Catherine Fontagné-Faucher

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 glucansucrase and dextran from Weissella sp. TN610 with potential as safe food additives.

Pear-derived Weissella sp. TN610 produced extracellular glycosyltransferase activity responsible for the synthesis of soluble exopolysaccharide from sucrose. Acid and dextranase-catalyzed hydrolysis revealed that the synthesized polymer was a glucan. According to (1)H and (13)C NMR analysis, the glucan produced by TN610 was a linear dextran made of 96% α-(1→6) and 4% α-(1→3) linkages. Zymogram analysis confirmed the presence of a unique glucansucrase of approximately 180 kDa in the cell-free supernatant from TN610. The crude enzyme, optimally active at 37°C and pH 5, has promising potential for application as a food additive since it catalyzes dextran synthesis in sucrose-supplemented milk, allowing its solidification. A 4257-bp product corresponding to the mature glucansucrase gene was amplified by PCR from TN610. It encoded a polypeptide of 1418 residues having a calculated molecular mass of 156.089 kDa and exhibiting 96% and 95% identity with glucansucrases from Lactobacillus fermentum Kg3 and Weissella cibaria CMU, respectively.

The Carbohydrate Metabolism Signature of Lactococcus lactis Strain A12 Reveals Its Sourdough Ecosystem Origin

ABSTRACT Lactococcus lactis subsp. lactis strain A12 was isolated from sourdough. Combined genomic, transcriptomic, and phenotypic analyses were performed to understand its survival capacity in the complex sourdough ecosystem and its role in the microbial community. The genome sequence comparison of strain A12 with strain IL1403 (a derivative of an industrial dairy strain) revealed 78 strain-specific regions representing 23% of the total genome size. Most of the strain-specific genes were involved in carbohydrate metabolism and are potentially required for its persistence in sourdough. Phenotype microarray, growth tests, and analysis of glycoside hydrolase content showed that strain A12 fermented plant-derived carbohydrates, such as arabinose and α-galactosides. Strain A12 exhibited specific growth rates on raffinose that were as high as they were on glucose and was able to release sucrose and galactose outside the cell, providing soluble carbohydrates for sourdough microflora. Transcriptomic analysis identified genes specifically induced during growth on raffinose and arabinose and reveals an alternative pathway for raffinose assimilation to that used by other lactococci.

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.

Inventory of the GH70 enzymes encoded by Leuconostoc citreum NRRL B‐1299 – identification of three novel α‐transglucosylases

Leuconostoc citreum NRRL B‐1299 has long been known to produce α‐glucans containing both α‐(1→6) and α‐(1→2) linkages, which are synthesized by α‐transglucosylases of the GH70 family. We sequenced the genome of Leuconostoc citreum NRRL B‐1299 to identify the full inventory of GH70 enzymes in this strain. Three new genes (brsA, dsrM and dsrDP) putatively encoding GH70 enzymes were identified. The corresponding recombinant enzymes were characterized. Branching sucrase A (BRS‐A) grafts linear α‐(1→6) dextran with α‐(1→2)‐linked glucosyl units, and is probably involved in the α‐(1→2) branching of L. citreum NRRL B‐1299 dextran. This is the first report of a naturally occurring α‐(1→2) branching sucrase. DSR‐M and DSR‐DP are dextransucrases that are specific for α‐(1→6) linkage synthesis and mainly produce oligomers or short dextrans with molar masses between 580 and 27 000 g·mol−1. In addition, DSR‐DP contains sequences that diverge from the consensus sequences that are typically present in enzymes that synthesize linear dextran. Comparison of the genome with five other L. citreum genomes further revealed that dsrDP is unique to L. citreum NRRL B‐1299. The presence of this gene in a prophage represents the first evidence of phage‐mediated horizontal transfer of genes encoding such enzymes in lactic acid bacteria. Finally, brsA and dsrM are located in a chromosomal region in which genes encoding strain‐specific GH70 enzymes are consistently located. This region may be a good target on which to focus in order to rapidly evaluate the diversity of GH70 enzymes in L. citreum strains.

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.