JPH032202A - Beta-glucan and production thereof - Google Patents

Abstract

NEW MATERIAL:beta-Glucans produced by culturing a microorganism belonging to Macrophomopsis genus and having a structure containing the following form of bonding. (A) All the D-glucopyranose residues of the main chain are beta-1,3 bond. (B) Branching at C-2 position and/or C-6 position of the D-glucopyranose residue of the main chain. (C) D-glucopyranose residues of side chains contain no beta-1,4 bond. USE:Useful as a thickening agent, an emulsifier, a stabilizer and a humectant in various fields such as food fields and cosmetic field. PREPARATION:A microorganism belonging to Macrophomopsis genus such as Macrophomopsis KAB 55 strain (FERM P-9366) is cultured at pH 5.0-8.0 and 20-35 deg.C for 3-7 days.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a β-glucan with a novel structure having high viscosity and a method for producing the same.

(Prior art and problems to be solved by the invention) Polysaccharides are used in a wide variety of fields, including the food industry, cosmetics industry, pharmaceutical industry, paper manufacturing industry, and chemical industry.

Conventionally, polysaccharides have been mainly supplied from higher plants, seaweed, etc., but recently polysaccharides (1mM) from microorganisms have been developed and started to be supplied from microorganisms, which can be stably supplied whenever needed.

Regarding polysaccharides produced by microorganisms, those produced by alkaline earth metals, bacteria belonging to the genus Xanthomonas and Pseudomonas, etc., and fungi belonging to the genus Plulania, Sclerotium, Albergil metal, etc. are known so far. . However, there are surprisingly few neutral polysaccharides that are stable to heat, have a high viscosity flow at room temperature, are not sticky, and are sensually superior.

Furthermore, looking at the structure of polysaccharides, homoglucans with β-1,3 glucoside bonds in the main chain, so-called β-1,3
Glucans are found in many fungi such as basidiomycetes, yeast, and filamentous fungi, and some are also produced in bacteria and algae.For example, lentinan from the fruiting bodies of shiitake mushrooms Papiman from the entity (1,
β-1,3 glucan from Basidiomycetes, including Schizophyllan from a culture of S. edulis 31), Ganoderan from a culture of Ganoderma (2), β-1,3 glucan (l) from baker's yeast cell wall, Scleroglucan 111 produced by Deuteromycetes Sufflerotium genus, Pestalotan+11 produced by Ascomycete Pestalotia genus, Curdlan produced by bacteria alkaline earth metals 33), β-1,3 glucan 141 produced by brown algae Laminaran, etc. be.

(1) “Isolation, cultivation, and identification of molds and the production and application of useful substances J. Mold Application Development Research Group (Dobundo) p. 354-369 (2) JP-A-6-0188402 (3) T., Harada and A. ,
jlsemura, Men, In5t,
Sci.

Ind, Res,, 0saka Univ, +
38 +37~49(4) M, Maeda an
d K, NiN15iza, J, Biocher
+.

l1, 199-206 (196B) However, among these, it is branched at the C-2 and/or C-6 position of the D-glucopyranose residue in the main chain, and the D-glucopyranose residue in the side chain is Pyranose residues include β-1,
There were no β-glucans without 4 bonds. Moreover, there were surprisingly few products that did not have a sticky feeling and were sensually superior.

The present invention aims to obtain new high viscosity polysaccharides that are stable and organoleptically resistant.

[Means to solve the problem]

The present invention relates to macrophomopsis (Macrophoa +
.

The present invention is a β-glucan with a novel structure, which is produced by culturing a microorganism belonging to the genus Psis, and has the following binding mode, and a method for producing the same.

(a) All D-glucopyranose residues in the main chain are β-1
, 3 bonds, and (b) c-2 and/or of D-glucopyranose residues in the main chain.
or branched at the C-6 position, and the D-glucopyranose residue in the (c) side chain has 61.4
There are no bonds.

The microorganism used in the present invention is Macrophomopsis (Mac).
For example, Macrophomopsis KA, which was deposited as FIKEN Entrustment No. 9366, belongs to the genus Macrophomopsis).
One example is the one named strain B55.

Details of the physical and chemical properties of this strain are available in Japanese Patent Application Laid-Open No. 64-63370.
It is stated in the No.

The novel multi-IJ of the present invention! Frequency has the following physical and chemical properties.

(1) Molecular weight: High performance liquid chromatography (hereinafter referred to as HPLC) using a 85Ahipak GS-710 column (exclusion limit molecular weight measured with pullulan: 10 million) using 50mM sodium chloride solution as the mobile phase.
When molecular sieving is performed, a single peak is observed near the exclusion limit. Therefore, its molecular size is 10 million Daltons or more, which indicates a large molecular size.

(2) Ultraviolet absorption spectrum: No absorption is shown.

(3) Infrared absorption spectrum; as shown in FIG. 1, it exhibits absorption at about 900 cm-' that is specific to β-glycosidic bonds.

(4) Solubility in solvents: Soluble in water, soluble in 0.5 N sodium hydroxide, 90% formic acid, insoluble in organic solvents such as methanol acetone, chloroform, and ethyl acetate.

(5) Color reaction A) Phenol-sulfuric acid reaction: + B) Iodine reaction: C) Carbazole-sulfuric acid reaction D) Ninhydrin reaction (6) Basic 1 Distinction between acidic and neutral: p of an aqueous solution of this substance
H is neutral.

(7) Color of substance: white.

(8) Types of constituent sugars: When hydrolyzed with 2,5 N trifluoroacetic acid for 8 hours and analyzed by HPLC using a TSK-gel Sugar AXG column (manufactured by Toyo Soda Co., Ltd.), it was found that the polysaccharide of the present invention It was found that glucose was the only major component.

(9) Degradability by enzymes: When this polysaccharide is treated with the above enzymes, each enzyme has 2.5 Units.
s, pH 5, 0, 37°C, O~24hr reaction)
, its ability to be hydrolyzed was determined by one-layer chromatography (developing solvent: n-butanol:acetic acid:ethyl ether:water = 9
: 6 : 3 : I) and AsahipakGS-
As a result of observation by 710Hr'LC, α-amylase,
It was not hydrolyzed at all by β-amylase and glucoamylase, and only laminarinase showed degradability in both analytical methods.

(10) Viscosity a) Viscosity: The solution of this polysaccharide becomes a highly viscous neutral solution. 1% when measuring the viscosity with a bismetron rotational viscometer
1200-1700 centipoise in aqueous solution (adapter No. 3).

The relationship between viscosity and concentration is as shown in Figure 2 (60 rotations, 30 seconds), and the viscosity is about twice that of existing xanthan gum.

) Stability against heat: shows stable high viscosity flow at room temperature,
It exhibits a constant viscosity between 5°C and 80°C.

It is also stable when heated in an autoclave at 121°C and 1 atm (20 minutes).

c) Stability against acids and alkalis: exhibits relatively stable viscosity in the pH range of 2 to 13.

2) Stability against salts It exhibits a certain viscosity and is stable in the presence of any salt such as diborate, acetate, sulfate, sodium salt, potassium salt, calcium salt, magnesium salt, etc.

(11) Binding mode After dissolving the present invention's polysaccharides in dimethyl sulfoxide, a methyl derivative is obtained by the Hakomori method using methyl sulfonyl carbanion and methyl iodide, which is hydrolyzed with acid, and the methylated sugar is converted to alditol and acetate. After induction, fixation and quantitative analysis using a combination of gas chromatography and mass spectrometry, the following products were observed.

2.3.4.6-Tetra-0-methyl-D-glucose is about 1.3 to 2.0 mol per 1.0 mol of 6-tetra-0-methyl-D-glucose, 4.6- About 0.2 to 0.6 mol of di-0-methyl-D-glucose, about 0.6 to 1.2 mol of 2.4-di-0-methyl-D-glucose.

4-Mono〇-methyl〇-glucose about 0.04 to 0.4 mol Judging from the above results comprehensively, the β-glucan of the present invention differs from conventionally known β-glucan in that it has no side chain. The main bonds of D-glucopyranose residues include β-1,
There is no 4-bond, and this side chain is a C-glucopyranose residue in the main chain that repeats β-1,3 bonds.
It is a neutral polysaccharide with a novel structure in which it is branched at the 2 and/or C-6 positions.

(12) Water evaporation test: Pour a certain amount of water into sample bottles for Group 4, paste a second base paper (a thin layer of waxed paper used in mimeograph machines) on the mouth of the bottle, and place hyaluronic acid on top of the base paper for each group. 1% solution, hyaluronic acid 0.5% solution.

Honda $1! 2 bottles of III% solution IJ! The amount of water evaporated was determined by applying a certain amount of a 0.5% solution of the same type and orally measuring the weight of the sample bottle.

Note that a paper coated with water was used as a control, and three samples in each group were used for measurement.

Moisture transpiration test (Measurements were made at 3 points per group, and the average value is shown.) This polysaccharide was found to be more effective in suppressing water transpiration than hyaluronic acid, which has a Y-retaining effect.

(13) Safety test Sensitization test of the polysaccharide of the present invention (MaXimizaLion
Method sample 4 degrees; induction 1% (slope 14 degrees 0.5% and 1%
), none of the 10 guinea pigs tested positive.

In addition, photosensitivity test of the polysaccharide of the present invention (Ad3uvant
-S trip method, sample concentration: induction 1%, induction 0.5
% and 1%), none of the 10 guinea pigs was found to be positive.

From the above, the present invention is multi-tJ! [is low sensitization and highly safe multi-1! It was similar.

(14) Other characteristic properties The polysaccharide of the present invention is tasteless and odorless. In addition, the polysaccharide of the present invention exhibits sensory properties when applied, as shown in the results of the sensory test in the application example described below (existing xanthan gum does not have the slippery feeling and has a smooth feel).

The culture method and purification method will be described below.

°Honda tl! Glucose, glycerin, maltose, and starch are the carbon sources used for culturing the microorganisms.

Lactose, si=11! Nitrogen sources include cornstarch liquor, yeast extract, dried yeast, oatmeal meat extract, casein hydrolyzate, ammonium salts, nitrates, and Pharmamedla (absorbent cottonseed flour). Other additives include inorganic salts such as sodium chloride, magnesium, calcium, and phosphoric acid. Furthermore, the medium contains iron, copper,
A trace amount of metal salt such as manganese may be included.

Cultivation can also be carried out in a conventional aqueous medium containing the above-mentioned culture medium by shaking culture, deep aeration culture, deep aeration culture, or single-rotation drum culture.

The culture conditions are pH 3.5 to 9.0, preferably pH 5.
0-8,0, culture temperature 10-40°C, preferably 20
Culture at ~35°C for usually 3-7 days. Multi-tJNM, the object of the present invention, can be obtained from the culture thus obtained. This culture solution is treated with an appropriate method such as filtration or centrifugation to remove the microbial cells, and a suitable precipitant is added to the resulting solution or supernatant, such as ethanol, methanol, isopropanol, propatool, acetone. Add an organic precipitant such as to precipitate the polysaccharide. This precipitate is separated by an appropriate method such as tlEs filtration or centrifugation, and then redissolved in water, followed by repeated precipitation with a precipitant, followed by dialysis and freeze-drying to produce purified multi-tM M is obtained.

〔Example〕

The present invention will be explained below with reference to Examples and Application Examples.

Example 1 Bacterial strain KAB55 belonging to the genus Macrophomopsis (Feikoken Contract No. 9366) was precultured for 3 days in a medium with the following composition, and 6 mff1 of this was inoculated into a 500 ml Erlenmeyer flask containing 100 mff1 of a medium with the same composition. The culture was then cultured at 25°C for 4 days with rotation at 120 revolutions/min.

(Composition) Glucose 100gP harsi
awedia 5 gKHL
POa Ig The obtained culture solution was centrifuged at 500 rotations/min for 20 minutes to remove bacterial cells, and an equal amount of 40% isopropanol was added to the supernatant to precipitate polysaccharides. This was centrifuged at 10,000 rpm for 5 minutes to obtain a polysaccharide. The obtained polysaccharide is dissolved in water again and the above operation is repeated to obtain a tasteless, odorless, white, highly viscous polysaccharide with 0
22 g was obtained.

Various measurements were carried out on this product to determine its physical and chemical properties. The results are as already described.

Example 2 Put the following medium 3042 into a 5ON jar fermenter,
Here, 17! of KAB55 precultured in the same manner as in Example 1 was added.
Inoculate and aerate at 25°C. Cultured in Ovvm for 4 days.

(&1N composition) G/L/:I-S 3000gP
har*asedia 1 5
0 gKH O PO430g MgSO4・7Hz 0 90g Application Example Comparative Example Emulsion The obtained culture solution was subjected to continuous centrifugation at 10,000 revolutions/min to remove bacterial cells, and an equal amount of 60% ethanol was added to the obtained supernatant to remove polysaccharides. was precipitated. This was purified by the same procedure as in Example 1, and the aqueous phase component heated to 80°C was added to the oil phase component heated to 80'C, and immediately stirred uniformly. After cooling, a milky lotion containing the polysaccharide of the present invention was obtained.

Furthermore, as a comparative example, emulsions were similarly prepared using existing xanthan gum instead of the polysaccharide of the present invention, and these were used in the following sensory test.

(Sensory test) Approximately 0.5 g of the applied and comparative emulsions described above were applied to each side of the left and right sides of the face on 20 female subjects, and the results were evaluated as follows: ``stickiness'' during application and ``skin smoothness'' after application. The evaluation item "smoothness" was evaluated by the test subject himself using the paired comparison method.

(3) Test results The results of a comparison between the above-mentioned application example and comparative example are shown in the table below.

It had a light and watery feel with no stickiness compared to (containing).

〔Effect of the invention〕

As mentioned above, Honda #M can be used as thickeners, emulsifiers, stabilizers, and preservatives in the fields of foods, cosmetics, etc.

[Brief explanation of drawings]

FIG. 1 is a diagram showing an infrared absorption spectrum of the polysaccharide of the present invention. Figure 2 shows the existing xanthan gum and the multi-tl! i
This is the time to show the relationship between concentration and viscosity. As is clear from the table, application examples (containing the polysaccharide of the present invention)
Comparative example (Existing xanthan gum T 2O00 = 10-Xanthan gum procedural amendment (voluntary) February 15, 1990 □ Director General Yoshi 1) Moon Yi IJl,
Display of the case 1999 Patent Application No. 137663 2 Name of the invention β-glucan and its manufacturing method 3 Person making the amendment Relationship to the case Patent applicant address 4-4゜, 5-17-4 Sumida, Sumida-ku, Tokyo Kanebo Co., Ltd. Patent Department, 1-5-90 Tomobuchi-cho, Bejima-ku, Osaka 534 Tel: (06) 921-1251 Number of claims increased by amendment None 6゜Contents of amendment (1) Page 3 of the specification In the fourth line, the text rAlpergillus genus j is corrected to "Aspergillus genus." (2) rSugerJ in page 7, line 10 of the specification
Correct the statement to rsgar4. (3) On page 13, line 13 of the specification, next to 1 for rorganic precipitant, insert r about 10 to 30% by weight.

Claims (2)

[Claims] (1) Macrophomops
is) A β-glucan with a novel structure having the following binding mode, which is produced by culturing a microorganism belonging to the genus. (a) All D-glucopyranose residues in the main chain are β-1
, 3 bonds, and (b) C-2 and/or of the D-glucopyranose residue in the main chain.
or branched at the C-6 position, and the (c) side chain D-glucopyranose residue has β'-1
, 4 bonds do not exist. (2) Cultivating microorganisms belonging to the genus Macrophomopsis,
From the culture, a novel structure of β with a binding mode as shown below was obtained.
- A method for producing β-glucan, which comprises collecting glucan. (a) All D-glucopyranose residues in the main chain are β-1
, 3 bonds, and (b) C-2 and/or of the D-glucopyranose residue in the main chain.
or branched at the C-6 position, and the (c) side chain D-glucopyranose residue has β-1,
There are no 4 bonds.