JPH0376578A - Immobilized yeast fungal cell - Google Patents

Abstract

PURPOSE:To enable obtaining of a sufficient strength and stability of an enzyme by immobilizing Sporobolomyces singularis ATCC24193 strain using gelatin treated with a protease and glutaraldehyde. CONSTITUTION:A wet strain of Sporobolomyces singularis ATCC24193, cultured in a culture medium containing lactose and recovered from a culture solution is mixed with gelatin treated by adding an endo type protease, such as trypsin or chymotrypsin, to an aqueous solution of gelatin originating from skin or bone of cattle, etc., and glutaraldehyde is then added and mixed therewith to immobilize the aforementioned strain. Post-treatments, such as drying, granulating and pulverizing, are then successively carried out to afford immobilized fungal cells. In order to obtain a galactooligosaccharide therewith, e.g. the resultant immobilized microbial cells are filled in a column of a suitable size and a raw material solution may be passed therethrough. Thereby, since enzymic activity is stable for a long period with a sufficient strength, the galactooligosaccharide can be continuously produced.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to immobilized yeast cells that can be used in the production of Bifidobacterium growth promoters and the like.

(Prior art) By immobilizing bacterial cells or enzymes used as reaction catalysts, these expensive catalysts can be efficiently reused in reactions without complicated recovery operations, and can be produced continuously. Development of immobilized microbial cells or enzymes is progressing.

On the other hand, Sporobolomyces singularis ATCC24
Strain 193 is used for the production of galactooligosaccharide, which is a growth factor for bifidobacteria, which are useful bacteria in the intestines (Japanese Patent Application Laid-Open No. 63-18
5373). For this reason.

Sporobolomyces singicolaris ATCC24193
If the bacterial strain can be immobilized, galactooligosaccharides can be produced efficiently and continuously.

Conventional methods for immobilizing bacterial cells and enzymes include diazo method, peptide method, alkylation method, carrier crosslinking method using silica particles and glutaraldehyde, ionic bonding method, crosslinking method using glutaraldehyde, and entrapment method using polyacrylamide (
"Immobilized Enzyme" Chibata Part, Kodansha Scientific)
etc. are known.

(Problem to be solved by the invention) However, Sporobolomyces singularis^T
Even if the CC24193 strain is immobilized by the above-mentioned immobilization method, the resulting immobilized cells are weak and turn into fine particles during continuous reactions, making it difficult to pass the raw material solution through, and also impairing enzyme activity. In this respect, the stability was not sufficient.

The present invention solves the above-mentioned drawbacks of the conventional technology, and the technical problem is to provide an immobilized yeast cell having sufficient strength and excellent enzyme stability. It is.

(Means for Solving the Problems) As a result of intensive studies to solve such problems, the present inventors discovered that Sporobolomyces singularis ATC
The inventors have discovered that by immobilizing C24193 with protease-treated gelatin and glutaraldehyde, immobilized yeast cells having sufficient strength and excellent enzyme stability can be obtained, and have thus arrived at the present invention.

That is, one aspect of the present invention is to use Sporobolomyces singularis.
is) An immobilized yeast cell obtained by immobilizing ATCC24193 strain with protease-treated gelatin and glutaraldehyde.

The immobilized yeast cells of the present invention are Sporobolomyces singularis.
aris) ATCC24193 strain was immobilized with protease-treated gelatin and glutaraldehyde.

The yeast cells used in the present invention are Sporobolomyces singularis ATCC24193, and the method for obtaining the above cells is not limited in any way.
For example, after culturing in a medium containing lactose or using glucose, sorbitol, maltose, sucrose, blackstrap molasses, etc. as a carbon source to sufficiently proliferate the bacterial cells, lactose is added, and the culture continues to produce β-galactosidase. After sufficient induction of the bacterial cells, cells can be obtained by commonly used methods such as centrifugation and filtration.

In the composition of the medium containing lactose, 0.1 as lactose
~30% by weight, preferably 5-20% by weight, is added to the nitrogen source 9 used for the culture, such as organic nitrogen sources such as peptone, casein, cornstarch liquor, meat extract, yeast extract, ammonium sulfate, ammonium chloride diurea, etc. 0.01-1 as an inorganic nitrogen source.

% by weight, preferably 0.5 to 5% by weight, of the phosphate compound 1 used as a mineral source, such as potassium phosphate 1st salt (KH, PO,), sodium phosphate 2nd salt hydrate (N . But that's fine.

In addition, the culture may be carried out using a commonly used liquid medium or solid medium, and may be either stationary culture, 1 aerated agitation culture, or shaking culture. The appropriate culture temperature and culture time are usually 20 to 28°C for 15 to 120 hours.

Thereafter, the bacterial cells recovered from the culture solution by conventional methods such as centrifugation and filtration may be immobilized by the following immobilization method.

That is, Sporobolomyces singularis AT[C
After mixing the wet mass of 24193 with gelatin treated with protease, 9 glutaraldehyde is added and mixed.

For example, in the present invention, gelatin can be treated with protease. This can be done by adding protease to an aqueous gelatin solution.

The gelatin used in the present invention includes, for example, those purified from the skin, bones, and cows of cows, pigs, and whales, and in general, many products made from cow skin and bones are commercially available. There are no particular limitations on its origin or degree of purification. The concentration of 6-gelatin in this aqueous gelatin solution is, for example, 5% (ill/V)
or more is appropriate, preferably 15% (W/V) or more,
Particularly preferred is 25 to 35% (W/V). The amount of protease to be added is 2, for example, 1 to 80 PUN (measured by the modified Anson-Hagiwara method, that is, the enzyme activity that shows coloration corresponding to IT tyrosine in 1 minute at 30°C is defined as 1 unit; It is called I PUN.)/100m
ff1 is suitable, and 5-50 PUN/100 ml is preferred. What is the reaction time at this time?

If the gelatin concentration is high and the amount of protease is low, the length will be long; if the gelatin concentration is low and the amount of protease is high, it will be short; and if the reaction temperature is high, it will be short, and if the reaction temperature is low, it will be long. /V), the amount of protease is 20PUN/100ml, and the temperature is 50℃.
When it is, 10 to 50 minutes is preferable.

Proteases used in the present invention include:

Any generally well-known endotype protease may be used, including well-known proteases such as trypsin, chymotrypsin, and pepsin.

In the present invention, the amount of protease-treated gelatin added is 0.1 to 0.5 (vt/
Wet bacterial cells wt) is appropriate, preferably 0.1 to 0.3 (wt/wet bacterial cells wt), and the amount of glutaraldehyde added is 0.02 (wt/wet bacterial cells) relative to wet bacterial cells. Wet bacterial cells wt) or higher is suitable, and 0.04 to 0.06 (
wt/wet bacterial cells wt) is preferred. At this time, if the amount of gelatin is small, the 6 particles are likely to be atomized, and if the amount of gelatin is large, the volume per unit becomes large and the activity per unit volume becomes low, which is not preferable. Furthermore, if the amount of glutaraldehyde is small, sufficient crosslinking will not occur.

This is not preferred because the resulting immobilized bacterial cells tend to become brittle.

The mixing time at this time is suitably 60 minutes or more, preferably 5 to 30 minutes. Also, the temperature when mixing should be a temperature that does not deactivate the enzyme.

That is, a temperature of 55°C or lower is appropriate, and a temperature of 10 to 40°C is preferable.

As for the processing after fixation in this way.

There are no particular limitations, and for example, drying, granulation, and pulverization may be performed in sequence.

Next, in order to obtain galactooligosaccharide using the obtained immobilized bacterial cells, for example, the immobilized bacterial cells obtained above may be packed into a column of an appropriate size, and the raw material solution may be passed therethrough.

(Example) Next, the present invention will be specifically explained using examples.

Reference Example 1 A culture medium having the following composition was placed in a 30f jar fermentor and sterilized.

Lactose 4000g Yeast extract 60g KH2P0. 20g M, S O < ・7Hz0 10g Vitamin B+ 60mg Vitamin B [160m
g Water 201 Next, Sporobolomyces singulari was precultured at 28°C for 96 hours in the same culture medium.
s) Inoculate ATCC24193 strain 11, pH 3,
57, culture was carried out for 96 hours at a temperature of 28° C., an aeration rate of 201/min, and an impeller rotation speed of 40 Or, p, m.

After completion of the culture, centrifugation was performed using a centrifuge model LAPX 202 manufactured by α-Laval to obtain 2.3 kg of wet bacterial cells.

Example 1 To 430 g of gelatin powder (manufactured by Ishizu Pharmaceutical Co., Ltd., 89138.Chemical Pure, originating from cow skin and bone), 107 g of water was added.
0107O was added and dissolved by heating, and the temperature was kept at 55°C. To this, 575 mg of protease 5P-10 (manufactured by Nagase Seikagaku Kogyo ■) was added, and after reacting for 30 minutes,
By boiling for a minute to inactivate the protease, 1.50 kg of protease-treated gelatin was obtained.

Add 1.32 kg of this protease-treated gelatin to 5.5 kg of wet bacterial cells obtained in Reference Example 1 at room temperature, mix thoroughly until the mixture becomes homogeneous, and then add 1.38 J of 25% glutaraldehyde. Mixed for 20 minutes at room temperature. The obtained mixture was processed using a granulator (manufactured by Fuji Baudal Co., Ltd., PBLL).
BTBRB
After pulverizing with MILL K II W-1), fine powder was removed by decanting with water to obtain 71 volumes of immobilized bacterial cells.

Next, the immobilized bacterial cells obtained in Example 1 were placed in a column with a diameter of 15 cm! (40cmH) and increase the column temperature to 50~
The temperature was adjusted to 53°C, and 30% lactose was passed through the flask at a flow rate of 11/hr to perform a galactooligosaccharide synthesis reaction.

This reaction was carried out continuously for 6 months, but the immobilized bacterial cells
The raw material solution could be passed through smoothly without becoming atomized.

The ratio of trisaccharides (galactooligosaccharides) to total sugars is used as an indicator of activity, and the initial ratio of trisaccharides to total sugars is 10
When the half-life of residual activity was determined to be 0, it was found to be stable at 870 days.

For comparison, immobilized bacterial cells were obtained in the same manner as above, except that gelatin treated with protease 1 was not added, and then the immobilized bacterial cells were packed in a column and a reaction was performed in the same manner as above. The immobilized bacterial particles were micronized after 3 days had passed after one infusion.

It caused a blockage in my eyes.

(Effects of the Invention) The immobilized yeast cells of the present invention have stable enzyme activity over a long period of time and have sufficient strength, so that they do not become microparticles, do not inhibit the passage of the raw material solution, and can be used for a long period of time. It becomes possible to continuously produce galactoolicosaccharides over a period of time.

Claims (1)

[Claims] (1) Sporobolomyces singularis
) Immobilized yeast cells obtained by immobilizing ATCC24193 strain with protease-treated gelatin and glutaraldehyde.