P. Resmini

Phosphopeptides from Grana Padano cheese: nature, origin and changes during ripening.

Casein phosphopeptides (CPP) which develop in Grana Padano cheese at different ages were isolated by precipitation with Ba2+ and analysed by HPLC. Profiles were complex throughout the period between 4 and 38 months. CPP in a cheese sample 14 months old were identified by a combination of fast atom bombardment-mass spectrometry and Edman degradation. They were found to consist of a mixture of components derived from three parent peptides, beta-CNf(7-28)4P, alpha s1-CNf(61-79)4P and alpha s2-CNf(7-21)4P. In total, 45 phosphopeptides were identified: 24 from beta-CN, 16 from alpha s1-CN and 5 from alpha s2-CN. The presence of aminopeptidase activity during cheese ripening was deduced from the presence of a number of CPP of different lengths with the loss of one or more residues from the N-terminus. The longest had C-terminal lysine and seemed to be progressively hydrolysed by carboxypeptidases A and B to shorter peptides. CPP in cheese appeared to be shortened plasmin-mediated products. Moreover, those most resistant to further hydrolysis contained at least three closely located phosphoserine residues. The anticariogenic activity of CPP is also discussed.

Gel electrophoresis and immunoblotting for the detection of casein proteolysis in cheese.

The whole N fraction of six samples of hard and semi-hard pressed cheeses was analysed using PAGE, polyacrylamide gel isoelectric focusing and immunoblotting with polyclonal antibodies against beta- and alpha s1-casein. The origin of some electrophoretic bands corresponding to peptides produced from the enzymic degradation of the casein fractions was established. A number of these peptides were also present in the in vitro hydrolysates of casein with plasmin and chymosin. Thus, it was also possible to determine which casein was the source of each peptide and which enzymes were active in cheese. Compared with the traditional Coomassie staining procedures, immunoblotting is more sensitive and specific, making the interpretation of each electrophoretic profile easy. Thus, it was also possible to obtain a clear picture of the state of each casein fraction in a cheese variety. Two main peptides were isolated from the pH 4.6-insoluble N fraction of Parmigiano-Reggiano using DEAE-cellulose chromatography and identified, from the amino acid sequence of the N- and C-terminal ends, as gamma 3-casein (beta-casein(f108-209)) and alpha s1-PL1 (alpha s1-casein(f80-199). In both cases, a Lys-X bond was hydrolysed, indicating the action of a trypsin-like enzyme in beta- and alpha s1-casein hydrolysis during the ripening of this variety of hard pressed cheese.

Evaluation of the stable reaction products of histidine with formaldehyde or with other carbonyl compounds in dairy products

Combined formaldehyde (FA) can be evaluated in cheese and other dairy products by determining spinacine (6-carboxy-1,2,3,4-tetrahydroimidazopyridine), an imino acid arising from the reaction of FA with the α-amino group of histidine. Other carbonyl compounds usually produced by fermentations can react themselves with histidine, forming molecules which interfere with the determination of spinacine. A sensitive (minimum detectable amount, 5 fmol spinacine) and interferencefree HPLC method, with precolumn derivatization with o-phtalaldehyde (OPA) and 9-fluorenylmethylchloroformate (FMOC), to evaluate these histidine reaction-compounds in dairy products is described. The clean-up of the FMOC-derivatized sample has been performed for the first time with solid-phase extraction (SPE) on an amino cartridge. The method was applied to samples of casein and differently ripened cheese, the origins of which were either known or commercial, in order to quantify the natural level of spinacine (0.7–2.7 ppm) probably deriving from biogenic FA, and to detect whether FA was used in processing.

Ein möglicher Mechanismus zur Bildung vonα-Aminobuttersäure während der Käsereifung

SummaryBy means of enzymatic extracts obtained from ripening Gorgonzola cheese and after incubation with14C-labelled arginine,α-aminobutyric acid formation has been investigated. The direct degradation of arginine to a-aminobutyric acid was pointed out by using arginine14C-5 and14COOH. Ornithine and glutamic acid, which have been indicated as possible precursors ofα-aminobutyric acid, are not involved in this mechanism.ZusammenfassungIn Bebrütungsversuchen von Enzymextrakten aus reifendem Gorgonzola wurde mit Hilfe des mit14C markierten Arginins die Bildung vonα-Aminobuttersäure studiert. Mit Hilfe von an C-5 und an COOH markiertem Arginin wurde der Abbau dieser Aminosäure inα-Aminobuttersäure bestätigt. Ornithin und Glutaminsiiure, ihre möglichen Vorläufer, sind nicht an diesem Prozeß beteiligt.