JPS5847492A - Immobilization and propagation of mold of microorganism - Google Patents

Abstract

PURPOSE:To carry out the immobilization and propagation of mold, by casting an aqueous solution of a suspension consisting of an aqueous solution of a polyvinyl alcohol having >= a specific saponification degree and >= a specified polymerization degree and a mold of microorganism into a molding mold, freezing, molding and dehydrating it, and, if necessary, adjusting it to a fixed water content, feeding the prepared gel to a medium. CONSTITUTION:An aqueous solution of a suspension consisting of an aqueous solution of a polyvinyl alcohol having a saponification degree of >=95mol% and a viscosity-average polymerization degree of >=1,500 and a lot of molds of microorganism such as one belonging to the genus Asperigillus, Pseudomonas, etc. is casted into a molding mold. The solution is frozen and molded at <=-6 deg.C and dehydrated in a vacuum into a dehydration ratio of >=5wt% without melting it. If necessary, it is immersed in water, and the water content is reached to 20-92wt%, to give a gel including a living body of microorganism. The gel is fed to a medium for propagating microorganism, the diameter of the mold colony for propagating microorganism formed by it is enlarged in a range of 50-550mum, and the mold is immobilized and propagated.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for immobilizing and propagating living microorganisms, and in particular, the present invention relates to a method for immobilizing and propagating living microorganisms, and in particular, the present invention relates to a method for immobilizing and propagating living microorganisms, and in particular, it involves encapsulating a single primary microorganism in a highly hydrous gel that is highly elastic and has excellent mechanical strength. After capture), it relates to a method for effectively propagating it.

The present invention provides a freeze-dried product (
The gel) encloses (captures and embeds) living microorganisms in the cavity and supplies a growth medium to the cavity, which causes the microorganism enclosing cavity to expand and to keep the size of this cavity constant. This is based on the fact that it has been found that effective growth and utilization of immobilized living microorganisms within the gel can be achieved by controlling the supply amount of the growth medium so as to keep it within a diameter of 550 μm. It is.

That is, in the present invention, a suspension consisting of an aqueous solution of polyvinyl alcohol having a saponification degree of 95 molar or more and a viscosity average degree of polymerization of 1,500 or more and living microorganisms is poured into a mold for molding, and then the suspension is heated to t-6. By freezing and molding at a temperature lower than °C, and then vacuum dehydrating the frozen and molded product to a dehydration rate of 5vtllG or more without thawing, that is, unfreezing, and immersing it in water as necessary, the water content is 20 to 92 wt- (
Wet body standard) After reaching K and obtaining a gel that encloses (embeds) living microorganisms, a microorganism growth medium is supplied to this gel, and the diameter of one proliferating microorganism colony formed by this is 50p.
, ~ 550 Expansion to the category of pfI@tl
- Provides a method for immobilizing and propagating living microorganisms with specific characteristics.

Attempts to trap (enclose) viable microorganisms in a gel and propagate them are already known, but as summarized in (1) to (6) below, each method has its drawbacks, and even better immobilization methods are known. Development of a propagation method has been desired.

(1): flno5 Ctherium simplex (Cor
yn・-bacterium simplex),
Also known as Bacter simplex with Arthro
bacter 51m1ex), Bacillus saptillus (l1acillus 5abtilus)
), Pseudomonas guchida (Pg+sudomona
After immobilizing bacteria such as S. sputida in polyacrylamide gel, they are grown by supplying a nutrient source (medium), and their respective activities, i.e., steroid-converting ability,
Methods for increasing α-amylase production ability, benzene oxidative decomposition ability, etc. are known (Naturet6%, Oct.
, 28.796 (1976), Bioteeh-B
ieeng-s 20.1267 (1978), Eur.
6p, J.

＾PPI-Micyoblol Biotechno
l, s 5, 233 (1?78), 4,75 (19
77)).

However, in these cases, the rate of increase in activity due to proliferation is only about 5 to 10 times.The monomer (acrylamide) that is the raw material for this immobilization carrier is highly toxic and is harmful to microorganisms (fermentation). and industry, l mutual, 92 (
1977), Blotech, Bio@ng, 2
0°1267 (1978)), and the radical initiators used for immobilization (monomer polymerization and polymer crosslinking) damage microorganisms (proteins) (8
ei@me...142.678 (1-963), mu dv
-Biochem lag.

Omi, 125 (1977), Agr, Biol, Ch@m
, 42.683 (1978), Chemical Engineering, Sat US2
6? (1979)), the Kerr immobilization gel itself is weak (Chemical Engineering, Soil 0,139 (1974)), and there are many points that are extremely unsatisfactory in practical use. (2) After immobilizing various microorganisms on agar, Proliferation has been widely practiced since ancient times, but this agar gel
It has poor mechanical strength, and while it can be used in the laboratory, it is difficult to use it industrially as an immobilization carrier. In addition, it has been pointed out that cracks occur from inside the gel due to the growth of microorganisms (B%ot@eh, B%oe
ng, 22°681 (198G)).

(5) Leuconostoc mesente poides (L.
uionostocugly・intestines・nt@roid@s)
f, after inclusion in polyvinyl alcohol/boric acid complex gel,
There is also an attempt to multiply this (Japanese Patent Application Laid-Open No. 54-15529
5). However, in this case, the boric acid complex (gel) produced from polyvinyl alcohol with a high degree of saponification is weak and difficult to mold. Due to the strong adhesiveness of the gel, it is difficult to maintain the shape after molding, making it unsuitable for practical use 0 (4) Saccharomyces S@r・Ma111
・) has been proposed to be grown after being immobilized on a gelatin membrane, but the activity increases by about 4 times, and the gel molded body is soft and its shape is unstable (Biot@
ch,Blo@ng,, 22.1755(
1980)).

(5) Some attempts have been made to propagate Satucharomyces cerevisiae after immobilizing it on calcium alginate gel. As is often the case with Kerr's natural polysaccharide gel, it is easy to cause tissue contamination, and it is necessary to apply slight acupressure by pinching it with your fingertips. When added, it immediately loses its shape and takes on a soft, pasty appearance, and the gel structure is destroyed by the phosphoric acid supplied as a nutritional source (Biot@ch,
Bioeng-, 19°587 (1977)).

(6) As a natural polysaccharide that has an acidic sulfate ester structure of polygalactose like agar, Nichiriki 2genan and Satucharomyces cerevisiae (5aeeba
r@mye@sC@r・Ma151m5), Saccharomyces number callusbergensis (8accharomyce
s earlab@rg@ns1m), Ac5tobaet@r 5
uboxy-mu nm), Serratia Φ marcescens (8@
rratimmarac@me@ms), Nizierisia coli (Kseh@r1chiac+eli), Brevibacterium flavum (Br. ma1-bacte)
+rium flavum), Streptomyces 7eochromogenes (8trsptomyees)
pha@oehromogenes) Brevi-baeteriu
Some attempts have been made to immobilize and then propagate the following species. However, this gel is weak (tensile strength <1KII/-) and further contains potassium chloride,
Even if it is hardened using hexamethylene diamine, glutaraldehyde, tannin, etc., the strength is only improved to the same level as polyacrylamide gel (1 to 1.5 K4/ai), and it is extremely weak. . Therefore, as microorganisms proliferate within the gel, cracks are generated and destroyed in the gel structure (J, 5olid Phas
@Bioshsm e 2. 225 (1977),
Enzym@Mi*rob-T@ehno1. lX
95 (1979), J, Ferm@nt, T@ch
no 1.88° (4) 327 (1980), polymer, main! , 238 (1980)) 0 As can be seen from these examples, all of the conventional carriers for immobilizing microorganisms have problems, and a better carrier (gel) is desired (polymer, main! , 238 (1980)) 0
Unlike these known methods, according to the present invention, a gel is generated during the process of freezing, molding, and dehydrating a mixed suspension of polyvinyl alcohol and live microorganisms. encapsulated in this gel. In the present invention,
This fixation (entrapping) process does not use acids, alkalis, radiation, radical generators, organic solvents, reaction reagents, etc., and also does not require any secondary curing treatment. The gel of the present invention is a rubber-like elastic body that is highly water-containing and has excellent permeability to carbon sources, nitrogen sources, oxygen gas, carbon dioxide gas, and other inorganic substances required for the activity of living microorganisms, and has a tensile strength. In terms of strength, the above-mentioned carrageenan (<14/j) or carrageenan subjected to secondary hardening treatment (1 to 15 m4
/d) 'I far exceeds strength (4-6 Kg/d)
above) t- has.

By supplying a growth medium to this gel, a single primary microorganism naturally proliferates.In the gel of the present invention, the microorganism-encompassing cavity is pushed and expanded as a result of this proliferation. Because the gel is extremely elastic and has excellent mechanical strength, the diameter force of the internal cavity of the gel ζ is 20 to 70 times (550 pm)
The gel structure of the present invention has the feature that it can withstand intense proliferation (swelling) reaching up to
When the culture reaches 50μ, a part of the proliferating bacteria passes through at least a part of the mesh of the cavity wall (gel tissue consisting of a fine network structure) and is discharged from the cavity, and at the discharge position (small cavity). , and then repeating the process of growth and expulsion 1. Finally, they are expelled from the gel from the cavity near the gel surface. Further, when the diameter of the growing colony exceeds about 550 pmt by further supplying a growth medium, the amount of bacterial cells shed increases significantly.

Therefore, the K to avoid loss (loss) of proliferating bacterial cells is:
It is preferable to keep the diameter of most of the proliferating bacterial colonies to 150 μm or less.

By the way, generally, at the same time as a microbial living cocoon proliferates, at least a portion thereof dies. Since it is undesirable for this autolyzed residue (mainly cell wall components) of the dead sterilized cells to continue to accumulate in the viable cell colony, in the gel of the present invention as well, it is removed from the gel intermittently or continuously. must be eliminated. However, since there is still no known method for selectively expelling only dead bacteria (residues from autolysis) out of the gel, it is unavoidable to remove part of the bacterial colony consisting of live and dead bacteria. must be allowed to flow out of the gel intermittently or continuously.

Regarding this, in the present invention, the colony diameter is
By reaching 50 pm or more, the above objective can be easily achieved.

Moreover, it is not preferable that the colony diameter exceeds 550 μm, as this will lead to the loss of a large amount of the proliferating microbial cells.

Therefore, in the present invention, the diameter of the bacterial colony is t-550.
By maintaining the temperature below μm, for example, 50 to 550 pm, it is possible to avoid wasteful loss of a large amount of proliferating viable bacterial cells, to effectively proliferate immobilized living bacterial cells, to make full use of them, and to kill the bacteria if necessary. The body can be expelled.

The diameter of the bacterial colony in the present invention can be easily controlled, for example, by controlling the amount of growth medium supplied, rather than by tracking and observing the expansion status of the colony.

On the other hand, an aqueous solution of polyvinyl alcohol was heated to 1
It has been well known for a long time that the phenomenon of increased viscosity or gelation is often observed when stored for a day to a week. However, as is often seen in the gels of natural polysaccharides mentioned above, this gel is soft, like agar, and moreover, even if it is simply stirred vigorously, it can be easily stirred by adding water or by heating it slightly. Since it dissolves, it is impossible to use it as a carrier for immobilizing microorganisms.

Furthermore, there is a method of mixing living microorganism cocoons into an aqueous polyvinyl alcohol solution and then air-drying the mixture to obtain a V membrane containing living microorganism cocoons, but this membrane has poor water resistance, is weak, and has a low microorganism trapping capacity. This membrane is supported (reinforced) with nylon cloth, etc.
Although there are some proposals to do so, most of the above-mentioned difficulties still remain.

In addition, as a feature of polyvinyl alcohol film,
Due to poor permeability of carbon sources, nitrogen sources, and inorganic materials required for microbial activity, the activity of immobilized microbial viable cells decreases (%Kaisho 52-14592, 软publicsho 55-354)
15, Plastic Materials Course 14, P, 155, P, 1
55 (Sho 45), Nikkan Kogyo Shimbun) 0 A polyvinyl alcohol crosslinking/gelation method is also well known, in which a polyvinyl alcohol aqueous solution is mixed with microorganisms or enzymes, then oxygen is excluded and cobalt 60 (r-ray) t'' is irradiated. be.

However, in this case, even if a radiation-damaged film-protecting substance such as glycerin is used in combination, it still has an adverse effect on microorganisms or enzymes, increases the cost of irradiation, and the resulting gel is soft and is often contaminated with other chemical reagents. Requires secondary curing treatment (Biot@ch,.

Bi**ng, 15, 407' (1975), Fermentation and Industry, 55.92 (1977)).

A method for producing a polyvinyl alcohol/silicic acid composite yeast membrane has also been proposed by adding acid to an aqueous suspension containing polyvinyl alcohol, tetraethyl silicate, and microorganisms and air drying the mixture, but this membrane is also weak.

In this case, even if the membrane is freeze-dried after adding an acid, the mechanical strength of the resulting membrane decreases and it is almost impossible to form the membrane.

In any case, since a step of adding an acid to the microorganism suspension to adjust the pH to 3 or less is included, the adverse effect on the microorganisms is often not negligible (% Publication No. 55-11311, Japanese Patent Publication No. 55-30358).

A method for immobilizing enzymes by gelling (freezing and solidifying) an aqueous solution of polyvinyl alcohol and enzyme at a low temperature has also been proposed (%iR Showa 5O-52276). However, the gel obtained by simple freezing and solidification does not exhibit elasticity, has extremely low mechanical strength, is a soft gel-like product (or mucus), is extremely sticky, and has poor water resistance. It dissolves poorly in water and the remainder turns into paste.

Furthermore, if the carrier is air-dried after freezing, solidifying, and thawing, only a weak wet film or a film with low water content is produced, which is not preferable as a carrier for immobilizing living microorganisms.

In addition, when attempting vacuum dehydration after freezing, solidifying, and thawing,
The molten liquid foams violently, often making it impossible to continue operation.Even if dehydration takes a long time, only a brittle, cloudy gel with almost no elasticity is obtained. Because of its low water content, it is not preferred as a carrier for immobilizing and propagating living microorganisms.

The gel used in the present invention is insoluble in water or Fi hot water, and is completely different from the gel produced by storage of polyvinyl alcohol as described above.

In addition, the gel of the present invention does not exhibit stickiness and is highly elastic,
Both its tensile strength and compressive strength are 4 noodles/- or more,
Furthermore, it has advantages over any of the above-mentioned (known) microorganism immobilization carriers in that the gel structure does not rupture even when the viable microorganisms grow vigorously. In addition, the gel of the present invention contains gamma rays, radical initiators, acids,
It has an advantage over all known methods in that it does not use alkali, chemical reagents, organic solvents, inorganic solvents other than water, etc. The degree of saponification of the polyvinyl alcohol used in the present invention is 95 molt or more, preferably 97 mol. The saponification degree is 80-88 moles, especially 8 moles or more.
Even if polyvinyl alcohol having a molecular weight of 57 or less is used, only a soft gel will be obtained, and the object of the present invention will not be achieved.

Further, in the present invention, polyvinyl alcohol having a viscosity average degree of polymerization of 1.500 or more is used. As the degree of polymerization of polyvinyl alcohol decreases, the mechanical strength of the resulting gel also decreases. Therefore, in the present invention, a commercially available product with a high degree of polymerization (degree of polymerization of approximately 1,700 to 2,600) is used. It's good.

In the present invention, first, an aqueous solution of polyvinyl alcohol is prepared. There is no particular limit to the degree of s, but for example, 1 to 20
The weight can be 7 to 15 wt, preferably 7 to 15 wt.

This #1 degree can be further increased to, for example, 90 wt, but the viscosity of the aqueous solution at room temperature is IQOOOO
Reaching more than eP L/% Also, viscosity increase or gelation may occur during storage, making it somewhat difficult to handle. Since this concentration can be set to 3 wt- or less, the time required for dehydration (drying), which will be described later, becomes longer and costs (dehydration power costs) increase.

In the present invention, the polyvinyl alcohol aqueous solution is sterilized prior to adding viable microorganisms to the polyvinyl alcohol aqueous solution. Sterilization processing conditions: 100℃
In some cases, the objective can be achieved with x5min, but heat resistance II
! For example, if it is contaminated with
Perform high pressure/steam sterilization for 1 m to 6 h. Ultraviolet irradiation sterilization can also be used, but since its effectiveness is limited to irradiated surfaces, it is desirable to use it in combination with the heat sterilization method described above. In any case, these treatments do not alter the quality of the materials used in the present invention and do not pose any hindrance to the implementation of the present invention.

The sterilized aqueous solution is then mixed with live microorganisms to be immobilized. Examples of viable microorganisms include molds such as Aspergillus, Rhizopus, Pseudomonas, Acetobacter, Streptomyces, Nijiyulicia, Satucharomyces, Candida, etc., such as filamentous genus, Actinosa 1, bacteria, yeast, algae, etc. Many living microorganisms can be targeted.

In this case, any of the following can be used: a frozen and dry-preserved pneumophilic cell, a culture solution for growing viable cells, or a concentrated suspension of living cells centrifuged from the growing culture solution. It is convenient to perform this addition (mixing) of living microorganisms at a temperature of about 10 to 35°C, but when fixing heat-resistant living cells, the operation is performed at a temperature of 135°C or higher depending on the heat resistance of each individual. You can also.

If the temperature is below 10°C, the viscosity of the aqueous solution increases and the mixing and dispersion of viable bacterial cells is slow.If this point is kept in mind, the above operation may be carried out at temperatures below 10°C.

The amount of viable microorganisms to be added (on a dry basis) is preferably kept at 7 times the amount of polyvinyl alcohol in the aqueous solution or less, from the viewpoint of immobilizing almost the entire amount of viable microorganisms. K to go through a dehydration (gelation) process
Therefore, 96 to 98 of living microorganisms can be reliably captured (enclosed and immobilized).

Freezing, molding, and dewatering process 1 (described later)! As a result of observing the gelatin of the present invention obtained through this process using a scanning electron microscope, it was found that, even when Salacharmyces cerevisiae was immobilized in an amount equivalent to 1/4 of the weight of polyvinyl alcohol, microbial cocoons were individually dispersed. They are each embedded in a cavity of 8-5 Ops, and the cavity wall is mainly composed of a network of 11-1 pm. Furthermore, the individually dispersed and embedded living microbial cells can easily and vigorously proliferate by the propagation operation described below. Therefore, in the present invention, rather than embedding a particularly large amount of living microorganisms in the gel in advance, it is preferable to leave room for expansion of the enclosing cavity due to proliferation, which will be described later.

That is, in the present invention, the amount of microbiology added to the immobilized tooth cutter can be 7 times or less than the amount of polyvinyl alcohol used, for example, 1 to 2,000, preferably 11 to 1.

15.000 2 In the present invention, care must be taken not to contaminate the thus obtained mixed suspension solution of polyvinyl alcohol and living microorganisms with bacteria, and also to avoid direct irradiation with germicidal lamps (ultraviolet light). At the same time, the suspended aqueous solution is injected into a mold for molding, frozen and molded. Here, the mold for molding is preferably one in the shape of the final use, but it may also be a container of any shape for obtaining a plate-like object, and these are also included in the mold for molding as referred to in the present invention. As a cooling agent for freezing, for example, salt-ice (
2,3 near 7) (-21°C), calcium chloride-ice (3
Cryogens such as 0nia0) (-55℃), dry ice-methyl alcohol (-72℃), liquid nitrogen (-1
96°C) to a mt lower than -6°C and freeze. If the cooling is insufficient, the shape of the gel obtained through the drying process described below will not match the initially expected shape, that is, the shape of the polyvinyl alcohol aqueous solution injection container or the mineral mold. 0 less strong
If liquid helium is used, it can be cooled down to -269°C, but for practical purposes, it is better to use a Freon refrigerator to cool down to, for example, -35°C or lower. Most of the living microorganisms are -20'C
Since it is undesirable to expose the material to temperatures around m degrees Celsius for a long time, it is preferable to rapidly cool it to below -20°C, for example, to -55-80°C. Freezing and molding at a low temperature such as kernels contributes to increasing the mechanical strength of the gel for supporting live microorganisms.1. , freezing and molding at temperatures between -20°C and -6°C is preferred.

In the freezing fist molding according to the present invention, a polyvinyl alcohol aqueous solution is solidified (frozen) and molded in a mold of an arbitrary shape, and then, if the mold has a -L self-cover or a to-en cover (or both), One or both of them can be removed and the molded product can be frozen and dehydrated while maintaining its shape.

Therefore, the shape of the gel of the present invention takes into consideration the diffusion between air, liquid, and solid phase, which is favorable for the growth and growth activities of immobilized living microorganisms, and any size and shape can be selected at the time of freezing. . Preferred shapes of molded bodies include ramchig rings, which are already used in the chemical industry for distillation columns, gas absorption columns, etc.
perforated plate@+5iev
@tray), telleratta
, Inter Yotsuku Saddle (1ntalox 5add
le), ball ring (pail rinsr)
It is possible to use a mold for molding such as. Also, the special request was made in 1983.
It is also possible to use a mold in the form of a zigzag or curved plate having protrusions as described in No. 51096. The gels with immobilized living microorganisms of the present invention obtained using these molding molds are all nutrient sources necessary for the growth and growth activities of the immobilized living microorganisms, as well as contact with the substrate and mass transfer. It is also superior to granular products, spherical products, plate-like crystals, film-like products, etc. in terms of low internal pressure loss when packed into a reaction tower.

Of course, granular forms are also included in the present invention, and methods such as forming a plate-like body and cutting it later are also included in the present invention. Considering the influence on viable microorganisms, α1~b should not be lowered to about -10°C, but after that, rapid cooling at 7~00°C/mi is preferable.

In the present invention, after confirming that the aqueous mixed suspension of polyvinyl alcohol and viable microorganisms that has been poured into the container or mold described above has been frozen, the kernels are subjected to a vacuum dehydration tube.

In this case, if the frozen/molded body is removed from the freezing chamber and transferred to the vacuum drying chamber and immediately suctioned and dehydrated, the sample will be cooled as water is removed (sublimation), so 4IK
Even without external cooling, the frozen/molded body does not melt.It is acceptable to heat the frozen/molded body to such an extent that the body does not melt, thereby promoting dehydration. In other words, there is no limit to the percentage of il [in the dehydration process] as long as the frozen/molded product is not thawed, that is, unfrozen.
This does not particularly affect the quality of the gel. In this dehydration step, the dehydration rate is set to 5 vi - or more, and the water content of the gel is, for example, 20 to 92 vi, preferably 60 to 90 vi.
t% (wet body basis).

Although the water content can be lower than ``Ik2011!'', even in this case, a water content of 50 to 90 Vt 9G can be achieved by immersing it in water as described below.

In the present invention, the frozen and molded body is subjected to a slight dehydration treatment (regardless of whether the polyvinyl alcohol is
(vacuum drying). In this case, the dehydration rate (weight reduction rate of the frozen/molded body) is, for example, 5 v t, or even 1
5wt- or more is adopted. In other words, as dehydration progresses, the gel strength decreases, so N
Desired J) It is best to select the main meeting according to your preference.

This dehydration step (freezing/drying) cannot be omitted.

That is, unless this is carried out, the highly elastic and highly hydrous gel of the present invention with excellent mechanical strength cannot be obtained.
As a result, the immobilized live microorganism gel is extremely soft.

In addition, if the frozen and molded product is melted and then dehydrated under reduced pressure without maintaining the frozen state, the foaming is so intense that it is often impossible to continue the operation, even if it takes a long time. Even if the frozen water is dehydrated, the vacuum dehydration method used in the present invention may be of any type as long as the frozen water can be easily dehydrated.
Below, Ir is preferably 11 ■Hg or less, more preferably α1■Hg or less, and usually used.

In the present invention, the frozen, molded, and dehydrated body is then allowed to stand at room temperature to thaw (thaw), thereby obtaining a highly elastic microorganism-immobilized gel. In this case, the melting operation is as follows:
1-b In some cases, 3-1.0110℃/m
It is also possible to form kernels due to rapid temperature rise in in. In any case, at temperatures above 60°C, a hard film rapidly forms on the surface of the gel, so regardless of the heat resistance of viable microorganisms, the thawing (thawing) operation temperature should be 40 to 50°C or lower. is desirable. After this thawing operation, the microorganism-immobilized gel can be easily taken out from the container or the supporting part of the mold. If necessary, this gel absorbs water by immersing it in water, and reaches a water content of 50 to 95 Vt (wet shell standard), or is still a strong elastic body, so it is not susceptible to the growth activity of microorganisms contained within the gel. suitable. Based on the above-mentioned findings from the scanning electron microscope and the above-mentioned water content, it is an excellent elastic body for a gel with such a high water content. High water content and mechanical strength have traditionally been considered to be compatible in developing medical polymers and selectively permeable membranes. The conventional air-dried film of an aqueous polyvinyl alcohol solution, or the soft gel obtained when the aforementioned aqueous polyvinyl alcohol solution is stored at 0 to 30°C, or when the aqueous polyvinyl alcohol solution is simply frozen and thawed. is completely different. As is clear from the fact that it holds a lot of moisture, the specific gravity of this gel is the same as that of buried water.
The gel of the present invention is not sticky as it only barely settles in water. Plate (8 x 8 x 2-), cylindrical (inner diameter 3-1 outer diameter 6, length 6), spherical (diameter 4), etc.rc
Even when 1 ton of IRmt and Tagel cypress was mixed in water at 50 °C for 10 days, no dust phenomena such as mutual adhesion or deformation were observed. Furthermore, even if konnyaku is soaked in tap water for a year, it will not dissolve and its elasticity and strength will not change. In the present invention, a single component of polyvinyl alcohol is used as a gel material (gelling component).However, it is important to note that inorganic or organic materials may coexist as long as it is dangerous to inhibit the gelation process of polyvinyl alcohol. There is no problem with the invention, and the coexisting amount can be, for example, 4 or less amounts of polyvinyl alcohol. On the other hand, substances that act on polyvinyl alcohol (or polyvinyl acetal as modified polyvinyl alcohol, polyvinyl butyral, etc.) to form a composite gel, and substances that react with polyvinyl alcohol to denature it, may coexist even in small amounts. Even so, it often has an unfavorable influence on the gel formation of the present invention (formation of a polyvinyl alcohol-monomer gel), making it difficult to produce a high water content gel with excellent mechanical strength. Examples of such substances include colloidal alkali silicates (U.S. Pat. No. 2.83 to 641 (1958
)), colloidal silica (U.S. Pat. No. 2,85!5661)
(1958)), alkaline colloidal silica (JP 54-153779), organosilicon compound (vinyl acetate resin, P, 93, Nikkan Kogyo Shimbunsha (1942)), tetraalkyl silicate (% publication 55-30558, special Publication No. 55-11311), boron, borax (French patent 745942 (1955)), phenol, naphthol, meta-cresol, pyrogallol, salicylanilide, disalicyl benzidide, resorcinol, polyamines (polymer chemistry, 1 U (105 )23, (1?54))
, kaolin (Nature m1
70.461 (1955)). These are avoided in the present invention because they form a complex gel with polyvinyl alcohol depending on the amount of coexistence.

In the present invention, when freezing microorganisms or biological tissues, or their suspension water, in an o-ff, which is essential for freezing and molding an aqueous suspension of living microorganisms, it is possible that these may be more or less susceptible to freezing damage. Receiving has been well known since ancient times. In order to avoid or significantly reduce freezing damage to this living body or its group 1 proteins, etc., the method of rapidly cooling to a low temperature of 130°C or lower is the hydroxyl group of carboxymethylcellulose, etc. A well-known method is to add a small amount of water-soluble polymeric substances and various freeze-drying protection agents. In the present invention, since the polyvinyl alcohol (gelling material) itself acts as a strong freeze/dry protection agent, most of the viable microorganisms are normally preserved and sufficient proliferation and growth activity is achieved as described below. However, a known freeze-drying protection agent may also be present.

Substances that reduce freeze-drying damage are generally 1-eel preservatives, eel-preservatives (Prot@etanL prot@ettoe
5ubstance), additives, additives (addi
tives, add%t%0! Ill 5ubsta
nce), medium, medium, adjuvant,
Dispersion medium (suspendedmelium), stabilizer (
Specific examples include magnesium ions, glycerin, dimethyl sulfoxide, honey, peptone, yeast extract with meat extract, skim milk, serum, albumin, L- or D-glutamic acid sodium salt, potassium salt, N-acetylglutamate, D- or L-arginine, DI, -2-pyrrolidone-5-carboxylic salt, polyvinylpyrrolidone, L-
Homoalginic acid, D-glucose, D-1 or L-aspartieacid, ascorbic acid, DL-) leonine (thr@onin@χD,
L-allothreonine (allothr@onins)
, gelatin, mucin, lactose, DL-malic acid, L-cysteine (cyst%n.), L-sorbitol, arabitol, pectin, gum arabic, mannose, ratatose, L-'I Resin D-74#dose, dextrin , dextran, sucrose, soluble starch, raffinose, citric acid, acetylglycine, D-xylitol, etc., as well as L-glutamate-skimmed milk (or dextran, soluble starch polyvinylpyrrolidone, carboxymethyl cellulose). Combination treatments of human gelatin, lactose) or dextran-ammonium chloride-thiourea-ascorbic acid, skim milk-7x:fulvin@(or sucrose), and glucose and serum are also known. As for the amount to be added, it is also possible to add α5 to 2- to the polyvinyl alcohol aqueous solution mentioned above, and add a force of about ζ 104, which is often sufficient to achieve the purpose.

According to the present invention, live microorganisms can be captured in a gel with high water content, high elasticity, and excellent mechanical strength.
By controlling the growth of the present invention, the microorganisms proliferate significantly and increase their activity, thereby achieving the immobilization and proliferation of living microorganisms, which is the objective of the present invention.

The fact that viable microorganisms multiply when supplied with a growth medium is not in itself a new fact; it is an extremely well-known principle from ancient times, but because the gel of the present invention is highly elastic, The living microorganisms embedded inside easily expand the enclosing cavity and multiply, growing into a huge colony.
Moreover, since the gel of the present invention has excellent mechanical strength, destruction (cracks) of the gel structure due to proliferation of viable microorganisms can be avoided. The gel of the present invention has both tensile strength and compressive strength of 4.
Noodles/- and above, natural polysaccharides (agar, kaladanan,
Pectin, etc., strength 11 cf/- or less)! Alternatively, a secondary hardening treatment product of carrageenan (strong, degree 1 to 1.511/cs
i) However, in the gel of the present invention, when the diameter of the growing bacterial colony reaches about 150 pm, some of the growing bacterial cells pass through the mesh of at least a part of the cavity wall and exit the cavity. The cells are discharged to the gel, and the process of growth and discharge is repeated in the discharge position (small cavity), and finally, they are discharged from the gel from the cavity near the gel surface. If this continues, the diameter of a colony of one proliferating body exceeds about 5507 gm, and as a result, the amount of loss of one body increases significantly. Therefore, in order to avoid the loss of a single proliferating body in the present invention, the diameter of the colony is usually set to 150 gm.
It is preferable to keep the number of deaths below 1.
In order to avoid the accumulation of bodies, it is possible to adopt a method of supplying a large amount of growth medium to reach a colony diameter of 150 μm or more. However, it is not preferable for the colony diameter to exceed 550 pmt, as this will result in a large amount of the aforementioned Toosho-proliferating bacterial cells being washed away. Therefore, in the present invention, the diameter of the bacterial colony is maintained below 550 p, for example, 50 to 550 μm, preferably 100 to 500 pvmK, to avoid wasteful loss of a large amount of growth, and to reduce the diameter of one bacterial colony intermittently or By continuously increasing the pressure to 150 to 550 pwa, some of the bacteria including dead bacteria (residues from autolysis) are removed from the gel from the nine-microbial colony. That is, in the present invention,
By controlling the supply of the growth medium so that the diameter of the growing bacterial colony remains below t-550 pg, wasteful loss of a large amount of growth is avoided.

In the present invention, a growth medium is supplied to the immobilized living microorganisms. μ The pole II [t of the gel is intermittently collected. μ For example, by scanning electron microscopy, the proliferating colony within the gel is observed and its diameter is 550 FIIl or less. When this point is reached, a large amount of viable bacterial cells can be eliminated by stopping the supply of the growth medium.

On the other hand, in order to increase the activity of immobilized live microorganisms, it is necessary to increase the number of live bacteria in the gel.The number of live bacteria that can be accommodated in a gel of 0 unit volume is of course limited, and its limit value ( jl
Density filling f) is based on the shape and size of viable bacterial cells.
It is calculated mathematically, for example, in Satucharomyces cerevisiae (spherical or elliptical, average about 5 μm), it is about 4.1
0 pieces/sg. In an actual gel, since the gel material also occupies some space, it is impossible to completely achieve the above limit value. It is possible to achieve a bacterial cell concentration extremely close to the above-mentioned limit value, within a range where the concentration of bacteria is very close to the above limit value. That is, when compared to polyvinyl alcohol, for example,

When Saccharomyces cerevisiae (based on dry bacterial body weight) is added, subjected to the comprehensive treatment of the present invention, and a growth medium is supplied for 3 to 4 days, single colonies with a diameter of about 400 μm are densely packed in the gel, and the yeast concentration is Approximately 10” pieces/--0 estimated to be gels
Furthermore, when Satucharomyces cerevisiae was supplied to polyvinyl alcohol as an additive layer growth medium to the extent of u'/, bacterial colonies with a diameter of about 200 μm were densely packed in the gel. -pcs/5ff
- Estimated to be gel.

The activity of the immobilized and proliferated specimen thus obtained does not drop sharply even after the supply of the growth medium is stopped, and the same activity is often maintained for 2 to 3 weeks. Most 1~
After 2 months, the diameter of KFi-body colonies decreases by about 20-40% and the activity decreases by about 30% in many cases. By supplying kernels for 12 to 24 hours, the activity and colony diameter of the immobilized bacterial cells are restored to their original values.

In the present invention, a diameter of 50 to 550 pW@,
By generating a large number of bacterial cell colonies of 100 to 5 QOp south, and then continuously supplying the growth medium intermittently or in small amounts, it is possible to maintain the initial bacterial cell activity. Characteristics: Even when the gel of the present invention is subjected to the above-mentioned operation for t-4 months or more, there is no change in the strength of the gel structure, and no cracking or destruction of the gel structure is observed.

By subjecting the polyvinyl alcohol gel of the present invention to a curing treatment for polyvinyl alcohol fibers or polyvinyl alcohol film, the mechanical strength of the gel is further increased slightly. Known hardening (crosslinking) treatments include aldehydes, dialdehydes, diincyanates, phenols, or metal compounds such as titanium, chromium, and zirconium, as well as borax, acrylonitrile, trimethylolmelamine, and evicoulohydride. Examples include methods using phosphorus, bis-(-monohydroxyethyl) sulfone, polyacrylic acid, dimethylol urea, maleic anhydride, etc. However, the gel of the present invention has the strength (load-bearing capacity) as described above. However, considering that the immobilized living microorganisms are often damaged by the above-mentioned auxiliary curing treatment and that the manufacturing cost of the gel is unnecessarily high, these curing treatments, which are often used for natural polysaccharides, In the present invention, it is preferable not to use such a treatment.Next, the present invention will be explained with reference to Examples.

Example 1 Commercially available polyvinyl alcohol (saponification degree 97 mol 1, viscosity average polymerization [2,200,491 viscosity of aqueous solution 1j (20 ° C.
)54eP) powder 85f (water content 6wt1g) was dissolved in 91st water.1. ,! LOvtl! Aqueous 1' [(pH
&9) was obtained◇Gin aqueous solution 5781 at 120℃
After being subjected to pressurized steam sterilization 1 treatment in an incubator and left to cool in a non-plane room, Satsukawa Myces 9 cerevisiae (8 aeeh
aromye*m e@revisims) -[12
20 - of suspension water (phosphate buffer pH 7) (culture solution concentrate) containing T was added and stirred for 7 ml. The polyvinyl alcohol concentration of this suspended aqueous solution was 7.6 wt.
%.

200 f of this suspended aqueous solution was poured into a Raschig ring (8 m x 8 m) mold for molding (665 molds) in a sterile room, and then cooled at -45°C x α5 h (a[l condensation and molding]). The cover was removed, and the lower cover supporting the molded body was subjected to vacuum dehydration treatment at 11-Hg for 6 hours.

After thawing, a gel of 132F (water content 88 wt IG, dehydration rate 35 wt1) was obtained.

This gel was mixed with α9-saline solution 15 (1
- As a result of being immersed in water for 6 hours, the molded gel absorbed water and became 1sar (
Do not reach a moisture content of 89 wt. The yeast Fi was not found in this immersion solution, and it was confirmed that the remaining amount of yeast was trapped in the Raschig ring.
The above Rashhili/g 130t-irregular secondary light cell, 120℃ x 15ml 1m pre-sterilized medium (Glucose 1-1Pyton LIL5Is1 Yeast Technology α5%
, malt extract RLS, potassium chloride 1%, pH 5.5
, 25° C.) from the bottom of the column at a flow rate of 120 sf/. Before and after this medium feeding operation, a very small amount of Raschig ring was collected and observed using a scanning electron microscope.The results showed that before the medium feeding started, only a small number of yeast were individually dispersed and encapsulated in the gel. Therefore, many yeast colonies (70 μm in diameter) could be easily observed after ζλ4h.

After 58b, we noticed that the diameter of each colony was 200 μm and the colonies were expanding and coming closer to each other, so we immediately stopped feeding the medium and added the substrate solution for ethyl alcohol synthesis (
Glucose IQwt, magnesium sulfate heptahydrate! O
ppm, PH! As a result of feeding from the bottom of the column at a flow rate of 80 ssg/h (L5.52°C), the ethyl alcohol concentration in the effluent reached 4 wt 1 (theoretical yield of 7711) K after 12 h. By continuing this operation for one month, the ethyl alcohol concentration of the top effluent decreased to 55 wt Is.The diameter of the viable bacterial colony in the regel was reduced to 140 μm. was fed again from the bottom of the column at a flow rate of 1 zo-/h, and the original colony diameter (200 μm) was restored after 12 hours. After that, the substrate solution for ethyl alcohol synthesis was fed into the tube at a flow rate of 80 d/h, and the effluent was concentrated with ethyl alcohol and then again subjected to AwtlGK. , viscosity average polymerization WL1, 700.4% aqueous solution viscosity (20°C
)26cP) powder (moisture content 7vtlG) was mixed with 9g of water.
LA was dissolved in 17t to make an aqueous solution of 8vt.

44 tons of this aqueous solution was first sterilized with pressurized steam at 120°C x 15 ml, then left to cool in a sterile room, and then transferred to Kluyma marxianus (Kluyma r.
omyoasmarxlanmlm) 1008 ft-
Pour 4 ft of suspension (culture solution) containing the mixture and stir for 7 ml. The polyvinyl alcohol concentration WL of this aqueous suspension solution was zSvt. In this suspended water 41tt1 cocoon-free chamber, a mold for molding (130
-58℃×[15h cooling (freezing/
After molding), the molded product was removed from the mold and subjected to vacuum dehydration for 6 hours. 23t after thawing (moisture content 85Wt'
A gel with a 4S dehydration rate of 43 wtl5) was obtained using a 9S saline solution 40sd''4C that had been sterilized in advance.
As a result of soaking for 6 hours, the molded gel absorbed water and became 27f (water content 8).
A glass column with a diameter of 3c11 and a height of 1051, which had reached 7wt. '
I's (151% PH", 25℃) t12d/h
It was introduced from the bottom of the tower at a flow rate of . As a result of observing the gel after 40 hours using a scanning electron microscope, a diameter of 450p was observed within the gel.
There were no fissures in the strange ζ gel structure observed in the colonies of m. After feeding the 0 medium for another 10 hours, the same observation was performed. As a result, when the diameter of the colonies exceeded 550 μwst, the effluent at the top of the column became turbid. I noticed that the yeast had started to bleed out a little. Therefore, it was necessary to control the colony diameter to 550 μm or less. Example 3 Commercially available polyvinyl alcohol (saponification, 1 viscosity average polymerization of 1,800,49 G
0°C) 29.5eP) powder 85t (moisture content 5vtIs
)t, LA 8wt dissolved in water 9132-aqueous solution (pH 7
).

18f of this aqueous solution was subjected to a pressurized water vapor treatment at 120°C x 20m1n, then allowed to cool in a sterile room, and then transferred to the 18f water solution.
ilis) a 2 ff of turbid water (culture solution, pH 7) containing 2 t of OQ was poured and stirred for 7'min.

The polyvinyl alcohol concentration of this suspended aqueous solution is 7wt-
It is.

Pour 18 ft of this aqueous suspension into a polyethylene container (bottom 6 x 6 tya) in a sterile room.
After cooling (freezing/molding) for 6 hours at ℃×α, vacuum dehydration was performed for 5 hours.

After thawing, 1a4f (moisture content 86 wt%, dehydration rate 4
A white opaque gel of 2 wt. As a result of soaking this in pre-sterilized A9-saline solution for 30sdk8h,
The gel absorbed water and reached 12t (water content 88wt56).

This gel was cut into a large number of pieces (8 m x 8 x 4 pieces) and then sterilized in a medium (5 ml of soluble starch, 1 ml of Pegton 1-1 meat extract, 1 ml of yeast extract, 2% sodium chloride A5-1 calcium chloride A0). , magnesium sulfate heptahydrate (101%,
After shaking for 48 hours in a thermostatic chamber at 30 to 33 degrees Celsius, a very small amount of the gel was collected and observed using a scanning electron microscope.
Many bacterial colonies of 70 m in size were observed, but no cracks were observed in the gel structure. Thereafter, while shaking for another 12 hours, similar observations were made, and it was found that after the colony diameter of the small 2's exceeded 550 μmk, the turbidity of the medium began to become noticeable, and a considerable amount of the 2's 2's were washed away. Therefore, it was confirmed that it was appropriate to separate the growth medium from the gel at the stage of 550 μm in diameter of each small colony.

Example 4 Commercially available polyvinyl alcohol (degree of saponification 97 mol, viscosity average degree of polymerization 1,700.4
6eP) powder 85t (moisture content 7vt'36)
t, 18wt-aqueous solution dissolved in water 912f (pH melon 9)
171 t of this aqueous solution obtained was subjected to pressurized steam sterilization at 120°C x 20 ml, and then left to cool in a sterile room.
Arthrobacter simplex (mrthro)
baeter simplex) (also known as Corynebacterium simplex, Coryne-baet@
rimm mimpl@x ) 1 sat suspension (tris(hydroxymethyl)aminomethane/hydrochloric acid buffer pH 7.5) 20 ft: Pour and stir for 7 ml. The polyvinyl alcohol concentration of this suspended aqueous solution is 7 W.
There is t To te.

After injecting 190 ft of this aqueous suspension into a mold for molding (630 pieces) Raschig rings (8 x 8 pieces) in a sterile room and cooling (ak condensation and molding) at -48°C x α5 hours,
The molded body was taken out and subjected to vacuum dehydration for 6 hours. After thawing,
A gel of 132t (water content 89wtl51 dehydration rate 5owt*) was obtained) This gel was diluted with a pre-sterilized α2% glucose aqueous solution (phosphate buffer, pH 7) 100-7.
As a result of immersion, the O which absorbed water into the molded gel and reached 1stf (water content 90vtlG) [11 diameter SeIm, height 60mm, the above Raschig ring 139ft - Irregular phototen LA synopsis Grednisolone (prednlsolona, '11β,
17α,21-trihydroxy-t4-pregnagen-420-dione) substrate solution for synthesis (cortisol cor
t1sol311β, 17ffs21-trihydroxy-4-lugnene-2o-dione (LIM, methyl asp co-h4%, pH 7, 34°C) was fed from the bottom of the column at a flow rate of t12°d/h, and was sterilized. Air was sent through the filter to the bottom of the column at a flow rate of 2005 g/h, and the effluent at the top of the column was produced for 2 to 20 h, and the concentration of prednisolone I
IL was only 3 wt- or less.

Next, a culture medium (15% peptone, 41 mM cortisol, 4 parts methyl alcohol, 30°C) was added to the bottom of the column.
40 at a flow rate of 8 sd/h, a very small amount of the gel was collected and observed using a scanning electron microscope. As a result, the diameter was 190 p.
, bacterial colonies were observed. Next, a substrate solution for prednisolone synthesis was fed from the bottom of the tower at a flow rate of 120 d/h, and air was fed into the bottom of the tower at a flow rate of 200 d/h through a sterile filter. ), thin layer chromatography (silica gelupe pyrene glycol-chloroform), high performance liquid chromatography (
As a result of analyzing the effluent by chloroform extraction), 20
Over the course of several days, prednisone concentration was 1.8-2.0w.
t% (yield 55-61 mol). However, 31
After a few days, this concentration decreased to t 5 wt %, and as a result of observing a very small amount of gel using an optical microscope, it was found that the bacterial colony had shrunk to a diameter of approximately 70 μm. Next, the culture solution (peptone α
5-, glucose (L29G, magnesium chloride 1ml
Calcium chloride 1mM, cortisol 1, methyl alcohol 4Is, phosphate buffer pH 7, 30C) 1 to 30
24 at a flow rate of d/h, a very small amount of the gel was collected, and as a result of observation with a scanning electron microscope, a bacterial colony with a diameter of 2201 mg was observed.

Thereafter, the 9 substrate solution was introduced again, and as in the previous case, ultraviolet absorption spectrum analysis of the effluent revealed that a glednisolone yield of 617 rupees had been achieved.

After repeating the feeding of the substrate solution and the intermittent feeding of the culture solution 8 times, the cavity containing the bacterial colony in the ζ gel was
No crack lines occurred at all.

Example 5 Commercially available polyvinyl alcohol (saponified [9114 mol-
1-Viscosity average polymerization Hzaoo, 4-Viscosity of aqueous solution [(zoc
)2ts@P) powder 84t (water content swt-)t dissolved in 916f of water I...8vt-aqueous solution (pH 7) was obtained.

This aqueous solution at 18F was subjected to pressure steam sterilization at 120°C x 20ml, and then allowed to cool in a sterile room.
2 ft of suspension water (phosphate buffer, pH 7) containing 20 wg of @sC@r・Ma1g1a・) was poured and stirred for 7 ml. The polyvinyl alcohol concentration of this suspended aqueous solution
is 7.2wt-.

In a sterile room, pour 18f of this suspended aqueous solution into Raschig ring (8 x 8) molds (59 molds).
After cooling (freezing and molding) at 8°C xo and sh, the molded body was taken out and vacuum dehydrated at LA 6h. After thawing, 1
A molded gel of α4f (water content 86 wt%, dehydration rate 42 wt%) was obtained. This was sterilized in advance (L91G91
As a result of immersion in α5o- for 6 hours, the gel absorbed water and reached 12t (water content aavt*).

The above Raschig ring 12F was placed in a glass cylinder with a diameter of 31 and a height of 105+, and the irregular side light was placed at 120°C x 2 in advance.
0 ml of sterilized substrate aqueous solution for ethyl alcohol synthesis (glucose 10 wt 1 G, magnesium sulfate heptahydrate 10100 pp PH!L 5.32°C) was added to 1
A force was introduced from the bottom of the column at a flow rate of 0 d/h (the ethyl alcohol concentration of the effluent was only 11 wt* (2- of the theoretical yield)). Next, a small amount of this molded gel was taken and a scanning type When observed using an electron microscope, no yeast colonies were observed, although the yeast were individually dispersed.
Subsequently, the culture medium (glucose 4-1
Yeast extract 1 ammonium chloride a2-1 magnesium sulfate heptahydrate αOI Ls, calcium chloride 1004L
After feeding P'5.5.28℃ for 55 hours at a flow rate of 5-/h, the amount of mold on the gel was measured and observed in the same manner.
Although many yeast colonies of 2Qp gas were observed, no cracks in the enclosing cavity were observed.Next, the aqueous substrate solution for ethyl alcohol synthesis was fed to the bottom of the column at a flow rate of 20 m/m, and the effluent was mixed with ethyl fluorol. (Gashamat analysis m> and glucose (dinitrosalicyl method) analysis of unity, cru; Dose J) The spermatozoa rate is +
Only 111-, ethyl alcohol 1! degree 4.7
It was learned that w t 'Ik (the theoretical yield of 92%) was reached.

After one month, the above ethyl alcohol concentration was '5.5 w
As a result of collecting a small amount of this gel and observing it with a scanning electron microscope, it was found that many yeast colonies had all shrunk to a diameter of 250p*.

After supplying the medium again to the bottom of the column 17 at a flow rate of t-5 m/h, the same observation revealed that there were a large number of colonies.
-I learned that both of them have returned to a diameter of 300 μm. Thereafter, the substrate aqueous solution for ethyl alcohol synthesis t-
Feed at a flow rate of 20 sd/1. ．． After 4 hours, the ethyl alcohol concentration of 4.7vt- in the effluent was reproduced.Through the above operations, only a trace amount of yeast was detected in the effluent, and not only yeast at the time of immobilization but also the column. Almost all of the yeast that proliferate within the gel are contained within the gel.

Comparative Example 1 In the polyvinyl alcohol aqueous solution 609 of Example 5, 12
After applying pressure steam sterilization at 0°C x 20 ml and leaving it to cool in a sterile room, 7 f'ft of suspension water (phosphate buffer) containing Satucharomyces cerevisiae a02f'i as in Example 5 was poured into this aqueous solution and stirred for 7 ml. 0 Next, this suspended aqueous solution 60f is placed on the bottom surface 10a+X 10aw
The adhesive film 14, which lacks any rigidity, was prepared by pouring it into a container and leaving it for two days to apply it to a wet cellon paper.
．． 6F (moisture content: 62 w) was obtained. This film (approximately 1.5 cm thick) was pre-washed and immersed in 20 cm of salt water. After 4 hours, the film was 1" thick.
7.5? (moisture content reached 67 wt). Cut this film into many pieces (8 x 8 x tswa)
After K cutting, culture solution 4rJ- of Example 5 was sterilized.
1. Pour into the tank and attach a cotton plug. After shaking for 4 hours in a thermostatic chamber at 30-32°C, a small amount of this film was taken and the inside of the film was observed using a scanning electron microscope, but no large colonies of yeast were observed, and only small colonies with a diameter of about 25μ bait were observed. The number of yeast bacteria in the film is estimated to be less than 101/-.

Example 50 Substrate aqueous solution 40+dt-Sakaro flask (50
G+d), sterilized at 120°C x 15m1n, left to cool in a sterile room, and the above film cut piece 17
After adding fe, a cotton plug was attached and the mixture was shaken in a constant temperature room at 30 to 32°C. After 24 h, the ethyl alcohol concentration was only [14v t - (15- of the theoretical yield),
I also learned that polyvinyl alcohol was eluted.

As described above, when using the known method using polyvinyl alcohol, the growth effect is poor, and the polyvinyl alcohol film has a problem in water resistance, making it difficult to obtain immobilized yeast with high glycolytic (ethyl alcohol production) activity.

Comparative Example 2 In place of the polyvinyl alcohol in Example 5, a commercially available polyvinyl alcohol having a degree of saponification of 93 mol, a viscosity average degree of polymerization of 1,700.4, and a viscosity of an aqueous solution (at 20° C.) of 50 cP was used, and the same procedure was carried out. Freezing/molding 1 tax water body 10f
(Water content: 85 wt %, dehydration rate: 44 VUI) After thawing, it softened even at 5°C, and in addition to a small amount of gel layer, a large amount of polyvinyl alcohol concentrated aqueous solution was observed to separate into layers. Therefore, it was impossible to pack this into a reaction column, and even when it was transferred to a flask, it completely lost its shape.

Comparative Example 3 Instead of polyvinyl alcohol in Example 5, saponification degree 9? , 2 mol - 1 ° viscosity average degree of polymerization 500.4 t A brittle gel similar to ζ agar 1 α sr which was operated in the same manner using a commercially available polyvinyl alcohol t with a viscosity of an aqueous solution (20 °C) a6eP and an aqueous solution of 18 F (Water content 84 wt 1, dehydration rate 42 wt%) was obtained, and it was not possible to fill the column.
l, Comparative Example 4 Same polymerization as Comparative Example 3 [500% polyvinyl alcohol aqueous solution concentrated [(-50vt-)], the aqueous solution 18t
A gel of 1a4f (water content: 84 wt%, dehydration wheel: 42 vtl) was obtained by the same procedure, but this gel significantly softened and lost its μ shape in water.

Comparative Example 5 Commercially available polyvinyl alcohol (saponified #t99 mole 1 viscosity average lidded [2,600,4-viscosity f of aqueous solution (20°C)
, 66 cP) powder (water content 8 wt%) was dissolved in 935 f of water to give w4L,,6 wt-. This aqueous solution 5
After applying pressurized water vapor to 4F and leaving it to cool, 5ft of the same yeast suspension water as in Example 5 was added [A-70℃ 57t, dehydration vehicle (0-1 water content 94wt9G) did not show any elasticity, and by immersing it overnight in water, the shape shifted and the water layer became turbid. Even if molding is performed, unless it is subsequently subjected to freezing and drying, the roux of the present invention will only form a soft gel with poor water resistance, and yeast will be immobilized and it may not be suitable for practical use. It is clear Comparative Example 6 After the freezing and molding process of Comparative Example 5, the molded body 54f1 was melted at room temperature, and then transferred to a vacuum dryer and dehydration under reduced pressure was attempted, but the molten liquid foamed violently and the operation was stopped. Next, as a measure against foaming, I took the molded product melted into the tube and collected (171). After applying this to the bottom of a polyethylene beaker (100 sd), I tried drying it under reduced pressure. As a result, the force ζ generated by 1f gel (water content 74 wt To, dehydration rate 72 wt 91) on the bottom of the beaker had poor elasticity and was immediately broken by a tensile stress of 6 oot/-.

Even if a polyvinyl alcohol aqueous solution is frozen and molded, it will not be melted (
Unless the gel is dehydrated (while maintaining the frozen state), the gel (yeast-containing gel) with high water content, high elasticity, and excellent mechanical strength of the present invention cannot be obtained.

Example 6 Commercially available polyvinyl alcohol (saponification [9h4 mol 1 viscosity average degree of polymerization 1,800.4-viscosity of aqueous solution [(20°C)
Dissolve 84f (29.5cP) powder (water content 5vt) in 916ft of water IC LA JLOwt CH aqueous solution CpH &
? ) was obtained.

This aqueous solution 1st was subjected to pressure steam sterilization at 120°C x 20ml, and then left to cool in an empty room.
@se@r・Ma1aia・) Pour 2tt of suspension water (phosphate buffer, TiH2) containing 14ft and stir for 7ml1n. This suspended aqueous solution of polyvinyl alcohol IlK
is 7.2vtLs 0 This suspended aqueous solution 18ft,
In a sterile room, a polyethylene container (bottom 4
4 es) and cooled (freeze/
After molding), vacuum dehydration was performed for 5 hours.

After thawing, a white opaque gel of 1 [L5F (water content 84 wt Ss dehydration rate 42 wt1) was obtained. As a result of immersing this in 40 dllfC6 of previously sterilized α9-salt water, the gel absorbed water and reached 12f (water content 86 wt'lG). The yeast was not detected in this soaking solution. Next, this gel was cut into many pieces (2cNX4awXA■) and washed with α9-saline solution 40-, which had been sterilized in advance.As a result of observing this washing solution with an optical microscope, a small number of the yeasts were observed, but the washing solution This was quantified based on the turbidity of the gel to ensure that at least 98% of the original yeast was entrapped in the gel.

Next, for this gel cut piece, mechanical strength [t-measured, tensile strength d Kf/cd, and compressive strength 4
It was Kf15# or above◇Also, even when I pinched it with my fingertips and pressed it intensely, it did not change shape at all and returned to its original shape.

The gel strength of Example 6 was measured in comparison with that of Comparative Examples 2-6.

Tensile strength Compressive strength (Kf/d) (jcf/ai) Example 6 (present invention) 6>4 Comparative example 2 Kuα2 Kua22
Comparison 3 <a2 <(12
Comparative example 4 <a2 (a
22 Comparison 5 ((L2
<(L22 comparison 6 α6
<a8 Gel for immobilizing and propagating microbial cocoons of the present invention 2>L
% conventionally known polyvinyl alcohol gel (Comparative Example 5,
It can be seen that the mechanical strength is significantly superior to Comparative Example 6 and polyvinyl alcohol gels not according to the present invention (Comparative Examples 2 to 4) K.

Comparative Example 7 (1) Sodium alginate dissolved at 5 wt% (19
56 Salt water 50m (35℃) contains Satsukalomy seven proboscis cereviche (15 ft- [L?'1 salt water 40d (5
After mixing 1 spherical gel (diameter 3 -
1 If, 000 pieces in total) were obtained.

(2) Add 20 mg (5 ml) of the above yeast suspension to α9 saline solution (35°C) in which 4 wt* of K-carrageenan has been dissolved.
5℃) was poured into a polystyrene moxibustion container with a 55IX x 5cm bottom surface, cooled to 20℃, and plate-shaped gel (thickness 2
．． 451) I got f.

(The above gel 2t(t2X2.4X[L2ow)t,
Hardening agent aqueous solution (5-potassium chloride) 2 Q- (30°C
) for 2 hours.

(4) 2f (4,2X2.4x@2) on the front ridge 2)
as) t, curing agent aqueous solution (hexamethylene diamine 0.
05 M, glutaraldehyde (LIM) 20+d(50
℃) for 2 hours and then washed with water.
36℃) to 40-20w of yeast suspension (1) (56℃
) was poured into a polystyrene container with a bottom surface of 5c IRX55+, cooled to 0°C, and a plate-shaped gel (thickness 2.
4 cm) t-obtained.

(6) α in which 4wt% of pectin (lemon type) powder was dissolved
9- Salt solution (36℃) 40- To (Rino's yeast suspension 20-
° (56 °C), mix, pour into a polystyrene container with a bottom surface of 5 cm x 5 cm, and cool to 55 °C.
A thickness of 2.4 cm) was obtained.

Above 11), (2 gels (3), (4), (5), and (6)) were all soft and brittle, and were crushed immediately by pinching them with the fingertips and applying light finger pressure.9. Both tensile strength did not withstand 1.5 V4/d and broke immediately.

Comparative example 8 Comparative example 7 (1), (2), (3), (4 pieces (5), (6
) gel strips (spheres or 3×3×5 m cubes) were delivered with paramedical growth medium in Example 5. 4-4) After 1 liter of observed rice feeding, cracks were observed in all the gels, and the effluent in each case was firmly suspended, confirming that the yeast had washed away.

Immediately, the supply of the medium was stopped and an aqueous solution of the substrate for ethyl alcohol synthesis was introduced instead, but the cracks in each gel did not disappear, and instead, they became worse! ! The number of spots increased and the yeast was washed away for more than 12 hours. In particular, Comparative Example 7 (to (3)
With gels (5) and (6), it was observed that the surface of the molded product became even softer and gradually fell off.

Procedural amendment November 17, 1980 Director General of the Patent Office Shima 1) Haru S+ Mr. 1, Indication of the case 1983 Patent Drama No. 143983 2, Name of the invention Method for immobilization and propagation of microorganism live aceae bodies 3, Amendment Relationship with the case involving a person who does
'6, Subject of amendment 7, Contents of amendment (1) The scope of claims is amended in Annex 9.

(2) Correct the following parts of the q# specification.

Claims: Saponification degree is 95 molar or more, and viscosity average polymerization degree i,
So.

The above-mentioned aqueous solution of polyvinyl alcohol and a suspension of living microorganisms were injected into a molding mold.
A 6υ manufactured sail is frozen and molded at one temperature, then vacuum dehydrated without thawing to a dehydration rate of 5w14 or more, and if necessary, immersed in water to obtain a water content of 20~ 92 wtfb (wet body standard) to obtain a gel that encloses (embeds) viable microorganisms, and then supplies a microorganism growth medium to this gel, which forms a large number of proliferating microbial cell colonies. A method for immobilizing and propagating living microorganisms, which is characterized in that the diameter of the microorganism is expanded to a range of 50 μS to 550 μS.

Claims (1)

[Claims] saponification degree of 95 mol or more, and viscosity average polymerization degree of 1,
Create a suspended aqueous solution consisting of an aqueous solution of polyvinyl alcohol of 500 or more and living microorganisms! Pour into the 1341 mold and keep it below -6℃! Freeze and mold the product, and then reduce the dehydration rate to 5uy without thawing
By vacuum dehydrating to more than tes and immersing in water as necessary, the water content is 20~? After obtaining a gel that has reached 2 wt % (wet body basis) and enclosing living microorganisms (package 11), a microorganism growth medium is supplied to this gel, and the diameter of the grown microorganism colony formed by this is fsQp Sui~5
Expansion to the range of 50 p@t-Featured microbial cell immobilization φ layer end method〇