An enzymatic production process for diglycerides

Abstract

This invention discloses an enzymatic production process for diglycerides, belonging to the field of bio-enzyme catalysis technology. The process employs a dual-enzyme combined catalytic system: immobilized lipase A is a copper oxide nanozyme complex, with porous silica treated with APTES ammoniation as the carrier; immobilized lipase B is a *Mucor* lipase immobilized on mesoporous silica material. An adsorption resin containing tertiary amine groups is added to the reaction system as a polar adsorbent to selectively adsorb excess glycerol and control the effective glycerol concentration. The copper oxide nanozyme complex is prepared by chemical precipitation of copper sulfate and sodium hydroxide, exhibiting lipase-like activity and excellent transesterification catalytic ability. This catalytic system achieves a vegetable oil conversion rate of over 87.7%, a diglyceride product purity of over 92.7%, a product yield exceeding 82.7%, and an enzyme activity retention rate exceeding 91.3%, providing a new technical route for the efficient preparation of functional oils.

Description

Technical Field

[0001] This invention belongs to the field of bio-enzyme catalysis technology, specifically an enzymatic production process for diglycerides. Background Technology

[0002] Currently, the industry typically uses fatty acid-glycerol ester exchange or vegetable oil-glycerol esterification reactions to improve yield and selectivity by immobilizing lipases and controlling water activity (e.g., by adding molecular sieves). However, the following problems still exist:

[0003] When glycerol is in excess, it is easy to generate monoglycerides (MAG) or triglycerides (TAG), which affects the purity of the product; the catalytic efficiency of a single enzyme is limited, making it difficult to balance conversion rate and selectivity; purification methods mostly rely on single-molecule distillation or membrane evaporation, which are insufficient in removing TAG or pigments, resulting in unsatisfactory product purity.

[0004] Furthermore, traditional immobilized lipases mostly utilize natural enzymes, resulting in high preparation costs, poor stability, and easy inactivation. In recent years, nanozyme technology has opened new avenues for the field of catalysis, particularly metal oxide nanozymes, which offer advantages such as low cost, high stability, and ease of preparation. However, the application of copper oxide nanozymes in diglyceride production has not yet been reported, mainly due to several technical obstacles. First, exposed CuO nanozymes lack the active site structure of lipases, leading to low catalytic efficiency and poor selectivity in transesterification reactions, making it difficult to achieve highly selective synthesis of diglycerides. Second, in the weakly acidic reaction environment of diglyceride production, Cu… 2+ The glycerol readily dissolves from the surface of CuO nanoparticles, leading to rapid catalyst deactivation and failing to meet the requirements of continuous industrial production. Simultaneously, current technologies lack effective methods for controlling glycerol concentration; excess glycerol tends to generate MAG or TAG, while insufficient glycerol results in low conversion rates, making it difficult to balance the contradiction between yield and selectivity. Furthermore, the limited contact interface between CuO nanozymes and lipid substrates leads to high mass transfer resistance, and the lack of effective dispersion and homogenization methods further contributes to low catalytic efficiency. Transesterification is extremely sensitive to water activity; existing single molecular sieve control methods struggle to precisely maintain the optimal water activity range under vacuum conditions, severely impacting reaction equilibrium and product distribution. Finally, traditional single purification methods (such as molecular distillation or thin-film evaporation) are ineffective at removing structurally similar TAGs and other impurities, failing to meet the requirements for high-purity diglyceride products. More importantly, current research focuses primarily on improving individual technological aspects, lacking a systematic integrated approach encompassing catalyst design, reaction condition control, and product separation and purification, hindering breakthroughs in overall process performance.

[0005] Therefore, there is an urgent need for a new technical solution to improve the yield and purity of DAG (diglyceride) by precisely controlling the glycerol concentration, using novel nanozyme catalysis, and advanced purification methods. Summary of the Invention

[0006] The purpose of this invention is to provide an enzymatic production process for diglycerides, which achieves high-selectivity, high-purity DAG preparation through an innovative catalytic system of copper oxide nanoenzyme complex, dynamic regulation of glycerol concentration by adsorption materials, synergistic effect of dual-enzyme tandem catalysis, precise control of vacuum and molecular sieves, and a combined purification process.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The enzymatic production process for diglycerides described in this invention includes the following steps:

[0009] (1) Vegetable oil, glycerol and immobilized lipase A are added to a reaction system containing polar adsorbent material for the first enzymatic hydrolysis. The polar adsorbent material selectively adsorbs excess glycerol and controls the effective concentration of glycerol in the reaction system to obtain the primary reaction product.

[0010] (2) The primary reaction product and the immobilized lipase B pretreated by high-speed homogenization were subjected to a second enzymatic hydrolysis under vacuum-mediated conditions. The water activity was controlled by molecular sieves, and the light phase product was collected to obtain crude diglyceride.

[0011] (3) The crude diglyceride was purified by short-path molecular distillation combined with thin-film evaporation to obtain a high-purity diglyceride product;

[0012] The immobilized lipase A is a copper oxide nanozyme complex, which uses CuO nanoparticles prepared by chemical precipitation as the active component. The CuO nanoparticles have a particle size of 3-8 nm and a specific surface area of ​​120-200 m² / g. After immobilization on a carrier, the CuO nanoparticles exhibit lipase-like activity and transesterification catalytic ability. The carrier has a pore size of 8-20 nm and a pore volume of 0.8-1.5 cm³ / g. The immobilized lipase B is a Mucor lipase immobilized on an organic mesoporous silica material. The polar adsorption material is an adsorption resin containing tertiary amine groups, and the pore size of the adsorption resin containing tertiary amine groups is 50-200 nm.

[0013] As a further explanation of the present invention, the adsorption resin containing tertiary amine groups has a suitable pore size structure (50-200 nm), providing ample space for molecular mass transfer. The tertiary amine groups, as polar active sites, preferentially adsorb polar glycerol molecules through hydrogen bonding and electrostatic interactions, while hydrophobic DAG, MAG, and FFA molecules have low affinity for the tertiary amine groups and are preferentially retained in the oil phase, thereby achieving selective separation of glycerol.

[0014] As a further explanation of the present invention, the design of the support pore size of 8-20 nm is based on the following considerations: the support pore size should be larger than the maximum size of CuO nanoparticles (8 nm) to ensure that the particles can effectively enter the support pores for immobilization; at the same time, the pore size should not be too large to maintain the mechanical strength of the support and the stable anchoring of CuO particles. The support pore volume is 0.8-1.5 cm³ / g. This range ensures an appropriate loading of CuO nanoparticles: if the pore volume is too small (<0.8 cm³ / g), the loading will be insufficient, affecting the catalytic efficiency; if the pore volume is too large (>1.5 cm³ / g), the mechanical strength of the support will decrease, and the particles will be unevenly dispersed. The preferred pore volume range allows the CuO nanoparticle loading to reach 5-15% of the support mass, achieving optimal catalytic performance.

[0015] Preferably, the vegetable oil is selected from at least one of soybean oil, rapeseed oil, peanut oil, olive oil, flaxseed oil, palm oil, and corn oil.

[0016] Preferably, the amount of polar adsorbent material used in step (1) is 3-8% of the mass of vegetable oil, the density of tertiary amine groups in the adsorbent resin containing tertiary amine groups is 1.2-3.5 mmol / g, the molar ratio of glycerol to vegetable oil is 1.5-3.0:1, and during the first enzymatic hydrolysis, the temperature is controlled at 45-65℃, the pH is 6.5-7.5, and the reaction is stirred at a stirring speed of 200-400 rpm for 4-8 hours.

[0017] Preferably, the preparation of the copper oxide nanozyme complex includes the following steps:

[0018] (a) Mix 0.1-0.5M copper sulfate aqueous solution with sodium hydroxide aqueous solution and control the pH to 11-12;

[0019] (b) Stir the reaction at 80-100℃ for 1-3 hours at a stirring speed of 100-300 rpm to form a mixture containing CuO nanoparticles;

[0020] (c) The mixture after the reaction in step (b) is separated by centrifugation, and the resulting solid is washed with deionized water to remove byproducts, thus obtaining CuO nanoparticles;

[0021] (d) The copper oxide nanoenzyme complex is prepared by immobilizing CuO nanoparticles on the support through physical adsorption or chemical bonding.

[0022] The CuO nanoparticles exhibit oxidoreductase and hydrolase-like activities under the reaction conditions.

[0023] The copper oxide nanozyme complex forms Cu-N coordination bonds through a support, anchoring CuO nanoparticles within the support pores (8-20 nm). The support is porous silica pre-ammoniated with 3-aminopropyltriethoxysilane (APTES), allowing CuO nanoparticles to chemically bond to the support via Cu–N coordination bonds, forming the copper oxide nanozyme complex. This structure causes localized alkalization of the CuO surface charge due to the support microenvironment, while the steric hindrance of the support pores hinders H⁺ from contacting the active sites, thereby inhibiting Cu⁺ activity under macroscopic pH conditions of 6.5-7.5. 2+ Dissolution;

[0024] This complex exhibits lipase-like activity and excellent transesterification catalysis.

[0025] Preferably, the high-speed homogenization pretreatment in step (2) includes: treating the immobilized lipase B suspension with a high-speed homogenizer with a rotation speed of 8000-15000 rpm for 2-5 min; in step (2), during the second enzymatic hydrolysis, the enzymatic hydrolysis is carried out for 2-4 h under the conditions of vacuum degree of 0.01-0.1 kPa and temperature of 35-50℃.

[0026] Preferably, the molecular sieve is a 4A molecular sieve or a 5A molecular sieve, used to control the water activity of the reaction system within the range of 0.2-0.6; the organic mesoporous silica material is an MCM-41 mesoporous silica material or an SBA-15 mesoporous silica material, and the specific surface area of ​​the organic mesoporous silica material is 600-1200 m² / g.

[0027] More preferably, the organic mesoporous silica material is MCM-41 mesoporous silica material or SBA-15 mesoporous silica material, wherein the MCM-41 mesoporous silica material is prepared using a cationic surfactant as a template agent, preferably hexadecyltrimethylammonium bromide (CTAB);

[0028] SBA-15 mesoporous silica material is prepared using triblock copolymers as template agents, preferably P123 triblock copolymers (EO20PO70EO20).

[0029] As a further explanation of the present invention, the molecular sieve (4A / 5A) is filled in the top of the reactor in the form of a fixed bed, physically isolated from the reaction liquid (e.g., separated by a sintered metal mesh). During vacuum dehydration, gaseous water molecules are selectively adsorbed after passing through the molecular sieve layer, while liquid water continues to evaporate due to Henry's law equilibrium, forming a dynamic equilibrium of gas-solid adsorption-liquid-gas evaporation, thus stabilizing the water activity within the range of 0.2-0.6.

[0030] As a further explanation of the present invention, the carrier (MCM-41 / SBA-15) for immobilized lipase B is formed by coating the surface with polyvinyl alcohol (PVA) to form an elastic protective layer (thickness ≈5 nm). Its shear modulus (≈1 GPa) is much higher than that of homogeneous shear stress (≈0.1 MPa at 8000-15000 rpm), and the flexible segments of PVA can disperse the impact force and prevent the carrier from breaking.

[0031] Preferably, in steps (1) and (2), the mass amounts of immobilized lipase A and immobilized lipase B are 1-4% and 2-6% of the mass of the vegetable oil, respectively; and the pH is maintained at 6.5-7.5 during the reaction.

[0032] Preferably, the vacuum-mediated conditions in step (2) are a two-step vacuum procedure: the first step is to react for 1-2 hours under a vacuum of 0.05-0.1 kPa to promote transesterification, and the second step is to react for 1-2 hours under a vacuum of 0.01-0.05 kPa and a temperature of 35-50°C to promote the separation of the light phase.

[0033] Preferably, in step (3), before using short-path molecular distillation combined with thin-film evaporation, the crude diglyceride is first decolorized with activated carbon, the amount of activated carbon being 0.5-2% of the crude product mass, the treatment temperature being 60-80℃, and the treatment time being 30-60min; then it is dehydrated using 3A molecular sieve, the amount of 3A molecular sieve being 1-3% of the crude product mass.

[0034] As a further explanation of the present invention, after the activated carbon is decolorized, the reaction solution is filtered through a 0.22 μm filter membrane to retain activated carbon particles and ensure the cleanliness of the filtrate from the subsequent molecular sieve dehydration treatment.

[0035] Preferably, the evaporation temperature of the short-path molecular distillation in step (3) is 100-140℃, the condensation temperature is 30-50℃, the system vacuum degree is 0.001-0.01Pa, and the scraping speed is 300-600rpm; the film evaporation temperature is 80-120℃, and the film thickness is 0.1-0.5mm.

[0036] The beneficial effects of this invention are as follows:

[0037] 1. Adsorption-dual-enzyme system synergistically improves yield and purity. Adsorption resin containing tertiary amine groups is used to regulate glycerol concentration, effectively inhibiting the formation of MAG and TAG; the dual-enzyme tandem design of copper oxide nanozyme and traditional lipase works synergistically to significantly improve substrate conversion efficiency, while improving the selectivity and final yield of diglyceride (DAG).

[0038] 2. High-speed homogenization and vacuum-molecular sieve strategy enhance catalytic efficiency. High-speed homogenization pretreatment significantly increases the contact interface between the enzyme and substrate, while vacuum control combined with molecular sieves allows for precise regulation of water activity, enabling accurate control of the reaction pathway, effectively promoting transesterification and light phase separation, and improving overall conversion rate and enzyme stability.

[0039] 3. Multi-stage composite purification process ensures high product quality. Utilizing a multi-stage combination of activated carbon decolorization, molecular sieve dehydration, short-path molecular distillation, and thin-film evaporation, it effectively removes TAG, pigments, and other impurities, achieving a high-quality diglyceride product with a purity exceeding 93%, meeting the standards for industrial production and food and pharmaceutical applications.

[0040] 4. Breakthrough in Catalytic Performance of Support-Immobilized Copper Oxide Nanoenzyme Composites. Through immobilization on a porous silica support treated with APTES, CuO nanoparticles are stably anchored via Cu-N coordination bonds. The confinement effect and microenvironment regulation of the support alter the electronic structure of the CuO surface, resulting in lipase-like activity. This composite system exhibits excellent transesterification catalysis in diglyceride synthesis, demonstrating higher thermal stability, pH stability, and organic solvent tolerance compared to traditional natural enzymes, with a 25-50% increase in specific activity. It provides an efficient and stable nanocatalytic solution for the preparation of functional oils. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to these embodiments. Those skilled in the art should recognize that the present invention covers all possible alternatives, improvements and equivalents within the scope of the claims.

[0042] Description of raw materials and equipment used in this invention:

[0043] Soybean oil, rapeseed oil, palm oil and olive oil: purchased from COFCO Group, iodine value 120-140;

[0044] Glycerin: analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0045] Mucor lipase: purchased from Novozymes, 43111M, 250 IUN / g;

[0046] Adsorption resin containing tertiary amine groups: purchased from Rohm and Haas, model IRA-400;

[0047] 3A, 4A and 5A molecular sieves: purchased from Shanghai Ruji Biotechnology Development Co., Ltd.;

[0048] Commercially immobilized lipase: purchased from Novozymes, Lipozyme RM IM;

[0049] All other reagents were of analytical grade and obtained through commercial purchase.

[0050] Example 1

[0051] An enzymatic process for the production of diglycerides includes the following steps:

[0052] (1) Take 100g of soybean oil, add glycerol at a molar ratio of 2.0:1 to soybean oil, add 5g of adsorption resin containing tertiary amine groups (average pore size 100nm, tertiary amine group density 2.0mmol / g) (accounting for 5% of the mass of soybean oil), and immobilize 2g of lipase A; react at 55℃ and stirring speed 300rpm for 6h, and maintain pH at 7.0 to obtain the primary reaction product.

[0053] (2) The primary reaction product was mixed with 4g of immobilized lipase B pretreated by high-speed homogenization. 4A molecular sieve was filled into the top of the reactor in the form of a fixed bed and physically isolated from the reaction liquid by a sintered metal mesh. A two-step vacuum procedure was adopted: the first step was to react for 1.5h under a vacuum of 0.1kPa and at a temperature of 42℃; the second step was to react for 1.5h under a vacuum of 0.03kPa and at a temperature of 42℃ to promote the separation of the light phase. The light phase product was collected to obtain crude diglyceride.

[0054] (3) The crude diglyceride was first decolorized with activated carbon (1% of the crude diglyceride mass) at 70°C for 45 minutes. After decolorization, it was filtered through a 0.22 μm filter membrane to retain activated carbon particles. Then, it was dehydrated with 3A molecular sieve (2% of the crude diglyceride mass) at 80°C for 2 hours. Short-path molecular distillation was used with an evaporation temperature of 120°C, a condensation temperature of 40°C, a vacuum of 0.005 Pa, and a scraping speed of 450 rpm. Subsequently, thin-film evaporation was carried out at a temperature of 100°C and a film thickness of 0.3 mm.

[0055] The method for preparing the immobilized lipase A includes the following steps:

[0056] Step 1: APTES amination pretreatment step; Take 10g of porous silica (average pore size 14nm, pore volume 1.2cm³ / g), treat it with 0.1M HCl solution for 2 hours to activate the surface hydroxyl groups, wash it with deionized water until neutral, and dry it at 80℃ for 4 hours; then disperse the activated porous silica (support) in 200mL of anhydrous toluene, add 5mL of 3-aminopropyltriethoxysilane (APTES), and reflux at 110℃ for 12 hours under nitrogen protection; after the reaction is completed, filter it, wash it with toluene, ethanol and deionized water in sequence, and dry it under vacuum at 120℃ for 6 hours to obtain the amination-treated porous silica support.

[0057] Step 2: Mix 200 mL of 0.2 M copper sulfate aqueous solution with 100 mL of 2 M sodium hydroxide aqueous solution, adjust the pH to 11.5 with NaOH, and stir the mixture at 90 °C for 2 hours at a stirring speed of 200 rpm. After the reaction is completed, centrifuge the mixture and wash the resulting mixture containing CuO nanoparticles three times with deionized water to obtain CuO nanoparticles with an average particle size of 6 nm and a specific surface area of ​​160 m² / g. The prepared CuO nanoparticles are chemically bonded to an aminated porous silica support through Cu-N coordination bonds to obtain a copper oxide nanoenzyme complex, i.e., immobilized lipase A.

[0058] The preparation method of MCM-41 mesoporous silica material is as follows: 2.0 g of hexadecyltrimethylammonium bromide (CTAB) is dissolved in 480 mL of deionized water, 14 mL of ammonia water is added, and after stirring for 30 min, 10 mL of tetraethyl orthosilicate (TEOS) is added dropwise. The mixture is reacted at 80 °C for 2 hours, filtered, washed, dried, and calcined at 550 °C for 6 hours to obtain MCM-41 mesoporous silica material with a specific surface area of ​​850 m² / g.

[0059] The method for preparing the immobilized lipase B includes the following steps:

[0060] Lipase B suspension was prepared by dissolving Mucor lipase at a concentration of 10 mg / mL in 0.1 M phosphate buffer (pH 7.0). The suspension was homogenized at 12,000 rpm for 3 min and then mixed with MCM-41 mesoporous silica material (specific surface area 850 m² / g) at a protein-to-carrier mass ratio of 1:10. Immobilization was achieved through physical adsorption. A 0.5 wt% aqueous solution of polyvinyl alcohol (PVA) with a molecular weight of 30,000–70,000 g / mol was prepared and the carrier was immersed for 2 hours to form an elastic protective layer with a thickness of about 5 nm. After drying, immobilized lipase B was obtained.

[0061] Example 2

[0062] An enzymatic process for the production of diglycerides includes the following steps:

[0063] (1) Take 100g of rapeseed oil, add glycerol at a molar ratio of 1.5:1 to rapeseed oil, add 3g of adsorption resin containing tertiary amine groups (average pore size 50nm, tertiary amine group density 1.2mmol / g) (accounting for 3% of the rapeseed oil mass), and immobilize 1g of lipase A; react at 45℃ and stirring speed 200rpm for 8h, and maintain pH at 7.0 to obtain the primary reaction product.

[0064] (2) The primary reaction product was mixed with 2g of immobilized lipase B pretreated by high-speed homogenization. 2g of 5A molecular sieve was packed into the top of the reactor in a fixed bed and physically isolated from the reaction liquid by a sintered metal mesh, and the water activity was controlled to 0.4. A two-step vacuum procedure was adopted: the first step was to react at 0.01kPa for 4h at 35℃; the second step was to react at 0.03kPa for 1.5h at 35℃ to promote the separation of the light phase. The light phase product was collected to obtain crude diglyceride.

[0065] (3) The crude diglyceride was first decolorized with activated carbon (0.5% of the crude diglyceride mass) at 60°C for 30 minutes. After decolorization, it was filtered through a 0.22 μm filter membrane to retain activated carbon particles. Then, it was dehydrated with 3A molecular sieve (1% of the crude diglyceride mass) at 80°C for 2 hours. Short-path molecular distillation was adopted: evaporation temperature 100°C, condensation temperature 30°C, vacuum degree 0.001Pa, and scraper rotation speed 300rpm. Then, thin film evaporation was carried out at 80°C with a film thickness of 0.1mm.

[0066] The method for preparing the immobilized lipase A includes the following steps:

[0067] Step 1: The APTES amination pretreatment process is the same as in Example 1.

[0068] Step 2: Mix 150 mL of 0.1 M copper sulfate solution with 75 mL of 2 M sodium hydroxide solution, adjust the pH to 12 with NaOH, and stir the mixture at 100 °C for 1 hour at a stirring speed of 200 rpm. After the reaction is complete, centrifuge the mixture and wash the mixture containing CuO nanoparticles three times with deionized water to obtain CuO nanoparticles with an average particle size of 3 nm and a specific surface area of ​​200 m² / g. The prepared CuO nanoparticles are chemically bonded to an aminated porous silica support through Cu-N coordination bonds to obtain a copper oxide nanoenzyme complex, i.e., immobilized lipase A.

[0069] Preparation method of SBA-15 mesoporous silica material: 4.0 g of triblock copolymer P123 (EO20PO70EO20) was dissolved in 120 mL of 2M HCl solution and stirred at 35 °C until completely dissolved; then 8.5 mL of tetraethyl orthosilicate (TEOS) was added dropwise, and after stirring at 35 °C for 20 hours, the mixture was transferred to a hydrothermal reaction at 100 °C for 24 hours; after filtration, washing, and drying, the template agent was removed by calcination at 550 °C for 6 hours to obtain SBA-15 mesoporous silica material with a specific surface area of ​​850 m² / g and an average pore size of about 6 nm.

[0070] The method for preparing the immobilized lipase B includes the following steps:

[0071] Lipase B suspension was prepared by dissolving Mucor lipase at a concentration of 10 mg / mL in 0.1 M phosphate buffer (pH 7.0). The suspension was homogenized at 12,000 rpm for 3 min and then mixed with SBA-15 mesoporous silica material (specific surface area 850 m² / g) at a protein-to-carrier mass ratio of 1:10. Immobilization was achieved through physical adsorption. A 0.5 wt% aqueous solution of polyvinyl alcohol (PVA) with a molecular weight of 30,000–70,000 g / mol was prepared and the carrier was immersed for 2 hours to form an elastic protective layer with a thickness of about 5 nm. After drying, immobilized lipase B was obtained.

[0072] Example 3

[0073] An enzymatic process for the production of diglycerides includes the following steps:

[0074] (1) 100g of palm oil was used, and glycerol was added at a molar ratio of 3.0:1 to glycerol. 8g of adsorption resin containing tertiary amine groups (average pore size 200nm, tertiary amine group density 3.5mmol / g) (accounting for 8% of the mass of palm oil) was added, and 4g of immobilized lipase A was added. The reaction was carried out at 65℃ and 400rpm for 4h, and the pH was maintained at 7.0 to obtain the primary reaction product.

[0075] (2) The primary reaction product was mixed with 6g of immobilized lipase B pretreated by high-speed homogenization. 6g of 4A molecular sieve was filled into the top of the reactor in the form of a fixed bed and physically isolated from the reaction liquid by a sintered metal mesh. The water activity was controlled to 0.6. A two-step vacuum procedure was adopted: the first step was to react at 0.05kPa for 2h at 42℃; the second step was to react at 0.03kPa for 1.5h at 42℃ to promote the separation of the light phase. The light phase product was collected to obtain crude diglyceride.

[0076] (3) The crude diglyceride was first decolorized with activated carbon (2% of the crude diglyceride mass) at 80°C for 60 minutes. After decolorization, it was filtered through a 0.22 μm filter membrane to retain activated carbon particles. Then, it was dehydrated with 3A molecular sieve (2% of the crude diglyceride mass) at 80°C for 2 hours. Short-path molecular distillation was used with an evaporation temperature of 140°C, a condensation temperature of 50°C, a vacuum of 0.01 Pa, and a scraping speed of 600 rpm. Subsequently, thin-film evaporation was carried out at a temperature of 120°C and a film thickness of 0.5 mm.

[0077] The method for preparing the immobilized lipase A includes the following steps:

[0078] Step 1: The APTES amination pretreatment process is the same as in Example 1.

[0079] Step 2: Mix 200 mL of 0.3 M copper sulfate solution with 150 mL of 2 M sodium hydroxide solution, adjust the pH to 12 with NaOH, and react at 90 °C for 2 hours. After the reaction is complete, centrifuge and wash the mixture containing CuO nanoparticles three times with deionized water to obtain CuO nanoparticles with a particle size of 8 nm and a specific surface area of ​​120 m² / g. The prepared CuO nanoparticles are chemically bonded to an aminated porous silica support through Cu-N coordination bonds to obtain a copper oxide nanoenzyme complex, i.e., immobilized lipase A.

[0080] The preparation method of the immobilized lipase B is the same as that in Example 1.

[0081] Example 4

[0082] An enzymatic production process for diglycerides differs from Example 1 only in step (1) taking 100g of a 1:1 mixture of soybean oil and olive oil, adding glycerol at a molar ratio of 2.5:1, adding 5g of an adsorption resin containing tertiary amine groups (average pore size 100nm, tertiary amine group density 2.0mmol / g) (accounting for 5% of the mass of the mixed oil), and immobilizing 2g of lipase A; reacting at 55℃ and a stirring speed of 300rpm for 6h, with the pH maintained at 7.0, to obtain the primary reaction product.

[0083] Comparative Example 1

[0084] An enzymatic production process for diglycerides differs from Example 1 only in that step (1) involves taking 100g of soybean oil and adding glycerol at a molar ratio of 2.0:1 to soybean oil, without adding an adsorption resin containing tertiary amine groups; reacting at 55°C and a stirring speed of 300rpm for 6 hours, with the pH maintained at 7.0, to obtain the primary reaction product.

[0085] Comparative Example 2

[0086] An enzymatic production process for diglycerides differs from Example 1 only in step (2) by mixing the primary reaction product with 4g of immobilized lipase B pretreated by high-speed homogenization, adding 4g of 4A molecular sieve (accounting for 4% of the total mass of the reaction system), controlling the water activity to 0.4, reacting at atmospheric pressure for 3h at a temperature of 42℃, and collecting the light phase product to obtain crude diglycerides.

[0087] Comparative Example 3

[0088] An enzymatic production process for diglycerides differs from Example 1 only in step (2) by mixing the primary reaction product with 4g of immobilized lipase B pretreated by high-speed homogenization, without adding molecular sieves to control water activity; a two-step vacuum procedure is adopted: the first step is to react at 0.1kPa for 1.5h at 42℃; the second step is to react at 0.03kPa for 1.5h to promote the separation of the light phase, and the light phase product is collected to obtain crude diglycerides.

[0089] Comparative Example 4

[0090] An enzymatic production process for diglycerides differs from Example 1 only in step (3): the crude diglycerides are first decolorized with 1% activated carbon at 70°C for 45 min, and then dehydrated with 3A molecular sieve (2% dosage). Short-path molecular distillation is used, with an evaporation temperature of 120°C, a condensation temperature of 40°C, a vacuum degree of 0.005 Pa, and a scraping speed of 450 rpm. The thin-film evaporation step is not performed.

[0091] Comparative Example 5

[0092] An enzymatic production process for diglycerides differs from Example 1 only in step (1): 100g of soybean oil is taken, and glycerol is added at a molar ratio of 2.0:1 to soybean oil. 5g of adsorption resin containing tertiary amine groups (average pore size 100nm, tertiary amine group density 2.0mmol / g) (accounting for 5% of the soybean oil mass) is added, and 2g of immobilized lipase A is added. The reaction is carried out at 55℃ and a stirring speed of 300rpm for 6h, with the pH maintained at 7.0, to obtain the primary reaction product.

[0093] The immobilized lipase A is a commercially available lipase immobilized on a conventional carrier.

[0094] Performance testing methods

[0095] (1) Determination of diglyceride purity: Gas chromatography was used with a DB-5 capillary column, injection port temperature of 350℃, detector temperature of 380℃, and column temperature programmed.

[0096] (2) Conversion rate determination: The conversion rate of vegetable oil raw materials is calculated by the changes in saponification value and iodine value. Conversion rate = [(initial saponification value - final saponification value) / initial saponification value] × 100%.

[0097] (3) Product yield: The percentage of the actual quality of diglyceride product obtained to the theoretical diglyceride yield based on the complete conversion of vegetable oil.

[0098] Yield (%) = (Actual mass of diglycerides obtained / Theoretical yield of diglycerides) × 100%

[0099] The theoretical diglyceride yield is calculated based on the molar number of vegetable oils, the stoichiometric ratio of the reaction, and the molecular weight of the diglyceride.

[0100] (4) Enzyme activity retention rate: the percentage of enzyme activity after the reaction compared to the initial enzyme activity. The results are shown in Table 1.

[0101] Table 1 Performance Tests

[0102] project Diglyceride purity (%) Conversion rate (%) Product yield (%) Enzyme activity retention rate (%) Example 1 94.8 91.5 86.2 93.7 Example 2 93.7 87.7 82.7 91.3 Example 3 93.5 89.2 84.1 92.0 Example 4 93.1 88.9 83.8 91.8 Comparative Example 1 78.5 65.3 62.8 85.2 Comparative Example 2 82.1 71.6 68.9 77.6 Comparative Example 3 80.3 69.8 66.4 76.3 Comparative Example 4 81.9 82.4 68.1 75.7 Comparative Example 5 81.7 75.2 65.8 78.4

[0103] The test results show that the diglyceride products prepared in the embodiments of the present invention have a purity of ≥93%, a conversion rate of ≥87%, a product yield of ≥82%, and an enzyme activity retention rate of ≥90%, all of which are superior to the comparative example.

[0104] Comparative Example 1, lacking a polar adsorbent, resulted in excessively high glycerol concentration, inhibiting enzyme activity. Comparative Examples 2 and 3, respectively, lacked vacuum conditions and water activity control, affecting reaction equilibrium and product separation. Comparative Example 4 showed incomplete purification, leading to low product purity. Comparative Example 5 used a traditional enzyme preparation, whose catalytic efficiency and stability were inferior to the copper oxide nanoenzyme complex. The results indicate that the synergistic effect of the various technical features of this invention significantly improves the production efficiency and product quality of diglycerides.

[0105] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An enzymatic production process for diglycerides, characterized in that, The method comprises the following steps: (1) performing first enzymolysis on plant oil, glycerol and immobilized lipase A in a reaction system containing polar adsorbent material, the polar adsorbent material selectively adsorbing excess glycerol, controlling the effective concentration of glycerol in the reaction system, and obtaining a primary reaction product; (2) performing second enzymolysis on the primary reaction product and high-speed homogenization pretreated immobilized lipase B under vacuum mediation, controlling water activity by using molecular sieves, and collecting light phase product to obtain crude diglyceride; (3) purifying the crude diglyceride by short path molecular distillation combined with thin film evaporation to obtain high-purity diglyceride product; In the method, the immobilized lipase A is a copper oxide nanocatalyst complex, the copper oxide nanocatalyst complex taking CuO nanoparticles prepared by chemical precipitation as an active component, the CuO nanoparticles having a particle size of 3-8 nm and a specific surface area of 120-200 m² / g, the CuO nanoparticles being fixed on a carrier by chemical bonding to have lipase-like activity and ester exchange catalytic ability, the carrier being porous silicon oxide which is preliminarily subjected to amino treatment with 3-aminopropyl triethoxysilane, the CuO nanoparticles being chemically bonded to the carrier through Cu-N coordination to form the copper oxide nanocatalyst complex, the carrier having a pore size of 8-20 nm and a pore volume of 0.8-1.5 cm³ / g, the immobilized lipase B being a Mucorales lipase immobilized on an organic mesoporous silica material, and the polar adsorbent material being an adsorption resin containing tertiary amine groups, the adsorption resin containing tertiary amine groups having a pore size of 50-200 nm.

2. The process for the enzymatic production of diglycerides according to claim 1, characterized in that: The plant oil is at least one selected from soybean oil, rapeseed oil, peanut oil, olive oil, linseed oil, palm oil and corn oil.

3. The process for the enzymatic production of diglycerides according to claim 1, characterized in that: In step (1), the polar adsorbent material has a mass dosage of 3-8% of the mass of the plant oil, the adsorption resin containing tertiary amine groups has a density of tertiary amine groups of 1.2-3.5 mmol / g, the molar ratio of glycerol to plant oil is 1.5-3.0:1, and / or In step (1), the first enzymolysis is controlled at a temperature of 45-65°C, a pH of 6.5-7.5, and a stirring speed of 200-400 rpm for 4-8 h.

4. The process for the enzymatic production of diglycerides according to claim 1, characterized in that: The preparation of the copper oxide nanocatalyst complex comprises the following steps: (a) mixing 0.1-0.5 M copper sulfate aqueous solution with sodium hydroxide aqueous solution to control the pH to be 11-12; (b) reacting at 80-100°C for 1-3 hours to form a mixed solution containing CuO nanoparticles; (c) centrifugally separating the mixed solution after reaction in step (b), and washing the obtained solid with deionized water to remove byproducts, to obtain CuO nanoparticles; (d) chemically bonding the CuO nanoparticles to the carrier to obtain the copper oxide nanocatalyst complex.

5. The process for the enzymatic production of diglycerides according to claim 1, characterized in that: In step (2), the high-speed homogenization pretreatment comprises: treating the suspension of immobilized lipase B with a high-speed homogenizer at a rotating speed of 8000-15000 rpm for 2-5 min; and / or, In step (2), the second enzymatic hydrolysis is carried out under the conditions of a vacuum degree of 0.01-0.1 kPa and a temperature of 35-50°C for 2-4 h.

6. The process for the enzymatic production of diglycerides according to claim 1, characterized in that: In step (2), the molecular sieve is 4A molecular sieve or 5A molecular sieve, and is used to control the water activity in the range of 0.2-0.

6. In the immobilized lipase B, the organic mesoporous silica material is MCM-41 mesoporous silica material or SBA-15 mesoporous silica material, and the specific surface area of the organic mesoporous silica material is 600-1200 m² / g.

7. The process for the enzymatic production of diglycerides according to claim 1, characterized in that: In steps (1) and (2), the mass amount of the immobilized lipase A and the immobilized lipase B is 1-4% and 2-6% of the mass of the vegetable oil, respectively.

8. The process for the enzymatic production of diglycerides according to claim 1, characterized in that: In step (2), the vacuum-mediated conditions are a two-step vacuum procedure: the first step is to promote ester exchange under a vacuum degree of 0.05-0.1 kPa for 1-2 h, and the second step is to promote light phase separation under a vacuum degree of 0.01-0.05 kPa for 1-2 h.

9. The process for the enzymatic production of diglycerides according to claim 1, characterized in that: In step (3), before short-path molecular distillation is combined with thin-film evaporation, the crude diglyceride is first subjected to decolorization treatment with activated carbon, the mass amount of the activated carbon is 0.5-2% of the mass of the crude diglyceride, the treatment temperature is 60-80°C, and the treatment time is 30-60 min; and then the crude diglyceride is subjected to dehydration treatment with 3A molecular sieve, the mass amount of the 3A molecular sieve is 1-3% of the mass of the crude diglyceride.

10. The process for the enzymatic production of diglycerides according to claim 1, characterized in that: In step (3), the evaporation temperature of the short-path molecular distillation is 100-140°C, the condensation temperature is 30-50°C, the system vacuum degree is 0.001-0.01 Pa, and the wiper film rotation speed is 300-600 rpm; the thin-film evaporation temperature is 80-120°C, and the film thickness is 0.1-0.5 mm.