JPS6044918B2 - Method for producing polysaccharides using microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to polysaccharides produced by spore-forming lactic acid bacteria and methods for producing the same, and more particularly, to novel water-soluble fructans produced by mesophilic spore-forming lactic acid bacteria and polysaccharides produced by mesophilic spore-forming lactic acid bacteria. Relating to a method of manufacturing.

It has been thought that so-called lactic acid bacteria, which metabolize glucose and convert more than 50% of consumed sugar into lactic acid, do not form endospores. Although metabolically it is no different from lactic acid bacteria, taxonomically it is an endospore-forming bacterium called Bacillac.
Bacteria belonging to the genus Eae) are called spore-forming lactic acid bacteria.
Spore-forming lactic acid bacteria include a group of thermophilic bacteria that produce dextrorotatory lactic acid and have an optimal growth temperature of 50°C, and a group of mesophilic bacteria that produce racemic lactic acid that is not levorotatory and have an optimal growth temperature of 45°C or lower. It is classified as a spore lactic acid bacterium.

Among the spore-forming bacteria, Bacillus subtilis
Among the lactic acid bacteria, levan produced by Leuconostoccus mesenterophides (
Leuconostocmesenteroides)
, Leuconostoccus dextranicum (Leucon)
Although it is known that bacteria such as L. ostocdextranicum produce polysaccharides, there have been no reports of spore-forming lactic acid bacteria producing polysaccharides.

Evening The present inventors discovered that Bacillus levoracticus (Ba.
4 belonging to cillus laev Olacticus)
that the strain produces insoluble glucans under the conditions described below, and together with it produces water-soluble fructans in solution;
and these four strains as well as other Bacillus revolactis, B. racemilactis
icus) and Sprolactobacillus (SpOrOIa
ctObacillLls) that produce water-soluble fructans, and that these glucans and fructans are polysaccharides different from the polysaccharides produced from the conventional spore-forming bacteria or lactic acid bacteria mentioned above, and that they are water-soluble. It was discovered that a water-soluble polysaccharide hydrolyzed from flutane and insoluble glucan exhibits physical properties similar to dextran and can be used as a plasma substitute, as well as exhibits some anticancer properties and can be used as a raw material for drugs, and the present invention was completed. did.

First, among the mesophilic lactic acid bacteria, Bacillus levoracticus (B.
M-4, -66, -68 and -71 strains belonging to A. acillus laev Olactic Lls produce water-soluble polysaccharides when cultured in a medium containing sucrose as a carbon source at 200C to 40C, preferably around 300C. but,
When using bacterial cells that have just sprouted from spores for inoculation,
Insoluble polysaccharides are produced, and when the culture medium in which insoluble polysaccharides themselves have already been produced after proliferation is inoculated twice as a seed and subcultured on a sucrose medium, insoluble polysaccharides are similarly produced and water-soluble polysaccharides are produced in the solution. It was discovered that a polysaccharide in the form of a sol was produced.

Furthermore, 0.1N sulfuric acid was added to this insoluble glucan and hydrolyzed over a boiling water bath.
200 (crosslinked dextran gel)! When filtered, K
It becomes water-soluble while maintaining a high molecular weight such that d becomes 0.

This insoluble polysaccharide was hydrolyzed with 1N sulfuric acid over boiling water for 5 hours, and then analyzed by paper chromatography.
After MS conversion and gas chromatography, a peak containing only glucose was obtained, and it was confirmed that the glucan was substantially composed only of glucose.

The water-soluble sol polysaccharide produced at the same time was precipitated by adding a mixture of 4 equal amounts of ethanol and ethyl ether, hydrolyzed with 0.1N sulfuric acid over a boiling water bath for 10 minutes, and then subjected to paper chromatography. Then, a peak containing only fructose was obtained by TMS and gas chromatography.
It was confirmed that this is a fructan composed essentially only of fructose. Note Tomari paper used for paper chromatography, Toyoro paper No. 5O, the solvent used is as follows, detection is aniline phthalate (a) n-propanol: ethyl acetate: water = 14:2:7 (b) n-butanol: acetic acid: water = 60:15:25 (c) n-Butanol reethanol: water = 4: 1:5 (upper layer used) (d) Phenol: 1% ammonia water = 4:1 In addition, Fig. 1 shows the results of gas chromatography for water-soluble polysaccharide hydrolyzate. This is shown in the chromatogram.

In the figure, 1 indicates the peak of n-eicosane and 4 indicates the peak of TMS-Flagdose, indicating that no sugar other than the corresponding sugar is contained.

It was surprising that the water-soluble polysaccharide was not a lower-order polymer of insoluble polysaccharides, but a separate polymer.

Furthermore, these 44 strains M-4, -66, -68 and -7
One strain produces completely insoluble polysaccharides when the seed mother is cultured with glucose and the carbon source of the main medium is sucrose; only water-soluble polysaccharides are produced; was exactly the same polysaccharide as the fructan co-produced with the above-mentioned insoluble polysaccharide.

Furthermore, among the mesophilic spore-forming lactic acid bacteria strains isolated and identified by the inventors, the following strain was confirmed to independently produce a sol-like water-soluble polysaccharide in a sucrose medium.

Note: Among the above, Bacillus levoracticus M-4 is ATCC Bacillus levoracticus M-1 is ATCC Bacillus levoracticus M-7 is Microkenbacterium 544* The four strains marked with 544* are polysaccharides that are insoluble depending on the culture conditions as described above. produce,
Depending on the conditions, some strains can only produce water-soluble polysaccharides.

The b strain listed in the above table produces only water-soluble polysaccharides when it is inoculated into a medium containing sucrose and cultured at 20°C to 40°C, preferably 30°C.

This water-soluble polysaccharide was hydrolyzed with 0.1N sulfuric acid over boiling water for 10 minutes, and then subjected to paper chromatography.
In addition, by TMS and gas chromatography, the first
Obtaining a peak identical to the fructose-only peak shown in the figure,
It was confirmed that the above-mentioned Bacillus revolacticus is the same as the fructan produced together with glucan, which is substantially composed only of fructan.

Next, the results of paper chromatography of the hydrolyzate in this case are shown.

1) Mesophilic spore-forming lactic acid bacteria used for the production of polysaccharides have the following properties.

Common characteristics Microscopic findings: 0.3-1.0X1.7-5.
0 micron rods.

They move singly or in short chains and are driven by circumferential hairs. 0 spores
．． It has an oval shape of 9 to 1.5 x 1.2 to 2.1 microns and is attached to one end of the cell or near one end. Gram staining: Positive Agar colonies: No or almost no growth occurs on agar media that do not contain fermentable carbon sources. Colonies on agar media that contain fermentable carbon sources are white or colorless and translucent and convex circular. , it will not spread.

Broth culture: No or very little growth occurs on a medium that does not contain a fermentable carbon source.

When it contains a fermentable carbon source, mixture or precipitation occurs, no bacterial membrane is formed, and acid is produced. Litmus milk: It remains unchanged or only slowly produces acid and does not undergo peptonization.

Gelatin: No change. Nitrate reduction, indole production, and hydrogen sulfide production were all negative.

Growth temperature: 20-40°C Carbon source availability: D-glucose, D-galactose,
Acid is produced from D-mannose, D-fructose, and maltose, no acid is produced from L-arabinose and D-xylose, and acid is not produced or is produced slowly from lactose.

Acid production from sucrose, dog starch, etc. differs depending on the strain. Other properties: By anaerobically injecting dextrose, more than 70% of the consumed sugar is converted to lactic acid, and no gas is generated anaerobically.

DNA(7)GO content is 45-46%. The individual properties of each strain are as follows.

Bacillus levoracticus (Bacillus laev)
Olactjcus): Has catalase and produces levorotatory lactic acid.

Strains that only produce water-soluble polysaccharides include M-1, M-7,
Strains that produce M-104, M-10\water-insoluble polysaccharides include M-66, M-4, M-68, and M-71. Among them, Bacillus revolacticus M-4 is ATCC2
3549, Bacillus revolacticus M-1 is ATC
C2349 Atsushi and Bacillus revolacticus M-7 have been deposited as FAIKEN Bacteria No. 544, respectively.

Bacillus racemilacticus (Bacillus racemicus)
emilacticus): has catalase and produces racemic lactic acid.

M-14 (Feikoken Bacteria No. 5
No. 46). SporOIactObaciIluslaevus
) : Produces levorotatory lactic acid, which has catalase.

M-87, M-88, M for making water-soluble polysaccharides
There is -118. Sporolactobacillus racemics (
SpOrOlactObacillLlsraCePi
CLlS): Does not have catalase and produces racemic lactic acid.

Examples of water-soluble polysaccharides include M-86 and M-103. Comparing the general properties of these polysaccharides of the present invention with known representative polysaccharides produced by spore-forming bacteria or lactic acid bacteria, the polysaccharides of the present invention differ in properties as follows. It is clear that it is a different polysaccharide. Next, the method for producing the polysaccharide of the present invention will be described.

As a carbon source for producing the polysaccharide of the present invention using these mesophilic spore-forming lactic acid bacteria, only sucrose is effective; other sugars such as D-xylose, L-arabinose, and D-
Galactose, D-glucose, D-mannose, D-
Flagdose, D-glucose + D-flagdose, maltose, lactose, trehalose, raffinose, merisitose, inulin, dextrin, soluble starch, rhamnose, α-methyl glycoside, mannitol, sorbitol, common oligosaccharides and polysaccharides such as sodium gluconate,
All sugars such as monosaccharides, sugar derivatives, and polyhydric alcohols cannot be used as raw materials for producing the polysaccharides of the present invention, and invert sugars are also not acceptable, and those to which xylose is added are extremely poor.

The suitable addition rate of sucrose is 1% to 10% (weight/volume, hereinafter, % means weight/volume % unless otherwise specified), particularly 2 to 8% for insoluble glucan production, and 2 to 8% for fructan production. A concentration of 2 to 4% is preferable, and a concentration of 10% or more is rather unsuitable.

All other nutrients used for culturing microorganisms can be used, but organic nitrogen sources such as yeast extract and peptone give preferable results, and inorganic nitrogen sources may be used as supplementary nitrogen sources.

In particular, magnesium phosphate, which is commonly used for culturing microorganisms, is required as an inorganic salt, and it is usually sufficient to use a commercially available salt mixture. The culture temperature should be 20 to 40 degrees Celsius, preferably around 30 degrees Celsius, and the pH should be in the range of 4 to 8. If the pH is below 4, polysaccharides will not be produced, and neutralization with calcium carbonate will not be favorable for glucan production, and fructans will not be produced. It produced good results.

However, if it becomes too alkaline, it tends to produce lactic acid, which is not preferable for producing polysaccharides. Although aeration brings about favorable results for the initial production of bacterial cells, it is possible to produce the polysaccharide of the present invention by a slight amount or by static fermentation. Bacillus
For Levoractis, M-4, -66, -68 or -7
If you use 1 and transfer the seed mother to a glucose medium and use it as the seed mother, glucan will not be produced, so in order to produce glucan, you should use a new seed mother that has germinated from the spores.
Alternatively, insoluble glucan can be obtained by producing gel-like glucan on a seed agar medium, inoculating it into a liquid medium as a seed medium, and subculturing it on a sucrose medium.

On the other hand, in the production of fructan by the lactic acid bacteria of the present invention, if the above-mentioned production strain is cultured in a sucrose medium, fructan will be formed unconditionally. However, in order to produce only soluble polysaccharides from the above-mentioned strains that produce insoluble polysaccharides, it is possible to use a seed mother that has been cultured for one passage in a glucose medium. Both are produced in the medium about 3 to 10 days after inoculation and culture, and in the case of insoluble glucan production, a solid gel-like mass is produced in the medium, separated from the liquid, and washed. Afterwards, a sticky liquid remains due to the sol-like polymer. Wash solid lumps with running water and lightly dissolve them with sulfuric acid (a). 1N, 10 minutes to 2 powders over boiling water) It becomes a smooth liquid with no viscosity, so it is centrifuged to remove bacterial cells, dialyzed, and freeze-dried. This substance is a pure white cotton-like substance, and although it is not hygroscopic, it is extremely soluble in water.When dissolved in water, it becomes a smooth and transparent liquid, making it easy to purify. Purification involves repeating the steps of solvent, precipitation by addition, and dissolution with water. The solvent used for precipitation is ethanol and ethyl ether or a mixture of equal amounts of methanol and ethyl ether. The yield of insoluble polysaccharide is about 25% based on sugar when recovered as a hydrolyzate, and when the sugar concentration is 8%, it is 20 gI0. As mentioned above, the viscous liquid that remains is not a solid low polymer of glucan but a fructan. In the case of the above 4 strains of Bacillus levoractis that only produce fructans, 1 strain of Bacillus racemiculacticus, 4 strains of Sporolactobacillus levus, 2 strains and 11 strains of Spororactobacillus racemicus, and Bacillus levoractis that produces insoluble polysaccharides. It was confirmed that the production of water-soluble polysaccharides produced when subculturing seeds in a glucose medium and culturing them in a sucrose medium also accumulated in about 3 to 10 days and did not increase after that. After separating and removing the bacterial cells, it can be purified by repeating dialysis, precipitation by adding a solvent, and dissolution in water.

Acetone is suitable as a solvent for precipitation, and the product is purified by repeating dialysis and acetone precipitation, followed by freeze-drying. The yield of this water-soluble polysaccharide is small, only a few percent per ton. The physical properties of the purified fructan product obtained as described above are as follows.

Purification method Culture solution → Centrifugation → Acetone precipitation of supernatant → Dissolution → Acetone precipitation → Dissolution → Dialysis → Lyophilization → Column chromatography 1 → (DEAE-cellulose (PH8.O) and Sepharose 2B) → Freeze drying 1 ．． Elemental analysis (Perkin-Elema 2nd model elemental analyzer) 2. Molecular weight Cepharose CL-? However, it flows out to Kav=o, and its molecular weight is 20×1Cf' or more.

The pattern is shown in FIG. 3. Melting point (this micro melting point measuring device can-52) 183-186°C 4. Specific optical rotation (PO-B Hitachi polarimeter) [α] starvation = -48 of 0.1
% aqueous solution 5. Ultraviolet absorption spectrum (Shimadzu double beam spectrophotometer grinder-200S) 0.1
% aqueous solution terminal absorption only 6. Infrared absorption spectrum (EPI-S? Hitachi infrared spectrophotometer) The spectrum diagram of KBr tablet shows furanose type absorption.

(Figure 3)7. Solubility Water 〉100m91m1
Methanol insoluble ethanol
Insoluble propanol
Insoluble Chloroform insoluble Acetone insoluble8.
Color reaction Iodide starch reaction 1 Fehring reaction 1 Aniline-phthalate reaction 1 Alkaline silver nitrate reaction 1 Orcinol method (pentose) − Phloroglucinol monohydrochloric acid method (
pentos)
1 Dishe' S CarbazOle method (acidic sugar)
-Dishe2s (deoxy sugar)
- Elson Morgan method (1) Anthrone method (10) Phenol sulfuric acid method
19. Basic, acidic, neutral
(TOAUM-5APH meter) 1% aqueous solution 20°C PH6.79-4.13 Neutral 10. Color of substance: white powder, odorless, tasteless 11. viscosity
Oswald viscometer (20±0.1 C) 1%
15.06 millipoise Reference dextran 1% liquid molecular weight 100,000-200,000 136,120,000-300,000 14.59 500,000 16.19 200,000 18.81 Water 10.09 Intrinsic viscosity 0.29 (25 ± 0.07C ) That is, it is clear at this stage that the water-soluble polysaccharide of the present invention is a polymer of fructofuranose.

The results of testing the antitumor properties of the water-soluble polysaccharide of the present invention are shown below (the toxicity of both was 12 m9/mouse (500 m
91k9) The test mice were ICR mice, 5 weeks old, obtained from Chaarsliba, and used at a weight of 24±1 g after 4 days of pre-breeding.

SarcOma-1180 was obtained from cancer nocenta strain mice that were passaged weekly into the above ICR mice.
Ascitic fluid was collected and I
2×1σ/0.25m1 subcutaneously in the right inguinal region of CR mice.
/ Mouse transplanted.

Treatment begins 1 day after tumor transplantation with each dose of physiological saline 0.
．． It was dissolved in 25 mL and administered by intraperitoneal injection once a day for 10 consecutive days, and each week the solid tumor area (major axis x minor axis),
On the last day of the 5th week, solids were extracted and the inhibition rate was calculated.

The results are shown in the table below. Based on this result, administer an appropriate amount (
Water-soluble polysaccharide 2.4m9/mouse-insoluble polysaccharide hydrolysis 6
m9/mouse), the inhibition rate was 60%. Example 1 Bacillus laev
M-4 strain ATCC23649, which has the ability and ability to produce water-insoluble and water-soluble polysaccharides.
Use.

A slant medium consisting of 0.5% peptone, 0.3% yeast dust, 0.05% dipotassium phosphate, 2% sucrose, and 1.5% agar was inoculated with M-4 spores and incubated at 30°C. When cultured for one day, a translucent gel-like water-insoluble polysaccharide mass is produced.

Remove the agar from the medium to create a medium containing 8% sucrose,
The translucent gel-like water-insoluble polysaccharide mass obtained from 100 mL of the agar medium was aseptically inoculated into 20e which had been sterilized by heating at 110'C for 5 days at 30°C. Sugar lumps grow large.

Cover this with a saran cloth and add the liquid. This Oki liquid was centrifuged at 10,000 rpm for 3 minutes to remove bacterial cells, and then treated with pronase using a conventional method. 10 g of activated carbon was added and mixed, left at 5°C for 12 hours, and filtered by suction. Add 20e of acetone to the Oki liquid to form a precipitate, dissolve the resulting precipitate in 500TL of pure water, put it in a cellophane bag and dialyze against pure water for 24 hours, add 500LILL of acetone to the dialysis solution. In addition, a precipitate was generated, and the precipitate portion was dissolved in a small amount of pure water and freeze-dried to obtain 24 g of a white lumpy water-soluble polysaccharide (fructan). The anticancer effect test results of this product are as described above.

Example 2 Bacillus revolacticus was added to a liquid medium 20e consisting of 1% corn stave liquor, 0.02% ammonium phosphate, and 5% sucrose.
lluslaevOlacticus)) M-1ATC
After inoculating C23493 and culturing at 30'C for 2 days, dissolve 1k9 of sucrose in 2' of pure water, heat 1 powder to 115°C to sterilize it, add the solution, and culture at 30°C for another 4 days. The obtained liquid was treated in the same manner as Oki liquid I in Example-1 to obtain 15 g of water-soluble polysaccharide (fructan).

Example-3 Bacillus laevolacticus M- of Example-2
When the same procedure was carried out using 7 Microtechnical Research Institute No. 544,
17 g of water-soluble polysaccharide (fructan) was obtained.

[Brief explanation of the drawing]

Figure 1 shows the TM of the water-soluble polysaccharide fructan hydrolyzate of the present invention.
Showing the gas chromatography pattern of S derivatives,
Figure 2 shows the same fructan, Cepharose CL-? Figure 3 shows the infrared absorption vector (KBr tablet) of the same fructan.

Claims (1)

[Scope of Claims] 1 A polymer belonging to the genus Bacillus, classified as mesophilic spore-forming lactic acid bacteria, and consisting essentially only of fructose, which has the following physical and chemical properties A) Molecular weight: 2
0x10^6 or more B) Melting point: 183-186℃ C) Specific rotation: [α] ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ (0.1% aqueous solution) D) Solubility: Water > 100 mg/ml Insoluble in methanol Insoluble in ethanol Insoluble in propanol Insoluble in chloroform Insoluble in acetone A method for producing a polysaccharide, which comprises collecting the polysaccharide. 2 The microorganism is Bacillus laevolacticus M
-4 (ATCC23549), M-1 (ATCC23
493), M-7 (Feikoken Bacterial Serial No. 544) or Bacillus racemi lacticus
milacticus) M-14 (Feikoken Bacterium 546
2. A method for producing a polysaccharide according to claim 1, which is