First application of an integrated microbiological and (meta)genomics approach to verify the biological origin and microbial safety of rennet products

Rennet is essential for cheese production, containing milk-clotting enzymes derived from animal, plant, or microbial sources. Accurate labelling of rennet’s biological origin is essential to respect consumer dietary choices, while microbial contamination may pose food safety risks. We developed an integrated strategy combining qPCR, metagenomics, culture-based methods, whole-genome sequencing, and mass spectrometry to verify the biological origin and microbial safety of 27 commercial products labelled as being of animal ( n = 11), plant ( n = 3), or microbial ( n = 13) origin. DNA matching the labelled production organism was detected in most products, although DNA belonging to other known rennet-producing organisms was also found in several products, including one plant-labelled product containing animal DNA. Additionally, viable B. cereus s.l. was detected in some animal rennet products. To evaluate the potential food safety impact of B. cereus s.l. contamination, rennet products were used to produce cheeses, analysed for B. cereus s.l. growth and cereulide. Bacterial growth and measurable cereulide levels were observed in one cheese, confirming potential food safety risks. To the best of our knowledge, this study provides the first integrated assessment of commercial rennet combining both conventional methods and (meta)genomics, demonstrating the potential of these approaches to identify authenticity issues and microbial safety risks.