Alicyclobacillin 24: a class III bacteriocin from Alicyclobacillus acidoterrestris targeting species associated with spoilage of acidic fruit-based products

The thermoacidophilic Alicyclobacillus (ACB) genus includes several species responsible for spoilage of acidic fruit-based products, yet bacteriocins produced by these bacteria remain unexplored. In this study, the spoilage strain Alicyclobacillus acidoterrestris DSM 3922T was explored as a potential bacteriocin producer, following genome analysis that revealed the presence of a putative geobacillin 26 (Geo26) family protein. Co-culture assays showed inhibitory activity against other ACB isolates, and proteomic analysis confirmed the secretion of a Geo26 homolog, designated alicyclobacillin 24 (Ali24). Ali24 was heterologously expressed with a C-terminal His-tag (Ali24-His), purified, and functionally characterized. Ali24-His exhibited a narrow antibacterial spectrum restricted to ACB species, including multiple spoilage-associated strains. SYTO9/propidium iodide staining of A. acidoterrestris MMB007 exposed to Ali24-His suggested a membrane associated antimicrobial mechanism. In addition, the antibacterial activity of Ali24-His was evaluated in industrial-relevant conditions, exhibiting effective inhibitory features under acidic pH and moderate heating (up to 50 °C), which are environments typical of fruit processing workflows, pre-pasteurization steps. Ali24-His stability was further assessed by dynamic light scattering and nano differential scanning fluorimetry revealing enhanced stability and reduced aggregation under acidic conditions, consistent with its activity profile. Overall, this work reports the first functional Class III bacteriocin from A. acidoterrestris and expands the repertoire and understanding of thermophile-derived bacteriocins with potential for sustainable control of ACB spoilage in the food industry.