JPS6046959B2 - Method for producing polysaccharides using microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a polysaccharide produced by a spore-forming lactic acid bacterium and a method for producing the same. The present invention relates to a thawed solubilized glucan and a method for producing the same.

It has been believed that so-called lactic acid bacteria, which metabolize glucose and the like and convert more than 50% of the consumed sugar into lactic acid, do not form endospores.

Although metabolically similar to lactic acid bacteria, taxonomically it is an endospore-forming bacterium called Bacllaceas.
) are called spore-forming lactic acid bacteria. Spore-forming lactic acid bacteria have an optimum temperature for growth of 50℃ and are a group of thermophilic bacteria that produce dextrorotatory lactic acid, and mesophilic spore-forming lactic acid bacteria that produce levorotatory or racemic lactic acid and have an optimum growth temperature of 45℃ or below. Classified. Among spore-forming bacteria, there is levan produced by Bacillus subtilis, and among lactic acid bacteria, Leuconostoc mesenteroides is produced.
roides), Leuconostoc, dextranicum
It is known that spore-forming lactic acid bacteria produce polysaccharides, but there are no reports yet of spore-forming lactic acid bacteria producing polysaccharides.

The present inventors discovered that Bacillus levoracticus (Bacj.
Four strains belonging to B. lluslasv Olacus produce insoluble glucans and water-soluble fructans in solution under the conditions described later, and these four strains together with other strains of Bacillus levoractis and B. racemilacticus
and SpOrOlactobacillus (SpOrOlactOba
11 strains such as strains of the genus S. 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.
The four strains M-4, -6eK-68 and -71 belonging to A. acjllus laev Olacticus produce water-soluble polysaccharides when cultured at 20°C to 40°C, preferably around 30°C, in a medium containing sucrose as a carbon source. However, when inoculation is performed using bacterial cells that have just germinated and grown from spores, insoluble polysaccharides are produced, and a medium in which insoluble polysaccharides themselves have already been produced after proliferation is inoculated as a seed and transferred to a saccharide culture medium. It was discovered that when subcultured, in addition to producing insoluble polysaccharides, water-soluble sol-like polysaccharides were also produced in the solution.

Furthermore, by adding 0.1N sulfuric acid to this insoluble glucan and hydrolyzing it over a boiling water bath, Cephadex G-2
When passed through p-filtration with 00 (crosslinked dextran gel), the molecular weight remains high enough to have a Kd of O and becomes water-soluble.

Therefore, it is clear that the molecular weight of the insoluble glucan 3 of the present invention is at least about 200,000 or more. This insoluble polysaccharide was hydrolyzed with 1N sulfuric acid over boiling water for 5 hours and then subjected to vapor chromatography and TMS, gas chromatography 4 to obtain a glucose-only peak. It was confirmed that

The water-soluble sol polysaccharide produced at the same time was precipitated by adding an equal mixture of ethanol and ethyl ether, hydrolyzed with 0.1N sulfuric acid over a boiling water bath for 10 minutes, and then analyzed by vapor chromatography. After TMS conversion, a peak containing only fructose was obtained by gas chromatography, and it was confirmed that the fructan was substantially composed only of fructose. Note: The p paper used for vapor chromatography was Toyo P Paper No. 5O, the solvent used is as follows, detection is aniline phthalate (a) n-propanol ethyl diacetate diwater = 14:2:7 (b) butanol diacetate: water = 60:15:25 (c) n-butanol Niethanol and water = 4:1:5 (upper layer used) (d) Phenol and 1% ammonia water = 4:1 The results of gas chromatography are shown in Figure 1A. ．． Shown in B.

A shows a chromatogram of an insoluble polysaccharide hydrolyzate, and B shows a chromatogram of a water-soluble polysaccharide hydrolyzate. In the figure, 1 indicates the peak of n-eicosane, 2 indicates the peak of TMS-α-glucose, 3 indicates the peak of TMS-β-glucose, and 4 indicates the peak of TMS-fructose.
This indicates that it does not contain sugars other than the relevant sugar.

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

Furthermore, when the seeds of these four strains M-4, -6 Miichizoku, and -71 are cultured with glucose and the carbon source of the main medium is sucrose, they do not produce any insoluble polysaccharides, and only water-soluble polysaccharides are produced. Only polysaccharides are produced and its Yanghe water? The polysaccharide was exactly the same as the fructan co-produced with the above-mentioned insoluble polysaccharide.

The mesophilic spore-forming lactic acid bacteria used in the production of polysaccharides according to the present invention have the following properties.

Common characteristics Microscopic findings: 0.3-1.0 x 1.7-5.
0 micron rods.

They move singly or in short chains and are driven by circumferential hairs. 0 spores
．． It has an elliptical shape of 9 to 1.5 x 1.2 to 2.1 microns, and grows at or near one end of the cell. Gram staining: positive agar colony: no or almost no growth on agar medium containing no fermentable carbon source.

The colonies on the agar medium containing the fermentable carbon source are white to colorless and translucent, convex, circular, and do not spread. Broth culture: Grows or hardly grows 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 remains unchanged or slowly produces acid, and does not undergo pebtonization.

Gelatin does not liquefy. Nitrate reduction, indole production, and hydrogen sulfide production were all negative.

Growth temperature: 20-40C Availability of carbon sources: D-glucose, D-galactol,
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, inulin, starch, etc. differs depending on the strain. Other properties: Metabolizes glucose anaerobically, converting more than 70% of consumed sugar to lactic acid, and does not generate gas anaerobically.

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

Bacillus levoracticus (Bacillus laev)
M-66. There are M-4, M-6 fox and M-71.

Among these, Bacillus revolacticus M-4 has been deposited as ATCC No. 23549. Bacillus racemilactic
us): Contains catalase and produces racemic lactic acid.

M-14 is one that produces water-soluble polysaccharides. Sporolactobacillus Slaevu
s): Produces levorotatory lactic acid with catalase.

There are M-87 and M-wo M-11ton M-118 that produce water-soluble polysaccharides. SpOrOlactobacillus racemi
cus): Produces racemic lactic acid with catalase.

M-8e1SM-103 is one that produces water-soluble polysaccharides. Comparing the general properties of these polysaccharides of the present invention with known typical polysaccharides produced by sporozoic bacteria or lactic acid bacteria, the polysaccharides of the present invention differ in properties as follows. It is clear that the polysaccharide is different from that of other polysaccharides.

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 our mesophilic spore-forming lactic acid bacteria, only sucrose is effective; other sugars include D-xylose, L-arabinose, D-galactose, and D-glucose. , D-mannose, D-fructose, D-glucose + D-fructose, maltose, lactose, trehalose, raffinose, merisitose, inulin, dextrin, soluble starch, rhamnose,
a-methyl glycoside, mannitol, sorbitol,
All sugars such as ordinary oligosaccharides such as sodium gluconate, polysaccharides, 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 possible.
Those to which xyrose was added were very poor.

The concentration of sucrose added is preferably in the range of 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 as a nitrogen source, organic nitrogen such as yeast extract, pebtone, etc. gives preferable results, and an inorganic nitrogen source may be used as an auxiliary nitrogen source.

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 is 20-40℃, preferably around 30℃, and the pH is 4-40℃.
A pH of 8 or lower is sufficient; polysaccharides are not produced at pH 4 or below, and neutralization with calcium carbonate is not favorable for glucan production, but good results were obtained for fructan production.

However, if it becomes too alkaline, it tends to produce lactic acid, which is not preferable for producing polysaccharides. Although aeration produces favorable results for the initial production of bacterial cells, it is possible to produce the polysaccharides of the present invention by a slight amount or by static fermentation. Bacillus
In the case of Levoractis, M-4, -6eIS-68 or -
71 and transplanted the seed mother into a glucose medium as the seed mother, glucan will not be produced. Therefore, to produce glucan, use a new seed mother that has germinated from spores, or use the seed mother Gel-like glucan is produced on a mother agar medium, inoculated into a liquid medium as a seed medium, and subcultured on a sucrose medium. When nourished, insoluble glucan is obtained.

On the other hand, in the case of fructan production by the above-mentioned lactic acid bacteria of the present invention, fructans are formed unconditionally when the above-mentioned production strain is cultured in a sucrose medium. But soluble from the above strains that produce insoluble polysaccharides! To produce only polysaccharides, a seed mother subcultured in a glucose medium may be used. Both types of glucan 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 formed in the medium, and it can be obtained by separating it from the liquid and washing it. 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, and when dissolved in water, it becomes a smooth and transparent liquid, making it easy to purify. For purification, precipitation by adding a solvent and dissolution with water are repeated.
The solvent used for precipitation is ethanol and ethyl ether or an isobaric mixture of methanol and ethyl ether. The yield of insoluble polysaccharide is recovered as a hydrolyzate and is 20 g/f when the sugar content is about 25% and the sugar concentration is 8%. As mentioned above, the viscous liquid that remains is not a solid low polymer of glucan but a fructan. The physical properties of the purified glucan hydrolyzate obtained as described above are as follows.

Purified hydrolyzate of test product 20m91m10.1N sulfuric acid (100°C5 powder)
Centrifugation → Dialysis → Freeze-drying → Column chromatography (DEAE cellulose (PH8) and Se7allose (cross-linked agarose) (CL-6B) → Freeze-drying One-element analysis (
2 (generation) type 2 molecular weight Cepharose CL-ω and Cepharose (cross-linked agarose) CL manufactured by Birkin-Elemer Co., Ltd.
−? The gel permeation method was used.

The patterns are shown in FIGS. 2 and 3, respectively.
640.00044.000 It is believed that there are molecules with various molecular weights in between.

3 Melting point (Tanuki-S2 micro melting point measuring device) 210-215℃ Decomposition 4 Specific rotation (PO-B type Hitachi polarimeter) 1% aqueous solution reference dextran (average molecular weight 1Cgj to 200,000) 5 Ultraviolet absorption spectrum (Shimadzu double Beam spectrophotometer (
UV-200S)) 1% aqueous solution No special absorption observed 6 Infrared absorption spectrum (EPI-S? Hitachi infrared spectrophotometer) KBr tablet The spectrum diagram is shown in Figure 4.

7 Solubility 8 Color reaction 9 Basic, acidic, neutral (TCAUM-5ArH meter) 1% aqueous solution 20°C PH6.
65-7.01 (neutral) White powder of 10 substances Odor Tasteless 11 Viscosity Ostwald viscometer (20±0.1 C) 1%
12.79 millipoise Reference dextran (1% liquid) Molecular weight Intrinsic viscosity 0.24 (25±0.0
2'C) In order to confirm the structure of the above purified product, the hydrolyzate was treated with the following various products and the results are shown in Table 3 below.

Note) Dextranase (E, C3, 2, l, ll
) Sigma D5884PH6. O, 37℃, 12 hours
Cellmase (E, C, 3, 2, l, 4) Sigma C7377PH5. O, 37) C, l Shima Amyloglucosidase (E, C3, 2, l, 3) Sigma A7255PH 4.5, 55 l C, l Shima From the above results, the hydrolyzate of insoluble polysaccharide has glucose. α-1,
Amylases, which are thought to have mostly 6-6 bonds and some β-1,4 bonds, act slightly, and α-glucosidase acts only when the enzyme amount is increased. Seem.

Therefore, the main repeating unit appears to be a homoglucan mainly composed of α-1,6 bonds.

However, the polymer before hydrolysis is completely insoluble in water, and in addition to the above-mentioned bonds, other bonding modes, such as α-1
, 3 bonds are likely to be cleaved to form a polymer of the above-mentioned hydrolyzate, which has physical properties similar to dextran and can be used as a plasma substitute by fractionation.

The results of antitumor activity tests on the polysaccharide hydrolyzate and the water-soluble polysaccharide of the present invention are shown below (the toxicity of both is 12mg).
/mouse (500m9/K9).

) The test mouse is an ICR mouse! ? ．． 5 weeks old, obtained from Charles River, bred for 4 days and used with 1 g of 24 soil.

SarcOma-180 ascites was collected from strain mice inherited from the cancer center that were passaged weekly into the ICR mice described above, and as a cell suspension of 8 x 103/ml.
2×l/0.25゜m subcutaneously in the groin area of CR mice
l/mouse was transplanted.

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

The results are shown in Table 4 below. Based on this result, administer an appropriate amount (
Water-soluble polysaccharide 2.4m9/mouse-insoluble polysaccharide hydrolyzate 6
The inhibition rate against Tn9/mouse was 60%. Example 1 Bacillus laev
M-4 strain ATCC23549, which has the ability to produce water-insoluble and water-soluble polysaccharides.
Use.

M-4 spores were inoculated onto a slant medium consisting of 0.5% Pefton, 0.3% yeast dust, 0.05% dipotassium phosphate, 2% sucrose, and 1.5% agar, 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,
When the translucent gel-like water-insoluble polysaccharide mass obtained from 100 ml of the agar medium was aseptically inoculated into 201 which had been heat sterilized at 110°C and incubated at 30°C for 5 days,
The polysaccharide mass grows large.

Cover this with a Saran cloth and let the liquid cool. Wrap the lumps left on the Saran cloth as they are,
After dialysis and washing with running water for 1 hour, a pure white mass is obtained. Add pure water to the washed mass to make a total volume of 5', add 24.5 g of pure sulfuric acid, and boil it for 50 minutes to obtain a clear liquid, so centrifuge continuously at 10,000 r'Pm. Remove the bacterial cells. Add a mixture of methanol 5f1 ethyl ether 5e to the supernatant, leave it overnight, decant the supernatant, add 200ml of pure water to the precipitate to dissolve it, put it in a cellophane bag and dialyze it under running water for 1 hour. Then, 200 ml of methanol and 200 ml of ethyl ether were added to the dialysis solution, mixed to form a precipitate, centrifuged at 10,000 r''Pm, and the bamboo precipitate was dissolved in a small amount of water and freeze-dried.
210 g of solubilized polysaccharide (glucan) in the form of a white lump that is extremely soluble in water was obtained. The anticancer effect test results of this product were as shown above.

[Brief explanation of the drawing]

Figure 1 is a gas chromatogram A of the acid hydrolyzed TMS derivative of the water-insoluble polysaccharide glucan of the present invention, and gas chromatogram B of the TMS derivative of the polysaccharide fructan produced together with it. Figure 2 is the acid hydrolyzed product. Figure 3 shows the gel overflow pattern by Sepharose CL-
2B gel filtration pattern, and Figure 4 shows the same infrared absorption spectrum (KBr tablet).

Claims (1)

[Claims] 1 Belongs to the genus Bacillus, α-1,
It is a polymer consisting only of glucose mainly having 6 bonds, and has the following physical and chemical properties A: Solubility: Insoluble in water, ethanol, and diethyl ether: B: 0.1N
Properties against hydrolysis with sulfuric acid (a) A water-soluble polysaccharide is obtained by treatment in boiling water for 15 minutes. This polysaccharide has a kd value of 0 when filtered through Cephadex G-200 (trademark of Pharmacia Co., Ltd.: crosslinked dextran gel). (b) The polysaccharide of (a) was further heated at 100°C for 5
By treating for 0 minutes (i) Molecular weight: approximately 64,000
The resulting polysaccharide has main peaks at 0 and about 44,000 (molecular weight calculated using cross-linked agarose gel). (ii) Melting point: decomposes at 210-215°C (purified lyophilized product, same hereinafter). (iii) Specific rotation: [α]_D^2^0175° (c
= 1% water) (iv) Fixed viscosity: 0.24 (25 ± 0.0
2° C.) in a nutrient medium containing sucrose, and collecting the polysaccharide from the culture. 2 Microorganisms belonging to the genus Bacillus are Bacillus and Bacillus laevo-lacticus.
The method for producing the polysaccharide according to claim 1, which is M-4 (ATCC23549).