Preparation method of multi-layer microcapsule for delivering fucoxanthin

Abstract

The invention discloses a preparation method of a multi-layer microcapsule for delivering fucoxanthin, and belongs to the technical field of food and drug processing. The preparation method comprises the following steps: S1, mixing krill oil with a co-emulsifier to obtain a microemulsion, adding fucoxanthin, stirring to obtain a krill oil self-microemulsion system, adding water, and mixing to obtain a fucoxanthin-loaded microemulsion; s2, mixing the obtained fucoxanthin-loaded microemulsion with chitosan, and adding a polysaccharide solution into the obtained mixed solution for mixing, so as to obtain a feed solution; and S3, drying the feed liquid to obtain the fucoxanthin-loaded microcapsule. The microcapsule wall material is prepared through electrostatic layer-by-layer deposition on the fucoxanthin-loaded microemulsion, and then spray drying is performed to obtain the fucoxanthin-loaded multilayer microcapsule, so that the bioavailability of fucoxanthin can be improved, and fixed-point release of fucoxanthin is realized.

Description

Technical Field

[0001] The present application relates to the field of food and drug processing technology, and in particular to a method for preparing a multilayer microcapsule for delivering fucoxanthin. Background Art

[0002] Fucoxanthin is a natural xanthophyll pigment extracted from algae. It has received extensive attention in recent years due to its anti-inflammatory, antioxidant, anti-cancer, and neuroprotective effects, especially in the food and pharmaceutical fields, where it has potential preventive and therapeutic effects on a variety of diseases. However, due to its highly unsaturated structure, fucoxanthin is very easy to oxidize and is not conducive to preservation. At the same time, its poor solubility in water reduces its bioavailability and limits its use. Therefore, a new formulation technology is needed to solve these problems.

[0003] In order to improve the dissolution rate and dissolution rate of fucoxanthin, increasing the specific surface area of ​​the particles is a common method. At the same time, the carrier can also protect fucoxanthin and enhance the stability of fucoxanthin. Therefore, microcapsules are the most suitable dosage form. At present, the research on the preparation of fucoxanthin microcapsules is not sufficient, the composition is relatively simple, and it cannot effectively improve the bioavailability and stability of fucoxanthin.

[0004] Electrostatic layer-by-layer deposition technology can be used to produce microcapsules of oil-in-water emulsion droplets, in which the wall material with positive charge is absorbed onto the negatively charged emulsifier at the droplet interface, which can increase the thickness of the microemulsion interface and better protect the encapsulated carrier material. Currently, there is no report on how to combine fucoxanthin with electrostatic layer-by-layer deposition technology in the prior art, and the present invention can fill the gap in related research. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for preparing multilayer microcapsules for delivering fucoxanthin. The present invention uses krill oil to form a microemulsion to encapsulate fucoxanthin, which can improve the bioavailability of fucoxanthin. The multilayer microcapsules are prepared by an electrostatic layer-by-layer deposition method. The thicker wall material of the microcapsule is wrapped on the surface of the microemulsion, which can better protect the loaded fucoxanthin, reduce dissolution in the gastric environment, and achieve the targeted release of fucoxanthin.

[0006] In one aspect, the present invention provides a method for preparing a multilayer microcapsule for delivering fucoxanthin, characterized in that the preparation method comprises the following steps:

[0007] S1: krill oil and glycerol are mixed to obtain krill oil microemulsion, fucoxanthin is added and stirred to obtain a krill oil self-microemulsion system, and water is added and mixed to obtain a microemulsion loaded with fucoxanthin;

[0008] S2: mixing the fucoxanthin-loaded microemulsion obtained above with chitosan, adding the polysaccharide solution to the obtained mixed solution and mixing them to obtain a feed solution;

[0009] S3: The feed liquid is dried to obtain microcapsules loaded with fucoxanthin.

[0010] Optionally, in step S1, the mass ratio of krill oil to glycerol is (1:5)-(1:10); and the mass fraction of fucoxanthin is 10-40wt% of the krill oil microemulsion.

[0011] Optionally, the co-emulsifier is any one or more of glycerol, propylene glycol, mono- and di-glycerol fatty acids.

[0012] Preferably, the co-emulsifier is glycerol.

[0013] Optionally, in step S1, the krill oil self-microemulsion system is mixed with water in a mass ratio of (1:1)-(1:5) to obtain a microemulsion loaded with fucoxanthin.

[0014] Optionally, the stirring rate in step S1 is 5000-20000 rpm, the stirring time is 3-10 min, and the stirring is uniform at 55-65 MPa.

[0015] Optionally, in step S2, the fucoxanthin-loaded microemulsion and the chitosan solution are mixed at a mass ratio of 1:1, and magnetically stirred for 10-20 minutes to obtain a mixed solution.

[0016] Optionally, the chitosan solution is prepared by dissolving chitosan powder in 1% acetic acid solution to fully dissolve the chitosan powder into a 1-4% chitosan solution.

[0017] Optionally, in step S2, the mixed solution and the polysaccharide solution are mixed in a mass ratio of 1:1, and magnetically stirred for 10-20 minutes to obtain a feed solution for spray drying.

[0018] Optionally, the polysaccharide solution is any one of laminarin and maltodextrin, and the concentration of the polysaccharide solution is 1-6%.

[0019] Preferably, the polysaccharide solution is laminarin, and the concentration of the polysaccharide solution is 2-4%.

[0020] Optionally, in step S3, the spray drying particle size is 0.5 mm, the air intake rate is 80%, the feed rate is 1 L / h, the inlet temperature is 180°C, and the outlet temperature is 85-90°C.

[0021] Another aspect of the present invention provides a multilayer microcapsule prepared by any one of the preparation methods described above, and application of the multilayer microcapsule in the manufacture of food, health products and drug delivery.

[0022] The beneficial effects of this application include but are not limited to:

[0023] 1. In the preparation method of the multilayer microcapsules for delivering fucoxanthin provided by the present invention, krill oil is firstly used to self-emulsify to form a microemulsion to combine with fucoxanthin. The microemulsion serves as the oil core of the microcapsule and can dissolve the fucoxanthin that is difficult to dissolve in water, thereby achieving the encapsulation of fucoxanthin and improving the bioavailability of fucoxanthin. In addition, the diameter of the droplets of the krill oil microemulsion beads is smaller than that of ordinary emulsions, and the nanoscale volume can provide a larger specific surface area, and the surface tension of the droplets is greater, which can further improve the solubility of fucoxanthin and is beneficial to the absorption and utilization of fucoxanthin.

[0024] 2. The present invention prepares multilayer microcapsules by an electrostatic layer-by-layer deposition method, wherein positively charged chitosan is used as a wall material and adsorbed on the negatively charged phospholipid surface layer on the surface of the microemulsion, and then negatively charged laminarin is adsorbed on the surface of the chitosan, and spray-dried to form multilayer microcapsules; the above-mentioned microcapsules have thicker wall materials, which can fully protect the internal oil core and the fucoxanthin therein from oxidation; in addition, the chitosan layer in the middle of the microcapsule has a strong positive charge in the acidic environment of the stomach, which can enhance the electrostatic adsorption of the outermost laminarin, improve the stability of the microcapsule, reduce the dissolution of the microcapsule in the stomach, and the electrostatic effect of the chitosan layer is weakened in the intestinal environment, and the microcapsule is decomposed, thereby achieving the fixed-point release of fucoxanthin.

[0025] 3. In the present invention, the oil core is made of krill oil microemulsion. Krill oil is rich in phospholipid polyunsaturated fatty acids, which can improve the solubility of fucoxanthin and facilitate its absorption by the digestive system. In addition, by delivering fucoxanthin in the form of microcapsules, powdered microcapsules can be made into a variety of solid dosage forms to meet the preparation needs of powders, tablets, capsules, etc., and broaden the application of fucoxanthin. DETAILED DESCRIPTION

[0026] In order to more clearly illustrate the overall concept of the present invention, a detailed description is given below by way of examples.

[0027] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0028] Hereinafter, a method for preparing a multilayer microcapsule for delivering fucoxanthin according to the present invention will be described in detail in conjunction with exemplary embodiments.

[0029] Example 1

[0030] A method for preparing multilayer microcapsules for delivering fucoxanthin comprises the following steps:

[0031] (1) Preparation of microemulsion loaded with fucoxanthin

[0032] Krill oil and glycerol are mixed in a mass ratio of 1:5 for self-emulsification to obtain a krill oil microemulsion, and 10wt% of fucoxanthin is added, and stirred at 5000rpm for 10 minutes using a high-speed homogenizer. After reaching a fluid state, a microfluidic device is immediately used to homogenize at 60MPa to obtain a krill oil self-microemulsification system loaded with fucoxanthin; the krill oil self-microemulsification system loaded with fucoxanthin is mixed with water in a mass ratio of 1:1 to obtain a microemulsion loaded with fucoxanthin.

[0033] (2) Preparation of microcapsule wall materials by electrostatic layer-by-layer deposition

[0034] The chitosan powder was dissolved in 1% acetic acid solution to obtain a 1% chitosan solution. The microemulsion loaded with fucoxanthin was mixed with the chitosan solution at a mass ratio of 1:1, and stirred slowly for 15 minutes with a magnetic stirrer to allow the chitosan to be adsorbed on the surface of the microemulsion droplets; the obtained solution was mixed with a 2% laminarin solution at a mass ratio of 1:1, and stirred slowly for 15 minutes with a magnetic stirrer to allow the laminarin to be adsorbed on the surface of the microcapsules to obtain a feed solution.

[0035] (3) Preparation of microcapsules by spray drying

[0036] The feed liquid was dried in a spray dryer equipped with a 0.5 mm nozzle atomizer, and was gently stirred during the spray drying process to finally obtain microcapsules loaded with fucoxanthin. The aspirator rate was adjusted to 80%, the feed rate was set to 1 L / h, the inlet temperature was set to 180°C, and the outlet temperature was set to 85°C.

[0037] Example 2

[0038] A method for preparing multilayer microcapsules for delivering fucoxanthin comprises the following steps:

[0039] (1) Preparation of microemulsion loaded with fucoxanthin

[0040] Krill oil and glycerol are mixed in a mass ratio of 1:8 for self-emulsification to obtain a krill oil microemulsion, and 20wt% of fucoxanthin is added, and stirred at 5000rpm for 10 minutes using a high-speed homogenizer. After reaching a fluid state, the mixture is immediately homogenized at 60MPa using a microfluidic device to obtain a krill oil self-microemulsification system loaded with fucoxanthin; the krill oil self-microemulsification system loaded with fucoxanthin is mixed with water in a mass ratio of 1:3 to obtain a microemulsion loaded with fucoxanthin.

[0041] (2) Preparation of microcapsule wall materials by electrostatic layer-by-layer deposition

[0042] The chitosan powder was dissolved in 1% acetic acid solution to obtain a 2% chitosan solution. The microemulsion loaded with fucoxanthin was mixed with the chitosan solution at a mass ratio of 1:1, and stirred slowly for 15 minutes with a magnetic stirrer to allow the chitosan to be adsorbed on the surface of the microemulsion droplets; the obtained solution was mixed with a 3% laminarin solution at a mass ratio of 1:1, and stirred slowly for 15 minutes with a magnetic stirrer to allow the laminarin to be adsorbed on the surface of the microcapsule to obtain a feed solution.

[0043] (3) Preparation of microcapsules by spray drying

[0044] The feed liquid was dried in a spray dryer equipped with a 0.5 mm nozzle atomizer, and was gently stirred during the spray drying process to finally obtain microcapsules loaded with fucoxanthin. The aspirator rate was adjusted to 80%, the feed rate was set to 1 L / h, the inlet temperature was set to 180°C, and the outlet temperature was set to 88°C.

[0045] Example 3

[0046] A method for preparing multilayer microcapsules for delivering fucoxanthin comprises the following steps:

[0047] (1) Preparation of microemulsion loaded with fucoxanthin

[0048] Krill oil and glycerol are mixed in a mass ratio of 1:10 for self-emulsification to obtain a krill oil microemulsion, and 40wt% of fucoxanthin is added, and stirred at 5000rpm for 10 minutes using a high-speed homogenizer. After reaching a fluid state, the mixture is immediately homogenized at 60MPa using a microfluidic device to obtain a krill oil self-microemulsification system loaded with fucoxanthin; the krill oil self-microemulsification system loaded with fucoxanthin is mixed with water in a mass ratio of 1:5 to obtain a microemulsion loaded with fucoxanthin.

[0049] (2) Preparation of microcapsule wall materials by electrostatic layer-by-layer deposition

[0050] The chitosan powder was dissolved in 1% acetic acid solution to obtain a 4% chitosan solution. The microemulsion loaded with fucoxanthin was mixed with the chitosan solution at a mass ratio of 1:1, and stirred slowly for 15 minutes with a magnetic stirrer to allow the chitosan to be adsorbed on the surface of the microemulsion droplets; the obtained solution was mixed with a 4% laminarin solution at a mass ratio of 1:1, and stirred slowly for 15 minutes with a magnetic stirrer to allow the laminarin to be adsorbed on the surface of the microcapsules to obtain a feed solution.

[0051] (3) Preparation of microcapsules by spray drying

[0052] The feed liquid was dried in a spray dryer equipped with a 0.5 mm nozzle atomizer, and was gently stirred during the spray drying process to finally obtain microcapsules loaded with fucoxanthin. The aspirator rate was adjusted to 80%, the feed rate was set to 1 L / h, the inlet temperature was set to 180°C, and the outlet temperature was set to 90°C.

[0053] Example 4

[0054] A method for preparing multilayer microcapsules for delivering fucoxanthin comprises the following steps:

[0055] (1) Preparation of microemulsion loaded with fucoxanthin

[0056] Krill oil and propylene glycol are mixed in a mass ratio of 1:6 for self-emulsification to obtain a microemulsion, and 30wt% of fucoxanthin is added, and stirred at 5000rpm for 10 minutes using a high-speed homogenizer. After reaching a fluid state, the mixture is immediately homogenized at 60MPa using a microfluidic device to obtain a krill oil self-microemulsification system loaded with fucoxanthin; the krill oil self-microemulsification system loaded with fucoxanthin is mixed with water in a mass ratio of 1:2 to obtain a microemulsion loaded with fucoxanthin.

[0057] (2) Preparation of microcapsule wall materials by electrostatic layer-by-layer deposition

[0058] The chitosan powder was dissolved in 1% acetic acid solution to obtain a 1.5% chitosan solution. The microemulsion loaded with fucoxanthin was mixed with the chitosan solution at a mass ratio of 1:1, and stirred slowly for 10 minutes with a magnetic stirrer to allow the chitosan to be adsorbed on the surface of the microemulsion droplets; the obtained solution was mixed with 1% maltodextrin solution at a mass ratio of 1:1, and stirred slowly for 10 minutes with a magnetic stirrer to allow the fucoidan to be adsorbed on the surface of the microcapsule to obtain a feed solution for spray drying.

[0059] (3) Preparation of microcapsules by spray drying

[0060] The feed liquid was dried in a spray dryer equipped with a 0.5 mm nozzle atomizer, and was gently stirred during the spray drying process to finally obtain microcapsules loaded with fucoxanthin. The aspirator rate was adjusted to 80%, the feed rate was set to 1 L / h, the inlet temperature was set to 180°C, and the outlet temperature was set to 85°C.

[0061] Example 5

[0062] A method for preparing multilayer microcapsules for delivering fucoxanthin comprises the following steps:

[0063] (1) Preparation of microemulsion loaded with fucoxanthin

[0064] Krill oil and mono- and di-glycerol fatty acid esters are mixed in a mass ratio of 1:7 for self-emulsification to obtain a microemulsion, and 25 wt% of fucoxanthin is added, and stirred at 5000 rpm for 10 minutes using a high-speed homogenizer. After reaching a fluid state, a microfluidic device is immediately used to homogenize at 60 MPa to obtain a krill oil self-microemulsification system loaded with fucoxanthin; the krill oil self-microemulsification system loaded with fucoxanthin is mixed with water in a mass ratio of 1:4 to obtain a microemulsion loaded with fucoxanthin.

[0065] (2) Preparation of microcapsule wall materials by electrostatic layer-by-layer deposition

[0066] The chitosan powder was dissolved in 1% acetic acid solution to obtain a 4% chitosan solution. The microemulsion loaded with fucoxanthin was mixed with the chitosan solution at a mass ratio of 1:1, and stirred slowly for 20 minutes with a magnetic stirrer to allow chitosan to be adsorbed on the surface of the microemulsion droplets; the obtained solution was mixed with a 6% maltodextrin solution at a mass ratio of 1:1, and stirred slowly for 20 minutes with a magnetic stirrer to allow maltodextrin to be adsorbed on the surface of the microcapsule to obtain a feed solution for spray drying.

[0067] (3) Preparation of microcapsules by spray drying

[0068] The feed liquid was dried in a spray dryer equipped with a 0.5 mm nozzle atomizer, and was gently stirred during the spray drying process to finally obtain microcapsules loaded with fucoxanthin. The aspirator rate was adjusted to 80%, the feed rate was set to 1 L / h, the inlet temperature was set to 180°C, and the outlet temperature was set to 90°C.

[0069] Comparative Example 1

[0070] In this comparative example, another comparative microcapsule loaded with fucoxanthin was prepared by the same preparation method as in Example 1. The difference from Example 1 is that krill oil was not added in step (1).

[0071] Comparative Example 2

[0072] In this comparative example, another comparative microcapsule loaded with fucoxanthin was prepared by the same preparation method as in Example 1. The difference from Example 1 is that glycerol is not added in step (1).

[0073] Comparative Example 3

[0074] In this comparative example, another comparative microcapsule loaded with fucoxanthin was prepared by the same preparation method as in Example 1. The difference from Example 1 is that the krill oil, glycerol and fucoxanthin were simply physically mixed.

[0075] Comparative Example 4

[0076] In this comparative example, another comparative microcapsule loaded with fucoxanthin was prepared by the same preparation method as in Example 1. The difference from Example 1 is that cellulose is used instead of chitosan in step (2).

[0077] Comparative Example 5

[0078] In this comparative example, another comparison microcapsule loaded with fucoxanthin was prepared by the same preparation method as in Example 1. The difference from Example 1 is that after the microemulsion loaded with fucoxanthin was prepared, electrostatic layer-by-layer deposition was no longer performed to prepare the microcapsule wall material.

[0079] Comparative Example 6

[0080] In this comparative example, another comparative microcapsule loaded with fucoxanthin was prepared by the same preparation method as in Example 1. The difference from Example 1 is that laminarin is not added in step (2).

[0081] Experiment 1 Effects of different preparation conditions on microemulsion

[0082] 1.1 Determination of the ratio of krill oil to glycerol

[0083] 1.1.1 Experimental method: krill oil and glycerol were mixed in proportion, and 10 wt% fucoxanthin was added and stirred evenly. A high-speed homogenizer was used to shear at 5000 rpm for 10 min, and a microfluidic device was used to homogenize at 60 MPa immediately after reaching a fluid state. The obtained krill oil self-microemulsion system loaded with fucoxanthin was mixed with water in a ratio of 1:2 to obtain a microemulsion loaded with fucoxanthin.

[0084] 1.1.2 Result analysis: Take microemulsion samples and measure the particle size of different samples using a laser particle size analyzer at room temperature.

[0085] Table 1. Particle size analysis of krill oil and glycerol at different weight ratios

[0086] Krill Oil: Glycerin Particle size / nm 1:0 Unable to form 1:1 Unable to form 1:2 Unable to form 1:3 Unable to form 1:4 Unable to form 1:5 452.15±56.32 1:6 379.22±31.28 1:8 276.47±27.61 1:10 227.53±16.82 1:12 1483.24±89.35 0:1 Unable to form

[0087] As shown in the experimental results in Table 1, due to the high free fatty acid content and poor fluidity of Antarctic krill oil, it is impossible to directly prepare a microemulsion as an oil phase. After adding glycerol, the krill oil-glycerol mixed system becomes more viscous at a certain ratio. According to the preparation method, the operation is carried out and different degrees of turbid emulsion are formed after adding water. When the ratio reaches 1:5, the microemulsion system is successfully prepared. A microemulsion with a smaller particle size and a more stable particle size can be formed in the mass ratio range of (1:5)-(1:10). When the ratio continues to increase, the particle size increases significantly. This is because the excess glycerol reduces the surface charge of the microemulsion, and the reduction of electrostatic repulsion leads to a higher average particle size, which is easy to break. When there is only glycerol in the system, a microemulsion cannot be prepared. Therefore, the mass ratio of krill oil and glycerol is preferably within the range of (1:5)-(1:10), which can ensure that the microemulsion loaded with fucoxanthin can form a stable particle size.

[0088] 1.2 Determination of fucoxanthin ratio

[0089] 1.2.1 Experimental method: The krill oil:glycerol ratio was determined to be 1:5, and the encapsulation efficiency and drug loading of fucoxanthin were tested by changing the weight ratio of fucoxanthin in the system.

[0090] 1.2.2 Result analysis: The specific values ​​of fucoxanthin encapsulation efficiency and drug loading are shown in the following table.

[0091] Table 2. Encapsulation efficiency and drug loading data of fucoxanthin microemulsion at different specific gravities

[0092] Fucoxanthin weight percentage Encapsulation rate / % Drug loading / % 5wt% 95.24±3.75 5.62±1.40 10wt% 96.38±3.21 12.74±4.40 20wt% 92.12±4.50 13.10±5.53 30wt% 91.48±1.80 13.68±2.91 40wt% 82.90±2.11 8.96±1.19 50wt% 75.30±1.26 6.37±2.25

[0093] As shown in the experimental results in Table 2, when the weight ratio of fucoxanthin is in the range of 5-30wt%, the encapsulation rate is above 90%, and when the weight ratio of fucoxanthin is in the range of 10-30wt%, the drug loading exceeds 10%, which meets the requirements of the use range. When the weight ratio of fucoxanthin exceeds 30wt%, both the encapsulation rate and the drug loading have a downward trend. Among them, when the weight ratio of fucoxanthin is 5wt%, although it has a higher encapsulation rate, the drug loading is low; based on the overall data, when the weight ratio of fucoxanthin is in the range of 10-40%, it can achieve an effective drug loading on the basis of a higher encapsulation rate.

[0094] 1.3 Determination of medium-water ratio

[0095] 1.3.1 Experimental method: krill oil and glycerol are mixed in a mass ratio of 1:1 to self-emulsify to obtain a microemulsion, and then 10wt% fucoxanthin is added for homogenization to obtain a krill oil self-microemulsification system loaded with fucoxanthin. The stable state of the emulsion is determined by changing the different ratios of the krill oil self-microemulsification system loaded with fucoxanthin and water.

[0096] 1.3.2 Result analysis: The dispersion states of the krill oil self-microemulsion system loaded with fucoxanthin and water at different proportions are shown in the following table.

[0097] Table 3. Analysis of the dispersion state of krill oil self-microemulsification system

[0098]

[0099] As shown in the experimental results in Table 3, when the mass ratio of self-microemulsion: water is in the range of (1:0.1)-(1:0.5), the amount of water added is small, and the loaded fucoxanthin microemulsion cannot be evenly dispersed in the water environment; as the water ratio increases, a clear and transparent microemulsion is formed; when the amount of water added exceeds 1:5, the concentration of krill oil as a surfactant is affected, and the microemulsion becomes turbid and forms stratification. The surfactant must maintain a certain concentration in the dispersion system to form a microemulsion. When the concentration is too low after excessive dilution with water, its aggregation morphology will change, and some surfactants will dissociate from the microemulsion and exist in the form of simple dispersion, which leads to the rupture of the microemulsion, causing some drugs to precipitate, resulting in turbidity of the emulsion. In summary, when the ratio of the krill oil self-microemulsion system loaded with fucoxanthin to water is in the range of (1:1)-(1:5), a uniformly dispersed, clear and transparent microemulsion can be obtained.

[0100] Experiment 2 Effects of different groups on the absorption of fucoxanthin by setting up different groups in various examples and comparative examples

[0101] 2.1 In vitro drug release

[0102] 2.1.1 Experimental method: In this experiment, the sample components prepared in Examples 1-5 and Comparative Examples 1-6 were placed in different dissolution media (artificial gastric juice with pH = 1.2, artificial intestinal juice with pH = 6.8) to conduct in vitro release behavior tests using the dialysis bag method.

[0103] The test samples prepared in each group of the embodiment and comparative example were respectively loaded into a dialysis bag with a molecular cutoff of 3500Da, and the dialysis bag was placed in a beaker. 100mL of artificial gastric juice was accurately measured and poured into the beaker, and the temperature was set to 37°C and the speed was 100rpm. 1mL was sampled at 30min, 1h, and 2h, and the corresponding volume of medium was replenished in time. After 2h, the dialysis bag was transferred to a 100mL beaker containing artificial intestinal fluid, and the release test was continued. 1mL was sampled at 6h, 12h, and 24h, and the corresponding volume of medium was replenished in time.

[0104] 2.1.2 Result analysis: The samples taken at each time point were centrifuged at 10,000 rpm for 10 min, then diluted with an appropriate amount of chromatographic methanol for pretreatment, filtered through a 0.22 μm microporous filter membrane, and the drug content was determined by HPLC to calculate the cumulative release rate.

[0105] (Cumulative release rate (%) = {concentration measured at the current sampling point × medium volume + (concentration measured at the previous sampling point × sampling volume)} ÷ dosage × 100%; total medium 100mL, dosage 5mg, take 1mL each time, and then add 1mL of fresh dissolution medium.)

[0106] Table 4. Release rates of different groups in artificial gastric juice and artificial intestinal juice

[0107]

[0108] As shown in the experimental results in Table 4, the simulated gastrointestinal environment has a great influence on the fucoxanthin content during the entire digestion stage. It can be seen from the data of the sample groups of Examples 1-5 and Comparative Examples 1-6 that the fucoxanthin microcapsules prepared in Examples 1-5 have thicker capsule wall materials, which can fully ensure that the oil core and the fucoxanthin therein are not oxidized; the chitosan layer in the middle can improve the stability of the microcapsules while reducing the dissolution of the microcapsules in the gastric environment. Comparative Examples 1, 2, and 3 all failed to form a microemulsion system, and because the electrostatic deposition effect of Comparative Examples 1 and 2 was not strong, relatively more fucoxanthin was released in the gastric juice. In Comparative Example 4, since cellulose carries a negative charge, it cannot adsorb with the negative charge of phospholipids in the microemulsion, and therefore is easily decomposed in gastric juice; in Comparative Example 5, the fucoxanthin in the microemulsion system can be released in gastric juice and intestinal juice in a gentle manner; in Comparative Example 6, no laminarin component is added, and adsorption protection cannot be formed on the surface of chitosan, increasing the dissolution of the microcapsules in gastric juice and releasing the fucoxanthin in advance. In summary, the multilayer microcapsules prepared by Examples 1-5 of the present application can be more stably released at a fixed point in the intestine, especially the multilayer microcapsules prepared by Examples 1-3 have a better stable release effect in the intestine.

[0109] 2.2 Body fat-lowering efficacy experiment

[0110] 2.2.1 Rats fed with high-fat diet for 8 weeks were randomly divided into 13 groups, namely blank control group, sample groups of Examples 1-5, and sample groups of Comparative Examples 1-6. Eight rats in each group were given equal amounts of samples. After 8 weeks of intervention, the rats were fasted overnight, weighed, and anesthetized with 3% sodium pentobarbital 40 mg / kg intraperitoneally. Visceral fat tissue specimens were separated, weighed, and cooled in liquid nitrogen for later use.

[0111] 2.2.2 Result analysis: The body weight and visceral fat weight of rats treated with the above different sample groups were weighed and analyzed.

[0112] Table 5. Body weight and visceral fat weight of rats in different treatment groups

[0113]

[0114]

[0115] As shown in the experimental data of Table 5, Examples 1-5 can significantly reduce the weight and visceral fat weight of rats, which shows that the microcapsule group prepared in this application can better control the fixed-point release of fucoxanthin and can increase the concentration of its active ingredients in the intestinal tract. In Comparative Examples 1, 2, and 3, it did not form a stable microemulsion system, and after entering the rat body, it could not well control the fixed-point release of fucoxanthin, and it would be released and decomposed in gastric acid in advance, and in Comparative Example 4, its cellulose could not form a stable package with the phospholipids in the emulsion; while in Comparative Example 5, no encapsulation effect on the microcapsules was formed, and it would be slowly released in gastric juice; Comparative Example 6 failed to form adsorption protection on the surface of chitosan, which would cause the early release of microcapsules. In summary, the microcapsule group prepared in this application is more conducive to controlling the fixed-point release of fucoxanthin in the intestines of rats, regulating the expression of thermogenic metabolic factors such as mitochondrial uncoupling protein 1 (UCP1) in adipose tissue, thereby accelerating the oxidation process of fatty acids, promoting the decomposition of visceral fat, and reducing the accumulation of fat in the viscera.

[0116] The above is only an embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing multilayer microcapsules for delivering fucoxanthin, characterized in that: The preparation method comprises the following steps: S1: krill oil is mixed with an emulsifier to obtain a krill oil microemulsion, fucoxanthin is added and stirred to obtain a krill oil self-microemulsification system, and water is added and mixed to obtain a fucoxanthin-loaded microemulsion; S2: mixing the fucoxanthin-loaded microemulsion obtained above with chitosan, adding the polysaccharide solution to the obtained mixed solution and mixing them to obtain a feed solution; S3: The feed liquid is dried to obtain microcapsules loaded with fucoxanthin.

2. The method for preparing multilayer microcapsules for delivering fucoxanthin according to claim 1, characterized in that: In the step S1, the mass ratio of krill oil to the co-emulsifier is (1:5)-(1:10), and the mass fraction of fucoxanthin is 10-40wt% of the krill oil microemulsion.

3. The method for preparing multilayer microcapsules for delivering fucoxanthin according to claim 1, characterized in that: In the step S1, the co-emulsifier is any one or more of glycerol, propylene glycol, mono- and di-glycerol fatty acid esters; preferably, the co-emulsifier is glycerol.

4. The method for preparing multilayer microcapsules for delivering fucoxanthin according to claim 1, characterized in that: In the step S1, the krill oil self-microemulsion system is mixed with water in a mass ratio of (1:1)-(1:5) to obtain a microemulsion loaded with fucoxanthin.

5. The method for preparing multilayer microcapsules for delivering fucoxanthin according to claim 1, characterized in that: In the step S2, the microemulsion loaded with fucoxanthin and the chitosan solution are mixed at a mass ratio of 1:1, and magnetic stirring is performed for 10-20 minutes to obtain a mixed solution.

6. The method for preparing multilayer microcapsules for delivering fucoxanthin according to claim 5, characterized in that: The preparation step of the chitosan solution is to dissolve chitosan powder with 1% acetic acid solution to fully dissolve it into a 1-4% chitosan solution.

7. The method for preparing multilayer microcapsules for delivering fucoxanthin according to claim 1, characterized in that: In the step S2, the mixed solution and the polysaccharide solution are mixed at a mass ratio of 1:1, and magnetically stirred for 10-20 minutes to obtain a feed solution.

8. The method for preparing multilayer microcapsules for delivering fucoxanthin according to claim 7, characterized in that: The polysaccharide solution is any one of laminarin and maltodextrin, and the concentration of the polysaccharide solution is 1-6%; Preferably, the polysaccharide solution is laminarin, and the concentration of the polysaccharide solution is 2-4%.

9. The method for preparing multilayer microcapsules for delivering fucoxanthin according to claim 1, characterized in that: In step S3, the spray drying particle size is 0.5 mm, the air intake rate is 80%, the feed rate is 1 L / h, the inlet temperature is 180° C., and the outlet temperature is 85-90° C.

10. A multilayer microcapsule obtained by the preparation method according to any one of claims 1 to 9, and use of the multilayer microcapsule in the manufacture of food, health products and drug delivery.

Images