Lactobacillus versmoldensis sp. nov., isolated from raw fermented ...

International Journal of Systematic and Evolutionary Microbiology (2003), 53, 513–517

DOI 10.1099/ijs.0.02387-0

Lactobacillus versmoldensis sp. nov., isolated from raw fermented sausage

Note

L. Kro¨ckel,1 U. Schillinger,2 C. M. A. P. Franz,2 A. Bantleon3 and W. Ludwig4 1

Bundesanstalt fu¨r Fleischforschung, E.-C.-Baumannstr. 20, D-95326 Kulmbach, Germany

Correspondence L. Kro¨ckel

2

[email protected]

3

Bundesforschungsanstalt fu¨r Erna¨hrung, Haid- und Neustr. 9, D-76131 Karlsruhe, Germany VFG Labor GmbH & Co. KG, Nordfeldstr. 19, D-33775 Versmold, Germany

4

Lehrstuhl fu¨r Mikrobiologie, TU Mu¨nchen, Am Hochanger 4, D-85350 Freising, Germany

Lactobacillus versmoldensis sp. nov. (KU-3T) was isolated from raw fermented sausages. The new species was present in high numbers, and frequently dominated the lactic acid bacteria (LAB) populations of the products. 16S rDNA sequence data revealed that the isolates are closely related to the species Lactobacillus kimchii DSM 13961T, Lactobacillus paralimentarius DSM 13238T, Lactobacillus alimentarius DSM 20249T and Lactobacillus farciminis DSM 20184T. DNA–DNA reassociation data, however, clearly distinguished the new isolates from these species; they showed a low degree of DNA relatedness with the type strains of this group of phylogenetically closely related lactobacilli. These results warrant separate species status for strain KU-3T, for which the name Lactobacillus versmoldensis sp. nov. is proposed. The type strain is KU-3T (=DSM 14857T =NCCB 100034T =ATCC BAA-478T).

Lactic acid bacteria (LAB) play an important role in the ripening process of raw fermented sausages. In European raw fermented sausages, strains of Lactobacillus sakei, Lactobacillus curvatus, Lactobacillus plantarum and Lactobacillus pentosus are widely used as starter organisms (Jessen, 1995). 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; Lu¨cke, 1998). Other LAB frequently isolated from fermented sausages include Lactobacillus alimentarius, Lactobacillus farciminis, Lactobacillus hilgardii and Carnobacterium spp. (Collins et al., 1993; Holzapfel, 1998; Lu¨cke, 1998; Schillinger & Lu¨cke, 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 salamitype raw fermented sausages. Strains L470, L476 and KU-19 Published online ahead of print on 16 August 2002 as DOI 10.1099/ ijs.0.02387-0. Abbreviations: LAB, lactic acid bacteria; RAPD, randomly amplified polymorphic DNA; rep-APD, repetitive primer-amplified polymorphic DNA. 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).

02387 G 2003 IUMS

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 & Lu¨cke, 1987). D(2)- 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 39 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,

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DNA was extracted as described by Bjo¨rkroth & 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).

profiles of the newly described species L. kimchii DSM 13961T or L. paralimentarius DSM 13238T (Fig. 1).

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).

According to 16S rRNA-based phylogenetic analyses, strain KU-3T, together with L. alimentarius DSM 20249T, L. farciminis DSM 20184T, L. kimchii DSM 13961T, Lactobacillus mindensis DSM 14500T and L. paralimentarius DSM 13238T (Cai et al., 1999; 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 maximumlikelihood 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.

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

DNA–DNA hybridization confirmed the species identity of the unknown isolates. DNA similarity between KU-3T and the isolates KU-4 and KU-9 was 101 and 100 %, respectively (101 % was obtained due to experimental error), while it was 60, 37, 30, 27, 17 and 5 % with L. paralimentarius DSM 13238T, L. kimchii DSM 13961T, L. alimentarius DSM 20249T, L. farciminis DSM 20184T,

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 maximumlikelihood 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.

Fig. 1. Genomic fingerprints of L. versmoldensis sp. nov. strains and related LAB strains, generated with BOX-rep-APD and M13-RAPD. 514

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Lactobacillus versmoldensis sp. nov.

Table 1. Differential characteristics of L. versmoldensis sp. nov. (KU-3T) Strains: 1, L. versmoldensis sp. nov. KU-3T (this study); 2, L. kimchii DSM 13961T (Yoon et al., 2000); 3, L. paralimentarius DSM 13238T (Cai et al., 1999); 4, L. farciminis DSM 20184T (Reuter, 1983); 5, L. alimentarius DSM 20249T (Reuter, 1983). +, Positive; 2, negative. Characteristic

Fig. 2. 16S rRNA-based tree reflecting the phylogenetic relationships of L. versmoldensis sp. nov. (KU-3T), its closest relatives and selected reference organisms. Bar, 10 % sequence divergence.

L. amylophilus DSM 20533T and L. sakei DSM 20017T, respectively, and thus below the threshold value of 70 % suggested for delineation of a new species (Stackebrandt & Goebel, 1994). 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(2)- and L(+)-lactic acid in approximately equal amounts or, as in the case of L. sakei, L(+)-lactate only http://ijs.sgmjournals.org

Fermentation of: Aesculin Cellobiose Lactose Melezitose Melibiose Ribose Salicin Sucrose Trehalose Xylose Growth at:* 8 ˚C 42 ˚C DNA G+C content (mol%)

1

2

3

4

5

2 2 + 2 + + 2 2 2 2

+ + 2 + 2 + + + + +

+ + 2 2 2 + + + + 2

+ + + 2 2 2 + + + 2

+ + 2 2 2 + + + + 2

+ 2 40?5

+ + + +D + + + + 35 37?2–38 34–36 36–37

*This study. DUsually negative (Reuter, 1983).

(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. The new isolates were obtained from typical quick-ripened industrial salami-style meat products, which had been stored for up to 50 days at room temperature, either vacuum-packaged (sausages A, B and C in Table 2) 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 (Lu¨cke, 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. g21. 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,


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Table 2. Details of sausages studied Sausage

Date of analysis

A: Provence-type premium cervelat B: Provence-type premium salami C: Poultry salami D: Milano-type salami

Age*

17 August 1999 17 August 1999 9 November 1999 11 November 1999

50 50 22 48

pHD

4?5 4?6 4?7 4?8

LAB starterd

L. L. L. L.

plantarum plantarum curvatus plantarum

Bacterial count§ TVC

LAB

MCC

6?0 7?0 6?0 7?5

8?0 7?6 7?0 7?9

6?0 5?0 6?0 5?5

*Age, days of storage at room temperature before analysis. DpH, measured at the time of microbial analysis. dAs communicated by the sausage producers. §Given as log10 [c.f.u. (g sausage)21]; TVC, mesophilic aerobic count on plate count agar (Merck); LAB, lactic acid bacteria on ROGOSA agar (Merck); MCC, Micrococcaceae on KRANEP agar (Merck).

which was indeed the case at the beginning of the storage period (not shown). The unexpectedly high abundance of L. versmoldensis in the examined sausages may be due to the low competitiveness of other LAB, including starter cultures as well as nonstarter LAB, under the specific conditions of sausage storage in combination with a surplus of fermentable sugar(s). Factors common to all four sausages were that they contained lactose and were intended for retail at room temperature in vacuum or modified-atmosphere packages. This kind of retail was not practiced before the mid-1980s in Germany, where most of the big surveys on the microflora of fermented sausages had been completed. This may explain why L. versmoldensis was not detected earlier. Description of Lactobacillus versmoldensis sp. nov. Lactobacillus versmoldensis (vers.mold.en9sis. 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?963?3 mm (0?961?6–6?0 mm) 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, greyishwhite 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%.

Acknowledgements We thank Jutta Popp and Dirk Vogel for their excellent technical assistance.

References Andrighetto, C., Zampese, L. & Lombardi, A. (2001). RAPD-PCR

Table 3. LAB flora of the sausages Taxa: 1, L. plantarum; 2, L. sakei/L. curvatus; 3, L. versmoldensis sp. nov. Data are bacterial counts given in log10 [c.f.u. (g sausage)21]. Sausage A B C D

1

2

3

5?2 7?0 5?5 7?1

5?2 6?5 6?7 7?0

8?0 7?5 7?2 7?8

characterization of lactobacilli isolated from artisanal meat plants and traditional fermented sausages of Veneto region (Italy). Lett Appl Microbiol 33, 26–30. Bjo¨rkroth, J. & Korkeala, H. (1996). Evaluation of Lactobacillus sake contamination in vacuum-packaged sliced cooked meat products by ribotyping. J Food Prot 59, 398–401. Cai, Y., Okada, H., Mori, H., Benno, Y. & Nakase, T. (1999).

Lactobacillus paralimentarius sp. nov., isolated from sourdough. Int J Syst Bacteriol 49, 1451–1455. Cancilla, M. R., Powell, I. B., Hillier, A. J. & Davidson, B. E. (1992).

Rapid genomic fingerprinting of Lactococcus lactis strains by 516

Downloaded from www.microbiologyresearch.org by International Journal of Systematic and Evolutionary Microbiology 53 IP: 185.107.94.33 On: Tue, 10 Oct 2017 12:27:46

Lactobacillus versmoldensis sp. nov. arbitrarily primed polymerase chain reaction with 32P and fluorescent labels. Appl Environ Microbiol 58, 1772–1775. Collins, M. D., Farrow, J. A. E., Phillips, B. A., Ferusu, S. & Jones, D. (1987). Classification of Lactobacillus divergens, Lactobacillus pisci-

cola, and some catalase-negative, asporogenous, rod-shaped bacteria from poultry in a new genus, Carnobacterium. Int J Syst Bacteriol 37, 310–316. Collins, M. D., Samelis, J., Metaxopoulos, J. & Wallbanks, S. (1993).

Lu¨cke, F.-K. (1998). Fermented sausages. In Microbiology of Fermented Foods, Vol. 2, pp. 441–583. Edited by B. J. B. Wood. London: Blackie. Ludwig, W. (1995). Sequence databases. In Molecular Microbial

Ecology Manual, Chapter 3.3.5, pp. 1–22. Edited by A. D. L. Akkermans, J. D. van Elsas & F. J. De Bruijn. Dordrecht: Kluwer. Ludwig, W. & Strunk, O. (1996). ARB: a software environment for sequence data (http://www.mikro.biologie.tu-muenchen.de).

Taxonomic studies on some leuconostoc-like organisms from fermented sausages: description of a new genus Weissella for the Leuconostoc paramesenteroides group of species. J Appl Bacteriol 75, 595–603.

Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid

De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative

Lactobacillus farciminis sp. nov., nom. rev. Syst Appl Microbiol 4, 277–279.

measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142. de Man, J. C., Rogosa, M. & Sharpe, M. E. (1960). A medium for the

cultivation of lactobacilli. J Appl Bacteriol 23, 130–135. Ehrmann, M. A., Mu¨ller, M. R. A. & Vogel, R. F. (2003). Molecular

extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151–156. Reuter, G. (1983). Lactobacillus alimentarius sp. nov., nom. rev. and

Schillinger, U. & Holzapfel, W. H. N. (1995). The genus

Carnobacterium. In The Genera of Lactic Acid Bacteria, pp. 307–326. Edited by W. H. N. Holzapfel & B. J. B. Wood. London: Blackie.

analysis of sourdough reveals Lactobacillus mindensis sp. nov. Int J Syst Evol Microbiol 53, 7–13.

Schillinger, U. & Lu¨cke, F.-K. (1987). Identification of lactobacilli

Hammes, W. P., Bantleon, A. & Min, S. (1990). Lactic acid bacteria in

Selenska-Pobell, S., Evguenieva-Hackenberg, E., Radeva, G. & Squartini, A. (1996). Characterization of Rhizobium ‘hedysari’ by

meat fermentation. FEMS Microbiol Lett 87, 165–174. Holzapfel, W. H. N. (1998). The Gram-positive bacteria associated

with meat and meat products. In The Microbiology of Meat and Poultry, pp. 35–84. Edited by A. R. Davies & R. G. Board. London: Blackie.

from meat and meat products. Food Microbiol 4, 199–208.

RFLP analysis of PCR amplified rDNA and by genomic PCR fingerprinting. J Appl Bacteriol 80, 517–528.

Hugas, M., Garriga, M., Aymerich, T. & Monfort, J. M. (1993).

Springer, N., Ludwig, W., Drozanski, V., Amann, R. & Schleifer, K.-H. (1992). The phylogenetic status of Sarcobium lyticum, an obligate

Biochemical characterization of lactobacilli from dry fermented sausages. Int J Food Microbiol 18, 107–113.

intracellular bacterial parasite of small amoebae. FEMS Microbiol Lett 96, 199–202.

Jessen, B. (1995). Starter cultures for meat fermentation. In Fermented Meats, pp. 130–159. Edited by G. Campbell-Platt & P. E. Cook. London: Blackie.

Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place

Kandler, O. & Weiss, N. (1986). Regular, nonsporing Gram-positive

rods. In Bergey’s Manual of Systematic Bacteriology, Vol. 2, pp. 1208–1234. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.

http://ijs.sgmjournals.org

for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849. Yoon, J.-H., Kang, S.-S., Mheen, T.-I. & 7 other authors (2000).

Lactobacillus kimchii sp. nov., a new species from kimchi. Int J Syst Evol Microbiol 50, 1789–1795.


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