Protein-coated DHA algal oil liposome as well as preparation method and application thereof

Abstract

The invention discloses a protein-coated DHA algal oil liposome as well as a preparation method and application thereof. While the storage stability of the DHA algal oil is prolonged, the DHA algal oil is slowly released in intestinal tracts, and the bioavailability is improved. The preparation method comprises the following steps: taking egg yolk lecithin and cholesterol as main wall materials, preparing a DHA algal oil liposome by using an ultrasonic-assisted film dispersion method, and coating the surface of the liposome with a protein solution to obtain the protein-coated DHA algal oil liposome. Protein is introduced into the structure of the DHA algal oil liposome, so that the DHA algal oil liposome has a first-level kinetic model release characteristic and a certain in-vivo slow release effect, the utilization degree of DHA algal oil is improved, the storage stability of the DHA algal oil nano-liposome at 4 DEG C is remarkably improved, and the problems that the encapsulation efficiency of fat-soluble substances in the traditional algal oil liposome is poor, and the DHA algal oil nano-liposome is difficult to absorb are effectively solved. The stability is poor, and stable and slow release in vivo cannot be realized.

Description

Technical Field

[0001] This invention relates to the field of liposome technology, specifically to a protein-coated DHA algal oil nanoliposome and its preparation method. Background Technology

[0002] DHA is an omega-3 long-chain polyunsaturated fatty acid that is highly susceptible to oxidation. During food processing and storage, this oxidation not only damages product quality but also reduces its nutritional value and functional properties. Furthermore, as a hydrophobic substance, DHA algal oil is unsuitable as an ingredient in most water-based foods and beverages. Therefore, improving the application of DHA algal oil in food processing and enhancing its stability and flavor acceptability has become an urgent problem to be solved.

[0003] As a commonly used nanomedicine carrier, liposomes have seen a shift in research focus in recent years, moving from gene therapy, the medical field, and the beauty industry to functional food technology and nutrition, due to their excellent biocompatibility, targeting ability, non-immunogenicity, and ease of surface modification. For example, Chinese utility model patent CN118104824A discloses "an algal oil DHA liposome and its preparation method," which uses phytosterols instead of cholesterol as the encapsulation material and rationally combines various excipients to prepare algal oil DHA liposomes using a simple preparation process. However, the in vivo sustained-release effect and stability of existing DHA algal oil liposomes cannot be effectively controlled, thus affecting the release and efficacy of DHA algal oil. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a DHA algal oil nanoliposome with sustained-release effect and its preparation method. Specifically, this invention uses egg yolk lecithin and cholesterol as wall materials to encapsulate DHA algal oil, and prepares nanoliposomes using an ultrasound-assisted thin-film dispersion method, with encapsulation efficiency and particle size as indicators. In vitro drug release experiments are conducted to simulate the release of DHA algal oil liposomes in the human body, thereby providing a protein-coated DHA algal oil liposome with high bioavailability and high stability.

[0005] This invention provides a method for preparing protein-coated DHA algal oil liposomes. Lecithin, cholesterol and DHA algal oil are dissolved in a solvent to obtain an organic phase, which is then rotary evaporated and hydrated to obtain a suspension. The suspension is then sonicated in an ice-water bath to obtain DHA algal oil liposomes. The obtained DHA algal oil liposomes are then dropped into a protein solution to obtain protein-coated DHA algal oil liposomes.

[0006] In a preferred embodiment, the lecithin is egg yolk lecithin, and further, the mass ratio of the DHA algal oil, cholesterol and egg yolk lecithin is 1:0.3-0.5:1.0-3.0.

[0007] In a preferred embodiment, the solvent is one or a mixture of two or more of anhydrous ethanol, chloroform, methanol or ethyl acetate.

[0008] In a preferred embodiment, Tween-80 is also added to the solvent, and the mass ratio of Tween-80 to DHA algal oil is 1:2.5 to 4.5.

[0009] In a preferred embodiment, the mass ratio of DHA algal oil to protein is 1:1.5 to 3.5.

[0010] In a preferred embodiment, the protein is one of lactoferrin, soy protein isolate, and whey protein isolate.

[0011] In a preferred embodiment, the algal oil DHA liposomes are prepared according to the following steps: S1: Weigh out egg yolk lecithin, cholesterol, Tween-80 and DHA algal oil according to the ratio, add solvent, and dissolve by sonication in a water bath; S2: After complete dissolution, the mixture solution is rotary evaporated at 35-45℃ to remove the solvent, and then hydrated to obtain a suspension; S3: The suspension is ultrasonicated in an ice-water bath. The ultrasonication is a programmed ultrasonication performed using an ultrasonic cell disruptor at 300-500W for 5-15 minutes with an interval of 3-6 seconds to obtain DHA algal oil nanoliposomes. S4: Add the DHA algal oil nanoliposomes to the protein solution and stir at 100-300 r / min for 60 min.

[0012] The present invention also provides a protein-coated DHA algal oil liposome, which is prepared by the above method.

[0013] As one embodiment of the present invention, the particle size of the DHA algal oil liposomes is 90.36±0.93nm, which falls within the nanoscale range.

[0014] As one embodiment of the present invention, the particle polydispersity (PDI) is 0.196±0.002; the particle uniformity is good.

[0015] As one embodiment of the present invention, the encapsulation efficiency of the DHA algal oil liposomes is 91.50 ± 0.39%.

[0016] As one embodiment of the present invention, the Zeta potential of the DHA algal oil liposomes is 35.20±0.35mV.

[0017] As one embodiment of the present invention, the DHA algal oil liposomes have been verified to have a significant sustained-release effect using a simulated gastrointestinal model.

[0018] As one embodiment of the present invention, the DHA algal oil liposomes exhibit good storage stability at 4°C.

[0019] This invention also provides the application of protein-coated DHA algal oil liposomes in the preparation of products that have antioxidant properties, improve memory, assist in improving memory, relieve visual fatigue, improve sleep, relieve physical fatigue, improve hypoxia tolerance, or help control body fat.

[0020] Furthermore, the product is food, health food, or medicine.

[0021] The present invention also provides a method for preparing protein-coated DHA fish oil liposomes, in which algal oil is replaced with fish oil in the above method.

[0022] The present invention also provides a protein-coated DHA fish oil liposome, which is prepared by the above method.

[0023] This invention also provides the application of protein-coated DHA fish oil liposomes in the preparation of products that have antioxidant properties, improve memory, assist in improving memory, relieve visual fatigue, improve sleep, relieve physical fatigue, improve hypoxia tolerance, or help control body fat.

[0024] The present invention also provides a method for preparing protein-coated EPA liposomes, wherein the algal oil in the above method is replaced with EPA oil.

[0025] The present invention also provides a protein-coated EPA liposome, which is prepared by the above method.

[0026] This invention also provides the application of protein-coated DHA fish oil liposomes in the preparation of products that have antioxidant properties, improve memory, assist in improving memory, relieve visual fatigue, improve sleep, relieve physical fatigue, improve hypoxia tolerance, help control body fat, lower blood lipids, lower blood pressure, or assist in lowering blood lipids and lowering blood pressure.

[0027] Furthermore, the product is food, health food, or medicine.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention introduces proteins into the structure of DHA algal oil liposomes, giving them first-order kinetic release characteristics and a certain degree of sustained-release effect in vivo, thus improving the utilization rate of DHA algal oil. Furthermore, the DHA algal oil nanoliposomes exhibit significantly improved storage stability at 4°C, effectively solving the problems of poor encapsulation efficiency and stability of lipid-soluble substances in traditional algal oil liposomes, which prevent stable and slow release in vivo. This provides a scientific basis for the widespread application of liposome technology in pharmaceuticals and food processing, expanding the application of liposome-encapsulated bioactive substances in pharmaceuticals or functional foods. Attached Figure Description

[0029] Figure 1 The average particle size of protein-coated DHA algal oil liposomes; Figure 2 Encapsulation efficiency of protein-coated DHA algal oil liposomes; Figure 3 Transmission electron microscopy image of protein-coated DHA algal oil nanoliposomes; Figure 4 The results of the storage stability test of protein-coated DHA algal oil liposomes are shown; where A is particle size; B is PDI; C is Zeta potential; and D is encapsulation efficiency. Figure 5 The figures represent in vitro release model curves of protein-coated DHA algal oil liposomes; where A is the zero-order kinetic equation; B is the first-order kinetic equation; C is the Higuchi planar diffusion equation; and D is the Retger-Peppas equation. Detailed Implementation

[0030] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments. The content mentioned in the embodiments is not intended to limit the present invention. Example 1

[0031] A method for preparing protein-coated DHA algal oil liposomes includes the following steps: 270 mg of egg yolk lecithin, 67.5 mg of cholesterol, 54 mg of Tween-80, and 180 mg of DHA algal oil were dissolved in 100 mL of anhydrous ethanol to obtain an organic phase. This organic phase was removed by rotary evaporation in water at 42 °C, yielding a suspension. The suspension was then sonicated for 10 min at 4 °C (400 W) with 5-second intervals to obtain DHA algal oil liposomes. These DHA algal oil liposomes were then added dropwise to a 15 mg / mL lactoferrin solution and stirred to obtain protein-coated DHA algal oil liposomes. Example 2

[0032] A method for preparing protein-coated DHA algal oil liposomes includes the following steps: 270 mg of egg yolk lecithin, 67.5 mg of cholesterol, 54 mg of Tween-80, and 180 mg of DHA algal oil were dissolved in 100 mL of anhydrous ethanol to obtain an organic phase. This organic phase was removed by rotary evaporation in water at 42 °C, yielding a suspension. The suspension was then sonicated for 10 min at 4 °C (400 W) with 5-second intervals to obtain DHA algal oil liposomes. These DHA algal oil liposomes were then added dropwise to a 15 mg / mL whey protein isolate solution and stirred to obtain protein-coated DHA algal oil liposomes. Example 3

[0033] A method for preparing protein-coated DHA algal oil liposomes includes the following steps: 270 mg of egg yolk lecithin, 67.5 mg of cholesterol, 54 mg of Tween-80, and 180 mg of DHA algal oil were dissolved in 100 mL of anhydrous ethanol to obtain an organic phase. This organic phase was removed by rotary evaporation in water at 42 °C, yielding a suspension. The suspension was then sonicated for 10 min at 4 °C (400 W) with 5-second intervals to obtain DHA algal oil liposomes. These DHA algal oil liposomes were then added dropwise to a 15 mg / mL soy protein isolate solution and stirred to obtain protein-coated DHA algal oil liposomes.

[0034] Comparative Example The difference from Example 1 is that the ultrasonically treated DHA algal oil liposomes are not added to the protein solution. Everything else is the same.

[0035] A method for preparing DHA algal oil liposomes includes the following steps: 270 mg of egg yolk lecithin, 67.5 mg of cholesterol, 54 mg of Tween-80, and 180 mg of DHA algal oil were dissolved in 100 mL of anhydrous ethanol to obtain an organic phase. This organic phase was then removed by rotary evaporation in water at 42 °C, yielding a suspension. This suspension was then sonicated for 10 min at 4 °C (400 W) with 5-second intervals to obtain DHA algal oil liposomes.

[0036] The performance of the DHA algal oil liposomes prepared in Examples 1-3 and the comparative example was tested. The specific process and results are as follows: 1. Particle size of protein-coated DHA algal oil liposomes Take 0.5 mL of the liposome samples prepared in the comparative example and Examples 1-3 respectively, dilute them 10 times, and then use a Malvern laser particle size analyzer to determine the particle size of algal oil DHA at 25°C. Perform three parallel measurements and take the average value.

[0037] The particle size results of the samples prepared in the comparative examples and Examples 1-3 are as follows: Figure 1As shown, the liposomes in Example 1 had the smallest particle size, at 88.94 nm. Compared with the liposomes in Examples 2 and 3, it can be seen that lactoferrin can be more tightly adsorbed to liposomes than whey protein isolate and soy protein isolate, effectively reducing the particle size of algal oil DHA.

[0038] 2. Encapsulation efficiency of protein-coated DHA algal oil liposomes The encapsulation efficiency of DHA algal oil in the liposome samples prepared in the comparative examples and Examples 1-3 was determined using organic solvent extraction. 1 mL of DHA algal oil liposomes was mixed with 4 mL of n-hexane solution, vortexed at room temperature, and then centrifuged at 6000 r / min for 10 minutes. The supernatant was collected, and this process was repeated twice, with the supernatants combined. The absorbance was then measured at the optimal wavelength of 274 nm. All samples were measured in triplicate, and the encapsulation efficiency was calculated using the following formula: EE = ( ×100% In the formula: m1 is the mass of DHA algal oil in the liposome suspension (initial addition amount); m2 is the mass of DHA algal oil in the supernatant.

[0039] The results are as follows Figure 2 As shown, the liposomes prepared in Examples 1 and 2 achieved an encapsulation rate of over 90% for DHA algal oil, and the encapsulation rates of Examples 1-3 were significantly higher than those of the comparative examples. The protein-coated liposomes introduced in this invention significantly improved the encapsulation rate of DHA algal oil compared to uncoated liposomes.

[0040] 3. Microscopic morphology of DHA algal oil liposomes The microstructure of liposomes was observed using negative staining. 10 μL of DHA algal oil liposomes from the comparative examples and Examples 1-3 were added to a copper grid. After standing for 5 min, excess sample around the grid was aspirated. The copper grid was then negatively stained with 2% (w / v) phosphotungstic acid solution for 5 min, and excess liquid around the grid was aspirated. The samples were then air-dried at room temperature, and the microstructure of the liposome samples was observed at 80 kV. For specific microstructure details, see [link to relevant documentation]. Figure 3 .

[0041] 4. Storage stability analysis of DHA algal oil liposomes The DHA algal oil liposomes prepared in Example 1 were placed at 4°C, and their particle size distribution, PDI, Zeta potential, and encapsulation efficiency were measured at 7, 14, 21, and 28 days to determine their stability. The test results are shown below. Figure 4 .

[0042] Figure 4The changes in (A) particle size, (B) PDI, (C) Zeta potential and (D) encapsulation efficiency of DHA algal oil liposome samples under 4℃ conditions are shown. As shown in the figure, under the condition of storage at 4℃ for 28 days, the particle size of protein-coated DHA algal oil liposomes decreased from 90.36±0.93nm to 80.65±1.97nm; the PDI increased from 0.196±0.002 to 0.257±0.002, and the PDI was still less than 0.3, indicating a stable state; after 28 days, the Zeta potential was still around 33mV, tending to stabilize; since the liposomes are in a dynamic equilibrium state after preparation, in the early stage of storage, components such as phospholipid molecules will undergo a certain degree of exchange and rearrangement, making the structure more compact, and the encapsulation rate shows a trend of first increasing and then decreasing. The encapsulation rate of protein-coated DHA algal oil liposomes can reach 95% after one week of storage, and the decrease is slow with the extension of storage time. After 21 days, the encapsulation rate is still around 91%, indicating that the DHA algal oil liposomes have good stability and can prevent DHA algal oil leakage.

[0043] 5. In vitro release experiment Transfer 10 mL of the DHA algal oil liposomes prepared in Example 1 into a pretreated dialysis bag (molecular weight cutoff of 1000 Da), seal the bag tightly, and immerse it in 200 mL of PBS buffer (pH=7.4) containing 1% Tween-80. Place the sample on a magnetic stirrer and rotate at 100 r / min at 37±1 °C. Subsequently, collect 10 mL of solution for measurement at intervals of 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 hours. After each sampling, rapidly replenish the PBS buffer release medium at the same temperature to ensure a constant total volume. Calculate the cumulative release rate according to the following formula:

[0044] Where V e V0 is the sampling volume (10 mL), V0 is the total volume (200 mL), and C is the total volume. l and C t , where m is the concentration (mg / L) of DHA algal oil in the solution at different release times, and m0 is the mass (mg) of oil encapsulated in the liposomes. Four release kinetic equations were selected for release analysis: zero-order kinetic equation, first-order kinetic equation, Higuchi planar diffusion equation, and Retger-Peppas equation. Curve fitting was performed to assess the accuracy of the fit, and then the in vitro release type of the liposomes was determined to elucidate the pattern of DHA algal oil liposome release.

[0045] Because DHA algal oil is poorly soluble in water, it is difficult to meet the leaky conditions required for in vitro release. Therefore, Tween-80 was used as a solubilizer in this experiment to increase the solubility of DHA algal oil in PBS buffer and meet the leaky conditions. To explore the release mechanism of DHA algal oil, its release kinetics were characterized by (A) zero-order kinetic equation, (B) first-order kinetic equation, (C) Higuchi plane diffusion equation, and (D) Retger-Peppas equation. The fitting equations and correlation coefficients of different models are shown below. Figure 5 As shown in Table 1, the correlation coefficient of the first-order kinetic equation fitting for Chlorella oil release was the highest, with the first-order fitting equation for protein-coated DHA algal oil liposomes being Q = 82.1829(1-exponential). -0.1214t ), R 2 =0.9865.

[0046] Table 1. Four release kinetic equations for Example 1 Fitting equation Fitting results <![CDATA[R 2 ]]> Zero-order dynamic equations Q = 1.4164t + 26.4578 0.8058 First-order dynamic equations <![CDATA[Q=82.1829(1-e -0.1214t )]]> 0.9865 Higuchi's Planar Diffusion Equation <![CDATA[Q=11.9160t 1 / 2 +8.8103]]> 0.9377 Retger-Peppas equations <![CDATA[Q=20.8560t 0.3752 ]]> 0.9611 Sampling time (t) and cumulative release percentage (Q) were used as the horizontal and vertical axes, respectively. Initially, the release rate of DHA from algal oil showed an upward trend, followed by a plateau, as shown in the figure. Figure 5 As shown, the correlation coefficient of the first-order kinetic equation fitting for DHA algal oil release was the highest, making it more suitable for describing the release of algal oil. In Example 1, the release rate of protein-coated DHA algal oil liposomes remained stable at 40.32 ± 2.2% in the first 6 hours. After 6 hours, the release rate gradually increased, reaching a cumulative release rate of 82.48 ± 0.99% at 48 hours. This indicates that liposomes, as a carrier of DHA algal oil, have a significant sustained-release effect.

[0047] Experimental Conclusion: This invention provides DHA algal oil nanoliposomes and their preparation method, aiming to prolong the storage stability of DHA algal oil while achieving slow release in the intestine and improving bioavailability. DHA algal oil nanoliposomes are prepared by encapsulating DHA algal oil using egg yolk lecithin and cholesterol as wall materials via ultrasound-assisted thin-film dispersion. Experiments showed that the storage stability of protein-coated DHA algal oil nanoliposomes was significantly improved, and the release mechanism of DHA algal oil nanoliposomes was revealed. The highest correlation coefficient was found when fitting the release of small DHA algal oil nanoliposomes using a first-order kinetic equation. The preparation method provided by this invention provides a scientific basis for the development of functional lipid carriers and the widespread application of liposome technology in food processing, expanding the application of liposome-encapsulated bioactive substances in functional foods.

[0048] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0049] It should be understood that the above detailed description of the technical solutions of the present invention with reference to preferred embodiments is illustrative and not restrictive. Those skilled in the art can modify the technical solutions described in the embodiments or make equivalent substitutions for some of the technical features based on reading this specification; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing protein-coated DHA algal oil liposomes, characterized in that, Lecithin, cholesterol, and DHA algal oil were dissolved in a solvent to obtain an organic phase. This phase was then rotary evaporated and hydrated to obtain a suspension. The suspension was then sonicated in an ice-water bath to obtain DHA algal oil liposomes. The obtained DHA algal oil liposomes were then added dropwise to a protein solution to obtain protein-coated DHA algal oil liposomes.

2. The method according to claim 1, characterized in that, The lecithin is egg yolk lecithin, and further, the mass ratio of the DHA algal oil, cholesterol and egg yolk lecithin is 1:0.3-0.5:1.0-3.

0.

3. The method according to claim 1, characterized in that, The solvent is one or a mixture of two or more of anhydrous ethanol, chloroform, methanol or ethyl acetate.

4. The method according to claim 1, characterized in that, The solvent also contains Tween-80, and the mass ratio of Tween-80 to DHA algal oil is 1:2.5 to 4.

5. and / or The mass ratio of DHA algal oil to protein is 1:1.5 to 3.

5.

5. The method according to claim 1, characterized in that, The protein is one of lactoferrin, soy protein isolate, and whey protein isolate.

6. The method according to claim 1, characterized in that, The algal oil DHA liposomes were prepared according to the following steps: S1: Weigh out egg yolk lecithin, cholesterol, Tween-80 and DHA algal oil according to the ratio, add solvent, and dissolve by sonication in a water bath; S2: After complete dissolution, the mixture solution is rotary evaporated at 35-45℃ to remove the solvent, and then hydrated to obtain a suspension; S3: The suspension is ultrasonicated in an ice-water bath. The ultrasonication is a programmed ultrasonication performed using an ultrasonic cell disruptor at 300-500W for 5-15 minutes with an interval of 3-6 seconds to obtain DHA algal oil nanoliposomes. S4: Add the DHA algal oil nanoliposomes to the protein solution and stir at 100-300 r / min for 60 min.

7. A protein-coated DHA algal oil liposome, prepared by the method of any one of claims 1-6.

8. The liposomes according to claim 7, characterized in that, The particle size of the DHA algal oil liposomes was 90.36±0.93 nm; and / or The polydispersity index (PDI) of the particles was 0.196 ± 0.002; the particle uniformity was good. and / or The encapsulation efficiency of the DHA algal oil liposomes was 91.50 ± 0.39%; and / or The zeta potential of the DHA algal oil liposomes was 35.20 ± 0.35 mV; and / or The DHA algal oil liposomes were verified to have a sustained-release effect using a simulated gastrointestinal model. and / or The DHA algal oil liposomes exhibited good storage stability at 4°C.

9. The present invention also provides the application of protein-coated DHA algal oil liposomes in the preparation of products that have antioxidant, memory-improving, memory-aiding, visual fatigue-relieving, sleep-improving, physical fatigue-relieving, hypoxia-resistant, or body fat-controlling properties; wherein the liposomes are prepared by the method of any one of claims 1-6.

10. A method for preparing protein-coated DHA fish oil liposomes, characterized in that, Replace the algal oil in claims 1-6 with fish oil.

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