JPH0669385B2 - Method for producing sugar with high content of branched oligosaccharide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Object of the Invention (Industrial field of application) The present invention is non-fermentative, has a caries-preventing effect, a bifidus-growing effect, and has an aqueous solution viscosity similar to that of sugar. The present invention relates to a method for industrially producing a saccharide having a high branched oligosaccharide content using an inexpensive enzyme. The sugar obtained by the production method of the present invention is advantageously used as a sweetener in the fields of food and drink, favourites, feed, feed, pharmaceuticals and the like.

(Prior Art) Branched oligosaccharides have long been used in sake as isomaltose, susbiose, kojibiose,
Panose, isomaltotriose and the like are known, and these branched oligosaccharides are also called non-fermentable sugars, and many studies have been published in relation to alcoholic beverages.

Further, in order to positively improve the quality of liquor, α-glucosidase (also referred to as truss glucosidase) involved in branched oligosaccharide production is prepared, and it is used as an enzyme preparation for four steps to control the content of branched oligosaccharide. Research on making a high four-stage saccharified solution and adding it to raw mash [Bryokyo: 74 , 53P (1979)], or using it for the production of starch sugar containing a large amount of maltose transfer sugar as a brewing saccharide. (Japanese Patent Sho 40-
No. 27319, Japanese Patent Publication No. 41-5918) were proposed,
All of them have high sugar production costs and cannot be used for purposes other than brewing, such as food and drink and feed.

Further, for the purpose of improving the taste of triple-brewed sake, it is proposed to produce a non-fermentable sugar using amylase and transglucosidase, which mainly produce maltose (JP-A-56-51).
No. 982) was also published. According to this, a branched oligosaccharide product having a content of non-fermentable sugars such as isomaltose, panose, isomaltotriose, and nigerose of about 50% can be obtained, but the product has a high limit dextrin content, Therefore, when it is used for purposes other than brewing, for example, for food and drink, it has a higher viscosity than sugar, which limits its use.

In recent years, sweeteners such as sugar tend to be shunned from the viewpoint of preventing dental caries, and the emergence of caries-preventing or low-cariogenic sweeteners has been demanded. Started to be noticed. For example, JP-A-58-76063 and JP-A-61-181354 describe physical properties of branched oligosaccharides, low cariogenicity, use in foods and drinks that utilize the bifidus bacteria growth effect, and the like, As a method for producing the branched oligosaccharide, a high molecular polysaccharide such as pullulan or dextran having many α-1,6 bonds is used as a substrate, and these are hydrolyzed with an appropriate enzyme or acid, and then molecular fractionation chromatograph Fractionation by e, ion exchange chromatography, etc.,
Although a method of drying under reduced pressure and pulverizing is described, this method is unsuitable for industrial production because substrates such as pullulan and dextran are very expensive. In addition, a method in which glucoamylase is allowed to act on a high-concentration glucose solution to generate branched oligosaccharides by utilizing the reverse synthesizing effect of glucoamylase, and then fractionation is also described, but this method requires a long time for the reaction. Required and not substantial.

Furthermore, in order to reduce the viscosity and increase the osmotic pressure of branched oligosaccharide products, a syrup containing a large amount of branched oligosaccharides is prepared using β-amylase, starch debranching enzyme and an enzyme that produces branched oligosaccharides by transfer action. Manufacturing method
61-219345) was also proposed, but in this method, the starch debranching enzyme was simply used in combination with the method described in JP-A-56-51982 to reduce the limit dextrin content. However, saccharification requires a long time and expensive starch debranching enzyme is required, which is industrially disadvantageous.

(Problems to be Solved by the Invention) The present invention provides a branched oligosaccharide-rich sugar having an excellent caries-preventing effect and a bifidus bacteria-proliferating effect, and having an aqueous solution viscosity similar to that of sugar using an inexpensive enzyme for a short time. Therefore, it is an object of the present invention to provide a method for industrially advantageous production.

(B) Structure of the Invention (Means for Solving Problems) The inventors of the present invention have conducted various studies in order to solve the above problems, and as a result, amylase and transfer of starch liquefaction liquid mainly producing maltose. In a known method of saccharification reaction in the presence of an enzyme that produces a branched oligosaccharide by action, if glucoamylase is further used in combination, the amount of the produced sugar is significantly reduced by a slight decrease in the branched oligosaccharide content. The present invention has been accomplished, and it has been found that the viscosity of the produced sugar aqueous solution can be lowered to a degree close to that of the sugar aqueous solution, and the present invention has been achieved.

That is, the method for producing a sugar having a high branched oligosaccharide content of the present invention is
In a method of saccharifying a starch liquefaction solution having a concentration of 25% or more and DE10 to 20 in the presence of an amylase mainly producing maltose and an enzyme producing a branched oligosaccharide by a transfer action, glucoamylase is further used. The method is characterized by

Regardless of the type of raw material starch used in the present invention, any of various types of starch can be used. The starch has a concentration of 25% or more and is liquefied within a range of DE10 to DE20 before the saccharification reaction.

Examples of the amylase that mainly produces maltose in the present invention include malt amylase, α-amylase of filamentous fungi and bacteria, β-amylase of actinomycetes and bacteria, and α-amylase of plant origin.

As the enzyme that produces a branched oligosaccharide by the transposition action in the present invention, α-glucosidase (also referred to as transglucosidase) is used, and its origin is not limited.
Specific examples thereof include filamentous fungal transglucosidases such as Aspergillus, Rhizopus, and Mucor.

As the glucoamylase in the present invention, those originating from Aspergillus or Razops are used.

As a typical embodiment of the production method of the present invention, a concentration of 25
% Or more and a DE10 to 20 starch liquefaction solution, an amylase mainly producing maltose, an enzyme producing a branched oligosaccharide by a transfer action, and a glucoamylase are added substantially simultaneously, and allowed to react at a predetermined temperature. , A method of simultaneously performing a branched oligosaccharide production reaction and a reduction reaction of produced oligosaccharides of 5 or more sugars, at a concentration of 25% or more, and DE10
To the starch liquefaction solution of ~ 20, first, amylase which mainly produces maltose and an enzyme which produces branched oligosaccharide by transfer action are added and reacted at a predetermined temperature to sufficiently produce branched oligosaccharide, and then, There are two methods of adding glucoamylase to reduce oligosaccharides having 5 or more sugars.

The reaction temperature in the production method of the present invention is usually 40 to 60 ° C, preferably 50 to 55 ° C, and the total reaction time is usually 20 to 35 hours, preferably 24 to 30 hours. Then, the reaction time after the addition of glucoamylase in the above method is usually 10
-24 hours, preferably 15-20 hours.

The branched oligosaccharide solution obtained by the production reaction of the present invention is
After performing treatments such as filtration, decolorization, and ion-exchange purification according to the usual methods, it is possible to concentrate in vacuum to obtain a high-concentration liquid product (syrup-like product), and add powdered base to the concentrate. Or spray-dried without addition to powder products.

Furthermore, the branched oligosaccharide solution obtained by the production reaction of the present invention is subjected to the following treatments (a) to (c) as necessary to obtain a highly pure branched oligosaccharide solution, and then the same as above. It is possible to obtain a highly pure branched oligosaccharide product by subjecting it to a purification treatment or spray drying.

(A) A process of adding salt to the sugar solution to crystallize double salt crystals of glucose-salt and removing glucose.

(B) Treatment of sugar solution using activated carbon column, molecular fraction chromatography such as gel filtration or ion exchange chromatography to remove sugars other than branched gorigo sugar.

(C) A process in which yeast is allowed to act on a sugar solution to remove sugars other than branched oligosaccharides by assimilating fermentation from the sugar solution.

(Examples, etc.) Hereinafter, examples and comparative examples will be described in more detail. The method for measuring the activity of the enzyme used in these examples is as follows.

Amylase Add 1 ml of enzyme solution to 10 ml of 1% soluble starch solution (pH 6.0),
The enzyme reaction was carried out at 40 ° C. for 30 minutes, and the reducing sugar was quantified by the Fehling-Lehmann-Schiol method, and the activity of producing 10 mg as glucose in 40 minutes at 40 ° C. was defined as 1 unit.

Transglucosidase 2% α-methyl-D-glucoside solution 1 ml and 0.02 N-acetic acid / sodium acetate buffer (pH 5.0) 1 ml mixed solution with enzyme solution
Add 0.5 ml and let the enzyme react at 40 ° C for 60 minutes.
Exactly 60 minutes later, the mixture is placed in a boiling aqueous solution, heated for 5 minutes, and cooled in running water. Using 0.1 ml of this solution, quantify the glucose produced by the glucose oxidase method and
The activity for producing 1 μg of glucose in 2.5 ml of the reaction solution for 60 minutes was defined as 1 unit.

Glucoamylase 0.56% Soluble starch solution Add 9 ml of enzyme solution to 40 ℃ 30
Enzyme reaction for minutes can be performed. Quantifying the reducing sugars produced by the enzymatic reaction by the Fehling-Lehmann-Sjohl method,
One unit was the activity of producing 10 mg of glucose at 40 ° C for 30 minutes.

Example 1 A corn starch liquefaction solution (concentration 30%, DE14.5) was adjusted to pH 5.5, and then koji surface α-amylase 8u / g-ds and Aspergillus niger transglucosidase 0600u /
After adding g-ds and saccharifying at 55 ° C for 24 hours, 0.45u / g-ds of glucoamylase (Aspergillus niger system) was added and further reacted without inactivating the enzyme. . The results are shown in Table 1.

As can be seen from Table 1, after saccharification for 24 hours, by adding glucoamylase without deactivating the enzyme and further performing a saccharification reaction, the total amount of branched 2-class compounds to branched tetrasaccharides is reduced by about 2. , About 11% sugars with 5 or more branched sugars
Could be reduced.

Example 2 A corn starch liquefaction solution (concentration 30.4%, DE13.0) was added to pH 5.5.
After adjusting to, aspergillus α-amylase 8u / g-ds, Aspergillus niger transglucosidase 600u / g
-Ds and glucoamylase (Aspergillus niger system) 0.225 to 0.45 u / g-ds were simultaneously added and the enzymatic reaction was carried out at 55 ° C. The results are shown in Table 2.

As can be seen from Table 2, even when glucoamylase is allowed to coexist in the reaction from the initial stage of the enzyme reaction, the content of the branched disaccharides to the branched tetrasaccharides is reduced without decreasing the content of the pentasaccharides or more. I was able to do it.

As is clear from Example 1 and Example 2 above, branching 2 sugars to branching 4 can be achieved by using glucoamylase in combination.
It is possible to effectively reduce the total content of saccharides of 5 or more sugars without substantially reducing the total content of saccharides, or with a very slight decrease. And since the branched oligosaccharide-rich sugar having a reduced sugar content of 5 sugars or more can bring the aqueous solution viscosity close to that of sugar,
It can be advantageously used for the same purpose as sugar.

Further, from Examples 1 and 2 described above, the proper addition amount of glucoamylase is 0.3 to 0.45u / in the case of α-amylase 8u / g-ds and transglucosidase 600u / g-ds.
g-ds, in the case of α-amylase 11.2u / g-ds and transglucosidase 840u / g-ds, 0225-0.
375u / g-ds.

Example 3 32% Corn Starch Slurry (pH 6.25) Heat-resistant α-
Amylase (Daiwa Kasei Co., Ltd., trade name Crystase T-5)
Was added to starch at 0.12%, cooked at 105 ° C for 10 minutes, and then heated at 95 ° C for 90 minutes to liquefy, to obtain a liquefied solution of DE13.5.

After cooling the liquefied liquid to 55 ° C and adjusting the pH to 5.5,
α-Amylase (Amano Pharmaceutical Co., trade name Biozyme-C) 0.
2% / ds, transglucosidase (Amano Pharmaceutical Co., Ltd.) 0.0
4% / ds, and glucoamylase (trade name GNL, Amano Pharmaceutical Co., Ltd.
-3000) 0.015% / ds was added at the same time, and saccharification reaction was carried out at 55 ° C for 30 hours.

The obtained saccharified solution was decolorized and subjected to ion exchange purification, and then concentrated in vacuo to obtain a sugar solution (syrup) having a concentration of 75%.
The sugar composition was as shown in Table 3. The aqueous solution viscosity of this sugar is as shown in FIG. It was similar to the aqueous solution viscosity of sugar. The osmotic pressure of this sugar aqueous solution was as shown in FIG.

Example 4 A starch liquefaction liquid obtained by treating in the same manner as in Example 3 was
After adjusting the temperature to 5.5 ° C and pH 5.5, the same α-amylase 0.2% / ds and the same transglucosidase 0.04% / ds used in Example 3 were simultaneously added and reacted at 55 ° C for 24 hours. The same glucoamylase as used in Example 3, 0.015% / ds, was further added without inactivating, and reacted for 16 hours.

The obtained saccharified solution was decolorized and purified by ion exchange, and then concentrated in vacuo to give a concentrated solution having a sugar concentration of 75%. Its sugar composition is third
It was as shown in the table.

Example 5 The same α-amylase 0.28% / ds used in Example 3,
The same transglucosidase 0.054% / ds and the same glucoamylase 0.0125% / ds were used, the saccharification time was changed to 24 hours, and the other reaction was performed in the same manner as in Example 3,
In the same manner, purification and vacuum concentration were performed to obtain a highly concentrated sugar solution having a sugar concentration of 75%. The sugar composition was as shown in Table 3.

Comparative Example A starch liquefaction liquid liquefied in the same manner as in Example 3 was added with the same α-amylase 0.1% / d as used in Example 3.
s and the same transglucosidase 0.02% / ds were added simultaneously and reacted at 55 ° C. for 20 hours. Decolorize the obtained sugar solution,
The sugar composition of the ion-exchange purified sugar solution is as shown in Table 3, and the content of branched pentasaccharides or higher was extremely high. The viscosity and permeation of this sugar solution in water were as shown in FIGS. 1 and 2.

Example 6 The purified solution of the sugar having a high branched oligosaccharide content obtained in Example 3 was spray-dried under the following conditions to give a powder product.

Sugar liquid concentration 45% Sugar liquid heating temperature 80 ℃ Spray dryer outlet temperature 60 ℃ Picture liquid amount 4.5 / hr Disk length 100mm Disk rotation speed 16,000rpm The sugar powder obtained had a water content of 2.2% and a rest at 25 ℃ and RH55%. The angle was 55 °. In addition, when the outlet temperature of the spray dryer in this drying was set to 65 ° C. or higher, the fluidity of the sugar powder was deteriorated and the sugar powder was deposited in the dryer.

Example 7 To a purified solution of a sugar having a high content of branched oligosaccharides having a concentration of 45% obtained in Example 3, 5% sodium caseinate and 25% dextrin were added to the solid content, and sprayed. Spray drying using the same conditions as in spray drying of Example 6 except that the dryer outlet temperature was 90 ° C,
A free flowing sugar powder product was obtained.

The sugar powder product had a water content of 2.4% and an angle of repose of 52 ° at 25 ° C and 55% RH.

Example 8 The sugar solution obtained in Example 3 was decolorized with activated carbon, purified with an ion exchange resin, and concentrated under vacuum to a concentration of 50%. Then, the obtained concentrated liquid was fractionated using a fractionation resin (Mitsubishi Kasei Kogyo Co., Ltd., trade name Diaion FRK-01) to obtain a highly pure branched oligosaccharide syrup. The fractionation conditions and the obtained sugar composition were as follows.

Fractionation conditions Column: 17 mmφ x 800 mm (filled with resin), with jacket, 60 ° C Separation resin: Diaion FRK-01 (Na type) Separation load: 45 g (dry matter) Flow rate: SV = 0.2 Sugar composition G ₁ 1.6% G ₂ 11.6% iG ₂ 33.7% G ₃ 1.3% P 9.5% iG ₃ 12.0% iG ₄ 12.3% iG ₅₊ 18.0% Branched oligosaccharide accounting 85.5% (e) Effect of the invention The production method of the present invention is cheaper than starch. It is possible to produce a sugar having a high content of branched oligosaccharides, which has an aqueous solution viscosity similar to that of sugar, industrially, advantageously, in a relatively short time using various enzymes.

And, the obtained high-branched oligosaccharide sugar has an extremely mellow sweetness, and it is in harmony with the sourness of various foods, etc., from salt to taste, astringency, umami, and bitter taste.
It can be used for sweetening or improving the taste of ordinary foods and drinks.

Further, this sugar having a high content of branched oligosaccharides is also excellent as a low or anti-cariogenic sweetener, a bifidus bacteria growth promoter, a moisturizer, a crystallization inhibitor, a food luster and a body-imparting agent.

[Brief description of drawings]

FIG. 1 is a saccharide having a high content of branched oligosaccharides obtained in Example 3,
FIG. 2 shows the relationship between the concentration of each aqueous solution and the viscosity of the sugar having a high content of branched oligosaccharide and the sugar obtained in Comparative Example. FIG. 2 shows the relationship between the concentration of each aqueous solution and the osmotic pressure. It was done.

Claims (4)

[Claims] 1. A method for saccharifying a starch liquefaction solution having a concentration of 25% or more and DE10 to 20 in the presence of an amylase mainly producing maltose and an enzyme producing a branched oligosaccharide by a transfer action, further comprising A method for producing a sugar having a high branched oligosaccharide content, which comprises using an amylase together. 2. The production method according to claim 1, wherein an amylase which mainly produces maltose, an enzyme which produces branched oligosaccharides by a transfer action and a glucoamylase are simultaneously added to the starch liquefaction liquid to carry out a saccharification reaction. 3. A starch liquefaction solution is first added with an amylase which mainly produces maltose and an enzyme which produces a branched oligosaccharide by a transfer action to cause a saccharification reaction to produce a branched oligosaccharide, and then a glucoamylase is added. The method according to claim 1, further comprising reacting. 4. The production method according to claim 1, 2, or 3, wherein a concentrated solution of the produced saccharified solution is spray-dried to obtain a powder product.