Polyamine production in mold-fermented cheese through metabolic interactions between mesophilic starter cultures and Penicillium candidum

Mold-fermented cheese is produced through sequential microbial processes in which mesophilic starter cultures initiate milk fermentation, followed by rennet-mediated coagulation and surface ripening by filamentous fungi such as Penicillium candidum . To investigate microbial contributions to polyamine-related metabolism during cheese ripening, we combined quantitative metabolite analysis with mass spectrometry imaging (MSI). In cheeses prepared with the CHN-19 mesophilic starter culture alone, putrescine (Put), spermidine (Spd), and γ-aminobutyric acid (GABA) did not detectably accumulate, whereas surface inoculation with P. candidum resulted in their appearance during ripening. MSI further revealed that arginine (Arg), ornithine (Orn), agmatine (Agm), and Spd were predominantly localized in the surface region penetrated by fungal hyphae. To determine whether acidification itself induces polyamine production, P. candidum was grown independently on a cheese-mimicking milk agar medium adjusted to pH 4.0–6.0. Polyamines were not detected under these conditions, indicating that acidic pH alone is insufficient for their production. Spd became clearly detectable following Arg supplementation, whereas Orn supported detectable Spd production only at a higher substrate concentration. These findings indicate that P. candidum is a major contributor to polyamine-related metabolism in this defined mold-fermented cheese model and that precursor availability, rather than low pH alone, is a key determinant of fungal polyamine production. Collectively, our results show that starter-associated changes in the cheese environment and fungal metabolism together shape the production and spatial distribution of polyamine-related metabolites during ripening.