Temperature and water activity interactively regulate the growth and aflatoxin biosynthesis of Aspergillus flavus in...

Dried red chili pepper is widely consumed yet prone to contamination by Aspergillus flavus and its carcinogenic aflatoxins, though how this fungus grows and produces toxins on this distinctive substrate remains poorly understood. This study examined the effects of temperature and water activity (a_w) on A. flavus growth and aflatoxin (AF) production in dried red chili pepper, and explored the associated molecular and metabolic responses through multi-omics analyses. Four isolates tested at 15-42 °C and a_w 0.86-0.98 showed optimal mycelial growth at 30-37 °C and a_w 0.98, whereas AF production peaked at 25-30 °C and a_w 0.95. Strain-dependent differences in toxigenic capacity among isolates with comparable growth rates demonstrated that growth rate alone does not predict aflatoxin output. The general polynomial model best described growth kinetics, outperforming the Gibson and Arrhenius-Davey models. Transcriptomic analysis revealed enrichment of differentially expressed genes in metabolic processes, catalytic activity, and membrane-associated functions; the early-pathway gene aflE (aryl alcohol dehydrogenase) and the AFG-specific gene aflU were downregulated at 37 °C, while laeA transcripts peaked at a_w 0.95, where AF accumulation was highest. Metabolomic profiling showed that elevated temperature reduced kainic acid (a precursor in kojic acid biosynthesis), citric acid, and nicotinamide ribotide, whereas elevated a_w increased glycine l-threonine, consistent with growth, and identified arginine, linoleic acid as key metabolites in the aw stress response. These findings provide insights into how temperature and a_w interactively regulate A. flavus growth and aflatoxin biosynthesis in dried red chili pepper, offering a theoretical basis for targeted contamination control during storage.