JPH09183803A - Spreading agent containing bacteria cellulose - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a spreading agent having a strong adhesive force to the surface of a crop, etc., being mixed and fixed on the surface and having excellent biodegradability without a problem of environmental safety and useful for also a cosmetic, etc., containing a bacteria cellulose. SOLUTION: This spreading agent contains preferably disaggregated bacteria cellulose, e.g. Acetobacter xylinum subsp. sucrofermentans (FERM P-13466). The disaggregation is performed by using mechanical shearing force, ultrasonic wave, high-pressure treatment, an acid hydrolysis, an enzyme hydrolysis or a bleaching agent, or their combination and preferably containing the bacteria cellulose obtained by an aeration stirring cultivation and having >=1.6×10<4> weight-average polymerization degree in terms of polystyrene.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a spreading agent containing a cellulosic substance (hereinafter referred to as “bacterial cellulose” or “BC”) which can be produced by culturing a cellulosic bacterium. .

[0002]

2. Description of the Related Art Generally, when a fertilizer and various agricultural chemicals such as plant hormones, insecticides, fungicides and herbicides are applied to agricultural crops and ornamental plants, a spreading agent is one of the auxiliary agents. It is used. Further, in many cases, various cosmetics also contain a spreading agent. The spreading agent has an effect of sufficiently adhering the drug to the surface of a crop or the like to which these various compositions are applied, and maintaining the effect for a long period of time, that is, the adhering drug gradually spreads and can exert its effect. It has the effect of Heretofore, carbohydrates such as monosaccharides and oligosaccharides, and cationic or anionic surfactants have been mainly used as the spreading agent. However, saccharides do not have sufficient adhesiveness, while surfactants have poor degradability,
Especially when used in a large amount, environmental safety problems such as fish toxicity and phytotoxicity have occurred. On the other hand, since BC (bacterial cellulose) is edible and tasteless and odorless, it is used in the food field, and because it has excellent water-based dispersibility and biodegradability, it retains the viscosity of foods, cosmetics, paints, etc. It has industrial value as a material fortification, water retention, food stability improvement, low calorie additive or emulsion stabilization aid. BC is characterized in that the fibril fragment width is as small as about two digits compared to cellulose produced from wood pulp or the like. Accordingly, the dissociated product of BC has various industrial uses as a reinforcing agent for polymers, especially aqueous polymers, based on such structural and physical characteristics of fibrils. A material obtained by solidifying such a cellulosic disagglomerated product into a paper or solid form exhibits a high tensile modulus, so that excellent mechanical properties based on the structural characteristics of fibrils are expected, and there are applications as various industrial materials. .

[0003]

Therefore, the present invention has a large adhesive force to the surface of crops, etc., and prevents the scattering and diffusion of, for example, microbial pesticides and the like fixed and mixed there, and the efficacy of the drug for a long period of time. While it can be sustained, it is excellent in biodegradability, there is no environmental safety problem such as phytotoxicity, and when used in cosmetics, etc., it has a good film-forming ability, giving a sufficient moist feeling and smooth feeling, It is intended to provide a spreader having excellent adhesion to the skin, water resistance and the like.

[0004]

That is, the present invention relates to a spreading agent containing bacterial cellulose. The amount of bacterial cellulose contained in the spreading agent can be appropriately adjusted by those skilled in the art according to the purpose and the like, and is usually 0.01
It is in the range of wt% to 3.0 wt%. In addition to the bacterial cellulose, the spreading agent of the present invention may contain a component that has been conventionally used as a spreading agent or a component such as a culture medium of a cellulose-producing bacterium. The spreader of the present invention, when used, fertilizer, as well as plant hormones,
Insecticides, fungicides and herbicides, and various agricultural chemicals such as microbial pesticides having the ability to suppress plant diseases such as Bacillus subtilis, or other agents contained in cosmetics and other components such as various microorganisms, and then applied, in addition to these It can also be applied by a route different from the various components of. Therefore, the present invention also relates to various compositions such as agricultural chemicals containing the spreader as a kind of auxiliary agent. The composition may be in a liquid state such as an aqueous suspension, or in a solid form such as a dry powder, a dry granule and a sheet.
These solids can be prepared by freeze-drying, spray-drying, squeezing, air-drying or the like of the aqueous suspension or the like using a conventionally known device. In particular, the composition of the present invention obtained by immobilizing various microorganisms on the inside and surface of a bacterial cellulose aggregate is obtained by stirring culture of the microorganism to be immobilized together with cellulose-producing bacteria, or by aeration stirring culture. It can be obtained by adding the microorganism (or its spores) to a suspension of BC, stirring the mixture, and then freeze-drying the culture solution or suspension.

Accordingly, the present invention further provides a method for immobilizing the microorganisms on the bacterial cellulose, which comprises stirring the suspension containing the bacterial cellulose and the microorganism obtained by the aeration-agitation culture, and the aeration-agitation culture. The present invention also relates to a microbial immobilizing agent composed of the above-mentioned bacterial cellulose and a microorganism immobilized by the microbial immobilizing agent. Those skilled in the art can appropriately select the conditions for preparing a bacterial cellulose aggregate containing various microorganisms such as the concentration of BC in the suspension and the number of added microorganisms in consideration of the purpose of use, the type of microorganisms and the like. The suspension may also contain other components such as various salts. The spreading agent and composition of the present invention can be sprayed and sprayed on, for example, the skin of human bodies or the leaves and stems of crops and the surface of soil, or can be applied using a brush or the like. Furthermore, the composition of the present invention can be inoculated into the soil by mixing with the soil. The spray operation itself can be performed using conventional spray equipment. That is, as a specific method of spraying, for example,
There are a pressure nozzle, a two-fluid nozzle, and the like, but the nozzle type has coarser particles than the two-fluid nozzle type. The method of spraying can be appropriately selected according to the purpose.

[0006] In the method of the present invention, the cellulosic material may be subjected to a defibration treatment. The disintegration phenomenon of bacterial cellulose is considered to be a phenomenon in which a stress generated inside the cellulose due to a mechanical external force or the like deforms or breaks it. Accordingly, the disaggregation treatment of bacterial cellulose can be performed by applying a mechanical external force to bacterial cellulose. Further, the disaggregation treatment can be carried out also by acid hydrolysis, enzymatic hydrolysis and bleach. The mechanical external force referred to herein includes, for example, stresses such as tension, bending, compression, torsion, impact, and shearing, but generally includes compression, impact, and shearing stress. When these mechanical external forces are actually applied to the bacterial cellulose, it can be achieved by using, for example, an ultrasonic oscillator such as a mixer, a polytron or a self-excited ultrasonic pulverizer.

In the disaggregation treatment by the mixer, the mechanical external force is mainly composed of the impact force caused by the collision between the stirring blade and the bacterial cellulose and the shear force generated by the displacement phenomenon due to the speed difference of the medium. In the disaggregation process using Polytron, the mechanical external force is the compressive force of bacterial cellulose sandwiched between the external teeth and the internal teeth, the impact force of the collision of high-speed rotating teeth with bacterial cellulose, the static external teeth and the high speed of stationary external teeth. The shear stress generated in the medium existing in the gap between the rotating internal teeth is mainly involved. In the disintegration by the ultrasonic pulverizer, the mechanical external force is mainly cavitation (cavity phenomenon) continuously generated in the medium due to the oscillation of the ultrasonic oscillating unit, and significant shear stress locally generated. The defibration treatment of the present invention can be performed by any method other than the above-mentioned specific examples as long as a certain load (mechanical external force) can be applied to the bacterial cellulose. Other disaggregation treatment conditions can be appropriately selected by those skilled in the art. Further, as described in Japanese Patent Application No. 7-160173, after the disaggregation treatment, the BC disintegrated material itself can be sieved with a screen having a predetermined opening for the purpose of adjusting the particle size. The porosity and particle size of the obtained particles can be adjusted by changing the concentration of the BC (disintegrated product) in the aqueous suspension and the size of the droplet during spraying.

Although the defibration treatment has been described above, the defibration treatment according to the present invention is carried out after stirring and culturing the cellulose-producing bacteria.
It is obvious to those skilled in the art that the present invention is not limited to an independent secondary operation performed on bacterial cellulose separated and purified from a culture solution. That is, the stirring operation has an action of disintegrating bacterial cellulose as described later, and in the stirring culture employed in the present invention, it is sufficiently possible to disintegrate the bacterial cellulose even by the stirring action for the purpose of culture. Because there is. Furthermore, bacterial cellulose obtained by stirring culture is separated,
The same can be said for the operations of washing, purifying and transporting, and it should be noted that additional disaggregation treatment in these operations is also included in the disaggregation treatment of the present invention.

[0009] Cellulose-producing bacteria used for the production of bacterial cellulose in the present invention include, for example, BPR2
Acetobacillus xylinum subspecies sucrofermentans ( Acetobac)
ter xylinum subsp. sucrofermentans ), Acetobacter xylinum ATCC23
768, Acetobacter xylinum ATCC 2376
9. Acetobacter pasteurianus ( A. pasteurianu)
s ) ATCC 10245, Acetobacter xylinum ATCC 14851, Acetobacter xylinum AT
CC11142 and Acetobacter xylinum ATC
Acetobacter such as C10821 (genus Acetobacter), in addition to genus Agrobacterium, genus Rhizobium, genus Sarsina, genus Pseudomonas, genus Achromobacter, genus Alcaligenes, genus Aerobactor, genus Azotobacter, and genus NTG ( Various mutant strains created by mutation treatment by a known method using nitrosoguanidine) and the like. In addition, BPR2001
The strain was deposited on February 24, 1993 at the Patented Microorganisms Depositary Center, National Institute of Bioscience and Human-Technology, Ministry of International Trade and Industry (Accession No. FERM P-13466), and then 199
Deposits based on the Budapest Treaty on the International Recognition of Deposits on Patent Proceedings dated February 7, 2004 (accession number FERM B
P-4545). Chemical mutation treatment methods using a mutagen such as NTG include, for example, Bio Factor
s, Vol. 1, p. 297-302 (1988) and J. Gen. Microbiol,
Vol. 135, p. 2917-2929 (1989). Therefore, those skilled in the art can obtain the mutant strain used in the present invention based on these known methods. The mutant strain used in the present invention can also be obtained by other mutation methods, for example, irradiation.

[0010] Among the cellulosic bacteria produced by the above-mentioned method, the weight average degree of polymerization in terms of polystyrene is 1.6 × 10 ⁴ or more by culturing with aeration and stirring.
Bacterial cellulose having a high degree of polymerization of preferably 1.7 × 10 ⁴ or more is produced, or is cultured by standing, so that the weight average degree of polymerization in terms of polystyrene is 2.0.
A strain producing bacterial cellulose having a high degree of polymerization of × 10 ⁴ or more is preferred. Among the bacteria producing bacterial cellulose having a high degree of polymerization that can be used in the present invention, BPR300
1A was deposited on June 12, 1995 with the Patented Microorganisms Depositary Center, National Institute of Bioscience and Human-Technology, Ministry of International Trade and Industry, and assigned accession number FERM P-14982. In general, it is known that the higher the degree of polymerization of a polymer, the higher the strength and elastic modulus of the polymer material. Similarly, in the case of bacterial cellulose, a film formed using bacterial cellulose having a high degree of polymerization as a raw material has a higher strength and elastic modulus than a film formed using bacterial cellulose having a relatively low degree of polymerization. Is high. Therefore, in the present invention, when it is desired to form a material having high strength and elastic modulus, the use of bacterial cellulose having a high degree of polymerization as described above provides a higher effect.

The weight-average degree of polymerization of various celluloses such as BC in the present invention can be measured by using GPC with RI as a detector.
It measures as follows using a system (Tosoh HLC-8020). Various cellulose samples were treated with fuming nitric acid-phosphorus pentoxide solution by WJ Alexander, RL Mitchell, Anal.
It is nitrated by the method of ytical chemistry 21, 12, 1497-1500 (1949). A nitrated cotton linter is used as a control. The cellulose nitrate is dissolved in THF (Wako Pure Chemicals first grade) at a concentration of 0.05%, and then filtered through a 1.0 μm pore size filter. THF is also used as the eluent for GPC. Flow rate is 0.5
ml / min, the pressure is 10-13 kgf / cm ² , and the sample injection volume is 100 μl. Column is TSKgel GMH
-Using HR (S) (7.5 ID x 300 mm x 2) and guard column (HHR (S)) (Tosoh Co., Ltd.) 35
Measure in ° C. The standard polystyrene (Tosoh) is used to calculate the molecular weight, and the relative molecular weight in terms of polystyrene is calculated. Elution time (t) and logarithm of molecular weight (log M) using polystyrene having a molecular weight of 2 × 10 ⁷ to 2630
For the cubic equation: (log M = At ³ + Bt ² + Ct
+ D) is approximated to create a standard curve.
Molecular weight: Tosoh's data processing machine (SC-8020)
The weight average molecular weight is calculated by a program (ver. From these molecular weight values, the weight average polymerization degree is calculated in consideration of the degree of substitution after nitration.

In the composition of the medium used for the culture, sucrose, glucose, fructose, mannitol, sorbitol, galactose, maltose, erythritol, glycerin, ethylene glycol, ethanol or the like may be used alone or in combination. You can Furthermore, starch hydrolyzate containing these, citrus molasses, beet molasses, beet juice, sugarcane juice,
Juices such as citrus fruits can also be used in addition to sucrose. Further, as the nitrogen source, an organic or inorganic nitrogen source such as ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, nitrate, urea and the like, or Bacto-Pepto can be used.
ne, Bacto-Soytone, Yeast-Ex
A nitrogen-containing natural nutrient source such as tract or soybean hydrolyzate may be used. Amino acids, vitamins, fatty acids, nucleic acids, 2,7,9-tricarboxy-1 as organic micronutrients
H-pyrrolo [2,3,5] -quinoline-4,5-dione,
Sulfurous acid pulp waste liquid, lignin sulfonic acid, etc. 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. Furthermore, inositol, phytic acid, pyrroloquinoline quinone (PQQ) (Japanese Patent Publication No. 5-1718; Mitsuo Takai, Paper and Paper Cooperative Journal, Vol. 42, No. 3, 237-2).
44), carboxylic acid or a salt thereof (Japanese Patent Application No. 5-1914).
No. 67), invertase (Japanese Patent Application No. 5-331491)
) And methionine (Japanese Patent Application No. 5-335564) can be added to the medium as appropriate. For example, when acetic acid bacterium is used as a production bacterium, the pH of the culture is controlled at 3 to 7, preferably around 5. The culture temperature is 10 to 40 ° C, preferably 25 to 3
Perform at 5 ° C. The oxygen concentration supplied to the culture device is 1 to
100%, preferably 21 to 80%. Those skilled in the art can appropriately select the composition ratio of each component in the medium, the inoculation of the cells into the medium, and the like, depending on the culture method. Bacterial cellulose has been conventionally known as a culture method for culturing microorganisms, that is, stationary, shaking or aeration and stirring culture, and also known as a culture operation method, so-called batch fermentation method, fed-batch batch fermentation method, It can be produced by a repeated batch fermentation method, a continuous fermentation method, or the like.
In addition, as the stirring means, for example, an impeller (a stirring blade), an air lift fermenter, a pump-driven circulation of a fermentation broth, a combination of these means, and the like are used.

[0014] The stirring culture is a culture method in which a culture solution is stirred while stirring, and the structure of bacterial cellulose, for example, the crystallization index is reduced due to the stirring action during the stirring culture. Changes to increase. As the stirring means, for example, an impeller, an airlift fermenter, a pump-driven circulation of fermentation broth, a combination of these means, and the like can be used. Furthermore, a method for producing a cellulosic substance by circulating a culture solution containing bacterial cells between a culture apparatus and a separation apparatus described in Japanese Patent Application No. 6-192287 in the name of the present applicant. And separating the cellulosic substance, which is a product, from the cells and the culture solution, and culturing the cellulosic bacteria described in Japanese Patent Application No. 6-192288 in the name of the present applicant. A method for producing a cellulosic material by continuously extracting a culture solution from a culture system and supplying a new culture solution having substantially the same volume as the amount withdrawn during the culture period. The production method is characterized in that the concentration of the cellulosic substance in the culture solution is kept low.

As a tank for performing the stirring culture,
For example, stirring tanks such as jar fermenters and tanks,
In addition, baffled flasks, Sakaguchi flasks, and air-lift type stirring tanks can be used, but are not limited thereto. In the stirring culture according to the present invention, at the same time as stirring,
Ventilation may be performed if necessary. The aeration referred to here may be any of an oxygen-containing gas such as air and an oxygen-free gas such as argon and nitrogen. These gases may be used by those skilled in the art according to the conditions of the culture system. Will be selected as appropriate. For example, in the case of anaerobic microorganisms, if an inert gas is ventilated, the culture solution can be stirred by the bubbles. In the case of aerobic microorganisms, the culture solution can be agitated while supplying oxygen necessary for the growth of the microorganisms by aerating an oxygen-containing gas.

The bacterial cellulose obtained by the stirring culture is separated from the culture solution by a centrifugation method, a filtration method, or the like.
Bacterial cellulose may be collected together with the cells,
Further, a treatment for removing impurities other than the cellulosic substance including bacterial cells contained in the substance can be performed. To remove impurities, washing with water, pressure dehydration, washing with diluted acid, washing with alkali, treatment with bleach such as sodium hypochlorite and hydrogen peroxide, treatment with cell lysing enzymes such as lysozyme, sodium lauryl sulfate, Impurities can be almost completely removed from the cellulosic material by performing a treatment with a surfactant such as deoxycholic acid, washing with heat in the range of room temperature to 200 ° C., alone or in combination. The cellulosic material thus obtained in 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 the heteropolysaccharide, components other than cellulose include hexoses such as mannose, fructose, galactose, xylose, arabinose, rhamnose, and glucuronic acid, pentoses, and organic acids. These polysaccharides may be a single substance, or two or more polysaccharides may be mixed by hydrogen bonding or the like.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to Examples, but this does not limit the present invention.

[0018]

【Example】

Example 1 Production and Disintegration of Bacterial Cellulose (1) Preparation of Seed Bacterial Solution (Proliferation of Microorganisms) Cellulose-producing microbes were multiplied by a flask culture method. Basic composition of 40 g / L fructose, 1.0 g / L potassium phosphate, 0.3 g / L magnesium sulfate, 3 g / L ammonium sulfate, 5 g / L bacto-peptone, 1.4 ml / L lactic acid, initial pH 5.0 Medium 100
BPR into a 750 ml Roux flask into which the
1 ml of a cryopreserved bacterial solution of the 2001 strain (FERM BP-4545) was inoculated, and statically cultured at 28 ° C. for 3 days in a constant temperature incubator. After the seed culturing, the Roux flask was shaken well, and the contents were subjected to gauze filtration under aseptic conditions to obtain a seed bacterial solution.

(2) Production of Bacterial Cellulose by Agitation Culture Aseptically, 60 ml of the above seed bacterial solution was aseptically inoculated into a sterilized small jar fermenter (total volume: 1000 ml) into which 540 ml of a stirring culture medium to be described later was inserted. 20 at 30 ° C
PH for 1 hour or 30 hours with 1N NaOH or 1N H
While controlling to 5.0 with ₂ SO ₄ , the stirring speed was initially 400 rpm, and the dissolved oxygen (DO) was 3.0.
Stirring culture was performed with a jar fermenter while automatically controlling the number of revolutions so as to fall within 0 to 21.0%. For the stirring culture, a medium having the following composition was used. 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, Bacto-Soytone (manufactured by Difco) 5 g / L and sono (soybean) 5 g / L Initial pH 5.0 After cultivation, the solids in the jar fermenter were collected, washed with water to remove the medium components, and then placed in a 1% NaOH aqueous solution at 110 ° C. The cells were removed by treatment for 20 minutes. Further, the produced cellulose was washed with water until the washing liquid became nearly neutral to obtain bacterial cellulose.

(3) Stirring culture with addition of carboxymethyl cellulose In the stirring culture described in (2), as the medium component,
Furthermore, carboxymethylcellulose was added at 5 g / l for culturing. The resulting culture was more viscous than the culture obtained in (2). (4) Production of Bacterial Cellulose (Comparative Example) by Static Culture 600 ml of a medium having the composition described in (2) was dispensed into a sterile petri dish by 30 ml each, and allowed to stand at 30 ° C. for 7 days. did. After completion of the culture, the bacterial cellulose formed on the surface of the Petri dish was washed with water to remove the medium components.

(5) Disaggregation treatment of bacterial cellulose Water was added to the washed bacterial cellulose obtained by the stirring culture method of (2), and suspended at a disaggregation concentration (dry weight of bacterial cellulose / volume) of about 0.2% by weight. A suspension was prepared. Similarly, water was added to the bacterial cellulose obtained by the static culture of (3) to prepare a suspension having a disaggregation treatment concentration (dry weight of bacterial cellulose / volume) of about 0.2% by weight. Next, these suspensions were disintegrated for 3 minutes at 25 ° C. using a stirrer (Blender manufactured by Auster). The rotation speed of the stirrer was set to the highest level. By this disaggregation treatment, a BC disintegration product (A) was obtained from the stirring culture, and a BC disintegration product (B) was obtained from the stationary culture. Water was added to the culture solution in the jar fermenter containing bacterial cellulose after completion of the stirring culture in (2) and (3) to adjust the bacterial cellulose content to 0.2%. Then, the mixture was stirred at 25 ° C. for 3
Disaggregated for a minute. The rotation speed of the stirrer was set to the highest level. By this disaggregation treatment, BC disaggregates (C) and (D) were prepared from the dilutions of the respective culture solutions. In addition,
The concentration of BC in the above was determined by centrifuging to remove a wet solid from the culture solution, and then in a 0.2 N sodium hydroxide solution (20 times the solid content) at 100 ° C.
After immersion for a time to remove bacterial cells and medium components other than bacterial cellulose, the cells were sufficiently washed and dried and calculated from the measured dry weight.

Example 2 2-Methoxy-4H-1,3,2-benzodioxaphosphorin-2-sulfide
An emulsion containing 25% of an active ingredient (trade name, salicion) was diluted 80-fold with the disaggregated product (A) prepared in Example 1 containing 0.2% of BC (hereinafter, referred to as drug solution (A)). . This diluted solution was sprayed on lilies and roses in Kawasaki City, Kanagawa Prefecture. The spraying was performed three times using a hand spray. As a comparative example, a liquid diluted with water alone was used (hereinafter, chemical (B)). The weather was fine. After spraying the medicinal solution, the spreading state of the medicinal solution on the lily was visually examined. The results are shown in Table 1.

[0023]

[Table 1] Table 1 Spreading state of liquid medicine on lilies ────────────────────────── Type of liquid medicine Spreading degree on stems and leaves ────────────────────────── Chemical liquid (A) ◎ − Chemical liquid (B) ○ + ───────────── ────────────── Degree of spread: ◎ Uniform spread, ○ Uniform even when spread Chemical liquid dripping: + liquid dripping, − liquid dripping at all

By blending and spraying bacterial cellulose into a chemical solution, a film was formed on the stems and leaves of lilies, and as a result, it was useful for improving the spread degree of the chemical solution and preventing dripping.

Example 3 As in Example 2, the chemical solution was sprayed on the roses. After drying for about 5 hours after spraying, a maximum rainfall of about 4 mm per hour was observed from the evening. After exposing the roses to the rain for 3 hours, the degree of spread of the chemical solution was visually inspected. In the case of the chemical solution (A), the coating was maintained and the chemical solution was spread even after rainfall, whereas in the case of the chemical solution (B), the chemical solution was not spread. Example 4 Allethrin against the larvae of Chaduga moth on Sancha flowers
At the same time as spraying a spray of insecticide and fungicide containing tetrachloroisophthalnitrile (Kadan D, manufactured by Fumakiller), the disintegrated product (B) having a BC concentration of 0.2% prepared in Example 1 was sprayed for about 5% using a hand spray. Sprayed for seconds. The spray amounts of both spray liquids were almost the same. As a comparative example, a spray of insecticide / bactericide and water were simultaneously sprayed.
The state of Chadokuga after spraying was visually observed. As a result, when the disintegrated product (B) was used, a bacterial cellulose film was formed on the body surface of the chadokuga after spraying.
Because the insecticide / fungicide adhered to the moth, the moth immediately stopped and died. On the other hand, in the case of the comparative example, the movement did not stop, and it was not confirmed whether the larva of the chadokuga escaped to the back side of the leaf and died.

Example 5 The same test as in Example 2 was carried out by using the disaggregated product (D) prepared in Example 1 instead of the disaggregated product (A). As a result, the same effect as that of the example 2 was obtained. Example 6 Using the disaggregated product (A) prepared in Example 1, a skin moisturizer was tested. As a test method, the same lotion, foundation cream, foundation,
I applied makeup in the order of the fun. After makeup, the left half of the face was sprayed twice with a 0.2% BC water suspension by hand spraying, and the right half was sprayed with water only twice by hand spraying. After 5 minutes, actual use evaluation was performed for each item shown in Table 2. In addition, in the case of the disintegrated material, the evaluation of each panel was described, but regarding the evaluation of the water spray as a comparative example, since all the panelists had the same evaluation for each item, the results were collectively described.

[0027]

[Table 2] Evaluation symbol ◎: extremely good, ○: good, Δ: normal, ×: poor,-: not evaluated

Example 7 The disaggregated product (A) prepared in Example 1 was treated by the same panelist as in Example 6 with 0.2% B in the left half of the face after washing and before makeup.
The C water suspension was hand sprayed twice and the right half was sprayed with water only twice. next,
The makeup was applied in the order of the same lotion, base cream, foundation, and white powder. After 5 minutes, actual use evaluation was performed for each item shown in Table 3. In the case of disaggregated products, the evaluation of each panel was described, but regarding the evaluation of water spray which is a comparative example, all panelists gave the same evaluation for each item, so the results are collectively shown.

[0029]

[Table 3] Evaluation symbol ◎: extremely good, ○: good, Δ: normal, ×: poor,-: not evaluated

As described above, by spraying the disaggregated material onto the skin to form a film, the formation of wrinkles due to water evaporation and drying from the skin is prevented, the moist feeling and suppleness of the skin are maintained, and wrinkles that have already been formed are formed. It became possible to make shallow. Further, by applying a BC water suspension by spraying or the like before or after normal make-up, it is possible to prevent the skin from drying without disturbing the make-up.

Example 8 Static of Cellulose Producing High Polymerization Cellulose
Incubation and Preparation of Cellulose (BC) BPR3001A was added to the medium 10 from glycerol stock.
Inoculation was carried out at a concentration of 1% in a 750 ml volume flask equipped with 0 ml, and the cells were cultured at 28 ° C. for 3 days. After the culture, the roux flask was shaken well to remove the cells from the cellulose membrane, and 3 ml of the bacterial solution was inoculated into a Petri dish (diameter: 90 mm) containing 27 ml of CSL-Fru medium and cultured at 28 ° C for 10 days. After the cultivation, the cellulose membrane of each bacterium was washed with running water and heated in about 500 ml of water at 80 ° C. for 20 minutes. After heating, each cellulose membrane was further washed with running water, and thereafter, about 500
Lysis was achieved by heating at 80 ° C. for 20 minutes in 0.1 ml NaOH. After lysis, each cellulose membrane is
Washing was carried out by heating in 00 ml of distilled water at 80 ° C. for 20 minutes. The same washing was performed 3 to 5 times while exchanging distilled water to obtain purified BC.

Example 9 Passage of high-polymerization degree cellulose-producing bacteria
Bacterial agitation culture and preparation of cellulose (BC) BPR3001A was prepared from glycerol stock by CSL-
1% of the cells were inoculated into a 750 ml roux flask charged with 100 ml of Fru medium and statically cultured at 28 ° C. for 3 days. After the culture, the roux flask was shaken well to remove the cells from the cellulose membrane, and 12.5 ml of the bacterial solution was added to 500 ml containing 112.5 ml of the medium.
The cells were inoculated into a ml flask and cultured at 28 ° C. and 180 rpm for 3 days. The culture was aseptically disaggregated with a blender, 60 ml of which was charged with 540 ml of CSL-Fru medium.
Inoculate the jar and adjust the pH to NH ₃ gas and 1N H ₂ S
While controlling the O ₄ at 4.9 to 5.1, the amount of dissolved oxygen (D
Main culture was performed while automatically controlling the rotation speed so that (O) was 3.0% or more. After the completion, the obtained culture solution was diluted about 5 times with acetate buffer and then centrifuged to collect the precipitate. The precipitate was diluted with distilled water to about 8 times the initial culture volume, heated at 80 ° C. for 20 minutes, and collected by centrifugation after heating. The precipitate is also 8 times the amount of 0.1N
Lysis was carried out by suspending in NaOH and heating at 80 ° C. for 20 minutes, and then the precipitate was recovered by centrifugation after lysis. Thereafter, the precipitate was suspended in 8 volumes of distilled water,
The cellulose was washed by heating for 1 minute and centrifuging after heating to collect the precipitate. Purified BC was obtained by performing the same washing three times.

Incidentally, the CSL-Fru used in the above embodiments.
Is as shown below.

[0034]

[Table 4]

[0035]

[Table 5] Vitamin mixture liquid compound mg / L inositol 200 niacin 40 pyridoxine HCl 40 thiamine HCl 40 calcium pantothenate 20 riboflavin 20 p-aminobenzoic acid 20 folic acid 0.2 biotin 0.2

[0036]

[Table 6] salts mixture _{FeSO 4 · 7H 2 O 360mg /} L CaCl 2 · 2H 2 O 1470mg / L Na 2 MoO 2 · 2H 2 O 242mg / L ZnSO 4 · 7H 2 O 173mg / L MnSO 4 · 5H 2 _{O 139mg / L CuSO 4 · 5H} 2 O 5mg / L

Example 10 The purified BC obtained in Examples 8 and 9 was disintegrated in the same manner as in the disaggregated product (A) described in Example 1 to obtain a disaggregated product. These are called disintegration products (E) and (F), respectively.
Using the disaggregated products (E) and (F), the same test as described in Example 2 was performed, and the same results as the disaggregated product (A) were obtained. Example 11 A sample for molecular weight analysis was prepared by drying the disaggregated material (A), the disaggregated material (B), the disaggregated material (E) and the disaggregated material (F) under reduced pressure at 80 ° C. for 12 hours. After nitration, the weight-average molecular weight was measured and the weight-average degree of polymerization was calculated according to the method already described for these samples. As a result, the weight-average degree of polymerization was 10,60 in terms of polystyrene, respectively.
0, 14,900, 22,500 and 17,400.

Example 12 (1) Preparation of BC sample Acetobacter xylinum ATCC 11142 was cultivated by aeration and stirring in the same manner as in Example 1, and then purified to prepare a BC sample. This BC sample was centrifuged, the supernatant was discarded, and the BC sample was stored in an undried state containing water. Collect this with a medicine spoon,
The BC sample was weighed in a polypropylene 50 ml tube. Put this tube in the vacuum dryer and put it under vacuum 8
Dry at 0 ° C. for 8 hours. After drying, the weight of the BC sample was measured, and the weight change caused by this change in the wet B
The dry weight of the C sample was converted.

(2) Preparation of bacterial cellulose aggregate (hereinafter abbreviated as "BC aggregate") and immobilization of spores By the method of (1), 0.5, 2.5, 5.
A BC sample of 0 g was weighed, sterilized water was added to make 200 ml, and the mixture was placed in a blender cup. This was steam sterilized in an autoclave at 120 ° C. for 20 minutes, the BC sample in the blender cup was homogenized at 10,000 rpm for 3 minutes, and placed in a 1 liter jar. Inside the jar, one propeller type blade (diameter 6.8 cm) was attached so as to be below the 500 ml liquid surface. In addition, sterilized (NH ₄ ) ₂ SO
Ammonium sulfate solution 270ml, which was dissolved ₄ 2.5g was added to the jar. Separately, a spore powder prepared from Bacillus subtilis RB14-C was suspended in 30 ml of sterile water in a polypropylene tube and added to a jar. At this time, the number of bacteria added to the jar was counted by colony counting. After the addition, the bacterial cellulose concentration was adjusted to 0.1, 0.5 and 1.0% w / v, and the (NH ₄ ) ₂ SO ₄ concentration was adjusted to 0.5%. This solution is about 7
The mixture was stirred at 0 rpm (= tip speed 25 cm / s) for 5 hours to obtain a BC aggregate suspension in which Bacillus subtilis was immobilized. Created BC
The aggregate was collected with a sterilized gauze and cooled in a -80 ° C freezer for 3 hours to be frozen. This is freeze-dried (FD-
1. Using Tokyo Rika Kikai Co., Ltd., it was dried at 0.05 Pa for 24 hours, and the dry weight of the BC aggregate was weighed. If not completely dried, it was dried for another 24 hours and weighed. After weighing, the mixture was homogenized at 10,000 rpm for 3 minutes, and the pulverized granular BC aggregates were put in a plastic petri dish and stored in a desiccator.

(3) Measurement of the number of Bacillus subtilis immobilized on bacterial cellulose About 0.05 g of the BC aggregate immobilized with Bacillus subtilis thus obtained was weighed and placed in a sterilized 50 ml Erlenmeyer flask. 0.8 ml of 20% cellulase solution and 7.2 ml of physiological saline were added to an Erlenmeyer flask containing BC aggregates, shaken with a vortex, and shaken at 37 ° C. and 120 spm for 1 hour to dissolve BC. The number of bacteria in this solution was counted by colony counting. The number of bacteria was converted to the number of bacteria per gram of BC aggregate, and the number of immobilized bacteria was determined from the total dry weight of the collected BC aggregates. Further, the efficiency of immobilization was also determined from the ratio of the number of bacteria immobilized on the BC aggregate and the number of bacteria added to the jar. Table 7 shows the measurement results of the number of added bacteria, the number of immobilized bacteria and the efficiency of immobilization at each BC concentration.

[0041]

[Table 7]

When the number of bacteria added was between 10 ^{10 and} 10 ¹¹ cells / ml, comparing the number of bacteria immobilized per 1 g of BC with the same number of bacteria added at each BC concentration, the BC concentration was 1.0.
The number of immobilized bacteria increased in the order of 0.5 and 0.1%. On the other hand, the relationship with the total number of bacteria actually immobilized on the BC aggregates was almost proportional regardless of the BC concentration. Furthermore, the highest immobilization efficiency was obtained when the BC concentration was 0.5%. Further, 70% or more of the Bacillus subtilis immobilized on the BC aggregates after freeze-drying were in the state of spores.

Example 13 Using the granular BC aggregates on which Bacillus subtilis was immobilized according to the present invention prepared in Example 12, the plant disease inhibitory ability was examined, and the present invention was spread as a microbial pesticide carrier. The usefulness of the adhesive was confirmed. Plant pathogen : Rhizoctonia solani is known to cause seedling wilt in various plants, and B. subt.
It has already been confirmed that ilis RB14-C suppresses seedling wilt caused by R. solani. R. solani K-1 is isolated from citrus , and mycelium grows at 10 to 35 ° C, and its optimum temperature is around 30 ° C. Medium used : Potato Dextrose for storage of R.solani K-1
Agar medium (hereinafter abbreviated as "PDA medium") was used. For culturing R. solani K-1, Potato Dextrose Polypepton medium (hereinafter abbreviated as “PDP medium”) used for culturing filamentous fungi was used. Table 8 shows the composition of PDA medium and PDP medium.

[0044]

[Table 8] Medium composition used ────────────────────────────── Medium composition (g / l) ────── ──────────────────────── PDA Potato infusion 200 (DIFCO) Glucose 20 Agar 15 (pH = 5.1) PDP Potato dextrose 24 Polypepton 1.0 (PH = 5.1)

Soil used : As a fertilizer, 50 g of lime superphosphate (P ₂ O ₅ 17%) (equivalent to P ₂ O ₅ 8.5 g) and 25 g of Rinkaan No. 42 (equivalent to N 3.5 g) were added to 4 kg of soil. Soil was used. Rinkaan No. 42 is (NH ₄ ) ₂ SO ₄ ,
A mixture containing 14% each of K ₂ SO ₄ and lime superphosphate. As the sterilized soil, the above soil was steam sterilized in an autoclave at 120 ° C. for 60 minutes four times (each time, at half-day intervals) and used in the experiment. Plants used : Tomatoes used, Ponderosa variety
(Takii Seed Co., Ltd.). A plant disease suppression test was carried out according to the procedure shown below. (1) Cultivation of plant pathogens and inoculation into soil For cultivation of plant pathogens, 40 ml of PDP medium was placed in a 200 ml Erlenmeyer flask and R. solani K-1 on a PDA medium plate stored at room temperature was about 5 mm square with a platinum loop. It was cut to the size of and inoculated. This Erlenmeyer flask was cultivated at 30 ° C. for 7 days to make a mycelium mat. After static culture, the mycelium mat was taken out with a spoon and homogenized with 40 ml of sterilized water at 4,000 rpm for 2 minutes. After homogenizing the mycelium mat, it was mixed with soil in a plastic tray so that the inoculation amount per pot would be 1/4 of the number of mycelial mats. This work was carried out 5 days before sowing, and the pots were allowed to stand in an artificial weather device for 5 days to allow the plant pathogens to settle in the soil. (2) Inoculation of Bacillus subtilis-immobilized bacterial cellulose into soil 0.1 g of the Bacillus subtilis-immobilized BC aggregate prepared in Example 12 was mixed into about 120 g of soil in a plastic tray (bacteria About 10 ⁷ cfu / g-dry soil). As a control, a pot in which 0.1 g of only BC aggregates prepared without immobilization of Bacillus subtilis was mixed in about 120 g of soil, a pot inoculated with spore powder of Bacillus subtilis (about 10 ⁷ cfu / g-dry soil) was also prepared. This work was carried out 3 days before sowing, and allowed to stand in an artificial weather device for 3 days to allow Bacillus subtilis to settle in the soil containing plant pathogens. The water content was set to 60% of the maximum water capacity of each soil, and sterilized water was replenished daily to 60% of the maximum water capacity.

(3) Surface sterilization of seeds and pre-germination Tomato seeds were immersed in 50 ml of 70% ethanol aqueous solution for 1 minute in a 200 ml beaker and then rinsed 5 times with distilled water. Further, it was immersed in 50 ml of 0.5% NaClO aqueous solution for 10 minutes, washed with sterilized water 10 times, and surface sterilized. The seeds after surface sterilization were placed on a 2.0% agar plate and allowed to stand in an artificial weather device for 3 days to germinate the seeds. (4) Plant test conditions Nine seeds germinated in (3) were sown on each soil that had been left to stand in an artificial weather device in advance at a position of about 1 cm from the soil surface. The pot in which the seeds were sown,
The temperature was set to 30 ° C. and the humidity was set to 80% by leaving it in the artificial weather device. The water content in the pot is 60% of the maximum water capacity of the soil.
And sterilized water was added every day for adjustment. Irradiation with a fluorescent lamp (about 12,000 LUX, 16 h / day) was performed. After growing for 14 days, count the diseased seedlings in each pot,
The morbidity was calculated. Further, the length of the tomato above the maximum growth point (stem length) and the length up to the leaf tips (leaf length) were measured for non-diseased plants, and the average value per plant was calculated.
Furthermore, only the above-ground part was harvested and dried overnight at 105 ° C. to measure the dry weight of the plant.

Results : Tables 9 and 10 show the results of statistical processing of the obtained experimental data. From Table 9, there was no significant difference in dry weight and morbidity between the pot containing only the pathogen and the pot containing BC. From Table 10, when the Bacillus subtilis immobilized on BC was inoculated, the morbidity was as low as when inoculated with only spores.

[0048]

[Table 9]

[0049]

[Table 10]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kasuko Kaneko 3-2-1 Sakado, Takatsu-ku, Kawasaki City, Kanagawa Prefecture Biopolymer Research Co., Ltd. 3-2-1 Biopolymer Research Co., Ltd. (72) Inventor Hiroshi Ogiya 3-2-1 Sakado, Takatsu-ku, Kawasaki-shi, Kanagawa Biopolymer Research Co., Ltd.

Claims (10)

[Claims] 1. A spreading agent containing bacterial cellulose. 2. The spreader according to claim 1, wherein the bacterial cellulose has been subjected to a disaggregation treatment. 3. The spreading agent according to claim 2, wherein the disaggregation treatment method is a method using mechanical shearing force, ultrasonic waves, high pressure treatment, acid hydrolysis, enzymatic hydrolysis or a bleaching agent, or a combination thereof. 4. The spreading agent according to claim 1, which contains bacterial cellulose having a polystyrene-equivalent weight average degree of polymerization of 1.6 × 10 ⁴ or more, which is obtained by aeration and agitation culture. 5. The spreading agent according to claim 1, which contains bacterial cellulose obtained by static culture and having a polystyrene-equivalent weight average degree of polymerization of 2.0 × 10 ⁴ or more. 6. A composition comprising a spreading agent containing bacterial cellulose obtained by aeration and agitation culture and a microorganism. 7. The composition according to claim 6, wherein the microorganism has a plant disease suppressing ability. 8. The composition according to claim 6, which is a dry powder or dry granule obtained by freeze-drying. 9. A method of immobilizing the microorganism on the bacterial cellulose, which comprises stirring the suspension containing the bacterial cellulose and the microorganism obtained by aeration and stirring culture. 10. A microorganism immobilization agent comprising bacterial cellulose obtained by aeration and agitation culture.