Characterisation of the non-coagulating-enzyme part of different milk-clotting preparations

The first step in cheese manufacture is the formation of a gel from milk. This step can be achieved by adding milk-clotting preparations prepared from various sources and different methods. Each preparation contains not only coagulating enzyme(s) but also an associated support. Our objective was to characterize this support and to identify differences between preparations according to their manufacturing process and sources. Twenty-four preparations representative of the diversity available for cheese manufacture were sampled 3 times over a period of 6 months: 10 commercial rennets, 9 artisanally-produced calf rennets, 2 recombinant chymosins and 3 microbial preparations. Preparations were analysed for sodium, chloride, phosphorus, ammonia, free amino groups and ash contents, pH value and the phosphotungstic-acid-soluble nitrogen fraction. The protein diversity was determined by SDS−polyacrylamide gel electrophoresis and particular bands identified by reversed-phase nano liquid chromatography coupled on line to tandem mass spectrometry. The preparations were clearly discriminated according to the composition of their support. Preparations from Cryphonectria parasitica had the highest content in ammonia and nitrogen fractions. Commercial rennets and preparations from Rhizomucor miehei had the highest values for mineral composition while traditional rennets had the lowest. Commercial rennets presented the highest variability in composition. This variability was also confirmed by electrophoresis performed at the same milk-clotting activity, in which numerous protein bands ranging from 10 kDa to 150 kDa were shown in contrast to other preparations. Among the identified proteins, bovine serum albumin was mainly present in commercial rennets as well as minor bovine proteins and as expected not in recombinant chymosins. The observed variability in the support between the investigated preparations was linked to the source of coagulating enzymes and within the same source was inherent to the method used to obtain preparations. Work is under progress to determine if support impacts the cheesemanufacturing process.