The transformation of milk into cheese occurs under the influence of many physicochemical, biochemical and microbiological processes, among which proteolysis plays a very important role. Proteolysis belongs to the most complex type of irreversible post-translational modification of proteins. Enzymatic proteolysis catalysts at different stages of cheese production are native milk enzymes, exo- and endopeptidases of starter and non-starter microorganisms, and milk-clotting enzymes. The article presents a brief overview of modern ideas about the properties, mechanism of action and specificity of the main representatives of enzymes that hydrolyze milk proteins at the stages of preparing milk for coagulation, during rennet coagulation and subsequent maturation of cheeses. These include the plasmin system of milk, enzymes of psychrotrophic bacteria and lactic acid microorganisms that enter milk both accidentally (non-starter microflora) and planned in the form of starter cultures from specially selected strains. Milk-clotting enzymes, having fulfilled their main function — milk coagulation — partially pass into cheese and, along with enzymes of starter microorganisms and plasmin, participate in proteolytic processes during cheese ripening. It is generally accepted that proteolysis in ripening cheeses is the most significant biochemical process that affects the formation of taste, aroma and texture along with lipolysis and glycolysis. The combination of proteolysis products (peptides, amino acids, amines, etc.) is individual for different types of cheese and varies depending on the technological parameters of production, including the duration of maturation. Proteolysis in cheeses has been studied by many scientists in various aspects. This review supplements the known information with new information, without claiming to be comprehensive.

The review presents the most common proteomics methods based on separation and identification of proteins, with examples of their use for studying the protein system of milk and dairy products. The essence of electrophoretic and chromatographic methods for separating proteins and peptides and their identification using Western blotting and mass spectrometry is described. The main types of electrophoresis methods (native, two-dimensional, in polyacrylamide gel with urea, immunoelectrophoresis, capillary) and chromatography methods (gas, liquid, ion exchange, gel filtration, affinity) are given. It is noted that the main direction of research using these methods, which has both scientific and applied significance, is the detection of falsifications in the composition of milk and dairy products. Such falsifications are associated with the deliberate addition of dry milk and cheese whey, as well as with the replacement of raw milk by milk from other animals, which is unacceptable in the production of cheeses with a protected designation of origin (PDO) or a protected geographical indication (PGI). Another area is research into the proteolytic processes that occur during the maturation of cheeses, on the basis of which their degree of maturity and species can be determined depending on the manufacturing technology. Given the wide range of cheeses from different countries, such research is clearly insufficient.

Cheese whey is a large-scale by-product of milk processing, characterized by high biological value. However, its irrational use can lead to a serious environmental burden. One of the ways to solve this problem is the isolation of proteins and their fractionation, the efficiency of which significantly depends on the method used. In this work, a comparative assessment of four methods of processing reconstituted cheese whey (6.0 % dry matter mass fraction) was carried out, such as chitosan precipitation (pH 4.5; 20 °C; 1 % gel; 20 min), thermal acid coagulation (pH 4.65; 90 °C; 15 min) using lactic, citric, phosphoric and hydrochloric acids, cryo-acid precipitation (pH 4.5; –18 °C; 24 h) and ultrafiltration (spiral membranes with a cutoff threshold of 10 kDa). The study determined the proportion of precipitated protein, the fractional composition of soluble nitrogenous compounds using gel filtration, and specific operating costs. Ultrafiltration provided the highest efficiency of protein separation (65.92 ± 0.59 %), which also turned out to be an order of magnitude cheaper (≈112 rubles/m3) than all chemical methods. Among the thermal acid options, citric (57.83 ± 0.51 %) and hydrochloric (56.63 ± 0.55 %) acids proved to be the most effective. Chitosan and cryo-acid treatment showed low overall efficiency (26.51 ± 0.20 % and 25.30 ± 0.15 %, respectively). Analysis of the fractional composition of the supernatant showed that thermal acid coagulation and ultrafiltration completely removed β-lactoglobulin and BSA. Chitosan precipitation preserved the native profile of all major whey proteins, removing only ballast impurities. The cryoacid method demonstrated unique selectivity, virtually completely removing peptides while preserving native α-lactalbumin and β-lactoglobulin. A step-by-step fractionation strategy is proposed: treatment with chitosan to remove impurities, cryoacid separation of peptides, followed by isolation of native proteins by ultrafiltration or the thermal acid method.

A one-pot synthesis of 2-(3-iminoisoindol-1-ylidene)-2-arylacetonitriles in yields of up to 95% by the cascade condensation of phthalonitrile with arylacetonitriles in a superbasic NaOH/DMSO medium was proposed. A method for the synthesis of (E)-2-(3-oxoisoindol-1-ylidene)-2-arylacetonitriles by the hydrolysis of 2-(3-iminoisoindol-1-ylidene)-2-arylacetonitriles in acetic acid was developed. The obtained (E)-2-(3-iminoisoindol-1-ylidene)-2-arylacetonitriles were shown to be active inhibitors of human monoamine oxidase [IC₅₀(MAO-A) for (E)-2-(3-iminoisoindol-1-ylidene)-2-(4-methylphenyl)acetonitrile 3.26 µM].