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Some taxonomical characteristics of encapsulated Lactobacillus sp. KPB-167B isolated from kefir grains and characterization of its extracellular polysaccharide
International Journal of Food Microbiology, 13 (1991) 257-264 © 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50
257
FOOD 00415
Some taxonomical characteristics of encapsulated Lactobacillus sp. KPB-167B isolated from kefir grains and characterization of its extracellular polysaccharide Haruhiko Yokoi 1, Takashi Watanabe 1 Yoshitsugu Fujii ~, Takao Mukai 2,,, Takahiro Toba 2 and Susumu Adachi 2,** 1 Hiratsuka Research Laboratory, Sumitomo Heavy Industries, Ltd., Yuhigaoka, Hiratsuka-shi, Kanagawa, Japan, and 2 Laboratory o f Animal Products Technology, Faculty of Agriculture, Tohoku Unit~ersity, Tsutsumidori-Amamiyamachi 1-1, Aoba-ku, Sendai-shi 981, Japan (Received 29 October 1990; accepted 29 March 1991)
A capsular polysaccharide-producing strain KPB-167B isolated from kefir grains was identified as a homofermentative Lactobacillus. The carbohydrate fermentation pattern and DNA base composition of the strain were different from those of other capsular Lactobacillus species previously isolated from kefir grains. The polysaccharide produced by Lactobacillus sp. KPB-167B was found similar to kefiran by 13C-NMR and methylation analysis. Lactobacillus sp. KPB-167B could grow and produce capsular polysaccharide in MRSL medium with better yield than L. kefiranofaciens, which suggested that it is suitable for kefiran production. Key words: Lactobacillus sp.; Kefir grains; Kefiran
Introduction
Kefir grains are used for making kefir, and consist of lactic acid bacteria and yeasts agglutinated with a water-soluble polysaccharide named kefiran (La Rivi6re et al., 1967). The chemical structure of kefiran was elucidated by Kooiman (1968) and Mukai et al. (1988), and its antitumor activity was reported by Shiomi et al. (1982) and Murofushi et al. (1983). The isolation of encapsulated bacteria from the kefir grains has been reported (Rosi and Rossi, 1978; Ohara, 1980; Ohta et al., 1984; Toba et al., 1986). Only Correspondence address: H. Yokoi, Hiratsuka Research Laboratory, Sumitomo Heavy Industries, Ltd., Yuhigaoka, Hiratsuka-shi, Kanagawa 254, Japan. * Present address: The School of Veterinary Med!:ine and Animal Sciences, Kitasato University, Sanbongi-Maeyachi 149-2, Towada-shi 034, Japan. ** Present address: Shokei Women's Junior College, Yurigaoka 4-10-1, Natori-shi 981-12, Japan.
258 T o b a et al. (1986) identified the encapsulated bacteria and revealed that homofermentative Lactobacillus kefiranofaciens produced kefiran as capsular material (Fujisawa et al., 1988; Mukai et al., 1990). Recently Yokoi et al. (1990) also isolated five encapsulated bacteria, KPB-167A, KPB-167B, KPB-167C, KPB-167D and KPB-167E on a newly developed whey medium. In this paper, some taxonomical characteristics of strain KPB-167B are examined, and the chemical structure of the extracellular polysaccharide is analysed to confirm that the isolate is a kefiran-producer.
Materials and Methods
Bacterial strain The encapsulated bacterial strain KPB-167B isolated from kefir grains from Chr. Hansen's Laboratory (Copenhagen, D e n m a r k ) was used (Yokoi et al., 1990).
Identification Cell shape, cell size, Gram-stain, India-ink-stain and physiological characteristics were observed for cells grown in a M R S L medium consisting of 1% trypton (Difco, Detroit, MI), 0.5% yeast extract (Difco), 1% meat extract (Extract Ehlrich, Wako Pure Chemical Industries Ltd., Osaka, Japan), 2% lactose monohydrate, 0.2% K 2 H P O 4, 0.2% triammonium citrate, 0.1% Tween 80, 0.5% sodium acetate, 0.028% MnSO 4 • 4 H 2 0 and 0.058% MgSO 4 • 7 H 2 0 , p H 6.5, in screw-capped tubes. Bacteria were cultivated for 5 days at 30 ° C after removal of air by passing carbon dioxide into the tubes for 30 s. For acid production from carbohydrates, M R S L medium without meat extract and lactose was used as a basal medium. The filter-sterilized carbon sources were added to the sterile basal medium to give a final concentration of 1%, except for esculine, whose final concentration was 0.25%. The final p H was measured after incubation for 10 days at 30 o C. Gas production resulting from glucose utilization was detected by the formation of bubbles in D u r h a m tubes. The configuration of lactic acid was determined enzymatically by methods described previously (Yokoi et al., 1990).
DNA base composition Cells of KPB-167B grown in M R S L medium were harvested in the late logarithmic phase, and washed twice with 0.15 M NaCI in 0.1 M ethylenediamine tetraaeetic acid, p H 8.0. D N A was isolated and purified according to a modified method of Marmur (1961). The purified D N A was hydrolysed into nucleosides using procedures described by T a m a o k a and Komagata (1984). The resulting deoxyribonucleosides were measured according to Kaneko et al. (1986) using a high-performance liquid chromatograph (HPLC) fitted with a reversed-phase column (4.6 m m i.d.x 150 mm) of Cosmosil 5C18 (Nakarai Tesque Co., Ltd., Kyoto, Japan). An equimolar mixture of four deoxyribonucleosides prepared from a
259 commercial standard mixture of four deoxyribonucleotides for analysis of G + C content (Yamasa Shoyu Co., Ltd) was used as a standard, using above-mentioned method.
Preparation of extracellular polysaccharide Extracellular polysaccharide in the culture supernatant was used for analysis, because its sugar composition is similar to capsular polysaccharides, and because of its higher yields (Yokoi et al., 1990). KPB-167B was cultivated in a MRSL medium for 5 days at 30 ° C. Polysaccharide in the supernatant was obtained by ethanol precipitation as described previously (Yokoi et al., 1990) and purified according to the following method. To degrade contaminating nucleic acids, 0.5% polysaccharide solution containing 5 mg/1 of nuclease PI (Yamasa Shoyu Co., Ltd.) and 0.1 mM ZnC1 z, pH 5.3, was incubated at 5 0 ° C for 2 h. Then remaining proteins were removed by the method of Sevag (1934). After dialysis against distilled water the polysaccharide was obtained as lyophilizate. The resulting polysaccharide was named KPB-P. Kefiran was extracted from kefir grains and purified as described previously (Mukai et al., 1988).
Analysis of monosaccharide composition KPB-P was hydrolysed with 2 N trifluoroacetic acid (TFA) at 100 ° C for 3 h. Monosaccharide composition was determined by H P L C analysis described previously (Yokoi et al., 1990).
Determination of molecular mass The molecular mass of KPB-P was determined by gel filtration chromatography. The chromatography was carried out with a Hitachi 655A-11 HPLC equipped with Asahipak GS-710 column (7.6 mm i.d. ×500 ram, Asahi Chemical Industry Co., Ltd., Kawasaki, Japan) at room temperature using 0.1 M NaCI as a solvent at a flow rate of 1.0 ml/min. Pullulan (Shodex S T A N D A R D P-82, Showa Denko K.K.) was used as a standard of molecular mass.
13C-nuclear magnetic resonance ( t3C-NMR) analysis 13C-NMR spectra of KPB-P and kefiran were obtained in D 2 0 at 70 ° C with a J E O L FX-400 spectrometer (Japan Electron Optical Laboratory Co., Akishima, Japan) operating at 100 MHz, and chemical shifts were recorded in ppm downfield to 3-(trimethylsilyl)-propanesulfonic acid sodium salt.
Methylation analysis KPB-P and kefiran were methylated according to the method of Kvernheim (1987). Extraction and hydrolysis of the permethylated polysaccharide, and reduction and acetylation were carried out according to Harris et al. (1984). The partially methylated alditol acetates were determined using a gas chromatographmass spectrometer as described previously (Mukai et al., 1988).
I
KPB- 167B
39
S-10-LR e
D(L)
D(L)
D(L)
D(L)
D(L)
D(L)
D(L)
+
-
±
.
45 ° C
+
+
+
+
-
-
-
-
-
biose
tose
+
Cello-
Mal-
+
+
+
+
+
tose
Lac-
Acid formation from
-
+
+
-
-
15 o C
G r o w t h at
-
.
-
-
-
-
.
lose
Treha-
.
.
.
inose
. -
-
-
+
+
+
.
biose
Meli-
. .
lose
Xy-
.
. nd
-
+
-
+
+
nose
Raffi-
.
nose
Rham-
Acid formation from Arab-
Ri-
nd
-
.
.
zitose
Mele-
-
nd
. .
bose
nd
nd
.
nd f
trin
Dex-
.
+
+
+
+
+
cose
Glu-
-
-
.
. .
nitol
Man-
-
.
nd
+
±
+
r No data included.
~ Data from Ohara
(1980).
d D a t a f r o m R o s i a n d R o s s i (1978),
c D a t a f r o m O h t a e t al. (1984).
h D a t a f r o m F u j i s a w a et al. (1988).
.
nose
Man-
-
-
.
.
bitol
Sor-
homofermentative
a B a s e d o n t h e c o n t e n t o f t . - ( + ) - l a c t i c a c i d as f o l l o w s : D , 0 t o 2 0 % ; D ( L ) , 20 to 4 0 % ; D L , 40 to 6 0 % ; L, 80 to 1 0 0 % .
39
34-35
F3M, F6M ~
A3-1, A3-3 c
MA2-1, MA2-2,
WT-6A, WT-7
WT-2B, WT-8,
L. kerifiranofaciens b
40
39
S-10-LR c
Lactobacillus sp.
39
34-35
D(L)
Lactic acid a
(mol%)
40
Isomer
of
G + C
F3M, F6M J
MA2-1, MA2-2, A3-1, A3-3 c
WT-6A, WT-7
WT-2B, WT-8,
L. kefiranofaciens b
KPB-167B
Lactobacillus sp.
Bacterial strain
content
o f p h e n o t y p i c c h a r a c t e r i s t i c s o f Lactobacillus sp. K B P - 1 6 7 B w i t h t h o s e o f o t h e r e n c a p s u l a t e d
from kefir grains
Comparison
TABLE
.
nd
nd
+
+
+
nd
nd
.
lin
.
Escu-
tose
Fruc-
.
+
+
+
+
+
nd
-
licin
Sal-
tose
Galac-
nd
-
dalin
Amyg-
+
+
+
+
+
crose
Su-
Lactobacillus s p e c i e s i s o l a t e d
261
Results and Discussion
Characteristics of the encapsulated strain KPB-167B was identified as homofermentative Lactobacillus with a G + C content of D N A of 40%. As shown in Table I, KPB-167B fermented glucose, mannose, fructose, galactose, sucrose, maltose and lactose. Arabinose, xylose, rhamnose, ribose, cellobiose, trehalose, melibiose, raffinose, melezitose, mannitol, sorbitol, esculin, salicin and amygdalin were not fermented. Sugar utilization of KPB-167B was not identical to those of any other homofermentative Lactobacillus species listed in Bergey's Manual of Systematic Bacteriology (Kandler and Weiss, 1986). The optimum growth temperature of KPB-167B was 30 ° C and the bacterium did not grow at 15 and 45 ° C. The optimum initial pH of a MRSL medium was around 5.5. Although it grew under an atmosphere of air or N e, growth was enhanced under an atmosphere of CO 2. The essential nutrient in a MRSL medium for KPB-167B was yeast extract, and the lack of trypton or meat extract allowed about 85% of the growth of control (data not included).
Structural analysis of extracellular polysaccharide from KPB-167B Gel filtration chromatography of KPB-P showed a single peak. The molecular mass was determined to be about 1.6 x 10 6 (Fig. 1). In elementary analysis, we did not detect nitrogen (unpublished results). Monosaccharides obtained by hydrolysis of KPB-P were composed of glucose and galactose. The molar ratio of glucose and galactose was 1.0:0.98 by H P L C analysis. Fig. 2 shows 13C-NMR spectra of KPB-P and kefiran from kefir grains. KPB-P was assumed to be kefiran because of the high similarity of the spectra. To verify this assumption, chemical structure of KPB-P was further investigated by methylation analysis. In Table III the partially methylated alditol acetates obtained from permethylated KPB-P and kefiran are compared. The O-methylsugars of KPB-P were identical to those of kefiran and their molar ratios were
Retention
time
(rain)
I
I
I
107 Molecular
106
105
I
104.
mass
Fig. 1. Gel filtration chromatogram of capsular polysaccharide from Lactobacillus sp. KPB-167B on a Asahipak GS-710 column (7.6 m m i.d. × 5 0 0 ram).
262
(A)
(B)
I
120
I
110
I
100
I
I
I
I
90
80
70
60
Chemical s h i f t
(ppm)
Fig. 2. Comparisonof 13C-NMR spectra of capsular polysaccharidefrom Lactobacillus sp. KPB-167B and kefiran from kefir grains. A. Polysaceharidefrom strain KPB-167B, B. Kefiran.
T A B L E II Methylation analysis of capsular polysaccharide from Lactobacillus sp. KPB-167B (KPB-P) and kefiran extracted from kefir grains Identified
Mode of linkage
O-methylsugar 2,3,4,6-tetra-O-methylglucose 2,3,6-tri-O-methylgalactose 2,3,6-tri-O-methylglucose 2,4,6-tri-O-methylgalactose 2,3,4-tri-O-methylglucose 2,3,4-tri-O-methylgalactose 3,4-di-O-methylgalactose
GIc-(1 ~ -4)-Gal-(l ~ -4)-Glc-(l ~ -3)-Gal-(1 ---* -6)-Glc-(1 --* -6)-Gal-(1 ~ -2,6)-Gal-( --*
Molar ratio a KPB-P
Kefiran
1.00 1.14 1.36 1.32 1.24 0.24 1.10
1.00 1.36 1.29 1.31 1.11 0.19 1.10
a Calculated from peak area of gas chromatograph-mass spectrogram.
263
almost identical. These results strongly suggest that KPB-P is kefiran and Lacto-
bacillus sp. KPB-167B is a kefiran-producing bacterium. Mukai et al. (1988, 1990) reported that kefiran extracted from kefir grains and capsular polysaccharide from L. kefiranofaciens K 1 contained 6-O-substituted galactose in the structure proposed by Kooiman (1968). In the structure of polysaccharide produced by Lactobacillus sp. KPB-167B, the presence also of 6-O-substituted galactose was confirmed by detecting 2,3,4-tri-O-methylgalactose in identified O-methylsugars. The phenotypic characteristics of other encapsulated homofermentative Lactobacillus species isolated from kefir grains are shown in Table I. Among these strains, only L. kefiranofaciens has been confirmed to be a kefiran-producer by structural analysis of capsular polysaccharide. Lactobacillus KPB-167B did not ferment either melibiose or raffinose, while Lactobacillus kefiranofaciens MA2-1, MA2-2, A3-1 and A3-3 ferment both sugars, F3M and F6M ferment only melibiose, and S-10-LR ferments only raffinose. The G + C content of DNA of Lactobacillus sp. KPB-167B are 5-6% higher than that of L. kefiranofaciens, but are nearly equal to those of F3M, F6M and S-10-LR. Although there are small differences in the G + C content and sugar utilization, Lactobacillus sp. KPB-167B could still belong to species L. kefiranofaciens because it produces kefiran. More detailed taxonomical investigations such as DNA homology experiments between these bacteria are necessary to verify this assumption. A procedure for mass production of kefiran is desired. However, microbial production has not been established except for the complicated procedure of kefiran extraction from kefir grains. As reported previously (Yokoi et al., 1990), Lactobacillus sp. KPB-167B produced kefiran in a higher yield in milk whey medium (300-400 rag/l) than L. kefiranofaciens. Although L. kefiranofaciens is able to grow and produce kefiran only in milk whey medium, it was found that Lactobacillus sp. KPB-167B grew and produced kefiran also in MRSL medium. These findings indicate that Lactobacillus sp. KPB-167B is suitable for the mass production of kefiran.
References Fujisawa, T., Adachi, S., Toba, T., Arihara, K. and Mitsuoka, T. (1988) Lactobacillus kefiranofaciens sp. nov. isolated from kefir grains. Int. J. Syst. Bacteriol. 38, 12-14. Harris, P.J., Henry, R.J., Blakeney, A.B. and Stone, B.A. (1984) An improved procedure for the methylation analysis of oligosaccharides and polysaccharides. Carbohydr. Res. 127, 59-73. Kandler, O. and Weiss, N. (1986) Genus Lactobacillus Beijerinck 1901, 212 AL. In: P.H.A. Sneath, N.S. Mair, M.E. Sharpe and J.G. Holt (Eds.), Bergey's Manual of Systematic Bacteriology, Vol. 2, Williams & Wilkins, Baltimore, MD, pp. 1209-1234. Kaneko, T., Katoh, K., Fujimoto, M., Kumagai, M. and Katayama-Fujimura, K. (1986) Determination of the nucleotide composition of a deoxyribonucleic acid by high-performance liquid chromatography of its enzymatic hydrolysate: a review. J. Microbiol. Methods 4, 229-240. Kooiman, P. (1968) The chemical structure of kefiran, the water-soluble polysaccharide of the kefir grain. Carbohydr. Res. 7, 200-211.
264 Kvernheim, A.L. (1987) Methylation analysis of polysaccharides with butyllithium in dimethyl sulfoxide. Acta Chem. Scand. Ser. B41, 150-152. La Rivi~re, J.W.M., Kooiman, P. and Schmidt, K. (1967) Kefiran, a novel polysaccharide produced in the kefir grain by Lactobacillus bred'is. Arch. Mikrobiol. 59, 269-278. Marmur, J. (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3, 208-218. Mukai, T., Toba, T., Itoh, T. and Adachi, S. (1988) Structural microheterogeneity of kefiran from kefir grains. Jpn. J. Zootech. Sci. 59, 167-176. Mukai, T., Toba, T., Itoh, T. and Adachi, S. (1990) Structural investigation of the capsular polysaccharide from Lactobacillus kefiranofaciens K t. Carbohydr, Res. 204, 227-232. Murofushi, M., Shiomi, M. and Aibara, K. (1983) Effect of orally administered polysaccharide from kefir grain on delayed-type hypersensitivity and tumor growth in mice. Jpn. J. Med. Sci. Biol. 36, 49-53. Ohara, N. (1980) Microbiological studies of kefir grains. Dissertation, Tokyo University of Agriculture, Japan. Ohta, T., Suzuki, Y. and Ohi, A. (1984) Studies on kefir. Sagami Jyoshidaigaku Kiyo 47, 19-25. Rosi, J. and Rossi, J. (1978) The kefir microorganisms: the lactic acid bacteria. Sci. Tecn. LattieroCaseari, 29, 291-305. Sevag, M.G. (1934) Eine neue physikalische Enteiweigungsmethode zur Darstellung biologisch wirksamer Substanzen. Biochem. Z. 273, 419-429. Shiomi, M., Sasaki, K., Murofushi, M. and Aibara, K. (1982) Antitumor activity in mice of orally administered polysaccharide from kefir grain. Jpn. J. Med. Sci. Biol. 35, 75-80. Tamaoka, J. and Komagata, K. (1984) Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol. Lett. 25, 125-128. Toba, T., Abe, S., Arihara, K. and Adachi, S. (1986) A medium for the isolation of capsular bacteria from kefir grains. Agric. Biol. Chem. 50, 2673-2674. Yokoi, H., Watanabe, T., Fujii, Y., Toba, T. and Adachi, S. (1990) Isolation and characterization of polysaccharide-producing bacteria from kefir grains. J. Dairy Sci. 73, 1684-1689.
257
FOOD 00415
Some taxonomical characteristics of encapsulated Lactobacillus sp. KPB-167B isolated from kefir grains and characterization of its extracellular polysaccharide Haruhiko Yokoi 1, Takashi Watanabe 1 Yoshitsugu Fujii ~, Takao Mukai 2,,, Takahiro Toba 2 and Susumu Adachi 2,** 1 Hiratsuka Research Laboratory, Sumitomo Heavy Industries, Ltd., Yuhigaoka, Hiratsuka-shi, Kanagawa, Japan, and 2 Laboratory o f Animal Products Technology, Faculty of Agriculture, Tohoku Unit~ersity, Tsutsumidori-Amamiyamachi 1-1, Aoba-ku, Sendai-shi 981, Japan (Received 29 October 1990; accepted 29 March 1991)
A capsular polysaccharide-producing strain KPB-167B isolated from kefir grains was identified as a homofermentative Lactobacillus. The carbohydrate fermentation pattern and DNA base composition of the strain were different from those of other capsular Lactobacillus species previously isolated from kefir grains. The polysaccharide produced by Lactobacillus sp. KPB-167B was found similar to kefiran by 13C-NMR and methylation analysis. Lactobacillus sp. KPB-167B could grow and produce capsular polysaccharide in MRSL medium with better yield than L. kefiranofaciens, which suggested that it is suitable for kefiran production. Key words: Lactobacillus sp.; Kefir grains; Kefiran
Introduction
Kefir grains are used for making kefir, and consist of lactic acid bacteria and yeasts agglutinated with a water-soluble polysaccharide named kefiran (La Rivi6re et al., 1967). The chemical structure of kefiran was elucidated by Kooiman (1968) and Mukai et al. (1988), and its antitumor activity was reported by Shiomi et al. (1982) and Murofushi et al. (1983). The isolation of encapsulated bacteria from the kefir grains has been reported (Rosi and Rossi, 1978; Ohara, 1980; Ohta et al., 1984; Toba et al., 1986). Only Correspondence address: H. Yokoi, Hiratsuka Research Laboratory, Sumitomo Heavy Industries, Ltd., Yuhigaoka, Hiratsuka-shi, Kanagawa 254, Japan. * Present address: The School of Veterinary Med!:ine and Animal Sciences, Kitasato University, Sanbongi-Maeyachi 149-2, Towada-shi 034, Japan. ** Present address: Shokei Women's Junior College, Yurigaoka 4-10-1, Natori-shi 981-12, Japan.
258 T o b a et al. (1986) identified the encapsulated bacteria and revealed that homofermentative Lactobacillus kefiranofaciens produced kefiran as capsular material (Fujisawa et al., 1988; Mukai et al., 1990). Recently Yokoi et al. (1990) also isolated five encapsulated bacteria, KPB-167A, KPB-167B, KPB-167C, KPB-167D and KPB-167E on a newly developed whey medium. In this paper, some taxonomical characteristics of strain KPB-167B are examined, and the chemical structure of the extracellular polysaccharide is analysed to confirm that the isolate is a kefiran-producer.
Materials and Methods
Bacterial strain The encapsulated bacterial strain KPB-167B isolated from kefir grains from Chr. Hansen's Laboratory (Copenhagen, D e n m a r k ) was used (Yokoi et al., 1990).
Identification Cell shape, cell size, Gram-stain, India-ink-stain and physiological characteristics were observed for cells grown in a M R S L medium consisting of 1% trypton (Difco, Detroit, MI), 0.5% yeast extract (Difco), 1% meat extract (Extract Ehlrich, Wako Pure Chemical Industries Ltd., Osaka, Japan), 2% lactose monohydrate, 0.2% K 2 H P O 4, 0.2% triammonium citrate, 0.1% Tween 80, 0.5% sodium acetate, 0.028% MnSO 4 • 4 H 2 0 and 0.058% MgSO 4 • 7 H 2 0 , p H 6.5, in screw-capped tubes. Bacteria were cultivated for 5 days at 30 ° C after removal of air by passing carbon dioxide into the tubes for 30 s. For acid production from carbohydrates, M R S L medium without meat extract and lactose was used as a basal medium. The filter-sterilized carbon sources were added to the sterile basal medium to give a final concentration of 1%, except for esculine, whose final concentration was 0.25%. The final p H was measured after incubation for 10 days at 30 o C. Gas production resulting from glucose utilization was detected by the formation of bubbles in D u r h a m tubes. The configuration of lactic acid was determined enzymatically by methods described previously (Yokoi et al., 1990).
DNA base composition Cells of KPB-167B grown in M R S L medium were harvested in the late logarithmic phase, and washed twice with 0.15 M NaCI in 0.1 M ethylenediamine tetraaeetic acid, p H 8.0. D N A was isolated and purified according to a modified method of Marmur (1961). The purified D N A was hydrolysed into nucleosides using procedures described by T a m a o k a and Komagata (1984). The resulting deoxyribonucleosides were measured according to Kaneko et al. (1986) using a high-performance liquid chromatograph (HPLC) fitted with a reversed-phase column (4.6 m m i.d.x 150 mm) of Cosmosil 5C18 (Nakarai Tesque Co., Ltd., Kyoto, Japan). An equimolar mixture of four deoxyribonucleosides prepared from a
259 commercial standard mixture of four deoxyribonucleotides for analysis of G + C content (Yamasa Shoyu Co., Ltd) was used as a standard, using above-mentioned method.
Preparation of extracellular polysaccharide Extracellular polysaccharide in the culture supernatant was used for analysis, because its sugar composition is similar to capsular polysaccharides, and because of its higher yields (Yokoi et al., 1990). KPB-167B was cultivated in a MRSL medium for 5 days at 30 ° C. Polysaccharide in the supernatant was obtained by ethanol precipitation as described previously (Yokoi et al., 1990) and purified according to the following method. To degrade contaminating nucleic acids, 0.5% polysaccharide solution containing 5 mg/1 of nuclease PI (Yamasa Shoyu Co., Ltd.) and 0.1 mM ZnC1 z, pH 5.3, was incubated at 5 0 ° C for 2 h. Then remaining proteins were removed by the method of Sevag (1934). After dialysis against distilled water the polysaccharide was obtained as lyophilizate. The resulting polysaccharide was named KPB-P. Kefiran was extracted from kefir grains and purified as described previously (Mukai et al., 1988).
Analysis of monosaccharide composition KPB-P was hydrolysed with 2 N trifluoroacetic acid (TFA) at 100 ° C for 3 h. Monosaccharide composition was determined by H P L C analysis described previously (Yokoi et al., 1990).
Determination of molecular mass The molecular mass of KPB-P was determined by gel filtration chromatography. The chromatography was carried out with a Hitachi 655A-11 HPLC equipped with Asahipak GS-710 column (7.6 mm i.d. ×500 ram, Asahi Chemical Industry Co., Ltd., Kawasaki, Japan) at room temperature using 0.1 M NaCI as a solvent at a flow rate of 1.0 ml/min. Pullulan (Shodex S T A N D A R D P-82, Showa Denko K.K.) was used as a standard of molecular mass.
13C-nuclear magnetic resonance ( t3C-NMR) analysis 13C-NMR spectra of KPB-P and kefiran were obtained in D 2 0 at 70 ° C with a J E O L FX-400 spectrometer (Japan Electron Optical Laboratory Co., Akishima, Japan) operating at 100 MHz, and chemical shifts were recorded in ppm downfield to 3-(trimethylsilyl)-propanesulfonic acid sodium salt.
Methylation analysis KPB-P and kefiran were methylated according to the method of Kvernheim (1987). Extraction and hydrolysis of the permethylated polysaccharide, and reduction and acetylation were carried out according to Harris et al. (1984). The partially methylated alditol acetates were determined using a gas chromatographmass spectrometer as described previously (Mukai et al., 1988).
I
KPB- 167B
39
S-10-LR e
D(L)
D(L)
D(L)
D(L)
D(L)
D(L)
D(L)
+
-
±
.
45 ° C
+
+
+
+
-
-
-
-
-
biose
tose
+
Cello-
Mal-
+
+
+
+
+
tose
Lac-
Acid formation from
-
+
+
-
-
15 o C
G r o w t h at
-
.
-
-
-
-
.
lose
Treha-
.
.
.
inose
. -
-
-
+
+
+
.
biose
Meli-
. .
lose
Xy-
.
. nd
-
+
-
+
+
nose
Raffi-
.
nose
Rham-
Acid formation from Arab-
Ri-
nd
-
.
.
zitose
Mele-
-
nd
. .
bose
nd
nd
.
nd f
trin
Dex-
.
+
+
+
+
+
cose
Glu-
-
-
.
. .
nitol
Man-
-
.
nd
+
±
+
r No data included.
~ Data from Ohara
(1980).
d D a t a f r o m R o s i a n d R o s s i (1978),
c D a t a f r o m O h t a e t al. (1984).
h D a t a f r o m F u j i s a w a et al. (1988).
.
nose
Man-
-
-
.
.
bitol
Sor-
homofermentative
a B a s e d o n t h e c o n t e n t o f t . - ( + ) - l a c t i c a c i d as f o l l o w s : D , 0 t o 2 0 % ; D ( L ) , 20 to 4 0 % ; D L , 40 to 6 0 % ; L, 80 to 1 0 0 % .
39
34-35
F3M, F6M ~
A3-1, A3-3 c
MA2-1, MA2-2,
WT-6A, WT-7
WT-2B, WT-8,
L. kerifiranofaciens b
40
39
S-10-LR c
Lactobacillus sp.
39
34-35
D(L)
Lactic acid a
(mol%)
40
Isomer
of
G + C
F3M, F6M J
MA2-1, MA2-2, A3-1, A3-3 c
WT-6A, WT-7
WT-2B, WT-8,
L. kefiranofaciens b
KPB-167B
Lactobacillus sp.
Bacterial strain
content
o f p h e n o t y p i c c h a r a c t e r i s t i c s o f Lactobacillus sp. K B P - 1 6 7 B w i t h t h o s e o f o t h e r e n c a p s u l a t e d
from kefir grains
Comparison
TABLE
.
nd
nd
+
+
+
nd
nd
.
lin
.
Escu-
tose
Fruc-
.
+
+
+
+
+
nd
-
licin
Sal-
tose
Galac-
nd
-
dalin
Amyg-
+
+
+
+
+
crose
Su-
Lactobacillus s p e c i e s i s o l a t e d
261
Results and Discussion
Characteristics of the encapsulated strain KPB-167B was identified as homofermentative Lactobacillus with a G + C content of D N A of 40%. As shown in Table I, KPB-167B fermented glucose, mannose, fructose, galactose, sucrose, maltose and lactose. Arabinose, xylose, rhamnose, ribose, cellobiose, trehalose, melibiose, raffinose, melezitose, mannitol, sorbitol, esculin, salicin and amygdalin were not fermented. Sugar utilization of KPB-167B was not identical to those of any other homofermentative Lactobacillus species listed in Bergey's Manual of Systematic Bacteriology (Kandler and Weiss, 1986). The optimum growth temperature of KPB-167B was 30 ° C and the bacterium did not grow at 15 and 45 ° C. The optimum initial pH of a MRSL medium was around 5.5. Although it grew under an atmosphere of air or N e, growth was enhanced under an atmosphere of CO 2. The essential nutrient in a MRSL medium for KPB-167B was yeast extract, and the lack of trypton or meat extract allowed about 85% of the growth of control (data not included).
Structural analysis of extracellular polysaccharide from KPB-167B Gel filtration chromatography of KPB-P showed a single peak. The molecular mass was determined to be about 1.6 x 10 6 (Fig. 1). In elementary analysis, we did not detect nitrogen (unpublished results). Monosaccharides obtained by hydrolysis of KPB-P were composed of glucose and galactose. The molar ratio of glucose and galactose was 1.0:0.98 by H P L C analysis. Fig. 2 shows 13C-NMR spectra of KPB-P and kefiran from kefir grains. KPB-P was assumed to be kefiran because of the high similarity of the spectra. To verify this assumption, chemical structure of KPB-P was further investigated by methylation analysis. In Table III the partially methylated alditol acetates obtained from permethylated KPB-P and kefiran are compared. The O-methylsugars of KPB-P were identical to those of kefiran and their molar ratios were
Retention
time
(rain)
I
I
I
107 Molecular
106
105
I
104.
mass
Fig. 1. Gel filtration chromatogram of capsular polysaccharide from Lactobacillus sp. KPB-167B on a Asahipak GS-710 column (7.6 m m i.d. × 5 0 0 ram).
262
(A)
(B)
I
120
I
110
I
100
I
I
I
I
90
80
70
60
Chemical s h i f t
(ppm)
Fig. 2. Comparisonof 13C-NMR spectra of capsular polysaccharidefrom Lactobacillus sp. KPB-167B and kefiran from kefir grains. A. Polysaceharidefrom strain KPB-167B, B. Kefiran.
T A B L E II Methylation analysis of capsular polysaccharide from Lactobacillus sp. KPB-167B (KPB-P) and kefiran extracted from kefir grains Identified
Mode of linkage
O-methylsugar 2,3,4,6-tetra-O-methylglucose 2,3,6-tri-O-methylgalactose 2,3,6-tri-O-methylglucose 2,4,6-tri-O-methylgalactose 2,3,4-tri-O-methylglucose 2,3,4-tri-O-methylgalactose 3,4-di-O-methylgalactose
GIc-(1 ~ -4)-Gal-(l ~ -4)-Glc-(l ~ -3)-Gal-(1 ---* -6)-Glc-(1 --* -6)-Gal-(1 ~ -2,6)-Gal-( --*
Molar ratio a KPB-P
Kefiran
1.00 1.14 1.36 1.32 1.24 0.24 1.10
1.00 1.36 1.29 1.31 1.11 0.19 1.10
a Calculated from peak area of gas chromatograph-mass spectrogram.
263
almost identical. These results strongly suggest that KPB-P is kefiran and Lacto-
bacillus sp. KPB-167B is a kefiran-producing bacterium. Mukai et al. (1988, 1990) reported that kefiran extracted from kefir grains and capsular polysaccharide from L. kefiranofaciens K 1 contained 6-O-substituted galactose in the structure proposed by Kooiman (1968). In the structure of polysaccharide produced by Lactobacillus sp. KPB-167B, the presence also of 6-O-substituted galactose was confirmed by detecting 2,3,4-tri-O-methylgalactose in identified O-methylsugars. The phenotypic characteristics of other encapsulated homofermentative Lactobacillus species isolated from kefir grains are shown in Table I. Among these strains, only L. kefiranofaciens has been confirmed to be a kefiran-producer by structural analysis of capsular polysaccharide. Lactobacillus KPB-167B did not ferment either melibiose or raffinose, while Lactobacillus kefiranofaciens MA2-1, MA2-2, A3-1 and A3-3 ferment both sugars, F3M and F6M ferment only melibiose, and S-10-LR ferments only raffinose. The G + C content of DNA of Lactobacillus sp. KPB-167B are 5-6% higher than that of L. kefiranofaciens, but are nearly equal to those of F3M, F6M and S-10-LR. Although there are small differences in the G + C content and sugar utilization, Lactobacillus sp. KPB-167B could still belong to species L. kefiranofaciens because it produces kefiran. More detailed taxonomical investigations such as DNA homology experiments between these bacteria are necessary to verify this assumption. A procedure for mass production of kefiran is desired. However, microbial production has not been established except for the complicated procedure of kefiran extraction from kefir grains. As reported previously (Yokoi et al., 1990), Lactobacillus sp. KPB-167B produced kefiran in a higher yield in milk whey medium (300-400 rag/l) than L. kefiranofaciens. Although L. kefiranofaciens is able to grow and produce kefiran only in milk whey medium, it was found that Lactobacillus sp. KPB-167B grew and produced kefiran also in MRSL medium. These findings indicate that Lactobacillus sp. KPB-167B is suitable for the mass production of kefiran.
References Fujisawa, T., Adachi, S., Toba, T., Arihara, K. and Mitsuoka, T. (1988) Lactobacillus kefiranofaciens sp. nov. isolated from kefir grains. Int. J. Syst. Bacteriol. 38, 12-14. Harris, P.J., Henry, R.J., Blakeney, A.B. and Stone, B.A. (1984) An improved procedure for the methylation analysis of oligosaccharides and polysaccharides. Carbohydr. Res. 127, 59-73. Kandler, O. and Weiss, N. (1986) Genus Lactobacillus Beijerinck 1901, 212 AL. In: P.H.A. Sneath, N.S. Mair, M.E. Sharpe and J.G. Holt (Eds.), Bergey's Manual of Systematic Bacteriology, Vol. 2, Williams & Wilkins, Baltimore, MD, pp. 1209-1234. Kaneko, T., Katoh, K., Fujimoto, M., Kumagai, M. and Katayama-Fujimura, K. (1986) Determination of the nucleotide composition of a deoxyribonucleic acid by high-performance liquid chromatography of its enzymatic hydrolysate: a review. J. Microbiol. Methods 4, 229-240. Kooiman, P. (1968) The chemical structure of kefiran, the water-soluble polysaccharide of the kefir grain. Carbohydr. Res. 7, 200-211.
264 Kvernheim, A.L. (1987) Methylation analysis of polysaccharides with butyllithium in dimethyl sulfoxide. Acta Chem. Scand. Ser. B41, 150-152. La Rivi~re, J.W.M., Kooiman, P. and Schmidt, K. (1967) Kefiran, a novel polysaccharide produced in the kefir grain by Lactobacillus bred'is. Arch. Mikrobiol. 59, 269-278. Marmur, J. (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3, 208-218. Mukai, T., Toba, T., Itoh, T. and Adachi, S. (1988) Structural microheterogeneity of kefiran from kefir grains. Jpn. J. Zootech. Sci. 59, 167-176. Mukai, T., Toba, T., Itoh, T. and Adachi, S. (1990) Structural investigation of the capsular polysaccharide from Lactobacillus kefiranofaciens K t. Carbohydr, Res. 204, 227-232. Murofushi, M., Shiomi, M. and Aibara, K. (1983) Effect of orally administered polysaccharide from kefir grain on delayed-type hypersensitivity and tumor growth in mice. Jpn. J. Med. Sci. Biol. 36, 49-53. Ohara, N. (1980) Microbiological studies of kefir grains. Dissertation, Tokyo University of Agriculture, Japan. Ohta, T., Suzuki, Y. and Ohi, A. (1984) Studies on kefir. Sagami Jyoshidaigaku Kiyo 47, 19-25. Rosi, J. and Rossi, J. (1978) The kefir microorganisms: the lactic acid bacteria. Sci. Tecn. LattieroCaseari, 29, 291-305. Sevag, M.G. (1934) Eine neue physikalische Enteiweigungsmethode zur Darstellung biologisch wirksamer Substanzen. Biochem. Z. 273, 419-429. Shiomi, M., Sasaki, K., Murofushi, M. and Aibara, K. (1982) Antitumor activity in mice of orally administered polysaccharide from kefir grain. Jpn. J. Med. Sci. Biol. 35, 75-80. Tamaoka, J. and Komagata, K. (1984) Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol. Lett. 25, 125-128. Toba, T., Abe, S., Arihara, K. and Adachi, S. (1986) A medium for the isolation of capsular bacteria from kefir grains. Agric. Biol. Chem. 50, 2673-2674. Yokoi, H., Watanabe, T., Fujii, Y., Toba, T. and Adachi, S. (1990) Isolation and characterization of polysaccharide-producing bacteria from kefir grains. J. Dairy Sci. 73, 1684-1689.