JPH0227981A - Gel-like artificial medium - Google Patents

Abstract

PURPOSE:To raise the safety of an artificial medium to living thing systems, etc., by dispersing synthetic stevensite having X-ray diffraction peaks at surface intervals of 16-26Angstrom in a state treated with ethylene glycol in water to prepare a gel-like artificial medium. CONSTITUTION:Synthetic stevensite comprising stevensite type magnesium sodium phyllositicate substantially containing only magnesium, sodium and silicon as metal components thereof and having X-ray diffraction peaks at surface intervals of 16-26Angstrom in a state treated with ethylene glycol is dispersed in water to prepare a gel-like artificial medium. The synthetic stevensite has substantially a chemical composition of a formula: MgxNaySi4O10(OH)2.Naz [x is >=2 and y is 0-0.1 under of a condition of z+y<3 and z is 0-1.0].

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a gel-like artificial medium necessary for sterile culture and tissue culture, and more specifically, the present invention relates to a gel-like artificial medium necessary for aseptic culture and tissue culture.
EnsiLe l is used as an active ingredient, and to this active ingredient, ingredients necessary for animal or plant tissue or growth and proliferation are added as necessary, and the mixture is colloidally dispersed in water or a mixed solution with a water-miscible organic solvent. This invention relates to a gel-like artificial medium.

(Prior Art) Agar has been commonly used as a bedding material for gel-like artificial media that have been used for animal or plant tissue culture.

Moreover, various organic compounds and inorganic compounds other than agar have been proposed.

For example, JP-A-60-2041)81 discloses a combination of carrageenan and galactomannan purified from seaweed, and JP-A-61-78:180 discloses a combination of carrageenan and galactomannan purified from seaweed. Iron oxide, bentonite, synthetic hectorite, etc. are disclosed.

(Problems to be solved) - The agar used in IilQ has poor transparency, solubility,
There are problems in terms of deterioration over time (oxidation and decay), price, etc., and improvements are particularly needed in terms of transparency.

Artificial media using other organic compounds are also susceptible to deterioration such as oxidation and putrefaction, and are inferior to inorganic compounds in terms of stability.

Bentonite, an inorganic compound, is a naturally occurring mineral, and even in the case of semi-synthetic products, natural clay minerals are used as the silicic acid raw material, and this is manufactured by alkali activation treatment. Cases are also based on natural minerals.

Therefore, contamination with impurities is unavoidable, and when this bentonite is swollen and dispersed in an aqueous medium, it becomes turbid, resulting in poor transparency and color tone. Furthermore, although bentonite aqueous dispersions exhibit relatively high viscosity, they also have a problem of being somewhat inferior in thixotropy, which is one of the rheological properties.

On the other hand, synthetic hectorite is known as a synthetic smectite mineral of the phyllosilicate class, and an aqueous dispersion of this synthetic hectorite has excellent transparency and thixotropic properties.

However, this hectorite contains lithium as a monovalent metal component and fluorine ion as an anion component, and since it contains these impurity components, there is concern that it may have an adverse effect on the organism or tissue that is to be cultured. However, its use is naturally limited.

In general, the steven site is expressed by the following formula +1), (Mga
, asMno, oaFeo, otlsi4010 (O
HI * ・(Ca, Mg) O, O?・・・・・・ (1
) This mineral is magnesium phyllosilicate, i.e. 1) 1g3sj
40+. (This is a compound in which part of the magnesium component of OHI 2 is replaced with another metal, and the other part is vacant.

Attempts to synthesize stevensite have already been known, for example, in Clays and Clay Minerals, Vol. 27, No. 4, pp. 253-260, p. 97.
9) describes that stevensite is synthesized by heat-treating a Seviolite-water system.

However, the naturally occurring stevensite is
1) As shown in the formula, stevensite contains coloring components such as iron and manganese, and stevensite obtained by the above-mentioned known synthesis method contains a considerable amount of unreacted seviolite. Therefore, neither is suitable as an artificial culture medium.

(Means for Solving the Problems) The present inventors have succeeded in producing synthetic stevensite consisting of only three metal components, Mg, Si, and Na, and a gel-like artificial medium containing such synthetic stevensite as an active ingredient. teeth,
It has been found that it has excellent properties such as transparency and solves the problems of the conventional artificial culture medium mentioned above.

That is, according to the present invention, the metal component is substantially magnesium,
A stevensite-type magnesium sodium phyllosilicate consisting only of sodium and silicon, which is characterized by a colloidal dispersion of synthetic stevensite in water, which has an X-ray diffraction peak at a lattice spacing of 16 to 26 when treated with ethylene glycol. A gel-like artificial medium is provided.

(Function) The synthetic stevensite used in the present invention contains Mg, S
It is made up of only three types of metal components, i. The liquid becomes clear without becoming milky.

As a result, when this synthetic stevensite is used as a gel-like artificial medium, its thixotropic properties work effectively as an artificial medium, and a gel-like artificial medium that does not contain impurities and has excellent transparency is prepared.

Furthermore, the synthetic stevensite used in the present invention is a stevensite-type magnesium sodium phyllosilicate whose metal components are essentially only ug, Si, and Na, and does not contain fluoride ions (F-), so it is suitable for living organisms. It is highly safe for cultured bodies such as living tissues and tissues.

Furthermore, when the synthetic stevensite is used in combination with a water-soluble polymer, the thixotropic properties are further significantly improved, and a suitable gel-like artificial medium is prepared.

(Preferred Embodiment of the Invention) A synthetic stevensite The synthetic stevensite preferably used for the purpose of the present invention substantially has the following formula, Mgx Nay S14010 (OH1z'Naz
`` ' In the formula (2), It has a chemical composition expressed as , which is a number up to 1.0.

The chemical composition of formula (2) above is determined as follows.

That is, from the compositional analysis of one synthetic compound, M per Si4 atom
Number of atoms of g (x) Total number of atoms of i5 and (Na) (X+
Z) is required.

Next, from the compositional analysis of the cation-exchanged product of the synthesized compound with ammonium ions, the N existing in the layer was determined.
The atom R(Y) of a is determined.

In this way, the number of atoms (x, y, z) in formula (2) can be determined. Here, Z is the commutativity N that exists between the layers
It is simply the sum of the number of atoms of a and the number of attached Na atoms (α).

The synthetic stevensite used in the present invention has the above formula (2)
, x+Y is less than 3, especially in the range of 2 or more.

Or, within a range that satisfies this condition, it is a value of 2 or more, and a range of 2.6 to 2.8 is preferable.

Y also has a value of 0 to 0.1 within a range that satisfies the above conditions, and preferably a value of 0 to 0.05.

The value of Z generally ranges from 0 to 1.

In addition, theoretically, the value of the cation exchange capacity ffi (Z-α) per molecular weight in the above compositional formula is calculated by the following formula, 12-a l =
Y + 2 (3-X-Yl = i:l) In the formula, a simply represents the number of Na atoms attached.

The synthetic stevensite used in the present invention exhibits an X-ray diffraction pattern characteristic of smectite karikami mineral. Figure 1 of the attached drawings shows
This is an XLS diffraction image of the above synthetic stevensite.

Furthermore, when smectite and smectite-containing mixed layer minerals are treated with ethylene glycol, bottom reflection of X-rays appears in 16 to 26 people. The synthetic stevensite used in the present invention has this characteristic, as shown in FIG. 1 of the accompanying drawings.

The synthetic stevensite described above is different from hectorite in that it is a hydrate of magnesium silicate belonging to the smectite family.

However, while the alkali metal component in the layer of this synthetic stevensite used in the present invention is Na,
In hectorite, the alkali metal component in the layer is Li, and in hectorite, the total atoms u fX + Yl of Mg and Li in the layer is 3, whereas in the synthetic stevensite, Mg and Na in the layer are Total atoms with! &
(The difference is that X+Yl is smaller than 3.

In the synthetic stevensite used in the present invention, the fact that the total number of atoms fX+Yl of Mg and Na in the layer is smaller than 3 means that 41
This indicates that some of the g atoms are substituted with Na, and the remaining parts are vacant. Furthermore, Mg
Other parts of the atoms may also be substituted with hydrogen atoms. The 5104 tetrahedral layer -M (< (N
Al Da octahedral layer - 5i04 Tetrahedral layer Basic layer structure has Na ions between the laminated layers. When water is mixed with synthetic stevensite, water is formed between the basic layers (plates). enters and swells, but eventually the basic layer disperses into a colloidal state and becomes a fluid state (state
).

When this (state I) is allowed to stand, a house of cards structure is formed due to attraction and repulsion between the basic layers, resulting in a highly thickened or gelled state (state II).

The shear stress required to return from state II to state (4) is gel stress.

The synthetic stevensite used in the present invention has this gel stress,
That is, the measurement method is as follows: Using a fan VG rotational viscometer at a solid content concentration of 4% by weight, rotate at 600 revolutions/minute for 10 seconds, stop the rotation, leave for 10 seconds, and then rotate at 3 revolutions/minute. The gel stress, defined as the shear stress (ffb/loft, 2) measured by
In particular, it is in the range of IO to 100, making it possible to form a stable gel state. This indicates that the synthetic stevensite used in the present invention has a large number of card house structures formed per unit volume and has a dense structure.

Further, the larger the value of this gel stress, the more thixotropic it is. From this, it is understood that the synthetic stevensite used in the present invention has significantly thixotropic properties.

Preparation of A-formed stevensite The synthetic stevensite used in the present invention is obtained by subjecting an aqueous mixture containing basic magnesium carbonate and a combination of sodium silicate or amorphous silica and sodium hydroxide to a hydrothermal treatment.

By selecting basic magnesium carbonate as the magnesium raw material, stevensite can be synthesized, and production with high yield and high purity becomes possible.

Any basic magnesium carbonate can be used, but if magnesium carbonate, magnesium hydroxide, or a mixture thereof is used, it is not possible to produce stevensite in high yield and purity.

As the basic magnesium carbonate, it is particularly desirable to use hydromagnesite, which has a chemical composition represented by the following formula (%) and an X-ray diffraction pattern assigned to AST152-513.

As the Si and Na component raw materials, a sodium silicate aqueous solution is advantageously used, but combinations of amorphous silica and sodium hydroxide and amorphous silica and sodium silicate can also be used. As the sodium silicate, sodium silicate of the formula % formula % (51 where n is a number from 1 to 5, particularly from 2.0 to 3.5) is used.Also, amorphous silica As the material, silica hydrosil, hydrogel, xerogel, wet amorphous silica, vapor phase amorphous silica, etc. are used.

The ratio of basic magnesium carbonate to sodium silicate or amorphous silica and sodium hydroxide is such that the atomic ratio of magnesium to silicate is Mg:Si in the range of 3 to 5:5. It is better to use more than the stoichiometric amount of sodium. When sodium silicate is used, an excess of sodium will be present in the system even without the addition of special sodium hydroxide.

Prior to the hydrothermal reaction, the raw materials used are mixed as uniformly as possible to form a homogenized aqueous slurry.
Desirable from the standpoint of improving yield and purity. This homogeneous mixing is preferably carried out under strong shear stirring (for this purpose, a high-speed multi-stage mixer, ball mill, sand mill, colloid mill, ultrasonic irradiation, etc. can be used).

In addition, a small amount of sodium silicate has the effect of uniformly dispersing basic magnesium carbonate in an aqueous solution, so when using sodium silicate as a raw material, it is necessary to disperse basic magnesium carbonate with sodium silicate in advance. Even if you add the remaining ingredients after mixing the slurry,
The purpose of uniform mixing can be achieved. The sodium silicate cum used in this case is preferably used in an amount of 0 to 10% by weight based on the aqueous slurry.

It is also desirable that the solid content concentration in the aqueous mixture is generally in the range of 1 to 30% by weight, particularly 5 to 15% by weight.

This mixture is charged into an autoclave and subjected to hydrothermal treatment.The hydrothermal treatment conditions may be relatively mild compared to conventional methods. ~100 kg/c
+s” (gauge), especially 6 to 40 kg/cI12
It is best to do this under pressure of G. The reaction time is generally 0.5
Synthetic stevensite obtained by a reaction in which a reaction time of 20 to 20 hours is sufficient is separated into individual liquids from the mother liquor, washed with water, and dried to obtain a product.

■-1 The synthetic stevensite used in the present invention can be prepared by combining water or water with alcohols such as methanol, ethanol, ethylene glycol, propylene glycol, and glycerin; ketones such as acetone and methyl ethyl ketone; methyl ether, ethyl ether, tetrahydrofuran, and dioxane. Ethers such as: Cellsolve solvents: Water-miscible organic solvents such as esters such as ethyl acetate, etc. Swell with any mixture.

Gives a clear colloidal thickened liquid or gel-like solid.

In this way, the stevensite synthesized here imparts thixotropic properties or imparts a transparent colloidal thickened liquid or gel-like solid when dispersed in water or a mixture with a water-miscible organic solvent. Therefore, tissue culture of animals and plants can be used as a bedding material for growing and culturing plants.

The mixing ratio of the synthesized stevensite and the mixture of water or a water-miscible organic solvent varies depending on the thixotropic properties required of the culture bed material, but in general,
A range of 0.5 to 10% by weight, in particular 1 to 8% by weight, based on the final water-containing culture bed is suitable.

In order to maintain the thixotropic properties by mixing the synthesized stevensite with a water-miscible organic solvent, the synthesized stevensite must be thoroughly mixed with water or a water-miscible organic solvent mixture, and the colloid must be mixed with water or a water-miscible organic solvent. It is important to form a dispersion liquid.

The conditions for forming a colloidal dispersion can be achieved by using conventional means of stirring at room temperature or under heating. Further, this dispersion can be performed under pressure, and can be performed simultaneously with the sterilization operation under heat and pressure required for the culture medium. -Temperature during radiation stirring ranges from room temperature to 200℃
It is preferable to keep the temperature in the range of ℃, and the stirring time is 5 to 12
It is preferable to set it in the range of 0 minutes.

The colloidal dispersion can be turned into a gel by leaving it to stand for at least 10 minutes.

According to the present invention, when this synthetic stevensite is mixed with water or a mixture of water-miscible organic solvents, if a water-soluble polymer is used in combination, the effect of imparting thixotropic properties can be further improved.

The combination ratio of synthetic stevensite and water-soluble polymer can vary over a wide range, but is generally between 10:0 and 2:0.
8. In particular, it is preferable to use a weight ratio of 9:1 to 5:5.

Water-soluble polymers include various starches, cyanoethylated starch, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose (CMC), l-laganth gum, gum arabic, sodium alginate, polyvinyl alcohol, partially saponified polyvinyl acetate, partially acetalized polyvinyl alcohol. , polyvinyl methyl ether, polyvinyl pyrrolidone, polyacrylamide, water-soluble acrylic resin, etc. or two.
Combinations of more than one species can be mentioned.

Among these, water-soluble cellulose derivatives such as carboxymethyl cellulose are preferred.

When the gel-like artificial medium of the present invention is used as a bed material for aseptic culture or tissue culture, it may contain other gelling agents, fillers, nutritional components, etc. as necessary.

For example, gelling agents include agar, carrageenan, phaseleran, etc., fillers include polysaccharides, etc., and nutritional components include glucose, carbohydrates such as sucrose, plant hormones (growth regulators), vitamins, amino acids, natural Fertilizers (coconut milk, bananas, potatoes, etc.), inorganic salts (commercially available fertilizers containing nitrogen, phosphoric acid, potassium, etc.), etc. Examples of specific formulations of these media are shown in the Examples.

A colloidal dispersion of this synthetic stevensite with a mixture of water-miscible organic solvents is used as a gel-like artificial medium.
Due to its excellent transparency and stable gel state, it can be used for the cultivation and preservation of various bacteria, yeast, and molds, animal and plant cell culture, cultivation of plant seedlings, embryos, organs, tissues, etc., and horticulture. It can be suitably used as a bedding material for the breeding and breeding of species, for various pathological research, etc.

(Effects of the Invention) According to the present invention, the metal component is substantially composed of only the three components Mg, Si, and Na, and a synthetic stevensite containing no fluoride ions (F-1) is used as a gel-like artificial medium and colloidal in water. By dispersing and using it in the form of a colloid, it is highly safe for living organisms and living tissues, has excellent transparency, and has the advantage of being able to form a gel state that is colloidal but has hardness.

(Example) The present invention will be explained with the following specific example. Note that this example is just one example of the many culture media that can be used, and only shows an example of how this culture medium can be usefully employed in a variety of fields.6 The test method for synthetic steven sites was as follows: Ta.

(1)X! 1 diffraction X-ray diffraction device manufactured by Rigaku Denki Co., Ltd. (X-ray generation device 40
36＾1. Goniometer-212501, counting device 50
The zero diffraction conditions using 71) are as follows.

Target Cu Filter Ni Detector SC Voltage 35KVP Current 15mA Count/Full Scale 8000C/S Time Constant
1 sec Scanning speed 2°/sin Chat speed
2 cm/n+in radiation angle
l.

Slit width 0.3m ■ Viewing angle 6° (2) xIi diffraction of ethylene glycol treated sample 1) 0
Collect 1.0 g of a sample that has been dried at ℃ for 2 hours.

Add 5 ml of 10% ethylene glycol aqueous solution to this using a whole pipette. Use a stirring rod (stir and then heat to 60°C.
Dry for 12 hours. Grind the dried material in an agate mortar.

The powder thus recovered was subjected to X-ray diffraction measurement under the following conditions.

Target Cu Filter Ni Detector SC Voltage 40KVP Current 20mA Count/Full Scale 2000C/S
Time constant: 2 sec Scanning speed: 1'/win Chart speed: 1 cm/win Radiation angle: 1/6° Slit width: 0.3 mm Glancing angle: 6° Measurement diffraction angle range: 1° to 12° (2θ) Plane spacing (d
) was calculated by the following formula from the diffraction angle (2θ) determined from the midpoint of the half width.

d=(E/2) 5in-' (θ) However, the tip is
The line wavelength is 1.542 people.

(3) Gel stress A sample solution in which synthetic stevensite powder was dispersed in water to a solid content concentration of 4% by weight was placed in a constant temperature bath at 25°C for 2 hours.
Store for 4 hours. This sample solution was mixed with a mixer for 15 minutes.
Disintegrate for 1 minute at a rotation speed of 00 rpm. Next, using a fan VG rotational viscometer, first rotate it at 600 rpm for 10 seconds, stop the rotation and leave it for 10 seconds, then rotate it at l rpm to determine the shear stress (lb/100 ft2).

(4) Transparency Add water to the test solution in which the gel stress was measured to prepare a sample solution with a solid content concentration of 2% by weight. This sample solution is placed in an optical cell with an optical path length of 10n+■, and the transmittance at 620° is measured using pure water as a comparison, and is expressed as transmittance %.

(5) Prepare a sample solution of 2% by weight based on the solid content concentration that changes over time, and use this sample solution 5
0g was placed in a 10On+1 Erlenmeyer flask, capped with aluminum foil, placed in an autoclave, and incubated at 120℃ for 2 hours.
Steam sterilize for 0 minutes.

Next, the sterilized test solution is transferred to a petri dish in a clean bench and left to cool.

After confirming that the sample liquid in the Petri dish has turned into a gel,
Invert the petri dish and store it in a 31'C incubator.

After 2 weeks, the gel plate in the petri dish was visually observed to see if there were any cracks, and those with no cracks were rated as "no change over time" (good).

A Steven site ;-.

Commercially available basic magnesium carbonate (TT manufactured by Tokuyama Soda) 14.
Put 5g (magnesia content: 6g) into about 150ml of water, add 54g of No. 3 sodium silicate (silica content: 12g), and stir to prepare a dispersion slurry.

This dispersed slurry is placed in an autoclave having an internal volume lβ. Hydrothermal treatment is carried out at 170° C. for 5 hours while stirring.

After the reaction was completed, the contents were taken out, filtered and dried to obtain 46 g of product.

This product was ground in a sample mill to obtain 20 g of synthetic stevensite powder, water was added to make 500 g, and the mixture was stirred and mixed to prepare a 4% by weight gelled liquid (A).

The X-ray diffraction spectrum of the synthetic stevensite powder and the X-ray diffraction spectrum of the ethylene glycol-treated powder are shown in Figures 1 and 2, respectively. Each characteristic was measured and the results are shown in Table 1.

Table 1 Water was added to the synthetic stevensite powder prepared by the above method to obtain a gel-like liquid with a solid content concentration of 10% by weight.

Add 1% by weight of carboxymethyl cellulose solution (manufactured by Dial Kagaku Kogyo Co., Ltd. 1) to 180 g of this gel-like liquid (50) 42
Add 0g and mix with stirring to obtain 4% by weight mixed gel liquid (B
) was prepared.

Powdered agar (manufactured by Wako Tsumugi Kogyo Kogyo) was selected as a comparative example of the first raw and blade.
Add 300 m 12 of water to 20 g of this agar, heat and stir,
Dissolved homogeneously.

Further, water was added to make the total amount 500 g to prepare an agar solution (C).

Test Example l Gel-like liquid prepared above, mixed gel-like liquid and *1
: * 2 : ○... No cracks, ×... Cracks, unable to return from gel state (state II) to fluid state (state ■).

Test Example 2 Mixed gel liquid prepared by the above method (80 g of Bl,
1 ml of Murashige Skoogl medium aqueous solution (main components: see Table 2) was added and mixed and stirred.

The resulting mixed solution had a diameter of 18.2 mm, had good gel strength as a tissue culture medium after being left for 30 minutes, and formed a gel-like medium plate for testing.

Matusig runners were placed on the test gel-like medium plate and kept at 25°C for 30 days to perform tissue culture.
The culture status was observed.

As a result, since the medium did not crack and was transparent, it was easy to observe the state of root growth in detail, confirming the superiority of the artificial medium made of the synthetic stevensite of the present invention.

Table 2 Murashige Skoog solution (1g/42)C
aC1z4Hz0 440 KN0. 190O NH,NO,1650 KIItPO4170 FeSO44H*0 27.8Nag-EDT
A 37.3Mn5O,・4H,022
,3 ZnSOn-7HzO8,6 H3B0. 6.2 Sucrose
30000 Test Example 3 To 150 g of the gel-like liquid (A) prepared by the above method, B
5. 300 ml of inorganic salt for medium (main components; see Table 3) was added and mixed and stirred.

The resulting mixed solution had a pH of 7.6, had good gel strength as a seed germination medium after being left for 30 minutes, and formed a gel-like medium plate for testing.

Seeds of Kaiwari radish were sown on the test gel-like medium plate and kept at 20°C for 7 days to observe the germination state of Kaiwari radish.

The germinated cotyledons were approximately 5c+ on this test medium plate.
It was often observed that the plants were growing smoothly and did not fall over even if they became elongated.

Moreover, due to the rheological properties of synthetic stevensite, this artificial medium made of synthetic stevensite can return from a gel-like state to a fluid state by externally applying stress such as vibration to the medium plate. We were able to take out the grown plants from this medium without damaging them, and were able to understand the characteristics of the present invention.

Table 3 Inorganic salt for B5 medium (g/l OH3,33 HNO34,00 1) 3Po4 1.73

[Brief explanation of the drawing]

FIG. 1 shows the x-ray diffraction spectrum of the synthetic stevensite prepared in the example of the present invention using α-rays on Cu-. FIG. ! This is a diffraction spectrum of s bottom surface reflection.

Claims (3)

[Claims] (1) Synthetic stevensite-type sodium phyllosilicate whose metal components are essentially only magnesium, sodium, and silicon, and which has an X-ray diffraction peak in the interplanar spacing of 16 to 26 Å when treated with ethylene glycol, is colloided in water. A gel-like artificial medium characterized by dispersion. (2) The synthetic stevensite is substantially of the following formula Mg_XN
a_YSi_4O_1_0(OH)_2・Na_Z In the formula, X and Y are a number of 2 or more, Y is a number of 0 to 0.1, and Z is larger than 0, under the condition that X+Y<3. The gel-like artificial medium according to claim 1, which has a chemical composition represented by the following, which is a number up to 1.0. (3) Synthetic stibunsite was measured using the following method, using a fan VG rotational viscometer at a solid content concentration of 4% by weight, rotating at 600 rpm for 10 seconds, stopping the rotation and leaving it for 10 seconds, then 3 rotations/min. Claim 1, wherein the gel stress defined by the shear stress (lb/100ft^2) measured by rotating the gel is 5 or more.
The described gel-like artificial medium.