JP2652049B2 - Method for producing galactooligosaccharide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a galacto-oligosaccharide having a Bifidobacterium growth promoting action, that is, a general formula Gal- (Gal) n-
The present invention relates to a method for producing a galactooligosaccharide represented by Glc (where Gal is a galactose residue, Glc is a glucose residue, and n is an integer of 1 to 4) with higher yield than lactose.

[Conventional technology]

As a method for producing galactooligosaccharides from lactose,
There are a method of allowing β-galactosidase of Aspergillus to act on lactose (Japanese Patent Publication No. 58-20266), a method of using a yeast belonging to the genus Cryptococcus (Japanese Patent Application Laid-Open No. 61-236790), and the like.
In the method utilizing the β-galactosyltransfer reaction by these β-galactosidases, the amount of galacto-oligosaccharide produced by the reaction is small, and the yield of galacto-oligosaccharide to lactose is low.
Only about 30%. One of the causes is considered to be an inhibitory effect of monosaccharides as a by-product. That is, β-
A monosaccharide is by-produced by a hydrolysis reaction that occurs in parallel with the galactosyl transfer reaction. Competes with the transfer reaction that produces sugar. Also, monosaccharides are known to inhibit the activity of β-galactosidase and reduce the rate of the transfer reaction. The amount of galactooligosaccharide production is maximized when lactose, which is the substrate for the reaction, is reacted until the concentration is less than about 1/2 of the charged concentration, but as the reaction proceeds, the concentration of monosaccharides in the reaction solution increases, It is considered that the action becomes stronger, which leads to a delay in the reaction time and other side reactions.

Furthermore, the reaction product contains a large amount of unreacted lactose, monosaccharides produced as a by-product of the hydrolysis reaction, and transfer disaccharides produced from the by-product monosaccharides. Low sugar content.

Therefore, a method for improving the oligosaccharide content of the reaction product by improving the yield of galacto-oligosaccharide relative to lactose has been studied, and as a result, glucose and galactose produced by the reaction when allowing Cryptococcus yeast to act on lactose have been studied. A method of obtaining a reaction product having a high galacto-oligosaccharide content by promoting a galacto-oligosaccharide production reaction while consuming the yeast with another yeast has been proposed (JP-A-62-130695). However, the method of promoting the reaction while erasing by consuming the monosaccharides by-produced in the reaction by the yeast is not sufficient because the microorganisms consume a large amount of glucose and galactose and cannot be used effectively. Is also not satisfactory. In addition, a large amount of yeast is required for the reaction, and an operation of removing the yeast after the reaction is completed, a purification operation of removing the metabolites of the yeast from the reaction purified product, a treatment of the waste yeast, and the like are also required.

Further, the above-described inhibition of the transfer reaction by the monosaccharide is more pronounced as the concentration of the monosaccharide is higher. Therefore, it hinders an attempt to increase the reaction efficiency by increasing the concentration of the raw material lactose in the reaction solution.

[Problems to be solved by the invention]

An object of the present invention is to solve the above-mentioned problems in a method for producing galactooligosaccharide from lactose using the action of β-galactosidase (or a microorganism containing the enzyme).

[Means for solving the problem]

The method for producing galactooligosaccharides provided by the present invention comprises a β-
When a galactosidase-containing microorganism or β-galactosidase is allowed to act on lactose to produce galactooligosaccharides, glucose isomerase is allowed to coexist in the reaction solution.

Glucose isomerase co-existed in the reaction solution by this production method sequentially converts glucose produced by the hydrolysis of lactose into fructose to prevent it from accumulating in the reaction solution and inhibiting the transfer reaction. I do. Therefore, it is necessary that glucose isomerase acts simultaneously with β-galactosidase,
If they are acted on sequentially, it is not possible to improve the yield of galactooligosaccharides.

The glucose isomerase to be used may be of any origin, for example, GODO-AGI (Jodo Shusei Co., Ltd.), Sweetzyme (Novo), TOYO-GI (Toyo Brewery Co., Ltd.) and the like can be used.

As β-galactosidase, any source may be used, for example, Aspergillus oryzae,
There are those derived from molds such as Aspergillus niger, those derived from yeasts such as Brera singularis, Candida, Kleberomyces, those derived from bacteria such as Bacillus circulans, Lactobacillus bulgaricus, Streptococcus thermophilus, and the like. ,Also,
Enzymes of Cryptococcus laurentii (JP-A-62-11)
1685) can also be used. Commercially available products include maxilac (Gist Brocade), lactase Y400 (Yakult Honsha), lactozyme (Novo), GODO-YNL
(Jodo Shuseisha) and Biolacta (Daiwa Kasei).

Both enzymes may be used as an enzyme solution or in the form of an immobilized enzyme. A microorganism containing each enzyme can also be used for the reaction, and in that case, the microorganism may be used in the state of an immobilized microorganism.

The amount of glucose isomerase used in combination is desirably an amount necessary for immediately converting glucose produced by the action of β-galactosidase to fructose, but it may be used in excess.

Reaction conditions such as pH and temperature are preferably selected according to their properties so that the two enzymes work well together. Usually, the transglycosylation reaction by β-galactosidase is
The preferred pH is 5-8, since it is possible from weakly acidic to near neutral and glucose isomerase acts from neutral to slightly alkaline. The reaction temperature is usually from 30 to 90 ° C., preferably from 40 to 70 ° C., but the enzyme is stabilized in a high-concentration sugar solution, so that a high reaction temperature can be employed.

The concentration of the raw material lactose at the start of the reaction may be in the range of about 4.5 to 90%, but β-galactosidase produces galacto-oligosaccharide at a higher rate as the lactose concentration is higher. Thus, a particularly preferred lactose concentration is about 20 including supersaturated conditions.
~ 80%. Also, isomerization by glucose isomerase progresses faster as the glucose concentration is higher. Therefore, it is desirable to supply lactose at the highest possible concentration.

When β-galactosidase and glucose isomerase used in the reaction have a metal requirement, a necessary metal is added within a range that does not inhibit the activities of other enzymes. Generally, glucose isomerase requires a magnesium salt for expression of activity, whereas β-galactosidase is stabilized by a magnesium salt or has no effect on the expression of activity. Is most appropriate.

The reaction time when the reaction is performed under the preferable reaction conditions as described above is as follows. First, in a batch reaction, the galacto-oligosaccharide, which continued to increase as the reaction time elapses, begins to decrease at a certain time due to the predominance of the hydrolysis reaction. Stop the reaction before and after. In the case of the immobilized enzyme reaction, the substrate supply rate is set so that the amount of galactooligosaccharide produced is maximized. In this case, since the isomerization reaction by glucose isomerase is an equilibrium reaction between glucose and fructose, the reaction in which β-galactosidase acts on lactose is a main factor affecting the galacto-oligosaccharide production rate. Therefore, the reaction time of β-galactosidase may be controlled in the presence of excess glucose isomerase. The same applies when a microorganism containing the required enzyme is used in the reaction, and no special precautions are required.

The reaction product may be subjected to processes such as microbial separation (when a microorganism is used), decolorization and purification, monosaccharide separation, concentration, and drying, if necessary, and then used as a Bifidobacterium growth promoting factor. .

〔The invention's effect〕

The galacto-oligosaccharide production method of the present invention is characterized in that β-galactosidase acts on lactose to produce galacto-oligosaccharide, and by-produced glucose is successively converted into fructose by glucose isomerase, and glucose is not accumulated in the reaction solution. Increase the yield of galactooligosaccharides by eliminating the competitive reaction in the transfer reaction
Second, the galacto-oligosaccharide yield can be improved by reducing the activity inhibiting action of β-galactosidase. And since the amount of transfer disaccharide and unreacted lactose which are difficult to separate from galacto-oligosaccharides can be reduced, purification after the reaction is easy, and the sugar mixture separated by the purification also has high sweetness of fructose. And galactose as a main component, which has the advantage of being highly useful as a sweetener.

〔Example〕

 Hereinafter, the present invention will be described with reference to examples.

Example 1 Lactose (350 g) was added to a 0.05 M potassium phosphate buffer (pH 6.0) and dissolved by heating to a total volume of 500 ml. To this were added 1500 units of Aspergillus oryzae-derived β-galactosidase / lactase Y-400 (Yakult Honsha Co., Ltd.) and 50 g of immobilized glucose isomerase / GODO-AGI (Godo Shusei Co., Ltd., 310 IGIU / g), followed by magnesium sulfate. Was added to a concentration of 5 mM and reacted at 60 ° C. with shaking overnight. After the immobilized glucose isomerase was filtered off, the reaction solution was heated to inactivate β-galactosidase. Next, 5 g of activated carbon was added to the reaction solution in which the enzymatic reaction was stopped, followed by decolorization treatment to obtain a colorless and transparent sugar solution.

Example 2 Lactose (30 g) was added to a 0.05 M potassium phosphate buffer (pH 6.0) and dissolved by heating to a total volume of 45 ml. To this, 27 units of β-galactosidase derived from Brera singularis, 5 g of immobilized glucose isomeraseGODO-AGI, 0.25 ml of 1M magnesium sulfate were added, and water was further added to the total amount.
The reaction was made up to 50 ml and shaken at 55 ° C. overnight. After the immobilized glucose isomerase was filtered off, the reaction solution was heated to inactivate β-galactosidase. Next, 1 g of activated carbon was added to the reaction solution in which the enzymatic reaction was stopped, followed by decolorization treatment to obtain a colorless and transparent sugar solution.

Example 3 Lactose (25 g) was added to a 0.05 M potassium phosphate buffer (pH 7.0) and dissolved by heating to a total volume of 45 ml. This is Bacillus
200 units of β-galactosidase bioactor (Daiwa Kasei Co., Ltd.) derived from circulans, 5 g of immobilized glucose isomerase / GODO-AGI, 0.25 ml of 1M magnesium sulfate
In addition, water was further added to make the total volume 5 ml, and the reaction was carried out at 55 ° C. with shaking overnight. After the immobilized glucose isomerase was filtered off, the reaction solution was heated to inactivate β-galactosidase. Next, 1 g of activated carbon was added to the reaction solution in which the enzymatic reaction was stopped, followed by decolorization treatment to obtain a colorless and transparent sugar solution.

Example 4 Lactose (25 g) was added to a 0.05 M potassium phosphate buffer (pH 7.0) and dissolved by heating to a total volume of 45 ml. To this, 300 units of β-galactosidase of Lactobacillus bulgaricus ATCC 11842 adjusted by a conventional method, 5 g of immobilized glucose isomerase GODO-AGI, 0.25 m of 1M magnesium sulfate were used.
l, water was further added to make a total volume of 50 ml, and the mixture was reacted at 50 ° C. with shaking overnight. After filtration of the immobilized glucose isomerase, the reaction solution was heated to inactivate β-glass tosidase. In the reaction solution after stopping the enzyme reaction,
Next, 1 g of activated carbon was added to perform a decolorizing treatment to obtain a colorless and transparent sugar solution.

The composition of the reaction product according to each of the above examples is summarized in the following table. The results of Comparative Examples performed in the same manner except that immobilized glucose isomerase was not used are also shown.

Claims (1)

(57) [Claims] 1. A microorganism containing β-galactosidase or β-galactosidase is allowed to act on lactose to give a general formula Gal- (Gal) n-Glc (where Gal is a galactose residue, Glc is a glucose residue, and n is A galacto-oligosaccharide represented by the following formula: wherein glucose isomerase is co-present in the reaction solution.