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 °C), intense green light, and hydrogen peroxide (H 2 O 2 ) 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 ‐based sterilization and reveal the individual contributions of different sterilization methods acting in tandem.