JPWO2017221905A1 - Melanin pigment non-producing or low-producing β-glucan-producing bacterium, its artificial production method, and β-glucan produced using the same, and its production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce β-glucan that is non-producing or low-producing melanin pigment that is mutated from a general β-glucan-producing bacterium and has the ability to produce β-glucan with zero or lower melanin-producing ability than the initial state before the mutation treatment Provide fungus.
[MEANS FOR SOLVING PROBLEMS] A gene mutation treatment step for carrying out a mutation treatment by a physical treatment or a chemical treatment for causing a gene mutation in Aureobasidium pullulans (black yeast), and a mutant obtained by the gene mutation treatment step In addition, in the culture applied with the tyrosinase pathway inhibitor and the culture applied with the polyketide pathway inhibitor, a mutant strain selection step of screening a strain in which no melanin production is observed is included. By this double screening, a melanin non-producing or low-producing β-glucan producing strain with the ability to suppress melanin pigment production and to produce β-glucan is produced. For example, a gamma ray irradiation process is used as the mutation process.
[Selection] Figure 7

Description

  The present invention is a β-glucan-producing incomplete bacterium, a melanin-nonproducing or low-producing β-glucan-producing bacterium obtained by an artificial technique from a microorganism belonging to the genus Aureobasidium that produces melanin pigment in the fermentation process, and its artificial Β-1,3 in which the degree of branching of β-1,6 bond is 50-100% with respect to β-1,3 bond using a glucan-producing microorganism that is a melanin non-producing or low-producing mutant strain thereof The present invention relates to a 1,6-glucan and a production method thereof.

In general, β-glucan is a generic name for a glucan having a glucose polymer main chain having a β-1,3-linkage, and in some proportion, β-1,6-branched glucose group or β-1,6-branched Those having a β-1,3-chain glucose polymer (β-1,3-1,6-glucan) and those having a β-1,4-bond partially (β-1,3-1,4- Glucan) is also known. These β-glucans are widely distributed from bacteria to basidiomycetes such as mushrooms, fungi such as yeast and mold, algae and protozoa, and higher organisms such as plants such as barley and oats (non-patent literature). 1).
Among β-glucans, β-1,3-1,6-glucan and the like are components (β-1,6-branch ratios) that are abundant in mushrooms (basidiomycetes) that inhabit the natural world. It is known that it is contained not only in their fruit bodies but also in cultured mycelium.

β-glucan is a useful substance and is known to have antitumor activity (Non-patent Document 1).
For example, each β-1,3-1,6-glucan represented by schizophyllan and lentinan extracted from Suehirotake and Shiitake has been approved as an anticancer agent, and is still used in combination with chemotherapy. It is reported as a compound having high physiological activity such as immunostimulatory effect and antitumor (Non-patent Document 2).

As one of the methods for producing β-glucan, a method using an Aureobasidium genus microorganism which is an incomplete bacterium is known. As a microorganism belonging to the genus Aureobasidium, for example, a technique using Aureobasidium pullulans (black yeast) is known. Aureobasidium pullulans is known to produce soluble β-1,3-1,6-glucan in the culture solution (the ratio of β-1,6-branch is 50% or more) (Non-patent Document 1). . That is, in the process of culturing Aureobasidium pullulans, β-1,3-1,6-glucan can be produced not as an insoluble cell wall component but as a soluble component in the culture solution. is there.
The obtained β-1,3-1,6-glucan has already been used as a food or cosmetic material, and research on the inclusion base of poorly water-soluble compounds due to its unique triple helical structure Has been done.

JP 2004-329077 A Japanese Patent No. 4573611 Japanese Patent No. 3722522 JP-A-7-51082 Basics and applications of β-glucan CMC Publishing Mushroom Chemistry and Biochemistry Academic Publishing Center Polymer processing, 36, 61 (1987) Bioresource Technology, 99, 7480 (2008) Food Function, 2,45 (2006) Development of functional carbohydrate materials and application to foods Acta Biochimica Polonica, 53, 429 (2006) J Coating Tech, 53, 23 (1981) BMC Genomics, 15, 549 (2014) J Biosci Bioeng, 97, 400 (2004) Journal of Biotechnology, 90, 115 (2012) Medical Mycology, 53, 10-18 (2014) H. Ishida et al., Appl. Microbiol. Biotechonol., 57, 131 Hideya Murakami, Tori Kawai: Brewery, 53, 88 (1958)) Masamichi Hara, Seinosuke Hatama: Brewery, 70, 169 (1975) Hiroshi Iizuka, Akira Yamaguchi, Journal of Japanese Society for Agricultural Chemistry, 26 (1952) No. 2, 71-74 Miyawaki et al., Japanese Agricultural Chemistry Society Annual Report 2007 Int. Immunopharmcol., 7,963 (2007). Agri.Biol.Chem., 47,1167 (1983) BioTechiniques, 21, 1030 (1996) Science and industry 64, 131 (1990)

However, the β glucan production method using Aureobasidium microorganisms which are β-1,3-1,6-glucan producing bacteria in the prior art has a point to be improved. This is a problem that melanin pigment is generated during the culture process, and black pigment is mixed into β-glucan.
Β-glucan using black yeasts produces black melanin pigments along with the production of β-glucan from the middle to the late stage of culture. Therefore, a high-purity β-1,3-1,6-glucan containing no pigment cannot be obtained.

In the prior art, several approaches are known as a method for suppressing melanin production in a culture solution in a β-glucan culture production process using Aureobasidium pullulans.
First, a method of adding a high concentration of vitamin C, oxalic acid, or the like to the culture solution is known (Non-patent Document 3).
Secondly, a method for controlling a nitrogen source in a culture solution is known (Non-Patent Document 4).
Thirdly, a special culture control method is known in which the induction of thick spore spore cells is synchronized and controlled by controlling the nitrogen source during culture and controlling the excess aeration amount (Patent Document 1).
However, in any of the above conventional methods, the cost is increased, a special production apparatus is required, and high-purity β-glucan cannot be mass-produced.

On the other hand, the conventional technique has a problem even in the recovery and purification step after the culture.
As a conventional recovery and production technique, the viscosity of the culture solution becomes a high viscosity of several thousand cP or more with the production of β-1,3-1,6-glucan. A recovery and purification method is known (such as adding sodium hydroxide or the like to the culture solution) to physically or chemically lower the viscosity (Patent Document 2, Non-Patent Document 5).
However, these cause a decrease in the molecular weight of β-glucan and damage the original physical properties. In addition, when high-concentration vitamin C is added during culture, vitamin C is denatured when subjected to high-temperature treatment or oxidation, and further pH is alkalinized, and the color of the whole culture solution changes from yellow to brown. In order to remove the components, complicated and excessive purification steps such as dialysis, activated carbon and the like are required. In addition, it is difficult to completely remove the pigment, and there is a problem that the obtained powder turns brown (Non-Patent Document 6).

  Here, if it is a special β-glucan-producing bacterium that has not been known so far, there is no melanin production or only a trace amount in the culture solution in the β-glucan culture production process. If β-glucan-producing bacteria having the characteristic of “low production” can be artificially obtained, it is possible to culture and produce high-purity β-glucan free from melanin pigment turbidity.

  The present invention relates to a melanin non-producing or low-producing β-glucan-producing bacterium, and provides a β-glucan-producing bacterium that is mutated from a general β-glucan-producing bacterium and has the characteristic of “melanin pigment non-producing or low production” The purpose is to do. The present invention also provides a method for artificially producing the “non-melanin-producing or low-producing β-glucan-producing bacterium”, a β-glucan produced using the method, and a method for producing the same.

In the study of “melanin pigment non-producing or low-producing β-glucan-producing bacteria”, the present inventor first studied the mechanism of melanin production in β-glucan-producing bacteria such as black yeast.
In general, two pathways are known as the main melanin synthesis pathways of microorganisms in vivo. One is a tyrosinase pathway with DOPA (3,4-dihydroxyphenylalanine) as an intermediate in which tyrosine or phenylalanine is oxidized, and the other is DHN (1,8-dihydroxynaphthalene) in which malonyl-CoA is converted by polyketide synthase. ) Via a polyketide route (Non-patent Document 7).
However, in the prior art, these have not yet been fully researched and cannot be said to have been fully elucidated.

Here, as a finding that the melanin synthesis pathway of microorganisms belonging to the genus Aureobasidium is reported, there is a report that microorganisms belonging to the genus Aureobasidium synthesize melanin through the polyketide pathway (Non-patent Document 8).
There is also a description of the melanin synthesis pathway in the literature (Non-patent Document 9) that has determined the genome sequence of microorganisms belonging to the genus Aureobasidium including four different Aureobasidium pullulans. According to the same literature, there are four strains in the genus Aureobasidium. Yes (Aureobasidium pullulans, Aureobasidium melanogenum, Aureobasidium subglaciale, Aureobasidium namibiae), it is described that genes of various enzyme proteins constituting the polyketide pathway could be identified in any of the four strain genomes.
Thus, it has been suggested that a melanin synthesis pathway of a polyketide pathway exists in the living body of β-glucan producing bacteria such as black yeast.

On the other hand, there is also knowledge that predicts the existence of the melanin synthesis pathway of the tyrosinase pathway.
For example, there is a report that the blackening phenomenon of sake lees due to melanin production during sake brewing is caused by the action of tyrosinase melB (essential enzyme of tyrosinase pathway) of Aspergillus oryzae used for production (non-patent literature) 10, Non-Patent Document 11). Furthermore, it is also known that the koji mold at the time of sake production has the presence or absence of melanin production in solid culture (stationary culture) and liquid culture (Non-patent Document 13).
Moreover, although it is another kind of fungi, five strains of the genus Aspergillus have both tyrosinase pathway and polyketide pathway as melanin synthesis pathways, and they are produced by any pathway when melanin pigment is produced. It has been shown that this condition varies depending on the species (Non-patent Document 12).

  Therefore, the inventor conducted a verification experiment based on the inference that not only the known polyketide pathway but also the tyrosinase pathway is involved as a melanin production pathway in microorganisms belonging to the genus Aureobasidium. That is, in order to examine the presence of the tyrosinase enzyme gene, the tyrosinase gene was identified and isolated and its base sequence was determined.

[Identification of tyrosinase gene of Aureobasidium microorganism]
First, the presence or absence of the tyrosinase enzyme gene was confirmed using the genome information of the Aureobasidium pullulans strain whose genome sequence was already decoded.
Based on genome sequence information registered in GenBank (public database of National Center for Biotechnology Information (NCBI) in the United States), homology search was performed using NCBI's Blast program server.
Using the amino acid sequence of the tyrosinase melB protein of Aspergillus oryzae as a query sequence for the presence or absence of tyrosinase homologous protein in the genome sequence of Aureobasidium pullulans and related strains, the genome of Aureobasidium pullulans EXF-150 was analyzed. A protein with a tyrosinase domain (accession number, KEQ79607) was found in the sequence.

Next, a protein blast search was performed on the genome sequence of Aureobasidium pullulans and its related strains again using the protein sequence of the Aureobasidium pullulans EXF-150 strain as a query sequence.Aureobasidium pullulans and Aureobasidium subglaciale, Aureobasidium namibiae The presence of homologous proteins was also confirmed in each strain (accession numbers in FIG. 20 are KEQ58869, XP_013348277, XP_013426151).
For these 4 strains for which genome sequence information is available, no other proteins showing significant homology were found, and it is considered that each strain has a single tyrosinase gene. From the genome sequence information linked to the information of individual homologous proteins found by these searches, the respective base sequences including the protein translation part (ORF) and the parts before and after it were obtained.

An alignment comparison analysis of the base sequences of the four tyrosinase genes found by homologous protein search was performed, and a plurality of PCR primer sequences were set centering on the highly conserved portions of the sequences. The base sequences of the PCR reaction products using the four primers a, b, c and d (FIG. 21) were finally determined.
As a result, the inventors found that a homologous protein of the tyrosinase gene (mel B) of Aspergillus exists in microorganisms belonging to the genus Aureobasidium.

Based on the knowledge obtained by this inventor, in producing β-glucan using microorganisms belonging to the genus Aureobasidium, in order to obtain high-purity β-glucan not contaminated with melanin pigment, the polyketide pathway and tyrosinase pathway, which are melanin production pathways, can be obtained. It has been determined that it is necessary to inhibit one of the pathways, or to make these two melanin production pathways fail in vivo, or to obtain a mutant strain that is impaired and does not exist.
In other words, when the production of melanin pigments of microorganisms belonging to the genus Aureobasidium occurs in synchronism with the fermentation production of β-1,3-1,6-glucan, melanin pigments are also mixed in, and the purity of the purified product is reduced. Coloring becomes a problem.
Therefore, the present inventor conducted research on a mutant strain in which two melanin production pathways are inactivated in order to obtain a high-purity β-glucan not contaminated with a melanin pigment.

In general, for microbial mutation, chemical mutagen treatment such as EMS (methyl ethanesulfonate) and NTG (nitrosoguanidine) and mutation methods by ultraviolet treatment are well known.
There have been few reports on related research on non-producing or low-producing strains of melanin pigments of microorganisms belonging to the genus Aureobasidium in the prior art.
First, there are reports of experiments that attempted to produce melanin non-producing or low-producing strains by ultraviolet treatment (Non-Patent Document 14, Non-Patent Document 15, Non-Patent Document 16). This is intended to solve the browning problem of sake lees derived from rice bran.
Secondly, there is a report of an experiment that attempted to produce a melanin non-producing or low-producing strain by chemical mutagen treatment (Non-patent Document 17, Patent Document 3). This is intended to solve the browning problem in food additives centering on the culture solution of black yeast.

However, the production of a melanin non-producing or low-producing strain by the first ultraviolet treatment and the production of a melanin non-producing or low-producing strain by the second chemical mutagen treatment both obtain a stable melanin non-producing or low producing strain. It has not reached. The reality is that the reproducibility is difficult, and it is necessary to repeat screening by trial and error to determine whether or not to produce melanin from a large number of mutant strains. Or a low production strain cannot be stably bred and mass-produced, and it must be said that it cannot be used industrially.
The above-mentioned conventional experimental report does not go beyond the scope of experiments conducted by trial and error, and reports on melanin-non-producing or low-producing strains that can be stably bred and mass-produced by inactivating or destroying two melanin production pathways. Does not exist.

Therefore, the inventor has earnestly continued research on effective mutation methods for microorganisms belonging to the genus Aureobasidium, and has found a method for effectively obtaining a melanin non-producing or low-producing strain.
In other words, after performing mutation treatment that damages both the tyrosinase pathway or the polyketide pathway or any of the melanin production pathways, a selection method for screening non-melanin producing or low producing strains in the presence of inhibitors of each pathway We found an effective method for generating combined mutants.
Specifically, the Aureobasidium genus microorganism is subjected to a mutation treatment of any one of irradiation treatment, chemical mutagen treatment, ultraviolet treatment, or a combination thereof, as a mutation treatment, and then each of the tyrosinase pathway and the polyketide pathway. Melanin non-producing or low-producing strains were obtained by a combination of methods for selecting melanin non-producing or low-producing strains based on inhibitors of the above pathway. Furthermore, by using the non-melanin-producing or low-producing strain, high-purity β-1,3-1,6-glucan that was simple and inexpensive in terms of fermentation control and medium composition was successfully produced.

In the case of the radiation mutation method as the mutation treatment, the type and size of the irradiation beam can be grasped scientifically, qualitatively and quantitatively, and the reproducibility is high. As an example, a mutation treatment by gamma ray irradiation was used, and the melanin non-producing or low-producing strain was successfully obtained by using the death rate as an index.
As analyzed by the present inventor, since melanin of microorganisms belonging to the genus Aureobasidium is involved not only in the polyketide pathway but also in the tyrosinase pathway, a technique for inactivating these two melanin production pathways was established, and two melanins were established. We succeeded in producing a melanin non-producing or low producing β-glucan producing strain in which the production pathway was inactivated.

The method for producing a melanin non-producing or low-producing β-glucan producing strain of the present invention,
A genetic mutation treatment step for carrying out a mutation treatment by a physical treatment or a chemical treatment for causing a gene mutation in an Aureobasidium genus microorganism, and a tyrosinase pathway inhibitor for a mutant strain obtained by the gene mutation treatment step And a mutant selection step of screening a strain in which melanin production is not observed by performing the applied culture and the polyketide pathway inhibitor, and the production ability of melanin pigment is zero or lower than the initial state before the mutation treatment and β A mutant strain having an ability to produce glucan is obtained.

Here, examples of the mutation treatment in the gene mutation treatment step include radiation irradiation treatment. The radiation treatment includes electromagnetic radiation (gamma rays) and particle rays (α rays, β rays, ion beams). As an example, a gamma ray irradiation treatment with cobalt 60 may be used. Melanin non-producing or low-producing mutants can be selected based on the death rate.
In addition, the mutation treatment in the gene mutation treatment step may include a mutation treatment by ultraviolet irradiation, a mutation treatment by administration of a chemical mutagen, or a mixture thereof.

The melanin non-producing or low producing β-glucan producing strain of the present invention is bred and produced as a mutant strain by the above production method. Screening for strains that do not produce melanin in cultures with tyrosinase pathway inhibitors and cultures with polyketide pathway inhibitors, so that all pathways that produce melanin are inactivated, so melanin contamination It has the ability to produce high-purity β-glucan.
By using this mutant strain, it is possible to obtain high-purity β-1,3-1,6-glucan in which melanin pigments are not mixed in the fermentation process of black yeast using a normal medium.

  According to the present invention, a melanin-non-producing or low-producing mutant strain of a microorganism belonging to the genus Aureobasidium is stably and efficiently obtained, and a high-purity β-1,3-1, using the non-melanin-producing or low-producing mutant strain is obtained. 6-glucan can be produced easily and inexpensively. That is, according to the method for fermentative production of β-1,3-1,6-glucan using the melanin non-producing or low-producing mutant strain of the present invention, since the melanin pigment is not mixed, the conventional technique is required to be complicated. A culture control method is not required, and a normal medium that does not contain vitamin C or the like can be used as a medium component. Therefore, β-1,3-1,6-glucan can be produced inexpensively and easily. The β-1,3-1,6-glucan and its solution obtained by the production method of the present invention have no browning and the like, are white and highly safe, and can be used as pharmaceuticals, cosmetics, and foods. is there.

It is a figure which shows the process of the manufacturing method of the melanin non-production or the low production beta glucan production strain of this invention, and a high purity beta glucan production method. It is the figure which showed the example of the NMR measurement value of what mixed (beta) -1,3-1,6-glucan and pullulan. It is a figure which shows the example of a composition of the agar medium which is a solid medium. It is a graph which shows the relationship between a radiation dose and the survival rate of a microbe. It is a figure which shows the mode of the strain obtained as a result of double screening. It is the figure which listed the polysaccharide production and viscosity of what was obtained by the screening of a melanin non-production or a low production mutant. It is a figure which shows the mode of melanin production of each strain in presence of the tyrosinase pathway, the polyketide pathway, and the inhibitor of each pathway. It is a figure which shows the example of a composition of the liquid medium for seeds. It is a figure which shows the example of a composition of the culture medium of this culture composition. It is a figure which shows the culture result of K-1 parent strain and mutant 1 (TS-1 strain). It is the figure which observed the color of the culture solution obtained by the culture result of K-1 parent strain and mutant 1 (TS-1 strain). It is a figure which shows the result of having performed the NMR analysis measurement of (beta) -1,3-1,6-glucan derived from the mutant 1 (TS-1 strain). It is a figure which shows the mode of the beta glucan producing bacteria (mutant strain 3 (TS-5 strain), mutant strain 4 (TS-9 strain)) obtained by mutating by irradiation and having no other melanin pigment producing ability. is there It is a figure which shows the mode of the strain (K-1W strain | stump | stock) obtained as a result of culture | cultivation in the chemical mutation agent processing method concerning Example 2. FIG. It is the figure which confirmed the presence or absence of the melanin production of each strain in presence of the inhibitor of a tyrosinase pathway and a polyketide pathway using K-1W strain | stump | stock. It is a figure which shows a mode that the chemical mutation agent process of the process 1 and the selection process of the strain of the process 2 were continued based on the K-1W strain. It is a figure which shows the mode of the melanin non-production or low production strain obtained based on the K-1W strain. It is a figure which shows the mode of the other melanin pigment non-production or low production beta-glucan production microbe obtained by making it mutate with a chemical processing agent. Mutant strain 1 (TS-1 strain), mutant strain 2 (TS-2 strain), mutant strain 3 (TS-5 strain), mutant strain 4 (TS-9 strain) obtained in Example 1 and Examples It is the figure which put together the property regarding the mutant 5 (K-1ww strain) obtained in 2, and the mutant 6 (UV-1 strain) obtained in Example 3. It is a figure which shows the blast search result of the tyrosinase homologous protein in Aureobasidium genus. It is a figure which shows the PCR primer for gene comparison of the tyrosinase gene sequence of Aureobasidium genus 4 strain, and gene amplification.

Hereinafter, embodiments of a β-glucan-producing bacterium that is a melanin non-producing or low-producing strain of the present invention, an artificial production method thereof, and a production method of β-glucan produced using the same will be described. The following embodiments and examples are merely examples, and do not limit the scope of the present invention.
The method for producing a melanin non-producing or low-producing β-glucan producing strain and the method for producing a high-purity β-glucan of the present invention include the steps shown in FIG. Each process will be described.

[Step 1] Gene mutation treatment As step 1, a mutation treatment (gene mutation treatment step) is performed by physical treatment or chemical treatment that causes a gene mutation in a microorganism belonging to the genus Aureobasidium.
[Step 2] Selection of candidates from surviving strains Next, after performing the “gene mutation treatment step” in step 1, strains that do not brown due to melanin even if cultured in the strain that survived in step 1 as step 2 Select.
[Step 3] Double screening Next, in Step 3, a strain in which melanin production is not observed in a culture in which a tyrosinase pathway inhibitor is applied and in a culture in which a polyketide pathway inhibitor is applied to a secondarily selected mutant strain. Screen (double screening).
[Step 4] Production of high-purity β-1,3-1,6-glucan As step 4, melanin non-producing or low-producing β-glucan produced by isolation and breeding of the strain obtained as a result of step 3 [TS1] β-1,3-1,6-glucan is produced with high purity and high production using bacteria. This step 4 can be stably repeated, and it is possible to achieve high purity and high production using a β-glucan producing bacterium having a melanin pigment production ability of zero or lower than the initial state before the mutation treatment and the ability to produce β-glucan. β-1,3-1,6-glucan can be produced.
Hereinafter, it demonstrates in order of each process.

[Step 1] Gene mutation treatment
The example of the gene mutation process process which performs the mutation process by the physical treatment or chemical treatment which makes the gene mutation with respect to Aureobasidium microbe is shown. In addition, although the mutation process by a radiation irradiation process was mentioned as an example here as an example, you may combine mutation methods, such as another chemical mutation agent process and an ultraviolet irradiation process.

[Strain used for mutation treatment]
The Aureobasidium genus microorganism to be subjected to the mutation treatment may be any strain that produces β-1,3-1,6-glucan, but is selected from the following two points.
The first condition of the strain is that which produces as little pullulan as possible.
Since there are strains that simultaneously produce a large amount of polysaccharides such as pullulan (α-glucan) depending on the strain, in order to obtain highly pure β-1,3-1,6-glucan, a strain that does not produce pullulan as much as possible preferable.
Therefore, it is preferable to use a strain that produces little pullulan and produces β-1,3-1,6-glucan.

The product of the strain can be evaluated by NMR measurement.
FIG. 2 shows an example of NMR measurement values of a mixture of β-1,3-1,6-glucan and pullulan.
β-1,3-1,6-glucan shows two signals of about 4.75 ppm and about 4.55 ppm in 1H NMR spectrum of a solution using 1N sodium hydroxide heavy aqueous solution as a solvent. These signals are attributed to β-1,3-linkage and β-1,6-linkage, respectively. In addition, since it is well known that the measured value of NMR changes due to a slight change in temperature condition and is accompanied by an error, “about 4.75 ppm” and “about 4.55 ppm” here are usually predicted. It means a numerical value including a fluctuation range (for example, ± 0.2) of a measured value within a range.
On the other hand, α-glucan such as pullulan has 3 peaks (4.95 ppm, 5.20 ppm, and 5.20 ppm) between about 4.9 ppm and 5.4 ppm in 1 H NMR spectrum of a solution using 1N sodium hydroxide heavy aqueous solution as a solvent. 28 ppm).
That is, by examining the measured value of NMR, the amount of α-glucan such as pullulan in the product obtained by culturing the strain can be evaluated. In addition, the degree of branching of β-1,3-1,6-glucan produced can be estimated from the signal ratio of β-1,6-bond / β-1,3-bond.

The second condition of the strain is that β-1,3-1,6-glucan obtained by culture is secreted into the culture solution outside the cells.
Mushrooms and baker's yeast produce β-1,3-1,6-glucan in the cell wall, but it is troublesome to collect and purify β-1,3-1,6-glucan from the cell wall. Moreover, since the cell wall of a microbial cell is destroyed, a continuous utilization of a strain is impossible. For example, continuous fermentation production of β-1,3-1,6-glucan is impossible.

The microorganism belonging to the genus Aureobasidium has the property of secreting β-1,3-1,6-glucan into the culture solution outside the cells by culturing. Therefore, β-1,3-1,6-glucan can be easily recovered, and the contents are not leaked because the cells are not damaged, and the purity is high. Moreover, it is preferable at the point which is water-soluble.
If it is an Aureobasidium genus microorganism, β-1,3-1,6-glucan having the degree of branching of 50-100% β-1,6-branch described above can be obtained, and the molecular weight is as high as 1 million or more. From a β-glucan having a molecular weight to a low-molecular β-glucan having a molecular weight of about several tens of thousands can be produced according to the culture conditions.
Of these, β-1,3-1,6-glucan produced by Aureobasidium pullulans is preferable.
In the examples described later, examples of breeding and production of melanin-non-producing or low-producing β-glucan-producing bacteria using Aureobasidium pullulans K-1 strain obtained from the Morinomiya Center, Osaka Industrial Technology Research Institute are shown.

[Gene mutation treatment]
Examples of gene mutation treatment include mutation treatment by irradiation treatment, mutation treatment by chemical mutagen treatment, and mutation treatment by ultraviolet irradiation treatment. It should be noted that the present invention can be applied even if these mutation treatments are used in combination.
First, an example of mutation processing by radiation irradiation processing will be described.
The radiation used for the mutation treatment may be anything that causes gene mutation. The radiation may include electromagnetic radiation (gamma rays), particle rays (α rays, β rays, ion beams) and the like. For example, there is one using γ rays. The supply source of γ rays is not particularly limited as long as it stably emits γ rays. An example is cobalt 60.
The irradiation dose (absorbed dose) may be 1 kGy or more and 30 kGy or less. Preferably, it is 5 kGy or more and 20 kGy or less. More preferably, it is 8 kGy or more and 15 kGy or less. For example, an irradiation dose at which the survival rate of the irradiated microorganism is about 10 −6 is used as a guide for the irradiation dose.
The temperature at the time of irradiation may be a temperature at which black yeast can grow or lower, and is 40 ° C. or lower, preferably 30 ° C. or lower, more preferably 25 ° C. or lower. To avoid freezing, keep it at 4 ° C or higher.
What is necessary is just to perform the variation | mutation process with respect to a black yeast by radiation irradiation on the said conditions, and to take out the surviving strain.
Next, an example of the mutation process by the chemical mutation agent process will be described.
The chemical mutagen used for the mutation treatment may be any one that causes gene mutation. For example, there is an example using ethyl methanesulfonic acid (EMS). A culture solution inoculated with Aureobasidium pullulans K-1 strain is prepared, ethyl methanesulfonic acid (EMS) is added, and the survival rate of the treated microorganism is caused to mutate with an index of about 99%. The surviving strain may be taken out by applying a mutation treatment to black yeast by administration of a chemical mutation agent under the above conditions.
Next, an example of the mutation process by the ultraviolet irradiation process will be described.
The ultraviolet light used for the mutation treatment has a strength that causes a gene mutation. The intensity of the ultraviolet rays and the irradiation time may be anything that causes genetic mutation. For example, irradiation is performed for 2 minutes using 15 watts and a wavelength of 260 nm. The ultraviolet irradiation treatment is preferably performed using an aseptic laboratory bench (clean bench). Mutation is caused with the survival rate of irradiated microorganisms as an index of about 99%. The surviving strain may be taken out by subjecting the black yeast to mutation treatment by ultraviolet irradiation under the above conditions.
As described above, mutation treatment for radiation treatment, mutation treatment by chemical mutagen treatment, mutation treatment by ultraviolet irradiation treatment, mutation treatment for black yeast by combinations thereof, etc. Take out.

[Step 2] Selection of candidates from surviving strains The selected mutant strain is cultured, and a strain that does not turn brown by culture, that is, a strain that does not produce melanin, is selected as a candidate strain to be subjected to the next double screening. .
Those that turn brown at this candidate selection stage (solid culture and liquid culture) are excluded because the melanin production pathway is activated and the ability to produce melanin is expressed. In addition, at this time, it cannot be determined that all melanin production pathways have lost activity for those that do not brown in this candidate selection culture treatment. In other words, those that do not brown in this candidate selection culture treatment are in a state where the melanin production pathway is not temporarily active, but only the tyrosinase pathway is inactivated, but the polyketide pathway is active. There are three types: those in which the polyketide pathway is inactivated but the tyrosinase pathway has not lost activity, and those in which both the tyrosinase pathway and the polyketide pathway are inactivated.

As a carbon source used for candidate selection culture, sucrose, glucose, fructose or the like is preferably used as a carbon source. As a method for adding a carbon source, an appropriate amount may be added at the start of culture, or fed continuously or stepwise.
As a nitrogen source used for the candidate culture, an inorganic salt such as nitrate or ammonium salt, or an organic nitrogen source such as peptone, yeast extract or potato extract can be used. Moreover, you may mix each. In addition, phosphates, minerals, and vitamins can be added as appropriate.

As a medium used for candidate selection, any medium that can grow black yeast such as a semi-synthetic medium or a potato dextrose medium may be used. For example, a Chapek medium using sucrose as a carbon source is suitable.
The aerobic conditions are used as the candidate culture conditions. The pH is preferably in the range of pH 3.0 to 6.5, preferably pH 3.5 to 5.0. Although pH control is not particularly required, since the pH tends to increase at the beginning of the culture, it can be cultured in a pH stat using an organic acid such as citric acid or an inorganic acid such as hydrochloric acid or phosphoric acid. The temperature may be up to 32 ° C, preferably 20 ° C to 30 ° C, more preferably 26 ° C to 28 ° C.
The cell culture phase (culture time) used for the mutation treatment is preferably from the logarithmic growth phase to the stable phase.

[Step 3] Double screening Screening with tyrosinase pathway inhibitors Strained non-producing or low-producing melanin strains are applied to a medium containing kojic acid (1000 μg / ml), an inhibitor of tyrosinase pathway. The melanin non-producing or low-producing strain (polyketide pathway inactivated strain) that does not brown is screened. By screening with an inhibitor of the tyrosinase pathway, a strain in which the polyketide pathway is inactivated can be selected, and those in which the activity of the polyketide pathway is not lost can be excluded.

Screening with Polyketide Pathway Inhibitors Applying to a medium containing phthalide (Phthalide, 250 μg / ml), an inhibitor of the polyketide pathway, and cultivating it, a non-brown melanin-producing or low-producing strain (tyrosinase pathway inactivated strain) Screen. Tricyclazole can also be used as an inhibitor of the polyketide pathway.
By screening with an inhibitor of the polyketide pathway, strains in which the tyrosinase pathway is inactivated can be selected, and those in which the activity of the tyrosinase pathway is not lost can be excluded.

The above two screenings may be performed serially, that is, one of the screenings may be performed first, and then the other screening may be performed on the selected strain, or may be performed in parallel, that is, from the same strain. Two experimental strains may be prepared by dividing the stock, and both screenings may be performed in parallel using each.
The strains remaining in the two screenings described above, ie, screening with inhibitors of the tyrosinase pathway and screening with inhibitors of the polyketide pathway, are strains that are inactivated in both the polyketide pathway and the tyrosinase pathway. It is confirmed that there is.
This strain is the melanin non-producing or low-producing β-glucan producing bacterium of the present invention.
At the stage of Step 3, a β-glucan producing bacterium having the ability to produce the melanin pigment of the present invention is zero or lower than the initial state before the mutation treatment and capable of producing β-glucan is obtained.

[Step 4] Production of high-purity β-1,3-1,6-glucan using non-producing or low-producing β-glucan-producing bacteria of the melanin of the present invention Next, the present invention manufactured by the above-described manufacturing method The production of β-glucan using non-producing or low-producing β-glucan-producing bacteria is also described.
Culturing in a β-1,3-1,6-glucan production medium using the non-producing or low-producing β-glucan-producing bacterium of the present invention obtained in the above steps 1 to 3, and producing a highly pure β-1, 3-1,6-glucan is obtained.
Here, a special culture method is not particularly required, and a production method of β-1,3-1,6-glucan by a microorganism belonging to the genus Aureobasidium may be applied.

  Various methods for culturing Aureobasidium microorganisms to produce β-1,3-1,6-glucan have been reported. In this section, it is assumed that the culture volume is several L or more as the culture medium volume and that the agitated aeration culture is performed using the agitated jar fermenter culture medium.

  The carbon source that can be used in the culture medium may be carbohydrates such as sucrose, glucose, fructose, gluconic acid, and xylose. The nitrogen source may be an inorganic nitrogen source such as ammonium sulfate, sodium nitrate or potassium nitrate, or an organic nutrient source such as peptone or yeast extract. These can also be used in combination. In order to increase the production of β-glucan, inorganic salts such as sodium chloride, potassium chloride, phosphate, magnesium salt, calcium salt, trace metal salts such as iron, copper, manganese, vitamins, etc. The addition is also an effective method.

In the culture of microorganisms belonging to the genus Aureobasidium, when a medium in which a high concentration of ascorbic acid is added to a chapec medium containing sucrose as a carbon source is used, a high concentration of β-1,3-1 is controlled while controlling melanin production. , 6-glucan is reported to be produced (Non-patent document 19, Non-patent document 21, Patent document 4). However, since the melanin non-producing or low-producing strain obtained this time has zero or lower melanin production ability than the initial state before the mutation treatment, the medium composition is such that the microorganism grows and β-1,3-1,6 -No particular limitation as long as it produces glucan. If necessary, a small amount of vitamins such as vitamin C and organic nutrient sources such as yeast extract and peptone may be added.
As conditions for aerobic cultivation of microorganisms belonging to the genus Aureobasidium in the above medium, about 10 to 45 ° C, preferably about 20 to 35 ° C, more preferably about 25 to 30 ° C, in addition, about 3 to 7, Preferably, a pH condition of about 3.5 to 5 is used.

  In order to effectively control the culture pH, it is also possible to control the pH of the culture solution with an alkali or an acid. Furthermore, you may add an antifoamer suitably for the defoaming of a culture solution. The culture time is usually about 1 to 10 days, preferably about 1 to 6 days, and thereby β-glucan can be produced. The culture time may be determined while measuring the production amount of β-glucan.

  When Aureobasidium microorganisms are cultured under aeration and agitation for about 4 to 6 days under the above conditions, β-glucan polysaccharide containing β-1,3-1,6-glucan as a main component is 0.1% (w / v) in the culture solution. ) To several percent (w / v). Β-1,3-1,6-glucan can be obtained as a precipitate by adding, for example, an organic solvent to a supernatant obtained by treating this culture with a centrifugation operation or the like. Further, it can be further produced with activated carbon, ion exchange resin or the like.

As Example 1, an example using a radiation irradiation method in the mutation treatment (Step 1) is shown.
The strain used in the experiment was Aureobasidium pullulans K-1 strain obtained from the Morinomiya Center, Osaka Industrial Technology Research Institute.
The K-1 strain of Aureobasidium pullulans has the ability to produce two types of β-1,3-1,6-glucan having a molecular weight of 2 million or more and about 1 million.
Aureobasidium microorganisms are limited to K-1 strains because other types of strain type cultures can be obtained through the National Institute of Technology (NITE), American Type Culture Collection (ATCC), etc. It is not used, but can be used over a wide range of microorganisms belonging to the genus Aureobasidium.

Here, it was confirmed that the Aureobasidium pullulans K-1 strain to be used has a tyrosinase gene.
Genomic DNA was prepared from Aureobasidium pullulans K-1 strain by protein denaturation with benzyl chloride and a method of removing polysaccharides by stepwise alcohol precipitation (Reference 20). Using this genomic DNA as a template, PCR reaction was performed with oligonucleotide primers (a and c pairs and b and d pairs) to amplify tyrosinase candidate genes. As a result of determining the base sequence of the PCR product, it was confirmed that the tyrosinase gene was present in the parent strain Aureobasidium pullulans K-1, and the specific base sequence was revealed.

[Step 1: Gene mutation treatment]
60 ml (pH 5.0) of a potato dextrose medium was placed in a 300 ml-volume baffled Erlenmeyer flask to prepare a medium that was steam sterilized at 121 ° C. This medium was used as a medium for growing Aureobasidium pullulans K-1 strain (black yeast) used in the experiment. The culture was aerobically performed at 27 ° C. with shaking culture.
Microorganisms in which the cells grew and the turbidity OD660 reached 10 or more and reached the logarithmic growth phase were used as test bacteria for the radiation mutation method. The cells were washed with a sterile medium (medium excluding sucrose, pH was adjusted to pH 5.0 with hydrochloric acid) shown in FIG. 3, and after collection by centrifugation (5000 rpm, 10 minutes), the same medium (carbon In a sterile medium not containing a source), the cells were suspended so that the turbidity OD660 of the cells was about 1. This bacterial cell diluted solution was irradiated with radiation cobalt 60 so as to be 10 kGy-18 kGy. The survival rate of the bacteria in this radiation dose range was about 10-6. FIG. 4 is a graph showing the relationship between the radiation dose and the survival rate of bacteria.

[Step 2] Selection of candidates from surviving strains Candidates were selected from surviving strains by culturing them in a solid medium (stationary culture) and a liquid medium. The agar medium shown in FIG. 3 was used as the solid medium (added 2% amount of agar). The pH was adjusted to 5 with hydrochloric acid.
The cells were applied to the solid medium and statically cultured in an incubator at 27 ° C. for about 1 week to 10 days.

FIG. 5 is a diagram showing the state of the bacterial strain obtained as a result of the culture in the double screening of solid culture and liquid culture. Among the obtained strains, only white strains were selected (FIG. 5 (a)).
Subsequently, the white strain was cultured in the same liquid medium (5 ml to 100 ml) under aerobic shaking at 27 ° C. for 5 to 10 days. Among the strains obtained as a result of the culture, strains that exhibited white color and showed viscosity were selected (FIG. 5 (b)).
The white strain obtained as a result of selection using the liquid medium (strain indicated by the arrow in FIG. 5B) is again cultured in 100 ml of the same liquid medium placed in a 500 ml baffled conical flask to produce β-glucan. And the presence or absence of melanin production, the relationship between β-1,3-1,6-glucan concentration and viscosity, and more promising strains were selected.

FIG. 6 is a list of polysaccharide production and viscosities of those obtained by screening melanin non-producing or low-producing mutant strains. The polysaccharide concentration was collected 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 was 66% (v / v) to precipitate the polysaccharide. Then, it melt | dissolved in ion-exchange water and quantified with the phenol sulfuric acid method. The viscosity was measured with a rotational viscometer. The viscosity of the culture solution was measured with a BM type rotational viscometer (manufactured by Toki Sangyo Co., Ltd.). A material having a very high viscosity of several hundred cP (mPa · s) to several thousand cP (mPa · s) at 30 ° C. was selected.
Here, two mutant strains were selected. Here, one of them is called “mutant strain 1 (TS-1 strain)” and the other one is called “mutant strain 2 (TS-2 strain)”.

[Step 3] Double screening Screening by culture in a medium to which an inhibitor of tyrosinase pathway was added and screening by culture in a medium to which an inhibitor of polyketide pathway was added were performed.

  FIG. 7 is a diagram showing the state of melanin production of each strain in the presence of tyrosinase pathway, polyketide pathway, and inhibitors of each pathway. Among the three areas in the petri dish, the strain in the upper left area is the parent strain before the gene mutation treatment (K-1 strain), and the strain in the upper right area is one of the mutant strains selected in step 2. “Mutant strain 1 (TS-1 strain)”, and “Mutant strain 2 (TS-2 strain)” which is one of the mutant strains selected in step 2 in the lower area.

In FIG. 7, the results of culturing under four conditions are shown side by side.
Potato dextrose agar medium is used for each, but four experimental media are prepared depending on whether or not an inhibitor is added.
The upper left is a control, and is a result of culturing on a potato dextrose agar medium to which no inhibitor is added.
The lower left is the result of culturing on a potato dextrose agar medium supplemented with 1000 μg / ml of kojic acid, an inhibitor of the tyrosinase pathway. The presence of kojic acid inhibits the function of the tyrosinase pathway in the strain.
The upper right is the result of culturing on a potato dextrose agar medium supplemented with 250 μg / ml of phthalide, an inhibitor of the polyketide pathway. The presence of phthalide inhibits the function of the polyketide pathway in the strain.
The lower right is the result of culturing on a potato dextrose agar medium supplemented with 200 μg / ml of tricyclazole, an inhibitor of the polyketide pathway. The presence of tricyclazole inhibits the function of the polyketide pathway in the strain.

The presence or absence of melanin production in each bacterium shown in FIG. 7 is evaluated.
First, the K-1 strain which is a parent strain not subjected to mutation treatment is evaluated.
From the culture results of the control, the parent strain K-1 has the ability to synthesize melanin.

From the culture results of addition of the polyketide pathway inhibitor phthalide, melanin production was inhibited by adding the polyketide pathway inhibitor, but some melanin production was observed. Tricyclazole, another inhibitor of the polyketide pathway, produced small amounts of brown melanin. From this experiment, it can be seen that the tyrosinase pathway is not completely inactivated in the parent strain K-1 and has some melanin pigment synthesis ability through the tyrosinase pathway.
From the culture results of addition of kojic acid as a tyrosinase pathway inhibitor, melanin pigments are actively synthesized even when a tyrosinase pathway inhibitor is added. From this experiment, it can be seen that the polyketide pathway is activated in the parent strain K-1 and has the ability to synthesize melanin by the polyketide pathway.

  Next, in “mutant strain 1 (TS-1 strain)”, the culture results of the control, the culture results of addition of the polyketide pathway inhibitor phthalide, the culture results of addition of tricyclazole, another inhibitor of the polyketide pathway, the tyrosinase pathway It can be seen that melanin production is inhibited in any of the results of the addition of kojic acid, which is an inhibitor of the above, and there is no ability to synthesize melanin completely.

On the other hand, in “mutant strain 2 (TS-2 strain)”, the culture result of the control, the culture result of addition of the polyketide pathway inhibitor phthalide, the culture result of addition of tricyclazole, another inhibitor of the polyketide pathway, the tyrosinase pathway It can be confirmed that the production of melanin pigment is suppressed to a small extent in any of the results of the culture with addition of kojic acid, which is an inhibitor, but the melanin production amount is not zero but slightly produced, and the tyrosinase pathway and the polyketide pathway It can be seen that some of the activity remains partially.
As a result of this double screening, “Mutant 1 (TS-1 strain)” can be selected as a mutant having no ability to synthesize melanin, and the production of melanin is partially suppressed. “Mutant 2 (TS-2 strain)” has been obtained as a mutant having remaining ability.

[Step 4]
[Step 4] Production of high-purity β-1,3-1,6-glucan using the melanin non-producing or low-producing β-glucan-producing bacterium of the present invention 100 ml of seed liquid medium having the composition shown in FIG. Put into a 500 ml Erlenmeyer flask with baffle, autoclaved at 121 ° C. for 15 minutes, and then sterilize the mutant strain that was previously obtained as a non-producing or low-producing melanin strain from the slant of the same medium composition. A seed culture solution was prepared by inoculating 1 platinum ear inoculum and culturing at 27 ° C. for 3 to 5 days with aerobic shaking and stirring.

  Next, 2 L (pH 3.7 to 4.0) of the main culture composition shown in FIG. 9 is placed in a 3 L jar fermenter culture apparatus (manufactured by Maruhishi Bioengineering) and pressurized steam at 121 ° C. for 15 minutes. Sterilized and 100 ml of the seed culture solution obtained as described above was inoculated aseptically, followed by aeration and agitation culture at 500 rpm, 27 ° C. and 1 L / min.

  Similarly, the culture was examined under the same conditions even in a medium not containing ascorbic acid in the main culture medium composition. In this case, the initial pH of the medium was controlled within the range of pH 3.7 to 4.0 using sodium hydroxide and hydrochloric acid. As a control, the parent strain K-1 was used, and the main culture medium was cultured under the same conditions in the same medium containing ascorbic acid.

FIG. 10 is a diagram showing the culture results of the K-1 parent strain and mutant strain 1 (TS-1 strain).
As shown in FIG. 10, the mutant strain 1 (TS-1 strain) was cultured in a state where melanin production was suppressed regardless of the presence or absence of ascorbic acid. Moreover, when each polysaccharide density | concentration ((beta) -1,3-1,6-glucan), a viscosity, and growth were measured, even if ascorbic acid was not added, the mutant strain had a favorable result.

FIG. 11 is a diagram observing the color of the culture solution obtained from the culture results of the K-1 parent strain and mutant strain 1 (TS-1 strain).
The left side of FIG. 11 is the color of the culture solution of the parent strain K-1 which is a control. It can be clearly seen that the culture is browned and melanin is mixed.
The center of FIG. 11 is the color of the culture solution of mutant strain 1 (TS-1 strain) when ascorbic acid is added. In the mutant strain 1 (TS-1 strain), no melanin pigment was mixed in the culture solution, but when ascorbic acid was added, the culture solution showed a slight yellowishness due to its influence.
The right side of FIG. 11 is the color of the culture solution of Mutant 2 (TS-2 strain) when no ascorbic acid is added. In the mutant strain 1 (TS-1 strain), no melanin production is observed in the culture solution, and there is no influence of ascorbic acid, so that the culture solution is white and has a beautiful color.
Thus, according to the β-glucan-producing bacterium (mutant strain 1 (TS-1 strain)) having the ability to produce β-glucan with zero melanin-producing ability of the present invention, no melanin pigment is mixed. White melanin-free β-1,3-1,6-glucan was obtained. In addition, according to β-glucan-producing bacteria (mutant strain 2 (TS-2 strain)) having the ability to produce β-glucan, the production ability of melanin pigment being suppressed lower than the initial state before the mutation treatment, A low melanin-containing β-1,3-1,6-glucan with reduced contamination was obtained.

Next, β-1, in a melanin-free culture obtained from a β-glucan producing bacterium (mutant strain 1 (TS-1 strain)) having the ability to produce β-glucan without producing melanin pigment. The purity of 3-1,6-glucan was also examined. FIG. 12 is a diagram showing the results of NMR analysis measurement of β-1,3-1,6-glucan derived from mutant strain 1 (TS-1 strain).
As described in the means for solving the above problems, in the 1H NMR spectrum of a solution using 1N sodium hydroxide heavy aqueous solution as a solvent, β-1,3-1,6-glucan is about 4.75 ppm and about 4 As shown in FIG. 12, two peaks of .55 ppm are shown, and α-glucan such as pullulan shows three peaks (4.95, 5.20, 5.28) between 4.9 and 5.4. In addition, a peak value peculiar to β-1,3-1,6-glucan is seen, but a peak value peculiar to pullulan is not seen. That is, it can be seen that high-purity β-1,3-1,6-glucan containing no pullulan is obtained. In the purity calculation from the weight, the purity was calculated to be 90% or more.
Thus, according to the melanin non-producing or low-producing β-glucan-producing bacterium of the present invention, highly pure β-1,3-1,6-glucan was produced.
Next, examples of other mutant strains that have been successfully created by repeating the above-described mutation treatment by irradiation and selection by double screening will be shown. FIG. 13 is a photograph showing β-glucan-producing bacteria (mutant strain 3 (TS-5 strain), mutant strain 4 (TS-9 strain)) having the ability to produce β-glucan with zero melanin pigment production ability. It is a copy. As shown in FIG. 13, the mutant strain 3 (TS-5 strain) and the mutant strain 4 (TS-9 strain) are also white. The purity of β-1,3-1,6-glucan in the culture solution obtained from these mutant 3 (TS-5 strain) and mutant 4 (TS-9 strain) was also examined. 1,3-1,6-glucan was produced. As described above, when the mutation treatment by irradiation is used, the production ability of the melanin pigment according to the present invention is zero or lower than the initial state before the mutation treatment and the ability to produce β-glucan is low or low. It can be seen that the production method of the production β-glucan production strain has high reproducibility.

Example 2 shows an example in which a chemical mutagen treatment method is used in the mutation treatment (step 1).
As in Example 1, the strain used in the experiment was Aureobasidium pullulans K-1 strain obtained from the Morinomiya Center, Osaka Industrial Technology Research Institute.
Since Aureobasidium microorganisms can be obtained from other types of strain type cultures, they are not limited to the K-1 strain and can be used over a wide range of Aureobasidium microorganisms.

[Step 1: Gene mutation treatment]
60 ml (pH 5.0) of a potato dextrose medium was placed in a 300 ml-volume baffled Erlenmeyer flask to prepare a medium that was steam sterilized at 121 ° C. This medium was used as a medium for growing Aureobasidium pullulans K-1 strain (black yeast) used in the experiment. The culture was performed aerobically at 27 ° C. with shaking culture.
Microorganisms in which the cells grew and the turbidity OD660 reached 10 or more and reached the logarithmic growth phase were used as test bacteria for the radiation mutation method. The bacterial cells were washed with a sterile medium (medium excluding sucrose, pH adjusted to pH 5.0 with hydrochloric acid) shown in FIG. 3 of Example 1, and collected again by centrifugation (5000 rpm, 10 minutes). The cells were suspended in the same medium (sterile medium containing no carbon source) so that the turbidity OD660 of the cells was about 1.
EMS was added to this microbial cell dilution so that it might become 4% (w / v), it was shaken for 30 minutes at 27 degreeC, and it processed using the death rate 99% as a parameter | index.

[Step 2] Selection of candidates from surviving strains Candidates were selected from surviving strains by culturing them in a solid medium (stationary culture) and a liquid medium. The agar medium shown in FIG. 3 of Example 1 was used as the solid medium (4% sucrose). The pH was adjusted to 5 with hydrochloric acid.
The cells were applied to the solid medium and statically cultured in an incubator at 27 ° C. for about 1 week to 10 days.

After 1 week to 10 days, screening was performed using a white colony mutant suitable for the purpose as an index. Among the strains obtained as a result of the culture, a strain that exhibited white color and showed viscosity was selected.
As a result, one white strain (non-melanin producing or low producing strain) was successfully isolated.
FIG. 14 is a diagram showing the state of the strain obtained as a result of the culture.

[Step 3] Double screening Screening by culture in a medium to which an inhibitor of tyrosinase pathway was added and screening by culture in a medium to which an inhibitor of polyketide pathway was added were performed in the same manner as in Example 1.
The presence or absence of melanin production was confirmed on an agar medium having the same composition containing 1000 μg / ml kojic acid (tyrosinase pathway inhibition) and 250 μg / ml phthalide (polyketide pathway inhibition) (FIG. 15). As a result, a mutant K-1W strain that was white in the medium containing kojic acid and gradually colored in the medium containing phthalide was obtained. This suggests that although the polyketide pathway is not functioning, it is a mutant strain in which the tyrosinase pathway remains functional.
Therefore, based on the K-1W strain, the chemical mutagen treatment in Step 1 and the strain selection process in Step 2 were continued (FIG. 16). As a result, a melanin non-producing or low-producing strain was successfully isolated even in a medium containing phthalide (FIG. 17). Here, this mutant strain is referred to as “mutant strain 5 (K-1ww strain)”.

[Step 4] Production of high-purity β-1,3-1,6-glucan using melanin-non-producing or low-producing β-glucan-producing bacteria of the present invention Mutant strain obtained by double screening (mutant strain 5 ( K-1ww strain)) was used to produce high-purity β-1,3-1,6-glucan in the same manner as in Step 4 shown in Example 1. In the main culture, 100 ml of the medium shown in FIG. 9 was added to a 500 ml baffled Erlenmeyer flask and aerobically shaken at 27 ° C. for 5 days. Β-glucan was recovered by ethanol precipitation in the same manner as described above. As a result, 512 mg of low-melanin-containing β-glucan with reduced melanin contamination was obtained.

Example 3 shows an example in which the ultraviolet irradiation mutation method is used in the mutation treatment (step 1).
As in Example 1, the strain used in the experiment was Aureobasidium pullulans K-1 strain obtained from the Morinomiya Center, Osaka Industrial Technology Research Institute.
Since Aureobasidium microorganisms can be obtained from other types of strain type cultures, they are not limited to the K-1 strain and can be used over a wide range of Aureobasidium microorganisms.

[Step 1: Gene mutation treatment]
60 ml (pH 5.0) of a potato dextrose medium was placed in a 300 ml-volume baffled Erlenmeyer flask to prepare a medium that was steam sterilized at 121 ° C. This medium was used as a medium for growing Aureobasidium pullulans K-1 strain (black yeast) used in the experiment. The culture was performed aerobically at 27 ° C. with shaking culture.
Microorganisms in which the cells grew and the turbidity OD660 reached 10 or more and reached the logarithmic growth phase were used as test bacteria for the radiation mutation method. The bacterial cells were washed with a sterile medium (medium excluding sucrose, pH adjusted to pH 5.0 with hydrochloric acid) shown in FIG. 3 of Example 1, and collected again by centrifugation (5000 rpm, 10 minutes). The cells were suspended in the same medium (sterile medium containing no carbon source) so that the turbidity OD660 of the cells was about 1.
Using a clean bench, this bacterial cell diluted solution was irradiated with ultraviolet rays (15 watts, 260 nm) for 2 minutes, and the treatment was performed using a death rate of 99% as an index. In addition, since the survival rate became 0% when irradiated for 5 minutes, the ultraviolet irradiation time was set to 2 minutes.

[Step 2] Selection of candidates from surviving strains Candidates were selected from surviving strains by culturing them in a solid medium (stationary culture) and a liquid medium. The agar medium shown in FIG. 3 of Example 1 was used as the solid medium (4% sucrose). The pH was adjusted to 5 with hydrochloric acid.
The cells were applied to the solid medium and statically cultured in an incubator at 27 ° C. for about 1 week to 10 days.

  After 1 week to 10 days, screening was performed using a white colony mutant suitable for the purpose as an index.

[Step 3] Double screening Screening by culture in a medium to which an inhibitor of tyrosinase pathway was added and screening by culture in a medium to which an inhibitor of polyketide pathway was added were performed in the same manner as in Example 1.
In this experiment, both a medium containing kojic acid and a medium containing phthalide were gradually colored, but a strain (UV-1 strain) in which melanin production was delayed was obtained. From this, it is presumed that the polyketide pathway and the tyrosinase pathway are mutant strains that have not lost function but have reduced melanin production ability. Here, this mutant strain is referred to as “mutant strain 6 (UV-1 strain)”.
FIG. 18 is a diagram showing a state of melanin non-producing or low-producing β-glucan producing bacteria mutated by ultraviolet irradiation treatment. As shown in FIG. 18, β-1,3-1,6-glucan containing low melanin with reduced melanin pigment contamination was obtained. Thus, even when ultraviolet irradiation treatment is used, melanin pigment non-production or low production with the ability to produce β-glucan is zero or lower than the initial state before the mutation treatment and the production ability of melanin pigment according to the present invention It can be seen that a β-glucan producing strain can be produced.

[Step 4] Production of high-purity β-1,3-1,6-glucan using melanin non-producing or low-producing β-glucan-producing bacterium of the present invention. Using mutant obtained as a result of double screening. High purity β-1,3-1,6-glucan was produced by the same method as in Step 4 shown in Example 1. 9 ml of a baffled conical flask with a baffle is added to the flask and aerobically shaken at 27 ° C. for 5 days. After sterilization, the supernatant is the same as in Example 1. Β-glucan was recovered by ethanol precipitation according to the above method. As a result, 503 mg of β-glucan was obtained.

Finally, Mutant 1 (TS-1 strain), Mutant 2 (TS-2 strain), Mutant 3 (TS-5 strain), Mutant 4 (TS-9 strain) obtained in Example 1, FIG. 19 shows a summary of the properties of mutant 5 (K-1ww strain) obtained in Example 2 and mutant 6 (UV-1 strain) obtained in Example 3.
In addition, the production β-glucan in the table was prepared by the method shown in Example 2. Each of the mutant strains and original strains in the table was added to 100 ml of baffled Erlenmeyer flask with 100 ml of medium shown in FIG. 9 and cultured aerobically at 27 ° C. for 5 days. The β-glucan was recovered by ethanol precipitation using the supernatant after sterilization treatment, and the production amount was measured. The column of NMR analysis describes the results of the method shown in Example 1 using the obtained β-glucan.
The preferred embodiments of the melanin non-producing or low-producing β-glucan-producing bacterium, the artificial production method thereof, and the production method of β-glucan produced using the same have been illustrated and described above. It will be understood that various modifications can be made without departing from the scope of the invention.

The aqueous dispersion of β-1,3-1,6-glucan and complex obtained by the production method of the present invention has high safety, such as pharmaceutical raw materials, quasi drugs, cosmetic raw materials, dyes, coloring agents, etc. It can be applied to various uses.

Claims (11)

A gene mutation treatment step for carrying out a mutation treatment by a physical treatment or a chemical treatment for causing a gene mutation in an Aureobasidium genus microorganism;
In the culture obtained by applying the tyrosinase pathway inhibitor and the culture obtained by applying the polyketide pathway inhibitor to the mutant strain obtained by the gene mutation treatment step, a mutant strain selection step of screening a strain with less melanin production,
A method for producing a melanin pigment non-producing or low-producing β-glucan producing strain having zero ability to produce melanin pigment or lower than the initial state before the mutation treatment and capable of producing β-glucan.   The method for producing a melanin non-producing or low-producing β-glucan producing strain according to claim 1, wherein the microorganism belonging to the genus Aureobasidium is Aureobasidium pullulans.   The method for producing a melanin non-producing or low-producing β-glucan producing strain according to claim 1 or 2, wherein the mutation treatment in the gene mutation treatment step includes irradiation treatment.   The method for producing a melanin non-producing or low producing β-glucan producing strain according to claim 3, wherein the irradiation treatment is an irradiation treatment with gamma rays, and a melanin non-producing or low producing mutant strain is selected based on the death rate.   5. The melanin pigment-free composition according to claim 1, wherein the mutation treatment in the gene mutation treatment step includes mutation treatment by ultraviolet irradiation, mutation treatment by administration of a chemical mutation agent, or a mixture thereof. A method for producing a production or low production β-glucan production strain. A gene mutation treatment process for applying a physical treatment or a chemical treatment for causing a gene mutation in a microorganism belonging to the genus Aureobasidium;
Obtained through a mutant selection process for screening a mutant strain obtained by the gene mutation treatment step and a strain in which melanin production is inhibited in a culture applied with a tyrosinase pathway inhibitor and a culture applied with a polyketide pathway inhibitor. A melanin non-producing or low-producing β-glucan-producing strain having zero or less ability to produce melanin or having an ability to produce β-glucan from the initial state before the mutation treatment.   The melanin non-producing or low-producing β-glucan producing strain according to claim 6, wherein at least one of the polyketide pathway or the tyrosinase pathway is inactivated in vivo.   A β- produced by culturing using a melanin non-producing or low-producing β-glucan producing strain obtained by the method for producing a melanin non-producing or low-producing β-glucan producing strain according to any one of claims 1 to 5. A method for producing 1,3-1,6-glucan.   A β-1,3-1,6-glucan that does not include a melanin pigment produced by the method for producing β-1,3-1,6-glucan according to claim 8.   A method for producing β-1,3-1,6-glucan produced by culturing using the melanin non-producing or low-producing β-glucan-producing strain according to claim 6 or 7. A β-1,3-1,6-glucan that does not include a melanin pigment produced by the method for producing β-1,3-1,6-glucan according to claim 10.