Study of cultivability and inter-species interactions within the microbial communities of the whey starter used for Parmigiano Reggiano cheese production.

The aim of this study was to investigate the role that different amino acid sources and interspecies interactions may play in the cultivability of lactic acid bacteria (LAB) from the natural whey starter (NWS), an undefined microbial consortium used for Parmigiano Reggiano PDO cheese production. Two NWS samples type-H (dominated by Lactobacillus helveticus) and type-D (dominated by Lactobacillus delbrueckii) were subjected to metagenomic analysis and viable cell counts on MRS agar supplemented with 12 different nitrogen sources. The media were evaluated for their ability to estimate the cultivable fraction (Log₁₀ CFU/mL), to maintain the viability and cultivability of isolates (percentage of viable isolates), and to preserve the original species ratios. All conditions altered species ratios and failed to cultivate L. delbrueckii subsp. lactis effectively, even in type-D whey where it dominated, indicating its recalcitrant to growth despite nitrogen supplementation. Cross-feeding experiments in milk were performed using three sets of axenic cultures, each composed of one strain of L. helveticus (Lh), L. delbrueckii (Ld), and S. thermophilus (St), tested in mono-culture, co-culture, and tri-culture combinations Tri-cultures, followed by Lh-St and Ld-St co-coltures, showed faster, more efficient acidification compared to monocultures, while the Lh-Ld combination did not. Analysis of lactate isomers revealed that St positively influences Ld without reciprocal benefit. Moreover, Ld and Lh showed negative interactions, likely due to sugar competition, as both are proteolytic. These findings demonstrate the complexity of microbial interactions in NWS and highlight the limitations of traditional culture-dependent methods in preserving microbial biodiversity ex situ.