title: Formation of Kokumi-Enhancing γ-Glutamyl Dipeptides in Parmesan Cheese by Means of γ-Glutamyltransferase Activity and Stable Isotope Double-Labeling Studies.
abstract: Recently, γ-glutamyl dipeptides (γ-GPs) were found to be responsible for the attractive kokumi flavor of Parmesan cheese (PC). Quantitation of γ-GPs and their parent amino acids in 13-, 24-, and 30-month ripened PC samples by LC-MS/MS and stable isotope dilution analysis (SIDA), in-cheese (13)C-labeling studies, followed by analysis of the γ-glutamyl transferase (GGT) activity revealed γ-GPs to be generated most efficiently after 24 months of ripening by a GGT-catalyzed transfer of the γ-glutamyl moiety of L-glutamine onto various acceptor amino acids released upon casein proteolysis. Following the identification of milk as a potential GGT source in PC, the functionality of the milk's GGT to generate the target γ-GPs was validated by stable isotope double-labeling (SIDL) experiments. Therefore, raw and heat-treated milk samples were incubated with L-glutamine-[U-(13)C] and acceptor amino acids (X) and the hetero- (γ-Glu-[(13)C5]-X) and homotranspeptidation products (γ-Glu-Gln-[(13)C10]) were quantitated by LC-MS/MS-SIDA using γ-Glu-Ala-[(13)C3] as the internal standard. High GGT activity to generate the γ-GPs and preference for L-phenylalanine and L-methionine as acceptor amino acids were found in raw milk and milk samples heat-treated for 10 min up to a maximum of 65 °C. In comparison, GGT activity and SIDL studies performed with inoculated Lactobacillus strains, including Lactobacillus harbinensis and Lactobacillus casei identified in PC by means of 16S rRNA gene sequencing, did not show any significant GGT activity and unequivocally demonstrated unpasteurized cow's milk, rather than microorganisms, as a key factor in γ-glutamyl dipeptide generation in Parmesan cheese.
authors: Chair for Food Chemistry and Molecular Sensory Science, Technische Universität München , Lise-Meitner-Straße 34, D-85354 Freising, Germany.
authors: Hedda
authors: H
authors: Hillmann
authors: Lehrstuhl für Technische Mikrobiologie, Technische Universität München , Weihenstephaner Steig 16, D-85350 Freising-Weihenstephan, Germany.
authors: Jürgen
authors: J
authors: Behr
authors: Lehrstuhl für Technische Mikrobiologie, Technische Universität München , Weihenstephaner Steig 16, D-85350 Freising-Weihenstephan, Germany.
authors: Matthias A
authors: MA
authors: Ehrmann
authors: Lehrstuhl für Technische Mikrobiologie, Technische Universität München , Weihenstephaner Steig 16, D-85350 Freising-Weihenstephan, Germany.
authors: Rudi F
authors: RF
authors: Vogel
authors: Chair for Food Chemistry and Molecular Sensory Science, Technische Universität München , Lise-Meitner-Straße 34, D-85354 Freising, Germany.|Bavarian Center for Biomolecular Mass Spectrometry , Gregor-Mendel-Straße 4, D-85354 Freising, Germany.
authors: Thomas
authors: T
authors: Hofmann
mesh_terms: D000818:Animals; D055162:Biocatalysis; D002417:Cattle; D002611:Cheese / Q000032:analysis* / Q000382:microbiology; D004151:Dipeptides / Q000378:metabolism; D005421:Flavoring Agents / Q000032:analysis / Q000378:metabolism*; D007553:Isotope Labeling; D007778:Lactobacillus / Q000378:metabolism; D008892:Milk / Q000737:chemistry / Q000201:enzymology* / Q000382:microbiology; D009587:Nitrogen Isotopes / Q000032:analysis; D053719:Tandem Mass Spectrometry; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
chemical_list: D004151:Dipeptides; D005421:Flavoring Agents; D009587:Nitrogen Isotopes; C047442:gamma-glutamylglutamine; D005723:gamma-Glutamyltransferase
keywords: GGT; Lactobacillus; Parmesan cheese; kokumi; stable isotope labeling; γ-glutamyl peptides; γ-glutamyl transferase