Lait 78 (1998) 425-438
DOI: 10.1051/lait:1998441

Evaluation of proteolysis in Parmesan cheese using electrophoresis and HPLC

Helmut Karl Mayer, Christa Rockenbauer and Hannes Mlcak

Department of Dairy Research & Bacteriology. University of Agriculture, Gregor Mendel-Strafie 33. A-II80 Vienna, Austria

Abstract - The proteolytic changes occurring during the ripening of Parmesan cheese were studied using urea-polyacrylamide gel electrophoresis (urea-PAGE) of caseins, HPLC analysis of free 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 |3-casein fractions. The relationship between the resulting coefficients (γ-Cn / β Cn) and the age of reference samples of Original Italian 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 (γ-Cn / β-Cn) were proposed for the verification of the required age of Parmesan cheese. The coefficients (γ-Cn / β-Cn) as well as the β-casein content of two additional series of references and of 117 commercial Parmesan samples 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, many 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 of FAA was also used to characterize the ripening process. Although FAA content of reference samples of Grana Padano showed a very high degree of variability, a distinct increase of FAA content could be observed 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 ripening 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 α_s]- and β-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 consequence of these findings. no accurate evaluation of the age of commercial Parmesan samples was possible 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 done to enable the evaluation of the extent of proteolysis and therefore the deduction of the age of commercial Parmesan samples analysed. © Inra / Elsevier, Paris

Résumé - Évaluation de la protéolyse du fromage parmesan par électrophorèse et HPLC
La protéolyse du parmesan a été suivie au cours de l'affinage par électrophorèse (urée-PAGE) des caséines, analyse par HPLC des acides aminés libres et détermination par Kjeldahl des fractions azotées solubles. L'analyse des profils protéiques obtenus par électrophorèse a permis d'établir un index d'affinage grâce auquel l'âge des échantillons de fromages du commerce peut être estimé. L'utilisation de cet index a en outre permis de montrer que beaucoup de parmesan vendu sous forme de râpé était de mauvaise qualité. L'analyse des acides aminés libres ne permet pas en revanche de déterminer correctement l'âge du fromage. © Inra / Elsevier, Paris

Key words: Parmesan cheese / proteolysis / electrophoresis / free amino acid / soluble nitrogen

Mots clés : fromage parmesan / protéolyse / électrophorèse / acide aminé libre / azote soluble

Corresponding author: Helmut Karl Mayer mayer@mail.boku.ac.at