JP3493243B2 - Culture method of useful bacteria - Google Patents

Description

Detailed Description of the Invention

[0001]

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.

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]

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.

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.

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] 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.

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] 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.

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] 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.

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.

Further, JP-A-3-32733 discloses an emulsifier of synthetic smectite group clay mineral such as synthetic stevensite.

[0013]

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.

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.

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]

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.

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.

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]

[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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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]

Preferred Embodiment of the Invention

(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.

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.

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. .

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.

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.

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. .

(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.

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.

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.

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.

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.

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.

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.

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.

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]

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.

(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]

[Table 1]

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]

[Table 2]

(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]

[Table 3]

(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]

[Table 4]

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]

[Table 5]

(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]

[Table 6]

(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]

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.

─────────────────────────────────────────────────── ─── 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)

(57) [Claims] 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. 2. The culture method according to claim 1, wherein the useful bacterium is carried on the clay mineral. 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. 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. 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. 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. 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. 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.