JPH0585158B2 - - Google Patents

Description

[Detailed description of the invention]

〓Field of Industrial Application〓 The present invention relates to an immobilization carrier that exhibits an excellent function in immobilizing acetic acid bacteria and a method for producing the same. It has excellent performance in immobilizing acetic acid bacteria in various reactors used for continuous fermentation of acetic acid, conversion of substances, etc. 〓Prior art〓 Conventionally, the present inventors disclosed in the application example of Japanese Patent Application No. 1-45781 that a porous chitosan molded product can be used as a carrier for immobilizing acetic acid bacteria. Chitosan granules are treated with an acid solution to perform crosslinking treatment. 〓Problem to be solved by the invention〓 In order to immobilize acetic acid bacteria, it is important to increase the affinity with the carrier.This makes it possible to increase the amount of acetic acid bacteria in the carrier, and It exhibits excellent performance when used in reactors for the conversion of substances using The porous chitosan molded article previously disclosed by the present inventors was treated with an acid, and hexamethylene bis-2 (2,3-epoxypropyldimethylammonium chloride), hexamethylene diisocyanate, and 4,4'-diphenylmethane were respectively treated. Porous chitosan molded products crosslinked with diisocyanate still have a low affinity for acetic acid bacteria, and therefore have the disadvantage of a low number of adsorbed bacteria. The object of the present invention is to provide porous chitosan particles with a high ability to immobilize acetic acid bacteria. <Means for Solving the Problems> The present invention is characterized in that the amount of phenyl groups introduced per 100 structural units of pyranose rings constituting the chitosan of the porous granules is 4 to 45, and the pore diameter of the porous granules is 3 to 45.
This relates to a carrier for immobilizing acetic acid bacteria having a diameter of 10 μm, and such a carrier for immobilizing acetic acid bacteria is prepared by coagulating and precipitating an acidic aqueous solution of low molecular weight chitosan in a basic aqueous solution to obtain porous chitosan particles, and then It is obtained by treatment with an acid solution and further reaction with phenyl isocyanate. In the present invention, low molecular weight chitosan with an average molecular weight of 10,000 to 230,000 is used as chitosan,
This is dissolved in an aqueous solution of acetic acid, dichloroacetic acid, formic acid, etc. alone or in combination to obtain an acidic chitosan aqueous solution. The concentration of chitosan in the chitosan acidic aqueous solution is 2~
It can be freely selected within a range of 20% (weight). At this time, polyvinyl alcohol, polyethylene glycol, etc., which are water-soluble polymer substances, may be added alone or in combination as a pore control agent to the chitosan acidic aqueous solution. In order to regenerate chitosan from the chitosan acidic aqueous solution and obtain porous chitosan granules, the chitosan acidic aqueous solution is dropped in a constant amount into the basic aqueous solution under pressure, for example, from a nozzle with a pore diameter of 0.1 to 0.25 mmφ. Porous chitosan granules are obtained. Basic substances added to the basic coagulation bath include:
Alkaline substances such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, ammonia, and ethylenediamine are used. To make a basic solution, water, polar alcohols such as methanol and ethanol, or water are used. The above-mentioned basic substance is added to a mixture of alcohols for use. The water in the obtained porous chitosan particles containing water is replaced using an alcohol such as methanol, ethanol, isopropyl alcohol, or a polar solvent such as acrylonitrile. Next, the surface of the porous chitosan particles is treated with an acid aqueous solution. Examples of acids used at this time include organic acids such as acetic acid, formic acid, and propionic acid, and mineral acids such as hydrochloric acid and nitric acid. The surface of the chitosan particles is treated with an aqueous acid solution for a short time, and then the acid is thoroughly washed away with water. At this time, if the granules contain water, it is preferable to use an aqueous acid solution, and if the granules contain a polar solvent, it is preferable to use a mixture of the polar solvent and the acid. The concentration of acid is
0.5-5.0% (by weight) is used, and acid treatment is
Perform at 25°C for 10 seconds to 5 minutes. However, the treatment conditions can be freely selected depending on the concentration of the chitosan acidic aqueous solution, the concentration of the basic coagulation bath, and the type and concentration of the acid used in the treatment so as to form large pores inside the granules on the surface. The porous chitosan particles obtained by the above acid treatment have the following properties:
Since the pore portion with a smaller diameter is removed, the pores have a larger diameter from the surface toward the inside. The water in the obtained porous chitosan particles is completely replaced with an organic solvent such as methanol, ethanol, dimethylformamide, dioxane, etc. Next, phenyl isocyanate is cross-linked with the amino groups in the porous chitosan particles at room temperature for 1 to 10 hours, and after the reaction is completed, unreacted phenyl isocyanate is removed again with the above-mentioned organic solvent and thoroughly washed with water. . At this time, the amount of phenyl isocyanate is 0.04 to 3.5 g per 1 g of dry weight of porous chitosan granules.
added. As mentioned above, in the present invention, phenyl isocyanate is introduced to obtain a carrier for immobilizing acetic acid bacteria. The reactivity is high, and the reactivity is significantly higher than that of compounds having an isocyanate group bonded to a linear alkyl group other than a phenyl group. This is because introduction of a hydrophobic group increases the hydrophobicity of the carrier, which has the effect of increasing affinity with acetic acid bacteria. Chitosan, which is a raw material, is often a copolymer of not only glucosamine but also N-acetylglucosamine, and its ratio (degree of deacetylation) is 50 to 100%. The amount of phenyl groups introduced is 4 to 100 structural units of pyranose rings constituting the porous granular chitosan.
45, it is stable in the entire pH range, and does not undergo any changes even when vinegar is produced by immobilizing acetic acid bacteria. Since a hydrophobic group is introduced, the affinity between the acetic acid bacteria and the carrier is significantly improved, and the number of acetic acid bacteria adsorbed during the immobilization operation can be significantly increased, allowing proliferative bacteria to proliferate on the surface and inside of the carrier. It also has excellent performance in retaining bacteria. In order to use porous chitosan particles into which phenyl groups have been introduced as a carrier for immobilizing acetic acid bacteria, the pore size must be between 3 and 3.
When the thickness is in the range of 10 μm, the amount of acetic acid bacteria adsorbed is high and the compressive elastic modulus is excellent. The amount of phenyl group introduced can be determined by the method detailed in the Examples below. 〓Example〓 Hereinafter, the present invention will be described in detail with reference to Examples.
The present invention is not limited to this range. The amount of phenyl group introduced, pore diameter, compressive elastic modulus, and number of acetic acid bacteria adsorbed described in Examples were measured by the following methods. Amount of phenyl group introduced: Accurately collect 50 ml (wet volume) of porous chitosan particles obtained by reacting phenyl isocyanate with a graduated cylinder, and replace with ethanol four times. Four more substitutions are made with ether. Remove the ether, dry it in a vacuum dessicator until the ether odor disappears, and measure its weight (Ag).Separately, 50 ml (wet volume) of porous chitosan granules that are not reacted with phenyl isocyanate are subjected to the above steps. and measure the dry weight (Bg). The amount of phenyl group introduced per 100 structural units of the pyranose ring was determined using the following formula. Amount of phenyl group introduced = {M×(AB)/(119×
B)}×100 However, M: Molecular weight in terms of pyranose ring, calculated from the following formula. M=[(161×DAC/100)+{203×(100−DAC)/100
} ] DAC: Degree of deacetylation (%) 119: Molecular weight of phenyl isocyanate 161: Molecular weight in terms of pyranose rings at 100% DAC 203: Molecular weight in terms of pyranose rings at 0% DAC Pore diameter The sample was rapidly cooled in liquid nitrogen, and - After freeze-drying at 50°C under a vacuum of 10 ^-7 Torr, measurements were taken using a scanning electron microscope. Compressive modulus Rheometer NRM-2010J-CW (Fudou Kogyo Co., Ltd.)
Pack the sample into a sample adapter with a diameter of 6.0 mmφ and a depth of 10 mm using a
The compressive elastic modulus was measured when the material was pushed in 4 mm at a speed of cm/min. Number of acetic acid bacteria adsorbed Acetobacter aceti (IFO-3823) was inoculated into 30 ml of PGY medium consisting of glycose, polypeptone, and yeast extract with 2% ethanol added.
Perform shaking culture at 30°C for 24 hours. Add 4 ml of this culture suspension to the previously sterilized sample and shake for 1 hour at 4°C to allow adsorption. After the adsorption is completed, take 100 ml of the supernatant, dilute it 4000 times, take 100 ml of the supernatant, spread it on a prepared plate medium, and after culturing for 48 hours, count the number of colonies that have appeared. For the culture suspension before adsorption, dilute it 8000 times and count the number of colonies using the same procedure. The number of adsorbed cells is calculated from the difference in the number of cells before and after adsorption. Example 1 60 g of chitosan with a degree of deacetylation of 76% and an average molecular weight of 51000 and polyethylene glycol (molecular weight
20000, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 6% acetic acid so that the total volume was 1000 ml. After solidifying and regenerating the acidic aqueous solution by dropping it into a mixed solution of 5.5% sodium hydroxide, 20% ethanol, and 74.5% water through a nozzle with a hole diameter of 0.25 mm,
Wash thoroughly with water until neutral, and the average particle size is approximately 1.2mmφ.
950 ml of porous chitosan granules with a pore diameter of 0.2 μm and a compressive modulus of 2.5×10 ⁶ dyn/cm ² were obtained. After removing the water adhering to the porous chitosan granules by suction filtration, 0.5%
A immersion treatment was performed in acetic acid aqueous solution 1 at 25° C. for 30 seconds. The pore diameter of this is 5 μm and the compressive modulus is 1.3×10 ⁶
It was dyn/ ^cm2 . After weighing and drying 100 ml (wet volume) of the granules, the dry weight was determined to be 7.3 g. Next, 5 tubes of 100 ml (wet volume) were collected from each tube, and the water was replaced with dimethylformamide to obtain 0.47 g, 2.37 g, 4.73 g, and 9.46 g of phenyl isocyanate.
and 23.65 g were added and reacted for 30 minutes. After the reaction,
Unreacted phenyl isocyanate was completely removed and replaced with dimethylformamide, and then washed with water to obtain five types of carriers for immobilizing acetic acid bacteria (samples 1 to 5). Amount of phenyl group introduced in samples 1 to 5, compressive elastic modulus,
The pore diameter was measured and the results are shown in Table 1.
In addition, after inoculating acetic acid bacteria IFO-3823 into 30 ml of PGY medium containing glycose, polypeptone, and yeast extract,
Add 2 ml of each of samples 1 to 5 to 4 ml of the culture suspension cultured at 30°C for 24 hours with shaking at 120 rotations/min, let the acetic acid bacteria adsorb at 50°C for 1 hour, and measure the number of adsorbed bacteria. It is shown in Table 1.

[Table] From the results in Table 1, the number of adsorbed acetic acid bacteria increases depending on the amount of phenyl groups introduced, while the pore diameter does not change due to the introduction of phenyl groups, but the compressive elastic modulus increases considerably due to the introduction of phenyl groups. It is clear that the carrier is excellent as a carrier for immobilizing acetic acid bacteria. Example 2 200 g of polyethylene glycol (average molecular weight 20,000, manufactured by Wako Pure Chemical Industries, Ltd.) was added to 130 g of chitosan with a deacetylation degree of 80% and an average molecular weight of 52,000, and a 6% acetic acid aqueous solution was added so that the total amount was 2,000 ml. dissolved in A certain amount of the acidic aqueous solution is dropped into a mixed solution consisting of 5% sodium hydroxide, 30% ethanol, and 65% water through a nozzle with a hole diameter of 0.25 mmφ to solidify and regenerate, and then thoroughly washed with water until it becomes neutral.
Porous chitosan granules 1960 with an average particle diameter of approximately 1.0 mmφ
Got ml. Next, three 400 ml (wet volume) each of the obtained porous chitosan granules were collected, the adhering water was removed by suction filtration, and each of 0.1, 0.25 and 0.25 ml of acetic acid aqueous solution was collected.
It was treated in 500 ml of 0.5% solution at 25°C for 30 seconds and washed with water until it became neutral. The wet volumes of the porous chitosan granules after washing with water were 396 ml, 268 ml, and 244 ml, respectively.
When the dry weights of 100 ml of the obtained granules were measured, they were 7.6 g, 7.0 g, and 6.9 g, respectively. Next, water was replaced with dimethylformamide for 100 ml (wet volume) of each granule, and phenyl isocyanate was added to each granule.
6.5 g, 4.5 g, and 4.0 g were added and reacted at room temperature for 1 hour to obtain samples 6 to 8. The amount of phenyl group introduced, compressive elastic modulus, and pore diameter of the obtained Samples 6 to 8 were measured in the same manner as in Example 1, and the results are shown in Table 2. Also, as in Example 1, acetic acid bacteria were adsorbed,
The number of adsorbed acetic acid bacteria was measured, and the results are shown in Table 2.

[Table] As is clear from Table 2, it is clear that when the pore diameter is 3 μm or less, the number of acetic acid bacteria adsorbed is significantly lower even if the amount of phenyl groups introduced is almost the same. Example 3 In 120 g of chitosan with a deacetylation degree of 78% and an average molecular weight of 50,000, 200 g of polyethylene glycol (average molecular weight 20,000, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 6% acetic acid so that the total amount was 2,000 ml. The acidic aqueous solution was mixed with 5.5% sodium hydroxide, 30% ethanol,
After solidifying and regenerating a fixed amount of chitosan particles by dropping them into a mixed solution consisting of 64.5% water through a nozzle with a pore size of 0.25 mmφ, the porous chitosan particles with an average particle size of 1.0 mmφ were washed thoroughly with water until they became neutral. ) was obtained. Next, 400 ml of each of the obtained porous chitosan granules
Three bottles (wet volume) were collected, and after removing the adhering moisture by suction filtration, they were treated with 500 ml of each 0.5, 1.0, and 5.0% aqueous acetic acid solution at 25°C for 30 seconds, and washed with water until they became neutral. , each wet volume is 260ml, 242
ml, 210ml. 100 each from the obtained granules
When I collected ml and measured the dry weight, it was 7.5
g, 7.2 g, and 6.8 g. Next, 100 ml (wet volume) of each of the obtained granules was collected, the water was replaced with dimethylformamide, 4.5 g, 4.0 g, and 4.0 g of phenyl isocyanate were added thereto, and the mixture was allowed to react at room temperature for 1 hour. After that, unreacted phenyl isocyanate was replaced with dimethylformamide, and then thoroughly washed with water to obtain Samples 9 to 11. The amount of phenyl group introduced, compressive elastic modulus, and pore diameter of the obtained sample were measured in the same manner as in Example 1, and the results are shown in Table 3. Further, in the same manner as in Example 1, acetic acid bacteria were adsorbed and the number of adsorbed acetic acid bacteria was measured, and the results are shown in Table 3.

[Table] As is clear from Table 3, when the pore diameter exceeds 15 μm, a significant decrease in the compressive elastic modulus is observed even when phenyl groups are introduced. Effects of the Invention The present invention involves molding an acidic aqueous solution of low molecular weight chitosan in a basic aqueous solution, treating it with the acid solution,
Furthermore, by reacting with phenyl isocyanate, the amount of phenyl groups introduced per 100 structural units of pyranose rings constituting the porous granular chitosan was reduced to 4.
〜45, and the pore size of the porous granules is 3〜45.
By setting the specific range of 10 μm, it is possible to increase the compressive elastic modulus required of the carrier and increase the amount of acetic acid bacteria adsorbed several times compared to conventional carriers (described in Japanese Patent Application No. 1-45781). According to the present invention, an excellent carrier for immobilizing acetic acid bacteria can be obtained.

Claims (1)

[Scope of Claims] 1. The amount of phenyl groups introduced per 100 structural units of pyranose rings constituting the chitosan of the porous granules is 4 to 4.
45, the carrier for immobilizing acetic acid bacteria, wherein the porous granules have a pore diameter of 3 to 10 μm. 2. A carrier for immobilizing acetic acid bacteria, characterized in that porous chitosan particles are obtained by coagulating and precipitating an acidic aqueous solution of low molecular weight chitosan in a basic aqueous solution, then treated with an acid solution, and further reacted with phenyl isocyanate. manufacturing method.