JP2000351790A - Production of fucose-containing oligosaccharide, or its composition and fucose-containing oligosaccharide or its composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a fucose-containing oligo-saccharide that is available from stably and readily available raw materials, has a clear structure and is useful over a wide variety of purposes, for example, food, chemical reagents, medicines and the like by extracting the component from specific seaweeds. SOLUTION: Fucoidan is extracted from seaweeds in Chordariaceae or Spermatochnaceae, hydrolyzed and the fucose-including oligosaccharide or its composition is separated and purified. In a preferred embodiment, Nemacystis decipiens (suringar) Kuckkusk is used as the above-stated seaweed, and the fucose-including oligosaccharide or its composition is fucosyl-α1,3-fucose or an oligosaccharide containing fucose. In one embodiment, the fucoidan is hydrolyzed with an acid, for example, by adding 0.01-12N hydrochloric acid so that the concentration of the fucoidan may become 0.01-10 wt.% and the pH may become 0.1-1.0 and the hydrolysis is carried out at 20-100 deg.C for about 0-24 hours.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a process for producing a fucose-containing oligosaccharide comprising fucose as a constituent sugar or a composition thereof,
And a specific fucose-containing oligosaccharide obtained by the above method or an oligosaccharide composition containing the same.

[0002]

2. Description of the Related Art In nature, fucose is contained in the form of fucoidan as a constituent of seaweed cell walls, and is involved in the body surface of oral species such as sea cucumber in animals and intercellular recognition in higher animals. Present as a constituent sugar of cell surface sugar chains.

[0003] In recent years, as the physiological functions of sugar chains have been elucidated, the physiological functions of oligosaccharides containing fucose have started to attract attention.

[0004] Fucoidan obtained from brown algae is a polymer having a large number of branches, and the type of constituent sugars varies depending on the type of seaweed, such as galactose, xylose, arabinose, mannose, and glucuronic acid.
Most fucoidans have a very complex structure, such as the ester sulfate bond being found at various positions in many fucoidans, and the structure has not yet been confirmed. Further, fucoidan is considered to have different constituent substances depending not only on the type of seaweed but also on the environment such as the stage of growth and the place of production.

From the results of structural analysis by methylation analysis of fucoidan extracted and purified from seaweed, it has been known that a disaccharide unit of fucosyl fucose is present (Journal of Chemical Society 2248-). 2254 (19
59)).

[0006] However, no report has been made on the extraction, separation and purification of fucose-containing oligosaccharides having a clear structure from seaweeds. A method for preparing fucose-containing oligosaccharides by chemical synthesis using monosaccharide fucose as a raw material has been reported. (Carb
ohydrate Research 4, 189-195 (1967), Carbohydrate
Research 37, 75-79 (1974), Carbohydrate Research
41, 308-312 (1975)).

[0007]

However, in the above-mentioned preparation method by chemical synthesis, the monosaccharide fucose used as a raw material must be prepared by complete hydrolysis of fucoidan, and multi-stage modification by organic synthesis reaction is required. The steps were very complicated and required low yields, such as the need for protection and deprotection reactions, and separation and purification using a chromatographic column for each step. Furthermore, these fucose-containing oligosaccharides cannot be used in foods because they are prepared by an organic synthesis reaction.

[0008] For this reason, there has been a demand for the development of an efficient method for preparing fucose-containing oligosaccharides that can be used in foods. However, it has been desired to stably produce oligosaccharides containing fucose with a clear structure using seaweed as a raw material. Was difficult.

[0009] Therefore, an object of the present invention is to provide a fucoidan, which is obtained from brown algae which is stable and easily available through aquaculture production, and has a clear structure and can be used for a wide range of applications such as foods, reagents and pharmaceuticals. A method for efficiently producing an oligosaccharide-containing or oligosaccharide composition containing the same,
And a specific fucose-containing oligosaccharide obtained by the method or an oligosaccharide composition containing the same.

[0010]

Means for Solving the Problems In order to achieve the above-mentioned object, the present inventors have found that among the seaweeds which are inexpensive, available in large quantities and easily, the sugar composition of fucoidan contained therein is relatively simple. Searched for things. And the family Sagamo (Chordariaceae) or Mozuku (Spermatochnacea)
e) By hydrolyzing the purified fucoidan obtained by extraction / separation from seaweed under suitable acidolysis conditions, it is possible to efficiently produce a specific fucose-containing oligosaccharide or an oligosaccharide composition containing the same. As a result, the present invention has been completed.

[0011] That is, the process for producing the fucose-containing oligosaccharide or the composition thereof of the present invention is carried out by the method of Chordariac family.
euc) or fucoidan obtained by extracting from seaweeds of the family Spermatochnaceae is subjected to acid hydrolysis, and then the fucose-containing oligosaccharide or a composition thereof is separated and purified.

In this case, it is preferable to use a mozuku (Nemacystis decipiens (Suringar) Kuckuck) belonging to the family Mozuku as the seaweed.

Further, the fucose-containing oligosaccharide or a composition thereof is a fucosyl-α1,3 represented by the following chemical formula 4.
-It is preferably fucose or an oligosaccharide composition containing it.

[0014]

Embedded image

Further, the fucose-containing oligosaccharide or a composition thereof is a xylosyl-β1,
It is preferably 2-fucose or an oligosaccharide composition containing it.

[0016]

Embedded image

Further, the fucose-containing oligosaccharide of the present invention or the composition thereof comprises the xylosyl-β represented by the above-mentioned formula (5).
It is 1,2-fucose or an oligosaccharide composition containing the same.

According to the present invention, a fucose-containing oligosaccharide having a well-defined structure or an oligosaccharide composition containing the same can be obtained as a partial hydrolyzate of fucoidan, which is a natural product, and these are used as food materials. Can be used. In addition, since a single oligosaccharide having a clear structure can be obtained as a fucose-containing oligosaccharide, it can be used as a substrate for measuring the activity of a fucoidan-related compound-degrading enzyme or as a raw material for synthesis in the field of sugar chain engineering.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION As a raw material of seaweed used in the present invention, fucoidan contained in the seaweed is preferably relatively simple, and is preferably stably available in large quantities. For example, when fucoidan containing a large amount of uronic acid is hydrolyzed, it is not preferable because the generated oligosaccharide has a high probability of containing a uronic acid residue, which extremely deteriorates the yield of the target oligosaccharide.

Specifically, brown algae containing fucoidan containing almost no uronic acid residue are preferred, and mozuku and Okinawa mozuku are particularly preferred. The mozuku may be a natural product or a cultured product, but it is preferable to use a cultured mozuku for stable supply and stable sugar composition. When used in the present invention, it is preferable that these collected seaweeds are washed lightly with water and used as they are.

-Method for extracting fucoidan In the present invention, as a method for extracting fucoidan from the above-mentioned raw material seaweed, an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid or the like, or acetic acid, citric acid, or shu An aqueous solution of an organic acid such as acid, succinic acid, formic acid, propionic acid,
H1-5, a method in which the raw material concentration is 0.1-3% by weight as a dry matter, and the mixture is extracted by stirring at 10-100 ° C for 0-24 hours. Preferably, hydrochloric acid is added to the above-mentioned raw material at a pH of 2 to 3 and the raw material concentration is 2 to 3 as a dry product.
3% by weight, and 15 to 2 at 20 to 30 ° C.
Extract for 0 hours. More preferably, the raw material is pulverized with a mixer or the like before extraction.

The extract thus obtained is filtered, neutralized to pH 7 to 8 with an alkaline agent, and concentrated under reduced pressure until the solid content concentration becomes 1 to 30% by weight. A crude fucoidan can be obtained by adding 2 to 4 times by weight of ethanol to the concentrated solution, refrigerated and allowed to stand for 0 to 24 hours, and then centrifuging to collect and dry the precipitate.

In the present invention, the purity of fucoidan used for acid hydrolysis is not necessarily required to be high, and the above crude extract can be used as long as the separation and purification of the fucose-containing oligosaccharide is not hindered. It is preferable to use purified fucoidan prepared as follows.

After dissolving the above crude fucoidan in 30 to 100 times by weight of demineralized water, cetylpyridinium chloride (hereinafter referred to as CP)
C), and the mixture is stirred at room temperature for 10 to 20 hours and then centrifuged to collect a precipitate. This sediment is
After dissolving in 4 times by weight of 4M calcium chloride solution, removing insolubles by filtration, adding 2 to 3 times by weight of ethanol, stirring, leaving still at 4 to 25 ° C for 2 to 8 hours, centrifuging to precipitate. Purified fucoidan can be obtained by collecting and drying the substance (Agricultural and Biological Chem
istry Vol. 30, No. 5, p. 495-499, 1966).

Hydrolysis of fucoidan Using fucoidan obtained as described above, preferably purified fucoidan as a raw material, inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, and succinic acid The hydrolysis is carried out in the presence of an organic acid such as formic acid, propionic acid and the like. Preferred examples of the hydrolysis conditions include 0.01 to 1
2N, preferably 0.2-1.0N hydrochloric acid, fucoidan concentration of 0.01-10% by weight, preferably 0.2-1.0%
At 1.0% by weight, the pH is added so as to be 0.1 to 1.0, preferably 0.1 to 0.5.
The hydrolysis reaction is carried out preferably at 75 to 85 ° C for 0 to 24 hours, preferably 0.5 to 2 hours.

After the completion of the reaction, the reaction solution is immediately cooled and neutralized to pH 7 to 8 with an alkali agent such as sodium hydroxide or sodium carbonate. The neutralized solution is decolorized with activated carbon, filtered, and desalted with an electrodialysis device to obtain a fucose-containing oligosaccharide composition.

Purification of Fucose-Containing Oligosaccharides The above-mentioned composition was used as “Dowex 1 × 4” (trade name, manufactured by The Dow Chemical Company), “DEAE-Seph”
adex A-25 "(trade name, manufactured by Amersham Pharmacia Biotech) and the like.
Collect the flow through fraction. The flow-through fraction is further passed through an activated carbon column to remove monosaccharides to obtain an oligosaccharide fraction.

The oligosaccharide fraction is subjected to, for example, HPLC preparative chromatography using an NH 2 column to fractionate and purify, whereby a fucose-containing oligosaccharide can be obtained.

One of the fucose-containing oligosaccharides obtained by such a method is the fucosyl-α represented by the above formula (4).
1,3-fucose, and another was confirmed to be xylosyl-β1,2-fucose represented by Chemical Formula 5.

The thus obtained fucoidan-containing oligosaccharide or composition thereof may be, for example, a substrate for measuring the activity of a fucoidan-related compound-degrading enzyme, a substrate for measuring the activity of α-fucosidase, a substrate for measuring the activity of β-xylosidase, an inhibitor, etc. For use as a medical material by incorporating it into reagents, diagnostic agents, synthetic polymers such as styrene, as a pharmaceutical composition or synthetic raw material, and as an intestinal environment improving substance for the purpose of promoting the growth of lactic acid bacteria For the purpose of adding and blending in foods, sanitary foods etc. for the purpose of preventing infection of pathogenic bacteria,
Or it can be used for toiletry products such as food additives, cosmetic materials, and materials for bath additives.

[0031]

EXAMPLES The present invention will now be described specifically with reference to examples, but the present invention is not limited by these examples.

Test Example An analysis of seaweeds was carried out on seaweeds (brown algae) available on the coast of Japan. Commercial sample (raw) as sample
Wakame, Hijiki, Kombu, Mozuku, Okinawa mozuku, and Hondawara, Yolemoku, Kajime and Alame collected from near seas were used.

After extracting and purifying fucoidan from each seaweed by a conventional method, and hydrolyzing and desalting each fucoidan with hydrochloric acid,
HPLC analysis was performed, and constituent sugar analysis (fucose content, neutral sugar composition) of each fucoidan was performed. as a result,
Mozuku and Okinawa mozuku were found to have relatively high fucose contents and low contents of other neutral sugars such as xylose, mannose and glucose. Further, uronic acid content, sulfate group content and the like were measured for mozuku and Okinawa mozuku, and the results are shown in Table 1.

From Table 1, it can be seen that Mozuku (Nemacystis decipiens)
Fucoidan derived from (Suringar) Kuckuck) has a low uronic acid content and a high fucose content in the neutral sugar composition, indicating that it is optimal as a raw material for preparing a fucose-containing oligosaccharide.

[0035]

[Table 1]

Example 1 (1) Extraction of fucoidan To 10 kg of raw mozuku, 30 L of 0.2N hydrochloric acid was added, and the mixture was crushed with a cooking mixer, followed by extraction at 25 ° C. for 18 hours with stirring. This solution was filtered to remove extraction residues, and then neutralized to pH 7.5 with 2N sodium hydroxide solution.
After removing the insoluble matter by filtration, the solution was concentrated by heating under vacuum until the liquid amount became 4 L. Eight liters of ethanol was added to this concentrated solution, stirred, refrigerated and allowed to stand for 18 hours, and then centrifuged (1
(000 × g, 15 minutes), and the precipitate was collected and dried under vacuum to obtain 153 g of crude fucoidan.

(2) Separation and purification of fucoidan 153 g of the obtained crude fucoidan was dissolved in 5 L of deionized water,
After adding 215 g of CPC and stirring at room temperature for 18 hours, the precipitate was collected by centrifugation (10,000 × g, 15 minutes). The precipitate was dissolved in 8 L of a 4 M calcium chloride solution, stirred for 18 hours, and then filtered using celite to remove insolubles. Ethanol 24 was added to the resulting solution.
L was added, stirred, cooled for 6 hours, and centrifuged in the same manner as above to collect a precipitate. This precipitate is deionized water 1.5
L and desalted using a dialysis tube. The dialysate was adjusted to pH 7.1 to 7.5 with 0.5N sodium hydroxide, and then freeze-dried to obtain 36.5 g of purified fucoidan.

(3) Hydrolysis of fucoidan 8 g of the above purified fucoidan was dissolved in 1.6 L of 0.5N hydrochloric acid and hydrolyzed at 80 ° C. for 1 hour with stirring. After cooling the reaction solution, pH was adjusted with 1 to 4N sodium hydroxide.
The mixture was neutralized to 7.5, 5 g of activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred for 1 hour to be decolorized. After filtering through a 0.45 μm membrane filter to remove activated carbon, desalting was performed with a micro-acyler (Asahi Kasei Kogyo) and freeze-dried to obtain 11.6 g of fucoidan hydrolyzate.

(4) Separation and Purification of Fucose-Containing Oligosaccharides After dissolving 2.9 g of the fucoidan hydrolyzate in 0.1 L of distilled water, "Dowex 1 × 4 (200-400mes)
h) "(trade name, manufactured by The Dow Chemical Company) and subjected to column chromatography to collect a flow-through fraction. This collected fraction was directly subjected to activated carbon-celite column chromatography under the following conditions to fractionate an oligosaccharide fraction.

Column: activated carbon (manufactured by Wako Pure Chemical): Celite (manufactured by Wako Pure Chemical) = 20 g: 20 g Flow rate: 1.6 ml / min Temperature: 25 ° C. Detection wavelength: 490 nm (phenol sulfate method) Solvent A: distilled water Solvent B: ethanol Concentration gradient: 0 to 30% B / 840 minutes The results are shown in FIG. In FIG. 1, the fraction (2) shown as F-1
The sugar fraction) is collected, concentrated, and further fractionated under the following conditions.
LC chromatography was performed.

Column: “Asahipak NH2P-90 (21.
5φ × 300 L) ”(trade name, manufactured by Showa Denko) Flow rate: 4 ml / min Temperature: 25 ° C. Detector: differential refractometer Solvent: water: acetonitrile = 1: 4 The results are shown in FIG. In FIG. 2, F-1a and F-1b
Were collected and lyophilized to obtain 3 mg of F-1a fraction and 2.7 mg of F-1b fraction.

(5) Nuclear magnetic resonance analysis Nuclear magnetic resonance analysis was performed on the F-1a fraction and the F-1b fraction. The results are shown in FIGS.

FIG. 3 shows that the F-1a fraction (fucosyl-α1,
It is a figure which shows CH COSY Spectrum of (3-fucose).
About the result measured at 30 ° C in heavy water, the vertical axis shows D of C13.
ownfield shift, H1 downfield shift on horizontal axis
The values are shown based on PS (sodium 3- (trimethylsilyl) -propionate). 77.08 with chemical shift value of C13
From the signals at ppm and 80.58 ppm, it can be seen that there is a bond at the carbon at the C-3 position, and from the chemical shift values at 97.97 ppm and 98.32 ppm, there is an α bond at the carbon at the C′-1 position. The number of carbon atoms and the sequence indicate that the product is a fucosyl fucose disaccharide, and thus fucosyl-α1,
3-Fucose was confirmed.

FIG. 4 shows the F-1b fraction (xylosyl-β
It is a figure which shows CH COSY Spectrum of (1, 2-fucose). The vertical axis shows C1 in the results measured at 30 ° C in heavy water.
The Downfield shift of No. 3 was shown on the horizontal axis, and the Downfield shift of H1 was measured by using TPS (sodium 3- (trimethylsilyl) -propionate) as a reference. From the signals of 82.55 ppm and 83.44 ppm in the chemical shift value of C13, there is a bond at the carbon at position C-2, and from the chemical shift values of 106.71 ppm and 106.76 ppm, there is a β bond at the carbon at position C'-1. I understand. The carbon number and the sequence revealed that the product was a xylosyl fucose disaccharide, and thus it was confirmed that the product was xylosyl-β1,2-fucose represented by the above Chemical Formula 5.

[0045]

As described above, according to the present invention,
The fucose-containing oligosaccharides or the oligosaccharide compositions containing the fucose-containing oligosaccharides, which are difficult to prepare by chemical synthesis, as shown in the chemical formulas 3 and 4, can be easily and efficiently obtained by hydrolyzing fucoidan, which is a natural product. Can be manufactured well.

The fucose-containing oligosaccharide or the oligosaccharide composition containing the same obtained by the present invention is obtained by hydrolyzing a natural product as described above, and has a clear structure. In addition to being usable, it can be used as a substrate for measuring the activity of a fucoidan-related compound-degrading enzyme, a synthetic raw material in the field of sugar chain engineering, and the like.

[Brief description of the drawings]

FIG. 1 is a view showing a chromatogram obtained by separating a fraction passing through a Dowex 1 × 4 column by activated carbon-celite column chromatography.

FIG. 2 is a view showing a chromatogram obtained by separating an F-1 fraction (a disaccharide fraction) by preparative HPLC chromatography.

FIG. 3 is a view showing an H1, C13-NMR spectrum of an F-1a fraction (fucosyl-α1,3-fucose).

FIG. 4 is a view showing an H1, C13-NMR spectrum of an F-1b fraction (xylosyl-β1,2-fucose).

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C07H 3/06 C07H 3/06 // A61K 35/80 A61K 35/80 Z F term (reference) 4B018 MD31 MD67 ME14 MF01 MF10 4C057 AA05 BB03 BB04 CC01 4C086 AA03 AA04 EA01 MA04 MA52 MA63 NA14 ZA66 ZB35 ZC20 4C088 AA13 BA12 CA14 CA22 ZA66 ZB35 ZC20

Claims (5)

[Claims] 1. A fucose-containing oligosaccharide or a fucose-containing composition characterized by separating and purifying a fucose-containing oligosaccharide or a composition thereof after acid hydrolysis of fucoidan obtained by extracting from seaweeds of the family Pineaceae (Chordariaceae) or Spermatochnaceae. A method for producing an oligosaccharide or a composition thereof. 2. As the seaweed, mozuku (Ne mozuku) of the family Mozuku
The method for producing a fucose-containing oligosaccharide or a composition thereof according to claim 1, wherein macystis decipiens (Suringar) Kuckuck) is used. 3. The fucose according to claim 1, wherein the fucose-containing oligosaccharide or a composition thereof is fucosyl-α1,3-fucose represented by the following chemical formula 1 or an oligosaccharide composition containing the same. A method for producing an oligosaccharide or a composition thereof. Embedded image 4. The method according to claim 1, wherein the fucose-containing oligosaccharide or the composition thereof is xylosyl-β1,2- represented by the following chemical formula 2.
The method for producing a fucose-containing oligosaccharide or a composition thereof according to any one of claims 1 to 3, which is fucose or an oligosaccharide composition containing the same. Embedded image 5. Xylosyl-β represented by the following chemical formula 3:
A fucose-containing oligosaccharide or a composition thereof, which is 1,2-fucose or an oligosaccharide composition containing the same. Embedded image