JP2006104439A - Beta-1,3-1,6-d-glucan and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a useful β-1,3-1,6-D-glucan aqueous solution. <P>SOLUTION: The β-1,3-1,6-D-glucan aqueous solution has two signals of 4.7 ppm and 4.5 ppm in<SP>1</SP>H NMR spectrum and shows viscosity of 50 cP ([mPa s]) or less in 0.5% (w/v) concentration at 30°C and pH 5.0. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention is a culture mainly comprising β-glucan produced by a microorganism, particularly a microorganism belonging to the genus Aureobasidium sp., Particularly β-1,3-1,6-D-glucan. The present invention relates to β-1,3-1,6-D-glucan, which is a polysaccharide useful as a soft drink, a health food material, a food thickener, or a medicine obtained from a liquid.

  β-glucan (β-1,3-D-glucan, or β-1,6-D-glucan, or β-1,3-1,6-D-glucan) is a mushroom that inhabits nature (basidiomycetes Recently, it is becoming clear that it is a component contained in a large number of fruit bodies and is contained not only in the fruit bodies but also in cultured mycelium. Β-glucan is known to have immunostimulatory activity and antitumor activity. For example, β-glucan extracted from Suehirotake, Kawaratake and Shiitake is sold as a pharmaceutical such as an anticancer drug (see Non-Patent Documents 1 and 2).

On the other hand, it is known that a microorganism belonging to the genus Aureobasidium sp., Which is an incomplete bacterium, also produces β-glucan outside the cell.
It has been reported that this microorganism produces β-1,3-1,6-D-glucan and fructooligosaccharide at the same time (see Patent Document 1) and produces pullulan together with β-glucan (see Patent Document 2). ing.

Aureobasidium sp. K-1 strain secreted and produced β-1,3-1,6-D-glucan outside the cell in a medium using sucrose as a carbon source (Non-patent Document 3) And the structure is that β- 1,3-D-glucan is the main chain, and D-glucose is bonded as a side chain with β-1,6-linkage, and a part thereof The side chain glucose residue is substituted with a sulfoacetic acid group (HO ₃ SCH ₂ COOH), and the molecular weight of this polysaccharide is estimated to be 2 million by gel filtration (GPC) (non-patented). References 3, 4 and Patent Reference 3).

  Β-1,3-1,6-D-glucan produced from microorganisms belonging to the genus Aureobasidium sp. Also has immunostimulatory activity, and this substance is a functional food (intestinal It has also been reported that it can be used for bifidobacteria growth, constipation prevention, immune enhancement) and intestinal regulating agents (see Patent Documents 2, 4, and 5).

In general, β-glucan aqueous solution has a single-helix or triple-helix structure, so that β-glucan easily forms a gel. Therefore, the β-glucan culture solution has high viscosity and its purification is extremely difficult ( Non-patent document 1). For example, β-glucan produced by microorganisms belonging to the genus Aureobasidium sp. Has high viscosity, and there are few methods for industrially separating, recovering and purifying bacterial cells and β-glucan from the culture solution. .
Currently reported methods are as follows.

1) Patent Document 1 discloses a method of obtaining a culture solution that can be used as a food or drink by sterilizing a culture solution of Aureobasidium sp. FERM P-4257 by heating and then filtering or centrifuging. Yes.
2) Patent Document 6 discloses a method for recovering insoluble β-1,3-glucan by subjecting insoluble β-1,3-glucan to an alkali heat treatment in the presence of an organic solvent.
3) Patent Document 7 discloses a method for recovering and purifying insoluble β-glucan by adding peroxide and hydroxide to a suspension of cells such as mushrooms and microorganisms to bring the pH to 10-12. .
4) In Patent Document 8, after sterilizing the culture solution, the cells are removed by centrifugation, ethanol is added to the supernatant so as to be 60% (v / v) or more, and β-glucan is precipitated, followed by filtration and recovery. A method for purifying β-glucan to be dried is disclosed.

However, in the method of Patent Document 1 of 1) above, the culture solution containing β-1,3-1,6-D-glucan at a high concentration has a very high viscosity at room temperature (several thousand cP ([mPa · s]) Above) It is industrially difficult to separate insoluble substances such as bacterial cells from the culture solution by filtration or centrifugation.
The methods described in Patent Documents 6-8 of the above 2), 3) and 4) are methods for extracting and recovering insoluble β-glucan mainly present on the cell wall of microorganisms such as mushrooms and yeast. The β-D-glucan obtained by the method described in Documents 6-8 is very difficult to dissolve in water and has a problem in use as a food material, particularly a beverage material, in terms of coloring.
Therefore, recently, insoluble β-glucan obtained from mushrooms and the like is subjected to high-pressure treatment (300--) in the presence of a general dispersing agent mainly using lecithin as an emulsifier after hot water or alkaline extraction and partial purification. 800 kgf / cm 2), and a method of forming ultrafine particles in a colloidal form is also disclosed (see Patent Document 9). However, this method requires a special pulverization treatment and an emulsifier.

Since the β-glucan aqueous solution has a high viscosity, only a low concentration can be obtained, and there are various problems associated therewith, and the development of a high concentration aqueous solution has been desired.
In addition, there is an increasing demand for powdery pure β-glucan, but it is difficult to remove cells, and only a method for producing low-purity powders containing cells, media, auxiliary agents for spray drying, alkalis, etc. Not known. In addition, in the case of the production method in which ethanol is precipitated from an aqueous solution (Patent Document 8), since the β-glucan concentration is low, it is difficult to say that it is a practical method from an industrial viewpoint.

JP-A-61-146192 Japanese Patent Laid-Open No. 62-201901 JP-A-7-51082 JP-A-6-340701 Japanese Patent Laid-Open No. 2002-204687 JP-A-5-308987 Japanese Patent Laid-Open No. 9-322795 JP-A-10-276740 International Publication No. 02/087603 Pamphlet Fragrance Journal, 5, 71-75 (1995) Nikkei Bio, No. 2, pp.91-94 (2003) Agric. Biol. Chem., 47, 1167-1172 (1983) Science and industry, 64, 131-135 (1990)

  The present inventors have developed a method for producing a low-viscosity β-glucan from a culture solution of a microorganism belonging to the genus Aureobasidium sp. (Japanese Patent Application No. 2003-106676). That is, an alkali is added at room temperature to a culture solution mainly composed of water-soluble β-1,3-1,6-D-glucan produced by microorganisms belonging to the genus Aureobasidium sp. The pH is adjusted to 12 or more, the viscosity is lowered, and then an acid is added to adjust the pH from the neutral range (pH 7.5) to the vicinity of the acidic range (pH 4 or less). A method for recovering and purifying an aqueous solution containing a low-viscosity and water-soluble β-1,3-1,6-D-glucan, characterized in that the liquid containing 3-1,6-D-glucan is separated at a high yield developed.

  As a result of examining the culture solution obtained by the above method developed by the present inventors (Japanese Patent Laid-Open No. 2004-092330) in more detail, the solubility, pH and temperature of β-1,3-1,6-D-glucan In addition, we found that there is a characteristic in molecular weight and particle size distribution, and further researched on the improvement of the production method, and improved storage stability and thermal stability in liquids such as gelation and aggregation, and further efficacy such as immunostimulating properties As a result, a β-1,3-1,6-D-glucan aqueous solution capable of significantly increasing the pH was found and the present invention was completed. Furthermore, the present inventors also found a method for efficiently producing high-purity β-1,3-1,6-D-glucan powder.

  The β-1,3-1,6-D-glucan provided by the present invention is present in an aqueous solution and in the form of fine particles dispersed at the same time, pH, metal ions (Na, Ca) It was found that stability was greatly affected by strength and temperature. Interestingly, the present particulate β-1,3-1,6-D-glucan has a relationship with solubility determined under various conditions such as pH, metal ion (Na, Ca) strength, temperature, etc. It was revealed that β-1,3-1,6-D-glucan exceeding the solubility range was finely divided (see Examples 5 and 6). The micronized β-1,3-1,6-D-glucan was found to be derived from a soluble glucan having a molecular weight of 10,000 to 500,000.

This micronized β-1,3-1,6-D-glucan causes aggregation and further gelation at 35 ° C. or higher in a state where there are many metal ions. For this reason, when used as a fluid food such as a soft drink, when heat treatment is performed at the time of heat sterilization, for example, at a pH of 3.5 and a temperature of 90 ° C., aggregation and further non-uniformity such as gelation are caused. I will. In the case of soft drinks, it is not preferable that turbidity and precipitation occur. It was found that these problems can be significantly improved by controlling the metal ion concentration for such problems.
On the other hand, in the case of an application in which gelation and thickening are actively aimed, gelation and thickening can be promoted by increasing the metal ion concentration on the contrary within the allowable range of the application. One object of the present invention is to provide a β-1,3-1,6-D-glucan aqueous solution with controlled heat and storage stability.

The present invention relates to an aqueous solution of β-1,3-1,6-D-glucan having a ¹ H NMR spectrum of 4.7 ppm and 4.5 ppm. The viscosity (BM type rotational viscometer, 12 rpm) at 30 ° C., pH 5.0, concentration 0.5% (w / v) of the aqueous solution is 50 cP ([mPa · s]) or less, preferably The present invention relates to a β-1,3-1,6-D-glucan aqueous solution characterized by being 40 cP, more preferably 30 cP or less.

The present invention also provides a β-1,3-1,6-D-glucan aqueous solution in which the molecular weight of the β-1,3-1,6-D-glucan is 10,000 to 500,000; Β-1,3-1,6-D-glucan aqueous solution containing fine particles β-1,3-1,6-D-glucan having a size of 05 to 2.0 μm; β-1,3-1,6-D -Β-1,3-1,6-D-glucan aqueous solution having a β-1,3 bond / β-1,6 bond ratio of glucan of 0.5 to 2.0; 1N sodium hydroxide heavy The β-1,3 bond / β-1,6 bond bond ratio based on the signal integration ratio of β-1,3 bond and β-1,6 bond in ¹ H NMR spectrum using aqueous solution as a solvent is 1.0. The β-1,3-1,6-D-glucan aqueous solution, which is ˜1.5; β-1,3-1,6-D-glucan having a sulfur-containing group, 6-D-G Β-1,3-1,6-D-glucan produced by a microorganism belonging to the genus Aureobasidium sp. Outside the cell, β-1,3-1,6-D-glucan The β-1,3-1,6-D-glucan aqueous solution; the β-1,3-1,6-D-glucan aqueous solution, wherein the microorganism is Aureobasidium pullulans; the microorganism is aureobasidium The β-1,3-1,6-D-glucan aqueous solution, which is an Aureobasidium pullulans GM-NH-1A1 strain or an Aureobasidium pullulans GM-NH-1A2 strain; β-1,3-1,6-D-glucan aqueous solution; the above β-1,3-1,6-D-glucan aqueous solution having a metal ion concentration, particularly Na ion and Ca ion concentration of 120 mg / 100 ml or less; Β-1,3-1,6-D-glucan obtained by removing fine particles β-1,3-1,6-D-glucan from the β-1,3-1,6-D-glucan aqueous solution It relates to an aqueous solution.
The present invention relates to fine particles β-1,3-1 obtained by removing water-dissolved β-1,3-1,6-D-glucan from the β-1,3-1,6-D-glucan aqueous solution. , 6-D-glucan; β-1, obtained by arbitrarily blending the β-1,3-1,6-D-glucan aqueous solution and the fine particles β-1,3-1,6-D-glucan, 3-1,6-D-glucan-containing liquid; and dry β-1,3-1,6-D-glucan obtained by drying the β-1,3-1,6-D-glucan aqueous solution. Related.

The present invention also includes a microorganism after adding an alkali or an aqueous solution thereof to a microorganism culture solution containing β-1,3-1,6-D-glucan to lower the viscosity of the culture solution to a pH of 12 or more. Β-1,3-1,6-D characterized by separating and removing insoluble matters and further removing the metal ions, particularly Na ion concentration and Ca ion, so that the metal ion concentration is 120 mg / 100 ml or less. -Preparation method of glucan aqueous solution; and the above β-1,3-1,6-D-glucan aqueous solution was dialyzed and concentrated, and alcohol was added to the concentrated solution to obtain β-1,3-1,6-D-glucan The resulting slurry of β-1,3-1,6-D-glucan / alcohol / water is separated, and the precipitated slurry is spray-dried. A method for preparing 1,6-D-glucan powder; The β-1,3-1,6-D-glucan aqueous solution described above is dialyzed, further concentrated under reduced pressure, and then the concentrated solution is spray-dried. High purity β-1,3-1,6-D- It also relates to a method for preparing glucan powder.
Furthermore, the present invention provides an anti-malignant neoplastic agent comprising a β-1,3-1,6-D-glucan aqueous solution or β-1,3-1,6-D-glucan as an active ingredient, Malignant tumor or anticancer agent, INF-γ production agent, therapeutic agent for diseases suppressed or cured based on INF-γ production effect, malignant neoplastic transfer inhibitor, allergic disease inhibitor, especially type I allergic disease inhibitor It also relates to agents and macrophage activators.

  The present invention also relates to a health food or health enhancer, an external preparation, and a cosmetic containing a β-1,3-1,6-D-glucan aqueous solution or β-1,3-1,6-D-glucan.

BEST MODE FOR CARRYING OUT THE INVENTION

The microorganism used in the present invention is not particularly limited as long as it can produce β-1,3-1,6-D-glucan, but is preferably a microorganism belonging to the genus Aureobasidium sp.
Various methods for culturing microorganisms belonging to the genus Aureobasidium sp. To produce β-1,3-1,6-D-glucan (hereinafter sometimes abbreviated as glucan) have been reported. ing. Examples of carbon sources that can be used include carbohydrates such as sucrose, glucose, fructose and starch, organic acids such as citric acid, malic acid, gluconic acid and lactic acid, and organic nutrient sources such as peptone and yeast extract. Examples of the nitrogen source include inorganic nitrogen sources such as ammonium sulfate, sodium nitrate, and potassium nitrate. In some cases, in order to increase the production amount of the β-glucan, inorganic salts such as sodium chloride, potassium chloride, phosphate, magnesium salt, calcium salt, trace metal salts such as iron, copper, manganese, etc. Adding vitamins is also an effective method.

  For example, when a microorganism belonging to the genus Aureobasidium sp. Is cultured in a medium in which ascorbic acid is added to a tourpec medium containing sucrose as a carbon source, a high concentration of β-1,3-1,6-D -It has been reported that glucan is produced (Non-patent Documents 3 and 4, Patent Document 3). However, the medium is not particularly limited as long as microorganisms grow and produce β-1,3-1,6-D-glucan. If necessary, organic nutrient sources such as yeast extract and peptone may be added.

As conditions for aerobic culture of microorganisms belonging to the genus Aureobasidium sp. In the above medium, the temperature is 10-45 ° C, preferably 20-35 ° C, and the pH is 3-7, preferably 3.5-5.
In order to effectively control the culture pH, it is also advantageous to control the pH of the culture solution with alkali or acid. Further, an antifoaming agent may be appropriately added for defoaming the culture solution. The culture time is usually preferably 1-10 days, and the β-glucan can be produced usually by culturing for 1-4 days. The culture time may be determined while measuring the production amount of the β-glucan.

When microorganisms belonging to the genus Aureobasidium sp. Were cultured under aeration and agitation for 4-6 days under the above conditions, the culture broth contained β-1,3-1,6-D-glucan as a main component. Glucan polysaccharide is contained in an amount of 0.1% to several% (w / v), and the viscosity of the culture solution is several hundred cP ([mPa · s ]) To a few thousand cP ([mPa · s]).
When an alkali is added to the culture solution while stirring at room temperature, the viscosity rapidly decreases. The alkali that can be used is not particularly limited as long as it can be solubilized in water, but sodium hydroxide has a final concentration of 0.5% (w / v) or more, preferably 1.25% (w / v) or more. It is necessary to add and stir well. That is, when an alkali is added and stirred so that the pH is 12 or more, preferably 13 or more, the viscosity of the culture solution is instantaneously reduced to several cP ([mPa · s]).

  The method for separating insoluble substances such as bacterial cells from the alkali-treated culture solution is not particularly limited as long as it can separate insoluble substances such as bacterial cells from the culture solution, and the supernatant is collected after the bacterial cells settle. (Decant method), centrifugation, total filtration using filter paper or filter cloth, filter press, and membrane filtration (ultrafiltration such as MF membrane). In the case of total filtration with filter paper or filter cloth, it is one means to use a filter aid such as celite. Industrially, removal of bacterial cells by a filter press is preferred.

  It should be noted that the acid may be added before separating insoluble matter such as bacterial cells to bring the pH from the neutral to the vicinity of the acidic range, or the pH may be changed from the neutral to the vicinity of the acidic range. Note that at normal temperature (15 to 35 ° C.), β-glucan polysaccharide is not gelled even if the pH is adjusted from near neutral to near acidic, and the viscosity remains low without increasing viscosity. It is. As described above, since the present invention does not increase the viscosity even when the viscosity of the high-viscosity β-glucan aqueous solution is reduced by alkali treatment and the pH is subsequently adjusted to acidic (pH 4>), the health food material, particularly The present invention provides a β-1,3-1,6-D-glucan aqueous solution useful as a health food beverage material, and also as an external preparation for cosmetics and the like.

Next, an example of blending health drinks is shown.

Next, the example of a lotion is shown.

  Examples of the alkali used for the alkali treatment include alkali carbonate aqueous solutions such as calcium carbonate aqueous solution, sodium carbonate aqueous solution, potassium carbonate aqueous solution and ammonium carbonate aqueous solution, alkali hydroxide aqueous solutions such as sodium hydroxide aqueous solution, potassium hydroxide aqueous solution and calcium hydroxide aqueous solution. There is no particular limitation as long as it is a commonly used alkali such as an aqueous ammonia solution. However, to use β-1,3-1,6-D-glucan for food, it is preferable to use an alkali recognized as a food additive.

  The acid used for neutralization and acid treatment is not particularly limited as long as it can normally neutralize alkali, such as hydrochloric acid, phosphoric acid, sulfuric acid, citric acid, malic acid, and gluconic acid. However, the acid recognized as a food additive is also preferable for use in food.

Here, the culture solution that has been subjected to alkali treatment, further neutralization treatment, or acid treatment may be used for food as it is, but it is preferable to remove metal ions in view of later overheating sterilization and storage stability.
For removal of metal ions, desalting using a UF membrane is used. The metal ion concentration is preferably 120 mg / 100 ml or less, more preferably 50 mg / 100 ml or less, and still more preferably 20 mg / 100 ml or less. For example, when the viscosity is lowered by alkali treatment using sodium hydroxide, the metal ions mentioned here are sodium ions.

Table 1 shows the results of thermal stability experiments conducted using the culture solution shown in Example 1 below. The culture solution in which the Na ion concentration was adjusted by buffer exchange and sodium chloride blending was allowed to stand in a 50 ° C. constant temperature oven, and the stability was examined. Here, the polysaccharide concentration was adjusted to 2 mg / ml, and the Ca ion was adjusted to 10 mg / 100 ml.
○ indicates no change, and × indicates that gelation, aggregation and precipitation occurred. Here, gelation is defined as a state where fluidity is lost.

Next, the same experiment was conducted for Ca ions. The results are shown in Table 2. After desalting with a UF membrane using the culture solution shown in Example 1, the Ca ion concentration was adjusted by blending calcium chloride. It was allowed to stand in a constant temperature oven at 50 ° C., and the stability was examined in the same manner. Here, the polysaccharide concentration was adjusted to 2 mg / ml, and the Na ion was adjusted to 16 mg / 100 ml.
○ indicates no change, and × indicates that gelation, aggregation and precipitation occurred. Here, gelation is defined as a state where fluidity is lost.

  This method of recovering and purifying β-1,3-1,6-D-glucan is mainly from a high molecular weight (molecular weight of 2 million or more) produced by any strain belonging to the genus Aureobasidium sp. (Molecular weight of several hundred to 20,000) Although it can be applied to β-glucan, it can be applied to β-1,3-1,6-D-glucan produced by all microorganisms and mushrooms. is there. Particularly preferred are Aureobasidium pullulans GM-NH-1A1 and Aureobasidium pullulans GM-NH-1A2 strains which are mutant strains of the Aureobasidium sp. K-1 strain It is. The original strain Aureobasidium sp. K-1 strain is known to produce several types of high molecular weight β-glucan (over 2 million and about 1 million β-glucan). Moreover, it is known that β-glucan produced by the Aureobasidium sp. K-1 strain has a sulfoacetic acid group (see Non-Patent Documents 3 and 4).

  The mutant strains GM-NH-1A1 and GM-NH-1A2 have high molecular weight β-glucan (particulate glucan) having an apparent main peak of 500 to 2.5 million and a main peak of 2 to 2 as shown in Examples. It is a strain that produces both 300,000 low molecular weight β-glucan. The properties of the GM-NH-1A1 and GM-NH-1A12 strains are as shown in the following table. As a result, this strain was identified as a strain belonging to Aureobasidium pullulans from the morphological properties of each strain and the base sequence of 28S rDNA. These strains are deposited as FERM BP-10294 and FERM BP-10295, respectively, at the National Institute of Advanced Industrial Science and Technology.

Next, as a result of measuring the particle size distribution of the main culture solution, β-1,3-1,6-D-glucan is not completely dissolved in the culture solution, and the primary particle size is partially 0. It was found to be composed of fine particles of about 0.05 to 2.0 μm.
Further, the particulate β-1,3-1,6-D-glucan-containing aqueous solution can be further stabilized by adding a known emulsifier such as lecithin or a stabilizer such as cyclic dextrin.

In order to produce dry β-1,3-1,6-D-glucan from the low-viscosity β-1,3-1,6-D-glucan aqueous solution of the present invention, known methods such as spray drying and freeze-drying are known. The drying method can be adopted. In this case, the low-viscosity β-1,3-1,6-D-glucan aqueous solution may contain bacterial cells and salts.
In the present invention, alkali or an aqueous solution thereof is added to a microorganism culture solution containing β-1,3-1,6-D-glucan to lower the viscosity, and then unnecessary substances containing microorganisms are separated and removed if necessary, followed by dialysis concentration. Then, alcohols are added to the concentrated solution with stirring, and the resulting slurry of β-1,3-1,6-D-glucan / alcohol / water is separated to give a sedimentation slurry concentration of 1.0% ( It is related with the preparation method of (beta) -1,3-1,6-D-glucan powder characterized by preparing and spray-drying so that it may become w / w) or more. According to this method, since the low-viscosity β-1,3-1,6-D-glucan aqueous solution can be concentrated and the volume can be reduced from the original fraction to a few tenths, The amount of alcohol added for purification can be drastically reduced. Unnecessary salt can be reduced by sufficiently performing dialysis as necessary. In addition, according to the present production method, since the high molecular weight β-1,3-1,6-D-glucan is preferentially precipitated when alcohol is added, inorganic salts such as sodium citrate can be removed, and the purity is improved. It is very effective for purification.

  As the microorganism culture solution, a culture solution mainly containing β-glucan produced by a microorganism belonging to the genus Aureobasidium outside the cell, particularly β-1,3-1,6-D-glucan is preferred. The microorganisms are preferably separated and removed by a filter press or centrifugation. Any alcohol can be used as long as it can precipitate β-1,3-1,6-D-glucan from the culture broth, but a particularly preferred alcohol is ethanol. The amount of ethanol used is preferably 1 time (volume ratio) or more, preferably 2 times or more, relative to the culture solution. After separation, alcohol can be recovered from the alcohol / water mixture by distillation or the like and recycled.

  On the other hand, the low-viscosity β-1,3-1,6-D-glucan powder of the present invention can be carried out by the following production method without using alcohol. In other words, alkali or an aqueous solution thereof is added to a microorganism culture solution containing β-1,3-1,6-D-glucan to lower the viscosity, and then unnecessary substances containing microorganisms are separated and removed if necessary, followed by dialysis concentration. It was found that a high-purity β-1,3-1,6-D-glucan powder can be obtained by further concentrating under reduced pressure, further concentrating under reduced pressure, and then spray drying the concentrated solution. By this method, there is no loss during alcohol recovery, no explosion-proof equipment is required, and high-purity β-1,3-1,6-D-glucan powder can be obtained easily and inexpensively. The process for producing β-1,3-1,6-D-glucan powder having such a vacuum concentration step is because the aqueous β-1,3-1,6-D-glucan solution of the present invention has low viscosity. It has become possible.

As shown in the Examples, the β-1,3-1,6-D-glucan aqueous solution of the present invention is a malignant neoplasm suppression experiment using Colon 26 colon cancer cells in mice. A biometastatic inhibitory effect was observed.
Therefore, the β-1,3-1,6-D-glucan or an aqueous solution thereof according to the present invention is useful as an anti-malignant neoplastic agent such as a malignant tumor therapeutic agent.
Furthermore, it was found that the number of NK positive cells and INF-γ positive cells in the intestinal mucosal tissue were significantly increased as compared with the β-glucan non-administered group, and IL-12 production in blood was also enhanced. It was. This suggests that the present β-1,3-1,6-D-glucan activates macrophages that are antigen-presenting cells.

Accordingly, the β-1,3-1,6-D-glucan or an aqueous solution thereof according to the present invention is also expected to have the following effect 1-3.
1. Antiviral and antibacterial infections:
INF-γ activates cellular RNAase (RNA degrading enzyme), further suppresses DNA synthesis, protein synthesis, and peptide synthesis, thereby inhibiting virus growth. Specifically, it is effective for the suppression of hepatitis A, B and C, cirrhosis, herpes disease, influenza and stomatitis, various malignant neoplasms (including viral ones) and their metastasis. It can be seen. It is also effective against antibacterial infections such as MRSA / VRE.
2. Type I allergy (anaphylactic reaction) inhibitors:
INF-γ inhibits the differentiation and activation of Th2 cells, which are one type of helper T cells involved in IgE antibody production. In addition, INF-γ has been reported to suppress IL4 production. As a result, it is presumed to suppress allergies (type I allergy) involving IgE antibodies. Specifically, hay fever, rhinitis allergies, allergic conjunctivitis, food allergies (gastrointestinal allergies such as urticaria, diarrhea), atopic dermatitis, bronchial asthma, anti-shock symptoms by drugs such as venom and penicillin Is done.
3. Type IV allergy inhibitors and autoimmune disease inhibitors:
2. As described in, self-components partially denatured by infection with viruses and bacteria become antigenic (self-antigen) and become autoimmune diseases. Specifically, rheumatoid arthritis, rheumatic fever, collagen disease, Sjogren's syndrome, Hashimoto's disease, Graves' disease, anemia such as autoimmune hemolytic anemia and pernicious anemia, insulin-dependent diabetes mellitus (type I diabetes), myasthenia gravis Symptoms and the like.
The β-1,3-1,6-D-glucan or an aqueous solution thereof according to the present invention is preferably administered orally. The dosage varies depending on symptoms, age, body weight, dosage form, number of administrations, etc., but is usually 1-200 mg per day (as β-1,3-1,6-D-glucan), preferably 5-100 mg Is done.

EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, it is not limited to these examples.
Example 1 Use of Aureobasidium pullulans GM-NH-1A1 strain 1) Culture production of β-glucan 1
A 100 ml liquid medium having the composition shown in Table 4 below was placed in a 500 ml shoulder flask, autoclaved at 121 ° C. for 15 minutes, and then Aureobasidium pullulans GM-NH-1A1 strain. One platinum ear was inoculated aseptically from the slant of the medium composition, and a seed culture solution was prepared by aeration-stirring culture at 130 rpm at 30 ° C. for 24 hours. Next, 200 L of medium having the same composition is put into a 300 L culture apparatus (manufactured by Maruhishi Bioengineer), autoclaved at 121 ° C. for 15 minutes, and 2 L of the seed culture solution obtained above is aseptically inoculated. , Aeration and agitation culture at 200 rpm, 27 ° C. and 40 L / min were performed. The pH was controlled within the range of pH 4.2 to 4.5 using sodium hydroxide and hydrochloric acid. The turbidity after 96 hours was 23 OD at OD 660 nm, and the polysaccharide concentration was 0.5% (w / v).
The polysaccharide concentration is determined by sampling several ml of the culture solution, centrifuging and removing the cells, and then adding ethanol to the supernatant so that the final concentration is 66% (v / v) to precipitate and recover the polysaccharide. The sample was dissolved in ion-exchanged water and quantified by the phenol sulfuric acid method.
Further, ethanol was added to the culture supernatant from which the cells had been removed so that the final concentration was 66%, and β-glucan was collected by precipitation. Thereafter, the sample was dissolved again in ion-exchanged water, centrifuged again, sodium chloride was added to the supernatant so that the final concentration was 0.9%, and β-glucan was again collected with 66% ethanol. This β-glucan recovery and purification operation was further repeated twice, and the obtained β-glucan aqueous solution was dialyzed against ion-exchanged water and then freeze-dried to obtain β-glucan powder. From the compositional analysis results of this β-glucan, the S content was 239 mg / kg, and the substituted sulfoacetic acid content calculated from this was 0.09%.

2) Alkali treatment When the culture solution obtained in Example 1 was measured at 30 ° C. and 12 rpm using a BM type rotational viscometer (manufactured by Tokyo Keiki Co., Ltd.), it was 1500 cP ([mPa · s]). The rotor used for the measurement was selected appropriately according to the viscosity. When 25% (w / w) sodium hydroxide was added to the culture solution so that the final concentration of sodium hydroxide was 2.4% (w / v) and stirred (pH 13.6), the viscosity decreased instantaneously. Subsequently, when the viscosity was measured after neutralizing to pH 5.0 with 50% (w / v) citric acid aqueous solution, the viscosity (30 ° C.) at that time was 20 cP ([mPa · s]). . Next, 1 wt% of KC Flock (manufactured by Nippon Paper Industries Co., Ltd.) was added to the culture broth as a filter aid, and the cells were removed using a Kamata filter press (manufactured by Kamata Kikai). About 230 L). The polysaccharide concentration was 0.5% (w / v), and the recovery rate was almost 100%.

3) Desalination of β-glucan aqueous solution The β-glucan aqueous solution (culture filtrate) is diluted to 0.3%, and then desalted with a UF membrane (molecular weight cut 50,000, manufactured by Nitto Denko). After the sodium ion concentration was dropped to 20 mg / 100 ml, the pH was adjusted to 3.5 with a 50% (w / v) aqueous citric acid solution. Subsequently, sterilization treatment was carried out at 95 ° C. for 3 minutes using a hot filling heating unit (manufactured by Nisaka Manufacturing Co., Ltd.) to obtain a final β-glucan aqueous solution. The concentration of β-glucan at this time was 0.22% (w / v) as measured by the phenol sulfuric acid method. Further, the total yield from the culture broth was about 73%. The obtained β-glucan aqueous solution was dialyzed against ion-exchanged water and then freeze-dried to obtain β-glucan powder. From the compositional analysis result of this β-glucan, the S content was 330 mg / kg, and the substituted sulfoacetic acid content calculated from this was 0.12%.

  Moreover, since the wavelength shift from 480 nm to about 525 nm could be confirmed by the Congo red method, the above-described culture filtrate that had been desalted contained glucan containing β-1,3 bonds. It was proved (K. Ogawa, Carbohydrate Research, 67, 527-535 (1978), supervised by Tadayuki Imanaka, development of the use of microorganisms, 1012-1015, NTS (2002)). The shift difference to the maximum value at that time was Δ0.48 / 500 μg polysaccharide.

15 ml of the culture filtrate was taken out, 30 ml of “ethanol” was added, and centrifuged at 4 ° C., 1000 rpm, 10 min to collect the precipitated polysaccharide. Wash with 66% ethanol, centrifuge at 4 ° C, 1000 rpm, 10 min, add 2 ml of ion-exchanged water and 1 ml of 1N sodium hydroxide aqueous solution to the precipitated polysaccharide, stir and dissolve at 60 ° C for 1 hour to dissolve the precipitate I let you. Next, after freezing at −80 ° C., vacuum lyophilization was performed overnight, and the dried powder was dissolved in 1 ml of heavy water to obtain a two-dimensional NMR sample. ¹ H NMR spectrum correlated with 106 ppm of two-dimensional NMR (13C-1H COSY NMR) 4.7 ppm and two signals in the vicinity of 4.5 ppm were obtained (see FIG. 1). As a result, it was proved that this β-glucan is β-1,3-1,6-D glucan (supervised by Tadayuki Imanaka, development of microbe utilization, 1012-1015, NTS (2002 )). From the integration ratio of each ¹ H NMR signal, it was found that the ratio of β-1,3 bond / β-1,6 bond was 1.15.
Further, when the ¹ H NMR spectrum was changed from 1N-NaOH deuterated solvent to dimethyl ethyl sulfoxide-d ₆ deuterated solvent and measured at 80 ° C., the peak of 4.7 ppm was 4.5 ppm and 4.5 ppm. Each peak shifted to 4.2 ppm.
The obtained β-1,3-1,6-D-glucan was hydrolyzed for several hours at 30 ° C. with exo-type β-1,3-glucanase (Cytalase M, manufactured by Keiai Kasei Co., Ltd.) (See Non-Patent Document 3 and Non-Patent Document 4). Degradable organisms were identified by HPLC analysis (acetonitrile / water, 70/30; 85 ° C; flow rate, 1 ml / min) using an amide column (TSK-GEL AMIDE-80, diameter 4.6 mm x 250 mm, manufactured by Tosoh Corporation). It was confirmed that glucose and gentiobiose were released as degradable organisms. Retention times for each were glucose, 5.7 minutes; gentiobiose, 8.5 minutes. From this, the structure of this glucan confirmed that one glucose molecule was branched into a side chain by β-1,6-linkage with respect to the main chain in which glucose was connected by β-1,3-linkage.

  Next, when the particle size of the culture solution was measured using a laser diffraction / scattering particle size distribution measuring apparatus (LA-920 manufactured by HORIBA), the particles were about 0.3 μm and 100 μm in size. I was seen. Subsequently, when particle size measurement was performed while irradiating with ultrasonic waves, the 100 μm peak disappeared as soon as it was seen, the 0.3 μm peak increased, and finally only 0.3 μm (see FIG. 2). 0.3 μm is a primary particle of β-1,3-1,6-D-glucan, and 100 to 200 μm is a secondary particle in which primary particles of β-1,3-1,6-D-glucan are aggregated. Think. Further, it was confirmed that the secondary particles disappeared in the same manner even when stirred by a magnetic stirrer and lightly shaken, and easily broken into primary particles. Therefore, the secondary particles are considered to be very loosely aggregated (slowly aggregated state).

  In addition, gel chromatography was performed using Toyopearl HW65 (column size: 75 cm × φ1 cm, excluded molecular weight: 2.5 million (dextran)) manufactured by Tosoh Corporation, using 0.1 M sodium hydroxide aqueous solution as an eluent, and dissolved β-1,3 The molecular weight of a β-1,3-1,6-D-glucan-containing liquid composed of primary particles of -1,6-D-glucan and β-1,3-1,6-D glucan was measured. The resulting polysaccharide has a molecular weight of 2 to 300,000 peaks derived from dissolved β-1,3-1,6-D-glucan and an apparent high molecular weight of 500 to 2.5 million derived from primary particles. It was found to consist of two types of molecular fractions. Here, a pullulan manufactured by Shodex was used as a molecular weight marker.

  On the other hand, in order to separate water-soluble β-1,3-1,6-D-glucan and fine particles, an aqueous solution of β-1,3-1,6-D-glucan prepared by this method (fine particles and solubilized glucan were mixed). When the product was filtered with a filter (0.2 μm) manufactured by Advantech, the polymer fraction of 500 to 2.5 million disappeared. That is, it was found that the polymer fraction corresponds to primary particles of β-1,3-1,6-D-glucan and secondary particles in which primary particles are aggregated. Therefore, the molecular weight of water-soluble β-1,3-1,6-D-glucan is considered to be 2 to 300,000.

Next, the thermal stability during heat sterilization was examined. After alkali treatment in Example 1, sterilization was performed, and after removing metal ions to 50 mg / 100 ml or less, the polysaccharide concentration was 0.2% (2 mg / ml), 0.40%, 0.52%, 0.77. % And 0.96%, followed by heat sterilization with an autoclave at 90 ° C. for 15 minutes. The results are shown in Table 5. Here, ◯ indicates no change, Δ indicates a part of the aggregation, and X indicates a non-fluid gel.
When the polysaccharide concentration was high, the tendency to aggregate easily was observed. When the sugar concentration exceeded 0.5% (5 mg / ml), a tendency of aggregation was observed.

Example 2 Use of Aureobasidium sp. K-1 Strain 1) Culture Production of β-Glucan 2
60 ml of the liquid medium having the composition shown in Table 4 was put into a 300 ml Erlenmeyer flask with baffle and autoclaved at 121 ° C. for 15 minutes, and then Aureobasidium sp. K-1 strain was obtained. Aseptically, one platinum ear was inoculated from a slant having the same medium composition, and a polysaccharide production experiment was conducted by aeration and agitation culture at 30 rpm for 30 hours at 30 ° C. The turbidity after 96 hours was 35 OD at OD 660 nm, and the polysaccharide concentration was 0.5% (w / v). The polysaccharide concentration was determined by the phenol-sulfuric acid method after centrifuging and removing ethanol from the culture solution, adding ethanol to 66% (v / v), precipitating it, dissolving it in ion-exchanged water. In addition, the S content was 2300 mg / kg from the compositional analysis result of this β-glucan, and the substituted sulfoacetic acid content calculated from this was 0.9%.

2) The properties of this polysaccharide were investigated in the same manner as in Example 1 below.
When the shift effect in Congo Red was examined, the maximum absorption was observed to shift from 480 nm to around 525 nm, and it was proved that glucan containing β-1,3 bonds was contained. The shift difference to the maximum value at that time was Δ0.50 / 500 μg polysaccharide. Two-dimensional NMR (13C-1H COSY NMR) analysis was also performed. From the result, it was proved that the obtained polysaccharide was β-1,3-1,6-D-glucan. From the integral ratio of each 1H signal, the ratio of β-1,3 bond / β-1,6 bond was 1.38. Furthermore, when the molecular weight was measured, the molecular weight was 10 to 3 million.
The obtained β-1,3-1,6-D-glucan was analyzed in the same manner as in Example 1, and the structure of this glucan was compared with the main chain in which glucose was connected by β-1,3-linkage. It was confirmed that one glucose molecule was branched to the side chain at the β-1,6-linkage.

  When the obtained culture broth was measured in the same manner as in Example 1 using a BM type rotational viscometer, the viscosity was 1900 cP ([mPa · s]) at 30 ° C. When 25% (w / w) sodium hydroxide was added to the culture solution so that the final sodium hydroxide concentration was 2.4% (w / v) and stirred (pH 13.4), the viscosity decreased instantaneously. did. Subsequently, the pH was adjusted to 4.8 with a 50% (w / v) aqueous citric acid solution. The viscosity at that time was 28 cP ([mPa · s]).

Example 3 Use of Aureobasidium pullulans GM-NH-1A2 Strain Aureobasidium pullulans GM-NH-1A1 strain to Aureobasidium pullulans GM-NH-1A2 strain Except for the change to, all were carried out by the method of Example 1. As a result, the produced polysaccharide concentration was 0.5% (w / v), and the viscosity at that time was 1300 cP ([mPa · s]). To the obtained culture broth, sodium hydroxide was added to a final concentration of 2.4% (w / w) in the same manner as in Example 2 (pH 13.6), followed by 50% (w / v). When the viscosity was measured in the same manner as in Example 1 after adjusting the pH to 5.0 with an aqueous citric acid solution, the viscosity decreased to 7 cP ([mPa · s]). The polysaccharide concentration at that time was 0.5% (w / v).

In the same manner as in Example 1, the properties of this polysaccharide were investigated.
When the shift effect in Congo red was examined, the maximum absorption was observed to shift from 480 nm to around 525 nm. The shift difference to the maximum value at that time was Δ0.45 / 500 μg polysaccharide. Two-dimensional NMR (13C-1H COSY NMR) analysis was also performed.
From the result, it was proved that the obtained polysaccharide was β-1,3-1,6-D-glucan. From the integral ratio of each 1H signal, the ratio of β-1,3 bond / β-1,6 bond was 1.23.
Further, when the molecular weight was measured, it was found that the molecular weight was composed of two types of a low molecular fraction having an average molecular weight of 2 to 300,000 peaks and a high molecular fraction having 500 to 2.5 million. Further, the S content was 229 mg / kg from the compositional analysis result of the present β-glucan, and the substituted sulfoacetic acid content calculated from this was 0.09%.

Example 4 Modification of Medium Composition All the procedures were performed in the same manner as in Example 1 except that the strain was changed to Aureobasidium pullulans GM-NH-1A2 and ascorbic acid in the medium of Table 4 was not added. . As a result, the produced polysaccharide concentration was 0.6%, and the viscosity at that time was 1500 cP ([mPa · s]). To the obtained culture broth was added in the same manner as in Example 2 so that the final concentration of sodium hydroxide was 2.4% (w / w) (pH 13.6), followed by 50% (w / v). When the viscosity was measured in the same manner as in Example 1 after adjusting the pH to 4.9 with an aqueous solution, the viscosity decreased to 7 cP ([mPa · s]). In the Congo red method, a shift in maximum absorption from 480 nm to around 525 nm was observed. The shift difference to the maximum value at that time was Δ0.45 / 500 μg polysaccharide.

In addition, two-dimensional NMR (13C-1H COSY NMR) analysis was performed in the same manner as in Example 1. As a result, it was proved that the obtained polysaccharide was β-1,3-1,6-D-glucan. From the integral ratio of each 1H signal, β-1,3 bond / β-1,6 bond was obtained. The ratio was 1.21.
Subsequently, when the molecular weight was measured by the same method as in Example 3, it was found that the molecular weight was composed of two kinds of low molecular fractions having an average molecular weight of 2 to 300,000 peaks and high molecular fractions of 50 to 250. .
The obtained β-1,3-1,6-D-glucan was analyzed in the same manner as in Example 1, and the structure of this glucan was compared with the main chain in which glucose was connected by β-1,3-linkage. It was confirmed that one glucose molecule was branched to the side chain at the β-1,6-linkage.

Example 5 Effect of pH on solubility The solubility of β-1,3-1,6-D-glucan varied greatly depending on the pH of the culture solution. As can be seen in Example 1, as a result of the particle size distribution measurement, it was found that most of β-1,3-1,6-D-glucan existing as particles has a size of 0.1 μm or more. Therefore, what can be passed through a filter (0.21 μm) manufactured by Advantech Co., Ltd. can be considered as fine particles that are dissolved and those that cannot pass through. Here, the abundance ratio (the weight of the polysaccharide in the culture solution that has passed 0.2 μm / the weight of the polysaccharide in the culture solution × 100 (%)) is defined as the solubility. FIG. 3 shows the solubility when the pH was changed after the polysaccharide concentration of the culture solution of Example 1 was adjusted to 0.2% (2 mg / ml).

Example 6 Effect of Temperature on Solubility The solubility of β-1,3-1,6-D-glucan varies greatly depending on the temperature. Here, the temperature was changed, and filtration was performed with a filter (0.2 μm) manufactured by Advantech in the same manner as in Example 5 to determine the solubility. FIG. 4 shows the solubility when the temperature was changed after the polysaccharide concentration of the culture solution of Example 1 was adjusted to 0.2% (2 mg / ml). Here, the abundance ratio (the weight of the polysaccharide in the culture solution that has passed 0.2 μm / the weight of the polysaccharide in the culture solution × 100 (%)) is defined as the solubility.

  As seen in Examples 5 and 6, dissolved β-1,3-1,6-D-glucan and fine particle β-1,3-1,6-D-glucan are selected by controlling pH and temperature. Can be prepared automatically.

Example 7 Characteristics of Powdered Glucan β-1,3-1,6-D Containing Fine Particles β-1,3-1,6-D-glucan Prepared by Alkali Treatment and Cell Removal Treatment of Example 1 -Polysaccharide glucan was precipitated by adding ethanol to the glucan aqueous solution so that the final concentration was 66% (v / v), and recovered by centrifugation. Subsequently, ethanol and water were removed by a freeze-drying method to obtain dry β-1,3-1,6-D-glucan. The yield at that time was 95% or more compared to the total sugar concentration before ethanol precipitation. Next, the obtained dry β-1,3-1,6-D-glucan was dissolved and dispersed in water so that the final concentration was 0.3% (w / v), and then Tosoh was used as shown in Example 1. When the molecular weight was measured by gel chromatography using Toyopearl HW65 (column size: 75 cm × φ1 cm, excluded molecular weight: 2.5 million (dextran)) manufactured by the same company with 0.1M sodium hydroxide aqueous solution as the eluent, the resulting polysaccharide was obtained. It has been found that the molecular weight of is composed of a low molecular fraction having a peak of 2 to 300,000 and a high molecular fraction having an apparent appearance of 500 to 2.5 million. Here, a pullulan manufactured by Shodex was used as a molecular weight marker.
On the other hand, in order to separate water-soluble β-1,3-1,6-D-glucan and fine particles, an aqueous solution of β-1,3-1,6-D-glucan prepared by this method (fine particles and solubilized glucan were mixed). When the product was filtered with a filter (0.2 μm) manufactured by Advantech, the polymer fraction of 500 to 2.5 million disappeared. Therefore, even if the β-1,3-1,6-D-glucan obtained by this method is dried, if it is redissolved, it is the same as β-1,3-1,6-D-glucan before drying. It was demonstrated to reproduce the physical behavior of

  As described above, low viscosity was successfully achieved by alkali-treating high-viscosity β-1,3-1,6-D-glucan produced by microorganisms belonging to the genus Aureobasidium. Β-1,3-1,6-D-glucan obtained by the method of the present inventors contains a water-soluble component and a fine particle dispersed component, and the ratio can be controlled by pH and temperature. It was also found that a culture solution having excellent storage stability and thermal stability can be obtained by controlling the concentration of metal ions in the desalting step. As a result, a method for recovering and purifying β-1,3-1,6-D-glucan with high yield can be provided.

Example 8 Production of high-purity β-1,3-1,6-D-glucan powder 90 L of culture solution (polysaccharide concentration 0.5% (5 mg / ml)) subjected to the alkali treatment of Example 1 was added to 50% citrate. After neutralizing with 9 kg of the acid aqueous solution, the cells were removed through a Kamata filter press 40D-4 pre-coated with 1.8 kg of filter aid (Nippon Paper Chemicals' powdered cellulose KC floc). The filtrate was concentrated to 9 L with an ultrafiltration spiral element (NTU3150-S4 manufactured by Nitto Denko). While stirring this concentrated solution, 18 L of ethanol was added to obtain a glucan / ethanol / water slurry. The viscosity of the slurry was 22 mPa · s (30 ° C.) with a BM viscometer. The mixture was allowed to stand at room temperature for 3 hours, and about 17 L of supernatant (ethanol / water) was removed. The viscosity of the remaining slurry was 45 mPa · s (30 ° C.). 10 L of this concentrated slurry was spray-dried using a Sakamoto Giken type spray dryer R-3 to obtain 360 g of β-1,3-1,6-D-glucan powder (recovery rate 80%). As a result of analyzing the NMR spectrum, the purity of the obtained β-1,3-1,6-D-glucan was 90% or more.
The β-1,3-1,6-D-glucan obtained by the above production method contains about 10% of easily water-soluble polysaccharides other than glucan, but could be easily purified by washing with water.

Example 9
100 L of the culture solution (polysaccharide concentration 5 mg / ml) subjected to alkali treatment in Example 1 was neutralized with 10 L of 50% aqueous citric acid solution, and then pre-coated with 1.8 kg of filter aid (Nippon Paper Chemicals Powdered Cellulose W-200). The bacterial cells were removed through a Kanda filter press 40D-4, and the filtrate was concentrated to 55 L of a half amount with an ultrafiltration spiral element (NTU-3150-S4 manufactured by Nitto Denko). The solution was finally diluted to 1/100 concentration and concentrated to 5 L by vacuum concentration, the viscosity of the concentrate reached 100 mPa · s and the solid content concentration reached 10%. Spray drying using an drying apparatus R-3 (inlet temperature 185 ° C., outlet temperature 120 ° C., rotation speed 33000 rpm), 310 g of β-1,3-1,6-D-glucan powder (Recovery rate: 62%) 0.5% of the obtained β-1,3-1,6-D-glucan powder was dissolved in water, and the polysaccharide concentration was measured. From this, the purity of this powder was estimated to be about 95% or more, and the obtained powder was light brown and had an average particle size of 25 μm. It was possible to carry out with simple stirring up to about 10%, and at 15%, it became a paste and did not fall even when the container was turned upside down.

Example 10 Malignant Neoplastic Inhibitory Activity and Immunostimulatory Activity (peritoneal administration)
Using β-1,3-1,6-D-glucan (hereinafter simply referred to as glucan) derived from the Aureobasidium pullulans GM-NH-1A1 strain prepared by the method shown in Example 1. In addition, malignant neoplasm inhibitory activity and malignant neoplastic metastasis inhibitory activity in mice were examined.
1. Methods and materials 1) Experimental materials
Matrigel (Reduced Growth Factor) was purchased from Becton Dickison. The glucan prepared in Example 1 was used, dialyzed with physiological saline, and sterilized by filtration through a 0.45 μm membrane. Then, 0.5 g / ml and 1.5 mg / ml glucan solutions were prepared and used for the experiment. 0.1 ml per 10 g body weight of mice was administered intraperitoneally.

2) Animals
Balb / c mice (male, 5 weeks old) were purchased from Clare Co., Ltd. and pre-bred for 1 week, and then healthy mice were used for the experiments.
3) Colon 26 colorectal cancer cells Colon 26 colorectal cancer cells distributed from Tohoku University Aging Research Institute Medical Cell Resource Center as malignant neoplasms were used after passage.
4) Creation of colon 26 colon cancer cell transplanted mice in the spleen
Colon 26 colon cancer cells (5 × 10 4 cells / ml) were suspended in a phosphate-saline buffer (PBS, pH 7.4), and 1 mg / ml Matrigel was added to the cell suspension. Under a Nembutal anesthesia, a small incision was made on the back of the Balb / c mouse to expose the spleen, and 0.2 ml of Colon 26 colon cancer cell suspension (1 × 10 4 cells / spleen) was injected into the spleen. Immediately thereafter, the small incision was sutured. From the day after colon 26 colon cancer was transplanted, the prepared glucan solution was intraperitoneally administered once a day at a ratio of 5 mg / kg body weight and 15 mg / kg body weight for 14 days. The normal group and the control group received saline alone. The day after the final administration (15 days after cancer transplantation), heparin was collected from the inferior vena cava under ether anesthesia. Further, the mice were sacrificed after blood collection, and the spleen, liver and thymus were removed and the weight of each tissue was measured. Colon 26 colon cancer cell colonies metastasized to the liver were counted.

Further, the small intestine was removed and fixed with 10% neutral formalin buffer. After embedding in paraffin, a pathological section of the small intestine was prepared. Each small intestine pathological section was processed by a conventional method and then immunostained using NK antibody and INF-γ antibody. And the number of NK positive cells and the number of INF-γ positive cells were counted.
Subsequently, IL-12 in blood of a mouse transplanted with colon 26 colon cancer in the spleen was measured. In the method, plasma was obtained after centrifugation of the blood, and IL-12 in the plasma was measured by an ELISA kit.

2. Experimental result 1) Effect of glucan on primary tumor in colon 26 colon cancer transplanted mouse into spleen As shown in Table 6, when colon 26 colon cancer was transplanted into spleen, cancer cells clearly grew in spleen However, the glucan administration group showed an inhibitory effect on the increase in cancer weight in the spleen. That is, an inhibitory effect of 33.2% was observed in the 15 mg / kg administration group.
2) Effect of glucan on liver metastasis in colon 26 colon cancer transplanted mice into the spleen As shown in Table 6, colon 26 colon cancer metastasis to the liver is evident when colon 26 colon cancer is transplanted into the spleen. And liver weight increased. On the other hand, in the intraperitoneal administration group of glucan, the number of cancer metastasis colonies to the liver was significantly suppressed in comparison with the colon 26 colon cancer transplanted mice group for metastasis to the liver. That is, the anticancer metastasis effect was 44.5% in the 5 mg / kg administration group and 31.2% in the 15 mg / kg administration group.

3) Influence of intraperitoneally administered glucan on intestinal immunity function in mice transplanted with colon 26 colon cancer cells in spleen As shown in Table 7, the number of NK positive cells in the small intestinal mucosa tissue transplanted colon 26 colon cancer in the spleen Intraperitoneal administration of 5 mg and 15 mg / kg glucan significantly restored NK positive cells in the small intestinal mucosal tissue to normal values. Further, intraperitoneal administration of 5 mg and 15 mg / kg of glucan significantly increased the number of INF-γ positive cells in the small intestinal mucosa as compared with the colon 26 transplanted mice.
4) Effect of intraperitoneally administered glucan on blood IL-12 levels in colon 26 colon cancer transplanted mice into the spleen As shown in Table 8, when colon 26 colon cancer was transplanted into the spleen, IL- 12 concentration increased. This fact is considered that the immune function was enhanced by the action of the biological defense system that tried to eliminate cancer. The intraperitoneal administration of glucan increased the blood concentration of IL-12 from the colon 26 colon cancer-transplanted mice at any dose.
The blood concentration of IL-12 was increased by intraperitoneal administration of glucan, suggesting that this glucan has the ability to activate macrophages that are antigen-presenting cells.

Example 11 Malignant Neoplastic Inhibitory Activity and Immunostimulatory Activity (About Oral Administration Experiment)
Using β-1,3-1,6-D-glucan (hereinafter simply referred to as glucan) derived from the Aureobasidium pullulans GM-NH-1A1 strain prepared by the method shown in Example 1. In addition, malignant neoplasm inhibitory activity and malignant neoplastic metastasis inhibitory activity in mice were examined.
1. Methods and Materials Experimental Materials 1) Matrigel (Reduced Growth Factor) was purchased from Becton Dickison. The glucan prepared in Example 1 was used, and after dialyzing with distilled water, 5 mg / ml and 4.5 mg / ml glucan solutions were prepared and used for the experiment. 0.1 ml per 10 g body weight of mice was orally administered.
2) Animals
Balb / c mice (male, 5 weeks old) were purchased from Clare Co., Ltd. and pre-bred for 1 week, and then healthy mice were used for the experiments.
3) Colon 26 colon cancer cells Colon 26 colon cancer cells distributed from Tohoku University Aging Research Institute Medical Cell Resource Center were used as malignant neoplasms.
4) Creation of colon 26 colon cancer cell transplanted mice in the spleen
Colon 26 colon cancer cells (5 × 10 4 cells / ml) were suspended in a phosphate-saline buffer (PBS, pH 7.4), and 1 mg / ml Matrigel was added to the cell suspension. Under a Nembutal anesthesia, a small incision was made on the back of the Balb / c mouse to expose the spleen, and 0.2 ml of Colon 26 colon cancer cell suspension (1 × 10 4 cells / spleen) was injected into the spleen. Immediately thereafter, the small incision was sutured. From the day after colon 26 colon cancer was transplanted, the prepared glucan was orally administered once a day at 50 mg / kg body weight and 450 mg / kg body weight for 14 days. The normal group and the control group received saline alone. The day after the final administration (15 days after cancer transplantation), heparin was collected from the inferior vena cava under ether anesthesia. Further, the mice were sacrificed after blood collection, and the spleen, liver and thymus were removed and the weight of each tissue was measured. Colon 26 colon cancer cell colonies metastasized to the liver were counted.

  Further, the small intestine was removed and fixed with a 10% neutral formalin buffer. After embedding in paraffin, a pathological section of the small intestine was prepared. Each small intestine pathological section was processed by a conventional method and then immunostained using an INF-γ antibody. The number of INF-γ positive cells was counted.

2. Experimental result 1) Effect of glucan on primary tumor in colon 26 colon cancer transplanted mouse to spleen As shown in Table 9 below, when colon 26 colon cancer is transplanted into spleen, cancer cells clearly grow in spleen However, the glucan administration group showed a significant inhibitory effect on the increase in cancer weight in the spleen. In other words, the inhibitory effect was 27.9% in the 50 mg / kg administration group and 23.9% in the 450 mg / kg administration group.
2) Effects of glucan on liver metastasis in mice transplanted with colon 26 colon cancer into the spleen As shown in Table 9 below, colon 26 colon cancer transplanted into the spleen clearly showed colon 26 colon cancer in the liver. Metastasis was observed and liver weight increased. On the other hand, in the oral administration group of glucan, the number of cancer metastasis colonies to the liver was significantly suppressed in comparison with the colon 26 colon cancer transplanted mice group for metastasis to the liver. That is, an anti-cancer metastasis effect of 37.3% was observed in the 50 mg / ml administration group and 4.9% in the 450 mg / kg administration group.

3) Effect of orally administered glucan on intestinal immunity function in mice transplanted with colon 26 colon cancer cells in spleen As shown in Table 10, when colon 26 colon cancer was transplanted into the spleen, INF-γ positive cells in the small intestinal mucosal tissue The number has decreased. With oral administration of 50 mg / kg glucan, the number of INF-γ positive cells was significantly increased as compared with colon 26 colon cancer-transplanted mice. The high dose (450 mg / kg) did not significantly increase with respect to the number of INF-γ positive cells.

Example 12 Allergy suppression effect Method 1) Allergy sensitization method by oral administration of ovalbumin
Balb / c mice (female, 4 weeks old) were allowed to ingest β-glucan-containing powdered diet (0, 0.25%, 0.5%, 1.0%) freely during the experiment (β-glucan-administered mice group, β -A mouse fed with a powder feed containing no glucan was taken as a control mouse group). During the breeding period, the amount of food consumed per cage (7 mice in a cage experiment) was measured every day, and the body weight of the mice was measured every week. From the second week after the start of ingestion of β-glucan, 1% ovalbumin aqueous solution was freely ingested for one week (primary antibody production, primary sensitization experiment). In the latter three days, a 2.5% ovalbumin aqueous solution was gavaged twice a day in the morning and evening (ovalbumin 5 mg x 2 times / mouse / day).
On the 3rd and 7th days after ingestion with an ovalbumin aqueous solution, 25 μg ovalbumin physiological saline solution containing 1.6 mg of aluminum hydroxide gel was intraperitoneally administered to induce immunization (secondary antibody production, secondary sensitization experiment). The day after the intraperitoneal administration of the ovalbumin twice, blood was collected from the inferior vena cava under ether anesthesia to obtain serum. The spleen, thymus, and small intestine were quickly removed, the spleen and thymus were weighed, and the removed small intestine was fixed with 10% formalin buffer to prepare pathological sections.
2) Measurement of serum cytokines and immunoglobulins Serum ovalbumin-specific immunoglobulins IgG, IgG1, IgG2a, IgA and IgE are ELISA using 96-well plate coated with ovalbumin and various anti-immunoglobulin antibodies. Measured by the method.
3) Immunohistological evaluation of the small intestine A formalin-fixed small intestine section was embedded in paraffin according to a conventional method, and a pathological section having a thickness of 5 μm was prepared. Thereafter, deparaffinization was performed, and immunostaining was performed using CD4, CD8, INF-γ, and IgA antibodies. Thereafter, the number of CD4 and CD8 positive cells and the number of INF-γ and IgA secreting cells were counted.

2. Results 1) Effect of β-glucan on body weight, intake, and systemic reactivity during ovalbumin-sensitized immune response Changes in body weight after 2 weeks in β-glucan-ingested mice and control mice, ovalbumin solution ingestion for 1 week Of body weight and body weight transition in the immunization induction by intraperitoneal administration of ovalbumin, compared with the group of mice not sensitized with ovalbumin (normal mouse group), the transition of body weight, administration of ovalbumin (1 There was no difference in the change in body weight after (secondary sensitization) and the change in weight after peritoneal administration of ovalbumin (secondary sensitization).
In the second and second sensitization by the intraperitoneal administration of ovalbumin in the first and second rounds, a significant decrease in food consumption was observed in the β-glucan-treated mice group compared to the normal mice group (the mice group not sensitized with ovalbumin). Was recognized. That is, immediate allergic symptoms were observed between the two. In the first ovalbumin sensitization, the amount of food consumed was decreased in the β-glucan-administered mouse group compared to the normal mouse group. In the second ovalbumin sensitization, the food intake decreased in the control mouse group, and allergic symptoms similar to those in the first time were observed, but in the β-glucan-treated mouse group (containing 1.0% and 0.5%), The amount of food intake increased compared to the control mouse group, indicating a food intake close to that of the normal mouse group. That is, allergic symptoms were dramatically alleviated.
With regard to the systemic reactivity associated with the first and second ovalbumin sensitization, the first and second ovalbumin sensitization (secondary sensitization) markedly observed the behavior of mouse shaking, napping and scumming. It was. The mice's tremor, nap, and tingling behavior due to the first ovalbumin sensitization were not recovered even after 12 hours of observation, and were not eliminated by the administration of β-glucan. The mice's tremor, nap, and tingling behavior due to the second ovalbumin sensitization were restored to normal behavior 4 hours after administration of ovalbumin by ingestion of β-glucan (containing 0.5% and 1.0%). It has been found.
2) Effect of β-glucan on spleen and thymus weight during ovalbumin-sensitized immune reaction The spleen weight was increased by ovalbumin sensitization compared to the normal group (mouse not administered ovalbumin). On the other hand, thymus weight decreased. In the β-glucan-administered mouse group, the spleen weight was further increased and the thymus weight was decreased in the β-glucan 1.0% dietary intake group as compared to the control mouse group. From this fact, it is considered that β-glucan administration increases immune function from the spleen (Table 11).

3) Effect of β-glucan on ovalbumin reaction blood immunoglobulin production (secondary antibody production) by ovalbumin sensitization Antibody production by IgG ovalbumin sensitization (IgG, IgG1 (Th1 cell-derived immunoglobulin), IgG2a ( Th2 cell-derived immunoglobulin), IgA and IgE) are significantly increased in the control mouse group as compared with the normal mouse group, and secondary antibody production is caused by oral administration of ovalbumin, causing an immune response. It has been found. In addition, the production of IgG, IgG1, IgG2a, and IgA was not affected by β-glucan intake compared to the control mouse group (Table 12).
On the other hand, IgE production related to the allergic reaction was observed to decrease in the β-glucan-administered mouse group (1.0% diet) (Table 12). This fact suggests that β-glucan may have an antiallergic effect.

4) Effect of β-glucan on small intestinal immune response by ovalbumin sensitization (immunohistological evaluation)
As shown in Table 13, the number of CD4 positive cells and CD8 positive cells of lymphocytes in the small intestine by ovalbumin intake was higher in the β-glucan intake group (0.5% and 1.0% mixed diet) than in the control mouse group. Significantly increased. This fact indicates that β-glucan further enhances the immune inflammatory reaction in the small intestine by ovalbumin sensitization, suggesting the possibility of acting as a biological defense reaction against foreign substances. That is, there is a possibility that Th4 cells of CD4 are activated and produce IFN-γ and the like, work on B cells to produce immunoglobulins, and eliminate pathogenic microorganisms. In addition, the increase in the number of CD8 positive cells may control excessive immune inflammatory reaction in the intestinal tract and eliminate excessive antigen entry.
CD4 positive cells are found in Th1 and Th2 cells, indicating that T helper cells are activated. Although the number of IgA-secreting cells in the β-glucan administration group was not significantly different from that in the control mouse group, there was a tendency to increase. On the other hand, CD8 positive cells show killer T cell activation induced by Th1 cell activation, and the increase in CD8 positive number significantly activates Th1 cell line compared to Th2 cell line. It suggests. This suggests that β-glucan suppresses allergic reactions.

Conclusion)
From the above results, it is suggested that ingestion of β-glucan has a function of controlling inflammation associated with immune reaction and maintaining a biological defense reaction against ovalbumin serving as a food antigen.

A two-dimensional NMR (13C-1H COSY NMR) spectrum is shown. The particle size distribution (median diameter 0.23 μm) after ultrasonic irradiation is shown. The relationship between solubility and pH (when the polysaccharide concentration is 0.2% at 25 ° C.) is shown. The relationship between solubility and temperature (when the polysaccharide concentration is 0.2% and pH is 4) is shown.

Claims (29)

An aqueous solution of β-1,3-1,6-D-glucan, and the ¹ H NMR spectrum of the solution using 1N sodium hydroxide heavy aqueous solution as a solvent has two signals of 4.7 ppm and 4.5 ppm. Β-1,3-1,6, wherein the aqueous solution has a viscosity of 50 cP ([mPa · s]) or less at 30 ° C., pH 5.0, and a concentration of 0.5% (w / v). -D-glucan aqueous solution.   The β-1,3-1,6-D-glucan aqueous solution according to claim 1, wherein the molecular weight of β-1,3-1,6-D-glucan is 10,000 to 500,000.   The β-1,3-1,6-D-glucan according to claim 1 or 2, comprising fine particles β-1,3-1,6-D-glucan having a primary particle diameter of 0.05 to 2.0 μm. Aqueous solution.   The β-1,3-1,6-D-glucan has a β-1,3 bond / β-1,6 bond binding ratio of 0.5 to 2.0. The β-1,3-1,6-D-glucan aqueous solution described. Β-1,3 bond / β-1,6 bond binding based on integral ratio of β-1,3 bond and β-1,6 bond signal in ¹ H NMR spectrum using 1N sodium hydroxide heavy aqueous solution as solvent The β-1,3-1,6-D-glucan aqueous solution according to any one of claims 1 to 3, wherein the ratio is 1.0 to 1.5.   The β-1,3-1,6-D-glucan aqueous solution according to claim 1, wherein β-1,3-1,6-D-glucan has a sulfur-containing group.   The β-1,3-1,6-D-glucan is β-1,3-1,6-D-glucan produced by a microorganism belonging to the genus Aureobasidium sp. The β-1,3-1,6-D-glucan aqueous solution according to any one of 1 to 6.   The β-1,3-1,6-D-glucan aqueous solution according to claim 7, wherein the microorganism is Aureobasidium pullulans.   The microorganism is Aureobasidium pullulans GM-NH-1A1 (deposit number: FERM P-19285, international deposit number FERM BP-10294), or Aureobasidium pullulans GM-NH-1A2 strain ( The β-1,3-1,6-D-glucan aqueous solution according to claim 8, which has a deposit number: FERM P-19286 and an international deposit number FERM BP-10295).   The β-1,3-1,6-D-glucan aqueous solution according to any one of claims 1 to 9, which does not contain microorganisms.   The β-1,3-1,6-D-glucan aqueous solution according to any one of claims 1 to 10, wherein the metal ion concentration is 120 mg / 100 ml or less.   The β-1,3-1,6-D-glucan aqueous solution according to claim 11, wherein the metal ions are Na ions and Ca ions.   Β-1, obtained by removing fine particles β-1,3-1,6-D-glucan from the β-1,3-1,6-D-glucan aqueous solution according to claim 1, 3-1,6-D-glucan aqueous solution.   Fine particles β obtained by removing water-soluble β-1,3-1,6-D-glucan from the β-1,3-1,6-D-glucan aqueous solution according to claim 1. -1,3-1,6-D-glucan.   A β-1,3-1,6-D-glucan aqueous solution obtained according to claim 13 and a β obtained by arbitrarily blending the fine particles β-1,3-1,6-D-glucan obtained according to claim 14 A liquid containing -1,3-1,6-D-glucan.   Dry β-1,3-1,6-D-glucan obtained by drying the β-1,3-1,6-D-glucan aqueous solution according to any one of claims 1 to 12.   An anti-malignant neoplasm comprising the β-1,3-1,6-D-glucan aqueous solution or β-1,3-1,6-D-glucan according to any one of claims 1 to 16 as an active ingredient.   A malignant neoplastic metastasis inhibitor comprising the β-1,3-1,6-D-glucan aqueous solution or β-1,3-1,6-D-glucan according to any one of claims 1 to 16 as an active ingredient. .   An INF-γ producing agent comprising the β-1,3-1,6-D-glucan aqueous solution or β-1,3-1,6-D-glucan according to any one of claims 1 to 16 as an active ingredient.   The β-1,3-1,6-D-glucan aqueous solution according to any one of claims 1 to 16 or INF-γ production containing β-1,3-1,6-D-glucan as an active ingredient Therapeutic agents for diseases that are suppressed or cured.   An allergy inhibitor comprising the β-1,3-1,6-D-glucan aqueous solution or β-1,3-1,6-D-glucan according to any one of claims 1 to 16 as an active ingredient.   A macrophage activator comprising the β-1,3-1,6-D-glucan aqueous solution or β-1,3-1,6-D-glucan according to any one of claims 1 to 16 as an active ingredient.   A health food or health enhancer containing the β-1,3-1,6-D-glucan aqueous solution or β-1,3-1,6-D-glucan according to any one of claims 1 to 16.   An external preparation containing the β-1,3-1,6-D-glucan aqueous solution or β-1,3-1,6-D-glucan according to any one of claims 1 to 16.   Cosmetics containing the β-1,3-1,6-D-glucan aqueous solution or β-1,3-1,6-D-glucan according to any one of claims 1 to 16.   An alkali or an aqueous solution thereof is added to a microorganism culture solution containing β-1,3-1,6-D-glucan, and the pH of the culture solution is reduced to 12 or more, and then the insoluble matter containing microorganisms is separated and removed. And a method for preparing an aqueous β-1,3-1,6-D-glucan solution, wherein the metal ions are removed so that the metal ion concentration is 120 mg / 100 ml or less.   27. The method for preparing an aqueous solution of β-1,3-1,6-D-glucan according to claim 26, wherein the metal ions are Na ion concentration and Ca ion.   The β-1,3-1,6-D-glucan aqueous solution of claims 1 to 10 is dialyzed and concentrated, and an alcohol is added to the concentrated solution to precipitate β-1,3-1,6-D-glucan. The obtained slurry of β-1,3-1,6-D-glucan / alcohol / water is separated, and the precipitated slurry is spray-dried, and the high purity β-1,3-1,6 -Preparation method of D-glucan powder.   The β-1,3-1,6-D-glucan aqueous solution of claims 1 to 10 is dialyzed, further concentrated under reduced pressure, and then the concentrated solution is spray dried. , 6-D-glucan powder preparation method.

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