How to form dark spots

Abstract

To provide a dark stain forming method that can efficiently form dark stain on a base material.SOLUTION: A dark stain forming method includes the following steps: step A of immersing a base material in a culture medium containing biofilm forming bacteria and dematiaceous fungi to perform culturing, thereby forming the biofilm on the base material; and step C of growing the dematiaceous fungi in the biofilm formed on the base material, to form dark stain.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for forming a darkening layer on a substrate, and also to a method for evaluating an anti-darkening effect and a method for screening a substance having an anti-darkening effect, using the method for forming a darkening layer. [Background technology]

[0002] Blackening is likely to occur in areas around water such as toilets, bathrooms, kitchens, and washrooms, and it is known that ring-shaped blackening is particularly likely to occur near the water surface in a toilet bowl. The mechanism by which blackening occurs is being clarified, and it is believed that first, bacteria attach and grow to form a biofilm, creating an environment in which black fungus can easily attach, and then the black fungus attaches to the biofilm and grows.

[0003] The blackening that occurs in living spaces not only causes visual discomfort, but also emits an unpleasant odor and can be a source of infection. Therefore, cleaning compositions containing antibacterial ingredients have been developed as cleaning agents to combat blackening.

[0004] On the other hand, when developing a cleaning agent to combat blackening, it is essential to evaluate the blackening inhibition effect. However, as mentioned above, blackening is a complex aggregate of biofilm and black fungus, so the blackening inhibition effect cannot be properly evaluated by only measuring the antibacterial activity of the cleaning agent. Therefore, in the past, in order to evaluate the blackening inhibition effect of a cleaning agent, it was necessary to conduct a field test in which the cleaning agent was used in an actual home and the occurrence of blackening was observed. However, there are problems with field tests, such as the long period of time required and the high cost of ensuring the number of subjects.

[0005] In recent years, a method has been developed to artificially form dark spots by forming a biofilm on a substrate, and then attaching and growing black fungi on the biofilm, and a laboratory test system capable of evaluating the effect of inhibiting dark spots has been constructed (see Non-Patent Document 1). However, the conventional method of forming dark spots has drawbacks such as the fact that depending on the type of bacteria in the biofilm, the growth of the black fungus does not progress, the dark spots formed are small, and it takes a long time for the formation of dark spots, making it impossible to form dark spots efficiently.

[0006] Against the background of such conventional technology, there is a strong need to establish a method for efficiently forming darkening on a substrate and to construct a laboratory test system capable of evaluating the anti-darkening effect. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Michitaka Muramatsu et al., "Establishment of a Laboratory Model for Black Spot Formation," Abstracts of the 2018 Annual Conference of the Society of Environmental Biotechnology, Published by the Society of Environmental Biotechnology, June 25, 2018, p. 41 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for forming a darkening layer on a substrate, which can efficiently form a darkening layer on the substrate. Another object of the present invention is to provide a method for evaluating an anti-darkening effect and a method for screening a substance having an anti-darkening effect, which utilize the method for forming a darkening layer. [Means for solving the problem]

[0009] The present inventors have conducted intensive research to solve the above problems, and have found that blackening can be efficiently formed by performing a biofilm formation step in which a substrate is immersed in a culture solution containing biofilm-forming bacteria and black fungi and then a blackening formation step in which the black fungi in the biofilm formed on the substrate are grown. The present inventors have also found that blackening can be formed more efficiently by performing a stress treatment step between the biofilm formation step and the blackening formation step to reduce the growth activity or viable cell count of the biofilm-forming bacteria in the biofilm. Furthermore, the present inventors have found that the use of the blackening formation method makes it possible to evaluate the anti-blackening effect and screen for substances having anti-blackening effect. The present invention has been completed through further research based on these findings.

[0010] That is, the present invention provides the following aspects. Item 1. A method for forming dark spots, comprising the following steps: A step A of forming a biofilm on a substrate by immersing the substrate in a culture solution containing biofilm-forming bacteria and black fungus and culturing the substrate; and Step C of generating black spots by growing black fungi in a biofilm formed on the substrate. Item 2. The method for forming black spots according to Item 1, comprising, prior to step C, step B of performing a stress treatment to reduce the proliferation activity or viable cell count of biofilm-forming bacteria in the biofilm formed in step A. Item 3. The method for forming a dark spot according to Item 2, wherein the stress treatment in step B is a drying treatment, an ultraviolet treatment, or a heating treatment. Item 4. The method for forming a dark spot according to any one of Items 1 to 3, wherein the culture medium used in the step A contains mannitol and / or bovine serum albumin. Item 5. The method for forming a black spot according to any one of Items 1 to 4, wherein after step A, the biofilm formed in step A is impregnated with a culture solution. Item 6. A method for evaluating the anti-darkening effect of a test sample, comprising: A step A of forming a biofilm on a substrate by immersing the substrate in a culture solution containing biofilm-forming bacteria and black fungus and culturing the substrate; and A step C of causing black spots by growing black fungi in a biofilm formed on a substrate, The evaluation method includes adding a test sample in at least one stage selected from the group consisting of during the step A, after the step A and before the step C, during the step C, and after the step C, and comparing the state of darkening under a condition in which the test sample is added with a condition in which the test sample is not added. Item 7. A method for screening a substance having an anti-darkening effect, comprising: A step A of forming a biofilm on a substrate by immersing the substrate in a culture solution containing biofilm-forming bacteria and black fungus and culturing the substrate; and A step C of causing black spots by growing black fungi in a biofilm formed on a substrate, A screening method comprising adding a candidate substance at at least one stage selected from the group consisting of during step A, after step A and before step C, during step C, and after step C, and comparing the state of the darkening formed under conditions in which the candidate substance is added and conditions in which the candidate substance is not added. Effect of the Invention

[0011] According to the present invention, since darkening can be efficiently formed on a substrate, a test material can be provided for evaluating the anti-darkening effect (the effect of suppressing the formation of darkening and the effect of removing darkening that has already been formed), and it becomes possible to efficiently develop cleaning agents and the like for preventing darkening. [Brief description of the drawings]

[0012] [Figure 1] 1 is a photograph showing the observation of black spots formed on a substrate in Test Example 1. [Diagram 2] 1 is a photograph showing the observation of black spots formed on a substrate in Test Example 2. [Diagram 3] 1 is a photograph showing the observation of black spots formed on a substrate in Test Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] 1. How dark spots form The method for forming black spots of the present invention is characterized by including a step A of forming a biofilm on a substrate by immersing the substrate in a culture solution containing a biofilm-forming bacterium and a black fungus and culturing the same, and a step C of causing the black fungus in the biofilm formed on the substrate to grow, thereby generating black spots. The method for forming black spots of the present invention will be described in detail below.

[0014] [Step A (biofilm formation step)] In step A, a substrate is brought into contact with a culture solution containing biofilm-forming bacteria and black fungus, and cultured to form a biofilm on the substrate.

[0015] The material of the substrate that will ultimately serve as a scaffold for the blackening may be appropriately selected depending on the test purpose, etc., and examples of the substrate include hard materials such as pottery, glass, stainless steel, ceramics, plastics, fibrous materials, etc. Among these, hard materials such as pottery, glass, stainless steel, ceramics, plastics, etc. are preferable.

[0016] The shape of the substrate may be appropriately set depending on the test purpose, etc., and may be, for example, a sheet, a block, a string, a granule, etc. Among these, from the viewpoint of ease of operation, etc., a sheet or a block is preferable, and a sheet is more preferable. The size of the substrate used in step A may be appropriately set depending on the test purpose, etc.

[0017] Furthermore, the substrate may be subjected to a coating treatment, etc., for adhering minerals, silicon, etc., if necessary.

[0018] Biofilm-forming bacteria are bacteria capable of forming a biofilm. The type of biofilm-forming bacteria used in step A is not particularly limited, and examples thereof include bacteria of the genus Rhizobium, Pseudomonas, Sphingomonas, Sphingopyxis, Sphingobium, Brevundimonas, Blastomonas, and Stenotrophomonas. , Porphyromonas, Aeromonas, Klebsiella, Flavobacterium, Ochrobactrum, Methylobacterium, Fusobacterium, Brevibacterium, Burkholderia, Enterobacter, nterbacter, Acinetobacter, Citrobacter, Aggregatibacter, Rhodobacter, Erwinia, Escherichia, Ralstonia, Salmonella, Serratia, Yersinia, Acidovorax idovorax, Prevotella, Tannerella, Alcaligenes, and other gram-negative bacteria; and Staphylococcus, Streptococcus, Enterococcus, Bacillus, Lactobacillus, Deinococcus, and other gram-positive bacteria.These bacteria may be used alone or in combination of two or more kinds.

[0019] Black fungi are fungi that are dark in color due to the production of melanin pigment. The type of black fungus used in step A is not particularly limited, and examples thereof include fungi of the genera Cladosporium, Aspergillus, Aureobasidium, Alternaria, Bipolaris, Cladophialophora, Exophiala, Fonsecaea, Phialophora, Rhinocladiella, Phoma, Scolecobasidium, Ulocladium, Epicoccum, Curvularia, and Chaetomium. These black fungi may be used alone or in combination of two or more.

[0020] In step A, the black fungus to be inoculated into the culture solution may be in the form of mycelium, but is preferably in the form of spores.

[0021] The composition of the culture medium is not particularly limited as long as it allows the growth of biofilm-forming bacteria, but may contain, for example, a carbon source, a nitrogen source, a sulfur source, a phosphorus source, a vitamin source, and a mineral source, and may be set according to the type of biofilm-forming bacteria used.

[0022] When the culture medium used in step A contains mannitol, the biofilm formed has a high production amount of extracellular proteins and a high viscosity, and has the same characteristics as a biofilm formed in an actual environment, and the blackening finally formed can be made to resemble that formed in an actual environment. Therefore, a suitable example of the culture medium is one containing mannitol. When the culture medium contains mannitol, the content thereof is, for example, 5 to 20 g / l, preferably 5 to 15 g / l, more preferably 7.5 to 12.5 g / l.

[0023] Furthermore, when the culture medium used in step A contains mannitol and does not contain a protein hydrolysate such as tryptone, the formed biofilm can more closely resemble the characteristics of a biofilm formed in an actual environment. Therefore, a preferred example of the culture medium is one that contains mannitol and does not contain a protein hydrolysate.

[0024] Furthermore, when the culture medium used in step A contains bovine serum albumin (BSA), it is possible to make the finally formed black spot appear deep black. Therefore, a suitable example of the culture medium is one containing BSA. When the culture medium contains BSA, the content thereof is, for example, 1 to 10 g / L, preferably 2 to 8 g / L, and more preferably 3 to 6 g / L.

[0025] The culture medium used in step A preferably contains yeast extract. When the culture medium contains yeast extract, the content is, for example, 2.0 to 6.0 g / l, preferably 2.0 to 5.0 g / l, and more preferably 2.5 to 3.5 g / l.

[0026] In step A, the seeding amount of the biofilm-forming bacteria in the culture solution (initial concentration of the biofilm-forming bacteria in the culture solution) may be appropriately set depending on the type of biofilm-forming bacteria used, the composition of the culture solution, the culture time, etc., but may be, for example, 1.0 × 10 3 ~1.0×108 cfu / ml, preferably 1.0×10 5 ~1.0×10 8 cfu / ml, more preferably 1.0×10 6 ~1.0×10 7 cfu / ml.

[0027] In step A, the seeding amount of the black fungus in the culture solution (initial concentration of the black fungus in the culture solution) may be appropriately set depending on the type of black fungus used, the composition of the culture solution, the culture time, etc., but may be, for example, 1 × 10 2 ~1×10 4 cfu / ml, preferably 3×10 2 ~5×10 3 cfu / ml, more preferably 5×10 2 ~2×10 3 cfu / ml.

[0028] In step A, examples of the culture methods for contacting a substrate with a culture solution containing biofilm-forming bacteria and black fungus include a method of immersing the substrate in the culture solution and incubating it; and a method of retaining the culture solution on the substrate and incubating it.

[0029] The culture conditions in step A may be appropriately set depending on the types of substrate, biofilm-forming bacteria, black fungus, and culture solution, so that a biofilm can be formed on the substrate. For example, the culture conditions may be a temperature condition of 22 to 32°C, preferably 25 to 30°C, and more preferably 28 to 30°C, for 60 to 100 hours, preferably 65 to 96 hours, and more preferably 72 to 84 hours.

[0030] Thus, a biofilm containing black fungus is formed on the substrate by carrying out step A. The formation of a biofilm on the substrate can be confirmed, for example, by staining with a staining solution such as crystal violet.

[0031] The biofilm formed in step A is subjected to a cleaning treatment such as washing with water as necessary, and then to the next step B or C.

[0032] [Process B (Stress treatment process)] Step B is an optional step in which a stress treatment is performed to reduce the proliferation activity or viable cell count of biofilm-forming bacteria in the biofilm. By performing the stress treatment (step B) to reduce the proliferation activity or viable cell count of biofilm-forming bacteria in the biofilm prior to step C, it becomes possible to generate blackening more efficiently.

[0033] The "stress treatment for reducing the proliferation activity or viable cell count of biofilm-forming bacteria in a biofilm" refers to a stress treatment applied to a biofilm in order to reduce the proliferation activity of biofilm-forming bacteria in the biofilm and / or reduce the viable cell count of biofilm-forming bacteria in the biofilm. The stress treatment in step B needs to be performed under conditions that do not kill the black fungus present in the biofilm.

[0034] The stress imposed on the biofilm by the stress treatment in the process may be within a range that can reduce the proliferation activity or bacterial count of the biofilm-forming bacteria in the biofilm without killing the black fungus, and may be set, for example, so that the viable count of the biofilm-forming bacteria in the biofilm is about 90% or less, preferably about 50% or less, more preferably about 25% or less, and particularly preferably about 10% or less, compared to before the stress treatment.

[0035] The stress treatment in step B is not particularly limited, so long as it does not kill the black fungus and reduces the proliferation activity of the biofilm-forming bacteria and / or reduces the viable cell count of the BF-forming bacteria in the biofilm. Specific examples of the stress treatment include drying treatment, ultraviolet light treatment, heating treatment, electromagnetic radiation treatment, chemical treatment, and gas treatment.

[0036] Drying treatment is a treatment that applies stress to a biofilm by evaporating water from the biofilm and drying it. Temperature conditions for the drying treatment include, for example, 15 to 80° C., preferably 15 to 50° C., more preferably 22.5 to 27.5° C., and even more preferably 24.5 to 25.5° C. The time for the drying treatment includes, for example, 0.5 to 2.5 hours, preferably 0.75 to 2 hours, and more preferably 1 to 1.5 hours.

[0037] The ultraviolet treatment is a treatment in which a biofilm is exposed to ultraviolet light to impose stress on the biofilm. The ultraviolet irradiation dose in the ultraviolet treatment is, for example, 150 mJ / cm 2 More than 170 mJ / cm 2 More preferably, 180 to 300 mJ / cm 2 Examples include:

[0038] Heat treatment is a treatment in which a biofilm is heated to apply stress to the biofilm. The temperature condition of the heat treatment is, for example, 40° C. or higher, preferably 40 to 50° C., and more preferably 45 to 50° C. The time of the heat treatment is, for example, 30 minutes or more, preferably 45 to 120 minutes, and more preferably 45 to 60 minutes.

[0039] Electromagnetic radiation treatment is a treatment that stresses the biofilm by exposing it to electromagnetic radiation such as infrared rays, visible light, ultraviolet rays, X-rays, and gamma rays. Chemical treatment is a treatment that stresses the biofilm by adding natural antibacterial agents such as herbs and spices, or organic chemical synthetic antibacterial agents such as alcohols and quaternary ammonium salts. Gas treatment is a treatment that stresses the biofilm by exposing it to gaseous ethylene oxide, hydrogen peroxide, etc.

[0040] Among these stress treatments, from the viewpoints of ease of operation and suppression of damage to the black fungus, drying treatment, ultraviolet light treatment, and low temperature treatment are preferable.

[0041] The biofilm-formed substrate after step B is subjected to a cleaning treatment such as washing with water, if necessary, and then to step C.

[0042] [Process C (blackening process)] In step C, black fungus is allowed to grow in the biofilm formed on the substrate, thereby causing black spots to form.

[0043] In step C, prior to growing the black fungus, the biofilm formed on the substrate may be impregnated with a culture solution necessary for the growth of the black fungus. By impregnating the biofilm with the culture solution in this way and then growing the black fungus, it is possible to generate blackening more efficiently. The culture solution to be impregnated into the biofilm may contain components necessary for the growth of the black fungus (carbon source, nitrogen source, sulfur source, phosphorus source, vitamin source, mineral source, etc.). A suitable example of the culture solution is potato dextrose broth. The culture solution can be impregnated into the biofilm by, for example, dropping or applying the culture solution onto the biofilm on the substrate film, or impregnating the substrate on which the biofilm is formed into the culture solution. In step C, in order to grow the black fungus after it has been attached to the biofilm and form black spots, the black fungus may be cultured under conditions that allow the black fungus to grow in the gas phase.

[0044] The culture conditions for growing the black fungus to cause blackening may be appropriately set depending on the type of black fungus used, and examples include a temperature condition of 20 to 30°C, preferably 22.5 to 27.5°C, and more preferably 22.5 to 25°C, for 70 to 200 hours, preferably 75 to 150 hours, and more preferably 80 to 100 hours.

[0045] By carrying out step C in this manner, dark spots are generated on the substrate, and a test material having dark spots formed on the substrate is obtained.

[0046] 2. Method for evaluating the anti-darkening effect The method for evaluating the anti-darkening effect of the present invention includes step A of forming a biofilm on the substrate by immersing the substrate in a culture solution containing a biofilm-forming bacterium and a black fungus and culturing the same, and step C of causing darkening by proliferating the black fungus in the biofilm formed on the substrate, and is characterized in that a test sample is added at at least one stage selected from the group consisting of during step A, after step A and before step C, during step C, and after step C, and the state of darkening is compared between a condition in which the test sample is added and a condition in which the test sample is not added.

[0047] The method for evaluating the anti-darkening effect of the present invention is a method for evaluating the anti-darkening effect of a test sample. The anti-darkening effect refers to the effect of suppressing the formation of darkening and the effect of removing formed darkening, and the method for evaluating the anti-darkening effect of the present invention can determine the presence or absence and the strength of the anti-darkening effect of a test sample.

[0048] The test sample is a sample that is to be evaluated for the presence or absence and strength of an anti-darkening effect, and may be a single compound or a composition containing two or more components (e.g., a cleaning composition for a toilet bowl).

[0049] In the method for evaluating the anti-darkening effect of the present invention, specific aspects of step A and step C are the same as those in the above-mentioned method for forming darkening.

[0050] In the method for evaluating the anti-darkening effect of the present invention, step B may be carried out between step A and step C, in which a stress treatment is carried out to reduce the proliferation activity or viable cell count of biofilm-forming bacteria in a biofilm. The specific embodiment of step B is the same as in the case of the above-mentioned method for forming darkening. When step B is carried out between step A and step C, a test sample may be added at least one stage selected from the group consisting of during step A, after step A and before step B, after step B and before step C, during step C, and after step C.

[0051] When the test sample is added during the step A, the culture solution containing the biofilm-forming bacteria, the black fungus, and the test sample may be brought into contact with the substrate and incubated, or the test sample may be added to the culture solution during incubation in a state in which the culture solution containing the biofilm-forming bacteria and the black fungus is brought into contact with the substrate. When the test sample is added after the step A and before the step B, the test sample may be added to the biofilm formed after the step A. When the test sample is added during the step B, the test sample may be added to the biofilm during the stress treatment in the step B. When the test sample is added after the step B and before the step C, the test sample may be added to the biofilm after the stress treatment in the step B. When the test sample is added during the step C, the test sample may be added to the biofilm either before the growth of the black fungus in the biofilm or at the stage of the growth of the black fungus. When the test sample is added after the step C, the test sample may be added to the black spot formed after the step C.

[0052] When a test sample is added during step A, after step A and before step B, after step B and before step C, and during step C, the state (size, number, etc.) of dark spots formed after step C is observed, and if the number of dark spots is smaller or the dark spots are smaller than the dark spots when the test sample is not added, the test sample is determined to have the effect of suppressing the formation of dark spots.

[0053] Furthermore, when a test sample is added after step C, the state of the dark spots (size, number, etc.) is observed over time after the addition of the test sample, and if the number of dark spots is reduced or the dark spots are smaller compared to when the test sample is not added, the test sample is determined to be effective in removing dark spots.

[0054] 3. Screening method for substances having anti-darkening effect The screening method of the present invention includes step A of forming a biofilm on a substrate by immersing the substrate in a culture solution containing a biofilm-forming bacterium and a black fungus and culturing the same, and step C of causing a black spot to form by proliferating the black fungus in the biofilm formed on the substrate, and is characterized in that a candidate substance is added at at least one stage selected from the group consisting of during step A, after step A and before step C, during step C, and after step C, and the state of the black spot formed under conditions where the candidate substance is added is compared with that under conditions where the candidate substance is not added.

[0055] The anti-darkening effect refers to the effect of suppressing the formation of darkening and the effect of removing darkening that has already formed, and the screening method of the present invention makes it possible to select from candidate substances those having an anti-darkening effect.

[0056] A candidate substance is a sample to be assessed for the presence or absence of an anti-darkening effect, and may be a single compound or a composition containing two or more components (e.g., a toilet bowl cleaning composition).

[0057] In the screening method of the present invention, specific aspects of steps A and C are the same as those in the above-mentioned method for forming dark spots.

[0058] In the screening method, step B of performing a stress treatment to reduce the proliferation activity or viable cell count of biofilm-forming bacteria in a biofilm may be carried out between steps A and C. Specific aspects of step B are the same as those in the blackening formation method. When step B is carried out between steps A and C, the candidate substance may be added at least one stage selected from the group consisting of during step A, after step A and before step B, after step B and before step C, during step C, and after step C.

[0059] When the candidate substance is added during the step A, the culture solution containing the biofilm-forming bacteria, the black fungus, and the candidate substance may be brought into contact with the substrate and incubated, or the candidate substance may be added to the culture solution during incubation in a state in which the culture solution containing the biofilm-forming bacteria and the black fungus is brought into contact with the substrate. When the candidate substance is added after the step A and before the step B, the candidate substance may be added to the biofilm formed after the step A. When the candidate substance is added during the step B, the candidate substance may be added to the biofilm during the stress treatment in the step B. When the candidate substance is added after the step B and before the step C, the candidate substance may be added to the biofilm after the stress treatment in the step B. When the candidate substance is added during the step C, the candidate substance may be added to the biofilm either before the growth of the black fungus in the biofilm or at the stage of the growth of the black fungus. When the candidate substance is added after the step C, the candidate substance may be added to the black spot formed after the step C.

[0060] When a candidate substance is added during step A, after step A and before step B, after step B and after step C, and during step C, the state (size, number, etc.) of dark spots formed after step C is observed, and if the number of dark spots is smaller or the dark spots are smaller than those under conditions in which the candidate substance is not added, the candidate substance is determined to have the effect of suppressing the formation of dark spots.

[0061] Furthermore, if a candidate substance is added after step C, the state of the dark spots (size, number, etc.) is observed over time after the addition of the candidate substance, and if the number of dark spots is reduced or the dark spots are smaller compared to the dark spots when the candidate substance is not added, the candidate substance is determined to have the effect of removing dark spots.

[0062] In the screening method of the present invention, a candidate substance determined to have an anti-darkening effect (an effect of suppressing the formation of darkening and / or an effect of removing darkening) is selected as a substance having an anti-darkening effect. EXAMPLES

[0063] The present invention will be described in detail below based on examples, but the present invention is not limited to these.

[0064] Test Example 1: Darkening test (1) 1. Method Biofilm formation process Biofilm-forming bacteria (environmental isolate, Rhizobium sp.) were precultured, and the preculture solution was added to the culture solution with the composition shown in Table 1 until the OD (600 nm) was 0.01 (bacterial count: 4.0 × 10 6 cfu / ml). In addition, spores of the black fungus Cladosporium halotolerans (NBRC111839) were added to the culture medium inoculated with the biofilm-forming bacteria at a concentration of 1.0 × 10 3 cfu / ml. Then, 2 ml of the culture solution containing the biofilm-forming bacteria and black fungus was dispensed into each well of the 12-well plate, and a cover glass (base material; length 1.8 cm, width 1.8 cm) was placed upright on each well, and a part of the cover glass was immersed in the culture solution in the well. After that, the 12-well plate was covered with a lid, and the 12-well plate was covered with a wrap film and subjected to stationary culture for 3 days in an incubator at 25°C or 30°C.

[0065] [Table 1]

[0066] The cover glass subjected to the static culture described above was taken out, and placed upright in each well of a 12-well plate into which 2 ml of 0.1 wt % crystal violet solution had been dispensed, and left to stand at room temperature for 10 minutes. As a result, it was confirmed that the cover glass was stained with crystal violet and a biofilm had been formed on the cover glass.

[0067] · Stress treatment (drying treatment) process The cover glass on which the biofilm was formed (hereinafter referred to as the BF-formed cover glass) (not stained) was washed with sterilized purified water, and then the moisture was wiped off with filter paper. The BF-formed cover glass was then placed in a safety cabinet and dried at 25°C for 1 hour.

[0068] · Darkening process The BF-formed cover glass after drying was immersed in a culture medium [PDB (potato dextrose broth); Bectone Dickinson], removed, and the PDB attached to the cover glass was wiped off with filter paper to impregnate the cover glass with PDB. In addition, a BF-formed cover glass after drying that was not impregnated with PDB was also prepared.

[0069] Two sheets of filter paper (7 cm in diameter) were placed on a round petri dish (9 cm in diameter), and 2 ml of water was added to the filter paper in the petri dish. A U-shaped glass rod, a slide glass, and a U-shaped staple (staple core) were then stacked on top of the filter paper in this order to assemble a black fungus culture set. Each of the BF-formed cover glasses (positioned so that the biofilm was on top) obtained above was placed on top of the U-shaped staple of the prepared black fungus culture set, and the petri dish was covered with a lid and then wrapped in paraffin film, and cultured at 25°C for 7 days.

[0070] 2.Results The results of observing the appearance of the cover glass after 2, 4, and 7 days of culture in the blackening generation process are shown in Figure 1. As a result, it was confirmed that blackening can be efficiently generated by culturing the substrate by contacting the substrate with a culture solution containing biofilm-forming bacteria and black fungus, forming a biofilm on the substrate, and then growing the black fungus in the biofilm in the gas phase, and that it is suitable as a test material for evaluating the anti-blackening effect. It was also confirmed that the efficiency of blackening generation is increased by using a culture solution containing BSA in the biofilm formation process. Furthermore, it was also clarified that the efficiency of blackening generation is increased by impregnating the BF-formed cover glass with a culture solution (PDB) and then subjecting it to the blackening generation process.

[0071] Test Example 2: Darkening test (2) 1. Method Biofilm formation process A BF-forming cover glass was prepared using a biofilm-forming bacterium (environmental isolate, Rhizobium sp.) and a black fungus (Cladosporium halotolerans (NBRC111839)) under the same conditions as in Test Example 1, except that a culture medium with the composition shown in Table 2 was used and the culture temperature was set at 25°C.

[0072] [Table 2]

[0073] · Stress treatment (drying treatment) process Each BF-formed cover glass obtained above was washed with sterilized purified water, and then the moisture was wiped off with filter paper. Then, the BF-formed cover glass was placed in a safety cabinet and dried at 25°C for 1 hour. For comparison, a BF-formed cover glass that was not dried was also prepared.

[0074] · Darkening process BF-formed cover glasses impregnated with PDB and subjected to stress treatment and unstress treatment were prepared under the same conditions as in Test Example 1. In addition, BF-formed cover glasses not impregnated with PDB and subjected to stress treatment and unstress treatment were also prepared.

[0075] A black fungus culture set was prepared under the same conditions as in Test Example 1. Each of the BF-formed cover glasses obtained above (positioned so that the biofilm was facing up) was placed on the U-shaped staple of the prepared black fungus culture set, the petri dish was covered with a lid, and then wrapped in paraffin film, and cultured at 25° C. for 3 days.

[0076] 2.Results The results of observing the appearance of the cover glass after the blackening generation process are shown in FIG. 2. From this result, it was found that blackening can be efficiently generated by culturing the substrate in contact with a culture solution containing biofilm-forming bacteria and black fungi to form a biofilm on the substrate, and then growing the black fungi in the biofilm in the gas phase. In particular, it was confirmed that the efficiency of blackening generation was significantly increased by performing a stress treatment (drying treatment) on the biofilm-forming bacteria in the biofilm after the biofilm formation process and before the blackening generation process. In addition, as in the results of Test Example 1, it was also revealed that the efficiency of blackening generation was increased by impregnating the BF-formed cover glass with a culture solution (PDB) and then subjecting it to the blackening generation process. Furthermore, it was also confirmed that the efficiency of blackening generation was increased by using a culture solution containing mannitol in the biofilm formation process.

[0077] Test Example 3: Darkening test (3) 1. Method Biofilm formation process A BF-forming cover glass was prepared using a biofilm-forming bacterium (environmental isolate, Rhizobium sp.) and a black fungus (Cladosporium halotolerans (NBRC111839)) under the same conditions as in Test Example 1, except that a culture solution A having the composition shown in Table 1 was used and the culture temperature was set at 25°C.

[0078] · Stress treatment (drying, UV treatment, and heating) process Each of the BF-formed cover glasses obtained above was washed with sterilized purified water, and then the moisture was wiped off with filter paper. Then, the BF-formed cover glasses were subjected to stress treatment under each of the conditions shown in Table 3.

[0079] To confirm the survival rate of the biofilm-forming bacteria, the biofilm was detached from the cover glass by stirring the BF-formed cover glass with and without stress treatment in a medium containing a surfactant, and the viable cell count of the biofilm-forming bacteria was measured. The measurement results are shown in Table 3.

[0080] [Table 3]

[0081] BF-formed cover glasses impregnated with PDB and subjected to stress treatment and unstress treatment were prepared under the same conditions as in Test Example 1. In addition, BF-formed cover glasses not impregnated with PDB and subjected to stress treatment and unstress treatment were also prepared.

[0082] A black fungus culture set was prepared under the same conditions as in Test Example 1. Each of the BF-formed cover glasses obtained above (positioned so that the biofilm was facing up) was placed on the U-shaped staple of the prepared black fungus culture set, the petri dish was covered with a lid, and then wrapped in paraffin film, and cultured at 25° C. for 3 days.

[0083] 2.Results The results of observing the appearance of the cover glass after the darkening process are shown in Figure 3. As a result, it was found that the stress treatment of the biofilm performed before the darkening process, not only drying treatment but also ultraviolet light treatment or heating treatment, can improve the efficiency of darkening.

Claims

1. A method for forming a dark spot, comprising the steps of: A step of forming a biofilm on a substrate by immersing the substrate in a culture solution containing a biofilm-forming bacterium and a black fungus and culturing the substrate, the culture solution containing mannitol and / or bovine serum albumin being used; and Step C is for causing black spots to develop by growing black fungi in a biofilm formed on the substrate.

2. A method for forming a dark spot, comprising the steps of: A step A includes a step of immersing a substrate in a culture solution containing a biofilm-forming bacterium and a black fungus and culturing the substrate to form a biofilm on the substrate; Step B is a step of performing a stress treatment for reducing the proliferation activity or viable cell count of the biofilm-forming bacteria in the biofilm formed in step A, the stress treatment being performed such that the viable cell count of the biofilm-forming bacteria in the biofilm is 90% or less compared to before the stress treatment; and Step C is for causing black spots to develop by growing black fungi in a biofilm formed on the substrate.

3. 2. The method for forming blackening according to claim 1, further comprising a step B, prior to the step C, of ​​performing a stress treatment to reduce the proliferation activity or viable cell count of the biofilm-forming bacteria in the biofilm formed in the step A, wherein the stress treatment is performed so that the viable cell count of the biofilm-forming bacteria in the biofilm is 90% or less compared to before the stress treatment.

4. The method for forming a darkening layer according to claim 2 or 3, wherein the stress treatment in the step B is a drying treatment, an ultraviolet treatment, or a heating treatment.

5. The method for forming a dark spot according to any one of claims 1 to 4, wherein the culture medium used in the step A contains mannitol and bovine serum albumin.

6. A method for forming black spots as described in claim 2, wherein the culture medium used in step A contains mannitol and / or bovine serum albumin.

7. The method for forming a blackening according to any one of claims 1 to 6, wherein after step A, the biofilm formed in step A is impregnated with a culture solution.

8. A method for evaluating the anti-darkening effect of a test sample, comprising the steps of: A step of forming a biofilm on a substrate by immersing the substrate in a culture solution containing a biofilm-forming bacterium and a black fungus and culturing the substrate, the culture solution containing mannitol and / or bovine serum albumin being used; and A step C of causing black fungus to grow in the biofilm formed on the substrate, thereby causing black spots to form; The evaluation method includes adding a test sample at at least one stage selected from the group consisting of during the step A, after the step A and before the step C, during the step C, and after the step C, and comparing the state of darkening under a condition in which the test sample is added with a condition in which the test sample is not added.

9. A method for screening a substance having an anti-darkening effect, comprising: A step of forming a biofilm on a substrate by immersing the substrate in a culture solution containing a biofilm-forming bacterium and a black fungus and culturing the substrate, the culture solution containing mannitol and / or bovine serum albumin being used; and A step C of causing black fungus to grow in the biofilm formed on the substrate, thereby causing black spots to form; A screening method comprising adding a candidate substance at at least one stage selected from the group consisting of during step A, after step A and before step C, during step C, and after step C, and comparing the state of the darkening formed under conditions in which the candidate substance was added and conditions in which the candidate substance was not added.

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