Method for maintaining indoor microbial diversity and device for maintaining indoor microbial diversity

Abstract

To provide a method and device that can maintain indoor microbial diversity in a simple manner.SOLUTION: Provided is a method for maintaining indoor microbial diversity, comprising spraying, indoors, a liquid composition containing Firmicutes bacteria that are nonpathogenic to humans and animals. Also provided is a method for maintaining indoor microbial diversity, comprising spraying, after spraying a disinfecting composition or a disinfecting gas, a liquid composition containing Firmicutes bacteria that are nonpathogenic to humans and animals.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for maintaining indoor microbial diversity and an apparatus for maintaining indoor microbial diversity, and more particularly to a method for maintaining indoor microbial diversity, which comprises a step of spraying indoors a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals, and an apparatus for maintaining indoor microbial diversity, which comprises a spraying means for spraying a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals. [Background technology]

[0002] The interior of a room (indoors) where humans live and work tends to be populated with microorganisms derived from human breath, droplets, skin, hair, etc. In other words, the indoor microbiome tends to be dominated by microorganisms of human origin and have low diversity. One possible reason for this is that when humans are active, microorganisms of human origin are constantly being supplied to the room through breathing, metabolism, etc.

[0003] Since some microorganisms derived from humans are pathogenic, the approach taken to improve the indoor microbial environment is to kill microorganisms in the room by disinfecting with alcohol or other agents. However, this approach does not fundamentally solve the problem because only bacteria resistant to alcohol and other agents survive, resulting in a loss of diversity, and the disinfecting effect is only temporary, with microorganisms, especially human-derived microorganisms, quickly multiplying.

[0004] As such, indoor microbial biodiversity tends to lose diversity. While the importance of biodiversity on a global scale has long been recognized, the present inventor has recently proposed that maintaining indoor microbial diversity is important for human health and immunity. The hygiene hypothesis is known, which states that if humans have frequent exposure to various microorganisms and viruses, for example through outdoor activities from childhood, their immune systems will become more tolerant, resulting in a lower rate of exacerbation of autoimmune diseases such as hay fever and asthma as they grow up. Based on this hygiene hypothesis, the present inventor believes that indoor microbial diversity affects human health and immunity. However, currently, no simple method for maintaining indoor microbial diversity is known.

[0005] Therefore, there is a need for a simple method to maintain indoor microbial diversity.

[0006] Patent Document 1 discloses a method for keeping surfaces, objects and / or installations free from pathogens, characterized in that a non-pathogenic bacterial strain is applied to the surface or the like.

[0007] However, Patent Document 1 does not describe maintaining microbial diversity indoors. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2006 / 125283 Brochure Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, an object of the present invention is to provide a method and apparatus that can maintain microbial diversity indoors in a simple manner. [Means for solving the problem]

[0010] As a result of intensive research into solving the above problems, the inventors discovered that the above problems could be solved by spraying a liquid composition containing microorganisms belonging to a specific group indoors, and thus completed the present invention.

[0011] That is, the present invention relates to the following [1] to

[11] . [1] A method for maintaining microbial diversity indoors, comprising the step of spraying a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals indoors. [2] A method for maintaining microbial diversity indoors, comprising the step of spraying a disinfecting composition or disinfecting gas, and then spraying a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals. [3] A method for maintaining microbial diversity indoors according to claim 1, wherein the bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals are bacteria of the genus Bacillus. [4] A method for maintaining microbial diversity indoors according to [1], which comprises spraying a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals once at predetermined time intervals. [5] The liquid composition containing the bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals contains the microorganisms in an amount of 1 x 10 5 A method for maintaining indoor microbial diversity that contains more than CFU / mL [1]. [6] A method for maintaining microbial diversity indoors according to [1], comprising spraying 0.1 to 3 ml of a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals once every 1 to 3 hours. [7] A method for maintaining microbial diversity in an indoor environment as described in [1], in which the product of phylogenetic diversity obtained by metagenomic sequencing of a sample collected by wiping the surface of the floor located in the center of the room and the Shannon index is compared before and after spraying, and the reduction rate from before spraying is maintained at 10% or less at any of the following times after spraying: 1 hour, 2 hours, 3 hours, 5 hours, 24 hours, 48 ​​hours, 72 hours, 1 week, or 14 days. [8] An indoor microbial diversity maintenance device comprising a spraying means for spraying a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals. [9] An indoor microbial diversity maintenance device comprising a spraying means for spraying a disinfecting composition or a disinfecting gas, and a spraying means for spraying a liquid composition containing microorganisms that are harmless to humans and animals.

[10] Bacteria from the phylum Firmicutes, which are used to maintain indoor microbial diversity.

[11] A composition for maintaining microbial diversity indoors, containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals. [Effects of the Invention]

[0012] The present invention makes it possible to provide a method and an apparatus that can maintain microbial diversity indoors in a simple manner. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a graph showing the results of an example, showing the variation in phylogenetic diversity (bacterial cell spray) in each sample. [Figure 2] 1 is a graph showing the results of an example, showing the fluctuation of the Shannon index (bacterial cell spray) for each sample. DETAILED DESCRIPTION OF THE INVENTION

[0014] <How to maintain indoor microbial diversity> The method for maintaining microbial diversity indoors according to the present invention is characterized by comprising a step of spraying a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals indoors.

[0015] [Microbial diversity] Biodiversity refers to the diverse living organisms present in ecosystems, biomes, or the entire planet. Microbial diversity refers to the diversity of microorganisms within biodiversity. Maintaining indoor microbial diversity means that people and pets living there will come into contact with a variety of microorganisms, which is expected to have a positive impact on health and immunity, similar to the hygiene hypothesis. Maintaining diversity also prevents certain microorganisms from multiplying and dominating. This, for example, can prevent odor-causing bacteria from dominating the indoor environment, thereby preventing bad odors.

[0016] Microbial diversity can be measured or evaluated by known methods. For example, samples are collected from indoor spaces or by wiping floors and walls, the 16S rRNA gene in the sample is amplified by PCR, and metagenomic sequencing is performed using a next-generation sequencer. Criteria for evaluating microbial diversity include, for example, the Shannon index and phylogenetic diversity. In the method of the present invention for maintaining indoor microbial diversity, when comparing the product of the phylogenetic diversity measured from a sample collected from the room and the Shannon index before and after spraying, it is preferable to maintain a reduction rate from before spraying of 10% or less at any time point, for example, 1 hour, 2 hours, 3 hours, 5 hours, 24 hours, 48 ​​hours, 72 hours, 1 week, or 14 days after spraying.

[0017] [Bacteria of the phylum Firmicutes] The present invention uses non-pathogenic Firmicutes bacteria. The inventors have discovered that non-pathogenic Firmicutes bacteria can be used to maintain indoor microbial diversity. "Non-pathogenic" means that they are not pathogenic to humans or animals with normal immune systems. In this case, animals preferably refer to pets such as dogs and cats, but may also include livestock such as cows and pigs. Therefore, pathogenic Firmicutes bacteria, such as Clostridium tetani, Clostridium botulinum, and Clostridium perfringens, are not used in the present invention. The Firmicutes phylum includes the classes Bacillales, Clostridium, Erysipelothrix, Negatiwicki, and Thermolysobacteria. Of these, the class Bacillus is preferred. The class Bacillus includes the orders Lactobacillales and Bacillales, with Lactobacillales being preferred. The Lactobacillales family includes the Aerococcaceae, Carnobacteriaceae, Enterococcusaceae, Lactobacillaceae, Leuconostocaceae, and Streptococcusaceae (Streptococcus family), with the Lactobacillaceae family being preferred. The Lactobacillaceae family includes the genus Lactobacillus. Only one type of non-pathogenic Firmicutes bacteria may be used, or two or more types may be used in combination. The bacteria used in the present invention are preferably bacteria known to be edible, bacteria known to be used in food production, or bacteria whose safety for humans has been confirmed.

[0018] [Liquid composition] The liquid composition used in the present invention is a liquid composition containing bacteria of the phylum Firmicutes. Preferably, it contains bacteria of the phylum Firmicutes and a solvent. The bacteria of the phylum Firmicutes may be in the form of cells or spores. Even if they are in the form of spores, they will become normal cells after spraying and are expected to contribute to the maintenance of microbial diversity. From the viewpoint of storage stability, the spore form is preferred. The solvent is not particularly limited as long as it can disperse the bacteria of the phylum Firmicutes without killing them, but it is preferable that it evaporates after a predetermined period of time has passed since spraying. Examples of the solvent include water, liquid medium, aqueous solvents such as ethanol, and mixtures thereof. The content of bacteria of the phylum Firmicutes in the liquid composition is preferably 1 x 105 CFU / mL or more, more preferably 1 x 10 5 ~1×10 6 CFU / mL.

[0019] The viscosity of the liquid composition is not particularly limited, but it is preferably low enough to be sprayable from a spray nozzle. It is preferable that:

[0020] [Scatter] The method for spraying the liquid composition is not particularly limited, and known chemical spraying devices, sprayers, atomizers, etc. can be used. Also usable are aerosol sprays, manual types, automatic types, hand sprays, pump-type spray products constructed by filling a container with a trigger pump, and aerosol products constructed by sealing the liquid composition and, if necessary, a propellant in a pressure-resistant container. Examples of propellants include liquefied petroleum gas (LPG), dimethyl ether (DME), nitrogen gas, carbon dioxide, nitrous oxide, and compressed air. Spray products and aerosol products may be one-push sprayers using a metered-dose injection valve or trigger-type sprayers. This allows an appropriate amount of liquid composition to be sprayed depending on the number of injections.

[0021] From the viewpoint of maintaining indoor microbial diversity, it is preferable to spray once within a predetermined time period. An automatic spraying device with a timer is preferred as a method for easily implementing such a spraying method. For example, the time is measured using a timer, and the liquid composition is sprayed once every preset time. More preferably, the liquid composition is sprayed once every 1 to 24 hours, and even more preferably, once every 1 to 8 hours. By spraying according to such a schedule, it is expected that indoor microbial diversity can be maintained more efficiently.

[0022] The spraying is preferably carried out into the air in the room from the viewpoint of widely dispersing the liquid composition throughout the room. It is also preferable to spray over a wide area rather than concentrating the spray on a single point. A dispersion aid such as a circulator can also be used. The liquid composition can also be sprayed by attaching a spraying device near the air outlet of an air conditioner, an electric fan, or the fan of a circulator.

[0023] The liquid composition is preferably sprayed in an amount of 0.1 ml to 10 ml, more preferably 0.1 ml to 5 ml, and particularly preferably 0.1 ml to 3 ml per spray. The larger the amount sprayed per spray, the greater the amount of Firmicutes bacteria that will be supplied to the room, and a higher effect can be expected. However, if the amount sprayed is too large, the effect will not be enhanced commensurate with the increase in the amount sprayed, and many droplets will remain without volatilizing after the liquid composition is sprayed.

[0024] The particle size of the droplets of the liquid composition to be sprayed is not particularly limited, but the smaller the particle size, the higher the dispersibility of the liquid composition, and the more widely the Firmicutes bacteria can be expected to be dispersed throughout the room. Meanwhile, the lower limit of the droplet size is determined by factors such as the nozzle shape of the spraying device. The particle size (e.g., D50) can be measured using a laser diffraction particle size distribution analyzer.

[0025] <Disinfecting composition or disinfecting gas> Another aspect of the present invention relates to a method for maintaining indoor microbial diversity, characterized by comprising the step of spraying a disinfecting composition or disinfecting gas, followed by spraying a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals. When the disinfecting composition or disinfecting gas is sprayed into a room, bacteria are non-specifically removed except for bacteria resistant to the disinfecting composition or disinfecting gas, resulting in a high occupancy rate of resistant bacteria and a decrease in diversity. Furthermore, the disinfecting effect of the disinfecting composition or disinfecting gas is temporary. Therefore, spraying the disinfecting composition or disinfecting gas is detrimental to maintaining indoor diversity. However, the present inventors have discovered that spraying a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals and useful for maintaining indoor diversity after spraying the disinfecting composition or disinfecting gas results in accelerated recovery of indoor diversity and is suitable for maintaining indoor diversity. By allowing non-pathogenic bacteria of the phylum Firmicutes to grow in the room after disinfection, drug-resistant bacteria and other bacteria can be prevented. It can suppress the occupation of harmful microorganisms and does not inhibit the growth of other microorganisms.

[0026] The disinfecting composition may be a known disinfecting composition, preferably a liquid disinfecting composition. Examples include an ethanol-containing liquid such as an aqueous solution containing 30 to 90% ethanol, an acetic acid-containing liquid, a citric acid-containing liquid, and hypochlorous acid water. The disinfecting composition may contain additives such as green tea extract, sodium lactate, glycerin, glycerin fatty acid ester, malic acid, and plant-derived antibacterial agents. The disinfecting composition preferably has disinfecting power but is harmless to humans and animals. The disinfecting gas may be any known disinfecting gas, such as ozone gas or chlorine dioxide gas. The disinfecting gas may also be a mixed gas.

[0027] In the present invention, it is preferable to spray a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals after a predetermined time has elapsed after spraying the disinfecting composition or disinfecting gas. It is preferable to spray a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals after 20 minutes to 3 hours have elapsed after spraying the disinfecting composition or disinfecting gas, more preferably after 0.5 to 2 hours have elapsed, and even more preferably after 1 to 2 hours have elapsed.

[0028] When the disinfecting composition is a liquid composition, its viscosity is not particularly limited, but it is preferably low enough to be sprayable from a spray nozzle.

[0029] [Scatter] The method for dispersing the disinfecting composition or disinfecting gas is not particularly limited, and known chemical dispersing devices, sprays, atomizers, etc. can be used. Also usable are aerosol sprays, manual types, automatic types, hand sprays, pump-type spray products constructed by filling a container with a trigger pump, and aerosol products constructed by sealing a liquid composition and, if necessary, a propellant in a pressure-resistant container. Examples of propellants include liquefied petroleum gas (LPG), dimethyl ether (DME), nitrogen gas, carbon dioxide, nitrous oxide, and compressed air. Spray products and aerosol products may be one-push sprayers using a metered-dose injection valve or trigger-type sprayers. This allows for the appropriate amount of disinfecting composition or disinfecting gas to be dispersed depending on the number of injections.

[0030] From the viewpoint of maintaining microbial diversity in a room, it is preferable to spray the disinfecting composition or disinfecting gas once within a predetermined time period. An automatic spraying device with a timer is preferred as a system for easily realizing such a spraying method. For example, the time is measured using a timer, and the disinfecting composition or disinfecting gas is sprayed once every preset time period. More preferably, the disinfecting composition or disinfecting gas is sprayed once every 24 to 96 hours, and even more preferably, once every 24 to 48 hours. By spraying according to such a schedule, it is expected that microbial diversity in a room can be maintained more efficiently. When the disinfecting composition or disinfecting gas is sprayed at regular intervals, it is also preferable to spray a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals at regular intervals.

[0031] The spraying is preferably carried out into the air in a room from the viewpoint of widely dispersing the disinfecting composition or disinfecting gas throughout the room. It is also preferable to spray over a wide area rather than concentrating the spray on a single point. A dispersion assisting means such as a circulator can also be used. The disinfecting composition or disinfecting gas can also be sprayed by attaching a spraying device near the air outlet of an air conditioner, an electric fan, or the fan of a circulator.

[0032] When the disinfecting composition or disinfecting gas is in a liquid form, the disinfecting composition or the disinfecting gas is sprayed in the following manner per spray: It is preferable to spray 0.1 ml to 10 ml of the disinfecting composition, more preferably 0.1 ml to 5 ml, and particularly preferably 0.1 ml to 3 ml. The larger the amount sprayed per time, the greater the amount of disinfecting composition supplied into the room, and a higher effect can be expected, but if the amount sprayed is too large, the effect will not be enhanced commensurate with the increase in the amount sprayed, and many droplets will remain without volatilization after the disinfecting composition is sprayed.

[0033] When the disinfecting composition to be sprayed is a liquid composition, the particle size of the droplets is not particularly limited.

[0034] <Indoor microbial diversity maintenance device> The apparatus for maintaining indoor microbial diversity of the present invention is characterized by comprising a spraying means for spraying a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals. The microbial diversity and the liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals are the same as those in the method for maintaining indoor microbial diversity of the present invention.

[0035] The spraying means of the indoor microbial diversity maintenance device of the present invention is not particularly limited, and a known spray nozzle or spraying mechanism can be used. In addition to the spraying means, the device of the present invention includes a tank for storing the liquid composition and a flow path for the liquid composition from the tank to the spraying means. Furthermore, spraying can be programmed to spray a fixed amount at regular intervals using electronic control. Furthermore, the device of the present invention can be used in conjunction with an air conditioning system to mix the liquid composition with the outside air and spray it indoors.

[0036] The indoor microbial diversity maintenance device of the present invention may include a spraying means for spraying a disinfecting composition or a disinfecting gas in addition to a spraying means for spraying a liquid composition containing Firmicutes bacteria. In this case, the tank also includes a tank for storing the disinfecting composition or the disinfecting gas in addition to a tank for storing the liquid composition containing Firmicutes bacteria. [Example]

[0037] Bacillus subtilis (Bacillus subtilis) in spore form, 1 × 10 5 A liquid composition was prepared consisting of an aqueous ethanol solution containing CFU / mL. The ethanol concentration was 8%. The liquid composition was sprayed in an amount of 1.0 mL per application into a private toilet room (0.8 tatami mats, room temperature: 25°C) in a reinforced concrete office building. The spraying was carried out into the air using a hand sprayer. The spraying was carried out once every 24 hours. Before and after spraying, the floor in the center of the room was swabbed over time to collect measurement samples. The swabs used were Isohelix Swab: SK-3, and the collected samples were stored in Isohelix BuccalFix Stabilization and Lysis Buffers: BFX / S1-05. The 16S rRNA gene of the measurement samples was amplified by PCR, followed by metagenomic sequencing using a next-generation sequencer to calculate phylogenetic diversity and the Shannon index. The results are shown in Figures 1 and 2. Figure 1 is a graph showing the change in the Shannon index over time, and Figure 2 is a graph showing the change in phylogenetic diversity over time. This experiment was conducted in two private toilets, designated "10F toilet" and "12F toilet," respectively. In Figures 1 and 2, "average" refers to the average values ​​for "10F toilet" and "12F toilet." Phylogenetic diversity is an index that evaluates diversity using a molecular phylogenetic tree by summing the branch lengths. The greater the number of phylogenetically distant species present, the higher the value. On the other hand, the Shannon index is calculated from the proportion of species in the entire sample; the more species present and the more evenly they are distributed, the higher the index. For example, Infect Drug Resist. 2019; 12: 501-5 As described in 10., it is known that conventional sanitation techniques using chemical detergents and disinfectants allow drug-resistant bacteria to survive, which in turn reduces indoor microbial diversity. It is also known that in tightly sealed rooms, microbial diversity tends to decrease unless special measures are taken. In mechanically ventilated rooms, phylogenetic diversity drops to an average of 10–15 (Steven W Kembel et al., The ISME Journal volume 6 pages 1469-1479 (2012)), while in confined spaces such as intensive care units (ICUs), the Shannon index dropped to an average of 3.8 (Alexander Mahnert et al., Nature Communications volume 10, number: 968 (2019)). In contrast, as is clear from the graphs in Figures 1 and 2, although it is known that microbial diversity in highly airtight rooms decreases over time, when a liquid composition containing Bacillus subtilis, a non-pathogenic bacterium of the phylum Firmicutes, was sprayed, the decrease in microbial diversity in the room was suppressed and microbial diversity was maintained.

Claims

1. A method for maintaining microbial diversity in a room, comprising the step of spraying a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals in the room.

2. A method for maintaining microbial diversity in an indoor environment, comprising the steps of spraying a disinfecting composition or disinfecting gas, and then spraying a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals.

3. The method for maintaining microbial diversity in an indoor environment according to claim 1, wherein the bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals are bacteria of the genus Bacillus.

4. 2. The method for maintaining microbial diversity in a room according to claim 1, wherein the liquid composition containing the bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals is sprayed once at a predetermined time interval.

5. The liquid composition containing the bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals contains the microorganism at a concentration of 1×10 ５ The method for maintaining indoor microbial diversity according to claim 1, wherein the indoor microbial diversity is at least 1 CFU / mL.

6. The method for maintaining microbial diversity in a room according to claim 1, wherein 0.1 to 3 ml of a liquid composition containing the bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals is sprayed once every 1 to 3 hours.

7. 2. The method for maintaining microbial diversity in a room according to claim 1, wherein the product of phylogenetic diversity and Shannon diversity index obtained by metagenomic sequencing of a sample collected by wiping the surface of a floor located in the center of the room is reduced by 10% or less from before spraying.

8. An apparatus for maintaining microbial diversity in an room, comprising a spraying means for spraying a liquid composition containing bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals.

9. An apparatus for maintaining microbial diversity in an room, comprising: a spraying means for spraying a disinfecting composition or a disinfecting gas; and a spraying means for spraying a liquid composition containing microorganisms that are harmless to humans and animals.

10. A bacterium from the phylum Firmicutes that maintains indoor microbial diversity.

11. A composition for maintaining microbial diversity in a room, comprising bacteria of the phylum Firmicutes that are non-pathogenic to humans and animals.

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