The resistance formation of spores in general and of Bacillus atrophaeus in particular has long been the focus of science in the bio-defense, pharmaceutical and food industries. In the food industry, it is used as a biological indicator (BI) for the evaluation of the inactivation effects of hydrogen peroxide in processing and end packaging lines' sterilization. Defined BI resistances are critical to avoid false positive and negative tests, which are salient problems due to the variable resistance of currently available commercial BIs. Although spores for use as BIs have been produced for years, little is known about the influence of sporulation conditions on the resistance as a potential source of random variability. This study therefore examines the dependence of spore resistance on the temperature, pH and partial oxygen saturation during submerged production in a bioreactor. For this purpose, spores were produced under different sporulation conditions and their resistance, defined by the D-value, was determined using a count reduction test in tempered 35% liquid hydrogen peroxide. The statistical analysis of the test results shows a quadratic dependence of the resistance on the pH, with the highest D‑values at neutral pH. The sporulation temperature has a linear influence on the resistance. The higher the temperature, the higher the D‑value. However, these factors interact with each other, which means that the temperature only influences the resistance when the pH is within a certain range. The oxygen partial pressure during sporulation has no significant influence. Based on the data obtained, a model could be developed enabling the resistance of BIs to be calculated, predicted and standardized depending on the sporulation conditions. BI manufacturers could thus produce BIs with defined resistances for the validation of sterilization effects in aseptic packaging/filling lines for the reliable manufacture of shelf-stable and safe food products.
Keywords: Bacillus atrophaeus; aseptic packaging; bioindicators; hydrogen peroxide; product safety; resistance; shelf life; spores; sporulation; sterilization.