JP2012046440A - Method for purifying oligosaccharide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain high purity oligosaccharide from garlic.SOLUTION: When an oligosaccharide is eluted from a garlic extract by using a weak anion exchange resin, only water is used as an eluate to absorb and remove a lectin therefrom.

Description

The present invention relates to a method for purifying garlic-derived oligosaccharides.

In recent years, various oligosaccharides derived from plants have been purified. For example, trisaccharides are present in small amounts in sugar beet, soybeans, etc., raffinose consisting of one molecule each of galactose, glucose, and fructose, and tetrasaccharides are present in two molecules of galactose, glucose, and fructose each present in soybeans and sugar. There is a stachyose consisting of. Oligosaccharides are not decomposed by human digestive enzymes and have been confirmed to proliferate so-called good bacteria such as bifidobacteria in the intestines. It's being used.

In raw garlic, sugar accounts for 26% to 30% of the total mass, and is about 80% in terms of dry matter. The main component is fructan, and galactan has been reported as a polysaccharide component of the cell wall. Further, glucose, fructose as monosaccharides, sucrose as disaccharides, fructofuranosyl sucrose as trisaccharides, and scorodose composed of 4 molecules of fructose as tetrasaccharides have been reported (see Non-Patent Document 1).

As a method for purifying this garlic-derived oligosaccharide, there are known purification methods proposed by the inventor by hot water extraction, ammonium sulfate fractionation, gel filtration and ion exchange chromatography, and freeze-drying (see Patent Document 1). ).

JP 2006-206572 A

Hiroshi Saito “Science of Garlic” Asakura Shoten, 2000, p.104

As described above, garlic-derived oligosaccharides are composed of various saccharides and are useful in various applications if they can be purified with high purity. However, the saccharide contained in the lyophilized product obtained by the above purification method is not necessarily as high as 64% of the total mass.
An object of the present invention is to provide a purification method capable of obtaining a high-purity oligosaccharide from garlic.

The present invention provides a method for purifying an oligosaccharide, characterized in that, when an oligosaccharide is eluted from a garlic extract using a weak anion exchange resin, only the water is used as an eluent and lectin is adsorbed and removed. provide.

In the present invention, fresh raw garlic is suitable as the garlic, but it is also possible to use a garlic that has been processed for storage, such as one that has been shade-dried.

In the present invention, the garlic extract refers to a solution in which oligosaccharides contained in garlic are dissolved.

In the present invention, the eluate is a solution used for eluting oligosaccharides through a column packed with a weak anion exchange resin. Distilled water is preferred as the eluent, but other purified water such as pure, ultrapure water, and ion exchange water can also be used.

Conventionally, in ion exchange chromatography, a buffer solution such as an acetate buffer solution or a phosphate buffer solution is generally used as an eluent. In the present invention, such a buffer solution is not used at all. In particular, oligosaccharides are eluted only with distilled water. As a result, high-purity oligosaccharides that could not be achieved conventionally have been purified. The reason for this is that when a buffer solution is used, a slight amount of a compound that appears to be a component of the buffer solution remains even after dialysis, which prevents complete removal of water during lyophilization. Conceivable.

By using water as the eluent, the oligosaccharide in the garlic extract is eluted without being adsorbed by the weak anion exchange resin, and only the lectin is adsorbed. Therefore, oligosaccharides and lectins can be separated and purified from the garlic extract even in a relatively short and small column.

Furthermore, the present invention provides a method for purifying an oligosaccharide, in which after the oligosaccharide is eluted, the lectin adsorbed and removed from the garlic extract is eluted by changing the eluate to sodium chloride.

The high-purity oligosaccharide obtained by the present invention can be used, for example, as a drug or a food material for activating immunity. In addition, the use of water instead of a buffer solution as an oligosaccharide eluate allows the dialysis step to be omitted, and the oligosaccharide purification cost can be reduced.

Graph showing ion exchange chromatography of a solution containing raw garlic extract

Embodiments of the present invention will be described below. Garlic is ground in a mixer with the same amount of distilled water. The treated product is filtered with a cotton cloth and the filtrate is collected in a flask or the like. The filtrate is heat treated and then returned to room temperature. The treatment solution is centrifuged to remove unnecessary substances and then salted out. This is centrifuged to obtain a precipitate. This precipitate is dissolved in distilled water and again centrifuged to remove unnecessary matter. The treatment liquid from which unnecessary substances have been removed is dialyzed against distilled water. The dialyzed processing solution is applied to a column equilibrated with distilled water (DEAE-TOYOPEARL 650M) and subjected to weak anion exchange chromatography. As an eluent, distilled water is used instead of a buffer solution such as a commonly used acetate buffer solution or phosphate buffer solution. Of the eluted treatment solution, a portion showing a high absorbance at 490 nm is collected and lyophilized.

The experimental result performed using the purification method of the oligosaccharide of this invention is shown. All were experiments to isolate and purify oligosaccharides from new garlic from Kumamoto Prefecture, and were performed three times on different days. Details of the experiment will be described based on the third data.

189 ml of distilled water was added to 189 g of new garlic from which the hard portion under the bulb was removed, and the mixture was pulverized for 2 minutes with a mixer. This was filtered using a cotton cloth, and the obtained solution was heated at 82 ° C. for 30 minutes in a thermostatic bath. This was cooled to room temperature, insoluble matter was removed by centrifugation (10000 rpm, 10 min, 4 ° C.), and 182 ml of supernatant was obtained. To this, 126 g of ammonium sulfate was added with stirring so as to be 80% saturation, and the mixture was allowed to stand at 4 ° C. for 15 minutes and then centrifuged again (10000 rpm, 10 min, 4 ° C.) to obtain a precipitate. This precipitate was dissolved in 100 ml of distilled water, and insoluble matter was removed by centrifugation (10000 rpm, 10 min, 4 ° C.).

The supernatant thus obtained was packed in a seamless cellulose tube and dialyzed against distilled water. The dialysis was performed by changing 3 l of distilled water 5 times over 2 days. When the solution obtained by dialysis was subjected to gel filtration by Sephadex G-75 column chromatography, the solution in the tube contained only oligosaccharide and lectin.

115 ml of the solution in the tube was applied to a DEAE-TOYOPEARL 650M column (4.2 × 19.0 cm) equilibrated with distilled water and eluted with distilled water. 17 ml was collected in each fraction, and the 26th and subsequent eluates were changed from distilled water to 1 M NaCl. 50 μl was collected from each fraction and saccharides were quantified by the phenol-sulfuric acid method. The result is shown in FIG.

The 12th to 18th fractions rich in carbohydrates were collected and lyophilized. The freeze-dried product was a pure substance, with 100% of the content being saccharides. As a result, 1028 mg of oligosaccharide was obtained. This oligosaccharide is a compound having a molecular weight of 1800 in which 10 fructose linked with β-2,1 are bonded to 1 glucose with β-1,2 bond.

The 37th to 43rd fractions showing high absorbance at 280 nm were collected, dialyzed against distilled water, and then lyophilized. This gave 303 mg of lectin. The DEAE-TOYOPEARL 650M column could be reused by washing with about 1 liter of distilled water.

The first and second experiments were performed using the same equipment and the same process. In the first round, 1248 mg of oligosaccharide and 349 mg of lectin were obtained for 155 g of raw garlic. In the second round, 1273 mg of oligosaccharide and 319 mg of lectin were obtained for 185 g of raw garlic.

The DEAE-TOYOPEARL 650M column is very short, 4.2 × 19.0 cm, but it is possible to separate and purify oligosaccharides and lectins by applying 115 ml of the solution to the eluent. This is because distilled water is used for elution without adsorbing oligosaccharide, and only lectin is adsorbed. The isoelectric point of lectin is 4.3, and it is negatively charged in distilled water, so it is adsorbed by DEAE-TOYOPEARL 650M.

Claims (2)

A method for purifying oligosaccharides, characterized in that, when an oligosaccharide is eluted from a garlic extract using a weak anion exchange resin, only the water is used as the eluent and the lectin is adsorbed and removed. The oligosaccharide purification method according to claim 1, wherein after the oligosaccharide is eluted, the lectin adsorbed and removed from the garlic extract is eluted by changing the eluate to sodium chloride.

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