KR101363293B1 - Micro reactor for synthesizing lactulose and method of synthesizing lactulose - Google Patents

Abstract

The present invention relates to a microreactor for lactulose synthesis and a method for producing lactulose, and specifically, it is economically prebiotics by reacting with a substrate including whey and fructose by beta-galactosidase immobilized in a microreactor. The present invention relates to a microreactor capable of producing lactulose, which is capable of biological production, and a method for producing lactulose.

Description

MICRO REACTOR FOR SYNTHESIZING LACTULOSE AND METHOD OF SYNTHESIZING LACTULOSE}

The present invention relates to a microreactor for lactulose synthesis and a method for producing lactulose.

Prebiotics are an indigestible substance that improves human health by selectively promoting the growth or activity of beneficial microorganisms present in the large intestine. Most of the substances showing the function of the prebiotics are saccharides, and some substances belonging to disaccharides, oligosaccharides and polysaccharides are included in this class. Lactoolulose, which is a derivative of lactose in the above disaccharides, represents a typical prebiotics function.

The lactulose is not decomposed or absorbed in the small intestine but is used by beneficial bacteria such as bifidobacteria and lactic acid bacteria present in the large intestine and is useful for the treatment and prevention of hepatic coma in constipation therapy and chronic contextual encephalopathy. It is a substance that can be safely used for a long time in the constipation of infants and the constipation during pregnancy or postpartum constipation.

At present, the production of lactulose is a chemical synthesis method and utilizes epimerization by alkali treatment. The chemical synthesis method is accompanied by an expensive multi-step purification process when it is applied as a pharmaceutical product or a special purpose food due to the production of various byproducts in addition to the reaction product lactulose, and the risk is high due to the reaction conditions at high temperature and high pressure, And the use of organic solvents may cause pollution.

On the other hand, enzymatic bioconversion synthesis has the advantage of high precision, specificity, selectivity in reaction, production in small scale devices, and environment-friendly. Therefore, in order to overcome the drawbacks of the chemical synthesis method, the enzyme which is a biocatalyst can replace the alkali catalyst.

However, in the bioconversion synthesis method, refined lactose is used as a substrate, resulting in low economic efficiency and lack of a continuous process.

Korean Patent Publication No. 1989-0009958 Korean Patent Publication No. 1992-0000779

The present invention is to solve the problems of the prior art as described above, it is an object of the present invention to provide a micro-reactor fixed to beta-galactosidase to synthesize lactulose continuously using whey and fructose as a substrate.

It is another object of the present invention to provide a method for continuously synthesizing lactulose by hydrolysis and sugar transfer reaction by immobilized beta-galactosidase.

The present invention provides a micro-reactor for lactulose synthesis in which beta-galactosidase to which a substrate including whey and fructose is reacted is immobilized using a multi-walled carbon nanotube as a linking group.

The present invention comprises the steps of preparing a reaction aqueous solution containing beta-galactosidase, buffer and lactose; Fixing the beta-galactosidase to the microreactor by injecting a solution containing the reaction aqueous solution and multi-walled carbon nanotubes into a microreactor; And it provides a lactulose production method comprising the step of reacting the substrate containing whey and fructose into the micro reactor.

The microreactor for lactulose synthesis of the present invention is economical by using whey, instead of expensive refined lactose, which is a substrate used for conventional lactulose production, and enables continuous production of lactulose.

In addition, the lactolulose production method of the present invention can improve the economical efficiency of the process by continuously synthesizing lactolose, and can recycle resources by actively utilizing lactose-containing waste liquid and the like, Can be used in the first half.

Further, by using whey as a substrate, it is possible to increase the utilization value of waste whey to be discarded, to reduce the cost of disposal and environmental purification, and to reduce the production cost of lactulose.

1 is a plan view showing a microreactor of the present invention.
Figure 2 is a schematic diagram showing a micro reaction system including a micro reactor of the present invention.
Figure 3 is a photograph showing the immobilization of beta-galactosidase on the surface of the microreactor of the present invention.
Figure 4 is a photograph showing the immobilization of beta-galactosidase on the surface of the microreactor of the present invention.
5 is a graph showing the concentration of lactulose according to the flow rate of the substrate injected into the microreactor.
6 is a graph showing the concentration of substrate and reaction product over time.

Hereinafter, the present invention will be described in detail.

The present invention provides a micro-reactor for lactulose synthesis in which beta-galactosidase to which a substrate including whey and fructose is reacted is immobilized using a multi-walled carbon nanotube as a linking group.

Whey is a remnant liquid after it has been clotted with milk and is also called whey or whey. It is usually produced as a by-product in the cheese or casein manufacturing process and is used for several commercial uses. Lactose, also known as lactose and lactose, is a typical carbohydrate in milk and is a disaccharide composed of one molecule of glucose and one molecule of lactose.

Galactosidase is an enzyme that hydrolyzes allyl and alkyl-D-galactoside to produce galactose. In addition to hydrolysis, it also acts as a galactosid transferase to transfer galactose residues to other sugars, alcohols and phenols. The galactosidase is classified into alpha-type and beta-type, and the beta-galactosidase is also referred to as lactase and acts on lactose in addition to beta-D-galactoside.

One of the prebiotics, an indigestible substance that selectively promotes the growth or activity of beneficial microorganisms present in the large intestine to improve human health, lactulose, which is not degraded or absorbed in the small intestine, It is mainly used for bifida bacterium and some lactic acid bacteria, and it is used for the treatment and prevention of hepatic coma in constipation therapy and chronic contextual encephalopathy. Especially, it can be safely used for constipation and postpartum constipation It is a substance.

The beta-galactosidase is immobilized on the surface of the microreactor to which the multi-walled carbon nanotubes, one of the carbon nanotubes, are adsorbed, and the multi-walled carbon nanotubes serve as a linker of the beta-galactosidase. do. Microreactors in which beta-galactosidase are immobilized can produce lactulose, one of the prebiotics, using whey as a substrate.

Specifically, the beta-galactosidase immobilized substrate containing whey and fructose is hydrolyzed to glucose and galactose by lactose contained in whey, and the lactulose reaction is performed by a sugar transfer reaction between galactose and fructose. Oss can be synthesized.

Through the immobilization of the beta-galactosidase, continuous production of lactulose is possible. In addition, it is possible to improve the economics by replacing expensive purified lactose with whey as a substrate that has been conventionally used for the synthesis of lactulose.

The whey contained in the substrate preferably contains lactose, that is, lactose at 10 to 30% (w / v). If the above-mentioned range is satisfied, the productivity of lactulose may be improved, under high temperature conditions. When the concentration of lactose exceeds 30% (w / v), there is a problem that byproducts are generated by the addition reaction, thereby lowering the productivity of lactulose.

The present invention comprises the steps of preparing a reaction aqueous solution containing beta-galactosidase, buffer and lactose; Fixing the beta-galactosidase to the microreactor by injecting a solution containing the reaction aqueous solution and multi-walled carbon nanotubes into a microreactor; And it provides a lactulose production method comprising the step of reacting the substrate containing whey and fructose into the micro reactor.

Specifically, in the pretreatment of the beta-galactosidase in order to immobilize the beta-galactosidase in the micro-reactor, a reaction aqueous solution containing beta-galactosidase, buffer and lactose is prepared. The buffer solution is preferably a phosphate buffer solution, and lactose is used as a pretreatment material. The lactose acts to protect the active site of the enzyme through reaction with beta-galactosidase. In other words, when not subjected to the pretreatment process by lactose, there is a problem in that covalent bonds are formed with multi-walled carbon nanotubes at the active site of the beta-galactosidase, thereby lowering the activity of the enzyme. Therefore, in order to overcome this, beta-galactosidase and lactose may be pretreated to undergo covalent bonding and dissociation at the active site, whereby multi-walled carbon nanotubes may form covalent bonds at sites other than the active site.

In the next step, the reaction aqueous solution prepared by pretreating the beta-galactosidase comprises mixing multi-walled carbon nanotubes and injecting them into a microreactor to immobilize the beta-galactosidase on the surface of the microreactor. At this time, the beta-galactosidase and multi-walled carbon nanotubes were mixed, and N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (N- (3-dimethylaminopropyl) -N'- It is preferable to add ethylcarbodiimide hydrochloride (EDC.HCl) and react.

The solution containing the beta-galactosidase is preferably immobilized in the microreactor by injecting it into the microreactor at a flow rate of 1 to 10µl / min at a temperature of 10 to 30 ° C.

The whey contained in the substrate preferably contains lactose, that is, lactose at 10 to 30% (w / v). If the above-mentioned range is satisfied, the productivity of lactulose may be improved, under high temperature conditions. When the concentration of lactose exceeds 30% (w / v), there is a problem that byproducts are generated by the addition reaction, thereby lowering the productivity of lactulose.

The step of injecting the substrate into the micro reactor to react the reaction is preferably performed at 37 to 57 ° C., and when the above range is satisfied, beta-galactosidase immobilized in the micro reactor is active as the substrate. The efficiency of conversion to lactulose is excellent.

It is preferred to inject the substrate into the micro reactor at a rate of 0.1 to 20 μl / min. If the above range is satisfied, the residence time of the substrate in the microreactor is increased to sufficiently react with the enzyme beta-galactosidase, thereby increasing the productivity of lactulose.

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

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1>

1. Micro reactor manufacture

1 is a plan view of a micro reactor, in which a microchannel is formed through etching of glass. That is, as shown in FIG. 1, twelve microchannels having a width of 390 μm, a depth of 150 μm, and a length of 13 mm were etched, and both ends of the microreactor were provided with an injection part and a discharge part having a diameter of 1 mm.

As a cover for protecting the microchannel, a piece of polydimethylsiloxane exposed to an oxygen plasma was used, and the microchannel and the polydimethylsiloxane fragment were brought into close contact with each other and heated at 80 ° C. for 15 minutes. Produced.

2. Functionalized Multiwall  Carbon nanotubes ( functionalized multi - walled carbon  manufacturing of nanotubes)

200 mg of multi-walled carbon nanotubes (Sigma, USA) were added to a solution of sulfuric acid and nitric acid in a volume ratio of 3: 1 and sonicated at 40 ° C. for 10 hours. Thereafter, the solution was washed several times with deionized water to prepare a multi-walled carbon nanotube solution functionalized with a carboxyl group.

3. Beta- Galactosidase  Pretreatment

10 mM (v / v) beta-galactosidase (Sigma, USA) derived from Kluyveromyces lactis and 100 mM lactose in 100 mM phosphate buffer (pH 7.2) 20 mL of reaction mixture was prepared by the addition. The reaction aqueous solution was reacted at 37 DEG C and 150 rpm for 1 hour to pre-treat beta-galactosidase.

4. Beta-in micro reactor Galactosidase  Immobilization

For immobilization of beta-galactosidase in the microreactor, an acetone solution of 15% (v / v) 3-aminopropyltriethoxysilane was added to the inside of the microreactor. Injected at a flow rate of 50 µl per minute for 2 hours at ℃, and then washed by distilled water was injected at a flow rate of 50 µl per minute for 1 hour at 25 ℃.

To a solution of 10 mL of a functionalized multi-walled carbon nanotube solution and 10 mL of the pretreated beta-galactosidase solution, 0.05 M of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (N -(3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC-HCl) was added and reacted at 4 ° C for 8 hours.

The mixed solution was injected into the micro reactor at a flow rate of 5 μl / min for 12 hours at 20 ° C., followed by injection of 100 mM phosphate buffer solution (pH 7.2) at a flow rate of 50 μl / min for 1 hour at 25 ° C. The reactor interior was washed to fix beta-galactosidase in the micro reactor.

5. Whey  Contained Of lactose solution  Produce

200 g of dried whey (Sigma, USA) was mixed with 100 mL of 50 mM phosphate buffer (pH 7.5), and the mixed solution was centrifuged at 10,000 rpm for 20 minutes. The supernatant was transferred to a new beaker and adjusted to pH 7.5. The acidity-adjusted solution was again centrifuged at 10,000 rpm for 20 minutes to prepare a supernatant, that is, a lactose solution (20%, w / v) for lactulose synthesis.

6. Micro Reaction System Including Micro Reactor

2 is a schematic diagram of a micro reaction system including a micro reactor. As shown in FIG. 2, the micro reaction system includes a syringe pump, a micro reactor and a water bath, and the flow of all the fluid entering the micro reactor is maintained constant by the syringe pump. And, the temperature control of the micro reactor can be controlled by a constant temperature water bath.

< Experimental Example  1: Surface Analysis of Micro Reactors>

Before and after immobilization of the beta-galactosidase on the surface of the microreactor of Example 1 was observed using a field emission scanning electron microscope (FE-SEM). As shown in FIGS. 3 and 4, the functionalized multi-walled carbon nanotubes were covered with beta-galactosidase to significantly reduce the roughness of the surface of the microchannels, thereby reducing the surface of the microreactor. It was confirmed that beta-galactosidase was immobilized.

< Experimental Example  2: change in flow rate Lactuloses  Effect on synthesis>

For continuous synthesis of lactulose in a micro-reactor, 20% (w / v) fructose was added to the 20% (w / v) lactose solution prepared in Example 1 to prepare a substrate solution.

The substrate solution was injected into the microreactor at flow rates of 1.0, 2.5, 5.0, 7.5 and 10.0 μl per minute at 47 ° C. to perform lactulose synthesis.

At this time, the concentrations of the lactose, fructose and fructose, and the products of glucose, galactose and lactulose were measured using high-performance liquid chromatography (HPLC). A high performance carbohydrate column (4.6 mm x 250 mm, Waters Co., USA) and a refractive index detector (RI detector) were used. The temperature of the column oven and the flow rate of the mobile phase were 35 ° C, And the mobile phase was prepared by using a solution having a ratio of deionized water to acetonitrile of 8: 2 (v / v).

As a result, as shown in FIG. 5, the synthesized lactulose gradually decreased with increasing flow rate. As a result, the longer the residence time of the substrate in the micro reactor, the better the yield.

< Experimental Example  3: Effect of long-term operation of enzyme microreactor on substrate consumption and product production>

A substrate solution was prepared in which 20% (w / v) lactose solution and 20% (w / v) fructose were mixed for continuous synthesis of lactulose in the microreactor of Example 1, and the substrate was 1.0 μl / min. Lactulose synthesis was performed for 48 hours at 47 ° C. at a flow rate, and the concentrations of lactose, fructose, glucose, galactose and lactulose were measured.

As a result, as shown in FIG. 6, the concentrations of the lactose and fructose, which are reactive substances, were decreased by the hydrolysis reaction and the sugar transfer reaction, whereas the concentrations of glucose, galactose, and lactulose which were hydrolyzate and the final reaction product were increased. It was.

Therefore, it was confirmed that the micro reactor was operated very stably for 48 hours, and showed an average of 1.29 g / L lactulose synthesis.

Through this, it was confirmed that lactulose can be continuously synthesized from lactose contained in whey using an enzyme microreactor immobilized with beta-galactosidase.

11: micro reactor 111: injection section
112: outlet 113: micro channel
114: wall 12: syringe pump
13: constant temperature water bath 14: reaction product

Claims (6)

A micro-reactor for lactulose synthesis, in which beta-galactosidase to which a substrate including whey and fructose is reacted is immobilized using a multi-walled carbon nanotube as a linkage. The micro-reactor for lactulose synthesis of claim 1, wherein the whey contains 10 to 30% (w / v) of lactose. Preparing an aqueous reaction solution comprising beta-galactosidase, a buffer solution and a lactose;
Fixing the beta-galactosidase to the microreactor by injecting a solution containing the reaction aqueous solution and multi-walled carbon nanotubes into a microreactor; And
Lactulose production method comprising the step of reacting the substrate containing whey and fructose into the micro reactor. 4. The method of claim 3, wherein the whey comprises lactose of 10 to 30% (w / v). The method of claim 3, wherein the step of injecting the substrate into the micro reactor to react the reaction at 37 to 57 ℃ temperature conditions. The method of claim 3, wherein the substrate is injected into the micro reactor at a rate of 0.1 to 20μl / min.

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