JPH0455672B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to an immobilized gel having a string-like or fibrous-like shape as a whole and a method for producing the same. More specifically, the present invention relates to a gel having a string-like or fibrous-like shape, and on which the outer surface of the gel immobilizes one or more types selected from microorganisms, enzymes, plant cells, animal cells, and other physiologically active substances. The present invention relates to an immobilized gel covered with a gel layer and a method for producing the same. The immobilized gel of the present invention has a string-like or fibrous-like shape as a whole, and the outer surface of the gel on which microorganisms, etc. are immobilized is covered with a gel layer, so that it remains stable even when cultured for a long period of time. This can prevent the leakage of converted microorganisms, etc. Further, if the immobilized gel of the present invention is used in culture etc. in the form of strings or fibers, separation of products becomes extremely easy, which will greatly contribute to the fermentation industry, pharmaceutical industry, etc. (Prior art) Generally, in order to immobilize microorganisms, enzymes, plant cells, animal cells, and other physiologically active substances, one or more of these are suspended in an aqueous solution of a substance with gelling ability such as sodium alginate. cloudy,
Drop it into a gelling agent such as Ca ⁺⁺ and gel it.
It is being fixed. (Problems to be Solved by the Invention) When microorganisms, etc. are immobilized using conventional technology, the microorganisms, etc. are mixed with an aqueous solution of a substance having gelling ability and gelled in a gelling agent. To,
The microorganisms and the like are uniformly dispersed in the gel, and some of the microorganisms and the like are present very close to the surface of the gel. If such an immobilized product is cultured in a culture solution, microorganisms near the surface will proliferate, leak from the surface of the gel, and be suspended in the culture solution. If microorganisms are suspended in the culture solution, the product cannot be separated directly, and the microorganisms must first be separated by filtration. Furthermore, a method has been proposed (Japanese Patent Laid-Open No. 59-232092) in which oxygen-immobilized particulate matter is enclosed in a gel, but the particulate matter requires a separation operation from the culture. (Means for Solving the Problems) The present inventors have conducted extensive research in search of a form that prevents microorganisms from leaking out of gelled immobilized materials and is extremely easy to operate. This problem could be solved by making the gel into a fibrous form and covering the outer surface of the fixed gel with a layer of gel alone. The present inventor has developed a gel layer that starts from the outer surface of a gel that is entirely string-like or fibrous and immobilizes one or more types selected from microorganisms, enzymes, plant cells, animal cells, and other physiologically active substances. This relates to an immobilized gel covered with In the present invention, the gel on which immobilized substances such as microorganisms are immobilized is string-like or fibrous-like as a whole, and a second gel coat is formed on the outer surface of the first gel, Since a gel layer is formed on the outer surface of the gel, leakage of microorganisms, etc. is prevented, and the separation of products can be easily and efficiently performed. . The method for producing the immobilized gel of the present invention involves immersing a gel in which microorganisms are immobilized in the form of strings or fibers in an aqueous solution of a substance that has gelling ability, and then pouring it into a gelling agent to form a gel. Although there are various methods such as a method of forming a layer, the following method is preferred in the present invention. That is, in the present invention, a mixed suspension of an aqueous solution of one or more selected from microorganisms, enzymes, plant cells, animal cells, and other physiologically active substances and a substance having gelling ability is produced from the inner tube of a double-tubular nozzle. Then, an aqueous solution of a substance having gelling ability is simultaneously and continuously flowed down into the gelling agent from the outer tube, gelled, and the whole becomes string-like or fibrous, and the gel is coated on the outer surface of the immobilized gel. This is a method for producing an immobilized gel characterized by forming a layer. In the present invention, the immobilized substances added to the aqueous solution of substances having gelling ability include bacteria, yeast,
Microorganisms such as molds and actinomycetes, enzymes such as protease and amylase, cell lines that produce various physiologically active substances, animal cells such as hybridomas that produce antibodies, plant cells that produce various physiologically active substances, mitochondria, microsomes, etc. Examples include intracellular organelles. These immobilized substances are mixed with an aqueous solution of a substance having gelling ability to form a mixed suspension. Substances with gelling ability include sodium alginate, pectin, chitosan, carrageenan,
There are agar, gelatin, etc. Sodium alginate is prepared as an aqueous solution of 0.1 to 10%, preferably 0.5 to 3%, and pectin is prepared as an aqueous solution of 1 to 10%.
Chitosan is prepared in an aqueous solution of 0.1 to 10%, preferably 0.5 to 3.0%, and carrageenan is prepared in an aqueous solution of 1 to 10%, preferably 2 to 4%. Agar and gelatin are prepared in an aqueous solution of 1 to 10%, preferably 2 to 4%, and carrageenan, agar and gelatin are heated to 40°C or higher before use. A mixed suspension obtained by mixing an immobilized substance with an aqueous solution of a substance capable of gelling is placed in a container communicating with the inner tube of the double-tubular nozzle. Further, an aqueous solution of a substance having gelling ability that does not contain immobilized substances is placed in a container that communicates with the outer tube of the double-tubular nozzle. The substance of the aqueous solution of the substance having gelling ability placed in the container communicating with the outer tube may be the same as or different from the substance having gelling ability placed in the container communicating with the inner tube. For example, a combination of sodium alginate (for the inner tube) and sodium alginate (for the outer tube), a combination of pectin (for the inner tube) and sodium alginate (for the outer tube), a combination of chitosan (for the inner tube) and chitosan (for the outer tube), and agar (for the inner tube). Examples include a combination of (for tubes) and sodium alginate (for outer tubes), and a combination of carrageenan (for inner tubes) and chitosan (for outer tubes). Furthermore, the concentration of the substance for the inner tube and the concentration for the outer tube may be the same or may be changed as appropriate. The point is that it is possible to produce an excellent immobilized gel that does not leak microorganisms even during culturing. A mixed suspension and an aqueous solution having gelling ability are simultaneously and continuously flowed down into the gelling agent from the inner tube and the outer tube. At the same time, if they are continuously dropped in a thread-like manner, a string-like or fibrous-like immobilized gel is produced. The gelling agent for the outer tube depends on the substance that has gelling ability, but for sodium alginate, calcium chloride, strontium chloride, barium chloride, aluminum chloride, iron chloride (bivalent, trivalent), etc.
0.01-1.0M of soluble polyvalent cations, such as
Preferably, a 0.05-0.5M aqueous solution is used, and for pectin, calcium chloride, strontium chloride,
Barium chloride, aluminum chloride, iron chloride (2
(trivalent, trivalent) 0.01 to 1.0M such as magnesium chloride,
Preferably, a 0.05-0.5M aqueous solution is used, and for chitosan, 0.01-1.0M such as polymerized phosphates such as sodium metaphosphate, sodium hexametaphosphate, and sodium polyphosphate, trivalent phosphates such as tripotassium phosphate, etc. Preferably, a 0.05-0.5M aqueous solution is used, and for carrageenan, potassium chloride, calcium chloride, ammonium chloride, etc.
A 0.1-0.5M aqueous solution is used, and for agar and gelatin, cooling water, cooling oil, etc. are used. (Effects) The string-like or fibrous immobilized gel obtained in the present invention has the outside of the gel containing the immobilized substance covered with a gel that does not contain the immobilized substance. There is no leakage of the gel, and it is also possible to prevent the gel from being destroyed due to the proliferation of microorganisms, cells, etc. In addition, the string-like or fibrous fixed gel of the present invention is quite durable and will not be destroyed even if it is used for culture, etc., so the separation operation after culture is easy and it can be used many times. It is possible. Next, examples of the present invention will be shown. Example 1 Add 2% commercially available baker's yeast to 100 ml of a 2% sodium alginate aqueous solution from the outer tube of a double tube, and 100 ml of a 2% sodium alginate aqueous solution from the inner tube.
(Dry weight/alg ml), the aqueous solution was simultaneously and continuously released into a 0.1M calcium chloride aqueous solution, and after being left for 1 hour, a 2 mm diameter
A fibrous double-immobilized yeast was obtained. When the fibrous immobilized gel is cut, it can be seen that it is covered with an outer layer of gel with an average thickness of 0.25 mm. The obtained fibrous gel was repeatedly cultured in batches in a medium containing 10% glucose, and the number of bacterial cells leaking during the long-term culture was measured at 0.D580 nm.
The results are shown in Table 1. As is clear from Table 1, it can be seen that very few bacterial cells leaked into the culture solution. Furthermore, when the fibrous gel was examined after culturing, it was found to have sufficient strength so that it would not break even when pulled by a finger. Separately, as a control, fibrous immobilized yeast with a diameter of 2 mm as in the conventional method was obtained using only the inner tube. The obtained fibrous gel was cultured in the same manner as above, and the bacterial cells leaking during the culture were measured at 0.D580 nm.
The results are shown in Table 1. As is clear from Table 1, a considerable amount of yeast leaked out and the culture solution became cloudy as these yeasts repeatedly multiplied in the culture solution. Furthermore, when we examined the fibrous gel after culturing, we found that it was easily cut when pulled by fingers.

[Table] (a): Control
(b): Double immobilized yeast example 2 4% carrageenan aqueous solution 100% from the outer tube of the double tube
ml, and add 2% (Dry weight/carrageenan) commercially available baker's yeast to the 4% carrageenan aqueous solution from the inner tube.
ml), both aqueous solutions were heated to 45°C and continuously released into a 0.1M potassium chloride aqueous solution cooled to 10°C. After standing for 1 hour,
A fibrous double-immobilized yeast was obtained. The obtained fibrous gel was cultured with shaking in a medium containing 10% glycose. Separately, when we prepared an uncoated fibrous gel as a control and cultured it in the same way, we found that bacterial cells leaked out and the medium became extremely cloudy, whereas the fibrous double-immobilized yeast Almost no leakage of bacterial cells was observed, and the strength of the fibrous gel was strong.

Claims (1)

[Scope of Claims] 1 The entire gel is string-like or fibrous, and the outer surface of the gel immobilizes one or more types selected from microorganisms, enzymes, plant cells, animal cells, and other physiologically active substances. The immobilized gel is covered with a layer of. 2 A mixed suspension of an aqueous solution of one or more types selected from microorganisms, enzymes, plant cells, animal cells, and other physiologically active substances and a substance having gelling ability is poured into the inner tube of the double-tubular nozzle, and then An aqueous solution of a substance with gelling ability is simultaneously and continuously flowed down into a gelling agent from a tube, gelled, and the whole becomes string-like or fibrous, and a gel layer is formed on the outer surface of the immobilized gel. A method for producing an immobilized gel, characterized by: