JPH0984594A - Production of panose liquid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently produce the subject glucide liquid useful for a sweetener, etc., capable of suppressing the dental caries by reacting a saccharified liquid with a high maltose content with an α-glucosidase, then reacting a glucoamylase therewith and treating the resultant reactional liquid with an ion exchange chromatography. SOLUTION: A saccharified liquid with a high content of maltose is reacted with an α-glucosidase to make the transfer reaction proceed until the panose content attains 15-35%/ds and a glucoamylase is further added to the first liquid containing the panose to make the hydrolysis of an oligosaccharide comprising 1,4-bonds proceed as much as possible within the range of <=40% apparent hydrolytic degree of the panose. The prepared second liquid containing the panose is then fractionated according to an ion exchange chromatography using an alkali(ne earth) metallic type strong acidic cation exchange resin to thereby readily provide a panose liquid at a high concentration useful as a sweetener, etc., capable of suppressing the dental caries with sugar in the method for producing the panose by fractionating the saccharified liquid containing panose according to the ion exchange chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a panose solution by chromatographically fractionating a panose-containing solution and a method for producing a panose solution and a panose powder which are expected as sweeteners for suppressing caries of sugar. . In particular, it relates to a method of easily obtaining a highly pure panose solution and a panose powder when a panose solution is produced by chromatographically fractionating a panose-containing solution.

As used herein, the term "transfer reaction" refers to a reaction involving a hydrolysis reaction and the formation of α-1,6 bonds and the like.

[0003]

2. Description of the Related Art In processed foods, prevention and reduction of tooth decay are major points of consumer needs.
BACKGROUND ART Sweeteners and food materials that reduce sugar (sucrose) that causes tooth decay and prevent tooth decay without using sugar have been used. Various oligosaccharides are commercially available as sweeteners for preventing tooth decay, but it has been pointed out that when applied to foods as a substitute for sugar, the taste of foods deteriorates. Therefore, there is a demand for the development of a sugar having a taste and physical properties more similar to those of sugar, which prevents tooth decay.

According to Professor Hamada (Osaka University Faculty of Dentistry), branched oligosaccharides having an α-1,6 bond contained in the hydrolysis of starch, particularly panose, do not act as a substrate for acid generation, and It was clarified that the sugar has a strong anti-cariogenic effect ("Microbiol. Immunol" 32 (1), 25-31,1988
), Panose has come to be expected as a sweetener for suppressing caries of sugar.

Panose is produced from an acid partial hydrolyzate of amylopectin, glycogen and pullulan, and in recent years, Thermoactinomyces vulgaris R.
There is a report that the α-amylase produced by -47 specifically produces panose using pullulan as a substrate (see “Ag
ric. Biol. Chem., 42, 1681 (1978) ”).

However, these methods use expensive pullulan as a raw material and the productivity of the pullulan-degrading enzyme produced by this bacterium is low, so that when considering industrial production,
There is a drawback that the cost is high.

Therefore, a panose-containing solution prepared by reacting α-glucosidase with maltose solution which can be easily prepared by adding various maltose-forming amylase (optionally in combination with debranching enzyme) from starch liquefaction solution ,
A method for producing a panose solution by chromatographic fractionation has been proposed (see Japanese Patent Publication No. 5-37037).

[0008]

However, as a result of the studies by the present inventors, the panose-containing liquid for chromatographic fractionation prepared by the above method has a panose content of at most 35% /
In addition to ds, glucose, which is a monosaccharide, contains a large amount of maltose, which is a disaccharide, and oligosaccharides other than other trisaccharides (5% of maltotriose, which is a trisaccharide,
near ds). That is, even if the panose content is increased to 35% / ds or more, the α-1,4 bond of panose is hydrolyzed and the ratio of isomaltose having the α-1,6 bond is increased. It is estimated that this is because of the reason.

When this panose-containing liquid was subjected to chromatographic fractionation, it was difficult to obtain a high-concentration (60% / ds or more) panose liquid, and at most about 50% / ds panose liquid was obtained.

This is because the disaccharides maltose and isomaltose are distributed in glucose compartments and panose compartments in approximately equal amounts, and oligosaccharides of three or more sugars are distributed in the panose compartment (see Table 3).

As a method for separating the panose component from the panose-containing liquid having a panose content of 35% / ds or less, paper chromatography, activated carbon column chromatography, column chromatography utilizing the principle of molecular sieve and the like can be used. However, these separation means are complicated in operation, poor in recovery rate, and difficult to scale up, and cannot be said to be industrially applicable.

In view of the above, an object of the present invention is to provide a method for producing a panose liquid and a panose powder, which makes it possible to easily obtain a high-concentration panose liquid.

[0013]

Means for Solving the Problems Accordingly, the present inventors have:
Diligently studying industrially applicable method for effectively removing glucose and oligosaccharide consisting of α-1,4 bond from a panose-containing solution having a panose content of 15 to 35% / ds to prepare a panose-rich liquid sugar Tried.

Then, a fixed amount of glucoamylase alone, a fixed amount of glucoamylase and α, which does not hydrolyze panose as much as possible into a liquid sugar containing panose of 15 to 35% / ds.
-It was found that oligosaccharides composed of α-1,4 bonds can be effectively hydrolyzed into glucose by the addition of a combined use of amylase and a fixed amount of combined use of glucoamylase and β-amylase, and treatment with these enzymes is performed. By removing the glucose component of the liquid by chromatography using an ion exchange resin, we succeeded in efficiently obtaining a liquid sugar with a high content of panose as shown in FIG. 1, and completed the present invention having the following constitution.

A method for producing a panose liquid by chromatographically fractionating a panose-containing liquid, wherein the maltose liquid is allowed to act with α-glucosidase, and the panose content is 15 to 35% /
To the first panose-containing solution which has undergone the rearrangement reaction to reach ds, glucoamylase or, if necessary, α-amylase or β-amylase is added in combination so that the apparent hydrolysis degree of panose is 40% or less. The second panose-containing liquid in which hydrolysis of the oligosaccharide composed of α-1,4 bonds has proceeded as much as possible is used as a panose-containing liquid for chromatographic fractionation.

[0016]

The action and effect of the invention: The first panose-containing liquid prepared by allowing α-glucosidase to act on maltose liquid has a low panose content of 35% / ds or less, and glucose and α
It contains oligosaccharides consisting of -1,4 bonds and branched oligosaccharides other than panose. Then, this first panose-containing liquid is glucoamylase alone or glucoamylase and α which easily hydrolyze other oligosaccharides containing α-1,4 bond such as maltose and maltotriose as compared with panose. -The second panose-containing liquid obtained by hydrolysis using a combination of amylase or β-amylase is almost fractionated into disaccharides (maltose etc.) and panose fractions which are fractionated in almost half in the panose fraction. The sugar composition ratio of oligosaccharides with three or more sugars (maltotriose, etc.) to panose decreases (see Tables 1 and 2).

Therefore, by chromatographically fractionating the second panose-containing solution, as a result, a panose solution having a panose content of 60% / ds or more, which has been difficult to obtain conventionally, can be easily obtained.

In addition, the second panose-containing liquid reduces the sugar composition ratio of oligosaccharides mainly composed of α-1,4 bonds, which are sugars causing acid generation by dental caries, due to hydrolysis,
The mixture ratio of such oligos in the panose solution after chromatographic fractionation is smaller than that in the first panose solution.

Therefore, when the panose liquid of the present invention is used in foods, it can be expected that the strong anti-cariogenic effect of panose is increased.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below.

(1) The maltose liquid which is the raw material liquid of the present invention has a maltose content of 60% / ds or more (desirably, 7%).
(0% or more), the production method thereof is not particularly limited, and a commercially available maltose solution may be used. Usually, a maltose-forming amylase (eg β-
Amylase, other new amylase, etc.) and a debranching enzyme (for example, prunalase, isoamylase, etc.) can be used in combination.

The kind of the raw material starch is not particularly limited, and any of above-ground stem starch such as corn starch and wheat starch, and subterranean stem starch such as potato starch and tapioca starch can be used.

As β-amylase, any β-amylase can be used regardless of its origin, but commercially available “Biozyme ML” (Amano Pharmaceutical Co., Ltd., 6,000).
0 U / g) and the like. In addition, the activity was displayed by using a 1% / ds soluble starch solution as a substrate,
One unit is the activity of producing 10 mg of glucose at 40 ° C. for 30 minutes.

As the pullulanase, any pullulanase can be used regardless of its origin, and commercially available "Amano" (manufactured by Amano Pharmaceutical Co., Ltd., 900 U / ml) and the like can be mentioned. The activity is indicated by using 0.5% pullulan solution as a substrate and reacting at 40 ° C. for 30 minutes with an enzyme amount of 1 micromolar corresponding to 1 micromole of glucose per minute. Use as a unit.

(2) The first panose-containing solution is prepared by allowing α-glucosidase to act on the maltose-rich solution to advance the transfer reaction until the panose content becomes 15 to 35% / ds. This first panose-containing liquid is a mixed liquid of branched oligosaccharides containing panose as a main component, glucose, and oligosaccharides composed of α-1,4 bonds.

Here, when the panose content is less than 15% / ds, it is difficult to obtain a target panose solution having a high concentration of 60% / ds or more, and when the panose content exceeds 35% / ds,
As mentioned above, it is difficult to obtain industrially.

As the above-mentioned α-glucosidase (transglucosidase), any α-glucosidase can be used regardless of its origin, but those of microbial origin and heat resistance are preferable. For example, the genus Aspergillus, Rh
An enzyme agent originating from the genus izopus can be used, and more specifically, “α-glucosidase L amano” (Amano Pharmaceutical Co., Ltd., 300,000 U / ml) and the like can be used.

The activity is displayed by using methyl-α-D-glucoside as a substrate, and the reaction solution 2.
One unit is the enzymatic force that produces 1 μg of glucose in 5 ml.

At this time, the addition amount of α-glucosidase is 50 to 1500 per 1 g of maltose solid content.
U / g · ds and transition time are inversely proportional to the amount added, and
Although it depends on the temperature, it is 3 to 36 hours.

(2) Next, glucoamylase alone or a combination enzyme of glucoamylase and α-amylase or glucoamylase and β-amylase is allowed to act on the above-mentioned first panose-containing liquid so that the apparent hydrolysis degree of panose is 40%. From the range of not more than%, the oligosaccharide composed of α-1,4 bond is hydrolyzed to glucose as much as possible (isomaltose composed of α-1,6 bond is hardly decomposed).

Here, the apparent hydrolysis degree of panose is 4
If it exceeds 0%, the panose content of the second panose-containing liquid becomes too low, and it is difficult to obtain a high-concentration panose-containing liquid when performing chromatographic fractionation. Possible hydrolysis of oligosaccharides is 1
Although it may be about 0%, it is preferably 60% or more.

As the glucoamylase, any glucoamylase can be used regardless of its origin, but those of microbial origin and thermostable are preferable. For example,
Enzyme agents originating from the genus Aspergillus and the genus Rhizopus can be used, and more specifically, “AMG” (manufactured by Novo Nordisk Bioindustry, 330 U / ml) and the like can be preferably used.

The activity is indicated as 2% / d as a substrate.
s Using soluble starch solution, 1 μg for 60 minutes at 40 ° C
The activity of producing glucose is defined as 1 unit.

As the α-amylase, any α-amylase can be used regardless of its origin, but a commercially available liquefied type “Spitase HS” (produced by Nagase Seikagaku Co., Ltd., 7,000 U / g) etc. can be mentioned. The activity is indicated by using 1% / ds soluble starch solution as a substrate, and 1 unit is the activity of reducing the blue value of 100 mg starch by 1% / ds at 40 ° C. for 1 minute.

As the β-amylase, the same one as the above-mentioned maltose-producing amylase can be used. Although any β-amylase can be used regardless of its origin, the commercially available "Biozyme ML" (manufactured by Amano Pharmaceutical Co., Ltd.,
6,000 U / g) and the like. In addition,
The activity is indicated by using 1% / ds-soluble starch solution as a substrate and the activity of producing 10 mg of glucose in 30 minutes at 40 ° C. as one unit.

Glucoamylase has the property of hydrolyzing panose (Starch Science Handbook, P106).
-Table 5.9) The panose content is reduced by acting on the panose containing solution. Therefore, when a branched oligosaccharide-containing liquid sugar such as panose is produced from starch and maltose, glucoamylase-free α-amylase, β-amylase and α-glucosidase are used. But,
As a result of intensive studies, the present inventors have found that oligosaccharides composed of α-1,4 bonds such as maltose are hydrolyzed to glucose by controlling the action conditions of glucoamylase to minimize the hydrolysis of panose. It succeeded in being able to do and came to complete this invention. Moreover, it was revealed that similar results can be obtained by hydrolysis with a combined enzyme of glucoamylase and α-amylase and β-amylase.

Here, the addition amount of glucoamylase varies depending on the reaction temperature and the hydrolysis time.
℃, hydrolysis time 24 hours 0.0 per 1g of solid content
2 to 0.30 U / g · ds (desirably 0.04 to 0.
23 U / g · ds). The addition amount of α-amylase used in combination is preferably 2 to 20 U / g · ds, and the addition amount of β-amylase is preferably 5 to 50 U / g · ds.

(3) The second panose-containing solution hydrolyzed with an enzyme to prepare a high panose solution is chromatographed using an ion exchange resin. The second panose solution is purified and concentrated by means of activated carbon filtration, an ion-exchange purification device (two-bed one-bed system), etc. during the chromatographic fractionation,
use.

As the ion exchange resin, a strongly acidic cation exchange resin is suitable for fractionating the glucose component and the panose fraction. Strong acid cation exchange resin
This is because it is suitable for fractionating monosaccharides and di- and trisaccharides.

The strong acid cation exchange resin used for the fractionation may be either an alkali metal type strong acid cation exchange resin or an alkaline earth metal type strong acid cation exchange resin, but preferably an alkali metal type strong acid. Cation exchange resin, particularly preferably sodium type strong acid cation exchange resin.

Specific examples thereof include sodium ion strongly acidic cation exchange resins of Diaion FRK series and Diaion "Unibeads" series.

Diaion is a trade name of Mitsubishi Kasei Co., Ltd. The liquid passing temperature to the column is 30 to 90.
C., preferably 50 to 70.degree. Further, the liquid passing rate is preferably in the range of SV = 0.01-0.10.

The separation operation of the present invention will be described in detail with reference to the chromatography apparatus shown in FIG. 2 which comprises four separation columns packed with a sodium-type strongly acidic cation exchange resin.

First, the enzyme treatment liquid (separated raw material liquid) is circulated in the packed bed apparatus from the first tower 11 through the second tower 13, the third tower 15 toward the fourth tower 17, and then the raw material. The liquid is supplied to the third column 15 to make the section flow down, and during this time, the second stage of extracting the solution rich in the panose component flowing out of the section to the outside of the system, and further the eluting water is supplied to the first column 11. To flow down the compartment, and during this time, the third stage of extracting the liquid rich in glucose component flowing out from the compartment to the outside of the system, and supplying the eluting water to the first tower 11 to flow down the compartment, The effluent of 11 flows into the second tower 13, the effluent of the second tower 13 flows into the third tower 15, and the panose-rich solution flowing out of the third tower 13 is withdrawn to the outside of the system. By the separation operation consisting of sequentially repeating the steps, the raw material liquid is Fractionated North-rich fraction and a glucose-rich fraction.

By this separation operation, the panose content is 60%.
It is possible to prepare a high panose solution of / ds or more.

[0046]

EXAMPLES Examples conducted in order to confirm the effects of the present invention will be described below.

Example 1 A cornstarch liquefied solution (DE 6.9, pH 5.5) having a concentration of 25% / ds was added with pullulanase "Amano" (manufactured by Amano Pharmaceutical Co., Ltd.) 0.045% / ds.
β-amylase "Biozyme ML" (manufactured by Amano Pharmaceutical Co., Ltd.)
A maltose solution having a maltose content of 73% / ds was prepared by adding 065% / ds and reacting at 55 ° C. for 24 hours.

Next, α-glucosidase L Amano (manufactured by Amano Pharmaceutical Co., Ltd., 300,000 U /
ml) 100 u / ds was added and the rearrangement reaction was carried out at 55 ° C. for 27 hours to prepare a first panose-containing solution having a panose content of 30.4% / ds.

Then, the liquid containing the first panose was adjusted to pH.
After preparation to 5.0, AMG enzyme which is glucoamylase (Novo Nordisk Industries, 330u /
0.10 U / g.ds was added to the reaction mixture, and hydrolysis was carried out at 55 ° C. for 24 hours. The hydrolyzed liquid is activated carbon filtered,
The second panose-containing liquid for chromatographic separation was obtained by purifying and concentrating with an ion-exchange purifier (two-bed mixed bed system).

Using the separation four-column type chromatography fractionation apparatus (packed with sodium type strongly acidic cation exchange resin "Diaion UBK 550") shown in FIG. 1, 60% / d
Chromatographic fractionation was performed at a liquid passing temperature of 60 ° C. The panose and glucose fractions of the chromatographic fraction are filtered with activated carbon, and the water content after purification with an ion exchange resin 3
The solution was concentrated to 0% / ds to obtain a panose solution and a glucose solution, respectively.

[Example 2] A cornstarch liquefied solution (DE 6.5, pH 5.5) having a concentration of 30% / ds, pullulanase "Amano" 0.045% / ds, biozyme ML
0.065% / ds was added and the reaction was carried out at 55 ° C. for 48 hours to prepare a maltose solution having a maltose content of 75% / ds. Then, α-glucosidase L Amano 100 u / ds was added to this maltose solution and the mixture was mixed at 55 ° C. for 3 times.
A rearrangement reaction was performed for 0 hours to prepare a first panose-containing solution.

Then, the first panose-containing liquid (pH 5.
0) AMG 0.1 u / g, and α-amylase spitase HS (manufactured by Nagase Seikagaku Corporation, 7,000 U / g)
g) 5 u / g was added and hydrolysis was carried out at 55 ° C. for 24 hours. The saccharified solution was purified and concentrated in the same manner as in Example 1 to obtain a second panose-containing solution for chromatographic fractionation.

Using a separation four-column type chromatographic separation device (packed with sodium type strongly acidic cation exchange resin "Diaion UBK 530") shown in FIG.
The ds chromatographic separation raw material liquid was subjected to chromatographic fractionation at a liquid passing temperature of 60 ° C. The panose fraction and the glucose fraction of the chromatographic fractionated liquid were purified and concentrated in the same manner as in Example 1 to obtain a panose liquid and a glucose liquid, respectively.

[Example 3] A cornstarch liquefied solution (DE 5.7, pH 5.5) having a concentration of 32% / ds was added to pullulanase "Amano" 0.45% / ds, "Biozyme M".
L ”0.065% / ds was added and the reaction was carried out at 55 ° C. for 48 hours to prepare a maltose solution having a maltose content of 74% / ds. Then, α-glucosidase L Amano 100u / ds was added to this maltose solution, and the temperature was 55 ° C.
A rearrangement reaction was carried out for 30 hours to prepare a first panose-containing solution.

Then, the first panose-containing liquid (pH
5.0) was added with 0.12 u / g of “AMG” and 10 u / g of β-amylase biozyme ML and hydrolyzed at 55 ° C. for 24 hours. The hydrolyzed solution was purified and concentrated in the same manner as in Example 1 to obtain a second panose-containing solution for chromatographic fractionation.

Using a separation four-column type chromatographic separation apparatus (packed with sodium type strongly acidic cation exchange resin "Diaion UBK550") shown in FIG. Chromatographic fractionation. The panose fraction and the glucose fraction of the chromatographic fractionated liquid were purified and concentrated in the same manner as in Example 1 to obtain a panose liquid and a glucose liquid, respectively.

[Application Example 4] The panose solution prepared in Example 3 was spray-dried under the following conditions to give a powder product.

Liquid sugar concentration 45% / ds Spray dryer outlet temperature 80 ° C. Liquid supply amount 4.5 l / hr Disk diameter 100 mm Disk rotation speed 16,000 rpm The sugar powder obtained had a water content of 2.5% / ds. The fluidity was extremely good.

<Test Results> For each example, the first panose-containing liquid, the second panose-containing liquid, and the purified and concentrated panose liquid / glucose liquid after chromatographic fractionation were analyzed for sugar composition by high performance liquid chromatography. The results are shown in Tables 1, 2 and 3, respectively.

From these results, the P category (panose solution) in each of the examples had a panose concentration of 60%.
It can be seen that the ratio of maltose and maltotriose containing α-1,4 bond is very small, about 5% / ds or less and 2% / ds or less, respectively.

Further, the panose content of the panose liquid prepared by changing the addition amount of glucoamylase in the above and Examples 1 to 3 was also measured. The results are shown in FIG.

From the results shown in FIG. 1, it can be seen that the combined use of α-amylase or β-amylase with glucoamylase makes it easy to obtain a panose solution having a higher panose content.

[0063]

[Table 1]

[0064]

[Table 2]

[0065]

[Table 3]

[Brief description of drawings]

FIG. 1 is a graph showing the panose content of a panose liquid obtained by subjecting a first panose-containing liquid to hydrolysis by varying the amount of enzyme added, followed by chromatographic fractionation, purification and concentration.

FIG. 2 is a schematic diagram of a chromatographic separation device used for the chromatographic fractionation of the present invention.

[Explanation of symbols]

 11 Chromatographic Separator First Tower 13 Chromatographic Separator Second Tower 15 Chromatographic Separator Third Tower 17 Chromatographic Separator Fourth Tower

Front page continuation (72) Inventor Chiaki Hamashima 114 Sosaku, Yokone-cho, Obu-shi, Aichi

Claims (5)

[Claims] 1. A method for producing a panose liquid by fractionating a panose-containing saccharified liquid (hereinafter abbreviated as “panose-containing liquid”) by ion exchange chromatography (hereinafter abbreviated as “chromatographic fractionation”). Then, α-glucosidase was allowed to act on the maltose-rich saccharified solution (hereinafter abbreviated as “maltose solution”), and the transfer reaction was advanced until the panose content became 15 to 35% / ds. The second panose-containing solution in which glucoamylase is added to promote hydrolysis of oligosaccharides composed of α-1,4 bonds as much as possible within the range where the apparent hydrolysis degree of panose is 40% or less is A method for producing a panose solution, which is a panose-containing solution for chromatographic fractionation. 2. A method for producing a panose liquid by chromatographically fractionating a panose-containing liquid, wherein the panose-containing liquid is allowed to react with maltose liquid by α-glucosidase to give a panose content of 15 to 35% / ds. To the first panose-containing liquid that has undergone the transfer reaction until, further, glucoamylase and α-amylase are added,
The second panose-containing solution in which the hydrolysis of the oligosaccharide consisting of α-1,4 bonds has been advanced as much as possible in the range where the apparent degree of hydrolysis of panose is 40% or less is the panose-containing solution for chromatographic fractionation. And a method for producing a panose liquid. 3. A method for producing a panose solution by chromatographically fractionating a panose-containing solution, wherein the panose-containing solution is allowed to react with maltose solution by α-glucosidase to give a panose content of 15 to 35% / ds. To the first panose-containing liquid that has undergone the rearrangement reaction until further, glucoamylase and β-amylase are added,
The second panose-containing solution in which the hydrolysis of the oligosaccharide consisting of α-1,4 bonds has been advanced as much as possible in the range where the apparent degree of hydrolysis of panose is 40% or less is the panose-containing solution for chromatographic fractionation. And a method for producing a panose liquid. 4. The strong acid cation exchange resin of alkali metal type or alkaline earth metal type according to claim 1, wherein the ion exchange resin used in the chromatographic fractionation is used. A method for producing a panose liquid. 5. The method for producing a panose powder according to claim 1, wherein the concentrated liquid of the fractionated panose liquid is spray-dried to give a powder product.