Isolation of bioactive components

Bioactive components, including proteins and peptides, were obtained as previously described [15]. The technology includes extraction of Sus scrofa thymus, spleen, and lymph nodes by water-salt solution (0.9% NaCl) containing 50 ppm deuterium, after which the extracts are purified and frozen at −40°C.

Due to the influence of the isotopic composition of water on the biological properties and total extra-activity of proteins and peptides, water with deuterium concentration of 50 ppm was used for the extraction [12]. Molecular composition was characterized as described by Fedulova et al. [13], including α-thymosin (C3VVV8_PIG), rho GDP-dissociation inhibitor 1 (ARHGDIA)+ Acetyl (Protein N-term), rho GDP-dissociation inhibitor 2 (ARHGDIB), serpin B9 (SERPINB9), tyrosine kinase-binding protein (TYRO) and calnexin (CNX), C-terminus cathepsin S (STSS), myeloid differentiation primary response protein (MyD88), and transgelin (TAGLN), among others.

Tissue-specific biomolecules immobilized into alginate capsules were prepared on the basis of the technology developed by Manjanna et al. [17]. In brief, sodium alginate (Ruskhim, Russia) was dissolved in distilled water containing protein-peptide complexes (with a protein concentration in the solution of 21±2 g/L) until a final concentration of alginate of 1.2% by weight was achieved using a laboratory dispersion unit (Laboteks, Russia). The resulting suspension was passed using a peristaltic pump (Shenchenlab 2015 MCSeries, China) with a tube diameter of 1.5 mm and nozzle with a hole diameter of 0.15 mm in a 2% solution of calcium chloride (AppliChem Panreac, Germany) with a final pH of the solution equal to 4.0 (adjustment to the target pH was performed with 1 M NaOH). Next, the obtained capsules that were continuously mixed for 30 min on an MM-5 magnetic stirrer (Russia), washed with distilled water, and freeze-dried (INEY-4, Russia) at a temperature of −40±2°C and pressure of 3.3 kPa.