Nano-microcapsule containing docosahexaenoic acid, preparation method therefor and use thereof, and animal feed or food

Abstract

A nano-microcapsule containing docosahexaenoic acid, a preparation method therefor and a use thereof, and animal feed or food. The nano-microcapsule comprises, from inside to outside, a core material, a core-coating wall material, and a gastro-soluble wall material, wherein the core material comprises DHA algal oil, an emulsifier, and an antioxidant, the core-coating wall material comprises zein, the gastro-soluble wall material comprises chitosan or polyacrylic resin IV, and the weight ratio of the DHA algal oil to the zein to the chitosan or the polyacrylic resin IV is 1:5-10:5-10.

Description

Nanocapsules containing docosahexaenoic acid, their preparation methods and applications, and animal feed or food.

[0001] This application claims priority to Chinese Patent Application No. 202411188665.X, filed on August 28, 2024, entitled "Nanocapsules Containing Docosahexaenoic Acid, Preparation Method and Application Thereof and Animal Feed or Food", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to a nanocapsule containing docosahexaenoic acid, its preparation method and application, and animal feed or food, belonging to the technical field of animal feed additives or food additives. Background Technology

[0003] Docosahexaenoic acid (DHA) is an essential omega-3 polyunsaturated fatty acid for humans, possessing extremely high nutritional value and participating in many important physiological processes. DHA is synthesized in very small amounts in the human body and must be obtained through diet. Among various sources, cow's milk has a unique advantage, serving as an important carrier for DHA supplementation. By adding DHA to feed and utilizing the dairy cow's metabolic mechanism, DHA can be enriched in milk to form an organic whole, creating native DHA milk, effectively avoiding the drawbacks of directly adding DHA to cow's milk. Improving the nutritional value and quality of milk through biofortification is an important direction for solving the problems in the DHA milk industry. However, the unique physiological structure of ruminants greatly limits the development of native DHA milk. DHA and other unsaturated fatty acids are easily destabilized by hydrogenation in the special digestive environment of the rumen, significantly affecting the enrichment of DHA in milk. It has been reported that rumen digestion is the digestive stage with the highest DHA loss rate during rumen digestion and absorption, with a loss rate exceeding 80%, severely limiting the application of DHA and other unsaturated fatty acids in ruminants. Therefore, the development of rumen-protected products containing unsaturated fatty acids has become a research hotspot in recent years.

[0004] Ruminants have four stomachs: the rumen, reticulum, omasum, and abomasum. The rumen is the primary site of DHA instability, with a pH generally around 6.8, while the abomasum (true stomach) typically has a pH below 1.5 and contains a large amount of pepsin. Therefore, utilizing the differences in physiological environments between the rumen, abomasum, and small intestine to specifically construct rumen-transferred carriers to reduce DHA loss is a very promising approach. Summary of the Invention

[0005] To address the aforementioned shortcomings and deficiencies, the present invention aims to provide a nanocapsule containing docosahexaenoic acid (DHA), its preparation method, applications, and animal feed or food. This invention utilizes pH-responsive materials and nano-coating technology to encapsulate or embed substances such as DHA. The resulting nanocapsules exhibit good resistance to rumen degradation and excellent release in the abomasum and small intestine, thereby improving the utilization rate of DHA in ruminants.

[0006] To achieve the above objectives, on the one hand, the present invention provides a nanocapsule containing docosahexaenoic acid (DHA), wherein the nanocapsule comprises, from the inside out, a core material, a core-encasing wall material, and a gastric-dissolving wall material. The core material contains DHA algal oil, an emulsifier, and an antioxidant. The core-encasing wall material contains amphiphilic proteins such as zein. The gastric-dissolving wall material contains pH-responsive materials such as chitosan or polyacrylic acid resin IV. The weight ratio of DHA algal oil, zein, and chitosan or polyacrylic acid resin IV is 1:5-10:5-10.

[0007] In one specific embodiment of the nano-microcapsules described above in this invention, the weight ratio of the DHA algal oil, emulsifier, and antioxidant is 100:1-2:1-2.

[0008] In one specific embodiment of the nano-microcapsules described above in this invention, the emulsifier includes Tween 80, etc., and the antioxidant includes tea polyphenols, etc.

[0009] In one specific embodiment of the nano-microcapsules described above in this invention, the gastric wall material is chitosan.

[0010] In one specific embodiment of the nanocapsules described above in this invention, the nanocapsules containing docosahexaenoic acid have a particle size of less than 400 nm.

[0011] On the other hand, the present invention also provides a method for preparing the above-described nanocapsules containing docosahexaenoic acid, wherein the preparation method includes:

[0012] S1: Dissolve zein in an ethanol-water solution and mix well to obtain a zein solution;

[0013] S2: Dissolve chitosan or polyacrylic acid resin IV in an aqueous acetic acid solution and mix well to obtain a gastric wall material solution;

[0014] S3: DHA algal oil, emulsifier and antioxidant are added to zein solution, and then nanoscale microcapsules are formed by high-speed shearing, homogenization and ultrasound.

[0015] S4: Slowly drip the material obtained in S3 into the gastric wall material solution and mix thoroughly;

[0016] S5: After vacuum drying to remove ethanol from the material obtained in S4, centrifuge or filter to obtain an emulsion;

[0017] S6: Spray dry the emulsion to obtain the nanocapsules containing docosahexaenoic acid.

[0018] As a specific embodiment of the preparation method described above in this invention, in S1, the volume concentration of ethanol in the ethanol-water solution is 75-85%, wherein the volume concentration is calculated based on the total volume of the ethanol-water solution.

[0019] As a specific embodiment of the preparation method described above in this invention, in S1, the concentration of zein in the zein solution is 10-40 mg / mL, wherein the concentration is calculated based on the total volume of the zein solution.

[0020] As a specific embodiment of the preparation method described above in this invention, in S2, the volume concentration of acetic acid in the aqueous acetic acid solution is 0.5-1%, wherein the volume concentration is calculated based on the total volume of the aqueous acetic acid solution.

[0021] As a specific embodiment of the preparation method described above in this invention, in S2, the mass concentration of chitosan or polyacrylic acid resin IV in the gastric wall material solution is 2-10 mg / mL, wherein the mass concentration is calculated based on the total volume of the gastric wall material solution.

[0022] In S1 and S2 of the preparation method described above in this invention, mixing and homogenization can be achieved by stirring.

[0023] As a specific embodiment of the preparation method described above in this invention, in S3, the formation of nanoscale microcapsules by high-speed shearing, homogenization, and ultrasound includes:

[0024] First, shear at high speed for 3-8 minutes at a rotation speed of 8000-12000 r / min, then sonicate for 5-15 minutes, wherein the ultrasonic frequency is 20-60 kHz and the power is 240-500 W; preferably, shear at high speed for 5 minutes at a rotation speed of 10000 r / min, then sonicate for 10 minutes, wherein the ultrasonic frequency is 40 kHz and the power is 240 W.

[0025] In step S4 of the preparation method described above, mixing and homogenization can also be achieved by stirring. In some embodiments of the present invention, the material obtained in step S3 can be slowly dripped into the gastric wall material solution while the solution is being stirred at high speed to achieve mixing and homogenization. In some embodiments of the present invention, the stirring speed in step S4 can be 500-2000 r / min, preferably 1000 r / min, and the stirring time can be 20-40 min.

[0026] In step S5 of the preparation method described above, the purpose of centrifugation or filtration is to remove unstable particles in order to obtain an emulsion.

[0027] As a specific embodiment of the preparation method described above in this invention, in S5, the vacuum drying can be rotary vacuum drying, and the vacuum drying time can be 15-30 min.

[0028] In a specific embodiment of the preparation method described above in this invention, in S5, the centrifugation speed is 1500-2500g and the centrifugation time is 5-15min, preferably 10min.

[0029] As a specific embodiment of the preparation method described above in this invention, in S6, the inlet air temperature of the spray drying is 160-190℃ and the outlet air temperature is 75-85℃.

[0030] In another aspect, the present invention also provides the application of the above-described nanocapsules containing docosahexaenoic acid as a food additive or animal feed additive.

[0031] In another aspect, the present invention also provides an animal feed comprising an animal feed additive, wherein the animal feed additive is the above-mentioned nanocapsules containing docosahexaenoic acid.

[0032] Finally, the present invention also provides a food product comprising a food additive, wherein the food additive is the aforementioned nanocapsules containing docosahexaenoic acid.

[0033] Compared with the prior art, the beneficial technical effects that the present invention can achieve include:

[0034] The nanocapsules containing docosahexaenoic acid provided by this invention have excellent rumen-passing performance and stability, and can be precisely released / responded to release in acidic (low pH) environments such as the abomasum and small intestine, which facilitates absorption by the abomasum and small intestine.

[0035] This invention uses high-purity DHA algal oil and other liquid lipids to prepare the core material of nanocapsules. The resulting nanocapsules have small particle size (less than 400 nanometers), high bioavailability, and facilitate efficient conversion of DHA.

[0036] The nanocapsules containing docosahexaenoic acid provided by this invention have a reasonable composition and ratio, and the nanocapsules have good granulation properties.

[0037] The preparation method of docosahexaenoic acid-containing nanocapsules provided by this invention is simple and controllable, consumes little energy, is conducive to safe production, has a short production cycle, and is suitable for large-scale production. In addition, the preparation method provided by this invention is also suitable for encapsulation of various rumen-soluble drugs or additives or additives with properties similar to DHA, such as EPA. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 is a process flow diagram of preparing nanocapsules containing docosahexaenoic acid in an embodiment of the present invention. Detailed Implementation

[0040] It should be noted that the term "comprising" and any variations thereof in the specification, claims, and accompanying drawings of this invention are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or devices.

[0041] The "range" disclosed in this invention is given in the form of a lower limit and an upper limit. It can be one or more lower limits and one or more upper limits, respectively. A given range is defined by selecting a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges defined in this way are composable, meaning that any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for specific parameters, it is also expected that ranges of 60-110 and 80-120 are also expected. Furthermore, if the listed minimum range values ​​are 1 and 2, and the listed maximum range values ​​are 3, 4, and 5, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5.

[0042] In this invention, unless otherwise specified, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this invention, and "0-5" is simply a shortened representation of these numerical combinations.

[0043] In this invention, unless otherwise specified, all embodiments and preferred embodiments mentioned in this invention can be combined with each other to form new technical solutions.

[0044] In this invention, unless otherwise specified, all technical features and preferred features mentioned in this invention can be combined with each other to form new technical solutions.

[0045] In this invention, unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, if the method includes steps (a) and (b), it means that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, if the method may also include step (c), it means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying tables, drawings, and embodiments. The embodiments described below are some, but not all, embodiments of this invention, and are only used to illustrate the invention, and should not be considered as limiting the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0047] Example 1

[0048] This embodiment provides a nanocapsule containing docosahexaenoic acid, as shown in Figure 1, and its preparation method includes:

[0049] S1: Add 8g of zein to an 80% ethanol-water solution and stir to mix well, so that the zein concentration is 20mg / mL, and obtain a zein solution.

[0050] S2: Selecting chitosan as the wall material: Dissolve 8g of chitosan in a 1% (v / v) aqueous solution of acetic acid, stir and mix well to maintain the chitosan concentration at 2mg / mL, and obtain a gastric wall material solution.

[0051] S3: 1.6g of high-purity DHA algal oil (DHA weight content of 69.2%), 16mg of tea polyphenols and 16mg of Tween 80 were sequentially added to the zein solution obtained in S1, so that the concentrations of DHA algal oil, tea polyphenols and Tween 80 were 4mg / mL, 0.04mg / mL and 0.04mg / mL, respectively. The mixture was first sheared at 10000r / min for 5min and then sonicated for 10min to form nanoscale microcapsules. This step yielded a nanoemulsion containing nanoscale microcapsules.

[0052] S4: Slowly drip the material obtained in S3 into the gastric wall material solution obtained in S2. While the material is dripping in, stir the solution at high speed to mix the material. After mixing is complete, continue stirring and hydrating for 30 minutes, with the stirring speed being 1000 r / min.

[0053] S5: The material obtained in S4 is dried under rotary vacuum at 40°C for 30 min to remove ethanol, then centrifuged at 2000g for 10 min to filter out unstable particles and obtain emulsion.

[0054] S6: The emulsion obtained in S5 is spray-dried, wherein the inlet air temperature of the spray dryer is 180℃ and the outlet air temperature is 80℃, and nanocapsules containing docosahexaenoic acid are collected.

[0055] Example 2

[0056] This embodiment provides a nanocapsule containing docosahexaenoic acid, which differs from Example 1 only in the amount of zein and chitosan used. In this embodiment, the amount of both is 16g.

[0057] Example 3

[0058] This embodiment provides a nanocapsule containing docosahexaenoic acid, which differs from Example 1 only in the amount of zein and chitosan used. In this embodiment, the amounts of the two are 16g and 8g, respectively.

[0059] Comparative Example 1

[0060] This comparative example provides an unprotected, high-purity DHA algal oil, wherein the DHA content is 69.2% by weight.

[0061] Comparative Example 2

[0062] This comparative example provides a nanocapsule containing docosahexaenoic acid, which differs from Example 1 only in that chitosan is not used and the amount of zein used is 3.2g.

[0063] Comparative Example 3

[0064] This comparative example provides a nanocapsule containing docosahexaenoic acid, which differs from Example 1 only in that chitosan is not used and the amount of zein used is 8g.

[0065] Comparative Example 4

[0066] This comparative example provides a nanocapsule containing docosahexaenoic acid, which differs from Example 1 only in that chitosan is not used and the amount of zein used is 16g.

[0067] Comparative Example 5

[0068] This comparative example provides a nanocapsule containing docosahexaenoic acid, which is prepared by a method including the following specific steps:

[0069] Step (1): Dissolve 8g of chitosan in a 1% acetic acid aqueous solution, stir and mix well to maintain the chitosan concentration at 2mg / mL, and obtain a chitosan solution.

[0070] Step (2): 1.6g of high-purity DHA algal oil (DHA weight content of 69.2%), 16mg of tea polyphenols and 16mg of Tween 80 were added to the chitosan solution obtained in step (1) in sequence, so that the concentrations of DHA algal oil, tea polyphenols and Tween 80 were 4mg / mL, 0.04mg / mL and 0.04mg / mL, respectively. First, the solution was sheared at 10000r / min for 5min, and then sonicated for 10min to form nanoscale microcapsules. The nanoemulsion containing nanoscale microcapsules was obtained in this step.

[0071] Step (3): Centrifuge the material obtained in step (2) at 2000g for 10min to filter out unstable particles and obtain an emulsion;

[0072] Step (4): Freeze-dry the emulsion obtained in step (3) and collect nanocapsules containing docosahexaenoic acid.

[0073] Comparative Example 6

[0074] This comparative example provides a nanocapsule containing docosahexaenoic acid, which is prepared by a method including the following specific steps:

[0075] Step (1): Add 8g of zein to an 80% ethanol-water solution and stir to make the zein concentration 20mg / mL, thus obtaining a zein solution.

[0076] Step (2): Dissolve 8g of sodium carboxymethyl cellulose in an aqueous solution of 1% acetic acid, stir and mix well to maintain the mass concentration of sodium carboxymethyl cellulose at 2mg / mL, and obtain sodium carboxymethyl cellulose solution.

[0077] Step (3): 1.6g of high-purity DHA algal oil (DHA weight content of 69.2%), 16mg of tea polyphenols and 16mg of Tween 80 were added to the zein solution obtained in S1 in sequence, so that the concentrations of DHA algal oil, tea polyphenols and Tween 80 were 4mg / mL, 0.04mg / mL and 0.04mg / mL, respectively. First, the mixture was sheared at 10000r / min for 5min, and then sonicated for 10min to form nanoscale microcapsules. The nanoemulsion containing nanoscale microcapsules was obtained in this step.

[0078] Step (4): Slowly drop the material obtained in step (3) into the sodium carboxymethyl cellulose solution obtained in step (2). While the material is being dropped in, the solution is stirred at high speed to mix the material. After mixing is complete, continue stirring and hydrating for 30 minutes, with the stirring speed being 1000 r / min.

[0079] Step (5): The material obtained in step (4) is dried under rotary vacuum at 40°C for 30 min to remove ethanol, then centrifuged at 2000g for 10 min to filter out unstable particles and obtain emulsion;

[0080] Step (6): Freeze-dry the emulsion obtained in step (5). From the appearance of the product, it can be seen that the product prepared by compounding sodium carboxymethyl cellulose and zein has poor coagulation and granulation properties and is easy to form a film.

[0081] Comparative Example 7

[0082] This comparative example provides a nanocapsule containing docosahexaenoic acid, which is prepared by a method including the following specific steps:

[0083] Step (1): Add 8g of ethyl cellulose to an 80% ethanol-water solution, stir and mix well to make the ethyl cellulose concentration 20mg / mL, and obtain an ethyl cellulose solution.

[0084] Step (2): Dissolve 8g of chitosan in a 1% acetic acid aqueous solution, stir and mix well to maintain the chitosan mass concentration at 2mg / mL, and obtain a chitosan solution.

[0085] Step (3): 1.6g of high-purity DHA algal oil (DHA weight content of 69.2%), 16mg of tea polyphenols and 16mg of Tween 80 were added to the zein solution obtained in S1 in sequence, so that the concentrations of DHA algal oil, tea polyphenols and Tween 80 were 4mg / mL, 0.04mg / mL and 0.04mg / mL respectively. First, the mixture was sheared at 10000r / min for 5min, and then sonicated for 10min to form nanoscale microcapsules. The nanoemulsion containing nanoscale microcapsules was obtained in this step.

[0086] Step (4): Slowly drop the material obtained in step (3) into the chitosan solution obtained in step (2). While the material is being dropped in, the solution is stirred at high speed to mix the material. After mixing is complete, continue stirring and hydrating for 30 minutes, with the stirring speed being 1000 r / min.

[0087] Step (5): The material obtained in step (4) is dried under rotary vacuum at 40°C for 30 min to remove ethanol and obtain an emulsion. The emulsion is unstable and prone to precipitation. From the appearance of the obtained product, it can be seen that the sample prepared by compounding ethyl cellulose and chitosan is unstable, prone to precipitation, and has a low product yield.

[0088] Comparative Example 8

[0089] This comparative example provides a nanocapsule containing docosahexaenoic acid, which differs from Example 1 only in that: chitosan is not used, the amount of zein used is 8g, and the emulsion obtained in S5 is freeze-dried in S6.

[0090] The appearance of the nanocapsules containing docosahexaenoic acid provided by Comparative Examples 8 and 5 shows that both zein and chitosan have good coagulation and granulation properties, and can spontaneously form particles with high-purity DHA algal oil. Meanwhile, according to the experimental data shown in Table 1 below, the rumen-passing efficiency of a single wall material is relatively low, while the rumen-passing efficiency of the composite material is higher.

[0091] Test Example 1

[0092] This test example examines the in vitro rumen-passing performance of the nanocapsules provided in Examples 1-3 and Comparative Examples 1-4 of this invention to determine their rumen-passing efficiency. Three parallel samples were set up for each sample. The specific steps are as follows:

[0093] Place an appropriate amount of nanocapsules and substrate (powder made from grinding dairy cow's daily feed) into an anaerobic fermentation tube, and add 15 mL of artificial rumen fluid (the volume ratio of fresh rumen fluid to rumen fluid is 1:2) into the anaerobic fermentation tube.

[0094] The fresh rumen fluid is taken from the rumen of dairy cows and includes:

[0095] Three hours after morning feeding, rumen contents from experimental cattle with rumen fistulas were collected in collection bottles and brought back to the laboratory for processing within half an hour. The rumen fluid was mixed thoroughly and filtered through four layers of gauze for later use.

[0096] The preparation method of rumen mimicry solution includes:

[0097] Prepare a trace element solution (solution A) containing 1.32 g / L CaCl2·2H2O, 1.00 g / L MnCl2·4H2O, 0.10 g / L CoCl2·6H2O, and 0.80 g / L FeCl3·6H2O.

[0098] Prepare a buffer solution (Solution B) containing 4 g / L NH4HCO3 and 35 g / L NaHCO3;

[0099] Prepare a macro-element solution (solution C) containing 9.45 g / L Na2HPO4·12H2O, 6.2 g / L KH2PO4, and 0.6 g / L MgSO4·7H2O;

[0100] Prepare a reducing agent solution (solution D) containing 95 mL of distilled water, 4 mL of 1 mol / L NaOH, 625 mg of NaS·9H2O, and 625 mg of L-cysteine ​​hydrochloride.

[0101] Preparation of rumen simulant solution: Add 0.1 mL of solution A, 208.1 mL of solution B, 208.1 mL of solution C, and 520.2 mL of distilled water to a narrow-necked flask, and gently shake the flask to mix the solutions thoroughly. Place the narrow-necked flask in a microwave oven and boil for about 10 minutes. Place a magnetic rod in the narrow-necked flask and place it on a magnetic stirrer, maintaining the temperature at 39°C, and continuously purging CO2 gas for about 1 hour to adjust the pH value to 6.8. After adjustment, add 62.4 mL of solution D and mix well to obtain the rumen simulant solution.

[0102] Artificial rumen fluid and all substrates (appropriate amounts of nanocapsules and powdered dairy cow feed) were loaded into an anaerobic fermentation tube. Carbon dioxide was introduced to fill the tube, which was then sealed and cultured in a 39°C water bath in the dark to simulate rumen digestion for 24 hours. For the first 12 hours, the tube was inverted and mixed at least once per hour, and for the next 12 hours, it was inverted and mixed every 3-4 hours to prevent flocculation of the analyte at the top, which could affect the experimental results. After 24 hours, the anaerobic fermentation tube was removed, and the DHA content was measured. The DHA loss rate was calculated using Equation 1). The experimental data are shown in Table 1 below.

[0103] DHA loss rate (%) = (C initial –Cdigesta ) / C initial ×100 (Formula 1);

[0104] In Equation 1), C initial This refers to the amount of DHA absorbed before rumen digestion; C digesta This refers to the remaining amount of DHA after simulated digestion.

[0105] Table 1. DHA loss rate after 24 hours of in vitro fermentation of different nanocapsules

[0106] As shown in Table 1 above, after simulating 24 hours of rumen digestion, the loss rate of unprotected DHA algal oil was as high as 68.18%. Compared with Comparative Example 1, the DHA loss rate of the zein-chitosan double-walled nanocapsules containing docosahexaenoic acid provided in Examples 1-3 of this invention was significantly reduced after 24 hours of in vitro fermentation, with the lowest loss rate being only 15.77%. This indicates that the zein-chitosan double-walled material in the nanocapsules provided in this invention effectively reduced the loss of DHA in the rumen, and the rumen passage rate of DHA in the DHA algal oil increased by 164.71%.

[0107] As can be seen from Table 1 above, the DHA loss rate of the nanocapsules provided in Comparative Examples 2-4 after 24 hours of in vitro fermentation was lower than that of Comparative Example 1, but still significantly higher than that of the zein-chitosan double-wall nanocapsules containing docosahexaenoic acid provided in Examples 1-3 of this invention. This indicates that the rumen-passing efficiency of single-wall nanocapsules containing docosahexaenoic acid is higher than that of unprotected DHA algal oil, but still significantly lower than that of composite-wall nanocapsules containing docosahexaenoic acid.

[0108] Test Example 2

[0109] This test example uses artificial rumen fluid and artificial gastric fluid to simulate the release of the nanocapsules provided in Example 1 of this invention in the real stomach, including:

[0110] 150 mg of the nanocapsules provided in Example 1 were placed in an anaerobic fermentation tube. 15 mL of artificial rumen fluid (the same as in Test Example 1) was added to the anaerobic fermentation tube to dissolve the nanocapsules in the artificial rumen fluid. Carbon dioxide was introduced to fill the tube space. The tube was sealed and cultured in a 39°C water bath in the dark for 12 hours to simulate rumen digestion.

[0111] Remove the fermentation tube, add pepsin (purchased from Maclean Biotechnology Co., Ltd., P816236) to a concentration of 8.5 mg / mL, then add 2 mol / L hydrochloric acid to adjust the pH to below 2 (this is equivalent to using artificial gastric fluid for simulation), seal, and incubate in a 39°C water bath shaker in the dark for 1 hour.

[0112] A comparison of the solubility of the nanocapsules in simulated rumen fluid and simulated gastric fluid reveals that in the neutral environment of the rumen, the carrier wall material has poor solubility, and the carrier of the nanocapsules provided in Example 1 of this invention is not easily broken down, thus DHA is not released. However, in the acidic environment of the real stomach, the carrier wall material dissolves rapidly, releasing DHA for further digestion. In other words, the nanocapsules provided in Example 1 of this invention maintain good stability in the rumen environment and do not dissolve, while their water solubility is enhanced in the simulated real stomach environment, allowing for effective release of the core material.

[0113] In summary, the experimental results show that the wall material of the nanocapsules containing docosahexaenoic acid provided in this embodiment of the invention is zein and chitosan. The combination of zein and chitosan has good processability and stability, and is easy to form particles. Moreover, the combination of the two effectively improves the rumen passage efficiency of the nanocapsules containing docosahexaenoic acid, proving that the two materials have a good synergistic effect.

[0114] The above description is merely a specific embodiment of the present invention and should not be construed as limiting the scope of the invention. Therefore, any substitution of equivalent components or equivalent changes and modifications made within the scope of protection of this patent should still fall within the scope of this patent. Furthermore, the technical features, technical features and technical inventions, and technical inventions in this invention can be freely combined and used.

Claims

1. A nanocapsule containing docosahexaenoic acid, wherein, The nanocapsule comprises, from the inside out, a core material, a core-encasing wall material, and a gastrolytic wall material. The core material contains DHA algal oil, an emulsifier, and an antioxidant. The core-encasing wall material is zein, and the gastrolytic wall material is chitosan. The weight ratio of DHA algal oil, zein, and chitosan is 1:5-10:5-10. The nanocapsules containing docosahexaenoic acid have a particle size of less than 400 nm.

2. The nanocapsule according to claim 1, wherein, The weight ratio of the DHA algal oil, emulsifier, and antioxidant is 100:1-2:1-2.

3. The nanocapsule according to claim 1 or 2, wherein, The emulsifier includes Tween 80, and the antioxidant includes tea polyphenols.

4. The method for preparing nanocapsules containing docosahexaenoic acid according to any one of claims 1-3, wherein, The preparation method includes: S1: Dissolve zein in an ethanol-water solution and mix well to obtain a zein solution; S2: Dissolve chitosan in an aqueous acetic acid solution and mix well to obtain a gastric wall material solution; S3: DHA algal oil, emulsifier and antioxidant are added to zein solution, and then nanoscale microcapsules are formed by high-speed shearing, homogenization and ultrasound. S4: Slowly drip the material obtained in S3 into the gastric wall material solution and mix thoroughly; S5: After vacuum drying to remove ethanol from the material obtained in S4, centrifuge or filter to obtain an emulsion; S6: Spray dry the emulsion to obtain the nanocapsules containing docosahexaenoic acid.

5. The preparation method according to claim 4, wherein, In S1, the concentration of zein in the zein solution is 10-40 mg / mL.

6. The preparation method according to claim 4 or 5, wherein, In S1, the volume concentration of ethanol in the ethanol-water solution is 75-85%, which is calculated based on the total volume of the ethanol-water solution.

7. The preparation method according to claim 4, wherein, In S2, the mass concentration of chitosan in the gastric wall material solution is 2-10 mg / mL.

8. The preparation method according to claim 4 or 7, wherein, In S2, the volume concentration of acetic acid in the aqueous acetic acid solution is 0.5-1%, where the volume concentration is calculated based on the total volume of the aqueous acetic acid solution.

9. The preparation method according to claim 4, wherein, In S3, the formation of nanoscale microcapsules through high-speed shearing, homogenization, and ultrasound includes: First, shear at high speed for 3-8 minutes at a rotation speed of 8000-12000 r / min, then sonicate for 5-15 minutes, with an ultrasonic frequency of 20-60 kHz and a power of 240-500 W.

10. The preparation method according to claim 4, wherein, In step S4, the material obtained in step S3 is slowly dripped into the gastric wall material solution while the solution is stirred at high speed to achieve uniform mixing of the material; wherein, the stirring speed is 500-2000 r / min and the stirring time is 20-40 min.

11. The preparation method according to claim 4, wherein, In S5, the vacuum drying time is 15-30 minutes.

12. The preparation method according to claim 4 or 11, wherein, In S5, the centrifugation speed is 1500-2500g, and the centrifugation time is 5-15min.

13. The preparation method according to claim 4, wherein, In S6, the inlet air temperature of the spray dryer is 160-190℃, and the outlet air temperature is 75-85℃.

14. The use of the nanocapsules containing docosahexaenoic acid as described in any one of claims 1-3 as a food additive or animal feed additive.

15. An animal feed comprising animal feed additives, wherein, The animal feed additive is the nanocapsule containing docosahexaenoic acid as described in any one of claims 1-3.

16. A food product containing food additives, wherein, The food additive is the nanocapsule containing docosahexaenoic acid as described in any one of claims 1-3.

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