Comparative analysis of the fatty acid and sterol profiles of widely consumed Mediterranean crustacean species

Food Chemistry 122 (2010) 292–299

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Analytical Methods

Comparative analysis of the fatty acid and sterol profiles of widely consumed Mediterranean crustacean species Katerina Tsape a, Vassilia J. Sinanoglou b, Sofia Miniadis-Meimaroglou a,* a b

Food Chemistry Laboratory, Department of Chemistry, University of Athens, Panepistimioupolis Zographou, 15701 Athens, Greece Laboratory of Food Analysis, Department of Food Technology, Technological Educational Institution of Athens, Greece

a r t i c l e

i n f o

Article history: Received 10 February 2009 Received in revised form 26 January 2010 Accepted 9 February 2010

Keywords: Crustaceans Neutral lipids PUFA x-3/x-6 Triacylglycerols Cholesterol

a b s t r a c t Comparative analysis of FA, NL and sterol profiles in the Nephrops norvegicus (langoustine), Palinurus vulgaris (lobster) and Penaeus kerathurus (shrimp) muscle and cephalothorax showed that C16:0, C16:1x-7, C18:0, C18:1x-9, C20:4x-6, EPA and DHA were found to be their major FA. Highest EPA occurred in langoustine muscle TL, DHA in both shrimp tissues TL while C20:4x-6 in lobster muscle and cephalothorax TL. Muscle and cephalothorax NL consisted mainly of sterols (42.5–54.4% and 13.7–43.1%) and triacylglycerols (35.4–45.8% and 44.6–59.4%). Cholesterol was the major sterol (70.90–98.58% and 97.10–98.31% of total sterols in muscle and cephalothorax respectively) followed by avenasterol (0.18–20.32% and 0.07– 0.70%) and b-sitosterol (0.29–7.30% and 0.23–0.75%). Lower concentrations of brassicasterol, stigmasterol, D7-stigmasterol, campesterol and campestanol were also found. The examined crustaceans muscle (edible part) was found to be a good x-3 PUFA source for the consumers, while the cephalothorax (which is usually discarded) could be used effectively as a source for x-3 PUFA production. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Marine lipids and especially the x-3 polyunsaturated fatty acids (x-3 PUFA) such as eicosapentaenoic acid (EPA, 20:5x-3) and docosahexaenoic acid (DHA, 22:6x-3) are believed be protective of human health in many ways. The consumption of x-3 polyunsaturated fatty acids (PUFA) reduces the risk of coronary heart disease and cancer, has both anti-athrogenic and anti-thrombotic effects as well as a role in the control of rheumatoid arthritis and hypertension. It also reduces the risk of diabetes and prevents cardiac arrhythmias (Mahaffey, 2004; Schmidt, 2003; Sidhu, 2003; Simopoulos, 2001). PUFA appear to play a useful role in the development of nervous (brain), photoreception (vision), and reproductive systems (Horrocks & Yeo, 1999). In humans, a-linolenic acid can be elongated and desaturated to a limited extent to EPA and DHA. Alternatively, these PUFA are mainly acquired from seafood thus, humans obtain the principal part of EPA and DHA by consuming fish and aquatic invertebrates such as molluscs and crustaceans (Schmidt, Arnesen, de Caterina, Rasmussen, & Kristensen, 2005). Cholesterol is the most abundant

sterol in the advanced invertebrates. Although cholesterol is essential for membrane structure as well as hormone and steroid biosynthesis, an excess concentration in plasma is considered to play a key role in the heart- and artery-related disease (Kannel, 1995), making it necessary to monitor cholesterol content in food. Crustaceans such as shrimp, langoustine and lobster represent some of the widely consumed species of the Mediterranean and comprise an important source of nutrients in the human diet (Chapelle, 1977; Sriket, Benjakul, Visessanguan, & Kijroongrojana, 2007). The present study focused on the comparative analysis of total and neutral lipid fatty acids, the neutral lipids composition as well as the sterol composition (since data concerning sterol composition of crustaceans are very limited) in the muscle and cephalothorax of the Nephrops norvegicus, Palinurus vulgaris and Penaeus kerathurus, the most popular crustacean species of the Mediterranean diet. 2. Materials and methods 2.1. Reagents and standards

Abbreviations: AA, arachidonic acid (C20:4x-6); DHA, docosahexaenoic acid (C22:6x-3); EPA, eicosapentaenoic acid (C20:5x-3); MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; FA, fatty acid(s); TAG, triacylglycerol(s); TL, total lipids. * Corresponding author. Tel.: +30 210 7274486; fax: +30 210 3624870. E-mail address: [email protected] (S. Miniadis-Meimaroglou). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.02.019

The lipid standards used (purity > 98%) were cholesterol, avenasterol, b-sitosterol, brassicasterol, campesterol, campestanol, stigmasterol, D7-stigmasterol, triolein and they were purchased from the Sigma Chemical Co. (Sigma–Aldrich Company, St. Louis,

K. Tsape et al. / Food Chemistry 122 (2010) 292–299

MO). Fatty acid methyl esters used as GC and GC/MS standards were: lauric acid M-E, cis-5,8,11,14,17-eicosapentaenoic acid ME, and cis-4,7,10,13,16,19-docosahexaenoic acid M-E, (purity P 98%) purchased from Sigma Chemical Co. (Sigma–Aldrich Company, UK); Matreya Bacterial Acid Methyl Esters CP™ Mix, Catalog No: 1114; Supelco™ 37 Component FAME Mix C4– C24, 100 mg Neat, Catalog No: 18919-1AMP; Supelco PUFA No: 1, Marine Source, 100 mg Neat, Catalog No: 47033. All solvents used for sample preparation were of analytical grade and the solvents used for GC and MS analyses were of HPLC grade from Merck (Darmstadt, Germany). Double distilled water was used throughout this work. Fll reagents used were of analytical grade and they were purchased from Mallinckrodt Chemical Works (St. Louis, MO) and from Sigma Chemical Co. (Sigma–Aldrich Company, St. Louis, MO). 2.2. Experimental animals – total lipid extraction and separation Eighteen adult N. norvegicus (langoustine) and 18 adult P. vulgaris (lobster) were collected from Argolicos Bay (one of the most important fishing places) in Greece. Furthermore, 18 adult P. kerathurus (shrimp) were caught in the North Aegean Sea (near Platamona Bay), in October. They were brought to the laboratory alive and individually measured for weight and length (with an average of 33.5 g and 17.5 cm/langoustine, 380 g and 25.5 cm/lobster, as well as 28.5 g and 16.7 cm/shrimp respectively). Then, samples of muscle and cephalothorax were taken; they were weighed, and afterwards separately homogenised. TL was extracted according to the Bligh and Dyer method (1959). After phase equilibration, the lower chloroform layer (TL) was removed and dried in a rotary vacuum evaporator at 32 °C. The extracted lipids were weighed in order to determine the TL, then redissolved in chloroform/methanol (9:1, v/v) and finally stored at 0 °C until used. Additionally, in order for that to be confirmed, aliquots were evaporated in preweighed vials to constant weight to determine the lipid content. To prevent oxidation t-butyl-hydroquinone was added to all samples during preparation. Total lipids were, afterwards, separated in neutral and polar lipids on pre-washed 500 mg silicic acid columns (Merck and Co., Kieselgel 60), by solid-phase extraction (SPE), using the modified method of Mastronicolis, German, and Smith (1996). Neutral lipid fractions were quantified by weight after being eluted from solidphase extraction columns. In order to determine the neutral lipids composition percentage, they were separated on silicic acid-coated quartz rods (Chromarods, Type SIII) and (they) quantified by passing the rods through a hydrogen flame ionisation detector (FID) operated with hydrogen flow-rate of 160 mL min1 and air flow-rate of 2 L min1 (Sinanoglou & Miniadis-Meimaroglou, 1998). Chromarods were developed with a solvent system consisting of n-hexane/diethylether/formic acid (80:20:2, v/v/v) to a height of 15 cm to detect the neutral lipids. The rods were then scanned in an Iatroscan MK-6 (TLC/FID – FPD Analyser) (Iatron Laboratories, Tokyo, Japan) equipped with a flame ionisation detector and connected to a personal computer for collecting the chromatograms. 2.3. Gas chromatography/mass spectrometry analysis of fatty acid methyl esters Fatty acids methyl esters of total lipids (TL) and neutral lipids (NL) were prepared according to the procedure described by Sinanoglou and Miniadis-Meimaroglou (1998). Both quantitative and qualitative analyses were performed on a Agilent 6890 Series Gas Chromatograph equipped with a flame ionisation detector. DB-23 capillary column (60 m  0.25 mm i.d., 0.15 lm film) [50%-cyano-

293

propyl)-methylpolysiloxane] (Agilent Technologies, Catalog No: 122–2361) was used. Helium was used as a carrier gas. The injector and detector temperature were 250 °C and 260 °C respectively. The temperature was programmed at 100 °C for 0 min, raised from 100 to 150 °C by a rate of 10 °C min1 and held constant at 150 °C for 0 min, then raised from 150 to 195 °C by a rate of 2 °C min1 and held constant at 195 °C for 5 min, then raised from 195 to 210 °C by a rate of 1 °C min1 and held at 210 °C for 0 min and finally raised from 210 to 240 °C by a rate of 10 °C min1 and held constant at 240 °C for 5 min. The duration of the analysis was 55.50 min. Individual fatty acids were quantified according to the formula:

lg ðFAÞ ¼ FAME peak area  FAME response factor; while FAME response factors were determined according to the FAME standards chromatograms under the same chromatogram conditions. A GC/MS analysis of the fatty acid methyl esters was also performed on a SHIMADZU 2010 GC/MS using a capillary column of high polarity EQUITY™ Catalog No: 28097-U (30 m  0.32 mm  0.25 lm film thickness). The temperature was programmed at 48 °C for 2 min, raised from 48 to 54 °C by a rate of 2 °C min1, held constant at 54 °C for 0 min, raised from 54 to 70 °C by a rate of 20 °C min1 and held at 70 °C for 1 min, then raised from 70 to 170 °C by 70 °C min1 held constant for 0 min, raised from 170 to 210 °C by a rate of 1 °C min1 and held at 210 °C for 5 min and, finally raised from 210 to 280 °C by a rate of 10 °C min1 and held at 280 °C for 5 min. The duration of the analysis was 65.23 min. Electron ionisation (EI) was produced by accelerating electrons from a hot filament through a potential difference usually of 70 eV (Sinanoglou & Miniadis-Meimaroglou, 1998). In both GC and GC/MS methods, the fatty acid methyl esters were identified by comparison of their retention times to those of the standard mixtures. 2.4. Isolation and determination of sterols A neutral lipid fraction containing sterols was isolated by silicic acid preparative thin layer chromatography on precoated silica gel 60 G plate (20  20 cm, 0.50 mm, Merck, Darmstadt, Germany). After spotting the sample, the plates were developed using solvent systems consisting of petroleum ether/diethyl ether/glacial acetic acid (either 70:30:1, 80:20:1, or 90:10:1, by vol.). The individual spots were visualised by exposure to iodine vapors and then, the sterols band on the plates was scrapped off and extracted from the silica gel using the solvent system of the Bligh–Dyer procedure, i.e. chloroform/methanol/water (2:2:1, by vol.). After phase separation, the chloroform extracts were evaporated until dryness. The residual sterol fractions were quantified by weight, then redissolved in chloroform/methanol (9:1, by vol.) and finally rechromatographed on HPTLC for purity confirmation. Afterwards the sterol composition was determined according to the method of (Yamashiro, Oku, Higa, Chinen, & Sakai, 1999) as follows: The sterol fractions were concentrated to dryness and then trimethylsilylated by heating them with BSTFA at 60 °C for 10 min. The reaction mixture was brought to dryness under a nitrogen stream, and the reaction products were extracted into a minimal volume of ethyl acetate. Quantitative and qualitative analysis of TMS sterols were performed on a HRGC Mega 2 Series 8560 MFC 800 (Fisons Instruments, Hellenic Labware, VG Biotech, UK) gas chromatograph, equipped with a EL 980 CE Instruments FID (flame ionization detector) (Hellenic Labware, VG Biotech, UK). TMS-derivatives of sterols were separated on a non-polar fused silica capillary column of chemically bonded methyl silicone liquid phase (CBP1-M50-025, 0.2 mm inner diameter  50 m length, Shimadzu). Column temperature was programmed from

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1 min held at 50 °C to final temperature of 300 °C at a rate of 10 °C min1. The carrier gas was helium with a flow-rate of 0.35 ml min1 (18 cm s1).

Table 1 Total lipids (% w/w), neutral lipids (% of TL) and total fatty acids (% of TL) in the examined crustaceans muscle and cephalothorax.

2.5. Schema of the experimental set up (procedure)

N. norvegicus

The experimental procedure of the ‘‘Comparative analysis of the fatty acid and sterol profiles of widely consumed Mediterranean crustacean species” described in the present study is depicted in Fig. 1.

P. vulgaris P. kerathurus

Muscle Cephalothorax Muscle Cephalothorax Muscle Cephalothorax

% TL (wet tissue)

% NL of TL

% TFA of TL

0.70 ± 0.05a 1.30 ± 0.08b 1.00 ± 0.10c 2.40 ± 0.08d 1.30 ± 0.06b 2.40 ± 0.05d

34.6 ± 0.1a 59.7 ± 0.2b 31.4 ± 0.3c 45.9 ± 0.2d 22.6 ± 0.1e 51.3 ± 0.2f

61.00 ± 0.31a 77.00 ± 0.58b 59.61 ± 0.35a 79.17 ± 0.23c 73.00 ± 0.52d 85.00 ± 0.46e

Means in the same column bearing different letters differ significantly (P < 0.05).

2.6. Statistical analysis Data were analysed with one-way ANOVA Post Hoc Tests and pairwise multiple comparisons were conducted with the Tukey’s honestly significant difference test. All data were analysed with the SPSS 10.0 statistical software. Data reported as means ± standard deviation of (at least) three replicate determinations of three different composite samples each including six adult crustaceans. 3. Results and discussion 3.1. Lipid content; neutral lipid profile and sterol composition The total lipid (TL) content in P. vulgaris (lobster), N. norvegicus (langoustine) and P. kerathurus (shrimp) muscles and cephalothorax was found to differ significantly (P < 0.05), ranging from 0.70 ± 0.05% to 1.30 ± 0.06% as well as from 1.30 ± 0.08% to 2.40 ± 0.05% respectively (Table 1). These values compared well, in general terms, with those reported by Bragagnolo and Rodriguez-Amaya (2001) as well as Krzynowek and Panunzio (1989) for other crustacean species.

The proportion of NL in TL of the above mentioned crustaceans cephalothorax was much higher (ranging from 45.9 ± 0.2 to 59.7 ± 0.02%) to that found in the muscle TL (ranging from 22.6 ± 0.1 to 34 ± 0.01%) (Table 1) since the latter mainly consisted of PL, as previously reported by the same research group (Garofalaki, Miniadis-Meimaroglou, & Sinanoglou, 2006; MiniadisMeimaroglou, Kora, & Sinanoglou, 2008. As shown in Table 1, the percentage of TFA, in cephalothorax TL of P. vulgaris, N. norvegicus and P. kerathurus was found to be higher than the ones in the muscle tissue TL, due to the fact that the latter was found to contain a higher percentage of TG (Table 2). The main components of the examined crustaceans NL were found to be TAG (triacylglycerols) and sterols, followed by waxes, sterol esters, diglycerides, monoglycerides, free alcohols and free fatty acids) (Table 2). The NL of N. norvegicus and P. vulgaris muscle and cephalothorax were found to contain significantly (P < 0.05) higher percentages of TAG compared to the ones in P. kerathurus muscle and cephalothorax NL, while the opposite was observed concerning the total sterol content (Table 2). The high percentages of TAG in the cephalothorax NL (59.4 ± 0.9%, 53.2 ± 1.5% and 44.6 ± 0.8% of

Muscle, cephalothorax of N. norvegicus, P. vul garis, P kerathurus Extracted according to Bligh-Dyer method

CH3OH/ water phase

CHCl3 phase (lipid-soluble)

Evaporated to dryness, weighing and redissolved in CHCL 3/CH3OH 9:1

TL of muscle and cephalothorax, GC/MS F.A of TL TL separation by SPE

PL fraction (muscle, cephalothorax) NL fraction (muscle, cephalothorax)

GC-MS FA of NL

Iatroscan F.I.D detector solvent system A

Preparative TLC solvent system A

NL components (muscle, ceph/ax) Isolated sterols Determination of Sterol Composition Fig. 1. Schema of the experimental set up (procedure) of the comparative analysis of the fatty acid and sterol profiles of widely consumed Mediterranean crustacean species.

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K. Tsape et al. / Food Chemistry 122 (2010) 292–299 Table 2 Neutral lipids profile of the muscle and cephalothorax of the crustaceans studied as determined by Iatroscan. Neutral lipids

N. norvegicus Muscle

P. vulgaris Cephalothorax

Muscle

P. kerathurus Cephalothorax

Muscle

Cephalothorax

Waxes % of TL % of NL

0.6 ± 0.1 1.7 ± 0.3a

4.1 ± 0.2 6.9 ± 0.7b

0.9 ± 0.1 2.9 ± 0.1c

4.3 ± 0.2 9.4 ± 1.4d

0.1 ± 0.0 0.5 ± 0.1e

Sterol esters % of TL % of NL

2.4 ± 0.1 6.8 ± 0.4a

6.0 ± 0.2 10.1 ± 0.8b

1.7 ± 0.1 5.5 ± 0.2c

3.9 ± 0.1 8.5 ± 0.9b

0.3 ± 0.1 1.3 ± 0.1d

2.1 ± 0.2 4.1 ± 0.1e

13.9 ± 0.2 40.1 ± 0.5a

35.5 ± 0.5 59.4 ± 0.9b

14.4 ± 0.3 45.8 ± 0.7c

24.4 ± 0.8 53.2 ± 1.5d

8.0 ± 0.1 35.4 ± 0.2e

22.9 ± 06 44.6 ± 0.8c

0.3 ± 0.1 1.0 ± 0.1a

1.5 ± 0.1 2.5 ± 0.2b

1.6 ± 0.1 3.5 ± 0.8c

0.7 ± 0.1 3.1 ± 0.1c

0.6 ± 0.1 1.2 ± 0.1a

15.1 ± 0.1 43.6 ± 0.7a

8.2 ± 0.1 13.7 ± 0.4b

13.3 ± 0.2 42.5 ± 0.5a

7.3 ± 0.1 16.0 ± 0.8c

12.3 ± 0.3 54.4 ± 0.5d

22.1 ± 0.2 43.1 ± 0.3a

4.4 ± 0.2 7.4 ± 0.8a

1.1 ± 0.1 3.3 ± 0.4b

4.3 ± 0.2 9.4 ± 0.5c

1.2 ± 0.1 5.3 ± 0.2d

3.6 ± 0.2 7.0 ± 0.2a

Triacylglycerols % of TL % of NL Free fatty acid % of TL % of NL Sterols % of TL % of NL

Free alcohols, diglycerides, monoglycerides % of TL 2.3 ± 0.1 % of NL 6.8 ± 0.3a

– –

– –

Means in the same row bearing different letters differ significantly (P < 0.05).

NL respectively) probably due to the fact that cephalothorax contains the hepatopancreas, the lipids of which are mainly composed of triglycerides (Chapelle, 1977). The high level of triglycerides in crustaceans may serve as an energy source during conditions of food shortage or possibly reflect greater feeding activity (Phleger, Nelson, Mooney, & Nichols, 2002). An estimation of the triacylglycerol quantity one may receive through diet showed that the consumption of equal quantities of each of these crustacean muscles, results in significantly (P < 0.05) different triacylglycerols intake (Table 3). The sterols composition of the examined crustaceans muscle and cephalothorax are shown in Table 3. Cholesterol was found to be the main sterol in both tissues of all the examined crustaceans. It was found to have the highest percentage in P. vulgaris muscle sterols (98.58 ± 0.19% of total sterols) as well as in cephalothorax sterols (97.1–97.36%) of all the above mentioned crustaceans (Table 3). Similarly high values of cholesterol in crustaceans’ sterols were also reported by Karakoltsidis, Zotos, and Konstantinides (1995). King, Childs, Dosett, Ostrander, and Monsen (1990) have reported that cholesterol constituted more than 90% of the shrimp sterols.

Cholesterol was also found to be the major sterol (80–100% of total sterols) followed by desmosterol (1–18%) in the Atlantic euphausiides, Euphausia superba, Euphausia tricantha, Euphausia frigida and Thysanoessa macrura (Phleger et al., 2002). Avenasterol was the second most abundant sterol in N. norvegicus muscle (20.32 ± 0.11% of total sterols) but markedly less in all other samples. b-Sitosterol was also determined in relatively high percentages in both N. norvegicus and P. kerathurus muscle (sterols). Low levels of brassicasterol, stigmasterol, D7-stigmasterol, campesterol, as well as campestanol (reduction product of campesterol) were also determined (Table 3). Crustaceans are incapable of de novo sterol synthesis, however they may biosynthesis cholesterol from other sterols such as bsitosterol, brassicasterol, ergosterol and campesterol; thus the sterols content in these depends on their diet. The different sterols composition in crustacean species indicates different feeding preferences or food sources (Phleger et al., 2002). Langoustine muscle contains a lees diverse sterol profile compared to the one in lobster and shrimp muscle while lobster cephalothorax contained a more diverse sterol profile compared to the one in langoustine and shrimp cephalothorax (Table 3).

Table 3 Total sterols, cholesterol, triglycerides and x-3 fatty acids content expressed as mg/100 g on a wet weight basis and sterol composition (w/w % of total sterols) in the muscle and cephalothorax of the examined crustacean. N. norvegicus

Total sterols Cholesterol Triglycerides x-3 Fatty acids

P. vulgaris

P. kerathurus

Muscle

Cephalothorax

Muscle

Cephalothorax

Muscle

Cephalothorax

105.73 ± 8.25a 74.97 ± 5.96a 97.37 ± 8.35a 146.54 ± 12.02a

106.65 ± 7.86a 103.56 ± 7.72b 461.77 ± 34.90c 325.21 ± 24.25b

133.13 ± 11.30b 131.26 ± 15.34c 144.20 ± 17.40b 137.16 ± 15.40a

175.25 ± 8.24c 170.63 ± 8.15d 586.03 ± 38.72d 419.26 ± 18.42c

159.90 ± 13.12c 144.29 ± 14.08c 104.01 ± 9.26a 301.61 ± 17.49b

530.40 ± 9.50d 521.44 ± 9.63e 549.60 ± 32.15d 462.96 ± 14.80d

97.10 ± 0.08b 0.61 ± 0.09a 0.43 ± 0.13a 0.74 ± 0.16a – 0.65 ± 0.19b

98.58 ± 0.19c 0.48 ± 0.15b 0.21 ± 0.07b 0.16 ± 0.03b – 0.29 ± 0.08c – 0.18 ± 0.14c

97.36 ± 0.07b 1.12 ± 0.14c 0.33 ± 0.16c 0.15 ± 0.17b 0.14 ± 0.06b 0.75 ± 0.05b 0.08 ± 0.08 0.07 ± 0.16c

90.24 ± 0.18d 0.61 ± 0.13a 0.80 ± 0.14d 0.32 ± 0.12c

98.31 ± 0.13e 1.11 ± 0.09e 0.22 ± 0.08d

Sterol composition % of total sterols Cholesterol 70.90 ± 0.11a Brassicasterol – Campesterol – Campestanol – Stigmasterol 1.63 ± 0.17a b-Sitosterol 7.21 ± 0.06a D7-Stigmasterol – Avenasterol 20.32 ± 0.11a

– 0.70 ± 0.15b

Means in the same row bearing different letters differ significantly (P < 0.05).

–

– 7.30 ± 0.07a

–

0.23 ± 0.22c –

0.71 ± 0.04b

0.11 ± 0.18c

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The total sterols found, on wet weight basis, were 0.105% and 0.106% for langoustine, 0.133% and 0.175% for lobster as well as 0.159% and 0.530% for shrimp, muscle and cephalothorax respectively. P. kerathurus muscle and cephalothorax were found to have the highest cholesterol content (144.29 ± 14.08 mg/100 g and 521.44 ± 9.63 mg/100 g of the wet tissue respectively) while N. norvegicus muscle and cephalothorax were found to have the lowest one (74.97 ± 5.96 mg/100 g and 103.56 ± 7.72 mg/100 g of wet tissue respectively) (Table 3). P. kerathurus cholesterol levels obtained in this study were lower than those reported by Engeseth and Gray (1990) with a mean of 262 mg/100 g for a non-identified species of shrimp, similar to those obtained by Johnston, Ghanbari, Wheeler, and Kirk (1983) with a mean of 201 mg/100 g for Penaeus aztecus, by Kritchevsky, Tepper, Ditullo, and Holmes (1967) with a mean of 200 mg/100 g for a non-identified species of shrimp and higher than those obtained by Bragagnolo and Rodriguez-Amaya (2001) with a general average mean of 127 mg/100 g for farmed freshwater prawn (Macrobrachium rosenbergii) and wild marine shrimps (Penaeus brasiliensis, Penaeus schimitti and Xiphopenaeus kroyeri) by Essien (1995) with a mean of 140 mg/100 g for Palaemometes spp. and by Luzia, Sampaio, Castellucci, and Torres (2003) with a mean of 165 mg/100 g for seabob shrimp X. kroyeri. After having taken into consideration the cholesterol values (Table 3), their contribution to the recommended maximum cholesterol intake can be calculated through the consumption of 100 g of N. norvegicus, P. vulgaris and P. kerathurus muscle. This contribu-

tion represented 24.99%, 43.75% and 48.09% of the recommended maximum cholesterol intake by the European Olive Oil Medical Information (300 mg per day) respectively, or 35.03%, 61.33% and 67.42% of the value recommended (214 mg per day) by Moschandreas and Kafatos (1999) respectively. 3.2. Fatty acids The fatty acid (FA) pattern of total lipids (TL) isolated from muscle and cephalothorax of N. norvegicus, P. vulgaris and P. kerathurus is shown in Table 4. The carbon chain length of the identified 39 fatty acids ranged from 14C to 24C. Furthermore, the number of double bonds ranged from 0 to 6. The main FA in N. norvegicus, P. vulgaris and P. kerathurus muscle and cephalothorax TL were found to be C16:0, C16:1x-7, C18:0, C18:1x-9, arachidonic acid, EPA and DHA, ranging from 77.95% to 93.76% of the TFA and from 66.19% to 90.99% of the TFA respectively (Table 4). The same fatty acids were also found to be the main FA (88.7% of TFA) in seabob shrimp X. kroyeri TL (Luzia et al., 2003). C16:0, C18:0, DHA and EPA were also the most abundant fatty acids in several shrimp species as reported by a number of other authors (Rosa & Nunes, 2003; Sriket et al., 2007; Yanar & Celik, 2005). The fatty acids profile of TL of the examined crustaceans muscle and cephalothorax presented a differentiation in the proportion as well as in the fatty acid species.

Table 4 Fatty acids composition [% (w/w)] in total lipids of the N. norvegicus, P. vulgaris and P. kerathurus muscle and cephalothorax. Fatty acids

N. norvegicus Muscle

C12:0 C14:0 C14:1x-5 C15:0 C15:1 C16:0 C16:1x-9 C16:1x-7 3-OHC12:0 Iso-C17:0 C17:0 C17:0cyclo 2-OHC14:0 C17:1x-7 C18:0 C18:1x-9 C18:1x-7 3-OHC14:0 C18:2x-6 cis C18:2x-6 trans C19:0 C18:3x-6 C19:0 cyclo C18:3x-3 C20:0 C20:1x-9 C20:2x-6 C20:3x-6 C20:4x-6 C20:3x-3 C22:0 C22:1x-9 C20:5x-3 C22:4x-6 C22:5x-6 C22:5x-3 C24:0 C24:1 C22:6x-3

– 1.16 ± 0.03a – – – 22.50 ± 0.17a – 5.01 ± 0.09a – – 1.47 ± 0.07a – – – 6.21 ± 0.07a 21.02 ± 0.14a – – 1.53 ± 0.02a – – 0.44 ± 0.01a – 0.55 ± 0.02a – 1.08 ± 0.03a – – 5.27 ± 0.11a – – – 15.30 ± 0.14a – – – – – 18.45 ± 0.18a

P. vulgaris Cephalothorax 0.12 ± 0.01 2.40 ± 0.04b – – – 19.29 ± 0.24b – 7.84 ± 0.12b – – 1.24 ± 0.03b – – – 5.25 ± 0.05b 21.55 ± 0.12a – – 1.89 ± 0.03b – – 0.54 ± 0.02b – 0.37 ± 0.03b 2.07 ± 0.02a 0.38 ± 0.01b – – 4.96 ± 0.09a – – – 13.70 ± 0.22b – – – – – 18.40 ± 0.15a

Means in the same row bearing different letters differ significantly (P < 0.05).

P. kerathurus

Muscle

Cephalothorax

Muscle

Cephalothorax

– 1.18 ± 0.02a 0.32 ± 0.01a 1.17 ± 0.03a – 10.86 ± 0.16c – 4.81 ± 0.08a – 0.36 ± 0.02a 1.71 ± 0.05c 1.23 ± 0.03a 0.56 ± 0.04a – 7.37 ± 0.05c 13.91 ± 0.21b 2.59 ± 0.14a – 2.07 ± 0.03c – – 0.60 ± 0.02c – 0.42 ± 0.02c 0.35 ± 0.02b 0.78 ± 0.06c 2.14 ± 0.06a – 22.76 ± 0.13b – – 0.99 ± 0.06a 11.17 ± 0.16c – – 0.61 ± 0.03a – 0.65 ± 0.02a 10.79 ± 0.09b

–

–

– 1.89 ± 0.05e 0.33 ± 0.02a 2.15 ± 0.03c 0.80 ± 0.04b 17.20 ± 0.13f – 5.17 ± 0.01ac 0.16 ± 0.02 1.96 ± 0.04d 2.73 ± 0.03e 1.82 ± 0.06c 0.93 ± 0.05c – 6.44 ± 0.08e 9.41 ± 0.15d 4.20 ± 0.12d 0.36 ± 0.01 1.07 ± 0.02d 0.32 ± 0.03b 0.25 ± 0.02 0.37 ± 0.04d 0.30 ± 0.01 0.40 ± 0.02c 2.49 ± 0.05e 1.88 ± 0.04f 2.07 ± 0.02d 0.20 ± 0.01b 8.65 ± 0.07e 0.58 ± 0.01 0.83 ± 0.02b 0.64 ± 0.04d 10.70 ± 0.12e – – 0.63 ± 0.02a 2.30 ± 0.04c – 10.38 ± 0.21b

1.98 ± 0.04c 0.76 ± 0.05b 2.90 ± 0.04b – 11.51 ± 0.20d 0.57 ± 0.05 5.78 ± 0.16c – 0.58 ± 0.03b 1.35 ± 0.02d – – 0.92 ± 0.03 5.62 ± 0.08d 11.32 ± 0.08c 5.89 ± 0.09b – 1.03 ± 0.01d 0.27 ± 0.01a – 0.52 ± 0.04b – 0.28 ± 0.01d 0.31 ± 0.02c 0.91 ± 0.05d 1.41 ± 0.01b 0.32 ± 0.04a 10.18 ± 0.21c – 0.89 ± 0.03a 0.52 ± 0.01b 13.90 ± 0.11b 0.81 ± 0.05 0.58 ± 0.04 – 1.81 ± 0.01a 1.87 ± 0.07b 7.88 ± 0.12c

0.68 ± 0.01d – 1.27 ± 0.06a 0.43 ± 0.02a 13.25 ± 0.11e – 5.63 ± 0.09c – 0.94 ± 0.02c 2.73 ± 0.02e 2.05 ± 0.03b 0.70 ± 0.02b – 6.56 ± 0.10e 11.14 ± 0.13c 4.81 ± 0.08c – 1.38 ± 0.04e – – – – 0.33 ± 0.01e 0.56 ± 0.05d 1.15 ± 0.05e 1.16 ± 0.03c – 11.04 ± 0.15d – – 0.37 ± 0.02c 17.28 ± 0.26d – – 1.12 ± 0.03b 2.21 ± 0.01b – 13.05 ± 0.16d

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The percentages of C16:0, C18:1x-9 and DHA in the TFA of N. norvegicus muscle and cephalothorax TL were significantly (P < 0.05) higher compared to the ones (in the TFA) of both P. vulgaris and P. kerathurus tissues TL. The percentage of arachidonic acid was found to have the highest value in the TFA of P. vulgaris muscle TL and the lowest in the TFA of N. norvegicus cephalothorax TL. Furthermore the percentages of C16:1x-7 and C18:0 in TFA of muscle and cephalothorax TL of these crustaceans showed insignificant differences. Some of the minor FA percentages (in the TFA) of these crustaceans, in both tissues TL, were found to differ significantly. C15:1x-7, C17:0, iso-C17:0, C17:0cyclo, 2-OHC14:0, C20:1x-9 and C24:0 were found in significantly (P < 0.05) higher percentages in the TFA of P. kerathurus (shrimp) muscle and cephalothorax TL compared to the same fatty acid percentages found in the TFA of both langoustine and lobster tissues TL. On the other hand, C14:0 was found in a significantly (P < 0.05) higher percentage in the TFA of these crustaceans cephalothorax TL compared to its percentage in the TFA of muscle TL of the examined crustaceans. The 16:0/18:0 ratios in the TFA of N. norvegicus, P. vulgaris and P. kerathurus muscle TL were found to be 3.62 ± 0.030, 1.47 ± 0.008 and 2.02 ± 0.010 respectively while in the cephalothorax TL they were found to be 3.67 ± 0.012, 2.05 ± 0.020 and 2.67 ± 0.014 respectively. The 18:1x-9/18:1x-7 ratios in TFA of the P. vulgaris muscle and cephalothorax TL were found to be 5.37 ± 0.10 and 2.32 ± 0.02 respectively, in P. kerathurus muscle and cephalothorax TL 1.92 ± 0.04 and 2.24 ± 0.05 respectively, while in N. norvegicus both tissues TFA of TL, C18:1x-7 was not detected. Furthermore, the EPA/DHA ratios in TFA of N. norvegicus, P. vulgaris and P. kerathurus muscle TL were found to be 0.83 ± 0.005, 1.04 ± 0.007 and 1.32 ± 0.009 respectively while in cephalothorax TL 0.74 ± 0.011, 1.76 ± 0.021 and 1.03 ± 0.006 respectively. High EPA/DHA ratios in the crustaceans may be due to metabolic rate variations of these two PUFA. An important dietary source of these PUFA for crustacean species is phytoplankton, especially diatoms and other phaeophytes, which are rich in PUFA, they synthesise (Phleger et al., 2002).

The sums of saturated fatty acids (RSFA), monounsaturated fatty acids (RMUFA) as well as polyunsaturated fatty acids (RPUFA) in muscle and cephalothorax TL of the examined crustaceans are presented in Table 5. While the distribution pattern of the sum of fatty acids in TFA in the examined crustaceans muscle TL as well as N. norvegicus cephalothorax TL was: PUFA > SFA > MUFA, in P. vulgaris and P. kerathurus cephalothorax TL the distribution pattern was PUFA > SFA = MUFA and SFA > PUFA > MUFA respectively (Table 5). The above data were quite similar to the ones reported by Bragagnolo and Rodriguez-Amaya (2001) for M. rosenbergii, P. brasiliensis, P. schimitti and X. kroyeri (RSFA 29–35%, RMUFA from 22% to 29%, RPUFA from 39% to 46% of TFA respectively), Bottino, Lilly, and Finne (1979) for P. aztecus (RSFA 30%, RMUFA 29% and RPUFA 41% of TFA) as well as Sriket et al. (2007) for black tiger shrimp (Penaeus monodon) and white shrimp (Penaeus vannamei) (RPUFA ranging 42.2–44.4% of TFA). The crustaceans are influenced by seasonality, showing higher saturated and unsaturated fatty acid contents in summer, whereas the origin and size of the organisms have no significant influence (Bragagnolo & Rodriguez-Amaya, 2001; Luzia et al., 2003). The PUFA/SFA ratios in the TFA of the muscle and cephalothorax TL in the examined crustaceans (ranging from 1.3 to 2.1 and from 0.8 to 1.7 respectively) were quite similar to the ones reported for freshwater prawn (M. rosenbergii) and wild marine shrimps (P. brasiliensis, P. schimitti and X. kroyeri) ranging from 1.2 to 1.5 (Bragagnolo & Rodriguez-Amaya, 2001). Erkkila, de Mello, Riserus, and Laaksonen (2008) reported that several studies have found an inverse association of the PUFA/ SFA ratio with cardiovascular outcomes, suggesting that a replacement of SFA with PUFA in the diet will decrease CVD. The quality of dietary fat in relation to cardiovascular disease forms the basis of the diet-heart hypothesis. Current recommendations on dietary fat now emphasise quality over quantity. The changes in dietary fat intake that have been occurring since the 1970s include a SFA reduction and a replacement of SFA with PUFA. The apparent protective effect of the PUFA/SAFA ratio increase supports these dietary changes in the prevention of CVD.

Table 5 Sums of SFA, MUFA and PUFA [% (w/w)] in the TL and NL of the examined crustaceans muscle and cephalothorax. Fatty acids

N. norvegicus RSFA RMUFA RPUFA Rx:3PUFA Rx:6PUFA x-3/x-6

Total lipids

Neutral lipids

Muscle

Cephalothorax

Muscle

Cephalothorax

31.34 ± 0.30Aa 27.11 ± 0.26Ab 41.54 ± 0.39Ac 34.30 ± 0.19A 7.24 ± 0.08A 4.74 ± 0.07A

30.36 ± 0.25Ba 29.77 ± 0.21Bb 39.86 ± 0.23Bc 32.47 ± 0.18B 7.39 ± 0.04A 4.39 ± 0.00B

30.73 ± 0.19ABa 34.17 ± 0.23Cb 35.10 ± 0.22Cc 28.67 ± 0.18C 6.43 ± 0.05B 4.45 ± 0.01B

35.30 ± 0.26Ca 36.93 ± 0.26Db 27.77 ± 0.23Dc 21.52 ± 0.16D 6.25 ± 0.04B 3.45 ± 0.05C

24.79 ± 0.18Aa 23.06 ± 0.26Ab 52.15 ± 0.25Ac 22.99 ± 0.15A 27.57 ± 0.12A 0.83 ± 0.01A

26.95 ± 0.29Ba 27.54 ± 0.18Ba 45.51 ± 0.23Bb 22.06 ± 0.17B 15.12 ± 0.18B 1.46 ± 0.01B

31.73 ± 0.16Ca 30.40 ± 0.21Cb 37.90 ± 0.17Cc 18.60 ± 0.08C 19.30 ± 0.14C 0.96 ± 0.00C

21.12 ± 0.15 Da 25.58 ± 0.16Db 53.30 ± 0.35Dc 18.45 ± 0.09C 9.73 ± 0.11D 1.89 ± 0.01D

30.95 ± 0.27Aa 23.54 ± 0.25Ab 45.35 ± 0.35Ac 31.77 ± 0.15A 13.58 ± 0.14A 2.34 ± 0.01A

42.40 ± 0.17Ba 22.40 ± 0.17Ab 35.10 ± 0.22Bc 22.69 ± 0.13B 12.41 ± 0.16B 1.83 ± 0.03B

50.33 ± 0.29Ca 27.99 ± 1.32Bb 21.80 ± 0.19Cc 12.87 ± 0.17C 8.93 ± 0.17C 1.44 ± 0.05C

59.99 ± 0.30 Da 25.16 ± 0.15Cb 14.79 ± 0.14Dc 9.38 ± 0.11D 4.82 ± 0.10D 1.93 ± 0.02D

P. vulgaris

RSFA RMUFA RPUFA Rx:3PUFA Rx:6MUFA

x-3/x-6 P. kerathurus

RSFA RMUFA RPUFA Rx:3PUFA Rx:6PUFA

x-3/x-6

Means in the same row bearing different capital letters differ significantly (P < 0.05). Means in the same column bearing different letters differ significantly (P < 0.05).

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As shown in Table 5, the total x-3 FA (in the TFA) of muscle and cephalothorax TL of N. norvegicus, P. vulgaris and P. kerathurus were found to be in significantly (P < 0.05) higher percentages compared to the total x-6 FA (in the TFA), except for P. vulgaris muscle TL, in which the latter was found in significantly (P < 0.05) higher percentage compared to x-3 FA (in the TFA) caused by the very high percentage of arachidonic acid (in the TFA). Similar results have been reported by Bragagnolo and Rodriguez-Amaya (2001) for M. rosenbergii, P. brasiliensis, P. schimitti and X. kroyeri (total x-3 FA ranging from 26% to 35% of TFA and total x-6 FA from 7.9% to 15% of TFA). As reported by the same authors (Sinanoglou & Miniadis-Meimaroglou, 1998), total x-3 FA in the mantle tissue TL of three Mediterranean cephalopods were also found in significantly higher percentages than total x6 FA (in the TFA) (ranging from 43.2% to 52.01% and 5.82% to 9.68% respectively). The consumption of equal quantities of the examined crustaceans results, in the case of P. kerathurus muscle, in a total x-3 fatty acid intake (calculated according to the fatty acid percentages from Table 1 and the Rx-3 proportion from Table 5), which is twice as much as the one derived from the P. vulgaris and N. norvegicus muscle respectively (Table 3). After having combined the values for x-3 PUFA (Table 3) and calculated the covering percentage of the RDA/RDI for adolescents and adults proposed by Health and Welfare Canada (1.1–1.8 g per day) as well as the one proposed by the UK Department of Health (250 mg marine x-3 PUFA per day), it appears, that the consumption of 100 g muscle tissue of langoustine, lobster and shrimp covers 8.13–13.31%, 7.62–12.47% and 16.76–27.41% respectively as well as 58.6%, 54.86% and 120.64% respectively of the x-3 PUFA RDA/RDI in the human diet. The RSFA, RMUFA, RPUFA, Rx-3 and Rx-6 percentages as well as the x-3/x-6 ratio in the TFA of N. norvegicus, P. vulgaris and P. kerathurus muscle and cephalothorax NL are shown in Table 5. P. kerathurus muscle and cephalothorax NL were found to contain significantly (P < 0.05) higher percentages of RSFA compared to the ones in TL of this shrimp as well as in the NL of both lobster and langoustine tissues. On the other hand the NL in langoustine both tissues as well as in lobster muscles were found to contain significantly higher percentages of RMUFA compared to their percentages in lobster cephalothorax as well as in shrimp muscle and cephalothorax NL. Furthermore, P. vulgaris muscle and cephalothorax NL as well as langoustine muscles NL were found to contain significantly higher percentages of RPUFA compared to the ones in P. kerathurus both tissues NL and langoustine cephalothorax NL. 4. Conclusions Careful examination of the data of the present study based on the comparative analysis of the lipid composition of three commercial crustaceans muscle and cephalothorax reveals that the FA profile of TL and NL, TAG content as well as sterols content and composition were found to differ significantly. Langoustine muscle presented a healthier lipid profile compared to lobster and shrimp, since it was found to have the highest x-3/x-6 ratio, thus providing consumers with the lowest cholesterol (74.97 mg/100 g of the wet tissue) and at the same time being a good source of x-3 fatty acids (especially EPA and DHA). Nevertheless all the examined crustaceans muscle provide the consumers with a satisfying amount of x-3 PUFA. Furthermore, cephalothorax of all of the above mentioned crustaceans (which is usually discarded) could be used effectively as a source for x-3 PUFA production since it contains significant x-3 PUFA amounts.

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