JPS58129001A - Novel immunoligically active polyglucide and its preparation - Google Patents

Abstract

PURPOSE:To obtain the titled novel polyglucide useful as an immune enhancer having low side effect, etc., having specific molecular weight and main repeating unit, by cultivating a bacterium belonging to the genus Klebsiella capable of producing polyglucide A29-PS in a medium, collecting the polyglucide from the culture. CONSTITUTION:A bacterium (FERM-P 6317) belonging to the genus Klebsiella, capable of producing A29-PS is cultivated in a medium, polyglucide A29-PS is formed and accumulated in the culture, it is collected from the culture, and, if necessary, it is partially decomposed with an acidic substance, and a partially decomposed product is collected to give the desired polyglucide having about 20,000-300,000 molecular weight and a main repeating unit of a saccharide part shown by the formula (D-GluA is D-glucuronic acid residue, D-Man is D-mannose residue; D-Gal is D-galactose residue; R is a group wherein hydroxyl groups of C4 and C6 of D-gal are replaced with pyruvic acid residue and hydroxyl groups of C2 and C3 are replaced with acetyl group).

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel polysaccharide A29-PS and a method for producing the same.

When certain types of bacterial endotoxins that exist in living organisms (such as ribopolysaccharides, which are endotoxins of Escherichia coli) are administered parenterally, they are inhibited by their cellular activation or reticulostimulatory effects. It is known to increase resistance to infection by various pathogens. In particular, enhancing the immune activity of cancer patients who are susceptible to infection by pathogens due to decreased immune capacity is an extremely effective treatment method.

However, conventional immune-enhancing treatments such as lipopolysaccharide, which is known as an intracellular toxin of Escherichia coli, are considered inappropriate as therapeutic agents because of their side effects of fever and toxicity.

The present inventors conducted various studies in order to obtain a biologically active substance that not only can impart strong resistance to various pathogens to living organisms but also has fewer side effects.
We have clarified the existence of polysaccharides in the culture solution of 29) that have a strong stimulating effect on the reticuloendothelial system and can increase the resistance of living organisms to non-specific infections, and have also investigated their chemical properties. This polysaccharide is considered to be a polysaccharide with a new structure, and this new polysaccharide was named 121-PS.
Based on these findings, the present inventors further determined that KiIIF
As a result of our research, we have completed the present invention.

The present invention is characterized in that: (1) the molecular weight is about 20,000 to 300,000, and the main repeating unit of the saccharide moiety is of the formula % (1 [wherein, D-Glum represents a D-glucuronic acid residue; D-
ManFiD-w is a base residue, D-Gal is a b-galactose residue, R is a D-galactose residue, the OC4 and Cgo hydroxyl groups are replaced by pyruvate residues, and the C2 and C30 hydroxyl groups are mainly acetyl residues. Polysaccharide A represented by ], each representing a group substituted with a group
29-PS (hereinafter abbreviated as "A29-P8J"), and (2) polysaccharide A29-PS producing bacteria belonging to the genus Talepjiev are cultured in a medium, and polysaccharide A29-PS is present in the culture. This is a method for producing polysaccharide A29-PS, which is characterized by producing and accumulating polysaccharide A29-PS, collecting it from a culture, partially decomposing it with an acidic substance if necessary, and collecting a partially decomposed product.

In the method of the present invention, A2 belonging to the genus Klepzieff
9-Pa production 1 is used. A representative example of this A29-ps producing bacterium is Klebsiella sp. strain A29, which was newly isolated by the present inventors from the soil of Nishinomiya City, Hyogo Prefecture.

The Society of American Bacteriology, Manual of Microbiology, A'
-Methods (Miorobiologioa1M@th
o? 18 1) 7 The 8001et7 0f
Am@ricanBaotsriologigs
The mycological properties of Klebsiella sp. A29 strain observed according to the method described in ) are those of a manure.

Microscopic findings 2 Short bacilli, 0.9-1.2 x 1.4-2.0 microns, present singly, with villi but no motility.

Guhum staining: Negative Colonies on agar: White, raised, convex circles Flesh culture: Mixed leakage, forming a fungal film Potato agar culture: Yellowish white, good growth Lydomus miμ
ri: peptonizes to produce acid Methyl red degree wide niacetiy, methip, force〃Pino- production: + Utilization of citrate: + Zeftin puncture culture: Rapidly liquefies reduction of nitrate: + Indo-... Production: + Production of hydrogen sulfide: + Growth temperature = 32°C to 37°C Availability of carbon sources: L-afvinose, D-glucose, D-gafuctose, D-mannose.

Acid and gas are produced from fuctose, octose, sucrose, maltose, sorbitol, and glycerin, but neither acid nor gas is produced from L-tumnose.

If the mycological properties of zero values are classified according to the description in Persis Manual μ op Deterministic Bacteriology, 8th edition, pp. 321-324 (1974), non-motile Ghum-negative short It is considered to belong to the Klebsiella genus K because of its properties such as being a bacillus, utilizing citric acid, producing acetyl-methoxy-pino-p, and being negative for methyl red reaction.

Furthermore, it is clearly different from the similar genus Enterobacter (Σnterobacter genus) due to its lack of motility.

However, the liquefaction of gelatin is extremely rapid;
Klebsiera pneumoniae (Klebsie-11a), which is positive for indole production.
pneumonia). From these points, this bacterial strain is recognized as belonging to a new bacterial species, and therefore Klebsiella
sp.

A29).

Klebsiella sp. A29 strain was transferred to Fermentation Research Institute (IN”O) K accession number I on January 11, 1930.
This microorganism was deposited as F O7tt/yr on July 2, 1932, and was given accession number FIRM to the Institute of Microbial Technology (IFR Engineering), Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
P, -63/7.

The properties of Taleguziev bacteria are easily changed, and can be mutated into spring crystals by, for example, X-ray irradiation, ultraviolet irradiation, radiation irradiation, artificial mutagenesis means using artificial mutating agents (eg, nitrosoguanidine, ethyleneimine, etc.). Even among such mutant strains, those that have the ability to produce Muku9-PaO,
All can be used in the method of the invention.

In the method of the present invention, the above-mentioned A29-PS producing bacteria belonging to the genus Craigjet is inoculated into a medium and cultured. The medium may be either liquid or solid, but a liquid medium is most conveniently used. Among cultures using liquid media,
Aerobic culture methods such as aerated agitation culture and deep aeration agitation culture are industrially advantageous. Ka in the medium, assimilable carbon source of Mu29-Ps production, i! Growth of Aspergillus Aspergillus and A2
Contains the substances necessary for the production of 9-ps. Examples of leg element sources include 7ctose and V kneecloth.

Various saccharides such as ma-dose, galactose, ricose, and mannose, and zero-saccharide aco-μ such as sopitose μ are advantageously used, and particularly high yields can be expected when fuctose is used. The concentration of these carbon sources multiplied by the medium K is 29
- Any concentration that allows P8 production strawberry to grow may be used, but usually about 5 to 10% (W/V) is convenient. Further, as the nitrogen source, for example, in addition to yeast extract, organic substances such as meat extract, defatted soybean, fish meal, cornstarch liquor, or ammonium sulfate salts can be used. In addition, small amounts of inorganic salts such as common salt, phosphates, metal salts such as 1M lead, manganese, and iron, and micronutrients are added to the medium as appropriate.

Culture conditions are selected depending on the composition of the medium, the bacterial strain used, etc., but the culture temperature is usually the optimum temperature for the bacteria used.
For example, the temperature is preferably about 25 to 32°C, and the liquid nature of the medium is preferably around neutrality. Culture time is approximately 30 hours to 200 hours.
It's time.

A2B-ps belonging to the genus Klepziev as described above.
When A29 is cultured, A29 is added to the medium as the culture progresses.
-PS is generated and stored. To collect the A29-Pa accumulated in the medium in this way, any means for collecting substitute products produced by microorganisms from the culture of the microorganisms can be conveniently used. A29-ps can be collected at any purity by using these means alone or repeatedly or in combination as appropriate.

For example, various physical chemistry differences such as solubility in solvents, partition ratio between two liquids, affinity for various adsorbents, dialysability, electrical transport, and reactivity to various reagents between active substances and impurities. A method that takes advantage of differences in physical properties is used. For example, A29-PS in the culture medium can be collected in high yield by purification assay (as described below).

That is, A29-PS obtained by the above-mentioned culture
If, for example, ethyl alcohol μ, acetone, etc. are added to the liquid obtained by removing insoluble matter such as bacterial cells from a culture of the producing bacteria, the desired mu-29-PS can be obtained as a precipitate. . Mu29- obtained in this way
Pg, like purified A29-PS described below, exhibits a strong resistance-enhancing effect against a wide range of infections when administered parenterally to living organisms, but it contains a small amount of inactive neutral carbohydrates. In order to further refine this product, K used the crude product as
A29-FB can be used as a purified product when buried with acidic carbohydrates and precipitated complexes that exhibit biological activity, such as cetacean, pyridium, crophyte, cetacean, trimethyl, ammonium, bromide, etc. Obtainable.

Although A29-PS is obtained as a sodium salt by the above method, it can also be converted into a free form by commonly used means. For example, by dissolving IJum filler in water and applying it to a strongly acidic resin [e.g. Amberphyte IR
-120 (manufactured by Rohm & ψ Haas, U, S, A
) or DOWEX 50 (manufactured by Dow Chemical Company, U, 8.M2), or passed through a cuff previously filled with the resin and washed with water. Free A29-Pa is obtained by adding several times the amount of acetone, etc. to a solution containing these passing liquids and washing liquid, precipitating it, dialyzing it against water, and freeze-drying it. It will be done.

The polysaccharide that can be collected from the culture medium according to the present invention is a polysaccharide with a new structure that has not been discovered so far, based on the properties described below. It was named -Ps.

A29-PS obtained in Example 1 described below has the following properties.

1. Uniformity: Gives a single peak by ultracentrifugation and Geyv'f5 filtration method.

2. Molecular weight: varies depending on culture and purification conditions, ranges from approximately 20,000 to 300,000 (ultracentrifugation method).

3.N content: hardly detected (less than 0.07%)
.

4. Phosphorus content: Not detected.

5. Specific optical rotation: [α] I+203′″~21o.

(C-1, Q, water) 6. Solubility: water, caustic soda solution below 11.

Easily soluble in formic acid, homeamide, and demethoxylic acid. Methanol, acetone, chloroform, acetic acid 5 tons
w @ K insoluble.

L Appearance, taste, odor: White, tasteless and odorless powder.

8. Jif! s summer response: A degree of phenol sulfuric acid degree: 1ili Dish (]) iaoh) power ~ Pazo A / degree response: positive ninhydrin degree of degree: negative 9, a〃force neutralization equivalent: carboxy When the y group is in a free form -, the result of neutralization titration with (LO5a) aOH is 738-740.

10! ! The ratio of these constituent walls is approximately 3-3.2:0.8-1.0:0.9-1.1. In addition, D-glucuronic acid is obtained by hydrolyzing A29-PS with acid and then reducing the aldehyde group of the uronic acid moiety)
The altonic acid (D-gluconic acid) produced was confirmed by gas chromatography as a trimethyl μVsu derivative after Tucton's death, but the uronic acid residues of KA29-PS were further reduced to the corresponding neutral sugar residues. It was also identified as D-glucose by subsequent hydrolysis.

The material also contains a small amount of pyruvic acid (0.
95-11.9%).

11. Constituent sugar residue: The constituent sugar residue is partially 0-acetylated, and its content is about 6.5 to 7.0 acetylated. Furthermore, about 2% of higher fatty acids, mainly consisting of β-hydroxy V-myristic acid, are bound to sugar residues through Es-D-μ bonds.

12. Infrared absorption spectrum: Infrared absorption spectrum/L/
(KBr) is the standard in FIG. Absorption near 84OcIII indicates the presence of α-bonds of D-series sugar residues,
The absorption at 1725m and 1250m is due to the acyl group mainly consisting of acetyl group.

13. Distinction between basic, neutral, and acidic: The free form is an acidic substance.

In addition, we investigated A2B-ps by chemical means such as methylation, periodic acid oxidation, complete ST, X decomposition, and relaxed Smith decomposition. It was recognized that it is a novel heteropolysaccharide with a structure. That is, (4) (1) the hydrolyzate of methylated 9A29-P8 was subjected to chromatography, gas chromatography (as an acetyl derivative after reduction to aldito), and mass spectrometry. 〃By means such as law 2, 4.
6-tree 0-methy~-D-mannose 1 mo/l', 2
, 4.6-) Lee O-methyl-D-gafuctose 2.1
9 moA/l, 2.3-di0-methyl-D-gafuctose Q, 68 moA/l and a trace amount of O2, 3,4,6-tetra-
Separate 0-methy/&-D-gactose. moreover,
By gas chromatography performed as a methyl glucoside derivative), the presence of 3-0-methyμ-D-glucuronic acid was discovered.

(ii) Reduction of A29-PSOD-glucuronic acid residue (
Therefore, in the methylation analysis after changing to D-glucose group,
2,4.6-tri〇-methyl-D-mannose 1.0
Mo~, 2,4,6-tri〇-methyl-D-gafuctose2. ．． 2mol/M, 2,3-di0-)flV-D-ga')9dose Q, 7w7v, 0.9 mol of 3,5-di0-methyl-D-glucose was obtained.

(iii) A29-PSOD-g/L1oyll, 11
7C4kt was heat-treated with dilute acid at pH 2.0 at 90°C for 2.5 hours to remove pyruvic acid as much as possible, and methylation analysis of the modified product revealed that it was 2,3-di-0-methy- Paralleling the decrease in D-gafuctose, new ftK2,3.

4. Since 6-tetra-0-methyμ-D-gafuctose was formed, the pyruvate group is the D-
It was estimated that ketal bonds were present at positions 04 and 06 of galactose residues.

(B) When A29-Ps is hydrolyzed with 0.5N sulfuric acid at loO'c for 2 hours, in addition to D-glucuronic acid, D-glucuronic acid and D-mannose force, α-(
1→3) Bound altobiuronic acid.

Both D-glucuronic acid, D-mannose and altobiuronic acid consisting of D-galactose were produced.

(C) A 29- P 8 is in the 11th state,
Although it is hardly oxidized by periodic acid, it is oxidized by periodic acid when the alkali (a, 51 NaOH, @ warm') Thea 501/ group is eliminated, and further, when it is completely hydrolyzed after reduction with NaBH, D-gafuctose, In addition to D-mannose and D-glucuronic acid, a small amount of slate
is produced, it is presumed that the acetiμ group, which is mainly an acetiμ group, is ester-bonded to C2 or C5 of the D-galactose residue at the end of the branch to which pyruvate is bound.

(D) A29-Pa subjected to the deacylation and carboxyl reduction operations described in Section 0 was subjected to relaxed Smith decomposition, and the methylation analysis of the polymer Smith decomposed polysaccharide revealed that 2.4.6-
) so 0-methyl-D-mannose 1 mo μ, 2,4.6
-) Di-0-me-thousand-D-gafuctose 1.7-2.0
In addition to 毫μ, 3,4.6-tri〇-methyμ-D-glucose (0.8 samples) was produced.

Judging comprehensively from the results of (4), CB), ((j), and (to) above, A29-Pa is (1) D-
Galactose.

D-glucuronic acid and D-mannose are composed of approximately 3:1:1 ratio, and (2) in the repeating unit, the main chain has 2 mo~α
-(1→3) bond D-galactose.

α-(1 → 3)-linked D-mannose residue of 1 cell, and α-D-gafuctose residue branched from C4 of α-(1 → 2)-linked D-glucuronic acid residue. are combined. (3) Methylation analysis after removal of pyruvate,
2,3- obtained from the methylated product of the original A29-Pa
Dimethy-D-galactose disappears and at the same time 2, 3, 4
．． From the appearance of 6-tetra-0-methyl-D-gafuctose, it is clear that pyruvate is located at positions 04 and 06 of the galactose terminal group. (4) o1u*(1″-3)Man and Q from partial hydrolyzate
Since luA→Man4Ga1 is obtained, these sugars H
It can be seen that there exists (5) Deacetylated postmortem relaxed Smith degradation causes the disappearance of D-galac and dosing residues that form branches, and methylation analysis of the remaining polysaccharide shows that D-glucuronic acid residues form (1-2) bonds. Since it is present in the main chain, a galactose residue forms a branch from the C4 position.

From these experimental results and considerations, it can be concluded that polymorphic A2g-ps
The main repeating unit of the sugar moiety has the following structure.

D-glu D-man D-gala D-gafukuronic acid Norsuctose Ctose D-gafuctose A29-Since the free form of PS is an acidic substance, it can also be made into a salt using a conventional method4. Examples include, for example, sodium salts, potassium salts, ammonium salts, and the like. Furthermore, in the method of the present invention,
It is also possible to further partially decompose A29-PS to obtain an appropriate partial decomposition product that reduces the molecular weight without reducing its immune activity and further reduces side effects such as pyrogenicity. good. That is, purified A29-Pg
The purpose can be achieved by partially decomposing K with an acidic substance. Examples of this acidic substance include mineral acids such as sulfuric acid, nitric acid, and hydrochloric acid, acetic acid, formic acid, and oxalic acid at dilute concentrations (concentration examples: approximately 0.01 to 1N).

Organic acids such as trifluoroacetic acid and amber phytoIR1
Cation exchange resins such as 20 and IRα 50 (manufactured by Rohm and Haas (USA)) are generally used. When A29-PS is heated to about 60 to 95°C in the presence of such an acidic substance, a partially decomposed product is obtained.
The intrinsic viscosity [η] decreases to about 0.10 to 0.30 (at 20°C).

Collection of the partially decomposed product is performed in the same manner as the collection method for A29-PS described above. A partially degraded product suitable for clinical use has a molecular weight reduced to about 1/5 to 1/10 that of the original purified product. The composition ratio of the two types of sugars constituting this partially decomposed product is exactly the same as the original polysaccharide, but
It is presumed that the AVA/group was partially eliminated from the results of infrared absorption and the like.

In this application, since this partial decomposition product also falls under the category of polysaccharide A29-Pa, it will also be referred to as ◆carbohydrate A29-P8.

As mentioned above, this polysaccharide A29-PS exhibits a strong resistance-enhancing effect against widespread NO infection cause 1 when administered parenterally, such as by intravenous or subcutaneous injection, to living organisms. Klebsi*1
1a pneumoniae), Pssuaomonasaerugl
nosa), Nijieri V A Koli (Esoh@ri-
ola ooli), Proteus vulgaris (
Protheths vulgaria), Staphylococcus aureus (8taph71ooooo
Some examples of experiments conducted using bacteria such as P. us aureus are as follows.

One group consisted of 10 mice weighing approximately 2Of, and one group was intraperitoneally administered with physiological saline alone, and the other group was intraperitoneally administered with 0.1we each of A29-ps 1W coral saline solution. , 3 days later Staphylococcus aureus (8taphyl
oooooua aureus) was infected by intravenous injection, and the number of survival days was determined. Two weeks after infection, the number of fresh pellets was 0/10 to 2/10 in the saline administration group, whereas it was 8/10 in the ice crystal A29-Pa administration group.
~9/10 and 9.

Cryolium 29-PS not only has the effect of increasing the body's resistance to infection, but also has a strong reticuloendothelial system cell activation effect. The immune-enhancing effects of ice crystals include: significantly promoting the blastogenesis of O-lymphocytes and mouse pancreatic-derived lymphocytes, increasing adjuvant activity, and significantly strengthening the predation effect of macrophages. From this, it is clear that it promotes lymphocyte function in the body.

Additionally, 30 minutes after intraperitoneal sensitization of mice with 0.1 ml of 20% sheep red blood cells, 100 μg of A29-ps dissolved in 0.5 ml of physiological saline was intraperitoneally administered to induce antibody production for 5 weeks. Comparing the ability using blood cell direct agglutination method,
It was found that A29-PS increases antibody production ability compared to the control.

The characteristics of A29-PS, which has such non-specific immune activity, as a therapeutic preparation include A29-PS, which is known to have a non-specific anti-infection effect similar to that of ice crystals. Compared to lipopolysaccharide, which is a toxin of Salmon. coli (]i:5oheriohia aoli) and Salmon. 11a, its pyrogenicity is less than 1/100 of that. For example, purified nine A29-P
When S was injected into the rabbit vein at a rate of 0.1 μg/#, 1 μg/#, and 10 μg/#, each of 0.1 μg/# and 10 μg/# within 24 hours.
-063°C, 0.6-1.2°C, and 2°C or less body temperature were only observed. Also ice crystal A29-P soI
, I) 5° is approximately 500 wf/# (mouse intraperitoneal injection).

As mentioned above, A29-PS has the effect of dramatically increasing the resistance of living organisms to various pathogenic bacteria, and has extremely few side effects on living organisms.

Moreover, a partial decomposition product of A29-PS also has the same effect as A29-PS.

Novel glycoside A2g-ps obtained by the method of the present invention
and・The partial decomposition product enhances the non-specific immunity of the living body without causing side effects such as pyrogenicity, and the I/a
significantly increases resistance to infection with I. In addition, this substance does not contain bacterial body structures such as conventionally known bacterial toxins, nor does it contain lipids, which are considered to be the cause of fever due to the so-called extracellular polysaccharide trade produced outside cells. Refining is extremely easy.

As mentioned above, the polysaccharide A29-ps of the present invention and its partial decomposition products enhance the non-specific immunity of living organisms and increase resistance to bacterial invasion, so they are effective against chronic or acute infections. patients with general immunodeficiency, congenital or acquired, such as those that occur during severe primary illness in old age, or patients with cancer who have undergone treatment that weakens their immune system, such as chemotherapy or radiation therapy. In some cases, blood-induced animals (e.g. mice) are used to suppress carcinogenesis when the carcinogenesis rate increases due to K, such as a decline in immune function (for example, aging).

(vii) Tablets manufactured by conventional methods for animals (cats, rats, dogs, cats, humans), *orally as decel preparations, or parenterally as injections manufactured by conventional methods. It can be administered to The dosage depends on the individual condition of the warm-blooded animal.
Depending on the species, age and dosage form, the preferred dosage is t1j'71-200 μg/# per day.

The present invention will be explained in more detail below using Examples, but the scope of the present invention is not limited thereby. However, the percentage of medium indicates the percentage of ZIF amount by weight.

Example 1 Lactose 5%, yeast extract 0.5%, K211[P
o.

0.05%, KH2PO, 0.05%, Mg50
．． 7H200,03%, CaC03o, 1% pH 7 medium 25011t was sterilized in an Erlenmeyer flask, and Kregziev sp, A29 strain (Engineering F
When O14145゜FΣRM 'I'-1,3/7) was inoculated and cultured at 27'C for 5 days, A2 g-ps was produced as lactose was consumed, and the culture became viscous. Ta.

The culture 11 thus obtained was heat sterilized at 80°C for 30 minutes and then centrifuged to remove bacterial cells and remaining CaC.
After O3 was removed, ethyl acetate was added to a concentration of 30%, and after death, centrifugation was performed again to obtain a clear supernatant. About 4 times the volume of ethyl acetate was added to this supernatant under stirring to obtain the desired A29-Pa as a precipitate. After washing this precipitate several times with ethiμaμcoμ,
When water is dissolved at 100vrlVc, dialyzed through a cellophane membrane, and the non-dialyzed portion is precipitated with ethyl alcohol, A29-
8f of crude mold of ps was obtained.

The A29-Pa crude product 2, Of thus obtained was dissolved in 1.51 g of water, and a 10% cetium pyridium crophyte solution was added thereto with stirring until no precipitate was formed.9. The collected precipitate was then dissolved in 10% saline water, centrifuged to remove trace amounts of insoluble matter, and about 3 times the volume of Ethia μcol was added to the supernatant to obtain a precipitate. .

When this is dissolved in water, dialyzed against distilled water, and then frozen and dried, purified polysaccharide A29-PS sodium salt 16F is obtained.

[α] +206° (C-1,0, water) Molecular weight 2 X
10' Components D-galactose 3.2 molar Sea mannose 1.0 molar to D-glucuronic acid 1.17 ruby to bic acid
11.9% 0-acetyl 6.7% Higher fatty acid ≦2.0% Example 2 A29-P82f obtained in Example 1 was mixed with 0.2M acetic acid 1
Melt 00sJK, heat at 95℃ for 1 hour, and cool it down.
Add alcohol-μ50-, remove a trace amount of precipitate by centrifugation, add about twice the volume of ethanol to the supernatant, and collect the resulting precipitate.] Partially degraded product ([η) -0
, 18) were obtained. The yield was about 70%. The constituent sugar ratio of this partially decomposed product was the same as that of the original A29-PB.

Weight pyrogenicity is 1γ/#, 0.0 at 10γ/9 administration
℃.

The temperature was 0.1°C. As a result of examining the infection resistance to Staphylococcus aureus using weight mice,
Two weeks after infection, the survival rate of the control μ group administered only with saline was 0/10 to 2/IOK, whereas in the group administered with weight,
The survival rate was 8/10 to 9/10.

Example 3 A29-PS2F obtained in Example 1 was mixed with 0.1M acetic acid 1
After melting 00sZK and heating it at 95°C for 3 hours, it was passed through a cuff of Sephadex G-50 (manufactured by Pharma Vya (Sweden)) to dissolve the partially decomposed product (rη) -0,15).
I got it. The yield was 90%.

Example 4 NineA29-Pa sodium salt 1f obtained in Example 1 was dissolved in 100-proof water, and Ampartite IR-120 (manufactured by Rohm & Haas, U.

The liquid was passed through a cuff filled with S, A, ), and then the cuff was washed with 50 wt of distilled water and combined with the passing liquid.

When about 3 times the volume of ethyl alcohol was added to this O solution, a precipitate was obtained. This was filtered into water KMI, dialyzed against distilled water, and then lyophilized to yield 129-PS (free form) 0.8F.
was gotten. The external absorption spectrum μ is shown in FIG.

[Brief explanation of drawings]

Figure 1 shows the external absorption spectrum μ of polysaccharide A29-PS sodium salt obtained in Example 1, and Figure 2 shows Example 4.
The external absorption spectra of the polysaccharide A29-PS (free W) obtained in .

Claims (2)

[Claims] (1) The molecular weight is about 20,000 to 300,000, and the main repeating unit of the saccharide moiety is the formula = 2 →・Totan-i←11・To blood meter ()-8)-<-D4
company ←) To F-(1→a↑ [In the formula, D-GluAFiD-glucuronic acid residue is D
-Man is a D-mannose residue, D-Gal is a D-galactose residue, R is a D-galactose residue in which the 04 and C6 hydroxyl groups are substituted with a pyruvate residue,
and 9 for C2 represents a group in which the hydroxyl group of C3 is mainly substituted with an acetyl group. ] Polysaccharide 2
9-PS. (2) Cultivate the polysaccharide 29-PS production plant belonging to the genus Klepziev in a medium, produce and accumulate KG carbohydrate 29-PS in the culture, collect it from the culture, and add it as necessary). A method for producing flea saccharide A29-ps, which comprises partially decomposing it with an acidic substance and collecting a partially decomposed product.