γ-Glu-Ala

The antigenic mosaic of the Archaebacterium Methanobacterium thermoautotrophicum, strain delta H, was analyzed with a panel of six monoclonal antibodies. Five antigenic determinants were identified. One contains N-acetyl-D-glucosamine, another contains N-acetyl-D-galactosamine, and a third contains gamma-glutamylalanine (gamma-Glu-Ala). These residues are not involved, at least as immunodominant epitopes, in the other two determinants, one of which contains L-talosaminuronic acid, a component of pseudomurein found only in Methanobacteriaceae. Each of the five determinants was recognized by one monoclonal antibody exclusively. A sixth antibody recognized a structure containing gamma-Glu-Ala that could be either a sixth determinant or a subdeterminant within the site already recognized as containing gamma-Glu-Ala. We postulate that two of the determinants are strain specific, three are species specific, and one is a common antigen.

Targeted quantitation of 65 candidate taste compounds and ranking on the basis of dose-over-threshold (DoT) factors, followed by taste re-engineering and omission experiments in aqueous solution as well as in a cheese-like model matrix, led to the identification of a total of 31 key tastants (amino acids, organic acids, fatty acids, biogenic amines, and minerals) with DoT factors ≥1.0 and a total of 15 subthreshold, but kokumi-enhancing, γ-glutamyl peptides in extraordinarily high concentrations of 20468 μmol/kg. Among the γ-glutamyl peptides, γ-Glu-Gly, γ-Glu-Ala, γ-Glu-Thr, γ-Glu-Asp, γ-Glu-Lys, γ-Glu-Glu, γ-Glu-Trp, γ-Glu-Gln, and γ-Glu-His have been identified for the first time in Parmesan cheese. The excellent match of the sensory profile of the taste recombinants and the authentic cheese demonstrated the identified taste compounds to be fully sufficient to create the characteristic taste profile of the Parmesan cheese. This molecular blueprint of a Parmesan's chemosensory signature might be a useful molecular target for visualizing analytically the changes in taste profiles throughout cheese manufacturing and opens new avenues for a more scientifically directed taste improvement of cheese by tailoring manufacturing parameters ("molecular food engineering").

Sixty-nine hybridomas were generated to produce monoclonal antibodies to species of methanogens representing three of the four families accepted at the present time: Methanobacteriaceae, Methanococcaceae, and Methanomicrobiaceae. The antibody of each of 29 hybridomas cross-reacted with a methanogen of the same species or genus as the immunizing (homologous) strain, whereas the antibody of the other 40 cell lines reacted only with the homologous strain. Inhibition-blocking experiments with compounds of known composition and structure were used to define the fine specificity of antibodies of four hybridomas representing the four genera of the methanogens used for immunization. The combining site of the monoclonal antibody against M. thermoautotrophicum delta H examined is specific for a structure in the pseudomurein involving N-acetyl-glucosamine but not the (1-3) linkage or the C-terminus gamma-Glu-Ala of the peptide, both of which are characteristic of pseudomurein, the cell-wall peptidoglycan distinctive of the Methanobacteriaceae. In contrast, the monoclonal antibody to the other methanogen of this family examined, M. arboriphilus DH1, recognizes a determinant involving gamma-Glu-Ala. Thus pseudomurein expresses at least two dissimilar antigenic determinants in different portions of the molecule. The monoclonal antibodies against M. vannielii SB and M. hungatei JF1, whose families do not possess pseudomurein, did not display specificity for analogues of pseudomurein.