## 3 Results and Discussion
### Purification and Characterization of \(\alpha\)AI-1
In Figure 6A, according to the LOC electrophoresis profiles of Pinto bean \"purified \(\alpha\)AI-1 protein\", electrophoretic peaks were detected at 24.92; 26.22; 26.79; 27.52; 28.51; 29.23; and 30.12 s retention times. The peaks of the pepsin were detected at 32.76 and 33.76 s retention times. The stomach sample of the control rat contained no peaks at retention times corresponding to the bean \(\alpha\)AI-1. Figure 5: Protein pattern of LOC electrophoresis of stomach and small intestinal digesta samples obtained from the acute rat model. **Lanes:** L. Molecular marker; 1. Pinto bean meal; 2. Pinto bean cooked meal; 3. Pinto bean “purified \(\alpha\)AI-1 protein”; 4. control stomach digesta; 5. Pinto bean meal stomach digesta; 6. Pinto bean meal small intestinal digesta; 7. Pinto bean cooked meal stomach digesta; 8. Pinto bean cooked meal small intestinal digesta; 9. control small intestinal digesta. In Figure 6B, in the case of Pinto bean meal stomach digesta, four peaks were detected at retention times of 24.36; 26,54; 27.81; and 29.54, which presumably correspond to the bean \(\alpha\)AI-1. In Figure 6C, Pinto bean cooked meal stomach digesta contained five peaks at t\({}_{\rm R}\) 24.87; 25.91; 26.55; 27.93; and 29.75 s in the region of the \(\alpha\)AI-1 peaks. The detected peaks indicate the presence of \(\alpha\)AI-1 in the stomach (resistant to pepsin digestion). However, the retention times of the in vivo small intestinal digestive enzymes were very close to the retention time of the bean \(\alpha\)AI-1; we could not identify the \(\alpha\)AI-1 from the small intestinal digesta by LOC electrophoresis. Therefore, we carried out the identification of \(\alpha\)AI-1 bands that were resistant to digestion by SDS-PAGE separation (Figure 7A,C) followed by immunoblot using anti-bean \(\alpha\)AI-1 rabbit IgG antibodies (Figure 7C,D). Figure 6: LOC electrophoretograms of stomach digesta samples obtained from the acute rat model. (**A**) Electrophoretograms of Pinto bean “purified \(\alpha\)AI-1 protein” (red), where the retention times of the \(\alpha\)AI-1 are 24.92; 26.22; 26.79; 27.52; 28.51; 29.23; and 30.12 sec (see in Table 1), and proteins extracted from stomach of control rat (blue). The retention times of the pepsin are 32.76 and 33.76 s. (**B**) Electrophoretograms of Pinto bean “purified \(\alpha\)AI-1 protein” (red) and Pinto bean meal stomach digesta (blue), where the retention times of the \(\alpha\)AI-1 are 24.36; 26.54; 27.81; and 29.54 s. (**C**) Electrophoretograms of Pinto bean “purified \(\alpha\)AI-1 protein” (red) and Pinto bean cooked meal stomach digesta (blue). The retention times of the \(\alpha\)AI-1 are 24.87; 25.91; 26.55; 27.93; and 29.75 s.  
Results showed that \(\alpha\)AI-1 released from Pinto bean meal stomach and small intestinal digesta (Figure 7B, lane 4, 5) and that released from Pinto bean cooked meal stomach and small intestinal digesta (Figure 7D, lane 4, 5) survived digestion in the immune-reactive form (see at 15 kDa). Besides the identification of the presence and the immune-reactive properties of \(\alpha\)AI-1 after in vivo digestion, we analyzed whether it retained its activity not only in the stomach but also in the small intestine. On native PAGE, the \(\alpha\)AI-1 activity in in vivo digested Pinto bean meal and Pinto bean cooked meal samples was analyzed (Figure 8). Similarly to gastric in vitro digestion, the \(\alpha\)AI-1 was still inhibitory-active in both stomach digesta samples. We assumed that both in vitro and in vivo gastric digestion circumstances probably led to the activation of the inactive pre-protein form of bean \(\alpha\)AI-1. Similarly to our results, [PERSON] et al. (2023) demonstrated that peptides of legume \(\alpha\)AI were active against \(\alpha\)-amylase after cooking and gastrointestinal digestion. They found that a <3 kDa peptide fraction was responsible for the main activity [19].