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1 Bundesanstalt für Fleischforschung, E.-C.-Baumannstr. 20, D-95326 Kulmbach, Germany
2 Bundesforschungsanstalt für Ernährung, Haid- und Neustr. 9, D-76131 Karlsruhe, Germany
3 VFG Labor GmbH & Co. KG, Nordfeldstr. 19, D-33775 Versmold, Germany
4 Lehrstuhl für Mikrobiologie, TU München, Am Hochanger 4, D-85350 Freising, Germany
Correspondence
L. Kröckel
Kr\|[ouml ]\|ckel. m-kroeckel{at}baff-kulmbach.de
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Published online ahead of print on 16 August 2002 as DOI 10.1099/ijs.0.02387-0.
The EMBL accession number for the 16S rRNA sequence of KU-3T is AJ496791.
A table showing species similarity based on substrate utilization is available as supplementary data in IJSEM Online (http://ijs.sgmjournals.org).
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TOPABSTRACTMAIN TEXTREFERENCES |
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). In sausage fermentations performed at 18–23 °C, the indigenous microflora is usually dominated by strains of L. sakei and L. curvatus (Hammes et al., 1990; Hugas et al., 1993; Lücke, 1998). Other LAB frequently isolated from fermented sausages include Lactobacillus alimentarius, Lactobacillus farciminis, Lactobacillus hilgardii and Carnobacterium spp. (Collins et al., 1993; Holzapfel, 1998; Lücke, 1998; Schillinger & Lücke, 1987). In this study we describe a new species, Lactobacillus versmoldensis sp. nov., which dominated the LAB flora of several German raw fermented sausages.
LAB strains were isolated from ROGOSA agar (Merck) during routine plate-count analyses of four German salami-type raw fermented sausages. Strains L470, L476 and KU-19 were isolated from sausages A, B and D, respectively, while KU-3T, KU-4 and KU-9 were from sausage C. Sausages A, B and D were products of a single factory, while sausage C was produced by another company. Reference strains included Lactobacillus kimchii DSM 13961T, Lactobacillus paralimentarius DSM 13238T, L. alimentarius DSM 20249T, L. farciminis DSM 20184T, Lactobacillus paracasei subsp. tolerans DSM 20258T, L. curvatus DSM 20010, Lactobacillus amylophilus DSM 20533T and L. sakei DSM 20017T and DSM 20494. All LAB strains used in this study were grown in MRS broth or on MRS agar (de Man et al., 1960) at 30 °C.
Differential plate counts and determination of sausage pH were performed using standard methods. The LAB were differentiated on the basis of morphological and metabolic traits (Schillinger & Lücke, 1987). D(-)- and L(+)-lactate were determined from culture supernatants after 4 days growth using an enzymic test kit (Roche Diagnostics). Maximum NaCl tolerance was determined 5 days after inoculation.
BOX-rep-APD (repetitive primer-amplified polymorphic DNA using an upstream primer complementary to the 3' half of the sense strand of the A subunit of the BOX element) fingerprinting was performed essentially as described by Selenska-Pobell et al. (1996). Total DNA from LAB, for use as a PCR template, was isolated as described by Cancilla et al. (1992). Species-specific amplicons were derived from comparisons between different LAB species and strains of individual species. For randomly amplified polymorphic DNA (RAPD) analysis, DNA was extracted as described by Björkroth & Korkeala (1996) and the M13 primer was used to generate the RAPD profiles (Andrighetto et al., 2001). BOX-rep-APD and RAPD patterns were analysed with Bionumerics software (Applied Maths). Similarity coefficients were calculated by using Pearson's product–moment correlation coefficient, and strains were grouped by using the unweighted pair group method with arithmetic averages (UPGMA).
16S rRNA-encoding DNA fragments were amplified in vitro and sequenced directly as described by Springer et al. (1992). The new 16S rRNA sequences were aligned with about 22 000 homologous full and partial sequences available in public databases (Ludwig, 1995), using the automated tools of the ARB software package (Ludwig & Strunk, 1996). Distance matrix, maximum-parsimony and maximum-likelihood methods were applied as implemented in the ARB software package. Different datasets were analysed, varying with respect to the sequences of outgroup reference organisms and the alignment positions selected according to their degrees of conservation.
For DNA–DNA hybridization experiments, DNA was isolated according to the guanidium thiocyanate method of Pitcher et al. (1989). The DNA G+C content (mol%) was determined from the thermal melting temperature of DNA using a Gilford Response spectrophotometer. DNA hybridization was determined spectrophotometrically from renaturation rates, according to the method of De Ley et al. (1970).
Genomic fingerprints of the unknown isolates produced by BOX-rep-APD, as well as by RAPD, showed a high degree of similarity: r=94·09 and 97·88, respectively (Fig. 1). They were clearly different from the profiles of other Lactobacillus species commonly found in raw sausages, including L. sakei and L. curvatus (data not shown), and they also did not show similarities to the profiles of the newly described species L. kimchii DSM 13961T or L. paralimentarius DSM 13238T (Fig. 1).
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; Ehrmann et al., 2003; Yoon et al., 2000), represents a subcluster among the Lactobacillaceae. However, strain KU-3T is a distant member of this group. The corresponding overall 16S rRNA sequence similarities for strain KU-3T and its closest relatives L. alimentarius DSM 20249T, L. farciminis DSM 20184T, L. kimchii DSM 13961T, L. mindensis DSM 14500T and L. paralimentarius DSM 13238T are 95·0, 95·0, 94·8, 95·6 and 94·9 %, respectively, whereas the latter organisms share 97·4 % or higher similarity with each other. The tree in Fig. 2 is based on the results of a maximum-parsimony analysis of more than 20 000 homologous sequences, at least 90 % complete with respect to the Escherichia coli standard, as stored in the ARB database (Ludwig & Strunk, 1996). The tree topology was evaluated and corrected according to the results of distance matrix and maximum-likelihood analyses. Only those alignment positions that shared identical nucleotides in at least 50 % of the members of the cluster containing strain KU-3T were included for tree reconstruction. Multifurcations indicated that a common relative branching order could not be resolved or was not supported by applying the alternative treeing methods.
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). On the basis of these results, we propose the novel species Lactobacillus versmoldensis sp. nov.
In agreement with the genotypic data, the physiological and biochemical test results clearly separated the unknown isolates from the other rod-shaped LAB. Their ability to grow on ROGOSA agar and the absence of gas production from glucose are typical reactions for the genus Lactobacillus and exclude Carnobacterium and Weissella, which also contain rod-shaped LAB, but are either non-aciduric or gas-producing (Collins et al., 1987, 1993; Kandler & Weiss, 1986; Schillinger & Holzapfel, 1995). Although the novel species is phylogenetically close to L. kimchii DSM 13961T, it can be easily differentiated from this species by its unique catabolic profile and growth physiology (Table 1). The DNA G+C content of 40·5 mol% is also higher than that of L. kimchii DSM 13961T and related species (35–38 mol%). The catabolic spectrum of the unknown sausage isolates was most similar to that of L. paracasei subsp. tolerans DSM 20258T (85 %) and least similar to that of L. kimchii DSM 13961T (45 %) (see supplementary data in IJSEM Online at http://ijs.sgmjournals.org). Similarities to L. curvatus DSM 20010, L. sakei DSM 20017T, L. alimentarius DSM 20249T, L. farciminis DSM 20184T and L. paralimentarius (DSM 13238T) were 79, 79, 61, 60 and 55 %, respectively. L. paracasei subsp. tolerans DSM 20258T can be readily differentiated from L. versmoldensis because it does not ferment melibiose or ribose. In contrast to typical representatives of L. curvatus and L. sakei, which in MRS broth either produce D(-)- and L(+)-lactic acid in approximately equal amounts or, as in the case of L. sakei, L(+)-lactate only (Kandler & Weiss, 1986), the new isolates produced 88–94 % L(+)-lactate. This, and the lack of growth at 4 °C, indicated that the new isolates could not belong to the L. curvatus/L. sakei cluster.
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) or in the presence of a reducing atmosphere consisting of 20–30 % CO2 and 20–80 % N2 (sausage D in Table 2). The sausages were 70 mm in diameter, and contained either beef and pork (sausages A, B and D) or poultry and pork (sausage C). This type of sausage is characterized by pH values in the range 4·5–4·8 and a relatively high water content of 40 %, corresponding to a water activity of 0·93 (Lücke, 1998). According to the manufacturers, the sausage ingredients included glucose, lactose and except for sausage C, also maltose, i.e. sugars that the new isolates were able to ferment. LAB counts were within the expected limits of 107–108 c.f.u. g-1. The new Lactobacillus species was discovered in this study because it dominated the LAB flora on ROGOSA agar and because of discrepancies between colony counts on plate count agar and ROGOSA agar, especially for sausages A and C (Table 3). This was surprising as the total mesophilic aerobic plate counts on non-selective nutrient agar from salami-type fermented sausages are usually about the same as on ROGOSA agar, which was indeed the case at the beginning of the storage period (not shown).
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Description of Lactobacillus versmoldensis sp. nov.
Lactobacillus versmoldensis (vers.mold.en'sis. N.L. masc. adj. versmoldensis pertaining to Versmold, the town in Germany where the strains were isolated).
Gram-positive, non-motile and non-spore-forming straight rods with rounded ends, 0·9x3·3 µm (0·9x1·6–6·0 µm) in size. Cells are found singly, in pairs and in small chains of generally four cells. Grows aerobically and anaerobically on ROGOSA and MRS agar, with better growth under anaerobic conditions. Grows better in MRS broth than on MRS agar. When transferred from MRS agar to MRS broth, a lag phase of up to 4 days may be observed. Cells aggregate during growth in MRS broth. Colonies on MRS agar after 3 days incubation at 30 °C are small (up to 1 mm in diameter), circular, convex with entire edges, greyish-white and catalase-negative. Growth occurs at 8–37 °C, but not at 4 or 42 °C. Homofermentative; no gas is produced from glucose. About 90 % of produced lactate is the L(+)-isomer. Ammonia is not produced from arginine. Maximum NaCl tolerance for growth in MRS broth is in the range 8–14 %. Galactose, lactose, maltose, glucose, melibiose and ribose are fermented. Arabinose, cellobiose, inulin, amygdalin, mannitol, melezitose, raffinose, rhamnose, sucrose, salicin, sorbitol, trehalose and xylose are not fermented.
The type strain, KU-3T (=DSM 14857T =NCCB 100034T =ATCC BAA-478T), was isolated from poultry salami. Reference strains are KU-4 and KU-9. The DNA G+C content of strain KU-3T is 40·5 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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