JPS62283988A - Production of maltooligosaccharide - Google Patents

Abstract

PURPOSE:To obtain the titled oligosaccharide in high yield at high temperature free from fear of sundry bacterial contamination without using any neutralizing reagents, by letting a heat- and acid-resistant alpha-amylase to act on polysaccharides having alpha-1,4glucoside bonding under specific conditions. CONSTITUTION:Polysaccharides having alpha-1,4glucoside bonding (preferably starch, amylose, amylopectin and partially decomposed material thereof) are subjected to action of a heat- and acid-resistant alpha-amylase (preferably alpha-amylase produced by bacteria belonging to the genus clostridium) at high temperature under acidic condition at <=100muM calcium concentration required for improvement of heat resistance, preferably at 2-6 pH and 70-90 deg.C within 20-25% range of decomposition ratio to obtain the aimed maltooligosaccharide-containing liquid mainly containing maltotriose and maltotetraose.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method for producing a malto-oligosaccharide solution containing maltotriose and maltotetraose as main components from starch.

[Conventional technology]

Recently, there has been a demand for the use of so-called oligosaccharides such as maltotriose, maltohexaose, and maltopentaose in the production of foods and medicines. However, the manufacturing technology has not yet been established, and urgent development is required.

In addition, as those related to oligosaccharide manufacturing technology, Japanese Patent Publications No. 59-4119, Japanese Patent Publication No. 59-37955, Japanese Patent Publication No. 59-37957, Japanese Patent Publication No. 60-188065, etc. can be mentioned.

[Problem that the invention seeks to solve]

The oligosaccharide-producing amylase used in the above-mentioned conventional technology is Bacillus sp.

Special Publication No. 59-37955, Special Publication No. 59-37957, Special Publication No. 59-4119) and Pseudomonas bacteria (
Pseudmonassp, JP-A-60-18806), etc. Since the optimal temperature for all of these enzymes is 60°C or lower, the enzymatic reaction for hydrolyzing starch into oligosaccharides is carried out at 60°C or lower. However, in this temperature range, bacteria may proliferate during the reaction, which may significantly reduce the quality of the product. For this reason, it is desirable to carry out the enzyme reaction at a high temperature range of 70° C. or higher, where normal bacteria cannot grow.

In addition, the optimal pH of these oligosaccharide-producing amylases
is in the neutral range of 6 or more, and decreases significantly in the acidic range. By the way, when starch is liquefied, it is usually made into a slurry with a concentration in the range of 10 to 40%, but the p of the slurry is
H is 5 or less, rarely 4 or less due to the organic acid contained in the raw starch.

For this reason, the pH is usually adjusted to 6 with slaked lime, calcium carbonate, etc.
The reaction is carried out after being neutralized as described above. As a result, not only the neutralizing agent is consumed, but also the load on the subsequent desalting process by the ion exchange tower increases.

Therefore, it is desirable to use acidic amylase whose optimum pH is in the range of 4 to 6 to carry out the enzymatic reaction without neutralizing the starch slurry.

On the other hand, in order to increase the thermal stability of enzymes, many enzymes require 0.5 to 20 mM of calcium ions (Japanese Patent Publication No. 59-37957). These enzymes are produced in water or tap water that does not contain calcium ions (usually with a calcium concentration of 100%).
(μM or less), the enzyme will be deactivated during the reaction and the expensive enzyme will be consumed. For this reason, a soluble calcium salt such as calcium chloride is usually added and reacted. However, as mentioned above, the addition of calcium salts
When obtaining the final oligosaccharide product, the load of the desalting process using the ion exchange tower increases. Therefore, enzymatic reactions using oligosaccharide-producing amylases that do not require the addition of calcium salts for stabilization are desirable.

The purpose of the present invention is to reduce the burden on the desalting process by adding chemicals for neutralizing raw materials and adding Ca ions for thermal stabilization of enzymes, and to conduct enzyme reactions at high temperatures without the risk of bacterial contamination. It is an object of the present invention to provide a method for producing maltooligosaccharides, which can be carried out and produce maltooligosaccharides at a high yield.

[Means for solving problems]

In order to achieve the above object, the present inventors searched for enzyme-producing microorganisms with the aim of obtaining an oligosaccharide-producing amylase that has excellent heat resistance, low calcium requirement, and high activity even in acidic conditions. I did it. As a result, thermophilic anaerobic bacteria belonging to the Clostridium genus (Clostridium genus R5-0001, Clostridium
ium sp, R3-0001+Feikoken Bacteria No. 791
No. 8) was found to produce oligosaccharide-producing α-amylase. Then, using this, α-1,4
As a result of extensive research into a method for producing oligosaccharides from polysaccharides having glycosidic bonds, the present invention was achieved.

The feature of the present invention is to use a novel oligosaccharide-producing heat-resistant and acid-resistant α-amylase to generate 200% of polysaccharides having α-1,4 glucosidic bonds under calcium concentration of 100 μm or less, high temperature, and acidic conditions. Hydrolyzes with a degradation rate of ~25%,
The objective is to produce an oligosaccharide-containing liquid. In particular, in hydrolysis, by adjusting the decomposition rate to 20-25%, maltotriose and maltotetraose are mainly used.
An oligosaccharide-containing liquid with low glucose and maltose contents can be obtained. Note that the decomposition rate here indicates the ratio of the amount of reducing sugar to the total amount of sugar in the reaction solution,
More details later.

Multiple $Js with α-1,4 glycosidic bonds that can be used in the present invention! The class is not particularly limited, and can be applied to, for example, starch, amylose, amylopectin, or partially decomposed products thereof obtained from potatoes, Japanese sesame seeds, corn, wheat, etc.

The addition of calcium salts to improve the heat resistance of enzymes
This is not necessary unless purified water such as distilled water or demineralized water is used as the charging water, and reagent-grade raw materials that have been completely decalcified are used. Therefore, if ordinary tap water is used, a subsequent desalination step for removing cations is not necessary.

When hydrolyzing, pH adjustment is not required unless the raw material is a partially decomposed product prepared by performing partial decomposition treatment under a neutral pH range or under strongly acidic conditions of pH 3.5 or less. is not necessary. This is because raw starch contains organic acids as impurities, so the
A 40% starch solution exhibits acidity with a pH of 5 or less. Therefore, even without pH adjustment, the optimum pH of α-amylase can be adjusted.
It falls within the range of H3.5 to 6.0.

The reaction temperature for hydrolysis may be any high temperature that does not cause contamination with bacteria, and considering the reaction rate and heat resistance of the enzyme used in the present invention, it is preferable to carry out the reaction at a temperature in the range of 70 to 90°C, particularly 80°C. preferable.

The amount of enzyme used in the hydrolysis reaction depends on the type of raw material polysaccharide in the reaction tank, residence time, reaction temperature, and pH.
etc., but if the processing time is fixed at 1 hour,
80°C, pF14. In order to hydrolyze potato starch to a decomposition rate of 25% at 100 ml, approximately 106 units are required per 1 kg of starch. Here, the unit of α-amylase activity is based on the Blue Value method, which will be explained in detail later.

The α-amylase-producing bacteria used in the present invention include bacteria belonging to the genus Clostridium (C1stridium).
ium sp R5-0001) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (Accession number: FERM P-7918).

First, the details of the mycological properties of this bacterium will be explained.

A. Morphological properties (1) Morphology of vegetative cells on agar plates in starch/peptone medium as described below.

When cultured for 2 days at 60°C in an anaerobic atmosphere, the vegetative cells are linear rods with a size of 0.4 to 0.8 x 2 to 5 μm. When cultured for 3 days or more, vegetative cells with the above-mentioned shape exist singly, and vegetative cells in chains also occur. The same holds true for liquid culture.

Composition of starch/peptone medium Soluble starch 1.5% peptone
0.5% yeast extract
0.5%KH2PO4.0.7% NaJPO40.35% Mg5Oa ・71.0 0.00
1% agar 2.0% Sodium thioglycolate 0.1% Tap water pH 6.4 (2) Spore formation Spore formation is observed in agar plate culture and liquid culture in starch/peptone medium.

B. Culture characteristics (1) Colony morphology Colonies in agar plate culture on starch/peptone medium have a flat circular shape with a slightly raised center and a golden edge at the periphery. No pigment formation is observed, and the surface is milky and opaque with gloss. It also has adhesive properties.

(2) Grows in agar plate culture and puncture culture in broth medium, producing colonies similar to those in starch/peptone medium.

Meat juice agar medium composition Meat extract 1.0% peptone
1.0% salt 0.2% sodium thioglycolate 0.1% agar
1.5% Distilled water pH 6.0 (3) Puncture culture of meat broth culture Grows with the generation of gas containing hydrogen and carbon dioxide, and as a result, the agar medium is divided into two or three places.

(4) Meat juice liquid culture It grows only in an anaerobic atmosphere, and the culture solution becomes cloudy.

Composition of meat juice medium Meat extract 1.0% peptone
1.0% salt - 0.2% sodium thioglycolate 0.1% distilled water pH 6.0 (5) No growth of meat juice/gelatin culture was observed.

Composition of meat juice/gelatin medium Meat extract 1.0% peptone
1.0% Salt 0.2% Gelatin 15% Sodium thioglycolate 0.1% Distilled water pH 6.0 (6) Lidomus Milk Culture Coagulates solidly with gas generation and turns red due to acid production.

C6 Physiological properties (1) Growth temperature range: Grows at 40°C to 63°C. No growth was observed at 30°C, but good at around 60°C.

(2) pH range for growth pH around 5-7°5.6 is good.

(3) Obligate anaerobic attitude towards oxygen (4) O-F test (modified method of Hugh La1fson) No growth was observed in air atmosphere, negative. Bacteria grow under the anaerobic conditions of the liquid paraffin overlay, producing acid and turning the culture solution yellow.

Medium Composition Peptone 0.2% Glucose
1.0% salt 0.5% hHPOt 0.0
3% sodium thioglycolate 0.1% bromcresol purple 0.002% agar
0.3% Distilled water pH 6.0 (5) Nitrate reduction negative.

(6) VP test negative.

(7) MR test Positive, turns red.

(8) Indole production It cannot be measured because it does not grow in peptone water.

(9) Generation of hydrogen sulfide Negative (in using Kligrer's medium).

(10) Starch hydrolysis positive. It breaks down not only soluble starch but also granular starch, including potato starch.

(11) Use of citric acid Negative (using Simmons medium).

(12) Use of ammonium salt It cannot be measured because it does not grow in peptone water.

(13) Extracellular production of pigment negative.

(14) Urease negative.

(15) Oxidase activity negative.

(16) Catalase activity negative.

(17) Assimilation of VA The table below shows the observation results of the assimilation of sugar and the presence or absence of gas generation using a Durham tube.

(Margins below this page) Chapter 1 Table (18) Growth on mineral salt medium Growth was not recognized.

(19) Generation of organic acids Table 2 shows the composition of organic acids produced from various media.

Composition of test liquid medium Carbon source 1.0% peptone
1.0% salt 0.2% sodium thioglycolate 0.1% distilled water pH 6.4 From these results, based on Holdeman's anaerobic bacteria classification manual, it was identified as a bacterium belonging to the genus Clostridium.

Next, the enzymatic properties of α-amylase for oligosaccharide production used in the present invention will be described.

The α-amylase activity was measured as follows.

Blue value method (Chemical Society of Japan wit: Experimental Chemistry Course Volume 24, Biochemistry ■, p279, Maruzen Shoten, 196
The glue refining power was measured according to 9). The starch-iodine compoex is produced as starch molecules are hydrolyzed.
This is based on the principle that the amount of blue color produced decreases in proportion to the decrease in molecular weight. First, 2d of 2 mg/ml starch solution and 0.1 M < citrate buffer (pH 4
,0)1+nZ was placed in a test tube and shaken in a 60°C water bath for 5 minutes. Next, culture filtrate 1rn1 was added as a crude enzyme solution and reacted for 30 minutes. After the reaction, 0.4-ml of the reaction solution was collected and immediately mixed with 2 d of 0.5 M acetic acid solution to stop the enzyme reaction. Next, 1- is 1/3 of 10m1
It was added to a 00ON iodine solution, and the absorbance at 680fII11 was measured using a spectrophotometer. On the other hand, the reaction solution immediately after adding the enzyme solution was sampled, colored in the same manner, and the absorbance was measured. Note that amylose with a degree of polymerization of about 2000 was used as starch.

α-amylase activity was calculated using the following formula.

α-amylase activity (unit) = (1) Preparation method Clostridium bacteria RS-0001 was inoculated into a liquid medium containing starch peptone and yeast extract, and cultured at 60°C for 1 to 3 days under anaerobic conditions. do. A so-called culture filtrate is obtained by removing insoluble substances such as bacterial cells from the culture solution by centrifugation or the like. In the main culture filtrate, 30 to 80 units/- of α-amylase are usually produced outside the bacterial cells, although differences can be seen depending on the culture conditions. Next, impurities were removed from the culture filtrate using appropriately known methods such as molecular sieve membrane filtration, salting out, ion exchange chromatography, and gel filtration chromatography, to obtain oligosaccharide-producing α-amylase.

(2) Optimum pH As shown in Figure 2, α-amylase used in the present invention
(curve 11) and α-amylase ■ (curve 12)
), the optimum pH range at 60°C is around 4, and the preferred pi (is 2-5, 7.2-6.3, respectively).
It has even higher activity on the acidic side than conventional acidic α-amylases. That is, at pH 2,
While conventional acidic α-amylase shows no activity at all, the α-amylase of the present invention exhibits 95% and 81% activity, respectively.
Shows high activity.

In addition, the following reaction system was used for the enzyme reaction.

Oxygen solution: 0.6 to 1.3 μg/ml Substrate: amylose 1/rnI Citrate buffer: 0.025 M As mentioned above, the α-amylase of the present invention is
- It is clear that it is a new α-amylase because its pH range of action is different from that of amylase.

(3) pH stability α-amylase I and ■ used in the present invention were
, 6, and 7 (0,025 M citrate buffer) at 60° C. for 30 minutes.

The reaction solution was diluted and adjusted to pH 4.0, and residual activity was measured using amylose as a substrate. As a result, the activity of both α-amylases was completely retained by the above pH treatment.

Therefore, the present α-amylase is characterized by high stability even in an acidic region.

(4) Optimal temperature As shown in Figure 3, α-amylase of the present invention (curve 11
) and (curve 12), the optimal temperatures at the optimal pH of 4 and 0 are both around 80°C. The preferred temperature (temperature range that has 80% of the activity at the optimum temperature) is 65-8
It is 7℃. In addition, citric acid buffer 0.025 was used for the reaction.
M was used.

(5) Thermostability α-amylase used in the present invention
Heat treatment was performed at 60 to 97°C in the presence of M calcium chloride, and residual activity was measured. Based on this, the activity half-life at each temperature was determined, and the results are shown in FIG. The half-life of 3tllI activity at 80°C and 90°C (no substrate added) is 8 hours and 0.5 hours, respectively, indicating excellent thermal stability. α-Amylase I also has an activity half-life of about 0.5 hours at 90°C, and has the same heat resistance as α-amylase II.

(6) Influence of metal salts on heat resistance Table 3 shows the influence of metal salts on the heat resistance of α-amylase ① used in the present invention. Various metal salts were added to an aqueous solution of α-amylase H at a concentration of 5mM, heat-treated, and the activity was measured. The activity after the heat treatment, that is, the residual activity, was expressed in % compared to before the heat treatment.

Heat treatment and activity measurement were performed under the following conditions.

Heat treatment conditions p H6,0 Heating temperature = 80℃ Holding time: 30 minutes Table 3 Activity measurement was performed under the following conditions after diluting the sample solution.

It has been confirmed that the addition of each metal salt at this concentration does not affect the activity measurement.

Activity measurement conditions pH 4,0 (0,025M < citrate buffer) Activity measurement temperature: 60°C From Table 3, it is clear that calcium ions have a protective effect, whereas sodium, potassium, and magnesium ions have a protective effect. , no significant protective effect was observed. On the other hand, nickel, cobalt, zinc, and manganese ions reduce heat resistance. It has also been confirmed that this α-amylase loses its thermostability when exposed to 0.5 μM EDTA.

As shown in curve 12 in Figure 5, the calcium requirement concentration required for thermostabilization of this α-amylase is 100 pM (4 ppm) to maintain 100% activity, and furthermore, at a calcium concentration of 1 μM or less, It also retains 65% activity. Therefore, this enzyme is sufficiently stabilized by the calcium concentration in tap water. Furthermore, α-amylase (2) also has a calcium requirement similar to that of α-amylase (2).

On the other hand, maltotriose-producing amylase of Bacillus subtilis requires 0.5 to 20 mM calcium (Japanese Patent Publication No. 37957/1983).

Therefore, the present amylase has significantly lower calcium requirement than conventionally known oligosaccharide-producing amylases.

(8) Molecular weight Although the molecular weight of the α-amylase of the present invention is unconfirmed, its behavior in Molecuie sieve membrane filtration indicates that the molecular weight is 20,
It is estimated to be over 000.

As is clear from the above, the oligosaccharide-producing α-amylase used in the present invention is significantly different from conventionally known oligosaccharide-producing amylases, particularly in terms of thermostability, operating pH, and calcium requirement.

In addition, the decomposition rate when hydrolyzing a polysaccharide having an α-1,4 glucosidic bond was calculated as follows. First, the total amount of sugar in the enzyme reaction solution was measured using the phenol/sulfuric acid method (separate volume, Proteins, Nucleic Acids, Enzymes, Biochemistry Experimental Methods XI, PTS, 1939).
Calculated based on maltose (October 2013, Kyoritsu Shuppan). Next, the amount of reducing sugar in the same solution was determined using the Nelson-Somogyi method (
Nelson-5o...ogyi, Reprint/Protein/Nucleic Acid/Enzyme, Biochemical Experimental Methods X1. pts, published in October 1962, Kyoritsu Publishing) based on maltose standards. The decomposition rate was expressed as a percentage of the amount of reducing sugar relative to the total amount of sugar.

〔Example〕

Hereinafter, examples of the present invention will be shown and explained in more detail.

Example 1 Soluble starch 1.5%, polypeptone 0.5%, yeast extract 0.5%, monopotassium phosphate 0.7%, dibasic phosphate
Soda 0.35%, Magnesium sulfate heptahydrate 0.0
15.75 kg of a liquid medium (pH 6,0) containing 1% sodium thiogelcholate, 0.1% sodium thiogelcholate, and tap water.
Dispense 3.15 kg each into 5 culture tanks with an internal volume of 51, and
Sterilize for 20 minutes at 20°C. To each test, 350 g of a bacterial cell suspension of the genus Clostridia isolated by the present inventors, which had been anaerobically cultured in the same medium as above, was added.

Next, a water seal trap is attached to the gas outlet, and after the gas phase inside the fermentation tank is sufficiently replaced with argon gas, the culture is carried out under anaerobic conditions. The pH of the culture solution is automatically adjusted to 6.0, and the temperature is also automatically adjusted to 60°C. After 22 hours of incubation, the cultures were combined at 6,0
Centrifuge at 00 rpm to remove bacterial cells.

This supernatant showed a specific activity of 60 units/g.

Next, after cooling 14.6 kg of the above supernatant liquid to 2°C,
300 g of cornstarch and 300 g of Celite (Wako Pure Chemical Industries, Ltd.) were added and brought into contact with each other for 10 minutes while stirring.Then, the cornstarch and Celite were collected by vacuum filtration, and further washed with pure water 31 cooled to 2°C.

Next, 0.05M Tris, hydrochloric acid buffer (p
The above cornstarch and celite were dispersed in the above buffer solution (65°C) and kept at 65°C on a water bath for 5 minutes, followed by solid-liquid separation by vacuum filtration.
℃), and the filtrate and washing solution were combined to obtain 41. Next, the filtrate was cooled to 2°C, filtered and concentrated through a molecular sieve membrane (molecular weight cut off: 20°000), and insoluble matter was further removed by filtration to obtain a concentrated liquid 400-. This concentrate showed an α-amylase activity of 1.5×10 3 units/−.

Next, the above concentrate was applied to diethylaminoethylated cross-linked agarose gel (DEAE Sepharose CL-6B).

It was purified by ion exchange chromatography (column size: 50 mm x 210 mm) using a column (manufactured by Pharmacia).

First, the above concentrated solution was precipitated twice with 0.05M IJS hydrochloric acid buffer (pH 7.5), and then insoluble materials were filtered and charged into a gel column buffered with the same buffer.
Washed. The sodium chloride concentration in the buffer was then developed in an increasing linear gradient. As a result, two peaks having α-amylase activity were observed at positions of 0.04M and 0.08M in sodium chloride concentration. 0.04M
The amount of activity of α-amylase I eluted at the position is 1
About 30% of the total activity, α-eluted at the 0.08M position.
The active fraction of amylase Ⅰ was approximately 60%, which was dialyzed in pure water and then lyophilized to obtain α-amylase I and α-amylase ②. The activity recovery rates based on the centrifuged supernatant of the culture solution were 17% and 32%, respectively. Next, amylose was hydrolyzed using α-amylase ① obtained above.

50 μm of amylose (EX-1 manufactured by Hayashibara Biochemical Research Institute) was dissolved in tap water by heating, and 1M acetic acid solution was added dropwise to adjust the pH to 4.
．． After adjusting to 0, the total amount was set to 3-. Next, dissolve 500 units of α-amylase in 2 ml of tap water, and add 1M acetic acid to the solution.
Adjusted to H4.0. Next, both of the above solutions were combined and reacted at 80° C. with shaking. A portion of the reaction solution was sampled at predetermined intervals, and the total amount of sugar and reduced IJ in the reaction solution were measured. The amount was measured. Furthermore, a part of the collected reaction solution was developed using a thin layer chromatography method using microcrystalline cellulose.
-Propertool/n-butanol/pyridine/water = 32
/14/31/23), the polysaccharide portion was scraped off, and the sugars contained in the cellulose were extracted and quantified by the phenol/sulfuric acid method. As a result, as shown in Figure 1, the hydrolysis reaction progresses rapidly until the decomposition rate reaches approximately 20%, and then
Decomposition of 0% or more became gradual. The main components of the hydrolyzate were maltotriose and maltotetraose. Of these, maltotetraose has a decomposition rate of 25%.
It gradually decreased after exceeding .

On the other hand, glucose and maltose increased in proportion to the reaction time from the start of the reaction. Therefore, maltotetraose is decomposed into glucose or maltose, and if the decomposition rate exceeds 25%, the amount of decomposition is considered to be greater than the amount of production.

The above results indicate that the present α-amylase is an oligosaccharide-producing α-amylase, and its main products are maltotriose and maltotetraose.

Furthermore, it is shown that in order for the reaction to proceed rapidly and to obtain maltotetraose in a high yield, the decomposition rate should be set to 20 to 25%.

Example 2 20 g of potato starch, 80 ml of tap water. After adding 10,000 units of α-amylase II prepared in Example 1, the container was immersed in a water bath at 80° C. under stirring to carry out a hydrolysis reaction. A portion of the reaction solution was appropriately sampled and the decomposition rate and sugar composition were examined using the method of Example 1.

As a result, the decomposition rate was 23% at 29 hours, and the sugar composition at that time was 35% maltotriose, 17% maltotetraose, % glycose, and 6% maltose.

Example 3 In a 200 ml autoclave equipped with a stirrer, 20 g of corn starch and 80 ml of tap water were added. and 12,500 units of α-amylase were added, and while stirring, steam at a gauge pressure of 2 kg/cal in the gas phase in the autoclave was first blown into the autoclave for 10 seconds with the exhaust valve open, then the exhaust valve was closed and the steam was blown into the cornstarch slurry. and increase the internal pressure to 1.1
kg/c++1 for 1 hour to carry out a liquefaction reaction. Then, after cooling to 80°C, α-amylase ns,
Further ooo units were added and hydrolyzed at 80°C. As a result, the decomposition rate was 21% after 10 hours, and the sugar composition at that time was 34% maltotriose and 1% maltotetraose.
5%, glucose 7%, and maltose 7%.

Example 4 In Example 2, a hydrolysis reaction was carried out in the same manner as in Example 2, using 20 g of amylopectin (manufactured by Wako Pure Chemical Industries, Ltd.) instead of potato starch. As a result, the decomposition rate was 2 at 25 hours.
1%, and the sugar composition at that point was 33% maltotriose, 18% maltotetraose, and 8% glucose.
%, maltose 6%.

Example 5 Tap water and 2,500 units of α-amylase II were added to 20 g of glutinous rice powder that had been milled and ground, and the mixture was heated to 80° C. under stirring.
It was held for 0 minutes. After immediately cooling to 50°C, 10,000 units of isoamylase (manufactured by Hayashibara Biochemical Research Institute, originating from Pseudomonas bacteria) was added, and the mixture was reacted at 50°C for 2 hours. Next, 5000 units of α-amylase I was further added, and the mixture was further hydrolyzed at 80°C. the result,
After 18 hours, the decomposition rate was 22%, and the sugar composition was 37% maltotriose, 18% maltotetraose, 6% glucose, and 5% maltose.

Example 6 α-amylase II 10' units were dissolved in water for 200 min.
m/closed. In addition, microporous glass particles having aminopropyl groups on the surface [Electronucreonics (El
Aminopropyl-CPG (manufactured by electro-Nucleonics Inc.) 20fIi was added thereto, and glutaraldehyde was further added to give a concentration of 0.25%, followed by contact at 20° C. for 5 hours with stirring. Next, the glass particles were taken out and washed with water to obtain immobilized α-amylase (2).

α-amylase activity of 6.5×10′ units was observed in 20 ml of the present immobilized particles.

Next, the immobilized particles were packed into a jacketed glass column measuring φ16×200 m. The immobilized particles were heated by passing constant temperature water at 80° C. into the jacket part.

A liquefied corn starch liquid 200-
Next, 20,000 units of the same isoamylase used in Example 5 was added to the liquefied solution and reacted at 50°C for 2 hours to prepare a dextrin solution.

The dextrin solution was passed through the column from above at a rate of 5 d/h to carry out a hydrolysis reaction. 24 hours after the start of liquid passage, the decomposition rate of the reaction solution flowing out from the bottom of the column was examined and found to be 24%. Also, the sugar composition is 38% maltotriose.

It contained 20% maltotetraose, 8% glucose, and 6% maltose.

Comparative Example 1 Bacillus subtilis (Bacillus 5ubti)
Amylose was hydrolyzed using the same method as in the example using maltotriose-producing α-amylase produced by lis. As a result, the hydrolysis reaction hardly progressed (decomposition rate 2%), and the amount of maltotriose in the reaction solution was 1% or less based on the raw material amylose. This revealed that the enzyme derived from Bacillus subtilis cannot withstand use at high temperatures.

Note that Table 4 shown below is a comparison table that summarizes the results of the above Examples and Comparative Examples.

〔Effect of the invention〕

According to the present invention, the oligosaccharide-producing enzyme reaction can be carried out without worrying about bacterial contamination at high temperatures, where the reaction could not be carried out with conventional oligosaccharide-producing amylase, and calcium addition and neutralization can be carried out. Since there is no need to add a chemical, the burden of the process of desalting the oligosaccharide-containing liquid can be reduced. Furthermore, by carrying out the hydrolysis for 8 cycles at a decomposition rate of 20 to 25%, there is no reduction in maltotetraose due to decomposition, so that malto-oligosaccharides can be obtained in high yield.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the relationship between oligosaccharide composition and decomposition rate in the oligosaccharide production reaction of one example of the present invention, and FIGS. 2, 3, 4, and 5 are used in the present invention. FIG. 2 is a diagram showing the characteristics of oligosaccharide-producing α-amylase. 1... Decomposition rate, 2... Maltotriose, 3...
Maltotetraose, 4...glucose, 5...maltose, 11...α-amylase I, 12...α
-Amylase ■. Patent applicant Hitachi, Ltd. Representative Patent attorney Yusuke Hiraki Reaction time (h) 1: Decomposition rate 4 Nigelcose 2: Maltotriose 5: Maltose 3: Maltotetraose Figure 2 Figure 3 Temperature ('C )

Claims (1)

[Claims] 1. In producing maltooligosaccharides mainly consisting of maltotriose and maltotetraose, heat-resistant and acid-resistant α-amylase is added to polysaccharides having α-1.4 glucosidic bonds to improve heat resistance. 1. A method for producing a maltooligosaccharide-containing solution, which comprises operating the solution at a decomposition rate of 20 to 25% under high temperature and acidic conditions at a required calcium concentration of 100 μM or less. 2. The α-amylase is an α-amylase produced by a bacterium belonging to the genus Clostridium,
A method for producing a maltooligosaccharide-containing liquid according to claim 1. 3. The method for producing a maltooligosaccharide-containing liquid according to claim 1, wherein the polysaccharide having an α-1.4 glucosidic bond is starch, amylose, amylopectin, or a partially decomposed product thereof. . 4. A method for producing an oligosaccharide-containing liquid according to any one of claims 1 to 3, characterized in that the reaction is carried out at a pH of 2 to 6 and a temperature of 70 to 90°C.