JPS6170994A - Production of cyclic(1-2)-beta-d-glucan - Google Patents

Abstract

PURPOSE:To obtain cyclic(1 2)-beta-D-glucan in high yield, by cultivating a bacterium in an assimilable nutritive medium while controlling the cultivation at a multiplication ratio smaller than the maximum multiplication ratio, and collecting the titled compound from the culture solution. CONSTITUTION:A mold (e.g., Agrobacterium radiobacter-A1-5, etc.) capable of producing cyclic(1 2), is cultivated in an assimilable nutritive medium preferably at 3-9pH at 20-40 deg.C while controlling the cultivation at a multiplication ratio (preferably <=0.05 average multiplication ratio) smaller than the maximum multiplication ratio (preferably >=0.08/hr), to give the aimed glucan.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing cyclic-(1-2)-β-D-glucan (hereinafter abbreviated as CG).
The present invention relates to a method for producing CG by fermentation by culturing d-producing bacteria while controlling the growth rate to be lower than its maximum growth rate.

[Prior Art] Conventionally, a method for producing CG using microorganisms involves culturing microorganisms belonging to the genus Rhizobium or Agrobacterium in a medium containing glucose, ammonium phosphate, potassium phosphate, etc. A method for producing and accumulating CG in a culture medium [J, Gen.

Microbiol, 128.1873 (1982)
and Carbohydr, Res, l 21.
31 (1983)] or as an improved method of the method,
Method for culturing mutant strains that no longer produce extracellular polysaccharides, etc. (JP-A-59-71686 and JP-A-820
92), etc. are known.

However, the CG manufacturing method using the above-mentioned known method does not necessarily provide satisfactory CG productivity.

As a result of various studies on CG production methods using fermentation methods, the present inventor found that CG productivity increases when microorganisms are cultured under conditions that are shifted from the optimal conditions for growth.
The present invention has now been completed.

[Structure of the Invention] The gist of the present invention is to cultivate microorganisms in an assimilable nutrient medium while controlling the growth rate to be lower than the maximum growth rate,
The present invention relates to a method for producing cyclic (1->2)-β-D-glucan, which comprises recovering cyclic (1-2)-β-D-glucan from this culture solution.

As the microorganism used in the present invention, any microorganism that belongs to the genus Rhizobium or the genus Agrobacterium and has the ability to produce CG can be used, for example,
Rhizobium melilotsutei IF013336, Rhizobium trifuori IF013337, Rhizobium japonicum IPO1333B, Rhizobium fusceoli A
I-I U 1133, Rhizobium faceori R
A-4 (FERM BP374), Rhizobium fatseori RA-8 (FERM BF279), Rhizobium fatseori RA-12 (FERM BF280)
, Agrobacterium radiobacter IFO1266
5. Agrobacterium radiobacter Al-5 (F
ERM BP373), Agrobacterium radiobacter AAl-7 (FERBP378), Agrobacterium radiobacter AA1-12 (FERBP37)
7), Agrobacterium rhizogenes IFO1325
9. Agrobacterium tumefaciens IF030
58, etc.

As a medium composition for culturing microorganisms, one containing a carbon source, a nitrogen source, an organic nutrient source, an inorganic substance, etc. as appropriate is used. As the carbon source and nitrogen source, any substance can be used as long as it can be assimilated by the bacterial strain used. Preferably, sugars are used as the carbon source, particularly glucose, maltose, sucrose, or molasses, and inorganic substances are used as the nitrogen source. Nitrogen sources, organic acid ammonium salts can be used. Yeast extract and corn methane liquor as organic nutritional sources 1. Meat extract, polypeptone, biotin, thiamin, etc. can be used. Ordinary inorganic salts can be used as the inorganic substance.

Cultivation is carried out aerobically in a batch, fed-batch or continuous manner. The average growth rate during the entire culture period is controlled to be lower than the maximum growth rate of the microorganism. Usually, pl(is 3
~9, the temperature is 20-40°C. The growth rate (hereinafter referred to as μ) is determined from the following formula by measuring the amount of bacterial cells at each culture time.

In batch or fed-batch culture, [where Xo = amount of bacterial cells at the time of initial initiation, X1→ is the amount of bacterial cells at the time of time cultivation, and t = culture time. ] In continuous culture, the amount of bacterial cells in the extract at tC time [where tc = continuous culture time]. ] The maximum growth rate (μmax) of the microorganism is preferably 0.08 hr −1 or more.

The average proliferation rate during the entire culture period is the value calculated from the above formula of μ during the entire period from the start of culture to the end in batch or fed-batch culture, and from the start to the end of continuous culture in continuous culture. .

As a method for controlling μ, any conditions may be used as long as the conditions do not inhibit CG production within the range in which the microorganisms used can grow. For example, culture conditions (temperature, pH, etc.), amounts of nutrients (carbon sources, nitrogen sources, phosphate sources, inorganic salts, etc.) added, or when the microorganisms used have nutritional requirements, vitamins, amino acids, nucleic acids, etc. Adjust the amount added. Alternatively, methods such as adding to the culture system a drug that suppresses the growth of microorganisms and does not inhibit CG production may be used. Examples of such drugs include capric acid, citric acid, malonic acid, succinic acid, organic acids such as formic acid, acetic acid, propionic acid, caprylic acid, and malic acid, or their salts, and alcoholic acids such as methanol, ethanol, propatool, etc. can be mentioned. These culture temperatures, pH1 medium compositions, and growth-inhibiting drugs can be used alone or in appropriate combinations. The method used to control μ varies depending on the type of microorganism used, the type of carbon source, etc., but methods of controlling by temperature, pH, etc. are advantageous. Growth control may be performed at any time during the culture period, and the period may be temporary or over the entire period. During culture, μ may change at any time, but ultimately it is preferable to control the average growth rate during the entire culture period to 0.05 hr−1 or less.

After removing microorganisms and/or polysaccharides, CG can be easily recovered from the culture solution by methods such as using an activated carbon column [Proceedings of the Japanese Society of Agricultural Chemistry, p. 585 (19,!14)]. I can do it.

[Example] Hereinafter, the present invention will be specifically explained with reference to Examples. In the examples, CG was confirmed and quantified using high performance liquid chromatography [J, Chromatogr, 265.
89 (1983)].

Example 1 Acrobacterium radiobacter A1-5 was grown in a medium C3fl/71. The seeds were inoculated into a large fermenter, and cultured for 5 days at an aeration rate of 1 vvm and a stirring rate of 60 rpm.

Medium composition Nigelcose 40g, (NH,)tHPo.

1.5g, KHtPo, 1.0g, Mg5O, 7H1
00.5g, CaCO55g - tap water 11. .

During culture, while measuring μ, the culture temperature is adjusted to the following (1) to (
3) to control μ so that it was approximately constant (pH was not adjusted, but in both cases it was 5 to 7.5).
changed between. ): (1) 30℃, (2) 33℃, (
3) The temperature was changed from 33°C to 30°C 1.5 days after the start of culture.

Table 1 shows the relationship between the average proliferation rate and the CG production amount during the entire culture period.

Table 1 Example 2 20 μg/i of biothione and 200 μg of thiamin in the medium
g/4 and Acrobacterium radiobacter A
Example 1 was repeated except that Rhizobium faceoli RA-12 was used in place of 1-5. Table 2 shows the relationship between the average proliferation rate and the CG production amount during the entire culture period.

Table 2 Procedure Auxiliary Device (Spontaneous) November 7, 1980 1, Case Indication 1988 Patent Application No. 193429 '5'2,
Name of the invention Process for producing cyclic (1→2)-β-D-glucan 3, Relationship with the case of the person making the amendment Patent applicant address: 111-chome, 12 stones, 39, Kita-ku, Osaka-shi, Osaka Prefecture, New Edition Kyuhill name (285) Daikin Industries, Ltd. Representative Yama 1) Minoru 4, Agent 5, Date of amendment order: Voluntary 7, The following parts are amended in the detailed explanation of the invention column of the statement of contents of the amendment. .

(1) 1st line at the end of page 2, “Unexamined Japanese Patent Publication No. 82092 J”
[Corrected as JP-A-59-82092J.

(2) Page 4, lines 10-11, “rAl-124”
A-12” was corrected.

that's all

Claims (5)

[Claims] (1) A method characterized by culturing microorganisms in an assimilable nutrient medium while controlling the growth rate to be lower than the maximum growth rate, and recovering cyclic (1→2)-β-D-glucan from this culture solution. Method for producing cyclic (1→2)-β-D-glucan. (2) The production method according to claim 1, wherein the microorganism is a bacterium belonging to the genus Rhizobium or the genus Agrobacterium. (3) The maximum growth rate of microorganisms is 0.08hr^-^1 or more, and the average growth rate during the entire culture period is 0.05hr.
^-^1 or less. The manufacturing method according to claim 1. (4) The production method according to claim 1, wherein the growth control method is to adjust the temperature, pH, the amount of inorganic salt in the culture medium, or the amount of auxotrophic substance of the bacteria used. (5) The production method according to claim 1, wherein the growth control method is to add an organic acid, a salt thereof, or an alcohol.