Enzymatic production process for diglyceride

Abstract

The invention discloses an enzymatic production process for diglyceride, and relates to the technical field of biological enzyme catalysis. According to the process, a double-enzyme combined catalysis system is adopted, wherein immobilized lipase A is a copper oxide nano-enzyme complex, and a carrier is porous silicon dioxide subjected to APTES amination treatment; the immobilized lipase B is mucor lipase immobilized by a meso-porous silicon material. Adsorption resin containing tertiary amine groups is added into a reaction system to serve as a polar adsorption material, excessive glycerin is selectively adsorbed, and the effective concentration of glycerin is regulated and controlled. The copper oxide nano-enzyme complex is prepared by chemical precipitation of copper sulfate and sodium hydroxide, and has lipase-like activity and excellent ester exchange catalytic ability. According to the catalytic system, the vegetable oil conversion rate reaches 87.7% or above, the diglyceride product purity reaches 92.7% or above, the product yield exceeds 82.7%, the enzyme activity retention rate exceeds 91.3%, and a new technical path is provided for efficient preparation of functional grease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological enzyme catalysis, and specifically relates to an enzymatic production process for diglyceride. BACKGROUND

[0002] At present, diglyceride in the industry is usually obtained by fatty acid and glycerol ester exchange or vegetable oil and glycerol esterification reaction, through the catalysis of immobilized lipase and the control of water activity (such as the addition of molecular sieve) to improve the yield and selectivity. However, the following problems still exist: When glycerol is excessive, monoglyceride (MAG) or triglyceride (TAG) is easily generated, affecting the purity of the product; the catalytic efficiency of a single enzyme is limited, and it is difficult to balance the conversion rate and selectivity; the purification method is mostly dependent on single molecular distillation or thin film evaporation, and the removal of TAG or pigment is insufficient, and the purity of the product is not ideal.

[0003] In addition, the traditional immobilized lipase mostly uses natural enzymes, and has the problems of high preparation cost, poor stability and easy deactivation. In recent years, nanoenzyme technology has opened up a new way for the catalysis field, and metal oxide nanoenzymes have the advantages of low cost, high stability and easy preparation. However, the application of copper oxide nanoenzyme in the production of diglyceride has not been reported, which is mainly limited by multiple technical obstacles. First, the naked CuO nanoenzyme lacks the active site structure of lipase, and has low catalytic efficiency and poor selectivity for ester exchange reaction, which makes it difficult to realize the high-selectivity synthesis of diglyceride. Secondly, in the weakly acidic reaction environment of diglyceride production, Cu 2+ is easy to dissolve out from the surface of CuO nanoparticles, leading to rapid deactivation of the catalyst and failing to meet the requirements of industrial continuous production. At the same time, the existing technology lacks effective means to regulate the concentration of glycerol, and when glycerol is excessive, it is easy to generate MAG or TAG, while when glycerol is insufficient, the conversion rate is low, and it is difficult to balance the contradiction between yield and selectivity. In addition, the contact interface between CuO nanoenzyme and oil substrate is limited, the mass transfer resistance is large, and there is a lack of effective dispersion and homogenization means, resulting in low catalytic efficiency. The ester exchange reaction is extremely sensitive to water activity, and the existing single molecular sieve control method is difficult to accurately maintain the optimal water activity range under vacuum conditions, which seriously affects the reaction equilibrium and product distribution. Finally, the traditional single purification method (such as molecular distillation or thin film evaporation) cannot effectively remove TAG and other impurities with similar structures, and cannot meet the requirements of high-purity diglyceride products. More importantly, the existing researches mostly focus on the improvement of single technical link, and lack of systematic technical integration scheme from catalyst design, reaction condition control to product separation and purification, which makes it difficult to realize the breakthrough of the overall process performance.

[0004] Therefore, there is an urgent need for a new technical scheme to improve the yield and purity of DAG (diglyceride) by accurately regulating the glycerol concentration, using new nanoenzyme catalysis and advanced purification method. SUMMARY

[0005] The application aims to provide an enzymatic production process for diglyceride, which realizes high-selectivity and high-purity DAG preparation through an innovative catalytic system of copper oxide nanocatalyst complex, dynamic regulation of glycerol concentration by adsorption material, double-enzyme series catalytic synergy, accurate control by vacuum and molecular sieve, and combined purification process.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows: The enzymatic production process for diglyceride provided by the application comprises the following steps: (1) adding plant oil, glycerol and immobilized lipase A into a reaction system containing polar adsorption material to perform first enzymolysis, the polar adsorption material selectively adsorbs excess glycerol to control the effective concentration of glycerol in the reaction system, and a primary reaction product is obtained.

[0007] (2) performing second enzymolysis of the primary reaction product and high-speed homogenization pretreated immobilized lipase B under vacuum mediation, controlling water activity by using a molecular sieve, and collecting light phase product to obtain a crude diglyceride product.

[0008] (3) purifying the crude diglyceride product by using short-path molecular distillation combined with thin film evaporation to obtain a high-purity diglyceride product. The immobilized lipase A is a copper oxide nanocatalyst complex, the copper oxide nanocatalyst complex takes CuO nanoparticles prepared by chemical precipitation as an active component, the CuO nanoparticles have a particle size of 3-8 nm and a specific surface area of 120-200 m² / g, the CuO nanoparticles have lipase-like activity and ester exchange catalytic ability after being immobilized by a carrier, 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 Mucorales lipase immobilized by an organic mesoporous silica material, and the polar adsorption material is an adsorption resin containing a tertiary amine group, the adsorption resin containing the tertiary amine group has a pore size of 50-200 nm.

[0009] As a further description of the application, the adsorption resin containing the tertiary amine group has a suitable pore size structure (50-200 nm) to provide sufficient space for molecular mass transfer. The tertiary amine group acts as a polar active site to preferentially adsorb polar glycerol molecules through hydrogen bond interaction and electrostatic interaction, while hydrophobic DAG, MAG and FFA molecules have low affinity with the tertiary amine group and are preferentially retained in the oil phase, so that selective separation of glycerol is realized.

[0010] As a further illustration of the present application, the design of the carrier with a pore size of 8-20 nm is based on the following considerations: the pore size of the carrier should be larger than the maximum size of CuO nanoparticles (8 nm) to ensure that the particles can effectively enter the carrier pores for immobilization; at the same time, the pore size should not be too large to maintain the mechanical strength of the carrier and the stable anchoring of CuO particles. The pore volume of the carrier is 0.8-1.5 cm³ / g, which ensures a suitable loading amount of CuO nanoparticles: if the pore volume is too small (<0.8 cm³ / g), the loading amount is insufficient, affecting the catalytic efficiency; if the pore volume is too large (>1.5 cm³ / g), the mechanical strength of the carrier decreases, and the particles are not uniformly dispersed. The preferred pore volume range allows the loading amount of CuO nanoparticles to reach 5-15% of the mass of the carrier, achieving the best catalytic performance.

[0011] 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.

[0012] Preferably, the amount of the polar adsorbent material used in step (1) is 3-8% of the mass of the vegetable oil, the density of the tertiary amine groups of 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°C, the pH is controlled at 6.5-7.5, and the stirring speed is controlled at 200-400 rpm for 4-8 h of stirring reaction.

[0013] Preferably, the preparation of the copper oxide nanoscale enzyme complex comprises the following steps: (a) mixing 0.1-0.5 M aqueous copper sulfate solution with aqueous sodium hydroxide solution to control the pH to 11-12; (b) stirring at 80-100°C for 1-3 hours at a stirring speed of 100-300 rpm to form a mixed solution containing CuO nanoparticles; (c) centrifuging the mixed solution after reaction in step (b) to obtain a solid, and washing the solid with deionized water to remove byproducts, thereby preparing CuO nanoparticles; (d) fixing the CuO nanoparticles on the carrier by physical adsorption or chemical bonding to prepare the copper oxide nanoscale enzyme complex; wherein the CuO nanoparticles exhibit oxidoreductase and hydrolytic enzyme-like activities under reaction conditions.

[0014] The copper oxide nanoscale enzyme complex forms a Cu-N coordination bond with the carrier, anchoring the CuO nanoparticles inside the carrier channel (8-20 nm). The carrier is porous silicon oxide that has been previously treated with 3-aminopropyl triethoxysilane (APTES) to form amino groups. The CuO nanoparticles are chemically bonded to the carrier through the Cu-N coordination bond to form the copper oxide nanoscale enzyme complex. This structure makes the CuO surface charge locally alkaline due to the carrier microenvironment, and the steric hindrance of the carrier channel prevents H⁺ from contacting the active center, thereby inhibiting the Cu 2+ dissolution; The complex has lipase-like activity and excellent transesterification catalytic ability.

[0015] Preferably, the high-speed homogenization pretreatment in step (2) includes: using a high-speed homogenizer with a rotation speed of 8000-15000 rpm to treat the immobilized lipase B suspension for 2-5 min; in step (2), the second enzymolysis 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.

[0016] Preferably, the molecular sieve is a 4A molecular sieve or a 5A molecular sieve, which is used to control the water activity of the reaction system in the range of 0.2-0.6; 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.

[0017] Further 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 cetyltrimethylammonium bromide (CTAB); The SBA-15 mesoporous silica material is prepared using a triblock copolymer as a template agent, preferably P123 triblock copolymer (EO20PO70EO20); As a further description of the present application, the molecular sieve (4A / 5A) is filled in the form of a fixed bed at the top of the reactor, which is physically isolated from the reaction liquid (such as separated by a sintered metal mesh). During vacuum dehydration, gas-phase water molecules are selectively adsorbed after passing through the molecular sieve layer, while liquid-phase water molecules continue to evaporate due to the Henry's law balance, forming a gas-solid adsorption-liquid-gas evaporation dynamic balance, which stabilizes the water activity in the range of 0.2-0.6.

[0018] As a further illustration of the present application, the carrier of the immobilized lipase B (MCM-41 / SBA-15) is formed with an elastic protective layer (thickness ≈ 5 nm) by surface polyvinyl alcohol (PVA) coating, and the shear modulus (≈ 1 GPa) is much higher than the homogeneous shear stress (≈ 0.1 MPa under 8000-15000 rpm), and the flexible segment of PVA can disperse the impact force to avoid the carrier from being broken.

[0019] Preferably, the mass dosage of the immobilized lipase A and the immobilized lipase B in steps (1) and (2) is 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 process.

[0020] Preferably, the vacuum-mediated condition in step (2) is a two-step vacuum procedure: the first step is to promote transesterification 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 and a temperature of 35-50℃ for 1-2 h.

[0021] Preferably, 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 dosage of the activated carbon is 0.5-2% of the mass of the crude product, the treatment temperature is 60-80℃, and the treatment time is 30-60 min; and then the crude diglyceride is subjected to dehydration treatment with 3A molecular sieves, the mass dosage of the 3A molecular sieves is 1-3% of the mass of the crude product.

[0022] As a further illustration of the present application, after the activated carbon is decolorized, the reaction solution is filtered through a filter membrane with a pore size of 0.22 μm to trap activated carbon particles, so as to ensure the cleanliness of the filtrate in the subsequent molecular sieve dehydration treatment.

[0023] Preferably, in step (3), the evaporation temperature of the short-path molecular distillation is 100-140℃, the condensation temperature is 30-50℃, the system vacuum degree is 0.001-0.01 Pa, and the scraping membrane rotation speed is 300-600 rpm; and the thin film evaporation temperature is 80-120℃, and the membrane thickness is 0.1-0.5 mm.

[0024] The present application has the following beneficial effects: 1. The adsorption-double enzyme system synergistically improves the yield and purity. The adsorption resin containing tertiary amine groups is used to regulate the glycerol concentration, effectively inhibiting the generation of MAG and TAG; and the double enzyme series design of the copper oxide nanometer enzyme and the traditional lipase synergistically improves the substrate conversion efficiency and increases the selectivity and final yield of diglyceride (DAG).

[0025] 2. High-speed homogenization and vacuum-molecular sieve strategy to enhance catalytic efficiency. High-speed homogenization pretreatment significantly increases the contact interface between enzyme and substrate, while vacuum control combined with molecular sieve precisely regulates water activity, which can precisely control the reaction path, effectively promote transesterification and light phase separation, and improve overall conversion rate and enzyme stability.

[0026] 3. Multistage composite purification process to ensure high-quality products. Using activated carbon decolorization, molecular sieve dehydration, short-path molecular distillation, and multi-stage membrane evaporation, TAG, pigments, and other impurities are effectively removed to achieve high-quality diglyceride products with a purity of more than 93%, meeting the standards for industrial production and food and pharmaceutical applications.

[0027] 4. Carrier immobilized copper oxide nanocatalyst complex to achieve catalytic performance breakthrough. Through APTES aminization treatment of porous silica carrier immobilization, CuO nanoparticles are stably anchored through Cu-N coordination bonding, and the carrier's confinement effect and microenvironment regulation change the surface electronic structure of CuO, obtaining a lipase-like activity. This complex system exhibits excellent transesterification catalytic ability in diglyceride synthesis, with higher thermal stability, pH stability, and organic solvent resistance than traditional natural enzymes, with a specific activity increase of 25-50%, providing an efficient and stable nanocatalyst solution for functional oil preparation. DETAILED DESCRIPTION

[0028] The application will be further described in conjunction with specific embodiments, but the application is not limited to these embodiments. Those skilled in the art should recognize that the application encompasses all possible alternatives, improvements, and equivalents within the scope of the claims.

[0029] Raw materials and equipment used in the application: Soybean oil, rapeseed oil, palm oil, and olive oil: purchased from COFCO Group, iodine value 120-140; Glycerol: analytical pure, purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; Mucorales lipase: purchased from Novozymes, 43111M, 250 IUN / g; Adsorption resin containing tertiary amine groups: purchased from Rohm & Haas, model IRA-400; 3A, 4A, and 5A molecular sieves: purchased from Shanghai Rujiyi Biological Technology Development Co., Ltd.; Carrier immobilized commercial lipase: purchased from Novozymes, Lipozyme RM IM; The remaining reagents are all analytical pure and obtained through commercial channels.

[0030] Example 1

[0031] An enzymatic production process for diglycerides, comprising the following steps: (1) Take 100 g of soybean oil, add glycerol at a molar ratio of glycerol to soybean oil of 2.0:1, add 5 g of adsorption resin containing tertiary amine groups (average pore size 100 nm, tertiary amine group density 2.0 mmol / g) (5% of the mass of soybean oil), and 2 g of immobilized lipase A; react at 55°C, stirring speed 300 rpm for 6 h, maintain pH at 7.0, and obtain a primary reaction product.

[0032] (2) Mix the primary reaction product with high-speed homogenization pretreated immobilized lipase B 4 g, fill 4A molecular sieves in the form of a fixed bed at the top of the reactor, and physically isolate them from the reaction liquid by a sintered metal mesh; use a two-step vacuum procedure: first step, 1.5 h at a vacuum degree of 0.1 kPa, temperature 42°C; second step, 1.5 h at a vacuum degree of 0.03 kPa, temperature 42°C, to promote light phase separation, collect the light phase product to obtain a crude diglyceride.

[0033] (3) The crude diglyceride is first decolorized with activated carbon (its mass usage is 1% of the mass of the crude diglyceride) at 70°C for 45 minutes, filtered through a 0.22 μm filter membrane after decolorization to intercept activated carbon particles, and then dehydrated with 3A molecular sieves (its mass usage is 2% of the mass of the crude diglyceride) at 80°C for 2 hours; 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 wiper film rotation speed of 450 rpm; then thin film evaporation is performed at a temperature of 100°C and a membrane thickness of 0.3 mm.

[0034] The preparation method of the immobilized lipase A comprises the following steps: Step one, APTES amination pretreatment step; take 10 g of porous silica (average pore size 14 nm, pore volume 1.2 cm³ / g), first treat with 0.1M HCl solution for 2 hours to activate the surface hydroxyl groups, wash with deionized water until neutral, and dry at 80°C for 4 hours; then disperse the activated porous silica (carrier) in 200 mL of anhydrous toluene, add 5 mL of 3-aminopropyl triethoxysilane (APTES), and reflux at 110°C for 12 hours under nitrogen protection; after the reaction is completed, filter, wash with toluene, ethanol, and deionized water in sequence, and vacuum dry at 120°C for 6 hours to obtain an aminated porous silica carrier.

[0035] Step two, 200 mL of 0.2 M copper sulfate aqueous solution was mixed with 100 mL of 2 M sodium hydroxide aqueous solution, and the pH was adjusted to 11.5 with NaOH. The mixture was stirred at 90°C for 2 hours at a stirring speed of 200 rpm. After the reaction was completed, the mixture was centrifuged, and the obtained CuO nanoparticle-containing mixture was washed with deionized water for 3 times 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 were chemically bonded to the amino-treated porous silica carrier through Cu-N coordination bonds to obtain a copper oxide nanoscale enzyme complex, i.e., immobilized lipase A.

[0036] The preparation method of the MCM-41 mesoporous silica material is as follows: cetyltrimethylammonium bromide (CTAB) 2.0 g was dissolved in deionized water 480 mL, ammonia water 14 mL was added, and tetraethyl orthosilicate (TEOS) 10 mL was added dropwise after stirring for 30 min. The mixture was reacted at 80°C for 2 hours, filtered, washed, dried, and calcined at 550°C for 6 hours to obtain the MCM-41 mesoporous silica material with a specific surface area of 850 m² / g.

[0037] The preparation method of the immobilized lipase B includes the following steps: The lipase B suspension was prepared by dissolving the Mucorales lipase in 0.1 M phosphate buffer (pH 7.0) at a concentration of 10 mg / mL, and then treated with a high-speed homogenizer at a speed of 12000 rpm for 3 min. Then, the MCM-41 mesoporous silica material (specific surface area 850 m² / g) was mixed with the enzyme protein and the carrier at a mass ratio of 1:10, and the immobilization was carried out by physical adsorption. A polyvinyl alcohol (PVA) with a molecular weight of 30,000-70,000 g / mol was prepared into a 0.5 wt% aqueous solution, and the carrier was immersed for 2 hours to form an elastic protective layer with a thickness of about 5 nm. After drying, the immobilized lipase B was obtained.

[0038] Example 2

[0039] An enzymatic production process for diglycerides includes the following steps: (1) Take 100 g of rapeseed oil, add glycerol at a molar ratio of 1.5:1, add 3 g of adsorption resin containing tertiary amine groups (average pore size 50 nm, tertiary amine group density 1.2 mmol / g) (3% of the mass of rapeseed oil), and 1 g of immobilized lipase A. The reaction was carried out at 45°C with a stirring speed of 200 rpm for 8 hours, and the pH was maintained at 7.0 to obtain a primary reaction product.

[0040] (2) The primary reaction product was mixed with 2 g of high-speed homogenized pretreated immobilized lipase B. 2 g of 5A molecular sieve was filled in the top of the reactor in the form of a fixed bed, which was physically isolated from the reaction solution 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 for 4 h at 0.01 kPa and a temperature of 35°C; the second step was to react for 1.5 h at 0.03 kPa and a temperature of 35°C, to promote the separation of the light phase, and the light phase product was collected to obtain the crude diglyceride.

[0041] (3) The crude diglyceride was first decolorized with activated carbon (the mass amount of which was 0.5% of the mass of the crude diglyceride) at 60°C for 30 min, and then filtered through a 0.22 μm filter membrane to intercept activated carbon particles. Then, 3A molecular sieve (the mass amount of which was 1% of the mass of the crude diglyceride) was used to dehydrate the product at 80°C for 2 h. Short-path molecular distillation was adopted: the evaporation temperature was 100°C, the condensation temperature was 30°C, the vacuum degree was 0.001 Pa, and the scraper membrane rotation speed was 300 rpm. Subsequently, thin film evaporation was performed at 80°C, and the membrane thickness was 0.1 mm.

[0042] The preparation method of the immobilized lipase A comprises the following steps: Step one: the APTES amination pretreatment step is the same as in Example 1.

[0043] Step two: 150 mL of a 0.1M copper sulfate solution was mixed with 75 mL of a 2M sodium hydroxide solution, and the pH was adjusted to 12 with NaOH. The mixture was stirred at 100°C for 1 hour at a stirring speed of 200 rpm. After the reaction was completed, centrifugal separation was performed, and the mixture containing CuO nanoparticles was washed with deionized water for 3 times 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 were chemically bonded to the aminated porous silica carrier through Cu-N coordination bonds to obtain a copper oxide nanosemiconductor complex, i.e., the immobilized lipase A.

[0044] The preparation method of the SBA-15 mesoporous silica material is as follows: 4.0 g of a triblock copolymer P123 (EO20PO70EO20) was dissolved in 120 mL of a 2M HCl solution, and stirring was performed at 35°C until complete dissolution. Then, 8.5 mL of tetraethyl orthosilicate (TEOS) was added dropwise, and stirring was performed at 35°C for 20 hours. Then, the mixture was transferred to a hydrothermal reactor for reaction at 100°C for 24 hours. Filtration, washing, and drying were performed, and the template agent was removed by calcination at 550°C for 6 hours to obtain the SBA-15 mesoporous silica material, which has a specific surface area of 850 m² / g and an average pore size of about 6 nm.

[0045] The preparation method of the immobilized lipase B comprises the following steps: Mucorales lipase was dissolved in 0.1 M phosphate buffer (pH 7.0) at a concentration of 10 mg / mL to prepare a lipase B suspension, which was treated with a high-speed homogenizer at 12,000 rpm for 3 min, then mixed with SBA-15 mesoporous silica material (specific surface area 850 m² / g) at a mass ratio of enzyme protein to carrier of 1:10, and immobilized by physical adsorption. A polyvinyl alcohol (PVA) with a molecular weight of 30,000-70,000 g / mol was prepared into a 0.5 wt% aqueous solution, the carrier was immersed for 2 hours to form an elastic protective layer with a thickness of about 5 nm, and the immobilized lipase B was obtained after drying.

[0046] Example 3

[0047] An enzymatic production process for diglycerides, comprising the following steps: (1) 100 g of palm oil was used, 8 g of adsorption resin containing tertiary amine groups (average pore size 200 nm, tertiary amine group density 3.5 mmol / g) (8% of the mass of palm oil) and 4 g of immobilized lipase A were added according to a molar ratio of glycerol to palm oil of 3.0:1, and the reaction was carried out at 65°C under stirring at a speed of 400 rpm for 4 h, with the pH maintained at 7.0, to obtain a primary reaction product.

[0048] (2) The primary reaction product was mixed with 6 g of high-speed homogenization pretreated immobilized lipase B, 6 g of 4A molecular sieves were filled in the form of a fixed bed at the top of the reactor, and were physically isolated from the reaction liquid by a sintered metal mesh, and the water activity was controlled to 0.6; a two-step vacuum procedure was used: the first step was at 0.05 kPa for 2 h at a temperature of 42°C, and the second step was at 0.03 kPa for 1.5 h at a temperature of 42°C, to promote the separation of the light phase, and the light phase product was collected to obtain a crude diglyceride.

[0049] (3) The crude diglyceride was first decolorized with activated carbon (its mass usage was 2% of the mass of the crude diglyceride) at 80°C for 60 minutes, then filtered through a 0.22 μm filter membrane to trap activated carbon particles, and then dehydrated with 3A molecular sieves (its mass usage was 2% of the mass of the crude diglyceride) 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 degree of 0.01 Pa, and a wiper film rotation speed of 600 rpm; then thin film evaporation was performed at a temperature of 120°C and a film thickness of 0.5 mm.

[0050] The preparation method of the immobilized lipase A comprises the following steps: Step one, the APTES amination pretreatment step is the same as in Example 1.

[0051] Step two, 200 mL of 0.3 M copper sulfate solution was mixed with 150 mL of 2 M sodium hydroxide solution, and the pH was adjusted to 12 with NaOH, and the reaction was carried out at 90°C for 2 hours; after the reaction was completed, centrifugal separation was carried out, and the mixed solution containing CuO nanoparticles was washed with deionized water 3 times to obtain CuO nanoparticles with a particle size of 8 nm and a specific surface area of 120 m² / g; the prepared CuO nanoparticles were chemically bonded and fixed on the amino-treated porous silica carrier through Cu-N coordination bonds to obtain a copper oxide nanoscale enzyme complex, i.e., immobilized lipase A.

[0052] The preparation method of the immobilized lipase B is consistent with that of Example 1.

[0053] Example 4

[0054] An enzymatic production process for diglycerides, which is only different from Example 1 in that step (1) 100 g of soybean oil and olive oil mixed at a ratio of 1:1 is taken, and glycerol is added at a molar ratio of glycerol to mixed oil of 2.5:1, 5 g (5% of the mass of the mixed oil) of adsorption resin containing a tertiary amine group (average pore size 100 nm, tertiary amine group density 2.0 mmol / g) and 2 g of immobilized lipase A are added; the reaction is carried out at 55°C and a stirring speed of 300 rpm for 6 h, and the pH is maintained at 7.0 to obtain a primary reaction product.

[0055] Comparative Example 1 An enzymatic production process for diglycerides, which is only different from Example 1 in that step (1) 100 g of soybean oil is taken, and glycerol is added at a molar ratio of glycerol to soybean oil of 2.0:1, and no adsorption resin containing a tertiary amine group is added; the reaction is carried out at 55°C and a stirring speed of 300 rpm for 6 h, and the pH is maintained at 7.0 to obtain a primary reaction product.

[0056] Comparative Example 2 An enzymatic production process for diglycerides, which is only different from Example 1 in that step (2) the primary reaction product is mixed with 4 g of high-speed homogenization pretreated immobilized lipase B, and 4 g (4% of the total mass of the reaction system) of 4A molecular sieves are added to control the water activity to 0.4; the reaction is carried out at normal pressure for 3 h at a temperature of 42°C, and the light phase product is collected to obtain a crude diglyceride product.

[0057] Comparative Example 3 An enzymatic production process for diglycerides, which is only different from Example 1 in that step (2) the primary reaction product is mixed with 4 g of high-speed homogenization pretreated immobilized lipase B, and no molecular sieves are added to control the water activity; a two-step vacuum procedure is adopted: the first step is carried out at 0.1 kPa for 1.5 h at a temperature of 42°C; the second step is carried out at 0.03 kPa for 1.5 h to promote the separation of the light phase, and the light phase product is collected to obtain a crude diglyceride product.

[0058] Comparative Example 4 An enzymatic production process for diglycerides, which differs from Example 1 only in that step (3) the crude diglyceride is first decolorized with 1% activated carbon at 70°C for 45 min, and then dehydrated with 3A molecular sieves (2% by weight). 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 wiper speed of 450 rpm. No thin-film evaporation step is performed.

[0059] Comparative Example 5 An enzymatic production process for diglycerides, which differs from Example 1 only in that step (1) 100 g of soybean oil is used, and 5 g (5% by weight of the soybean oil) of adsorption resin containing tertiary amine groups (average pore size 100 nm, tertiary amine group density 2.0 mmol / g) is added, along with 2 g of immobilized lipase A, at a molar ratio of glycerol to soybean oil of 2.0:1. The primary reaction product is obtained after 6 h of reaction at 55°C and a stirring speed of 300 rpm, with the pH maintained at 7.0.

[0060] The immobilized lipase A is a commercial lipase immobilized on a conventional carrier.

[0061] Performance test method (1) Diglyceride purity determination: gas chromatography is used, with a DB-5 capillary column, an injection port temperature of 350°C, a detector temperature of 380°C, and a column temperature program.

[0062] (2) Conversion rate determination: the conversion rate of the vegetable oil raw material is calculated from the change in saponification value and iodine value, and the conversion rate = [(initial saponification value - final saponification value) / initial saponification value] x 100%.

[0063] (3) Product yield: the percentage of the actual mass of diglyceride product obtained relative to the theoretical diglyceride yield based on complete conversion of the vegetable oil.

[0064] Yield (%) = (actual mass of diglyceride obtained / theoretical diglyceride yield) x 100% The theoretical diglyceride yield is calculated based on the number of moles of vegetable oil, the stoichiometric ratio of the reaction, and the molecular weight of diglyceride.

[0065] (4) Enzyme activity retention rate: the percentage of the enzyme activity after the reaction relative to the initial enzyme activity. The results are shown in Table 1.

[0066] Table 1 Performance test Item Glyceride 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 It can be seen from the test results that the purity of the diglyceride product prepared by the embodiment of the application is all ≥93%, the conversion rate is ≥87%, the product yield is ≥82%, and the enzyme activity retention rate is ≥90%, and all indexes are better than those of the comparative examples.

[0067] The polar adsorbent material is not used in Comparative Example 1, resulting in too high glycerol concentration and inhibiting enzyme activity; Comparative Examples 2 and 3 lack vacuum condition and water activity control respectively, affecting reaction equilibrium and product separation; Comparative Example 4 is not completely purified, and the product purity is low; and Comparative Example 5 uses traditional enzyme preparation, and the catalytic efficiency and stability are not as good as the copper oxide nanometer enzyme complex. The results show that the synergistic effect of the technical features of the application significantly improves the production efficiency and product quality of diglyceride.

[0068] The above is only the preferred specific embodiment of the application, but the protection scope of the application is not limited to this, any person skilled in the art can make equivalent replacement or change according to the technical scheme and the inventive concept of the application within the technical range disclosed by the application, which should be covered in the protection scope of the application.

Claims

1. An enzymatic production process for diglycerides, characterized in that, Includes the following steps: (1) Vegetable oil, glycerol and immobilized lipase A are subjected to a first enzymatic hydrolysis in a reaction system containing a polar adsorbent material. 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. (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. (3) The crude diglyceride product was purified by short-path molecular distillation combined with thin-film evaporation to obtain a high-purity diglyceride product; 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.

2. The enzymatic production process for diglycerides as described in claim 1, characterized in that: 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.

3. The enzymatic production process for diglycerides as described in claim 1, characterized in that: In step (1), the mass amount of the polar adsorbent material is 3-8% of the mass of the vegetable oil; the density of the 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 / or, In step (1), during the first enzymatic hydrolysis, the temperature is controlled at 45-65℃, the pH is 6.5-7.5, and the reaction is carried out at a stirring speed of 200-400 rpm for 4-8 hours.

4. The enzymatic production process for diglycerides as described in claim 1, characterized in that: The preparation of the copper oxide nanozyme complex includes the following steps: (a) Mix 0.1-0.5M copper sulfate aqueous solution with sodium hydroxide aqueous solution and control the pH to 11-12; (b) React at 80-100℃ for 1-3 hours to form a mixed solution containing CuO nanoparticles; (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; (d) The copper oxide nanoenzyme complex is prepared by immobilizing CuO nanoparticles on the carrier through physical adsorption or chemical bonding.

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

6. The enzymatic production process for diglycerides as described in claim 1, characterized in that: In step (2), the molecular sieve is a 4A molecular sieve or a 5A molecular sieve, used to control the water activity within the range of 0.2-0.6; In the immobilized lipase B, the organic mesoporous silica material is either 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 enzymatic production process for diglycerides as described in claim 1, characterized in that: In steps (1) and (2), the amounts of immobilized lipase A and immobilized lipase B are 1-4% and 2-6% of the mass of the vegetable oil, respectively.

8. The enzymatic production process for diglycerides as described in claim 1, characterized in that: The vacuum-mediated conditions described 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 to promote the separation of the light phase.

9. The enzymatic production process for diglycerides as described in claim 1, characterized in that: 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 used is 0.5-2% of the mass of the crude diglyceride, the treatment temperature is 60-80℃, and the treatment time is 30-60 min. Then, it is dehydrated using 3A molecular sieves. The amount of 3A molecular sieves used is 1-3% of the mass of the crude diglyceride.

10. The enzymatic production process for diglycerides as described in claim 1, characterized in that: 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.