## 4 In Silico Studies on the Biological Activities of Meat Peptides--Practical Approach
Nevertheless, as shown by previous studies [14], only a part of potentially active molecules was released (or was resistant to enzymatic degradation) as a result of the action of pepsin, trypsin, and chymotrypsin. These studies were based on calculations of the frequency of bioactive peptides, which corresponds to the ratio of the number of bioactive fragments with selected activity in a dietary protein sequence (a), and the number of amino acid residues of protein (N) (parameter A = a/N, BIOPEP-UWM). These incidence values, however, consider that all bioactive sequences could be released simultaneously from the respective dietary protein and that they are equivalent in terms of their bioactive properties. However, in these in silico analyses, neither the potency of the peptides, nor the overlapping amino acid residues, were considered (the common amino acid residue may be part of different peptides, as mentioned earlier). This trend was also confirmed in an in vitro analysis, where the anti-radical activity of hydrolyses after gastric digestion (compared to undigested protein extracts, expressed as the concentration of peptides that can inhibit 50% of the enzyme activity, IC\({}_{50}\)) of dry-cured pork loin with probiotic lactic acid bacteria (LAB) strains was reduced [60]. Interesting conclusions were drawn by [PERSON] et al. [34], who proved that the in vitro hydrolysis of bovine collagen with papain led to the production of ACE inhibitory peptides, and the most active of them was identified as a pentapeptide (GPRGF). However, in predictive studies, this peptide was not found to be an ACE-inhibitory peptide during in silico hydrolysis by papain. According to the authors, this was mainly due to complete hydrolysis, which was not achieved by in vitro hydrolysis. This example shows that in silico analyses, despite many advantages, are not free from disadvantages, and the obtained results must be confirmed with laboratory tests, and within the broader perspective on living organisms, in order to demonstrate their impact on human health. A different approach was presented by [PERSON] et al. [32], who first used an in vivo approach to evaluate the kinetics of peptide release during the hydrolysis of beef proteins in the stomachs of animals (gastric digestion steps). Subsequently, the peptides generated from the hydrolysis of the meat in the stomachs were identified and quantifiedusing a spectrometric method, followed by in silico digestion mimicking the action of enzymes (intestinal digestion steps). The pool of peptides that was identified as bioactive included an ACE inhibitor, an antioxidative peptide, a glucose-uptake-stimulating peptide, an antithrombotic peptide, a DPP-IV inhibitor, a regulator of the stomach mucosal membrane activity, a dipeptidyl carboxypeptidase inhibitor, and a hypotensive peptide (the list of peptides is presented in Table 1). In addition, studies have shown that certain proteins are hydrolyzed more slowly in vivo in the stomach, such as proteins involved in muscle contraction, than those noted in in vitro or in silico studies. The explanation may be that these proteins (e.g., myosin, actin, and myosin-binding protein C) form a complex (here: an actomyosin) that can reduce the availability of proteolytic enzymes to their cleavage sites located inside the protein. In silico analysis is performed on linear protein sequences (equivalent to the primary protein structure). In vivo, the structure of proteins is not so clear. There may be a quaternary form when it is a large intact protein moiety, or an intermediate form (e.g., alpha helix, beta sheet) when proteolytic enzymes are activated. It is not always possible to cleave the peptide bonds at the enzyme-specific cleavage sites within the chain. This is especially true for globular proteins, in which part of the polypeptide chain, called \"the core\", is hidden in the depths of the molecule and is not influenced by proteases of narrow specificities.