Optimization and validation of Bacillus subtilis spore inactivation regimes in supercritical carbon dioxide: Pure gas, hydrogen peroxide, peracetic acid

Introduction. Healthcare-associated infections (HAIs) continue to pose a significant challenge to public health. The existing sterilization methods possess a number of limitations regarding their applicability to heat-sensitive materials and the possibility of residual toxicity or material damage. Sterilization using supercritical carbon dioxide, particularly in combination with oxidative additives, appears to be a promising alternative capable of providing the required level of microbial safety at low temperatures and moderate pressures. However, the available data show significant variability depending on experimental conditions, indicating the need for optimization and validation of processing regimes.

Objective. Optimization and validation of Bacillus subtilis spore sterilization regimes in an experiment using supercritical carbon dioxide (scCO₂), including pure scCO₂, scCO₂ with hydrogen peroxide (H₂O₂), and scCO₂ with peracetic acid (PAA).

Materials and methods. Experiments were conducted using a prototype test bench for investigating sterilization regimes. Bacillus subtilis subsp. spizizenii ATCC 6633 NCTC 10400 was used as the biological indicator. The study involved the contamination of titanium discs with spores followed by their treatment under the three regimes (pure scCO₂, scCO₂ + H₂O₂, scCO₂ + PAA). Further, microbiological analysis with enumeration of surviving spores and statistical processing of the results were carried out. For each regime, central composite experimental designs were implemented, varying temperature, pressure, and exposure duration. Efficacy was assessed based on the log-reduction of viable spores. Validation series included 10 independent replicates each.

Results. Gas-dynamic treatment with pure scCO₂ in the range of 35–60°C, 70–120 atm, and 60–120 min proved ineffective (maximum log-reduction ≤ 0.8). The addition of oxidizing agents significantly increased the degree of inactivation: for the scCO₂ + H₂O₂ (200 ppm) regime, the optimal parameters were 37.9°C, 120 atm, 30 min (log-reduction 4.4 ± 0.3; CV = 7.3%); for scCO₂ + PAA (50 ppm) — 45°C, 94 atm, 10 min (log-reduction 6.0 ± 0.3; CV = 4.9%), which meets the requirements for the sterility assurance level (SAL) for medical devices. The PAA regime provided a statistically significant higher efficacy compared to H₂O₂ (t-test, p < 0.001).

Conclusions. The sterilization methodology based on supercritical carbon dioxide with the addition of hydrogen peroxide or peracetic acid ensures effective inactivation of Bacillus subtilis spores under parameters compatible with the low-temperature processing of heat-sensitive medical materials, thus meeting modern sterility requirements for medical devices.

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