Lipid classes and fatty acids composition of the roe of wild Silurus glanis from subalpine freshwater

Food Chemistry 232 (2017) 163–168

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Lipid classes and fatty acids composition of the roe of wild Silurus glanis from subalpine freshwater Francesco Saliu ⇑, Barbara Leoni, Roberto Della Pergola Earth and Environmental Science Department, University of Milano Bicocca, Piazza della Scienza 1, Milano 20126, Italy

a r t i c l e

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Article history: Received 1 February 2017 Received in revised form 15 March 2017 Accepted 3 April 2017 Available online 4 April 2017 Keywords: Fish roe Silurus glanis Lipid Fatty acid DHA

a b s t r a c t The fat content and the fatty acid composition of the roe of European catfish (Silurus glanis) wild specimens captured in the Lambro river basin (Como, Northern Italy) were investigated in order to assess their nutritional value. Total extracted lipid values on roe weight were 5.8–6.3%. Phospholipids (40.2–43.6%) and triacylglycerols (31.8–34.7%) were the most represented lipid classes. Relevant amount of cholesteryl esters (14.3–15.6%) were also detected. The main fatty acids were palmitic, oleic and cis-4,7,10,13,16,19docosahexaenoic acid (DHA). Total saturated fatty acids were 28.1–30.9%, monounsaturated fatty acids were 28.2–30.6%, and polyunsaturated fatty acids (PUFAs) were 26.7–29.1%. The good found content of phospholipids and x3-polyunsaturated fatty acids encourage further investigation for the exploitation of Silurus glanis roe in food products and/or supplements. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Seafood consumption is nowadays widely promoted as health beneficial, mainly due to the presence of phospholipids (PL) and polyunsaturated fatty acids (PUFAs) which may be useful for the prevention of cardiovascular diseases and Alzheimer (Sahena et al., 2009). A particular emphasis is placed on the x3-PUFAs, ci s-5,8,11,14,17-eicosapentaenoic acid (EPA; C20:5-n3) and cis-4,7, 10,13,16,19-docosahexaenoic acid (DHA; C22:6-n3), which were showed to be helpful also for alleviation of rheumatoid arthritis symptoms (Innis, 2008), and the improvement of brain and eye function in infants (Connor, 2000; Ruxton, Reed, Simpson, & Millington, 2004). Under this respect, fish roe are an interesting food source, because consumed after simple curing procedure, which slightly affects their nutritional value (Bledsoe, Bledsoe, & Rasco, 2003; Rosa et al., 2009). Sturgeon caviar (‘‘black caviar”) is by far the most known example of seafood obtained from fish roe, which display a millenary history and is actually marketed at high luxury price. Similar products are obtained from the salted roe of salmon (‘‘red caviar”, known as ‘‘Ikura” in Japan), lumpfish, herring (included in various Scandinavian specialties), cod, flying fish, capelin and pollock (Shirai, Higuchi, & Suzuki, 2006). In Italy, ‘‘Bottarga” is an appreciated artisan regional specialty (Sardinia and Sicily)

⇑ Corresponding author. E-mail address: [email protected] (F. Saliu). http://dx.doi.org/10.1016/j.foodchem.2017.04.009 0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.

obtained from the whole roe pouch of mullet or tuna dried and cured in sea salt (Scano et al., 2008, 2013). Among these products, variation in the total amount of crude lipid, in the proportion of the different lipid class and in the fatty acids profile was reported (Kaitaranta & Ackman, 1981). For instance herring, capelin and cod eggs were found to be rich in polar lipids (>65%); salmon and whitefish displayed the highest content of triacylglycerols (>50%), mullet was found to be very rich in wax esters (>40%) (Bledsoe et al., 2003; Iyengar & Schlenk, 1967). Very high level of EPA and DHA were reported for trout (Mahmoud, Linder, Fanni, & Parmentier, 2008), herring (Huynh, Kitts, Hu, & Trites, 2007), and tuna (Intarasirisawat, Benjakul, & Visessanguan, 2011). All these differences affect directly the flavor and the texture of the roe products, their oxidative stability and their nutritional quality. Moreover, they must be carefully taken in account in order to perform correct risk/benefit analysis, especially when the consumption is aimed to reach the recommended EPA and DHA dietary intakes (Domingo, Bocio, Falco, & Llobet, 2007; Sidhu, 2003; WHO/FAO, 2003). To the best of our knowledge, in the current scientific literature no data are available for the roe of European catfish (Siluris glanis), the largest European freshwater fish, native from Eastern Europe rivers (Danube and Volga) and become invasive after the introduction for recreational fishing and aquaculture in various European countries, included Italy (Linhart et al., 2002). Previous studies showed a good food value for the fillets of young specimens (Ozogul, Ozogul, & Alagoz, 2007; Steffens,

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2016; Vasconi et al., 2015) and the importance of the rearing condition in order to avoid off-flavors problems (Hallier, Prost, & Serot, 2005). Exploitation of the eggs for caviar preparation is also reported for some east European countries (Copp et al., 2009) but, differently from the fillets, we were not able to find a list of international commercial producer by a web based research. Since the absolute fecundity of mature female of range from 14,600 to 354,000 eggs, (relative fecundity range 7–42 eggs g 1 wt) and the size of the eggs is generally large: 0.7–3.5 mm depending on gonad maturation, (Copp et al., 2009), the utilization on large scale should be technically feasible. In Italy European catfish is fished only for recreational purpose, and due to the growing concern about possible negative ecological impact related to its diffusion, local regulation force the anglers to do not release the fished specimens, but kill and take care of the disposal (at their expense). It is a matter of debate if promoting a change on the perception of its commercial value and encouraging a professional exploitation (for fillets, caviar or nutritional supplement production) could limit the presence and lower the pressure on the subalpine aquatic ecosystem, as observed for some population in their native habitats. Prompted by all these consideration, in this study we aimed to preliminary asses lipid and fatty acid composition of the roe of some wild specimens captured in northern Italy freshwater by means of chromatographic (TLC, GC and LC) and spectroscopic (13C NMR, MS) techniques. Results were compared to those regarding ‘‘popular” fish roe, available in the current literature. 2. Materials and methods 2.1. Sample Roes of Silurus glanis were collected from adult female specimens captured in streams and shallow lakes of the Lambro river basin (Como, Northern Italy) by using an electro-fishing boat. The sample characteristics and fish species examined in this study are shown in Table 1. Fishes were measured for fork length and body weight, placed on ice and transported to the lab for dissection. Gonads were kept at 20 °C until analysis. 2.2. Chemicals Analytical grade solvents were purchased from Sigma Aldrich (Milan, Italy). A Supelco 37 FAME mix standard solution (10 mg/ ml) from Supelco was used for FA quantitative determination. Tridecanoic acid, triolein, cholesterol, phosphatidylcholine, cholesteryl palmitate, oleyl oleate and cis-4,7,10,13,16,19-docosahexae noic acid from Sigma Aldrich (Milan, Italy) were used as reference standard. The reagents 14% BF3 in MeOH and N,O-bis(trimethylsi lyl)trifluoroacetamide (BSTFA) with trimethylchlorosilane (TMCS) 99:1 from Supelco (Bellefonte, USA) were used for the derivatization before GC analysis.

2.3. Proximate analysis and sulphur content Samples were submitted to proximate analysis for the determination of moisture, protein, fat and ash contents accordingly to AOAC methods 950.46, 928.08, 960.39 and 920.153 (AOAC, 2000). Sulphur content was determined on 2–3 mg of dry matter placed on tin capsule using a CHNS-O analyser (PerkinElmer Instruments, Milan, Italy). 2.4. Lipid extraction and lipid class analysis Lipids were extracted according to Folch procedure (Folch, Lees, & Sloane-Stanley, 1957): gonads (10 g) were minced and homogenized with 90 ml of a mixture of chloroform–methanol (2:1, by volume) containing 0.01% BHT as antioxidant for 15 min. The mixture was filtered through a filter paper (Whatman International Ltd., Maidstone, UK). The filtrate was them transferred into a separating funnel and washed three times. The collected organic phase was treated with anhydrous sodium sulphate (1–2 g) and filtered again. The solvent was then evaporated at 25 °C using a rotary evaporator (Rotavapor, model R-14, Büchi, Switzerland) and residual eliminated by flushing with nitrogen. The total amount of lipid extracted was determined gravimetrically. The constituent lipids classes were then separated by silica gel column chromatography (Kieselgel60, Merck and Co. Ltd., Darmstadt, Germany) and quantified by gravimetric recovery of the fractions. Specifically, steryl esters (SE) and wax esters (WE) were eluted with dichloromethane and n-hexane, 2:3; triacylglycerols (TAG) with dichloromethane; diacylglycerols (DAG)/monacylglycerols (MAG) with dichloromethane–diethyl ether 35:1; sterols (ST) with dichloromethane–ether 9:1, free fatty acids (FFA) with dichloromethane–methanol 10:1, phospholipids with dichlorome thane–methanol from 1:1 to 1:20. Identification was confirmed by comparison to the Rf values of authentic standards using thin-layer chromatography (TLC; thickness 0.25 mm for analysis, Kieselgel60, Merck and Co. Ltd., Darmstadt, Germany) and by ESI-MS/MS infusion analysis with a API2000 QTRAP ESI-MS/MS system (AB SCIEX, Milano, Italy). Instrument setup was heater temperature 350 °C, Ion Spray voltage = 4.0 kV, curtain gas (N2, 98%) 20 psi, nebulizer gas = 30 psi. Analyses were carried out in enhanced product ion operating mode, selected mass range 1600–150 amu, scan rate 1000 amu/s, declustering potential = 100 V, collision energy 50 V. Syringe pump flow was 10 mL/min. 2.5. Determination of fatty acid composition and total cholesterol content Separation of fatty acids, fatty alcohols and cholesterol was obtained by mild saponification (KOH, 10 N) accordingly to described method (Rosa et al., 2009).

Table 1 Description of the specimens of Silurus glanis submitted to the gonads lipid analysis. ID

Age (months)a

Weight (g)

Length (mm)

Sex

Gonad weigt (g)

Gonad maturation stageb

X2 X4 X9 X15 X23

69 69 57 21 50

4500 2000 3900 323 3200

840 620 840 375 780

F F F F F

87 36 87 54 63

III II-III IV V V

I = immature, small gonads; II = resting, ovaries firmer, smaller and lighter in colour (pale red); III = maturing, ovaries bigger and oval, tiny eggs visible with naked eyes. IV = mature, biggest ovaries, orange yellow translucent eggs clearly visible; V = ripe and running, ovaries smooth and less firm, eggs extruded with slight pressure; VI = spent, ovaries opaque and empty deflated sacs. a Estimated by the analysis of pectoral fin rays. b Estimated by morphology according to Nikolskii, 1963 (modified by Gandolfi, Zerunian, Torricell, & Marconato, 1991).

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Fatty acids were methylated to obtain the corresponding fatty acid methyl esters (FAMEs) in a screw capped vial with 1.0 mL of 14% BF3 in methanol for 30 min at 50 °C by using tridecanoic acid as internal standard. The analysis was therefore carried out with a gas chromatograph Agilent HP-6890 (Hewlett–Packard, Palo Alto, USA) equipped with a flame ionisation detector. Chromatographic separation was performed on a StabilwaxÒ (Restek Corporation, Bellefonte, USA) column (30 m, 0.32 mm ID, 0.25 mm) by using Helium as carrier gas at 2 mL/min flow rate. The oven temperature was hold at 160 °C for 3 min and then raised to 250 °C at 4 °C/min. Injector temperature was 260 °C, injection volume was 1 mL and the split ratio was 40:1. Fid temperature was set up at 300 °C, air flow 400 mL/min, H2 flow 30 mL/min. FAME were identified by comparing the retention times with those of the standard 37 component mixture. Peak area was calculated and results expressed as % of total fatty acids (FA). Cholesterol was analyzed after derivatization (Klatt, Mitchell, & Smith, 1995; Ulberth & Reich, 1992) on a HP-5 5% methyl siloxane capillary column (34 m long, ID 0.25 mm, 0.25 mm film thickness) using helium as carrier gas, 2.0 mL/min; initial temperature was 180 °C with increments of 5 C/min up to 280 °C. For both FA and cholesterol determination, analysis repeated three times and the date reported represent the average values. Actual values for all data fall within a range of 3% from the average. 2.6. GC–MS analysis Extracts obtained from the mild saponification were also submitted to a qualitative GC–MS analysis, performed as preliminary screening for the presence of organic contaminants in the lipid fraction. An Agilent 6890 chromatograph connected to an Agilent 5973 mass selective detector was used. Samples were injected using a 15:1 split ratio. The injection volume was 1.0 lL. An HP-5 fusedsilica capillary column (stationary phase 100% dimethylpolysiloxane, 30 m, 0.25-mm inner diameter, 0.25-lm film thickness) was used for chromatographic separation, using helium as a carrier gas (1.4 mL/min). The oven temperature was held at 45 °C for 4 min and then raised at 4 °C/min to a final temperature of 300 °C, then held for additional 15 min. The MS system was operated in electron impact mode with an electron voltage of 70 eV and a source temperature of 260 °C. Data were acquired in full-scan mode (50–550m/z). 2.7. LC–MS analysis Triacylglycerols were identified accordingly to previously described method (Saliu, Degano, & Colombini, 2014) on a 1200 Infinity HPLC (AgilentTechnologies, USA) with Agilent Poroshell 120 EC-C18 column (3.0 mm  50 mm, 2.7 mm), coupled by a Jet Stream ESI interface (Agilent) and a Quadrupole-Time of Flight tandem mass spectrometer 6530Infinity Q-ToF detector (Agilent Technologies).

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3. Results and discussion 3.1. Proximate composition Proximate compositions for the five sample of gonads from wild Silurus glanis are reported in Table 2. The gonads contained 47.2– 59.1% of moisture, 31.9–39.2% of protein, 5.9–6.7% of lipid and 1.2–1.4% of ash. Noteworthy, these compositions appear comparable with the composition reported for the fish roe used in the preparation of popular food products. Relative amount of total crude lipids, were found higher in chum salmon (12–20%), Caspian sturgeons (11– 18%) and pink salmon (10–15%); whereas lower values (around 4–5%) were reported for Alaska pollock, Pacific cod and herring roe (Balaswamy, Rao, Rao, Rao, & Jyothirmayi, 2009; Bledsoe et al., 2003). Also total sulphur is in line with the values found in salmon roe (Bekhit, Morton, Dawson, Zhao, & Lee, 2009) and tuna gonads (Intarasirisawat et al., 2011). Since the total sulphur is used an indicator for the organic compounds containing it, which are capable to spread off-odor when undergo degradation, Silurus glanis roe seems to be a safe lipid source also under this respect. 3.2. Lipid classes In Table 3, the lipid classes compositions obtained by column chromatography separation of the lipid extracts of Silurus glanis gonads are reported. Phospholipids were the major component by weight, accounting for 50–55% of the total lipids. Triacylglycerols were found as the second dominant lipid class, ranging from 31.9% to 39.2%. Stearyl esters, sterols and diglycerides were also represented. These compositions result very similar to those found in common fish roe used for caviar products, such as like salmon and capelin. In literature (Bledsoe et al., 2003), higher relative level of phospholipids (>70%) were reported only for few species (herring, cod, roach) while high values of WE/SE (>50%) must be considered unique since found only in few species (burbot, perch, mullet). Peak assignments on 13C NMR/ATP spectra of the lipid extracts (Table S1) performed accordingly to literature data (Falch, Storseth, & Aursand, 2006; Scano et al., 2008), and ESI-MS/MS infusion analysis on the different fractions, confirmed these composition data and give some additional information. In particular, phosphatidylcholines (Fig. S2) and cholesteryl esters (Fig. S3) displaying various fatty acyl substitutions were recognized in the WE/SE and PL fraction respectively, whereas no signals related to wax esters and phosphatidylserine were detected. Moreover, the presence of only one NMR signal ascribable to the C3 carbon atom of the steroid ring highlighted at 72.2 ppm (Table S1), suggested that the most of cholesterol was in the form of cholesteryl esters. 3.3. Fatty acid and cholesterol

2.8. NMR analysis Extracted lipids were diluted in CDCl3 and placed in 5 mm NMR tubes for the analysis. NMR experiments were performed on a Avance Bruker 500 MHz instrument (Bruker Biospin GmBH, Germany) using standard Bruker pulse sequences. The free induction decay of each spectrum was acquired with a 1s acquisition time, pulse width of 10 kHz, and 2 s relaxation delay. NOE-suppressed, proton-decoupled 13C NMR and ATP (attached proton test) spectra were recorded. 50,792 scans were collected The spectra were referenced internally to TMS. Zero filling and a line broadening of 0.3 Hz was applied prior to Fourier transformation to minimize the noise.

The fatty acid compositions and the cholesterol amounts found in the roe of the 5 specimens of Silurus glanis are reported in Table 4. All the samples showed a good content of polyunsaturated fatty acid (PUFAs, 26.7–29.1%), with a moderate amount of saturated fatty acid (SFA, 28.8–33.0%) and monounsaturated fatty acid (MUFA, 28.1–30.9). The PUFA/SFA ratio was in all the cases higher than the minimum recommended value for human diet of 0.45 (Howe, Meyer, Record, & Baghurst, 2005). Palmitic acid (16:0) was the most abundant SFA (20.5–21.8) and oleic acid (C18:1 n-9) was the main MUFA (16.3–22.1). Considerable amount of palmitoleic acid (C16:1 x-7) was also detected (4.7–7.7%).

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Table 2 Proximate analysis for the gonads of wild Silurus glanis. Lipid Class (% wet basis)

X2

X4

X9

X15

X23

Moisture Protein (%N *5.7) Lipids Ash S

50.2 32.4 6.0 1.2 0.35

59.1 33.1 5.9 1.4 0.30

50.1 31.9 6.3 1.2 0.32

47.2 39.2 6.1 1.3 0.38

50.9 32.2 6.7 1.3 0.34

Table 3 Lipid classes composition (% on extracted lipid) found in Silurus glanis gonads. Lipid Class

X2

X4

X9

X15

X23

WE/SE TAG DAG/MAG Sterols Free fatty acids Phospholipid

14.9 33.4 5.8 0.4 – 40.2

14.7 34.0 5.6 0.3 0.2 43.6

14.3 34.7 5.2 0.2 – 42.9

15.6 31.8 5.5 0.3 – 41.2

14.6 33.7 5.1 0.4 – 43.2

Table 4 Fatty acid composition in the roe samples, expressed as (%) of total FA.

a

Fatty acid

X2

X4

X15

X23

12:0 14:0 15:0 16:0 17:0 18:0 20:0 22:0 23:0 24:0 P SFA 14:1 15:1 16:1 17:1 18:1 x9 20:1 22:1 x9 24:1 P MUFA 18:2 x6 18:3 x3 18:6 x3 20:2 20:3 x3 20:3 x6 20:4 x6 (AA) 20:5 x3 (EPA) 22:2 22:6 x3 (DHA) P PUFA x6/x3 DHA/EPA PUFA/SFA Cholesterola

0.2 1.4 0.2 20.8 0.2 6.0 0.0 0.0 0.0 0.0 28.8 0.0 0.0 4.7 0.2 19.9 2.5 0.9 0.0 28.2 1.3 0.4 0.1 0.8 0.1 0.4 3.8 5.9 0.0 13.9 26.7 0.27 2.36 0.93 635

0.1 1.0 0.2 22.5 0.3 6.2 0.0 0.0 0.0 0.0 30.3 0.0 0.0 4.9 0.2 20.4 3.0 0.7 0.0 29.2 1.6 0.5 0.1 0.7 0.1 0.3 4.0 6.1 0.0 13.6 27.0 0.29 2.23 0.89 597

0.1 1.8 0.3 20.5 0.4 9.7 0.1 0.0 0.1 0.0 33.0 0.1 0.0 7.7 0.3 16.3 2.9 0.8 0.0 28.1 1.2 0.5 0.1 0.8 0.1 0.4 3.9 7.0 0.0 13.4 27.4 0.26 1.91 0.83 652

0.2 1.6 0.2 21.8 0.2 6.1 0.0 0.0 0.0 0.0 30.1 0.0 0.1 5.0 0.2 22.1 2.6 0.9 0.0 30.6 1.6 0.4 0.1 0.8 0.1 0.4 3.9 5.8 0.0 15.9 29.1 0.26 2.74 0.96 628

Expressed as mg/100 g of roe.

Among PUFAs, DHA was the most represented (14.4–15.9%) with EPA (5.8–7.0%) and arachidonic acid (3.8–4.0%). The total content of x6 fatty was particular low, as a typical for fish roes, with a x6/x3 ratio less than 0.30. The DHA/EPA ratio was comprised between 1.91 and 2.71%. These fatty acid compositions were similar to the composition found in fish roe used for seafood preparation. Comparable level of palmitic acid, palmitoleic acid and oleic acid were in fact reported for sturgeon (Nieminen, Westenius, Halonen, & Mustonen, 2014) and salmon (Bekhit et al., 2009). In addition,

the relative content of DHA and EPA was comparable to the one found in sturgeon (Nieminen et al., 2014) and herring roe (Huynh et al., 2007). Stearic acid and arachidonic acid relative values were higher than in sturgeon, but comparable to the values reported for tuna roes (Intarasirisawat et al., 2011). In summary, due to the good content of x3-PUFA and the proportion in x6/x3, the roe of European catfish results particular interesting as a source of these nutrients for the associated health benefits. Total cholesterol levels, however, were particularly high: 597– 652 mg/100 g roe, which correspond approximately 10–11% of

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Fig. 1. A = Representative base peak chromatogram obtained from LC-ESI-QTOF analysis of Silurus glanis gonads lipid extract (sample x23). B = Mass Spectrum of (ODhO). Molecuar ion [ODhO + Na]+ at m/z = 953.75, and related fragments [OO]+ at m/z = 603.53; [OO + Na]+ at m/z = 625.51, [ODh]+ at m/z = 649.52, [ODh + Na]+ at m/z = 671.50 are evidenced. C = Mass spectrum of PnOO. Molecuar ion [PnOO + Na]+ at m/z = 879.74, and related fragments [PnO]+ at m/z = 575.50, [PnO + Na]+ at m/z = 597.48, [OO]+ at m/ z = 603.53, [OO + Na]+ at m/z = 625.51 are evidenced. Positional assignment are tentatively given on the basis of the detected DAG and RCO+ fragments.

total lipid. These values are similar to those found in other fish roes such as salmon, cod and tuna, which are in general very rich in cholesterol, either in the free and ester form (Ortega & Mourente, 2010). Since the recommendation for cholesterol intake is do not exceed 300 mg/day (Wardlaw & Insel, 1996), the use of Silurus glanis roe for x3-PUFAs integration seems to be more likeable after the application of an enrichment procedure, such as like is currently operated for the preparation of molecular distilled squid oil supplements (Liang & Hwang, 2000; Saliu, Longhin, Salanti, Degano, & Della, 2016). 3.4. LC–MS LC–MS analysis of the lipid extracts showed a variety of TAGs, with ECN number comprised between 38 and 54 (Table S2). Due to the high content of polyunsaturated species, the precise specification of the constitutional features of TAGs in fish oil and similar materials is still considered a challenge (Murphy & Axelsen, 2011). However for some well resolved TAG, the recognition of the constituents fatty acids and also of their position in the glycerol backbone was enabled by exploiting the preferred fragmentation pathway obtained by the high resolution low-energy CID tandem mass spectrometry (Fig. 1). In particular, we have been able to recognize TAGs derived from palmitic, oleic, steraric, palmitoleic and x3-PUFAs.

non targeted screening for the presence of organic contaminants and toxin. Being Silurus glanis at the higher levels of the food web, targeted specific study regarding the possible presence of contaminants, in relationship with the provenance from different fishing areas, are needed. No data for the gonads are available in the current literature. 4. Conclusions Silurus glanis roes from northern Italy were submitted to analysis for the determination of lipid content. Results showed interesting amount of x3-polyunsaturated fatty acids which are widely considered beneficial in human diet especially in the prevention of cardiovascular diseases. Pospholipid and triacylglycerol were the most represented lipid classes. High levels of cholesterol, mainly in the cholesteryl ester form were also found. Acknowledgements The work was supported by University of Milano Bicocca - FAR 2015. We thank Dr Alessandro Marieni (Centro Studi Biologia e Ambiente) for technical support during Silurus glanis capture operation and our students Monica Moreschi, Daniele Sanvito, Simone Cornago, Alice Wappner and Lucia Scarduelli for help in the experiments.

3.5. GC–MS analysis

Appendix A. Supplementary data

No organic species different from fatty acids, fatty alcohols and cholesterol were highlighted trough the qualitative full scan GC– MS on the saponified lipid extracts, performed as preliminary

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2017. 04.009.

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