JPH0244513B2 - BISEIBUTSUSEIKINTAINOKOTEIKAHO - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for immobilizing live microorganisms, in particular, by freezing and molding an aqueous suspension containing polyvinyl alcohol and living microorganisms, followed by vacuum dehydration (drying). The present invention relates to a method for immobilizing living microorganisms in a gel. polyvinyl alcohol, polyacrylamide,
Methods of immobilizing (capturing, entrapping) living microorganisms on alginic acid, carrageenan, agar, etc. are already known, but as summarized in (1) to (8) below, all of them have drawbacks, and even better methods can be used. It has been desired to develop a new immobilization method. (1) There is a method to obtain a membrane containing viable microorganisms by mixing viable microorganisms in an aqueous polyvinyl alcohol solution and air drying, but this membrane has poor water resistance, is soft, and has a low microbial trapping capacity. . Although some proposals have been made to support (reinforce) this membrane with nylon cloth or the like, most of the above-mentioned difficulties still remain unsolved. In addition, a feature of polyvinyl alcohol film is that it is a carbon source required for microbial activity.
Due to poor permeability to nitrogen sources, inorganic substances, etc., the activity of immobilized living microorganisms decreases (JP-A-52-145592, JP-A-55-35415, Plastic Materials Course 14 , P.135, P. .133 (1972), Nikkan Kogyo Shimbunsha). (2) After mixing the polyvinyl alcohol aqueous solution and microorganisms or enzymes, remove oxygen and make cobalt 60
A polyvinyl alcohol crosslinking/gelation method using (γ-ray) irradiation is also well known. However, in this case, even if a radiation protection 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 (Biotech.Bioeng.,
15, 607 (1973), Fermentation and Industry, 35 , 92 (1977)). (3) It has been well known for a long time that when boric acid or a boric acid aqueous solution is added to a polyvinyl alcohol aqueous solution, it immediately gels, and methods for immobilizing microorganisms or enzymes that utilize this principle have also been attempted. However, the gel produced from highly saponified polyvinyl alcohol is weak and difficult to mold. This problem can be slightly improved by using polyvinyl alcohol with a low degree of saponification, but since it is a sticky gel, it is difficult for molded products such as shredded pieces to maintain their shape (Japanese Patent Laid-Open No. 1983 -135295, Special Publication Showa 55-51552). (4) 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 it. In any case, a step of adding acid to the microorganism suspension to adjust the pH to 3 or less is included, and the adverse effects on the microorganisms are often not negligible (Japanese Patent Publications No. 55-11311, No. 55-30358). (5) A method for immobilizing enzymes by gelling (freezing and solidifying) an aqueous solution of polyvinyl alcohol and enzymes at low temperatures has also been proposed (Japanese Patent Application Laid-Open No.
50−52296). However, although the gel is produced by simple freezing and solidification treatment, it exhibits no elasticity and has extremely low tensile and compressive strengths. Furthermore, if the film is air-dried after freezing, solidifying, and thawing, only a soft film similar to the case (1) above is obtained. Note that when attempting vacuum dehydration after twisting, solidification, and melting, the molten liquid bubbles so violently that it often becomes impossible to continue the operation. Not shown. Moreover, only a fairly brittle white cloudy gel is produced, and its water content is low. (6) Another well-known immobilization method is to deoxidize a mixed suspension of acrylamide, N,N'-methylenebisacrylamide, and viable microorganisms, and then irradiate the solution with radiation or add a radical generator. However, it is well known that the residual monomer in the resulting gel is highly toxic, and the residual monomer and radiation or radicals can cause significant damage to living microorganisms, and the gel has low mechanical strength. , often requiring a secondary curing treatment (Biotech.Bioeng., 20 , 1264
(1978)). (7) Another well-known immobilization method is to apply calcium ions or aluminum ions to an aqueous mixed suspension of sodium alginate and live microorganisms (to form a gel).
Not only does it have poor mechanical strength (especially compressive strength), it is often attacked and destroyed by potassium dihydrogen phosphate buffer (PH7), which is used as a medium component for living microorganisms (Biotech.Bioeng. 19 , 387
(1977)). (8) The above alginate (poly-1,4-β-D
Typical examples of naturally occurring polysaccharides include locust bean gum (D-mannose, D-galactose) and pectin (poly-1,4-α-D-galacturonic acid). ), agar (acidic sulfuric ester of polygalactose), carrageenan (also acidic sulfuric ester of polygalactose, etc.), and of course, like the above-mentioned alginate, these can also be used as carriers for immobilizing living microorganisms. However, a common drawback of gels obtained from these naturally occurring polysaccharides is the fact that they do not have sufficient mechanical strength, and often require an auxiliary (secondary) curing treatment. Polysaccharide aqueous solutions often solidify (gel) at room temperature, and when mixed with living microorganisms, they must be heated to 40 to 50°C or higher, which is harmful to many living microorganisms. Also, in the auxiliary (secondary) curing process mentioned above, damage to living microorganisms may be caused, for example, by heating above 50°C or using chemical reagents, solvents, etc. Also, as often found in natural products,
The quality of these products is not always consistent depending on the place of production or the time of harvest, and there are many unknowns regarding their chemical composition and structure. Agar has been used for immobilization (culture carrier) of living microorganisms for a long time, and carrageenan, which is most similar in chemical structure to this, was naturally proposed as a carrier for living microorganisms, and agar It has been confirmed that it performs a similar function. However, the above-mentioned difficulties are no exception to these polysaccharides, and the development of even better carriers is desired (Enzyme Microb. Technol.
1, 95 (1979), Kobunshi, 29 , 238 (1980)). As a result of studies aimed at overcoming the shortcomings of the prior art, the present inventors obtained a highly elastic and highly water-containing gel from an aqueous suspension containing polyvinyl alcohol and live microorganisms, and found that microorganisms were contained in this gel. It was discovered that viable bacterial cells could be almost completely encapsulated (captured) without any damage, and the present invention, which is highly effective, was completed. Already, the present invention provides an aqueous suspension solution obtained by adding living microorganisms to an aqueous solution of polyvinyl alcohol with a saponification degree of 95 mol% or more and a viscosity average degree of polymerization of 1500 or more, into a container of any shape or for molding. Inject it into a mold, freeze and mold it at a temperature lower than -6°C, then vacuum dry the molded product to a dehydration rate of 5wt% or more without melting, and immerse it in water if necessary. The present invention provides a method for immobilizing live microbial cells with characteristics. According to the present invention, a gel is generated during the process of freezing, molding, and drying a mixed suspension of polyvinyl alcohol and viable microorganisms, and almost all of the viable microorganisms are trapped (captured) in this gel. ) to be done. In the present invention, the immobilization process does not use acids, alkalis, radiation, radical generators, organic solvents, reaction reagents, etc., and does not require secondary curing treatment, so microorganisms are hardly damaged. It is not affected (avoiding death, of course), and is captured as a viable bacterial body.
The original activity of living microorganisms is maintained as is. Conventional microbial immobilization methods using reactive reagents or gamma rays often kill all or most of the microorganisms. In the present invention, viable microorganisms can be immobilized without being damaged by γ rays or chemical reagents (or reaction solvents). The immobilization carrier (gel) of the present invention is highly water-containing, and also contains a carbon source necessary for the activity of living microorganisms.
It is a rubber-like elastic body that has excellent permeability to nitrogen sources, oxygen gas, carbon dioxide gas, and other inorganic substances, and also has excellent mechanical strength. 1 of an aqueous solution of polyvinyl alcohol at 0 to 30°C.
The phenomenon of increased viscosity or gelation is often observed after storage for a few days to a week.
It has been well known since ancient times. However, this gel, as often seen in gels of the above-mentioned naturally occurring polysaccharides,
For example, it is soft like agar, and can be dissolved simply by stirring vigorously, by adding water and stirring, or by slightly warming it. On the other hand, the gel of the present invention is insoluble in water or hot water, and is completely different from the above-mentioned known gels. This indicates that the present invention provides a novel gel that is completely different from the conventional knowledge regarding the gelation of known polyvinyl alcohol aqueous solutions or the gelation by chemical treatment of polyvinyl alcohol aqueous solutions described above. There's a story. The degree of saponification of the polyvinyl alcohol used in the present invention needs to be 95 mol% or more, preferably 97 mol% or more. Even if polyvinyl alcohol having a saponification degree of 80 to 88 mol %, particularly 85 mol % or less, is used, only a soft gel is obtained, and the object of the present invention is not achieved. Further, in the present invention, polyvinyl alcohol having a viscosity average degree of polymerization of 1500 or more is used. Since the degree of polymerization of polyvinyl alcohol decreases, the mechanical strength of the resulting gel also decreases, so in the present invention, it is preferable to use products with a high degree of polymerization (degree of polymerization of about 1700 to 2600) that are normally commercially available. In the present invention, first, an aqueous solution of polyvinyl alcohol is prepared. The concentration is not particularly limited, but may be, for example, 1 to 20 wt%, preferably 7 to 15 wt%. Although this concentration can be further increased to, for example, 90 wt%, the viscosity of the aqueous solution at room temperature reaches 10,000 cP or more, and the viscosity may increase or gelatinize during storage, making it somewhat difficult to handle. Although this concentration can be lowered 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. As for sterilization processing conditions, the objective may be achieved at 100℃ x 5 minutes, but if it is contaminated with heat-resistant bacteria, 120℃
Perform high pressure/steam sterilization at ℃ x 15 min to 6 h. Ultraviolet irradiation sterilization can also be used, but its effectiveness is limited to irradiated surfaces, so it is preferable to use it in conjunction 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 (i.e., filamentous fungi) such as Aspergillus, Rhizopus, Pseudomonas, Acetobacter, Streptomyces, Escherichia, Satucharomyces, and Candida, actinobacteria,
Many living microorganisms such as bacteria, yeast, and algae can be targeted. In this case, any of lyophilized and preserved live microbial cells, a growth culture solution of live microbial cells, or a concentrated suspension of live microbial cells centrifuged from the growth culture solution can be used. It is most convenient to perform this addition (mixing) of living microorganisms at a temperature of about 10 to 35°C, but when fixing heat-resistant living bacteria, temperatures above 35°C may be used, depending on the heat resistance of each. It can also be operated. At temperatures below 10°C, the viscosity of the water solution increases and the mixing and dispersion of viable bacterial cells becomes slow; however, if this point is kept in mind, the above operations may be carried out at temperatures below 10°C. It is preferable to keep the amount of viable microorganisms added (on a dry basis) to less than 7 times the weight of the polyvinyl alcohol in the aqueous solution, from the viewpoint of immobilizing almost the entire amount of viable microorganisms. By going through the drying (gelation) process, it is possible to reliably capture (enclose and immobilize) 96 to 98% of viable microorganisms. As a result of observing the gel obtained through the freezing, molding, and drying processes described below using a scanning electron microscope, the inside of the gel is porous and contains a solid phase (polyvinyl alcohol that is insoluble in cold and hot water) and a liquid phase (microbial growth). It was inferred that the water was in a labyrinthine network of channels, with the bacteria (suspended water) being intricately intertwined. The width of the channel ranges from 1/2 to 100 μm, and it meanders continuously in a complicated manner. Therefore, after some viable microorganisms (1 to 10 μm) have been captured in this continuous meandering waterway, by infiltrating the culture solution (nutrient medium) into this aqueous phase, the microbial growth is naturally increased. Bacterial cells can be grown. Since the gel obtained by the present invention has such advantages in terms of its internal structure, there is no limit to the amount of viable microorganisms added (initial immobilized amount); It can be selected arbitrarily within the range of culture. In the present invention, care is taken to prevent contamination of the mixed suspension of polyvinyl alcohol living microbial cells obtained in this manner, and in addition, germicidal lamps (ultraviolet rays) are used.
The suspended aqueous solution is injected into a container of any shape or into a desired mold, and is frozen and molded, taking care not to directly irradiate it. In this case, as a cooling agent, for example, a cold agent such as salt-ice (23:77) (-21℃), calcium chloride-ice (30:70) (-55℃),
Or dry ice-methyl alcohol (-72
℃), liquid nitrogen (-196℃), etc., to a temperature lower than -6℃ to 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 mold, and the mechanical strength of the monkey will be affected. Inferior. If liquid helium is used, it can be cooled down to -269°C, but it is uneconomical and has no advantage in gel quality, so in practice a Freon refrigerator is used, for example -
It is best to cool it to below 20℃, or even below -35℃. Since it is undesirable for most living microorganisms to be exposed to temperatures around -20 to -30°C for long periods of time, it is preferable to rapidly cool them to below -30°C, for example, to -35 to -80°C. Freezing and molding at such low temperatures contributes to increasing the mechanical strength of the gel for supporting live microorganisms, and
It is more preferable to freeze and mold the product at a temperature between . In the freezing and molding process according to the present invention, the polyvinyl alcohol aqueous solution is solidified (frozen) and molded in a mold of an arbitrary shape, and then the top cover or bottom cover (or both) of the mold is removed to change the shape of the molded product. It can be frozen and dried while being preserved. The cooling rate during freezing and molding is as follows:
-10℃ considering the effect on viable microorganisms mentioned above.
Slow cooling at a rate of about 0.1 to 7°C/min may be sufficient up to a certain extent, but after that, rapid cooling at a rate of 7 to 1000°C/min is preferable. In the present invention, after confirming that the aqueous mixed suspension of polyvinyl alcohol and live microorganisms poured into the container or mold is frozen, it is vacuum dried. In this case, if the frozen/molded body is removed from the freezing chamber, transferred to the vacuum drying chamber, and immediately suctioned and dehydrated, the sample will be cooled as moisture is removed (sublimation), so external cooling is especially recommended. Even if it is not applied, the frozen/molded body will not thaw. There is no problem in heating the frozen/molded product to such an extent that it does not melt, and this can promote dehydration. In other words, there is no particular restriction on the temperature of the dehydration step as long as the frozen/molded product is not thawed, and this does not particularly affect the quality of the gel. In this dehydration process, the dehydration rate is
5wt% or more, for example, the water content of the gel is 20~
Reach 92wt%. The moisture content can also be 20% or less. In the present invention, the frozen and molded product is subjected to a slight dehydration treatment (vacuum drying) regardless of the concentration of polyvinyl alcohol. In this case, the dehydration rate is 5wt% or more, 15wt% or more. That is, as the dehydration progresses, the gel strength increases significantly, so it is preferable to select the amount of dehydration depending on the desired gel strength. This dehydration step (freezing/drying) cannot be omitted. In other words, unless this is done,
The high water content gel of the present invention, which is highly elastic and has excellent mechanical strength, cannot be obtained, and therefore, the gel of immobilized live microorganisms is extremely weak. In addition, when using a method that depressurizes and dehydrates the frozen/molded product after thawing it without maintaining the frozen state, the foaming is so intense that it is almost impossible to continue the operation.
Even if dehydration takes a long time, only a cloudy gel with poor elasticity will be formed. Next, the frozen, molded, and dried product is allowed to stand at room temperature and thawed (thawed) to obtain a highly elastic microorganism-immobilized gel. In this case, the melting operation includes slow heating at 1 to 3°C/min,
Taking into consideration the heat resistance of viable microorganisms, rapid heating at a rate of 3 to 1000° C./min may be possible in some cases. In any case, at temperatures above 60°C, a hard film will rapidly form on the surface of the gel, so regardless of the heat resistance of viable microorganisms, the thawing (thawing) operation temperature should be below 40-50°C. is desirable.
After this thawing operation, from the container or mold support,
The microorganism-immobilized gel can be easily taken out. This gel is primed in water and has a water content of 50 to 95wt.
% (wet body standard), but since it is still a strong elastic body, it is difficult for the microorganisms contained within the gel to grow.
Suitable for activities. Findings from the above-mentioned scanning electron microscope and the above-mentioned water content (50-95wt%)
As is clear from this, most of the interior of the gel is occupied by pores (aqueous phase). The water content of this gel is not as high as that of, for example, Konniyaku (water content of about 97 wt%, polysaccharide wet gel), but it is higher than the water content of living cells and living tissues such as humans and animals (70 to 90 wt%). Similar in strength and elasticity to konnyaku, agar, alginic acid, carrageenan, guar gum,
It is far superior to polysaccharide gels such as locust bean gum, agarose, and tragacanth gum, and resembles living tissues such as humans and animals. The gel of the present invention exhibits elasticity despite containing a large amount of water, and although it temporarily changes when squeezed tightly, it immediately returns to its original shape and does not lose its shape. Moreover, in this case, almost no leaching of the water content is observed; for example, in a gel with a water content of 90 wt%, 2
Even if a compressive stress of Kg/cm ² is applied, the amount of leached (flowing) water is only 1 to 2% of the contained water, and the tensile strength is 4 Kg/cm ² , making it difficult for gels with such high water content to is an extremely excellent elastic body. High water content and mechanical strength have traditionally been considered incompatible challenges in developing medical polymers and selectively permeable membranes, but the gel of the present invention
It has the above-mentioned high water content and strength, and can be used for conventional air-drying films of aqueous polyvinyl alcohol solutions, when storing the above-mentioned aqueous polyvinyl alcohol solutions at 0 to 30°C, or when simply freezing and thawing polyvinyl alcohol aqueous solutions. This is completely different from the soft gel obtained. As is clear from the fact that it strongly retains a large amount of water, the apparent specific gravity of this gel is
It is almost the same as water and barely settles in water. The gel of the present invention is non-tacky. Plate (8mm
×8mm×2mm), cylindrical (inner diameter 3mm, outer diameter 6mm,
Even when approximately 10 g of gel formed into a sphere (4 mm in length) and spherical (4 mm in diameter) is stirred in 50 ml of water for 10 days, no phenomena such as mutual adhesion or deformation are observed. Furthermore, even after being soaked in tap water for a year, it did not dissolve and its elasticity and strength did not change (this is in sharp contrast to, for example, when konnyaku is soaked in tap water for several days, it loses its shape severely). be). In the present invention, a single polyvinyl alcohol component is used as a gel material (gelling component). However, the coexistence of inorganic or organic substances that are completely unrelated to the gelling phenomenon of polyvinyl alcohol does not affect the present invention, and the amount of such coexistence can be, for example, 1/2 or less of the amount 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, even if they coexist in small amounts. Even so, it often has an unfavorable effect on the gel formation of the present invention (formation of a gel of a single component of polyvinyl alcohol), making it difficult to produce a high water content gel with excellent mechanical strength. Examples of such substances include colloidal alkali silicates (US Pat. No. 2,833,661 (1958)), colloidal silica (US Pat. No. 2,833,661 (1958)), and alkaline colloids, which are known to interact with polyvinyl alcohols. Silica (Japanese Unexamined Patent Publication No. 54-153779), organosilicon compound (vinyl acetate resin, P93, Nikkan Kogaku Shimbunsha)
Y1962)), tetraalkyl silicate (Special public interest
-30358, Special Publication No. 55-11311) boron, borax (French patent 743942 (1933)), phenol, naphthol, meta-cresol, pyrogallol, salicylanilide, disalicyl benzidide, resorcinol, polyamines (polymer chemistry, 11 , (105) 23
(1954)) Kaolin (Natura, 170 , 461)
(1955)), etc. These are avoided in the present invention because they form a composite gel with polyvinyl alcohol depending on the amount of coexistence. In the present invention, as described above, it is essential to freeze and mold an aqueous suspension of living microorganisms. In general, it has been well known for a long time that when microorganisms, biological tissues, or suspensions thereof are frozen, they suffer some degree of freezing damage. In order to avoid or significantly reduce this freezing damage to living organisms, their tissues, proteins, etc., in addition to the method of rapid cooling to a temperature below -30℃ described above, hydroxyl group-containing aqueous solutions such as carboxymethylcellulose can be used. A well-known method is to coexist a small amount of a polymeric substance or various freeze-drying protection agents. In the present invention, since polyvinyl alcohol (gelling material) itself acts as a strong freeze/dry protection agent, most of the viable microorganisms are normally protected, ensuring sufficient growth and activity as described below. However, a known freeze-drying protection agent may also be present. Substances that reduce freeze-drying damage are generally protective agents, protective substances, additives.additional substances,
It is called a medium, medium, adjuvant, suspended medium, stabilizer, etc., and specific examples include magnesium ion, glycerin, dimethyl sulfoxide, etc. The microorganism-immobilized gel molded product obtained by the present invention is, for example, a 2 mm to 5 mm cubic gel or an 8 mm x 8 mm Raschig ring, which has an optimal temperature and PH value for the growth and activity of immobilized microorganisms. When added to an aqueous substrate solution (medium) and brought into contact in a flask, an activity of approximately 85 to 97% is observed compared to the original (non-immobilized) viable microbial cells.
If the size of this gel molded product is further increased, the above relative ratio tends to decrease, but in such cases, known substrate diffusion penetration enhancers such as lauryl alcohol sulfate, cetyl Surfactants such as pyridium chloride and cetyltrimethylammonium bromide can be used without deteriorating the properties of the gel, such as its mechanical strength and appearance. By subjecting the polyvinyl alcohol gel of the present invention to a curing treatment for polyvinyl alcohol fibers or polyvinyl alcohol films, the mechanical strength of the gel is further increased slightly. This known hardening (crosslinking) treatment includes, for example, aldehydes, dialdehydes, diisocyanates, phenols, or metal compounds such as titanium, chromium, and zirconium, as well as borax, acrylonitrile, trimethylolmelamine, and shrimp chloride. Examples include methods using hydrin, bis-(β-hydroxyethyl) sulfone, polyacrylic acid, dimethylol urea, maleic anhydride, and the like.
However, considering that the gel of the present invention has the strength (load resistance) as described above, and that the immobilized living microorganisms are often damaged by the above-mentioned auxiliary curing treatment, The present invention does not require these curing treatments. Next, the present invention will be explained based on examples. Example 1 Commercially available polyvinyl alcohol (saponification degree 97 mol%, viscosity average degree of polymerization 1700, viscosity of 4% aqueous solution (20
℃) 26cP) powder (moisture content 7wt%), water
It was dissolved in 914g to make an 8.0wt% aqueous solution. 44 g of this aqueous solution was subjected to pressure steam sterilization at 120°C for 20 minutes, and then left to cool in a sterile room.
4 g of suspension water (phosphate buffer, pH 7) containing 0.8 g of Saccharomyces cerevisiae was poured into this and stirred for 7 minutes. The polyvinyl alcohol concentration of this aqueous suspension was 7.3 wt%. In a sterile room, 40 g of this suspension water was injected into molds (for 130 pieces) of Raschig rings (outer diameter 8 mm, inner diameter 4 mm, length 8).
After cooling (freezing and molding) for -33℃ x 0.5h,
The molded body was taken out and vacuum dried for 6 hours. After thawing, 23 g (moisture content 84 wt%, dehydration rate ≡ weight loss rate of frozen and molded body = 43 wt%) of molded gel (Raschig ring containing yeast) was obtained. This gel was soaked in 40 ml of previously sterilized 0.9% saline solution for 6 hours. As a result of soaking,
The molded gel absorbed water and reached 27 g (water content 87 wt%). Moreover, the yeast is not detected in this immersion liquid. Therefore, it was found that almost the entire amount of yeast was entrapped (captured) within the Raschig ring. Glass cylinder (column) with a diameter of 3 cm and a height of 10 cm.
was filled with 27 g of the Raschig ring mentioned above, and an aqueous substrate solution for ethyl alcohol synthesis (glucose) that had been sterilized at 120°C for 20 minutes
10wt%, magnesium sulfate heptahydrate 60ppm, PH
As a result, the ethyl alcohol concentration in the effluent after 12 hours reached 4 wt% (77% of the theoretical yield). After this operation was continued for 12 days, the ethyl alcohol concentration of the top effluent was still 4 wt. Example 2 84 g of powder (water content 5 wt%) of commercially available polyvinyl alcohol (saponification degree 98.4 mol%, viscosity average degree of polymerization 1800, viscosity of 4% aqueous solution (20°C) 29.5 cP),
It was dissolved in 916 g of water to obtain an 8.0 wt% aqueous solution (PH6.9). 18 g of this aqueous solution was subjected to pressurized steam sterilization at 120°C for 20 minutes, and then left to cool in a sterile room.
2 g of suspension water (phosphate buffer, pH 7) containing 0.4 g of Saccharomyces cerevisiae was poured into this and stirred for 7 minutes. The polyvinyl alcohol concentration of this aqueous suspension was 7.2 wt%. Pour 18 g of this aqueous suspension into a polyethylene container (bottom 6 x 6 cm) in a sterile room at -53°C.
After cooling (freezing/molding) for 0.6 h, vacuum drying was performed for 5 h. After thawing, 10.5g (moisture content 84wt)
A white opaque gel with a dehydration rate of 42 wt% was obtained.
Add this to 30ml of sterilized 0.9% saline solution.
After soaking for 6 hours, the gel absorbed 12g of water (water content
86wt%). The yeast was not detected in this soaking solution. Next, this gel was cut into many pieces (8 mm x 8 mm x 4 mm) and washed with 40 ml of sterilized 0.9% saline solution. When this washing solution was observed with an optical microscope, a small number of the yeasts were observed. , we quantified this based on the turbidity of the wash solution and found that at least 98% of the initial yeast gel (strips)
I knew that it was definitely included. Glucose 5wt%, magnesium sulfate heptahydrate
40ml of 60ppm aqueous substrate solution for alcohol synthesis,
After collecting in Sakaguchi flask (500ml), 120℃×20min
After being sterilized and left to cool in a sterile room, 12g of the above-mentioned cut gel (immobilized yeast) was put into this, a cotton plug was attached, and the product was shaken in a constant temperature room at 30-33℃. 1.9 wt% ethyl alcohol was detected in the substrate aqueous solution after 24 h. In this case, no outflow of yeast into the aqueous substrate solution was observed. On the other hand, 40 ml of the aforementioned substrate aqueous solution was added to 12 g of suspended water containing 0.4 g of Satucharomyces cerevisiae, the same strain as the above-mentioned yeast, and shaken in the same manner. As a result, the ethyl alcohol concentration was 2.2 wt% (theoretical yield) after 24 hours. reached 86%). Therefore, the yeast encapsulated in the gel of the present invention remains immobilized in the gel during the alcohol fermentation operation, and its glycolytic activity (ethyl alcohol production ability) is comparable to that of the original yeast (non-immobilized yeast). It is clear that up to 90%. Comparative Example 1 60 g of the 8.0 wt% polyvinyl alcohol aqueous solution of Example 2 was subjected to pressure steam sterilization at 120°C for 20 minutes, and after cooling, the same strain of Satucharomyces cerevisiae as in Example 2 was added to this aqueous solution in a sterile room. 7 g of suspension water (phosphate buffer) containing 1.4 g of Saccharomyces cerevisiae was poured and stirred for 7 minutes. Next, add 60 g of this suspended aqueous solution to the bottom 10
By pouring it into a 10cm container and leaving it for 2 days, a soft sticky film similar to wet cellophane paper will form.
14.5g (moisture content 62wt%) was obtained. This film (approximately 1.5 mm thick) was immersed in 20 ml of 0.9% saline solution that had been sterilized in advance, and after 4 hours the film was 17.2
g (moisture content 67 wt%). A considerable amount of yeast was found in this immersion solution, and from the results of specific gravity analysis,
It was found that 15% of the yeast was transferred to the immersion solution without being encapsulated in the polyvinyl alcohol film. Next, this film was cut into many pieces (8 mm x 8 mm x 1.5 mm) and washed with 84 ml of previously sterilized 0.9% saline (the yeast that leaked into this washing solution was
(approximately 1wt%). 40 ml of the substrate aqueous solution of Example 2 was placed in a Sakaguchi flask (500 ml), sterilized at 120°C for 15 minutes, left to cool in a sterile room, and 17 g of the above-mentioned cut film fragments were put there, followed by a cotton sinter stopper. The mixture was shaken in a constant temperature room at 30-33℃, but the ethyl alcohol concentration after 24 hours was only 0.4wt% (15% of the theoretical yield), and some polyvinyl alcohol was found to have eluted. . As described above, when following the known method using polyvinyl alcohol, the loss of yeast during the immobilization process is more significant than in Example 2, and the glycolysis (ethyl alcohol production) of the immobilized yeast is extremely slow. Moreover, the obtained film is soft. Comparative Example 2 Instead of polyvinyl alcohol in Example 2,
The same procedure was carried out using commercially available polyvinyl alcohol having a degree of saponification of 93 mol%, a viscosity average degree of polymerization of 1700, and a viscosity of a 4% aqueous solution (at 20° C.) of 30 cp. 10 g of frozen, molded, and dried material (moisture content: 83 wt%, dehydration rate: 44 wt%) was obtained, but after thawing, it became soft even at 5°C, and in addition to a small amount of gel layer, a large amount of polyvinyl alcohol concentrated aqueous solution formed. allowed to separate. At the same time, we also learned that a large amount of yeast had leaked into this separated liquid phase. Comparative Example 3 Instead of polyvinyl alcohol in Example 2,
Saponification degree 99.2 mol%, viscosity average polymerization degree 500, 4%
Using commercially available polyvinyl alcohol with an aqueous solution viscosity (20℃) of 5.6 cP, per 18 g of the 18 wt aqueous solution,
Same operation, but 10.5g of a brittle gel similar to agar
(Water content: 84 wt%, dehydration rate: 42 wt%), it showed almost no elasticity and was soft. Comparative Example 4 The concentration of the same polyvinyl alcohol aqueous solution with a degree of polymerization of 500 as in Example 3 was increased to 30 wt%, and 18 g of the aqueous solution was subjected to the same operation to obtain 10.4 g (water content 84 wt%).
%, dehydration rate 42wt%), but this gel significantly softened and lost its shape in water.
At the same time, yeast leaked into the water. Comparative Example 5 Commercially available polyvinyl alcohol (saponification degree 99.4 mol%, viscosity average degree of polymerization 2600, viscosity of 4% aqueous solution (20
℃), 66cP) powder (moisture content 8wt%), water
It was dissolved in 93.5g to give a concentration of 6wt%. This aqueous solution was subjected to pressure steam sterilization, left to cool, and then 3 g of yeast suspension water similar to that in Example 1 was added, and frozen at -70°C for 0.5 h.
After the molding process, a soft gel (37 g, dehydration rate 0%, water content 94 wt%) was obtained by leaving it at room temperature for 2 hours.
It showed no elasticity and lost its shape after being immersed in water overnight, the water layer became cloudy, and a large amount of yeast leaked out into the water. In addition, the soft gel before immersion in water is only 100
It was easily broken under a tensile stress of g/cm ² . That is, even if a polyvinyl alcohol aqueous solution is frozen and molded, unless it is subsequently frozen and dried, a soft gel with poor water resistance will be produced compared to the gel of the present invention. It is clear that immobilizing yeast is not suitable for practical use. Comparative Example 6 After the freezing and molding process of Comparative Example 5, 34 g of the molded product was melted at room temperature, and then transferred to a vacuum dryer and drying under reduced pressure was attempted, but the molten liquid bubbled violently and the operation had to be stopped. It didn't happen. Next, as a measure against foaming, collect 1/10 (3.7 g) of the above molded body melt and place it in a polyethylene beaker (100 ml).
After applying it to the bottom of the machine, I tried drying it under reduced pressure. This results in
1 g of gel (water content 74wt%, dehydration rate 72wt%) was formed on the bottom of the beaker, but its elasticity was poor;
Moreover, it was immediately cut by a tensile stress of 600 g/cm ² . On the contrary, the gel of Example 2 of the present invention (yeast-containing gel) did not break at a tensile stress of 4 Kg/cm ² .
Furthermore, even if a compressive stress of 2 kg/cm ² was applied to this, the original shape was almost completely recovered after this stress was removed. In this way, even if the polyvinyl alcohol aqueous solution is frozen and molded, unless it is dehydrated without being thawed (maintaining the frozen state), the high water content and elasticity of the present invention can be obtained. , gel with excellent mechanical strength (yeast-containing gel)
It turns out that you can't get it. The elasticity and strength of the gel (gel containing microorganisms) are both important physical properties in the operation of filling the gel into a reactor (tower), as well as the expansion of the pores within the gel due to the growth of microorganisms within the gel. This has an extremely important meaning from the viewpoint of (tensile stress on the gel material near the proliferated area and compressive stress on the gel material sandwiched between different proliferated areas). That is, a carrier gel for immobilizing live microorganisms is required to have both gel elasticity sufficient to accommodate the intense growth of microorganisms and strength capable of withstanding the expansion of pores. As described in Comparative Example 6, the gel of this Comparative Example is still insufficient in this respect compared to the gels of Examples 1 and 2 of the present invention. Example 3 Commercially available polyvinyl alcohol (saponification degree 97 mol,
Viscosity average degree of polymerization 1700, 4% aqueous solution (20℃) 26cP)
86g of powder (water content: 7wt%) was dissolved in 914g of water to obtain an 8.0wt% aqueous solution (PH6.8). 170 g of this aqueous solution was subjected to pressure steam sterilization at 120°C for 20 minutes, and then left to cool in a sterile room, where 4 g of Erwinia herbicola were sterilized.
Pour 20g of suspension water (phosphate buffer) containing
Stir for 7 minutes. The polyvinyl alcohol concentration of this suspended aqueous solution is 7 wt%. In a sterile room, 190 g of this suspended aqueous solution was poured into a mold for molding Raschig rings (8 mm x 8 mm) (630 molds), and cooled to -53°C for 0.5 h (freezing and molding).
After that, the molded body was taken out and vacuum dried for 6 hours. After thawing, 133 g of gel (water content: 86 wt%, dehydration rate: 30 wt%) was obtained. This gel was immersed in 100 ml of pre-sterilized 0.9% saline for 6 hours, resulting in a molded gel that absorbed 139 g of water (water content 87 wt%).
reached. The bacteria are not detected in this immersion liquid. Therefore, it can be seen that almost the entire amount of bacteria was encapsulated within the Raschig ring. An aqueous tyrosine synthesis substrate solution (phenol) was filled into an acrylic resin cylinder (column) with a diameter of 3 cm and a height of 60 cm, filled with 139 g of the above Raschig rings, and sterilized at 120°C for 20 min.
0.1%, sodium nitrite 0.2%, ammonium acetate 5%, sodium pyruvate 3%, pyridoxal phosphate 100ppm, ethylenediaminetetraacetate 300ppm, pH 8, 30℃) 150ml/
After 10 hours, the concentration of β-tyrosine (β-(p-hydroxyphenyl)alanine) in the effluent was 600 ppm (yield 30 mol%).
reached. Example 4 Commercially available polyvinyl alcohol (saponification degree 97 mol%, viscosity average degree of polymerization 2200, viscosity of 4% aqueous solution (20
℃) 54cP) powder (moisture content 6wt%), water
It was dissolved in 915 g to obtain an 8.0 wt% aqueous solution (PH6.9). 380 g of this aqueous solution was subjected to pressure steam sterilization at 120°C for 20 minutes, and then left to cool in a sterile room.
Here, suspension water containing 4 g of Lactobacillus brevis (tris (hydroxymethyl) aminomethane buffer,
PH8.0) was added and stirred for 7 minutes. The polyvinyl alcohol concentration of this suspended aqueous solution is 7.6wt
%. 200g of this suspended aqueous solution was put into molds for molding Raschig rings (8 mm x 8 mm) (665 pieces) in a sterile room.
After cooling (freezing and molding) at -45°C for 0.5 h, the upper cover of the mold was removed and the lower cover supporting the molded body was vacuum dried for 6 h. After thawing, 130 g of gel (water content 87 wt%, dehydration rate 35%) was obtained. This gel was pre-sterilized with 0.9%
As a result of immersion in 150 ml of saline solution for 6 hours, the molded gel absorbed water and reached 139 g (water content 88 wt%). The bacteria were not observed in this immersion solution, indicating that almost all of the bacteria were trapped within the Raschig ring. The column of Example 3 was randomly packed with 139 g of the above-mentioned molded gel, and an aqueous substrate solution for fructose synthesis (glucose 5 wt%, manganese sulfate tetrahydrate 0.01 mole/
, 62° C., PH 6.3) was fed from the bottom of the column at a flow rate of 12 ml/h, and the effluent after 35 hours had a pH of 6.7 and a fructose concentration of 2.0 wt% (yield: 40 mol%). Example 5 In Example 4, Streptomyces phaeochromogenes was used instead of Lactobacillus brevis,
Moreover, the amount added is 1/1 of that in Example 4.
200, that is, 20 ml of suspension water (culture solution) containing 0.002 g of bacteria. Mixed suspension aqueous solution of polyvinyl alcohol and bacteria
Freeze, mold, and dry 200g in the same way to obtain 128g of gel.
(moisture content: 87 wt%, dehydration rate: 36 wt%). Add this molded gel to 150 ml of pre-sterilized 0.9% saline solution.
As a result of soaking in water for 6 hours, the molded gel absorbed 137g of water.
(moisture content reached 88wt%). This gel (137 g) was packed irregularly into the column of Example 3, and the culture solution (xylose 1%, peptone 1%, meat extract 1%, yeast extract 0.3%, Salt 0.5
%, magnesium sulfate heptahydrate 0.06%, 30℃)
was fed from the bottom of the column over a period of 48 h at a flow rate of 120 ml/h. Before and after this culture solution (medium) feeding operation, a part of the filling (Raschig ring) was collected and observed using a scanning electron microscope. As a result, almost no bacteria were observed in the gel before the culture medium feeding started. On the contrary, after feeding the medium, bacterial colonies (approximately 50μ
m, approximately 300,000 bacteria) were observed in various locations within the gel. Next, a substrate solution for fructose synthesis (glucose 5%, magnesium sulfate 0.01M, PH8.3, 65℃)
was fed from the bottom of the tower at a flow rate of 90 ml/h, resulting in 28 h
The fructose concentration in the subsequent effluent is 2 wt% (yield 40
mol%) and the pH value was 6.3. In addition, even if the above-mentioned culture (proliferation) operation was omitted and the substrate solution was immediately introduced for 28 hours, it was confirmed that the fructose in the effluent was only 0.02 wt%. In this way, in the present invention, since living microorganisms can be immobilized, it is naturally possible to grow them within the gel.

Claims (1)

[Claims] 1. A suspended aqueous solution obtained by adding living microorganisms to an aqueous solution of polyvinyl alcohol with a saponification degree of 95 mol% or more and a viscosity average degree of polymerization of 1500 or more is poured into a container of any shape or a mold for molding, This is frozen and molded at a temperature lower than -6℃, and then
The dehydration rate of this molded body is 5wt% without melting.
A method for immobilizing live microorganisms, which is characterized by vacuum drying as described above and, if necessary, immersing them in water.