JPH10108690A - Measurement of oxygen permeability coefficient in membrane material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring an oxygen permeability coefficient easily, economically, efficiently and in high accuracy. SOLUTION: Microorganisms are cultured for a given time in such a state as to lay a membrane material sample to be tested on the surface of a flat plate-like nutrient medium suspended with the microorganisms, and the proliferation level of the microorganisms right under the sample is compared with that in the case with no sample to determine the aimed oxygen permeability coefficient of the test sample.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring an oxygen permeability coefficient of a film material such as a contact lens, artificial skin, or a household vegetable wrap.

[0002]

2. Description of the Related Art Membrane materials are widely used in various industrial fields. The diversification of utilization technologies utilizing membrane functions is remarkable. There are various utilization forms related to the permeation of low molecular substances such as gas and ions in the membrane material as follows. In the electronics industry, water-soluble separation membranes are used to produce sterile water, in the food industry, sterile filtration membranes are used to produce wine, etc., ultrafiltration membranes are used to concentrate juices, and in the chemical industry, particles of chemical solution particles are used. Microfiltration membranes are used for removal, and ultrafiltration membranes are used for the preparation of sterile water and sterile air in the fermentation industry, and are widely used in various industrial fields. In addition, the use of oxygen selectively permeable membranes (oxygen-enriched membranes) for use in the medical field utilizing gas permeability and gas selective permeability is also active.

[0003] New materials have been developed one after another, which make use of the gas permeation function to enable membrane separation of gas. The gas permeation coefficient of a gas separation membrane is generally expressed in terms of the permeated gas amount per unit per unit area, per unit time and per unit thickness of the membrane in a standard state of 0 ° C. and 1 atm.

[0004] Materials having excellent oxygen permeability can be used in advanced fields as medical materials such as blood bags.
Materials having excellent oxygen permeability are also used for contact lenses for correcting vision. This contact lens for vision correction requires an advanced oxygen transmission function to supply oxygen to the corneal epithelium. It has been pointed out that when the oxygen permeability coefficient of the contact lens material becomes poor, inhibition of the corneal metabolic system occurs, glycogen disappears, lactic acid accumulates, and in rare cases, even swelling and edema of the corneal epithelium occurs.

[0005] The main methods for measuring the oxygen permeability coefficient of a contact lens material film are: 1) analysis by gas chromatography, 2) electrode method,
3) Although there are three types of polarography methods, each method has the following problems.

[0006] 1) Gas chromatography method: A method in which a high pressure side, a vacuum side, and a chamber are provided with a sample membrane separated, and the concentration of gas permeating from a high pressure side to a low pressure side through a test sample is analyzed by gas chromatography. . As the measuring system, a high-performance and large-scale measuring device such as a gas permeability measuring device and a gas chromatography for detecting a permeated gas is required. Measurement requires chemical knowledge and skills. To measure the oxygen permeability coefficient with high accuracy requires skill in measurement techniques. Since the pressure on the low pressure side gradually increases due to the gas flowing from the high pressure side, gas permeation is only possible within a certain range of the pressure increase on the low pressure side. In the reference cell dimensions, the transmission area and the dimensions of the sample film need to be set to 15.2 cm ² and 60 mmφ, respectively, and there are severe restrictions on the shape of the sample. The upper limit of the film thickness is set to 1 mm. Therefore, it is difficult to say that it is simple and economical, and it is not possible to sufficiently measure the oxygen permeability coefficient of a film material having an arbitrary sample shape.

2) Electrode method: A method in which a spacer and a sample are sandwiched between an electrode portion and a fixing ring, and dissolved oxygen permeating the sample membrane in a 0.9% saline solution at 35 ° C. is measured with an oxygen electrode. is there. By measuring the normal current value from the electrode, the oxygen permeability coefficient can be measured. Since three sets of materials must be measured for five samples of the same material but different thicknesses, it is difficult to measure oxygen permeability coefficient simply. As described above, there are restrictions on sample preparation, and it is not always an easy measurement method.

3) Polarography: The sample film is separated into a mixed gas side of nitrogen and water vapor and a mixed gas side of oxygen and water vapor. Oxygen permeates through the sample membrane to the nitrogen and water vapor sides. This transmitted oxygen is measured by a polarographic oxygen electrode. A highly accurate measurement device is required, and measurement techniques and skills are required.

As described above, the size of a sample is limited to measure the oxygen permeability coefficient of a contact lens, and the measurement of the oxygen permeability coefficient requires skill and experience, and is simple and accurate. The emergence of a measurable technology has been strongly desired.

Further, when the wearing state of the soft contact lens is viewed in detail, the side where the contact lens contacts the cornea (hereinafter abbreviated as “back surface”) is filled with tear fluid, and the side opposite to the cornea (hereinafter abbreviated as “front surface”). ) Is in contact with the air phase containing water vapor. That is, the contact lens is worn so as to be in direct contact with the corneal epithelium, and oxygen is supplied to the corneal epithelium via tear fluid. Polymethyl methacrylate, which is a contact lens material, is used for hard contact lenses and does not have good hygroscopicity. However, when blinking, tear fluid between the contact lens and the cornea is exchanged. This replenishes the cornea with oxygen. Therefore, in order to discuss the oxygen permeability of a soft contact lens material used in a water-containing state for a hard contact lens lacking in hygroscopicity, the humidity of the air phase on one surface is different from the humidity of the air phase on the other surface. Although it is necessary to discuss the oxygen permeability coefficient when a contact lens is placed in an environment, it has been difficult to evaluate the oxygen permeability coefficient considering the humidity difference between the back and front sides of the contact lens due to technical difficulties in measurement. It was not possible.

In order to discuss the gas permeability of a membrane material such as a contact lens, artificial skin or household vegetable wrap, sufficient information cannot be obtained by the conventional method of measuring the gas permeability coefficient. That is, since the inside of the film material is in a high humidity environment and the outside is in a low humidity environment, the difference in the moisture absorption of the film material between the inner and outer surface layers affects gas transmission. Therefore, it is desired to develop a method for measuring the oxygen permeability coefficient in consideration of the difference in humidity between inside and outside.

In the present invention, one of the test samples is set so as to be in contact with the microorganism culture site. As for the humidity of the gas phase in contact with the surface side, a gas phase containing arbitrary water vapor can be prepared by continuously blowing sterile air having different dehumidification degrees. Thus, it is extremely important in practical use to measure the oxygen permeability coefficient when one of the samples is in contact with the gas phase and the other is in contact with the liquid phase. In addition, the conventional measurement technique only measures the oxygen transmission coefficient specific to the contact lens,
Evaluation of practical oxygen supplementation for the cornea (EO
P; Equivalent oxygen percentage
Considering the importance of the tag, a device capable of such a measurement was required. Nevertheless, the reason why the evaluation was difficult is that the development of a measuring device that meets the purpose has not been advanced. The emergence of a simple evaluation method for practical oxygen supplementation is desired.

In addition, the conventional measurement requires a large and precise measuring device, and it is only possible to evaluate the oxygen permeability coefficient of one sample by one measurement. There was a problem in the reproducibility of measurement results such as variations. Therefore, it has been desired to develop a technique for economically and efficiently measuring the oxygen permeability coefficient even with a small amount of sample.

[0014]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problems of the prior art and provides a method for simply, economically, efficiently and accurately measuring the oxygen permeability coefficient of a membrane material. Make it an issue. Another object of the present invention is to provide a method for measuring an oxygen permeability coefficient in a film composition in consideration of a humidity difference between the front and back surfaces of a film material.

[0015]

Means for Solving the Problems The present inventors have focused on the fact that aerobic microorganisms need sufficient oxygen to grow, and have studied the growth of bacteria in a medium immediately below a test sample that comes into contact with a bacterial medium. Since the amount is closely related to the oxygen permeability coefficient of the test sample, it has been found that the oxygen permeability coefficient of the test sample can be easily evaluated by evaluating the amount of bacterial growth, and the present invention has been completed. That is, according to the present invention, the microorganisms are cultured for a certain period of time with the test sample placed on the surface of a plate-shaped nutrient medium in which the microorganisms are suspended, and the amount of microorganism growth immediately below the test sample and the test sample are determined. There is provided a method for measuring the oxygen permeability coefficient of a membrane material, wherein the method is to determine the oxygen permeability coefficient of the test sample by comparing the growth rate of the microorganism with a medium without the medium.

In the method of the present invention, microorganisms are used. However, from the viewpoints of not infecting humans and animals and being safe for a measurer, the use of aerobic microorganisms, especially aerobic phytopathogenic bacteria, is preferred. preferable. Any aerobic microorganism can be used. Such aerobic microorganisms need not be cultured at a constant temperature of 37 ° C., unlike general cell culture. In the method of the present invention, a culture temperature of about 20 to 30 ° C. is sufficient, so that the merit of using a plant pathogenic bacterium as a microorganism is great.

In the method of the present invention, any medium can be used as long as it is a nutrient medium in which bacteria can be grown. King B medium (hereinafter abbreviated as KB medium), potato decoction / glucose medium, semi-synthetic medium, etc. Wakimoto's medium is preferred. In addition, in the method of the present invention, a solid plate medium is used as a medium, but the solidifying agent does not inhibit the growth of bacteria and can be used with any solidifying agent as long as it has high transparency, agar, duran gum, Gelatin is particularly preferred.

When an aerobic microorganism is mixed with a medium and cultured, the medium, which is not turbid and is transparent before the microorganism grows, becomes cloudy in 1 to 2 days as the cells grow, and the clarity of the culture (hereinafter, referred to as (Abbreviated as medium clarity). When a test sample having excellent oxygen permeability is placed on the surface of the culture medium, bacteria can easily grow immediately below the test sample. Therefore, the culture medium becomes cloudy with the growth of bacteria, and the amount of light transmitted through the culture medium decreases. However, when the test sample is impermeable to oxygen, the culture medium remains transparent because bacteria cannot grow below the sample. Therefore, since the transparency of the culture medium is inversely related to the growth rate of the bacteria, the oxygen permeability coefficient of the test sample can be easily evaluated by comparing the transparency of the culture medium with the transparency of the culture medium immediately below the test sample.

In the present invention, the oxygen permeability coefficient is measured by bringing a test sample into contact with a culture medium for culturing microorganisms and measuring the transparency of the culture medium immediately below the test sample. The back surface of the test sample is in contact with the liquid medium, that is, the surface of the water-containing medium,
The surface is in contact with a dry or gas phase containing any humidity. The present invention is to measure the oxygen permeability coefficient of a test sample in terms of the growth state of microorganisms from the difference in culture medium transparency, and is characterized in that it can be easily evaluated without using special equipment.

In the present invention, since a large number of small pieces of a test sample can be simultaneously set in a medium in which microorganisms are suspended in a petri dish, it is particularly efficient and economical when the oxygen permeability coefficient is evaluated for various kinds of samples. Become a target.

In the present invention, the measurement can be performed in a state where one side of the test sample is in contact with a culture medium for culturing microorganisms and the other side is in contact with a gas phase containing arbitrary humidity. In order to bring the surface of the test sample into contact with the gas phase of high humidity, a gas phase of 100% humidity can be obtained by sealing the microorganism culturing vessel. Further, the humidity of the culture container can be controlled by continuously feeding the sterilized air containing any humidity into the microorganism culture container by the dehumidification adjusting device.

The method for measuring the oxygen permeability coefficient according to the present invention is very effective for evaluating gas permeability of biomaterials. For example, the lower part of the artificial skin that touches the living tissue is in a wet state with bodily fluid exudate due to a burn, while the upper part is in a dry state because it is in contact with a dry atmospheric phase (air phase). The method for evaluating the oxygen permeability coefficient according to the present invention can also appropriately evaluate the oxygen permeability coefficient of the artificial skin material under such circumstances.

Examples of aerobic plant pathogenic bacteria used in the method of the present invention include tomato canker, lettuce rot, and cruciferous vegetable black rot. As the phytopathogenic fungi to grow making spores, forming a black spores pathogenic fungi of rice, Inegoma leaf blotch fungus, which is frequently used in bioassays, as well as the spores among Fusarium spp Mulberry blight fungus F. soluni
Can be exemplified. Examples of plant pathogens and their scientific names that can be used in the present invention are shown below. .. Corynebacterium michiganese pv michiganese tomato canker Pseudomonas cichorii various Rot (lettuce) Xanthomonas campestris pv campestris cruciferous vegetables black rot fungus plant pathogenic fungi: Biporaris leersiae rice sesame leaf blight fungus Fusarium solani f.sp. mori mulberry bud blight

The clarity of the medium is measured after a test sample is set on a nutrient medium in which microorganisms are suspended and cultured for a certain period of time. If the test sample is opaque, remove it from the culture medium,
If the medium is transparent, the transmitted light passing through the medium is photographed in a state where black or white paper is placed on the opposite side, and the transparency of the medium can be evaluated by image processing.
In addition, the amount of light transmitted through the medium can be easily evaluated by measuring the exposure time of a photograph linked thereto with a low magnification microscope.

The relationship between the transparency of the bacterial culture in contact with the sample and the oxygen permeability coefficient of the test sample can be determined for any test sample by preparing a calibration curve between the degree of bacterial growth inhibition and the oxygen permeability coefficient. The degree of bacterial growth inhibition reveals the oxygen permeability coefficient of the test sample. Also, by preparing two or three standard membranes and placing them as a control, it is possible to correct the difference in growth inhibition due to the microorganism concentration and culture conditions. By preparing a calibration curve of the thickness of the test sample and the degree of inhibition of bacterial growth, the thickness can be easily corrected whether the film thickness is small or large, and the oxygen permeability coefficient for each thickness can be easily evaluated. In the present invention, in order to measure the oxygen permeability coefficient of the test sample, it is not necessary to strictly control the size of the sample as in the conventional method, so that the sample may be in small pieces or in any form. Also, unlike the conventional method, the sample does not need to be firmly attached by applying force with a jig of the oxygen permeability coefficient measuring device, so the test sample is a film, powder, gel containing water, fibrous material, etc. Various forms such as a lump and a lump can be similarly used. Among such samples, the power of the present invention is particularly exhibited when the gel is easily deformed when a force is applied.

When the surface of the test sample has irregularities and the surface of the medium becomes uneven and the transparency becomes uneven, the measurement can be made by dropping a drop of water, placing a transparent film on top and flattening. Conventionally, there are various devices for measuring the oxygen permeability coefficient, but only one sample data can be obtained in one measurement. However, according to the present invention, a plurality of small pieces of the test sample are placed on the surface of a nutrient medium in which microorganisms are suspended. By setting, many measurements can be performed at once.

[0027]

The present invention will be described below with reference to examples. The evaluation in the examples was based on the following method. A. Evaluation method of bacterial growth inhibition degree After heating and dissolving, 15 ml of semi-synthetic wakimoto medium or King B medium kept at 55 ° C. and 2 ml of test bacteria (concentration of 10 ⁹ to 10 ¹⁰ cells / ml) were mixed, poured into a Petri dish, and plated. Hardened. A section of the test sample cut into a disk or a square (1 cm ² ) was placed on the plate medium mixed with the bacterial solution, and carefully placed on the medium with forceps so as to bring the entire test sample into contact with the medium. After maintaining the temperature at 20 to 25 ° C. for 2 days, the degree of bacterial growth inhibition in the medium immediately below the test sample was evaluated in four steps according to the following criteria. ++: Strong (microorganisms hardly grow, medium transparency is high and the appearance of transparent glass) ++: Slightly strong +: Weak (microorganisms grow slightly, so medium transparency is about the middle between transparent glass and ground glass) ± : Slight (growth of bacteria is about 1/5, and medium transparency is about ground glass)-: Bacteria grow well and medium is opaque. If the test sample is transparent, immediately below the sample set on the surface of the medium Of the medium can be directly evaluated by a transmission method without removing the test sample. If the test sample is opaque, after culturing in a medium containing bacteria for a certain period of time, the opaque test sample is peeled off, and the amount of bacterial growth can be evaluated by evaluating the transparency of the medium immediately below the test sample.

B. Method for evaluating antimicrobial activity against microorganisms A test sample was placed on the bottom of a Petri dish in advance, and 2 ml of a PSA medium and a spore solution (concentration of 10 ⁵ to 10 ⁶ / ml) of the test bacteria, which had been heated and dissolved at 55 ° C., were placed thereon. The mixture was gently poured and solidified into a plate shape, and observed in the same manner as in the evaluation method of A above. A sample was determined to have antibacterial activity when bacterial growth was inhibited immediately above the sample.

C. Medium transparency After setting a transparent membrane sample and test sample that do not pass oxygen at all to the medium in which the microorganisms are suspended, and culturing for a certain period of time,
Observe the medium with the interlocking microscope. The indicated value of the light meter in each sample was measured, and the culture medium transparency was calculated by the following equation (1). Medium transparency (%) = (1− (ba) / (ca)) × 100 (1) where a is an exposure meter for a transparent membrane sample (eg, polyvinylidene chloride) that does not substantially pass oxygen. The indicated value or the indicated value of the light meter of the medium before culture, b indicates the indicated value of the light meter immediately below the test sample, and c indicates the indicated value of the light meter of the medium in which the bacteria were grown without the membrane sample.

Example 1 A test sample having a known oxygen permeability coefficient was set in a medium containing tomato canker bacterial, and the degree of bacterial growth inhibition in the medium immediately below the test sample was evaluated. King B medium was used as the medium. The results of the antibacterial evaluation are those maintained at 25 ° C. for 2 days after setting. Table 1 shows the obtained results. In addition,
The unit of the oxygen permeability coefficient quoted from the literature (the latest medical materials development and utilization handbook, 1986) is cc / m ² · hr · atm.
It is.

[0031]

[Table 1] * Unit: cm / m ^{2 ·} hr · atm.

When a silicon sheet having the best oxygen permeability among the test samples in the present invention is placed on the surface of a culture medium, oxygen necessary for the growth of bacteria is sufficiently supplied via the test sample. Therefore, bacterial growth is very good and no bacterial growth inhibition is observed. As shown in Table 1, as the test sample has a higher oxygen permeability coefficient, the growth of the bacteria in the nutrient medium immediately below the sample is better, so that the culture becomes more turbid as the bacteria grow and the transparency of the medium decreases. The oxygen permeability of the test sample can be evaluated by quantifying the transparency of the medium. The medium clarity in the medium immediately below the test sample was evaluated as follows. Using a Nikon microscope, the indicated value of the exposure time (s) of the light meter was read by the following method. The magnification of the microscope is 4 × 2.5. Set the culture dish on the microscope to the microscope, and set the intensity of the illumination lamp light source under the microscope so that the exposure time under the medium that does not grow bacteria or under the polyvinylidene chloride membrane that substantially blocks oxygen is almost 1.00 seconds. Is adjusted for convenience. Next, each test sample in contact with the medium is placed in the field of view of the microscope, and the respective exposure times are measured. Finally, the exposure time of the background (where bacteria grow and become opaque) with no sample placed thereon is finally measured. Is measured.

Example 2 As an additional investigation of Example 1, the presence or absence of antibacterial properties of a test sample was examined. A section of the test sample No. 1-4 is placed on the bottom of the Petri dish, and a KB medium containing tomato canker bacterium is poured from above with sufficient care so that the test sample does not rise from above, and cultured for 2 days. The bacterial growth inhibition was examined.
As a result, the bacteria grew well in the medium immediately above the test samples of sample numbers 1 to 4, and no bacterial growth inhibition by the sample was observed. Thus, since the sample did not have antibacterial properties, it can be determined that the bacterial growth inhibition degree shown in Table 1 is based on oxygen permeation.

Example 3 The number of the cellulose tube membranes of Sample No. 3 used in Example 1 was changed and set in a KB medium, and the bacterial growth inhibition was evaluated using tomato canker as a test bacterium. Table 2 shows the obtained results.

[0035]

[Table 2] Thickness; μm

By preparing a calibration curve showing the relationship between the thickness of the sample membrane and the degree of bacterial growth inhibition, the oxygen permeability at a constant thickness of the sample membrane can be easily corrected and displayed regardless of the film thickness.

Example 4 Silk fibroin membranes having different oxygen permeability coefficients were prepared, and the oxygen permeability was examined as follows. The rear silk wire was taken out from the mature silkworm, liquid silk fibroin was dispersed in distilled water, and diluted about 0.85 times with distilled water. A silk fibroin aqueous solution was spread on a polyethylene membrane in a thermostat set at 25 ° C. and evaporated to dryness to prepare a silk fibroin membrane.
Next, in order to make the obtained membrane water-insoluble, the membrane was treated with a 50% by weight aqueous solution of methanol for 1, 10, and 30 minutes, taken out, and then dried at room temperature for 24 hours. . The value of the oxygen permeability coefficient of the membrane was determined by the oxygen electrode method. That is, the silk fibroin membrane was attached to the sensitive part of the oxygen electrode, the electrode was immersed in oxygen-saturated water at 30 ° C., and the oxygen transmission coefficient value was measured from the obtained electrode value. The thickness of the film was about 40 μm. However, the unit of the oxygen permeability coefficient is cm ³ (STP) · cm / cm ² · s ·
cmHg). Table 3 shows the obtained results. Table 3 also shows the oxygen transmission coefficient measured by the oxygen electrode method.

[0038]

[Table 3] ** Unit: cm ³ (STP) cm / m ² s cm Hg

The high transparency of the medium after the cultivation means that the oxygen permeability of the sample membrane is low, which means that the bacteria are difficult to grow. When a silk film is immersed in a methanol solution, the treatment tends to impede oxygen as the treatment time increases. This is considered to be due to the fact that the form of silk fibroin molecules usually shifts from a random coil type to a β type by the insolubilization treatment, and the aggregation structure of the molecules becomes dense accompanying this, thereby decreasing the oxygen permeability coefficient. As can be seen from Table 3, the same result as the oxygen permeability coefficient measured by the oxygen electrode method was obtained in this method.

[0040]

According to the present invention, the oxygen permeability coefficient of a test sample is determined by bringing a test membrane into contact with the surface of a culture medium containing microorganisms, culturing them for a certain period of time, and measuring the proliferation of the microorganisms using a biological technique. It is characterized by the simplicity of measuring. Since the oxygen permeability coefficient of the test sample is not measured by a conventional physicochemical method, a measuring device for measuring the oxygen permeability coefficient is unnecessary. Furthermore, the method of the present invention has an economically superior effect as the number of test samples increases. This is because by setting a large number of small pieces of the test sample on the surface of the medium in which the microorganisms are suspended, the oxygen permeability coefficient of the sample can be evaluated at the same time. It is a measurement method. The present invention has an unprecedented feature in that it allows measurement of the oxygen permeability coefficient of a test sample in which one is in contact with a solution phase and the other is in contact with a gas phase containing arbitrary humidity. Further, since it is not necessary to strictly control the size of the sample, the sample may be in any form or in any form. Therefore, the test sample may be a film, a powder, a gel containing water, or a fibrous material. It is also superior to the conventional method in that various forms such as lumps can be similarly used.
It has the characteristic that it can be measured even for gels that are easily deformed when a force is applied.

Claims (3)

[Claims] 1. A microorganism is cultured for a certain period of time with a test sample placed on a surface of a plate-shaped nutrient medium in which the microorganism is suspended, and the amount of microorganism growth immediately below the test sample and no test sample are present. A method for measuring an oxygen permeation coefficient of a membrane material, comprising determining an oxygen permeation coefficient of the test sample by comparing the amount of microbial growth in a culture medium. 2. The method for measuring the oxygen permeability coefficient of a membrane material according to claim 1, wherein an aerobic microorganism is used. 3. The membrane material according to claim 1, wherein the measurement is performed in a state in which one side of the sample is in contact with a liquid phase or a solid phase and the other side is in contact with a gas phase containing arbitrary humidity. How to measure the transmission coefficient.