*2.7. Protein Fractionation*

The protein composition of pig brain was fractionated according to Hashimoto et al. [15]. The non-protein nitrogenous (NPN) compound fraction, water-soluble protein fraction, saltsoluble protein fraction, alkali-soluble protein fraction, and stromal protein fraction were

separated from the pig brain proteins due to their varied solubilities. Briefly, the ground sample (20 g) were homogenized in 200 mL of phosphate buffer (15.6 mM Na2HPO4, 3.5 mM KH2PO4), pH 7.5 with an IKA Labortechnik homogenizer. The homogenate was centrifuged (5000× *g*/15 min/4 ◦C) using an RC-5B plus centrifuge. The residue was homogenized and centrifuged again after being mixed with 200 mL of the same buffer. These two supernatants were combined, and TCA was added to obtain a final concentration of 5% (*w/v*). The resulting precipitate was collected by filtration and referred to as the water-soluble protein fraction. The filtrate was used as the NPN fraction. For the above residue, 10 vol of phosphate buffer (15.6 mM Na2HPO4, 3.5 mM KH2PO4) containing 0.45 mM KCl, pH 7.5 was added. The mixture was homogenized and centrifuged (5000× *g*/15 min/4 ◦C). The procedure was carried out twice more. Both supernatants were combined and used as the salt-soluble protein fraction. The precipitate obtained was added with 5 vol of 0.1 M NaOH and stirred for 10 h at 4 ◦C. The mixtures were then centrifuged (5000× *g*/15 min/4 ◦C). The supernatant was given as the alkali soluble protein fraction. The final precipitate was used as the stromal protein fraction. The nitrogen distribution was estimated after each fraction by the Kjeldahl method [9].

### *2.8. Amino Acid Profile and Fourier Transform Infrared (FTIR) Spectroscopy*

According to Chinarak et al. [16], the amino acid profile of the pig brain was measured. In a 10 mL crimp seal glass vial, freeze-dried samples were combined with 5 mL of hydrolysis solution (6 M HCl, 5% thioglycolic acid, and 1% phenol) and tightly sealed. One 1 mL of the sample was centrifuged at 10,000× *g* for 10 min) after being hydrolyzed at 110 ◦C for 18 h. The supernatant (100 μL) was then neutralized with 1 M sodium carbonate. An aliquot of 25 μL was transferred to a 2 mL GC glass vial, which was then filled with 50 μL of 200 nM norleucine as an internal standard. After drying for around 1–2 h at 60 ◦C, 50 μL of dichloromethane was added and dried for another 30 min to eliminate any remaining water. Then, a derivatizing agent, *N*-tert-Butyldimethylsilyl-*N*-methyltrifluoroacetamide, with 1% *tert*-Butyldimethylchlorosilane (50 μL) and acetonitrile (50 μL), were mixed with the samples and subjected to incubate in a hot-air oven (100 ◦C/4 h). The sample (2 μL) was analyzed using a Shimadzu GCMS-TQ8050 NX (Kyoto, Japan) after cooling to room temperature.

A horizontal attenuated total reflectance (ATR) trough plate crystal cell (45◦ ZnSe; 80 mm long, 10 mm wide and 4 mm thick) (Pike Technology, Inc., Madison, WI, USA), equipped with a Bruker Model Vector 33 FTIR spectrometer (Bruker Co., Ettlingen, Germany), was used to perform FTIR analysis on the freeze-dried pig brain and pig brain lipid. In the mid-infrared region (500–4000 cm<sup>−</sup>1), 16 scans, at a resolution of 4 cm<sup>−</sup>1, were used to capture FTIR spectra at room temperature (26–29 ◦C). A reference air spectrum was acquired as a background. Analysis of spectral data was carried out using the OPUS 3.0 data collection software program [17].

### *2.9. Fatty Acid Profile of Total Lipid, Neutral Lipid Fraction, and Polar Lipid Fraction*

According to Estefanell et al. [18], the neutral and polar fractions of total lipids were fractionated by adsorption chromatography on silica cartridges (Sep-pak; Waters S.A., MA, USA) using 30 mL chloroform and 20 mL chloroform/methanol (49:1, *v/v*) as neutral lipid solvents, followed by a 30 mL methanol rinse to yield the polar fraction. Fatty acid methyl esters (FAME) in the samples (total, neutral, and polar lipids) were determined using a gas chromatography/quadrupole time of flight (GC/Q-TOF) mass spectrometer (GC 7890B/MSD 7250, Agilent technologies, USA) coupled to the PAL auto sampler system (CTC Analytics AG, Switzerland). The MassHunter software was used to collect MS data (Version 10.0, Agilent Technologies, Santa Clara, CA, USA). The complete method and optimal condition can be obtained from the report of Chinarak et al. [16].
