## 3 Results and Discussion
### Acetylcholinesteraseerase Inhibitory Actzivity of LPH  
The importance of acetylcholinesteraseinesterase currently represents the major clue pointing to the cause of [PERSON]'s disease and comprises much of the scientific attention and research. The protein structure of the enzymes suggests their blocking by peptides of suitable structure; however, it was so far neglected by the scientific community until now. Nowadays, only few studies have examined whether enzymatic hydrolyses of dietary proteins can block the enzyme [83; 84]. More investigations are needed to clarify the relationships between particular amino acids and their order on a peptide chain with AChE-inhibitory effectiveness both in vitro and in vivo. The findings of our study have shown that lupin proteins could be considered as a highly prominent precursor of peptides with anti-AChE activity. Presented in Figure 2 are the results from the analysis of LPH against acetylcholinesterasecholinesteraseerase inhibitory activity. According to the results in Figure 2, all of the papain protein hydrolyses exhibited acetylcholinesteraseiciency inhibitory activity. The effect of E\(\cdot\)S ratio and duration of hydrolysis was tested by two-way ANOVA and Tukey HSD multiple comparisons test (Results in Supplementary file). Both factors are considered as the combination of them has significant impact (\(p\)-value \(\leq\) 0.05, results in Supplementary file) on the manifestation of the activity. Statistically significant difference was calculated between the three different E\(\cdot\)S ratios. The tests between the hydrolysis duration levels showed \(p\)-value \(\leq\) 0.05 only between 30-60 and 30-120. Sample **P1** exhibited the highest AChE inhibition (IC\({}_{50}\) = 199.63 mg/g protein), which is suggestive of the presence of peptides with significant affinity for the enzyme. We may assume that the AChE-inhibitory effects of papain protein hydrolyses could be also the result of the synergistic interactions between various peptides. The study on hempseed protein hydrolyses derived by the action of alcalase, papain, and pepsin reported active peptides in the range 6-11 mg/mL (IC\({}_{50}\)) [49]. The analysis of molecular weight distribution suggested that the active peptides possess ultra-low molecular weight (<1000 Da). Some papers have documented specific proteins and amino acids as relevant to the inhibition of AChE [47]. The same authors assumed that the presence of high concentration of lysine (39.62-40.18%) in the active  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline
**Microorganism** & **P1** & **P2** & **P3** & **P4** & **P5** & **P6** & **P7** & **P8** & **P9** \\ \cline{2-10} _E. coli_ ATCC 8739 & 0.9 \(\pm\) 0.01 4 & 1 \(\pm\) 0.06 \({}^{\circ}\) & 0.9 \(\pm\) 0.00 4 & 0.8 \(\pm\) 0.01 & 0.6 \(\pm\) 0.00 5 & 1.1 \(\pm\) 0.00 5 & 1.3 \(\pm\) 0.00 4 & 0.7 \(\pm\) 0.01 6 & 1.1 \(\pm\) 0.01 5 \\ \hline _S. enterica_ NCTC 6017 & - & - & - & - & - & - & - & - & - \\ _S. aureus_ ATCC 25093 & - & - & - & 0.7 & - & - & - & - & - \\ _L. monocytogenes_ & - & - & - & - & - & - & - & - & - \\ NCTC 11994 & - & - & - & - & - & - & - & - & - \\ \hline \end{tabular}
\end{table}
Table 4: Antibacterial activity of lupin protein hydrolysate.  
hydrolysates could be also a contribution. Nevertheless, further studies are needed to clarify the kinetics of inhibition. Future research on Alzheimer's disease should take the inhibitory peptides into perspective to demonstrate their potential efficacy as therapeutic agents and/or functional food additives. While in vivo studies are required to definitively determine the positive impact of adding hydrolysates to functional meals, it is crucial to do previous in vitro research to identify lupin hydrolysates with AChE inhibitory activity.