Characterisation of the ripening process by untargeted metabolite profiling and proteomics
SINTEF Industry, NTNU and NOFIMA
Non-targeted metabolite analysis by mass spectrometry (MS) was employed to determine the ripening rates at different water activities in small-scale laboratory experiments, and to follow the progress of the ripening in production samples. Approximately thousand metabolites were detected. Of these, 90-95 % had molecular masses below 800 Da, and more than 60 % below 500 Da. In the order of 150 metabolites were putatively annotated. In addition to free amino acids and muscle metabolites, the analysis revealed the time-profiles of di- and tripeptides, as well as a high number of compounds generated by further conversion of amino acids, muscle metabolites, and probably lipids. Statistical processing of the data sets showed that ripening of fresh and unsalted, dried meat, generated other profiles than salted samples.
Larger peptides were identified by LC-MS/MS -based proteomics, in collaboration with CSIC in Spain. Hams from four different times of processing: raw hams, post-salted hams (3 months of processing), hams selected in the middle of the production (12 months of processing) and hams at the end of the processing (24 months) were analysed. A high number of peptides and their development with time were identified.
Samples from the small-scale drying experiment described above, were analysed by proteomics analyses using 2D electrophoresis and MALDI-TOF MS in order to study the influence of RH and salt on the initial protein hydrolysis. Clear differences between drying conditions were observed, but further analyses are required in order to explain the results.
The metabolite analyses showed large changes the first 10-12 months of the processing, and only very small further changes, in accordance with the proteolysis index, which reached a value of 4.5 % with non-significant differences between 12 and 24 months of ripening. Determination of the activity of the proteolytic enzymes also confirmed these findings. Cathepsin H activity decreased until negligible values after 3 months of processing, whereas cathepsins B and B + L were inactive at 12 months. AAP was the most active aminopeptidase whereas RAP and MAP were active just during the first 12 months of processing.
Unfreezable water content and glass transition temperature were investigated using differential scanning calorimetry (DSC) technique with non-significant differences in the temperature of glass transition for 12 and 24 months of processing.