KR100945306B1 - A method of production for high purity galactooligosacchraide - Google Patents

Abstract

The present invention relates to a method for preparing galactooligosaccharide, which produces galactoligosaccharide of high purity by treating two kinds of beta-galactosidase which are different in the main catalytic reaction from lactose or lactose-containing substance. In particular, the method of the present invention High content of 4'-galactosyl lactose was obtained by sequentially reducing the content of residual undegraded lactose by sequentially using beta-galactosidase with high metastatic capacity and beta-galactosidase with high lactose hydrolysis ability. It is possible to obtain a high yield of galactoligosaccharides contained as a high yield has the advantage that it is very useful industrially.

Galactooligosaccharide, 4'-galactosyl lactose, beta-galactosidase

Description

A manufacturing method of high purity galactoligosaccharide {A METHOD OF PRODUCTION FOR HIGH PURITY GALACTOOLIGOSACCHRAIDE}

The present invention is to obtain a high-purity galactoligosaccharide containing high content of 4'-galactosyl lactose by treating two kinds of beta-galactosidase, the main catalytic reaction is different in lactose or lactose-containing substance It relates to a method for producing galactooligosaccharide.

Galactooligosaccharide is a generic term for oligosaccharides produced by the action of beta-galactosidase on lactose, and the general formula “Gal- (Gal) n-Glc (Gal means galactose residue, Glc means glucose residue). N represents an integer of 1 to 4). In addition, transition disaccharides, such as galactose and galactose, or disaccharides having different beta bonds such as beta-1,2, beta-1,3, beta-1,4 and beta-1,6, are also present. Galactooligosaccharides are included in the category of galactooligosaccharides because they have the same physiological functionality.

The galactooligosaccharide is a component of the oligosaccharide present in breast milk, and has the following advantages. First, it functions to selectively propagate Bifidobacterium bacteria and lactic acid bacteria, which are useful bacteria that reproduce in the human intestine. In particular, 4'-galactosyl lactose compared to raffinose or lacchurose Bifidobacterium aldoresentis ( Bifidobacterium) aldolescentis ), Bifidobacterium bifidum), Bifidobacterium breve (Bifidobacterium breve), bifidobacteria tumefaciens Infante Tees (Bifidobacterium infantis), BP tobak Te Solarium ronggeom (Bifidobacterium It can be used only by Bifidobacterium bacteria such as longum ), and other enteropathogenic bacteria are hardly available to select, and it is helpful to maintain intestinal lactic acid balance when ingesting 4'-galactosyl lactose. (Ohtsuka K. et al . , Bifidus Vol 2 pp. 143--149 (1989).

Second, it is a low-calorie low calorie material that can be widely used in diet control such as diet. Third, it promotes the absorption of minerals such as calcium and magnesium in the body. In particular, 4'-galactosyl lactose improves the bioavailability of iron (D. Perez-Conesa et al. , Food Science and Technology International, Vol. 13, pp. 69-77 (2007).

Fourth, unlike common sugars such as sugar does not cause tooth decay. In particular, 4'-galactosyl lactose is the causative agent of tooth decay, Streptococcus It is not used by bacteria such as mutans and insoluble glucan is not produced, which is characterized by low tooth decay (S. Hata. et al. , Oral Microbiology and Immunology Vol 16 pp. 57-62 ( 2001). Fifth, it is much better oligosaccharide in terms of heat and pH stability than fructooligosaccharide, isomaltooligosaccharide and the like. In addition, galactooligosaccharide is used in fermented milk and infant formula as an excellent material in terms of health function. In particular, 4'-galactosyl lactose, which is a representative oligosaccharide of breast milk, is currently required for industrial use as a component of infant formula in order to reproduce the metabolic function of breast milk, and a high concentration of 4'-galactosyl lactose is used. There is a need for development of a method for producing the same.

Such galactooligosaccharides are industrially produced by beta-galactosidase (β 3.2gal).

The beta-galactosidase (EC 3.2.1.23) is an enzyme that hydrolyzes beta-galactosidic bonds, and is also referred to as lactase because it mainly performs a reaction of breaking beta-galactosidic bonds of lactose. do. The beta-galactosidase is present in the small intestine of a mammal, leaves, seeds and fruits of plants, and also exists in various fungi, yeasts, microorganisms and the like. Recently, beta-galactosidase extracted from fungi, yeasts and microorganisms has been used commercially.

The beta-galactosidase not only performs the reaction of hydrolysis of the lactose beta-galactosidase to galactose and glucose, but also the galactosyl transfer reaction that transfers the decomposed galactose residues to hydroxyl groups of other sugars. That is, the sugar transfer reaction can be performed.

The lactose hydrolysis reaction of the beta-galactosidase is as follows.

(E: beta-galactosidase, Gal-Glc: lactose, E-Gal: galactose enzyme intermediate, Glc: glucose, Gal: galactose, H ₂ O: water)

In addition, the sugar transition reaction to produce 4-galactosyl lactose, which is a representative component of galactooligosaccharide of beta-galactosidase, is as follows.

(E: beta-galactosidase, Gal-Glc: lactose, E-Gal: galactose enzyme intermediate, Glc: glucose, Gal: galactose, Gal-Gal-Glc: 4 'galactosyl lactose)

Industrially, the former is mainly used in the production of low lactose milk for lactose intolerant patients by hydrolyzing milk lactose, and the latter in the manufacture of galactooligosaccharide.

The sugar transfer reaction is reported to be different depending on the origin of the microorganism. Specifically, Kluyveromyces lactis or Kluyeromyces beta-galactosidase derived from fragilis ) has a lower glycemic potential compared to lactose hydrolysis, and thus, when reacted with lactose or a lactose-containing substance, it is reported that the amount of galactooligosaccharide produced is low. Aspergillus oryzae ), Bacillus circulans , derived enzymes have high levels of sugar metabolism and have been reported to produce high amounts of galactooligosaccharides when reacted with lactose or lactose-containing substances (MA. Boon et al . Enzyme and Microbial Technology.Vol. 26 pp. 271-281 (2000).

As a representative production method of the galacto-oligosaccharides are reported to the prior art described in U.S. Patent No. 4,435,389, the milk sugar in Aspergillus duck chair (Aspergillus oryzae ) is a method for producing galacto-oligosaccharides by treatment with beta-galactosidase-derived galactosyl transfer reactions, that is, sugar transfer reactions. The manufacturing method is a representative method for producing galactooligosaccharides using a sugar transfer reaction of microorganism-derived beta-galactosidase, but by the above method, the production rate of galactoligosaccharides is 30% (w / w) Very low, there is a problem that a large amount of unreacted lactose and monosaccharides in the reaction product. That is, since the galactoligosaccharide mixed liquid produced by the manufacturing method contains a large amount of lactose and monosaccharides, the galactooligosaccharide mixed liquid has a problem that its use is limited due to lactose intolerance and lactose allergy caused by a large amount of lactose. There is a problem that the calorie of food added with galactooligosaccharide mixed solution is increased.

Japanese Patent Application No. 1987-003186 proposes a method of treating galactooligosaccharide mixture obtained by treatment with microbial-derived beta-galactosidase with a strongly acidic cationic resin in order to solve the problems of the prior art as described above. Specifically, the manufacturing method described in Japanese Patent Application No. 1987-003186 further includes a step of treating with a strongly acidic cationic resin, thereby removing a large amount of unreacted lactose and monosaccharides by 85% (w / w) or more. A method for producing high purity galactoligosaccharides is provided. According to the preparation method, although high purity galactooligosaccharide can be prepared, when performing a chromatography of the galactooligosaccharide mixture produced by the microbe-derived beta-galactosidase, lactose and Since there is a substantial portion of overlapping galactooligosaccharide components, the higher the content of the unreacted lactose in the mixed solution, there is a problem that the purity and yield of the obtained galactoligosaccharide is lower. That is, when 85% (w / w) of high purity galactooligosaccharide is prepared by the above method, the separation yield of galactooligosaccharide is due to 40% (w / w) or more of the unreacted lactose present in the galactooligosaccharide mixture. That is, there is a problem that the productivity is lowered.

In order to solve the problems of the prior art, various methods have been proposed to reduce the content of unreacted lactose in the galactoligosaccharide mixture or increase the content of galactoligosaccharide. As a representative example, Japanese Patent No. 2739335 describes the content of galactooligosaccharides in a galactooligosaccharide mixture by sequentially treating two beta-galactosidases with different microbial origins, thereby reducing the amount of galactoligosaccharides degraded during the reaction. Although a method of increasing the amount of unreacted lactose remaining in the galactoligosaccharide mixture was 17 to 24% (w / w), it did not solve the fundamental problem of the prior art.

The prior art by sequentially using two beta-galactosidase of different microbial origin, by increasing the production amount of galactoligosaccharide or preventing the re-decomposition of galactoligosaccharides of the galactoligosaccharide in the galactoligosaccharide mixture Although the effect of increasing the content is recognized, the galactooligosaccharide content of the galactooligosaccharide product manufactured and marketed by the prior art is still less than 70% (w / w) based on the total sugar content in the product, and the lactose content is Since it is more than 20% (w / w) based on the total sugar content, there is a problem that the use is limited by lactose intolerance or lactose allergy. In addition, as described above does not propose a method for treating a large amount of unreacted lactose present in the galactooligosaccharide mixture, there is a problem that can not be produced in high yield when producing a high purity galactooligosaccharide.

Therefore, unreacted in the galactooligosaccharide mixture prepared using beta-galactosidase in order to industrially mass-produce galactooligosaccharides having a high nutritional value and can be used for various purposes as an industrial material. There is a need to develop a method for producing galactooligosaccharide, which produces a high yield of galactoligosaccharide by lowering lactose content.

In order to solve the problems of the prior art as described above, the present invention lowers the unreacted lactose content of the galactooligosaccharide mixture prepared by beta-galactosidase of microbial origin, it is used in patients with lactose intolerance or lactose intolerance It is an object of the present invention to provide a method for producing galactooligosaccharide having a high content of 4'-galactosyl lactose in galactooligosaccharide, as well as preparing galactooligosaccharide of as high purity as possible.

In order to achieve the above object, the present invention is an unreacted reaction of the galactooligosaccharide mixture by sequentially using beta-galactosidase having high beta-galactosyl transfer ability and beta-galactosidase having high lactose hydrolysis ability. After lowering the lactose content, the present invention provides a method for preparing galacto-oligosaccharide, which is performed by chromatography to prepare galacto-oligosaccharide. The production method of the present invention has the advantage of producing a high-purity galactooligosaccharide in high yield by significantly lowering the content of lactose remaining in the galactoligosaccharide mixture prepared by the enzymatic reaction.

In addition, the production method of the present invention has the advantage that the content of 4'-galactosyl lactose can be produced galactooligosaccharides of 35% (w / w) or more based on the total sugar content.

More specifically, the present invention

a) treating the lactose or lactose-containing substance with a beta-galactosidase having high beta-galactosyl transfer ability to produce a galactooligosaccharide mixed solution (first reaction);

b) decomposing unreacted lactose by treating beta-galactosidase having high lactose hydrolyzing ability to the galactooligosaccharide mixture obtained in step a) (secondary reaction); And

c) two beta-galactos with different main catalytic reactions, including the step of separating high purity galactoligosaccharides by performing a strong acid cation exchange resin chromatography on the galactoligosaccharide mixture obtained from the unreacted lactose obtained in step b). It provides a high-purity galactoligosaccharide manufacturing method for treating a sidase.

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

The present invention, by reacting the substrate of the primary reaction with a high initial lactose concentration to beta-galactosidase with high metastatic capacity to further increase the galactoligosaccharide production ability, the substrate of the secondary reaction in which the lactose concentration is lower than the initial lactose concentration It is characterized in that to produce a high-purity galactoligosaccharides of high purity by selecting the reactor and the enzyme in each reaction step to further increase the lactose hydrolysis ability by reacting the high hydrolytic beta-galactosidase with respect to Galactooligosaccharide production method.

In the present invention, galactooligosaccharide refers to a generic term for oligosaccharides produced by acting beta-galactosidase on lactose. For example, the galactooligosaccharide is a general formula Gal- (Gal) n-Glc (In the general formula, Gal means a galtose residue, Glc means a glucose residue, and n means an integer of 1 to 4). It may be a saccharide represented by), preferably the galactoligosaccharide is lactose 4'-galactosyl lactose beta-1,4 bonded to lactose, 4'-galactosyl lactose to beta-1, It may be one or more selected from the group consisting of four linked tetrasaccharides to six sugars and transition disaccharides.

The 4'-galactosyl lactose BP gambling compared to raffinose or rakchyuroseu Te Solarium Al Dore sentiseu (Bifidobacterium aldolescentis ), Bifidobacterium bifidum ) or Bifidobacterium It can be used by only Bifidobacterium bacteria such as Beve ), and other intestinal harmful bacteria are rarely used, which has been reported to have an advantage in maintaining the balance of intestinal lactic acid bacteria (Ohtsuka K. et. al . , Bifidus Vol 2 pp. 143--149 (1989)), which improves bioavailability of iron (D. Perez-Conesa) et al . , Food Science and Technology International, Vol. 13, pp. 69-77 (2007). In addition, Streptococcus is a causative agent of tooth decay. It is not used by bacteria such as mutans ) and insoluble glucan is not produced, which is characterized by low tooth decay (S. Hata. et . al . , Oral Microbiology and Immunology Vol 16 pp. 57-62 (2001)), 4'-galactosyl lactose, a representative oligosaccharide of breast milk, is essential for use as a component of infant formula to reproduce the metabolic function of breast milk.

In the present invention, the beta-galactosidase is hydrolyzed to galactose and glucose using a lactose having a beta-galactoside bond as a substrate, and at the same time, galactose is transferred to lactose to perform galactoligosaccharide production. do. In addition, the lactose hydrolysis capacity and the transfer capacity is different from each other, and various beta-galactosidase present in nature differs in the lactose hydrolysis capacity and the transfer capacity.

That is, beta-galactosidase produces galactose enzyme intermediates containing galactose residues from lactose substrates, and different products, namely different kinds, depending on substances (galactose receptors) that accept galactose residues of galactose enzyme intermediates. Of galactooligosaccharide is produced, and as the reaction proceeds, the reaction product including the produced galactooligosaccharide is further decomposed and recombined to produce various results. However, when comparing the content of the resulting product up to a point in time, that is, the point of the galactoligosaccharide before the decomposition of galactoligosaccharide in the reactant shows the highest content, the lactose hydrolysis capacity and the transfer capacity of the beta-galactosidase Can be compared.

Therefore, it is possible to select beta-galactosidase having high metastatic capacity and beta-galactosidase having high hydrolytic ability by comparing the resulting products.

In the present invention, the beta-galactosidase having a high beta-galactosyl transfer ability in step a) proceeds with the reaction of lactose or lactose-containing substance with a substrate, resulting in decomposition of the entire reaction product, namely, galactoligosaccharide in the reactant. When the galactooligosaccharide content before the start of the highest content, the galactoligosaccharide content in the reactant means a beta-galactosidase higher than the galactose content in the reactant (see Fig. 1-a).

The beta-galactosyl transition high ability beta-galactosidase azepin preferably, Aspergillus age (Aspergillus niger ), Aspergillus oryzae , Streptococcus Thermophilus , Lactobacillus bulgaricus , Cryptococcus laurentii , and Bacillus succulant ( Bacillus) circulans ) may be beta-galactosidase derived from any one selected from the group consisting of.

More preferably, it may be a beta-galactosidase derived from Cryptococcus Lorenti or Bacillus sucrose, and most preferably, it is not only high in beta-galactosyl transfer capacity but also high in 4'-galactosyl lactose generating ability. Beta-galactosidase from beta-galactosidase, Bacillus succurance.

Beta-galactosidase may be affected by galactoligosaccharide production, depending on the initial lactose substrate concentration.

In the present invention, the lactose-containing material of step a) refers to a substance containing lactose, and if the lactose concentration of the lactose-containing material is too high, there is a problem that the lactose is difficult to dissolve because of high concentration and high viscosity, and lactose is difficult to dissolve. If the lactose concentration of the containing material is too low, the ability to produce galactooligosaccharide is low, preferably 20% (w / w) to 70% (w / w), more preferably 30% (w / w) based on the total composition. w) to 60% (w / w), most preferably 40% (w / w) to 50% (w / w).

In the present invention, the galactooligosaccharide mixture obtained by treating the beta-galactosidase having high beta-galactosyl transfer ability in step a) may include the galactooligosaccharide, unreacted lactose and monosaccharide. The galactoligosaccharide mixed solution may include 4'-galactosyl lactose, and preferably, the content of 4'-galactosyl lactose is 20% (w / w) based on the total sugar content of the galactoligosaccharide mixed solution )

In the present invention, step a) of making the galactooligosaccharide mixed solution may be performed at a temperature and pH range at which the enzyme is not inactivated, and preferably, the step a) is performed at an optimum pH and temperature range of the enzyme used. Can be performed. Step a) may be preferably performed at pH 5 to pH 9.5, more preferably at pH 5.5 to pH 6.5. In addition, the step a) may be performed at 50 ° C to 70 ° C, more preferably 55 ° C to 65 ° C.

The end point of step a) may be determined by the unreacted lactose content of the galactooligosaccharide mixture, which is a product of step a).

If the unreacted lactose content is less than 30% (w / w) based on the total sugar content of the galactoligosaccharide mixed solution, which is the product of step a), the hydrolysis reaction is low because the content of lactose used as the substrate in the secondary reaction is low. There is a problem that recombination of 4'-galactosyl lactose does not occur. On the other hand, if the unreacted lactose content is 50% (w / w) or more based on the total sugar content of the galactoligosaccharide mixed solution, there is a problem in that the yield of the final galactoligosaccharide is reduced and the yield is lowered.

Therefore, the step a) is preferably when the unreacted lactose content in the galactooligosaccharide mixture, the product of step a), is 30% (w / w) to 50% (w / w) based on the total sugar content. May be terminated, more preferably between 35% (w / w) and 45% (w / w).

The reaction time of step a) may be adjusted by the amount of beta-galactosidase having high beta-galactosyl transfer ability. Specifically, when the amount of the enzyme is large, the content of lactose drops quickly to reduce the reaction time, and when the amount of the enzyme is small, the reaction time is long. For example, the amount of the enzyme may be 0.1 g to 3 g based on 1 kg of the substrate solids, and the reaction time of step a) may vary depending on the amount of beta-galactosidase having high beta-galactosyl transfer ability. It may be 48 hours.

When the step a) is completed, the galactooligosaccharide mixture is treated for 10 minutes to 2 hours, preferably 30 minutes to 1 hour, under conditions of 70 ° C. to 100 ° C., thereby increasing the beta-galactosyl transfer ability. After deactivating the activity of galactosidase, step b) may be performed.

In the present invention, the high hydrolyzable beta-galactosidase of step b) is when the lactose or lactose-containing substance is reacted with a substrate, and the entire reaction result, that is, decomposition of galactoligosaccharides in the reactant starts. Highest galactooligosaccharide content At the time point showing the content, it means a beta-galactosidase in which the galactose content in the reactant is higher than the galactoligosaccharide content in the reactant (see Fig. 1-b).

Preferably, the beta-galactosidase having high lactose hydrolysis capacity specifically decomposes only unreacted lactose in the galactoligosaccharide mixture and does not decompose galactoligosaccharides, in particular, does not decompose 4'-galactosyl lactose. be-galactosidase of an enzyme origin different from step a).

The high lactose hydrolyzate beta-galactosidase is preferably Kluyveromyces galactose derived from lactis ) or Kluyeromyces fragilis may be more hydrolysable beta-galactosidase, which is more produced, and most preferably beta derived from Kluberomyces lactis . May be galactosidase.

In the case of using beta-galactosidase derived from Krubermises lactis, at least 20% (w / w) of 4'-galactosyl lactose is based on the total sugar content generated in step a). In step b), the content is maintained without decomposing, thus preparing galactooligosaccharides containing 40% (w / w) or more of 4'-galactosyl lactose based on the total sugar content in the resulting galactoligosaccharides. There is an advantage to this.

In the present invention, the step b) of decomposing the unreacted lactose may be performed at a temperature and pH range at which the enzyme is not inactivated, and preferably, step b) is performed at an optimum pH and temperature range of the enzyme used. Can be performed. Step b) may be preferably performed at pH 5 to pH 8, more preferably at pH 6 to pH 7. In addition, the step b) may be preferably performed at 30 ° C to 50 ° C, more preferably at 35 ° C to 45 ° C.

The duration of step b) is not limited, but if the amount of unreacted lactose in the galactoligosaccharide mixture, the reaction product of step b), is less than 10% (w / w) based on the total sugar content, step b) After that, the separation rate and recovery rate of the chromatography to be performed can be increased, and the lactose content of the final product using the galactooligosaccharide obtained by the chromatography is low, so that lactose intolerance and lactose allergy are not induced.

Therefore, the end point of step b) may preferably be terminated when the unreacted lactose content in the galactooligosaccharide mixture is 10% (w / w) based on the total sugar content, more preferably the unreacted It can be terminated when the lactose content is 5% (w / w) or less based on the total sugar content.

C) performing chromatography to obtain high purity galactoligosaccharides from the galactooligosaccharide mixture, which is a product of step b) obtained by treating unreacted lactose by treating beta-galactosidase having high lactose hydrolyzing ability. The steps can be performed.

The chromatography performed in step c) may preferably be calcium or sodium type strong acid cation exchange resin chromatography.

Separation of sugar constituents by the strongly acidic cation exchange resin chromatography is performed in the order of high to small degree of polymerization of sugars by molecular size exclusion effect. That is, the sugar solution is eluted in the order of galactooligosaccharide (more than trisaccharide), disaccharide (lactose, transition 2 sugar), monosaccharide (glucose, galactose). Therefore, in case of reducing the amount of disaccharide, the disaccharide eluted in the middle part, the peak of lactose is decreased in chromatography during elution and divided into two major peaks of galactoligosaccharide and monosaccharide, so the purity and separation rate of galactooligosaccharide are separated. Can increase.

Before separation into a strong acid cation exchange resin, the galactooligosaccharide mixture obtained in two enzymatic reactions may be further decolorized with activated carbon or desalted with ion exchange resin. When the concentration of sugar solution is low, It can be concentrated under reduced pressure. The resin used for separation of the mixed solution of galactooligosaccharide may vary depending on the sugar composition to be separated. The strong acid cation exchange resin is preferably a Na-type or Ca-type resin having a -SO ₃ -group as an exchanger based on a styrene divinylbenzene copolymer. In the specific example of this invention, the product of Mitsubishi Chemical Co., Ltd. UBK530 (Na type), UBK555 (Ca type), etc. can be used as a strongly acidic cation exchange resin.

If the temperature of the strong acid cation exchange resin separation system is below 50 ° C., there is a risk of microbial contamination, and the viscosity of the sugar solution is low, which causes a load in the separation operation, and if the temperature is above 90 ° C., the sugar solution may brown and become difficult to purify. The temperature of the separation system may be 50 ℃ to 90 ℃, preferably 60 ℃ to 80 ℃, it is preferable to maintain a constant temperature of the separation system.

The concentration of galactooligosaccharide mixture, which is the product of step b) obtained by treating unreacted lactose by treating beta-galactosidase having high lactose hydrolysis capacity supplied to the column, that is, the product of the galactoligosaccharide mixture, which is the product of the second reaction. Since the amount of the reaction solution added to the resin affects the resolution of the resin, the concentration of the added solution is preferably about 10% (w / w) to about 60% (w / w), and is a product of the secondary reaction. The amount of the galactoligosaccharide mixed solution is preferably 2% (v / v) to 20% (v / v) of the resin amount.

Step c) may be performed by supplying a mixture of galactooligosaccharides, the product of the secondary reaction, to a column to adsorb saccharides, and eluting the sugar solution by flowing water at SV = 0.1 to 2.0. The galactoligosaccharide mixed solution, which is the product of the secondary reaction eluted, is sequentially collected into the galactooligosaccharide fraction and the monosaccharide fraction.

The additional advantage of low lactose content in the galactooligosaccharide mixture, which is the product of the secondary reaction, can be obtained in the same manner as in the various separation methods such as the batch type as well as the pseudo mobile phase method using a calcium or sodium type strong acid cationic resin. Application is possible. When the strong acid cationic exchange resin separation system is operated in a pseudo mobile phase method capable of a continuous process compared to a batch type, mass production is possible, and the resin utilization rate is high, thereby increasing economic efficiency.

The separated galactooligosaccharide can be commercialized in the liquid phase after concentration under reduced pressure, and can also be powdered through vacuum drying.

When the steps a), b) and c) are performed, galactoligosaccharides having a purity of 90% (w / w) to 95% (w / w) can be obtained in a yield of 40% to 50%. , 95% (w / w) of galactoligosaccharides can be obtained in a yield of 40% to 45%, the content of 4'-galactosyl lactose in the galactoligosaccharides is 35% based on the total sugar content (w / w) to 55% (w / w), preferably 40% (w / w) to 50% (w / w), unreacted lactose remaining in the galactoligosaccharide mixture The content of is from 1% (w / w) to 10% (w / w), preferably from 1% (w / w) to 5% (w / w), more preferably from 1% (w / w) 2.5% (w / w). The purity means the percentage of the galactooligosaccharide content (solid content) to the total sugar content (solid content), and the yield is the weight of the galactooligosaccharide fraction (solid content) relative to the weight of the total oligosaccharide content (solid content). Means percentage. Specifically, the purity can be obtained by the formula "galactoligosaccharide content (solid content basis) / total sugar content (solid content basis) x 100 (%)", the yield is the "galactoligosaccharide fraction (solid content basis) ) / Weight of oligosaccharide total solids (based on solids) x 100 (%) ".

As described above, the galactooligosaccharide production method according to the present invention reduces the content of the unreacted lactose in the galactoligosaccharide mixture prepared by the enzymatic reaction and performs chromatography, thereby yielding high yield at the same purity as compared to the conventional production method. Galactooligosaccharide can be prepared, and the content of galactoligosaccharide is also excellent in functionality, and the content of 4'-galactosyl lactose, which is attracting the most attention in the industry, is very high compared to the conventional galactooligosaccharide product. Since the lactose content supplied to the column is low when performing the chromatography, it is possible to obtain a galactooligosaccharide product having a purity of 90% (w / w) or more by batch separation method using a strong acid cationic resin as well as pseudo mobile phase method. Its industrial use value will be very large.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example

Example 1 Contents of Lactose, Galactooligosaccharide, and Galactose Changed When Beta-galactosidase with High Metastasis Capacity or Beta-galactosidase with High Lactose Hydrolysis Capacity was Treated

0.12 g of lactose was dissolved in 145 g of distilled water, and 0.32 g of beta-galactosidase (Biolacta N5, Amano Enzyme) derived from Bacillus Circulans having high beta galactosyl transfer reaction in a 40% (w / w) lactose solution. 3.2 g of the substrate solids 1 kg) was added and reacted at pH 6.0 and 60 ° C. for 24 hours. The lactose content, galactoligosaccharide content and galactose content change during the reaction are shown in Fig. 1-a.

As shown in FIG. 1-a, the content of galactoligosaccharide and galactose was 55.06%, and the content of galactose was 3.07%, respectively, at 16 hours of reaction when galactooligosaccharide was decomposed.

Dissolve 95g of lactose in 950g of distilled water and 2.85g of beta-galactosidase (Lactozym, Novozyme) derived from Krubermises lactis with high hydrolysis ability in 10% (w / w) lactose solution (compared to 1kg of substrate solids). 30g) was added and reacted for 24 hours at pH 6.5, 40 ℃. The lactose content, galactoligosaccharide content, and galactose content change during the reaction are shown in Fig. 1-b.

As shown in FIG. 1-b, the content of galactoligosaccharide and galactose was 20.12%, and the content of galactose was 33.17%, respectively, at 16 hours of reaction when galactooligosaccharide was decomposed.

Example  2: Metastatic  High beta- Galactosidase  When processing, lactose, 4'- Galactosyl  Changes in Lactose and Galactose Content

Using a Erlenmeyer flask, 105 g of lactose was dissolved in 145 g of distilled water to prepare three lactose solutions of 40% (w / w), and then beta derived from Bacillus circulans having a high beta galactosyl transition reaction to the lactose solution. PH was added by adding galactosidase (Biolacta N5, Amano Enzyme) 0.025g (0.25g vs. 1kg substrate solids), 0.08g (0.8g vs. 1kg substrate solids), and 0.32g (3.2g vs. 1kg substrate solids) The reaction was carried out at conditions of 6.0 and 60 ° C for 24 hours.

The lactose content, 4′-galactosyl lactose content and galactose content change during the reaction are shown in FIGS. 2-a, 2-b and 2-c, respectively.

As shown in FIGS. 2-a, 2-b and 2-c, 4'-galactosyl in each case using 0.25 g, 0.8 g and 1.6 g of beta-galactosidase relative to 1 kg of substrate solids The remaining lactose content at the highest lactose content was 41.7% (w / w), 42.2% (w / w) and 43.5% (w / w), respectively, based on the total sugar content. The contents were 1.0% (w / w), 0.9% (w / w) and 0.9% (w / w), respectively.

Example 3: Galactooligosaccharide Enzyme Reaction According to Lactose Concentration of Initial Lactose Containing Substance

Example  3-1

After dissolving 631.6 g of lactose in 868.4 g of distilled water to prepare a lactose solution of 40% (w / w), the beta-galactosidase derived from Bacillus Circulans having high beta-galactosyl transferability into the lactose solution was obtained. N5, Amano Enzyme Co.) 0.15g (0.25g compared to 1kg of substrate solids) was added to perform the enzyme reaction at pH 6.0, 60 ℃ for 24 hours. After completion of the enzyme reaction treated with beta-galactosylase having high beta-galactosyl transfer ability, the enzyme was inactivated by treatment at 90 ° C. for 30 minutes. The sugar composition of the galactooligosaccharide mixture obtained by the beta-galactosidase having high beta-galactosyl transfer ability was 43.3% (w / w) (4'-galactosyl) based on the total sugar content. Lactose 22.8% (w / w), lactose 37.7% (w / w), glucose 17.8% (w / w), galactose 1.2% (w / w). 250 g of the reaction solution in which the beta-galactosidase having high beta-galactosyl transfer ability was inactivated was dispensed into a Erlenmeyer flask, and then beta-galactosidase derived from Krubermises lactis having high lactose hydrolysis capacity (Lactozym). , Novozyme) was added 0.32ml (based on 3.2ml compared to 1kg of substrate solids) and the enzyme reaction was performed for 48 hours at pH 6.5, 40 ℃. The sugar composition of the galactoligosaccharide mixed solution obtained by treating the high lactose hydrolyzable beta-galactosidase was 50.9% (w / w) (4'-galactosyl lactose 24.3%) based on the total sugar content. (w / w)), lactose 4.0% (w / w), glucose 32.6% (w / w) and galactose 12.5% (w / w).

Example  3-2

A lactose solution of 50% (w / w) was prepared by dissolving 789.5 g of lactose in 710.5 g of distilled water, and then a beta-galactosidase derived from Bacillus Circulans having high beta-galactosyl transfer ability in the lactose solution. N5, Amano Enzyme Co.) 0.19g (0.25g compared to 1kg substrate solids) was added to perform the enzyme reaction at pH 6.0, 60 ℃ for 24 hours. After completion of the enzyme reaction treated with beta-galactosylase having high beta-galactosyl transfer ability, the enzyme was inactivated by treatment at 90 ° C. for 30 minutes. The sugar composition of the galactooligosaccharide mixture obtained by the beta-galactosidase having high beta-galactosyl transfer ability was 43.6% (w / w) (4'-galactosyl) based on the total sugar content. Lactose 23.8% (w / w), lactose 40.7% (w / w), glucose 15.4% (w / w), galactose 0.4% (w / w). 250 g of the reaction solution in which the beta-galactosidase having high beta-galactosyl transfer ability was inactivated was dispensed into a Erlenmeyer flask, and then beta-galactosidase derived from Krubermises lactis having high lactose hydrolysis capacity (Lactozym). , Novozyme Co.) was added 0.38ml (based on 3.2ml compared to 1kg of substrate solids) to perform the enzyme reaction for 48 hours at pH 6.5, 40 ℃. The sugar composition of the galactoligosaccharide mixed solution obtained by treating the high lactose hydrolyzable beta-galactosidase was 48.2% (w / w) of galactooligosaccharide based on the total sugar content (of which 4'-galactosyl Lactose 24.5% (w / w), lactose 3.4% (w / w) glucose 33.5% (w / w) and galactose 14.5% (w / w).

Example  4

Example  4-1

Beta-galactosidase Nagase F (high lactose hydrolyzate derived from Krubermises lactis) was added to 250 g of the reaction solution inactivated beta-galactosidase having high beta-galactosyl transfer ability prepared in Example 3-1. Nagase Enzyme) 1.1ml was added and reacted at pH 6.5 and 40 ° C. The sugar composition of the galactooligosaccharide mixture obtained by treating the beta-galactosidase (Nagase F) and reacting for 3 hours was 50.9% (w / w) (4'-galacto) based on the total sugar content. Silactose 24.5% (w / w), lactose 4.7% (w / w) glucose 31.6% (w / w) and galactose 11.9% (w / w).

Example  4-2

Beta-galactosidase Maxilact (DSM) having high lactose hydrolytic activity derived from Krubermises lactis in 250 g of the reaction solution inactivated beta-galactosidase having high beta-galactosyl transfer ability prepared in Example 3-1. G) was added and reacted at pH 6.5 and 40 ° C. The sugar composition of the galacto-oligosaccharide mixture obtained by treating the beta-galactosidase (Maxilact) for 16 hours and reacting for 4 hours was based on the total sugar content of 42.7% (w / w) (4'-galactosyl) Lactose 23.2% (w / w), lactose 2.5% (w / w), glucose 35.4% (w / w) and galactose 18.6% (w / w).

Example  5: according to the type of strongly acidic cation exchange resin Galactooligosaccharide  detach

Example  5-1: Na  By strong acid cation exchange resin Galactooligosaccharide  detach

Activated charcoal treatment and ion purification were performed on the galactooligosaccharide mixed solution treated with beta-galactosidase having high lactose hydrolysis ability prepared in Example 4-1. 10% (w / w) of the purified galactooligosaccharide mixture and 30 ml were loaded on a column (2.6 cm inner diameter, 40 cm long jacketed column) filled with a strong acid cation exchange resin UBK 530 (Na type, Mitsubishi Corporation), and then solvent Phosphorus spilled water. In this case, the temperature of the jacket column was 70 ° C., and the injection speed of the sugar solution and the solvent was SV = 0.56. 5 ml of the liquid from the end of the column was separated and each sugar composition was confirmed by HPLC. The results are shown in FIG. 3. As shown in FIG. 3, when the galacto-oligosaccharide was separated into a galactoligosaccharide fraction and a monosaccharide fraction such that the purity of the galactoligosaccharide was 95% (w / w), the sugar composition of the whole composition was as shown in Table 1 below. The total yield of galactooligosaccharide fraction was 44.2%.

Example  5-2: Ca  By strong acid cation exchange resin Galactooligosaccharide  detach

Activated carbon treatment and ion purification were performed on the galactooligosaccharide mixture solution treated with beta-galactosidase having high lactose hydrolysis capacity prepared in Example 3-1. 10% (w / w) of the purified galactooligosaccharide mixture, 30 ml, was loaded on a column (2.6 cm inner diameter, 40 cm long jacketed column) filled with a strong acid cation exchange resin UBK 555 (Ca type, Mitsubishi Corporation), and then solvent Phosphorus spilled water. In this case, the temperature of the jacket column was 70 ° C., and the injection speed of the sugar solution and the solvent was SV = 0.56. 5 ml of the liquid from the end of the column was separated and each sugar composition was confirmed by HPLC, and the results are shown in FIG. 4. As shown in FIG. 4, when the galactooligosaccharide fraction and the monosaccharide fraction were separated such that the purity of the galactooligosaccharide was 95% (w / w), the sugar composition of the whole composition was as shown in Table 2 below. The total yield of galactooligosaccharide fraction was 43.6%.

Comparative Example 1 Composition of Galactooligosaccharide Isolated from High Acid Cation Exchange Resin Treated with Beta-galactosidase with High Beta-Galactosyl Transferability

       After dissolving 210.5 g of lactose in 288.5 g of distilled water to prepare a lactose solution (40% (w / w)), the beta-galactosidase derived from Bacillus circulans having high beta-galactosyl transfer ability in the lactose solution (Biolacta N5). , Amano Enzyme Co.) 0.2g (0.5g compared to 1kg substrate solids) was added to perform the enzyme reaction for 48 hours at pH 6.0, 60 ℃. After completion of the enzyme reaction, the enzyme was inactivated by treating at 90 ° C. for 30 minutes. The sugar composition of the galactooligosaccharide mixture in which the enzyme was inactivated was 54.6% (w / w) (4'-galactosyl lactose 11.3% (w / w)) based on the total sugar content, and lactose 23.4% ( w / w) and 22.4% (w / w) of monosaccharides, and the lactose content did not drop below 20% (w / w) even when the enzyme reaction was further performed for a long time.

After performing activated carbon treatment and ion purification on the galactoligosaccharide mixture obtained through the enzymatic reaction, 10% (w / w) and 30 ml of the purified galactooligosaccharide mixture were subjected to a strong acid cation exchange resin UBK 530 (Na type, Mitsubishi Corporation). ) Was loaded into a column (2.6 cm inner diameter, jacket type column having a length of 40 cm), and then water, a solvent, was poured. In this case, the temperature of the jacket column was 70 ° C., and the injection speed of the sugar solution and the solvent was SV = 0.56. 5 ml of the liquid from the end of the column was separated and each sugar composition was confirmed by HPLC, and the results are shown in FIG. 5. As shown in FIG. 5, when the galactooligosaccharide fraction and the monosaccharide fraction were separated such that the purity of the galactooligosaccharide was 95% (w / w), the sugar composition of the entire composition was as shown in Table 3 below. The total yield of galactooligosaccharide fraction was 22.2%.

FIG. 1 shows the lactose content, galactoligosaccharide content and galactose content consumed when treated with two beta-galactosidase enzymes having different main catalytic reactions to lactose-containing substances.

1-a is a lactose content and the resulting galactose produced when the lactose-containing material is treated with beta-galactosidase having a high metastatic capacity of 3.2 g relative to 1 kg of substrate solids, according to an embodiment of the present invention. It is a graph showing the content of oligosaccharides and galactose according to the reaction time,

1-b shows that lactose content and galactose produced when treated with lactose-containing material alone with beta-galactosidase having a high hydrolysis capacity of 30 g relative to 1 kg of substrate solids, according to one embodiment of the present invention It is a graph showing the content of oligosaccharides and the content of galactose according to the reaction time.

2 shows the amount of lactose consumed and the amount of 4′-galactosyl lactose produced according to the content of high-transferase beta-galactosidase treated for lactose-containing substances.

Figure 2-a shows that when the lactose-containing material is treated with beta-galactosidase having a high transfer capacity of 0.25 g relative to 1 kg of substrate solids, lactose content and 4'-gal produced It is a graph showing the content of lactosyl lactose according to the reaction time,

Figure 2-b is when the lactose-containing material is treated with beta-galactosidase having a high transfer capacity of 0.8g relative to 1kg of substrate solids according to an embodiment of the present invention, the lactose content and 4'-gal produced It is a graph showing the content of lactosyl lactose according to the reaction time,

Figure 2-c shows the lactose content consumed and the 4'-gal produced when the lactose-containing material was treated with beta-galactosidase having a high metastatic capacity of 1.6 g relative to 1 kg of substrate solids according to one embodiment of the present invention. It is a graph showing the content of lactosyl lactose according to the reaction time.

3 is a galactoligosaccharide prepared by treating lactose-containing material with beta-galactosidase with high beta-galactosyl transfer ability and beta-galactosidase with high lactose hydrolysability according to an embodiment of the present invention. It is a graph which shows the chromatogram which separated the mixed liquid using Na type | mold strong acid cation exchange resin.

4 is a galactoligosaccharide mixed solution prepared by treating lactose-containing substance with beta-galactosidase having high beta-galactosyl transfer ability and beta-galactosidase having high lactose hydrolytic ability according to an embodiment of the present invention. Is a graph showing the chromatogram separated using Ca type strong acid cation exchange resin.

5 is a galactoligosaccharide mixed solution prepared by treating lactose-containing material with beta-galactosidase having high beta-galactosyl transfer ability only according to an embodiment of the present invention using Na-type strong acid cation exchange resin. It is a graph showing one chromatogram.

Claims (11)

a) The amount of unreacted lactose in the galactooligosaccharide mixture obtained by treating beta-galactosidase having high beta-galactosyl transfer ability to lactose or lactose-containing substance is 30% (w / w) based on the total sugar content. Making a mixture of galactoligosaccharides by terminating the reaction at 50% (w / w); b) The lactose oligosaccharide mixture obtained in step a) is treated with beta-galactosidase having high lactose hydrolysis ability, so that the content of unreacted lactose in the galactooligosaccharide mixture is less than 10% (w / w) based on the total sugar content. Decomposing the unreacted lactose by ending the reaction at the time; And c) separating galactooligosaccharides by performing strong acid cation exchange resin chromatography on the galactoligosaccharide mixture solution in which the unreacted lactose obtained in step b) is decomposed. A method for producing galactoligosaccharides of high purity, wherein two major beta-galactosidases, each of which contains a main catalytic reaction sequentially, are treated. The method of claim 1, The manufacturing method is galactooligosaccharide which can be produced in a yield of 40% (w / w) to 50% (w / w) galactoligosaccharides of purity 90% (w / w) to 95% (w / w) Manufacturing method. The method of claim 1, The galactooligosaccharide is a galactooligosaccharide, which is a galactoligosaccharide comprising a galactoligosaccharide comprising 35% (w / w) to 55% (w / w) of 4'-galactosyl lactose, based on the total sugar content. The method according to any one of claims 1 to 3, The lactose or lactose-containing substance has a lactose content of 20% (w / w) to 70% (w / w) based on the total composition. The method of claim 1, wherein the beta-galactosidase having a high beta-galactosyl transfer capacity in step a) is reacted with a lactose or a lactose-containing substance with a substrate, thereby providing the highest content of galactoligosaccharide in the reactant. At a visible time point, the galactooligosaccharide content in the reactant is a beta-galactosidase higher than the galactose content in the reactant. The method of claim 5, wherein the beta-galactosidase having high beta-galactosyl transfer ability is Aspergillus niger , Aspergillus oryzae , Streptococcus thermophilus Beta-galactosyl metastases derived from any one of the group consisting of Streptococcus Thermophilus , Lactobacillus bulgaricus , Cryptococcus laurentii and Bacillus circulans Galacto-oligosaccharide manufacturing method which is a galactosidase. According to claim 1, wherein the high lactose hydrolyzate beta-galactosidase of step b) proceeds with the reaction of the lactose or lactose-containing substance to the substrate, the point at which the highest content of galactoligosaccharides in the reactant , A method for producing galactooligosaccharide, which is a beta-galactosidase, wherein the content of galactose in the reactant is higher than the content of galactoligosaccharide in the reactant. The lactose hydrolyzate of claim 7, wherein the beta-galactosidase having high lactose hydrolysis ability is a galactosidase derived from Kluyveromyces lactis or Kluyeromyces fragilis . Galacto oligosaccharide manufacturing method that is a high resolution galactosidase. The galactooligosaccharide production method according to claim 1, wherein the strongly acidic cation exchange resin is a Na-type resin or a Ca-type ion exchange resin having an -SO ₃ -group as an exchanger based on a styrenedivinylbenzene copolymer. delete delete

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