JPH10215892A - Production of oligosaccarides composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an oligosaccaride composition useful as a health food material with functions, such as low sweetness and low calorific value and resistance to decomposition and cariogenicity, by binding a saccaride except glucose to panose through an enzymatic reaction. SOLUTION: This oligosaccaride is obtained by making one kind of saccaride selected from the group consisting of monosaccarides except glucose, sugar alcohols and oligosaccarides coexist with a saccaride solution containing pullulan or panose, followed by subjecting the system to an enzyme capable of forming panose with pullulan as substrate; wherein coexistence of a monosaccaride produces a heterooligosaccaride as a tetrose with the monosaccaride bound to the reduced terminal(s) of panose, coexistence of a sugar alcohol a reduced oligosaccaride as a tetrose with sorbitol bound to the reduced terminal(s) of panose through α-1,6 linkage, coexistence of a sugar alcohol except sorbitol a reduced oligosaccaride as a tetrose with the sugar alcohol bound to the reduced terminal(s) of panose, and coexistence of an oligosaccaride an oligosaccaride as a pentose or higher saccaride with the oligosaccaride bound to the reduced terminal(s) of panose.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention provides an oligosaccharide composition containing a hetero- or reduced oligosaccharide composed of tetrasaccharide or an oligosaccharide composed of pentasaccharide or more, which is industrially useful as a food material having excellent functionality. On how to do it.

[0002]

2. Description of the Related Art Carbohydrates have been conventionally used as a sweetness source or a nutrient source.
Sugars having functional properties such as bifidobacteria growth factor and hard caries have been developed and used as health food materials.

[0003] Among these new oligosaccharides, the one with the highest production is isomalto-oligosaccharide. Isomaltooligosaccharides are highly evaluated for not only the above-mentioned functions but also for improving taste quality. Moreover, since it is produced by an enzymatic method using starch as a raw material, it is one of the factors that is highly valued because it is the least expensive oligosaccharide recently developed.

Focusing on the excellent functionality of isomaltoligosaccharides, Sakano, one of the present inventors, first examined the transglycosylation reaction of an enzyme that decomposes pullulan to produce panose, and examined the novel oligosaccharide isoform. Maltosyl isomaltose (IMI
M) and filed a patent application (Japanese Unexamined Patent Publication No. 7-258).
No. 91).

On the other hand, among oligosaccharides recently developed and used as food materials, there are many types of heterooligosaccharides such as galactooligosaccharides, coupling sugars, fructooligosaccharides, nectar oligosaccharides, soybean oligosaccharides and raffinoses. It is added to foods taking advantage of their respective functions.

[0006] Soybean oligosaccharides and raffinose are produced from oligosaccharides contained in soybeans and sugar beets, whereas
Galactooligosaccharide is an oligosaccharide composition obtained by reacting lactose with β-galactosidase, and is composed of a mixture of galactosyl lactose and galactose-glucose n (n is 1 to 4). Coupling sugar is an oligosaccharide composition obtained by allowing cyclodextrin synthase (CGTase) to act on starch and sucrose, and is composed of a mixture of glucose n-sucrose (n is 1 to 4). Fructooligosaccharide is an oligosaccharide composition obtained by reacting fructofuranosidase on sugar, and is composed of a mixture of sucrose-fructose n (n is 1 to 4). Nectar oligosaccharide is an oligosaccharide composition obtained by allowing fructofuranosidase to act on sugar and lactose, and contains lactosylfructoside as a main component.

As described above, many heterooligosaccharides are produced by enzymatic methods using natural oligosaccharides which are easily available as raw materials, and are used as oligosaccharide compositions having several similar structures.

However, a hetero-oligosaccharide in which a hexose such as fructose or mannose other than glucose is bonded to the reducing end of panose, a kind of isomaltose, has not been known so far. Furthermore, there is no known heterooligosaccharide in which pentose of xylose or arabinose is bonded to the reducing end of panose.

Further, a reduced oligosaccharide in which sorbitol is α-1,6-linked to the reducing end of panose is not known. Furthermore, a reduced oligosaccharide in which a sugar alcohol other than sorbitol, such as xylitol or erythritol, is bonded to the reducing end of panose is not known. Further, there is no known pentasaccharide or more heterooligosaccharide in which a disaccharide such as sucrose, lactose, cellobiose, and trehalose is bonded to the reducing end of panose.

[0010]

SUMMARY OF THE INVENTION The present inventors have studied the enzymatic action of amylase in detail and found that panose is produced using pullulan as a substrate by allowing glucose to coexist in a sugar solution containing pullulan or panose. It has been found that a novel isomaltooligosaccharide, IMIM, and a known isomaltosyl maltose (IMM) are produced by the action of an enzymatic enzyme, and a patent application was previously filed (JP-A-7-25891).
issue).

By the way, if a sugar other than glucose is bound to panose, a novel oligosaccharide having both the properties of an isomaltooligosaccharide and the properties of a heterooligosaccharide will be produced. Therefore, when the binding properties of sugars other than glucose to panose were examined, it was surprisingly found that hetero-oligosaccharides and hexasaccharides composed of hexasaccharides, pentoses, and sugar alcohols excluding glucose and tetrasaccharides in which sugar alcohols were bonded to the reducing end of panose. It was found that sugar was formed. Furthermore, they have also found that oligosaccharides such as disaccharides and the like are linked to panose to produce oligosaccharides of pentasaccharide or more, and have completed the present invention.

That is, an object of the present invention is to provide a novel method for producing a hetero-oligosaccharide or a reduced oligosaccharide having both the properties of an isomalt-oligosaccharide and the properties of a hetero-oligosaccharide which are useful as industrial food materials and have excellent functionality. Is what you do.

[0013]

The first invention of the present application is to coexist a monosaccharide other than glucose in a sugar solution containing pullulan or panose, and to add an enzyme for producing panose using pullulan as a substrate to act. And a method for producing an oligosaccharide composition comprising a heterooligosaccharide comprising a tetrasaccharide in which a monosaccharide is bonded to a reducing end of panose.

According to the second invention of the present application, a sugar alcohol is made to coexist with a sugar solution containing pullulan or panose, and an enzyme for generating panose using pullulan as a substrate is added thereto to act. -1,6-
This is a method for producing an oligosaccharide composition containing a reduced oligosaccharide comprising a bound tetrasaccharide.

[0015] The third invention of the present application is to coexist a sugar alcohol other than sorbitol in a sugar solution containing pullulan or panose, add an enzyme capable of producing panose using pullulan as a substrate, and act on the sugar alcohol. This is a method for producing an oligosaccharide composition containing a reduced oligosaccharide consisting of a tetrasaccharide to which a sugar alcohol is bound.

[0016] The fourth invention of the present application is to coexist an oligosaccharide in a sugar solution containing pullulan or panose, add an enzyme that generates panose using pullulan as a substrate, and make it act.
This is a method for producing an oligosaccharide composition containing an oligosaccharide composed of five or more saccharides in which an oligosaccharide is bonded to a reducing end of panose.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION In the first invention of the present application, at least one selected from the group consisting of fructose, mannose, xylose and arabinose is preferably used as a coexisting monosaccharide.

In the second invention of the present application, sorbitol is used as the coexisting sugar alcohol. In the third invention of the present application, as the sugar alcohol to be coexisted,
Preferably, at least one selected from the group consisting of xylitol and erythritol is used.

In the fourth invention of the present application, the oligosaccharide to be co-present is preferably lactose, sucrose,
At least one selected from the group consisting of trehalose and cellobiose is used.

The enzyme used in the present invention is not particularly limited as long as it is an α-amylase which hydrolyzes an α-1,4-glucoside bond of pullulan to produce panose. Examples of such α-amylase include, for example, high-temperature actinomycetes T
An enzyme produced by hermoactinomyces vulgarisR-47 (JP-A-7-25891) can be mentioned.

Shimizu et al., Thermoactinomyces v
ulgaris R-47 α-amylase (TVA I) was found to degrade pullulan to produce panose (M. Shimi
zu et al., Agric. Biol. Chem., 42 , 1681 (1978)). Next, one of the present inventors, Sakano, conducted a detailed study on the enzymatic action of TVA I and clarified its properties.
(Y. Sakano et al., Agric. Biol. Chem., 46 , 1121 (19
82); Y. Sakano et al., Agric. Biol. Chem., 46 , 142.
3 (1982); Y. Sakano et al., Agric. Biol. Chem., 47 ,
2211 (1983); Y. Sakano et al., Agric. Biol. Chem.,
49 , 3391 (1985)).

Furthermore, Sakano found that α-amylase (TVA II) different from TVA I was produced in Escherichia coli cloned by fractionating a plasmid from the gene of this bacterium by the shotgun method (Y . Sakano et al., Biosci. Biot
ech. Biochem., 57 , 395 (1993)). After that, by constructing an expression system that enhances the protein synthesis ability of TVA II to greatly increase the production of this enzyme by Escherichia coli,
A method for producing a high-purity enzyme that is easily crystallized by extraction operations such as heating and ultrasonic treatment has been developed (Japanese Patent Laid-Open No. 7-1995).
No. 25891).

TVA II is an α-amylase having a different base sequence and amino acid sequence from TVA I and a different physicochemical property from the conventional enzyme (Y. Sakano et al., Biosci. B.
iotech. Biochem., 57 , 395 (1993)).

The physicochemical properties of TVA II are as follows. (1) Action (a) It mainly produces maltose from starch. (B) Generate panose from pullulan. (C) hydrolyzing pullulan in the presence of glucose, with the following formula (I):

[0025]

Embedded image

An isomaltooligosaccharide represented by the following formula (II):

[0027]

Embedded image

To produce the isomaltooligosaccharide represented by

(2) Substrate specificity The α-1,4-glucosidic bond of starch and pullulan is hydrolyzed to produce maltose and panose, respectively.
The generated panose is not decomposed, and produces the isomaltooligosaccharide represented by the formula (I) and the isomaltooligosaccharide represented by the formula (II) in the presence of glucose. Decomposes the isomaltosyl maltose represented by the formula (II).

(3) Optimum pH and pH stability There is an optimum pH of 6 to 7 using pullulan as a substrate.
It is stable in H6-9.

(4) Optimum temperature and thermal stability There is an optimum temperature of 45 to 55 ° C using pullulan as a substrate,
Stable up to 50 ° C.

(5) The molecular weight is about 60,000 (S
DS polyacrylamide gel slab electrophoresis). TVA II hydrolyzes pullulan in the presence of glucose,
Generate IMIM and IMM. That is, IMIM in which glucose is α-1,6-linked and glucose are added to the reducing end of panose produced by hydrolysis of pullulan.
An oligosaccharide composed of two kinds of 1,4-linked IMM tetrasaccharides is produced. In addition, the generated IMM is decomposed by the present enzyme, but the IMIM is not decomposed and accumulates in the reaction solution.

That is, the present enzyme is an enzyme that mainly produces maltose from starch and panose from pullulan.
It is a unique enzyme that binds glucose, which is not an original product, to the reducing end of panose by a sugar transfer reaction.

[0034] If the enzyme has the ability to carry out a glycosyltransfer reaction on glucose which is not the original product, it may be possible to bind sugars other than glucose. Thus, instead of glucose, fructose, the same hexose, was allowed to react with TVAII in the presence of pullulan. When the reaction solution was subjected to thin layer chromatography on silica gel, in addition to panose with Rf = 0.68, Rf =
Spots appeared at the positions of 0.55 and Rf = 0.47.

The produced sugar is separated by silica gel G60.
(Merck) by thin layer chromatography, using n-butanol: ethyl alcohol: water = 5: 5:
It was developed twice using 2.5. Rf indicates a relative development distance when the development distance of glucose is set to 1.

By the way, when glucose coexists with pullulan, the main products are IMIM (Rf = 0.43) and panose (R
f = 0.68) and IMM (Rf = 0.61). Fructose R
Since f is 0.98 and shows almost the same development distance as glucose, and the spot with Rf = 0.47 has almost the same development distance as IMIM, the product with high color development rate and short development distance is located at the reducing end of panose. Fructose is α-1,
6-linked hetero-oligosaccharide of tetrasaccharide. The spot having a long development distance of Rf = 0.55 is a tetrasaccharide hetero-oligosaccharide in which fructose is bound to the reducing end of panose at a position different from α-1,6-.

That is, when fructose is co-existed with pullulan and reacted with TVA II, two types of heterooligosaccharides composed of tetrasaccharide having fructose bonded to the reducing end of panose are generated in addition to panose, and these heterooligosaccharides are contained. An oligosaccharide composition was obtained.

Next, when TVA II was reacted in the presence of pullulan with mannose instead of glucose, as in the case of fructose, Rf = 0.62 and Rf
The product was observed at the position of f = 0.47. Mannose Rf
Is 1.02 and shows almost the same development distance as glucose, and the spot with Rf = 0.47 has almost the same development distance as IMIM.Therefore, the product with a high color development rate and a short development distance has mannose at the reducing end of panose. Is α-1,6-
A linked tetrasaccharide hetero-oligosaccharide. Since the spot with Rf = 0.62 is closer to the panose development distance than the IMM, it is difficult to distinguish it from the panose. However, since a longer spot was obtained compared to panose alone, it is considered that a tetrasaccharide hetero-oligosaccharide having α-1,4-linked mannose at the reducing end of panose was generated. That is, two types of heterooligosaccharides composed of tetrasaccharides in which mannose was bonded to the reducing end of panose were produced, and an oligosaccharide composition containing these heterooligosaccharides was obtained.

As described above, TVA II not only binds glucose to panose to produce isomatooligosaccharides consisting of tetrasaccharides of IMIM and IMM, but also converts hexoses such as fructose and mannose other than glucose to panose. It has been clarified that a heterooligosaccharide composed of tetrasaccharide is produced by binding to the reducing end side, and an oligosaccharide composition containing these heterooligosaccharide as a main component is produced.

Hetero-oligosaccharides in which hexoses other than glucose are bound to panose, one of the isomaltooligosaccharides, have not been known at all. These hetero-oligosaccharides are also difficult to degrade because TVA II cannot degrade the transglycosylation product IMIM. Hetero-oligosaccharides that TVAII is difficult to degrade are difficult to degrade even with many other amylases. One of the functions of oligosaccharides is that they are hardly decomposable. Hetero-oligosaccharides, in which panose and a monosaccharide other than glucose are bonded, are attracting attention as functions of hardly degradable oligosaccharides.

Not only is a heterooligosaccharide having fructose and mannose bound to the reducing end of panose, but also galactose is bound to the reducing end of panose.
It has been recognized that heterooligosaccharides composed of sugars are produced.

Since hexoses other than glucose are bound to panose, pentoses may also be bound to panose. Next, when pentose xylose was coexisted with pullulan instead of glucose, TVA II was reacted, and a product was unexpectedly found at the position of Rf = 0.59. This developed position was almost the same as that of the IMM (Rf = 0.61), and it was confirmed that a hetero-oligosaccharide composed of a tetrasaccharide having xylose 1,4-linked to the reducing end of panose was generated.

Since xylose is a pentose and does not have a carbon at the sixth position, a tetrasaccharide having xylose 1,4-bonded to the reducing end of panose is produced. Similarly,
When arabinose coexists with pullulan and reacts, Rf
= 0.60, and the formation of a heterooligosaccharide consisting of a tetrasaccharide in which arabinose was bound to the reducing end of panose was confirmed.

As described above, TVA II binds a pentose such as xylose and arabinose to the reducing end of panose to produce a hetero-oligosaccharide consisting of tetrasaccharides, and produces an oligosaccharide composition containing these hetero-oligosaccharides. It became clear. It is recognized that not only a hetero-oligosaccharide in which xylose and arabinose are bound to panose is formed, but also a ribose is formed in a hetero-oligosaccharide consisting of a tetrasaccharide bonded to the reducing end of panose.

Hetero-oligosaccharides in which pentose is bonded to the reducing end of panose, one of the isomaltooligosaccharides, have not been known at all. These hetero-oligosaccharides are also TVA
II is considered to be difficult to decompose, and the hetero-oligosaccharide in which panose and pentose are linked also attracts attention as a function as a hardly decomposable oligosaccharide.

We have further noticed that TVA II produces unexpected oligosaccharides. Sorbitol, a sugar alcohol, coexists with pullulan instead of glucose
Upon reaction with TVAII, a product was observed at the position of Rf = 0.41. The development distance of sorbitol is Rf = 0.90 and I
Since the deployment distance of the MIM is Rf = 0.43, Rf = 0.41
Is an α-saccharide with sorbitol at the reducing end of panose.
It is a reduced oligosaccharide consisting of 1,6-linked tetrasaccharides. No reduced oligosaccharide in which sorbitol is α-1,6-linked to panose has been known at all.

Further, when xylitol and erythritol are allowed to react with pullulan as sugar alcohols instead of sorbitol, the reaction is carried out.
A product is observed at the position of 0.58 and Rf = 0.47.In the case of erythritol, the development distance is close to panose.Rf = 0.65 and Rf =
The product was observed at the position of 0.56. Each of them is a reduced oligosaccharide composed of a tetrasaccharide in which a sugar alcohol is bonded to the reducing end of panose. A reduced oligosaccharide in which a sugar alcohol other than sorbitol is bound to panose, one of the isomalt oligosaccharides, has not been known at all.

As described above, TVA II binds sugar alcohols such as sorbitol, xylitol and erythritol to the reducing end of panose to produce reduced oligosaccharides consisting of tetrasaccharides, and oligosaccharide compositions containing these reduced oligosaccharides It became clear to produce.

When a sugar alcohol is bonded to the reducing end of panose, it becomes a reduced oligosaccharide having no reducing property, and an oligosaccharide having excellent heat stability can be obtained. Moreover, many of these reduced oligosaccharides are considered to be difficult to decompose many amylases, and the reduced oligosaccharides in which panose and sugar alcohol are bonded are attracting attention for their function as hardly decomposable oligosaccharides.

Next, the present inventors carried out a reaction on the assumption that disaccharides may also bind to panose. TVA II with sucrose coexisting with pullulan instead of glucose
The product was observed at the same Rf = 0.43 as that of IMIM. Although oligosaccharides in which sucrose is bound to panose are pentasaccharides, tetrasaccharide IMI
It is the same as the development distance of M.

This indicates that the development distance of sucrose is Rf = 0.94, which is close to the development distance of glucose, that sucrose is not decomposed by TVA II, that the development distance of disaccharides described later is shorter than that of sucrose, Since the development distance of the transfer product is shorter than the development distance of the sucrose sugar transfer product, the oligosaccharide appearing at the position of Rf = 0.43 is not IMIM, but pentasaccharide with sucrose bound to the reducing end of panose. Is a hetero-oligosaccharide.

Further, when lactose, trehalose and cellobiose are reacted with pullulan in the presence of pullulan as disaccharides, lactose is at the position of Rf = 0.29, trehalose is at the position of Rf = 0.20, and cellobiose is at the position of Rf = 0.20.
The product was observed at the position of Rf = 0.35.

As described above, TVA II was shown to form a disaccharide such as sucrose, lactose, trehalose and cellobiose at the reducing end of panose to produce a heterooligosaccharide consisting of a pentasaccharide. It has been clarified that an oligosaccharide composition containing as an ingredient is produced.

There is no known heterooligosaccharide in which a disaccharide other than maltose and isomaltose is bound to the reducing end of panose, which is one of the isomaltoligosaccharides. If sucrose or trehalose is bonded to the reducing end of panose, an oligosaccharide having excellent heat stability can be obtained because it has no reducing property. Moreover, many of these hetero-oligosaccharides are also difficult to degrade amylases, and hetero-oligosaccharides in which panose and oligosaccharides are bonded are noted for their function as hardly degradable oligosaccharides.

The first invention of the present application is to dissolve a sugar selected from the group consisting of pullulan or panose and a monosaccharide such as fructose, mannose, xylose, and arabinose, and act on an enzyme which produces panose using pullulan as a substrate. Is a method for producing an oligosaccharide composition containing a hetero-oligosaccharide, which can be easily carried out. Furthermore, the use of galactose or ribose in addition to the above-described monosaccharides has been confirmed to produce hetero-oligosaccharides composed of tetrasaccharides. Not only pentoses and hexoses but also any monosaccharides other than glucose can be used. Similarly, an oligosaccharide composition containing a hetero-oligosaccharide consisting of tetrasaccharide can be produced.

Further, the second and third inventions of the present application dissolve a sugar selected from the group consisting of pullulan or panose and sugar alcohols such as sorbitol, xylitol, erythritol and act on an enzyme which produces panose using pullulan as a substrate. This is a method for producing an oligosaccharide composition containing a reduced oligosaccharide, which can be easily carried out. Furthermore, the production of reduced oligosaccharides has been confirmed even when mannitol is used in addition to the above-mentioned sugar alcohols, and an oligosaccharide composition containing reduced oligosaccharides consisting of tetrasaccharides is similarly produced using any of the sugar alcohols. can do.

Further, the fourth invention of the present application is characterized in that an enzyme capable of producing panose using pullulan as a substrate is obtained by dissolving pullulan or panose and a sugar selected from the group consisting of disaccharides such as sucrose, lactose, trehalose and cellobiose. This is a method for producing an oligosaccharide composition containing a pentasaccharide oligosaccharide as a component, which can be easily carried out. Furthermore, the production of hetero-oligosaccharides has been confirmed even using raffinose, which is a trisaccharide, in addition to the above-mentioned disaccharides. With any of the oligosaccharides, the oligosaccharide in which the oligosaccharide is bonded to the reducing end of panose is used. Oligosaccharide compositions containing the same can be produced.

It is also recognized that when maltose is used in place of glucose, panosyl maltose is produced, and when isomaltose is used, panosyl isomaltose is produced. In addition, 2 such as maltitol and lactitol
Sugar alcohols have also been found to produce reduced oligosaccharides consisting of pentasaccharides linked to panose.

As the enzyme used here, the above-mentioned TVA II
But also TVA I and neopluranase
(N. Kuriki et al., J. Bacteriol., 170 , 1554 (1988);
N. Kuriki et al., J. Bacteriol., 173 , 6147 (1991))
An α-amylase that produces panose from isopululane can be used.

For example, when TVA II is used, a compound such as pullulan, which is one substrate of the reaction, and fructose, which is the other substrate, are dissolved at 50 ° C., and the pH is adjusted to 6.5 with an aqueous sodium hydroxide solution. The substrate concentration is adjusted to 20% by weight of pullulan and 10% by weight of saccharides such as fructose. By adding TVA II thereto and reacting at 50 ° C. for 3 days, an oligosaccharide composition containing the target oligosaccharide can be obtained in high yield.

One substrate of the reaction is pullulan or panose, and starch, amylopectin, etc., which are raw materials of panose, also serve as substrates. As for the concentration of pullulan or panose, the higher the concentration, the better.
A range of 30% by weight is preferred. Needless to say, pullulan or panose powder may be added during the reaction, or the reaction may be carried out in a partially undissolved state at 30% by weight or more from the beginning to increase the concentration of the finally produced oligosaccharide.

The saccharide such as fructose, which is the other substrate, is usually added to the reaction solution from the beginning of the reaction, but may be added in the middle of the reaction. TVA II
Cannot hydrolyze panose, and the transfer reaction of the compound such as fructose proceeds in the presence of panose,
After the high-concentration pullulan solution undergoes enzymatic degradation and its viscosity decreases, the concentration of the target oligosaccharide can be increased by adding the above-mentioned compound such as fructose.

As conditions for the enzyme reaction, the concentration of the enzyme varies depending on the type of the enzyme.
% By weight. The pH and temperature of the reaction can be used under any conditions as long as they are within the working pH and working temperature range of the enzyme.
Conditions of 80 ° C. are used. The reaction time is appropriately set according to the purpose of use of the product, but in many cases, 30 minutes to 96 hours is preferable.

The sugar solution containing the oligosaccharide obtained by the reaction can be directly used as a sugar solution through a purification procedure such as decolorization with activated carbon and desalting with an ion exchange resin according to a conventional method. When the obtained oligosaccharide is a reducing sugar, it can be used as a sugar alcohol by a reduction treatment.

Further, if necessary, the desired oligosaccharide can be obtained by using gel filtration chromatography, carbon column chromatography, ion exchange resin column chromatography, fractionation by membrane treatment or crystallization. It can be obtained with high purity.

[0066]

EXAMPLES The present invention will be described below in more detail with reference to examples, but the technical scope of the present invention is not limited by the following examples.

Example 1 The enzyme used was disclosed in
It was produced according to the method described in JP-A-891. Thermoac
Escherichia coli N transformed from E. coli MV1184 with TVA II gene excised from tinomyces vulgaris R-47
One loopful of K699311 (FERM P-13717) was inoculated into a medium (100 mL medium containing 1 g peptone, 0.5 g yeast extract, and 0.5 g salt) and cultured at 37 ° C. for 16 hours to perform seed culture. 1.
To 100 mL of a medium containing 6 g peptone, 1.0 g yeast extract, 0.5 g salt, and 5 mg ampicillin, 1 mL of the obtained seed culture solution was added, and main culture was carried out in a 500 mL Sakaguchi flask at 37 ° C. for 16 hours with reciprocal shaking. In addition, 0.1 mL of 0.5M isopropyl-β-D-thiogalactoside was added to the medium during 5 hours of culture. The culture was centrifuged (2,000 g, 20 minutes) to collect the cells, and a 100 mM Tris-HCl buffer (pH 7.20) containing 5 mM CaCl ₂ was added.
5) Suspended. The suspension cells were heat-treated at 80 ° C. for 30 minutes, crushed by sonication, and centrifuged (7,500 g,
10 minutes), the supernatant was recovered. The resulting supernatant is 1
It contains more than 00mg of protein and was used in the reaction as a TVA II enzyme solution. The measurement of the enzyme protein was performed by the method of Lowry.

Water was added to 112 g of pullulan (100 g of solid content (BD)) to make 450 g, and the mixture was dissolved by heating to 50 ° C. with good stirring, and then adjusted to pH 6.5 by adding a 10% by weight aqueous sodium hydroxide solution. It was adjusted. To this, add 50 g of fructose and stir evenly, then add 100 mg of TVA II and add 50 mg.
The mixture was reacted at 72 ° C. for 72 hours to obtain an oligosaccharide composition solution.

As shown in the thin layer chromatogram of FIG.
In addition to fructose (Rf = 0.98) and panose (Rf = 0.68), Rf
= 0.55 and spot at Rf = 0.47. Furthermore, the oligosaccharide composition was subjected to high performance liquid chromatography (H
According to analysis by PLC), 26% of the hetero-oligosaccharide was composed of a tetrasaccharide having fructose bound to the reducing end of panose, and 7% had fructose bound to the reducing end of panosyl panose.
Hetero-oligosaccharide composed of sugar was contained at 7%.

The HPLC analysis conditions are as follows. Column: Shodex SUGAR KS-802 (300 x 8 mm, 2 pcs) Solvent: Water Flow rate: 1.0 mL / min Temperature: 80 ° C Detector: Differential refractometer

(Example 2) Pullulan 112 g (BD 100 g)
Was added to water to make 450 g, and the mixture was dissolved by heating to 50 ° C. while stirring well, and then adjusted to pH 6.5 by adding a 10% by weight aqueous sodium hydroxide solution. To this, add 50 g of mannose and stir evenly, then add 100 mg of TVA II and add 50 mg.
The mixture was reacted at 72 ° C. for 72 hours to obtain an oligosaccharide composition solution.

As shown in the thin layer chromatogram of FIG.
Mannose (Rf = 1.02), panose (Rf = 0.68) and Rf =
A spot was observed at the position of 0.62 and Rf = 0.47. HP
Analysis by LC revealed that 23% of the hetero-oligosaccharide was composed of a tetrasaccharide having mannose bound to the reducing end of panose, and 10% was hetero-oligosaccharide composed of 7 sugars having mannose bound to the reducing end of panosyl panose.

(Example 3) Pullulan 112 g (BD 100 g)
Was added to water to make 450 g, and the mixture was dissolved by heating to 50 ° C. while stirring well, and then adjusted to pH 6.5 by adding a 10% by weight aqueous sodium hydroxide solution. Xylose (50 g) is added to the mixture, and the mixture is stirred uniformly. Then, TVA II (100 mg) is added.
The mixture was reacted at 72 ° C. for 72 hours to obtain an oligosaccharide composition solution.

As shown in the thin layer chromatogram of FIG.
Xylose (Rf = 1.11), panose (Rf = 0.68) and Rf =
A spot was observed at the position of 0.59. Analysis by HPLC revealed that xylose was bound to the reducing end of panose.
Hetero-oligosaccharides composed of saccharides were contained in 9%, and hetero-oligosaccharides composed of heptasaccharides in which xylose was bound to the reducing end of panosylpanose were contained in 4%.

(Example 4) 112 g of pullulan (100 g of BD)
Was added to water to make 450 g, and the mixture was dissolved by heating to 50 ° C. while stirring well, and then adjusted to pH 6.5 by adding a 10% by weight aqueous sodium hydroxide solution. Arabinose 50g
And stirred uniformly, then add 100 mg of TVA II and add 5 mg.
The mixture was reacted at 0 ° C. for 72 hours to obtain an oligosaccharide composition solution.

As shown in the thin layer chromatogram of FIG.
Arabinose (Rf = 1.00), Panose (Rf = 0.68) and Rf
A spot was observed at the position of = 0.60. When analyzed by HPLC, it contained 3% of a hetero-oligosaccharide consisting of a tetrasaccharide in which arabinose was bound to the reducing end of panose.

(Example 5) Pullulan 112 g (BD 100 g)
Was added to water to make 450 g, and the mixture was dissolved by heating to 50 ° C. while stirring well, and then adjusted to pH 6.5 by adding a 10% by weight aqueous sodium hydroxide solution. 50g sorbitol
And stirred uniformly, then add 100 mg of TVA II and add 5 mg.
The mixture was reacted at 0 ° C. for 72 hours to obtain an oligosaccharide composition solution.

As shown in the thin layer chromatogram of FIG.
Sorbitol (Rf = 0.90), panose (Rf = 0.68) and Rf
A spot was observed at the position of = 0.41. Analysis by HPLC revealed that 15% of the reduced oligosaccharides consisted of tetrasaccharides having sorbitol bound to the reducing end of panose, and 6% contained 7% reduced oligosaccharides consisted of 7 sugars having sorbitol bound to the reducing end of panosylpanose.

(Example 6) 112 g of pullulan (100 g of BD)
Was added to water to make 450 g, and the mixture was dissolved by heating to 50 ° C. while stirring well, and then adjusted to pH 6.5 by adding a 10% by weight aqueous sodium hydroxide solution. 50g xylitol
And stirred uniformly, then add 100 mg of TVA II and add 5 mg.
The mixture was reacted at 0 ° C. for 72 hours to obtain an oligosaccharide composition solution.

As shown in the thin layer chromatogram of FIG.
Xylitol (Rf = 0.97), panose (Rf = 0.68) and Rf
= 0.58 and Rf = 0.47. H
Analysis by PLC revealed that 17% of the reduced oligosaccharides consisted of tetrasaccharide with xylitol bound to the reducing end of panose, and 9% contained 7% reduced oligosaccharides of xylitol bound to the reducing end of panosyl panose.

Example 7 112 g of pullulan (100 g of BD)
Was added to water to make 450 g, and the mixture was dissolved by heating to 50 ° C. while stirring well, and then adjusted to pH 6.5 by adding a 10% by weight aqueous sodium hydroxide solution. Erythritol 50
g, and the mixture was stirred uniformly. Then, 100 mg of TVA II was added and reacted at 50 ° C. for 72 hours to obtain an oligosaccharide composition solution.

As shown in the thin layer chromatogram of FIG.
In addition to erythritol (Rf = 0.97) and panose (Rf = 0.68)
Spots were observed at Rf = 0.65 and Rf = 0.56.
When analyzed by HPLC, reduced oligosaccharides composed of tetrasaccharide having erythritol bound to the reducing end of panose were contained in 23%, and reduced oligosaccharides composed of heptasaccharide having erythritol bound to the reducing end of panosylpanose were contained in 7%.

(Example 8) 112 g of pullulan (100 g of BD)
Was added to water to make 450 g, and the mixture was dissolved by heating to 50 ° C. while stirring well, and then adjusted to pH 6.5 by adding a 10% by weight aqueous sodium hydroxide solution. 50g sucrose to this
And stirred uniformly, then add 100 mg of TVA II and add 5 mg.
The mixture was reacted at 0 ° C. for 72 hours to obtain an oligosaccharide composition solution.

As shown in the thin layer chromatogram of FIG.
Sucrose (Rf = 0.94), panose (Rf = 0.68) and Rf
= 0.43 and spots at Rf = 0.21. H
Analysis by PLC revealed that 15% of the heterooligosaccharide consisting of pentasaccharide with sucrose bound to the reducing end of panose was contained.

Example 9 112 g of pullulan (100 g of BD)
Was added to water to make 450 g, and the mixture was dissolved by heating to 50 ° C. while stirring well, and then adjusted to pH 6.5 by adding a 10% by weight aqueous sodium hydroxide solution. Lactose (50 g) is added to the mixture, and the mixture is stirred uniformly, and then TVA II (100 mg) is added.
The mixture was reacted at 72 ° C. for 72 hours to obtain an oligosaccharide composition solution.

As shown in the thin layer chromatogram of FIG.
In addition to lactose (Rf = 0.77) and panose (Rf = 0.68), Rf =
A spot was observed at the position of 0.29. Analysis by HPLC showed that lactose was bound to the reducing end of panose.
Hetero-oligosaccharide composed of sugar was 39%, and reduced oligosaccharide composed of octasaccharide having lactose bound to the reducing end of panosylpanose was 18%.

Example 10 112 g of pullulan (100 g of BD)
), Add water to make 450g, and stir well at 50 ° C.
The solution was heated and dissolved, and then adjusted to pH 6.5 by adding a 10% by weight aqueous sodium hydroxide solution. This is trehalose
After adding 50 g and uniformly stirring, 100 mg of TVA II was added and reacted at 50 ° C. for 72 hours to obtain an oligosaccharide composition solution.

As shown in the thin layer chromatogram of FIG.
Trehalose (Rf = 0.86), panose (Rf = 0.68) and Rf
A spot was observed at the position of 0.20. Analysis by HPLC revealed that 9% of the heterooligosaccharide consisted of a pentasaccharide in which trehalose was bound to the reducing end of panose.

Example 11 112 g of pullulan (100 g of BD)
), Add water to make 450g, and stir well at 50 ° C.
The solution was heated and dissolved, and then adjusted to pH 6.5 by adding a 10% by weight aqueous sodium hydroxide solution. This is cellobiose
After adding 50 g and uniformly stirring, 100 mg of TVA II was added and reacted at 50 ° C. for 72 hours to obtain an oligosaccharide composition solution.

As shown in the thin layer chromatogram of FIG.
Cellobiose (Rf = 0.85), panose (Rf = 0.68) and Rf
A spot was observed at the position of = 0.35. When analyzed by HPLC, 39% of the hetero-oligosaccharide was composed of pentasaccharide having cellobiose bound to the reducing end of panose, and 18% was reduced oligosaccharide composed of octasaccharide having cellobiose bound to the reducing end of panosyl panose.

[0091]

The oligosaccharide composition produced according to the present invention is an oligosaccharide consisting of hexose or pentose other than glucose and pansaccharide which is one of isomaltoligosaccharides, or tetrasaccharide in which a sugar alcohol is bonded, Or, it contains pentasaccharide or more oligosaccharides in which oligosaccharides are bound to panose, and has low sweetness, low calories, hardly decomposable, Bifidobacterium growth factor,
It is a sugar composition having high utility as a health food material having functions such as intractable caries. Therefore, according to the present invention, an oligosaccharide composition having high utility value in the food industry where functionality is required can be produced.

[Brief description of the drawings]

FIG. 1 is a thin-layer chromatograph of a produced sugar in Examples 1 to 4. Separation conditions are silica gel G60 (Merck)
Was developed twice using n-butanol: ethyl alcohol: water = 5: 5: 2.5 as a developing solvent. Panose, standard sugar (glucose = G1, maltose = G2,
(Malt triose = G3, maltotetraose = G4)
5 is a chromatogram of a reaction product of only each monosaccharide, TVA II and pullulan alone, and a reaction product of TVA II coexisting with pullulan, glucose, fructose, maltose, xylose, and arabinose.

FIG. 2 is a thin-layer chromatograph of produced sugars in Examples 5 to 7. Separation conditions are silica gel G60 (Merck)
Was developed twice using n-butanol: ethyl alcohol: water = 5: 5: 2.5 as a developing solvent. A reaction product of only panose, standard sugars (G1, G2, G3, G4) and each sugar alcohol, and only TVA II and pullulan;
5 is a chromatogram of a reaction product in which pullulan and glucose, sorbitol, xylitol, and erythritol coexist in TVA II.

FIG. 3 is a thin-layer chromatograph of produced sugars in Examples 8 to 11. Separation was performed twice using silica gel G60 (Merck) and developing solvent using n-butanol: ethyl alcohol: water = 5: 5: 2.5. Reaction products of panose, standard sugars (G1, G2, G3, G4) and each oligosaccharide alone, TVA II and pullulan alone, reaction products in the presence of pullulan and glucose, sucrose, lactose, trehalose and cellobiose, respectively It is a chromatogram of a product.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Toshiyuki Kimura 9th Yawata Kaigandori, Ichihara-shi, Chiba Oji Cornstarch Co., Ltd. (72) Inventor Yoshiyo Suzuki 9th Yawatakaigandori, Ichihara-shi, Chiba Within the Development Laboratory Co., Ltd. (72) Inventor Naoto Arai 9 at Oji Corn Starch Co., Ltd.

Claims (8)

[Claims] 1. A monosaccharide other than glucose is allowed to coexist in a sugar solution containing pullulan or panose, and an enzyme for generating panose using pullulan as a substrate is added thereto to act, whereby the monosaccharide is bound to the reducing end of panose. A method for producing an oligosaccharide composition comprising a heterooligosaccharide consisting of a tetrasaccharide. 2. The method according to claim 1, wherein the monosaccharide to be bound is fructose, mannose, xylose or arabinose. 3. Sorbitol is coexistent in a sugar solution containing pullulan or panose, and an enzyme for producing panose using pullulan as a substrate is added thereto to act. The sorbitol is α-1,6-linked at the reducing end of panose. A method for producing an oligosaccharide composition comprising a reduced oligosaccharide comprising a reduced tetrasaccharide. 4. A sugar solution other than sorbitol is allowed to coexist with a sugar solution containing pullulan or panose, and an enzyme for generating panose using pullulan as a substrate is added thereto to act, whereby the sugar alcohol is bound to the reducing end of panose. 4
A method for producing an oligosaccharide composition comprising a reduced oligosaccharide comprising a saccharide. 5. The method according to claim 4, wherein the sugar alcohol to be bound is xylitol or erythritol. 6. An oligosaccharide coexisting in a sugar solution containing pullulan or panose, and an enzyme for generating panose using pullulan as a substrate is added thereto to act, and the pentasaccharide or more having an oligosaccharide bonded to the reducing end of panose. A method for producing an oligosaccharide composition comprising an oligosaccharide comprising a saccharide. 7. The method according to claim 6, wherein the oligosaccharide to be bound is lactose, sucrose, trehalose or cellobiose. 8. The method according to claim 1, wherein the enzyme that produces panose using pullulan as a substrate is TVA II.
The method described in the section.