Assessment of residual plant DNA in bulk milk for Grana Padano PDO production by a metabarcoding approach

The aim of this study was to evaluate the ability of DNA metabarcoding, by rbcl as barcode marker, to identify and classify the small traces of plant DNA isolated from raw milk used to produce Grana Padano (GP) cheese. GP is one of the most popular Italian PDO (Protected Designation of Origin) produced in Italy in accordance with the GP PDO specification rules that define which forage can be used for feeding cows. A total of 42 GP bulk tank milk samples were collected from 14 dairies located in the Grana Padano production area. For the taxonomic classification, a local database with the rbcL sequences available in NCBI on September 2020/March 2021 for the Italian flora was generated. A total of 8,399,591 reads were produced with an average of 204,868 per sample (range 37,002–408,724) resulting in 16, 31 and 28 dominant OTUs at family, genus and species level, respectively. The taxonomic analysis of plant species in milk samples identified 7 families, 14 genera and 14 species, the statistical analysis conducted using alpha and beta diversity approaches, did not highlight differences among the investigated samples. However, the milk samples are featured by a high plant variability and the lack of differences at multiple taxonomic levels could be due to the standardisation of the feed rationing, as requested by the GP rules. The results suggest that DNA metabarcoding is a valuable resource to explore plant DNA traces in a complex matrix such as milk.