TWI696426B - Method for inhibiting retrogradation of amylaceous substances, and method for producing starch with lower retrogradation than before introduction of trehalose structure - Google Patents

Abstract

The present invention aims to provide a method of inhibiting the aging of starch without providing changes in physical properties other than the aging of starch, that is, without significantly changing the structure of starch, by providing a method for inhibiting aging of starch The above problem is solved by this method. The aging-inhibiting method of starch is characterized by introducing a trehalose structure into the reducing end of the starch by causing the glycosylated trehalose-generating enzyme to function in the aging starch. However, the aging properties of the starch are lower than before the introduction of the trehalose structure.

Description

Method for inhibiting starch aging, and method for manufacturing starch with lower aging property than before introduction of trehalose structure

The present invention relates to a method for inhibiting starch aging, a method for producing starch with reduced aging properties, and an aging inhibitor.

Starch is mainly a high-molecular-weight glucan stored in the cells of high-equivalent seeds or underground stems, and is generally a mixture of amylose and amylopectin. The amylose is an α-1,4 glucan having a structure in which glucose is linearly bonded with α-1,4 bonds. On the other hand, amylopectin has various positions on the straight chain of α-1,4 glucan, usually α-1,4 glucan with a degree of glucose polymerization of 6 or more branched with α-1,6 bonds structure. Starch is excellent in processability and storage, and is low in price. Therefore, it is used as a so-called starch containing starch or a partial decomposition product of starch obtained by hydrolyzing starch with acid or enzyme. The main raw material, in addition, as a thickening stabilizer or colloidal stabilizer, etc., for the purpose of improving the physical properties of food and maintaining quality, and as glucose, high fructose corn syrup (high-fructose corn syrup), malt oligosaccharides, syrup and other raw materials, and widely used in industry. However, the starchy system has the property of swelling and gelatinizing if its aqueous dispersion is heated, but it does not dissolve with time, that is, it will age, and if the starch ages, it will not only damage its content. Food taste or flavor can also cause defects such as being difficult to digest in the human body. Therefore, the scope of utilization of starch in food and other industries is naturally limited. In view of this situation, in the past, for the purpose of imparting aging resistance to starch, many attempts have been made to change the structure of the molecules constituting the starch, especially to change the structure enzymatically. The impact of security can be expected, so vigorously discuss.

For example, Patent Document 1 discloses a method of suppressing the aging of starch by causing branching enzyme (EC 2.4.1.18) to function in starch and introducing a branch structure into the starch.

In addition, Patent Document 2 discloses that by branching enzyme, 4-α-glucan transferase (EC 2.4.1.25) or cyclomaltodextrin glucan transferase (EC 2.4.1.19) in starch A method for inhibiting the aging of starch by introducing an inner branch ring structure and an outer branch structure into the starch.

Furthermore, it is disclosed in Patent Document 3 that a ring {→6)-α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→6 )-α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→} structured cyclic tetrasaccharide enzymes play a role in amyloid, in which The non-reducing end is introduced into the branch structure to inhibit the aging of starch.

However, enzymatic changes of the structure of starch are used to suppress The conventional methods of starch aging are all introduced into the starch structure of the branch structure. Even if the starch structure can be given aging resistance, by introducing the branch structure, the structure of each molecule constituting the starch will also change, and then there are At this time, the molecular weight distribution of the whole starch will change greatly, and as a result, there will be a defect that the physical properties such as the viscosity of the starch before the change are greatly changed. Therefore, starch with reduced aging property by the introduction of branch structure has advantages in reducing aging property, but if it is simply used instead of the existing starch, the physical properties of the resulting product will often Therefore, it is difficult to obtain a product having the same physical properties as the product obtained by using the existing starch while reducing the aging property by introducing the branch structure. Under such circumstances, it is desirable to provide a method for suppressing the aging of starch by not changing the physical properties other than the aging properties of starch as much as possible, that is, without significantly changing the structure of starch or the molecular weight distribution of the entire starch. .

[Prior Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open No. 60-075295

[Patent Document 2] Japanese Patent No. 3107358

[Patent Document 3] Japanese Patent No. 4893980

[Effect of invention]

The present invention aims to provide a method for suppressing the aging of starch without significantly changing the structure of starch or the molecular weight distribution of the entire starch, that is, without significantly changing the physical properties other than the aging properties of starch. In addition, the present invention is aimed at providing a method for producing starch with suppressed aging and an aging inhibitor.

In order to solve the above-mentioned problems, the present inventors have made efforts to study various enzymes that function in starch, and unexpectedly discovered that if aging starch is used to make glycosyl trehalose-generating enzymes work, The introduction of a trehalose structure at the reducing end eliminates the need to significantly change the structure of individual molecules or the molecular weight distribution of the entire starch, and aging is significantly suppressed, thus completing the present invention.

That is, the present invention solves the above-mentioned problems by providing a method for inhibiting the aging of starch, which is characterized in that the glucosyl trehalose-generating enzyme is produced by the aging starch It functions to introduce the trehalose structure to the reducing end of the starch, and the aging property of the starch is lower than before the trehalose structure is introduced.

In addition, the present invention solves the above-mentioned problem by providing a method for producing starch that has aging properties lower than that before introduction of a trehalose structure. This method is characterized in that it contains sugar groups contained in aging starch Steps in which the trehalose-generating enzyme functions, and the step of introducing the obtained starch into the trehalose structure at the reducing end for purification, before The step of performing purification does not include the step of recovering the starchy precipitate.

Furthermore, the present invention solves the above-mentioned problem by providing a starchy aging inhibitor, which contains a glycosyl trehalose-producing enzyme as an active ingredient.

The method for inhibiting starch aging of the present invention is because it is not necessary to greatly change the structure of each molecule constituting starch or the molecular weight distribution of the entire starch, and it can impart aging resistance to starch, so even if aging resistance is imparted The physical properties of the starch are expected to expand the use of starch in various fields including the food field.

The present invention provides a method for inhibiting starch aging, which is characterized by introducing a trehalose structure into the reducing end of the starch by introducing a glycosyl trehalose-generating enzyme into the starch with aging properties. The aging of starch is lower than before the introduction of trehalose structure.

The aging in this specification means that, as is known, starch that is gelled or dissolved by heating with water will change into an insoluble state in water with time. The aging of starch is observed as the cloudiness or solidification of the solution.

The starch in this manual means starch, constituent starch Amylose or amylopectin, partially decomposed starch after partially hydrolyzed by hydrolyzing enzymes such as acid, α-amylase or isoamylase in starch, and further, partially decomposed starch thus obtained In this process, glycotransferases such as branching enzymes or α-glucosyltransferases are allowed to function to introduce a partial decomposition product of starch in a branched structure, or a mixture of these.

In addition, the origin or source of the starch system is not particularly limited. For example, it may be corn flour, rice flour, wheat flour, barley flour, rye flour, teff flour, oat flour, soybean flour, chickpea Flour, pea flour, mung bean flour, buckwheat flour, amaranth powder, chestnut powder, acorn flour, banana starch and other ground starch, or tapioca starch, potato flour, taibai powder, sweet potato starch, lotus root powder, fern powder, etc. The underground starch may be a starch prepared by adding one or more types of starch selected from these as a base.

The starches that are the subject of the method for inhibiting starch aging of the present invention have aging starches, and the aging starches have a degree of difference. The introduction of trehalose structure at the reducing end improves the aging properties. That is, the aging property is lower than before the introduction of the trehalose structure to the reduced end. However, although it also depends on the type or origin of amyloid to exert its effect of inhibiting aging due to the introduction of trehalose structure at its reducing end, in general, amylose has a weight average molecular weight of 3,000 to 5,000 Those with a weight average molecular weight of 12,000 to 120,000 are more suitable as an amylopectin system. On the other hand, when the starch is a common starch or a partial decomposition product of a mixture of amylose and amylopectin, amylose and amylopectin are mixed together Therefore, it can be said that it is preferable to use a weight average molecular weight of 3,000 to 120,000 in the whole starch. On the other hand, even in amylose with a weight average molecular weight of less than 3,000 or more than 5,000, the aging inhibition effect due to the introduction of the trehalose structure at the reducing end is poor, but it does not completely fail. That is, in amylopectin with a weight average molecular weight of less than 12,000 or more than 120,000, the effect of inhibiting aging due to the introduction of the trehalose structure at the reducing end is poor, but it does not completely fail. Therefore, the weight average molecular weight is Amylose or amylopectin outside the above range can also be suitably used in cases where the required aging suppression effect is low. Similarly, in the case of amylose in which amylose and amylopectin are mixed together, even if the weight average molecular weight is less than 3,000 or more than 120,000, the suppression of the introduction of the trehalose structure at the reducing end is suppressed Although the aging effect is poor, it does not completely fail. Therefore, it can be suitably used in cases where the required aging suppression effect is low.

In addition, the aging starch that is the subject of the present invention is generally starch with a ratio of maltose in the solids per unit of β-amylase digest of 30% by mass or more. That is, the β-amylase system hydrolyzes starch in a maltose unit from the non-reducing end, an exotropic enzyme, and stops the hydrolysis reaction before the branch portion of the α-1,6-glycoside linkage of the starch. Therefore, the ratio of maltose in the solids per unit of β-amylase digest can be used as an indicator of the content of the linear structure from the non-reducing end of the starch to the branch. The content of the chain structure is large and it is easy to aging, so it can also be used as one of the indexes of starch aging.

Therefore, starch with a maltose content of 30% by mass or more per unit of β-amylase digest solids is aging starch, and the introduction of trehalose structure at the reducing end of such starch can be more The aging inhibiting effect is remarkably exerted. Therefore, amylum having a maltose content of 30% by mass or more per unit of β-amylase digest solids is suitable as the object of the aging inhibiting method of the present invention. On the other hand, even if the proportion of maltose in the solids per unit of β-amylase digest is less than 30% by mass of amylose, the aging-inhibiting effect due to the introduction of trehalose structure to the reducing end is not It will not work at all, so there is no problem as the object of the aging suppression method of the present invention.

The glycosyl trehalose-generating enzyme in this specification means the enzyme assigned by the enzyme number EC 5.4.99.15, which is glucose at the reducing end of the glucan with a polymerization degree of 3 or more linked by α-1,4 bonds It functions to convert the bonding mode from α-1,4 bond to α-1,1 bond, thereby introducing trehalose-structured enzyme at the reducing end.

In the present invention, the origin of the glycosyl trehalose-producing enzyme system is not particularly limited as long as it has the activity of introducing a trehalose structure at the reducing end of starch, for example, it may be of the genus Actinoplanes or Arthrobacter Arthrobacter, Bradyrhizobium, Brevibacterium, Corynebacterium, Curtobacterium, Flavobacterium, Gryllotalpicola ), Metallosphaera, Micrococcus, Mycobacterium ( Mycobacterium), Rhizobium, Sulfolobus, Terrabacter, Anabaena (Algae of the Candidae family), and Candida (Nostoc, Candidae family) Any of the genus-derived glycosyl trehalose-producing enzymes or variants thereof selected from the group consisting of algae may also be glycosyl trehalose-producing enzymes obtained by genetic recombination. However, among the aforementioned microorganisms, they are selected from the group consisting of Arthrobacter, Brevibacterium, Brevibacterium, Flavobacterium, Micrococcus, Mycobacterium, Rhizobium, Sulfolobus, and Geobacterium Any one of the genera is a glycosyl trehalose-generating enzyme derived from microorganisms or a variant thereof. Among them, the glycosyl trehalose-generating enzyme from Arthrobacter sp. S34 disclosed in Japanese Patent No. 3956884, Japan Arthrobacter ‧ Species Q34, Rhizobium ‧ Species M-11, Brevibacterium ‧ Helovolum, Flavobacterium ‧ Acuatim, Micrococcus luteus, Micrococcus rosea Micrococcus roseus), Curtobacterium citreum, Mycobacterium smegmatis, or glycosyl trehalose-producing enzyme from Terrabactertumescens, acidophilus disclosed in Japanese Patent No. 3557287 Sulfolobus acidocaldarius, or sugar-based trehalose-producing enzymes from Sulfolobus solfataricus, or variants of these can be suitably used, and these can also be obtained by genetic recombination technology .

In addition, the activity of the glycosyl trehalose-producing enzyme can be described, for example, in paragraph 0026 of Japanese Patent No. 3956884 The method, that is, a method using maltopentose as a substrate to measure the reduction of the reducing power accompanying the action of the glycosyl trehalose-producing enzyme to the introduction of the trehalose structure at the reducing end is measured. The unit of the activity of the glycosyl trehalose-producing enzyme referred to in this specification is the above-mentioned activity measurement method, which reduces the amount of enzyme equivalent to the reducing power of maltopentose in 1 minute in 1 minute.

The ratio of the trehalose structure introduced into the reducing end of the starch by the action of the glycosyl trehalose-producing enzyme (hereinafter, referred to as "trehalose structure introduction rate") can be estimated according to the following formula 1. That is, the starch to which the trehalose structure is introduced at the reducing end is analyzed by size exclusion chromatography, etc., and the number-average molecular weight is obtained, and the glucose obtained by making the glucose amylase fully function in the starch The amylase digest is analyzed by high-speed liquid chromatography, etc., and the mass ratio of trehalose in the solid content of the glucose amylase digest per unit is obtained. Next, the number average molecular weight of the starch after introduction of the trehalose structure at the reducing end can be multiplied by the ratio of trehalose in the solids per unit of glucoamylase digest, and divided by 342 which is the molecular weight of trehalose. The obtained value was regarded as the introduction rate of trehalose structure. In the present invention, the introduction rate of the trehalose structure of the starchy substance that causes the glycosyl trehalose-generating enzyme to function is preferably 20% or more, more preferably 30% or more, and even more preferably 40% or more.

Formula 1

The action amount of the aforementioned glycosyl trehalose-generating enzyme is not particularly limited, and it is usually appropriately selected within the range of 0.1 to 100 units per 1 g of starch solids. In addition, the temperature and pH at which the glycosyl trehalose-producing enzyme functions can be appropriately selected within the range in which the enzyme reaction proceeds. For example, the reaction temperature is 10 to 90°C, and the reaction pH is preferably in the range of pH 3 to 9. In addition, the reaction time can be appropriately selected according to the degree of progress of the enzyme reaction, for example, as long as it is selected from the range of 0.1 to 100 hours.

In addition, as long as the trehalose structure introduced at the reducing end is not lost, together with the glycosyl trehalose-generating enzyme, or after the glycosyl trehalose-generating enzyme functions, the purpose of further reducing the aging property is There is no limit to the use of enzymes introduced into branching structures in starches such as starch branching enzymes or α-glucosyltransferases.

In addition, the present invention provides a method for producing starch with reduced aging property than before introduction of a trehalose structure. The method is characterized in that it is contained in an aging starch to make a glycosyl trehalose-producing enzyme function. And the step of purifying the obtained starch with a trehalose structure introduced into the reducing end. The aforementioned step of purifying does not include the step of recovering the starchy precipitate.

In addition, the aforementioned purification step does not include the step of recovering the starchy precipitate, which means that the starch with aging properties When the starch with reduced aging properties obtained by making the glycosyl trehalose-generating enzyme function is purified, the starch is aged, or the object is precipitated by adding an organic solvent, thereby precipitating the starch The steps for recycling are not necessary.

In the past, aging starches, including amylose, are aging and easily insoluble, so the step of recovering the precipitate during purification is necessary. The step of recovering the precipitated starch is due to Cumbersome and time-consuming, so it is one of the reasons for the price increase of products. According to the manufacturing method of the present invention, the starchy structure in which the trehalose structure is introduced at the reducing end is not easy to age. Therefore, the starchy reaction solution or the like can be directly filtered or continuously purified by column chromatography to take the starchy structure. After the troublesome purification step of recovering the precipitate, there is an advantage that the product can be manufactured at a lower price than the conventional manufacturing method.

The starch system obtained by introducing the trehalose structure at the reducing end obtained by the present invention is less likely to be aged than ordinary starch, and the other physical properties are the same as the starch before the change. Therefore, it can be suitably used as aging The usual starchy substitute with no reduction in sex. In addition, there is no limit to the use of mixing with normal starches whose aging properties are not reduced. Furthermore, since the starch obtained by introducing the trehalose structure into the reducing end of the present invention has reduced aging properties, it can be suitably used even in products whose starch utilization is restricted due to aging properties.

The starch obtained by the present invention with the trehalose structure introduced at the reducing end has higher aging resistance than that before the introduction of the trehalose structure, and can maintain the original starch quality of the trehalose structure before the introduction Physical properties other than aging, so it can be used as a substitute for ordinary starch, or mixed with ordinary starch, for example, it can provide energy supply, thickness, viscosity, polishing, shape retention, It is used for foods (for example, sterilized bagged foods, frozen foods, instant foods, pickles) for the purpose of preventing freezing denaturation, improving eating taste, improving taste, adjusting water activity, preventing water separation, preventing crystal precipitation, and imparting moisture retention. Food, boiled, refined products, dairy products, Japanese confectionery, western-style confectionery, snack foods, frozen desserts, liquid foods, weaned foods, health foods, etc.), or beverages (for example, refreshing drinks, carbonated drinks, fruit drinks, vegetable juices) , Milk beverages, coffee, black tea beverages, oolong tea beverages, green tea beverages, barley tea beverages, alcoholic beverages, etc.), cosmetics (lotions, essences, lotions, creams, base makeup, mascara, lipstick, nail polish, skin cleansing , Facial cleanser, mouthwash, shampoo, repair, conditioner, hair products, perfume, etc.). In addition, the starch obtained by the present invention in which the trehalose structure is introduced at the reducing end has better solubility than ordinary starch, so it can be suitably used as various liquids (for example, soup or Japanese soup, seasoning sauce, soy sauce) , Vinegar, mirin, fat, milk, coffee, black tea, oolong tea, green tea, barley tea, alcohol, etc.) powdered base material, and can be suitably used as excipients such as lozenges.

As a typical example of the aging starch, amylose can be mentioned. Amylose has the property of forming a spiral structure in solution. It is well known that the inclusion of various hydrophobic low-molecular substances in the spiral cavity forms an inclusion complex, but amylose itself is easy to age, so its use The scope is limited. Obtained by the present invention The amylose with a trehalose structure introduced at the reducing end is not much different from the normal amylose in structure, so it has the same helix formation energy as the normal amylose. It is more difficult to age, so it can be suitably used as an insoluble or poorly water-soluble pharmaceutical ingredient or functional food ingredient, oil, coloring, flavoring, etc. solubilizer or stabilizer, masking agent, sustained release agent, powdered substrate . In addition, it is not limited as a substitute for normal amylose or mixed with normal amylose for utilization. Therefore, according to the present invention, it is possible to provide a stabilizer, a masking agent, a slow-release agent, or a powdered base material containing amylose having a trehalose structure introduced at the reducing end as an active ingredient.

Amylose, which is a typical example of amylose having aging properties, may be amylose obtained by any manufacturing method, for example, isoamylase (EC 3.2.1.68) or branched starch Amylose obtained by the action of starch debranching enzymes such as amylase (EC 3.2.1.41), or cyclodextrin glucan transferase (EC 2.4.1.19) in cyclodextrin The obtained amylose, starch starch sucrase (EC 2.4.1.4) functioning in sucrose The obtained amylose, α-glucan phosphorylase (EC 2.4.1.1) in glucose-1-phosphate Amylose obtained by functioning, amylose derived from starch synthesis enzyme (EC 2.4.1.21) in ADP-glucose, and maltose transferase (EC 2.4.1.25) obtained in dextrin The resulting amylose etc.

In addition, the present invention provides an aging inhibitor of starch containing glycosyl trehalose-producing enzyme as an active ingredient. Glycosyl The trehalose-generating enzyme acts by converting glucan with a degree of polymerization of 3 or more connected via the α-1,4 bond, and can convert the bonding mode of the glucose residue at its reducing end from the α-1,4 bond Forming α,α-1,4 bonds, the trehalose structure is introduced at the reducing end, and the starch system with trehalose structure introduced at the reducing end is not easy to age, so the enzyme system can use itself as an aging inhibitor of starch.

The starchy aging inhibitor of the present invention can be blended with other components according to the purpose within the range where the activity of the glycosyl trehalose-producing enzyme as an active ingredient can be stably maintained. For example, acids or bases, salts, carbohydrates, amino acids, proteins, etc. may also be blended. In addition, the morphology of the aging inhibitor may be powder, granules, lozenges, liquid, and the like.

Hereinafter, the present invention will be described in more detail based on experiments.

<Experiment 1: Effect of introduction of trehalose structure at the reduced end of amylose on aging properties>

In order to investigate the effect of the introduction of the trehalose structure on the reduced end of amylose on aging, the trehalose structure was introduced on the reduced end of amylose, and the aging property of the amylose introduced with the trehalose structure was evaluated.

<Experiment 1-1: Preparation of amylose with trehalose structure introduced at the reducing end>

The amylose causes the glycosyl trehalose-generating enzyme to function, and the trehalose structure is introduced at the reducing end, and the amylose having the trehalose structure introduced at the reducing end is prepared.

First, a test-grade short-chain amylose powder showing a weight-average molecular weight of 3,200 (trade name "Amylose EX-I", manufactured by Linyuan Co., Ltd., hereinafter referred to as "amylose 1"), and display Amylose powder with a weight average molecular weight of 5,000 (prepared by Linyuan Co., Ltd., hereinafter referred to as "amylose 2") was dissolved in pure water so as to have a solid concentration of 1% by mass and adjusted to pH 5.8 , 2 units of glycosyl trehalose-producing enzyme derived from Arthrobacter sp. S34 (Arthrobacter sp.) S34 obtained by the method described in Example 2-2 of Japanese Patent No. 3956884 is added per 1 g of solid matter at 50°C The reaction was carried out for 48 hours, and then the enzyme was deactivated by heating at 100°C for 10 minutes. Next, by adding sodium hydroxide to each of the obtained reaction solutions, the pH was adjusted to 11, and the heat treatment was performed at 100°C for 1 hour, and the non-reducing amylose with a trehalose structure introduced at the reducing end was added. The remaining reducing sugar is decomposed. Each of the obtained treatment liquids was decolorized using activated carbon, desalted using an ion exchange resin, and then freeze-dried to obtain powders of amylose 1 and 2 having a trehalose structure introduced at the reducing end.

Then, for the amylose 1 and 2 used as raw materials, and the amylose 1 and 2 with a trehalose structure introduced at the reducing end, the weight-average molecular weight, reducing power, and solids per unit of β-amylase digest were measured. The ratio and solubility of maltose in the material. The results are shown in Table 1. In addition, various analysis systems are performed by the following methods.

<weight average molecular weight>

Amylose or amylose with a trehalose structure introduced at the reducing end was dissolved so as to have a solid concentration of 1% by mass, adjusted to pH 7.0, and then subjected to size exclusion chromatography. Next, weight average molecular weights were calculated based on the calibration curve prepared by analyzing the Pullulan standard for molecular weight measurement (manufactured by Linyuan Co., Ltd.) in the same manner. In addition, the size exclusion chromatography system is used when the column is connected with two "TSK GEL α-M" (manufactured by TOSOH Co., Ltd.), and 10 mM phosphate buffer (pH 7.0) is used for the eluate. The temperature was 40°C and the flow rate was 0.3 ml/min. The detection was performed using a differential refractometer "RID-10A" (manufactured by Shimadzu Corporation).

<reducing power>

The amylose and amylose with a trehalose structure introduced at the reducing end were supplied to the anthrone-sulfuric acid method, respectively, and the total sugar amount was measured based on a calibration curve prepared using glucose as a standard sugar. Next, amylose and amylose with a trehalose structure introduced at the reducing end were supplied to the Somogyi-Nelson method, based on a calibration curve prepared using glucose as the standard sugar While measuring the amount of reducing sugar. Then, the ratio of the amount of reducing sugar to the amount of total sugar of amylose and amylose with a trehalose structure introduced at the reducing end was calculated as the reducing power.

<Proportion of maltose in solids per unit of β-amylase digest>

After amylose was dissolved so as to have a solid concentration of 1% by mass and adjusted to pH 5.0, 50 units of β-amylase was added per 1 g of solid (trade name "# 1500", manufactured by Nagase Chemtex Co., Ltd. ), after acting at 50°C for 20 hours, heating at 100°C for 10 minutes stops the enzyme reaction. Next, the obtained β-amylase digest was subjected to high-speed liquid chromatography, and the ratio of the peak area of maltose to the total peak area of the chromatography was determined as the solid content per unit of β-amylase digest. The proportion of maltose. In addition, the high-speed liquid chromatography system is used when two "MCI gel CK04SS" (manufactured by Mitsubishi Chemical Co., Ltd.) are connected to the column, and ultrapure water is used for the eluate at a column temperature of 80°C and a flow rate of 0.4 ml/ The conditions are divided, and the detection is performed using a differential refractometer "RID-10A" (manufactured by Shimadzu Corporation).

<solubility>

In pure water or a 1% by mass trehalose aqueous solution, amylose was excessively added and amylose with a trehalose structure introduced at the reducing end was heated at 100°C for 10 minutes, and then allowed to stand at 25°C for 16 hours. And prepare a saturated solution. After that, the mass of the solid matter contained in 1 g of each saturated solution at 25° C. was measured according to the normal method of drying loss, and the value was converted into a value per 100 g of water to obtain the solubility. In addition, regarding the solubility with respect to a 1% by mass trehalose aqueous solution, a value obtained by subtracting the solid content of the added trehalose from the mass of the solid content contained in 100 g of water is taken as the solubility.

As is clear from Table 1, the reducing powers of amylose 1 and 2 used in the raw materials are 6.0% and 3.8%, respectively. In contrast, amylose 1 and 2 with trehalose structure are introduced at the reducing end. The reducing power is 0.1% and 0.2% respectively. Since there is no substantial showing reducing power, it can be It was confirmed that the introduction rate of the amylose 1 and 2 series trehalose structure into which the trehalose structure was introduced at the reducing end was substantially 100%. In addition, as in the measurement of the weight average molecular weight, the result of confirming the molecular weight distribution by size exclusion chromatography is the same as that of the amylose 1 and amylose 2 before and after the action of the glycosyl trehalose-producing enzyme. No significant changes were observed in the molecular weight distribution. In addition, the ratio of amylose 1 and 2 to the maltose in the solids per unit of β-amylase digest is 88.6 to 91.0% by mass.

The interesting thing is that due to amylose 1 and 2, the solubility for 100g of water is 4.5g and 1.7g, respectively. In contrast, amylose 1 and 2 with trehalose structure introduced at the reducing end, for 100g of water The solubility is 7.4g and 4.6g, respectively. Therefore, amylose with trehalose structure is introduced at the reducing end. Compared with the amylose used in the raw material, the solubility is improved by 1.6 to 2.7 times. Here, since the weight-average molecular weights of the amylose 1 and 2 with the trehalose structure introduced at the reducing end are 3,700 and 5,200, respectively, and the molecular weight of the trehalose is 342, it can be estimated that the straight chain with the trehalose structure introduced at the reducing end Starch 1 and 2 contained 0.7 g (=7.4×(342/3700)) and 0.3 g (=4.6×(342/5200)) of trehalose in 100 g of the saturated solution at 25° C. as the structure of trehalose. On the other hand, since the solubility of amylose 1 and 2 to 100 g of a 1% by mass trehalose aqueous solution is 4.8 g and 1.5 g, respectively, and the solubility to 100 g of water, respectively, the free seaweed is added only to amylose For sugar, no effect of increasing the solubility of amylose 1 and 2 was observed. Based on the above, the conclusion is that due to the introduction of trehalose structure at the reducing end of amylose The solubility will increase. These results describe that the introduction of trehalose structure at the reducing end of starch can significantly improve the solubility of the starch. Since the aging of starch is caused by the insolubilization of the molecules that constitute it, it can be speculated that The introduction of trehalose structure at the reduced end can impart aging resistance to starch.

<Experiment 1-2: Evaluation of aging properties of amylose with trehalose structure introduced at the reducing end>

The aging property was investigated for the amylose with trehalose structure introduced at the reduced end obtained in Experiment 1-1, and the influence of the introduction of the trehalose structure for the reduced end of amylose on the aging property was evaluated.

The amylose 1 used in Experiment 1-1, the amylose 1 with trehalose structure introduced at the reducing end obtained in Experiment 1-1, and these at 20:80, 40:60, 60: The mixtures mixed in the ratio of 40 and 80:20 were suspended in pure water so as to have a solid concentration of 1% by mass, heated and dissolved at 100°C for 10 minutes, and then stored at 4°C. At the start of storage, after 30 minutes, after 1 hour, after 2 hours, after 4 hours, after 1 day, after 2 days, and after 4 days, measure the turbidity of the solution by a spectrophotometer (manufactured by Shimadzu Corporation) Absorbance at a wavelength of 650 nm) to determine the increase in turbidity from the start of storage. In addition, for amylose 2 used in experiment 1-1, amylose 2 with a trehalose structure introduced at the reducing end obtained in experiment 1-1, and this was set at 20:80, 40:60, The mixture mixed in the ratio of 60:40 and 80:20 is measured in the same way, and the time from the start of storage is obtained The increase in turbidity. The results are shown in Tables 2 and 3, respectively. In addition, if the increase in the turbidity exceeds 2.0, it is indicated as "over 2.0" in the table, and the subsequent measurement of the turbidity is not performed, and is indicated by a slash in the table.

As is clear from Table 2, the solution of amylose 1 showed an increase in turbidity of more than 2.0 after 1 day, that is, 24 hours after storage. In contrast, the trehalose structure was introduced at the reducing end It takes 4 days (96 hours) for amylose 1 to show that the turbidity exceeds 2.0, and the rate of aging is reduced to a maximum of 1/4. Furthermore, even when the mixing ratio of amylose 1 with trehalose structure introduced at the reducing end was 20%, as seen from the measured values after 2 hours and 4 hours, the increase in turbidity was inhibition. The higher the mixing ratio of amylose 1 in which trehalose structure was introduced at the reducing end of the entire system, it was observed that the aging rate was significantly reduced.

Also, as is clear from Table 3, the solution of amylose 2 showed an increase in turbidity of more than 2.0 after being stored for 1 hour. In contrast, amylose 2 with a trehalose structure was introduced at the reducing end It takes one day (24 hours) to show that the turbidity exceeds 2.0, and the rate of aging is reduced to 1/24 at the maximum. Furthermore, even if the mixing ratio of amylose 2 with a trehalose structure introduced at the reducing end was 20%, as seen from the measured value after 1 hour, the increase in turbidity was suppressed, and the trehalose structure was introduced at the reducing end The higher the mixing ratio of amylose 2, the aging rate can be observed to be significantly reduced.

From these results, it is clear that by introducing the trehalose structure to the reduced end of amylose, the aging rate is reduced, that is, aging resistance can be imparted, and the effect is even for relatively low molecules with a weight average molecular weight of about 3,000 The amylose can also be confirmed. Furthermore, it is understood that as long as the mixing ratio of the amylose introduced with the trehalose structure is at least 20% or more, the aging will be suppressed. The amylose 1 and 2 used in the above experiment did not have a trehalose structure, the introduction rate of trehalose structure was 0%, and the amylose 1 and 2 with the trehalose structure introduced at the reducing end were as shown in experiment 1-1 In general, the introduction rate of trehalose structure is substantially 100%, so as long as the mixing ratio of the two is at least 20% or more, the fact that aging can be suppressed means that if the introduction rate of trehalose structure is observed, it is only necessary to use at least 20% or more The introduction rate of trehalose structure into the reducing end of amylose into the trehalose structure can inhibit the aging of starch.

In addition, in general, starch with a large weight average molecular weight is relatively easy to age. In contrast, amylose 1 and 2 with a trehalose structure introduced at the reducing end, compared to the control with no trehalose structure introduced at the reducing end Amylose 1 and 2, even if the weight average molecular weight of each is somewhat larger, the aging rate will also decrease. This result faithfully shows that the remarkable effect of improving the aging resistance of the present invention is not obtained by a decrease in molecular weight, but by the introduction of a trehalose structure at the reduced end of amylose.

<Experiment 2: Effect of introduction of trehalose structure on the reducing end of amylopectin with different weight-average molecular weight on aging properties>

In experiment 2, in order to investigate the effect of the introduction of the trehalose structure at the reducing end on the aging of amylopectin, the partial decomposition product of waxy corn starch consisting essentially of amylopectin was reduced. The trehalose structure was introduced at the end, and the aging property was evaluated.

As a partial decomposition product of waxy corn starch, use commercially available "Pinedex# 100", "Pinedex# 1", "Pinedex# 2", and "Pinedex# 4" (all sold by Songgu Chemical Co., Ltd.) 4 Species (hereinafter, each is referred to as amylopectin 1 to 4), and for each of them, the weight average molecular weight and solids per unit of β-amylase digest are determined by the same method as experiment 1-1 The ratio of maltose. Amylopectin 1 to 4 were dissolved in pure water so as to become a solid concentration of 30% by mass, 2 units of glycosyl trehalose-producing enzyme used in Experiment 1 were added per 1 g of solid, and the enzyme was made at 50°C. After 22 hours of action, the enzyme reaction was stopped by heating at 100°C for 10 minutes. Thereafter, the obtained The solutions were stored at 4°C. For the turbidity of the solution (absorbance at a wavelength of 650 nm), at the beginning of storage, after 1 day, after 2 days, after 4 days, after 8 days, after 16 days, and after 32 days, by spectrophotometer (Manufactured by Shimadzu Corporation) Each measurement was performed to determine the increase in turbidity from the start of storage. In addition, in order to compare the aging inhibitory effect caused by the introduction of the trehalose structure at the reducing end, the same method was used for those who treated amylopectin 1 to 4 in the same way except that no glycosyl trehalose-producing enzyme was added. The increase in turbidity from the start of storage is indicated. The results are shown in Table 4. In addition, if the increase in the turbidity exceeds 2.0, it is indicated as "over 2.0" in the table, and the subsequent measurement of the turbidity is not performed, and is indicated by a slash in the table.

As can be seen from Table 4, the relatively low molecular weight amylopectin 4 with a weight average molecular weight of 2,660 was only stored at 4°C for 16 days, and no turbidity was observed regardless of whether the glycosyl trehalose-generating enzymes worked or not. Aging means aging, but when stored for 32 days, the sugar group No increase in turbidity was observed even after the trehalose-generating enzyme was activated. In contrast, those with a sugar-based trehalose-generating enzyme that did not function did observe a slight increase in turbidity, even when the weight average molecular weight was 2,660. It is also confirmed that pullulan 4, which has a low molecular and original aging property, has only a small amount of introduction of the trehalose structure at the reduced end, but still exerts an aging suppression effect. On the other hand, the amylopectin 1 with a weight average molecular weight of 118,000 showed aging property with a turbidity exceeding 2.0 when stored at 4°C for 4 days. On the other hand, the glycosyl trehalose-generating enzyme acts as a branch It takes 8 days for the amylose 1 series to reach a turbidity of more than 2.0, which obviously inhibits aging. Similarly, the amylopectin 2 series with a weight-average molecular weight of 23,400 showed a turbidity of more than 2.0 when stored at 4°C for 2 days. In contrast, the amylopectin 2 series in which glycosyl trehalose-generating enzymes function It took 8 days for the turbidity to reach more than 2.0. Furthermore, the amylopectin 3 series with a weight average molecular weight of 12,100 showed a turbidity of 0.6 when stored at 4°C for 32 days. In contrast, glycosyl trehalose-producing enzymes The functional pullulan 3 series did not observe an increase in turbidity even when stored for 32 days, and in either case, a significant aging inhibitory effect was confirmed. In addition, as in Experiment 1-1, the molecular weight distribution was confirmed by size exclusion chromatography. Before and after the action of the glycosyl trehalose-producing enzyme, no molecular weight distribution of amylopectin 1 to 4 was observed. Significant changes. In addition, it can be confirmed that amylopectin 1 to 3, which is significantly inhibited by glucosyl trehalose-producing enzymes and trehalose structure introduced at the reducing end, is significantly inhibited from aging, is a solid substance per unit of β-amylase digest The proportion of maltose in is 32.7 to 45.3% by mass, Either one is more than 30% by mass.

From these results, it became clear that in amylopectin with a ratio of maltose in the solids per unit of β-amylase digest of 30% by mass or more, if the weight-average molecular weight becomes 12,000 or more, the sugar-based algae The sugar-generating enzyme acts to introduce a trehalose structure at the reducing end, which significantly reduces the aging rate, that is, the effect of imparting aging resistance is significantly exerted. The effect is even for a relatively high molecular weight such as a weight average molecular weight of 120,000 Amylopectin can also be fully utilized.

<Experiment 3: Effect of introduction of trehalose structure at the reducing end of a mixture of amylose and amylopectin on aging>

In Experiments 1 and 2, it was revealed that by introducing the trehalose structure to the reducing ends of amylose and amylopectin, it is possible to suppress such aging. Therefore, in Experiment 3, in order to investigate the effect of the introduction of the trehalose structure at the reducing end on the aging of the mixture of amylose and amylopectin, the ratio of amylose to amylopectin was contained at a ratio of approximately 1:4. The partial decomposition product of cassava starch, starch, was introduced into the trehalose structure at the reduced end, and the aging property was evaluated.

First, cassava starch was suspended in pure water so as to have a solid concentration of 30% by mass, and calcium chloride was added to a final concentration of 1 mM, and then adjusted to pH 6.0 to prepare a starch suspension. To the obtained starch suspension, 10 units of α-amylase (trade name "Speedase HK", manufactured by Nagase Chemtex Co., Ltd.) were added per 1 g of solid matter, while flowing in a continuous liquefaction apparatus The solution was passed through at a rate of 1 L/min, while heating at 100°C for 25 minutes, and then heating at 140°C for 5 minutes to stop the enzyme reaction, and a solution of starch partial decomposition product 1 was obtained. Next, add 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1.0 unit of α-amylase per 1 g of solid matter to the obtained solution of starch partial decomposition product 1 (trade name "Kleistase E5C", Nagase chemtex Co., Ltd.), after the reaction was carried out at 50°C for 22 hours, the enzyme reaction was stopped by heating at 100°C for 20 minutes, and solutions of starch partial decomposition products 2 to 8 were obtained, respectively. Thereafter, the resulting solutions of the starch partial decomposition products 1 to 8 were decolorized using activated carbon, and after desalting using an ion exchange resin, they were freeze-dried and powdered. The weight average molecular weight and the ratio of maltose in solids per unit of β-amylase digest were determined for the powders of the partially decomposed starches 1 to 8 obtained in the same manner as in Experiment 1-1.

The obtained starch partial decomposition products 1 to 8 were dissolved in pure water so as to become a solid concentration of 30% by mass, and 2 units of glycosyl trehalose-generating enzyme used in Experiment 1 were added per 1 g of solid to 50 After allowing it to act for 22 hours at ℃, it was heated at 100 ℃ for 10 minutes to stop the enzyme reaction. Thereafter, it was stored at 4°C, and at the start of storage, after 30 minutes, after 1 hour, after 2 hours, and after 3 days, the aging property was visually evaluated according to the criteria shown below.

"-": No change

"White turbidity": the solution is in a white turbid state due to the formation of insolubles due to aging

"Curing": aging progresses, the solution is in a cured state

In addition, in order to compare the aging inhibition effect caused by the introduction of the trehalose structure at the reduced end, the same treatment was performed for those who treated amylopectin 1 to 4 in the same way except that no glycosyl trehalose-producing enzyme was added. . The results are shown in Table 5.

As is clear from Table 5, even at a high solids concentration of 30% by mass and a low temperature of 4°C, the glycosyl trehalose-generating enzyme functions to have a weight average molecular weight of 45,300 to 49,800 For starch partial decomposition products 1 to 3 for at least 30 minutes, for starch partial decomposition products 4 with a weight average molecular weight of 40,700 for at least 1 hour, for starch partial decomposition products 5 with a weight average molecular weight of 34,400 for at least 2 hours, In the starch partial decomposition product 6 with a weight average molecular weight of 20,200 for at least 3 days, its aging will be suppressed. On the other hand, for the partial decomposition products 7 and 8 of the starch with a weight average molecular weight of less than 15,000, no matter whether the glycosyl trehalose-producing enzyme works or not, it does not change even if stored for 3 days, and no observation is made. Ageing. In addition, as in Experiment 1-1, the molecular weight distribution was confirmed by size exclusion chromatography. Before and after the action of the glycosyl trehalose-producing enzyme, there was no observation on the molecular weight distribution of starch partial decomposition products 1 to 8. To a big change. In addition, it was confirmed that the partial decomposition products of starch 1 to 6 whose aging is inhibited by introducing the trehalose structure into the reducing end by the action of the glycosyl trehalose-producing enzyme are solids per unit of β-amylase digest The ratio of maltose is 56.5 to 59.2% by mass.

From these results, it became clear that even partial decomposition products in the mixed portion of amylose and amylopectin like cassava starch differed in degree due to the weight average molecular weight, but by making glycosyl trehalose-producing enzymes play a role Function, introduce trehalose structure at the reducing end, and reduce the aging rate, that is, it can impart aging resistance.

If the insights obtained from experiments 1 to 3 are combined, then The reducing end of the starch is introduced into the trehalose structure, which can inhibit its aging. The amylose with a weight average molecular weight of 3,000 to 5,000 and the amylopectin with a weight average molecular weight of 12,000 to 120,000 have a particularly significant effect, even in this The same effect is also achieved in the mixture. From the above, the entire starchy system can be tested. The glucosyl trehalose-generating enzyme acts in the aging starch with a weight average molecular weight of 3,000 to 120,000, and the trehalose structure is introduced at its reducing end, which can be significantly suppressed. Ageing.

In addition, in Experiments 1 to 3, each of amylose 1 and 2, amylopectin 1 to 3, and starch partial decomposition products 1 to 6 due to the aging inhibitory effect due to the introduction of trehalose structure at the reducing end were significantly observed The ratio of maltose in the solids per unit of β-amylase digest is 32.7 to 91.0% by mass, therefore, it can be speculated that as long as the weight-average molecular weight is 3,000 to 120,000, and the solids per unit of β-amylase digest The proportion of maltose is 30% by mass or more. Specifically, it shows that the starch is 30% by mass or more and 100% by mass or less. Due to the introduction of a trehalose structure at the reducing end, aging can be particularly significantly suppressed.

<Experiment 4: Analysis of introduction rate of trehalose structure>

In Experiments 2 and 3, the pull-in starches 1 to 3 and the starch partial decomposition products 1 to 6 in which aging inhibition caused the glycosyl trehalose-generating enzyme to function significantly were observed, and the introduction rate of trehalose structure was investigated.

First, pullulan 1 to 3 and partial starch decomposed products 1 to 6 functioning as a glycosyl trehalose-producing enzyme to become a solid concentration 1% by mass dissolved in deionized pure water, and supplied to size exclusion chromatography, and based on the Pullulan standard for molecular weight determination (Pullulan) standard product (manufactured by Linyuan Co., Ltd.) The calibration curve is made to calculate the respective number average molecular weight. In addition, the size exclusion chromatography system is used when the column is connected with two "TSK GEL α-M" (manufactured by TOSOH Co., Ltd.), and 10 mM phosphate buffer (pH 7.0) is used for the eluate. The temperature was 40°C and the flow rate was 0.3 ml/min. The detection was performed using a differential refractometer "RID-10A" (manufactured by Shimadzu Corporation).

Next, in a solution having a solid content concentration of 1% by mass of amylopectin 1 to 3 and starch partial decomposition products 1 to 6 functioning as a glycosyl trehalose-producing enzyme, 50 units of glucoamylase per 1 g of solid content ( Trade name "Glucozyme # 20000", manufactured by Nagase chemtex), pH 5.0, after acting at 50°C for 20 hours, heating at 100°C for 10 minutes to stop the enzyme reaction. Then, the obtained glucoamylase digest is analyzed by high-speed liquid chromatography, and the ratio of the peak area of trehalose to the total peak area of the chromatogram is obtained and used as the solid matter per unit of glucoamylase digest The proportion of maltose. In addition, the high-speed liquid chromatography system uses two "MCI gel CK04SS" (manufactured by Mitsubishi Chemical Co., Ltd.) connected as a column, and uses ultrapure water in the eluate at a column temperature of 80°C and a flow rate of 0.4ml/ The conditions are divided, and the detection is performed using a differential refractometer "RID-10A" (manufactured by Shimadzu Corporation).

Next, the introduction of the trehalose structure is obtained according to the aforementioned formula 1 rate. That is, the ratio of the number average molecular weight of the partial decomposition product of starch due to the action of glycosyl trehalose-producing enzymes multiplied by the mass of trehalose in the solids per unit of glucose amylase digest, and divided by the algae The molecular weight of sugar is 342, and the introduction rate of trehalose structure is determined. The results are shown in Table 6.

As is clear from Table 6, in experiments 2 and 3, the trehalose structure introduction rate of amylopectin 1 to 3 and partial starch decomposed products 1 to 6 in which aging-suppressed glycosyl trehalose-generating enzymes function was significantly observed. 41 to 98%. These results prove that as long as the introduction rate of the trehalose structure is at least 20% by mass or more, it is the result of Experiment 1-2 that can sufficiently suppress aging.

Although the following examples illustrate the present invention in further detail, the technical scope of the present invention should not be affected by such examples. Limited interpretation.

[Example 1] <Manufacture of partial decomposition products of starch containing amylose with trehalose structure introduced at the reducing end>

In the partial decomposition of starch, glycosyl trehalose-generating enzymes and starch debranching enzymes are made to act, and a partial decomposition product of starch containing amylose having a trehalose structure introduced at the reducing end is produced. The details are described below.

After the powders of starch partial decomposition products 6 to 8 prepared in Experiment 3 were dissolved in pure water so as to have a solid concentration of 30% by mass, the pH was adjusted to 5.8, and 2 units of Japan were added per 1 g of solid. Glycosyl trehalose-producing enzyme derived from Arthrobacter sp. S34 obtained by the method described in Example 2-2 of Patent No. 3956884, and 400 units of isoamylase (made by Linyuan Co., Ltd.) per 1 g of solid matter At 50°C for 48 hours. By heating the obtained reaction solution at 100° C. for 10 minutes, the enzyme reaction was stopped, and then, as a purification step, decolorization by activated carbon and desalination by ion exchange resin were performed. Then, the resulting solution was freeze-dried to obtain powders containing partially decomposed products A to C of amylose with a trehalose structure introduced at the reducing end. In addition, in the above purification step, the partial decomposition products A to C of amylose-containing starch will not age without dissolving, so it is not necessary to recover the precipitate, but directly in solution To be purified. With respect to the obtained amylose A to C containing amylose having a trehalose structure introduced at the reducing end, the weight average molecular weight was determined in the same manner as in Experiment 1-1. The results are shown in Table 7.

As shown in Table 7, the obtained starch partial decomposition products A to C containing amylose having a trehalose structure introduced at the reducing end are those having a weight-average molecular weight of 2,350 to 5,020 and having remarkable aging resistance. In addition, the partial decomposition products A to C of amylose containing trehalose structure introduced at the reducing end are as described above, and will not age without dissolving. Therefore, in the purification step, there is no need to go through the trouble of recovering the precipitate Compared with the normal amylose, it can be produced in a very simple way.

The powder obtained in this way contains starch partial decomposition products A to C of the amylose structure with trehalose introduced at the reducing end. When dissolved in water, it is less likely to age than normal amylose, so there is no cause of aging It can be used without any doubt about insolubilization, and can be suitably used as an insoluble or poorly water-soluble drug or a functional food ingredient, oil, coloring, flavoring, etc. compounds, solubilizers or stabilizers, masking agents, sustained release agents, powders化Substrate. Also, as a substitute for the usual amylose, or It is not limited to mixing with normal amylose to utilize.

[Example 2] <Manufacturing of a partial decomposition product of starch containing starch with a trehalose structure introduced at the reducing end>

After dissolving the powder of starch partial decomposition product 6 prepared in Experiment 3 in pure water so as to have a solid concentration of 30% by mass, the pH was adjusted to 5.8, and 2 units of Japanese Patent No. 3956884 were added per 1 g of solid. The glycosyl trehalose-producing enzyme derived from Arthrobacter sp. S34 obtained by the method described in Example 2-2 of Gazette was allowed to act at 50°C for 48 hours. By heating the obtained reaction solution at 100° C. for 10 minutes, the enzyme reaction was stopped, and then, as a purification step, decolorization by activated carbon and desalination by ion exchange resin were performed. Then, the obtained solution was lyophilized to obtain a powder containing a starch partial decomposition product of a starchy structure having a trehalose structure introduced at the reducing end and having a weight average molecular weight of 20,000. In addition, in the above purification step, the partial decomposition products A to C of the amylose-containing starch are not aged and do not dissolve. Therefore, it is possible to directly purify the solution without recovering the precipitate. The powder of the starch partial decomposition product obtained as described above is introduced into the trehalose structure at the reducing end, so the aging property is remarkably reduced, and it is used as a substitute for the starch partial decomposition product or mixed with the usual starch partial decomposition product for utilization Nothing is limited.

[Example 3] <Aging inhibitor of starch>

The glycosyl trehalose-producing enzyme derived from Arthrobacter sp. S34 obtained by the method described in Example 2-2 of Japanese Patent No. 3956884 was dialyzed against a 2% by mass trehalose aqueous solution, and then concentrated to a protein concentration After 4 mg/ml, freeze-drying was performed to obtain a powder of glycosyl trehalose-producing enzyme. This product has an enzyme activity of about 10,000 units per 1g, and can be suitably used as an aging inhibitor for starch.

[Example 4] <cake>

After 500 parts by mass of glutinous rice flour and 500 parts by mass of Japanese rice flour were uniformly mixed, 700 parts by mass of water was added for mixing, and steam was steamed for 40 minutes. Next, the steamed material was mixed with a mixer (ACM20LVW, Aikosha Co., Ltd.) to make the dough about 55°C, and then 360 parts by mass of sucrose and trehalose (registered trademark "TREHA", Linyuan Co., Ltd.) were added. (Commercial sales) 240 parts by mass, and 10 parts by mass of the aging inhibitor obtained in Example 3, and then further mixed for 3 minutes, put into a plastic container with an inner diameter of 60 mm and a height of 22 mm, formed, cooled and stored . This product has a trehalose structure introduced into the reducing end of starch by the action of glycosyl trehalose-generating enzyme as an active ingredient of aging inhibitors, so aging will be suppressed The softness is continuous and extended, and it is a high-quality cake with a good taste.

[Example 5] <Hagi Cake>

Dissolve 350 parts by mass of maltose (registered trademark "SUNMALT", sold by Linyuan Commercial Co., Ltd.) and 150 parts by mass of trehalose (registered trademark "TREHA", sold by Linyuan Commercial Co., Ltd.) in warm water to prepare a concentration of 70 Sugar solution with mass% and keep warm at 55℃. Next, 1000 parts by mass of glutinous rice previously immersed in water was steamed in a steamer by a conventional method and cooled to 55° C., then 500 parts by mass of the aforementioned sugar solution and 5 parts by mass of the aging inhibitor obtained in Example 3 were uniformly stirred. Put it in a heat preservation container for about 1 hour, keep it at 45~50℃, take it out, and use the bean paste to prepare the Hagi cake. This product is introduced with a trehalose structure at the reducing end of starch by the action of glycosyl trehalose-generating enzyme as an active ingredient of aging inhibitors, therefore, aging will be suppressed even if it is thawed after refrigeration or cryopreservation There is also no occurrence of water separation, etc., but it is a high-quality Hagi cake whose softness can be maintained immediately after preparation.

[Industry availability]

As described above, according to the present invention, the glycosyl trehalose-generating enzyme is activated in the starch with aging properties, and the trehalose structure is introduced at the reducing end without significantly changing the structure of the starch or the overall molecular weight distribution. , And can inhibit its aging. The introduction of the present invention has The starch of the trehalose structure is a starch with reduced aging, and can be easily replaced with the existing starch. In addition, the starch obtained by introducing the trehalose structure obtained in the present invention has a reduced aging property, so that the utilization of the starch can be expanded to a product in which the utilization of the starch is restricted due to the aging. The present invention is truly an invention that has greatly contributed to this field.

Claims (8)

A method for inhibiting starch aging, which is characterized in that glycosyl trehalose-generating enzymes function in aging starch, and a trehalose structure is introduced at the reducing end of the starch. The method for inhibiting aging according to claim 1, wherein the weight average molecular weight of the aforementioned starch with aging property is 3,000 to 120,000. The method for suppressing aging according to claim 1 or 2, wherein the starch with aging properties is maltose of 30% by mass or more per unit digest of solids by digestion with β-amylase. The method for inhibiting aging according to claim 1 or 2, wherein the glycosyl trehalose-producing enzymes are derived from the genera Actinoplanes, Arthrobacter, Bradyrhizobium, and Brevibacterium, Corynebacterium, Curtobacterium, Flavobacterium, Gryllotalpicola, Metallosphaera, Micrococcus, One selected from Mycobacterium, Rhizobium, Sulfolobus, Terrabacter, Anabaena, and Nostoc Enzymes of microorganisms. The method for suppressing aging according to claim 1 or 2 is to introduce a trehalose structure at the reducing end of the aging starch with the introduction rate of trehalose structure of 20% or more. A method for producing starch with lower aging properties than before introduction of a trehalose structure, which is characterized in that it includes a step of introducing trehalose structure into a reducing end by introducing a glycosyl trehalose-generating enzyme into the aging starch And a step of purifying the starch obtained by introducing a trehalose structure into the reducing end. The step of purifying does not include the step of recovering the starchy precipitate. The manufacturing method according to claim 6, wherein the weight-average molecular weight of the aging starch is 3,000 to 120,000. The manufacturing method according to claim 6 or 7, wherein the aging starch is maltose produced by the digestion with β-amylase to produce 30% by mass or more of solids per unit digest.