Abstract
Koji- and tempeh-based fermentations, central to miso and soy sauce production, rely on filamentous fungi such as Aspergillus oryzae and Rhizopus oryzae to transform grains and legumes into foods with enhanced flavor and digestibility. In this study, we adapted this fermentation framework to grasshoppers (Locusta migratoria manilensis) using two independently evaluated systems. In the first system, pure cultures of A. oryzae and R. oryzae were applied to 100% grasshopper substrate. In the second, independently evaluated system, traditional matrix-based starters (soy koji and soy tempeh) were incorporated into a 50:50 grasshopper–soy composite substrate. Each system was assessed relative to its own Day 0 baseline. In the 100% grasshopper system, fermentation increased the degree of hydrolysis from 1.3% to > 16%, elevated free amino acids 3–5 fold (including glutamate, alanine, and leucine), and improved in vitro protein digestibility relative to the unfermented control. Proteomic profiling revealed reduced abundance of structural and allergen-associated proteins following fermentation. In the composite system, fermentation similarly promoted proteolysis, enhanced digestibility relative to its initial composite baseline, and diversified amino acid pools within the mixed substrate. Across both systems, volatile analysis identified the formation of pyrazines, esters, alcohols, ketones, and organic acids absent at Day 0, reflecting coordinated amino acid and lipid metabolism. Collectively, these results demonstrate that fermentation, whether using traditional starters or defined fungal cultures, can transform grasshopper into a more digestible, less allergenic, and more flavorful ingredient, thereby advancing its potential for sustainable food innovation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-45428-x.