Method for extracting phospholipid from rapeseed oil residues
Through the combination of temperature-sensitive vesicles combined with microbial treatment, the problems of low phospholipid extraction efficiency and poor purity in rapeseed oil feet are solved, and efficient and environmentally friendly phospholipid extraction is achieved and its emulsification characteristics are improved. It is suitable for food, medicine and cosmetics fields.
Patent Information
- Application Number
- CN202510642538.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-19
- Publication Date
- 2025-07-25
AI Technical Summary
The prior art is difficult to extract phospholipids from rapeseed oil feet efficiently and environmentally friendly, and the traditional methods have problems such as high solvent residues, complex process, poor selectivity and difficult purification.
The temperature-sensitive vesicles are treated with microbial treatment. The temperature-sensitive vesicles selectively adsorb phospholipids at low temperatures, and can be controlledly released at high temperatures. Combined with Yarrow's lipolytica and Rhesus opaque to degrade impurities, achieving efficient extraction of phospholipids.
It improves the extraction efficiency and purity of phospholipids, improves the emulsification characteristics of phospholipids, avoids contamination from organic solvent extraction, and enhances the emulsification stability and application effect of phospholipids.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of food processing, and particularly to a method for extracting phospholipids from rapeseed oil foots. Background Art
[0002] Rapeseed oil foots are by-products generated during the refining process of rapeseed oil, and are rich in components such as phospholipids, free fatty acids, and glycerides. As a natural active substance with high added value, phospholipids have a wide range of applications in the fields of food, medicine, cosmetics, etc., such as emulsifiers, liposome carriers, nutritional supplements, etc. Traditional phospholipid extraction methods (such as organic solvent extraction, acid / alkali treatment, column chromatography, etc.) have problems such as high solvent residues, complex processes, low recovery rates, etc., and it is difficult to selectively separate specific phospholipid components. In addition, phospholipids in rapeseed oil foots often coexist with impurities such as neutral lipids and pigments, further increasing the purification difficulty.
[0003] In the prior art, although solvent extraction methods (such as ethanol, acetone, etc.) can partially separate phospholipids, they have poor selectivity and are prone to protein denaturation; enzymatic hydrolysis methods (such as phospholipase treatment) are mild in conditions but have high costs; while intelligent materials based on temperature or pH response (such as thermosensitive polymers) show potential in the separation field, but have not been effectively applied to the extraction of phospholipids from oil foots. In addition, microbial fermentation methods can degrade oils and release phospholipids through specific strains (such as Yarrowia lipolytica, Rhodococcus opacus), but there is less research on the synergistic optimization with physical extraction technologies. Therefore, developing an efficient and environmentally friendly method for extracting rapeseed oil foot phospholipids, combining the selective capture of thermosensitive materials with microbial directional degradation, is of great significance for improving the purity, emulsifying properties, etc. of phospholipids. Summary of the Invention
[0004] Technical problems to be solved: Aiming at the above technical problems, the purpose of the present invention is to provide a method for extracting phospholipids from rapeseed oil foots, which uses thermosensitive vesicles combined with microbial treatment to synergistically improve the extraction efficiency and purity of phospholipids, and at the same time effectively improves the emulsifying properties of phospholipids; uses thermosensitive vesicles for selective adsorption and controllable release of phospholipids, adsorbing phospholipids at a low temperature of 25°C: N-isopropylacrylamide (PNIPAM) modified on the surface of thermosensitive vesicles remains in an expanded state below the phase transition temperature (35 - 40°C), showing a hydrophilic extended state, with the hydrophobic core exposed, and the vesicles are in an open state, capable of efficiently adsorbing phospholipids; releasing phospholipids by shrinking at a high temperature of 40°C: when the temperature rises to 40°C, PNIPAM undergoes a phase transition, the polymer shrinks, the hydrophobic groups expose water molecules, resulting in the volume contraction of the vesicles, thereby compressing the phospholipid molecules inside the vesicles and releasing them, achieving mild and controllable separation, improving the phospholipid yield, and avoiding the pollution caused by organic solvent extraction.
[0005] Technical solution: A method for extracting phospholipids from rapeseed oil foots, comprising the following steps: S1. Degumming treatment of rapeseed oil: Heat rapeseed oil to 60 °C, add water accounting for 2% of the mass of rapeseed oil and stir, centrifuge at 5000 - 6000 rpm for 15 min, remove the aqueous phase to obtain degummed rapeseed oil; S2. Mix the degummed rapeseed oil with an ethyl acetate - ethanol mixed solvent at a volume ratio of 1:(3 - 4) evenly to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles and 0.1 M NaCl to the diluted rapeseed oil, and oscillate at 25 °C for 2 - 3 h; S4. Centrifuge at a speed of 100,000 g for 25 - 35 min, collect the vesicles loaded with phospholipids, and wash with ethanol to remove surface non - polar impurities; S5. Resuspend the vesicles loaded with phospholipids with PBS buffer, heat to 40 °C and maintain for 30 min, centrifuge to remove the vesicles to obtain a phospholipid - enriched solution; S6. Adjust the pH of the phospholipid - enriched solution to 6.8 - 7.5, add Yarrowia lipolytica, and react at 25 - 30 °C for 2 h; S7. Centrifuge to remove the cells of Yarrowia lipolytica, adjust the pH to 7.2, inoculate Rhodococcus opacus, and oscillate at 28 °C and 200 - 300 rpm for 12 h; S8. Inactivate the cells at 90 °C, precipitate the phospholipids with cold acetone, centrifuge and then dry in vacuo to obtain phospholipids.
[0006] Further, in the ethyl acetate - ethanol mixed solvent in step S2, the volume ratio of ethyl acetate to ethanol is 4:1.
[0007] Further, in step S3, the mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is (4 - 5):1.
[0008] Further, the phase transition temperature of the thermosensitive vesicles in step S3 is 35 - 40 °C; The preparation method is as follows, by weight: S31. Take 10 - 30 mg of phosphatidylcholine with a purity of not less than 98% and dissolve it in 10 mL of chloroform, rotate and evaporate to form a uniform lipid film, and dry in vacuo; S32. Add ultrapure water at 55 - 60 °C for hydration, vortex and oscillate in a 55 °C water bath for 1 - 2 min until the solution becomes turbid, and let it stand for hydration for 30 - 40 min to form a multilamellar liposome solution; S33. Subject the multilamellar liposome solution to 3 freeze - thaw cycles, and extrude it through an extruder 10 - 15 times through a polycarbonate membrane with a pore size of 200 nm to obtain vesicles with a particle size of 100 - 200 nm; S34. Disperse the vesicles in a PBS solution containing 3 wt% - 5 wt% N-isopropylacrylamide, add 0.2 wt% - 0.5 wt% glutaraldehyde and 0.1 M HCl, adjust the pH to 4 - 5, and stir and react at 25°C in the dark for 6 - 7 h; S35. Ultracentrifuge at 10,000 rpm for 30 min to remove unreacted monomers and obtain thermosensitive vesicles.
[0009] Furthermore, the solid-liquid dosage ratio of ultrapure water in step S32 to the lipid membrane in step S31 is (5 - 20) mg: 1 mL.
[0010] Furthermore, the temperatures of the freeze-thaw cycle in step S33 are -80°C / 55°C respectively.
[0011] Furthermore, the PBS buffer solution in step S5 contains 5 mM EDTA.
[0012] Furthermore, the OD600 of Yarrowia lipolytica in step S6 is 0.6 - 1.0.
[0013] Furthermore, the OD600 of Rhodococcus opacus in step S7 is 0.8 - 0.9.
[0014] Application of the phospholipids extracted by the above extraction method in food, medicine and cosmetics.
[0015] Beneficial effects: 1. The present invention combines thermosensitive vesicles with microbial treatment, synergistically improving the extraction efficiency and purity of phospholipids, and effectively improving the emulsifying properties of phospholipids.
[0016] 2. The present invention uses thermosensitive vesicles for selective adsorption and controllable release of phospholipids. At low temperature (25°C), phospholipids are adsorbed: N-isopropylacrylamide (PNIPAM) modified on the surface of thermosensitive vesicles remains in an expanded state below the phase transition temperature (35 - 40°C), showing a hydrophilic extended state, with the hydrophobic core exposed, and the vesicles are in an open state, capable of efficiently adsorbing phospholipids; at high temperature (40°C), phospholipids are released by contraction: when the temperature rises to 40°C, PNIPAM undergoes a phase transition, the polymer contracts, the hydrophobic groups expose water molecules, resulting in the shrinkage of the vesicle volume, thus compressing the phospholipid molecules inside the vesicles and releasing them, achieving mild and controllable separation, increasing the phospholipid yield, and avoiding pollution caused by organic solvent extraction.
[0017] 3. During the preparation of thermosensitive vesicles in the present invention, the liposomes are homogenized to control the vesicle size within 100 - 200 nm, increasing the specific surface area and enhancing the phospholipid adsorption capacity.
[0018] 4. The present invention uses glutaraldehyde to crosslink PNIPAM, enabling the vesicles to maintain a stable structure even after multiple temperature cycles and being suitable for repeated use.
[0019] 5. The present invention enhances the ionic strength by adding 0.1 M NaCl, promoting the entry of phospholipids into the vesicle bilayer through hydrophobic interactions and increasing the phospholipid binding rate.
[0020] 6. In the present invention, the mixed solvent is an ethyl acetate - ethanol mixed solvent with a ratio of 4:1, which has both polar and non - polar characteristics, improving the solubility of phospholipids and reducing toxicity at the same time.
[0021] 7. The phospholipids extracted by the present invention have very good emulsifying properties. Enriched by thermosensitive vesicles, they can protect the natural structure of phospholipids, improve purity. Low - temperature adsorption can avoid the oxidation or degradation of phospholipids caused by high temperature and maintain the integrity of their amphiphilic structure; gentle phase change releases phospholipids, reducing the risk of chemical modification, ensuring emulsifying activity. At the same time, the vesicles selectively adsorb phospholipids, can remove impurities such as triglycerides, reduce competitive adsorption at the interface, and improve the emulsification efficiency. Its HLB value is more stable, can form a more compact arrangement at the oil - water interface, and enhance the stability of the emulsion. Yarrowia lipolytica can degrade saturated fatty acids (such as palmitic acid, stearic acid), increase the proportion of unsaturated fatty acids (such as oleic acid, linoleic acid), improve the fluidity of the phospholipid membrane, making it easier to spread during emulsification and reducing the interfacial tension. Rhodococcus opacus further degrades short - chain fatty acids and glycolipids, reducing the interference of small - molecule impurities on the emulsification membrane; the increase in the proportion of unsaturated fatty acids makes the emulsification membrane formed by phospholipids more flexible and stable. The phospholipids obtained by the present invention have a complete phospholipid molecular structure, can adsorb more effectively at the oil - water interface, form a stable emulsification system, and can be better applied to the food industry, pharmaceutical / cosmetic industry. Detailed implementation manners
[0022] The present invention proposes a method for extracting phospholipids from rapeseed oil foot. To make the purpose, technical solution and effects of the present invention clearer and more definite, the following will further elaborate on the present invention in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0023] Example 1 The preparation method of thermosensitive vesicles is as follows: S31. Take 20 mg of high - purity phosphatidylcholine (purity 98.6%) and dissolve it in 10 mL of chloroform, rotate and evaporate to form a uniform lipid film, and dry it under vacuum; S32. Take 5 mg of the lipid film, add 1 mL of ultrapure water at 55 °C for hydration, vortex and shake in a 55 °C water bath for 2 min until the solution becomes turbid, and let it stand for 35 min for hydration to form a multi - vesicular liposome solution; S33. The multi-chamber liposome solution was subjected to 3 freeze-thaw cycles (-80 °C / 55 °C) and extruded 15 times through an extruder with a polycarbonate membrane having a pore size of 200 nm to obtain vesicles with a particle size of 100 - 200 nm; S34. The vesicles were dispersed in a PBS solution containing 4 wt% N-isopropylacrylamide, 0.3 wt% glutaraldehyde and 0.1 M HCl were added, the pH was adjusted to 4.5, and the reaction was stirred in the dark at 25 °C for 7 h; S35. Ultracentrifugation was carried out at 10,000 rpm for 30 min to remove unreacted monomers and obtain temperature-sensitive vesicles.
[0024] Example 2 The preparation method of the temperature-sensitive vesicles is as follows: S31. Take 20 mg of high-purity phosphatidylcholine (purity 98.6%) and dissolve it in 10 mL of chloroform, rotary evaporate to form a uniform lipid film, and vacuum dry; S32. Take 10 mg of the lipid film, add 1 mL of ultrapure water at 55 °C for hydration, vortex and shake in a 55 °C water bath for 2 min until the solution becomes turbid, and let it stand for 35 min for hydration to form a multi-chamber liposome solution; S33. The multi-chamber liposome solution was subjected to 3 freeze-thaw cycles (-80 °C / 55 °C) and extruded 15 times through an extruder with a polycarbonate membrane having a pore size of 200 nm to obtain vesicles with a particle size of 100 - 200 nm; S34. The vesicles were dispersed in a PBS solution containing 4 wt% N-isopropylacrylamide, 0.4 wt% glutaraldehyde and 0.1 M HCl were added, the pH was adjusted to 4.5, and the reaction was stirred in the dark at 25 °C for 7 h; S35. Ultracentrifugation was carried out at 10,000 rpm for 30 min to remove unreacted monomers and obtain temperature-sensitive vesicles.
[0025] Example 3 The preparation method of the temperature-sensitive vesicles is as follows: S31. Take 20 mg of high-purity phosphatidylcholine (purity 98.6%) and dissolve it in 10 mL of chloroform, rotary evaporate to form a uniform lipid film, and vacuum dry; S32. Take 10 mg of the lipid film, add 1 mL of ultrapure water at 55 °C for hydration, vortex and shake in a 55 °C water bath for 2 min until the solution becomes turbid, and let it stand for 35 min for hydration to form a multi-chamber liposome solution; S33. The multi-chamber liposome solution was subjected to 3 freeze-thaw cycles (-80 °C / 55 °C) and extruded 15 times through an extruder with a polycarbonate membrane having a pore size of 200 nm to obtain vesicles with a particle size of 100 - 200 nm; S34. Disperse the vesicles in a PBS solution containing 3 wt% N - isopropylacrylamide, add 0.5 wt% glutaraldehyde and 0.1 M HCl, adjust the pH to 4.5, and stir the reaction in the dark at 25 °C for 7 h; S35. Ultra - centrifuge at 10,000 rpm for 30 min to remove unreacted monomers and obtain temperature - sensitive vesicles.
[0026] Example 4 The preparation method of the temperature - sensitive vesicles is as follows: S31. Take 10 mg of high - purity phosphatidylcholine (purity 98.6%) and dissolve it in 10 mL of chloroform. Rotate and evaporate to form a uniform lipid film, and then dry it under vacuum; S32. Take 10 mg of the lipid film, add 1 mL of ultrapure water at 55 °C for hydration. Vortex and oscillate in a 55 °C water bath for 2 min until the solution becomes turbid, and then let it stand for 35 min for hydration to form a multi - vesicular liposome solution; S33. Subject the multi - vesicular liposome solution to 3 freeze - thaw cycles (-80 °C / 55 °C), and extrude it 15 times through an extruder with a polycarbonate membrane with a pore size of 200 nm to obtain vesicles with a particle size of 100 - 200 nm; S34. Disperse the vesicles in a PBS solution containing 5 wt% N - isopropylacrylamide, add 0.4 wt% glutaraldehyde and 0.1 M HCl, adjust the pH to 4.5, and stir the reaction in the dark at 25 °C for 7 h; S35. Ultra - centrifuge at 10,000 rpm for 30 min to remove unreacted monomers and obtain temperature - sensitive vesicles.
[0027] Example 5 The preparation method of the temperature - sensitive vesicles is as follows: S31. Take 30 mg of high - purity phosphatidylcholine (purity 98.6%) and dissolve it in 10 mL of chloroform. Rotate and evaporate to form a uniform lipid film, and then dry it under vacuum; S32. Take 20 mg of the lipid film, add 1 mL of ultrapure water at 55 °C for hydration. Vortex and oscillate in a 55 °C water bath for 2 min until the solution becomes turbid, and then let it stand for 35 min for hydration to form a multi - vesicular liposome solution; S33. Subject the multi - vesicular liposome solution to 3 freeze - thaw cycles (-80 °C / 55 °C), and extrude it 15 times through an extruder with a polycarbonate membrane with a pore size of 200 nm to obtain vesicles with a particle size of 100 - 200 nm; S34. Disperse the vesicles in a PBS solution containing 4 wt% N - isopropylacrylamide, add 0.4 wt% glutaraldehyde and 0.1 M HCl, adjust the pH to 4.5, and stir the reaction in the dark at 25 °C for 7 h; Centrifuge at 10,000 rpm for 30 min to remove unreacted monomers and obtain temperature-sensitive vesicles.
[0028] Example 6 The preparation method of temperature-sensitive vesicles is as follows: S31. Dissolve 30 mg of high-purity phosphatidylcholine (purity 98%) in 10 mL of chloroform, rotate and evaporate to form a uniform lipid film, and dry in vacuum; S32. Take 20 mg of the lipid film, add 1 mL of ultrapure water at 58 °C for hydration, vortex and shake in a water bath at 55 °C for 1.5 min until the solution becomes turbid, and let it stand for 30 min for hydration to form a multi-lamellar liposome solution; S33. Subject the multi-lamellar liposome solution to 3 cycles of freezing-thawing (-80 °C / 55 °C), and extrude it 12 times through an extruder with a polycarbonate membrane with a pore size of 200 nm to obtain vesicles with a particle size of 100 - 200 nm; S34. Disperse the vesicles in a PBS solution containing 4 wt% N-isopropylacrylamide, add 0.2 wt% glutaraldehyde and 0.1 M HCl, adjust the pH to 4.5, and stir and react in the dark at 25 °C for 7 h; S35. Centrifuge at 10,000 rpm for 30 min to remove unreacted monomers and obtain temperature-sensitive vesicles.
[0029] Example 7 The preparation method of temperature-sensitive vesicles is as follows: S31. Dissolve 30 mg of high-purity phosphatidylcholine (purity 98%) in 10 mL of chloroform, rotate and evaporate to form a uniform lipid film, and dry in vacuum; S32. Take 20 mg of the lipid film, add 1 mL of ultrapure water at 60 °C for hydration, vortex and shake in a water bath at 55 °C for 1 min until the solution becomes turbid, and let it stand for 40 min for hydration to form a multi-lamellar liposome solution; S33. Subject the multi-lamellar liposome solution to 3 cycles of freezing-thawing (-80 °C / 55 °C), and extrude it 10 times through an extruder with a polycarbonate membrane with a pore size of 200 nm to obtain vesicles with a particle size of 100 - 200 nm; S34. Disperse the vesicles in a PBS solution containing 4 wt% N-isopropylacrylamide, add 0.2 wt% glutaraldehyde and 0.1 M HCl, adjust the pH to 5, and stir and react in the dark at 25 °C for 6 h; S35. Centrifuge at 10,000 rpm for 30 min to remove unreacted monomers and obtain temperature-sensitive vesicles.
[0030] Comparative Example 1 The difference between this comparative example and Example 5 is as follows: A PBS solution containing 1% N-isopropylacrylamide was used, specifically as follows: The preparation method of the temperature-sensitive vesicles is as follows: S31. Dissolve 30 mg of high-purity phosphatidylcholine (purity 98.6%) in 10 mL of chloroform, rotate and evaporate to form a uniform lipid film, and dry it under vacuum; S32. Take 20 mg of the lipid film, add 1 mL of ultrapure water at 55 °C for hydration, vortex and shake in a water bath at 55 °C for 2 min until the solution becomes turbid, and let it stand for 35 min for hydration to form a multi-lamellar liposome solution; S33. Subject the multi-lamellar liposome solution to three freeze-thaw cycles (-80 °C / 55 °C), and extrude it through an extruder 15 times through a polycarbonate membrane with a pore size of 200 nm to obtain vesicles with a particle size of 100-200 nm; S34. Disperse the vesicles in a PBS solution containing 1 wt% N-isopropylacrylamide, add 0.4 wt% glutaraldehyde and 0.1 M HCl, adjust the pH to 4.5, and stir and react in the dark at 25 °C for 7 h; S35. Ultracentrifuge at 10,000 rpm for 30 min to remove unreacted monomers and obtain temperature-sensitive vesicles.
[0031] Comparative Example 2 The difference between this comparative example and Example 5 is as follows: The hydration temperature in step S32 is 45 °C, specifically as follows: The preparation method of the temperature-sensitive vesicles is as follows: S31. Dissolve 30 mg of high-purity phosphatidylcholine (purity 98.6%) in 10 mL of chloroform, rotate and evaporate to form a uniform lipid film, and dry it under vacuum; S32. Take 20 mg of the lipid film, add 1 mL of ultrapure water at 45 °C for hydration, vortex and shake in a water bath at 45 °C for 2 min until the solution becomes turbid, and let it stand for 35 min for hydration to form a multi-lamellar liposome solution; S33. Subject the multi-lamellar liposome solution to three freeze-thaw cycles (-80 °C / 45 °C), and extrude it through an extruder 15 times through a polycarbonate membrane with a pore size of 200 nm to obtain vesicles with a particle size of 100-200 nm; S34. Disperse the vesicles in a PBS solution containing 4 wt% N-isopropylacrylamide, add 0.4 wt% glutaraldehyde and 0.1 M HCl, adjust the pH to 4.5, and stir and react in the dark at 25 °C for 7 h; S35. Ultracentrifuge at 10,000 rpm for 30 min to remove unreacted monomers and obtain temperature-sensitive vesicles.
[0032] Comparative Example 3 The difference between this comparative example and Example 5 is that glutaraldehyde is not added, specifically as follows: The preparation method of the thermosensitive vesicles is as follows: S31. Take 30 mg of high-purity phosphatidylcholine (purity 98.6%) and dissolve it in 10 mL of chloroform, rotate and evaporate to form a uniform lipid film, and dry it under vacuum; S32. Take 20 mg of the lipid film, add 1 mL of ultrapure water at 55 °C for hydration, vortex and shake in a 55 °C water bath for 2 min until the solution becomes turbid, and let it stand for 35 min for hydration to form a multi-lamellar liposome solution; S33. Subject the multi-lamellar liposome solution to freeze-thaw cycles (-80 °C / 55 °C) 3 times, and extrude it 15 times through an extruder with a polycarbonate membrane with a pore size of 200 nm to obtain vesicles with a particle size of 100 - 200 nm; S34. Disperse the vesicles in a PBS solution containing 4 wt% N-isopropylacrylamide, add 0.1 M HCl, adjust the pH to 4.5, and stir and react in the dark at 25 °C for 7 h; S35. Ultracentrifuge at 10,000 rpm for 30 min to remove unreacted monomers and obtain thermosensitive vesicles.
[0033] Comparative Example 4 The difference between this comparative example and Example 5 is that the freeze-thaw cycle is 1 time, specifically as follows: The preparation method of the thermosensitive vesicles is as follows: S31. Take 30 mg of high-purity phosphatidylcholine (purity 98.6%) and dissolve it in 10 mL of chloroform, rotate and evaporate to form a uniform lipid film, and dry it under vacuum; S32. Take 20 mg of the lipid film, add 1 mL of ultrapure water at 55 °C for hydration, vortex and shake in a 55 °C water bath for 2 min until the solution becomes turbid, and let it stand for 35 min for hydration to form a multi-lamellar liposome solution; S33. Subject the multi-lamellar liposome solution to freeze-thaw cycles (-80 °C / 55 °C) 1 time, and extrude it 15 times through an extruder with a polycarbonate membrane with a pore size of 200 nm to obtain vesicles with a particle size of 100 - 200 nm; S34. Disperse the vesicles in a PBS solution containing 4 wt% N-isopropylacrylamide, add 0.4 wt% glutaraldehyde and 0.1 M HCl, adjust the pH to 4.5, and stir and react in the dark at 25 °C for 7 h; S35. Ultracentrifuge at 10,000 rpm for 30 min to remove unreacted monomers and obtain thermosensitive vesicles.
[0034] Performance measurement: (1) Phase transition temperature: Detection method: DLS temperature ramp scanning, observe the sudden change in particle size; (2) Phospholipid loading efficiency: Detection method: Ultraviolet spectrophotometry (measuring the phospholipid content in vesicles); Phosphomolybdic acid colorimetry (total phospholipid quantification), calculation formula: ; (3) Temperature-controlled release performance: Detection method: Dialysis bag method, measuring the release rate within 2 hours at 40 °C; (4) Recycling performance: Measuring the retention rate of phospholipid adsorption after 5 cycles of use, and the results are shown in Table 1 below: Table 1
[0035] As can be seen from Table 1 above, in Comparative Example 1, since the NIPAM concentration was reduced to 1 wt%, the phase transition hysteresis occurred due to the decrease in the thermosensitive polymer, resulting in an increase in the phase transition temperature; due to the decrease in the crosslinking density, the loading efficiency decreased; due to the weakening of the thermosensitive response, the release rate decreased significantly; due to insufficient structural stability, the recycling retention rate decreased (75.8%). In Comparative Example 2, the hydration temperature was 45 °C, and due to the low temperature, the lipid membrane was not fully hydrated, resulting in a significant reduction in the phospholipid loading efficiency; due to the loose vesicle structure, the phase transition temperature became higher; due to the poor integrity of the vesicles, the release rate and recycling retention rate decreased. In Comparative Example 3, without glutaraldehyde, due to the lack of effective crosslinking, the phase transition temperature could not be measured (the thermosensitivity was lost); due to the lack of fixation of phospholipids, the loading efficiency was extremely low; due to the inability of the vesicles to exist stably, the recycling retention rate was only 20.1%. In Comparative Example 4, freeze-thawing was carried out once, and due to insufficient liposome encapsulation, the loading efficiency and recycling retention rate decreased, and due to the poor homogeneity of the vesicles, the release rate decreased.
[0036] After comparing the characteristics of the thermosensitive vesicles in each example, the thermosensitive vesicles prepared in Example 5 were selected for subsequent phospholipid extraction experiments.
[0037] Example 8 A method for extracting phospholipids from rapeseed oil foot, comprising the following steps: S1. Take 50 g of rapeseed oil, heat it to 60 °C, add 1 g of water and stir, centrifuge at 5500 rpm for 15 min, remove the aqueous phase, and obtain degummed rapeseed oil; S2. Mix 10 mL of degummed rapeseed oil with 35 mL of an ethyl acetate-ethanol mixed solvent, and the volume ratio of ethyl acetate to ethanol in the ethyl acetate-ethanol mixed solvent is 4:1, to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles to the diluted rapeseed oil, and the mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is 4:1, 0.1 M NaCl, and shake at 25 °C for 2.5 h; S4. Centrifuge at 100,000 g for 30 min, collect the vesicles loaded with phospholipids, and wash with ethanol to remove surface non-polar impurities; S5. Resuspend the phospholipid-loaded vesicles with PBS buffer containing 5 mM EDTA, heat to 40 °C and maintain for 30 min, centrifuge to remove the vesicles, and obtain a phospholipid-enriched solution; S6. Adjust the pH of the phospholipid-enriched solution to 7.0, add Yarrowia lipolytica with OD 600 = 0.6, and react at 28 °C for 2 h; S7. Centrifuge to remove the cells of Yarrowia lipolytica, adjust the pH of the released solution to 7.2, inoculate Rhodococcus opacus with OD 600 = 0.9, and shake at 28 °C and 250 rpm for 12 h; S8. Inactivate the cells at 90 °C, precipitate the phospholipids with cold acetone, centrifuge and dry in vacuo to obtain the phospholipids.
[0038] Example 9 A method for extracting phospholipids from rapeseed oil foots, comprising the following steps: S1. Take 50 g of rapeseed oil, heat to 60 °C, add 1 g of water and stir, centrifuge at 5500 rpm for 15 min, remove the aqueous phase, and obtain degummed rapeseed oil; S2. Mix 10 mL of degummed rapeseed oil with 35 mL of an ethyl acetate-ethanol mixed solvent evenly. The volume ratio of ethyl acetate to ethanol in the ethyl acetate-ethanol mixed solvent is 4:1 to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles to the diluted rapeseed oil. The mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is 4:1, 0.1 M NaCl, and shake at 25 °C for 2.5 h; S4. Centrifuge at 100,000 g for 30 min, collect the phospholipid-loaded vesicles, and wash with ethanol to remove surface non-polar impurities; S5. Resuspend the phospholipid-loaded vesicles with PBS buffer containing 5 mM EDTA, heat to 40 °C and maintain for 30 min, centrifuge to remove the vesicles, and obtain a phospholipid-enriched solution; S6. Adjust the pH of the phospholipid-enriched solution to 7.0, add Yarrowia lipolytica with OD 600 = 0.9, and react at 28 °C for 2 h; S7. Centrifuge to remove the cells of Yarrowia lipolytica, adjust the pH of the released solution to 7.2, inoculate Rhodococcus opacus with OD 600 = 0.9, and shake at 28 °C and 250 rpm for 12 h; S8. Inactivate the cells at 90 °C, precipitate the phospholipids with cold acetone, centrifuge and dry in vacuo to obtain the phospholipids.
[0039] Example 10 A method for extracting phospholipids from rapeseed oil foots, comprising the following steps: S1. Take 50 g of rapeseed oil, heat it to 60 °C, add 1 g of water and stir. Centrifuge at 5500 rpm for 15 min, remove the aqueous phase to obtain degummed rapeseed oil; S2. Mix 10 mL of degummed rapeseed oil with 35 mL of ethyl acetate - ethanol mixed solvent evenly. The volume ratio of ethyl acetate to ethanol in the ethyl acetate - ethanol mixed solvent is 4:1 to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles to the diluted rapeseed oil. The mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is 5:1, 0.1 M NaCl, and shake at 25 °C for 2.5 h; S4. Centrifuge at 100,000 g for 30 min, collect the vesicles loaded with phospholipids, and wash with ethanol to remove surface non - polar impurities; S5. Resuspend the vesicles loaded with phospholipids with PBS buffer containing 5 mM EDTA, heat to 40 °C and maintain for 30 min, then centrifuge to remove the vesicles to obtain a phospholipid - enriched solution; S6. Adjust the pH of the phospholipid - enriched solution to 7.0, add Yarrowia lipolytica with OD 600 = 0.9, and react at 28 °C for 2 h; S7. Centrifuge to remove the cells of Yarrowia lipolytica, adjust the pH of the released solution to 7.2, inoculate Rhodococcus opacus with OD 600 = 0.8, and shake at 28 °C and 250 rpm for 12 h; S8. Inactivate the cells at 90 °C, precipitate the phospholipids with cold acetone, centrifuge and then dry in vacuo to obtain phospholipids.
[0040] Example 11 A method for extracting phospholipids from rapeseed oil foot, comprising the following steps: S1. Take 50 g of rapeseed oil, heat it to 60 °C, add 1 g of water and stir. Centrifuge at 5500 rpm for 15 min, remove the aqueous phase to obtain degummed rapeseed oil; S2. Mix 10 mL of degummed rapeseed oil with 30 mL of ethyl acetate - ethanol mixed solvent evenly. The volume ratio of ethyl acetate to ethanol in the ethyl acetate - ethanol mixed solvent is 4:1 to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles to the diluted rapeseed oil. The mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is 4:1, 0.1 M NaCl, and shake at 25 °C for 2.5 h; S4. Centrifuge at 100,000 g for 30 min, collect the vesicles loaded with phospholipids, and wash with ethanol to remove surface non - polar impurities; S5. Resuspend the vesicles loaded with phospholipids with PBS buffer containing 5 mM EDTA, heat to 40 °C and maintain for 30 min, then centrifuge to remove the vesicles to obtain a phospholipid - enriched solution; S6. Adjust the pH of the phospholipid-enriched solution to 7.0, add Yarrowia lipolytica with OD 600 = 0.9, and react at 28 °C for 2 h; S7. Centrifuge to remove the cells of Yarrowia lipolytica, adjust the pH of the released solution to 7.2, inoculate Rhodococcus opacus with OD 600 = 0.8, and shake at 28 °C and 250 rpm for 12 h; S8. Inactivate the cells at 90 °C, precipitate the phospholipids with cold acetone, centrifuge and then dry in vacuo to obtain the phospholipids.
[0041] Example 12 A method for extracting phospholipids from rapeseed oil foot, comprising the following steps: S1. Take 50 g of rapeseed oil, heat it to 60 °C, add 1 g of water and stir, centrifuge at 5500 rpm for 15 min, remove the aqueous phase to obtain degummed rapeseed oil; S2. Mix 10 mL of degummed rapeseed oil with 40 mL of an ethyl acetate-ethanol mixed solvent evenly. The volume ratio of ethyl acetate to ethanol in the ethyl acetate-ethanol mixed solvent is 4:1 to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles to the diluted rapeseed oil. The mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is 4:1, 0.1 M NaCl, and shake at 25 °C for 2.5 h; S4. Centrifuge at a speed of 100,000 g for 30 min, collect the vesicles loaded with phospholipids, and wash with ethanol to remove surface non-polar impurities; S5. Resuspend the vesicles loaded with phospholipids with a PBS buffer solution containing 5 mM EDTA, heat to 40 °C and maintain for 30 min, centrifuge to remove the vesicles to obtain a phospholipid-enriched solution; S6. Adjust the pH of the phospholipid-enriched solution to 7.0, add Yarrowia lipolytica with OD 600 = 0.8, and react at 28 °C for 2 h; S7. Centrifuge to remove the cells of Yarrowia lipolytica, adjust the pH of the released solution to 7.2, inoculate Rhodococcus opacus with OD 600 = 0.9, and shake at 28 °C and 250 rpm for 12 h; S8. Inactivate the cells at 90 °C, precipitate the phospholipids with cold acetone, centrifuge and then dry in vacuo to obtain the phospholipids.
[0042] Example 13 A method for extracting phospholipids from rapeseed oil foot, comprising the following steps: S1. Take 50 g of rapeseed oil, heat it to 60 °C, add 1 g of water and stir, centrifuge at 5500 rpm for 15 min, remove the aqueous phase to obtain degummed rapeseed oil; S2. Mix 10 mL of degummed rapeseed oil with 35 mL of ethyl acetate - ethanol mixed solvent evenly. The volume ratio of ethyl acetate to ethanol in the ethyl acetate - ethanol mixed solvent is 4:1 to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles to the diluted rapeseed oil. The mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is 4:1, 0.1 M NaCl, and shake at 25 °C for 2.5 h; S4. Centrifuge at a speed of 100,000 g for 30 min, collect the vesicles loaded with phospholipids, and wash with ethanol to remove surface non - polar impurities; S5. Resuspend the vesicles loaded with phospholipids with PBS buffer containing 5 mM EDTA, heat to 40 °C and maintain for 30 min, then centrifuge to remove the vesicles to obtain a phospholipid - enriched solution; S6. Adjust the pH of the phospholipid - enriched solution to 7.0, add Yarrowia lipolytica with OD 600 = 1.0, and react at 28 °C for 2 h; S7. Centrifuge to remove the cells of Yarrowia lipolytica, adjust the pH of the release solution to 7.2, inoculate Rhodococcus opacus with OD 600 = 0.9, and shake at 28 °C and 250 rpm for 12 h; S8. Inactivate the cells at 90 °C, precipitate the phospholipids with cold acetone, centrifuge and then dry in vacuum to obtain phospholipids.
[0043] Example 14 A method for extracting phospholipids from rapeseed oil foot, comprising the following steps: S1. Take 50 g of rapeseed oil, heat it to 60 °C, add 1 g of water and stir, centrifuge at 5000 rpm for 15 min, remove the aqueous phase to obtain degummed rapeseed oil; S2. Mix 10 mL of degummed rapeseed oil with 35 mL of ethyl acetate - ethanol mixed solvent evenly. The volume ratio of ethyl acetate to ethanol in the ethyl acetate - ethanol mixed solvent is 4:1 to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles to the diluted rapeseed oil. The mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is 4:1, 0.1 M NaCl, and shake at 25 °C for 2 h; S4. Centrifuge at a speed of 100,000 g for 25 min, collect the vesicles loaded with phospholipids, and wash with ethanol to remove surface non - polar impurities; S5. Resuspend the vesicles loaded with phospholipids with PBS buffer containing 5 mM EDTA, heat to 40 °C and maintain for 30 min, then centrifuge to remove the vesicles to obtain a phospholipid - enriched solution; S6. Adjust the pH of the phospholipid - enriched solution to 6.8, add Yarrowia lipolytica with OD 600 = 1.0, and react at 25 °C for 2 h; S7. Centrifuge to remove Yarrowia lipolytica cells, adjust the pH of the release solution to 7.2, inoculate Rhodococcus opacus with OD 600 = 0.9, and shake at 28 °C and 200 rpm for 12 h; S8. Inactivate the cells at 90 °C, precipitate phospholipids with cold acetone, centrifuge and then dry under vacuum to obtain phospholipids.
[0044] Example 15 A method for extracting phospholipids from rapeseed oil foots, comprising the following steps: S1. Take 50 g of rapeseed oil, heat it to 60 °C, add 1 g of water and stir, centrifuge at 6000 rpm for 15 min, remove the aqueous phase to obtain degummed rapeseed oil; S2. Mix 10 mL of degummed rapeseed oil with 35 mL of an ethyl acetate-ethanol mixed solvent evenly. The volume ratio of ethyl acetate to ethanol in the ethyl acetate-ethanol mixed solvent is 4:1 to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles to the diluted rapeseed oil. The mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is 4:1, 0.1 M NaCl, and shake at 25 °C for 3 h; S4. Centrifuge at a speed of 100,000 g for 35 min, collect the vesicles loaded with phospholipids, and wash with ethanol to remove surface non-polar impurities; S5. Resuspend the vesicles loaded with phospholipids with PBS buffer containing 5 mM EDTA, warm up to 40 °C and maintain for 30 min, centrifuge to remove the vesicles to obtain a phospholipid-enriched solution; S6. Adjust the pH of the phospholipid-enriched solution to 7.5, add Yarrowia lipolytica with OD 600 = 1.0, and react at 30 °C for 2 h; S7. Centrifuge to remove Yarrowia lipolytica cells, adjust the pH of the release solution to 7.2, inoculate Rhodococcus opacus with OD 600 = 0.9, and shake at 28 °C and 300 rpm for 12 h; S8. Inactivate the cells at 90 °C, precipitate phospholipids with cold acetone, centrifuge and then dry under vacuum to obtain phospholipids.
[0045] Comparative Example 5 The difference between this comparative example and Example 13 is that thermosensitive vesicles and biological treatment steps are not used, and the details are as follows: A method for extracting phospholipids from rapeseed oil foots, comprising the following steps: S1. Take 50 g of rapeseed oil, heat it to 60 °C, add 1 g of water and stir, centrifuge at 5500 rpm for 15 min to obtain degummed rapeseed oil; S2. Directly precipitate phospholipids with cold acetone; S3. Centrifuge and then dry under vacuum to obtain phospholipids.
[0046] Comparative Example 6 The difference between this comparative example and Example 13 is that thermosensitive vesicles are not used for treatment, and the specific steps are as follows: The method for extracting phospholipids from rapeseed oil foot includes the following steps: S1. Take 50 g of rapeseed oil, heat it to 60 °C, add 1 g of water and stir, centrifuge at 5500 rpm for 15 min, remove the aqueous phase, and obtain degummed rapeseed oil; S2. Add PBS buffer, adjust the pH to 7.0, add Yarrowia lipolytica with OD 600 = 0.6, and react at 28 °C for 2 h; S3. Centrifuge to remove the cells of Yarrowia lipolytica, adjust the pH of the released solution to 7.2, inoculate Rhodococcus opacus with OD 600 = 0.9, and shake at 28 °C and 250 rpm for 12 h; S4. Inactivate the cells at 90 °C, precipitate the phospholipids with cold acetone, centrifuge and then dry in vacuo to obtain phospholipids.
[0047] Comparative Example 7 The difference between this comparative example and Example 13 is that Yarrowia lipolytica is not used for treatment, and the specific steps are as follows: The method for extracting phospholipids from rapeseed oil foot includes the following steps: S1. Take 50 g of rapeseed oil, heat it to 60 °C, add 1 g of water and stir, centrifuge at 5500 rpm for 15 min, remove the aqueous phase, and obtain degummed rapeseed oil; S2. Mix 10 mL of degummed rapeseed oil with 35 mL of ethyl acetate-ethanol mixed solvent evenly. The volume ratio of ethyl acetate to ethanol in the ethyl acetate-ethanol mixed solvent is 4:1 to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles to the diluted rapeseed oil. The mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is 4:1, 0.1 M NaCl, and shake at 25 °C for 2.5 h; S4. Centrifuge at 100,000 g for 30 min, collect the vesicles loaded with phospholipids, and wash with ethanol to remove surface non-polar impurities; S5. Resuspend the vesicles loaded with phospholipids with PBS buffer containing 5 mM EDTA, heat to 40 °C and maintain for 30 min, centrifuge to remove the vesicles, and obtain a phospholipid enrichment solution; S6. Adjust the pH of the phospholipid enrichment solution to 7.2, inoculate Rhodococcus opacus with OD 600 = 0.9, and shake at 28 °C and 250 rpm for 12 h; S7. Inactivate the cells at 90 °C, precipitate the phospholipids with cold acetone, centrifuge and then dry in vacuo to obtain phospholipids.
[0048] Comparative Example 8 The difference between this comparative example and Example 13 is that no Rhodococcus opacus treatment is adopted, specifically as follows: The method for extracting phospholipids from rapeseed oil foot includes the following steps: S1. Take 50 g of rapeseed oil, heat it to 60 °C, add 1 g of water and stir, centrifuge at 5500 rpm for 15 min, remove the aqueous phase, and obtain degummed rapeseed oil; S2. Mix 10 mL of degummed rapeseed oil with 35 mL of ethyl acetate - ethanol mixed solvent evenly. The volume ratio of ethyl acetate to ethanol in the ethyl acetate - ethanol mixed solvent is 4:1 to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles to the diluted rapeseed oil. The mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is 4:1, 0.1 M NaCl, and shake at 25 °C for 2.5 h; S4. Centrifuge at 100,000 g for 30 min, collect the vesicles loaded with phospholipids, and wash with ethanol to remove surface non - polar impurities; S5. Resuspend the vesicles loaded with phospholipids with PBS buffer containing 5 mM EDTA, raise the temperature to 40 °C and maintain for 30 min, centrifuge to remove the vesicles, and obtain a phospholipid - enriched solution; S6. Adjust the pH of the phospholipid - enriched solution to 7.0, add Yarrowia lipolytica with OD 600 = 0.6, and react at 28 °C for 2 h; S7. Centrifuge to remove the Yarrowia lipolytica cells, inactivate the cells at 90 °C, precipitate the phospholipids with cold acetone, centrifuge and then dry in vacuo to obtain phospholipids.
[0049] Comparative Example 9 The difference between this comparative example and Example 13 is that no treatment with two kinds of bacteria is adopted, specifically as follows: The method for extracting phospholipids from rapeseed oil foot includes the following steps: S1. Take 50 g of rapeseed oil, heat it to 60 °C, add 1 g of water and stir, centrifuge at 5500 rpm for 15 min, remove the aqueous phase, and obtain degummed rapeseed oil; S2. Mix 10 mL of degummed rapeseed oil with 35 mL of ethyl acetate - ethanol mixed solvent evenly. The volume ratio of ethyl acetate to ethanol in the ethyl acetate - ethanol mixed solvent is 4:1 to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles to the diluted rapeseed oil. The mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is 4:1, 0.1 M NaCl, and shake at 25 °C for 2.5 h; S4. Centrifuge at 100,000 g for 30 min, collect the vesicles loaded with phospholipids, and wash with ethanol to remove surface non - polar impurities; S5. Resuspend the phospholipid-loaded vesicles with PBS buffer containing 5 mM EDTA, heat to 40 °C and maintain for 30 min, centrifuge to remove the vesicles, and obtain a phospholipid-enriched solution; S6. Adjust the pH to 9.0 with 0.1 M NaOH solution and hydrolyze at 60 °C for 1 h; S8. Neutralize the pH, precipitate the phospholipids with cold acetone, centrifuge and dry in vacuo to obtain phospholipids.
[0050] Performance test: Phospholipid purity test method: High performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD); Phospholipid recovery rate: Mass balance method Calculation formula: ; Fatty acid residue detection method: Gas chromatography-mass spectrometry (GC-MS), and the results are shown in Table 2 below: Table 2
[0051] As can be seen from Table 2 above, Examples 8-13 all showed high purity (>96%) and high recovery rate (>93%), and Example 13 was the best. The phospholipid purity (78.6%) and recovery rate (70.2%) of Comparative Example 5 were the lowest, and the fatty acid residue was as high as 52.7 ppm. The purity of Comparative Example 6 decreased to 85.4%, indicating that the enrichment effect of vesicles on phospholipids was significant. The fatty acid residues of Comparative Examples 7-8 (19.3-25.1 ppm) were higher than that of Example 13 (11.9 ppm), proving that the synergistic degradation of the two bacteria could more thoroughly remove fatty acids with different chain lengths. The purity (89.1%) and fatty acid residue (35.9 ppm) of Comparative Example 9 were lower than those of the Examples, proving that Yarrowia lipolytica and Rhodococcus opacus played a key role in removing fatty acids, and the chemical method could not achieve the directional degradation effect of biological treatment.
[0052] Determination method of peroxide value (PV): Accelerated oxidation: Store in an oven at 60 °C for 7 days, take 1 g of phospholipids and dissolve them in 30 mL of acetic acid-isooctane (3:2), add 0.5 mL of saturated KI solution, react in the dark for 1 min, add 30 mL of distilled water, and titrate with starch indicator until the blue color disappears. The calculation formula is:
[0053] Determination method of thiobarbituric acid reactive substances (TBARS): Take 0.5 g of phospholipids and 5 mL of 0.02 M TBA solution (containing 0.2% BHT), boil in a water bath for 40 min, cool and measure the absorbance at 532 nm. Using malondialdehyde as the standard curve, the results are expressed as μM malondialdehyde equivalent / g sample; the results are shown in Table 3 below: Table 3
[0054] As can be seen from Table 3 above, the peroxide values (3.85, 2.67 meq / kg) and TBARS (2.32, 1.14 μM / g) of Comparative Examples 5-6 are significantly higher than those of Example 13 (1.09 meq / kg, 0.35 μM / g), indicating that the low-temperature operation of thermosensitive vesicles and mild treatment of microorganisms can effectively protect phospholipids from oxidative damage; from Comparative Examples 7-9, it can be seen that Yarrowia lipolytica secretes lipase to specifically hydrolyze short-chain fatty acids (C12-C16), while Rhodococcus mainly degrades long chains (C18+), and the two bacteria cover the entire chain length range.
[0055] Emulsion preparation: 1% phospholipid solution (w / v) and soybean oil (1:1, v / v), homogenize at 10,000 rpm for 1 min.
[0056] Method: Spectrophotometry (pH 7.0 PBS buffer system) Method for measuring emulsifying activity EAI: Immediately take 50 μL of the emulsion and dilute it 100 times, measure the absorbance at 500 nm (A0). Calculation formula:
[0057] Method for measuring emulsifying stability ESI: After standing for 30 min, measure A at the same position. 30 , calculate the retention rate:
[0058] The measurement results of emulsifying activity EAI and emulsifying stability ESI are shown in Table 4: Table 4
[0059] As can be seen from Table 4 above, the emulsifying activity (28.7 m 2 / g) and stability (45.4%) of Comparative Example 5 are the worst, while those of Example 13 (47.5 m 2 / g, 91.2%) are the best, proving that the present invention significantly improves the interfacial properties of phospholipids through vesicle enrichment and microbial modification. From Comparative Example 8, it can be seen that Rhodococcus removes hydrophobic long-chain fatty acids, reduces their competitive adsorption at the oil-water interface, and improves the stability of the phospholipid membrane.
Claims
1. A method for extracting phospholipids from rapeseed oil foot, characterized in that, It includes the following steps: S1. Degumming treatment of rapeseed oil: Heat rapeseed oil to 60 °C, add water accounting for 2% of the mass of rapeseed oil and stir, centrifuge at 5000 - 6000 rpm for 15 min, remove the aqueous phase to obtain degummed rapeseed oil; S2. Mix the degummed rapeseed oil with an ethyl acetate - ethanol mixed solvent at a volume ratio of 1:(3 - 4) evenly to obtain diluted rapeseed oil; S3. Add thermosensitive vesicles and 0.1 M NaCl to the diluted rapeseed oil, and oscillate at 25 °C for 2 - 3 h; S4. Centrifuge at a speed of 100,000 g for 25 - 35 min, collect the vesicles loaded with phospholipids, and wash with ethanol to remove surface non - polar impurities; S5. Resuspend the vesicles loaded with phospholipids with PBS buffer, heat to 40 °C and maintain for 30 min, centrifuge to remove the vesicles to obtain a phospholipid - enriched solution; S6. Adjust the pH of the phospholipid - enriched solution to 6.8 - 7.5, add Yarrowia lipolytica, and react at 25 - 30 °C for 2 h; S7. Centrifuge to remove the cells of Yarrowia lipolytica, adjust the pH to 7.2, inoculate Rhodococcus opacus, and oscillate at 28 °C and 200 - 300 rpm for 12 h; S8. Inactivate the cells at 90 °C, precipitate the phospholipids with cold acetone, centrifuge and then dry in vacuum to obtain phospholipids.
2. The method for extracting phospholipids from rapeseed oil foot according to claim 1, characterized in that, In the ethyl acetate - ethanol mixed solvent in step S2, the volume ratio of ethyl acetate to ethanol is 4:
1.
3. The method for extracting phospholipids from rapeseed oil foot according to claim 1, characterized in that, In step S3, the mass ratio of the diluted rapeseed oil to the thermosensitive vesicles is (4 - 5):
1.
4. The method for extracting phospholipids from rapeseed oil foot according to claim 1, characterized in that, In step S3, the phase transition temperature of the thermosensitive vesicles is 35 - 40 °C; The preparation method is as follows, by weight: S31. Take 10 - 30 mg of phosphatidylcholine with a purity of at least 98% and dissolve it in 10 mL of chloroform, rotate and evaporate to form a uniform lipid film, and dry in vacuum; S32. Add ultrapure water at 55 - 60 °C for hydration, vortex and oscillate in a 55 °C water bath for 1 - 2 min until the solution becomes turbid, and let it stand for 30 - 40 min for hydration to form a multi - vesicular liposome solution; S33. Subject the multi - vesicular liposome solution to 3 freeze - thaw cycles, and extrude it through an extruder 10 - 15 times through a polycarbonate membrane with a pore size of 200 nm to obtain vesicles with a particle size of 100 - 200 nm; S34. Disperse the vesicles in a PBS solution containing 3 wt% - 5 wt% N - isopropylacrylamide, add 0.2 wt% - 0.5 wt% glutaraldehyde and 0.1 M HCl, adjust the pH to 4 - 5, and stir and react at 25 °C in the dark for 6 - 7 h; S35. Ultra - centrifuge at 10,000 rpm for 30 min to remove unreacted monomers to obtain thermosensitive vesicles.
5. The method for extracting phospholipids from rapeseed oil foot according to claim 4, wherein, In step S32, the solid - liquid dosage ratio of the ultrapure water to the lipid film in step S31 is (5 - 20) mg:1 mL.
6. The method for extracting phospholipids from rapeseed oil foot according to claim 4, characterized in that, In step S33, the temperatures of the freeze - thaw cycles are - 80 °C / 55 °C respectively.
7. The method for extracting phospholipids from rapeseed oil foot according to claim 1, characterized in that, In step S5, the PBS buffer contains 5 mM EDTA.
8. The method for extracting phospholipids from rapeseed oil foot according to claim 1, wherein, The OD of Yarrowia lipolytica in the step S6 600 = 0.6 - 1.
0.
9. The method for extracting phospholipids from rapeseed oil foot according to claim 1, characterized in that, The OD of Rhodococcus opacus in the step S7 600 = 0.8 - 0.
9. Application of the phospholipids extracted by the extraction method according to any one of claims 1 - 9 in food, medicine and cosmetics.