The influence of frying technique, cooking oil and fish species on the changes Bárbara Nieva-Echevarrı́a, Encarnación Goicoechea
occurring in fish lipids and oil during shallow-frying, studied by ¡ce:sup
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The influence of frying technique, cooking oil and fish species on the changes occurring

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in fish lipids and oil during shallow-frying, studied by 1H NMR

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Bárbara Nieva-Echevarría, Encarnación Goicoechea, María J. Manzanos, María D.

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MA Guillén*
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Food Technology, Faculty of Pharmacy, Lascaray Research Center, University of the Basque
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Country (UPV/EHU). Paseo de la Universidad nº 7, 01006 Vitoria, Spain, Telf: 34-945-013081. E-

mail: mariadolores.guillen@ehu.eus
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Running title header: 1H NMR study on fish shallow-frying

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Abstract

Fillets of farmed gilthead sea bream (Sparus aurata) and European sea bass

(Dicentrarchus labrax) were pan- and microwave-fried using extra-virgin olive and

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sunflower oils. Moreover, both oils were heated under the same conditions in the absence

of food. Proton Nuclear Magnetic Resonance spectroscopy (1H NMR) was employed to

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study the changes occurring both in the frying oils and in the fish lipids, in terms of main

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and of some minor components. These results confirm that there is a migration of main and

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some minor lipidic compounds between oil and fish, this exchange being linked to the

proportion of each component in the original lipidic medium. The occurrence of trans-2-
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alkenals, alkanals, cis,trans- and trans,trans-2,4-alkadienals, trans-9,10-epoxystearate and

1,2-diglycerides in all these systems was quantified. No signals related to primary oxidation
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compounds were detected. Pan-frying provoked a greater oil thermo-oxidation degree than
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microwave-frying did. Regarding aldehyde formation, extra-virgin olive oil is safer and
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more suitable than sunflower oil for shallow-frying.

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Keywords: fish lipids, extra-virgin olive oil, sunflower oil, microwave- and pan-frying,
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H NMR, lipid exchange.

Chemical compounds studied in this article:

Docosahexaenoic acid (PubChem CID: 445580); Eicosapentaenoic acid (PubChem CID:
446284); Linoleic acid (PubChem CID: 5280450); Oleic acid (PubChem CID: 445639);
Δ7-avenasterol (PubChem CID: 12795736); β-sitosterol (PubChem CID: 222284);
campesterol (PubChem CID: 173183); cholesterol (PubChem CID: 5997);
phosphatidylcholine (PubChem CID: 5287971); trans-9,10-epoxystearic acid (PubChem
CID-12235226).

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1. INTRODUCTION

Frying is a fast and simple culinary technique employed world-wide which consists of

cooking food above the boiling point of water, by means of partial (shallow-frying) or total

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(deep-frying) immersion in edible oils or fats that are liquid at frying temperature. Although

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fried foods are highly appreciated for their sensory characteristics, they must also satisfy

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health-related consumer concerns (Oztop, Sahin, & Sumnu, 2007). In comparison with

other cooking techniques, greater changes were reported in fish lipid composition after

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frying; although it must be noted that different results were found, depending on whether
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the frying method was shallow or deep (Moradi et al., 2011). In general, these changes

which take place during food frying have been attributed to food moisture loss, absorption
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of culinary oil/fat into the food, leaching of liposoluble molecules out of the food, lipolysis
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under the action of moisture, and oxidation of triglycerides due to the generation of free

radicals. In fact, the frying conditions, the nature of the culinary oil/fat and the fish lipid
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content have proved to be decisive parameters for lipid changes in the fried food and in the

frying oil (Bakar, Rahimabadi, & Che Man, 2008; Moradi et al., 2011; Martínez-Yusta &
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Guillén, 2014abc, 2015).

Although some exceptions can be found (Sanchez-Muniz , Viejo, & Medina, 1992;

Sioen et al., 2006; Amira et al., 2010; Martínez-Yusta & Guillén, 2014abc, 2015), it must

be noted that most previous studies of fish frying have focused only on the changes

occurring in fish lipids, disregarding those taking place at the same time in the frying oil. In

addition, most of them reported only the changes occurring in major but not minor lipid

components. Therefore, in this context, as many aspects remain unknown, it is of

paramount importance to study the frying process from a global point of view, in order to

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understand all the changes that it provokes not only in food, but also in the frying oil/fat.

As for the methodology employed, most of the main lipid component studies were

carried out by transformation of the triglycerides into their corresponding fatty acid methyl

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esters (FAMEs) and subsequent analysis using a gas chromatograph (GC) and a flame

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ionization detector (FID) (Echarte, Zulet & Astiasaran, 2001; Al-Saghir et al., 2004; Bakar

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et al., 2008; Weber et al., 2008; Ozogul, Ozyurt & Kuley Boga, 2009; Mnari Bhouri et al.,

2010; Amira et al., 2010) or a mass spectrometer (MS) (Kalogeropoulos, Andrikopoulos, &

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Hassapidou, 2004; Gladyshev et al., 2006). This is a multistep time-consuming
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methodology, which involves disadvantages such as the possible oxidation of

polyunsaturated acyl groups (Eder, 1995). Furthermore, in most of the studies the
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occurrence of oxidation reactions during frying was studied using classical methods like
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Peroxide Value (PV), Acid Value (AV), Thiobarbituric Acid Reactive substances test

(TBARS) or Conjugated Dienes (CD) (Echarte et al., 2001; Al-Saghir et al., 2004; Bakar et
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al., 2008; Weber et al., 2008), whose limited value and lack of specificity have been widely

commented on in the last 40 years (Connell, 1975; Addis, 1986; Saito & Udagawa, 1992;
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Haywood et al., 1995; Frankel, 2005).

Recently, Proton Nuclear Magnetic Resonance spectroscopy (1H NMR) has been

employed to study the evolution of the composition of extra-virgin olive, sunflower and

linseed oils submitted to frying temperatures in an industrial fryer in the absence of food for

prolonged periods of time (Guillén & Uriarte, 2009, 2012a,b,c). Likewise, using extra-

virgin olive, sunflower and soybean oils, the evolution of the frying media and of fried food

lipids (doughnuts, pork adipose tissue and farmed salmon (Salmo salar) fillets) in an

industrial deep-fryer have been subject of attention (Martínez-Yusta & Guillén, 2014a,b,c,

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2015). These 1H NMR studies on vegetable oils of different natures submitted to frying

conditions, in the absence and in the presence of food, reported the non-detection of

hydroperoxides associated with conjugated double bonds in the oils. These results were in

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agreement with the absence of significant changes in the PV found by some authors in

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olive, sunflower, palm and soybean oils heated at 189-200°C for 10 h/day for 10 days

(Dobarganes & Perez-Camino, 1988). Thus, it can be inferred that parameters like PV and

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CD values are not useful or suitable to monitor the changes in edible oils used for food

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frying. The 1H NMR technique can provide much more information than these and other

classical methods, not only concerning the molecular nature of the lipidic components, but
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also their concentration, in just one fast run without any sample modification.
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In this context, the influence of the frying technique, the cooking oil and the fish species
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on the changes occurring in fish lipids and in the oil during shallow-frying will be studied

by 1H NMR. Special attention will be paid to the possible lipid migration between the two
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systems involved (oil/fish fillet) and to the possible occurrence of reactions such as thermo-

oxidation and hydrolysis. As far as we know, this is the first time that 1H NMR has been
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employed for a detailed study of food shallow-frying.

2. MATERIALS AND METHODS

2.1. Oil samples subjects of study

Two cooking oils, extra-virgin olive oil (named evo) and sunflower oil (named s), were

acquired in a local supermarket.

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In order to evaluate the effect of heating on the oils, extra-virgin olive and sunflower oils

were submitted to the same frying conditions in the microwave-oven and in the pan in the

absence of food. These heated samples were named: evoP, extra-virgin olive oil submitted

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to pan-frying conditions without food; evoM, extra-virgin olive oil submitted to

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microwave-frying conditions without food; sP, sunflower oil submitted to pan-frying

conditions without food; and sM, sunflower oil submitted to microwave-frying conditions

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without food.

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After being used to fry fish fillets, oil samples were also collected and named evoPA,
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extra-virgin olive oil used to pan-fry sea bream (Sparus aurata); evoPL, extra-virgin olive

oil used to pan-fry sea bass (Dicentrarchus labrax); sPA, sunflower oil used to pan-fry S.
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aurata; sPL, sunflower oil used to pan-fry D. labrax; evoMA, extra-virgin olive oil used to
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microwave-fry S. aurata; evoML, extra-virgin olive oil used to microwave-fry D. labrax;

sMA, sunflower oil used to microwave-fry S. aurata; and sML, sunflower oil used to
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microwave-fry D. labrax. It must be noted that oils were used just once for frying and never

reused.
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2.2. Fish lipid samples subjects of study

Eight specimens of farmed gilthead sea bream (Sparus aurata) (A) and eight specimens

of farmed European sea bass (Dicentrarchus labrax) (L) were acquired in a local

supermarket on the day of the experiment. Just before frying, fishes were gutted, cleaned

and filleted. The average weight of sea bream fillets was 332.7±21.7 g and that of sea bass

fillets 295.4±29.6 g; all fillets presented very similar dimensions (width and length). From

each specimen, one fillet was submitted to cooking and the other one was kept raw (R) as a

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control. The lipid extracts obtained from the control fillets were named AR, lipids of raw

sea bream (S. aurata), and LR, lipids of raw sea bass (D. labrax).

The lipid extracts obtained from shallow-fried fish fillets were named: APevo, lipids of

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S. aurata pan-fried in extra-virgin olive oil; LPevo, lipids of D. labrax pan-fried in extra-

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virgin olive oil; APs, lipids of S. aurata pan-fried in sunflower oil; LPs, lipids of D. labrax

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pan-fried in sunflower oil; AMevo, lipids of S. aurata microwave-fried in extra-virgin olive

oil; LMevo, lipids of D. labrax microwave-fried in extra-virgin olive oil; AMs, lipids of S.

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aurata microwave-fried in sunflower oil; and LMs, lipids of D. labrax microwave-fried in
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sunflower oil.

2.3. Shallow-frying techniques

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Two different shallow-frying techniques were employed, conventional pan-frying (P)

using a domestic pan (28 cm internal diameter Ø) over an electric heating unit, and
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microwave-frying (M) using a domestic ceramic baking dish (28 cm internal diameter Ø) in

a household microwave oven (Samsung Combi CE 117KB) operating at 900 W. In order to

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obtain comparable results and to mimic domestic conditions, some experimental conditions

were the same in both techniques: oil temperature (170ºC), cooking time (2.5 min each

fillet side) and oil surface/oil volume ratio (28 cm diameter Ø /100 ml). These conditions

were maintained for all frying experiments. Before fish frying, oil temperature was checked

with a dual purpose infrared and penetration thermomether (104-IR, Testo instruments,

Cabrils, Spain), that can measure both oil/food surface and core temperatures. One fish

fillet was fried each time and two independent experiments were carried out for consistency

of results. The mean core temperature reached in pan-fried fillets was 60±5ºC and in

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microwave-fried ones 95±3ºC; crust formation was observed on the surface of the former

but not on the latter. After cooking, all fried fillets were drained for 15 s to remove excess

oil and then minced in a grinder, vacuum-packed and stored at -80ºC for up to 24 h for

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subsequent study.

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2.4. Fish lipid extraction method

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Lipids of fish fillets before and after frying were extracted using carbon disulphide as

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solvent (CS2, HPLC grade, Sigma-Aldrich, St. Louis, MO, USA) in a proportion of 1:2

(w/v) in an ultrasonic bath for 1 h, as in previous studies (Guillén & Ruiz, 2004). This
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solvent was selected because of its ability to extract lipids and its high volatility.

Afterwards, solvent was eliminated by means of a rotary evaporator under reduced pressure
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at room temperature in order to avoid lipid oxidation.

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2.5. Proton Nuclear Magnetic Resonance spectra acquisition

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The 1H NMR spectra both of the oils unheated, heated and after their use in shallow-

frying, as well as of the fish lipids extracted from raw and fried fillets, were recorded on a
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Bruker Avance 400 spectrometer operating at 400 MHz. As in previous edible oil studies

carried out in our laboratory (Guillén & Ruiz, 2004), 200 μl of lipid samples were mixed in

a 5 mm diameter tube with 400 μl deuterated chloroform (CDCl3), which contains 0.2% of

non-deuterated chloroform, and a small proportion of tetramethylsilane (TMS) used as

reference compound for calibrating chemical shift at 0.0 ppm internal reference

(Euroisotop, Paris, France). In order to select the most appropriate values to obtain accurate

quantitative results in the shortest possible period of time, a very broad range of recycling

times and relaxation delays were tested in the acquisition of the 1H NMR spectra. Thus, the

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acquisition parameters selected as being the most appropriate were the following: spectral

width 6410 Hz, relaxation delay 3 s, number of scans 64, acquisition time 4.819 s and pulse

width 90º. Each lipid sample was analyzed in duplicate. 1H NMR spectra were plotted at a

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fixed value of absolute intensity to be valid for comparative purposes. Spectra were

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processed using MestreNova program (Mestrelab Research, Santiago de Compostela,

Spain).

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Table 1 shows the chemical shifts of the several 1H NMR signals, their assignment to the

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different kinds of protons in the sample and their multiplicities, in agreement with previous
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studies (Haywood et al., 1995; Guillén & Ruiz, 2003, 2004; Guillén et al., 2008; Chou et

al., 2010; Sopelana, Arizabaleta, Ibargoitia, & Guillén, 2013). Some of these signals are
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shown in Figures 1 and 2.

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2.6. Determination from 1H NMR data of the molar percentage of main acyl groups and
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of the concentration of some minor components, hydrolytic and thermo-oxidation

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compounds in the frying oils and in fish lipids

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As the area of the 1H NMR signal is proportional to the number of protons that generate

it, and because the proportionality constant is the same for all types of hydrogen atoms, it is

possible to determine in an accurate way the absolute concentration and also the molar

percentages of the different kinds of acyl group chains present in the oils and in fish lipids.

These determinations were carried out in agreement with previous studies (Guillén et al.,

2008; Martínez-Yusta & Guillén, 2014abc, 2015). It is worth considering that the

contribution diglycerides present in the oils and of phosphatidylcholine in fish lipids is very

small (due to the fact that their molar abundance is a hundred times lower than that of

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triglycerides). Thus, the molar percentage of total omega-3 acyl groups was determined using the

equation ω-3%= 100(4AB)/ (9AI) [eq. 1], in function of the area (A) of signals B and I (see Table

1). In the same way, the molar percentages of other acyl groups were determined, like

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docosahexaenoic (C22:6ω3) acyl groups DHA%= 100AF2/ (3AI) [eq. 2]; eicosapentaenoic (EPA,

C20:5ω3) plus arachidonic (ARA, C20:4ω6) acyl groups EPA+ARA%= 100(2AD2)/ (3AI) [eq. 3]

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(but as the ARA content in these farmed fishes is usually very low (Orban, Nevigato, Lena, Casini,

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& Marzetti, 2003), the whole area of signal D2 was considered due to EPA); diunsaturated omega-6

acyl groups, mainly linoleic (C18:2ω6), DUω-6%= 100(2AG)/ (3AI) [eq. 4]; total unsaturated (U)

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acyl groups U%= 100(2AE + AF2)/ (6AI) [eq. 5]; saturated plus modified (S+M) acyl groups were
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calculated by difference to 100%; oleic (C18:1ω9) plus other unsaturated (O+OU) acyl groups, the

latter being mainly other monounsaturated, ARA and other minor unsaturated acyl groups,

O+OU%= (U%) – (ω-3%) – (ω-1%) – (DUω-6%) [eq. 6]; and finally, omega-1 acyl groups ω-1%=
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100(2AP)/ (3AI) [eq. 7]. Due to the overlapping of signals D1-D2 and G-H, the spectra of pure

standard trieicosapentaenoin, triarachidonin, trilinolein and trilinolenin, acquired from Larodan AB

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(Malmö, Sweden), were recorded and taken into account for a correct determination of A D2 and
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AG.
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Moreover, quantification of some minor components present in the frying oils (β-sitosterol plus

Δ5-campesterol (Sit+Camp) and Δ7-avenasterol) and in fish lipids (cholesterol and

phosphatidylcholine), expressed as millimoles per mole of triglyceride (TG), was also carried out

using the following equations: St (mmol/molTG)= 1000(4ASt)/(3AI) [eq. 8]; Phosphatidylcholine

(mmol/molTG)= 1000(4AO)/(9AI) [eq. 9], where ASt is the area of the signal of the methylic proton

at the carbon atom C-18 of each sterol (Sit+Camp, Δ7-avenasterol, cholesterol) and AO the area of

signal O (see Table 1).

Regarding hydrolytic and thermo-oxidation compounds, the concentrations of 1,2-diglycerides,

aldehydes (Ald) and (E)-9,10-epoxystearate, expressed also as mmol/mol of TG, were also

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determined as follows: 1,2-diglycerides(mmol/molTG)= 1000(2A3.72)/ AI [eq. 10] where A3.72 is the

area of the signal at 3.72 ppm due to -CH2OH protons of 1,2-diglycerides; Ald(mmol/molTG)=

1000(4AAld)/ AI [eq. 11] where AAld is the area of signals Q, R, S or T (see Table 1); (E)-9,10-

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epoxystearate(mmol/molTG)= 1000(2A2.63)/ AI [eq. 12] where A2.63 is the area of the signal at 2.63

ppm due to -CHOHC- protons of (E)-9,10-epoxystearate. All this quantitative information is shown

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in Tables 2 and 3.Thus, the molar percentage of the different kinds of acyl groups in the oils

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and in fish lipids were calculated: total omega-3 (ω-3), docosahexaenoic (DHA),

eicosapentaenoic (EPA), diunsaturated omega-6 (DUω-6, which includes mainly linoleic),

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total unsaturated (U), saturated plus modified (S+M), oleic plus other unsaturated (O+OU,
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which are mainly other monounsaturated, arachidonic and other minor unsaturated acyl

groups) and omega-1 (ω-1) acyl groups. It should be noted that as the arachidonic content
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in these farmed fishes is usually very low (Orban et al., 2003), the whole area of signal D2
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was considered due to EPA acyl groups.

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Moreover, quantification of some minor components present in the frying oils, like β-
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sitosterol plus Δ5-campesterol (Sit+Camp) and Δ7-avenasterol, and in fish lipids, such as
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cholesterol and phosphatidylcholine, expressed as millimoles per mole of triglyceride (TG),

was also carried out using the following equations:

St (mmol/molTG)=1000(4ASt)/(3AI) [eq. 1]

Phosphatidylcholine (mmol/molTG)=1000(4AO)/(9AI) [eq. 2]

where ASt is the area of the signal of the methylic proton at the carbon atom C-18 of

each sterol (Sit+Camp, Δ7-avenasterol, cholesterol), AI that of signal I, and AO the area of

signal O (see Table 1). Regarding hydrolytic and thermo-oxidation compounds, the

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concentrations of 1,2-diglycerides, aldehydes and (E)-9,10-epoxystearate, expressed also as

mmol/mol of TG, were also determined, in agreement with previous studies (Martínez-

Yusta & Guillén, 2014abc, 2015). All this quantitative information is shown in Tables 2

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and 3.

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2.7. Statistical Analysis

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Statistical analysis was performed using the Statistical package SPSS v.19 (IBM, NY,

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USA). The significance of the differences on the several determinations among groups

were determined by one-way and by two-way variance analysis (ANOVA) followed by

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post hoc Tukey b test at 0.05 threshold.
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3. RESULTS AND DISCUSSION

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Farmed gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus
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labrax) were shallow-fried under domestic conditions using two frying methods
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(microwave- and pan-frying) and two cooking oils (sunflower and extra-virgin olive); these

latter were never reused. The changes which occurred in the main and minor components of

fish lipids and of the oils used for frying, either in the absence or in the presence of fish

fillets, were studied by 1H NMR.

3.1. Changes in the molar percentages of acyl groups. Causes and consequences.

In this section the initial acyl group composition of the oils and the fish lipids will be

considered, followed by a description of the changes observed in the acyl groups of the oils

submitted to heating without food, of the oils used to fry fish, and of the fried fish lipids. In
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addition, the causes and consequences of these phenomena will be discussed.

3.1.1. Unheated oils and raw fish lipids

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The 1H NMR spectra of sunflower oil (see s in Figure 1) and extra-virgin olive oil (see

evo in Figure 2) before heating or their use in frying, can be seen to contain the typical

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signals related to these kinds of vegetable oils (Guillén & Ruiz, 2003) and their assignment

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is shown in Table 1. The spectrum of sunflower oil shows that this oil is especially rich in

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linoleic acyl groups, which is evidenced by the high intensity of the triplet at 0.890 ppm

(signal A), the peaks at 2.038 and 2.056 (signal E) and the triplet at 2.765 ppm (signal G).
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The signals related to oleic acyl groups show lower intensities, namely the triplet at 0.880

ppm (signal A), the shoulder at 1.270 ppm (signal C) and the peaks at 2.002 and 2.017 ppm
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(signal E), whereas the signals due to linolenic acyl groups are absent (signal B). In the 1H
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NMR spectrum of extra-virgin olive oil (see Figure 2), the signals related to oleic acyl
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groups show the highest intensities, whereas those associated with linoleic and linolenic
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acyl groups are much lower. Table 2 shows the average molar percentages of these main
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acyl groups initially present in both oils, together with their standard deviation.

Regarding the initial composition of the lipids of both raw fish, the profile of their 1H

NMR spectra are quite similar. As the spectra of sea bass (D. labrax, LR, Fig. Figure 1)

and sea bream (S. aurata, AR, Fig. Figure 2) lipids show, before frying both contain higher

proportions of saturated plus monounsaturated than of linoleic acyl groups (see the triplets

at 0.880 and 0.890 of signal A), as well as ω-3 acyl groups including EPA and DHA

(signals B, D2, F2) and ω-1 acyl groups (signal P). This is observable in a direct way from

the AR and LR spectra. In spite of this, the lipids of both fishes differ in the molar

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percentage of some of the above-mentioned components. As shown in Table 3, one of the

most significant differences is the higher molar percentage of ω-3 acyl groups in AR than

in LR, whereas the opposite is true for diunsaturated ω-6 acyl groups (mainly linoleic).

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Different DHA/EPA ratios are observed in both species, being near 2 in AR and near 1 in

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LR.

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3.1.2. Heated oils

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When sunflower and extra-virgin olive oils were submitted to heating conditions in the

absence of fish fillets, some changes could be observed in the intensity of their main 1H
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NMR spectral signals. Figure 1 shows the spectrum of sunflower oil submitted to heating

conditions in the pan (sP) and Figure 2 that of extra-virgin olive oil submitted to heating
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conditions in the microwave oven (evoM), but in order to notice the changes related to acyl
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groups composition further enlargement is needed. The quantitative data reported on Table
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2 evidence that in each kind of vegetable oil there is a certain decrease in its main
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unsaturated acyl group: oleic in evo and linoleic in s. Thus, in the case of extra-virgin olive
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oil the oleic molar percentage (O+OU) slightly decreases from 81.0% to 80.2-80.7% (see

evoP, evoM in Table 2). In the case of sunflower oil, the linoleic (DUω-6) molar

percentage decreases more intensely, from 59.1% to 55.7-57.1% (see sP, sM), especially in

that submitted to heating in the pan. Simultaneous to this loss of the main unsaturated acyl

group in the heated oils, there is an increase of the saturated plus modified (S+M) acyl

groups molar percentage. Moreover, in heated extra-virgin olive oil there is also a decrease

in the DUω-6%, especially in evoP. These results suggest that heating in the pan provokes

a slightly greater degradation of acyl groups than heating in the microwave-oven.

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In addition to these changes observed in the acyl groups molar percentages, in some

heated oil spectra signals of the aldehydic protons of secondary oxidation compounds,

which can be supported either on small molecules or on modified acyl chains, appeared. It

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must also be pointed out that during the 5 minute heating experiment a certain amount of

aldehydes of low molecular weight may have escaped into the atmosphere and that 1H

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NMR spectroscopy only detects the aldehydic groups present in the liquid phase, either of

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low or high molecular weight. Both phenomena, the changes in the acyl groups molar

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percentages and the formation of aldehydes, can be explained by the occurrence of thermo-

oxidation reactions in the heated oils. Nevertheless, it must be noted that in none of the 1H
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NMR spectra of the heated oils were signals of hydroperoxy protons or of conjugated

dienic protons observed. This indicates that if these primary oxidation compounds are
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formed, they are degraded so quickly that their concentration is not detectable by 1H NMR,
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in agreement with previous studies on oils submitted to frying temperatures in the absence
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of food (Guillén & Uriarte, 2009, 2012a,b,c).

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In the spectra of sunflower oil heated in the pan and in the microwave oven (see sP in
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Fig. Figure 1 and data on sP, sM in Table 2) the formation of (E)-2-alkenals (signal Q),

(E,E)- 2,4-alkadienals (signal R), (Z,E)-2,4-alkadienals (signal S) and alkanals (signal T)

was observed, (E,E)-2,4-alkadienals being in the highest concentrations, followed by

similar amounts of (E)-2-alkenals and alkanals. Regarding the appearance of aldehydic

proton signals in heated extra-virgin olive oil spectra, alkanals and (E)-2-alkenals were only

detected after pan-heating (see evoP in Table 2). The absence of formation of (E,E)- and

(Z,E)-2,4-alkadienals is an important difference, because the higher the unsaturation degree

of aldehydes, the higher their reactivity and toxicity. The lack of formation of aldehydes in

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extra-virgin olive oil heated in the microwave-oven suggests that heating in a pan provokes

a higher thermo-oxidation degree than heating in a microwave-oven, in agreement with that

above-commented on acyl groups. These results also evidenced that, from an aldehyde

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formation point of view, extra-virgin olive oil is safer than sunflower oil for heating at

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frying temperatures.

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In addition, it is well-known that when oils are submitted to frying temperatures, acyl

groups can also evolve to give epoxides. In these heated samples the only one detected was

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(E)-9,10-epoxystearate, and then only in the spectrum of extra-virgin olive oil heated in the
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pan (see evoP in Table 2). This fact confirms again the greater thermo-oxidation provoked

in the oil when heating in a pan than in a microwave-oven. Kalogeropoulos, Salta, Chiou,
D

& Andrikopoulos (2007) studied the formation and distribution of epoxides derived from
TE

oleic and linoleic acyl groups when deep- and pan-frying potatoes in cottonseed, sunflower,

vegetable shortening, palm and virgin olive oils (reusing the oils for eight successive frying
P
CE

sessions). They concluded that in all the oils (E)-9,10-epoxystearate was the main epoxide

generated, in much higher concentrations in virgin olive oil than in sunflower oil,
AC

unsurprisingly as it is derived from oleic acyl groups. However, it must be noted that no

studies on the possible toxicity of this epoxide can be found in the literature, whereas there

are many on that of epoxyoleates, derived from linoleic acyl groups. Moreover,

Kalogeropoulos et al. (2007) reported that regardless the nature of the oil, higher

concentrations of total epoxides were generated in the oils after their use for pan-frying

than for deep-frying.

3.1.3. Oils used for fish shallow-frying

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Concerning the changes occurred in the acyl groups molar percentages of the oils used

just once to pan- or microwave-fry either sea bream (S. aurata, A) or sea bass (D. labrax,

L) fillets, greater changes were observed in their 1H NMR spectra (see sPL in Fig. Figure 1

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and evoMA in Fig. Figure 2), in comparison with those of the heated oils, because some

signals change their intensities and other new ones appear. Thus, in the 1H NMR spectra of

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both kinds of oils a decrease of the intensity of the signals of the corresponding main

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unsaturated acyl group is observed without further enlargement. In the case of the 1H NMR

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spectra of extra-virgin olive oil used to shallow-fry fish, those signals due to oleic acyl

groups decrease (see evoAM evoMA spectrum of Fig. Figure 2), reaching a molar
MA
percentage (O+OU) of a similar value regardless of the frying technique and the fish

species (from 81.0% in evo to 71.8-73.2%). The fact that the lowered molar percentage of
D

the main unsaturated acyl group of the oils used for shallow-frying fish is higher than that
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in the corresponding heated oil, can be explained not only by the occurrence of oil thermo-
P

oxidation (as in the heated oils), but also, and mainly, by the oil/fish lipid migration effect,
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in agreement with previous studies (Martinez-Yusta & Guillén, 2014abc, 2015). In the case
AC

of sunflower oil, the signals related to linoleic acyl groups decrease (see sPL spectrum of

Fig. Figure 1), reaching lowest molar percentage values after frying sea bream fillets, as

Table 2 shows (from 59.1% in s to 46.0% in sAM sMA and 49.1% in sAP SPA). This fact

could be explained by the lower initial content of linoleic acyl groups in sea bream than in

sea bass fillets (see DUω-6% in AR and LR in Table 3), because in this shallow-frying

lipid exchange the oil is mainly enriched by the lipidic components that are in higher

concentration in the fish fillet, and vice versa.

Moreover, in the spectra of all the frying oils those signals due to specific fish ω-3 acyl

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groups, namely eicosapentaenoic (EPA, signal D2) and docosahexaenoic (DHA, signal F2),

appear, evidencing the leaching of triglycerides containing these acyl groups out of the fish

fillet into the frying oil. Signals related to total ω-3 acyl groups (signal B, peaks at 2.091

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and 2.111 ppm of signal E, signal H) increase their intensity in extra-virgin olive oil

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spectra, and appear in those of sunflower oils. Table 2 shows that regardless of the oil

nature and the frying method, the increase of the molar percentage of total ω-3 acyl groups

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is higher after shallow-frying sea bream fillets (evoMA, evoPA, sMA, sPA) than sea bass

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fillets (evoML, evoPL, sML, sPL), which can be explained by the higher initial content in

total ω-3 acyl groups in sea bream than in sea bass fillets (see AR, LR in Table 3).
MA
Regarding specific fish ω-3 acyl groups, the molar percentages of DHA and EPA in both

frying oils after frying sea bass fillets are very similar (≈1%), whereas after frying sea
D

bream fillets the molar percentage of DHA is near double (≈2%) than that EPA, which is
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also in agreement with the initial lipid composition of both fish species (see AR, LR in
P

Table 3). In addition, signal P appears in the spectra of all the oil samples used for frying,
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due to the migration of fish triglycerides containing unsaturated ω-1 acyl groups. It is
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noteworthy that this migration effect is much more evident in food shallow-frying than in

deep-frying studies, as could be expected due to the food:oil volume ratio (Martínez-Yusta

& Guillén, 2014a,b,c, 2015).

Table 2 also shows that after frying fish in both vegetable oils there is a slight increase in

the molar percentage of saturated plus other modified (S+M) acyl groups. In addition, there

is a small increase in the molar percentages of oleic acyl groups (O+OU) in sunflower oil

samples (see sMA, sPA, sML, sPL) and of linoleic (DUω-6) ones in extra-virgin olive oil

samples (see evoMA, evoPA, evoML, evoPL), this latter being more noteworthy after

18
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shallow-frying sea bass fillets, which initially were richer in linoleic acyl groups than sea

bream fillets (see DUω-6% in LR and AR in Table 3), as mentioned before.

When it comes to the occurrence of thermo-oxidation reactions, it must be noted that no

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aldehydes were detected either in the spectra of extra-virgin olive oil after their use for fish

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frying, (see evoAM evoMA, evoAP evoPA, evoLM evoML and evoLP evoPL in Table 2)

SC
or in those of sunflower oils submitted to microwave-frying (see sMA, sML in Table 2).

However, in the spectra of sunflower oil employed for sea bass pan-frying (E,E)-2,4-

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alkadienals and (E)-2-alkenals appeared and in that employed for sea bream pan-frying
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only (E,E)-2,4-alkadienals (see sPL in Fig. Figure 1 and sPA, sPL in Table 2). Therefore, it

is confirmed that, both in the absence and in the presence of food, pan-frying provokes a
D

higher thermo-oxidation degree than microwave-frying, in terms of aldehyde formation. In

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addition, the fact that (E)-2-alkenals are only detected in the sunflower oil employed to

pan-fry sea bass fillets (sPL) and not in that used for sea bream (sPA), can be explained by
P
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the influence of the fish species. Sea bream contains a lower molar percentage of DUω-6

acyl groups (mainly linoleic) and a higher one of ω-3 acyl groups than sea bass (see AR
AC

and LR in Table 3). It is well-known that during frying the degradation of linoleic acyl

groups generates (E)-2-alkenals of high molecular weight, mainly (E)-2-heptenal and (E)-2-

hexenal, whereas the degradation of polyunsaturated ω-3 acyl groups, like linolenic, gives

rise mainly to the formation of (E)-2-alkenals of lower molecular weight, like (E)-2-

butenal, that are very volatile and escape more easily towards the atmosphere due to their

lower boiling points (Guillén & Uriarte, 2012d). Thus, the fact that sea bass lipids are richer

in linoleic acyl groups than sea bream lipids, can explain why (E)-2-alkenals are detected in

the sunflower used to pan-fry the former (sPL) and not in that corresponding to the latter

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(sPA). This deduction was confirmed by the results obtained in the simultaneous study of

the headspace composition of these samples, carried out by Solid-Phase Microextraction

followed by Gas Chromatography-Mass Spectrometry (SPME-GC/MS) (data not published

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yet). In sPL headspace the abundances of (E)-2-heptenal and (E)-2-hexenal were double

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than in sPA headspace, that of (E)-2-butenal being much lower in both samples. In this

context, several authors have drawn special attention to the use of oils rich in

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polyunsaturated acyl groups, like sunflower, for food frying or heating processes, and the

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consequent possible ingestion of toxic aldehydes, because they clearly pose health hazards

worthy of increased clinical and public concern (Haywood et al., 1995; Grootveld, Silwood,
MA
& Claxson, 1999). In previous studies on deep-frying with oils of different nature it was

shown that both the nature and concentrations of the aldehydes detected in the frying media
D

are dependent on the composition in acyl groups of the oil and food lipids involved in the
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process and the heating time (Guillén & Uriarte, 2009, 2012a,b,c; Martínez-Yusta &
P

Guillén, 2014abc, 2015).

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Moreover, it must be noted that these aldehydes detected in the 1H NMR spectra of the
AC

sunflower oils used for fish pan-frying (sPA, sPL) are in much lower concentrations than

those detected in the heated sunflower oils (see sM, sP in Table 2). This fact could be

explained by several reasons. One is that when the fish fillet is introduced into the system,

the oil temperature goes down for a while and as a consequence the thermo-oxidation

process can be reduced, whereas in the absence of food it maintains constant (≈170ºC) for

the 5 minute heating experiment. Another is that the presence of the fish fillet provokes a

greater movement of the surrounding oil, which facilitates the escape of volatile aldehydes

to the atmosphere. A third is the occurrence of Maillard type reactions between aldehydes

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and fish proteins, in agreement with previous studies (Weber et al. 2008). Finally, there is a

potential dilution effect due to the leaching of fish lipids into the frying media.

3.1.4. Lipids of shallow-fried fish fillets

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As for the changes occurring in the acyl groups of fish fillets after pan- or microwave-

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frying in sunflower or extra-virgin olive oils, the spectra of all fried fish lipid samples

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contained those signals related to total ω-3, EPA and DHA acyl groups in lower intensities

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than there were in the spectra of the corresponding raw fish lipids (see LPs and LR in Fig.

Figure 1, AMevo and AR in Fig. Figure 2). Table 3 reports quantitative data on this
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decrease and it can be explained mainly by the oil/fish lipid migration effect, in agreement

with that commented on above concerning the increase of these acyl groups in the frying
D

oils (Table 2). As can be observed in Table 3, fried fish lipid samples also showed lower
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molar percentages of saturated plus modified (S+M) and of ω-1 acyl groups than before
P

frying. Moreover, regardless of the frying technique employed, in all fried fish lipid
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samples there is an increase in the molar percentage of the main unsaturated acyl group of
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the corresponding oil employed, namely linoleic (DUω-6) in the case of sunflower and

oleic (O+OU) in that of extra-virgin olive oil. All these changes in fried fish lipid

composition are explained by the composition of the frying oil that is absorbed by the fillet,

in such a way that fried fish lipids become richer in those acyl groups that are in higher

concentration in the frying oil than in fish lipids, but poorer in those acyl groups that are in

higher concentration in the raw fish lipids than in the original oils.

In addition to the oil uptake, due to the high tendency of fish polyunsaturated acyl

groups to undergo oxidation, some changes observed in the acyl groups of fish lipids could

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also be attributed to the occurrence of thermo-oxidation reactions. Nevertheless, no 1H

NMR signals related to primary oxidation compounds, aldehydes or epoxides were detected

in the fish lipid spectra. This absence evidences that during shallow-frying there is very

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little thermo-oxidation of fish lipids, if any, and that if aldehydes present in the frying oils

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are absorbed by the fillet, they must have reacted with fish proteins through Maillard type

reactions.

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3.1.5. Lipid migration during frying

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Bearing in mind all the above, it is evident that under the conditions of this study lipid
MA
exchange between the fish fillet and the culinary oil occurs. The molar percentages of the

different kinds of acyl groups, before and after frying, in the lipids of both systems (oil and
D

fish) are given in Tables 2 and 3. Assuming that the loss of lipids due to thermo-
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degradation processes is small in both systems, the estimation of the contribution of the
P

migrated fish lipids (oil) to the final molar percentage of the different acyl groups in fried
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oil (fried fish lipids) can be calculated. The obtained results are given in Table 4, together
AC

with the fat content of fish fillets, before and after frying.

Thus, considering all the frying techniques, oils and fish species, a contribution ranging

from 19.4 to 28.1% of the migrated fish lipids to the final molar percentage of the different

kinds of acyl groups in the fried oil was estimated. The highest contribution of fish lipids to

the changes observed in the fried oil is observed in sea bream specimens pan fried in evo

(≈25% contribution) and in those microwave-fried in sunflower oil (≈28% contribution). In

fact, before frying these fish fillets contained the highest initial fat content (≈42 and 44g,

respectively) and as a result of the frying process underwent the highest fat loss (≈ -16g in

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both cases), evidencing a remarkable leaching of their lipids. By contrast, the smallest

contribution of the oil was observed in sea bass fillets pan- and microwave-fried in

sunflower oil (≈19.5%), which are those fish fillets showing the lowest initial fat content

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(≈19-22g). In the other fish specimens, regardless of the species, the oil used and the frying

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method, the contribution of fish lipids to the molar percentage of the different kinds of acyl

groups in fried oil is about 21-22%. These contributions can be considered important taking

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into account that the amount of original oil used in all the frying experiments is of 100ml

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(much higher than the total amount of fat present initially in fish fillets (≈19-44g)) and that

a part of this culinary oil is absorbed by the fish fillet, thus contributing to final fried fish
MA
lipid composition.
D

As Table 4 shows, the contribution of migrated oil to the molar percentage of the
TE

different acyl groups in the fried fish lipids is also important, ranging in most of the cases

from 15 to 25%. This contribution is somewhat lower than that of fish lipids to the fried oil.
P
CE

The exception is the case of sea bass fillets microwave-fried in sunflower oil, in which the

contribution of the oil is very high (≈43%). These results are in agreement with the fact that
AC

the latter sea bass fillets showed the lowest initial fat content (≈19 g) and that they were the

only ones in which the total fat content increased after frying (≈7g), highlighting that when

microwave-frying these fillets the oil uptake took place to a greater extent than the leaching

out of the fish lipids (see Table 4).

3.2. Changes in the concentration of sterols and phosphatidylcholine. Causes and

consequences

This section focuses on the changes occurring in the concentration of certain minor

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components, in both oils and fish lipids, as consequence of the frying process. Before

frying, some of them are present only in the oils (Δ7-avenasterol, β-sitosterol, Δ5-

campesterol) and others, only in fish lipids (cholesterol, phosphatidylcholine).

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Concerning sterols, the detection and quantification of Δ7-avenasterol is possible by

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signal L at 0.54 ppm and that of β-sitosterol plus Δ5-campesterol by signal N at 0.684 ppm,

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due in all cases to the methylic protons in C-18. If cholesterol is also present in the sample,

this latter signal N overlaps with signal M at 0.682 ppm due to the same protons in

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cholesterol. The concentrations of Δ7-avenasterol and of β-sitosterol plus Δ5-campesterol
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(Sit+Camp) in the original oils are given in Table 2 and that of cholesterol in the raw fish

lipids in Table 3. With the simple oil heating, the concentration of sterols in both sunflower
D

and extra-virgin olive oils remained almost unchanged (see evoM, evoP, sM, sP in Table
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2). However, after being used for fish frying, a slight diminution in the concentration of Δ7-

avenasterol in the fried sunflower oil was observed (see sMA, sPA, sML, sPL in Table 2).
P
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This fact shows that a possible migration of a certain amount of this sterol to fish during

frying has occurred. This is confirmed by the appearance of Δ7-avenasterol in fried fish
AC

lipids, as Table 3 shows (see AMs, APs, LMs and LPs).

As for β-sitosterol plus Δ5-campesterol present in both original sunflower oil and extra-

virgin olive oils, during frying a degree of migration to the fish fillet similar to that of Δ7-

avenasterol can be expected. Simultaneously, the migration of fish cholesterol to the frying

oil can also occur, being even higher than that of β-sitosterol plus Δ5-campesterol from the

oil to the fish fillet. This is in agreement with the higher concentration observed of β-

sitosterol+Δ5-campesterol+cholesterol in both fried sunflower and extra-virgin olive oils

than that of β-sitosterol+Δ5-campesterol in the original oils. From these results, it could be

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expected that the concentration of β-sitosterol+Δ5-campesterol+cholesterol in fried fish

lipids would be smaller than the concentration of cholesterol in the raw fish lipids;

however, data in Table 3 shows the opposite (see AMevo, APevo, LMevo, LPevo and

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AMs, APs, LMs, LPs). Taking into account that the degree of migration of β-sitosterol

plus Δ5-campesterol from the oil to the fish fillet is small, the increase observed in the

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concentration of β-sitosterol+Δ5-campesterol+cholesterol in fried fish lipids can be mainly

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attributed to a higher extraction after thermal treatment of cholesterol from structures to

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which it is bounded in fish meat, as it has been pointed out by other authors (Larsen, Quek,

& Eyres, 2010).

MA
In short, migration of sterols from fish to oil and vice versa occurs in such way that fish
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meat is enriched in healthy vegetable sterols and oils used to fish frying are enriched in
TE

cholesterol. Furthermore, in lipids extracted from fried fish, a higher concentration of

cholesterol is found than in those extracted from raw fish.

P
CE

As for phosphatidylcholine, its concentration in fish lipids before and after frying was
AC

determined using the area of signal O, related to the methylic protons in the choline group.

A remarkable increase in the intensity of this signal was noticed by comparing the spectra

of fish lipid samples before and after frying (see LR, LPs in Figure 1 and AR, AMevo in

Figure 2). Indeed, from data reported in Table 3, it can be observed that the concentration

of this compound in all fried fish lipid samples is higher (≈5.3-11.3 mmol/mol TG) than

that initially present in raw fish lipids (≈1.4-1.7 mmol/mol TG). It must be noted that since

phosphatidylcholine is a well-known structural component of cell membranes, this increase

could be related to a more exhaustive extraction from fried fish muscle, due to the changes

occurring after thermal processing. However, the occurrence of this fish minor component

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in oils after frying was not detected, being its migration to the frying oil much more

restricted than that of other fish lipid minor components, probably due to its polarity.

3.3. Changes in the concentration of 1,2-diglycerides. Causes and consequences

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In addition to triglycerides, small amounts of di- and/or monoglycerides can also be

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present in food lipids. Only 1,2-diglycerides were detected in the oils and fish lipids

SC
involved in this study. Their quantification can be made from the intensity of the doublet at

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3.73 ppm due to the protons in C-3 of the glyceryl backbone. In the original sunflower and

extra-virgin olive oils the concentration of 1,2-diglycerides was 4.9 and 11.2 mmol/mol
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TG, respectively (see Table 2) and in raw fish lipids much lower concentrations were

detected (see AR and LR in Table 3).

D
TE

It has been observed that the heating provokes only in extra-virgin olive oil a decrease of

1,2-diglycerides concentration (up to 8-9 mmol/mol TG), which is more accentuated with
P
CE

the fish frying process (up to 6-7 mmol/mol TG). However, these changes are much smaller

in sunflower oil after both heating and fish frying processes. As the evolution of 1,2-
AC

diglycerides in fish lipids during frying does not follow a clear trend (see Table 3), the

changes observed in their concentration in oils during fish frying are probably associated

with hydrolytic or degradation processes, in addition to lipid exchange between fish fillet

and oil.

4. CONCLUSIONS

During fish shallow-frying under domestic conditions, migration of fish lipids to

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culinary oil occurs, as does migration of the oil components to the fish fillet. As

consequence, the composition of the oils used for frying changes, becoming richer in those

acyl groups that are in higher concentration in fish lipids than in the original oil and poorer

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in those acyl groups that are in smaller concentration in fish lipids than in the original oil.

Thus, after frying extra-virgin olive oil is richer than the original oil in ω-3, ω-1, DUω-6

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and saturated acyl groups and poorer in ω-9 groups. Likewise, after frying sunflower oil is

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richer than the original in all kinds of acyl groups except for DUω-6 groups. In addition,

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after frying fish both oils are enriched, although to a much lesser extent, in cholesterol in

relation to the original composition. Concerning fish lipids, their composition also changes
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during frying, becoming richer in those acyl groups that are in higher concentration in the

frying oil than in fish lipids, while poorer in those acyl groups and minor components that
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are in higher concentration in the raw fish lipids than in the original oils.
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In addition to lipid migration, the submission of the oils to high temperatures in the
P
CE

presence of oxygen provokes some thermo-oxidation to a very low extent. Sunflower oil

shows a smaller resistance to degradation than extra-virgin olive oil, not only during
AC

heating but also during frying, as expected. Heating by microwave provokes a lower

thermodegradation than heating in a pan. The number and concentration of secondary

oxidation products found in oils after frying are lower than those found after heating. No

thermooxidation was observed in extra-virgin olive oil used for fish frying. The highest

concentration of secondary oxidation compounds was found in sunflower oil heated in the

pan, followed by that used to pan-fry sea bass.

The frying technique, the nature of the cooking oil and the fish species have been

evidenced to have a great influence on the changes occurring during food shallow-frying.

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The selection of the cooking oil is of paramount importance due to its impact on the fish

lipid profile and on the possible generation of toxic compounds in the oil during frying,

which can have a great influence on food safety and human health.

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ACKNOWLEDGEMENTS

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This work has been supported by the Spanish Ministry of Economy and Competitiveness

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(MINECO, AGL2012-36466), by the Basque Government (EJ-GV, GIC10/85-IT-463-10)

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and by the Unit for Education and Research “Food Quality and Safety” (UPV/EHU-UFI-

11/21). B. N-E. thanks the UPV/EHU for a predoctoral fellowship.

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REFERENCES

Addis, P. B. Occurrence of lipid oxidation products in foods. Food and Chemical

Toxicology (1986), 24(10-11), 1021-30.
Al-Saghir, S., Thurner, K., Wagner, K.-H., Frisch, G., Luf, W., Razzazi-Fazeli, E., &

PT
Elmadfa, I. (2004). Effects of different cooking procedures on lipid quality and
cholesterol oxidation of farmed salmon fish (Salmo salar). Journal of Agricultural and
Food Chemistry, 52(16), 5290-5296.

RI
Amira, M. B., Hanene, J. H., Madiha, D., Imen, B., Mohamed, H., & Abdelhamid, C.
(2010). Effects of frying on the fatty acid composition in farmed and wild gilthead sea

SC
bream (Sparus aurata). International Journal of Food Science & Technology, 45(1),
113-123.
Bakar, J., Rahimabadi, E. Z., & Che Man, Y. B. (2008). Lipid characteristics in cooked,

NU
chill-reheated fillets of Indo-Pacific king mackerel (Scomberomorous guttatus). LWT -
Food Science and Technology, 41(10), 2144-2150.
Chou, T. H., Chen, J. J., Lee, S. J., Chiang, M. Y., Yang, C. W., & Chen, I. S. (2010).
Cytotoxic Flavonoids from the Leaves of Cryptocarya chinensis. Journal of Natural
MA
Products, 73(9), 1470-1475.
Connell, J. J. (1975). Control of Fish Quality, 1st Edn., Fishing NewBooks, Oxford (UK).
Dobarganes, M. C., & Perez-Camino, M. C. (1988). Fatty acid composition: a useful tool
for the determination of alteration level in heated fats. Revue Francaise des Corps
D

Gras, 35(2), 67-70.

TE

Echarte, M., Zulet, M. A., & Astiasaran, I. (2001). Oxidation process affecting fatty acids
and cholesterol in fried and roasted salmon. Journal of Agricultural and Food
Chemistry, 49(11), 5662-5667.
P

Eder, K. (1995). Gas chromatographic analysis of fatty acid methyl esters. Journal of
Chromatography B: Biomedical Sciences and Applications, 671(1+2), 113-131.
CE

Frankel, E. N. (2005). Lipid Oxidation, 2nd Edn., Oily Press, Davis (USA).
Gladyshev, M. I., Sushchik, N. N., Gubanenko, G. A., Demirchieva, S. M., & Kalachova,
G. S. (2006). Effect of boiling and frying on the content of essential polyunsaturated
AC

fatty acids in muscle tissue of four fish species. Food Chemistry, 101, 1694-1700.
Grootveld, M., Silwood, C. J. L., & Claxson, A. W. D. (1999). Warning: thermally-stressed
polyunsaturates are damaging to health. Food Chemistry, 67(2), 211-213.
Guillén, M. D., & Ruiz, A. (2003). Edible oils: Discrimination by 1H nuclear magnetic
resonance. Journal of the Science of Food and Agriculture, 83(4), 338-346.
Guillén, M. D., & Ruiz, A. (2004). Study of the oxidative stability of salted and unsalted
salmon fillets by 1H nuclear magnetic resonance. Food Chemistry, 86(2), 297-304.
Guillén, M., Carton, I., Goicoechea, E., & Uriarte, P. S. (2008). Characterization of cod
liver oil by spectroscopic techniques. New approaches for the determination of
compositional parameters, acyl groups, and cholesterol from 1H nuclear magnetic
resonance and Fourier transform infrared spectral data. Journal of Agricultural and
Food Chemistry, 56(19), 9072-9079.
Guillén, M. D., & Uriarte, P. S. (2009). Contribution to further understanding of the
evolution of sunflower oil submitted to frying temperature in a domestic fryer: Study by
1
H nuclear magnetic resonance. Journal of Agricultural and Food Chemistry, 57(17),
7790-7799.

29
 ACCEPTED MANUSCRIPT

Guillén, M. D., & Uriarte, P. S. (2012a). Study by 1H NMR spectroscopy of the evolution
of extra virgin olive oil composition submitted to frying temperature in an industrial
fryer for a prolonged period of time. Food Chemistry, 134(1), 162-172.
Guillén, M. D., & Uriarte, P. S. (2012b). Monitoring by 1H nuclear magnetic resonance of
the changes in the composition of virgin linseed oil heated at frying temperature.
Comparison with the evolution of other edible oils. Food Control, 28(1), 59-68.

PT
Guillén, M. D., & Uriarte, P. S. (2012c). Simultaneous control of the evolution of the
percentage in weight of polar compounds, iodine value, acyl groups proportions and

RI
aldehydes concentrations in sunflower oil submitted to frying temperature in an
industrial fryer. Food Control, 24(2), 50-56
Guillén, M. D., & Uriarte, P. S. (2012d). Aldehydes contained in edible oils of a very

SC
different nature after prolonged heating at frying temperature: Presence of toxic
oxygenated α,β unsaturated aldehydes. Food Chemistry, 131(3), 915-926.
Haywood, R. M., Claxson, A. W. D., Hawkes, G. E., Richardson, D. P., Coumbarides, G.,

NU
Hawkes, J., Lynch, E. J., & Grootveld, M. C. (1995). Detection of aldehydes and their
conjugated hydro-peroxydiene precursors in thermally-stressed culinary oils and fats:
investigations using high resolution proton NMR spectroscopy. Free Radical Research,
MA
22(5), 441-482.
Kalogeropoulos, N., Andrikopoulos, N. K., & Hassapidou, M. (2004). Dietary evaluation of
Mediterranean fish and molluscs pan-fried in virgin olive oil. Journal of the Science of
Food and Agriculture, 84(13), 1750-1758.
D

Kalogeropoulos, N., Salta, F. N., Chiou, A., & Andrikopoulos, N. K. (2007). Formation and
distribution of oxidized fatty acids during deep- and pan-frying of potatoes. European
TE

Journal of Lipid Science and Technology, 109(11), 1111-1123.

Larsen, D., Quek, S. Y., & Eyres, L. (2010). Effect of cooking method on the fatty acid
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profile of New Zealand king salmon (Oncorhynchus tshawytscha). Food Chemistry,

119(2), 785-790.
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Martínez-Yusta, A., & Guillén, M. D. (2014a). Deep-frying food in extra-virgin olive oil: a
study by 1H Nuclear Magnetic Resonance of the influence of food nature on the
evolving composition of the frying medium. Food Chemistry, 150, 429-437.
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Martínez-Yusta, A., & Guillén, M. D. (2014b). A study by 1H nuclear magnetic resonance

of the influence on the frying medium composition of some soybean oil-food
combinations in deep-frying. Food Research International, 55, 347-355.
Martínez-Yusta, A., & Guillén, M. D. (2014c). Deep-frying. A study of the influence of the
frying medium and the food nature, on the lipidic composition of the fried food, using
1
H nuclear magnetic resonance. Food Research International, 62, 998-1007.
Martínez-Yusta, A., & Guillén, M. D. (2015). Monitoring compositional changes in
sunflower oil-derived deep-frying media by 1H Nuclear Magnetic Resonance. European
Journal of Lipid Science and Technology, ahead of print. DOI: 10.1002/ejlt.201500270
Mnari Bhouri, A., Jrah Harzallah, H., Dhibi, M., Bouhlel, I., Hammami, M., & Chaouch, A.
(2010). Nutritional fatty acid quality of raw and cooked farmed and wild sea bream
(Sparus aurata). Journal of Agricultural and Food Chemistry, 58(1), 507-512
Moradi, Y., Bakar, J., Motalebi, A. A., Syed Muhamad, S. H., & Che Man, Y. (2011). A
review on fish lipid: composition and changes during cooking methods. Journal of
Aquatic Food Product Technology, 20(4), 379-390.
Orban, E., Nevigato, T., Lena, G. D., Casini, I., & Marzetti, A. (2003). Differentiation in
the lipid quality of wild and farmed seabass (Dicentrarchus labrax) and gilthead sea
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bream (Sparus aurata). Journal of Food Science, 68(1), 128-132.

Ozogul, Y., Ozyurt, G., & Kuley Boga, E. (2009). Effects of cooking and reheating
methods on the fatty acid profile of sea bream treated with rosemary extract. Journal of
the Science of Food and Agriculture, 89(9), 1481-1489.
Oztop, M. H., Sahin, S., & Sumnu, G. (2007). Optimization of microwave frying of potato
slices by using Taguchi technique. Journal of Food Engineering, 79(1), 83-91.

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Saito, H., & Udagawa, M. (1992). Application of NMR to evaluate the oxidative
deterioration of brown fish meal. Journal of the Science of Food and Agriculture, 58,

RI
135-137.
Sanchez-Muniz, F. J., Viejo, J. M., & Medina, R. (1992). Deep-frying of sardines in
different culinary fats. Changes in the fatty acid composition of sardines and frying fats.

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Journal of Agricultural and Food Chemistry, 40(11), 2252-2256.
Sioen, I., Haak, L., Raes, K., Hermans, C., De Henauw, S., De Smet, S., & Van Camp, J.
(2006). Effects of pan-frying in margarine and olive oil on the fatty acid composition of

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cod and salmon. Food Chemistry, 98(4), 609-617.
Sopelana, P., Arizabaleta, I., Ibargoitia, M. L., & Guillén, M. D. (2013). Characterisation of
the lipidic components of margarines by 1H Nuclear Magnetic Resonance. Food
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Chemistry, 141(4), 3357-3364.
Weber, J., Bochi, V. C., Ribeiro, C. P., Victório, A. de M., & Emanuelli, T. (2008). Effect
of different cooking methods on the oxidation, proximate and fatty acid composition of
silver catfish (Rhamdia quelen) fillets. Food Chemistry, 106(1), 140-146.
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FIGURE CAPTIONS

Figure 1. 1H NMR spectra of sea bass (D. labrax) lipids before (LR) and after being pan-

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fried in sunflower oil (LPs), together with those corresponding to sunflower oil before (s),
after being heated in the pan without fish (sP) and after being used once to pan-fry sea bass

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(sPL). Some spectral regions are properly enlarged and the signal letters agree with those in

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Table 1.

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Figure 2. 1H NMR spectra of sea bream (S. aurata) lipids before (AR) and after being
microwave-fried in extra-virgin olive oil (AMevo), together with those corresponding to
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extra-virgin olive oil before (evo), after being heated in the microwave-oven without fish
(evoM) and after being used once to microwave-fry sea bream (evoMA). Some spectral
regions are properly enlarged and the signal letters agree with those in Table 1.
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Figure 1
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Figure 2

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Table 1. Chemical shift assignments and multiplicities of the 1H NMR signals in CDCl3 of the main acyl
groups, some minor compounds and some oxidation compounds present in the samples subject of study.
The signal letters agree with those given in Figures 1 and 2.

Chemical Functional group

Signal shift Multiplicity

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(ppm) Type of protons Compound
Main acyl groups

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A 0.88 t -CH3 saturated, monounsaturated
ω-9 and/or ω-7 acyl groups
0.89 t -CH3 unsaturated ω-6 acyl groups

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B 0.97 t -CH3 unsaturated ω-3 acyl groups

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C 1.19-1.42 m* -(CH2)n- acyl groups
D1 1.61 m -OCO-CH2-CH2- acyl groups, except for DHA,
EPA and arachidonic acyl groups
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D2 1.69 m -OCO-CH2-CH2- EPA and arachidonic acyl groups
E 1.92-2.15 m** -CH2-CH=CH- acyl groups, except for -CH2- of DHA
acyl group in β-position in relation to
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carbonyl group
F1 2.26-2.36 dt -OCO-CH2- acyl groups, except for DHA acyl groups
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F2 2.37-2.41 m -OCO-CH2-CH2- DHA acyl groups

2.77 t =HC-CH2-CH= diunsaturated ω-6
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acyl groups
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H 2.77-2.90 m =HC-CH2-CH= polyunsaturated ω-6 and ω-3

acyl groups
I 4.22 dd,dd ROCH2-CH(OR’)-CH2OR’’ glyceryl group in triglycerides
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J 5.27 m ROCH2-CH(OR’)-CH2OR’’ glyceryl group in triglycerides

K 5.28-5.46 m -CH=CH- acyl groups
Minor compounds
L 0.54 s -CH3 (C-18) Δ7-avenasterol
M 0.68 s -CH3 (C-18) cholesterol
N 0.68 s -CH3 (C-18) β-sitosterol and Δ5-campesterol
O 3.35 s -N(CH3)3 phosphatidylcholine
P 4.95-5.07 dq,dq -HC=CH2 unsaturated ω-1 acyl groups
Oxidation compounds
Q 9.49 d -CHO (E)-2-alkenals

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R 9.52 d -CHO (E,E)-2,4-alkadienals

S 9.60 d -CHO (Z,E)-2,4-alkadienals
T 9.75 t -CHO alkanals
Abbreviations: t: triplet; m: multiplet; *overlapping of multiplets of methylenic protons in the different acyl groups

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either in β-position, or further, in relation to double bonds, or in γ-position, or further, in relation to the carbonyl
group; DHA: docosahexaenoic acyl group; EPA: eicosapentaenoic acyl group; **overlapping of multiplets of the α-
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DHA acyl groups in β-position in relation to the carbonyl group; d: doublet; s: singlet; q: quartet.

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Table 2. Molar percentages (%) of acyl groups and concentration (mmol/mol of triglyceride) of some minor components and thermo-oxidation
products, determined by 1H NMR, in the oils employed in this study before, after being heated in the microwave-oven and in the pan, and after

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shallow-frying sea bream (S. aurata) or sea bass (D. labrax) fillets, either by microwave-frying or by conventional pan-frying. Different letters within
each column of both oils indicate a statistically significant difference (p<0.05).

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Extra-virgin olive oil (evo) Sunflower oil (s)

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Compositional parameters
evo evoM evoP evoMA evoPA evoML evoPL s sM sP sMA sPA sML sPL
Acyl groups (%)
ω-3 0.5±0.1a 0.6±0.0a 0.5±0.0a 4.1±0.7bc 4.7±0.6b 3.5±0.2c 3.1±0.3c 5.4±0.1a 4.2±0.1b 2.5±0.3c 2.5±0.1c

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--- --- ---
a a b b
DHA --- --- --- 1.8±0.3 2.1±0.3 1.1±0.0 1.0±0.1 --- --- --- 2.6±0.1a 2.0±0.1b 0.9±0.1c 0.9±0.1c
a a a a
EPA* --- --- --- 1.2±0.1 1.5±0.3 1.2±0.1 1.3±0.2 --- --- --- 1.5±0.2a 1.1±0.0b 1.0±0.1b 1.0±0.0b

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a a a b b c c
DUω-6 5.7±0.2 5.4±0.2 5.2±0.3 7.4±0.3 7.3±0.2 8.8±0.6 8.5±0.1 59.1±0.7 57.1±0.1 55.7±0.3 46.0±1.0c 49.1±1.1d 51.3±0.8d 51.3±0.9d
a ab b
a a a b b b
O+OU 81.0±0.3 80.7±0.1 80.2±0.2 73.2±1.1 71.8±0.7 72.6±0.8 72.9±0.9 30.4±0.6a 31.1±0.6a 32.3±0.1a 33.2±0.7a 31.9±0.9a 33.1±0.5a 32.6±1.1a
b
a a a
ω-1 --- --- --- 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0a --- --- --- 0.2±0.0a 0.1±0.0b 0.1±0.0b 0.1±0.0b
a a b c d c
U 87.3±0.0 86.7±0.1 85.9±0.1 84.8±0.2 84.0±0.4 84.9±0.1 84.6±0.5 89.5±0.1 88.2±0.6 88.0±0.4 84.8±0.1c 85.3±0.0c 86.9±0.0d 86.5±0.1d
c a b b

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a a b c d c
S+M 12.7±0.0 13.3±0.1 14.1±0.1 15.2±0.2 16.0±0.4 15.1±0.1 15.4±0.5c 10.5±0.1a 11.8±0.6b 12.0±0.4b 15.2±0.1c 14.7±0.0c 13.1±0.0d 13.5±0.1d
a a a b b b
O+OU 81.0±0.3 80.7±0.1 80.2±0.2 73.2±1.1 71.8±0.7 72.6±0.8 72.9±0.9b 30.4±0.6a 31.1±0.6a 32.3±0.1a 33.2±0.7a 31.9±0.9a 33.1±0.5a 32.6±1.1a
a a a
ω-1 --- --- --- 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0a --- --- --- 0.2±0.0a 0.1±0.0b 0.1±0.0b 0.1±0.0b
Minor components
(mmol/molTG)
Δ7-avenasterol
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--- --- --- --- --- --- ---
a a b ac c ac ac
Sit+Camp** 2.7±0.1 2.7±0.1 2.3±0.2 3.0±0.2 3.2±0.0 2.9±0.0 3.0±0.1 2.5±0.1a 2.5±0.3a 2.3±0.1a 3.1±0.1b 2.8±0.2ab 2.8±0.2ab 2.5±0.0a
a b bc de e cd de
1,2-diglycerides 11.2±0.1 8.8±0.0 8.0±0.3 6.9±0.4 6.1±0.7 7.3±0.1 7.0±0.4 4.9±0.5a 4.7±0.1a 4.8±0.3a 4.7±1.4a 4.3±0.3a 4.6±0.4a 4.0±0.3a
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Thermo-oxidation products
(mmol/molTG)
(E)-2-alkenals --- --- 1.7±0.0 --- --- --- --- --- 1.4±0.1a 1.5±0.0a --- --- --- 0.7±0.2b
a a b
(E,E)-2,4 alkadienals --- --- --- --- --- --- --- --- 2.2±0.0 2.1±0.0 --- 0.9±0.2 --- 1.1±0.2b
a b
(Z,E)-2,4-alkadienals --- --- --- --- --- --- --- --- 0.5±0.1 0.9±0.2 --- --- --- ---
Alkanals --- --- 1.8±0.0 --- --- --- --- --- 1.5±0.1a 1.5±0.1a --- --- --- ---
(E)-9,10-epoxystearate --- --- 1.8±0.1 --- --- --- --- --- --- --- --- --- --- ---
Abbreviations: M: microwave-frying; P: pan-frying; A: sea bream; L: sea bass; ω-3: omega-3; DHA: docosahexaenoic; EPA: eicosapentaenoic; DUω-6: diunsaturated omega-6; O+OU: oleic plus other
unsaturated acyl groups (monounsaturated, arachidonic and other minor unsaturated); ω-1: omega-1; U: total unsaturated; S+M: saturated plus modified; O+OU: oleic plus other unsaturated acyl
groups (monounsaturated, arachidonic and other minor unsaturated); ω-1: omega-1; TG: triglycerides; Sit+Camp: β-sitosterol and Δ5-campesterol.
(*) Arachidonic acyl groups not considered, because their content in these farmed fishes is usually very low (Orban et al., 2003).
(**) For fried samples this value includes, in addition to β-sitosterol and Δ5-campesterol, a certain amount of cholesterol migrated from the fish lipids to the frying oil.
* For fried samples this value includes, in addition to β-sitosterol and Δ5-campesterol, a certain amount of cholesterol migrated from the fish lipids to the frying oil.

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Table 3. Molar percentages (%) of acyl groups and concentration (mmol/ mol of triglyceride) of some minor
components of fish lipids extracted from sea bream (S. aurata) and sea bass (D. labrax) fillets before and after
shallow-frying, either by microwave-frying or by conventional pan-frying using extra-virgin olive oil and
sunflower oil, determined by 1H NMR. Different letters within each column of lipid extracts indicate a
statistically significant difference (p<0.05).

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Compositional Farmed sea bream (A, S. aurata) Farmed sea bass (L, D. labrax)
parameters AR AMevo APevo AMs APs LR LMevo LPevo LMs LPs

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Acyl groups (%)
ω-3 19.6±0.3a 15.0±0.6b 16.8±0.2cd 17.2±0.1c 16.5±0.1d 15.3±0.5a 11.5±0.7b 12.2±0.2b 9.7±0.8c 12.6±0.9b

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a b c c c a b b c
DHA 9.1±0.2 7.0±0.3 7.9±0.1 8.0±0.0 7.8±0.1 5.4±0.3 4.1±0.4 4.2±0.1 3.5±0.2 4.6±0.4b
EPA* 4.9±0.1a 3.8±0.1b 4.2±0.1c 4.2±0.2c 4.1±0.1c 5.5±0.2a 4.1±0.2b 4.2±0.0b 3.2±0.5c 4.2±0.2b
DUω-6 12.6±0.4a 10.8±0.2b 11.5±0.3b 19.9±0.8c 20.2±0.6c 18.5±0.3a 15.8±0.4b 16.7±0.4ab 35.6±2.6c 26.3±0.4d

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a b c d d a b b c
O+OU 43.4±0.5 52.8±0.3 49.1±1.2 41.3±0.4 41.2±0.6 43.5±0.5 52.3±2.0 50.8±1.2 37.4±1.3 41.1±0.7a
ω-1 0.5±0.0a 0.3±0.0b 0.4±0.0b 0.4±0.0b 0.4±0.0b 0.4±0.0a 0.3±0.0b 0.3±0.0b 0.3±0.0b 0.3±0.0b
U 76.1±0.2a 78.9±0.2b 77.8±0.8c 78.8±0.6b 78.2±0.1bc 77.6±0.6a 79.7±0.9b 80.0±0.8b
MA 83.0±0.4c 80.3±0.2b
S+ M 23.9±0.2a 21.1±0.2b 22.2±0.8c 21.2±0.6b 21.8±0.1bc 22.4±0.6a 20.3±0.9b 20.0±0.8b 17.0±0.4c 19.7±0.2b
a b c d d a b b c
O+OU 43.4±0.5 52.8±0.3 49.1±1.2 41.3±0.4 41.2±0.6 43.5±0.5 52.3±2.0 50.8±1.2 37.4±1.3 41.1±0.7a
ω-1 0.5±0.0a 0.3±0.0b 0.4±0.0b 0.4±0.0b 0.4±0.0b 0.4±0.0a 0.3±0.0b 0.3±0.0b 0.3±0.0b 0.3±0.0b
Minor components
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(mmol/molTG)
Δ7-avenasterol --- --- --- 0.4±0.1a 0.4±0.1a --- --- --- 0.9±0.1a 0.6±0.1b
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Cholesterol* * 4.7±0.6a 8.0±0.3b 5.8±0.3c 7.1±0.9bd 6.1±0.4d 4.3±0.6a 4.9±0.2a 6.4±1.1b 6.7±0.5b 6.1±0.2b
Phosphatidylcholine 1.4±0.3a 7.7±0.6b 5.4±0.3c 7.3±1.3b 5.8±0.3c 1.7±0.7a 5.3±0.3b 8.6±3.2cd 11.3±0.3c 6.8±0.1bc
a b ac c c a a a a
1,2- diglycerides 1.8±0.4 2.8±0.1 1.7±0.1 1.3±0.3 1.3±0.2 0.9±0.4 1.9±0.6 1.9±0.5 1.7±0.5 1.0±0.3a
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Abbreviations: R: raw; M: microwave-frying; evo: extra-virgin olive oil; P: pan-frying; s: sunflower oil; ω-3: omega-3; DHA: docosahexaenoic; EPA:
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eicosapentaenoic; DUω-6: diunsaturated omega-6; O+OU: oleic plus other unsaturated acyl groups (monounsaturated, arachidonic and other minor
unsaturated); ω-1: omega-1; U: total unsaturated; S+M: saturated plus modified; O+OU: oleic plus other unsaturated acyl groups (monounsaturated,
arachidonic and other minor unsaturated); ω-1: omega-1; TG: triglycerides.
* For fried samples this value includes, in addition to cholesterol, a certain amount of β-sitosterol and Δ5-campesterol migrated from the frying oil to
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the fish fillet.

(*) Arachidonic acyl groups not considered, because their content in these farmed fishes is usually very low (Orban et al., 2003).
(**) For fried samples this value includes, in addition to cholesterol, a certain amount of β-sitosterol and Δ5-campesterol migrated from the frying oil
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Table 4. Fat content (g) of fish fillets before and after shallow-frying, together with the estimation by 1H
NMR of the contribution of migrated fish lipids (oil) to fried oil (fried fish lipids), expressed as molar
percentage of acyl groups.

Studied frying systems Fat content of fish fillet (g) Contribution of the Contribution of the

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fish lipids migrated oil migrated to
Oil Fish Frying technique Before frying After frying to the fried oil (%) fried fish lipids (%)
evo Sea bream Pan frying 41.9±2.3 26.3±3.4 24.8±3.3 15.2±0.5

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evo Sea bream Microwave frying 28.8±1.5 20.3±2.9 21.6±2.5 25.0±0.8
evo Sea bass Pan frying 26.3±4.7 24.8±7.3 22.2±4.2 19.8±4.4

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evo Sea bass Microwave frying 31.4±3.1 30.6±2.1 22.9±2.0 23.0±2.1
s Sea bream Pan frying 33.2±1.2 24.1±1.3 21.5±8.1 16.2±0.6
s Sea bream Microwave frying 43.8±2.2 28.1±2.9 28.1±5.5 16.1±3.2

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s Sea bass Pan frying 22.4±4.7 18.7±0.8 19.4±4.0 19.5±2.2
s Sea bass Microwave frying 18.7±6.5 25.7±4.9 19.5±2.4 42.7±4.5
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Graphical Abstract

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Highlights

 The study of fish shallow-frying is undertaken for the first time by 1H NMR.

 Migration of main and minor lipidic compounds between oils and fish occurs.

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 Thermo-oxidation and hydrolysis reactions in oils and fish lipids are studied.

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 Pan-frying provokes higher thermo-oxidation than microwave-frying.

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 As for aldehyde formation, extra-virgin olive oil is safer than sunflower oil.

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