JP2004002229A

JP2004002229A - Method for sterilization

Info

Publication number: JP2004002229A
Application number: JP2002159917A
Authority: JP
Inventors: Tetsuya Okano; 岡野　哲也; Shigeru Tamura; 田村　成
Original assignee: Kao Corp
Current assignee: Kao Corp
Priority date: 2002-05-31
Filing date: 2002-05-31
Publication date: 2004-01-08

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly safe method for sterilization with which high sterilizing effects on bacterial spores are obtained by a simple treatment with excellent operating efficiency. <P>SOLUTION: The method for sterilization comprises treating the bacterial spores with a germination inducer (an amino acid, a saccharide, an alkali metal ion, adenosine, etc.) and then treating the resultant bacterial spores with a bactericide (benzalkonium chloride, hexamethylenebiguanidine hydrochloride, sodium hypochlorite, an alcohol, etc.). <P>COPYRIGHT: (C)2004,JPO

Description

【０００１】
【発明の属する技術分野】
本発明は、細菌芽胞を効率的に殺菌する方法に関する。
【０００２】
【従来の技術】
従来、広範な環境における殺菌消毒剤として、次亜塩素酸ナトリウム、次亜塩素酸カルシウム、ジクロロイソシアヌル酸ナトリウム等の塩素系殺菌剤が広く用いられている。中でも次亜塩素酸ナトリウム等の次亜塩素酸塩は、価格面と効果の点で汎用されているが、医療、食品工業等、種々の分野で要求される微生物の殺菌、滅菌に対して、更にその効力を向上させるための多くの提案がなされている。例えば、特開昭５７−６１０９９号には、次亜塩素酸塩、アルカリ性物質及び特定の第四級アンモニウム塩型カチオン界面活性剤を、それぞれ特定の重量比で含有する液体殺菌漂白剤組成物が開示されている。また、特開平７−２３３３９６号には、次亜塩素酸ナトリウム塩、陰イオン界面活性剤、アルカリ剤、アニオン界面活性剤及びキレート剤を含有する人工透析機等の医療機器用の殺菌洗浄剤が開示されている。
【０００３】
しかしながら、従来の次亜塩素酸塩系殺菌剤は、一般細菌やカビ（菌糸）については効果があるものの、薬品耐性のより高い細菌芽胞に対して簡易な操作では十分な効果が期待できない。
【０００４】
細菌芽胞を死滅させる方法として、湿熱による加熱加圧（物理的殺菌法）や、グルタルアルデヒド等のアルデヒド類や過酢酸の適用（化学的殺菌法）などが知られているが、装置が大がかりであったり、殺菌以外の弊害が生じたりして何れも汎用性の高いものとは言えなかった。特開平５−３１７３９１号、特開平５−２２７９２５号、特開平６−１５３８８０号には、細菌芽胞を対象とした殺菌方法が開示されているが、簡易な方法で高い殺菌効果が得られるとは言いがたい。
【０００５】
【発明が解決しようとする課題】
本発明は、簡易な処理により、高い殺菌効果が得られ、且つ安全性、作業性に優れた殺菌方法を提供することを目的とする。
【０００６】
【課題を解決するための手段】
本発明は、細菌芽胞を発芽誘起物質で処理した後、殺菌剤で処理する殺菌方法に関する。また、本発明は、細菌芽胞の発芽誘起物質を含む第１の剤と、殺菌剤を含む第２の剤とからなる、上記本発明の殺菌方法に用いられる２剤型殺菌剤に関する。
【０００７】
【発明の実施の形態】
細菌芽胞とは、増殖に適さない環境において作られる耐久性を有する休眠細胞であり、菌体の外側には多重の層状外殻を有している。このような細菌芽胞は薬剤や熱などに対する耐久性が非常に高く、一般的な殺菌では完全に死滅させることは困難である。
【０００８】
しかし、本発明では、まず、細菌芽胞を発芽誘起物質で処理して発芽を促進して栄養型細胞に変態させ、次いでこの栄養型細胞を殺菌することで、一般的な殺菌剤でも十分な殺菌効果が得られる。すなわち、本発明は、細菌芽胞を発芽誘起物質で処理して発芽させ栄養型細胞に変態させた後、該栄養型細胞を殺菌剤で処理する殺菌方法である。本発明の方法は、簡易に且つ確実に細菌芽胞を死滅させる方法として有用である。
【０００９】
本発明に用いられる発芽誘起物質としては、アミノ酸、糖、アルカリ金属イオン、アデノシンから選ばれる一種以上が挙げられる。アミノ酸は、Ｌ−アミノ酸が好ましく、Ｌ−アラニン、Ｌ−アスパラギン、Ｌ−チロシン、Ｌ−プロリン及びＬ−リシンから選ばれる一種以上であるが好ましい。糖は、ブドウ糖及び果糖から選ばれる一種以上が好ましい。アルカリ金属イオンは、カリウムイオンが好ましい。アルカリ金属イオンは水中で該イオンを放出可能な物質、例えば塩化カリウム、リン酸カリウム、硝酸カリウムなどを含有する水溶液により適用される。
【００１０】
一般に、発芽誘起物質を０．００１〜１０００ｍＭ（モル濃度、以下同様）、更に０．０１〜１０００ｍＭ、特に０．１〜１００ｍＭの濃度で含有する水溶液を、５〜６０分、更に３０〜６０分、細菌芽胞に接触させることで、細菌芽胞は発芽し、栄養型細胞に変態する。その際、発芽誘起物質を含有する水溶液の温度は限定しないが、１０〜６０℃が好ましく、特に２５〜４５℃が好ましい。
【００１１】
本発明では、発芽誘起物質による処理は、細菌芽胞に発芽誘起物質を含有する水溶液を一定時間接触させることが好ましい。該水溶液を細菌芽胞に接触させる方法は限定されないが、散布、噴霧、浸漬、充填等の方法が挙げらる。該処理終了後、対象部位を必要に応じて水洗し、殺菌剤による処理を行う。発芽誘起物質による処理から殺菌剤による処理までの時間は特に限定しないが短い方が好ましい。
【００１２】
また、本発明において、殺菌剤による処理は一般細菌を殺菌する際の一般的な方法に準ずることができるが、通常、殺菌剤を１〜１００００ｐｐｍ（重量比、以下同様）、更に１０〜１００００ｐｐｍ、特に１０〜１０００ｐｐｍの濃度で含有する水溶液を、１〜６０分、更に５〜３０分、栄養型細菌に接触させることで行われる。殺菌剤水溶液の温度も限定しないが、１０〜７０℃が好ましく、特に２０〜６０℃が好ましい。殺菌剤水溶液を栄養型細胞に接触させる方法は、前記した発芽誘起物質を含有する水溶液を細菌芽胞に接触させる方法に準ずるが、殺菌剤による処理では、適当な担体に水溶液を含浸させたもので対象物を拭き取る方法も可能である。
【００１３】
殺菌剤は、カチオン系殺菌剤、ハロゲン系殺菌剤及びアルコール系殺菌剤から選ばれる一種以上が好ましい。
【００１４】
カチオン系殺菌剤としては、４級アンモニウム塩系殺菌剤、例えば塩化ジデシルジメチルアンモニウムのような炭素数８〜１６の長鎖アルキル基を２つ有するジ長鎖アルキルジメチルアンモニウム塩、塩化ベンザルコニウム、ビグアナイド系殺菌剤、例えばクロルヘキシジン、ポリヘキサメチレンビグアニジン等が挙げられ、中でも、塩化ベンザルコニウム、塩化ジデシルジメチルアンモニウム、ポリヘキサメチレンビグアニジンが好ましい。なお、ビグアナイド系殺菌剤は、適宜無機酸や有機酸で中和して用いても良い。
【００１５】
ハロゲン系殺菌剤としては、塩素系化合物、臭素系化合物、ヨウ素系化合物からなるものが挙げられ、中でも、次亜塩素酸塩、塩素化イソシアヌル酸塩、亜塩素酸塩、二酸化塩素等の塩素系化合物が好ましい。これらの塩はナトリウム塩が好ましい。
【００１６】
アルコール系殺菌剤としては、エタノール等の低級アルコールを種々の濃度で含有する処方のものが知られているが、本発明では、エタノールを５０％（体積比）以上含有するものが好ましい。
【００１７】
殺菌剤の水溶液は、より良い殺菌効果を引き出すために、それぞれの殺菌剤に適したｐＨに調整されることが好ましい。例えば、カチオン系殺菌剤の場合、水溶液のｐＨ（２５℃）は１〜１４、更に２〜１３、特に３〜９が好ましく、ハロゲン系殺菌剤の場合、水溶液のｐＨ（２５℃）は３〜１２、更に５〜１２、特に６〜１１が好ましく、アルコール系殺菌剤の場合、水溶液のｐＨ（２５℃）は２〜１０、更に３〜９、特に５〜８が好ましい。このようなｐＨに調整するために、殺菌剤水溶液は、ｐＨ調整剤として有機酸もしくは無機酸又はそれらの塩を含有することが好ましい。
【００１８】
また、殺菌剤水溶液は界面活性剤を含有することができる。界面活性剤としては、両性界面活性剤、陽イオン界面活性剤（殺菌剤を除く）及び非イオン界面活性剤から選ばれる１種以上が好ましい。
【００１９】
本発明の殺菌方法は、芽胞に対する効果が高いため、幅広い分野での殺菌方法として有用である。例えば、病院、養護施設、食品加工工場、クリーニング施設、厨房等の壁、床、窓等あるいはそれらで用いられる器具、備品、及び製品用（例えば飲料液用）容器の殺菌に用いられる。しかも、本発明の殺菌方法は、耐性の強い特に芽胞に対しても優れた効果を示し、且つ安全性、作業性に優れる。
【００２０】
本発明の殺菌方法は、細菌芽胞の発芽誘起物質を含む第１の剤（第１剤）と、殺菌剤を含む第２の剤（第２剤）とからなる２剤型殺菌剤により実施できる。第１剤と第２剤の形態や有効分濃度は問わない。
【００２１】
第１剤と第２剤の剤型は、製剤安定性や使用しやすさの見地から、液体系でも粉末や顆粒のような固体系でも良い。主に細菌芽胞の発芽誘起物質からなる第１剤は液体もしくは固体の形状で、その有効成分濃度は１〜１００重量％、更に１〜７０重量％、特に１〜５０重量％が好ましい。主に殺菌剤からなる第２剤は液体の形状でその有効成分濃度は１〜９０重量％、更に５〜９０重量％、特に５〜８０重量％が好ましい。
【００２２】
【実施例】
実施例１〜１０及び比較例１〜５
芽胞形成細菌である、枯草菌（Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　ＡＴＣＣ６６３３）とセレウス菌（Ｂａｃｉｌｌｕｓ　ｃｅｒｅｕｓ　ＩＦＯ１３４９４）を、それぞれＳＣＤ寒天培地（日本製薬（株）製）に３０℃で約４週間前培養した後、寒天培地上に形成されたコロニーを適量かきとって１ｍｌの滅菌水に懸濁し、検鏡して細菌芽胞の形成を確認した。この懸濁液を２回遠心洗浄後、適量の滅菌水で約１０^８〜１０^９ｃｅｌｌ／ｍｌの菌濃度に調整した（芽胞液１）。この芽胞液１を１ｍｌ試験管に取り、その中に、表１、２示す濃度で芽胞発芽誘起物質を含有する水溶液を１ｍｌ添加し、４０℃（液温）で３０分間作用させた。なお、比較例１〜５では水のみを添加した。
【００２３】
その後、０．２μｍポアサイズのメンブレンフィルター（アドバンテック（株））にて濾過し、フィルター上の芽胞を１ｍｌの滅菌水で捕集し芽胞液を得た（芽胞液２）。芽胞液２を０．１ｍｌとり、表１、２の濃度で殺菌剤を含有する水溶液２ｍｌに接種し、２５℃にて１２０秒作用させた。
【００２４】
その後、直ちに、芽胞液２を含む殺菌剤水溶液の０．１ｍｌを、１．０％チオ硫酸ナトリウムを加えたＳＣＤＬＰ培地（日本製薬（株））中に添加して、殺菌剤を不活性化した（芽胞液３）。なお、殺菌剤がアルコールを含む場合、希釈することにより不活性化した。
【００２５】
芽胞液３を、標準寒天培地に０．２ｍｌ塗抹して、３５℃で３６時間培養して、培地上に形成されたコロニー数をカウントした。コロニー数が少ないほど、殺菌効果が高いことを意味する。結果を表１、２に示す。
【００２６】
【表１】

【００２７】
【表２】

【００２８】
【発明の効果】
本発明によれば、簡易な操作で細菌芽胞を効率的に殺菌することができる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for efficiently killing bacterial spores.
[0002]
[Prior art]
Conventionally, chlorine-based disinfectants such as sodium hypochlorite, calcium hypochlorite, sodium dichloroisocyanurate and the like have been widely used as disinfectants in a wide range of environments. Above all, hypochlorites such as sodium hypochlorite are widely used in terms of price and effect, but medical, food industry, etc., for sterilization and sterilization of microorganisms required in various fields, Many proposals have been made to further improve its efficacy. For example, JP-A-57-61099 discloses a liquid germicidal bleach composition containing hypochlorite, an alkaline substance and a specific quaternary ammonium salt type cationic surfactant in a specific weight ratio. It has been disclosed. JP-A-7-233396 discloses a sterilizing detergent for medical equipment such as an artificial dialysis machine containing sodium hypochlorite, an anionic surfactant, an alkali agent, an anionic surfactant and a chelating agent. It has been disclosed.
[0003]
However, although the conventional hypochlorite fungicides are effective for general bacteria and mold (hyphae), sufficient effects cannot be expected by simple operations on bacterial spores having higher drug resistance.
[0004]
As methods for killing bacterial spores, heating and pressurization with wet heat (physical sterilization method) and application of aldehydes such as glutaraldehyde and peracetic acid (chemical sterilization method) are known, but the equipment is large-scale. In any case, adverse effects other than sterilization occurred, and none of them was highly versatile. JP-A-5-317391, JP-A-5-227925, and JP-A-6-153880 disclose sterilization methods for bacterial spores. However, a high sterilization effect can be obtained by a simple method. It's hard to say.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a sterilization method which can achieve a high sterilization effect by simple processing and is excellent in safety and workability.
[0006]
[Means for Solving the Problems]
The present invention relates to a sterilization method in which bacterial spores are treated with a germination inducer and then treated with a bactericide. The present invention also relates to a two-part disinfectant used in the disinfection method of the present invention, comprising a first agent containing a germination-inducing substance of bacterial spores and a second agent containing a disinfectant.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
Bacterial spores are durable, dormant cells that are created in an environment unsuitable for growth and have multiple layered outer shells outside the cells. Such bacterial spores have extremely high durability against drugs, heat, and the like, and it is difficult to completely kill them by general sterilization.
[0008]
However, in the present invention, first, bacterial spores are treated with a germination-inducing substance to promote germination to be transformed into vegetative cells, and then these vegetative cells are sterilized. The effect is obtained. That is, the present invention is a sterilization method in which a bacterial spore is treated with a germination-inducing substance, germinated and transformed into a vegetative cell, and then the vegetative cell is treated with a bactericide. The method of the present invention is useful as a method for simply and reliably killing bacterial spores.
[0009]
Examples of the germination-inducing substance used in the present invention include one or more selected from amino acids, sugars, alkali metal ions, and adenosine. The amino acid is preferably an L-amino acid, and more preferably one or more selected from L-alanine, L-asparagine, L-tyrosine, L-proline and L-lysine. The sugar is preferably at least one selected from glucose and fructose. The alkali metal ion is preferably a potassium ion. The alkali metal ion is applied by an aqueous solution containing a substance capable of releasing the ion in water, for example, potassium chloride, potassium phosphate, potassium nitrate and the like.
[0010]
In general, an aqueous solution containing a germination-inducing substance at a concentration of 0.001 to 1000 mM (molarity, the same applies hereinafter), further 0.01 to 1000 mM, particularly 0.1 to 100 mM is used for 5 to 60 minutes, further 30 to 60 minutes. Upon contact with the bacterial spores, the bacterial spores germinate and transform into vegetative cells. At this time, the temperature of the aqueous solution containing the germination-inducing substance is not limited, but is preferably 10 to 60 ° C, particularly preferably 25 to 45 ° C.
[0011]
In the present invention, the treatment with a germination-inducing substance is preferably performed by bringing an aqueous solution containing a germination-inducing substance into contact with bacterial spores for a certain period of time. The method for bringing the aqueous solution into contact with the bacterial spores is not limited, and examples thereof include methods such as spraying, spraying, dipping, and filling. After the completion of the processing, the target site is washed with water as necessary, and is treated with a disinfectant. The time from the treatment with the germination-inducing substance to the treatment with the fungicide is not particularly limited, but a shorter one is preferred.
[0012]
In the present invention, the treatment with a bactericide can conform to a general method for disinfecting general bacteria. Usually, however, the bactericide is used in an amount of 1 to 10000 ppm (weight ratio, the same applies hereinafter), further 10 to 10000 ppm, In particular, it is carried out by contacting an aqueous solution containing a concentration of 10 to 1000 ppm with vegetative bacteria for 1 to 60 minutes, further for 5 to 30 minutes. The temperature of the germicide aqueous solution is not limited, but is preferably from 10 to 70C, particularly preferably from 20 to 60C. The method of contacting the fungicide aqueous solution with the vegetative cells conforms to the method of contacting the aqueous solution containing the germination-inducing substance with the bacterial spores described above. A method of wiping the object is also possible.
[0013]
The fungicide is preferably at least one selected from a cationic fungicide, a halogen-based fungicide and an alcohol-based fungicide.
[0014]
As the cationic fungicide, a quaternary ammonium salt fungicide, for example, a dilong chain alkyldimethylammonium salt having two long chain alkyl groups having 8 to 16 carbon atoms such as didecyldimethylammonium chloride, benzalkonium chloride And biguanide fungicides such as chlorhexidine and polyhexamethylenebiguanidine, among which benzalkonium chloride, didecyldimethylammonium chloride and polyhexamethylene biguanidine are preferred. The biguanide bactericide may be appropriately neutralized with an inorganic acid or an organic acid before use.
[0015]
Examples of the halogen-based germicides include those composed of chlorine-based compounds, bromine-based compounds, and iodine-based compounds. Among them, hypochlorites, chlorinated isocyanurates, chlorites, and chlorine-based compounds such as chlorine dioxide Compounds are preferred. These salts are preferably sodium salts.
[0016]
As the alcohol-based bactericide, a formulation containing various concentrations of a lower alcohol such as ethanol is known, but in the present invention, a bactericide containing 50% or more (by volume) of ethanol is preferable.
[0017]
The aqueous solution of the germicide is preferably adjusted to a pH suitable for each germicide in order to obtain a better germicidal effect. For example, in the case of a cationic disinfectant, the pH of the aqueous solution (25 ° C.) is preferably 1 to 14, more preferably 2 to 13, and particularly preferably 3 to 9. In the case of a halogen disinfectant, the pH of the aqueous solution (25 ° C.) is 3 to 10. 12, more preferably 5 to 12, particularly preferably 6 to 11, and in the case of an alcoholic bactericide, the pH (25 ° C.) of the aqueous solution is preferably 2 to 10, more preferably 3 to 9, particularly preferably 5 to 8. In order to adjust the pH to such a level, the aqueous germicide solution preferably contains an organic or inorganic acid or a salt thereof as a pH adjuster.
[0018]
Further, the germicide aqueous solution may contain a surfactant. The surfactant is preferably at least one selected from amphoteric surfactants, cationic surfactants (excluding bactericides), and nonionic surfactants.
[0019]
The sterilization method of the present invention is effective as a sterilization method in a wide range of fields because of its high effect on spores. For example, it is used for sterilizing walls, floors, windows, etc. of hospitals, nursing homes, food processing factories, cleaning facilities, kitchens and the like, or instruments, fixtures, and products (eg, beverages) containers used therein. Moreover, the sterilization method of the present invention exhibits an excellent effect even on highly resistant spores, and is excellent in safety and workability.
[0020]
The disinfection method of the present invention can be carried out by a two-part disinfectant comprising a first agent (first agent) containing a germination inducer of bacterial spores and a second agent (second agent) containing a disinfectant. . The form and effective concentration of the first and second agents are not limited.
[0021]
The dosage form of the first agent and the second agent may be a liquid system or a solid system such as a powder or granules from the viewpoint of formulation stability and ease of use. The first agent mainly composed of a germination-inducing substance of bacterial spores is in a liquid or solid form, and its active ingredient concentration is 1 to 100% by weight, preferably 1 to 70% by weight, particularly preferably 1 to 50% by weight. The second agent mainly composed of a bactericide is in a liquid form, and its active ingredient concentration is preferably 1 to 90% by weight, more preferably 5 to 90% by weight, particularly preferably 5 to 80% by weight.
[0022]
【Example】
Examples 1 to 10 and Comparative Examples 1 to 5
The spore-forming bacteria Bacillus subtilis ATCC 6633 and Bacillus cereus IFO13494 were pre-cultured on SCD agar medium (manufactured by Nippon Pharmaceutical Co., Ltd.) at 30 ° C. for about 4 weeks, and then on the agar medium. An appropriate amount of the colony formed in the above was scraped off, suspended in 1 ml of sterilized water, and examined by microscopy to confirm the formation of bacterial spores. The suspension was centrifugally washed twice, and then adjusted to a bacterial concentration of about 10 ⁸ to 10 ⁹ cells / ml with an appropriate amount of sterilized water (spore fluid 1). 1 ml of this spore solution 1 was placed in a test tube, and 1 ml of an aqueous solution containing a spore germination-inducing substance at the concentrations shown in Tables 1 and 2 was added thereto and allowed to act at 40 ° C. (liquid temperature) for 30 minutes. In Comparative Examples 1 to 5, only water was added.
[0023]
Thereafter, the mixture was filtered through a membrane filter having a pore size of 0.2 μm (Advantech Co., Ltd.), and the spores on the filter were collected with 1 ml of sterilized water to obtain a spore fluid (spore fluid 2). 0.1 ml of spore fluid 2 was taken, inoculated into 2 ml of an aqueous solution containing a bactericide at the concentrations shown in Tables 1 and 2, and allowed to act at 25 ° C. for 120 seconds.
[0024]
Immediately thereafter, 0.1 ml of an aqueous germicide solution containing spore fluid 2 was added to an SCDLP medium (Nippon Pharmaceutical Co., Ltd.) supplemented with 1.0% sodium thiosulfate to inactivate the germicide. (Spore fluid 3). When the bactericide contained alcohol, it was inactivated by dilution.
[0025]
0.2 ml of the spore liquid 3 was smeared on a standard agar medium and cultured at 35 ° C. for 36 hours, and the number of colonies formed on the medium was counted. The smaller the number of colonies, the higher the bactericidal effect. The results are shown in Tables 1 and 2.
[0026]
[Table 1]

[0027]
[Table 2]

[0028]
【The invention's effect】
According to the present invention, bacterial spores can be efficiently killed by a simple operation.

Claims

A sterilization method in which bacterial spores are treated with a germination inducer and then treated with a bactericide.

The sterilization method according to claim 1, wherein the germination-inducing substance is at least one selected from amino acids, sugars, alkali metal ions, and adenosine.

The sterilization method according to claim 2, wherein the amino acid is at least one selected from L-alanine, L-asparagine, L-tyrosine, L-proline and L-lysine.

The sterilization method according to claim 2 or 3, wherein the sugar is at least one selected from glucose and fructose.

The sterilization method according to any one of claims 2 to 4, wherein the alkali metal ion is a potassium ion.

The two-part disinfectant used in the disinfection method according to any one of claims 1 to 5, comprising a first agent containing a germination-inducing substance of bacterial spores and a second agent containing a disinfectant.