## 4 In Silico Studies on the Biological Activities of Meat Peptides--Practical Approach
The authors identified the peptide sequences by peptidomics (by liquid chromatography-electrospray ionization-mass spectrometry (LCESI-MS), and then subjected the identified sequences to in silico analysis (PeptideRanker and BIOPEP). It was observed that collagen peptides (with PeptideRanker bioactivity scores of 0.6-0.8) and high-score peptides (>0.8) may contribute to bioactivities [58]. The \"Potential Biological Activity Profile\" is another tool available in the BIOPEP-UWM database and is used to discover the different bioactivities of individual protein fragments. This database provides the opportunity to discover the potential properties of the bioactive peptides in a total of 55 different activities, including those improving health (antioxidant, antihypertensive, antidiabetic, anticancer, memory-improving, and immune-system-support properties) as well as those harmful to health (anorectic, opioid, toxic, or embryotoxic) (data for November 2021). It was previously used to predict the potential of a protein to release biologically active peptides (DPP-IV and ACE inhibitors) from muscle proteins [22; 30; 54]. Regardless of the analyzed meat protein particles, on the basis of the quoted literature sources, many different biological activities of peptides, according to their structures, were determined, which confirms the high potential of meat and meat products to modulate the physiological functions of the human body. Among them, the inhibitory activity of the enzymes, DPP-IV and ACE-I, and the antioxidant activity were most often determined (Table 1). Figure 4: Workflow for assessing the biological potential of food proteins—scheme. Enzymatic hydrolysis is the most important method for obtaining bioactive peptides from food products and is part of the prediction of the release of bioactive peptides, as shown in Figure 2. Therefore, after determining which proteins are potentially the best sources, the next step is the hydrolytic release of the peptides from the precursor protein. For example, a total of 29 different proteases (using BIOPEP-UWM tools) were tested to evaluate the most effective enzyme for releasing peptides acting as ACE inhibitors from bovine collagen, and papain was indicated as the most effective one [34]. [PERSON] et al. [30] assessed six proteases (trypsin (EC 3.4.21.4), pepsin (EC3.4.26.1), papain (EC 3.4.22.2), bromelain (EC 3.4.22.4), ficain (EC3.4.22.3), and thermolysin (EC 3.4.24.27)) for in silico hydrolysis using the programs, ExPASy PeptideCutter [59] and BIOPEP [23]. Five proteins of pork or bovine byproducts were evaluated as the substrates for use in the generation of prolyl endopeptidase inhibitory peptides [30]. The variety of enzyme preparations, and the different methods for obtaining hydrolyses (using a single enzyme or a combination thereof), offer great opportunities for the selection of the most advantageous enzyme before performing the in vitro steps. In this context, the use of in silico methods enables quick and economical design proteolysis processes and facilitates the selection of the most effective enzyme for cleaving peptide bonds. Depending on the expected results, in silico hydrolysis analysis can be performed using many different enzymes, and the only limitation is the degree of development of the database tools. However, according to the INFOGEST concept, hydrolysis that imitates the digestive processes of the human digestive system is particularly relevant. As shown in Table 1, the protein sequences were often hydrolyzed in silico with enzymes that are equivalent to human digestive proteases, namely, pepsin (EC 3.4.23.1), trypsin (EC 3.4.21.4), and chymotrypsin (EC 3.4.21.1). This integrated approach allows for the simultaneous discovery of the complete biological activity profiles of peptides released from proteins by using different proteolytic enzymes. In general, it seems that peptides released from proteins retain biological activity after the in silico hydrolysis step.