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1 BCCM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
2 Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing (IMDO), Department of Applied Biological Sciences, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium
3 Laboratory of Dairy Research, Agricultural University of Athens (AUA), GR-11855 Athens, Greece
4 Laboratory of Microbiology, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
Correspondence
M. Vancanneyt
Marc.Vancanneyt{at}UGent.be
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ABSTRACT |
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Published online ahead of print on 4 October 2004 as DOI 10.1099/ijs.0.63274-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains LMG 22198T, LMG 22199 and LMG 22200T are AJ632157, AJ422039 and AJ632158, respectively.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Vogel et al., 1999). Sourdough fermentations improve dough properties, bread texture and flavour, retard the staling process and protect bread from mould and bacterial spoilage (Corsetti et al., 1998; Hammes & Gänzle, 1998; Rosenquist & Hansen, 1998). Because of their artisanal and region-dependent handling, sourdoughs are a huge source of diverse LAB species and strains.
Lactobacillus brevis is one of the common taxa found in several sourdoughs such as artisanal Greek wheat sourdoughs (De Vuyst et al., 2002) and Belgian sourdoughs (our ongoing study). In the latter two surveys, most strains identified as L. brevis showed the characteristic taxonomic properties of the species. Three isolates were aberrant in their phenotypic and genotypic properties and were assigned as L. brevis-like. In the present study, their taxonomic position was investigated.
The three strains studied were isolated on different occasions from three different artisanal wheat sourdoughs. One strain, ACA-DC 3411 t1 (=LMG 22199), was isolated in 1997 from Greek sourdough (De Vuyst et al., 2002). The strain was purified after suspension (1 : 10, w/v) and serial dilutions in saline (0·9 % NaCl, w/v), plating on MRS agar supplemented with 2 % (w/v) maltose and incubation at 30 °C for 48 h. Two further strains, LMG 22198T and LMG 22200T, were isolated in 2003 from Belgian sourdoughs. They were purified after suspension (1 : 10, w/v) and serial dilution in peptone/water (0·1 %, w/v), plating on MRS agar (pH 5·4) supplemented with 1 % (w/v) maltose and fructose and 0·1 % (w/v) cycloheximide and incubation at 37 °C for 48 h. Bacteriological purity of all isolates was checked by plating and examining living and Gram-stained cells. Cultivation conditions for further experiments and for maintenance of the sourdough isolates and reference strains listed in Table 1 were MRS agar (pH 5·4) and incubation at 30 °C for 24–48 h, unless indicated otherwise.
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. Densitometric analysis, normalization and interpolation of protein profiles, and a numerical analysis were performed using the GELCOMPAR software package, versions 3.1 and 4.0, respectively (Applied Maths). After comparison with an in-house database, comprising profiles of nearly all recognized LAB species, the three isolates were identified as related to the species L. brevis. Among isolates of L. brevis, strain-specific differences were observed in the varying position of dominant protein bands in the molecular mass range 30–65 kDa and may indicate the presence of an S-layer on the outside of the bacterial cell (Boot et al., 1996). The latter dense bands largely influenced the numerical analysis; after omitting this variable region from the cluster analysis, the three new isolates LMG 22198T, LMG 22199 and LMG 22200T occupied a distinct subgroup in a dendrogram, as shown in Fig. 1. Twelve recognized L. brevis strains, including the type strain LMG 6906T, constituted a homogeneous and separate cluster. Lactobacillus spicheri LMG 21871T, the closest phylogenetic neighbour of L. brevis (Meroth et al., 2004), occupied a unique and distinct position. Among the three new sourdough isolates, a close relationship was observed between LMG 22198T and LMG 22199, whereas strain LMG 22200T showed a more distant relationship.
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. 16S rRNA gene amplification, purification and sequencing were performed as described by Vancanneyt et al. (2004) with the following modifications. PCR-amplified 16S rRNA gene sequences were purified using a NucleoFast 96 PCR clean-up kit (Macherey-Nagel). Sequencing reactions were performed using a BigDye Terminator cycle sequencing kit (Applied Biosystems) and purified using a Montage SEQ96 sequencing reaction clean-up kit (Millipore). Electrophoresis of sequence reaction products was performed using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Sequence assembly was performed using the program AUTOASSEMBLER (Applied Biosystems). The 16S rRNA gene sequences (continuous stretches of 1518 bp) and sequences of strains retrieved from EMBL were aligned and a phylogenetic tree was constructed by the neighbour-joining method using the BIONUMERICS software package, version 3.50 (Applied Maths). Unknown bases were discarded for the analyses. Bootstrapping analysis was undertaken to test the statistical reliability of the topology of the neighbour-joining tree using 500 bootstrap resamplings of the data (Fig. 2). Comparison of the newly determined complete sequences revealed sequence similarities higher than 99·7 %. Comparison with deposited sequences available in the EMBL database classified the strains in the Lactobacillus buchneri group (Schleifer & Ludwig, 1995) with nearest neighbours L. spicheri and L. brevis (sequence similarities of 98·2–98·0 % and 97·6–97·5 %, respectively).
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). AFLP fingerprinting was performed as described by Thompson et al. (2001) with the following modifications: EcoRI/TaqI was used as the restriction enzyme combination and primer combination E01/T01 (both having an adenosine extension at the 3'-end) was applied for selective PCR. The resulting electrophoretic patterns were analysed with the GELCOMPAR software package, version 4.2 (Applied Maths) using a Dice coefficient and the UPGMA linkage cluster analysis. Fig. 3 shows a dendrogram and confirmed the grouping of strains obtained after protein analysis (Fig. 1). Twelve recognized L. brevis strains grouped in a single separate cluster. The type strain of L. spicheri had a unique position and the three L. brevis-like isolates also grouped together. In this last cluster, the Greek isolate LMG 22199 and one Belgian isolate LMG 22198T showed highest sequence similarity, linked to the second Belgian isolate LMG 22200T.
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, using a combination of glass beads and enzymes, but with the following modifications. Volumes were increased tenfold for application on large scale. Vortexing with beads of the SDS-treated cells was done for 30 s. After addition of 16·5 ml buffer (10 mM Tris/HCl, 100 mM EDTA, pH 8·0) and 5 ml 5 M NaCl and gentle shaking, the suspension was incubated at 65 °C for 10 min. Subsequent chloroform/isoamyl alcohol extraction, precipitation, spooling of DNA on a glass rod, washing with ethanol and RNase treatment was performed as described by Marmur (1961). For determination of the DNA G+C content, DNA was enzymically degraded into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture was then separated by HPLC using a Waters SymmetryShield C8 column maintained at 37 °C. The solvent was 0·02 M (NH4)H2PO4 (pH 4·0) with 1·5 % acetonitrile. Non-methylated 
-phage DNA (Sigma) was used as the calibration reference. DNA G+C contents of strains LMG 22198T, LMG 22199 and LMG 22200T were 54, 53 and 51 mol%, respectively. These values were close to the value of 55 mol% determined for the type strain of L. spicheri, and significantly higher than the value of 46 mol% for the type strain of L. brevis. The values of the latter two reference species confirmed those described in the literature (Meroth et al., 2004).
DNA–DNA hybridizations were performed between the L. brevis-like strains LMG 22198T, LMG 22199 and LMG 22200T and the type strains of L. brevis and L. spicheri (DNA was prepared as described above). The microplate method was used as described by Ezaki et al. (1989) and Goris et al. (1998), using an HTS7000 Bio Assay Reader (Perkin Elmer) for fluorescence measurements. Biotinylated DNA was hybridized with unlabelled ssDNA, which was bound non-covalently to microplate wells. Hybridizations were performed at 44 °C in hybridization mixture (2x SSC, 5x Denhardt solution, 2·5 % dextran sulphate, 50 % formamide, 100 µg denatured salmon sperm DNA ml–1, 1·25 µg biotinylated probe DNA ml–1). The three sourdough isolates had hybridization levels of 15–18 % with the type strain of L. spicheri and 6–8 % with the type strain of L. brevis. The hybridization level between LMG 22198T and LMG 22199 was 75 %; this indicates that the two strains comprise a single species. LMG 22199 and LMG 22200T had a hybridization value of only 47 %, indicating separate species status for the latter strain.
Growth characteristics and colony morphology were investigated on MRS agar (pH 5·4) after 24 h of incubation at 37 °C under aerobic conditions and are given below in the species description.
Conventional biochemical tests were performed on the three L. brevis-like strains and the type strains of L. brevis and L. spicheri. Growth characteristics were determined in MRS broth (pH 5·4; Oxoid). Except for L. brevis LMG 6906T, all strains grew at 15 °C and in the presence of 5 % NaCl, but not at 45 °C. Four strains, excluding LMG 22200T, grew in 6 % NaCl, and only LMG 22198T and LMG 22199 grew in the presence of 7 % NaCl. All strains were facultatively anaerobic and produced gas from 2 % glucose and 2 % gluconate in MRS broth (pH 5·4, without addition of triammonium citrate). Gas production from glucose for L. spicheri was not confirmed by Meroth et al. (2004). Arginine hydrolysis was tested in a medium containing 0·5 % tryptone, 0·5 % yeast extract, 0·3 % arginine, 0·05 % glucose and 0·2 % K2HPO4 (pH 7·0), with phenol red as indicator; a positive reaction was recorded for all five strains tested. Metabolites from glucose were lactate, acetate and ethanol, as determined by HPLC (Waters). The proportions of D- and L-lactate were determined enzymically (R-Biopharm) and the isomers were in a ratio of 3 : 7 for the L. brevis-like strains LMG 22198T, LMG 22199 and LMG 22200T, 1 : 1 for L. spicheri LMG 21871T and 1 : 4 for L. brevis LMG 6906T.
Carbohydrate fermentation tests were carried out using API 50 CHL galleries following the manufacturer's instructions (bioMérieux). Strains were cultivated at 37 °C for 24 h under aerobic conditions on MRS agar (pH 5·4). Table 2 shows that only a single characteristic, acid formation from D-arabitol, allowed differentiation between the three L. brevis-like strains and recognized L. brevis strains and L. spicheri. All other features for all taxa are consistently positive or negative or are strain-dependent. None of these tests distinguished LMG 22200T from LMG 22198T and LMG 22199.
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Description of Lactobacillus acidifarinae sp. nov.
Lactobacillus acidifarinae (a.ci.di.fa'ri.nae. L. adj. acidus sour; L. n. farina flour; N.L. gen. n. acidifarinae pertaining to sour flour).
Cells are rod-shaped, occur singly or in pairs and in chains, 2–20 µm in length and 1·0 µm wide, Gram-positive, catalase-negative, non-spore-forming and non-motile. After 24 h, colonies are beige in colour, circular with a rough and wrinkled surface and approximately 1 mm in diameter. Growth occurs at 15 °C but not at 45 °C. Growth occurs at 5 % NaCl. Facultatively anaerobic and produces DL-lactic acid heterofermentatively with acetic acid and ethanol as other metabolites from glucose. Gas is produced from glucose and gluconate. Arginine is deaminated. Acid is produced from L-arabinose, D-arabitol, galactose, gluconate, N-acetylglucosamine, D-glucose, D-fructose, maltose, mannitol, ribose and D-xylose. Negative for acid production from D-arabinose, L-arabitol, adonitol, amygdalin, arbutin, cellobiose, dulcitol, aesculin, erythritol, D-fucose, L-fucose, 
-gentiobiose, 2- and 5-ketogluconate, methyl 
-D-glucoside, glycerol, glycogen, inositol, inulin, lactose, D-lyxose, D-mannose, methyl -D-mannoside, melezitose, melibiose, D-raffinose, rhamnose, sucrose, salicin, starch, sorbitol, L-sorbose, D-tagatose, trehalose, D-turanose, xylitol, L-xylose and methyl -xyloside. The DNA G+C content is 51 mol%.
The type strain, LMG 22200T (=R-19065T=CCM 7240T), was isolated from a Belgian artisanal wheat sourdough.
Description of Lactobacillus zymae sp. nov.
Lactobacillus zymae (zy'mae. N.L. n. zyma from Gr. n. zume leaven, sourdough; N.L. gen. n. zymae of sourdough).
Cells are rod-shaped, occur singly or in pairs and in chains, 2–20 µm in length and 1·0 µm wide, Gram-positive, catalase-negative, non-spore-forming and non-motile. After 24 h, colonies are beige in colour, circular with a rough and wrinkled surface and approximately 1 mm in diameter. Growth occurs at 15 °C but not at 45 °C. Growth occurs at 7 % NaCl. Facultatively anaerobic and produces DL-lactic acid heterofermentatively with acetic acid and ethanol as other metabolites from glucose. Gas is produced from glucose and gluconate. Arginine is deaminated. Both strains produce acid from L-arabinose, D-arabitol, gluconate, N-acetylglucosamine, D-glucose, D-fructose, maltose and ribose. Negative for acid production from D-arabinose, L-arabitol, adonitol, amygdalin, arbutin, cellobiose, dulcitol, aesculin, erythritol, D-fucose, L-fucose, -gentiobiose, 2- and 5-ketogluconate, methyl -D-glucoside, glycerol, glycogen, inositol, inulin, D-lyxose, D-mannose, methyl -D-mannoside, melezitose, D-raffinose, rhamnose, sucrose, salicin, starch, sorbitol, L-sorbose, D-tagatose, trehalose, D-turanose, xylitol, L-xylose and methyl -xyloside. Results are strain-dependent for acid production from galactose, lactose, mannitol, melibiose and D-xylose. The DNA G+C content is 53–54 mol%.
The type strain, LMG 22198T (=R-18615T=CCM 7241T), was isolated from a Belgian artisanal wheat sourdough.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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