JP2006014709A - Standard testing specimen for antimicrobial property testing, method for producing the same and method for antimicrobial property testing - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a standard testing specimen for an evaluating antimicrobial effect, which more accurately ascertaining variation in evaluation. <P>SOLUTION: The standard testing specimen for antimicrobial property testing comprises a substrate 1, a first binding layer 2 that is composed of first compounds formed on a fixed area of the substrate 1 and bonded to the surface of the substrate 1, a second binding layer 4 that is composed of second compounds which are arranged in a fixed amount per unit area on the first binding layer 2, specifically bonded to each of the first compounds and in which each molecule retains a fixed amount of antimicrobial components 6 and the antimicrobial components 6 retained in an exposed state on each molecule of the second compounds. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a standard test piece for antibacterial test, a production method thereof, and an antibacterial test method.

  In recent years, antibacterial processed products have been produced and sold to suppress the growth of bacteria on the surface and provide a hygienic use situation. An antibacterial processed product is obtained by applying an antibacterial process to a substrate made of fiber, plastic, metal, ceramics, paint, rubber, wood, paper or the like. For antibacterial processing, silver, copper, zinc and other metals alone or compounds, antibacterial agents supporting these metals on inorganic carriers, natural antibacterial agents such as hinokitiol and chitosan, pyridine and imidazole organic Antibacterial components such as antibacterial agents are used.

  As a standard for evaluating the antibacterial effect of these antibacterial processed products, in Japan, in response to the antibacterial product guidelines published by the Life-related New Functional Processing Product Roundtable of the Ministry of Economy, Trade and Industry (former Ministry of International Trade and Industry) “Antibacterial processed products-antibacterial test method / antibacterial effect” is prescribed. In this standard, Staphylococcus aureus and Escherichia coli are inoculated into antibacterial processed products and unprocessed products, and after culturing them, the difference in the logarithmic value of each viable cell count is defined as the antibacterial activity value. The antibacterial effect of antibacterial processed products is evaluated by this antibacterial activity value. In addition, antibacterial tests were added to the accreditation category in the testing company accreditation system (JNLA) based on the Industrial Standardization Act, and laboratory accreditation in this field was started.

  On the other hand, JNLA has been conducting on-site inspections of already-accredited test operators for new applications since July 2001 in response to the international standard for laboratory accreditation being revised from ISO / IEC Guide 25 to ISO / IEC 17025: 1999. Since January 2002, ISO / IEC 17025 [JIS Q 17025: 2000] has been applied to all test operators, and at the same time, a policy to apply the “Requirements for Test Uncertainty” has been announced.

  The concept of “measurement uncertainty” in laboratories is unfamiliar so far, and the policies of each country are not yet clearly defined in the fields of electricity and physics. In addition, in the field of bioassay using microorganisms, it is considered to be the most difficult field in the world, and even in antibacterial testing, a guide for estimating uncertainty in the operation of the laboratory accreditation system. There is a need for possible embodiments.

  Currently, the production base and technology of antibacterial processed products in Japan are rapidly transferring to overseas countries such as Asia, Europe and the United States, so the standards related to antibacterial processed products are changed from national standards (JIS Z 2801: 2000) to international standards (ISO It is required to step up to (standard) in Japan and overseas, and redesign of the test method considering the uncertainty (ISO standardization work of the test method) is necessary. On the other hand, in the international market, data from reliable laboratories that meet international standards is required, and data obtained from one laboratory can be accepted all over the world (One-Stop- The benefits of testing are enormous. For this purpose, a standard test specimen for antibacterial test that can perform a homogeneous and stable antibacterial test regardless of the technical level of the laboratory is indispensable.

  From the above, currently, a standard test piece for antibacterial test described in Patent Document 1 is being studied. As shown in FIG. 5A, this standard test piece for antibacterial test is applied with a water-soluble resin 92 containing a certain amount of an antibacterial component 91 on the surface of a certain area of a substrate 90 made of polyethylene terephthalate (PET) film. It is a thing. For example, when a JIS antibacterial test is performed using this standard test piece for antibacterial test, the water-soluble resin 92 is ideally dissolved by the bacterial solution as shown in FIG. It is considered that component 91 contributes to the antibacterial action. For this reason, in this case, it is considered that the uncertainty of measurement becomes clearer than before, and the antibacterial effect can be evaluated accurately and with high reproducibility.

JP 2002-95497 A

  However, in the above-described conventional standard test piece for antibacterial test, in reality, as shown in FIG. 5 (C), the water-soluble resin 92 does not necessarily dissolve uniformly in the bacterial solution. However, the antibacterial effect may be evaluated while being retained in the water-soluble resin 92. In this case, the variation in evaluation cannot be accurately determined, making it difficult to adopt as a global standard.

  This invention is made | formed in view of the said conventional situation, Comprising: It is set as the problem which should be solved to provide the standard test piece for antimicrobial effect evaluation which can pinpoint the variation of evaluation more correctly.

  The inventors have intensively studied to solve the above problems. In this case, according to “Hyundai Kagaku” (Tokyo Chemical Dojin, April 2002, pp. 32-37), sterol-binding proteins such as Vibrio cholerae hemolysin have sterol-binding properties. , Bind on the sterol to form a layer of protein. Such proteins are considered to be arranged in a constant amount per unit area on the substrate. In addition, it was considered that such proteins can retain a certain amount of antibacterial components. Thus, the present invention has been completed.

  The standard test piece for antibacterial test of the present invention includes a substrate, a first binding layer formed on a certain area of the substrate and made of a first compound bonded to the surface of the substrate, and the first binding layer. A second binding layer composed of a second compound arranged in a constant amount per unit area, specifically binding to each first compound and capable of holding a certain amount of an antimicrobial component in each molecule; The antibacterial component held in an exposed state for each molecule of two compounds.

  In the standard test piece for antibacterial test of the present invention, the first bonding layer made of the first compound is formed on a certain area of the substrate. The second compound that specifically binds to each first compound and each molecule can hold a certain amount of the antibacterial component is arranged on the first binding layer in a certain amount per unit area. . And these 2nd compounds hold | maintain a fixed amount of antimicrobial components in an exposed state. Therefore, in this standard test piece for antibacterial test, the bacterial solution comes into contact with a certain amount of antibacterial components, and the antibacterial effect is evaluated in this state. For this reason, the standard test for antibacterial tests of the present invention itself does not cause variations in evaluation.

  Therefore, according to the standard test piece for an antibacterial test of the present invention, it becomes possible to more accurately determine the variation in evaluation other than the test piece.

  The standard test piece for antimicrobial test of the present invention can be produced by the production method of the present invention. In the method for producing a standard test piece for antibacterial test of the present invention, a substrate preparing step for preparing a substrate and a first bonding layer made of a first compound bonded to the surface of the substrate are formed on a certain area of the substrate. A fixed amount per unit area on the first binding layer, and a second compound capable of specifically binding to each first compound and capable of holding a certain amount of antibacterial component in each molecule. A second binding layer forming step arranged in a step, and an antibacterial component holding step for holding the antibacterial component in an exposed state for each molecule of the second binding layer.

  In the substrate preparation process, a substrate is prepared. This substrate preferably has a hydrophobic surface. As the substrate, glass having a polished surface, polyethylene, acrylic, a polyvinylidene difluoride (PVDF) film, mica, or the like can be used. In order to make the surface of the substrate hydrophobic, the surface can be coated with silicone, fluorine, or the like.

  In the first bonding layer forming step, a first bonding layer made of the first compound bonded to the surface of the substrate is formed on a certain area of the substrate. A sterol can be adopted as the first compound. As sterol, cholesterol (colesterol), diosgenin (diosgenin), campesterol (campesterol), ergosterol (ergosterol), etc. are employable. When forming a 1st coupling layer with these sterols, after dissolving sterol in highly volatile solvents, such as chloroform, and making it a sterol liquid, a sterol liquid can be apply | coated on the said hydrophobic substrate.

  In the second binding layer forming step, the second compound that specifically binds to each first compound and each molecule can hold a certain amount of antibacterial component is arranged on the first binding layer in a certain amount per unit area. Let As the second compound, a sterol-binding protein can be employed. As a sterol-binding protein, Vibrio cholerae hemolysin and the like can be employed. Vibrio cholerae hemotoxin is not only cytotoxic, but must be purified from Vibrio cholerae, a pathogenic bacterium. Therefore, it is necessary to identify a region having only sterol-binding ability of Vibrio cholerae hemolysin using genetic engineering techniques, and to produce only this region in large quantities using Escherichia coli.

  When arranging a second binding layer composed of a sterol-binding protein on the first binding layer in a constant amount per unit area, prepare a solution in which the sterol-binding protein is dispersed in a phosphate buffer or a tris buffer, By thinly applying this solution to the substrate having the first binding layer, the sterol-binding protein can be specifically bound to the sterol molecules of the first binding layer to form a thin layer or a single protein film. . In the second binding layer thus obtained, an amino group, a carboxyl group, an SH group, or a chemical substance having specific binding ability is located on the surface.

  In the antibacterial component holding step, the antibacterial component is held in an exposed state for each molecule of each second binding layer. This antibacterial component includes inorganic antibacterial agents such as silver and copper, which have high protein binding properties, as well as alcohols, phenols, aldehydes, carboxylic acids, esters, ethers, nitriles, peroxides, Epoxy, halogen, pyridine / quinoline, triazine, isothiazolone, imidazole, thiazole, anilide, biguanide, disulfide, thiocarbamate, peptide protein, surfactant, organometallic, etc. Organic antibacterial agents and organic-inorganic hybrid antibacterial agents can be employed. If the functional group is a phenolic, carboxylic acid, aldehyde or peptide protein antibacterial agent, it can be quantitatively and stably bound from the reactivity with protein and selective reaction.

  The antibacterial test method of the present invention is characterized by using the standard test piece for antibacterial test of the present invention. In this case, since the standard test for antibacterial test of the present invention itself does not cause a variation in evaluation, the uncertainty of measurement becomes clearer than before, and the antibacterial effect is evaluated accurately and with high reproducibility. Is possible

  DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments embodying the present invention will be described below with reference to the drawings.

(1) Substrate Preparation Step As shown in FIG. 1, a substrate 1 of 20 (mm) × 20 (mm) × 0.2 (mm) was prepared. The substrate 1 is made of a polyvinylidene difluoride (PVDF) film.

(2) First Bonding Layer Formation Step Cholesterol was dissolved in chloroform as a solvent so as to have a concentration of 100 to 500 (nmol / μl) to prepare a sterol solution. Then, this sterol solution was smeared on the substrate 1 using a triangular spreader coated with a silicon tube, and as shown in FIG. 2, a first bonding layer 2 made of sterol was formed. Moreover, the 1st coupling layer 2 can also be formed on the board | substrate 1 by passing the board | substrate 1 in a sterol liquid. Thus, the first substrate 3 was obtained.

(3) Second bonding layer forming step As a sterol-binding protein, Vibrio cholerae hemolysin (molecular mass; 65 kDa) is used. Using a genetic engineering technique, a region having only styrol-binding ability of Vibrio cholerae hemolysin was identified (a region of about 250 residues from which 400 residues on the C-terminal side had been deleted). Mass produced. Then, a solution (100 ng to 1 μg / ml) in which sterol-binding protein is dispersed in a phosphate buffer solution (pH 7.2) is prepared, each first substrate 3 is submerged in 1 ml of this solution, and allowed to stand at room temperature for 1 hour. I put it. After each first substrate 3 was taken out of the solution, it was immersed in 10 ml of a phosphate buffer solution, vibrated for 5 minutes, and washed. This operation was repeated 10 times to wash out non-specifically bound sterol-binding proteins. As a result, as shown in FIG. 3, the sterol-binding protein 4a having a length of about 10 nm (details are unknown) is vertically bound to the first binding layer 2 on the surface of the first substrate 3, and the sterol concentration The second binding layer 4 consisting of a quantitatively and three-dimensionally uniform arrangement of sterol-binding proteins 4a depending on the first substrate 3 could be applied. The second binding layer 4 thus obtained has an amino group, a carboxyl group, an SH group, or a chemical substance having a specific binding ability located on the surface. Thus, the second substrate 5 was obtained.

(4) Antibacterial component holding step Glutaraldehyde (Glutar alddhyde) is prepared as an antibacterial component, and the antibacterial component 6 is added via the chemical substance of the second bonding layer 4 on the second substrate 5 as shown in FIG. Combined. Thereby, the antibacterial component 6 was held in an exposed state for each protein 4a, and a standard test piece for an antibacterial test was made.

  If an antibacterial test is performed with the obtained standard test piece for antibacterial test, the bacterial solution comes into contact with a certain amount of the antibacterial component 6, and the antibacterial effect is evaluated in this state. For this reason, the standard test for antibacterial testing itself does not cause variation in evaluation.

  Therefore, according to these standard test pieces for antibacterial tests, it becomes possible to more accurately determine the variation in evaluations other than the test pieces. For this reason, in the antibacterial test, the measurement uncertainty becomes clearer than before, and the antibacterial effect can be evaluated with high accuracy and high reproducibility.

  The present invention can be used for antibacterial tests.

It is a perspective view of a board | substrate concerning an Example. 1 is a perspective view of a substrate having a first bonding layer according to an embodiment. FIG. 4 is a perspective view of a substrate having a first bonding layer and a second bonding layer according to an embodiment. It is a perspective view of the standard test piece for an antimicrobial test which concerns on an Example. The standard test piece for antibacterial tests concerning a prior art example is shown, a figure (A) is a schematic cross section of a test piece, a figure (B) is a schematic cross section of a test piece under an ideal test, and figure (C) is a reality. It is a schematic cross section of the test piece during a typical test.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Board | substrate 2 ... 1st coupling layer 4 ... 2nd coupling layer 6 ... Antibacterial component

Claims (7)

A substrate,
A first bonding layer formed on a certain area of the substrate and made of a first compound bonded to the surface of the substrate;
A second compound comprising a second compound arranged on the first binding layer in a constant amount per unit area, specifically binding to each of the first compounds, and each molecule capable of holding a fixed amount of an antimicrobial component. A bonding layer;
An antibacterial test standard piece comprising the antibacterial component held in an exposed state for each molecule of the second compound.   The antibacterial test standard piece according to claim 1, wherein the substrate has a hydrophobic surface, the first compound is a sterol, and the second compound is a sterol-binding protein.   3. The standard test piece for antibacterial test according to claim 2, wherein the sterol-binding protein is Vibrio cholerae hemolysin. A substrate preparation process for preparing a substrate;
Forming a first bonding layer made of a first compound bonded to the surface of the substrate on a certain area of the substrate;
Second binding layer formation in which a second compound that specifically binds to each first compound and each molecule can hold a certain amount of antibacterial component is arranged on the first binding layer in a certain amount per unit area Process,
An antibacterial component holding step for holding the antibacterial component in an exposed state for each molecule of each second binding layer.   5. The antibacterial test standard piece according to claim 4, wherein the substrate has a hydrophobic surface, the first compound is sterol, and the second compound is sterol-binding protein. Method.   6. The method for producing a standard test piece for antibacterial test according to claim 5, wherein the sterol-binding protein is Vibrio cholerae hemolysin.   An antibacterial test method, which is performed using the standard test piece for antibacterial test according to any one of claims 1 to 3.

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