Salicylic acid nano-liposome cold damage resistant preservative

Abstract

The invention provides a salicylic acid nano-liposome cold damage resistant preservative and application thereof in prevention and control of fruit and vegetable cold damage, a preparation method of the salicylic acid nano-liposome cold damage resistant preservative comprises the following steps: dissolving salicylic acid, phosphatidylcholine and ergosterol in ethanol to form a uniform mixed solution; carrying out rotary evaporation on the obtained mixed solution to remove the organic solvent to form a uniform lipid film; adding a preheated phosphate buffer solution containing Tween 80 into the obtained lipid film, and hydrating for 10-20 minutes; and carrying out ultrasonic treatment and the like on the obtained product to prepare the salicylic acid nano-liposome with uniform particle size. The salicylic acid nano-liposome disclosed by the invention has a good fresh-keeping effect and slow-release performance, and the bioavailability of salicylic acid can be effectively improved, so that efficient protection on cold injury of fruits and vegetables is realized. The preparation process is simple, the production cost is low, and the application prospect is wide.

Description

Technical Field

[0001] This application belongs to the technical fields of fruit and vegetable preservation and nano - preparation technology. Specifically, this application provides a salicylic acid nano - liposome cold - injury inhibitor and its application in preventing and controlling cold injury of fruits and vegetables. Background Art

[0002] Cold injury is a non - freezing injury caused by low - temperature stress during the storage of fruits and vegetables, commonly found in tropical and subtropical fruits and vegetables, such as bananas, tomatoes, cucumbers and young ginger. Once fruits and vegetables suffer from cold injury, problems such as epidermal browning, texture softening, water loss and tissue structure damage will occur, further accelerating decay, shortening the shelf life, and seriously affecting their sensory quality and commercial value. According to statistics, the loss rate of fruits and vegetables in the post - harvest storage and transportation link in China is as high as 20% - 30%, and cold injury is one of the important factors causing fruit and vegetable losses. Cold injury not only affects the economic benefits of farmers and producers, but also increases the cost burden at the logistics, retail and consumer ends. At the same time, it exacerbates food waste and restricts the sustainable development of the fruit and vegetable industry. Therefore, developing efficient, safe and green technologies for preventing and controlling cold injury of fruits and vegetables has important economic value and social significance.

[0003] Salicylic acid, also known as o - hydroxybenzoic acid, is a low - molecular - weight phenolic substance with the function of inducing plant stress resistance. Research shows that exogenous salicylic acid treatment can regulate the activity of antioxidant enzymes in fruits and vegetables, stabilize the cell membrane structure and regulate the content of osmoregulatory substances, thereby enhancing the tolerance of fruits and vegetables to low - temperature stress and slowing down the occurrence of cold injury. However, salicylic acid has disadvantages such as poor water solubility, easy degradation and low bioavailability, making it difficult to achieve a lasting and stable effect in practical applications. Moreover, high - concentration use may also cause toxic reactions to fruit and vegetable tissues, limiting its application scope.

[0004] In recent years, nano-carrier delivery systems have provided new ideas for the stable delivery of active substances. Among them, liposomes, as spherical bilayer vesicles composed of phospholipids and cholesterol, have strong encapsulation ability and good biocompatibility, showing good application prospects in the field of food and agricultural product preservation. Patent CN 116138386A discloses a preparation method of p-coumaric acid nano-liposomes and its antibacterial and fresh-keeping application. Using soy lecithin and cholesterol as membrane materials, the p-coumaric acid nano-liposomes are prepared by the thin film dispersion combined with ultrasonic method. However, the encapsulation efficiency of this liposome is only 40% - 60%, mainly limited by the high unsaturation of soy lecithin, resulting in insufficient stability of the vesicle membrane structure, thus limiting the effective embedding of active ingredients. Patent CN 110754522A discloses a preparation method of a citral liposome fruit and vegetable antibacterial preservative, also using soy lecithin and cholesterol as membrane materials to prepare citral liposomes. Although it improves the volatility and stability of citral to a certain extent, it still has problems of unstable membrane structure and low encapsulation efficiency, and it is difficult to meet the long-term fresh-keeping requirements.

[0005] Traditional liposomes have certain limitations. Commonly used soy lecithin contains a large amount of unsaturated fatty acids, and its double bond structure is easily oxidized and damaged, resulting in disordered phospholipid arrangement, making the liposome membrane structure loose, and reducing the integrity and stability of the vesicles. At the same time, cholesterol is also prone to oxidation, further affecting the storage stability and overall performance of liposomes. In addition, there is no relevant report on the preparation of salicylic acid liposomes and their application in preventing and controlling cold injury of fruits and vegetables in the existing technology. Therefore, it is of great research significance and broad application prospects to develop a salicylic acid nano-liposome preservative with high stability, excellent slow-release property and suitable for cold injury prevention and control. Summary of the Invention

[0006] On the one hand, the present application provides a salicylic acid nano-liposome cold injury-preventing preservative, and the salicylic acid nano-liposomes are prepared according to the following method:

[0007] (1) Take salicylic acid, phosphatidylcholine and ergosterol, dissolve them in ethanol to form a uniform mixed solution;

[0008] (2) Rotate evaporate the organic solvent from the mixed solution obtained in step (1) to form a uniform lipid film;

[0009] (3) Add a preheated phosphate buffer solution containing Tween 80 to the lipid film obtained in step (2), and hydrate for 10 - 20 minutes to initially form liposomes;

[0010] (4) Ultrasonically treat the product obtained in step (3) to promote the homogenization of liposomes;

[0011] (5) Process the product obtained in step (4) through a high-pressure homogenizer to further improve the uniformity and stability of the liposomes;

[0012] (6) Extrude the product obtained in step (5) to obtain salicylic acid nano-liposomes with uniform particle sizes.

[0013] Further, the phosphatidylcholine is a mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine at a mass ratio of 3:1.

[0014] Further, in step (1), the mass ratio of phosphatidylcholine to ergosterol is 2 - 6:1.

[0015] Further, in the mixed solution formed in step (1), the concentration of salicylic acid is 10 - 25 mol / L.

[0016] Further, in step (2), the organic solvent is removed by rotary evaporation at 40 - 50 °C.

[0017] Further, in step (3), a phosphate buffer solution preheated to 45 - 55 °C with a pH of 6.8 - 7.4 is added to the lipid film obtained in step (2), and hydrated for 10 - 20 minutes to initially form liposomes.

[0018] Further, in the phosphate buffer solution in step (3), it contains 0.25% - 2.0% w / v of Tween 80.

[0019] Further, in step (4), the product obtained in step (3) is ultrasonically treated at 50 - 60 °C for 5 - 25 minutes to promote the homogenization of liposomes.

[0020] Further, in step (5), when processing through the high-pressure homogenizer, the pressure is controlled at 800 - 1200 bar and circulated 3 - 5 times.

[0021] Further, in step (6), the product obtained in step (5) is successively extruded through membranes with pore sizes of 400 nm and 200 nm to obtain salicylic acid nano-liposomes with uniform particle sizes.

[0022] This application also claims to protect the preparation method of the above salicylic acid nano-liposome cold injury-preventing preservative.

[0023] On the other hand, this application provides the use of the above salicylic acid nano-liposome cold injury-preventing preservative in the preparation of cold injury-preventing products.

[0024] On the other hand, this application provides the use of the above salicylic acid nano-liposome cold injury-preventing preservative in the cold injury prevention of fruits and vegetables.

[0025] Further, the fruits and vegetables are young ginger.

[0026] Further, in the application, a liquid containing the above-mentioned salicylic acid nano-liposomes is sprayed on fruits and vegetables to be refrigerated, and the volume ratio of the liquid to the mass of the fruits and vegetables is 1:7 - 1:10.

[0027] The beneficial effects of this application include:

[0028] The present invention uses the thin film dispersion combined with ultrasonic method to prepare salicylic acid nano-liposomes, with phosphatidylcholine and ergosterol as the membrane materials, and constructs a stable phospholipid bilayer structure through the self-assembly process of liposomes. Phosphatidylcholine is 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, which contain fatty acids with different chain lengths and have different hydrophilic-lipophilic properties. The mixing of the two is beneficial to simulate the natural lipid bilayer and make the liposomes have better stability. In addition, the ergosterol used has good structural stability, which can improve the membrane structure of phosphatidylcholine in the liposomes and make the lipid bilayer have good rigidity and fluidity. This method has the advantages of simple process flow, convenient operation, and no need for complex equipment, and is suitable for large-scale production. Compared with the traditional method, this preparation process significantly reduces the production cost, improves the economic feasibility and industrial application potential.

[0029] The salicylic acid nano-liposomes of the present invention have a uniform vesicle structure, the particle size is distributed in 100 - 200 nm, and the encapsulation efficiency is 70 - 80%. The obtained liposomes have good dispersibility and physical stability, without obvious aggregation or leakage, and can maintain their structural integrity and functional performance for a long time.

[0030] By embedding salicylic acid in the phospholipid bilayer, the present invention not only effectively improves the limitations of poor water solubility and easy degradation of salicylic acid, but also realizes its slow release effect in plants and prolongs the action time. At the same time, the liposome structure can enhance the penetration ability of salicylic acid in plant tissues, improve its bioavailability, thereby enhancing its ability to regulate plant metabolism and stress resistance reactions, and improving the stability and persistence of the fresh-keeping effect.

[0031] The application effect of the salicylic acid nano-liposomes of the present invention in the cold storage and fresh-keeping of tender ginger is remarkable. Compared with free salicylic acid, the salicylic acid nano-liposomes can more effectively reduce the chilling injury index during cold storage, slow down the tissue softening, browning and cell structure damage caused by low temperature stress, improve the cold resistance and storage quality of tender ginger, thereby enhancing its market competitiveness and commercial value. The popularization and application of this technology can promote the standardization upgrade of tender ginger fresh-keeping technology, and has good application prospects and industrial transformation potential for improving the industrial added value and promoting the intensive processing of related agricultural products. Description of the Drawings

[0032] Figure 1Transmission electron micrograph of salicylic acid nano-liposomes of Example 1 of the present invention;

[0033] Figure 2 Particle size distribution map and Zeta potential map of salicylic acid nano-liposomes of Example 1 of the present invention;

[0034] Figure 3 Showing the effect of Example 1 of the present invention on the chilling injury index during the low-temperature storage of young ginger. Detailed implementation mode

[0035] Preparation of salicylic acid nano-liposome 1 in Example 1

[0036] Dissolve phosphatidylcholine (phosphatidylcholine is a mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with a mass ratio of 3:1, the same in the following examples), ergosterol, and salicylic acid in absolute ethanol. The mass ratio of phosphatidylcholine to ergosterol is 4:1, and the concentration of salicylic acid is 15 mol / L. Remove ethanol from the mixed solution by rotary evaporation at 45 °C until a thin film is formed. Add 25 mL of PBS solution (0.01 mol / L, pH 7.4) containing 0.5% (w / v) Tween 80. After sonication for 10 minutes, use a liposome extruder to extrude the liposome solution through membranes with pore sizes of 400 nm and 200 nm in sequence to obtain salicylic acid nano-liposomes.

[0037] Preparation of salicylic acid nano-liposome 2 in Example 2

[0038] Dissolve phosphatidylcholine, ergosterol, and salicylic acid in absolute ethanol. The mass ratio of phosphatidylcholine to ergosterol is 2:1, and the concentration of salicylic acid is 10 mol / L. Remove ethanol from the mixed solution by rotary evaporation at 45 °C until a thin film is formed. Add 25 mL of PBS solution (0.01 mol / L, pH 7.4) containing 0.25% (w / v) Tween 80. After sonication for 5 minutes, use a liposome extruder to extrude the liposome solution through membranes with pore sizes of 400 nm and 200 nm in sequence to obtain salicylic acid nano-liposomes.

[0039] Preparation of salicylic acid nano-liposome 3 in Example 3

[0040] Dissolve phosphatidylcholine, ergosterol, and salicylic acid in absolute ethanol. The mass ratio of phosphatidylcholine to ergosterol is 6:1, and the concentration of salicylic acid is 25 mol / L. Remove the ethanol from the mixed solution by rotary evaporation at 45 °C until a thin film is formed. Add 25 mL of PBS solution (0.01 mol / L, pH 7.4) containing 2% (w / v) Tween 80. After sonication for 25 minutes, use a liposome extruder to extrude the liposome solution through membranes with pore sizes of 400 nm and 200 nm in sequence to obtain salicylic acid nano-liposomes.

[0041] Example 4 Characterization of Salicylic Acid Nano-Liposomes

[0042] The characterization method of the salicylic acid nano-liposomes obtained in Example 1 of the present invention is as follows:

[0043] (1) Observation of the morphology of salicylic acid nano-liposomes: Drop 10 μL of the liposomes to be tested onto the surface of a copper grid and dry for 60 s until a thin film of liposomes is formed on the copper grid surface. Then, stain the liposomes with 10 μL of 1% phosphotungstic acid solution (pH = 6.5) for 2 minutes, and subsequently observe the liposome morphology with a transmission electron microscope.

[0044] It can be seen from Figure 1 that the salicylic acid nano-liposomes exhibit a typical spherical or nearly spherical structure, with a smooth surface, clear vesicle-like characteristics, and no obvious aggregation or rupture phenomena, indicating that the salicylic acid nano-liposomes have good dispersibility and structural integrity.

[0045] (2) Determination of particle size distribution, polydispersity index (PDI), and Zeta potential: Dilute the sample 10 times with 0.01 mol / L PBS solution before measurement, and use a nanoparticle size and zeta potential analyzer to measure the average particle size and Zeta potential of the salicylic acid nano-liposomes.

[0046] As Figure 2 shown, the particle size of the salicylic acid nano-liposomes is 129 nm, the PDI is 0.04, and the Zeta potential is -28.07 mV. This indicates that the salicylic acid nano-liposomes are uniformly distributed and the system has good stability.

[0047] Tested by the same method, the salicylic acid nano-liposomes prepared in Example 2 and Example 3 also have similar structural characteristics, showing a nano-scale uniform and stable distribution.

[0048] Example 5 Inhibitory Effect of Salicylic Acid Nano-Liposomes on Chilled Injury of Young Ginger

[0049] The fresh young ginger was disinfected with 1% (v / v) sodium hypochlorite solution for 2 minutes, then washed with tap water and air-dried naturally at room temperature. The washed and disinfected young ginger was randomly divided into 3 groups: distilled water (control group), salicylic acid treatment group, and the treatment group with 15 mmol / L salicylic acid-loaded nano-liposomes prepared in Example 1. The samples of each group were air-dried after spraying treatment, then packed in polyethylene bags (0.03 mm thick) and stored in a cold storage at 6 ± 1 °C. The storage period was 25 days, and fresh samples were taken every 5 days to determine the chilling injury index of the young ginger.

[0050] Determination of the chilling injury index: The water-soaked patch area of the young ginger was classified according to a scale of 1 to 5 to define the chilling injury level as follows: 1 = 0%; 2 ≤ 25%; 3 = 25% - 50%; 4 = 50% - 80%; 5 ≥ 80%. The CI index was calculated according to the following formula: Chilling injury index (%) = ∑(number of young ginger with chilling injury × chilling injury level) / (5 × total number of young ginger) × 100.

[0051] The experimental results showed that the salicylic acid-loaded nano-liposomes provided by the present invention had a significant inhibitory effect on the chilling injury of young ginger, could effectively maintain the appearance quality of young ginger during storage, significantly reduce the chilling injury index ( Figure 3 ), delay the increase of weight loss rate, and maintain the color of young ginger, delay browning, thus significantly improving the storage quality and commercial value of young ginger.

Claims

1. A salicylic acid nano-liposome cold injury-preventing and freshness-preserving agent, characterized in that, The salicylic acid nano-liposome cold injury-resistant preservative is prepared according to the following method: (1) Take salicylic acid, phosphatidylcholine and ergosterol, dissolve them in ethanol to form a uniform mixed solution; (2) Rotate and evaporate the organic solvent from the mixed solution obtained in step (1) to form a uniform lipid film; (3) Add preheated phosphate buffer solution containing Tween 80 to the lipid film obtained in step (2), and hydrate for 10 - 20 minutes to initially form liposomes; (4) Ultrasonically treat the product obtained in step (3) to promote the homogenization of liposomes; (5) Treat the product obtained in step (4) with a high-pressure homogenizer to further improve the uniformity and stability of liposomes; (6) Extrude the product obtained in step (5) to obtain a salicylic acid nano-liposome cold injury-resistant preservative with uniform particle size.

2. The salicylic acid nano-liposome cold injury-resistant preservative according to claim 1, wherein the phosphatidylcholine is a mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with a mass ratio of 3:

1.

3. The salicylic acid nano-liposome cold injury-resistant preservative according to claim 1 or 2, wherein the mass ratio of phosphatidylcholine to ergosterol in step (1) is 2 - 6:

1.

4. The salicylic acid nano-liposome cold injury-resistant preservative according to claim 3, wherein the concentration of salicylic acid in the mixed solution formed in step (1) is 10 - 25 mol / L.

5. The salicylic acid nano-liposome cold injury-resistant preservative according to any one of claims 1 - 4, wherein in step (2), the organic solvent is removed by rotary evaporation at 40 - 50 °C.

6. The salicylic acid nano-liposome cold injury-resistant preservative according to any one of claims 1 - 5, wherein in step (3), add preheated phosphate buffer solution with a pH of 6.8 - 7.4 at 45 - 55 °C to the lipid film obtained in step (2), and hydrate for 10 - 20 minutes to initially form liposomes.

7. The salicylic acid nano-liposome cold injury-resistant preservative according to claim 6, wherein the phosphate buffer solution in step (3) contains 0.25% - 2.0% w / v of Tween 80.

8. The salicylic acid nano-liposome cold injury-resistant preservative according to any one of claims 1 - 7, wherein in step (4), the product obtained in step (3) is ultrasonically treated at 50 - 60 °C for 5 - 25 minutes to promote the homogenization of liposomes.

9. The salicylic acid nano-liposome cold injury-resistant preservative according to any one of claims 1 - 8, wherein when treating with a high-pressure homogenizer in step (5), the pressure is controlled at 800 - 1200 bar and circulated 3 - 5 times.

10. The salicylic acid nano-liposome cold injury-resistant preservative according to any one of claims 1 - 9, wherein in step (6), the product obtained in step (5) is extruded through membranes with pore sizes of 400 nm and 200 nm in sequence to obtain salicylic acid nano-liposomes with uniform particle size.

11. Use of the salicylic acid nano-liposome cold injury-resistant preservative according to any one of claims 1 - 10 in the preparation of cold injury-resistant preservation products.

12. Use of the salicylic acid nano-liposome cold injury inhibitor as claimed in any one of claims 1-10 in cold injury prevention of fruits and vegetables.

13. The use according to claim 12, wherein the fruit and vegetable is young ginger.

14. The use according to claim 12 or 13, wherein in the use, a liquid containing the above-mentioned salicylic acid nano-liposome cold injury inhibitor is sprayed on the fruits and vegetables to be refrigerated, and the volume ratio of the liquid to the mass of the fruits and vegetables is 1:7-1:10.

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