## 2 Materials and Methods
### LC-MS Analysis  
Liquid Chromatography-Mass Spectrometry (LC-MS) was employed to analyze the fatty acid composition and characterize the bioactive polar lipids present in all TAC extracts derived from both apple juice and apple pomace, following the methodology outlined by [PERSON] et al. [7]. Shortly afterwards, each dried extract was redissolved in 500 \(\upmu\)L of a dichloromethane/methanol mixture (1:2, \(v/v\)) and centrifuged at 13,000 rpm for 6 min (accuSpin 17R, Fisher Scientific Ltd., Dublin, Ireland). The resulting clear supernatants were filtered using 3 kDa ultracentrifuge filters (Amicon Ultra 3K, Merck Millipore Ltd., Darmstadt, Germany). To determine the fatty acid profile in these filtrates, 10 \(\upmu\) of each sample was injected into an HPLC system (Agilent 1260 series, Agilent Technologies Ireland Ltd., Little Island, Co., Cork, Ireland) equipped with a Q-TOF mass spectrometer (Agilent 6520) utilizing electrospray ionization (ESI) as a source. Fatty acid separation was conducted using an Agilent C18 Poroshell 120 column (2.7 \(\upmu\)m, 3.0 \(\times\) 150 mm) with gradient elution. Mobile phase A consisted of 2 mM ammonium acetate in water, while mobile phase B contained 2 mM ammonium acetate in 95% acetonitrile. The initial flow rate for the mobile phase was set at 0.3 mL/min for the first 5 min, increased to 0.6 mL/min at 10 min, and maintained at this flow rate until the conclusion of the analysis. The mass spectrometer scanned \(m/z\) values ranging from 50 to 1100, using reference masses of 1033.988 and 112.9855 to monitor scans in negative ionization mode. Instrument parameters included a capillary voltage of 3500 V, skimmer and fragmentation voltages of 65 V and 175 V, respectively, and a drying gas flow rate of 5 L/min with a nebulizer pressure of 30 psi and a temperature of 325 \({}^{\circ}\)C.  
The LC-MS method was validated by a comparison of the Molecular Weight (Mr) and retention times (RT) of several standards of common saturated and unsaturated fatty acids, including lauric (C12:0), myristic (C14:0), palmitic (C16:0), stearic (C18:0), oleic (OA,C18:In-9 cis), linoleic (LA, C18:2n-6 cis), gamma-linolenic (GLA, C18:3n-6), alpha-linolenic (ALA, C18:3n-3), arachidonic (ARA, C20:4n-6), eicosapentaenoic (EPA, 20:5n-3), docosapentaenoic (DPA, 22:5n-3) and docosahexaenoic (DHA, 22:6n-3) acids (Sigma, Ireland) [7; 9]. These standards facilitated the identification of fatty acids in the lipid extracts, based on their precise mass values. Each discovered fatty acid's peak area was averaged across three samples, and the resulting relative content was noted. However, as peak areas do not correspond to absolute fatty acid quantities, the results should be interpreted cautiously.  
The identification of free fatty acids (FFAs) and polar lipids (including phospholipids and glycolipids) was achieved through a combination of survey, precursor, daughter and neutral loss scans. The identities of the detected bioactive polar lipid molecules at specific elution times were further confirmed using the LIPID MAPS: Nature Lipidomics Gateway database (www.lipidmaps.org, accessed on 20 December 2024), by selecting the lowest delta values and cross-referencing with LC-MS results from the fatty acid composition analysis of saponified phospholipids, as previously described by [PERSON] et al. [7].