Tempoyak) in Malaysia

International Journal of Systematic and Evolutionary Microbiology (2002), 52, 927–931 DOI: 10.1099/ijs.0.02091-0 Lactobacillus durianis sp. nov., isolated from an acid-fermented condiment (tempoyak) in Malaysia 1 Department of Veterinary J. J. Leisner,1 M. Vancanneyt,2 K. Lefebvre,2 K. Vandemeulebroecke,2 Microbiology, Royal 2 1 3 2 Veterinary and B. Hoste, N. Euras Vilalta, G. Rusul and J. Swings Agricultural University, Stigbøjlen 4, 1870 Frederiksberg C., Author for correspondence: Jørgen Leisner. Tel: j45 3528 2768. Fax: j45 3528 2757. Copenhagen, Denmark e-mail: jjl!kvl.dk 2 BCCM/LMG Bacteria Collection, Laboratory of Microbiology, University Lactic acid bacteria (LAB) are the predominant micro-organisms in tempoyak, a of Ghent, K. L. Malaysian acid-fermented condiment. In a study on the diversity of LAB in this Ledeganckstraat 35, product, three isolates could not be identified using SDS-PAGE of whole-cell B-9000 Ghent, Belgium proteins or API 50 CH. The taxonomic position of the three isolates was 3 Department of Food clarified in the present study. 16S rDNA sequencing classified a representative Science, Universiti Putra Malaysia, 43400 Serdang, strain in the genus Lactobacillus, clearly separated from all known species, and Selangor DE, Malaysia most closely related to the Lactobacillus reuteri phylogenetic group. DNA–DNA hybridization experiments and an extensive phenotypic description confirm that the strains represent a single and separate novel species among the obligately heterofermentative lactobacilli. The three isolates are distinguished at the intra-species level by plasmid profiling, pulsed-field gel electrophoresis of macro-restriction fragments and biochemical features. The name Lactobacillus durianis sp. nov. is proposed for the novel taxon and the type strain is LMG 19193T (l CCUG 45405T). Keywords: lactic acid bacteria, identification, tempoyak, durian fruit, Malaysia INTRODUCTION 16S rDNA sequence analysis. Genomic DNA of strain LMG 19193T was extracted by using the DNA isolation protocol During a study on the biodiversity of lactic acid of Niemann et al. (1997). A fragment of the 16S rDNA bacteria (LAB) from a traditional Malaysian acid- (corresponding to positions 8–1541 in the Escherichia coli fermented condiment, tempoyak, three similar strains numbering system) was amplified by PCR using the con- served primers pA (5 -AGAGTTTGATCCTGGCTCAG- were isolated that could not be identified to the species h 3h) and pH (5h-AAGGAGGTGATCCAGCCGCA-3h). The level when applying SDS-PAGE of whole-cell proteins PCR product was purified using a Qiaquick PCR purifi- or API 50 CH strips (Leisner et al., 2001). In a cation kit (Qiagen), according to the manufacturer’s instruc- polyphasic approach, the three strains were studied tions. Sequencing was performed using an Applied Bio- extensively and it was found that they constitute a systems 377 DNA Sequencer and the protocols of the novel species that is described in the present paper. manufacturer using the ABI Prism Dye Terminator Cycle Sequencing ready reaction kit. The eight sequencing primers METHODS used are listed in Coenye et al. (1999). Sequence assembly was performed by using the program (Ap- Bacterial strains. Strains LMG 19193T and LMG 19196 were plied Biosystems). Phylogenetic analysis was performed isolated from a single batch of tempoyak and strain LMG using the software package (Applied Maths) 19195 from a second batch, both obtained from the same after including the consensus sequence in an alignment of producer at a night market in Serdang, Selangor DE, small ribosomal subunit sequences collected from the in- Malaysia. The isolation procedure of the isolates has been ternational nucleotide sequence library EMBL. This align- described previously (Leisner et al., 2001). All three strains ment was calculated pairwise using an open gap penalty of were deposited in the BCCM\LMG Bacteria Collection 100% and a unit gap penalty of 0%. A similarity matrix was (http:\\www\belspo.be\bccm\lmg.htm). created by homology calculation with a gap penalty of 0%; ................................................................................................................................................. unknown bases were discarded. The resulting tree was Abbreviation: LAB, lactic acid bacteria. constructed using the neighbour-joining method. The EMBL accession number for the 16S rDNA sequence of strain LMG DNA base compositions. High-molecular-mass native DNA T 19193T is AJ315640. was prepared from strains LMG 19193 and LMG 19196. 02091 # 2002 IUMS Printed in Great Britain 927 J. J. Leisner and others Cells were cultivated in MRS broth (Oxoid) for 24 h at nisin (produced by Lactococcus lactis ATCC 11454), diver- 28 mC. DNA was extracted from 0n75–1n25 g wet weight of gicin A (produced by Carnobacterium divergens NCFB cells using the protocol described by Pitcher et al. (1989) with 2855), enterocin P (produced by Enterococcus faecium LMG the following modifications: the washed cell pellet was 19827 and LMG 19828), enterocins L50A and L50B resuspended and lysed in a buffer (10 mM Tris\HCl, (produced by Enterococcus faecium LMG 19828) and an " 100 mM EDTA, pH 8 0) containing RNase (200 µgml− ; unidentified enterocin (produced by Enterococcus faecium n " Sigma), proteinase K (500 µgml− ; Merck) and lysozyme ATCC 19434T). −" (50 mg ml ; SERVA) for 1 h at 37 mC before the addition of Pulsed field gel electrophoresis (PFGE). Lysis of cells, DNA GES reagent. DNA was enzymically degraded into nucleo- restriction with ApaI and electrophoresis were performed as sides as described by Mesbah et al. (1989). The nucleoside described by Ojeniyi et al. (1991, 1996). Similarities between mixture obtained was then separated by HPLC using a PFGE patterns were calculated by use of Jaccard’s coef- Waters SymmetryShield C8 column thermostatted at 37 mC. ficient (Sj). The solvent was 0n02 M NH%H#PO% (pH 4n0) with 1n5% Plasmid profiles. Mini-scale extraction of plasmids was done acetonitrile. Non-methylated lambda phage DNA (Sigma) as described by Klaenhammer (1984) except that samples was used as the calibration reference. were not heated at 62 mC for 1 h after addition of lysis buffer DNA–DNA hybridization experiments. High-molecular-mass and that the phenol used for the extraction was not saturated native DNA was prepared as described above for deter- with NaCl. Agarose gel electrophoresis was done on a 0n8% mination of the DNA base composition. DNA–DNA agarose gel (A-9539; Sigma) in TAE buffer (40 mM Tris hybridizations were performed with photobiotin-labelled acetate, 1 mM EDTA) at 100 V for 2n5 h. Plasmid sizes were probes in microplate wells as described by Ezaki et al. (1989), estimated by using Escherichia coli strains V517 and 39R361, using a HTS7000 Bio Assay Reader (Perkin Elmer) for the which contain plasmids of known sizes (Macrina et al., 1978; fluorescence measurements. Biotinylated DNA was hybrid- Rochelle et al., 1985; Threlfall et al., 1986). ized with single-stranded unlabelled DNA, non-covalently bound to microplate wells. Hybridizations were performed RESULTS AND DISCUSSION at 40 mC in hybridization mixture (2i SSC, 5i Denhardt’s T solution, 2 5% dextran sulfate, 50% formamide, 100 µg Three analogous strains of LAB, LMG 19193 ,LMG n " denatured salmon sperm DNA ml− , 1250 ng biotinylated 19195 and LMG 19196, isolated from tempoyak " probe DNA ml− ). (Leisner et al., 2001), could not be identified by whole- Biochemical and physiological tests. Cell morphology and cell protein fingerprinting and their patterns were motility were examined using phase-contrast microscopy on distinct from those of all species of LAB available in cultures grown in All-Purpose-Tween broth (APT; Difco " the database (data not shown). A polyphasic study was Laboratories, 1984) with glucose (10 g l− ) or glucose plus performed in order to verify whether these strains −" -xylose (both 10 g l ) as substrates. Under the same con- represent a novel species. ditions, growth was tested at different temperatures (7, 10, T 15, 21, 25, 30, 37, 42 and 45 mC). Growth at different salinities One representative strain, LMG 19193 , was selected (APT agar containing 6n5 or 8% NaCl) was tested at 30 mC. for 16S rDNA sequence analysis and represented a The strains were tested for production of gas from glucose separate branch in the genus Lactobacillus, linked " " (10 g l− ) or gluconic acid (10 g l− ) in APT broth without most closely to the Lactobacillus reuteri phylogenetic added citric acid. Production of ammonia from arginine was " group (Fig. 1; Schleifer & Ludwig, 1995). Highest investigated in a medium containing tryptose (5 g l− ), yeast " " extract (5 g l− ), potassium phosphate (2 g l− ), glucose (0n5 −" −" gl ) and arginine (3 g l ) with an initial pH of 7n0. Dissimilarity (%) Reduction of tetrazolium was examined on SBM agar 04 (Wilkinson & Jones, 1977). The ability to acidify La-broth to a pH value below 4n15 was investigated at 25 and 30 mC (Shaw & Harding, 1984). Growth on acetate agar (Oxoid) L. durianis LMG 19193T (AJ315640) T was tested under microaerophilic conditions at 30 mC. 62 99 L. fermentum ATCC 14931 (M58819) Nitrate reduction was tested at 30 mC in Nutrient broth L. mucosae CCUG 43179T (AF126738) −" (Oxoid) supplemented with KNO (1 g l ) and -xylose 93 L. reuteri DSM 20016T (X76328) " $ − L. vaginalis NCTC 12197T (X61136) (10 g l ). The Voges–Proskauer test was performed on Clark 99 & Lubbs medium (Barrow & Feltham, 1993) at 25 and 44 L. pontis LTH 2587T (X76329) 100 T 30 mC. The strains were tested for production of acids from 46 L. frumenti DSM 13145 (AJ250074) T carbohydrates and related compounds in API 50 CH strips 51 96 L. oris DSM 4864 (X94229) using API CHL medium according to the instructions of the 85 L. panis DSM 6035T (X94230) manufacturer (bioMe! rieux). The strips were incubated at 100 L. buchneri DSM 20057T (M58811) 30 mC and read after 1, 3, 4 and 10 days. Production and L. casei NCDO 161T (X61135) concentration of lactic acid isomers and acetic acid were L.

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