JP2022024332A - Method for producing isomaltose - Google Patents

Abstract

To provide a method for efficiently producing isomaltose.SOLUTION: A method for producing isomaltose comprises the steps of: producing isomaltose by combining a 6-α-glucosyltransferase, which has an activity of acting on starch or a partial starch decomposition product to produce a branched α-glucan having a branched structure in which D-glucose is α-1,6 bound to a hydroxyl group at position 6 of a non-reducing terminal glucose residue of an α-1,4 glucan chain thereof, with isopullulanase and acting it on starch or starch partial decomposition products; and collecting the produced isomaltose.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for producing isomaltose, specifically, a method for producing isomaltose from starch or a partially decomposed starch product in which a glycosyltransferase and isopullulanase are combined.

In isomaltose (6-O-α-D-glucosyl-D-glucose), two molecules of D-glucose are bound via α-1,6 glucosidic bonds (hereinafter referred to as "α-1,6 bonds"). It is a reducing disaccharide having a similar structure, and is a saccharide that is difficult to crystallize and has excellent moisturizing properties. Isomaltose is contained in a small amount in fermented foods and the like, and has been conventionally used in various foods and cosmetics in the form of a mixture with D-glucose, maltose, panose and the like.

As a method for producing isomaltose, α-glucosidase (also known as “transglucosidase”) derived from Aspergillus is allowed to act on maltose (maltose) obtained by decomposing starch with β-amylase, and isomaltose and isomaltose. Although a method of producing isomaltoligosaccharides such as aus, panose, and isomalttetraose and chromatose fractionating them to obtain isomaltose is known, isomaltoligosaccharides obtained by an enzymatic reaction. The content of isomaltose in the contained sugar is usually only about 26% by mass per solid substance, and it is not easy to isolate isomaltose from the isomalto-oligosaccharide mixture. Further, even if isomaltoxtranase (EC 3.21.94) is allowed to act on the isomaltoligosaccharide-containing sugar to produce isomaltose, the content of isomaltose in the reaction solid is still high. Usually, it is as low as less than 40% by mass. On the other hand, as another method for producing isomaltose, a method of allowing isomalt dextranase to act on a product obtained by partially decomposing dextran with an acid (Patent Document 2) is known. However, although this method using dextran as a raw material has a high yield of isomaltose, it is not easy to manufacture and obtain a special α-1,6 glucan called dextran, so isomaltose as a general-purpose product is industrially manufactured. It wasn't done yet.

The same applicant as the present application combined the 6-α-glucosyl transferase (also known as α-isomaltose glucosaccharide-producing enzyme) found as a novel enzyme in Patent Document 3 with the isomalt dextranase as a raw material. An efficient method for producing isomaltose, which is characterized by simultaneously acting on starch or a partially decomposed product of starch, has been established and disclosed. The 6-α-glucosyl transferase acts on starch or a partial decomposition product of starch, and D-glucose binds α-1,6 to the 6-position hydroxyl group of the non-reducing terminal glucose residue of the α-1,4 glucan chain. A branched α-glucan having a branched structure (also known as α-isomaltosylglucosaccharide) is an enzyme having an activity of producing a branched α-glucan, which is obtained by acting on a starch or a partially decomposed product of glucose. , Α-1,4 Glucan chain non-reducing terminal A branched structure in which D-glucose is α-1,6 bonded to the 6-position hydroxyl group of the glucose residue, that is, isomaltose at the non-reducing end of the α-1,4 glucan chain. It is a sugar having a structure (also known as α-isomaltosyl gluco sugar). When isomalt dextranase is allowed to act on this, isomaltose can be released and produced because the α-1,4 bond to which the isomaltosyl group is bound is specifically hydrolyzed. By alternately repeating the reactions of these two enzymes, isomaltose can be efficiently produced from starch or a partial decomposition product of starch. According to this production method, a sugar composition containing about 63% by mass of isomaltose per solid substance can be obtained from the starch partial decomposition product by an enzymatic reaction in which 6-α-glucosyl transferase and isomalt dextranase are combined. Furthermore, when starch debranching enzyme is used in combination with the combination of these enzymes, a sugar composition containing about 70% by mass of isomaltose per solid substance can be obtained, so that isomaltose can be efficiently produced. However, in this method using isomalt dextranase, when the concentration of raw starch or starch partial decomposition product during the enzymatic reaction is increased to 30% by mass or more of the industrial production level, the production of isomaltose is reduced and starch is produced. Even when debranching enzyme is used in combination, there are drawbacks such as the isomaltose content per solid matter in the reaction solution is reduced to 55% by mass or less, and a large amount of isomalt dextranase is required for the enzyme reaction. Yes, it has not been put into practical use.

If an enzyme that can be replaced with isomalt dextranase and has a better isomaltose-producing ability can be found in the method for producing isomaltose in combination with the above-mentioned 6-α-glucosyltransferase, starch or starch or It was considered that isomaltose could be produced more efficiently using the partially decomposed starch as a raw material.

Among the above-mentioned sugars having an isomaltose structure at the non-reducing end of the α-1,4 glucan chain, the lowest molecular weight sugar is panose (62 ^- O-α-D-glucosyl-maltose). The isomalt dextranase is an enzyme that hydrolyzes panose into isomaltose and D-glucose. Isopullulanase (EC) is an enzyme that catalyzes this reaction in addition to isopullulanase. Although 3.21.57, Non-Patent Document 1) is known, only isomalt dextranase is disclosed in Patent Document 3, and a production method utilizing isopullulanase has been completely tried. do not have.

Japanese Unexamined Patent Publication No. 61-219345 Japanese Unexamined Patent Publication No. 63-2164943 International Publication No. 2002/08874 Pamphlet

Sakano et al., Biochemistry Journal (Biochem.J.), Vol. 323, pp. 757-764 (1997).

An object of the present invention is to provide an efficient method for producing isomaltose.

The present inventors have focused on isopullulanase, which has an action similar to that of isopullulanase on panose, as an enzyme useful for the production of isomaltose, and in the method for producing isomaltose disclosed in Patent Document 3. When isomalt dextranase was replaced with isopullulanase and tested, surprisingly, it was found that isomaltose can be produced significantly more efficiently than the method using isomalt dextranase, and 6-α-glucosyltransferase and iso The present invention was completed by establishing a method for producing isomaltose using a combination with isopullulanase.

That is, the present invention has a branched structure in which D-glucose is α-1,6 bonded to the 6-position hydroxyl group of the non-reducing terminal glucose residue of the α-1,4 glucan chain that acts on starch or starch partial decomposition products. A step of producing isomaltose by combining a 6-α-glucosyl transferase having an activity of producing a branched α-glucan with isopluranase and allowing it to act on starch or a starch partial decomposition product, and an isomaltose produced. The above problem is solved by providing a method for producing isomaltose, which comprises a step of collecting.

According to the method for producing isomaltose of the present invention, isomaltose can be efficiently produced from starch or a partially decomposed starch product as a raw material, and isomaltose can be produced on an industrial scale. In addition, the establishment of this isomaltose production method facilitates the production of isomaltitol, which is a reduced product of isomaltose. It will be possible to supply.

It is a schematic diagram which shows the outline of the isomaltose production reaction in the isomaltose production method of this invention.

The present invention acts on starch or a partially decomposed starch product, and has a branched structure in which D-glucose is α-1,6 bonded to the 6-position hydroxyl group of the non-reducing terminal glucose residue of the α-1,4 glucan chain. Isomaltose by combining 6-α-glucosyl transferase (also known as α-isomaltosyl glucosaccharide-producing enzyme) having an activity to produce α-glucan and isopluranase and acting on starch or starch partial decomposition products. The present invention relates to a method for producing isomaltose, which comprises a step of producing glucose and a step of collecting the produced isomaltose.

The outline of the isomaltose production reaction in the isomaltose production method of the present invention is shown in FIG. 1 as a schematic diagram. This method is an isomaltose in which 6-α-glucosyl transferase (also known as α-isomaltosyl glucosaccharide-producing enzyme) and isomalt dextranase disclosed in Patent Document 3 by the same applicant as the present application are combined. In the production method, isopullulanase is used instead of isomalt dextranase. Isomalt dextranase is an activity that hydrolyzes isomalttriose to produce isomaltose and D-glucose, and isomaltose that hydrolyzes dextran, which is α-1,6 glucan, from the non-reducing end in isomaltose units. Has an activity to produce isomaltose and D-glucose, and an activity to hydrolyze pullulan to produce isopanose (6-O-α-maltosyl-D-glucose). On the other hand, the isopulranase used in the method for producing isomaltose of the present invention has an activity of hydrolyzing panose into isomaltose and D-glucose and an activity of hydrolyzing purulan to produce isopanolose. Although it is the same as isomalt dextranase in that it does not hydrolyze the α-1,6 bond, it does not have the activity of hydrolyzing isomaltose and the activity of hydrolyzing dextran. It is an enzyme that is decisively different from maltxtranase. When 6-α-glucosyl transferase and isopulranase are combined and acted on starch or starch partial decomposition products, the action of 6-α-glucosyl transferase causes the non-reducing terminal glucose residue of the α-1,4 glucan chain. Branched α-glucan having a branched structure in which D-glucose is α-1,6 bonded to the 6-position hydroxyl group, that is, a sugar having an isomaltose structure at the non-reducing end of the α-1,4 glucan chain (also known as α-). Isomaltose (glucose glucosaccharide) is produced, and then isomaltose is produced by the specific hydrolysis of the α-1,4 bond to which the isomaltosyl group at the terminal is bound by isopluranase. Then, by repeating this reaction, isomaltose will be accumulated in the reaction solution.

The 6-α-glucosyl transferase (also known as α-isomaltosylglucosyl glucosaccharide-producing enzyme) that can be used in the method for producing isomaltose of the present invention is not particularly limited as long as it has the above-mentioned enzyme activity, but is suitable. Examples of the enzyme include an enzyme derived from Bacillus globisporus C9, Bacillus globisporus C11 or Bacillus globisporus N75 disclosed in International Publication No. 2002/010361 by the same applicant as the present application, and Arthrobacter. Examples include enzymes derived from Arthrobacter globiformis A19 or Arthrobacter ramosus S1. The 6-α-glucosyltransferase used in the present invention is not limited by the degree of purification thereof, and may be a crude enzyme, a partially purified enzyme or a purified enzyme as long as it does not interfere with the desired isomaltose production reaction. .. Bacillus globisporus is currently classified as Paenibacillus filicis based on the homology (identity) of the base sequence of 16S rDNA.

The enzymatic activity of the 6-α-glucosyltransferase used in the present invention can be measured by the following method disclosed in Patent Document 3 by the same applicant as the present application. That is, maltotriose was dissolved in 100 mM acetate buffer (pH 6.0) so as to have a concentration of 2% (w / v) to prepare a substrate solution, and 0.5 mL of an enzyme solution was added to 0.5 mL of the substrate solution. , Enzymatic reaction at 35 ° C. for 60 minutes, boiling the reaction solution for 10 minutes to stop the reaction, and then isomaltosylmartose (63 ^- α-D-glucosylmaltotri) mainly produced in the reaction solution. Of Aus) and Martose, Martose is quantified by high performance liquid chromatography (hereinafter abbreviated as "HPLC"). HPLC was performed on a "YMC Pack ODS-AQ303" column (manufactured by YMC Co., Ltd.) using deionized water as an eluent at a column temperature of 40 ° C. and a flow rate of 0.5 mL / min. Is detected using a differential refractometer "RI-8012" (manufactured by Tosoh Corporation). One unit (U) of activity of 6-α-glucosyltransferase is defined as the amount of enzyme that produces 1 μmol of maltose per minute under the above conditions.

The isopullulanase that can be used in the method for producing isomaltose of the present invention has an activity of hydrolyzing panose to produce isomaltose and D-glucose, and an activity of hydrolyzing pullulan to produce isopullanase. As long as it is an enzyme that has and does not act on isomaltose and dextran, it is not particularly limited by the source, but for example, isopullulanase derived from Asbergillus niger is commercially available as an enzyme agent. Therefore, it can be advantageously used in the production method of the present invention.

In addition, the same applicant as in the present application, in Japanese Patent Application Laid-Open No. 2004-261132, "pullulan decomposition" in which a microorganism belonging to the genus Aureobasidium, which is also a pullulan-producing bacterium, hydrolyzes pullulan in the culture supernatant to produce isopullulanase. They have found that they produce "enzymes", ie isopullulanase. Isopullulanase produced by this aureobasidium genus microorganism can also be advantageously used in the isomaltose production method of the present invention.

The method for measuring the activity of isopullulanase used in the present invention is not particularly limited, and based on the above-mentioned activity of isopullulanase, panose is used as a substrate and the activity of hydrolyzing panose to produce isomaltose and D-glucose is measured. Alternatively, pullulan can be used as a substrate to measure the activity of hydrolyzing pullulan to produce isopullanose. The enzymatic activity of isopullulanase described in the present specification is obtained by dissolving purulan as a substrate in a formic acid buffer (pH 3.5) having a concentration of 100 mM so as to have a concentration of 1.0% (w / v) to obtain a substrate solution. , The enzyme solution prepared by diluting with the same buffer solution was added to the substrate solution and reacted at 40 ° C., and the reducing power of isopullulanase produced by the hydrolysis of purulan was measured in terms of D-glucose using D-glucose as a standard product. .. One unit (U) of isopullulanase activity was defined as the amount of enzyme that produced a reducing power equivalent to 1 μmol of D-glucose per minute under the above conditions.

In the method for producing isomaltose of the present invention, as the raw material substrate, for example, ground starch such as corn starch, rice starch, wheat starch, underground starch such as potato starch, sweet potato starch, tapioca starch and their partial hydrolyzate ( Starch partial decomposition product) can be preferably used. The starch partial decomposition product is usually obtained by suspending the above-mentioned above-ground or underground starch in water and having a concentration of 10% by mass or more, more preferably 15% by mass to 65% by mass, still more preferably 20% by mass or more. It can be obtained as 50% by mass starch milk, which is heated to gelatinize and then liquefied (partially decomposed) with an acid or heat-resistant α-amylase. The degree of liquefaction is preferably set relatively low, and is usually preferably less than DE (Dextrose Equivalent) 15 and preferably less than DE10, more preferably in the range of DE9 to 0.1. In the case of liquefying with an acid, for example, a method of liquefying with an acid agent such as hydrochloric acid, phosphoric acid or oxalic acid and then neutralizing to a desired pH with an alkaline agent such as calcium carbonate, calcium oxide or sodium carbonate is usually used. adopt.

In the isomaltose production reaction of the method for producing isomaltose of the present invention, the concentration of starch or starch partial decomposition product in the reaction solution is not particularly limited, but the solid matter concentration is usually 40% by mass or less, preferably 35% by mass. The following are suitable, and isomaltose can be advantageously produced under these conditions. The reaction temperature may be up to the temperature at which the isomaltose production reaction by 6-α-glucosyltransferase and isopullulanase proceeds, that is, 40 ° C. A temperature of 35 ° C. or lower, more preferably around 30 ° C. is used. The reaction pH is usually adjusted to the range of 4.0 to 6.0, preferably pH 5.0 to 5.5. The amount of enzyme used and the reaction time are closely related, and the amount of enzyme used and the reaction time may be appropriately adjusted according to the progress of the desired enzyme reaction.

In the step of producing the isomaltose of the method for producing isomaltose of the present invention, further, if necessary, a starch debranching enzyme such as isoamylase or pullulanase, α-amylase, cyclomaltdextrin glucanotransferase ( It is also advantageous to use one or more enzymes selected from CGTase) and glucoamylase in combination. In particular, the starch debranching enzyme is an enzyme that specifically hydrolyzes (branches) the α-1,6 bond of the branched structure via the α-1,6 bond in the starch or the starch partial decomposition product as a raw material. Therefore, when used in combination, the α-1,6 branch of starch or starch partial decomposition product, which is a raw material substrate, is decomposed, and the 6-α-glucosyl transfer enzyme proceeds from the non-reducing terminal side of starch or starch partial decomposition product. It has the effect of increasing the efficiency of the isomartose production reaction with isopulranase and increasing the amount of isomaltose produced. Further, since α-amylase and CGTase hydrolyze starch or partially decomposed starch, it is possible to suppress the residual high molecular starch in the reaction solution of the isomaltose production reaction. Furthermore, CGTase changes from maltose and maltotriose, which are relatively difficult for 6-α-glucosyltransferase to react, to maltotetraose and maltopentaose, which have a higher degree of polymerization and are relatively easy to react, due to a catalyzed disproportionation reaction. Since it has an activity to produce it, it also has the effect of increasing the efficiency of the isomaltose production reaction and further increasing the amount of isomaltose produced.

Patent Document 3 (International Publication No. 02/088374 pamphlet) discloses a method for producing isomaltose in which the 6-α-glucosyl transfer enzyme and isomalt dextranase are combined, but as a starch debranching enzyme. In the production method using isoamylase in combination with the above, the isomaltose content per reaction solid of the reaction product obtained from the raw material starch partial decomposition product is about 70% by mass at the maximum under the condition of the substrate concentration of 5% by mass, but the substrate. It is described that under the conditions of the concentrations of 30% by mass and 40% by mass, the maximum is about 55% by mass and about 50% by mass, respectively, and that the production of isomaltose is significantly reduced when the substrate concentration is increased. The production of isomaltose decreases as the substrate concentration increases because the action of isomalt dextranase on the substrate decreases. On the other hand, in the production method of the present invention in which isopullulanase is replaced with isopullulanase, isomaltose is slightly produced as the substrate concentration increases in the case of isopullulanase as shown in the section of the experiment described later. Although it decreases, the isomaltose content per solid matter of the reaction product can be maintained at 70% by mass or more even under the condition of the substrate concentration of 30% by mass or more. Isomaltose can be produced more efficiently than tranase.

Further, the present invention further adds to the above-mentioned method for producing isomaltose, a step of reducing isomaltose by hydrogenation to convert it into isomaltitol, and a step of collecting the converted isomaltitol. It also relates to a method for producing isomaltitol. The isomaltose or isomaltose-containing sugar obtained by the above method for producing isomaltose can be converted into isomaltitol or an isomaltitol-containing substance in high yield by hydrogenation under a reduction catalyst. Specifically, for example, a Raney nickel catalyst is added to an isomartose-containing aqueous solution having a solid substance concentration of 40 to 60%, this is placed in a pressure-resistant container, hydrogen is filled in the container, pressure is applied, and the temperature is 100 to 120. Add hydrogen by stirring at ° C until hydrogen is no longer consumed. At this time, isomaltose is reduced to isomaltose, and other reducing sugars contained in the isomaltose-containing sugars, such as D-glucose, maltose, maltotriose, and other reducing starch partial water additions. The decomposition product is also reduced to sugar alcohol. Whether the obtained isomartol-containing solution is separated from a lane nickel catalyst, decolorized with activated carbon according to a conventional method, desalted with an H-type or OH-type ion exchange resin, purified, and concentrated to form a syrup-like product. Further, this is dried to obtain a powder. If necessary, further, for example, fractionation by column chromatography such as ion exchange column chromatography, activated carbon column chromatography, silica gel column chromatography, crystallization, separation using an organic solvent such as alcohol and acetone, membrane separation method, etc. It is also possible to purify by appropriately combining one kind or two or more kinds of methods to obtain high-purity isomartol. Crystals of isomaltitol are known, and even higher purity products can be produced by crystallization.

The reaction product or its reduced product (hydrogenated product) obtained by the above-mentioned method for producing isomaltose of the present invention can be used as it is as an isomaltose-containing sugar solution or an isomaltitol-containing solution, but is generally used. , Further purified and used. As the purification method, a usual method used for purification of sugar and sugar alcohol may be appropriately adopted. For example, decolorization with activated charcoal, desalting with H-type or OH-type ion exchange resin, ion exchange column chromatography, activated charcoal column. Fractionation by column chromatography such as chromatography, silica gel column chromatography, separation by organic solvent such as alcohol and acetone, separation by membrane having appropriate separation performance, and contaminating sugar without using isomartose or isomartol. A purification method such as fermentation treatment with a microorganism that assimilate and decompose the quality, for example, yeast, can be appropriately adopted.

In particular, as a purification method in mass production, adoption of ion exchange column chromatography is preferable, and for example, strongly acidic cations disclosed in JP-A-58-23799, JP-A-58-72598 and the like. It is possible to advantageously produce an isomaltose-containing saccharide and an isomaltose-containing substance in which contaminants are removed by column chromatography using an exchange resin and the content of the target substance is improved. At this time, it is optional to adopt any of the fixed floor method, the moving floor method, and the pseudo moving floor method.

The aqueous solution containing the isomaltose-containing sugar and the isomaltitol-containing substance thus obtained can usually be concentrated into a syrup-like product. The syrup-like product may be further dried to form an amorphous solid product or an amorphous powder product.

The isomaltose-containing sugars and isomaltitol-containing substances obtained by the production method of the present invention can be used as sweeteners, taste improvers, quality improvers, stabilizers, discoloration inhibitors, excipients, etc. with other components. It can be advantageously used in various compositions such as combinations, foods and drinks, tastes, feeds, feeds, cosmetics, pharmaceuticals, and industrial products.

As a method for incorporating the isomaltose-containing sugar and isomaltitol-containing substance obtained by the production method of the present invention into various compositions as described above, it is possible to include them in the process until the product is completed. Well, for example, known methods such as mixing, kneading, dissolution, melting, dipping, permeation, spraying, coating, coating, spraying, injecting, crystallization, solidification and the like are appropriately selected. The amount is usually 0.1% by mass or more, preferably 1% by mass or more.

Hereinafter, the present invention will be described in detail by experiments.

<Experiment: Production of isomaltose from a partially degraded starch product that combines 6-α-glucosyltransferase and isopullulanase>
A commercially available isopullulanase enzyme preparation is combined with a 6-α-glucosyltransferase derived from Bacillus globisporus N75 disclosed in International Publication No. 2002/010361 to act on a starch partial decomposition product to form an isomaltose. I tried to manufacture it.

<Experiment 1: Effect of combined use of various enzymes in isomaltose production reaction>
Using a partially decomposed starch product (trade name "Paindex # 100", sold by Matsutani Chemical Industry Co., Ltd.) as a raw material substrate, it is dissolved in a 20 mM acetate buffer (pH 5.5) containing 1 mM calcium chloride so that the final concentration is 5% by mass. It was used as a substrate solution. Next, 1 U of 6-α-glucosyltransferase (Hayashihara Preparation Co., Ltd.) per 1 g of substrate solid, 64 U of commercially available isopullulanase (derived from Aspergillus niger, sold by Megazyme), and 1,000 fu of isoamylase (sold by Megazyme). Hayashihara Preparation Co., Ltd.) and 0.5U cyclomaltodextrin glucanotransferase (CGTase, Hayashihara Preparation Co., Ltd.) were added in the combinations shown in Table 1, and hinokithiol was added as an antiseptic to a final concentration of 60 ppm. After the addition, the reaction was carried out at 30 ° C. for 72 hours. After completion of the reaction, the enzyme was inactivated by heating at 100 ° C. for 10 minutes, and then the sugar composition of each reaction solution was measured by HPLC under the following conditions to determine the isomaltose content per solid substance of the reaction solution. The results are shown in Table 1.

<HPLC conditions>
Column; "MCIgel CK04SS" (manufactured by Mitsubishi Chemical Corporation)
Two columns are connected in series and used;
Eluent: Ultrapure water Column temperature: 80 ° C
Flow rate: 0.4 mL / min Detection: Differential refractometer RID-10A (manufactured by Shimadzu Corporation)

As shown in Table 1, when 6-α-glucosyl transferase and isopullulanase are allowed to act on the starch partial decomposition products alone, isomaltose is hardly produced, whereas 6-α-glucosyl transferase and isopullulanase are produced. When the above was allowed to act in combination, isopullulanase was efficiently produced only by the combination of these two enzymes, and the isopullulanase content per solid reaction solution reached 75.0% by mass. Further, it was found that when isoamylase, which is a starch debranching enzyme, was used in combination with the combination of these enzymes, the isomaltose content per solid reaction solution was slightly increased to 76.3% by mass. Furthermore, it was also found that when CGTase was further used in combination with these enzyme combinations, the isomaltose content per reaction solution solid was 77.1% by mass, which was slightly further increased. The effect of increasing the yield of isomaltose by the combined use of isoamylase or CGTase was small.

<Experiment 2: Effect of substrate concentration on isomaltose formation reaction>
The substrate concentration used for the isomaltose production reaction was changed to 5, 10, 20, 30 or 40% by mass, and the enzyme reaction was carried out under the same conditions as in Experiment 1 except that all four types of enzymes used in Experiment 1 were allowed to act. The isomaltose content per solid substance of each obtained reaction solution was measured by the above-mentioned HPLC analysis. The results are shown in Table 2.

As can be seen in Table 2, the isomaltose content per solid matter of the reaction solution reached about 78% by mass in the range of the substrate concentration of 5 to 10% by mass. The isomaltose content per solid matter of the reaction solution under the conditions of the substrate concentrations of 20, 30 and 40% by mass was 76.6, 73.6 and 70.0% by mass, respectively, and the isomaltose as the substrate concentration increased. Although the content gradually decreased, it was maintained at 70% by mass or more.

The results of Experiments 1 and 2 show that according to the method for producing isomaltose of the present invention using a combination of 6-α-glucosyltransferase and isopullulanase, other enzymes can be appropriately used even under relatively high substrate concentration conditions. When used in combination, a reaction product having an isomaltose content of 70% by mass or more per solid substance can be obtained, which indicates that isomaltose can be efficiently produced from starch or a partially decomposed product of starch.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to these examples.

<Manufacturing of isomaltose from starch>
The corn starch is made into starch milk having a concentration of about 30% by mass, and 0.1% by mass of calcium carbonate is added to the starch to adjust the pH to 6.5, and heat-resistant α-amylase (trade name "Tarmamir 60L", manufactured by Novo) is added to the starch. 0.3% by mass was added per unit, and the mixture was reacted at 95 ° C. for 15 minutes, then autoclaved at 120 ° C. for 20 minutes, and further cooled to about 40 ° C. to prepare a starch liquefied solution having a DE of about 4. The pH of this starch liquefied solution was adjusted to 5.5, and 1 U of 6-α-glucosyltransferase (derived from Bacillus globisporus N75, Hayashihara-like product) and 64 U of isopullulanase (derived from Aspergillus niger) per 1 g of starch solids. (Sold by Megazyme), 1,000 fu starch debranching enzyme (isoamylase, Hayashihara preparation product), and 0.5 U cyclomalt dextrin pullulanase (CGTase, Hayashihara preparation product) are added, and further. After adding sodium pyrosulfite as a preservative to a final concentration of 0.01% by mass, the mixture was reacted at 40 ° C. for 72 hours. After completion of the reaction, the enzyme was inactivated by heating at 96 ° C. for 30 minutes, and then the sugar composition of the reaction solution was measured by HPLC. As a result, D-glucose 3.8% by mass and isomaltose 72.4% by mass were measured. The amount of other oligosaccharides was 23.8% by mass. This isomaltose-containing sugar solution can be advantageously used as a sweetener, a carbon source for fermentation, a reagent, a chemical product, a raw material for pharmaceuticals, an intermediate, etc. as an isomaltose-containing syrup after decolorization, desalting, and concentration.

<Manufacturing of isomaltose from partial starch decomposition products>
A partially decomposed starch product (trade name "Paindex # 100", sold by Matsutani Chemical Industry Co., Ltd.) was dissolved in a 20 mM acetate buffer (pH 5.5) containing 1 mM calcium chloride so as to have a final concentration of 20% by mass. Isopullulanase crude enzyme obtained by culturing 1 U of 6-α-glucosyltransferase (Hayashihara Preparation Co., Ltd.) and aureobasidium pullulans IFO6353 per 1 g of substrate solid by a conventional method and centrifuging the culture solution. Add 64U of the solution as isopullulanase, 1,000fu of isoamylase (Hayashihara preparation product), and 0.5U of CGTase (Hayashihara preparation product), and add sodium pyrosulfate as a preservative to the final concentration of 0. After adding to 01% by mass, the mixture was reacted at 30 ° C. for 72 hours. After completion of the reaction, the filtrate obtained by inactivating the enzyme by heating at 96 ° C. for 30 minutes, cooling and filtering is decolorized with activated carbon according to a conventional method, and decolorized with H-type and OH-type ion exchange resins. It was salted and purified, further concentrated and dried to obtain an isomaltose-containing powder having an isomaltose content of 75.4% by mass. This product has excellent moisturizing property, low sweetness, osmotic pressure controllability, excipient property, shine-imparting property, moisturizing property, viscosity, sugar crystallization prevention property, fertility resistance, starch aging prevention property, etc. Therefore, it can be advantageously used for various foods and drinks, health foods, feeds, feeds, cosmetics, pharmaceuticals, luxury goods and the like.

<High-purity isomaltose>
The isomaltose-rich syrup obtained by the method of Example 1 is diluted with water, and column chromatography is performed using a strong acid cation exchange resin trade name "Amberlite CR-1310", Na ⁺ type, manufactured by Organo Corporation). Was done. That is, the resin was filled in 10 stainless steel columns with a jacket having an inner diameter of 12.5 cm, and these columns were connected in series to make the total length of the resin layer 16 m. While maintaining the temperature inside the column at 40 ° C., 1.5% (v / v) of the diluted solution was added to the amount of resin, and warm water at 40 ° C. was flowed through SV0.2 to fractionate the eluate. The isomaltose fraction was collected and purified while monitoring the sugar composition of the above by the HPLC method to obtain an isomaltose-containing liquid. The yield of isomaltose per solid was about 80%. This solution was decolorized, desalted, and concentrated according to a conventional method to obtain an isomaltose syrup having a concentration of about 75%. This product contained about 96.7% by mass of isomaltose per solid substance. This product is difficult to crystallize and has excellent moisturizing properties, low sweetness, osmotic pressure controllability, formability, shine-imparting property, moisturizing property, viscosity, sugar crystallization prevention property, fertility resistance, and starch aging prevention. Since it has sex and the like, it can be advantageously used for various foods and drinks, health foods, feeds, feeds, cosmetics, pharmaceuticals, luxury foods and the like.

<Manufacturing of crystalline isomaltitol>
Activated Raney nickel was added to the high-purity isomaltitol syrup obtained in Example 3, hydrogenated by a conventional method, then Raney nickel was removed, decolorized with activated carbon, and desalted and purified with H-type and OH-type ion exchange resins. A high isomaltitol-containing syrup containing 1.2% by mass of sorbitol, 96.5% by mass of isomaltitol, and 2.3% by mass of other sugar alcohols per solid matter was obtained in a yield of about 90% per solid matter. Obtained. This isomaltitol-rich syrup is concentrated to a concentration of about 75% by mass, this concentrate is placed in a crystallization can, and crystalline isomaltitol powder as a seed crystal is added in an amount of 0.1% by mass per solid substance at a temperature of 25 ° C. It was held for about 20 hours to crystallize isomaltitol. Subsequently, the nectar was separated using a centrifuge, and the crystalline isomaltitol was recovered. The isomaltitol crystals were vacuum dried at 80 ° C. for 20 hours to obtain crystalline isomaltitol. This product contained 0.2% by mass of sorbitol, 99.3% by mass of isomaltitol, and 0.5% by mass of other sugar alcohols per solid substance. This product is non-reducing, non-moisture-absorbing, low-sweetness, osmotic pressure-regulating, morphogenic, shining, moisturizing, viscous-imparting, sugar crystallization-preventing, refractory, starch aging. Since it has preventive properties, it can be advantageously used for various foods and drinks, health foods, health supplements, feeds, feeds, cosmetics, pharmaceuticals, luxury foods and the like.

<Sweetness>
8 parts by mass of powdered isomaltose-containing powder obtained by the method of Example 2, 2 parts by mass of trehalose dihydrous crystal-containing powder (registered trademark "Treha", sold by Hayashihara Co., Ltd.), α-glycosyl stevioside (trade name "αG") Sweet ”, sold by Toyo Refinery Co., Ltd.) 0.1 parts by mass and L-aspartyl-L-phenylalanine methyl ester (trade name“ Aspartame ”) 0.1 parts by mass are uniformly mixed and subjected to a granule molding machine for granular sweetness. I got a fee. This product has excellent sweetness and has about twice the sweetness of sugar. This product is a sweetening composition containing isomaltose, which is difficult to crystallize and has excellent moisturizing properties. In addition, this product is stable under normal temperature storage without fear of deterioration.

<Cosmetic cream>
2 parts by mass of polyoxyethylene glycol monostearate, 5 parts by mass of self-emulsifying glycerin monostearate, 2 parts by mass of crystalline isomartol obtained by the method of Example 4, α-glucosyllutin (trade name "αG rutin", (Sold by Toyo Refinery Co., Ltd.) 2 parts by mass of liquid paraffin, 10 parts by mass of glycerin trioctanoate and an appropriate amount of preservative are heated and dissolved according to a conventional method, and 2 parts by mass of L-lactic acid and 1,3-butylene glycol are dissolved therein. 5 parts by mass and 66 parts by mass of purified water were added, and the mixture was emulsified by homogenizer, and an appropriate amount of fragrance was further added and mixed by stirring to produce a cosmetic cream. This product has antioxidant properties, high stability, and can be advantageously used as a high-quality sunscreen, skin-beautifying agent, fair-skinning agent, and the like.

<Injury treatment plaster>
To 100 parts by mass of syrup-like high-purity isomaltose and 300 parts by mass of maltose obtained by the method of Example 3, 50 parts by mass of methanol in which 3 parts by mass of iodine was dissolved was added and mixed, and a 10% (w / v) pullulan aqueous solution was further added. 200 parts by mass was added and mixed to obtain a traumatic therapeutic plaster showing moderate elongation and adhesion. This product is a high commercial value plaster that prevents the volatilization of iodine and methanol by isomaltose and has little change over time. In addition, this product not only has a bactericidal action by iodine, but also acts as an energy supply agent for cells by maltose, so that the healing period is shortened and the wound surface is healed cleanly.

<Tablet>
After thoroughly mixing 60 parts by mass of the powdered crystalline isomaltitol obtained by the method of Example 4 and 4 parts by mass of cornstarch with 10 parts by mass of aspirin, the mixture is tableted by a tableting machine according to a conventional method to have a thickness of 5. A 25 mm tablet of 680 mg per tablet was produced. This product utilizes the excipient of crystalline isomaltitol powder, has no hygroscopicity, has sufficient physical strength, and has extremely good disintegration in water.

The establishment of the method for producing isomaltose of the present invention using a combination of 6-α-glucosyltransferase and isopullulanase is to produce isomaltose on an industrial scale using starch or a partially decomposed starch product as a raw material, and further to isomaltose. It enables toll production and is of great significance not only in the sugar industry but also in the food, cosmetics and pharmaceutical industries related to it.

Claims (5)

A branched α-glucan having a branched structure in which D-glucose is α-1,6 bonded to the 6-position hydroxyl group of the non-reducing terminal glucose residue of the α-1,4 glucan chain that acts on starch or starch partial decomposition products. It includes a step of producing isomaltose by combining 6-α-glucosyl transferase having an activity to produce and isopluranase and acting on starch or a partially decomposed product of starch, and a step of collecting the produced isomaltose. How to make isomaltose. The method for producing isomaltose according to claim 1, wherein the 6-α-glucosyl transfer enzyme is an enzyme derived from a microorganism belonging to the genus Bacillus or a microorganism belonging to the genus Arthrobacter. The method for producing isomaltose according to claim 1 or 2, wherein the isopullulanase is an enzyme derived from a microorganism belonging to the genus Aspergillus or a microorganism belonging to the genus Aureobasidium. Any of claims 1 to 3 in which one or more enzymes selected from starch debranching enzyme, α-amylase, cyclomaltodexstring lucanotransferase and glucoamylase are further used in the step of producing isomaltose. The method for producing isomaltose described in Crab. The method for producing isomaltose according to any one of claims 1 to 4, further comprising a step of reducing isomaltose by hydrogenation to convert it into isomaltitol and a step of collecting the converted isomaltitol. A method for producing additional isomaltitol.

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