A phospholipase-based micromotor reactor-based oil degumming method

Abstract

The application belongs to the field of oil and fat biological refining, and particularly relates to an oil and fat degumming method based on a phospholipase micromotor reactor. The application discloses an enzymatic degumming method for edible vegetable oil, algal oil and animal oil and fat. In view of the pain points of low degumming efficiency, long time consumption, easy deactivation and difficult recovery of enzymes in the traditional enzymatic degumming method, a core-shell mesoporous silica microsphere is designed, an emulsion method is used to graft alkyl and alkyl amine on both sides of the microsphere respectively, a Janus carrier with asymmetric chemical environment is constructed, and phospholipase A1, A2, B or C is solidified by physical adsorption to prepare a phospholipase micromotor reactor. In the degumming process, the micromotor cooperates with the 'interface activation-strengthening mass transfer-dynamic self-cleaning' mechanism, realizes self-driven movement in the interface microscale range by virtue of the substrate concentration gradient, significantly improves the phospholipase enzyme activity and stability, and realizes efficient degumming of oil and fat. The method has high oil yield, green process and is suitable for industrial popularization.

Description

Technical Field

[0001] This invention belongs to the field of biorefining of oils and fats, specifically relating to a highly efficient degumming method for oils and fats based on a phospholipase micromotor reactor. Background Technology

[0002] Phospholipids are the main colloidal impurities in oil refining, and excessive residues can deteriorate the flavor and stability of oils. The main components of phospholipids are hydrated phospholipids and non-hydrated phospholipids. Existing hydration degumming methods can effectively remove hydrated phospholipids, but non-hydrated phospholipids are highly hydrophobic, and even after acid degumming, the phosphorus content of the oil remains at 15~80 mg / kg, which is difficult to meet the requirements for deep degumming of vegetable oils, algal oils, and animal fats.

[0003] Enzymatic degumming involves the specific hydrolysis of the Sn-1 or Sn-2 ester bonds of phospholipids by phospholipases (PLA1, PLA2, PLB, PLC, etc.), generating highly hydrophilic lysophospholipids and glycerophospholipids, which can be easily removed by hydration, reducing phosphorus content to <10 mg / kg. This method is characterized by mild conditions and few byproducts. This technology has significant application value in improving the economic and environmental benefits of the oil and fat industry. However, free enzymes tend to remain in the oil, making them unrecoverable and increasing costs. While conventional immobilization solves the recovery problem, it introduces mass transfer resistance, prolonging reaction time and reducing degumming efficiency. Immobilizing phospholipases retains their unique high efficiency and specificity, improves their stress resistance and stability, allows for reuse, and reduces costs. However, it increases mass transfer resistance between the substrate and enzyme, prolonging reaction time and reducing degumming efficiency. Therefore, there is an urgent need in this field for a novel degumming system that can maintain high enzyme activity, be reusable, and enhance interfacial mass transfer.

[0004] Research has found that enzyme molecules can utilize the energy released during substrate transformation in the enzymatic reaction to drive their own movement when a substrate is present. When an enzyme is immobilized on one side of an anisotropic Janus support, the mechanical forces generated by its catalytic reaction cannot cancel each other out, thus exhibiting autonomous movement and significantly improving its efficiency. This autonomous movement drives the flow of surrounding liquids, molecules, and microparticles, forming a local substrate concentration gradient field, which in turn promotes substrate mass transfer. Currently, the development of enzyme-driven micromotors is booming, with catalase (CAT), glucose oxidase (Gox), urease, and trypsin all being used to construct micromotors. However, research on phospholipase-driven micromotors remains lacking. Summary of the Invention

[0005] In view of the above, this invention provides a method for degumming oils based on a phospholipase micromotor reactor to address the shortcomings of the prior art. Phospholipases A1, A2, B, or C are asymmetrically physically adsorbed and immobilized onto the surface and pores of a Janus carrier. During the phospholipase-catalyzed hydrolysis of phospholipids, the micromotor experiences an imbalance of mechanical force, driving it to continuously move towards regions with high phospholipid concentrations, forming a dynamic cycle of "interface positioning - product diffusion - motor migration," thereby improving degumming efficiency.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for degumming oils based on a phospholipase micromotor reactor specifically includes the following steps:

[0008] 1) Add 0.5 g of HMSS particles to DDAB aqueous solution (60 mg / mL), followed by 5.0 g of solid paraffin; heat the mixture at 80 ℃ for 30 min to completely melt the paraffin; stir the mixture vigorously at 10000 rpm for 160 seconds in a homogenizer; after the emulsion cools to room temperature, centrifuge to separate the obtained particles, wash repeatedly with deionized water several times, and dry at room temperature overnight to obtain paraffin@HMSS colloid.

[0009] 2) The above colloidal particles were dispersed in a methanol solution containing 0.15~0.25 mmol of triamine (diethylenetriaminepropyltrimethoxysilane). After ultrasonic dispersion, the mixture was placed in a shaker at 30 °C and shaken at 150 rpm for 8~12 h. After the reaction, the particles were separated by centrifugation, washed with methanol and chloroform to remove unreacted triamine and residual paraffin, and dried in an oven at 50 °C for 6 h to obtain Janus-HMSS-N3 particles. The Janus-HMSS-N3 particles were dispersed in a toluene solution containing 0.25~0.5 mmol of n-octyltrichlorosilane. After ultrasonic dispersion, the mixture was stirred at room temperature for 2~6 h. After the reaction, the particles were separated by centrifugation, washed multiple times with ethanol, and dried in an oven at 50 °C to finally obtain Janus-HMSS-C8 / N3 particles.

[0010] Furthermore, the carrier (Janus-HMSS-C8 / N3) has a core-shell structure with an average particle size of 200 nm and a pore size of 4 nm. After asymmetric modification, it exhibits a water contact angle of 139°, thus stabilizing water-in-oil microemulsion systems.

[0011] 3) Add free phospholipase to phosphate buffer to prepare an enzyme solution; mix the enzyme solution with the Janus-HMSS-C8 / N3 prepared above, fix and dry to obtain the phospholipase micromotor reactor PL@Janus-HMSS-C8 / N3, the immobilized enzyme loading is 110.5~123.5 mg / g, and the enzyme activity is 100.0~106.7 U / g.

[0012] It should be noted that the constructed Janus-HMSS-C8 / N3 with chemically asymmetric environment provides a differentiated microenvironment for the selective immobilization of phospholipase. At the same time, it can stabilize the microemulsion system and increase the contact area between the enzyme and the substrate. During the phospholipase-catalyzed hydrolysis of phospholipids, the unbalanced mechanical force obtained by the micromotor drives it to continuously move towards the high phospholipid concentration region, forming a dynamic cycle of "interface localization-product diffusion-motor migration", which significantly improves the overall catalytic efficiency.

[0013] 4) Enzymatic degumming process: The oil is pretreated before enzymatic degumming. The pretreated mixture is cooled to the reaction temperature, and 4% (w / v) NaOH solution is added dropwise to adjust the pH of the system. Then, deionized water and immobilized enzyme (phospholipase micromotor reactor PL@Janus-HMSS-C8 / N3) are added according to the oil weight ratio. After homogenization and stirring, the reaction is continued. After the reaction is completed, the immobilized enzyme particles are separated and collected, washed and dried, and the above steps are repeated to carry out multiple rounds of enzyme-catalyzed reaction for oil degumming.

[0014] Furthermore, the free phospholipase is one or more combinations of PLA1, PLA2, PLB, and PLC; the pH of the phosphate buffer is 6.0, and the protein content of the prepared enzyme solution is 6 mg / mL; the 30 ml enzyme solution is mixed with 0.45 g of Janus-HMSS-C8 / N3, the ratio of carrier mass to enzyme solution volume is 15:1 (m / v, mg / mL), the immobilization time is 60 min, and the temperature is 30 ℃.

[0015] Furthermore, the pretreatment step involves heating 30 g of oil in a water bath to 70 °C; then adding 0.2 mL of citric acid solution (45 g / L) and homogenizing at 10,000 rpm for 1 min; and then continuously stirring the mixture at 500 rpm at 70 °C for 20 min.

[0016] It should be noted that the phospholipase micromotor reactor described herein transforms HMSS, which possesses a symmetrical chemical environment, into Janus-HMSS-C8 / N3, which possesses an asymmetrical chemical environment, through asymmetric grafting of monomers C8 and N3. This results in different microenvironments during the immobilization of the free enzyme, providing conditions for asymmetric immobilization of phospholipase. The different adsorption capacities of phospholipase on both sides of the Janus-HMSS-C8 / N3 particles lead to asymmetric immobilization of phospholipase. Therefore, the imbalance of mechanical forces obtained during the phospholipase-catalyzed phospholipid hydrolysis process gives the micromotor its self-driving capability based on the substrate concentration gradient. In the micro-aqueous oil degumming system, PL@Janus-HMSS-C8 / N3 can stabilize the microemulsion system, allowing hydrolysis products to actively diffuse into the aqueous phase. The phospholipase micromotor reactor actively tends towards the high phospholipid concentration region at the interface, improving catalytic efficiency.

[0017] Furthermore, the oil is one of arachidonic acid crude oil, rapeseed oil, corn oil, soybean oil, and walnut oil.

[0018] Furthermore, the reaction temperature is 35~55 ℃; the pH of the reaction system is adjusted to 4.0~4.5; the amount of deionized water added is 0.5~2.5% of the oil weight; the amount of enzyme added is 15~75 mg; and the reaction time of the enzymatic degumming is 60~120 min.

[0019] Furthermore, the oil phase is separated using a continuous centrifuge to obtain degummed oil. The precipitate is washed several times with a buffer solution to remove phospholipids, and the immobilized enzyme is recovered. After washing with n-hexane to remove residual substrate and product, it can be recycled more than 5 times.

[0020] The dephosphorization rates of the degummed oils prepared by the method of this invention are all above 95%. After 60 min of reaction, the phosphorus content of crude ARA oil decreased from 442.4 mg / kg to 9.1 mg / kg; the phosphorus content of rapeseed oil decreased from 204.0 mg / kg to 6.9 mg / kg; the phosphorus content of soybean oil decreased from 434.2 mg / kg to 5.8 mg / kg; the phosphorus content of corn oil decreased from 404.2 mg / kg to 15.9 mg / kg; and the phosphorus content of walnut oil decreased from 175.1 mg / kg to 6.0 mg / kg.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention provides a method for degumming oils based on a phospholipase micromotor reactor. The method operates under mild reaction conditions (35-55℃, pH 4.0-4.5), with water added at only 0.5-2.5% of the oil weight. Within 60 minutes, the phosphorus content of various oils and crude oils can be reduced to below 16 mg / kg. The immobilized enzyme can be recycled more than 5 times and can be recovered by simple centrifugation. It has outstanding advantages such as high catalytic efficiency, strong substrate adaptability, simple operation, and environmental friendliness, providing an efficient, economical, and sustainable degumming solution for oil refining. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 The images show SEM (a) and TEM (b) images of the carrier material obtained in Example 1 of this invention.

[0025] Figure 2 The infrared spectra of HMSS, Janus-HMSS-N3, Janus-HMSS-C8 / N3, and PL@Janus-HMSS-C8 / N3 and PLB of the present invention are shown.

[0026] Figure 3 This is a graph showing the reusability results of enzymatic degumming in Example 4 of the present invention. Detailed Implementation

[0027] 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in the embodiments of this application, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in this application is merely for describing particular implementations and is not intended to limit the scope of this disclosure.

[0029] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0030] To better illustrate the content of this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0031] Without conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solution belongs to the content disclosed in the embodiments of this application.

[0032] This invention discloses a method for degumming oils based on a phospholipase micromotor reactor.

[0033] It should be noted that, in the following embodiments, the phosphorus content in the vegetable oil of the present invention was determined according to the first method of molybdenum blue colorimetric method in GB / T 5537-2008.

[0034] Figure 1 SEM / TEM showed that Janus-HMSS-C8 / N3 consists of uniform spheres with core-shell mesoporous structures and a particle size of about 200 nm.

[0035] Figure 2 The FT-IR spectrum shows 801 cm⁻¹ -1 The absorption peak at 3431 cm⁻¹ is attributed to the symmetric stretching vibration of Si-O-Si, a peak also detected in HMSS materials, Janus particles, and immobilized enzyme samples. -1 The broad peak at 2929 cm⁻¹ corresponds to the NH stretching vibration. After amino functionalization, the peak shape of the Janus particle in this region is significantly broadened, indicating that a new -NH₂ group may have been introduced into the system. -1 and 2852 cm -1 The characteristic peaks at [location] originate from the antisymmetric and symmetric stretching vibrations of the CH bond, respectively, confirming that the octyl group has been successfully grafted onto the HMSS surface. After phospholipase B immobilization, a peak at 1100 cm⁻¹ was observed in the infrared spectrum of the immobilized enzyme. -1 1410 cm -1 and 1652 cm -1 The characteristic absorption peaks of phospholipase B provide strong evidence of successful enzyme immobilization.

[0036] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention.

[0037] Example 1

[0038] 0.5 g of HMSS particles were added to a DDAB aqueous solution (60 mg / mL), followed by 5.0 g of solid paraffin. The mixture was heated at 80 °C for 30 min to completely melt the paraffin. The mixture was then vigorously stirred in a homogenizer at 10,000 rpm for 160 seconds. After the emulsion cooled to room temperature, the particles were separated by centrifugation, washed several times with deionized water, and dried at room temperature overnight to obtain paraffin@HMSS colloid.

[0039] The above colloidal particles were dispersed in a methanol solution containing 0.15 mmol of triamine (diethylenetriaminepropyltrimethoxysilane). After ultrasonic dispersion, the mixture was placed in a shaker at 30 °C and shaken at 150 rpm for 12 h. After the reaction, the particles were separated by centrifugation, washed with methanol and chloroform to remove unreacted triamine and residual paraffin, and dried in an oven at 50 °C for 6 h to obtain Janus-HMSS-N3 particles. The Janus-HMSS-N3 particles were dispersed in a toluene solution containing 0.5 mmol of n-octyltrichlorosilane, ultrasonically dispersed, and stirred at room temperature for 2 h. After the reaction, the particles were separated by centrifugation, washed multiple times with ethanol, and dried in an oven at 50 °C to finally obtain Janus-HMSS-C8 / N3 particles.

[0040] Phospholipase B was added to phosphate buffer at pH 6.0 to prepare an enzyme solution with a protein concentration of 6 mg / mL. 30 mL of the enzyme solution was mixed with 0.45 g of Janus-HMSS-C8 / N3, with a carrier mass to enzyme solution volume ratio of 15:1 (m / v, mg / mL). The immobilization time was 60 min at 30 ℃. After centrifugation and drying, the phospholipase micromotor reactor PL@Janus-HMSS-C8 / N3 was obtained.

[0041] The immobilization capacity of PL@Janus-HMSS-C8 / N3 was determined to be 123.5 mg / g, and the enzyme activity was 106.7 U / g.

[0042] Example 2

[0043] 0.5 g of HMSS particles were added to a DDAB aqueous solution (60 mg / mL), followed by 5.0 g of solid paraffin. The mixture was heated at 80 °C for 30 min to completely melt the paraffin. The mixture was then vigorously stirred in a homogenizer at 10,000 rpm for 160 seconds. After the emulsion cooled to room temperature, the particles were separated by centrifugation, washed several times with deionized water, and dried at room temperature overnight to obtain paraffin@HMSS colloid.

[0044] The above colloidal particles were dispersed in a methanol solution containing 0.2 mmol of triamine (diethylenetriaminepropyltrimethoxysilane). After ultrasonic dispersion, the mixture was placed in a shaker at 30 °C and shaken at 150 rpm for 8 h. After the reaction, the particles were separated by centrifugation, washed with methanol and chloroform to remove unreacted triamine and residual paraffin, and dried in an oven at 50 °C for 6 h to obtain Janus-HMSS-N3 particles. The Janus-HMSS-N3 particles were dispersed in a toluene solution containing 0.5 mmol of n-octyltrichlorosilane. After ultrasonic dispersion, the mixture was stirred at room temperature for 4 h. After the reaction, the particles were separated by centrifugation, washed multiple times with ethanol, and dried in an oven at 50 °C to finally obtain Janus-HMSS-C8 / N3 particles.

[0045] Phospholipase B was added to phosphate buffer at pH 6.0 to prepare an enzyme solution with a protein concentration of 6 mg / mL. 30 mL of the enzyme solution was mixed with 0.45 g of Janus-HMSS-C8 / N3, with a carrier mass to enzyme solution volume ratio of 15:1 (m / v, mg / mL). The immobilization time was 60 min at 30 ℃. After centrifugation and drying, the phospholipase micromotor reactor PL@Janus-HMSS-C8 / N3 was obtained.

[0046] The immobilization capacity of PL@Janus-HMSS-C8 / N3 was determined to be 115.6 mg / g, and the enzyme activity was 103.3 U / g.

[0047] Example 3

[0048] 0.5 g of HMSS particles were added to a DDAB aqueous solution (60 mg / mL), followed by 5.0 g of solid paraffin. The mixture was heated at 80 °C for 30 min to completely melt the paraffin. The mixture was then vigorously stirred in a homogenizer at 10,000 rpm for 160 seconds. After the emulsion cooled to room temperature, the particles were separated by centrifugation, washed several times with deionized water, and dried at room temperature overnight to obtain paraffin@HMSS colloid.

[0049] The above colloidal particles were dispersed in a methanol solution containing 0.25 mmol of triamine (diethylenetriaminepropyltrimethoxysilane). After ultrasonic dispersion, the mixture was placed in a shaker at 30 °C and shaken at 150 rpm for 10 h. After the reaction, the particles were separated by centrifugation, washed with methanol and chloroform to remove unreacted triamine and residual paraffin, and dried in an oven at 50 °C for 6 h to obtain Janus-HMSS-N3 particles. The Janus-HMSS-N3 particles were dispersed in a toluene solution containing 0.25 mmol of n-octyltrichlorosilane, ultrasonically dispersed, and stirred at room temperature for 6 h. After the reaction, the particles were separated by centrifugation, washed multiple times with ethanol, and dried in an oven at 50 °C to finally obtain Janus-HMSS-C8 / N3 particles.

[0050] Phospholipase B was added to phosphate buffer at pH 6.0 to prepare an enzyme solution with a protein concentration of 6 mg / mL. 30 mL of the enzyme solution was mixed with 0.45 g of Janus-HMSS-C8 / N3, with a carrier mass to enzyme solution volume ratio of 15:1 (m / v, mg / mL). The immobilization time was 60 min at 30 ℃. After centrifugation and drying, the phospholipase micromotor reactor PL@Janus-HMSS-C8 / N3 was obtained.

[0051] The immobilization capacity of PL@Janus-HMSS-C8 / N3 was determined to be 110.5 mg / g, and the enzyme activity was 100.0 U / g.

[0052] Example 4

[0053] 30 g of crude rapeseed oil (phosphorus content 204 mg / kg) was placed in an Erlenmeyer flask, preheated in a 70 ℃ water bath, and 0.2 mL of citric acid solution (45 g / L) was added. The mixture was homogenized at high speed (10,000 rpm, 1 min) and stirred continuously at 500 rpm for 20 min at 70 ℃. After the solution cooled to 45 ℃, 4% (w / v) NaOH solution was added dropwise to adjust the pH of the system to 4.0, and the mixture was stirred at 500 rpm for 5 min. 1% deionized water and 30 mg of the phospholipase micromotor reactor prepared in Example 1 were added according to the oil weight ratio. After homogenization, the mixture was reacted at 500 rpm for 60 min. The degummed oil and the phospholipase micromotor reactor were separated by centrifugation, and the phosphorus content was determined.

[0054] The degummed oil was found to contain 6.9 mg / kg of phosphorus, with a dephosphorization rate of 96.7%.

[0055] Repeated use of immobilized enzyme PL@Janus-HMSS-C8 / N3 After five cycles, the dephosphorization rate of crude rapeseed oil can still reach 96%.

[0056] Example 5

[0057] 30 g of crude rapeseed oil (phosphorus content 204 mg / kg) was placed in an Erlenmeyer flask, preheated in a 70 ℃ water bath, and 0.2 mL of citric acid solution (45 g / L) was added. The mixture was homogenized at high speed (10,000 rpm, 1 min) and stirred continuously at 500 rpm for 20 min at 70 ℃. After cooling the solution to 35 ℃, 4% (w / v) NaOH solution was added dropwise to adjust the pH to 4.5, and the mixture was stirred at 500 rpm for 5 min. 0.5% deionized water and 15 mg of the phospholipase micromotor reactor prepared in Example 2 were added according to the oil weight ratio. After homogenization, the mixture was reacted at 500 rpm for 60 min. The degummed oil and the phospholipase micromotor reactor were separated by centrifugation, and the phosphorus content was determined.

[0058] The degummed oil was found to contain 7.2 mg / kg of phosphorus, with a dephosphorization rate of 96.4%.

[0059] Example 6

[0060] 30 g of crude rapeseed oil (phosphorus content 204 mg / kg) was placed in an Erlenmeyer flask, preheated in a 70 ℃ water bath, and 0.2 mL of citric acid solution (45 g / L) was added. The mixture was homogenized at high speed (10,000 rpm, 1 min) and stirred continuously at 500 rpm for 20 min at 70 ℃. After cooling the solution to 55 ℃, 4% (w / v) NaOH solution was added dropwise to adjust the pH to 4.5, and the mixture was stirred at 500 rpm for 5 min. 2.5% deionized water and 75 mg of the phospholipase micromotor reactor prepared in Example 3 were added according to the oil weight ratio. After homogenization, the mixture was reacted at 500 rpm for 120 min. The degummed oil and the phospholipase micromotor reactor were separated by centrifugation, and the phosphorus content was determined.

[0061] The degummed oil was found to contain 6.5 mg / kg of phosphorus, with a dephosphorization rate of 96.8%.

[0062] Example 7

[0063] 30 g of crude arachidonic acid oil (phosphorus content 442.4 mg / kg) was placed in an Erlenmeyer flask, preheated in a 70 °C water bath, and 0.2 mL of citric acid solution (45 g / L) was added. The mixture was homogenized at high speed (10,000 rpm, 1 min) and stirred continuously at 500 rpm for 20 min at 70 °C. After cooling the solution to 45 °C, 4% (w / v) NaOH solution was added dropwise to adjust the pH to 4.0, and the mixture was stirred at 500 rpm for 5 min. 1% deionized water and 30 mg of the phospholipase micromotor reactor prepared in Example 1 were added according to the oil weight ratio. After homogenization, the mixture was reacted at 500 rpm for 60 min. The degummed oil and the phospholipase micromotor reactor were separated by centrifugation, and the phosphorus content was determined.

[0064] The degummed oil was found to contain 9.1 mg / kg of phosphorus, with a dephosphorization rate of 97.9%.

[0065] Example 8

[0066] 30 g of crude soybean oil (phosphorus content 434.2 mg / kg) was placed in an Erlenmeyer flask, preheated in a 70 ℃ water bath, and 0.2 mL of citric acid solution (45 g / L) was added. The mixture was homogenized at high speed (10,000 rpm, 1 min) and stirred continuously at 500 rpm for 20 min at 70 ℃. After cooling the solution to 45 ℃, 4% (w / v) NaOH solution was added dropwise to adjust the pH to 4.0, and the mixture was stirred at 500 rpm for 5 min. 1% deionized water and 30 mg of the phospholipase micromotor reactor prepared in Example 1 were added according to the oil weight ratio. After homogenization, the mixture was reacted at 500 rpm for 60 min. The degummed oil and the phospholipase micromotor reactor were separated by centrifugation, and the phosphorus content was determined.

[0067] The degummed oil was found to contain 5.8 mg / kg of phosphorus, with a dephosphorization rate of 98.6%.

[0068] Example 9

[0069] 30 g of crude corn oil (phosphorus content 404.2 mg / kg) was placed in an Erlenmeyer flask, preheated in a 70 °C water bath, and 0.2 mL of citric acid solution (45 g / L) was added. The mixture was homogenized at high speed (10,000 rpm, 1 min) and stirred continuously at 500 rpm for 20 min at 70 °C. After cooling the solution to 45 °C, 4% (w / v) NaOH solution was added dropwise to adjust the pH to 4.0, and the mixture was stirred at 500 rpm for 5 min. 1% deionized water and 30 mg of the phospholipase micromotor reactor prepared in Example 1 were added according to the oil weight ratio. After homogenization, the mixture was reacted at 500 rpm for 60 min. The degummed oil and the phospholipase micromotor reactor were separated by centrifugation, and the phosphorus content was determined.

[0070] The degummed oil was found to contain 15.9 mg / kg of phosphorus, with a dephosphorization rate of 96.0%.

[0071] Example 10

[0072] 30 g of crude walnut oil (phosphorus content 175.1 mg / kg) was placed in an Erlenmeyer flask, preheated in a 70 °C water bath, and 0.2 mL of citric acid solution (45 g / L) was added. The mixture was homogenized at high speed (10,000 rpm, 1 min) and stirred continuously at 500 rpm for 20 min at 70 °C. After cooling the solution to 45 °C, 4% (w / v) NaOH solution was added dropwise to adjust the pH to 4.0, and the mixture was stirred at 500 rpm for 5 min. 1% deionized water and 30 mg of the phospholipase micromotor reactor prepared in Example 1 were added according to the oil weight ratio. After homogenization, the mixture was reacted at 500 rpm for 60 min. The degummed oil and the phospholipase micromotor reactor were separated by centrifugation, and the phosphorus content was determined.

[0073] The degummed oil was found to contain 6.0 mg / kg of phosphorus, with a dephosphorization rate of 96.6%.

[0074] To further demonstrate the beneficial effects of the present invention and to better understand the present invention, the following comparative examples further illustrate the properties and application performance of the oil degumming method based on a phospholipase micromotor reactor disclosed in the present invention. However, these should not be construed as limiting the present invention. Other methods obtained by those skilled in the art based on the above-described invention and their applications are also considered to fall within the protection scope of the present invention.

[0075] Comparative Example 1

[0076] 0.5 g of HMSS particles were added to a toluene solution containing 0.15 mmol of triamine. After ultrasonic dispersion, the mixture was placed in a shaker at 30 °C and shaken at 150 rpm for 12 h. After the reaction was completed, the particles were separated by centrifugation and washed several times with ethanol. The washed particles were dried in an oven at 50 °C to finally obtain HMSS-N3 particles.

[0077] Phospholipase B was added to phosphate buffer to prepare an enzyme solution with a protein content of 6 mg / mL; 30 mL of the enzyme solution was mixed with 0.45 g of HMSS-N3 and dried to obtain the phospholipase micromotor reactor PL@HMSS-N3.

[0078] The same method as in Example 4 was used for degumming, and 30 mg of immobilized enzyme PL@HMSS-N3 was added. After reacting for 60 min, samples were taken to determine the phosphorus content.

[0079] The degummed oil was found to contain 15.7 mg / kg of phosphorus, with a dephosphorization rate of 92.3%.

[0080] Comparative Example 2

[0081] 0.5 g of HMSS particles were added to a toluene solution containing 0.5 mmol of n-octyltrichlorosilane. After ultrasonic dispersion, the mixture was stirred and reacted at room temperature for 2 h. After the reaction was complete, the particles were separated by centrifugation and washed several times with ethanol. The washed particles were then dried in a 50 °C oven to obtain HMSS-C8 particles.

[0082] Phospholipase B was added to phosphate buffer to prepare an enzyme solution with a protein content of 6 mg / mL; 30 mL of the enzyme solution was mixed with 0.45 g of HMSS-C8 and dried to obtain the phospholipase micromotor reactor PL@HMSS-C8.

[0083] The same method as in Example 4 was used for degumming, and 30 mg of immobilized enzyme PL@HMSS-C8 was added. After reacting for 60 min, samples were taken to determine the phosphorus content.

[0084] The degummed oil was found to contain 12.2 mg / kg of phosphorus, with a dephosphorization rate of 94.0%.

[0085] Comparative Example 3

[0086] 0.5 g of HMSS particles were added to a toluene solution containing 0.15 mmol of triamine and 0.5 mmol of n-octyltrichlorosilane. After ultrasonic dispersion, the mixture was placed in a shaker at 30 °C and shaken at 150 rpm for 12 h. After the reaction was completed, the particles were separated by centrifugation and washed several times with ethanol. The washed particles were dried in a 50 °C oven to finally obtain HMSS-C8 / N3 particles.

[0087] Phospholipase B was added to phosphate buffer to prepare an enzyme solution with a protein content of 6 mg / mL; 30 mL of the enzyme solution was mixed with 0.45 g of HMSS-C8 / N3 and dried to obtain the phospholipase micromotor reactor PL@HMSS-C8 / N3.

[0088] The same method as in Example 4 was used for degumming, and 30 mg of immobilized enzyme PL@HMSS-C8 / N3 was added. After reacting for 60 min, samples were taken to determine the phosphorus content.

[0089] The degummed oil was found to contain 14.9 mg / kg of phosphorus, with a dephosphorization rate of 92.7%.

[0090] Comparative Example 4

[0091] The same method as in Example 4 was used for degumming, and 0.2 mL of free phospholipase B was added to replace the immobilized enzyme. After reacting for 60 min, a sample was taken to determine the phosphorus content.

[0092] The degummed oil was found to contain 12.1 mg / kg of phosphorus, with a dephosphorization rate of 94.0%.

[0093] Table 1. Phosphorus content and dephosphorization rate of degummed products using different carrier enzyme methods

[0094] .

[0095] 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 phospholipase-based micro-motor reactor-based oil degumming method, characterized by, Comprising the following steps: 1) Preparation of core-shell mesoporous silica Janus microspheres Janus-HMSS-C8 / N3 by Pickering emulsion template method: HMSS particles and solid paraffin are added to a double decyl dimethyl ammonium bromide (DDAB) aqueous solution, heating is performed to completely melt the paraffin, homogenization is performed to form a Pickering emulsion, and the HMSS is adsorbed at the colloidal interface; after the emulsion is cooled to room temperature, centrifugation, washing, and drying are performed to obtain paraffin@HMSS colloids; the colloidal particles are dispersed in a methanol solution containing diethylenetriamine propyl trimethoxysilane, shaking bed oscillation reaction is performed, centrifugation, washing, and drying are performed to obtain Janus-HMSS-N3 particles; the Janus-HMSS-N3 particles are dispersed in a toluene solution containing n-octyltrichlorosilane, stirring reaction is performed at room temperature, and the other side is grafted with an alkyl group to obtain Janus-HMSS-C8 / N3 particles; In step 1), the addition amount of the HMSS particles is 0.5 g, the concentration of the DDAB aqueous solution is 60 mg / mL, the addition amount of the solid paraffin is 5.0 g, the heating temperature is 80 ℃, and the homogenization condition is homogenization at 10,000 rpm for 160 seconds. In step 1), the shaking bed oscillation reaction is oscillation reaction at 150 rpm for 8-12 h at 30 ℃; the amount of diethylenetriamine propyl trimethoxysilane in the methanol solution is 0.15-0.25 mmol, the amount of n-octyltrichlorosilane in the toluene solution is 0.25-0.5 mmol; and the stirring reaction time is 2-6 h. 2) Preparation of a phospholipase micro-motor reactor: free phospholipase is added to a phosphate buffer to prepare a free phospholipase solution; the Janus-HMSS-C8 / N3 prepared in step 1) is added to the free phospholipase solution for mixing, and a phospholipase micro-motor reactor PL@Janus-HMSS-C8 / N3 is obtained after fixation and drying; 3) Enzymatic degumming: the pretreated oil mixture is cooled to the reaction temperature, the pH value of the system is adjusted, deionized water and the phospholipase micro-motor reactor PL@Janus-HMSS-C8 / N3 are added, and homogenization is performed to uniformly mix the reaction system, and continuous stirring reaction is performed; after the reaction is completed, centrifugation, washing, and drying are performed to obtain degummed oil and immobilized enzyme particles.

2. The phospholipase-based micro-motor reactor-based oil degumming method according to claim 1, characterized in that, In step 2), the free phospholipase is one or a combination of PLA1, PLA2, PLB, and PLC; the pH of the phosphate buffer is 6.0, and the protein content of the prepared enzyme solution is 6 mg / mL; the ratio of the mass of the carrier to the volume of the enzyme solution is 15:1, m / v, mg / mL, the immobilization time is 60 min, and the temperature is 30 ℃.

3. The phospholipase-based micro-motor reactor-based oil degumming method according to claim 1, characterized in that, In step 3), the pretreatment step is as follows: 30 g of oil is heated to 70 ℃ in a water bath; then 0.2 mL of a citric acid solution with a concentration of 45 g / L is added, and homogenization is performed at 10,000 rpm for 1 min; the mixture is continuously stirred at 500 rpm for 20 min at 70 ℃.

4. The phospholipase-based micro-motor reactor-based oil degumming method according to claim 1 or 3, characterized in that, The oil and fat is one of arachidonic acid crude oil, rapeseed oil, corn oil, soybean oil and walnut oil.

5. The phospholipase-based micro-motor reactor-based oil degumming method according to claim 1, characterized in that, In step 3), the reaction temperature is 35-55 ℃; the pH of the reaction system is adjusted to 4.0-4.5; the amount of deionized water added is 0.5-2.5% of the oil weight; the amount of immobilized enzyme added is 15-75 mg; and the reaction time of the enzymatic degumming is 60-120 min.

6. The phospholipase-based micro-motor reactor-based oil degumming method according to claim 1 or 5, characterized in that, In step 3), the degummed oil is separated from the oil phase by using a continuous centrifuge, the precipitate is washed several times with a buffer solution to remove phospholipids, and the immobilized enzyme is recovered; after washing with n-hexane to remove residual substrates and products, the immobilized enzyme can be recycled for more than 5 times.

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