JPH03127989A - Immobilized microbial cell - Google Patents

Abstract

PURPOSE:To remarkably increase the permeability of oxygen into a gel by including and immobilizing microbial cells with a gelatinizable polysaccharide in the form of thin filamentous gel. CONSTITUTION:The objective immobilized microbial cell containing microbial cells in a state included and immobilized in a gel is a thin filamentous gel having a diameter of <=0.5mm and derived from microbial cells and a gelatinizable polysaccharide. The polysaccharide is preferably alginic acid, its salt. kappa-carrageenan, etc. The gel can be formed e.g. by contacting a liquid mixture containing the microbial cells and alginic acid or its salt with a coagulation liquid containing polyvalent metal ion such as calcium ion, cooling a mixed liquid containing microorganisms and kappa-carrageenan, contacting the above mixed liquid with ions such as ammonium ion or potassium ion and an amine or contacting with a watermiscible organic solvent such as ethanol or acetone.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to microorganisms that have been comprehensively immobilized by gels that have the property of gelatinization, and further relates to microorganisms that have been immobilized entrappingly from gels that have the property of gelatinization. A microbial cell immobilized product entrappingly immobilized by a filamentous alginate gel obtained by contacting the containing liquid mixture with a coagulation liquid, and a κ-carrageenan obtained by cooling a mixed liquid of microbial cells and κ-carrageenan. The present invention relates to immobilized microbial cells entrappingly immobilized with a gel.The present invention also relates to a method for producing microbial products using these immobilized microbial cells.

[Conventional technology]

In recent years, it has become common practice to immobilize microorganisms, enzymes, etc. on various carriers and utilize them in food production, pharmaceutical production, wastewater treatment, and the like. For immobilization of enzymes, carrier binding method is used to immobilize microorganisms,
For immobilization of animal and plant cells, multi-I! Comprehensive immobilization methods are often used.

The method of comprehensively immobilizing microbial cells with alginate gel or carrageenan gel is known as an excellent method that allows immobilization in a short time under mild processing conditions. Normally, when entrapping and immobilizing microbial cells with alginate gel, the microbial cells are suspended in an aqueous solution of alginate and then inserted into a coagulating solution containing polyvalent metal ions such as calcium ions using a syringe, etc. A method is used in which a gel is formed into a spherical or thread-like gel by dropping it using a gel, and conventionally, particles with a particle size of about 2 m [11 to 5 M] have been obtained.

In addition, when entrapping and immobilizing microbial cells in a κ-carrageenan gel, a suspension of cough microorganisms in an aqueous solution of carrageenan is cooled, or ammonium ions, potassium ions, calcium ions, , contact with ions such as magnesium ions, iron (III) ions, or amines, or contact with water-miscible organic solvents such as ethanol or acetone, and form it into a spherical or thread-like gel like alginate gel. It is taken.

Regarding the production of alginic acid fibers using an alginic acid aqueous solution and metal ions, there is, for example, Japanese Patent Application Laid-open No. 5,616,9809 (fibers for food processing), which is a method for making alginic acid fibers for processed foods in which an alginic acid aqueous solution and metal ions are reacted under high shear force. It is a fruit-bulp-shaped fiber that does not contain immobilized microorganisms, and the fiber diameter is not specified.

There is a report on the immobilization of microorganisms in the form of filaments using alginate gel, such as JP-A-57-163484 (invertase immobilization method). Although there is a description of an example in which it is extruded into an aqueous solution and immobilized in the form of a thread, the diameter is not specified.
Furthermore, it is not intended to improve oxygen permeability.

Further, in Japanese Patent Publication No. 1-27720 (Method for producing alginate fiber paper containing enzymes and/or microorganisms), an alginic acid aqueous solution containing enzymes or microorganisms is spun into a coagulating liquid from a nozzle with a diameter of 0.5 mm or less, -Although the degree is considered to be a fibrous gel, the fiber length /
Relating to a method for producing fiber paper for the purpose of increasing the specific surface area, with the essential condition of cutting the gel to shorten the fibers so that the fiber diameter is 150 (in the example, the length is 3 mm) It is not intended to improve oxygen permeability.

[Problem to be solved by the invention]

One of the problems when immobilizing microbial cells using a polysaccharide gel that has a gelling property is the oxygen permeability of the gel. That is, the area near the surface of the gel is aerobic, but the inside is anaerobic. Therefore, it is generally believed that when aerobic microorganisms are immobilized in a polysaccharide gel that has a gelling property, the bacterial cells inside the gel die rapidly due to lack of oxygen. On the other hand, several research examples of bioreactors utilizing the properties of this gel have been reported. For example, 11°Ta
naka, llKurosawa+ tl, Mura
kami et al.'s rEthanolProduction
from 5tarch by a Cojmmobi
lizedMixed Culture System
of Aspergillus awamorian
d Zymomonas mobilis...Biot
echnoloHy and Bi.

engineering+ 28:1761-1768
('86) J., H., Kurosawa.

H, [shikawa+ H, Tanaka et al.'L-
Lactic Ac1d Pro-duction f
ron+ 5tarch by Coimmobili
zed MixedCulture System
of Aspergi mouth us awamori and
Streptococcus Iactis・-Bio
Technology and Bi.

engineering+ 31:183-187('
88) J, etc., but when aerobic bacteria and anaerobic bacteria are immobilized at the same time, the aerobic bacteria Asp, aw
amori contains anaerobic bacteria Zym and mo on the gel surface layer.
This is a research report showing that Bilis, St, and Iactis are immobilized inside the gel and function.

However, when aerobic microorganisms are comprehensively immobilized or microorganisms that perform specific functions under aerobic conditions are immobilized, the oxygen permeability of the gel is improved and sufficient oxygen is supplied to the microorganisms. There is no doubt that if this technology is successful, the applications of immobilized biocatalysts will expand and they will greatly contribute to the production of useful substances by microorganisms. It also greatly contributes to the immobilization of animal and plant cells.

The present inventors have developed a multi-IJ film with conventional gelling properties.
! ? The present invention was completed as a result of intensive research to solve the problem of oxygen deficiency inside the gel of immobilized microorganisms immobilized by i-gel, and its purpose is to By using a polysaccharide gel, preferably an alginate gel, or a carrageenan gel, the "oxygen permeability" to the inside of the gel is significantly increased, and the cell activity is equivalent to that of non-immobilized cells (Free Cell). The purpose of the present invention is to provide an immobilized microbial cell material.

The term "oxygen permeability" is hereinafter used to mean "the property of allowing oxygen to pass through to the inside of an object."

[Means to solve the problem]

The present inventors have already discovered the above-mentioned 'L-Lactic ac
idProduction from 5tarch
by Coi+nmobilized MixedCu
lture System of Aspergill
us atvamori and Streptococcus
In the report on ``Cus Iactis,'' when aerobic bacteria and anaerobic bacteria were immobilized simultaneously on alginate gel beads, aerobic bacteria were placed near the surface of the gel, and anaerobic bacteria were placed inside the gel, depending on the difference in oxygen demand. It has been confirmed that so-called ``separation'' will take place.

It has also been confirmed from microscopic photographs of gel beads that the immobilization of aerobic bacteria is limited to about 0.3 degrees from the surface layer. Based on these findings, the present inventors have repeatedly investigated the shape of immobilized bacterial cells.

The present invention provides multi-tJ that has the property of gelling with microbial cells.
! It has been found that this product is obtained by immobilizing microbial cells in the form of thin filaments with a diameter of 0.5 mm or less, has excellent oxygen permeability into the gel interior, and can be used for fermentation production. Further, the present invention provides a gel obtained by contacting a mixed solution containing microbial cells and alginic acid or a salt thereof with a coagulation solution containing polyvalent metal ions such as calcium ions, which has a diameter of 0.5+ nm or less. This is an immobilized microbial cell in which the microbial cells contained in the thin filamentous gel are encircled and immobilized within the gel, and the immobilized bacterial cells are formed into a thin filamentous body with a diameter of 0.5 mm or less. This shows that the oxygen permeability of the gel is significantly improved.

In addition, the present invention cools a mixed solution containing microbial cells and κ-carrageenan, or cools it or cools the mixture containing ions such as ammonium ions, potassium ions, calcium ions, sinus ions, magnesium ions, iron (I[[) ions, etc.] and amines. A microorganism in which the microbial cells are entrappingly immobilized in the gel, and the gel obtained by contacting with a water-miscible organic solvent such as ethanol or acetone is a thin filamentous gel with a diameter of 0.5 mm or less. A bacterial cell immobilized product,
Since the immobilized bacterial cells are formed into thin filaments with a diameter of 0.5111ff+ or less, the gel exhibits an extremely excellent oxygen permeability effect.

Furthermore, the present invention provides a method for producing bacterial cells, which comprises bringing the immobilized microbial cells described above into contact with a culture liquid and maintaining them under aerobic conditions to produce bacterial products in the culture liquid. It's in the way things are produced.

In the present invention, polysaccharides with gelling properties used for entrapping immobilization of bacterial cells include alginic acid and its salts, carrageenan, agar, pectin, konjac powder,
Chitosan etc. are applied.

These are normally used in concentrations of 1-5%.

The microorganism to be immobilized may be any aerobic microorganism such as yeast, bacteria, or mold.

The immobilization method is not limited, but it is possible to use polyimide, which has the property of forming a gel with microbial cells. A desirable method is to extrude a mixture containing the above substances into a coagulating liquid through a thin nozzle or injection needle to form a gel.

In the present invention, the diameter of the immobilized filamentous gel must be 0.5 mm or less. If it exceeds 0.5 mm, sufficient oxygen permeability to the interior cannot be ensured, and the proportion of microorganisms that die will rapidly increase.

The immobilized bacterial cells of the present invention can be used repeatedly when placed in a culture tank, or can be filled into a column and a culture solution passed therethrough to produce substances.

Since the gel of the immobilized microbial cells of the present invention has good oxygen permeability, when used for material production under aerobic conditions, the material productivity is comparable to that of non-immobilized microbial cells (Free Ce11). It has the outstanding advantage of showing .

Furthermore, although the immobilized filamentous gel of the present invention has an extremely small diameter, it also has the advantage of being easier to prepare than granular gel.

〔Effect of the invention〕

According to the present invention, a microbial cell immobilized product entrappingly immobilized with a polysaccharide gel having gelling properties with excellent oxygen permeability into the gel interior, and a microbial cell product efficiently produced using the microbial cell immobilized material. We were able to provide a method for better production.

〔Example〕

Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples.

Example I Using 100 volumes of MR3 medium, S. 1actis 5ub
sp.

diacetylactis strain 3022 at 30°C
Incubate for 20 hours.

Centrifuge the main culture solution (5000 rpm, 4°C915
Collect bacteria using a sodium alginate solution (
Sodium alginate concentration 2.5%). The MR3 medium contains 10i peptone, 10g meat extract, 5g yeast extract, 20g glucose, and Tween 801. Om
l, K2HPO42,Og, CHzCOONa・3
11zO5g, ammonium citrate 2g 1Mg5
On 4tlz0200mg, MnSO4・4H,0
Mix 50mg+ and distilled water 10100O, pH 6,
0 to 6.5 and sterilized at 120°C for 15 minutes.

An immobilized filamentous gel having a diameter of about 0.2 ml was prepared by spinning a mixed solution of sodium alginate and 3022 strain of bacterial cells into an O.1M calcium chloride solution using a syringe needle. Thread-like gels with small diameters were easier to prepare than granular gels.

Transfer 10all of this immobilized filamentous gel to 10ml of fresh MR8 medium.
0d and shaken at 30°C (1255 trokes).
/5in) diacetyl was produced. In addition,
As a control, an MR3 culture solution containing an amount of bacterial cells equivalent to 10 d of immobilized gel was collected by centrifugation and used as non-immobilized bacterial cells (Free Cell).

This immobilized filamentous gel and non-immobilized bacterial cells (Free Ce1
Figure 1 shows the amount of diacetyl produced by 1). From this result, oxygen permeability was improved by forming a thin filamentous gel, and when cultured with shaking, non-immobilized bacterial cells (Free
It can be confirmed that the same amount of diacetyl as Ce11) is produced.

In addition, 5treptococcu used in this example
slactiSSubsp, diacetylacti
The present inventors have already demonstrated that when cultured with shaking (i.e., aerobic culture), the diacetyl production activity and amount of diacetyl produced by the S. 3022 strain are significantly higher than when cultured statically. There is (Kaneko Tsutomu r)
Strep-tococcus Iactis 5ub
"About the diacetyl-producing ability of the sp.
It has been deposited as No. 08 (FERM P-11008) and can be easily accessed by a third party.

(Comparative example) Here, S, ]actis 5u used in Example 1
bsp.

diacetylactis strain 3022 was comprehensively immobilized with alginate gel using a conventional method, and using the obtained immobilized bead-like gel with a particle size of approximately 3 mm, a diacetyl production test was conducted using non-immobilized bacterial cells (Free Ce1l) as a control. I did it.

Bacterial cells prepared in the same manner as in Example 1 are mixed with a sodium alginate solution (sodium alginate concentration 2.5%).

This mixed solution was added dropwise to a 0.1M calcium chloride solution to obtain a fixed bead-like gel having a particle size of about 311II11.

This immobilized bead-shaped gel is added to fresh MR3 medium 100 ml.
d and shaken at 30°C (1255 trokes/
m1n) The amount of diacetyl produced was measured. As a control, an MR3 culture solution containing an amount of bacterial cells corresponding to LO ml of immobilized beaded gel was collected by centrifugation, and this was used as non-immobilized bacterial cells (Free Cell).

Immobilized bead-shaped gel and non-immobilized bacterial cells (Free Ce1
As shown in FIG. 2, there was a large difference in the amount of diacetyl produced by 1).

Despite having the same amount of bacterial cells, the amount of diacetyl produced by the immobilized bead-like gel is higher than that of non-immobilized bacterial cells (
It is clear that there is a problem with the oxygen permeability of the bead-like gel since it is only about 1/2 to 173 of Free Ce11).

Example 2 Bacterial cells prepared in the same manner as in Example 1 were mixed with a κ-carrageenan solution (ni-carrageenan concentration: 3.4%).

Inject this mixture into 2% potassium chloride using a syringe needle. A filamentous gel having a diameter of approximately 0.3 M was prepared by spinning into 8 liquids. Using 10 ml of this fixed filamentous gel, diacetyl was added once in the same manner as in Example 1.

The amount of diacetyl produced after 6 hours of culture was 20 mg/l: MR
3 medium, and non-immobilized bacterial cells (Fr
ee Ce1l) at 24 mg/f; the production amount was approximately the same as that of the MR3 medium.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the amount of diacetyl produced by immobilized filamentous gel and non-immobilized bacterial cells by shaking culture. FIG. 2 is a graph showing the amount of diacetyl produced by immobilized bead-like gel and non-immobilized bacterial cells by shaking culture.

Claims (1)

[Claims] 1. A gel obtained from a polysaccharide that has the property of gelling with microbial cells is a thin filamentous gel with a diameter of 0.5 mm or less, in which microbial cells are encircled and immobilized. Immobilized microbial cells. 2. The gel obtained by contacting a mixed solution containing microbial cells and alginic acid or its salt with a coagulating solution containing polyvalent metal ions such as calcium ions is a thin filamentous gel with a diameter of 0.5 mm or less. A microbial cell immobilized product in which microbial cells consisting of are comprehensively immobilized in a gel. 3. Cooling the liquid mixture containing microbial cells and κ-carrageenan, or contacting it with ions such as ammonium ions and potassium ions, or amines, or contacting it with a water-miscible organic solvent such as ethanol and acetone. The gel obtained by this method is a thin filamentous gel with a diameter of 0.5 mm or less, and the microbial cells are entrappingly immobilized within the gel. 4. Bringing the immobilized microbial cell according to any one of claims 1, 2, and 3 into contact with a culture solution and maintaining it under aerobic conditions to produce a microbial product in the culture solution. A method for producing a bacterial cell product characterized by: