D. Hemme

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.

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.

Peptide utilization by Lactococcus lactis and Leuconostoc mesenteroides

C. FOUCAUD, D. HEMME AND M. DESMAZEAUD. 2001. To explain the competition for nitrogenous nutrients observed in mixed strain cultures of Lactococcus lactis and Leuconostoc mesenteroides, the utilization of peptides as a source of essential amino acids for growth in a chemically defined medium was compared in 12 strains of dairy origin. Both species were multiple amino acid auxotrophs and harboured a large set of intracellular peptidases. Lactococcus lactis can use a wide variety of peptides up to 13 amino acid residues whereas Leuc. mesenteroides assimilated only shorter peptides containing up to seven amino acids. Growth was limited by the transport of peptides and not by their hydrolysis. The nutritional value of peptides varied with the strains and the composition of the peptides, L. lactis being advantaged over Leuc. mesenteroides.

Effect of the addition of extracts of thermophilic lactobacilli on acid production by Streptococcus thermophilus in milk.

Soluble extracts of 20 strains of thermophilic lactobacilli (Lactobacillus helveticus, L. lactis and L. bulgaricus) were prepared and added to milk for the culture of 10 strains of Streptococcus thermophilus. Acid production was stimulated in 64.5% of cases for 9 of these 10 strains. The L. helveticus extracts were the most stimulatory, but the same extracts did not always strongly stimulate each strain of Str. thermophilus. The stimulatory effects observed varied with the volume of extract and the strain of Str. thermophilus. The exception was Str. thermophilus 385, which was never stimulated. The stimulatory effects observed were due to aminopeptidases present in the lactobacillus extracts and were not related to a general caseinolytic activity. The possible addition of such extracts to milk for cooked hard cheese is discussed.

Glutamate uptake in Lactobacillus delbrueckii subsp. bulgaricus CNRZ 208 and its enhancement by a combination of Mn2+ and Mg2+

Aims: To demonstrate the mechanism of glutamate uptake in the dairy strain Lactobacillus delbrueckii subsp. bulgaricus CNRZ 208, and to characterize key aspects of the system .

Characterization of amino acid transport in the dairy strain Leuconostoc mesenteroides subsp. mesenteroides CNRZ 1273

Aims: To identify and characterize amino acid transport in Leuconostoc mesenteroides. 
Methods and Results: The transport of labelled amino acids was measured in whole cells of Leuc. mesenteroides CNRZ 1273. Systems were operative under physiological conditions of growth, energy dependent and differed from peptide transport. Some of the systems were shared by several amino acids. Kinetic analysis indicated the presence of three transport systems with very high (VH), high (H) and low affinity (H) for the 11 amino acids studied. The Kt values (µmol l−1) ranged from 0·088 to 0·815 (VH), 6–390 (H) and 320–4500 (L) and the Vmax values [nmol s−1 (g dry weight)−1] from 0·015 to 0·8 (VH), 15–95 (H) and 90–470 (L). 
Conclusions: The study showed the presence of three transport systems in Leuc. mesenteroides for all amino acids tested, some of them being shared by several amino acids. 
Significance and Impact of the Study. The findings are discussed with reference to the growth of Leuc. mesenteroides in milk as pure or in mixed‐strain culture with Lactococcus lactis.

Acidification test based on the use of biomass for screening of Leuconostoc. Application to Ln. mesenteroides strains isolated from French raw milk cheeses

Unlike that of other lactic acid bacteria, the growth of Leuconostoc spp. in milk is poor and the resulting acidification cannot be used to distinguish different strains. An acidification test based on the use of high initial numbers (10 9 cfu/ml) has been developed and proved to be an efficient tool for discriminating between 110 Leuconostoc strains isolated from French raw milk cheeses. The pH values after 24 h ranged from 6.55 to 4.05 and distinguished four acidification groups. All 34 % of the strains that acidified milk to at least pH 5.1, coagulating it and should be considered as Lac + . The differences in rates and degrees of acidification could not be related to the proteolytic activity which was, from all 27 representative strains tested, similar to that of Lactococcus lactis Prt - variants.

Valine transport and biodiversity of Leuconostoc wild strains from french raw milk cheeses

The rate of L-valine transport in whole cells of Leuconostoc was at the maximum at 30 degrees C, pH 6.0 in the presence of an energy source. Transport was inhibited by 40-55%, in the presence of the ionophores (valinomycin, nigericin or monensin), and uncouplers (carbonyl cyanide-m-chloro-phenylhydrazone or 2,4-dinitrophenol) confirming the previously described delta p-driven branched-chain amino acid transport system described in cytoplasmic membranes (Winters et al., 1991, Appl. Environ. Microbiol., 57, 3350-3354). Sulfhydryl group reagents (p-chloro-mercuribenzoate, iodoacetate and N-ethyl maleimide) all inhibited valine transport by 60-70%, indicating that valine is actively transported at high valine concentration. Three kinetically distinguishable transport systems were identified for each strain using whole cells, confirming results obtained with membranes. L-valine transport Kt and Vmax could be an additional tool to estimate the biodiversity of 18 Leuconostoc strains belonging to the dominant flora of French raw milk cheeses. Kt values varied from 20 to 510 nmol/l for the very high affinity system, from 26 to 427 pmol/l for the high affinity system and from 0.65 to 4.40 mmol/l for the low affinity system. No correlation existed between valine transport rates and a particular strains ability to acidify milk or complex media, suggesting that valine transport is not a growth-limiting function in species of the genus Leuconostoc.