A preparation method of galacto-oligosaccharide

Abstract

The invention discloses a method for preparing galacto-oligosaccharides, comprising the following steps: S1, using a lactose solution as a raw material, loading a barrier layer with beta-galactosidase immobilized by calcium pectin gel-chitosan, wherein the calcium pectin gel-chitosan as a carrier is composed as follows: chitosan is assembled on the surface of calcium pectin gel particles by electrostatic adsorption, and the beta-galactosidase is cross-linked to the chitosan; S2, circulating, wherein the lactose solution flows through the surface of the calcium pectin gel-chitosan and contacts with the beta-galactosidase for reaction; S3, pumping out the reaction solution, separating the galacto-oligosaccharides using a continuous simulated moving bed technology, and recovering the separated lactose to continue the reaction; the invention uses the calcium pectin gel particles as the core, gathers chitosan on the surface of the gel particles, further adsorbs and cross-links the beta-galactosidase, and cooperates with the liquid circulation during the reaction process to effectively improve the conversion rate of the galacto-oligosaccharides.

Description

Technical Field

[0001] The present 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 galactose groups and 1 glucose group. The galactose groups are connected 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 connected by β-(1,4).

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

[0004] Chinese invention patent publication number CN111978423B discloses a method for preparing high-purity galacto-oligosaccharides. The method utilizes six-bed cis-simulated moving bed chromatography separation technology and employs a sodium-type macroporous cationic resin, which achieves superior separation of galacto-oligosaccharides compared to calcium-type cationic resins. This method enables the precise separation of glucose, lactose, galactose, and galacto-oligosaccharides, yielding a high-purity galacto-oligosaccharide product exceeding 98%. While existing technologies primarily focus on the separation and purification of galacto-oligosaccharides, the separation, recovery, and reuse of enzymes remain pressing challenges. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing galacto-oligosaccharides. The method 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:

[0007] S1. Lactose solution is pumped into a reaction tank as a raw material, and n-1 partitions are formed in the reaction tank by separating the layers along the axial direction of the tank body by n layers of screens, to load β-galactosidase immobilized by calcium pectin gel-chitosan, wherein n is an integer ≥3;

[0008] The composition of the pectin calcium gel-chitosan as a carrier is as follows:

[0009] Chitosan is assembled on the surface of calcium pectin gel particles by electrostatic adsorption, and β-galactosidase cross-links chitosan;

[0010] The ratio of the mass of the immobilized β-galactosidase input to the volume of the lactose solution is 18 mg to 25 mg / 1 L;

[0011] S2, the cycle temperature is 45°C to 52°C, and the cycle time is 10 hours to 14 hours;

[0012] The lactose solution flows through the surface of calcium pectin gel-chitosan and comes into contact with β-galactosidase for reaction;

[0013] S3. The reaction liquid is pumped out and separated using continuous simulated moving bed technology to obtain oligosaccharides, and the separated lactose is recovered to continue the reaction.

[0014] Preferably, the method for preparing β-galactosidase immobilized by calcium pectin gel-chitosan comprises the following steps:

[0015] S11, placing the calcium pectin gel microparticles in a chitosan solution with a pH of 4.0 to 4.5, stirring, and allowing the chitosan to accumulate on the surface of the calcium pectin gel particles to form a polyelectrolyte complex;

[0016] S12, adding β-galactosidase to the system, and adsorbing the β-galactosidase via chitosan;

[0017] S13, the beta-galactosidase adsorbed by the pectin calcium gel-chitosan obtained by S12 is placed in a glutaraldehyde aqueous solution and cross-linked to obtain the beta-galactosidase of pectin calcium gel-chitosan solidification. The present invention uses pectin calcium gel particles as the core structure for loading chitosan, and subsequent glutaraldehyde cross-linked chitosan and beta-galactosidase are coordinated, wherein glutaraldehyde cross-linking includes chitosan distributed on the surface of pectin calcium gel particles, and chitosan is the structure of the shell structure stabilizing pectin calcium gel particles as a carrier; In the production process of oligomeric galactose, due to the non-mobility of water in pectin high gel particles, lactose solution flows through the surface of pectin calcium 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 space flow channel with three-dimensional structure formed by carrier stacking, i.e. flows through the carrier surface, promotes the contact of lactose with enzyme, the carrier obtained by the present invention can be used repeatedly, and maintains a relatively stable conversion efficiency.

[0018] The mass ratio of the calcium pectin gel that swells to form calcium pectin gel microparticles by water absorption to chitosan is preferably (1.1 to 1.3): 1. The present invention utilizes the calcium pectin gel particles to effectively load chitosan and improves the stability of the calcium pectin gel particles through chitosan.

[0019] The preferred mass ratio of β-galactosidase to chitosan is (0.8 mg to 1.0 mg):1 g. In the present invention, β-galactosidase is distributed in a three-dimensional flow channel, and the flow of the lactose solution is coordinated to ensure sufficient contact between the lactose and the enzyme.

[0020] The preferred process conditions for adsorbing β-galactosidase by chitosan in step S12 are as follows:

[0021] The adsorption was carried out on a shaker at 10-15°C and 200-280 r / min for 3 to 5 hours.

[0022] The method for preparing calcium pectin gel microparticles preferably comprises the following steps:

[0023] S111, using deionized water to prepare a low-ester pectin and glucose mixture, and adjusting the pH of the mixture to 3.0;

[0024] S112, heating the mixed solution obtained in S111 in boiling water;

[0025] S113, adding calcium chloride to the mixed solution heated in S112, and stirring rapidly and evenly; placing the resulting mixed solution in an environment of 3°C to 5°C to cool and obtain calcium pectin gel;

[0026] S114, freeze-drying the calcium pectin gel, crushing it, and sieving it through 250-300 mesh;

[0027] S115. Place the calcium pectin powder obtained in S114 in an acid solution to swell and obtain calcium pectin gel micron particles.

[0028] Preferably, step S115 wherein the calcium pectin gel particles absorb water and swell to form calcium pectin gel micron particles comprises the following steps:

[0029] A1. Soak the calcium pectin gel particles obtained by drying in step S114 in an aqueous solution of pyrrole monomer at 4° C. for 4 to 8 hours to allow the pyrrole monomer to evenly penetrate the calcium pectin gel particles.

[0030] 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 being permeated into gel particles inside by the mode of infiltration by pyrrole monomer, along with the dispersed uniform distribution of moisture, ferric nitrate causes pyrrole as initiator and can self-polymerize into polypyrrole so as to be formed by polypyrrole to the stable structure of calcium pectin gel particles;And because pyrrole is with water continuous penetration, therefore polypyrrole effectively runs through gel particles, stabilizing gel particle structure;Gel particles are then put into chitosan solution, chitosan electrostatic adsorption is on gel particle surface, then glutaraldehyde is utilized to realize the crosslinking between chitosan and chitosan and chitosan and enzyme, obtains the carrier for enzyme fixation of structural stability.

[0031] Preferably, in step S2, the liquid circulation pump circulates the reaction liquid at a flow rate of 50 mL / min to 80 mL / min.

[0032] The sieve is preferably a steel mesh with mesh size of 100 to 200. The present invention utilizes the steel mesh in combination with the particle shape of the carrier to further effectively disperse the lactose solution. The solution flows through the small holes of the steel mesh at an instantaneous increase in flow rate, has a greater interaction force with the carrier, and is more easily exposed to the enzyme.

[0033] 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, making it convenient to put the immobilized enzyme into the reaction tank.

[0034] By adopting the above technical solution, the beneficial effects of the present invention are:

[0035] The present invention uses calcium pectin gel particles as cores, gathers 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 oligogalactosidose. During the conversion of lactose, n-1 partitions obtained by layer-by-layer separation along the axial direction of the tank body in the reaction tank are loaded with β-galactosidase immobilized by calcium pectin gel-chitosan, thereby preventing a large amount of calcium pectin gel-chitosan from stacking, ensuring that the lactose solution flows along the distribution of the enzyme, and facilitating mass transfer of reactants and products. The carrier structure for immobilizing the β-galactosidase in the present invention is stable, has good biocompatibility, and can be reused. Through the cooperation of the screen, flow rate changes between micro-ranges are formed during the circulation of the reaction solution, thereby improving the conversion efficiency of oligogalactosidose. DETAILED DESCRIPTION

[0036] In order to further explain the technical solution of the present invention, the present invention is described in detail below through specific embodiments.

[0037] Example 1

[0038] This embodiment discloses a method for preparing galacto-oligosaccharide, comprising the following steps:

[0039] S1. 500 g / L lactose solution was pumped into a 20 L reaction tank as a raw material. β-galactosidase immobilized by calcium pectin gel-chitosan was loaded into three compartments in the reaction tank separated layer by layer along the axial direction of the tank by four layers of sieves;

[0040] The composition of the pectin calcium gel-chitosan as a carrier is as follows:

[0041] Chitosan is assembled on the surface of calcium pectin gel particles by electrostatic adsorption, and β-galactosidase cross-links chitosan;

[0042] S2, the cycle temperature is 45°C to 48°C, and the cycle time is 14 hours;

[0043] The lactose solution flows through the surface of calcium pectin gel-chitosan and comes into contact with β-galactosidase for reaction;

[0044] In step S2, the liquid circulation pump circulates the reaction solution at a flow rate of 50 mL / min.

[0045] S3. The reaction liquid is pumped out and separated using continuous simulated moving bed technology to obtain oligosaccharides, and the separated lactose is recovered to continue the reaction.

[0046] The ratio of the mass of the immobilized β-galactosidase to the volume of the lactose solution was 18 mg / 1 L;

[0047] The preparation method of β-galactosidase immobilized by calcium pectin gel-chitosan in this embodiment comprises the following steps:

[0048] S11, placing the calcium pectin gel microparticles in a chitosan solution with a pH of 4.0, stirring, and allowing the chitosan to accumulate on the surface of the calcium pectin gel particles to form a polyelectrolyte complex;

[0049] In this embodiment, the mass ratio of the calcium pectin gel that swells to form calcium pectin gel microparticles by absorbing water to chitosan is 1.1:1.

[0050] S12, adding β-galactosidase to the system, and adsorbing the β-galactosidase via chitosan;

[0051] In this embodiment, the mass ratio of β-galactosidase to chitosan is 0.8 mg:1 g.

[0052] S13. The calcium pectin gel-chitosan-adsorbed β-galactosidase obtained in S12 is placed in a 2% glutaraldehyde aqueous solution for cross-linking for 3 hours to obtain calcium pectin gel-chitosan-solidified β-galactosidase.

[0053] In step S12 of this embodiment, the process conditions for adsorbing β-galactosidase by chitosan are as follows:

[0054] The adsorption was carried out on a shaker at 10°C and 200 r / min for 3 hours.

[0055] The preparation method of calcium pectin gel microparticles in this embodiment comprises the following steps:

[0056] S111. Prepare a mixture of low-ester pectin and glucose using deionized water, wherein the mass fraction of low-ester pectin (35% degree of esterification, 88% total galacturonic acid) is 1% and the mass fraction of glucose is 10%. Adjust the pH of the mixture to 3.0.

[0057] S112, placing the mixture obtained in S111 in boiling water and heating for 2 minutes;

[0058] 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; cooling the resulting mixed solution in an environment of 3° C. to 5° C. for 24 hours to obtain a calcium pectin gel;

[0059] S114, freeze-dried calcium pectin gel, crushed, and sieved through 250 mesh;

[0060] S115. The calcium pectin powder obtained in S114 is placed in an aqueous solution to absorb water to obtain calcium pectin gel micron particles.

[0061] 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;

[0062] 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.

[0063] Example 2

[0064] The main difference between this embodiment and embodiment 1 is that:

[0065] In this embodiment, step S115 of the calcium pectin gel particles absorbing water and swelling to form calcium pectin gel micron particles includes the following steps:

[0066] A1. Soak the calcium pectin gel particles obtained by drying in step S114 in a pyrrole monomer aqueous solution at 4° C. for 6 hours to allow the pyrrole monomer to evenly penetrate the calcium pectin gel particles.

[0067] The mass ratio of dry calcium pectin gel particles to pyrrole monomers was 10:1;

[0068] A2. Soak the hydrogel obtained in step A1 in a FeCl3 (0.089 mol / L) aqueous solution to crosslink the pyrrole monomers into polypyrrole.

[0069] Example 3

[0070] The main differences between this embodiment and embodiment 2 are shown in Table 1 and Table 2.

[0071] Example 4

[0072] The main differences between this embodiment and embodiment 2 are shown in Table 1 and Table 2.

[0073] Example 5

[0074] The main differences between this embodiment and embodiment 2 are shown in Table 1 and Table 2.

[0075] Table 1 Process parameters for preparing galacto-oligosaccharides in Examples 1 to 5

[0076]

[0077] Table 2 Process parameters for immobilizing β-galactosidase in Examples 1 to 5

[0078]

[0079] Comparative Example 1

[0080] This comparative example discloses a method for preparing galacto-oligosaccharide, comprising the following steps:

[0081] 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;

[0082] S2, the cycle temperature is 45°C to 48°C, and the cycle time is 10 hours;

[0083] S3. Separate the reaction solution using continuous simulated moving bed technology to obtain galacto-oligosaccharides, and recover the separated lactose to continue the reaction.

[0084] Comparative Example 2

[0085] This comparative example discloses a method for preparing galacto-oligosaccharide, comprising the following steps:

[0086] S1. A 500 g / L lactose solution was pumped into a 20 L reaction tank as a raw material. The reaction tank was then loaded with chitosan-immobilized β-galactosidase in three compartments separated by four layers of mesh along the axial direction of the tank.

[0087] S2, the cycle temperature is 45°C to 48°C, and the cycle time is 10 hours;

[0088] When the lactose solution flows through the surface of chitosan, it comes into contact with β-galactosidase and reacts;

[0089] In step S2, the liquid circulation pump circulates the reaction solution at a flow rate of 50 mL / min.

[0090] S3. The reaction liquid is pumped out and separated using continuous simulated moving bed technology to obtain oligosaccharides, and the separated lactose is recovered to continue the reaction.

[0091] The ratio of the mass of the immobilized β-galactosidase to the volume of the lactose solution was 18 mg / 1 L;

[0092] The preparation method of chitosan-immobilized β-galactosidase in this embodiment comprises the following steps:

[0093] S11. Add chitosan solution with a pH of 4.0 dropwise into a large amount of 1 mol / L NaOH aqueous solution, let it stand for reaction, then wash the chitosan microspheres with distilled water until neutral, and collect the microspheres by filtration.

[0094] S12. The chitosan microspheres prepared in S11 were placed in a glutaraldehyde aqueous solution, allowed to react, and β-galactosidase was added thereto at a mass ratio of β-galactosidase to chitosan of 0.8 mg:1 g. The chitosan adsorbed the β-galactosidase and cross-linked in a 2% glutaraldehyde aqueous solution for 3 hours to obtain chitosan-solidified β-galactosidase.

[0095] Table 3 Conversion of lactose into galacto-oligosaccharides

[0096]

[0097] 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.

[0098] Table 4 Structural stability of enzyme carriers immobilized by soaking in Examples 1 to 5 and Comparative Example 2

[0099]

[0100] As shown in Table 4, the carrier for immobilizing enzymes of the present invention has a stable structure after compression and good biocompatibility, and is suitable for the preparation of galacto-oligosaccharides.

[0101] 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 conversion retention rate were shown in Table 5.

[0102] Table 5 Conversion rate of the fifth preparation of galacto-oligosaccharides by immobilized enzyme in Examples 1 to 5 and Comparative Example 2

[0103]

[0104] In conjunction with Table 5, it can be seen that the present invention uses gel as the inner core of enzyme carrier to cooperate with carrier surface chitosan in the solid state in neutral aqueous solution, and the immobility of water in pectin high gel particles, realizes the lactose solution for controlling flow to gather and flow through from the distribution position of stacking immobilized enzyme, lactose solution fully flows through from the enzyme surface, ensures that lactose solution is fully contacted with beta-galactosidase, promotes the contact of lactose with enzyme;The present invention is used for the carrier of immobilized enzyme to have stable structure, good compression resistance, while ensuring that enzyme is contacted with lactose solution, the carrier obtained by the present invention can be used repeatedly, and maintains relatively stable conversion efficiency.Compared to pure chitosan as enzyme carrier in comparative example 2, the chitosan balls with porous structure are difficult to ensure that lactose solution is fully enriched relative to enzyme in the flow process, and compared to Examples 1 to 5 of the present invention, the dispersibility of enzyme load is also limited, therefore, the production method of oligomeric galactose proposed by the present invention has high conversion rate and can be repeatedly used. Further comparing Example 1 with Examples 2 to 5, Examples 2 to 5 penetrate the pyrrole monomer into the interior of the gel particles by infiltration, and with the uniform dispersion of water, ferric nitrate is used as an initiator to trigger the polymerization of pyrrole into polypyrrole, thereby stabilizing the structure of the calcium pectin gel particles through the formation of polypyrrole; the gel particles are then added to a chitosan solution, and chitosan is electrostatically adsorbed on the surface of the gel particles. Glutaraldehyde is then 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 more stable conversion efficiency.

Claims

1. A method for preparing galacto-oligosaccharide, characterized in that: The following steps are involved: S1. Lactose solution is pumped into a reaction tank as a raw material, and n-1 partitions are formed in the reaction tank by separating the layers along the axial direction of the tank body by n layers of screens, to load β-galactosidase immobilized by calcium pectin gel-chitosan, wherein n is an integer ≥3; The composition of the pectin calcium gel-chitosan as a carrier is as follows: Chitosan is assembled on the surface of calcium pectin gel particles by electrostatic adsorption, and β-galactosidase cross-links chitosan; S2, the cycle temperature is 45°C to 48°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 oligogalactose is obtained by using continuous simulated moving bed technology, and the separated lactose is recovered and continued to react; The preparation method of β-galactosidase immobilized by calcium pectin gel-chitosan comprises the following steps: S11, placing the calcium pectin gel microparticles in a chitosan solution with a pH of 4.0 to 4.5, stirring, and allowing the chitosan to accumulate on the surface of the calcium pectin gel particles to form a polyelectrolyte complex; The calcium pectin gel particles absorb water and swell to form calcium pectin gel micron particles, comprising the following steps: A1. Soak the calcium pectin gel particles obtained by drying in step S114 in an aqueous solution of pyrrole monomer at 4° C. for 4 to 8 hours to allow the pyrrole monomer to evenly penetrate the calcium pectin gel particles. A2, soaking the hydrogel obtained in step A1 in an aqueous FeCl3 solution to crosslink the pyrrole monomers into polypyrrole; S12, adding β-galactosidase to the system, and adsorbing the β-galactosidase via 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.

2. The preparation method according to claim 1, wherein: The mass ratio of dry calcium pectin gel that swells to calcium pectin gel microparticles by water absorption to chitosan is (1.1 to 1.3):

1.

3. The preparation method according to claim 1, wherein: The mass ratio of β-galactosidase to chitosan is (0.8 mg to 1.0 mg):1 g.

4. The preparation method according to claim 1, wherein: The process conditions for adsorbing β-galactosidase by chitosan in step S12 are as follows: The adsorption was carried out on a shaker at 10-15°C and 200-280 r / min for 3 to 5 hours.

5. The preparation method according to claim 1, wherein: The preparation method of calcium pectin gel micron particles comprises the following steps: S111, using deionized water to prepare a low-ester pectin and glucose mixture, 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 evenly; placing the resulting mixed solution in an environment of 3°C to 5°C to cool and obtain calcium pectin gel; S114, freeze-drying the calcium pectin gel, crushing it, and sieving it through 250-300 mesh; S115. The calcium pectin powder obtained in S114 is placed in an aqueous solution to absorb water to obtain calcium pectin gel micron particles.

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

7. The preparation method according to claim 1, wherein: The screen is a steel wire mesh with a mesh size of 100 to 200.

8. The preparation method according to claim 7, characterized in that: Each screen is threadedly connected to the inner wall of the reaction tank.

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