Evaluation, characterisation and preservation of microbial diversity in traditional type I sourdough

Traditional Type I sourdoughs are complex microbial ecosystems in which lactic acid bacteria and yeasts determine the technological, sensory, nutritional, and functional properties of bread, including leavening performance, acidification, flavour development, digestibility, and shelf life. Preserving and studying these microbial communities is therefore essential to safeguard traditional bread identity, ensure process reproducibility, and support biotechnological innovation. However, their stability may be compromised by taxonomic and functional drift induced by conventional backslopping practices and industrial standardization. Despite their relevance, the scientific literature has historically lacked in: i) analytical characterization and in vitro simulation of the impact of traditional Type I sourdough on bread digestibility; ii) standardized protocols for the long-term preservation of sourdough microbiomes as intact functional units in public microbial biological centre (mBRC); iii) analytical characterization of yeast biodiversity as marker of traditional bread identity. To fill these gaps, a narrative review established the state-of-the-art in preservation technologies and regulatory compliance, confirming that current methods are focused on pure culture preservation rather than on the microbiome as a functional unit. Second, three Sardinian sourdoughs (SD81, SD82, SD84) were characterized using metabarcoding (16S rRNA and ITS) and culture-dependent biotyping (RAPD and rep-PCR); their nutritional impact was then assessed through a static in vitro digestion model by measuring in vitro protein digestibility (IVPD), predicted glycemic index (pGI), as well as peptide and amino acid profiles. Third, a microbiome cryopreservation workflow was validated at -80°C over 12 months for the three previously analysed sourdough, monitoring functional recovery through community-level physiological profiling (CLPP) with the OmniLog© system. Finally, the "TRIGU" citizen science project in Villaurbana enabled the collection and high-resolution typing of 130 yeast isolates form traditional sourdoughs by combining MALDI-TOF MS, inter-δ analysis, and microsatellite typing. Results showed that microbial diversity directly modulates bread quality: the SD81 starter significantly increased IVPD (79% vs. 70%), while SD82 reduced the pGI to approximately 59 and synthesized high levels of the prebiotic mannitol (51.1 mM). All sourdoughs achieved a 52–63% reduction in phytic acid content, enhancing mineral bioaccessibility. Cryopreservation effectively maintained metabolic fingerprints, and five backslopping cycles proved sufficient to restore core technological functions. The Villaurbana study revealed high intraspecific yeast diversity, identifying possible endemic Torulaspora delbrueckii lineages and Saccharomyces cerevisiae strains that are genetically divergent from those deposited in international collections. The results obtained provide validated protocols for the biobanking of sourdough microbiomes (TRL 3–4). Future perspectives include advancing towards higher technology readiness levels (TRL 5–6), with the development of clean-label starter consortia representative of local microbial biodiversity.