*2.3. Proximate Composition*

Moisture (A.O.A.C method number 950.46), crude protein (A.O.A.C method number 928.08, Kjeldahl factor of 6.25), fat (A.O.A.C method number 963.15), ash (A.O.A.C method number 920.153), and carbohydrate (calculated by the difference) were all investigated in the proximate composition of pig brain [9]. The results were expressed in grams per 100 g of fresh weight (fw).

### *2.4. Determination of Total Phospholipid and Total Cholesterol Contents*

Bligh and Dyer's method [10] was used to extract lipid from the pig brain. Samples (25 g) were homogenized with 200 mL of a mixture of chloroform, methanol, and distilled water (1:2:1, *v/v/v*) at 9500 rpm for 2 min at 4 ◦C using an IKA Labortechnik homogenizer (Selangor, Malaysia). Then, 50 mL chloroform was added to the homogenate, and the mixtures were homogenized at the same speed for 1 min. After that, 25 mL distilled water was added, and the mixtures were homogenized at the same speed for 30 s. The homogenate was centrifuged at 3000× *g* for 15 min at 4 ◦C using an RC-5B plus centrifuge (Sorvall, Norwalk, CT, USA) and then transferred to a separating flask. The chloroform phase was drained into a 125 mL Erlenmeyer flask containing around 2–5 g of sodium sulfate, agitated well, and filtered through Whatman No. 4 filter paper (GE Healthcare Bio-Sciences Corp., Pittsburgh, PA, USA) into a round-bottom flask. A rotary evaporator (Model N-100, Eyela Ltd., Tokyo, Japan) was used to evaporate the solvent at 40 ◦C.

The total phospholipid content of the extracted oil was determined using a modified Stewart method [11]. Oil samples (20 μL) were dissolved in chloroform to obtain a final volume of 2 mL. Then, 1 mL of thiocyanate reagen<sup>t</sup> (a mixture of 0.10 M ferric chloride hexahydrate and 0.40 M ammonium thiocyanate) was added. The lower layer was removed after 1 min of vigorous mixing and the absorbance at 488 nm was determined. Phosphatidylcholine (0–50 ppm) was used to create a standard curve. The total phospholipid content was measured in g/100 g fw.

The cholesterol content of the oil samples was determined using a modified version of Beyer and Jensen's method [12]. The oil sample (0.1–0.2 g) was saponified using 2% alcoholic KOH for 10 min. The unsaponified fraction was extracted with 2 × 10 mL hexane. The extracts were rinsed with 5 mL distilled water and dried at 45 ◦C in a W350 Memmert temperature-controlled water bath (Schwabach, Germany). The dried extract was resuspended in 3 mL of glacial acetic acid, and 2 mL coloring reagen<sup>t</sup> was added. To prepare the coloring reagent, the stock reagen<sup>t</sup> of 10% (*w/v*) FeCl3·6H2O in glacial acetic acid was made and then 1 mL of the stock reagen<sup>t</sup> was diluted with 100 mL of concentrated H2SO4. The absorbance of the reaction mixture was read at 565 nm against a glacial acetic acid blank using a UV–Vis spectrophotometer (UV-1900, Shimadzu, Kyoto, Japan). A standard curve was prepared using cholesterol in glacial acetic acid at 0 to 120 mg/L. The total cholesterol content was measured in g/100 g fw.

### *2.5. Total Heme Protein Content and Color Measurement*

Using Chaijan and Undeland's method [13], the total heme protein content was assessed and stated in g of hemoglobin per 100 g of sample. A sample and 3 volumes of 0.1 M phosphate buffer, pH 7 containing 5% SDS (*w/v*) was homogenized at 13,500 rpm for 20 s. The homogenate was heated in a water bath (85 ◦C) for 1 h and cooled under running tap water for 10 min. The solution was then centrifuged (5000× *g*/15 min/25 ◦C). The absorbance of the supernatant was read at 535 nm using a UV–Vis spectrophotometer with phosphate buffer as a blank. A standard curve of bovine hemoglobin (0–20 μM) was used.

A portable Hunterlab ColorFlex® EZ device (Hunter Assoc. Laboratory; Reston, VA, USA) was used to collect colorimetric data of the pig brain in triplicate. A white and black standard were used to calibrate the device. The measurement modes tristimulus *L*\* (lightness), *a*\* (redness/greenness), and *b*\* (yellowness/blueness) were chosen. According to Chen et al. [14], the redness index (*a*\*/*b*\*) was computed.
