Rennets differing in chymosin-to-pepsin ratio shape the metabolomic and sensory profile of Grana Padano PDO cheese during ripening

Utilizing different chymosin and pepsin ratios in cheesemaking may represent a potential strategy to shape the sensory profile of hard cheeses. This study investigated the impact of rennet with varying chymosin and pepsin ratios on the chemical profile and sensory attributes of Grana Padano PDO cheese at different ripening times (10 to 20 months). The research involved the analysis of hard cheese manufactured with distinct calf chymosin percentages (99 %, 95 %, and 83 %), exploiting sensory analyses and untargeted metabolomics to identify marker compounds correlating with specific sensory traits. The results demonstrated that varying the rennet composition significantly affected sensory profile; in particular, the rennet made by 83 % chymosin and 17 % pepsin generated a more complex sensory profile starting from 12 months. AMOPLS and ASCA analysis on untargeted metabolomics signatures revealed that ripening time was the only significant factor when compared with rennet type and the interaction ripening x rennet. Finally, at more advanced ripening times, 3-methylbutanoic acid and homoethone were significantly up-accumulated in cheese samples manufactured with higher pepsin percentages, likely explaining sensory outcomes. This study provides valuable insights into using rennet to tailor the sensory qualities of hard cheeses, underscoring the importance of enzyme selection in cheese manufacturing to drive innovation in the dairy industry.