JPH07191A - Production of highly branched oligosaccharide - Google Patents

Abstract

PURPOSE:To produce a highly branched oligosaccharide by enzymatic reaction. CONSTITUTION:A substrate having a carbohydrate concentration of >=20% is treated with >=25 U/g-substrate of neopullulanase or of neopullulanase together with amylase.

Description

Detailed Description of the Invention

[0001]

The present invention relates to two or more α-1,6-
A branched oligosaccharide having a glucosidic bond and having one or more α-1,4-glucosidic bonds (herein,
It is referred to as a hyperbranched oligosaccharide). Highly branched oligosaccharides are growth factors of bifidobacteria or functional oligosaccharides showing caries resistance, and are intermediate between branched oligosaccharides having a relatively low degree of polymerization of up to about 4 and branched polysaccharides such as pullulan. It can be expected as a food material showing properties. 1 to 12 show the structures of highly branched oligosaccharides that can be produced by the present invention.

[0002]

BACKGROUND OF THE INVENTION Highly branched oligosaccharides could be produced by synthetic chemistry. However, it was never manufactured biochemically. The α-glucosidase conventionally used for producing a branched oligosaccharide is an oligosaccharide having only two or more α-1,6 bonds (isomaltotriose (the structure is shown in FIG. 13),
Isomaltotetraose (Fig. 14 shows its structure)
Etc.), but is highly branched oligosaccharide, that is, it has two or more α-1,6 bonds and one or more α-1,6
A branched oligosaccharide having a 4-glucoside bond could not be produced.

In the patent application No. 258,575, filed on Sep. 9, 1991, No. 258575 (the title of the invention, a method for producing a sweetener containing isopanose), the present inventors have made neopullulanase act on a substrate. A sweetener containing isopanose was prepared. However, the method did not detect highly branched oligosaccharides.

[0004]

The substrate used in the present invention is
α-1,4-glucosidic bond only or α-1,4 and α
It is a carbohydrate having a -1,6-glucoside bond. For example, starch, dextrin, starch syrup, and various maltooligosaccharides. Dextrins liquefy starch with a bacterial liquefied α-amylase or, in addition, bacterial saccharified or fungal α
-It can be produced by decomposing with amylase.

The neopullulanase used in the present invention hydrolyzes pullulan to mainly produce panose, and α-
It is an enzyme that catalyzes 1,4 and α-1,6-glycosidyl transfer reactions. For example, Bacillus stearo
neopullulanase (Jour-na) derived from thermophilus TRS40 (Deposit No. 9609 of the Institute of Microbiology and Biotechnology)
l of Bacteriology Volume 170, Volume 1
554, described in 1988), Thermo
α-derived from actinomyces vulgalis
Amylase (Agricultural and Bi)
Logi-cal Chemistry, Volume 42,
(Pp. 1681, published in 1978), Baci
llus stearothermophilus K
P1064 pullulan hydrolase (Applied
Microbiology and Biotechn
biology, vol. 21, p. 20, published in 1985) and Bacteroides theatio.
taomicron 95-1 neopullulanase (Jo
urinal of Bacteriology, No. 17
Volume 3, page 2962, published in 1991), and so on.

The α-amylase used in the present invention is not special. A commercially available product may be used. No special means is required for the action of the enzyme and the action of the two types of enzymes in combination. Normal pH and temperature may be used. The enzyme does not have to be immobilized.

The liquid after the reaction is decolorized with activated carbon or purified with an ion exchange resin as required to obtain a product.
Further, if necessary, a product obtained by fractionating and concentrating by precipitation with ethanol or activated carbon column chromatography can be produced.

[0008]

[Function] Neopullulanase is characterized by not only transferring only glucosyl group to α-1,6, but also more than maltosyl group by α
-1,6 transition. This is different from the characteristic of α-glucosidase in that only a glucosyl group is transferred by α-1,6. The production of highly branched oligosaccharides utilizes the transfer reaction and condensation reaction of neopullulanase. These reactions require high concentration of substrate and high concentration of enzyme.

(Experiment 1) To the saccharified cornstarch solution (DE10) having various concentrations, 25 U of neopullulanase was added.
The mixture was mixed at a ratio of g-substrate and reacted at 50 ° C. and pH 6.0 for 32 hours. Table 1 shows how the production ratio of hyperbranched oligosaccharides in all the products changes depending on the concentration of the corn starch saccharified solution (substrate).

[Table 1]

(Experiment 2) A cornstarch saccharified solution (DE10) having a concentration of 30% was mixed with neopullulanase in various ratios to various substrates and reacted at 50 ° C. and pH 6.0 for 32 hours. Table 2 shows how the production ratio of hyperbranched oligosaccharides in all products changes depending on the ratio of neopullulanase (enzyme) to the substrate.

[Table 2]

[0011]

【Example】

(Example 1) Corn starch saccharification liquid (DE10) 30
25 U / g-substrate neopullulanase was added to the 50% solution and reacted at 50 ° C. for 48 hours. As a result of quantifying the reaction product by high performance liquid chromatography, syrup containing highly branched oligosaccharide shown in Table 3 was obtained.

[Table 3]

Example 2 25 U / g-substrate neopullulanase and 5 U / g-substrate liquefied α-amylase derived from bacteria were added to a 30% dextrin solution, and the mixture was allowed to stand at 50 ° C. for 4 hours.
The reaction was carried out for 8 hours. As a result of quantifying the reaction product by high performance liquid chromatography, syrup containing highly branched oligosaccharide shown in Table 4 was obtained.

[Table 4]

[0013]

[Effect] According to the present invention, highly branched oligosaccharides that could not be produced by biochemical synthesis and conventional enzymatic reactions could be produced.

[Brief description of drawings]

FIG. 1 Glcα1-6Glcα1-4Glcα1
-6Glc structure

FIG. 2 Glcα1-4Glcα1-6Glcα1
-4Glcα1-6Glc structure

FIG. 3 Glcα1-6Glcα1-4Glcα1
Structure of -6Glcα1-4Glc

FIG. 4 Glcα1-4Glcα1-6Glcα1
Structure of -4Glcα1-6Glcα1-4Glc

FIG. 5: Glcα1-6Glcα1-4Glcα1
Structure of -4Glcα1-6Glcα1-4Glc

FIG. 6 Glcα1-4Glcα1-4Glcα1
Structure of -6Glcα1-4Glcα1-6Glc

FIG. 7: Glcα1-6Glcα1-4Glcα1
Structure of -6Glcα1-4Glcα1-6Glc

FIG. 8 Glcα1-4Glcα1-6Glcα1
-4Glcα1, 6Glcα1-4Glcα1-6Gl
structure of c

FIG. 9: Glcα1-6Glcα1-4Glcα1
-6Glcα1-4Glcα1-6Glcα1-4Gl
structure of c

FIG. 10: Glcα1-4Glcα1-4Glcα1
-6Glcα1-4Glcα1-6Glcα1-4Gl
structure of c

FIG. 11: Glcα1-4Glcα1-6Glcα
1,4Glcα1-4Glcα1-6Glcα1-4G
structure of lc

FIG. 12: Glcα1-4Glcα1-6Glcα1
-4Glcα1-6Glcα1-4Glcα1-4Gl
structure of c

FIG. 13 Structure of Isomaltotriose

FIG. 14: Structure of isomalttetraose

[Explanation of symbols]

 1 glucose 2 α-1,4-bond 3 α-1,6-bond 4 reducing terminal glucose

Claims (2)

[Claims] 1. 25 U / g for a substrate having a sugar concentration of 20% or more
-A method for producing highly branched oligosaccharides, characterized in that neopullulanases above the substrate are allowed to act 2. A method for producing a highly branched oligosaccharide, which comprises allowing a substrate having a sugar concentration of 20% or more to act in combination with α-amylase and 25 U / g-substrate or more of neopullulanase.