JPH07278170A - New oligosaccharide and its production - Google Patents

Abstract

PURPOSE:To efficiently produce a xylulose-bonded oligosaccharide having an activity as a Lactobacillus growth factor, hopefully expected to be utilized as a sweetening, a food material as a functional food, a medical material, etc., at low cost. CONSTITUTION:This method for production of a xylulose-bonded oligosaccharide is carried out by allowing an enzyme (and a yeast) having a fructose transition activity to act on a glucosylxylulosidecontaining solution which is a donor substrate in the presence of a new xylulose-bonded oligosaccharide having a structure in which a D-xylulose is bonded through a beta-bond to a lactose, an L-fucose or an L-sorbose and an aldose or a ketose as an acceptor substrate to transfer and bond a xylulosyl group other than a glucose.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel oligosaccharide and a method for producing the same, and more specifically, in the presence of aldose or ketose, an enzyme having a fructose transfer activity is allowed to act on glucosylxyluloside to transfer a xylulosyl group other than glucose. The present invention relates to a novel xylulose-conjugated oligosaccharide obtained by conjugation and a method for producing the same.

[0002]

2. Description of the Related Art In recent years, various oligosaccharides synthesized by utilizing the glycosyl transfer activity of a sugar hydrolase have been used as food materials or pharmaceutical materials. In particular, researches using oligosaccharides as growth factors of Bifidobacteria known as human intestinal useful bacteria have been actively conducted, and for example, fructooligosaccharides, isomaltooligosaccharides, galactooligosaccharides, lactosucrose, and N-acetylglucosamine are used. The oligosaccharides contained therein are widely used in the food field as those having a function of growing Bifidobacteria.

Xylulose is an important member of pentose metabolism, and its physiological significance is great. However, oligosaccharides containing xylulose are glucosyl xyluloside (J. Am.
m. Soc., 68, 1465, 1946).
The conventional method for producing glucosyl xyluloside is α-
These are a method of allowing sucrose phosphorylase to act on a mixed solution of D-glucose-1-phosphate and D-xylulose, and a method of allowing sucrose phosphorylase to act on a mixed solution of sucrose and D-xylulose. However, recently, a method of allowing a xylose isomerase and sucrose phosphorylase to act on a mixed solution of sucrose and D-xylose (Japanese Patent Laid-Open No. 3-30023)
191786) was developed and enabled industrial synthesis. Further, the subsequent studies by the present inventors have revealed that this glucosyl xyluloside is a selective growth factor for Bifidobacterium.

[0004]

Up to now, there have been no reports on other oligosaccharides containing xylulose and a method for producing the same, and they have fructose transfer activity using aldose or ketose and glucosylxyluloside as raw materials. A xylulose-bonded oligosaccharide obtained by transferring an xylulosyl group to glucose in addition to glucose and a method for producing the same have not yet been reported.

An object of the present invention is to obtain a novel substance by using the xylulose transfer reaction of an enzyme having a fructose transfer activity and to establish a method for producing the same.

[0006]

[Means for Solving the Problems] In view of the above-mentioned circumstances, the present inventors have conducted diligent studies on enzymatic synthesis of oligosaccharides that bind xylulose, and as a result, aldose or ketose and glucosylxylloside-containing liquid Furthermore, they have found that a novel xylulose-bonded oligosaccharide can be efficiently produced by causing an enzyme having a fructose transfer activity to act and transfer-bond a xylulosyl group in addition to glucose, and completed the present invention.

That is, the present invention is a donor substrate in the presence of a novel xylulose-binding oligosaccharide in which lactose, L-fucose or L-sorbose is β-bonded to D-xylulose and aldose or ketose as an acceptor substrate. It is intended to provide a method for producing a xylulose-bonded oligosaccharide, which comprises reacting a glucosylxyluloside-containing solution with an enzyme having a fructose transfer activity to transfer and bond a xylulosyl group in addition to glucose.

The novel xylulose-bonded oligosaccharide of the present invention is xylulose β-bonded to the reducing hydroxyl group of aldose or ketose, and is composed of glucosylxylloside which is a xylulose residue donor and the acceptor of the xylulose residue. Β-fructofuranosidase is allowed to act on a mixture containing aldose or ketose, which is the body,
It is obtained by β-bonding xylulose to a reducing hydroxyl group of aldose or ketose.

The present invention will be described in detail below, focusing on a method for producing a novel oligosaccharide. In the present invention, aldose or ketose is an acceptor substrate, specifically, an aldose or ketose that can newly generate a xylulose-bonded oligosaccharide other than glucosylxyluloside, which is a xylulose donor, by an enzyme having a fructose transfer activity, Monosaccharides or oligosaccharides that are aldoses or ketose other than glucose are desirable. For example, D-xylose, D-galactose, D-, L-arabinose, L-sorbose, D-, L-fucose, maltose, cellobiose, xylobiose, isomaltose,
Lactose, maltotriose, isomaltotriose, panose, isopanose and the like are suitable, and preferred are lactose, L-fucose and L-sorbose. These acceptor substrates may be used alone or in combination of two or more. Further, it may be a partial hydrolyzate of a polysaccharide such as starch, cellulose, arabinogalactan, xyloglucan, fucoidan.

The glucosyl xyluloside used in the present invention is, as described above, a method of allowing sucrose phosphorylase to act on a mixed solution of α-D-glucose-1-phosphate and D-xylulose, of sucrose and D-xylulose. It can be easily produced by a method of allowing sucrose phosphorylase to act on the mixed solution, or a method of allowing xylose isomerase and sucrose phosphorylase to act on a mixed solution of sucrose and D-xylose.

The enzyme having a fructose transfer activity used in the present invention decomposes glucosylxylloside when acted on a solution containing an acceptor substrate and glucosylxyluloside coexisting, and accepts the xylulosyl group. Any enzyme can be used as long as it can be transferred to a body substrate to generate a new xylulose-linked oligosaccharide, and those of various origins can be used. Normally, Arthrobacter (A rthrobacte r sp.) Microorganisms, Aspergillus niger (A spergillus n iger), but Aureobasidium Day Umm genus (A ureobasidium) of microbial origin, such as microbial β- fructofuranosidase can be used , Of these, β-fructofuranosidase derived from Arthrobacter microorganisms is preferable, and Arthrobacter
An enzyme derived from SPK-1 (FERM BP-3192) strain is suitable. These enzymes may be crude enzymes, and may be used in a free state or an immobilized state.

Enzyme activity is 20% containing 40% xylose.
% Sucrose solution (50 mM phosphate buffer pH 6.5) 200
Add 200 μl of the enzyme solution diluted appropriately to 40 μl,
After reacting for 10 minutes, a part of the reaction solution was put in boiling water to inactivate the enzyme by heat, and then the amount of glucose and fructose released by the F kit (glucose / fructose; Boehringer Mannheim Yamanouchi) was calculated. The amount of transferred fructose was calculated by subtracting the amount of fructose from the amount. One unit of enzyme is 1 μmo per minute.
It was defined as the amount of enzyme for transferring 1 fructosyl group.

In order to produce the target xylulose-linked oligosaccharide, β-fructofuranosidase is reacted with a substrate solution in which aldose or ketose (acceptor substrate) and glucosylxyluloside (donor substrate) are allowed to coexist. You can do it. The molar ratio of the acceptor substrate and the donor substrate during the reaction is usually 1:50 to 50: 1, preferably 1: 5 to 5.
It is 5: 1. In addition, the total substrate concentration of the acceptor substrate and the donor substrate (total sugar concentration in the examples) is usually 1 to 90% (W.
/ W), preferably 10 to 50% (W / W). The pH and temperature at the time of reaction may be any condition as long as β-fructofuranosidase acts to produce xylulose-linked oligosaccharide, and the pH is usually 4 to 9, preferably 5.5 to 7.
0, the temperature is selected in the range of 20 to 70 ° C, preferably 30 to 60 ° C. The amount of enzyme used is closely related to the reaction time, and the reaction is usually 0.1 hour to 1 week, preferably 1 hour.
The amount of enzyme should be such that it is completed in ~ 100 hours,
It is not limited to these.

When β-fructofuranosidase is allowed to react with a substrate solution in which aldose or ketose and glucosylxyluloside coexist, glucose is produced as a by-product, which inhibits β-fructofuranosidase and binds xylulose. The yield of oligosaccharides may decrease. In order to improve this, it is also possible to simultaneously act β-fructofuranosidase and yeast on the substrate solution so as to assimilate glucose by-produced into yeast to increase the yield of xylulose-binding oligosaccharide. The yeast used in this case may be any yeast as long as it does not assimilate any of the acceptor substrate, the donor substrate, and the novel xylulose-binding oligosaccharide and selectively assimilates glucose. As such yeast, for example, Saccharomyces yeast, Candida yeast, etc. are used. The conditions for glucose assimilation by yeast vary depending on the type of yeast used, heat resistance, sugar resistance, and other properties, but usually the total sugar concentration in the reaction solution is 10 to 40% (W / W), And pH
It is suitable that the temperature is 4.5 to 7.0 and the temperature is 30 to 40 ° C. However, it can generally be carried out under the reaction conditions of β-fructofuranosidase.

In order to separate and purify the novel xylulose-bonded oligosaccharide thus produced from the reaction solution, a known method may be applied. For example, after heat inactivating the enzyme,
After decolorization and desalting using activated carbon or an ion exchange resin, if necessary, it can be purified to a high purity by a chromatographic operation such as activated carbon column chromatography to obtain a xylulose-bound oligosaccharide.

[0016]

EXAMPLES Next, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto. Example 1 Preparation of lactosyl xyluloside 5 g of lactose and 5 g of glucosyl xyluloside were dissolved in a McIlbein buffer (pH 6.5), and β-fructofuranosidase derived from a microorganism belonging to the genus Arthrobacter was added to 200 g per 1 g of glucosyl xyluloside. A unit was added to make the total sugar concentration 50% (W / W), and the reaction was carried out at 55 ° C. for 20 hours to obtain a reaction liquid containing 20% of the transfer reaction product (A). A high performance liquid chromatogram of the above reaction liquid using YMC-Pack AQ is shown in FIG. The reaction solution is heated to stop the enzymatic reaction, and then decolorized using activated carbon and an ion exchange resin.
After desalting, YMC-Pack AQ SH-343-
The transfer reaction product (A) was isolated by preparative HPLC using 5 (20 mm × 30 cm, manufactured by YMC, column temperature 30 ° C.) with water (flow rate 6.0 ml / min) as a solvent. Then, lyophilization was performed to obtain 1.9 g of a transfer reaction product (A) having a purity of 98%.

When the transfer reaction product (A) is acid-hydrolyzed, D-glucose, D-galactose and D-
It was shown to consist of xylulose. In addition, FAB-
As a result of measuring the molecular weight by MS, the molecular weight was 474. FIG. 2 shows the ¹³ C-NMR spectrum. From the above results, it was found that the transfer reaction product (A) is lactosyl xyluloside in which D-xylulose is β-2,1-linked to the glucose residue at the reducing end of lactose. The specific rotation of this substance is [α] _D +43 (25 ℃, water)
Met.

Example 2 Preparation of fucosyl xyluloside 30 g of L-fucose and 60 g of glucosyl xyluloside were dissolved in water in advance and the pH was adjusted to 6.5 with 1N-NaOH. The original β-fructofuranosidase was added to 500 units per 1 g of glucosyl xyluloside, and the total sugar concentration was 36% (W /
The reaction solution was designated as W) and reacted at 35 ° C. for 70 hours to obtain a reaction solution containing 8% of the transfer reaction product (B). YMC- of the above reaction solution
Figure 3 shows a high-performance liquid chromatogram using Pack AQ.
It was shown to. After the reaction solution is heated to stop the enzymatic reaction, it is decolorized and desalted using activated carbon and an ion exchange resin, and then YMC
-Pack AQ SH-343-5 (20 mm x 30
cm, manufactured by YMC Co., Ltd., column temperature 30 ° C.), and preparative HPL using water (flow rate 6.0 ml / min) as a solvent.
The rearrangement reaction product (B) was isolated at C. Then, freeze-drying was performed to obtain a transfer reaction product (B) having a purity of 98% at 7.0.
g was obtained.

When this transfer reaction product (B) was acid-hydrolyzed, it was shown to consist of L-fucose and D-xylulose. Further, the molecular weight was 296 as a result of measuring the molecular weight by FAB-MS. FIG. 4 shows the ¹³ C-NMR spectrum. From the above results, the transfer reaction product (B) has L-fucose containing D-xylulose of β-2,1.
It was found to be the bound fucosyl xyluloside.
The specific rotation of this substance is [α] _D- 71.4 (25
℃, water).

Example 3 Preparation of sorbosylxylloside 5 g of L-sorbose and 5 g of glucosylxyluloside were pre-dissolved in the same manner as in Example 1 and the pH was adjusted to 6.5 with 1N-NaOH, and then althuro was used. 200 units of β-fructofuranosidase derived from a bacterium of the genus Bacterium was added per 1 g of glucosylxyluloside, and the total sugar concentration was 40% (W /
W) and reacted at 55 ° C. for 20 hours to obtain a reaction liquid containing 20% of the transfer reaction product (C). YMC of the above reaction solution
A high performance liquid chromatogram using -Pack AQ is shown in FIG. After the reaction liquid is heated to stop the enzymatic reaction, it is decolorized and desalted using activated carbon and an ion exchange resin, and then YM
C-Pack AQ SH-343-5 (20 mm x 3
0 cm, manufactured by YMC Co., column temperature 30 ° C.)
Preparative HP using water (flow rate 6.0 ml / min) as a solvent
The transfer reaction product (C) was isolated by LC. Then, freeze-drying was performed to obtain a transfer reaction product (C) having a purity of 98% as 1.
6 g were obtained.

When the transfer reaction product (C) was acid-hydrolyzed, it was shown to consist of L-sorbose and D-xylulose. The molecular weight was 312 as a result of measuring the molecular weight by FAB-MS. Fig. 6 Fig. ¹³ C-NM
The spectrum of R is shown. From the above results, the transfer reaction product (C) has L-sorbose and D-xylulose β-.
It was found to be a sorbosyl xyluloside with 2,2 linkages. The specific rotation of this substance is [α] _D- 93.8.
(25 ° C., water).

Example 4 Preparation of lactosyl xyluloside (simultaneous reaction of yeast) 50 g of lactose and 50 g of glucosyl xyluloside were dissolved in a McIlbein buffer (pH 6.5), and β-fructofuranosidase derived from a microorganism belonging to the genus Arthrobacter was dissolved. Yeast (Saccharomyces cerevisiae) of 200 units per 1 g of glucosyl xyluloside and 50 mg per total substrate sugar
Was added to adjust the total sugar concentration to 40% (W / W), and the reaction was carried out for 30 hours while adjusting the pH to 6 to 7 at 35 ° C.
As a result, a reaction solution containing 58% of lactosyl xyluloside was obtained. After stopping the enzymatic reaction and glucose assimilation of the yeast by heating the reaction solution, the yeast was removed by centrifugation, decolorized and desalted using activated carbon and an ion exchange resin, and then freeze-dried to obtain 83 g of lactosylxyluro. A powdered product of Sid was obtained.

Example 5 Using 117 representative strains of human-derived intestinal bacteria, the assimilation of various xylulose-binding oligosaccharides was tested.
For comparison, glucose and 1-kestose were similarly tested. The test strains were Bifidobacterium ( Bifidobacterium ) microorganisms (20 strains), and plain harmless bacteria (usually harmless but in some cases harmful) Bacteroides (16 strains).
Strains), Eubacterium ( 6 strains), Fusobacterium (4 strains), P
eptostreptococcus (6 strains), Enterococcus (5 strains), E
scherichia (5 strains), other (30 strains), as harmful bacteria
Clostridium ( 25 strains) was used. First, a frozen bacterial strain was streak-cultured on a BL agar plate, and an operation of obtaining an isolated colony was repeated twice to obtain a pure cultured bacterial strain. BL agar plate is 5% of defibrinated horse horse made by Kojin Co., Ltd. on BL agar medium made by Nissui Pharmaceutical.
Was used. The culture was carried out anaerobically at 37 ° C. for 48 hours using an anaerobic incubator (manufactured by SANYO / FORMA), an atmosphere of a mixed gas of CO ₂ 10%, H ₂ 10% and N ₂ balance. next,
Fildes solution Anatomically cultivated and subcultured in GAM broth supplemented with GAM broth at 37 ° C for 24 hours, and 0.03 ml of this culture solution was added to anaerobic medium [PYF semi-fluid agar medium to obtain various concentrations of various sugars.
0.5% was added and sterilized by autoclaving at 115 ° C. for 20 minutes] 1.5 ml was inoculated using an automatic multi-species inoculation device (MD-120 LIFETEC).

The culture is carried out in an anaerobic incubator (SAN
YO / FORMA), and the atmosphere is CO ₂ 10
%, H ₂ 10%, N ₂ balance mixed gas is used.
The pH of the medium after culturing was measured anaerobically at 7 ° C. for 4 days. In addition, pH measurement and data processing are based on BIS-
120 (manufactured by LIFETEC) was used.

Composition of PYF semi-liquid agar medium (pH 7.2) Trypticase (BBL) 10.0 g Yeast extract (Difco) 10.0 g Fildes solution ^* 40.0 ml Salts solution ^** 40.0 ml L-Cysteine hydrochloride monohydrate 0.5 g Agar 1.5g Purified water 920.0ml

* Fildes solution Saline 150 ml Concentrated hydrochloric acid 6 ml Horse blood 50 ml Pepsin (1: 10,000, Difco) 1 g After digesting the above ingredients at 55 ° C overnight, 20% NaOH solution 1
After adjusting the pH to 7.6 after adding 2 ml. ** Salts solution CaCl ₂ Anhydrous 0.2g MgSO ₄ 0.2g K ₂ HPO ₄ 1.0g KH ₂ PO ₄ 1.0g NaHCO ₃ 10.0g NaCl 2.0g Purified water 1000.0ml

The presence or absence or assimilation of the assimilation ability of the strain was judged according to the following criteria. That is, the degree of pH decrease was used as a scale, and the larger the pH, the better the growth of enterobacteria, and the higher the assimilation capacity of the carbohydrate in the medium was determined.

[0028]

The results of assimilation are shown in Table 1 (No. 1, 2, 3)
Table 2 shows the results of the utilization rate. The assimilation rate (%) indicates the assimilation tendency of the microorganism to which the test strain belongs.
It was calculated from the number of strains that assimilated sugar / total number of strains.

As is apparent from the table, lactosylxylloside, fucosylxylloside and sorbosylxylloside are assimilated by bifidobacteria, but by other plain harmless or harmful bacteria. The result was that it was a difficult sugar. Therefore, the xylulose-linked oligosaccharide of the present invention can be used as a carbohydrate for growing Bifidobacterium.

[0031]

[Table 1]

[0032]

[Table 2]

[0033]

[Table 3]

[0034]

[Table 4]

[0035]

INDUSTRIAL APPLICABILITY The novel xylulose-binding oligosaccharide of the present invention has activity as a bifidobacteria growth factor.
According to the method of the present invention, these xylulose-bound oligosaccharides can be produced inexpensively and efficiently, and useful sweeteners, functional foods such as food materials, and pharmaceutical materials are expected to be used. It

[Brief description of drawings]

FIG. 1 is a high performance liquid chromatograph of the reaction solution of Example 1.

FIG. 2 is a graph showing a ¹³ C-NMR spectrum of lactosyl xyluloside.

FIG. 3 is a high performance liquid chromatograph of the reaction solution of Example 2.

FIG. 4 is a graph showing a ¹³ C-NMR spectrum of fucosyl xyluloside.

5 is a high performance liquid chromatograph of the reaction liquid of Example 3. FIG.

FIG. 6 is a graph showing a ¹³ C-NMR spectrum of sorbosyl xyluloside.

[Explanation of symbols]

 X1: carbon 1-position of xylulose X2: carbon 2-position of xylulose X3: carbon 3-position of xylulose X4: carbon 4-position of xylulose G1: carbon 1-position of glucose Gal1: carbon 1-position of galactose Gal4: Galactose 4th carbon F1: 1st fucose 1st carbon F6: Fucose 6th carbon S1: Sorbose 1st carbon S2: Sorbose 2nd carbon S6: Sorbose 6th carbon Me: Methanol ( Internal standard substance)

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Sumio Kitahata 621-440 Noda, Kumatori-cho, Sennan-gun, Osaka Prefecture (72) Inventor Hirofumi Nakano 3-14-3 Hattori Nishimachi, Toyonaka City, Osaka Prefecture (72) Inventor Masao Kondo 13-46 Daikokucho, Tsurumi-ku, Yokohama-shi, Kanagawa Shimizu Port Sugar Sugar Co., Ltd. (72) Inventor Hajime Taniguchi 2-194-4 Kariya-cho, Ushiku-shi, Ibaraki (72) In Ren Hashimoto Tsurumi-ku, Yokohama-shi, Kanagawa 13-46 Daikokucho Shimizu Port Sugar Co., Ltd.

Claims (8)

[Claims] 1. A novel xylulose-binding oligosaccharide in which D-xylulose is β-bonded to lactose, L-fucose or L-sorbose. 2. The novel oligosaccharide according to claim 1, which is a lactosyl xyluloside in which D-xylulose is β-2,1-linked to a glucose residue at the reducing end of lactose. 3. L-fucose contains D-xylulose β-
The novel oligosaccharide according to claim 1, which is a 2,1-linked fucosyl xyluloside. 4. D-xylulose is β in L-sorbose.
The novel oligosaccharide according to claim 1, which is a sorbosyl xyluloside having a 2,2 bond. 5. An enzyme having fructose transfer activity is allowed to act on a liquid containing glucosylxyluloside as a donor substrate in the presence of aldose or ketose as an acceptor substrate to transfer and bond a xylulosyl group in addition to glucose. And a method for producing a xylulose-linked oligosaccharide. 6. A xylulose-binding oligosaccharide characterized in that in the presence of aldose or ketose as an acceptor substrate, an enzyme having fructose transfer activity and yeast are simultaneously acted on a solution containing glucosylxyluloside as a donor substrate. Production method. 7. The method for producing a novel oligosaccharide according to claim 5 or 6, wherein the acceptor substrate is lactose, L-fucose or L-sorbose. 8. An enzyme having a fructose transfer activity is Arthrobacter sp. K-1.
The method according to any one of claims 5 to 7, which is β-fructofuranosidase derived from (FERM BP-3192).