JP2784054B2 - Method for producing high-purity aldosyl fructoside - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing high-purity aldosylfructoside, and more specifically, to efficiently produce aldosylfructoside from a sugar solution containing other saccharides together with aldosylfructoside. The present invention relates to a method for producing high-purity aldosyl fructoside by fractionation and elution.

[Problems to be solved by conventional technology and invention]

Specific examples of aldosyl fructoside include xylosyl fructoside (hereinafter abbreviated as “XF”), lactosyl fructoside (hereinafter abbreviated as “LF”), galactoside fructoside (hereinafter “Gal F”). Abbreviated as ".)
And the like.

These aldosyl frucsides can be converted into enzymes (levansucrase) produced by microorganisms such as Bacillus subtilis or sucrose in the presence of xylose, lactose and galactose, respectively, or enzymes produced by microorganisms such as Arthrobacter (β). -Fructofuranosidase). In the case of XF, it has anti-cariogenic properties, and in the case of LF and Gal F, it has the property of selectively proliferating Bifidobacteria, so that it is expected to be widely used in the pharmaceutical and food fields.

Separation of aldosyl fructoside and other sugars using activated carbon column chromatography, first eluting and removing monosaccharides with water, then increasing the ethanol concentration stepwise or continuously to elute sucrose and other unintended sugars A method of projecting and recovering a target aldosyl fructoside after removal is known. As another method, a method of separating and purifying using an alkali metal or alkaline earth type acidic cation exchange resin is known.

However, the fractionation method using activated carbon uses a large amount of ethanol, so that the production cost is too high for industrial use. Further, there is a disadvantage that it takes a long time to elute the protein by increasing the ethanol concentration stepwise or continuously, and the efficiency is not good. Therefore, it is extremely difficult to industrially produce high-purity aldosyl fructoside. In addition, it is extremely difficult to produce high-purity aldosyl fructoside because fractionation is insufficient with a fractionation method using a strongly acidic cation exchange resin.

[Means for solving the problem]

Therefore, the present inventors have considered such circumstances, and as a result of repeated studies to develop a method for producing an efficient and practical high-purity aldosyl fuclatoside, using a chemically modified silica carrier, The present inventors have found that it is possible to separate only with water and completed the present invention.

That is, the present invention is characterized in that a sugar solution containing at least one of glucose, fructose, sucrose and aldose together with aldosylfructoside is brought into contact with a chemically modified silica carrier to separate and elute aldosylfructoside. And a method for producing high-purity aldosylfructoside.

The aldosyl fructoside referred to in the present invention includes xylosyl fructoside, galactosyl fructoside, lactosyl fructoside, sorbosyl fructoside, fucosyl fructoside, kestose, stachyose, erulose, raffinose, isomaltosyl fructoside and arabinosyl fructoside. Means any of the lactosides.

The chemically modified silica carrier used in the present invention has a structure in which a silanol group of silica gel is substituted with a linear alkyl group having 8 to 18 carbon atoms. Such a chemically modified silica carrier is prepared by Preparative-C18 (manufactured by Forters),
It is commercially available under the trade name of YMC-PACKODS-A, YMC-PACK ODS-AQ (manufactured by Yamamura Chemical Laboratory). Among these, particularly preferred are those which can use a water solvent and have a long life YMC-PAC.
K ODS-AQ.

In the present invention, water is used as a mobile phase. Although a low-concentration alcohol or acetonitrile can be used as the mobile phase, water alone may be used in consideration of the cost required for fractionation and the like.

The conditions for fractionation and elution of altosyl fructoside differ depending on the target aldosyl fructoside, but the column temperature is usually room temperature.In the case of aldosyl fructoside with a short elution time, raise the column temperature. Can elute faster. The mobile phase, that is, the flow rate of water, does not need to be particularly limited because a silica carrier having excellent end pressure properties is used.
It is preferable that SV is 0.03 to 0.06 in the preparative column.

Aldosyl fructoside eluted in this way is
In particular, it is high in purity without performing other operations such as purification.

〔Example〕

Next, examples are shown, and the present invention will be described in more detail and specifically.

Example 1 (I) Preparation of β-fructofuranosidase Arthrobacter sp.K-1 (FERM P-10736) was mixed with corn steep liquor 5 w / v%, sucrose 3 w / v%, and ammonium phosphate 0.4 w. / v% and magnesium sulfate ・ 7
The bacteria were inoculated into a sterilized liquid medium 12 consisting of 0.1 w / v% of water salt, and cultured with aeration and stirring at 37 ° C. for 24 hours.

The obtained culture was centrifuged to obtain a crude enzyme preparation having a fructose transfer activity of about 600,000 units. Here, one unit of the activity is 40 w / v containing 0.1 M phosphate buffer at pH 6.5.
200 μl of 20% w / v sucrose solution and 200 μl of appropriately diluted enzyme solution were added and reacted at 40 ° C. for 10 minutes.100 μl of the reaction solution was taken, added to 1.9 ml of boiling water and kept for 10 minutes. Then, the reaction is stopped, and then glucose and fructose are quantified using an F kit (manufactured by Boehringer Mannheim Yamanouchi), the amount of fructose transferred to xylose is calculated, and 1 μmol of fructose is transferred per minute.

(II) Preparation of raw sugar solution Equivalent amounts of xylose and sucrose were dissolved at a concentration of 40 w / w%, and β-fructranosidase obtained in (I) was added at a ratio of 10 units per gram of solid material. , PH 6.5,
The reaction was performed at a temperature of 60 ° C. for 20 hours. Activated carbon was added to the reaction solution, which was then heated and kept at 100 ° C. for 15 minutes. Next, the mixture was filtered and desalted and purified using H-type and OH-type ion exchange resins. This sugar solution had a concentration of 20 w / w% and contained about 28% of XF.

(III) YMC-PACK ODS-AQ (15 μm, 20φ × 50
(0 mm, manufactured by Yamamura Chemical Laboratory), and the fractionation operation was performed at room temperature. 100 μm of the raw sugar solution obtained in the above (II) was loaded on a kamula, and each sugar was eluted by flowing water at 6 m / min. The eluate was monitored with a differential refractometer to obtain the elution pattern shown in FIG. XF eluted in a completely different place from the monosaccharides and sucrose, and was recovered with a purity of about 100% and a recovery of about 100%.

Example 2 (I) Preparation of Raw Sugar Solution The same procedure was carried out except that the enzyme obtained in Example 1 (I) was used and xylose was replaced with lactose in the preparation of the raw sugar solution in Example 1 (II). Was performed to prepare a raw sugar solution. This sugar solution had a concentration of 20 w / w% and contained about 28% LF.

(II) YMC-PACK ODS-AQ (15 μm, 20φ × 50
(0 mm, manufactured by Yamamura Chemical Laboratory), and the fractionation operation was performed at room temperature. 100 μm of the raw sugar solution obtained in the above (I) was loaded on a kamula, and water was passed at 6 ml / min. To protrude each sugar. The protrusion liquid was monitored by a differential refractometer, and collected by a fraction collector. This operation was repeated three times to analyze the fraction. The elution pattern is shown in FIG. The sugar composition (%) of each fraction is shown in the table below.

As is clear from the table, the recovery rate was 85.2% and the purity was 95.6%.
Got LF.

Example 3 (I) Preparation of Raw Sugar Solution Using the enzyme obtained in (I) of Example I, (I)
A raw sugar solution was prepared in the same manner as in I) except that xylose was replaced by galactose and the reaction time was changed to 6 hours. This sugar solution has a concentration
At 20% w / w, it contained about 16% GalF.

(II) Fractionation of GalF Using YMC ODS-AQ (50 μm, manufactured by Yamamura Chemical Laboratory) as a packing material, a plastic column (500φ × 300 mm, manufactured by Yamamura Chemical Laboratory) and a main column (500φ × 1000 mm, Yamamura Chemical Laboratory) ) And fractionated. The fractionation operation was performed at room temperature. The raw sugar solution obtained in the above (I) is applied to a column at 1500
ml was loaded, and water was passed at a flow rate of 6000 ml / min. to protrude each sugar. The eluate was monitored with a differential refractometer and analyzed with a fraction collector. The elution pattern is shown in FIG.

 GalF with a purity of 84.3% was obtained with a recovery rate of 58.8%.

〔The invention's effect〕

ADVANTAGE OF THE INVENTION According to this invention, aldosyl fructoside of high purity can be industrially manufactured efficiently at low cost. The aldosyl fructoside obtained according to the present invention has anti-cariogenic and bifidobacterial growth activity, and thus is useful in the fields of medicines and foods.

[Brief description of the drawings]

FIG. 1 is a curve diagram showing the elution pattern of XF obtained in Example 1. FIG. 2 is a curve diagram showing an elution pattern of LF obtained in Example 2. FIG. 3 is a curve diagram showing an elution pattern of GalF obtained in Example 3.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Kozo Hara 2-6-3-104 Namiki, Kanazawa-ku, Yokohama, Kanagawa Prefecture (72) Inventor Jin Hashimoto 4-31-10, Imaizumidai, Kamakura City, Kanagawa Prefecture (58) Survey Field (Int.Cl. ⁶ , DB name) C07H 1/06 C07H 3/04 CA (STN) REGISTRY (STN) WPIDS (STN)

Claims (2)

(57) [Claims] A sugar solution containing at least one of glucose, fructose, sucrose and aldose together with aldosyl fructoside is brought into contact with a chemically modified silica carrier to separate and elute aldosyl fructoside. A method for producing high-purity aldosyl fructoside. 2. The method of claim 1, wherein the aldosyl fructoside is xylosyl fructoside, galactosyl fructoside, lactosyl fructoside, sorbosyl fructoside, fucosyl fructoside, kestose, stachyose, erulose, raffinose, isomaltosyl fructoside, and arabinosyl fructoside. The method for producing a high-purity aldosyl fructoside according to claim 1, which is one of lactosides.