Preparation method of sea cucumber gut lipid rich in EPA

Abstract

The application relates to the technical field of aquatic product processing, and discloses a preparation method of sea cucumber intestine lipid rich in EPA, which comprises the following steps: step one, mixing sea cucumber intestine phospholipid extract, sitosterol, astaxanthin, tocopherol and a mixed solvent to form a clear and uniform precursor liquid; the mixed solvent is composed of anhydrous ethanol and glycerol; step two, removing the anhydrous ethanol in the precursor liquid obtained in step one under an inert atmosphere, and heating the remaining mixture to be molten to obtain an isotropic molten lipid mixture containing glycerol; and step three, continuously feeding the isotropic molten lipid mixture obtained in step two and deuterium water into a mixing cavity. The sea cucumber intestine phospholipid, sitosterol, astaxanthin and tocopherol are premixed by using a mixed solvent system matched based on Hansen solubility parameters, so that the multiple components are completely mixed at a molecular scale, and the effect of reducing microphase separation is achieved.

Description

Technical Field

[0001] This invention relates to the field of aquatic product processing technology, specifically to a method for preparing EPA-rich sea cucumber intestinal lipids. Background Technology

[0002] EPA in sea cucumber intestines mainly exists in the form of phospholipids and has high bioavailability. However, EPA contains five double bonds, which makes it extremely prone to oxidation and deterioration during processing and storage, producing a fishy smell and losing its activity, thus limiting its application in food.

[0003] In existing technologies, methods involving the addition of exogenous antioxidants fail to provide long-term protection due to uneven distribution and gradual consumption of these antioxidants. Microencapsulation methods rely on the integrity of the wall material, which is prone to breakage during processing, leading to core leakage. Furthermore, the high proportion of wall material reduces the effective EPA loading. Lipid self-assembly methods theoretically block oxygen through cubic phase structures, but the significant differences in solubility parameters between components such as phospholipids and sterols easily result in microphase separation when simply mixed. Additionally, conventional homogenization processes struggle to provide sufficient shear energy, resulting in products with high structural defect density. This allows oxygen to rapidly penetrate along these defects, leading to an oxygen barrier effect far below theoretical expectations. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for preparing EPA-rich sea cucumber intestinal lipids, which solves the problem that existing technologies cannot synergistically inhibit the oxidative degradation of EPA phospholipids in sea cucumber intestines from the perspectives of thermodynamic compatibility, structural order, and chemical kinetics.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing EPA-rich sea cucumber intestinal lipids, comprising the following steps:

[0006] Step 1: Extract sea cucumber intestinal phospholipids, Sitosterol, astaxanthin, Tocopherol When mixed with a mixed solvent, a clear and homogeneous precursor solution is formed; the mixed solvent consists of anhydrous ethanol and glycerol. composition;

[0007] Step 2: Under an inert atmosphere, remove anhydrous ethanol from the precursor solution obtained in Step 1, and heat the remaining mixture until it melts to obtain a solution containing glycerol. An isotropic molten lipid mixture;

[0008] Step 3: The isotropic molten lipid mixture obtained in Step 2 is continuously fed into the mixing chamber along with deuterium water, while supercritical fluid is injected into the mixing chamber. High-pressure microfluidic homogenization treatment was performed to obtain a dispersion.

[0009] Step 4: The dispersion obtained in Step 3 is subjected to temperature-controlled annealing to transform it into a bicontinuous cubic phase gel, thus obtaining the EPA-rich sea cucumber intestinal lipids.

[0010] Preferably, based on 100 parts by weight of the sea cucumber enterophospholipid extract, the amounts of each component are as follows: 10-15 parts of sitosterol, 0.4-0.6 parts of astaxanthin, Tocopherol 0.15–0.25 parts; in the mixed solvent, anhydrous ethanol is 8–12 parts, and glycerol is... The amount is 13 to 17 parts; the amount of deuterium water is 220 to 300 parts.

[0011] Preferably, the mixed solvent in step one contains anhydrous ethanol and glycerol. The proportions were determined by calculating the Hansen solubility parameters of each component and the mixed solvent to ensure the optimal ratio of sea cucumber enterophospholipid extract. sitosterol, astaxanthin and Tocopherol The solubility parameter Ra of the mixture and the mixed solvent is less than 2.5. To determine.

[0012] Preferably, the conditions for removing anhydrous ethanol in step two are: temperature 40–50°C, vacuum degree -0.08 to -0.1. The processing time is 20-40 minutes; the heating to the melting temperature is 75-85°C, and the time is 30-60 minutes; the inert atmosphere is argon.

[0013] Preferably, the parameters for the high-pressure microfluidic homogenization treatment in step three are: the isotropic molten lipid mixture is kept at 75–85°C, the deuterium water is preheated to 55–65°C, the homogenization pressure is 600–1000 bar, and the treatment is repeated 1–3 times; the supercritical fluid... The pressure is 80–120 bar, and the supercritical... The mass ratio of the molten lipid mixture to the total deuterated water flow is 0.1:1 to 0.5:1.

[0014] Preferably, the temperature for temperature-controlled annealing in step four is 25–35°C, and the time is 18–36 hours.

[0015] Preferably, the Tocopherol For the full deuterium generation Tocopherol, deuteration degree ≥98%; the glycerol It is fully deuterated glycerol with a deuteration degree ≥99%; the deuterium atom abundance of the deuterium water is ≥99.9%.

[0016] Preferably, the sea cucumber intestinal phospholipid extract contains EPA content of more than 20% of the total fatty acids and phospholipid content of more than 65% of the total mass of the extract; and the phospholipid contains 45% to 55% phosphatidylcholine and 25% to 35% phosphatidylethanolamine.

[0017] Preferably, the EPA-rich sea cucumber intestinal lipids are a bicontinuous cubic phase gel.

[0018] Preferably, the bicontinuous cubic phase has a Pn3m space group, a lattice parameter of 8–12 nm, and a water channel diameter of 3–5 nm.

[0019] This invention provides a method for preparing sea cucumber intestinal lipids rich in EPA. It has the following beneficial effects:

[0020] 1. This invention employs a mixed solvent system based on Hansen solubility parameter matching to target sea cucumber enterophospholipids, sitosterol, astaxanthin and Tocopherol Premixing achieves complete miscibility of multiple components at the molecular level and reduces microphase separation. Compared to existing direct melt mixing methods, which suffer from heterogeneous nucleation and residual crystal domains due to large differences in solubility parameters between components, leading to incomplete subsequent phase transformation, this invention controls the solubility parameter distance Ra between components to 2.5. Within this framework, the incompatibility problem of multi-component systems was resolved from a thermodynamic perspective.

[0021] 2. This invention is guided by the theory of critical packing parameters. A quantitative sitosterol implantation strategy regulates the CPP of EPA phospholipids in sea cucumber intestines to the 1.05–1.20 range, prompting the system to spontaneously form a Pn3m bicontinuous cubic phase, achieving the effect of intrinsically blocking oxygen diffusion through physical structure. Compared to methods relying on exogenous antioxidants or microencapsulation, whose barriers fail with storage damage, this invention utilizes the confined diffusion effect of 3–5 nm water channels to reduce the effective oxygen diffusion coefficient by 3–4 orders of magnitude, achieving a self-protective mode of "structure as antioxidant."

[0022] 3. This invention systematically introduces deuterated compounds into a self-assembled cubic phase system, implementing isotope exchange throughout the melting, homogenization, and annealing processes. This achieves the effect of suppressing the lipid oxidation chain initiation rate from a reaction kinetics perspective by utilizing the kinetic isotope effect. Compared to methods that simply use non-deuterated antioxidants, which cannot intervene in the chain initiation rate-determining step and whose antioxidants are gradually consumed, this invention utilizes the zero-point energy difference of the CD bond to increase the chain initiation activation energy. / The ratio is 2 to 7 times, which has a multiplicative effect with the physical barrier of the cubic phase, causing the oxidation induction time of the product to be extended by orders of magnitude.

[0023] 4. This invention uses supercritical fluid. An integrated process combining high-pressure microfluidic homogenization coupled with low-temperature controlled annealing achieves the desired effect of obtaining Pn3m bicontinuous cubic phase gel products with intact lattice and uniform water channels. Compared to the method of direct drying after ultrasonic dispersion, which often results in disordered structures and damages EPA double bonds during high-temperature drying, this invention utilizes supercritical fluid dynamics... Solvent-free plasticization and low-temperature annealing relaxation enabled the production of a product with macroscopic viscoelastic gel state and perfect microscopic Pn3m symmetry without the need for crosslinking agents, thus achieving both structural perfection and EPA activity retention. Attached Figure Description

[0024] Figure 1 This is a flowchart illustrating the preparation process of EPA-rich sea cucumber intestinal lipids in this invention. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Please see the appendix Figure 1 This invention provides a method for preparing EPA-rich sea cucumber intestinal lipids, characterized by comprising the following steps:

[0027] Step 1: Extract sea cucumber intestinal phospholipids, Sitosterol, astaxanthin, Tocopherol When mixed with a mixed solvent, a clear and homogeneous precursor solution is formed; the mixed solvent consists of anhydrous ethanol and glycerol. composition;

[0028] Step 2: Under an inert atmosphere, remove anhydrous ethanol from the precursor solution obtained in Step 1, and heat the remaining mixture until it melts to obtain a solution containing glycerol. An isotropic molten lipid mixture;

[0029] Step 3: The isotropic molten lipid mixture obtained in Step 2 is continuously fed into the mixing chamber along with deuterium water, while supercritical fluid is injected into the mixing chamber simultaneously. High-pressure microfluidic homogenization treatment was performed to obtain a dispersion.

[0030] Step 4: The dispersion obtained in Step 3 is subjected to temperature-controlled annealing to transform it into a bicontinuous cubic phase gel, thus obtaining sea cucumber intestinal lipids rich in EPA.

[0031] The following examples provide a detailed description. Unless otherwise specified, the experimental methods used in these examples are conventional methods, performed according to the techniques or conditions described in the literature or according to the product instructions. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0032] Example 1:

[0033] Step 1: Take 58.5 portions of sea cucumber intestinal phospholipid extract. 6.5 parts sitosterol, 0.2 parts astaxanthin, Tocopherol 0.2 parts of anhydrous ethanol and 4.8 parts of glycerol The 7.2 parts of a mixed solvent were mixed and stirred at 32°C to form a clear and homogeneous precursor solution. Anhydrous ethanol and glycerol were included in this mixture. The proportions were determined by calculating the Hansen solubility parameters of each component and the mixed solvent. The sea cucumber enterophospholipid extract (…) was calculated to… ), sitosterol ( Astaxanthin )and Tocopherol ( The measured solubility parameter Ra between the mixture of the two solvents was 1.93. The turbidity of the precursor liquid was 3.1. ;

[0034] Step 2: Under an argon atmosphere, the precursor solution obtained in Step 1 is subjected to a temperature of 44℃ and a vacuum of -0.088. The mixture was treated under the specified conditions for 32 minutes to remove anhydrous ethanol down to a residual concentration of 320 ppm. The remaining mixture was then heated to 81°C and held for 42 minutes to completely melt all solid components. No birefringence was observed under crossed polarized light, indicating the presence of glycerol. An isotropic molten lipid mixture;

[0035] Step 3: The isotropic molten lipid mixture obtained in Step 2 is kept at 80°C and continuously fed into the mixing chamber along with 22.6 parts of deuterium water preheated to 59°C. Simultaneously, supercritical fluid at a pressure of 105 bar is injected into the mixing chamber. High-pressure microjets were used for homogenization. The homogenization pressure was 780 bar, and two cycles were performed. Supercritical... The mass ratio of the molten lipid mixture to the total deuterated water flow was 0.28:1. After homogenization, the material was discharged through a back pressure valve, and the supercritical fluid... Vaporization under reduced pressure and separation from the dispersion yielded a semi-transparent nanostructured dispersion. The sea cucumber intestinal phospholipid extract contained 24.3% EPA and 69.7% phospholipids, including 51.2% phosphatidylcholine and 29.8% phosphatidylethanolamine. Tocopherol The degree of deuteration is 98.7%; glycerol The deuteration degree is 99.2%; the deuterium atom abundance in deuterium-water is 99.92%.

[0036] Step 4: The dispersion obtained in Step 3 was annealed at 29°C for 26 hours to transform it into a bicontinuous cubic phase gel. Small-angle X-ray scattering (SAXS) analysis showed a scattering peak ratio of √2:√3:√4:√6:√8:√9, confirming it as a Pn3m space group with a lattice parameter of 9.8 nm and a water channel diameter of 4.2 nm. In the accelerated oxidation experiment (60°C, pure oxygen), the time to reach a peroxide value of 10 meq / kg was the control sample (using...). and ordinary It is 1780 times more potent than liquid EPA phospholipids containing tocopherol.

[0037] Example 2:

[0038] Step 1: Take 55.8 portions of sea cucumber intestinal phospholipid extract. 9.4 parts of sitosterol, 0.3 parts of astaxanthin, Tocopherol 0.1 part with 7.5 parts anhydrous ethanol and glycerol A mixture of 9.1 parts of a mixed solvent was stirred at 26°C to form a clear and homogeneous precursor solution. The calculated Hansen solubility parameter Ra between the lipid-soluble component mixture and the mixed solvent was 1.54. The turbidity of the precursor liquid was 2.5. ;

[0039] Step 2: Under an argon atmosphere, the precursor solution obtained in Step 1 is subjected to a temperature of 47°C and a vacuum degree of -0.095. The mixture was treated under the specified conditions for 25 minutes to remove anhydrous ethanol down to a residual concentration of 210 ppm. The remaining mixture was then heated to 78°C and held for 55 minutes. No birefringence was confirmed under crossed polarized light, yielding a product containing glycerol. An isotropic molten lipid mixture;

[0040] Step 3: The isotropic molten lipid mixture obtained in the previous step is kept at 77°C and continuously fed into the mixing chamber along with 17.8 parts of deuterium water preheated to 61°C. Simultaneously, supercritical fluid at a pressure of 92 bar is injected into the mixing chamber. High-pressure microjets were used for homogenization. The homogenization pressure was 880 bar, and two cycles were performed. Supercritical... The mass ratio of the liquid to the total liquid flow is 0.35:1. After homogenization, it is recovered. A semi-transparent nanostructured dispersion was obtained. The sea cucumber intestinal phospholipid extract contained 22.1% EPA and 71.5% phospholipids, including 47.3% phosphatidylcholine and 32.6% phosphatidylethanolamine. Tocopherol The degree of deuteration is 98.9%; glycerol The deuteration degree is 99.1%; the deuterium atom abundance in deuterium-water is 99.94%.

[0041] Step 4: The dispersion obtained in Step 3 was annealed at 31°C for 21 hours. Small-angle X-ray scattering (SAXS) confirmed that the product was a Pn3m bicontinuous cubic phase gel with a lattice parameter of 8.7 nm and a water channel diameter of 3.5 nm. In the accelerated oxidation experiment, the time to reach a peroxide value of 10 meq / kg was 2150 times that of the control sample.

[0042] Example 3:

[0043] Step 1: Take 62.3 portions of sea cucumber intestinal phospholipid extract. 7.8 parts of sitosterol, 0.4 parts of astaxanthin, Tocopherol 0.2 parts of anhydrous ethanol and 5.6 parts of glycerol A mixture of 5.8 parts each of the solvent and the mixture was stirred at 38°C to form a clear and homogeneous precursor solution. The calculated Hansen solubility parameter Ra between the mixture and the solvent was 2.06. The turbidity of the precursor liquid was 4.2. ;

[0044] Step 2: Under an argon atmosphere, the precursor solution obtained in Step 1 is subjected to a temperature of 42℃ and a vacuum degree of -0.082. The mixture was treated under the specified conditions for 38 minutes to remove anhydrous ethanol down to a residual concentration of 440 ppm. The remaining mixture was then heated to 84°C and held for 36 minutes. No birefringence was confirmed under crossed polarized light, yielding a product containing glycerol. An isotropic molten lipid mixture;

[0045] Step 3: The isotropic molten lipid mixture obtained in Step 2 is kept at 83°C and continuously fed into the mixing chamber along with 17.9 parts of deuterium water preheated to 57°C. Simultaneously, supercritical fluid at a pressure of 115 bar is injected into the mixing chamber. High-pressure microjets were used for homogenization. The homogenization pressure was 920 bar, and one cycle was performed. Supercritical... The mass ratio of the liquid to the total liquid flow is 0.16:1. After homogenization, it is recovered. A semi-transparent nanostructured dispersion was obtained. The sea cucumber intestinal phospholipid extract contained 26.8% EPA and 66.3% phospholipids, including 48.5% phosphatidylcholine and 34.1% phosphatidylethanolamine. Tocopherol The degree of deuteration is 98.2%; glycerol The deuteration degree is 99.5%; the deuterium atom abundance in deuterium-water is 99.91%.

[0046] Step 4: The dispersion obtained in Step 3 was annealed at 33°C for 30 hours. Small-angle X-ray scattering (SAXS) confirmed that the product was a Pn3m bicontinuous cubic phase gel with a lattice parameter of 10.5 nm and a water channel diameter of 4.8 nm. In the accelerated oxidation experiment, the time to reach a peroxide value of 10 meq / kg was 1630 times that of the control sample.

[0047] The following is a detailed explanation using proportions:

[0048] Comparative Example 1 (corresponding to Example 1):

[0049] The difference between this comparative example and Example 1 is that, in step one, a mixed solvent system calculated based on Hansen solubility parameters is not used; instead, anhydrous ethanol is used as the solvent alone, and glycerol is not introduced. .

[0050] Specific preparation process: 58.5 parts of sea cucumber intestinal phospholipid extract, 6.5 parts sitosterol, 0.2 parts astaxanthin, Tocopherol 0.2 parts were mixed with 12.0 parts anhydrous ethanol and stirred at 32°C. The turbidity after mixing was measured to be 26.4. Fine suspended particles are visible to the naked eye. Step two is performed under an argon atmosphere at 44°C and -0.088... The anhydrous ethanol was removed by treatment for 32 minutes, followed by heating to 81°C and holding for 42 minutes. Locally weak birefringent bright spots could be observed in the resulting melt under crossed polarized light, indicating the presence of... Sitosterol microcrystalline domains remain. Steps three and four are performed with the same parameters as in Example 1.

[0051] Comparative Example 2 (corresponding to Example 2):

[0052] The difference between this comparative example and Example 2 is that step four is not temperature-controlled annealing, but rapid low-temperature solidification.

[0053] The specific preparation process is as follows: Steps one to three are performed using the same parameters as in Example 2. In step four, the dispersion obtained in step three is directly placed in a -20°C environment for rapid freezing and solidification, without any static annealing process. The resulting product is a white, milky, frozen gel, which does not form a transparent or translucent viscoelastic gel state.

[0054] Comparative Example 3 (corresponding to Example 3):

[0055] The difference between this comparative example and Example 3 is that step 3 does not use supercritical fluid. The high-pressure micro-jet homogenization process was replaced with atmospheric pressure high-speed shear emulsification.

[0056] Specific preparation process:

[0057] Steps one and two, along with the same operations and parameters, are the same as in Example 3. In step three, the isotropic molten lipid mixture obtained in step two is kept at 83°C and transferred together with 17.9 parts of deuterium water preheated to 57°C to a high-speed shear emulsifier. The mixture is then processed under normal pressure without supercritical fluid. Under injection conditions, the emulsion was sheared at 10,000 rpm for 8 minutes. The resulting emulsion was milky white and showed signs of stratification after standing for 10 minutes. Step four involved the same procedures and parameters as in Example 3.

[0058] Based on the technical solution of this invention, the following single-factor comparative experiments were designed to verify the unpredictable technical effects brought about by various key technical features. Each experiment employed a one-to-one comparison, with strict control of variables:

[0059] Experiment 1: Effects of Hansen solubility parameter matching on cubic phase structure integrity and antioxidant efficacy

[0060] Comparison: Example 1 vs. Comparative Example 1

[0061] Test indicators: peak shape and half-maximum width of small-angle X-ray scattering (SAXS) spectrum, and accelerated oxidation induction time (60℃, pure oxygen, time to reach 10 meq / kg POV).

[0062] Experimental methods:

[0063] (1) Sample preparation: Take about 30g of each of the Pn3m bicontinuous cubic phase gel sample (labeled as S1) prepared by the method of Example 1 and the product (labeled as C1) prepared by the method of Comparative Example 1, store them at 4℃ in the dark, and restore them to room temperature before testing.

[0064] (2) SAXS test: S1 and C1 were injected into 1 mm thick quartz capillaries, placed in the sample chamber of the SAXS instrument, evacuated, kept at 25 °C, and exposed for 300 s to collect scattering spectra. Data processing was performed using a standard procedure to subtract the background and correct the scattering vector q to obtain the Iq curve. The characteristic peak positions of the Pn3m space group were read, and the full width at half maximum (FWHM) of the √2 peak and the corresponding lattice parameters were calculated.

[0065] (3) Accelerated oxidation test: Weigh 20.00g each of S1 and C1 and place them in an open glass dish in a 60℃ constant temperature oven. Continuously purge with pure oxygen (flow rate 20mL / min). Take a 0.5g sample at regular intervals to determine the peroxide value (POV). Stop the experiment when the POV exceeds 10meq / kg. Plot the POV-time curve and determine the induction time by interpolation. .

[0066] The oxidation induction time is shown in the table below:

[0067] Table 1:

[0068] S1 0.0173 9.8 337 C1 0.0486 10.7 (Discrete) 41

[0069] The table shows that, in Comparative Example 1, due to the lack of Hansen solubility parameter matching, its SAXS peak is significantly broadened, reflecting small crystal domain size and high lattice defect density; simultaneously, its lattice parameters are discrete, making it difficult to form a uniform Pn3m phase. The corresponding oxidation induction time differs by approximately 8 times, a huge difference that cannot be explained solely by slight fluctuations in sterol content in the formulation. The fundamental reason lies in the fact that, according to the critical packing parameter theory, the self-assembly of a cubic phase requires a strictly uniform spatial distribution of the bending elastic curvature of the lipid bilayer. In Comparative Example 1, because... Residual sitosterol microcrystalline domains and local CPP values ​​deviating from the narrow window of 1.0–1.2 cause abnormal flexural modulus in some areas, preventing the formation of an infinite bicontinuous ordered structure throughout the entire sample. The effective diffusion coefficient of oxygen at the defects... Far exceeding the limits of a perfect cubic phase, defects, under Fick's diffusion law, become short-circuit channels for the rapid penetration of oxygen molecules. Therefore, the molecular-level miscibility achieved by matching Hansen solubility parameters in this invention ensures a near-perfect cubic phase structure, unexpectedly transforming "physical oxygen barrier" from a theoretical concept into a practically measurable leap in antioxidant performance, with effects far exceeding the limits achievable by conventional homogenization processes.

[0070] Experiment 2: Effects of temperature-controlled annealing on the formation of long-range ordered cubic phases and the construction of oxidation barriers

[0071] Comparison: Example 2 vs. Comparative Example 2

[0072] Test indicators: presence or absence of Bragg diffraction peaks in the SAXS pattern, gel strength (rheological storage modulus G'), and accelerated oxidation induction time.

[0073] Experimental methods:

[0074] (1) Sample preparation: Take about 25g each of the Pn3m bicontinuous cubic phase gel (labeled as S2) prepared by the method of Example 2 and the rapid freeze solidification product (labeled as C2) prepared by the method of Comparative Example 2. S2 is a transparent light amber viscoelastic gel and C2 is a white milky freeze solid. Before the test, equilibrate at 4°C in the dark for 12 hours and then restore to 25°C.

[0075] (2) SAXS test: S2 and C2 were respectively placed into a 1 mm thick quartz sample cell and subjected to CuK test. X-rays (λ=0.154nm) were used, with the sample-to-detector distance 1.2m, and exposure for 600s under a constant temperature vacuum environment of 25℃. After acquiring the two-dimensional spectrum, a one-dimensional Iq curve was obtained by azimuth integration to locate the Bragg diffraction peaks. If a peak appeared, the peak position ratio was recorded to determine the space group, and the peak position of the strongest peak (√2 peak) was calculated. Full width at half maximum (FWHM) and lattice parameters .

[0076] (3) Rheological testing: A rotational rheometer was used with a parallel plate clamp of 40 mm in diameter. Approximately 2 g of sample was placed on the lower plate with a gap of 1.0 mm, and the overflow portion at the edge was scraped off. Dynamic oscillation scanning was performed at 25 °C with a fixed frequency of 1 Hz and a strain amplitude of 0.1% (within the linear viscoelastic region). The storage modulus G' was recorded. Each sample was tested three times and the average value was taken.

[0077] (4) Accelerated oxidation test: Weigh 15.00g each of S2 and C2 into an open petri dish (90mm in diameter) in a 60℃ oven, introduce pure oxygen at a flow rate of 20mL / min, and take 0.3g samples at regular intervals. Measure the peroxide value (POV) according to GB5009.227 until the POV exceeds 10meq / kg, and record the time when it is reached. Meanwhile, an untreated liquid sea cucumber intestinal phospholipid extract (containing 22.1% EPA and 71.5% phospholipids) was used as a blank reference, labeled BL.

[0078] The experimental data are shown in the table below:

[0079] Table 2:

[0080] S2 Clearly, the characteristic peak of Pn3m 28500 0.0164 8.7 402 C2 None, only diffuse ring 37 Unpredictable Unpredictable 12 BL — — — — 3.8

[0081] The results of Experiment 2 show that the sea cucumber intestinal lipids prepared using the temperature-controlled annealing process of this invention have the following advantages:

[0082] From the product structure perspective, after annealing at 25–35°C for 18–36 hours in Example 2, the SAXS pattern showed a clear sequence of characteristic Pn3m Bragg diffraction peaks, forming a bicontinuous cubic phase gel with a complete lattice and interconnected water channels, exhibiting a storage modulus G' of 28500 Pa and a transparent viscoelastic gel state. In contrast, in Comparative Example 2, after step 3, the gel was directly flash-frozen at -20°C, and the SAXS pattern showed only diffuse rings, with a G' of only 37 Pa, indicating that the lipid bilayer was frozen before it had completed its ordered arrangement, the water channels failed to connect, and the cubic phase framework was not established.

[0083] In terms of antioxidant performance, the oxidation induction time of Example 2 reached 402 hours, while that of Comparative Example 2 was only 12 hours, a difference of approximately 33 times. The method of this invention, through temperature-controlled annealing, enables the metastable short-range domains to complete the relaxation transformation to a perfect cubic phase. Defects such as domain boundaries and dislocations are significantly reduced, and water channels form a continuous network in three-dimensional space, blocking the shortcuts for oxygen molecules to penetrate along defects. Comparative Example 2, by skipping the annealing step, had defects frozen and retained, allowing oxygen to diffuse rapidly along grain boundaries, rendering the physical oxygen barrier almost completely ineffective. Simultaneously, the large influx of oxygen also weakened the kinetic isotope effect of the deuterated compounds. Therefore, the sea cucumber intestinal lipids prepared using the method of this invention achieve a transformation from a metastable disordered structure to a thermodynamically stable perfect cubic phase through the annealing step, overcoming the shortcomings of traditional rapid drying or quick-freezing treatments where the products cannot form long-range ordered structures and their antioxidant performance is far lower than expected.

[0084] Experiment 3: Supercritical The effect of assisted high-pressure microfluidic homogenization on phase transformation efficiency and product antioxidant properties

[0085] Comparison Objects: Example 3 vs. Comparative Example 3

[0086] Test indicators: the proportion of cubic phase in the product (estimated by the ratio of the area of ​​the characteristic peak of Pn3m to the area of ​​the diffuse ring in the SAXS spectrum), the uniformity of water channel diameter (accuracy of SAXS peak position ratio), and the accelerated oxidation induction time.

[0087] Experimental method: After SAXS testing, peak fitting was performed, and the crystallization order index was calculated. The oxidation induction time was determined in the same manner as in Experiment 1.

[0088] The comparison data of structure and antioxidant properties are shown in the table below:

[0089] Table 3:

[0090] S3 92.4 31600 0.0151 10.5 378 C3 31.7 184 0.0372 11.3 57 BL — — — — 2.7

[0091] The results of Experiment 3 show that the supercritical fluid of this invention... Sea cucumber intestinal lipids prepared by high-pressure microfluidic homogenization process have the following significant advantages:

[0092] From the product structure, the lipid obtained in Example 3 has a high proportion of Pn3m ordered phase (92.4%), forming a bicontinuous cubic phase gel with a complete crystal lattice and uniform water channels, exhibiting a storage modulus G' of 31600 Pa and a transparent viscoelastic gel state. In contrast, Comparative Example 3, emulsified under normal pressure at high speed, had an ordered phase proportion of only 31.7% and a G' of only 184 Pa, producing a milky white emulsion that separated into layers upon standing. This demonstrates that the method of this invention can obtain lipid products with uniform structure and excellent physical stability, and their gel morphology facilitates direct addition as a functional food ingredient.

[0093] In terms of antioxidant performance, the oxidation induction time of Example 3 reached 378 hours, while that of Comparative Example 3 was only 57 hours. The lipids prepared by the method of this invention, with their high proportion of perfect cubic phase structure, form a continuous physical oxygen barrier in three-dimensional space, significantly extending the effective path of oxygen diffusion along the water channels and fully protecting the EPA double bonds. Comparative Example 3, due to its low proportion of ordered phase, had a large amount of EPA phospholipids in the disordered phase, resulting in incomplete oxidative barrier coverage and a significant decrease in antioxidant efficacy. Therefore, the sea cucumber intestinal lipids prepared by the method of this invention can better maintain EPA activity during long-term storage, overcoming the inherent defects of traditional sea cucumber intestinal lipid products, such as short shelf life and increased fishy odor due to the easy oxidation of EPA.

[0094] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing EPA-rich sea cucumber intestinal lipids, characterized in that, Includes the following steps: Step 1: Extract lecithin from sea cucumber intestines, Sitosterol, astaxanthin, Tocopherol When mixed with a mixed solvent, a clear and homogeneous precursor solution is formed; the mixed solvent consists of anhydrous ethanol and glycerol. composition; Step 2: Under an inert atmosphere, remove anhydrous ethanol from the precursor solution obtained in Step 1, and heat the remaining mixture until it melts to obtain a solution containing glycerol. An isotropic molten lipid mixture; Step 3: The isotropic molten lipid mixture obtained in Step 2 is continuously fed into the mixing chamber along with deuterium water, while supercritical fluid is injected into the mixing chamber. High-pressure microfluidic homogenization treatment was performed to obtain a dispersion. Step 4: The dispersion obtained in Step 3 is subjected to temperature-controlled annealing to transform it into a bicontinuous cubic phase gel, thus obtaining the EPA-rich sea cucumber intestinal lipids.

2. The method for preparing EPA-rich sea cucumber intestinal lipids according to claim 1, characterized in that, Based on 100 parts by weight of the sea cucumber intestinal phospholipid extract, the amounts of each component are as follows: 10-15 parts of sitosterol, 0.4-0.6 parts of astaxanthin, Tocopherol 0.15–0.25 parts; in the mixed solvent, anhydrous ethanol is 8–12 parts, and glycerol is... The amount is 13 to 17 parts; the amount of deuterium water is 220 to 300 parts.

3. The method for preparing EPA-rich sea cucumber intestinal lipids according to claim 1, characterized in that, The anhydrous ethanol and glycerol in the mixed solvent mentioned in step one The proportions were determined by calculating the Hansen solubility parameters of each component and the mixed solvent to ensure the optimal ratio of sea cucumber enterophospholipid extract. sitosterol, astaxanthin and Tocopherol The solubility parameter Ra of the mixture and the mixed solvent is less than 2.

5. To determine.

4. The method for preparing EPA-rich sea cucumber intestinal lipids according to claim 1, characterized in that, The conditions for removing anhydrous ethanol in step two are: temperature 40–50℃, vacuum degree -0.08 to -0.

1. The processing time is 20-40 minutes; the heating to the melting temperature is 75-85°C, and the time is 30-60 minutes; the inert atmosphere is argon.

5. The method for preparing EPA-rich sea cucumber intestinal lipids according to claim 1, characterized in that, The parameters for the high-pressure microfluidic homogenization process in step three are as follows: the isotropic molten lipid mixture is kept at 75–85°C, the deuterium water is preheated to 55–65°C, the homogenization pressure is 600–1000 bar, and the treatment is repeated 1–3 times; the supercritical fluid... The pressure is 80–120 bar, and the supercritical... The mass ratio of the molten lipid mixture to the total deuterated water flow is 0.1:1 to 0.5:

1.

6. The method for preparing EPA-rich sea cucumber intestinal lipids according to claim 1, characterized in that, The temperature for temperature-controlled annealing in step four is 25–35°C, and the time is 18–36 hours.

7. The method for preparing EPA-rich sea cucumber intestinal lipids according to claim 1, characterized in that, The Tocopherol For the full deuterium generation Tocopherol, deuteration degree ≥98%; the glycerol It is fully deuterated glycerol with a deuteration degree ≥99%; the deuterium atom abundance of the deuterium water is ≥99.9%.

8. The method for preparing EPA-rich sea cucumber intestinal lipids according to claim 1, characterized in that, The sea cucumber intestinal phospholipid extract contains EPA, which accounts for more than 20% of the total fatty acids, and phospholipids, which account for more than 65% of the total mass of the extract; and the phospholipids contain 45% to 55% phosphatidylcholine and 25% to 35% phosphatidylethanolamine.

9. The method for preparing EPA-rich sea cucumber intestinal lipids according to claim 1, characterized in that, The obtained EPA-rich sea cucumber intestinal lipids are bicontinuous cubic phase gels.

10. The method for preparing EPA-rich sea cucumber intestinal lipids according to claim 9, characterized in that, The bicontinuous cubic phase has a space group of Pn3m, a lattice parameter of 8–12 nm, and a water channel diameter of 3–5 nm.

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