JPH0339673B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immobilized yeast product having proliferation ability and a method for producing the same.

There is growing interest in producing ethanol by fermentation as a new energy source to replace petroleum. However, although ethanol has been produced from sugar using yeast, it is a batch reaction and the fermentation reaction rate is slow, so there is now a need for continuous production and faster reaction technology.

In recent years, methods for immobilizing microorganisms including yeast have been studied, and immobilization methods using synthetic or natural polymeric substances as carriers have been proposed. The inventors of the present application have already proposed a method for effectively immobilizing microorganisms and enzymes without significantly losing their original activity by mixing the microorganisms and enzymes with monomers and subjecting them to radiation polymerization at low temperatures. In immobilization methods using the so-called comprehensive method, including the method proposed by the present inventors, microorganisms, enzymes, etc. are mixed with polymerizable monomers or natural polymers dissolved at elevated temperatures, and this is then exposed to light. Alternatively, a method of solidifying and producing a fixed substance using means such as radiation irradiation, use of a catalyst, or cooling has been adopted. Therefore, in such conventional comprehensive methods, there is a risk that microorganisms and enzymes are already present in the process of manufacturing the immobilization carrier and may be deactivated by the immobilization reaction.

The purpose of the present invention is to not only eliminate the risk of such deactivation when immobilizing yeast, but also to ensure that the yeast proliferates on the surface or inside the carrier and the number per unit volume is much higher than that during immobilization. Therefore, it is an object of the present invention to provide an immobilized yeast that has a significantly increased activity and proliferation ability, and also to provide a method for producing such an immobilized yeast.

As a result of intensive research to achieve this objective, the inventors of the present application first produced a porous carrier by various means in the absence of yeast, and then shook this porous carrier in a culture solution containing yeast. By adsorbing yeast onto the surface of a porous carrier and then continuing to shake it, we developed a method for immobilizing yeast while growing it. A porous carrier is prepared by polymerizing a yeast immobilized substance having the ability to perform the polymerization and a polymerizable monomer containing water or a non-polymerizable solvent at room temperature or below, and after sufficiently swelling the carrier with water, it is injected into a yeast culture solution. We have arrived at a method for producing an immobilized yeast product with proliferation ability, which consists of shaking to adsorb yeast onto the surface of a carrier, and further shaking in a yeast culture solution.

In this way, the method of the present invention involves first producing an immobilization carrier through various chemical reactions in the absence of yeast, and then subjecting a small amount of polymerizable monomer to various chemical reactions in the presence of yeast. or immobilized without using any polymerizable monomer or chemical reaction. Therefore, the method of the present invention has the advantage that yeast is less likely to be inactivated than the conventional inclusion method in which yeast is already present when the carrier is prepared and the yeast is likely to be inactivated by the reaction. Furthermore, unlike most conventional methods that utilize only the yeast present at the time of immobilization, the present invention allows yeast to grow on or within the immobilized material to increase the number of yeast per unit volume compared to that at the time of immobilization. It has the characteristic of significantly increasing the activity of the immobilized product.

In the present invention, appropriate flexibility of the porous carrier is essential for the carrier to actively proliferate the adsorbed yeast and to have the receptivity to accept the increase in yeast volume due to yeast proliferation. This is achieved by soaking the prepared porous carrier in water for a long period of time to sufficiently swell it. In addition, the pore size of the porous carrier was determined by the size limit of yeast.
It is desirable that it is Å or more.

Next, the method of the present invention will be specifically explained.

First, the polymerizable monomer used in the method of the present invention is one or more monomers that belong to the following group [A] and can be in a stable supercooled state without crystallizing even at room temperature to low temperature. Or, monomers of [B] group which do not have supercooling property by themselves, but when mixed with [Å] group monomers at 30% or less, the mixed monomers as a whole maintain supercooling property. are: [A] Group monomers: hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxypropyl methacrylate, hydroxypropyl acrylate, hydroxybutyl methacrylate, hydroxybutyl acrylate, hydroxypentyl methacrylate, hydroxypentyl acrylate, hydroxyhexyl methacrylate, hydroxyhexyl acrylate , hydroxyheptyl methacrylate, hydroxyheptyl acrylate, glycidyl methacrylate, glycidyl acrylate, diethylene glycol dimethacrylate, diethylene glycol diacrylate, tetraethylene glycol dimethacrylate, tetraethylene glycol diacrylate, polyethylene glycol dimethacrylate, polyethylene glycol diacrylate, ethylene glycol dimethacrylate, Ethylene glycol diacrylate, propylene glycol dimethacrylate, propylene glycol diacrylate, dipropylene glycol dimethacrylate, dipropylene glycol diacrylate, polypropylene glycol dimethacrylate, polypropylene glycol diacrylate, butylene glycol dimethacrylate, butylene glycol diacrylate,
Pentanediol dimethacrylate, pentanediol diacrylate, hexanediol methacrylate, hexanediol diacrylate, heptanediol dimethacrylate, heptanediol diacrylate, neopentyl glycol dimethacrylate, neopentyl glycol diacrylate, trimethylolethane trimethacrylate, trimethylolethane Triacrylate, trimethylolpropane trimethacrylate, trimethylolpropane triacrylate, glycerol monomethacrylate, glycerol monoacrylate, glycerol dimethacrylate, glycerol diacrylate, glycerol trimethacrylate, glycerol triacrylate, diethylaminoethyl methacrylate, diethylaminoethyl acrylate, diethylaminobutyl methacrylate , diethylaminobutyl acrylate,
Dimethylaminoethyl methacrylate, dimethylaminoethyl acrylate, dimethylaminobutyl methacrylate, dimethylaminobutyl acrylate, benzyl methacrylate, methoxypolyethylene glycol methacrylate,
Methoxypolyethylene glycol acrylate, ethoxypolyethylene glycol methacrylate, ethoxypolyethylene glycol acrylate, methoxypropylene glycol methacrylate, methoxypropylene glycol acrylate, ethoxypropylene glycol methacrylate, ethoxypropylene glycol acrylate, etc.

[B] Group monomers: vinyl acetate, vinyl propionate, vinyl butyrate, methyl methacrylate, methyl acrylate, ethyl methacrylate, ethyl acrylate, propyl methacrylate, propyl acrylate, butyl methacrylate, butyl acrylate, pentyl methacrylate, pentyl acrylate, hexyl methacrylate , hexyl acrylate, lauryl methacrylate, lauryl acrylate, stearyl methacrylate, stearyl acrylate, styrene, vinyltoluene, sulfonated styrene, acrylic acid, methacrylic acid, aminostyrene, vinylpyrrolidone, acrylamide, methacrylamide, methylenebisacrylamide, methylolacrylamide, acrylonitrile , methacrylonitrile, diallyl phthalate, itaconic acid, maleic anhydride, diallyl isophthalate, diallyl succinate, diallyl itaconate,
divinylbenzene, triallyl dianurate,
etc.

Next, the non-polymerizable solvent used in the present invention is a [C] group solvent as follows: [C] group: acetone, methyl ethyl ketone, ethylene glycol diethyl ether, methyl formate, ethyl formate, methyl acetate, glycol Carbonate, ethyl acetate, ethylene glycol monomethyl ether acetate, ethylene glycol monoethyl ether acetate, glycol diacetate, glycol monomethyl ether, glycol monoethyl ether, glycol monobutyl ether, diethylene glycol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, methyl alcohol, ethyl alcohol, n
-propyl alcohol, isopropyl alcohol, ethylene glycol, sec-butyl alcohol, etc.

The polymerizable monomers shown in group [A] and group [B] are
It is mixed with water or a non-polymerizable solvent shown in group [C], and a polymeric porous carrier is produced by a polymerization reaction under irradiation with light or ionizing radiation at room temperature or below. Next, it is desirable to soak this porous carrier in water for several days to sufficiently swell it with water, and then sterilize it using an autoclave or the like.

By shaking the porous carrier prepared as described above in a yeast culture solution, yeast is effectively absorbed and immobilized on the surface of the porous carrier. The porous carrier on which yeast has been adsorbed and immobilized is further shaken in a yeast culture solution to maintain conditions suitable for aerobic yeast growth, allowing the adsorbed yeast to invade inside the porous carrier while proliferating. do. Through the above process, yeast is immobilized on the surface and inside of the porous carrier while maintaining an active growth state. It is essential for the porous carrier to have appropriate flexibility when yeast invades the inside of the porous carrier while multiplying, and this flexibility allows the walls of the pores inside the carrier to deform appropriately. Allow for an increase in yeast volume due to yeast growth.

In carrying out the method of the present invention, the temperature employed as the polymerization reaction temperature is room temperature 25°C to -
It is 196℃.

In carrying out the method of the present invention, yeast is used after being cultured in a preculture solution containing, for example, sugar, peptone, yeast extract, and salt at a pH of 4 to 5.

In the method of the present invention, the radiation sources employed are light from low-pressure or high-pressure mercury lamps, X-rays, gamma rays, beta rays, electron beams, alpha rays, mixed radiation from chemical reactors, spent fuel, nuclear fission products. Any gamma ray from The irradiation dose can be selected relatively freely since the yeast is not irradiated with light or radiation during the irradiation for producing the porous carrier, but it is necessary to cure the polymerizable monomer by at least 50%. Perform irradiation of at least the required 1×10 ⁴ R. In the case of radiation, an irradiation dose of 1×10 ⁴ R to 1×10 ⁷ R is desirable at a dose rate of 1× ^{10 3} to 10 ⁹ R/hour.

Instead of polymerization by irradiation with light or ionizing radiation in the method of the invention, redox catalysts or other catalysts can be used to carry out the polymerization. However, in irradiation polymerization using light or ionizing radiation, there is no possibility that catalyst residues and the like remain in the immobilized material as in the case of catalytic polymerization, and the polymerization is carried out favorably by the growth of yeast. Furthermore, in radiation polymerization, polymerization can be easily carried out in a short period of time, and various properties can be changed over a wide range by combining various monomers.

EXAMPLES The present invention will be explained in more detail with reference to Examples below. Ethanol produced in the examples was quantified using alcohol dehydrogenase.

Example 1 20 ml of water was added to 5 ml of 2-hydroxyethyl acrylate (HEA) and 5 ml of methoxypolyethylene glycol methacrylate (M-23G) as monomers, and after thorough mixing, the r-ray from ⁶⁰ Co was heated 1x at -78°C. A porous carrier was prepared by irradiating with 10 ⁶ R, cut into small pieces, and then shaken in water for 4 days to fully swell in water. Yeast was precultured under aerobic conditions at 30°C for 24 hours in a preculture medium containing 1% glucose, 0.1% molasses, 0.5% peptone, 0.3% yeast extract, and 0.3% malt extract. 1ml of 12% glucose, 1% molasses, 0.15% yeast extract, 0.25%
_NH4Cl , 0.55% _{K2HPO4 ,} 0.025% _MgSO4 .
Mixed with 20 ml of complete medium containing _7H2O , 0.1% NaCl, 0.001% _CaCl2 and 0.3% lactic acid. The porous carrier swollen in water was placed in a complete medium containing yeast, and aerobic conditions were maintained by shaking. After maintaining aerobic conditions for a certain period of time, a portion of the immobilized material is gently shaken with an equal volume of complete medium to produce ethanol through a fermentation reaction, and the amount of alcohol produced in the first hour is This was used as an indicator of the activity of the immobilized product.
The activity was also measured in the same manner as with the immobilized product using a mixture of a small amount of a complete medium suspension of non-immobilized yeast obtained by the same procedure except that an immobilized carrier was added, and the same volume of the complete medium. The results are shown in FIG. 1 for comparison. As the culture continues under aerobic conditions, the activity of the immobilized yeast (curve A) increases rapidly, and the activity of the unimmobilized yeast (curve B) increases rapidly.
The increase in activity reached 13 times, and the high activity was maintained for more than 20 days. Furthermore, FIG. 2 shows the results of fermentation reaction using the immobilized product not being stopped after 1 hour but being allowed to continue until all the sugars were converted to ethanol. Immobilized yeast (curve A) gives a 100% ethanol yield in 150 minutes, but unimmobilized yeast (curve B) gives only 10% ethanol yield within the same time period. Much higher activity was obtained in immobilized yeast than in yeast that did not.

[Brief explanation of the drawing]

FIGS. 1 and 2 are graphs comparing the activity of the immobilized yeast obtained in Example 1 with the activity of unimmobilized yeast. In FIG. 1, the horizontal axis is culture time (hr), and the vertical axis is alcohol concentration (%). In FIG. 2, the horizontal axis is fermentation time (minutes), and the vertical axis is alcohol yield (%).

Claims (1)

[Claims] 1. A polymerizable monomer is mixed in a dispersed state in water or a non-polymerizable solution and polymerized at a temperature below room temperature to produce an organic polymer porous carrier, and the porous carrier is After sufficiently swelling with water, the porous carrier is shaken in a yeast culture solution to adsorb and solidify the yeast on the surface, and the porous carrier with the yeast adsorbed and immobilized is further shaken in the yeast culture solution to provide aerobic conditions. 1. A method for producing an immobilized yeast product having the ability to proliferate, which comprises adsorbing and immobilizing yeast on the surface and inside of the porous carrier while maintaining yeast in a proliferative state by maintaining conditions suitable for the multiplication of yeast. 2. The method for producing an immobilized yeast having proliferation ability according to item 1, wherein the polymerization is carried out by irradiation with light or ionizing radiation. 3 In the case of polymerization by irradiation with ionizing radiation, the irradiation dose is 1x10 ³ to 1x10 ⁹ at a dose rate of 1x10 ⁴ to 1x10 9
2. A method for producing an immobilized yeast having a proliferation ability of 1x10 ⁷ R. 4. The method for producing an immobilized yeast having proliferation ability according to item 1, wherein the polymerization is carried out in the presence of a catalyst. 5. The method for producing an immobilized yeast having proliferation ability according to item 4, wherein the catalyst is a redox catalyst.