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In the present work, a novel method for monitoring sterilisation processes with gaseous H2O2 in combination with heat activation by means of a specially designed calorimetric gas sensor was evaluated. Therefore, the sterilisation process was extensively studied by using test specimens inoculated with Bacillus atrophaeus spores in order to identify the most influencing process factors on its microbicidal effectiveness. Besides the contact time of the test specimens with gaseous H2O2 varied between 0.2 and 0.5 s, the present H2O2 concentration in a range from 0 to 8% v/v (volume percent) had a strong influence on the microbicidal effectiveness, whereas the change of the vaporiser temperature, gas flow and humidity were almost negligible. Furthermore, a calorimetric H2O2 gas sensor was characterised in the sterilisation process with gaseous H2O2 in a wide range of parameter settings, wherein the measurement signal has shown a linear response against the H2O2 concentration with a sensitivity of 4.75 °C/(% v/v). In a final step, a correlation model by matching the measurement signal of the gas sensor with the microbial inactivation kinetics was established that demonstrates its suitability as an efficient method for validating the microbicidal effectiveness of sterilisation processes with gaseous H2O2.
To gain insight on chemical sterilization processes, the influence of temperature (up to 70 °C), intense green light, and hydrogen peroxide (H₂O₂) concentration (up to 30% in aqueous solution) on microbial spore inactivation is evaluated by in-situ Raman spectroscopy with an optical trap. Bacillus atrophaeus is utilized as a model organism. Individual spores are isolated and their chemical makeup is monitored under dynamically changing conditions (temperature, light, and H₂O₂ concentration) to mimic industrially relevant process parameters for sterilization in the field of aseptic food processing. While isolated spores in water are highly stable, even at elevated temperatures of 70 °C, exposure to H₂O₂ leads to a loss of spore integrity characterized by the release of the key spore biomarker dipicolinic acid (DPA) in a concentration-dependent manner, which indicates damage to the inner membrane of the spore. Intensive light or heat, both of which accelerate the decomposition of H₂O₂ into reactive oxygen species (ROS), drastically shorten the spore lifetime, suggesting the formation of ROS as a rate-limiting step during sterilization. It is concluded that Raman spectroscopy can deliver mechanistic insight into the mode of action of H₂O₂-based sterilization and reveal the individual contributions of different sterilization methods acting in tandem.
An H2O2 sensor for the application in industrial sterilisation processes has been developed. Therefore, automated sterilisation equipment at laboratory scale has been constructed using parts from industrial sterilisation facilities. In addition, a software tool has been developed for the control of the sterilisation equipment at laboratory scale. First measurements with the developed sensor set-up as part of the sterilisation equipment have been performed and the sensor has been physically characterised by optical microscopy and SEM.