## 4 Conclusions  
In vitro digestion experiments are widely used to assess the digestibility and structural changes of food components under simulated gastrointestinal conditions. In this study, we assessed the effect of in vitro and in vivo digestion on Hungarian putative intraveutical common bean (Pinto, Huanita, Bush, and Red Kidney) derived \(\alpha\)AI-1 tested in matrix (flour/native and cooked bean meal) and/or purified form. Purified \(\alpha\)AI-1 proteins showed that, after 60-min in vitro gastric digestion, \(\alpha\)AI-1s remained resistant, retained their immune reactivity, but lost their inhibitory activity. When the bean flour (bean meal) was studied and not the purified protein extract, the activity was detectable.  
Our results were comparable with clinically controlled trials by [PERSON] et al. [9], where purified \(\alpha\)AI did not inhibit effectively the carbohydrate digestion, only the partially purified preparation. Other studies [15; 19; 30] also revealed \(\alpha\)AI activity in protein hydrolysates or peptide extracts instead of purified \(\alpha\)AI.  
Knowing this information, we used bean meal (native and cooked) in a next step in rat feeding experiments and tested in vivo the fate of \(\alpha\)AI-1 in the stomach and then in the small intestine. Immune-reactive \(\alpha\)AI-1 bands were detected but the activity was not detectable in the small intestine, in contrast to the stomach.  
We could conclude from both gastric in vitro and in vivo experiments that bean flour and even the cooked bean flour containing \(\alpha\)AI-1 precursor became active against the alpha  
Figure 8: Monitoring the effect of in vivo digestion (for 60 min) on the bioactivity of \(\alpha\)AI-1 in bean meals by native-PAGE. **Lanes**: 1. Pinto bean “purified \(\alpha\)AI-1 protein”; 2. Pinto bean meal stomach digesta; 3. Pinto bean meal small intestinal digesta; 4. Pinto bean cooked meal stomach digesta; 5. Pinto bean cooked meal small intestinal digesta; 6. wheat “purified \(\alpha\)AI-1 protein”; 7. Pinto bean “purified \(\alpha\)AI-1 protein”; 8. control meal stomach digesta; 9. control meal small intestinal digesta.  
amylase digestive enzyme in the gastrointestinal tract, as the physiological circumstances during digestion in the stomach could activate the inactive protein form of the bean \(\alpha\)AI-1.  
The study of alpha-amylase inhibitors can, therefore, provide new opportunities and valuable data not only in nutrition science but also in drug development, the food industry, and biochemistry.  
Conceptualization, A.M.; methodology, A.J., A.N., and K.T.; software, A.J., A.N. and K.T.; validation, A.M., A.J., A.N. and K.T.; formal analysis, A.M., A.N. and K.T.; investigation, A.N., A.J. and K.T.; resources, A.M.; data curation, A.M.; writing--original draft preparation, A.M.; writing--review and editing, K.T. and I.D.; visualization, A.M.; supervision, A.M.; project administration, A.M. and I.D.; funding acquisition, A.M. All authors have read and agreed to the published version of the manuscript.  
The research was supported with the GMSAFOOD EU7 (EU FP7/2007-13 SCP, no FP7 211820) \"Biomarkers for post market monitoring of short and long-term effects of genetically modified organisms on animal and human health\" Fund.  
Not applicable.  
The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.  
This work was supported by the Research Excellence Program of the Hungarian University of Agriculture and Life Sciences. Furthermore, the authors are grateful to the COST FA1403 Action \"Interindividual variation in response to consumption of plant food bioactives and determinants involved (POSITIVE)\" for the support of the research.  
The authors declare no conflicts of interest.