JPH0739386A - Production of bacterial cellulose - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the subject edible compound by culturing a microorganism, belonging to the genus Acetobacter and having the ability to produce a cellulosic substance in a culture medium containing a carboxylic acid (salt) which is a factor in promoting the production of the cellulose and then collecting the resultant product, CONSTITUTION:This method for producing a bacterial cellulose is to inoculate a microorganism, belonging to the genus Acetobacter and having the ability to produce a cellulosic substance (e.g. Acetobacter xylinum ATCC23768) in a culture medium containing a carboxylic acid (e.g. lactic acid) or a salt thereof as a factor in promoting the production of cellulose added thereto, carry out the stationary culture in a low- temperature incubator at 28 deg.C for 3 days, then filter the culture solution through a gauze, subsequently centrifuge the filtrate, separate the microbial cell, add a sterilized physiological saline solution to the separated microbial cell, provide a seed microbial solution, inoculate the prepared seed microbial solution into the same culture medium as that used in culturing the seed, perform the main culture at 28 deg.C for 4 days while shaking the culture medium and then collect the cellulose and cellulosic substance, produced and accumulated in the culture medium. This bacterial cellulose is useful for retaining the viscosity of a food, a cosmetic, a coating, etc., and usable as a food additive, an emulsion stabilizer, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD 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, it relates to the production of a cellulosic substance by the microorganism in a medium containing a specific organic acid as a cellulose production promoting factor.

Since this cellulosic material is edible and used in the food field and has excellent water-based dispersibility, it retains the viscosity of foods, cosmetics, paints, etc., strengthens the raw material of food materials, retains water, and stabilizes foods. It has industrial utility value as a property improving agent, a low calorie additive or an emulsion stabilizing aid.

The disaggregated material of the cellulosic material has various industrial uses as a reinforcing agent for polymers, especially aqueous polymers, based on the structural and physical characteristics of microfibrils. Since such a disaggregated material shows a high tensile elastic modulus, a substance obtained by solidifying the cellulosic disaggregated material into a paper or solid form is expected to have excellent mechanical properties based on the structural characteristics of microfibrils, and various industrial materials. There is an application as.

[0004]

2. Description of the Related Art Conventionally, a method of culturing a microorganism belonging to the genus Acetobacter to produce cellulose has been known (hereinafter, this microorganism is referred to as "cellulose-producing acetic acid bacterium"). For example, JP-A-62-265990,
This is described in JP-A-61-222101 and the like. As a nutrient medium which is considered to be suitable when culturing cellulose-producing acetic acid bacteria, carbon sources, 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) is known. However, when shaking or aeration-agitation culture was carried out in the above nutrient medium, the yield of cellulose obtained was low and the production rate was not always satisfactory. In addition to the above-mentioned nutrient medium, a medium containing corn steep liquor (CSL), malt extract, etc. is known. Specific components contained in these natural nutrients (peptone, yeast extract, CSL, malt extract, etc.) Is not known to be involved in promoting cellulose production. The currently known factors for promoting cellulose production by specific nutrients in the medium include inositol, phytic acid, and pyrroloquinoline quinone (PQQ).
(Japanese Examined Patent Publication No. 5-1718; Mitsuo Takai, Journal of Paper and Paper Technology,
Vol. 42, No. 3, pp. 237-244), etc.
The amount of cellulose produced was still insufficient, and their effects in shaking or aeration-agitation culture were not clear.

[0005]

Studies on organic acids and acetic acid bacteria have been reported by Takeuchi et al., J. Ferment. Technol.
Vol.46, No.4, pp. 288-291, 1968, Minamiba et al., Journal of Japan Society of Food Industry, Volume 32, No. 5, 331-337, 19
Although it is described in 1985 etc., these documents are studies on the effect of acetic acid bacteria on acid production, and no reference is made to the effect of these organic acids to promote cellulose production by cellulose-producing acetic acid bacteria. Absent.

It is an object of the present invention to develop a method for producing a cellulosic substance at a low cost by using a cellulosic acetic acid bacterium to improve the productivity.

[0007]

[Means for Solving the Problems] The present inventors have conducted various studies to achieve the above-mentioned object, and are microorganisms belonging to the genus Acetobacter and capable of producing a cellulosic substance (that is, cellulose-producing acetic acid). It was found that the productivity of the cellulosic substance is remarkably improved by culturing the bacterium) in a medium to which a specific organic acid is added, as compared with the conventional production method.

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

The microorganism used in the present invention may be any microorganism as long as it belongs to the genus Acetobacter and produces a cellulosic substance. For example, Acetobacter sp. BPR200
1 strain, Acetobacter xylinum
num) ATCC23768, Acetobacter xylinum ATCC23769, Acetobacter xylinum A
Examples include TCC10821.

The specific carboxylic acid or salt thereof used in the method of the present invention acts as a cellulose production promoting factor on the cellulose biosynthesis system of the microorganism to remarkably improve the productivity of cellulose. That is, as shown in Examples described later, the amount of cellulose accumulated in the medium reaches about 1.5 times to about 12 times that in the case where no carboxylic acid is added. In the present specification, the "carboxylic acid or its salt as a cellulose production promoting factor" refers to a carboxylic acid or salt that increases the amount of accumulated cellulose by about 1.5 times or more as compared with the case where the carboxylic acid or its salt is not added. . The carboxylic acid that can be used in the present invention may be either a saturated or unsaturated carboxylic acid, and examples of the saturated carboxylic acid include acetic acid, lactic acid, pyruvic acid,
Examples thereof include succinic acid, malic acid, gluconic acid and glucuronic acid, and examples of unsaturated carboxylic acids include fumaric acid, maleic acid, acrylic acid and benzoic acid. Although the number of carbon atoms and the number of carboxyl groups of these carboxylic acids are not particularly limited, they are preferably water-soluble. Preferred carboxylic acids are lactic acid, pyruvic acid and acetic acid. When the carboxylic acid is used as a salt, it is not particularly limited, but a salt of an alkali metal or an alkaline earth metal can be usually used.
These carboxylic acids or salts thereof may be used alone or in combination of two or more.
The addition amount of the carboxylic acid or its salt is not particularly limited, but is preferably 1 mmol to 200 mmol / L. In the present invention, the carboxylic acid or a salt thereof can significantly improve the cellulose productivity by only adding a trace amount or a small amount as a cellulose production promoting factor, rather than adding it to the medium as a main carbon source.

In the medium composition, as the carbon source, choucloth, glucose, fructose, mannitol, sorbitol, galactose, maltose, erythritol, gadnit, glycerin, ethylene glycol, ethanol and the like are used alone or in combination. Furthermore, starch hydrolysates containing these substances, citrus molasses, beet molasses, beet juice, sugar cane juice, fruit juices including citrus fruits, and the like can be used.

Ammonium sulfate as a nitrogen source,
Ammonium salts such as ammonium chloride and ammonium phosphate, nitrates, organic or inorganic nitrogen sources such as urea may be used, or Bact-Peptone, Ba.
ct-Soytone, Yeast-Extract,
Nitrogen-containing natural nutrient sources such as soybean concentrate 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 using an auxotrophic mutant strain that requires amino acids and the like for growth, it is necessary to supplement the required nutrients. 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 3 to 7, preferably 5
Control near.

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

The oxygen concentration 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 any of stationary culture, shaking culture and aeration-agitation culture may be used. It is also one of the features of the method of the present invention that it does not affect the cellulose productivity even if it is cultured under shaking or aeration stirring.

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

To remove impurities, washing with water, pressure dehydration, washing with dilute acid, washing with alkali, treatment with a polar organic solvent such as toluene and ethyl acetate, treatment with a bleaching agent such as sodium hypochlorite and hydrogen peroxide, lysozyme Treatment with microbial cell lysing enzyme, etc., treatment with surfactant such as sodium lauryl sulfate, deoxycholic acid, etc.
Impurities can be removed from the cellulose-like substance by subjecting it to heating treatment in the range of ° C alone or in combination.

The thus-obtained cellulosic material of 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 glucan such as β-1,3, β-1,2. In the case of heteropolysaccharides, constituents other than cellulose include mannose, fructose, galactose, xylose,
Hexoses such as arabinose, rhamnose, glucuronic acid,
Examples include pentose sugar and organic acids.

In addition, these polysaccharides may be a single substance, or two or more kinds of polysaccharides may be mixed by hydrogen bonding or the like.

[0023]

The present invention will be described in more detail by the following examples.

Culturing Conditions Cellulose-producing acetic acid bacteria were cultivated using the flask culture method and the jar-famenter 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, 750 ml Roux flask filled with 100 ml of basic medium of initial pH 5.0 composition, inoculated with 1 ml of cryopreserved bacterial solution of cellulose-producing acetic acid bacterium, 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 the contents were gauze-filtered under aseptic conditions to separate the cellulose pieces and the bacterial cells. Then, the cells were centrifuged at 10,000 rpm for 15 minutes to separate the medium components and the bacterial cells (+ microcellulose), and the bacterial cells (+ microcellulose) were washed once or twice with sterile physiological saline, and the centrifugation was repeated. It was A required amount of sterile physiological saline was added to the washed bacterial cells, and after stirring, this was used as a seed bacterial solution.

Next, 7.5 ml of the seed bacterial solution was added to the study medium 6
The cells were inoculated into a 300 ml spiral baffle flask containing 7.5 ml. Using a shaking culture machine, amplitude 2c
Main culture was carried out for 4 days while rotating and shaking under the conditions of m, rotation speed 180 rpm, and temperature 28 ° C.

The quantification of the produced bacterial cellulose was performed as follows. That is, the solid substance in each flask was washed with water to remove the medium components, and then treated in a 1% NaOH aqueous solution at 110 ° C. for 20 minutes to remove the bacterial cells, and further, the cellulose was washed until the washing liquid became nearly neutral. Was washed with water, vacuum dried at 80 ° C., and the dry weight was measured.

Jar-famentor culture method 750 ml Ro infused with 100 ml of the above basic medium
The ux flask was inoculated with 1 ml of a cryopreserved bacterial solution of cellulose-producing acetic acid bacteria, and static culture was carried out at 28 ° C. for 3 days in a low temperature incubator. After the stationary culture was completed, the Roux flask was shaken well, and the content was subjected to gauze filtration under aseptic conditions to separate cellulose pieces and cells. 7.5 ml of the obtained bacterial solution
Was inoculated into a 300-ml spiral baffle flask containing 67.5 ml of a basic medium, and seed culture was carried out for 3 days while shaking with a shaking incubator under the conditions of an amplitude of 2 cm, a rotation speed of 180 rpm, and a temperature of 28 ° C. .

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 contents were used as a seed bacterial solution and used in 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.

60 ml of the above seed bacterial solution was aseptically inoculated into a small jar fermentor (total volume of 1,000 ml) in which 540 ml of sterilized study medium was placed, and the mixture was incubated at 30 ° C. for 20 minutes.
Time or 30 hours, pH 1N NaOH or 1N
While controlling the pH to 5.0 with H ₂ SO ₄ , the stirring speed was initially 400 rpm, and the dissolved oxygen amount (D
Main culture was performed while automatically controlling the rotation speed so that (O) was within 3.0 to 21.0%.

After the completion of the culture, the solid matter in the jar fermenter was collected and washed with water to remove the medium components, and then 1% Na
The cells were removed by treatment in an aqueous OH solution at 110 ° C. for 20 minutes. Further, the produced cellulose was washed with water until the washing liquid became nearly neutral, and then dried under vacuum at 80 ° C. for 12 hours to measure the dry weight.

Example 1 Lactic acid in various organic nitrogen sources
Addition effect Acetobacter species BPR2001 strain as a cellulose-producing acetic acid bacterium, and the following composition containing the following different organic nitrogen sources as the study medium: fructose 40 g / L, KH ₂ PO ₄ 1.0 g / L, MgSO ₄ 0. 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 soybean concentrate (acid hydrolyzed concentrated solution of soybean protein), milk acid 0 or 1.4 ml / L, initial pH of 5.0, the main culture is performed for 2 days or 4 according to the above flask culture method. The test was carried out for a day to evaluate the amount of accumulated cellulose in the presence or absence of lactic acid.

The results are shown in Table 1.

[0034]

[Table 1]

As can be seen from the results in Table 1, when lactic acid was added to the medium, the amount of accumulated cellulose was about 5 times to about 12 in any organic N source as compared with the case where no lactic acid was added.
Doubled. The amount of accumulated cellulose due to the organic N source used was Bact-Peptone <Bact after 4 days of culture.
-Soytone <Yeast-Extract <bean concentrated.

Example 2 Effect of addition of carboxylic acid other than lactic acid
Fruit Bact-Soytone as an organic nitrogen source, acetic acid and pyruvic acid in addition to lactic acid as a carboxylic acid 10 m
The culture was carried out in the same manner as in Example 1 except that the addition of mol / L or no addition was performed, and the effect of addition of a carboxylic acid 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 even when acetic acid and pyruvic acid were used as carboxylic acids, the amount of accumulated cellulose increased by about 2 to 6 times as compared with the case of no addition. When evaluated after 4 days of culture, the degree of cellulose accumulation increased in the order of acetic acid <pyruvic acid <lactic acid.

Example 3 Effect on Cellulose Accumulation
Effect of Rubonic Acid Addition Bact-Soytone as an organic nitrogen source, and lactic acid 0.1 ml / L, 0.5 ml / L, 1.0 ml / L
L, 5.0 ml / L, 10.0 ml / L, 15.0 ml
The amount of cellulose accumulated at each addition amount was evaluated by culturing for 4 days in the same manner as in Example 1 except that the addition amount of / L was used.

The results are shown in Table 3.

[0042]

[Table 3]

The results in Table 3 show that the amount of lactic acid added was 0.1 ml / L.
From 1 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 Bacteria affecting 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 carried out 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.

The results are shown in Table 4.

[0046]

[Table 4]

From the results shown in Table 4, although the substantial amount of accumulated cellulose differs among the three strains used, the addition of lactic acid increased the accumulated amount of cellulose to about 2.5 times to about 7 times in all the strains. Increased. In particular, Acetobacter
Species BPR2001 strain and Acetobacter xylinum ATCC 23769 showed high cellulose production.

Example 5 Cellulose-Producing Acetobacter Di
Afermentor culture Acetobacter species BPR2001 strain as a cellulose-producing acetic acid bacterium, 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 ) ₂ SO ₄ 3 g / L, Bact-Soytone 5 g / L, Lactic acid 0 or 1.4 ml / L, Initial pH 5.0 The main culture was performed for 20 hours or 30 hours according to the above jarfamenter culture method. Over time, the amount of cellulose accumulated in the presence or absence of lactic acid was evaluated.

The results are shown in Table 5.

[0050]

[Table 5]

The results shown in Table 5 show that when the fermentation conditions such as pH and dissolved oxygen content are controlled using a jar fermenter, the production of cellulose is significantly improved even when lactic acid is not added, but when lactic acid is added, no cellulose is added. It is shown that the amount of accumulated cellulose is further improved by about 1.5 times to about 2.5 times that in the case.

[0052]

INDUSTRIAL APPLICABILITY 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 by adding a trace amount or a small amount of carboxylic acid or its salt to the medium. This shows that this method can produce cellulose efficiently and cheaply.

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[Procedure amendment]

[Submission date] August 18, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction content]

The microorganism used in the present invention may be any microorganism as long as it belongs to the genus Acetobacter and produces a cellulosic substance. For example, Acetobacter sp. BPR200
1 strain (FERM P-13466) , Acetobacter
Xylinum (Acetobacter xylinum) ATCC2376
8, Acetobacter xylinum ATCC 23769,
Acetobacter xylinum ATCC 10821 and the like.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0010

[Correction method] Change

[Correction content]

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

Claims (4)

[Claims] 1. A microorganism belonging to the genus Acetobacter and capable of producing a cellulosic substance is cultivated in a medium to which a carboxylic acid or a salt thereof as a cellulose production promoting factor is added to produce a cellulose and a cellulosic substance in the medium. A method for producing cellulose, which comprises 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 in a medium in an amount of 1 to 2.
3. The method according to claim 1, wherein the amount added is 00 mmol / L.
The method described. 4. The method according to any one of claims 1 to 3, wherein the culture is static, shaking, or aeration-agitation culture.