPA, and DHA. PUFA contents in different marine fishes and methods for their extraction and
fractionation, in terms of fatty acid constituents in the form of methyl esters, are covered in this review. Emphasis is
given to the fractionations of EPA and DHA by means of supercritical fluid extractions (SFE). The advantages of SFE
compared to conventional methods are discussed in this review. PUFAs are usually extracted at about 10 to 30 MPa
and at 40 to 80 ◦ C. SFE is a promising and currently the best technique to extract PUFAs, especially EPA and DHA,
from marine and freshwater fish.

Introduction
Fish oils are a readily available source of long-chain polyun-
saturated fatty acids (PUFAs), especially those of the n-3 se-
ries, mainly cis-5,8,11,14,17-eicosapentaenoic acid (EPA; C20:5)
and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA; C20:6)
(Table 1). Recognition of the roles played by these fatty acids
in human health and nutrition (Bang and Dyerberg 1972; Braden
and Carrol 1986; Simopoulos 1991) and the resulting growth in
new markets have stimulated much interest in methods of extract-
ing and concentrating them from natural sources. These PUFAs
occur as triglycerides (TG), also called triacylglycerols, in fish oils
at levels between 10% and 25% (Haraldsson and others 1995).
The nature and quantity of fish lipids vary according to species
and habitats (Shamsudin and Salimon 2006). It is well known
that fish lipids are the main sources of PUFAs, especially EPA and
DHA (Osman and others 2001). These 2 fatty acids cannot be
synthesized by the human body and must be obtained from the
diet (Linko and Hayakawa 1996).
Fish oil is the main source of ω-3 fatty acids, therefore, the fatty
acid composition of fish oil is very important for understanding
its functional properties (von Schacky and others 1999; Nestel
MS 20080594 Submitted 8/3/2008, Accepted 12/4/2008 . Authors Sahena,
Zaidul, Jinap, Saari, Jahurul, and Abbas are with Faculty of Food Science
and Technology, Univ. Putra Malaysia, 43400 UPM Serdang, Selangor DE,
Malaysia. Author Norulaini is with School of Distant Education, Univ. Sains
Malaysia, 11800 USM, Pulau Pinang, Malaysia. Direct inquiries to author
Zaidul (E-mail: zaidul@food.upm.edu.my).
2000) and, in particular, the correct ratio between ω-3 and ω-6
fatty acids, which is very important. Fish oils typically contain
unsaturated straight-chain fatty acids, ranging from C14 to C22,
having from 1 to 6 double bonds. Fish oil derivatives in the form
of ω-3 fatty acids are increasingly demanded as pharmaceutical
products, food additives, and dietary health supplements. Inter-
est in ω-3 fatty acids (such as C18:3 alpha-linolenic acid [ALA]),
C20:5 eicosapentaenoic acid (EPA), and C22:6 docosahexaenoic
acid (DHA) began several years ago and now there is an extensive
scientific literature supporting their positive role in human health,
because they can intervene in the prevention and modulation of
certain diseases that are common in many populations; research
studies have shown that ω-3 can reduce the risk of heart disease
and high blood pressure, prevent blood clots, protect against can-
cer, and even alleviate depression (Shahar and others 1994; von
Schacky and others 1999). Among the compounds of interest are
concentrates of EPA (5cis, 8cis, 11cis, 14cis, 17cis eicosapen-
taenoic acid) and DHA (4cis, 7cis, 10cis, 13cis, 16cis, 19cis do-
cosahexaenoic acid) (Table 1). They have pharmaceutical value
in the prevention of atherosclerosis, heart attack, hypertension,
and cancer (FAO 1998). Clinical trials have shown that fish oil
supplementation is effective in the treatment of many disorders
including rheumatoid arthritis (Kremer 2000) and diabetes. Also,
results of clinical and epidemiological research suggest that EPA
and DHA, found only in fish and seafood, have extremely ben-
eficial properties for the prevention of human coronary artery
disease (Leaf and Weber 1998).
Along with this knowledge, an increasing interest in suitable
commercial processes to recover PUFAs in concentrated form


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Vol. 8, 2009—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 59
CRFSFS: Comprehensive Reviews in Food Science and Food Safety
Table 1 --- Polyunsaturated fatty acids.
Fatty acids name Commercial name Chemical name

Omega-3 fatty acids

16:3 (n-3) all-cis 7,10,13-hexadecatrienoic acid
18:3 (n-3) Alpha-linolenic acid (ALA) all-cis-9,12,15-octadecatrienoic acid
18:4 (n-3) Stearidonic acid (STD) all-cis-6,9,12,15,-octadecatetraenoic acid
20:3 (n-3) Eicosatrienoic acid (ETE) all-cis-11,14,17-eicosatrienoic acid
20:4 (n-3) Eicosatetraenoic acid (ETA) all-cis-8,11,14,17-eicosatetraenoic acid
20:5 (n-3) Eicosapentaenoic acid (EPA) all-cis-5,8,11,14,17-eicosapentaenoic acid
22:5 (n-3) Docosapentaenoic acid (DPA, Clupanodonic acid) all-cis-7,10,13,16,19-docosapentaenoic acid
22:6 (n-3) Docosahexaenoic acid (DHA) all-cis-4,7,10,13,16,19-docosahexaenoic acid
24:5 (n-3) Tetracosapentaenoic acid all-cis-9,12,15,18,21-tetracosapentaenoic acid
24:6 (n-3) Tetracosahexaenoic acid (Nisinic acid) all-cis-6,9,12,15,18,21-tetracosahexaenoic acid
Omega-6 fatty acids
18:2 (n-6) Linoleic acid all-cis-9,12-octadecadienoic acid
18:3 (n-6) Gamma-linolenic acid (GLA) all-cis-6,9,12-octadecatrienoic acid
20:2 (n-6) Eicosadienoic acid all-cis-11,14-eicosadienoic acid
20:3 (n-6) Dihomo-gamma-linolenic acid (DGLA) all-cis-8,11,14-eicosatrienoic acid
20:4 (n-6) Arachidonic acid (AA) all-cis-5,8,11,14-eicosatetraenoic acid
22:2 (n-6) Docosadienoic acid all-cis-13,16-docosadienoic acid
22:4 (n-6) Adrenic acid all-cis-7,10,13,16-docosatetraenoic acid
22:5 (n-6) Docosapentaenoic acid (Osbond acid) all-cis-4,7,10,13,16-docosapentaenoic acid
Omega-9 fatty acids,
mono and polyunsaturated
18:1 (n-9) Oleic acida cis-9-octadecenoic acid
20:1 (n-9) Eicosenoic acida cis-11-eicosenoic acid
20:3 (n-9) Mead acid all-cis-5,8,11-eicosatrienoic acid
22:1 (n-9) Erucic acida cis-13-docosenoic acid
24:1 (n-9) Nervonic acida cis-15-tetracosenoic acid
a Monounsaturated.

can be monitored. The conventional methods for their extraction,

fractionation, and purification include vacuum distillation, urea
crystallization, hexane extraction, and conventional crystalliza-
tion, all having the disadvantages of requiring high-temperature
processing, resulting in loss or decomposition of the thermally la-
bile compounds, or employing flammable or toxic solvents (Staby
and Mollerup 1993). Therefore, extraction with supercritical flu-
ids as solvents, especially carbon dioxide, offers new opportu-
nities for the solution of separation problems (Brunner and Riha
2000). Supercritical carbon dioxide is a promising solvent for the
extraction and fractionation of edible oils containing labile PU-
FAs. Extraction can be carried out at lower pressure and lower
temperature. Besides, supercritical fluid extraction (SFE) offers
new opportunities thanks to the fact that CO 2 is a nontoxic, non-
flammable, inexpensive, and clean solvent. However, the pur-
poses of this review were to identify the higher content of PUFAs,
especially EPA and DHA, in cheaper fish sources, and their better
methods of separation, purification, and fractionation.

Chemistry
Polyunsaturated fatty acids (PUFAs) are classified according
to the position of double bonds either from the methyl end or
the carboxyl end. In the “omega” or “n” convention, the double
bonds are counted from the methyl end of the carbon chain. A Figure 1 --- Family of ω-3 fatty acids (source: Mishra and
majority of PUFAs of biological importance belong to the ω-6 others 1993).
(arachidonic acid) and ω-3 (eicosapentaenoic acid) groups. The
1st double bond in ω-3 fatty acids starts at the 3rd carbon atom rupted) cis-type double-bond configuration, the carbon chain is
from the methyl end. The fatty acids belonging to this family are bent at the site of double bonds and the molecule shows some
α-linolenic acid (ALA, 18:3), eicosapentaenoic acid (EPA, 20:5), crumpling. These fatty acids originate in unicellular phytoplank-
docosahexaenoic acid (DHA, 22:6 ω-3), and docosapentaenoic tons and seaweeds and accumulate in fish (Ackman 1980). The
acid (DPA, 22: 5). The chemical structures of these fatty acids precursor of long-chain ω-3 fatty acids is ALA, which is subse-
are shown in Figure 1. Due to nonconjugated (methylene inter- quently chain-elongated and desaturated to EPA and DHA. Both
60 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 8, 2009
PUFAs in fish . . .
EPA and DHA occur as constituents of fish triglycerides and phos-
pholipids. Figure 2 shows the pathway of omega-3 and omega-6
fatty acid synthesis, where the PUFAs are either n-3 or n-6 fatty
acids. ALA and linoleic acid are the precursors of n-3 and n-6
fatty acids, respectively, and are converted to different long-chain
PUFAs by sequential desaturation and elongation.
PUFAs in Disease
The importance of a balanced PUFA intake has been recog-
nized by health organizations throughout the world over the past
decade. There is now some consensus that PUFAs should form a
bare minimum 3%, and preferably 10% to 20%, of the total lipid
intake, and that the 6- to 3-series ratio should ideally be around
4:1 or 5:1. Although the biological effects of eicosanoids are
undisputed, the same can not be said for their PUFA precursors,
and most of the diverse pharmacological effects that have been
proposed for PUFAs have yet to be proved. Also, the importance
of PUFA intakes as compared with other nutritional factors, ge-
netic determinants, and environmental influences in the initiation
and progress of diseases is not clear.
The pioneering epidemiological work of Dyerberg and Bang
(1979) and Dyerberg (1986) on Greenland Eskimos suggested
a possible link in low incidence of heart diseases to the
consumption of seafood. Since then, many studies have been
published on the role of ω-3 fatty acids in human health and
diseases. Pamela (2001) reported that EPA and DHA have bio-
chemical effects in the prevention and treatment of several dis-
orders and diseases, such as coronary heart disease, rheumatoid
Vol. 8, 2009—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY
arthritis, asthma, cancers, diabetes, and others. At present, the
major sources of EPA and DHA are from the cool deep-sea fish
oil such as menhaden, cod, sardine, anchovy, and others (Sham-
sudin and Salimon 2006). These fatty acids have been reported
to have beneficial effects in cardiovascular diseases, autoimmune
disorders, and various inflammations (Weaver and Holub 1988).
The beneficial effects of ω-3 fatty acids are explained through
altered zicosanoid metabolism in the circulatory system leading
to production of prostaglandins considerably weaker in inducing
platelet aggregation than those produced from ω-6 fatty acids.
The physiological effects of ω-3 fatty acids are: (1) lowering of
plasma triglycerides; (2) increased aggregation time for platelets;
(3) decreased viscosity of blood; (4) decreased blood pressure;
(5) reduction of arthrosclerosis; (6) reduction of inflammation
arthritis, psoriasis, asthma; (7) reduction in tumors. Prevention
of cancer is still debated as unsaturated fatty acids produce free
carbonyl compounds, which are tumorigenic. Detailed treatment
of the medical aspects has been the subject of many publications
(Kinsella 1986, 1987, 1990; Lees and Karel 1990).
An increase in PUFA consumption carries an elevated risk of
exposure to toxic oxidation products, which are implicated in
cancer, and thrombotic and inflammatory diseases. In particu-
lar, there is concern that current intakes of dietary antioxidants
are insufficient to cope with an increased polyunsaturate load,
and that serious health hazards could be posed by an eleva-
tion of in vivo PUFA-peroxide production. Hence, any moves
designed to increase dietary polyunsaturates must be accompa-
nied by measures to ensure that antioxidant levels are adequately
maintained.
Figure 2 --- Metabolic pathway of
omega-6 and omega-3 fatty acid
synthesis (source: Siddiqui and
others 2008).
61
CRFSFS: Comprehensive Reviews in Food Science and Food Safety
PUFAs in Human Health within other phospholipids (Lagarde 2008). Author also reported
that DHA, the end-product of the n-3 family fatty acid, is an abun-
PUFAs are long-chain fatty acids containing 2 or more double
dant component in the brain phospholipids, and a major nutrient
bonds. The potential applications of PUFAs are found in ther-
of marine lipids. DHA is also a fairly good substrate of lipoxy-
apeutics, foods, and nutrition. They can be found in animals,
genases, especially the n-9 and n-6 ones. Hydroxy derivatives,
plants, algae, fungi, and bacteria. They are produced commer-
that is, docosanoids, exhibit potent biological activities, which
cially from selected seed plants and some marine sources. PUFAs
may explain part of the potential benefit of DHA in the brain and
are grouped into 2 series on the basis of the position of the termi-
vascular bed (Lagarde 2008).
nal double bond being 3C or 6C from the terminal carbon atom of
The eicosanoid pathway in mammals begins with the
the fatty acid chain. Some examples are: 3-series PUFA linolenic
phospholipase-mediated release of PUFAs from membrane phos-
acid (LA) and alpha-linolenic acid (ALA). Six-series PUFA gamma-
pholipids and is followed by cyclooxygenase-catalyzed reac-
linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic
tions that give rise to such major classes of metabolites as
acid (EPA), and docosahexaenoic acid (DHA). They provide struc-
prostaglandins, thromboxanes, lipoxins, and leukotrienes. In
tural and functional characteristics and are involved in a wide
1971, John Vane discovered that asiprin inhibits the cyclooxy-
range of biological components including membranes phospho-
genase enzyme and thus inhibits prostaglandin synthesis. This
lipids. They are involved in regulating architecture, dynamics,
is the basis for aspirin as an anti-inflammatory agent. Also,
phase transitions, and permeability of membranes, and control
prostaglandins and thromboxanes are involved in regulating
of membrane-associated processes. Also, they are involved in
blood clot formation so “baby” aspirins are prescribed for patients
regulating membrane-bound proteins such as ATPase, transport
at risk of heart disease/stroke, because a low dose of aspirin dis-
proteins, and histocompatibility complexes. In addition, PUFAs
turbs the PG/TX balance and inhibits clot formation. Present-day
regulate expression of some genes, including those coding for
eicosanoid-related ailments can be traced to changes in human
fatty-acid synthase, nitric-oxide synthase, and sodium-channel
nutrition. Research indicates that intake of PUFAs was evenly bal-
proteins. Thus, they have an impact on cellular biochemical ac-
anced 10000 years ago with 3- and 6-series PUFAs. Since then,
tivities, transport processes, and cell-stimulus responses. They are
reductions in 3-series PUFAs have created an imbalance in most
involved in physiological processes including immune responses
modern societies. This can be attributed to a move away from
and cold adaptation, and are implicated in pathological condi-
tions such as cardiovascular disease. hunter–gatherer nutrition resulting in a large rise in 6-series PUFA
intake from the increased consumption of particular plants and
Equally important, PUFAs serve as precursors for conversion
animal products from intensive agriculture, which has reduced
into metabolites that regulate critical biological functions. In
levels of other 3-series PUFA types.
plants, they are transformed by a variety of enzymes into oxy-
Church and others (2008) summarized that excess and de-
genated molecules, ranging from simple aldehydes to oxylipins.
ficient dietary ω-3 fatty acids during pregnancy and lactation
These act as anti-infection agents, wound-response mediators,
caused delayed neural transmission along the brainstem audi-
and regulatory, hormonal, and chemotactic agents, as well as
aroma and taste compounds. In humans, PUFA metabolism and tory pathway as evidenced by prolonged auditory brainstem re-
sponse (ABR) latencies in a 24-d-old rat offspring. The excess
eicosanoid function became important when it was discovered
offspring also showed postnatal growth retardation and a trend
that arachidonate is the precursor for prostaglandins. Ecosanoids
for increased postnatal mortality. Their study’s investigation of
are a diverse group of hormones including prostaglandins, throm-
both deficient and excess ω-3 fatty acids consumption during
boxanes, and leukotrienes. Research shows that eicosanoid hor-
mones are fundamental to proper maintenance of homeostasispregnancy and lactation has important health implications. Cur-
rent United States consumption of ω-3 fatty acids is lower than
and are linked to important physiological and pathophysiolog-
national and international recommendations (Akabas and Deck-
ical conditions. Figure 3 shows that the DHA, from the blood
elbaum 2006) and excess ω-3 fatty acids is being consumed vol-
or the conversion of linolenic acid (LNA) in the liver, may be
untarily (Rump and others 2001; Thorsdottir and others 2004)
esterified in phosphatidylcholine (PC) and secreted in lipopro-
teins as PC-DHA, which can be converted into lysoPC-DHA byand being given as treatment for preterm birth (Sattar and oth-
ers 1998; Smuts and others 2003). Church and others (2007,
the endothelial lipase (Lagarde 2008). It can be speculated that
2008) also reported that both excess and deficient amounts of
lysoPC-DHA might alternatively be produced in and secreted by
dietary ω-3 fatty acids during pregnancy and lactation can cause
the liver. LysoPC-DHA is taken up by the brain to release DHA
or for being converted into PC-DHA, with DHA redistributedpostnatal growth retardation as well as sensory and neurologi-
cal abnormalities in the offspring. Even though supplementing
one’s diet with modest amounts of ω-3 fatty acids can be ben-
eficial, consuming or administering large amounts of ω-3 fatty
acids to pregnant women or nursing infants could have harmful
consequences. Despite such caveats, some recent publications
are still recommending that pregnant women and infants should
consume higher than normal amounts of ω-3 fatty acids (Akabas
and others 2006; Jensen 2006). Church and others (2008) dis-
agree with this above statement. Moreover, recent literature re-
views have failed to find convincing evidence that prenatal fish
oil treatments improve pregnancy outcome (Makrides and others
2006) and failed to find that postnatal ω-3 fatty acids supplemen-
tation during nursing/bottle-feeding has lasting effects on infant
brain and visual functions (Hadders-Algra and others 2007). In
light of such findings and because excess ω-3 fatty acids dur-
ing pregnancy and lactation can have adverse neurological and
developmental effects, we conclude that using large amounts of
Figure 3 --- DHA, from the blood or the conversion of LNA dietary ω-3 fatty acids during pregnancy and lactation has little
or may be esterified in PC (source: Lagarde 2008). merit, is potentially harmful to the offspring, and should not be
62 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 8, 2009
PUFAs in fish . . .
advocated in clinical or public practice. Instead, more research
is needed to determine: (1) how much perinatal ω-3 fatty acids is
too much, too little, and just right for the developing human and
(2) the long-term consequences with regard to the fetal program-
ming of neurodevelopmental and sensory impairments and the
adult-onset diseases of hypertension, diabetes, age-related neural
degeneration, and a shortened life span (Barker 2004).
n-3 PUFAs exert many of their beneficial effects upon the car-
diovascular system via their effects on several cellular processes.
n-3 PUFAs improve the plasma lipid profile. Siddiqui and oth-
ers (2008) reported that intake of n-3 PUFAs (4 g/d for 6 wk)
reduced TG levels by 18% to 20% but had a minimal impact on
low-density lipoprotein cholesterol or high-density lipoprotein
cholesterol (HDL-C) (Mori and others 2000). In contrast to these
studies, long-term treatment of hypertriglyceridemic patients with
n-3 PUFAs (4 g/d for 16 wk) led to a significant reduction in TG
by 47%, while TG levels rose by 16% with placebo (corn oil).
This effect of n-3 PUFAs was associated with a decrease in total
cholesterol: HDL ratios (20%) and a modest increase in HDL-
C (13%) (Harris and others 1997). Similar results were also re-
ported in another study where hypertriglyceridemic patients were
treated with n-3 PUFAs (4 g/d) for 6 mo (Abe and others 1998).
It appears from different studies (McKenney and Sica 2007) that
higher levels of n-3 PUFAs for longer duration have beneficial ef-
fects on plasma lipid profile. n-3 PUFAs also have antiatherogenic
actions. Eritsland and others (1996) reported that n-3 PUFA sup-
plementation (4 g/d for 1 y) in postcoronary artery bypass graft
patients was associated with a reduced frequency of vein graft
occlusions. This n-3 PUFA effect was not linked to an influence
on serum lipoproteins because serum cholesterol levels were not
altered by n-3 PUFA supplementation. Furthermore, there was
no association between the reduction in serum TG and vein graft
occlusion. These studies, therefore, concluded that the n-3 PUFA
effect on new plaque development appeared to be due to an-
tithrombotic as well as antiatherosclerotic properties of n-3 fatty
acids. Furthermore, Thies and others (2003) reported that n-3
PUFA supplementation (1 to 4 g/d for an average of 42 d) in heart
patients prior to undergoing carotid endarterectomy resulted in a
rapid incorporation of n-3 PUFAs into advanced atherosclerotic
plaques, which was associated with structural changes consistent
with increased plaque stability. The antiatherosclerotic effects of
n-3 PUFAs appear to be mediated through their anti-inflammatory
effects on platelets and endothelial cells. Platelets, through their
interaction with the vascular endothelium, play a critical role in
atherogenesis (Ross 1993). Mori and others (1997) observed that
human consumption of n-3-PUFAs (3 to 4 g/d) for 3 wk reduced
platelet aggregation induced by collagen and platelet-activating
factor (PAF) regardless of whether n-3 PUFAs were ingested as
daily fish meals or fish oil capsules. Similarly, Agren and others
(1997) reported that consuming moderate amounts of n-3 PUFAs
for 15 wk in the form of a fish diet (0.38 g EPA + 0.67 g DHA/d)
or fish oil (1.33 g EPA + 0.95 g DHA) also inhibited platelet
aggregation but did not affect hemostatic factors. Notably, EPA-
free DHA oil (1.68 g/d) was not effective in decreasing in vitro
platelet aggregability (Agren and others 1997). The ineffective-
ness of DHA implies that modulation of platelet aggregation
by n-3 PUFAs may be mediated through the eicosanoid path-
way rather than being a direct effect of fatty acids on platelets.
Furthermore, in the patients with elevated TG levels, prolonged
treatment with n-3 PUFAs (4 g/d for 7 mo) was associated with
reduced levels of soluble adhesion molecules (sICAM-1 and sE-
selectin) (Abe and others 1998). Soluble adhesion molecules lack
membrane-spanning and cytoplasmic domains that are present in
the membrane-bound forms, but their levels have been noted to
be elevated in pathological conditions in which tissue expressions
of the membrane bound forms of adhesion molecules are known
Vol. 8, 2009—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY
to be unregulated (Ballantyne and others 1994). It is difficult to
measure the membrane expression of adhesion molecules in the
human vasculature after n-3 PUFA supplementation. Evidence
for n-3 PUFA effects on cell membrane expression of adhesion
molecules is derived from in vitro experiments. DHA treatment
to human adult saphenous vein endothelial cells at concentra-
tions (10 μM) compatible with nutritional supplementation of
this fatty acid to individuals consuming a normal Western diet
reduced surface expression of adhesion molecules (De Caterina
and others 2000). It appears from these studies that one of the
beneficial effects of n-3 PUFAs on the cardiovascular system is
mediated through its antiatherosclerosis properties. Furthermore,
n-3 PUFAs also improve vascular functions. However, dietary
supplementation with fish oil (5 g EPA+DHA/d for 3 wk) sig-
nificantly improved endothelium-dependent coronary vasodila-
tion in heart transplant recipients without altering the responses
to endothelium-independent vasodilation. These improved vas-
cular functions play a small role in reducing hypertension. A
metaregression analysis of 36 randomized trials on fish oil sup-
plementation (mean consumption of 3.7 g/d for a median of 12
wk) in largely overweight and hypertensive subjects showed only
a small antihypertensive effect (Geleijnse and others 2002). Fur-
thermore, it is suggested that DHA is likely more favorable in
lowering blood pressure and heart rate than EPA (Mori 2006).
In conclusion, observational studies, human intervention trials,
animal models, and cell culture studies suggest that n-3 PUFAs
have beneficial effects on the cardiovascular system. The molec-
ular and cellular effects of n-3 PUFAs for mediating the beneficial
cardiovascular effects are not fully known. Siddiqui and others
(2008) reported that the beneficial effects of fish oil are attributed
to their n-3 polyunsaturated fatty acid (PUFA/omega-3 fatty acids)
content, particularly EPA (20:5, n-3), and DHA (22:6, n-3). Di-
etary supplementation of DHA and EPA influences the fatty acid
composition of plasma phospholipids that, in turn, may affect
cardiac cell functions in vivo. Recent studies have demonstrated
that long-chain omega-3 fatty acids may exert beneficial effects by
affecting a wide variety of cellular signaling mechanisms. Path-
ways involved in calcium homeostasis in the heart may be of
particular importance. L-type calcium channels, the Na+− Ca2+
exchanger, and mobilization of calcium from intracellular stores
are the most obvious key signaling pathways affecting the cardio-
vascular system; however, recent studies now suggest that other
signaling pathways involving activation of phospholipases, syn-
thesis of eicosanoids, regulation of receptor-associated enzymes,
and protein kinases also play very important roles in mediating
n-3 PUFA effects on cardiovascular health.
There is direct evidence that suggests n-3 PUFAs are very
potent agents in regulating intracellular calcium levels. Earlier
studies have shown that EPA and DHA (5 μM) can prevent ar-
rhythmias, fibrillation, and contracture in isolated rat cardiac my-
ocytes induced by toxic concentrations of ouabain (Hallaq and
others 1990), a cardiac glycoside that binds to the α-subunit of
membrane-bound Na, K-ATPase (Wallick and others 1974). Ad-
dition of either oxygenase inhibitors or antioxidants did not alter
the effects of n-3 PUFAs on ouabain-induced cardiac arrhythmia
(Hallaq and others 1990). This observation suggests that n-3 PUFA
incorporation into the phospholipids of cell membranes may have
prevented the toxicity caused by ouabain, and its presence was
associated with fewer rises in cytosolic free calcium (Hallaq and
others 1990). Furthermore, EPA (2-10 μM) exhibited a similar
protective effect against ouabain toxicity when cardiomyocytes
were incubated for 3 to 5 d in the presence of these n-3 PU-
FAs (Hallaq and others 1992). Two other PUFAs, linoleic acid
(18:2, n-6), and linolenic acid (18:3, n-3), also exhibited similar
but less potent effects compared with EPA. In contrast, neither
oleic acid (18:1, n-9) nor saturated fatty acids (18:0, 14:0, 12:0)
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CRFSFS: Comprehensive Reviews in Food Science and Food Safety
affected contraction rate (Hallaq and others 1992). These studies
have shown that the beneficial effects of fish oil in preventing fatal
arrhythmias in myocardial ischemia are at least in part mediated
by modulating the dihydropyridine-sensitive L-type calcium cur-
rent (Hallaq and others 1992). Consistent with this observation,
treatment of cardiomyocytes with DHA or EPA prevented the in-
crease in calcium influx by Bay K8644, an established agonist for
L-type calcium channels (Hallaq and others 1992), strongly sug-
gesting that L-type calcium channels are the target site for these
fatty acids. Similarly, it has been demonstrated that n-3 PUFAs
modulate calcium current through the L-type calcium channels,
and these effects occur within minutes of adding EPA or DHA
to the perfusing medium of the cultured cardiac myocytes (Leaf
1995). Moreover, it has also been shown that the delayed rec-
tifier K+ channel is inhibited by n-3 PUFAs (Honore and oth-
ers 1994). The combined effect of this is suggested to reduce
electrical excitability, making arrhythmias less likely (Kang and
others 1995). Both EPA and DHA are known to be antiarrhyth-
mic. They depress surface membrane electrical excitability (Kang
and others 1995) and inhibit spontaneous release of Ca2+ from
overloaded cardiac SR (Negretti and others 2000). The effect of
n-3 PUFAs on the L-type Ca2+ channel appears to be due to
their direct binding to the channel proteins. This fact is supported
by a recent study investigating the link between n-3 PUFA con-
tent of the plasma membrane and ion channel activity (Pound
and others 2001), which suggested that n-3 PUFA concentrations
required for antiarrhythmic action were too low to produce a
significant change in the overall arrangement of the phospho-
lipids within cardiac membranes. Similarly, the effect is quickly
reversed when free PUFAs are extracted from the cells by adding
dilapidated BSA to the bathing medium (Kang and Leaf 1996).
These observations imply that n-3 PUFAs are neither fully incor-
porated into membrane phospholipids nor covalently bound to
any constituents of the myocyte to produce the antiarrhythmic
effect (Kang and Leaf 1996). These studies, therefore, suggest that
n-3 PUFAs exert antiarrhythmic effects by direct interactions with
SL ion channels rather than indirectly by perturbing membrane
phospholipids packing.
Pharmaceutical and Food Applications
Evidence of the possible medical effects of PUFA deficiencies
have, coupled with the growing acceptance of “pharmafoods”
by consumers, brought these compounds to the attention of food
and pharmaceutical companies, which have been quick to ex-
ploit markets in the biomedical and pharmafood areas. A variety
of specialty PUFA lipids are available for uses, ranging from an-
tiaging, antithrombotic, anti-inflammatory, anticholesterolemic,
and anticancer drugs to immunostimulant and immunosup-
pressant therapeutics. However, their efficiencies remain to be
verified. Unregulated applications for esters, glycerides, and
phospholipids, such as health food additives for foods, nutritional
formulas, and cosmetics ingredients, have also increased. The
most obvious commercial impact of PUFAs has been in health
supplements, with a host of plant- and fish-derived GLA, EPA,
and DHA products now being available in the marketplace for
uncontrolled dietary use.
This raises a question whether PUFAs should be treated as
exceptional nutritional products or as pharmaceuticals. As they
occur in foodstuffs, there is a case for considering them as “gen-
erally recognized as safe” food components; on the other hand,
they are biologically active. In view of the serious disease states
that may result from eicosanoid imbalances, there is justification
to regard them as biomedical additives. Despite limited under-
standing of PUFA biochemistry and many therapeutic, nutritional,
and regulatory issues that remain to be resolved, the market po-
64 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 8, 2009
tential for PUFA in the food, health-supplement, cosmetics, and
pharmaceutical sectors is obvious. In addition to such “first gener-
ation” applications, it is predicted that high-value PUFA-derived
eicosanoids aimed at treating specific disease conditions will
appear in the therapeutics market, once biotechnological ap-
proaches are in place for their production.
PUFAs in Fishes
Mishra and others (1993) reported that the total world produc-
tion of fish oil in 1988 was 1.5 million metric tons (FAO Yearbook
of Fishery Statistics; Commodities, 1988). Most of this production
has been used in various food and pharmaceutical formulations.
Typical food uses of hydrogenated or partially hydrogenated fish
oils are production of salad oils, frying oils, table margarines,
low-calorie spreads, and shortenings in bakery products (Bimbo
1990; Bimbo and Crowther 1991). Most of the production from
the United States is exported overseas, mainly to Europe, which
is the major fish oil consumer. Currently, it is known that fatty
acids of fish flesh are the most beneficial for human health due
to its high proportion of unsaturated fatty acids. Fish lipids are
well known to be rich in long-chain n-3 PUFAs, especially EPA
and DHA (Osman and others 2007). Research has also indicated
that fatty acid composition of the fish differs due to climatic in-
fluence, diet, age, maturity, and type of species (Kinsella 1988;
Ratkowsky and others 1996; Saito and others 1999; Haliloğlu and
others 2004; Çelik and others 2005). 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 (C18:2 n-6) and linolenic (C18:3
n-3) acids and EPA (Haliloğlu and others 2004; Çelik and oth-
ers 2005). The plankton of marine feeds presents low levels of
n-6 PUFA, of which EPA and DHA are the predominant acids
(Justi and others 2003). Thus, marine fish are distinguished by
high concentrations of n-3 since they feed on plankton while
freshwater fish mainly contains n-6 fatty acids.
The fatty acid compositions of some marine fishes are listed in
Table 2 (Osman and others 2007). The most abundant PUFA in
the fin fishes (head, middle, and tail) was C22:6 (DHA) (8.18%
to 23.95%), while C20:5 (EPA) was also present in significant
proportion (7.13% to 19.61%), with n-6 fatty acids also present
in significant proportion. C18:2 n-6 ranged from 5.47 to 16.11,
while C20:4 n-6 had a range from 0.34 to 13.41 (Table 2). How-
ever, Suriah and others (1995) reported that the make-up of fatty
acids of fin fish from seawater is slightly different when compared
to that of freshwater fish, where the concentrations of MUFAs are
higher than the saturated and PUFAs. Other researchers have also
reported that freshwater fish have lower contents of PUFAs (Vlieg
and Body 1988). The differences can be due to the fact that fresh-
water fishes feed mainly on vegetation and plant materials while
marine fishes feed on zooplanktons, which are rich in PUFAs.
According to Piggott and Tucker (1990), the n-6:n-6 ratio is
a better index in identifying nutritional value of fish oils of dif-
ferent species. Osman and others (2007) reported that in terms
of individual PUFA contents most of the fin fish analyzed had
higher contents of AA and DHA than menhaden oil (Table 2).
As for DHA content, all of the fishes studied had higher percent-
ages of DHA, as compared to menhaden oil (7.9%). The highest
level was found in “bagi” (Aacnthurs nigrosis) (23.95%), which
was 3 times higher than in menhaden oil. However, for the EPA,
most of the species studied had lower concentrations (7.51% to
11.53%) than the EPA concentration of menhaden oil (12.5%).
This finding is in agreement with the data reported by Osman and
others (2001) and Wang and others (1990). All these researchers
revealed that marine fish were rich in n-3, especially EPA and
DHA.
PUFAs in fish . . .

Osman and others (2001) studied fatty acid composition of
selected marine fish in Malaysian waters and have summarized
the total fatty acid contents (Table 3). Authors reported that the
PUFA contents were much higher (56% to 92%) than the satu-
rated fatty acids (3.63% to 11.4%), and MUFAs were lowest (1%
to 10%) in marine fish. The trend is different when compared to
an earlier study on freshwater fish, where the concentrations of
MUFAs were higher than the saturated and PUFAs (Suriah and
others 1995). Other researchers have also shown that freshwater
fish have lower contents of PUFAs (Vlieg and Body 1988). The
difference can be attributed to the fact that freshwater fishes feed
largely on vegetation and plant materials, whereas marine fish
staple diets are mainly zooplanktons, rich in PUFAs. Menhaden
oil, which is marine fish oil, has been proposed by the U.S. FDA
(1997) as a PUFA supplement. The concentrations of ω-3 PUFA
(29.7% to 48.4%) and other PUFAs (27.7% to 40%) in the study
of FDA (1997) were found to be much greater than found in the
standard menhaden oil (ω-3 PUFA, 28.9%; other PUFAs 16.6%,
whereas ω-6 PUFAs are 14.3%). The values for MUFAs (1.37% to
9.12%) and saturated fatty acids (3.63% to 16.38%) were lower
than those of menhaden oil (MUFA, 19.4%; saturated, 20.2%)
(Table 3). This study has shown that marine fish were richer in
ω-3 PUFAs (29.7% to 48.4%) than ω-6 PUFAs (11% to 20%).
Suriah and others (1995), O’Dea and Sinclair (1982), and Suzuki
and others (1986) reported on freshwater and cultured fish, had
shown opposite results, where the levels of ω-3 PUFAs were lower
than ω-6 PUFAs. However, Wang and others (1990) reported sim-
ilar findings, in that marine fish were rich in ω-3, especially DHA
and EPA.
The contents of arachidonic acid (AA), EPA, and DHA of the
fish analyzed ranged from 0.19% to 0.68%, 0.82% to 6.76%, and
9.36% to 28.6%, respectively. Fish that had higher contents of AA

Table 2 --- Fatty acid profiles of fin fish caught in Pulau Tuba, Langkawi, Malaysia.
Tenggiri
Duri Kintan Jenahak Kerisi Kembong batang Kebasi Bagi Yu 9 Laban
Fatty Marine Pseudo- Golden Threadfin Indian Spanish Anodon- Aacnthurs Lizard Tongue Menhaden
acids (%) catfish rhombus snappers breams mackerel mackerel tostoma Nigrosis fish soles oil

C10:0 0.08 0.03 0.02 0.14 0.03 0.11 0.80 0.07 0.06 0.16 0.00
C14:0 7.41 5.00 2.23 2.72 11.58 2.94 3.43 1.42 1.45 4.72 9.00
C15:0 3.30 1.32 0.72 0.97 0.77 2.20 1.67 1.49 0.88 8.59 9.02
C16:0 31.63 19.96 26.85 27.49 19.21 19.90 18.97 21.59 19.32 17.62 19.1
C17:0 2.44 1.89 1.76 1.82 1.06 2.54 2.09 2.01 1.78 1.81 0.00
C18:0 10.56 9.91 9.32 11.53 7.89 10.18 9.67 8.98 8.98 4.48 2.83
C20:0 0.78 4.40 1.67 0.92 0.62 0.64 0.42 0.39 0.55 0.15 1.00
Total 55.59 39.21 42.29 45.09 38.55 38.71 37.05 35.90 33.02 37.52 40.95
C18:1 n-9 13.46 9.02 11.80 12.86 8.46 7.01 8.19 8.99 9.13 2.39 14.29
C18:1 n-13 0.91 0.84 0.64 0.54 0.52 0.55 0.48 1.01 0.74 0.10 1.21
C20:1 n-9 0.51 1.34 3.51 0.82 1.19 0.79 0.54 1.01 0.32 4.56 0.20
C20:1 n-13 0.11 0.02 0.21 0 0 0.10 0 0.20 0 1.05 0.00
Total 14.99 11.22 16.16 14.22 6.17 8.45 9.21 11.21 10.19 8.11 15.70
C18:3 n-3 1.10 1.38 1.46 0.55 0.58 0.87 0.64 0.2 0.44 1.32 2.00
C18:2 n-6 8.30 7.83 8.14 5.47 9.69 5.55 11.69 9.49 16.11 12.08 1.00
C20:3 n-3 2.00 4.45 0.71 0.51 0.54 9.13 1.09 0.55 0.34 0.35 0.00
C20:4 n-6: AA 0.34 1.05 5.24 4.38 4.15 13.41 5.21 7.16 1.87 4.52 0.47
C20:5 n-3: EPA 7.51 10.81 8.81 9.44 16.08 7.13 10.72 11.53 14.89 19.61 12.50
C22:6 n-3: DHA 8.18 20.06 17.19 17.34 19.25 14.75 21.40 23.95 19.46 16.48 7.90
Total 27.43 48.58 41.55 37.69 51.29 50.84 50.75 52.88 53.11 54.36 23.87
n-3:n-6 2.17 4.13 2.11 2.83 2.63 1.68 2.00 2.18 1.95 2.27 8.66
Others 1.99 1.01 0.29 3 3.99 2 2.9 0.14 3.68 2.91 1.95
Source: Osman and others 2007.

Table 3 --- Total fatty acid contents (%) in fish oils.

PUFA PUFA Other Total
Species Saturated MUFA ω-6 ω-3 PUFA PUFA

Black pomfret (Parastromateus niger ) 11.4 3.64 13.5 30.5 30.6 74.4
Silver pomfret (Pampus argenteus) 9.17 3.27 13.5 31.7 36.3 81.5
Hardtail scad (Magalapsis cordyla) 9.45 3.60 11.7 48.4 27.7 86.9
Indian mackerel (Rastrelliger kanagurta) 7.71 3.25 20.0 33.4 35.3 56.8
Whiptail stingray (Gymnura spp.) 3.63 3.88 18.0 38.9 34.5 91.5
Yellow striped scad (Selarides leptolejus) 16.1 11.4 12.0 41.3 32.3 85.5
Striped sea catfish (Plotosus spp.) 10.1 1.37 18.0 32.0 34.0 84.0
Fourfinger threadfin (Eleutheronema tradactylum) 16.3 3.96 19.78 29.7 40.0 89.5
Fringescale sardine (Clupea fimbriata) 9.88 9.12 11.0 30.9 35.0 76.9
Spanish mackerel (Scomberomorus commersonii ) 6.39 4.08 11.1 47.9 32.8 86.7
Menhaden oil 20.8 19.43 14.3 28.9 16.6 59.8
Source: Osman and others 2001.
MUFA = mono-unsaturated fatty acid; PUFA = poly-unsaturated fatty acid.

Vol. 8, 2009—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 65

CRFSFS: Comprehensive Reviews in Food Science and Food Safety
than the menhaden oil (0.47%) were striped sea catfish (0.68%), China, and the Ryukyu Islands. They have entered the eastern
silver pomfret (0.60%), and black pomfret (0.52%). Based on an Mediterranean Sea through the Suez Canal. Indian mackerel is
earlier study (Suriah and others 1995), in some cases the con- a very important species. Catches are usually recorded as Ras-
tents of AA in marine fishes were lower than that of freshwater trelliger spp. or combined with R. brachysoma. In the last 25
fishes. The DHA contents of all the fish studied were higher than y, the world catch for R. kanagurta alone fluctuated between
the amounts found in menhaden oil (6.20%), and the highest about 96000 t in 1975 and a peak of 351193 t in 1994; since
level, found in hardtail scad (28.6%), was almost 5 times higher. 1984, catches reported to FAO as Rastrelliger spp. have exceeded
The amounts of EPA in the species studied (0.82% to 6.76%) 300000 t annually. In the Western Indian Ocean area most of the
were lower than in the menhaden oil (8.54%). However, the catches (about 185000 t in 1995) are identified as R. kanagurta,
other richest and cheapest source of PUFAs is Indian mackerel. while in the Eastern Indian Ocean 224000 t are reported as Ras-
Charles and others (2003) chose mackerel waste to extract PUFAs trelliger spp. and 43000 t as R. kanagurta. Instead, in the Western
because it is an underutilized fish with a limited range of value- Central Pacific, which ranks as the area of major catches for
added products developed from it. This is probably because of Rastrelliger species, 252000 t are not identified at the species
the highly perishable nature of the species. Being so oily makes level, 104000 t are identified as R. kanagurta, and 26000 t as R.
the fish very prone to postmortem spoilage, such as rancidity, and brachysoma. Indian mackerel are caught with purse seines, encir-
presents processing challenges (Banks 1967). However, in light cling gillnets, high-opening bottom trawl, lift nets, and bamboo
of the nutritional value of marine oils, the oily nature of mackerel stake traps. The total catch reported for this species to FAO for
tissues presents opportunities. It is important to increase the in- 1999 was 302387 t. The countries with the largest catches were
formation base available on mackerel to enhance its exploitation. India (146367 t) and the Philippines (53606 t).
The baseline characteristics of mackerel oil in Table 4 indicate
that the distribution of oil in the tissues is not proportional and
the skin yields are the highest at about 38% compared to 9% Conventional Extraction of PUFAs from Fish
for viscera and muscle. This disparity was evident irrespective The production of fish oil deals with the separation of fatty
of the solvent extraction system used. Consequently, the use of substances (lipids) from other constituents of the fish. Generally,
chloroform/methanol (2:1, vol/vol) for oil extraction was discon-separation starts from the preparation of the raw material up to
tinued after the preliminary analysis, since there were no inher-the purification of the product, which is the final stage of the
ent advantages over hexane/isopropanol (3:2, vol/vol) that would process. One of the methods used industrially in obtaining fish
oil is hydraulic pressing; a batch process whereby the oil is ob-
justify the risk of toxicity associated with exposure to both chlo-
roform and methanol. Since mackerel skins are usually discarded tained or expressed by hydraulic pressing a mass of moderately
cooked oil-bearing fish. A recent development is in the extrac-
during processing and they have high oil content, skins are a most
suitable material for producing PUFAs because they will gener- tion of oil from oil-bearing material using a solvent or solvent
ate comparatively high yields (about 38%, w/w) of oil for small system. Solvent extraction, which is also referred to as leaching,
inputs, thus reducing bulk handling and transportation. Indian is a process whereby soluble constituents, present either as solids
mackerel are widespread in the Indo-West Pacific from South or liquids are removed by the use of solvents. In fact, solvent ex-
Africa, Seychelles, and Red Sea areas and east through Indone- traction techniques are among the most commonly used methods
sia and off northern Australia to Melanesia, Micronesia, Samoa, of isolating lipids from food samples (including fish) and of deter-
mining the total lipid content of foods. The principle is based on
the fact that lipids are soluble in organic solvents, but insoluble
Table 4 --- Fatty acid contents (wt%) of saponified mack- in water, hence providing a convenient method of separating the
erel oil before and after urea complexation. lipid components in the food samples from water-soluble com-
Mackerel oil
ponents such as protein, carbohydrates, minerals, and water itself
(McClements 2003). For a successful extraction of oil, a sample
Fatty acid Muscle Viscera Skin must undergo specific preparations prior to solvent extraction
(McClements 2003). In practice, the efficiency of solvent extrac-
14:0 5.1 ± 0.9 1.7 ± 1.5 4.9 ± 0.2 tion depends on the polarity of the lipids present compared to the
14:1 <0.5 <0.5 <0.5 polarity of the solvent. Polar lipids (such as glycolipids or phos-
16:0 19.9 ± 2.1 17.5 ± 0.5 15.3 ± 0.4 pholipids) are more soluble in polar solvents (such as alcohols)
16:1 5.8 ± 0.8 3.8 ± 0.2 6.9 ± 0.1 than in nonpolar solvents (such as hexane). On the other hand,
18:0 3.6 ± 0.9 5.9 ± 0.2 2.9 ± 0.1 nonpolar lipids (such as triacylglycerols, triglycerides) are more
18:1 9.1 ± 1.1 29.8 ± 2.2 10.9 ± 1 soluble in nonpolar lipid than in polar ones. Soxhlet extraction is
18:2 1.3 ± 0.9 0.5 ± 0.1 1.3 ± 0.1 one of the most commonly used methods for determination of to-
18:3 2.2 ± 0.8 <0.5 2.6 ± 0.8 tal lipids in dried samples. It is fairly simple to carry out and is the
20:0 0.7 ± 0.2 <0.5 <0.5 officially recognized method for a wide range of fat content de-
20:1 9.4 ± 1.7 5.1 ± 0.5 9.3 ± 0.7 terminations. The main disadvantages of the technique are that:
20:2 <0.5 <0.5 <0.5 a relatively dry sample is needed (to allow the solvent to pen-
20:4 <0.5 0.5 ± 0.2 <0.5 etrate), it is destructive, and it is time-consuming (McClements
20:5 8.0 ± 0.1 6.3 ± 0.7 8.5 ± 0.2 2003). Shamsudin and Salimon (2006) extracted fish lipids from
22:1 13.6 ± 1.3 5.1 ± 0.7 12.1 ± 1.1 the local marine fish aji-aji of Malaysian waters using a mixture of
22:6 9.7 ± 0.2 7.9 ± 0.5 10.2 ± 0.7 chloroform:methanol (2:1) by Soxhlet extraction according to An-
24:0 1.1 ± 0.8 1.82 ± 0.1 1.3 ± 0.1 drade and others (1995) and Saify and Akhtar (2003). The crude
Others 10.5 14.1 13.8 extract was purified by adding an aqueous 0.88% KCl solution
Total PUFA 21.2 15.2 22.6 in 8:4:3 proportions of chloroform:methanol:KCl following the
Total MUFA 37.9 43.8 39.2 extraction method of Nordback and others (1998). The authors
found that aji-aji fish contain about 18.3% crude lipids and about
Total n-3 19.9 14.2 21.3 16.8% lipid matter after purification process. The results showed
Source: Charles and others 2003. that aji-aji fish is high in lipid content. Among local marine fish
66 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 8, 2009
PUFAs in fish . . .
species studied, aji-aji fish were found to contain average val-
ues in lipid percentages, ranging from 10% to 21% (Osman and
others 2001). Shamsudin and Salimon (2006) also reported that
PUFAs in the omega-3 class exhibit higher percentages than PU-
FAs in the omega-6 class in aji-aji fish oil. DHA (about 8%)
contained in aji-aji fish oil is higher than EPA (only about 1%)
comparatively. However, their percentages are much lower com-
pared to EPA (about 13%) and DHA (about 12.6%) contained in
menhaden fish oil (cold water fish). Lipid contents and fatty acid
compositions of marine fishes differ with regard to species, sex,
age, size, reproductive status, geographic location, and season
(Piggott and Tucker 1990). Fish oil is a major source of PUFAs,
mainly EPA and DHA; and they are important for human health
and nutrition, as well as for the fish itself. The role of PUFAs in
fish is to maintain cellular structure and function, and also to
regulate normal cell growth and development (Cejas and others
2004). Under normal circumstances, fish obtains the PUFAs from
the diet, mainly from phytoplankton.
Several methods have been reported for concentrating PUFAs
in marine oils, with varied yields. Among methods that concen-
trate PUFAs as TGs, without prior hydrolysis, are solvent fraction-
ation, winterization, and molecular distillation (Haraldsson and
others 1995). Concentrations of up to 30% EPA and DHA are fea-
sible using these methods. However, higher levels (65% to 80%)
are attainable by processes combining either hydrolysis or esteri-
fication with methods such as supercritical fluid extraction, urea
complexation, and molecular distillation. Concentrations beyond
90% are possible with high-performance liquid chromatogra-
phy (HPLC; Haraldsson and others 1995). Urea complexation
has been applied extensively to concentrate PUFAs from various
sources including marine and vegetable oils (Ackman and others
1988; Traitler and others 1988; Hayes and others 1998; Ju and
others 1998). Ratnayake and others (1988) demonstrated pilot-
scale (20 kg) urea complexation for concentrating n-3 PUFAs.
Compared with the other methods for producing PUFA concen-
trates, urea fractionation allows handling of large quantities of
materials in simple equipment. Since the process requires only
limited use of less toxic organic solvents such as ethanol, it is
environmentally friendly. It is also cost-effective because urea
is relatively inexpensive. Urea forms complexes with molecules
containing linear alkyl chains, which act as a template with which
urea molecules complex in spiral-shaped structures during sev-
eral hours of cooling (Hayes and others 1998). Separation of urea
complexes from the nonurea complexing fraction effectively re-
moves saturated and long-chain MUFAs and enriches the liquid
extract in unsaturated FAs. Fish processing could generate wastes
of up to about 50% of the body weight of the processed fish
based on the body components of interest to the processor (Babbit
1990). Wastes from fish processing are used to produce fishmeal,
with oil as a by-product, or to remediate soil. The oil content of
fish waste lies between 1.4% and 40.1% (Babbit 1990) depend-
ing on the species and tissue. Fish processing waste is therefore
an important source of fish oil that could serve as a good source
of PUFAs while adding value to the waste. This study was under-
taken to extract and characterize the oil from mackerel processing
waste, which is comprised mainly skin, viscera, and muscle tis-
sues, and to assess the possibility of concentrating PUFAs from
the oil extracted from these tissues using urea complexation.
However, conventional fish processing for removal of oil from
proteins involves cooking, pressing, and/or liquid extraction. Re-
moval of lipids with organic solvents causes protein denatura-
tion and loss of functional properties (Pariser and others 1978).
Adeniyi and Bawa (2008) extracted mackerel (Scomber scrom-
brus) oil using standard medium fish that were pretreated by
washing, drying, particle size reduction, and acid hydrolysis be-
fore transferring into the Soxhlet extraction apparatus for contin-
Vol. 8, 2009—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY
uous extraction with petroleum ether (Macrae and others 1993).
Refining of the extracted crude oil was carried out through
degumming, neutralization, deodorization, filtering, and bleach-
ing using methods as outlined by Hall (1992). The refined oil was
then characterized to determine the moisture content and values
of acid, saponification, iodine, peroxide, refractive index, and
specific gravity by employing the methods specified by Intl. Stan-
dards Organization (ISO 1975, 1988, 1998). Graham and others
(2007) reported that current omega-3 long chain-PUFA sources
(predominantly oceanic fish oils) are in serious decline; more-
over, environmental contamination of marine environments has
resulted in the presence of potentially toxic substances such as
heavy metals and dioxins in fish oils. Thus, there is a very obvious
requirement for an alternative and sustainable source of fish oils
for use in human nutrition.
Enzymatic Extraction
Mahmoud and others (2008) fractionated lipids from rainbow
trout (Oncorhynchus mykiss) roe were extracted by chemical and
enzymatic methods. Figure 4 shows a scheme for the procedure
of enzymatic extraction of trout roe. The authors reported en-
zymatic hydrolysis by Alcalase, Neutrase, and Protamex yielded
3 fractions after centrifugation: a light phase (oil), an intermedi-
ate fraction (water-soluble material) and a residual heavy phase
(Figure 4). The hydrolysis was performed within 120 min with
the 3 tested enzymes and the degree of hydrolysis was calculated
according to the pH-Stat method. Using the different proteases,
some discrepancies were noticeable among the hydrolysis de-
gree (DH) values. Alcalase was the most efficient (7.8% DH)
compared to Neutrase (3.2%) and Protamex (2.3%). Neverthe-
less, the total nitrogen contents of the water phase were in the
same range for Alcalase and Protamex, indicating that Alcalase
may lead to a high quantity of small peptides, while Protamex
and Neutrase may cut larger peptide fragments. Moreover, the
DH from Alcalase on fish roe (7.8%) can be compared with the
19% obtained with the same enzyme in trout fillets. The low yield
of fish roe proteolysis could lie in the specific composition of the
chorion, which must resist fungal and microbial attacks for days
in water. Also, the insolubility of the chorion might be due to the
formation of isopeptide bonds involving the side chains of Glu
and Lys or Arg residues of its constituent proteins. Heavy fractions
of molecular mass of more than 2000 g/mol represented about
25% of total peptides of the hydrolysate of trout roe treated with
Alcalase. Another remarkable result concerned the oil release af-
ter the different enzymatic extractions. Alcalase remained the best
tool for oil release, with 38.5% of total lipid content, compared
to 29.6% and 18.3% for Protamex and Neutrase, respectively.
Such observation is consistent with the fact that a high degree of
hydrolysis is needed for a good oil extraction. The authors also
compared the melting points and enthalpies of the oils fractions
obtained by solvent extraction against enzymatic processing, in-
cluding the supernatant neutral oil and the lipids contained in
the heavy phase. Melting points stood around −15 to −18 ◦ C,
indicating that the lipid fraction contained a high proportion of
unsaturated fatty acids, assessed by the low enthalpies of fusion,
which are characteristic of low-melting oils. However, it is inter-
esting to notice that the melting point of the total oil extracted
by solvents was an average of that of the lipids contained in the
supernatant oil and in the heavy phase after enzymatic treatment.
This indicated that there is a partition of the oil composition be-
tween both phases during the enzymatic treatment.
Considering the enzymatic extraction, it can be stressed that
the supernatant oil is mainly composed of neutral lipids (97.5%),
while the lipids contained in the heavy phase are mainly polar
lipids (73.3%). This is probably due to their amphipatic properties
67
CRFSFS: Comprehensive Reviews in Food Science and Food Safety
that facilitate their linkage with hydrophilic material. The anal- Extraction and Fractionation of PUFAs from Fish Using
ysis of the polar lipid fraction resulting from thin layer chro- Supercritical CO 2
matography (TLC)-flame-ionization detector (FID) determinations Supercritical fluid extraction and fractionation of fish oil fatty
also showed that solvents do not extract the same amounts of acids have been studied by many researchers during the last 20 y
major phospholipids than a protease. While both solvent meth- (Esqúıvel and others 1997; Borch-Jensen and Mollerup 1999;
ods released similar amounts of phosphatidylcholine (PC), phos- Brunner and Riha 2000; Létisse and others 2006; Perretti and
phatidylethanolamine (PE) and phosphatidylinositol (PI), it ap- others 2007). In general, pressure, temperature, CO 2 rate, and
pears that the major phospholipid released by Alcalase was time seemed to be important parameters for the optimization of
mainly PC accounting for 89.6% of total polar lipids. As the extraction. Several studies showed the influence of parameters
enzymatic process was carried out at pH 8 with no pH lowering which included pressure, temperature, CO 2 rate, and time. Pres-
at the end of the reaction time, a positive charge could persist
sure, temperature, and time have been studied by Özden (2000),
on the choline moiety, which exhibit a pKa of 13.9. Therefore,
pressure and temperature by Esqúıvel and others (1997) and Fleck
PC can engage strong interactions with the residual proteins frag-
and others (1998), while temperature, pressure, and CO 2 rate
ments contained in the heavy phase. The fatty acid composition
have been studied by Dunford and others (1998). However, frac-
of neutral oil and polar lipids obtained by solvent and proteolytic
tionation of fish oil fatty acid ethyl esters was investigated with
extractions compared to literature are shown in Table 5. As stated
the aim of obtaining a lipid fraction enriched in ω-3 fatty acids
above, the overall PUFA content was high and stood around 40%
and with a suitable EPA/DHA ratio. Perretti and others (2007)
to 50% of total fatty acids in total lipids: 46.4% and 41.8% by sol-
reported the possibility of modifying the original fatty acid ethyl
vent extraction and enzymatic treatment, respectively, compared
ester concentrations by optimizing the extraction conditions in
to literature: 41.8% (Haliloğlu and others 2003). According to
terms of pressure, temperature, and supercritical carbon dioxide
Shirai and others (2006), the polar lipid content was nearly 50%,
flow rate. The authors conducted 2-h runs using different pres-
to be compared with the 53.4%. It is noticeable that DHA reached
sures (100, 140, 150, and 300 bar) and different liquid CO 2 flow
29.7% of total fatty acids in the polar lipids fraction. This con-
rates (2.5, 3.5, 5, and 10 kg/h), keeping the temperatures of the
tent was one of the highest DHA levels found in animals tissues,
3 column sections at 40, 50, and 60 ◦ C, respectively, starting
unless in the heavy fraction resulting from salmon head prote-
from the bottom. They stated that supercritical fluid fractiona-
olysis by Alcalase, 2.4 L for example, where the concentration
tion appears to be a useful processing technique for changing
of DHA is about 33% (Alder-Nissen 1977). Major phospholipids
the composition of lipids in order to obtain high-value functional
were represented by PC and PE in the solvent extract. They ap-
products. The use of proper fractionation temperatures and pres-
peared slightly different regarding their fatty acid composition.
sures along with the column influenced the solvent-to-feed ratio
For instance, PC contained 23% palmitic acid compared with
to obtain fractions with suitable composition for market require-
11.4% in PE. Despite that, the overall PUFA content was the
ments.
same (41.4% total fatty acids), EPA reached.

Figure 4 --- Scheme of the enzymatic

hydrolysis of trout roe (source:
Mahmoud and others 2008).

68 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 8, 2009

PUFAs in fish . . .

Dunford and others (1997) studied supercritical CO 2 extrac-
tion of oil and residual proteins from Atlantic mackerel (Scomber
scombrus). They reported that supercritical fluid extraction of fish
muscle may be an alternative technique to produce high-quality
fish meal and oil; and it has been used for fish processing. The
concentration of ω-3 fatty acids in fish oil using supercritical
carbon dioxide (SC-CO 2 ) was studied extensively by Eisenbach
(1984), Krukonis (1988), Rizvi and others (1988), Nilsson and oth-
ers (1988, 1989), and Higashidate and others (1990). However,
oil extraction directly from high-fat fish muscle has attracted less
attention than fish oil fractionation. Extraction of ground freeze-
dried Antarctic krill with SC-CO 2 at 25 to 40 MPa and 40 to
80 ◦ C yielded oils which contained no phospholipids (PL) but
triglycerides (TG) with an eicosapentaenoic acid (EPA) content of
11% (Yamaguchi and others 1986). Ikushima and others (1986)
extracted freeze-dried mackerel (Scomber japonicus) powder us-
ing SC-CO 2 at 4.9 to 24.5 MPa and 40 ◦ C. The oil yield increased
much with pressure compared to using 5-h hexane extraction. In
their study, a kinetic model was developed to describe transport
phenomena within the solids during the SCFE process. SC-CO 2
and SC-CO 2 /ethanol (EtOH) mixtures have been used to remove
lipids from trout muscle with a moisture content of 70% (w/w)
(Hardardottir and Kinsella 1988). Those authors reported that
lipid removal did not improve when pressure and temperature
were increased from 13.8 to 34.5 MPa and from 40 to 50 ◦ C,
probably due to the very high moisture content of the feed.
SC-CO 2 and SC-CO 2 /EtOH extraction removed 78% and 97%
of the lipids and 97% and 99% of cholesterol, respectively. The
moisture content of the SC-CO 2 -extracted muscle (13.1%) was
substantially lower than the initial moisture content of 74.9%, in-
dicating removal of moisture with the lipids (Hardardottir and
Kinsella 1988). Temelli and others (1994, 1995) investigated
SC-CO 2 extraction of freeze-dried Atlantic mackerel (Scomber
scombrus), studying in particular the changes in proteins due to
high-pressure oil extraction. Extraction conditions of 34.5 MPa
and 35 ◦ C resulted in high oil yield and minimal changes in
residual proteins. Water binding potential and pH of residual
proteins, changes in sarcoplasmic proteins and ω-3 fatty acid
composition of the SC-CO 2 -extracted oil were reported. In most
of the cited studies (Ikushima and others 1986; Temelli and others
1994, 1995), fish samples were freeze-dried prior to SC-CO 2 ex-
traction of oil to improve extraction efficiency. Hardardottir and
Kinsella (1988) froze trout muscle pieces in liquid nitrogen and
ground them to increase surface area. Moisture of the feed mate-
rials may interfere with SC-CO 2 extraction of desired components
from the sample matrix. This was shown to be very important dur-
ing the SC-CO 2 extraction of lipids from muscle (King and others
1989). Extraction of intact muscle presents difficulties due to its
fibrous structure and high moisture content (Wehling and others
1992). King and others (1989) reported higher extraction rates for
meat products, which were comminuted and dehydrated prior
to SC-CO 2 extraction of lipids. However, optimization of sam-
ple moisture content for SC-CO 2 lipid extraction from muscle
tissues and its effect on proteins has not been reported. Corre-
lation of moisture content with water activity in such biological
samples should also be studied. Water activity would be a crit-
ical factor affecting all component interactions during SC-CO 2
extraction.
Dunford and others (1997) experimented with mackerel sam-
ples with moisture contents of 3.8%, 10.2%, 26%, and 64% us-
ing SC-CO 2 extractions at 35 ◦ C and 34.5 MPa for 5 h (CO 2
flow rate was 2.2 ± 0.4 g/min). The researchers character-
ized the lipid composition of SC-CO 2 extracts and the residual
oil. They also analyzed changes in sarcoplasmic proteins after

Table 5 --- Fatty acid compositions of neutral oil and polar lipids obtained by solvent and proteolytic extractions from
rainbow trout roe (Mahmoud and others 2008).
Solvent extraction Enzymatic extraction
Trout roe Oil from
Fatty acid total lipids Ref.a TAG Ref.b PL Ref.c Oil heavy phase

14:0 3.0 ± 0.1 3.1 3.8 ± 0.9 5.8 1.8 ± 0.1 2.1 3.8 ± 0.0 2.4 ± 0.0
16:0 16.1 ± 0.0 15.2 15.4 ± 2.2 9.3 14.0 ± 0.2 12 13.7 ± 0.0 17.2 ± 0.2
16:1 n-7 6.2 ± 0.0 5.9 7.3 ± 1.3 8.0 1.6 ± 0.1 2.3 6.8 ± 0.1 5.2 ± 0.1
18:0 5.1 ± 0.0 3.1 4.1 ± 0.2 2.0 11.2 ± 0.9 8.9 3.4 ± 0.0 6.4 ± 0.0
18:1 n-9 15.6 ± 0.1 22.41 20.1 ± 1.1 20.8 7.9 ± 0.3 10.7 16.0 ± 0.1 15.2 ± 0.0
18:1 n-7 4.3 ± 0.2 − 5.4 ± 0.1 3.1 6.1 ± 0.0 3.4 3.4 ± 0.2 4.3 ± 0.1
18:2 n-6 4.2 ± 0.0 9.6 4.9 ± 0.5 1.3 1.7 ± 0.0 0.5 4.7 ± 0.0 3.4 ± 0.1
20:1 n-9 3.4 ± 0.0 0.9 4.0 ± 0.3 1.2 5.6 ± 0.3 0.3 1.1 ± 0.0 0.8 ± 0.0
20:4 n-6 1.9 ± 0.0 3.1 1.6 ± 0.1 2.2 3.7 ± 0.4 2.7 2.4 ± 0.0 3.7 ± 0.0
20:5 n-3 11.5 ± 0.0 4.7 9.7 ± 1.5 14.9 10.9 ± 0.7 16.1 11.3 ± 0.0 10.9 ± 0.1
22:5 n-3 4.8 ± 0.0 1.2 4.1 ± 0.1 5.5 4.1 ± 0.1 6.2 4.4 ± 0.0 4.9 ± 0.0
22:6 n-3 24.0 ± 0.3 20.7 17.4 ± 3.4 14.3 29.7 ± 0.7 27.9 19.0 ± 0.2 25.6 ± 0.1
Total SFA 24.2 ± 0.1 21.9 23.3 ± 2.9 17.1 27.0 ± 1.0 23.0 20.8 ± 0.0 26 ± 0.3
Total MUFA 29.4 ± 0.2 33.9 36.7 ± 2.1 33.1 21.2 ± 0.1 16.7 27.4 ± 0.2 25.5 ± 0.0
Total PUFA 46.4 ± 0.3 41.8 37.7 ± 4.6 38.2 50.1 ± 1.1 53.4 41.8 ± 0.2 48.6 ± 0.2
Total n-9 19.0 ± 0.1 − 24.1 ± 0.8 22.0 13.5 ± 0.1 11.0 17.1 ± 0.1 16.0 ± 0.0
Total n-7 10.4 ± 0.2 − 12.6 ± 1.4 11.1 7.8 ± 0.1 5.7 10.2 ± 0.3 9.5 ± 0.0
Total n-6 6.1 ± 0.0 15.7 6.5 ± 0.4 3.5 5.4 ± 0.3 3.2 7.1 ± 0.0 7.1 ± 0.0
Total n-3 40.3 ± 0.3 26.0 31.2 ± 5.0 34.7 44.7 ± 1.5 50.2 34.7 ± 0.2 41.5 ± 0.2
n-3/n-6 6.6 ± 0.1 1.6 4.8 ± 1.1 9.9 8.2 ± 0.8 15.6 4.9 ± 0.0 5.9 ± 0.0
DHA/EPA 2.1 ± 0.0 4.4 1.8 ± 0.1 0.9 2.7 ± 0.1 1.7 1.7 ± 0.0 2.3 ± 0.1
TAG = triacylglycerols; PL = phospholipids. Data are given as mean ± S.E.M. (n = 3).
a Fatty acid composition of the total lipids from unfertilized eggs of rainbow trout (Ustadi and others 2005).
b Fatty acid composition of triacylglycerols (%) from Ikura (salted salmon roe, Bligh, and Dyer extraction) (Shirai and others 2006).
c Fatty acid composition of phospholipids (%) from Ikura (salted salmon roe, Bligh, and Dyer extraction) (Shirai and others 2006).

Vol. 8, 2009—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 69

CRFSFS: Comprehensive Reviews in Food Science and Food Safety
SC-CO 2 extraction at different moisture contents using capillary 323.15 K at 24.5 MPa showed no variation in the values for sol-
electrophoresis (CE). Previous studies with Atlantic mackerel had ubility. The highest values for solubility found in this study were
shown that the highest ω-3 fatty acid concentration in the extract about 7 g oil/kg CO 2 at 29.4 MPa and the different tempera-
with minimal damage to proteins was attained under those con- tures, and higher values can be expected at higher pressures. For
ditions (Temelli and others 1994, 1995). The amounts of extract Atlantic mackerel oil, the highest solubility observed was 14.21
obtained at 64% and 26% moisture were similar. The amount ex- g/kg CO 2 at 34.5 MPa and 308.15 K (Temelli and others 1995).
tracted increased when the moisture content of mackerel samples The behavior of the solubility values obtained in this study was
was decreased to 10.2% from the original level of 64% prior to consistent, considering that the solubility of a solute is directly
SC-CO 2 extraction. Further dehydration of samples from 10.2% dependent on the density of the solvent, and that increments in
to 3.8% did not increase the amount extracted with SC-CO 2 . The pressure at constant temperature promote an increase in the den-
amount of oil extracted increased from 0.3 to 2.5 g when the sity of the solvent. In the pressure range studied, the solubility did
moisture content was decreased from 64% to 26%. However, the not show a crossover point for retrograde behavior. Knowledge
extract yield was similar (2.5 to 2.7 g) for samples with 10.2% of the solubility of the sample under study is important to esti-
and 3.8% moisture. Only 10% of the oil in the feed was extracted mate the economic viability of the process, due to the fact that
at the original moisture content (64%, w/w). The low oil recovery the greater the value for solubility the lower the consumption of
was due to 2 reasons: (a) high moisture content acted as a barrier solvent. In general, the solubility of oils can be estimated by cor-
to diffusion of SC-CO 2 into and diffusion of oil out of the matrix,relations as proposed by Del Valle and Aguilera (1998). Corrêa
and (b) the pasty consistency of chopped muscle samples reduced and others (2008) also fractionated fish oil with babassu fat using
SC-CO 2 -sample contact. These results indicated that it was not SC-CO 2 . In order to better visualize how the fatty acid compo-
necessary to dry mackerel samples to less than 26% moisture sition of the triacylglycerols, the molar mass, and the degrees of
to achieve higher oil yields from SC-CO 2 extraction. This would unsaturation influenced the selectivity of the solvent, a mixture
result in saving time (freeze drying is a very slow process) and containing equal parts of fish oil and babassu fat was prepared,
energy. Furthermore, shorter drying times would reduce the risk and the fractionation analyzed. The possibility of babassu fat to
of quality deterioration of proteins. influence the equilibrium and the fractionation of fish oil was also
Corrêa and others (2008) extracted fish oil using CO 2 and analyzed. Babassu fat was chosen because it contains a different
reported that ω-3 fatty acid has a better possibility of fraction- fatty acid composition in comparison with the fish oil, being
ation under the operational conditions ranging from 306.15 to composed of approximately 80% of saturated fatty acids (C8:0 to
329.15 K and from 7.8 to 29.4 MPa. This result indicates that C18:0) and 20% of unsaturated C18:1 and C18:2 (Gioielli and
the CO 2 is more selective in the zone near its critical point to others 1998). Corrêa and others (2008) also reported that the sol-
fractionate the triacylglycerols of fish oil. However, the solubil- ubility of the mixture of fish oil and babassu fat (1:1) was higher
ity of 0.52 g/kg CO 2 obtained for the fish oil at 301.15 K and than the solubility of the fish oil, due to the content of shortchain
7.8 MPa was much lower that the solubility under the other op- fatty acids (lower molar mass) from the babassu fat. This behav-
erational conditions (Table 6). The low value for solubility can ior can be verified in the literature (Soares and others 2007). To
limit the process because of the high consumption of solvent. verify if the babassu fat has some influence on the separation of
Table 6 shows the solubility of the oil in SC-CO 2 as a function the compounds of interest, the solubility of the fatty acids EPA
of temperature and pressure or as a function of temperature and and DHA in the extracts of fish oil and in the mixture of fish oil
density. Since the light phase of the equilibrium mixture for the and babassu fat (1:1) were analyzed under the operational con-
systems under study was only slightly concentrated in the solute, ditions of 7.8 MPa and 301.15 K. The extracts obtained under
the density was considered to be that of pure CO 2 and they were these distinctive situations resulted in triacylglycerols containing
calculated by an empirical equation (Huang and others 1985), 11.7% and 1.45% of EPA, corresponding to values for solubility
which reproduces the experimental values of IUPAC (Angus and of 0.061 and 0.032 g (EPA)/kg CO 2 . In an analogous way, they
others 1976). The results show that the solubility of the fish oil contained 11.24% and 1.32% of DHA, with solubility values cor-
increased with increase in pressure at constant temperature, the responding to 0.058 and 0.029 g (DHA)/kg CO 2 , confirming that
effect of temperature being less meaningful. With an increase the solubility values for the triacylglycerols containing EPA and
from 313.15 to 323.15 K at 19.6 MPa, the solubility decreased DHA reduced to half the original value in the mixture (fish oil and
by about 18%, and the same increase in temperature at 29.4 babassu fat). This behavior indicates that the babassu fat did not
MPa resulted in a decrease in solubility of only about 7%. An in- promote a change in the fractionation of the fish oil. However, the
crease from 303.15 to 313.15 K at 29.4 MPa and from 313.15 to researchers concluded that ω-3 fatty acid composition of the fish
oil used in this study was high, almost 1/3 of the total fatty acids,
and it was highly probable that the fatty acids of interest would
Table 6 --- Fish oil solubility in SC-CO 2 . be present in almost all the triglyceride molecules, as evidenced
by the difficulty in fractionating them. Thus it is possible that it
Temperature Pressure CO 2 density Solubility could be used to enrich fish oil containing a lower ω-3 fatty acid
(K) (MPa) (kg/m3 ) (g/kg) composition.
Several studies also on the supercritical extraction of fish oil
303.15 29.4 946 7.0 ± 0.3 and mainly the fractionation of EPA and DHA as fatty acid ethyl
313.15 19.6 837 2.8 ± 0.1 esters from fish oils are shown in Table 7, such as the SC-
24.5 877 3.5 ± 0.1 CO 2 extraction of oil from sardine (Létisse and others 2006);
29.4 907 7.1 ± 0.3 the influence of moisture from the matrix (Atlantic mackerel) on
323.15 14.7 694 2.0 ± 0.1 the oil yield and on the changes in sarcoplasmic proteins of the
19.6 780 2.3 ± 0.1 matrix after SC-CO 2 extraction (Dunford and others 1997); the
24.5 831 3.6 ± 0.1 SC-CO 2 fractionation of EPA and DHA as fatty acid ethyl es-
29.4 867 6.6 ± 0.2 ters (Riha and Brunner 1999; Alkio and others 2000; Jaubert and
306.15 7.8 574 0.45 ± 0.02 others 2001; Espinosa and others 2002; Gironi and Maschietti
301.15 7.8 733 0.52 ± 0.03 2006; Jachmanián and others 2007; Perretti and others 2007);
Source: Corrêa and others 2008. and the phase equilibrium for SC-CO 2 and fish oil, including free
70 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 8, 2009
PUFAs in fish . . .

fatty acids, cholesterol, wax esters, and di- and triacylglycerols is denoted as pressure swing (PS) technique. Researchers applied
(Borch-Jensen and Mollerup 1997). the PS technique and were able to get higher yield with least
amount of CO 2 . Extractions were performed at 353.2 K and at
pressures from 10 to 25 MPa. Results were compared with contin-
Future Development of Fish Oil Extraction Method uous extractions, in which supercritical CO 2 was flowed through
Using SC-CO 2 the packed bed of solids for a given time period. For the PS ex-
By far it is obvious that the SFE method is very advantageous tractions, some intact or bound oil could be extracted from the
and environmentally friendly over other conventional either sol- 3rd PS step at 15 MPa, while for continuous extractions pressures
vent or enzyme extraction methods for recovering PUFAs, but of 20 MPa were required to obtain comparable yields. In the PS
the major problem is the SFE method consumes a lot of CO 2 . To extractions, disruption of the oil glands in palm kernel granules
overcome this problem, Zaidul and others (2007) introduced the probably lead to higher yields obtained at 20 and 25 MPa and
pressure swing technique for the separation of palm kernel oil this was confirmed with SEM micrographs. However, almost all of
(PKO) from undehulled ground palm kernel using supercritical the oil was extracted using combined PS and continuous extrac-
CO 2 . The pressurization of CO 2 into the sample, with holding tion at 25 MPa. The researchers developed a simple correlation
to penetrate into the sample matrix, and then depressurization, based on the kinetic mass transfer model, which allows one to

Table 7 --- SC-CO 2 extraction of fish oil and of fractionation of EPA and DHA (Corrêa and others [2008]).
Material Process T (◦ C) P (bar) Analysis Observation Source

Sardine heads SC-CO 2 extraction of 50 to 80 100 to 350 Oil yield, EPA, and Better results at 75 ◦ C (a)
oil DHA composition 300 bar
Atlantic mackerel SC-CO 2 extraction of 35 345 Oil yield, changes in Better results at 10.2% (b)
oil protein. 3.8% to 64% of moisture
of moisture
Commercial mixture of SC-CO 2 fractionation 40 to 60 100 to 300 EPA and DHA Possibility to obtain (c)
fish oil fatty acid ethyl of fatty acid ethyl EPA/DHA ratio EPA and a DHA and
esters, 64% esters in a 3-stage ω-3/ω-6 ratio solvent EPA/DHA ratio for
(EPA+DHA) column mass flow rate market purpose
influence
Fatty acid ethyl esters SC-CO 2 extraction of 40 to 70 86.3 to 180.4 Ethyl esters Increase of DHA (d)
from hake liver oil ethyl esters composition solubility content from 17.5%
selectivity of CO 2 for to 55%
EPA and DHA
Fatty acid ethyl esters SC-CO 2 extraction of 42 to 70 101 to 172 Thermodynamic Better results were (e)
from a mixture of ethyl esters modeling process obtained at 70 ◦ C
sardine, anchovy, simulation 167 MPa
and mackerel oils
(95.7%) EPA and High-pressure 30 to 80 47 to 242 Vapor–liquid equilibria For each system, 70 (f)
(93.6%) DHA ethyl variable-volume data for CO 2 –EPA equilibrium pressures
esters view cell ethyl ester and for (dew or bubble) were
CO 2 –DHA ethyl measured
ester thermodynamic
modeling
13 different fatty acid High-pressure 40 to 80 90 to 250 Thermodynamic An equation of state is (g)
ethyl ester mixtures variable-volume modeling sufficient to correlate
derived from sardine view cell vapor–liquid phase equilibrium
oil equilibria data for
CO 2 –ethyl ester
mixtures
Complex mixtures of Simulation models for - - Thermodynamic A group contribution (h)
fish oil fatty acid ethyl process units modeling. Simulation equation of state
esters (GC-EOS) was used.
Fatty acid ethyl esters Supercritical fluid 65 145 Concentration DHA ester (i)
mixture from tuna oil chromatography concentrates up to
95% purity were
obtained
Fish oil from the sand High-pressure 20 to 120 60 to 650 Vapor–liquid equilibria The fish oil may be (j)
eel (Ammodytes variable-volume data for free fatty deodorized at
lancea) view cell acid, cholesterol, and temperatures above
triacylglycerol 353 K and pressures
selectivity of CO 2 at 350 to 500 bar
(a) Létisse and others 2006; (b) Dunford and others 1997; (c) Perretti and others 2007;
(d) Jachmanián and others 2007; (e) Gironi and Maschietti 2006; (f) Jaubert and others 2001;
(g) Riha and Brunner 1999; (h) Espinosa and others 2002; (i) Alkio and others 2000;
(j) Borch-Jensen and Mollerup 1997.

Vol. 8, 2009—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 71

CRFSFS: Comprehensive Reviews in Food Science and Food Safety
estimate the minimum amount of CO 2 required for a given yield.
However, the model that was developed by Zaidul and others
(2007) could be applied to extract and fractionate the fish oil es-
pecially PUFA (omega-3, 6 fatty acids) with minimum amount of
CO 2 usage for a wide range of conditions and would provide the
means for studying other pressure, temperature, and flow rates,
including temperature and pressure programming.
PUFA Production at Industrial-Scale by the Supercritical
Fluid (SFC) Method
Alkio and others (2000) reported a systematic procedure for
developing supercritical fluid (SFC) separation method for pro-
ducing EPA and DHA ethyl ester concentrates. The development
was done in preparative, laboratory-scale batches using the spe-
cific production rate of EPA and DHA ethyl esters as the target
function, which was maximized. The specific production rate
was the hourly production in grams of the desired compound per
kilogram of stationary phase. Their experimental material was
tuna oil, which is a low-value by-product of the fishmeal indus-
try. A series of preparative SFC runs were carried out at estimated
optimal conditions to obtain the real production rate. They ap-
plied pure CO 2 (without co-solvents) to study the technical and
economic feasibility of producing EPA and DHA ethyl ester con-
centrates from by-product fish oil. The fatty acids in the tuna oil
were converted to ethyl esters by transesterification with abso-
lute ethyl alcohol. In the transesterification, 50 g tuna fish oil
was first dried with anhydrous Na 2 SO 4 . Its water content after
drying was 0.24 wt%. Dried oil was filtered and refluxed for 1.5
h with 350 g absolute ethyl alcohol. Freshly made sodium alco-
holate was used as catalyst. The mixture was then extracted with
n-hexane to obtain the fatty acid esters. The composition of the
resulting fish oil ethyl ester mixture was analyzed by GC–MS and
GC–FID. After transesterification, the DHA content of the oil was
higher, the EPA content unchanged, and the oleic and palmitic
acid contents lower than in the original oil. The authors suspect
that the repeated extraction of the ethanol containing aqueous
layer with hexane did not remove all the lighter fatty acid esters.
However, the slight change of the fatty acid composition during
transesterification does not influence the SFC process develop-
ment.
Alkio and others (2000) reported the most critical impurities
in obtaining pure DHA were other C22 esters. The separation
between EPA and DHA was incomplete, but due to the marginal
amount of EPA in the starting material, DHA did not interfere with
DHA. At all load ratio levels, the 1st DHA fractions also contained
18:0 and 18:1 esters. This indicates that the low unsaturated C18
esters tend to tail. This was not observed with C20 esters or with
C18 esters of a higher degree of unsaturation. The separation
of EPA from DHA, judged feasible by Reichmann and Brunner
(1996), was complete at each load ratio. The calculated SFC
separation factor between EPA and DHA was good, α = 1.40.
In the preparative runs, the measured production rate at 90%
(wt) purity was 0.85 g DHA-ethyl ester/(kg stationary phase ×
h). At 80 wt% purity the production rate was 1.9 g DHA ethyl
ester/(kg stationary phase × h). The purest EPA fractions at 5 and
2.5 g/kg load ratios contained, respectively, 33 and 53.8 wt%
EPA. Reducing the load to 1.25 g/kg did not increase EPA purity.
Similarly to the preparation of DHA, also in the separation of
EPA the main impurities were stearic (18:0) and oleic (18:1) acid
esters. At 50 wt% purity, the specific production rate of EPA was
0.23 g/(kg × h).
However, the production of DHA- and EPA-ethyl ester con-
centrates from transesterified tuna oil at 80 to 95 and 50 wt% re-
spective purities requires only one supercritical chromatographic
step. The flowsheet of an industrial SFC process, with descrip-
72 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 8, 2009
tions of main equipment, has been described by Aaltonen and
others (1998). An SFC process, which produces 1000 kg DHA-
ethyl ester and 400 kg EPA-ethyl ester concentrates per year re-
quires that 2.6 tons of CO 2 per hour is circulated in the process.
The octadecylsilane-grafted silica (ODS) stationary phase require-
ment is 160 kg, which would preferably be packed in four parallel
600-mm-i.d. columns. Major equipment for such an SFC process
costs about U.S. $2 million. In assuming that the stationary phase
would have to be replaced once a year, the total SFC operating
costs are U.S. $550/kg DHA and EPA concentrate. The purifica-
tion cost is sensitive to the lifetime of the stationary phase. The
cost almost equals the US$200 to 500 range reported in 1994
by KD Pharma (Lembke and Engelhardt 1994) and is consider-
ably less than the US$4000/DHA concentrate (95%) reported by
Shisheido Corp. (Anonymous 1996) where a proprietary silver-
containing stationary phase was used.
Conclusions
The potentiality to obtain health benefits of PUFAs especially
for the family of omega-3 fatty acids (linolenic acid, arachidonic
acid, EPA, and DHA) have been steadily increasing. Those fatty
acids have been identified to have a role in ameliorating various
human diseases. Marine fishes, especially Indian mackerel, of-
fer higher amounts of PUFAs (EPA and DHA) compared to other
marine fishes. The extraction and purification methods for those
fatty acids are summarized in this review. Emphasis is given on
recent advances in technological developments, particularly su-
percritical fluid extraction (SFE) from marine fish, especially from
Indian mackerel, and their fatty acid compositions. Use of SFE
technology that offers suitable extraction and fractionation ap-
pears to be promising for the production of omega-3 fatty acid
concentrates for the food and pharmaceutical industries. A brief
overview is provided of the principles and advantages of using
supercritical fluids for extraction and of chromatographic meth-
ods involving direct extraction of oil rich in EPA and DHA, as
well as of methods used for concentrating these fatty acids in the
ester form.
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