## 2 Materials and Methods
### Chemical Analyses Related to Quality Loss  
The pH values of the mackerel muscle were monitored throughout the storage period via a 6 mm diameter insertion electrode ([PERSON], Barcelona, Spain). Trimethylamine (TMA) formation was measured via the picrate spectrophotometric method (410 nm) ([PERSON], DU 640; London, UK), as described by [PERSON] et al. [46]. This involved the preparation of a 5% trichloroacetic acid extract from fish white muscle (10 g in 25 mL), with the results expressed as mg TMA-N\(\cdot\)kg\({}^{-1}\) muscle. Lipids were extracted from mackerel white muscle via the [PERSON] and Dyer [47] method, which involves single-phase solubilization with a chloroform-methanol (1:1) mixture. The results were calculated as g lipid\(\cdot\)kg\({}^{-1}\) muscle. Lipid quantification followed the [PERSON] and Allen [48] method, with the lipid content expressed as g\(\cdot\)kg\({}^{-1}\) muscle. Free fatty acid (FFA) content was determined from the lipid extract of fish muscle via the [PERSON] and Tinsley [49] method on the basis of complex formation with cupric acetate-pyridine and subsequent spectrophotometric assessment at 715 nm. The results are expressed as g FFAs\(\cdot\)kg\({}^{-1}\) lipids. The peroxide value (PV) of the lipid extract was measured spectrophotometrically at 520 nm, following the method described in previous research [50]. The results are expressed as meq. active oxygen\(\cdot\)kg\({}^{-1}\) lipids. The thiobarbituric acid (TBA) index (TBA-i) was determined following the methodology described by [PERSON] [51]. The content of TBA-reactive substances (TBARSs) was determined by spectrophotometry at 532 nm, and the results obtained were expressed as mg malondialdehydedehyde\(\cdot\)kg\({}^{-1}\) muscle. The formation of fluorescent compounds was determined in the lipid extract obtained from fish muscle using an LS 45 fluorimeter (Perkin Elmer Espana; Tres Cantos, Madrid, Spain), proceeding to its reading at wavelengths of 393/463 nm and 327/415 nm, as described in a previous work [52]. Relative fluorescence (RF) was determined following the formula: RF = F/F\({}_{\text{st}}\), where F is defined as the fluorescence measured at each excitation/emission wavelength pair, and F\({}_{\text{st}}\) represents the fluorescence intensity of a quinine sulfate solution (1 ug-mL\({}^{-1}\) in 0.05 M H\({}_{2}\)SO\({}_{4}\)) at the corresponding wavelength pair. The results obtained were presented as the fluorescence ratio (FR), which was calculated from the ratio between the two RF values: FR = RF\({}_{393/463\,\text{nm}}\)/RF\({}_{327/415\,\text{nm}}\). To determine the lipid profile of the samples, FA methyl esters (FAMEs) were previously obtained from the lipid extracts using acetyl chloride in methanol. Subsequently, the FAMEs obtained were analyzed by gas chromatography (Perkin-Elmer 8700 chromatograph, Madrid, Spain) [53]. An SP-2330-fused silica capillary column (0.25 mm i.d. \(\times\) 30 m, Supelco, Inc., Bellefonte, PA, USA) was used for these determinations, using temperature adjusted to increase from 145 \({}^{\circ}\)C to 190 \({}^{\circ}\)C at 1.0 \({}^{\circ}\)C min\({}^{-1}\) and from 190 \({}^{\circ}\)C to 210 \({}^{\circ}\)C at 5.0 \({}^{\circ}\)C min\({}^{-1}\), maintained at 210 \({}^{\circ}\)C for 13.5 min. Nitrogen was used as a carrier gas at 10 psig, and detection was performed with a flame ionization detector at 250 \({}^{\circ}\)C. A programmed temperature vaporizing injector was used in split mode (150:1) and heated from 45 \({}^{\circ}\)C to 275 \({}^{\circ}\)C, increasing 15 \({}^{\circ}\)C min\({}^{-1}\). Peaks corresponding to FAMEs were identified by means of comparing their retention times with those of standard mixtures (Qualmix Fish and Supelco 37 Component FAME Mix, Supelco, Inc., Bellefonte, PA, USA). The peak areas were automatically integrated, using C19:0 as an internal standard for quantitative purposes. The content of each FA was calculated as g FA\(\cdot\)100 g\({}^{-1}\) total FAs. The polyene index (PI) was calculated as the FA ratio: C20:5u3 + C22:6u3 to C16:0.