JP2835152B2 - Arabinoxylo-oligosaccharide - Google Patents

Description

The present invention relates to a novel arabinoxylo-oligosaccharide.

[Content of the invention]

Oligosaccharides have recently attracted attention as one of the functional food materials, and various oligosaccharides have been developed and commercialized.

Under such circumstances, the present inventors have studied oxygen treatment of hemicellulose derived from wheat bran. As a result, when the hemicellulose is hydrolyzed with endoxylanase, an oligosaccharide mixture containing various oligosaccharides is produced, and in the oligosaccharide mixture, four xylose molecules and two arabinose molecules are located at specific positions. We discovered that a novel arabinoxylo-oligosaccharide attached was involved and isolated it.

Therefore, the present invention provides the following formula: Is an arabinoxylo-oligosaccharide represented by

As is clear from the above formula, the arabinoxylo-oligosaccharide of the present invention has a xylose molecule having a 6-membered ring structure of 4
It has a structure in which two arabinose molecules having a five-membered ring structure are adjacently bonded to the third xylose from the reducing end of the main chain by bonding in an individual chain to form a main chain.

The arabinoxylo-oligosaccharide of the present invention can be prepared by hydrolyzing hemicellulose derived from wheat bran with endoxylanase to produce an oligosaccharide mixture, and isolating the arabinoxylo-oligosaccharide from the oligosaccharide mixture. In this case, as a bran-derived hemicellulose to be subjected to endoxylanase treatment, water-insoluble hemicellulose,
Preferably, one containing at least one of a water-soluble, 60% ethanol-insoluble hemicellulose and a water-soluble, anion exchanger non-adsorbable hemicellulose,
Even if endoxylanase is allowed to act on% ethanol-soluble hemicellulose, hydrolysis is not performed smoothly and oligosaccharides including the arabinoxylo-oligosaccharide of the present invention are not efficiently produced. The above water-insoluble hemicellulose, water-soluble 60% ethanol-insoluble hemicellulose (hereinafter simply referred to as "60% ethanol-insoluble hemicellulose") and water-soluble anion exchanger non-adsorbable hemicellulose (hereinafter "anion exchanger non-adsorbed hemicellulose") )) Can be used, and the preparation method and the like thereof are not particularly limited, but those hemicelluloses can be prepared, for example, by the following method.

(I) Water-insoluble hemicellulose: Wheat bran is extracted with an aqueous alkali solution, and then the aqueous alkali solution is neutralized with an acid, and the precipitated fraction is separated and recovered as water-insoluble hemicellulose. In this case, after performing an extraction treatment of wheat bran using an alkaline aqueous solution of about 0.15 to 0.30 N at a temperature of about 40 to 70 ° C, water-insoluble hemicellulose that precipitates when the wheat bran is neutralized with hydrochloric acid or the like. It is good to collect.

Hemicellulose obtained from wheat bran by alkali extraction or the like is once recovered as a solid content, dissolved in water, and its water-insoluble fraction is separated and recovered as water-insoluble hemicellulose. In this case, about 25 to 35% by weight of the bran-derived hemicellulose is recovered as water-insoluble hemicellulose.

(Ii) 60% ethanol-insoluble hemicellulose: 60% water-soluble hemicellulose prepared by any method
Dissolve in ethanol aqueous solution and separate and collect insoluble fraction.

(Iii) Anion exchanger non-adsorbed hemicellulose: A solution obtained by dissolving water-soluble hemicellulose in a buffer solution is passed through an anion exchanger to recover a liquid containing the non-adsorbable fraction, and this liquid is subjected to an ultrafiltration membrane. And freeze-dried to obtain the desired anion exchanger-free adsorbed hemicellulose.
In this case, as a buffer, a Tris-HCl buffer or a phosphate buffer having a pH of about 7.5 to 8.5 is used, and a solution obtained by dissolving about 150 to 250 ml of water-soluble hemicellulose in 20 ml of the buffer is used as a cellulose. Anion exchangers with a diethylaminoethyl group (hereinafter referred to as “DEAE”) bound to
The solution containing non-adsorbed hemicellulose is recovered from the lower part of the anion exchanger by passing the solution at a flow rate of 1.5 ml / min.

Then, in obtaining the arabinoxylo-oligosaccharide of the present invention, at least, for example, at least one of water-insoluble hemicellulose derived from wheat bran, 60% ethanol-insoluble hemicellulose and anion exchanger non-adsorbed hemicellulose prepared as described above. One or a hemicellulose obtained by extracting wheat bran with an alkali or the like is hydrolyzed with endoxylanase to produce an oligosaccharide mixture.

The endoxylanase used at this time is also called β-xylanase, but any endoxylanase can be used. When commercially available oxygen is used, endoxylanase contained in cell wall decomposed oxygen or the like may be used because it is difficult to obtain endoxylanase alone. Examples include "Cellulase Onozuka" manufactured by Yakult, "Pectriase Y-23" manufactured by Seishin Pharmaceutical Co., Ltd.,
Examples include endoxylanase contained in "Mecerase" manufactured by Sanko Junyaku Co., Ltd. Among them, the endoxylanase contained in "Cellulase Onozuka" manufactured by Yakult is particularly preferable because it has a high enzyme activity and can obtain an oligosaccharide mixture in a high yield.

Endoxylanase may be used in a free state or immobilized on a carrier, and the hydrolysis treatment with endoxylanase may be performed by a continuous method or a batch method. The treatment is carried out by appropriately selecting various conditions such as the origin of the endoxylanase, its use amount, temperature, pressure, pH, time and the like during the treatment.

In the present invention, as an indication of the end of the hydrolysis treatment of hemicellulose with endoxylanase, the enzyme is inactivated at an appropriate time when the viscosity of the aqueous solution containing hemicellulose sharply decreases at first and then gradually decreases.

For example, when the above-mentioned hemicellulose is converted into a solution having a concentration of about 20 mg / ml and an aqueous solution having a pH of about 5 to 6, to which about 0.2 units / ml of endoxylanase is added and hydrolyzed at a temperature of about 50 ° C. for about 8 hours, Can be obtained.

The oligosaccharide mixture produced as described above is a mixture (mixed oligosaccharide) of a xylooligosaccharide having only xylose bonded thereto and an arabinoxylooligosaccharide having xylose and arabinose bonded, and is usually a xylooligosaccharide and an arabinoxylooligosaccharide. And in substantially the same proportion.
The mixture oligosaccharide, xylose or xylose and arabinose are bonded 2-6, formula X _n A _m (wherein, X: xylose, A: arabinose, n: number of bonds xylose, m: arabinose And n is usually 2 to 2.
4, m = 0 to 2) is a mixture represented by, arabinoxylo oligosaccharides of the present invention is obtained as one of oligosaccharide represented by X ₄ A _2.

The arabinoxylo-oligosaccharides of the present invention can be isolated by directly treating the hydrolyzed solution with the endoxylanase containing various oligosaccharides or monosaccharides produced as described above, or once the oligosaccharides are hydrolyzed from the hydrolyzed solution. Or a mixture of monosaccharides may be separated and recovered, and then isolated therefrom.

In order to directly treat the hydrolyzed solution with endoxylanase to isolate the arabinoxylo-oligosaccharide of the present invention, for example, a method comprising the following steps (a) to (g) can be adopted.

Isolation method of arabinoxylo-oligosaccharides (a) Desalting a hydrolyzed solution containing various oligosaccharides and the like through an ion exchange resin (for example, Amberlite MB-3 manufactured by Organo Kogyo Co., Ltd.); (b) (C) filtering the supernatant using a microfilter having a pore size of 0.45 μm, and then concentrating the solution by an evaporator to form a concentrated solution; (d) concentrating the supernatant. The solution is subjected to gel filtration chromatography (for example,
Column diameter 2.6 cm, column length 90 cm; column packing Toyopearl HW-40S manufactured by Tosoh Corporation; (e) High performance liquid chromatography (HPLC) for separation under specific conditions; (f) Purpose and Until the unity of the fraction to be confirmed is confirmed (d)
(G) The structure of the substance is determined at the stage where unity is confirmed, and it is confirmed that the target substance is obtained.

Separation and recovery of the oligosaccharide mixture from the hydrolyzed solution by endoxylanase can be performed, for example, by the following methods (1) to (4).

The oligosaccharide-containing hydrolyzate is desalted by passing it through an ion-exchange resin (for example, Amberlite MB-3), centrifuged, and the supernatant is filtered through a microfilter having a pore size of 0.45 μm. When the activated carbon adsorbing section is eluted with 70% ethanol, the eluate of the oligosaccharide mixture containing a small amount of monosaccharide is recovered. A method for obtaining an oligosaccharide mixture containing a monosaccharide.

In the case of this method, if the activated carbon adsorbing section is treated with 5% ethanol to elute and remove the monosaccharides before eluting the activated carbon adsorbing section with 70% ethanol, the 70%
From the solution eluted with ethanol, a high-purity oligosaccharide containing no monosaccharide can be obtained.

A method of centrifuging an oligosaccharide-containing hydrolyzate, drying the supernatant obtained by treating the supernatant with an ultrafiltration membrane, and collecting an oligosaccharide mixture containing a small amount of monosaccharide.

The oligosaccharide-containing hydrolyzate is desalted by passing it through an ion exchange resin (for example, Amberlite MB-3), centrifuged, and the supernatant is subjected to a microfilter (pore size: 0.45 μm).
Through. The filtrate is concentrated to about 5 ml by an evaporator and subjected to gel filtration chromatography (for example, Toyopearl HW-40S). In the eluate, except for the high molecular fraction having a molecular weight of about 2,000 or more, the oligosaccharide fraction appearing thereafter is collected, and the solid oligosaccharide is recovered by freeze-drying or the like. A single oligosaccharide can also be obtained by finely separating the eluate from gel filtration chromatography.

Among the above methods, particularly the method is effective when water-insoluble hemicellulose, 60% ethanol-insoluble hemicellulose and anion exchanger non-adsorbed hemicellulose are used as raw materials, and it is possible to obtain oligosaccharides with high purity in high yield. it can.

Further, the method is effective when hemicellulose derived from wheat bran is used as a raw material as it is, and high-molecular-weight water-soluble hemicellulose hydrolyzed with endoxylanase by activated carbon treatment is smoothly separated as an activated carbon non-adsorbing section. .

For example, in order to isolate the arabinoxylo-oligosaccharide of the present invention from the oligosaccharide mixture separated and recovered by the above methods (1) to (4), the oligosaccharide mixture is dissolved in water to form an aqueous solution, and this aqueous solution is used. (C) to above
The step (g) may be performed.

Since the arabinoxylo-oligosaccharide of the present invention was prepared from hemicellulose derived from wheat bran, arabinose, one of its constituent units, is L-arabinose, as in hemicellulose derived from wheat bran.

The arabinoxylo-oligosaccharide of the present invention is a white solid, has high hygroscopicity, has high solubility in water, and has various characteristics of showing almost no sweetness. And can be used effectively for other purposes.

Hereinafter, the present invention will be described specifically by way of examples, but the present invention is not limited thereto. All percentages in the examples below are by weight.

Reference Example 1 2 kg of selected wheat bran is dispersed in warm water 20 at 50 ° C. and stirred for 5 minutes. Thereafter, the solid content was recovered by filtration with a centrifugal filter (manufactured by Tanabe Iron Works), and 3 kg of the obtained solid content was 0.2 N at 70 ° C.
Stir for 90 minutes in aqueous sodium hydroxide solution 20 to dissolve. After allowing to cool, a 0.8N aqueous hydrochloric acid solution 5 is gradually added with stirring to neutralize. The neutralized solution was centrifuged at 5,000 G for 10 minutes, and the supernatant and the precipitate were recovered to obtain 190 g of water-insoluble hemicellulose (total sugar content 32.1%, protein content 10.0%, and other components 57.9%).

Reference Example 2 Water 20 was added to the supernatant liquid 20 obtained in Reference Example 1 for dilution, and while maintaining the solution temperature at 50 ° C., a tubular ultrafiltration membrane NTU 3520 manufactured by Nitto Denko (Model P-18, 0.76m ² membrane area, inside diameter
(11.5 mm) through a tube and treated for 3 hours under the conditions of a pressure of 8 kg · f / cm ² and a flow rate of 13 / min. At this time, the same amount of water as the membrane permeating solution is always replenished into the tube, and the amount of the membrane treatment solution is kept constant. After 3 hours, the supply of water was stopped and the same conditions as above (pressure 8 kg · f / c
Concentration is started at m ² , flow rate 13 / min) and concentrated without considering the decrease in flux.
Repeat until / ml. The treated solution is eluted into 500 cc of cation exchange resin (IR-120E) manufactured by Organo at a flow rate 10 times the ion exchange resin capacity per hour, and then to the anion exchange resin (IRA-93) manufactured by the same company at the same flow rate. Shed. The aqueous solution obtained after the ion exchange treatment is freeze-dried under vacuum at a temperature of 30 ° C. and a degree of vacuum of 0.1 Torr or less, and about 150 g of water-soluble hemicellulose is obtained.
I got

Ethanol was added to a solution obtained by dissolving 50 g of this water-soluble hemicellulose in water 0.7, and the ethanol concentration of the solution was reduced.
The mixture was adjusted to 60%, stirred for about 20 minutes and allowed to stand. The resulting precipitate was washed with 60% ethanol and freeze-dried to obtain 20 g of 60% ethanol-insoluble hemicellulose.

Reference Example 3 50 g of the water-soluble hemicellulose prepared in Reference Example 2 was
0 mmol (mM) of Tris-HCl buffer (pH 8.5) 5 and dissolved in an anion exchanger (DEAE-Sepharose CL).
(-6B: Pharmacia) was passed through a column packed with 0.2 to collect the liquid containing the non-adsorbed fraction. The liquid was dried to obtain 20 g of hemicellulose without adsorbing anion exchanger.

Example 1 Water-insoluble hemicellulose prepared in Reference Example 1 above was used.
5 g was added to 50 ml of a sodium phosphate buffer (pH 6.0) to prepare a 1% solution of water-insoluble hemicellulose.

Then, the solution was preliminarily heated to 50 ° C., and 6 mg (50 units of xylanase) of cellulase “Onozuka RS” (manufactured by Yakult) was added thereto.
Time went on. Then boil to inactivate the enzyme,
2 ml was collected and centrifuged at 3000 rpm for 10 minutes. Supernatant 1m
l through a tube of a tubular ultrafiltration unit (Molcut II LCC type: membrane area 13 cm ² , inner diameter 17 mm, manufactured by Nippon Millipore Co., Ltd.) under pressure (2 kg / cm ² ), and the resulting effluent is HPLC analysis was performed under the following conditions.

HPLC analysis Injection volume: 20μ Column type: Shodex KS-802 + KS-801 Eluent: pure water Flow rate: 0.7 ml / min Temperature: 80 ° C Detector: differential refractometer As a result, the chromatogram shown in Fig. 1 Grams were obtained. Each fraction of peaks 1 to 8 in FIG. 1 was acid-hydrolyzed and then subjected to HPLC again to examine the composition of constituent sugars. The total sugar content was determined by the phenol-sulfuric acid method, and the reducing sugar content was determined by the Somogyi-Nelson method to determine the degree of polymerization of each peak.

 The results are shown in Table 1 below.

Next, the above fraction of peak 1 was collected and subjected to the above gel filtration chromatography and HP separation until identity was confirmed.
The LC analysis was repeated, and identity was confirmed when gel filtration chromatography and HPLC analysis were repeated twice.

Here, the confirmation of the unity was performed by adding 10 mg of the compound sample to 1.0 m
A 1% solution was prepared by dissolving in 1 l of pure water and subjected to HPLC and paper chromatography as shown in the following.

HPLC-1 Injection volume: 20μ Column type: Shodex KS-801 (Showa Denko KK) Eluent: Pure water Flow rate: 0.7 ml / min Temperature: 80 ° C Detector: Differential refractometer Principle: Gel Filtration + ligand exchange This resulted in a single peak at a retention time of 7.5 minutes.

HPLC-2 Injection volume 20μ Column type: Senshu Pack-NH ₂ (manufactured by Senshu Kagaku) Eluent: acetonitrile: water = 65:35 Flow rate: 1.0 ml / min Temperature: room temperature Detector: differential refractometer Principle: Distribution As a result, a single peak was obtained at a retention time of 12.6 minutes.

Paper chromatography Paper Toyo Filter Paper No.50 Liquid volume used: 1 μ Separation liquid: butanol: pyridine: water = 6: 4: 4 Color former: silver nitrate As a result, a single spot was obtained at an Rf value around 0.24.

Next, the compound whose unity was confirmed as described above was subjected to the following (1) measurement of the degree of polymerization, (2) complete hydrolysis with a strong acid, (3) partial hydrolysis with a dilute acid, and (4) methylation analysis. , (5) Mass spectral analysis (SIMS method) and (6) ¹³ C-NMR analysis were used to determine the structure.

(1) Measurement of degree of polymerization: 10 mg of a compound sample was dissolved in 1.0 ml of pure water to prepare a 1% solution, the total sugar content of this solution was determined by the phenol-sulfuric acid method, and the amount of reducing sugar was determined by the Somogyi Nelson method. Determined to be 9.6 mg / ml and 1.6 mg / ml, respectively. From these values, it was found that the compound was a hexasaccharide.

(2) Complete hydrolysis with strong acid: 5 mg of the compound sample was dissolved in 1 ml of 2N trifluoroacetic acid and hydrolyzed at 100 ° C for 2 hours. After cooling, trifluoroacetic acid was removed with an evaporator, and the obtained dried product was dissolved by adding 0.5 ml of pure water, and analyzed by the above-described HPLC-1 method. As a result, the constituent sugars of the compound were only xylose and arabinose, and the area ratio between xylose and arabinose was about 2: 1. When this result is combined with the result of the above (1), it is understood that the compound is an oligosaccharide composed of 4 molecules of xylose and 2 molecules of arabinose.

(3) Partial hydrolysis with dilute acid: Dissolve 5 mg of the compound sample in 1 ml of 0.1 N sulfuric acid, and add
After hydrolyzing at 2 ° C. for 2 hours, the mixture was neutralized with barium carbonate.
Next, the mixture was centrifuged, and the supernatant was passed through a microfilter having a pore size of 0.45 μm, and the filtrate was analyzed by the HPLC-1 method described above. As a result, it was confirmed that a xylotetraose peak appeared at a retention time of 8.5 minutes. confirmed.
From this, it was found that the oligosaccharide composed of 4 molecules of xylose and 2 molecules of arabinose confirmed by the above (1) and (2) was an oligosaccharide in which 2 molecules of arabinose were bound to xylotetraose.

(4) Methylation analysis: After 2 mg of the compound sample was methylated by the Hakomori method, it was concentrated to dryness by an evaporator. The resulting dried product was hydrolyzed using 88% formic acid and 1N trifluoroacetic acid. This was concentrated to dryness by an evaporator, reduced with sodium borohydride, then acetylated, and analyzed by gas chromatography. As a result, 2,3,4-trimethylxylitol monoacetate, 2,3,5-trimethylarabinitol monoacetate, 2,3-dimethylxylitol diacetate and xylitol tetraacetate were obtained.

Further, the compound sample was reduced with sodium borohydride to prepare a reduced sample, and the reduced sample 2 m
Using g, methylation, hydrolysis,
Reduction and gas chromatography analysis were performed. As a result, 1,2,3-trimethylxylitol monoacetate, 2,3,4-trimethylxylitol monoacetate,
2,3,5-trimethylarabinitol monoacetate, 2,3
-Dimethylxylitol diacetate and xylitoltetraacetate were obtained.

From the above, that arabinose is not bound to any of the reducing end and xylose at the non-reducing end,
It was found that arabinose was bound to positions 2 and 3 of xylose.

(5) Mass spectrum analysis (SIMS method): 10 mg of a compound sample was mixed with 0.1 ml of glycerol, and then applied to a stainless steel sample table. Xenon gas was applied to this to ionize it, supplemented with ions generated by a mass spectrometer (M-80: manufactured by Hitachi, Ltd.), and processed by a data processor (M-80).
003: Hitachi, Ltd.). As a result, a parent peak having a mass of 833 and a fragment peak having a mass of 265 were obtained.

(6) ¹³ C-NMR analysis: In order to determine the position of xylose to which arabinose was bound in the above compound, ¹³ C-NMR spectra of xylotetraose and the above compound were measured, and both spectra were compared.

That is, 90 mg of xylotetraose and 30 mg of each of the above compounds were dissolved in 0.6 ml of heavy water, and then dioxane was added as an internal standard. This was placed in a sample tube having a diameter of 5 mm, and analyzed using an NMR analyzer (JEOL GSX-270W; manufactured by JEOL Ltd.) under the conditions of a resonance frequency of 67.9 MHz, a magnetic field strength of 6.3 T, and a temperature of 27 ° C. .

As a result, as for xylotetrate, ¹³ C in FIG.
-The NMR spectrum was measured for the above compound using ¹³ C-
NMR spectra were obtained.

According to the ¹³ C-NMR spectrum, the carbon at the 1-position of the first xylose from the reducing end of xylotetraose (
-C1), the first carbon of the second xylose (noted as -C1), the first carbon of the third xylose (noted as -C1), and the fourth xylose. The NMR signal for the carbon at position 1 (denoted by -C1) and the corresponding xylose of each of the above compounds, 1
Table 2 shows a comparison of the NMR signal for the carbon at the 2nd position.

When arranged in the order of the absolute value of the difference, >>>>>
Which indicates that arabinose is bound to the third xylose from the reducing end.

When the results of the above (1) to (6) were combined, it was found that the compound isolated as peak 1 in FIG. 1 was a novel arabinoxylo-oligosaccharide having the following structure.

The yield of the arabinoxylo-oligosaccharide was 4% based on the weight of the water-insoluble hemicellulose used as the raw material.
Met.

Example 2 The same treatment, analysis and structure determination as in Example 1 were conducted except that the 60% ethanol-insoluble hemicellulose prepared in Reference Example 2 was used instead of the water-insoluble hemicellulose. The arabinoxylo-oligosaccharide having two arabinose molecules bound to the third xylose from the reducing end of the same xylotetraose main chain was obtained.

The yield of this arabinoxylo-oligosaccharide was about 3% based on the weight of the 60% ethanol-insoluble hemicellulose used as the raw material.

Example 3 Except that the water-insoluble hemicellulose was replaced with the anion exchanger non-adsorbed hemicellulose prepared in Reference Example 3 above, the treatment, analysis and structure determination were performed in the same manner as in Example 1. An arabinoxylo-oligosaccharide in which two arabinose molecules are bonded to the third xylose from the reducing end of the same xylotetraose main chain as obtained in 1 was obtained.

The yield of the arabinoxylo-oligosaccharide was about 3% based on the weight of the anion exchanger non-adsorbed hemicellulose used as the raw material.

[Brief description of the drawings]

Accompanying drawings Figure 1 is a diagram showing the Clamato grams of each fraction obtained by HLPC analysis of the oligosaccharide mixtures produced in Example 1, FIG. 2 of xylotetraose ¹³
FIG. 3 is a diagram showing a C-NMR spectrum, and FIG. 3 is a diagram showing a compound ^{13 of the} oligosaccharide mixture fraction produced in Example 1 which was purified from the peak 1 fraction and confirmed to have unity.
It is a figure which shows a C-NMR spectrum.

────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Kaoru Shiiba 78-6 Shimoshingashi, Kawagoe-shi, Saitama Prefecture (72) Inventor Hiroyoshi Hara 2-10-10 Higashi-Mizuhodai, Fujimi-shi, Saitama 29 (58) Field surveyed (Int. Cl. ⁶ , DB name) C08B 37/00 C12P 19/14 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. The following formula: An arabinoxylo-oligosaccharide represented by: