JP3493243B2 - Culture method of useful bacteria - Google Patents
Culture method of useful bacteriaInfo
- Publication number
- JP3493243B2 JP3493243B2 JP04666595A JP4666595A JP3493243B2 JP 3493243 B2 JP3493243 B2 JP 3493243B2 JP 04666595 A JP04666595 A JP 04666595A JP 4666595 A JP4666595 A JP 4666595A JP 3493243 B2 JP3493243 B2 JP 3493243B2
- Authority
- JP
- Japan
- Prior art keywords
- oil
- clay mineral
- culture
- clay
- useful
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【0001】[0001]
【産業上の利用分野】本発明は、水と油の二液相を乳化
させて成る培地に放線菌、糸状菌などを培養させ有用物
を発酵生産する培養方法に関し、より詳細には培養菌が
栄養分(原料)とする油と培養菌が生息する水とを含む
培養液(培地)を安定に乳化させ、培養効率を向上させ
るために用いる粘土鉱物から成る乳化剤に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culturing method for fermentatively producing a useful substance by culturing actinomycetes, filamentous fungi, etc. in a medium obtained by emulsifying two liquid phases of water and oil, and more specifically, culturing bacteria. Relates to an emulsifier composed of a clay mineral, which is used to stably emulsify a culture solution (medium) containing oil as a nutrient (raw material) and water in which a culture bacterium lives, and improve the culture efficiency.
【0002】更に本発明は、セピオライト等の繊維状粘
土鉱物が水と油を乳化安定化させる特性を利用して、油
脂、石油等を原料とする発酵生産並びに動植物油を含む
廃棄物及び廃水を微生物により効率良く処理する方法に
関する。Furthermore, the present invention utilizes the property of fibrous clay minerals such as sepiolite to emulsify and stabilize water and oil, thereby producing fermentative production from fats and oils and petroleum as well as waste and wastewater containing animal and vegetable oils. The present invention relates to a method for efficiently treating with a microorganism.
【0003】[0003]
【従来の技術】従来より互いに混じりあわない水と油の
二液相の系に乳化剤(界面活性剤)を添加し、攪拌混合
等の操作により安定に乳化させて得られるエマルジョン
(乳化状態)は、化粧品、農薬、医薬品、食料品、塗
料、接着剤等の工業分野に広く利用され、我々の生活に
密接に関係している。2. Description of the Related Art An emulsion (emulsified state) obtained by adding an emulsifier (surfactant) to a system of two liquid phases of water and oil, which have been immiscible with each other, and stably emulsifying the mixture by operations such as stirring and mixing has been known. It is widely used in the industrial fields of cosmetics, agricultural chemicals, pharmaceuticals, foods, paints, adhesives, etc. and is closely related to our lives.
【0004】一方、発酵工業においては、放線菌、糸状
菌などの有用菌を培養菌として用いて、工業的に有用物
であるイタコン酸、グルコン酸、クエン酸、乳酸などの
有機酸、セファマイシンC、ペニシリン、セファロスポ
リンCなどの抗生物質、グルコアミラーゼ、リパーゼな
どの酵素などが多く生産されている。On the other hand, in the fermentation industry, useful bacteria such as actinomycetes and filamentous fungi are used as culture bacteria to produce industrially useful organic acids such as itaconic acid, gluconic acid, citric acid and lactic acid, and cefamycin. Many antibiotics such as C, penicillin, cephalosporin C, enzymes such as glucoamylase and lipase are produced.
【0005】ところがこれらの有用菌を有用物を生産す
る培養生産菌として利用するためには、菌が生息する水
が必要である。またこれらの菌は、工業的に原料となる
大豆油、ナタネ油、牛脂等の油脂或いは石油系の油、疎
水性の有機物等を広く原料(栄養源)として繁殖する。However, in order to utilize these useful bacteria as culture-producing bacteria for producing useful products, water in which the bacteria live is necessary. In addition, these fungi widely propagate using oils and fats such as soybean oil, rapeseed oil and beef tallow, or petroleum-based oils and hydrophobic organic substances, which are industrially raw materials, as raw materials (nutrient sources).
【0006】従って有用菌が生息する水と菌が栄養源と
する油とは互いに混じりあわない二液相から成る系であ
って、菌と栄養源の油との接触は極めて困難である。よ
ってこの系を工業的に効率の良い培養生産系にする手段
としては、例えば菌が生息する水相(分散媒)中に原料
となる油相(分散質)を微細粒子として分散させ、この
系を安定な乳化状態にできれば、両者の接触が高まり培
養効率を著しく向上させることが期待される。[0006] Therefore, the water in which useful bacteria inhabit and the oil as a nutrient source for the bacteria are systems consisting of two liquid phases that are immiscible with each other, and it is extremely difficult to contact the bacteria with the oil as a nutrient source. Therefore, as a means to make this system an industrially efficient culture production system, for example, an oil phase (dispersoid) as a raw material is dispersed as fine particles in an aqueous phase (dispersion medium) in which bacteria inhabit, and this system is dispersed. If a stable emulsified state can be obtained, it is expected that contact between the two will increase and the culture efficiency will be remarkably improved.
【0007】この乳化状態を安定化させる手段として、
一般的には界面活性剤(乳化剤)の利用が検討される
が、従来よりこれらの培養生産系に上述する目的として
乳化剤、即ち界面活性剤を使用し生産効率を高めるとい
う技術は、今だ実績のないのが実状である。As means for stabilizing the emulsified state,
Generally, the use of surfactants (emulsifiers) is considered, but the technique of using emulsifiers, ie surfactants, for the above-mentioned purpose in these culture production systems to improve the production efficiency has been proven to date. There is no such thing.
【0008】その主たる要因として、
界面活性剤といえば、多くは有機系の化合物である
ことから菌の栄養分にもなる。
その結果、目的外の生成物が副産される可能性があ
る。
また界面活性剤が消費され、乳化状態が崩れる恐れ
がある。
また界面活性剤が水相或いは油相のどちらかに連続
相を形成し、培養生産系から有用生産物の分離が不可能
になる。
更にまた有機化合物系の界面活性剤には、毒性や安
全性等の面からその使用に際してはさらなる制限を受け
る。
等の技術的且つ安全性等からなる諸問題により、界面活
性剤による乳化技術の導入が、いまだ実用化されていな
いものと想定される。[0008] As the main factor, most of the surfactants are organic compounds, so they also serve as nutrients for the bacteria. As a result, unintended products may be by-produced. Further, the surfactant may be consumed, and the emulsified state may be destroyed. In addition, the surfactant forms a continuous phase in either the aqueous phase or the oil phase, making it impossible to separate useful products from the culture production system. Furthermore, organic compound type surfactants are further limited in their use in terms of toxicity and safety. It is assumed that the introduction of emulsification technology using a surfactant has not yet been put into practical use due to various technical and safety problems.
【0009】また発酵生産物の分離精製にあたっては、
生成物及び糸状菌等の培養菌がお互いに絡み合い、更に
は培養生成物の増加等に伴う培養液粘度の高まり、又菌
を遠心分離、濾過等により分離するのに非常に困難を来
たしており、商業生産上の大きな障害になっているのが
現在の発酵工業の実状である。When separating and refining the fermentation product,
Products and cultured fungi such as filamentous fungi are entangled with each other, further increasing the viscosity of the culture solution due to the increase of culture products, and it has become very difficult to separate the bacteria by centrifugation, filtration, etc. However, it is the current state of the fermentation industry that is a major obstacle to commercial production.
【0010】近年これらの諸問題を解決するために、バ
イオリアクターとして担体に菌を固定させた固定化微生
物をバイオ触媒に用いるプロセスが報告されている。そ
の固定化の担体として、セルロース、アルギン酸カルシ
ウム、ポリアクリル酸アミドなどの有機物担体が検討さ
れているようであるが、学問的成果はともかく、未だほ
とんど実用化されていないのが実状である。[0010] In recent years, in order to solve these problems, a process has been reported in which an immobilized microorganism having a bacterium immobilized on a carrier is used as a bioreactor as a bioreactor. As the carrier for immobilization, an organic carrier such as cellulose, calcium alginate, polyacrylic acid amide, etc. seems to be studied, but it is the fact that it has not been practically used, despite the academic achievement.
【0011】一方、本発明者らは有機物担体が持つ種々
の問題を解消することを目的に、鎖状粘土鉱物に有用な
糸状菌を固定化させ、有用物を生産する糸状菌の新規な
培養方法を開発し、特願平5−288753号として提
案している。On the other hand, for the purpose of solving various problems of the organic carrier, the present inventors have immobilized a filamentous fungus which is useful on a chain clay mineral and have a novel culture of the filamentous fungus which produces a useful substance. A method was developed and proposed as Japanese Patent Application No. 5-288753.
【0012】又、特開平3−32733号公報には、合
成スチブンサイト等の合成スメクタイト族粘土鉱物系の
乳化剤が開示されている。Further, JP-A-3-32733 discloses an emulsifier of synthetic smectite group clay mineral such as synthetic stevensite.
【0013】[0013]
【発明が解決しようとする課題】本発明においては、上
述したごとく、これらの有用菌を培養生産菌に利用して
有用物の生産性を著しく向上させるためには、第一に菌
が生息するに十分な水が必要である。第二にこれらの菌
が、工業的に原料であり、菌が好んで栄養源とする大豆
油、ナタネ油等の油脂或いは疎水性ではあるが石油系の
有機物等を必要とすることから、本発明の目的を果たす
ために必要な条件は、これらの水と油の二相を限りなく
一相に近似させることである。In the present invention, as described above, in order to remarkably improve the productivity of useful substances by utilizing these useful bacteria as culture-producing bacteria, the bacteria inhabit first. Need enough water for. Second, these fungi are industrial raw materials, soybean oil that the fungus prefers as a nutrient source, oils and fats such as rapeseed oil or hydrophobic but requires petroleum-based organic matter, etc. The condition necessary for fulfilling the purpose of the invention is to make these two phases of water and oil approximate to one phase infinitely.
【0014】従って、効率的培養生産にあっては、有用
菌が生息する水相と、有用菌が工業的に原料とする油相
から成る培養生産系を安定に乳化させ、且つそのために
必要とする乳化剤が固相系であれば、水相に油相を微細
粒子として分散させられ、発酵生産速度が高まり、且つ
乳化剤が固体であることから菌の分離が改善され、発酵
生産物の回収、精製等の培養効率が著しく向上させら
れ、しかもこの固体の乳化剤が無機系であれば、上記の
ごとく乳化剤が有機化合物であるが故に使用制限される
諸問題も解消されるであろう。Therefore, in efficient culture production, it is necessary to stably emulsify a culture production system comprising an aqueous phase in which useful bacteria live and an oil phase in which useful bacteria are industrially used as a raw material, and for that purpose. If the emulsifier to be a solid phase system, the oil phase is dispersed as fine particles in the aqueous phase, the fermentation production rate is increased, and the separation of the bacteria is improved because the emulsifier is solid, the fermentation product is recovered, If the culture efficiency of purification or the like is remarkably improved, and the solid emulsifier is an inorganic type, various problems that the use of the emulsifier is restricted because it is an organic compound will be solved.
【0015】以上から本発明の目的は、発酵生産におけ
る有用菌が、効率良く有用物を生産するに必要とする無
機系固体の乳化剤を提供することにある。From the above, it is an object of the present invention to provide an inorganic solid emulsifier which is required by a useful bacterium in fermentation production to efficiently produce a useful product.
【0016】[0016]
【課題が解決するための手段】本発明によれば、上記目
的を達成するために、放線菌、糸状菌などの有用菌の培
養において、油相と水相から成る培養液に粘土鉱物を添
加し、或いはこの有用菌を固定化させた該粘土鉱物を添
加し、該培養液を安定に乳化させ、培養液中の油相を微
細な粒子に分散させて菌体と効率よく接触させることを
特徴とする、該粘土鉱物から成る乳化剤及びこの乳化剤
を用いる有用菌の培養方法が提供される。According to the present invention, in order to achieve the above object, clay minerals are added to a culture solution consisting of an oil phase and an aqueous phase in the cultivation of useful bacteria such as actinomycetes and filamentous fungi. Alternatively, the clay mineral to which this useful bacterium has been immobilized is added to stably emulsify the culture solution, and the oil phase in the culture solution is dispersed into fine particles to efficiently contact the cells. An emulsifying agent comprising the clay mineral and a method for cultivating a useful bacterium using the emulsifying agent are provided.
【0017】本発明によればまた、繊維状又はその収束
体であって、その繊維径が60乃至400Åで、繊維長
が0.2乃至400μmの範囲にあるセピオライトト等
の繊維状粘土鉱物からなる乳化剤が提供される。According to the present invention, a fibrous clay mineral such as sepiolite or the like, which is fibrous or a bundle thereof and has a fiber diameter of 60 to 400 Å and a fiber length of 0.2 to 400 μm, is used. An emulsifier is provided.
【0018】本発明の別の態様では、上記繊維状粘土鉱
物がセピオライトであって、このセピオライトにあらか
じめ有用菌を固定化させた後、乳化剤及びバイオ触媒と
して培養液との接触を回分、半回分、反復回分或いは連
続のいずれかの方法で行ない、培養液を安定に乳化させ
ながら有用物を生産し、該有用物及び該粘土鉱物のセピ
オライトを含む該培養液から乳化剤のセピオライトを分
離し、有用生産物を効率良く回収する培養方法が提供さ
れる。In another embodiment of the present invention, the fibrous clay mineral is sepiolite, and useful bacteria are immobilized in advance on the sepiolite, and then contact with a culture solution as an emulsifier and a biocatalyst is carried out in a batch or semi-batch manner. , A batch method or a continuous method to produce a useful product while stably emulsifying the culture liquid, and separating the emulsifier sepiolite from the culture liquid containing the useful product and the clay mineral sepiolite, A culture method for efficiently recovering a product is provided.
【0019】[0019]
【作用】一般に発酵生産の多くは回分培養で行なわれて
おり、発酵が終了すれば菌体は分離され廃棄処分され、
菌体の再利用が行なわれていないばかりでなく、その菌
体の合成に使用された基質分が毎回ロスし、その菌の廃
棄にかかるコストも無視できない。従ってこの菌体をな
るべく再利用する観点から、従来から理論的にその効果
が期待される反復回分培養が種々開発されたが、未だ工
業的に実用性の乏しいものであった。[Function] Generally, most fermentation production is carried out by batch culture, and when fermentation is completed, the bacterial cells are separated and discarded.
Not only is the microbial cell not reused, but the substrate used for the synthesis of the microbial cell is lost each time, and the cost of disposing of the bacterium cannot be ignored. Therefore, from the viewpoint of reusing these bacterial cells as much as possible, various types of repeated batch cultures have been developed, which are theoretically expected to be effective, but they have not been industrially practically useful.
【0020】そこで本発明者等は、すでに上述したごと
くセピオライト系の鎖状粘土鉱物に糸状菌を担持させ、
糸状菌が安定に固定化されたバイオ触媒、及びこれを用
いた反復回分培養プロセスを開発し特許を出願すると共
に、セピオライトが微生物と油脂を同時に吸着し発酵が
著しく促進される事実を知った。Therefore, the present inventors have already supported the filamentous fungus on the sepiolite chain clay mineral as described above,
We have developed a biocatalyst in which filamentous fungi are stably immobilized, and an iterative batch culture process using the biocatalyst, and applied for a patent. At the same time, we learned that sepiolite adsorbs microorganisms and fats and oils at the same time and fermentation is significantly accelerated.
【0021】より詳細には、本発明はホルマイト系の鎖
状粘土鉱物であるセピオライトが、培養液(培地)系に
おいて糸状菌が繊維状のセピオライトの表面又はその収
束体間隙に固定化され、その周辺に微細な粒子として油
分が分散していることを見出し、この知見に鑑み繊維状
のセピオライトを水相と油相(大豆油)から成る系に添
加し、機械的に細粒化させたところ、この系が乳化状態
で安定化するという知見に基づくものである。More specifically, according to the present invention, sepiolite, which is a holmite chain-like clay mineral, has filamentous fungi immobilized on the surface of fibrous sepiolite or in the space between converging bodies thereof in a culture solution (medium) system. It was found that oil was dispersed as fine particles in the periphery, and in view of this finding, fibrous sepiolite was added to a system consisting of an aqueous phase and an oil phase (soybean oil), and mechanically granulated. , Is based on the finding that this system stabilizes in an emulsified state.
【0022】即ち、セピオライト等の粘土鉱物が機械的
に細粒化された油分を吸着し、又は上記収束体間隙に油
分を取り込み、細粒化された状態で油分を安定にする作
用(乳化作用)を有する事実から本発明に至ったもので
ある。That is, clay minerals such as sepiolite adsorb mechanically atomized oil components or take oil components into the converging body gaps to stabilize the oil components in the atomized state (emulsification action). ) Has led to the present invention.
【0023】従来より、水相と油相からなる乳化状態
(エマルジョン)を安定化するために乳化剤としてこの
系に加えられる界面活性剤は、比較的低分子の水溶性有
機系化合物であり、
(1)分散粒子と連続相の界面張力を低下させて、分散
相の機械的細粒化を容易にさせる。
(2)水と油界面に配向吸着し、保護膜を作り粒子の凝
集と合一を阻止する。
等の機能により分散粒子の融合を抑制させ、エマルジョ
ンを安定化させるが、この為に界面活性剤を大量に使用
しなければならないとされている。Conventionally, a surfactant added to this system as an emulsifier for stabilizing an emulsified state (emulsion) composed of an aqueous phase and an oil phase is a water-soluble organic compound having a relatively low molecular weight. 1) The interfacial tension between the dispersed particles and the continuous phase is lowered to facilitate mechanical atomization of the dispersed phase. (2) Orientationally adsorbs on the interface between water and oil to form a protective film and prevent aggregation and coalescence of particles. It is said that the fusion of dispersed particles is suppressed and the emulsion is stabilized by such functions as described above, but a large amount of a surfactant must be used for this purpose.
【0024】これに対してゼラチン、寒天、卵黄、アラ
ビヤゴムなどの高分子物質も乳化安定作用を持つ。この
ような高分子は、一部は界面に吸着して粒子を保護し、
一部は液中に枠組式構造(Scaffolding structure)を作
り、エマルジョン粒子を捕獲し安定化させると言われて
いる。On the other hand, high molecular substances such as gelatin, agar, egg yolk and arabic gum also have an emulsion stabilizing action. Some of these polymers are adsorbed at the interface to protect the particles,
Some are said to create a framework structure in the liquid (Scaffolding structure) to capture and stabilize the emulsion particles.
【0025】またこれらの低分子乳化剤と高分子乳化剤
との両者の性質を利用して形成されるエマルジョンのタ
イプには、水が連続相である牛乳のような水中油滴型
(O /W型)、油が連続相であるマーガリンのような
油中水滴型(W/O型)又は(W/O)/W型のような
多重型エマルジョン等がある。The type of emulsion formed by utilizing the properties of both the low molecular weight emulsifier and the high molecular weight emulsifier includes oil-in-water type (O / W type) such as milk in which water is a continuous phase. ), A water-in-oil type (W / O type) such as margarine in which oil is a continuous phase, or a multiple type emulsion such as (W / O) / W type.
【0026】一方、本発明で乳化剤に用いられるホルマ
イト系粘土であるセピオライト、パリゴスカイト等に代
表される鎖状粘土鉱物は、その外観形状が繊維状又はそ
の収束体であり、その基本構造は八面体層の骨格を形成
する主成分が、マグネシウムから成る3−八面体型ケイ
酸マグネシウム粘土鉱物である。On the other hand, chain clay minerals represented by sepiolite, palygoskite, etc., which are the holmite-based clays used as emulsifiers in the present invention, have a fibrous appearance or a converging body, and their basic structure is an octahedron. The 3-octahedral magnesium silicate clay mineral composed of magnesium is the main component forming the skeleton of the layer.
【0027】上記の鎖状粘土鉱物は同じくケイ酸マグネ
シウム粘土鉱物であるタルクのような二次元の層状結晶
構造物とは異なり、三次元の鎖状の結晶構造を有し、そ
の外観形状の繊維及びその収束体の間隙にできる空孔が
BET法で60乃至600m2/gの比表面積を有し、ま
たその収束体構造を有するが故に、吸着作用を有する多
孔質の鎖状粘土鉱物である。更にセピオライト等は、同
じく多孔質粘土鉱物で、ケイ酸アルミニウム粘土鉱物で
あるモンモリロナイトに代表される通常の層状結晶構造
物とは異なり、水溶系で膨潤しないことも大きな特徴で
ある。Unlike the two-dimensional layered crystal structure such as talc which is also a magnesium silicate clay mineral, the above chain clay mineral has a three-dimensional chain crystal structure, and the fiber of its appearance shape. And the pores formed in the gaps of the converging body have a specific surface area of 60 to 600 m 2 / g by the BET method, and because they have the converging body structure, they are porous chain clay minerals having an adsorbing action. . Further, sepiolite and the like are also porous clay minerals, and unlike ordinary layered crystal structures represented by montmorillonite, which is an aluminum silicate clay mineral, are notable for not swelling in a water-soluble system.
【0028】また本発明において鋭意検討したところ、
上記のセピオライトの他に、繊維状形態をして一般的に
は石綿と言われている蛇紋石又は蛇紋石類縁鉱物、更に
は工業的に利用価値があり、外観形状が板状又は鱗片状
である3−八面体型スメクタイト粘土鉱物等も本発明の
乳化剤として好適に使用されることが分かった。Further, as a result of intensive studies in the present invention,
In addition to the above-mentioned sepiolite, it is a fibrous form and is generally called asbestos serpentine or serpentine-related minerals, and also has industrial utility value, and its appearance shape is plate-like or scale-like. It has been found that certain 3-octahedral smectite clay minerals are also suitably used as the emulsifier of the present invention.
【0029】更に検討したところ、セピオライト等の3
−八面体型ケイ酸マグネシウム系粘土鉱物以外のカオリ
ナイト等の2−八面体型1:1系粘土、セリサイト等の
2−八面体型2:1系粘土及び酸性白土、ベントナイト
等の2−八面体型スメクタイトなども本発明の乳化剤と
して好適に使用されることが分かった。As a result of further examination, 3 such as sepiolite
2-octahedral 1: 1 clays such as kaolinite other than octahedral magnesium silicate clay minerals, 2-octahedral 2: 1 clays such as sericite, and acid clay, bentonite and the like 2- It was found that octahedral smectite and the like are also suitably used as the emulsifier of the present invention.
【0030】本発明において、上記した天然及び合成の
粘土鉱物が、水と油の系を安定に乳化させる機能を持つ
ことについて、その詳細は不明であるが以下のように整
理することができる。In the present invention, the details of the fact that the above-mentioned natural and synthetic clay minerals have the function of stably emulsifying the water-oil system can be summarized as follows, although the details are unknown.
【0031】これらの構造的特徴と後述する水と大豆油
から成る系での乳化性を評価した結果から、本発明の乳
化剤として実施例で使用したセピオライト、石綿、サポ
ナイト、ヘクトライト、酸性白土、ベントナイト、カオ
リン、セリサイト等は何れも極めて安定な乳化状態を形
成する。From these structural characteristics and the results of evaluation of the emulsifying property in the system consisting of water and soybean oil described below, the sepiolite, asbestos, saponite, hectorite, acid clay used in the examples as the emulsifier of the present invention, Bentonite, kaolin, sericite, etc. all form an extremely stable emulsified state.
【0032】即ち、セピオライト、サポナイト、酸性白
土、ベントナイト、カオリン、セリサイト等の粘土鉱物
の外観形状は繊維状及びその収束体、鱗片状、層状で或
いは膨潤する等から液中で絡み、収束、カードハウス等
の枠組式構造(Scaffolding structure) を作り易く、物
理的にエマルジョン粒子を捕獲し易いものと想定され
る。That is, the appearance of clay minerals such as sepiolite, saponite, acid clay, bentonite, kaolin, and sericite is fibrous and their aggregates, scales, layers, or swelling. It is assumed that it is easy to form a framework structure such as a card house, and it is easy to physically capture emulsion particles.
【0033】更にまた該粘土鉱物は、いずれも比較的に
比表面積の大きい多孔質粘土鉱物であり、その構造内に
ナトリウム等のカチオンが挟雑しており、酸性白土、ベ
ントナイト等の多孔質粘土の陽イオン交換容量(CE
C)70乃至150meq/100gからこれらの粘土
鉱物のシリカ四面体層は親水性であろうし、一方セピオ
ライト、ハロイサイト、セリサイト、サポナイト等のC
ECが5乃至50meq/100gの範囲にあることか
らすると、CECが小さいことが親油性を阻害させない
要因と想定され、本発明においてはCECが5乃至12
0meq/100gの範囲にあり、好ましくは10乃至
100meq/100gで、更に好ましくは20乃至7
0meq/100gである粘土鉱物が本発明の乳化剤と
して有効である。Furthermore, each of the clay minerals is a porous clay mineral having a relatively large specific surface area, and cations such as sodium are intercalated in the structure, and porous clay such as acid clay and bentonite. Cation exchange capacity (CE
C) From 70 to 150 meq / 100 g, the silica tetrahedral layers of these clay minerals will be hydrophilic, while C such as sepiolite, halloysite, sericite, saponite etc.
Since the EC is in the range of 5 to 50 meq / 100 g, it is assumed that the small CEC is a factor that does not inhibit the lipophilicity. In the present invention, the CEC is 5 to 12
It is in the range of 0 meq / 100 g, preferably 10 to 100 meq / 100 g, and more preferably 20 to 7
A clay mineral of 0 meq / 100 g is effective as the emulsifier of the present invention.
【0034】以上から、本発明の乳化剤であるセピオラ
イト、カオリナイト、セリサイト、酸性白土等の粘土鉱
物は、基本的に親水性であり、その構造体に親油性サイ
トがあり、外観形状が繊維状で且つその収束体構造であ
ることから、本発明の乳化剤の無機高分子であるセピオ
ライトのように、上記の有機高分子乳化剤であるゼラチ
ン、寒天のごとく、セピオライトの一部は界面に吸着し
て粒子を保護し、一部は、液中に枠組式構造に相当する
収束体間隙又は、セピオライト繊維の絡みがエマルジョ
ン粒子を捕獲し、培養液を乳化状態で安定化させている
ものと想定される。From the above, clay minerals such as sepiolite, kaolinite, sericite and acid clay, which are emulsifiers of the present invention, are basically hydrophilic and have a lipophilic site in their structure, and the appearance shape is fiber. Because of its shape and its convergent structure, like sepiolite which is an inorganic polymer of the emulsifier of the present invention, a part of sepiolite is adsorbed on the interface like gelatin and agar which are the above organic polymer emulsifiers. It is assumed that, in part, the gaps between converging bodies corresponding to the framework structure in the liquid or the entanglement of the sepiolite fibers capture the emulsion particles and stabilize the culture liquid in the emulsified state. It
【0035】[0035]
(無機乳化剤)本発明に使用する無機乳化剤は、外観形
状が繊維状、鱗片状、層状で水−油のいずれかの相に微
細に分散するに適した形態及び粒度であれば、下記する
天然又は合成の粘土鉱物群から任意に選ぶことができ
る。(Inorganic emulsifier) The inorganic emulsifier used in the present invention has a natural appearance as long as it has a fibrous, scale-like, or layered form and is suitable for finely dispersing in any of the water-oil phases and has a particle size as described below. Alternatively, it can be arbitrarily selected from the group of synthetic clay minerals.
【0036】カオリン、ハロイサイト等のカオリン鉱
物、イライト、セリサイト等の雲母粘土鉱物、酸性白
土、ベントナイト、サポナイト、ヘクトライト、スチブ
ンサイト等のスメクタイト粘土鉱物及びセピオライト、
パリゴルスカイト、クリソタイル等の繊維状粘土鉱物類
から選ばれた、少なくとも一種の粘土鉱物を本発明の乳
化剤として使用することができる。Kaolin minerals such as kaolin and halloysite, mica clay minerals such as illite and sericite, smectite clay minerals such as acid clay, bentonite, saponite, hectorite and stevensite, and sepiolite,
At least one clay mineral selected from fibrous clay minerals such as palygorskite and chrysotile can be used as the emulsifier of the present invention.
【0037】また必要において、上記粘土鉱物100重
量部当たり蛇紋石類縁鉱物のペコラアイト、ネポーアイ
ト、グリーナライト、カリオピライト、アメサイト、バ
ーチェリン、ブリンドリアイト、ケリアイト、クロステ
ダイト、タルク系のタルク、ケロライト、ウィレムスア
イト、ピメライト、ミネソタアイト、緑泥石系のクリノ
クロア(Mg緑泥石)、FeMg緑泥石、ニマイト、ペナンタ
イト、混合層鉱物の3−八面体型緑泥石/3−八面体型
スメクタイト、2−八面体型雲母/2−八面体型スメク
タイト、2−八面体型緑泥石/2−八面体型スメクタイ
ト、カオリン/モンモリロナイト等から選ばれるどれか
一種を1乃至20重量部の範囲で使用することができ
る。In addition, if necessary, per 100 parts by weight of the above clay mineral, serpentine-related minerals such as pecoraite, nepoite, greenalite, caryopyrite, amethite, birchellin, brindriaite, kellyite, closterite, talc-based talc, kerolite, Willemsite, pimelite, minnesotaite, chlorite clinochlore (Mg chlorite), FeMg chlorite, nimite, penanthite, mixed-layer mineral 3-octahedral chlorite / 3-octahedral smectite, 2-8 It is possible to use any one selected from the group consisting of tetrahedral mica / 2-octahedral smectite, 2-octahedral chlorite / 2-octahedral smectite, kaolin / montmorillonite, etc. within the range of 1 to 20 parts by weight. .
【0038】本発明では、上記の3−八面体型ケイ酸マ
グネシウム粘土鉱物の中から、世界的に各地に産出し比
較的入手しやすい鎖状粘土鉱物で、その形状が黙視観察
で見て繊維状であり、顕微鏡観察で見てその収束体であ
るセピオライトが好適に使用され、次いで顕微鏡観察で
見て鱗片状、層状の粘土鉱物で入手しやすいサポナイ
ト、ヘクトライト、酸性白土、ベントナイト等のスメク
タイト及びカオリン、セリサイト等が本発明の乳化剤と
して好適に使用され、またこれらの粘土鉱物は単独或い
は二種以上の組合わせで使用してもよい。In the present invention, among the above-mentioned 3-octahedral magnesium silicate clay minerals, chain clay minerals produced in various places around the world and relatively easy to obtain, the shapes of which are fibers observed by visual observation. It is a shape, and sepiolite that is its convergent body is suitably used by microscopic observation, and then smectites such as saponite, hectorite, acid clay, bentonite that are easily available as scale-like, layered clay minerals by microscopic observation. And kaolin, sericite, etc. are preferably used as the emulsifier of the present invention, and these clay minerals may be used alone or in combination of two or more kinds.
【0039】また本発明の乳化剤のBET比表面積は3
0乃至700m2 /gの範囲にあるが、この比表面積が
高すぎると乳化組成物の粘性を高める要因になることか
ら、好適には比表面積が80乃至400m2 /gの範囲
にあり、また吸油量は特に必要としないが、少なくとも
30至150ml/100gの範囲にある上記粘土鉱物
であれば好適に使用される。The emulsifier of the present invention has a BET specific surface area of 3
The specific surface area is in the range of 0 to 700 m 2 / g, but if the specific surface area is too high, it becomes a factor to increase the viscosity of the emulsion composition. Therefore, the specific surface area is preferably in the range of 80 to 400 m 2 / g. Although the oil absorption amount is not particularly required, the above clay mineral in the range of at least 30 to 150 ml / 100 g is preferably used.
【0040】本発明では、これらの粘土鉱物を乳化剤と
して使用する前に、ボールミル、ハンマーミキサーでの
解砕、スーパーミキサー、擂解機等での練込み粉砕及び
アトマイザー、ジェットミル等での衝撃粉砕により又は
必要に応じて焼成及び化学処理等で弱い一次粉砕処理を
施し、収束体としての鎖状構造、及び鱗片状としての積
層構造を部分的に解繊またはほぐしてから使用すること
がより好ましい。In the present invention, before using these clay minerals as an emulsifier, crushing with a ball mill, a hammer mixer, kneading crushing with a super mixer, a disintegrator, etc. and impact crushing with an atomizer, jet mill, etc. It is more preferable to perform weak primary crushing treatment by firing or chemical treatment, etc., if necessary, and to partially disintegrate or loosen the chain structure as a converging body and the laminated structure as scales before use. .
【0041】(乳化組成物及び乳化方法)本発明におけ
る乳化組成物とは、糸状菌等の有用菌が培養、発酵等に
おいて生の生産菌として有用物を生産する水相−油相−
乳化剤から成る培地組成物である。この培地で菌が、栄
養源として且つ工業的に原料として好んで食べる油相成
分としては、大豆油、ナタネ油、パーム油、パーム核
油、ひまわり油、ベニ花油、胡麻油、アマニ油、棉実
油、オリーブ油、ヤシ油、コーン油、米油、桐油、アマ
ニ油、落花生油、ヒマシ油、等の植物油、イワシ、ニシ
ン、イカ、サンマ等の魚油、牛脂、豚脂、鯨油等の動物
油、石油系の脂肪族(例えば炭素数6乃至17のもの)
及び芳香族炭化水素類油、炭素数4以上のアルコール類
を挙げることができる。(Emulsified Composition and Emulsifying Method) The emulsified composition in the present invention means an aqueous phase-oil phase-wherein useful bacteria such as filamentous fungi produce useful products as raw production bacteria in culture, fermentation and the like.
A medium composition comprising an emulsifier. As the oil phase component that the fungus eats in this medium as a nutrient source and industrially as a raw material, soybean oil, rapeseed oil, palm oil, palm kernel oil, sunflower oil, safflower oil, sesame oil, linseed oil, cotton Vegetable oil such as seed oil, olive oil, coconut oil, corn oil, rice oil, tung oil, linseed oil, peanut oil, castor oil, etc., fish oil such as sardines, herring, squid, saury etc., animal oil such as beef tallow, lard, whale oil, etc. Petroleum-based aliphatic (for example, having 6 to 17 carbon atoms)
And aromatic hydrocarbon oils and alcohols having 4 or more carbon atoms.
【0042】本発明において、水相は純粋な水のみでも
よいが、有用菌としての放線菌、糸状菌が前もって含有
していてもよく、これらの有用菌は特に限定されるもの
ではなく、例えばクエン酸、グルコン酸等を生産するAs
pergillus niger 、イタコン酸を生産する Aspergillus
terreus、グルコアミラ−ゼを生産するAspergillus aw
amori 抗生物質セファマイシンCを生産するStreptmyce
s clavuligenes、セファロスポリンCを生産するCephlo
sporium polgaleum 等を挙げることができる。又前述
したとおり本発明においては、これらの有用菌を本発明
による粘土鉱物からなる乳化剤に予め固定化しておける
ことも本発明の大きな特徴である。従って本発明におけ
る乳化組成物には、当然のごとくこれらの有用菌が含有
されていてもよい。In the present invention, the aqueous phase may be pure water only, but actinomycetes and filamentous fungi as useful bacteria may be contained in advance, and these useful bacteria are not particularly limited. As producing citric acid, gluconic acid, etc.
pergillus niger, Aspergillus producing itaconic acid
terreus, Aspergillus aw that produces glucoamylase
Streptmyce producing amori antibiotic cefamycin C
Cephlo, which produces cephalosporin C
Examples include sporium polgaleum. Further, as described above, in the present invention, it is a great feature of the present invention that these useful bacteria can be immobilized in advance on the emulsifier made of the clay mineral according to the present invention. Therefore, the emulsion composition of the present invention may naturally contain these useful bacteria.
【0043】更に本発明における水相には、上記有用菌
の他に、必要に応じてアミノ酸、炭水化物、蛋白質、各
種の水溶性無機塩類等が包含されていてもよい。Further, the aqueous phase in the present invention may contain amino acids, carbohydrates, proteins, various water-soluble inorganic salts and the like, if necessary, in addition to the above-mentioned useful bacteria.
【0044】上記した有機物は水とは通常の条件では均
一相を形成しない常温で、液体又は固体であるが本発明
では、これらの有機物の少なくとも何れか一種以上を油
相として使用するに、本発明においては種々の培養反応
の目的及び方法から特に限定されないが、一般的に水相
と油相との容量比が5:95乃至99:1の範囲におい
て、水相と上記粘土鉱物からなる乳化剤とを重量比で5
0:50乃至99:1の範囲で添加して、攪拌等の乳化
操作により両者を本発明の目的を満足させるに十分な安
定度の、W/O乃至O/W型の乳化状態にすることが出
来る。The above-mentioned organic substances are liquid or solid at room temperature which does not form a homogeneous phase with water under normal conditions, but in the present invention, at least one or more of these organic substances are used as an oil phase. The invention is not particularly limited in view of various purposes and methods of the culture reaction, but generally, an emulsifier comprising an aqueous phase and the above clay mineral in a volume ratio of the aqueous phase to the oil phase of 5:95 to 99: 1. And 5 by weight
Add in the range of 0:50 to 99: 1, and emulsify both of them into a W / O to O / W type emulsified state with stability sufficient to satisfy the object of the present invention. Can be done.
【0045】本発明での乳化方法としては、上記三者の
添加順序には特にこだわらなくてもよいが、油と水との
両相の配合割合又はその用途、使用条件による油の種類
にもよるが、好ましくは油に乳化剤を添加して攪拌後、
水を加えて攪拌乳化させる方法が選ばれるが、より好ま
しくは水に乳化剤を添加して攪拌後、油を加えて攪拌乳
化させる方が、より安定した乳化組成物を得ることがで
きる。In the emulsification method of the present invention, the order of addition of the three is not particularly limited, but the mixing ratio of both phases of oil and water, its application, and the type of oil depending on the use conditions. However, preferably after adding an emulsifier to the oil and stirring,
A method of adding water and stirring and emulsifying is selected, but more preferably, a more stable emulsified composition can be obtained by adding an emulsifier to water and stirring and then adding oil and stirring and emulsifying.
【0046】また本発明における乳化操作は、一般的に
採用される攪拌等の物理的な方法でよく、実験室的には
通常の家庭用ミキサー程度の剪断力を有する機械力で数
十秒乃至数分間の処理時間でよく、工業的には大量に処
理できる代表的なものとしてホモジナイザー、コロイド
ミル、ジェットフローミキサー、ボーテーター等を挙げ
ることができるが、本発明では必ずしもこのような物理
的方法に限定されるものではなく、必要に応じて反転乳
化法、ゲル乳化法、HLB−温度乳化法等の化学的方法
をも採用されるものである。The emulsification operation in the present invention may be carried out by a generally employed physical method such as stirring, and in the laboratory, a mechanical force having a shearing force equivalent to that of an ordinary household mixer is used for several tens of seconds to A treatment time of a few minutes is sufficient, and industrially, a large amount can be treated by a homogenizer, a colloid mill, a jet flow mixer, a voterator, etc., but in the present invention, such a physical method is not necessarily used. The method is not limited, and if necessary, a chemical method such as an inversion emulsification method, a gel emulsification method, an HLB-temperature emulsification method or the like may be adopted.
【0047】また培養にあたっては、有用菌と油脂等を
含有する培養培地に本発明の粘土鉱物から成る乳化剤を
添加して、上記のようにして形成された乳化組成物を通
常の通気攪拌槽、充填床型リアクター、エアーリフト型
気泡塔等のリアクターを使用して発酵生産物の特性に応
じて回分、半回分、反復回分或いは連続のいずれかの方
式で培養することができる。In culturing, the emulsifier composed of the clay mineral of the present invention is added to the culture medium containing useful bacteria, fats and oils, and the emulsified composition formed as described above is treated with a conventional aeration stirring tank, Using a reactor such as a packed bed type reactor or an air lift type bubble column, the fermentation product can be cultured in a batch, semi-batch, repetitive batch or continuous manner depending on the characteristics.
【0048】本発明による無機系個体の乳化剤は、微生
物が生息する水と微生物が栄養源とする油からなる二相
を乳化させ、菌による培養効率を向上させ、且つ乳化剤
が無機個体であるが故に分離回収性が高く、よって本乳
化技術は有用物質を生産する目的の他にも、油脂等の油
を含有する廃棄物及び工場廃水の処理にも有効に利用さ
れるものである。The inorganic solid emulsifier according to the present invention emulsifies two phases consisting of water inhabited by microorganisms and oil used by the microorganisms as a nutrient source to improve the culture efficiency by the fungus, and the emulsifier is an inorganic solid. Therefore, it has high separation and recovery properties, and thus the present emulsification technology is effectively used not only for producing useful substances but also for treating wastes containing oil such as fats and oils and industrial wastewater.
【0049】また本発明によれば、無機系個体の乳化剤
を介して水−油の乳化液系で、所謂通常のカビを繁殖さ
せる応用の可能性が期待されるものである。Further, according to the present invention, it is expected that the so-called ordinary mold can be propagated in a water-oil emulsion system via an inorganic solid emulsifier.
【0050】[0050]
【実施例】本発明を次の例で更に詳細に説明する。な
お、本発明に使用した物性の測定方法、評価方法及び本
発明の乳化剤である粘土鉱物種の物性は以下の通りであ
る。The invention is explained in more detail in the following examples. The physical property measuring methods and evaluation methods used in the present invention and the physical properties of the clay mineral species as the emulsifier of the present invention are as follows.
【0051】(1)粒度
コールターカウンター(コールターエレクトロニクス社
製 TA−II型)法により測定した。
(2)比表面積
カルロエルバ社製Sorptomatic Serie
s1800を使用し、BET法により測定した。
(3)吸油量
JIS K−5101、19 に準じて測定した。
(4)陽イオン交換容量(CEC)
日本鉱物協会東海支部無機砂型研究部会発行の試験方法
TISK−413記載の方法に準拠して測定した。
(5)乳化状態の評価方法
家庭用のジューサーミキサーに所定量の水、油、粘土鉱
物(100μm以下に粉砕したもの)を入れ、1分間混
合し、有栓メスシリンダーに移し、所定時間放置後に混
合液の乳化状態を目視観察し、次の三段階で評価とし
た。
A:水と油がほぼ均質に乳化分散している。
B:一週間経過で乳化が若干不安定となる。
C:水と油の相が明らかに分離し、乳化状態が見られな
い。
なお、油の乳化状態を目視観察し易いように、予め油を
ズダンブラック−Bで染色しておいた。
(6)乳化剤(粘土鉱物種)の諸物性を表1に示した。(1) Particle size Measured by a Coulter counter (TA-II type manufactured by Coulter Electronics Co.). (2) Specific surface area Sorptomatic Series manufactured by Carlo Erba
It was measured by the BET method using s1800. (3) Oil absorption amount Measured according to JIS K-5101,19. (4) Cation Exchange Capacity (CEC) Test Method Published by Inorganic Sand Mold Research Group, Tokai Branch, Japan Mineral Society It was measured according to the method described in TISK-413. (5) Evaluation method of emulsified state A predetermined amount of water, oil and clay mineral (crushed to 100 μm or less) are put in a household juicer mixer, mixed for 1 minute, transferred to a graduated cylinder with a stopper, and allowed to stand for a predetermined time. The emulsified state of the mixed liquid was visually observed and evaluated in the following three stages. A: Water and oil are almost uniformly emulsified and dispersed. B: The emulsification becomes slightly unstable after one week. C: The water and oil phases were clearly separated and no emulsified state was observed. The oil was dyed with Sudan Black-B in advance so that the emulsified state of the oil could be visually observed. (6) Various physical properties of the emulsifier (clay mineral type) are shown in Table 1.
【0052】[0052]
【表1】 [Table 1]
【0053】(実施例1〜8)家庭用ジューサーミキサ
ー水900mlと、「表1」に示す粘土鉱物を、各々2
0gを入れ30秒間混合した後、脱酸大豆油を100m
l入れ、さらに30秒間混合し、1000mlの有栓メ
スシリンダーに入れ10分間放置後、乳化状態を目視観
察で評価し、その結果を表2に示した。その結果、脱酸
大豆油と上記8種の粘土鉱物を混合した処、乳化状態は
良好であった。ただし酸性白土、ベントナイト、セリサ
イトが僅かに劣る結果となった。セピオライトを始めと
する本発明に用いる粘土鉱物は、水と油の乳化剤として
適しているといえる。Examples 1 to 8 900 ml of household juicer mixer water and 2 times each of the clay minerals shown in "Table 1" were used.
Add 0g and mix for 30 seconds, then add deoxidized soybean oil to 100m
1 ml, mixed for another 30 seconds, placed in a graduated cylinder of 1000 ml with a stopper, and allowed to stand for 10 minutes. The emulsified state was evaluated by visual observation. The results are shown in Table 2. As a result, when the deoxidized soybean oil and the above eight types of clay minerals were mixed, the emulsified state was good. However, the results were slightly inferior to acid clay, bentonite, and sericite. It can be said that the clay minerals used in the present invention such as sepiolite are suitable as emulsifiers for water and oil.
【0054】[0054]
【表2】 [Table 2]
【0055】(実施例9〜11)家庭用ジューサーミキ
サーに水990mlと脱酸大豆油を10ml入れ30秒
間混合した後、市販セピオライト20gを入れ、更に1
分間混合し、1000ml有栓メスシリンダーに入れ1
0分間放置後、乳化状態を目視観察し評価した。更に混
合比を水:脱酸大豆油=500ml:500mlと10
0ml:900mlに設定し、実施例1と同様にして評
価し、その結果を表3に示した。その結果、水と油の混
合比を変えても乳化状態に変わりなく全ての例で評価は
Aで、乳化状態は良好であった。(Examples 9 to 11) 990 ml of water and 10 ml of deoxidized soybean oil were placed in a household juicer mixer and mixed for 30 seconds, and then 20 g of commercially available sepiolite was added, and further 1 part was added.
Mix for 1 minute and put in a graduated cylinder with 1000 ml stopper 1
After standing for 0 minute, the emulsified state was visually observed and evaluated. Furthermore, the mixing ratio is water: deoxidized soybean oil = 500 ml: 500 ml and 10
The amount was set to 0 ml: 900 ml, evaluated in the same manner as in Example 1, and the results are shown in Table 3. As a result, even if the mixing ratio of water and oil was changed, the emulsified state did not change and the evaluation was A in all examples, indicating that the emulsified state was good.
【0056】[0056]
【表3】 [Table 3]
【0057】(実施例12〜19)家庭用ジューサーミ
キサーに水900mlと脱酸菜種油100mlを入れ3
0秒間混合した後、市販のセピオライト20gを入れ、
さらに1分間混合し、1000mlの有栓メスシリンダ
ーに入れ、1時間放置後、乳化状態を目視観察し評価し
た。同様にして脱酸コーン油、牛脂、nーヘキサン、ト
ルエン、ブチルアルコール、脱酸大豆油とn−ヘキサン
の混合油、脱酸大豆油とブチルアルコールの混合油、の
乳化状態を評価し、その結果を表4に示した。(Examples 12 to 19) 900 ml of water and 100 ml of deoxidized rapeseed oil were put into a home juicer mixer, and 3
After mixing for 0 seconds, add 20 g of commercially available sepiolite,
The mixture was further mixed for 1 minute, placed in a graduated cylinder with 1000 ml of a stopper, and allowed to stand for 1 hour. Then, the emulsified state was visually observed and evaluated. Similarly, the emulsified state of deoxidized corn oil, beef tallow, n-hexane, toluene, butyl alcohol, a mixed oil of deoxidized soybean oil and n-hexane, a mixed oil of deoxidized soybean oil and butyl alcohol was evaluated, and the result was evaluated. Is shown in Table 4.
【0058】[0058]
【表4】 [Table 4]
【0059】(実施例20〜21)水、油、粘土鉱物を
混合する際の添加順序を変えた場合、乳化状態がどうな
るか検討した。任意の油として脱酸大豆油、任意の粘土
鉱物として、セピオライトを選択して実施した。家庭用
ジューサーミキサーに水500mlと脱酸大豆油500
mlを入れ、1分間混合した後、セピオライト20gを
入れ、さらに1分間混合し1000mlの有栓メスシリ
ンダーに入れ、1時間放置後、乳化状態を目視観察し評
価した。次に家庭用ジューサーミキサーに水500ml
とセピオライト20gを入れ1分間混合した後、脱酸大
豆油500mlを入れ、更に1分間混合し、1000m
l有栓メスシリンダーに入れ1時間放置後、乳化状態を
目視観察しその結果を表5に示した。その結果、油と粘
土鉱物の添加順序に関わり無く、両例とも評価はAで乳
化状態は良好であった。Examples 20 to 21 It was examined what happens to the emulsified state when the order of addition when mixing water, oil and clay minerals is changed. Deoxidized soybean oil was selected as an arbitrary oil, and sepiolite was selected as an arbitrary clay mineral. Household juicer mixer 500 ml water and deoxidized soybean oil 500
After adding 1 ml and mixing for 1 minute, 20 g of sepiolite was added, and further mixed for 1 minute and placed in a graduated cylinder of 1000 ml with a stopper, and allowed to stand for 1 hour. Then, the emulsified state was visually observed and evaluated. Next, 500 ml of water in a household juicer mixer
Add 20g of sepiolite and mix for 1 minute, then add 500ml of deoxidized soybean oil and mix for another 1 minute, 1000m
After being placed in a graduated cylinder with 1 stopper and left for 1 hour, the emulsified state was visually observed and the results are shown in Table 5. As a result, the evaluation was A and the emulsified state was good in both cases regardless of the order of addition of the oil and the clay mineral.
【0060】[0060]
【表5】 [Table 5]
【0061】(実施例22〜24)二種の粘土鉱物をス
ーパーミキサーを用いて下記の重量割合で混合し、得ら
れたM−1、M−2、M−3の乳化剤20gを家庭用ミ
キサー中の水900mlにそれぞれ添加し、30秒間混
合した後、それぞれ脱酸大豆油100mlを入れ、更に
30秒間混合した後、1000mlの有栓メスシリンダ
ーに入れ10分間放置してその乳化性を評価した。
M−1; 市販セピオライト:酸性白土 = 4:1
M−2; 市販セピオライト:ベントナイト= 4:1
M−3; 市販セピオライト:セリサイト = 4:1
その結果、表−6から明らかなように乳化剤として二種
の粘土鉱物を混ぜて用いても乳化状態は変わり無く、三
例ともその評価はAであった。(Examples 22 to 24) Two kinds of clay minerals were mixed with a supermixer at the following weight ratios, and 20 g of the obtained emulsifiers M-1, M-2 and M-3 were mixed in a household mixer. Each of them was added to 900 ml of water and mixed for 30 seconds, then 100 ml of deoxidized soybean oil was added, respectively, further mixed for 30 seconds, placed in a graduated cylinder with 1000 ml of stopper, and left for 10 minutes to evaluate its emulsification property. . M-1; Commercial sepiolite: acid clay = 4: 1 M-2; Commercial sepiolite: bentonite = 4: 1 M-3; Commercial sepiolite: sericite = 4: 1 As a result, as shown in Table-6, an emulsifier. As a result, the emulsified state did not change even if two kinds of clay minerals were mixed and used, and the evaluation was A in all three cases.
【0062】[0062]
【表6】 [Table 6]
【0063】(実施例25)
(前培養液の調製)次いで本発明による乳化方法を培地
に用いて、ストレプトマイセスアウレオファシン(St
reptomyces aureofaciens)を
培養し、抗生物質クロルテトラサイクリンを生産回収し
た。クロルテトラサイクリン生成能を有するストレプト
マイセス アウレオファシン(Streptomyce
s aureofaciens)NRRL−2209よ
り単胞子分離で選別した菌株のスラントより1白金耳
を、500ml容三角フラスコに入れた培地100ml
(シュクロース30g/l、硫酸アンモン2g/l、炭
酸カルシウム7g/l、CSL16.5ml/l、PH
6.8に調整し、1kg/cm2で15分滅菌)に植菌し、3
0℃で2日間ロータリーシェーカー(170rpm)で
振とう培養した。これを前培養液とした。
(本培養液の調製)次いで本培養液を調製した。家庭用
ジューサーミキサーに水450mlと市販のセピオライ
ト10gを入れ30秒間混合した後、脱酸大豆油を50
ml入れ、さらに30秒間混合し、1000ml容の三
角フラスコに入れ、滅菌用オートクレーブで1kg/cm2で
15分滅菌した。その後30℃まで放冷し、前培養液を
10%(v/v)となるように添加し、30℃で7日間
ロータリーシェーカー(170rpm)で振とう培養し
た。この培養終了液を遠心分離することにより抗生物質
クロルテトラサイクリンを回収した。なお、粘土鉱物を
添加せずに、同様の培養反応を行ない比較評価した。以
上の結果、クロルテトラサイクリンの生成量は1日〜3
日の間で、粘土鉱物を添加しないものが、100〜35
0μg/mlであったのに対し、本発明による乳化培地
で培養したものは、200〜1100μg/mlと約2
倍強の値を示したことから、乳化条件下でクロルテトラ
サイクリンの生産速度が増大し、収率が向上したものと
思われる。(Example 25) (Preparation of preculture liquid) Next, using the emulsification method according to the present invention as a medium, Streptomyces aureofacin (St
reptomyces aureofaciens) was cultured, and the antibiotic chlortetracycline was produced and recovered. Streptomyces aureofacin (Streptomyce) capable of producing chlortetracycline
aureofaciens) NRRL-2209 1 platinum loop from slant of the strain selected by monospore separation in 100 ml medium containing 500 ml Erlenmeyer flask
(Sucrose 30 g / l, ammonium sulfate 2 g / l, calcium carbonate 7 g / l, CSL 16.5 ml / l, PH
Adjusted to 6.8, sterilized at 1 kg / cm 2 for 15 minutes, and inoculated into 3
The cells were cultivated with shaking on a rotary shaker (170 rpm) for 2 days at 0 ° C. This was used as a preculture liquid. (Preparation of Main Culture Solution) Next, a main culture solution was prepared. After adding 450 ml of water and 10 g of commercially available sepiolite to a household juicer mixer and mixing for 30 seconds, deoxidized soybean oil was added to 50
ml, mixed for 30 seconds, placed in a 1000 ml Erlenmeyer flask, and sterilized in an autoclave for sterilization at 1 kg / cm 2 for 15 minutes. Then, the mixture was allowed to cool to 30 ° C., the preculture liquid was added so as to be 10% (v / v), and shake culture was carried out at 30 ° C. for 7 days on a rotary shaker (170 rpm). The antibiotic chlortetracycline was recovered by centrifuging the culture completed liquid. In addition, the same culture reaction was performed without adding the clay mineral, and comparative evaluation was performed. As a result, the amount of chlorotetracycline produced was 1 to 3 days.
100-35 without clay minerals added during the day
In contrast to 0 μg / ml, the one cultured in the emulsification medium according to the present invention had a concentration of 200 to 1100 μg / ml, which was about 2 μm.
Since the value was more than doubled, it is considered that the production rate of chlorotetracycline increased under the emulsification condition and the yield improved.
【0064】[0064]
【発明の効果】本発明によれば、有用物の効率的培養生
産にあたって、有用菌が生息する水相と有用菌が栄養源
に好んで食べ、工業的に原料である大豆油等の油相に粘
土鉱物を添加し、或いはこの有用菌を固定化させた該粘
土鉱物を添加し、該培養液を安定に乳化させ、培養液中
の油相を微細な粒子に分散させて、菌体と油が効率よく
接触させることを特徴とする該粘土鉱物から成る無機系
の乳化剤を提供することができ、且つ乳化剤が無機の固
体であるが故に菌の分離が改善され、発酵生産物の回
収、精製等の培養効率が著しく向上させられることが、
可能になる乳化剤を用いる有用菌の培養方法を提供する
ことができた。INDUSTRIAL APPLICABILITY According to the present invention, in efficient culture production of useful substances, an aqueous phase in which useful bacteria live and an oil phase such as soybean oil which is a raw material industrially used by the useful bacteria as a nutrient source. Clay mineral is added to, or the clay mineral in which this useful bacterium is immobilized is added, the culture solution is stably emulsified, and the oil phase in the culture solution is dispersed into fine particles to obtain bacterial cells. It is possible to provide an inorganic emulsifier consisting of the clay mineral, which is characterized in that oil is efficiently contacted, and since the emulsifier is an inorganic solid, the separation of bacteria is improved and the fermentation product is recovered. That the culture efficiency such as purification can be significantly improved,
It was possible to provide a method for culturing useful bacteria using an enabled emulsifier.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 小堺 裕子 東京都中央区日本橋室町4丁目1番21号 水 澤化学工業株式会社社内 (58)調査した分野(Int.Cl.7,DB名) C12N 1/38 C12N 11/14 BIOSIS/WPI(DIALOG)─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Yuko Kosakai 4-1-21, Nihombashi Muromachi, Chuo-ku, Tokyo Mizusawa Chemical Co., Ltd. In-house (58) Fields investigated (Int.Cl. 7 , DB name) C12N 1/38 C12N 11/14 BIOSIS / WPI (DIALOG)
Claims (8)
いて、油相と水相から成る培養液にカオリン、ハロイサ
イト等のカオリン粘土鉱物、イライト、セリサイト等の
雲母粘土鉱物、酸性白土、ベントナイト、サポナイト、
ヘクトライト、スチブンサイト等のスメクタイト粘土鉱
物及びセピオライト、パリゴルスカイト、クリソタイル
等の繊維状粘土鉱物群の中から選ばれた少なくとも一種
の粘土鉱物種を添加し、該培養液を乳化させることを特
徴とする有用菌の培養方法。1. In the culturing of useful bacteria such as actinomycetes and filamentous fungi, kaolin clay minerals such as kaolin and halloysite, mica clay minerals such as illite and sericite, acid clay, in a culture solution comprising an oil phase and an aqueous phase, Bentonite, saponite,
Hectorite, stephensite and other smectite clay minerals and sepiolite, palygorskite, at least one clay mineral species selected from the fibrous clay mineral group such as chrysotile is added, and is characterized by emulsifying the culture solution useful Method of culturing bacteria.
いる請求項1記載の培養方法。2. The culture method according to claim 1, wherein the useful bacterium is carried on the clay mineral.
C)が10乃至110meq/100gの範囲にある請
求項1記載の培養方法。3. The cation exchange capacity (CE) of the clay mineral
The culture method according to claim 1, wherein C) is in the range of 10 to 110 meq / 100 g.
体の繊維径が60乃至400Åで、繊維長が0.2乃至
400μmの範囲にある請求項1記載の培養方法。4. The culture method according to claim 1, wherein the fibers of the fibrous clay mineral or a bundle thereof have a fiber diameter of 60 to 400 Å and a fiber length of 0.2 to 400 μm.
95乃至99:1の範囲において、水相と上記粘土鉱物
との重量比が50:50乃至99:1の範囲であること
を特徴とする請求項1記載の培養方法。5. The volume ratio of the aqueous phase to the oil phase in the culture solution is 5:
The method according to claim 1, wherein the weight ratio of the aqueous phase to the clay mineral is in the range of 50:50 to 99: 1 in the range of 95 to 99: 1.
類油、炭素数4以上のアルコール類から選ばれた少なく
とも1種である請求項1記載の培養方法。6. The culture method according to claim 1, wherein the oil phase is at least one selected from vegetable oil, animal oil, petroleum hydrocarbon oil, and alcohols having 4 or more carbon atoms.
半回分、反復回分或いは連続のいずれかの方法で、培養
液を乳化安定化させる請求項1記載の培養方法。7. The contact between the clay mineral and the culture solution is batchwise,
The culture method according to claim 1, wherein the culture solution is emulsion-stabilized by any of a semi-batch method, a repetitive batch method, and a continuous method.
有用物及び上記粘土鉱物を含む該培養液から該粘土鉱物
を分離する請求項1乃至7記載の培養方法。8. The culture method according to claim 1, wherein the clay mineral is separated from the culture solution containing the useful material and the clay mineral after the useful material is produced in the culture solution.
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