International Centre for Brewing & Distilling, Department of Biological Sciences, Heriot-Watt University, Edinburgh EH14 4AS, Scotland, UK1
Department of Biotechnology, Royal Institute of Technology,S-100 44 Stockholm, Sweden2
Author for correspondence: Fergus G. Priest. Tel: +44 131 451 3464. Fax: +44 131 451 3009. e-mail: f.g.priest{at}hw.ac.uk
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ABSTRACT |
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Keywords: 16S rRNA, lactobacilli, taxonomy, whisky, ribotyping, random-amplified polymorphic DNA
Abbreviations: RAPD, random-amplified polymorphic DNA
The GenBank accession number for the 16S rRNA sequence of strain R7-84 is AF071856.
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INTRODUCTION |
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The origins of these bacteria within the distillery are unclear. The lactic flora could become resident within the pipework and vessels of the plant and comprise a stable population of strains selected from the raw materials by the particular practices adopted in that distillery. In such circumstances, the flora would vary between different distilleries. Alternatively, the lactic acid bacteria could be flushed out by the cleaning and sterilization procedures and regularly reintroduced into the distillery with the raw materials during mashing. Since the malt is sourced from relatively few suppliers, in this case the flora would be expected to be similar between geographically distinct distilleries.
Several attempts have been made to identify the lactic acid bacteria in Scotch whisky fermentations using traditional schemes (MacKenzie & Kenny, 1965 ; Bryan-Jones, 1975
; Makanjuola & Springham, 1984
) and numerical taxonomy of phenotypic characters (Priest & Barbour, 1985
). These studies have been partially successful and, while many isolates could not be identified, some were equated with established species such as Lactobacillus brevis, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum and Lactobacillus plantarum (Bryan-Jones, 1975
; Priest & Barbour, 1985
). With the introduction of molecular systematics, in particular 16S rDNA sequencing and phylogenetic analysis of the data, the classification and identification of lactic acid bacteria has been revolutionized (Schleifer & Ludwig, 1995
). Moreover, molecular methods provide high discriminatory power for epidemiological typing of lactobacilli (Rodtong & Tannock, 1993
; Tynkkynen et al., 1999
). In this paper, we describe the use of these approaches to identify lactic acid bacteria isolated from fermentation samples from 23 malt whisky distilleries from various geographical locations in Scotland (Fig. 1
) and show that a distillery does develop its own stable population of lactobacilli.
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METHODS |
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Molecular methods.
Chromosomal DNA was prepared using a modification of the method described by Johansson et al. (1995) . Bacteria from 100 ml MRS broth grown for 16 h were harvested by centrifugation at 8000 g and washed in 10 ml TES buffer (50 mM NaCl, 100 mM Tris/HCl, 70 mM EDTA, pH 8·0) and recentrifuged. The pellet was resuspended in 3 ml TES supplemented with 25% sucrose and 50 mg lysozyme ml-1 (Sigma). Mutanolysin (Sigma) dissolved in 0·1 M potassium phosphate buffer (pH 8·0) was added to 140 units ml-1 and the suspension was incubated at 42 °C for 2 h to effect lysis. Thereafter the published method was followed and the DNA pellet was dissolved in 500 µl 0·1x SSC.
Ribotype patterns were prepared using 3 µg HindIII-digested DNA, electrophoresed in 1% agarose at a constant voltage of 50 V in Tris/acetate buffer. Southern blots were hybridized with a digoxigenin-labelled probe, derived from the amplified 16S rRNA gene of Bacillus sphaericus as described previously (Jahnz et al., 1996 ). For chromosomal DNADNA hybridization (Nielsen et al., 1995
), the probe DNA was labelled using random-priming with digoxigenin-labelled dUTP (Roche) and hybrids were detected with a chemiluminescent substrate (CSPD, Roche) which was fixed on Kodak X-Omat film. Developed films were scanned and quantified using the NIH image analysis programme available at http://scrc.cit.nih.gov/imaging/. The density of each spot was recorded and the percentage hybridization calculated relative to the homologous reaction. Renaturation was at 62 °C (non-stringent conditions) and 72 °C (stringent) for 16 h.
Random-amplified polymorphic DNA (RAPD)-PCR was based on purified DNA (about 500 ng) or boiled cell extract as template. For the latter, a single colony on MRS agar was transferred to a microfuge tube and suspended in 15 µl 1x PCR reaction buffer (Bioline). The cells were boiled for 10 min, transferred to ice for 10 min and centrifuged to remove cell debris. The supernatant (5 µl) was used as the template with Bioline Taq polymerase and the primer KS3, 5'-GGCATGACCT-3' (Du Plessis & Dicks, 1995 ). The amplification programme consisted of 1 cycle at 94 °C for 5 min; 4 cycles of 94 °C for 45 s, 30 °C for 2 min, 72 °C for 1 min; 45 cycles of 94 °C for 1 min, 36 °C for 1 min, 72 °C for 2 min; followed by 1 cycle of 72 °C for 10 min. The reaction was carried out in a Perkin Elmer GeneAmp PCR System 2400 thermocycler. PCR products were electrophoresed at 15 V for 14 h in horizontal 1·5% (w/v) agarose gels and stained in ethidium bromide.
In vitro amplification and sequence determination of the 16S rRNA gene.
Primers used for PCR and 16S rDNA sequencing were those described earlier (De Silva et al., 1998 ). Products were subjected to cycle sequencing comprising 25 cycles of denaturation at 96 °C for 15 s, annealing at 50 °C for 30 s and elongation at 60 °C for 60 s. The MegaBACE ET-dye-terminator kit (Amersham Pharmacia Biotech) was used for the generation of Sanger dideoxy fragments. Excess dye-terminators were removed by ethanol precipitation, the reactions were dried and 10 µl de-ionized formamide was added to each reaction. The microtitre plate was inserted at the cathode stage in a MegaBACE 96-capillary DNA sequencing unit. Injection was performed at 3 kV for 50 s and the sequencing products were subsequently separated by electrophoreses at 5 kV for 120 min.
Phylogenetic analysis.
The trace files were edited and merged into a 16S rRNA gene contig using the Sequencher assembly program (Gene Codes). The resulting primary structures were aligned manually by using the Genetic Data Environment (GDE) software (Smith, 1992 ). The alignment contained previously described 16S rRNA gene sequences from Lactobacillus species and relatives, as retrieved from GenBank and those which can be downloaded in the form of an alignment based on secondary structure models from The Ribosomal Database Project (RDP-II; Maidak et al., 1999
). Phylogenetic calculations were performed using algorithms implemented in PHYLIP version 3.573 (Felsenstein, 1993
).
Nucleotide accession numbers.
The GenBank accession numbers for the 16S rRNA sequences of the reference strains used for phylogenetic calculations are given in the phylogenetic tree (Fig. 4).
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RESULTS |
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The difficulties in distinguishing L. casei, L. paracasei and Lactobacillus zeae were resolved by sequencing the U1 to U2 region of the 16S rRNA genes in which discriminatory signature codons have been identified (Mori et al., 1997 ). This identified all the isolates as L. paracasei but with two variants; strains R1-20, R1-69, R14-42 and R16-76 matched perfectly with the signatures for L. paracasei proposed by Mori et al. (1997)
, while strains R1-1 and R1-44 contained a unique polymorphism (20% A, 80% G) in position 110 according to the numbering for L. casei (Mori et al., 1997
) but in other respects matched the L. paracasei signature.
Two strains, R7-84 representing ribotype 7 and R15-103 representing ribotype 15, revealed identical partial 16S rRNA sequences (Table 1) but showed a sequence similarity of only 97% to previously described species when searching the relevant databases. Therefore, the virtually complete 16S rRNA gene sequence of strain R7-84 was determined and used for deciphering evolutionary relationships to related organisms. Phylogenetic trees were constructed from a starting alignment comprising about 100 published and unpublished 16S rRNA gene sequences from members of Lactobacillus and relatives. Different subsets of the alignment for final phylogenetic calculations were generated by removing gaps, ambiguously aligned positions and by applying a nucleotide consistency filter of 50%, i.e. positions for which a certain nucleotide composition could not be observed in more than half of the sequences. All trees showed an overall agreement with regard to topology and a representative phylogenetic tree inferred by the neighbour-joining method from a distance matrix corrected for multiple nucleotide substitutions at single locations by the one-parameter model of Jukes & Cantor (1969)
is shown in Fig. 4
. The tree was computed from the resulting matrix that was corrected by only removing gapped positions. Bootstrap percentage values have been given at the nodes of the tree which were statistically supported to
80%. A close relationship was found between strain R7-84 and the obligately heterofermentative species of the genus Lactobacillus belonging to the L. buchneri group or cluster 2 (Collins et al., 1991
). In all trees strain R7-84 repeatedly formed a tightly held entity with L. kefiri ATCC 35411T, L. buchneri ATCC 4005T and L. hilgardii ATCC 8290T, with 16S rDNA similarities of 98·4%, 98·1% and 97·3%, respectively.
Lactobacillus strain R7-84 represents a new species
Since the 16S rDNA sequence similarity values were above the proposed limit of 97% for species definition suggested by Stackebrandt & Goebel (1994) , DNA hybridization experiments were used to evaluate the status of strain R7-84 and relatives. DNA from strain R7-84 was labelled with digoxigenin and hybridized under stringent (72 °C) and non-stringent (62 °C) conditions with DNA from related distillery isolates, as judged by RFLP patterns (Fig. 1
) and phylogenetically closely related type strains of the genus Lactobacillus. The results (Table 3
) revealed close genomic similarity between strains R7-84 and R7-9 (79% at 62 °C renaturation temperature and 78% at 72 °C) but DNA from strain R7-84 hybridized poorly with the reference strains of L. buchneri, L. hilgardii and L. kefiri. In addition, one of the two ribotype 15 strains (R15-103) shared 73% and 44% hybridization with strain R7-84 at non-stringent and stringent temperatures respectively but strain R15-101 was unrelated to the other three distillery isolates and is not considered further here. These results suggested that strains R7-9, R7-84 and R15-103 should be classified in a new species.
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DISCUSSION |
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The recovery of L. paracasei from fermentations has not been reported previously, although in the past isolates may have been confused with L. casei which has been identified using traditional phenotypic schemes as a distillery bacterium (Bryan-Jones, 1975 ; Makanjuola & Springham, 1984
). Indeed, much confusion surrounds L. casei and L. paracasei. A request for the name L. paracasei to be rejected as a synonym of L. casei (Dellaglio et al., 1991
) was rejected by the Judicial Commission (Wayne, 1994
). Subsequent detailed analysis of 16S rRNA sequences (Mori et al., 1997
), ribotyping and RAPD analyses (Tynkkynen et al., 1999
; Ward & Timmins, 1999
) revealed the distinctiveness of the two species. Our results support the signature sequences for L. paracasei determined by Mori et al. (1997)
with the exception that we found a polymorphism in some strains, which comprised a transition of guanine to adenine at position 110 in a minority of rrn operons.
Partial 16S rRNA sequence analysis is an accurate approach to identification of Lactobacillus species (Walter et al., 2000 ). Performing BLAST searches using between 300 and 900 nt sequence resulted in unambiguous identifications usually at the 99100% level although in some instances, particularly with L. fermentum strains, it was slightly lower (Table 1
). Further 16S rDNA sequence analysis and DNA reassociation studies should reveal whether this variation is due to species heterogeneity or to the existence of subspecies or even novel species.
Ribotyping has been recommended for subspecific discrimination of lactobacilli in several studies including strains of L. casei and relatives (Tynkkynen et al., 1999 ), Lactobacillus helveticus strains from cheese (Giraffa et al., 2000
), and various species isolated from the vagina (Zhong et al., 1998
). Ribotyping of distillery isolates had its deficiencies in this study, largely because the range of ribotype patterns within a species varied considerably. The 16 strains of L. fermentum were fairly evenly distributed among 11 ribotypes, which provided for powerful discrimination. L. fermentum appears to be a genomically diverse species as judged by both ribotyping (Zhong et al., 1998
) and RAPD analysis (Hayford et al., 1999
). The 11 strains of L. brevis isolated from distillery fermentations were assigned to just four ribotypes, consistent with the heterogeneity detected by RAPDs in strains isolated from wine (Sohier et al., 1999
). L. paracasei was the most conserved species among the distillery organisms, in that 14 strains from 12 geographically dispersed distilleries all belonged to ribotype 1. However, these ribotype 1 strains could be distinguished by RAPD (Fig. 3
), which is consistent with the extensive heterogeneity detected among L. paracasei strains from cheese revealed by RAPD analysis (Fitzsimons et al., 1999
). This suggests that there is no one perfect method for typing lactobacilli, and that different approaches will be most effective with different species, dependent on the genetic structure of that species. A combination of the two typing procedures was optimal and allowed us to demonstrate a wide diversity of lactobacilli in the 23 distilleries which were investigated in this study.
It was important to establish if our sampling of the various distilleries represented a stable microbial flora or a transient population. Extended sampling at Glenkinchie distillery showed that strains of L. brevis, L. fermentum, L. paracasei and an unidentified bacterium were generally present (Table 2). There was marked stability of these strains, as judged by ribotyping, and when particular types were not detected this may have reflected low relative numbers rather than a complete absence. A new bacterium was probably introduced with a change of malt (L. fermentum ribotype 22) but subsequently lost when the malt was changed back to the original cultivar. Similarly, the annual closure period accompanied by extensive cleaning and maintenance removed some types but most had returned by the final visit. Thus the distillery has a stable, resident flora that may be influenced by raw materials.
Strains of ribotype patterns 7 and 15 could not be identified as a validly described Lactobacillus species. The RFLPs were very similar (Fig. 2) suggesting closely related organisms. A virtually complete 16S rRNA sequence of strain R7-84 placed the organism in the L. buchneri cluster (Collins et al., 1991
; Schleifer & Ludwig, 1995
). L. buchneri and other members of this group including L. hilgardii (Sohier et al., 1999
), L. kefiri (Kandler & Kunath, 1983
) and the more distantly related L. lindneri (Banks, 1998
) are associated with alcoholic fermentations. This is consistent with the growth of these strains in relatively high alcohol concentrations (Scotch whisky fermentations contain up to about 8%, v/v, ethanol) indicating a possible idiosyncratic phenotypic feature of the L. buchneri subclade. Strains R7-84 and R7-9 had the same ribotype pattern and shared almost 80% DNA hybridization at both stringent and non-stringent hybridization temperatures (Table 3)
. On the other hand, strain R15-103 is more difficult to classify. DNA hybridization at the optimal reassociation temperature with DNA from strain R7-84 (73%) indicated that it is likely to be a member of the same species as the ribotype 7 strains. Moreover, it shared identical DNA sequence over the 440 bases of the U2 to U5 region of the 16S rRNA gene. However, the low hybridization value with strain R7-84 at stringent temperature (44%) suggests substantial mismatching of hybrids and is inconsistent with it being included in the same species (Stackebrandt & Goebel, 1994
). On balance, we suggest that it be included in the same taxon as strain R7-84 until evidence to the contrary is presented. Strain R15-101 is unusual since it has the same ribotype as strain R15-103 but appears to belong to a different species by DNA hybridization. Further hybridizations using different probe DNAs are needed to determine the taxonomic position of this strain.
The three strains were phenotypically similar to other members of the L. buchneri group. All were heterofermentative, produced ammonia from arginine and DL-lactic acid from glucose, features likely to have been harboured by their latest common ancestor. However, the distillery isolates could be distinguished from the other members of the phylogenetic group by a selection of sugar fermentation tests (Table 4). We therefore recommend that strains R7-9, R7-84 and R15-103 should be included in a new species for which we propose the name Lactobacillus ferintoshensis after the estate in the north of Scotland that during the 18th century gave its name to one of the first brands of Scotch whisky, Ferintosh.
Description of Lactobacillus ferintoshensis sp. nov.
Lactobacillus ferintoshensis (fe.rin.to.shen'sis. M.L. adj. ferintoshensis from Ferintosh, a Scottish estate famous for its whisky).
Cells are rod-shaped and occur singly or in pairs or in short chains, 34 µm in length and about 1 µm wide. After 48 h incubation on modified MRS agar (MRS agar supplemented with vitamins), colonies are 25 mm in diameter, circular, shiny and creamy white in colour. Cells stain Gram-positive and are non-spore forming and non-motile. Catalase-negative. Grows at 15 °C but not 45 °C. Heterofermentative, producing DL-lactic acid from glucose. Acid is produced from N-acetylglucosamine, L-arabinose, galactose, glucose, fructose, maltose, melibiose, melezitose, ribose, salicin, sucrose, trehalose, D-xylose and in some cases from cellobiose, mannitol, mannose, -methyl D-glucoside,
-methyl D-mannoside, raffinose, sorbitol, sorbose, tagatose and turanose. Acid is not produced from adonitol, D-arabinose, D- and L-arabitol, dulcitol, erythritol, D- and L-fucose, gentiobiose, glycerol, glycogen, gluconate, inositol, inulin, 2- and 5-ketogluconate, lactose, rhamnose, L-xylose and xylitol. Aesculin but not starch is hydrolysed. Some strains hydrolyse arbutin. The type strain is positive for the variable reactions given above with the exception of acid from melezitose, melibiose, raffinose, tagatose and turanose for which it is negative. Habitat: malt whisky fermentations. The type strain is strain R7-84T which has been deposited with the Collection de LInstitut Pasteur as CIP 106749T.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 August 2000;
revised 5 January 2001;
accepted 10 January 2001.