KR20090120347A - NOVEL PAENIBACILLUS POLYMYXA MUT5 AND METHOD FOR PRODUCTION OF beta-GLUCAN USING THE SAME - Google Patents

Abstract

PURPOSE: A method for producing a beta-glucan with a novel Paenibacillus polymyxa MUT5 is provided to optimize massive production process and reduce production cost. CONSTITUTION: A method for producing a beta-glucan comprises a step of fixing pH concentration of medium to 8.0 containing 0.15% of yeast extract as nitrogen source and 14% of sucrose as carbon source and a step of inoculating Paenibacillus polymyxa MUT5(deposit number KCCM 10943P) in a medium.

Description

Novel Paenibacillus polymyxa MUT5 and method for production of β-glucan using the same

The present invention relates to a novel F. Panaxillis polymyxa MUT5 strain having high beta-glucan productivity and a method for producing beta-glucan using the strain.

Polysaccharides are additives for processing and preserving foods.They are functional additives for agriculture, moisturizing and skin improvement in the food sector, soil repair, seedlings for seedling, and plant cultivation media. It is widely used in pharmaceutical fields such as cloning, physiological active agents, drug delivery systems, and substitute serum agents, and in the industrial fields of fibers, films, paper, paints, and inks.

In particular, β-glucan, a β-1,3 glycoside-linked glucose polymer, not only promotes immune response but also has various physiological activities such as anti-cancer, anti-inflammatory, and antioxidant. Usage in the industry continues to increase.

Beta-glucan is extracted from cell walls of plants, mushrooms and yeasts, but has a low purity and low recovery rate. Therefore, in order to overcome these problems, an attempt was made to produce extracellular polysaccharides by separating Paenibacillus polymyxa JB115, a microorganism that produces glucan as extracellular polysaccharide.

Bacterial-derived glucan can be produced in large quantities by optimizing strain breeding or fermentation conditions, thereby stably producing a large amount of glucan.

In the present invention, the glucan production strain of Paniccillus polymixes ( Panebacillus) for reducing the production cost by increasing the yield in the mass production of glucan polymyxa ) JB115 (KACC 91305P) was added to artificial mutants to select high glucan mutant strains were used to complete the optimized glucan mass production method.

It is an object of the present invention to provide a novel beta-glucan high production mutant obtained from the glucan producing strain Fanibacillus polymix yarn JB115.

Still another object of the present invention is to provide a method for producing beta-glucan, using a novel Favabacillus polymyxa MUT5 strain that can reduce the production cost by increasing the yield in the production of beta-glucan.

The present invention relates to a novel F. Panaxilas polymix yarn MUT5 ( Paenibacillus ) having high beta-glucan productivity. polymyxa MUT5 ) strain (Accession Number: KCCM 10943P).

In addition, the present invention, after fixing the initial pH of the medium containing sucrose (sucrose) as a carbon source, the yeast extract (yeast extract) as a nitrogen source to 8.0, and incubated by inoculating the novel F. Provided is a method for producing beta-glucan using a novel F. Panybacilli polymyxa MUT5 strain.

According to the present invention it is possible to optimize the mass production process of beta-glucan has an excellent effect of reducing the production cost through increased yield in the production of beta-glucan.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention relates to a Paenibacillus polymyxa MUT5 strain (Accession No .: KCCM 10943P), a beta-glucan high production mutant.

Optimal production conditions of beta-glucan according to the present invention is fixed to the initial pH of the medium containing sucrose (sucrose) as a carbon source, the yeast extract (yeast extract) as a nitrogen source to 8.0, then the beta-glucan high production mutant Fanny It is characterized by inoculating Bacillus polymyxa MUT5 in the culture medium.

Preferably, in the method for producing beta-glucan, the weight ratio (C: N) of the carbon source and the nitrogen source may be 18: 1.

In addition, preferably, the sucrose may be 14% and the yeast extract may be 0.15% based on the total weight of the medium.

Hereinafter, preferred examples and experimental examples are provided to aid in understanding the present invention, but the scope of the present invention is not limited by the following examples and experimental examples.

Example 1 Selection of Beta-Glucan High Production Mutant

1. Use medium and strain

The parent strain for the development of β-glucan high-producing mutants is Paenibacillus. was used to polymyxa JB115 (KACC91305P), selection medium for the mutant selection are aniline blue agar medium (20g / L sucrose, 5g / L yeast extract, 20g / L agar, 0.05g / L aniline blue, 3g / L CaCO 3 ) Was used.

2. Isolation of Mutantism

Mutation was UV irradiation to induce high β-glucan high mutant strains. That is, after incubating the strain of P. polymyxa JB115 for 18 hours in nutrient borth, diluting the culture solution to 10 ^-3 mL, smearing 100 μL in aniline blue agar medium, and then applying a 20 cm high UV lamp (504 nm) for 5 minutes. After irradiation, the cells were cultured at 30 ° C., and dark blue of the colonies generated was selected and subcultured to nutrient agar.

3. Glucan production test of parent strain and mutant strain

P. polymyxa JB115, a β-glucan-producing parent strain, and the medium for β-glucan production of mutant strains were mineral salt medium (MSM, KH ₂ PO ₄ 1.74; CaCl _{2 ·} H ₂ O 0.015; K ₂ HPO ₄ 0.49; MnCl _{2 · H 2 O 0.01; Na 2} SO 4 · H 2 O 3.7; Citrate 0.21; MgCl 2 · H 2 O 0.25; NH 4 Cl 1.5; FeCl 3 · H 2 O 0.024, 10 % by weight of a carbon source in g / L) Sucrose was added and used for about 18 hours pre-cultivation in nutrients and diluted with sterile distilled water so that the cell concentration (OD ₆₀₀ ) of the preculture was 1.5, then inoculated into glucan production medium and incubated at 30 ° C. for 3 days. To produce glucan. β-glucan separation was carried out by centrifugation of the culture solution at 5,000 rpm for 15 minutes to recover the culture supernatant, and added three times the volume of cold ethanol to the culture supernatant and then precipitated at 4 ℃. Precipitated β-glucan was shown by measuring the dry weight of normal pressure heating, that is, dried at 105 ℃ for 24 hours.

4. Selection of Beta-Glucan High Production Mutants

After UV irradiation with P. polymyxa JB115 (KACC91305P), four strains of visually dark blue color were selected from aniline blue agar medium (see FIG. 1). A mutant strain, MUT5, MUT7, MUT8, and P. polymyxa JB115 (KACC91305P), the parent strain, were pre-incubated in nutrient broth, and 1 mL (OD ₆₀₀ : 1.5) of each preculture was added to 10 wt% sucrose mineral. After incubation in salt medium and incubated for 3 days, the beta-glucan produced in the culture supernatant was measured. MUT5, MUT7, and MUT8 showed higher beta-glucan productivity than P. polymyxa JB115 (KACC91305P), especially P. polymyxa. In the case of MUT5 (KCCM10943P), glucan productivity was improved by 150% or more than the parent strain, and the best beta-glucan productivity was 13.2 g / L (see Table 1 below).

Strains β-glucan (g / L) Paenibacillus poplymyxa JB115 ( Wilde type) 8.8 MUT5 13.2 MUT7 9.6 MUT8 10.5

Experimental Example 1 Investigation of Optimal Conditions for Beta-Glucan Production and Production of Glucan by Batch Culture

1. Investigation of carbon source, nitrogen source and pH condition

Optimal carbon irradiation by a carbon source for fructose, glucose, lactose, sucrose on mineral salt medium is added 2% by weight were measured and β-glucan productivity, optimum nitrogen source NH ₄ Cl instead of in the mineral salt medium for the selected mutant attention glucan producing NaNO ₃ , NH ₄ Cl and (NH ₄ ) ₂ SO ₄ were used as inorganic nitrogen sources, and peptone, tryptone, and yeast extract were added as organic nitrogen sources, and β-glucan production was investigated by dry weight method. The optimum pH condition for β-glucan production of the selected mutant strains was measured at the initial pH of 4-10.

In order to investigate the optimal carbon source for glucan mutant strains for β-glucan production, we investigated the productivity of β-glucan at 2 wt% concentrations of four carbon sources, including glucose, indicating that fructose and lactose are not suitable as substrates for the synthesis of extracellular polysaccharides. Contrary to reports, P. polymyxa MUT5 (KCCM10943P) showed good β-glucan productivity in fructose, glucose lactose, and sucrose. In particular, P. polymyxa MUT5 (KCCM10943P) was excellent in β-glucan productivity at 4.6 g / L in sucrose (see FIG. 2). Sucrose has also been reported as a suitable substrate for the production of extracellular polysaccharides in Pseudomonas mendocina and Agrobacterium sp. Sucrose is cheaper than other carbon sources and is economical as a substrate for industrial fermentation. Therefore, sucrose is suitable for mass production of β-glucan of P. polymyxa MUT5 (KCCM10943P). The optimal nitrogen source for β-glucan production of P. polymyxa MUT5 (KCCM10943P) was yeast extract, which produced 9.3 g / L of β-glucan. In addition, β-glucan productivity was better in the organic nitrogen source except for peptone, which was consistent with the report that the production of extracellular polysaccharide was better when using the organic nitrogen source (see FIG. 3).

2. Investigation of pH Effect on Glucan Production

β-glucan production of P. polymyxa MUT5 (KCCM10943P) with pH increased from pH 6 to β-glucan production and peaked at pH 8, and then decreased (see FIG. 4). Agrobcaterium sp. Was reported to have a good yield when the initial pH was below 6, and P. polymyxa MUT5 (KCCM10943P) was different from this report. However, similar to the report that β-glucan productivity was the highest at pH 7.5 in Bacillus sp., PH of β-glucan production was different by production strain.

3. Influence of Sucrose and Yeast Extract Concentration on Glucan Production

As a result of investigating β-glucan production according to sucrose concentration, which is an optimal carbon source, sucrose was the highest at 23.2 g / L at 14 wt% of sucrose and 0.15 wt% of nitrogen-containing yeast extract (see FIGS. 5 and 6). The production of extracellular polysaccharides is controlled by the ratio of C and N. P. polymyxa MUT5 has a β-glucan productivity of up to 18: 1 at a maximum weight ratio of C and N. It was promoted and similar to other reports. Increased β-glucan biosynthesis due to nitrogen source restriction has been reported to increase β-glucan biosynthesis as isoprenoid lipids are used for the synthesis of extracellular polysaccharides instead of synthesizing polysaccharides for cell composition as cell growth begins to decrease. . Therefore, the deficiency of nitrogen source is an essential phenomenon to increase β-glucan productivity.

4. Experiment of Glucan Production in 5L Jar Fermenter by Batch Culture

In order to confirm the reproducibility of β-glucan production for the industrial use of the selected mutant strains, sterilization at 121 ° C. for 20 minutes in the optimum β-glucan medium condition investigated in 5L jar ferment, and then batch culture for 3 days at the optimum pH Was carried out. At this time, the pH was not adjusted, the stirring speed was 200 rpm, the air flow was adjusted to 2.0 vvm. Also, silica-antifoam (Sigma, USA) was used as antifoam to remove the foam.

Sucrose was added 14% by weight as carbon source and 0.15% by weight of yeast extract instead of amonium chloride, and the initial pH was set at 8.0. P. polymyxa, a high-glucan mutant, was produced in 5L jar fermenter. MUT5 (KCCM10943P) preculture was inoculated at 1% concentration and incubated at 30 ° C. for 5 days, and β-glucan productivity was examined. As a result P. polymyxa The β-glucan productivity from MUT5 (KCCM10943P) was the highest at 23.8 g / L on the 3rd day (see FIG. 7), and similar to the β-glucan productivity in the flask culture, it was confirmed that the industrial glucan was reproducible. .

FIG. 1 is a photograph showing beta-glucan production by Fanibacilli polymyxa JB115 (wild type, CON) and beta-glucan production mutant bacteria.

Figure 2 is a graph showing the effect of the carbon source on the production of beta-glucan by the mutant strains of Pa.

Figure 3 is a graph showing the effect of the nitrogen source on the production of beta-glucan by the mutant strain of Pa.

FIG. 4 is a graph showing the effect of pH on the production of beta-glucan by the mutant strain of Pannibacillus polymix MUT5.

FIG. 5 is a graph showing the effect of sucrose concentration on the production of beta-glucan by the mutant strain of Pannibacillus polymix MUT5.

6 is a graph showing the effect of the concentration of yeast extract on the production of beta-glucan by the mutant strains of Pannivacillus polymyxa MUT5.

Figure 7 is a graph showing the effect of the incubation time on the production of beta-glucan by the mutant strain of Pannibacillus polymix MUT5.

Claims (4)

Beta-glucan (β-glucan) and the novel Bacillus Waist poly miksa MUT5 (Paenibacillus polymyxa has a productivity MUT5 ) strain (Accession Number: KCCM 10943P). After the initial pH of the medium containing sucrose as a carbon source and the yeast extract as a nitrogen source is fixed to 8.0, the novel F. ni. Polysaccharide is characterized by inoculating the culture of the strain of claim 1 on the medium. Method for producing beta-glucan using the MIX Corporation MUT5 strain. The method of claim 2, wherein the weight ratio (C: N) of the carbon source and the nitrogen source is 18: 1. The method of claim 2, wherein the sucrose is 14% and the yeast extract is 0.15% based on the total weight of the medium.

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