3.4.2. Nuclear Magnetic Resonance Hydrogen Spectroscopy (1H NMR)

A variety of solvents were first screened and three were finally identified for comparison, namely D2O, CD4O and a mixture of D2O:CD4O = 1:1. The best solution for QPI is D2O:CD4O = 1:1 solvent mixture (Figure 4A). Further study of the effect of different heat treatment temperatures on the 1H 600 MHz NMR spectra of QPI (Figure 4B). The peak at 0 ppm is the internal standard (sodium,2,2,3,3-tetradeuterio-3-trimethylsilylpropanoat -e), 4.50–4.80 ppm is the water peak, and 0.75–3.81 ppm is the amino characteristic range of protein [51,52]. The spectra of all QPIs in the range of 0.75 ppm to 3.81 ppm are very complete and clear, which is helpful for further data processing and analysis. In this study, the 1H 600 MHz NMR spectra of QPI were subjected to a series of operations such as phase correction, baseline correction, calibration, peak calibration, integration and normalization in the range of 0.75–3.81 ppm in units of 0.51 ppm to obtain the NMR integral spectra (Figure S2). The results showed that only the normalized relative percentages in the range of 3.30–3.81 ppm were dependent on the heat treatment temperature of QPI, which gradually decreased with increasing temperature in the range of 60–121 ◦C, and had a minimum value of 34.97 ± 0.82% at the heat treatment condition of 121 ◦C for 30 min, which was significantly lower than the untreated 8.46% (*p* < 0.05). Its variation in the interval from 0.75 to 1.26 ppm was not only dependent on temperature, but also at 121 ◦C, 30 min had a minimum value of 23.25 ± 0.34%, significantly lower than the untreated 2.47% (*p* < 0.05). Its variation in the interval of 1.77–2.28 ppm, 2.28–2.79 ppm and 2.79–3.30 ppm was not dependent on temperature and reached maximum values of 10.85 ± 0.07%, 2.26 ± 0.38% and 5.42 ± 0.96% at the heat treatment condition of 121 ◦C for 30 min, respectively, which were significantly higher than the untreated 24.57%, 4.51 times and 7.54% (*p* < 0.05) (Table 1). This indicates that 0.75–1.26 ppm, 3.30–3.81 ppm, 1.77–2.28 ppm, 2.28–2.79 ppm and 2.79–3.30 ppm are all susceptible to heat treatment.

Heat treatment temperature has a significant effect on the 1H NMR spectrum, turbidity and flexibility of QPI (Figure 4C). The 1H NMR of 1.77–2.28 ppm and 2.28–2.79 ppm showed highly significant positive correlations (*p* < 0.01) with heat treatment temperature and turbidity, and 3.30–3.81 ppm showed highly significant negative correlations (*p* < 0.01) with turbidity and significant negative correlations (*p* < 0.05) with heat treatment temperature. In addition, 0.75–1.26 ppm also had a significant negative correlation with temperature (*p* < 0.05), while flexibility had a significant positive correlation with temperature (*p* < 0.05) and turbidity had a highly significant positive correlation with temperature (*p* < 0.001). This suggests that heat treatment affects the protein characteristic 1.77 to 2.79 ppm region of quinoa, which in turn alters the turbidity of the QPI. However, studies on the influence of heat treatment on 1H NMR spectra and microscopic substances are still in the initial stage, and the specific mechanisms and principles are not well understood, and further in-depth studies are needed.

**Figure 4.** Effect of different solvents on dissolution of quinoa protein isolate (**A**), heat treatment temperatures on 1H NMR spectra of QPI (**B**), correlation between the influence of heat treatment on QPI 1H NMR spectrum and temperature, flexibility and turbidity (**C**).

**Table 1.** Effect of different heat treatment temperatures on the relative percentage content of QPI's 1H NMR integrated spectra after normalization.


Different letters (a–e) represent significant differences in the same column (*p* < 0.05).

#### *3.5. Hydrolysis Degree during In Vitro Digestion*

Degree of hydrolysis (DH) is defined as the proportion of cleaved peptide bonds in the protein hydrolysate [53]. In this study, the trend of hydrolysis of QPI after heat treatment was similar to that of untreated QPI, and the hydrolysis of QPI after heat treatment at 90 and 121 ◦C for 30 min was lower than that of untreated QPI during the whole digestion process, among which the hydrolysis of QPI after treatment at 121 ◦C was the smallest, which was 25.53% after simulated in vitro digestion for 240 min, significantly lower than that of untreated QPI at 20.64% (Figure 5A). Previous studies found that the hydrolysis of soybean isolate protein (SPI) decreased under heat treatment conditions greater than 85 ◦C [54]. Reduction in protein digestibility of QPI after heating treatment has generally been attributed to the formation of cross-linked protein polymers, which are resistant to proteolysis [55]. Therefore, after heat treatment of the QPI suspension, pepsin became less effective in enzymatic digestion and its digestibility was lower than that of untreated protein [6]. This is consistent with the results of reduced digestibility of chicken breast proteins after heat treatment at 121 ◦C [56]. Previous studies have shown that heat treatment is an important process for the complete digestion of food proteins. These processes significantly affect the protein structure and thus its digestive resistance [57]. Therefore, we speculate that the QPIs treated at 90 ◦C and 121 ◦C may have structures that are difficult to degrade enzymatically and may inhibit the activity of digestive enzymes.

**Figure 5.** Effect of different heat treatment temperatures on the degree of hydrolysis (**A**) and total amino acid content (**B**) of QPI during in vitro digestion. Different letters indicate significant differences (*p* < 0.05).

#### *3.6. Total Amino Acid Content during In Vitro Digestion*

This study found that the content of each amino acid at the end point of pepsin digestion (120 min) was significantly lower than that of trypsin digestion end point (240 min) at the same temperature (Figure 5B). The amino acid content increased gradually with the prolongation of digestion time, and the amino acid content in the trypsin digestion stage was significantly higher than that in the pepsin digestion stage. The content of amino acids in the in vitro digestion products of untreated QPI was higher than that of the heat treated products. The content of amino acids in the in vitro digestion products of QPI after treatment at 121 ◦C was the lowest, and the content at the end of digestion was 16.60 mg/g (Table 2), which was significantly lower than that of untreated 27.85%. Previous studies have found that the amino acid content of heat-treated king oyster mushroom protein was significantly reduced after digestion in vitro [58]. Amino acid content of in vitro digested gluten protein was significantly reduced after microwave heating [59]. Furthermore, Giménez et al. [60] indicated that low levels of His, Pro and Ala in protein hydrolyzates were associated with a decrease in their antioxidant potential. Similar results were found in soy protein isolates [61]. In this study, the contents of His, Pro and Ala in the in vitro digestion products of QPI treated at 90 ◦C and 121 ◦C were significantly lower than those of untreated. Therefore, we speculate that the high heating temperature might cause

serious damage to amino acids and irreversible decomposition, resulting in the reduction of content. This result confirms the reason why heat treatment leads to a decrease in the antioxidant activity of QPI in vitro.

**Table 2.** Total amino acid content in vitro digestion products of quinoa protein isolate after different heat treatment.

