JPH0468896B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for strengthening alginic acid gels or immobilized gels. More particularly, the present invention relates to a method of treating an alginic acid gel or immobilized gel formed with calcium ions with strontium ions to strengthen the gel or immobilized gel. The strengthened gel or immobilized gel in the present invention can withstand long-term biochemical reactions without being weakened by culture medium containing phosphoric acid, etc., and is of great benefit to the biochemical community. There is. (Prior art) Generally, enzymes, microorganisms, animal cells, etc. are added to a sodium alginate solution, and the solution is dropped into a solution containing calcium ions or spun to form a gel, and then subjected to biochemical reactions, etc. It is well known to use (Problem to be solved by the invention) However, the gel strength of the conventional gel obtained by reacting sodium alginate and calcium ions is significantly reduced due to various effects contained in the reaction solution or changes in pH. There were problems in that the gel was dissolved due to the presence of chelating agents such as phosphate ions. Therefore, in order to improve the strength of the gel, a large amount of calcium ions are added to the reaction solution, but the large amount of calcium ions causes enzymes,
A problem arises in that the biochemical activities of microorganisms, animal cells, etc. are inhibited. (Means for Solving the Problems) As a result of intensive research to make alginate gel strong enough to withstand long-term culture, the present inventors have developed an alginate gel gelled using calcium ions. By treating with strontium ions, we succeeded in obtaining a strong alginate gel. The present invention is a method for producing a reinforced gel, characterized in that a sodium alginate solution is shaped into a suitable shape with calcium ions, and then treated with strontium ions to strengthen it. The present invention also provides a method for producing a strengthened immobilized gel, which comprises shaping a sodium alginate solution containing a biochemically active substance into an appropriate shape with calcium ions, and then treating and strengthening it with strontium ions. It is. The sodium alginate used in the present invention is obtained by chemically extracting alginic acid contained in algae and converting it into a sodium salt, and any commercially available sodium alginate can be used. However, alginic acid is D-mannuronic acid (M) and L
- guluronic acid (G), and the ratio of both components (M/G ratio) has a great influence on the physical properties. There are parts consisting only of M, parts consisting only of G, and parts where M and G are mixed, but it is mainly the function of the G block that combines with the metal ions of the gelling agent to form a strong gel. It has become clear that In the present invention, the ratio of D-mannuronic acid (M) and L-guluronic acid (G) constituting alginic acid, that is, the M/G ratio is 0.1 to 0.8, preferably 0.1 to 0.2.
It is best to select and use the following. Sodium alginate with such a low M/G ratio can be found in seaweeds with high G content, or in parts that contain a large amount [stem (stem)], and in the harvest season (5 to 5 days).
By selecting and adjusting the extraction (August), or by gifting one with a high G content,
It can be obtained by special preparation. When the M/G ratio is within the above range, the gel strength is high. The present invention is characterized in that the alginate gel treated with calcium ions is treated with strontium ions to strengthen the gel. As the calcium ion, calcium nitrate, calcium acetate, calcium lactate, calcium butyrate, calcium chloride, etc. are used. As the strontium ion, water-soluble salts such as strontium chloride, strontium nitrate, and strontium acetate are used, and strontium chloride is particularly easily available and easy to use. Specifically, a solution of sodium alginate with or without biochemically active substances is molded into granules, fibers, etc. with a solution containing calcium ions, and then the molded gel is treated with a solution containing strontium ions. It is strengthened against phosphoric acid, etc. Some or most of the calcium that replaced sodium is replaced with strontium, or strontium is newly replaced with remaining potassium,
Furthermore, combinations of these may occur, and the gel as a whole is thought to be strengthened against phosphoric acid, etc.
The details of why the gel is strengthened against phosphoric acid and the like by gelling with calcium ions and further treating with strontium ions are unknown. The concentration of sodium alginate is 0.5 (W/V)
% to 8 (W/V)% concentration is good, more preferably
The concentration is 0.8 (W/V)% to 3 (W/V)%. Biochemically active substances added to the sodium alginate solution include enzymes, microorganisms, animal cells, and plant cells. Enzymes include alcohol dehydrogenase,
D-amino acid oxidase, oxidoreductase such as catalase, transferase such as transketolase, adenylate kinase, hexokinase, hydrolase such as β-galactosidase, penicillinase, lipase, esterase, fumarase, aspartase, threonine Examples include lyase enzymes such as aldolase and β-tyrositase, isomerase enzymes such as glucose isomerase and alanine isomerase, and ligase enzymes such as glutathione synthase and glutamine synthase. Microorganisms are not particularly limited as long as they have enzymatic activity such as bacteria, yeast, molds, and actinomycetes. Animal cells include cell lines that produce various physiologically active substances and antibodies. There are hybridomas that produce bioactive substances, and there are also plant cells that produce various physiologically active substances. Biochemically active form is 0.01-50%, preferably 0.1
~20% is added to the sodium alginate solution and mixed. However, in the case of animal cells, 1×10 ⁴ to 1 ml of sodium alginate solution
×10 ⁹ , preferably 1 × 10 ⁵ to 5 × 10 ⁸ are added. A sodium alginate solution with or without the addition of a biochemically active substance is gelated by methods 1 to 3 below. The concentration of calcium ions used for gelation is about 0.01 to 1.0 molar, preferably
The concentration is about 0.02 to 0.3 molar. 1 Bead-shaped gel can be obtained by dropping it into an aqueous solution containing calcium ions from a nozzle such as a syringe or pipette. 2. A fibrous gel can be obtained by continuously discharging an aqueous solution containing calcium ions from a nozzle such as a syringe or pipette. 3. A membrane-like gel can be obtained by casting it on a flat plate or impregnating it into filter paper or gauze, and then contacting it with calcium ions in an aqueous solution. The resulting gel is treated by immersing it in a solution containing strontium ions or by spraying the solution. The concentration of strontium ions is 0.01~
The amount is about 1.0 mol, preferably about 0.02 to 0.3 mol.
Processing time: 1 minute to 1 hour, pH 3 to 11, temperature 4 to 50
It is about ℃. The alginate gel reinforced with strontium ions of the present invention has strong resistance to phosphoric acid. This method is particularly effective when animal cell lines, hybridomas, etc. are contained in the gel, since a phosphate buffer is used as the culture medium. In other words, those that have been strengthened with strontium ions will not disintegrate even if immersed in a phosphate buffer for a long period of one week or more, but those that have not been strengthened with strontium ions will completely disintegrate within one day of being immersed in a phosphate buffer. It's something you end up doing. Next, examples of the present invention will be shown. Example 1 A hybridoma culture suspension obtained by fusion of commercially available mouse cells (manufactured by Dainippon Pharmaceutical KK) B lymphocytes by a conventional method was added to 100 ml of a 2% sodium alginate solution.
Add and mix to make 1.0×10 ⁶ gel/g.
The mixture was filled into a syringe, added dropwise to a 0.1M calcium chloride solution in ice water, and left to stand for 60 minutes to obtain a granulated gel. The resulting gel was incubated at 37°C for 3 days in serum-free RDF medium.
The cells were cultured statically. Furthermore, the granulated gel was divided into five parts and placed in ice water.
0 min, 5 min, 10 min in 0.1M strontium chloride solution
Soaked for 1 minute, 20 minutes, and 30 minutes. Each of the obtained reinforced gels was statically cultured in a serum-free RDF medium at 37°C for 3 days, and the amount of glucose consumed was measured. The results are shown in Table 1.

[Table] When each gel was immersed in 250mM phosphate buffer (PH7.2) after 3 days, the strontium-untreated gel dissolved within 30 minutes, but the strontium-treated gel remained dissolved even after 1 week. There was no change in shape. Also, strontium treatment had no effect on the cells. Example 2 4.7% commercially available young hamster kidney cells (manufactured by Dainippon Pharmaceutical KK) were added to 100ml of 2% sodium alginate solution.
The mixture was added and mixed at a concentration of ×10 ⁶ pieces/g, filled into a syringe, added dropwise to a 0.1M calcium chloride solution in ice water, and left to stand for 60 minutes to obtain a granulated gel. The resulting gel was packed in a column and cultured in circulation at 30°C for 65 days while replacing 300 ml of BME medium supplemented with 3% glucose every day. Glucose consumption was measured. After 65 days, the gel was taken out and placed in 0.1 M strontium chloride in ice water. Immerse in the solution for 20 minutes, then repack into the column and add 300 ml of BME medium supplemented with 3% glucose.
The cells were cultured in circulation at 30°C for another 10 days, changing the amount every day, and the amount of glucose consumed was measured. The results are shown in Figure 1, which shows that treatment with strontium chloride has no effect on the cells.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the effect of treatment with strontium chloride on cells in Example 2.

Claims (1)

[Claims] 1. A method for producing a reinforced gel, which comprises shaping a sodium alginate solution into an appropriate shape with calcium ions, and then treating and strengthening it with strontium ions. 2. A method for producing a strengthened immobilized gel, which comprises shaping a sodium alginate solution containing a biochemically active substance into an appropriate shape with calcium ions, and then treating and strengthening it with strontium ions. 3. Claim 1, wherein the sodium alginate is sodium alginate with an M/G ratio of 0.1 to 0.8.
A method for producing a gel or immobilized gel according to item 2 or 2. 4. The method for producing an immobilized gel according to claim 2, wherein the biochemically active substance is an enzyme, a microorganism, an animal cell, or a plant cell.