JP4595074B2 - Novel glucan and method for producing the same - Google Patents

Description

The present invention relates to a novel α-1,3-glucan and β-1,3-glucan derived from a filamentous fungus culture, a production method thereof, and the like.

Oligosaccharides in which glucose is α-1,3-glucoside-linked have recently found value as oligosaccharides having physiological activity. Nigerose, a disaccharide of α-1,3-glucoside, has low sweetness and refined sweetness, has heat resistance and moisture retention, and is rich in sake, miso, miso, and other foods that use koji molds. It is also recognized as a factor of body taste, and further, immunostimulatory action and anti-cariogenic functionality are attracting attention. Nigeritol, a sugar alcohol derived from nigerose, is a low-calorie sweetener that is difficult to digest and absorb and is also non-cariogenic.

In Patent Document 1 and Non-Patent Document 1, hydrolysis is performed using enzymes, acids, or the like using nigeran (linear glucan linked alternately by α-1,3 bonds and α-1,4 bonds) or erucinan as a substrate. Thus, a method for producing nigerooligosaccharides such as nigerose has been proposed.

Patent Document 2 describes a method for producing nigerose acetate by acidolysis of a cyclized tetrasaccharide having a structure in which four glucoses are alternately linked by α-1,3 and α-1,6 bonds. ing. Patent Document 3 discloses a method for producing α-1,3-glucooligosaccharide having a degree of polymerization of 2 to 4 by reacting mutanase with mutan.

Furthermore, in Non-Patent Document 3, Histoplasma capsultum, Paracocciodioides brasiliensis, Blastomyces dermatitidis ( Blastomyces dermatitidis ) are human infectious dimorphic yeasts. ), A decrease in pathogenicity is observed when α-1,3-glucan is reduced. This is thought to be because when the cell surface is covered with α-1,3-glucan and β-1,3-glucan is masked, it grows in the body without being recognized by the human immune system. Yes. From this point as well, α-1,3-glucan and β-1,3-glucan are versatile polysaccharides with different functions, and it is desired to obtain them in large quantities at low cost. A method for producing such a polysaccharide in a stable and large amount from an organism has not been established.

Japanese Patent Laid-Open No. 55-19004 Japanese Patent Laid-Open No. 2004-23887 JP 2005-185182 A S. A. Baker et al., "Journal of the Chemical Society", pages 2448-2454, 1957 Kenichi Ishibashi et al., `` FEMS Immunology and Medical Microbiology '' Vol. 42, pp. 155-166, 2004 A. Beauvais et al., “Applied and Environmental Microbiology”, Volume 71, pp. 1531–1538, 2005

Although the usefulness of α-1,3-gluco-oligosaccharides containing nigerose is widely recognized, the production of these oligosaccharides requires high-purity α-1,3-glucan nigeran as a substrate. It is an industrial problem that nigerang and erucinan are very expensive and can be obtained only in small quantities. Furthermore, in the case of mutan or erucinane mainly composed of nigelan or α-1,3-glucan and containing α-1,6 linkage, it is mostly present in a mixture with other polysaccharides. It was difficult to purify only a large amount easily.

On the other hand, as shown in Non-patent Documents 2 and 3, the filamentous fungus β-1,3-glucan containing the genus Aspergillus is a mixture with other polysaccharide components such as α-1,3-glucan. Although known, their separation and purification is not easy.

The present invention has been made in view of such prior art, and is free of α-1,3-glucan and α-1,3-glucan from the filamentous fungus. It is an object to provide a method for producing β-1,3-glucan, and α-1,3-glucan and β-1,3-glucan obtained by the production method.

As a result of intensive research aimed at solving the above-mentioned problems, the present inventor has intensively studied a simple purification method for both glucans from a culture of koji mold that can be cultured in large quantities, has a food experience, and is safe. Soluble proteins and soluble polysaccharides are removed from the culture containing sucrose by hot water extraction and weak alkali extraction, and purified α-1,3-glucan is added to the strong alkali-soluble extract fraction, and the supernatant of the second strong alkali extraction ( The present invention was completed by succeeding in obtaining purified β-1,3-glucan for the extract and residue classification.

That is, the present invention relates to the following aspects.
[1] obtained from a culture of filamentous fungi, consisting only of α-1,3 bonds having an average molecular weight of 500 to 99,000, preferably 30,000 to 35,000, for example about 32,580 α-1,3-glucan.
[2] A method for producing the α-1,3-glucan including the following steps:
(A) a hot water extraction treatment of the filamentous fungus culture,
(B) a step of subjecting the residue obtained in (a) to a weak alkali extraction treatment,
(C) a step of subjecting the weak alkali-insoluble part (residue) obtained in (b) to a strong alkali extraction treatment, and (d1) neutralizing the strong alkali-soluble part (extract) obtained in (c), Dialyzing and concentrating to prepare a precipitate consisting of α-1,3-glucan (AS-2 fraction).
[3] Obtained from a culture of filamentous fungi, average molecular weight: 5,000 to 990,000, preferably 230,000 to 280,000, eg about 253,000, β-1,3 / 1,6 branch Β-glucan based on β-1,3-glucan having a degree: 0-20%, preferably 1-10%, for example about 6%.
[4] A method for producing the β-1,3-glucan including the following steps:
(A) a hot water extraction treatment of the filamentous fungus culture,
(B) a step of subjecting the residue obtained in (a) to a weak alkali extraction treatment,
(C) a step of subjecting the weak alkali-insoluble part (residue) obtained in (b) to a strong alkali extraction treatment,
(D2) A step of subjecting the strong alkali insoluble part (residue) obtained in (c) to a strong alkali extraction treatment again, and (e) suspending the strong alkali insoluble part (residue) obtained in (d2) in water. Step of preparing a residue (AI fraction) consisting of β-1,3-glucan by turbidity, neutralization and dialysis.

In the present invention, “ α-1,3-glucan consisting only of α-1,3 bond ” means a bond other than 1,3-glycoside bond in various analysis results described in the examples in this specification. Is not detected and it means that it is a linear glucan substantially free of β-1,3-glucan. In addition, “β-glucan mainly composed of β-1,3-glucan” is similarly a small proportion of the above-mentioned degree based on various analysis results described in the examples in this specification. Of β-1,3 / 1,6. The mixing ratio of α-1,3-glucan in such β-glucan is 10% or less, desirably 5% or less.

In the present invention, the “filamentous fungus culture” refers to a filamentous fungus culture using rice bran, sake lees, soy sauce lees, shochu, miso, wheat bran in addition to the cultured fungal fungi themselves. Various fermented products (food and raw materials) containing filamentous fungi that are produced by culturing filamentous fungi such as enzyme extraction residues and filamentous fungus liquid cultures, that is, filamentous fungi-derived products are also included. Moreover, the filamentous fungus culture used in the production method of the present invention can be in any state (form) such as freeze-dried cells.

According to the present invention, as a result of examining the extraction conditions of polysaccharides from fungal cell walls, high-purity α-1,3-glucan and α-1,3, which are substantially composed only of α-1,3 bonds. -Preparation of β-1,3-glucan based on β-1,3-glucan from which glucan has been significantly removed is possible, and both glucans can be substantially separated and supplied stably It is.

There is no restriction | limiting in particular in the kind of filamentous fungi used by this invention, Arbitrary bacteria well-known to those skilled in the art can be used. Considering the fact that both glucans are contained in a significant amount, food experience, safety and mass production are possible, representative examples are Aspergillus oryzae, Aspergillus sojae (Aspergillus sojae). ), Aspergillus niger, Aspergillus kawachi, Aspergillus shirousami, Aspergillus awamori, natural mutants of these filamentous fungi, artificial mutants, And mutants produced by genetic manipulation. Artificial mutants and genetically engineered mutants can be prepared using any method or means known to those skilled in the art. These filamentous fungi can also be cultured using any method or means known to those skilled in the art.

The conditions for each step of the production method of the present invention will be described in detail below. An outline of the production method of the present invention is shown in FIG.

The hot water extraction treatment of the filamentous fungus culture in (a) is performed by subjecting the filamentous fungus culture to an appropriate pretreatment such as pulverization, and then, for example, an appropriate buffer such as 0.1 M phosphoric acid and water. It is preferably suspended at 50 to 150 ° C., preferably 70 ° C. or more, more preferably 80 ° C. or more for 0.5 to 2 hours. For example, the temperature can be about 120 ° C. for about 60 minutes in an autoclave. In order to remove more soluble proteins and soluble polysaccharides, it is preferable to perform such hot water extraction treatment a plurality of times. In addition, it is possible to efficiently remove polysaccharides such as starch contained in the culture medium by using industrial amylase, glucoamylase and xylanase in combination.

The step of subjecting the residue obtained in (a) in (b) to weak alkali extraction is, for example, 4-15 ° C., for example, about 4 ° C., 12 under weak alkali conditions (pH 11 or more) such as 0.5M NaOH. It is preferable to carry out for ~ 36 hours, for example, 12 hours.

The step of subjecting the weak alkali-insoluble part (residue) obtained in (b) to a strong alkali extraction treatment in (c) is, for example, 4-15 ° C., for example, about 4 under strong alkaline conditions (pH 11 or higher) such as 2M NaOH. It is preferable to carry out at 12 ° C. for 12 to 36 hours, for example, about 12 hours.

In the step (d2), the strong alkali insoluble part (residue) obtained in (c) is again subjected to a strong alkali extraction treatment (second strong alkali extraction treatment step) under basically the same conditions as in the step (c). Although it can be done, it can preferably be longer, for example about 24 hours. It is also possible to perform this process multiple times.

The neutralization operation in the steps (d1) and (e) can be performed using an appropriate acid such as acetic acid. The dialysis operation is preferably performed using purified water such as Milli-Q. The concentration operation can be performed by any method known to those skilled in the art, such as centrifugation and filtration.

The separation of the alkali-insoluble part and the alkali-soluble part that is performed after each alkali extraction step is, for example, a centrifugal operation under an appropriate condition such as 10,000 xg for 20 minutes, and an arbitrary known to those skilled in the art such as filtration. It can be done by the method.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the technical scope of the present invention is not limited to these descriptions, and various modifications that can be easily conceived by those skilled in the art based on the descriptions in this specification. Various modified or modified embodiments are also included in the present invention.

(1) Acquisition of α-1,3-glucan and β-1,3-glucan from the aerial mycelium cell wall
A sterilized nylon filter net (Nippon Rikagaku Kikai Co., LTD., Pore size: 63 μm) was layered on the Czapek-Dox agar medium so as to be in close contact with the plate using sterile tweezers. The nylon filter net was spot-inoculated with 1 × 10 ⁴ conidia suspension and cultured at 30 ° C. for 105 hours.

After culturing, the nylon filter net with the mycelium attached thereto was peeled off using tweezers, and placed in liquid nitrogen and immediately frozen. At this time, only the microbial cells are frozen, and the nylon filter net is not frozen. The frozen cells were scraped from the net using a spatula and collected in a 500 ml beaker. To remove conidia, pour about 200 ml of sterilized water into a beaker containing mycelia, treat with an ultrasonic treatment device (Iuchi) for 20 minutes, filter with a funnel equipped with Miracloth (CALBIOCHEM), and several times Washed with. The mycelium remaining on Miracloth was freeze-dried to obtain freeze-dried cells.

5 g of freeze-dried cells were pulverized in a mortar until pouring with liquid nitrogen. The crushed cells were suspended in 200 ml of 0.1 M phosphate buffer (pH 7.0), transferred to four 80 ml centrifuge tubes, and then extracted with hot water at 120 ° C. for 1 hour in an autoclave. The extract was centrifuged at 10,000 x g for 20 minutes, and fractionated into hot water-soluble (HW) and hot water-insoluble parts. The same operation was performed again on the hot water insoluble part.

The hot water soluble part was dialyzed against Milli-Q water to obtain the HW-1 fraction. The hot water insoluble part was suspended in 200 ml of 0.5 N cold NaOH water, stirred at 4 ° C. for 12 hours and extracted with dilute alkali. After extraction, transferred to a 80 ml centrifuge tube tube 4, dilute alkali-soluble fraction by centrifugation 10,000 xg, 20 min (d ilute a lkali-soluble fraction : DA) and was fractionated in dilute alkali insoluble portion. The dilute alkali-insoluble part was further suspended in 200 ml of 2N cold NaOH water, and stirred at 4 ° C. for 12 hours for strong alkali extraction. After extraction, transferred to a 80 ml centrifuge tube tube 4, strong alkali-by centrifugation 10,000 xg, 20 min soluble portion: and partitioned with strong alkali insoluble portion (a lkali- s oluble fraction AS) . The strong alkali-insoluble part was suspended again in 200 ml of 2N cold NaOH water, and stirred at 4 ° C. for 24 hours for strong alkali extraction. After extraction, transferred to a 80 ml centrifuge tube tube 4, the second strong alkali-soluble fraction by centrifugation at 10,000 xg, 20 min: was partitioned alkali-insoluble portion and (s econd a lkali- s oluble fraction SAS).

Alkali-insoluble portion, was suspended with 200 ml of cold Mill-Q, was neutralized with acetic acid while cooling in ice, and dialyzed against Milli-Q water, alkali-insoluble portion (a lkali- i nsoluble fraction: AI ) Each alkali-soluble part was neutralized with acetic acid while cooling in ice, dialyzed against Milli-Q water, and centrifuged at 10,000 xg for 10 minutes. -1 fraction and precipitation were DA-2, AS-2, SAS-2 fractions.

All fractions were lyophilized and the weight of each fraction was measured. The dry weight ratios of the fractions thus obtained are shown in Table 1. As a result, it was revealed that the cell wall of the aerial mycelium is fractionated mainly into the AS-2 fraction (24.9%) and the AI fraction (49.8%). Thereafter, the quantification of the constituent sugars contained in this fraction and the polysaccharide structure were analyzed.

  The total sugar (hexose) content of AS-2 and AI fractions was measured. The total amount of sugar in the sample was measured by modifying the phenol-sulfuric acid method [Dubois M et al. “Nature” (Nature. 168: 167 1951)]. After suspending approximately 1 mg of sample in 500 μl of Milli Q water, add 1 ml of 2.5% (w / v) phenol, and then add 2.5 ml of concentrated sulfuric acid directly to the liquid surface and vortex. Mix well. After standing for 30 minutes, it was diluted as appropriate and the absorbance at 490 nm was measured. A standard curve was prepared using a glucose solution as the standard sugar, and the total amount of sugar in the sample was calculated based on the standard curve.

  Among the main constituent sugars of the cell wall, neutral sugars such as glucose, galactose and mannose were quantified by the following method.

A 25 mg sample of the AS-2, AI fraction was weighed into a medium test tube, dissolved in 0.5 ml of 25 NH ₂ SO _{4 and} allowed to stand overnight at 4 ° C. To the acid concentration 1 N, 12 ml of water was added and mixed, and the mixture was completely hydrolyzed at 100 ° C. for 12 hours. The hydrolyzate was neutralized by adding an excess amount of barium carbonate, and the resulting barium sulfate precipitate was removed by centrifugation at 16,500 × g for 5 minutes. The supernatant was filtered through TOYO paper No. 5C and then desalted using cation exchange resin Dowex 50W-X2 (H ⁺ type, Muromachi Chemical Industries). At the stage of this desalting operation, hexosamine produced by hydrolysis of chitin and the like is adsorbed on the resin and removed. After the desalting operation, the resin was removed by filtration, and the filtrate was used as a neutral sugar fraction. Then, HPLC was used to quantify glucose, galactose, and mannose in the neutral sugar fraction. Glucose, galactose, and mannose as standard sugars were applied at 60, 80, and 100 μg, respectively, and the retention time and peak area were determined to prepare a standard curve. The sample was pretreated with a Millex-LG (MILLIPORE) filter and then applied. The elution time and peak area of the peak were compared with the standard sugar, and the type and amount of neutral sugar in the sample were calculated. The measurement was performed under the following conditions.

Table 3 shows the measurement results of the total sugar (hexose) content of the AS-2 and AI fractions and the ratios of glucose, galactose and mannose contained in the total sugar content. As a result, it was revealed that all the neutral sugars contained in the AS-2 fraction were glucose. In the AI fraction, 88% was glucose.

Chitin, the main constituent sugar of the cell wall, is the amount of hexosamine produced by hydrolysis of hydrochloric acid. The Blix method, an improved version of the Elson-Morgen method [Blix G, Acta chemica Scandinavica 2 Volume: 467 to 473 1948)]. About 5 mg of AS-2, AI fraction was weighed into an Eppendorf tube, suspended in 400 μl of 4 N HCl, and hydrolyzed with a heat block at 96 ° C. for 12 hours. After hydrolysis, 400 μl of water was added and left at room temperature for 1 hour. Cell wall residues that were not degraded were precipitated by centrifugation.

2-20 μl of the supernatant after centrifugation was filled up to 200 μl with sterile water to prepare a sample. 400 μl of acetylacetone reagent (acetylacetone: 1.25 N Na ₂ CO ₃ = 3: 100) was added to each sample and reacted at 90 ° C. for 1 hour. After cooling with running water, the test tube containing 4 ml of special grade ethanol was transferred, and 400 μl of Ehrlich reagent (p-dimethylaminobenzamide 1.6 g, conc. HCl 30 ml, special grade ethanol 30 ml) was added and stirred well. After standing for 1 hour or longer, the absorbance at 530 nm was measured. A standard curve was created using glucosamine hydrochloride as the standard sugar, and the amount of hexosamine in the sample solution was calculated based on this standard curve. Table 4 shows the proportion of hexosamine (chitin) in the AS-2 and AI fractions.

In order to study the polysaccharide structure of AS-2 and AI fractions, methylation analysis, ¹³ C-NMR analysis and β-1,3-glucanase enzyme treatment method were used in the AI fraction. Were methylated and treated with β-1,3-glucanase enzyme.

AS-2 fraction analysis (methylation analysis)
The methylation method was in accordance with the Hakomori method [Hakomori S “Journal of Biochemistry (Tokyo)” (J. Biochem [Tokyo]. 55: 205-208 1964). In addition, all the concentrates were dried using Concenrator TC-8 (TAITEC) 1 mL of DMSO was added to 10 mg of sample polysaccharide and dissolved in a sonic bath. Carbanion (MSC) (1 ml) was added and the atmosphere was replaced with nitrogen, and the mixture was allowed to react in a sonic bath at 50 ° C or lower for 5 hours with occasional agitation, followed by addition of 1 ml of methyl iodide in cold water and in a Sonic bath. The mixture was reacted for 2 hours in the same manner at 30 ° C or lower, dialyzed against running water overnight, and the oily portion was concentrated to dryness.In the case of oligosaccharide, the reaction solution was diluted with water and extracted with chloroform, After washing several times with a small amount of water, the solution was concentrated to dryness.

1 M of 2 M TFA was added to the methylated sample, and the mixture was reacted for 5 min in a Sonic bath, then hydrolyzed at 120 ° C. for 90 min in an autoclave, and concentrated to dryness. Next, 1 ml of 0.2 M ammonia was added to make it weakly alkaline, 10 mg of NaBH ₄ was added, 10 min Sonic was applied, and the mixture was allowed to stand and subjected to a reduction reaction overnight at room temperature.

Dowex-50w x2 (H + type) was added to neutrality until no bubbles were generated, and filtered with TOYO paper No. 5C. The filtrate was concentrated to dryness and concentrated to dryness while adding approximately 0.5 ml of 99% methanol. This concentration and drying was repeated 5 times, then 0.5 ml of acetic anhydride and pyridine were added, reacted at 100 ° C. for 1 hour, acetylated, concentrated to dryness, 0.5 ml of toluene was added, and the mixture was concentrated and dried twice. This was dissolved in 100 μl of acetone, and 2 to 10 μl of it was subjected to gas chromatography and analyzed. The sample was subjected to gas chromatography under the conditions shown in Table 5 below.

Shizophylan (derived from Shirohirotake) as a standard substance by the method of “Journal of Biochemistry (Tokyo). 90: 1093 to 1100” such as Shida M. et al. The elution time of each partially methylated alditol acetate was determined using chromatograms of the prepared products, maltoheptaose (Seikagaku Corporation), and dextran T70 (Amersham Pharmacia). Schizophyllan has a chemical ratio that when methylated, the ratio of partially methylated sugar is 2,3,4,6-tetramethyl: 2,4,6-trimethyl: 2,4-dimethyl = 1: 2: 1 It is a β-1,3 / β-1,6-glucan with a structure. The ratio of peak areas of the obtained chart was corrected by a response factor in consideration of the peak area of schizophyllan, and the amount ratio of each bond was determined.

[Preparation of MSC (methyl sulfinyl calbanion)]
Weigh 300 mg of NaH in a capped test tube, add 3 ml of DMSO and replace with N ₂ , then warm to 50 ° C in a sonic bath and react for about 1 hour until the solution turns pale green . During the reaction, the generated hydrogen was released by loosening the cap from time to time. The prepared MSC was stored at −20 ° C. in the dark.

  The results of methylation analysis are shown in Table 6. From this result, it was shown that the AS-2 fraction is a linear glucan composed of 1,3-glycosidic bonds. Multiply the ratio of tetramethyl and trimethyl by the response factor (Rf) based on the peak area of schizophyllan to give an average chain length of alkali-soluble glucan (AS-2 fraction) of 311 x Rf = 311 x Calculated as (2 / 3.1) = 201.

AS-2 fraction analysis ( ¹³ C-NMR analysis)
In order to investigate whether the 1,3-glycoside bond of the AS-2 fraction is α-bond or β-bond, ¹³ C-NMR analysis of AS-2 fraction was performed. 8 mg of AS-2 fraction was dissolved in 0.6 ml of DMSO-d6 (Wako), suspended well, and then centrifuged at 3,000 xg for 5 minutes. The supernatant was analyzed by ¹³ C-NMR under the conditions shown in Table 7 below. The results are shown in FIG.

  Only 6 signals from the 6 carbons of glucose were detected from the AS-2 fraction, with chemical shifts of 100.36, 83.49, 72.57, 71.47, 70.02, 60.80 ppm (A). This is the value of 99.49, 82.96, 71.85, 70.72, 69.43, 60.30 ppm of the chemical shift of α-1,3 glucan fractionated and purified from the cell wall of S. pombe [Sugawara T “Carbohydrate research 339: 2255-2265 2004)] (B). For reference, only 6 signals were detected from TFA-treated curdlan (β-1,3-glucan), and chemical shifts were 103.02, 86.31, 76.32, 72.83, 68.39, 60.87 ppm. This was in good agreement with the chemical shifts 102.82, 86.110, 76.24, 72.67, 68.30, and 60.80 ppm of β-1,3 glucan obtained from the cell wall of S. pombe. From the above results, it was shown that the AS-2 fraction mainly consists of α-1,3-glucan.

AS-2 fraction analysis (Endo-β-1,3-glucanase treatment)
In order to examine the abundance of β-1,3-glucan in the AS-2 fraction, endo-β-1,3-glucanase treatment was performed. Endo-β-1,3-glucanase prepared by Kanazawa Institute of Technology, Dr. Sano was used as described in JP-A-2005-43146.

The powdered AS-2 fraction ground in a mortar was suspended in 50 mM sodium acetate buffer (pH 5.0) to a final concentration of 3 mg / ml. Thereto was added 50 μl (1 mg / ml) of purified endo-β-1,3-glucanase, and after stirring, the mixture was reacted at 37 ° C. for a fixed time. After the reaction, centrifuge at 16,500 xg for 1 minute, and determine the amount of free reducing sugar in 100 μl of the supernatant using the Nelson-Somogyi method [Nelson N, The Journal of Biological Chemistry]. (The Journal of biological chemistry 153: 375-380 1944, Somogyi M) The Journal of biological chemistry Volume 195: 19-22 1952)] An equal amount of TFA-degrading curdlan (β-1,3-glucan) was used as a substrate as a control.

The Nelson-Somogyi method was performed as shown in Table 8 below. 100 μl of alkaline copper reagent was added to 100 μl of sample solution and heated in boiling water for 10 minutes. After rapid cooling, 100 μl of molybdenum arsenic reagent was added and left for 30 minutes or longer, and the absorbance at 500 nm was measured. A standard curve was prepared using glucose.

The results of endo-β-1,3-glucanase treatment of the AS-2 fraction are shown in Table 9. Reducing sugars were released from curdlan, a straight-chain β-1,3-glucan, by decomposition, whereas no reducing sugars were released from the A-2 fraction. From the above, it was clarified that the AS-2 fraction is substantially composed only of α-1,3-glucan, with substantially no β-1,3-glucan. From the above, it was found that α-1,3-glucan free of β-1,3-glucan was obtained with high purity from the filamentous fungal cell wall. From the average chain length of 201, it was shown that the average molecular weight of this α-1,3-glucan was 32,580.

Analysis of AI fraction (methylation analysis)
The AI fraction was subjected to methylation analysis in the same manner as the AS-2 fraction. The results are shown in Table 6. Since the AI fraction is mainly composed of 2,4,6-trimethyl peaks showing 1,3 glycosidic bonds, it was revealed that the glucan in the AI fraction is 1,3 glucan. In addition, a 2,4-dimethyl-like peak indicating 1,3 / 1,6 branched glucose was confirmed, and from the ratio of this branched glucose to linear glucose, 1,3-glucan present in the AI fraction averaged 17 glucose. It was shown that there was a degree of branching at the rate of one per residue. That is, 1,3-glucan contained in the AI fraction was shown to have 6% 1,3 / 1,6 branching degree.

Analysis of AI fraction (Endo-β-1,3-glucanase treatment)
In order to investigate whether the glucan present in the AI fraction is α-linked or β-linked, the 1,3-glycoside bond was examined by endo-β-1,3-glucanase treatment.
In order to remove the chitin contained in the AI fraction so that the enzyme can act easily, chitinase treatment was performed. Chitinase is a plasmid pHT012 expressing the Bacillus circulans chitinase A1 described in Niigata, Watanabe T. et al., Journal of Bacteriology (J. Bacteriol.) 172: 4017-4022 1990. A product obtained by roughly purifying from E. coli JM109 strain obtained from the University of Agriculture, Professor Takeshi Watanabe and transformed from E. coli JM109 strain was used.

The chitinase treatment of the AI fraction was performed as follows. The AI fraction was placed in a mortar and pulverized with a pestle while pouring liquid nitrogen until powdered. After freeze-drying this pulverized AI fraction, 300 mg was weighed into a 50 ml Falcon tube, and 37 ° C in 0.1 M Sodium Phosphate buffer (pH 6.0) containing 800 μg chitinase A1 and 0.05% sodium azide. Chitinase treatment was performed for 2 days. After the treatment, it was centrifuged at 10,000 xg and 4 ° C for 20 minutes, and the same chitinase treatment was further performed twice on the precipitate. The precipitate after chitinase treatment was washed five times by suspending in 40 ml MQ and centrifuging to remove salt and chitinase, and the precipitate was lyophilized.

The chi-nase-treated AI fraction was treated with endo-β-1,3-glucanase in the same manner as the AS-2 fraction. The results are shown in Table 9. From the fact that free reducing sugar was confirmed in the supernatant of the reaction solution, it was shown that 1,3-glucan in the AI fraction was mainly β-1,3-glucan.

Analysis of AI fraction (measurement of average degree of polymerization)
The average degree of polymerization of β-1,3-glucan was measured by quantifying the reducing end group of the polysaccharide and determining the ratio with the amount of glucose contained in the sample. The quantification of the reducing end group was determined by reducing the reducing end glucose to sorbitol with sodium borohydride (NaBH ₄ ) and measuring the amount of sorbitol by an enzymatic method. [Carbohydrate research, such as Manners DJ (Carbohydrate research 17: 109-114, 1971)]

Chitinase-treated AI fraction (40 mg) was suspended in 5 ml of 0.05 M NaOH, 80 mg of NaBH ₄ was added, sonicated for 10 minutes, and left at room temperature overnight. In order to decompose excess NaBH ₄ , 0.2 ml of concentrated hydrochloric acid was added to adjust to pH 4 or less, and the solution was concentrated to dryness. After adding 3 ml of methanol and concentrating to dryness 5 times, it was dialyzed overnight against distilled water. After dialysis, lyophilization was performed to prepare a glucan in which the reducing terminal glucose was reduced to sorbitol. The reduction with NaBH ₄ was performed twice, and it was confirmed by the Nelson-Somogyi method that reduction of NaBH ₄ did not decrease even if the reduction with NaBH ₄ was repeated further. This reduced glucan is completely hydrolyzed by the method described above, the amount of glucose is quantified by HPLC, and the sorbitol derived from the reducing end is quantified by F-kit (manufactured by Roche). = The average degree of polymerization was determined as glucose / sorbitol (μmol).

The average degree of polymerization (DPn) of the fraction obtained by measuring chitinase-treated AI fraction by measuring the amount of sorbitol produced by NaBH ₄ reduction was DPn = 1000 / 0.64 +1 = 1564. Therefore, β-1,3-glucan contained in the AI fraction had a branching degree of 6% and an average molecular weight of 253,000.

(2) Production of α-1,3-glucan and β-1,3-glucan from Aspergillus-derived products As an example of Aspergillus-derived products, α-1,3-glucan and β-1 from rice bran used for making miso Therefore, the acquisition of 3-glucan was shown.
100 g of rice bran was weighed and freeze-dried. After freeze-drying, the rice bran was crushed to a powder while pouring liquid nitrogen, and then freeze-dried again. The lyophilized sample was suspended in 500 ml of 0.1 M phosphate buffer (pH 7.0), and extracted with hot water at 120 ° C. for 1 hour in an autoclave. The extract was centrifuged at 10,000 xg for 20 minutes, and fractionated into a hot water soluble part and a hot water insoluble part. The same operation was performed again on the hot water insoluble part. The hot water insoluble part was suspended in 500 ml of 0.5 N cold NaOH water, stirred at 4 ° C. for 12 hours and extracted with dilute alkali. After extraction, it was fractionated into a dilute alkali-soluble part and a dilute alkali-insoluble part by centrifugation at 10,000 xg for 20 minutes. The dilute alkali-insoluble part was further suspended in 500 ml of 2N cold NaOH water, and stirred at 4 ° C. for 12 hours for strong alkali extraction. After extraction, it was fractionated into a strong alkali-soluble part and a strong alkali-insoluble part by centrifugation at 10,000 xg for 20 minutes.

The strong alkali-soluble part was neutralized with acetic acid while cooling in ice, dialyzed against Milli-Q water, and centrifuged at 10,000 xg for 10 minutes to fractionate into supernatant and precipitate. The precipitate fraction was freeze-dried to obtain α-1,3-glucan.

In order to confirm that no β-1,3-glucan was present in the α-1,3-glucan obtained, ¹³ C-NMR analysis of this fraction was conducted in the same manner as in Example 1. .

The chemical shifts obtained were 99.75, 82.88, 78.69, 73.08, 71.95, 70.86, 69.44, 60.19 ppm, and 99. 75, 82. 88, 71.95, 70.86, 69.44, 60.19 ppm of these chemical shifts were carried out. The AS-2 fraction obtained in Example 1 almost coincided with the chemical shift (described in paragraph “0051”). In addition, since the characteristic chemical shifts around 103 and 86 ppm at the C1 and C3 positions of β-1,3-glycosidic bond were not detected, α-1,3-glucan prepared from rice bran was measured by NMR. It was shown that β-1,3-glucan was not mixed at the analysis level. 78.69 ppm and 73.08 ppm were suggested to be derived from starch contained in rice bran.
From this result, as described in paragraph “0017”, in order to remove more polysaccharides contained in the culture medium, it can be efficiently performed by performing a hot water extraction treatment a plurality of times or in combination with an enzyme such as amylase. Therefore, it was attempted to remove the starch mixed in the α-1,3-glucan fraction by hot water extraction.

50 mg of α-1,3-glucan fraction was weighed and well suspended in 50 ml of Milli-Q water, followed by hot water extraction at 120 ° C. for 1 hour in an autoclave. The extract was filtered and fractionated into a hot water soluble part and a hot water insoluble part. The obtained hot water insoluble fraction was thoroughly washed with Milli-Q water and freeze-dried.

  When an iodine starch reaction was carried out on the obtained hot water soluble part, a color reaction was shown, which revealed that starch was mixed in the α-1,3-glucan fraction. In addition, in order to check the contamination rate, add 300 μl of iodine-potassium iodide solution (0.15% iodine, 1.5% potassium iodide solution) to 140 μl of hot water soluble part, stir well, and absorb the absorbance at 700 nm. Was measured. Moreover, the standard curve was created using commercially available starch, and the starch amount in a sample was computed based on this. As a result, it was revealed that 15.1 μg of starch was present per 140 μl of hot water soluble part. That is, the starch contamination rate with respect to the α-1,3-glucan fraction was 10.8%.

The hot water insoluble fraction of the freeze-dried α-1,3-glucan fraction was prepared again in the same manner as in Example 1 and analyzed by ¹³ C-NMR under the same conditions.

  As a result, only 6 signals were detected, and chemical shifts were 99.80, 82.96, 71.98, 70.90, 69.46 and 60.23 ppm, respectively. Chemical shifts around 78 and 73 ppm, which were thought to be derived from starch contained in rice bran, were not observed. From the above, it was shown that the starch mixed in the α-1,3-glucan fraction was removed by hot water extraction. That is, it was shown that it is possible to obtain α-1,3-glucan free from starch and β-glucan by reheated water extraction of the AS-2 fraction.

The strongly alkali-insoluble part was again suspended in 500 ml of 2N cold NaOH water, and stirred at 4 ° C. for 48 hours for strong alkali extraction. After extraction, it was fractionated into a strong alkali-soluble part and a strong alkali-insoluble part by centrifugation at 10,000 xg for 20 minutes. The alkali-insoluble part was suspended in 500 ml of cold Mill-Q, neutralized with acetic acid while cooling in ice, dialyzed against Milli-Q water, freeze-dried, and β-1,3-glucan Acquired.
Table 10 shows the weight ratio of the α-1,3-glucan fraction and the β-1,3-glucan fraction.

The outline | summary including each process of the manufacturing method of this invention is shown. The results of AS-2 fraction analysis ( ¹³ C-NMR analysis) are shown.

Claims (10)

A method for producing an α-1,3-glucan having only an α-1,3 bond and having an average molecular weight of 30,000 to 99,000 including the following steps:
(A) a hot water extraction treatment of the filamentous fungus culture,
(B) a step of subjecting the residue obtained in (a) to a weak alkali extraction treatment,
(C) a step of subjecting the weak alkali-insoluble part (residue) obtained in (b) to a strong alkali extraction treatment, and (d1) neutralizing the strong alkali-soluble part (extract) obtained in (c), Dialyzing and concentrating to prepare a precipitate consisting of α-1,3-glucan. Comprising the following steps, the production method of the alpha-1,3-glucan of claim 1, wherein:
(A) a step of subjecting the filamentous fungus culture to hot water extraction treatment at 50 to 150 ° C. for 0.5 to 2 hours;
(B) a step of subjecting the residue obtained in (a) to a weak alkali extraction treatment at 4 to 15 ° C. and 12 to 36 hours,
(C) A step of subjecting the weak alkali-insoluble part (residue) obtained in (b) to a strong alkali extraction treatment at 4 to 15 ° C. for 12 to 36 hours, and a strong alkali-soluble matter obtained in (d1) (c) A step of preparing a precipitate consisting of α-1,3-glucan by neutralizing a part (extract), dialyzing, and centrifuging. The production method according to claim 1 or 2 , wherein α-1,3-glucan has an average molecular weight of 30,000 to 35,000. The production method according to claim 3 , wherein α-1,3-glucan has an average molecular weight of 32,580. Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, Aspergillus kawachi, Aspergillus shirousami awamori), and natural mutants of these filamentous fungi, artificial mutants, and genetically engineered mutants. The production method according to any one of claims 1 to 4 . Β-, mainly composed of β-1,3-glucan having an average molecular weight of 5,000 to 990,000 and a β-1,3 / 1,6 degree of branching of 0 to 20%, including the following steps: Glucan production method:
(A) a step of subjecting the filamentous fungus culture to a hot water extraction treatment;
(B) a step of subjecting the residue obtained in (a) to a weak alkali extraction treatment,
(C) a step of subjecting the weak alkali-insoluble part (residue) obtained in (b) to a strong alkali extraction treatment,
(D2) A step of subjecting the strong alkali insoluble part (residue) obtained in (c) to a strong alkali extraction treatment again, and (e) suspending the strong alkali insoluble part (residue) obtained in (d2) in water. Turbidity, neutralization and dialysis to prepare a residue consisting of β-1,3-glucan. The method for producing β-glucan according to claim 6 , comprising the following steps:
(A) a step of subjecting the filamentous fungus culture to a hot water extraction treatment at 50 to 150 ° C. for 0.5 to 2 hours;
(B) a step of subjecting the residue obtained in (a) to a weak alkali extraction treatment at 4 to 15 ° C. and 12 to 36 hours,
(C) a step of subjecting the weak alkali-insoluble part (residue) obtained in (b) to a strong alkali extraction treatment at 4 to 15 ° C. for 12 to 36 hours,
(D2) A step of subjecting the strong alkali-insoluble part (residue) obtained in (c) to a strong alkali extraction treatment again at 4 to 15 ° C. for 24 to 72 hours, and (e) the strength obtained in (d2) A step of suspending an alkali-insoluble part (residue) in water, neutralizing, and dialysis to prepare a residue composed of β-1,3-glucan. The method for producing β-glucan according to claim 6 or 7 , wherein the β-glucan has an average molecular weight: 230,000 to 280,000 and β-1,3 / 1,6 branching degree: 1 to 10%. The method for producing β-glucan according to claim 8 , wherein the β-glucan has an average molecular weight: 253,000 and a β-1,3 / 1,6 branching degree: 6%. Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, Aspergillus kawachi, Aspergillus shirousami awamori), and natural mutants of these filamentous fungi, artificial mutants, and genetically engineered mutants of β-glucan according to any one of claims 6 to 9 Production method.

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