Michèle Nardi

Production of Methanethiol from Methionine by Brevibacterium linens CNRZ 918

The conditions under which Brevibacterium linens CNRZ 918, a strain isolated from the surface smear flora of Gruyère de Comté cheese, produced methanethiol from methionine were studied. Demethiolation was estimated from the methanethiol production capacity of resting cells. Methionine was demethiolated mainly during the exponential growth phase of the organism during which time the cells were rod-shaped and had a generation time of 5 h, and the medium became alkaline. At the end of growth (pH 9) the cells were coccoid, and produced only very little methanethiol. The production of methanethiol required the presence of methionine in the culture medium, this reflecting the probable induction of the enzyme systems involved. Glucose favoured growth and inhibited production of methanethiol. Lactate favoured both growth and methanethiol production. Resting rod cells also produced methanethiol from structural analogues of methionine and from methionine-containing peptides. The apparent kinetic constants of the production of methanethiol for rod and coccoid cells were respectively Km = 14 mM and 46 mM, Vmax = 208 nkat g-1 and 25 nkat g-1. The optimum temperature and pH for production were 30 degrees C and pH 8. Azide or malonate favoured the production of methanethiol by resting cells, whereas chloramphenicol had no effect.

Identification and Functional Analysis of the Gene Encoding Methionine-γ-Lyase in Brevibacterium linens

ABSTRACT The enzymatic degradation of l-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. l-Methionine-γ-lyase (MGL) can convert l-methionine to methanethiol (MTL), α-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that l-methionine degradation via γ-elimination is a key step in the formation of VSCs in B. linens.

Induction of methanethiol production by Brevibacterium linens CNRZ 918

SUMMARY: A non-inducing medium (NID) was defined for studying the induction of methanethiol production by Brevibacterium linens CNRZ 918. The lowest L-methionine concentration capable of inducing maximal methanethiol production by the cells was 7 mM. The peptides L-Ala-L-Met and L-Met-L-Ala induced greater methanethiol production than free L-methionine. D-Methionine, L-cysteine, S-methyl-L-cysteine and L-ethionine were poor inducers. Culture temperature affected the duration of induction. An Na+ concentration of 1 M in the culture medium led to maximal methanethiol production capacity of both cells and cell extracts. L-Methionine and L-ethionine were the best substrates for the crude soluble cells extract (with release of methanethiol and ethanethiol respectively). Neither the derivatives of L-methionine that acted as inducers, nor D-methionine, were substrates for demethiolation. Demethiolation activity of the crude extract was thermolabile, not stimulated by Na+ and strongly inhibited by Zn2+, Mn2+ and Cu2+. The shortest generation time obtained for B. linens CNRZ 918 in NID medium + L-methionine was 4 h at 26 °C. Only coccoid forms were present when the culture temperature was 30 °C. The presence of L-methionine in the medium favoured their appearance. The strain grew best in the presence of 1% NaCl but tolerated concentrations up to 15%. The induction of methanethiol production was due to the induction of L-methionine-γ-demethiolase. The level of induction was probably related to the intracellular concentration of L-methionine. The transport system of L-methionine by B. linens CNRZ 918 was constitutive and Na+ dependent.

Accelerating cheese proteolysis by enriching Lactococcus lactis proteolytic system with lactobacilli peptidases

Abstract The knowledge available on the genetics and proteolytic system of lactic acid bacteria makes it possible to genetically engineer starters with increased proteolytic properties. Our objective was to identify the best available strains capable of accelerating or modulating casein proteolysis during cheese ripening. To attain this goal, we used Lactococcus lactis strains expressing 5 different Lactobacillus peptidases to ripen a cheese model. At the end of ripening, free amino acids were quantitatively and qualitatively analysed. We identified the mixture of prolidase, PepQ, and X-prolyl dipeptidyl peptidase, PepX, as well as the peptidase PepW as the most efficient peptidases to increase, up to 3-fold, the overall level of amino acids at the end of ripening. The levels of threonine, asparagine, glycine, methionine, valine, glutamine, isoleucine and proline in particular increased (more than 3.5 fold). Grouping the amino acids produced according to the specific aroma compounds that each may give rise to following an enzymatic or chemical conversion, revealed that expression of PepW or PepX and PepQ increased the amounts of all groups of amino acids while expression of PepQ or PepN increased more especially those of aromatic amino acids/proline and glutamic acid, respectively. The combination of increased proteolysis and conversion of amino acids into aroma compounds now needs to be tested. In addition, the role of proline and its derived compounds in the overall flavour of cheese should be investigated.

Modulation of casein proteolysis by lactococcal peptidase gene inactivation

Abstract Peptidases from lactic acid bacteria participate in cheese ripening by hydrolysing peptides and liberating free amino acids, which are precursors of aroma compounds. Using a pseudo-curd model and lactococcal mutants, negative for different peptidases, as ripening agents, we determined some of the key peptidases in the ripening process. The total level of amino acids quantified after 28 days of ripening was significantly reduced when the general aminopeptidase PepN was absent. The situation was aggravated when the tripeptidase PepT was also missing. The deficiency in the proline-specific aminopeptidase PepX more specifically decreased the level of free proline in the pseudo-curds after 28 days of ripening. These three peptidases, which are also necessary for optimal lactococcal growth in milk, can be considered as peptidases of technological importance. On the other hand, the suppression of the other peptidases tested (PepF1, PepF2, PepC, PepP) did not significantly affect amino acid pools in the conditions of the test.

Genetic transformation of Brevibacterium linens strains producing high amounts of diverse sulphur compounds.

By its numerous properties and importance in cheese technology (production of colour, flavour, bacteriocins and resistance to salt) Brevibacterium linens is a major cheese ripening bacteria. However, the genetic approach of such biological functions has been hindered, up to now, by the lack of tools necessary to realise genetic modifications in this species. Our objective was to demonstrate that it is possible to genetically modify several strains exhibiting interesting technological properties, especially the production of sulphur compounds. We worked with a phenotypically and genetically diverse collection of 11 strains. We genetically transformed several Brevi. linens with acceptable rates with plasmids classically used to transform lactic acid bacteria and other Gram+ bacteria. These results open up new prospects to investigate the most interesting Brevi. linens metabolic pathways both at the biochemical and genetic level.

Identification of the gene responsible for the synthesis of volatile sulfur compounds in Brevibacterium linens

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSC) is believed to be essential for flavour development in cheese. L-methionine γ-lyase (MGL) can convert L-methionine to methanethiol (MTL) α-ketobutyrate and ammonia. The MGL gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSC. The disruption of the MGL gene, achieved in strain ATCC 9175, resulted in a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via γ-elimination is a key step to form VSC in B. linenes.