Harnessing biodiversity in dairy cultures: fifty years of Lactobacillus helveticus evolution

This study investigates the genetic and phenotypic biodiversity of Lactobacillus helveticus (LH) strains isolated 50 years apart from natural whey starters (NWS) in traditional dairy productions and never revitalized, in comparison with recently isolated LH strains from the same ecological niche. A preliminary in vitro screening of 40 LH strains (previously tested for the absence of antibiotic resistance and amino-biogenic properties) revealed significant biodiversity between the two groups. Older strains showed higher growth rates and shorter lag phases when cultivated in both broth medium and milk under traditional fermentation conditions, suggesting better fitness and adaptation. To evaluate LH technological potential, the most promising strains were tested in a miniaturised cheese model, simulating the traditional process conditions. Older strains showed decreased cell viability and produced distinct volatile compounds after two weeks of ripening, with higher levels of alcohols and lower amounts of acetic acid and acetoin. Based on these results, two old and two recent strains were selected for pilot-scale cheese trials, co-inoculated with Lactobacillus delbrueckii subsp. lactis UPCCO5365 to mimic traditional NWS cultures using a defined protocol for cooked hard cheese production. The samples (approx 1 kg) were ripened for three weeks at 18 °C. Culture-dependent and culture-independent techniques were applied to study microbial dynamics showing strain-specific differences. In addition, SPME-GC-MS demonstrated distinct aroma profiles, highlighting different metabolic performances within dairy fermentations. This work offered a solid base for the valorization of LH strains in tailored dairy applications, providing important insights into the adaptive evolution of this species in these environments. This research is supported by EU-NextGenerationEU (Piano Nazionale di Ripresa e Resilienza (PNRR)), program Prin 2022 with the project “A 50 years evolutionary history of Lactobacillus helveticus from traditional dairy environments: biodiversity of strains as an opportunity for technological exploitation and new products-Helv4DairHy” (CUP J53D23010690006).