## 2 Materials and Methods
### Antioxidant Assay of Porcine By-Products Hydrolyzates  
#### 2.5.1 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Activity  
The DPPH radical scavenging activity of the hydrolyzates was determined in triplicate [15]. Four different concentrations (0.25, 0.5, 0.75, and 1 mg/mL) of samples were prepared, and 1.5 mL of the diluted sample, 1.5 mL of 99.5% (\(v/v\)) ethanol, and 375 uL of 0.02% (\(w/v\)) ethanolic 0.5 mM DPPH solution were mixed. As a control, 1.5 mL of DDW was added instead of the diluted sample. The mixture was incubated in a dark room (22 \({}^{\circ}\)C) for 50 min and centrifuged at 2500\(\times\)\(g\) for 10 min to spin down insoluble fractions. The absorbance of the supernatant was read at 517 nm against a blank using aspectrophotometer (Libia S22 UV/Vis Spectrophotometer, Biochrom Ltd., Cambridge, UK), and DPPH radical scavenging activity was calculated using the following equation:  
\[\text{DPPH radical scavenging activity (\%)}=\text{((Ab $-$ As)/Ab)}\times 100\]  
where Ab is the absorbance of control and As is the absorbance of each sample. The DPPH radical IC\({}_{50}\) value, which was the sample concentration required to achieve 50% inhibition, was calculated based on the results. 2.5.2. 2-Azino-bis-3-ethylbenzothazoline-6-sulfonic Acid (ABTS) Radical  
The ABTS radical-scavenging activity of the hydrolyzates was performed in triplicate [16]. Forty microliters of serially diluted samples (1, 5 and 10 mg/mL) or DDW (as control) were mixed with 5 mL of ABTS\({}^{+}\) reagents (1:1 mixture of 7.4 mM ABTS reagent and 2.6 mM potassium persulfate) and reacted at room temperature (dark condition) for 6 min. The absorbance of the reactant was read at 734 nm using the spectrophotometer. The ABTS radical scavenging activity was calculated using the following equation:  
\[\text{ABTS radical scavenging activity (\%)}=\text{((Ab$-$ As)/Ab)}\times 100\]  
where Ab is the absorbance of the control and As represents the absorbance of each sample. Based on the results, the ABTS radical IC\({}_{50}\) value, defined as the sample concentration at 50% inhibition, was calculated. 2.5.3. Hydroxyl Radical Scavenging Activity  
The hydroxyl (OH) radical scavenging activity of the hydrolyzates was determined in triplicate according to the method described by [PERSON] et al. [16]. One milliliter of serially diluted sample (1, 2.5, and 5 mg/mL) or DDW (as control) was mixed with 1 mL of 6 mM hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)), and the mixture was incubated at 37 \({}^{\circ}\)C for 10 min. After incubation, 1 mL of 6 mM salicylic acid was added and reacted at 37 \({}^{\circ}\)C for 30 min. The absorbance of the reactant was read at 510 nm, and hydroxyl radical scavenging activity was calculated using the following equation. \[\text{Hydroxyl radical scavenging activity (\%)}=\text{((Ab$-$ As)/Ab)}\times 100\]  
where Ab is the absorbance of the control and As is the absorbance of each sample. The OH radical IC\({}_{50}\) value, the sample concentration at 50% inhibition, was calculated based on the results. 2.5.4. Reducing Power  
The reducing power of the hydrolyzates was determined in triplicate [17]. Two milliliters of diluted sample (1 mg/mL) were mixed with 2 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 2 mL of 1% potassium ferricyanide. The mixture was incubated at 50 \({}^{\circ}\)C for 20 min, and then 2 mL of 10% trichloroacetic acid (TCA) was mixed. Two milliliters of aliquots from the reactant were mixed with 2 mL of DDW and 0.4 mL of 0.1% (_w/v_) ferric chloride and then incubated at 50 \({}^{\circ}\)C for 10 min. The absorbance of the final reactant was read at 700 nm using the spectrophotometer. 2.6. Statistical Analysis  
The experimental design of this study was a completely randomized block design with three independent batches. Data were expressed as mean \(\pm\) standard deviation.