Method for preparing lactulose using immobilized β-galactosidase

Abstract

Provided is a method for preparing lactulose using immobilized β-galactosidase. The method comprises the following steps: pretreating a macroporous weakly basic styrene-based anion exchange resin; mixing the pretreated resin with a β-galactosidase solution and adding same to a buffer, adsorbing same using a shaker, and leaving same to stand after adsorption; then adding a glutaraldehyde solution for cross-linking, and discarding the supernatant to obtain an immobilized enzyme column; adding a magnesium ion solution to a mixed solution of lactose and fructose, and then converting same by means of the immobilized enzyme column to obtain a lactulose conversion solution; and sequentially performing decolorization, ion exchange, vacuum concentration, and sequential simulated moving bed chromatography separation on the lactulose conversion solution to obtain a lactulose solution.

Description

A method for preparing lactulose using immobilized β-galactosidase

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411650854.4, filed on November 19, 2024, entitled “A method for preparing lactulose by immobilized β-galactosidase”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of functional sugar preparation technology, and in particular to a method for preparing lactulose by immobilizing β-galactosidase. Background Technology

[0004] Lactulose (C12H12O11; FW: 342.30 Da), also known as 4-O-β-D-galactopyranosyl-D-fructose, is a non-absorbable disaccharide composed of D-galactose and D-fructose linked by a β-1,4 glycosidic bond. Lactulose is a white, irregularly crystalline powder. Pure lactulose has a pure sweetness with a cool, mellow taste, and its sweetness is 48%-62% that of sucrose. It cannot be metabolized by digestive enzymes in animals and can therefore be directly fermented by microorganisms in the gastrointestinal tract. It is known as a "Bifidobacterium growth factor" and has beneficial functions such as regulating intestinal flora balance, serving as an indirect nutritional supplement, treating constipation, lowering blood ammonia, enhancing immunity, and reducing blood endotoxin levels. It is widely used in the food, pharmaceutical, and animal feed industries.

[0005] Currently, the main methods for producing lactulose are chemical and enzymatic methods. Chemical methods for preparing isomerized lactose are simple to operate and have a high conversion rate. However, the final product generally includes lactulose, residual lactose, D-galactose, and D-fructose, which are not only difficult to separate but also often have a dark color. The generation of byproducts and pigments further reduces the yield of lactulose. While enzymatic synthesis of lactulose can overcome the shortcomings of chemical synthesis, its low conversion rate severely limits the large-scale production and widespread application of this product. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a method for preparing lactulose using immobilized β-galactosidase. The preparation process of this invention uses immobilized β-galactosidase with a relative enzyme activity of 65-78%, and converts lactose and fructose as raw materials. Finally, the obtained lactulose solution is separated by chromatography using a cation exchange resin column-decolorizing resin column-anion exchange resin column to obtain lactulose with a purity of 97% and a conversion rate of ≥60%.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing lactulose using immobilized β-galactosidase, comprising the following steps:

[0009] S1. Preparation of immobilized enzyme column:

[0010] Amberlite IRA-93 macroporous weakly basic styrene-based anion exchange resin was sequentially soaked in ethanol solution, NaCl solution, HCl solution, and NaOH solution. After each soaking, the soaking solution was filtered out and the resin was washed with deionized water until neutral to obtain pretreated resin.

[0011] The pretreated resin was mixed with β-galactosidase enzyme solution, Tris-hCl buffer was added and the mixture was shaken for adsorption. After adsorption, the mixture was allowed to stand. Then, glutaraldehyde solution was added for cross-linking, and the supernatant was discarded to obtain the immobilized enzyme column.

[0012] S2. Add magnesium ion solution to a mixture of lactose and fructose and then convert it through an immobilized enzyme column to obtain lactulose conversion solution;

[0013] S3. The lactulose inversion solution is subjected to decolorization, ion exchange, vacuum concentration, and continuous simulated moving bed chromatography to obtain a lactulose solution.

[0014] Preferably, in S1, the concentration of the ethanol solution is 95 wt%; the concentration of the NaCl solution is 10 wt%; the concentration of the HCl solution is 10 wt%; and the concentration of the NaOH solution is 10 wt%.

[0015] Preferably, the soaking time in S1 is 10 to 20 hours.

[0016] More preferably, the soaking time for the ethanol solution is 15-20 hours, the soaking time for the NaCl solution is 10-15 hours, the soaking time for the HCl solution is 10-15 hours, and the soaking time for the NaOH solution is 10-15 hours.

[0017] Preferably, the mass-to-volume ratio of the pretreated resin to the β-galactosidase solution in S1 is 1 g: 5-7 mL; more preferably, it is 1 g: 5 mL.

[0018] Preferably, the concentration of Tris-hCl buffer in S1 is 50 mmol / L and the pH is 8.0; the concentration of glutaraldehyde solution is 2 wt%.

[0019] Preferably, the relative enzyme activity of β-galactosidase in the immobilized enzyme column in S1 is 65-78%.

[0020] Preferably, the process parameters in S1 satisfy one or more of the following conditions:

[0021] The adsorption time was 10–13 h, and the adsorption temperature was 28–32 °C.

[0022] The settling time is 25–35 minutes, and the settling temperature is 2–5℃;

[0023] The cross-linking time is 5.5–6.5 h, and the cross-linking temperature is 23–28 °C.

[0024] Preferably, the mass concentration of the lactose and fructose mixture in S2 is 50-55%, more preferably 50%; the molar ratio of lactose to fructose is 1:1 to 1:1.5, more preferably 1:1.

[0025] The pH of the lactose and fructose mixture is further preferably 7.

[0026] Preferably, the magnesium ion concentration in the mixed liquid system after the addition of magnesium ions in S2 is 1 mM; the magnesium ion source is MgCl2 or MgSO4.

[0027] Preferably, in step S2, the flow rate of the lactose and fructose mixture through the immobilized enzyme column is 2.0 BV / hour, and the temperature is 60–70°C, more preferably 70°C.

[0028] Preferably, each process in S3 is one or more of the following:

[0029] Decolorization was performed using a granular activated carbon column. Activated carbon was added to the lactulose solution and stirred at 70-90℃ for 20-40 minutes. The amount of activated carbon added was 1-2% of the dry weight of lactose. The solution was then filtered through an Ama filter at a working pressure of 0.2-0.4 MPa to obtain a decolorized lactose solution.

[0030] The ion exchange process involves sequentially passing the decolorized material through a cation exchange column, a decolorizing resin column, and an anion exchange column. The material temperature is 65-70℃, and the flow rate is 2 BV / h. The cation exchange resin is LX-150 strong acid cation exchange resin, the decolorizing resin is D750 macroporous adsorption resin, and the anion exchange resin is LX-360 weak base anion exchange resin.

[0031] The vacuum concentration process uses a six-effect evaporator to concentrate the ion-exchange material, with a vacuum degree of 0.06-0.09 MPa and a concentration temperature of 50-80℃.

[0032] The continuous simulated moving bed chromatography separates water and material in a ratio of 2:1 to 5:1, with a feed rate of 0.04 to 0.08 kg / L resin / h, a temperature of 65 to 70°C, a switching time of 1080 s, and a feed flow rate of 0.35 to 0.45 L / h. The simulated moving bed uses calcium-type cation exchange resin.

[0033] Beneficial technical effects:

[0034] 1. This invention immobilizes β-galactosidase and uses a resin-based immobilized enzyme column to ensure full contact between the enzyme and the material, eliminating the need for enzyme inactivation. The relative enzyme activity of β-galactosidase reaches 65-78%. By immobilizing β-galactosidase, the optimal temperature range of the enzyme is improved, with the reaction temperature reaching 60-70℃. This temperature range can avoid contamination by miscellaneous bacteria during production and promote the forward reaction of the enzyme preparation.

[0035] 2. This invention continuously enhances the activity of β-galactosidase by adding magnesium ion solution to the substrate, thereby prolonging the use of β-galactosidase. The lactulose conversion rate is ≥60%, the lactulose yield is ≥92%, and the lactulose solution obtained has an ipilactose content of ≤3.5%.

[0036] 3. This invention uses a series of cation-decolorizing resin-anion resin for decolorization, which increases the transmittance of the solution to ≥90%, significantly reduces the color of the material to ≤100 RBU, simplifies the refining process, and is beneficial for industrial production. Detailed Implementation

[0037] The present invention will now be described in detail through specific embodiments. These embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the reagents and materials used are commercially available.

[0038] Amberlite IRA-93 macroporous weakly basic styrene-based anion exchange resin was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.; β-galactosidase (produced from Aspergillus oryzae BLCY-006, deposited on December 5, 2018 at the China General Microbiological Culture Collection Center, accession number CGMCC No. 16965).

[0039] Example 1:

[0040] (1) Soak the resin in 95% ethanol solution (2 times the volume of Amberlite IRA-93 macroporous weakly basic styrene-based anion exchange resin) for 18 hours. After filtering out the ethanol solution, wash the Amberlite IRA-93 macroporous weakly basic styrene-based anion exchange resin with deionized water. Then soak the resin in 10% NaCl solution for 12 hours. After filtering out the NaCl solution, wash the resin with deionized water until the solution pH is 7. Then soak the resin in 2% HCl solution for 12 hours. After filtering out the HCl solution, wash the resin with deionized water until the solution pH is 7. Then soak the resin in 5% NaOH solution for 12 hours. After filtering out the NaOH solution filtrate, wash the resin with deionized water until the solution pH is 7. Filter out the excess liquid to complete the resin pretreatment.

[0041] (2) The pretreated resin and the β-galactosidase enzyme solution to be immobilized were mixed in a certain proportion, and Tris-hCl buffer (pH 8.0) with a mass concentration of 50 mmol / L was added. The mixture was adsorbed in a shaker at 30℃ for 12 h, and then allowed to stand at 4℃ for 30 min. Then, 2% glutaraldehyde solution was added for cross-linking at a mass concentration of 25℃ for 6 h to complete the preparation of immobilized β-galactosidase. After immobilization, the supernatant was discarded to obtain the immobilized enzyme. The volume-to-mass ratio of the enzyme solution to the resin was 1 g: 5 mL.

[0042] Enzyme activity assay: Prepare a 1 mg / mL solution of o-nitrobenzene-β-D-galactoside (ONPG) using phosphate buffer (pH 7.0, 20 mmol / L) as solvent; preheat the ONPG solution at 50 °C for 10 min; take 3 mL of ONPG solution, add 0.1 g of immobilized enzyme, and react at 50 °C for 15 min; add 2 mL of 1 mol / L Na2CO3 aqueous solution to the reacted solution to inactivate the enzyme; measure the absorbance at 420 nm.

[0043] Enzyme activity unit (U): Under certain conditions, the amount of enzyme required for β-galactosidase to hydrolyze ONPG to generate 1 μmol of o-nitrophenol (ONP) in 1 min.

[0044] When the ratio of resin to added enzyme was 1g:5mL, the relative enzyme activity was 77.89% according to the enzyme activity assay.

[0045] (3) The immobilized enzyme described in step (2) is packed into an immobilization column. A mixture of lactose and fructose is passed through the immobilization column, and MgCl2 solution is added to the mixture (the magnesium ion concentration in the mixture after addition is 1 mM) to obtain a lactulose inversion solution. The mass concentration of the lactose and fructose mixture is 50%, and the molar ratio of lactose to fructose is 1:1. The flow rate of the lactose and fructose mixture is 2.0 BV / hour, the temperature is 70℃, and the pH of the lactose and fructose mixture is 7.

[0046] (4) The lactulose inversion solution from step (3) is decolorized, ion-exchanged, vacuum concentrated, and separated by continuous simulated moving bed chromatography to obtain a lactulose solution.

[0047] Decolorization was performed using a granular activated carbon column. Activated carbon was added to the lactulose solution and stirred at 80°C for 30 minutes. The amount of activated carbon added was 2% of the dry weight of the lactulose. The solution was then filtered through an Ama filter at an operating pressure of 0.3 MPa to obtain a decolorized lactulose solution with a color of 2382.15 RBU.

[0048] The ion exchange process involves sequentially passing the decolorized material through a cation exchange column, a decolorizing resin column, and an anion exchange column. The material temperature is 70°C, and the flow rate is 2 BV / h. The cation exchange resin is LX-150 strong acid cation exchange resin; the decolorizing resin is D750 macroporous adsorption resin; and the anion exchange resin is LX-360 weak base anion exchange resin. The solution color is 20.25 RBU.

[0049] The vacuum concentration process uses a six-effect evaporator to concentrate the ion-exchange material, with a vacuum degree of 0.09 MPa and a concentration temperature of 75°C.

[0050] The continuous simulated moving bed separation water-to-material ratio is 3:1, the feed rate is 0.06 kg / L resin / h, the temperature is 70℃, the switching time is 1080 s, and the feed flow rate is 0.4 L / h. The simulated moving bed uses calcium-type cation exchange resin.

[0051] Under the conditions of this embodiment, the lactulose conversion rate is 68%, the lactulose yield is 97%, the obtained lactulose solution has an ipilactose content of 3.0%, and the transmittance of the solution is 92%.

[0052] Example 2:

[0053] (1) Soak the resin in 95% ethanol solution (2 times the volume of Amberlite IRA-93 macroporous weakly basic styrene-based anion exchange resin) for 15 hours. After filtering out the ethanol solution, wash the Amberlite IRA-93 macroporous weakly basic styrene-based anion exchange resin with deionized water. Then soak the resin in 10% NaCl solution for 12 hours. After filtering out the NaCl solution, wash the resin with deionized water until the solution pH is 7. Then soak the resin in 2% HCl solution for 12 hours. After filtering out the HCl solution, wash the resin with deionized water until the solution pH is 7. Then soak the resin in 5% NaOH solution for 12 hours. After filtering out the NaOH solution filtrate, wash the resin with deionized water until the solution pH is 7. Filter out the excess liquid to complete the resin pretreatment.

[0054] (2) The pretreated resin and the β-galactosidase enzyme solution to be immobilized were mixed in a certain proportion, and Tris-hCl buffer (pH 8.0) with a mass concentration of 50 mmol / L was added. The mixture was adsorbed in a shaker at 30℃ for 12 h, and then allowed to stand at 4℃ for 30 min. Then, 2% glutaraldehyde solution was added for cross-linking at a mass concentration of 25℃ for 6 h to complete the preparation of immobilized β-galactosidase. After immobilization, the supernatant was discarded to obtain the immobilized enzyme. The volume-to-mass ratio of the enzyme solution to the resin was 1 g: 7 mL.

[0055] Enzyme activity assay: Prepare a 1 mg / mL solution of o-nitrobenzene-β-D-galactoside (ONPG) using phosphate buffer (pH 7.0, 20 mmol / L) as solvent; preheat the ONPG solution at 50 °C for 10 min; take 3 mL of ONPG solution, add 0.1 g of immobilized enzyme, and react at 50 °C for 15 min; add 2 mL of 1 mol / L Na2CO3 aqueous solution to the reacted solution to inactivate the enzyme; measure the absorbance at 420 nm.

[0056] Enzyme activity unit (U): Under certain conditions, the amount of enzyme required for β-galactosidase to hydrolyze ONPG to generate 1 μmol of o-nitrophenol (ONP) in 1 min.

[0057] When the ratio of resin to added enzyme was 1g:7mL, the relative enzyme activity was 67.42% according to the enzyme activity assay.

[0058] (3) The immobilized enzyme described in step (2) is packed into an immobilization column. The lactose and fructose mixture is passed through the immobilization column, and MgCl2 solution is added to the mixture (the magnesium ion concentration in the mixture after addition is 1 mM) to obtain a lactulose inversion solution. The mass concentration of the lactose and fructose mixture is 55%, and the molar ratio of lactose to fructose is 1:1.5. The flow rate of the lactose and fructose mixture is 2.0 BV / hour, the temperature is 60℃, and the pH of the lactose and fructose mixture is 7.

[0059] (4) The lactulose inversion solution from step (3) is decolorized, ion-exchanged, vacuum concentrated, and separated by continuous simulated moving bed chromatography to obtain a lactulose solution.

[0060] Decolorization was performed using a granular activated carbon column. Activated carbon was added to the lactulose solution and stirred at 90°C for 20 minutes. The amount of activated carbon added was 1.5% of the dry weight of the lactulose. The solution was then filtered through an Ama filter at an operating pressure of 0.2 MPa to obtain a decolorized lactulose solution with a color of 2489.67 RBU.

[0061] The ion exchange process involves sequentially passing the decolorized material through a cation exchange column, a decolorizing resin column, and an anion exchange column. The material temperature is 70°C, and the flow rate is 2 BV / h. The cation exchange resin is LX-150 strong acid cation exchange resin; the decolorizing resin is D750 macroporous adsorption resin; and the anion exchange resin is LX-360 weak base anion exchange resin. The solution color is 39.75 RBU.

[0062] The vacuum concentration process uses a six-effect evaporator to concentrate the ion-exchange material, with a vacuum degree of 0.06 MPa and a concentration temperature of 65°C.

[0063] The continuous simulated moving bed chromatography separation has a water-to-material ratio of 5:1, a feed rate of 0.08 kg / L resin / h, a temperature of 65℃, a switching time of 1080 s, and a feed flow rate of 0.35 L / h. The simulated moving bed uses calcium-type cation exchange resin.

[0064] Under the conditions of this embodiment, the lactulose conversion rate is 65%, the lactulose yield is 94%, the obtained lactulose solution has an ipilactose content of 3.1%, and the transmittance of the solution is 91%.

[0065] Example 3:

[0066] (1) Soak the resin in 95% ethanol solution (2 times the volume of Amberlite IRA-93 macroporous weakly basic styrene-based anion exchange resin) for 20 hours. After filtering out the ethanol solution, wash the Amberlite IRA-93 macroporous weakly basic styrene-based anion exchange resin with deionized water. Then soak the resin in 10% NaCl solution for 12 hours. After filtering out the NaCl solution, wash the resin with deionized water until the solution pH is 7. Then soak the resin in 2% HCl solution for 12 hours. After filtering out the HCl solution, wash the resin with deionized water until the solution pH is 7. Then soak the resin in 5% NaOH solution for 12 hours. After filtering out the NaOH solution filtrate, wash the resin with deionized water until the solution pH is 7. Filter out the excess liquid to complete the resin pretreatment.

[0067] (2) The pretreated resin and the β-galactosidase enzyme solution to be immobilized were mixed in a certain proportion, and Tris-hCl buffer (pH 8.0) with a mass concentration of 50 mmol / L was added. The mixture was adsorbed in a shaker at 30℃ for 12 h, and then allowed to stand at 4℃ for 30 min. Then, 2% glutaraldehyde solution was added for cross-linking at a mass concentration of 25℃ for 6 h to complete the preparation of immobilized β-galactosidase. After immobilization, the supernatant was discarded to obtain the immobilized enzyme. The volume-to-mass ratio of the enzyme solution to the resin was 1 g: 6 mL.

[0068] Enzyme activity assay: Prepare a 1 mg / mL solution of o-nitrobenzene-β-D-galactoside (ONPG) using phosphate buffer (pH 7.0, 20 mmol / L) as solvent; preheat the ONPG solution at 50 °C for 10 min; take 3 mL of ONPG solution, add 0.1 g of immobilized enzyme, and react at 50 °C for 15 min; add 2 mL of 1 mol / L Na2CO3 aqueous solution to the reacted solution to inactivate the enzyme; measure the absorbance at 420 nm.

[0069] Enzyme activity unit (U): Under certain conditions, the amount of enzyme required for β-galactosidase to hydrolyze ONPG to generate 1 μmol of o-nitrophenol (ONP) in 1 min.

[0070] When the ratio of resin to added enzyme was 1g:6mL, the relative enzyme activity was 69.82% according to the enzyme activity assay.

[0071] (3) The immobilized enzyme described in step (2) is packed into an immobilization column. The lactose and fructose mixture is passed through the immobilization column, and MgCl2 solution is added to the mixture (the magnesium ion concentration in the mixture is 1 mM after addition) to obtain a lactulose inversion solution. The mass concentration of the lactose and fructose mixture is 50%, the molar ratio of lactose to fructose is 1:1.5, the flow rate of the lactose and fructose mixture is 2.0 BV / hour, the temperature is 65℃, and the pH of the lactose and fructose mixture is 7.

[0072] (4) The lactulose inversion solution from step (3) is decolorized, ion-exchanged, vacuum concentrated, and separated by continuous simulated moving bed chromatography to obtain a lactulose solution.

[0073] Decolorization was performed using a granular activated carbon column. Activated carbon was added to the lactulose solution and stirred at 70°C for 40 minutes. The amount of activated carbon added was 1% of the dry weight of the lactulose. The solution was then filtered through an Ama filter at an operating pressure of 0.4 MPa to obtain a decolorized lactulose solution with a color of 2478.65 RBU.

[0074] The ion exchange process involves sequentially passing the decolorized material through a cation exchange column, a decolorizing resin column, and an anion exchange column. The material temperature is 65°C, and the flow rate is 2 BV / h. The cation exchange resin is LX-150 strong acid cation exchange resin; the decolorizing resin is D750 macroporous adsorption resin; and the anion exchange resin is LX-360 weak base anion exchange resin. The solution color is 45.66 RBU.

[0075] The vacuum concentration process uses a six-effect evaporator to concentrate the ion-exchange material, with a vacuum degree of 0.08 MPa and a concentration temperature of 65°C.

[0076] The continuous simulated moving bed chromatography separation has a water-to-material ratio of 2:1, a feed rate of 0.04 kg / L resin / h, a temperature of 70℃, a switching time of 1080 s, and a feed flow rate of 0.45 L / h. The simulated moving bed uses calcium-type cation exchange resin.

[0077] Under the conditions of this embodiment, the lactulose conversion rate is 63%, the lactulose yield is 92%, the obtained lactulose solution has an ipilactose content of 3.5%, and the transmittance of the solution is 90%.

[0078] Comparative Example 1:

[0079] This comparative example prepares lactulose according to the preparation method of Example 1, except that in step 4, the ion exchange process involves passing the decolorized material sequentially through a cation exchange column and an anion exchange column. All other steps and operations are the same as in Example 1.

[0080] The resulting solution had a color of 1678.59 RBU. Under these comparative conditions, the lactulose conversion rate was 68%, the lactulose yield was 97%, the lactulose solution had an ipilactose content of 3.3%, and the transmittance of the solution was 75%.

[0081] Comparative Example 2:

[0082] This comparative example prepares lactulose according to the preparation method of Example 1, except that in step 4, the ion exchange process involves passing the decolorized material sequentially through a cation exchange column, an anion exchange column, and a decolorizing resin column. All other steps and operations are the same as in Example 1.

[0083] The resulting solution had a color of 1234.88 RBU. Under these comparative conditions, the lactulose conversion rate was 68%, the lactulose yield was 97%, the lactulose solution had an ipilactose content of 3.4%, and the transmittance of the solution was 79%.

[0084] Comparative Example 3:

[0085] This comparative example prepared lactulose according to the preparation method of Example 1, except that in step 3, the mass concentration of the lactose and fructose mixture was 45%, and other metal ions were added to the lactose and fructose mixture (after addition, the concentration of other metal ions in the mixture was 1 mM, excluding Mg). 2+ Except for those in Table 1, all other steps and operations are the same as in Example 1.

[0086] The results of the obtained lactulose are shown in Table 1 below. The results show that, compared with Example 1, other metal ions have poor activation of β-galactosidase, which reduces the conversion rate of lactulose.

[0087] Table 1. Conversion rate of lactulose after adding different metal ions

[0088] Comparative Example 4:

[0089] This comparative example prepares lactulose according to the preparation method of Example 1, except that the mass concentration of the lactose and fructose mixture in step 3 is 60%, and no metal ions are added to the lactose and fructose mixture. All other steps and operations are the same as in Example 1.

[0090] Under these comparative conditions, the lactulose conversion rate was 53%, the lactulose yield was 82%, the obtained lactulose solution had an ipilactose content of 5.2%, and the transmittance of the solution was 85%.

[0091] Comparative Example 5:

[0092] This comparative example prepared lactulose according to the preparation method of Example 1, except that in step 2, the volume-to-mass ratio of enzyme solution to resin was fixed at 1g:8mL, and the relative enzyme activity was 62.44%.

[0093] In step 3, the molar ratio of lactose to fructose is 1:0.8, and in step 4, the material temperature for ion exchange is 60°C. All other steps and operations are the same as in Example 1.

[0094] Under these comparative conditions, the lactulose conversion rate was 48%, the lactulose yield was 80%, the obtained lactulose solution had an ipilactose content of 4.7%, and the transmittance of the solution was 88%.

[0095] Comparative Example 6:

[0096] This comparative example prepared lactulose according to the preparation method of Example 1, except that in step 2, the volume-to-mass ratio of enzyme solution to resin was fixed at 1g:4mL, and the relative enzyme activity was 60.37%.

[0097] In step 3, the molar ratio of lactose to fructose is 1:1.6, and in step 4, the material temperature for ion exchange is 75°C. All other steps and operations are the same as in Example 1.

[0098] Under these comparative conditions, the lactulose conversion rate was 55%, the lactulose yield was 86%, the obtained lactulose solution had an ipilactose content of 4.3%, and the transmittance of the solution was 84%.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing lactulose using immobilized β-galactosidase, characterized in that, Includes the following steps: S1. Preparation of immobilized enzyme column: Amberlite IRA-93 macroporous weakly basic styrene-based anion exchange resin was sequentially soaked in ethanol solution, NaCl solution, HCl solution, and NaOH solution. After each soaking, the soaking solution was filtered out and the resin was washed with deionized water until neutral to obtain pretreated resin. The pretreated resin was mixed with β-galactosidase enzyme solution, Tris-hCl buffer was added and the mixture was shaken for adsorption. After adsorption, the mixture was allowed to stand. Then, glutaraldehyde solution was added for cross-linking, and the supernatant was discarded to obtain the immobilized enzyme column. S2. Add magnesium ion solution to a mixture of lactose and fructose and then convert it through an immobilized enzyme column to obtain lactulose conversion solution; S3. The lactulose inversion solution is subjected to decolorization, ion exchange, vacuum concentration, and continuous simulated moving bed chromatography to obtain a lactulose solution.

2. The method according to claim 1, characterized in that, In S1, the concentration of the ethanol solution is 95 wt%; the concentration of the NaCl solution is 10 wt%; the concentration of the HCl solution is 10 wt%; and the concentration of the NaOH solution is 10 wt%.

3. The method according to claim 1, characterized in that, The soaking time in S1 is 10-20 hours.

4. The method according to claim 1, characterized in that, The mass-to-volume ratio of the pretreated resin to the β-galactosidase solution in S1 is 1 g: 5-7 mL.

5. The method according to claim 1, characterized in that, The concentration of Tris-hCl buffer in S1 is 50 mmol / L, pH is 8.0, and the concentration of glutaraldehyde solution is 2 wt%.

6. The method according to claim 1, characterized in that, The relative enzyme activity of β-galactosidase in the immobilized enzyme column in S1 is 65-78%.

7. The method according to claim 1, characterized in that, The process parameters in S1 satisfy one or more of the following conditions: The adsorption time was 10–13 h, and the adsorption temperature was 28–32 °C. The settling time is 25–35 minutes, and the settling temperature is 2–5℃; The cross-linking time is 5.5–6.5 h, and the cross-linking temperature is 23–28 °C.

8. The method according to claim 1, characterized in that, The mass concentration of the lactose and fructose mixture in S2 is 50-55%; the molar ratio of lactose to fructose is 1:1 to 1:1.

5.

9. The method according to claim 1, characterized in that, After the addition of magnesium ions in S2, the magnesium ion concentration in the mixed liquid system is 1 mM; the magnesium ion source is MgCl2 or MgSO4.

10. The method according to claim 1, characterized in that, In S2, the mixture of lactose and fructose is passed through the immobilized enzyme column at a flow rate of 2.0 BV / hour and a temperature of 60–70 °C.

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