JPH0591891A - Production of saccharide compound - Google Patents

Abstract

PURPOSE:To obtain a saccharified compound having a dental carries preventing effect and a sweetness in high purity from glucose-1-phosphoric acid and sugar alcohol by using sucrose phosphorylase without requiring any complicated isolation and purification treatment. CONSTITUTION:Glucose-1-phosphoric acid or sucrose is made to react with a sugar alcohol (e.g. xylitol or arabitol) using sucrose phosphorylase to provide the objective saccharide compound in which glucose and sugar alcohol are bound. The reaction is preferably carried out at 35-45 deg.C and pH6-7 for 8-15hr. Furthermore, as the sucrose phosphorylase, e.g. enzyme derived from Leuconostoc mesenteroides is used.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing a sugar compound having a caries-preventing effect and which is very useful as a sweetener, and more specifically, glucose-1-phosphate or sucrose using sucrose phosphorylase. And a sugar alcohol, the method of obtaining a sugar compound having glucose and a sugar alcohol bound to each other and having a high purity in a high purity without performing a complicated isolation and purification operation.

[0002]

2. Description of the Related Art Conventionally, a sugar compound in which glucose and a sugar alcohol are bound is efficiently produced from glucose-1-phosphate or sucrose and a sugar alcohol using a sugar-phosphorylating enzyme sucrose phosphorylase. The method is not yet known. On the other hand, there is known a method for producing a sugar compound in which glucose and sugar alcohol are bound from glucose and sugar alcohol using a sugar-degrading enzyme α-glucosidase (see Japanese Patent Laid-Open No. Hei 2-163092). Generally, when a saccharide compound is produced using glucosidase, galactosidase, amylase, cyclodextrin synthase or fructosyl transferase, the reaction product is obtained as a mixture of oligosaccharides having different degrees of polymerization, and it is difficult to obtain a highly pure oligosaccharide. There is a problem.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have to perform a complicated isolation and purification operation of glucose-1-phosphate or sucrose and sugar alcohol from a sugar compound in which glucose and sugar alcohol are bound. As a result of various studies in order to obtain it, glucose-1-phosphate or sucrose and sugar alcohol are added to the solution containing the glycosyl phosphate-degrading enzyme, sucrose phosphorylase, to obtain a high-purity object. The present invention has been completed based on this finding, knowing that the sugar compound can be obtained in good yield.

[0004]

That is, the present invention uses sucrose phosphorylase in order to produce a sugar compound in which glucose and sugar alcohol are bound from glucose-1-phosphate or sucrose and sugar alcohol. It is a method for producing a sugar compound, which is characterized by utilizing an enzymatic reaction.

[0005]

The present invention will be described in detail below.

First, in order to carry out the present invention, a predetermined amount of glucose-1-phosphate or sucrose and sugar alcohol are mixed in a reaction solution, and sucrose phosphorylase is added to the mixture to carry out the reaction.

As the sugar alcohol used here,
Examples include xylitol, arabitol, adonitol, glycerol, sorbitol and the like.

Sucrose phosphorylase is an enzyme that acts on sucrose in the presence of inorganic phosphate to produce glucose-1-phosphate and fructose, or catalyzes its reverse reaction.

[0009] And, as its origin, for example, Leuconostoc (Leuconostoc)
mesenteroides), Pseudomonas saccha
rophila), Pseudomonas putrefacien (Pseudomonas putrefacien)
s), Clostridium pasteurianum (Clos
tridium pasteurianum, Acetobacter xy
linum), Pullularia pullulans (Pullul)
aria pullulans) etc. are known [Biotechnology and Bioengineering (Biotechnol. Bioeng.,) Vo.
l. 29, Pp8-15, 1987], but is not limited thereto.

In carrying out the present invention, first, a relatively high concentration reaction solution containing glucose-1-phosphate or sucrose and sugar alcohol is prepared. The concentration range of this preparation liquid is 5 to 100% by weight, more preferably 20 to 100%. Further, the blending ratio of the mixture is such that the sugar alcohol to glucose-1-phosphate or sucrose is 100: 1 by weight.
The range is 1: 100, more preferably 1:10 to 10 :.
The range is 1.

The amount of sucrose phosphorylase added to the reaction solution is 1 unit or more, preferably 50 to 100 units, per 1 gram of the mixture (substrate) of glucose-1-phosphate or sucrose and sugar alcohol. ..

One unit is determined according to the method described in JP-A-3-4785 "Method for producing sucrose phosphorylase".

The pH of the enzyme reaction is 5 to 8, preferably 6 to 7.5, the temperature is 20 to 50 ° C., preferably 35 to 45 ° C., and the time is 1 to 24 hours, preferably Is 8 to 15 hours.

The reaction solution thus obtained is adsorbed on a column packed with activated carbon and adsorbed on the column, and then eluted with an ethanol solution to easily separate the target sugar compound as a single compound. I can.

[0015]

INDUSTRIAL APPLICABILITY The present invention is to add a sugar-phosphorylating enzyme, sucrose phosphorylase, to a solution containing glucose-1-phosphate or sucrose and sugar alcohol. , The desired α-
Sugar compounds such as D-glucopyranosyl-xylitol, α-D-glucopyranosyl-arabitol and α-D-glucopyranosyl-adnitol can be obtained in good yield.

Therefore, the operation of isolating and purifying the desired sugar compound from the reaction solution is very simple, and the sugar compound obtained in the present invention has a caries preventive effect (low caries property). It is characterized by being indigestible (low calorie) and is very useful as a sweetener.

Therefore, it is also suitable for use in chocolate, cookies and biscuits.

Further, it is resistant to heat, and unlike reducing substances such as starch syrup and maltose, there is no strict restriction on the addition time and heating temperature, so that there is an advantage that workability can be improved.

Furthermore, these compounds are difficult to be used for molds (blue mold, koji mold, black mold, hair mold), yeasts, etc., and lactic acid drinks, fermented foods, pickled vegetables, boiled foods, and other high-moisture foods. It is expected that the use of this product for fermentation suppresses fermentation, improves the shelf life of foods, and improves the shelf life of products.

Hereinafter, the present invention will be described more specifically by showing examples.

[0021]

Example 1 40 g of glucose-1-phosphate and 40 g of xylitol (sugar alcohol) were placed in a beaker having a capacity of 300 ml, and 0.1 mol of ME was added thereto.
Dilute with S buffer (pH 6.8) to prepare a 100 ml reaction solution, and add 6000 units of sucrose phosphorylase (manufactured by Seishin Pharmaceutical Co., Ltd.) to the mixture at 42 ° C. for 15 minutes.
The sugar compound formation reaction is carried out for a time, then at 100 ° C. for 5
The enzyme reaction was stopped by heat treatment for a minute to obtain an enzyme reaction treatment liquid. Next, the obtained reaction solution was passed through an activated carbon column (inner diameter: 6 cm, length: 50 cm), water was passed therethrough to wash off unreacted xylitol, and then an ethanol 5% solution was passed through, A solution containing the target α-D-glucopyranosyl-xylitol was obtained. The reaction solution also contains unreacted glucose-1-phosphate, but when it comes into contact with activated carbon, it is strongly adsorbed in this and does not elute even with an ethanol solution.

The thus-obtained solution containing α-D-glucopyranosyl-xylitol was then concentrated under reduced pressure by a rotary evaporator.
A concentrate containing 8 ml of syrupy α-D-glucopyranosyl-xylitol was obtained.

The above concentrate was analyzed by high performance liquid chromatography (TSK-gel Amide-80) to measure the purity of α-D-glucopyranosyl-xylitol, which was 98%, and the yield was 12.7 g. there were.

The structure was determined by the following analytical method, and it was confirmed that the obtained compound was α-D-glucopyranosyl-xylitol. That is,
Hydrochloric acid was added to the syrupy concentrate to give a final concentration of 2 N solution, and 2 ml of this solution was reacted at 90 ° C. for 4 hours, and the reaction solution was subjected to high performance liquid chromatography (TSK-
As a result of analysis by gel Amide-80), peaks of glucose and xylitol were obtained.

When the structure of the above syrup-like concentrated liquid was analyzed by nuclear magnetic resonance analysis, it was confirmed that the constituent sugars were glucose and xylitol.

Furthermore, it was confirmed by a mass spectrum that the molecular weight was 304.

From the above results, the obtained sugar compound was α-
It was confirmed to be D-glucopyranosyl-xylitol.

[0028]

Example 2 40 g of sucrose and 40 g of xylitol (sugar alcohol) were placed in a beaker having a volume of 300 ml and diluted with 0.1 mol of MES buffer (pH 6.8) to give 100 ml of reaction solution. 6000 units of sucrose phosphorylase (manufactured by Morishin Pharmaceutical Co., Ltd.) was prepared, and a sugar compound-forming reaction was carried out at 42 ° C. for 15 hours, followed by heat treatment at 100 ° C. for 5 minutes to stop the enzymatic reaction. ..

Next, 40 units of sucrose-degrading enzyme (invertase) (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the obtained reaction solution to completely decompose sucrose into glucose and fructose, which was then activated carbon column. (Inside diameter 6 cm, length 50 cm), water was passed through to decompose glucose and fructose, and unreacted xylitol was washed off, and then 5% ethanol solution was passed through it. A solution containing α-D-glucopyranosyl-xylitol was obtained.

The thus-obtained solution containing α-D-glucopyranosyl-xylitol was then concentrated under reduced pressure using a rotary evaporator.
A concentrate containing 3 ml of syrupy α-D-glucopyranosyl-xylitol was obtained.

The above concentrate was analyzed by high performance liquid chromatography (TSK-gel Amide-80) to measure the purity of α-D-glucopyranosyl-xylitol, which was 95%, and the yield was 20.8 g. there were. It was.

The structure was determined by the following analytical method, and it was confirmed that the obtained compound was α-D-glucopyranosyl-xylitol. That is,
Hydrochloric acid was added to the syrupy concentrate to give a final concentration of 2 N solution, and 2 ml of this solution was reacted at 90 ° C. for 4 hours, and the reaction solution was subjected to high performance liquid chromatography (TSK-
As a result of analysis by gel Amide-80), peaks of glucose and xylitol were obtained.

The structure of the syrup-like concentrated liquid was analyzed by nuclear magnetic resonance analysis, and it was confirmed that the constituent sugars were glucose and xylitol.

Furthermore, it was confirmed by a mass spectrum that the molecular weight was 304.

From the above results, the obtained sugar compound was α-
It was confirmed to be D-glucopyranosyl-xylitol.

[0036]

[Application Example 1] In dental caries, sucrose is converted into water-insoluble glucan such as dextran by dental caries in the oral cavity, and this glucan covers the surface of the tooth in a thin film, and passes through this thin film to allow the tooth surface to pass through. It has been clarified that the sugars that reach the temperature of 1 are subjected to anaerobic fermentation to produce organic acids, and attack the tooth enamel. Therefore, it is desired from the viewpoint of preventing dental caries to suppress the formation of water-insoluble glucan and organic acid in the oral cavity as much as possible.

"Anti-cariogenicity test (water-insoluble glucan production test)" Caries bacteria, Streptococcus mutans (S
treptococcus mutans) IFO139
Brain Heart Infusion
Broth (Brain Heart Infusion)
Broth) (manufactured by Nissui Pharmaceutical Co., Ltd.) was inoculated into a medium composed of a 3.5% aqueous solution, and statically cultured at 37 ° C. for 18 hours to obtain a preculture liquid.

Next, 3 sections of the above-mentioned medium consisting of 3.5% aqueous solution were prepared, 5% sucrose was added to the first section, and the second section was obtained in this Example 1. 5%
α-D-glucopyranosyl-xylitol was added, and 5% sucrose and 5% α-D-glucopyranosyl-xylitol obtained in this Example 1 were added to the third section, and 10 ml of each of the preculture solution was added. 0.
The cells were inoculated with 2% and statically cultured at 37 ° C. for 18 hours, and the amount of insoluble glucan produced in the obtained culture solution was measured by the following method. The results are summarized in Table 1.

[0039]

[Measurement method of insoluble glucan] After culturing, the culture solution was gently removed and washed with water, and then 0.5% of solid matter adhering to the peripheral wall of the glass test tube.
1 ml of normal NaOH was added, and the mixture was kept at 37 ° C. for 1 hour to dissolve.

The amount of glucan in this solution was measured by the phenol-sulfuric acid method.

From the results shown in Table 1, it can be seen that, in Category 1, sucrose is converted into water-insoluble glucan such as dextran by a dental caries in a test tube to produce a causative agent of dental caries. On the other hand, Category 2 is not converted by the dental caries at all and no water-insoluble glucan is generated in the test tube.
That is, it is understood that the α-D-glucopyranosyl-xylitol obtained by the present invention does not produce glucan, which is a causative substance of caries. Further, it is understood that in Category 3, α-D-glucopyranosyl-xylitol obtained according to the present invention has an action of suppressing glucan production from sucrose.

"Anti-cariogenicity test (organic acid production test)" Caries bacteria, Streptococcus mutans (Strept
ococcus mutans) IFO 13955 strain inoculated with Brain Heart Infusion Bro
(th) (manufactured by Nissui Pharmaceutical Co., Ltd.) was inoculated into a medium consisting of a 3.5% aqueous solution, and statically cultured at 37 ° C. for 18 hours to prepare a culture solution 700.
I got milliliters.

Then, this culture solution was centrifuged, and the obtained bacterial cells were washed with a 0.9 (W / V)% NaCl aqueous solution to obtain viable bacterial cells, which were suspended in 1.5 ml of distilled water. As a result, a viable cell preparation liquid was obtained.

Next, 0.2 ml of this viable cell preparation liquid was added to Stephan's buffer (pH 7.0).
(Stephan, RM et al., Jour.
nalf Dental Research, Vol
26, pp. 15-41 1947) 1.5 ml and 0.3 ml of an aqueous solution containing 30 mg of sugar are mixed, the mixture is kept at 37 ° C. for 30 minutes, and then pH is measured to give an index of organic acid production. did. The results are shown in Table 2.

[0046]

From the results in Table 2, it is considered that Category 1 has a sharp decrease in pH in a short time, and thus sucrose is rapidly metabolized by dental caries to produce organic acid. On the other hand, in the category 2 of the present invention, the pH is hardly lowered, and therefore the organic acid is hardly generated, so it is understood that α-D-glucopyranosyl-xylitol is hardly metabolized by the dental caries.

[0048]

Experimental Example 1 Glucose-1-phosphate (10 g) and xylitol (sugar alcohol) (10 g) were placed in a beaker having a capacity of 100 ml, and 0.1 mol of ME was added thereto.
Dilute with S buffer (pH 6.8) to prepare 40 ml reaction solution, add 1500 units of sucrose phosphorylase (manufactured by Seishin Pharmaceutical Co., Ltd.), and perform sugar compound formation reaction at 42 ° C. for 15 hours, Then, the enzyme reaction was stopped by heating at 100 ° C. for 5 minutes to obtain an enzyme reaction-treated solution. Next, the enzyme reaction-treated solution obtained above was subjected to high performance liquid chromatography (TSK-gel Amide-80).
The ratio of the amount of α-D-glucopyranosyl-xylitol to the amount of oligosaccharides having a higher molecular weight than that produced was calculated from the peak area. For comparison, 10 grams of glucose and 10 grams of xylitol (sugar alcohol)
Gram and 100ml beaker,
This was diluted with 0.05 mol phosphate buffer (pH 6.8) to prepare a 40 ml reaction solution, and 1500 units of α-glucosidase (manufactured by Toyobo Co., Ltd.) derived from Saccharomyces was added to this at 42 ° C. The sugar compound-forming reaction was performed for 15 hours, and then heat treatment was performed at 100 ° C. for 5 minutes to stop the enzyme reaction and obtain an enzyme reaction treatment liquid. Next, the enzyme reaction-treated solution obtained above was subjected to high performance liquid chromatography (T
SK-gel Amide-80),
The ratio of the production amount of α-D-glucopyranosyl-xylitol (disaccharide) to the oligosaccharide having a larger molecular weight than that (3 or more sugars) was calculated from the peak area. Table 3 shows the above two results. Note 1) A: α-D-glucopyranosyl-xylitol Note 2) B: Oligosaccharide with a large molecular weight of 3 sugars or more Note 3) The values in column B are the values when A is 100 in each category. .. From the results of Table 3, in the method of producing a sugar compound in which glucose and a sugar alcohol are bound from glucose and a sugar alcohol using the glycolytic enzyme α-glucosidase, the reaction product is other than the target sugar compound. In addition, there is a problem that oligosaccharide fractions having different degrees of polymerization are also obtained as impurities and it is difficult to obtain a desired oligosaccharide with high purity. On the other hand, according to the present invention, since sucrose phosphorylase is used, it can be seen that the desired oligosaccharide having high purity can be efficiently obtained.

Claims (3)

[Claims] 1. An enzyme reaction using sucrose phosphorylase is used for producing a sugar compound in which glucose and sugar alcohol are bound from glucose-1-phosphate or sucrose and sugar alcohol. A method for producing a sugar compound. 2. The production method according to claim 1, wherein xylitol, arabitol, adonitol, or glycerol is used as the sugar alcohol. 3. The sugar compound is α-D-glucopyranosyl-
The production method according to claim 1, which is xylitol, α-D-glucopyranosyl-arabitol, or α-D-glucopyranosyl-adnitol.