JPH05317075A - Production of oligosaccharide - Google Patents

Abstract

PURPOSE:To obtain an oligosaccharide extremely effective in promoting the proliferation of bifidus bacteria, especially arabinooligosaccharide, with a simple operation in high efficiency by hydrolyzing a water-insoluble fibrous substance originated from wheat flour with a cell wall digesting enzyme. CONSTITUTION:A water-insoluble fibrous substance originated from wheat flour is hydrolyzed with a cell wall digesting enzyme to obtain an oligosaccharide. The water-insoluble fibrous substance is preferably a red cake recovered in solid state from a wheat starch waste liquid discharged in the production of starch from wheat flour, from the viewpoint of the effective utilization of red cake. The hydrolysis is carried out preferably by using 5-50 unit of the cell wall digesting enzyme in terms of xylanase based on 1g of the water-insoluble fibrous substance at 50-60 deg.C and pH5-6. The reaction time is preferably <1hr.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a method for producing oligosaccharides. Specifically, the present invention provides a method capable of efficiently producing an oligosaccharide extremely effective for promoting the growth of Bifidobacterium by a simple operation.

[0002]

2. Description of the Related Art In recent years, intestinal flora (bacterial flora)
Have become known to be deeply related to human health, and interest in intestinal bacteria is increasing. Among the intestinal bacteria, bifidobacteria are known to exhibit beneficial physiological effects such as suppressing the growth of intestinal putrefactive bacteria and pathogenic bacteria, and various attempts have been made to increase bifidobacteria. .. As one of them, glucose, galactose, a method of growing bifidobacteria using fructooligosaccharides or soybean oligosaccharides having hexoses such as fructose as constituent sugars has been proposed, but these saccharides are bifidobacteria and although being digestion by beneficial bacteria of lactic acid bacteria growing them, Bacteroides fragilis (Bacteroides fragilis) bacteria and Bacteroides vulgatus (Bacteroides vulgatus) bacteria, such harmful fungi is digestion by harmful bacteria such as Escherichia coli It also has the drawback that it also has a growth promoting action.

The present inventors have conducted research on oligosaccharides which can selectively grow bifidobacteria without growing harmful bacteria. Among oligosaccharides, xylose and arabinose mainly consist of pentose components. It was found that oligosaccharides can grow useful bifidobacteria while suppressing the growth of harmful bacteria, and previously filed an invention relating to a bifidobacteria growth promoter containing arabinoxylo oligosaccharide as an active ingredient (Japanese Patent Application No. No. 3-208770).

The above-mentioned Japanese Patent Application No. 3-2087 filed by the present inventors.
The arabinoxylo-oligosaccharide used as the bifidobacteria promoter of No. 70 is also disclosed in Japanese Patent Application No.
It can be produced by the method described in JP-A-162788 (JP-A-4-53496) and JP-A-3-282400. The former method (JP-A-4-53496) hydrolyzes water-insoluble hemicellulose obtained from wheat bran, water-soluble 60% ethanol-insoluble hemicellulose or water-soluble anion-exchanger non-adsorptive hemicellulose with endoxylanase. Is a method for producing an oligosaccharide mixture containing arabinoxylo-oligosaccharide, and the latter method (Japanese Patent Application No. 3-282400) is a method for producing an arabinoxylan-containing site such as a skin of a Gramineae plant at a temperature in the presence of water. 1
It is a method for producing arabinoxylo-oligosaccharide by subjecting a plant cell wall-disintegrating enzyme to action after heat treatment under pressure at 0 to 145 ° C. and a pressure of 1 to 4 atm.

All of the above-mentioned methods make it possible to smoothly produce useful oligosaccharides mainly composed of arabinose and xylose from wheat bran, which has been conventionally used mainly for livestock feed. It has a great technical meaning. However, the former method is to extract hemicellulose from wheat bran by alkali extraction or the like and hydrolyze the resulting hemicellulose with an enzyme, so that it is used for the extraction step and extraction of hemicellulose from wheat bran. Since it is necessary to neutralize the existing alkali and to remove salt produced by the neutralization, the process is complicated, and the cost cannot be denied. In addition, the latter method has an advantage that an arabinoxylo-oligosaccharide can be directly obtained from the skin of a grass family plant such as wheat bran without performing the extraction treatment of hemicellulose. Need.

On the other hand, in the production of wheat starch, water is added to wheat flour and kneaded to produce a dough, and the dough is washed with water and the washed product is divided into water-insoluble gluten and a starch-containing emulsion. A method of separating and recovering starch from the contained emulsion is generally adopted. In that case, most of the emulsion after separating and collecting the starch is conventionally discarded as a wheat starch waste liquid, and only a part of the emulsion remains in a liquid state or in a solid state and used as a feed for livestock. However, it has been difficult to handle it in terms of wastewater treatment.

[0007] In particular, from the wheat starch waste liquor generated in the process of producing wheat starch from wheat flour, a colored water-insoluble fibrous material called red meal mainly consisting of fine fragments of wheat bran and fibers contained in the endosperm A white water-insoluble fibrous substance mainly called white meal is obtained, and the portion corresponding to white meal is the food according to the previous application by the present inventors (Japanese Patent Application No. 2-418026). Although effective utilization as a fiber and a smooth recovery method thereof have been found, there has been no effective use of red cake until now, and its treatment has been a problem.

[0008]

SUMMARY OF THE INVENTION Under the above circumstances, the present inventors have
Oligosaccharides containing arabinose and xylose, which have useful bifidobacterial growth-promoting activity without showing growth-promoting activity of harmful bacteria, especially arabinoxylo-oligosaccharides undergo complicated steps such as alkali extraction and pressure heat treatment. Further, we have conducted further research in order to develop a method that can be efficiently manufactured with a simple operation. At the same time, the present inventors have continued to study the effective utilization of the water-insoluble fiber contained in the wheat starch waste liquor, particularly the fiber recovered as red cake.

As a result, when the water-insoluble fiber contained in the wheat starch waste liquid discharged during the production of starch and gluten from wheat flour is used and the cell wall-degrading enzyme is directly acted on this fiber, alkali extraction results. The present invention has been completed by finding that a useful oligosaccharide of interest, which is rich in arabinose and xylose, can be produced extremely easily and efficiently without any recovery of hemicellulose or heat treatment under pressure.

Therefore, the present invention is a method for producing an oligosaccharide characterized by hydrolyzing a water-insoluble fiber derived from wheat flour with a cell wall degrading enzyme.

In the present invention, as the water-insoluble fiber derived from wheat flour, any fiber derived from wheat flour and insoluble in water can be used, and the method for collecting and obtaining the water-insoluble fiber is not particularly limited. However, as the water-insoluble fiber,
It is desirable to use the water-insoluble fiber contained in the wheat starch waste liquid discharged during the production of starch from wheat flour. Among them, using a fiber called so-called red cake, in particular, the point of effective utilization of the red cake, which has not been known for its useful application, and further, in the oligosaccharides obtained from it, the bifidobacteria of It is preferable because it contains a large amount of arabinose and xylose useful for growth.

In the production of oligosaccharides by acting a cell wall degrading enzyme on the water-insoluble fiber contained in the wheat starch waste liquid, the cell wall-degrading enzyme is directly applied to the wheat starch waste liquid containing the water-insoluble fiber in a liquid state. Good. However, in addition to this method, a method of recovering a water-insoluble fiber composed of red meal, white meal, or a mixture thereof in a solid form from a wheat starch waste liquid, and causing a cell wall degrading enzyme to act on the recovered solid fiber However, salts and water-soluble proteins and the like are removed, and as a result, an oligosaccharide having a high purity can be obtained, which facilitates the subsequent purification of the oligosaccharide, which is particularly preferable.

The method for recovering the water-insoluble fiber from the wheat starch waste liquor as a solid such as red meal, white meal or a mixture thereof is not particularly limited. For example, wheat starch may be produced from wheat flour by a conventionally known method, and a solid water-insoluble fibrous substance such as red meal, white meal or a mixture thereof produced as a by-product at that time may be used as it is. Alternatively,
The water-insoluble fiber may be recovered in the following manner according to the method of the above-mentioned Japanese Patent Application No. 2-418026 by the present inventors. Particularly, in the case of the following method, it is a raw material of the present invention. A water-insoluble fiber can be obtained easily and efficiently.

According to the method of Japanese Patent Application No. 2-418026
Water-insoluble fibrous material recovery method Water is added to wheat flour and kneaded to produce a dough or emulsion, which is washed with water, and then the washed product is separated into gluten and a starch-containing emulsion to give a starch-containing milk. The starch is separated and recovered from the suspension. Water is added to the residue (liquid) after the starch has been separated to make a water-diluted emulsion, which is then subjected to a centrifugal separation treatment to contain colored solids equivalent to red meal and those corresponding to white meal. Of the white solid and the colored solid, the white emulsion and the water, and the dried solid and the white emulsion are dried to give red cake and white cake. Each of which is used as the water-insoluble fiber in the present invention.

In the present invention, the water-insoluble fiber derived from wheat flour is hydrolyzed with a cell wall degrading enzyme to produce an oligosaccharide. The cell wall degrading enzyme used in the present invention may be any as long as it has xylanase activity,
For example, "Cellulase Onozuka" manufactured by Yakult Co., "Pectolyase Y-23" manufactured by Seishin Pharmaceutical Co., Ltd., and "Mecerase" manufactured by Sanko Junyaku Co., Ltd. may be mentioned.

The cell wall degrading enzyme may be used in a free state or immobilized on a carrier, and the hydrolysis treatment with the cell wall degrading enzyme may be carried out by a continuous method or a batch method. The treatment is performed by appropriately selecting various conditions such as the origin of the cell wall degrading enzyme, the amount used, the temperature, pressure, pH and time during the treatment. When water-insoluble fiber is recovered from wheat starch waste liquor as solids such as white meal and red meal, and the solid matter is used for hydrolysis treatment with a cell wall degrading enzyme, solids consisting of the water-insoluble fiber It is preferable to disperse or suspend the substance in water and perform the enzyme treatment.

Although not limited thereto, generally, 1 to 100 units, preferably 5 to 50 units, of a cell wall-degrading enzyme is used as xylanase per 1 g of water-insoluble fibrous material at 30 to 70 ° C., preferably Is 50-60
PH of 4 to 7, preferably pH 5 to 6, at a temperature of ℃ 4
When the hydrolysis reaction is performed for less than 1 hour, preferably less than 1 hour, the target oligosaccharide can be obtained in good yield and at low cost.

As one measure of completion of the hydrolysis reaction, there can be mentioned a point of time when the viscosity of the suspension containing the water-insoluble fibrous material has stopped decreasing, and at that time point, the enzyme is inactivated by heating or the like. Good. When it is desired to control the reaction conditions more precisely, it is advisable to perform it while analyzing the composition of the enzyme reaction product by high performance liquid chromatography (HPLC method) or the like.

The oligosaccharide-containing hydrolyzate obtained as described above is treated by combining one or more means of separation such as ultrafiltration membrane, activated carbon, gel filtration chromatography, ion exchange resin and the like. , The target oligosaccharide mainly containing arabinoxylo-oligosaccharide can be separated and recovered.

Specific examples of the method for separating and recovering oligosaccharides from the hydrolyzed liquid are as follows, but of course the invention is not limited thereto. After centrifuging the oligosaccharide-containing hydrolyzate, the supernatant is filtered through a microfilter (for example, pore size 0.45 μm), and the filtrate is dried to recover oligosaccharides mainly composed of arabinoxylo-oligosaccharide. After the oligosaccharide-containing hydrolyzate is centrifuged, the supernatant is treated with an ultrafiltration membrane and the resulting effluent is dried to recover oligosaccharides mainly composed of arabinoxylo-oligosaccharide.

After the oligosaccharide-containing hydrolyzate is centrifuged to remove solids, it is desalted by passing through an ion exchange resin, and the supernatant is microfiltered (pore size: 0.45 μm).
m) and then passed through an activated carbon column to divide into activated carbon adsorption section and non-adsorption section, then activated carbon adsorption section to 70
Elute with% ethanol and dry the eluate to recover oligosaccharides, which consist primarily of arabinoxylo-oligosaccharides. After centrifuging the oligosaccharide-containing hydrolyzate, the supernatant is passed through a microfilter (for example, pore size 0.45 μm), and the filtrate is concentrated and gel filtration chromatography (for example, Toyopearl HW-40s manufactured by Tosoh Corporation). After that, the eluate is finely separated and then dried to recover a single oligosaccharide.

An oligosaccharide produced by the method of the present invention,
In particular, the oligosaccharides obtained by the above-mentioned ~ recovery methods are arabinoxylo-oligosaccharides in which xylose and arabinose are bound and / or xylooligosaccharides in which only xylose is bound, and further monosaccharides such as xylose, arabinose and glucose. A small amount is included. The mixed oligosaccharide contains a large amount of arabinoxylo-oligosaccharide (usually about 70 to 80%), and the arabinoxylo-oligosaccharide has a composition formula (Xyl) n (Ara) m (in the formula, Xyl: xylose). , Ara: arabinose, n: xylose bond number,
m: the number of arabinose bonds), usually n
= 2-10, m = 1-10.
The mixed oligosaccharides may be used in the form of a mixture, or may be separated and collected into a single oligosaccharide by the above-mentioned recovery method or the like and used.

When wheat bran or the like is used, oligosaccharides cannot be obtained by directly acting the cell wall degrading enzyme, whereas in the present invention using water-insoluble fiber derived from wheat flour Although the rationale for obtaining sugar directly and easily is not clear, it is presumed that the reason is as follows.

That is, the water-insoluble fibers derived from wheat flour such as red meal and white meal recovered from the wheat starch waste liquor used in the present invention are mainly contained in fine fragments of wheat bran or wheat endosperm generated during milling. It is made of water-insoluble fiber. Since these water-insoluble fibers are contained in the final product, wheat flour, which has undergone the sieving process in the milling process, ordinary wheat flour with a large grain size that is sieved in the first stage of the milling process is used. Generally, 200 μm
It is composed of fine particles having a finer particle size. Therefore, it has a large surface area and is in a state of being easily contacted with an enzyme, and further, the bond between cellulose and hemicellulose in the water-insoluble fiber is partially broken, and it is not as strong as wheat bran. Moreover, when wheat starch or gluten is collected from wheat flour, it undergoes steps such as kneading and washing with water, so that it is sufficiently hydrated, and the cell wall-degrading enzyme easily penetrates into the cell wall of the water-insoluble fibrous material. Is becoming As a result, the hydrolysis is smoothly performed by directly acting the cell wall-degrading enzyme on the water-insoluble fiber derived from wheat flour without performing pretreatment such as extraction treatment of hemicellulose with alkali or heat and pressure treatment. It is considered that the desired oligosaccharide can be obtained.

Since the oligosaccharides produced by the present invention are mainly composed of arabinoxylo-oligosaccharides, they do not have a growth promoting action on harmful bacteria such as Bacteroides fragilis, Bacteroides vulgatus and Escherichia coli. Bifidobacterium, which is a useful bacterium, can be selectively grown and can be usefully used in functional foods and other applications.

[0026]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto. All percentages in the following examples are by weight. In addition, in the following examples, the total sugar amount was determined by the phenol-sulfuric acid method. Further, the content of oligosaccharide and the sugar composition in the obtained product were measured by the following methods.

[Measurement of Oligosaccharide Content] After dissolving 50 mg of the product obtained in the following example in 1 ml of pure water, HPLC analysis was carried out under the following conditions to obtain its chromatogram, and each chromatogram was analyzed. The oligosaccharide content was determined from the peak area ratio. HPLC analysis conditions : Injection volume: 20 μl Column: Ultrahydrogel 250 × 2 (manufactured by Waters) Flow rate: 0.8 ml / min Temperature: 70 ° C. Detector: Differential refractometer

[Measurement of Sugar Composition] To 20 mg of the product obtained in each of the following examples, 2 ml of 2N trifluoroacetic acid was added, hydrolyzed at 100 ° C. for 2 hours, and then acid was removed under the above conditions. HPLC analysis was carried out to determine the sugar composition.

Reference Example 1 [Recovery of water-insoluble fiber derived from wheat flour] Water-insoluble fiber was recovered from wheat flour by the following method according to Japanese Patent Application No. 2-418026. That is, 6 parts of water was added to 10 parts of wheat flour (protein content 16.0%) and kneaded at a temperature of about 20 ° C. for 20 minutes using a kneader kneader to produce a dough. Next, 60 parts of water was added to 16 parts of this dough, and water was washed for 60 minutes at a water temperature of about 20 ° C. using a water washing device (uniaxial screw conveyor). As a result of this water washing step, wheat gluten in the dough was separated as a water-insoluble solid substance, so the gluten solid substance was removed and the aqueous phase consisting of the starch-containing emulsion was recovered. Then, the starch-containing emulsion recovered above is treated using a 300-mesh vibrating screen to separate the starch and leave the residue on the screen.
A strike-like residue (water content about 95%) was recovered.

Next, twice the amount of water was added to the paste-like residue recovered in the above step to prepare a water-diluted emulsion having a solid content concentration of about 2%. In addition, a continuous centrifuge [Sharpless Super Decanter P-660
Type: manufactured by Tomoe Kogyo Co., Ltd.], the water-diluted emulsion supply rate is 2000 kg / h, the rotation speed of the rotary cylinder is 5000 rp.
m (2130G), conveyor rotation speed 3000 rpm
By centrifuging with, the colored water-insoluble fiber corresponding to red meal is discharged from the outlet on the upstream side, while the white emulsion containing the water-insoluble fiber corresponding to white meal is discharged from the outlet on the downstream side. It was recovered at a rate of 1900 kg / h. Each of the colored water-insoluble fibrous material and the white emulsion recovered above was dried to obtain so-called red cake and white cake. The general analytical values and neutral sugar composition of the red meal and white meal obtained above were as shown in Table 1 below.

Reference Example 2 Red meal and white meal were prepared in the same manner as in Reference Example 1 except that the type of wheat flour was changed, and their general analytical values and sugar composition were examined. It was as shown in 1. For reference, Table 1 also shows general analysis values and neutral sugar composition of wheat bran.

[0032]

[Table 1] General analysis value Sugar composition Moisture Ash protein Glc ¹⁾ Xyl ²⁾ Ara ³⁾ (%) (%) (%) (%) (%) (%) Reference Example 1 Red meal 10.0 1.5 7.2 20.0 48.3 31.5 White meal 10.0 0.6 6.0 65.2 20.2 14.6 Reference Example 2 Red meal 4.1 1.2 6.9 51.0 30.9 48.2 White meal 4.5 0.7 4.3 63.4 23.1 13.5 Wheat bran 12.6 5.1 16.4 32.1 42.9 25.0 1) glucose, 2) xylose, 3) arabinose

As can be seen from Table 1 above, red meal and white meal, which are water-insoluble fibers derived from wheat flour, have a smaller content ratio of substances that become impurities in the preparation of oligosaccharides such as ash and protein, as compared with wheat bran. However, it is preferable that it is preferable as a raw material for oligosaccharide production. Glucose is considered to be mainly derived from wheat starch. Furthermore, although the ratios of glucose, xylose and arabinose in red meal differ considerably between Reference Example 1 and Reference Example 2, this is due to the degree of separation from starch during the preparation of red meal. Seem. Glucose becomes an impurity during the production of oligosaccharides containing arabinoxylo-oligosaccharide as a main component, and therefore it is preferable to separate as much as possible during the production of water-insoluble fiber such as red cake. When the red meal is prepared by the method of the above-mentioned Japanese Patent Application No. 2-418026 by the present inventors, the proportion of glucose contained in the red meal is small, and the red meal is the water-insoluble fiber according to the present invention. Suitable for quality.

Example 1 Red meal 5 prepared in Reference Example 1
g in 50 mM acetate buffer at pH 5.5,
100 ml of a 5% (w / v) suspension of red cake was prepared. After heating this to 50 ° C, cellulase Onozuka RS (manufactured by Yakult Honsha) 5 mg (42 unit as xylanase)
s) was added and the hydrolysis reaction was performed for 30 minutes. Then
The enzyme was inactivated by boiling at 100 ° C for 10 minutes. After allowing to cool, it was centrifuged at 9000 G for 30 minutes, the obtained supernatant was filtered through a microfilter having a pore size of 0.45 μm, and the filtrate was freeze-dried to obtain a solid. The yield (%), total sugar amount, oligosaccharide content and sugar composition of the solid matter obtained here were measured by the methods described above. The results are shown in Table 2 below.
Shown in.

Example 2 An oligosaccharide was produced in the same manner as in Example 1 except that the white meal prepared in Reference Example 1 was used. The results are shown in Table 2.

Example 3 Red meal 5 prepared in Reference Example 1
g in 50 mM acetate buffer at pH 5.5,
100 ml of a 5% (w / v) suspension of red cake was prepared. This was heated to 60 ° C., 70 μl of Termamyl 120L (manufactured by NOVO) (10 KNU as amylase) was added, and the reaction was carried out for 1 hour, followed by centrifugation at 9000 G for 30 minutes to remove the supernatant. After newly adding 50 mM acetate buffer at pH 5.5 and adjusting the volume to 100 ml, the mixture was heated to 50 ° C., 5 mg of cellulase Onozuka RS (42 units as xylanase) was added, and a hydrolysis reaction was performed for 30 minutes. It was Then, the enzyme was inactivated by boiling at 100 ° C. for 10 minutes. After allowing to cool, it was centrifuged at 9000 G for 30 minutes, the obtained supernatant was filtered through a microfilter having a pore size of 0.45 μm, and the filtrate was freeze-dried to obtain a solid. The yield (%), total sugar amount, oligosaccharide content and sugar composition of the solid matter obtained here were measured by the methods described above. The results are shown in Table 2 below.

Example 4 1.5 kg of red meal prepared in Reference Example 1 was suspended in 50 mM acetate buffer at pH 5.5 to obtain 15 liters of a 10% (w / v) suspension of red meal. Was prepared. This was placed in a fermenter MBU-50 (manufactured by Tokyo Rikakiki Co., Ltd.) and heated to 60 ° C. To this, 21 ml of Termamyl 120 L (3000 KN as amylase
U) was added and reacted for 1 hour, and then centrifugal filter cloth [Co., Ltd.
Tanabe Iron Works] was used to remove the reaction solution. The residue was put into the fermentor MBU-50 again, and the pH was newly adjusted to 50 at 50.
The volume was adjusted to 15 liters by adding mM acetate buffer. Then, the mixture was heated to 50 ° C., 1.5 g of cellulase Onozuka RS (12450 units as xylanase) was added, and a hydrolysis reaction was performed for 30 minutes. Then 100 ℃
The enzyme was inactivated by boiling for 10 minutes. After allowing to cool, a filtrate was prepared with a centrifugal filter cloth, and 1% (w / v) of a filter aid (Showa Chemical Industry Co., Ltd .; Radiolite 2000) was added to the filtrate.
In addition, suction filtration was performed with a filter having a pore size of 5 μm. The obtained filtrate was spray-dried with a spray dryer to obtain a dried product. The yield (%), total sugar amount, oligosaccharide content and sugar composition of the dried product obtained here were measured by the above-mentioned methods. The results are shown in Table 2 below.

By the way, the chromatogram of the dried product obtained in Example 4 when it was subjected to the above-mentioned HPLC analysis was as shown in FIG. On the other hand, when HPLC analysis was performed in the same manner using a substance having a known molecular weight, in the case of pullulan having a molecular weight of 5800, the position was 19.20 minutes.
For glucose having a molecular weight of 180, a peak appeared at a position of 27.69 minutes, and for xylose and arabinose having a molecular weight of 150, a peak appeared at a position of 28.55 minutes. From this, in the chromatogram of FIG. 1, peaks 1 to 3 having a larger molecular weight on the left side of the peak 4 (molecular weight 180) almost corresponding to glucose corresponded to oligosaccharides, and the production of oligosaccharides was confirmed. ..

Comparative Example 1 50 g of a freeze-dried product of wheat bran that had been washed with water was suspended in 50 mM acetate buffer at pH 5.5 to prepare 1 liter of a 5% (w / v) suspension of wheat bran. .. After heating this to 50 ℃, cellulase Onozuka RS 50mg (420 unit as xylanase
s) was added and the hydrolysis reaction was performed for 30 minutes. Then
The enzyme was inactivated by boiling at 100 ° C for 10 minutes. After allowing to cool, the mixture was centrifuged at 9000 G for 30 minutes, Radiolite 2000 was added to the resulting supernatant at a ratio of 1% (w / v), and the mixture was filtered through a filter having a pore size of 5 μm, and the filtrate was freeze-dried. A solid was obtained. The yield (%), total sugar amount, oligosaccharide content and sugar composition of the solid matter obtained here were measured by the methods described above. The results are shown in Table 2 below.

Comparative Example 2 50 g of a freeze-dried product of wheat bran washed with water was suspended in 50 mM acetate buffer at pH 5.5 to prepare 1 liter of a 5% (w / v) suspension of wheat bran. .. This is autoclaved at 120 ° C, 2.
Pressure heat treatment was performed at 1 atm for 10 minutes. After allowing to cool, the mixture was heated to 50 ° C., 50 mg of cellulase Onozuka RS (420 units as xylanase) was added, and a hydrolysis reaction was performed for 30 minutes. Then, the enzyme was inactivated by boiling at 100 ° C. for 10 minutes. After allowing to cool, centrifuge at 9000 G for 30 minutes, and add 1% of Radiolite 2000 to the obtained supernatant.
(W / v) was added and the mixture was filtered through a filter having a pore size of 5 μm, and the filtrate was freeze-dried to obtain a solid. The yield (%), total sugar amount, oligosaccharide content and sugar composition of the solid matter obtained here were measured by the methods described above. The results are shown in Table 2 below.

[0041]

[Table 2] Actual Example Comparative Example 1 2 3 4 1 2 Solids yield (%) 40.1 32.5 30.4 28.8 3.6 23.7 Total sugar amount (%) 88.7 86.3 88.1 90.0-85.8 Oligosaccharide content 16.4 11.8 16.3 15.1-12.1 Sugar composition Glc ¹⁾ (%) 43.1 47.8 25.0 28.2-25.7 Xyl ²⁾ (%) 38.7 33.3 51.9 48.7 − 57.2 Ara ³⁾ (%) 18.2 18.9 23.1 23.1 − 17.1 Total (%) 100.0 100.0 100.0 100.0 − 100.0 1) glucose, 2) xylose, 3) arabinose

From the results shown in Table 2 above, Examples 1 to 1 of the present invention were conducted.
In No. 4, although the water-insoluble fiber derived from wheat flour is only directly hydrolyzed by the cell wall degrading enzyme without pretreatment, the pretreatment such as pressure heating of the fiber is performed. It can be seen that an oligosaccharide containing a large amount of arabinose and xylose can be obtained in a higher yield as compared with Examples 1 and 2. Moreover, when red meal is used as the water-insoluble fiber derived from wheat flour, it is possible to obtain an effective oligosaccharide having a low glucose content and a high arabinose and xylose content by promoting the growth of bifidobacteria.

[0043]

INDUSTRIAL APPLICABILITY According to the method of the present invention, an oligosaccharide containing a large amount of arabinose and xylose, which does not show the effect of promoting the growth of harmful bacteria and has the effect of promoting the growth of bifidobacteria, which is a useful bacterium, particularly arabinoxylo-oligosaccharide, It is possible to manufacture with a simple operation and a high yield, without going through complicated pretreatment steps such as extraction and heat treatment under pressure. Furthermore,
According to the present invention, it is possible to effectively use the water-insoluble fiber in the wheat starch waste liquid, which has been difficult to handle up to now, in particular, the fiber recovered as red meal, and there has been almost no effective use in the past. The water-insoluble fiber derived from wheat flour can be converted into useful oligosaccharides.

[Brief description of drawings]

1 is a diagram showing a chromatogram of each fraction obtained by HPLC analysis of the product obtained in Example 4. FIG.

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[Procedure amendment]

[Submission date] July 1, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0032

[Correction method] Change

[Correction content]

[0032]

[Table 1] General analysis value Sugar composition Moisture Ash protein Glc ¹⁾ Xyl ²⁾ Ara ³⁾ (%) (%) (%) (%) (%) (%) Reference Example 1 Red meal 10.0 1.5 7.2 20.2 48.3 31.5 White meal 10.0 0.6 6.0 65.2 20.2 14.6 Reference Example 2 Red meal 4.1 1.2 6.9 51.0 30.9 18.1 White meal 4.5 0.7 4.3 63.4 23.1 13.5 Wheat bran 12.6 5.1 16.4 32.1 42.9 25.0 1) glucose, 2) xylose, 3) arabinose

Claims (1)

[Claims] 1. A method for producing an oligosaccharide, which comprises hydrolyzing a water-insoluble fiber derived from wheat flour with a cell wall degrading enzyme.