[A study of soy protein proteolysis by digestive enzymes and description of the peptide profile of the obtained hydrolysates].

Abstract

The study of proteolysis of plant proteins is attracting increasing attention. When proteolytic enzymes act on plant proteins, peptides with different biological activities can be released. Proteolytic hydrolysis, being a natural mechanism of protein digestion in the body, has been successfully adapted for use in food technology in order to modify the functional and biological properties of protein ingredients. The purpose of this work was to study the effect of digestive enzymes on soy protein in vitro and analyze the peptide profile of the obtained hydrolysates using in silico instruments.

Material and methods: The proteolysis of soy protein isolate (with a protein content of 89%) was carried out using the enzymes trypsin (at pH 7.8±0.1), pepsin (at pH 2.0±0.1), as well as their sequential action - hydrolysis first with pepsin (for 2 hours), and then with trypsin (2 hours). The degree of hydrolysis was calculated as the ratio of the concentration of amine nitrogen released during proteolysis to the total nitrogen content in the suspension before hydrolysis. The dominant peptides were identified using the nano- LC-MS/MS method. The databases BIOPEP-UWM, AntiBP3, Antifp, AntiCP 2.0, and AntiTbPred were used to predict the biological activity of the dominant peptide sequences in hydrolysates. The toxicity of the peptides was assessed using ToxinPred3.0. AutoDock Vina 1.2.7 software was used for molecular docking.

Results: When treated with trypsin (200 U/g) at an enzyme dosage of 4% for 4 hours, the degree of hydrolysis was 5.38%, when treated with pepsin (420 U/g) at the same enzyme dosage for 4 hours, taking into account optimal pH values, the degree of hydrolysis was 7.06%. With sequential proteolysis by these enzymes, a hydrolysis rate of 7.71% was achieved. Based on mass spectrometric data, 37 of the most common peptide sequences present in hydrolysates were analyzed in silico. The dominant properties predicted for the discovered peptide sequences were the ability to inhibit angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV), as well as oncostatic properties. The binding energy of some peptides to DPP-IV and ACE enzymes was also estimated using molecular docking. A low binding energy to DPP-IV (-8.154 kcal/mol) was observed in the ATISDQPRGSY peptide, while the LAIPVNKPGRF, TTVPPHSVQVHTTTHRYEAGVPPARF and VSIIDTNSLENQLDQMPRRF peptides had a low binding index to ACE (<-8.5 kcal/mol), indicating a potentially high inhibitory activity of these peptides against DPP-IV and ACE.

Conclusion: The present study describes a number of new bioactive peptides from soy that can be formed during proteolysis by pepsin and trypsin. The data obtained form the basis for further investigation of the biological activity of the obtained hydrolysates and the identified peptides, which in the future may be useful for the development of technologies for functional products and components from soy protein. Another area of further research (in view of the origin of the enzymes under study) may be the search for identified peptides in conditions close to human digestion, which will expand knowledge about the mechanisms of the physiological effect of soy protein when consumed. It is advisable to direct further research to the chemical synthesis of the peptides found in this research in order to study their properties in vitro and in vivo.