1. Original article
Impact of different preservation treatments on lipids of the
smooth clam Callista chione
Christos D. Papaioannou,1
Vassilia J. Sinanoglou,2
* Irini F. Strati,3
Charalampos Proestos,1
Vasiliki R. Kyrana4
& Vladimiros P. Lougovois4
1 Food Chemistry Laboratory, Department of Chemistry, National and Kapodistrian University of Athens, Panepistimioupolis Zographou,
15701 Athens, Greece
2 Instrumental Food Analysis Laboratory, Department of Food Technology, Technological Educational Institution of Athens, Agiou
Spyridonos, 12210 Egaleo, Greece
3 Laboratory of Food Chemistry and Technology, School of Chemical Engineering, National Technical University of Athens, Iroon
Polytechniou 5, Zografou, 15780 Athens, Greece
4 Fisheries Laboratory, Department of Food Technology, Technological Educational Institution of Athens, Agiou Spyridonos, 12210 Egaleo,
Greece
(Received 3 June 2015; Accepted in revised form 9 September 2015)
Summary The soft clam Callista chione is highly appreciated among marine inhabitants for nutritional, commercial
and economic reasons. This work aims to determine the lipid classes’ profile of C. chione and the effect of
different preservation treatments on them. C. chione meat was found to be a rich source of dietary phos-
pholipids (PhLs), x-3 fatty acids and carotenoids, encouraging the exploitation of the species as human
food. Among treatments, only marinating reduced the PhLs content. Parboiling and freezing resulted in a
significant decrease in unsaturated fatty acids, while marinating caused partial replacement of polyunsatu-
rated with monounsaturated fatty acids. Lipid quality indices remained favourable for a healthy diet.
With the exception of astaxanthin, the rest of the carotenoids identified were susceptible to processing
and frozen storage. Parboiling and freezing of the vacuum-packed meats for up to 4 months would be
most appropriate, among the treatments applied, for preserving the lipid quality of C. chione.
Keywords Callista chione, carotenoids, fatty acids, freezing, marinating.
Introduction
Bivalve molluscs constitute a very important fishery
resource worldwide, with the principal commercial spe-
cies (oysters, mussels, scallops and clams) amounting
to 1.8 million tonnes from marine and freshwater cap-
ture fisheries and 12.8 million tonnes from aquaculture
(FAO, 2013). Clams account for more than 38% of
the global production and in terms of economic value
represent the second most important group next to
scallops. Bivalve molluscs are considered highly nutri-
tional commodities and suitable for cardioprotective
diets, providing significant amounts of x-3 fatty acids,
essential amino acids, important macro- and trace ele-
ments, vitamin B12, low cholesterol content and low
atherogenic and thrombogenic indices (Karnjanapra-
tum et al., 2013; Anacleto et al., 2014). The carpet
shell clams (Ruditapes decussatus, Ruditapes philip-
pinarum) and Venus clams (Chamelea gallina, Venus
striatula) are among the most preferred bivalve mol-
luscs for raw human consumption, while razor clams
(Ensis siliqua, Ensis arcuatus) are the favoured species
in the canning sector (Fernandez-Tajes Mendez,
2007; Anacleto et al., 2014). However, as the natural
beds of several commercial species are now under pres-
sure (Cross et al., 2014), other clams, for example the
smooth clam Callista chione, also have attracted atten-
tion and are becoming increasingly important (Leon-
tarakis Richardson, 2005). Callista chione
(Linnaeus, 1758), widely distributed in the Mediter-
ranean and the East Atlantic, is a shallow-burrowing
species that inhabits sandy sediments in coastal waters
and feeds by selectively filtering suspended particles
out of the surrounding water, including microalgae,
bacteria and detritus (Ezgeta-Balic et al., 2011). It is
harvested commercially in France, Portugal, Spain
(Catalan Sea), Italy (Andriatic Sea) and Greece
*Correspondent: Fax: +30-2105314874;
e-mails: v_sinanoglou@yahoo.gr, vsina@teiath.gr
International Journal of Food Science and Technology 2015
doi:10.1111/ijfs.12972
© 2015 Institute of Food Science and Technology
1

2. (Aegean, Ionian and Cretan Seas) (Leontarakis
Richardson, 2005; Ezgeta-Balic et al., 2011). In
Mediterranean, C. chione represents the prominent
bivalve species in terms of biomass (Leontarakis
Richardson, 2005). Callista chione are held in a live
state through distribution and retailing; all soft parts
of the animal, including the foot, mantle and viscera,
are most frequently eaten raw. So far, most studies
on C. chione have focused on biological behaviour
and growth rate, population dynamics in relation to
harvesting area and the effect of dredge design on the
size and quality of the catch (Leontarakis Richard-
son, 2005; Ezgeta-Balic et al., 2011). Apparently, no
study has examined compositional aspects or the
influence of processing and storage on nutritionally
important constituents of C. chione. Shellfish are fre-
quently placed on the market as marinated semipre-
serves, luxury smoked products or individually quick-
frozen (IQF) cooked meats exhibiting long storage
life. Deterioration of the susceptible polyunsaturated
fatty acids during processing and subsequent storage
directly affects product quality, including flavour, col-
our, texture and nutritional value. The main objective
of this study was to characterise the lipid classes and
fatty acid profile of freshly harvested C. chione meat
and to identify the major carotenoid pigments con-
tributing to the colour of the animal flesh. An addi-
tional task was to investigate the impact of
marinating and freezing on lipid and carotenoid sta-
bility, in view of the growing interest for extending
postharvest shelf life of the shellfish and increasing
product variety.
Materials and methods
Chemicals, standards and solvents
The lipid standards were as follows: cholesteryloleate,
cholesterol, tristearoyl-glycerol, oleic acid, 1,
2-distearoyl-glycerol, 1-monostearoyl-rac-glycerol,
phosphatidylcholine (PC), phosphatidylethanolamine
(PE), lysophosphatidylcholine (l-PC), lysophos-
phatidylethanolamine (l-PE), phosphatidylinositol (PI),
phosphatidylserine (PS) and sphingomyelin (Sigma-
Aldrich Company, St. Louis, MO, USA). Fatty acid
methyl esters standards were as follows: Supelco TM
37 Component FAME Mix C4-C24, Supelco PUFA
No.1, Marine Source and conjugated linoleic acid
methyl esters standard mixture (Sigma-Aldrich Com-
pany, St. Louis, MO, USA). The carotenoid standards
were as follows: trans-lutein, trans-zeaxanthin, trans-
canthaxanthin, trans-astaxanthin, trans-b-cryptoxan-
thin and trans-b-carotene (Sigma-Aldrich Company).
All solvents used for GC-FID, HPLC-DAD and thin-
layer chromatography–flame ionisation detection
(TLC-FID) analyses were of HPLC grade from Merck
(Darmstadt, Germany). All reagents used were of ana-
lytical grade (Sigma-Aldrich Company).
Raw material, preparation and sampling
Commercial size live specimens of C. chione
(30–40 clams kgÀ1
) were obtained from a licensed dredg-
ing vessel operating in FAO Zone 37.3.1 (western–central
Aegean), in a production area classified by the competent
authority as being of ‘class A’ and free of marine biotox-
ins. Live bivalves collected from the particular area are
placed on the market for direct human consumption,
according to the provisions of Regulation (EC) 853/2004.
Six sampling repetitions were conducted from October till
November 2014. The shellfish were packed in insulated
polystyrene boxes and transported to the laboratory.
Upon arrival, for each sampling repetition, 300 randomly
chosen specimens (8.5 kg live weight) were divided in two
lots, A and B. The specimens of lot A (50 clams) were
manually sucked by cutting the adductor muscles with a
knife; the sucked meats were drained and subjected to fur-
ther chemical analysis. The specimens of lot B (250 clams)
were packed in wire mesh baskets and immersed in boil-
ing water for 4 min, followed by cooling in icy water
(0 °C) for 1 min. The parboiled meats were removed from
the open shells by hand and spray-washed with tap water
to remove any remaining sand or grit. Parboiled meats
from approximately 50 clams (sublot B1) were sampled
for further analysis, while the rest of the quantity (par-
boiled meats from 200 clams) was divided into two sub-
lots, B2 and B3, which were further subjected to
preservation treatments, freezing and marinating, respec-
tively. Parboiled meats in sublot B2 were IQF in an Arm-
field FT 34-MKII plate freezer (Armfield Ltd.,
Hampshire, UK) (plate temperature À36 °C, freezing
time 1 h) and vacuum-packaged in food grade polya-
mide/polyethylene (20/80) lm barrier pouches
(30 9 35 cm) of low gas permeability. The pouches were
heat-sealed in a Multivac model A 300 vacuum-packaging
machine (Bury, Lancashire, UK) and stored at À18 °C.
For the marinating process, the parboiled meats in sublot
B3 were first allowed to stand for 3 days in a vinegar/salt
solution containing 3% NaCl and 2% acetic acid. After
draining, the meats were packed into glass jars and cov-
ered with vinegar containing 3% acetic acid. The jars were
sealed and stored at 4 °C. Chemical analyses of the frozen
(sublot B2) and marinated (sublot B3) clam meats were
performed at 4 and 6 months of storage.
Total lipid extraction
Total lipids (TLs) were extracted according to the
Bligh Dyer method (1959), and their contents were
calculated gravimetrically. The lipid samples were
transferred to amber vials and kept under an atmo-
sphere of nitrogen at 0 °C, until use.
© 2015 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2015
Effects of preservation on C. chione lipids C. D. Papaioannou et al.2

3. Iatroscan analysis of neutral and polar lipids
Lipid classes were separated and quantified by TLC-
FID, using an Iatroscan MK-6 TLC/FID – FPD Ana-
lyzer (Iatron Laboratories, Tokyo, Japan) as described
before (Sinanoglou et al., 2013).
Gas chromatography analysis of fatty acid methyl esters
The analysis of fatty acid methyl esters (FAME) was
performed according to Sinanoglou et al. (2013), using
an Agilent 6890 Series Gas Chromatograph (Agilent
Technologies, Palo Alto, CA, USA) equipped with
flame ionisation detector. Individual FAMEs were
identified by comparing their retention times with
those of authentic standard mixtures.
Indices calculations
The atherogenic index (AI) and thrombogenic index
(TI) were calculated according to the Ulbrich South-
gate (1991) equations:
AI ¼ ½12 : 0 þ ð4 Â 14 : 0Þ þ 16 : 0Š=ðx
À 3PUFA þ x6PUFA þ MUFAÞ
TI ¼ ð14 : 0 þ 16 : 0 þ 18 : 0Þ=ð0:5MUFA
þ 0:5x À 6PUFA þ 3x À 3PUFA
þ x À 3PUFA=x À 6PUFAÞ
The peroxidisability index (PI) was calculated
according to the equation proposed by Erickson
(1992):
PI ¼ ð0:025 Â monoenesÞ þ ð1 Â dienesÞ þ ð2 Â trienesÞ
þ ð4 Â tetraenesÞ þ ð6 Â pentaenesÞ þ ð8
 hexaenesÞ:
Hypocholesterolaemic (hI) and hypercholestero-
laemic (HI) fatty acids were calculated according to
the Santos-Silva et al. (2002) equations:
hI ¼ C18 : 1x À 9 þ C18 : 2x À 6 þ C20 : 4x À 6
þ C18 : 3x À 3 þ C20 : 5x À 3 þ C22 : 5x À 3
þ C22 : 6x À 3
HI ¼ C14 : 0 þ C16 : 0:
Carotenoid analysis
Spectrophotometric determination of carotenoids
The total carotenoid content of the lipid extracts was
determined spectrophotometrically using the calibra-
tion curve of absorbance vs. carotenoid concentration
of trans-astaxanthin standard solutions, at 478 nm. A
double-beam ultraviolet–visible (UV–vis) spectropho-
tometer (Hitachi U-3210; Hitachi, Ltd., Tokyo, Japan)
was used. The concentrations of the trans-astaxanthin
standards were in the range of 0.25–10.0 lg mLÀ1
,
and the calibration curve was expressed by the equa-
tion: y = 0.167x + 0.0096 (R2
= 0.998, n = 6), where y
denotes absorbance and x means concentration
(lg mLÀ1
). The limits of detection (LOD) (lg mLÀ1
)
and the limits of quantification (LOQ) (lg mLÀ1
) were
calculated from the equations (LOD = 3.3 9 r/S) and
(LOQ = 10 9 r/S), respectively, where S is the slope
of the calibration curve and r is the standard devia-
tion of the response (Strati et al., 2012). The LOD and
LOQ were 0.48 and 0.88 lg mLÀ1
, respectively. The
total carotenoid content was expressed as mg of trans-
astaxanthin per g of TLs.
HPLC-DAD analysis of carotenoids
Total lipids were analysed by high-performance liquid
chromatography (HPLC) coupled to a diode array
detector (DAD) (Hewlett Packard Series 1100, Wald-
bronn, Germany) equipped with an YMC (Tokyo,
Japan) C30 column (250 9 4.6 mm I.D., 5 lm parti-
cle), as described by Strati et al. (2012). The identifica-
tion of carotenoids was carried out by comparing the
retention times and absorption spectra with those of
reference standards. Quantification was performed as
described before (Strati et al., 2012). Carotenoids were
expressed as mg per g of TLs.
Statistical analysis
Duplicate measurements obtained from six indepen-
dent trials were combined and analysed by one-way
ANOVA post hoc tests. Pair-wise multiple comparisons
were conducted using Tukey’s significant difference test
at P 0.05. All statistical calculations were per-
formed using the SPSS statistical software for Win-
dows (IBM SPSS Statistics, version 19.0, Chicago, IL,
USA).
Results and discussion
Neutral and polar lipids
The TL content of the raw edible portion of C. chione,
including the foot, viscera, mantle and siphons, was
similar to the values reported for other clam species
(Orban et al., 2006; Laxmilatha, 2009). No significant
change in lipid content was induced by the processing
treatments applied (Table 1).
Neutral lipids (NL) were quantified as the minor TL
class, both in raw and treated meats (24.18–36.51% of
TL). NL mainly consisted of triglycerides (TG), fol-
lowed by cholesterol (Table 1). After the parboiling
treatment, no significant (P 0.05) changes in NL
© 2015 Institute of Food Science and Technology International Journal of Food Science and Technology 2015
Effects of preservation on C. chione lipids C. D. Papaioannou et al. 3

4. proportions were observed, apart from the detection
of minor levels of hydrocarbons (HC). By contrast,
the marinating process induced a significant
(P 0.05) increase in NL proportion, which resulted
mainly from the decrease of polar lipid proportion.
During storage of the marinated and frozen meats, the
TG proportion decreased significantly and this change
was accompanied by a gradual increase in monoglyc-
erides (MG) and free fatty acids (FFA). In general, an
increase in hydrolysis products such as HC, MG and
FFA may be attributed to the degradation of TG dur-
ing preservation treatments (Vittadini et al., 2003).
However, as the parboiling process was meant to cause
only partial coagulation of the muscle proteins, render-
ing the shells open, residual activity from endogenous
lipases might have been involved as well.
Polar lipids (PL) mainly consisted of PC, followed
by PE and PS (Table 1). No significant (P 0.05) dif-
ferences in the PL proportion were observed between
raw and parboiled samples, apart from the presence of
l-PC (Table 1), resulting from the hydrolysis of the
acyl group at the SN-1 position of PC. Frozen storage
of the vacuum-packed, parboiled meats had no
(P 0.05) effect either on the PL or on the individual
phospholipid (PhL) proportions. Given the fact that
phospholipases in marine invertebrate muscles are
responsible for PhL hydrolysis (Sriket et al., 2007),
parboiling is expected to retard degradation by deacti-
vating the enzymes involved. On the contrary, the
marinating process significantly (P 0.05) reduced
the PL proportion, due to an outstandingly high
decrease in PE levels (Table 1). Significant levels of l-
PE and l-PC were found in the marinated meats after
4 and 6 months of storage. Furthermore, a significant
(P 0.05) decrease in PS proportion was observed in
the marinated samples after storage for 6 months. The
hydrolysis of PL caused by the marinating process
could be attributed to the presence of acetic acid in
the vinegar/salt solution, leading to the formation of
l-PE and l-PC. Lysophospholipids may degrade further
to water-soluble compounds, which are not detectable
in the lipid fraction (Nakayama et al., 1981). This
would explain why the increase in l-PE proportion
observed in the present study did not compensate for
the corresponding loss in PE.
Concerning the nutritional value of C. chione meat,
the contents of PC, PE and PS (calculated from
Table 1) were 0.32 Æ 0.01, 0.27 Æ 0.02 and
0.11 Æ 0.01 g per 100 g of wet tissue, respectively. As
the recommended dietary intake of choline is 400–
550 mg per day, C. chione meat seems to be a good
source of this nutrient. Choline constitutes 13% (w/w)
of the PC, and dietary PC is almost completely
absorbed by the human intestine (Fischer et al., 2005).
Fatty acids
The fatty acid profile of the raw and processed meats
is shown in Table 2. GC-FID analysis revealed the
presence of 40 fatty acids. SFA were predominant in
all samples, followed by polyunsaturated (PUFA) and
monounsaturated (MUFA) fatty acids. PUFA have
Table 1 Moisture and total fat content (g/100 g wet tissue) and individual neutral and polar lipid profile (% of TL) in total lipids of raw, par-
boiled, frozen vacuum-packed and marinated Callista chione meat
Raw Parboiled
Frozen, vacuum-packed
(4 months)
Frozen, vacuum-packed
(6 months)
Marinated
(4 months)
Marinated
(6 months)
Moisture 80.55 Æ 0.36a 81.52 Æ 1.10a 78.32 Æ 2.08a 80.07 Æ 0.38a 83.57 Æ 0.59b 82.00 Æ 1.79ab
Fat 0.92 Æ 0.14a 0.73 Æ 0.10a 0.75 Æ 0.07a 0.81 Æ 0.05a 0.78 Æ 0.03a 0.80 Æ 0.10a
NL (% of TL) 24.18 Æ 3.48a 23.92 Æ 2.35a 24.98 Æ 1.96a 23.53 Æ 1.08a 36.51 Æ 1.96b 34.50 Æ 0.69b
HC nd 0.98 Æ 0.23 nd nd nd nd
TG 13.10 Æ 1.34a 11.34 Æ 0.94a 13.08 Æ 2.54a 7.64 Æ 1.53d 21.60 Æ 2.54b 17.27 Æ 1.28c
FFA nd nd 1.00 Æ 0.23a 2.67 Æ 0.20b 1.59 Æ 0.23c 2.58 Æ 0.47b
Cholesterol 11.08 Æ 2.38ab 11.60 Æ 1.46ab 10.39 Æ 1.72a 11.86 Æ 0.62a 12.62 Æ 0.64ab 13.39 Æ 0.54b
MG nd nd 0.51 Æ 0.09b 1.36 Æ 0.13c 0.70 Æ 0.02a 1.26 Æ 0.26c
PL (% of TL) 75.82 Æ 3.48a 76.08 Æ 2.35a 75.02 Æ 1.96a 76.47 Æ 1.08a 63.49 Æ 1.96b 65.50 Æ 0.69b
PE 29.35 Æ 0.91a 30.08 Æ 1.47a 29.00 Æ 1.48a 30.84 Æ 0.54a 9.74 Æ 1.48b 5.53 Æ 0.38c
PS 11.82 Æ 0.82a 11.06 Æ 1.36a 11.12 Æ 1.83a 11.14 Æ 0.49a 10.98 Æ 1.03a 8.24 Æ 1.03b
l-PE nd nd nd nd 7.44 Æ 0.56a 15.18 Æ 1.25b
PC 34.65 Æ 2.29a 32.45 Æ 0.48a 34.90 Æ 2.79a 34.49 Æ 0.64a 29.75 Æ 1.46a 32.20 Æ 2.63a
l-PC nd 2.49 Æ 0.26a nd nd 5.58 Æ 0.48b 4.33 Æ 0.63c
HC, hydrocarbons; TG, triglycerides; FFA, free fatty acids; MG, monoglycerides; PE, phospatidylethanolamine; PS, phosphatidylserine; l-PE,
lysophospatidylethanolamine; PC, phosphatidylcholine; l-PC, lysophosphatidylcholine; nd, not detected.
Results represent means Æ SD (N = 6 9 2).
Means in the same row bearing different letters differ significantly (P 0.05).
© 2015 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2015
Effects of preservation on C. chione lipids C. D. Papaioannou et al.4

5. been reported as the dominant fatty acid group in
commercial bivalve species such as mussels (Khan
et al., 2006), oysters (Lira et al., 2013), scallops
(Soudant et al., 1996) and clams (Orban et al., 2006;
Karnjanapratum et al., 2013). However, the relative
proportions of saturated and unsaturated fatty acids
(UFA) vary according to taxonomy, season, food
availability, water temperature and stage of reproduc-
tive cycle (Soudant et al., 1996; Orban et al., 2006;
Anacleto et al., 2014).
Table 2 Fatty acid profile (% of total FAME) in total lipids of raw, parboiled, frozen and marinated Callista chione meats
Fatty acids Raw Parboiled
Frozen,
vacuum-packed
(4 months)
Frozen,
vacuum-packed
(6 months)
Marinated
(4 months)
Marinated
(6 months)
C14:0 3.30 Æ 0.28a 4.20 Æ 0.31ab 4.75 Æ 0.43ab 4.99 Æ 1.03b 7.13 Æ 0.85c 7.33 Æ 0.02c
C15:0 0.54 Æ 0.01a 0.73 Æ 0.05b 0.70 Æ 0.03b 0.78 Æ 0.02bc 0.83 Æ 0.03c 0.85 Æ 0.03c
C16:0 24.36 Æ 0.35a 28.22 Æ 0.47b 26.51 Æ 0.15ab 28.43 Æ 1.86b 31.06 Æ 0.76c 32.08 Æ 0.35c
iso-C16:0 0.44 Æ 0.02a 0.42 Æ 0.03a 0.29 Æ 0.25a 0.42 Æ 0.01a 0.51 Æ 0.19a 0.34 Æ 0.05a
iso-C17:0 0.78 Æ 0.04a 1.07 Æ 0.01bd 1.14 Æ 0.20d 1.05 Æ 0.04bd 1.37 Æ 0.07 cd 1.50 Æ 0.01c
anteiso-C17:0 0.76 Æ 0.10a 1.09 Æ 0.03b 0.90 Æ 0.06ac 1.09 Æ 0.05b 0.90 Æ 0.03ac 0.93 Æ 0.03c
cyclo-C17:0 1.09 Æ 0.17a 1.62 Æ 0.02b 0.88 Æ 0.03ac 1.62 Æ 0.10b 0.66 Æ 0.04 cd 0.63 Æ 0.03d
C17:0 1.26 Æ 0.34a 1.87 Æ 0.03b 1.44 Æ 0.07ab 1.86 Æ 0.14b 1.27 Æ 0.07a 1.40 Æ 0.10a
C18:0 5.51 Æ 0.02abd 5.95 Æ 0.33b 4.83 Æ 0.27de 5.55 Æ 0.18ab 4.10 Æ 0.39ce 4.29 Æ 0.19ce
C19:0 1.09 Æ 0.01ac 1.18 Æ 0.01ad 1.18 Æ 0.10ad 1.33 Æ 0.11d 0.87 Æ 0.10bc 0.91 Æ 0.01c
C20:0 1.88 Æ 0.07a 1.50 Æ 0.04bc 1.70 Æ 0.01ac 1.61 Æ 0.11ac 1.41 Æ 0.23bc 1.23 Æ 0.03b
C21:0 0.34 Æ 0.03a 0.34 Æ 0.01a 0.41 Æ 0.01b 0.30 Æ 0.00a 0.33 Æ 0.03a 0.34 Æ 0.03a
C22:0 0.27 Æ 0.07a 0.00 Æ 0.00b 0.03 Æ 0.06b 0.02 Æ 0.04b 0.14 Æ 0.12ab 0.11 Æ 0.02b
C23:0 0.74 Æ 0.04a 0.65 Æ 0.04a 0.63 Æ 0.08a 0.73 Æ 0.10a 0.33 Æ 0.03b 0.25 Æ 0.04b
C24:0 1.33 Æ 0.05a 1.16 Æ 0.06a 1.17 Æ 0.09a 1.22 Æ 0.19a 0.74 Æ 0.09b 0.63 Æ 0.08b
Σx:0 (SFA) 43.68 Æ 1.01a 49.99 Æ 0.66b 46.57 Æ 0.68d 51.01 Æ 2.61c 51.66 Æ 1.03c 52.83 Æ 0.58c
C14:1 0.79 Æ 0.04a 1.01 Æ 0.12ab 0.91 Æ 0.02ab 1.12 Æ 0.10b 1.10 Æ 0.18b 1.08 Æ 0.04b
C15:1x-5 0.07 Æ 0.02a 0.00 Æ 0.00a 0.00 Æ 0.00a 0.08 Æ 0.05a 0.08 Æ 0.05a 0.06 Æ 0.03a
C16:1x-7 4.43 Æ 0.35a 5.44 Æ 0.27a 6.98 Æ 0.08c 5.06 Æ 0.49a 10.73 Æ 1.06b 12.22 Æ 0.49b
C17:1x-7 0.36 Æ 0.04ab 0.43 Æ 0.01b 0.35 Æ 0.01a 0.49 Æ 0.03b 0.24 Æ 0.03c 0.28 Æ 0.05ac
C18:1 trans-9 0.20 Æ 0.04ab 0.27 Æ 0.04ab 0.09 Æ 0.15b 0.27 Æ 0.04ab 0.34 Æ 0.03a 0.30 Æ 0.05a
C18:1x-9 10.06 Æ 1.09a 5.87 Æ 0.34b 6.29 Æ 0.18b 5.69 Æ 0.19b 8.78 Æ 1.18ac 7.01 Æ 0.50bc
C18:1x-7 1.90 Æ 0.25a 1.62 Æ 0.02b 2.10 Æ 0.12a 1.60 Æ 0.07b 2.53 Æ 0.15c 2.67 Æ 0.02c
C20:1x-9 0.68 Æ 0.01abc 0.64 Æ 0.01abc 0.78 Æ 0.15b 0.80 Æ 0.12b 0.51 Æ 0.08a 0.49 Æ 0.01ac
C22:1x-9 2.28 Æ 0.11a 2.25 Æ 0.16a 2.02 Æ 0.02a 2.47 Æ 0.43a 1.05 Æ 0.09b 0.93 Æ 0.06b
C22:1x-11 1.20 Æ 0.09a 1.06 Æ 0.05a 0.95 Æ 0.03a 1.20 Æ 0.23a 0.54 Æ 0.06b 0.46 Æ 0.04b
C24:1x-9 0.04 Æ 0.08ab 0.00 Æ 0.00a 0.00 Æ 0.00a 0.01 Æ 0.02a 0.12 Æ 0.05b 0.05 Æ 0.01ab
Σx:1 (MUFA) 22.01 Æ 0.96a 18.59 Æ 0.12b 20.46 Æ 0.22d 18.78 Æ 0.17b 26.02 Æ 0.80c 25.56 Æ 0.92c
C18:2cis-9trans-11 0.23 Æ 0.01a 0.10 Æ 0.09ab 0.05 Æ 0.08b 0.15 Æ 0.02ab 0.13 Æ 0.01ab 0.11 Æ 0.01ab
C18:2x-6 3.45 Æ 0.41a 1.41 Æ 0.01bc 1.71 Æ 0.03b 1.15 Æ 0.08c 1.84 Æ 0.14b 1.92 Æ 0.22b
C18:3x-6 0.34 Æ 0.03a 0.40 Æ 0.15a 0.22 Æ 0.19a 0.53 Æ 0.39a 0.36 Æ 0.07a 0.35 Æ 0.01a
C18:3x-3 0.76 Æ 0.02a 0.54 Æ 0.02b 0.74 Æ 0.02a 0.44 Æ 0.06b 0.73 Æ 0.08a 0.80 Æ 0.01a
C18:4x-3 0.38 Æ 0.08a 0.52 Æ 0.04abc 0.56 Æ 0.06bc 0.45 Æ 0.06ab 0.49 Æ 0.08abc 0.62 Æ 0.04c
C20:3x-6 1.40 Æ 0.10a 1.53 Æ 0.08a 1.75 Æ 0.01b 1.53 Æ 0.02a 1.41 Æ 0.12a 1.45 Æ 0.04a
C20:4x-6 0.25 Æ 0.01a 0.23 Æ 0.01a 0.39 Æ 0.09b 0.24 Æ 0.02a 0.28 Æ 0.02a 0.30 Æ 0.01ab
C20:3x-3 3.22 Æ 0.06a 3.28 Æ 0.15a 3.03 Æ 0.11a 3.27 Æ 0.31a 1.92 Æ 0.19b 2.08 Æ 0.16b
C20:5x-3 4.43 Æ 0.31a 4.77 Æ 0.06a 5.94 Æ .018b 4.17 Æ 0.19a 4.55 Æ 0.55a 5.06 Æ 0.60ab
C22:2x-6 0.50 Æ 0.03ac 0.38 Æ 0.02ab 0.51 Æ 0.10c 0.35 Æ 0.03b 0.38 Æ 0.03ab 0.37 Æ 0.02b
C22:4x-6 0.35 Æ 0.01ace 0.40 Æ 0.02abc 0.44 Æ 0.11c 0.19 Æ 0.04de 0.57 Æ 0.14bc 0.01 Æ 0.01d
C22:5x-6 2.01 Æ 0.15a 1.73 Æ 0.17ab 1.50 Æ 0.11b 1.80 Æ 0.28ab 0.61 Æ 0.07c 0.72 Æ 0.11c
C22:5x-3 1.78 Æ 0.01a 1.67 Æ 0.05a 1.54 Æ 0.01a 1.85 Æ 0.30a 0.91 Æ 0.07c 0.76 Æ 0.09c
C22:6x-3 15.20 Æ 0.01a 14.44 Æ 0.48a 14.58 Æ 0.42a 14.10 Æ 1.54a 8.15 Æ 0.92b 7.06 Æ 0.66b
Σx:n (PUFA) 34.31 Æ 0.07a 31.42 Æ 0.56b 32.97 Æ 0.82d 30.22 Æ 2.72bd 22.32 Æ 1.82c 21.61 Æ 1.49c
Σx:3 25.78 Æ 0.46a 25.23 Æ 0.38a 26.39 Æ 0.66a 24.28 Æ 2.14a 16.75 Æ 1.65b 16.37 Æ 1.53b
Σx:6 8.30 Æ 0.52a 6.09 Æ 0.27bcd 6.53 Æ 0.23d 5.79 Æ 0.62bcd 5.45 Æ 0.18c 5.12 Æ 0.04bc
SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.
Results represent means Æ SD (N = 6 9 2).
Means in the same row bearing different letters differ significantly (P 0.05).
© 2015 Institute of Food Science and Technology International Journal of Food Science and Technology 2015
Effects of preservation on C. chione lipids C. D. Papaioannou et al. 5

6. Among SFA, palmitic acid (C16:0) predominated,
followed by stearic (C18:0) and myristic (C14:0) acids.
Oleic (C18:1x-9) and palmitoleic (C16:1x-7) acids were
the predominant MUFA, while docosahexaenoic acid
(DHA, C22:6x-3) was found in the highest proportion
among PUFA, followed by eicosapentaenoic (EPA,
C20:5x-3) and linoleic (C18:2x-6) acids (Table 2).
Parboiling induced a significant decrease (P 0.05)
in UFA and an increase (P 0.05) in SFA propor-
tion (Table 2). Marinating significantly (P 0.05)
increased the proportions of both SFA and MUFA,
while it decreased PUFA. On the other hand, freezing
of the vacuum-packed parboiled meats had the oppo-
site effect of gradually increasing (P 0.05) UFA and
SFA proportions with storage time. Concerning the
SFA, palmitic acid (C16:0) proportion significantly
increased by parboiling, marinating and frozen storage
for 6 months. The proportion of C14:0 was almost
doubled by the marinating process, whereas C18:0
decreased significantly. All treatments caused a signifi-
cant (P 0.05) decrease in C18:1x-9 proportion; the
greatest losses were observed after parboiling and fro-
zen storage of the meats. Moreover, palmitoleic acid
proportion increased significantly (P 0.05) after
marinating, but was not affected by either parboiling
or frozen storage (Table 2). Concerning PUFA, DHA
proportion decreased significantly (P 0.05) in the
marinated samples, whereas linoleic acid decreased
almost by half after all treatments. By contrast, EPA
proportion did not change in the various treatments.
The x-3 and x-6 fatty acid proportions of the treated
samples changed in a similar manner with DHA and
linoleic acid, respectively. A possible explanation for
this finding is that the vinegar/salt solution caused the
acidic hydrolysis of PhLs and the liberation of their
main fatty acid constituents, that is PUFA (Nakayama
et al., 1981). Regarding the effect of the applied treat-
ments on the fatty acid profile of C. chione meats, it
seems that marinating had a negative impact with
respect to PUFA, especially on the proportion of x-3
fatty acids.
Lipid quality indices
Polyunsaturated/saturated (PUFA/SFA), monounsatu-
rated/saturated (MUFA/SFA), x-3/x-6 and hI/HI (h/
H) fatty acid ratios are widely used to evaluate the
nutritional value of lipid (Orellana et al., 2009). The
MUFA/SFA ratio decreased significantly (P 0.05)
after parboiling and frozen storage, but it was not
affected by marinating (Table 3), as this process
caused a simultaneous increase in both MUFA and
SFA proportions. Although the PUFA/SFA ratio
showed a significant decrease in all treatments applied,
it remained close to or exceeded the recommended
value of 0.45 (Williams, 2000). Thus, all samples
exhibited a desirable fatty acid balance, as indicated
by the high MUFA/SFA and PUFA/SFA ratios
observed. The recommended x-3/x-6 fatty acid ratio
for health benefits is above 0.25 (Raes et al., 2004);
however, this ratio is 10 times lower in typical Western
diets (Simopoulos, 2003). All samples demonstrated
high x-3/x-6 ratios (3), irrespectively of the preserva-
tion treatment applied (Table 3). Frozen storage of the
vacuum-packed parboiled meats exhibited the highest
x-3/x-6 ratio. The h/H ratio is an index of the choles-
terolaemic effect of the lipid, representing the effect of
individual fatty acids on cholesterol metabolism (San-
tos-Silva et al., 2002). This ratio differed significantly
(P 0.05) between treatments, with less favourable
values being observed in marinated samples (Table 3).
The peroxidisability index, representing the
Table 3 Lipid quality indices of raw, parboiled, frozen vacuum-packed and marinated Callista chione meat
Lipid quality
indices Raw Parboiled
Frozen,
vacuum-packed
(4 months)
Frozen,
vacuum-packed
(6 months)
Marinated
(4 months)
Marinated
(6 months)
HI 27.66 Æ 0.63a 32.41 Æ 0.16b 31.27 Æ 0.47ab 33.42 Æ 2.89b 38.19 Æ 1.23c 39.41 Æ 0.36c
hI 35.94 Æ 1.15a 28.94 Æ 0.13bd 31.20 Æ 0.56d 27.64 Æ 1.74bc 25.24 Æ 1.69ce 22.91 Æ 0.63e
h/H 1.30 Æ 0.07a 0.89 Æ 0.01b 1.00 Æ 0.03b 0.83 Æ 0.12bc 0.66 Æ 0.06c 0.58 Æ 0.02c
AI 0.67 Æ 0.04a 0.90 Æ 0.01b 0.85 Æ 0.04b 1.00 Æ 0.17b 1.24 Æ 0.09c 1.31 Æ 0.02c
TI 0.35 Æ 0.00a 0.42 Æ 0.01b 0.37 Æ 0.01a 0.44 Æ 0.06b 0.62 Æ 0.06c 0.65 Æ 0.05c
PI 190.95 Æ 1.09a 182.94 Æ 4.79a 190.19 Æ 5.54a 176.74 Æ 17.25a 118.54 Æ 10.43b 111.53 Æ 10.37b
MUFA/SFA 0.50 Æ 0.03a 0.37 Æ 0.01b 0.44 Æ 0.01c 0.37 Æ 0.02b 0.50 Æ 0.01a 0.48 Æ 0.01a
PUFA/SFA 0.79 Æ 0.02a 0.63 Æ 0.02b 0.71 Æ 0.03d 0.60 Æ 0.08b 0.43 Æ 0.04c 0.41 Æ 0.03c
x-3/x-6 3.12 Æ 0.25a 4.15 Æ 0.12b 4.04 Æ 0.06b 4.20 Æ 0.19b 3.07 Æ 0.20a 3.20 Æ 0.32a
hI, Hypocholesterolaemic; HI, hypercholesterolaemic; AI, atherogenic index; TI, thrombogenic index; PI, peroxidisability index; SFA, saturated fatty
acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.
Results represent means Æ SD (N = 6 9 2).
Means in the same row bearing different letters differ significantly (P 0.05).
© 2015 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2015
Effects of preservation on C. chione lipids C. D. Papaioannou et al.6

7. relationship between fatty acid composition of a tissue
and its susceptibility to oxidation (Erickson, 1992), sig-
nificantly (P 0.05) decreased only after marinating,
probably as a result of PUFA level reduction. The TI
remained below the recommended value (1.0) for a
healthy diet (Valfre et al., 2003), despite the significant
increase observed in the marinated samples. The AI
exceeded the value of 1.0 only in marinated meats.
The higher AI vs TI values observed in the marinating
process are related to the significant increase of myris-
tic acid proportion with simultaneous decrease of stea-
ric acid.
Carotenoids
The HPLC-DAD analysis used to identify the carote-
noid composition in C. chione TL revealed the pres-
ence of six carotenoids (Table 4). In the raw meats,
canthaxanthin and astaxanthin predominated, followed
by b-carotene, lutein, b-cryptoxanthin and zeaxanthin.
Total carotenoid content of the raw, parboiled, frozen
and marinated samples was also determined by
UV–vis spectroscopy, and the results were in good
agreement with the ones obtained by HPLC-DAD
analysis (Table 4). With the exception of b-cryptoxan-
thin, parboiling significantly affected the carotenoid
content. Most of the carotenoids were degraded,
whereas astaxanthin content increased significantly. In
marinated samples stored for 4 months, astaxanthin
content exhibited a slight increase, which became sig-
nificant (P 0.05) after 6 months of storage. Can-
thaxanthin was reduced (P 0.05) in the marinated
samples at 4 months of storage, whereas it was not
detected after 6 months. Lutein, zeaxanthin, b-cryptox-
anthin and b-carotene were not detected at either 4 or
6 months of storage. During frozen storage of the
vacuum-packed parboiled meats, astaxanthin content
increased (P 0.05), while lutein, zeaxanthin,
b-cryptoxanthin, b-carotene and canthaxanthin con-
tents were gradually degraded.
Carotenoids are found in invertebrates in free form,
as carotenoproteins, or bound to esters, glycosides and
sulphates (Elde et al., 2012). Astaxanthin and canthax-
anthin tend to form complexes with certain proteins,
with the best known examples, a-crustacyanin and
b-crustacyanin being responsible for the blue col-
oration of several invertebrates’ tissues. These com-
plexes exhibit bathochromic shift in the light
absorption spectrum in relation to the free carotenoid
(Liaaen-Jensen Kildahl-Andersen, 2008; Elde et al.,
2012) and therefore cannot be detected spectrophoto-
metrically in the specific absorption spectral range of
440–480 nm. As crustacyanin complexes absorb at dif-
ferent wavelength from astaxanthin, the significant
increase of astaxanthin observed after different process
treatments (parboiling, marinating and freezing) was
possibly due to the dissociation of these proteins,
releasing astaxanthin.
Conclusion
The lipid constituents of the soft clam C. chione were
determined, and the impact of typical preservation
treatments (parboiling, marinating and freezing) on
their nutritional quality was evaluated. Among treat-
ments, only marinating caused a significant decrease in
PE, which was accompanied by the production of l-PE
and l-PC. The PUFA/SFA ratio was significantly
decreased by the various treatments, whereas an
increase in MUFA proportion was observed in the
marinated samples. The highest x-3/x-6 ratios were
observed in parboiled and frozen, vacuum-packed
meats. Marinating process had the most adverse
impact on lipid quality indices, even though beneficial
Table 4 Carotenoid content expressed as mg per g of total lipids of raw, parboiled, frozen and marinated Callista chione meat
Carotenoids Raw Parboiled
Frozen, vacuum-packed
(4 months)
Frozen, vacuum-packed
(6 months)
Marinated
(4 months)
Marinated
(6 months)
trans-lutein 0.13 Æ 0.01a 0.06 Æ 0.01b 0.06 Æ 0.01b 0.05 Æ 0.01b nd nd
trans-zeaxanthin 0.08 Æ 0.01a nd nd nd nd nd
trans-astaxanthin 0.21 Æ 0.01a 0.36 Æ 0.02b 0.42 Æ 0.02c 0.50 Æ 0.03e 0.23 Æ 0.01ad 0.25 Æ 0.01d
trans-canthaxanthin 0.29 Æ 0.02a 0.13 Æ 0.01b 0.07 Æ 0.01c 0.09 Æ 0.01c 0.12 Æ 0.01b nd
trans-b-cryptoxanthin 0.11 Æ 0.01a 0.11 Æ 0.01a 0.08 Æ 0.01b nd nd nd
trans-b-carotene 0.17 Æ 0.02a 0.13 Æ 0.01b 0.12 Æ 0.01b 0.14 Æ 0.02b nd nd
Total carotenoids* 0.99 Æ 0.04a 0.79 Æ 0.04b 0.75 Æ 0.04b 0.78 Æ 0.05b 0.35 Æ 0.03c 0.25 Æ 0.01d
Total carotenoids†
1.00 Æ 0.09a 0.79 Æ 0.08b 0.69 Æ 0.06b 0.85 Æ 0.10ab 0.37 Æ 0.06c 0.26 Æ 0.06c
nd, not detected.
Results represent means Æ SD (N = 6 9 2).
Means in the same row bearing different letters differ significantly (P 0.05).
*Sum of the carotenoids content identified by HPLC expressed as mg per g of total lipids.
†
Sum of the carotenoids content identified spectrophotometrically expressed as mg per g of total lipids.
© 2015 Institute of Food Science and Technology International Journal of Food Science and Technology 2015
Effects of preservation on C. chione lipids C. D. Papaioannou et al. 7

8. values for a healthy diet were retained. With the
exception of astaxanthin, all the carotenoids identified
were very susceptible to all treatments. Parboiling pro-
cess and freezing of the vacuum-packed meats would
be the most appropriate treatments for preserving the
quality of lipid constituents for up to 4 months. The
marinating process would provide an alternative long-
term preservation treatment, also adding unique fla-
vour to the final product. From a nutritional point of
view, C. chione was found to be a rich source of diet-
ary PhLs and x-3 fatty acids, which are beneficial for
consumer health, encouraging the full exploitation of
the species as human food.
Acknowledgment
The present work was partially financed by the Special
Research Fund Account of the National and Kapodis-
trian University of Athens (Project No. 11400/2015).
Conflict of interest
The authors declare no conflict of interests.
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