Hervé Robert

Biodiversity of exopolysaccharides produced from sucrose by sourdough lactic acid bacteria.

The distribution and diversity of natural exopolysaccharides (EPS) produced from sucrose by thirty heterofermentative lactic acid bacteria strains from French traditional sourdoughs was investigated. The EPS production was found to be related to glucansucrase and fructansucrase extracellular activities. Depending on the strain, soluble and/or cell-associated glycansucrases were secreted. Structural characterization of the polymers by 1H and 13C NMR spectroscopy analysis further demonstrated a high diversity of EPS structures. Notably, we detected strains that synthesize glucans showing amazing variations in the amount of alpha-(1-->2), alpha-(1-->3) and alpha-(1-->6) linkages. The representation of Leuconostoc strains which produce putative alternan polymers and alpha-(1-->2) branched polymers was particularly high. The existence of glucan- and fructansucrase encoding genes was also confirmed by PCR detection. Sourdough was thus demonstrated to be a very attractive biotope for the isolation of lactic acid bacteria producing novel polymers which could find interesting applications such as texturing agent or prebiotics.

Biodiversity of lactic acid bacteria in French wheat sourdough as determined by molecular characterization using species-specific PCR.

The lactic acid microflora of nine traditional wheat sourdoughs from the Midi-Pyrénées area (South western France) was previously isolated and preliminary characterized using conventional morphological and biochemical analysis. However, such phenotypic methods alone are not always reliable and have a low taxonomic resolution for identification of lactic acid bacteria species. In the present study, a total of 290 LAB isolates were identified by PCR amplification using different sets of specific primers in order to provide a thorough characterization of the lactic flora from these traditional French sourdoughs. Overall, the LAB isolates belonged to 6 genera: Lactobacillus (39%, 8 species), Pediococcus (38%, 1 species), Leuconostoc (17%, 2 species), Weissella (4%, 2 species), Lactococcus (1%, 1 species) and Enterococcus (<1%, 1 species) and 15 different species were detected: L. plantarum, L. curvatus, L. paracasei, L. sanfranciscensis, L. pentosus, L. paraplantarum, L. sakei, L. brevis, P. pentosaceus, L. mesenteroides, L. citreum, W. cibaria, W. confusa, L. lactis and E. hirae. Facultative heterofermentative LAB represent more than 76% of the total isolates, the main species isolated herein correspond to L. plantarum and P. pentosaceus. Obligate heterofermentative lactobacilli (L. sanfranciscencis, L. brevis) represent less than 3% of the total isolates whereas Leuconostoc and Weissella species represent 21% of the total isolates and have been detected in eight of the nine samples. Detection of some LAB species was preferentially observed depending on the isolation culture medium. The number of different species within a sourdough varies from 3 to 7 and original associations of hetero- and homofermentative LAB species have been revealed. Results from this study clearly confirm the diversity encountered in the microbial community of traditional sourdough and highlight the importance of LAB cocci in the sourdough ecosystem, along with lactobacilli.

Characterization of dextran-producing Weissella strains isolated from sourdoughs and evidence of constitutive dextransucrase expression

The study of exopolysaccharide production by heterofermentative sourdough lactic acid bacteria has shown that Weissella strains isolated from sourdoughs produce linear dextrans containing α-(1→6) glucose residues with few α-(1→3) linkages from sucrose. In this study, several dextran-producing strains, Weissella cibaria and Weissella confusa, isolated from sourdough, were characterized according to carbohydrate fermentation, repetitive element-PCR fingerprinting using (GTG)(5) primers and glucansucrase activity (soluble or cell-associated). This study reports, for the first time, the characterization of dextransucrase from Weissella strains using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ polymer production (after incubation with sucrose) from enzymatic fractions harvested from both sucrose and glucose culture media. Results demonstrate that dextransucrase activity was mainly soluble and associated with a constitutive 180-kDa protein. In addition, microsequencing of the active dextransucrase from W. cibaria LBAE-K39 allowed the design of specific primers that could detect the presence of glucansucrase encoding genes similar to GTFKg3 of Lactobacillus fermentum Kg3 and to DSRWC of W. cibaria CMU. This study hence indicates that sourdough Weissella strains synthesize original dextransucrase.

Characterization of glucan-producing Leuconostoc strains isolated from sourdough.

Sourdough was previously demonstrated to be a fruitful biotope for isolation of lactic acid bacteria producing exopolysaccharides and more accurately diverse glycan polymers which have interesting applications as texturing agents or prebiotics. Characterization of polymers by (1)H and (13)C NMR spectroscopy analysis demonstrated that these strains could synthesize glucans of high structural variety and containing different amounts of α-(1→2), α-(1→3) and α-(1→6) linkages. In this study, fifteen glucan-producing Leuconostoc mesenteroides and L. citreum strains from sourdoughs were characterized according to carbohydrate fermentation, rep-PCR fingerprinting using (GTG)(5) primers and glycansucrase activity (soluble or cell-associated). Enzyme characterization using SDS-PAGE and in situ polymer production after incubation with sucrose correlated with synthesis of classical or α-(1→2) branched dextrans, alternan and levan. In addition, the presence of genes coding for alternansucrase was detected by PCR and partially characterized by sequence analysis. We thus provide new information on the biodiversity of glucan production by sourdough Leuconostoc strains.

Characterization of a novel dextransucrase from Weissella confusa isolated from sourdough

Weissella confusa and Weissella cibaria isolated from wheat sourdoughs produce, from sucrose, linear dextrans due to a single soluble dextransucrase. In this study, the first complete gene sequence encoding dextransucrase from a W. confusa strain (LBAE C39-2) along with the one from a W. cibaria strain (LBAE K39) were reported. Corresponding gene cloning was achieved using specific primers designed on the basis of the draft genome sequence of these species. Deduced amino acid sequence of W. confusa and W. cibaria dextransucrase revealed common structural features of the glycoside hydrolase family 70. Notably, the regions located in the vicinity of the catalytic triad (D, E, D) are highly conserved. However, comparison analysis also revealed that Weissella dextransucrases form a distinct phylogenetic group within glucansucrases of other lactic acid bacteria. We then cloned the W. confusa C39-2 dextransucrase gene and successfully expressed the mature corresponding enzyme in Escherichia coli. The purified recombinant enzyme rDSRC39-2 catalyzed dextran synthesis from sucrose with a Km of 8.6xa0mM and a Vmax of 20xa0μmol/mg/min. According to 1H and 13C NMR analysis, the polymer is a linear class 1 dextran with 97.2xa0% α-(1→6) linkages and 2.8xa0% α-(1→3) branch linkages, similar to the one produced by W. confusa C39-2 strain. The enzyme exhibited optimum catalytic activity for temperatures ranging from 35 to 40xa0°C and a pH of 5.4 in 20xa0mM sodium acetate buffer. This novel dextransucrase is responsible for production of dextran with predominant α-(1→6) linkages that could find applications as food hydrocolloids.

Genome Sequence of Weissella confusa LBAE C39-2, Isolated from a Wheat Sourdough

Weissella confusa is a rod-shaped heterofermentative lactic acid bacterium from the family of Leuconostocaceae. Here we report the draft genome sequence of the strain W. confusa LBAE C39-2 isolated from a traditional French wheat sourdough.

Genome Sequences of Three Leuconostoc citreum Strains, LBAE C10, LBAE C11, and LBAE E16, Isolated from Wheat Sourdoughs

Leuconostoc citreum is a key microorganism in fermented foods of plant origin. Here we report the draft genome sequence for three strains of Leuconostoc citreum, LBAE C10, LBAE C11, and LBAE E16, which have been isolated from traditional French wheat sourdoughs.

Overview of the glucansucrase equipment of Leuconostoc citreum LBAE-E16 and LBAE-C11, two strains isolated from sourdough

The whole set of putative glucansucrases from Leuconostoc citreum LBAE-E16 and LBAE-C11 was retrieved from the draft genome sequence of these two sourdough strains previously suggested as alternan producers. Four and five putative glycoside hydrolase family 70 (GH70) encoding genes were identified in the genome sequence of strain C11 and E16, respectively. Some putative genes have high sequence identity to known Leuconostoc dextransucrases. Molecular and biochemical data confirmed that L. citreum C11 could be considered as a new alternan-producing strain, unlike strain E16. In the latter, two new putative glucansucrases with unusual structural features were retrieved. In particular, the GSE16-5 gene encodes for a protein of 2063 amino acids with a theoretical molecular mass of 229 kDa that shares 61% identity with the alternansucrase (ASR) of L. citreum NRRL B-1355, due to the presence of seven APY repeats identified in the C-terminal peptide sequence. Cloning and expression of the corresponding coding sequence revealed synthesis of a low molecular weight (10(4) Da) linear dextran polymer with glucosyl residues only linked by α-1,6 linkages. This novel GH70 enzyme may thus be viewed as a natural chimeric enzyme resulting from the addition of the ASR C-terminal region in a dextransucrase.

Complete Genome Sequence of Leuconostoc citreum Strain NRRL B-742

ABSTRACT Leuconostoc citreum belongs to the group of lactic acid bacteria and plays an important role in fermented foods of plant origin. Here, we report the complete genome of the Leuconostoc citreum strain NRRL B-742, isolated in 1954 for its capacity to produce dextran.

Characterization of Glycansucrase Activities from Leuconostoc Mesenteroides LM17 and URE 13 Strains

ABSTRACT 14 original strains from traditional Bulgarian fermented food have been studied in order to characterize new orginal glycosyltransferase activities In order to characterize the whole spectrum of functional glycosyltransferases in the studied strains, the EPS produced in liquid media where first purified and hydrolysed and the global enzymatic activities measured by DNS assay. Activities present in the culural supernatant and cell associated were determined using SD_SPAGE and in situ Periodic Acid Schiffs staining after incubation with sucrose and raffinose. Among the studied strains Lm17 and URE 13 strains shown interesting activities. Three polymers bands were detected during in situ EPS production in presence of sucrose associated respectively to ∼300 kD (only in URE 13) and 180 kDa and 120 kDa (in both Lm 17 and URE13) proteins The 180KDa band could correspond to a glucansucrase activity of these strains. Analysis of zymogram incubated in raffinose as substrate and hydrolysis of the EPS produced in liquid culture are in good correlation with the hypothesis of a fructosyltransferase activity that could be attributed to a 120 kDa extracellular and cell-associated protein‥ GTF specific motifs (YG repeats from the Glucan Binding Domain) from Lm 17 and URE 13 strains were amplified by PCR and sequenced. They share a high percentage of identity to each other, and to the YG-repeats from ATCC 8293 and NRRL B-512F. A conserved motif from the catalytic core of FTF genes from Lm 17 and URE 13 strains was also shown to be very conserved between these two strains and with sequences of levansucrase from Ln. mesenteroides NRRL B-512F.