JPH0649715B2 - Gluco-oligosaccharide having inositol residue bound to the terminal and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel and useful glucooligosaccharide having an inositol residue at the terminal and a method for producing the same.

[Conventional technology]

Recently, various oligosaccharides have been provided as biochemical reagents,
For example, it has attracted attention as a substrate for measuring amylase activity, a cariogenic sweetener, a growth promoting substance for Bifidobacterium, and the like.

Among these, the substrates for measuring amylase activity include those targeting maltotetraose or maltopentaose (JP-A-50-56998) and those targeting nitro or halogenated aromatic glycosides (JP-B-61). -78) is known. Further, an oligosaccharide having 1 to 4 molecules of fructose bound to sucrose as a carious sweetener (Japanese Patent Laid-Open No. 56-154967), and O-β-D- as a growth promoting substance for Bifidobacterium. Galactose pyranosyl-
(1 → 4)-[O-β-D-galactopyranosyl- (1 →
6)]-D-Glucose oligosaccharide (JP-A-58-99)
No. 497) is known.

On the other hand, some of these oligosaccharides contain α-
A method for producing by reacting an enzyme such as amylase has been proposed (Japanese Patent Publication No. 56-22520).

The above oligosaccharides have different functions, and it can be seen that oligosaccharides produced differently have various functions. Therefore, development of new oligosaccharides and a method for producing the same is desired due to such a variety of functions of oligosaccharides.

[Object of the Invention]

The present invention is to meet the above demands, and is a novel oligosaccharide which has not been published in the literature, and is used not only as a biochemical reagent but also as a substrate for measuring amylase activity and a growth promoting substance for Bifidobacterium. The purpose of the present invention is to propose a glyco-oligosaccharide useful as such and a method for producing the same.

[Structure of Invention]

The present invention is a glucooligosaccharide having an inositol residue attached to the next terminal and a method for producing the same.

(1) General formula (Glc _n Glc-Ino [I] (In the formula, Glc represents a glucose residue, Ino represents an inositol residue, n represents 0 or an integer of 1 to 7, and Glc-Glc Represents an α-1,4-glycoside bond, and Glc-Ino represents a glycosidic bond formed by the 1-position hydroxyl group of Glc and the hydroxyl group of Ino.) A glucooligosaccharide having an inositol residue bonded to the end.

(2) Cyclodextrin and inositol are reacted in the presence of cyclodextrin glucanotransferase, a general formula (Glc _n Glc-Ino [I] (In the formula, Glc represents a glucose residue, Ino represents inositol. Represents a residue, n represents 0 or an integer of 1 to 7, and Glc-Glc represents an α-1,4-glycoside bond, and Glc-Ino represents a glycoside bond between the 1-position hydroxyl group of Glc and the hydroxyl group of Ino. The method for producing a glucooligosaccharide in which an inositol residue is bound to the end represented by.

The glucooligosaccharide having an inositol residue attached to the end according to the first aspect of the present invention is represented by the above general formula [I], and contains one glucose having Glc as a residue or α-
It is an oligosaccharide in which the hydroxyl group of inositol having Ino as a residue is linked to the hydroxyl group at the 1-position of the terminal glucose of 2 to 7 glucose oligomers having 1,4 glucoside bonds by a glycoside bond. The position of the hydroxyl group of inositol that is bound by a glycoside bond is arbitrary.

Examples of the oligosaccharide represented by the general formula [I] include the following compounds.

Compound (1) A compound (n = 0 in the general formula [I]) in which a glucose residue is glycosidic-bonded to an inositol residue.

Compound (2) A compound in which a maltose residue is glycoside-linked to an inositol residue (n = 1 in the general formula [I]).

Compound (3) A compound in which a maltotriose residue is glycoside-bonded to an inositol residue (n = 2 in the general formula [I]).

Compound (4) A compound in which a maltotetraose residue is glycosidic bonded to an inositol residue (n = 3 in the general formula [I]).

Compound (5) A compound in which a maltopentaose residue is glycoside-bonded to an inositol residue (n = 4 in the general formula [I]).

Compound (6) A compound in which a maltohexaose residue is glycoside-bonded to an inositol residue (n = 5 in the general formula [I]).

Compound (7) A compound in which a maltoheptaose residue is glycoside-linked to an inositol residue (n = 6 in the general formula [I]).

Compound (8) A compound in which a maltooctaose residue is glycosidic-bonded to an inositol residue (n = 7 in the general formula [I]).

In the general formula [I], the inositol residue represented by Ino can be any one as long as the object of the present invention can be achieved.
Although not limited to a particular isomer, preferred examples include residues derived from myo-inositol widely found in microorganisms such as animals, plants and yeast.

The physicochemical properties of the glucooligosaccharide of the present invention are shown below.

Physicochemical properties: (A) Solubility in solvent All oligosaccharides of n = 0 to 7 in the formula [I] are soluble in water, but insoluble in acetone, chloroform and benzene and hardly soluble in hydrous alcohol. is there.

(B) Color reaction All the olig sugars of n = 0 to 7 of the formula [I] show ammonia / silver nitrate reaction negative phenol / sulfuric acid method positive anthrone reaction positive.

(C) Color tone Each of the above oligosaccharides is white in a dry powder form.

(D) Acidic, Basic, and Neutral Each oligosaccharide is neutral.

(E) Infrared absorption spectrum Among the above oligosaccharides, the n = 0 oligosaccharide of the formula [I] is
The infrared absorption spectrogram measured by the KBr tablet method is as shown in FIG. An absorption band appears at almost the same position for oligosaccharides with n = 1 to 7.

(F) Proton Nuclear Magnetic Resonance Spectra Among the above oligosaccharides, the proton nuclear magnetic resonance spectrogram of the oligosaccharide of formula [I] n = 0 measured at 270 MHz with heavy water (D ₂ O) as a solvent is shown in FIG. Is the street.

(G) ¹³ C Nuclear Magnetic Resonance Spectra Among the above oligosaccharides, oligosaccharides of n = 0 in the formula [I] are prepared by using deuterated water (D ₂ O) as a solvent and sodium trimethylsilylpropionate (TSP) as a standard substance. The ¹³ C nuclear magnetic resonance spectrogram measured by the complete coupling method at 68 MHz is shown in FIG.

(H) Specific optical power Similarly, the specific optical power of the n = 0 oligosaccharide of the formula [I] is ▲ [α] ²⁵ _D ▼ + 82.70 (C = 1.0, H ₂ O).

(I) Confirmation of constituent sugars (i) Confirmation of the n-number of oligosaccharides represented by the formula [I] is carried out by preparing a solution of the test oligosaccharide so that the final hydrochloric acid concentration is 1N, It can be carried out by treating for about 90 minutes to completely hydrolyze the glycosidic bond and calculating the content ratio of glucose and inositol produced.

The molar ratio of glucose to inositol in each oligosaccharide represented by the formula [I] is as follows.

In formula [I], when n = 0, 1/1, when n = 1, 2/1, n = 2
3/1 when n = 3, 4/1 when n = 3, 5/1 when n = 4, n =
6 for 5; 7/1 for n = 6; 8/1 for n = 7.

(Ii) When each n> 0 oligosaccharide of the formula [I] is treated with glucoamylase, glucose is sequentially released, and n = 0 oligosaccharide is obtained as a final product.

From this, it can be confirmed that Glc-Glc constituting the oligosaccharide of the present invention is composed of an α-1,4-glycoside bond (Nature, 181 , 770 (1958)).
It can be confirmed that the inositol residue is attached to the end.

The oligosaccharide specified above can be produced by the production method of the second invention of the present invention. in this way,
Cyclodextrin and inositol are reacted in the presence of cyclodextrin glucanotransferase (hereinafter referred to as CGTase) to produce a glucooligosaccharide having an inositol residue at the end.

The cyclodextrin used as a substrate in the present invention is D-glucose formed by α-1,4-glycosidic bond to form a ring. Α-cyclodextrin consisting of 6 glucose residues, 7 glucose residues And β-cyclodextrin composed of 8 glucose residues, and any of them may be used depending on the type of enzyme, the value of n of the target oligosaccharide, and the like.

The other substrate of the present invention, inositol, is not particularly limited as described above, but myo-inositol is preferable.
The molar ratio of cyclodextrin to inositol is preferably about 1: 1 to 10.

As the CGTase (EC2.4.1.19) used in the above reaction, any CGTase (EC2.4.1.19) can be used, so long as it can achieve the object of the present invention, regardless of the type of microorganism produced. CG like this
Examples of Tase include Bacillus orvensis ( Bacill
us ohbensis ), B. megateri
um ), Bacillus macerans ( B. macerans ), Bacillus circulans ( B. circulans ), and other known CGTases obtained from cultures can be preferably used, and those derived from Bacillus obensis are particularly preferable. . The amount of CGTase added to the reaction substrate is 200-300u / g-
It is about the substrate.

The reaction conditions vary slightly depending on the type of CGTase used, and are not particularly limited, but generally, the temperature is 30 to 70 ° C, pH 6.0 to
7.5, the reaction time may be 1 hour or more, preferably 1 to 24 hours.

By the above reaction, CGTase acts on cyclodextrin, transposition of glucose residue occurs using inositol as an acceptor, and a glucooligosaccharide in which an inositol residue is bound to the terminal of formula [I] is produced. At this time, the bond between the inositol and the glucose residue is a glycosidic bond, and it is presumed that the residue at the 1-position of glucose as a donor is bonded to the hydroxyl group of inositol having a configuration corresponding to the 4-position of glucose. It Further, the value of n of the oligosaccharide produced increases as the number of glucose residues of the substrate cyclodextrin increases, and for example, when γ-cyclodextrin is used, n = 7 oligosaccharides are produced and glucose Residues are liberated to produce smaller n values.

Thus, in the reaction solution, oligosaccharides with different numbers of constituent glucose residues having inositol residues bound to the ends and their side reaction products are mixed, so it is possible to separate and collect each oligosaccharide as necessary. it can.

As the separation / collection means, known means used for separation / collection of various sugars can be used, and examples thereof include gel filtration, ion exchange, and chromatography using an adsorption carrier.

The identification of each oligosaccharide represented by the formula [I] is carried out by separating the above reaction solution with a preparative aminopropyl column in the same separation mode as the analytical HPLC column, and confirming each preparative fraction with the aforementioned constituent sugars. It may be identified based on the method.

The oligosaccharide of the formula [I] produced by the above is useful as a general biochemical reagent, a substrate for measuring amylase activity, a growth promoting substance for Bifidobacterium, and the like.

Generally, when an oligosaccharide is used as a substrate for measuring the malamase activity, for example, α-amylase is used as a coupling enzyme for α-amylase.
-Using glucosidase, the activity of α-amylase can be measured by the following method.

The glucose produced here can be quantified by, for example, glucose oxidase / peroxidase / dye system or hexokinase / phosphoglucomutase / glucose-6-phosphate dehydrogenase / NADH system to convert the activity of α-amylase.

As described above, the oligosaccharide of the present invention can be used in place of conventional maltopentose and is useful as a substrate for measuring amylase activity.

When the oligosaccharide is used as a growth promoting substance for Bifidobacterium, instead of glucose used in a conventional culture medium, or by culturing by adding the oligosaccharide to the medium together with this glucose,
It is possible to promote the growth of Bifidobacterium.

Since the oligosaccharide of the present invention is indigestible, when it is ingested, it reaches the intestine directly and is utilized by the bifidobacteria in the intestine, so that the intestinal flora can be maintained in a normal balance.

〔The invention's effect〕

According to the first aspect of the present invention, a glucooligosaccharide in which an inositol residue is bound to a novel and useful terminal, which has not been published in the literature, can be obtained. Further, according to the second aspect of the present invention, a glucooligosaccharide having an inositol residue bound to the terminal can be efficiently produced by a simple method.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to Examples. In the examples,% is% by weight unless otherwise specified.

(I) Production of Gluco-oligosaccharide with Inositol Residue Bonded to the Terminal As an enzyme reaction substrate, β-cyclodextrin 50 g and
Using 50 g of myo-inositol, a 25 mM McIlvaine buffer (McIvaine buffe
r) (pH 6.0) is added to
After adding 250 u / g-substrate of CGTase, the mixture was reacted at 60 ° C. for 20 hours. After the reaction was completed, it was boiled for 15 minutes to inactivate the enzyme.

Then, this reaction solution was cooled, and the inactivated enzyme was removed by filtration as a sample, and a preparative aminopropyl group-supported silica packed column (YMCA-643, 20 mmφ × 250 mm) was used.
Chromatographic separation was performed using 2 Ls, internal volume 157 m, manufactured by Yamamura Chemical Co., Ltd. The chromatographic conditions are as follows.

(Conditions) Sample concentration: 2 w / v% (diluted with distilled water) Sample injection amount: 2.0 m (solid content 40 mg) Eluent: CH ₃ CN / H ₂ O (1/1; volume ratio) Liquid speed: 2 m / min Detector: RI Detector The chromatogram obtained above is shown in FIG.

The first peak shown in FIG. 4 is glucose (content 2
%) And the second peak was inositol (21%), which was confirmed by HPLC analysis after fractionation. After the third peak, the constituent sugars were confirmed by the above-mentioned hydrochloric acid treatment after fractionation. For example, for the fourth peak, after the start of separation
60 minutes (120m) from 170 minutes to 230 minutes were collected, and this operation was repeated 5 times to obtain a total of 600 peak NO.4 areas.
m fractions were collected. When this liquid was concentrated and dried, it was taken out and weighed. The sample injection amount was 10 m (solid content 200 m
The dry solid (= peak NO.4 component) obtained from g) was 44 mg. This dried solid was dissolved in 1N hydrochloric acid (10 m) in a flask and heated to boil. At this time, a cooling pipe was connected to the upper part of the flask to maintain the liquid amount. After 1.5 hours, heating was terminated, and the cooled liquid was desalted with a regenerated anion exchange resin. HPLC analysis of the thus obtained peak NO.4 component acid hydrolyzate revealed that the peak NO.4 component acid hydrolyzate consisted of glucose 63.5% and inositol 36.5%. The ratio (= molar ratio) was 2: 1.

When the other peaks were similarly collected and analyzed,
The third peak is Glc-Ino (content 34%), the fourth peak is (Glc ₂ Ino (22%) and the fifth peak is (Glc ₃ Ino (11%)). The peak is (Glc ₄ Ino
(6%), 7th peak (Glc ₅ Ino (3%),
The eighth peak was identified to be each oligosaccharide having (Glc ₆ Ino (1%)). The physicochemical properties of each oligosaccharide obtained are as described above.

(Ii) Growth-promoting effect of Bifidobacterium The assimilation ability of the oligobacteria obtained as described above by Enterobacter in vitro was examined by the following method.

Bifido bacteria medium (polypeptone 1.0%, meat extract 0.5%, yeast extract 0.5%, glucose 1.0%, K ₂ HPO
₄ 0.3%, Tween80 0.1%, pH 7.0) basal medium consisting of a composition excluding glucose, to each medium added with each oligosaccharide of the present invention and other saccharides as a control at a concentration of 1%,
Bifidobactetium adolescentis JCM129, a representative Bifidobacterium
Inoculate the bacterial solution of 5 to 10 ⁴ to 10 ⁵ / m, and incubate at 37 ℃ for 2
Anaerobic culture was carried out for 4 hours. The growth of the fungus was determined by measuring the turbidity at 610 nm with the turbidity of glucose set to 100 and the relative growth of the fungus in other saccharides. The results are shown in Table 1.

From Table 1, among the oligosaccharides of the present invention, (Glc) ₂ -Ino, (Glc) ₃ -Ino, and (Glc) ₄ -Ino show a higher growth rate than glucose and have a large effect of promoting the growth of Bifidobacterium. I understand. Among them, (Glc) ₃ -Ino exhibits the highest effect, and a growth promoting effect about twice that of glucose is recognized.

[Brief description of drawings]

Fig. 1 is the infrared absorption spectrogram of Glc-Ino, 2nd
The figure shows the proton nuclear magnetic resonance spectrogram of Glc-Ino,
Fig. 3 shows the ¹³ C nuclear magnetic resonance spectrogram of Glc-Ino,
FIG. 4 is a chromatogram of preparative HPLC of the enzyme reaction solution in the examples.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroshi Morita 7-1 Morinosato Wakamiya, Atsugi City, Kanagawa Prefecture Kurita Industry Co., Ltd. (72) Inventor Nobuhiro Oda 7-1 Morinosato Wakamiya, Atsugi City, Kanagawa Prefecture Kurita Industry Co., Ltd. (72) Inventor Mikiharu Okumura 3-4-7 Nishi-Shinjuku, Shinjuku-ku, Tokyo Kurita Industry Co., Ltd.

Claims (6)

[Claims] 1. A general formula (Glc _n Glc-Ino [I] (wherein Glc represents a glucose residue, Ino represents an inositol residue, n represents 0 or an integer of 1 to 7, and Glc -Glc represents an α-1,4-glycoside bond, and Glc-Ino represents a glycosidic bond formed by the 1-position hydroxyl group of Glc and the hydroxyl group of Ino.), Which is an inositol residue-bound glucooligosaccharide. 2. The glucooligosaccharide according to claim 1, wherein the inositol residue is derived from myo-inositol. 3. A general formula (Glc _n Glc-Ino [I] (wherein Glc represents a glucose residue and Ino is represented by Ino), wherein cyclodextrin and inositol are reacted in the presence of cyclodextrin glucanotransferase. Represents an inositol residue, n represents 0 or an integer of 1 to 7, Glc-Glc represents an α-1,4-glycoside bond, and Glc-Ino represents a glycoside formed by the 1-position hydroxyl group of Glc and the hydroxyl group of Ino. The method for producing a glucooligosaccharide in which an inositol residue is bound to the end represented by 4. The method according to claim 3, wherein the cyclodextrin is α-, β- or γ-cyclodextrin. 5. The method according to claim 3 or 4, wherein the inositol is myo-inositol. 6. The method according to any one of claims 3 to 5, wherein the cyclodextrin glucanotransferase is derived from Bacillus orvensis, Bacillus megaterium, Bacillus macerans or Bacillus circulans. Method.