Optimization of methods for isolation and identification of peptides isolated from Hermetia illucens larvae

Background and Objectives: The development of resistance of microorganisms to existing antibacterial agents requires constant updating of existing drugs and research in the search for alternative sources of active substances. In recent years, the problem of the emergence of microorganisms resistant to all existing antimicrobial drugs has become systematic and requires significant attention from researchers to search for alternative sources of active substances. The main problem in the development of drugs based on antimicrobial peptides is the search for optimal solutions in the preparation of these substances. Therefore, optimization and search for methods of isolation, analysis and control of protein fractions of water-soluble peptides used for the subsequent development of antibacterial drugs based on them is an urgent task. Materials and Methods: Optimal conditions and methods have been selected for the preparative production of water-soluble peptides isolated from the biomass of Hermetia illucens larvae. Optimization and search of methods for isolation, analysis and control of protein fractions of these water-soluble peptides will ensure the accuracy of the results and obtain optimal amounts of protein fractions. Results: It has been found that the use of molecular sieves makes it possible to obtain a mixture of three peptides with a difference in chromatographic retention time of less than 1 minute, which has been confirmed by three parallel experiments on the isolation and purification of peptides. During HPLC it has been noted that protein fractions 1 and 2 have similar values and the first and third protein fractions have a smaller difference in retention time, which is why there is no complete separation of these chromatographic peaks. Comparison of the percentage of the area of the peptides obtained allows us to talk about the possibility of obtaining peptides of the same size from H. illucens larvae by HPLC, and in combination with DLS to obtain protein fractions with very similar physicochemical and physical characteristics, since this type of chromatography separates substances according to their size. Conclusion: The use of high-performance liquid chromatography makes it possible to establish the reproducibility of the method of isolation of antimicrobial peptides by cold extraction with water and further stages of protein purification, salting and molecular sieve chromatography, which, in correlation with DLS analysis, makes it possible to reliably identify the peptides obtained, and the developed technology of isolation and purification makes it possible to produce these proteins on an industrial scale at low cost.