Metabolomic Analysis of Fermented Nori Powders: Divergence of Betaine Structural Analogs Production by Three Types of koji Fungal Fermentation

Fermenting seaweed with <i>koji</i> fungi transforms its chemical composition, generating bioactive compounds absent in the raw material. We previously reported that the fungal fermentation of the edible red alga <i>Pyropia yezoensis</i> (Nori) produces betaine structural analogs (such as betaine, stachydrine, and carnitine), which are of particular interest because of their physiological roles and potential health benefits. Using metabolomic profiling, we compared non-fermented Nori with powders fermented by three industrially important fungi: <i>Aspergillus luchuensis</i> mut. <i>kawachii</i> (white <i>koji</i> fungus)<i>, Aspergillus oryzae</i> (yellow <i>koji</i> fungus)<i>,</i> and <i>Monascus purpureus</i> (red <i>koji</i> fungus). All fermentations enhanced the levels of betaine and carnitine, but stachydrine production was unique to the yellow <i>koji</i> fungus. Precursor patterns revealed distinct metabolic strategies: the yellow <i>koji</i> fungus exhibited an intermediate detectable choline oxidation route and strong proline methylation, the white <i>koji</i> fungus rapidly converted choline without intermediate accumulation, and the red <i>koji</i> fungus favored carnitine and proline but produced little stachydrine. Fermentation also shifted the methylation balance toward a state that supports methyl-dependent pathways. These findings reveal clear species-specific strategies for the production of betaine structural analogs, providing a mechanistic basis for understanding the metabolic divergence among <i>koji</i> fungi and guiding the targeted design of functional seaweed products.