Role of Microbial Interactions in the Impaired Cultivability of Thermophilic Lactic Acid Bacteria in Natural Whey Starter for Parmigiano Reggiano PDO Cheese Production

Marianna Cristofolini¹Maria Ronsivalle¹Maria Pramazzoni²Giulia Zaccarini¹Valentina Pizzamiglio³Lisa Solieri^1*

• ¹Department of Life Sciences, Universita degli Studi di Modena e Reggio Emilia - Sede di Reggio Emilia, Reggio Emilia, Italy
• ²Dipartimento di Scienze Agrarie, Alimentari e Agro-ambientali, University of Pisa, Pisa, Italy
• ³Consorzio del formaggio Parmigiano Reggiano, Reggio Emilia, Italy

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, Lactobacillus delbrueckii subsp. lactis, 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 Lactobacillus helveticus, 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 L. delbrueckii subsp. lactis 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 L. helveticus, even when L. delbrueckii dominated the community according to metagenomic profiling (type-D NWS). Supplementation of MRS medium with cysteine and formic acid enabled the recovery of viable L. delbrueckii subsp. lactis isolates, accounting for 35% of the strains obtained from type-D NWS. Cross-feeding experiments further revealed that co-culturing L. delbrueckii with the formate-producing Streptococcus thermophilus significantly enhanced milk acidification compared with monocultures, indicating a beneficial metabolic interaction. In contrast, no such improvement was observed in the presence of L. helveticus, likely due to negative interactions with L. delbrueckii subsp. lactis. Accordingly, the impaired cultivability of L. delbrueckii subsp. lactis could thus be partially alleviated either in co-culture with S. thermophilus or under axenic conditions mimicking natural metabolite exchange between these species.