Aspergillus oryzae (AO), known as the Koji mold, is essential in East Asian fermentation for foods like sake, shoyu, amazake, and miso. AO is "Generally Recognized as Safe" (GRAS) due to its non-pathogenic nature and lack of aflatoxin production. This work focuses on an AO extract's postbiotic potential. A two-stage fermentation process with a significant pH increase in the second phase enhances antioxidative capacity, phenolic content, and induces changes in the primary and secondary metabolite profile. In human reporter cell lines, post-stress AO samples showed immunomodulatory activities, unlike pre-stress AO samples. In Drosophila melanogaster, a 5% supplementation with post-stress AO extract significantly improved heat tolerance and reproduction. Gene expression analyses indicated notable changes, particularly in metallothioneins expression. This study highlights the importance of pH elevation during AO fermentation, leading to changes in bioactivity and metabolite profiling.

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