JPS63309149A - Production of gel or immobilized gel - Google Patents

Abstract

PURPOSE:To obtain a reinforced gel or immobilized gel capable of withstanding biochemical reaction, etc., for a long period, by treating a gel or immobilized gel of alginic acid with strontium ions. CONSTITUTION:A solution of sodium alginate at about 0.1-0.8, preferably about 0.1-0.2 ratio (M/G) of D-mannuronic acid (M) to L-gluronic acid (G) containing, as desired, a biochemical activator, such as an enzyme, microorganism, animal or plant cell, added thereto is molded into a suitable form with calcium ions. The resultant gel or immobilized gel of alginic acid is then dipped in a solution containing strontium ions, e.g. solution containing the strontium ions in about 0.01-1.0mol. concentration or the above-mentioned solution is sprayed thereon.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for strengthening alginic acid gels or immobilized gels.

More specifically, 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 gel or immobilized gel strengthened 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. .

(Prior art) Generally, enzymes, microorganisms, animal cells, etc. are added to a sodium alginate solution, and the solution contains calcium ions.
It is well known that it can be applied dropwise or spun to form a gel and used for biochemical reactions and the like.

(Problems to be Solved by the Invention) However, the gel strength of the conventional gel obtained by reacting sodium alginate and calcilam ion is significantly reduced due to the influence of various salts contained in the reaction solution or changes in pH. There have been problems in that the gel is dissolved due to the presence of chelating agents such as phosphate ions.

Therefore, in order to improve the strength of the gel, large amounts of calcium ions are added to the reaction solution, but there is a problem that large amounts of calcium ions inhibit the biochemical activities of enzymes, microorganisms, animal cells, etc. It happens.

(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. A strong alginate gel was successfully obtained by treatment with strontium ions.

The present invention is a method for producing a reinforced gel, which is characterized in that a sodium alginate solution is shaped into an appropriate shape with calcium ions, and then treated with strontium ions to be strengthened.

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 a sodium salt obtained by extracting alginic acid contained in algae by chemical manipulation, and any commercially available sodium alginate can be used. However, alginic acid is composed of mannuronic acid (M) and L-guluronic acid (CG), and the ratio of these two components (M/G ratio) has a great influence on the physical properties.

Then, a part consisting only of M, a part 1M consisting only of G
Although there are parts where G and G are mixed, it is clear that it is mainly the function of the G block that forms a strong gel by combining with the metal ions of the gelling agent.

In the present invention, D-mannuronic acid (
It is preferable to select and use one having a ratio of M) to L-guluronic acid (G), that is, an M/G ratio of 0.1 to 0.8, more preferably 0.1 to 0.2.

Sodium alginate with such a low M/G ratio can be used in seaweeds with high G content or in parts that contain a large amount [stem].
It can be obtained by special preparation, such as by extracting selected stems and the season (May to August), or by blending those with a high G content.

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.

Calcium ions include calcium nitrate, calcium acetate, calcium lactate, and 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. , phosphoric acid, etc.

Some or most of the calcium that replaced sodium may be replaced with strontium, strontium may be newly replaced with remaining sodium, or a combination of these may occur, resulting in the gel being strengthened against phosphoric acid, etc. as a whole. However, 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)%
A concentration of ~8 (W/V)% is good, more preferably 0.8 (
The concentration is from 3(W/V)% to 3(W/V)%.

Biochemically active substances added to the sodium alginate solution include enzymes, microorganisms, animal cells, and plant cells.

Examples of enzymes include oxidoreductases such as alcohol dehydrogenase, D-amino acid oxidase, and catalase, transferases such as transketolase, adenylate kinase, and hexokinase, and hydrolyzing enzymes such as β-galactosidase, penicillinase, lipase, and esterase. Enzymes, fumarase, aspartase, threonine aldolase, β
- Lyase enzymes such as tyrosidase, isomerase enzymes such as glucose isomerase and alanine isomerase, and glue gauze 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 plant cells that produce various physiologically active substances.

The 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, l x 10' to l x 10' for 1++1 sodium alginate solution.
.

Preferably, I x 10' to 5 x 108 pieces 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 0.01-1
．． The concentration is about 0 molar, preferably about 0.02 to 0.3 molar.

! A 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. Syringe in an aqueous solution containing calcium ions,
A fibrous gel can be obtained by continuously discharging it from a nozzle such as a pipette.

3. After casting on a flat plate or impregnating filter paper or gauze, a membrane-like gel can be obtained by contacting with calcium ions in an aqueous solution.

The gel thus obtained is treated by immersing it in a solution containing strontium ions or by spraying the solution. The concentration of strontium ions is about 0.01 to 1.0 mol, preferably about 0.02 to 0.3 mol.

Processing time is 1 minute to 1 hour, PI (3 to 11, temperature 4 to
The temperature is about 50°C.

The alginate gel reinforced with strontium ions of the present invention has strong resistance to phosphoric acid.

When animal cell lines, hybridomas, etc. are contained in the gel, it is particularly effective because a phosphate buffer is used as the culture medium. Materials that do not disintegrate when immersed in a buffer solution, but are not reinforced with strontium ions, completely disintegrate within one day after being immersed in a phosphate buffer solution.

Next, examples of the present invention will be shown.

Example 1 A hybridoma culture suspension in which B lymphocytes were fused to commercially available mouse cells (manufactured by Dainippon Pharmaceutical Co., Ltd.) using a conventional method was added to 100 mA of a 2% sodium alginate solution.
Add and mix to make 0'kg/g gel, fill in a syringe, drop into 0.1M calcium chloride solution in ice water,
One granulated gel was obtained after being left for 0 minutes.

The obtained gel was statically cultured at 37° C. for 3 days in serum-free RDF medium.

Furthermore, the granulated gel was divided into 5 parts and added to 0°IN strontium chloride solution in ice water for 5 minutes and 10 minutes.

It was immersed for 20 minutes and 30 minutes, respectively.

Each of the obtained reinforced gels was incubated in serum-free RDF medium for 3 days at 37°C.
The cells were cultured statically and the amount of glucose consumed was measured.

The results are shown in Table 1.

After 3 days, each gel was soaked in 250 wM phosphate buffer (pH 7).
, 2), the strontium-untreated gel dissolved within 30 minutes, but the strontium-treated gel did not change its shape even after one week.
Strontium treatment had no effect on the cells.

Example 2 Commercially available young hamster kidney cells (Kl from Dainippon Pharmaceutical Co., Ltd.) were added to 2% sodium alginate solution 100IIQ at 4.7X.
The mixture was added and mixed at a concentration of 106 gels/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 into a column and 3% glucose added to BM.
3 for 65 days, changing 300 mQ of E medium every day.
Circulating culture was carried out at 0°C, and glucose consumption was measured.

After 65 days, the gel was taken out, immersed in a 0.1M strontium chloride solution in ice water for 20 minutes, and then packed into a column again and cultured in circulation at 30°C for another 10 days, replacing 300 tQ of BME medium supplemented with 3% glucose every day. Glucose consumption 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 drawings]

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 molding 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. The method for producing a gel or immobilized gel according to claim 1 or 2, wherein the sodium alginate is sodium alginate with an M/G ratio of 0.1 to 0.8. 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.