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Food Chemistry 131 (2012) 328336

Contents lists available at ScienceDirect

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


sixth species that contributed most to global catches in 2006
Total lipid content of sh samples is an important parameter (FAO, 2009).
used in biochemical, physiological, and nutritional studies. Many The lipid group consists of many types of lipids with different
efforts are being made in order to include sh in human diet due chemical composition. Lipids are usually classied into two
to its numerous benecial effects on health which are related to groups: the neutral or non-polar lipids (triglycerides, diglycerides,
its high protein and unsaturated fatty acid contents. However, monoglycerides, sterols, etc.) and the more polar lipids (free fatty
the reduction of sh stocks, restrictions on harvesting, the associ- acids, phospholipids, sphingolipids, etc.) (Manirakiza, Covaci, &
ated operation costs and the need for waste reduction have con- Schepens, 2001).
tributed to the growing interest in maximising the use of shery Lipid content has been estimated using Soxhlet which is the of-
resources producing sh derivatives such as llet and minced sh. cial recommended method (AOAC, 2005). Several alternative meth-
These products are without smell, without spines and ready to eat ods were developed for total lipid extraction in sh samples. Folch,
or heat and serve foods (FAO/WHO, 2005). Less, and Sloane (1957) optimised a method using a chloroform/
The process of freezing has been widely employed to maintain methanol/water phase system which, under various modications,
sh properties (FAO/WHO, 2005). However, the efciency of freez- continues to be considered the classical and most reliable mean
ing conservation is not always achieved due to the occurrence of for quantitative lipid extraction (Iverson, Lang, & Cooper, 2001). As
lipid hydrolysis and oxidation which are directly related to the pro- previously referred (Priego-Capote, Ruiz-Jimnez, & Luque de Cas-
duction of off-avours and odours and inuence sh acceptance. tro, 2007), conventional fat extraction methods (Bligh and Dyer
Some studies have been published about the changes in different (1959); modied Bligh & Dyer (Smedes, 1999); modied Folch
sh species during the frozen storage (Aubourg, Lehmann, & Gal- method (Lin, Liu, Yang, & Lee, 2004); Roese-Gottlied methodology
lardo, 2002; Aubourg, Pieiro, & Gonzlez, 2004; Aubourg & Ugli- (Manirakiza et al., 2001) and Hara & Radin procedure (Hara & Radin,
ano, 2002; Le Bihan, Perrin, & Koueta, 2007; Serdaroglu & 1978)) have not been greatly improved, despite modications in sol-
Felekoglu, 2005). However, none was focused on chub mackerel vent mixtures and laboratory practice. Long preparation times with
re-extraction steps to ensure complete lipid isolation are still re-
Corresponding author. Tel.: +351 222078927; fax: +351 222003977. quired. In the interest of economy and environment, new technolo-
E-mail address: beatoliv@ff.up.pt (M.B.P.P. Oliveira). 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 329

Extraction (PLE), Focused Microwave-Assisted Soxhlet Extraction 823-B-00-07 (2000). Homogenised samples of minced sh were
(FMASE) (Priego-Capote et al., 2007), Dynamic Ultrasound Assisted immediately analysed and placed individually in 50 ml polycar-
Extraction (DUAE) (Ruiz-Jimnez & Luque de Castro, 2004) and bonate containers and stored at 20 C until further analysis.
Microwave-Integrated Soxhlet (MIS) (Virot, Tomao, Colnagui, Visi-
noni & Chemat, 2007; Virot, Tomao, Ginies, Visinoni & Chemat, 2.3. Moisture determination
2008a, 2008b) have been reported for total fat extraction. Micro-
wave-assisted extraction (MAE) is the process by which microwave Water content (moisture) of each sh sample was determined
energy is used to heat solvents in contact with solid samples and to gravimetrically using homogenised samples (approximately 10 g,
partition compounds of interest from the sample into the solvent. accurately weighed), and dried for 3 h in an oven heated at
Studies dealing with MAE of lipids are scarce but show that it holds 105 C, until constant weight according to the Portuguese standard
great promise to improve lipid yields and repeatability/reproduc- procedure NP 2282 (1991). Analyses were performed in triplicate
ibility, requiring, however, to be tested on a wider range of sample and results were expressed as g water per g wet muscle.
matrices (ElKhori, Par, Blanger, & Prez, 2007; Virot et al., 2007;
Virot et al., 2008a, 2008b). Furthermore, previous works (Batista, 2.4. Lipid extraction methods
Vetter, & Luckas, 2001; Virot et al.,2007; Virot et al., 2008a, 2008b)
concluded that MAE is a well-suited alternative to conventional Eight different lipid extraction methods were carried out as fol-
Bligh and Dyer and Soxhlet methods for the extraction of lipids fol- lows: Soxhlet (AOAC, 2005), Bligh & Dyer (Bligh and Dyer (1959)),
lowed by determination of the fatty acid prole. Results showed that modied Bligh & Dyer (Smedes, 1999), Folch (Folch et al., 1957),
total fats and oils extracted by MAE were qualitatively similar to modied Folch (Lin et al., 2004), Hara & Radin (Hara & Radin,
those obtained by conventional methods. No signicant difference 1978), Roese-Gottlieb (Manirakiza et al., 2001), and MAE. Quadru-
was obtained between fatty acid composition of each extract allow- plicate of each sh composite were analysed by the eight tested
ing to conclude that the method is effective and valuable for extrac- methods immediately after purchase and after one, two, three,
tion of fats and oils from food products. four, ve, six and nine months of freezing storage. The lipid content
The present study aimed (i) to evaluate the total lipid content of was gravimetrically determined.
the most consumed sh species in Portugal, namely horse macker-
el (Trachurus trachurus), chub mackerel and sardine (Sardina pil- 2.4.1. Soxhlet (S)
chardus); (ii) to study the stability of total lipids content of Extraction was performed according to AOAC (2005). One gram
minced llets during frozen storage. Different lipid extraction of homogenised sh and 2 g of Na2SO4 were placed in a Soxhlet
methods were tested: MAE, Soxhlet, Bligh & Dyer, modied Bligh extractor (Soxtest, Trade Raypa Soxhlet, SX-6, Spain). Thirty mil-
& Dyer, Folch, modied Folch, Hara & Radin and Roese-Gottlieb. liliters of n-hexane were passed through, in hot extraction mode
A MAE method was proposed as a viable alternative to conven- at 140 C for 4 h. The extract was dried in an oven (Selecta, P,
tional lipid extraction techniques for sh samples. This promising Spain) at 104 C, until constant weight.
technique supports sustainable development, as it requires less en-
ergy and solvent than conventional processes, whilst generating 2.4.2. Bligh & Dyer (BD)
fewer wastes (ElKhori et al., 2007). Methods were compared for Five grams of sample, 20 mL of methanol (MeOH) and 10 mL of
their lipid extraction efciencies. 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-
2. Experimental
ters of distilled water were added and the mixture was vortexed
again for 2 min. The layers were separated by centrifugation (Sig-
2.1. Chemicals
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.
All solvents used in extraction procedures were of analytical
A second extraction was done with 20 mL of 10% (v/v) MeOH in
grade and obtained from Merck (Darmstadt, Germany) and Panreac
CHCl3 by vortexing for 2 min. After centrifugation, the CHCl3 phase
(Barcelona, Spain). Sodium sulphate anhydrous (Panreac, Barce-
was added to the rst extract. Evaporation was done in a rotary
lona, Spain) was used as drying agent.
evaporator at 45 C and the residue was further dried in an oven
at 104 C for 1 h.
2.2. Sample collection and characterisation
2.4.3. Modied Bligh & Dyer (BDm)
Fresh individuals of the selected sh species originated from This method is a modication of the Bligh & Dyer procedure in
Atlantic Ocean were purchased from different local markets in Por- which MeOH and CHCl3 is replaced by 2-propanol and cyclohex-
to region (NW Portugal) during 2009. Horse mackerel specimens ane, respectively. A mixture of water:2-propanol:cyclohexane
were purchased in winter. For chub mackerel and sardine, individ- (11:8:10, v/v/v) was used as solvent. A second extraction with
uals were purchased during two seasons winter and spring. Sea- 20 mL of 10% (v/v) 2-propanol in cyclohexane was done. After cen-
son, length, weight and gender were recorded for each individual. trifugation, the cyclohexane phase was added to the rst extract.
Each sh specimen, from a total of 64, was headed, eviscerated, The advantage of cyclohexane over CHCl3 is its lower density,
skinned and lleted manually. Spines still present in tissues after which consequently separated on the top of the extraction mix-
lleting were carefully removed. Each sample further analysed ture. Evaporation was done in a rotary evaporator at 45 C and
(composite) was constituted by the edible parts of several individ- the residue was further dried in an oven at 104 C for 1 h.
uals and had a minimum mass of 200 g. Homogenisation was
performed mechanically with a Ufesa blender (Brio 400 WMAX, 2.4.4. Folch (F)
PD-5310, Spain) until a smooth paste was obtained. Male and fe- Five grams of sample were mixed with 100 mL of CHCl3:MeOH
male samples were kept separated (sh gender was determined (2:1, v/v), centrifuged for 10 min at 3000 rpm and ltered. Subse-
by visual examination of the gonads). The number of specimens, quently, 5 mL of distilled water was added to the ltrate and the
composites and biometric characterisation are indicated in Table 1. new mixture was shaken vigorously. The nal biphasic system
Sampling was performed in accordance to the US EPA Guide No 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 Composites LenghtA (cm) WeightA (g) MoistureA,* (%, g/g)
Sardine Winter F 9 2 20.3 0.8 72.1 8.7 81.3 0.1
M 6 1 19.9 1.1 71.3 16.4 82.4 0.6
Spring F 4 1 21.1 0.5 78.0 3.7 74.3 0.6
M 21 2 20.5 0.7 76.6 8.1 75.8 0.1
Chub mackerel Winter F 4 1 27.6 1.3 155.0 20.0 78.5 0.4
M 6 2 28.3 2.3 170.0 40.7 79.5 1.0
Spring F 6 2 35.6 3.2 379.0 111.4 68.6 0.1
M 3 1 30.8 2.0 217.0 50.9 75.6 0.7
Horse mackerel Winter F 2 1 31.5 0.7 283.0 29.7 77.3 0.3
M 3 2 31.5 3.1 295.0 75.2 75.5 0.4
A
Mean values standard deviation of the mean.
*
n = 3.

The upper aqueous phase was eliminated. The lower CHCl3 phase One gram of homogenised sh sample and 2 g of Na2SO4 were
was ltered through Na2SO4, collected and evaporated in a rotary weighted for a quartz extraction vessel (GreenChem Plus) of a
evaporator at 45 C and the residue was further dried in an oven Microwave Accelerated Reaction System, MARS-X, 1500 W (CEM,
at 104 C for 1 h. Mathews, NC, USA), congured with a 14 position carousel (Santori-
us BasicPlus, P 211D, Germany). During operation, both temperature
2.4.5. Modied Folch (Fm) and pressure were monitored in a single vessel. Magnetic stirring in
Ethyl acetate and ethyl alcohol were mixed at the same ratio as each extraction vessel and a sensor registering the solvent leaks in
CHCl3 and MeOH in the Folch method (Folch et al., 1957). 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:
2.4.6. Hara & Radin (HR) magnetron power 100%; time to reach settings 10 min; extraction
One gram of sample was added to 18 mL of n-hexane:isopropa- duration 20 min; medium speed stirring, extraction temperature
nol (3:2, v/v), homogenised for 30 s and ltered. The residue was 90 C and maximum vessel pressure cut off 1.38  106 Pa. After cool-
washed three times with 2 mL portions of n-hexane:isopropanol ing, the extract was ltered through Whatman GF/C lter paper
(3:2, v/v), by resuspending the residue each time and letting the (England) lled with Na2SO4 and collected into an evaporator tube
solvent soak for 2 min before applying air pressure. Nonlipid com- (Normax, internal diameter 24 mm, external length 145 mm, Portu-
pounds in the extract were removed by mixing the pooled ltrates gal). The extract was evaporated in a rotary evaporator (Buchi Rota-
for at least 1 min with 12 mL of aqueous Na2SO4 (prepared from 1 g vapor, R-200, Switzerland) at 45 C and the residue was further dried
of the anhydrous salt and 15 mL of water). The two formed layers in an oven at 104 C for 1 h.
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
2.5. Statistical methods
evaporated in a rotary evaporator at 45 C. The residue was further
dried in an oven at 104 C for 1 h.
In order to evaluate extraction efciencies, means of extracted
fat per composite were calculated (on wet and dry weight bases
2.4.7. Roese-Gottlieb (RG) but to simplify the discussion only concentrations in wet weight
This method consists of total lipid extraction with petroleum (ww) are presented), yielding 120 means per method. These means
ether after hydrolysis of the bounded lipids. To 1 g sample, 6 mL were plotted for comparison purposes. The average mean per
of boiling water were added and the mixture was vortexed for method (grand mean per method) was then computed averaging
1 min and allowed to cool. One millilitre of 25% ammonia solution respective composite means, and students t tests were used to
was added and vortexed for 2 min, then 7.5 mL of MeOH were compare the methods extraction efciencies (grand means).
added to the mixture and vortexed for 2 min. To this last mixture, For the evaluation of each methods repeatability, the standard
a rst addition of 17 mL of diethyl ether and a second addition of deviations within composites were calculated and plotted for com-
17 mL of petroleum ether were added and shaken vigorously. parison purposes. Composite standard deviations were squared,
The mixture was centrifuged and the upper layer was transferred averaged over each method, and the square root of the average
to a tarred beaker. Concentration was performed with a rotary was calculated as the residual standard deviation (RSD) for each
evaporator at 45 C and further drying in an oven at 104 C for 1 h. method. F tests were used to compare pairs of methods in relation
to their RSD.
2.4.8. Microwave-assisted extraction (MAE) Coefcients of variation were calculated for each composite and
The optimum extraction conditions were based on a previous plotted for comparison purposes. A general coefcient of variation
study concerning fat composition of Portuguese bakery products (CV) for each extraction method was obtained by division of the
(Casal et al., 2007). For performing this survey, a MAE procedure respective RSD by the methods grand mean.
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 3. Results and discussion
is typically found at high levels in shery products acting as a polar
solvent and producing coextractables which complicate analysis, Figs. 13 present the results obtained for chub mackerel, sar-
anhydrous sodium sulphate was mixed with homogenised samples dine and horse mackerel, respectively. In each gure, i.e., for each
before MAE. Several different anhydrous sodium sulphate/sample sh species, results are presented by season, within each season
ratios were tested, maintaining a total mass of 1 g. The best results by gender, within gender by frozen storage time. Regarding the
were obtained when using 1:2 (w/w) ratio. 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, of fact, all results show a pattern common to all frozen times. As
three, four, ve, six and nine months of freezing storage, respec- within all frozen times the extraction methods appear in the same
tively. Most of frozen products containing sh have an expiry date order, observed patterns are related to extraction methods.
of six months and consequently the lipid content was analysed Observing parts a) of the three gures, it is seen that within
month per month during this period. each time period with eight points, higher extractions correspond
Within each storage time there are (i) eight points representing to points appearing in positions 1, 3, 5 and 7, corresponding to
the extraction methods MAE, S, BD, BDm, F, Fm, HD and RG, by this methods MAE, BD, F and HR, whilst within time periods with six-
order, or (ii) sixteen points representing the extraction methods in teen points higher extractions correspond to points appearing in
duplicate, following the same order, i.e., MAE, MAE, S, S, . . ., RG, RG, positions 1 and 2 (MAE), 5 and 6 (BD), 9 and 10 (F) and 13 and
in accordance with the design shown in column with header Com- 14 (HR). Points appearing in positions 2 and 4 in the case with
posites of Table 1. no duplicates (S and BDm) and in positions 3 and 4 and 7 and 8
In parts (a) of all gures, each point represents the average of in the case with duplicates (S,S and BDm,BDm), correspond to
four lipid extractions using a particular extraction method. In parts the methods showing the lesser extraction efciencies. As the line
(b) points represent the corresponding standard deviations, and in plot pattern is repeated within frozen times, it can be said that
parts (c) the corresponding coefcients of variation. In all gures, each methods extraction efciency is stable and independent of
values (points) are presented as a line plot. This presentation is all factors considered, i.e., sh species, gender, season and time
articial, but makes it easier to observe any pattern. As a matter 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- deviations by the corresponding means, one obtains the coef-
crease in lipid content observed during six months of frozen stor- cients of variation which are shown in parts (c) of Figs. 13. It is
age, being more accentuate at the end of nine months probably due now clear that in all situations considered in this work, these coef-
to oxidation reactions. The same pattern of variation is observed cients are consistently lower for methods MAE, BD, F and HR, and
with data on dry weight base (results not shown) which clearly ex- eventually RG, always around 0.05 or below.
clude the possibility of being moisture interference. Considering all data available and displayed in the above men-
In parts (b) of Figs. 13, repeating patterns are again observed. tioned gures, methods MAE, BD, F and HR show the best efcien-
In these gures each point represents the standard deviation of the cies (in terms of higher amounts of extracted fat on wet weight
four replicates used for each condition. As all standard deviations basis and on dry (results not shown) weight basis) and also the
correspond to the average extractions shown in parts a), it can be highest repeatabilities/reproducibilities (expressed by lower coef-
easily seen that methods MAE, BD, F and HR are again superior cient of variation). However, these conclusions are based solely
than the others, because they show a higher repeatability (ex- on observation of gures, and a more reliable statistical examina-
pressed by lower standard deviation). tion is necessary, as it is reported in the following paragraphs.
In Figs. 13, parts (b), it is also seen that, as it is typical, when- Table 2 reports the general results obtained for the statistical
ever the mean extracted values increase, the corresponding stan- comparison of extraction methods. In Table 2a, the rst row shows
dard deviations also increase. However, dividing the standard 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 tical differences in RSD values between pairs of methods were
gures for extraction efciencies and must be analysed together compared through F tests, shown in Table 2c. It is seen that the
with Table 2b, where t tests for the signicance of differences be- lower RSD, equal to 0.117, was found for MAE, being signicantly
tween methods, taken two at a time, are shown. It is seen that lower than all other methods. The only methods that, although
although the average extracted fat with MAE was always the high- worse, approach MAE, are the F (p = 0.025) and BD (p = 0.004)
est, the observed differences are not signicant in relation to BD, F methods (highlighted in bold in Table 2c). All others are signi-
and HR (p always greater than 0.8, highlighted in bold in Table 2b). cantly worse (p < 0.000). These RSD values are general gures for
The group of methods that proved to extract less fat were BDm, RG methods standard deviations in several situations, being a good
and S, being comparable between them (p always greater than 0.9, indicator of the methods reproducibilities. The division of any
highlighted in italic in Table 2b). The differences between these RSD by the corresponding grand mean yields the coefcient
two groups of methods are signicant, with p values around of variation (CV), shown in the third line of Table 2a, expressed
0.05. Intermediate seems to be Fm, which is statistically not differ- in percentage. This is a standard measure of repeatability/repro-
ent from the rst group (0.80 < p < 0.60) and also not different from ducibility. Taking into consideration Figs. 13 and Table 2, it be-
BDm (p = 0.09). comes clear that although the efciency of methods MAE, BD, F
The RSD values are presented in the second line of Table 2a. Like and HR are the highest and are not statistically different between
the grand means, the RSD values were based on all composites themselves, differences exist in terms of variability, with MAE
(over species, gender, season and frozen storage times). The statis- 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 tent varying from 0.18% to 1.57%. The same pattern of variation
extraction method. Remaining methods, RG, S, Fm and BDm have was observed for the same species by Soyer and Sahin (1999).
low efciencies and low reproducibility, mainly S and BDm with Fat content levels of sardines varied from 0.62% to 4.42%.
CV equal to 15.1% and 17.6%, respectively. According to some authors, the fat content of sardine shows impor-
The information in Figs. 13 is complemented with Table 3, the tant seasonal changes during the year, which is typical of pelagic
latter reporting ANOVA results for the performance of all methods species. Bandarra, Batista, Nunes, Empis, and Christie (1997) found
in each sh species. For each case, the amounts of extracted fat are a total lipid content ranging between 1.2% and 18.4% in sardines
considered dependent on four factors (extraction method, gender, harvested in the Portuguese coast, from Northeast Atlantic Ocean,
season and frozen time), enabling the evaluation of the signicance monthly during a year. Passi, Cataudella, Di Marco, De Simone, and
of the observed differences between extraction efciencies per Rastrelli (2002) determined the total fat content in the white mus-
method and the effects and interactions between the main factors cle tissues of 21 species of teleosts and they obtained a value of
considered during frozen storage. 4.81 0.73% for sardine from central Tyrrhenian Sea, Italy. Caponio
As discussed previously in relation to gures, these tables show et al. (2004) determined the total lipid content in sardine captured
that there are signicant differences in the amounts of fat ex- from Ionian Sea, and obtained a range of 0.50.8%. Zlatanos and
tracted (on wet weight basis and on dry (results not shown) weight Laskaridis (2007) obtained a minimum fat content at the end of
basis). These differences depend on the extraction method used winter (3.88 0.2 g/100 g dry weight) and a maximum at the end
and frozen time. Interactions between the main factors are almost of spring beginning of summer (11.86 0.6 g/100 g dry weight)
always highly signicant. It is important to emphasise that the li- for sardines purchased from the Central Market of Thessaloniki
pid content of these species varies throughout the year particularly (northern Greece).
with their life cycle, due to their feeding growth and nonfeeding Concerning horse mackerel, the values obtained (3.225.01%)
maturation cycles (Caponio, Lestingi, Summo, Bilancia, & Laudadio, are in range with those reported by Losada, Pieiro, Barros-Velz-
2004). quez, and Aubourg (2005) (1.23.7%) for horse mackerel from Gali-
The determined fat contents for chub mackerel (0.828.99%) are cian Atlantic coast and by Simeonidou, Govaris, and Vareltzis
in accordance to those found by Stamatis and Arkoudelos (2007) (1998) (4.39 1.3%) for specimens originated from the southeast
and Karakoltsidis, Zotos, and Constantinides (1995) for Scomber co- coast of Halkidiki peninsula of North Greece. Aubourg and Ugliano
lias japonicus (4.54.9%, Stamatis and Arkoudelos, 2007; and (2002) determined a total fat content varying from 1.5% to 3.0% in
4.0 0.6%, Karakoltsidis et al., 1995) from the Aegean Sea (Greece). horse mackerel from the Northeast Atlantic. Variations in chemical
Celik (2008) reported for chub mackerel from the Iskenderun Bay composition in marine shes are closely related to nutrition, living
on the north-eastern Mediterranean coast of Turkey, a lipid con- 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

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