JPH0441598B2 - - Google Patents

Description

[Detailed description of the invention]

[Background of the Invention] Technical Field The present invention relates to a carrier for retaining microorganisms and its use,
That is, it relates to an immobilized microorganism obtained by immobilizing microorganisms on this carrier. In the applied microbial industry, a technique is known in which microorganisms are immobilized on a suitable holding carrier, ie, used as immobilized microorganisms. Immobilized microorganisms can be used in the same way as industrial catalysts, such as when used as a so-called bioreactor, so it is possible to expand the usage of microorganisms, and immobilized microorganisms have a relatively high substrate resistance. Immobilized microorganisms are an alternative to conventional developments, as they can be used at high concentrations and overcome the problems that often arise when using low microbial concentrations (e.g., the need for long reaction times). is expected. In particular, the production of alcoholic beverages (especially beer), which is one of the typical applied microbial industries, can be said to be particularly amenable to the use of immobilized microorganisms (ie, yeast). It goes without saying that the holding carrier used for immobilized microorganisms is important, but conventional carriers have not always been fully satisfactory. For example, a method using immobilized yeast is known as an attempt to manufacture alcoholic beverages in a short period of time using small-scale equipment. In this method, polysaccharide particles such as alginate gel beads have generally been used as carriers for retaining microorganisms from the viewpoint of food hygiene and other reasons. However, in the case of manufacturing alcoholic beverages using immobilized yeast in which yeast is immobilized on conventional polysaccharide granules (alginate gel beads, etc.), the strength of the polysaccharide granules is weak, and for example, in a large-scale packed bed,
Consolidation deformation of the granules caused by being pressed against perforations or the like that direct the granules in the layer impedes liquid flow and reduces the fermentation ability of the immobilized yeast. In cases where alcoholic beverages are produced under low pH conditions (e.g. 3 or less) or in the presence of various ions (e.g. phosphate ions), where polysaccharide granules swell during fermentation and the durability of immobilized yeast decreases (e.g. In the case of some types of wine), it is not necessarily sufficient to produce alcoholic beverages stably over a long period of time due to problems such as the elution and disintegration of the polysaccharide that is the immobilized carrier. On the other hand, porous ceramics (e.g. porous titania, porous alumina,
Porous silica) is known. The bond between these porous ceramics and microorganisms is generally weak, and the number of microorganisms that can be immobilized per unit volume of the carrier is small, and once immobilized, they are quickly detached. Crosslinking reagents (eg glutaraldehyde) have been used. However, the use of such a crosslinking reagent in the production of foods (alcoholic beverages) cannot be said to be preferable in terms of food hygiene. Furthermore, to the best of the inventors' knowledge,
It appears that no attempt has been made to date to produce alcoholic beverages using immobilized yeast, which is obtained by immobilizing yeast on porous ceramics. [Summary of the Invention] Summary The present inventors conducted various tests and research on methods for immobilizing yeast and methods for producing alcoholic beverages using immobilized yeast, and as a result, they discovered that highly porous foam glass was used as a carrier for retaining microorganisms. The inventors have discovered that the above-mentioned problems can be effectively solved by using the present invention, and have completed the present invention based on this discovery. That is, the present invention generally relates to a carrier for retaining microorganisms, and the carrier for retaining microorganisms according to the present invention has a structure in which a large number of bubbles existing inside and on the surface layer have walls between the bubbles and between the bubbles on the surface side and the outside. It has open cells with fine openings that communicate with it, has a water absorption rate of 50% or more, and has a central pore diameter (volume) of 1.0.
~50μ and a pore volume of 1.0 to 5.0 ml/g. The present invention also relates to the use of this carrier, and the immobilized microorganism according to the present invention has a structure in which the walls of a large number of bubbles existing inside and on the surface communicate between the bubbles and between the bubbles on the surface side and the outside. Contains open cells with fine openings, water absorption rate of 50% or more, median pore diameter (volume) of 1.0 to 50μ, and pore volume of 1.0
It is characterized in that microorganisms are immobilized on a microorganism holding carrier made of highly porous foamed glass having a density of ~5.0 ml/g. Effects The highly porous foam glass used as the immobilization carrier in this invention has excellent strength and durability, and
It has many pores of, for example, 0.3 to 2000μ on the surface and inside, and microorganisms such as yeast can grow and adhere not only on the surface of the carrier but also inside it.
Therefore, microorganisms can be maintained at an extremely high concentration without using a crosslinking reagent. Further, by appropriately selecting the median pore diameter and pore volume of the highly porous glass foam, the number of microorganisms to be immobilized can be adjusted over a wide range. If this invention is utilized in the production of alcoholic beverages, the following advantages can be obtained. (a) Since immobilized yeast has excellent strength and durability and does not swell, shrink, or deform, it is possible to produce alcoholic beverages continuously over a long period of time, or to manufacture alcoholic beverages in batches using the same immobilized yeast repeatedly. be. Furthermore, since the material of the immobilization carrier is glass, it is stable even in a wide PH range and in the presence of various ions, and therefore, the present invention is also applicable to alcoholic beverages produced under such conditions. Furthermore, even in the continuous production of alcoholic beverages using a packed bed type reactor, the compressive strength of the immobilized yeast is high and the specific gravity can be adjusted over a wide range, so highly efficient operation can be achieved with low pressure loss within the reactor. (b) The material of the immobilization carrier is glass, and unlike the case of conventional porous ceramics, there is no need to use a crosslinking reagent during immobilization, which is preferable in terms of food hygiene. Moreover, since yeast can be immobilized at a high concentration, alcoholic beverages can be rapidly produced by fermentation at a high yeast concentration. (c) Since the material of the immobilization carrier is glass and is stable to heat, drugs, etc., the carrier can be sterilized by heating and pressure or by drugs. For example, by simultaneously heat-pressure sterilizing the brewing raw material liquid and highly porous foam glass supplied into the reaction tank, and supplying aseptically cultured yeast to this, yeast immobilization and alcoholic beverage production, which is preferable from a sanitation perspective, can be carried out. Manufacture is possible. [Specific Description of the Invention] Microorganism Retaining Carrier The microorganism retaining carrier according to the present invention is defined as described above. Highly porous foam glass as used in the present invention is glass containing many fine bubbles in the surface layer and/or inside, and as is clear from its high water absorption rate of 50% or more. At least a majority of the cells are open cells. In addition, the products manufactured by the preferred manufacturing method described below contain many bubbles with many air permeable pores in the cell walls, and it is believed that this pore structure also contributes to the high water absorption rate. be done. Foam glass is already well known, but conventional foam glass is usually manufactured by adding a foaming agent to glass powder, heating it to soften and foam it, and then cooling and solidifying it. As is clear from the fact that it is used as a material, its cells are mainly closed cells, and its water absorption rate is low, ranging from several percent to 30%. The manufacturing method and preferred specific examples of the carrier for retaining microorganisms according to the present invention are as described later in connection with the production of alcoholic beverages in which this carrier is typically used. However, if the microorganism to be immobilized differs, preferred specific examples for the microorganism will generally differ, and therefore, one appropriate for the given microorganism must be selected. Therefore, when the target microorganism is yeast, the pore conditions of the carrier are as described below, but when the target microorganism is, for example, a bacterium of the genus Zymomonas, the median pore diameter (volume) is about 0.4 to 10μ. , the pore volume is
The amount is preferably about 0.5 to 2.0 ml/g. The definitions and measurement methods of water absorption, median pore diameter (volume), and pore volume are as follows. Water absorption rate (%): After placing the foam glass in water and keeping it in a vacuum state (76 mmHg strength) for about 10 minutes, when the pressure is returned to normal, the volume of water absorbed into the foam glass is defined as X, and the volume of water absorbed into the foam glass is If the volume (including the empty volume where water is not absorbed within 1 minute when immersed in water at normal pressure) is defined as Y, then X/Y. Median pore diameter (volume) (μ): When the volume of each pore in one foam glass is accumulated in descending order of pore diameter, the pore corresponding to 1/2 of the total pore volume It is the diameter of the pore (μ). In this specification,
30,000 (PSIa) from vacuum state by mercury intrusion method
Pore diameter (μ) corresponding to the injection pressure that gives 1/2 the amount of mercury injected when mercury is injected up to
It is shown. Pore volume (ml/g): The volume (ml) of vented air in 1 g of foam glass. It is indicated by the amount of mercury intrusion. Further, although the carrier of the present invention can have various shapes, a typical shape is granular, and in this case, the diameter of the particles refers to the maximum dimension of the particles. Immobilized Microorganisms There can be various types of microorganisms to be immobilized on the carrier according to the present invention. Specific examples include yeast, lactic acid bacteria, Zymomonas bacteria, and others. The immobilized microorganism according to the present invention is typically a yeast, the details of which will be described later regarding the production of alcoholic beverages. Other microorganisms can also be immobilized in the same manner as yeast. The immobilized microorganism according to the present invention generally has a high loading rate on a carrier and is less likely to be detached from the carrier. In the case of yeast, a typical microorganism, the number of yeast per unit surface area is
10 ⁶ cells/cm ² or more, and the yeast detachment rate is 1% or less (the measurement method will be described later). Utilization of Immobilized Microorganisms (Part 1) The immobilized microorganisms according to the present invention can be used for applications that focus on the unique properties of the microorganisms. For example, when the microorganism is yeast, a typical usage mode is the production of alcoholic beverages using sugars derived from various sources as substrates, the details of which will be described later. In addition, if the microorganism is a Zymomonas bacterium,
A typical application is the production of ethanol using sugars, especially waste molasses, as substrates. Utilization of immobilized microorganisms (Part 2) One of the typical immobilized microorganisms according to the present invention is immobilized yeast, which is utilized in the production of alcoholic beverages. Brewing Raw Material Liquid The brewing raw material liquid contains a substrate for the yeast used, and is usually a solution or dispersion containing sugar as a substrate. Specific examples of such brewing raw material liquids include wort for beer and whiskey, fruit juice for wine, and mash (parts other than yeast) for sake and Otsu-type shochu. Immobilized Yeast In this application of the invention, immobilized yeast immobilized on highly porous foam glass is used. As the yeast to be immobilized, Saccharomyces cerevisiae and other yeasts commonly used in the production of alcoholic beverages can be used. Highly porous foam glass as a carrier for immobilizing yeast has a median pore diameter (volume) of 1.0 to 50μ, preferably 20 to 40μ, and a pore volume of 1.0 to 5.0ml/g, preferably 2.0 to 3.0ml/g. , water absorption rate 50-90%, preferably
50-85%, bulk specific gravity 0.1-0.5, preferably 0.2-
A value of 0.35 is appropriate. If either the median pore diameter value (volume) or the pore volume value is extremely small, or if both are small, the amount of yeast immobilized will be small, and the median pore diameter value will decrease. If the volume is too large, the immobilized yeast will easily detach. Additionally, if either the median pore diameter value (volume) or the pore volume value is extremely large, or if both are large, the strength and durability of the immobilized yeast will be impaired. . Therefore, the properties of the foam glass may be appropriately selected within the above range so as to obtain an immobilized yeast suitable for the purpose. The water absorption rate and apparent specific gravity are determined according to the median pore diameter value (volume) and pore volume value. The shape and size of the foam glass may be selected as appropriate, such as granular, plate-like, rod-like (prismatic, cylindrical), cylindrical, polygonal, etc., depending on the type of fermenter (reactor), etc., but depending on the reaction efficiency and ease of handling. From the viewpoint of size, particles with a diameter of 0.5 to 15 mm, preferably 1.5 to 15 mm, particularly 2 to 6 mm are preferred. As described above, the diameter in this case means the longest diameter for objects that are not on a true sphere. The materials for the above bubble glass include silica glass, soda lime glass, aluminoborosilicate glass, borosilicate glass, aluminosilicate glass,
There are lead glass, etc., and it can be changed as necessary. From an economical point of view, inexpensive soda-lime glass is desirable. The highly porous foam glass used in this invention is, for example, 5 to 20
Granular foam glass having a water absorption rate of % is immersed in hot water or an alkaline solution to elute the soluble alkali components and silica in the foam glass, thereby creating openings in the surface layer and closed cells of the foam glass. , this can be manufactured. In addition, 5 to 10% of the glass powder is mixed with a blowing agent and metal oxides with high melting points, such as alumina, silica, and zirconia.
It can also be produced by adding it, firing it, and then rapidly cooling it to generate fine bubbles.
-Refer to each publication No. 154099). Immobilization of yeast onto highly porous foam glass is carried out by contacting a liquid in which yeast is suspended with highly porous foam glass for a certain period of time or more. Any dispersion medium for suspending the yeast can be used as long as it causes no food hygiene problems and does not adversely affect the activity of the yeast. Usually, brewing raw material liquid, fermentation liquid, water, or a mixture thereof is used. The yeast concentration of the yeast suspension may be appropriately selected so as to obtain the expected yeast concentration in the immobilized yeast. Usually, the yeast concentration is set higher than the expected yeast concentration in the immobilized yeast. When the dispersion medium contains a brewing raw material liquid, the yeast can also be immobilized on the foam glass while being cultured and grown in the dispersion medium. Therefore, in this case, it is also possible to set the initial yeast concentration to a low value. The contact time is usually
The yeast concentration in the dispersion medium is sufficient for about 1 to 2 days, and the amount of yeast immobilized does not often increase even if the contact is made for longer than that (the amount of yeast that is finally immobilized mainly depends on the amount of yeast used). This is determined by the characteristic values of foam glass (pore volume, central pore diameter (volume), etc.). However, when immobilization is performed while culturing and proliferating yeast in a dispersion medium containing a brewing raw material liquid, the propagation time of the yeast should also be considered. Contact between the foam glass and the yeast suspension can be carried out in any manner as long as sufficient contact between the two is achieved. For example, when immobilizing yeast on granular foam glass, the granular foam glass is immersed in a yeast suspension and left standing or stirred, or the granular foam glass is packed into a cylindrical column and the yeast is suspended in it. There are methods such as circulating a liquid (for specific immobilization methods, see Examples below). Production of alcoholic beverages The production of alcoholic beverages in the application example of the present invention is carried out by bringing the above-mentioned immobilized yeast into contact with the brewing raw material liquid and fermenting the brewing raw material liquid, and any suitable mode may be adopted. can. For example, fermentation can be carried out by supplying the brewing raw material liquid in a batch manner to a reaction tank containing immobilized yeast granules as a fixed bed, non-fixed bed, or fluidized bed, or a method in which the brewing raw material liquid is continuously passed through. There is a method in which fermentation is carried out (once or multiple times). For details on these methods, see the so-called bioreactor method in "Enzyme Engineering" (edited by Saburo Fukui, Ichiro Chibata, and Shuichi Suzuki) (Tokyo Kagaku Doujin), D. Williams,
DMMunnecke: Biotech.and Bioeng. 23 1813−
25 (1981) etc. The filling rate of immobilized yeast in the reaction tank can be determined arbitrarily, but in order to take advantage of the characteristics of immobilized yeast and rapidly produce alcoholic beverages, a high yeast concentration, specifically 0.4w/ preferably v% or more (however, w is the dry weight (g) of the yeast contained in the immobilized yeast filled in the reaction tank, and v is the volume of the brewing raw material liquid in the case of a batch system ( ml), or in the case of a continuous system, the volume (ml) of the reaction tank filled with immobilized yeast.) The fermentation conditions and other conditions may be subject to modification when implementing this application example of the present invention. It is essentially the same as the conventionally known one. The resulting fermented liquor is already an alcoholic beverage in itself, but it would normally be further aged to form the final product. Experimental Examples The following examples are for specifically explaining the present invention. Example 1 (Manufacture of foam glass beads) 2% CaCO ₃ was added as a foaming agent to powdered glass obtained by wet-pulverizing a soda-lime glass bottle for 24 hours.
The granulated product is heated at a temperature of 820 to 850℃.
Bake for 100-150 seconds. The foam glass thus obtained was immersed in hot water at 70° C. for 4 days to produce highly porous granular foam glass. Table 1 shows the properties of the foam glass thus obtained.
Shown below.

[Table] Example 2 (Immobilization of yeast) Brewer's yeast (Saccharomyces cerevisiae) was added to the wort whose sugar content was adjusted to 11°P at a concentration of 6.0x.
The cells were suspended at 10 ⁸ cells/ml. The foamed glass beads produced in Example 1 were immersed in this at a ratio of approximately 15% (volume of foamed glass beads/volume of wort), and the mixture was heated to 8°C.
The yeast was immobilized by batch stirring at 200 rpm for about 48 hours. Table 2 shows the number of immobilized yeast and the detachment rate.

[Table] *1, *2, *3 These are defined as below. Number of yeast per unit volume = x/y Number of yeast per unit surface area = x/z Number of yeast per unit weight area = x/w However, x, y, z, and w are defined as below. It is. Number of yeast immobilized in one immobilized yeast:
xcells Volume of foam glass used as an immobilization carrier (including the empty volume where water is not absorbed within 1 minute when immersed in water at normal pressure): yml Volume of foam glass used as an immobilization carrier Surface area (Surface area when foam glass is not considered a porous material, but is assumed to have a flat surface.For example, when foam glass is spherical with a diameter of rcm, it is 4πr ² cm ² ): zcm ² fixed Weight of foam glass used as carrier: wg *4 Desorption rate was determined by the following method. In a 500ml beaker, inner diameter 33mm, height 95mm
A cylindrical basket (with a square mesh of 1.5 mm on each side) was placed so that the center of the basket and the center of the beaker matched and the distance between the bottom of the basket and the bottom of the beaker was 2 cm. . Into this basket, put 15 pieces of granular immobilized yeast produced as above,
400 (ml) of water was placed in a beaker and stirred for 48 hours at a stirring speed of 700 rpm using a stirrer bar with a length of 5 cm. The number of yeast immobilized on one immobilized yeast before stirring is X (cells/one immobilized yeast), and the number of yeast immobilized on one immobilized yeast after stirring is Y
(cells/1 immobilized yeast), the detachment rate Z
(%) is Z(%)=(1-Y/X)×100. Note that this measurement method is applied when yeast is immobilized by molding foam glass as an immobilization carrier into a spherical shape with a diameter of 1.5 to 15 mm. Example 3 (Manufacture of alcoholic beverages) Pour 200 ml of wort whose sugar content was adjusted to 11.0P into a 250 ml graduated cylinder, add 30 ml of immobilized yeast beads No. 2 obtained in Example 2, and let stand at 8°C for 50 hours. At the same time, beer was brewed in batches. Table 3 shows the properties of the beer obtained.

[Table] Example 4 (Production of alcoholic beverages) A cylindrical column with an inner diameter of 3.5 cm and a height of 30 cm was packed with immobilized yeast beads No. 3 obtained in Example 2 so as to be almost full. This column has a sugar content of 11.0°P.
Beer was continuously brewed by flowing 10 ml of wort per hour at 8°C. There were no particular problems with the strength, durability, etc. of the immobilized yeast, and beer with the properties shown in Table 4 was stably obtained for more than 10 days.

[Table] Example 5 (Manufacture of foam glass beads) 3% CaCO ₃ was added as a foaming agent to hard glass (borosilicate glass) crushed to an average particle size of 22.0μ,
Granulated to an average size of 2.00 to 2.38 mmφ, 200 ml at 870°C
It was fired for a few seconds to create granular foam glass. Regarding this granular foam glass, patent application No. 60-154099
Alkali treatment was carried out according to the invention of the present invention. That is,
As a result of immersing the above granular foam glass in a 3% aqueous solution of NaOH (120°C) for 4 hours in an autoclave at 120°C, highly porous foam glass having the following physical properties could be obtained.

[Table] *Measurement method: Example 6 (Bacteria fixation) Yeast extract 1.0 (w/v)%, Sucrose 10.0
(w/v)% in a liquid medium (PH6-7) containing Zymomonas mobilis:
ATCC 10988) was suspended at a concentration of 1.8×10 ⁶ cells/ml. The foam glass beads prepared in Example 5 were immersed in this at a ratio of about 5% (w/v), and the temperature was kept at 30°C.
Zymomonas mobilis was immobilized by static culture for 24 hours. Table 6 shows the number of immobilized Zymomonas mobilis bacteria.

【table】

[Table] *See note to Example 2

Claims (1)

[Claims] 1. Contains continuous cells with fine openings in the walls of a large number of cells existing inside and on the surface layer to communicate between the cells and between the cells on the surface side and the outside, and has a water absorption rate.
A carrier for retaining microorganisms, characterized in that it consists of highly porous foam glass having a median pore diameter (volume) of 1.0 to 50 μ and a pore volume of 1.0 to 5.0 ml/g. 2. The carrier for holding microorganisms according to claim 1, wherein the highly porous foam glass is granular with a diameter of 0.5 to 15 mm. 3. Contains open cells with fine openings in the walls of many cells existing inside and on the surface layer, which communicate between the cells and between the cells on the surface layer and the outside, and has a high water absorption rate.
Microorganisms are immobilized on a microorganism holding carrier made of highly porous foam glass having a median pore diameter (volume) of 1.0 to 50 μ and a pore volume of 1.0 to 5.0 ml/g. Immobilized microorganisms. 4. The immobilized microorganism according to claim 3, wherein the microorganism is yeast. 5. The immobilized microorganism according to claim 4, wherein the number of yeast per unit surface area is 10 ⁶ cells/cm ² . 6. Claims 4 to 6 are granular with a diameter of 1.5 to 15 mm, and have a yeast detachment rate of 1 or less.
The immobilized microorganism according to item 5. 7. The immobilized microorganism according to claim 3, wherein the microorganism is Zymomonas mobilis. 8 Highly porous foam glass has a median pore diameter (volume)
The immobilized microorganism according to any one of claims 3 to 7, which has a pore volume of 0.4 to 50 μ and a pore volume of 0.5 to 5.0 ml/g. 9. The immobilized microorganism according to any one of claims 3 to 8, wherein the highly porous foam glass is granular with a diameter of 0.5 to 15 mm.