Assessment of a Diterpene-Rich Rosemary (Rosmarinus officinalis L.) Extract as a Natural Antioxidant for Salmon Pâté Formulated with Linseed

The use of natural plant extracts with standardised antioxidant properties is a growing strategy to stabilise food products. The use of a rosemary lipophilic extract (RLE), obtained from the by-product of high-yield selected plants and rich in polyphenols (334 mg/g, with diterpenes such as carnosic acid and carnosol as main compounds), is here proposed. Four RLE doses (0, 0.21, 0.42 and 0.63 g/kg) were tested in a salmon pâté formulated with sunflower oil and linseed, which was pasteurised (70 °C for 30 min) and subjected to storage at 4 °C and 600 lux for 42 days. Rosemary diterpenes resisted pasteurisation without degrading and showed antioxidant activities during the shelf-life of pasteurised pâté. RLE addition led to increased peroxide value (from 3.9 to 5.4 meq O2/kg), but inhibited formation of secondary oxidised lipids such as malondialdehyde (from 1.55 to 0.89 mg/g) and cholesterol oxidation products (from 286 to 102 µg/100 g) and avoided discolouration (slight brownness) in the refrigerated pâté. However, this did not entail relevant changes in fatty acid content or in the abundance of volatile organic compounds from oxidised lipids. Increasing the RLE dose only improved its antioxidant efficacy for some oxidation indexes. Thus, the oxidative deterioration of these types of fish emulsion can be naturally controlled with rosemary extracts rich in diterpenes.


Introduction
Fish emulsions, such as salmon pâté, represent a valuable dietary source of polyunsaturated fatty acids (PUFA), in particular, of some essential long-chain n-3 PUFA, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [1]. The intake of these two PUFA has been correlated with a lesser risk of cardiovascular diseases [2], neurodegenerative diseases and inflammatory disorders [3]. However, PUFA are particularly prone to oxidation and can be degraded during manufacturing and storage of fish pâté due to oxidising factors such as oxygen, iron, salt, heating and mechanical treatments [4,5]. Lipid oxidation can generate secondary compounds, such as malondialdehyde (MDA) and cholesterol oxidation products (COP), together with volatile organic compounds (VOC), including hydrocarbons, aldehydes, ketones and alcohols, among others, responsible for flavour and rancidity [6]. An excessive intake of oxidised lipids may increase the risk of some pathologies such as metabolic and neurodegenerative diseases and some cancers [7,8]. Oxidation reactions involving other pâté components may also lead to some changes in colour and pH. Therefore, it is justified that fish pâté, a product rich in unsaturated fat,

Experimental Design
A randomised statistical design was performed for the experiment. Three doses of RLE were tested in a PUFA-enriched salmon pâté formulated with linseed: (i) Low (R21): 0.21 g/kg, (ii) Medium (R42): 0.42 g/kg and (iii) High (R63): 0.63 g/kg. R63 treatment provided 150 mg carnosic acid + carnosol per kg pâté, the maximum dose authorised by EC [22]. Lipid oxidation (primary and secondary) and some related parameters (colour and pH) were assessed in salmon pâté just after pasteurisation and further displayed for 42 days. Sample size was n = 9 (3 pâté jars × 3 fabrication batches). A two-way ANOVA (Repeated Measures Design) was performed to determine the effects of treatments (RLE and storage time) on the dependent variables. The Tukey's range test was used. Data were analysed with the Statistix 8.0 software for Windows (Analytical Software, Tallahassee, FL, USA).

Obtention of Rosemary Lipophilic Extract (RLE)
Selected high-yielding rosemary plants from a germplasm bank were cultivated and harvested in the experimental farm of the Institute of Agricultural and Food Research and Development (IMIDA, Murcia, Spain). RLE was obtained from the leaf by-product that had been previously distilled with water steam and extracted with water to obtain the essential oil and the RWE, respectively [27]. Then, 3 g of rosemary by-product were mixed with 450 mL acetone:water (1:1, v/v) and kept under constant stirring (30 • C for 90 min). The mix was centrifuged in a Digecen 21 R centrifuge (Orto Alresa, Madrid, Spain) (4560× g and 5 • C for 10 min) and the supernatant was then filtered (Whatman No. 4). The acetone fraction was removed by applying a vacuum at 40 • C in a Syncore Polyvap R-96 evaporation system (Buchi, Flawil, Switzerland), and the water fraction was removed using a lyophilisation system (Lyobeta 15, Telstar, Terrassa, Spain) (100 mbar and −80 • C for 24 h). Lyophilized RLE, a yellow-greenish dry powder with an intense herbal flavour, was stored at −80 • C until further use.

Salmon Pâté Manufacturing
Salmon pâté ingredients are shown in Table 1. Norwegian fresh salmon (Salmo salar) loins were purchased from Nordlaks Produkter AS (Murcia, Spain) and stored at −20 • C for 48 h. Thawed loins (at 4 • C/24 h) without skin and bones were cut in cubes (2 cm 3 ) using a knife and cooked in water (2:1; w:v) at 90 • C for 10 min under stirring in a cooking robot (Taurus Mycook 1.6, Lérida, Spain). Before homogenising, cooked salmon cubes were minced for 10 min, skimmed milk was dissolved in water at 50 • C and linseed was hydrated at 1:2 (w:v) in a Silent Crusher homogeniser at 9500 rpm for 1 min. All ingredients (including flavouring, thickening, dye and RLE) were homogenised together for 10 min. Once paste was obtained, sterile glass jars (33-mL capacity) were manually filled and pasteurised in a water bath at 70 • C for 30 min. The jars were cooled in a water bath at 5 • C for 30 min and then kept for up to 42 days in a refrigerated display cabinet (Mod. 103899, Difri, Xirivella, Valencia, Spain) at 4 • C, 30% RH and white continuous illumination (600 lux). The antimicrobial efficiency of these pasteurisation conditions was checked in a previous trial [28]. Samples were stored at −80 • C before analyses.

Proximate Composition
Crude protein content was determined by the Kjeldahl method (reference 955.04; AOAC, 2000) [32]. Crude ash content was estimated by incineration (at 550 • C/24 h) in a furnace (reference 923.03; AOAC, 2000) [32]. Moisture content was determined with an infrared thermobalance (Model MA 50.R; Radwag, Radom, Poland). Available carbohydrates were estimated by weight difference. The energy value was calculated using standard conversion factors: 4 kcal/g for protein and carbohydrate and 9 kcal/g for fat, respectively [33].

Lipid Extraction for Analyses
Pâté fat was extracted by the Folch method with some modifications [34]. First, 15 g of sample were mixed with chloroform:methanol (2:1, v/v) and 0.003% (w/v) BHT and then mixed with 1 M KCl. The organic phase was separated and dried in a Hei-vap rotavapor (Heidolph, Schwabach, Germany) at 30 • C and 337-470 mm Hg pressure. Fat content was gravimetrically determined (total lipids) and stored at −80 • C until analyses.

Peroxide Value (PV)
PV indicated the amount of hydroperoxides of fat pâté (AOCS Cd 8-53) [35]. In brief, 0.5 g fat was homogenised for 1 min with 10 mL chloroform, 15 mL of glacial acetic acid Antioxidants 2022, 11, 1057 5 of 17 and 1 mL saturated potassium iodide water solution. The sample was kept in darkness for 5 min, mixed with 75 mL water and titrated with 0.002 N sodium thiosulphate solution in an automatic Titrino equipped with a 0160451100 PT WOC combined electrode (Metrohm Hispania, Madrid, Spain). Results were expressed as meq O 2 /kg fat.

Conjugated Dienes (CD) and Trienes (CT)
CD and TC were determined according to Pegg (2001) [36], with some modifications. A 0.1 g sample was dissolved in 10 mL isooctane and absorbance was measured at 233 nm (CD) and 268 nm (TC) in a UV/Vis spectrophotometer (Spectronic Unicam, New York, NY, USA). Results were expressed as µmol/g.

Sterols and Cholesterol Oxidation Products (COP)
Sterols were extracted and purified according to Cardenia et al. (2015) [34]. About 200 mg of lipid extract containing internal standards for quantification of cholesterol and COP (141.12 µg of betulinol (Sigma Chemical, St. Louis, MO, USA) and 13.38 µg of 19-hydroxycholesterol (Steraloids, Newport, RI, USA), respectively) were cold saponified. Then, 100 µL of the unsaponifiable fraction were used for determining sterol composition, while the rest (900 µL) was subjected to SPE-NH 2 for COP purification. Both sterol and COP fractions were derivatized with 1 mL of a silylating mixture (pyridine:hexamethyldisilazane: trimethylchlorosilane, 5:2:1, v/v/v), left standing at 40 • C for 20 min, dried under a nitrogen stream, re-dissolved in n-hexane (300 µL and 20 µL for sterols and COP, respectively) and injected into a Fast gas-chromatograph/mass-spectrometer (Fast GC/MS) as reported by Cardenia et al. (2012) [38], with slight modifications. For COP analysis, the split ratio was 1:15. Mass spectra and retention times of chemical standards (Sigma Chemical; Steraloids (Newport, RI, USA); Avanti Polar Lipids (Alabaster, AL, USA)) were compared with the chromatographic peaks of fish pâté for correct identification of sterols and COP. Both sterols and COP were quantified in the SIM acquisition mode, using calibration curves for each chemical compound, and their results were expressed as mg/100 g of pâté and µg/100 g of pâté, respectively. The proportion of total cholesterol oxidation (OR) was calculated as follows: OR = [(Total COP/Total cholesterol) × 100]/1000 [34]. Nine independent replicates were made for each sample.

Volatile Organic Compounds (VOC)
VOC were determined by headspace solid phase-micro-extraction (HS-SPME) according to Ortuño et al. (2021) [39], with some modifications. First, 5 g pâté were placed in amber 20-mL screw-capped vials (Agilent Technologies, Frankfurt, Germany) and flushed with nitrogen (at 275 kPa for 5 s). Before HS-SPME, the vials were kept in a water bath (at 40 • C for 10 min). VOC extraction was carried out in a water bath MPS 2XL autosampler (at 40 • C for 45 min) with continuous shaking (250 rpm) (Gerstel, Mülheim an der Ruhr, Germany). Analyses were performed with a 7890B Agilent gas chromatograph coupled to a 5977 A MSD mass spectrometer (GC-MS) using a VF-WAXms capillary column (30 m . Operating conditions were: needle immersion depth: 2.5 cm; vaporisation chamber diameter: 0.75 mm; desorption time inside the injector: 5 min; injection port and ionisation source temperature: 250 and 280 • C, respectively. The oven program was: 40 • C to 150 • C at 2.5 • C/min and then taken to 250 • C at 10 • C/min. Identification of individual compounds was performed by comparison of the spectra with the NIST 98 mass spectrometry library (NIST, Gaithersburg, MDN) and information obtained in previous trials [39]. Results (relative abundance) were expressed as Arbitrary Units (AU) × 10 6 .
Salmon pâté contained 12 g/100 g total protein, 19 g/100 g total lipids and 61 g/100 g moisture, contributing with 270.6 kcal/100 g (Table 3). Data regarding general lipid oxidation, CIELab colour and pH are shown in Table 4. RLE addition did not affect PV on day 0, but R63 pâté presented the highest PV on day 42, while not impacting CD and CT values on day 0 and 42. Thus, PV discriminated the effects of RLE on primary lipid oxidation better than CD and CT, which provide similar information. Chill storage did not affect PV but actually decreased CD and CT values. The antioxidant effects of RLE addition on lipids were confirmed by TBARS. MDA levels were higher in the untreated pâté on day 0 and 42 than in the pâté with RLE (at any dose). No differences were found in the MDA levels among R21, R42 and R63 pâtés. Similarly, adding RLE did not affect colour on day 0, while pâtés with more RLE (R42 and R63) had lower values of L* and a* and higher values of b* and h* than the untreated pâté on day 42. Therefore, salmon pâté underwent some discolouration (slight browning) during chill storage that might be inhibited using RLE. Regardless of the dose used, RLE slightly decreased the pâté pH on days 0 and 42. Overall, the pH values hardly decreased by around 0.2 after chill storage.     Twenty-two FA were quantified (g FA/100 g fat) ( Table 5). The most abundant class was MUFA (22.7-26.2 g/100 g fat) with C18:1t n-9 as main FA (18.3-22.2 g/100 g fat), followed by PUFA (17.3-20.2 g/100 g fat), with C18:2c n-6 (11.8-13.8 g/100 g fat) and C18:3α n-3 (2.1-2.7 g/100 g fat) as major FA, and by SFA (9.2-10.6 g/100 g fat), with C16:0 as predominant FA (3.6-4.6 g/100 g fat). Levels of EPA and DHA were around 1 and 1.4 g/100 g fat, respectively. RLE addition did not affect the FA levels on day 0 and 42, while chill storage only led to some changes in minor FA. TFA content was similar for the untreated and the RLE pâtés on day 0 and 42. Consequently, nutritional FA ratios (n-6/n-3 and P/S) were unaffected by RLE addition or chill storage.

Discussion
Recovery of rosemary polyphenols depends on the raw materials, solvents and operating conditions applied to obtain the extracts [19,40]. Different studies agree that carnosic acid and carnosol are the most abundant polyphenols present in RLE [19,21,40]. The reported concentration ranges are 47-179 mg/g for carnosic acid, 5-28 mg/g for carnosol and 50-200 mg/g for total polyphenols [19,41]. Thus, the RLE tested in the present study was particularly rich in polyphenols (333 mg/g), which was one of the main objectives of the plant's selection. As a purity criterion, European Union Directive 2010/67/UE [42] establishes that the RLE for food application must contain at least 10% (w:w) of carnosic acid plus carnosol, a percentage widely exceeded by the RLE used in this experiment. Assessment of the stability of carnosic acid and carnosol provides an idea of how rosemary antioxidants behave in food matrices. Under oxidising conditions, carnosic acid is transformed into carnosol [21,43], which can be regenerated or not by other antioxidants acting in the food matrix. As observed in the experimental data, both diterpenes were quite resistant to the pasteurisation conditions applied, as has also been reported for carnosic acid in pork liver pâté [24]. In general, rosemary diterpenes are quite resistant to the cooking procedures applied in food [24,41]. In the present study, the degradation of carnosic acid and carnosol mainly occurred during chill storage. This was expected, since salmon pâté was aerobically homogenised, which favours the presence of occluded oxygen, and jars were kept under refrigeration and fluorescent lighting (600 lux) for 42 days. Doolaege et al. (2012) [24] found that a part (6-32 mg/kg) of the added carnosic acid (250-750 mg/kg) degraded in liver pâté kept at 4 • C for 48 h. There were no available data on the stability of rosemary polyphenols in other studies on the antioxidant properties of RE in meat and fish products [11,23].
The formulation used for salmon pâté in the present study enabled the reduction of fat content and caloric intake compared to commercial fish pâtés (30% fat and 330 kcal/100 g pâté) [44][45][46]. Salmon pâté reflected the FA and sterol profiles of the different fat sources used as ingredients (salmon muscle, sunflower oil and linseed). The FA profile was similar to those reported for other fish pâtés [45][46][47]. The relevance of the different FA classes (MUFA > PUFA > SFA) is coherent with the above-mentioned fat sources, since salmon flesh and linseed are rich in MUFA and PUFA [30,48], while sunflower oil is rich in PUFA [49]. Salmon muscle is rich in cholesterol, while β-sitosterol is the main phytosterol in sunflower oil and linseed [50,51]. Sterol content tended to decrease in all pâtés following chilled storage, likely due to the oxidation reactions involving lipids. The cholesterol content of the salmon pâté coincides with that reported by Echarte et al. (2004) [47]. From a nutritional standpoint, salmon pâté showed a n-6/n-3 ratio (2.3-2.6) below the recommended level of 4 [52], resulting in a balanced dietary source of n-3 FA. An adequate n-6/n-3 ratio in the diet contributes to optimising bioavailability, metabolism and incorporation of FA into membrane phospholipids [30]. Likewise, the P/S ratio (1.79-1.97) was above the minimum recommended (0.5-0.7) [52], which is considered a good nutritional trait. An increase in the P/S ratio can lead to reduced plasma total cholesterol [52], while, at the technological level, it is a useful indicator of fat oxidation susceptibility, which mainly affects PUFA. In the present study, pâté lipids, integrated by a high proportion of PUFA, were quite stable during chill storage, perhaps partly due to linseed, a thickening agent containing hydrocolloids that might help stabilising the emulsion [53].
Rosemary polyphenols, containing benzene rings able to act as free radical scavengers, fulfilled their antioxidant role delaying lipid oxidation in the pâté; however, in quantitative terms, the addition of up to 210 mg/kg of rosemary polyphenols does not seem enough to protect a fatty fish emulsion (19 g fat/100 g pâté, of which, 4.7 g MUFA and 3.4 g PUFA) against oxidation. Lipid oxidation reactions involve three stages: initiation, propagation and termination. Some techniques measure the loss of initial reactants (such as oxygen, lipid and antioxidants), others the formation of primary oxidation products (such as hydroperoxides and conjugated dienes) and others the formation of secondary oxidation products (such as alcohols, aldehydes, hydrocarbons and ketones) [54]. Thermal exposure in the presence of oxygen induces lipid oxidation, which forms hydroperoxides and causes double bond displacement or isomerisation, resulting in an increased production of CD and CT from unsaturated FA [55]. As oxidation advances in pâté during the display period, secondary oxidised lipids are formed to the detriment of the early oxidised lipids, even though they may still be generated. This may explain why primary oxidation stabilised or decreased after chill storage, as indicated by the reduction observed in the CD and CT values. The reactive chemical species that gave rise to CT evolved towards other secondary compounds such as TBARS [56]. PV was the sole index that discriminated the antioxidant activity of rosemary polyphenols in the early stages. As observed in Table 4, R63 pâté clearly had more hydroperoxides than the other samples on day 42, suggesting that the formation of secondary oxidised lipids such as TBARS and VOC was inhibited. Rosemary polyphenols may inhibit the formation of oxidised lipids through hydrogen donation or preventing the cleavage of lipid hydroperoxides [55].
TBARS is a widely used index with technological (flavour and rancidity) and nutritional (toxicity) implications that measures the levels of aldehydes and other secondary oxidised lipids in the pâté. Lipid oxidation is enhanced by the thermal treatment and favoured by fat unsaturation and the presence of oxygen. Salmon pâté presented an incipient lipid oxidation just after pasteurisation. The antioxidant role of RLE during pâté preparation was modest, since it only slightly inhibited the formation of MDA and was not dose-dependent. Salmon was cooked before adding RLE, which would explain why there were no marked differences regarding MDA levels among the different freshly pasteurised pâtés. TBARS formation continued during further retail display. On day 42, MDA level practically doubled in the pâté formulated without RLE, reaching TBARS values near 2 mg MDA/kg, the threshold value from which rancidity is sensorially detected in cooked meat [57]. In contrast, RLE clearly inhibited MDA formation in the chill-stored pâté. However, increasing RLE dose did not improve results. In other studies, lipid oxidation was inhibited when using different RLE in pork liver pâté [24,58], chicken pâté [5] or RWE in frozen salmon [18] and refrigerated sardines [26], confirming the present findings.
The antioxidant effect of RLE was also confirmed for cholesterol. The COP profile found in this study is similar to that reported by Echarte et al. (2004) [47] for salmon pâté, where the most abundant COP originated from the monomolecular oxidation reaction pathway involving B ring (i.e., 7-oxysterols), while epoxy and triol derivatives, generated from a bimolecular reaction pathway, were found in smaller quantities (β-EC and triol) or even undetected (α-EC) in the pâtés with RLE. The amounts of total COP formed do not correspond to the observed decrease in cholesterol, which might be due to the reaction of COP with amino groups from amino acids, peptides and proteins leading to the formation of Schiff bases [59] and/or the formation of mid-polarity sterol oxidation products [60] that cannot be determined under the analytical conditions used. Total COP and OR showed the same trend as TBARS, with values in untreated samples that were about twice as much as those found in pâtés with RLE at 0 and 42 days of chilled storage. Cholesterol oxidation is known to be favoured in the presence of oxygen and during chilled display [61], but RLE addition was able to greatly hinder COP formation, without a dose-dependent effect. RLE addition did not initially affect pâté colour, as also reported in pork liver pâté with RLE [25]. It is unlikely that a small amount of RE can alter the colour of an orange salmon pâté that was also coloured with capsanthin. Other studies on pork liver pâté made with tea and grape seed extracts [62] and with seaweed extracts [56] reported some changes in CIELab colour due to the contribution of pigments such as chlorophylls [63] or by the action of polyphenol oxidases able to condense quinones, forming dark compounds [62,64]. As these enzymes are inactivated by heating, it is unlikely that enzymes from a rosemary by-product (distilled with hot water steam) remain active in a thermally treated pâté. The untreated pâté showed some discolouration on day 42, as indicated by the increased Hue angle, a browning index, thus evidencing a general oxidation of product. Discolouration processes affecting the cooked-chilled muscle products may be ascribable to metmyoglobin formation, the denaturation of myofibrillar proteins producing colour changes when interacting with myoglobin [18,65] and lipid oxidation [25]. Similarly, colour could be stabilised in pork liver pâtés with RLE [24,25] or with beer residue, chestnut leaves and peanut skin [66], as well as in frozen salmon with rosemary whole extract [18].
The pH of pâté was slightly lower when RLE was incorporated into its formulation. Similar results were found in pork liver pâté made with RE [24] and with other vegetable extracts [62,66]. The natural acidity of plant extracts may decrease the pH of treated pâtés [62]. In the present study, this effect was not dose-dependent, which suggests that some components of RLE were implicated in chemical reactions involving small changes in pH. The pH dropped slightly more (by 0.2) after storage in all pâtés, formulated with and without RLE, likely due to oxidation phenomena, since microbial events often result in more pronounced changes in pH and, in addition, the efficacy of pasteurisation treatment had been checked previously [28]. Moreover, carnosol and carnosic acid may present bacteriostatic activities, as these can alter the lipidic order of phospholipidic membranes [67].
A relevant part of the VOC identified in headspace corresponded to C5-C8 aliphatic aldehydes and alcohols. Hexanal has been identified as the most abundant VOC in sunflower and linseed oil blends, together with other VOC (propanal, pentanal, 1-penten-3-one, 1-pentanol, octanal, 1-octen-3-one, 1-octen-3-ol and (E,Z)-2,4-heptadienal) that contribute to oil aroma [68]. In addition, saturated and unsaturated aliphatic VOC can be generated from the thermal degradation and oxidation of C18-C20 MUFA and C18-C22 PUFA present in fish lipids. In fact, propanal and hexanal are the most abundant VOC detected in cooked salmon aroma [69,70]. Benzene derivatives at different oxidation stages (e.g., toluene; benzaldehyde and benzoic acid) can also be formed in cooked salmon from aromatic precursors [69,70]. BHT can be used to stabilise carotenoid dyes, which might explain why BHT appeared in pâté headspace. α-Pinene and eugenol are volatile terpenes that can proceed from rosemary by-products [71] or can be directly formed in cooked fish [69,70]. Unlike the TBARS trend, the VOC levels, except for 1-pentanol, did not allow discrimination of the antioxidant effect of RLE on salmon pâté lipids. For instance, inhibition by RLE of hexanal, a well-known VOC generated by cooking, could not be proven. The VOC profile of salmon pâté suggests that vegetable VOC from sunflower and linseed predominated over VOC from fish in headspace under the conditions used for HS-SPME. The number of VOC identified in salmon pâté was actually modest compared to those reported for cooked salmon [69,70]. Moreover, many characteristic C9-C11 aliphatic aldehydes (e. g. nonanal and nonenal) of flesh origin were not detected. In general, the VOC detected in salmon pâté seems to provide little information about lipid oxidation and would thus be of little interest as lipid oxidation markers. In any case, sampling of different manufacturing batches might have increased intra-group variability in parameters (FA, sterols and others) related to the composition of pâté, making treatment effects less evident.

Conclusions
Food applications for rosemary extracts require standardising their polyphenol content. The crop of high-yielding selected rosemary plants can help in improving and standardising these extracts. As reported in the present study, the rosemary lipophilic extract used is rich in polyphenol antioxidants, particularly diterpenes, and provides good results in salmon pâté, a product prone to oxidation. Rosemary diterpenes resist pasteurisation without degrading and can act as antioxidants during the shelf-life of pasteurised pâté. As a result, lipids are stabilised against oxidation and salmon pâté contains less secondary oxidation compounds, in all likelihood being healthier. However, this did not entail relevant changes in the fatty acid content or relative abundance of volatile organic compounds from lipids. Therefore, oxidative deterioration of these types of fish emulsions can be naturally controlled with rosemary extracts rich in diterpenes, thus representing a valid alternative for the formulation of clean label seafood products. It is not necessary to reach the maximum quantity of rosemary diterpenes (150 mg/kg) authorized for fish products to obtain a suitable antioxidant effect in this PUFA-enriched pâté. Most of the oxidation indices improved in the salmon pâté when a third of the aforementioned dose was used.