Biotechnological and microbiological aspects of development of capsule form for fecal microbiota transplantation

Aim: To evaluate the feasibility of freeze-drying the substance of monkey gut microbiota and the use of serial acid-tolerant capsules for the preparation of a finished form for fecal microbiota transplantation (FMT). Materials and Methods: To prepare a liquid substance for fecal microbiota transplantation, monkey feces were homogenised in physiological solution, then centrifuged to obtain the supernatant, which was stabilised by adding 5% sucrose. The obtained suspension was poured into vials and frozen at -80 °C. After that, the substance was dried in Labconco's FreeZone 6 freeze-drying unit, crushed and filled into capsules using a PRESSORE 100 capsule machine. To protect the capsules from the acidic environment of the stomach, tests were carried out using barium sulphate. The capsules were examined radiologically by introducing them into the mouths of the animals and taking radiographs of the gastrointestinal tract in two projections. Molecular genetic studies were carried out according to the methodological instructions for working with nucleic acids. Intestinal suspensions of microbiota were prepared using sterile phosphate buffer. Nucleic acid extraction and PCR were performed using DNA-Sorb-B reagents and the COLONOFLOR-16 kit, with detection on a CFX-96 device. Results: The preparation of capsules for fecal microbiota transplantation requires a dry active substance of the microbiota. The lyophilisation process was carried out at -37°C for 32 hours. DR capsTM ACID-RESISTANT CAPSULES have been used to protect the active microbiota from gastric juice, but initial studies have shown poor protection. To improve protection, a new acid enzyme resistant capsule was developed that preserved the microbiota well in acidic medium and dissolved in alkaline medium. The study of the microbial composition by PCR with fluorescence detection confirmed that the number and composition of microorganisms remained unchanged after freeze-drying, indicating that their activity was preserved. Conclusion: Our own studies have shown that it is possible to freeze-dry the substance of the monkey gut microbiota and produce capsules with enterosolubilised shells suitable for targeted delivery in fecal microbiota transplantation.