Role of microbial interactions in the impaired cultivability of thermophilic lactic acid bacteria in natural whey starter for Parmigiano Reggiano PDO cheese production

Natural whey starter (NWS) cultures play a pivotal role in the production of Parmigiano Reggiano (PR) Protected Designation of Origin (PDO) cheese; however, their microbial ecology and functional dynamics remain only partially understood. In particular, <i>Lactobacillus delbrueckii</i> subsp. <i>lactis</i>, a dominant species in type-D NWS communities, exhibits impaired cultivability that limits its isolation and characterization. Consequently, most studies have focused on strain variability within <i>Lactobacillus helveticus</i>, which is predominant in type-H NWS communities. In this study, we evaluated the effects of 14 different medium supplementations on the recovery and maintenance of <i>L. delbrueckii</i> subsp. <i>lactis</i> isolates from two PR NWS samples representatives of type-D and type-H communities. Although most supplementations increased lactobacilli plate counts compared with the control MRS medium, they failed to sustain cell viability during the purification for culture collection establishment. Moreover, these media altered species ratios in favor of <i>L. helveticus</i>, even when <i>L. delbrueckii</i> dominated the community according to metagenomic profiling (type-D NWS). Supplementation of MRS medium with cysteine and formic acid enabled the recovery of viable <i>L. delbrueckii</i> subsp. <i>lactis</i> isolates, accounting for 35% of the strains obtained from type-D NWS. Cross-feeding experiments further revealed that co-culturing <i>L. delbrueckii</i> with the formate-producing <i>Streptococcus thermophilus</i> significantly enhanced milk acidification compared with monocultures, indicating a beneficial metabolic interaction. In contrast, no such improvement was observed in the presence of <i>L. helveticus</i>, likely due to negative interactions with <i>L. delbrueckii</i> subsp. <i>lactis.</i> Accordingly, the impaired cultivability of <i>L. delbrueckii</i> subsp. <i>lactis</i> could thus be partially alleviated either in co-culture with <i>S. thermophilus</i> or under axenic conditions mimicking natural metabolite exchange between these species.