A method for preparing diacylglycerol by using an immobilized enzyme of ultrafine pulverized plant protein

Abstract

This invention discloses a method for preparing diglycerides using an immobilized enzyme from ultrafine pulverized plant protein, relating to the field of food engineering technology. The method employs a high-energy media mill to co-mill plant protein and chitosan to prepare a composite material with a porous network structure. This composite material is then mixed with a lipase solution to immobilize the lipase within the composite material, obtaining an immobilized enzyme. This immobilized enzyme is used as an emulsifier, and vegetable oil as a reaction substrate is added. A Pickering emulsion is prepared using a high-speed shear mill, followed by catalytic hydrolysis of the vegetable oil in a constant-temperature water bath to obtain the diglyceride product. This system improves enzyme catalytic efficiency, achieving a diglyceride content of over 45% after 30 minutes of reaction, and maintaining over 40% even after eight repetitions. The immobilized enzyme carrier of this invention uses inexpensive natural food raw materials, is simple to prepare, highly safe, produces minimal pollution, and allows for continuous production. Furthermore, the prepared Pickering emulsion exhibits good stability, which is beneficial for the clean production of diglycerides and has excellent industrialization potential.

Description

Technical Field

[0001] This invention relates to the field of food engineering technology, and more specifically, to a method for preparing diglycerides from ultrafine pulverized plant protein using an immobilized enzyme process. Background Technology

[0002] Diacylglycerol (DAG) is an important functional oil component, hailed as a "healthy fat." Studies have shown that diacylglycerol's metabolic pathway in the human body differs from that of ordinary triglycerides. After consumption, it is less likely to accumulate as fat in the body, exhibiting significant physiological effects such as lowering serum triglycerides, inhibiting visceral fat accumulation, and preventing obesity and hyperlipidemia. Given its numerous health benefits, converting abundant and inexpensive common oils (such as soybean oil and rapeseed oil) into functional oil bases rich in diacylglycerol has become an important research direction in the field of oil processing and functional foods.

[0003] Currently, the main methods for preparing diglycerides include chemical methods and enzymatic methods. Chemical methods are typically carried out under high temperature, high pressure, and strong acid / alkali conditions. While the reaction rate is relatively fast, they suffer from problems such as numerous byproducts, dark-colored products, easy generation of harmful substances, severe equipment corrosion, and difficulties in subsequent separation and purification, making it difficult to meet the production requirements of green and healthy foods. In contrast, enzymatic methods have attracted much attention due to their mild reaction conditions, high substrate specificity, fewer byproducts, and high product safety. In enzymatic methods, compared to glycerol hydrolysis reactions that require the addition of expensive exogenous co-substrates (such as glycerol), the preparation of diglycerides using lipase-catalyzed lipid hydrolysis directly utilizes water in the system as a reactant, offering advantages such as low raw material costs and simple process control, making it more suitable for industrial production. However, in conventional enzymatic catalysis, because lipases are usually water-soluble while lipid substrates are hydrophobic, their poor compatibility leads to high mass transfer resistance, limiting further improvement in reaction efficiency. Furthermore, the difficulty in separating the enzyme from the product after the reaction restricts the recycling of the enzyme.

[0004] Pickering emulsions are emulsion systems that use solid particles instead of traditional molecular surfactants to stabilize the oil-water interface. Using them as a biocatalytic reaction medium provides a large oil-water interfacial area, enriching lipases at the interface and significantly increasing the contact probability between the oil substrate and the lipase, effectively solving the key problem of low mass transfer efficiency in enzymatic reactions. However, existing Pickering emulsion catalytic systems still have certain limitations: on the one hand, commonly used inorganic particle-immobilized enzyme emulsifiers (such as silica and magnetic nanoparticles), although possessing good mechanical strength, suffer from complex preparation processes, high cost, poor biocompatibility, and potential safety hazards in food systems; on the other hand, traditional small-molecule surfactants, while exhibiting good emulsification effects, result in emulsions with poor thermodynamic stability and are difficult to demulsify. In contrast, proteins and polysaccharides, as natural food components, possess good biocompatibility, biodegradability, and non-toxicity. Immobilized enzyme materials prepared by protein-polysaccharide composites can not only effectively immobilize lipases to improve their stability, but also act as particulate emulsifiers to stabilize the oil-water interface, thus constructing highly efficient interfacial biocatalytic microreactors. This approach is expected to address the shortcomings of existing technologies in terms of safety and economy.

[0005] In summary, to address the problems existing in the current enzymatic preparation of diglycerides, such as low enzyme-substrate mass transfer efficiency, insufficient safety and economy of existing carrier materials, and difficulties in recycling the reaction system, it is of great theoretical and industrial significance to develop a green, safe, industrially scalable protein-polysaccharide-immobilized enzyme Pickering emulsion interface catalytic system with both emulsifying and catalytic functions.

[0006] Therefore, how to develop a method for preparing diglycerides using an enzyme immobilized from ultrafine pulverized plant protein is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a method for preparing diglycerides by immobilizing plant protein with an enzyme through ultrafine grinding.

[0008] A method for preparing diglycerides from ultrafine pulverized plant protein using an immobilized enzyme process includes the following steps:

[0009] (1) Plant protein and chitosan were co-ground using a high-energy media mill, and food-grade plant protein-chitosan composite material with a porous network structure was prepared by mechanical force. (2) The food-grade plant protein-chitosan composite material obtained in step (1) is mixed with a lipase solution prepared with phosphate buffer, and the lipase is immobilized in the composite material through non-covalent interaction to obtain an immobilized enzyme based on the plant protein-chitosan food-grade composite carrier. The concentration of the lipase solution is 4-12 mg / mL, and the volume ratio of the food-grade plant protein-chitosan composite material to the lipase solution is 1:(0.4-1). (3) The immobilized enzyme based on the plant protein-chitosan food-grade composite carrier obtained in step (2) is used as an emulsifier. The reaction substrate plant oil is added to it, and Pickering emulsion is prepared by high-speed shearing machine. The plant oil is hydrolyzed by constant temperature water bath to obtain diglyceride product. The Pickering emulsion is an O / W system with an oil phase volume fraction of 0.3-0.7 and a constant temperature water bath temperature of 35-55 ℃.

[0010] Furthermore, in step (1), the plant protein includes one or more of soybean protein, pea protein, peanut protein, walnut protein, sunflower seed protein, oat protein, rice protein, corn protein or quinoa protein, preferably, the plant protein is pea protein.

[0011] Further, in step (1), the preparation method of the food-grade plant protein-chitosan composite material includes the following steps: dispersing plant protein in water, adjusting its pH to 5.0-10.0 with NaOH, hydrochloric acid or citric acid, and stirring evenly to obtain a plant protein solution; dissolving chitosan in an acetic acid solution with a mass fraction of 0.1%-2.0%, adjusting its pH to 5.0-10.0 with NaOH to obtain a chitosan solution; the mass ratio of plant protein to chitosan is (1-10):1; the mass fraction of the plant protein solution is 1.0-6.0%, and the mass fraction of the chitosan solution is 1.0-6.0%.

[0012] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: If the pH is too high, chitosan cannot dissolve sufficiently, and its composite effect with plant protein will be greatly reduced, easily forming composite materials with large particle sizes, which is detrimental to its dispersion stability. In addition, the positive charge effect generated by protonation of chitosan will be greatly weakened, which is not conducive to the adsorption and fixation of lipase. If the pH is too low, plant protein cannot be fully expanded, and cannot provide more sites for binding with chitosan. It is also not conducive to the immobilization of lipase and the stability of the emulsion. If the mass ratio of plant protein to chitosan is too high, the adsorption effect of lipase will decrease. If the mass ratio is too low, it is not conducive to the formation of composite materials with uniform particle size distribution, and the physical treatment effect is not significant.

[0013] Furthermore, in step (1), the system temperature is kept below 30℃ during the high-energy media milling process; the specific process parameters of the high-energy media mill are: grinding bead diameter 0.6-2.0 mm, grinding chamber filling degree 60-75%, rotation speed 100-1500 r / min, and grinding time 50-400 min.

[0014] The beneficial effects of adopting the above-mentioned further technical solution are as follows: When the filling level is too low, the mechanical stress on the material in the grinding chamber is insufficient, and the grinding effect is reduced. Conversely, when the filling level is too high, the material has poor flowability in the chamber, which is also not conducive to material processing. Grinding time has a significant impact on the particle size, surface area, and microstructure of plant protein-chitosan composite materials.

[0015] Furthermore, in step (1), the particle size of the food-grade plant protein-chitosan composite material is 0.4-200.0 μm, and the specific surface area is 1000-6000 m². 2 / kg, with a contact angle of 35-80°, and has a network-like porous structure with chitosan as the connecting backbone and plant proteins distributed within it.

[0016] Furthermore, in step (2), the pH of the phosphate buffer is 5.0-10.0; the lipase is one or more of Candida antarctica lipase, Pseudomonas cepacia lipase, Aspergillus niger lipase, Michelia niger root hair lipase or Thermophilus sparsely cottony lipase.

[0017] Furthermore, in step (2), the mixing speed is 200-1000 rpm and the mixing temperature is 4-30℃.

[0018] The beneficial effects of adopting the above-mentioned further technical solution are as follows: Too low a lipase concentration will affect the rate of the catalytic reaction, while too high a concentration may lead to uneven distribution of lipase on the plant protein-chitosan composite material, causing self-aggregation and thus hindering the binding of the substrate to the enzyme active site. Furthermore, by screening the amount of lipase added, the lipase activity can be prevented from decreasing due to the saturation of binding sites on the plant protein-chitosan composite material.

[0019] Further, in step (3), the immobilized enzyme loading is 20-130 mg / g, and the enzyme activity is 400-550 U / g; the diffusion rate at the immobilized enzyme interface ( K diff The value is 0.82-1.19 mNm. -1 s 0.5 Permeation rate ( K p The value is 2.36 × 10 -4 -2.52×10 -4 s -1 rearrangement rate ( K r The value is 13.72 × 10 -4 -14.95×10 -4 s -1It exhibits superior interfacial adsorption behavior compared to free enzymes; the vegetable oil is one or more of rapeseed oil, soybean oil, rice bran oil, perilla seed oil, corn oil, walnut oil, peanut oil, camellia seed oil, sesame oil, flaxseed oil, or sunflower seed oil.

[0020] Furthermore, in step (3), the high-speed shear machine rotates at 8000-30000 rpm / min, and the shearing time is 1-3 min; the Pickering emulsion is an O / W system, the volume-weighted average diameter of the emulsion droplets is 50-80 μm, and the number of emulsion droplets per unit volume is 3.73 × 10⁻⁶. 6 -1.53×10 7 The apparent viscosity of the Pickering emulsion is 1.5 × 10⁻⁶. 4 -4.0×10 4 The energy storage modulus (G') is greater than the loss modulus (G"), and the loss factor (tanδ) is 0.1-0.5, exhibiting excellent stability; the hydrolysis reaction time is 15-90 min.

[0021] The beneficial effects of adopting the above-mentioned further technical solution are: excessively high water bath temperature can easily cause enzyme activity loss, while excessively low temperature is not conducive to enzyme catalysis.

[0022] Furthermore, in step (3), the content of diglycerides in the diglyceride product ranges from 40% to 55%.

[0023] The beneficial effects of adopting the above-mentioned further technical solution are that the reaction system can be reused for at least 8 catalytic cycles.

[0024] The beneficial effects of the present invention are as follows: (1) The present invention uses natural food ingredients plant protein and chitosan as carriers to prepare immobilized enzymes, and further uses them as catalysts and emulsifiers to construct a food-grade Pickering emulsion enzyme catalytic system. The selected immobilized enzyme carrier raw materials are widely available, inexpensive and readily available, green, safe and pollution-free. Through physical modification equipment - high-energy media mill, plant protein and chitosan are co-ground to prepare food-grade composite materials with submicron particle size and high positive charge. The operation is convenient, the steps are simple, the safety is controllable, the processing capacity is large and continuous production is possible.

[0025] (2) The plant protein-chitosan composite material prepared by the present invention has a porous network structure, which not only provides more binding sites for lipase, but also more effectively protects the active conformation of lipase, so as to maintain its excellent catalytic activity and stability.

[0026] (3) The present invention utilizes the Pickering emulsion interface to catalyze the preparation of diglycerides. It not only has a large catalytic reaction interface, but also the plant protein-chitosan composite material positions the lipase on the interface of the emulsion droplets, which greatly increases the contact probability between the enzyme and the substrate, shortens the mass transfer distance, and significantly improves the reaction efficiency and utilization rate of the enzyme.

[0027] (4) The Pickering emulsion reaction system constructed in this invention has the dual characteristics of separating products and recovering catalysts, and still has high relative activity (greater than 85%) after at least 8 reaction cycles.

[0028] (5) The production process of diglycerides in this invention results in products with high safety, which are suitable for fields with high safety requirements such as pharmaceuticals, food and health products.

[0029] In summary, this method, based on high-energy media milling technology, offers advantages such as low cost, environmental friendliness, high catalytic efficiency, good stability, and continuous industrial production capability. This invention utilizes natural food-grade components to prepare a composite material with a network-porous structure through pure mechanical force, immobilizing lipase within it. This lipase is then applied to Pickering emulsion interfacial catalysis, effectively enhancing the stability of the enzyme structure and increasing the probability of enzyme-substrate contact. Simultaneously, the large oil-water interface provided by the emulsion accelerates mass transfer efficiency, promoting the efficient synthesis of diglycerides. Furthermore, using a plant protein-chitosan complex as the immobilization carrier ensures readily available raw materials, good emulsification performance, and recyclability.

[0030] This system can improve enzyme catalytic efficiency, achieving a diglyceride content of over 45% after 30 minutes of reaction, and maintaining over 40% even after 8 repetitions. The immobilized enzyme carrier of this invention is made from inexpensive natural food raw materials, is simple to prepare, highly safe, produces little pollution, and can be produced continuously. Furthermore, the resulting Pickering emulsion has good stability, which is beneficial for the clean production of diglycerides, and has good industrialization potential. Attached Figure Description

[0031] Figure 1 Figures (a) and (b) are SEM and TEM images of the pea protein-chitosan composite material obtained in Example 1, respectively. Figure (c) is a particle size distribution diagram of the pea protein-chitosan composite material obtained in Example 1. Figure (d) is a contact angle diagram of the pea protein-chitosan composite material obtained in Example 1.

[0032] Figure 2The images show laser confocal microscopy images of the distribution of FITC-labeled pea protein-chitosan composite material and Cy5-labeled lipase at the Pickering emulsion droplet interface, where (a) is a laser confocal microscopy image of the distribution of FITC-labeled pea protein-chitosan composite material at the Pickering emulsion droplet interface, and (b) is a laser confocal microscopy image of the distribution of Cy5-labeled lipase at the Pickering emulsion droplet interface.

[0033] Figure 3 This is a comparison diagram of the preparation of diglycerides using Pickering emulsion catalysis in Example 1 and Comparative Example 5, showing the immobilized enzyme and the free enzyme.

[0034] Figure 4 The volume-weighted average diameter (D) of the Pickering emulsion droplets stabilized by immobilized and free enzymes in Example 1 and Comparative Example 5 is shown. [4,3] ).

[0035] Figure 5 The number of emulsion droplets per unit volume of the Pickering emulsion stabilized by immobilized and free enzymes in Example 1 and Comparative Example 5.

[0036] Figure 6 The following are rheological data graphs of Pickering emulsions in Example 1 and Comparative Example 5: (a) shows the relationship between apparent viscosity and shear rate; (b) shows the frequency scan of storage modulus (G') and loss modulus (G'); and (c) shows the loss factor. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] It should be noted that the experimental measurement content and methods are as follows: 1. Determination of glyceride composition: The composition of glycerides was determined using a high-temperature gas chromatograph. After the hydrolysis reaction was completed, 10 μL of the sample was added to 1 mL of n-hexane, vortexed, centrifuged, filtered through a 200 μL organic filter membrane, and then placed in a sample vial for analysis. The glyceride composition of the vegetable oil was determined using a gas chromatograph equipped with a flame ionization detector and a fused silica capillary column, and the diglyceride content was calculated using the area normalization method. Detailed gas chromatograph parameters were as follows: high-purity helium as carrier gas, total gas flow rate of 2 mL / min, injection and detector temperatures of 320 °C and 350 °C, respectively; initial column temperature held at 170 °C for 2 min, then increased to 380 °C at a rate of 5 °C / min and held for 6 min; split ratio of 50:1.

[0039] 2. Reusability test: After one enzymatic reaction, the upper oil phase was removed by centrifugation at 8000 rpm / min for 10 min and subjected to molecular distillation. The fresh oil phase was then added to the aqueous phase and subjected to high-speed shearing before entering the next Pickering emulsion interface catalytic reaction to prepare diglycerides. The reusability of the system was characterized by measuring the diglyceride content in each cycle.

[0040] 3. Distribution of pea protein-chitosan composite material with immobilized lipase in emulsion: The distribution of FITC-labeled pea protein-chitosan composite material (green) and Cy5-labeled lipase (blue) on Pickering emulsion was observed.

[0041] 4. The high-energy media mill used in the following experiments is model XCFB-4T, manufactured by Beijing Collaborative Innovation Food Technology Co., Ltd.

[0042] Example 1 (1) Preparation of pea protein-chitosan composite material: 0.36 kg of pea protein powder was dispersed in 8.64 kg of deionized water, and the pH was adjusted to 6.0 with hydrochloric acid to obtain a 4% (w / w) pea protein solution. 0.12 kg of chitosan was dissolved in 2.88 kg of 0.1% acetic acid solution, and the pH was adjusted to 6.0 with NaOH to obtain a 4% (w / w) chitosan solution. The pea protein solution and chitosan solution prepared above were then poured into the stirring tank of a high-energy media mill and mixed evenly. The milling was carried out under the following parameters: grinding bead diameter 0.6 mm, grinding chamber filling degree 60%, rotation speed 1200 r / min, grinding time 300 min, and system temperature below 30 ℃. A composite material with a pea protein to chitosan mass ratio of 3:1 was prepared.

[0043] (2) Preparation of immobilized enzyme from pea protein-chitosan composite material: Pseudomonas cepacia lipase was dissolved in a phosphate buffer solution at pH 6.0 to prepare a lipase solution of 9 mg / mL. This solution was then mixed with the pea protein-chitosan composite material from step (1) at a ratio of 1:0.6 (V / V) and magnetically stirred at 30 ℃ and 600 rpm for 30 min to prepare immobilized lipase (immobilized loading of 90 mg / g, enzyme activity of 500 U / g). The interfacial adsorption behavior of this immobilized enzyme is shown in Table 1, and the diffusion rate (… K diff The value is 1.095 mNm. -1 s 0.5 Permeation rate ( K p The value is 2.507 × 10 -4 s -1 rearrangement rate ( K r The value is 14.900 × 10 -4 s -1 It is superior to free enzymes.

[0044] (3) Construction of Pickering emulsion catalytic system: The immobilized lipase obtained in step (2) was mixed with rapeseed oil and sheared at 12000 rpm for 1.5 min using a high-speed shear mill to prepare the Pickering emulsion catalytic system, wherein the oil phase volume fraction was 0.4%. The volume-weighted average diameter of the emulsion droplets was 64.25 μm. Figure 4 The number of droplets per unit volume is 7.20 × 10⁻⁶. 6 indivual( Figure 5 The apparent viscosity is 1.98 × 10⁻⁶. 4 mPa·s ( Figure 6 (Figure (a)).

[0045] (4) Preparation of diglycerides: The Pickering emulsion system constructed in step (3) was placed in a constant temperature water bath at 50 °C and hydrolyzed for 30 min without mechanical stirring.

[0046] The microstructure of the pea protein-chitosan composite material is as follows: Figure 1 Figure (a) Figure 1 As shown in Figure (b), the particle size is as follows Figure 1 As shown in Figure (c), the range is 0.4–2.5 μm, and the specific surface area is 5515 m². 2 / kg, contact angle as Figure 1 As shown in Figure (d), the contact angle is 65.69°, and it has a network-like porous structure with chitosan as the connecting backbone and plant proteins distributed in it.

[0047] The distribution of immobilized lipase at the emulsion droplet interface is as follows: Figure 2 As shown in Table 1, the diglyceride content in the Pickering emulsion catalytic system reached 48.25% after 30 min of reaction. After 8 cycles of reuse, the diglyceride content still reached 43.67% after 30 min of reaction. The reusability results of this Pickering emulsion interfacial catalytic system are detailed in Table 1.

[0048] Example 2 The grinding time in step (1) of Example 1 was changed from 300 min to 100 min, while the remaining steps were the same as in Example 1. The pea protein-chitosan composite material has a particle size of 0.6-14 μm and a specific surface area of ​​2612 m². 2 / kg, with a contact angle of 42.15°. The immobilized lipase loading was 53 mg / g, and the enzyme activity was 357 U / g. This immobilized enzyme at the interface... K diff It is 0.894 mNm -1 s 0.5 , K p 2.259×10 -4 s -1 , K r It is 13.587×10 -4 s -1 The volume-weighted average diameter of the emulsion droplets is 70.08 μm, and the number of droplets per unit volume is 5.55 × 10⁻⁶. 6 One, with an apparent viscosity of 1.76 × 10⁻⁶. 4 The diglyceride content of this Pickering emulsion catalytic system reached 45.30% after 30 min of reaction. A reusability test of the system showed that after 8 cycles of recycling, the diglyceride content still reached 40.77% after 30 min of reaction.

[0049] Example 3 The *Pseudomonas cepacia* lipase in step (1) of Example 1 was changed to *Thermophilus spp.* lipase, while the remaining steps were the same as in Example 1. The immobilized lipase loading was 88 mg / g, and the enzyme activity was 473 U / g. This immobilized enzyme at the interface... K diff It is 0.982 mNm -1 s 0.5 , K p 2.145×10 -4 s -1 , K r 14.016×10 -4 s -1The diglyceride content of the Pickering emulsion catalytic system reached 47.61% after 30 minutes of reaction. A reusability test of the system showed that after eight cycles of recycling, the diglyceride content still reached 43.12% after 30 minutes of reaction.

[0050] Example 4 The concentration of the lipase solution in step (2) of Example 1 was changed from 9 mg / mL to 6 mg / mL, while the remaining steps were the same as in Example 1. The prepared immobilized lipase had a loading of 65 mg / g and an enzyme activity of 440 U / g. This immobilized enzyme at the interface... K diff It is 1.051 mNm -1 s 0.5 , K p 2.357×10 -4 s -1 , K r 14.118×10 -4 s -1 The diglyceride content of the Pickering emulsion catalytic system reached 46.27% after 30 min of reaction. A reusability test of the system showed that after eight cycles of recycling, the diglyceride content still reached 41.96% after 30 min of reaction.

[0051] Example 5 The volume ratio of pea protein-chitosan composite material to lipase solution in step (2) of Example 1 was changed from 1:0.6 to 1:1, while the remaining steps were the same as in Example 1. The prepared immobilized lipase had a loading of 110 mg / g and an enzyme activity of 450 U / g. The immobilized enzyme at the interface... K diff It is 1.216 mNm -1 s 0.5 , K p It is 2.484×10 -4 s -1 , K r 14.853×10 -4 s -1 The diglyceride content of the Pickering emulsion catalytic system reached 46.36% after 30 min of reaction. A reusability test of the system showed that after eight cycles of recycling, the diglyceride content still reached 41.08% after 30 min of reaction.

[0052] Example 6 The rapeseed oil in step (3) of Example 1 was replaced with rice bran oil, while the remaining steps were the same as in Example 1. The volume-weighted average diameter of the Pickering emulsion droplets was 60.91 μm, and the number of emulsion droplets per unit volume was 8.45 × 10⁻⁶. 6 The apparent viscosity of the emulsion is 2.30 × 10⁻⁶. 4 The diglyceride content of this Pickering emulsion catalytic system reached 53.48% after 30 min of reaction. A reusability test of the system showed that after 8 cycles of recycling, the diglyceride content still reached 48.15% after 30 min of reaction.

[0053] Example 7 The oil phase volume fraction in step (3) of Example 1 was changed from 0.4 to 0.6, while the remaining steps were the same as in Example 1. The volume-weighted average diameter of the Pickering emulsion droplets was 71.92 μm, and the number of emulsion droplets per unit volume was 5.14 × 10⁻⁶. 6 The apparent viscosity of the emulsion is 3.15 × 10⁻⁶. 4 The Pickering emulsion interface catalytic system achieved a diglyceride content of 45.54% after 30 min of reaction. Reusability testing showed that after eight cycles, the diglyceride content remained at 40.22% after 30 min of reaction.

[0054] Example 8 The reaction temperature in step (4) of Example 1 was changed from 50 °C to 35 °C, while the remaining steps were the same as in Example 1. After 30 min of reaction, the diglyceride content of the Pickering emulsion catalytic system reached 47.92%. The reusability of the system was tested, and after 8 cycles of recycling, the diglyceride content after 30 min of reaction still reached 42.58%.

[0055] Comparative Example 1 The preparation method of the pea protein-chitosan composite material in Example 1 was modified by omitting the high-energy medium grinding treatment in step (1). The unground pea protein-chitosan composite material was used for Pickering emulsion catalysis to prepare diglycerides. Other operating steps were the same as in Example 1. Compared with Example 1, the unground composite material could not form a complex porous network structure, with a particle size of 6.0-400.0 μm and a specific surface area of ​​820 m². 2 / kg, with a contact angle of 35.83°. The prepared immobilized lipase had a loading of 35 mg / g and an enzyme activity of 210 U / g. This immobilized enzyme at the interface... K diff It is 0.623 mNm -1 s 0.5 , Kp 2.098×10 -4 s -1 , K r 13.106×10 -4 s -1 The volume-weighted average diameter of the Pickering emulsion droplets was 88.51 μm, and the number of droplets per unit volume was 2.75 × 10⁻⁶. 6 The apparent viscosity of the emulsion is 1.04 × 10⁻⁶. 4 The diglyceride content of the Pickering emulsion catalytic system decreased to 30.45% after 30 min of reaction. A reusability test was conducted on the system, and after four cycles, the diglyceride content was 15.84% after 30 min of reaction.

[0056] Conclusion: When the high-energy media milling step is omitted, the protein and chitosan undergo only simple physical mixing, failing to form the submicron-scale porous network framework described in this invention. This not only leads to a sharp decrease in the specific surface area of ​​the carrier and a significant decline in enzyme loading and activity, but also reduces its ability to stabilize Pickering emulsions as a particulate emulsifier, causing severe droplet aggregation. This indicates that high-energy media milling is a crucial step in endowing the composite material with excellent emulsifying properties and efficient interfacial catalytic performance.

[0057] Comparative Example 2 The pea protein-chitosan composite material in Example 1 was changed to a single pea protein component; all other operating steps remained the same as in Example 1. Compared with Example 1, the pea protein in this example had a particle size of 1.0-100.0 μm and a specific surface area of ​​2018 m². 2 The immobilized lipase had a loading capacity of 29 mg / g and an enzyme activity of 193 U / g. The volume-weighted average diameter of the Pickering emulsion droplets was 68.32 μm, and the number of droplets per unit volume was 5.99 × 10⁻⁶. 6 The apparent viscosity of the emulsion is 1.79 × 10⁻⁶. 4 The diglyceride content of the Pickering emulsion catalytic system decreased to 27.56% after 30 min of reaction. A reusability test was conducted on the system, and after three cycles, the diglyceride content was 16.25% after 30 min of reaction.

[0058] Conclusion: Immobilization of enzymes using only pea protein significantly reduced enzyme loading and emulsion catalytic performance, with the resulting diglyceride content being only 57% of that in Example 1. This indicates that chitosan is an important component for improving the immobilization performance of materials.

[0059] Comparative Example 3 The concentration of the lipase solution in step (2) of Example 1 was changed from 9 mg / mL to 20 mg / mL, while other operating steps remained the same as in Example 1. Compared with Example 1, the immobilized lipase had a loading capacity of 120 mg / g and an enzyme activity of 400 U / g. This immobilized enzyme at the interface... K diff It is 1.087 mNm -1 s 0.5 , K p It is 2.391×10 -4 s -1 , K r 13.958×10 - 4 s -1 The volume-weighted average diameter of the Pickering emulsion droplets was 61.15 μm, and the number of droplets per unit volume was 8.35 × 10⁻⁶. 6 The apparent viscosity of the emulsion is 1.88 × 10⁻⁶. 4 The diglyceride content in the Pickering emulsion catalytic system decreased to 43.23% after 30 min of reaction. A reusability test was conducted on the system, and after 8 cycles, the diglyceride content was 39.10% after 30 min of reaction.

[0060] Conclusion: When the lipase concentration was increased to 20 mg / mL, the enzyme loading increased, but the enzyme activity decreased to 400 U / g, and the final diglyceride content was lower than in Example 1. This indicates that excessively high free enzyme concentrations are not conducive to improving enzyme catalytic activity; on the contrary, they can hinder the contact between the substrate and the enzyme's active site and may even disrupt the enzyme's native conformation, leading to a phenomenon of "high loading, low activity." Therefore, this invention controls the enzyme concentration within a specific range, which not only avoids enzyme waste and reduces costs but also ensures that the immobilized enzyme maintains excellent catalytic activity.

[0061] Comparative Example 4 The volume ratio of pea protein-chitosan composite material to lipase solution in step (2) of Example 1 was changed from 1:0.6 to 1:2, while other operating steps remained the same as in Example 1. Compared with Example 1, the immobilized lipase had a loading capacity of 123 mg / g and an enzyme activity of 406 U / g. This immobilized enzyme at the interface... K diff It is 1.090 mNm -1 s 0.5 , K p It is 2.473×10 -4 s -1 , K rIt is 14.187×10 -4 s -1 The volume-weighted average diameter of the Pickering emulsion droplets was 60.21 μm, and the number of droplets per unit volume was 8.75 × 10⁻⁶. 6 The apparent viscosity of the emulsion is 1.95 × 10⁻⁶. 4 The diglyceride content in the Pickering emulsion catalytic system decreased to 44.83% after 30 min of reaction. A reusability test was conducted on the system, and after 8 cycles, the diglyceride content was 39.81% after 30 min of reaction.

[0062] Conclusion: Changing the volume ratio of the composite material to the lipase solution to 1:2 was detrimental to improving enzyme activity, and the content further decreased after 8 cycles of use, with all indicators being inferior to Example 1. This indicates that in the Pickering emulsion interfacial catalytic system, the number of effective binding sites on the carrier is one of the factors determining catalytic efficiency, rather than the absolute total amount of enzyme in the system. Therefore, this invention, by limiting the volume ratio of the two components, ensures efficient immobilization and catalytic reaction of the lipase, avoiding negative interference from excessive enzyme in the reaction system.

[0063] Comparative Example 5 The immobilized enzyme used to stabilize the Pickering emulsion system in step (3) of Example 1 was changed to a free lipase; all other operating steps remained the same as in Example 1. Compared with Example 1, the free enzyme at the interface... K diff It is 0.504 mNm -1 s 0.5 , K p It is 2.187×10 -4 s -1 , K r 13.033×10 -4 s -1 The interfacial adsorption kinetics are poor. The volume-weighted average diameter of the emulsion droplets is 109.73 μm, and the number of emulsion droplets per unit volume is 1.45 × 10⁻⁶. 6 The apparent viscosity of the emulsion is 6.94 × 10⁻⁶. 2 The diglyceride content in the Pickering emulsion catalytic system decreased to 43.86% after 30 min of reaction. A reusability test of the system showed that after only three cycles, the diglyceride content decreased to 22.18% after 30 min of reaction.

[0064] Conclusion: Although the free enzyme system achieved a diglyceride content of 43.86% in the first reaction, the content plummeted to 22.18% after three cycles, while Example 1 maintained a high activity of 43.67% after eight cycles. This indicates that the immobilized enzyme emulsion catalytic system not only overcomes the limitations of traditional mass transfer and solves the problem of difficult reuse of free enzymes, but also greatly enhances the conformational stability of the enzyme through interfacial immobilization, demonstrating excellent potential for industrial application.

[0065] Comparative Example 6 The oil phase volume fraction in step (3) of Example 1 was changed from 0.4 to 0.8, while other operating steps remained the same as in Example 1. Compared with Example 1, the volume-weighted average diameter of the Pickering emulsion droplets was 79.26 μm, and the number of emulsion droplets per unit volume was 3.84 × 10⁻⁶. 6 The apparent viscosity of the emulsion is 3.86 × 10⁻⁶. 4 The diglyceride content in the Pickering emulsion catalytic system decreased to 34.22% after 30 min of reaction. A reusability test was conducted on the system, and after eight cycles, the diglyceride content was 29.37% after 30 min of reaction.

[0066] Conclusion: Increasing the oil phase volume fraction to 0.8, which exceeds the range defined in this invention, significantly reduced the diglyceride content to 34.22%. This indicates that in the Pickering emulsion interface catalytic system, the oil-water phase ratio must be controlled within a specific range to avoid insufficient solid particle emulsifier due to an excessively high oil phase ratio, resulting in incomplete coverage of the oil-water interface, emulsion instability and aggregation, and hindering the enzymatic hydrolysis reaction.

[0067] Comparative Example 7 The Pickering emulsion system in step (3) of Example 1 was changed to a biphasic reaction system, that is, the high-speed shearing machine treatment was omitted, and the other operation steps were the same as in Example 1. Compared with Example 1, the content of diglycerides in this biphasic system was only 10.85% after 30 min of reaction.

[0068] Conclusion: When the high-speed shearing process was omitted and the reaction system was changed from a Pickering emulsion to a traditional oil-water biphase system, the diglyceride content decreased the most, by only 10.85%. This fully demonstrates the significant advantages of immobilized enzyme Pickering emulsion interfacial catalysis. This system creates an oil-water interfacial microreactor with a huge surface area, precisely locating and enriching the enzyme at the reaction site, thus breaking through the mass transfer bottleneck in enzymatic catalysis.

[0069] Comparative Example 8 The reaction temperature in step (4) of Example 1 was changed from 50 °C to 25 °C, while other operating steps remained the same as in Example 1. Compared with Example 1, the content of diglycerides in the Pickering emulsion catalytic system decreased to 33.24% after 30 min of reaction. The reusability of the system was tested, and after 8 cycles of recycling, the content of diglycerides after 30 min of reaction was 29.83%.

[0070] Conclusion: When the reaction temperature was reduced to 25 °C, the initial reaction content of diglycerides decreased significantly to 33.24%, which was significantly lower than that in Example 1. This indicates that although the Pickering emulsion system constructed in this invention has largely eliminated mass transfer resistance, the enzymatic hydrolysis reaction is still essentially limited by the catalytic characteristics of lipase. Excessively low reaction temperatures not only weaken the thermal motion of molecules within the system and drastically reduce the effective collision frequency, but more importantly, the low temperature reduces the conformational flexibility of the lipase molecules, making it impossible to reach the optimal activation state required for catalytic lipoprotein bond cleavage. Therefore, this invention controls the temperature within a specific range, which helps to maximize its catalytic potential while ensuring the stability of the enzyme protein structure.

[0071] Table 1 Results of Reusability Tests

[0072] Table 2. Interfacial adsorption kinetics data of immobilized enzyme and free enzyme in Example 1 and Comparative Example 5.

[0073] Conclusion: In summary, this invention successfully constructed a novel Pickering emulsion interface catalytic system based on ultrafine pulverized plant protein, providing an innovative technical solution for the efficient and green preparation of diglycerides. This invention overcomes the inherent bottleneck of "interfacial mass transfer obstruction" between the aqueous enzyme and oil substrate in traditional enzymatic catalysis. Through high-energy media milling physical co-grinding technology, the immobilized enzyme material is endowed with the dual functions of "particulate emulsifier" and "biocatalyst." In this system, lipase is precisely anchored at the large oil-water interface, achieving efficient synthesis of diglycerides (≥45%) within 30 min without mechanical stirring. Simultaneously, the porous network structure of the carrier protects the enzyme conformation, resulting in excellent reusability and stability; the catalytic activity remains above 85% after 8 consecutive cycles. Compared to existing chemical methods, traditional free enzyme methods, and Pickering catalytic systems based on inorganic particles, this invention utilizes inexpensive, readily available, and absolutely safe natural food ingredients from the source, achieving a fully green preparation chain encompassing "low-cost and controllable carrier preparation—efficient enzyme immobilization—interfacial catalysis enhancement—easy product separation and recovery." This technology not only effectively avoids food safety risks and environmental pollution problems, but also perfectly meets the urgent needs of the modern functional oil industry for large-scale, continuous, and clean production with extremely low energy consumption and extremely high production efficiency, possessing extremely high scientific and technological achievement transformation value and broad industrial application prospects.

[0074] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for the enzymatic production of diglycerides by ultrafine pulverized plant protein immobilized enzymes, characterized in that, Includes the following steps: (1) Plant protein and chitosan were co-ground using a high-energy media mill, and food-grade plant protein-chitosan composite material with a porous network structure was prepared by mechanical force. (2) The food-grade plant protein-chitosan composite material obtained in step (1) is mixed with a lipase solution prepared with phosphate buffer, and the lipase is immobilized in the composite material through non-covalent interaction to obtain an immobilized enzyme based on the plant protein-chitosan food-grade composite carrier. The concentration of the lipase solution is 4-12 mg / mL, and the volume ratio of the food-grade plant protein-chitosan composite material to the lipase solution is 1:(0.4-1). (3) The immobilized enzyme based on the plant protein-chitosan food-grade composite carrier obtained in step (2) is used as an emulsifier. The reaction substrate plant oil is added to it, and Pickering emulsion is prepared by high-speed shearing machine. The plant oil is hydrolyzed by constant temperature water bath to obtain diglyceride product. The Pickering emulsion is an O / W system with an oil phase volume fraction of 0.3-0.7 and a constant temperature water bath temperature of 35-55 ℃.

2. The method for preparing diglycerides from ultrafine pulverized plant protein using an immobilized enzyme according to claim 1, characterized in that, In step (1), the plant protein includes one or more of the following: soybean protein, pea protein, peanut protein, walnut protein, sunflower seed protein, oat protein, rice protein, corn protein, or quinoa protein.

3. The method for preparing diglycerides from ultrafine pulverized plant protein using an immobilized enzyme according to claim 1, characterized in that, In step (1), the preparation method of the food-grade plant protein-chitosan composite material includes the following steps: dispersing plant protein in water, adjusting its pH to 5.0-10.0 with NaOH, hydrochloric acid or citric acid, and stirring evenly to obtain a plant protein solution; dissolving chitosan in an acetic acid solution with a mass fraction of 0.1%-2.0%, adjusting its pH to 5.0-10.0 with NaOH to obtain a chitosan solution; the mass ratio of plant protein to chitosan is (1-10):1; the mass fraction of the plant protein solution is 1.0-6.0%, and the mass fraction of the chitosan solution is 1.0-6.0%.

4. The method for preparing diglycerides by ultrafine pulverization of plant protein immobilized with enzymes according to claim 1, characterized in that, In step (1), the system temperature is kept below 30℃ during high-energy media grinding. The specific process parameters of the high-energy media mill are: grinding bead diameter 0.6-2.0 mm, grinding chamber filling degree 60-75%, rotation speed 100-1500 r / min, and grinding time 50-400 min.

5. The method for preparing diglycerides by ultrafine pulverization of plant protein immobilized with enzymes according to claim 1, characterized in that, In step (1), the food-grade plant protein-chitosan composite has a particle size of 0.4-200.0 μm, a specific surface area of 1000-6000 m 2 / kg, a contact angle of 35-80°, and a network-like porous structure with chitosan as a connecting framework and plant protein distributed therein.

6. The method for preparing diglycerides by ultrafine pulverization of plant protein immobilized with enzymes according to claim 1, characterized in that, In step (2), the pH of the phosphate buffer is 5.0-10.0; the lipase is one or more of Candida antarctica lipase, Pseudomonas cepacia lipase, Aspergillus niger lipase, Michelia niger root hair lipase or Thermophilus sparsely cottony lipase.

7. The method for preparing diglycerides by ultrafine pulverization of plant protein immobilized with enzymes according to claim 1, characterized in that, In step (2), the mixing speed is 200-1000 rpm and the mixing temperature is 4-30℃.

8. The method for preparing diglycerides by ultrafine pulverization of plant protein immobilized with enzymes according to claim 1, characterized in that, In step (3), the immobilized enzyme loading is 20-130 mg / g, and the enzyme activity is 400-550 U / g; the diffusion rate at the immobilized enzyme interface... K diff The value is 0.82-1.19 mNm. -1 s 0.5 Permeation rate K p 2.36×10 -4 -2.52×10 -4 s -1 rearrangement rate K r It is 13.72×10 -4 -14.95×10 -4 s -1 It exhibits superior interfacial adsorption behavior compared to free enzymes; the vegetable oil is one or more of rapeseed oil, soybean oil, rice bran oil, perilla seed oil, corn oil, walnut oil, peanut oil, camellia seed oil, sesame oil, flaxseed oil, or sunflower seed oil.

9. The method for preparing diglycerides by ultrafine pulverization of plant protein immobilized with enzymes according to claim 1, characterized in that, In step (3), the high-speed shearing machine has a rotation speed of 8000-30000 rpm / min and a shearing time of 1-3 min; the Pickering emulsion is an O / W system, the volume-weighted average diameter of the emulsion droplets is 50-80 μm, the number of emulsion droplets per unit volume is 3.73×10 6 -1.53×10 7 ; the Pickering emulsion has an apparent viscosity of 1.5×10 4 -4.0×10 4 mPa·s, a storage modulus G' and a loss modulus G" and a loss factor tanδ of 0.1-0.5, and excellent stability; and the hydrolysis reaction time is 15-90 min.

10. The method for preparing diglycerides by ultrafine pulverization of plant protein immobilized with enzymes according to claim 1, characterized in that, In step (3), the content of diglycerides in the diglyceride product ranges from 40% to 55%.

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