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In this study, we investigated the altered enzymatic activities and metabolite profiles of koji fermented using varying permutations of Aspergillus oryzae and/or Bacillus amyloliquefaciens . Notably, the protease and β-glucosidase activities were manifold increased in co-inoculated (CO) koji samples (co-inoculation of A. oryzae and B. amyloliquefaciens ). Furthermore, gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling indicates that levels of amino acids, organic acids, sugars, sugar alcohols, fatty acids, nucleosides, and vitamins were distinctly higher in CO, SA (sequential inoculation of A. oryzae , followed by B. amyloliquefaciens ), and SB (sequential inoculation of B. amyloliquefaciens , followed by A. oryzae ). The multivariate principal component analysis (PCA) plot based on GC-MS datasets indicated a clustered pattern for MA and MB ( koji samples inoculated either with A. oryzae or B. amyloliquefaciens ) across PC2 (20.0%). In contrast, the CO, SA, and SB metabolite profiles displayed segregated patterns across PLS1 (22.2%) and PLS2 (21.1%) in the partial least square discriminant analysis (PLS-DA) model. Intriguingly, the observed disparity in the levels of primary metabolites was engendered largely by higher relative levels of sugars and sugar alcohols in MA, SA, and CO koji samples, which was commensurate with the relative amylase activities in respective samples. Collectively, the present study emphasizes the utility of integrated biochemical and metabolomic approaches for achieving the optimal permutation of fermentative inocula for industrial koji preparation.

Varying Inocula Permutations (Aspergillus oryzae and Bacillus amyloliquefaciens) affect Enzyme Activities and Metabolite Levels in Koji