JP2904568B2 - Gas permeable culture stopper - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention has a higher gas permeability than gas-permeable culture stoppers, especially conventional products, and does not decrease in gas permeability even during long-term use. Accordingly, the present invention relates to a gas-permeable culture stopper that is useful for long-term culture of bacteria.

(Conventional technology) As a culture stopper used for culturing bacteria, a cotton stopper obtained by rolling cotton to an appropriate hardness has been conventionally used. However, in this method, the hardness of the cotton stopper is insufficient. If the density of the cotton yarn is insufficient, the bacteria will invade and the effect of preventing the invasion of the bacteria will be incomplete.On the contrary, if the cotton thread is hardened and the density of the cotton yarn is increased, the air permeability will decrease, and it will be cultured. There is a problem that the bacteria to be killed are killed, and therefore, the operation of making the cotton plug requires a great deal of skill.

Therefore, a rubber foam is used as the culture stopper, and a silicone rubber-made stopper is widely used because it can withstand sterilization treatment at a high temperature during many uses.

(Problems to be Solved by the Invention) However, as for the culture stopper using the silicone rubber foam, the silicone rubber foam is obtained as an independent foam in which the foaming space does not form a chain, and has a low gas permeability. Therefore, it is necessary to apply pressure to the foam with a roll or the like to break closed cells and form a chain to improve gas permeability. In this case, the formation of chains is not stable, and the gas permeability varies depending on the product. This is also due to the fact that excessive destruction of closed cells in order to improve gas permeability results in gas permeability. However, at the same time, there is a problem that the effect of preventing invasion of various bacteria is reduced. Since there is a case where the gas permeability is reduced due to the adhesion and the culture may be hindered, there is also a disadvantage that the culture stopper needs to be replaced during the culture.

(Means for Solving the Problems) The present invention relates to a gas-permeable culture stopper capable of solving such disadvantages, which comprises a zeolite having molecular sieve characteristics dispersed in a rubber material. It is characterized by the following.

That is, the present inventors have made various studies to develop a gas-permeable culture stopper that does not invade various bacteria, provides stable high gas permeability, and does not decrease in gas permeability even after long-term use. Assuming that the culture stopper is a zeolite having molecular sieve properties dispersed in a rubber material, the gas permeability becomes large, and it is found that this gas permeability does not decrease even after long-term use. However, if this zeolite is subjected to hydrophobic treatment with organosilazane, gas permeability does not decrease due to water absorption or the like, and a gas-permeable culture stopper having an antibacterial effect can be obtained by doping zeolite with metal ions. Furthermore, if this rubber material is formed into a foam during molding, the gas permeability is further improved, and the followability of the molded body to a complicated shape is improved. The present invention has been completed by sure.

 This will be described in more detail below.

(Operation) The present invention relates to a gas-permeable culture stopper that has higher gas permeability and does not decrease in gas permeability even after long-term use.

The gas-permeable culture stopper of the present invention is characterized in that zeolite having molecular sieve characteristics is dispersed and arranged in a rubber material.

The rubber material may be the one conventionally used in the production of this type of culture stopper, and thus includes silicone rubber, acrylic rubber (ACM), ethylene propylene diene rubber (EPDM), fluoro rubber (FKM), Units such as urethane rubber (U) and chlorosulfonated ethylene rubber (CSM), or a polymer of two or more of these units, and a copolymer such as acrylic silicone rubber are exemplified. Since it is necessary to perform heat treatment at a temperature of, for example, 100 ° C. or more for several minutes to several tens of minutes during sterilization, it is preferable to use a silicone rubber having good heat resistance, and this is also a compression set. When the strain is large, the distortion reduces the adhesiveness around the plug, and there is a possibility that bacteria and the like may enter. Therefore, it is preferable that the compression set is small.

In addition, since the zeolite compounded in this rubber material has pores on its surface, it only needs to exhibit molecular sieve characteristics, and when the zeolite is expanded with a large particle diameter, it becomes larger than the wall thickness. There is a risk that it will fall off due to deformation during use, and if it falls off, it will be in the form of a large hole and it will not be possible to prevent the invasion of various bacteria, and if this particle size is too small, it will be necessary to obtain high gas permeability. Since it is necessary to increase the blending amount, it is preferable that the pore size is in the range of 0.1 to 100 μm. However, it may be used in combination of several kinds having different particle sizes. In addition, when the zeolite absorbs moisture in the air and becomes water-containing, the gas permeability of the culture stopper using the zeolite decreases, so that the zeolite is preferably low in water absorption. If the Si / Al atomic ratio in is less than 10, the water absorption becomes extremely high.
It is good to be 0 or more.

The gas-permeable culture stopper of the present invention is made by mixing the above-mentioned rubber material with the above-mentioned zeolite and molding a rubber material obtained by dispersing the zeolite. No improvement can be obtained, and if the amount is too large, the rubber material becomes hard and the compression set becomes large.
It is preferably in the range of ~ 85% by volume. The mixing and dispersion of the zeolite into the rubber material can be performed by using a known mixing and dispersing apparatus, for example, an oven-type two-roll or three-roll, a pressure-type kneader, a Hensiel mixer, a Banbury mixer, a spiral mixer, an extruder, and the like. Good.

Further, the thus obtained zeolite is blended, molding of the dispersed rubber material may be performed by a known method,
Therefore, after mixing and dispersing a predetermined amount of zeolite into a rubber material, a crosslinking agent, a lubricant, an antioxidant, etc. may be added as necessary, and then molded. In the case of a single molded body as shown in (1), this composition may be placed in a mold and molded by applying pressure and heating, or covered with a stopper of a culture vessel in order to enhance its mountability. In order to obtain a cap type as shown in FIG. 2, the outer periphery of the cap type is formed of an unfoamed rubber material, and the composition of the present invention is formed in advance in a sheet shape. The sheet may be adhered and fixed, or the sheet may be integrally formed at the same time as the molding of the unfoamed body at the outer periphery, and according to this, the thickness of the gas-permeable core portion can be reduced, so that Fig. 1 shows the gas permeation Can be improved as compared with that of Gutaipu, which can be obtained the same effect as a hollow plug type as illustrated in FIG. 3,
Each of the culture stoppers manufactured in this manner is made of a rubber material mixed with zeolite having molecular sieve characteristics. It has a structure in which large molecules such as resin cannot enter, and the inside is hollow, which has adsorptive properties, so that conventional silica, titanium oxide, calcium carbonate,
Where a filler such as carbon is added to a rubber material, the gas permeability does not change or tends to decrease, whereas a culture stopper obtained by molding a rubber material containing this zeolite is a zeolite containing Since the chain apparently reduces the film thickness due to the chain, the gas permeability is advantageously increased.

However, since the gas permeable culture stopper of the present invention also absorbs water, its gas permeability is reduced. Therefore, it is preferable to improve the hydrophobicity. This treatment has at least one silazane bond in the molecule because the water repellent covers the fine pores on the zeolite surface with a thick film and greatly reduces its gas permeability. It is good to use organosilazane. This organosilazane has the formula {Si-NR-Si} (where R is a hydrogen atom or carbon 1
, For example, a compound in which a hydrogen group of silazane is substituted with a hydrophobic organic group. Represented by disilazane, trisilazane, 1,1,3,3-tetramethyldisilazane, hexamethyldisilazane, heptamethyldisilazane, 1,3-divinyl-1,1,3,3-tetramethyldisilazane, 1 , 3-Bis (chloromethyl) -1,1,3,3
-Tetramethyldisilazane, 1,1,3,3,5,5-hexamethylcyclotrisilazane, 1,1,3,3,5,5,7,7-octamethylcyclotetrasilazane, 1,3- Bis- (2-perfluorooctyl) ethyl-1,1,3,3-tetramethyl-1,3-
Examples include disilazane and the like, into which a hydrophobic trimethylsiloxy group can be introduced. It is preferable to use 1,3-bis (perfluoroalkyl) ethyl-1,1,3,3-tetramethyl-1,3-disilazane having a perfluoroaralkylsiloxy group introduced therein.

This hydrophobization treatment with organosilazane converts this to HMDS
For example, zeolite, HMDS and water may be put into a closed container and stirred, and according to this, HMDS has a boiling point (vapor pressure of 76 mmHg, 50 ° C) of 126 ° C, and a density of 0.77 ° C.
g / cm ² , 0.7cp viscosity liquid. One HMDS molecule easily reacts with two hydrophilic -OH groups ion-adsorbed on the zeolite surface to release ammonia. Can be easily and uniformly introduced into the zeolite surface in a monomolecular manner, but the treatment amount of the organosilazane used here varies depending on the particle size of the zeolite used, and tends to be unclear,
In this treatment, an organosilazane is added in excess to completely replace the --OH group on the zeolite surface with a trimethylsiloxy group, and then the atmosphere above the boiling point of the organosilazane used, the boiling point of the organosilazane being the boiling point of water10
When the temperature is lower than 0 ° C., the treatment may be completed by introducing the mixture into an atmosphere at 100 ° C. or higher and volatilizing excess organosilazane and water.

In addition, the gas-permeable culture stopper of the present invention preferably has an antibacterial effect in order to prevent a decrease in gas permeability due to the adhesion of culture bacteria during long-term use. It is preferable that zeolite mixed with the material is doped with a metal ion having a high antibacterial effect, for example, a metal ion such as silver, zinc, or copper. As a method for doping the metal ions, the zeolite particles are immersed in an aqueous solution of an appropriate concentration such as a nitrate or a sulfate of the metal, stirred, doped with metal ions, dried, and dried.
Heat treatment at a high temperature around 0 ° C (JP-B 63-540)
The antibacterial zeolite thus obtained may be subjected to the hydrophobizing treatment with the above-mentioned organosilazane, or the zeolite subjected to the hydrophobizing treatment and the zeolite described above may be used. As described above, the antibacterial zeolite may be combined with the rubber material and combined with the rubber material, whereby the advantage of preventing the growth of bacteria and mold can be provided.

In addition, the gas-permeable culture stopper of the present invention may be obtained by mixing zeolite into a rubber material, adding a foaming agent to the mixture, and foaming the mixture at the time of molding, so that the intended culture stopper is made of a foam. According to this, the gas permeability of the culture stopper can be further improved. This foaming agent needs to be appropriately selected depending on the heating and pressurizing conditions in molding and the type of the crosslinking agent to be used, but it may be any of a general inorganic foaming agent and an organic foaming agent such as a hydrazine derivative. Examples of the inorganic blowing agent include sodium bicarbonate and ammonium carbonate. Examples of the organic blowing agent include azodicarbonamide (ADCA), azobisisobutyronitrile (AIBN), and dinitrosopentamethylenetetramine (DP).
T), p-Toluenesulfonyl hydrazide (TSH), p, p'-
Oxybis (benzenesulfonyl hydrazide) (OBSH)
For example, a urea-based resin composition may be added as a foaming aid.

(Examples) Next, examples of the present invention will be described. Parts in the examples indicate parts by weight.

Example 1 100 parts of liquid dimethylpolysiloxane KE103 [trade name of Shin-Etsu Chemical Co., Ltd.] and CAT103 [Shin-Etsu Chemical Co., Ltd.]
Trade name] 20 parts by weight of an organopolysiloxane composition consisting of 5 parts, a hydrophobic zeolite / silicalite having an average particle size of about 1.5 μm and an infinite Si / Al ratio [manufactured by UCC, USA] Trade name] 79% by weight of powder and azobisisobutyronitrile (AIBN) as blowing agent [Otsuka Chemical Co., Ltd.
1.0% by weight in a crusher, and then mixed three times using a three-roll mill to obtain a completely dispersed putty-like composition.

Next, this compound was put into a mold for making the product shown in FIG. 1 equipped with a press machine, and about 50 kg / cm ² at 180 ° C.
Heated and pressurized 20 minutes condition to perform the curing foamed molded, when the molded article was removed from the mold, was heated 200 ° C. × 2 hours be complete crosslinking, D ₁ in FIG. 1 =
A plug-type gas-permeable culture stopper for a 100 ° C. flask having 32 mmφ, D ₂ = 22 mmφ, L ₁ = 20 mm, and L ₂ = 40 mm was obtained.

Then, the culture stopper for gas permeation was attached to a 100 cc Erlenmeyer flask, and the air flow rate of this with a 16 mm water column was measured with a free flow meter (LAS-S302), which was about 500 cc / min. The results show that the closed cell type obtained from the conventional rubber composition not containing zeolite had a ventilation rate of 100 cc / min. The closed cells were crushed 4 to 7 times using a metal roll. However, it was confirmed that the air permeability was clearly superior to that of the above, which had an air permeability of 50 to 100 cc / min.

Example 2 A varnish having a solid content concentration of about 50% and a viscosity of 2,000 p in toluene was added to the putty-like blend containing the zeolite used in Example 1 without adding AIBN, and this was placed on a fluororesin separator. Casting was performed using a knife coater so that the dried film became approximately 200 μm, and this was heated at 80 ° C. for 5 minutes to disperse the toluene, and then dried.
The resin was cured by heating at 0 ° C. for 30 minutes.

Then, the membrane was peeled off from the separator, and the gas permeability was measured using a gas permeability measuring device, Gas Palm 100 (trade name, manufactured by JASCO Corporation). ^{Pair = 1,500 × 10 -8 cm 3} · cm / c
m ² · sec · cmHg, indicating that the gas permeability was improved about 300 times as compared with the silicone rubber alone.

Therefore, this film is made of silicone rubber
In the hollow portion of the outer peripheral frame shown in FIG. 3 and FIG. 3, a one-component RTV rubber, KE-42 (trade name, manufactured by Shin-Etsu Chemical Co., Ltd.) is used as shown in the drawing. As a result, cap-type and hollow plug-type culture stoppers having excellent gas permeability were obtained.

Example 3 To 100 parts of zeolite / silicalite used in Example 1, 10 parts of hexamethyldisilazane (HMDS) -LS-7150 [trade name of Shin-Etsu Chemical Co., Ltd.] and 5 parts of pure water were added, and this mixture was added. Was placed in a ball mill container, mixed and crushed for 1 hour, and the mixture was left in a ball mill container at room temperature for 72 hours.
The mixture was heat-treated in an atmosphere at a temperature of 150 ° C., which is a boiling point of 126 ° C. or higher, for 2 hours to diffuse excess HMDS and water to produce hydrophobic silicalite.

Next, a plug-type gas-permeable culture stopper was prepared in the same manner as in Example 1 except that the zeolite / silicalite in Example 1 was changed to the hydrophobized silicalite thus obtained. However, this showed a result of 500 cc / min as in Example 1.

However, after the culture stopper thus obtained and the culture stopper obtained in Example 1 were immersed in water at 40 ° C. for 12 hours, moisture was removed using a centrifuge, and their gas permeability was examined. However, the gas permeability of the culture stopper obtained in Example 1 is
The gas permeation of the culture stopper obtained in Example 3 was 400 cc / min or more, and the decrease in gas permeability due to the adsorption of water was very low.

Example 4 The zeolite / silicalite of Example 1 was replaced with 50
% Of synthetic zeolite doped with Ag having a particle size of 2 to 5 μm.
A gas permeable culture stopper was prepared in the same manner as in Example 1 except that Bacte Killer (trade name manufactured by Kanebo Co., Ltd.) was used, and the gas permeability of the stopper was measured by the same method as in Example 1. The result was 300 cc / min.

Then, using this culture stopper and the culture stopper obtained in Example 1, the yeast was subjected to static culture, and this was cultured at 7 ml / tub.
e. The test was carried out at a culture temperature of 37 ° C. for one week. After the culture was completed, the gas permeability of each culture stopper was measured in the same manner as in Example 1. The gas permeation of the culture stopper prepared in Example 1 was measured. Although the rate dropped to 100 cc / min or less, the gas permeation rate of the culture stopper made using the Ag-doped zeolite prepared in Example 4 was 100 to 200 cc / min. It was confirmed that the transmittance was lower than that of Example 1.

(Effect of the Invention) The present invention relates to a gas-permeable culture stopper, which is characterized in that zeolite having molecular sieve characteristics is dispersed and arranged in a rubber material as described above. Since zeolite has a number of fine pores on its surface, when it is mixed with rubber material and molded, the gas permeability is high and this gas permeability does not decrease even after long-term use. The stopper can be easily obtained,
This has the advantage that the process of crushing closed cells is not required as in the past, and this product can be made to permeate even if it absorbs moisture if zeolite has been treated with organosilazane in advance to make it hydrophobic. If the zeolite is doped with metal ions in advance, the culture stopper will have an antibacterial effect, so even if used for a long period of time, the gas permeability will decrease due to the culture bacteria In addition, when a foaming agent is added to the rubber material at the time of molding to form the molded body into a foamed body, the gas permeability can be further improved.

[Brief description of the drawings]

FIG. 1 is a longitudinal sectional view of a plug type gas permeable culture stopper of the present invention, FIG. 2 is a longitudinal sectional view of a kip type gas permeable culture stopper of the present invention, and FIG. 3 is a hollow plug type gas permeable stopper of the present invention. 1 shows a longitudinal sectional view of a sex culture stopper.

Claims (4)

(57) [Claims] 1. A gas-permeable culture stopper wherein zeolite having molecular sieve characteristics is dispersed and arranged in a rubber material. 2. The gas-permeable culture stopper according to claim 1, wherein the zeolite has been hydrophobized with organosilazane. 3. The gas-permeable culture stopper according to claim 1, wherein the zeolite has been given an antibacterial effect by metal ion doping. 4. The gas-permeable culture stopper according to claim 1, wherein the rubber material is formed by foam molding.