Grease degumming method based on phospholipase micromotor reactor

Abstract

The invention belongs to the field of biological refining of grease, and particularly relates to a grease degumming method based on a phospholipase micromotor reactor. The invention discloses an enzymatic degumming method of edible vegetable oil, algal oil and animal fat, which aims at the pain points of low degumming efficiency, long time consumption, easy enzyme inactivation and difficult recovery of the traditional enzymatic degumming, designs core-shell mesoporous silica microspheres, adopts an emulsion method to respectively graft alkyl and alkylamine on two sides of the microspheres, constructs a Janus carrier with asymmetric chemical environment, and improves the degumming efficiency of the edible vegetable oil, the algal oil and the animal fat. And immobilizing phospholipase A1, A2, B or C through physical adsorption to prepare the phospholipase micromotor reactor. In the degumming process, a micromotor cooperates with an interface activation-enhanced mass transfer-dynamic self-cleaning mechanism, self-driven movement in an interface micro-scale range is realized by virtue of a substrate concentration gradient, the enzyme activity and stability of phospholipase are remarkably improved, and efficient degumming of grease is realized. The method is high in oil yield, green in process and suitable for industrial popularization.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of oil refining, and particularly relates to an oil efficient degumming method based on a phospholipase micromotor reactor. BACKGROUND

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

[0003] Enzymatic degumming is to hydrolyze the ester bond of Sn-1 or Sn-2 of phospholipids by phospholipase (PLA1, PLA2, PLB, PLC, etc.), to generate corresponding lysophospholipids with strong hydrophilicity and glycerol acyl phospholipids, which can be easily removed by hydration, and the phosphorus content can be reduced to <10 mg / kg, and the conditions are mild and the by-products are few. This technology has great application value in improving the economic and environmental benefits of the oil industry. However, free enzymes are prone to remain in oil, which cannot be recycled and reused, thus increasing the cost. Although conventional immobilization solves the recycling problem, it causes mass transfer resistance, prolongs the reaction time, and reduces the degumming efficiency. The immobilized enzyme prepared by immobilizing phospholipase retains the high efficiency and specificity of phospholipase, improves the enzyme resistance and stability, realizes repeated use, and reduces the use cost, but increases the mass transfer resistance between the substrate and the enzyme, prolongs the reaction time, and reduces the degumming efficiency. Therefore, there is an urgent need in the art for a new degumming system that can maintain high enzyme activity, be reusable, and strengthen the interface mass transfer.

[0004] Research has found that when there is a substrate, the enzyme molecules can use the energy released during the conversion of the substrate in the enzymatic reaction process to drive their own movement. When the enzyme is fixed on one side of the Janus carrier with anisotropy, the mechanical force generated by the catalytic reaction cannot be balanced, so as to show autonomous movement and significantly improve the movement efficiency. This autonomous movement will drive the flow of surrounding liquid, molecules and micro-particles, forming a local substrate concentration gradient field, which in turn promotes the mass transfer of the substrate. At present, the development of enzyme-driven micromotors is in its infancy, and catalase (CAT), glucose oxidase (Gox), urease (Urease) and trypsin (Trypsin) have been used to construct micromotors. However, the research on phospholipase-driven micromotors is still in the blank. SUMMARY

[0005] In view of the above, the present application provides a phospholipase-based micro-motor reactor for oil degumming method to address the above-mentioned deficiencies in the prior art. The phospholipase A1, A2, B or C is asymmetrically physically adsorbed to the surface and pores of the Janus carrier. During the catalytic hydrolysis of phospholipids by phospholipase, the mechanical force imbalance obtained drives the micro-motor to continuously move towards the high phospholipid concentration area, forming a dynamic cycle of "interface positioning-product diffusion-motor migration", thereby improving the degumming efficiency.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] A phospholipase-based micro-motor reactor for oil degumming method, specifically comprising the following steps:

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

[0009] 2) Disperse the above colloidal particles in a methanol solution containing 0.15-0.25 mmol of triamine (diethylenetriamine propyl trimethoxysilane), and after ultrasonic dispersion, place the mixture in a 30°C shaking bed and oscillate at 150 rpm for 8-12 h; after the reaction is completed, centrifuge the particles, wash with methanol and chloroform to remove unreacted triamine and residual paraffin, and dry the particles in a 50°C oven for 6 hours to obtain Janus-HMSS-N3 particles; disperse the Janus-HMSS-N3 particles in a toluene solution containing 0.25-0.5 mmol of n-octyltrichlorosilane, ultrasonic dispersion, and then stir at room temperature for 2-6 h; after the reaction is completed, centrifuge the particles, wash with ethanol several times, and dry the washed particles in a 50°C oven to obtain Janus-HMSS-C8 / N3 particles.

[0010] Further, the carrier (Janus-HMSS-C8 / N3) has a core-shell structure, an average particle size of 200 nm, and a pore size of 4 nm. After asymmetric modification, the water contact angle is 139°, and the oil-in-water microemulsion system can be stabilized.

[0011] 3) adding free phospholipase into phosphate buffer to prepare an enzyme solution; mixing the enzyme solution with the Janus-HMSS-C8 / N3 prepared above, fixing and drying to obtain a phospholipase micro-motor reactor PL@Janus-HMSS-C8 / N3, the immobilized enzyme loading being 110.5-123.5 mg / g, and the enzyme activity being 100.0-106.7 U / g.

[0012] It should be noted that the Janus-HMSS-C8 / N3 with asymmetric chemical environment provides a differential microenvironment for the selective immobilization of phospholipase, and at the same time, it can stabilize the microemulsion system and increase the contact area between the enzyme and the substrate; during the catalytic hydrolysis of phospholipids by phospholipase, the mechanical force imbalance obtained by the micro-motor drives it to continuously move towards the high-phospholipid concentration area, forming a dynamic cycle of "interface positioning-product diffusion-motor migration", which significantly improves the overall catalytic efficiency.

[0013] 4) Enzymatic degumming process: first, pretreat the oil, then perform enzymatic degumming. Cool the pretreated mixture to the reaction temperature, adjust the pH of the system by adding 4% (w / v) NaOH solution, then add deionized water and immobilized enzyme (phospholipase micro-motor reactor PL@Janus-HMSS-C8 / N3) according to the oil weight ratio, homogenize and mix uniformly, then continuously stir the reaction; after the reaction is completed, separate and collect the immobilized enzyme particles, wash and dry them, then repeat the above steps to perform multiple rounds of enzymatic catalytic reaction for oil degumming.

[0014] Further, 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 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°C.

[0015] Further, the pretreatment step is to heat 30 g of oil in a water bath to 70°C; then add 0.2 mL of citric acid solution (45 g / L) and homogenize at 10,000 rpm for 1 min; continuously stir the mixture at 500 rpm at 70°C for 20 min.

[0016] It should be noted that the phospholipase micro-motor reactor is converted from HMSS with chemical environment symmetry to Janus-HMSS-C8 / N3 by asymmetric grafting monomers C8 and N3, which causes different microenvironments when the enzyme is immobilized, and provides conditions for asymmetric immobilization of phospholipase; the Janus-HMSS-C8 / N3 particles have different abilities to adsorb phospholipase on both sides, which leads to asymmetric immobilization of phospholipase. Therefore, during the catalytic hydrolysis of phospholipids by phospholipase, the mechanical force imbalance is obtained, so that the micro-motor has the self-driving ability relying on the substrate concentration gradient. The PL@Janus-HMSS-C8 / N3 can stabilize the microemulsion system in the micro-water degumming system of oil, the hydrolysis product actively diffuses to the water phase, and the phospholipase micro-motor reactor actively tends to the high phospholipid concentration area at the interface, thereby improving the catalytic efficiency.

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

[0018] Further, the reaction temperature is 35-55 DEG C, the pH of the reaction system is adjusted to 4.0-4.5, the addition amount of deionized water is 0.5-2.5% of the oil weight, the addition amount of the enzyme is 15-75 mg, and the reaction time of the enzymatic degumming is 60-120 min.

[0019] Further, the degummed oil is separated from the oil phase by using a continuous centrifuge, the phospholipids are removed by washing the precipitate with a buffer solution for several times, and the immobilized enzyme is recovered. After washing with n-hexane to remove residual substrates and products, the enzyme can be recycled for more than 5 times.

[0020] The dephosphorization rate of the degummed oil prepared by the method of the application is as high as 95% or more, and when the reaction time is 60 min, the phosphorus content of ARA crude oil is reduced from 442.4 mg / kg to 9.1 mg / kg, the phosphorus content of rapeseed oil is reduced from 204.0 mg / kg to 6.9 mg / kg, the phosphorus content of soybean oil is reduced from 434.2 mg / kg to 5.8 mg / kg, the phosphorus content of corn oil is reduced from 404.2 mg / kg to 15.9 mg / kg, and the phosphorus content of walnut oil is reduced from 175.1 mg / kg to 6.0 mg / kg.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] The application provides a phospholipase micromotor reactor-based oil degumming method, the method has mild reaction conditions (35-55 DEG C, pH 4.0-4.5), the water addition amount is only 0.5-2.5% of the oil weight, the phosphorus content of various oils and crude oils can be reduced to below 16 mg / kg within 60 minutes, the immobilized enzyme can be recycled for more than 5 times, and can be recovered by simple centrifugation, has the advantages of high catalytic efficiency, strong substrate adaptability, simple operation and green environmental protection, and provides an efficient, economical and sustainable degumming solution for oil refining. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creative labor.

[0024] Figure 1 SEM (a) and TEM (b) diagrams of the carrier material obtained in Example 1 of the present application.

[0025] Figure 2 Infrared spectrograms of the present application HMSS, Janus-HMSS-N3, Janus-HMSS-C8 / N3 and PL@Janus-HMSS-C8 / N3, PLB.

[0026] Figure 3 The reusability result diagram of the enzymatic degumming in Example 4 of the present application. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0028] The special word "embodiment" here as "exemplary" explained any embodiment does not have to be interpreted as superior or better than other embodiments. In the performance index test of the embodiments of the present application, unless otherwise specified, the conventional test method in the art is adopted. It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the disclosure of the present application.

[0029] Unless otherwise defined, all 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 belongs; methods and techniques similar to but not specifically described herein are deemed to be part of the general knowledge of the skilled artisan.

[0030] For a better understanding of the present application, numerous specific details are given in the following embodiments. It will be understood by those skilled in the art that the present application can be practiced without some of the specific details, and indeed, other methods, means, instruments, devices, etc. that are well known in the art are omitted so as not to obscure the main idea of the present application.

[0031] The technical features disclosed in the embodiments of the present application can be combined arbitrarily without conflict, and the resulting technical solutions belong to the disclosure of the embodiments of the present application.

[0032] The application discloses a phospholipase micro-motor reactor-based oil degumming method.

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

[0034] Figure 1 The SEM\TEM of Janus-HMSS-C8 / N3 shows that the Janus-HMSS-C8 / N3 is a uniform sphere with a core-shell mesoporous structure, and the particle size is about 200 nm.

[0035] Figure 2 The FT-IR spectrum of Janus-HMSS-C8 / N3 shows that the absorption peak at 801 cm -1 is attributed to the symmetric stretching vibration of Si-O-Si, and the peak is detected in the HMSS material, Janus particles and immobilized enzyme sample. The wide peak at 3431 cm -1 corresponds to the N-H stretching vibration, and after the amino functionalization, the peak shape of the Janus particles in this region is obviously widened, indicating that new -NH2 groups may be introduced into the system. The characteristic peaks at 2929 cm -1 and 2852 cm -1 are respectively derived from the anti-symmetric and symmetric stretching vibration of C-H bond, which confirms that the octyl has been successfully grafted to the surface of the HMSS. After the immobilization of phospholipase B, the characteristic absorption peaks of phospholipase B such as 1100 cm -1 , 1410 cm -1 and 1652 cm -1 are observed in the infrared spectrum of the immobilized enzyme, which fully proves the successful immobilization of the enzyme.

[0036] For better understanding of the present application, the following examples are further illustrated in detail below, but can not be understood as limiting the present application, and some non-essential improvements and adjustments made by those skilled in the art according to the above content of the application are also regarded as falling within the scope of protection of the present application.

[0037] Example 1

[0038] 0.5 g of HMSS particles were added to a DDAB aqueous solution (60 mg / mL), followed by the addition of 5.0 g of solid paraffin; the mixture was heated at 80 ℃ for 30 min to completely melt the paraffin; the mixture was subjected to vigorous stirring in a homogenizer at 10,000 rpm for 160 s; after the emulsion was cooled to room temperature, the particles were separated by centrifugation and washed repeatedly with deionized water for several times, and dried at room temperature overnight to obtain paraffin@HMSS colloids.

[0039] The above colloidal particles were dispersed in a methanol solution containing 0.15 mmol of triamine (diethylenetriamine propyl trimethoxysilane), and after ultrasonic dispersion, the mixture was placed in a 30 ℃ shaking bed and oscillated at 150 rpm for 12 h; after the reaction was completed, the particles were separated by centrifugation, washed with methanol and chloroform to remove unreacted triamine and residual paraffin, and the particles were dried in a 50 °C oven for 6 hours to obtain Janus-HMSS-N3 particles; the Janus-HMSS-N3 particles were dispersed in a toluene solution containing 0.5 mmol of n-octyltrichlorosilane, and after ultrasonic dispersion, the reaction was stirred at room temperature for 2 h; after the reaction was completed, the particles were separated by centrifugation, washed with ethanol several times, and the washed particles were dried in a 50 °C oven to finally obtain Janus-HMSS-C8 / N3 particles.

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

[0041] It was determined that the loading capacity of PL@Janus-HMSS-C8 / N3 was 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 the addition of 5.0 g of solid paraffin; the mixture was heated at 80 °C for 30 min to completely melt the paraffin; the mixture was subjected to vigorous stirring in a homogenizer at 10,000 rpm for 160 s; after the emulsion was cooled to room temperature, the resulting particles were centrifugally separated, repeatedly washed with deionized water for several times, and dried at room temperature overnight to obtain paraffin@HMSS colloids.

[0044] The colloidal particles were dispersed in a methanol solution containing 0.2 mmol of triamine (diethylenetriamine propyl trimethoxysilane), and after ultrasonic dispersion, the mixture was placed in a 30 °C shaking bed and oscillated at 150 rpm for 8 h; after the reaction was completed, the particles were centrifugally separated, washed with methanol and chloroform to remove unreacted triamine and residual paraffin, and dried in a 50 °C oven 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, and after ultrasonic dispersion, the reaction was carried out at room temperature for 4 h; after the reaction was completed, the particles were centrifugally separated, washed with ethanol for several times, and the washed particles were dried in a 50 °C oven to finally obtain Janus-HMSS-C8 / N3 particles.

[0045] Phospholipase B was added to a phosphate buffer with a pH of 6.0 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 Janus-HMSS-C8 / N3, the ratio of carrier mass to enzyme solution volume was 15:1 (m / v, mg / mL), the immobilization time was 60 min, the temperature was 30 °C, and the phospholipase micro-motor reactor PL@Janus-HMSS-C8 / N3 was obtained after centrifugation and drying.

[0046] It was determined that the loading capacity of PL@Janus-HMSS-C8 / N3 was 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 the addition of 5.0 g of solid paraffin; the mixture was heated at 80 °C for 30 min to completely melt the paraffin; the mixture was subjected to vigorous stirring in a homogenizer at 10,000 rpm for 160 s; after the emulsion was cooled to room temperature, the resulting particles were centrifugally separated, repeatedly washed with deionized water for several times, and dried at room temperature overnight to obtain paraffin@HMSS colloids.

[0049] The above colloidal particles were dispersed in a methanol solution containing 0.25 mmol of triamine (diethylenetriamine propyl trimethoxysilane), and after ultrasonic dispersion, the mixture was placed in a 30 ℃ shaking bed and oscillated at 150 rpm for 10 h; after the reaction was completed, the particles were centrifuged, washed with methanol and chloroform to remove unreacted triamine and residual paraffin, and the particles were placed in a 50 °C oven for drying for 6 hours to obtain Janus-HMSS-N3 particles; the Janus-HMSS-N3 particles were dispersed in a toluene solution containing 0.25 mmol of n-octyltrichlorosilane, and after ultrasonic dispersion, the reaction was stirred at room temperature for 6 h; after the reaction was completed, the particles were centrifuged and washed with ethanol several times, and the washed particles were dried in a 50 °C oven to obtain Janus-HMSS-C8 / N3 particles.

[0050] Phospholipase B was added to a phosphate buffer with a pH of 6.0 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 Janus-HMSS-C8 / N3, the ratio of carrier mass to enzyme solution volume was 15:1 (m / v, mg / mL), the immobilization time was 60 min, the temperature was 30 ℃, and after centrifugation and drying, a phospholipase micro-motor reactor PL@Janus-HMSS-C8 / N3 was obtained.

[0051] It was determined that the loading capacity of PL@Janus-HMSS-C8 / N3 was 110.5 mg / g, and the enzyme activity was 100.0 U / g.

[0052] Example 4

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

[0054] It was determined that the degummed oil contained 6.9 mg / kg of phosphorus, and the dephosphorization rate was 96.7%.

[0055] Repeated use of immobilized enzyme PL@Janus-HMSS-C8 / N3 After 5 times, the dephosphorization rate of the crude rapeseed oil can still reach 96%.

[0056] Example 5

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

[0058] It was determined that the phosphorus content of the degummed oil was 7.2 mg / kg, and the dephosphorization rate was 96.4%.

[0059] Example 6

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

[0061] It was determined that the phosphorus content of the degummed oil was 6.5 mg / kg, and the dephosphorization rate was 96.8%.

[0062] Example 7

[0063] 30 g arachidonic acid crude oil (containing phosphorus 442.4 mg / kg) was placed in a conical 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 10,000 rpm for 1 min, and stirred at 500 rpm for 20 min at 70 ℃. After the solution was cooled to 45 ℃, 4% (w / v) NaOH solution was added to adjust the pH of the system to 4.0, and stirred at 500 rpm for 5 min. 1% deionized water and 30 mg of phospholipase micro-motor reactor prepared in Example 1 were added according to the proportion of oil weight, and homogenized. After stirring at 500 rpm for 60 min, the degummed oil and phospholipase micro-motor reactor were separated by centrifugation, and the phosphorus content was determined.

[0064] It was determined that the phosphorus content of the degummed oil was 9.1 mg / kg, and the dephosphorization rate was 97.9%.

[0065] Example 8

[0066] 30 g soybean oil crude oil (containing phosphorus 434.2 mg / kg) was placed in a conical 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 10,000 rpm for 1 min, and stirred at 500 rpm for 20 min at 70 ℃. After the solution was cooled to 45 ℃, 4% (w / v) NaOH solution was added to adjust the pH of the system to 4.0, and stirred at 500 rpm for 5 min. 1% deionized water and 30 mg of phospholipase micro-motor reactor prepared in Example 1 were added according to the proportion of oil weight, and homogenized. After stirring at 500 rpm for 60 min, the degummed oil and phospholipase micro-motor reactor were separated by centrifugation, and the phosphorus content was determined.

[0067] It was determined that the phosphorus content of the degummed oil was 5.8 mg / kg, and the dephosphorization rate was 98.6%.

[0068] Example 9

[0069] 30 g of corn oil crude oil (containing 404.2 mg / kg of phosphorus) was placed in a conical 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 10,000 rpm for 1 min, and stirred at 500 rpm for 20 min at 70 ℃. After the solution was cooled to 45 ℃, 4% (w / v) NaOH solution was added to adjust the pH of the system to 4.0, and stirred at 500 rpm for 5 min. 1% deionized water and 30 mg of phospholipase micro-motor reactor prepared in Example 1 were added according to the proportion of oil weight, and homogenized. After stirring at 500 rpm for 60 min, the degummed oil and phospholipase micro-motor reactor were separated by centrifugation, and the phosphorus content was determined.

[0070] The phosphorus content of the degummed oil was determined to be 15.9 mg / kg, and the dephosphorization rate was 96.0%.

[0071] Example 10

[0072] 30 g of walnut oil crude oil (containing 175.1 mg / kg of phosphorus) was placed in a conical 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 10,000 rpm for 1 min, and stirred at 500 rpm for 20 min at 70 ℃. After the solution was cooled to 45 ℃, 4% (w / v) NaOH solution was added to adjust the pH of the system to 4.0, and stirred at 500 rpm for 5 min. 1% deionized water and 30 mg of phospholipase micro-motor reactor prepared in Example 1 were added according to the proportion of oil weight, and homogenized. After stirring at 500 rpm for 60 min, the degummed oil and phospholipase micro-motor reactor were separated by centrifugation, and the phosphorus content was determined.

[0073] The phosphorus content of the degummed oil was determined to be 6.0 mg / kg, and the dephosphorization rate was 96.6%.

[0074] In order to further prove the beneficial effects of the present application and better understand the present application, the following comparative examples further illustrate the properties and application performance of the phospholipase micro-motor reactor-based oil degumming method disclosed in the present application, but should not be understood as limiting the present application. The method properties obtained by other determination experiments and the application according to the above properties made by those skilled in the art are also considered to fall within the scope of the present application.

[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 30 °C shaker to oscillate at 150 rpm for 12 h. After the reaction was completed, the particles were separated by centrifugation and washed with ethanol for several times. The washed particles were placed in a 50 °C oven to dry, and finally HMSS-N3 particles were obtained.

[0077] Phospholipase B was added to phosphate buffer, configured to have a protein content of 6 mg / mL enzyme solution; 30 ml of enzyme solution was mixed with 0.45 g of HMSS-N3, and after drying, phospholipase micro-motor reactor PL@HMSS-N3 was obtained.

[0078] Degumming was carried out in the same way as in Example 4, 30 mg of immobilized enzyme PL@HMSS-N3 was added, and the phosphorus content was determined after 60 min of reaction.

[0079] It was determined that the phosphorus content of the degummed oil was 15.7 mg / kg, and the dephosphorization rate was 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, stirring was carried out at room temperature for 2 h. After the reaction was completed, the particles were separated by centrifugation and washed with ethanol for several times. The washed particles were placed in a 50 °C oven to dry, and finally HMSS-C8 particles were obtained.

[0082] Phospholipase B was added to phosphate buffer, configured to have a protein content of 6 mg / mL enzyme solution; 30 ml of enzyme solution was mixed with 0.45 g of HMSS-C8, and after drying, phospholipase micro-motor reactor PL@HMSS-C8 was obtained.

[0083] Degumming was carried out in the same way as in Example 4, 30 mg of immobilized enzyme PL@HMSS-C8 was added, and the phosphorus content was determined after 60 min of reaction.

[0084] It was determined that the phosphorus content of the degummed oil was 12.2 mg / kg, and the dephosphorization rate was 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 30 °C shaker to oscillate at 150 rpm for 12 h. After the reaction was completed, the particles were separated by centrifugation and washed with ethanol for several times. The washed particles were placed in a 50 °C oven to dry, and finally HMSS-C8 / N3 particles were obtained.

[0087] Phospholipase B was added into phosphate buffer, configured as 6 mg / mL protein content of enzyme solution; 30 ml of enzyme solution was mixed with 0.45 g of HMSS-C8 / N3, and after drying, phospholipase micro-motor reactor PL@HMSS-C8 / N3 was obtained.

[0088] Degumming was carried out in the same way as in Example 4, 30 mg of immobilized enzyme PL@HMSS-C8 / N3 was added, and after 60 min of reaction, the sample was taken to determine the phosphorus content.

[0089] It was determined that the phosphorus content of the degummed oil was 14.9 mg / kg, and the dephosphorization rate was 92.7%.

[0090] Comparative Example 4

[0091] Degumming was carried out in the same way as in Example 4, 0.2 mL of free phospholipase B was added instead of immobilized enzyme, and after 60 min of reaction, the sample was taken to determine the phosphorus content.

[0092] It was determined that the phosphorus content of the degummed oil was 12.1 mg / kg, and the dephosphorization rate was 94.0%.

[0093] Table 1 Phosphorus content and dephosphorization rate of different carrier enzyme degumming

[0094] .

[0095] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended 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 were added to a double dodecyl dimethyl ammonium bromide (DDAB) aqueous solution, heated to completely melt the paraffin, homogenized to form a Pickering emulsion, and the HMSS was adsorbed at the colloidal interface; after the emulsion was cooled to room temperature, it was washed and dried to obtain paraffin@HMSS colloids; the colloidal particles were dispersed in a methanol solution containing diethylenetriamine propyl trimethoxysilane, and shaken on a shaker for reaction, washed and dried to obtain Janus-HMSS-N3 particles; the Janus-HMSS-N3 particles were dispersed in a toluene solution containing n-octyltrichlorosilane, and stirred at room temperature for reaction to graft alkyl on the other side, and obtain Janus-HMSS-C8 / N3 particles; 2) Preparation of phospholipase micro-motor reactor: the Janus-HMSS-C8 / N3 prepared in step 1) was added to a free phospholipase (PL) solution, mixed, fixed, and separated to obtain a phospholipase micro-motor reactor PL@Janus-HMSS-C8 / N3; 3) Enzymatic degumming: the pretreated oil mixture was cooled to the reaction temperature, the pH value of the system was adjusted, deionized water and the phospholipase micro-motor reactor PL@Janus-HMSS-C8 / N3 were added, and the mixture was homogenized and continuously stirred for reaction; after the reaction was completed, centrifugal separation was 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 1), the addition amount of the HMSS particles was 0.5 g, the concentration of the DDAB aqueous solution was 60 mg / mL, the addition amount of the solid paraffin was 5.0 g, the heating temperature was 80°C, and the homogenization condition was 10,000 rpm for 160 seconds.

3. The phospholipase-based micro-motor reactor-based oil degumming method according to claim 1 or 2, characterized in that, In step 1), the shaking reaction was performed at 30°C with 150 rpm for 8-12 h; the amount of diethylenetriamine propyl trimethoxysilane in the methanol solution was 0.15-0.25 mmol, and the amount of n-octyltrichlorosilane in the toluene solution was 0.25-0.5 mmol; the stirring reaction time was 2-6 h.

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

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

6. The phospholipase-based micro-motor reactor-based oil degumming method according to claim 1 or 5, characterized in that, The oil was one of arachidonic acid crude oil, rapeseed oil, corn oil, soybean oil, and walnut oil.

7. 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 enzymatic degumming is 60-120 min.

8. The phospholipase-based micro-motor reactor-based oil degumming method according to claim 1 or 7, characterized in that, In step 3), the degummed oil is separated from the oil phase by 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.

9. The phospholipase-based micro-motor reactor-based oil degumming method according to claim 1, characterized in that, After degumming, the phosphorus content of the oil is reduced to ≤16 mg / kg, and the dephosphorization rate is ≥95%. For arachidonic acid crude oil, the phosphorus content is reduced from 442.4 mg / kg to 9.1 mg / kg after 60 min of reaction; for rapeseed oil, from 204.0 mg / kg to 6.9 mg / kg; for soybean oil, from 434.2 mg / kg to 5.8 mg / kg; for corn oil, from 404.2 mg / kg to 15.9 mg / kg; and for walnut oil, from 175.1 mg / kg to 6.0 mg / kg.

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