JPH06343487A - Separation and purification of neotrehalose - Google Patents

Abstract

PURPOSE:To obtain a neotrehalose by industrially advatageous separation and purification method characterized by simple operations using a column chromatography without decomposing intermixed other disaccharides, to say nothing of monosaccharides, trisaccharides and higher ones. CONSTITUTION:The method of separating and purifying a neotrehalose characterized by (l<1>) applying a mixture solution of the trehalose to a column chromatography consists of a stationary phase of a silica gel bonded with a long chain alkyl group and a moving phase of water or an aqueous solution of organic solvents, and (2) after applying the above solution to a column chromatography of a salt type strongly acidic cation exchange resin, applying the elute solution to the column chromatography which consists of a stationary phase of a silica gel bonded with a long chain alkyl group and a moving phase of water or an aqueous solution of organic solvents.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for separating and purifying neotrehalose. More specifically, it relates to a method for separating and purifying neotrehalose by column chromatography using silica gel having a long-chain alkyl group chemically bonded as a stationary phase. Neotrehalose has an O-α-D-glucopyranosyl β-D-glucopyranoside structure or O-β-
It has long been known as a disaccharide having a D-glucopyranosyl α-D-glucopyranoside structure. Neotrehalose has a sweetness that is about 1/4 that of sucrose, is non-reducing, is water-soluble, but is stable rather than hygroscopic / deliquescent, and can be orally ingested into various beverages, processed foods, and tastes. object,
It can be used for feed, pharmaceuticals, etc.

[0002]

2. Description of the Related Art As a method for producing neotrehalose, a chemical method (Journal of the Chemical Societ
y, 1931, 2847-2850 and ibid, 1951, 285-287), but the operation is complicated and the yield is extremely low, which is not practical. As a method of preparing from a natural product, the disaccharide-containing fraction obtained by subjecting the non-fermentable sugar contained in koji juice to carbon chromatography is treated with phenylhydrazine, the precipitate is filtered off, and the sugar in the liquid is acetylated, There is a method of obtaining an octaacetate crystal of a disaccharide and then deacetylating it (Journal of Japan Society for Agricultural Chemistry, Volume 30, 119 to 123 (1959).
)), The content of koji soup is low, and the purification process is complicated, which makes it unsuitable for industrial production.

On the other hand, as a biochemical method using inexpensive starch as a raw material, a method for producing neotrehalose together with the trisaccharide centose by reacting cyclodextrin synthase with a substrate selected from starch and its degradation product is proposed. (Japanese Patent Laid-Open No. 4-179490). In this method, a large amount of components other than neotrehalose are mixed in the reaction solution, and it is particularly difficult to separate neotrehalose from the same disaccharides such as maltose, isomaltose and kojibiose. Therefore, in order to obtain high-purity neotrehalose, disaccharide components other than neotrehalose are decomposed and neutralized by alkali treatment and then fractionated by column chromatography, but other components are decomposed for purification. There are problems such as wasting resources and complicated operation.

Therefore, an object of the present invention is to provide a method for separating and purifying neotrehalose from a mixed solution containing neotrehalose without decomposing other components.

[0005]

In order to solve the above-mentioned problems, the inventors of the present invention use a difference in the interaction between both phases of a substance in a stationary field consisting of a filler (stationary phase) and a solvent (mobile phase). The separation method by liquid column chromatography utilizing the difference in the moving speed was examined, especially focusing on the structures of neotrehalose and other disaccharides.

Regarding the separation of disaccharides by liquid chromatography, Nikolov et al. Examined the elution time of disaccharides using acetonitrile / water as a mobile phase by normal phase chromatography using a packing in which an amino group is chemically bonded to silica gel. (J. Chrom., 207 (1985) 51-57),
The elution positions of the disaccharides are close to each other, and particularly neotrehalose cannot be an effective means for separating neotrehalose from these disaccharides because the elution positions of maltose and kojibiose are close to each other. Also, the use of acetonitrile as the mobile phase has a cost disadvantage.

As related to the present invention, Norm was determined by reverse phase partition chromatography using a silica gel column (ODS column) in which octadecyl groups were chemically bonded.
an et al. investigated the separation of a mixture of maltooligosaccharides or isomaltooligosaccharides with water as the mobile phase (J. Chrom., 209).
(1981) 439-444) does not refer to the separation of disaccharides from each other, and there is no report on the separation of neotrehalose and other saccharides using an ODS column. Therefore, it was unclear whether neotrehalose could be separated from a mixture of other disaccharides.

Neotrehalose is a molecule in which two glucose molecules are bound by α, β-1,1. Maltose (α-1,4) and nigerose (α), which are also composed of two glucose molecules.
-1,3), kojibiose (α-1,2), isomaltose (α-1,6) and the like have a different three-dimensional structure. Due to this difference in the three-dimensional structure, the distribution of the hydrophobic and hydrophilic parts is different, and it is considered that even disaccharides having glucose as a constituent sugar differ in the strength of the hydrophobicity or hydrophilicity present on the molecular surface due to the difference in the bond. . Therefore,
The present inventors believe that it is possible to separate by using a mobile phase (eluent) having a hydrophobic substituent as a stationary phase (separating agent) for column chromatography, and elution is carried out for an ODS column. As a chemical, water and an aqueous solution of an organic solvent having a different concentration were used and earnestly studied. As a result, they have found that neotrehalose can be separated from other disaccharides by using water or an aqueous alcohol solution as a mobile phase.

Furthermore, separation of monosaccharides such as neotrehalose and glucose and trisaccharides such as centose or more is O.
Although it is possible to use a DS column, if the ratio of monosaccharides and trisaccharides is high in the sugar composition of the mixture containing neotrehalose, the monosaccharides should be prepared beforehand by column chromatography using a salt-type strongly acidic cation exchange resin. It was confirmed that the removal of trisaccharides and trisaccharides reduces the load on the ODS column and enables more efficient separation and purification.

That is, the present invention provides 1) a method for separating and purifying neotrehalose, which comprises subjecting a mixed solution containing neotrehalose to column chromatography using silica gel having a long-chain alkyl group chemically bonded as a stationary phase. 2) The method for separating and purifying neotrehalose according to 1 above, which uses silica gel chemically bound to an octadecyl group, 3) After subjecting the mixed solution containing neotrehalose to column chromatography with a salt-type strongly acidic cation exchange resin, 4. A method for separating and purifying neotrehalose, which comprises subjecting silica gel having a long-chain alkyl group chemically bonded thereto to a stationary phase as a stationary phase, and 4) using silica gel having an octadecyl group chemically bonded, as described in 3 above. Neotrehalose separation and purification method, and 5) Water or mobile phase There is provided a method for the separation and purification neotrehalose according to any one of 1 to 4 using the machine solvent solution.

The mixed solution containing neotrehalose, which is a raw material for the separation and purification method according to the present invention, is a mixture containing neotrehalose, for example, a method in which cyclodextrin synthase is allowed to act on a substrate selected from starch and its degradation products. It may be prepared by any method capable of obtaining a liquid.

The present inventors have confirmed that it is possible to separate neotrehalose and other disaccharides by using silica gel having an octadecyl group chemically bonded as a stationary phase column packing material. The same effect can be expected on silica gel bonded with a long-chain alkyl group other than octadecyl group. Therefore, a separation method using silica gel having a long-chain alkyl group other than octadecyl group bonded thereto is also included in the present invention.

The silica gel column which can be used in the separation and purification method of the present invention includes octadecyl group as a long chain alkyl group, hexadecyl group, tetradecyl group, dodecyl group,
Examples thereof include those chemically bound to a decyl group and the like, but octadecyl group is preferred. Such a silica gel column chemically bonded to a long-chain alkyl group can be prepared, for example, by subjecting silica (silica sol) treated with hydrochloric acid, washed with water and dried to toluene with dimethyldichlorosilane (DMC) according to the following process formula.
S) is refluxed in a solution in which the solution is dissolved and then filtered under reduced pressure, and then a predetermined amount of silicon monomer RSiCl ₃ (R
Represents a long-chain alkyl group. ) Is added and refluxed, followed by washing with water and drying.

[0014]

[Chemical 1]

For example, a silica gel column chemically bonded with an octadecyl group is available from YMC Corporation YMC-.
The products commercially available as the Pack series can be used.

As the mobile phase, water, an organic solvent, or a mixture thereof can be used. Here, as the organic solvent, methanol, ethanol, acetonitrile or the like is used. The higher the polarity of the aqueous organic solvent solution, the better the separation of the disaccharides from each other. Therefore, it is preferred to use water as the mobile phase. Also, water is more advantageous in price than organic solvents.

In the present invention, neotrehalose and other disaccharides can be separated by using a silica gel column having a long-chain alkyl group chemically bonded as a stationary phase and using water or an organic solvent of each concentration as a mobile phase. . In particular, when water is used as the mobile phase, the elution times of neotrehalose and maltose, isomaltose, cordiose, nigerose and other disaccharides having glucose as a constituent sugar differ greatly. Therefore, neotrehalose can be efficiently separated from the mixed solution containing neotrehalose, and other disaccharides can be recovered alone or as a mixed solution.

With respect to monosaccharides and saccharides of three or more,
Similarly, it can be separated and recovered because it differs from the elution position of neotrehalose, but if the ratio of monosaccharides and trisaccharides in the neotrehalose-containing liquid is high, column chromatography using a salt-type strongly acidic cation exchange resin can It is more effective to reduce the ratio and then carry out the above separation operation. That is, by utilizing the fact that the elution position of neotrehalose differs from that of monosaccharides and trisaccharides by column chromatography using a salt-type strongly acidic cation exchange resin, the long-chain alkyl group is chemically treated after increasing the neotrehalose concentration. Chromatography on a bound silica gel column is performed.

[0019]

As described in detail above, according to the present invention, neotrehalose is mixed with monosaccharides and trisaccharides and other disaccharides such as maltose, isomaltose, kojibiose, nigerose, sucrose and lactose. Neotrehalose can be easily separated and purified from the mixed solution by column chromatography without decomposing other components, paving the way for the industrial production of neotrehalose. Neotrehalose separated and purified by the method of the present invention is a high-purity product containing almost no other components, and syrup, freeze-drying, spray-drying,
In the form of crystals, it can be used for various beverages, processed foods, favorite foods, feeds, pharmaceuticals and the like.

[0020]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

Example 1: Elution time of each standard by ODS column ODS column (YMC Co., Ltd., trade name YMC
-Pack ODS-AQ, 4.6 x 250 mm) prepared with about 1% maltose, kojibiose, nigerose, isomaltose, trehalose, sucrose,
10 μl of lactose and neotrehalose preparations were injected and the elution time of each was examined. Mobile phase is distilled water and 5,
The elution time shown in Table 1 was obtained as a result of detection with a differential refractometer using 10% methanol at a flow rate of 0.5 ml / min.

[0022]

[Table 1]

Example 2 (1) Preparation of Neotrehalose-Containing Solution Maltose was used at a final concentration of 20%, and a cyclodextrin synthase derived from Bacillus macerans (manufactured by Amano Pharmaceutical Co., Ltd., trade name Contizyme) was used as a substrate. 1,000 THU per gram, pH 6.0, 1 at 50 ° C
After reacting for 0 days, it was deactivated. Furthermore, this solution was adjusted to pH 4.5 and 50 ° C., and glucoamylase (Novo Nordisk Bioindustry Co., Ltd., trade name AMG) was used.
300 L) was added in an amount of 1.5 U per gram of substrate, and the mixture was reacted for 24 hours and then inactivated. This solution was purified with activated carbon and an ion exchange resin and then concentrated to 50% concentration. This solution contained approximately 15% neotrehalose per solids.

(2) Concentration of neotrehalose with salt type strongly acidic cation exchange resin The solution obtained in Example 2 (1) was used as a salt type strongly acidic cation exchange resin (trade name UBK-53 manufactured by Mitsubishi Kasei Co., Ltd.).
0, resin amount 11.8 liters), glucose 10% based on solid content, neotrehalose 28%, disaccharides such as maltose except neotrehalose 52%, trisaccharide 1
A sugar solution having a sugar composition of 0% was obtained.

(3) Preparation of neotrehalose The solution obtained in Example 2 (2) was concentrated to a concentration of 50%, and 5 g of the solid content was added to an ODS column (YMC Co., Ltd., trade name YMC-Pack ODS-). A
Q, 10 × 100 cm × 2). Demineralized water was used as the mobile phase, and the solution was passed at a flow rate of 250 ml / min. As a result, 0.9 gram of neotrehalose fraction was collected as a solid content. The separation pattern at this time is shown in FIG.

(4) Identification of neotrehalose fraction The neotrehalose fraction obtained in Example 2 (3) was concentrated and lyophilized.
When hydrolyzed with sulfuric acid, 2 mol of glucose was produced from 1 mol of the standard product. In addition, as a result of measuring the specific optical rotation and the melting point,
It was identified as neotrehalose by showing the following values and being close to the literature values and the values of the crystal product obtained from this fraction. Specific rotation [α] _D ²⁰ + 94.3 ° (C = 1, H
₂ O) Melting point 145 ° C

[Brief description of drawings]

FIG. 1 is a separation pattern of ODS (stationary phase) -water (mobile phase) of a mixed solution containing neotrehalose obtained by reacting cyclodextrin synthase with maltose as a substrate.

Claims (5)

[Claims] 1. A method for separating and purifying neotrehalose, which comprises subjecting a mixed solution containing neotrehalose to column chromatography using silica gel having a long-chain alkyl group chemically bonded as a stationary phase. 2. The method for separating and purifying neotrehalose according to claim 1, wherein silica gel having an octadecyl group chemically bonded is used. 3. A mixed solution containing neotrehalose is subjected to column chromatography using a salt-type strongly acidic cation exchange resin to obtain a surface fraction having a high neotrehalose content, and then silica gel having a long-chain alkyl group chemically bonded thereto is used as a stationary phase. A method for separating and purifying neotrehalose, which comprises subjecting to column chromatography. 4. The method for separating and purifying neotrehalose according to claim 3, wherein silica gel chemically bound with an octadecyl group is used. 5. The method for separating and purifying neotrehalose according to any one of claims 1 to 4, wherein water or an aqueous solution of an organic solvent is used as a mobile phase.