2.9.3. Degree of Hydrolysis

The hydrolysis degree of QPI was determined by o-phthalaldehyde (OPA) method. The hydrolyzed supernatant (400 μL) was mixed with OPA (3 mL), and the absorbance was measured at 340 nm immediately after 2 min of reaction. Deionized water and serine standard solution (0.9516 mM) were used as blank and stand, respectively [31]. The degree of hydrolysis was calculated as follows:

$$SerineNH\_2 = \frac{OD\_{sample} - OD\_{blank}}{OD\_{stand} - OD\_{blank}} \times 0.9516 \times \frac{N \times V}{X \times P} \tag{3}$$

where *SerineNH2* is the serine content per gram of protein, mmol/g, *X* is the quality of the sample, g, *p* is the protein content of the sample, %, *N* is dilution times, *V* is volume of supernatant, L.

$$DH\left(\%\right) = \frac{\left(ServiceNH\_2 - \beta\right)/a}{h\_{tot}} \times 100\tag{4}$$

where *β* and α are constants 0.4 and 1, respectively, *htot* is number of peptide bonds of protein, mmol/g, the *htot* of quinoa protein is 7.4 mmol/g.

## 2.9.4. Total Amino Acid Content of In Vitro Digestion Products

The enzymolysis solution (2 mL) was mixed with 6 M HCl (8 mL) and placed in a digestive tube then filled with high-purity nitrogen for 3 min. The bottle plug was quickly sealed and digested at 110 ◦C for 24 h. It was taken out after cooling. All the hydrolysates were transferred to 25 mL volumetric flask. The digestive solution (2 mL) was dried in an oven at 60 ◦C, and a few solid or stains were left at the bottom. The residue was redissolved with 2 mL of ultrapure water, then dried again, repeated for 3 times, and diluted to 5 mL. Digestion solution (2 mL) was filtered with 0.22 μm aqueous membrane into sample bottles for automatic amino acid analyzer (LA8080 Hitachi High-Tech Science Co., Ltd. Naka Office, Tokyo, Japan) testing [32].

#### *2.10. Data Analysis*

Experimental data are presented as mean ± standard deviation (calculated by Excel 2007). Origin 2021 was used for plotting graphs. SPSS 17.0 was used for one-way ANOVA and multiple comparisons. The response surface results were analysed and graphs were plotted using Design-Expert 8.0. MestReNova was used to analyze the 1H NMR spectrum.
