JPH0873505A - Polysaccharide substance tpsb, its production, and its use - Google Patents

Abstract

PURPOSE: To obtain a polysaccharide substance TPSB which inhibits the multipli cation of B lymphocytes and is useful as an inhibitor for B lymphocyte multipli cation by separating TPBS from the product of culturing TPBS-producing lactic acid bacteria belonging to the genus Lactobacillus. CONSTITUTION: The substance TPSB has the following physicochemical properties: results of elemental analysis such that C=41.2±5, H=6.8±0.8, and O=52.0π6; a mol.wt. of about 2,500,000-500,000; a specific rotation [α]<25> D of 58-68 (C=0.3%); such an m.p. that it starts browning at about 220 deg.C and turns dark brown at about 250 deg.C; peaks of the IR spectrum at 3410, 3932, 2899, 1646, 1419, 1363, 1078, 1034, 894, and 788 (cm<-1> ); such a solubility that it is sol. in water and insol. in methanol, ethanol, acetone, and ether; Molisch's reaction, positive; anthrone reaction, positive; cysteine-sulfuric acid reaction, negative; carbazole-sulfuric acid reaction, negative; Elson-Morgan reaction, negative; ninhydrin reaction, negative; a pH of the 0.3% aq. soln. of 7.5; the form of white fiber; and a ratio of glucose to galactose of (1.0:0.8)-(1.0:1.2).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel polysaccharide substance TPSB having a useful physiological activity, its production method and use.

[0002]

2. Description of the Related Art Many studies have been conducted on polysaccharide substances produced by lactic acid bacteria, and their structural diversity has been reported [FEMS Microbiology Reviews 87 (1990) 113-13.
0]. Furthermore, some reports have been made on its physiological activity (Japanese Patent Laid-Open Nos. 63-61002 and 58-2).
04001).

[0003]

DISCLOSURE OF THE INVENTION The present inventors have been conducting research on lactic acid bacteria found in foods, and as a result, Lactobacillus helveticus TAKE-01 (Lactobacillus
helveticus TAKE-01) found that the novel polysaccharide substance TPSB was produced, and succeeded in isolating it. When the physiological activity of TPSB thus isolated was studied, it was found that it showed a B lymphocyte proliferation inhibitory effect. As diseases caused by B lymphocyte abnormalities, autoimmune diseases such as malignant lymphoma mainly caused by abnormal proliferation, systemic lupus erythematosus due to autoantibodies produced by abnormal activity, and rheumatoid arthritis are well known. For these diseases, it is effective to suppress the proliferation of B lymphocytes, but at present, no drug that specifically inhibits the proliferation of B lymphocytes is used. The novel polysaccharide substance TPSB of the present invention is considered to be an excellent B lymphocyte proliferation inhibitor due to its B lymphocyte proliferation inhibitory effect.

[0004]

The present invention provides a novel polysaccharide substance TPSB having the following physicochemical properties and the TP.
The present invention provides a method for producing TPSB, which comprises separating and collecting TPSB from a culture of SB-producing lactic acid bacteria. Furthermore, the present invention provides a B lymphocyte proliferation inhibitor containing TPSB or a derivative thereof as an active ingredient.

Physicochemical properties of the novel polysaccharide substance TPSB (1) Elemental analysis value C: 41.2 ± 5 H: 6.8 ± 0.8 O: 52.0 ± 6 (2) Molecular weight 2,500,000 to 500,000 (3 ) specific rotation _{^{[α] 25 D = 58~68 (}} C = 0.3%) (4) begins to change from near the melting point 220 ° C. brown, becomes dark brown at around 250 ° C.. (5) Infrared absorption spectrum (KBr method) 3410, 2932, 2899, 1646, 1419,
1363, 1149, 1078, 1034, 894, 7
88 cm ^-1

(6) Solubility in solvent Soluble in water, insoluble in methanol, ethanol, acetone, ether (7) Color reaction (a) Maurish reaction + (b) Anthrone reaction + (c) Cysteine-sulfuric acid reaction -(D) Carbazole-sulfuric acid reaction- (e) Elson-Morgan reaction- (f) Ninhydrin reaction- (8) Basic, acidic or neutral 0.3% aqueous solution shows pH 7.5. (9) Color of substance The freeze-dried product has a white fiber shape. (10) Types of composition and composition ratio Glucose: Galactose = 1.0: 0.8 to 1.2

The novel polysaccharide substance TPSB can be obtained by culturing a TPSB-producing bacterium. A typical example of the TPSB-producing bacterium is Lactobacillus helveticus TAKE-01 (Lactobacillus helveticus), which is a lactic acid bacterium belonging to the genus Lactobacillus found by the present inventors.
TAKE-01), which is July 1, 1994.
Deposited under the deposit number FERM P-14411 at the Institute of Biotechnology, Institute of Biotechnology, dated to date.

Lactobacillus helveticus TAK
E-01 (Lactobacillus helveticus TAKE-01)
The mycological properties of the are shown below. Bacterial morphology Bacillus Gram stain + Fermentation type Homo-type lactic acid Optical DL GC content (%) 40.2 Catalase reaction-Growth at 15 ° C-Glucose-fermenting amydarin-mannose + arabinose-merezitose-cellobiose-meleviose-esculin-raffinose- Fructose + rhamnose-galactose + ribose-glucose + salicin-gluconate-sorbitol-lactose + sucrose-maltose-trehalose + mannitol-xylose-

From the above mycological properties, Lactobacillus
It was identified as a lactic acid bacterium belonging to Helvetecus. The polysaccharide-producing bacterium belonging to the genus Lactobacillus is irradiated with ultraviolet rays, X-rays, radiation, etc., treated with various mutagens,
It is possible to mutate by other means, and in the present invention, not only the TAKE-01 strain but also such a mutant strain or a naturally obtained mutant strain, which has the ability to produce TPSB, is used. , All can be used in the present invention.

The polysaccharide substance TPSB is collected from the culture after culturing the polysaccharide substance TPSB-producing bacterium in a medium. The culture medium may be liquid or solid, but it is suitable to use a liquid medium when treating a large amount. A carbon source, a nitrogen source, an inorganic substance, and a micronutrient that can be assimilated by the producing bacterium are appropriately added to the medium. As the carbon source, glucose, galactose, lactose, a substance that can be assimilated by the production bacterium such as fructose, as the nitrogen source,
Yeast extract, dried yeast, soybean protein, various protein hydrolysates, and the like are used. Furthermore, salts containing sodium, potassium, calcium, etc., metal salts containing iron, manganese, etc., salts such as phosphoric acid, citric acid, acetic acid, etc., and other vitamins (eg, B1, B2, calcium pantothenate, nicotinic acid, etc.) ), Nucleic acids (eg, purines, pyrimidines, etc.) are used as appropriate, but skim milk powder,
More preferred are media containing dairy products such as milk whey, milk casein and its hydrolysates. The culture conditions include T
Any culture condition may be used as long as it produces PSB, and the pH of the medium is pH 4.5 to 7.0, particularly pH.
5.5 to 6.5 are preferable, and the culture temperature is 25 to 48.
Culturing at 1 ° C for 1 to 7 days is preferred.

In order to collect the desired polysaccharide substance TPSB from the culture, a general method for collecting water-soluble polysaccharides produced by microorganisms such as centrifugation, solvent fractionation, gel filtration, ion exchange chromatography and dialysis is used. These may be performed alone or in combination as appropriate. For example, a supernatant fraction and a precipitation fraction are obtained from a culture of a polysaccharide TPSB-producing bacterium by centrifugation. In the precipitation section, after extraction with water, hot water, etc., a supernatant is obtained by centrifugation. During these centrifugation operations,
If necessary, a deproteinizing agent such as trichloroacetic acid may be added to carry out an efficient deproteinizing operation from the supernatant.
A precipitate is obtained by adding an organic solvent such as ethyl alcohol or acetone to the supernatant thus obtained from the supernatant fraction and the precipitation fraction. The obtained precipitate is dissolved in water, the insoluble matter is removed by a method such as centrifugation and filtration, and then an organic solvent such as ethyl alcohol and acetone is added again to obtain a precipitate. The obtained precipitate is dissolved in an appropriate buffer such as Tris-hydrochloric acid buffer and loaded on a column packed with an ion exchanger such as diethylaminoethyl cellulose buffered with the same buffer. Subsequently, the same buffer solution is flown, the non-adsorbed fraction is separated, dialyzed in ion-exchanged water, and the dialyzed solution is freeze-dried to obtain the purified polysaccharide substance TPSB.

The polysaccharide substance TPSB thus obtained can be used as it is or as a derivative thereof in the form of a drug, food or the like as the B lymphocyte proliferation inhibitor of the present invention. The derivative of the polysaccharide substance TPSB may be any TPSB derivative exhibiting a B lymphocyte proliferation inhibitory action, an enzymatically modified product,
It includes chemical modifications, physical modifications, low molecular weight compounds and the like. For example, enzymatically modified derivatives obtained by acting enzymes such as β-galactosidase, α-galactosidase, α-glucosidase, β-glucosidase, etc. alone or in combination, periodate oxidation, mild acid hydrolysis, sodium borohydride, etc. Chemically modified derivative obtained by performing reduction treatment with, ultrasonic irradiation treatment, high-speed shearing treatment,
Examples thereof include physically modified derivatives obtained by performing high-pressure treatment and the like. When used as a medicine or the like, the administration form of the polysaccharide substance TPSB or its derivative may be oral administration or parenteral administration. For oral administration, dosage forms such as capsules, tablet granules, syrups, and powders are possible, and the polysaccharide substance TPSB is supplemented with additives such as excipients,
Binders, disintegrants, lubricants, coloring agents, flavoring agents, stabilizers and the like may be added. Among them are sucrose, fructose, lactose, glucose, mannitol, sorbitol, starch, crystalline cellulose, carboxymethyl cellulose, dextrin,
Examples thereof include magnesium stearate and talc precipitated calcium carbonate.

For parenteral administration, the polysaccharide substance TPS
B or its derivative can be dissolved or suspended in a conventional diluent and used as a solution, emulsion, suspension, suppository, and other appropriate dosage forms. Diluents include distilled water, physiological saline, Ringer's solution, aqueous glucose solution, and the like. Furthermore, in the pharmaceutical preparation, as an additive, an emulsifier, a suspending agent, a solubilizing agent, a stabilizer, a preservative, a soothing agent, a tonicity agent, a buffering agent, a pH adjusting agent, a coloring agent, a covering agent. Agents and the like may be included. These formulations can be manufactured by a conventional method.

Further, it is possible to provide the polysaccharide substance TPSB or its derivative in the form of various foods such as lactic acid bacterium drink, yogurt, jelly, dressing, powder and granular foods. In that case, it is also possible to add the isolated TPSB or its derivative, or to use the NPS-producing bacterium culture solution as it is.

[0015]

EXAMPLES Next, the present invention will be described more specifically by showing Examples and Test Examples of the present invention. Example 1 Lactobacillus helveticus TAKE-01 was inoculated into 1 kg of 10% skim milk powder medium, and static culture was carried out at 32 ° C. for 3 days. The resulting culture solution was vigorously stirred, trichloroacetic acid was added thereto to a concentration of 7%, and the mixture was centrifuged (7,000 rpm, 15 minutes) to obtain a supernatant fraction and a precipitate fraction. 300 ml of water was added to the obtained precipitate fraction, and the mixture was extracted by stirring at room temperature for 1 hour, and then trichloroacetic acid was added to the extract solution at a concentration of 7% while stirring vigorously.
The resulting solution was centrifuged (7,000 rpm, 15
Min) and a supernatant was obtained. After combining the obtained supernatant fraction and the supernatant fraction from the culture solution, ethyl alcohol was added to a final concentration of 50.
% To form a precipitate, which is collected by centrifugation (10,000 rpm, 15 minutes), dissolved in ion-exchanged water, and the insoluble substance is centrifuged (1
At 0,000 rpm for 15 minutes). After repeating this operation three times, the supernatant was dialyzed against running water for 1 day, and the Tris-hydrochloric acid buffer solution was added to the dialyzed solution to give a solution of 0.
It was adjusted to be 02M Tris-hydrochloric acid buffer (pH 8.7). Next, the column was loaded on a diethylaminoethylcellulose column buffered with the same buffer, and the same buffer was flowed to collect the non-adsorbed fraction. The resulting non-adsorbed fraction was dialyzed in running water for 1 day and then in ion-exchanged water for 2 days. The obtained dialysate is freeze-dried and purified TPSB is
80 mg was obtained.

The physicochemical properties of the obtained TPSB are as follows. (1) Elemental analysis value (105 ° C. for 4 hours under reduced pressure) C: 41.2 H: 6.8 O: 52.0 (2) Molecular weight The molecular weight is Asahipak GS710 (7.6 × 500 mm, manufactured by Asahi Kasei Kogyo Co., Ltd.). When it was measured by a gel filtration method using HPLC, the molecular weight was about 2,500,000 to 500,000. The conditions of HPLC are shown below. Conditions: temperature 30 ° C. eluate 100 mM sodium nitrate solution flow rate 1.0 ml / min

(3) Specific optical rotation (dried under reduced pressure at 105 ° C. for 4 hours) [α] ²⁵ _D = 61.6 (C = 0.3 water) (4) Melting point: Around 220 ° C., a brown color starts to change to 250. Turns blackish brown at around ℃. (5) Infrared absorption spectrum (KBr method) FIG. 1 shows the infrared absorption spectrum of the obtained purified TPSB. (6) Solubility in solvent Soluble in water, insoluble in methanol, ethanol, acetone, ether (7) Color reaction (a) Maurish reaction + (b) Anthrone reaction + (c) Cysteine-sulfuric acid reaction-(d ) Carbazole-sulfuric acid reaction- (e) Elson-Morgan reaction- (f) Ninhydrin reaction-

(8) Basic, acidic, and neutral 0.3% aqueous solution has a pH of 7.5. (9) Color of substance The freeze-dried product has a white fiber shape. (10) Types of composition and composition ratio 2N trifluoroacetic acid was added to 5 mg of the obtained purified TPSB sample, which was hydrolyzed at 100 ° C. for 12 hours, and then concentrated to dryness under reduced pressure to remove trifluoroacetic acid. , Wakowp
As a result of analysis by HPLC using ac Wakobeads T-131-E (7.8 × 300 mm, manufactured by Wako Pure Chemical Industries, Ltd.),
The ratio of glucose: galactose = 1: 0.8 to 1.2 was detected. The conditions of HPLC are shown below. Conditions: Temperature 60 ° C. Eluent 10 ⁻⁴ M sodium hydroxide solution Flow rate 0.5 ml / min

Example 2 A TPSB-producing bacterial culture cultivated in 10% skim milk powder solution (1 kg) at 32 ° C. for 3 days was sterilized at 70 ° C. for 5 minutes, and the solid content was adjusted to 25% with an excipient such as dextrin. Then, it is pulverized by a method such as spray drying or freeze drying to obtain a fermented powder containing TPSB. Based on the obtained fermented powder, the granular food made by the following formulation,
It contains about 36 mg% of TPSB. Fermented powder 50.0g Raffinose 30.0g Glucose 20.0g

Example 3 Commercially available milk (500 ml) was sterilized three times at 90 ° C. for 30 minutes, and a TPSB-producing bacterium was cultured at 32 ° C. for 3 days. A sour cream dressing prepared by the following formulation using the obtained culture solution of TPSB-producing bacteria contains about 14 mg% of TPSB. Culture liquid 100.00 g Fresh cream 20.00 g Sugar 8.00 g Salt 1.00 g Sodium glutamate 0.20 g 5'Ribnucleotide sodium 0.02 g White pepper 0.05 g Mustard seasoning 0.13 g

Example 4 10% of commercially available milk was sterilized twice at 90 ° C. for 30 minutes twice, and 5% gelatin was added to commercially available milk with respect to a solution obtained by culturing TPSB-producing bacteria at 32 ° C. for 3 days. The solution dissolved by heating at a concentration and the fructose-glucose liquid sugar were stirred and mixed at the following ratios and cooled. The obtained jelly food has a TSPB of 55 m.
It has about g%. Culture solution 10 kg 4% gelatin commercial milk solution 10 kg Fructose-glucose liquid sugar 1.4 kg TPSB 0.01 kg

Example 5 A culture solution of a TPSB-producing bacterium, which was cultivated in a 10% skim milk powder solution (1 kg) at 32 ° C. for 3 days, was sterilized at 70 ° C. for 5 minutes,
The sterilized lactic acid bacterium beverage prepared with the following formulation contains 9 parts of TPSB.
Contains about mg%. Culture solution 500g Sugar 180g Pectin 40g Yogurt flavor 10g Water 270g

Example 6 Tablets were produced by directly mixing the ingredients in the following amounts and then directly pressurizing them. 1 tablet (800 mg)
About 64 mg of TPSB is contained. TPSB 16g Lactose 175g Crystalline cellulose 1.2g Talc 8g

Test Example 1 In this test example, TPSB obtained in Example 1 was used to examine the effect of TPSB on mouse splenic lymphocyte proliferative response, thereby verifying the effect of TPSB to suppress B lymphocyte proliferation. Spleens were aseptically removed from mice (C57BL / 6, female, 10 weeks old), the spleens were crushed in RPMI 1640 medium, and passed through # 200 mesh to obtain a spleen cell suspension. After measuring the cell number of the spleen cell suspension with an automatic hemocytometer, the cell number was adjusted to a concentration of 1 × 10 ⁷ / ml with RPMI 1640 medium and seeded in a 24-well tissue culture plate with 200 microliters per well. did. Concanavalin A, a T lymphocyte proliferation stimulator, dissolved in RPMI 1640 medium at a concentration of 8 microgram / ml, and lipopolysaccharide, a B lymphocyte proliferation stimulator, at a concentration of 200 microgram / ml, RPMI
Solution dissolved in 1640 medium or RPMI 164
0 medium was added to the spleen cell suspension inoculated with 200 microliters per well, and the T lymphocyte stimulation group,
B lymphocyte stimulation group and non-stimulation group were used. RPM for these 3 groups
I 1640 medium (control) or TPSB 8 mg /
Add 400 microliters of each solution dissolved in RPMI 1640 medium at a concentration of ml to 37 ° C.
The cells were cultured in the 5% carbon dioxide incubator for 3 days. 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium bromide was added 5 hours before the end of the culture.
A solution of RPMI 1640 medium at a concentration of mg / ml was added in an amount of 40 microliters per well, and a 20% sodium dodecyl sulfate solution was added to each well at the end of the culture at a concentration of 200 μl.
After adding microliter, the mixture was allowed to stand at 37 ° C. for 1 day, and the proliferative reaction of spleen cells was determined by measuring the absorbance of the culture medium at 570 nm. The results are shown in Table 1.

[0025]

[Table 1]

Although the effect of TPSB on the T lymphocyte proliferative reaction was slight, TPSB remarkably suppressed the B lymphocyte proliferative reaction, and the effect of TPSB to suppress the B lymphocyte proliferative effect was recognized.

Example 7 170 mg of TPSB obtained in Example 1 was dissolved in 50 ml of 0.02M sodium acetate buffer (pH 4.5),
25 mg of β-galactosidase (manufactured by Sigma) derived from Aspergillus oryzae was added, and the mixture was reacted with stirring at 30 ° C. for 72 hours. Then, 20 mg of the above-mentioned enzyme was further added, and the mixture was reacted with stirring at 30 ° C. for 24 hours. After completion of the reaction, the reaction solution was neutralized with sodium hydroxide, heated at 100 ° C. for 10 minutes, and then cooled to inactivate the enzyme. The reaction solution was centrifuged (12,000 rpm for 15 minutes) to obtain a supernatant. The obtained supernatant was dialyzed against running water for 2 days and further dialyzed for 1 day in ion-exchanged water, and the dialyzed solution was freeze-dried to obtain 120 mg of enzyme-decomposed TPSB. Obtained enzymatically decomposed TPS
When the type and composition ratio of the constituent sugars of B were measured by the method described in Example 1, glucose: galactose = 1.
It was detected at a ratio of 0: 0.7 to 0.6.

Example 8 150 mg of TPSB obtained in Example 1 was dissolved in 75 ml of ion-exchanged water, and then 75 ml of 0.1 M sodium metaperiodate was added to adjust the total amount to 150 ml, and the mixture was placed at 4 ° C. in the dark. The reaction was stirred for one day. After completion of the reaction, 20 ml of ethylene glycol was added, and then dialyzed against running water for 2 days. After the dialysis was completed, 100 mg of sodium borohydride was added to the dialyzed solution, and a reduction reaction was performed overnight. After the reaction was completed, about 5 ml of acetic acid was added to adjust the solution to be weakly acidic, and the solution was dialyzed against running water for 2 days and the dialyzed solution was freeze-dried to obtain 105 mg of TPSB periodate redox product.

Example 9 2 g of TPSB obtained in Example 1 was dissolved in 200 ml of ion-exchanged water, and then Ultrasonic Processor VP
Using -30 (manufactured by Taitec Co., Ltd.), while cooling, ultrasonic irradiation was performed for 40 minutes in a cycle of irradiation for 42 seconds and stop for 18 seconds to obtain a low molecular weight TPSB solution. The resulting solution was freeze-dried to obtain low molecular weight TPSB. The obtained low molecular weight TPSB was 10,000 to 500,000 as measured by the molecular weight measurement method described in Example 1.

Test Example 2 In this test example, TPSB obtained in Example 1, enzymatically-degraded TPSB obtained in Example 7, TPSB periodate redox product obtained in Example 8, and low yield obtained in Example 9 were used. Using molecularized TPSB, the effects of TPSB, enzyme-degraded TPSB, TPSB periodate redox product, and low molecular weight TPSB on the B lymphocyte proliferation inhibitory effect on the mouse spleen lymphocyte proliferation reaction were verified. Spleens were aseptically removed from mice (C57BL / 6, female, 10 weeks old), the spleens were crushed in RPMI 1640 medium, and passed through # 200 mesh to obtain a spleen cell suspension.
After measuring the cell number of the spleen cell suspension with an automatic hemocytometer, the cell number was adjusted to 1 × 10 ⁷ / ml with RPMI.
Prepared in 1640 medium and seeded 50 microliters per well of 96-well tissue culture plate. Concanavalin A, a T lymphocyte proliferation stimulant, 8 microgram / ml
Of the B lymphocyte proliferation stimulant lipopolysaccharide dissolved in the RPMI 1640 medium at a concentration of 200 microgram / ml, anti-mouse immunoglobulin M of the B lymphocyte proliferation stimulant RP at a concentration of 200 microgram / ml
Solution dissolved in MI 1640 medium or RPMI
1640 medium was added to each spleen cell suspension in which 50 microliters had been seeded per well to make a T lymphocyte stimulation group, a B lymphocyte stimulation group (1), a B lymphocyte stimulation group (2), and a non-stimulation group. . RPMI 164 for these 4 groups
0 medium (control) or TPSB dissolved in RPMI 1640 medium at a concentration of 2 mg / ml, enzyme-degraded TP
SB dissolved in RPMI 1640 medium at a concentration of 2 mg / ml, TPSB periodate redox product at 2 mg / ml
A solution prepared by dissolving the low molecular weight TPSB in RPMI 1640 medium at a concentration of ml and RPMI 16 at a concentration of 2 mg / ml.
100 microliters of each solution dissolved in 40 medium was added to each well, and the solution was placed in a 5% carbon dioxide incubator at 37 ° C for 2
Cultured for a day. 3 hours before the end of the culture, 3- (4,
5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium at a concentration of 5 mg / ml in RPMI
10 microliters of the solution dissolved in 1640 medium was added per well, and at the end of the culture, 50% of 20% sodium dodecyl sulfate solution was added per well.
After standing at 0 ° C for 1 day, the proliferation reaction of spleen cells was determined by measuring the absorbance of the culture medium at 550 nm with a microplate reader. Table 2 is a table showing the results. TPSB and enzyme-degraded TPS that affect T lymphocyte proliferation-stimulating reaction
B, TPSB Periodate redox product, low molecular weight TPSB
Was slightly affected, but TPSB, enzymatically decomposed TPS
B, TPSB Periodate redox product, low molecular weight TPSB
Markedly suppressed the B lymphocyte proliferation reaction, and TPSB, enzyme-degraded TPSB, TPSB periodate redox product, and low molecular weight TPSB had B lymphocyte proliferation inhibitory effects.

[0031]

[Table 2]

[0032]

INDUSTRIAL APPLICABILITY According to the present invention, a novel polysaccharide substance TPSB having an excellent B lymphocyte proliferation inhibitory effect is provided. The polysaccharide substance can be easily isolated and collected from the culture solution of the TPSB-producing bacterium. The isolated TPSB can be added to existing foods without damaging the taste, appearance, etc. because it is in the form of a preparation or a colorless and odorless substance. Since it can be used for existing foods and the like, it is suitable for the prevention and treatment of the abnormal proliferation of B lymphocytes, the development of malignant lymphomas caused by abnormal activity, autoimmune diseases, etc. It can be used as a research reagent.

[Brief description of drawings]

FIG. 1 is a drawing showing an infrared absorption spectrum of the polysaccharide substance TPSB of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location (C12P 19/04 C12R 1: 225) (72) Inventor Tetsuo Nunome 2-13, Koto, Kawanishi-shi, Hyogo No. 15 Maison Kimura No. 305 (72) Inventor Hiroaki Kusaka 3-3-1-2 Akuraminami, Takarazuka-shi, Hyogo Prefecture (72) Inventor Shin-shi Kasano 3-12-4 Naruohama, Nishinomiya-shi, Hyogo Prefecture Inside the Research Institute, Kisho Corporation

Claims (4)

[Claims] 1. A polysaccharide substance TP having the following physicochemical properties:
SB. (1) Elemental analysis value C: 41.2 ± 5 H: 6.8 ± 0.8 O: 52.0 ± 6 (2) Molecular weight about 2,500,000 to 500,000 (3) Specific rotation [α] ²⁵ _D = 58 -68 (C = 0.3%) (4) Melting point A brown color starts to change from around 220 ° C to a brown color around 250 ° C. (5) Infrared absorption spectrum (KBr method) 3410, 2932, 2899, 1646, 1419,
1363, 1149, 1078, 1034, 894, 7
88cm ^-1 (6) Solubility in solvent Soluble in water, insoluble in methanol, ethanol, acetone, ether (7) Color reaction (a) Maurish reaction + (b) Anthron reaction + (c) Cysteine-sulfuric acid reaction -(D) Carbazole-sulfuric acid reaction- (e) Elson-Morgan reaction- (f) Ninhydrin reaction- (8) Basic, acidic or neutral 0.3% aqueous solution shows pH 7.5. (9) Color of substance The freeze-dried product has a white fiber shape. (10) Types of composition and composition ratio Glucose: Galactose = 1.0: 0.8 to 1.2 2. A method for producing the polysaccharide substance TPSB, which comprises culturing a TPSB-producing bacterium belonging to the genus Lactobacillus in a medium, allowing the polysaccharide substance TPSB to be produced and accumulated in the culture, and collecting the TPSB. 3. A TPSB-producing bacterium belonging to the genus Lactobacillus is Lactobacillus helveticus TAKE-0.
1 (Lactobacillus helveticus TAKE-01) (FE
The production method according to claim 2, which is RM P-14411). 4. A B lymphocyte proliferation inhibitor containing the polysaccharide substance TPSB or a derivative thereof as an active ingredient.