Evaluation of proteolysis in Parmesan cheese using electrophoresis and HPLC

Abstract

The proteolytic changes occurring during the ripening of Parmesan cheese were studied using urea-polyacrylamide gel electrophoresis (urea-PAGE) of caseins, HPLC analysis offree amino acids (FAA) and Kjeldahl determination of soluble nitrogen fractions. An electrophoretic ripening index for the evaluation of proteolysis in Parmesan cheese was established. The separation of caseins by alkaline PAGE (12 % T, 2.6 % C, pH 8.9, 5 M urea) was followed by the densitometric analysis of the y- and ~-casein fractions. The relationship between the resulting coefficients (y-Cn/~-Cn) and the age of reference samples of Original ltalian Grana Padano (6-22 months) was linear up to 15 months, allowing an evaluation of the extent of proteolysis and therefore a deduction of the age of the Parmesan samples analysed. Threshold levels (y-Cn/~-Cn) were proposed for the verification of the required age of Parmesan cheese. The coefficients (y-Cn/~-Cn) as weil as the 13-casein content of two addition al series of references and of 117 commercial Parmesan sampI es are presented. Commercial Parmesan samples retailed as a loaf or as prepacked slices were generally found to fulfill the requirements concerning endoproteolytic changes during ripening. However, man y grated Parmesan samples taken from retail outlets in Austria showed poor quality, which was probably due to the adulteration with products with low proteolysis (e.g., cheese rind, very young cheese). HPLC analysis ofFAA was also used to characterize the ripening process. Although FAA content ofreference samples of Grana Padano showed a very high degree of variability, a distinct increase of FAA content could be obser- ved during the ripening period. However, two series of reference samples, which had completely different electrophoretic casein patterns, could not be distinguished by HPLC analysis of the total amount of FAA. As an experiment, two loaves of Parmesan cheese were removed from the ripe- ning room at an age of 4 and 2 months and were subsequently stored at 6 C for 18 months. During the storage period, endoproteolytic changes were slowed down and breakdown of C:XS1-and ~-casein as weil as the accumulation of degradation products proceeded at a reduced rate, and could be detected using urea-PAGE casein patterns, but not by HPLC analysis of FAA content. In conse- quence of these findings, no accurate evaluation of the age of commercial Parmesan samples was pos- sible by means of the total amount of FAA. Since Kjeldahl determination of WSN and HPLC analysis of FAA content give insufficient information about the distinct endoproteolytic changes, which were found to be typical for Parmesan cheese, urea-PAGE of the casein fraction has to be do ne to enable * Oral communication at the IDF symposium 'Ripening and quality of cheeses', Besancon, France,