JP5922628B2 - Method for producing antifungal agent and antifungal method - Google Patents

Description

  The present invention relates to an antifungal agent used for prevention or removal of wrinkles generated on a ceiling, wall or floor of a building, and in particular, not only removes generated wrinkles but also prevents wrinkles from occurring over a long period of time. The present invention relates to an antifungal agent and an antifungal method.

Inside the building, organic substances such as polysaccharides may adhere to the building over time, and the organic substances may become nutrients and spiders may be generated and proliferated.
It is also known that wrinkles are caused by fungi contained in pets or pigeon feces (malignant pathogens: WHO is considered a substance that may cause P3 level infection). In order to prevent the occurrence of such wrinkles, measures such as netting the windows of buildings are taken, but the current situation is that the net cannot sufficiently prevent fungal invasion.
As for the koji growing on the building material, it becomes a cause of deterioration of the building material by being oxidized and neutralized by the gluconic acid fermenting bacteria. niger (Aspergillus niger), especially in Japan, is harmful to resin and board. Versicolor (Aspergillus / barge color), Penicillium (Penicillium) that pollutes all food, clothing, and living such as building materials, living environment, food, Eurotium (Eurotium, Japanese name: Kawakiko-koji mold) that propagates on imported wood, tatami mats, boards, plywood, etc., building materials・ A. of osmotic pressure which causes wear of industrial products. restrictus (Aspergillus resteractus), Aureobasidium (Aureobasilium, Japanese name: black yeast-like fungus) that propagates in vinyl chloride resin in bathrooms and places with high humidity and causes damage to building materials, produces aflatoxin Fusarim (Fusarium, Japanese name: red mold), which is toxic to animals and plants, Cladosporium (Cladosporium, Japanese name: high resistance to ultraviolet rays and heating that causes deterioration, decay and allergens of wood, paint and resin. Black mold), Chaetomium (chaytium, Japanese name: genus Trichoderma), which is abundant in the soil of wetlands, and is said to be a precursor of the toxic aflatoxin, and wood-rotting fungus Trichoderma (Trichoderma), which are common in Japan.

Such sputum not only detracts from the aesthetics of the interior of the building, but has also been linked to allergies and sick house syndrome. In particular, in the United States, in recent years, there have been a number of lawsuits for damage to health caused by drought in houses and public facilities, and the number is increasing.
In addition, due to recent climate change, high temperature and humidity in Japan has become remarkable, and drastic changes have been seen in the growth and damage status of cocoons. As such, problems such as allergy, sick house, and mycosis caused by hemorrhoids are becoming increasingly serious.

In order to remove generated wrinkles, conventionally, a cleaning agent mainly containing a chlorine-based or oxygen-based bleaching agent (for example, Patent Literatures 1 and 2) and a cleaning agent mainly containing an alcohol component (for example, Patent Literature 3) 4, 5), and a method of adding a synthetic substance such as TBZ (product developed by Merck as an agrochemical) to emulsion coating.
However, chlorine bleach such as sodium hypochlorite, and those mainly composed of bleach such as hydrosulfite are acidic detergents, although they have excellent bleaching power and soot removal performance. When it is mixed with toxic chlorine gas is generated in the human body, the chlorine odor is too strong. In particular, it is known that urea or the like adhering to a floor or a wall of a toilet or the like causes a chemical reaction with the above-described cleaning agent to generate trihalomethane (carcinogenic substance). Further, bleaching agents such as halogens only bleached dirt and soot pigments, and did not eliminate the soot itself.
Further, in the case of an oxygen bleaching agent containing hydrogen peroxide as a main component, the oxygen bleaching agent alone cannot sufficiently remove and prevent soot. Therefore, it was necessary to use in combination with other bleach activators (for example, Patent Document 2). In addition, oxygen-based bleaching agents could not prevent the generation of soot only by temporarily bleaching dirt and soot itself. On the contrary, cleaning agents with a low biodegradation rate have the disadvantage of creating an environment that encourages the growth of sputum.

  Moreover, although the cleaning agent which has alcohol as a main component has the bactericidal power with respect to a wrinkle, the effect which prevents the reproduction | regeneration of dirt and a wrinkle is low. That is, since the alcohol as the main component has high volatility, the duration of the effect is short, and there is a drawback that an environment in which wrinkles are generated immediately after the application of the cleaning agent is caused. In addition, when a hiba material is used as a building material, it is known that the unique texture of hiba disappears when a cleaning agent mainly composed of alcohol is used. Furthermore, since alcohol is not permeable, it is also problematic that the cleaning agent does not reach the roots of the bushes behind the wood or cement material.

In addition, detergents formulated with synthetic substances such as TBZ have a short duration of effect and are difficult to dissolve in water, etc., so there is a great risk of damaging eyes and skin by splashing in the air. Since it could be the cause, there was a disadvantage that secondary damage occurred.
The present inventor has invented a cleaning agent using Bacillus subtilis in view of various problems as described above (see Patent Document 6). The cleaning agent according to this patented invention was able to remove wrinkles generated on the surface of building materials such as vinyl cloth.
However, it has been found that the cleaning agent according to the above-mentioned patented invention does not exhibit a complete effect for removing and preventing wrinkles generated on walls and ceilings formed of wood, cement material or the like. As a result of studying such a new problem, the present inventor has found that the cleaning agent according to the above-mentioned patented invention does not reach the root of the ridge in the back of such a wall material or the like. And the reason was ascertaining that the minute gonococcus having a size of about 0.75 μm was caused by entering the back of the wood or cement material to form roots.

JP 2000-169306 A JP-A-2-225600 JP 2010-5875 A JP 2005-336087 A JP 2003-342103 A Japanese Patent No. 4260576

  Conventional antifungal agents against bacterial spores have been highly toxic oxidizing agents, alkylating agents or protein denaturing agents. As with antibiotics, conventional anti-fungal agents that inhibit protein synthesis, DNA / RNA synthesis, cell wall synthesis, respiratory and electron transport systems, and metabolic systems such as transport and excretion develop resistant bacteria through a very simple process. Resulting in.

Therefore, an object of the present invention is to provide an antifungal agent that is a physiologically active substance having an antifungal effect against a wide variety of bacterial species. In particular, an object of the present invention is to provide an antifungal agent having a remarkable antifungal effect against microorganisms that form spores.
Another object of the present invention is to prevent the occurrence of wrinkles by not only removing the generated wrinkles but also preventing the development of resistant bacteria over a long period of time.
Furthermore, an object of the present invention is to provide an antifungal agent that uses only an enzyme produced by a microorganism and an antibacterial active substance while suppressing excessive growth of bacteria that are the main component of the cleaning agent.

In order to solve the above problems, the present inventor has employed an antifungal agent and an antifungal method having the following configurations.
(1) Antifungal agent used for prevention or removal of wrinkles generated on the ceiling, wall or floor of a building, and at least spore-like Bacillus subtilis and microbial cells belonging to Pseedomonas Is an antifungal agent characterized by being a liquid or semi-fluid substance.
(2) The anti-fungal agent having the spore-state Bacillus subtilis and the microbial cells belonging to the genus Pseudomonas is composed of a bioactive substance that produces an enzyme and an antibacterial active substance from which proliferative cells are excluded. The antifungal agent described in (1) above, which is characterized.
(3) The anti-fungal agent is a detergent having 3.0 to 7.0 × 10 ⁹ / ml spores of Bacillus subtilis and 1.0 to 3.0 × 10 ⁹ / ml Pseudomonas sp. The antifungal agent described in (1) above, which is characterized in that it exists.

(4) An anti-fungal method used for prevention or removal of wrinkles occurring on the ceiling, wall or floor of a building, comprising: a spore-like Bacillus subtilis and a microbial cell belonging to Pseedomonas An antifungal method comprising a step of applying and / or spraying a contained liquid or semi-fluid antifungal agent.
(5) The anti-fungal agent cultivates spores of Bacillus subtilis and Pseudomonas sp. To form colonies, and then separates them into a culture solution and cells using a centrifuge, and the cells are solid-cultured. The antifungal method described in (4) above, which is a physiologically active substance obtained by filtering the extracted suspension after extraction with ethanol or absolute alcohol.
(6) The step of applying and / or spraying the anti-fungal agent using the culture solution obtained by centrifugation before the step of applying and / or spraying the anti-fungal agent. It is an anti-mold method described in (5).

By adopting the antifungal agent and the antifungal method according to the present invention, an antifungal effect can be exhibited against a wide range of bacterial species. In particular, it has an excellent effect of exerting a remarkable antifungal effect against microorganisms that form spores.
That is, by adopting the antifungal agent and the antifungal method according to the present invention, it is possible to effectively remove wrinkles generated on the ceiling, wall or floor of a building.

In addition, by adopting the antifungal agent and the antifungal method according to the present invention, not only the generated wrinkles can be removed, but also the development of resistant bacteria can be prevented and the occurrence of wrinkles can be prevented over a long period of time. .
Furthermore, by adopting the antifungal agent and the antifungal method according to the present invention, it is possible to suppress the excessive growth of the fungus that is the main component of the antifungal agent and to use only the enzyme produced by the microorganism and the antibacterial active substance. Has an effect.

The anti-fungal agent and the anti-fungal method according to the present invention are characterized by containing two types of bacteria, a spore-shaped Bacillus subtilis and a microbial cell belonging to Pseedomonas.
Here, Bacillus subtilis in the spore state is a kind of eubacteria called Bacillus subtilis, which is an aerobic Gram-positive gonococcus of 0.7 to 0.8 μm × 2.0 to 3.0 μm and is in the spore state. Say something. Bacillus subtilis is one of the resident bacteria (airborne bacteria) scattered in the soil and in the air, and is often isolated from the surface of withered grass. Although careful attention is required for the culture, the present inventor planted Bacillus subtilis that is widely present in the soil on a tryptosoy agar medium, cultured at 37 ± 1 ° C. for 3 days, left at low temperature, and diluted solution It is cultured by suspending and collecting in a suspension. However, the spore-shaped Bacillus subtilis according to the present invention may be cultured by other methods and is not limited to the above-described culture method.

  Next, the Pseudomonas spp. Is particularly preferably Psuedomonas stutzeri, which is a Gram-negative bacilli and is a genus of aerobic eubacteria. This is because Pseudomonas stazzelli has the property of powerfully degrading pollutants and colloidal dirt. Any strain may be used as long as it has an action of decomposing cocoons growing on building materials. Among strains currently recognized by the present inventor, Pseudomonas NBRC (NITE Biological Resource Center) 14165 is used. Most preferably it is used. Such strains can grow and have a safety level of hazard level 1 (hazard level 1 is a level that can be handled by general consumers). Of course, this invention is not limited to the said strain, What is necessary is just a strain which has the capability to process an artificial synthetic organic substance.

The anti-fungal agent according to the present invention is characterized by comprising a bioactive substance that produces an enzyme and an antibacterial active substance from which proliferative cells are excluded.
Here, the bioactive substance which produces the enzyme and antibacterial active substance from which the proliferating microbial cells were excluded can be produced by the following method. A culture solution of Bacillus subtilis and Pseudomonas spp. In a spore state is centrifuged at 20,000 rpm for 30 minutes and separated into a culture solution and cells, and then the cells are solid-cultured and dissolved and extracted with ethanol or anhydrous alcohol. Thereafter, the cells that can grow can be eliminated by filtering the extracted suspension. In this way, it is possible to suppress the excessive growth of bacteria that are the main components of the antifungal agent, and to use only the enzymes and antibacterial active substances produced by the microorganisms.

The antifungal agent thus obtained can effectively remove generated wrinkles. In addition, since the growth of bacterial cells is suppressed, the effect as an antifungal agent is sustained, so that the expression of resistant bacteria can be effectively suppressed and the occurrence of wrinkles can be prevented over a long period of time. Furthermore, it has an excellent effect of not harming human and animal health.
On the other hand, as an antifungal agent according to the present invention, 3.0 to 7.0 × 10 ⁹ / ml spores of Bacillus subtilis and 1.0 to 3.0 × 10 ⁹ / ml of Pseudomonas are used. It is preferable to do this.
If the spores of Bacillus subtilis is less than 3.0 × 10 ⁹ / ml, or less than 1.0 × 10 ⁹ / ml, the effect of the present invention may not be sufficiently exhibited. On the other hand, the number of spores of Bacillus subtilis is more than 7.0 × 10 ⁹ / ml, or Pseudomonas sp. Is more than 3.0 × 10 ⁹ / ml because growth by pure culture becomes difficult with current technology. is there. In particular, depending on the upper limit value, a product exceeding the above value may be produced and used by devising a pure culture method.

Sprouting Bacillus subtilis and Pseudomonas spp. In the above numerical range are centrifuged at 20,000 rpm for 30 minutes, and separated into a culture solution and cells. The culture solution thus obtained can be added to the cleaning agent. For example, 5.0 × 10 ⁹ / ml Bacillus subtilis and 1.9 × 10 ⁹ / ml Pseudomonas spp. Are mixed in soapy water, adjusted to 10 to 15 ppm, added with 3% baking soda to remove dirt. And an antifungal agent mainly used for sterilization can be obtained.

The antifungal agent according to the present invention can be produced as follows. The following production method is an example of the method for producing the antifungal agent according to the present invention, and does not limit the present invention. Of course, the antifungal agent according to the present invention may be produced by other methods.
First, Bacillus subtilis in a spore state of 5.0 × 10 ⁹ / ml is cultured for a certain period under suitable conditions. After confirming the colony, it is centrifuged at 20,000 rpm for 30 minutes by a centrifuge to separate into a culture solution and cells. The separated cells are cultured in solid, and dissolved and extracted with ethanol or absolute alcohol.
By filtering the extracted suspension using a 0.2 μm filtration membrane, a physiologically active substance produced from Bacillus subtilis in which excessive growth of the fungus is suppressed can be obtained.
By filtering the extracted suspension, it is possible to eliminate the cells that can grow. As a result, it is possible to produce only an enzyme and an antibacterial active substance by a microorganism from which proliferating cells are excluded. By eliminating the proliferative cells, excessive growth of the fungus that is the main component of the antifungal agent can be suppressed, so there are changes in the environment including high temperature and high humidity, and effects such as sealing It is possible to continue to exhibit anti-fungal and antibacterial effects over a long period of time in all environments of clothing, food and housing. Further, as long as the antifungal agent is present, the antifungal effect and the like are maintained semipermanently. Furthermore, a safe antifungal agent can be obtained without toxicity to human bodies and animals.

At the same time, 1.9 × 10 ⁹ / ml of Pseudomonas is cultured for a certain period under suitable conditions. After confirming the colony, it is centrifuged at 20,000 rpm for 30 minutes by a centrifuge to separate into a culture solution and cells. The separated cells are cultured in solid, and dissolved and extracted with ethanol or absolute alcohol.
By filtering the extracted suspension using a 0.2 μm filter membrane, a physiologically active substance produced from Pseudomonas spp. In which excessive growth of the fungus is suppressed can be obtained.
A mixture of a physiologically active substance produced from Bacillus subtilis and a physiologically active substance produced from Pseudomonas at a ratio of 1: 1 is used as an antifungal agent of 0.3 ppm to 1.0 ppm. I was able to.

On the other hand, the obtained 0.3 ppm to 1.0 ppm antifungal agent is admixed as an additive, whereby an adhesive, a house cleaner, a mold remover, a paint, a resin wax, a stucco, a clay wall, a cement, Application to food cleaner, beeswax for white wood (protectant for white wood), dish cleaner, animal cleaner, human cleaner (finger, body, face, head hair, stain, athlete's foot, white hair, wrinkle) can do. For example, by adding 0.3% to 1.0 ppm of an antifungal agent at 1% in purified water, it can be used as a fungicide for spraying and spraying on areas where wrinkles are likely to occur. A detergent intended to remove vegetables, tableware, hand sanitizers, pollutants, agricultural chemicals, etc. by adding 1% of 0.3 to 1.0 ppm antifungal agent to pure water soap water Can be used as Furthermore, by adding 1% of alcohol and 0.3ppm to 1.0ppm anti-fungal agent to pure water, it is used as a hygiene management product for the purpose of sterilization in stores, kitchens, cooking utensils, fingers, etc. be able to. In particular, since the anti-fungal agent according to the present invention has no growth of bacteria, it can be used for hygiene management purposes in food factories and restaurant kitchens.
The obtained anti-fungal agent of 0.3 ppm to 1.0 ppm is used as a coating agent as a cleaning agent for wood, paper, vinyl cloth, tile, tile joint, resin board, plywood, tatami mat, solar panel, etc. can do.

Next, the function and effect of the antifungal agent according to the present invention will be described.
The antifungal agent according to the present invention promotes the rupture of hyphae by deforming and swelling the hyphae of mycelia of fungi and filamentous fungi. For such a phenomenon, the inventor decided that the antifungal agent according to the present invention acts on the biological membrane, dissolves in the mycelial lipid waste, forms a complex with the cation, and increases the ion permeability. I think it is caused.
Therefore, since the antifungal agent according to the present invention is not due to protein synthesis inhibition like conventionally used antibiotics and antifungal agents, it is hardly toxic and can suppress the appearance of resistant bacteria.
Therefore, by adopting the antifungal agent according to the present invention, it is possible to prevent the emergence of resistant bacteria and to exert an antifungal effect against a wide range of bacterial species.

Next, an embodiment of the antifungal agent according to the present invention will be described.
Embodiment 1
Bacillus subtilis in a spore state of 5.0 × 10 ⁹ / ml was cultured for a certain period under suitable conditions. Further, 1.9 × 10 ⁹ / ml of Pseudomonas was cultured for a certain period under suitable conditions.
After each colony was confirmed, the mixture was centrifuged at 20,000 rpm for 30 minutes with a centrifuge to separate into a culture solution and cells.
In this way, a first antifungal agent consisting of a culture solution produced from Bacillus subtilis and a first antifungal agent consisting of a culture solution produced from Pseudomonas sp. Were produced.
The bacterial cells separated by the centrifuge were each solid-cultured and dissolved and extracted with ethanol.
The extracted suspension was filtered using a 0.2 μm filter membrane to obtain a physiologically active substance produced from Bacillus subtilis and a physiologically active substance produced from Pseudomonas sp.
In this way, a second antifungal agent composed of a physiologically active substance produced from Bacillus subtilis and a second antifungal agent composed of a physiologically active substance produced from Pseudomonas sp. Were produced.
Next, an example of the antifungal method using the antifungal agent according to the present invention will be described. The following antifungal method is an example of the antifungal method according to the present invention and does not limit the present invention.

(First step)
A first cleaning agent was prepared by mixing an aqueous solution containing hydrogen peroxide and acidic nonionic water in the following ratio.
Aqueous solution containing H ₂ O ₂ 0.15% by weight
Acidic non-ionic water 99.85% by weight
(The above components and proportions may vary depending on environmental conditions such as temperature and humidity and the type and degree of odor-causing substances, and the present invention is not limited thereto. The same applies to the following antifungal agents. .)
The first cleaning agent is weakly acidic and water-soluble, and chemically decomposes (vaporizes) into water and oxygen in about 6 hours, so there is no risk of secondary contamination.
The first cleaning agent was sprayed or applied to an object generated on the ceiling, wall or floor of the building. Depending on the object, this first step may be omitted.

(Second step)
The two types of culture solutions (first antifungal agents) obtained above, that is, the first antifungal agent produced from Bacillus subtilis and the first antifungal agent produced from Pseudomonas sp. A second cleaning agent mixed in step 1 was prepared.
Amisoles CDE in soapy water, PEG approx. 1%
Baking soda 0.3%
Bacillus subtilis culture filtrate 10-15ppm
10-15 ppm of Pseudomonas culture filtrate (The ratio of Bacillus subtilis culture filtrate to Pseudomonas culture filtrate was 1: 1)
The said 2nd cleaning agent was sprayed or apply | coated to the location apply | coated at said 1st process.

(Third process)
A mixed agent obtained by mixing a second anti-fungal agent composed of a physiologically active substance produced from Bacillus subtilis and a second anti-fungal agent composed of a physiologically active substance produced from Pseudomonas at a mixing ratio of 1: 1. Prepared. An antifungal agent was produced by adding 0.3 to 1 ppm of such a mixture to purified water.
The said antifungal agent was apply | coated to the location apply | coated at the said 2nd process. Furthermore, the said antifungal agent was injected to the symmetry plane and space using the compressor.

  When the target was observed by the above-mentioned method, it was found that a wide range of bacterial species such as Aspergillus niger, Aspergillus barge color, Penicillium, Kawakuji mushroom, Aspergillus reustlicus, black yeast-like fungus, red mold, black mold, black mold, trichoderma It was found that the anti-epileptic effect is exerted. In addition, such an antidepressant effect was proved by the anti-mold test by NPO corporation mold consultation center. In addition, the effect of reoccurrence of dirt and wrinkles has been confirmed at present, but we will add that the reoccurrence of wrinkles is being followed for 10 years.

Claims (4)

After culturing microbial cells belonging to spores of Bacillus subtilis and Pseudomonas (Pseudomonas), they were separated into a culture solution and cells by centrifugation, and then spores of Bacillus subtilis and Pseudomonas (Pseudomonas) From the physiologically active substance producing the enzyme and the antibacterial active substance from which the proliferating bacterial body was eliminated by solid-culturing the microbial cell belonging to the group, dissolving and extracting the suspension with alcohol, and filtering the suspension wherein the obtaining a Do that liquid or semi-fluidized material, a ceiling of a building, a manufacturing method of the antifungal agents used in the prevention or elimination of fungi that occur in the wall or floor. Before the centrifugal separation, and Bacillus subtilis spores state of 3.0~7.0 × 10 ⁹ / ml, and a Pseudomonas bacteria 1.0~3.0 × 10 ⁹ / ml, and mixed The method for producing an antifungal agent according to claim 1, wherein the antifungal agent is cultured . After culturing microbial cells belonging to the spore-like Bacillus subtilis and Pseudomonas (Pseudomonas), forming colonies, and separating them into a culture solution and cells by centrifugation, the spores of Bacillus subtilis and Pseudomonas Microbial cells belonging to fungus (Pseudomonas) are solid-cultured, the suspension is dissolved and extracted with alcohol, and the suspension is filtered to produce enzymes and antibacterial active substances that are free from proliferating cells. the liquid or semi-fluid state substance ing from physiologically active substance, a ceiling of a building, antifungal methods for preventive or removal of fungi, characterized in that it comprises a step of coating and / or spraying the wall or floor. A step of applying and / or spraying an anti-fungal agent using a culture solution obtained by centrifugation before the step of applying / spraying the liquid or semi-fluid substance. Item 4. The antifungal method according to Item 3.