JP2774594B2 - Immobilized yeast cells - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an immobilized yeast cell which can be used for producing a growth promoter for Bifidobacterium and the like.

(Prior art) By immobilizing cells or enzymes used as a catalyst for the reaction, these expensive catalysts can be efficiently reused in the reaction without complicated recovery operations, and can be produced continuously. Therefore, the development of immobilized cells or enzymes has been promoted.

On the other hand, Sporoboromyces singularis ATCC 24193 strain can be used for the production of galacto-oligosaccharide which is a growth factor of bifidobacterium, a useful intestinal bacterium (JP-A-63-185373). For this reason, if sporoboromyces singularis ATCC 24193 can be immobilized, galacto-oligosaccharides can be produced efficiently and continuously.

Conventional methods for immobilizing cells and enzymes include the diazo method,
Peptide method, alkylation method, carrier cross-linking method using silica particles and glutaraldehyde, ionic bonding method, cross-linking method using glutaraldehyde, inclusive method using polyacrylamide ("Immobilized enzyme" Ichiro Chibatake, Kodansha Scientific) are known. ing.

(Problems to be solved by the invention) However, Sporoboromyces singularis ATCC
Even when the 24193 strain is immobilized by the above-mentioned immobilization method, the immobilized cells obtained have a low strength, become micronized in a continuous reaction, making it difficult to pass the raw material solution, and in terms of enzyme activity. But its stability was not enough.

An object of the present invention is to solve the above-mentioned drawbacks of the prior art, and 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 have found that Sporoboromyces singularis ATCC 2
The present inventors have found that immobilization of 4193 with protease-treated gelatin and glutaraldehyde can provide immobilized yeast cells having sufficient strength and excellent enzyme stability, and have reached the present invention.

That is, the gist of the present invention is an immobilized yeast cell obtained by immobilizing Sporobolomyces singularis ATCC 24193 with protease-treated gelatin and glutaraldehyde.

The immobilized yeast cells of the present invention are obtained by immobilizing Sporobolomyces singularis ATCC 24193 with protease-treated gelatin and glutaraldehyde.

The yeast cell used in the present invention is Sporoboromyces singularis ATCC 24193, and the method for obtaining the above-mentioned cells is not limited at all.
Culture in a medium containing lactose, or grow the cells sufficiently using glucose, sorbitol, maltose, sucrose, molasses, etc. as a carbon source, add lactose, continue culturing, and obtain sufficient β-galactosidase. After induction, the cells can be obtained by commonly used methods such as centrifugation and filtration.

In the composition of the medium containing lactose, 0.1% lactose
3030% by weight, preferably 5-20% by weight, of a nitrogen source used for the culture, for example, an organic nitrogen source such as peptone, casein, cornstay liquor, meat extract, yeast extract and the like, and ammonium sulfate, ammonium chloride, urea and the like. 0.
01 to 10 wt%, preferably 0.5 to 5 wt%, phosphoric acid compound used as a mineral source, such as potassium phosphate first salt (KH ₂ PO ₄₎ sodium phosphate second hydrate (N _a2 HPO
₄ · 12H ₂ O) as a 0.05 to 5% by weight, preferably 0.1 to 1
% By weight, and vitamins and the like may be added as necessary.

The culture method may be any of stationary culture, aeration-agitation culture, and shake culture in a commonly used liquid medium or solid medium. The culturing temperature and culturing time are usually appropriate at 20 to 28 ° C for 15 to 120 hours.

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

That is, Sporoboromyces singularis ATCC 241
After mixing the 93 wet strains with the protease-treated gelatin, glutaraldehyde is added and mixed.

To treat gelatin with a protease in the present invention, for example, protease may be added to an aqueous gelatin solution.

Examples of the gelatin used in the present invention include bovine,
Pigs, whales and other skins, bones, tendons, etc. are used as raw materials and refined. In general, many are derived from bovine hides and bones. It is not limited. The gelatin concentration in this aqueous gelatin solution is suitably, for example, 5% (W / V) or more, preferably 15% (W / V) or more, and more preferably 25 to 35%.
% (W / V) is preferred. The amount of protease to be added is, for example, 1 to 80 PUN (measured by the modified Anson-Hagiwara method, that is, an enzyme activity that exhibits a color corresponding to 1γ of tyrosine per minute at 30 ° C. for 1 minute is defined as 1 unit. 1P
It is called UN. ) / 100 ml is suitable, and 5-50 PUN / 100 ml is preferred. At this time, the reaction time was high when the gelatin concentration was high.
When the amount of protease is small, it tends to be longer, the gelatin concentration is low, and when the amount of protease is large, it tends to be short. When the reaction temperature is high, it tends to be short, and when it is low, it tends to be long. Protease amount is 20PUN / 100ml
When the temperature is 50 ° C., it is preferably 10 to 50 minutes.

The protease used in the present invention may be any protease that is generally known as an endo-type protease, such as trypsin,
Well-known proteases such as chymotrypsin and pepsin are exemplified.

In the present invention, an appropriate amount of the protease-treated gelatin to be added is 0.1 to 0.5 (wt / wet cell wt), preferably 0.1 to 0.3 (wt / wet cell wt), based on the wet cells. The amount of glutaraldehyde to be added is suitably 0.02 (wt / wet cell wt) or more based on the wet cell.
04-0.06 (wt / wet cell wt) is preferred. At this time, if the amount of gelatin is small, the particles are likely to be finely divided, while if the amount of gelatin is large, the volume is increased, and the activity per unit volume is undesirably reduced. Also, if the amount of glutaraldehyde is small, it is not preferable because crosslinking is not sufficiently performed and the obtained immobilized cells tend to become brittle.

An appropriate mixing time at this time is 60 minutes or less.
5 to 30 minutes are preferred. The temperature at which the mixture is mixed is suitably a temperature at which the enzyme is not inactivated, that is, 55 ° C or lower, and preferably 10 to 40 ° C.

The treatment after immobilization in this way is not particularly limited. For example, drying, granulation, and pulverization may be performed sequentially.

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

(Examples) Next, the present invention will be specifically described with reference to examples.

Reference Example 1 A medium having the following composition was placed in a 30-liter jar fermenter and sterilized.

Lactose 4000g yeast extract _{60g KH 2 PO 4 20g M g} SO 4 · 7H 2 O 10g vitamin B ₁ 60 mg vitamin B ₆ 60 mg water 20l then Suporoboromisesu-Shingyurasu were precultured 96 hrs at 28 ℃ in a medium having the same composition ( Sporobolomyces singulari
s) Inoculation of ATCC 24193 strain 1, pH 3.57, temperature 28 ° C,
The culture was performed for 96 hours at an aeration rate of 20 l / min and an impeller rotation speed of 400 rpm.

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

Example 1 1070 ml of water was added to 430 g of gelatin powder (89138, manufactured by Ishizu Pharmaceutical Co., Chemical Pure, originating from cow skin and bone), dissolved by heating, and kept at 55 ° C. To this, 575 mg of protease SP-10 (manufactured by Nagase Seikagaku Corporation) was added, reacted for 30 minutes, and then boiled for 10 minutes to inactivate the protease, resulting in 1.50 kg of protease-treated gelatin.
I got

1.32 kg of this protease-treated gelatin was added to 5.5 kg of the wet cells obtained in Reference Example 1 at room temperature, mixed well until almost uniform, then 1.38 l of 25% glutaraldehyde was added and mixed at room temperature for 20 minutes. did. The obtained mixture was granulated with a granulator (Fuji Paudal, PELLETER EXDS-60), air-dried overnight, and crushed (Fuji Paudal, SAM).
After crushing with PLE-MILL KIIW-I), fine powder was removed by a gradient with water to obtain 7 liters of immobilized cells.

Next, 7 l (40 cm H) of the immobilized cells obtained in Example 1 were packed in a 15 cm diameter column, the column temperature was adjusted to 50 to 53 ° C., and 30% lactose was passed through at a flow rate of 7 l / hr. And a galactooligosaccharide synthesis reaction was performed.

This reaction was performed continuously for 6 months, but the immobilized cells were not micronized and the raw material solution could be passed smoothly.

The activity of the trisaccharide (galacto-oligosaccharide) in the total sugar is used as an indicator of the activity.
When the half-life of the residual activity was calculated at 0, it was stable at 870 days.

For comparison, immobilized cells were obtained in the same manner as above, except that gelatin treated with protease was not added, and the reaction was performed by packing the immobilized cells in a column as described above. However, three days after the passage, the immobilized bacterial cell particles were made fine and clogged.

(Effect of the Invention) Since the immobilized yeast cells of the present invention have a stable enzyme activity for a long period of time and have sufficient strength, they are not micronized, and the flow of the raw material solution is not hindered. For the production of galactooligosaccharides continuously.

Claims (1)

(57) [Claims] 1. Sporobolomyces singularis (Sporob)
(olomyces singularis) Immobilized yeast cells obtained by fixing ATCC 24193 strain with protease-treated gelatin and glutaraldehyde.