Role of Aspergillus neutral protease II in heat-induced soy sauce sediment formation

Soy sauce is produced from fermented soybeans, wheat, and salt by koji molds (Aspergillus oryzae and Aspergillus sojae) together with salt-tolerant lactic acid bacteria and yeast. Pasteurization, an essential production step, causes protein insolubilization and produces heat-induced sediment that must be removed because it impairs product appearance. Although koji mold-derived proteins have been suggested to contribute to sediment formation, the underlying mechanisms remain unclear. In this study, we aimed to identify the key protein responsible for heat-induced sediment formation. Pasteurizing soy sauce in the presence of a metal chelator prevented sediment formation, and sediment reappeared upon reintroduction of divalent metal ions, indicating that heat-induced aggregation requires metal ion availability. This dependence is consistent with the involvement of neutral protease II (NpII, also known as DeuA or NptB), a thermostable zinc metalloprotease. Soy sauce brewed with A. oryzae and A. sojae strains deficient in NpII showed no sediment formation. Furthermore, in soy sauce brewed with the NpII-disruptant strains, the addition of a non-thermostable protease preparation after pasteurization restored sediment formation, demonstrating that partial proteolysis occurring only after heat treatment is crucial for sediment generation. Proteomic analysis revealed that > 98% of the proteins in heat-induced sediment originated from koji molds and exhibited highly similar profiles across species and strains. To the best of our knowledge, this is the first study to demonstrate that improvement of koji mold strains can prevent detectable heat-induced sediment formation in soy sauce brewing, contributing to more sustainable manufacturing by eliminating the sediment removal process. KEY POINTS: Neutral protease II promotes heat-induced sediment formation in soy sauce. Heat-induced sediment proteins were > 98% koji mold-derived. Sediment formation occurs via protease-mediated hydrophobic aggregation.