## 3 Results and Discussion
### Purification and Characterization of \(\alpha\)AI-1
We suppose that pepsin digestion helps the pre-protein degradation, which leads to the activation of the active form (15 kDa protein) function. [PERSON] et al. [9] published that a partially purified white bean product was active against salivary, intraudodenal, and intra-ilecal amylase in vitro, while the crude preparation of the purified bean amylase inhibitors had insufficient enzyme-inhibiting activity, which corresponds to our finding as well. These results lead to the conclusion that it is better to consume \(\alpha\)AI-1 in matrix (flour) because it retains its activity in this form (while purified \(\alpha\)AI-1 loses its activity). [PERSON] et al. [15] showed, in the case of protein hydrolysate from lima bean (_Phaseolus luntatus_ L), that the highest \(\alpha\)AI activity was in a \(>\)10 kDa fraction, followed by a 3-5 kDa fraction. Peptide fraction \(<\)3 kDa from Pinto bean was found to have high \(\alpha\)AI activity (62.1%) by [PERSON] et al. [30]. The Effect of In Vivo Acute Rat Digestion Model on the Presence, Immune Reactivity, and Bioactivity of \(\alpha\)AI-1  
Our previous studies have so far concluded that \(\alpha\)AI-1 retained its biological activity in matrix (flour), while it was lost it in its purified form during gastric digestion (see Figure 3). We wanted to confirm the validity of these results in vivo in rats as well, furthermore, to study the effect of the intestinal digestive processes on the resistance, immune reactivity, and bioactivity of \(\alpha\)AI-1. Therefore, it could be considered that the bioactive \(\alpha\)AI-1 (in flour) could affect carbohydrate metabolism due to the inhibition of pancreatic amylase. For this purpose, an acute rat digestion model was used, where the duration of digestion was 60 min (see Section 2.4). Pinto bean meal and a highly heat-treated sample, i.e., Pinto bean cooked meal, were given as feed for the rats. As regards the use of cooked sample, with heat treatment (cooking), we wanted to abolish the undesirable effects of  
Figure 4: Monitoring the effect of gastric in vitro digestion (for 60 min) on the bioactivity of the “purified \(\alpha\)AI-1 protein” of common beans by native PAGE. **Lanes (A) (“purified \(\alpha\)AI-1 proteins”):** 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; 6. wheat “purified \(\alpha\)AI-1 protein” (undigested). **Lanes (B) (flours)**: 1. Huanita Dry bean flour digested for 60 min; 2. Bush bean flour digested for 60 min; 3. Red Kidney bean flour digested for 60 min; 4. Pinto bean flour digested for 60 min; 5. wheat flour (undigested). the potential harmful components of beans such as lectin and trypsin inhibitors. On the other hand, we have taken into account the results obtained in Section 2.10, based on the investigation of the effect of heat treatment on the activity of \(\alpha\)AI. \(\alpha\)AI-1 lost its inhibitory activity at 100 \({}^{\circ}\)C (Table 2); however, during in vitro pepsin digestion in simulated gastric fluid, we concluded that the pre-protein in the bean \"\(\alpha\)AI-1 extract\", which was present in a higher proportion in the simulated gastric fluid, could be activated during digestion. Therefore, we chose to perform the feeding experiment with Pinto bean cooked meal (treated at 100 \({}^{\circ}\)C) too, as we wanted to find out whether or not the pre-protein could be activated in the heated form. There is need to characterize the temperature-inactivation profile of the inhibitor further, since many potential products in which it can be incorporated would require heat treatment during processing [31]. The fate of \(\alpha\)AI-1 released during digestion was assessed by LOC electrophoresis (Figures 5 and 6).