Brevibacterium enzymes as biological tools for ochratoxin A detoxification in dairy foods

The origin of ochratoxin A (OTA) in cheeses is mainly due to mould growth during the ripening process, and to a lesser extent, to the use of OTA-contaminated milk in cheese production. Bacterial smear-ripened cheeses developed a smear microbiota on their rind during ripening that greatly contributes to its typical aroma and texture. Bacteria from the Brevibacterium genus belong to the typical smear microbiota of cheeses. Type strains from Brevibacterium species frequently isolated from cheese and milk products were able to transform OTA into much less toxic ochratoxin α (OTα) and L-phenylalanine. Protein searches allowed the identification of a protein annotated as amidohydrolase in these OTA-degrader Brevibacterium strains. The OTA-hydrolytic activity of the identified amidohydrolase was demonstrated by the heterologous production of this protein from B. linens DSM 20425^T (BlOTA). In vitro assays revealed that BlOTA transformed OTA into less toxic OTα, as well as ochratoxin B. When compared with other previously described OTA-degrading amidohydrolases, BlOTA exhibited optimal activity at a higher pH (8.0), while showing similar high temperature for optimal activity (55 °C) and thermostability; in addition, a clear preference for substrates with Phe, Tyr or Leu amino acid residues at the C-terminal position was clearly observed. BlOTA efficiently detoxifies OTA-contaminated bovine milk, without provoking changes on its free amino acid composition. Moreover, in silico predictions revealed that BlOTA is a non-allergenic, non-antigenic, and poorly immunogenic protein. Therefore, the QPS status possessed by cheese-Brevibacterium species, as well as the characteristics exhibited by BlOTA, make them suitable tools for the biological detoxification of OTA in dairy and food products.