JP2008253164A - Method for evaluating antimicrobial performance of photocatalytic material for general environmental microorganism - Google Patents
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Abstract
Description
本発明は、光触媒材料の抗菌性能を評価する方法に関するものである。 The present invention relates to a method for evaluating the antibacterial performance of a photocatalytic material.
光触媒の中で、酸化チタンは化学的に安定で、紫外光照射によって強い酸化力を生じる。この酸化力によって、有機物を酸化分解したり、微生物を殺菌したりすることができる。近年、光触媒技術の研究開発が活発に行われ、抗菌タイルなど、一部実用化されている。その一方で、十分な効果がない製品、いわゆるまがいものが市場に多く流通しないように、光触媒技術の信頼の確立が求められている。 Among photocatalysts, titanium oxide is chemically stable and generates strong oxidizing power when irradiated with ultraviolet light. With this oxidizing power, organic substances can be oxidatively decomposed and microorganisms can be sterilized. In recent years, research and development of photocatalytic technology has been actively conducted, and some antibacterial tiles have been put into practical use. On the other hand, establishment of trust in photocatalytic technology is demanded so that products that do not have sufficient effects, so-called false things, do not circulate in the market.
こうした状況下において、使用する光触媒の性能を正しく評価する必要がある。例えば抗菌性能評価の場合、従来の方法は、大腸菌、黄色ブドウ球菌などの菌を用いて、培地に出現する細菌コロニーの数から抗菌性能を評価するものであり（特許文献１〜３等）、光触媒の場合も、同様の細菌コロニーをカウントする方法が用いられている。
例えば、特許文献１に記載された方法は、光触媒粉末の懸濁液に、大腸菌懸濁液を添加し、これに紫外線を照射して光触媒反応を開始させ、該反応液を経時的にサンプリングして、希釈液を作成し、それぞれの希釈液を用いて培地上で培養し、形成されたコロニーをカウントして生菌数を測定するというものである。
しかしながら、従来の方法は、特定の細菌以外の雑菌の影響を排除するための設備を必要とした。そして主な用途としては、医療現場や公衆衛生の場で用いられるものに限られていた。また、培地上での再培養に約１日、コロニーのカウントに数十分と、その評価結果が得られるまでに長い時間を要するものであった。さらに、光触媒の抗菌性能を評価する場合には、紫外線照射による光触媒反応をさせる必要があるが、供試菌自体が、紫外線照射に対して耐性が弱いという欠点を有している。
For example, in the method described in Patent Document 1, an E. coli suspension is added to a suspension of photocatalyst powder, and this is irradiated with ultraviolet rays to initiate a photocatalytic reaction, and the reaction solution is sampled over time. Then, a diluted solution is prepared, cultured on the medium using each diluted solution, the formed colonies are counted, and the number of viable bacteria is measured.
However, the conventional method requires equipment for eliminating the influence of various bacteria other than the specific bacteria. And the main uses were limited to those used in the medical field and public health field. Moreover, it took about a day for the re-culture on the medium, several tens of minutes for the count of colonies, and it took a long time to obtain the evaluation result. Furthermore, when the antibacterial performance of the photocatalyst is evaluated, it is necessary to carry out a photocatalytic reaction by ultraviolet irradiation, but the test bacteria themselves have a drawback that they are weakly resistant to ultraviolet irradiation.
本発明は、以上のような事情に鑑みてなされたものであって、雑菌排除などの特別な設備を必要とせず、簡便で迅速かつ定量性のある抗菌性能評価方法を提供することを目的とするものである。 The present invention has been made in view of the circumstances as described above, and does not require special equipment such as elimination of germs, and aims to provide a simple, rapid and quantitative antibacterial performance evaluation method. To do.
発明者らは、上記目的を達成すべく鋭意研究を重ねた結果、以下の知見を得た。すなわち、酸化チタン光触媒反応は、紫外光照射により誘起され、抗菌性能を発現するが、この光触媒の抗菌性能を環境分野や産業用途に応用する場合には、周囲の光が強いことを考慮して、従来民生用途で試供菌とされてきた大腸菌や黄色ブドウ球菌より一般性があって紫外光に強い菌を使う方が良い。また、生死の判別を簡便に評価できるように、例えば細胞内の色素や化学発光機能が死滅に伴い変化する菌を探索することは重要である。
そこで、我々はまず、微細藻類のシアノバクテリアが持つクロロフィル色素の酸化分解反応に注目した。シアノバクテリアは、海水・淡水・陸上において広く生息しており、日光がよく当たる自然環境の細菌として代表種となり得る。また、クロロフィルをもつことから緑色を呈しており、死ぬとまもなくクロロフィルが分解され色変化が起こることから、死亡判定を吸光度変化や蛍光強度変化で評価することができる可能性がある。
As a result of intensive studies to achieve the above object, the inventors have obtained the following knowledge. In other words, the titanium oxide photocatalytic reaction is induced by ultraviolet light irradiation and exhibits antibacterial performance. However, when the antibacterial performance of this photocatalyst is applied to the environmental field and industrial applications, it is considered that ambient light is strong. It is better to use bacteria that are more general and resistant to ultraviolet light than Escherichia coli and Staphylococcus aureus that have been used as samples for conventional consumer use. In addition, it is important to search for bacteria whose intracellular pigment or chemiluminescence function changes with death so that life / death discrimination can be easily evaluated.
Therefore, we first focused on the oxidative degradation reaction of chlorophyll pigments possessed by microalgae cyanobacteria. Cyanobacteria are widely inhabited in seawater, freshwater, and land, and can be representative species of bacteria in the natural environment where they are exposed to sunlight. Moreover, since it has chlorophyll, it has a green color, and as soon as it dies, chlorophyll is decomposed and a color change occurs. Therefore, there is a possibility that the death determination can be evaluated by a change in absorbance or a change in fluorescence intensity.
本発明は、これらの知見に基づいて完成に至ったものであり、以下のとおりのものである。
（１）抗菌性材料の抗菌性能を評価する方法であって、色素合成能を有する細菌を含有する液と該液中にさらに抗菌性材料を含有する液との、液中の色素変化を指標として用いることを特徴とする抗菌性能評価方法。
（２）前記抗菌性材料が光触媒であって、該光触媒を含有する液に紫外線を照射することを特徴とする上記（１）の抗菌性能評価方法。
（３）前記細菌が、シアノバクテリアである上記（１）又は（２）の抗菌性能評価方法。
（４）色素合成能を有する細菌を含有する液からなる抗菌性能評価用試薬。
（５）前記細菌が、シアノバクテリアである上記（４）の抗菌性能評価用試薬。
The present invention has been completed based on these findings, and is as follows.
(1) A method for evaluating the antibacterial performance of an antibacterial material, which is an indicator of pigment change in a liquid between a liquid containing a bacterium capable of synthesizing a dye and a liquid further containing an antibacterial material in the liquid An antibacterial performance evaluation method characterized by being used as:
(2) The antibacterial performance evaluation method according to (1) above, wherein the antibacterial material is a photocatalyst, and the liquid containing the photocatalyst is irradiated with ultraviolet rays.
(3) The antibacterial performance evaluation method according to (1) or (2) above, wherein the bacterium is cyanobacteria.
(4) A reagent for evaluating antibacterial performance comprising a liquid containing bacteria having pigment synthesis ability.
(5) The reagent for evaluating antibacterial performance according to (4), wherein the bacterium is cyanobacteria.
本発明の方法は、再培養操作が不要で、試験中も無菌操作が全く不要で、簡便なリアルタイム計測が可能であり、クロロフィルの吸収スペクトルや蛍光スペクトルを測定することにより、呈色反応でありながら定量性のある、有益な抗菌性能評価法を可能とするものである。また、シアノバクテリアを用いた場合には、紫外線照射に耐性があるという優れた利点がある。本発明の方法によれば、医療現場や公衆衛生の場に限られず、広く環境全般について実施することが可能となる。 The method of the present invention does not require re-culture operation, does not require aseptic operation even during the test, allows simple real-time measurement, and is a color reaction by measuring the absorption spectrum and fluorescence spectrum of chlorophyll. However, it enables quantitative and useful antibacterial performance evaluation methods. Further, when cyanobacteria are used, there is an excellent advantage that they are resistant to ultraviolet irradiation. According to the method of the present invention, the present invention is not limited to medical sites and public health sites, and can be widely implemented for the entire environment.
本発明の抗菌性能評価方法は、色素合成能を有する細菌を含有する液と該液中にさらに抗菌性材料を含有する液との、液中の色素変化を指標として用いることを特徴とする。
第１図は、本発明の評価方法の概要を示す図である。
The antibacterial performance evaluation method of the present invention is characterized in that a change in pigment in a liquid between a liquid containing bacteria having pigment synthesis ability and a liquid further containing an antibacterial material is used as an index.
FIG. 1 is a diagram showing an outline of the evaluation method of the present invention.
シアノバクテリア（Synechocystis sp. PCC 6803）は、酸素発生型光合成を行う菌であり、増殖培地ＢＧ１１で増殖し、クロロフィルａ、β−カロテン、フィコエリスリンなどの色素を合成するが、死滅すると、まもなく細胞内部の色素が分解される。
本発明では、この色素の分解反応に伴って、色素がもつ自家蛍光特性或いは吸光特性の減少を指標として抗菌性材料の抗菌性能を評価するものである。
本発明においては、色素がもつ自家蛍光特性或いは吸光特性の減少を汎用な分光器で計測することで、定量的に殺菌率の判定をすることが可能となる。
本発明の評価方法は、上記シアノバクテリア以外に、緑藻（淡水・海水）、紅藻（海水）、褐藻（淡水）にも広く適用できる。
In the present invention, the antibacterial performance of the antibacterial material is evaluated by using, as an index, a decrease in the autofluorescence characteristic or light absorption characteristic of the dye accompanying the decomposition reaction of the dye.
In the present invention, it is possible to quantitatively determine the sterilization rate by measuring the decrease in autofluorescence characteristics or light absorption characteristics of the dye with a general-purpose spectrometer.
The evaluation method of the present invention can be widely applied to green algae (freshwater / seawater), red algae (seawater), and brown algae (freshwater) in addition to the cyanobacteria.
本発明において、こうした液中の色素変化を指標として抗菌性能を評価する場合、その液は、色素合成能を有する細菌及び／又は抗菌性材料を含有する液そのものであっても、或いは、これらの液体から前記細菌及び／又は前記抗菌性材料をフィルタによる除去等の公知の方法を用いて除去した後の液体であってもよい。 In the present invention, when the antibacterial performance is evaluated using the pigment change in such a liquid as an index, the liquid may be a liquid itself containing a bacterium capable of synthesizing a dye and / or an antibacterial material, or these liquids may be used. The liquid after removing the bacteria and / or the antibacterial material from the liquid by a known method such as removal by a filter may be used.
また、本発明の方法に用いる抗菌性能評価用試薬は、色素合成能を有する細菌を含有する液であればよく、該細菌の培養液そのものであっても、或いは、該培養液を希釈したものであってもよい。
本発明の抗菌性能評価用試薬を用いて抗菌性材料の抗菌性能を評価するには、該試薬中に抗菌性材料を添加し、或いは、抗菌性材料を含有する液中に該試薬を添加し、添加前後の色素変化を指標として用いる。
In addition, the antibacterial performance evaluation reagent used in the method of the present invention may be a liquid containing a bacterium capable of synthesizing pigments, and may be a culture solution of the bacterium itself or a dilution of the culture solution. It may be.
In order to evaluate the antibacterial performance of an antibacterial material using the antibacterial performance evaluation reagent of the present invention, an antibacterial material is added to the reagent, or the reagent is added to a liquid containing the antibacterial material. The pigment change before and after the addition is used as an index.
以下、以下、本発明を実施例によってさらに具体的に説明するが、本発明はこれら実施例により何ら限定されるものではない。
（サンプルの調整）
ＢＧ１１液体培地で２８℃で通気培養を行った1週間以内のシアノバクテリア（倍化時間約１日）の培養液を、１０，０００ｒｐｍで５分間遠心をかけ、上清を捨て超純粋による洗浄作業を２回行った。次に、吸光度（波長７３０ｎｍ）が０．２になるように超純水菌液を調整した。
光触媒として、市販のアナターゼ酸化チタンゾル（石原産業製 ＳＴＳ−２１、４０ｗｔ％スラリー液）用い、これに前記菌液と攪拌子をいれて、攪拌を行いながらキセノン灯からの紫外線（波長４００ｎｍ以下、０．５ｍＷ／ｃｍ２）を照射した。
同様にして、酸化チタンを添加しない菌液、紫外線照射を行わない菌液、酸化チタン添加も紫外線照射も行わない菌液を調整して、全部で４つの条件のサンプルを用い、生菌数の経時変化を調べた。
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely below, this invention is not limited at all by these Examples.
(Sample adjustment)
A culture solution of cyanobacteria (doubling time of about 1 day) aerated in a BG11 liquid medium at 28 ° C. for 1 week is centrifuged at 10,000 rpm for 5 minutes, and the supernatant is discarded and washing with ultrapure Was performed twice. Next, the ultrapure water bacterial solution was adjusted so that the absorbance (wavelength 730 nm) was 0.2.
As a photocatalyst, a commercially available anatase titanium oxide sol (STS-21, manufactured by Ishihara Sangyo Co., Ltd., 40 wt% slurry liquid) was used. .5 mW / cm 2 ).
In the same manner, a bacterial solution not added with titanium oxide, a bacterial solution not subjected to ultraviolet irradiation, and a bacterial solution not subjected to addition of titanium oxide or ultraviolet irradiation were prepared, and samples of four conditions were used in total. The change with time was examined.
（シアノバクテリア（Synechocystis sp. PCC 6803）に対する酸化チタン光触媒の抗菌効果の確認）
菌の生死の判定を、ＬＩＶＥ／ＤＥＡＤ BacLight Bacterial Viability Kit（Invitrogen社）を用いた生死判定で行なった。
図２はその結果を示す写真であり、左側が生きている細菌のもので、緑色に発光しているが、右側の死んだ細菌のものは、赤く発光している。
図３は、光触媒反応後のサンプルについて、LIVE/DEAD BacLight Bacterial Viability Kit（invitrogen）を用いた生死判定を示すものであって、６時間の紫外線照射により、光触媒反応が進行すると、９０％以上の死滅率が得られた。
(Confirmation of antibacterial effect of titanium oxide photocatalyst against cyanobacteria (Synechocystis sp. PCC 6803))
The determination of viability of the fungus was performed by viability determination using a LIVE / DEAD BacLight Bacterial Viability Kit (Invitrogen).
FIG. 2 is a photograph showing the results. The left side is for living bacteria and emits green light, while the right side for dead bacteria is red.
FIG. 3 shows a life / death determination using the LIVE / DEAD BacLight Bacterial Viability Kit (invitrogen) for the sample after the photocatalytic reaction. When the photocatalytic reaction proceeds by UV irradiation for 6 hours, 90% or more The death rate was obtained.
（シアノバクテリアの死滅に伴う菌液の色変化の確認）
図４は、光触媒反応によるクロロフィルの退色を確認した結果を示すものである。
図に示すとおり、酸化チタンを添加して６時間の紫外線照射を行った菌液（ＴｉＯ２＋、ＵＶ６ｈｒ）のみ、クロロフィルの退色が進み、緑色から青色に変化したが、酸化チタンを添加しなかった菌液（ＴｉＯ２−、ＵＶ６ｈｒ）、紫外線照射を行わなかった菌液（ＴｉＯ２＋、ＵＶ−）、及び酸化チタンの添加も紫外線照射も行わなかった菌液（ＴｉＯ２−、ＵＶ−）は、いずれも緑色のままであった。
酸化チタンを添加して６時間の紫外線照射を行った菌液（ＴｉＯ２＋、ＵＶ６ｈｒ）について、紫外線照射前後の色素スペクトルの変化を測定した。
図５は、吸収スペクトル測定の結果を示す図である。該図に示されているとおり、クロロフィル色素の吸収ピークが消失し、クロロフィルが分解されたことが明らかとなった。
この結果は、前記のＬＩＶＥ／ＤＥＡＤａｓｓａｙによる評価と対応しており、殺菌率を呈色反応で定量的に評価できることを示すものである。
(Confirmation of color change of bacterial liquid accompanying the death of cyanobacteria)
FIG. 4 shows the result of confirming chlorophyll fading due to the photocatalytic reaction.
As shown in the figure, only the bacterial solution (TiO2 +, UV6hr) that was irradiated with ultraviolet rays for 6 hours after adding titanium oxide, the chlorophyll faded and changed from green to blue, but no titanium oxide was added. The liquid (TiO2-, UV6hr), the bacterial liquid that was not irradiated with ultraviolet light (TiO2 +, UV-), and the bacterial liquid (TiO2-, UV-) that was neither added with titanium oxide nor irradiated with ultraviolet light were all green. It remained.
For a bacterial solution (TiO2 +, UV6hr) which was irradiated with ultraviolet rays for 6 hours after adding titanium oxide, the change in the dye spectrum before and after the ultraviolet irradiation was measured.
FIG. 5 is a diagram showing the results of absorption spectrum measurement. As shown in the figure, the absorption peak of the chlorophyll pigment disappeared, and it was revealed that chlorophyll was decomposed.
This result corresponds to the evaluation by the above-mentioned LIVE / DEADassay, and shows that the bactericidal rate can be quantitatively evaluated by a color reaction.
（シアノバクテリアの液体培養液中における光触媒効果）
ＢＧ１１液体培地を１／１０に希釈して、２８℃で通気培養を行った。酸化チタンは石原産業製のＳＴＳ−２１液を水で希釈して用いた。
２０ｍｌのサンプル液に対して、キセノン灯からの紫外線（波長４００ｎｍ以下、０．５ｍＷ／ｃｍ２）を照射して、酸化チタンの有無による違いを調べた。
その結果を図６に示す。
図６に示すとおり、光触媒の入ったサンプルでは、１日経過後、ガラス容器の壁面や通気用のチューブに細菌が付着する現象が確認されたが、３日後には、すべて剥がれ落ちて、沈殿となった。
この結果は次のように解釈できる。すなわち、シアノバクテリアは、まず紫外光による酸化ストレスから身を守るため、酸化チタンやガラス容器などの無機物を核に付着・凝集して、バイオフィルム化することで、外的要因の強度を軽減しようとするが、その後、光触媒酸化反応により、バイオフィルムが分解されることを示している。
(Photocatalytic effect of cyanobacteria in liquid culture)
BG11 liquid medium was diluted to 1/10 and aerated culture was performed at 28 ° C. Titanium oxide was prepared by diluting STS-21 solution manufactured by Ishihara Sangyo with water.
A 20 ml sample solution was irradiated with ultraviolet rays (wavelength 400 nm or less, 0.5 mW / cm 2 ) from a xenon lamp, and the difference due to the presence or absence of titanium oxide was examined.
The result is shown in FIG.
As shown in FIG. 6, in the sample containing the photocatalyst, after 1 day, it was confirmed that bacteria adhered to the wall surface of the glass container and the ventilation tube. became.
This result can be interpreted as follows. In other words, cyanobacteria will reduce the strength of external factors by first attaching and aggregating inorganic substances such as titanium oxide and glass containers to the core to form a biofilm in order to protect themselves from oxidative stress caused by ultraviolet light. However, it is shown that the biofilm is then decomposed by the photocatalytic oxidation reaction.
本発明の方法及び試薬を用いることにより、光触媒製品の防藻効果やバイオフィルム形成阻害効果などの評価法の確立が期待される。 By using the method and reagent of the present invention, it is expected to establish an evaluation method for the anti-algae effect and biofilm formation inhibition effect of the photocatalyst product.
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