JPH0568236B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for producing cellulosic substances produced by microorganisms that belong to the genus Acetobacter and have the ability to produce cellulosic substances. This cellulosic substance is edible and used in the food field, and has excellent aqueous dispersibility, so it can be used to maintain the viscosity of foods, cosmetics, or paints, strengthen food raw materials, retain moisture, improve food stability, etc. It has industrial utility as a low-calorie additive or emulsion stabilization aid. Furthermore, the disaggregated cellulosic material has various industrial uses as a reinforcing material for polymers, particularly water-based polymers, based on the structural and physical characteristics of microfibrils. Since such disaggregated products exhibit high tensile modulus, materials obtained by solidifying the cellulosic disaggregated products into paper-like or solid forms are expected to have excellent mechanical properties based on the structural characteristics of microfibrils, and are suitable for various industrial applications. It has applications as a material. (Problems to be Solved by the Present Invention) The problems to be solved by the present invention are that in static fermentation, the production of cellulosic substances is rapid at the initial stage of culture, but as the thickness increases, the production slows down. Productivity was poor. However, the object of the present invention is to develop a method for producing cellulosic substances stably, rapidly, efficiently, and inexpensively by continuously or intermittently adding a medium. (Natural technology) A method of producing cellulose using a microorganism belonging to the genus Acetobacter and having the ability to produce cellulose has been known. The culture method for producing cellulose is mainly a static culture method, and a culture method adding a liquid medium has not been studied. In the static culture method, cellulose production slows down as the cellulose membrane becomes thicker, and long-term culture is required to produce thick cellulose. Furthermore, it has been difficult to produce cellulosic materials with different hardnesses or to produce multilayered cellulosic materials. (Means for Solving the Problems) The present inventors have developed a method of continuously or intermittently adding a liquid medium or a liquid medium containing microorganisms capable of producing cellulose, compared to the conventional static culture method. It has been found that the productivity of cellulosic substances can be significantly improved by culturing the cellulosic substances. Also,
We learned that by changing the addition rate, it is possible to produce cellulosic materials with different hardness. Furthermore, it was discovered that a multilayered cellulosic material can be produced by adding a medium intermittently. The present invention was completed based on this knowledge. That is, the microorganism used in the present invention belongs to Acetobacter and may be any microorganism that produces cellulosic substances. One example is Acetobacter aceti subspice xylinum.
xylinum) ATCC10821. As the carbon source, sucrose glucose, fructose, mannitol, sorbitol, galactose, maltose, erythritol, cadnite, glycerin, ethylene glycol, ethanol, acetic acid, etc. are used alone or in combination. Furthermore, starch hydrolysates containing these substances, citrus molasses, beet molasses, beet juice, sugarcane juice, fruit juices including citrus fruits, etc. can be used. As the nitrogen source, organic or inorganic nitrogen sources such as ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, nitrates, urea, and peptone are used. As organic micronutrients, amino acids, vitamins, fatty acids, nucleic acids, peptones containing these, casamino acids, yeast extracts, etc. are used, and in addition to these, 2,7,9,-tricarboxy-1H hyolo[2, 3-5]-quinoline-
Addition of 4,5-dione also has an effect. When using an auxotrophic mutant strain that requires amino acids and the like for growth, it is necessary to supplement the required nutrients. Inorganic salts include phosphates,
Magnesium salts, calcium salts, iron salts, manganese salts, cobalt salts, molybdates, red blood salts, chelated metals, etc. are used. Any method for adding the medium used in the present invention can be used. For example, a dripping method using a tube or a spraying method using a shower nozzle can be used. The addition rate may be any rate as long as it is less than or equal to the maximum production rate. Air has a constant flow rate of 1/100 to 2/1 VVm for 1 to 30 days.
Supply to the static culture tank within the range of . The oxygen concentration supplied to the culture tank may be 5 to 100%, preferably 20 to 40%. The pH of the culture is controlled within the range of 3 to 6. Desirably, the pH may be 3 to 5. All organic and inorganic acids and alkalis can be used for PH control. The culture is carried out at a temperature of 10 to 40°C, preferably 25 to 35°C. When cultured using the above method, a cellulosic substance produced in the form of a plate is soft when the culture addition rate is fast, and a hard plate-like cellulosic substance is produced when the culture addition rate is slow. Also, by adding culture medium every 6 to 72 hours, a thin, multilayered cellulosic material is produced. The cellulosic material produced by the method of the present invention is subjected to a treatment to remove substances other than the cellulosic material, including bacterial cells contained in the material. To remove impurities, water washing, pressure dehydration, dilute acid washing, alkaline washing, treatment with polar organic solvents such as toluene and ethyl acetate, treatment with bleaching agents such as sodium hypochlorite and hydrogen peroxide, and treatment with lysozyme etc. Impurities are removed from cellulosic materials by treatment with bacterial cell lytic enzymes, treatment with surfactants such as sodium lauryl sulfate and deoxycholic acid, and heating washing at temperatures ranging from room temperature to 200℃, either alone or in combination. I can do it. The cellulosic substance obtained in this way as used in the present invention refers to the following. The cellulosic substances of the present invention include those containing cellulose and heteropolysaccharides having cellulose as a main chain, and those containing glucans such as β-1,3 and β-1,2. In the case of heteropolymer, the constituents other than cellulose are mannose, fructose, galactose, xylose, arabinose, rhamnose,
These include hexoses, pentoses, and organic acids such as glucuronic acid. Note that these polysaccharides may be a single substance, or two or more types of polysaccharides may be mixed together due to hydrogen bonding or the like. Example 1 Seuculose 5%, monopotassium phosphate 0.3%,
Add 400 ml of a medium containing 0.05% magnesium sulfate (heptahydrate), 0.8% casamino acid (manufactured by Difco), and a pH of 5.
The mixture was poured into a 500ml Sakaguchi flask and sterilized at 120°C for 30 minutes. Acetobacter aceti subspice xylinum ATCC10821 grown for 6 days at 30°C on a slanted agar medium with the above composition and 2% agar added.
was inoculated into the above-mentioned sterilized medium and cultured with shaking at 30°C for 2 days to prepare a seed mother. Yeast extract (Difco
Medium 135 with 0.50% of
ml into a glass shear tray with an outer diameter of 24 cm, and then add 15 ml of seed mother as well and ventilate to 1/10 VV.
After culturing at 30°C for 24 hours, the above medium with 0.5% yeast extract was added at 10 μ/cm ² hr70 μ.
It was added from the top using a tube at a rate of /cm ² ·hr.
The culture was terminated when the amount of medium added reached 400 ml. After measuring the elasticity of the produced cellulosic material, it was thoroughly washed with a wire cloth repeatedly. then 105
It was dried at ℃ until it reached a constant weight and measured. the result,
The hardness of the produced cellulosic material is 850 g/cm ² at a medium addition acceleration of 10 μ/cm ² hr, which is 70 μ
/cm ² ·hr, the elasticity was 39 g/cm ² .

[Table] The production rate was 80 ml/dl·day when the medium addition rate was 10 μ/cm ² ·hr, and 20 ml/dl·day when the medium addition rate was 70 μ/cm ² ·hr. In addition, 500 ml of the same medium as above was aseptically placed in a glass shear tray with an outer diameter of 24 cm, and 50 ml of seed mother was also placed in it, and the mixture was heated at 30°C with ventilation of 1/10VVm.
Cultured for 1 day. The cellulosic material produced is elastic
It was in the form of a plate weighing 95 g/cm ² and having a thickness of 3 mm. This cellulosic material was thoroughly washed repeatedly in a washing machine, dried at 105° C. until it reached a constant weight, and then measured. The result was 108g. Example 2 Seuucrose 5%, potassium phosphate 0.3%,
Add 400 ml of a medium containing 0.05% magnesium sulfate (heptahydrate), 0.8% casamino acid (manufactured by Difco), and a pH of 5.
The mixture was poured into a 500ml cap flask and sterilized at 120°C for 30 minutes. Acetopacter aceti subspice xylinum ATCC10821 grown for 6 days at 30°C on a slanted agar medium with the above composition and 2% agar added.
was inoculated into the above-mentioned sterilized medium and cultured with shaking at 30°C for 2 days to prepare a seed mother. Add yeast extract (Difco
Add 0.50% (manufactured by S.A.) and adjust the pH to 4.
Pour 135 ml into a glass shear tray with an outer diameter of 24 cm aseptically, and add 15 ml of seed mother as well and ventilate to 1/10.
After culturing at 30°C for 24 hours at VVm, 0.5% yeast extract was added to the above medium and cultured while uniformly adding it to the top surface using a spray nozzle at a rate of 10 μ/cm ² hr. The culture was terminated when the amount of medium added reached 350 ml. After measuring the elasticity of the produced cellulosic material, it was washed thoroughly by repeated washing. Thereafter, it was dried at 105°C until it reached a constant weight and measured. As a result, the weight was 2.10g.
The hardness of the produced cellulosic material was measured using a rheometer and was found to have an elasticity of 840 g/cm ² . Example 3 Seuculose 5%, monopotassium phosphate 0.3%
Add 400 ml of a medium containing 0.05% magnesium sulfate (heptahydrate), 0.8% casamino acid (manufactured by Difco), and a pH of 5.
The mixture was poured into a 500ml Sakaguchi flask and sterilized at 120°C for 30 minutes. Acetopacter aceti subspice xylinum grown for 6 days at 30°C on a slanted agar medium with the above composition and 2% agar added.
Inoculate ATCCC10821 into the above sterilized medium for 30 minutes.
It was cultured with shaking at ℃ for 2 days and used as a seed mother. A medium prepared by adding 0.05% phytic acid (manufactured by Ajinomoto) to the above medium was adjusted to pH 4, sterilized at 120°C for 30 minutes, and then placed in a glass shear tray with an outer diameter of 24 cm, 135 ml, and placed in a 15 ml seed mother with ventilation 1. After culturing at 30°C for 24 hours at /10VVm, 100ml of the above medium was added from above at 24 hour intervals and cultured repeatedly for 10 days. The cellulosic material produced is 1mm to 1.5mm
It became a thick plate shape and produced 11 sheets. The generated cellulosic material is thoroughly washed in the washing machine repeatedly.105
It was dried at ℃ until it reached a constant weight and measured. the result,
The amount of cellulosic material produced was 3.40 g. Pour 1035 ml of the same medium as above into a glass shear tray with an outer diameter of 24 cm, and add 115 ml of seed mother.
The cells were cultured at 30°C for 11 days. The cellulosic material produced was in the form of a plate with a thickness of 5 mm. The generated cellulosic material is washed thoroughly by repeated washings.
It was dried at 105°C until it reached a constant weight and measured. As a result, the amount produced was 2.28g.

[Table] Example 4 Seuculose 5%, monopotassium phosphate 0.3%,
Add 400 ml of a medium containing magnesium sulfate (0.05% heptahydrate, 0.8% casamino acid (manufactured by Difeo), pH 5).
The mixture was poured into a 500ml Sakaguchi flask and sterilized at 120°C for 30 minutes. Acetopacter aceti subspice xylinum ATCC10821 grown for 6 days at 30°C on a slanted agar medium with the above composition and 2% agar added.
was inoculated into the above-mentioned sterilized medium and cultured with shaking at 30°C for 2 days to prepare a seed mother. Enzyme extract (Difco
Co., Ltd.) 0.50% was added to adjust the pH to 4. Medium 50ml
was aseptically placed in a glass beaker with an outer diameter of 12 cm, and 5 ml of seeds were also added thereto, and cultured at 30°C for 24 hours with ventilation at 1/10VVm. Thereafter, 50 ml of the above medium mixed with 10% (V/V) seed mother and stored refrigerated at 5°C was added aseptically from above every 24 hours, and cultured for 10 days. The cellulosic material produced was in the form of plates with a thickness of 1.5 to 2.0 mm, and 11 plates were produced. The produced cellulosic material was thoroughly washed repeatedly in a washing machine, dried at 105°C until it reached a constant weight, and then measured. As a result, 11 thin plates with cellulosic properties having an average weight of 93 mg were obtained. Example 5 Seuculose 5%, monopotassium phosphate 0.3%,
Add 400 ml of a medium containing 0.05% magnesium sulfate (heptahydrate), 0.8% casamino acid (manufactured by Difeo), and a pH of 5.
The mixture was poured into a 500ml Sakaguchi flask and sterilized at 120°C for 30 minutes. Acetobacter aceti subspice xylinum ATCC10821 grown for 6 days at 30°C on a slanted agar medium with the above composition and 2% agar added.
was inoculated into the above-mentioned sterilized medium and cultured with shaking at 30°C for 2 days to prepare a seed mother. Yeast extract (Difco
Add 0.50% (manufactured by S.A.) and adjust the pH to 4.
Pour 135 ml aseptically into a glass shear tray with an outer diameter of 24 cm, add 15 ml of seeds, and ventilate 1/2.
After culturing at 30℃ for 24 hours at 10VVm, the above medium with 0.5% yeast extract was added at 10μ/cm ² hr.
It was added in a mist form from the top at a speed of 2.7 liters and cultured. As a result, the thickness of the plate-like cellulosic material produced was 6 cm. The cellulosic material was washed thoroughly by repeated washing, dried at 105°C until it reached a constant weight, and then measured. The result was 51.3g. Example 6 Seuculose 5%, monopotassium phosphate 0.3%,
Add 400 ml of a medium containing 0.05% magnesium sulfate (heptahydrate), 0.8% casamino acid (manufactured by Difeo), and a pH of 5.
The mixture was poured into a 500ml Sakaguchi flask and sterilized at 120°C for 30 minutes. Acetobacter aceti subspice xylinum ATCC10821 grown for 6 days at 30°C on a slanted agar medium with the above composition and 2% agar added.
was inoculated into the above-mentioned sterilized medium and cultured with shaking at 30°C for 2 days to prepare a seed mother. Yeast extract (Difco
0.50% (manufactured by Seishin Corporation) was added and the pH was adjusted to 4. medium
Aseptically put 50ml into a glass beaker with an outer diameter of 12cm, add 5ml of seeds, and vent to 1/10.
The cells were cultured in VVm at 30°C for 18 hours. The above medium was then pumped for 30 h at 18 h intervals using a tube pump.
ml was added 10 times and culture was continued. As a result, the cellulosic material took on a multilayered shape. The produced cellulosic material was peeled off one by one and thoroughly washed repeatedly, placed on a glass flat plate, dried at 50°C until it reached a constant weight, and measured. As a result, the obtained cellulosic material had a thickness of 2μ and a weight of 42mg.
I got 11 pieces.

Claims (1)

[Claims] 1. Inoculate a microorganism that belongs to the genus Acetobacter and has the ability to produce cellulosic substances into a medium, and while culturing it by static culture, add the medium or the microorganism that belongs to the genus Acetobacter and has the ability to produce cellulosic substances and the medium on the surface. A method for producing a cellulosic material, which comprises adding a mixed liquid continuously or intermittently to produce and accumulate a thick or thin sheet-like cellulosic material, and collecting this material.