JP2011050320A - Method for preserving microorganism, member for preserving microorganism, and method for producing porous sheet-formed material for preserving microorganism - Google Patents

Method for preserving microorganism, member for preserving microorganism, and method for producing porous sheet-formed material for preserving microorganism Download PDF

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JP2011050320A
JP2011050320A JP2009202613A JP2009202613A JP2011050320A JP 2011050320 A JP2011050320 A JP 2011050320A JP 2009202613 A JP2009202613 A JP 2009202613A JP 2009202613 A JP2009202613 A JP 2009202613A JP 2011050320 A JP2011050320 A JP 2011050320A
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sheet
microorganisms
resin
multilayer structure
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JP5667352B2 (en
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Takuzo Imaizumi
卓三 今泉
Naomi Goto
直美 後藤
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Futamura Chemical Co Ltd
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14Scaffolds; Matrices
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00Means for pre-treatment of biological substances
    • C12M45/22Means for packing or storing viable microorganisms
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00Constructional details, e.g. recesses, hinges
    • C12M23/20Material Coatings

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for preserving microorganisms, enabling the preservation of microorganisms inexpensively and simply, and also a quick and large amount of culture by using a communicating porous material, a member for preserving microorganisms by attaching the microorganisms with the same material, and further a method for producing the porous sheet-formed material for preserving microorganisms. <P>SOLUTION: This porous sheet-formed material for preserving microorganism is provided by forming the communicated porous material 13 of communicating cavity parts 12 with each other within a resin substrate material, forming a porous multiple-layered structural material by laminating the porous sheet-formed material 10A with opening cavity parts on the surface of the resin substrate material, and immersing the same structural material in the suspension of the microorganisms to attach the microorganisms in the cavity parts of the porous multiple-layered structural material, and is produced by mixing granular materials to be dissolved, capable of being dissolved afterwards in a resin material, molding as a predetermined sheet-formed material, and then dissolving the granular materials to be dissolved, to form the porous material having cavity parts therein. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

本発明は、微生物の保存方法及び微生物保存部材、並びに微生物保存用多孔質シート状物の製造方法に関し、特に多孔質体の内部に備わる空洞部を利用して微生物を保持することに特徴を備える。   The present invention relates to a method for preserving microorganisms, a microorganism storage member, and a method for producing a porous sheet-like material for preserving microorganisms, and is particularly characterized in that microorganisms are retained using a cavity provided in a porous body. .

微生物を保存する方法として、主に凍結法、継代培養法、凍結乾燥法・乾燥法等が挙げられる。安定的に微生物を保存する場合は凍結法が最良である。ただし、液体窒素をはじめ、専用の冷凍設備、保存用の薬品等を必要とし、保存自体に要する経費もかさむ。また、低温耐性の低い菌種には向かない場合もある。   Methods for preserving microorganisms mainly include freezing methods, subculture methods, freeze-drying methods and drying methods. The freezing method is the best when storing microorganisms stably. However, it requires liquid nitrogen, dedicated refrigeration equipment, storage chemicals, etc., and the cost required for storage itself increases. Moreover, it may not be suitable for bacterial species with low low temperature tolerance.

継代培養法は生存もしくは休眠状態の微生物をそのまま新しい培地に移すため、凍結や乾燥に弱い菌種の他、ほぼ全種類の菌種の保存に適する。しかしながら、継代作業の多くは人手に依存する。移植する菌種が増えるほど手間を要する。また、継代を繰り返す内に微生物の形質転換が生じ保存中の菌種の性質が変化して、保存微生物の正確な形質を再現できなくなるおそれもある。凍結乾燥法は、微生物を休止状態にして保存できるため、比較的安価かつ簡便な保存方法として広く利用されている。多種類の微生物を多量に保存する用途に向く。乾燥法についても同様である。ただし、乾燥耐性の低い菌種には向かない場合がある。   The subculture method transfers living or dormant microorganisms as they are to a new medium, and is therefore suitable for the preservation of almost all types of bacterial species in addition to those that are vulnerable to freezing and drying. However, much of the subculture work depends on manpower. The more strains to be transplanted, the more time is required. In addition, transformation of microorganisms may occur during repeated passages and the nature of the bacterial species being preserved may change, making it impossible to reproduce the exact traits of the preserved microorganisms. The freeze-drying method is widely used as a relatively inexpensive and simple storage method because microorganisms can be stored in a resting state. Suitable for storing a large amount of many kinds of microorganisms. The same applies to the drying method. However, it may not be suitable for bacterial species with low drought tolerance.

比較的簡単に微生物を保存するため、次のような器具、装置等が提案されている。例えば、ゲル化した吸水性樹脂粒子の表面に微生物が保持されると、吸水性樹脂粒子は吸水によりゲル化して膨潤し互いに密着状態となる。微生物はゲル化して膨潤した吸水性樹脂粒子により取り囲まれ、空気中の酸素から遮断されると共に乾燥から保護される微生物保存剤が提案されている(特許文献1参照)。マルチウェルプレートにメンブレンシートを介在させ、ここに微生物等を含んだ培養液を滴下し、メンブレンシートの両面をマルチウェルプレートに貼り付けたフィルムで同シートを密封し保護可能とする容器が提案されている(特許文献2参照)。粘着テープの粘着面を外側とするように巻き付けたテープ状物により微生物を付着、採取し、その付着部分に培養シート、保護シートを重ねて手軽に保存用サンプルを作成する装置も提案されている(特許文献3参照)。   In order to store microorganisms relatively easily, the following instruments and devices have been proposed. For example, when microorganisms are held on the surface of the gelled water-absorbent resin particles, the water-absorbent resin particles are gelled by water absorption and swell and become in close contact with each other. Microorganism preservatives have been proposed in which microorganisms are surrounded by gelled and swollen water-absorbent resin particles, which are shielded from oxygen in the air and protected from drying (see Patent Document 1). A container has been proposed in which a membrane sheet is interposed in a multi-well plate, a culture solution containing microorganisms, etc. is dropped here, and the sheet is sealed and protected with a film in which both sides of the membrane sheet are attached to the multi-well plate. (See Patent Document 2). There has also been proposed an apparatus for easily preparing a sample for storage by attaching and collecting microorganisms with a tape-like material wound so that the adhesive surface of the adhesive tape is on the outside, and stacking a culture sheet and a protective sheet on the attached part. (See Patent Document 3).

特許文献1の微生物保存剤にあっては、その性質上乾燥は厳禁である。特許文献3の装置は採取が主目的であるため、保存性能は必ずしも十分ではない。特許文献2の微生物保存に供する器具は多品種少量の微生物の保存に適しており、取り扱いも容易である。その一方、特定(少品種)の微生物を大量かつ安価に保存することができる方法、器材、所定の保存を終えて再び微生物を速やかにしかも大量に培養することが可能な方法、器材等について、必ずしも満足できる手法、装置等は見出されてはいない。   In the microorganism preservative of Patent Document 1, drying is strictly prohibited due to its properties. Since the main object of the device of Patent Document 3 is collection, the storage performance is not always sufficient. The instrument used for preserving microorganisms of Patent Document 2 is suitable for preserving a small variety of microorganisms and is easy to handle. On the other hand, for a method (equipment) capable of preserving a large amount of specific (small varieties) microorganisms at low cost, equipment, a method capable of cultivating microorganisms quickly and in large quantities again after completing a predetermined preservation, equipment, etc. No satisfactory method or apparatus has been found.

このようなことから、特定種類の微生物に関して安価かつ簡便に保存が可能であること、保存後の培養に際し、迅速かつ大量の菌数を容易に得ることの両面を併せ持つ方法等が探求されてきた。   For this reason, a method has been sought that can be both inexpensively and easily stored for specific types of microorganisms, and that it is easy to obtain a large number of bacteria quickly and easily during culturing after storage. .

特開平7−111887号公報Japanese Patent Laid-Open No. 7-111887 特開2007−325536号公報JP 2007-325536 A 特開平5−292997号公報Japanese Patent Laid-Open No. 5-292997

その後、発明者は以前より開発を進めていた樹脂製の連通多孔質体に着目し、その微生物保存用途への応用について鋭意検討を重ねた。その結果、発明者が提案した連通多孔質体は微生物保存においても有効であることを見出した。   After that, the inventor paid attention to the resin-made continuous porous body, which has been developed for a long time, and conducted extensive studies on its application to the preservation of microorganisms. As a result, the inventors have found that the continuous porous material proposed by the inventor is also effective in microbial preservation.

本発明は、前記の点に鑑みなされたものであり、連通多孔質体を用いることにより微生物の安価かつ簡便に保存すると共に迅速かつ大量の培養も可能とする微生物の保存方法と、これに微生物を付着した微生物保存部材、さらに微生物保存用多孔質シート状物の製造方法を提供する。   The present invention has been made in view of the above-described points, and a microorganism storage method capable of preserving microorganisms inexpensively and easily while using a continuous porous body, and enabling rapid and large-scale cultivation, and microorganisms And a method for producing a porous sheet for preserving microorganisms.

すなわち、請求項1の発明は、樹脂基材の内部に空洞部同士が連通した連通多孔質体を形成すると共に該樹脂基材の表面に前記空洞部が開口している多孔質シート状物を多層化して多孔質多層構造体を形成し、前記多孔質多層構造体を微生物の懸濁液中に浸漬して前記多孔質多層構造体の前記空洞部内に微生物を付着させることを特徴とする微生物の保存方法に係る。   That is, the invention of claim 1 is a porous sheet-like material in which a continuous porous body in which cavities communicate with each other is formed inside a resin base material and the cavities are open on the surface of the resin base material. A microorganism characterized by forming a porous multilayer structure by multilayering, and immersing the porous multilayer structure in a suspension of microorganisms to attach the microorganism to the cavity of the porous multilayer structure Related to the storage method.

請求項2の発明は、前記多孔質多層構造体を微生物の懸濁液中に浸漬した後に該多孔質多層構造体を乾燥して乾燥多孔質多層構造体とする請求項1に記載の微生物の保存方法に係る。   According to a second aspect of the present invention, the porous multilayer structure is dipped in a suspension of microorganisms, and then the porous multilayer structure is dried to obtain a dry porous multilayer structure. It relates to the storage method.

請求項3の発明は、前記乾燥多孔質多層構造体の表面をろう、パラフィン、またはホットメルト樹脂のいずれかにより被覆する請求項2に記載の微生物の保存方法に係る。   The invention according to claim 3 relates to the method for preserving microorganisms according to claim 2, wherein the surface of the dry porous multilayer structure is coated with either wax, paraffin or hot melt resin.

請求項4の発明は、前記多孔質多層構造体を微生物の懸濁液中に浸漬する前に、前記多孔質多層構造体を保護剤溶液中に浸漬して該保護剤溶液を乾燥する請求項1ないし3のいずれか1項に記載の微生物の保存方法に係る。   According to a fourth aspect of the present invention, before the porous multilayer structure is immersed in a suspension of microorganisms, the porous multilayer structure is immersed in a protective agent solution to dry the protective agent solution. It concerns on the preservation | save method of the microorganisms of any one of 1-3.

請求項5の発明は、前記多孔質多層構造体が巻き取りロール形状である請求項1ないし4のいずれか1項に記載の微生物の保存方法に係る。   The invention of claim 5 relates to the method for preserving microorganisms according to any one of claims 1 to 4, wherein the porous multilayer structure is in the form of a take-up roll.

請求項6の発明は、前記多孔質シート状物の内部に中空管路が形成されている請求項1ないし5のいずれか1項に記載の微生物の保存方法に係る。   A sixth aspect of the present invention relates to the method for preserving microorganisms according to any one of the first to fifth aspects, wherein a hollow pipe line is formed inside the porous sheet-like material.

請求項7の発明は、前記樹脂基材が生分解性樹脂よりなる請求項1ないし6のいずれか1項に記載の微生物の保存方法に係る。   The invention according to claim 7 relates to the method for preserving microorganisms according to any one of claims 1 to 6, wherein the resin substrate is made of a biodegradable resin.

請求項8の発明は、請求項1ないし7のいずれか1項に記載の微生物の保存方法により、前記多孔質多層構造体の多孔質内に微生物を付着させたことを特徴とする微生物保存部材に係る。   The invention of claim 8 is a microorganism storage member characterized in that microorganisms are adhered to the inside of the porous multilayer structure by the microorganism storage method according to any one of claims 1 to 7. Concerning.

請求項9の発明は、樹脂基材の内部に空洞部同士が連通した連通多孔質体を形成すると共に該樹脂基材の表面に前記空洞部が開口している多孔質シート状物を多層化して多孔質多層構造体を形成し、前記多孔質多層構造体を微生物の懸濁液中に浸漬して前記多孔質多層構造体の前記空洞部内に微生物を付着させるための微生物保存用多孔質シート状物の製造方法であって、事後的に溶解可能な粒状被溶解物を樹脂材料に混入して所定のシート状物に成形し、この後に前記粒状被溶解物を溶解することにより前記シート状物の内部に空洞部を有する多孔質体を形成することを特徴とする微生物保存用多孔質シート状物の製造方法に係る。   The invention of claim 9 is to form a continuous porous body in which cavities communicate with each other inside a resin base material, and multilayer a porous sheet-like material having the cavities open on the surface of the resin base material. Forming a porous multilayer structure, and immersing the porous multilayer structure in a suspension of microorganisms to attach microorganisms to the hollow portion of the porous multilayer structure. A method for producing a sheet, wherein a granular material to be dissolved afterwards is mixed into a resin material and formed into a predetermined sheet material, and then the granular material is dissolved to dissolve the granular material. The present invention relates to a method for producing a porous sheet-like material for preserving microorganisms, wherein a porous material having a cavity is formed inside the material.

請求項10の発明は、前記シート状物に占める前記空洞部の体積割合を、少なくとも50%以上とする請求項9に記載の微生物保存用多孔質シート状物の製造方法に係る。   The invention of claim 10 relates to the method for producing a porous sheet-like material for preserving microorganisms according to claim 9, wherein a volume ratio of the hollow portion in the sheet-like material is at least 50% or more.

請求項11の発明は、前記粒状被溶解物が、水、酵素、または有機溶剤のいずれかによって除去される請求項9又は10に記載の微生物保存用多孔質シート状物の製造方法に係る。   Invention of Claim 11 concerns on the manufacturing method of the porous sheet-like material for microorganism preservation | save of Claim 9 or 10 with which the said granular to-be-dissolved material is removed with either water, an enzyme, or an organic solvent.

請求項12の発明は、前記粒状被溶解物を前記樹脂材料に混入して所定のシート状物を成形するに際し、事後的に溶解可能な管路予定被溶解物を混入し、所定のシート状物に成形した後に前記粒状被溶解物及び前記管路予定被溶解物を溶解して、前記シート状物の内部に空洞部と共に中空管路を有する多孔質体を形成する請求項9ないし11のいずれか1項に記載の微生物保存用多孔質シート状物の製造方法に係る。   In the invention of claim 12, when forming the predetermined sheet-like material by mixing the granular material to be mixed with the resin material, a predetermined pipe-shaped material to be dissolved afterwards is mixed, 12. A porous body having a hollow pipe and a hollow portion is formed inside the sheet-like material by melting the granular material to be melted and the pipeline to-be-melted material after being formed into a product. The method for producing a porous sheet-like material for preserving microorganisms according to any one of the above.

請求項13の発明は、前記粒状被溶解物を前記樹脂材料に混入して所定のシート状物を成形し、前記シート状物同士を貼り合わせる際の接合面に事後的に溶解可能な管路予定被溶解物を載置して前記シート状物同士からなる接合物を成形した後に前記粒状被溶解物及び前記管路予定被溶解物を溶解して、前記接合物の内部に空洞部と共に中空管路を有する多孔質体を形成する請求項9ないし11のいずれか1項に記載の微生物保存用多孔質シート状物の製造方法に係る。   In a thirteenth aspect of the present invention, the granular material to be dissolved is mixed into the resin material to form a predetermined sheet-like material, and a conduit that can be subsequently dissolved on the joint surface when the sheet-like materials are bonded together. After the object to be melted is placed and a joined product composed of the sheet-like materials is formed, the material to be melted in granular form and the object to be melted in the pipeline are melted, and the inside of the joined object together with the hollow portion It concerns on the manufacturing method of the porous sheet-like material for microorganism preservation | save of any one of Claim 9 thru | or 11 which forms the porous body which has an empty pipe line.

請求項14の発明は、前記管路予定被溶解物が、水、酵素、または有機溶剤のいずれかによって除去される請求項12又は13に記載の微生物保存用多孔質シート状物の製造方法に係る。   The invention of claim 14 is the method for producing a porous sheet-like material for preserving microorganisms according to claim 12 or 13, wherein the pre-dissolved material to be ducted is removed by any one of water, an enzyme, and an organic solvent. Related.

請求項15の発明は、前記樹脂材料が生分解性樹脂よりなる請求項9ないし14のいずれか1項に記載の微生物保存用多孔質シート状物の製造方法に係る。   The invention of claim 15 relates to the method for producing a porous sheet for preserving microorganisms according to any one of claims 9 to 14, wherein the resin material comprises a biodegradable resin.

請求項1の発明に係る微生物の保存方法によると、樹脂基材の内部に空洞部同士が連通した連通多孔質体を形成すると共に該樹脂基材の表面に前記空洞部が開口している多孔質シート状物を多層化して多孔質多層構造体を形成し、前記多孔質多層構造体を微生物の懸濁液中に浸漬して前記多孔質多層構造体の前記空洞部内に微生物を付着させるため、微生物の安価かつ簡便な保存を可能とし、しかも、保存後に迅速かつ大量の培養も可能とすることができる。   According to the method for preserving microorganisms according to the first aspect of the present invention, a porous body is formed in which a communicating porous body in which cavities communicate with each other is formed inside a resin substrate, and the cavity is open on the surface of the resin substrate. Forming a porous multilayer structure by multilayering a porous sheet, and immersing the porous multilayer structure in a suspension of microorganisms to attach microorganisms to the cavity of the porous multilayer structure In addition, microorganisms can be stored inexpensively and easily, and rapid and large-scale culture can be performed after storage.

請求項2の発明に係る微生物の保存方法によると、請求項1の発明において、前記多孔質多層構造体を微生物の懸濁液中に浸漬した後に該多孔質多層構造体を乾燥して乾燥多孔質多層構造体とするため、乾燥により水分含量を減らすことができ、保存中の微生物の懸濁液の腐敗を避けることができる。また、保存時の汚染要因を除去することができる。   According to the method for preserving a microorganism according to the invention of claim 2, in the invention of claim 1, after the porous multilayer structure is immersed in the suspension of the microorganism, the porous multilayer structure is dried and dried to be porous. In order to obtain a multi-layered structure, the moisture content can be reduced by drying, and the microbial suspension during storage can be prevented from decaying. Moreover, the contamination factor at the time of storage can be removed.

請求項3の発明に係る微生物の保存方法によると、請求項2の発明において、前記乾燥多孔質多層構造体の表面をろう、パラフィン、またはホットメルト樹脂のいずれかにより被覆するため、乾燥多孔質多層構造体の内外の酸素の流通を抑えることができ、保存期間を延ばすことができる。   According to the method for preserving microorganisms according to the invention of claim 3, in the invention of claim 2, since the surface of the dry porous multilayer structure is coated with either wax, paraffin, or hot melt resin, dry porous The circulation of oxygen inside and outside the multilayer structure can be suppressed, and the storage period can be extended.

請求項4の発明に係る微生物の保存方法によると、請求項1ないし3のいずれか1項に記載の発明において、前記多孔質多層構造体を微生物の懸濁液中に浸漬する前に、前記多孔質多層構造体を保護剤溶液中に浸漬して該保護剤溶液を乾燥するため、保護剤により微生物の極度の乾燥が防止され、適度な水分及び栄養の維持、浸透圧の変化の緩和に都合がよい。   According to the method for preserving microorganisms according to the invention of claim 4, in the invention according to any one of claims 1 to 3, before the porous multilayer structure is immersed in a suspension of microorganisms, Since the porous multilayer structure is immersed in the protective agent solution to dry the protective agent solution, the protective agent prevents extreme drying of the microorganisms, maintaining appropriate moisture and nutrients, and mitigating changes in osmotic pressure. convenient.

請求項5の発明に係る微生物の保存方法によると、請求項1ないし4のいずれか1項に記載の発明において、前記多孔質多層構造体が巻き取りロール形状であるため、多孔質シート状物は巻き取られて容易に多層化でき、しかも十分な巻き取り長さを確保することができ、微生物の保存量を多くすることができる。また、巻き取りロール形状となることにより省スペース化を図ることができ、ロール単位による管理がしやすくなる。   According to the method for preserving microorganisms according to the invention of claim 5, in the invention of any one of claims 1 to 4, the porous multilayer structure is in the form of a take-up roll. Can be wound up and easily multi-layered, and a sufficient winding length can be ensured, and the amount of microorganisms stored can be increased. Moreover, space-saving can be attained by becoming a winding roll shape, and management by a roll unit becomes easy.

請求項6の発明に係る微生物の保存方法によると、請求項1ないし5のいずれか1項に記載の発明において、前記多孔質シート状物の内部に中空管路が形成されているため、多孔質シート状物自体の吸液、浸透性能が向上する。   According to the method for preserving a microorganism according to the invention of claim 6, in the invention according to any one of claims 1 to 5, since a hollow pipe is formed inside the porous sheet-like material, The liquid absorption and permeation performance of the porous sheet is improved.

請求項7の発明に係る微生物の保存方法によると、請求項1ないし6のいずれか1項に記載の発明において、前記樹脂基材が生分解性樹脂よりなるため、使用後に回収の必要性が無くなり、環境への負荷を低減することができる。   According to the method for preserving a microorganism according to the invention of claim 7, in the invention according to any one of claims 1 to 6, the resin base material is made of a biodegradable resin. It can be eliminated and the load on the environment can be reduced.

請求項8の発明に係る微生物保存部材によると、請求項1ないし7のいずれか1項に記載の微生物の保存方法により、前記多孔質多層構造体の多孔質内に微生物を付着させたため、微生物の保存のみに留まらず、その後の培養や使用時の利便性を図ることができる。   According to the microorganism storage member according to the invention of claim 8, the microorganism is attached to the inside of the porous multilayer structure by the microorganism storage method according to any one of claims 1 to 7. In addition to storage, it is possible to achieve convenience during subsequent culture and use.

請求項9の発明に係る微生物保存用多孔質シート状物の製造方法によると、樹脂基材の内部に空洞部同士が連通した連通多孔質体を形成すると共に該樹脂基材の表面に前記空洞部が開口している多孔質シート状物を多層化して多孔質多層構造体を形成し、前記多孔質多層構造体を微生物の懸濁液中に浸漬して前記多孔質多層構造体の前記空洞部内に微生物を付着させるための微生物保存用多孔質シート状物の製造方法であって、事後的に溶解可能な粒状被溶解物を樹脂材料に混入して所定のシート状物に成形し、この後に前記粒状被溶解物を溶解することにより前記シート状物の内部に空洞部を有する多孔質体を形成するため、微生物保存用多孔質シート状物を容易に製造することができる。   According to the method for producing a microorganism-preserving porous sheet-like material according to the invention of claim 9, a continuous porous body in which the cavities communicate with each other is formed inside the resin base material, and the cavity is formed on the surface of the resin base material. Forming a porous multilayer structure by multilayering a porous sheet-like material having an open portion, and immersing the porous multilayer structure in a suspension of microorganisms to form the cavity of the porous multilayer structure A method for producing a microorganism-preserving porous sheet for adhering microorganisms in a part, a granular material to be dissolved afterwards is mixed in a resin material and formed into a predetermined sheet-like material. Since the porous body having a cavity is formed inside the sheet-like material by dissolving the granular material to be dissolved later, the porous sheet-like material for preserving microorganisms can be easily produced.

請求項10の発明に係る微生物保存用多孔質シート状物の製造方法によると、請求項9の発明において、前記シート状物に占める前記空洞部の体積割合を、少なくとも50%以上とするため、空洞部同士が連通した連通多孔質体を容易に形成することができる。   According to the method for producing a porous sheet-like material for preserving microorganisms according to the invention of claim 10, in the invention of claim 9, the volume ratio of the hollow portion in the sheet-like material is at least 50% or more, A communicating porous body in which the hollow portions communicate with each other can be easily formed.

請求項11の発明に係る微生物保存用多孔質シート状物の製造方法によると、請求項9又は10の発明において、前記粒状被溶解物が、水、酵素、または有機溶剤のいずれかによって除去されるため、除去の効率が良く、多孔質シート状物製造の量産性に優れる。   According to the method for producing a porous sheet for preserving microorganisms according to the invention of claim 11, in the invention of claim 9 or 10, the granular material to be dissolved is removed by any of water, an enzyme, or an organic solvent. Therefore, the removal efficiency is good and the mass productivity of the production of the porous sheet material is excellent.

請求項12の発明に係る微生物保存用多孔質シート状物の製造方法によると、請求項9ないし11のいずれか1項に記載の発明において、前記粒状被溶解物を前記樹脂材料に混入して所定のシート状物を成形するに際し、事後的に溶解可能な管路予定被溶解物を混入し、所定のシート状物に成形した後に前記粒状被溶解物及び前記管路予定被溶解物を溶解して、前記シート状物の内部に空洞部と共に中空管路を有する多孔質体を形成するため、中空管路を有する多孔質体の製造工程を簡略化することができる。   According to the method for producing a porous sheet-like material for preserving microorganisms according to the invention of claim 12, in the invention of any one of claims 9 to 11, the granular material to be dissolved is mixed into the resin material. When molding a predetermined sheet-like material, mix the planned melt-dissolved material that can be dissolved afterwards, and dissolve the granular melt and the planned melt-dissolved material after forming into a predetermined sheet-like material. And since the porous body which has a hollow pipe line with a cavity part in the inside of the said sheet-like material is formed, the manufacturing process of the porous body which has a hollow pipe path can be simplified.

請求項13の発明に係る微生物保存用多孔質シート状物の製造方法によると、請求項9ないし11のいずれか1項に記載の発明において、前記粒状被溶解物を前記樹脂材料に混入して所定のシート状物を成形し、前記シート状物同士を貼り合わせる際の接合面に事後的に溶解可能な管路予定被溶解物を載置して前記シート状物同士からなる接合物を成形した後に前記粒状被溶解物及び前記管路予定被溶解物を溶解して、前記接合物の内部に空洞部と共に中空管路を有する多孔質体を形成するため、製造工程を簡略化することができる。また、正確な形状の中空管路を微生物保存用の多孔質シート状物の内部に形成することができる。   According to the method for producing a porous sheet-like material for preserving microorganisms according to the invention of claim 13, in the invention of any one of claims 9 to 11, the granular material to be dissolved is mixed into the resin material. A predetermined sheet-like material is formed, and a predetermined melt-dissolved duct material is placed on the joining surface when the sheet-like materials are bonded to each other to form a joined material composed of the sheet-like materials. In order to form a porous body having a hollow portion and a hollow portion inside the joined product by dissolving the granular material to be melted and the pipeline planned material to be melted, the manufacturing process is simplified. Can do. In addition, it is possible to form a hollow pipe having an accurate shape inside a porous sheet-like material for preserving microorganisms.

請求項14の発明に係る微生物保存用多孔質シート状物の製造方法によると、請求項12又は13の発明において、前記管路予定被溶解物が、水、酵素、または有機溶剤のいずれかによって除去されるため、除去の効率が良く、中空管路を備えた多孔質シート状物製造の量産性に優れる。   According to the method for producing a porous sheet-like material for preserving microorganisms according to the invention of claim 14, in the invention of claim 12 or 13, the to-be-dissolved material for pipelines is either water, an enzyme, or an organic solvent. Since it is removed, the removal efficiency is good, and the mass productivity of the production of a porous sheet-like material having a hollow pipe line is excellent.

請求項15の発明に係る微生物保存用多孔質シート状物の製造方法によると、請求項9ないし14のいずれか1項に記載の発明において、前記樹脂材料が生分解性樹脂よりなるため、使用後に回収の必要性が無くなり、環境への負荷を低減することができる。   According to the method for producing a porous sheet for preserving microorganisms according to the invention of claim 15, in the invention according to any one of claims 9 to 14, the resin material is made of a biodegradable resin. There is no need for collection later, and the burden on the environment can be reduced.

第1実施形態の連通多孔質体の断面模式図である。It is a cross-sectional schematic diagram of the communicating porous body of 1st Embodiment. 第2実施形態の連通多孔質体の断面模式図である。It is a cross-sectional schematic diagram of the communication porous body of 2nd Embodiment. 多孔質多層化構造体の例示図である。It is an illustration figure of a porous multilayered structure. 微生物の保存方法の第1工程図である。It is 1st process drawing of the preservation | save method of microorganisms. 微生物の保存方法の第2工程図である。It is a 2nd process figure of the preservation | save method of microorganisms. 微生物保存部材の断面模式図である。It is a cross-sectional schematic diagram of a microorganism preservation member. 微生物保存部材の使用状態を示す第1の模式図である。It is a 1st schematic diagram which shows the use condition of a microorganisms preservation | save member. 微生物保存部材の使用状態を示す第2の模式図である。It is a 2nd schematic diagram which shows the use condition of a microorganisms preservation | save member. 微生物保存用多孔質シート状物の第1製造方法の概略図である。It is the schematic of the 1st manufacturing method of the porous sheet-like material for microorganisms preservation | save. 微生物保存用多孔質シート状物の第2製造方法の概略図である。It is the schematic of the 2nd manufacturing method of the porous sheet-like material for microorganism preservation. 微生物保存用多孔質シート状物の第3製造方法の概略図である。It is the schematic of the 3rd manufacturing method of the porous sheet-like material for microorganism preservation. 実施例1の微生物保存用多孔質シート状物内部の電子顕微鏡写真である。2 is an electron micrograph of the inside of a porous sheet-like material for preserving microorganisms of Example 1. FIG. 図12の保護剤溶液を含浸した後の電子顕微鏡写真である。It is an electron micrograph after impregnating the protective agent solution of FIG. 図13の酵母を保存した後の電子顕微鏡写真である。It is an electron micrograph after preserve | saving the yeast of FIG. 図14を拡大した電子顕微鏡写真である。It is the electron micrograph which expanded FIG. 図15を拡大した電子顕微鏡写真である。It is the electron micrograph which expanded FIG.

本発明の微生物の保存方法とは、多孔質を内部構造として備えたシート状物(多孔質シート状物)が用意され、これを多層化して多孔質多層構造体に形成される。その後、多孔質シート状物の多孔質に由来する空洞部内に微生物が付着、保持され、当該微生物が保存される方法である。はじめに図1、図2を用い微生物保存部材の一部となる多孔質シート状物10A(第1実施形態),10B(第2実施形態)の構造から説明する。多孔質の構造体は、多層化しやすくする便宜からシート状あるいはフィルム状等の薄肉形状の部材として形成される。図1、図2はシート状物の1枚の断面形状を開示する。   In the method for preserving microorganisms of the present invention, a sheet-like material (porous sheet-like material) having a porous structure as an internal structure is prepared, and this is multilayered to form a porous multilayer structure. Thereafter, the microorganism is attached and held in the cavity derived from the porous structure of the porous sheet-like material, and the microorganism is stored. First, the structure of porous sheet-like materials 10A (first embodiment) and 10B (second embodiment), which are part of the microorganism storage member, will be described with reference to FIGS. The porous structure is formed as a thin-walled member such as a sheet or film for the convenience of facilitating multilayering. 1 and 2 disclose the cross-sectional shape of one sheet.

図1の断面模式図に開示の第1実施形態の多孔質シート状物10Aは、その樹脂基材11の内部に空洞部12同士を連通して有し、連通多孔質体13として形成される(詳細は後出の電子顕微鏡写真参照)。当該多孔質シート状物10Aの表面14には開口部15が形成される。開口部15を通じて連通多孔質体内部の空洞部12に微生物の懸濁液等が浸透する。なお、開口部を拡張するため、連通多孔質体の表面を研磨等により削除することもできる。   A porous sheet 10 </ b> A according to the first embodiment disclosed in the schematic cross-sectional view of FIG. 1 is formed as a continuous porous body 13 having the cavity portions 12 communicating with each other inside the resin base material 11. (For details, see the electron micrograph below). An opening 15 is formed on the surface 14 of the porous sheet-like material 10A. A microorganism suspension or the like permeates into the cavity 12 inside the communicating porous body through the opening 15. In addition, in order to expand an opening part, the surface of a communicating porous body can also be deleted by grinding | polishing etc.

基材11内部の空洞部12の形状は、特段限定されることはなく、球形状、楕円体、紡錘体、多角形状体等の適宜である。空洞が略球形状の場合には、大きさSzは直径であり、楕円体、紡錘体等であれば、大きさはそれらの最大長となる。空洞部12の形状、大きさは、保持する微生物の菌種、形態、保持量等により適宜である。後述する製造方法から把握されるように、空洞部は粒状被溶解物の大きさに依存する。微生物の懸濁液等は毛細管現象を通じて空洞部に浸透すること、及び微生物が空洞部内に存在する必要上微生物の体長よりも大きいことが求められる。そのため、空洞部12の大きさSzは、約1μm〜5mmの範囲内となる。   The shape of the hollow portion 12 inside the base material 11 is not particularly limited, and may be a suitable shape such as a spherical shape, an ellipsoid, a spindle, or a polygonal shape. When the cavity has a substantially spherical shape, the size Sz is a diameter. When the cavity is an ellipsoid, a spindle, or the like, the size is the maximum length thereof. The shape and size of the cavity portion 12 are appropriate depending on the species, form, retention amount, etc. of the microorganism to be retained. As can be understood from the manufacturing method described later, the hollow portion depends on the size of the granular material to be dissolved. It is required that the suspension of microorganisms or the like penetrates into the cavity through capillary action, and that the microorganism is present in the cavity so that it is larger than the length of the microorganism. Therefore, the size Sz of the cavity 12 is in the range of about 1 μm to 5 mm.

図示の多孔質シート状物10Aにおいて、連通多孔質体13内の空洞部12同士は連通口16によって互いに接触した連通構造として形成されている。空洞部同士を互いに連通させた多孔質体を得る最も単純な方法は、空洞部自体の数を増やすことである。これは、空洞部形成の元となる粒状被溶解物(後述の製造方法を参照)の添加量自体を増やすことにより実現できる。そのため、多孔質シート状物10Aに占める空洞部12の体積割合は、少なくとも50%以上、好ましくは70%以上となる。ただし、強度確保の点から空洞部の体積割合の上限は約85%となる。空洞部同士の連通が進むことにより連通多孔質体内部の表面積が広がる。このため、内表面に保持できる微生物の量も増加する。   In the illustrated porous sheet-like material 10 </ b> A, the hollow portions 12 in the communicating porous body 13 are formed as a communicating structure in which they are in contact with each other through the communicating port 16. The simplest method for obtaining a porous body in which cavities communicate with each other is to increase the number of cavities themselves. This can be realized by increasing the addition amount of the granular material to be dissolved (see the manufacturing method described later) that is the basis for forming the cavity. Therefore, the volume ratio of the hollow portion 12 in the porous sheet-like material 10A is at least 50% or more, preferably 70% or more. However, the upper limit of the volume ratio of the cavity is about 85% from the viewpoint of securing the strength. As the communication between the cavities proceeds, the surface area inside the communicating porous body increases. For this reason, the amount of microorganisms that can be held on the inner surface also increases.

図2の模式図に開示する多孔質シート状物10Bにおいては、図2(a)参照のとおり、連通多孔質体13はその内部に空洞部12に加え、さらに中空管路17が形成されている構造である。空洞部等の大きさ、構造は第1実施形態とほぼ同様である。中空管路17が備えられているため、中空管路17と空洞部12との連通(中空管路同士も含む)による管路連通口18、表面14における中空管路17の管路開口部19も新たに生じる。中空管路17は、長さ、形状、太さ、断面形状等は適宜である。ただし、極端に太くなると多孔質シート状物を構成できなくなるため、断面径は概ね約10μm〜3mmの範囲内である。中空管路17は後述の製造方法における管路予定被溶解物の溶解に起因する。   In the porous sheet-like material 10B disclosed in the schematic diagram of FIG. 2, as shown in FIG. 2 (a), the communicating porous body 13 includes a hollow portion 17 in addition to the hollow portion 12 therein. It is a structure. The size and structure of the cavity and the like are substantially the same as those in the first embodiment. Since the hollow pipe 17 is provided, the pipe 18 is connected to the hollow pipe 17 and the hollow portion 12 (including the hollow pipes), and the pipe of the hollow pipe 17 on the surface 14 is provided. A road opening 19 is also newly generated. The hollow conduit 17 has an appropriate length, shape, thickness, cross-sectional shape, and the like. However, since it becomes impossible to constitute a porous sheet-like material when it becomes extremely thick, the cross-sectional diameter is generally in the range of about 10 μm to 3 mm. The hollow pipe line 17 is caused by dissolution of the pipe line planned dissolution material in the manufacturing method described later.

多孔質シート状物10Bに占める空洞部12の体積割合も、少なくとも50%以上、好ましくは70%以上となる。同様に強度確保の点から空洞部の体積割合の上限は約85%となる。なお、中空管路の占める体積割合も影響するため、多孔質シート状物10Aよりは空洞部の割合は抑えられる。   The volume ratio of the hollow portion 12 in the porous sheet-like material 10B is also at least 50% or more, preferably 70% or more. Similarly, the upper limit of the volume ratio of the cavity is about 85% from the viewpoint of securing the strength. In addition, since the volume ratio which a hollow pipe line occupies influences, the ratio of a cavity part is suppressed rather than 10 A of porous sheet-like materials.

多孔質シート状物内の中空管路は図2(b)に開示の不規則な配置(いわゆるランダムな管路の配置)とするほか、当該多孔質シート状物の面方向に中空管路を揃えた配置や分岐した管路(ともに図示せず)に形成することも可能である。多孔質シート状物10Bのように空洞部と中空管路を併せ持つことにより、多孔質シート状物自体の吸液、浸透性能は向上する。   In addition to the irregular arrangement disclosed in FIG. 2B (so-called random pipe arrangement), the hollow pipe in the porous sheet is hollow in the surface direction of the porous sheet. It is also possible to form an arrangement in which the paths are aligned or a branched pipe line (both not shown). By having both a hollow portion and a hollow pipe line like the porous sheet-like material 10B, the liquid absorption and permeation performance of the porous sheet-like material itself is improved.

図1及び図2にて開示の多孔質シート状物10A,10Bの構造は、微生物保存用多孔質シート状物の一例であり、シート状物自体の大きさ、形状、シートの肉厚の他、空洞部や中空管路の大きさ、形状、種類にも変更、改良が加えられる。そこで、図3に多孔質シート状物を多層化した多孔質多層構造体20の例を示す。適用する多孔質シート状物は第1実施形態の10Aまたは第2実施形態の10Bのいずれでもよく、その他の形態とすることもできる。   The structure of the porous sheet-like material 10A, 10B disclosed in FIGS. 1 and 2 is an example of the porous sheet-like material for preserving microorganisms. In addition to the size and shape of the sheet-like material itself and the thickness of the sheet, Changes and improvements are also made in the size, shape, and type of the cavity and the hollow pipe. FIG. 3 shows an example of the porous multilayer structure 20 in which the porous sheet-like material is multilayered. The porous sheet to be applied may be either 10A of the first embodiment or 10B of the second embodiment, and may take other forms.

図3(a)は多孔質シート状物10A,10Bを幾重にも巻き取ることにより多層化した多孔質多層構造体20であって、巻き取りロール形状21(巻きロール体)である。多孔質シート状物を長く形成すれば巻き取りは簡単である。図示では巻き芯22の周りに多孔質シート状物は巻き取られる。この巻き芯の使用は任意である。図3(b)は巻き取りロール形状の多孔質多層構造体の断面の部分拡大図である。各々の多孔質シート状物は巻き取られて容易に多層化し、ひとつの塊となる。巻き取りロール形状とする利点は必要時に必要量を切り分けて使用することができる。十分な巻き取り長さを確保することにより、微生物を保存した後、大量培養を所望する際に都合がよい。特にロール形状となることによって、保存時の省スペース化が可能となる。また、ロール単位で微生物の保存等の管理を行うため、作業性においても優れる。   FIG. 3A shows a porous multilayer structure 20 in which the porous sheet-like materials 10A and 10B are multi-layered by winding up a plurality of layers, and has a winding roll shape 21 (winding roll body). Winding is easy if the porous sheet-like material is formed long. In the drawing, the porous sheet is wound around the winding core 22. The use of this winding core is optional. FIG.3 (b) is the elements on larger scale of the cross section of the porous multilayer structure of a winding roll shape. Each porous sheet is wound up and easily multi-layered to form one lump. The advantage of having a winding roll shape can be used by dividing the required amount when necessary. By securing a sufficient winding length, it is convenient when a large-scale culture is desired after the microorganism is stored. In particular, the roll shape enables space saving during storage. Moreover, since management of the preservation | save etc. of microorganisms is performed per roll, it is excellent also in workability | operativity.

図3(c)は多孔質シート状物10A,10Bを1枚ずつ重ねて多層化した多孔質多層構造体であって枚葉多層形状23(枚葉多層体)である。各多孔質シート状物は結束バンド24によりひとつの枚葉多層形状にまとめられる。図3(d)は多孔質シート状物10A,10Bを一定の長さで折り返しながら多層化した多孔質多層構造体であって蛇腹形状25(蛇腹多層体)である。これは巻き取りロール形状21の変形である。一度にまとまった量が必要となる場合やすぐにシート状物を広げたい場合に適する。図3(e)は多孔質シート状物10A,10Bの一枚をいったんV字状に折り曲げ、広がった部分同士を互いに噛み合わせることにより何重にも重ねて多層化した多孔質多層構造体であって折り曲げ重ね形状26(曲げ重ね体)である。これは多孔質シート状物を1枚ずつ取り出して使用したい場合に都合がよい。   FIG. 3C shows a porous multilayer structure in which the porous sheet-like materials 10A and 10B are stacked one by one, and has a single-wafer multilayer shape 23 (single-wafer multilayer body). Each porous sheet-like material is collected into a single sheet multi-layer shape by a binding band 24. FIG. 3D shows a porous multilayer structure in which the porous sheet-like materials 10A and 10B are multilayered while being folded back at a certain length, and has a bellows shape 25 (bellows multilayer body). This is a modification of the winding roll shape 21. This is suitable when a large amount is required at once or when it is desired to immediately expand the sheet. FIG. 3 (e) shows a porous multilayer structure in which one of the porous sheet-like materials 10A and 10B is once bent into a V shape, and the expanded portions are meshed with each other to overlap each other. Thus, it is a folded and stacked shape 26 (bent and stacked body). This is convenient when it is desired to take out and use the porous sheet material one by one.

図示のように多孔質シート状物を重ねて多層化して多孔質多層構造体を構成する場合、多孔質シート状物が単層であるときよりも、外気(主に空気)に触れる面積は総じて少なくなる。このため、保存期間中、保持されている微生物が被る酸素ストレスの影響は緩和される。なお、多孔質シート状物を多層化して多孔質多層構造体に加工する際の折り曲げ方、折り畳み方は所望の用途に応じて適宜であり、図示の例に拘束されない。   As shown in the figure, when a porous multilayer structure is configured by stacking a porous sheet-like material, the area in contact with the outside air (mainly air) is generally larger than when the porous sheet-like material is a single layer. Less. For this reason, the influence of the oxygen stress which the microorganisms hold | maintained during a preservation | save period is eased. In addition, the folding method and folding method when the porous sheet-like material is multilayered and processed into a porous multilayer structure are appropriate depending on the desired application, and are not restricted by the illustrated example.

図4ないし図6に基づいて多孔質多層構造体を用いて行う微生物の保存方法を説明する。図4の第1工程図においては、はじめに多孔質多層構造体が用意され(S1)、これに対して滅菌が行われる(S11)。滅菌は、薬品添加、ガス薫蒸、乾熱、水蒸気加熱、放射線照射等の適宜である。これと並行して保存目的の微生物の懸濁液が調製される(S2)。懸濁液中の微生物濃度は当該微生物に応じて適宜である。   A method for preserving microorganisms using a porous multilayer structure will be described with reference to FIGS. In the first process diagram of FIG. 4, a porous multilayer structure is prepared first (S1), and sterilization is performed on this (S11). Sterilization is appropriate as chemical addition, gas fumigation, dry heat, steam heating, radiation irradiation and the like. In parallel with this, a suspension of microorganisms for preservation is prepared (S2). The microorganism concentration in the suspension is appropriate depending on the microorganism.

多孔質多層構造体の滅菌後、当該多孔質多層構造体は微生物の懸濁液中に浸漬される(S12)。多孔質多層構造体内の全ての空洞部が微生物の懸濁液中に浸るように、多孔質多層構造体の全体が微生物の懸濁液中に沈められる。   After sterilization of the porous multilayer structure, the porous multilayer structure is immersed in a suspension of microorganisms (S12). The entire porous multilayer structure is submerged in the microbial suspension so that all cavities in the porous multilayer structure are immersed in the microbial suspension.

浸漬に伴い必要に応じて減圧が行われる(S13)。微生物の懸濁液は減圧可能な密閉容器に入れられており、多孔質多層構造体の浸漬後、真空ポンプ等を用いて密封容器内の空気が抜かれる(脱気)。多孔質多層構造体内の空洞部内の空気、多孔質多層構造体の撥水効果、微生物の懸濁液の表面張力等の影響から、単に浸漬のみでは多孔質多層構造体内部へ懸濁液は浸透しないことが多い。また、多層化構造であることも浸透が容易でない要因である。そのため、減圧下で多孔質多層構造体内の空洞部内の空気を脱気することが必要となる場合がある。減圧時の真空度は装置の大きさ、処理量、時間に応じて適宜である。こうして、多孔質多層構造体の空洞部内に微生物が付着される。   Along with the immersion, pressure reduction is performed as necessary (S13). The suspension of microorganisms is put in a sealed container that can be decompressed, and after the porous multilayer structure is immersed, the air in the sealed container is vented (degassed) using a vacuum pump or the like. Suspension penetrates into the porous multilayer structure simply by dipping due to the effects of air in the cavity in the porous multilayer structure, the water repellent effect of the porous multilayer structure, the surface tension of the suspension of microorganisms, etc. Often not. In addition, the multilayer structure is also a factor that is not easily penetrated. Therefore, it may be necessary to deaerate the air in the cavity in the porous multilayer structure under reduced pressure. The degree of vacuum at the time of depressurization is appropriate according to the size of the apparatus, the processing amount, and the time. Thus, microorganisms adhere to the cavity of the porous multilayer structure.

減圧により多孔質多層構造体内の空洞部内部に微生物の懸濁液が満たされた後、減圧は終了し、密閉容器より多孔質多層構造体は懸濁液から引き上げられ、続いて乾燥が行われて乾燥多孔質多層構造体となる(S14)。乾燥条件は保存の対象となる微生物の乾燥耐性、処理時間等が考慮される。乾燥により水分含量を減らすことができ、以降の保存に際し微生物の懸濁液の腐敗を避けることができる。保存時における微生物の増殖要因を除去する必要があるためである。   After the microbial suspension is filled in the cavity of the porous multilayer structure by decompression, the decompression is completed, and the porous multilayer structure is pulled out of the suspension from the sealed container, followed by drying. Thus, a dry porous multilayer structure is obtained (S14). Drying conditions take into consideration the drying resistance, processing time, etc. of the microorganisms to be stored. Drying can reduce the water content and avoid rot of the microorganism suspension during subsequent storage. This is because it is necessary to remove the growth factor of microorganisms during storage.

前記の乾燥多孔質多層構造体に至る工程によって微生物保存部材(P1)の完成とすることができる。微生物の保存期間等をさらに伸ばすことに加え、嫌気性等の空気環境下では生存が困難な微生物の場合、乾燥多孔質多層構造体表面からの酸素の流通、通気を抑制ないし阻止する必要がある。その場合、多孔質多層構造体の空洞部内に微生物が付着後乾燥されてなる乾燥多孔質多層構造体の表面は、ろう(蝋)、パラフィン、またはホットメルト樹脂のいずれかにより被覆され(S15)、微生物保存部材として完成する。   The microorganism preservation member (P1) can be completed by the process leading to the dry porous multilayer structure. In addition to further extending the storage period of microorganisms, in the case of microorganisms that are difficult to survive in an anaerobic environment such as anaerobic, it is necessary to suppress or prevent oxygen flow and aeration from the surface of the dry porous multilayer structure. . In that case, the surface of the dried porous multilayer structure obtained by drying microorganisms after adhering to the cavity of the porous multilayer structure is coated with either wax (wax), paraffin, or hot melt resin (S15). Completed as a microorganism preservation member.

ろう(蝋)、パラフィン、またはホットメルト樹脂のいずれかよりなる被覆剤(コート剤)により乾燥多孔質多層構造体の表面全体がまんべんなく被覆(コート)されることにより、乾燥多孔質多層構造体の内外の酸素の流通、通気を抑えることができる。空気環境下、常温、常圧での微生物保存部材の保存が容易となる。微生物保存部材を真空容器に封入して不活性ガスを充填する手間が必要なく、保存に要する負担が大きく低減される。   The entire surface of the dry porous multilayer structure is uniformly coated (coated) with a coating (coating agent) made of either wax (wax), paraffin, or hot melt resin. Circulation and ventilation of oxygen inside and outside can be suppressed. It is easy to store the microorganism storage member at room temperature and normal pressure in an air environment. There is no need to fill the microorganism storage member in a vacuum container and fill with an inert gas, and the burden required for storage is greatly reduced.

被覆に用いるろう(蝋)は高級脂肪酸と高級1価アルコールのエステルであり、パラフィンはアルカン系炭化水素(Cn2n+2として表される。)である。ホットメルト樹脂は、ホットメルト接着剤として利用される樹脂であり、例えば、エチレン・酢酸ビニル系樹脂、ポリウレタン樹脂、ポリエステル系樹脂等であり、樹脂の融点が80℃〜120℃ほどの比較的低融点の樹脂である。 Wax used for the covering (wax) are esters of higher fatty acids and higher monohydric alcohols, paraffin is (expressed as C n H 2n + 2.) Alkane hydrocarbons. The hot melt resin is a resin used as a hot melt adhesive, for example, an ethylene / vinyl acetate resin, a polyurethane resin, a polyester resin, and the like, and the melting point of the resin is relatively low, about 80 ° C. to 120 ° C. It is a melting point resin.

図4の第1工程図に基づいて得た微生物保存部材(P1)は、図6(a)の断面模式図として示すことができる。同図は、前出の多孔質シート状物10Aから形成した微生物保存部材(P1)である。各部位の符号は前記同様である。連通多孔質体13内の空洞部12同士は連通口16によって互いに接触している。微生物の懸濁液は、連通口16を通じて個々の空洞部12内に流入する。乾燥の後、懸濁液中の微生物Moは空洞部12の内壁12wに貼り付いて取り残される。そして、当該図示の状態のまま、保存に供される。なお、被覆剤については図示を省略している。   The microorganism preservation member (P1) obtained based on the first step diagram of FIG. 4 can be shown as a schematic cross-sectional view of FIG. The figure is a microorganism preservation member (P1) formed from the porous sheet-like material 10A described above. The code | symbol of each site | part is the same as the above. The hollow portions 12 in the communicating porous body 13 are in contact with each other through the communication port 16. The suspension of microorganisms flows into the individual cavities 12 through the communication port 16. After drying, the microorganisms Mo in the suspension are stuck to the inner wall 12w of the cavity 12 and left behind. And it is provided for preservation | save with the state of the said illustration. Note that illustration of the coating agent is omitted.

図5の第2工程図においても、はじめに多孔質多層構造体が用意され(S1)、これに対して滅菌が行われる(S21)。次に、保護剤溶液が調製され、加熱もしくは濾過により滅菌される。   Also in the 2nd process figure of Drawing 5, a porous multilayered structure is prepared first (S1), and sterilization is performed to this (S21). Next, a protective agent solution is prepared and sterilized by heating or filtration.

滅菌後の多孔質多層構造体は、微生物の懸濁液中への浸漬(S25)の前に保護剤溶液中に浸漬される(S22)。多孔質多層構造体内の全ての空洞部が保護剤溶液中に浸るように、多孔質多層構造体の全体が保護剤中に沈められる。そして、減圧(S23)、乾燥(S24)が行われる。保護剤溶液の浸漬後に行われる減圧(S23)の理由は、前記の第1工程図における減圧(S13)と同様に多孔質多層構造体内への浸透性を高めるためである。その後の乾燥(S24)は保護剤溶液中の余分な水分を蒸発させるためである。なお、樹脂の種類によっては減圧が省略される場合があり、保護剤溶液や微生物の種類により乾燥が省略される場合もある。   The porous multilayer structure after sterilization is immersed in the protective agent solution (S22) before being immersed in the suspension of microorganisms (S25). The entire porous multilayer structure is submerged in the protective agent so that all cavities in the porous multilayer structure are immersed in the protective agent solution. And pressure reduction (S23) and drying (S24) are performed. The reason for the reduced pressure (S23) performed after immersion of the protective agent solution is to increase the permeability into the porous multilayer structure as in the reduced pressure (S13) in the first step diagram. The subsequent drying (S24) is for evaporating excess water in the protective agent solution. Note that the decompression may be omitted depending on the type of the resin, and drying may be omitted depending on the type of the protective agent solution or the microorganism.

保護剤は微生物の極度の乾燥の防止、適度な水分及び栄養の維持、浸透圧の変化の緩和、目的外の微生物の増殖抑制等の目的で用いられる。例えば、保護剤はグルコース、ラクトース、スクロース、トレハロース等の糖類、ポリビニルアルコール、グリセリン、スキムミルク、ポリペプトン、さらに各種塩類、目的外微生物の成育を阻害する抗生物質等のいずれかあるいはそれらを混合した溶液である。保護剤は保存対象となる微生物の種類に応じた組成となり、例示以外の成分も当然に含められる。   The protective agent is used for the purpose of preventing extreme drying of microorganisms, maintaining appropriate moisture and nutrients, mitigating changes in osmotic pressure, and suppressing the growth of undesired microorganisms. For example, the protective agent is a sugar, such as glucose, lactose, sucrose, trehalose, polyvinyl alcohol, glycerin, skim milk, polypeptone, various salts, antibiotics that inhibit the growth of undesired microorganisms, or a mixture thereof. is there. The protective agent has a composition corresponding to the type of microorganism to be stored, and naturally includes components other than those exemplified.

図5の第2工程図における乾燥(S24)以降の微生物の懸濁液中への浸漬(S25)、減圧(S26)、乾燥(S27)、被覆(S28)の各工程は、図4の第1工程図における(S12)ないし(S15)の各工程と共通するため、詳細を省略する。   Each step of immersion (S25), pressure reduction (S26), drying (S27), coating (S28) in the suspension of microorganisms after drying (S24) in the second step diagram of FIG. Details are omitted because they are common to the steps (S12) to (S15) in the one-step diagram.

この第2工程図に基づく微生物の保存方法においても、前記同様、乾燥多孔質多層構造体に至るまで工程によって微生物保存部材(P2)の完成とすることができる。また、多孔質多層構造体の空洞部内に微生物が付着後乾燥されてなる乾燥多孔質多層構造体の表面の全体にろう(蝋)、パラフィン、またはホットメルト樹脂のいずれかによって被覆した微生物保存部材(P2)とすることもできる。いずれを選択するべきかについては、内部に保存される微生物、保護剤の選択等の条件による。   Also in the microorganism storage method based on the second process diagram, the microorganism storage member (P2) can be completed through the processes up to the dry porous multilayer structure as described above. Also, a microorganism storage member in which the entire surface of the dried porous multilayer structure obtained by adhering microorganisms in the cavity of the porous multilayer structure and dried is coated with either wax, paraffin, or hot melt resin. (P2) can also be used. Which should be selected depends on conditions such as selection of microorganisms stored inside and protective agents.

図5の第2工程図に基づいて得た微生物保存部材(P2)は、図6(b)の断面模式図として示すことができる。同図は、前出の多孔質シート状物10Aから形成した微生物保存部材(P2)である。各部位の符号は前記同様である。連通多孔質体13内の空洞部12同士は連通口16によって互いに接触している。まず、保護剤溶液は、連通口16を通じて個々の空洞部12内に流入する。乾燥により適度に水分が蒸発して保護剤溶液の被膜Praが空洞部12の内壁12wに形成される。続いて、微生物の懸濁液も連通口16を通じて個々の空洞部12内に流入する。乾燥の後、懸濁液中の微生物Moは、空洞部12の内壁12wに形成された保護剤溶液の被膜Praに貼り付いて取り残される。そして、当該図示の状態のまま、保存に供される。なお、被覆剤については図示を省略している。   The microorganism preservation member (P2) obtained based on the second process diagram of FIG. 5 can be shown as a schematic cross-sectional view of FIG. This figure is a microorganism preservation member (P2) formed from the porous sheet-like material 10A described above. The code | symbol of each site | part is the same as the above. The hollow portions 12 in the communicating porous body 13 are in contact with each other through the communication port 16. First, the protective agent solution flows into the individual cavities 12 through the communication port 16. Moisture evaporates moderately by drying, and a film Pra of the protective agent solution is formed on the inner wall 12 w of the cavity portion 12. Subsequently, the suspension of microorganisms also flows into the individual cavities 12 through the communication port 16. After drying, the microorganisms Mo in the suspension are left on the coating Pra of the protective agent solution formed on the inner wall 12w of the cavity 12. And it is provided for preservation | save with the state of the said illustration. Note that illustration of the coating agent is omitted.

図示並びに説明のとおり多孔質多層構造体の多孔質内に微生物が付着されてなる微生物保存部材について、図7、図8を用い代表的な使用例を説明する。図7(a)は、前出の図3(a),(b)と同様の巻き取りロール形状21の多孔質多層構造体20であり、微生物保存部材の一例である。巻き取りロール形状自体の大きさは任意である。これより、必要量の微生物保持シート27が切り取られる。図7(b)のように、切り取られた微生物保持シート27はシャーレ31内の培地上に載置される。すると、微生物保持シート内の空洞部に保持されている微生物が培地側に移動し、増殖し始める。図7(c)の例では、切り取られた微生物保持シート27はマイヤー32(例えば図示の坂口フラスコ等の培養容器)の液体培地中に投入される。同様に、微生物保持シート内の空洞部に保持されている微生物が培地側に移動し、増殖し始める。   As shown in the drawings and description, a typical example of use of a microorganism storage member in which microorganisms are attached in the porous structure of a porous multilayer structure will be described with reference to FIGS. FIG. 7A shows a porous multilayer structure 20 having a winding roll shape 21 similar to that shown in FIGS. 3A and 3B, and is an example of a microorganism storage member. The size of the winding roll shape itself is arbitrary. Thus, a necessary amount of the microorganism holding sheet 27 is cut off. As shown in FIG. 7B, the cut microorganism holding sheet 27 is placed on the culture medium in the petri dish 31. Then, the microorganisms held in the cavity in the microorganism holding sheet move to the medium side and start to grow. In the example of FIG. 7C, the cut microorganism holding sheet 27 is put into a liquid medium of a Mayer 32 (for example, a culture container such as a Sakaguchi flask shown in the figure). Similarly, the microorganisms held in the cavity in the microorganism holding sheet move to the medium side and start to grow.

このように、比較的簡便な部材によって微生物の保存及び培養に用いられる。図示の巻き取りロール形状の多孔質多層構造体については、表面へのろう、パラフィン、ホットメルト樹脂による被覆は適宜である。ただし、いったん切り出して微生物保持シートを使用した後、保持されている微生物が被る損傷を避けるため、再び、多孔質多層構造体の表面にろう、パラフィン、またはホットメルト樹脂を被覆することもある。   Thus, it is used for preservation and culture of microorganisms by a relatively simple member. For the porous multi-layer structure in the form of a take-up roll shown in the figure, the surface is appropriately coated with wax, paraffin, or hot melt resin. However, after cutting out and using the microorganism holding sheet, the surface of the porous multilayer structure may be coated again with wax, paraffin, or hot melt resin in order to avoid damage to the held microorganisms.

図8(a)は、前出の図3(c)と同様の枚葉多層形状23の多孔質多層構造体20であり、微生物保存部材の一例である。枚葉多層形状の一枚当たりの大きさや束の厚さは適宜である。図8(b)は、家庭、飲食店、工場、各種商業施設等に設置されている汚水浄化槽33(グリーストラップ、曝気槽等でもよい)に枚葉多層形状の中から何枚かの微生物保持シート28が投入された様子を示す。この場合の微生物保持シートには、汚物34等の分解効率の良い菌種が保持される。さらに、図8(c)は、河川、湖沼、海洋等の油濁汚染が生じた際の油濁の分解除去に供される例である。図示では、海洋35において船舶等から流出した重油等の油濁36がオイルフェンス37により囲まれている。そこで、何枚もの微生物保持シート28が油濁海域内に散布される。この場合の微生物保持シートには、油濁36等の分解効率の良い菌種が保持される。   FIG. 8A shows a porous multilayer structure 20 having a single-wafer multilayer shape 23 similar to FIG. 3C described above, and is an example of a microorganism storage member. The size per sheet and the thickness of the bundle are appropriate. Fig. 8 (b) shows the retention of some microorganisms from the single-wafer multi-layer shape in a sewage purification tank 33 (which may be a grease trap, an aeration tank, etc.) installed in homes, restaurants, factories, and various commercial facilities. A state in which the sheet 28 is loaded is shown. In this case, the microorganism holding sheet holds bacterial species having high decomposition efficiency such as the filth 34. Further, FIG. 8 (c) is an example used for decomposing and removing oil spillage when oil spillage occurs in rivers, lakes, oceans, and the like. In the drawing, an oil spill 36 such as heavy oil that has flowed out of a ship or the like in the ocean 35 is surrounded by an oil fence 37. Therefore, a number of microorganism holding sheets 28 are dispersed in the oily sea area. In this case, the microorganism holding sheet holds the bacterial species having good decomposition efficiency such as the oil turbidity 36.

図7の例は、微生物保持シートを適量ずつ複数回にわたり切り分けて用いるため、小規模での使用が想定される。対照的に、図8の例は、一度に大量の微生物保持シートの使用が想定される大規模での使用が想定される。このように、微生物保存部材は目的、用途等を勘案して多様な形態で製造、提供される。むろん、図7、図8の例示以外にも各種の好適な用途に用いられる。例えば、鉱山等における鉱物の回収、浄化等のバクテリアルリーチングに用いられる。さらに、空洞部を有する多孔質シート状物の構造特性を生かして直接バイオリアクターとして用いることも可能である。従って、所望の微生物を保持し保存する部材であると共に、これを使用する際の利便性も高めることができる。   In the example of FIG. 7, the microorganism holding sheet is used by cutting out an appropriate amount a plurality of times, so that it is assumed to be used on a small scale. In contrast, the example of FIG. 8 is assumed to be used on a large scale where a large amount of microorganism holding sheets are assumed to be used at one time. As described above, the microorganism storage member is manufactured and provided in various forms in consideration of the purpose and application. Of course, it is used for various suitable uses other than the illustration of FIG. 7, FIG. For example, it is used for bacterial leaching such as mineral recovery and purification in mines. Furthermore, it is also possible to directly use it as a bioreactor taking advantage of the structural characteristics of the porous sheet-like material having a cavity. Therefore, it is a member for holding and storing a desired microorganism, and the convenience when using it can be enhanced.

開示の実施形態の微生物保持シートを形成する樹脂基材、及び後出の樹脂材料としては、広義に有機高分子化合物が用いられる。有機高分子化合物は、微生物保持シートとしての安定性、加工容易性、価格等が重視される場合に選択されることが多い。後記する製造方法からも明らかなように、シート状とする場合に都合が良く、用途も広いためである。有機高分子化合物として、例えば、ポリオレフィン樹脂、ポリアミド樹脂、あるいはポリエステル樹脂等が用いられる。また、有機高分子化合物においても生分解性能を考慮した動植物、微生物由来の天然有機高分子化合物、合成の生分解性樹脂も用いられる。   An organic polymer compound is used in a broad sense as the resin base material forming the microorganism holding sheet of the disclosed embodiment and the resin material described later. The organic polymer compound is often selected when stability as a microorganism holding sheet, ease of processing, price, and the like are important. As will be apparent from the manufacturing method described later, this is convenient in the case of forming a sheet and is versatile. As the organic polymer compound, for example, polyolefin resin, polyamide resin, or polyester resin is used. For organic polymer compounds, animals and plants that take biodegradability into consideration, natural organic polymer compounds derived from microorganisms, and synthetic biodegradable resins are also used.

ポリオレフィン樹脂を例示すると、エチレン単独重合体、エチレンとプロピレン、1−ブテン、1−ペンテン、1−ヘキセン、4−メチル−1−ペンテン等の1種または2種以上のα−オレフィンとのランダムまたはブロック共重合体、エチレンと酢酸ビニル、アクリル酸、メタクリル酸、アクリル酸メチルとの1種または2種以上のランダムまたはブロック共重合体、プロピレン単独重合体、プロピレンとプロピレン以外のエチレン、1−ブテン、1−ペンテン、1−ヘキセン、4−メチル−1−ペンテン等の1種または2種以上のα−オレフィンとのランダムまたはブロック共重合体、1−ブテン単独重合体、アイオノマー樹脂、さらに前記したこれら重合体の混合物等のポリオレフィン系樹脂、石油樹脂及びテルペン樹脂等の炭化水素系樹脂である。   Examples of polyolefin resins include ethylene homopolymer, random and one or more α-olefins such as ethylene and propylene, 1-butene, 1-pentene, 1-hexene, 4-methyl-1-pentene, etc. Block copolymer, one or more random or block copolymers of ethylene and vinyl acetate, acrylic acid, methacrylic acid, methyl acrylate, propylene homopolymer, ethylene other than propylene and propylene, 1-butene , 1-pentene, 1-hexene, 4-methyl-1-pentene, etc., one or more random or block copolymers with α-olefin, 1-butene homopolymer, ionomer resin, and Hydrocarbon resins such as polyolefin resins such as mixtures of these polymers, petroleum resins and terpene resins Resin.

ポリアミド樹脂を例示すると、ナイロン6、ナイロン66、ナイロン11、ナイロン12、ナイロン610、ナイロン6/66、ナイロン66/610及びナイロンMXD等のポリアミド系樹脂である。   Examples of the polyamide resin include polyamide resins such as nylon 6, nylon 66, nylon 11, nylon 12, nylon 610, nylon 6/66, nylon 66/610, and nylon MXD.

ポリエステル樹脂を例示すると、ポリエチレンテレフタレート、ポリブチレンテレフタレート及びポリエチレンナフタレート等のポリエステル系樹脂である。   Examples of the polyester resin include polyester resins such as polyethylene terephthalate, polybutylene terephthalate, and polyethylene naphthalate.

その他に利用可能な樹脂として、ポリメチルメタクリレート等のアクリル系樹脂、ポリスチレン、スチレン−アクリロニトリル共重合体等のスチレン−アクリロニトリル系樹脂、PTFE等のフッ素樹脂、ポリイソプレン系樹脂、SBR等のブタジエン系のゴム、ポリイミド樹脂、ポリビニルアルコール、エチレン−ビニルアルコール共重合体等の水素結合性樹脂、ポリカーボネート樹脂、塩化ビニル樹脂、塩化ビニリデン樹脂、ポリエーテルイミド樹脂、フェノール樹脂、メラミン樹脂、エポキシ樹脂、尿素樹脂、シリコーン樹脂、ポリケトン樹脂等を挙げることができる。   Other usable resins include acrylic resins such as polymethyl methacrylate, styrene-acrylonitrile resins such as polystyrene and styrene-acrylonitrile copolymers, fluorine resins such as PTFE, polyisoprene resins, and butadiene resins such as SBR. Rubber, polyimide resin, hydrogen bonding resin such as polyvinyl alcohol, ethylene-vinyl alcohol copolymer, polycarbonate resin, vinyl chloride resin, vinylidene chloride resin, polyetherimide resin, phenol resin, melamine resin, epoxy resin, urea resin, Examples thereof include silicone resins and polyketone resins.

生分解性樹脂は、動物、植物からの産生物をほぼそのまま利用した化合物と、この化合物を出発原料として適宜調製した樹脂素材の両方が含まれる。前者の天然物には、コラーゲン、デンプン、アルギン酸(架橋物等)、キチン、キトサン、天然ゴム、アラビアゴム、ダンマル、コパール、ロジン、グッタベルカ等である。後者の樹脂素材には、羊毛等のケラチン由来のタンパク質樹脂、例えばバチルス属等の細菌から産生されるポリ−3−ヒドロキシ酪酸、あるいはポリ−3−ヒドロキシ吉草酸、並びに両分子からなる共重合体、カゼインプラスチック、大豆タンパクプラスチック、セルロースアセテート(アセチルブチルセルロース)、セルロースアセテートブチレート、カルボキシメチルセルロース、ニトロセルロース、加えてセルロース由来のビスコースより調製される再生セルロース、デンプンから調製されるポリ乳酸等、種々の樹脂が該当する。さらに、これら以外にも、微生物的生分解性能に優れたポリカプロラクトン、ポリエチレンサクシネート、ポリブチレンサクシネート等も含めることができる。   Biodegradable resins include both compounds that use products from animals and plants almost as they are and resin materials that are appropriately prepared using this compound as a starting material. Examples of the former natural products include collagen, starch, alginic acid (cross-linked product, etc.), chitin, chitosan, natural rubber, gum arabic, dammar, copal, rosin, and Guttavelca. The latter resin material includes protein resins derived from keratin such as wool, for example, poly-3-hydroxybutyric acid or poly-3-hydroxyvaleric acid produced from bacteria such as Bacillus, and a copolymer comprising both molecules , Casein plastic, soy protein plastic, cellulose acetate (acetylbutylcellulose), cellulose acetate butyrate, carboxymethylcellulose, nitrocellulose, in addition to regenerated cellulose prepared from viscose derived from cellulose, polylactic acid prepared from starch, etc. Various resins are applicable. In addition to these, polycaprolactone, polyethylene succinate, polybutylene succinate and the like excellent in microbial biodegradability can also be included.

列記の生分解性樹脂を微生物保存部材の樹脂基材、樹脂材料とすることにより、前出の図8に開示の使用環境下で事後分解消滅すると考えられる。このため、使用後に回収の必要性が無くなり、環境への負荷を低減することができる。   By using the biodegradable resin listed as the resin base material or resin material of the microorganism storage member, it is considered that the post-degradation and disappearance will occur under the use environment disclosed in FIG. For this reason, the necessity of collection | recovery after use is lose | eliminated and the load to an environment can be reduced.

これまでに述べた微生物保持シートに関し、そのもととなる微生物保存用の多孔質シート状物を第1ないし第3製造方法例に従って説明する。図9の工程断面模式図(第1製造方法例)に開示のように、はじめに、事後的に溶解可能な粒状被溶解物が樹脂材料中に混入され、所定のシート状物に成形される(混合・成形工程)。図9(a)のとおり、成形体101において、樹脂材料からなる基材110中に粒状被溶解物120は適度に分散されている。むろん、この成形段階では、粒状被溶解物の溶解は始まっておらず、ほぼ混入時の形状を維持している。   Regarding the microorganism-holding sheet described so far, a porous sheet-like material for preserving microorganisms will be described according to first to third manufacturing method examples. As disclosed in the process cross-sectional schematic diagram of FIG. 9 (first manufacturing method example), a granular material to be dissolved afterwards is first mixed in the resin material and formed into a predetermined sheet-like material ( Mixing and forming process). As shown in FIG. 9A, in the molded body 101, the granular material 120 is appropriately dispersed in the base material 110 made of a resin material. Of course, at this molding stage, the dissolution of the granular material to be dissolved has not started, and the shape at the time of mixing is almost maintained.

混合・成形工程の後、成形体101内の粒状被溶解物120を溶解することによって、当該成形体内に空洞部を形成した多孔質体が得られる(多孔質形成工程)。図9(b)では、成形体101内の粒状被溶解物120の溶解に伴い粒状被溶解物は徐々に縮小する。最終的に図9(c)のように、成形体101内の粒状被溶解物120は完全に消失し、その空間は空洞部12となる。こうして基材110が取り残され多孔質体13を得ることができる。第1製造方法例に基づく多孔質シート状物10Aの完成である。粒状被溶解物120の溶解についての詳細は後述する。   After the mixing / molding step, the granular material to be dissolved 120 in the molded body 101 is dissolved to obtain a porous body in which a cavity is formed in the molded body (porous formation step). In FIG.9 (b), a granular to-be-melted material shrinks gradually with melt | dissolution of the to-be-melted granular material 120 in the molded object 101. FIG. Finally, as shown in FIG. 9C, the granular melt 120 in the molded body 101 disappears completely, and the space becomes the cavity 12. In this way, the base material 110 is left and the porous body 13 can be obtained. The porous sheet-like material 10A is completed based on the first manufacturing method example. Details of the dissolution of the granular material 120 will be described later.

図10の工程断面模式図(第2製造方法例)は、微生物保存用の多孔質シート状物10Bの製造方法例を開示する。はじめに図10(a)のとおり、事後的に溶解可能な粒状被溶解物205と樹脂材料201が混入され、樹脂混合物210が得られる。図10(b)のように、樹脂混合物210から所定のシート状物を成形するに際し、事後的に溶解可能な管路予定被溶解物220が混入される。図10(c)においては、樹脂材料が固化して成形体240が生じた後、当該成形体の内部に含まれている粒状被溶解物205及び管路予定被溶解物220が溶解、除去される。図10(d)のとおり、シート状物の内部に空洞部と共に中空管路を有した多孔質体250(管路形成多孔質体)が生じる。管路予定被溶解物220を樹脂混合物210と合わせる際には、図示のように適当に並べて樹脂混合物210内に埋没させてもよく、あるいは双方を一緒に混練して均一に分散させてもよく、双方の混練は適宜である。このように、簡単に中空管路を有した多孔質体を形成することができる。粒状被溶解物205及び管路予定被溶解物220の溶解についての詳細も後述する。   The process cross-sectional schematic diagram of FIG. 10 (2nd manufacturing method example) discloses the manufacturing method example of the porous sheet-like material 10B for microorganism preservation. First, as shown in FIG. 10A, a granular material 205 that can be dissolved afterwards and a resin material 201 are mixed, and a resin mixture 210 is obtained. As shown in FIG. 10B, when a predetermined sheet-like material is formed from the resin mixture 210, a planned pipeline dissolution material 220 that can be dissolved later is mixed. In FIG. 10C, after the resin material is solidified to form a molded body 240, the granular melt 205 and the planned pipeline melt 220 contained in the molded body are dissolved and removed. The As shown in FIG. 10D, a porous body 250 (pipe-line-forming porous body) having a hollow pipe along with a hollow portion is generated inside the sheet-like material. When the pre-dissolved material 220 to be melted is combined with the resin mixture 210, they may be arranged side by side as illustrated and buried in the resin mixture 210, or both may be kneaded together and uniformly dispersed. The kneading of both is appropriate. Thus, a porous body having a hollow pipe can be easily formed. Details of the dissolution of the granular material to be dissolved 205 and the pipeline to-be-melted material 220 will be described later.

図11の工程断面模式図(第3製造方法例)は、微生物保存用の多孔質シート状物10Bの別の製造方法例を開示する。まず、図11(a)のとおり、事後的に溶解可能な粒状被溶解物205と樹脂材料201が混入され、樹脂混合物210が得られる。図10(b)では、いったん樹脂混合物210から所定の混合樹脂シート状物が成形される。図11(c)のように、成形後に生じた混合樹脂シート状物215の一面側に管路予定被溶解物220が載置される。続いて図11(d)のように、混合樹脂シート状物215上に管路予定被溶解物220が載置されている接合面(貼り合わせ面213)上に再度混合樹脂シート状物215が重ね合わされ、接合物216が得られる。管路予定被溶解物220は両混合樹脂シート状物215の間に挟まれて存在する。   The process cross-sectional schematic diagram (third manufacturing method example) of FIG. 11 discloses another manufacturing method example of the porous sheet-like material 10B for preserving microorganisms. First, as shown in FIG. 11A, a granular material 205 that can be dissolved later and the resin material 201 are mixed, and a resin mixture 210 is obtained. In FIG. 10B, a predetermined mixed resin sheet is once formed from the resin mixture 210. As shown in FIG. 11 (c), the planned melted pipe material 220 is placed on the one surface side of the mixed resin sheet-like material 215 generated after molding. Subsequently, as shown in FIG. 11 (d), the mixed resin sheet material 215 is again formed on the joint surface (bonding surface 213) on which the planned melted material 220 is placed on the mixed resin sheet material 215. The bonded product 216 is obtained by superimposing. The planned melted material 220 is sandwiched between the two mixed resin sheet-like materials 215.

図11(e)では、接合物216の樹脂材料が固化した後、当該成形体の内部に含まれている粒状被溶解物205及び管路予定被溶解物220が溶解される。そして、シート状の接合物の内部全体に空洞部を有すると共に、接合物の内部、つまり接合面(貼り合わせ面)に中空管路を有した多孔質体260(管路形成多孔質体)が生じる。図11の第3製造方法例にあっては、混合樹脂シート状物215の内部に別途管路予定被溶解物220を適量添加、混練して、空洞部のみの場合よりも多孔質シート状物内部の空間を拡張することも可能である。混合樹脂シート状物上の接合面(貼り合わせ面)への管路予定被溶解物の載置は、図示の並べ置く他に、管路予定被溶解物のもととなる樹脂状物を接合面(貼り合わせ面)に塗布もしくは描画することもできる。従って、当該図11の製造方法の場合、正確な形状の中空管路を微生物保存用の多孔質シート状物の内部に形成することができる。   In FIG.11 (e), after the resin material of the joining thing 216 solidifies, the granular to-be-dissolved object 205 and the pipe line to-be-dissolved object 220 contained in the inside of the said molded object are melt | dissolved. And the porous body 260 (pipe-line-forming porous body) which has a hollow part in the inside of a joined body, ie, a joining surface (bonding surface), while having a hollow part in the whole inside of a sheet-like joined body. Occurs. In the third production method example of FIG. 11, an appropriate amount of the pre-dissolved pipe 220 to be melted is added to the inside of the mixed resin sheet 215 and kneaded, so that the porous sheet is better than the case of only the cavity. It is also possible to expand the internal space. In addition to placing the pipe line to be melted on the joining surface (bonding surface) on the mixed resin sheet, the resinous material that is the source of the pipe to be melted is joined. It is also possible to apply or draw on the surface (bonding surface). Therefore, in the case of the manufacturing method of FIG. 11, a hollow pipe having an accurate shape can be formed inside the porous sheet-like material for preserving microorganisms.

管路予定被溶解物の形状について、図2の第2実施形態等の短い中空管路を所望するのであれば、後出の繊維状の管路予定被溶解物を適当な長さに砕いて細かくしてから用いられる。図10,図11の第2実施形態等の中空管路を所望するのであれば、繊維状の管路予定被溶解物は適当な長さに裁断してから用いられる。   If a short hollow pipe line such as the second embodiment of FIG. 2 is desired for the shape of the pipe line to-be-dissolved material, the following fibrous pipe line to-be-dissolved material is crushed to an appropriate length. It is used after making it fine. If a hollow pipe line such as the second embodiment shown in FIGS. 10 and 11 is desired, the fibrous material to be dissolved in the pipe line is cut into an appropriate length before being used.

事後的に溶解可能な粒状被溶解物と樹脂材料との混練、あるいは事後的に溶解可能な管路予定被溶解物と樹脂材料との混練は、互いにほぼ均一に混ぜ合わせる必要がある。このため、製造規模に応じて公知のブレンダーやニーダー等が用いられる。樹脂の種類に応じて、硬化しないように加温しながら混練される。   The kneading of the granular material to be dissolved that can be dissolved afterwards and the resin material, or the kneading of the material to be dissolved and the material to be melted after the piping, and the resin material must be mixed almost uniformly. For this reason, a well-known blender, a kneader, etc. are used according to a manufacturing scale. Depending on the type of resin, it is kneaded while warming so as not to cure.

以上列記した樹脂材料と粒状被溶解物(管路予定被溶解物を含む場合もある)を含んでなるシート状物について、これらの成形には、押出成形、ブロー成形、プレス成形等の適宜樹脂加工分野の公知成形手法が用いられる。この結果、所望の成形形状が得られる。これらの他に、冷間静水圧プレス(CIP)、テープキャスティング法等を用いても良い。   About the sheet-like material comprising the resin materials listed above and the granular material to be dissolved (which may include the material to be dissolved in the pipeline), for these moldings, appropriate resins such as extrusion molding, blow molding, press molding, etc. Known molding techniques in the processing field are used. As a result, a desired molded shape is obtained. In addition to these, a cold isostatic press (CIP), a tape casting method, or the like may be used.

または、溶液キャスト法、Tダイ法、チューブラー法、カレンダー法等の公知の方法が使用される。樹脂材料を熱可塑性樹脂とするフィルムは、無延伸とする他、機械的物性等から、延伸フィルムとしてもよい。延伸フィルムを製造する際の延伸方法には、ロール−一軸延伸、圧延、逐次二軸延伸、同時二軸延伸、チューブラー延伸等の公知の方法が使用できる。特に、逐次二軸延伸、同時二軸延伸が、厚薄精度、機械的物性等の点で優れているため好ましい。   Alternatively, a known method such as a solution casting method, a T-die method, a tubular method, or a calendar method is used. In addition to non-stretching, the film using a resin material as a thermoplastic resin may be a stretched film in view of mechanical properties. As a stretching method for producing a stretched film, known methods such as roll-uniaxial stretching, rolling, sequential biaxial stretching, simultaneous biaxial stretching, and tubular stretching can be used. In particular, sequential biaxial stretching and simultaneous biaxial stretching are preferred because they are excellent in terms of thickness accuracy and mechanical properties.

延伸フィルムとする場合にあっては、前出の粒状被溶解含有物を温水中等で適度に加温しながら、管路予定被溶解物を引き延ばすようにすると、成形が容易になる。管路予定被溶解物も形状によっては粒状被溶解含有物と共に一緒に押し出され薄く広げることもでき、成形方法は自由である。   In the case of using a stretched film, molding can be facilitated by stretching the pre-dissolved material to be ducted while appropriately heating the above-mentioned granular material to be dissolved in warm water or the like. Depending on the shape of the material to be melted in the pipeline, the material to be melted can be extruded together with the granular material to be melted and spread thinly, and the molding method is free.

前出の図9に示しているように、粒状被溶解物同士は互いに接触した構造として形成されている。空洞部同士を互いに連通させた多孔質シート状物を得ることは、空洞部形成の元となる粒状被溶解物の添加量自体を増やすことにより実現できる。図示の第1ないし第3製造方法例に基づいて微生物保存用多孔質シート状物を形成するに際し、シート状物に占める空洞部の体積割合を少なくとも50%以上、好ましくは70%以上としている。つまり、樹脂材料に添加、混合される粒状被溶解物の体積割合を少なくとも50%以上、好ましくは70%以上に制御することで、前記の空洞部の体積割合とすることができる。ただし、単位体積あたりの樹脂材料に占める粒状被溶解物の量(体積割合)が増えすぎると、微生物保存用多孔質シート状物としての強度が低下し、脆弱化するため、ほぼ85%が上限となる。なお、管路予定被溶解物を添加して中空管路を形成する場合、その量は適宜調整される。   As shown in FIG. 9 above, the granular materials to be dissolved are formed as a structure in contact with each other. Obtaining a porous sheet-like material in which the cavities communicate with each other can be realized by increasing the amount of the granular material to be dissolved that is the basis for forming the cavities. When forming a porous sheet-like material for preserving microorganisms based on the first to third production method examples shown in the drawing, the volume ratio of the cavity portion in the sheet-like material is at least 50%, preferably 70% or more. That is, by controlling the volume ratio of the granular material to be added and mixed to the resin material to at least 50% or more, preferably 70% or more, the volume ratio of the hollow portion can be obtained. However, if the amount (volume ratio) of the granular material to be dissolved in the resin material per unit volume is excessively increased, the strength as a porous sheet-like material for preserving microorganisms decreases and becomes brittle. It becomes. In addition, when adding a to-be-dissolved material of a pipe line and forming a hollow pipe line, the quantity is adjusted suitably.

図示の微生物保存多孔質シート状物の第1ないし第3製造方法例に用いられる事後的に溶解可能な粒状被溶解物は、将来的にシート状の多孔質体として形成されたときに空洞部に置換される。つまり、空洞部の大きさはそのもととなる粒状被溶解物の大きさに近似する。空洞部の大きさは、約1μm〜5mmの範囲内であることから、粒状被溶解物の大きさも約1μm〜5mmの範囲内に合致する程度の大きさとなる。粒状被溶解物の形状には特段の制約は無く、球状、紡錘状、角形状等の適宜である。   The granular material to be dissolved after use used in the first to third examples of the method for producing a microorganism-preserving porous sheet shown in the figure is a cavity when it is formed as a sheet-like porous material in the future. Is replaced by That is, the size of the hollow portion approximates the size of the granular material to be dissolved. Since the size of the hollow portion is in the range of about 1 μm to 5 mm, the size of the granular material to be dissolved also becomes a size that matches the range of about 1 μm to 5 mm. There is no special restriction | limiting in the shape of a granular to-be-dissolved material, and spherical, spindle shape, square shape, etc. are appropriate.

図示の第2,第3製造方法例に用いられる事後的に溶解可能な管路予定被溶解物は、将来的にシート状の多孔質体として形成されたとき、当該多孔質体の内部に中空管路に置換される。中空管路は、微生物の懸濁液を前出の多孔質多層構造体内部に浸透しやすくするために形成される。そのため、中空管路自体の管径、管路長等は必ずしも厳密ではなく、既述の説明、さらには用途、目的、耐久性、樹脂基材の組成に応じて適宜となる。   The post-dissolvable pre-dissolvable material to be used for the second and third production methods shown in the figure is formed inside the porous body when formed in the future as a sheet-like porous body. Replaced with an empty pipeline. The hollow duct is formed to facilitate the penetration of the microorganism suspension into the porous multilayer structure. Therefore, the pipe diameter, the pipe length, and the like of the hollow pipe itself are not necessarily strict, and are appropriate according to the above-described explanation, and further, the use, purpose, durability, and composition of the resin base material.

粒状被溶解物の事後的な溶解可能性を満たすため、粒状被溶解物は水、酵素、または有機溶剤のいずれかによって溶解、除去される組成物からなる。また、管路予定被溶解物の事後的な溶解可能性を満たすため、管路予定被溶解物も水、酵素、または有機溶剤のいずれかによって溶解、除去される組成物である。粒状被溶解物、管路予定被溶解物の溶解、除去に当たり、同一の溶媒あるいは異なる溶媒を用いてもよい。   In order to satisfy the subsequent dissolution possibility of the granular material to be dissolved, the granular material to be dissolved is composed of a composition that is dissolved or removed by any one of water, an enzyme, and an organic solvent. In addition, in order to satisfy the possibility of subsequent dissolution of the pre-dissolved material in the pipeline, the pre-dissolved material in the conduit is a composition that is dissolved and removed by either water, an enzyme, or an organic solvent. The same solvent or different solvents may be used for dissolving and removing the granular material to be dissolved and the pipeline-specific material to be dissolved.

粒状被溶解物、管路予定被溶解物の溶解に際し、水を溶媒として前記のシート状物(成形体や接合物)は水中に浸漬、あるいは散水等が行われる。前記のシート状物(成形体や接合物)内部の粒状被溶解物、管路予定被溶解物のいずれかもしくは両方は溶解される。水には、温水、熱水、亜臨界水も含まれる。また、酸・アルカリのpH値の調整や適宜の塩類の溶解液も含まれる。これらは総称して水系の溶剤である。   In dissolving the granular material to be dissolved and the pipe line scheduled material to be dissolved, the sheet-like material (molded product or bonded product) is immersed in water or sprinkled with water as a solvent. Either or both of the granular material to be melted and the pipe line material to be melted inside the sheet-like product (molded product or bonded product) are dissolved. Water includes warm water, hot water, and subcritical water. Moreover, adjustment of the pH value of an acid / alkali and a solution of an appropriate salt are also included. These are generically water-based solvents.

具体的に水溶性の粒状被溶解物とは、グルコースの結晶、氷砂糖、あるいは粉糖(糖の凝固物)等の糖類の結晶である。塩類の結晶の場合、例えば、塩化ナトリウムの結晶、みょうばんの結晶、硝酸カリウムの結晶等である。石灰岩や炭酸カルシウム結晶を水溶性の被溶解物とすることも可能である。この場合、水系の溶媒として希塩酸を用い溶解が行われる。それぞれは所定の粒子径に粉砕、分級される。微生物保存用の多孔質シート状物は、前出の水系の溶媒に不溶、もしくは難溶な性質の樹脂材料から選択される。水溶性の管路予定被溶解物とは、ゼラチン繊維をはじめ、ポリビニルアルコール(PVA)、ポリビニルピロリドン(PVP)等から調製される繊維状物である。   Specifically, the water-soluble granular material to be dissolved is a crystal of sugar such as glucose crystal, rock sugar, or powdered sugar (sugar coagulum). In the case of salt crystals, for example, sodium chloride crystals, alum crystals, potassium nitrate crystals, and the like. Limestone or calcium carbonate crystals can be used as a water-soluble substance to be dissolved. In this case, dissolution is performed using dilute hydrochloric acid as an aqueous solvent. Each is pulverized and classified to a predetermined particle size. The porous sheet-like material for preserving microorganisms is selected from resin materials that are insoluble or hardly soluble in the aqueous solvent described above. The water-soluble material to be dissolved in the pipeline is a fibrous material prepared from gelatin fiber, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) and the like.

水系の溶剤の別形態として、粒状被溶解物、管路予定被溶解物は酵素により分解され溶解可能となる物質から選択される。すなわち当該酵素の基質が用いられる。使用する酵素は、アミラーゼ、プルラナーゼ、セルラーゼ、リパーゼ、プロテアーゼ(ペプチダーゼ)等の加水分解酵素から適切に選択され、基質に応じて単一種の酵素、あるいは複数種の酵素が使用される。酵素と被溶解物との対応は両者間の基質特異性に依存する。アミラーゼ、プルラナーゼ、セルラーゼ等による場合、基質となる被除去物は糖鎖化合物となる。リパーゼは直鎖カルボン酸、トリグリセリド、パラフィン等の油脂類の分解に用いられる。プロテアーゼ(ペプチダーゼ)はタンパク質、あるいはペプチド結合、アミド結合を有する高分子化合物、ポリ乳酸等の加水分解に用いられる。   As another form of the water-based solvent, the granular material to be dissolved and the pipeline target material to be dissolved are selected from substances which are decomposed by an enzyme and can be dissolved. That is, the enzyme substrate is used. The enzyme to be used is appropriately selected from hydrolases such as amylase, pullulanase, cellulase, lipase, and protease (peptidase), and a single type of enzyme or a plurality of types of enzymes are used depending on the substrate. The correspondence between the enzyme and the substance to be lysed depends on the substrate specificity between them. In the case of amylase, pullulanase, cellulase, etc., the substance to be removed becomes a sugar chain compound. Lipase is used for the decomposition of fats and oils such as linear carboxylic acids, triglycerides and paraffins. Proteases (peptidases) are used for hydrolysis of proteins, polymer compounds having peptide bonds or amide bonds, polylactic acid, and the like.

具体例を明示すると、基質となる粒状被溶解物がデンプンの粒子であり、管路予定被溶解物がゼラチン繊維、デンプン繊維、ポリビニルピロリドン、ポリビニルアルコール、水溶性アクリル樹脂等の繊維である場合、酵素にはα,β−アミラーゼ、加えてプルラナーゼ等が選択される。同時に、多孔質体を形成するシート状物は、前記のアミラーゼ等の加水分解を受けない、あるいは受けにくい組成とする必要がある。例えば、基材はポリエチレン、ポリプロピレン等の樹脂素材となる。他に管路予定被溶解物には植物の繊維である木綿糸、麻糸、または絹糸やポリグルタミンの繊維等を用いることもできる。   If a specific example is specified, the granular material to be dissolved as a substrate is starch particles, and the planned material to be dissolved in the pipeline is gelatin fiber, starch fiber, polyvinyl pyrrolidone, polyvinyl alcohol, water-soluble acrylic resin, or the like, As the enzyme, α, β-amylase, pullulanase and the like are selected. At the same time, the sheet-like material forming the porous body needs to have a composition that does not undergo hydrolysis of the amylase or the like, or is difficult to receive. For example, the base material is a resin material such as polyethylene or polypropylene. In addition, the cotton line, hemp thread, silk thread, polyglutamine fiber, etc., which are plant fibers, can also be used for the to-be-dissolved material.

デンプン粒子の形態や粒径は植物種によって異なり、粒径は約1〜100μmである。例えば、馬鈴薯デンプンの粒子は平均粒径約30〜40μmの楕円形であり、コーンスターチ粒子は平均粒径13〜15μm程度でその形状はやや角張っている。他に、タピオカデンプンの粒子は約5〜15μm、食用カンナのデンプン粒子は約30〜40μmの粒径である。出来上がるシート状物の厚さに応じてこれらのデンプン粒子が粒状被溶解物として選択される。さらに、前記のデンプンの粒子径を上回る大きさの空洞部を形成する場合には、米や麦等の粒をそのまま使用してもよく、あるいはデンプン粉を練って粒状とした粒子体を使用してもよい。なお、酵素分解を容易にするため、成形体は分解するデンプンの糊化温度以上の温水浴中にて加温され、デンプンの糊化(アルファ化)が促進される場合もある。   The form and particle size of starch particles vary depending on the plant species, and the particle size is about 1 to 100 μm. For example, potato starch particles have an elliptical shape with an average particle size of about 30 to 40 μm, and corn starch particles have an average particle size of about 13 to 15 μm, and the shape thereof is somewhat angular. In addition, tapioca starch particles are about 5-15 μm, and edible canna starch particles are about 30-40 μm. Depending on the thickness of the finished sheet, these starch particles are selected as the granular material to be dissolved. Furthermore, when forming a cavity having a size larger than the above-mentioned starch particle size, grains such as rice and wheat may be used as they are, or a granulated granule obtained by kneading starch powder is used. May be. In order to facilitate enzymatic degradation, the molded body may be heated in a warm water bath that is equal to or higher than the gelatinization temperature of the starch to be decomposed to promote starch gelatinization (pregelatinization) in some cases.

酵素処理に供する酵素溶液は、当該酵素の活性が最適に反映される至適温度、至適pHに維持される。粒状被溶解物、管路予定被溶解物(つまり基質)の酵素加水分解物により、酵素溶液自体のpH等が変化することもあり得るため、適宜の緩衝液が添加されることもある。また、酵素加水分解物が反応阻害剤としても作用する懸念もあり得ることから、連続処理、回分処理を適式に組み合わせて行われる。併せて、用途に応じ、必要により残存する酵素の失活を行う場合もある。例えば、アルコール、高塩溶液、酸や塩基の溶液へ浸す他、加熱することもある。なお、樹脂材料の性質によるものの、速度反応論を加味して、至適温度を高めとする酵素の選択が好ましい。酵素処理を利用する利点は、水に不溶、難溶、あるいは含水に伴ってゲル化や粘調化する溶解物を用い、より速い処理速度により空洞部、中空管路の形成が可能となることである。   The enzyme solution subjected to the enzyme treatment is maintained at an optimal temperature and an optimal pH at which the activity of the enzyme is optimally reflected. Since the pH and the like of the enzyme solution itself may change due to the enzyme-hydrolyzed product of the granular material to be dissolved and the pipeline-specific material to be dissolved (that is, the substrate), an appropriate buffer may be added. In addition, since the enzyme hydrolyzate may also act as a reaction inhibitor, the continuous treatment and the batch treatment are performed in an appropriate combination. In addition, depending on the application, the remaining enzyme may be deactivated if necessary. For example, it may be heated in addition to dipping in an alcohol, high salt solution, acid or base solution. Although depending on the properties of the resin material, it is preferable to select an enzyme that increases the optimum temperature in consideration of the kinetics. The advantage of using enzyme treatment is that it is possible to form cavities and hollow pipes at higher processing speeds by using a solution that is insoluble, sparingly soluble, or gelled or thickened with water content. That is.

水系の溶剤(酵素を含む)に加えて有機溶剤も粒状被溶解物、管路予定被溶解物の溶解、除去のために加えられる。有機溶剤の種類は、メタノール、エタノール、イソプロパノール、ブタノールをはじめとする各種アルコール類、ジメチルエーテル、ジエチルエーテル、メチルエチルエーテル等のエーテル類、他にアセトン、メチルエチルケトン等のケトン類、酢酸エチル、他にアセトニトリル等、また、へキサン、シクロヘキサン、オクタン、ベンゼン、トルエン、キシレン、ピリジン、クロロホルム、テトラクロロエチレン、シリコーンオイル、テルペン類、リモネン等のいずれであっても良い。これらは、単独種で用いることもできるが、シート状物を形成する樹脂基材及び各被溶解物の溶解性に鑑み複数種の有機溶剤を混合調整して用いることができる。シート状物を形成する樹脂基材と各被溶解物が共に油溶性成分である場合であっても、被溶解物側のみ特に有機溶剤に溶解しやすい樹脂種を選択し、シート状物を形成する樹脂基材の溶解が進行する以前に溶剤を除去することも考えられる。各被溶解物を有機溶剤に溶出させた後、多孔質体は適宜乾燥される。   In addition to the aqueous solvent (including the enzyme), an organic solvent is also added to dissolve and remove the granular material to be dissolved and the material to be dissolved in the pipeline. The types of organic solvents are methanol, ethanol, isopropanol, butanol and other alcohols, ethers such as dimethyl ether, diethyl ether and methyl ethyl ether, ketones such as acetone and methyl ethyl ketone, ethyl acetate, and acetonitrile. Or any of hexane, cyclohexane, octane, benzene, toluene, xylene, pyridine, chloroform, tetrachloroethylene, silicone oil, terpenes, limonene, and the like. These can be used alone, but a plurality of types of organic solvents can be mixed and used in view of the solubility of the resin base material forming the sheet-like material and each material to be dissolved. Even if the resin base material that forms the sheet-like material and each material to be dissolved are both oil-soluble components, the resin type that is particularly soluble in the organic solvent is selected only on the material-to-be-dissolved side to form the sheet-like material. It is also conceivable to remove the solvent before the dissolution of the resin base material to proceed. After eluting each substance to be dissolved in an organic solvent, the porous body is appropriately dried.

有機溶剤により溶解、除去される粒状被溶解物、管路予定被溶解物には、例えば、ポリスチレン、ポリ乳酸、ポリカプロラクトン、ポリエチレンサクシネート、ポリブチレンサクシネート等の樹脂が挙げられる。   Examples of the granular material to be dissolved and removed by the organic solvent include the resins such as polystyrene, polylactic acid, polycaprolactone, polyethylene succinate, and polybutylene succinate.

シート状物(成形体や接合物)からの粒状被溶解物、管路予定被溶解物の溶解(分解)、除去は、簡単に調達できる水、酵素、または有機溶剤のいずれかとなるため、製造経費を軽減することができる。同時に、これらにより溶解、除去される組成であるため、多孔質構造体を製造する際の量産性に優れる。   Manufacture is possible because the dissolution (decomposition) and removal of the granular material to be dissolved from the sheet-like material (molded product and bonded product) and the material to be dissolved in the pipeline are either water, enzymes, or organic solvents that can be easily procured. Expenses can be reduced. At the same time, since the composition is dissolved and removed by these, it is excellent in mass productivity when producing a porous structure.

これまでの説明から明らかであるように、本発明の微生物の保存方法及び微生物保存部材に供する微生物保存用多孔質シート状物は樹脂材料を構造骨格として形成されていることから、軽量であり、取り扱いが便利である。とりわけ、シート状であるため、折り曲げや積層等が容易である。微生物の保存期間、使用目的に応じてシート状物の厚さや大きさを自由に設定できるため、保持可能な微生物量の調節も容易である。また、多孔質のシート状物を採用したことにより、微生物が付着可能な内部空間を広げることができ、単位体積当たりの微生物の保持量が高められる。   As is clear from the above description, the microorganism-preserving porous sheet-like material to be used for the microorganism-preserving method and the microorganism-preserving member of the present invention is formed from a resin material as a structural skeleton, and is lightweight. Handling is convenient. In particular, since it is in the form of a sheet, it can be easily folded and laminated. Since the thickness and size of the sheet-like material can be freely set according to the storage period of microorganisms and the purpose of use, it is easy to adjust the amount of microorganisms that can be retained. In addition, by adopting a porous sheet-like material, the internal space to which microorganisms can adhere can be expanded, and the amount of microorganisms retained per unit volume can be increased.

[使用材料]
発明者らは、実施例1ないし4の微生物保存用多孔質シート状物を構成する樹脂材料として、直鎖状低密度ポリエチレン(宇部丸善ポリエチレン株式会社製:ユメリット ZM033)(以下同樹脂を「LLDPE樹脂」と略記する。)、エチレンビニルアルコール樹脂(日本合成化学工業株式会社製:ソノアール4412)(以下同樹脂を「EVOH樹脂」と略記する。)、生分解性樹脂であるポリ乳酸樹脂(和光純薬工業株式会社製:PLA−0020)(以下同樹脂を「PLA樹脂」と略記する。)の3種類の樹脂を用いた。
[Materials used]
The inventors have used a linear low density polyethylene (Ube Maruzen Polyethylene Co., Ltd .: Umerit ZM033) (hereinafter referred to as “LLDPE”) as a resin material constituting the porous sheet for preserving microorganisms of Examples 1 to 4. Abbreviated as “resin”), ethylene vinyl alcohol resin (manufactured by Nippon Synthetic Chemical Industry Co., Ltd .: Sonoir 4412) (hereinafter the resin is abbreviated as “EVOH resin”), polylactic acid resin (Japanese Three types of resins, manufactured by Kojun Pharmaceutical Co., Ltd .: PLA-0020) (hereinafter, the resin is abbreviated as “PLA resin”) were used.

連通多孔質体を形成する空洞部の基となる事後的に溶解可能な粒状被溶解物として馬鈴薯デンプン(東海澱粉株式会社製:丸特士幌(平均粒径約30μm))を用い、また、管路予定被溶解物としてポリビニルピロリドン(BASF社製:Luvitec K90)を用いた。粒状被溶解物の溶解に際し、耐熱α−アミラーゼ(大和化成株式会社製:クライスターゼT10S)を用いた。   Potato starch (manufactured by Tokai Starch Co., Ltd .: Marutoshijihoro (average particle size of about 30 μm)) is used as a granular material that can be dissolved afterwards, which is the basis of the cavity forming the continuous porous body. Polyvinylpyrrolidone (manufactured by BASF: Luvitec K90) was used as the road to be dissolved. A heat-resistant α-amylase (manufactured by Daiwa Kasei Co., Ltd .: Christase T10S) was used for dissolving the granular material to be dissolved.

保護剤溶液は、ポリビニルアルコール(日本合成化学工業株式会社製:ゴーセノールNM−11Q)の10%水溶液と、トレハロース(株式会社林原製:トレハ)の10%水溶液とを等量ずつ混合し調製した。   The protective agent solution was prepared by mixing equal amounts of a 10% aqueous solution of polyvinyl alcohol (Nippon Gosei Chemical Co., Ltd .: Gohsenol NM-11Q) and a 10% aqueous solution of trehalose (Hayashibara Co., Ltd .: Treha).

[使用微生物]
保存耐性の評価に際し、酵母(Saccharomyces cerevisiae);財団法人日本醸造協会が供給する清酒用酵母(きょうかい7号)を用いた。また、大腸菌(Escherichia coli);NBRC3301株を用いた。
[Microbe used]
In the evaluation of storage tolerance, yeast (Saccharomyces cerevisiae); yeast for sake (Kyokai No. 7) supplied by the Japan Brewing Association was used. Also, Escherichia coli; NBRC3301 strain was used.

[実施例1の微生物保存用多孔質シート状物の作成]
粒状被溶解物(馬鈴薯デンプン)70重量部をLLDPE樹脂30重量部に混入し、170℃に加熱し樹脂を溶融しながら混錬してLLDPE樹脂混練物を得た。LLDPE樹脂混練物をステンレス鏡面板内に注入し、150℃を維持しながら10MPaで5分間押圧して加熱プレス成形した。成形後、冷却、裁断してLLDPE樹脂シート状物(縦5cm×横30cm,厚さ500μm)を得た。
[Preparation of Porous Sheet for Preserving Microorganisms of Example 1]
70 parts by weight of a granular material to be dissolved (potato starch) was mixed in 30 parts by weight of LLDPE resin, and the mixture was heated to 170 ° C. and kneaded while melting the resin to obtain an LLDPE resin kneaded product. The LLDPE resin kneaded material was poured into a stainless steel mirror face plate, and pressed at 10 MPa for 5 minutes while maintaining 150 ° C. to be hot press molded. After molding, the product was cooled and cut to obtain an LLDPE resin sheet (length 5 cm × width 30 cm, thickness 500 μm).

耐熱α−アミラーゼを1重量%含み、80℃に加温した熱水浴中にLLDPE樹脂シート状物を2時間浸漬した後、40℃の超音波浴中に5分間浸漬した。続いて、吸引びんにブフナー漏斗を接続すると共にこの漏斗上に載置し、吸引しながら蒸留水を通水した。ブフナー漏斗を通過した通過液が糖類の呈色反応を示さなくなるまで蒸留水の通水洗浄を続けた。洗浄後、80℃の乾燥機内で24時間乾燥した。こうして、LLDPE樹脂シート状物から粒状被溶解物を溶解して除去し、シート状物の内部に空洞部を有する微生物保存用多孔質シート状物(実施例1)を作成した。   The LLDPE resin sheet-like material was immersed in a hot water bath containing 1% by weight of heat-resistant α-amylase and heated to 80 ° C. for 2 hours, and then immersed in an ultrasonic bath at 40 ° C. for 5 minutes. Subsequently, a Buchner funnel was connected to the suction bottle and placed on the funnel, and distilled water was passed through while sucking. Washing with distilled water was continued until the liquid passing through the Buchner funnel showed no saccharide color reaction. After washing, it was dried in a dryer at 80 ° C. for 24 hours. In this way, the granular material to be dissolved was dissolved and removed from the LLDPE resin sheet material to prepare a porous sheet material for preservation of microorganisms (Example 1) having a cavity inside the sheet material.

図12は実施例1の微生物保存用多孔質シート状物内部を500倍に拡大して示す電子顕微鏡写真である。写真から自明なとおり、微生物保存用多孔質シート状物は内部に無数の空洞部を有している。空洞部の形状は、粒状被溶解物(実施例では馬鈴薯デンプン)の粒形状に依存する。そして、それぞれの空洞部同士は互いに連通している。従って、微生物の懸濁液や保護剤溶液の浸透、流入が可能であることがわかる。   FIG. 12 is an electron micrograph showing the inside of the porous sheet-like material for preserving microorganisms of Example 1 magnified 500 times. As is obvious from the photograph, the porous sheet-like material for preserving microorganisms has innumerable cavities inside. The shape of the hollow portion depends on the particle shape of the granular material to be dissolved (potato starch in the examples). And each cavity part is mutually connected. Therefore, it can be seen that the microbial suspension and the protective agent solution can permeate and flow in.

[実施例2の微生物保存用多孔質シート状物の作成]
粒状被溶解物(馬鈴薯デンプン)70重量部をEVOH樹脂30重量部に混入し、170℃に加熱し樹脂を溶融しながら混錬してEVOH樹脂混練物を得た。EVOH樹脂混練物をステンレス鏡面板内に注入し、170℃を維持しながら10MPaで5分間押圧して加熱プレス成形した。成形後、冷却、裁断してEVOH樹脂シート状物(縦5cm×横30cm,厚さ500μm)を得た。
[Preparation of Porous Sheet for Preserving Microorganisms of Example 2]
70 parts by weight of a granular material to be dissolved (potato starch) was mixed in 30 parts by weight of EVOH resin, and the mixture was heated to 170 ° C. and kneaded while melting the resin to obtain an EVOH resin kneaded product. The EVOH resin kneaded material was poured into a stainless steel mirror face plate and pressed at 10 MPa for 5 minutes while maintaining 170 ° C. to be hot press molded. After molding, cooling and cutting were performed to obtain an EVOH resin sheet (length 5 cm × width 30 cm, thickness 500 μm).

EVOH樹脂シート状物からの粒状被溶解物の溶解、除去は、実施例1と同様とした。そして、シート状物の内部に空洞部を有する微生物保存用多孔質シート状物(実施例2)を作成した。   The dissolution and removal of the granular material to be dissolved from the EVOH resin sheet were the same as in Example 1. And the porous sheet-like material for microorganism preservation (Example 2) which has a cavity part inside a sheet-like material was created.

[実施例3の微生物保存用多孔質シート状物の作成]
粒状被溶解物(馬鈴薯デンプン)70重量部をPLA樹脂30重量部に混入し、170℃に加熱し樹脂を溶融しながら混錬してPLA樹脂混練物を得た。PLA樹脂混練物をステンレス鏡面板内に注入し、170℃を維持しながら10MPaで5分間押圧して加熱プレス成形した。成形後、冷却、裁断してPLA樹脂シート状物(縦5cm×横30cm,厚さ500μm)を得た。
[Production of Porous Sheet Material for Preserving Microorganisms of Example 3]
70 parts by weight of a granular material to be dissolved (potato starch) was mixed in 30 parts by weight of PLA resin, kneaded while melting the resin by heating to 170 ° C. to obtain a PLA resin kneaded product. The PLA resin kneaded product was poured into a stainless steel mirror face plate and pressed at 10 MPa for 5 minutes while maintaining 170 ° C. to be hot press molded. After molding, it was cooled and cut to obtain a PLA resin sheet (length 5 cm × width 30 cm, thickness 500 μm).

PLA樹脂シート状物からの粒状被溶解物の溶解、除去は、実施例1と同様とした。そして、シート状物の内部に空洞部を有する微生物保存用多孔質シート状物(実施例3)を作成した。   The dissolution and removal of the granular material to be dissolved from the PLA resin sheet was the same as in Example 1. And the porous sheet-like material for microorganism preservation (Example 3) which has a cavity part inside a sheet-like material was created.

[実施例4の微生物保存用多孔質シート状物の作成]
メタノールにポリビニルピロリドン(PVP)を溶解してPVPの10%(w/v)溶液とした。同PVP溶液を卓上用塗布装置(武蔵エンジニアリング株式会社製:商品名「SHOT mini 100S」)に装着した内径0.2mmのニードル付きシリンジ内に充填した。実施例1にて作成した厚さ300μmのLLDPE樹脂シート状物の表面に対し、卓上用塗布装置からPVP溶液を直線上に、1cmずつ互いの直線の間隔を空けて吐出した。適当な本数のPVP溶液の直線が並んだところで、オーブンにより80℃で乾燥した。こうして、LLDPE樹脂シート状物の接合面(貼り合わせ面)に管路予定被溶解物を載置した。
[Production of Porous Sheet Material for Preserving Microorganisms of Example 4]
Polyvinylpyrrolidone (PVP) was dissolved in methanol to give a 10% (w / v) solution of PVP. The PVP solution was filled into a syringe with a needle having an inner diameter of 0.2 mm attached to a tabletop coating apparatus (manufactured by Musashi Engineering Co., Ltd .: trade name “SHOT mini 100S”). On the surface of the LLDPE resin sheet having a thickness of 300 μm prepared in Example 1, the PVP solution was discharged from the desktop coating apparatus in a straight line by 1 cm at intervals of each straight line. When an appropriate number of straight lines of the PVP solution were arranged, they were dried at 80 ° C. in an oven. In this way, the pipe line to-be-dissolved material was placed on the bonding surface (bonding surface) of the LLDPE resin sheet.

続いて、管路予定被溶解物を載置したLLDPE樹脂シート状物の接合面(貼り合わせ面)に、実施例1にて作成した厚さ300μmのLLDPE樹脂シート状物を被せ、150℃を維持しながら10MPaで5分間押圧して加熱プレス成形した。成形後、冷却、裁断してLLDPE樹脂接合物(縦5cm×横30cm,厚さ500μm)を得た。   Subsequently, the LLDPE resin sheet-like material having a thickness of 300 μm created in Example 1 was placed on the joining surface (bonding surface) of the LLDPE resin sheet-like material on which the pipeline to-be-dissolved material was placed, and 150 ° C. was applied. While maintaining, pressing was performed at 10 MPa for 5 minutes to perform hot press molding. After molding, the product was cooled and cut to obtain an LLDPE resin bonded product (length 5 cm × width 30 cm, thickness 500 μm).

LLDPE樹脂接合物からの粒状被溶解物及び管路予定被溶解物の溶解、除去は、実施例1と同様とした。そして、シート状物の内部に空洞部と共に中空管路を有する微生物保存用多孔質シート状物(実施例4)を作成した(厚さ約500μm)。   The dissolution and removal of the granular material to be dissolved and the pipe line predetermined material from the LLDPE resin joined product were the same as in Example 1. And the porous sheet-like material for microorganism preservation (Example 4) which has a hollow pipe line with a hollow part inside the sheet-like material was created (thickness of about 500 μm).

[多孔質多層構造体の作成と保護剤溶液の含浸]
前記の実施例の中から、実施例1及び実施例4の微生物保存用多孔質シート状物を直径約1cmのガラス管により巻き取り端をテープにより接着して、巻き取りロール形状の多孔質多層構造体に仕上げた。実施例1の微生物保存用多孔質シート状物から形成した巻き取りロール形状の多孔質多層構造体をPS1とし、同様に実施例4由来の多孔質多層構造体をPS4とする。前出の保護剤溶液の入ったビーカー内に多孔質多層構造体(PS1,PS4)を沈め、浮き上がらないようにステンレスメッシュで重しをした。保護剤溶液の入ったビーカーごと真空デシケーターに移し真空ポンプを用いて真空デシケーター内を脱気し、各実施例に由来する多孔質多層構造体内部に保護剤溶液を浸透させた。その後、保護剤溶液のビーカーから多孔質多層構造体を取り出し、余分な保護剤溶液をふき取り、オーブンにより80℃で乾燥した。
[Preparation of porous multilayer structure and impregnation with protective agent solution]
Among the above-mentioned examples, the porous sheet-like material for preserving microorganisms of Examples 1 and 4 is bonded to the winding end with a glass tube having a diameter of about 1 cm with a tape to form a porous multilayer in the form of a winding roll. Finished with a structure. A porous multilayer structure in the form of a take-up roll formed from a porous sheet-like material for preserving microorganisms of Example 1 is designated PS1, and similarly, a porous multilayer structure derived from Example 4 is designated PS4. The porous multilayer structure (PS1, PS4) was submerged in a beaker containing the above-mentioned protective agent solution, and weighted with a stainless mesh so as not to float. The beaker containing the protective agent solution was transferred to a vacuum desiccator, and the inside of the vacuum desiccator was deaerated using a vacuum pump, and the protective agent solution was permeated into the porous multilayer structure derived from each example. Then, the porous multilayer structure was taken out from the beaker of the protective agent solution, the excess protective agent solution was wiped off, and dried at 80 ° C. in an oven.

図13は、前掲図12の実施例1の微生物保存用多孔質シート状物に対し保護剤溶液を浸透させ、500倍に拡大して示す電子顕微鏡写真である。微生物保存用多孔質シート状物内部の空洞部が保護剤により覆われている様子がわかる。   FIG. 13 is an electron micrograph showing the protective agent solution permeated into the porous sheet for preserving microorganisms of Example 1 of FIG. It can be seen that the cavity inside the porous sheet-like material for preserving microorganisms is covered with a protective agent.

[微生物懸濁液の調製及びその含浸]
〈大腸菌の培養、懸濁液の調製〉
前出の大腸菌を寒天培地(Difco社製:NAAgar(スラント用))に接種し培養した。寒天培地にて培養した大腸菌から白金耳2回分の画線を分取し、1/500の濃度に希釈した液体培地(Difco社製:NB培地)9mLの中に懸濁した。液体培地の大腸菌懸濁液の吸光度を吸光光度計により測定した(波長:660nm)。吸光度と大腸菌数の相関から、同懸濁液中の大腸菌の濃度を1mL当たり107〜108個と推定した。これをさらに前記の液体培地により希釈して105個/mLの大腸菌濃度の懸濁液とした。
[Preparation of microorganism suspension and its impregnation]
<Culture of Escherichia coli, preparation of suspension>
The aforementioned E. coli was inoculated into an agar medium (Difco: NAAgar (for slant)) and cultured. Two lines of platinum ears were collected from E. coli cultured on an agar medium and suspended in 9 mL of a liquid medium (manufactured by Difco: NB medium) diluted to a concentration of 1/500. The absorbance of the E. coli suspension in the liquid medium was measured with an absorptiometer (wavelength: 660 nm). From the correlation between the absorbance and the number of E. coli, the concentration of E. coli in the suspension was estimated to be 10 7 to 10 8 per mL. This was further diluted with the above liquid medium to obtain a suspension with an E. coli concentration of 10 5 cells / mL.

〈酵母の培養、懸濁液の調製〉
前出の酵母を寒天培地(Difco社製:YMAgar)に接種し培養した。寒天培地にて培養した酵母から白金耳2回分の画線を分取し、液体培地(酵母エキス3g、麦芽エキス3g、ポリペプトン5g、グルコース10g、クロラムフェニコール0.05g、及び蒸留水1Lからなる。)9mL内に懸濁した。液体培地の酵母懸濁液の吸光度を吸光光度計により測定した(波長:660nm)。吸光度と酵母数の相関から、同懸濁液中の酵母の濃度を1mL当たり107〜108個と推定した。これをさらに液体培地により希釈して105個/mLの酵母濃度の懸濁液とした。
<Yeast culture, suspension preparation>
The yeast described above was inoculated into an agar medium (Difco: YMAgar) and cultured. From the yeast cultured on the agar medium, two streaks of platinum ears were collected, and from the liquid medium (3 g yeast extract, 3 g malt extract, 5 g polypeptone, 10 g glucose, 0.05 g chloramphenicol, and 1 L distilled water. It was suspended in 9 mL. The absorbance of the yeast suspension in the liquid medium was measured with an absorptiometer (wavelength: 660 nm). From the correlation between the absorbance and the number of yeasts, the concentration of yeast in the suspension was estimated to be 10 7 to 10 8 per mL. This was further diluted with a liquid medium to obtain a suspension having a yeast concentration of 10 5 cells / mL.

[多孔質多層構造体内への微生物の付着]
実施例1、実施例4の各微生物保存用多孔質シート状物からなる保護剤付きの多孔質多層構造体(PS1,PS4)のそれぞれについて、50%エタノール溶液中に沈めて当該多孔質多層構造体の空洞部内にエタノール溶液を浸透させた。滅菌シャーレに取り出し蓋をして、減圧乾燥後、オーブンにより80℃で乾燥した。
[Adherence of microorganisms in porous multilayer structure]
Each of the porous multilayer structures (PS1, PS4) with a protective agent made of the porous sheet-like material for preserving microorganisms of Examples 1 and 4 was immersed in a 50% ethanol solution and the porous multilayer structure. The ethanol solution was infiltrated into the cavity of the body. The sterilized petri dish was taken out, covered, dried under reduced pressure, and dried in an oven at 80 ° C.

前出の大腸菌懸濁液100mLを200mLのビーカーに入れ、このビーカーに多孔質多層構造体(PS1,PS4)のそれぞれを沈め、浮き上がらないようにステンレスメッシュで重しをした。懸濁液の入ったビーカーごと真空デシケーターに移し真空ポンプを用いて真空デシケーター内を脱気し、多孔質多層構造体(PS1,PS4)のそれぞれの内部に懸濁液を浸透させた。懸濁液のビーカーから多孔質多層構造体を取り出し、減圧乾燥機により25℃、2時間乾燥した。こうしてPS1,PS4の各多孔質多層構造体の空洞部内に大腸菌を付着させた。酵母についても前記大腸菌の付着と同様の手順、条件で処理を行い、PS1の多孔質多層構造体の空洞部内に酵母を付着させた。以下、PS1の大腸菌付着物を「PS1E」、PS4の大腸菌付着物を「PS4E」、PS1の酵母付着物を「PS1S」と表記する。   The aforementioned E. coli suspension (100 mL) was placed in a 200 mL beaker, and each of the porous multilayer structures (PS1, PS4) was submerged in this beaker, and weighted with a stainless mesh so as not to float. The beaker containing the suspension was transferred to a vacuum desiccator, and the inside of the vacuum desiccator was deaerated using a vacuum pump, so that the suspension was infiltrated into each of the porous multilayer structures (PS1, PS4). The porous multilayer structure was taken out from the suspension beaker and dried at 25 ° C. for 2 hours with a vacuum dryer. Thus, Escherichia coli was adhered in the cavity of each porous multilayer structure of PS1 and PS4. The yeast was also treated in the same procedure and conditions as for the attachment of E. coli, and the yeast was allowed to adhere in the cavity of the porous multilayer structure of PS1. Hereinafter, the PS1 E. coli adhering material is referred to as “PS1E”, the PS4 E. coli adhering material is referred to as “PS4E”, and the PS1 yeast adhering material is referred to as “PS1S”.

[多孔質多層構造体内への微生物の付着、パラフィンによる表面被覆]
保存時の多孔質多層構造体表面からの酸素の流通、通気の抑制ないし阻止の効果も併せて評価するため、パラフィンによる被覆を試みた。100mLのビーカーに融点42〜44℃のパラフィン(米山薬品工業株式会社製)を50g入れ、オートクレーブを用いてパラフィンを滅菌し液温を50℃に冷ました。その後、PS1及びPS4の空洞部内に大腸菌を付着した多孔質多層構造体(PS1E及びPS4E)をビーカー内のパラフィン中に投入して表面全体をパラフィンにより覆い、直ちに取り出した。取り出した後、滅菌シャーレに収容して封止し当該滅菌シャーレごと以下の条件で保存した。PS1Eのパラフィン被覆物を「PS1EC」、PS4Eのパラフィン被覆物を「PS4EC」と表記する。
[Adherence of microorganisms in porous multilayer structure, surface coating with paraffin]
In order to evaluate the effect of suppressing or preventing the flow of oxygen from the surface of the porous multilayer structure during storage and the suppression of airflow, a coating with paraffin was attempted. 50 g of paraffin (manufactured by Yoneyama Pharmaceutical Co., Ltd.) having a melting point of 42 to 44 ° C. was put into a 100 mL beaker, and the paraffin was sterilized using an autoclave and the liquid temperature was cooled to 50 ° C. Thereafter, porous multilayer structures (PS1E and PS4E) having E. coli adhered in the hollow portions of PS1 and PS4 were put into paraffin in a beaker, the entire surface was covered with paraffin, and immediately taken out. After taking out, it accommodated in the sterile petri dish, it sealed, and the said sterile petri dish was preserve | saved on the following conditions. The PS1E paraffin coating is referred to as “PS1EC”, and the PS4E paraffin coating is referred to as “PS4EC”.

[保存期間、保存条件]
大腸菌を付着した多孔質多層構造体(PS1E,PS4E)について、−60℃、4℃にて、4日間、14日間、一部1ヶ月間密封容器内で保存した。同大腸菌を付着した多孔質多層構造体(PS1EC,PS4EC)について、4℃にて、4日間、14日間密封容器内で保存した。また、酵母を付着した多孔質多層構造体(PS1S)について、−60℃、4℃にて、4日間、14日間密封容器内で保存した。
[Retention period and storage conditions]
The porous multilayer structure (PS1E, PS4E) attached with E. coli was stored in a sealed container at −60 ° C. and 4 ° C. for 4 days, 14 days and partly for 1 month. The porous multilayer structure (PS1EC, PS4EC) attached with the same E. coli was stored in a sealed container at 4 ° C. for 4 days and 14 days. Further, the porous multilayer structure (PS1S) attached with yeast was stored in a sealed container at −60 ° C. and 4 ° C. for 4 days and 14 days.

酵母を付着した多孔質多層構造体(PS1S)の4℃、4日間経過後の内部の様子を観察した。図14は酵母付着のPS1Sの500倍に拡大して示す電子顕微鏡写真である。前出の図12,13の写真(同倍率)との比較からわかるように、微生物保存用多孔質シート状物のどの空洞部にも付着物(すなわち酵母)が存在していることがわかる。図15は同じ多孔質多層構造体(PS1S)の1000倍に拡大した電子顕微鏡写真である。多孔質多層構造体の空洞部の内部に細かな粒状物が付着している。さらに、図16は空洞部を5000倍に拡大した電子顕微鏡写真である。多孔質多層構造体の空洞部の内表面に隙間なく酵母が付着している様子がわかる。このように、多孔質多層構造体の空洞部に微生物が付着可能であることが確認できた。   The state of the inside of the porous multilayer structure (PS1S) attached with yeast after 4 days at 4 ° C. was observed. FIG. 14 is an electron micrograph magnified 500 times that of PS1S adhering to yeast. As can be seen from the comparison with the above-mentioned photographs of FIGS. 12 and 13 (same magnification), it can be seen that deposits (that is, yeast) are present in any cavity of the porous sheet-like material for preserving microorganisms. FIG. 15 is an electron micrograph of the same porous multilayer structure (PS1S) magnified 1000 times. Fine particulate matter adheres to the inside of the cavity of the porous multilayer structure. Further, FIG. 16 is an electron micrograph in which the cavity is magnified 5000 times. It can be seen that the yeast adheres to the inner surface of the cavity of the porous multilayer structure without any gaps. Thus, it was confirmed that microorganisms can adhere to the cavity of the porous multilayer structure.

[微生物の生存確認]
各温度で保存した多孔質多層構造体(PS1E,PS4E,PS1EC,PS4EC,PS1S)について、前記の期間を経た後、多孔質多層構造体の巻き取りをほどいてもとのシート状とし、長さ方向の中間部分を1cmの幅で切り取った(縦5cm×横1cmのサンプル切片となる。)。PS1EC,PS4ECについては表面を覆うパラフィンを剥がし、同様に多孔質多層構造体の巻き取りをほどいてもとのシート状とし、サンプル切片を得た。
[Confirmation of microbe survival]
With respect to the porous multilayer structure (PS1E, PS4E, PS1EC, PS4EC, PS1S) stored at each temperature, after passing through the above period, the porous multilayer structure is unwound to obtain the original sheet shape, and the length A middle part in the direction was cut out with a width of 1 cm (a sample slice of 5 cm long × 1 cm wide). For PS1EC and PS4EC, the paraffin covering the surface was peeled off, and the porous multilayer structure was similarly unwound to obtain the original sheet shape, thereby obtaining a sample slice.

大腸菌の生存確認では、試験管に前出の大腸菌の培養に用いた液体培地(Difco社製:NB培地)を15mL入れ、ここにPS1E,PS4E,PS1EC,PS4ECに由来するそれぞれのサンプル切片を投入し、モルトン栓で蓋をした。続いて、試験管ごと真空デシケーターに移し真空ポンプを用いて真空デシケーター内を脱気し、サンプル切片の内部に液体培地を浸透させた。そして、インキュベーターにて35℃で24時間培養した。   For confirmation of the survival of E. coli, 15 mL of the liquid medium (Difco: NB medium) used for the above-mentioned culture of E. coli was placed in a test tube, and each sample slice derived from PS1E, PS4E, PS1EC, and PS4EC was added thereto. And capped with a Molton stopper. Subsequently, the whole test tube was transferred to a vacuum desiccator, the inside of the vacuum desiccator was deaerated using a vacuum pump, and the liquid medium was permeated into the sample section. And it culture | cultivated at 35 degreeC for 24 hours with the incubator.

酵母の生存確認では、試験管に前出の酵母の培養に用いた液体培地を15mL入れ、ここにPS1Sに由来するそれぞれのサンプル切片を投入し、モルトン栓で蓋をした。続いて、試験管ごと真空デシケーターに移し真空ポンプを用いて真空デシケーター内を脱気し、サンプル切片の内部に液体培地を浸透させた。そして、インキュベーターにて25℃で24時間培養した。   In the confirmation of the survival of the yeast, 15 mL of the liquid medium used for the yeast culture described above was put in a test tube, and each sample slice derived from PS1S was put therein, and capped with a Molton stopper. Subsequently, the whole test tube was transferred to a vacuum desiccator, and the inside of the vacuum desiccator was deaerated using a vacuum pump, and the liquid medium was permeated into the sample section. And it culture | cultivated at 25 degreeC for 24 hours with the incubator.

[培養液の吸光度の測定]
各温度、各経時日数のサンプル切片を投入して培養した培養液を1mLずつ分取し、吸光光度計により660nmの吸光度を測定し、培養液の濁りの程度を調べた。培養液のみの吸光度よりも吸光度が上昇した培養液については、微生物が生存しているものとして評価した。
[Measurement of absorbance of culture solution]
1 mL each of the culture broth cultured by introducing sample slices of each temperature and each elapsed time was measured and the absorbance at 660 nm was measured with an absorptiometer to examine the degree of turbidity of the culture broth. A culture solution having an absorbance higher than that of the culture solution alone was evaluated as a living microorganism.

[シャーレによる培養の評価]
大腸菌の場合、デソキシコレート寒天培地を20mL注入したシャーレに、各温度、各経時日数のサンプル切片を投入して培養した培養液を1mLずつ分注し、35℃で24時間インキュベートした。酵母の場合、前出の寒天培地(Difco社製:YMAgar)のシャーレに、各温度、各経時日数のサンプル切片を投入して培養した培養液を1mLずつ分注し、25℃で5日間インキュベートした。
[Evaluation of culture by petri dish]
In the case of Escherichia coli, 1 mL of a culture solution obtained by introducing sample sections of each temperature and each elapsed time into a petri dish into which 20 mL of desoxycholate agar medium was injected was incubated at 35 ° C. for 24 hours. In the case of yeast, 1 mL each of the culture solution in which the sample sections of each temperature and each aging period are put in a petri dish of the above-mentioned agar medium (Difco: YMAgar) is cultured and incubated at 25 ° C. for 5 days. did.

各温度で保存した多孔質多層構造体(PS1E,PS4E,PS1EC,PS4EC,PS1S)の所定期間経過後の生存評価は、表1ないし表3のとおりである。表1は大腸菌を付着した多孔質多層構造体(PS1E及びPS1EC)、表2は大腸菌を付着した多孔質多層構造体(PS4E及びPS4EC)、表3は酵母を付着した多孔質多層構造体(PS1S)の保存結果である。表中“○”は生存が確認できたサンプルであり、“×”は生存が確認できなかったサンプルである。   Tables 1 to 3 show the survival evaluation of the porous multilayer structure (PS1E, PS4E, PS1EC, PS4EC, PS1S) stored at each temperature after a predetermined period. Table 1 shows porous multilayer structures (PS1E and PS1EC) attached with E. coli, Table 2 shows porous multilayer structures (PS4E and PS4EC) attached with E. coli, and Table 3 shows porous multilayer structures (PS1S) attached with yeast. ). In the table, “◯” indicates a sample whose survival was confirmed, and “×” indicates a sample whose survival was not confirmed.

Figure 2011050320
Figure 2011050320

Figure 2011050320
Figure 2011050320

Figure 2011050320
Figure 2011050320

[結果と考察]
−60℃の冷凍状態の保存では長期保存が可能であることが確認できた。これは冷凍休眠状態となったためと考える。しかし、4℃における大腸菌の付着、保存の場合、生存日数は、酵母と比較して短いことが判明した。おそらく、大腸菌は酵母よりも体長が小さく、細胞膜の構造が酵母より脆弱であるため保存時の酸素の影響等により死滅したことが予想される。なお、多孔質多層構造体のPS1EとPS4Eとの比較では性能の差は生じなかった。また、多孔質多層構造体のPS2,PS3については、PS1とほぼ同様の構造であることから、PS1と同様の結果を示すことが予想できる。
[Results and discussion]
It was confirmed that long-term storage was possible when stored in a frozen state at −60 ° C. This is thought to be due to the frozen dormancy state. However, in the case of attachment and storage of E. coli at 4 ° C., it was found that the survival days were shorter than that of yeast. Probably, E. coli is shorter than yeast and its cell membrane structure is more fragile than yeast, so it is expected that it was killed by the influence of oxygen during storage. Note that there was no difference in performance between the porous multilayer structure PS1E and PS4E. Further, since PS2 and PS3 of the porous multilayer structure are substantially the same as PS1, it can be expected to show the same result as PS1.

実施例における多孔質多層構造体への微生物の付着、保存の試験、評価によると、保存性能の差異は微生物種に大きく依存するものと考える。そこで、広汎な種類の微生物の保存に対応できるようにするためには、多孔質多層構造体内部の空洞部の大きさの調整、保存時の酸素流通の抑制等の課題も明らかとなった。   According to the test and evaluation of microbial adhesion and storage to the porous multilayer structure in the examples, it is considered that the difference in storage performance largely depends on the microbial species. Therefore, in order to be able to cope with the preservation of a wide variety of microorganisms, problems such as the adjustment of the size of the cavity inside the porous multilayer structure and the suppression of oxygen circulation during storage have been clarified.

上記の点を踏まえ、大腸菌を付着し、その後表面をパラフィンで被覆した多孔質多層構造体(PS1EC,PS4EC)によると、4℃の温度下におけるPS1E及びPS4Eと比較して保存効率の向上を確認することができた。すなわち、多孔質多層構造体に微生物を付着させた後、その表面をパラフィンで被覆することは、保存時の酸素流通に伴う微生物への影響を軽減していることを示唆する。   Based on the above points, according to the porous multilayer structure (PS1EC, PS4EC) to which Escherichia coli was attached and then coated with paraffin on the surface, improved storage efficiency was confirmed compared to PS1E and PS4E at a temperature of 4 ° C. We were able to. That is, after attaching microorganisms to the porous multilayer structure, covering the surface with paraffin suggests that the influence on microorganisms due to oxygen circulation during storage is reduced.

本発明の微生物の保存方法は多孔質多層構造体内部の空洞部に特徴を有し、これを利用して微生物を保存する用途に都合がよい。本発明の微生物保存部材は多孔質多層構造体を用いるため、微生物の担持、保存し、そのまま微生物を利用する用途に好適である。本発明の微生物保存用多孔質シート状物の製造方法は多孔質多層構造体の製造を容易に可能とすることができ、量産の足がかりとなる。   The method for preserving microorganisms of the present invention has a feature in the cavity inside the porous multilayer structure, which is convenient for use for preserving microorganisms. Since the microorganism storage member of the present invention uses a porous multilayer structure, it is suitable for applications in which microorganisms are supported and stored and directly used. The method for producing a porous sheet-like material for preserving microorganisms of the present invention can easily produce a porous multilayer structure, which is a foothold for mass production.

10A,10B 多孔質シート状物
11 樹脂基材
12 空洞部
12w 空洞部の内壁
13 連通多孔質体
16 連通口
17 中空管路
20 多孔質多層構造体
21 巻き取りロール形状(巻きロール体)
101,240 成形体
110 樹脂材料からなる基材
120,205 粒状被溶解物
220 管路予定被溶解物
P1,P2 微生物保存部材
Mo 微生物
Pra 保護剤溶液の被膜
DESCRIPTION OF SYMBOLS 10A, 10B Porous sheet-like material 11 Resin base material 12 Cavity part 12w Inner wall of a cavity part 13 Communication porous body 16 Communication port 17 Hollow pipe line 20 Porous multilayer structure 21 Winding roll shape (winding roll body)
101,240 Molded body 110 Base material made of resin material 120,205 Granular material to be melted 220 Pipe line material to be melted P1, P2 Microorganism preservation member Mo Microbe Pra Protective agent coating

Claims (15)

樹脂基材の内部に空洞部同士が連通した連通多孔質体を形成すると共に該樹脂基材の表面に前記空洞部が開口している多孔質シート状物を多層化して多孔質多層構造体を形成し、前記多孔質多層構造体を微生物の懸濁液中に浸漬して前記多孔質多層構造体の前記空洞部内に微生物を付着させることを特徴とする微生物の保存方法。   A porous porous structure is formed by forming a continuous porous body in which cavities communicate with each other inside a resin base material and multilayering a porous sheet-like material having the cavities open on the surface of the resin base material. A method for preserving microorganisms, comprising forming and immersing the porous multilayer structure in a suspension of microorganisms to allow the microorganisms to adhere to the cavity of the porous multilayer structure. 前記多孔質多層構造体を微生物の懸濁液中に浸漬した後に該多孔質多層構造体を乾燥して乾燥多孔質多層構造体とする請求項1に記載の微生物の保存方法。   The method for preserving microorganisms according to claim 1, wherein the porous multilayer structure is dipped in a suspension of microorganisms and then dried to obtain a dry porous multilayer structure. 前記乾燥多孔質多層構造体の表面をろう、パラフィン、またはホットメルト樹脂のいずれかにより被覆する請求項2に記載の微生物の保存方法。   The method for preserving microorganisms according to claim 2, wherein the surface of the dry porous multilayer structure is coated with any one of wax, paraffin, and hot melt resin. 前記多孔質多層構造体を微生物の懸濁液中に浸漬する前に、前記多孔質多層構造体を保護剤溶液中に浸漬して該保護剤溶液を乾燥する請求項1ないし3のいずれか1項に記載の微生物の保存方法。   4. The method according to claim 1, wherein the porous multilayer structure is dipped in a protective agent solution and the protective agent solution is dried before the porous multilayer structure is immersed in the microorganism suspension. The method for preserving a microorganism according to Item. 前記多孔質多層構造体が巻き取りロール形状である請求項1ないし4のいずれか1項に記載の微生物の保存方法。   The method for preserving microorganisms according to any one of claims 1 to 4, wherein the porous multilayer structure has a winding roll shape. 前記多孔質シート状物の内部に中空管路が形成されている請求項1ないし5のいずれか1項に記載の微生物の保存方法。   The method for preserving microorganisms according to any one of claims 1 to 5, wherein a hollow conduit is formed inside the porous sheet-like material. 前記樹脂基材が生分解性樹脂よりなる請求項1ないし6のいずれか1項に記載の微生物の保存方法。   The method for preserving microorganisms according to any one of claims 1 to 6, wherein the resin substrate is made of a biodegradable resin. 請求項1ないし7のいずれか1項に記載の微生物の保存方法により、前記多孔質多層構造体の多孔質内に微生物を付着させたことを特徴とする微生物保存部材。   A microorganism storage member characterized in that microorganisms are attached to the inside of the porous multilayer structure by the microorganism storage method according to any one of claims 1 to 7. 樹脂基材の内部に空洞部同士が連通した連通多孔質体を形成すると共に該樹脂基材の表面に前記空洞部が開口している多孔質シート状物を多層化して多孔質多層構造体を形成し、前記多孔質多層構造体を微生物の懸濁液中に浸漬して前記多孔質多層構造体の前記空洞部内に微生物を付着させるための微生物保存用多孔質シート状物の製造方法であって、
事後的に溶解可能な粒状被溶解物を樹脂材料に混入して所定のシート状物に成形し、この後に前記粒状被溶解物を溶解することにより前記シート状物の内部に空洞部を有する多孔質体を形成することを特徴とする微生物保存用多孔質シート状物の製造方法。
A porous porous structure is formed by forming a continuous porous body in which cavities communicate with each other inside a resin base material and multilayering a porous sheet-like material having the cavities open on the surface of the resin base material. A method for producing a porous sheet-like material for preserving microorganisms, wherein the porous multilayer structure is formed and immersed in a suspension of microorganisms to allow the microorganisms to adhere to the hollow portions of the porous multilayer structure. And
A porous material having a hollow portion inside the sheet-like material by mixing a granular material to be dissolved afterwards into a resin material and forming it into a predetermined sheet-like material, and then dissolving the granular material to be dissolved. A method for producing a porous sheet-like material for preserving microorganisms, comprising forming a material.
前記シート状物に占める前記空洞部の体積割合を、少なくとも50%以上とする請求項9に記載の微生物保存用多孔質シート状物の製造方法。   The method for producing a porous sheet-like material for preserving microorganisms according to claim 9, wherein a volume ratio of the hollow portion in the sheet-like material is at least 50% or more. 前記粒状被溶解物が、水、酵素、または有機溶剤のいずれかによって除去される請求項9又は10に記載の微生物保存用多孔質シート状物の製造方法。   The method for producing a porous sheet-like material for preserving microorganisms according to claim 9 or 10, wherein the granular material to be dissolved is removed by any one of water, an enzyme, and an organic solvent. 前記粒状被溶解物を前記樹脂材料に混入して所定のシート状物を成形するに際し、事後的に溶解可能な管路予定被溶解物を混入し、所定のシート状物に成形した後に前記粒状被溶解物及び前記管路予定被溶解物を溶解して、前記シート状物の内部に空洞部と共に中空管路を有する多孔質体を形成する請求項9ないし11のいずれか1項に記載の微生物保存用多孔質シート状物の製造方法。   When forming the predetermined sheet-like material by mixing the granular material to be dissolved in the resin material, the granular material to be melted after the pipe can be dissolved afterwards and molded into the predetermined sheet-like material. 12. The porous material having a hollow pipe line together with a hollow portion inside the sheet-like material is formed by dissolving the material to be melted and the material to be melted in the pipe line. Of producing a porous sheet-like material for preserving microorganisms. 前記粒状被溶解物を前記樹脂材料に混入して所定のシート状物を成形し、前記シート状物同士を貼り合わせる際の接合面に事後的に溶解可能な管路予定被溶解物を載置して前記シート状物同士からなる接合物を成形した後に前記粒状被溶解物及び前記管路予定被溶解物を溶解して、前記接合物の内部に空洞部と共に中空管路を有する多孔質体を形成する請求項9ないし11のいずれか1項に記載の微生物保存用多孔質シート状物の製造方法。   The granular material to be melted is mixed into the resin material to form a predetermined sheet material, and a planned pipeline material to be melted later is placed on the joint surface when the sheet materials are bonded together. Then, after forming the joined product composed of the sheet-like materials, the granular material to be melted and the pipeline-scheduled material to be melted are dissolved, and a porous material having a hollow pipe and a hollow portion inside the joined material. The manufacturing method of the porous sheet-like material for microorganism preservation | save of any one of Claim 9 thru | or 11 which forms a body. 前記管路予定被溶解物が、水、酵素、または有機溶剤のいずれかによって除去される請求項12又は13に記載の微生物保存用多孔質シート状物の製造方法。   The method for producing a porous sheet-like material for preserving microorganisms according to claim 12 or 13, wherein the material to be dissolved in the pipeline is removed with water, an enzyme, or an organic solvent. 前記樹脂材料が生分解性樹脂よりなる請求項9ないし14のいずれか1項に記載の微生物保存用多孔質シート状物の製造方法。   The method for producing a porous sheet-like material for preserving microorganisms according to any one of claims 9 to 14, wherein the resin material comprises a biodegradable resin.
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