Andrias davidianus oil extraction and microcapsule preparation method and its application in antioxidant products

Abstract

The application discloses a giant salamander oil extraction and microcapsule preparation method and application of the giant salamander oil in antioxidant products, and comprises the following steps: after pretreatment of giant salamander fat tissue, tea polyphenol cellulose inclusion compound, citric acid and EDTA-2Na, adding protease for enzymolysis, then adding phytic acid degumming, and carrying out enzyme inactivation and separation to obtain the giant salamander oil. The giant salamander oil is prepared into liposomes, and then mixed with a wall material solution, granulation is carried out to obtain microcapsules. The application realizes efficient extraction of the giant salamander oil, and comprehensively improves the antioxidant performance and water dispersion performance of the giant salamander oil microcapsules, and is suitable for development of giant salamander oil antioxidant products.

Description

Technical Field

[0001] This invention belongs to the field of food and pharmaceutical technology, specifically relating to the method of extracting and preparing microcapsules from giant salamander oil and its application in antioxidant products. Background Technology

[0002] The tail of the giant salamander is rich in adipose tissue, accounting for approximately 5% to 8% of its body weight, making it an important raw material for extracting functional oils. Studies have shown that giant salamander oil is rich in various unsaturated fatty acids and has significant anti-inflammatory, lipid-regulating, and brain-development-promoting physiological functions. Giant salamander oil can be extracted using an aqueous enzymatic method. This method utilizes proteases to hydrolyze proteins, breaking down the protein membrane formed by the binding of phospholipids and proteins outside the fat cells, thus releasing the oil. Compared to traditional methods such as high-temperature cooking, pressing, and solvent extraction, the aqueous enzymatic method offers advantages such as milder conditions, effective protection of the active ingredients in the oil from high-temperature degradation, high oil extraction rate, no organic solvent residue, simple operation, and high production safety. However, conventional aqueous enzymatic methods lack effective pretreatment methods, resulting in insufficient cell disruption, limited enzymatic hydrolysis efficiency, and longer extraction times. Furthermore, the resulting oil-water emulsion often exhibits high stability, making demulsification difficult, leading to low oil separation efficiency and easy oil residue in both the aqueous and solid phases, resulting in a high extraction loss rate. Moreover, the large surface area of ​​oil in contact with air during enzymatic hydrolysis makes it prone to oxidation and deterioration, ultimately resulting in poor antioxidant properties of the extracted giant salamander oil, which limits its application. Summary of the Invention

[0003] The purpose of this invention is to provide a method for extracting and preparing microcapsules from giant salamander oil and its application in antioxidant products, so as to solve at least one aspect of the problems and defects mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] The method for extracting oil from giant salamanders includes the following steps:

[0006] The giant salamander fat tissue was pretreated with tea polyphenol cellulose inclusion complex, citric acid and EDTA-2Na, then enzymatically hydrolyzed with protease, and degummed with phytic acid. After enzyme inactivation and separation, the giant salamander oil was obtained.

[0007] As a further improvement of the present invention, the tea polyphenol cellulose inclusion complex is obtained by mixing modified microcrystalline cellulose with tea polyphenols and then crosslinking it with chitosan quaternary ammonium salt and sodium tripolyphosphate.

[0008] As a further improvement of the present invention, the modified microcrystalline cellulose is obtained by using at least one of oxygen modification, enzymatic hydrolysis modification and phosphoric acid modification of microcrystalline cellulose.

[0009] As a further improvement of the present invention, the modified microcrystalline cellulose is obtained by treating microcrystalline cellulose with oxygen plasma at 30~50W for 1~10 min.

[0010] As a further improvement of the present invention, the modified microcrystalline cellulose is obtained by modifying microcrystalline cellulose by enzymatic hydrolysis with cellulase.

[0011] As a further improvement of the present invention, the amount of cellulase used is 1 to 3% of the mass of microcrystalline cellulose.

[0012] As a further improvement of the present invention, the cellulase hydrolysis temperature is 40~60 ℃.

[0013] As a further improvement of the present invention, the cellulase hydrolysis pH is 4.0~6.0, preferably 4.5~5.0.

[0014] As a further improvement of the present invention, the modified microcrystalline cellulose is obtained by soaking microcrystalline cellulose in a phosphoric acid solution with a mass concentration of 0.2-0.7%, wherein the mass concentration of the phosphoric acid solution is preferably 0.4-0.6%.

[0015] As a further improvement of the present invention, the particle size of the modified microcrystalline cellulose is 10~40 μm, preferably 20~30 μm.

[0016] As a further improvement of the present invention, the tea polyphenol cellulose inclusion complex is prepared by the following method:

[0017] Modified microcrystalline cellulose and tea polyphenols were dispersed in an ethanol aqueous solution, stirred, filtered, and dried to obtain tea polyphenol-loaded cellulose.

[0018] The tea polyphenol-loaded cellulose was mixed with chitosan quaternary ammonium salt, and sodium tripolyphosphate was added for cross-linking to obtain the tea polyphenol cellulose inclusion complex.

[0019] As a further improvement of the present invention, the particle size of the tea polyphenol cellulose inclusion complex is 100~200 μm, preferably 120~160 μm.

[0020] As a further improvement of the present invention, the mass ratio of the modified microcrystalline cellulose to tea polyphenols is 4~7:1.

[0021] As a further improvement of the present invention, the mass ratio of tea polyphenol-loaded cellulose to chitosan quaternary ammonium salt is 5~8:1, preferably 7:1.

[0022] As a further improvement of the present invention, the mass ratio of chitosan quaternary ammonium salt to sodium tripolyphosphate is 2~4:1, preferably 3:1.

[0023] As a further improvement of the present invention, the tea polyphenol cellulose inclusion complex further includes polyethylene glycol, preferably low molecular weight polyethylene glycol, and more preferably polyethylene glycol 400.

[0024] As a further improvement of the present invention, the mass ratio of tea polyphenol-loaded cellulose to polyethylene glycol is 8~12:1, preferably 10:1.

[0025] In a preferred embodiment of the present invention, microcrystalline cellulose is treated with oxygen plasma modification, enzymatic hydrolysis modification, and phosphoric acid modification. Oxygen plasma treatment can etch the surface of microcrystalline cellulose and directionally introduce oxygen-containing active functional groups, effectively destroying its dense crystalline structure, increasing the specific surface area, and improving surface hydrophilicity, providing more active sites for subsequent enzymatic hydrolysis reactions; further enzymatic hydrolysis modification and phosphoric acid modification introduce phosphate groups, enhance the surface charge density, and improve cell adsorption capacity and compatibility.

[0026] As a further improvement of the present invention, the mass ratio of the giant salamander adipose tissue to the tea polyphenol cellulose inclusion complex is 100:5~10, preferably 100:5~8.

[0027] As a further improvement of the present invention, the mass ratio of the giant salamander's adipose tissue to citric acid is 1000:2~4.

[0028] As a further improvement of the present invention, the mass ratio of the giant salamander adipose tissue to EDTA-2Na is 1000:0.3~0.7, preferably 1000:0.5.

[0029] As a further improvement of the present invention, the pretreatment temperature is 40~50 ℃.

[0030] As a further improvement of the present invention, the pretreatment time is 30~60 min.

[0031] As a further improvement of the present invention, the protease is selected from at least one of a complex protease and papain, preferably a mixture of a complex protease and papain.

[0032] As a further improvement of the present invention, the protease comprises a complex protease and papain in a mass ratio of 1:1 to 2.

[0033] As a further improvement of the present invention, the complex protease contains 9-15 wt% neutral protease and 2-5 wt% alkaline protease.

[0034] As a further improvement of the present invention, the mass ratio of the giant salamander adipose tissue to the protease is 1000:10~15.

[0035] As a further improvement of the present invention, the mass ratio of the giant salamander adipose tissue to phytic acid is 1000:2~3.

[0036] As a further improvement of the present invention, the temperature for enzymatic hydrolysis of the protease is 40~60 °C.

[0037] As a further improvement of the present invention, the enzymatic hydrolysis time of the protease is 1-4 h.

[0038] As a further improvement of the present invention, the pH of the protease hydrolysis is 6.0~7.2.

[0039] In a preferred embodiment of the present invention, the adipose tissue of the giant salamander is pretreated before enzymatic hydrolysis. Citric acid is used to adjust the osmotic pressure of the cells, and a low pH environment promotes the dissolution of calcium and magnesium in the adipose tissue, assisting in subsequent degumming. EDTA-2Na chelates the dissolved metal ions, blocking lipid oxidation catalysis. The inner layer of the tea polyphenol cellulose inclusion complex modified microcrystalline cellulose is treated with oxygen plasma to form a rough surface. The phosphate groups introduced by enzymatic hydrolysis and phosphoric acid modification enhance the surface charge density, improve cell adsorption capacity and compatibility, further disrupt the structure of adipocytes, and increase cell membrane permeability.

[0040] During the enzymatic hydrolysis stage, the tea polyphenol cellulose inclusion complex continues to play a flocculation role, adsorbing negatively charged cell debris and proteins produced by enzymatic hydrolysis and promoting oil droplet aggregation; at the same time, the tea polyphenols in the inclusion complex are slowly released under heating conditions, blocking lipid oxidation during enzymatic hydrolysis.

[0041] After enzymatic hydrolysis, the chelation of phytic acid with metal ions in phospholipid molecules promotes the precipitation of insoluble complexes of phospholipids, achieving deep degumming; at the same time, it helps to disrupt the interface of oil-in-water emulsions, promoting the demulsification and separation of oils.

[0042] The microcapsule preparation method uses giant salamander oil obtained by any of the extraction methods described above as raw material.

[0043] As a further improvement of the present invention, the microcapsule preparation method includes converting the giant salamander oil into liposomes, mixing them with a wall material solution, and granulating them to obtain the microcapsules.

[0044] As a further improvement of the present invention, the liposomes also include soybean lecithin and cholesterol.

[0045] As a further improvement of the present invention, the liposomes also include at least one of vitamin E and tea polyphenols.

[0046] As a further improvement of the present invention, the wall material solution includes maltodextrin, sodium octenyl succinate starch and carboxymethyl chitosan.

[0047] As a further improvement of the present invention, the mass ratio of giant salamander oil to soybean lecithin and cholesterol in the liposomes is 10:20~25:3~5.

[0048] As a further improvement of the present invention, the mass ratio of maltodextrin, sodium octenyl succinate starch and carboxymethyl chitosan in the wall material solution is 2~3:1~2:1.

[0049] In the microcapsule preparation process, maltodextrin provides water solubility and film-forming properties, the hydrophobic group of sodium octenyl succinate starch can anchor liposome oil droplets, the hydrophilic group improves the stability of the water-oil interface, and carboxymethyl chitosan improves antioxidant stability and bioavailability. The giant salamander oil in the liposomes is encapsulated by phospholipids, and the addition of vitamin E and tea polyphenols forms multiple antioxidant barriers.

[0050] As a further improvement of the present invention, the microcapsule preparation method includes the following steps:

[0051] Soybean lecithin, cholesterol, and PBS buffer were mixed, and then the giant salamander oil was added and ultrasonically dispersed to obtain giant salamander oil liposomes.

[0052] The giant salamander oil liposomes were mixed and homogenized with the wall material solution, and then dried to obtain the microcapsules.

[0053] Application of giant salamander oil obtained by any of the extraction methods described above or microcapsules obtained by any of the preparation methods described above in antioxidant products.

[0054] The present invention has at least the following beneficial effects:

[0055] By using citric acid, EDTA-2Na, and a specially formulated tea polyphenol-cellulose inclusion complex, cell disruption, flocculation, and antioxidant functions are achieved. Phytic acid is then used for deep degumming and to assist in demulsification, enabling efficient water-enzymatic extraction and protection of giant salamander oil, resulting in giant salamander oil with excellent antioxidant effects. Furthermore, through multiple encapsulation processes, giant salamander oil liposomes are constructed. Finally, a composite wall material is formed using maltodextrin, sodium octenyl succinate starch, and carboxymethyl chitosan, comprehensively enhancing the antioxidant and water-dispersible properties of the giant salamander oil microcapsules. Attached Figure Description

[0056] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings.

[0057] Figure 1 The curves showing the DPPH free radical scavenging ability of the giant salamander oil prepared in Examples 1-4 at different concentrations are shown.

[0058] Figure 2 Curves showing the scavenging capacity of giant salamander oil prepared in Examples 1-4 at different concentrations against •OH free radicals;

[0059] Figure 3 Curves showing the O2-• free radical scavenging ability of the giant salamander oil prepared in Examples 1-4 at different concentrations;

[0060] Figure 4 The figure shows the effect of giant salamander oil microcapsules on H2O2-induced ROS production in zebrafish.

[0061] Figure 5 The figure shows the effect of giant salamander oil microcapsules on H2O2-induced oxidative damage in zebrafish.

[0062] Figure label: ### indicates P < 0.001 compared to the control group;

[0063] This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group. Detailed Implementation

[0064] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0065] Therefore, the following detailed description of embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0066] The adipose tissue of the giant salamander was taken from the tail of a healthy 3-year-old artificially bred giant salamander.

[0067] The complex protease was provided by Novozymes (China) Biotechnology Co., Ltd., model Protamex 1.6, with an enzyme activity of 1.6 AU-N / g. By mass fraction, it consists of 88% sodium chloride (CAS no. 7647-14-5), 9% protease (neutral, CAS no. 9080-56-2), 2% protease (subtilisin, CAS no. 9014-01-1), and 1% water (CAS no. 7732-18-5).

[0068] Papain enzyme activity ≥800,000 U / g.

[0069] Example 1

[0070] The method for extracting oil from giant salamanders includes the following steps:

[0071] S1. Cut 1 kg of giant salamander fat tissue into 5 mm³ pieces, add 4 kg of water, 80 g of tea polyphenol cellulose inclusion complex, 3 g of anhydrous citric acid and 0.5 g of EDTA-2Na, and stir in a water bath at 45 ℃ for 30 min.

[0072] S2. Adjust the pH to 6.8 with 1 mol / L NaOH, add 7 g of complex protease and 7 g of papain, stir at 200 rpm at 50 ℃ for 2 h for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, add 2 g of phytic acid and stir for 10 min.

[0073] S3, inactivate enzymes in 85 ℃ water bath for 10 min, filter and separate, centrifuge the filtrate at 10000 rpm for 15 min, collect the upper oil phase to obtain giant salamander oil with a mass of 231 g.

[0074] Tea polyphenol cellulose inclusion complex was prepared by the following method:

[0075] Microcrystalline cellulose was placed in a plasma treatment instrument, pure oxygen was introduced, and the treatment was carried out at 40W for 5 min. Then, it was dispersed in deionized water at a solid-liquid ratio of 1g:10mL. The pH of the system was adjusted to 4.5-5.0, and 2% (by weight) of cellulase was added. The mixture was stirred at 50℃ and 200 rpm for 3 h for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the temperature was raised to 90℃ and kept at 10 min to inactivate the enzyme. After filtration, the mixture was soaked in a 0.5% (by weight) phosphate solution at 25℃ for 40 min. Then, it was washed with deionized water until neutral, dried, and pulverized to a D50 of 20-30 μm to obtain modified microcrystalline cellulose.

[0076] Tea polyphenols were dispersed in a 50% ethanol aqueous solution at a solid-liquid ratio of 1g:15mL. Modified microcrystalline cellulose with a mass of 6 times that of tea polyphenols was added. The mixture was stirred and adsorbed at 30℃ for 2 h, filtered, and dried under vacuum at 40℃ to obtain tea polyphenol-loaded cellulose.

[0077] Add 200 g of tea polyphenol-loaded cellulose, 30 g of chitosan quaternary ammonium salt and 20 g of polyethylene glycol 400 to a 1% acetic acid solution per liter, and stir at 30°C for 1 h; then add 100 mL of sodium tripolyphosphate aqueous solution with a concentration of 100 mg / mL, and continue stirring for 30 min; allow to stand for crosslinking for 2 h, filter, vacuum dry at 40°C, and pulverize through a 100-mesh sieve to obtain tea polyphenol-cellulose inclusion complex.

[0078] Example 2

[0079] The difference from Example 1 is as follows:

[0080] In step S1, the amount of tea polyphenol cellulose inclusion complex added was adjusted to 50 g, and the amount of anhydrous citric acid added was adjusted to 2 g.

[0081] In step S2, the amount of papain added was adjusted to 10 g, and the amount of phytic acid added was adjusted to 3 g.

[0082] Giant salamander oil was obtained, with a mass of 224 g.

[0083] Example 3

[0084] The difference from Example 1 is as follows:

[0085] Anhydrous citric acid is not added in step S1;

[0086] Phytic acid is not added in step S2;

[0087] Polyethylene glycol 400 is not added during the preparation of tea polyphenol cellulose inclusion complex;

[0088] Giant salamander oil was obtained, with a mass of 206 g.

[0089] Example 4

[0090] The difference from Example 1 is as follows:

[0091] No tea polyphenol cellulose inclusion complex is added in step S1;

[0092] In step S2, the amount of compound protease added was adjusted to 10 g, and the amount of papain added was adjusted to 10 g.

[0093] Giant salamander oil was obtained, with a mass of 220 g.

[0094] Test Example 1

[0095] The DPPH free radical scavenging ability, •OH free radical scavenging ability, and O2-• free radical scavenging ability of the giant salamander oil obtained in Examples 1-4 were tested. The results are as follows: Figures 1-3 As shown.

[0096] The results showed that the giant salamander oil prepared in Examples 1 and 2 had good scavenging rates against DPPH free radicals, •OH free radicals and O2-• free radicals, indicating that cell destruction, metal ion chelation, flocculation separation and deep degumming were achieved during the extraction process in Examples 1 and 2, ensuring good oil extraction effect and antioxidant effect.

[0097] In Example 3, inadequate cell pretreatment and oil posttreatment led to decreased enzymatic hydrolysis efficiency, increased lipid oxidation, and higher oil loss.

[0098] In Example 4, no tea polyphenol cellulose inclusion complex was used in the extraction process. Although the increased enzyme content compensated for the extraction rate, the oil was exposed to air for a longer period of time, resulting in increased oxidation loss and a decrease in the antioxidant properties of the giant salamander oil.

[0099] Example 5

[0100] Giant salamander oil microcapsules were prepared by the following method:

[0101] D1. Mix maltodextrin, sodium octenyl succinate starch and carboxymethyl chitosan (carboxylation degree ≥80%) in a mass ratio of 5:3:2, add to deionized water in a solid-liquid ratio of 1g:10mL, and stir at 55℃ and 400 rpm for 3 h to obtain the wall material solution.

[0102] D2. Mix and dissolve 20 g of soybean lecithin and 4 g of cholesterol in 80 mL of anhydrous ethanol. Remove the solvent by vacuum rotary evaporation at 45 °C. Add 200 mL of PBS buffer (pH=7.4) and hydrate by stirring at 45 °C for 30 min to obtain a lipid suspension. Mix 10 g of giant salamander oil prepared in Example 1, 0.5 g of vitamin E and 0.5 g of tea polyphenols, add to the lipid suspension, and sonicate at 200 W for 15 min (pulse mode, 3 seconds on and 3 seconds off) to obtain giant salamander oil liposomes.

[0103] D3. Mix the giant salamander oil liposomes and the wall material solution at a ratio of 1g:60mL, homogenize at 8000rpm for 8min to obtain an emulsion; spray dry the emulsion at a feed rate of 400mL / h, an inlet air temperature of 140℃, and an outlet air temperature of 60℃ to obtain giant salamander oil microcapsules.

[0104] Example 6

[0105] The difference from Example 5 is as follows:

[0106] In step D1, the mass ratio of maltodextrin, sodium octenyl succinate starch, and carboxymethyl chitosan is adjusted to 3:1:1.

[0107] Example 7

[0108] The difference from Example 5 is as follows:

[0109] In step D2, the amount of soy lecithin was adjusted to 25 g and the amount of cholesterol was adjusted to 3 g.

[0110] Example 8

[0111] The difference from Example 5 is as follows:

[0112] In step D3, the ratio of giant salamander oil liposomes to wall material solution was adjusted to 1g:50mL.

[0113] Example 9

[0114] The difference from Example 5 is as follows:

[0115] In step D1, no carboxymethyl chitosan is added, and the wall material is a mixture of maltodextrin and sodium octenyl succinate starch in a mass ratio of 5:3.

[0116] No cholesterol is added in step D2.

[0117] Example 10

[0118] Giant salamander oil microcapsules were prepared by the following method:

[0119] D1, Same as step D1 in Example 5;

[0120] D2. Mix and dissolve 20 g of soybean lecithin and 4 g of cholesterol in 80 mL of anhydrous ethanol. Then add 10 g of giant salamander oil prepared in Example 1, 0.5 g of vitamin E and 0.5 g of tea polyphenols and mix. Disperse the mixture by ultrasonication at 200 W for 15 min (pulse mode, 3 seconds on and 3 seconds off) to obtain the giant salamander oil phase.

[0121] D3. Mix the giant salamander oil liposomes and the wall material solution at a ratio of 1g:60mL, homogenize at 8000rpm for 8min to obtain an emulsion; spray dry the emulsion at a feed rate of 400mL / h, an inlet air temperature of 140℃, and an outlet air temperature of 60℃ to obtain giant salamander oil microcapsules.

[0122] The salamander oil microcapsules obtained in Examples 5-10 were subjected to accelerated oxidation stability analysis, and the trend of peroxide value change is shown in Table 1.

[0123] The detection method is as follows: the giant salamander oil microcapsules are loaded into brown wide-mouth bottles, the sample volume is 1 / 2 of the bottle capacity, and the bottles are placed in an oven at 60±1℃ to accelerate oxidation. Samples are taken on days 0 (initial), 7, 14 and 21 to determine the peroxide value.

[0124] Table 1

[0125]

[0126] The results showed that the POV of the giant salamander oil microcapsules prepared in Examples 5-8 was below 20 meq / kg after 21 days, indicating good oxidative stability. In Example 9, the composition of the core and wall materials was altered, resulting in insufficient liposome stability and outer antioxidant barrier function, leading to a significant increase in the oxidation rate of the prepared giant salamander oil microcapsules. In Example 10, no liposomes were used; the oil and aqueous phases were directly mixed and dried to form microcapsules, resulting in poor antioxidant properties.

[0127] Stability tests were conducted on the giant salamander oil microcapsules prepared in Examples 5-10. 1 g of giant salamander oil microcapsule was weighed, 100 mL of deionized water was added, and the mixture was magnetically stirred at 300 rpm for 5 min. The mixture was then allowed to stand at 25 ℃ for 24 h, 7 days, and 30 days. The changes in the solution were observed, and the results are shown in Table 2.

[0128] Table 2

[0129]

[0130] The results showed that the giant salamander oil microcapsules prepared in Examples 5-8 maintained good dispersion stability within 7 days. After 30 days, the giant salamander oil microcapsules prepared in Examples 6-8 showed only slight turbidity, which could be dispersed and clarified after gentle shaking. In Example 9, the composition of the wall material was changed, which affected the dispersibility of the giant salamander oil microcapsules in water and reduced their stability. The microcapsules formed by the oil-water mixture in Example 10 exhibited extremely poor dispersibility.

[0131] Test Example 2

[0132] The salamander oil microcapsules prepared in Examples 5-10 were used in zebrafish experiments to verify their antioxidant effects in vivo.

[0133] 1. Experimental Methods

[0134] Adult wild-caught AB-type zebrafish used in the experiment were purchased from the Institute of Hydrobiology, Chinese Academy of Sciences. The broodstock zebrafish were reared in a laboratory-controlled, temperature-controlled, recirculating aquaculture system. The water temperature was maintained at 28 ± 0.5℃, the pH at 7.0 ± 1.0, and the dissolved oxygen level above 6 mg / L. The photocycle of the system was 14 hours of light followed by 10 hours of darkness. During the daily rearing process, the zebrafish were fed brine shrimp twice daily, in the morning and evening. The water circulation system was shut off when the zebrafish were feeding and reopened after feeding.

[0135] Zebrafish embryos were obtained through breeding. The night before spawning, female and male zebrafish were transferred to a mating tank at a 1:1 ratio, separated by a partition and covered. The mating tank was placed in a constant-temperature incubator at 28.5℃, kept in darkness overnight according to the zebrafish's photocycle. The next morning, light was introduced according to the photocycle while the partition was removed, and the zebrafish began mating and spawning. Approximately one hour after spawning, the fertilized eggs were transferred to a culture dish containing 0.5 mg / L methylene blue solution and continued to be cultured at 28.5℃ for a period of time. At the 4-hour postfertilization (hpf) stage, normally developing zebrafish embryos were selected under an optical microscope for subsequent experiments.

[0136] Embryos at 8 hours post-fertilization were collected and transferred to 12-well plates, with 25 embryos per group. Experimental groups 5-10 were cultured in embryo-water containing 800 μg / ml of the giant salamander oil microcapsules prepared in Examples 5-10, while the model and control groups used the same volume of embryo-water. After 1 hour of incubation, H2O2 was added to the embryo-water of both experimental and model groups at a final concentration of 5 mM, and incubation continued until 24 hours post-fertilization. The embryo-water was then replaced, and incubation continued for another 2 days. Surviving juveniles were selected for testing.

[0137] 2. Detection Method

[0138] (1) ROS detection

[0139] The effects of H2O2 on reactive oxygen species (ROS) in zebrafish are mainly manifested in its interference with cell membrane, mitochondrial function, and the antioxidant system. H2O2 induces excessive ROS production within cells by penetrating the cell membrane, thereby triggering lipid peroxidation, protein oxidation, and DNA damage. ROS levels were detected using the DCFH-DA fluorescent probe, with changes in fluorescence intensity reflecting the ROS level. A ROS detection kit was used to assess cellular ROS levels. DCFH-DA was pre-diluted to 10 μM in fish tank water, added to working solution, and incubated at 37°C in the dark for 20 min. The cells were then rinsed three times with fish tank water before imaging using a fluorescence microscope.

[0140] (2) Determination of malondialdehyde (MDA) content

[0141] Malondialdehyde (MDA) is the main end product of cell membrane lipid peroxidation, and its content is a key indicator for measuring the level of oxidative stress and the degree of cell membrane damage. Zebrafish samples were taken and, at a ratio of body weight (g):volume (ml) = 1:9, were added to physiological saline and freeze-ground. After centrifugation, the supernatant was collected. Following the instructions of the MDA assay kit, reagents were added sequentially to a 96-well plate as shown in Table 3.

[0142] Table 3

[0143]

[0144] After thorough mixing, heat in a 100℃ constant temperature metal bath for 15 min, then cool to room temperature (25℃) in a water bath. Centrifuge the tubes at 25℃ and 12000 rpm for 10 min, then add 200 μL of the supernatant to a 96-well plate, and measure the absorbance at 532 nm using a microplate reader. Finally, calculate the malondialdehyde concentration according to the manufacturer's instructions.

[0145] 3. Test Results

[0146] The results of the study on the effect of giant salamander oil microcapsules on H2O2-induced ROS production in zebrafish are as follows: Figure 4As shown in the figure. Compared with the control group, the green fluorescence intensity in zebrafish treated with H2O2 in the model group was significantly enhanced, indicating that H2O2 can induce zebrafish to produce more ROS, thereby promoting oxidative stress in zebrafish. Compared with the model group, the ROS fluorescence intensity of zebrafish pretreated with giant salamander oil microcapsules in the experimental group was reduced, indicating that giant salamander oil microcapsules can alleviate H2O2-induced ROS production. Among them, the ROS fluorescence intensity of zebrafish in experimental group 7 was the lowest, indicating that the giant salamander oil microcapsules prepared by the method in Example 7 have a better effect on reducing H2O2-induced ROS production in zebrafish.

[0147] The effect of giant salamander oil microcapsules on malondialdehyde (MDA) content in zebrafish, such as Figure 5 As shown in the figure. The results showed that the MDA content in the model group was significantly higher than that in the control group, indicating successful modeling. Compared with the model group, the MDA content in experimental groups 5-8 was significantly lower, the MDA content in experimental group 9 was not significantly lower, and the MDA content in experimental group 10 was basically the same as that in the model group. This indicates that the giant salamander oil microcapsules prepared in Examples 5-8 can effectively inhibit H2O2-induced oxidative damage.

[0148] The above description is merely an example and illustration of the structure of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A method for extracting oil from giant salamanders, characterized in that, Includes the following steps: After pretreating the adipose tissue of the giant salamander with tea polyphenol cellulose inclusion complex, citric acid and EDTA-2Na, the oil was obtained by adding protease for enzymatic hydrolysis, adding phytic acid for degumming, and then inactivating enzymes and separating the oil. The tea polyphenol cellulose inclusion complex is obtained by mixing modified microcrystalline cellulose with tea polyphenols, and then crosslinking it with chitosan quaternary ammonium salt and sodium tripolyphosphate.

2. The method for extracting giant salamander oil according to claim 1, characterized in that, The modified microcrystalline cellulose is obtained by using at least one of oxygen modification, enzymatic hydrolysis modification and phosphoric acid modification on microcrystalline cellulose; And / or, the tea polyphenol cellulose inclusion complex further includes polyethylene glycol; And / or, the mass ratio of the giant salamander adipose tissue to the tea polyphenol cellulose inclusion complex is 100:5~10; And / or, the mass ratio of the giant salamander's adipose tissue to citric acid is 1000:2~4; And / or, the mass ratio of the giant salamander adipose tissue to EDTA-2Na is 1000:0.3~0.7; And / or, the pretreatment temperature is 40~50 ℃.

3. The method for extracting giant salamander oil according to claim 1, characterized in that, The protease is selected from at least one of complex protease and papain; And / or, the mass ratio of the giant salamander's adipose tissue to protease is 1000:10~15; And / or, the mass ratio of the giant salamander's adipose tissue to phytic acid is 1000:2~3; And / or, the enzymatic hydrolysis temperature of the protease is 40~60 °C; And / or, the enzymatic hydrolysis time of the protease is 1~4 h.

4. A method for preparing microcapsules, characterized in that, The giant salamander oil obtained by the extraction method according to any one of claims 1 to 3 is used as the preparation raw material.

5. The microcapsule preparation method according to claim 4, characterized in that, This includes converting the giant salamander oil into liposomes, mixing them with a wall material solution, and then granulating them to obtain microcapsules.

6. The microcapsule preparation method according to claim 5, characterized in that, The liposomes also contain soybean lecithin and cholesterol; And / or, the liposomes further include at least one of vitamin E and tea polyphenols; And / or, the wall material solution includes maltodextrin, sodium octenyl succinate starch, and carboxymethyl chitosan.

7. The microcapsule preparation method according to claim 6, characterized in that, Includes the following steps: Soybean lecithin, cholesterol, and PBS buffer were mixed, and then the giant salamander oil was added and ultrasonically dispersed to obtain giant salamander oil liposomes. The giant salamander oil liposomes were mixed and homogenized with the wall material solution, and then dried to obtain the microcapsules.

8. The microcapsule preparation method according to claim 6, characterized in that, The mass ratio of giant salamander oil to soybean lecithin and cholesterol in the liposomes is 10:20~25:3~5; And / or, the mass ratio of maltodextrin, sodium octenyl succinate starch, and carboxymethyl chitosan in the wall material solution is 2~3:1~2:

1.

9. The application of giant salamander oil obtained by the extraction method according to any one of claims 1 to 3 or microcapsules obtained by the preparation method according to any one of claims 4 to 8 in the preparation of antioxidant products.

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