Biocompatibility of Orally Delivered Microencapsulated Fibrinolytic Proteases from Bacillus tequilensis for Biomedical Application

Developing safe and effective fibrinolytic enzymes for oral delivery is still a major challenge and concern in translational biotechnology. Although microbial proteases have gained increasing attention, the in vivo evidence of their systemic safety after oral administration remains limited. This study was designed to evaluate the systemic biocompatibility of a spray-dried microencapsulated fibrinolytic protease derived from Bacillus tequilensis Holothuria scabra Fermented Intestine 5 (HSFI-5) following repeated oral administration in mice. The strain was isolated from the fermented intestine of a sea cucumber captured from the seawaters of Nusa Tenggara, Indonesia. The extracellular enzyme from B. tequilensis HSFI-5 was partially purified and subsequently encapsulated via spray-drying using either maltodextrin (M) or Arabic gum (AG) as wall materials. Proteolytic activity was retained after microencapsulation (0.26–0.28 U/mL; ~5.52 units per milligram of protein [U/mg]). Thirty male C3H mice ( n = 6 per group) received daily oral administration for four weeks. No significant differences were observed in body weight (BW), lipid profile, or endpoint inflammatory markers (IL–6, IL–1β, and C-reactive protein [CRP]) between treated and control groups ( p > 0.05), although ΔIL-1β (change from baseline) showed an isolated statistical difference ( p = .047). Hematological test results showed no consistent patterns suggestive of toxicity. Statistical analysis using one-way and two-way Analysis of Variance ( ANOVA) confirmed the absence of treatment-related effects ( p > 0.05). In conclusion, the repeated oral administration of the tested microencapsulated protease formulation did not induce detectable systemic inflammatory or metabolic disturbances under the conditions of this study. This work provides initial in vivo evidence supporting the biological compatibility of orally delivered microbial protease formulations and contributes to the development of marine-derived bioactive enzymes for biotechnological applications.