Helv4DairHy

Lactobacillus helveticus (LH) is a homofermentative lactic acid bacterium associated with natural whey starters (NWS) used in the production of traditional cheeses such as Parmigiano Reggiano PDO. This species displays remarkable phenotypic and genotypic diversity, even among strains inhabiting the same ecosystem. Over time, technological evolution and environmental selective pressures associated with cheese manufacturing may have reshaped LH populations, leading to the replacement of historical strains by better-adapted variants.

Since the mechanisms driving the selection of LH biotypes in this environment remained largely unexplored, the Helv4DairHy project investigated the genotypic and phenotypic diversity of L. helveticus strains isolated from Parmigiano Reggiano natural whey starters approximately 50 years apart. The project relied on a unique historical collection of LH strains isolated in the 1970s from traditional dairies in the Reggio Emilia area and preserved without propagation for over five decades, together with a recent collection isolated in 2023 from the same production area and representative of the current biodiversity of this ecological niche.

Through an integrated approach combining microbial ecology, comparative genomics, phenotypic characterization and progressive validation in dairy systems, the project investigated the evolutionary adaptation of L. helveticus to the dairy environment while identifying strains with promising technological properties for dairy applications. Approximately one hundred strains were initially screened using a "funnel" strategy to identify representative isolates displaying the highest diversity and technological potential. Selected strains were subsequently characterized at the phenotypic and genomic levels, evaluated in innovative miniaturized cheese models reproducing key steps of Parmigiano Reggiano manufacture, and finally validated through pilot-scale cheese production and laboratory-scale fermented milk manufacture.

Results achieved

The project successfully achieved all its scientific objectives, providing new insights into the long-term evolution of Lactobacillus helveticus within the Parmigiano Reggiano natural whey starter ecosystem. Genotypic analysis of approximately one hundred strains revealed a clear separation between historical and contemporary isolates, demonstrating that the bacterial population has undergone significant evolutionary changes over the last five decades. Comparative genome analysis of 34 representative strains further showed that historical isolates possess larger and more diverse genomes, whereas recent isolates display a more homogeneous genomic architecture with reduced accessory gene content, supporting the hypothesis of progressive adaptation and domestication within the dairy environment.

Phenotypic characterization demonstrated a high safety profile for both historical and recent isolates, with limited antibiotic resistance, absence of histamine production and only low tyramine production in two historical strains. Comparative analyses of growth kinetics, acidification behaviour and physiological traits highlighted substantial differences between historical and contemporary populations, enabling the selection of strains with the highest technological potential for further investigation.

A dedicated miniaturized cheese model was successfully developed to reproduce the main technological steps of Parmigiano Reggiano manufacture under controlled laboratory conditions. This innovative food model enabled rapid screening of selected strains, demonstrating marked strain-

dependent differences in growth, persistence and aroma formation, and provided the basis for selecting the best-performing candidates for validation at larger scale.

Four selected strains were subsequently evaluated in pilot-scale short-ripened cheese manufacture using standardized production protocols specifically developed within the project. Cheese prototypes were characterized for microbiological, physicochemical and quality-related parameters, including volatile organic compounds, biogenic amines and microbial persistence through both culture-dependent and molecular approaches. In parallel, selected strains were employed for laboratory-scale fermented milk production, where they showed distinct performances in milk acidification, viability during refrigerated storage, aroma development and proteolytic activity. In particular, one historical strain exhibited faster acidification, superior persistence and greater peptide production than the recent isolates, demonstrating the technological value retained by historical microbial biodiversity.

The project generated valuable scientific resources, including publicly available genome sequences and the preservation of the characterized strains within the University of Parma Culture Collection (UPCC), ensuring their long-term accessibility to the scientific community. Project outcomes have been disseminated through national and international conferences and are being consolidated into scientific publications. Overall, Helv4DairHy demonstrated that historical microbial collections represent not only an exceptional resource for understanding microbial evolution and domestication in traditional dairy ecosystems, but also a valuable reservoir of technologically relevant biodiversity for the development of tailored starter cultures supporting innovative and sustainable dairy production.

D.D. del MUR n. 104 del 02/02/2022

CUP: J53D23010690006

Coordinator: Università degli studi di Bologna

Ruolo UNIBO: PI

Sostegno finanziario UE: € 206.848,00

Ambiti di ricerca: Microbiologia agroambientale e degli alimenti

Scientific Officer: Giulia Tabanelli

Duration: 12/10/2023 - 12/10/2025

Research group: Giulia Tabanelli, Fausto Gardini.