JPS63173590A - Carrier for supporting microorganism and use thereof - Google Patents

Abstract

PURPOSE:To provide a carrier for supporting microorganism, composed of a highly porous foam glass having a specific water-absorption, having excellent strength and durability and useful in the field of applied microbial industry, etc. CONSTITUTION:Powder of glass such as silica glass is added with a foaming agent, heated to effect simultaneous softening and foaming and solidified by cooling to obtain the objective carrier for supporting microorganism. The carrier has a water-absorption of >=50wt.%, a central pore diameter (volume) of 1.0-50mu and a pore volume of 1.0-5.0ml/g. If necessary, the carrier is made to contact for 1-2 days with a suspension produced by dispersing yeast cells, etc., in a dispersant such as a fermentation liquid to obtain immobilized microorganism containing >=10<6> cells of yeast per 1cm<2> of the surface of the carrier and having an elimination ratio of the yeast of <=1%.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Technical Field The present invention relates to a carrier for holding microorganisms and its use, that is, 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 attractive option for future development, as they can be used at high concentrations and overcome some of the problems that 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 produce 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 production of alcoholic beverages using immobilized yeast in which yeast is immobilized on conventional polysaccharide granules (alginate gel beads, etc.), Due to the compaction deformation of the granules caused by being pressed against the perforated plate that supports the granules in the fermentation chamber, the fermentation ability of the immobilized yeast is reduced due to the liquid flow being hindered. swell,
The durability of immobilized yeast decreases, ■ under low pH conditions (e.g. 3
(below) or when producing alcoholic beverages in the presence of various ions (for example, phosphate ions) (for example, in the case of certain types of wine), the polysaccharide that is the immobilized carrier may elute.
However, due to problems such as disintegration during heating, it is not necessarily sufficient to produce alcoholic beverages stably over a long period of time.

On the other hand, porous ceramics (e.g. porous titania, porous alumina, porous silica) can be used as carriers for retaining microorganisms.
It has been known. The bond between these porous ceramics and microorganisms is generally weak, and the number of microorganisms immobilized per unit volume of the carrier is small, or the microorganisms are easily detached even after immobilization. cross-linking reagents (eg glutaraldehyde) have been used. However, the use of such a crosslinking reagent in the production of food (alcoholic beverages) cannot be said to be desirable in terms of food hygiene.

As far as the present inventors know, no attempt has been made to date to produce alcoholic beverages using immobilized yeast in which yeast is immobilized on porous ceramics.

[Summary of the invention]

Summary The present inventors have conducted various tests and research on methods for immobilizing yeast and methods for producing alcoholic beverages using immobilized yeast, and have found that the above-mentioned problems can be overcome by using highly porous foam glass as a carrier for retaining microorganisms. They have found that the problem can be solved effectively, 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 is characterized in that it is made of highly porous foam glass having a water absorption rate of 50% or more. be.

The present invention also relates to the use of this B1 body, and the immobilized microorganism according to the present invention is a microorganism that is immobilized on a microorganism holding carrier made of highly porous foam glass with a water absorption rate of 50% or more. It is characterized by:

Effects The highly porous foam glass used as the immobilization carrier in this invention has excellent strength and durability, and has many pores of, for example, 0.3 to 2000μ on the surface and inside, and is resistant to microorganisms such as yeast. Microorganisms can grow and adhere not only on the surface of the carrier but also inside it, and therefore microorganisms can be retained 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 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. moreover,
Even in the continuous production of alcoholic beverages using a packed bed type glue reactor, since the compressive strength of the immobilized yeast is high and the specific gravity can be adjusted over a wide range, highly efficient operation with low pressure loss within the reactor is possible.

(b) Food hygiene - 1 - preferred because the abrasive material of the immobilization carrier is glass and there is no need to use a crosslinking reagent during immobilization unlike in the case of conventional porous ceramics. Moreover, since yeast can be immobilized at a high concentration, alcoholic beverages can be rapidly produced by fermentation at a high yeast concentration.

(c) The material of the immobilization carrier is glass and is stable against heat, drugs, etc., so the carrier can be sterilized by heating and pressure or by drugs. For example, by simultaneously heating and pressurizing the brewing raw material liquid and highly porous foam glass supplied into the reaction tank, and supplying aseptically cultured yeast thereto, yeast immobilization and alcoholic beverages are possible, which is preferable for sanitation. It is possible to manufacture

[Detailed description of the invention]

Carrier for retaining microorganisms The carrier for retaining microorganisms according to the present invention is as defined above.

The highly porous foamed glass referred to in the present invention is glass containing a large number of fine bubbles in the surface layer and/or inside, and as is clear from its high water absorption rate of 50% or more, these 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 a few percent to about 30 percent.

The manufacturing method and preferred specific examples of the carrier for microbial storage F and +7 according to the present invention are as described later in connection with the production of alcoholic beverages in which the carrier is typically used.

However, if the microorganism to be immobilized differs, preferred specific examples for the microorganism will generally differ, so an appropriate one should be selected for the given microorganism.

Therefore, when the counter-symbol organism is yeast, the pore conditions of the carrier are as described below, but when the counter-symbol organism is a bacterium such as Zymomonas, the median pore diameter (volume) is 0.4. ~10μ, pore volume 0.5~2. 0rA
It is preferably about I/g.

In addition, the definition of water absorption rate, median pore diameter (volume), and pore volume and the i11+1 standard method are as follows.

Water absorption rate (%): Place the bubble glass in water and place it in a vacuum state (76
The volume of water absorbed into the foam glass when the pressure is returned to normal pressure after keeping it for about 10 minutes (approximately (including the volume of voids where water is not absorbed) 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 Hole diameter (μ)
It is. In this specification, the pore diameter (μ) corresponds to an injection pressure that is half of the large amount of mercury when mercury is injected from a vacuum state to 30,000 (PSIa) by the mercury intrusion method. It is shown by .

Pore volume (ml/g): It is the ventilation pore volume (1) in 1 g of foam glass, and in this specification, when mercury is injected from a vacuum state to 30,000 (PSIa) by mercury intrusion method. 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 one in which the microorganism is yeast, and the details thereof are as described later in connection with 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, which is a typical microorganism, the number of yeast per unit surface area is 106 cells/c+J or more, and the detachment rate of yeast is 1% or less (the measurement method will be described later).

Utilization of immobilized microorganisms (-) The immobilized microorganisms according to the present invention can be used for uses that take advantage of the unique properties of the microorganisms.

For example, when the microorganism is yeast, the typical usage is to produce alcoholic beverages using sugars from various sources as substrates,
The details will be described later.

Furthermore, when the microorganism is a Zymomonas genus bacterium, a typical usage mode is the production of ethanol using sugar, especially blackstrap molasses, as a substrate.

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,
It is usually a solution or dispersion containing sugar as substrate. Specific examples of such brewing raw material liquids include wort for beer and whisky, fruit juice for wine, and mash (parts other than yeast) for sake and 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 RJ
Median pore diameter (volume) 1.0-50μ, preferably 20
~40μ, pore volume 1.0-5.0ml/g-preferably 2.0-3.0ml/g, water absorption 50-90%, preferably 50-85%, bulk specific gravity 0.1-0, 5, preferably 0.2 to 0.35. 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 (volume) will be small. ) is too large, the immobilized yeast will easily detach. Furthermore, 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 bulk specific gravity are determined by the median pore diameter value (
volume) and pore volume. The shape and size of the foam glass can be selected as appropriate, such as granular, plate-like, rod-like (prismatic, cylindrical), honeycomb, polygonal, etc., depending on the type of fermenter (reactor), etc., but depending on the reaction efficiency and ease of handling. From the point of magnitude, the diameter is 0. 5-15mm, preferably 1
．． 5-15m+i.

Particularly preferred is a granular material with a content of 2 to 61111%. As described above, the diameter in this case means the longest diameter for objects that are not on a true sphere.

Examples of the material for the bubble glass mentioned above include silica glass, soda lime glass, aluminoborosilicate glass, borosilicate glass, aluminosilicate glass, lead glass, etc., and 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 can be obtained by, for example, immersing granular foam glass having a water absorption rate of 5 to 20% produced by a conventional method in hot water or an alkaline solution to remove the soluble alkali in the foam glass. It can be produced by eluting the components and silica to create openings in the surface layer and closed cells of the foam glass.

In addition, glass powder contains foaming agents and metal oxides with high melting points,
For example, it can also be produced by adding 5 to 10% of alumina, silica, zirconia, etc., firing it, and then rapidly cooling it to generate fine bubbles.
No. 0, Japanese Patent Application Publication No. 61-6141 and Patent Application No. 1988-154
(Refer to each publication No. 099).

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. The dispersion medium for suspending yeast is
Any material can be used as long as it does not cause food hygiene problems and does not adversely affect yeast activity.

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 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. Normally, the contact time is about 1 to 2 days depending on the yeast concentration of the dispersion medium, and even if the contact time is longer than that, the amount of immobilized yeast often does not increase (finally immobilized yeast The amount of yeast is mainly determined by the characteristic values of the foam glass used (pore volume value, median pore diameter value (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, there are two methods: immersing the granular foam glass in a yeast suspension and leaving it to stand or stirring; or filling a cylindrical column with granular foam glass and pouring the yeast suspension into this. There are methods such as circulation (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, in a reaction tank containing immobilized yeast granules as a fixed bed, a non-fixed bed, or a fluidized bed,
There is a method in which fermentation is carried out by supplying the brewing raw material liquid in a batch manner, and a method in which fermentation is carried out by passing the brewing raw material liquid continuously (once or several times). For details on these methods, see the so-called bioreactor method in "Enzyme Engineering" (edited by Sambe Fukui, Part Chibata, and Shu Suzuki (Tokyo Kagaku Dojin), D. Wlllams, D.M.
Munneeke: r31otech, and
Reference may be made to, for example, M. Nloeng, 231813-25 (1981). 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, is required. /v% or more (however, W is the dry weight U (g) of the yeast contained in the immobilized yeast filled in the reaction tank, and V is the brewing raw material in the case of a batch system) The volume of the liquid (ml), or in the case of a continuous method, the volume of the reaction tank filled with the immobilized yeast (ml).) The fermentation conditions and other conditions are as follows when implementing this application example of the present invention.
The present invention is essentially the same as that known in the art, with some necessary modifications.

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 intended to specifically explain the present invention.

Example 1 (Manufacture of foamed glass peas) Powdered glass obtained by 24-hour crushing of soda-lime glass bottles was granulated by adding 29.6% of CaCO3 as a foaming agent, and granulated at a temperature of 820 to 850°C. 100
Baked for ~150 seconds. The foam glass thus obtained was immersed in hot water at 70° C. for 40 hours to produce highly porous granular foam glass.

Table 1 shows the properties of the foam glass thus obtained.

Honkawa method: Based on mercury intrusion method. "Pore Sipe 9310" sold by Shimabara Seisakusho Co., Ltd. (Micromer L, USA)
ics) Usage Example 2 (Immobilization of yeast) Brewer's yeast (Saccharomyces cerevisiae) was added to wort whose sugar content was adjusted to 11'P at a concentration of 6.0 x 108 eel.
The mixture was suspended at Is/ml. The foamed glass peas produced in Example 1 were soaked in this at a ratio of about 15% (volume of foamed glass peas/volume of wort), and the mixture was heated at 8°C for about 48 hours.
Yeast was immobilized by batch stirring at 20 rpm. Table 2 shows the number of immobilized yeast and the detachment rate.

Book 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 something.

Number of yeast immobilized on one immobilized yeast: eells Volume of foam glass used as an immobilization carrier (including the volume of voids where water is not absorbed within 1 minute when immersed in water at normal pressure) ): ml Surface area of the foam glass used as an immobilization carrier (Surface area when foam glass is not considered a porous material, but is assumed to have a flat surface.For example, if foam glass is spherical with a diameter of rein. Sometimes 4πr2cI112):cj Weight of foam glass used as immobilization carrier; g *4 The desorption rate was determined by the following method. 50
Place a cylindrical basket (with a square mesh of 1.5 m + e on each side) with an inner diameter of 33 mm and a height of 95 cm in a 0 m1 beaker, so that the center of the basket coincides with the center of the beaker.
It was installed so that the distance between the bottom of the basket and the bottom of the beaker was 20111. In this basket, put 15 pieces of granular immobilized yeast produced as above, and in the beaker, put 40 pieces of water.
0 (ml) and stirred for 48 hours at a stirring rotation speed of 70 rpm using a stirrer bar with a length of 5 CI+.

The number of yeast immobilized on one immobilized yeast before stirring is X
(cells/1 immobilized yeast), the number of yeast immobilized on one immobilized yeast after stirring is Y(calls
/ immobilized yeast), the detachment rate Z (%) is Z (%
)-(1-Y/X)X100.

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) 200ml of wort that has been adjusted to a sugar content of 11.0P is
The mixture was placed in a 0 ml measuring cylinder, 30 ml of 2 was added to the immobilized yeast beads obtained in Example 2, and the mixture was left standing at 8° C. for 50 hours to perform batch beer brewing. Table 3 shows the properties of the beer obtained.

Example 4 (Production of alcoholic beverages) Example 2 was placed in a cylindrical column with an inner diameter of 3.5 cm and a height of 30 cso.
The immobilized yeast beads No. 3 obtained in the above were filled almost to the same volume. This column has a sugar content of 11.0'P.
The wort adjusted to
Beer was continuously brewed.

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 over 10 or more sips.

Example 5 (Manufacture of foam glass peas) 3% Ca COa was added as a foaming agent to hard glass (borosilicate glass) crushed to an average particle size of 22.0μ, and the average particle size was 22.0μ.
．． The granules were granulated to a size of 00 to 2.38 m+*φ and fired at 870° C. for 200 seconds to produce granular foam glass.

Regarding this granular foam glass, patent application No. 60-154099
Alkali treatment was carried out according to the invention. That is, 120
A 3% aqueous solution of NaOH (120 °C) in an autoclave
℃) for 4 hours, it was possible to obtain highly porous foam glass having the following physical properties.

Table 5 Example 6 of foam glass properties (immobilization of bacteria) Yeast extract 1.0 (W/V)%, sucrose TO,
Zymomonas mobilis (Zya+ol!lonasmob) was added to a liquid medium (pH 6-7) consisting of O (w/v)%.
ilis: ATCC10988) at a concentration of 168x 10
It was suspended to a concentration of 6 cells/ml. The foam glass peas produced in Example 5 were immersed in this at a ratio of about 5% (W/V) and cultured stationary at 30°C for 24 hours to immobilize Zymomonas mobilis.

Table 6 shows the number of immobilized Zymomonas mobilis bacteria.

Claims (1)

[Claims] 1. A carrier for holding microorganisms, which is made of highly porous foam glass having a water absorption rate of 50% or more. 2. Highly porous foam glass has a central pore diameter (volume) of 1.0
The carrier for holding microorganisms according to claim 1, which has a pore volume of 1.0 to 5.0 ml/g. 3. Any one of claims 1 to 2, wherein the highly porous foam glass is granular with a diameter of 0.5 to 15 ml.
Microorganism retention carrier described in Section 1. 4. An immobilized microorganism, characterized in that the microorganism is immobilized on a microorganism holding carrier made of highly porous foam glass having a water absorption rate of 50% or more. 5. The immobilized microorganism according to claim 4, wherein the microorganism is yeast. 6. The number of yeast per unit surface area is 10^6 cells/c
The immobilized microorganism according to claim 5, which has a particle size of m^2 or more. 7. The immobilized microorganism according to any one of claims 5 to 6, which is granular with a diameter of 1.5 to 15 mm and has a yeast detachment rate of 1% or less. 8. The immobilized microorganism according to claim 4, wherein the microorganism is Zymomonas mobilis. 9. Highly porous foam glass has a median pore diameter (volume) of 0.4
~50μ and a pore volume of 0.5 to 5.0 ml/g, any one of claims 4 to 8
Immobilized microorganisms described in section. 10. The immobilized microorganism according to any one of claims 4 to 9, wherein the highly porous foam glass is granular with a diameter of 0.5 to 15 mm.