Sporulation temperature and pH influence the heat and high hydrostatic pressure resistance of Alicyclobacillus acidoterrestris spores through proteomic remodeling

Alicyclobacillus acidoterrestris is a major spoilage organism in acidic beverages, owing to the exceptional resistance of its spores to both thermal and non-thermal preservation processes. However, how sporulation environments regulate subsequent stress responses and the underlying molecular mechanisms remain poorly understood. In this study, we investigated the effects of sporulation temperature (37–53 °C) and pH (2.5–5.5) on the heat resistance, high hydrostatic pressure (HHP) resistance, and proteomic profiles of A. acidoterrestris spores. Higher sporulation temperature enhanced heat resistance, accompanied by remodeling of membrane-associated proteins and increased abundance of sporulation regulators and maturation-related proteins, including Spo0A, SpoIIID, and SpoVAD. Spores produced at pH 2.5 also exhibited enhanced heat and HHP resistance, whereas pH 5.5 affected the expression of cortex-, coat-, and germination-related proteins resulting in reduced spore resistance. Across different sporulation conditions, YlmC/YmxH family protein showed a condition-dependent abundance pattern and was the only identified protein significantly associated with both 200 MPa and 500 MPa resistance phenotypes. Notably, greater heat resistance did not translate into uniformly higher pressure resistance; sporulation conditions tuned the HHP response by modulating the abundance of germination receptors and SpoVA components. These results demonstrate that sporulation temperature and pH act as developmental cues that modulate spore maturation and stress-response strategies. This study provides new insights into how environmental conditions shape spore heterogeneity and influence the persistence of spoilage spores during food processing and preservation.