Metabolic gene-targeted monitoring of non-starter lactic acid bacteria during cheese ripening

Long ripened cheeses, such as Grana Padano (GP), a Protected Designation of Origin (PDO) Italian cheese, harbor a viable microbiota mainly composed of non-starter lactic acid bacteria (NSLAB), which contribute to the final characteristics of cheese. The NSLAB species Lactobacillus rhamnosus, Lb. casei and Lb. paracasei are frequently found in GP, and form a closely related taxonomic group (Lb. casei group), making it difficult to distinguish the three species through 16S rRNA sequencing. SpxB, a metabolic gene coding for pyruvate oxidase in Lb. casei group, was recently used to distinguish the species within this bacterial group, both in pure cultures and in cheese, where it could provide an alternative energy source through the conversion of pyruvate to acetate. The aim of this work was to study the evolution of the metabolically active microbiota during different stages of GP ripening, targeting 16S rRNA to describe the whole microbiota composition, and spxB gene to monitor the biodiversity within the Lb. casei group. Furthermore, activation of pyruvate oxidase pathway was measured directly in cheese by reverse transcription real time PCR (RT–qPCR). The results showed that Lb. casei group dominates throughout the ripening and high-throughput sequencing of spxB allowed to identify four clusters inside the Lb. casei group. The dynamics of the sequence types forming the clusters were followed during ripening. Pyruvate oxidase pathway was expressed in cheese, showing a decreasing trend over ripening time. This work highlights how the composition of the microbiota in the early manufacturing stages influences the microbial dynamics throughout ripening, and how targeting of a metabolic gene can provide an insight into the activity of strains relevant for dairy products.