*2.2. Experimental Analysis*

#### 2.2.1. Inhibition Activity Assay of Linoleic Acid Oxidation

The ability of amino acids and peptides to inhibit lipid oxidation was determined using a linoleic acid emulsified model. Briefly, amino acids or peptides were dissolved in 50 mM phosphate buffer (pH = 7.0) and then added to a linoleic acid emulsion. In previous studies, different incubation temperatures were designed for linoleic acid oxidation assessment at 20 ◦C [11], 40 ◦C [4,12], and 60 ◦C [13], respectively. However, most studies selected 40 ◦C for linoleic acid oxidation assay. Subsequently, the reaction mixture was then incubated

in tubes at 40 ◦C in dark for 48 h. The degree of linoleic acid oxidation was measured at 500 nm after mixing with 30% ammonium thiocyanate (dissolved in water) and ferrous chloride solution (dissolved in 3.5% HCl). Effect on the oxidation of linoleic acid was described as inhibition rate (IR), which was calculated by Equation (1). GSH was used as control and the results were expressed as GSH equivalents.

$$\text{IR\%} = (1 - A\_{\text{sample}} / A\_{\text{blank}}) \times 100 \tag{1}$$

where A means absorbance value of samples (Asample) and blank (Ablank) at 500 nm.

#### 2.2.2. Superoxide Radical Scavenging Activity Assay

The superoxide radical scavenging activity was measured at room temperature by monitoring the inhibition effect of pyrogallol auto-oxidation described by Li et al. [14]. Sample solutions (0.1 mL) were incubated at 25 ◦C for 10 min after added 2.8 mL Tris HCl-EDTA buffer (0.1 M, pH 8.0). The optical density was measured at 325 nm every 10 s for 240 s after mixing with pyrogallol solution (3 mM). The slopes represented rates of pyrogallol auto-oxidation. Superoxide radical scavenging activity was calculated by Equation (2). GSH was used as the control and the results were expressed as GSH equivalents.

$$\mathbf{K} = (\mathbf{V}\_{\mathbf{c}} - \mathbf{V}\_{\mathbf{s}}) / \mathbf{V}\_{\mathbf{c}} \times 100 \tag{2}$$

where K means superoxide radical scavenging activity assay, %; Vc indicates auto-oxidation rate of pyrogallol; Vs. stands for oxidation rate of pyrogallol after adding the sample.
