JP4831313B2 - Carrier for immobilizing chitosan-based microorganisms having magnetism and method for producing the same - Google Patents

Description

  The present invention relates to a carrier for immobilizing a chitosan-based microorganism having a magnetic property that can maintain performance as a carrier for immobilizing microorganisms and that can be collected by magnetic force, and a method for producing the same.

In recent years, bioreactor systems, such as precision organic synthesis using microorganisms, have become increasingly popular with major technological advances related to biotechnology. As the immobilization carrier, the use of a granular porous material, in particular, a microorganism immobilization carrier derived from chitosan has been actively used.
The microorganism-immobilized carrier derived from chitosan has a) that the material is naturally derived and excellent in safety, and b) that the large pores are uniformly present from the surface to the inside of the carrier compared to the synthetic resin-based carrier. It has excellent diffusion, c) has an amino group advantageous for immobilization by enzyme covalent bond and heavy metal chelate, and d) chitosan itself has high affinity with enzymes and microorganisms. It has many advantages, such as a large amount that can be immobilized, and examples thereof include granular porous chitosan disclosed in JP-B-63-54285.

  Recently, the use of chitosan-derived microorganism-immobilized carriers has been activated mainly on the beaker scale for the above-mentioned reasons, but there are an increasing number of examples intended for application to marine, river, and wastewater treatment. In the near future, large-scale and open space cases can be assumed. However, when used in such a wide space, in the conventional system, the dissipation of the carrier is large, and it is a problem that a large facility is required for collection. In addition, in the operation at the beaker scale, the development of a carrier that can be easily separated into a solid and a liquid is desired for the purpose of application to an apparatus for storing microorganisms.

For the purpose of collecting the carrier, natural polysaccharides such as chitosan, and a method of kneading a magnetic substance or magnetic particles in their derivatives, a method of molding after mixing the magnetic substance or magnetic particles, and There are methods such as coating the surface of magnetic materials and magnetic particles with natural polysaccharides. For example, in JP-A-3-278834 (Patent Document 1), a method in which magnetic fine particles are dispersed in a chitosan solution and simultaneously coagulated and regenerated to form a chitosan composite is disclosed in JP-A-10-99843 (Patent Document 2). Have proposed a method in which a magnetic material is used as a nucleus and the surface thereof is coated with a chitosan mixture, but these are intended for application as adsorbents of compounds having a relatively low molecular weight. It was unsuitable as a carrier for immobilizing various substances and culturing. For example, when considering from the viewpoint of performance, the microorganism-immobilized support has a large pore diameter ranging from 5 μm to a maximum of 100 μm, and macropores are formed inside the microorganism-immobilized support. However, the above method cannot cope with immobilization of macro substances or use for culture. In addition, the above method has a large amount of equipment contamination and loss during molding, and a dedicated manufacturing facility is necessary to manufacture the magnetic carrier. There was a problem.
JP-A-3-278834 JP-A-10-99843

  An object of the present invention is to provide a chitosan-based microorganism immobilization carrier capable of being collected by magnetic force while maintaining the performance as a microorganism immobilization carrier and a method for producing the same.

In order to solve the above-mentioned problems, the present inventors have intensively studied, and as a result, have reached the present invention.
That is, the first invention in the present invention, as in the monoferric ion and ash content increment from magnetic products made of ferric ion is in the range of 4% to 25%, the particulate porous cross-linked chitosan surface A support for immobilizing chitosan-based microorganisms having magnetism formed by forming magnetized particles. The second invention is a method in which granular porous crosslinked chitosan is mixed in a solution containing ferrous ions and ferric ions. A method for producing a carrier for immobilizing a chitosan-based microorganism having magnetism, characterized in that it is treated with an alkaline aqueous solution to form magnetized particles in granular porous crosslinked chitosan. A third invention is the immobilization of magnetic chitosan-based microorganisms in which the ratio of the number of moles of ferrous chloride hydrate in the treatment solution of the granular porous crosslinked chitosan is 25 mol% to 75 mol% of the number of moles of hydrate. This is a method for producing a chemical carrier.

  Since the carrier for immobilizing chitosan-based microorganisms of the present invention has a microorganism immobilizing ability and magnetism at a level that can be used practically, it is a pure culture carrier for microorganisms, a carrier for searching for new microorganisms, It is a carrier that can be used and applied for various uses in the field of biotechnology such as collection, activated sludge substitute, and precision organic synthesis, and can be collected by magnetic force after use.

The present invention is described in detail below.
As the granular porous chitosan used as the base in the present invention, it is preferable to use the granular porous chitosan disclosed in Japanese Patent Publication No. 1-16420. In the microorganism-immobilized enzyme carrier, it is necessary that the large pores are uniformly present from the surface layer to the center of the carrier. The appropriate pore size is 5 μm or more and has no upper limit, but is preferably 1,000 μm or less from the viewpoint of production stability.

  The granular porous chitosan for immobilizing microorganisms as a base in the present invention must have a cross-linked structure or be insolubilized in a general-purpose solvent. Although the crosslinking agent and modifier at the time of crosslinking are not particularly limited, examples thereof include diisocyanate compounds, bisepoxide compounds, and acid anhydrides. In particular, hexamethylene diisocyanate, 4,4′-diphenylmethane diisocyanate, and modified products thereof, 3-chloro-2-hydroxypropyltrimethylammonium chloride, polyethylene glycol diglycidyl ether compound (the chain length of the ethylene glycol portion is 1 or more) Arbitrary), a polypropylene glycol diglycidyl ether compound (the chain length of the propylene glycol portion is 1 or more is arbitrary), and acetic anhydride, succinic anhydride, and the like are preferable.

In the present invention, when to be formed in particulate porous cross-linked chitosan by post-processing the magnetic particles, in a solution containing a first monoferric ion and ferric ion, it is necessary to process the particulate porous cross-linked chitosan. Ferrous ions and ferric ions have high affinity with chitosan-based adsorption carriers, and the system tends to be uniform. As the ion source, any of general-purpose and commercially available salts corresponding to these iron ions such as hydrochloride, sulfate, citrate, and acetate can be used. From the viewpoint of the above, each chloride is preferable.

The solution containing the first monoferric ion and ferric ion that put the present invention, ferrous alone chloride, or preferably the ferrous chloride is mixed aqueous solution of ferric chloride, the mixture first chloride The ratio of ferrous iron is preferably in the range of 25 mol% to 75 mol% when calculated from the number of moles of hydrate. Within this range, both the performance as a microorganism-immobilized support and the performance of a support capable of being collected by magnetic force are combined, so that the object of the present invention can be sufficiently satisfied. When it is out of this range, that is, when it is prepared only with ferric chloride or when ferrous chloride is less than 25 mol%, the treated particles do not adhere around the magnet even if it is close to the magnet, or attached. The purpose of this case cannot be fulfilled, such as weakness.

Charge of a mixture of put that salts of ferrous and ferric chloride in the present invention, when taking the granular porous cross-linked chitosan in the wet state of 25ml example, is preferably at least 0.003 mol, more Preferably it is in the range of 0.003 mol or more and 0.02 mol or less. In this case, the increase in the ash content (measurement method is described in detail below) due to the magnetized material is preferably 4% or more, more preferably 4% or more and 15% or less. If it is less than 4%, it is satisfactory as a microorganism-immobilized carrier, but it is weak in magnetism and does not adhere to the magnet even if it is brought close to it, so that the object of the present invention cannot be achieved. On the contrary, if it exceeds 15%, the performance as a magnetic material will be manifested, but the number of immobilized bacteria will decrease, and even if the charging amount is set to a very high concentration, the ash content will stop rising at about 25%. From the viewpoint of economy, it is not preferable . After mixing the particulate porous cross-linked chitosan in a solution containing a first monoferric ion and ferric ion, an alkaline aqueous solution is treated with an alkaline aqueous solution, but to form magnetic particles, particulate, porous crosslinked chitosan, which is used when the Any conventional aqueous solution of a basic substance can be used, but it is preferable to use aqueous ammonia or aqueous sodium hydroxide. The amount of the aqueous alkali solution may be appropriately selected so that the pH of the reaction system is 8 or more. However, if the amount of aqueous ammonia is excessively used, the cross-linking groups and modifying groups of the granular porous cross-linked chitosan Desorption and shrinkage of the pores occur, and the number of immobilized bacteria on the magnetic carrier may decrease rapidly, which is not preferable.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples. In Examples and Comparative Examples, the reaction unevenness, magnetism, the form of the microorganism-immobilized support, the ash content, and the number of immobilized bacteria were measured or evaluated by the following methods.
(1) Reaction unevenness (appearance evaluation) The color tone of the carrier prepared by the method shown in the examples was visually observed, and the reaction unevenness was evaluated from the unevenness. A case where there was no color unevenness and excellent uniformity was evaluated as ◯, a case where there was color unevenness but within a range acceptable in quality, Δ, and a case where color unevenness was large and quality was unacceptable as x.
(2) Adhesion to magnet (appearance evaluation) Transfer the carrier prepared by the method shown in the example to a glass petri dish, add RO water (reverse osmosis water) until the carrier is submerged, and then rotate at a magnetic flux density of 0.3T. A child take-out rod (manufactured by ASONE Co., Ltd.) was inserted into the system, and the state of adhesion to the magnet and the state of being attracted were evaluated by visual observation. The evaluation results are indicated by ○ when attached, and × when not attached or cannot be taken out of the system.

(3) Scanning electron microscope (SEM) observation The carrier prepared by the method shown in the examples was washed with RO water, methanol, ethanol, and t-butyl alcohol in this order, solvent exchanged, and lyophilized. After freeze-drying, gold was sputtered onto the gel according to a conventional method, and observed at 300 times using JEOL JSM6060LV. In addition, for cells grown with granular porous cross-linked chitosan, after washing with 0.1 M phosphate buffer (pH 7.0), cross-linking treatment with glutaraldehyde was performed for the process and observed at 1000 times did.
(4) Ash content measurement In a hot air circulating drier, a sample completely dried under conditions of 105 ° C and 4 hours was placed in a ceramic crucible with a known tare weight and then ashed under conditions of 600 ° C and 5 hours in an electric furnace. Turned into. After ashing, it was immediately put into a desiccator, allowed to cool to room temperature in the desiccator, weighed, and determined the ash content from the following formula.
Ash content (%) = calcined residue weight (g) / absolute dry sample weight (g) × 100
(5) Pre-culture of yeast yeast 25 ml of 108 medium (aqueous solution comprising glucose 10 g / l, peptone 5 g / l, yeast extract 3 g / l, malt extract 3 g / l L) in an autoclave at 121 ° C. for 20 minutes After being sterilized, the mixture was allowed to cool to room temperature, inoculated with 1-platinum yeast yeast (Candida albicans NBRC1385), and cultured at 24 ° C. for 24 hours with shaking (70 rpm).

(6) Cultivation of yeast yeast in carrier After putting 2.5 g of chitosan beads (water content about 80%) into a 100 ml Erlenmeyer flask, 25 ml of 108 medium was added, and the condition of 121 ° C. for 20 minutes in an autoclave Sterilized with. After allowing to cool to room temperature, 50 μl of the preculture was added to the system and cultured at 24 ° C. for 40 hours with shaking (shaking speed: 70 rpm).
(7) Quantification of the number of inhabiting yeasts After the cultured beads were washed with physiological saline, 20 immobilized beads were taken and placed in a 10 ml graduated cylinder together with 1 ml of physiological saline. The beads were broken using a glass rod in the cylinder, irradiated with 40 Hz ultrasonic waves for 10 minutes, and then diluted to 10 ml with physiological saline. The number of yeasts was counted according to a conventional method using a hemocytometer (manufactured by Nippon Clinical Instrument Co., Ltd.).

[Example 1]
As a granular porous crosslinked chitosan, 25 ml of Fujibo Holdings' brand name Chitopearl HP-5020 (particle size 800 to 1,200 μm, specific surface area 20 to 30 m ² / g) was weighed in a plastic sealed container, and a 20 μm sieve was obtained. The water was lightly removed with a dropper attached to the tip. The whole amount of a solution prepared by dissolving 1.625 g of ferrous chloride / tetrahydrate and 2.5 g of ferric chloride / hexahydrate in 45 g of RO water was added here. Mix well. Subsequently, the reaction system was heated to 60 ° C. using a shaking water bath and shake mixed at 130 strokes / minute for 2 hours. After elapse of a predetermined time, a solution obtained by diluting 3.75 g of 28% ammonia water (manufactured by Wako Pure Chemical Industries, Ltd.) with 21.25 g of RO water was added to the reaction system while bubbling air and mixed well. At this point, the system turned brown. Thereafter, the temperature of the reaction system was raised to 60 ° C. again using a shaking water bath, and the mixture was shaken and mixed at 130 strokes / minute for 1 hour. After a predetermined time, the product was obtained by filtration and washing with water. Storage was performed in RO water at 5 ° C.

  FIG. 1 shows the state of the granular porous crosslinked chitosan before treatment, and FIG. 2 shows the state of the carrier for immobilizing microorganisms after the formation of magnetized particles according to the present invention. 1 and 2 are scanning electron microscope (SEM) photographs. As can be seen from the photographs, the porous structure inherent to the granular porous crosslinked chitosan was maintained as it was after the treatment. When the carrier for immobilizing microorganisms of the present invention was baked and incinerated, the ash content was evaluated to be 10.5%. The ash content before magnetization was almost zero. The observation of the remaining components of the crucible after firing and ashing the carrier for immobilizing microorganisms is a scanning electron micrograph as shown in FIG. 3, but the pore structure remains although shrinkage is observed by firing. The structure accurately traced the shape of the granular porous crosslinked chitosan.

  When the magnet was brought close to the wet granular porous crosslinked chitosan after the treatment, the granular porous crosslinked chitosan was attracted to and adhered to the magnet. Therefore, it can be said that granular porous crosslinked chitosan having magnetism was obtained. When yeast yeast was used as a model compound and its immobilization amount was evaluated, it was 8.81 × 10 5 cells / grain. When the growth of yeast yeast in the carrier for immobilizing microorganisms of the present invention was observed with a scanning electron microscope, it was as shown in the photograph in FIG. 4 and sufficiently maintained the performance as a carrier for immobilizing microorganisms. The evaluation results are summarized in Table 1 described below.

[Example 2]
The same operation as in Example 1 was performed except that the amount of each iron chloride and the amount of 28% ammonia water were reduced to 1/5. As a result, as shown in Table 1, a carrier satisfying the object of the present invention was obtained.

Example 3
The same operation as in Example 1 was performed except that 75 mol% of ferrous chloride and tetrahydrate and 25 mol% of ferric chloride and hexahydrate were used as the iron ion source. As a result, as shown in Table 1, a carrier satisfying the object of the present invention was obtained.

Example 4
The same operation as in Example 1 was performed except that 25 mol% of ferrous chloride / tetrahydrate and 75 mol% of ferric chloride / hexahydrate were used as the iron ion source. As a result, as shown in Table 1, a carrier satisfying the object of the present invention was obtained.

[Comparative Example 1]
The same operation as in Example 1 was performed except that only ferrous chloride tetrahydrate was used as the iron ion source. As a result, reaction unevenness was observed in the appearance of the carrier .
[Comparative Example 2]
The same operation as in Example 1 was performed except that the amount of each iron chloride and the amount of 28% ammonia water were doubled. As a result, as shown in Table 1, magnetization was possible, but a rapid decrease in the number of immobilized bacteria was observed, and a carrier satisfying the object of the present invention was not obtained.
[Comparative Example 3]
The same operation as in Example 1 was performed except that the amount of each ferric chloride charged and the amount of 28% ammonia water were quadrupled. As a result, as shown in Table 1, although magnetization was possible, a sharp decrease in the number of immobilized bacteria was observed, and a carrier satisfying the object of the present invention could not be obtained. Further, the ash content was almost the same as that of Comparative Example 1 and stopped to increase, which was not preferable from the viewpoint of economy.
[Comparative Example 4]
The same operation as in Example 1 was performed except that the charged amount of each iron chloride and the charged amount of 28% ammonia water were reduced to 1/10. As a result, as shown in Table 1, no decrease in the number of immobilized bacteria was observed, but the ash content decreased, adhesion to the magnet was not observed, and a carrier satisfying the object of the present invention was not obtained. .
[Comparative Example 5]
The same operation as in Example 1 was performed except that 100 mol% of ferric chloride hexahydrate was used as the iron ion source. As a result, as shown in Table 1, no adhesion to the magnet was observed, and no carrier satisfying the object of the present invention was obtained.

  From the results of Table 1, the ratio of ferrous chloride is in the range of 25 mol% to 100 mol% of the number of moles of hydrate, and the ash content of magnetized particles composed of ferrous ions and ferric ions. It has become clear that the increment is in the range of 4% to 25%, and that the amount of ammonia water charged is appropriate.

  The carrier for immobilizing a chitosan-based microorganism having magnetic properties of the present invention is advantageous in that it can easily control the pore size and can process the required amount, so that it can cope with small lots and various varieties as well as production stability. Moreover, since it has the ability to immobilize microorganisms and magnetism at a level that can be used practically, pure culture support for microorganisms, support for searching for new microorganisms, collection of magnetic bacteria, substitute for activated sludge, precision organic synthesis field, etc. It can be used and applied for various uses in the bio-related technical field.

It is a scanning electron micrograph which shows the cross section of the granular porous bridge | crosslinking chitosan before a process. It is a scanning electron micrograph which shows the cross section of the support | carrier for microorganisms fixation of this invention. It is a scanning electron micrograph which shows the cross section of the crucible residual component after baking and ashing the support | carrier for microorganisms fixation of this invention. It is a scanning electron micrograph of yeast yeast growing inside the carrier for immobilizing microorganisms of the present invention.

Claims (3)

As ash content increment of the monoferric ions and magnetic particles consisting of ferric ion is in the range of 4% to 25%, made by forming a magnetic particles in the particulate porous cross-linked chitosan, having magnetic A carrier for immobilizing chitosan-based microorganisms. The particulate porous cross-linked chitosan, were mixed in a solution containing a first monoferric ions and ferric ions, it is treated with an alkaline aqueous solution, a magnetic, characterized in that to form the magnetic particles in the particulate porous cross-linked chitosan A method for producing a carrier for immobilizing chitosan-based microorganisms. The magnetic chitosan-based microorganism according to claim 2, wherein the ratio of the number of moles of ferrous chloride hydrate in the treatment liquid of the granular porous crosslinked chitosan is from 25 mol% to 75 mol% of the number of moles of hydrate. A method for producing an immobilizing carrier.