JP6885574B2 - Method for producing a microbial cell adsorption medium to which a multivalent cationic substance is bound - Google Patents
Method for producing a microbial cell adsorption medium to which a multivalent cationic substance is bound Download PDFInfo
- Publication number
- JP6885574B2 JP6885574B2 JP2016569467A JP2016569467A JP6885574B2 JP 6885574 B2 JP6885574 B2 JP 6885574B2 JP 2016569467 A JP2016569467 A JP 2016569467A JP 2016569467 A JP2016569467 A JP 2016569467A JP 6885574 B2 JP6885574 B2 JP 6885574B2
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- water
- polyethyleneimine
- insoluble carrier
- pei
- carrier
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Images
Classifications
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- A01M—CATCHING, TRAPPING OR SCARING OF ANIMALS; APPARATUS FOR THE DESTRUCTION OF NOXIOUS ANIMALS OR NOXIOUS PLANTS
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- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
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- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
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- A01N33/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic nitrogen compounds
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- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
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- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
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- A23L3/3526—Organic compounds containing nitrogen
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- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
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- D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
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Description
本願発明は、微生物菌体を吸着除去させる資材及びその製造方法に関する。また、当該資材を用いた食品等の被保存物の保存方法に関する。 The present invention relates to a material for adsorbing and removing microbial cells and a method for producing the same. Further, the present invention relates to a method for preserving a preserved object such as food using the material.
人類の生命活動の中で、環境中の微生物を制御する事は極めて大きな意義を有している。人類は、微生物を利用することによって様々の利益を得て来たが、一方では微生物との戦いに勝利する事によって発展して来た歴史を持っており、殺菌・抗菌能を有する物質の開発とそれらの効率的な利用は人類にとって極めて重要な課題である。 Controlling microorganisms in the environment is extremely significant in human life activities. Human beings have gained various benefits by using microorganisms, but on the other hand, they have a history of developing by winning the battle with microorganisms, and developing substances with bactericidal and antibacterial properties. And their efficient use is an extremely important issue for humankind.
現在までに、抗生物質（生物が生産する抗菌物質）を始め、種々の微生物制御物質が開発され実用に供せられている。しかし、それらの物質は、何れも水溶性であり、水の存在する条件下で使用すると逸散してしまうので、工業生産の場で持続的に安定して効力を発揮させることが困難であった。 To date, various microbial control substances have been developed and put into practical use, including antibiotics (antibacterial substances produced by living organisms). However, all of these substances are water-soluble and dissipate when used under the condition of water, so it is difficult to sustainably and stably exert their effects in the field of industrial production. It was.
抗菌・殺菌能を有する物質を水不溶性担体に結合させて持続的に抗菌・殺菌効果を保持させる技術も開発されているが（特許技術 １）、現在までに開発されている抗菌・殺菌物質を担持させた資材は、抗菌・殺菌物質の安定性や製品の安定性、価格、あるいは汎用性の面で改良の余地が残されていた。 A technology has also been developed in which a substance having antibacterial and bactericidal activity is bound to a water-insoluble carrier to maintain the antibacterial and bactericidal effect continuously (patented technology 1). There was room for improvement in the supported materials in terms of antibacterial and bactericidal substance stability, product stability, price, and versatility.
本発明は上記事情を鑑みてなされたものであり、優れた抗菌効果、特に優れた除菌効果を発揮する微生物菌体吸着資材及びその製造方法を提供することを目的とする。
また、当該微生物菌体吸着資材を微生物菌体が繁殖し得る被保存物に接触させた状態で保存する被保存物の保存法を提供することを目的とする。The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a microbial cell adsorbing material exhibiting an excellent antibacterial effect, particularly an excellent sterilizing effect, and a method for producing the same.
Another object of the present invention is to provide a method for preserving a preserved material in which the microbial cell adsorbing material is stored in a state of being in contact with a preserved object on which the microbial cells can propagate.
本願発明は、安定性、殺菌活性及び価格的に、より優れた抗菌活性を有する水不溶性資材を提供するもので、その価値は既存の資材を遥かに凌駕する大きな意義を有している。
多価カチオン性物質の一種であるポリエチレンイミンが、従来、殺菌力を持つとされてきたが（非特許文献１）、その活性は実用に利用出来るものではなく、学問的に解析すれば殺菌活性を否定出来ない程度のものであった。The present invention provides a water-insoluble material having more excellent antibacterial activity in terms of stability, bactericidal activity and price, and its value is of great significance far surpassing existing materials.
Polyethylenimine, which is a kind of polyvalent cationic substance, has been conventionally considered to have bactericidal activity (Non-Patent Document 1), but its activity is not practically usable, and bactericidal activity can be analyzed academically. Was undeniable.
本願発明者は、ポリエチレンイミンがアメリカ食品医薬品局（ＦＤＡ）にて「間接食品添加物」として食品の包装に使用されていることに着目し、これに抗菌性を付与出来れば、機能性の包装材を製造出来るものと考えて検討を加え、水不溶性の物質に結合させて固定化すると、水溶液中の微生物菌体を吸着除去する機能を持たせ得ることを発見して、詳細な検討を加え本願発明を完成するに至った。 The inventor of the present application has focused on the fact that polyethyleneimine is used in food packaging as an "indirect food additive" by the US Food and Drug Administration (FDA), and if antibacterial properties can be imparted to this, functional packaging. We considered that the material could be manufactured, and found that if it was bound to a water-insoluble substance and immobilized, it could have a function of adsorbing and removing microbial cells in an aqueous solution, and a detailed study was added. The invention of the present application has been completed.
本発明者らは、（１）分子量200〜1000,000のポリエチレンイミン（以下、PEIと称する）を水不溶性担体に結合させた結合体（以下、PEI-担体と称する）が微生物菌体を吸着する強い活性を有することを発見し、この知見に基づき本願発明を完成させた。すなわち、PEIを結合させた水不溶性担体が水を含む溶液中の細菌や酵母菌体を能動的に吸着して当該液体中から微生物を除去することを発見し、この知見に基づき本願発明を完成させた。水不溶性担体としては、セルロースを含有する物質、有機酸が結合したポリマー（より具体的にはペクチン、アルギン酸など）、カルボニル基を有する物質（より具体的には、ポリエステル、ポリアミド）、ウレタン結合を有する物質、ヒドロキシルを有する物質、ガラス質または粘度鉱物（より具体的にはケイ酸化合物など）を例示することができ、さらに具体的には、布、木材、ポリエステルなどのプラステイック類を例示することができる。 The present inventors have (1) adsorbed microbial cells by a conjugate (hereinafter referred to as PEI-carrier) in which polyethyleneimine having a molecular weight of 200 to 1000,000 (hereinafter referred to as PEI) is bound to a water-insoluble carrier. It was discovered that it has a strong activity of polyethylenimine, and the invention of the present application was completed based on this finding. That is, it was discovered that a water-insoluble carrier to which PEI was bound actively adsorbs bacteria and yeast cells in a solution containing water to remove microorganisms from the liquid, and based on this finding, the present invention was completed. I let you. Examples of the water-insoluble carrier include a substance containing cellulose, a polymer to which an organic acid is bound (more specifically, pectin, alginic acid, etc.), a substance having a carbonyl group (more specifically, polyester, polyamide), and a urethane bond. Substances having, substances having hydroxyl, vitreous or viscous minerals (more specifically, silicic acid compounds, etc.) can be exemplified, and more specifically, plastics such as cloth, wood, polyester, etc. can be exemplified. Can be done.
また、本発明者らは、（２）多価カチオン性抗菌剤を水不溶性担体に結合させた結合体（以下、PAA-担体と称する）にあっても微生物を吸着する強い活性を有することを発見し、この知見に基づき本願発明を完成させた。多価カチオン性抗菌剤としては、ポリリジン、プロタミン、ハイジニア、キトサン、クロルヘキシジン、ポリヘキサメチレンビグアニジドなどを例示でき、１種又は２種以上を選択して用いることができる。 In addition, the present inventors have stated that (2) even in a conjugate in which a polyvalent cationic antibacterial agent is bound to a water-insoluble carrier (hereinafter referred to as PAA-carrier), it has a strong activity of adsorbing microorganisms. It was discovered, and the invention of the present application was completed based on this finding. Examples of the polyvalent cationic antibacterial agent include polylysine, protamine, hygiene, chitosan, chlorhexidine, polyhexamethylene biguanidide, and the like, and one or more of them can be selected and used.
さらに本発明者らは、（３）PEI-担体やPAA-担体に、抗菌性を有する物質を結合させることによって新規な微生物菌体吸着媒を提供するものである。
（３−１）抗菌性を有する物質としては、上述の多価カチオン性抗菌剤を含む。Furthermore, the present inventors provide a novel microbial cell adsorption medium by binding a substance having antibacterial activity to (3) PEI-carrier or PAA-carrier.
(3-1) The substance having antibacterial activity includes the above-mentioned multivalent cationic antibacterial agent.
（３−２）抗菌性を有する物質としては、上記の多価カチオン性抗菌剤以外の物質も用いることができる。例えば、チンクピリチオン、ヒノキチオール、グルコサミン、アセチルグルコサミン、キチン、ポリアミン、タンニンなどを例示することができる。また、銀イオン、銀錯体、銅イオン、銅錯体、チタン化合物、チタン錯体など抗菌性の物質を用いることもできる。これらの抗菌性を有する物質は、１種又は２種以上を選択して用いることができる。 (3-2) As the substance having antibacterial activity, a substance other than the above-mentioned multivalent cationic antibacterial agent can also be used. For example, tinkpyrithione, hinokitiol, glucosamine, acetylglucosamine, chitin, polyamines, tannins and the like can be exemplified. Further, antibacterial substances such as silver ion, silver complex, copper ion, copper complex, titanium compound and titanium complex can also be used. As these antibacterial substances, one kind or two or more kinds can be selected and used.
本発明は、PEI-担体やPAA-担体が、これらの抗菌性を有する物質を含有することによって、新規な固定化抗菌材を製造することを可能とした点で極めて利用性の高い技術である。
（４）また、本発明はPEI-担体からなる微生物菌体吸着媒、抗菌性を有する物質を結合させたPEI-担体からなる微生物菌体吸着媒、PAA-担体からなる微生物菌体吸着媒、若しくは抗菌性を有する物質を結合させたPAA-担体からなる微生物菌体吸着媒を、食品などの微生物が繁殖し得る被保存物に接触させた状態で保存することを特徴とする新規有用な微生物繁殖性被保存物の保存法を提供するものである。The present invention is an extremely versatile technique in that the PEI-carrier and PAA-carrier contain these antibacterial substances to enable the production of a novel immobilized antibacterial material. ..
(4) Further, the present invention comprises a microbial cell adsorption medium composed of a PEI-carrier, a microbial cell adsorption medium composed of a PEI-carrier to which a substance having antibacterial activity is bound, and a microbial cell adsorption medium composed of a PAA-carrier. Alternatively, a novel useful microorganism characterized in that a microbial cell adsorption medium composed of a PAA-carrier to which a substance having antibacterial activity is bound is stored in a state of being in contact with an object to be preserved in which microorganisms such as food can grow. It provides a method for preserving reproductive substances.
本発明の微生物菌体吸着媒は、水を含む溶液・気体中の微生物菌体を連続して吸着除去することができ、優れた抗菌効果、特に優れた除菌効果を発揮する。
本発明の微生物菌体吸着媒を、例えば、包装用資材として用いた場合にあっては、被保存物の微生物菌体の繁殖を抑えることができる。The microbial cell adsorption medium of the present invention can continuously adsorb and remove microbial cells in a solution or gas containing water, and exhibits an excellent antibacterial effect, particularly an excellent sterilizing effect.
When the microbial cell adsorption medium of the present invention is used, for example, as a packaging material, it is possible to suppress the growth of microbial cells in the preserved material.
以下、本発明の実施の形態の一例をとりあげて説明する。
ここで、「抗菌」とは菌の繁殖を抑制することを意味し、「殺菌」とは菌などの病原体を死滅させることを意味し、「除菌」とは菌を取り除くことを意味し、「静菌」とは菌の増殖を一時的に抑制することを意味する。Hereinafter, an example of the embodiment of the present invention will be taken up and described.
Here, "antibacterial" means to suppress the growth of bacteria, "sterilization" means to kill pathogens such as bacteria, and "eradication" means to remove bacteria. "Bacteriostatic" means to temporarily suppress the growth of bacteria.
本発明の微生物菌体吸着媒は、分子量200〜1000,000のポリエチレンイミンを水不溶性担体に結合させた結合体である。また、本願発明の微生物菌体吸着媒は、多価カチオン性抗菌剤を水不溶性担体に結合させた結合体である。
本発明に用いられるポリエチレンイミンは、その分子量（平均分子量）が200〜1000,000である。この分子量に限られるものではないが、200以上であることが本願発明に係る機能および製造操作の点から好ましい。The microbial cell adsorption medium of the present invention is a conjugate obtained by binding polyethyleneimine having a molecular weight of 200 to 1000,000 to a water-insoluble carrier. The microbial cell adsorption medium of the present invention is a conjugate obtained by binding a polyvalent cationic antibacterial agent to a water-insoluble carrier.
The polyethyleneimine used in the present invention has a molecular weight (average molecular weight) of 200 to 1000,000. The molecular weight is not limited to this, but 200 or more is preferable from the viewpoint of the function and the manufacturing operation according to the present invention.
本発明に用いられる多価カチオン性抗菌剤は、ポリリジン、プロタミン、ハイジニア、キトサン、クロルヘキシジン、ポリヘキサメチレンビグアニジドなどを例示することができる。多価カチオン性抗菌剤は、１種又は２種以上を選択して用いることができる。
本発明に用いられる水不溶性担体は、ポリエチレンイミンや多価カチオン性抗菌剤などのポリカチオン性化合物を結合させ得る水不溶性の物質であれば、何れの物質でも使用できるが、水不溶性のポリアニオン性物質、カルボニル化合物、ポリエステル化合物、ウレタン化合物など化学合成高分子物質、天然高分子物質の何れでも原料と出来る。形態とすれば、バルクポリマーもしくはフィルム、繊維、ウエブ、織布、布帛、不織布、繊維、シート、ダンボール、木材、粒状物（木材チップ、オガクズ、そば殻、モミガラなど）及びそれらを用いる構造物などポリカチオン性化合物を結合出来る資材であればいずれでも用いることができる。Examples of the polyvalent cationic antibacterial agent used in the present invention include polylysine, protamine, hygiene, chitosan, chlorhexidine, polyhexamethylene biguanidide and the like. As the multivalent cationic antibacterial agent, one kind or two or more kinds can be selected and used.
The water-insoluble carrier used in the present invention can be any water-insoluble substance that can bind a polycationic compound such as polyethyleneimine or a polyvalent cationic antibacterial agent, but is water-insoluble polyanionic. Any of chemically synthesized polymer substances such as substances, carbonyl compounds, polyester compounds and urethane compounds, and natural polymer substances can be used as raw materials. In terms of morphology, bulk polymers or films, fibers, webs, woven fabrics, fabrics, non-woven fabrics, fibers, sheets, cardboard, wood, granules (wood chips, sawdust, buckwheat husks, fir husks, etc.) and structures using them, etc. Any material that can bind a polycationic compound can be used.
ポリエチレンイミンを水不溶性担体に結合させて、微生物菌体を吸着する結合体、すなわち微生物菌体吸着媒を得る方法は、特に制限されない。例えば、ポリエチレンイミンを水に溶解したポリエチレンイミン溶液を用いて水不溶性担体に結合させる方法や、ポリエチレンイミンを樹脂等の水不溶性担体の原料に添加し成形して結合させる方法等が挙げられる。液状のポリエチレンイミンそのものに水不溶性担体を浸漬したり塗布したりしてポリエチレンイミンを水不溶性担体に結合させてもよい。 The method of binding polyethyleneimine to a water-insoluble carrier to obtain a conjugate that adsorbs microbial cells, that is, a microbial cell adsorption medium is not particularly limited. For example, a method of binding polyethyleneimine to a water-insoluble carrier using a polyethyleneimine solution in which polyethyleneimine is dissolved in water, a method of adding polyethyleneimine to a raw material of a water-insoluble carrier such as a resin, molding and binding, and the like can be mentioned. The water-insoluble carrier may be dipped or applied to the liquid polyethyleneimine itself to bind the polyethyleneimine to the water-insoluble carrier.
ここで、ポリエチレンイミンと水不溶性物質との結合のメカニズムは確定されてはいないが、化学結合による担持であると考えている。
ポリエチレンイミン溶液を用いて水不溶性担体に結合させる方法にあっては、ポリエチレンイミン溶液に水不溶性担体を一定時間浸漬させて結合させる方法や、ポリエチレンイミン溶液を水不溶性担体の表面に噴霧して結合させる方法、ポリエチレンイミン溶液を水不溶性担体の表面に塗布して結合させる方法等が挙げられる。その後、必要に応じて乾燥させる。乾燥の際の条件については、特に制限されない。Here, the mechanism of the bond between polyethyleneimine and the water-insoluble substance has not been determined, but it is considered to be supported by a chemical bond.
In the method of binding to the water-insoluble carrier using the polyethyleneimine solution, the water-insoluble carrier is immersed in the polyethyleneimine solution for a certain period of time to bond, or the polyethyleneimine solution is sprayed onto the surface of the water-insoluble carrier to bond. Examples thereof include a method of applying a polyethyleneimine solution to the surface of a water-insoluble carrier and binding the solution. Then, if necessary, it is dried. The conditions for drying are not particularly limited.
ポリエチレンイミン溶液を用いて水不溶性担体に結合させる場合、溶液のｐＨは7.5であることが好ましい。ｐＨ調整剤は炭酸水を用いて実施することができる。 When bound to a water-insoluble carrier using a polyethyleneimine solution, the pH of the solution is preferably 7.5. The pH adjuster can be carried out using carbonated water.
また、ポリエチレンイミン溶液中のポリエチレンイミン濃度は、0.1〜20重量％の範囲であることが好ましく、0.2〜1.0重量％の範囲であることがより好ましい。0.25重量％以上の濃度であることが、十分な効果を得る点で有利である。また、ポリエチレンイミンを2.0重量％以上の濃度とすることも可能であるが、濃度を高めてもそれに見合う効果の向上が困難であると考えられる。 The concentration of polyethyleneimine in the polyethyleneimine solution is preferably in the range of 0.1 to 20% by weight, more preferably in the range of 0.2 to 1.0% by weight. A concentration of 0.25% by weight or more is advantageous in obtaining a sufficient effect. Further, although it is possible to set the concentration of polyethyleneimine to 2.0% by weight or more, it is considered that it is difficult to improve the effect corresponding to the concentration even if the concentration is increased.
ポリエチレンイミンを樹脂等の水不溶性担体の原料に添加し成形して結合させる方法にあっては、ポリエチレンイミンと水不溶性担体の原料とを混練し、射出成型、押出し成形、ロール成形、圧縮成形等の成形方法にて、プレート、フィルム、シート、繊維、あるいは種々の形態に成形することができる。水不溶性担体の原料に対し5.0〜10重量％のポリエチレンイミンを混練することが好ましい。 In the method of adding polyethyleneimine to the raw material of a water-insoluble carrier such as resin, molding and bonding, polyethyleneimine and the raw material of the water-insoluble carrier are kneaded, and injection molding, extrusion molding, roll molding, compression molding, etc. It can be molded into a plate, a film, a sheet, a fiber, or various forms by the molding method of. It is preferable to knead 5.0 to 10% by weight of polyethyleneimine with respect to the raw material of the water-insoluble carrier.
多価カチオン性抗菌剤を水不溶性担体に結合させて、微生物菌体を吸着する結合体を得る方法は、ポリエチレンイミンを水不溶性担体に結合させて、微生物菌体を吸着する結合体を得る方法と同様の方法を用いることができる。
多価カチオン性抗菌剤と水不溶性担体との結合も、ポリエチレンイミンと水不溶性物質との結合と同様、化学結合による担持であると考えている。The method of binding a polyvalent cationic antibacterial agent to a water-insoluble carrier to obtain a conjugate that adsorbs microbial cells is a method of binding polyethyleneimine to a water-insoluble carrier to obtain a conjugate that adsorbs microbial cells. The same method as above can be used.
It is considered that the bond between the polyvalent cationic antibacterial agent and the water-insoluble carrier is supported by a chemical bond as well as the bond between polyethyleneimine and the water-insoluble substance.
多価カチオン性抗菌剤を水などに溶解した多価カチオン性抗菌剤溶液を用いて水不溶性担体に結合させる場合、溶液のｐＨは、多価カチオン性抗菌剤の種類に応じて適宜設定すればよい。例えば、多価カチオン性抗菌剤としてポリリジンを用いる場合にあっては、その溶液のｐＨを9.5とすることが好ましく、多価カチオン性抗菌剤としてキトサンを用いる場合にあっては、その溶液のｐＨを1.0〜6.0の範囲内とすることが好ましい。 When binding a polyvalent cationic antibacterial agent to a water-insoluble carrier using a polyvalent cationic antibacterial agent solution dissolved in water or the like, the pH of the solution may be appropriately set according to the type of the polyvalent cationic antibacterial agent. Good. For example, when polylysine is used as the polyvalent cationic antibacterial agent, the pH of the solution is preferably 9.5, and when chitosan is used as the polyvalent cationic antibacterial agent, the pH of the solution is used. Is preferably in the range of 1.0 to 6.0.
また、多価カチオン性抗菌剤溶液中の多価カチオン性抗菌剤の濃度は、多価カチオン性抗菌剤の種類に応じて適宜設定すればよいが、例えば、多価カチオン性抗菌剤としてポリリジンを用いる場合にあっては、0.2重量％と1重量％とで実施することができ、例えば、多価カチオン性抗菌剤としてキトサンを用いる場合にあっては、0.25〜1.0重量％の範囲であることがより好ましい。 The concentration of the polyvalent cationic antibacterial agent in the polyvalent cationic antibacterial agent solution may be appropriately set according to the type of the polyvalent cationic antibacterial agent. For example, polylysine may be used as the polyvalent cationic antibacterial agent. When used, it can be carried out at 0.2% by weight and 1% by weight . For example, when chitosan is used as a polyvalent cationic antibacterial agent, it shall be in the range of 0.25 to 1.0% by weight. Is more preferable.
さらに、多価カチオン性抗菌剤を樹脂等の水不溶性担体の原料に添加し成形して結合させる方法にあっては、多価カチオン性抗菌剤と水不溶性担体の原料とを混練し、射出成型、押出し成形、ロール成形、圧縮成形等の成形方法にて、プレート、フィルム、シート、繊維、あるいは種々の形態に成形することができる。水不溶性担体の原料に対する多価カチオン性抗菌剤を混練する割合は、多価カチオン性抗菌剤の種類に応じて適宜設定すればよい。 Further, in the method of adding a polyvalent cationic antibacterial agent to a raw material of a water-insoluble carrier such as a resin, molding and binding the polyvalent cationic antibacterial agent, the polyvalent cationic antibacterial agent and the raw material of the water-insoluble carrier are kneaded and injection molded. It can be molded into a plate, a film, a sheet, a fiber, or various forms by a molding method such as extrusion molding, roll molding, or compression molding. The ratio of kneading the multivalent cationic antibacterial agent to the raw material of the water-insoluble carrier may be appropriately set according to the type of the polyvalent cationic antibacterial agent.
なお、両方の結合について、その詳細なメカニズムは不明であるが、本発明者らは下記のメカニズムによるものと考えている。多価カチオン性抗菌剤にあってはその一例としてポリリジンを例に説明する。
１）リグニン等とアミンの反応
リグニン等のフェノール性水酸基を有する化合物はアミンと下式の反応をなす。The detailed mechanism of both bindings is unknown, but the present inventors consider that the mechanism is as follows. Polylysine will be described as an example of the multivalent cationic antibacterial agent.
1) Reaction of lignin and the like with an amine A compound having a phenolic hydroxyl group, such as lignin, reacts with an amine according to the following formula.
セルロースを水酸化ナトリウムで処理すると、６位の水酸基（下記の式、化４中で〇で示した「ＯＨ」）部位の水素がＮａに入れ替わることが知られている。
３）カルボニル基とポリエチレンイミンの反応
ポリエチレンイミンはアルデヒド、ケトン、エステル、ウレタン結合等のカルボニル基と、下式の反応をなす。3) Reaction of carbonyl group and polyethyleneimine Polyethyleneimine undergoes the following reaction with carbonyl groups such as aldehydes, ketones, esters, and urethane bonds.
アミンは弱酸性において、アルデヒド、ケトン、エステル、ウレタン結合等のカルボニル基と下式の反応をなす。
抗菌性を有する物質には、上述の多価カチオン性抗菌剤を含む。また、抗菌性を有する物質として、上記の多価カチオン性抗菌剤以外の物質を用いることができる。例えば、チンクピリチオン、ヒノキチオール、グルコサミン、アセチルグルコサミン、キチン、ポリアミン、タンニンなどを用いることができる。また、銀イオン、銀錯体、銅イオン、銅錯体、チタン化合物、チタン錯体などの金属物質を用いることもできる。これらの抗菌性を有する物質は、１種又は２種以上を選択して用いることができる。 Substances having antibacterial properties include the above-mentioned multivalent cationic antibacterial agents. Further, as the substance having antibacterial activity, a substance other than the above-mentioned multivalent cationic antibacterial agent can be used. For example, tinkpyrithione, hinokitiol, glucosamine, acetylglucosamine, chitin, polyamines, tannins and the like can be used. Further, metal substances such as silver ion, silver complex, copper ion, copper complex, titanium compound and titanium complex can also be used. As these antibacterial substances, one kind or two or more kinds can be selected and used.
抗菌性を有する物質を水不溶性担体に結合させるには、水不溶性担体にポリエチレンイミンを結合させて微生物菌体を吸着する結合体を得る方法や、水不溶性担体に多価カチオン性抗菌剤を結合させて微生物菌体を吸着する結合体を得る方法と同様の方法を用いることができる。 To bind an antibacterial substance to a water-insoluble carrier, a method of binding polyethyleneimine to the water-insoluble carrier to obtain a conjugate that adsorbs microbial cells, or binding a polyvalent cationic antibacterial agent to the water-insoluble carrier. A method similar to the method for obtaining a conjugate that adsorbs microbial cells can be used.
抗菌性を有する物質を水不溶性担体に結合させるに際し、ポリエチレンイミンを結合させた水不溶性担体にさらに抗菌性を有する物質を結合させてもよいし、ポリエチレンイミンと抗菌性を有する物質とを同時に水不溶性担体に結合させてもよい。また、多価カチオン性抗菌剤を結合させた水不溶性担体にさらに抗菌性を有する物質を結合させてもよいし、多価カチオン性抗菌剤と抗菌性を有する物質を同時に水不溶性担体に結合させてもよい。 When binding the antibacterial substance to the water-insoluble carrier, the water-insoluble carrier to which polyethyleneimine is bound may be further bound to the antibacterial substance, or polyethyleneimine and the antibacterial substance may be simultaneously combined with water. It may be bound to an insoluble carrier. Further, a substance having antibacterial activity may be further bound to the water-insoluble carrier to which the polyvalent cationic antibacterial agent is bound, or the polyvalent cationic antibacterial agent and the substance having antibacterial property are simultaneously bound to the water-insoluble carrier. You may.
本発明の微生物菌体吸着媒は、水を含む溶液中の微生物菌体を吸着し除去することができる。また、微生物菌体は、水を含む溶液中以外に存在するものであってもよく、例えば、食品中の微生物菌体を吸着し除去することもできる。
ここで、水を含む溶液とは、水や水溶液等が挙げられる。また、本発明の微生物菌体は、大腸菌、黄色ブドウ球菌、枯草菌等の菌類、カンジタ菌等の酵母菌体が挙げられる。The microbial cell adsorption medium of the present invention can adsorb and remove microbial cells in a solution containing water. Further, the microbial cells may be present in a solution other than the one containing water, and for example, the microbial cells in food can be adsorbed and removed.
Here, examples of the solution containing water include water, an aqueous solution, and the like. In addition, examples of the microbial cells of the present invention include fungi such as Escherichia coli, Staphylococcus aureus and Bacillus subtilis, and yeast cells such as Candida.
また、本発明の微生物菌体吸着媒を微生物菌体が繁殖し得る被保存物に接触させて保存することができる。
被保存物としては、おにぎりや総菜、エビ等の食品や花の球根等の植物等が挙げられる。また、この被保存物に対して用いられる本発明の微生物菌体吸着媒の形態としては、おにぎりなどの包装に使用するフィルムの形態を例示できる。また、エビ、花の球根などの包装に使用するオガクズの形態を例示し得る。これらの例示以外、被保存物の種類や態様に応じて、本発明の微生物菌体吸着媒は、本明細書に記載の種々の形態の水不溶性担体にPEIなどを結合させたものとして実施することができる。これにより、被保存物の微生物菌体の繁殖を抑え、ひいては被保存物の賞味期限や保存期限の延長も期待できる。In addition, the microbial cell adsorption medium of the present invention can be stored in contact with an object to be preserved in which microbial cells can propagate.
Examples of the preserved material include foods such as rice balls, delicatessen and shrimp, and plants such as flower bulbs. Further, as the form of the microbial cell adsorption medium of the present invention used for this object to be preserved, the form of a film used for packaging rice balls and the like can be exemplified. In addition, the form of sawdust used for packaging shrimp, flower bulbs, and the like can be exemplified. In addition to these examples, the microbial cell adsorption medium of the present invention is carried out assuming that PEI or the like is bound to various forms of water-insoluble carriers described in the present specification, depending on the type and mode of the object to be preserved. be able to. As a result, the growth of microbial cells of the preserved material can be suppressed, and the expiration date and the preservation period of the preserved product can be expected to be extended.
以下に本発明を実施例に基づいて、より具体的に説明するが、本発明はこれらに限定されるものではない。
尚、本願発明の抗微生物活性の評価に使用した微生物は、次の通りである。Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto.
The microorganisms used for the evaluation of the antimicrobial activity of the present invention are as follows.
（１）大腸菌：Eschelichia coli IFO 03301
（２）黄色ブドウ球菌：Staphylococcus aureus JCM 20624
（３）枯草菌：Bacillus subtilis JM101
（４）酵母：Candida albicans JCM 1542
（実施例１）PEI-担体の製造
PEIの水不溶性担体への結合の条件を検討し、本発明では次に述べる方法を確立し、以下、此の方法に準じて本発明を実施した。(1) Escherichia coli: Eschelichia coli IFO 03301
(2) Staphylococcus aureus JCM 20624
(3) Bacillus subtilis: Bacillus subtilis JM101
(4) Yeast: Candida albicans JCM 1542
(Example 1) Production of PEI-carrier
The conditions for binding PEI to a water-insoluble carrier were examined, and the following method was established in the present invention, and the present invention was subsequently carried out according to this method.
PEI（30wt％水溶液、日本触媒株式会社製、P-1000（粘度法による数平均分子量（Ｍｎ）は70,000））を炭酸水でpH 7.5に調整し、PEI濃度を0.5〜１％になるように蒸留水で希釈し、此れに水不溶性担体として綿布（綿100％ の一辺 5 cm の正方形）を浸漬して30分間、室温で静置した後、PEIが結合されたPEI-担体を取り出し、余剰のPEI液を水洗などで除去後、65℃で3時間乾燥させて、PEI担体を製造した。 Adjust PEI (30 wt% aqueous solution, manufactured by Nippon Catalyst Co., Ltd., P-1000 (number average molecular weight (Mn) by viscosity method is 70,000)) to pH 7.5 with carbonated water so that the PEI concentration becomes 0.5 to 1%. Dilute with distilled water, soak a cotton cloth (100% cotton square with a side of 5 cm) in it as a water-insoluble carrier, allow it to stand at room temperature for 30 minutes, and then remove the PEI-carrier to which PEI is bound. After removing the excess PEI solution by washing with water or the like, it was dried at 65 ° C. for 3 hours to produce a PEI carrier.
綿布に結合させたPEI量は、1.75％アシッドオレンジ7（和光純薬株式会社製）溶液（以下、オレンジ７溶液とする）による発色度で測定した。次に、細菌吸着活性による方法によりPEI結合量を測定した。すなわち、微生物菌体である黄色ブドウ球菌の水溶液（650〜660 nm における濁度:1.0）3 mlに100ｍgのPEI-担体を入れ、10 分間、室温で静置し、上清の濁度（650nm)の変化と、生菌数（cfu、コロニー形成数）をNutrient Broth(以下、NB培地、DIFCO社製）寒天平板上で30℃で24時間培養して測定した。対照として、PEIを結合させていない綿布を上記と同じ方法で処理した液を用いた。 The amount of PEI bound to the cotton cloth was measured by the degree of color development with a 1.75% acid orange 7 (manufactured by Wako Pure Chemical Industries, Ltd.) solution (hereinafter referred to as orange 7 solution). Next, the amount of PEI bound was measured by a method based on bacterial adsorption activity. That is, 100 mg of PEI-carrier was placed in 3 ml of an aqueous solution of Staphylococcus aureus (turbidity at 650 to 660 nm), which is a microbial cell, and allowed to stand at room temperature for 10 minutes, and the turbidity of the supernatant (650 nm). ) And the number of viable bacteria (cfu, number of colonies formed) were measured by culturing on a Nutrient Broth (hereinafter, NB medium, manufactured by DIFCO) agar plate at 30 ° C. for 24 hours. As a control, a liquid obtained by treating a cotton cloth to which PEI was not bound in the same manner as described above was used.
図１に、綿布にPEIを結合させる際のPEI濃度とPEIの結合量、及び黄色ブドウ球菌の除去能との関係を示す。
発色度測定の結果、PEI液中のPEI濃度に比例して綿布にPEIが結合されていることを確認した。FIG. 1 shows the relationship between the PEI concentration when PEI is bound to cotton cloth, the amount of PEI bound, and the ability to remove Staphylococcus aureus.
As a result of the color development measurement, it was confirmed that PEI was bound to the cotton cloth in proportion to the PEI concentration in the PEI solution.
上清の濁度（650nm)の変化は1.0以上であった。
また、PEI液中のPEI濃度が0.２５wt％以上のPEI液中で綿布にPEIを結合させたPEI担体にあっては、目視で確認したところ、測定培地上にコロニーが形成されておらず、上清の濁度の変化と相まって当該溶液中の黄色ブドウ球菌を吸着除去するものであると考えた。The change in turbidity (650 nm) of the supernatant was 1.0 or more.
In addition, in the PEI carrier in which PEI was bound to cotton cloth in a PEI solution having a PEI concentration of 0.25 wt% or more in the PEI solution, no colonies were formed on the measurement medium as a result of visual confirmation. It was considered that Staphylococcus aureus in the solution was adsorbed and removed in combination with the change in the turbidity of the supernatant.
（試験例１）PEI-担体への微生物菌体吸着活性１
100 ml 容の三角フラスコに滅菌したNB培地40 mlを入れ、黄色ブドウ球菌を104cfu/mlとなるよう接種して30℃で24時間、振盪培養した。このNB培地に、実施例1の方法で製造したPEI-担体（PEI液中のPEI濃度0.5％ wt％、綿100％ の一辺 5 cm の正方形の綿布：PEI-綿布と称する）を無菌的に投入して、10分間、室温で振盪した。その結果を図２に示す。PEI-綿布を投入した三角フラスコ中の培地が透明であり、PEI-綿布を投入した場合、黄色ブドウ球菌はNB培地中から消失していることが観察され、対照であるPEIを結合させていない綿布を投入した三角フラスコ中の培地が濁っているのが観察されたことから、PEI-綿布が黄色ブドウ球菌を吸着除去する機能を持つと考えられた。(Test Example 1) PEI-Morganism cell adsorption activity on carrier 1
40 ml of sterilized NB medium was placed in a 100 ml Erlenmeyer flask, and Staphylococcus aureus was inoculated to 10 4 cfu / ml and cultured at 30 ° C. for 24 hours with shaking. The PEI-carrier produced by the method of Example 1 (PEI concentration in PEI solution 0.5% wt%, 100% cotton square cotton cloth with a side of 5 cm: referred to as PEI-cotton cloth) is sterilely applied to this NB medium. It was charged and shaken at room temperature for 10 minutes. The result is shown in FIG. The medium in the Erlenmeyer flask containing PEI-cotton cloth was transparent, and when PEI-cotton cloth was added, Staphylococcus aureus was observed to disappear from the NB medium, and the control PEI was not bound. Since it was observed that the medium in the Erlenmeyer flask containing the cotton cloth was turbid, it was considered that PEI-cotton cloth had a function of adsorbing and removing Staphylococcus aureus.
（試験例２）PEI-担体への微生物菌体吸着活性２
この現象を更に明確にする目的で、次に述べる試験を行った。
100 ml 容の三角フラスコに滅菌した蒸留水20 mlを入れ、実施例1の方法で製造したPEI-担体（水溶液中のPEI濃度0.5wt％、綿100％ の一辺 5 cm の正方形の綿布：PEI-綿布と称する）を無菌的に投入した。これに培養液（30℃で24時間培養、104〜105 cfu/ml cfu/ml）を滅菌蒸留水で100倍に希釈して、その0.1 ml を接種して、25℃、120 rpmで30分間振盪培養した。培養液（Ｓ１）を適宜希釈し、NB寒天平板培地上に塗抹して25℃で24時間培養して形成されるコロニーを計測した。次に、100 ml 容の三角フラスコに滅菌した蒸留水20 mlを入れ、培養液（Ｓ１）から取り出したPEI-綿布を投入し、25℃、120 rpmで30分間振盪し、振盪後の蒸留水（Ｓ２）を適宜希釈し、NB寒天平板培地上に塗抹して25℃で24時間培養して形成されるコロニーを計測した。次に、100 ml 容の三角フラスコに滅菌した蒸留水20 mlを入れ、Ｓ２から取り出したPEI-綿布を投入し、25℃、120 rpmで30分間振盪し、振盪後の蒸留水（Ｓ３）を適宜希釈し、NB寒天平板培地上に塗抹して25℃で24時間培養して形成されるコロニーを計測した。次に、Ｓ３から取り出したPEI-綿布をNB培地20mlに浸漬し、25℃、120 rpmで一夜振盪培養し、振盪培養した培地（Ｓ４）の650nmにおける濁度を測定した。次に、Ｓ４から取り出したPEI-綿布をNB培地20mlに浸漬し、25℃、120 rpmで一夜振盪培養し、振盪培養した培地（Ｓ５）の650nmにおける濁度を測定した。対照として、PEIを結合させていない綿布を上記と同じ方法で処理した液を用いた。以下、図３に示す手順で処理し、PEI-担体は取り出してNB液体培地に浸漬し、25℃、150 rpmで一夜振盪培養して、菌の増殖をモニターしたところ、表１に示す結果が得られた。これらの結果は、PEI-担体は、黄色ブドウ球菌体を水溶液中から吸着除去し、吸着した細菌はPEI-担体を水洗しても溶出されないが、培地中に投入して培養すると吸着した菌体の一部は増殖することを示唆した。此の事実から、実用上では、PEI-担体は細菌の殺菌力より吸着除去力を活用することが有利であると推測される。(Test Example 2) PEI-Morganism cell adsorption activity on carrier 2
For the purpose of further clarifying this phenomenon, the following tests were conducted.
20 ml of sterilized distilled water was placed in a 100 ml Erlenmeyer flask, and the PEI-carrier produced by the method of Example 1 (PEI concentration in aqueous solution 0.5 wt%, 100% cotton square cotton cloth with a side of 5 cm: PEI -Called cotton cloth) was aseptically added. Dilute the culture solution (cultured at 30 ° C for 24 hours, 10 4 to 10 5 cfu / ml cfu / ml) 100-fold with sterile distilled water, inoculate 0.1 ml of the solution, and inoculate at 25 ° C and 120 rpm. The cells were shake-cultured for 30 minutes. The culture solution (S1) was appropriately diluted, smeared on an NB agar plate medium, and cultured at 25 ° C. for 24 hours to measure colonies formed. Next, 20 ml of sterilized distilled water was placed in a 100 ml Erlenmeyer flask, PEI-cotton cloth taken out from the culture solution (S1) was added, and the mixture was shaken at 25 ° C. and 120 rpm for 30 minutes. (S2) was appropriately diluted, smeared on an NB agar plate medium, and cultured at 25 ° C. for 24 hours to measure colonies formed. Next, put 20 ml of sterilized distilled water in a 100 ml Erlenmeyer flask, add the PEI-cotton cloth taken out from S2, shake at 25 ° C. and 120 rpm for 30 minutes, and add the distilled water (S3) after shaking. The colonies formed by diluting appropriately, smearing on NB agar plate medium and culturing at 25 ° C. for 24 hours were measured. Next, the PEI-cotton cloth taken out from S3 was immersed in 20 ml of NB medium, and shake-cultured at 25 ° C. and 120 rpm overnight, and the turbidity of the shake-cultured medium (S4) at 650 nm was measured. Next, the PEI-cotton cloth taken out from S4 was immersed in 20 ml of NB medium, and shake-cultured at 25 ° C. and 120 rpm overnight, and the turbidity of the shake-cultured medium (S5) at 650 nm was measured. As a control, a liquid obtained by treating a cotton cloth to which PEI was not bound in the same manner as described above was used. Hereinafter, the treatment was performed according to the procedure shown in FIG. 3, the PEI-carrier was taken out, immersed in NB liquid medium, and cultured with shaking at 25 ° C. and 150 rpm overnight to monitor the growth of the bacteria. The results shown in Table 1 were obtained. Obtained. These results show that the PEI-carrier adsorbs and removes Staphylococcus aureus cells from the aqueous solution, and the adsorbed bacteria are not eluted even if the PEI-carrier is washed with water, but the adsorbed bacteria cells are adsorbed when they are put into a medium and cultured. Some of them suggested that they proliferate. From this fact, it is presumed that, in practical use, it is advantageous for the PEI-carrier to utilize the adsorption / removal power rather than the bactericidal power of bacteria.
実施例１ の方法に準じて、種々の水不溶性担体を用いてPEI-担体の製造を試みたところ、表２に示す水不溶性担体などがPEI-担体を形成する好適な素材である事が明らかとなった。表２は、種々の水不溶性担体１ｇに結合させたPEI量を、オレンジ7溶液による発色度で測定し、１ｇの木綿布に結合させたPEI量を１００として種々の水不溶性担体１ｇに結合させたPEI量を比較したものである。
すなわち、水不溶性担体としては、（ａ）ポリエチレンおよびポリエステル類あるいはそれらを含む織物、編み物、不織布（単織・混織布）、混紡の布帛、（ｂ）獣毛（例えば、羊毛）あるいはそれらを含む織物、編み物、不織布（単織・混織布）、混紡の布帛、（ｃ）木綿繊維を含む不織布、織物、編み物、布帛、（ｄ）絹糸あるいはそれらを含む織物、編み物、不織布（単織・混織布）、混紡の布帛、さらに（ｅ）モミガラ、ソバ殻、稲ワラ、麦ワラ、オガ屑、木材、ボール紙など紙製品などの木質系繊維類、（ｆ）ガラスなどケイ酸含有物、（ｇ）天然ゴム（ラッテクス）の群を好適に用い得ることが明確となった。これらの群は、１種又は複数種を併用することができ、各群中の物質に関しても１種又は複数種を併用することができる。さらにこれらの群に属する物質を含む物質を水不溶性担体として用いることもできる。なお、ポリエチレンおよびポリエステル類あるいはそれらを含むものにあっては、その形態が繊維に限られず、プレート、フィルム、シート等の形態であってもよい。 That is, as the water-insoluble carrier, (a) polyethylene and polyesters or woven fabrics containing them, knitting, non-woven fabrics (single woven / mixed woven fabrics), blended fabrics, (b) animal hair (for example, wool) or them are used. Textiles, knitting, non-woven fabrics (single-woven / mixed-woven fabrics), blended fabrics, (c) non-woven fabrics containing cotton fibers, textiles, knitting, fabrics, (d) silk threads or textiles containing them, knitting, non-woven fabrics (single-woven) -Mixed woven fabric), blended fabric, (e) wood-based fibers such as fir, buckwheat husk, rice straw, wheat straw, ogas waste, wood, cardboard and other paper products, (f) silicic acid-containing materials such as glass , (G) It has been clarified that the group of natural rubber (latex) can be preferably used. One or more of these groups can be used in combination, and one or more of the substances in each group can be used in combination. Furthermore, substances containing substances belonging to these groups can also be used as water-insoluble carriers. In addition, polyethylene and polyesters or those containing them are not limited to fibers, and may be in the form of a plate, a film, a sheet or the like.
種々のPEI-担体の一例として、水不溶性担体としてキッチンペーパー（セルロース製）を用いてPEI-担体（以下、PEI-キッチンペーパー担体）を製造し、その大腸菌と枯草菌に対する抗菌活性を試験した。すなわち、実施例１ の方法に準じてPEI-キッチンペーパー担体（PEI液中のPEI濃度0.5wt％、一辺 5 cm の正方形のキッチンペーパー）を製造し、この4cm2を切り取り、（Ａ）枯草菌あるいは（Ｂ）大腸菌の増殖を試験例１又は２で述べた方法に準じてモニターした。その結果を図４に示すが、PEI- キッチンペーパー担体を投入した培地では、菌のコロニーが全く形成されず、菌が存在しないことが明らかである一方、対照とした無処理のキッチンペーパーを投入した培地では、両菌株ともに正常な菌の増殖が認められた。この結果から、PEI-キッチンペーパーがグラム陰性、グラム陽性を問わず細菌を除菌する活性を持つことが明らかとなった。
（実施例４）PEI-オガクズ担体の製造
実施例1の綿布に替えて、実施例１の方法に準じてオカクズを用いてPEI-オガクズ担体（PEI液中のPEI濃度0.5wt％、２００ｍｇのオガクズ）を製造した。オガクズに結合させたPEI量は、オレンジ7溶液による発色度で測定した。オレンジ７溶液で染色した、PEI-オガクズ担体と対照のPEIを結合させていないオガクズの写真を図５に示す。(Example 4) Production of PEI-sawdust carrier PEI-sawdust carrier (PEI concentration 0.5 wt% in PEI solution, 200 mg sawdust) using sawdust according to the method of Example 1 instead of the cotton cloth of Example 1. ) Was manufactured. The amount of PEI bound to sawdust was measured by the degree of color development with the Orange 7 solution. FIG. 5 shows a photograph of sawdust stained with Orange 7 solution and not bound with PEI-sawdust carrier and control PEI.
（試験例４）PEI-オガクズ担体の抗菌活性
このPEI-オガクズ担体の抗菌活性を評価する目的で、PEI-オガクズ担体200mg をNB培地中に添加し、これに大腸菌、黄色ブドウ球菌、あるいは酵母カンヂダ・アルビカンスを105 cfu/mlとなるように接種した。これらを25℃、120 rpmで一夜振盪培養し、650 nmで濁度を測定して、菌の増殖を評価した。その結果、表３に示す様に、PEI-オガクズ担体も抗菌活性を示すことが明らかになった。(Test Example 4) Antibacterial activity of PEI-Ogakuzu carrier For the purpose of evaluating the antibacterial activity of this PEI-Ogakuzu carrier, 200 mg of PEI-Ogakuzu carrier was added to NB medium, and Escherichia coli, Staphylococcus aureus, or yeast Candida was added thereto.・ Albicans was inoculated to 10 5 cfu / ml. These were cultured with shaking at 25 ° C. and 120 rpm overnight, and the turbidity was measured at 650 nm to evaluate the growth of the fungus. As a result, as shown in Table 3, it was clarified that the PEI-sawdust carrier also exhibited antibacterial activity.
PEI-担体の抗（除）菌機能を増強させる目的で、殺菌活性が認められている銀-ヒスチジン錯体（Ag-His、日本曹達株式会社製)あるいは、ポリリジン（三栄源株式会社製）をPEI-担体に結合させてPEI-担体複合体を製造した。すなわち、実施例１に準じて作成したPEI-担体（水溶液中のPEI濃度0.5wt％、一辺 5 cm の正方形のキッチンペパー）を5重量％のAg-Hisあるいは1重量％ポリリジン水溶液（pH 9.5）に1時間浸漬した後、液中から取り出し、蒸留水による洗浄操作を２回繰り返した後60℃1時間で乾燥させた。また、PEIを結合させていないキッチンペパーを対照として同様の処理を施した。
For the purpose of enhancing the anti-bacterial function of the PEI-carrier, silver-histidine complex (Ag-His, manufactured by Nippon Soda Co., Ltd.) or polylysine (manufactured by Saneigen Co., Ltd.), which has been confirmed to have bactericidal activity, is used as PEI. -The PEI-carrier complex was prepared by binding to a carrier. That is, the PEI-carrier (PEI concentration 0.5 wt% in the aqueous solution, square kitchen pepper with a side of 5 cm) prepared according to Example 1 was mixed with 5% by weight Ag-His or 1% by weight polylysine aqueous solution (pH 9.5). After immersing in the solution for 1 hour, the solution was taken out of the solution, and the washing operation with distilled water was repeated twice and then dried at 60 ° C. for 1 hour. In addition, the same treatment was performed using kitchen pepper to which PEI was not bound as a control.
（試験例５ ）PEI-担体複合体の抗菌活性
こうして作成したPEI-担体複合体の殺菌活性は大腸菌を塗抹した寒天平板培地を用いて検定した。具体的には、大腸菌（104 cfu/ml）を塗抹した寒天平板培地の上にPEI-担体複合体と対照をそれぞれ載置し、25℃、24時間培養した（ハロー試験）。ハロー試験の結果を図６に示すが、PEI-担体（PEI-担体をディスク状に裁断したペーパーデイスク）に銀-ヒスチジン錯体あるいはポリリジンを結合させた場合に抗菌活性の増強が認められた。(Test Example 5) Antibacterial activity of PEI-carrier complex The bactericidal activity of the PEI-carrier complex thus prepared was tested using an agar plate medium smeared with Escherichia coli. Specifically, E. coli (10 4 cfu / ml) on the smeared agar plate medium the PEI- carrier complex as controls were respectively placed, 25 ° C., and cultured for 24 hours (halo test). The results of the halo test are shown in FIG. 6. When a silver-histidine complex or polylysine was bound to a PEI-carrier (a paper disk obtained by cutting a PEI-carrier into a disk shape), an enhancement of antibacterial activity was observed.
なお、図６において「ポリリジンのみ担持」の試験片の周囲には、ハローが確認されていない。これは、次の理由によると考えられる。ポリリジンは、微生物菌体と接触することによって細胞表層に損傷を与えることで抗菌効果を発揮する。したがって、水溶液の状態では菌がが死滅するが、担体と結合させるとポリリジンは担体外では菌との接触面が少なく、寒天培地表面での殺菌速度よりも増殖速度が大きいので、殺菌によるハローは検出することが出来ないと考えられる。一方、銀イオンの場合は、寒天との接触面での銀イオン濃度が大で、殺菌活性が菌の増殖速度より大きく培地中に浸出するため、ハローが検出できるものと考えられる。但し、銀イオンの場合、その担持濃度に比べると、ハローの形成は小さいものである。 In FIG. 6, no halo was confirmed around the test piece "supporting only polylysine". This is considered to be due to the following reasons. Polylysine exerts an antibacterial effect by damaging the cell surface layer by contacting with microbial cells. Therefore, the bacteria are killed in the state of an aqueous solution, but when combined with the carrier, polylysine has few contact surfaces with the bacteria outside the carrier, and the growth rate is higher than the sterilization rate on the surface of the agar medium. It is considered that it cannot be detected. On the other hand, in the case of silver ions, the silver ion concentration on the contact surface with agar is high, and the bactericidal activity is greater than the growth rate of the bacteria and leaches into the medium, so that it is considered that halo can be detected. However, in the case of silver ions, the formation of halos is small compared to the supported concentration.
（実施例６）ポリリジン-綿布担体の製造
多価アニオン性抗菌剤の一つである、ポリリジン50％含有のサンキーパーNo. 381（三栄源株式会社製、サンキーパー381、ｐＨ9）を蒸留水に溶解し、水酸化カリウム液でpH 11以上に調製した後、蒸留水を加えてポリリジン1重量％溶液を調製した。この溶液に水不溶性担体として綿100％の綿布（精練品、5-cm 角の正方形）を浸漬して一夜室温で静置した。ポリリジンが結合されたポリリジン-綿布担体を取り出し、水洗により余剰のポリリジンを除去した後、60℃で3時間乾燥した。結合させたポリリジン量は、1.75％オレンジ7（和光純薬株式会社製）溶液による発色度で測定した。オレンジ７溶液で染色した、ポリリジン-綿布担体と対照であるポリリジンを結合させていない綿布の写真を図７に示す。(Example 6) Production of polylysine-cotton cloth carrier Sunkeeper No. 381 (manufactured by Saneigen Co., Ltd., Sunkeeper 381, pH 9) containing 50% polylysine, which is one of the multivalent anionic antibacterial agents, is used in distilled water. After dissolving and adjusting the pH to 11 or higher with a potassium hydroxide solution, distilled water was added to prepare a polylysine 1 wt% solution. A 100% cotton cloth (refined product, 5-cm square) was immersed in this solution as a water-insoluble carrier and allowed to stand at room temperature overnight. The polylysine-cotton cloth carrier to which polylysine was bound was taken out, excess polylysine was removed by washing with water, and then dried at 60 ° C. for 3 hours. The amount of bound polylysine was measured by the degree of color development with a 1.75% orange 7 (manufactured by Wako Pure Chemical Industries, Ltd.) solution. A photograph of a polylysine-cotton carrier and a control polylysine-free cotton cloth stained with Orange 7 solution is shown in FIG.
（試験例６）ポリリジン-綿布担体の抗菌性
ポリリジン-綿布担体の抗菌性は、次の方法で測定した。すなわち、上記の方法で製造したポリリジン-綿布担体を20 mlの滅菌水に浸漬し、これらの浸漬液に大腸菌、黄色ブドウ球菌、あるいは酵母を、細胞数が103〜104 cfu/ml になる様に接種し、25℃、120 rpmで振盪した。１時間後と１２時間後との２度、培養液を0.1 ml採取し、それぞれをNB寒天培地上に接種し、30℃で24時間培養して寒天培地上に形成されたコロニーを計測してcfuとした。表４に示すように、ポリリジン-綿布担体は強い抗菌性を示し、この条件で何れの微生物も完全に殺滅されることが明らかになった。(Test Example 6) Antibacterial property of polylysine-cotton cloth carrier The antibacterial property of polylysine-cotton cloth carrier was measured by the following method. That is, the polylysine-cotton cloth carrier produced by the above method is immersed in 20 ml of sterile water, and Escherichia coli, Staphylococcus aureus, or yeast is added to these immersion solutions to bring the cell number to 10 3 to 10 4 cfu / ml. And shaken at 25 ° C. and 120 rpm. Collect 0.1 ml of the culture solution twice, 1 hour and 12 hours later, inoculate each on the NB agar medium, inoculate at 30 ° C. for 24 hours, and measure the colonies formed on the agar medium. It was cfu. As shown in Table 4, the polylysine-cotton cloth carrier showed strong antibacterial properties, and it was revealed that all microorganisms were completely killed under these conditions.
実施例６の方法に準じて、実施例 6の綿布に替えて種々の水不溶性担体へのポリリジンの結合を調べたところ、PEI の場合と同様に、本願発明で開発した条件では、種々の水不溶性担体にポリリジンを結合させ得ることが明らかになった。表５に、本発明による方法での種々の水不溶性担体へのポリリジンの結合を示した。表５は、種々の水不溶性担体１ｇに結合させたポリリジン量を、オレンジ7溶液による発色度で測定し、１ｇの木綿布に結合させたPEI量を１００として種々の水不溶性担体１ｇに結合させたポリリジン量を比較したものである。
キトサン（キミカキトサン、キミカ社製）1 gを、80 ml の蒸留水に懸濁し、撹拌しながら、これに炭酸ガスを通気してキトサンを溶解させて、キサトン溶液（ｐＨ3.0）に綿布（精練品、5-cm 角）を浸漬して室温で一夜静置した。この綿布を引き出して水洗で余剰のキトサンを除去した後、60℃で3時間乾燥した。この操作により、綿布にキトサンが吸着していることは、実施例1に述べた、色素染色法で確認した。オレンジ７溶液で染色した、キトサン-綿布担体と対照であるキトサンを結合させていない綿布の写真を図８に示す。
（試験例７）キトサン-綿布担体の抗菌活性
このキトサン-綿布担体の大腸菌に対する抗菌活性を試験例6と同様の手法で測定して結果を表６に示す。これらの結果からキトサン-綿布担体は静菌活性を有するものと考えた。(Test Example 7) Antibacterial activity of chitosan-cotton cloth carrier The antibacterial activity of this chitosan-cotton cloth carrier against Escherichia coli was measured by the same method as in Test Example 6, and the results are shown in Table 6. From these results, it was considered that the chitosan-cotton cloth carrier had bacteriostatic activity.
次に微生物の吸着能に関して検討した。すなわち、
大腸菌、黄色ぶどう球菌、またはカンジダ・アルビカンスを104 cfu/mlとなるようにNB培地に接種して２５℃、１２０ｒｐｍにて一夜振盪培養した。これらの培養液を滅菌水で２０倍に希釈し、その２０ｍｌを１００ｍｌ容の三角フラスコに採取し、上記の方法で作製したキトサン-綿布担体（５ｃｍ×５ｃｍ）２枚を添加し、２５℃、１２０ｒｐｍにて３０分間振盪した。これらの液と、キトサン-綿布担体無添加の希釈培養液の650 nmの吸光度を測定、比較して微生物菌体の吸着能を測定した。
Escherichia coli, Staphylococcus aureus, or Candida albicans was inoculated into NB medium at 10 4 cfu / ml and cultured with shaking at 25 ° C. and 120 rpm overnight. These cultures were diluted 20-fold with sterilized water, 20 ml of the culture solution was collected in a 100 ml Erlenmeyer flask, and two chitosan-cotton cloth carriers (5 cm × 5 cm) prepared by the above method were added to the mixture at 25 ° C. The mixture was shaken at 120 rpm for 30 minutes. The absorbance at 650 nm of these solutions and the diluted culture solution without the addition of chitosan-cotton carrier was measured and compared to measure the adsorption capacity of microbial cells.
その結果を表８に示すが、キトサン-綿布担体を添加して振盪したことによって、菌液の吸光度は著しく減少した。これらの結果から、キトサン-綿布担体は大腸菌、黄色ぶどう球菌、およびカンジダ・アルビカンスなど、細菌や酵母の菌体を吸着し除去する機能を有することが明らかになった。 The results are shown in Table 8. The absorbance of the bacterial solution was significantly reduced by adding the chitosan-cotton cloth carrier and shaking. From these results, it was clarified that the chitosan-cotton cloth carrier has a function of adsorbing and removing bacterial cells such as Escherichia coli, Staphylococcus aureus, and Candida albicans.
精練した綿布（使用綿糸番手：20番手[たて、よこ共]）、織物タイプ：平織り、サンプルの大きさ：幅8cm×長さ8cm、質量：0.68 g を実施例 １に示した方法で処理してPEI-担体を製造した（PEI液中のPEI濃度1wt％）。このPEI-担体を0.2重量％のポリリジン（三栄源株式会社製、サンキーパー381、pＨ9.5）水溶液に浸漬して30分間室温に放置した後、よく絞って余剰のポリリジン液を除去し、水洗、室温で風乾してポリリジン・PEI-綿布複合体（以下、ポリリジン・PEI-綿布複合体）を製造した。
A scoured cotton cloth (cotton thread count used: 20 counts [vertical and horizontal]), woven fabric type: plain weave, sample size: width 8 cm x length 8 cm, mass: 0.68 g was processed by the method shown in Example 1. To produce a PEI-carrier (PEI concentration in PEI solution 1 wt%). This PEI-carrier is immersed in a 0.2 wt% polylysine (Saneigen Co., Ltd., Sunkeeper 381, pH 9.5) aqueous solution and left at room temperature for 30 minutes, then squeezed well to remove excess polylysine solution and washed with water. , Air-dried at room temperature to produce a polylysine-PEI-cotton cloth composite (hereinafter, polylysine-PEI-cotton cloth composite).
（試験例９）ポリリジン・PEI-綿布複合体の抗菌活性
このポリリジン・PEI-綿布複合体（１cm2)を20 mlの殺菌生理食塩水に浸漬して抗菌活性を測定した。すなわち、この浸漬液に大腸菌、黄色ブドウ球菌あるいは酵母を、細胞数が104〜105 cfu/ml になる様に接種し、25℃、120 rpmで1時間振盪培養した。この培養液を0.1 ml採取し、NB寒天培地上に接種し、30℃で24時間培養して寒天培地上に形成されたコロニーを計数してcfuとした。また、25℃、120 rpmで1時間振盪した培養液を一夜培養した後（1時間の振盪培養を含む）、650nm における濁度を測定して増殖度とした。その結果を表９に示すが、ポリリジン・PEI-綿布複合体を含む水中で３株の微生物菌体は完全に死滅した除菌状態を実現したことが明確になり、ポリリジン・PEI-複合体が抗菌材として有効であることが立証された。(Test Example 9) Antibacterial activity of polylysine-PEI-cotton cloth complex This polylysine-PEI-cotton cloth complex (1 cm 2 ) was immersed in 20 ml of bactericidal physiological saline to measure the antibacterial activity. That is, E. coli this immersion liquid, Staphylococcus aureus or yeast, cell number was inoculated so as to become 10 4 ~10 5 cfu / ml, 25 ℃, and 1 hour shaking culture at 120 rpm. 0.1 ml of this culture solution was collected, inoculated on an NB agar medium, cultured at 30 ° C. for 24 hours, and the colonies formed on the agar medium were counted to obtain cfu. In addition, after culturing the culture solution shaken at 25 ° C. and 120 rpm for 1 hour overnight (including shaking culture for 1 hour), the turbidity at 650 nm was measured to determine the degree of proliferation. The results are shown in Table 9. It was clarified that the three strains of microbial cells in water containing the polylysine / PEI-cotton cloth complex were completely sterilized, and the polylysine / PEI-complex was sterilized. It has been proven to be effective as an antibacterial material.
（試験例１０）ポリリジン・PEI-綿布複合体の安定性の評価（反復使用）
100-ml容の三角フラスコに蒸留水20mlを入れ、加圧滅菌した。この滅菌水に、予め75％エタノールで滅菌した300mgのポリリジン・PEI-綿布複合体（以下、検体と呼称する）を浸漬し、25℃、120rpmで振盪して、洗浄した（反復回数９回）。各回において、下記の方法で液中の抗菌活性を測定した。反復回数９回で、抗菌活性が検出されなくなった。(Test Example 10) Evaluation of stability of polylysine / PEI-cotton cloth complex (repeated use)
20 ml of distilled water was placed in a 100-ml Erlenmeyer flask and sterilized under pressure. 300 mg of polylysine-PEI-cotton cloth complex (hereinafter referred to as a sample) sterilized with 75% ethanol in advance was immersed in this sterilized water, shaken at 25 ° C. and 120 rpm, and washed (repeated times 9 times). .. At each time, the antibacterial activity in the liquid was measured by the following method. After 9 repetitions, antibacterial activity was no longer detected.
各回の液中の抗菌活性の測定は、大腸菌（E.coli IFO 3301）を含む、表９に示す３種の被検体として用いた。被検体を細胞数が103〜104 cfu/ml になる様にNB培地に接種し、25℃で１夜振盪培養し、培養液を生理食塩水で1,000倍に希釈し、上記の洗浄液10ml に0.1mlを接種して25℃、120rpmで振盪培養した後、NB寒天培地上で培養してcfuを計測した。The antibacterial activity in the liquid was measured each time, and it was used as three kinds of subjects shown in Table 9 including E. coli (E. coli IFO 3301). Inoculate the subject into NB medium so that the number of cells becomes 10 3 to 10 4 cfu / ml, incubate with shaking at 25 ° C overnight, dilute the culture solution 1,000-fold with physiological saline, and 10 ml of the above washing solution. Was inoculated with 0.1 ml and cultured with shaking at 25 ° C. and 120 rpm, and then cultured on NB agar medium to measure cfu.
これによって、反復回数９回において抗菌活性の完全な消失を確認した上で、ポリリジン・PEI-綿布複合体の抗菌活性を計測したものである。
すなわち、上記の検体を20mlの滅菌水に浸漬して、これに被検菌を103〜104ヶ/mlになる様に植菌して、25℃、120rpmで1時間振盪し、NB寒天培地上でcfuを測定して抗菌活性を評価した。As a result, the antibacterial activity of the polylysine-PEI-cotton cloth complex was measured after confirming the complete disappearance of the antibacterial activity after 9 repetitions.
That is, the above sample is immersed in 20 ml of sterilized water, inoculated with the test bacteria to 10 3 to 10 4 cells / ml, shaken at 25 ° C. and 120 rpm for 1 hour, and NB agar. The antibacterial activity was evaluated by measuring cfu on the medium.
この測定の結果を表１０に示すが、この条件では８回まで、実用的には9回まで反復使用可能であるとする結果を得た。 The results of this measurement are shown in Table 10, and it was obtained that the measurement can be repeated up to 8 times under these conditions and up to 9 times in practice.
また、試験例１０で示した様に、本発明に係る微生物菌体吸着媒は、反復して利用可能な従来に例を見ない新規な実用性の高い抗菌資材である。 Further, as shown in Test Example 10, the microbial cell adsorption medium according to the present invention is a novel and highly practical antibacterial material that can be repeatedly used and is unprecedented.
Claims (3)
前記水不溶性担体はセルロース製のキッチンペーパーであり、
前記ポリエチレンイミンと前記水不溶性担体との結合及び前記ポリリジンと前記水不溶性担体との結合は、それぞれ化学結合による担持であり、
前記ポリエチレンイミンの30wt％水溶液を炭酸水でpH 7.5に調整し、pH 7.5に調整された前記水溶液中のポリエチレンイミン濃度を0.5wt％になるように蒸留水で希釈し、0.5wt％ポリエチレンイミン水溶液に前記水不溶性担体を浸漬して、前記水不溶性担体に前記ポリエチレンイミンを結合させ、1重量％ポリリジン水溶液（pH 9.5）に前記ポリエチレンイミンを結合させた前記水不溶性担体を浸漬して、前記ポリエチレンイミンを結合させた前記水不溶性担体に前記ポリリジンを結合させることを特徴とする微生物菌体吸着媒の製造方法。 By binding polylysine and polyethyleneimine having a molecular weight of 70,000 to a water-insoluble carrier, a conjugate that adsorbs microbial cells is obtained.
The water-insoluble carrier is a paper towel made cellulose,
The bond between the polyethyleneimine and the water-insoluble carrier and the bond between the polylysine and the water-insoluble carrier are supported by chemical bonds, respectively.
The 30 wt% aqueous solution of polyethyleneimine was adjusted to pH 7.5 with carbonated water, and the concentration of polyethyleneimine in the aqueous solution adjusted to pH 7.5 was diluted with distilled water so as to be 0.5 wt%, and the 0.5 wt% polyethyleneimine aqueous solution was diluted. the water-insoluble carrier by immersing, by coupling the polyethylene imine to the water-insoluble carrier, by immersing the 1 wt% polylysine aqueous solution of the water-insoluble carrier bound with the polyethyleneimine (pH 9.5) in the polyethylene method for producing a microbial cell adsorbent, wherein the benzalkonium bound the polylysine to the water-insoluble carrier bound with imine.
前記水不溶性担体はセルロース製のキッチンペーパーであり、
前記ポリエチレンイミンと前記水不溶性担体との結合は、化学結合による担持であり、
前記ポリエチレンイミンの30wt％水溶液を炭酸水でpH 7.5に調整し、pH 7.5に調整された前記水溶液中のポリエチレンイミン濃度を0.5wt％になるように蒸留水で希釈し、0.5wt％ポリエチレンイミン水溶液に前記水不溶性担体を浸漬して、前記水不溶性担体に前記ポリエチレンイミンを結合させ、5重量％の銀-ヒスチジン錯体水溶液に前記ポリエチレンイミンを結合させた前記水不溶性担体を浸漬して、前記ポリエチレンイミンを結合させた前記水不溶性担体に前記銀-ヒスチジン錯体を結合させることを特徴とする微生物菌体吸着媒の製造方法。 By binding a silver-histidine complex and polyethyleneimine having a molecular weight of 70,000 to a water-insoluble carrier, a conjugate that adsorbs microbial cells is obtained.
The water-insoluble carrier is a cellulose kitchen paper.
The bond between the polyethyleneimine and the water-insoluble carrier is supported by a chemical bond.
The 30 wt% aqueous solution of polyethyleneimine was adjusted to pH 7.5 with carbonated water, and the concentration of polyethyleneimine in the aqueous solution adjusted to pH 7.5 was diluted with distilled water so as to be 0.5 wt%, and the 0.5 wt% polyethyleneimine aqueous solution was diluted. the water-insoluble carrier by immersing the polyethylene imine is bound, 5 wt% of silver to the water-insoluble carrier - and immersing the water-insoluble carrier bound with the polyethyleneimine histidine complex solution, the polyethylene the silver to the water-insoluble carrier bound with imine - method for producing microbial cell adsorbent, wherein the benzalkonium bound histidine complex.
前記水不溶性担体は綿布であり、The water-insoluble carrier is a cotton cloth.
前記ポリエチレンイミンと前記水不溶性担体との結合及び前記ポリリジンと前記水不溶性担体との結合は、それぞれ化学結合による担持であり、The bond between the polyethyleneimine and the water-insoluble carrier and the bond between the polylysine and the water-insoluble carrier are supported by chemical bonds, respectively.
前記ポリエチレンイミンの30wt％水溶液を炭酸水でpH 7.5に調整し、pH 7.5に調整された前記水溶液中のポリエチレンイミン濃度を1wt％になるように蒸留水で希釈し、1wt％ポリエチレンイミン水溶液に前記水不溶性担体を浸漬して、前記水不溶性担体に前記ポリエチレンイミンを結合させ、0.2重量％のポリリジン（pH 9.5）水溶液に前記ポリエチレンイミンを結合させた前記水不溶性担体を浸漬して、前記ポリエチレンイミンを結合させた前記水不溶性担体に前記ポリリジンを結合させることを特徴とする微生物菌体吸着媒の製造方法。The 30 wt% aqueous solution of polyethyleneimine was adjusted to pH 7.5 with carbonated water, the concentration of polyethyleneimine in the aqueous solution adjusted to pH 7.5 was diluted with distilled water so as to be 1 wt%, and the above was added to the 1 wt% polyethyleneimine aqueous solution. The water-insoluble carrier is immersed, the polyethyleneimine is bound to the water-insoluble carrier, and the water-insoluble carrier to which the polyethyleneimine is bound is immersed in a 0.2 wt% polylysine (pH 9.5) aqueous solution, and the polyethyleneimine is immersed. A method for producing a microbial cell adsorption medium, which comprises binding the polylysine to the water-insoluble carrier to which the polylysine is bound.
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