JP2933960B2 - Method for producing branched oligosaccharide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] <Industrial application field> The present invention relates to a method for producing a branched oligosaccharide by a novel application technique of an enzyme agent, and more particularly to a food or drink as a general sweetener. The present invention relates to a method for producing a branched oligosaccharide used in various fields such as a culture raw material such as a medicine, a bifidobacterium growth factor, a low cariogenic sweetener, and a low calorie sweetener as a functional saccharide or a functional saccharide.

<Prior Art> Branched oligosaccharides are also referred to as non-fermentable sugars, and have been studied in detail as fermented foods, particularly as oligosaccharides present in sake, a traditional liquor since ancient times in Japan. That is, isomaltose (a disaccharide having an α-1,6 glucosidic bond in the molecule),
Nigerose (a disaccharide having an α-1,3 glucoside bond in the molecule), kojibiose (a disaccharide having an α-1,2 glucoside bond in the molecule) or panose (a disaccharide having an α-1,2 glucoside bond in the molecule)
Trisaccharide having -1.6 and α-1,4 glucosidic bond), isomalttriose (trisaccharide having α-1,6 glucosidic bond in the molecule) and the like.

Conventionally, saccharides have been used for various properties and energy sources mainly for sweetness, but in addition to the sweetness of consumers, maintenance of health, functions useful for promotion, for example, sugars that are unlikely to become tooth decay, Alternatively, saccharides which are sweet but hardly obese, or saccharides which are desirable growth factors for enteric bacteria are required. Branched oligosaccharides are attracting attention because of such characteristics.

For the conventional method for producing branched oligosaccharides, see
-27319, JP-B-41-5918, JP-B-46-24057, and the like, and also JP-A-56-51982 and JP-A-61-124.
389, JP-A-61-219345, JP-A-63-291588, etc., but the basics of these production methods are as follows:
It is mainly composed of amylase that produces maltose, and an enzyme having a sugar-transferring action is acted on the amylase. That is, in this reaction, glucose generated by the action of the transferase to maltose is transferred to glucose or maltose as an acceptor to produce isomaltose or panose, but the generation of branched oligosaccharides proceeds. As a result, glucose is produced as a by-product, so that the amount of glucose after the reaction is relatively large, and since the main component of the substrate is maltose having a glucose polymerization degree of 2, the branched oligosaccharides generated are also relatively large in branched disaccharides and branched 3 sugars. It was characterized by relatively less than sugars.

<Problems to be Solved by the Invention> Although the branched oligosaccharides obtained by the above-mentioned production methods have many effective effects as described above, this property is particularly effective for branched oligosaccharides of more than branched trisaccharides. Under such circumstances, the present invention provides an efficient and economical industrial production method of a branched oligosaccharide having a low glucose content and a high branched oligosaccharide content and containing a branched trisaccharide or more as a main component. Things.

[Configuration of the invention]

<Means for Solving the Problems> The present invention provides an amylase that is mainly composed of maltotriose or an amylase that is mainly composed of maltotetraose, by allowing an enzyme having a sugar transfer activity to act on the amylase. Having successfully solved the problem, the present invention will be described in detail below.

Starch is a natural high molecular compound in which glucose is polymerized by α-1,4 and α-1,6 bonds.When this starch is hydrolyzed with an acid or an enzyme, oligosaccharides of various bonding modes and degrees of polymerization are obtained depending on the decomposition conditions. Sugar can be obtained. Glucose is α
Among the maltooligosaccharides polymerized by a -1,4 glucoside bond, a maltooligosaccharide product mainly containing maltose having a degree of glucose polymerization of 2, maltotriose having a degree of glucose polymerization of 3, and malitotetraose having a degree of glucose polymerization of 4, has been put on the market. ing. The first step in the production of these maltooligosaccharides is liquefaction of starch. An appropriate amount of α-amylase is added to the starch emulsion, mixed with the above using a jet cooker, immediately heated to 105 to 107 ° C., and heated for about 5
Detained for up to 10 minutes, released to the atmosphere, and given decomposition rate (DE)
After the reaction is advanced to the above, various oligosaccharide-forming amylase is allowed to act. For example, as an amylase produced mainly from maltose used in the case of maltose production,
Actinobacterial and bacterial β-amylases, plant-derived β-amylases and mold α-amylases are known.

Examples of the amylase produced by maltotriose as a main component in the present invention include, for example, amylase of Bacillus and amylase of Bacillus sp.
Further, as the amylase produced mainly from maltotetraose, for example, amylase of Pseudomonas genus and amylase of Bacillus genus are used, but their origin does not matter.

Examples of the enzyme having a sugar transfer activity in the present invention include:
For example, a transglucosidase (also referred to as α-glucosidase) of the genus Aspergillus, Rhuzopus, Mucor or the like is used, but its origin does not matter.

In the present invention, an enzyme having a sugar-transferring action is allowed to act on an amylase formed mainly of maltotriose or an amylase formed mainly of maltotetraose using a starch liquefied liquid as a substrate. It may be added at the same time as the aose or maltotetraose-forming amylase, or may be added after the maltotriose or maltotetraose-forming amylase is allowed to act beforehand. If necessary, a branching enzyme such as pullulanase or isoamylase may be allowed to act simultaneously with the maltotriose or maltotetraose-forming amylase, as a preferred method for producing a branched oligosaccharide of the present invention. The amount of enzyme is 0.01 to 3.0% (w / w) of maltotriose or maltotetraose-forming amylase per substrate (solid), and g of substrate (solid).
Per transglucosidase is about 30 to 3,000 units, but when the amount of enzyme is large, the reaction time is short, and when the amount is small, the reaction time is long.

The starch used in the present invention is corn starch, potato starch, sweet potato starch, tapioca starch and the like, and is not limited as long as it is a starch usually used for saccharification.

These starches are usually made from a starch liquefied liquid liquefied in the range of DE 8 to 25, and simultaneously or stepwise a maltotriose or maltotetraose-forming amylase and a transferase are allowed to act thereon. The substrate concentration may be 10% or more, and other saccharification conditions include a normal temperature of 40%.
It works at ~ 65 ° C and pH 4-8 for 30-90 hours.

By the above method, the glucose content is low, the branched oligosaccharide content is high, and the branched oligosaccharides having a branched trisaccharide or more as a main component can be efficiently produced. It is possible to produce branched oligosaccharides in a state of form, and, if necessary, to act on a strongly acidic cation exchange resin as a separating agent to separate and remove unbranched oligosaccharides such as glucose, thereby producing high-purity branched oligosaccharides.

<Examples> Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1 A 30% (w / w) DE12 starch liquefied solution (using corn starch) was adjusted to a temperature of 55 ° C. and a pH of 6.0, and added with maltotriose-forming amylase of the genus Bacillus at 0.8% per solid.
(W / w) and Aspergillus transglucosidase were added at 450 units per gram of solid and saccharified for 42 hours.
After completion of the saccharification, a heat treatment was performed at 85 ° C. for 5 minutes, followed by filtration, ion exchange purification, activated carbon treatment, and concentration to obtain a branched oligosaccharide. Table 1 shows the results.

Example 2 A liquefied solution of 30% (w / w) DE12 starch (using corn starch) was adjusted to a temperature of 50 ° C. and a pH of 6.5, and a maltotetraose-forming amylase of Bacillus sp.
% (W / w) and saccharified for 40 hours. Then temperature 55 ° C, p
It was adjusted to H5.0, and transglucosidase of Aspergillus sp. Was added thereto to 450 units per g of solid, and saccharified for 24 hours. After completion of the saccharification, a heat treatment was performed at 85 ° C. for 5 minutes, followed by filtration, ion exchange purification, activated carbon treatment, and concentration to obtain a branched oligosaccharide. Table 1 shows the results.

Example 3 Under the same conditions as in Example 1, the branching enzyme pullulanase was used. That is, a 30% (w / w) DE12 starch liquefied liquid (using corn starch) was adjusted to a temperature of 55 ° C. and a pH of 6.0, and a maltotriose-forming amylase of the genus Bacillus was added at 0.8% (w / w) per solid. ) And Aspergillus transglucosidase at 450 units per g solid and Bacillus pullulanase at 0.1% (w / w) per solid.
Saccharified for 42 hours. After completion of the saccharification, a heat treatment was performed at 85 ° C. for 5 minutes, followed by filtration, ion exchange purification, activated carbon treatment, and concentration to obtain a branched oligosaccharide.

 Table 1 shows the results.

COMPARATIVE EXAMPLE 30% (w / w) DE10 starch liquefied liquid (using corn starch) was adjusted to a temperature of 55 ° C. and a pH of 5.5, to which maltogenic amylase of malt was added at 0.3% (w / w) and Aspergillus per solid. The genus transglucosidase was added for 450 units per gram of solid and saccharified for 4 hours. After saccharification, 85
A heat treatment was performed at 5 ° C. for 5 minutes, followed by filtration, ion exchange purification, activated carbon treatment, and concentration to obtain a branched oligosaccharide.

The results are shown in Table 1. Each of the branched oligosaccharides obtained from Examples 1 to 3 has a higher glucose content, a smaller amount of branched oligosaccharides, and more than branched trisaccharides than the branched oligosaccharides of Comparative Examples. Was also significantly less.

[Effects of the Invention] As described in detail above, according to the present invention, a branched oligosaccharide having a low glucose content, a high branched oligosaccharide content, and a branched trisaccharide or more as a main component is efficiently and economically produced in a large amount. And an industrial production method for production.

The branched oligosaccharide obtained by this production method can be used as a general sweetener in foods and drinks, or as a functional saccharide, as a raw material for culturing pharmaceuticals, bifidobacterial growth factor, low cariogenic sweetener, low calorie sweetness. It is a branched oligosaccharide that is effective in various fields such as materials.

Continuing on the front page (72) Inventor Takana Kimura 622, Oyagi-cho, Takasaki City, Gunma Prefecture Inside Gunei Kagaku Kogyo Co., Ltd. (58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 19/18 C12P 19/14

Claims (1)

(57) [Claims] A liquefied solution of starch having a DE of 8 to 25 is used as a substrate, and an amylase produced mainly with maltotriose or an amylase produced mainly with maltotetraose is used as a substrate. Producing a branched oligosaccharide, characterized by reacting the enzyme with the enzyme simultaneously or stepwise at a substrate concentration of 10% or more, at a temperature of 40 to 65 ° C., at a pH of 4 to 8, for 30 to 90 hours. Method.