EVALUATION OF PROTEOLYSIS IN PARMESAN CHEESE USING ELECTROPHORESIS AND HPLC

The proteolytic changes occurring during the ripening of Parmesan chee se were studied using urea-polyacrylamide gel electrophoresis (urea-PA GE) of caseins, HPLC analysis of free amino acids (FAA) and Kjeldahl d etermination of soluble nitrogen fractions. An electrophoretic ripenin g index for the evaluation of proteolysis in Parmesan cheese was estab lished. 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 gamm a- and beta-casein fractions. The relationship between the resulting c oefficients (gamma-Cn/beta-Cn) and the age of reference samples of Ori ginal Italian Grana Padano (6-22 months) was linear up to 15 months, a llowing an evaluation of the extent of proteolysis and therefore a ded uction of the age of the Parmesan samples analysed. Threshold levels ( gamma-Cn/beta-Cn) were proposed for the verification of the required a ge of Parmesan cheese. The coefficients (gamma-cn/beta-Cn) as well as the beta-casein content of two additional series of references and of 117 commercial Parmesan samples are presented. Commercial Parmesan sam ples retailed as a loaf or as prepacked slices were generally found to fulfill the requirements concerning endoproteolytic changes during ri pening. However, many grated Parmesan samples taken from retail outlet s in Austria showed poor quality, which was probably due to the adulte ration with products with low proteolysis (e.g., cheese rind, very you ng cheese). HPLC analysis of FAA was also used to characterize the rip ening process. Although FAA content of reference samples of Grana Pada no showed a very high degree of variability, a distinct increase of FA A content could be observed during the ripening period. However, two s eries of reference samples, which had completely different electrophor etic casein patterns, could not be distinguished by HPLC analysis of t he total amount of FAA. As an experiment, two leaves of Parmesan chees e were removed from the ripening room at an age of 4 and 2 months and were subsequently stored at 6 degrees C for 18 months. During the stor age period, endoproteolytic changes were slowed down and breakdown of alpha(S1)- and beta-casein as well 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 con sequence of these findings, no accurate evaluation of the age of comme rcial Parmesan samples was possible by means of the total amount of FA A. Since Kjeldahl determination of WSN and HPLC analysis of FAA conten t give insufficient information about the distinct endoproteolytic cha nges, which were found to be typical for Parmesan cheese, urea-PAGE of the casein fraction has to be done to enable the evaluation of the ex tent of proteolysis and therefore the deduction of the age of commerci al Parmesan samples analysed. (C) Inra/Elsevier, Paris.