Assessing the authenticity of animal rennet using δ¹⁵N analysis of chymosin

Chymosin is a protease that curdles the milk casein. Animal rennet was the first discovered source of chymosin and its use is mandatory for the production of PDO cheeses such as Parmigiano Reggiano and Grana Padano. Of the alternatives, fermentation-produced chymosin is the most competitive because it functions in a similar way, but is much cheaper. Analytical tools are necessary in order to distinguish the 2 types of chymosin and verify the compulsory use of animal rennet in the production of PDO cheeses. In this work, a method to analyse ¹⁵N/¹⁴N in chymosin after extraction was developed. The δ¹⁵N values of animal rennet range from 5.7‰ to 8‰, whereas the δ¹⁵N values of fermentation-produced chymosin are significantly lower, ranging from -5.3‰ to 2.2‰. A threshold value of 5.7‰ was defined for authentic animal rennet. Addition of fermentation-produced chymosin to animal rennet, or its complete substitution, can be therefore detected.