JPH06141879A - Method for purifying cyclic inulooligosaccharide - Google Patents

Abstract

PURPOSE:To efficiently obtain a cyclic inulooligosaccharide with good selectivity by adsorbing only a cyclic inulooligosaccharide on a siloxane-based silica carrier and then eluting the cyclic inulooligosaccharide with an aqueous solution of an alcohol. CONSTITUTION:A cyclic inulooligosaccharide is adsorbed from a saccharide liquid containing the cyclic inulooligosaccharide, a monosaccharide, a disaccharide and a straight-chain oligosaccharide on a siloxane-based silica carrier. The cyclic inulooligosaccharide adsorbed on the carrier is then readily eluted with an aqueous solution of an alcohol to afford the objective cyclic inulooligosaccharide at about 87% recovery ratio.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying cyclic inulooligosaccharides, and more particularly to a method for selectively separating and purifying cyclic inulooligosaccharides by using a specific carrier.

[0002]

2. Description of the Related Art Conventionally, the problems to be solved by the invention
Glucose is α- (1-
4) Glucoside-bonded cyclodextrins are known. Cyclodextrin has been attracting attention as an inclusion agent for foods, and contributes to high added value of foods for the purpose of retaining flavor components, masking specific odors, removing bitterness, and preventing oxidation. It is also used in various fields other than food. However, cyclodextrin has a low solubility in water, and its application to the fields of foods and pharmaceuticals is currently limited.

In recent years, Uchiyama et al. Added Bacillus circulans MZ No. 3 to inulin, which is a polysaccharide containing fructose as a main component. It has been reported that a cyclic inulooligosaccharide is obtained by reacting an enzyme produced by No. 31 (Microtechnology Research Institute, No. 9943) (JP-A-2-252701) (C.
arbohydrate Research, 192
83-90, 1989). Cyclic inulooligosaccharides are sugars in which fructose molecules are cyclically linked by β- (2-1) bonds, and cycloinulohexaose consisting of 6 fructose residues, cycloinuloheptaose consisting of 7 The existence of cycloinuro octaose consisting of individual pieces is known. This cyclic inulooligosaccharide has attracted attention as a new functional sugar different from cyclodextrin because of its extremely high solubility in water. When cyclic inulooligosaccharide is produced by reacting cyclic inulooligosaccharide-producing enzyme (cycloinulooligosaccharide fructanotransferase = CFTase, hereinafter sometimes referred to as CFTase) with inulin or the like, it is present in the reaction solution. Other contaminants, for example, unreacted fructan and by-products such as fructose and other monosaccharides, disaccharides and linear oligosaccharides during the reaction are unavoidable, and a step of separating and purifying cyclic inulooligosaccharides is required. ing. However, as a conventional method for separating and purifying only cyclic inulooligosaccharide from a sugar solution containing cyclic inulooligosaccharide, only a method of fractionating and collecting using activated carbon is known (JP-A-2-252701). . Moreover, the method for purifying cyclic inulooligosaccharides using activated carbon requires elution by changing the alcohol concentration stepwise, which is not an efficient method in terms of yield. It cannot be said that the examination of the purification method is sufficient, and it is difficult to industrially produce it. Therefore,
It has been desired to develop an efficient method for purifying cyclic inulooligosaccharides.

[0004]

[Means for Solving the Problems] Therefore, the present inventors have developed a method for efficiently purifying cyclic inulooligosaccharides from a sugar solution composed of cyclic inulooligosaccharides, monosaccharides, disaccharides and linear oligosaccharides. As a result of intensive studies, other contaminants present in the reaction solution in the process of reacting fructan with CFTase to produce cyclic inulooligosaccharides,
For example, siloxane silica gel is very efficient and selective for obtaining cyclic inulooligosaccharides by removing unreacted fructans, monosaccharides such as fructose, which are by-produced during the reaction, disaccharides, and linear oligosaccharides. It has been completed that the present invention has been found to be adsorbed with and easily eluted with alcohol.

That is, the gist of the present invention is to contact a sugar solution consisting of cyclic inulooligosaccharides, monosaccharides, disaccharides, and linear oligosaccharides with a siloxane-based silica carrier to selectively adsorb only cyclic inulooligosaccharides. Then, a method for purifying a cyclic inulooligosaccharide characterized in that the cyclic inulooligosaccharide adsorbed on the carrier is eluted with an aqueous alcohol solution.

The present invention will be described below. First, examples of cyclic inulooligosaccharides that can be purified according to the present invention include cyclic inulooligosaccharides such as cycloinulohexaose, cycloinuloheptaose, and cycloinulooctaose. Examples of the linear oligosaccharides to be removed according to the present invention include sugars such as fructooligosaccharides in which the reducing end is glucose and about several to several tens of fructose are bound. Examples of monosaccharides include fructose and glucose, and examples of disaccharides include sucrose and inulobiose.

The cyclic inulooligosaccharide producing enzyme may be a producing enzyme known per se. For example, JP-A-2-2527
Bacillus circulans MZ No. 31, (Microtech Lab
), Bacillus circulans MCI-2554 described in Japanese Patent Laid-Open No. 237496/1992
1940) and the like.

To obtain a sugar solution consisting of cyclic inulooligosaccharides, monosaccharides, disaccharides and linear oligosaccharides, that is, a solution containing cyclic inulooligosaccharides, an extract of a plant having a high inulin content such as Jerusalem artichoke, burdock and chicory is obtained. And / or a bacterium that produces a cyclic inulooligosaccharide-producing enzyme in a solution containing inulin as the sole carbon source, such as Bacillus circulans MZ No. 31 (Microtechnological Research Institute No. 9943), Bacillus circulans MCI-2554 (Microtechnical Research Institute No. 1)
1940) -derived cyclic inulooligosaccharide-producing enzyme. At that time, the microbial cells themselves may be allowed to act, or the enzyme may be extracted from the microbial cells and then,
Alternatively, they may be immobilized and then acted on. Alternatively, a bacterium that produces a cyclic inulooligosaccharide-producing enzyme in an extract of a plant having a high inulin content such as Jerusalem artichoke, burdock, chicory, and / or a culture medium containing inulin as a sole carbon source, for example, Bacillus circulans MCI.
It is also possible to obtain cyclic inulooligosaccharides in the supernatant obtained by culturing -2554 (Microtechnology Research Institute No. 11940).

When obtaining cyclic inulooligosaccharides in the culture supernatant, for example, inulin is contained as a carbon source in an amount of about 1-40%, and in addition, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract are also used as nitrogen sources. , Peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. In addition, if necessary, the present bacterium is inoculated into a medium containing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other inorganic salts that generate other ions, and shake culture is performed. At this time, the culturing temperature is 20 to 37 ° C., and the culturing time is 12 to 120.
Time is good. The resulting culture solution is centrifuged to remove bacteria, and the enzyme in the supernatant is inactivated by heat treatment.
The liquid thus obtained is a cyclic inulooligosaccharide-containing solution.

When the enzyme is allowed to act, for example, the culture solution cultivated by the above method may be sterilized by centrifugation and the obtained culture filtrate may be allowed to act, or the filtrate may be further subjected to ammonium sulfate (65). (% Saturation) may be added for salting out, and the deposited precipitate may be obtained by centrifugation and suspended in a small amount of water, followed by dialysis to allow the resulting crude enzyme solution to act. This culture filtrate or crude enzyme solution is used, for example, at pH 6.0.
The desired cyclic inulooligosaccharide-containing solution is obtained by allowing inulin having a concentration of about 5% or more to act for 30 minutes or more at 30 to 60 ° C. in the 0.01-0.1 M phosphate buffer solution adjusted to 1. .

Of course, a purified enzyme may be allowed to act in place of the supernatant or the crude enzyme solution. Such a purified enzyme is, for example,
DEAE-Toyopearl 650M, QAE-T
After purification by ion exchange chromatography using an oyopearl 650M column (manufactured by Tosoh Corporation), T
Obtained by performing gel filtration chromatography using oyopearl HW55F, in which case an enzyme preparation showing a single band can be obtained by electrophoresis.

In purifying cyclic inulooligosaccharides from these cyclic inulooligosaccharide-containing solutions, a siloxane-based silica carrier is washed with acetone, methanol or the like by a predetermined method. The silica carrier is not particularly limited, but silica gel (ODS) chemically bound with an octadecyl group is particularly preferably used. Specifically, S
ilica Gel ODS ・ G-5, Silica
Examples include Gel ODS.Q-3 (Fuji Gel Sales Co., Ltd.) and Cosmo Seal 75C18-OPN (Nacalai Tesque Co., Ltd.). The washed silica support is then gradually replaced with water. These are contacted with a cyclic inulooligosaccharide-containing solution and washed with water. Then 5-80
% Of alcohol, the extract is desalted and desalted, and then concentrated to dryness to obtain the desired cyclic inulooligosaccharide. It can also be carried out by packing the gel in a column. 1 / of the resin packed in the column
After the liquid containing 100 to 1/10 the amount of cyclic inulooligosaccharide is placed and the monosaccharide, disaccharide and linear oligosaccharide are eluted with water,
Elution is performed with an 80% aqueous alcohol solution. The eluate is
Any type may be used as long as it dissociates the cyclic inulooligosaccharide from the resin. For example, a 25% methanol aqueous solution is used. Since a cyclic inulooligosaccharide is obtained in this eluted fraction, the desired cyclic inulooligosaccharide can be obtained by decolorizing and desalting this fraction and concentrating to dryness.

[0013]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto unless it exceeds the gist thereof. Example 1 Inulin 4%, yeast extract 0.2%, sodium nitrate 0.5%, magnesium sulfate, potassium chloride 0.05%, potassium phosphate 1% 0.05%, ferric chloride 0.001% 150 ml of the contained medium was adjusted to pH 7.0 and steam sterilized at 120 ° C. for 20 minutes. One platinum loop of Bacillus circulans MCI-2554 was inoculated into this sterilized medium, and shake culture was performed at 160 rpm at 30 ° C. for 30 hours. The OD value of the culture solution at 660 nm was about 5. After the culture is completed, the cells are removed by centrifugation,
A culture filtrate was obtained. The obtained culture filtrate was used as a crude enzyme solution.
3 g of inulin was finally added to 100 ml of the crude enzyme solution so that the concentration became 3%, and the mixture was reacted at 37 ° C. for 8 hours. After the reaction, analysis by high performance liquid chromatography (HPLC) revealed that cycloinulohexaose was 10.37 g / L,
Cycloinuloheptaose was obtained at a concentration of 5.19 g / L. The reaction solution contains cycloinulohexaose,
In addition to cycloinuloheptaose, cycloinulooctaose, unreacted inulin, linear oligosaccharides such as fructooligosaccharides, and fructose are included.

Adsorption of cyclic inulo-oligosaccharides was examined using the above reaction solution for resins having the physical properties shown in Table 1. The resin was washed with methanol and gradually replaced with water. 20 ml of resin thoroughly washed with water was packed in the column. After 100 ml of water was passed through the column, 20 ml of the reaction liquid was passed at sv = 1 for adsorption. 20 ml of the reaction solution contained 0.207 g of cycloinulohexaose. Then, the unadsorbed substance was eluted with water and then with 25% ethanol. Cycloinulohexaose in the ethanol eluate was quantified by HPLC. As a result, 0.180 g of cycloinulohexaose was obtained with a recovery rate of 87%. Comparative Examples 1 to 4 As shown in Table 1, purification was carried out by adsorption in the same manner as in the method described in Example 1 except that the type of resin was changed. The obtained recovery rates are shown in Table 1.

[0015]

[Table 1] Table 1 ──────────────────────────────────── Eluent Cycloinuro obtained Hexaose (ml) (g) Recovery rate (%) Remarks ──────────────────────────────────── Example 1 35 0.180 87.0 ODS Siloxane Cosmosil 75C18-OPN Silica Carrier (Nacalai Tesque, Inc.) Comparative Example 1 46 0.080 38.6 SepaBeads (Synthetic Adsorption Resin) SP800 (Mitsubishi Kasei Co., Ltd.) Comparative Example 2 45 0.090 43.5 Diaion (Synthetic Adsorption Resin) HP20 (Mitsubishi Kasei Co., Ltd.) Comparative Example 3 35 0.110 53.1 Diaion (Synthetic Adsorption Resin) HP21 (Mitsubishi Kasei Co., Ltd.) Comparative Example 4 40 0.085 41.0 Chromatography (Activated Carbon) Activated Carbon ( (Wako Pure Chemical Industries)

[0016]

INDUSTRIAL APPLICABILITY According to the present invention, when a cyclic inulooligosaccharide is purified from a cyclic inulooligosaccharide-containing solution, a siloxane silica carrier can be used to obtain the cyclic inulooligosaccharide selectively and in high yield. . The obtained cyclic inulooligosaccharide can be expected to be used not only in the field of food but also in a wide range of fields.

Claims (2)

[Claims] 1. A sugar solution composed of cyclic inulooligosaccharides, monosaccharides, disaccharides and linear oligosaccharides is brought into contact with a siloxane-based silica carrier to selectively adsorb only the cyclic inulooligosaccharide and then to the carrier. A method for purifying cyclic inulooligosaccharides, which comprises eluting cyclic inulooligosaccharides with an aqueous alcohol solution. 2. The method for purifying a cyclic inulooligosaccharide according to claim 1, wherein the sugar solution is contacted with the siloxane-based silica carrier by passing the sugar solution through a column packed with the siloxane-based silica carrier. .