Appl Microbiol Biotechnol (1999) 53: 6974

Springer-Verlag 1999

ORIGINAL PAPER

L. Micheli D. Uccelletti C. Palleschi V. Crescenzi

Isolation and characterisation of a ropy Lactobacillus strain producing the exopolysaccharide keran

Received: 6 April 1999 / Received revision: 30 July 1999 / Accepted: 13 August 1999

Abstract A capsular-polysaccharide-producing strain, LM-17, was isolated from ker grains and was identied as a slime-forming, rod-shaped Lactobacillus. According to 1H- and 13C-NMR spectral data, the exopolysaccharide produced by the isolated bacterial strain is identical to the glucogalactan extracted from ker grains and therefore known as keran. The keran produced was characterised by means of viscosity, optical rotatory power, circular dichroism and IR spectral measurements. A batch procedure was set up for the culture and extraction of the exopolysaccharide in laboratory conditions, resulting in a yield of 2 g/l puried keran from the culture supernatant of the LM-17 strain.

Introduction
Bacterial growth is often accompanied by production of exopolysaccharides (EPS), which have relevant ecological and physiological functions. There is increasing interest in the study of these molecules because of their potential applications in food and pharmaceutical productions. One interesting fermentation product in the dairy industry is ker, an ancient milk beverage produced in the Caucasian regions. Ker grains are gelatinous and irregular particles with a diameter of about 810 mm; they resemble cauliower orets and contain a complex microora mainly composed of yeast and lactobacilli. This mixed population causes an acid alcoholic fermentation of milk. At least 24% of the dry material consists
L. Micheli V. Crescenzi (8) Department of Chemistry, University of Rome ``La Sapienza'', Ple Aldo Moro 5, I-00185 Rome, Italy e-mail: crescenzi@axrma.uniromal.it Tel.: +390649913630 D. Uccelletti C. Palleschi Department of Developmental and Cell Biology, University of Rome ``La Sapienza'', Rome, Italy

of keran, the capsular polysaccharide of the Lactobacillus sp. present in the grains. This polysaccharide is regarded as safe since ker has long been consumed, and it has been recently reported to have antibacterial, antifungal and antitumoral activity (Shiomi et al. 1982; Cevikbas et al. 1994). In addition, an enhancing eect on the production of interferon b cortisol and noradrenaline in human cell lines has been reported and a possible use as a stress-reducing food component has been hypothesized (Kabayama et al. 1997). Keran is a water-soluble branched glucogalactan containing approximately equal amounts of D-glucose and D-galactose residues (Fig. 1) (La Riviere et al. 1967; Marshall and Cole 1984). This polymer is quite resistant to hydrolysis and forms gels in aqueous solutions containing ethanol (Mukai et al. 1991). Extraction of keran from ker grains on an industrial scale is dicult and expensive: a simpler procedure for mass production of this polymer is therefore of interest. Several eorts have been directed to the isolation of capsule-forming lactic acid bacteria in order to achieve the production of EPS in a pure culture. Various isolates have been reported and described as Lactobacillus ker (Kandler and Kunath 1983), Lactobacillus keranofaciens (Fujisawa et al. 1988), Lactobacillus sp. KPB167B (Yokoi et al. 1991), Lactobacillus kergranum and Lactobacillus paraker (Takizawa et al. 1994). This nonexhaustive listing indicates that the complex taxonomic relationships among the bacterial species of ker have not been completely explored. In addition, the inuence of the geographical origin of ker grains is also to be taken into account (Ottogalli et al. 1973; Angulo et al. 1993; Pintado et al. 1996). The chemical structure of keran extracted from ker grains and from isolated Lactobacillus species was elucidated by Kooiman (1968) and by Mukai et al. (1988, 1990a). The structure proposed is a branched hexa- or heptasaccharide repeating unit that is itself composed of a regular pentasaccharide unit to which one or two sugar residues are randomly linked. In this context we report the isolation of a Lactobacillus sp. able to produce keran as a capsular polysac-

70 Fig. 1 The chemical structure of keran, the water-soluble polysaccharide of the ker grains

charide, the molecular characterisation of the strain by the sequence of the 16S rDNA and the structural characterisation of the polysaccharide produced.

Materials and methods

Culturing of ker grains Ker grains utilised in this study came from a local diary of a central region of Italy. The grains were grown for 1 week at 28 C in skimmed milk without stirring. The medium was changed daily and the grains were washed with sterile water. Isolation of polysaccharide-producing bacteria The ker grains washed with sterile distilled water were homogenised with a Waring blender. Ropy strains were isolated by plating diluted homogenates of ker grains on MRSL medium (Yokoi and Watanabe 1992) with the following additions: 7 mM CaCl2, 0.04% MnSO4 4H2O and 0.07% MgSO4; the pH was adjusted to 6.5. The plates were incubated at 28 C for 56 days in an anaerobic atmosphere (Oxoid complete anaerobic jar assembly). The isolates retained their ropy character on the MRSL medium upon repeated transfers. DNA sequence analyses Cells of isolated ropy strains grown in MRSL medium in anaerobic conditions at 28 C were harvested after 6 days. Chromosomal DNA was isolated and puried according to Marmur (1961) with minor modications. Samples of this DNA (100 g) were then used for the polymerase chain reaction (PCR) (Expand High Fidelity PCR System, Boehringer Manneheim) to obtain the amplied 16S rDNA. To this end, the oligonucleotides fD1 (5-AGAGTTTGATCCTGGCTCAG-3) and r (P1 + P2) (5-ACGGT/CTACCTTGTTACGACTT-3) were employed according to Weisburg et al.

(1991). The DNA fragment amplied was cloned in the TA vector (Invitrogen) and sequenced on both strands according to the Sanger method. Retrieval of the sequences related to the 16S rDNA of the isolated ropy strain was achieved by using the Blast program of the GCG package (Devereux et al. 1984). The alignment of 22 16S rDNA species was generated with the program Clustalw (GCG package), using the default parameters. Evolutionary distances between all pairs of taxa were calculated with the DNAdist program of the Phylip package (Felsenstein 1981); the resulting distance matrix was then used to construct an unrooted tree with the program Neighbor-Joining (Phylip package). Extraction of polysaccharide from ker grains Before the extraction, the grains were washed with distilled water. Two methods were used: (a) extraction with hot water; (b) extraction with hot water after homogenisation (Yokoi and Watanabe 1992). a. Extraction in hot water A weighed amount of wet ker grains was treated in boiling water (1:100) for 1 h with stirring. The mixture was then cooled and centrifuged at 16 000 g for 15 min. The procedure was repeated once again with the remaining sediment. The polysaccharide dissolved in the combined supernatants was precipitated out by addition of an equal volume of cold ethanol and left at 4 C overnight. The precipitate was redissolved in hot water (1:100) for 1 h at 70 C with stirring and the precipitation procedure was repeated twice. The precipitate was nally dissolved in 100 ml distilled water, dialysed against distilled water until the conductivity reached 1.2 lS and lyophilised. b. Extraction with hot water after homogenisation A weighed amount of wet ker grains was washed with distilled water by sedimentation; the sediment was then homogenised with a Waring blender. The homogenised material was boiled in water

71 (1:100) for 1 h under stirring either with or without a centrifugation step at 16 000 g for 15 min. The following steps were the same as for the previous extraction procedure. Extraction of polysaccharide from the isolated strain The strain LM-17 was inoculated into 20 ml MRSL modied liquid medium and grown at 28 C for 10 days under a CO2 atmosphere. The culture was centrifuged at 16 000 g for 15 min and the polysaccharide was extracted from both supernatant and cells. The polysaccharide in the supernatant was precipitated by adding an equal amount of cold ethanol at 4 C overnight. The precipitate was collected by centrifugation at 16 000 g for 15 min and dissolved in distilled water, precipitated with ethanol three times, dialysed against water (until the conductivity reached 1.2 lS) and lyophilised. The polysaccharide from the cells was extracted with hot water for 1 h. The supernatant was obtained by centrifugation at 16 000 g for 15 min and precipitated by adding an equal volume of cold ethanol at 4 C overnight. The precipitate was collected by centrifugation and puried as previously described. The lyophilised material was dissolved in distilled water (0.3%), the pH was adjusted to 7.5 with 1 M NaOH and porcine pancrease (Sigma) was added (1% of the lyophilised weight). The enzymatic treatment was carried out at 34 C for 24 h with stirring (Wang 1996). The mixture was centrifuged at 16 000 g for 15 min. The supernatant was added with an equal volume of cold ethanol. The following purication steps were carried out as described above. Analytical procedures Preparation of the keran solutions Lyophilised samples, obtained under the conditions described above, were dissolved in distilled water or sodium chloride (0.1 M) as appropriate. Data were collected using EPS solutions at concentrations not exceeding 0.5% (w/v) since there are solubility problems at concentrations higher than 1%. Sonication This procedure was carried out by a Vibra Cell Sonicator (Sonic & Materials, model VC) at 20 MHz. Two cycles for 30 min each at 0 C and at 100 W were applied to EPS solutions (0.4% w/v in water) produced by ker grains. The products obtained were ltered (58 lm), dialysed against distilled water until conductivity reached 1.2 lS and lyophilised. Intrinsic viscosity The viscosity of the solutions was measured with a Schott-Geraete instrument, tted with a Ubbelohde capillary viscometer (0.53 mm) at 20 C (grel = 1.22). Circular dichroism The spectra were recorded with a Jasco J500-A instrument. Determinations were performed at room temperature. Polarimetry A Perkin-Elmer 241 polarimeter at k = 589 nm and at 20 C (0.01 C) with a DT1 thermostat (Het Lab Coratory Equipment) was used. NMR and IR spectroscopy The spectra were recorded with a Bruker Ac 2000 (300 MHz) NMR spectrometer and a Perkin Elmer series FTIR spectrometer. NMR determinations were made at 353 K for C and H, the polymer being dissolved in D2O to give 1% (w/v) solutions. Sequence data The sequence of the 16S rDNA from the isolated bacterial strain (LM-17) can be retrieved under the EMBL accession number AJ132757.
13 1

Results
Isolation and characterisation of the isolated ropy strain Ker grains were locally produced and employed for the extraction of the polysaccharide keran (see Materials and methods). The same grains were homogenised, diluted and plated to obtain single colonies on modied MRSL plates. Mucoid colonies were re-streaked several times to assess the homogeneity of the isolate and the consistency of the mucoid phenotype. The isolates consisted of gram-positive rod-shaped bacteria surrounded by a capsule, as revealed by the India ink staining (not shown). Cultures of a selected isolate (LM-17) strain were then employed for the extraction of the capsular exopolysaccharide (see next section). In order to achieve a better characterisation of the isolated strain we sought to obtain the nucleotide sequence of the corresponding 16S rRNA gene. To this end we extracted the total DNA from the LM-17 strain and ran a PCR amplication using the oligonucleotide primers fD1 and r (P1 + P2) (Weisburg et al. 1991). This primer pair is capable of amplifying the 16S rDNA of a wide variety of bacterial taxa. The amplied fragment was cloned and sequenced, resulting in a nearly complete 16S rDNA sequence of 1472 nucleotides. A comparative sequence analysis was performed and an unrooted phylogenetic tree was derived from the multiple alignment with the most similar 16S rDNA of several Lactobacillus spp. (see Fig. 2). The LM-17 isolate clearly resulted in a strain related to Lactobacillus casei, although the classication of the L. casei group is not yet stable and has been recently revised (Mori et al. 1997). The only other ker-related strain with available 16S rDNA sequence was Lactobacillus keri; the analysis programs with the default settings placed this strain quite apart from our isolate. Extraction of EPS The two procedures for extracting the EPS from the grains gave dierent results. The extraction in hot water (procedure a) consistently resulted in a better yield, typically around 50% of the initial wet weight of grains. The extraction procedure, including homogenisation of the grains, did not improve the yield and resulted in a

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Properties of the puried EPS The intrinsic viscosity, [g], was obtained by combining the Huggins and Kraemer extrapolations (Morris and Ross Murphy 1981). The measures were carried out on the EPS puried from the grains following various procedures (see Materials and methods) and from the supernatant of the LM-17 cultures. Maximum viscosity was reached by EPS extracted from integral grains in water ([g] = 5.68 0.5 dl/g). The viscosity of the polysaccharide produced from the LM-17 culture was instead reduced. This reduction was probably due to the presence of contaminating glycosidases in the commercial proteases employed in the purication procedure from the culture broth (our unpublished observations). Determinations of optical activity and rotational strength (ellipticity) were carried out with the 1% (w/v) sample solutions. The specic rotating power of the EPS extracted from grains, [a]D, was +64 at 20 C and that of the EPS produced by the isolated strain was +63. The values observed were very similar to published results (La Riviere et al. 1967; Mukai et al. 1990a,b). The dichroic spectra showed absence of ellipticity, which is the expected behaviour of a polymer without chromophore groups able to absorb in the range of k analysed. These measures are a good index for determining the structural identity between the two polymers extracted in this laboratory and the EPS classied in the literature as keran (La Riviere et al. 1967; Kooiman 1968). A qualitative investigation was carried out with IR spectroscopic analyses in order to investigate the purity and the structure of the samples. The absence of the protein absorption (also conrmed by CD spectra) indicated a good degree of purity. A characteristic ngerprint was observed at specic k for the puried EPS. The spectra of the polymer from grains (Fig. 3, curve a) and from LM-17 culture (Fig. 3, curve b) were perfectly superimposable. The primary structure of EPS was conrmed by means of its 13C- and 1H-NMR spectra (Yokoi et al. 1991). In the 1H-NMR spectra of EPS from ker grains (Fig. 4c) and from the culture supernatant (Fig. 4d), we observed a peak at 5.1 ppm for an anomeric b hydrogen,
mean viscosity is the mathematical mean of the Huggins and Kraemer values (Young and Lovell, 1991) u H KH Mean 5.45 5.68 0.43 2.69 2.95 4.06 4.28 3.12 0.6 0.5 0.1 0.4 0.6 0.6 0.6 0.3 NaCl 0.1 M H2O H2O NaCl 0.1 M H2O NaCl 0.1 M H2O H2O Solvent

Fig. 2 Phylogenetic analysis based on 16S rDNA data from 22 Lactobacillus species. The analysis was performed with the DNAdist and Neighbor-Joining programs of the Phylip package. The scale bar is proportional to the estimated nucleotide divergence

signicant reduction of the intrinsic viscosity of the recovered polysaccharide (see Table 1). This problem could be partially reversed by removing low-molecularmass fractions with an additional centrifugation step; however, this caused a further reduction of the nal yield. The EPS was also puried from the culture medium of the LM-17 strain. We consistently obtained a yield of 2 g/l when the strain was grown on the modied MRSL broth.
Table 1 Viscosity of exopolysaccharide (EPS) solutions; the polysaccharides were extracted from kers grains and from the LM-17 strain. Bidistilled water was used in the solvent in all cases. The Source of EPS Intrinsic viscosity (dl/g) Huggins Grains Integral Sonicate Homogenised Homogenised and centrifuged Isolated strain 5.38 5.64 0.43 2.66 2.95 3.99 4.24 3.08

Kraemer 5.51 5.72 0.43 2.71 2.94 4.14 4.33 3.14

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The very high similarity of the C- and H-NMR spectra demonstrated the identity of the primary structure between the sample extracted from ker grains and that from the supernatant of LM-17 culture.

13

Discussion
A bacterial strain isolated from ker grains and belonging to Lactobacillus sp. showed its ability to produce an EPS, which, according to structural studies, was identical to the one puried from ker grains. The ker grains utilised were produced in the central regions of Italy by a local dairy and this origin has to be taken into account, since dierences in the microora of grains have been reported, depending on the dierent sources of the grains (La Riviere et al. 1969; Ottogalli et al. 1973; Angulo et al. 1993) and on dierent techniques employed during processing. The bacterial strain studied was therefore characterised by determining the nucleotide sequence of the gene coding for the 16S rRNA; this approach is now regarded as a powerful technique for assessing the phylogenetic relationship among micro-organisms, as exemplied by the reorganisation of the L. casei group (Mori et al. 1997). This analysis placed the LM-17 strain in the L. casei group; this implies that it is not very related to L. keri, which could be ascribed to the dierent geographical origin of the starting ker grains. On the basis of the reported data, we propose that the strain isolated during this work is the producer of the

Fig. 3 IR spectra of keran from ker grains (a) and capsular polysaccharide from LM-17 strain (b)

at 4.82 ppm, 4.77 ppm, 4.52 ppm, 4.50 ppm and 4.45 ppm for several anomeric a hydrogens and at 4.65 ppm for an anomeric a hydrogen, assigned to a sugar on a lateral branch. Similar conclusions were drawn from 13C spectra (Fig. 4a EPS from grains; Fig. 4b EPS from culture supernatant), in particular from the absorption of the C-glycosidic bond.
Fig. 4ad Comparison of 13C- and 1H-NMR spectra of capsular polysaccharide from the LM-17 strain (a, c) and keran from ker grains (b, d) respectively

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EPS constituting the matrix of the ker grains studied. It is worth noticing that the spectroscopic data agree with those reported for a polysaccharide extracted from the Lactobacillus K1 strain (Mukai et al. 1990a; Yokoi et al. 1991). The results obtained conrm that the primary structure of the polymer studied consists of galactose and glucose in molar ratio of 1.1:0.9, as proposed by Mukai and co-workers for keran (1990b). This is therefore the rst report characterising the bacterial strain producing the exopolysaccharide keran at the molecular level with the nucleotide sequence of the gene encoding 16S rRNA. Another interesting result was that keran could be obtained by using a pure culture in the modied MRSL medium, obtaining a reproducible yield of 2 g/l from the culture supernatant. This productivity compared well with the yield of 1.97 g/l reported for the supernatant of Lactobacillus sp. KPB-167B (Yokoi and Watanabe 1992). It is also worth noticing that LM-17 strain was cultured in the MRLS-modied medium suggested by Yokoi and Watanabe (1992) under standard laboratory conditions and no attempts were made to ameliorate the process; we did not, for example, control the pH during the growth. The isolated strain could therefore represent a promising starting point for developing an industrial process for the production of keran.
Acknowledgements The authors thank Mr. Francesco Castelli for expert technical assistance. This work has been carried out with nancial support of the Italian Ministry for Universities and Research, MURST (``conanziato-1997'').

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