| Abstract [eng] |
The rapidly growing global population leads to an increased protein demand in food and feed systems. Proteins play a crucial role in supporting essential functions within cells, tissues, organs, and various biological systems. In recent years, the popularity of plant-based proteins has grown due to their benefits over animal-based proteins. Plants provide high levels of proteins without the high content of fats which is often related to animal sourced proteins. Moreover, plant-based proteins are more environmentally friendly than animal-based proteins, requiring less energy and water for production. Protein hydrolysate is the most nutritious form of proteins. It contains a mixture of short-chain peptides and free amino acids. Traditional chemical hydrolysis methods are performed under hazardous conditions that can influence protein structure and functional properties. Oppositely, enzymatic hydrolysis is performed under mild conditions, it is easily controllable due to the specificity of enzymes and environmentally safe method. Protein hydrolysates can be applied in the food industry as functional foods and in the pharmaceutical industry due to the presence of bioactive peptides. These bioactive peptides can exhibit various beneficial features, including antioxidant activity, antihypertensive activity, antimicrobial activity, etc. In this study, the main aim was to develop a biocatalytic technology for the hydrolysis of plant-based proteins. The study consisted of two parts. The first part was setting the parameters for the hydrolysis of alfalfa meal using commercial enzymes. ATR-FTIR spectroscopy method was developed to assess the hydrolysis process and to determine the degree of hydrolysis (DH). Vilzim Pro AL3000 protease was defined to the best performer and used for further hydrolysis of alfalfa meal. The highest DH was obtained when the reaction was performed under pH 7.0 at 35 °C for 4 h. However, it was observed that during hydrolysis, microbial contamination occurs. The application of antimicrobial agents revealed that sodium nitrite inhibits the growth of microorganisms. The second part of the study was the development of expression system for heterologous recombinant protease in Pichia pastoris yeast. Two different cloning systems were applied for serratopeptidase gene cloning – the conventional cloning method and the GoldenPiCS approach. The first cloning method was applied successfully, and expression of recombinant protein was achieved. However, for the GoldenPiCS approach further investigation is required. |
