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.