JP2807575B2 - Arabinoxylo-oligosaccharide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel arabinoxylo-oligosaccharide.

[0002]

Description 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 on enzymatic 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.

Accordingly, the present invention provides the following formula:

Embedded image Is an arabinoxylo-oligosaccharide represented by

As is apparent from the above formula, the arabinoxylo-oligosaccharide of the present invention comprises a xylose molecule having a 6-membered ring structure bonded to four chains to form a main chain, and the reducing end of the main chain. Has a structure in which two arabinose molecules having a 5-membered ring structure are adjacently bonded to the third xylose.

The arabinoxylo-oligosaccharide of the present invention is produced by hydrolyzing hemicellulose derived from wheat bran with endoxylanase to produce an oligosaccharide mixture, and isolating the arabinoxylo-oligosaccharide from the oligosaccharide mixture. it can. In this case, the bran-derived hemicellulose subjected to the endoxylanase treatment includes at least one of water-insoluble hemicellulose, water-soluble 60% ethanol-insoluble hemicellulose, and water-soluble anion exchanger non-adsorbable hemicellulose. It is preferable to use it. Even if endoxylanase is allowed to act on 60% ethanol-soluble hemicellulose, hydrolysis is not performed smoothly and oligosaccharides including the arabinoxylo-oligosaccharide of the present invention are not efficiently produced. The above-mentioned 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-adsorbent”) Any of these can be used as the “hemicellulose”, and its preparation method and the like are not particularly limited, but those hemicelluloses can be prepared, for example, by the following method.

(I) Water-insoluble hemicellulose: Wheat bran is subjected to an extraction treatment with an aqueous alkali solution, 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 the wheat bran is extracted using an alkaline aqueous solution of about 0.15 to 0.30 at a temperature of about 40 to 70 ° C., it is precipitated when neutralized with hydrochloric acid or the like. Preferably, the water-insoluble hemicellulose is recovered.

[0008] 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: water-soluble hemicellulose prepared by an arbitrary method
Dissolve in 0% 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 is passed through an anion exchanger, and a liquid containing the non-adsorbable fraction is recovered. After concentrating with an outer filtration membrane, freeze-drying is performed to obtain the desired anion exchanger-free adsorbed hemicellulose. In this case, a Tris-HCl buffer or a phosphate buffer having a pH of about 7.5 to 8.5 is used as a buffer, and a water-soluble hemicellulose of about 150 to 2 is used for 20 ml of the buffer.
The solution in which 50 ml is dissolved is passed at a flow rate of 0.5 to 1.5 ml / min through an anion exchanger in which a diethylaminoethyl group (hereinafter referred to as "DEAE") is bonded to cellulose, and the solution is passed from the bottom of the anion exchanger. The liquid containing non-adsorbed hemicellulose is recovered.

In order to obtain the arabinoxylo-oligosaccharide of the present invention, first, for example, a water-insoluble hemicellulose derived from wheat bran prepared as described above, 6
At least one of 0% ethanol-insoluble hemicellulose and anion exchanger non-adsorbed hemicellulose, or hemicellulose obtained by extracting wheat bran with alkali or the like is hydrolyzed with endoxylanase to produce an oligosaccharide mixture. Let it.

The endoxylanase used at that time is as follows:
Although it is also called β-xylanase, any endoxylanase can be used. When a commercially available enzyme is used, endoxylanase contained in a cell wall degrading enzyme or the like may be used because it is difficult to obtain an endoxylanase alone. Examples thereof include endoxylanase contained in "Cellulase Onozuka" manufactured by Yakult, "Pectlase Y-23" manufactured by Seishin Pharmaceutical Co., Ltd., "Mecerase" manufactured by Sanko Junyaku Co., Ltd. Among them, the endoxylanase contained in Yakult's "Cellulase Onozuka" has high enzyme activity,
This is preferable because an oligosaccharide mixture can be obtained in a high yield.

[0013] 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 endoxylanase, the amount used, temperature, pressure, pH, time and the like during the treatment.

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

For example, the above-mentioned hemicellulose is added at a concentration of about 20%.
mg / ml solution, aqueous solution of pH about 5-6,
When 2 units / ml of endoxylanase is added and hydrolyzed at a temperature of about 50 ° C. for about 8 hours, hydrolyzed solutions containing various oligosaccharides can be obtained.

The oligosaccharide mixture produced as described above is
It is a mixture (mixed oligosaccharide) of a xylooligosaccharide to which only xylose is bound and an arabinoxyloligosaccharide to which xylose and arabinose are bound, and usually contains xylooligosaccharide and arabinoxyloligosaccharide in almost the same ratio. The mixture oligosaccharide, xylose or xylose and arabinose are bonded 2-6, formula X _n A _m (wherein, X: xylose, A: arabinose,
n is the number of bonds of xylose, m is the number of bonds of arabinose, and is usually a mixture represented by n = 2 to 4, m = 0 to 2), and the arabinoxylo-oligosaccharide of the present invention is X ₄ A ₂
Is obtained as one kind of oligosaccharide represented by

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

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 employed.

[0019]Isolation of arabinoxylo-oligosaccharides  (a) Hydrolyzing solution containing various oligosaccharides and the like
Exchange resin (for example, Amber manufactured by Organo Kogyo Co., Ltd.)
Desalting through a light MB-3); (b) the desalted liquid is filtered and centrifuged;
And then concentrated by an evaporator to concentrate.
(D) forming the concentrate into a gel permeation chromatography (eg,
Column diameter 2.6cm, column length 90cm; column packing material
(Tosoh Corporation Toyopearl HW-40s); (e) High performance liquid chromatography (HPLC)
Separation under specific conditions; (f) until the identity of the desired fraction is confirmed (d)-
(e) Repeat the operation; (g) When the identity is confirmed, determine the structure of the substance
Go and confirm that the desired product is obtained.

The separation and recovery of the oligosaccharide mixture from the hydrolyzed solution by endoxylanase can be performed, for example, by the following methods:
It can be performed by the method of.

The oligosaccharide-containing hydrolyzate is desalted by passing it through an ion exchange resin (eg, Amberlite MB-3), centrifuged, and the supernatant is filtered through a microfilter having a pore size of 0.45 μm. When the activated carbon adsorption section is separated into an activated carbon adsorption section and a non-adsorption section through an activated carbon column, and the activated carbon adsorption section is eluted with 70% ethanol, an eluate of an oligosaccharide mixture containing a small amount of monosaccharides is recovered. To obtain an oligosaccharide mixture containing a small amount of monosaccharides.

In the case of this method, the activated carbon adsorption classification is 70
Before the elution with 5% ethanol, the activated carbon adsorption section is treated with 5% ethanol to elute and remove monosaccharides in advance. Is obtained.

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

The oligosaccharide-containing hydrolyzate is desalted through an ion exchange resin (eg, Amberlite MB-3), centrifuged, and the supernatant is passed through a microfilter (pore size: 0.45 μm). The filtrate was evaporated with an evaporator for about 5
and gel filtration chromatography (eg Toyope
arl HW-40S). Of the eluate, a molecular weight of about 200
The oligosaccharide fraction appearing thereafter is removed except for the high molecular fraction of 0 or more, 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.

The above method is particularly effective when water-insoluble hemicellulose, 60% ethanol-insoluble hemicellulose and anion exchanger non-adsorbed hemicellulose are used as raw materials, and high-purity oligosaccharides can be obtained in high yield. Obtainable.

The above 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 which is not hydrolyzed by endoxylanase by activated carbon treatment can be separated smoothly as a non-adsorbed section of activated carbon. Is done.

For example, in order to isolate the arabinoxylo-oligosaccharide of the present invention from the oligosaccharide mixture separated and recovered by the above-mentioned methods, the oligosaccharide mixture is dissolved in water to form an aqueous solution. Use above
The steps (c) to (g) may be performed.

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

The arabinoxylo-oligosaccharide of the present invention is a white solid, has high hygroscopicity, high solubility in water,
In addition, it has various properties of showing almost no sweetness, and based on such properties, can be effectively used for functional foods and other uses.

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

REFERENCE EXAMPLE 1 Selected wheat bran (2 kg) was dispersed in 20 l of warm water at 50 ° C.
Stir for 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 added to 20 l of a 0.2N aqueous sodium hydroxide solution at 70 ° C. and stirred for 90 minutes to dissolve. After cooling, 5 l of 0.8N hydrochloric acid aqueous solution
Is added slowly under stirring to neutralize. Neutralization solution 5,000
The mixture was centrifuged at 0 G for 10 minutes, and then the supernatant and the precipitate were recovered, and 190 g of water-insoluble hemicellulose (total sugar content: 32.1%, protein content: 10.0%, other components: 57.9%)
I got

REFERENCE EXAMPLE 2 20 l of the supernatant liquid obtained in the above reference example 1 was diluted by adding 20 l of water, and while maintaining the solution temperature at 50 ° C, a tubular ultrafiltration membrane NTU 3520 (P- 18 kg, membrane pressure 0.78 m ² , inner diameter 11.5 mm)
The treatment is performed for 3 hours under the conditions of cm ² and a flow rate of 13 l / 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, supply of water was stopped, and concentration was started under the same conditions as above (pressure: 8 kg · f / cm ² , flow rate: 13 l / min), and concentration was performed without considering a decrease in flux. Perform until about 10 mg / ml. The treated solution was treated with a cation exchange resin (IR-120, manufactured by Organo Corporation).
E) Ion exchange resin capacity of 10 per hour to 500 cc
Elution is performed at twice the flow rate, and then the mixture is flown at the same flow rate over an anion exchange resin (IRA-93) manufactured by the company. The aqueous solution obtained after the ion exchange treatment was freeze-dried under vacuum at a temperature of 30 ° C. and a degree of vacuum of 0.1 Torr or less to obtain about 15 water-soluble hemicellulose.
0 g was obtained.

Ethanol was added to a solution prepared by dissolving 50 g of this water-soluble hemicellulose in 0.7 l of water to adjust the ethanol concentration of the solution to 60%. The solution was stirred for about 20 minutes and allowed to stand. The resulting precipitate was washed with 60% ethanol and freeze-dried under vacuum 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 dissolved in 5 l of 20 mM (mM) Tris-HCl buffer (pH 8.5), and this solution was anion-exchanged (DEAE- Sepharose CL-6B (manufactured by Pharmacia) was passed through a column packed with 0.2 l to recover the liquid containing the non-adsorbed fraction. The liquid was dried to obtain 20 g of hemicellulose without adsorbing anion exchanger.

Example 1 The water-insoluble hemicellulose prepared in Reference Example 1 was used in an amount of 0.1%.
5 g was added to 50 ml of a sodium phosphate buffer (pH 6.0) to prepare a 1% solution of water-insoluble hemicellulose.

Next, the solution was preliminarily heated to 50 ° C., and then cellulase “Onozuka RS” (manufactured by Yakult) was added to the solution.
mg (50 units as xylanase) was added, and hydrolysis was carried out for 4 hours while maintaining the same temperature. Thereafter, the enzyme was inactivated by boiling, and 2 ml of the solution was collected and centrifuged at 3000 rpm for 10 minutes. 1 ml of the supernatant is applied to a tubular ultrafiltration unit (Molcut II LCC type: membrane area 1.3 cm ² , inner diameter 17 m)
m: Pressurized inside the pipe of Nippon Millipore Co., Ltd. (2kg)
/ Cm ² ) and the resulting effluent is filtered under H 2 under the following conditions:
PLC analysis was performed.

[0037]HPLC analysis  Injection volume: 20 μl Column type: Shodex KS-802 + KS-801 Eluent: pure water Flow rate: 0.7 ml / min Temperature: 80 ° C. Detector: differential refractometer Grams were obtained. Figure
Acid hydrolysis of each fraction of peaks 1 to 8 of 1
Was again subjected to HPLC to examine the composition of the constituent sugars. Also
Check total sugar content by enol sulfuric acid method
The amount of reducing sugars was determined by the Son method and the degree of polymerization of each peak was determined.

The results are shown in Table 1 below.

[0039]

[Table 1]

Next, the above fraction of peak 1 was collected and the above gel filtration chromatography and HPLC analysis were repeated until unity was confirmed. The gel filtration chromatography and HPLC analysis were repeated. Unity was confirmed after two repetitions.

Here, the unity was confirmed by dissolving 10 mg of the compound sample in 1.0 ml of pure water to prepare a 1% solution.
This was performed by HPLC and paper chromatography shown in the following.

HPLC-1 Injection volume: 20 μl Column type: Shodex KS-801 (manufactured by Showa Denko KK) Eluent: pure water Flow rate: 0.7 ml / min Temperature: 80 ° C. Detector: differential refractometer Principle: Gel filtration + ligand exchange As a result, a single peak was obtained at a retention time of 7.5 minutes.

HPLC-2 Injection volume: 20 μl Column type: Senshu Pack-NH ₂ (manufactured by Senshu Kagaku) Eluent: acetonitrile: water = 65: 35 Flow rate: 1.0 ml / min Temperature: room temperature detection Apparatus: 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 Amount of liquid used: 1 μl Separation liquid: butanol: pyridine: water = 6: 4: 4 Color former: silver nitrate As a result, a single spot was obtained near an Rf value of 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) The structure was determined using five methods, methylation analysis and (5) ¹³ C-NMR analysis.

(1) Measurement of degree of polymerization: 10 m of a compound sample
g 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. / Ml
And 1.6 mg / ml. From these values, it was found that the compound was a hexasaccharide.

(2) Complete hydrolysis with a strong acid: 5 mg of a compound sample was dissolved in 1 ml of 2N trifluoroacetic acid.
Hydrolysis at 100 ° C. for 2 hours. After cooling, trifluoroacetic acid was removed by 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: 5 mg of a compound sample was dissolved in 1 ml of 0.1 N sulfuric acid,
And then neutralized with barium carbonate. Subsequently, the mixture was centrifuged, the supernatant was passed through a microfilter having a pore size of 0.45 μm, and the filtrate was analyzed by the above-mentioned HPLC-1 method. A xylotetraose peak was observed at a retention time of 8.5 minutes. I confirmed that it would appear. From this, the oligosaccharide consisting of 4 molecules of xylose and 2 molecules of arabinose confirmed by the above (1) and (2) was added to xyltetratetraose by arabinose 2
The molecule was found to be an attached oligosaccharide.

(4) Methylation analysis: Compound sample 2 mg
Was methylated by the Hakomori method and 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. Using 2 mg of the reduced sample, methylation was performed in the same manner as described above.
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 I got

From the above, it was found that arabinose was not bound to either the reducing end or the non-reducing end of xylose, and that arabinose was bound to the 2- and 3-positions of xylose.

[0052] (5) ¹³ C-NMR analysis: To determine the position of the xylose arabinose in said compound is bound, ¹³ C-NMR of xylotetraose and the compound
The spectra 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 dioxane was added as an internal standard. this,
The sample was placed in a sample tube having a diameter of 5 mm, and an NMR analyzer (JEOL G
(SX-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.

[0054] As a result, the ¹³ C-NMR spectrum of FIG. 2 for xylotetraose, was obtained a ¹³ C-NMR spectrum of Figure 3 for the compound.

With respect to the ¹³ C-NMR spectrum, the 1st carbon of xylose (denoted by -C1) and the 1st carbon of xylose (denoted by -C1) of the first xylose from the reducing end of xylotetraose ), The first position carbon of the third xylose (denoted by -C1) and the first position carbon of the fourth xylose (denoted by -C1), and the corresponding NMR signal of the above compound Table 2 shows a comparison of the NMR signal for each carbon at position 1 of xylose.

[0056]

[Table 2]

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.

From the results of the above (1) to (5), it was found that the compound isolated as the peak 1 in FIG. 1 was a novel arabinoxylo-oligosaccharide having the following structure.

[0059]

Embedded image

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

Example 2 The procedure of Example 1 was repeated, except that the water-insoluble hemicellulose was replaced with the 60% ethanol-insoluble hemicellulose prepared in Reference Example 2 above. The above arabinoxylo-oligosaccharide having two arabinose molecules bound 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 60% ethanol-insoluble hemicellulose used as the raw material.

Example 3 Processing, analysis and structure determination were carried out in the same manner as in Example 1 except that the anion exchanger non-adsorbed hemicellulose prepared in Reference Example 3 was used instead of the water-insoluble hemicellulose. An arabinoxylo-oligosaccharide in which two arabinose molecules were bonded to the third xylose from the reducing end of the xylotetraose main chain, which was also obtained in Example 1, was obtained.

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

[Brief description of the drawings]

FIG. 1 shows the HLP of the oligosaccharide mixture produced in Example 1.
It is a figure which shows the chromatogram of each fraction obtained by C analysis.

FIG. 2 is a diagram showing a ¹³ C-NMR spectrum of xylotetraose.

FIG. 3 is a view showing a ¹³ C-NMR spectrum of a compound of which identity was confirmed by purifying the fraction of peak 1 in the oligosaccharide mixture fraction produced in Example 1.

────────────────────────────────────────────────── ─── 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

Claims (1)

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