Novel assay for the determination of residual coagulant activity in cheese

A new method for determining residual coagulant activity in cheese was developed. Cheese samples were dispersed in 0.1 M trisodium citrate, under which conditions chymosin was stable for at least 5h. On incubation of such dispersions with a synthetic heptapeptide substrate (Pro-Thr-Glu-Phe-[NO₂-Phe]-Arg-Leu) at 37°C and pH 3.2, a single peptide ([NO₂-Phe]-Arg-Leu) was produced from the substrate by the action of coagulant in the cheese and quantified by reverse-phase HPLC with detection at 300 nm. This peptide was produced at a constant rate. The concentration of this peptide was proportional to the amount of active enzyme present and the assay was capable of measuring chymosin activities in buffer as low as 3.66 x10^-4 rennet units (RU) ml^-1. The assay was highly repeatable with a coefficient of variation of 4.3%, and was applied to a number of commercial cheeses for which residual coagulant activities in the range 11.1-20.1 RU kg^-1 were determined. The assay developed in this study is a simple, rapid, sensitive and reproducible method for determining residual coagulant activity in cheese and other dairy products.