JPH08116992A - Examination of antimicrobial property - Google Patents

Abstract

PURPOSE: To provide an antimicrobial efhect-examining method capable of quantitatively judging ability to catch a microbial cell with a fibrous raw material and/or a product and ability to suppress proliferation of caught microbial cell. CONSTITUTION: This method for examining antimicrobial properties comprises combinedly using both the measurement of catching activity of microbial cell comprising dispersing a microbial cell attaching to a raw material and/or a product into a liquid culture medium or a buffer and recovering, cleaning and culturing the dispersed microbial cell and examining viable microbe numbers and the measurement of antimicrobial activity comprising measuring biochemical activity of a microorganism attached to the raw material and/or the product and examining survival property of the attached microorganism, in measuring an antimicrobial activity of the fibrous raw material and/or the product to judge antimicrobial activity of the raw material and/or the product. One or more biochemical activities selected from a catalase activity, an indole producing activity and a gelatin liquefying activity are preferably used as the biochemical activities.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibacterial test method, and more particularly to a method for measuring the antibacterial activity of fibrous materials and / or products.

[0002]

2. Description of the Related Art In recent years, as the social demand for comfort and cleanliness of living environment has increased, interest in antibacterial properties of textiles such as clothing has increased. In addition, interest in the comfort of the indoor environment, which accounts for most of the living time, has increased,
In addition to conventional dust removal, the performance of air filters in air conditioners is required to have various functions. Above all, the function of suppressing the growth of microorganisms on the air filters, that is, the antibacterial property is required. It was In addition, nosocomial infections become a problem in hospitals, and not only ordinary hand washing sterilization and sterilization of the environment such as hospitals, but also antibacterial function of textiles such as sheets, bedspreads and lab coats in hospitals to prevent contact infection. However, the antibacterial function of the air filter for air conditioning is strongly required to prevent air infection.

As a method for measuring the antibacterial property of a textile product, for example, there is an antibacterial deodorant processed product certification standard established by the Textile Product Processing Council. This is because the antibacterial component eluted from the test piece added to the culture solution diffuses into the culture solution in both the bacterial count measurement method and the shake flask method, which are defined as the processing effect evaluation test method for the antibacterial and deodorant processed product. This is to measure the effect of suppressing the growth of bacterial cells. The Hello Test method, which is proposed as a method for daily quality control in the same certification standard, also observes a transparent zone around a sample piece that is closely attached to agar inoculated with the target microorganism. Similar to the method and shake flask method, the antibacterial component eluted from the sample piece diffuses into the agar medium and the effect of suppressing the growth of bacterial cells is measured.

In addition, as a method similar to the above-mentioned Hello Test method, the sample piece adhered to the agar inoculated with the target microorganism was removed after 2 hours, and the presence or absence of the bacterial growth inhibition zone at the position where the sample piece was placed, As in the method of measuring antibacterial activity in large and small (textile processing technology, p.190, acquaintance library, 1989),
There is also a method of measuring the antibacterial effect against bacteria that are in direct contact and the effect that the antibacterial component eluted from the sample piece diffuses into the agar medium and suppresses the growth of bacterial cells. As described above, the conventional methods for measuring the antibacterial activity of fibrous materials and / or products are mainly those in which the drug carried on the fibrous materials and / or products diffuses in a water-containing medium such as a medium to exert antibacterial activity. It is to quantify the function to be exerted.

[0005]

As described above, the conventional methods for measuring the antibacterial activity of fibrous materials and / or products include microbial bacteria adhered mainly by contact in clothing, sheets, bed covers, gauze and the like. It can be said that this is a method suitable for determining the potential antibacterial activity against the body, but whether there is a bactericidal or bacteriostatic effect on the adhered bacterial cells in a relatively low moisture condition close to the state of use, It is not possible to judge whether the body may be released from the fibrous material and / or product and again contaminate the other.

[0006] In particular, the function of the air filter is that the air filter is arranged so as to shut off the air flow that is forcibly created, and captures microbial cells and spores floating in the air.
It is required to have both the removing effect and the effect of suppressing the growth of the captured bacterial cells on the air filter.
This is an important function in order to prevent the bacterial cells grown on the air filter from being released and spreading to the entire building through conditioned air, or to prevent the infection of workers when the air filter is replaced. . In addition, cloths used for wiping off instruments, desks, and stands to remove bacteria in hospitals and research facilities also have a function to prevent the spread of microbial contamination so that the bacterial cells do not separate and contaminate others, and the captured bacteria The function of suppressing the growth of the body on the cloth is required.

That is, as a method for measuring the antibacterial activity of a fibrous material and / or product, particularly an air filter or its material, a drug carried on the fibrous material and / or product is conventionally used as a medium. The method of quantifying the function of exerting antibacterial activity by diffusing in a water-containing medium has not been sufficient. Therefore, the present invention has been made in view of the above points, the ability to capture the bacterial cells of the fibrous material and / or product,
It is another object of the present invention to provide an antibacterial test method capable of quantitatively determining the ability to suppress the growth of captured bacterial cells.

[0008]

In order to solve the above-mentioned problems, in the present invention, in measuring the antibacterial activity of a fibrous material and / or product, microbial cells adhered to the fibrous material and / or product After dispersion treatment in a liquid medium or buffer, the dispersed bacterial cells are collected, washed and cultured to check the viable cell count, and the biocapacity of the microorganisms attached to the material and / or product is measured. This is an antibacterial test method characterized by determining the antibacterial activity of the material and / or product in combination with the activity measurement and the antibacterial activity measurement for examining the viability of attached microorganisms.

As a method for attaching microbial cells to the fibrous material and / or product to be tested, any method can be used as long as a fixed amount of cultured bacterial cells can be evenly attached onto the sample piece. However, the simplest method is to use a method in which a fixed amount of a culture solution having a cell concentration of about 1.0 × 10 ⁸ cells / ml is dropped on a sample piece and air-dried. As a method for dispersing the adherent cells in a liquid medium or a buffer solution, any of the methods generally used for dispersion treatment of cells can be used as long as it is a method of applying physical force from outside the container. However, it is preferable to use both dispersion by mechanical stirring with a test tube stirrer and dispersion by ultrasonic waves.

As a method for collecting and washing the bacterial cells dispersed in a liquid medium or a buffer solution, any method can be used as long as it is a method generally used for collecting and washing the bacterial cells, but the most convenient method. For this, a method can be used in which the microbial cell dispersion is subjected to suction filtration using a membrane filter and then the microbial cells on the filter are washed with water or a buffer solution.
The cells collected here are washed in order to prevent the antibacterial agent carried on the fibrous material and / or the product to be tested from diffusing into the medium and exhibiting antibacterial activity. . As a method for measuring the viable cell count of the cells recovered and washed by this method, after immersing the membrane filter in a liquid medium for culturing, a method for measuring the turbidity of the culture solution, or in the liquid medium for measuring the turbidity It can be measured by a method in which the cells are dispersed by immersing the filter and then appropriately diluted according to a conventional method to form colonies on the agar plate medium. At this time, as a control, by measuring the viable cell count of the bacterial cell culture solution of the same amount as the bacterial cells added to the sample piece, by determining the difference between the number of control bacterial cells and the number of bacterial cells released and dispersed from the sample piece, The number of bacterial cells captured on the sample piece can be quantitatively obtained.

On the other hand, as a method for measuring the biochemical activity of the microorganisms adhered to the fibrous material and / or product to be tested and checking the viability of the adhered microorganisms, the activity of the enzyme possessed by the microorganisms is measured. Any method can be used so long as it is easy to operate and relatively easy to determine the activity, such as catalase activity, indole forming activity and gelatin liquefaction activity. Catalase is widely distributed in aerobic cells of animals, plants and microorganisms, hydrogen peroxide decomposition reaction, H ₂ O ₂ + H ₂ O ₂ → O ₂ + 2H ₂ O
It is an enzyme that catalyzes. Since an enzyme gas is generated as a reaction product, when a hydrogen peroxide solution is dropped on a sample piece to which the target bacterial cells are attached, if the catalase activity is positive, foaming phenomenon is observed, so it can be easily determined. it can. At this time, as a control, the same amount of the bacterial cell culture solution was dropped on the fibrous material and / or product piece having no antibacterial activity, and after air-drying, the state of foaming when the hydrogen peroxide solution was dropped was observed, and the sample piece The inhibition rate of catalase activity can be determined from the degree of foaming. However, since the fibrous material and / or the antibacterial agent carried on the product used for the test may catalyze the decomposition of hydrogen peroxide, hydrogen peroxide solution should also be used as a control for sample pieces to which the cell culture solution is not added. It is necessary to drop to confirm the presence or absence of foaming.

[0012] Catalase is the most preferable biochemical activity test method used in the method of the present invention because it is widely distributed in aerobic cells of microorganisms and the activity can be easily determined. It cannot be used when the antibacterial drug itself carried on the material and / or product catalyzes the decomposition of hydrogen peroxide. In this case, when a microorganism having an indole forming ability or a gelatin liquefying ability is targeted, a method for examining these activities can be used. Indole is a decomposition product of tryptophan, which is a type of amino acid, and its presence or absence can be determined by utilizing the fact that it reacts with the Kovac reagent to give a red color. As a specific method, bacterial cells are attached to a fibrous material and / or product sample piece to be tested, air-dried, the sample piece is immersed in a modified SIM medium, and after culture, a Kovac reagent is dropped. By determining the presence or absence of indole production, inhibition of the indole producing activity of adherent cells can be determined.

On the other hand, gelatin is one in which the triple helix structure of collagen, which is a type of protein, is broken, and when a concentrated gelatin aqueous solution gels when cooled, it has a gelling ability by being degraded by proteolytic enzymes. Lose and liquefy.
Therefore, the fibrous material and / or product sample pieces to be tested are allowed to adhere to the cells having gelatin liquefaction ability, and after air-drying,
When the sample piece is immersed in the gelatin-containing medium before gelation and cultured, the inhibition of the gelatin liquefaction activity can be determined because the liquefaction phenomenon does not occur when the gelatin liquefaction ability is inhibited. Both the indole-forming activity and the gelatin liquefying activity may be inhibited by the antibacterial agent carried on the fibrous material and / or product used in the test, so do not drop the cell culture solution as a control. It is necessary to perform the same experiment on the sample piece to confirm the presence or absence of activity inhibition.

In order to confirm whether or not the operation of the antibacterial drug carried on the fibrous material and / or the product is such that the drug component diffuses in the aqueous solution and exerts the antibacterial activity, A conventional hello test method can be used. As described above, in the antibacterial test method of the present invention, in measuring the antibacterial activity of the fibrous material and / or the product, the bacterial cell-capturing power of the material / or the product and the adhesion to the material / or the product are measured. In combination with antibacterial activity measurement for measuring the biochemical activity of microorganisms, it is characterized by determining the antibacterial activity of the material / or product, especially by an antibacterial test method suitable for evaluation of air filters etc. is there.

[0015]

EXAMPLES The present invention will be specifically described below based on examples and reference examples, but the present invention is not limited thereto. Reference Example 1 Inhibitory circle method First, 50 ml of a base layer medium was solidified on an Eiken No. 1 square petri dish, and a flat plate was prepared by superimposing 15 ml of a seed layer on which test bacteria were suspended. As a test bacterium, as a Gram-positive bacterium, Bacillus subtillis ATCC6
633, as a Gram-negative bacterium, Escherichia coli (Es
cherichia coli) NIHJ, Aspergillus fumigatus TIMM00 as a filamentous fungus
63 was used. The medium is B. B. subtilis
lis) ATCC6633, and E. E. coli NI
Nutrient Agar medium was used as HJ for AJ. A. fumigatus TIMM006
A glucose-added Yeast Nitrgen Base (Difco) medium was used as 3.

The fibrous material used for the test is 1 cm × 2 cm
A sample piece cut into a corner was prepared, and this was brought into close contact with the flat plate to perform culture, and an inhibition circle generated by inhibiting the growth of the bacteria was observed. This test is a test corresponding to the above-mentioned conventional hello test method. As samples, 5 kinds of antibacterial processed fiber materials and fiber materials before antibacterial processing were used for the experiment. The results of the experiment are shown in Table 1. In this test, formation of an inhibition circle was observed in Samples A, D and E.

[0017]

[Table 1] Note) Blocking circle size +++: Large ++: Medium +: Small −: Do not form blocking circle

Reference Example 2 Broth Culture Method B. 1 μl of 25 μl (about 10 ⁶ cells) of bacterial cell liquid obtained by culturing subtilis ATCC6633 in a liquid medium (Nutrient Broth, NB medium) for 18 hours at 37 ° C.
The sample was cut into cm square pieces, dropped on the sample piece of the fibrous material, and air-dried for 5 seconds to attach the bacterial cells to the sample piece. This bacterial cell-attached sample piece was added to a test tube containing 2.5 ml of NB medium, mixed with a test tube stirrer for 15 seconds, and further sonicated for 30 seconds to disperse the bacterial cells, and then at 37 ° C. After culturing for 36 hours, the turbidity of the culture solution was observed. As samples, 5 kinds of antibacterial processed fiber materials and fiber materials before antibacterial processing were used for the experiment. As a control, B. B. subtillis A
25 μl of TCC6633 cell fluid was directly added to NB medium 2.5
The turbidity was observed by adding the solution to a test tube containing ml and subjecting to the same dispersion treatment and culturing. Table 2 shows the results of the experiment.
In this test, the growth of bacteria was not observed in all five kinds of antibacterial processed fiber materials.

From the results of the blocking circularity test of Reference Example 1, it is predicted that Samples A, D and E have an antibacterial action due to the drug eluted from the sample pieces, but the results of Reference Example 1 and the results of this test are compared. Overall, in Samples A, D, and E, the bacteria attached to the sample piece were either growth-inhibited in the adhered state, or the growth was inhibited by the drug eluted in the culture medium after being released from the sample piece. It was not possible to judge whether it was a mistake.

[0020]

[Table 2] Note) Bacterial growth +: Growth-: Growth not observed

Example 1 Bacterial capture power test method B. B. subtillis ATCC6633 was cultured in a liquid medium (Nutrient Broth, NB medium) for 18 hours at 37 ° C., and the bacterial cell fluid was diluted 3 times, and 25 μl thereof (about 10
⁶ cells) was dropped into a 1 cm × 1 cm square sample piece of the fibrous material to be tested, and air-dried for 5 seconds to attach the bacterial cells to the sample piece. The bacterial cell-attached sample piece was added into a test tube containing 2.5 ml of a phosphate buffer solution, mixed with a test tube stirrer for 15 seconds, sonicated for 30 seconds, and again tested with a test tube stirrer.
After mixing for 0 seconds to disperse the bacterial cells, the pore size is 0.45 μm
Suction filtration was carried out using the membrane filter of No. 1 and the bacterial cells on the filter were washed with 20 ml of phosphate buffer. This washing operation is for removing the antibacterial drug component eluted from the test fibrous material in the phosphate buffer. After washing, the microbial cell-attached filter was added to a test tube containing 2.5 ml of NB medium, mixed for 15 seconds with a test tube agitator, and further sonicated for 30 seconds to disperse the microbial cells, and then 37 ° C.
Culturing was performed for 18 hours and after 8 days, the turbidity of the culture solution was observed.

As samples, 5 kinds of antibacterial processed fiber materials and fiber materials before antibacterial processing were used for the experiment. As a control, B. Twenty-five microliters of B. subtillis ATCC6633 cell fluid was added directly into a test tube containing 2.5 ml of phosphate buffer solution, and treated and cultured in the same manner to observe the turbidity. The results of the experiment are shown in Table 3. In this test, in Samples A, C and E, the growth of bacteria was not recognized even after 8 days. Further, in Samples B and D, growth of bacteria was not observed after 18 hours, but growth of bacteria was observed after 8 days. From the results of this test, all five types of antibacterial processed fiber materials have either a function of trapping bacterial cells or a function of immediately inhibiting the growth of bacterial cells in the state where the bacterial cells adhere, and the function is that of samples A and C. And E were found to be particularly excellent.

[0023]

[Table 3] Note) Bacterial growth +: Growth-: Growth not observed

Example 2 Biochemical activity test method Catalase activity test B. 1 μm of 25 μl (about 3 × 10 ⁶ cells) of bacterial cell liquid obtained by culturing B. subtillis ATCC6633 in a liquid medium (Nutrient Broth, NB medium) for 18 hours at 37 ° C.
× Drop it on the test fibrous material sample piece cut into 1 cm squares,
The cells were attached to the sample piece by air-drying for 5 seconds. A drop of hydrogen peroxide solution was dropped onto the cell-attached sample piece, and the state of foaming was observed. Further, in order to confirm the hydrogen peroxide decomposition catalytic action of the test fibrous material itself, one drop of hydrogen peroxide water was dropped on the test fibrous material sample piece cut into 1 cm × 1 cm squares, and the foaming state was observed. . As samples, 5 kinds of antibacterial processed fiber materials and fiber materials before antibacterial processing were used for the experiment. The results of the experiment are shown in Table 4. In this test, strong inhibitory activity was observed for samples A and B, and weak inhibitory activity was observed for samples D and E. In addition, since the raw material itself had a catalytic activity for decomposing hydrogen peroxide in Sample C, the catalase activity of the cells could not be examined.

Indole production activity test Escherichia coli NIHJ was cultured in a liquid medium (Nutrient Broth, NB medium) at 37 ° C. for 18 hours, and 25 μl of bacterial cell fluid (about 3 × 10 ⁶ cells) was added to 1 cm.
× Drop it on the test fibrous material sample piece cut into 1 cm squares,
The cells were attached to the sample piece by air-drying for 5 seconds. A modified SIM medium 2.5 m in which this microbial cell-attached sample piece was added with sodium thiosulfate, ammonium citrate, L-cystine, L-cysteine and tryptophan using NB medium as a basal medium
1 was added to the test tube and cultured at 37 ° C. for 18 hours. The Kovac reagent was dropped into this cell culture medium and the state of coloration was observed. As samples, 5 kinds of antibacterial processed fiber materials and fiber materials before antibacterial processing were used for the experiment. The results of the experiment are shown in Table 4. In this test, strong inhibitory activity was observed for samples A and E, and weak inhibitory activity for samples B and D.

Gelatin Liquefaction Activity Test B. 1 μm of 25 μl (about 3 × 10 ⁶ cells) of bacterial cell liquid obtained by culturing B. subtillis ATCC6633 in a liquid medium (Nutrient Broth, NB medium) for 18 hours at 37 ° C.
× Drop it on the test fibrous material sample piece cut into 1 cm squares,
The cells were attached to the sample piece by air-drying for 5 seconds. The bacterial cell-attached sample piece was added to a test tube containing 2.5 ml of gelatin medium and cultured at 37 ° C. for 90 hours to observe the state of gelatin liquefaction. As samples, 5 kinds of antibacterial processed fiber materials and fiber materials before antibacterial processing were used for the experiment. The results of the experiment are shown in Table 4. In this test, inhibitory activity was observed in all 5 kinds of antibacterial processed fiber materials.

From the results of this biochemical activity test, the gram-positive bacteria B. B. subtillis ATCC6633
In contrast, all five types of antibacterial processed fiber materials have the function of inhibiting the growth of bacterial cells attached to the fiber, and the gram-negative bacterium Escherichia coli.
hia coli) Samples A, D, and E for NIHJ
It was found that the cells have a function of inhibiting the growth in the state where the cells adhere to the fibers. Combining the results of Example 1 microbial cell-capturing ability test and Example 2 biochemical activity test, Samples A, D, and E were excellent in the function of inhibiting the growth of microbial cells attached to the fiber, and It was found that it is unlikely that the bacterial cells attached to the cells would be released in a viable state and would again contaminate the others. In addition, although the samples B and C were determined to have no antibacterial activity in the blocking circularity test (Reference Example 1), they had a function of inhibiting the growth of the bacterial cells in a state where they were attached to the fibers, and the bacterial cells trapped. It was found that the function to do so is excellent, and it is unlikely that the bacterial cells once attached to the fibers are released and contaminate the other cells again.

[0028]

[Table 4] Note) Catalase activity ++: Strong foaming is recognized +: Weak foaming is recognized −: No foaming Indole-forming activity ++: Strong coloration is recognized ±: Slight coloration is recognized −: No coloration Gelatin Liquefaction activity +: Liquefaction is observed −: No liquefaction

[0029]

As described above, by using the antibacterial test method of the present invention, it is possible to obtain a relatively high moisture content close to the use condition which cannot be judged by the conventional method for measuring the antibacterial activity of fibrous materials and / or products. Make an accurate judgment as to whether or not there is a bactericidal or bacteriostatic effect on adhered bacterial cells in a low state, and whether the adhered bacterial cells may be released from the fibrous material and / or product and contaminate other materials again. We were able to. Further, by using the antibacterial test method of the present invention, a supported drug, which is judged to have no antibacterial activity by the conventional method for measuring the antibacterial activity of fibrous materials and / or products, is a medium. Attached to a fibrous material and / or product that has the function of exerting antibacterial activity when the drug is bound to the fibrous material and / or product instead of diffusing into a water-containing medium to exert antibacterial activity It was possible to make an accurate judgment as to whether or not there was a bactericidal or bacteriostatic effect on the cells.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Kenichi Sasaki 4-2-1 Honfujisawa, Fujisawa-shi, Kanagawa Inside the EBARA Research Institute (72) Inventor Naoshi Kawakami 4-2-1, Fujisawa, Kanagawa No. 1 Incorporated EBARA Research Institute (72) Inventor Kazuhiko Okamura 2502-1 Fujisawa, Fujisawa City, Kanagawa Prefecture (72) Inventor Michiko Sakamoto 3-7-15 Matsubayashi, Chigasaki City, Kanagawa Prefecture

Claims (2)

[Claims] 1. When measuring the antibacterial activity of a fibrous material and / or product, the microbial cells adhered to the material and / or product are dispersed in a liquid medium or a buffer solution, and the dispersed microbial cells are recovered. , Measurement of bacterial cell-capturing ability by washing, culturing and culturing to determine the number of viable cells, and measurement of biochemical activity of microorganisms attached to the material and / or product, and measurement of antibacterial activity for examining viability of the attached microorganisms. Is used together to determine the antibacterial activity of the material and / or product. 2. The biochemical activity is a catalase activity,
The antibacterial test method according to claim 1, wherein the antibacterial test method is one or more selected from an indole forming activity and a gelatin liquefaction activity.