JPS59148794A - Production of high-purity glucooligosaccharide - Google Patents

Abstract

PURPOSE:A mixed solution of saccharides is passed through a column filled with an alkali or alkaline earth metal type strong acidic cationic exchange resin and eluted with water to enable mass production of high-purity oligosaccharides used as reagents, diagnostics and drugs in high yield. CONSTITUTION:A mixed solution containing glucooligosaccharide of 3-10 glucose polymerization degree (GPD) (the objective oiligosaccharide), saccharides of 1 or more larger polymerization degree than the objective (L saccharides) and other sacchaides of 1 or more smaller polymerization degree (S saccharides) is passed through a column filled with a strong-acid cationic exchange resin of alkali metal or alkaline earth metal type. Then, elution is effected with water to fractionate into in order the fraction of high content of the L saccharides, the fraction of high content of the objective oiligosaccharide and the L saccharide, the one of the objective oligosaccharides, the one of the objective and S saccharide and the one of S saccharide. Then, the fraction of the objective saccharide is collected.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing high purity glucooligosaccharide having a glucose polymerization degree selected from the range of 3 to 10.

In recent years, glucooligosaccharides selected from the range of glucose polymerization degrees of 3 to 10 have attracted attention as reagents, diagnostic agents, medicines, etc., and the demand for them is rapidly increasing.

Glucooligosaccharides are contained in partial hydrolysates of starch, pullulan, ercinan, etc., but these partial hydrolysates are usually not only glucooligosaccharides of interest selected from the range of polymerization degree of 3 to 10. It is mixed with a sugar mixture containing a large amount of other oligosaccharides. Therefore, the purity of the target gluco-olibosaccharide is low, and it is extremely difficult to use it as it is for uses such as reagents, diagnostic agents, and medicines.

On the other hand, purification methods such as fractional precipitation using an organic precipitant and selective adsorption/desorption using an activated carbon column are known as methods for collecting the desired glucooligosaccharide with high purity from such a sugar mixture.

There is. However, it is extremely difficult to industrially produce large quantities of highly purified target glucooligosaccharides using these methods.

The present inventors have conducted intensive research on a method for industrially mass-producing a highly pure glucooligosaccharide selected from the range of glucose polymerization degree of 3 to 10.

As a result, when producing a target glucooligosaccharide with a glucose polymerization degree of 3 to 10 (hereinafter referred to as "target oligosaccharide"), saccharides with a glucose polymerization degree of 1 or more (hereinafter referred to as "L-saccharide") can be obtained along with the target oligosaccharide. ) and a saccharide with a glucose polymerization degree of 1 or more (hereinafter referred to as S sugar) - is poured into a column packed with an alkali metal type or alkaline earth metal type strongly acidic cation exchange resin, Then, it was eluted with water, and the L Hodaka-containing fraction, L
Fractionation is performed in the order of sugar/target oligosaccharide-rich fraction, target oligosaccharide-rich fraction, target oligosaccharide/S Hotaka-containing fraction, and S&I-high content fraction, and the target oligosaccharide-rich fraction is collected. It was discovered that the objective oligosaccharide of high purity can be easily produced by this method, and that it is suitable as an industrial production method, and the present invention was completed.

In addition, the cation exchange resin to be used is one with a crosslinking degree of 6% or less when the target oligosaccharide is selected from the range of glucose polymerization degree of 3 to 5, and when the target oligosaccharide is selected from the range of glucose polymerization degree of 6 to 10. found that a crosslinking degree of 4% or less is suitable.

In addition, when flowing the sugar mixture containing the target oligosaccharide through the column, high purity can be achieved by flowing together the already obtained L-saccharide/target oligosaccharide-rich fraction and the target oligosaccharide/S-high content fraction. The present invention was completed based on the finding that the desired oligosaccharide can be stably produced at high concentration and high yield.

The sugar mixture containing the target oligosaccharide used in the present invention (hereinafter referred to as raw sugar solution) has a target oligosaccharide content of about 10 to 80 W% (hereinafter, unless otherwise specified, the content % is , 9 w/w% based on solid matter), and is a sugar solution that does not substantially contain ketose, and according to the present invention, a highly purified target oligosaccharide with a target oligosaccharide content of 90% or more can be obtained. Any material that can be collected with high yield may be used.

When the target oligosaccharide is maltotriose, for example,
If the maltotriose-containing partial hydrolyzate obtained by treating starch, pullulan, ercinan, etc. with an acid or enzyme is maltotetraose, for example, starch can be treated with echinomaltotetraohitolase (EC3, 2,1
, 60) is maltopentaose, for example, fX derived from bacteria belonging to the genus Bacillus is added to starch.
- When the partial aO hydrolysis product containing maltopentaose obtained by the action of amylase (gC3,2,1,1') is maltohexaose, for example, starch,
When the maltohexaose-containing partial hydrolyzate obtained by treating α-cyclodextrin with acid or enzyme is maltoheptaose, for example, starch, β-
A partial hydrolyzate containing maltoheptaose obtained by the action of an acid or an enzyme on cyclodextrin is added to a mixture of α-cyclodextrin and glucose with cyclodextrin glucanotransferase (
When the maltoheptaose-containing sugar mixture obtained by the action of EC2, 4, 1, 19) is maltooctaose, for example, starch, γ-tuclodex l-'
A maltooctaose-containing partial hydrolyzate obtained by reacting acid or an enzyme with a maltooctaose, or a mixture of α-cyclodextrin and maltose or a mixture of β-cyclodextrin and glucose, is combined with maltooctaose-containing glucanotransferase. When the sugar mixture containing maltooctaose obtained by reacting is maltononaose, for example, a mixture of β-cyclodextrin and maltose, or a mixture of When the maltononaose-containing sugar mixture obtained by the action of cyclodextrin glucanotransferase on the mixture is maltodecaose, for example, a mixture of β-cyclotextrin and maltotriose or a mixture of γ-nclodextrin A sugar mixture containing maltodecaose obtained by reacting 7 clodext and ring glucanotransferase on a mixture of maltose and maltose is used.

In addition, the target oligosaccharide is a branch sugar, for example, 3-0-α-
In the case of maltotriosylmaltotriose, a sugar mixture containing 3-0-α-maltotriosylmaltotriose obtained by reacting pullulan with pullulanase (gc 3.2.1.41) is used. .

Also used in the present invention. Alkali metal type or alkaline earth metal type strongly acidic cation exchange resins include alkali metal salt types such as Na+ type and K+ type of styrene-divinylbenzene crosslinked copolymer resins with AI sulfone groups, or Ca type and Mg type. It has been found that one or more types of alkaline earth metal salt types such as the following are used as appropriate, and among them, those with a degree of crosslinking of 6% or less are suitable. The degree of crosslinking in the present invention refers to the weight percentage (%) of divinylbenzene based on the total monomers used when producing a styrene-divinylbenzene crosslinked copolymer. Product name: DOWEX 50W x 1, DOWEX 50W x 2, DOWEX 50
WX4, product name Amberly manufactured by Rohm & Haas)
C()-120, trade name XT-1022E manufactured by Tokyo Organic Chemical Industry Co., Ltd., XT-'1007, trade name Diaion SK IB1 manufactured by Mitsubishi Chemical Industries, Ltd.
Diaion SK 102, Diaion SK 1
04 etc. These resins are not only excellent in fractionating a fraction containing a high content of the target oligosaccharide, but also have excellent heat resistance and abrasion resistance, and are extremely advantageous for mass production of highly purified target oligosaccharides.

In the present invention, resin having a particle size of about 0.01 to 0.65 mm may be used by filling a column. The length of the resin layer filled in the column is preferably 9 m or more in total length, and in this case, even if it is 9 m or more for one column or 9 m or more by connecting two or more columns in series. good. The material and shape of the column can be freely selected as long as the purpose of the present invention can be achieved. That is, the material can be made of glass, plastic, stainless steel, etc., and the shape can be cylindrical, rectangular, etc., so that the liquid passing through the filled resin layer can flow as laminar as possible. can.

Furthermore, the method of implementing the present invention will be described in more detail.

An alkali metal type or alkaline earth metal type strongly acidic cation exchange resin is suspended in water and packed into a column so that the total length of the resin layer is usually 9 m or more. While maintaining the temperature inside this column at 45-85°C, a concentration of about 40-7
Add 0% raw sugar solution to the resin at about 1 to 50 v/v%,
This was eluted by flowing water at a flow rate of SVVO21 to 20 using an ascending or descending method, and the raw sugar solution was used as a fraction containing L.
Fractionation is performed in the order of sugar/target oligosaccharide-rich fraction, target oligosaccharide-rich fraction, target oligosalt/S hotaka-containing fraction, and S hotaka-containing fraction, and the purpose is, ]-1+) high gosaccharide content. All you have to do is collect the fraction.

At this time, the eluate is usually collected at approximately 1% of the resin used.
This is carried out every ~20v/v%, but this may be automated and distributed to the above-mentioned fractions.

In addition, when fractionating the raw sugar solution by flowing it through a column, the already obtained L-saccharide/target oligosaccharide-rich fraction and the target oligosalt/S-high-content fraction can be placed before and after the raw sugar solution, or before and after the raw sugar solution. By flowing it together with the sugar solution, the amount of water required for fractionation can be reduced, and the target oligosalt in the raw sugar solution can be collected with high purity, high concentration, and high recovery rate, which is advantageous.

In general, the sequence is to flow the already obtained L-saccharide/target oligosaccharide-rich fraction, then flow the raw sugar solution, and then flow the target oligosalt/S hotaka-rich fraction that has already been obtained. It is preferable to adopt it.

The target oligosaccharide-rich fraction collected in this way can be used as it is, but if necessary, it can be purified by decolorizing and desalting according to conventional methods, and further, for example, concentrated. It can be made into a sill, but it can also be dried and powdered to collect the powder.

The highly purified target oligosalts produced in this way can be advantageously used not only as reagents, diagnostic agents, and medicines, but also as compounding agents for various foods and drinks, cosmetics, and raw materials for chemicals. can.

Next, the present invention will be explained in detail through experiments.

Actually, Experiment 1 Preparation of raw sugar solution 1-A Maltotriose preparation Kawahara sugar solution A 10% aqueous solution of pullulan (sold by Hayashibara Shoji Co., Ltd., PF grade) was prepared with pullulanase (Hayashibara Biochemical Co., Ltd.) at pH 60 and temperature of 50°C. (Sold by the Institute) was added to pullulangram at a ratio of 5,000 units/1% and reacted for 24 hours. The reaction solution was kept at 95°C for 10 minutes, then cooled and filtered. The resulting P solution was decolorized with activated carbon according to a conventional method, and purified by desalting with H-type and OH-type ion exchange resins. Then, it is further processed to produce a sugar solution with a concentration of 60% with a yield of 8.
Obtained at 3%.

The sugar composition of this sugar solution was 3.9% of saccharides with a degree of glucose polymerization of 2 or less, 717% of maltotriose, and 244% of saccharides with a degree of glucose polymerization of 4 or more.

1-B W for preparing maltopentaose, sugar solution 6% potato starch milk is heated and gelatinized, then adjusted to pH 45 and temperature 50°C, and isoamylase (manufactured by Hayashibara Biochemical Research Institute Co., Ltd.) is added to the starch. It was added at a rate of 2,500 units per duram and reacted for 200 hours. The reaction solution is P
Adjust to H6.0 and autoclave (120℃) for 10
1. minutes, then cooled to 45'C. To this, α-amylase (manufactured by Novo, trade name Termameal 60L) was added at a ratio of 150 units per gram of starch.
A 24-hour reaction was performed. The reaction solution was autoclaved (1
20°C) K: After 20 minutes, the mixture was cooled and purified and concentrated in the same manner as in Experiment 1-A to obtain a 55% concentration sugar solution with a yield of 91%.

The sugar composition of this sugar solution was 47.5% of saccharides with a degree of glucose polymerization of 4 or less, 40.3% of maltopentaose, and 22% of saccharides with a degree of glucose polymerization of 6 or more.

1-C Maltohexaose Preparation Kawahara Sugar Solution 0, IN-Dissolve α-cyclotextrin in sulfuric acid to a concentration of 10%, keep at 100°C for 60 minutes for partial hydrolysis, and then dissolve in sodium hydroxide solution. It was peaceful.

Concentrate this liquid to about 1/3 volume and add 5v/
Experiment 1
-A was purified and concentrated in the same manner as in A to obtain a sugar solution with a concentration of 55% at a yield of 87%.

The sugar composition of this sugar solution (d1 was 31.7% of @s with a degree of polymerization of kunonocose of 5 or less, 60.1% of maltohexaose, and 82% of saccharides with a degree of glucose polymerization of 7 or more).

1-D Raw sugar for maltothecaose preparation (1) Cyclotextrin glucano "Preparation of lancepherase Bacillus stearothermophilus"
stearothermophilus ) F E
I(, M-P Nn 2222 with soluble starch 2 W/V%, nitric acid 7%, =-U A l w
/V X −, rif 1 #z potassium Q, l w/v%
, magnesium sulfate 7 water + K O, 05 w/v%,
The cells were inoculated into 10 t of a sterilized liquid medium consisting of corn staple liquor 〇, 5 W/V% and calcium carbonate IW/v%, and incubated with aeration at 50° C. for 3 days.

The obtained culture solution was centrifuged, and the supernatant was salted out with saturated ammonium sulfate to determine the activity of Schiflobuchis l-IJ glucanotransferase (Pc 2.4.1.19).
A crude enzyme preparation having , ooo units was obtained. One unit of activity herein refers to 0.0000000000000 containing 0.02M acetate buffer and 2 x 10-"M calcium chloride at pH 5.5.
3 Add 2 ml of appropriately diluted enzyme solution OJ to 5 ml of 3 W/W% Norupuru starch solution, react at 40°C for 10 minutes, then take 90.5 ml of the reaction and add 0.02 N
- Mix with 15 mJ of sulfuric acid aqueous solution to stop the reaction, and add 0.0% to this reaction stop solution. IN iodine potassium iodide solution Q
This refers to the amount of enzyme that completely eliminates the coloration of iodine in 15 mg of soluble starch by adding 0.2 rnl to develop color, then measuring the absorbance at 66] nm, and reacting at 40°C for 10 minutes.

(11) Preparation of raw sugar solution Ncrodextrin glucanotransferase prepared by the method (1) above is added to an aqueous solution containing 10% β-cyclodextrin and % maltotriose at a rate of 5 units per cyclodextrinodarum. In addition, PH55,
After reacting at a temperature of 70°C for 6 hours, it was kept at 95°C for 15 minutes, then cooled and filtered.

5 v/v% bromobenzene was added to the obtained Pe, left in a cold room overnight with stirring, and then filtered. The resulting P solution was purified and concentrated in the same manner as in Experiment 1-A. A sugar solution with a concentration of 45% was obtained in a yield of 75%.

The sugar composition of this sugar solution was 719% saccharides with a degree of glucose polymerization of 9 or less, 153% maltodecaose, and 128% saccharides with a degree of glucose polymerization 11 or more.

Experiment 2 Effect of strongly acidic cation exchange resin on fractionation of raw sugar solution Using the raw sugar solution prepared in Experiment 1, the influence of the degree of crosslinking of the strongly acidic cation exchange resin on its fractionation was investigated.

As the strongly acidic cation exchange resin, a commercially available product (Na type) was used after adjusting the average particle size to 0.1 to 0.3 mm, and the types thereof are shown in Table 1.

Table 1 Crosslinking degree and resin used The column was made of jacketed stainless steel and had an inner diameter of 2.2Lyn, and was filled with resin so that the resin layer length was lom. While maintaining the temperature inside the column at 70°C, add a raw sugar solution with a concentration of 40% to the resin at IOV/V%,
Furthermore, hot water at 70° C. was flowed at a flow rate of Svo, 4, and the eluted sugar solution was fractionated and collected in the order of elution.

When the elution of sugar from the column was nearing completion, injection of hot water into the column was stopped, and the previously fractionated sugar solution was added to the column in the order of elution, followed by hot water in the same manner. Repeat this operation 5 times (however, in the case of maltodecaose Kawahara sugar solution, repeat 8 times) to obtain the L-hodaka-containing fraction, L-sugar,
The target oligosaccharide is eluted and fractionated in the following order: the target oligosaccharide high sugar content fraction, the target oligosaccharide high content fraction, the target oligosaccharide/S high content fraction, and the S high content fraction, and the target oligosaccharide contains 90% or more of high purity target oligosaccharide. I collected the mountains and valleys.

The results are shown in Table 2, where the percentage of the target oligosaccharide content in the obtained high purity fraction with respect to the target oligosaccharide content in the raw sugar solution used was defined as the target oligosaccharide recovery rate.

As is clear from the results in Table 2, high-purity target oligosaccharides with a glucose polymerization degree in the range of 3 to 10 can be obtained by eluating and fractionating the raw sugar solution in the above method using a strongly acidic cation exchange resin. It was found that it could be collected with a high recovery rate of over 80%.

Among them, the degree of glucose polymerization of the target oligosaccharide is 3 to 5.
In this case, it is preferable to use a strongly acidic cation exchange resin with a degree of crosslinking of 6% or less, and in cases where the degree of glucose polymerization is 6 to 10, it is preferable to use a strongly acidic cation exchange resin with a degree of crosslinking of 4X or less. found.

Below, we will discuss a few examples of older sisters.

Example 1 The sugar solution with a maltotriose content of 717% prepared in Maltotriose Experiment 1-A was used as a raw sugar solution. The resin used was an alkali metal type strongly acidic cation exchange resin (manufactured by Tokyo Organic Chemical Industry Co., Ltd., trade name XT-1007, Na type, degree of crosslinking 6%), and the resin was placed in a jacketed stainless steel column with an inner diameter of 5.4 crn. The resin layer was filled with a water suspension, and four columns were connected so that the liquid flowed in series, making the total length of the resin layer 20 m.

While maintaining the column temperature at 55°C, add 5% v/v of raw sugar solution to the resin, and add 55°C hot water to it by SV O.
, 13 to collect a maltotriose-rich fraction with a maltotriose content of 90% or more.

The maltotriose recovered in this maltotriose-rich fraction is approximately 83% of the maltotriose in the raw sugar solution.
%Met.

Example 2 Maltotriose First, during the first fractionation, the raw sugar solution was mixed with the resin by 2
Fractionation was carried out in the same manner as in Example 1, except that 0 v/v% was used. The elution pattern of the fractionated product is shown in the figure.

In the figure, A indicates a fraction containing L saccharide, B indicates a fraction containing high L sugar/target oligosaccharide, C indicates a fraction containing high target oligosaccharide, and D indicates a fraction containing target oligosaccharide/S saccharide. E indicates the fraction containing S Hotaka.

The elution order of the fractions was A, BXClD, and E. Among them, fraction C (fraction containing high maltotriose) was collected, and fractions A and E were removed.

For the second and subsequent fractionations, add about 10 v/v% of the raw sugar solution and fraction C to the same column based on the fraction B1 resin, and then add warm water at 55°C as in Example 1. A maltotriose-rich fraction with a maltotriose content of 90% or more was collected.

The fractionation operation from the second time onwards was repeated a total of 20 times and the average result for each time was calculated, and the recovery rate of maltotriose was as high as about 94%.

Actual M Example 3 Maltopentaose Prepared in Experiment 1-B Maltopentaose content 40.3
% sugar solution was used as raw sugar solution. Resin (resin: alkaline earth metal type strong acid cation exchange resin (manufactured by Dow Chemical Company, trade name DOWEX 50 W X 4, Mg type, degree of crosslinking 4%)
), and the total length of the resin layer was 30 mm in the same column as in Example 1.
It was filled so that it became m.

While maintaining the column internal temperature at 75°C, 6.6 V/'V% raw sugar solution was added to the resin, and 75°C hot water was added to the resin.
It was fractionated by flowing at a flow rate of Vo, 13.

The obtained fractionated product was applied to the elution yt column again and fractionated in the same manner, and a maltopentaose-rich fraction with a maltopentaose content of 90% or more was collected. The recovery rate of maltopentaose was about 85%. there were.

Actual example 4. Maltohexaose fruit; Maltohexaose content prepared in Test 1-C: 60.
A 1% sugar solution was used as a raw sugar solution. The resin is an alkali metal type strong acid cation exchange resin (manufactured by Mitsubishi Chemical Industries, Ltd., trade name Diaion SK), 04, K+ type, degree of crosslinking 4%).
The same column as in Example 3 was used.

While maintaining the column internal temperature at 60°C, 7 v/v% raw sugar solution was added to the resin, and 60°C hot water was added to it by SVo,
It was fractionated by flowing at a flow rate of 12, and the maltohexaose content was 9.
A fraction with a high maltohexaose content of 0% or more was collected.

The recovery rate of maltohexaose is approximately 86% based on the raw sugar solution.
%Met.

Example 5 Maltohexaose First, during the first fractionation, the raw sugar solution was mixed with the resin at a ratio of 1
Fractionation was carried out in the same manner as in Example 4, except that 0 v/v% was used. The elution pattern of the fractionated products was similar to that shown in the figure, and the fractions were fractionated in the order of A, B, C, D, and E. Among them, fraction C (fraction containing high maltohexaose) was collected, and fractions A and E were removed.

For the second and subsequent fractionations, the raw sugar solution and fraction C were added to the same column at a concentration of about 6.6 v/v% based on fraction B1 resin, and 60°C hot water was added to Example 4. A maltohexaose-rich fraction having a maltohexaose content of 90% or more was collected in the same manner as above.

The fractionation operation from the second time onwards was repeated a total of 50 times and the average result per time was determined, and the recovery rate of maltohexaose was about 93%.

Example 6 3-O-α-maltotriosylmaltotriose reagent pullulan (sold by Hayashibara Biochemical Research Institute Co., Ltd.)
% aqueous solution, and at a pH of 6.0 and a temperature of 45° C., pullulanase (sold by Hayashibara Biochemical Research Institute) was added at a ratio of 2,500 units per pullulan gram, and the mixture was allowed to react for 9 hours. The reaction solution was kept at 95°C for 10 minutes and then cooled, methanol was added to it to a concentration of 80 v/v%, left for two nights, filtered, and the resulting R solution was concentrated under reduced pressure to a concentration of 50%. A raw sugar for preparing 3-0-α-maltotriblastyl maltotriose was obtained.

The sugar composition of this sugar solution is 444% saccharides with a glucose polymerization degree of 5 or less, 257% 3-〇-α-maltotriosylmaltotriose, and 299% saccharides with a glucose polymerization degree of 7 or more.
%Met.

The resin is an alkali metal type strongly acidic cation exchange resin (manufactured by Daucumical Co., Ltd., trade name Dauenox 50WX2, N
Type a, crosslinking degree 2%) was used and packed in the same column as in Example 1.

While maintaining the temperature inside the column at 60°C, add 5v/v% raw sugar solution to the resin, and add 60°C warm water to it at 5V-0.
It was fractionated by flowing at a flow rate of 2.

The obtained fractionated products were applied to the column again in the order of elution and fractionated in the same manner to obtain a high 3-0-α-maltotriosylmaltotriose content of -tc+o% or more. Fractions were collected.

The recovery rate of 3-0-α-maltotriotulmaltotriose was about 82%.

Example 7. Maltoheptaose The same procedure as in Experiment 1-C was used except that β-7 clodextrin was used instead of α-cyclodextrin and bromobenzene was used instead of tetrachloroethane, and the concentration was 55%.
A maltoheptaose-prepared Kawahara sugar solution was prepared.

The sugar composition of this sugar solution was 213% saccharides with a degree of glucose polymerization of 6 or less, 673% maltoheptaose, and 114% saccharides with a degree of glucose polymerization 8 or more.

The resin is an alkaline earth metal type strongly acidic cation exchange resin (
Manufactured by Mitsubishi Chemical Industries, Ltd., product name: Diaion SK1
'02, Ca type, degree of crosslinking 2%) was used.

The column used and the fractionation operation were carried out in the same manner as in Example 4, and the recovery rate of maltoheptaose was about 89%.

Example 8. Maltoheptaose First, during the first fractionation, the raw sugar solution was mixed with 1 part of the resin.
Fractionation was carried out in the same manner as in Example 7, except that 0 v/v% was used.

The elution pattern of the fractionated products was similar to that shown in the figure, and the fractions were fractionated in the order of A, B, C, D, and E.

Among them, fraction C (fraction containing high maltoheptaose) was collected, and fractions A and E were removed.

The second and subsequent fractionation operations were performed in the same manner as in Example 5, and a maltoheptaose-rich fraction with a maltoheptaose content of 90% or more was collected.

The fractionation operation from the second time onwards was repeated 0 times, and the average result of each time was determined, and the recovery rate of maltoheptaose was about 96%.

Example 9 The sugar solution with a maltodecaose content of 153% prepared in Maltodecaose Experiment 1-D was used as a raw sugar solution. The resin used is an alkali metal type strong acid cation exchange resin (manufactured by Dow Chemical Company, trade name DOWEX 50W x 1, K type, degree of crosslinking 1%), and the inner diameter is 2.
．． The water suspension was packed into a column with a coating tube (No. 2). At this time, the resin layer is packed into four columns with a length of 5 m, and the four columns are connected so that the liquid flows in series, making the total length of the resin layer 2.
It was set to 0m.

While maintaining the column internal temperature at 65-℃, 5v/v% raw sugar solution was added to the resin, and 65℃ warm water was added to it by SVo,
It was fractionated by flowing at a flow rate of 2.

The obtained fractionated product was applied to the eluate column again and fractionated in the same manner, and a maltodecaose-rich fraction with a maltodecaose content of 90% or more was collected.

The recovery rate of maltodecaose was about 88%.

[Brief explanation of drawings]

The figure is a diagram showing an example of an elution pattern of a raw sugar solution. The symbols in the figure are explained as follows. A is the fraction containing L sugar, B is the fraction high in L sugar and target oligosaccharide, and the fraction high in target oligosaccharide is the fraction containing target oligosaccharide and S panicle.E is the fraction containing S spikelet Patent applicant Co., Ltd. Hayashibara Biochemistry Research Institute Procedural Amendment February 20, 1980 E. Commissioner of the Patent Office Kazuo Wakasugi 1. Indication of the case 1988 Patent Application No. 19550 2. Title of the invention Process for producing high-purity glucooligosaccharide 3. Amendment Patent applicant 4 Item 5 of ``Detailed explanation of the invention in the specification subject to the amendment'' Contents of the amendment (1) Book Mei'a, page 39, line 1 to page 10, line 2 The statement ``Also, the alkali metal type used in the present invention...is extremely advantageous for mass production.'' will be corrected in a humanistic manner. [In fact, the alkali metal type or alkaline earth metal type strongly acidic cation exchange resin used in the present invention (is the Na + type, K + type of styrene-divinylbenzene crosslinked copolymer resin bonded with a sulfone group)] One or more types of alkali metal salt types such as (r;:Ca'' type, \-1g4+ type, etc.) are appropriately pressurized and commercially available (e.g. Product name: DOWEX 50WX1, DOWEX 50＼■ Manufactured by Dow Chemical Company
×2, Dawesos 50W ×4, product name Amberlite cG-120 manufactured by Rohm & Haas, product name X'l'-1022E, manufactured by Tokyo Organic Chemical Industry Co., Ltd., XT
-1007, Diaion 5KIB, Diaion SK'102, and Diaion SK 104 manufactured by Mitsubishi Chemical Industries, Ltd. Among them, the degree of crosslinking is 6%
The following resins were found to be suitable. The degree of weight gain as used in the present invention refers to the weight E>percentage (X) of divinylbenzene based on all monomers charged when producing a styrene-divinylbenzene crosslinked copolymer. These resins are not only effective in fractionating fractions containing a high content of target oligosaccharides, but also have excellent heat resistance and abrasion resistance, and are extremely advantageous for mass production of highly purified target oligosaccharides. J

Claims (1)

[Claims] (1) Glucooligo whose degree of glucose polymerization is selected from the range of 3 to 1O! (hereinafter referred to as the "target oligosaccharide"), together with the target oligosaccharide, a saccharide with a degree of glucose polymerization greater than or equal to the target oligosaccharide (hereinafter referred to as the "L sugar") and a saccharide with a degree of glucose polymerization one or more lower than the target oligosaccharide (hereinafter referred to as the "S sugar") are prepared. The sugar mixture containing the saccharide is passed through a column packed with an alkali metal type or alkaline earth metal type strongly acidic cation exchange resin, and then eluted with water to separate the two fractions containing L-hodaka and the fraction high in L-sugar and target oligosaccharide. , the desired oligosaccharide-high content fraction, the desired oligosaccharide/S hotaka containing fraction, and the S hotaka containing fraction are fractionated in this order, and the desired oligosaccharide-rich fraction is collected, and the degree of glucose polymerization is 3. A method for producing a high purity glucooligosaccharide selected from the range of 1 to 10. (2) When the target oligosaccharide is selected from the range of glucose polymerization degree of 3 to 5, the high purity according to claim 1, characterized in that a cation exchange resin with a degree of crosslinking of 6% or less is used. A method for producing glucooligosaccharide. (3) The target oligo wall is polymerized with crucose.
The method for producing high-purity glucooligosaccharides in one batch according to claim 1, characterized in that when selected from the range of The target oligosaccharide-rich components solidify the target oligosaccharide wall.
Claims 1 and 2 are characterized in that they contain 90% or more of St. Claim 1, characterized in that when the sugar mixture is passed through the column, all of the already obtained L Ig/target oligosaccharide Hodaka' containing fraction and the target oligosaccharide/S sugar high content fraction are passed. , the method for producing high-purity glucooligosaccharide according to item 2, item 3, or item 4. (6) When flowing the sugar mixture containing the target oligosaccharide through the column, the already obtained L-saccharide/target oligosaccharide Claim 1, characterized in that after flowing the high sugar content fraction, flowing the sugar mixture containing the target oligosaccharide, and then flowing the already obtained target oligosaccharide/S hot-containing fraction. ,
The method for producing a high purity glucooligosaccharide as described in item 2, item 3, or item 4. (7) Claims 1, 2, 3, and 4, characterized in that the sugar mixture containing the target oligosaccharide contains 910 to 80% of the target oligosaccharide based on the solid content. section,
The method for producing a high purity glucooligosaccharide according to item 5 or 6.