## 4 In Silico Studies on the Biological Activities of Meat Peptides--Practical Approach
Because of the abovementioned considerations, the stage of in silico enzymatic hydrolysis was treated as a preliminary (predictive) study, which enabled the planning of laboratory-scale studies using in vitro methods. The data obtained demonstrate the need for the further exploration and development of effective hydrolysis protocols in order to obtain the necessary information for the development of new therapeutic formulations or functional foods. It must also be mentioned that in silico hydrolysis is also an integral part of proteomic processes, and the most frequently repeated activity in the literature is the in vitro hydrolysis of dietary proteins, and their evaluation by chromatography combined with mass spectrometry (MS). A key step in MS-based proteomics is the identification of peptides in sequence databases on the basis of their fragmentation spectra. For example, the search engine, Andromeda, uses the MaxQuant computational proteomics platform (freely available at www.maxquant.org, accessed on 1 June 2021) [61] or Mascot (usually the data are processed by Mascot Distiller and then by Mascot Search, and the results are then compared to the Uniprot database) [35; 36; 60]. According to [PERSON] et al. [62], MS is a very good method for quantifying bioactive peptides. However, although this technique has high sensitivity and accuracy, there are several problems with its use for the detection and quantification of bioactive peptides. The concentration of some peptides in food products may be below the detection limit. Another problem arises during hydrolysis. This process, occurring before MS, generates many peptides without biological activity from endogenous proteins, which may interfere with the signals from bioactive peptides and prevent the correct determination of their concentration [63; 64]. The sequence of peptides identified by peptidomics from meat or meat products enables the accurate assessment of their bioactivity by in silico methods. In silico analyses allow not only for the search for, and the evaluation of, the biological activity of molecules (peptides), but they also provide the opportunity to better understand the properties of the biomolecules, including their functions in the human body. The computer-assisted molecular modeling software seems to be helpful in this respect. Molecular modeling software are used as effective tools for predicting the interaction of a test compound with a target molecule. By using a theoretical simulation method (i.e., molecular docking), it is possible to predict the binding of peptides to the specific sites (e.g., receptors and active sites of enzymes) of proteins, and to quantify the binding energy by using a scoring system [29; 65]. Generally, this approach consists of searching for the optimal (with the lowest energy) ligand-binding mode in the receptor structure (interaction of the ligand with the receptor--most often with a protein). The main assumptions of molecular docking, as well as the types of this approach and its possible application in the context of food, are presented as an example in [66]. Regarding food of animal origin,the molecular docking of two peptides (VGPV and GPRGF), derived from bovine collagen hydrolyzed with Alcalase(r) and papain, confirmed that these peptides influenced the ACE molecule and, additionally, indicated that the inhibitory mechanism of these peptides was noncompetitive [67]. Docking simulations were also carried out using ACE and different peptides from dry-cured pork ham. Molecular modeling showed that KPVAAP, KAAAATP, and KPGRP are stabilized by both hydrogen and hydrophobic interactions with at least one residue of the three pockets, which is consistent with the typical competitive inhibition model [24]. Moreover, two novel ACE inhibitory peptides (EACF and CDF) were screened from rabbit meat proteins using in silico methods. Molecular docking experiments revealed that EACF established eight H-bond interactions in the S1 and S2 pockets, and a metal-acceptor interaction with Zn 701.