In Lactococcus lactis, which is widely used as a starter in the cheese industry, the first step of aromatic and branched-chain amino acid degradation is a transamination which is catalyzed by two major aminotransferases. We have previously purified and characterized biochemically and genetically the aromatic aminotransferase, AraT. In the present study, we purified and studied the second enzyme, the branched-chain aminotransferase, BcaT. We cloned and sequenced the corresponding gene and used a mutant, along with the luciferase gene as the reporter, to study the role of the enzyme in amino acid metabolism and to reveal the regulation of gene transcription. BcaT catalyzes transamination of the three branched-chain amino acids and methionine and belongs to class IV of the pyridoxal 5-phosphate-dependent aminotransferases. In contrast to most of the previously described bacterial BcaTs, which are hexameric, this enzyme is homodimeric. It is responsible for 90% of the total isoleucine and valine aminotransferase activity of the cell and for 50 and 40% of the activity towards leucine and methionine, respectively. The original role of BcaT was probably biosynthetic since expression of its gene was repressed by free amino acids and especially by isoleucine. However, in dairy strains, which are auxotrophic for branched-chain amino acids, BcaT functions only as a catabolic enzyme that initiates the conversion of major aroma precursors. Since this enzyme is still active under cheese-ripening conditions, it certainly plays a major role in cheese flavor development.

No abstract

The pure water extraction of Comtk cheese solubles and their chromatographic separation facilitate sensory analysis experiments with the fractions directly collected from the column. Two extracts, one obtained after ultrafiltration, the other after chromatography on Sephadex G25, have been separated on Toyopearl HW40S. The sensory evaluation of the obtained fractions shows in the two cases that some fractions have interesting flavors. However, physicochemical analysis shows that organoleptic characteristics are linked more to the presence of amino acids and salts than to the presence of peptides. Apart from monosodium glutamate, most of the amino acids are present in the fractions at concentrations lower than their threshold values measured in water. Therefore, the hypothesis of synergistic effects among amino acids or between amino acids and other kinds of compounds, such as salts, can be raised. Moreover, the fractions containing free amino acids are also generally the most aromatic. The nature and the origin of these volatile compounds responsible for these aromas have to be determined.

Thirteen low-molecular-weight phosphopeptides were isolated from the water-soluble fraction of Comtd cheese. The sample was fractionated and purified by gel permeation chromatography and reverse-phase HPLC. The peptide sequences were identified by Edman degradation and primary molecular structure was confirmed by mass spectrometry. The different peptides purified correspond to fragments of the sequence Vall3-Lys 28 of p-casein and of the sequence Glu S-Lys 21 of CQ casein. These fragments probably originated from an initial proteolysis of the two caseins by plasmin, followed by further endopeptidase aminopeptidase and, possibly, carboxypeptidase digestions. Partial dephosphorylation of some p-casein fragments was observed. These peptides probably influence the flavor profile of comt6 cheese.

Isolation, purification and identification of some low-molecular-weight compounds from a water-soluble extract of Comtt? cheese was achieved. We used liquid CO1 extraction, subsequent fractionation (by gel permeation and reserve-phase HPLC) and mass spectometry. Major constituents were N-acyl amino acids (N-ace@ methionine, N-propionyl methionine, N-propionyl leucine, N-propionyl phenylalanine), diketopiperazines (cyclo(Pro-Pro), cyclo(Pro-Val), cyclo(Pro-Phe), cy clo(Pro-Leu), cyclo(Pro-Ala)) and non-peptide compounds (theobromine, 4-methyl-t-thiazoleethanol, uracil, (56)dihydro-uracil, thymine). These compounds were synthesized and tasted. Most were bitter. N-propionyl methionine had a cheese flavor but was not detected when added to a simulated cheese at 180 mg/L.