Phospholipid-epicatechin liposome and preparation method and application thereof

Abstract

The application discloses a phospholipid-epicatechin liposome and a preparation method and application thereof, and relates to the field of food preservation technology. The preparation method of the phospholipid-epicatechin liposome comprises the following steps: taking a functional phospholipid fraction as a membrane material, taking epicatechin as an active component, and preparing the phospholipid-epicatechin liposome by an ethanol injection-ultrasonic method; and the functional phospholipid fraction is prepared by taking low-value sturgeon caviar as raw material, extracting by chloroform-methanol and purifying by silica gel column chromatography. The phospholipid-epicatechin liposome provided by the application can be applied to sturgeon caviar preservation, can effectively delay product brightness attenuation, inhibit abnormal accumulation of volatile basic nitrogen and malondialdehyde, retard excessive degradation of protein, and maximally retain key functional fatty acids such as EPA and DHA in the caviar.

Description

Technical Field

[0001] This invention relates to the field of food preservation technology, and in particular to a phospholipid-epicatechin liposome, its preparation method, and its application. Background Technology

[0002] Sturgeon caviar is a high-end processed seafood product, rich in high-quality protein, phospholipids, and polyunsaturated fatty acids such as EPA and DHA. Its nutritional value and economic added value are outstanding, but the inherent physicochemical properties of the raw material make its storage and preservation challenging. Caviar matrix has a high water activity and rich nutritional components, making it highly susceptible to multi-pathway quality deterioration under conventional refrigerated storage and transportation conditions. On the one hand, the unsaturated fatty acids within the system are continuously oxidized by oxygen and endogenous lipoxygenases, constantly generating harmful oxidizing substances such as malondialdehyde. This not only destroys the product's inherent nutritional value but also produces a pungent, fishy odor. On the other hand, endogenous proteases in the fish meat continuously decompose large protein molecules, causing an abnormal increase in volatile basic nitrogen and free amino acid content, resulting in soft, mushy meat, a dull, yellowish color, a significant decline in the product's sensory grade, and severely shortening its shelf life.

[0003] Currently, the mainstream preservation methods in the industry are divided into two categories: physical preservation and exogenous additive preservation. Low-temperature refrigeration can only slow down the rate of spoilage, but it cannot inhibit lipid oxidation and protein degradation at the root. Long-term cold chain will also increase storage and transportation costs. Although high-salt pickling can inhibit bacteria, excessive salt will destroy the original flavor and nutritional structure of caviar. Although artificially synthesized preservatives have antioxidant capabilities, they have the disadvantages of strict control over the amount added to food and low consumer acceptance. Most existing preservation materials only focus on a single antioxidant or antibacterial function, making it difficult to simultaneously meet the multiple needs of color protection, nutrient retention, and flavor stabilization.

[0004] Meanwhile, the sturgeon processing industry continuously generates a large amount of low-value waste caviar byproducts that are of insufficient quality and cannot be directly sold commercially. For a long time, there has been a lack of technology for the resource utilization of these byproducts, leading to their mostly direct disposal as waste. This not only wastes high-quality phospholipid resources but also creates environmental pressure. Current phospholipid extraction methods mostly use general chemical raw materials, resulting in high raw material costs. There is a lack of industrialization strategies for the targeted preparation of functional phospholipids from aquatic processing byproducts. The industry lacks an integrated technical solution that can simultaneously achieve high-value conversion of byproducts and long-term preservation of finished caviar. Developing novel natural-derived preservation carriers has become an urgent technological need in the field of aquatic product preservation. Summary of the Invention

[0005] The purpose of this invention is to provide a phospholipid-epicatechin liposome, its preparation method, and its application, to solve the problems existing in the prior art. The phospholipid-epicatechin liposome provided by this invention can be used for the preservation of sturgeon caviar, effectively delaying the decline in product brightness, inhibiting the abnormal accumulation of volatile basic nitrogen and malondialdehyde, blocking excessive protein degradation, and maximizing the retention of key functional fatty acids such as EPA and DHA in caviar.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing phospholipid-epicatechin liposomes, comprising the steps of using functional phospholipid fractions as membrane material, using epicatechin as active component, and preparing the phospholipid-epicatechin liposomes by ethanol injection-ultrasound method; The functional phospholipid fraction was prepared from low-value sturgeon caviar as raw material by chloroform-methanol extraction and silica gel column chromatography purification.

[0007] Furthermore, the ethanol injection-ultrasound method includes the following steps: The functional phospholipid fraction and epicatechin were dissolved in ethanol at a mass ratio of (5-20):1 to form an organic phase; the organic phase was injected into the aqueous phase and subjected to ultrasonic treatment and static aging treatment to obtain the phospholipid-epicatechin liposomes.

[0008] Furthermore, the conditions for the ultrasonic treatment are: ice bath, power 300-500 W, treatment time 5-10 min; and / or The settling and ripening process takes 4 to 12 hours.

[0009] Preferably, the mass ratio of the functional phospholipid fraction to the epicatechin is 10:1; the conditions for ultrasonic treatment are: ice bath, power 400 W, treatment for 8 min; and the time for static aging treatment is 8 h.

[0010] Furthermore, the chloroform-methanol extraction includes the following steps: Low-value sturgeon caviar powder was added to a chloroform / methanol mixture with a volume ratio of 2:1 and subjected to ultrasonic extraction. Water was added and the mixture was centrifuged to separate the layers. The lower layer was collected and the upper aqueous phase was repeatedly extracted with chloroform. The collected lower layers were combined, the solvent was removed by rotary evaporation, and precipitated with acetone. The precipitate was obtained by centrifugation and dried with nitrogen to obtain crude chloroform-methanol phospholipids.

[0011] Furthermore, the silica gel column chromatography purification uses 200-300 mesh silica gel, the sample loading amount to silica gel mass ratio is 1:50, and the eluent is a dichloromethane / methanol mixture with a volume ratio of 3:2.

[0012] The present invention also provides a phospholipid-epicatechin liposome prepared according to the above preparation method.

[0013] The present invention also provides the use of the above-mentioned phospholipid-epicatechin liposomes in (1) or (2): (1) Preparation of a preservative for sturgeon caviar; (2) Preservation of sturgeon caviar.

[0014] The present invention also provides a sturgeon caviar preservative comprising the above-mentioned phospholipid-epicatechin liposome.

[0015] The present invention also provides a method for preserving sturgeon caviar, comprising the step of adding the above-mentioned phospholipid-epicatechin liposome to sturgeon caviar for preservation.

[0016] Furthermore, the phospholipid-epicatechin liposome is added to the sturgeon caviar at an amount of 0.02 to 0.20 mL / g, and the addition method includes, but is not limited to, surface spraying, dripping, stirring, and coating; the preferred addition amount is 0.05 to 0.15 mL / g.

[0017] The present invention discloses the following technical effects: This invention develops a novel food preservation material using low-value sturgeon caviar byproducts as raw materials and caviar-homogeneous phospholipids as carriers to prepare phospholipid-epicatechin liposomes. It leverages the extraction and purification of phospholipids from byproducts to achieve resource utilization of waste materials, effectively filling the technological gap in high-value conversion of aquatic byproducts and composite preservation of caviar. This invention obtains purified phospholipids enriched with EPA and DHA polyunsaturated phospholipid components through column chromatography screening. These phospholipids are suitable for caviar preservation and caviar matrix compatibility, reducing the introduction of exogenous lipid carriers. Furthermore, these phospholipids exhibit excellent biocompatibility, showing no cytotoxicity within commonly used food additive concentrations, thus conforming to food industry application standards. This invention uses epicatechin as the active ingredient to construct the liposome structure. Epicatechin forms hydrogen bonds with the phospholipids through phenolic hydroxyl groups and polar heads, improving the orderliness and storage stability of the bilayer membrane. The liposomes prepared by this invention have uniform particle size, excellent dispersibility, minimal fluctuations in membrane hydrophobicity during storage, and a slow increase in peroxide value, demonstrating outstanding storage stability and free radical scavenging ability. When this liposome is applied to the preservation of sturgeon caviar, it can effectively delay the decline in product brightness, inhibit the abnormal accumulation of volatile basic nitrogen and malondialdehyde, block excessive protein degradation, and retain key functional fatty acids such as EPA and DHA in caviar to the greatest extent. Compared with conventional preservation methods, this invention can achieve a synergistic improvement in multiple preservation effects, including sensory, nutritional, and antioxidant properties. Furthermore, by reusing homologous phospholipids derived from caviar byproducts, it reduces dependence on exogenous preservation carriers, thus combining economic value with food safety. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Statistical graphs showing the purity and recovery rate of phospholipids under different silica gel mesh sizes (A), sample loading amounts (B), and eluent ratios (C); Figure 2 Heatmaps of cluster analysis of phosphatidylcholine metabolites from commercially available phosphatidylcholine (EPC), crude phosphatidylcholine (RPL), and column chromatography-purified phosphatidylcholine (PPL); Figure 3 The graph shows the cytotoxicity test results of commercially available phosphatidylcholine (A) and the phospholipid (B) prepared in this invention; Figure 4 The images are transmission electron microscope images of liposomes; where AD represents EPC-empty, EPC-EC, EPC-BHA, and EPC-BHT, respectively; and ad represents PPL-empty, PPL-EC, PPL-BHA, and PPL-BHT, respectively; the scale bar is 1 μm. Figure 5 The images are atomic force microscopy images of liposomes; where AD represents EPC-empty, EPC-EC, EPC-BHA, and EPC-BHT, respectively; ad represents PPL-empty, PPL-EC, PPL-BHA, and PPL-BHT, respectively; the scale bar is 4 μm. Figure 6 Statistical graph showing the scavenging rates of different EPC and PPL liposomes against ABTS radicals (A) and DPPH radicals (B); Figure 7 The changes in membrane hydrophobicity of different EPC liposomes (A) and PPL liposomes (B) during storage at 4℃ from 0 to 4 W are shown in the figure. Figure 8 The graph shows the peroxide value changes of empty liposomes (A) and liposomes loaded with active ingredients (B) stored at 4°C for 0-4 W. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1: Screening of methods for crude phospholipid extraction from sturgeon caviar Acetone-ethanol extraction method: First, weigh 100 g of low-value sturgeon caviar freeze-dried powder into a beaker, add acetone at a material-to-liquid ratio of 1:10 (g:mL) for defatting, and stir at 300 rpm for 1.5 h. After defatting, filter; the filter cake is acetone-insoluble, and the filtrate is an acetone solution containing sturgeon caviar oil. Place the filter cake in a 40℃ constant temperature drying oven and dry for 1.5 h to obtain defatted caviar powder. Weigh the defatted caviar powder into a beaker, add 93.77% ethanol solution at a material-to-liquid ratio of 1:16 to extract phospholipids. After extraction, filter and separate; the supernatant is rotary evaporated at 40℃ to recover the ethanol. The obtained phospholipids are crude phospholipids extracted with acetone-ethanol.

[0026] Chloroform-methanol extraction method: First, weigh 100 g of low-value sturgeon caviar freeze-dried powder, add 300 mL of chloroform / methanol (chloroform:methanol = 2:1, v / v), sonicate at room temperature for 30 min, then add 100 mL of ultrapure water, centrifuge at 7000 r / min for 10 min, and collect the lower layer liquid. Add 200 mL of chloroform to the upper layer sample, repeat the extraction 3 times, combine the collected lower layer liquids, and rotary evaporate at 40℃ to obtain an oily lipid mixture. After cooling, add 100 mL of acetone at 4℃, centrifuge at 8000 r / min for 10 min, remove the supernatant, and blow dry the precipitate with nitrogen to obtain phospholipids. The obtained phospholipids are crude chloroform-methanol extracted phospholipids.

[0027] As shown in Table 1, the purity of crude phospholipids obtained by the chloroform-methanol extraction system was 69.86%, while the purity of phospholipids obtained by the acetone-ethanol system was only 39.96%. Therefore, the chloroform-methanol extraction method was chosen.

[0028] Table 1 Purity of crude phospholipids Note: Data are expressed as mean ± standard deviation (n=3); different superscript letters in the same column indicate significant differences between samples. P <0.05).

[0029] Example 2: Optimization of purification conditions for functional phospholipid fractions derived from sturgeon caviar Crude phospholipids extracted with chloroform-methanol were purified by silica gel column chromatography. The crude phospholipids were completely dissolved in a small amount of dichloromethane solution and slowly added dropwise to the column, ensuring silica gel surface equilibrium. The eluents containing the target phospholipids were combined and concentrated under reduced pressure at 35-40 °C to remove dichloromethane and methanol. The eluents were then dried under nitrogen to constant weight to obtain the column-purified functional phospholipid fraction derived from sturgeon caviar. Single-factor purification experiments were conducted using activated silica gel powders of 100-200 mesh, 200-300 mesh, and 300-400 mesh, sample / silica gel mass ratios of 1 / 40, 1 / 50, and 1 / 60, and dichloromethane / methanol volume ratios of 2:3, 3:2, and 1:1.

[0030] Depend on Figure 1 As shown in Figure A, the purity of the purified phospholipids obtained from the three mesh sizes of silica gel powder were all above 90%, and column chromatography elution significantly improved the purity of the phospholipids. Specifically, the phospholipid purity after purification with 200-300 mesh silica gel powder reached 96.69%, significantly different from the other two groups. The phospholipid purities after purification with 100-200 mesh and 300-400 mesh silica gel powder were 90.82% and 92.95%, respectively. Furthermore, the recovery rate of phospholipids purified with 200-300 mesh silica gel powder (28.51%) was also significantly higher than the other two groups. Therefore, 200-300 mesh silica gel powder is the preferred choice.

[0031] Depend on Figure 1 As shown in Figure B, the change in the loading ratio has no significant effect on the purity of phospholipids, and the silica gel corresponding to the three loading amounts has a similar adsorption capacity for the samples. Among them, the recovery rate of phospholipids purified by loading at 1 / 50 is slightly higher, at 28.51%. Therefore, the loading amount of 1 / 50 is preferred for subsequent optimization.

[0032] Depend on Figure 1 As shown in Figure C, the purity and recovery rate of the purified phospholipids decreased with increasing methanol ratio in the eluent. The highest purity and recovery rate were achieved when the dichloromethane:methanol ratio was 3:2. At a elution ratio of 2:3, the purity of the purified phospholipids was significantly lower than the other two groups, at 86.41%; the recovery rate was 20.42%, which was not significantly different from the 1:1 elution ratio group, but lower than the 3:2 elution ratio group. Therefore, a dichloromethane / methanol ratio of 3:2 is the preferred eluent ratio for column chromatography purification.

[0033] Using 200-300 mesh silica gel, a sample / silica gel mass ratio of 1 / 50, and a dichloromethane / methanol volume ratio of 3:2 as elution conditions, functional phospholipid fractions—column chromatography purified phospholipids (PPL) with a purity of 96.69% were prepared.

[0034] Example 3: Changes in the content of fatty acids and molecular species of functional phospholipids in sturgeon caviar-derived functional phospholipid fractions. The free fatty acid composition of crude phospholipids (RPL) and column chromatography-purified phospholipids (PPL) obtained in Example 2 was determined and is shown in Table 2. It was found that compared with crude phospholipids, the content of most fatty acids in column chromatography-purified phospholipids decreased or even completely eluted. Although the content of EPA and DHA also decreased significantly, they were still the main fatty acid components of purified phospholipids. Purification achieved relative enrichment of EPA and DHA.

[0035] Table 2. Fatty acid composition and enrichment characteristics before and after column chromatography Using commercially available phosphatidylcholine (EPC) and crude phospholipids (RPL) as controls, the changes in molecular species of sturgeon caviar phospholipids before and after purification were determined, and a total of 147 PC molecular species were identified. Figure 2The 50 most abundant PC species were presented. The results showed that column chromatography purification did not result in proportional enrichment or loss of each PC species, but rather exhibited significant structure selectivity. Some highly unsaturated PC species actually increased in content after purification, becoming the dominant components in PPL, such as PC (22:6 / 21:0), which increased from 6.36% to 8.01%. Conversely, some low-unsaturated PC species disappeared completely during purification, such as PC (22:1 / 22:6) and PC (18:1 / 24:1). Furthermore, the abundance changes of PC species in PPL and RPL were significantly higher than in EPC, especially in PC species containing polyunsaturated fatty acids such as EPA and DHA. Caviar-derived phospholipids showed rich diversity; for example, PC (19:0 / 22:6), PC (15:2 / 22:6), and PC (15:0 / 22:5) were detected only in RPL and PPL, while EPC was almost completely absent in these species. It is worth noting that the preferential retention of highly unsaturated PC molecules during column chromatography purification means that PPL still retains a considerable proportion of flavor precursors. In subsequent applications, it can not only play the role of the liposome's antioxidant barrier function, but also provide a material basis for flavor regulation during caviar preservation. On the other hand, the removal of low-unsaturated PC molecules after purification helps to delay the oxidative deterioration of the liposomes themselves and extend the effective window of preservation.

[0036] Example 4: Safety and compositional characteristics of functional phospholipid fractions The safety of purified phospholipids (column-chromatographically purified phospholipids prepared in Example 2) was evaluated using HepG2 cells. The HepG2 cell line is commonly used to study liver and foreign body metabolism, substance toxicity, genotoxicity, cytotoxicity, and drug targeting. Given the presence of residual toxic organic solvents, especially chloroform, which can enter the bloodstream through skin contact, respiratory tract, and digestive tract, causing gastrointestinal damage, dermatitis, and hepatotoxicity, the HepG2 cell line was chosen to evaluate the toxicity of column-chromatographically purified phospholipids (PPL), using commercially available phosphatidylcholine (EPC) as a reference. According to the international standard ISO 10993-5:2009, when the cell viability after treatment with the extract drops to <70% of the blank, it is considered to have potential cytotoxicity.

[0037] Depend on Figure 3As can be seen, within the concentration range of 12.5-300 μg / mL, the cell viability of both phospholipids was above 70% (EPC 87.59%-99.94%, PPL 80.34%-99.88%), indicating that neither exhibited significant toxicity to HepG2 cells within this concentration range and possessed good biocompatibility. When the concentration increased to 500 μg / mL, the cell viability of EPC decreased to 69.62%, below the safety threshold of 70%; the viability of PPL at the same concentration was 65.12%. At concentrations of 600 μg / mL and 1000 μg / mL, the viability of the two materials further decreased to approximately 54% and 15%-21%, respectively, exhibiting significant dose-dependent cytotoxicity. At a test concentration of 1000 μg / mL, the cell viability of PPL (20.67%) was slightly higher than that of EPC (14.72%), revealing that PPL has lower cytotoxicity and better biocompatibility, and has the potential to be further applied to liposome encapsulation.

[0038] Example 5: Preparation and Optimization of Liposomes 1. Preparation of liposomes Using the functional phospholipid fraction obtained in Example 2 or the commercially available control phosphatidylcholine (EPC) as the membrane material, and epicatechin (EC), butylated hydroxyanisole (BHA), and 2,6-di-tert-butyl-p-cresol (BHT) as active components, liposomes were prepared via ethanol injection-ultrasound method. The mass ratio of membrane material to active components was controlled at (5–20):1. Ultrasound conditions included an ice bath, a power of 300–500 W, a treatment time of 5–10 min, and a maturation time of 4–12 h. After optimization of the preparation conditions, the target liposomes were finally prepared according to the following optimal preparation method: The membrane material and active components were mixed in ethanol at a mass ratio of 10:1 to form an organic phase; the organic phase was injected into the aqueous phase at a constant rate to form an initial liposome suspension; then ultrasonic treatment was performed under ice bath conditions of 400 W for 8 min; finally, the suspension was allowed to stand at 4℃ for 8 h to obtain the target liposomes.

[0039] The potential of epicatechin, butylated hydroxyanisole, and 2,6-di-tert-butyl-p-cresol to form liposomes with phospholipids was investigated according to the above-mentioned optimal preparation method. At the same time, empty vector groups EPC-empty and PPL-empty without active components were set as controls.

[0040] 2. Screening of active components and selection of final liposomes Table 3 shows that the average particle size of the EPC group was smaller than that of the PPL group. The empty EPC particle size was only 177.93 nm, and the particle size further decreased after loading BHA / EC, with EPC-BHA particle size at 98.89 nm and EPC-EC particle size at 110.42 nm. In the PPL group, the particle size of the antioxidant-loaded PL liposomes was smaller than that of the empty PPL group, with PPL-EC having the smallest particle size at 245.30 nm. The addition of antioxidants significantly reduced the particle size of the liposomes. The particle size of the EPC group was all below 200 nm, and all liposomes were monolayer vesicles. The PDI of each group was less than 0.3, indicating good dispersibility of the liposome suspension. All liposomes carried a negative charge, and the absolute value of the liposome potential increased significantly after loading with antioxidants, especially the EC-loaded liposomes, with the EPC-EC liposome reaching a zeta potential of -8.20 and the PPL-EC liposome reaching a zeta potential of -16.67. Overall, the antioxidant EC showed the best effect in improving liposome particle size, PDI, and ζ potential.

[0041] Table 3. Criteria for screening active components and selecting the final liposomes The above results demonstrate that EC is not an arbitrarily selected active component. Although BHA exhibits superior performance in some thermal stability indicators, considering the overall results of particle size, potential, antioxidant activity, and storage stability, EC is more suitable as the preferred active component in the final application scheme of this invention.

[0042] like Figure 4 As shown, the morphology of liposomes was observed using transmission electron microscopy (TEM). All liposome samples exhibited nanoscale dimensions, with most particles forming spherical vesicles with clear boundaries. The particle size distribution was consistent with the results from the nanoparticle size analyzer. In the EPC group, EPC-EC and EPC-BHA had the smallest and most uniform particle size, while EPC-unloaded and EPC-BHT had slightly larger particle sizes and exhibited some aggregation, resulting in poor dispersibility. In the PPL group, the vesicles were relatively large, possibly due to the complex phospholipid composition of PPL, containing multiple polar head groups and fatty acid chains. In comparison, both EC and BHA-loaded liposomes maintained good spherical structure and dispersibility, while BHT loading had a more significant impact on liposome morphology, especially in PPL liposomes, showing increased particle size, irregular morphology, and a tendency to aggregate.

[0043] Figure 5The results of atomic force microscopy (AFM) measurements are presented. EPC-empty liposomes are uniformly spread on the mica sheet surface with a relatively uniform particle size distribution and no aggregation, indicating that EPC-empty liposomes have good structural stability and dispersibility. After EC loading, EPC liposomes still maintain a spherical morphology, but the surface roughness of the particles increases slightly, and the edges of some liposomes become slightly blurred. EC embedding may have caused local changes in physicochemical properties. In contrast, liposomes with BHA embedding are more uniformly distributed and have good compatibility with EPC. After BHT loading, some EPC liposome particles show a tendency to increase in size, and slight adhesion exists in some areas. BHT embedding also has a certain impact on the liposome membrane structure. Compared with the EPC group, PPL liposomes have a slightly wider particle size distribution and some particles are uneven in size, but overall, the dispersibility is good and there is no obvious aggregation, indicating that sturgeon caviar-extracted mixed phospholipids also have the ability to form stable liposomes. After EC loading, PPL-EC liposomes also exhibit some irregular shapes and a small amount of aggregation.

[0044] Overall, both TEM and AFM showed that EPC liposomes had better structural stability than PL liposomes after being loaded with antioxidants, exhibiting smaller particle size and more uniform distribution. PPL liposomes, due to the presence of multiple phospholipid components, had a more complex membrane structure, with a slightly wider particle size distribution, but they could still form a stable vesicle structure.

[0045] The results of ABTS free radical scavenging rate detection for each liposome are as follows: Figure 6 As shown in Figure A, the scavenging rates of both EPC-empty and PPL-empty liposomes were relatively low, at 3.67% and 2.99%, respectively, but they still exhibited certain antioxidant activity. A significant difference existed between the two groups, with the EPC group showing superior activity compared to the PPL group. Compared to the empty group, liposomes loaded with EC, BHA, and BHT showed significantly increased ABTS free radical scavenging rates, indicating that the addition of antioxidants can enhance the antioxidant performance of liposomes. Specifically, the scavenging rates of EPC-EC and PPL-EC reached 86.81% and 70.12%, respectively, significantly higher than those of the BHA and BHT groups. Significant differences also existed between the EPC and PPL groups for liposomes loaded with the same antioxidants. Under the same antioxidant conditions, the antioxidant activity of the EPC group liposomes was significantly higher than that of the PPL group, which may be related to the stronger antioxidant activity of EPC itself and the compatibility between phospholipids and antioxidants.

[0046] The DPPH free radical scavenging rate of each liposome is as follows: Figure 6As shown in Figure B, the free radical scavenging rate of DPPH also showed a similar trend. Compared with the empty loading group, the DPPH scavenging rate of EPC-EC (17.45%) was about 25 times higher than that of EPC-empty loading (0.66%), and the DPPH scavenging rate of PPL-EC (31.13%) was about 8 times higher than that of PPL-empty loading (3.39%). EC liposomes still showed the strongest antioxidant activity, followed by BHA > BHT. However, unlike the free radical scavenging results of ABTS, under the same antioxidant conditions, the PPL group showed a higher DPPH scavenging rate than the EPC group. This may be due to the fact that PPL is rich in polyunsaturated fatty acids, has strong membrane fluidity and a large hydrophobic interlayer gap, and DPPH can more easily penetrate into the hydrophobic region of the membrane to undergo a highly efficient hydrogen atom transfer (HAT) reaction, thereby achieving free radical scavenging.

[0047] Each liposome was stored at 4°C for 4 weeks. During this period, membrane hydrophobicity and peroxide value (POV) were measured to assess its storage stability. The results are shown below. Figures 7-8 .

[0048] like Figure 7As shown, the overall fluorescence intensity of the EPC group liposomes was higher than that of the PPL group, indicating stronger hydrophobicity and more stable membrane structure. In the empty-load group liposomes, the membrane hydrophobicity of the EPC-empty-load group did not change significantly before and after storage, remaining relatively stable at 817.57 at 0 W and 819.63 at 4 W. The membrane hydrophobicity of the PPL-empty-load group decreased significantly from the second week onwards, fluctuating considerably from 321.97 at 0 W to 330.87 at 2 W, and then dropping to 300.93 at 4 W. This is attributed to the lower saturation of PPL itself, resulting in a less dense membrane layer arrangement. The membrane hydrophobicity of EC-loaded EPC and PPL liposomes changed little during storage, remaining essentially the same at 0 W and 4 W. The fluorescence intensities of the EPC group were 819.53 and 826.10, respectively, while those of the PPL group were 339.80 and 342.37, respectively. This is attributed to the hydrogen bonds formed between the phenolic hydroxyl groups of EC and the phospholipid heads, which limited changes in membrane structure. The BHA-loaded EPC and PPL liposomes exhibited the lowest fluorescence intensities at the initial storage period (0 W), at 615.67 and 297.93, respectively. After 1 and 3 W of storage, the fluorescence intensities increased, possibly due to structural rearrangement, but remained at the lowest position, indicating the weakest hydrophobicity. After 4 W of storage, the fluorescence intensities decreased again. The EPC group showed an increase compared to 0 W, reaching 672.97, but the PPL group dropped to a lower intensity of 276.43, possibly because the PPL membrane structure is more loose and fluid, allowing smaller BHA molecules to more easily insert into the hydrophobic layer and disrupt the orderly stacking of fatty acid chains. EPC-BHT and PPL-BHT consistently exhibited significantly higher membrane hydrophobicity than the empty loading group and other active component groups during storage, with fluorescence intensities of 874.87 and 355.43, respectively, at 0 W. This is likely due to the high hydrophobicity of BHT.

[0049] like Figure 8As shown, the POV of almost all liposomes increased with prolonged storage time, and the POV of the empty liposome group was significantly higher than that of the antioxidant-loaded liposomes. The incorporation of antioxidants into the liposomes significantly affected the formation of hydrogen peroxide. The POV increase rate of EPC-empty liposomes was significantly higher than that of PPL-empty liposomes. This may be because the compact membrane structure of EPC-empty liposomes accelerated the chain reaction of lipid oxidation. Only the POV of EPC-EC and EPC-BHT decreased with prolonged storage time, and these two groups of liposomes also exhibited low POV levels at 0 W storage, at 5.98 μg / mL and 5.80 μg / mL, respectively, demonstrating good oxidative stability. The decrease in POV is due to the easy decomposition of unstable primary oxidation product hydrogen peroxide, which forms carbonyl compounds. However, the POV of BHT-PPL-bound liposomes during storage was significantly higher than that of other antioxidant liposomes, reaching 8.36 μg / mL at 0 W, consistent with previous findings that antioxidants like EPC have better compatibility with PPL. While the POV of the PPL-EC group continued to rise during storage, the increase was slow, and its POV was similar to that of the EPC-EC and EPC-BHT groups, exhibiting the lowest POV (4.40 μg / mL) at 0 W, indicating that PPL-EC liposomes have better antioxidant inhibition capabilities.

[0050] Example 6: Application of Sturgeon Caviar in Cold Storage and Preservation The PPL-EC or EPC-EC liposomes prepared in Example 5 were uniformly sprayed onto the surface of sturgeon caviar at an addition rate of 0.1 mL / g (i.e., 0.1 mL of liposomes per 1 g of sturgeon caviar) and stored at 4°C. A blank control group without liposome spraying was also included. After 20 days of storage, the effects on... L Brightness value, as well as the contents of volatile basic nitrogen (TVB-N), malondialdehyde (MDA), total free amino acids, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), were measured. The results are shown in Table 4.

[0051] After 20 days of storage, the brightness value of the PPL-EC treatment group was 29.50, significantly higher than that of the control group (22.51); TVB-N decreased from 18.43 mg / 100 g in the control group to 15.86 mg / 100 g; MDA decreased from 70.02 nmol / g to 65.46 nmol / g; total free amino acids decreased from 977.24 mg / 100 g to 866.06 mg / 100 g; and EPA and DHA increased to 15.21 mg / g and 48.03 mg / g, respectively.

[0052] The EPC-EC treatment group also showed good preservation and nutrient retention effects, but the overall effect was not as good as that of PPL-EC. At 20 days, the brightness value of the EPC-EC treatment group was 24.40, TVB-N was 17.96 mg / 100 g, MDA was 69.67 nmol / g, and EPA and DHA were 13.74 mg / g and 44.56 mg / g, respectively.

[0053] Based on the above experimental results, it can be concluded that PPL-EC treatment of sturgeon caviar can delay the decline in brightness, inhibit the increase of volatile basic nitrogen, inhibit the accumulation of malondialdehyde, slow down protein degradation, and maintain the content of EPA and DHA.

[0054] Table 4 Key endpoint data for the preservation effect of sturgeon caviar (20 days) The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing phospholipid-epicatechin liposomes, characterized in that, The process includes the steps of preparing the phospholipid-epicatechin liposomes by using functional phospholipid fractions as membrane materials and epicatechin as the active component via ethanol injection-ultrasound method. The functional phospholipid fraction was prepared from low-value sturgeon caviar as raw material by chloroform-methanol extraction and silica gel column chromatography purification.

2. The preparation method according to claim 1, characterized in that, The ethanol injection-ultrasound method includes the following steps: The functional phospholipid fraction and epicatechin were dissolved in ethanol at a mass ratio of (5-20):1 to form an organic phase; the organic phase was injected into the aqueous phase and subjected to ultrasonic treatment and static aging treatment to obtain the phospholipid-epicatechin liposomes.

3. The preparation method according to claim 2, characterized in that, The conditions for the ultrasonic treatment are: ice bath, power 300-500 W, treatment time 5-10 min; and / or The settling and ripening process takes 4 to 12 hours.

4. The preparation method according to claim 3, characterized in that, The mass ratio of the functional phospholipid fraction to the epicatechin is 10:1; the conditions for ultrasonic treatment are: ice bath, power 400 W, treatment for 8 min; and the time for static aging treatment is 8 h.

5. The preparation method according to claim 1, characterized in that, The chloroform-methanol extraction includes the following steps: Low-value sturgeon caviar powder was added to a chloroform / methanol mixture with a volume ratio of 2:1 and subjected to ultrasonic extraction. Water was added and the mixture was centrifuged to separate the layers. The lower layer was collected and the upper aqueous phase was repeatedly extracted with chloroform. The collected lower layers were combined, the solvent was removed by rotary evaporation, and precipitated with acetone. The precipitate was obtained by centrifugation and dried with nitrogen to obtain crude chloroform-methanol phospholipids.

6. The preparation method according to claim 1, characterized in that, The silica gel column chromatography purification uses 200-300 mesh silica gel, the sample loading amount to silica gel mass ratio is 1:50, and the eluent is a dichloromethane / methanol mixture with a volume ratio of 3:

2.

7. A phospholipid-epicatechin liposome prepared by the preparation method according to any one of claims 1-6.

8. The use of the phospholipid-epicatechin liposome as described in claim 7 in (1) or (2): (1) Preparation of a preservative for sturgeon caviar; (2) Preservation of sturgeon caviar.

9. A caviar preservative, characterized in that, Including the phospholipid-epicatechin liposome as described in claim 7.

10. A method for preserving sturgeon caviar, characterized in that, The step includes adding the phospholipid-epicatechin liposomes of claim 7 to sturgeon caviar for preservation.

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