Influence of Temperature, Light, and H₂O₂ Concentration on Microbial Spore Inactivation: In-Situ Raman Spectroscopy Combined with Optical Trapping

To gain insight on chemical sterilization processes, the influence of temperature (up to 70 degrees 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 degrees 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.