Cod liver oil pure chitosan microspheres as well as preparation method and application thereof
Pure chitosan microspheres made of cod liver oil are prepared by self-assembly technology at mild temperature, which solves the problems of high preparation temperature and the use of cross-linking agents in the existing technology, and realizes cod liver oil microspheres with high stability and high embedding rate, which are suitable for food and medicine.
Patent Information
- Application Number
- CN202510980847.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-16
- Publication Date
- 2025-10-14
AI Technical Summary
The existing microsphere preparation method has the disadvantages of too high preparation temperature, requiring cross-linking agents and being unsuitable for heat-sensitive fat-soluble compounds. It is difficult to control safety and has poor encapsulation effect, which makes cod liver oil easily oxidized and has a short shelf life.
Using self-assembly technology at mild temperature, cod liver oil and chitosan solution are mixed to form an emulsion, which is then squeezed into a sodium dodecyl sulfate solution to dry and stand, and then immersed in a sodium hydroxide solution for washing to prepare cod liver oil pure chitosan microspheres without using a cross-linking agent.
The cod liver oil microspheres have achieved high biosafety, high stability and high encapsulation rate, good antibacterial properties, solved the problem of easy oxidation of cod liver oil, and expanded its application in food and medicine.
Smart Images
Figure CN120771126A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of chitosan microspheres, and particularly relates to cod liver oil pure chitosan microspheres and a preparation method and application thereof. Background Art
[0002] Cod liver oil, extracted from fish livers, has a complex composition. Besides being rich in EPA and DHA, it's also rich in natural vitamins like vitamin A (VA) and vitamin D (VD). VA and VD in cod liver oil can improve the human visual system and promote bone growth, respectively, making them important natural vitamins that the human body, especially children, needs to regularly supplement. Therefore, cod liver oil is commonly used in daily life to supplement the body's VA and VD needs. However, the polyunsaturated fatty acids in cod liver oil are easily oxidized, leading to an increased peroxide value and rancidity, which reduces its food safety and commercial value. Therefore, a common processing method is to process cod liver oil into products such as cod liver oil capsules.
[0003] Microsphere technology is a technique that uses film-forming materials to coat solids or liquids to form tiny particles. Microsphereization can solve the problems of cod liver oil's easy oxidation, short shelf life, and difficulty in preparing powdered preparations, thereby expanding the use of cod liver oil in the food and pharmaceutical fields. Chitosan has the advantages of high biosafety and strong antibacterial effects, and is commonly used in the preparation of various microspheres. Methods for preparing microspheres include spray drying, emulsion crosslinking, ion gelation, and precipitation coagulation. However, these methods have problems such as high preparation temperatures and the need for crosslinking agents. They are not suitable for heat-sensitive, fat-soluble compounds such as cod liver oil. The use of crosslinking agents such as formaldehyde or glutaraldehyde is toxic to the human body, safety is difficult to control, the preparation process is complex, and the embedding effect is poor. Therefore, there is an urgent need to provide a method for preparing cod liver oil microspheres with high embedding efficiency, high safety, and high stability. Summary of the Invention
[0004] In order to solve the problems existing in the prior art, the first purpose of the present invention is to provide a method for preparing pure chitosan microspheres of cod liver oil without using a cross-linking agent. By using self-assembly technology at a mild temperature, no cross-linking agent is required, the preparation process is simple, and the embedding effect is good.
[0005] The second object of the present invention is to provide cod liver oil pure chitosan microspheres prepared by the above preparation method.
[0006] The third object of the present invention is to provide the use of the above-mentioned cod liver oil pure chitosan microspheres in the preparation of food and medicine.
[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0008] The present invention provides a method for preparing cod liver oil pure chitosan microspheres without using a cross-linking agent, comprising the following steps:
[0009] The fish liver oil and the chitosan solution are stirred uniformly at a volume ratio of 1:(2-6) to obtain an emulsion m1; the emulsion m1 is extruded into a sodium dodecyl sulfonate solution to obtain fish liver oil chitosan microspheres ms2; the fish liver oil chitosan microspheres ms2 are dried and placed in the sodium dodecyl sulfonate solution to stand to obtain fish liver oil chitosan microspheres ms3; the fish liver oil chitosan microspheres ms3 are immersed in a sodium hydroxide solution, and then washed with water to neutral to obtain fish liver oil chitosan microspheres ms4.
[0010] Preferably, the concentration of the chitosan solution is 3-5 g / 100 mL, and the solvent of the chitosan solution is 2% acetic acid.
[0011] Preferably, the concentration of the sodium dodecyl sulfonate solution is 10-20 mg / mL, and the temperature of the sodium dodecyl sulfonate solution is 60-90°C.
[0012] Preferably, the drying temperature is 40-60°C, and the time is 50-70 min.
[0013] Preferably, the standing time is 2-5 days.
[0014] Preferably, the concentration of the sodium hydroxide solution is 0.5-2 M.
[0015] Preferably, the fish liver oil chitosan microspheres ms3 are immersed in the sodium hydroxide solution for 10-30 min.
[0016] Preferably, the water washing is normal pressure filtration water washing.
[0017] The application further provides fish liver oil pure chitosan microspheres prepared by the preparation method.
[0018] The application further provides application of the fish liver oil pure chitosan microspheres in preparation of food and medicine.
[0019] Compared with the prior art, the technical scheme of the application has the following beneficial effects:
[0020] The application forms the hollow microspheres wrapping fish liver oil only by pure chitosan through self-assembly technology at a mild temperature without using a crosslinking agent, the preparation process is simple, the stability of the fish liver oil can be improved, the fish liver oil pure chitosan microspheres prepared have high biological safety, good antibacterial property and high embedding rate. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 : fish liver oil chitosan microsphere reaction process schematic diagram;
[0022] Figure 2 : infrared spectrum of fish liver oil pure chitosan microspheres, chitosan, fish liver oil and sodium dodecyl sulfonate.
[0023] Figure 3 : Peroxide value of cod liver oil microspheres in each group;
[0024] Figure 4 : Cutting diagram of cod liver oil pure chitosan microspheres prepared in Example 3;
[0025] Figure 5 : Cutting diagram of cod liver oil pure chitosan microspheres prepared in Comparative Example 1;
[0026] Figure 6 : Cutting diagram of cod liver oil pure chitosan microspheres with sand yellow aqueous solution dripped inside. DETAILED DESCRIPTION
[0027] The present invention provides a method for preparing cod liver oil pure chitosan microspheres without using a cross-linking agent, comprising the following steps:
[0028] The cod liver oil and chitosan solution are uniformly stirred in a volume ratio of 1:(2-6) to obtain an emulsion m1; the emulsion m1 is squeezed into a sodium dodecyl sulfate solution to obtain cod liver oil chitosan microspheres MS2; the cod liver oil chitosan microspheres MS2 are dried, placed in a sodium dodecyl sulfate solution and allowed to stand to obtain cod liver oil chitosan microspheres MS3; the cod liver oil chitosan microspheres MS3 are immersed in a sodium hydroxide solution and then washed with water until neutral to obtain cod liver oil chitosan microspheres MS4.
[0029] The preparation process of cod liver oil pure chitosan microspheres of the present invention is as follows: Figure 1 shown.
[0030] The chitosan solution of the present invention has a concentration of 3 to 5 g / 100 mL, preferably 4 g / 100 mL, and the solvent of the chitosan solution is 2% acetic acid. The 2% acetic acid of the present invention is obtained by mixing 2 mL of acetic acid with 100 mL of purified water. The chitosan solution of the present invention is preferably prepared by dissolving chitosan in a 2% acetic acid solution.
[0031] The volume ratio of the cod liver oil and chitosan solution is preferably 1:3, 1:4 or 1:5. After the cod liver oil and chitosan solution are uniformly stirred to obtain emulsion m1, the emulsion m1 is preferably squeezed into the sodium lauryl sulfate solution using a syringe.
[0032] The concentration of the sodium dodecyl sulfate solution of the present invention is 10-20 mg / mL, preferably 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL or 19 mg / mL, and the temperature of the sodium dodecyl sulfate solution is 60-90° C., preferably 65° C., 70° C., 75° C., 80° C. or 85° C.
[0033] The present invention dries the cod liver oil chitosan microspheres MS2 at a drying temperature of 40 to 60° C., preferably 45° C., 50° C. or 55° C., and a drying time of 50 to 70 minutes, preferably 55 minutes, 60 minutes or 65 minutes.
[0034] The present invention places dried cod liver oil chitosan MS2 in a sodium dodecyl sulfate solution and allows the solution to stand for 2 to 5 days, preferably 3 to 4 days, to obtain cod liver oil chitosan microspheres MS3. The chitosan solution in the cod liver oil chitosan MS2 is allowed to stand in the sodium dodecyl sulfate solution for more than 2 days, thereby self-assembling to form thick spherical shells, thereby achieving a better cod liver oil encapsulation effect.
[0035] The invention immerses the cod liver oil chitosan microspheres ms3 in a sodium hydroxide solution with a concentration of 0.5 to 2 M, preferably 1 to 1.5 M; and immerses the cod liver oil chitosan microspheres ms3 in the sodium hydroxide solution for 10 to 30 minutes, preferably 15 minutes, 20 minutes or 25 minutes.
[0036] In the present invention, the cod liver oil chitosan microspheres are removed from the sodium hydroxide solution and then washed with water, preferably by filtration under normal pressure until the water is neutral, to obtain cod liver oil chitosan microspheres MS4. The cod liver oil chitosan microspheres MS4 obtained in the present invention are pure cod liver oil chitosan microspheres prepared without using a crosslinking agent.
[0037] The present invention also provides cod liver oil pure chitosan microspheres prepared by the above preparation method. The cod liver oil pure chitosan microspheres have high biological safety, good antibacterial property and high embedding rate.
[0038] The present invention also provides the use of the cod liver oil pure chitosan microspheres in the preparation of food and medicine.
[0039] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0040] In the following examples, unless otherwise specified, all methods are conventional.
[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0042] Example 1
[0043] 4 g of chitosan was dissolved in 100 mL of 2% acetic acid solution (obtained by mixing 2 mL of acetic acid and 100 mL of purified water) to prepare a 4% chitosan solution; cod liver oil and 4% chitosan solution were stirred evenly in a volume ratio of 1:3 to obtain emulsion m1; the emulsion m1 was squeezed into a 10 mg / mL sodium dodecyl sulfate solution at 90°C using a syringe to form cod liver oil chitosan microspheres ms2; the cod liver oil chitosan microspheres ms2 were taken out, dried at 60°C for 1 hour, placed in a 10 mg / mL sodium dodecyl sulfate solution at 90°C, allowed to stand for 3 days, and taken out to obtain cod liver oil chitosan microspheres ms3; the cod liver oil chitosan microspheres ms3 were immersed in a 0.5 M sodium hydroxide solution for 10 minutes, taken out, filtered and washed with water at normal pressure until neutral, to obtain cod liver oil pure chitosan microspheres (ms4).
[0044] Example 2
[0045] 3 g of chitosan was dissolved in 100 mL of 2% acetic acid solution (obtained by mixing 2 mL of acetic acid and 100 mL of purified water) to prepare a 3% chitosan solution; cod liver oil and 3% chitosan solution were stirred evenly in a volume ratio of 1:2 to obtain emulsion m1; the emulsion m1 was squeezed into a 20 mg / mL sodium dodecyl sulfate solution at 70°C using a syringe to form cod liver oil chitosan microspheres ms2; the cod liver oil chitosan microspheres ms2 were taken out, dried at 50°C for 1 hour, placed in a 20 mg / mL sodium dodecyl sulfate solution at 70°C, allowed to stand for 2 days, and taken out to obtain cod liver oil chitosan microspheres ms3; the cod liver oil chitosan microspheres ms3 were immersed in a 2M sodium hydroxide solution for 20 minutes, taken out, filtered and washed with water at normal pressure until neutral, to obtain cod liver oil pure chitosan microspheres (ms4).
[0046] Example 3
[0047] 5 g of chitosan was dissolved in 100 mL of 2% acetic acid solution (obtained by mixing 2 mL of acetic acid and 100 mL of purified water) to prepare a 5% chitosan solution; cod liver oil and 5% chitosan solution were stirred evenly in a volume ratio of 1:6 to obtain emulsion m1; the emulsion m1 was squeezed into a 15 mg / mL sodium dodecyl sulfate solution at 60°C using a syringe to form cod liver oil chitosan microspheres ms2; the cod liver oil chitosan microspheres ms2 were taken out, dried at 40°C for 1 hour, placed in a 15 mg / mL sodium dodecyl sulfate solution at 60°C, allowed to stand for 4 days, and taken out to obtain cod liver oil chitosan microspheres ms3; the cod liver oil chitosan microspheres ms3 were immersed in a 1.5 M sodium hydroxide solution for 30 minutes, taken out, filtered and washed with water at normal pressure until neutral, to obtain cod liver oil pure chitosan microspheres (ms4).
[0048] Comparative Example 1
[0049] 5 g of chitosan was dissolved in 100 mL of 2% acetic acid solution (obtained by mixing 2 mL of acetic acid and 100 mL of purified water) to prepare a 5% chitosan solution; cod liver oil and 5% chitosan solution were stirred evenly in a volume ratio of 1:6 to obtain an emulsion m1; the emulsion m1 was squeezed into a 15 mg / mL sodium dodecyl sulfate solution at 60°C using a syringe to form cod liver oil chitosan microspheres ms2; the cod liver oil chitosan microspheres ms2 were taken out, immersed in a 1.5 M sodium hydroxide solution for 30 minutes, taken out, filtered and washed with water at normal pressure until neutral, and cod liver oil pure chitosan microspheres (ms4) were obtained.
[0050] Test Example 1
[0051] 1. Comparison of the infrared spectra of the cod liver oil pure chitosan microspheres (cod liver oil chitosan microspheres), chitosan, cod liver oil and sodium lauryl sulfate prepared in Example 3. The results are as follows: Figure 2 The results showed that the cod liver oil chitosan microspheres did not contain the characteristic peak of sodium dodecyl sulfate, indicating that sodium dodecyl sulfate had been completely removed from the cod liver oil chitosan microspheres.
[0052] 2. Determination of microsphere embedding efficiency
[0053] The embedding efficiency of the cod liver oil pure chitosan microspheres prepared in Comparative Example 1 and Examples 1 to 3 was tested.
[0054] Surface oil: Accurately weigh 3g of dry microsphere sample (M) into a dried conical flask, add 30mL of petroleum ether with a boiling range of 30-60°C, shake and extract for 5 minutes, filter through filter paper, and then wash the conical flask and filter paper with 10mL of petroleum ether. The filtrate is collected in a dry constant weight conical flask (M1). After evaporating the filtrate in a 60°C water bath, transfer it to a forced air drying oven and dry it at 60°C to constant weight (M2). Perform three parallel tests, and the test data is the average of the three times.
[0055] Surface oil = (M2-M1) / M×100%;
[0056] Where M is the mass of the sample (g), M1 is the mass of the conical flask (g), and M2 is the mass of the dried conical flask and oil (g).
[0057] Total oil: Accurately weigh 2g of dry sample (M) into a 50mL test tube. Dissolve in 10mL of dilute hydrochloric acid. Transfer to a 100mL stoppered graduated cylinder. Add 30mL of ether. Stopper and shake for 1 minute. Let stand for 10 minutes. Unstopper and rinse the stopper and cylinder with 20mL of an equal mixture of petroleum ether and ether. When the upper layer of liquid is clear, aspirate the supernatant and place it in a dry, constant-weight conical flask (M1). Add another 10mL of ether to the stoppered graduated cylinder, shake, let stand, and then aspirate the upper layer of ether and place it in the original conical flask. Evaporate the conical flask in a water bath and dry in an oven at (105±1)°C to a constant weight (M2). Perform three parallel tests, and the test data is the average of the three replicates.
[0058] Total oil = (M2-M1) / 2×100% where M1 is the mass of the conical flask (g) and M2 is the mass of the conical flask and oil after drying (g).
[0059] Embedding rate = (1-surface oil / total oil) x 100%. The statistical results of the embedding rate are shown in Table 1.
[0060] Table 1 Embedding rate results of each group (%)
[0061] batch Comparative Example 1 Example 1 Example 2 Example 3 one 59.12 92.54 90.43 95.83 two 58.48 91.65 91.56 96.42 three 58.20 92.17 90.33 94.88 average value 58.60 92.12 90.77 95.71
[0062] The results showed that after standing for more than 2 days, the embedding rate of chitosan self-assembled microspheres was significantly improved, and increasing the chitosan concentration was also beneficial to improving the embedding rate.
[0063] 3. Determination of microsphere peroxide value
[0064] The peroxide values of the cod liver oil, comparative example 1 (comparative example 1), and the cod liver oil pure chitosan microspheres prepared in Examples 1 to 3 were detected.
[0065] Determination of peroxide value: Take the dried microspheres as samples and place them in an environment of (40±2)℃ and relative humidity of 75%±5% for 1, 3, 6, 9, 12, 18, 24 and 30 days to observe the changes in the peroxide value of the samples. The peroxide value of microsphere cod liver oil was determined in accordance with GB / T5009.37-2003 "Analysis method of edible vegetable oil hygiene standard". The results are as follows: Figure 3 shown.
[0066] The results showed that in the same period of time, the peroxide value of cod liver oil in self-assembled cod liver oil chitosan microspheres was much smaller than that of cod liver oil without any protective measures and cod liver oil chitosan microspheres without static treatment, indicating that the self-assembled statically treated chitosan microspheres have a very obvious protective effect on cod liver oil.
[0067] 4. Microsphere cutting observation
[0068] The cod liver oil pure chitosan microspheres prepared in Example 3 and Comparative Example 1 were cut and observed.
[0069] The cod liver oil pure chitosan microspheres prepared in Example 3 were cut as follows Figure 4 The results show that the diameter of the cod liver oil pure chitosan microspheres is about 1mm. When the microspheres are cut open, the cod liver oil inside can be seen, and they are hollow. This indicates that after self-assembly during static treatment, all the liquid chitosan forms a membrane, entrapping the cod liver oil inside.
[0070] The cod liver oil pure chitosan microspheres prepared in Comparative Example 1 were cut as follows Figure 5 The results show that the diameter of the cod liver oil pure chitosan microspheres is about 1 mm, and the chitosan inside the microspheres is liquid. After cutting the microspheres, the microspheres deform and the internal liquid overflows. This indicates that self-assembly without static treatment, the microspheres are in a liquid state after cutting without the protection of the shell.
[0071] For the convenience of observation, the cod liver oil pure chitosan microspheres prepared in Example 3 were dripped with a safflower aqueous solution (a 0.45 mm needle was used to inject the safflower aqueous solution into the microspheres). Figure 6 As shown, the results show that the interior of the microspheres is uniformly red, proving that they are hollow microspheres with obvious spherical shells.
[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for preparing pure chitosan microspheres from cod liver oil without using a crosslinking agent, characterized in that: The following steps are involved: The cod liver oil and chitosan solution are uniformly stirred in a volume ratio of 1:(2-6) to obtain an emulsion m1; the emulsion m1 is squeezed into a sodium dodecyl sulfate solution to obtain cod liver oil chitosan microspheres MS2; the cod liver oil chitosan microspheres MS2 are dried, placed in a sodium dodecyl sulfate solution and allowed to stand to obtain cod liver oil chitosan microspheres MS3; the cod liver oil chitosan microspheres MS3 are immersed in a sodium hydroxide solution and then washed with water until neutral to obtain cod liver oil chitosan microspheres MS4.
2. The preparation method according to claim 1, characterized in that The concentration of the chitosan solution is 3-5 g / 100 mL, and the solvent of the chitosan solution is 2% acetic acid.
3. The preparation method according to claim 1, characterized in that The concentration of the sodium dodecyl sulfate solution is 10-20 mg / mL, and the temperature of the sodium dodecyl sulfate solution is 60-90° C.
4. The preparation method according to claim 1, characterized in that The drying temperature is 40-60° C. and the drying time is 50-70 minutes.
5. The preparation method according to claim 1, characterized in that The standing time is 2 to 5 days.
6. The preparation method according to claim 1, characterized in that The concentration of the sodium hydroxide solution is 0.5-2M.
7. The preparation method according to claim 1, characterized in that The cod liver oil chitosan microspheres MS3 are immersed in a sodium hydroxide solution for 10 to 30 minutes.
8. The preparation method according to claim 1, characterized in that The water washing is normal pressure filtration washing.
9. Cod liver oil pure chitosan microspheres prepared by the preparation method according to any one of claims 1 to 8.
10. Use of the cod liver oil pure chitosan microspheres according to claim 9 in the preparation of food and medicine.