JP3109841B2 - Process for producing maltohexaose and maltoheptaose-containing starch sugar - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to maltohexaose,
The present invention relates to a method for producing starch sugar containing maltoheptaose at a high content.

[0002]

2. Description of the Related Art In recent years, with the improvement of eating habits, demand for foods that are not too sweet is increasing. Conventionally, relatively high molecular weight starch sugars such as various dextrins have been used to suppress sweetness. However, when dextrin is added to foods, there is a problem that powdery odor is present, solubility is poor, viscosity of the product is increased, and physical properties are deteriorated.

Hitherto, various types of amylase have been applied to starch to produce starch sugars such as starch syrup consisting of glucose, isomerized sugar, maltose, cyclodextrin and high molecular weight dextrin on an industrial scale.

In recent years, oligosaccharides located between these monosaccharides and disaccharides and high molecular weight dextrins have attracted attention as low-sweet starch sugars, and some studies have been reported. For example, "Starch Science" (Keinum Kainuma, Vol. 28, p. 9)
2, 1981) report various amylases that specifically produce trisaccharide to hexasaccharide oligosaccharides.

[0005] In addition, "Starch Science" (Kanaya Kenichi et al., 24th edition)
Vol., P. 42, 1977) shows that malt α-amylase contains maltohexaose at an early stage of the saccharification reaction at a low substrate concentration.
It is reported to accumulate maltoheptaose.

[0006]

As shown in the report by Keiji Kainuma, various amylases that specifically produce maltooligosaccharides are known, but specifically produce oligosaccharides of maltoheptaose or more. The enzyme has not yet been discovered. In addition, although maltohexaose-forming amylase has been reported as one of the maltooligosaccharide-forming amylase, there is a problem that the enzyme producing ability is low and the enzyme cost is high.

Also, in the report of Kenichi Kanaya et al., Maltohexaose and maltoheptaose are not the final products of the enzymatic reaction but are reaction intermediate products. Decomposed into sugar. In addition, there is a problem that when the concentration of the substrate is increased to a production condition that is practically cost-effective, those reaction intermediate products are decomposed more quickly into low-polymerized sugars. Furthermore, in the actual production process, when a starch liquefaction liquid as a substrate is charged into a saccharification tank, it is continuously performed over several hours while adding an enzyme, so that the liquid charged first and the liquid charged last are added. In such a case, since the saccharification reaction time varies, it is difficult to control and stop the enzymatic reaction at the beginning of the reaction.

As described above, at present, a method for producing starch sugar having a high content of maltohexaose and maltoheptaose has not been established.

Therefore, an object of the present invention is to provide a low sweetness
It is an object of the present invention to provide a method for producing starch sugar having a higher content of maltohexaose and maltoheptaose as starch sugars having a lower viscosity than dextrin.

[0010]

In general, when starch is hydrolyzed by the action of enzymes such as glucoamylase, β-amylase and exo-type oligosaccharide-forming amylase, which are exo-type enzymes, these enzymes are used. Acts on the starch from the non-reducing end to break the α-1,4-glucosidic bond, so that the hydrolysis reaction stops at the branch where the α-1,6-glucosidic bond exists. Or slow. In this case, α-1,6- is added by adding a debranching enzyme.
It is known that cleavage of the glucosidic bond eliminates the obstacle and increases the yield of the target hydrolyzate.
However, in this case, the amount of addition of the debranching enzyme may be the minimum necessary.If added excessively, the amylose which has been cut and becomes linear may age or a reverse synthesis reaction by the debranching enzyme may occur. On the contrary, the yield of the target hydrolyzate is reduced.

On the other hand, it is known that maltohexaose accumulates about 30% when starch is hydrolyzed by the action of α-amylase derived from Bacillus subtilis, which is an endo-type enzyme. Acts randomly from inside the starch molecule, so that the addition of the branching enzyme has no remarkable effect, and the production of low-molecular sugars such as glucose and maltose increases.

However, the present inventors have conducted intensive studies on a method for producing a starch sugar containing maltohexaose and maltoheptaose at a high content in order to achieve the above object. As a result, the starch was converted to malt α-amylase. In the case of hydrolysis, the presence of the debranching enzyme in a predetermined activity unit or more ensures that the maltohexaose and maltoheptaose contents are not significantly reduced even when the reaction is continued for a long time, and that the malthexaose and maltoheptaose contents are notably reduced. The present inventors have discovered a novel fact that maltohexaose and maltoheptaose can be obtained in higher yields by accumulating and accumulating a large excess of the debranching enzyme. It was completed.

That is, maltohexaose of the present invention,
The method for producing maltoheptaose-containing starch sugar is such that, when hydrolyzing starch or starch-based saccharide with malt α-amylase, a debranching enzyme composed of pullulanase and / or isoamylase is present in a predetermined activity unit or more. Features.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments.

In the present invention, the starch or starch-based saccharide used as a raw material is not particularly limited, and various ones can be used. For example, potato starch, sweet potato starch, corn starch, waxy corn starch, cassava starch and the like are preferably used. Can be Alternatively, a liquefied starch solution obtained by liquefying these may be used. The liquefied starch liquid can be prepared according to a conventional method. For example, it can be prepared by heating and gelatinizing starch and then adding α-amylase.
The liquefied starch solution has a concentration of 10 to 35 W / W% and a glucose equivalent of 2.
To 10 are preferably used.

In the present invention, starch or starch-based saccharide is hydrolyzed with malt α-amylase in the presence of a debranching enzyme.

It is preferable to use 0.5 to 50 units of malt α-amylase per 1 g of starch or starch-based saccharide. The unit of α-amylase in malt in the present invention is a unit measured by using a neoamylase test (trade name, manufactured by Daiichi Pure Chemicals Co., Ltd.), and the measuring method will be described later.

The debranching enzyme is an enzyme that cleaves the α-1,6-glucoside bond in starch, and pullulanase and / or isoamylase are preferably used. The origin of these enzymes is not particularly limited, and for example, pullulanase (H. Bender and K. Wallenfels, Biochemical) derived from Klebsiella pnumoniae
J., 334, 79 (1961)), an isoamylase of Pseudonas amyloderamosa origin (T. Harada et al. Appl. Microbiol., 16, 1439 (1968)).
And the like). Among these, pullulanase is more preferable in consideration of the optimum pH of malt α-amylase.

The amount of the debranching enzyme to be added is 0.1% in the case of pullulanase per unit of malt α-amylase in terms of activity ratio.
225 units or more are preferable, and in the case of isoamylase, 22.5 units or more are preferably used. When pullulanase and isoamylase are used in combination, the amount is preferably such that the total of the value obtained by multiplying the activity unit of pullulanase by 100 with respect to 1 unit of malt α-amylase and the activity unit of isoamylase is 22.5 or more. .

The activity unit of pullulanase is the amount of enzyme that produces a reducing power of 1 μmol of glucose equivalent per minute under the conditions of pH 6.0 and 40 ° C. using pullulan as a substrate. The activity unit of isoamylase is , Biochem.Biophys.Ac
ta, 212, 458-469 (1970). These measuring methods will be described later.

[0021] When hydrolyzing starch or starch-based saccharide with malt α-amylase in the presence of a debranching enzyme,
The H and temperature conditions are pH 4.5-6.5 and temperature 40-70 ° C. in order to maintain the activity of malt α-amylase and branching enzyme.
Is preferred.

The enzyme reaction time varies depending on the amount of malt α-amylase added, but is usually preferably 3 to 72 hours.

After the enzymatic reaction is completed as described above, the enzyme is deactivated by heating to 80 ° C. or higher, decolorized with activated carbon according to a conventional method, desalted through an ion exchange resin column, and concentrated. Thus, a starch sugar solution containing maltohexaose and maltoheptaose at high contents can be obtained.

[0024]

In the present invention, the reason is evident by simultaneously reacting malt α-amylase and a debranching enzyme consisting of pullulanase and / or isoamylase having a predetermined activity unit or more on starch or starch-based carbohydrate. However, as shown in the data of Examples described later, even if the reaction is continued for a long time, the content of maltohexaose and maltoheptaose is remarkably generated without decreasing, and stably accumulates. Moreover, as the amount of the branching enzyme added increases, maltohexaose and maltoheptaose accumulate at higher contents. Therefore, maltohexaose,
A starch sugar containing maltoheptaose at a high content can be obtained.

FIG. 1 shows the relative sweetness of various maltooligosaccharides with the sugar being 100. FIG.
In, "G1" is glucose, "G2" is maltose, "G3"
Maltotriose, "G4" is maltotetraose, "G5" is maltopentaose, "G6" is maltohexaose, "G7"
Indicates maltoheptaose. As can be seen from FIG.
Maltoheptaose (G7) has a lower sweetness than monosaccharides and disaccharides.

FIG. 2 shows the relationship between the concentration of various maltooligosaccharides and the intrinsic viscosity. In FIG. 2,
"G1" to "G2" indicate the same maltooligosaccharide as described above, and "G8"
Is maltooctaose, "G9" is maltononaose, "G1
"0" indicates maltodecaose. As can be seen from FIG. 2, maltohexaose (G6), maltoheptaose (G7)
Has a lower viscosity than sugars having a degree of polymerization of maltooctaose (G8) or higher.

Therefore, starch starch containing maltohexaose and maltoheptaose at a high content has a low sweetness,
It can be used as low viscosity starch sugar. That is, by using the starch sugar of the present invention, foods that have been desired in recent dietary habits and are not too sweet can be obtained without impairing the physical properties of the product.

[0028]

EXAMPLES In the present invention, the activities of malt α-amylase and branching enzyme were measured by the following methods.

(1) Method for measuring malt α-amylase activity Malt α-amylase activity was determined using “Neo-amylase test” (trade name, manufactured by Daiichi Pure Chemicals Co., Ltd.) as follows. It was measured.

In a test tube, take 100 μl of the sample, and add 4 ml of distilled water.
And warm in a thermostat at 37 ° C for 5 minutes.

Next, 45 mg of blue starch and bovine serum albumin
One tablet containing 3 mg is added and immediately mixed vigorously with a mixer for about 10 seconds until the tablet disintegrates.

Thereafter, the mixture was heated in a constant temperature bath at 37 ° C. for 30 minutes to carry out the reaction, and immediately thereafter, 0.5N sodium hydroxide reagent 1.
Add 0 ml and mix vigorously to stop the reaction.

The reaction solution is centrifuged or filtered to obtain a supernatant or a filtrate, and the absorbance is measured at a wavelength of 620 nm. The obtained value was converted to "Fadebas Humylase H" with known activity.
Is converted to activity units (IU) using the calibration curve prepared by the above.

(2) Method of Measuring the Activity of Pullulanase Using pullulan as a substrate, the amount of an enzyme that produces a reducing power of 1 μmol of glucose equivalent per minute under the conditions of pH 6.0 and 40 ° C. is defined as one unit.

(3) Method for measuring the activity of isoamylase The activity of isoamylase was determined by the method described in (Biochem. Biophys. Acta, 21).
2, 458-469 (1979)).

That is, 0.5 ml of 1% soluble glutinous rice starch
0.1 ml of 0.5 M acetate buffer and 0.1 ml of enzyme solution at 40 ° C.
And react for 60 minutes. 0.5 ml of the obtained reaction solution was collected,
Add 0.5 ml of 0.01 M iodine-0.1 M potassium iodide solution,
After standing at room temperature for 15 minutes, dilute with water to 12.5 ml.
Measure the absorbance at 610 nm in a cm-wide cell.

Using the inactivated enzyme, the same measurement as above is performed to make a blank, and the amount of the enzyme that changes the absorbance by 0.1 for 60 minutes is defined as one unit.

Example 1 (Measurement of change over time in maltohexaose and maltoheptaose contents when pullulanase was used as a branching enzyme) Corn starch was liquefied using α-amylase by a conventional method, and the concentration was 25 W / W. %, A liquefied liquid having a glucose equivalent of 8 was obtained.

To this liquefied liquid, malt α-amylase was added
2 units were added to 1 g of starch, and pullulanase (manufactured by Amano Pharmaceutical Co., Ltd.) was added in an amount of 0, 0.45,
It was added in 4.0 units and hydrolyzed under conditions of pH 6.0 and temperature of 55 ° C. for 41 hours to saccharify.

At 17 hours, 24 hours and 41 hours after the start of the reaction, the respective sugar compositions were measured. The sugar composition is
Aminex (Aminex)
nex) Analysis was performed using an HPX-42A column (manufactured by Bio-Rad).

Table 1 shows the results. In the table, each result is indicated as follows. In the following examples, “G1” to “G7” indicate various maltooligosaccharides as described above, and “G8 ≦” indicates a high molecular dextrin of maltooctaose or higher.

[0042]

[Table 1]

From the results shown in Table 1, when pullulanase was not added, maltohexaose (G6) produced at the beginning of the reaction,
Maltoheptaose (G7) is an intermediate product of the reaction, which is decomposed with the lapse of reaction time.After 41 hours, it decreases to 24.2%, and high-molecular-weight dextrin of maltooctaose (G8) or more is 36% or more. It turns out that it remains without decomposing.

On the other hand, when pullulanase was added, the content of maltohexaose and maltoheptaose in the initial stage of the reaction was maintained after 41 hours of reaction, and
It can be seen that when the unit is added, it is about 10% higher than when no unit is added. Further, when pullulanase was added, the amount of high molecular dextrin of maloctaose or more was reduced.

Therefore, pullulanase can be used for malt α-
By adding 0.45 units to 2 units of amylase, that is, 0.225 units to 1 unit of malt α-amylase,
It can be seen that the maltohexaose and maltoheptaose contents can be stably kept constant.

Further, it can be seen that the effect is larger when adding 4.0 units, which is almost 10 times that of adding 0.45 unit of pullulanase, and the effect is larger when the amount of the added pullulanase is larger. This result indicates that the conventional amount of addition of the debranching enzyme is preferably the minimum necessary.If a large amount is added, aging of amylose or reverse synthesis reaction may occur, and the yield of the desired final product may decrease. It is a new fact that is different from the fact that there is.

Example 2 (Measurement of Time-Dependent Changes in Maltohexaose and Maltoheptaose Contents when Isoamylase is Used as a Branching Enzyme) Potato starch was liquefied using α-amylase by a conventional method and the concentration was 20 W A liquefied liquid having a glucose equivalent of 3 / W% was obtained.

To this liquefied liquid, malt α-amylase was added
20 units were added to 1 g of starch, and isoamylase (manufactured by Hayashibara) was added in an amount of 0, 450, 40 to 1 g of starch.
Changed to 00 units and added at pH 5.5 and 55 ° C.
It was saccharified by hydrolysis for 4.5 hours.

At 1.5 hours, 2.4 hours, and 4.5 hours after the start of the reaction, the respective sugar compositions were measured. The sugar composition analysis was performed in the same manner as in Example 1. Table 2 shows the results.

[0050]

[Table 2]

From the results shown in Table 2, it was found that the case where no isoamylase was added was similar to the case where no pullulanase was added in Example 1.
It can be seen that maltohexaose and maltoheptaose generated in the initial stage of the reaction are rapidly decomposed and reduced with the elapse of the reaction time, but high-molecular-weight dextrin of maltooctaose or more remains without being decomposed.

On the other hand, when isoamylase is added, maltohexaose and maltoheptaose are produced stably, and their contents do not decrease even after long-term reaction. It can be seen that only the number decreases.

In the case of isoamylase, at least 450 units of isoamylase are used for 20 units of malt α-amylase.
That is, it is found that the content of maltohexaose and maltoheptaose can be stably maintained at a constant level by adding 22.5 units or more of isoamylase to 1 unit of malt α-amylase.

Further, it can be seen that the effect can be enhanced by increasing the amount of isoamylase added, in sympathy with the pullulanase of Example 1.

Example 3 (Measurement of change with time in maltohexaose and maltoheptaose contents when pullulanase and isoamylase are used in combination as branching enzymes) Corn starch was liquefied using α-amylase by a conventional method. A liquefied liquid having a concentration of 25 W / W% and a glucose equivalent of 6 was obtained.

To this liquefied liquid, malt α-amylase was added
Two units were added to 1 g of starch, and pullulanase (manufactured by Amano Pharmaceutical Co., Ltd.) and isoamylase (manufactured by Hayashibara Co., Ltd.) were added in combination. The amounts of addition were 0.225 units of pullulanase and 22.5 units of isoamylase, and 2.0 units of pullulanase and 20 units of isoamylase per gram of starch.
The system was used in combination with 0 unit, and the reaction was carried out in a system to which neither was added.

The mixture was hydrolyzed under conditions of pH 5.5 and temperature of 55 ° C. for 41 hours to saccharify, and the sugar composition was measured in the same manner as in Example 1. Table 3 shows the results.

[0058]

[Table 3]

From the results shown in Table 3, when no debranching enzyme was added, as in Examples 1 and 2, maltohexaose and maltoheptaose produced at the beginning of the reaction were rapidly decomposed with the lapse of reaction time. It can be seen that the high molecular weight dextrin of maltoocta or higher remains without being decomposed.

On the other hand, when pullulanase and isoamylase are added, maltohexaose and maltoheptaose are produced stably, and their contents do not decrease even after long-term reaction. It can be seen that only molecular dextrin is reduced. The debranching enzyme is 0.225 units of pullulanase and 22.5 units of isoamylase for 2 units of malt α-amylase, that is,
1 unit of malt α-amylase, 0.2% pullulanase.
By adding 1125 units and 11.25 units of isoamylase, the effect can be sufficiently exhibited, and it can be seen that the effect is further enhanced by increasing the amount of addition.

Further, when the amounts of the debranching enzymes for keeping the maltohexaose and maltoheptaose contents of Examples 1 and 2 stable and constant were examined, the maltohexaose and maltoheptaose contents were found to be stable. It can be seen that the amount of debranching enzyme kept constant is approximately one unit of pullulanase corresponds to 100 units of isoamylase. Therefore,
When both are used in combination as a debranching enzyme, the activity unit of pullulanase is 100 units per unit of α-amylase of malt.
By adding so that the sum of the doubled value and the activity unit of isoamylase becomes 22.5 or more, the maltohexaose and maltoheptaose contents are stably maintained regardless of the addition ratio of pullulanase and isoamylase. We can see that we can do it.

Example 4 (Production of starch syrup having high maltohexaose and maltoheptaose contents) Corn starch was liquefied using α-amylase by a conventional method, and a liquefied liquid having a concentration of 25 W / W% and a glucose equivalent of 7 was obtained. Obtained.

After 4 l of this liquefied liquid was adjusted to pH 6.0, 2 units of malt α-amylase were added per 1 g of starch.
4 units of pullulanase (manufactured by Amano Pharmaceutical Co., Ltd.)
It was hydrolyzed at 55 ° C. for 41 hours to cause a saccharification reaction.

After the completion of the reaction, the solution was heated to 80 ° C. and heat-treated for 30 minutes to inactivate the enzyme. Purification was carried out using activated carbon and an ion-exchange resin by a conventional method, and the mixture was concentrated to a concentration of 71 W / W%. 1830 g of starch sugar syrup was obtained.

The sweetness of this starch syrup is determined by the sweetness of sugar.
The relative sweetness was evaluated to be 100, and the result was 20, indicating that a low-sweet syrup of starch syrup could be obtained.

Example 5 The sugar composition, viscosity, and powder of the starch sugar starch syrup (G6 and G7-containing starch syrup) having a high content of maltohexaose and maltoheptaose obtained in Example 4 and dextrin having the same glucose equivalent. The odors were compared.

The sugar composition was analyzed by high performance liquid chromatography in the same manner as in Example 1. Table 4 shows the results.

[0068]

[Table 4]

From the results shown in Table 4, G6 prepared in Example 4
The G7-containing starch syrup has a higher maltohexaose and maltoheptaose content than dextrin, indicating that the sugar composition is more suitable for obtaining desired properties.

The viscosity of a 30 W / W% solution was measured at 20 ° C.
It measured using the B-type viscometer. Table 5 shows the results and the presence or absence of powder odor.

[0071]

[Table 5]

From the results shown in Table 5, it can be seen that the syrup containing G6 and G7 has a lower viscosity and no powdery odor as compared with dextrin having the same glucose equivalent.

[0073]

As described above, according to the present invention,
It is possible to produce low-malt, low-viscosity starch sugars with high maltohexaose and maltoheptaose contents. Therefore, it can be used as a starch sugar that can meet the demand for foods that are not too sweet in recent dietary habits without impairing the physical properties of the product.

[Brief description of the drawings]

FIG. 1 is a table showing the relative sweetness of maltooligosaccharides (G1 to G7) with respect to sugar.

FIG. 2 is a chart showing the relationship between the concentration of maltooligosaccharides (G1 to G10) and the intrinsic viscosity.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Takehiro Umino 2954 Imaizumi, Fuji-shi, Shizuoka Prefecture 2-201, Nisshinkinomiya Company House (72) Inventor Masahiro Arima 3 Kirin Brewery, Miyahara-cho, Takasaki-shi, Gunma Pharmaceutical Development Inside the laboratory (72) Inventor Kozo Tanabe 3 Kirin Brewery, Miyahara-cho, Takasaki City, Gunma Prefecture Inside the Pharmaceutical Development Laboratory (72) Inventor Katsuhiko Yamada 6-26-1, Jingumae, Shibuya-ku, Tokyo Inside Kirin Brewery Co., Ltd. (56) References JP-A-63-74489 (JP, A) JP-A-1-218598 (JP, A) JP-A-63-42696 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name ) C12P 19/00-19/16 A23L 1/09 BIOSIS (DIALOG)

Claims (4)

(57) [Claims] 1. The method of claim 1 wherein starch or starch-based saccharide is converted to α-malt of malt.
In the process for producing maltohexaose and maltoheptaose-containing starch sugars, which are reacted in the presence of a debranching enzyme when hydrolyzing with amylase, the malt α-maltose is expressed by the activity ratio α-
A method for producing maltohexaose and maltoheptaose-containing starch sugar, wherein at least 0.225 unit of pullulanase is used as the branching enzyme per unit of amylase. 2. The method of claim 1 wherein the starch or starch-based saccharide is converted to malt α-
In the process for producing maltohexaose and maltoheptaose-containing starch sugars, which are reacted in the presence of a debranching enzyme when hydrolyzing with amylase, the malt α-maltose is expressed by the activity ratio α-
A method for producing maltohexaose and maltoheptaose-containing starch sugar, wherein 22.5 units or more of isoamylase is used as the branching enzyme per unit of amylase. 3. The method of claim 1 wherein the starch or starch-based saccharide is converted to α-malt of malt.
In the process for producing maltohexaose and maltoheptaose-containing starch sugars, which are reacted in the presence of a debranching enzyme when hydrolyzing with amylase, the malt α-maltose is expressed by the activity ratio α-
Maltohexaose used in an amount such that the total of the activity unit of the isoamylase and the value obtained by multiplying the activity unit of the pullulanase by 100 times the activity unit of the pullulanase with respect to 1 unit of the amylase is 100. A process for producing maltoheptaose-containing starch sugar. 4. The method according to claim 1, wherein the hydrolysis is carried out at pH 4.5 to 6.5,
The method for producing maltohexaose and maltoheptaose-containing starch sugar according to any one of claims 1 to 3, which is carried out at a temperature of 40 to 70 ° C.