Lipid stability: fatty acids profile and lipid oxidation

Fifteen fatty acids (FA) were identified in the salmon samples under study, as can be seen in Table 5.4. The FA composition did not change significantly between samples, showing a moderate content of polyunsaturated fatty acids (PUFAs, 17.6-22.1 g/100 g lipids), with a low amount of saturated fatty acids (SFA, 9.6-11.6 g/100 g lipids) and a high content of monounsaturated fatty acids (MUFA, 37.4-42.4 g/100 g lipids). The PUFA/SFA ratio was between 1.77 and 1.97, which were in all the cases higher than the minimum recommended value for human diet of 0.45 [22].

The total SFA composition was not significantly (p > 0.05) affected by storage (Table 5.4). The source of total SFAs mainly came from myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0), with FA contents in the initial salmon samples of 1.14 ± 0.12, 7.57 ± 1.04 and 2.05 ± 0.16 g/100 g lipids, respectively. Among SFAs, palmitic acid (C16:0) was largely predominant in all samples (7.15-8.71 g/100 g lipids), followed by stearic acid (C18:0) (1.57-2.06 g/100 g lipids). Similar results were observed for other fish species, such as saithe (Pollachius virens) and hoki (Macruronus novaezelandiae) [23], and Atlantic mackerel (Scomber scombrus) [24].

MUFA predominated in the FA profile (Table 5.4), with palmitoleic acid (C16:1n-7), vaccenic acid (C18:1n-7), oleic acid (C18:1n-9), gadoleic acid (C20:1n-9) and eruric acid (C22:1n-9) showing initial values of 1.59 ± 0.28, 3.56 ± 0.55, 29.29 ± 7.11, 4.84 ± 0.58 and 0.64 ± 0.06 g/100 g lipids, respectively. Among the MUFAs, oleic acid (C18:1n-9) showed the higher amount, followed by gadoleic acid (C20:1n9). Gonçalves et al. (2017) [25] also reported oleic acid as the predominant FA in farmed Chilean salmon samples, with concentrations within the range of 29.8-33.1 g/100 g lipids, being in agreement with the values obtained in the present work. Furthermore, no significant difference (p > 0.05) in MUFA content were found among the samples, regardless of storage condition and storage time.

The highest amount of PUFA was associated to n-6 compounds (10.6-13.3 g/100 g lipids), followed by n-3 compounds in a lower content (6.3-9.3 g/100g lipids). These results were similar to Gonçalves et al. (2017) [25] for farmed Chilean salmon. These results are associated to the feeding of farmed fish caused by the substitution of fish diets with other

alternatives with low quantities of n-3 and high levels of n-6 compounds (vegetal source) and SFA (animal source) [25]

Total n-3 PUFA were mainly composed of eicosatetraenoic acid (C20:4n-3), eicosapentaenoic acid (EPA, C20:5n-3), docosapentaenoic acid (C22:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), presenting an initial FA content of 0.74 ± 0.10, 1.98 ± 0.16, 0.94 ± 0.11 and 4.51 ± 0.70 g/100 g lipids, respectively (Table 5.4). From the identified n-3 PUFAs, only DHA showed significant variation (p < 0.05) during storage. HS samples showed tendency to have higher DHA content than the AP samples (mainly at AP/5 ºC for 15 days and AP/25 ºC for 5 days), with values between 4.24 and 5.39 g/100 g lipids, compared to values between of 3.23 and 4.21 g/100g lipids for AP samples. DHA is very unstable toward oxidation due to the high number of double bonds in its chemical structure, so for AP samples the decrease of this PUFA might be due to its susceptibility to oxidation at RT (25 ºC after 5 days) and under refrigeration (5 ºC for longer storage times, 15 days).

These results are in agreement with those obtained by Chaijan et al. (2005) [26] and Tenyang et al. (2017) [27] who observed a decrease in PUFA content during refrigerated storage of sardine (Sardinella gibbosa) and catfish oil, respectively. The reason for the different results for DHA at AP and HS might be related to the different behaviour of the samples concerning lipid oxidation (see below) and should be further studied. Furthermore, several n-6 PUFAs were detected in the salmon samples: linoleic acid (C18:2n-6), eicosadienoic acid (C20:2n-6) and docosadienoic acid (C22:2n-(C20:2n-6), with values for the initial salmon samples of 9.04 ± 1.63, 0.95 ± 0.15 and 2.24 ± 0.45 g/100 g lipids, respectively. Total n-6 PUFAs did not show any significant variation (p > 0.05) for all samples.

Table 5.4 – Fatty acids profile ⁽¹⁾ (wt.%, g FA/100 g of total lipids) of Atlantic salmon stored at 75 MPa/25 ºC, at atmospheric pressure (AP) at 5 ºC and AP/25 ºC for different storage times. Different letters along each row denote significant differences (p < 0.05); absence of letters indicates not statistically significant differences.

Free fatty acids ⁽¹⁾ Fresh fish 75 MPa/25 ºC AP/5 ºC AP/25 ºC

0 days 5 days 15 days 30 days 5 days 15 days 5 days

C14:0 1.14 ± 0.12 1.27 ± 0.13 1.17 ± 0.06 1.05 ± 0.19 1.01 ± 0.10 1.30 ± 0.14 1.14 ± 0.02 C16:0 7.57 ± 1.04 8.71 ± 0.61 8.24 ± 0.50 7.15 ± 1.41 6.78 ± 0.97 7.37 ± 0.16 7.86 ± 1.29 C18:0 2.05 ± 0.16 1.57 ± 1.14 2.06 ± 0.12 1.82 ± 0.30 1.84 ± 0.18 1.91 ± 0.20 2.02 ± 0.35

∑ SFA 10.76 ± 1.22 11.55 ± 1.02 11.47 ± 0.58 10.02 ± 1.90 9.62 ± 1.17 10.57 ± 0.36 10.01 ± 2.08 C16:1n-7 1.59 ± 0.28 1.59 ± 0.24 1.57 ± 0.13 1.22 ± 0.16 1.34 ± 0.09 1.68 ± 0.12 1.49 ± 0.09 C18:1n-7 3.56 ± 0.55 3.70 ± 0.31 3.09 ± 0.11 2.99 ± 1.09 3.34 ± 0.76 3.54 ± 0.81 3.76 ± 0.14 C18:1n-9 29.29 ± 7.11 28.06 ± 3.29 29.80 ± 1.62 28.59 ± 1.06 29.69 ± 3.20 31.47 ± 5.33 28.53 ± 1.97 C20:1n-9 4.84 ± 0.58 4.79 ± 0.23 4.46 ± 0.15 3.90 ± 0.70 4.36 ± 0.55 4.89 ± 0.74 4.62 ± 0.67 C22:1n-9 0.64 ± 0.06 0.80 ± 0.13 0.71 ± 0.02 0.71 ± 0.18 0.66 ± 0.11 0.80 ± 0.21 0.76 ± 0.04

∑ MUFA 39.91 ± 8.45 38.94 ± 4.13 39.63 ± 1.83 37.42 ± 1.09 39.39 ± 4.40 42.38 ± 6.64 37.72 ± 2.76 C20:4n-3 0.74 ± 0.10 0.76 ± 0.04 0.74 ± 0.07 0.64 ± 0.05 0.66 ± 0.06 0.71 ± 0.08 0.72 ± 0.13 C20:5n-3 (EPA) 1.98 ± 0.16 2.24 ± 0.29 2.12 ± 0.12 1.86 ± 0.36 1.80 ± 0.27 1.90 ± 0.05 2.01 ± 0.46 C22:5n-3 0.94 ± 0.11 0.94 ± 0.12 0.93 ± 0.04 0.86 ± 0.16 0.90 ± 0.08 0.95 ± 0.07 0.87 ± 0.11 C22:6n-3 (DHA) 4.51 ± 0.70^abc 5.39 ± 0.41^a 4.93 ± 0.34^ab 4.24 ± 0.92^abc 4.21 ± 0.65^abc 3.52 ± 0.32^bc 3.23 ± 0.55^c

∑ n-3 PUFA 8.17 ± 0.89^ab 9.33 ± 0.76^a 8.72 ± 0.40^ab 7.59 ± 1.48^ab 7.57 ± 0.99^ab 7.08 ± 0.40^ab 6.31 ± 1.26^b C18:2n-6 9.04 ± 1.63 9.49 ± 0.50 8.87 ± 0.27 7.78 ± 1.25 8.35 ± 1.10 9.86 ± 1.49 9.11 ± 0.69 C20:2n-6 0.95 ± 0.15 0.97 ± 0.03 0.92 ± 0.04 0.83 ± 0.15 0.89 ± 0.06 0.99 ± 0.11 0.93 ± 0.05 C22:2n-6 2.24 ± 0.45 2.31 ± 0.08 2.24 ± 0.18 2.02 ± 0.21 2.14 ± 0.10 2.43 ± 0.42 2.16 ± 0.18

∑ n-6 PUFA 12.23 ± 2.12 12.77 ± 0.59 12.02 ± 0.48 10.63 ± 1.60 11.37 ± 1.25 13.28 ± 2.00 11.26 ± 1.76 PUFA/SFA 1.90 ± 0.17 1.93 ± 0.22 1.81 ± 0.10 1.82 ± 0.04 1.97 ± 0.06 1.92 ± 0.14 1.77 ± 0.07 n-6/n-3 ratio 1.51 ± 0.27 1.38 ± 0.16 1.38 ± 0.10 1.41 ± 0.08 1.51 ± 0.09 1.88 ± 0.30 1.79 ± 0.14

Also, no significant changes were observed for the n-6/n-3 ratio (Table 5.4), with values between 1.38 and 1.88. According to Simopoulos (2008) [28], a lower ratio of n-6/n-3 ratio is desirable (approximately 1) to reduce the risk of many chronic diseases, such as secondary prevention of cardiovascular diseases, reduction of rectal cell proliferation in patients with colorectal cancer, suppression of inflammation in patients with rheumatoid arthritis, and beneficial effects on patients with asthma.

Polyene index evolution in the salmon loins is a measure of the variation of long chain (LC)‐PUFAs (DHA and EPA) during storage, relative to a saturated fatty acid representative of marine products such as salmon (C16:0), being a good index to evaluate lipid oxidation (Figure 5.4) [29]. Salmon stored at AP/25 ºC for 5 days presented already a decrease (p <

0.05) from an initial value of 0.86 ± 0.03 to 0.68 ± 0.04. However, at AP/5 ºC, the polyene index decreased also significantly (p < 0.05) but only after 15 days to a value of 0.73 ± 0.03 (Figure 5.4). On the other hand, HS did not cause changes (p > 0.05) on polyene index during storage (between 0.85 and 0.88). The decrease of polyene index during storage verified for samples stored under AP showed that oxidation mechanisms are active during storage.

Figure 5.4 – Polyene index of Atlantic salmon stored at 75 MPa/25 ºC, AP/5 ºC and AP/25 ºC during 30, 15 and 5 days, respectively. Different letters denote significant differences (p < 0.05) between salmon samples stored at different conditions and time (a-b).

There is no information regarding the effect of HS/RT on FA profile of fish products, but recently Otero et al. (2019) [19] reported that hyperbaric cold storage (HS at low

temperature, 50 MPa/5 ºC during 12 days) did not reveal significant changes on the fatty acid composition of Atlantic mackerel during storage, as well as in samples stored at AP/5 ºC. These authors verified no changes on polyene index for 12 days, neither at AP/5 ºC or at 50 MPa/5 ºC [19]. In the present study, most of FA were also not affected by storage conditions; only DHA content was higher after 30 days at HS/RT than at AP, reflecting a higher n-3 PUFAs preservation. Consequently, polyene index was maintained in HS/RT samples, and decreased for AP samples (AP/25 ºC after 5 days and AP/5 ºC after 15 days).

FA variations under HS/RT were already studied in different food products, such as ready-to-eat cod meal “Bacalhau com natas” (50-150 MPa/~21 ºC during 12 h) [30], raw bovine meat (50-150 MPa/~21 ºC during 12 h) [31], and whey cheese (100 MPa/17 ºC during 10 days) [32], revealing no consistent pattern to a possible effect of HS/RT, possibly due to the short storage periods (maximum of 10 days).

5.3.1.3.2. Lipid oxidation evolution

Primary (peroxides values), secondary (thiobarbituric acid reactive substances, TBARS) and tertiary (fluorescence ratio) lipid oxidation was assessed to evaluate the rancidity development on salmon loins during 30 days of storage (Figure 5.5). Initial fresh salmon samples presented peroxide values, TBARS and a fluorescence ratio of 4.76 ± 0.50 mg Fe (III)/kg lipids, 0.21 ± 0.14 µg MDA/g fish and 0.04 ± 0.01, respectively, which are in agreement with previous work (Chapter 4 – Section 4.2.1). Peroxides values (Figure 5.5a) did not show significative changes (p > 0.05) during the 30 days of storage in all conditions, showing values between 3.55 and 4.76 mg Fe (III)/kg lipids. Concerning to TBARS (Figure 5.5b), there was a pronounced increase for samples stored at 75 MPa/25 ºC, reaching a value of 1.01 ± 0.12 µg MDA/g fish (5-fold) after 5 days of storage, remaining thereafter constant (p > 0.05) until 30^th day. However, a higher increase was observed in the previous study (Chapter 4 ), when Atlantic salmon muscle portions were stored at 75 MPa/25 ºC, revealing an increment of TBARS values in about 29-fold after 25 days of storage. In the present study, salmon loins were vacuum-packaged, which allowed obtaining lower TBARS values. Lower TBARS values (between 0.13 and 0.27 µg MDA/g fish) were observed for the samples stored under AP (5 and 25 ºC, after 15 and 5 days, respectively).

Figure 5.5 - Primary lipid oxidation (a; peroxides values in mg Fe (III)/kg lipids), secondary lipid oxidation (b; thiobarbituric acid reactive substances, TBARS in µg MDA/g fish) and tertiary lipid oxidation (c; fluorescent compounds in fluorescence ratio) of Atlantic salmon stored at 75 MPa/25 ºC, at atmospheric pressure (AP) at 5 ºC and AP/25 ºC during 30, 15 and 5 days, respectively.

Different letters denote significant differences (p < 0.05) between salmon samples stored at different conditions and time (a-c). For primary lipid oxidation (a) no significant differences were observed in all cases.

Moreover, the fluorescence ratio increased (p < 0.05) for AP samples stored at 25 ºC about 40-fold the initial value, showing a fluorescence ratio of 1.54 ± 0.19 after 5 days of storage (Figure 5.5c). For AP/5 ºC, there was also a slightly increase (p> 0.05) of about 9-fold after 5 days of storage, compared to those obtained in fresh salmon, decreasing after 15 days. For HS, 75 MPa/25 ºC, fluorescent compounds did not change (p > 0.05) during 30 days of storage and were much lower compared to the other two storage conditions.

The lipid oxidation mechanism that occur during salmon storage was already described in the previous study (Chapter 4). Basically, lipid oxidation in fish is a complex chain of reactions, which originates primary and secondary products, which can react with amino constituents (proteins, peptides, free amino acids, and phospholipids), producing interaction compounds (fluorescent compounds). Generally, AP samples (being at 25 ºC more pronounced than at 5 ºC) showed lower TBARS values and higher fluorescent compounds, as a result of lipid oxidation mechanisms from secondary and tertiary oxidations, respectively. On the other hand, HS samples showed high TBARS values and lower fluorescent compounds, indicating that oxidation extent was lower when compared to AP samples. These results indicate that under HS, no tertiary compounds formation occurs, produced from the interaction between secondary products and amino constituents of the muscle, resulting in higher TBARS but a lower fluorescence ratio value.