Scientific and experimental assessment of the biosafety of a recombinant cytokine-producing strain as part of a biocorrective agent

Introduction Recombinant probiotic microorganisms that express immunoregulatory proteins are considered promising tools for the local correction of inflammatory bowel diseases. However, their practical application requires a comprehensive biosafety assessment, including an analysis of microbiological purity, toxicity, genetic stability and the potential risk of horizontal gene transfer. Objective The aim was to perform a comprehensive experimental biosafety assessment of a biocorrective agent based on the recombinant L. plantarum 8PAЗ (B-11007) (pLacDualIL10/IL22) strain, which produces interleukins IL-10 and IL-22. Methods Microbiological purity, haemolysis, antibiotic sensitivity, biogenic amines, strain viability and activity after lyophilisation, acute toxicity in rats and calves, colonisation and genetic properties were assessed. Studies were carried out in three biological replicates. Statistical processing was performed using Student’s t -test for two-group comparisons, and one-way ANOVA with Tukey’s post hoc test was performed for multiple-group comparisons ( p < 0.05). Results No foreign microflora were detected in any batch of the biocorrective agent (detection limit <10 CFU/g). The strain was characterised by γ-haemolysis, and it retained a typical antibiotic susceptibility profile and did not produce biogenic amines (less than 0.5 mg/kg). After lyophilisation, the viability recovery rate was 72%, and after 12 months of storage, the viable cell concentration remained at 1.9 × 10 12 CFU/g. IL-10 and IL-22 concentrations after recovery were 47.3 ± 5.2 and 38.6 ± 4.1 pg/mL, respectively. In an acute toxicity study in rats and calves, no lethal outcomes, clinical signs of intoxication or statistically significant changes in haematological and biochemical parameters were detected ( p > 0.05). The recombinant strain demonstrated transient intestinal colonisation without signs of long-term persistence. Analysis of the plasmid construct confirmed the absence of the tra and mob genes and the oriT site, while modelling of the gastrointestinal environment revealed degradation of over 90% of the plasmid DNA during the gastric stage and the absence of intact DNA after the intestinal phase. Conclusion The biocorrective agent meets microbiological purity requirements, exhibits high strain integrity and lacks acute toxicity (hazard class IV). The available evidence suggests that it has a low risk of horizontal transfer of genetic material.