*2.4. Optimisation of Quinoa Protein Extraction Process*

### 2.4.1. Single-Factor Test for Ultrasound-Assisted Alkaline Extraction

The basic conditions for extraction were: ultrasonic time 90 min, solid-liquid ratio 1:15, ultrasonic temperature 45 ◦C, and pH 10; for single-factor test, one of these parameters was changed at a time keeping the other three parameters constant. The different conditions tested were ultrasonic time (30, 60, 90, 120, 150 and 180 min), solid-liquid ratio (1:5, 1:10, 1:15, 1:20, 1:25 and 1:30; *w:v*), ultrasonic temperature (25, 35, 45, 55, 65 and 75 ◦C) and pH (8.0, 9.0, 10.0, 11.0, 12.0 and 13.0), with extraction rate of QPI as the index. The experiments were performed in triplicates.

#### 2.4.2. Response Surface Optimisation Test

On the basis of the single-factor test, the four parameters for quinoa protein extraction including ultrasonic time (A), solid-liquid ratio (B), ultrasonic temperature (C) and pH(D) were optimised. The response surface test scheme was designed according to the Box-Behnken method in the Design-Expert 8.0.6 software, as shown in Tables S1 and S2.

#### 2.4.3. Comparison between Ultrasound-Assisted and Traditional Alkaline Extraction Methods

The extraction rate and purity of QPI obtained by the ultrasound-assisted alkaline extraction method and the traditional alkaline extraction method were compared and the optimum technological conditions were determined.

#### *2.5. Isoelectric Point*

The isoelectric point of QPI was determined according to the method of Pedroche et al. [24]. Under the optimal extraction conditions of QPI, eight equal portions of protein extract supernatant were collected, and the pH was adjusted to 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 with 1 M HCl, respectively. The optimum pH of acid precipitation was determined by measuring the extraction rate of the protein. The pH corresponding to the maximum protein extraction rate is the isoelectric point of QPI.

#### *2.6. Heat Treatment*

QPI was dissolved in phosphate buffer (0.2 M, pH 7.0), while stirring for 2 h at ambient temperature (25 ◦C) and stored it overnight at 4 ◦C to enhance hydration. The QPI solution with a concentration of 5% was treated at different temperatures of 60, 70, 80, 90, 100, and 121 ◦C for 5, 10, 20, and 30 min (Employed each temperature at different times), and then quickly cooled with ice water for 5 min. Samples were stored at 4 ◦C and generally analyzed within 2 d of treatment [25].

#### *2.7. Flexibility and Turbidity Measurement*

Trypsin solution (250 μL, 0.1%) was mixed with heat-treated QPI solution (14 mL, 0.1%), and after 5 min incubation at 38 ◦C, 4 mL of trichloroacetic acid solution (5%) was added. After centrifugation, the supernatant was taken, and its absorbance was measured at 280 nm, indicating flexibility [26]. The heat-treated QPI solution was diluted to 3 mg/mL, and the turbidity was measured by measuring its absorbance at 400 nm after shaking and mixing [27].
