13C NMR, GC and HPLC characterization of lipid components of the salted and dried mullet (Mugil cephalus) roe “bottarga”

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Chemistry and Physics of Lipids 151 (2008) 69–76

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C NMR, GC and HPLC characterization of lipid components of the salted and dried mullet (Mugil cephalus) roe “bottarga” P. Scano a,∗ , A. Rosa b , F. Cesare Marincola a , E. Locci a , M.P. Melis b , M.A. Dess`ı b , A. Lai a b

a Dipartimento di Scienze Chimiche, Universit` a di Cagliari, Cagliari, Italy Dipartimento di Biologia Sperimentale, Universit`a di Cagliari, Cagliari, Italy

Received 5 June 2007; received in revised form 27 September 2007; accepted 2 October 2007 Available online 7 October 2007

Abstract 13

C NMR spectroscopy, in conjunction with HPLC and GC techniques, has been used to study the molecular composition of lipids extracted from commercial products of bottarga. To this goal, both the saponifiable and unsaponifiable fractions of lipid extracts were also examined by 13 C NMR. Among the major lipid classes wax esters (WE) showed a concentration of more than 50 mol%, triacylglycerols (TAG) and phospholipids (PL) represented a minor fraction. Concentrations up to 29 mol% of free fatty acids (FFA) were found. The most represented fatty alcohol was 16:0 that accounted for more than 50%, among fatty acids the most represented were 16:1 n-7, 22:6 n-3, 18:1 n-9, 16:0, and 20:5 n-3, in particular the n-3 polyunsaturated fatty acids (PUFA) averaged 40 mg/g of the edible portion. 13 C NMR spectroscopy put in evidence that cholesterol was present in its free and esterified forms and its total content was measured as ca. 10 mg/g of the edible portion. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Fish roe; Bottarga; NMR; Lipids; Wax esters; n-3 PUFA

1. Introduction A wide spectrum of fish roe products are consumed throughout the world. Among them, mullet (Mugil cephalus) roe is regarded as a delicacy. The salted and dried products are known as “karasumi” in Japanese and “bottarga” in Italian (Bledsoe et al., 2003). Among the Mediterranean countries, Sardinia (Italy) has a long tradition in making a high quality bottarga that is becoming increasingly popular in the international market. Bottarga is the final product of a number of treatments on the ovaries of mullets. To obtain a good bottarga is an art that requires a great deal of skills; nowadays different procedures exist, from the more traditional to the industrial ones; in the latter drying is conducted in rooms with controlled humidity and temperature. The final product can be sold as whole ovaries

∗ Corresponding author at: Dipartimento di Scienze Chimiche, Universit` a di Cagliari, Cittadella Universitaria di Monserrato, S.S. 554 Bivio per Sestu, 09042, Monserrato, Cagliari, Italy. Tel.: +39 070 675 4391; fax: +39 070 675 4388. E-mail address: [email protected] (P. Scano).

0009-3084/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2007.10.001

under vacuum packaging, or grated in jar. Bottarga has an amber colour and its unique chewy mouth feel is due to the peculiar lipid composition, rich in WE (Bledsoe et al., 2003). To date, a limited number of investigations have been performed on the lipid composition of fresh and/or salted and dried mullet roes. The proximate lipid content of fresh mullet roes was estimated to be 13.7%, and in the salted derivates, because of the drying process, this value was observed to increase to 23.7% (Lu et al., 1979). The lipid composition of fresh ovaries of mullets caught in the Gulf of Mexico was extensively studied by liquid and gas chromatographic methods (Iyengar and Schlenk, 1967). It was found that the extracted lipids exhibit a high portion of WE (ca. 60–70%), that follow the general rule of marine WE (Nevenzel, 1969; Joh et al., 1995), i.e. are composed by saturated and unsaturated fatty acids, esterified mainly to saturated and monounsaturated fatty alcohols of C14 to C18 chain-length. In addition, TAG, PL, cholesterol (Cho), cholesterol esters (CE), and free fatty acids and alcohols were found in the lipid extracts (Iyengar and Schlenk, 1967). Moreover, the fatty acids composition of salted mullet roe was reported by Lu et al. (1979), and recently, the fatty acids and the fatty alcohols composition of only the WE fraction in commercial prepara-

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tions of bottarga has been studied by GC (Bernasconi et al., 2007). To our knowledge, an exhaustive study of the lipid composition of salted and dried mullet roes is lacking from the literature. To this goal, we used the high resolution 13 C NMR spectroscopy together with HPLC and GC techniques to investigate the lipid extracts of different commercial samples of bottarga produced in Sardinia. The NMR technique was used to obtain semiquantitative and multi-component information about the lipid composition of the extracted oil, avoiding invasive manipulations of the sample. The potentiality of this technique in the study of the molecular components of complex lipid systems has been successfully demonstrated (Casu et al., 1991; Sacchi et al., 1993; Aursand et al., 1993, 2007; Pollesello et al., 1996; Scano et al., 1999, 2005, 2006; Siddiqui et al., 2003; Gribbestad et al., 2005; Falch et al., 2006), and recently biochemical changes in cod gonads during storage were studied by 1 H and 13 C NMR (Falch et al., 2007). The GC and HPLC analyses of the unsaponifiable and saponifiable fractions of the extracted oil provided detailed information on the individual fatty acids, fatty alcohols and cholesterol in bottarga. In parallel, both fractions were also analyzed by NMR, in order to support, besides the GC data, the assignment of resonances in the NMR spectrum of the pure lipid matrix. 2. Materials and methods 2.1. Samples Commercial products of grated mullet roe (bottarga di muggine) manufactured by two different companies located in Sardinia (Italy) were acquired at a local supermarket. Ingredients were reported in the label as mullet roe and salt. 2.2. Chemicals All solvents used, of the highest available purity, were purchased from Merck (Darmstadt, Germany). Deuterated chloroform (CDCl3 ), l-␣-phosphatidylcholine, oleyl oleate, triolein, fatty alcohols and fatty acids methyl esters standard compounds were purchased from Sigma–Aldrich (Milan, Italy). The reagents 14% BF3 in MeOH and hexamethyldisilazane (HMDS)–trimethychlorosilane (TMCS)–pyridine (3:1:9) were purchased from Supelco (Bellefonte, USA). Desferal (deferoxamine methanesulfonate) was purchased from CIBA-Geigy (Basel, Switzerland). All the other chemicals used in this study were of analytical grade. 2.3. Lipid extraction and preparation of cholesterol, fatty alcohols and fatty acids Lipids were extracted from grated mullet bottarga by the Folch et al. (1957) procedure. An aliquot of total lipid extract recovered from the lower chloroform phase was analyzed by 13 C NMR.

The total lipids were quantified by the method of Chiang et al. (1957). Separation of cholesterol, fatty alcohols and fatty acids was obtained by mild saponification. A 5 mg portion of lipids from each sample was dissolved in 5 ml of ethanol and 100 ␮l of Desferal solution (25 mg/ml of H2 O), 1 ml of a water solution of ascorbic acid (25%, w/v), and 0.5 ml of 10N KOH were added. The mixtures were left in the dark at room temperature for 14 h. After addition of 10 ml of n-hexane and 7 ml of H2 O, samples were centrifuged for 1 h at 900 × g (Rosa et al., 2005). The hexane extract containing the unsaponifiable fraction (cholesterol and fatty alcohols) was collected and the solvent was evaporated; an aliquot was stored at −20 ◦ C until NMR analysis. A portion of the residue was dissolved in 1 ml of MeOH and injected into the HPLC system. Aliquot of dried fatty alcohols was converted to trimethylsilyl ether by a mixture of TMCS, HMDS, and anhydrated pyridine (1:3:9, v/v/v) (200 ␮l) for 2 h at room temperature, and trimethylsilyl ethers were applied to capillary gas chromatography. After addition of further 10 ml of n-hexane to the mixtures, samples were acidified with 37% HCl to pH 3–4 and then centrifuged for 1 h at 900 × g. The hexane extract with fatty acids, i.e. the saponifiable phase, was collected and after solvent evaporation an aliquot was stored at −20 ◦ C until NMR analysis. A portion of the dried residue was dissolved in 1 ml of CH3 CN with 0.14% (v/v) CH3 COOH and aliquots of the samples were injected into the HPLC system. Aliquot of dried fatty acids was methylated with 1 ml of 14% BF3 in MeOH (Christie, 1993) for 30 min at room temperature. After addition of 4 ml of nhexane and 2 ml of H2 O, samples were centrifuged for 20 min at 900 × g. The hexane phase with fatty acids methyl esters was collected, the solvent was evaporated, the residue was dissolved in 250 ␮l of n-hexane and aliquots of the samples were injected into the GC system. The recovery of fatty acids, fatty alcohols, and cholesterol was calculated by using an external standard mixture. All solvents evaporation was performed under vacuum. 2.4. HPLC analyses Analyses of cholesterol and unsaturated fatty acids were carried out with a Agilent Technologies 1100 liquid chromatograph (Agilent Technologies, Palo Alto, USA) equipped with a diode array detector. Cholesterol, detected at 203 nm, was measured using a Chrompack column (Chrompack, Middelburg, The Netherlands), Inertsil 5 ODS-3, 150 mm × 3 mm, and MeOH as mobile phase, at a flow rate of 0.4 ml/min. Analyses of unsaturated fatty acids and conjugated dienes fatty acids hydroperoxides (HP), detected at 200 and 234 nm, respectively, were carried out with a Chrompack column, Inertsil 5 ODS-2, 150 mm × 4.6 mm with a mobile phase of CH3 CN/H2 O/CH3 COOH (70/30/0.12, v/v/v) at a flow rate of 1.5 ml/min. The identification of cholesterol, fatty acids, and HP was made using standard compounds and second derivative as well as conventional UV spectra, generated using the Agilent Chemstation A.10.02 software (Rosa et al., 2005).

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2.5. GC analyses Fatty acids methyl esters and trimethylsilyl derivatives of fatty alcohols were measured on a gas chromatograph Hewlett-Packard HP-6890 (Hewlett-Packard, Palo Alto, USA) with flame ionization detector and equipped with a cyanopropyl methyl-polysiloxane HP-23 FAME column (30 m × 0.32 mm × 0.25 ␮m) (Hewlett-Packard). Nitrogen was used as carrier gas at a flow 2 ml/min. The oven temperature was set at 175 ◦ C, injector temperature 250 ◦ C, and detector temperature 300 ◦ C. The fatty acids methyl esters and trimethylsilyl derivatives of fatty alcohols were identified by comparing the retention times with those of standard compounds. The percentage composition of individual fatty acids and alcohols were

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calculated using a calibration curve with components injected at different concentrations, using the Hewlett-Packard A.05.02 software. 2.6. NMR analysis The extracted lipids (300 mg), the dried saponifiable and unsaponifiable fractions (100 mg each) were collected and separately dissolved in 0.6 ml of CDCl3 . The samples were placed in 5 mm NMR tubes, stored at −20 ◦ C, and analyzed within 2 days. NMR experiments were performed at 25 ◦ C on a Varian VXR-300 and on a Varian UNITY INOVA 400 spectrometers, operating at the frequency of 75.42 and 100.56 MHz for carbon, respectively. NOE-suppressed, proton-decoupled 13 C

Fig. 1. The 75 MHz 13 C NMR spectra (180–0 ppm) with interpretation of the more relevant resonances of (a) bottarga lipid extract, with enlargement of the spectral regions between 178.0 and 176.5, 75.0 and 52.5 ppm; (b) saponifiable and (c) unsaponifiable fractions of the extract. All samples are diluted in CDCl3 .

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NMR spectra were recorded. The free induction decay of each spectrum was acquired with a 2 s acquisition time, a sweep width of 26 kHz, a 45◦ pulse angle and a 20 s relaxation delay, 10240 scans were collected. Zero filling and a line broadening of 0.3 Hz were applied prior to Fourier transformation to minimize the noise, but not at expense of resolution. 13 C spectra of reference standard compounds were also obtained using CDCl3 as solvent. All chemical shifts, cited in ppm, were referred to tetramethylsilane (TMS). Compositional data of the two products, reported as mol% of the total lipid extracted, were expressed as means and standard deviations over six samples of each bottarga product. 3. Results and discussion 3.1.

13 C

NMR analysis of the extracted lipids

A representative 13 C NMR spectrum of the lipid extracts from bottarga is reported in Fig. 1a. Peak assignments were performed with the aid of literature data (Gunstone, 1993; Pollesello et al., 1996; Siddiqui et al., 2003; Scano et al., 2006; Christie,

2007) and by recording spectra of standard compounds. In some cases, validation of the peak attribution was achieved by adding standard compounds to the lipid solution and re-recording the NMR spectrum under the same conditions. The most significant assignments for each class of compounds are described below. 3.1.1. Carbonyl region (190–160 ppm) In this spectral region the peaks in the range 180–177 ppm were attributed to carboxyl carbons of FFA (docosahexaenoic acid DHA, eicosapentaenoic acid EPA, and other fatty acids, see inset Fig. 1a). The intense group of signals centred at 173.84 belongs to the carbonyl carbons in WE, while the remaining peaks between 173.7 and 172.0 ppm were assigned to carbonyl carbons of PL, TAG, and CE. 3.1.2. Olefinic region (140–120 ppm) In Fig. 2a an expanse of the olefinic region between 132 and 126 ppm is reported. Here, the intense signals at 131.97 and 126.97 ppm were attributed to the ␻-3 and ␻-4 carbon atoms in n-3 PUFA (in their acid or alcoholic form), respectively. The remaining olefinic carbons of n-3 PUFA resonate between 129.5

Fig. 2. Olefinic region (132.5–126.5 ppm), with interpretation of the more relevant resonances, of the 75 MHz saponifiable and (c) unsaponifiable fractions of the extract. All samples are diluted in CDCl3 .

13 C

NMR spectra of (a) bottarga lipid extract, (b)

P. Scano et al. / Chemistry and Physics of Lipids 151 (2008) 69–76

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Fig. 3. 13 C NMR spectrum of bottarga lipid extract in CDCl3 after the addition of DHA (22:6 n-3). Enhanced peaks with respect to the spectrum of the original lipid matrix, indicated by numbers, are assigned to the following functional groups of DHA: peak 1 belongs to the carboxyl carbon, peaks 2–9 to olefinic carbons and in particular peaks 3 and 8 to C4 and C5, respectively. Peak 10 was assigned to C2, peak 11 to CH CH2 CH , peak 12 belongs to C3, peak 13 and peak 14 to ␻-2 and ␻-1 carbons, respectively.

and 127.3 ppm. In this spectral region, signals from the free and the esterified form of DHA and EPA were identified by spiking the sample with authentic standard compounds (as an example the 13 C NMR spectrum of a sample of extracted oil with the addition of DHA is reported in Fig. 3). In particular, the resonances at 129.16 and 127.67 ppm were assigned to C4 and C5 of free DHA respectively (Aursand and Grasdalen, 1992), while the signal at 129.38 ppm corresponds to C5 of the esterified DHA. The signals at 128.95 and 128.74 ppm were attributed to C5 and C6 of free EPA and the peaks at 128.89 and 128.79 ppm were ascribed to esterified EPA. The two peaks at 130.42 and 127.50 ppm were ascribed to the ␻-6 and ␻-7 olefinic carbons of arachidonic acid (AA) and of other long chain n-6 PUFA, the low intensity of these signals indicating a small amount of n-6 PUFA in the lipid mixture. In the region between 130.0 and 129.5 ppm, characteristic of monounsaturated fatty acids (MUFA), the two main groups of signals centred at 129.94 and 129.69 ppm were ascribed to C9 and C10 of the 9 monoenes, respectively, and the peaks at 129.87 and 129.79 ppm were assigned to C11 and C12 of the 11 (Scano et al., 1999). At lower fields with respect to the resonances ascribable to the olefinic carbons of fatty acids and alcohols, two small peaks at 140.68 and 139.54 ppm were observed (Fig. 1a) and assigned to the C6 involved in the double bond of the cyclopentaphenanthren-3-ol ring of cholesterol in its free and esterified form, respectively. In parallel, the high field resonances at 122.47 and 121.48 ppm were ascribed to the C5 olefinic carbons of CE and Cho, respectively (Pollesello et al., 1996; Falch et al., 2007). The chemical shift differences of C5 and C6 in CE compared to Cho are due the different chemical environment induced by the nearby ester bond in C1 position. 3.1.3. N CH , O CH region (75–53 ppm) In this spectral region the signals from aliphatic carbons directly bound to an oxygen or to a quaternary nitrogen appear. These resonances are representative of all the main classes of fatty compounds and were then used for quantitative purpose. The most prominent cluster of peaks centred at 64.46 ppm

belongs to the O CH2 groups of the alcoholic moiety of WE. The peaks at 68.80 and 61.98 ppm were attributed to the O CH and O CH2 functional groups of TAG, respectively, while the N (CH3 )3 of the choline head group in phosphatidylcholine (PC) resonates at 54.38 ppm. Finally, the small peaks at 71.56 and 73.57 ppm were ascribed to the C1 carbon atom of Cho and CE, respectively. 3.1.4. Up-field aliphatic region (35–11 ppm) In this spectral region the peak at 28.57 ppm was attributed to the O CH2 CH2 group of WE and the well-resolved signal at 20.45 ppm to the ␻-2 methylene group of all the n-3 fatty acids and alcohols. The signal at 11.79 ppm was ascribed to the CH3 group (C18) of Cho and CE. No signal characteristic of trans double bonds in fatty acids at ∼30 and ∼32 ppm (Christie, 2007) was found, according to previous data obtained by infrared spectroscopy (Iyengar and Schlenk, 1967). 3.2. Semi-quantitative NMR data Table 1 shows the composition of the major lipid classes estimated by integration of suitable 13 C NMR resonances of the extracted oil. All samples showed similar contents of TAG, Table 1 Lipid class composition of bottarga products calculated from the integrated areas of 13 C NMR spectra Percentage of total lipids (mol%)a Product 1 WE TAG FFA Cho CE PC

63.6 6.7 19.4 2.7 1.5 6.1

± ± ± ± ± ±

2.7 0.8 3.9 0.4 0.7 1.1

Product 2 51.2 8.7 28.8 3.6 1.9 5.8

± ± ± ± ± ±

0.7 0.6 0.5 0.1 1.2 0.2

Relative contents were measured from normalized areas of the following peaks: 64.46 ppm for WE, 61.98 ppm for TAG, range 180–177 ppm for FFA, 71.56 ppm for Cho, 73.57 ppm for CE, 54.38 ppm for PC. a Mean and standard deviation over six samples.

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Cho, CE and PC, while different quantity of FFA and WE were detected in the samples of product 1 with respect to those of product 2. The high FFA content (means of 19.4 and 28.8 mol%, for products 1 and 2, respectively) found in all samples is to be ascribed to hydrolysis processes on the esterified molecules of the original lipid matrix during manufacturing and storage, according to previous investigations on fish roes (Basby et al., 1998; Falch et al., 2007). Therefore, due to the high WE content of bottarga lipids, in addition to the release of FFA from the original lipid matrix also the free fatty alcohol counterparts from the hydrolysis processes are to be expected. However, due to the marked overlapping, the typical resonances of free alcoholic derivatives were not identified in the 13 C NMR spectra, but in the corresponding 1 H NMR spectra (spectra not shown) at 3.62 ppm a triplet signal appears, attributed to the HO CH2 CH2 group of free fatty alcohols. On the other hand, NMR signals originating from residual fractions of TAG and PC after their hydrolysis were below detection. 3.3. 13 C NMR spectra of the saponifiable and unsaponifiable fractions Further information on the molecular composition of the lipid extracts of bottarga were gained from the analysis of the 13 C NMR spectra of both the saponifiable (Figs. 1b and 2b) and the unsaponifiable (Figs. 1c and 2c) fractions. As expected, in the former spectrum fatty acids from hydrolysis of WE, TAG, PL, and CE, together with the original FFA were detected, while in the latter spectrum the alcoholic lipid derivatives, i.e. Cho and the alcoholic moieties of WE, were found. From the comparison of these spectra with that of the pure extracted lipids (Figs. 1a and 2a) the following considerations can be drawn: • The carbonyl region of the saponifiable matter exhibited an enhancement of resonances from FFA (180–177 ppm), while, as expected, no carbonyl peaks were observed in the spectrum of the unsaponifiable fraction. • In the olefinic region of the unsaponifiable fraction only the typical resonances (spectral range between 130.0 and 129.5 ppm, Fig. 2c) of monoenes were found. The saponifiable fraction showed, in addition to MUFA, also a number of resonances ascribed to PUFA, among which DHA and EPA, were clearly detectable. Furthermore, the unsaponifiable fraction lacked in the olefinic signals of CE detected in Fig. 1a, while the characteristic resonances at 140.71 and 121.65 ppm of C6 and C5 in Cho were enhanced. • In the unsaponifiable fraction the intense peak at 63.09 ppm (Fig. 1c) was ascribed to the HO CH2 functional group of fatty alcohols, exhibiting an up-field shift with respect to the corresponding peak of the esterified form (64.46 ppm in Fig. 1a); in parallel, the signal at 32.80 ppm, attributed to the HO CH2 CH2 group, resulted down-field shifted as compared to the corresponding esterified form at 28.57 ppm. These attributions are in agreement with previous studies (Gunstone, 1993).

Table 2 Fatty alcohols composition (%) of bottarga products by GC Fatty alcohol

Product 1

14:0 15:0 16:0 18:0 16:1 n-7 18:1 n-7 18:1 n-9

10.9 3.1 52.8 6.8 10.4 3.3 3.9

SFA MUFA

73.6 ± 0.5 17.7 ± 0.2

± ± ± ± ± ± ±

0.1 0.0 0.4 0.1 0.1 0.1 0.1

Product 2 7.3 3.3 51.7 7.9 9.4 3.6 4.4

± ± ± ± ± ± ±

0.1 0.2 0.6 0.1 0.3 0.1 0.1

70.2 ± 0.5 17.3 ± 0.4

SFA, saturated fatty alcohols; MUFA, monounsaturated fatty alcohols. Mean and standard deviation over six samples.

3.4. Fatty alcohols and fatty acids analyses by GC Quali-quantitative information on the individual fatty acids and fatty alcohols that compose the major lipid classes of bottarga were obtained by GC analysis. The analyzed samples exhibited similar fatty acids and alcohols profiles. The content of fatty alcohols present in the unsaponifiable matter is reported in Table 2 as percentage of the total amount of alcohols. There was no considerable variation between the analyzed samples. As found in previous investigations (Iyengar and Schlenk, 1967; Bernasconi et al., 2007) only saturated (C14 to C18 chain-length) and monounsaturated (16:1 and 18:1 isomers) alcohol derivatives were found, in particular, the samples were characterized by a high amount of 16:0 (about 52%). The composition of fatty acids present in the saponifiable matter, that comprise the original FFA and those derived from induced hydrolysis of the intact molecules, of the two bottarga products are shown in Table 3 and expressed as percentage of total fatty acids. They showed a concentration of approximately 16–18% of saturated fatty acids (mainly 16:0), 35% of MUFA (mainly 16:1 n-7, and 18:1 n-9), and 34-37% of PUFA. In particular, the total content of the n-3 derivatives EPA and DHA amounted to ca. 22%. Low concentrations of n-6 fatty acids (4–5%) were found, thus resulting in an elevate (n-3)/(n-6) ratio, in agreement with previous works on fish roes (Wiegand, 1996; Falch et al., 2006). Finally, the very low contents of 20:1 and longer monounsaturated fatty acids and alcohols found in the samples suggest that the strong selection against the incorporation of these monoenes in fish eggs (Wiegand, 1996) exists also in mullet roes. 3.5. Main lipid characteristics An average lipid content of 270 mg/g of the edible portion for bottarga samples was measured. By HPLC, the total cholesterol was measured as mean content of 9.3 and 10.5 mg/g of the edible portion for samples of products 1 and 2, these values represented ca. 3–4% of total lipids, that is, a concentration lower than in chicken egg (Yannakopoulos et al., 2005). Furthermore, the highly unsaturated fatty acids n-3 content in the edible portion was detected as follows: 20.5 and 23.2 mg/g of DHA, 13.5 and 9.9 mg/g of EPA, and a minor amount of 22:5 n-3 (9.7 and

P. Scano et al. / Chemistry and Physics of Lipids 151 (2008) 69–76 Table 3 Fatty acids composition (%) of bottarga products by GC Fatty acid

Product 1

Product 2

12:0 14:0 15:0 16:0 18:0 20:0 16:1 n-7 18:1 n-7 18:1 n-9 20:1 n-9 16:2 16:3 16:4 18:2 n-6 18:3 n-3 18:3 n-6 18:4 n-3 20:3 n-3 20:3 n-6 20:3 n-9 20:4 n-6 20:5 n-3 22:4 n-6 22:5 n-3 22:6 n-3

0.03 ± 0.01 2.07 ± 0.11 0.44 ± 0.08 10.90 ± 0.19 3.18 ± 0.12 0.15 ± 0.07 17.92 ± 0.43 7.22 ± 0.20 10.61 ± 0.13 0.16 ± 0.02 0.70 ± 0.03 1.46 ± 0.14 1.98 ± 0.45 1.27 ± 0.04 0.57 ± 0.02 0.40 ± 0.01 1.42 ± 0.03 Trace 0.25 ± 0.05 0.11 ± 0.05 1.69 ± 0.08 9.40 ± 0.17 0.49 ± 0.06 5.95 ± 0.12 11.62 ± 0.20

0.04 ± 0.01 1.75 ± 0.06 0.52 ± 0.02 12.69 ± 0.28 3.07 ± 0.07 0.14 ± 0.08 14.71 ± 0.35 6.23 ± 0.11 13.70 ± 0.18 0.37 ± 0.04 0.56 ± 0.02 0.63 ± 0.18 Trace 1.58 ± 0.14 0.97 ± 0.05 0.38 ± 0.08 1.16 ± 0.05 0.18 ± 0.03 0.15 ± 0.00 0.29 ± 0.15 2.21 ± 0.10 6.73 ± 0.31 0.48 ± 0.14 3.96 ± 0.14 15.28 ± 1.03

SFA MUFA PUFA

16.77 ± 0.27 35.90 ± 0.32 37.27 ± 0.36

18.22 ± 0.43 35.01 ± 0.27 34.47 ± 1.48

SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Mean and standard deviation over six samples.

by HPLC as HP, was comparable to that of fresh marine oils (Alamed et al., 2006). To note, FFA can originate from hydrolysis processes on the original lipid matrix and previous studies on fresh (Falch et al., 2007) and salted (Basby et al., 1998) fish eggs demonstrated that the extent of this deterioration process is strongly influenced by storage conditions. This finding can represent a serious drawback when either the fresh roe or the final bottarga products are submitted to long-term storage. Due to the high content of WE in lipid mixture valuable information were also given by the analysis of the 13 C NMR spectra of both the saponifiable and unsaponifiable matters, and the comparison with the more detailed HPLC and GC data allowed a complete characterization of the fatty alcohols and fatty acids that compose the WE, TAG, PL, and CE. The two commercial products here examined show similar composition of fatty acids and alcohols as calculated by GC technique. Semi-quantitative NMR data indicate a different content of WE and FFA, this result suggests that probably product 2 underwent major hydrolysis process compared to product 1. The bottarga samples were found to be rich in n-3 PUFA, as DHA and EPA, that have been recognised as having an important role in health (Ruxton et al., 2004). Considering the high content of WE in bottarga, we can suppose that a significant amount of these n-3 fatty acids are WE components, as previously observed (Bernasconi et al., 2007). In the light of a recent study that demonstrated that WE enriched in n-3 fatty acids have a low degree of susceptibility to oxidation (Gorreta et al., 2002), bottarga may be regarded as stable natural source of health beneficial n-3 fatty acids.

5.5 mg/g), as mean values over six samples for products 1 and 2, respectively. The oxidation status of fatty acids was also measured by HPLC detection of HP. The level of HP in the bottarga samples was ca. 0.1 ␮mol/g of the edible portion (0.4 nmol/mg of lipid).

Acknowledgments

4. Conclusions

References

In the present work, by the conjunct use of NMR and chromatographic techniques, we have exhaustively studied the major intact lipid classes and the individual fatty acids and alcohols of the lipid extracted from commercial products of bottarga manufactured in Sardinia. We have assigned the most important resonances in the 13 C NMR spectrum of this complex lipid mixture and the major lipid classes were measured as mol%. The more represented lipid components were WE. In agreement with previous studies on fish eggs (Bledsoe et al., 2003; Falch et al., 2006) among the PL classes, only PC was found. Besides cholesterol, also cholesterol esters, probably formed during storage of the fresh eggs (Falch et al., 2007), were detected in the 13 C NMR spectra. Furthermore, a relatively high quantity of FFA was reported, and the presence of DHA and EPA in their free form can be inferred from the occurrence of specific resonances in the carbonyl, olefinic and aliphatic spectral regions of the 13 C NMR spectra. In spite of the high content of FFA the level of oxidative products, measured

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We thank Nicoletta Zinnarosu for her expert NMR technical assistance, and Stefano Rocca for helpful discussions on manufacturing mullet roes.

Alamed, J., McClements, D.J., Decker, E.A., 2006. Influence of heat processing and calcium ions on the ability of EDTA to inhibit lipid oxidation in oil-inwater emulsions containing omega-3 fatty acids. Food Chem. 95, 585–590. Aursand, M., Grasdalen, H., 1992. Interpretation of the 13 C spectra of omega-3 fatty acids and lipid extracted from white muscle of Atlantic Salmon (Salmo salar). Chem. Phys. Lipids 62, 239–251. Aursand, M., Rainuzzo, J.R., Grasdalen, H., 1993. Quantitative high-resolution 13 C and 1 H nuclear magnetic resonance of ␻-3 fatty acids from white muscle of Atlantic Salmon (Salmo salar). JAOCS 70, 971–981. Aursand, M., Standal, I.B., Axelson, D.E., 2007. High-resolution 13 C nuclear magnetic resonance spectroscopy pattern recognition of fish oil capsules. J. Agric. Food Chem. 55, 38–47. Basby, M., Jeppesen, V.F., Huss, H.H., 1998. Chemical composition of fresh and salted Lumpfish (Cyclopterus lumpus) roe. J. Aquat. Food Prod. Technol. 7, 7–21. Bernasconi, R., Bolzacchini, E., Galliani, G., Gugliersi, F., Rindone, B., Rindone, M., Tacconi, M.T., Terraneo, A., 2007. Determination of the content of wax esters in some sea food and their molecular composition. A comparison with ␻-3 enriched wax esters. Lebensm-Wiss. Technol. 40, 569–573. Bledsoe, G.E., Bledsoe, C.D., Rasco, B., 2003. Caviars and fish roe products. Crit. Rev. Food Sci. Nutr. 43, 317–356.

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P. Scano et al. / Chemistry and Physics of Lipids 151 (2008) 69–76

Casu, M., Anderson, G.J., Choi, G., Gibbons, W.A., 1991. NMR lipid profiles of cells, tissues and body fluids-1D and 2D proton NMR of lipids from rat liver. Magn. Res. Chem. 29, 594–602. Chiang, S.D., Gessert, C.F., Lowry, O.H., 1957. Colorimetric determination of extracted lipids. An adaptation for microgram amounts of lipids obtained from cerumen. Curr. List. Med. Lit. Res. Rep. 33, 56–64. Christie, W.W., 1993. Preparation of ester derivatives of fatty acids for chromatographic analysis. In: Advantage in Lipid Methodology—Two. The Oily Press, Dundee, Scotland, pp. 69–111. Christie, W.W., The Lipid Library, www.lipidlibrary.co.uk and the literature therein cited. Accessed April 2007. Falch, E., Storseth, T.R., Aursand, M., 2006. Multi-component analysis of marine lipids in fish gonads with emphasis on phospholipids using high resolution NMR spectroscopy. Chem. Phys. Lipids 144, 4–16. Falch, E., Storseth, T.R., Aursand, M., 2007. High resolution NMR for studying lipid hydrolysis and esterification in cod (Gadus morhua) gonads. Chem. Phys. Lipid. 147, 46–57. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipid from animals tissues. J. Biol. Chem. 226, 497–509. Gorreta, F., Bernasconi, R., Galliani, G., Salmona, M., Tacconi, M.T., Bianchi, R., 2002. Wax esters of n-3 polyunsaturated fatty acids: a new stable formulation as a potential food supplement. 1—Digestion and absorption in rats. Lebensm-Wiss. Technol. 35, 458–465. Gribbestad, I.S., Aursand, M., Martinez, I., 2005. High resolution 1 H magnetic resonance spectroscopy of whole fish, fillets and extracts of farmed atlantic salmon (Salmo salar) for quality assessment and compositional analyses. Aquaculture 250, 445–457. Gunstone, F.D., 1993. High-resolution 13 C spectra of long-chain acids, methyl esters, glycerol esters, wax esters, nitriles, amides, alcohols and acetates. Chem. Phys. Lipids 66, 189–193. Iyengar, R., Schlenk, H., 1967. Wax esters in mullet (Mugil cephalus) roe oil. Biochem. 6, 396–402. Joh, Y.G., Brechany, E.Y., Christie, W.W., 1995. Characterization of wax esters in the roe oil of amber fish, Seriola aureovittata. J. Am. Oil Chem. Soc. 22, 707–713.

Lu, J.Y., Ma, Y.M., Williams, C., Chung, R.A., 1979. Fatty and amino acid composition of salted mullet roe. J. Food Sci. 44, 676–677. Nevenzel, J.C., 1969. Occurrence, function and biosynthesis of wax esters in marine organisms. Lipids 5, 308–319. Pollesello, P., Eriksson, O., Hockerstedt, K., 1996. Analysis of total lipid extracts from human liver by 13 C and 1 H nuclear magnetic resonance spectroscopy. Anal. Biochem. 236, 41–48. Rosa, A., Deiana, M., Corona, G., Atzeni, A., Incani, A., Dess`ı, M.A., 2005. Cholesterol as target of Fe-NTA induced peroxidation in rats tissue. Toxicol. Lett. 157 (1), 1–8. Ruxton, C.H.S., Reed, S.C., Simpson, M.J.A., Millington, K.J., 2004. The health benefits of omega-3 polyunsaturated fatty acids: a review of the evidence. J. Hum. Nutr. Diet. 17, 449–459. Sacchi, R., Medina, I., Aubourg, S.P., Giudicianni, I., Paolillo, L., Addeo, F., 1993. Quantitative high-resolution 13 C-NMR analysis of lipids extracted from the white Muscle of Atlantic Tuna (Thunnus alalunga). J. Agric. Food Chem. 41, 1247–1253. Scano, P., Casu, M., Lai, A., Saba, G., Dess`ı, M.A., Deiana, M., Corongiu, F.P., Bandino, G., 1999. Recognition and quantitation of cis-vaccenic and eicosenoic fatty acids in olive oils by 13 C nuclear magnetic resonance spectroscopy. Lipids 34, 757–759. Scano, P., Maxia, C., Maggiani, F., Crnjar, R., Lai, A., Sirigu, P., 2005. A histological and NMR study of the melon of the striped dolphin (Stenella coeruleoalba). Chem. Phys. Lipids 134, 21–28. Scano, P., Cesare Marincola, F., Locci, E., Lai, A., 2006. 1 H and 13 C NMR studies of melon and head blubber of the striped Dolphin (Stenella coeruleoalba). Lipids 41, 1039–1048. Siddiqui, N., Sim, J., Silwood, C.J.L., Toms, H., Iles, R.A., Grootveld, M., 2003. Multicomponent analysis of encapsulated marine oil supplements using high-resolution 1 H and 13 C NMR techniques. J. Lipid Res. 44, 2406– 2427. Wiegand, M.D., 1996. Composition, accumulation and utilization of yolk lipids in teleost fish. Rev. Fish Biol. Fisheries 6, 259–286. Yannakopoulos, A., Tserveni-Gousi, A., Christaki, E., 2005. Enhanced egg production in practice: the case of Bio-Omega-3 Egg. Int. J. Poult. Sci. 4, 531–535.