Multiple factors contribute to guaiacol production by Alicyclobacillus acidoterrestris and Alicyclobacillus suci in apple juice, as revealed by transcriptomic and phenotypic analyses.

UNLABELLED: Alicyclobacillus acidoterrestris and Alicyclobacillus suci are guaiacol-producing bacteria that can spoil fruit juices. To study the genetic underpinnings of guaiacol production in juice, transcriptomics was performed on multiple strains of A. suci and A. acidoterrestris under guaiacol production conditions. While guaiacol production was consistently higher by A. acidoterrestris than by A. suci, the gene expression levels associated with guaiacol production (e.g., vdcC, vdcB, vdcD) showed significant variability across strains of both species, suggesting that other factors beyond vdcC transcription influence the amount of guaiacol produced. A. acidoterrestris exhibited a maximum growth rate at 40°C, but produced the most guaiacol at 25°C (16.2 ± 0.6 µg/L, day 14), suggesting that guaiacol is not only linked to growth. When adjusting pH, A. acidoterrestris produced the most guaiacol at pH 4.0 at 14 days (3.2 ± 2.2 µg/L). A. suci also showed the highest amount of guaiacol production at 25°C (3.6 ± 0.3 µg/L, day 7). In pH-adjusted apple juice, A. suci showed consistent growth across all pH levels, yet produced the most guaiacol at pH 3.0 (2.1 ± 0.08 µg/L, day 7). Importantly, guaiacol levels did not consistently correlate with bacterial growth, underscoring the limitations of enumeration-based spoilage assessments. Comparative strain analysis revealed species-specific differences, with A. acidoterrestris producing significantly more guaiacol in apple juice than A. suci, a trend not reflected in media-based PECA assays. These findings highlight the need to evaluate spoilage risk in relevant food matrices under realistic storage conditions to provide beverage producers with insights for improved mitigation strategies.

IMPORTANCE: Beverage spoilage caused by microbes such as Alicyclobacillus poses a significant challenge for the food industry, leading to substantial economic losses and increased food waste. While it is known that some species, specifically those that produce the off-flavor compound guaiacol, are responsible for spoilage, current risk assessment practices often fail to accurately predict spoilage potential. These practices are often flawed because they rely on simple bacterial enumeration or use media-based assays without relevance to a specific food matrix, which do not reflect real-world conditions. Testing the presence or absence of these bacteria without assessment of spoilage can lead to false positives and overly conservative product disposition decisions. This study highlights the need for a more nuanced approach by demonstrating that the relationship between bacterial growth, gene expression, and guaiacol production is not straightforward as it impacts juice spoilage.