Food Chemistry 131 (2012) 328336

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Lipid content of frozen sh: Comparison of different extraction methods

and variability during freezing storage
Maria Joo Ramalhosa a,b, Paula Paga a, Simone Morais a, M. Rui Alves b,c, Cristina Delerue-Matos a,
Maria Beatriz Prior Pinto Oliveira b,
a
Requimte, Instituto Superior de Engenharia do Porto, Rua Dr. Antnio Bernardino de Almeida 431, 4200-072 Porto, Portugal
b
Requimte, Servio de Bromatologia, Faculdade de Farmcia, Universidade do Porto, Rua Anbal Cunha 164, 4099-030 Porto, Portugal
c
ESTG, Instituto Politcnico de Viana do Castelo, Av. Atlntico, 4900-348 Viana do Castelo, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: In this work, a microwave-assisted extraction (MAE) methodology was compared with several conven-
Received 26 October 2010 tional extraction methods (Soxhlet, Bligh & Dyer, modied Bligh & Dyer, Folch, modied Folch, Hara &
Received in revised form 29 May 2011 Radin, Roese-Gottlieb) for quantication of total lipid content of three sh species: horse mackerel (Tra-
Accepted 31 July 2011
churus trachurus), chub mackerel (Scomber japonicus), and sardine (Sardina pilchardus). The inuence of
Available online 5 August 2011
species, extraction method and frozen storage time (varying from fresh to 9 months of freezing) on total
lipid content was analysed in detail.
Keywords:
The efciencies of methods MAE, Bligh & Dyer, Folch, modied Folch and Hara & Radin were the highest
Fish
Extraction methods
and although they were not statistically different, differences existed in terms of variability, with MAE
Lipids showing the highest repeatability (CV = 0.034). Roese-Gottlieb, Soxhlet, and modied Bligh & Dyer meth-
Fat content ods were very poor in terms of efciency as well as repeatability (CV between 0.13 and 0.18).
2011 Published by Elsevier Ltd.

1. Introduction (Scomber japonicus; whole sh or minced llets) which was the

Total lipid content of sh samples is an important parameter
used in biochemical, physiological, and nutritional studies. Many
efforts are being made in order to include sh in human diet due
to its numerous benecial effects on health which are related to
its high protein and unsaturated fatty acid contents. However,
the reduction of sh stocks, restrictions on harvesting, the associ-
ated operation costs and the need for waste reduction have con-
tributed to the growing interest in maximising the use of shery
resources producing sh derivatives such as llet and minced sh.
These products are without smell, without spines and ready to eat
or heat and serve foods (FAO/WHO, 2005).
The process of freezing has been widely employed to maintain
sh properties (FAO/WHO, 2005). However, the efciency of freez-
ing conservation is not always achieved due to the occurrence of
lipid hydrolysis and oxidation which are directly related to the pro-
duction of off-avours and odours and inuence sh acceptance.
Some studies have been published about the changes in different
sh species during the frozen storage (Aubourg, Lehmann, & Gal-
lardo, 2002; Aubourg, Pieiro, & Gonzlez, 2004; Aubourg & Ugli-
ano, 2002; Le Bihan, Perrin, & Koueta, 2007; Serdaroglu &
Felekoglu, 2005). However, none was focused on chub mackerel
Corresponding author. Tel.: +351 222078927; fax: +351 222003977.
E-mail address: beatoliv@ff.up.pt (M.B.P.P. Oliveira).
sixth species that contributed most to global catches in 2006
(FAO, 2009).
The lipid group consists of many types of lipids with different
chemical composition. Lipids are usually classied into two
groups: the neutral or non-polar lipids (triglycerides, diglycerides,
monoglycerides, sterols, etc.) and the more polar lipids (free fatty
acids, phospholipids, sphingolipids, etc.) (Manirakiza, Covaci, &
Schepens, 2001).
Lipid content has been estimated using Soxhlet which is the of-
cial recommended method (AOAC, 2005). Several alternative meth-
ods were developed for total lipid extraction in sh samples. Folch,
Less, and Sloane (1957) optimised a method using a chloroform/
methanol/water phase system which, under various modications,
continues to be considered the classical and most reliable mean
for quantitative lipid extraction (Iverson, Lang, & Cooper, 2001). As
previously referred (Priego-Capote, Ruiz-Jimnez, & Luque de Cas-
tro, 2007), conventional fat extraction methods (Bligh and Dyer
(1959); modied Bligh & Dyer (Smedes, 1999); modied Folch
method (Lin, Liu, Yang, & Lee, 2004); Roese-Gottlied methodology
(Manirakiza et al., 2001) and Hara & Radin procedure (Hara & Radin,
1978)) have not been greatly improved, despite modications in sol-
vent mixtures and laboratory practice. Long preparation times with
re-extraction steps to ensure complete lipid isolation are still re-
quired. In the interest of economy and environment, new technolo-
gies such as Supercritical Fluid Extraction (SFE), Pressurised Liquid

0308-8146/$ - see front matter 2011 Published by Elsevier Ltd.

doi:10.1016/j.foodchem.2011.07.123
 M.J. Ramalhosa et al. / Food Chemistry 131 (2012) 328336
Extraction (PLE), Focused Microwave-Assisted Soxhlet Extraction
(FMASE) (Priego-Capote et al., 2007), Dynamic Ultrasound Assisted
Extraction (DUAE) (Ruiz-Jimnez & Luque de Castro, 2004) and
Microwave-Integrated Soxhlet (MIS) (Virot, Tomao, Colnagui, Visi-
noni & Chemat, 2007; Virot, Tomao, Ginies, Visinoni & Chemat,
2008a, 2008b) have been reported for total fat extraction. Micro-
wave-assisted extraction (MAE) is the process by which microwave
energy is used to heat solvents in contact with solid samples and to
partition compounds of interest from the sample into the solvent.
Studies dealing with MAE of lipids are scarce but show that it holds
great promise to improve lipid yields and repeatability/reproduc-
ibility, requiring, however, to be tested on a wider range of sample
matrices (ElKhori, Par, Blanger, & Prez, 2007; Virot et al., 2007;
Virot et al., 2008a, 2008b). Furthermore, previous works (Batista,
Vetter, & Luckas, 2001; Virot et al.,2007; Virot et al., 2008a, 2008b)
concluded that MAE is a well-suited alternative to conventional
Bligh and Dyer and Soxhlet methods for the extraction of lipids fol-
lowed by determination of the fatty acid prole. Results showed that
total fats and oils extracted by MAE were qualitatively similar to
those obtained by conventional methods. No signicant difference
was obtained between fatty acid composition of each extract allow-
ing to conclude that the method is effective and valuable for extrac-
tion of fats and oils from food products.
The present study aimed (i) to evaluate the total lipid content of
the most consumed sh species in Portugal, namely horse macker-
el (Trachurus trachurus), chub mackerel and sardine (Sardina pil-
chardus); (ii) to study the stability of total lipids content of
minced llets during frozen storage. Different lipid extraction
methods were tested: MAE, Soxhlet, Bligh & Dyer, modied Bligh
& Dyer, Folch, modied Folch, Hara & Radin and Roese-Gottlieb.
A MAE method was proposed as a viable alternative to conven-
tional lipid extraction techniques for sh samples. This promising
technique supports sustainable development, as it requires less en-
ergy and solvent than conventional processes, whilst generating
fewer wastes (ElKhori et al., 2007). Methods were compared for
their lipid extraction efciencies.
2. Experimental
2.1. Chemicals
All solvents used in extraction procedures were of analytical
grade and obtained from Merck (Darmstadt, Germany) and Panreac
(Barcelona, Spain). Sodium sulphate anhydrous (Panreac, Barce-
lona, Spain) was used as drying agent.
2.2. Sample collection and characterisation
Fresh individuals of the selected sh species originated from
Atlantic Ocean were purchased from different local markets in Por-
to region (NW Portugal) during 2009. Horse mackerel specimens
were purchased in winter. For chub mackerel and sardine, individ-
uals were purchased during two seasons winter and spring. Sea-
son, length, weight and gender were recorded for each individual.
Each sh specimen, from a total of 64, was headed, eviscerated,
skinned and lleted manually. Spines still present in tissues after
lleting were carefully removed. Each sample further analysed
(composite) was constituted by the edible parts of several individ-
uals and had a minimum mass of 200 g. Homogenisation was
performed mechanically with a Ufesa blender (Brio 400 WMAX,
PD-5310, Spain) until a smooth paste was obtained. Male and fe-
male samples were kept separated (sh gender was determined
by visual examination of the gonads). The number of specimens,
composites and biometric characterisation are indicated in Table 1.
Sampling was performed in accordance to the US EPA Guide No
329
823-B-00-07 (2000). Homogenised samples of minced sh were
immediately analysed and placed individually in 50 ml polycar-
bonate containers and stored at 20 C until further analysis.
2.3. Moisture determination
Water content (moisture) of each sh sample was determined
gravimetrically using homogenised samples (approximately 10 g,
accurately weighed), and dried for 3 h in an oven heated at
105 C, until constant weight according to the Portuguese standard
procedure NP 2282 (1991). Analyses were performed in triplicate
and results were expressed as g water per g wet muscle.
2.4. Lipid extraction methods
Eight different lipid extraction methods were carried out as fol-
lows: Soxhlet (AOAC, 2005), Bligh & Dyer (Bligh and Dyer (1959)),
modied Bligh & Dyer (Smedes, 1999), Folch (Folch et al., 1957),
modied Folch (Lin et al., 2004), Hara & Radin (Hara & Radin,
1978), Roese-Gottlieb (Manirakiza et al., 2001), and MAE. Quadru-
plicate of each sh composite were analysed by the eight tested
methods immediately after purchase and after one, two, three,
four, ve, six and nine months of freezing storage. The lipid content
was gravimetrically determined.
2.4.1. Soxhlet (S)
Extraction was performed according to AOAC (2005). One gram
of homogenised sh and 2 g of Na2SO4 were placed in a Soxhlet
extractor (Soxtest, Trade Raypa Soxhlet, SX-6, Spain). Thirty mil-
liliters of n-hexane were passed through, in hot extraction mode
at 140 C for 4 h. The extract was dried in an oven (Selecta, P,
Spain) at 104 C, until constant weight.
2.4.2. Bligh & Dyer (BD)
Five grams of sample, 20 mL of methanol (MeOH) and 10 mL of
chloroform (CHCl3) were added and vortexed (Nahita vortex, 681/
5, Spain) during 2 min in a conical ask. After that, 10 mL of CHCl3
were added and shaken vigorously during 2 min. Eighteen millili-
ters of distilled water were added and the mixture was vortexed
again for 2 min. The layers were separated by centrifugation (Sig-
ma, Santorius, 2-16, Germany) for 10 min at 2000 rpm. The lower
layer was transferred to a pear-shaped ask with a Pasteur pipette.
A second extraction was done with 20 mL of 10% (v/v) MeOH in
CHCl3 by vortexing for 2 min. After centrifugation, the CHCl3 phase
was added to the rst extract. Evaporation was done in a rotary
evaporator at 45 C and the residue was further dried in an oven
at 104 C for 1 h.
2.4.3. Modied Bligh & Dyer (BDm)
This method is a modication of the Bligh & Dyer procedure in
which MeOH and CHCl3 is replaced by 2-propanol and cyclohex-
ane, respectively. A mixture of water:2-propanol:cyclohexane
(11:8:10, v/v/v) was used as solvent. A second extraction with
20 mL of 10% (v/v) 2-propanol in cyclohexane was done. After cen-
trifugation, the cyclohexane phase was added to the rst extract.
The advantage of cyclohexane over CHCl3 is its lower density,
which consequently separated on the top of the extraction mix-
ture. Evaporation was done in a rotary evaporator at 45 C and
the residue was further dried in an oven at 104 C for 1 h.
2.4.4. Folch (F)
Five grams of sample were mixed with 100 mL of CHCl3:MeOH
(2:1, v/v), centrifuged for 10 min at 3000 rpm and ltered. Subse-
quently, 5 mL of distilled water was added to the ltrate and the
new mixture was shaken vigorously. The nal biphasic system
was allowed to separate by centrifugation (10 min, 3000 rpm).
330 M.J. Ramalhosa et al. / Food Chemistry 131 (2012) 328336
Table 1
Sample characterisation.
Species Capture season Gender Specimen number
Sardine Winter F 9
M 6
Spring F 4
M 21
Chub mackerel Winter F 4
M 6
Spring F 6
M 3
Horse mackerel Winter F 2
M 3
A
Mean values standard deviation of the mean.
*
n = 3.
The upper aqueous phase was eliminated. The lower CHCl3 phase
was ltered through Na2SO4, collected and evaporated in a rotary
evaporator at 45 C and the residue was further dried in an oven
at 104 C for 1 h.
2.4.5. Modied Folch (Fm)
Ethyl acetate and ethyl alcohol were mixed at the same ratio as
CHCl3 and MeOH in the Folch method (Folch et al., 1957).
2.4.6. Hara & Radin (HR)
One gram of sample was added to 18 mL of n-hexane:isopropa-
nol (3:2, v/v), homogenised for 30 s and ltered. The residue was
washed three times with 2 mL portions of n-hexane:isopropanol
(3:2, v/v), by resuspending the residue each time and letting the
solvent soak for 2 min before applying air pressure. Nonlipid com-
pounds in the extract were removed by mixing the pooled ltrates
for at least 1 min with 12 mL of aqueous Na2SO4 (prepared from 1 g
of the anhydrous salt and 15 mL of water). The two formed layers
have each one around 18 mL in volume. No precipitate was visible
at the interface. Lipids were in the upper n-hexane-rich layer and
evaporated in a rotary evaporator at 45 C. The residue was further
dried in an oven at 104 C for 1 h.
2.4.7. Roese-Gottlieb (RG)
This method consists of total lipid extraction with petroleum
ether after hydrolysis of the bounded lipids. To 1 g sample, 6 mL
of boiling water were added and the mixture was vortexed for
1 min and allowed to cool. One millilitre of 25% ammonia solution
was added and vortexed for 2 min, then 7.5 mL of MeOH were
added to the mixture and vortexed for 2 min. To this last mixture,
a rst addition of 17 mL of diethyl ether and a second addition of
17 mL of petroleum ether were added and shaken vigorously.
The mixture was centrifuged and the upper layer was transferred
to a tarred beaker. Concentration was performed with a rotary
evaporator at 45 C and further drying in an oven at 104 C for 1 h.
2.4.8. Microwave-assisted extraction (MAE)
The optimum extraction conditions were based on a previous
study concerning fat composition of Portuguese bakery products
(Casal et al., 2007). For performing this survey, a MAE procedure
was validated for biscuit fat extraction, using petroleum ether:ace-
tone (2:1, v/v) (Casal et al., 2007). For sh samples, and since water
is typically found at high levels in shery products acting as a polar
solvent and producing coextractables which complicate analysis,
anhydrous sodium sulphate was mixed with homogenised samples
before MAE. Several different anhydrous sodium sulphate/sample
ratios were tested, maintaining a total mass of 1 g. The best results
were obtained when using 1:2 (w/w) ratio.
Composites LenghtA (cm) WeightA (g) MoistureA,* (%, g/g)
2 20.3 0.8 72.1 8.7 81.3 0.1
1 19.9 1.1 71.3 16.4 82.4 0.6
1 21.1 0.5 78.0 3.7 74.3 0.6
2 20.5 0.7 76.6 8.1 75.8 0.1
1 27.6 1.3 155.0 20.0 78.5 0.4
2 28.3 2.3 170.0 40.7 79.5 1.0
2 35.6 3.2 379.0 111.4 68.6 0.1
1 30.8 2.0 217.0 50.9 75.6 0.7
1 31.5 0.7 283.0 29.7 77.3 0.3
2 31.5 3.1 295.0 75.2 75.5 0.4
One gram of homogenised sh sample and 2 g of Na2SO4 were
weighted for a quartz extraction vessel (GreenChem Plus) of a
Microwave Accelerated Reaction System, MARS-X, 1500 W (CEM,
Mathews, NC, USA), congured with a 14 position carousel (Santori-
us BasicPlus, P 211D, Germany). During operation, both temperature
and pressure were monitored in a single vessel. Magnetic stirring in
each extraction vessel and a sensor registering the solvent leaks in
the interior of the microwave oven were also used. After adding to
each sample 30.0 mL of petroleum ether:acetone (2:1, v/v) the ves-
sels were closed. The operational parameters were the following:
magnetron power 100%; time to reach settings 10 min; extraction
duration 20 min; medium speed stirring, extraction temperature
90 C and maximum vessel pressure cut off 1.38  106 Pa. After cool-
ing, the extract was ltered through Whatman GF/C lter paper
(England) lled with Na2SO4 and collected into an evaporator tube
(Normax, internal diameter 24 mm, external length 145 mm, Portu-
gal). The extract was evaporated in a rotary evaporator (Buchi Rota-
vapor, R-200, Switzerland) at 45 C and the residue was further dried
in an oven at 104 C for 1 h.
2.5. Statistical methods
In order to evaluate extraction efciencies, means of extracted
fat per composite were calculated (on wet and dry weight bases
but to simplify the discussion only concentrations in wet weight
(ww) are presented), yielding 120 means per method. These means
were plotted for comparison purposes. The average mean per
method (grand mean per method) was then computed averaging
respective composite means, and students t tests were used to
compare the methods extraction efciencies (grand means).
For the evaluation of each methods repeatability, the standard
deviations within composites were calculated and plotted for com-
parison purposes. Composite standard deviations were squared,
averaged over each method, and the square root of the average
was calculated as the residual standard deviation (RSD) for each
method. F tests were used to compare pairs of methods in relation
to their RSD.
Coefcients of variation were calculated for each composite and
plotted for comparison purposes. A general coefcient of variation
(CV) for each extraction method was obtained by division of the
respective RSD by the methods grand mean.
3. Results and discussion
Figs. 13 present the results obtained for chub mackerel, sar-
dine and horse mackerel, respectively. In each gure, i.e., for each
sh species, results are presented by season, within each season
by gender, within gender by frozen storage time. Regarding the
storage time, time zero (t0) was considered for determinations in
 M.J. Ramalhosa et al. / Food Chemistry 131 (2012) 328336 331

Fig. 1. Data relative to chub mackerel (Scomber japonicus), organised by season, gender and frozen time. Within each frozen time, for winter females and Spring males, points
represent the extraction methods MAE, S, BD, BDm, F, Fm, HR and RG, by this order. For winter males and Spring females, the order is the same, but values are duplicated
(MAE, MAE, S, S, . . .).

fresh sh and t1, t2, t3, t4, t5, t6 and t9 for analysis after one, two,
three, four, ve, six and nine months of freezing storage, respec-
tively. Most of frozen products containing sh have an expiry date
of six months and consequently the lipid content was analysed
month per month during this period.
Within each storage time there are (i) eight points representing
the extraction methods MAE, S, BD, BDm, F, Fm, HD and RG, by this
order, or (ii) sixteen points representing the extraction methods in
duplicate, following the same order, i.e., MAE, MAE, S, S, . . ., RG, RG,
in accordance with the design shown in column with header Com-
posites of Table 1.
In parts (a) of all gures, each point represents the average of
four lipid extractions using a particular extraction method. In parts
(b) points represent the corresponding standard deviations, and in
parts (c) the corresponding coefcients of variation. In all gures,
values (points) are presented as a line plot. This presentation is
articial, but makes it easier to observe any pattern. As a matter
of fact, all results show a pattern common to all frozen times. As
within all frozen times the extraction methods appear in the same
order, observed patterns are related to extraction methods.
Observing parts a) of the three gures, it is seen that within
each time period with eight points, higher extractions correspond
to points appearing in positions 1, 3, 5 and 7, corresponding to
methods MAE, BD, F and HR, whilst within time periods with six-
teen points higher extractions correspond to points appearing in
positions 1 and 2 (MAE), 5 and 6 (BD), 9 and 10 (F) and 13 and
14 (HR). Points appearing in positions 2 and 4 in the case with
no duplicates (S and BDm) and in positions 3 and 4 and 7 and 8
in the case with duplicates (S,S and BDm,BDm), correspond to
the methods showing the lesser extraction efciencies. As the line
plot pattern is repeated within frozen times, it can be said that
each methods extraction efciency is stable and independent of
all factors considered, i.e., sh species, gender, season and time
in frozen storage.
332 M.J. Ramalhosa et al. / Food Chemistry 131 (2012) 328336

Fig. 2. Data relative to sardine (Sardina pilchardus), organised by season, gender and frozen time. Within each frozen time, for Spring females and winter males, points
represent the extraction methods MAE, S, BD, BDm, F, Fm, HR and RG, by this order. For Spring males and winter females, the order is the same, but values are duplicated
(MAE, MAE, S, S, . . .).

The results of this work allow further to identify a residual de-
crease in lipid content observed during six months of frozen stor-
age, being more accentuate at the end of nine months probably due
to oxidation reactions. The same pattern of variation is observed
with data on dry weight base (results not shown) which clearly ex-
clude the possibility of being moisture interference.
In parts (b) of Figs. 13, repeating patterns are again observed.
In these gures each point represents the standard deviation of the
four replicates used for each condition. As all standard deviations
correspond to the average extractions shown in parts a), it can be
easily seen that methods MAE, BD, F and HR are again superior
than the others, because they show a higher repeatability (ex-
pressed by lower standard deviation).
In Figs. 13, parts (b), it is also seen that, as it is typical, when-
ever the mean extracted values increase, the corresponding stan-
dard deviations also increase. However, dividing the standard
deviations by the corresponding means, one obtains the coef-
cients of variation which are shown in parts (c) of Figs. 13. It is
now clear that in all situations considered in this work, these coef-
cients are consistently lower for methods MAE, BD, F and HR, and
eventually RG, always around 0.05 or below.
Considering all data available and displayed in the above men-
tioned gures, methods MAE, BD, F and HR show the best efcien-
cies (in terms of higher amounts of extracted fat on wet weight
basis and on dry (results not shown) weight basis) and also the
highest repeatabilities/reproducibilities (expressed by lower coef-
cient of variation). However, these conclusions are based solely
on observation of gures, and a more reliable statistical examina-
tion is necessary, as it is reported in the following paragraphs.
Table 2 reports the general results obtained for the statistical
comparison of extraction methods. In Table 2a, the rst row shows
methods grand means (the average extracted fat taken over all
 M.J. Ramalhosa et al. / Food Chemistry 131 (2012) 328336 333

Fig. 3. Data relative to horse mackerel (Trachurus trachurus) in winter, organised by gender and frozen time. Within each frozen time, for females points represent the
extraction methods MAE, S, BD, BDm, F, Fm, HR and RG, by this order. For males the order is the same, but values are duplicated (MAE, MAE, S, S, . . .).

species, gender, season and frozen time). These values are general
gures for extraction efciencies and must be analysed together
with Table 2b, where t tests for the signicance of differences be-
tween methods, taken two at a time, are shown. It is seen that
although the average extracted fat with MAE was always the high-
est, the observed differences are not signicant in relation to BD, F
and HR (p always greater than 0.8, highlighted in bold in Table 2b).
The group of methods that proved to extract less fat were BDm, RG
and S, being comparable between them (p always greater than 0.9,
highlighted in italic in Table 2b). The differences between these
two groups of methods are signicant, with p values around
0.05. Intermediate seems to be Fm, which is statistically not differ-
ent from the rst group (0.80 < p < 0.60) and also not different from
BDm (p = 0.09).
The RSD values are presented in the second line of Table 2a. Like
the grand means, the RSD values were based on all composites
(over species, gender, season and frozen storage times). The statis-
tical differences in RSD values between pairs of methods were
compared through F tests, shown in Table 2c. It is seen that the
lower RSD, equal to 0.117, was found for MAE, being signicantly
lower than all other methods. The only methods that, although
worse, approach MAE, are the F (p = 0.025) and BD (p = 0.004)
methods (highlighted in bold in Table 2c). All others are signi-
cantly worse (p < 0.000). These RSD values are general gures for
methods standard deviations in several situations, being a good
indicator of the methods reproducibilities. The division of any
RSD by the corresponding grand mean yields the coefcient
of variation (CV), shown in the third line of Table 2a, expressed
in percentage. This is a standard measure of repeatability/repro-
ducibility. Taking into consideration Figs. 13 and Table 2, it be-
comes clear that although the efciency of methods MAE, BD, F
and HR are the highest and are not statistically different between
themselves, differences exist in terms of variability, with MAE
showing lowest variability (CV = 3.35%), followed by methods F
334 M.J. Ramalhosa et al. / Food Chemistry 131 (2012) 328336

Table 2
Comparison of extraction efciencies and precisions (BD Bligh & Dyer; BDm modied Bligh & Dyer; F Folch; Fm modied Folch; HR Hara & Radin; MAE Microwave-
assisted extraction; RG Roese-Gottlieb; S Soxhlet).

BD BDm F Fm HR MAE RG S
(a)
Mean 3.454 2.918 3.478 3.381 3.434 3.500 2.944 2.917
RSD 0.132 0.513 0.128 0.443 0.149 0.117 0.159 0.441
CV% 3.835 17,570 3.690 13,097 4.344 3.352 5.394 15,105
(b) BDm 1.95
0.052
F 0.08 2.06
0.934 0.040
Fm 0.26 1.70 0.34
0.799 0.090 0.733
HR 0.07 1.87 0.16 0.18
0.943 0.062 0.877 0.856
MAE 0.16 2.13 0.08 0.42 0.23
0.873 0.034 0.938 0.677 0.817
RG 1.94 0.09 2.05 1.67 1.85 2.12
0.054 0.926 0.042 0.096 0.065 0.035
S 2.04 0.00 2.16 1.78 1.96 2.23 0.10
0.042 0.997 0.032 0.076 0.051 0.027 0.920
BD BDm F Fm HR MAE RG
(c) BDm 14.98
0.000
F 1.07 15.96
0.243 0.000
Fm 11.17 1.34 11.91
0.000 0.001 0.000
HR 1.27 11.81 1.35 8.82
0.005 0.000 0.001 0.000
MAE 1.28 19.10 1.20 14.25 1.62
0.004 0.000 0.025 0.000 0.000
RG 1.44 10.42 1.53 7.78 1.13 1.83
0.000 0.000 0.000 0.000 0.085 0.000
S 11.06 1.35 11.79 1.01 8.73 14.10 7.70
0.000 0.000 0.000 0.456 0.000 0.000 0.000
BD BDm F Fm HR MAE RG

(b) t Tests comparing pairs of extraction methods for their efciency. In each cell, t value above, signicance level below. (c) F tests comparing pairs of extraction methods for
their repeatibility. In each cell, F value above, signicance level below.

and BD. HR, with a CV lower than 5% can also be considered a good
extraction method. Remaining methods, RG, S, Fm and BDm have
low efciencies and low reproducibility, mainly S and BDm with
CV equal to 15.1% and 17.6%, respectively.
The information in Figs. 13 is complemented with Table 3, the
latter reporting ANOVA results for the performance of all methods
in each sh species. For each case, the amounts of extracted fat are
considered dependent on four factors (extraction method, gender,
season and frozen time), enabling the evaluation of the signicance
of the observed differences between extraction efciencies per
method and the effects and interactions between the main factors
considered during frozen storage.
As discussed previously in relation to gures, these tables show
that there are signicant differences in the amounts of fat ex-
tracted (on wet weight basis and on dry (results not shown) weight
basis). These differences depend on the extraction method used
and frozen time. Interactions between the main factors are almost
always highly signicant. It is important to emphasise that the li-
pid content of these species varies throughout the year particularly
with their life cycle, due to their feeding growth and nonfeeding
maturation cycles (Caponio, Lestingi, Summo, Bilancia, & Laudadio,
2004).
The determined fat contents for chub mackerel (0.828.99%) are
in accordance to those found by Stamatis and Arkoudelos (2007)
and Karakoltsidis, Zotos, and Constantinides (1995) for Scomber co-
lias japonicus (4.54.9%, Stamatis and Arkoudelos, 2007; and
4.0 0.6%, Karakoltsidis et al., 1995) from the Aegean Sea (Greece).
Celik (2008) reported for chub mackerel from the Iskenderun Bay
on the north-eastern Mediterranean coast of Turkey, a lipid con-
tent varying from 0.18% to 1.57%. The same pattern of variation
was observed for the same species by Soyer and Sahin (1999).
Fat content levels of sardines varied from 0.62% to 4.42%.
According to some authors, the fat content of sardine shows impor-
tant seasonal changes during the year, which is typical of pelagic
species. Bandarra, Batista, Nunes, Empis, and Christie (1997) found
a total lipid content ranging between 1.2% and 18.4% in sardines
harvested in the Portuguese coast, from Northeast Atlantic Ocean,
monthly during a year. Passi, Cataudella, Di Marco, De Simone, and
Rastrelli (2002) determined the total fat content in the white mus-
cle tissues of 21 species of teleosts and they obtained a value of
4.81 0.73% for sardine from central Tyrrhenian Sea, Italy. Caponio
et al. (2004) determined the total lipid content in sardine captured
from Ionian Sea, and obtained a range of 0.50.8%. Zlatanos and
Laskaridis (2007) obtained a minimum fat content at the end of
winter (3.88 0.2 g/100 g dry weight) and a maximum at the end
of spring beginning of summer (11.86 0.6 g/100 g dry weight)
for sardines purchased from the Central Market of Thessaloniki
(northern Greece).
Concerning horse mackerel, the values obtained (3.225.01%)
are in range with those reported by Losada, Pieiro, Barros-Velz-
quez, and Aubourg (2005) (1.23.7%) for horse mackerel from Gali-
cian Atlantic coast and by Simeonidou, Govaris, and Vareltzis
(1998) (4.39 1.3%) for specimens originated from the southeast
coast of Halkidiki peninsula of North Greece. Aubourg and Ugliano
(2002) determined a total fat content varying from 1.5% to 3.0% in
horse mackerel from the Northeast Atlantic. Variations in chemical
composition in marine shes are closely related to nutrition, living
area, sh size, catching season, water temperature, water salinity,
 M.J. Ramalhosa et al. / Food Chemistry 131 (2012) 328336 335

Table 3
Evaluation of factors affecting extraction efciencies: ANOVA with Sigma-restricted parameterisation.

Sum squares Degrees. freedom Mean squares F p

(a) Results for chub mackerel (Scomber japonicus)
Intercept 13731.83 1 13731.83 61434.41 0.00000
Method 207.53 7 29.65 132.63 0.00000
Gender 2431.84 1 2431.84 10879.75 0.00000
Season 5156.98 1 5156.98 23071.66 0.00000
Frozen-time 87.29 7 12.47 55.79 0.00000
MethodGender 56.14 7 8.02 35.88 0.00000
MethodSeason 47.33 7 6.76 30.25 0.00000
GenderSeason 1941.52 1 1941.52 8686.12 0.00000
MethodFrozen-time 4.51 49 0.09 0.41 0.99990
GenderFrozen-time 10.85 7 1.55 6.94 0.00000
SeasonFrozen-time 11.94 7 1.71 7.63 0.00000
Error 322.09 1441 0.22
(b) Results for sardine (Sardina pilchardus)
Intercept 8860.798 1 8860.798 109418.6 0.00000
Method 68.006 7 9.715 120 0.00000
Gender 0.306 1 0.306 3.8 0.05198
Season 1336.865 1 1336.865 16508.4 0.00000
Frozen-time 80.819 7 11.546 142.6 0.00000
MethodGender 1.844 7 0.263 3.3 0.00198
MethodSeason 17.94 7 2.563 31.6 0.00000
GenderSeason 13.498 1 13.498 166.7 0.00000
MethodFrozen-time 10.943 49 0.223 2.8 0.00000
GenderFrozen-time 1.11 7 0.159 2 0.05748
SeasonFrozen-time 1.019 7 0.146 1.8 0.08384
Error 116.693 1441 0.081
(c) Results for horse mackerel (Trachurus trachurus)
Intercept 9850.102 1 9850.102 81203.93 0.00000
Method 17.1 7 2.443 20.14 0.00000
Gender 174.894 1 174.894 1441.82 0.00000
Frozen-time 65.871 7 9.41 77.58 0.00000
MethodGender 0.95 7 0.136 1.12 0.34893
MethodFrozen-time 25.416 49 0.519 4.28 0.00000
GenderFrozen-time 1.873 7 0.268 2.21 0.03208
Error 83.576 689 0.121

seasonal and sexual variations as well as other environmental con- References

ditions (Stamatis and Arkoudelos, 2007).
4. Conclusion
Different extraction methods vary in their lipid extraction ef-
ciency and thus provide different lipid amounts. MAE was the
method with best extraction efciency and repeatability
(CV = 0.034). Methods BD, F, Fm and HR also performed well. Meth-
ods S, BDm and RG were very poor in terms of efciency and
repeatability for S and BDm. Furthermore, because of the inherent
difculties in the phase separation and the high number of manip-
ulations used in conventional extractions, MAE may be advised as a
convenient automated method for lipid extraction from sh sam-
ples. MAE has the advantages of requiring less laboratory material,
solvent volume and time. Also, lower toxicity solvents are applied.
MAE is easier to perform with several samples in parallel since 14
samples may be extracted simultaneously.
In conclusion, MAE is appropriate for sh fat extraction reach-
ing total lipid content similar to or higher than ofcial methods
with higher repeatability.
The stability of total lipid content during freezing and frozen
storage was considered. The results of this study showed that the
lipid content of minced sh remains almost unchanged over six
months, identifying this period as the shelf life time of this frozen
product, especially if produced with fatty sh.
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