JP7235077B2 - Anti-mold and anti-algae test method - Google Patents

Description

The present invention relates to a test method for evaluating antifungal properties and antialgal properties. In particular, the present invention relates to a test method for evaluating the resistance of exterior materials and the like to microbial contamination by mold and algae that develop on the surface over time as a laboratory test.

Observation of dirt on the outer wall reveals that mold and algae are growing there, and research and development are being carried out to prevent or suppress such microbial contamination over the long term.

In general, demonstration tests under outdoor exposure are conducted as a means of evaluating antifungal properties or antialgae properties (for example, JP-A-2019-196440: Patent Document 1). Outdoor exposure tests have the problem that the test period ranges from half a year to several years and the uncertainty of reproducibility due to outdoor environmental factors. Under such circumstances, a laboratory test that reproduces the outdoor exposure test was required to improve the efficiency of research and development.

JIS Z2911:2018 "Mold Resistance Test for Industrial Products" (Non-Patent Document 1) is known as a lab test method for evaluating antifungal properties. In this test method, the surface of a specimen made of an industrial product or the like is inoculated with a mixed spore liquid of fungi, cultured for one week or longer, and the degree of fungal propagation is visually evaluated.

In addition, JIS R1705:2016 is defined as an antifungal test method for photocatalyst products (Non-Patent Document 2). In this test method, mold spore liquid is inoculated on the surface of a photocatalytic specimen, left to stand for a predetermined time under light irradiation with sufficient ultraviolet irradiance, and the number of spores that can germinate and grow is evaluated. It is a way to

Further, as a method for evaluating algae resistance by laboratory tests, for example, JP-A-2018-150278 is known (Patent Document 2). In this test method, a test specimen is inoculated with a culture solution of algae, adhered to a film, cultured under ultraviolet and visible light conditions for 1 to 4 weeks, and the amount of algal growth is visually evaluated.

However, the above conventional methods also have room for improvement. Non-Patent Literature 1 and Patent Literature 2 are methods for judging the reproduction of test organisms by visual inspection, and require one week or longer for culturing. Moreover, both the test organisms and the test environment are different from the actual site where microbial contamination occurs, and it is difficult to say that the exposure test is sufficiently reproduced.

In addition, some of the present inventors have discovered that cerium oxide with specific properties has the effect of suppressing germination and elongation of hyphae without killing mold spores (PCT/JP2020). /009574), but in the case of such a surface provided with cerium oxide, the test described in Non-Patent Document 2 would result in a determination of no mildew resistance. The conventional test method described in Non-Patent Document 2 cannot evaluate antifungal properties based on such new findings.

JP 2019-196440 A JP 2018-150278 A PCT/JP2020/009574 application

JIS Z2911:2018 JIS R1705:2016

The present invention has been made in view of the above circumstances. The objective is to develop a test method that enables the reproducible evaluation of the resistance of materials such as interior and exterior materials to stains caused by mold and algae over time (i.e., anti-mold and anti-algae properties) over a period of several days. is to provide

By using xerophilic fungi or xerophilic fungi, the present inventors have found that it is possible to quickly and reproducibly determine the anti-mold and anti-algae properties of exterior materials as laboratory tests. I found out. In addition, some of the present inventors have previously reported that algae grow by germinating fungal spores and then adhering to elongated or branched hyphae (Patent Document 3). It has been found that the mechanism of algae propagation can also be reproduced in the test method according to the present invention.

That is, the present invention
A test method for evaluating the anti-algae property of a member,
The surface of the member is inoculated with spores of a fungus belonging to xerophilic fungi or xerophilic fungi, and after culturing, the breeding rate of algae on the surface is estimated from the breeding rate of the fungus.

According to this test method, the anti-algae property of materials such as exterior materials, which are usually exposed to the outside air, exposed to light, and occasionally rain and water, can be tested as a laboratory test with good reproducibility and It is possible to judge quickly.

Another aspect of the present invention is
A test method for evaluating the anti-mold property or anti-algae property of a member,
The surface of the member is inoculated with spores of a fungus belonging to xerophilic fungi or xerophilic fungi, cultured, and then the breeding degree of the fungus is measured.

According to this test method, not only the anti-algae property but also the anti-mold property of materials such as exterior materials, which are usually exposed to the outside air, exposed to light, and occasionally rain and water, can be tested in the laboratory. As a test, it is possible to make determinations with good reproducibility and speed.

Furthermore, another aspect of the present invention is
A test method for evaluating the antifungal property or antialgae property of a member, comprising the following steps.
(1) a step of preparing a test body comprising the member;
(2) inoculating mold spores onto the surface of the specimen;
(3) culturing the inoculated mold;
(4) measuring the growth rate of the mold after the culturing;
wherein said inoculation is carried out by applying spores of said mold and a liquid medium to said surface,
It is applied such that the carbon source contained in the liquid medium is more than 0.00 mg/cm ² and less than 0.04 mg/cm ² .

According to this test method, it is possible to perform a highly accurate quantitative evaluation of the breeding rate by suppressing the supply amount of the carbon source, which is a nutrient for fungi during culture.

According to the present invention, it is possible to reproducibly evaluate the resistance (i.e., anti-mold property or anti-algae property) of members such as interior and exterior materials against aging stains due to mold and algae in a test for several days. It becomes possible to provide a test method.

It is a schematic diagram of a preservation Petri dish used in the test method of the present invention. Fig. 2 is an optical microscope photograph showing the degree of hyphal elongation of "0: ungerminated spores". It is an optical microscope photograph showing the elongation degree of hyphae "1: some spores have germinated, but hyphae are several 100 µm or less". It is an optical microscope photograph showing the elongation degree of hyphae "2: germination of spores was observed, and hyphae partially elongated to several 100 μm or more". It is an optical micrograph showing that the elongation degree of the mycelia is "3: Most of the spores germinate and the mycelia spread all over." 1 is a graph showing the relationship between ATP values and fungal growth in lab tests. 1 is a graph showing the relationship between laboratory tests and field tests on ATP values. It is the graph which showed the relationship between an ATP value and a color difference. 4 is a graph showing the relationship between the ATP value in the laboratory test and the ATP value in the outdoor exposure test for each type of liquid mixture used.

1. Elements Constituting the Invention
Element
The members used in the test method of the present invention are members constituting interior and exterior materials of buildings, housing equipment, agricultural materials, and the like. These members are inorganic materials such as metal materials and ceramics, organic materials such as synthetic resin products and wood, and composite materials thereof. Specific examples include tiles, sanitary ware, tableware, ceramic products such as calcium silicate plates and cement extrusion plates, glass, mirrors, wood, and resins. In addition, examples of members when expressed as applications include building exterior materials, building interior materials, window frames, window glass, structural members, vehicle exteriors, dustproof covers for articles, traffic signs, various display devices, advertising towers, Soundproof walls for roads, soundproof walls for railways, bridges, guardrails, tunnel interiors and coatings, insulators, solar cell covers, solar water heater heat collection covers, vinyl greenhouses, vehicle lighting covers, housing equipment, toilet bowls, bathtubs, washbasins Tables, lighting fixtures, lighting covers, kitchen utensils, dishwashers, dish dryers, sinks, cooking ranges, kitchen hoods, ventilation fans, protective films, etc. These members also include members on which a film is formed by printing, coating, coating, lamination, or the like.

In the present invention, the member is evaluated whether it has antifungal properties or antialgal properties, and to what extent it has these properties. The members to be evaluated in the present invention are those to which an antibacterial agent, antifungal agent, or antialgae agent is added to the surface, photocatalyst materials, etc., which are positively provided with antifungal or antialgal properties. It may be a thing, or it may be something that is not actively provided with those properties.

In the present invention, the member is preferably a non-water-absorbing member impermeable to water, and the surface to be evaluated is preferably flat. The size of the member is also not particularly limited, but according to one aspect of the present invention, the surface to be evaluated should have a size of at least 25 mm×25 mm.

Specimen The specimen is preferably obtained by appropriately cutting out a member. The specimen preferably has a flat surface to be evaluated. It is preferable that the specimen be a square of 25±2 mm square and be the standard size. By doing so, three substrates can be accommodated in a petri dish with a diameter of 90 mm. If it is difficult to cut out a square of 25±2 mm square, it may be used as a specimen if the surface area is at least 400 mm ² . According to the test method of the present invention, the size of the specimen is not limited to this, and may be, for example, 50 mm square. Also, the shape is preferably square, but is not limited to this and may be circular. It is preferable that the thickness of the specimen does not come into contact with the petri dish lid. When using a 90 mm petri dish (height 13.2 mm) for the evaluation of the exterior material, the thickness of the specimen is preferably 4 mm or less.

Mold (Dry-resistant mold or Xerophilic mold)
The fungi used in the test method of the present invention are xerophilic fungi or xerophilic fungi in the evaluation of exterior materials. Drought-resistant fungi and xerophilic fungi are based on the definitions described in Kosuke Takatori, "Illustrated Illustrated Fungi Inspection and Operation Manual at a Glance", Techno System Co., Ltd., 1991, 1st edition, page 142. These molds are preferably molds isolated from members such as exterior materials on which microbial stains are formed in an outdoor environment. Identification of the genus and species of fungi is performed by ITS rDNA analysis at Technosurgarabo Co., Ltd.

Said xerophilic mold or xerophilic mold is preferably a mold isolated from the site where the microbial soil is formed. Moreover, both the xerophilic fungi and the xerophilic fungi described above preferably have the property that spores adhere to the surface of the specimen. Moreover, it is preferable that the mold has a property that algae adhere to the hypha. Said algae are single-celled organisms classified as microalgae, preferably isolated algae taken from the same environment as the isolated mold. Examples of such algae include Protococcus sp. Moreover, Nothophoma sp. is a preferred example of the fungi having the above properties.

The fungi used in the test method of the present invention are xerophilic fungi or xerophilic fungi in the evaluation of members (such as cabinets) that are used indoors in environments where they are not exposed to water. The definitions of xerophilic fungi and xerophilic fungi are the same as above. These molds are preferably molds isolated from sites where microbial soils have formed in indoor environments. Identification of the genus and species of fungi is performed by ITS rDNA analysis at Technosurgarabo Co., Ltd. Here, the term "water-free environment" means an environment in which the member is not expected to be exposed to water during normal use.

Both the xerophilic fungi and xerophilic fungi described above preferably have the property that spores adhere to the surface of the specimen. A preferred example of the mold having the above properties is Eurotium sp., which is isolated from a member indoors on which microbial soiling has formed.

(hygroscopic mold)
As the mold used in the test method of the present invention, it is also possible to use hygroscopic mold when conducting a mold resistance test of a member used in a wet indoor environment such as a bathroom. It is preferable to use any one selected from the group of Cladosporium sp., Scolecobasidium sp. and Phoma sp. as hygroscopic fungi. A hygroscopic mold can be used that has been isolated from a humid environment such as a bathroom.

Spores and Suspensions Thereof According to a preferred embodiment of the invention, a spore suspension is used in the test method for inoculating the component or specimen with mold spores. Preparation of the spore suspension is as follows. The mold is inoculated onto potato dextrose agar (PDA) slants and pre-cultured at 28±2° C. for 7-21 days. Pour a wetting solution (sterilized purified water containing 0.005 wt% Tween 80) into the preculture test tube, pipette to remove spores from the culture surface, and filter through several sheets of sterilized gauze or cotton wool. . After filtration, the spores are sufficiently dispersed using a test tube mixer. Further, a spore concentration is adjusted to 1.0×10 ⁵ /mL using a wetting solution to obtain a spore suspension. The prepared spore suspension should preferably be stored in a refrigerator during the test and used on the same day.

Liquid Medium The liquid medium used in the test method of the present invention contains a carbon source and inorganic salts as nutrients. It is preferable that this liquid medium be a synthetic medium in that germination of fungal spores and elongation of mycelia can be regulated. Here, the carbon source is preferably carbohydrate, more preferably composed of carbohydrate. In the present invention, the saccharide is preferably a water-soluble saccharide, more preferably at least one selected from monosaccharides, disaccharides and oligosaccharides, and more preferably sucrose. Saccharides with a lower degree of polymerization yield test results with superior speed and quantification. In the present invention, the carbon source does not include surfactants used as spore dispersants.

According to a preferred embodiment of the test method of the present invention, the amount of carbon source in the liquid medium is greater than 0.00 mg/cm ² and less than 0.04 mg/cm ² . This amount of carbon source is characterized by being lower than in known test methods. That is, in the present invention, the liquid medium preferably has a low carbon source concentration. The test method of the present invention comprises a step of inoculating mold spores onto the surface of a member or specimen. is preferably applied to said surface. The concentration of the carbon source contained in this mixed solution is preferably more than 0 g/L and less than 3 g/L, more preferably more than 0 g/L and 2 g/L or less. By reducing the amount of carbon source supplied compared to the conventional method using a medium containing a high concentration of carbon source as a nutrient, it is possible to quantitatively evaluate the degree of fungal propagation with high accuracy.

As a specific example of the liquid medium, a diluted Czapekdox (Cz) medium is used. Preparation of the medium is as follows. Add _{3g of} NaNO3, _1g of _K2HPO4 , 0.5g of _MgSO4.7H2O , 0.5g _of KCl, 0.01g _{of FeSO4.7H2O} , and 20g of sucrose to 1000mL of purified water, and then dissolve them completely. , sterilize by autoclaving. After sterilization, filter filtration is performed, and the filtrate is used as the stock solution of the Cz medium. This undiluted solution is diluted with sterilized tap water so that the sucrose concentration is within the above range, and used for the test.

2. Operation procedure The test method of the present invention is a test method for evaluating the anti-mold property or anti-algae property of a member. is characterized by measuring This test method can be rephrased as a test method including the following steps. i.e.
(1) preparing a specimen;
(2) a step of inoculating mold spores on the surface of the specimen;
(3) culturing the (spores of) the inoculated mold;
(4) a step of measuring the growth rate of fungi after culturing;
is. Each step will be described in detail below.

(1) Preparation of specimen This is the process of adjusting the size of the specimen appropriately and then cleaning it. Cleaning is subdivided into cleaning and sterilizing processes.

Washing the test piece Wash the front and back of the test piece with tap water to remove cutting waste generated in the process of cutting out the test piece. If washing with running water causes softening of the specimen or dissolution or elution of components, treat with an air spray or the like.

After lightly wiping the entire surface of the sterilized test piece two or three times with absorbent cotton soaked with 99.5% ethanol, the test piece is thoroughly dried. If the alcohol treatment causes changes such as softening of the specimen, dissolution of the coating on the surface, or elution of components, etc., and it is judged to affect the test results, other appropriate methods such as those described below should be used. used or used directly for testing without sterilization.

In order to remove contaminant organic matter on the surface of the sterilization test specimen when the test specimen is a photocatalytic material , it is irradiated with an ultraviolet fluorescent lamp at an intensity of 1000 to 1500 μW/cm ² for 14 to 18 hours from the day before inoculation with mold spores. After that, before inoculation with the test spore solution, sterilization treatment is performed for 15 minutes each on the back and front sides in this order with a sterilization lamp in a clean bench.

Other Appropriate Methods Sterilize by irradiating the surface with a germicidal lamp in a clean bench for 15 minutes and the back for 15 minutes.

(2) Inoculation of fungal spores This is a step of applying fungal spores and a liquid medium to the surface of the specimen. A spore suspension and a liquid medium are used in this inoculation step.

Mixed solution: Mixed solution of spore suspension and liquid medium In the test method of the present invention, the mold spores and the liquid medium may be applied separately to the surface of the specimen. In addition, it is preferable to use a mixed liquid obtained by mixing a spore suspension and a liquid medium. The mixed solution is more preferably a mixture of the Cz medium diluted with sterilized tap water and the spore suspension in equal amounts. The prepared mixture should be stored in a refrigerator during the test and used on the same day.

Application to the surface of the specimen (in the case of evaluation of exterior materials or members used in an indoor environment where water is not splashed)
0.1 mL of the mixed liquid is sampled with a pipette and dropped onto the surface of the specimen. If the surface of the specimen is water-repellent, spread the dropped test spore solution over the entire surface by pipetting so as not to touch the surface. It is preferable to apply such an operation such that the carbon source contained in the liquid medium is more than 0.00 mg/cm ² and less than 0.04 mg/cm ² , more preferably 0.02 mg/cm ² . . By reducing the amount of carbon source supplied, it becomes possible to quantitatively evaluate the degree of fungal propagation with high accuracy.

When using xerophilic fungi or xerophilic fungi, it is preferable to then leave the specimens inoculated with the mixture in a clean bench and dry them at 25° C. for 3 hours. At that time, the air inside the clean bench is agitated by a fan.

After a predetermined time, visually confirm that no liquid remains on the surface of the specimen. Avoiding the presence of moisture on the surface improves correlation with test results from actual outdoor exposure.

(In the case of evaluation of materials used in indoor humid environments (e.g. bathroom interior materials))
It is preferred to carry out the test using hygroscopic molds. In this case, 0.1 mL of the mixed solution is dropped on the surface of the specimen, and a 20 mm square film (KOKUYO, VF-10) is adhered on the dropped mixed solution to prevent drying. For the adhesive film, it is preferable to use a material that does not affect the growth of microorganisms, does not absorb water, and has a smooth surface. This is because such a film has good adhesion between the mixture and the film. It is preferable to apply such an operation such that the carbon source contained in the liquid medium is more than 0.00 mg/cm ² and less than 0.04 mg/cm ² , more preferably 0.02 mg/cm ² . . By reducing the amount of carbon source supplied, it becomes possible to quantitatively evaluate the degree of fungal propagation with high accuracy.

(3) Cultivation Cultivation for evaluation of exterior materials or members used in an indoor water-free environment is performed under conditions of humidity 70% to 90%, temperature 24 to 28° C., and culture time 48±4 hours. In the case of evaluation of interior materials, culture is performed in an incubator under conditions of humidity of 90% or more, temperature of 28° C., and culture time of 48±4 hours.

In the case of evaluation of exterior materials and photocatalyst materials, after drying the liquid mixture on the surface of the specimen, the specimen is placed in a humidity-controlled storage petri dish. For the storage petri dish, place a sterilized humidity control filter paper on the bottom of the sterilized petri dish, add 8 mL of sterile tap water, place a glass U tube so that the filter paper does not come into direct contact, and place a slide glass on top. A schematic diagram of the storage petri dish is shown in FIG. In addition, the preserved petri dish is treated with a germicidal lamp for 15 minutes before being used for the test. Place the specimen on a glass slide.

(Light irradiation conditions)
When evaluating a photocatalyst material, light irradiation is performed in order to evaluate antifungal properties based on the action of reactive oxygen species generated by photoexcitation on fungi. A storage petri dish is placed at a position on the floor of the light irradiation apparatus where predetermined ultraviolet illuminance and visible light illuminance can be obtained. As light irradiation conditions, the following four conditions are selected according to the purpose, and the cells are cultured at a temperature of 26±2° C. for 48 hours.
・Ultraviolet light continuous irradiation Illuminance: 0.50 mW/cm ²
・Visible light continuous irradiation Illuminance: 5000lx
・Intermittent mixed light irradiation (intermittent light and dark conditions)
Ultraviolet irradiation and visible light irradiation are simultaneously applied for a predetermined period of time, and then the irradiation is stopped for the same period of time to create a dark environment. This light irradiation and intermittent irradiation in the dark are repeated, and the cells are cultured for a total time of 48±2 hours.
The light irradiation time and dark place time are preferably 10 to 12 hours each.
・Dark place Install in a dark room environment with light shielding.

In the case of evaluation of a member used in an indoor environment free from water exposure, after drying the mixed liquid on the surface of the specimen, the specimen is placed in a humidity-controlled storage petri dish. For the storage petri dish, place a sterilized humidity control filter paper on the bottom of the sterilized petri dish, add 8 mL of sterile tap water, place a glass U tube so that the filter paper does not come into direct contact, and place a slide glass on top. A schematic diagram of the storage petri dish is shown in FIG. In addition, the preserved petri dish is treated with a germicidal lamp for 15 minutes before being used for the test. Place the specimen on a glass slide.

(Culture conditions)
A storage petri dish is placed in a light-shielded darkroom environment, and cultured at a temperature of 26±2° C. for 48 hours.

In the case of evaluation of a member used in a humid indoor environment, put the test sample in which the mixed liquid was adhered with a film in a petri dish, let it stand in a humidity-controlled plastic container (Sun Platec, Tight Box No. 5), and put it in an incubator. Incubate at 26±2° C. for 48±2 hours. Place a sterilized wiper or the like with high water absorption in the plastic container and pour sterilized tap water to adjust the humidity.

(4) Measurement of Fungal Breeding Rate This is a step of measuring the fungal breeding rate on the surface of the specimen after culturing. The degree of fungal growth is measured by evaluating either or both of mycelial elongation and ATP value.

Microscopic observation of spores or mycelia adhering to the surface of the mycelial elongation specimen. It is desirable to use an optical microscope that allows observation within a magnification range of 200 to 500 times. Evaluation of the degree of mycelial elongation is preferably carried out by observing the germination of fungal spores and the state of mycelial elongation in a field of view at a magnification of 340 using a reflected illumination microscope (ECLIPSE LV100ND, Nikon).

According to one preferred embodiment of the present invention, based on observations, the spore germination mycelial elongation is expressed as follows.
0: Spores not yet germinated 1: Some spores germinated, but hyphae are short (several tens to several hundred μm) State 2: Spores germinated, and hyphae partially elongated to several hundred μm or more State 3: Most of the spores have germinated and the hyphae have grown all over.

ATP value (definition of ATP value)
In the present invention, the "ATP level" is defined as the amount of luminescence proportional to the total amount of ATP and AMP using an enzymatic cycling method combining luminescence reaction by luciferase and pyruvate orthophosphate dikinase.

(Quantification of ATP value)
An ATP wiping test system manufactured by Kikkoman Corporation is used for quantifying the ATP value. After culturing, the surface of the specimen is wiped with the company's "Lucipac (registered trademark) Pen", inserted into the company's "Lumitester (registered trademark) PD-30", luciferase catalyzes, luciferin, oxygen, and The amount of light emitted by the reaction of ATP is measured and converted into an ATP value per unit area of the coated body surface.

Evaluation of fungicidal property As a result of the test method according to the present invention, if the degree of mycelial elongation is low, it means that the member has high fungicidal property. Also, if the ATP value is 300 RLU/cm ² or less, it can be determined that the member has antifungal properties. For example, when the ATP value is 300 RLU/cm ² or less, the mycelial elongation inhibitory effect is exhibited, and when the ATP value is 100 RLU/cm ² or less, the antifungal property can be identified as the germination inhibitory effect.

The conventional antifungal test method, in which the degree of fungal propagation was visually evaluated, required a culture period of one month or more. However, the test method of the present invention provides results in a matter of days, and results can be obtained quickly. Furthermore, according to the present invention, by using the ATP value, it is possible to evaluate the degree of fungal propagation by the absolute value of the ATP value, so that the antifungal property can be evaluated more quantitatively than the conventional test method. becomes possible.

Evaluation of anti-algae property As described above, regarding the attachment mechanism of algae outdoors, algae germinate from fungal spores, then attach to elongated and branched hyphae and propagate. Therefore, if it is possible to effectively suppress the growth of fungi over a long period of time, it is also possible to prevent the growth of algae on the surfaces of members outdoors. That is, by measuring the degree of fungal propagation by the test method of the present invention, it is possible to evaluate the anti-algae property of exterior materials and the like.

For example, in the test method according to the present invention, if the ATP value is 300 RLU/cm ² or less, it can be determined that the member has anti-algae properties.

The invention is further illustrated by the following examples, but the invention is not limited to these examples.

Materials A primer mainly containing an epoxy resin was applied to a substrate/aluminum substrate and dried at room temperature for 24 hours. After that, an enamel paint containing a silicone-modified acrylic resin and a white pigment was further applied and dried at room temperature for 24 hours to obtain a substrate.

Cerium oxide particles 1-1 Water dispersion of cerium oxide (fluorite type, basic, cerium oxide concentration 10wt%, average crystallite size 6nm)

Silica particles 2-1 Water-dispersed colloidal silica (Na dispersion, _SiO2 concentration 30wt%, average particle size 25nm)
Titanium oxide particles 3-1 Titanium oxide aqueous dispersion (anatase type, basic, TiO ₂ concentration 17.5 wt%, average particle size 45 nm)
・3-2 Titanium oxide aqueous dispersion (rutile type, basic, TiO ₂ concentration 6.0 wt%, average particle size 35 nm)

Dispersion medium: Purified water Additive: Polyether-modified silicone surfactant

1) cerium oxide water dispersion , 2) water-dispersed colloidal silica, 3) titanium oxide water dispersion, 4) dispersion medium, and 5) Additives were mixed to obtain a coating composition. The concentration of film-forming components in the coating composition was 5.5% by weight. Here, the concentration of the film-forming component is the concentration of the total amount (charged amount) of the metal oxides 1) to 3) in the coating composition. For reference, the coating composition was heated to 400° C., then slowly cooled to room temperature, and the concentration when the weight became constant was measured.

Test 1: Relationship between ATP level and mold growth
Test 1 (1): Relationship between ATP value and mold growth in laboratory evaluation Coating composition C1 was applied to the surface of a substrate heated to 55°C by air spray so as to be 12.5 g/m ² . and dried at room temperature to form a surface layer. The coated body thus obtained (coated body 19) was used for evaluation.

First, a drought-tolerant fungus ( Nothophoma sp.) isolated from an exterior material highly contaminated with microorganisms at the outdoor exposure site was precultured on a potato dextrose agar slant medium at 28° C. for 7 to 14 days. The spores obtained by the preculture are suspended in sterile purified water containing 0.005 wt% Tween 80, diluted with sterile purified water to a spore concentration of 1×10 ⁵ /mL, and the spore suspension is obtained. A liquid was prepared. This inoculum was mixed with 10% Czapekdox liquid medium in equal amounts to prepare a mixture.

Next, the coated body was cut into a size of 25 mm x 25 mm to obtain a test body. This specimen was sterilized by irradiation with a germicidal lamp, and 0.1 mL of the mixed solution was dropped on the coated surface (that is, the surface of the specimen), and then the entire surface was smeared to inoculate with mold spores. After the coated body after smearing was left at room temperature for 3 hours and visually confirmed that the surface was dry, it was left to stand in a dark place in an environment with a temperature of 28°C and a relative humidity of 100%. , cultivated mold. Incubation time: 0 hour, 17 hours, 24 hours, and 40 hours test specimens were measured for fungal propagation. The degree of fungal propagation was measured by evaluating the degree of mycelial elongation and quantifying the ATP value.

An ATP wiping test system manufactured by Kikkoman Corporation was used for quantification of the ATP value. Wipe the surface of the specimen with the company's "Lucipac (registered trademark) Pen", insert it into the company's "Lumitester (registered trademark) PD-30", and luciferase-catalyzed reaction of luciferin, oxygen, and ATP. The amount of luminescence was measured and converted to the ATP value per unit area of the surface of the specimen.

The degree of mycelial elongation was observed using a reflected illumination microscope (ECLIPSE LV100ND, Nikon) in a field of view of 340x magnification. It was evaluated by Figs. 1 to 4 show typical fungal propagation conditions for each of them.

(Mycelium elongation)
0: spores are not germinated (Fig. 2),
1: Some spores have germinated, but the hyphae are short (several tens to several hundred μm) (Fig. 3),
2: A state in which spore germination was observed and hyphae were partially elongated to several hundred μm or more (FIG. 4).
3: Most of the spores have germinated, and the mycelium has grown all over (Fig. 5).
In addition, when the mycelial elongation is 3, dullness or black fungal stains on the surface of the sample caused by fungal growth can be visually observed.

FIG. 6 shows the relationship between the ATP value and the degree of mycelial elongation.

There was a high correlation between the ATP value and the degree of mycelial elongation, and the higher the ATP value, the longer the fungal mycelia. It was found that the ATP value corresponds to the degree of fungal propagation from the spore state to germination and mycelial elongation.

Test 1 (2): Relationship between ATP value after lab test and ATP value after outdoor exposure test In order to compare ATP values in the lab test and outdoor exposure test, C1 to C5 were used as coating compositions. , five types of coated bodies having different compositions of the surface layer were produced. The conditions for preparing the coated body were the same as in Test 1 (1) above. Table 2 shows the correspondence between the coated body and the coating composition used.

The outdoor exposure test was conducted in an environment surrounded by forests in the Tokai area. The painted body shown in Table 2 was placed facing north in the test site, exposed to the outdoors for one month, and the ATP value was measured.

The lab tests were performed according to the following procedure. That is, the coated body was cut into a size of 25 mm×25 mm to obtain a test body. This specimen was sterilized by sterilizing with a germicidal lamp, and 0.1 mL of the mixed solution prepared in the same manner as in Test 1 (1) was dropped on the coated surface (that is, the surface of the specimen), and then the entire surface was smeared. The coated body after smearing was left at room temperature for 3 hours, and after visually confirming that the surface was in a dry state, it was left to stand in an environment with a temperature of 28°C and a relative humidity of 100% to culture the mold. . During the incubation period, a BLB lamp (FL40SBLB, manufactured by Sankyo Denki Co., Ltd.) was used so that the UV intensity on the coated body surface was 0.5 mW/cm ² using a UVR-2 UV intensity meter manufactured by Topcon Technohouse. Irradiation was performed at intervals of 12 hours. ATP values were quantified after a total of 48 hours of irradiation and non-irradiation.

The ATP value was quantified by the same method as described for the ATP value quantification in Test 1(1) above.

FIG. 7 shows the relationship between the ATP value after the laboratory test and the ATP value after the outdoor exposure test.

No algae adhered or propagated on each painted body surface subjected to the one-month exposure test, and only mold spore adherence, germination, and hyphal elongation were observed. Moreover, the ATP value confirmed by the laboratory test showed a high correlation with the ATP value after the outdoor exposure test.

Coated body 1 showed a significantly high ATP value both in the laboratory test and the outdoor exposure test. On the other hand, the other 4 samples had low ATP values in both tests, and the order of the ATP values in the laboratory test (painted body 1>>painted body 5>painted body 2>painted body 4≒painted body 3) was the order of the outdoor exposure test. was almost the same as

The ATP value is effective as an index for measuring the antifungal effect, and can be treated as an index of the extent of the influence of the coated body surface on mold spores. A coated body surface that can keep the ATP value low effectively exhibits an antifungal effect.

Test 2: Relation between fungal growth (ATP value) and algae growth The relationship between the ATP value in the laboratory test and the algae growth in the outdoor exposure test was evaluated. The painted bodies used were the same 5 samples as in Test 1 (2) above. evaluated.

The laboratory test was conducted in the same manner as Test 1 (2), except that the light irradiation conditions were changed. The light irradiation conditions were as follows.

A BLB lamp (FL40SBLB, manufactured by Sankyo Electric Co., Ltd.) and a white fluorescent lamp (FLR40SW/M/36-B, manufactured by Hitachi Appliances, Inc.) were simultaneously irradiated for 12 hours. Ultraviolet intensity meter manufactured by Topcon Technohouse: UVR-2 The ultraviolet intensity on the surface of the coated body is 0.5 mW/cm ² , and illuminance meter manufactured by Topcon Technohouse: IM-5. The illuminance on the body surface was 5000lx. After light irradiation, the dark condition was set to 12 hours, and intermittent irradiation was performed at intervals of 12 hours.

In the follow-up observation, after one month of exposure, mold spores germinated on the painted body corresponding to the comparative example, and the mycelium was observed to extend all over, but algae adhesion was not confirmed. . In this test, the coated body that showed a high ATP value in the laboratory test began to adhere to algae after the growth of mold mycelium, and after 6 months, greenish stains were visible. rice field.

The degree of contamination after 6 months was evaluated using a spectrophotometer CM-2600d manufactured by Konica Minolta Japan, Inc. Based on JIS Z8730 (2009), it was quantified as the color difference ΔE ^* between the coated surface before outdoor exposure and after 6 months in the L ^* a ^* b ^* color system.

FIG. 8 shows the relationship between the ATP value in the laboratory test and the color difference after the outdoor exposure test.

According to FIG. 8, ATP value and color difference showed good correlation.

It was suggested that a coated body that can keep the ATP value low is effective not only in designing antifungal effects but also in designing antialgal effects. Considering the above findings and the stain generation process in the outdoor exposure test, there is a close relationship between the anti-mold effect and the anti-algae effect. is thought to be important for the expression of Therefore, it can be said that the ATP value is an index of the degree of influence of the coated body surface on mold spores, that is, it is effective not only as an index of the antifungal effect but also as an index of the antialgal effect.

Test 3: Antifungal property evaluation test using hygroscopic fungi Cladosporium sp., which is an isolated hygroscopic fungus collected from bathrooms of ordinary households using unit baths, was used as the fungus. As a test piece, a resin piece (made of ABS resin, 25 mm×25 mm) containing no component such as an antifungal agent that inhibits the growth of microorganisms was used. The mold spores were inoculated by applying the mixture to the surface of the specimen as in test 1(1), followed by drying for 3 hours. After drying, it was placed in a storage petri dish and cultured at 25° C. for 48 hours in the dark. On the other hand, using the same test fungi and specimens, after inoculation of the mixed solution, the mixture was allowed to stand in a humidity-controlled container in a film-adhered state, and cultured at 28° C. for 48 hours. The tightness of the film prevented the culture from drying out during the incubation period. For the specimens after culturing under these two conditions: dry condition and wet condition, evaluation of mycelial elongation and quantification of ATP value were performed. Table 3 shows the results.

When drying treatment was performed after inoculation with the test spore solution, the ATP value was about 100 RLU/cm ² and the mycelial elongation was low. On the other hand, when the film was adhered and the test spore fluid was retained, the ATP value exceeded 1000 RLU/cm ² and the mycelial elongation was 3. Dry conditions are not suitable for the growth of hygroscopic fungi, and it is preferable to maintain moist conditions by film adhesion.

Based on the above test results, the anti-mold and anti-algae properties of materials such as exterior materials, which are usually exposed to the outside air, are exposed to light, and are occasionally splashed with water such as rain, are evaluated by laboratory tests. It has been found that it is preferable to use xerophilic fungi or xerophilic fungi for culture, and to keep the surface of the specimen dry during culture. In addition, in order to evaluate antifungal properties in a humid environment such as a bathroom by laboratory tests, it is preferable that hygroscopic fungi be used and that the surface of the test specimen be in a wet state during cultivation. I found out.

Test 4: Relationship between mold growth (ATP value) and carbon source concentration as nutrients The ATP values in the lab tests and the ATP values in the outdoor exposure tests were compared.

Using C1, C2, C4, and C6 as coating compositions, four types of coated bodies with different surface layer compositions were produced. The conditions for producing the coated body were the same as in Test 1 (1) above.

(Lab test)
These coated bodies were cut to 25 mm×25 mm to obtain test bodies. These specimens were sterilized by irradiation with a germicidal lamp.

Tzapexdox liquid medium was prepared and diluted with sterile tap water to make 2-fold, 5-fold and 10-fold dilutions, respectively. A spore suspension prepared in the same manner as in Test 1 (1) was mixed with these diluted liquid media at a volume ratio of 1:1 to prepare three mixed solutions. As a control, the spore suspension was mixed with the same volume of sterilized tap water to prepare a mixture containing no carbon source.

After 0.1 mL of the mixed solution was dropped onto the above sterilized specimen, the entire surface was smeared to inoculate with mold spores. After leaving the coated body after smearing for 3 hours at room temperature and visually confirming that the surface is dry, leave it for 48 hours in an environment with a temperature of 28 ° C and a relative humidity of 100% to remove mold. was cultured. Light irradiation was performed under the same conditions as in Test 2 during the culture period.

The ATP value of the cultured specimen was quantified in the same manner as in Test 1 (1) above.

(Outdoor exposure test)
The prepared coated body was subjected to an exposure test in the same manner as in Test 1 (2) above, and the ATP value was quantified in the same manner as in Test 1 (1) above. Moreover, visual observation was also performed.

(result)
FIG. 9 shows the relationship between the ATP value after the laboratory test and the ATP value after the outdoor exposure test for each type of mixed solution used.

In an outdoor exposure test, a coated body containing 100% cerium oxide using C2 and a coated body containing 10% titanium oxide using C4 had an ATP value of around 100 RLU/cm2 and had high antifungal properties. showed that. The coated body containing 1% titanium oxide using C6 and the coated body containing 100% silica using C1 had a high ATP value of 800 RLU/cm2, and mold growth was observed. .

In laboratory tests, when the sucrose concentration in the mixed liquid was 7.5 g/L and 3.0 g/L, only the coated body containing 100% silica was around 1000 RLU/cm2, and the other three test bodies were several hundred to several hundred. It was 10 RLU/cm2, and the correlation between the laboratory test and the outdoor exposure test was low.

When the sucrose concentration in the mixed solution was 1.5 g/L, the ATP values in the laboratory test and the outdoor exposure test showed a high correlation.

When the sucrose concentration in the mixed liquid was 0.0 g/L, the ATP value of all specimens in the laboratory test was low, and the growth of fungi tended to be suppressed, resulting in poor experimental accuracy.

From the above test results, in order to reproduce the antifungal property in outdoor exposure, it is preferable that the sugar concentration in the mixed liquid in the laboratory test is more than 0 g/L and less than 3 g/L.

Test 5: Antifungal property evaluation test using xerophilic fungi As the fungus, Eurotium sp., which is an isolated xerophilic fungus collected from indoor cabinets of houses, was used. A washed float plate glass (25 mm×25 mm) was used as a test piece. The mold spores were inoculated by applying the mixture to the surface of the specimen as in test 1(1), followed by drying for 3 hours. After drying, it was placed in a storage petri dish and cultured at 25° C. for 48 hours in the dark. On the other hand, using the same test fungi and specimens, immediately after inoculation of the mixed solution, they were placed in a humidity control container and cultured at 28° C. for 48 hours. The culture solution did not dry out during the culture period because it was left still in the humidity-controlled container immediately after inoculation. For the specimens after culturing under these two conditions: dry condition and wet condition, evaluation of mycelial elongation and quantification of ATP value were performed. Table 4 shows the results.

Claims (11)

A test method for evaluating the anti-mold property or anti-algae property of a member,
a step of inoculating the surface of the member with spores of a fungus belonging to xerophilic fungi or xerophilic fungi, culturing the fungi, and then measuring the degree of proliferation of the fungi; Or at least comprising a step of evaluating algae resistance ,
said inoculation is carried out by applying spores of said mold and a liquid medium to said surface;
A test method, wherein the carbon source contained in the liquid medium is more than 0.00 mg/cm ² and less than 0.04 mg/cm ² . 2. The test method according to claim 1 , wherein said xerophilic mold or xerophilic mold is a mold isolated from a site where microbial soil is formed. 3. The test method according to claim 1 or 2 , wherein the mold is Nothophoma sp. , which has the property that algae adhere to the hyphae of the mold. The test method according to any one of claims 1 to 3 , wherein after said inoculation, said surface is dried and then said cultivation is carried out. 5. The test method according to claim 4, wherein the culturing is performed in a humidified state. The inoculation is carried out by applying a mixed liquid in which the mold spores are dispersed in a liquid medium to the surface,
The test method according to any one of claims 1 to 5 , wherein the mixed liquid contains more than 0 g/L and less than 3 g/L of carbon source in the liquid medium. A test method for evaluating the anti-mold or anti-algae properties of a member comprising the steps of:
(1) a step of preparing a test body comprising the member;
(2) inoculating mold spores onto the surface of the specimen;
(3) culturing the inoculated mold;
(4) measuring the growth rate of the mold after the culturing;
and
wherein said inoculation is carried out by applying spores of said mold and a liquid medium to said surface,
A test method, wherein the carbon source contained in the liquid medium is more than 0.00 mg/cm ² and less than 0.04 mg/cm ² . The inoculation is carried out by applying a mixed liquid in which the mold spores are dispersed in a liquid medium to the surface,
8. The test method according to claim 7, wherein the mixed liquid contains more than 0 g/L and less than 3 g/L of carbon source in the liquid medium. The test method according to any one of claims 1 to 8 , wherein the carbon source is carbohydrate. 10. The test method according to any one of claims 1 to 9 , wherein the breeding degree of the mold is evaluated by the ATP value. The test method according to any one of claims 1 to 10 , wherein the culture is intermittently irradiated with light.

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