Preparation method of galactooligosaccharide

Abstract

The invention discloses a preparation method of galactooligosaccharide, which comprises the following steps: S1, taking a lactose solution as a raw material, loading beta-galactosidase immobilized by calcium pectate gel-chitosan in an interlayer, wherein the composition mode of the calcium pectin gel-chitosan serving as the carrier is as follows: the chitosan is assembled on the surfaces of calcium pectin gel particles through electrostatic adsorption, and the beta-galactosidase is cross-linked to the chitosan; s2, circulation is carried out; a lactose solution flows through the surface of the calcium pectate gel-chitosan and is subjected to a contact reaction with beta-galactosidase; s3, pumping out the reaction liquid, separating by using a continuous simulated moving bed technology to obtain galactooligosaccharide, and recovering separated lactose to continue the reaction; the calcium pectin gel particles are used as cores, chitosan is gathered on the surfaces of the gel particles, beta-galactosidase is further adsorbed and crosslinked, and liquid circulation in the reaction process is matched, so that the conversion rate of galactooligosaccharide is effectively increased.

Description

Technical Field

[0001] The invention relates to the technical field of galacto-oligosaccharide preparation, in particular to a method for preparing galacto-oligosaccharide by utilizing immobilized biological enzymes. Background Art

[0002] Galacto-oligosaccharide is an important functional sugar. Its molecule is composed of 2 to 10 galactosyl groups and 1 glucose group. The galactosyl groups are linked by β-(1,3), β-(1,4) and β-(1,6) bonds, with β-(1,4) bonds being the main ones. Galactose and glucose are linked by β-(1,4).

[0003] The common preparation methods of galacto-oligosaccharides in the prior art mostly use enzymatic synthesis of galacto-oligosaccharides, using lactose in milk as a substrate and catalyzed by β-galactosidase for synthesis. However, the GOS product synthesized by the enzymatic method is a mixture, including substances such as glucose, galactose and lactose, and needs to be separated and purified to obtain galacto-oligosaccharides. Generally, the purity of galacto-oligosaccharides is 85.03%, and the second column can reach 89.39%.

[0004] The Chinese invention patent with the authorization announcement number CN111978423B discloses a method for preparing high-purity oligogalactose, which uses a six-bed cis-simulated moving bed chromatography separation technology and a sodium-type macroporous cationic resin. Compared with a calcium-type cationic resin, the separation effect of oligogalactose is better, and the fine separation of glucose, lactose, galactose and oligogalactose can be achieved to obtain a high-purity oligogalactose product of more than 98%. It can be seen that the existing technology is mostly aimed at the separation and purification of oligogalactose, and the separation, recovery and reuse of enzymes are also one of the problems that need to be solved urgently. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing galacto-oligosaccharides. The present invention uses calcium pectin gel particles as cores, aggregates chitosan on the surface of the gel particles, further adsorbs and cross-links β-galactosidase, and cooperates with liquid circulation during the reaction process to effectively improve the conversion rate of galacto-oligosaccharides.

[0006] To solve this technical problem, the technical solution of the present invention is: a method for preparing galacto-oligosaccharide, comprising the following steps: S1, pumping lactose solution as raw material into a reaction tank, and loading β-galactosidase immobilized by calcium pectin gel-chitosan into n-1 partitions obtained by layer-by-layer separation along the axial direction of the tank body through n layers of screens, wherein n is an integer ≥ 3; The composition of the calcium pectin gel-chitosan as a carrier is as follows: Chitosan was assembled on the surface of calcium pectin gel particles by electrostatic adsorption, and β-galactosidase was cross-linked to chitosan; The ratio of the mass of the immobilized β-galactosidase to the volume of the lactose solution is 18 mg to 25 mg / 1L; S2, the cycle temperature is 45°C to 52°C, and the cycle time is 10 hours to 14 hours; The lactose solution flows through the surface of calcium pectin gel-chitosan and comes into contact with β-galactosidase for reaction; S3. The reaction liquid is pumped out, and galacto-oligosaccharides are separated using continuous simulated moving bed technology, and the separated lactose is recovered to continue the reaction.

[0007] Preferably, the method for preparing β-galactosidase immobilized by calcium pectin gel-chitosan comprises the following steps: S11, placing calcium pectin gel microparticles in a chitosan solution, wherein the pH value of the chitosan solution is 4.0 to 4.5, and stirring, so that chitosan is enriched on the surface of the calcium pectin gel particles to form a polyelectrolyte complex; S12, adding β-galactosidase to the system, and adsorbing the β-galactosidase by chitosan; S13, the beta-galactosidase adsorbed by the calcium pectin gel-chitosan obtained by S12 is placed in a glutaraldehyde aqueous solution for cross-linking, and the beta-galactosidase solidified by calcium pectin gel-chitosan is obtained. The present invention uses calcium pectin gel particles as the core structure for loading chitosan, and the subsequent glutaraldehyde cross-linking chitosan and beta-galactosidase are matched, wherein the glutaraldehyde cross-linking includes chitosan distributed on the surface of the calcium pectin gel particles, and the shell structure of chitosan as a carrier stabilizes the structure of the calcium pectin gel particles; in the production process of oligomeric galactose, due to the immobility of water in pectin high gel particles, lactose solution flows through the surface of calcium pectin gel-chitosan, so as to ensure the contact of lactose solution with beta-galactosidase, and the present invention utilizes the flow of lactose, flows through the carrier stack to form a space flow channel with a three-dimensional structure, i.e. flows through the carrier surface, improves the contact of lactose with the enzyme, and the carrier obtained by the present invention can be used repeatedly, and maintains a relatively stable conversion efficiency.

[0008] Preferably, the mass ratio of calcium pectin gel that is swollen into calcium pectin gel microparticles by water absorption to chitosan is (1.1 to 1.3): 1. The present invention utilizes calcium pectin gel particles to effectively load chitosan, and improves the stability of calcium pectin gel particles through chitosan.

[0009] The preferred mass ratio of β-galactosidase to chitosan is (0.8 mg to 1.0 mg): 1 g. The present invention distributes β-galactosidase in a three-dimensional flow channel, and coordinates the flow of lactose solution to ensure sufficient contact between lactose and enzyme.

[0010] The process conditions for adsorbing β-galactosidase by chitosan in step S12 are preferably as follows: 10℃-15℃, 200r / min-280r / min shaking table for adsorption, the shaking time is 3 hours to 5 hours.

[0011] The method for preparing calcium pectin gel microparticles preferably comprises the following steps: S111, preparing a low-ester pectin and glucose mixture using deionized water, and adjusting the pH of the mixture to 3.0; S112, heating the mixed solution obtained in S111 in boiling water; S113, adding calcium chloride to the mixed solution heated in S112, and stirring rapidly and uniformly; placing the obtained mixed solution in an environment of 3°C to 5°C to cool and place to obtain calcium pectin gel; S114, calcium pectin gel is freeze-dried, crushed, and sieved through 250-300 mesh; S115, placing the calcium pectin powder obtained in S114 in an acid solution to swell and obtain calcium pectin gel micron particles.

[0012] Preferably, step S115 wherein the calcium pectin gel particles absorb water and swell to form calcium pectin gel micron particles comprises the following steps: A1. At 4° C., the calcium pectin gel particles obtained by drying in step S114 are immersed in a pyrrole monomer aqueous solution for 4 to 8 hours to allow the pyrrole monomer to evenly penetrate the calcium pectin gel particles; A2, the hydrogel obtained by step A1 is immersed in 0.089mol / L ferric nitrate aqueous solution, and pyrrole monomer is cross-linked to polypyrrole. The present invention is by infiltrating pyrrole monomer into gel particles by the way of infiltration, along with the uniform distribution of water dispersion, ferric nitrate as initiator triggers pyrrole can be self-polymerized into polypyrrole so as to form the stability of calcium pectin gel particle structure by polypyrrole;And because pyrrole is continuously infiltrated with water, therefore polypyrrole effectively runs through gel particles, stabilizing gel particle structure;Then gel particles are put into chitosan solution, chitosan electrostatic adsorption on gel particle surface, then glutaraldehyde is used to realize the crosslinking between chitosan and chitosan and chitosan and enzyme, and the carrier for enzyme fixation of stable structure is obtained.

[0013] Preferably, in step S2, the liquid circulation pump circulates the reaction liquid at a flow rate of 50 mL / min to 80 mL / min.

[0014] Preferably, the sieve is a steel mesh with meshes of 100 to 200. The present invention utilizes the steel mesh in combination with the particle form of the carrier to further effectively disperse the lactose solution, the flow rate of the solution through the small holes of the steel mesh increases instantly, the interaction force with the carrier is greater, and it is easier to contact the enzyme.

[0015] As a further improvement, each screen is threadedly connected to the inner wall of the reaction tank. The screen of the present invention can be detachably connected to the inner wall of the reaction tank, which is convenient for the input of the immobilized enzyme.

[0016] By adopting the above technical solution, the beneficial effects of the present invention are: The invention uses calcium pectin gel particles as cores, gathers chitosan on the surface of the gel particles, further adsorbs and cross-links beta-galactosidase, and cooperates with liquid circulation in the reaction process to effectively improve the conversion rate of oligogalactose. In the conversion process of lactose, n-1 partitions obtained by layer-by-layer separation by n layers of screens along the axial direction of the tank body in the reaction tank are loaded with beta-galactosidase immobilized by calcium pectin gel-chitosan, so that a large amount of calcium pectin gel-chitosan is prevented from stacking, and the lactose solution is guaranteed to flow along the distribution of the enzyme, which is beneficial to the mass transfer of reactants and products. The carrier used for fixing the beta-galactosidase in the invention has a stable structure, good biocompatibility, and can be reused. Through the cooperation of the screen, the flow rate change between micro-ranges is formed in the circulation process of the reaction solution, so the conversion efficiency of oligogalactose is improved. DETAILED DESCRIPTION

[0017] In order to further explain the technical solution of the present invention, the present invention is described in detail below through specific embodiments.

[0018] Example 1 This embodiment discloses a method for preparing galacto-oligosaccharide, comprising the following steps: S1, 500 g / L lactose solution is pumped into a 20 L reaction tank as a raw material, and three compartments in the reaction tank are separated layer by layer by four layers of screens along the axial direction of the tank body to load β-galactosidase immobilized by calcium pectin gel-chitosan; The composition of the calcium pectin gel-chitosan as a carrier is as follows: Chitosan was assembled on the surface of calcium pectin gel particles by electrostatic adsorption, and β-galactosidase was cross-linked to chitosan; S2, the cycle temperature is 45°C to 48°C, and the cycle time is 14 hours; The lactose solution flows through the surface of calcium pectin gel-chitosan and comes into contact with β-galactosidase for reaction; In step S2, the liquid circulation pump circulates the reaction solution at a flow rate of 50 mL / min.

[0019] S3. The reaction liquid is pumped out, and galacto-oligosaccharides are separated using continuous simulated moving bed technology, and the separated lactose is recovered to continue the reaction.

[0020] The ratio of the mass of the immobilized β-galactosidase to the volume of the lactose solution was 18 mg / 1L; The preparation method of β-galactosidase immobilized by calcium pectin gel-chitosan in this embodiment comprises the following steps: S11, placing calcium pectin gel microparticles in a chitosan solution, wherein the pH value of the chitosan solution is 4.0, and stirring, so that chitosan is enriched on the surface of the calcium pectin gel particles to form a polyelectrolyte complex; In this embodiment, the mass ratio of the calcium pectin gel that is swollen to form calcium pectin gel microparticles by absorbing water to chitosan is 1.1:1.

[0021] S12, adding β-galactosidase to the system, and adsorbing the β-galactosidase by chitosan; In this embodiment, the mass ratio of β-galactosidase to chitosan is 0.8 mg:1 g.

[0022] S13, placing the calcium pectin gel-chitosan adsorbed β-galactosidase obtained in S12 in a 2% glutaraldehyde aqueous solution for cross-linking for 3 hours to obtain calcium pectin gel-chitosan solidified β-galactosidase.

[0023] In step S12 of this embodiment, the process conditions for adsorbing β-galactosidase by chitosan are as follows: The adsorption was carried out on a shaker at 10°C and 200 r / min for 3 hours.

[0024] The preparation method of calcium pectin gel microparticles in this embodiment comprises the following steps: S111. Prepare a low-ester pectin and glucose mixture using deionized water, wherein the mass fraction of low-ester pectin (esterification degree 35%, total galacturonic acid 88%) is 1%, and the mass fraction of glucose is 10%; adjust the pH of the mixture to 3.0; S112, placing the mixed solution obtained in S111 in boiling water and heating for 2 minutes; S113, adding calcium chloride to the mixed solution heated in S112, with the mass ratio of calcium ion to low-ester pectin being 30 mg:1 g, and stirring rapidly and evenly; placing the obtained mixed solution in an environment of 3° C. to 5° C. and cooling for 24 hours to obtain calcium pectin gel; S114, calcium pectin gel is freeze-dried, crushed, and sieved through 250 mesh; S115, placing the calcium pectin powder obtained in S114 in an aqueous solution to absorb water to obtain calcium pectin gel micron particles.

[0025] The calcium pectin gel particles obtained by drying in step S114 are immersed in an aqueous solution with a pH value of 4 for 4 hours to obtain calcium pectin gel particles; The screen used in this embodiment is a 100-mesh steel wire mesh, and each screen is threadedly connected to the inner wall of the reaction tank.

[0026] Example 2 The main difference between this embodiment and embodiment 1 is that: In this embodiment, step S115 of the calcium pectin gel particles absorbing water and swelling to form calcium pectin gel micron particles comprises the following steps: A1. At 4° C., the calcium pectin gel particles obtained by drying in step S114 are immersed in a pyrrole monomer aqueous solution for 6 hours to allow the pyrrole monomer to evenly penetrate the calcium pectin gel particles; The mass ratio of dry calcium pectin gel particles to pyrrole monomer was 10:1; A2. Soak the hydrogel obtained in step A1 in FeCl 3 (0.089 mol / L) aqueous solution, pyrrole monomers are cross-linked to form polypyrrole.

[0027] Example 3 The main differences between this embodiment and embodiment 2 are shown in Table 1 and Table 2.

[0028] Example 4 The main differences between this embodiment and embodiment 2 are shown in Table 1 and Table 2.

[0029] Example 5 The main differences between this embodiment and embodiment 2 are shown in Table 1 and Table 2.

[0030] Table 1 Process parameters for preparing galacto-oligosaccharides in Examples 1 to 5

[0031] Table 2 Process parameters for immobilizing β-galactosidase in Examples 1 to 5

[0032] Comparative Example 1 This comparative example discloses a method for preparing galacto-oligosaccharide, comprising the following steps: S1. Pump 500 g / L lactose solution as raw material into a 20 L reaction tank, and add β-galactosidase into the reaction tank; the mass ratio of the added β-galactosidase to the volume of the lactose solution is 25 mg / 1 L; S2, the cycle temperature is 45°C to 48°C, and the cycle time is 10 hours; S3. Separate the reaction solution using continuous simulated moving bed technology to obtain galacto-oligosaccharides, and separate lactose to recover and continue the reaction.

[0033] Comparative Example 2 This comparative example discloses a method for preparing galacto-oligosaccharide, comprising the following steps: S1, 500 g / L lactose solution is pumped into a 20 L reaction tank as a raw material, and the three compartments in the reaction tank are separated layer by layer by four layers of screens along the axial direction of the tank body and loaded with β-galactosidase immobilized by chitosan; S2, the cycle temperature is 45°C to 48°C, and the cycle time is 10 hours; When the lactose solution flows through the surface of chitosan, it comes into contact with β-galactosidase and reacts; In step S2, the liquid circulation pump circulates the reaction solution at a flow rate of 50 mL / min.

[0034] S3. The reaction liquid is pumped out, and galacto-oligosaccharides are separated using continuous simulated moving bed technology, and the separated lactose is recovered to continue the reaction.

[0035] The ratio of the mass of the immobilized β-galactosidase to the volume of the lactose solution was 18 mg / 1L; The preparation method of chitosan-immobilized β-galactosidase in this embodiment comprises the following steps: S11, dripping a chitosan solution with a pH of 4.0 dropwise into a large amount of 1 mol / L NaOH aqueous solution, allowing to react, then washing the chitosan microspheres with distilled water until neutral, and collecting the microspheres by filtration.

[0036] S12, placing the chitosan microspheres of S11 in a glutaraldehyde aqueous solution, allowing the reaction to stand, adding β-galactosidase, the mass ratio of β-galactosidase to chitosan being 0.8 mg:1 g, the chitosan adsorbing β-galactosidase, cross-linking in a 2% glutaraldehyde aqueous solution for 3 hours, to obtain chitosan-solidified β-galactosidase.

[0037] Table 3 Conversion of lactose into galacto-oligosaccharides

[0038] The compressive strength of the particle structure of the immobilized enzyme was further tested after different immersion times. When sampling, 30 particles were placed on the sample table of the compressive strength meter with tweezers. The average value of the test results was taken as the compressive strength. The specific data are shown in Table 4.

[0039] Table 4 Structural stability of enzyme carriers immobilized by soaking in Examples 1 to 5 and Comparative Example 2

[0040] As shown in Table 4, the carrier for immobilizing enzymes of the present invention has a stable structure after being subjected to compression and has good biocompatibility, and is suitable for the preparation of galacto-oligosaccharides.

[0041] The immobilized enzymes obtained in Examples 1 to 5 and Comparative Example 2 were used to prepare galacto-oligosaccharides and were reused 5 times. Compared with the first use, the galacto-oligosaccharide content and the conversion retention rate were shown in Table 5.

[0042] Table 5 Conversion rate of the fifth preparation of galacto-oligosaccharides by immobilized enzyme in Examples 1 to 5 and Comparative Example 2,

[0043] It can be seen from Table 5 that the present invention uses gel as the inner core of the enzyme carrier to cooperate with the carrier surface chitosan in a solid state in a neutral aqueous solution, and the immobility of water in the pectin high gel particles, and the lactose solution controlling the flow gathers and flows from the distribution position of the stacked immobilized enzyme, and the lactose solution fully flows from the enzyme surface, ensuring that the lactose solution is fully in contact with β-galactosidase, and promoting the contact of lactose with the enzyme; the carrier for the immobilized enzyme of the present invention has a stable structure and good compression resistance, and at the same time ensures that the enzyme is in contact with the lactose solution, and the carrier obtained by the present invention can be used repeatedly and repeatedly, and a relatively stable conversion efficiency is maintained. Compared with pure chitosan as an enzyme carrier in Comparative Example 2, the chitosan sphere with a porous structure is difficult to ensure that the lactose solution is fully enriched relative to the enzyme during the flow process, and compared with Examples 1 to 5 of the present invention, the dispersibility of the enzyme load is also limited, therefore, the production method of oligosaccharides proposed by the present invention has a high conversion rate and can be repeatedly used. Further comparing Example 1 with Examples 2 to 5, Examples 2 to 5 penetrate pyrrole monomer into the interior of gel particles by infiltration, and with the uniform dispersion of water, ferric nitrate is used as an initiator to trigger pyrrole to polymerize into polypyrrole by itself, thereby stabilizing the structure of calcium pectin gel particles through the formation of polypyrrole; the gel particles are then put into a chitosan solution, and chitosan is electrostatically adsorbed on the surface of the gel particles, and glutaraldehyde is used to achieve crosslinking between chitosan and chitosan and between chitosan and enzyme, thereby obtaining a structurally stable carrier for enzyme fixation. As the number of cycles increases, Examples 2 to 5 have a more stable conversion efficiency.

Claims

1. A method for preparing galacto-oligosaccharide, characterized in that: The following steps are involved: S1, pumping lactose solution as raw material into a reaction tank, and loading β-galactosidase immobilized by calcium pectin gel-chitosan into n-1 partitions obtained by layer-by-layer separation along the axial direction of the tank body through n layers of screens, wherein n is an integer ≥ 3; The composition of the calcium pectin gel-chitosan as a carrier is as follows: Chitosan was assembled on the surface of calcium pectin gel particles by electrostatic adsorption, and β-galactosidase was cross-linked to chitosan; S2, the cycle temperature is 45°C to 52°C, and the cycle time is 10 hours to 14 hours; The lactose solution flows through the surface of calcium pectin gel-chitosan and comes into contact with β-galactosidase for reaction; S3. The reaction liquid is pumped out, and galacto-oligosaccharides are separated using continuous simulated moving bed technology, and the separated lactose is recovered to continue the reaction.

2. The preparation method according to claim 1, characterized in that: The preparation method of β-galactosidase immobilized by calcium pectin gel-chitosan comprises the following steps: S11, placing calcium pectin gel microparticles in a chitosan solution, wherein the pH value of the chitosan solution is 4.0 to 4.5, and stirring, so that chitosan is enriched on the surface of the calcium pectin gel particles to form a polyelectrolyte complex; S12, adding β-galactosidase to the system, and adsorbing the β-galactosidase through chitosan; S13, placing the calcium pectin gel-chitosan adsorbed β-galactosidase obtained in S12 in a glutaraldehyde aqueous solution for cross-linking to obtain calcium pectin gel-chitosan solidified β-galactosidase.

3. The preparation method according to claim 2, characterized in that: The mass ratio of dry calcium pectin gel that swells to become calcium pectin gel microparticles by water absorption to chitosan is (1.1 to 1.3):

1.

4. The preparation method according to claim 2, characterized in that: The mass ratio of β-galactosidase to chitosan is (0.8 mg to 1.0 mg):1 g.

5. The preparation method according to claim 2, characterized in that: The process conditions for adsorbing β-galactosidase by chitosan in step S12 are as follows: 10℃-15℃, 200r / min-280r / min shaking table for adsorption, the shaking time is 3 hours to 5 hours.

6. The preparation method according to claim 2, characterized in that: The preparation method of calcium pectin gel microparticles comprises the following steps: S111, preparing a low-ester pectin and glucose mixture using deionized water, and adjusting the pH of the mixture to 3.0; S112, heating the mixed solution obtained in S111 in boiling water; S113, adding calcium chloride to the mixed solution heated in S112, and stirring rapidly and uniformly; placing the obtained mixed solution in an environment of 3°C to 5°C to cool and place to obtain calcium pectin gel; S114, calcium pectin gel is freeze-dried, crushed, and sieved through 250-300 mesh; S115, placing the calcium pectin powder obtained in S114 in an aqueous solution to absorb water to obtain calcium pectin gel micron particles.

7. The preparation method according to claim 6, characterized in that: Step S115 wherein the calcium pectin gel particles absorb water and swell to form calcium pectin gel micron particles comprises the following steps: A1. At 4° C., the calcium pectin gel particles obtained by drying in step S114 are immersed in a pyrrole monomer aqueous solution for 4 to 8 hours to allow the pyrrole monomer to evenly penetrate the calcium pectin gel particles; A2. Soak the hydrogel obtained in step A1 in an aqueous FeCl3 solution, and cross-link the pyrrole monomers into polypyrrole.

8. The preparation method according to claim 1, characterized in that: In step S2, the liquid circulation pump circulates the reaction liquid at a flow rate of 50 mL / min to 80 mL / min.

9. The preparation method according to claim 1, characterized in that: The sieve is a steel wire mesh with a mesh size of 100 to 200.

10. The preparation method according to claim 9, characterized in that: Each screen is threadedly connected to the inner wall of the reaction tank.

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