JP2766165B2 - Method for producing bacterial cellulose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a cellulosic substance produced by a microorganism belonging to the genus Acetobacter and capable of producing a cellulosic substance. More specifically, the present invention relates to the production of cellulosic substances by a microorganism in a medium containing a specific organic acid as a cellulose production promoting factor.

This cellulosic substance is edible and used in the food field, and is excellent in water-based dispersibility, so that it retains the viscosity of foods, cosmetics or paints, strengthens food raw materials, retains moisture, maintains food stability. It is industrially useful as an additive for improving caloric properties, as a low-calorie additive, or as an emulsion stabilizing aid.

[0003] In addition, the disintegrated product of the cellulosic substance has various industrial uses as a reinforcing agent for polymers, particularly aqueous polymers, based on the structural and physical characteristics of microfibrils. Since such a defibrated material has a high tensile modulus, a material obtained by solidifying the cellulosic deflated product into a paper or solid form is expected to have excellent mechanical properties based on the structural characteristics of microfibrils, and various industrial materials. As an application.

[0004]

2. Description of the Related Art A method for producing cellulose by culturing a microorganism belonging to the genus Acetobacter has been known (this microorganism is hereinafter referred to as "cellulose-producing acetic acid bacterium"). For example, JP-A-62-265990,
Japanese Patent Application Laid-Open No. 61-221201 discloses such a method. As a nutrient medium that is considered to be suitable for culturing cellulose-producing acetic acid bacteria, a carbon source, peptone, yeast extract,
Schramm / Hestri consisting of sodium phosphate and citric acid
n medium (Schramm et al., J. General Biology, 11 , pp. 123
129, 1954). However, when the nutrient medium was shaken or aerated with stirring, the resulting cellulose production was low and the production rate was not always satisfactory. In addition, a medium containing corn steep liquor (CSL), malt extract, or the like in addition to the above nutrient medium is known. Specific components contained in these natural nutrients (peptone, yeast extract, CSL, malt extract, and the like) are known. Is not known to be involved in promoting cellulose production. Inositol, phytic acid, pyrroloquinoline quinone (PQQ) are currently known as factors promoting cellulose production by specific nutrients in the medium.
(Japanese Patent Publication No. 5-1718; Mitsuo Takai, Journal of Paper and Paper Technology Association,
Vol. 42, No. 3, pp. 237-244), etc.
The amount of cellulose produced was still insufficient, and their effects in shaking or aeration and stirring cultures were not clear.

[0005]

Research on organic acids and acetic acid bacteria is described in Takeuchi et al., J. Ferment. Technol.
Vol. 46, No. 4, pp. 288-291, 1968, Nanba et al., Journal of the Food Science Society of Japan, Vol. 32, No. 5, pp. 331-337, 19
Although these are described in 1985, etc., these studies have studied the effects of acetic acid bacteria on acid production, and there is no mention of the effect of these organic acids on the production of cellulose by cellulose-producing acetic acid bacteria. Absent.

An object of the present invention is to develop a method for improving the productivity of a cellulosic substance by using a cellulose-producing acetic acid bacterium and producing it at low cost.

[0007]

Means for Solving the Problems The present inventors have conducted various studies in order to achieve the above object, and have found that microorganisms belonging to the genus Acetobacter and having the ability to produce cellulosic substances (ie, cellulose-producing acetate). It has been found that by culturing Bacteria in a medium to which a specific organic acid is added, the productivity of cellulosic substances is remarkably improved as compared with the conventional production method.

That is, the present invention provides a method for culturing a microorganism belonging to the genus Acetobacter and having a cellulosic substance-producing ability in a medium to which a carboxylic acid or a salt thereof is added as a cellulose production promoting factor. The present invention provides a method for producing cellulose, comprising producing and accumulating a sexual substance and collecting the substance.

The microorganism used in the present invention belongs to the genus Acetobacter and may be any microorganism that produces cellulosic substances. For example, Acetobacter sp. BPR200
1 strain (FERM P-13466) , Acetobacter
Acetobacter xylinum ATCC 2376
8, Acetobacter xylinum ATCC 23767,
Acetobacter xylinum ATCC10821 and the like.

[0010] The specific carboxylic acid or its salt used in the method of the present invention acts on the cellulose biosynthesis system of the microorganism as a cellulose production promoting factor, thereby significantly improving the productivity of cellulose. That is, as shown in Examples described later, the amount of accumulated cellulose in the medium reaches about 1.5 to about 12 times as compared with the case where no carboxylic acid is added. As used herein, the term "carboxylic acid or a salt thereof as a cellulose production promoting factor" increases the amount of accumulated cellulose as compared to the case where no carboxylic acid or a salt thereof is added , preferably about 1.5 times. It refers to a carboxylic acid or salt that increases the above. The carboxylic acid that can be used in the present invention may be either a saturated or unsaturated carboxylic acid, and examples thereof include acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, gluconic acid, and glucuronic acid as saturated carboxylic acids, and And unsaturated carboxylic acids such as fumaric acid, maleic acid, acrylic acid and benzoic acid. These carboxylic acids are not particularly limited in the number of carbon atoms and the number of carboxyl groups, but are preferably water-soluble. Preferred carboxylic acids are lactic acid, pyruvic acid,
Acetic acid. When a carboxylic acid is used as a salt, it is not particularly limited, but usually an alkali metal or alkaline earth metal salt can be used. These carboxylic acids or salts thereof may be used alone or in combination of two or more. The amount of the carboxylic acid or salt thereof is not particularly limited, but is preferably 1 mmol to 20
0 mmol / L. In the present invention, the productivity of cellulose can be remarkably improved only by adding a small amount or a small amount of a carboxylic acid or a salt thereof as a cellulose production promoting factor, rather than adding it to a medium as a main carbon source.

In the medium composition, as a carbon source, shoe cloth, glucose, fructose, mannitol, sorbitol, galactose, maltose, erythrit, gadunit, glycerin, ethylene glycol, ethanol and the like are used alone or in combination. Further, starch hydrolyzate, citrus molasses, beet molasses, beet juice, sugarcane juice, fruit juices including citrus fruits and the like containing these can be used.

As a nitrogen source, ammonium sulfate,
An organic or inorganic nitrogen source such as ammonium salt such as ammonium chloride and ammonium phosphate, nitrate, urea may be used, or Bact-Peptone, Ba may be used.
ct-Soytone, Yeast-Extract,
Nitrogen-containing natural nutrients such as soybean may be used. Amino acids, vitamins, fatty acids, nucleic acids, as organic micronutrients
2,7,9-Tricarboxy-1H pyrrolo [2,3-
5] -Quinoline-4,5-dione may be added.

When an auxotrophic mutant that requires an amino acid or the like for growth is used, it is necessary to supplement the required nutrient. As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, cobalt salts, molybdates, red blood salts, chelate metals and the like are used.

The pH of the culture is between 3 and 7, preferably 5
Control near.

The culture temperature is 10 to 40 ° C., preferably 25
Perform in the range of ~ 35 ° C.

The concentration of oxygen supplied to the culture tank is 1 to 100.
%, Preferably 21 to 80%.

In the method of the present invention, the culture method is not limited, and may be any of stationary, shaking, and aeration-agitation culture. One of the features of the method of the present invention is that the cellulosic productivity is not affected even if the culture is performed under shaking or aeration and stirring.

The cellulosic substance produced by the method of the present invention may be collected as it is, or may be subjected to a treatment for removing substances other than cellulosic substances such as bacterial cells contained in the substance.

To remove impurities, washing with water, dehydration under pressure, washing with diluted acid, washing with alkali, treatment with a polar organic solvent such as toluene and ethyl acetate, treatment with a bleach such as sodium hypochlorite and hydrogen peroxide, lysozyme Etc., treatment with a surfactant such as sodium lauryl sulfate, deoxycholic acid, etc.
Impurities can be removed from the cellulose-like substance by performing heating washing in the range of ° C alone or in combination.

The cellulosic material thus obtained in the present invention is as follows.

The cellulosic substance of the present invention includes cellulose, a substance containing a heteropolysaccharide having cellulose as a main chain, and a substance containing glucans such as β-1,3, β-1,2 and the like. Components other than cellulose in the case of heteropolysaccharide are mannose, fructose, galactose, xylose,
Hexoses such as arabinose, rhamnose, glucuronic acid,
Pentose and organic acids.

These polysaccharides may be a single substance, or two or more polysaccharides may be mixed due to hydrogen bonding or the like.

[0023]

The present invention will be described in more detail with reference to the following examples.

Culture conditions Cellulose-producing acetic acid bacteria were cultured using a flask culture method and a jar fermenter culture method.

Flask culture method Fructose 40 g / L, monopotassium phosphate 1.0 g /
L, magnesium sulfate 0.3 g / L, ammonium sulfate 3 g / L, bacto-peptone 5 g / L, lactic acid 1.4 m / L
L, inoculated with 1 ml of a cryopreserved bacterial solution of cellulose-producing acetic acid bacteria in a 750 ml Roux flask into which 100 ml of a basic medium having an initial pH of 5.0 was placed, and allowed to stand at 28 ° C. for 3 days in a low-temperature incubator. Culture was performed. After the seed culture, the Roux flask was shaken well, and then the contents were filtered with a gauze under aseptic conditions to separate cellulose fragments and cells. Next, the mixture is centrifuged at 10,000 rpm for 15 minutes to separate the medium component and the cells (+ microcellulose), and the cells (+ microcellulose) are washed with sterile physiological saline once or twice, and centrifugation is repeated. Was. A required amount of sterile physiological saline was added to the washed cells, and after stirring, this was used as a seed cell solution.

Next, 7.5 ml of the seed bacterium solution was added to the medium 6 for examination.
The cells were inoculated into a 300 ml spiral baffle flask into which 7.5 ml had been inserted. Using a shaking incubator, amplitude 2c
Main culture was performed for 4 days while rotating and shaking under the conditions of m, rotation speed of 180 rpm, and temperature of 28 ° C.

The amount of the produced bacterial cellulose was determined as follows. That is, the solids in each flask were washed with water to remove the medium components, and then treated in a 1% aqueous NaOH solution at 110 ° C. for 20 minutes to remove bacterial cells. Was washed with water and vacuum dried at 80 ° C., and the dry weight was measured.

Jar fermenter culturing method 750 ml of Ro with 100 ml of the above basic medium
The ux flask was inoculated with 1 ml of a cryopreserved bacterial solution of a cellulose-producing acetic acid bacterium and allowed to stand still at 28 ° C. for 3 days in a low-temperature culture machine. After completion of the stationary culture, the Roux flask was shaken well, and the content was filtered under a sterile condition with a gauze to separate the cellulose fragments from the cells. 7.5 ml of the obtained bacterial solution
Was inoculated into a 300 ml spiral baffle flask into which 67.5 ml of a basic medium had been inserted, and seed culture was carried out for 3 days using a shaking incubator under the conditions of an amplitude of 2 cm, a rotation speed of 180 rpm, and a temperature of 28 ° C. while rotating and shaking. .

After completion of the culture, the contents of the flask were transferred to a sterile blender and crushed at 10,000 rpm for 3 minutes. The crushed content was used as a seed bacterial solution and used for the following main culture. In addition, 10,
After centrifugation at 000 rpm for 20 minutes, it was confirmed that lactic acid did not remain in the obtained supernatant.

The above seed cell solution (60 ml) was aseptically inoculated into a small jar fermenter (total volume: 1,000 ml) into which 540 ml of a sterilized test medium was inserted, and the mixture was incubated at 30 ° C. for 20 minutes.
PH for 1 hour or 30 hours
While controlling the pH to 5.0 with H ₂ SO ₄ , and at the initial rotation of 400 rpm, the amount of dissolved oxygen (D
Main culture was performed while automatically controlling the number of revolutions so that O) was within 3.0 to 21.0%.

After the completion of the culture, the solid matter in the jar fermenter was accumulated, and the medium was removed by washing with water.
The cells were treated in an aqueous OH solution at 110 ° C. for 20 minutes to remove the cells. Further, the resulting cellulose was washed with water until the washing liquid became nearly neutral, and then vacuum-dried at 80 ° C. for 12 hours to measure the dry weight.

Example 1 Lactic acid in various organic nitrogen sources
Additive effect Acetobacter sp. BPR2001 strain as a cellulose-producing acetic acid bacterium, and the following composition containing different organic nitrogen sources as a study medium: fructose 40 g / L, KH ₂ PO ₄ 1.0 g / L, MgSO ₄ . 3 g / L, (NH ₄ ) ₂ SO ₄ 3 g / L, organic nitrogen (N) source 5 g / L, Bact-Peptone (manufactured by Difco); Bact-Soytone (manufactured by Difco); Yeast-Extract (manufactured by Difco) ); Or a soybean protein (acid hydrolyzed concentrated solution of soybean protein), lactic acid 0 or 1.4 ml / L, initial pH 5.0 using a culture medium for 2 days or 4 days according to the above flask culture method. The test was performed for one day, and the amount of accumulated cellulose in the presence or absence of lactic acid was evaluated.

The results are shown in Table 1.

[0034]

[Table 1]

As can be seen from the results in Table 1, when lactic acid is added to the medium, the amount of accumulated cellulose is about 5 times to about 12 times in any organic N source as compared with the case where lactic acid is not added.
Doubled. The amount of cellulose accumulated by the organic N source used was determined as Bact-Peptone <Bact after 4 days of culture.
-Soytone <Yeast-Extract <Deno concentration.

Example 2 Effect of adding carboxylic acid other than lactic acid
The Bact-Soytone as fruit organic nitrogen source and in addition to acetic acid and pyruvate lactate as the carboxylic acid 10m
Culture was carried out in the same manner as in Example 1 except that mol / L was added or not, and the effect of addition of carboxylic acids other than lactic acid on the amount of accumulated cellulose was examined.

The results are shown in Table 2.

[0038]

[Table 2]

From Table 2, it was found that when acetic acid and pyruvic acid were used as the carboxylic acids, the amount of accumulated cellulose increased about 2 to 6 times as compared with the case where no carboxylic acid was added. When evaluated 4 days after culture, the degree of cellulose accumulation increased in the order of acetic acid <pyruvic acid <lactic acid.

Example 3 Effect of mosquito on cellulose accumulation
Influence of added amount of rubonic acid Bact-Soytone as an organic nitrogen source and 0.1 ml / L, 0.5 ml / L, 1.0 ml /
L, 5.0 ml / L, 10.0 ml / L, 15.0 ml
After culturing for 4 days in the same manner as in Example 1 except that the amount of / L was used, the amount of accumulated cellulose at each added amount was evaluated.

Table 3 shows the results.

[0042]

[Table 3]

The results in Table 3 show that the amount of lactic acid added was 0.1 ml / L.
From 10.0 to 10.0 ml / L, the amount of accumulated cellulose gradually increases, but even if the amount of lactic acid added is further increased, the amount of accumulated cellulose tends to decrease.

Example 4 Effects of Bacteria on Cellulose Accumulation
Bac strains influence cellulose productivity Acetobacter xylinum ATCC23768 and Acetobacter xylinum ATCC23769 other Acetobacter sp BPR2001 strain as acetic acid bacteria, and as organic nitrogen sources
Culture was performed for 4 days in the same manner as in Example 1 except that t-Soytone was used, and the amount of accumulated cellulose was measured for each strain.

Table 4 shows the results.

[0046]

[Table 4]

From the results in Table 4, it can be seen that although the substantial amount of cellulose accumulated differs among the three strains used, the cellulose accumulation was increased by about 2.5 to about 7 times by adding lactic acid in any of the strains. Increased. In particular, Acetobacter
The species BPR2001 strain and Acetobacter xylinum ATCC 23969 showed high cellulose production.

Example 5 Cellulose-producing acetic acid bacteria
Acetobacter sp. BPR2001 strain as a cellulosic acid-producing bacterium for fermenter culture, and the following composition as a study medium: fructose 40 g / L, KH ₂ PO ₄ 1.0 g / L, MgSO ₄ 0.3 g / L, (NH ₄ ) Main culture was performed for 20 hours or 30 according to the above jar fermenter culture method using a medium of 3 g / L of ₂ SO _{4, 5} g / L of Bact-Soytone, 0 or 1.4 ml / L of lactate, and an initial pH of 5.0. Over time, the amount of cellulose accumulated in the presence or absence of lactic acid was evaluated.

Table 5 shows the results.

[0050]

[Table 5]

The results in Table 5 show that when fermentation conditions such as pH and dissolved oxygen content are controlled using a jar fermenter, cellulose production is significantly improved even when lactic acid is not added. This indicates that the amount of accumulated cellulose is further improved about 1.5 to about 2.5 times the case.

[0052]

According to the present invention, in the production of cellulose using the fermentation method according to the present invention, the cellulose productivity is remarkably improved as compared with the conventional method only by adding a small amount or a small amount of a carboxylic acid or a salt thereof to the medium. This indicates that the present method can produce cellulose efficiently and inexpensively.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 19/04 CA (STN)

Claims (4)

(57) [Claims] 1. A microorganism belonging to the genus Acetobacter and having a cellulosic substance-producing ability is cultured in a medium to which a carboxylic acid or a salt thereof as a cellulose production promoting factor is added to produce a cellulose or a cellulosic substance in the medium. A method for producing cellulose, comprising accumulating and collecting the substance. 2. The method according to claim 1, wherein the carboxylic acid or a salt thereof is lactic acid, pyruvic acid or acetic acid, or a salt thereof. 3. A carboxylic acid or a salt thereof is added to a medium in an amount of 1 to 2 times.
3. The method according to claim 1, wherein said compound is added in an amount of 00 mmol / L.
The described method. 4. The method according to claim 1, wherein the culture is stationary, shaking, or aeration / agitation culture.