## 3 Results and Discussion
### Purification and Characterization of \(\alpha\)AI-1
The \(\beta\)-lactoglobulin still remained undigested even after 60 min, similarly to the 15 kDa protein (active \(\alpha\)AI-1), while the 30 kDa protein band (precursor protein) had already been destroyed after 2-min popsin digestion. So, these results indicated that the active \(\alpha\)AI-1 inhibitor was resistant to popsin. The explanation for this phenomenon is that legume proteins--similarly to \(\beta\)-lactoglobulin--have high \(\beta\)-sheet content that can be related to their resistance to gastrointestinal digestion [16]. As the active \(\alpha\)AI-1 (15 kDa) of Pinto bean was still present after 60 min digestion, the \"purified \(\alpha\)AI-1 protein\" of the other common bean varieties (Huanita Dry, Bush, and Red Kidney) have already been submitted under 60-min digestion. The results on their protein profile showed the same as before in the case of Pinto bean, where all active \(\alpha\)AI-1s at 15 kDa remained undigested, while the 30 kDa protein band (precursor protein) was destroyed (Figure 3A). It was a positive phenomenon that \(\alpha\) AI-1 remained active in the gastric phase, but it still needed further analyses regarding whether it remained in active form in the small intestine or not (see later in the in vivo experiment). Immunoblotting, i.e., the use of an antigen-specific antibody, can be used to determine the presence and molecular weight of the proteins of interest. In order to confirm our previous results, immunoblotting was performed. We wondered whether the remaining 15 kDa protein would retain its immune reactivity (antigenicity) or not. We observed that these proteins remained immune-reactive against anti-bean \(\alpha\)AI-1 rabbit IgG antibodies (Figure 3B). Besides monitoring the resistance and immune reactivity of \(\alpha\)AI-1, we have investigated whether the digestion-resistant and antigenic \(\alpha\)AI-1 protein (15 kDa) could retain its bioactivity towards \(\alpha\)-amylase even after gastric digestion or not. For the analyses of the \(\alpha\)AI-1 inhibitory activity of the \"purified \(\alpha\)AI-1 protein\" of the four common bean varieties that had been gastric digested in vitro (for 60 min), native PAGE was conducted (Figure 4). Pinto-bean-derived \(\alpha\)AI-1 extract and wheat-derived \(\alpha\)AI-1 extract were used as positive controls (undigested). Native PAGE is preferable for enzyme activity testing because proteins retain their native, active form, which is essential for measuring their activity. Native PAGE separation is based not only on molecular weight but also on electrical charge and natural conformation, which can be useful for preserving and testing enzyme activity. Figure 3: Monitoring the effect of gastric in vitro digestion (for 60 min) on the resistance to digestion of the “purified \(\alpha\)AI-1 protein” of common beans by 6/15% SDS-PAGE (**A**) and on the immune reactivity of those by immunoblot using anti-bean \(\alpha\)AI-1 rabbit IgG antibodies (**B**). **Lanes (A,B):** 1. Pinto bean \"purified \(\alpha\)AI-1 protein\" (undigested); 2. Huanita Dry bean \"purified \(\alpha\)AI-1 protein\" digested for 60 min; 3. Bush bean “purified \(\alpha\)AI-1 protein” digested for 60 min; 4. Red Kidney bean “purified \(\alpha\)AI-1 protein” digested for 60 min; 5. Pinto bean “purified \(\alpha\)AI-1 protein” digested for 60 min. The pattern in Figure 4A shows that the active \(\alpha\)AI-1 protein (15 kDa) of the \"purified \(\alpha\)AI-1 protein\" extract of all bean varieties have lost their activity after the 60-min digestion, while the inhibitor activities were still detected in the bean flour samples after the 60-min digestion (Figure 4B). This can be explained by the fact that the bean flours still contain the pre-protein of the \(\alpha\)AI-1 (30 kDa) after 60-min digestion, while, in the case of the \"purified \(\alpha\)AI-1 protein\" sample, the 30 kDa precursor protein degraded after 60 min digestion (see Figure 1).