Optimizing germination conditions for Alicyclobacillus acidoterrestris spores, and achieving efficient inactivation via high hydrostatic pressure using a germination-inactivation strategy

The 'germination-inactivation' strategy can potentially eliminate Alicyclobacillus acidoterrestris spores by exploiting the loss of their resistance that occurs during germination. However, the germination behavior of A. acidoterrestris spores remains incompletely characterized. In this study, the germination responses of A. acidoterrestris spores to various stimuli, including L-alanine, AGFK (L-asparagine, D-glucose, D-fructose, and KCl), and high hydrostatic pressure (HHP) were comprehensively examined. Surprisingly, it was observed that A. acidoterrestris spores germinate in acidic buffer, even in the absence of L-alanine or AGFK. Optimal acidic-induced germination efficiency was achieved at pH 4.0 following heat shock (70 °C, 15 min), and incubation at 37 °C. While heat treatment (65–75 °C) accelerated germination, acidic conditions (pH 3.0–4.0) were strictly necessary to initiate the process. Meanwhile, HHP-induced germination can also occur under acidic conditions (pH 3.0–5.0), demonstrating the acidophilic nature of A. acidoterrestris spores. Notably, spores subjected to HHP (200 or 500 MPa, 3 min, 30 °C) underwent spontaneous germination during subsequent incubation at 37 °C, irrespective of the medium's pH. Building on these findings, a combined 'HHP-incubation-pasteurization' approach was developed that achieved up to a 5.3 ± 0.51 log reduction in A. acidoterrestris spores within acidic systems. The results of this study provide a valuable foundation for the development of efficient sterilization techniques in acidic juice processing.