Method for preparing lactulose from immobilized beta-galactosidase

Abstract

The invention provides a method for preparing lactulose from immobilized beta-galactosidase, and belongs to the technical field of functional sugar preparation.The method comprises the following steps that an immobilized enzyme column is prepared, specifically, macroporous alkalescent styrene anion exchange resin is soaked and washed with an ethanol solution, a NaCl solution, an HCl solution and a NaOH solution in sequence; the pretreated resin and beta-galactosidase liquid are mixed, a buffer solution is added, shaking table adsorption is performed, and standing is performed after adsorption; adding a glutaraldehyde solution for cross-linking, and discarding the supernatant to obtain an immobilized enzyme column; adding a magnesium ion solution into a mixed solution of lactose and fructose, and then performing conversion through an immobilized enzyme column to obtain a lactulose conversion solution; the lactulose conversion liquid is sequentially subjected to decoloration, ion exchange, vacuum concentration and continuous simulated moving bed chromatographic separation to obtain a lactulose solution, by means of the method, the lactulose conversion rate is larger than or equal to 60%, the lactulose yield is larger than or equal to 92%, the preparation process is simple, and the cost is low.

Description

Technical Field

[0001] The invention relates to the technical field of functional sugar preparation, and in particular to a method for preparing lactulose by using immobilized beta-galactosidase. Background Art

[0002] Lactulose (Lactulose: C12H12O11; FW: 342.30Da), also known as 4-Ο-β-D-pyranosyl-D-fructose, isomerized lactose or lactulose, is a non-absorbable disaccharide composed of D-galactose and D-fructose bonded by β-1,4 glycosidic bonds. Lactulose is a white irregular crystalline powder. Pure lactulose has a pure sweetness, with a cool and mellow feeling. Its sweetness is 48%-62% of sucrose. It cannot be metabolized by digestive enzymes in animals, so it can be directly fermented by microorganisms in the gastrointestinal tract. It is called "Bifidobacterium Proliferation Factor" and has beneficial functions such as adjusting the balance of intestinal flora, serving as an indirect nutritional supplement, treating constipation, reducing blood ammonia, enhancing the body's immunity, and reducing blood endotoxin levels. It is widely used in food, medicine, feed and other fields.

[0003] At present, the methods for producing lactulose mainly include chemical method and enzymatic method. The chemical method for preparing isomerized lactose is mainly simple to operate and has a high conversion rate. The disadvantage is that the final product generally includes lactulose, residual lactose, D-galactose and D-fructose. The product is not only difficult to separate, but also often has a darker color. The production of by-products and pigments reduces the yield of lactulose. Although the enzymatic synthesis of lactulose can overcome the shortcomings of chemical synthesis, the enzymatic production of lactulose has a low conversion rate, which seriously limits the large-scale production and promotion and application of the product. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for preparing lactulose by using immobilized β-galactosidase. The preparation process of the present invention adopts immobilized β-galactosidase, wherein the relative enzyme activity of β-galactosidase reaches 65-78%, and lactose and fructose are used as raw materials for conversion. Finally, the obtained lactulose liquid is passed through a cation resin column-decolorization resin column-anion resin column and chromatographic separation to obtain lactulose with a purity of 97%, and the conversion rate is ≥60%.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing lactulose by using immobilized β-galactosidase, comprising the following steps:

[0007] S1. Preparation of immobilized enzyme column:

[0008] Amberlite IRA-93 macroporous weakly basic styrene anion exchange resin is sequentially soaked in ethanol solution, NaCl solution, HCl solution, and NaOH solution, and after each soaking, the soaking solution is filtered out and washed with deionized water until neutral to obtain a pretreated resin;

[0009] The pretreated resin and β-galactosidase enzyme solution are mixed, a Tris-HCl buffer is added, and the mixture is adsorbed on a shaker, and the mixture is allowed to stand after adsorption; a glutaraldehyde solution is then added for cross-linking, and the supernatant is discarded to obtain an immobilized enzyme column;

[0010] S2. adding magnesium ion solution to the mixture of lactose and fructose and then converting it through an immobilized enzyme column to obtain a lactulose conversion solution;

[0011] S3. The lactulose conversion liquid is sequentially subjected to decolorization, ion exchange, vacuum concentration, and continuous simulated moving bed chromatography to obtain a lactulose solution.

[0012] Preferably, the concentration of the ethanol solution in S1 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 %.

[0013] Preferably, the soaking time in S1 is 10 to 20 h.

[0014] More preferably, the immersion time in the ethanol solution is 15 to 20 h, the immersion time in the NaCl solution is 10 to 15 h, the immersion time in the HCl solution is 10 to 15 h, and the immersion time in the NaOH solution is 10 to 15 h.

[0015] Preferably, the mass volume ratio of the pretreated resin to the β-galactosidase enzyme solution in S1 is 1 g: (5-7) mL; more preferably 1 g: 5 mL.

[0016] Preferably, the concentration of the Tris-HCl buffer in S1 is 50 mmol / L, and the pH is 8.0; the concentration of the glutaraldehyde solution is 2 wt%.

[0017] Preferably, the relative enzyme activity of β-galactosidase in the immobilized enzyme column in S1 is 65-78%.

[0018] Preferably, the process parameters in S1 satisfy one or more of the following conditions:

[0019] The adsorption time is 10-13 h and the adsorption temperature is 28-32 °C;

[0020] The standing time is 25~35 min, and the standing temperature is 2~5 ℃;

[0021] The cross-linking time is 5.5~6.5 h, and the cross-linking temperature is 23~28 ℃.

[0022] 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 1:1-1:1.5, more preferably 1:1.

[0023] It is further preferred that the pH of the lactose and fructose mixture is 7.

[0024] Preferably, after the magnesium ions in S2 are added, the magnesium ion concentration in the mixed solution system is 1 mM; the source of the magnesium ions is MgCl2 or MgSO4.

[0025] Preferably, the flow rate of the mixed solution of lactose and fructose in S2 passing through the immobilized enzyme column is 2.0 BV / h, and the temperature is 60-70°C, more preferably 70°C.

[0026] Preferably, each process in S3 is one or more of the following:

[0027] The decolorization is carried out by using a granular activated carbon column, adding activated carbon to the lactulose solution, stirring at 70-90°C for 20-40 min, wherein the amount of activated carbon added is 1-2% of the dry basis mass of lactose, and then filtering through an Ama filter, wherein the working pressure of the Ama filter is 0.2-0.4 Mpa, to obtain a decolorized lactose solution;

[0028] The ion exchange process is to pass the decolorized material through a cation exchange column, a decolorization resin column, and an anion exchange column in sequence, with a material temperature of 65-70°C and a flow rate of 2BV / h; the cation resin is LX-150 strong acid cation resin; the decolorization resin is D750 macroporous adsorption resin; and the anion resin is LX-360 weak base anion resin;

[0029] The vacuum concentration process uses a six-effect evaporator to concentrate the material after separation, with a vacuum degree of 0.06-0.09Mpa and a concentration temperature of 50-80°C;

[0030] The continuous simulated moving chromatography separation water-to-material ratio is 2:1-5:1, the feed amount is 0.04-0.08 kg / L resin / h, the temperature is 65-70 °C, the switching time is 1080 s, the feed flow rate is 0.35-0.45 L / h, and the simulated moving bed uses a calcium type cationic resin.

[0031] Beneficial technical effects:

[0032] 1. The present invention immobilizes β-galactosidase, uses resin as a carrier to run an immobilized enzyme column, allows the enzyme to fully contact with the material, eliminates the enzyme inactivation operation, and the relative enzyme activity of the β-galactosidase reaches 65-78%; by immobilizing the β-galactosidase, the optimal temperature range of the enzyme is increased, and the reaction temperature reaches 60-70°C. This temperature range can avoid contamination of miscellaneous bacteria during production and promote the forward reaction of the enzyme preparation.

[0033] 2. The present invention adds a magnesium ion solution to the substrate so that new magnesium ions are continuously introduced, thereby continuously enhancing the activity of β-galactosidase and prolonging the use of β-galactosidase. The lactulose conversion rate is ≥60%, the lactulose yield is ≥92%, and the obtained lactulose solution has a low lactose content of ≤3.5%.

[0034] 3. The present invention uses cation resin-decolorization resin-anion resin series decolorization to increase the light transmittance of the solution by ≥90%, greatly reduce the material chromaticity, the chromaticity is ≤100 RBU, simplify the refining process, and is conducive to industrial production. DETAILED DESCRIPTION

[0035] The present invention will be described in detail below by specific examples. These examples are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples, unless otherwise specified, are conventional methods. Unless otherwise specified, the reagents and materials used can all be obtained through commercial channels.

[0036] Amberlite IRA-93 macroporous weakly basic styrene anion exchange resin was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.; β-galactosidase (produced from Aspergillus oryzae BLCY-006, preserved in the General Microbiology Center of China National Microbiological Culture Collection on December 5, 2018, with the collection number CGMCC No. 16965).

[0037] Embodiment 1:

[0038] (1) Soak the resin in 95% ethanol solution (2 times the volume of Amberlite IRA-93 macroporous weakly basic styrene anion exchange resin) for 18 h. After filtering out the ethanol solution, wash the Amberlite IRA-93 macroporous weakly basic styrene anion exchange resin with deionized water. Then soak it in 10% NaCl solution for 12 h. After filtering out the NaCl solution, wash the resin with deionized water until the pH of the solution is 7. Then soak the resin in 2% HCl solution for 12 h. After filtering out the HCl solution, wash the resin with deionized water until the pH of the solution is 7. Then soak the resin in 5% NaOH solution for 12 h. After filtering out the filtrate of the NaOH solution, wash the resin with deionized water until the pH of the solution is 7. Filter out the excess liquid to complete the pretreatment of the resin.

[0039] (2) The pretreated resin and the β-galactosidase solution to be immobilized were mixed in proportion, and a 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 °C for 12 h, and then allowed to stand at 4 °C for 30 min after adsorption. A 2% glutaraldehyde solution was then added for cross-linking at a temperature of 25 °C and a cross-linking time of 6 h to complete the preparation of immobilized β-galactosidase. After the immobilization was completed, the supernatant was discarded to obtain the immobilized enzyme. The volume mass ratio of the enzyme solution to be immobilized to the resin was 1 g:5 mL.

[0040] Determination of enzyme activity: prepare a 1 mg / mL o-nitrobenzene-β-D-galactoside (ONPG) solution 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.

[0041] Enzyme activity unit (U): Under certain conditions, the amount of enzyme required for β-galactosidase to hydrolyze ONPG to produce 1 µmol of o-nitrophenol (ONP) within 1 min.

[0042] The enzyme activity was determined to be 77.89% when the ratio of resin to added enzyme was 1 g:5 mL.

[0043] (3) The immobilized enzyme described in step (2) is filled into an immobilized column, a mixture of lactose and fructose is passed through the immobilized column, and a MgCl2 solution is added to the mixture (after addition, the magnesium ion concentration in the mixture is 1 mM) to obtain a lactulose conversion 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 / h, the temperature is 70°C, and the pH of the lactose and fructose mixture is 7.

[0044] (4) The lactulose conversion liquid of step (3) is subjected to decolorization, ion exchange, vacuum concentration, and continuous simulated moving bed chromatography to obtain a lactulose solution.

[0045] The decolorization was carried out by using a granular activated carbon column, and activated carbon was added to the lactulose solution, and stirred at 80°C for 30 minutes, wherein the amount of activated carbon added was 2% of the dry basis weight of lactulose. The solution was then filtered through an Ama filter, and the working pressure of the Ama filter was 0.3 Mpa to obtain a decolorized lactulose solution, and the chromaticity of the solution was 2382.15 RBU.

[0046] The ion exchange process is to pass the decolorized material through a cation exchange column, a decolorization resin column, and an anion exchange column in sequence, with a material temperature of 70°C and a flow rate of 2BV / h. The cation resin is LX-150 strong acid cation resin, the decolorization resin is D750 macroporous adsorption resin, the anion resin is LX-360 weak base anion resin, and the solution chromaticity is 20.25 RBU.

[0047] The vacuum concentration process uses a six-effect evaporator to concentrate the material after separation, with a vacuum degree of 0.09 Mpa and a concentration temperature of 75 °C.

[0048] The continuous simulated moving chromatography separation water-to-material ratio is 3:1, the feed amount is 0.06 kg / L resin / h, the temperature is 70°C, the switching time is 1080 s, the feed flow rate is 0.4 L / h, and the simulated moving bed uses calcium type cationic resin.

[0049] Under the conditions of this embodiment, the lactulose conversion rate is 68%, the lactulose yield is 97%, the obtained lactulose solution has a 3.0% picolinose content, and the light transmittance of the solution is 92%.

[0050] Embodiment 2:

[0051] (1) Soak the resin in 95% ethanol solution (2 times the volume of Amberlite IRA-93 macroporous weakly basic styrene anion exchange resin) for 15 h. After filtering out the ethanol solution, wash the Amberlite IRA-93 macroporous weakly basic styrene anion exchange resin with deionized water. Then soak it in 10% NaCl solution for 12 h. After filtering out the NaCl solution, wash the resin with deionized water until the pH of the solution is 7. Then soak the resin in 2% HCl solution for 12 h. After filtering out the HCl solution, wash the resin with deionized water until the pH of the solution is 7. Then soak the resin in 5% NaOH solution for 12 h. After filtering out the filtrate of the NaOH solution, wash the resin with deionized water until the pH of the solution is 7. Filter out the excess liquid to complete the pretreatment of the resin.

[0052] (2) The pretreated resin and the β-galactosidase solution to be immobilized were mixed in proportion, and a 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 °C for 12 h, and then allowed to stand at 4 °C for 30 min after adsorption. A 2% glutaraldehyde solution was then added for cross-linking at a temperature of 25 °C and a cross-linking time of 6 h to complete the preparation of immobilized β-galactosidase. After the immobilization was completed, the supernatant was discarded to obtain the immobilized enzyme. The volume mass ratio of the enzyme solution to be immobilized to the resin was 1 g:7 mL.

[0053] Determination of enzyme activity: prepare a 1 mg / mL o-nitrobenzene-β-D-galactoside (ONPG) solution 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.

[0054] Enzyme activity unit (U): Under certain conditions, the amount of enzyme required for β-galactosidase to hydrolyze ONPG to produce 1 µmol of o-nitrophenol (ONP) within 1 min.

[0055] The enzyme activity was determined to be 67.42% when the ratio of resin to added enzyme was 1 g:7 mL.

[0056] (3) The immobilized enzyme described in step (2) is filled into the immobilized column, the mixed solution of lactose and fructose is passed through the immobilized column, and the MgCl2 solution is added to the mixed solution (after addition, the magnesium ion concentration in the mixed solution system is 1 mM) to obtain a lactulose conversion solution. The mass concentration of the mixed solution of lactose and fructose is 55%, and the molar ratio of lactose to fructose is 1:1.5. The flow rate of the mixed solution of lactose and fructose is 2.0 BV / h, the temperature is 60°C, and the pH of the mixed solution of lactose and fructose is 7.

[0057] (4) The lactulose conversion liquid of step (3) is subjected to decolorization, ion exchange, vacuum concentration, and continuous simulated moving bed chromatography to obtain a lactulose solution.

[0058] The decolorization was carried out by using a granular activated carbon column, and activated carbon was added to the lactulose solution, and stirred at 90°C for 20 minutes, wherein the amount of activated carbon added was 1.5% of the dry basis weight of lactulose. The solution was then filtered through an Ama filter, and the working pressure of the Ama filter was 0.2 Mpa to obtain a decolorized lactulose solution, and the chromaticity of the solution was 2489.67 RBU.

[0059] The ion exchange process is to pass the decolorized material through a cation exchange column, a decolorization resin column, and an anion exchange column in sequence, with a material temperature of 70°C and a flow rate of 2BV / h. The cation resin is LX-150 strong acid cation resin, the decolorization resin is D750 macroporous adsorption resin, and the anion resin is LX-360 weak base anion resin. The solution chromaticity is 39.75 RBU.

[0060] The vacuum concentration process uses a six-effect evaporator to concentrate the material after separation, with a vacuum degree of 0.06 Mpa and a concentration temperature of 65 °C.

[0061] The continuous simulated moving chromatography separation water-to-material ratio is 5:1, the feed amount is 0.08 kg / L resin / h, the temperature is 65°C, the switching time is 1080 s, the feed flow rate is 0.35 L / h, and the simulated moving bed uses calcium type cationic resin.

[0062] Under the conditions of this embodiment, the lactulose conversion rate is 65%, the lactulose yield is 94%, the obtained lactulose solution has a pilolactose content of 3.1%, and the light transmittance of the solution is 91%.

[0063] Embodiment 3:

[0064] (1) Soak the resin in 95% ethanol solution (2 times the volume of Amberlite IRA-93 macroporous weakly basic styrene anion exchange resin) for 20 h. After filtering out the ethanol solution, wash the Amberlite IRA-93 macroporous weakly basic styrene anion exchange resin with deionized water; then soak it in 10% NaCl solution for 12 h. After filtering out the NaCl solution, wash the resin with deionized water until the pH of the solution is 7; then soak the resin in 2% HCl solution for 12 h. After filtering out the HCl solution, wash the resin with deionized water until the pH of the solution is 7; then soak the resin in 5% NaOH solution for 12 h. After filtering out the filtrate of the NaOH solution, wash the resin with deionized water until the pH of the solution is 7, filter out the excess liquid, and complete the pretreatment of the resin.

[0065] (2) The pretreated resin and the β-galactosidase solution to be immobilized were mixed in proportion, and a 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 °C for 12 h, and then allowed to stand at 4 °C for 30 min after adsorption. A 2% glutaraldehyde solution was then added for cross-linking at a temperature of 25 °C and a cross-linking time of 6 h to complete the preparation of immobilized β-galactosidase. After the immobilization was completed, the supernatant was discarded to obtain the immobilized enzyme. The volume mass ratio of the enzyme solution to be immobilized to the resin was 1 g:6 mL.

[0066] Determination of enzyme activity: prepare a 1 mg / mL o-nitrobenzene-β-D-galactoside (ONPG) solution 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.

[0067] Enzyme activity unit (U): Under certain conditions, the amount of enzyme required for β-galactosidase to hydrolyze ONPG to produce 1 µmol of o-nitrophenol (ONP) within 1 min.

[0068] The enzyme activity was determined to be 69.82% when the ratio of resin to added enzyme was 1 g:6 mL.

[0069] (3) The immobilized enzyme described in step (2) is filled into the immobilized column, the mixed solution of lactose and fructose is passed through the immobilized column, and the MgCl2 solution is added to the mixed solution (after addition, the magnesium ion concentration in the mixed solution system is 1 mM) to obtain a lactulose conversion solution. The mass concentration of the lactose and fructose mixed solution is 50%, the molar ratio of lactose to fructose is 1:1.5, the flow rate of the lactose and fructose mixed solution is 2.0 BV / h, the temperature is 65°C, and the pH of the lactose and fructose mixed solution is 7.

[0070] (4) The lactulose conversion liquid of step (3) is subjected to decolorization, ion exchange, vacuum concentration, and continuous simulated moving bed chromatography to obtain a lactulose solution.

[0071] The decolorization was carried out by using a granular activated carbon column, and activated carbon was added to the lactulose solution, and stirred at 70°C for 40 minutes, wherein the amount of activated carbon added was 1% of the dry basis weight of lactulose. The solution was then filtered through an Ama filter, and the working pressure of the Ama filter was 0.4 Mpa to obtain a decolorized lactulose solution, and the chromaticity of the solution was 2478.65 RBU.

[0072] The ion exchange process is to pass the decolorized material through a cation exchange column, a decolorization resin column, and an anion exchange column in sequence, with a material temperature of 65°C and a flow rate of 2BV / h. The cation resin is LX-150 strong acid cation resin, the decolorization resin is D750 macroporous adsorption resin, and the anion resin is LX-360 weak base anion resin. The solution chromaticity is 45.66 RBU.

[0073] The vacuum concentration process uses a six-effect evaporator to concentrate the material after separation, with a vacuum degree of 0.08 Mpa and a concentration temperature of 65 °C.

[0074] The continuous simulated moving chromatography separation water-to-material ratio is 2:1, the feed amount is 0.04 kg / L resin / h, the temperature is 70°C, the switching time is 1080 s, the feed flow rate is 0.45 L / h, and the simulated moving bed uses calcium type cationic resin.

[0075] Under the conditions of this embodiment, the lactulose conversion rate is 63%, the lactulose yield is 92%, the obtained lactulose solution has a pilolactose content of 3.5%, and the light transmittance of the solution is 90%.

[0076] Comparative Example 1:

[0077] In this comparative example, lactulose was prepared according to the preparation method of Example 1, except that in step 4, the ion exchange process was to pass the decolorized material through a cation exchange column and an anion exchange column in sequence, and the other steps and operations were the same as those of Example 1.

[0078] The chromaticity of the obtained solution was 1678.59 RBU. Under the conditions of this comparative example, the lactulose conversion rate was 68%, the lactulose yield was 97%, the lactulose content of the obtained lactulose solution was 3.3%, and the light transmittance of the solution was 75%.

[0079] Comparative Example 2:

[0080] In this comparative example, lactulose was prepared according to the preparation method of Example 1, except that in step 4, the ion exchange process was to pass the decolorized material through a cation exchange column, an anion exchange column, and a decolorizing resin column in sequence, and the other steps and operations were the same as those of Example 1.

[0081] The chromaticity of the obtained solution was 1234.88 RBU. Under the conditions of this comparative example, the lactulose conversion rate was 68%, the lactulose yield was 97%, the lactulose content of the obtained lactulose solution was 3.4%, and the light transmittance of the solution was 79%.

[0082] Comparative Example 3:

[0083] In this comparative example, lactulose was prepared according to the preparation method of Example 1, except that in step 3, the mass concentration of the mixed solution of lactose and fructose was 45%, and other metal ions were added to the mixed solution of lactose and fructose (the concentration of other metal ions in the mixed solution system after addition was 1 mM, except Mg 2+ Except for the above, see Table 1 for details), the other steps and operations are the same as those in Example 1.

[0084] The obtained lactulose is shown in Table 1 below. The results show that, compared with Example 1, other metal ions have poorer activation of β-galactosidase, which reduces the conversion rate of lactulose.

[0085] Table 1 Conversion rate of lactulose after adding different metal ions

[0086] Metal ions <![CDATA[K ＋ ]]> <![CDATA[Li ＋ ]]> <![CDATA[Ca 2+ ]]> <![CDATA[Na ＋ ]]> <![CDATA[Co 2+ ]]> <![CDATA[Fe 2+ ]]> <![CDATA[Mn 2+ ]]> Lactulose conversion rate 41 % 41.5 % 42 % 38 % 51 % 50.8 % 55 %

[0087] Comparative Example 4:

[0088] In this comparative example, lactulose was prepared according to the preparation method of Example 1, except that in step 3, the mass concentration of the mixed solution of lactose and fructose was 60%, no metal ions were added to the mixed solution of lactose and fructose, and the other steps and operations were the same as those of Example 1.

[0089] Under the conditions of this comparative example, the lactulose conversion rate was 53%, the lactulose yield was 82%, the obtained lactulose solution had a lactose content of 5.2%, and the light transmittance of the solution was 85%.

[0090] Comparative Example 5:

[0091] In this comparative example, lactulose was prepared according to the preparation method of Example 1, except that in step 2, the volume mass ratio of the immobilized enzyme solution to the resin was 1 g:8 mL, and the relative enzyme activity was 62.44%.

[0092] In step 3, the molar ratio of lactose to fructose is 1:0.8, in step 4, the material temperature of the ion exchange is 60° C., and the other steps and operations are the same as those in Example 1.

[0093] Under the conditions of this comparative example, the lactulose conversion rate was 48%, the lactulose yield was 80%, the obtained lactulose solution had a lactose content of 4.7%, and the light transmittance of the solution was 88%.

[0094] Comparative Example 6:

[0095] In this comparative example, lactulose was prepared according to the preparation method of Example 1, except that in step 2, the volume mass ratio of the immobilized enzyme solution to the resin was 1 g:4 mL, and the relative enzyme activity was 60.37%.

[0096] In step 3, the molar ratio of lactose to fructose is 1:1.6, and in step 4, the material temperature of the ion exchange is 75° C. Other steps and operations are the same as those in Example 1.

[0097] Under the conditions of this comparative example, the lactulose conversion rate was 55%, the lactulose yield was 86%, the lactulose content of the obtained lactulose solution was 4.3%, and the light transmittance of the solution was 84%.

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing lactulose by immobilizing β-galactosidase, characterized in that: The following steps are involved: S1. Preparation of immobilized enzyme column: Amberlite IRA-93 macroporous weakly basic styrene anion exchange resin is sequentially soaked in ethanol solution, NaCl solution, HCl solution, and NaOH solution, and after each soaking, the soaking solution is filtered out and washed with deionized water until neutral to obtain a pretreated resin; The pretreated resin and β-galactosidase enzyme solution are mixed, a Tris-HCl buffer is added, and the mixture is adsorbed on a shaker, and the mixture is allowed to stand after adsorption; a glutaraldehyde solution is then added for cross-linking, and the supernatant is discarded to obtain an immobilized enzyme column; S2. adding magnesium ion solution to the mixture of lactose and fructose and then converting it through an immobilized enzyme column to obtain a lactulose conversion solution; S3. The lactulose conversion liquid is sequentially 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 The concentration of the ethanol solution in S1 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 immersion time in S1 is 10 to 20 h.

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

5. The method according to claim 1, characterized in that The concentration of the Tris-HCl buffer in S1 is 50 mmol / L, and the pH is 8.0; the concentration of the 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 meet one or more of the following conditions: The adsorption time is 10-13 h and the adsorption temperature is 28-32 °C; The standing time is 25~35 min, and the standing temperature is 2~5 ℃; The cross-linking time is 5.5~6.5 h, and the cross-linking temperature is 23~28 ℃.

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 1:1~1:1.

5.

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

10. The method according to claim 1, characterized in that The flow rate of the mixed solution of lactose and fructose in S2 through the immobilized enzyme column is 2.0 BV / h, and the temperature is 60-70°C.