Preparation method of phytosterol delivery system

Abstract

The invention discloses a preparation method of a phytosterol delivery system, and the preparation method of the phytosterol delivery system comprises the following steps: diluting a vegetable fat body with water, and preheating to obtain a fat body diluent; adding phytosterol into the grease body diluent, carrying out primary stirring and ultrasonic treatment, and then heating and stirring to obtain a phytosterol grease body mixed solution; and cooling the phytosterol grease body mixed solution, and then centrifuging to take an upper-layer paste, so as to obtain a grease body loaded with phytosterol, namely the phytosterol delivery system. According to the phytosterol delivery system prepared by the invention, the contact between the phytosterol and the outside is reduced, the activity of the phytosterol is favorably maintained, and the bioavailability and functional activity of the phytosterol are improved; meanwhile, the unique structure formed by wrapping the phytosterol with the vegetable fat body can enhance the water solubility of the phytosterol, and can interact with the absorption mechanism in the intestinal tract, so that the absorption efficiency of the phytosterol can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and in particular to a method for preparing a phytosterol delivery system. Background Art

[0002] Phytosterols are a class of natural compounds with important physiological activities. They are widely found in plants and play many key roles in maintaining human health. Phytosterols can lower blood cholesterol levels and reduce the risk of cardiovascular disease. A large number of studies have shown that adequate intake of phytosterols can effectively prevent the intestinal absorption of cholesterol and promote cholesterol excretion, thereby regulating blood lipids and protecting heart and blood vessel health. In addition, phytosterols also have potential biological activities such as antioxidant, anti-inflammatory, and anti-tumor activities, which are of great significance for preventing chronic diseases and maintaining the normal physiological functions of the body.

[0003] However, phytosterols face some challenges in practical applications. First, phytosterols have extremely poor water solubility, which makes their bioavailability in the human body low. Since they are difficult to dissolve in aqueous environments, the absorption and metabolism of phytosterols are greatly limited. Secondly, phytosterols are prone to oxidation and degradation during storage and processing, reducing their activity and stability. In addition, how to effectively deliver phytosterols to target tissues or organs so that they can exert their optimal physiological effects is also a problem that needs to be solved urgently. Summary of the invention

[0004] The main purpose of the present invention is to propose a method for preparing a phytosterol delivery system, aiming to provide a method that can effectively improve the bioavailability and functional activity of phytosterols.

[0005] To achieve the above object, the present invention provides a method for preparing a phytosterol delivery system, the method for preparing the phytosterol delivery system comprising the following steps:

[0006] Diluting the vegetable oil with water and preheating it to obtain an oil dilution liquid;

[0007] Adding phytosterols to the oil body dilution liquid, subjecting to initial stirring and ultrasonic treatment, and then heating and stirring to obtain a phytosterol oil body mixed liquid;

[0008] The phytosterol oil body mixture is cooled, and then centrifuged to obtain the upper paste to obtain the phytosterol-loaded oil body, that is, the phytosterol delivery system is prepared.

[0009] Optionally, in the step of diluting the vegetable oil body with water and preheating the vegetable oil body to obtain an oil body dilution solution:

[0010] The vegetable fat bodies include pumpkin seed fat bodies.

[0011] Optionally, the extraction solvent of the pumpkin seed oil body includes any one of a NaHCO3 solution, a PBS solution (i.e., a phosphate buffer solution), and a Tris-HCl solution (i.e., a buffer solution prepared with tris(hydroxymethyl)aminomethane hydrochloride).

[0012] Optionally, in the step of diluting the vegetable oil body with water and preheating the vegetable oil body to obtain an oil body dilution solution:

[0013] The mass of the water is 2 to 10 times the mass of the vegetable oil.

[0014] Optionally, in the step of diluting the vegetable oil body with water and preheating the vegetable oil body to obtain an oil body dilution solution:

[0015] The preheating method includes water bath heating, the preheating temperature is 70-80° C., and the heating time is 1-2 min.

[0016] Optionally, in the step of adding phytosterols to the oil body dilution solution, subjecting the mixture to initial stirring and ultrasonic treatment, and then heating and stirring to obtain a phytosterol oil body mixture solution:

[0017] The mass of the phytosterol is 1-20% of the mass of the oil body diluent.

[0018] Optionally, in the step of adding phytosterols to the oil body dilution solution, subjecting the mixture to initial stirring and ultrasonic treatment, and then heating and stirring to obtain a phytosterol oil body mixture solution:

[0019] The stirring rate of the initial stirring is 200-300 rpm, and the stirring time is 3-5 min.

[0020] Optionally, in the step of adding phytosterols to the oil body dilution solution, subjecting the mixture to initial stirring and ultrasonic treatment, and then heating and stirring to obtain a phytosterol oil body mixture solution:

[0021] The ultrasonic power of the ultrasonic treatment is 200-400W, and the ultrasonic time is 2-3min.

[0022] Optionally, in the step of adding phytosterols to the oil body dilution solution, subjecting the mixture to initial stirring and ultrasonic treatment, and then heating and stirring to obtain a phytosterol oil body mixture solution:

[0023] The heating method of the heating and stirring includes water bath heating, the heating temperature of the heating and stirring is 60-100° C., and the heating time is 15-25 minutes.

[0024] Optionally, the phytosterol oil body mixture is cooled, and then centrifuged to obtain an upper paste to obtain the phytosterol-loaded oil body, i.e., in the step of preparing the phytosterol delivery system:

[0025] The cooling method includes ice water bath cooling.

[0026] Optionally, the phytosterol oil body mixture is cooled, and then centrifuged to obtain an upper paste to obtain the phytosterol-loaded oil body, i.e., in the step of preparing the phytosterol delivery system:

[0027] The centrifugal speed of the centrifugation is 4000-8000 rpm, and the centrifugal time is 10-30 min.

[0028] In the technical solution provided by the present invention, a phytosterol delivery system is formed by encapsulating phytosterols with plant oil bodies, which reduces the contact of phytosterols with the outside world, thereby helping to maintain the activity of phytosterols and improve the bioavailability and functional activity of phytosterols; at the same time, the unique structure formed by the encapsulation of phytosterols by plant oil bodies can enhance the water solubility of phytosterols and may interact with the absorption mechanism in the intestine, thereby helping to improve the absorption efficiency of phytosterols; in addition, the preparation method provided by the present invention has a simple process, is easy to realize industrial mass production, and the prepared phytosterol delivery system has high stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 A schematic flow chart of an embodiment of a method for preparing a phytosterol delivery system provided by the present invention;

[0031] Figure 2 is the amount of NO released in the cell inflammation model;

[0032] Figure 3 is the amount of IL-6 released in the cell inflammation model;

[0033] Figure 4 is the amount of TNF-α released in the cellular inflammation model.

[0034] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0035] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0036] Phytosterols face some challenges in practical applications. First, the water solubility of phytosterols is extremely poor, which makes their bioavailability in the human body low. Since they are difficult to dissolve in aqueous environments, the absorption and metabolism of phytosterols are greatly limited. Secondly, phytosterols are prone to oxidation and degradation during storage and processing, reducing their activity and stability. In addition, how to effectively deliver phytosterols to target tissues or organs so that they can exert their best physiological effects is also a problem that needs to be solved urgently.

[0037] After in-depth research, the inventors found that using the lipid body structure naturally present in oil seeds to achieve the encapsulation and delivery of specific substances may effectively improve the bioavailability and functional activity of phytosterols. Furthermore, after a lot of practice and experiments, the inventors invented a method for preparing a phytosterol delivery system. The phytosterol delivery system is constructed with plant lipid bodies, which can improve the biological activity and water solubility of phytosterols and improve the absorption rate of phytosterols by organisms. Specifically, Figure 1 Shown is an embodiment of the preparation method of the plant sterol delivery system provided by the present invention.

[0038] See also Figure 1 As shown, in this embodiment, the preparation method of the phytosterol delivery system comprises the following steps:

[0039] Step S10, diluting the vegetable oil with water and preheating the mixture to obtain an oil dilution solution;

[0040] Step S20, adding phytosterols to the oil body dilution liquid, performing initial stirring and ultrasonic treatment, and then heating and stirring to obtain a phytosterol oil body mixed liquid;

[0041] Step S30, cooling the phytosterol oil body mixture, and then centrifuging to obtain the upper paste to obtain the phytosterol-loaded oil body, that is, preparing the phytosterol delivery system.

[0042] First, the plant oil body is diluted with distilled water, and then preheated to obtain an oil body dilution liquid as a transmission carrier; then, phytosterol is added to the oil body dilution liquid, stirred, and after the phytosterol is completely immersed in the oil body dilution liquid, ultrasonic treatment is performed to make the phytosterol evenly distributed in the system, and then heated to increase the solubility of the phytosterol to obtain a phytosterol oil body mixed liquid; finally, the heated phytosterol oil body mixed liquid is immediately placed in a low-temperature environment to quickly cool it, and then centrifuged to obtain an upper paste to obtain a phytosterol-loaded oil body, thereby completing the preparation of the phytosterol delivery system.

[0043] In the technical solution provided by the present invention, a phytosterol delivery system is formed by encapsulating phytosterols with plant oil bodies, which reduces the contact of phytosterols with the outside world, thereby helping to maintain the activity of phytosterols and improve the bioavailability and functional activity of phytosterols; at the same time, the unique structure formed by the encapsulation of phytosterols by plant oil bodies can enhance the water solubility of phytosterols and may interact with the absorption mechanism in the intestine, which helps to improve the absorption efficiency of phytosterols, thereby improving the anti-inflammatory activity of phytosterols; in addition, the preparation method provided by the present invention has a simple process, is easy to realize industrial mass production, and the prepared phytosterol delivery system has high stability.

[0044] Furthermore, the specific advantages of the phytosterol delivery system prepared by the method provided by the present invention are reflected in: after the gastrointestinal simulated digestion experiment, the bioavailability of phytosterols was significantly improved from 45.76% to 75.74%, and the cholesterol-lowering activity was significantly enhanced from 44.79% to 50.21%; in the cell inflammation model experiment, the inhibitory ability of phytosterols on the release of crude inflammatory mediators by LPS-stimulated inflammatory cells was significantly enhanced. The above research results all show that the phytosterol delivery system constructed by encapsulating phytosterols in plant oil bodies provided by the present invention effectively improves the bioavailability and functional activity of phytosterols.

[0045] Plant lipid bodies are important subcellular organelles for storing lipids and energy in plant cells. They have a unique structure. The lipid bodies are composed of a hydrophobic core of neutral lipids and a layer of phospholipid monolayer outside the core. The phospholipid monolayer has specific protein modifications. In some embodiments of the present invention, the plant lipid bodies in step S10 include pumpkin seed lipid bodies, which are rich in a large amount of phenolic substances that can produce a synergistic effect with the loaded phytosterols, further improving the functional activity of the phytosterols.

[0046] The inventors have found through experimental research that since the phospholipids and proteins on the surface of the plant oil body are both hydrophilic and hydrophobic, the extraction effect of the plant oil body under different solvents is different. In order to better select oil bodies suitable for delivering plant sterols, the inventors have conducted a large number of experimental comparisons and preferentially selected the extraction solvents of the pumpkin seed oil body, including any one of NaHCO3 solution, PBS solution (i.e., phosphate buffer solution), and Tris-HCl solution (i.e., a buffer solution prepared with tris(hydroxymethyl)aminomethane hydrochloride). Furthermore, the pH value of different extraction solvents is preferably 8.3-8.4 for the NaHCO3 solution, 7.4-7.5 for the PBS solution, and 8.4-8.5 for the Tris-HCl solution.

[0047] In some embodiments of the present invention, the mass of the water in step S10 is 2 to 10 times the mass of the vegetable oil, for example, it can be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, etc.

[0048] In some embodiments of the present invention, the preheating temperature in step S10 is 70-80°C, for example, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, etc. In some specific embodiments of the present invention, the preheating temperature is preferably 75°C. Further, the preheating heating method and heating time can be adjusted according to the amount of material and the heating state of the material during actual operation. The heating method and set heating time used in laboratory small-scale production and industrial mass production may be different. The specific heating method and heating time are based on the overall uniform heating of the material to the aforementioned limited heating temperature range. Specifically, taking small-scale production in the laboratory as an example, in some embodiments of the present invention, the preheating method includes water bath heating, which makes the heating more uniform, requires simple equipment, and is energy-saving; the preheating time is 1 to 2 minutes, for example, it can be 1 minute, 1 minute 10 seconds, 1 minute 20 seconds, 1 minute 30 seconds, 1 minute 40 seconds, 1 minute 50 seconds, 2 minutes, and the like.

[0049] In some embodiments of the present invention, the mass of the phytosterols in step S20 is 1-20% of the mass of the oil body diluent, for example, it can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc. In some specific embodiments of the present invention, the mass of the phytosterols is preferably 10% of the mass of the oil body diluent.

[0050] In step S20, the plant sterols and the oil body diluent are preliminarily mixed by the stirring. The stirring speed and stirring time of the stirring can be adjusted according to the amount of materials and the mixing state of the materials during actual operation. The stirring conditions set for small-scale laboratory preparation and industrial mass production may be different. The specific stirring conditions are based on the uniform mixing of the materials visible to the naked eye. Specifically, taking the small-scale laboratory preparation as an example, in some embodiments of the present invention, the stirring speed of the stirring in step S20 is 200-300rpm, for example, it can be 200rpm, 210rpm, 220rpm, 230rpm, 240rpm, 250rpm, 260rpm, 270rpm, 280rpm, 290rpm, 300rpm, etc., and the stirring time of the stirring is 3-5min, for example, it can be 3min, 3min 30s, 4min, 4min 30s, 5min, etc.

[0051] Furthermore, in step S20, the phytosterols are more evenly distributed in the oil body diluent through the ultrasonic treatment. The ultrasonic conditions of the ultrasonic treatment can be adjusted according to the amount of material and the mixing state of the material during actual operation. The ultrasonic conditions set for small-scale laboratory preparation and industrial mass production may be different. The specific ultrasonic conditions are based on achieving sufficient and uniform mixing of the materials. Specifically, taking a small amount of preparation in the laboratory as an example, in some embodiments of the present invention, the ultrasonic power of the ultrasonic treatment in step S20 is 200-400W, for example, it can be 200W, 210W, 220W, 230W, 240W, 250W, 260W, 270W, 280W, 290W, 300W, 310W, 320W, 330W, 340W, 350W, 360W, 370W, 380W, 390W, 400W, etc.; the ultrasonic time of the ultrasonic treatment is 2-3min, for example, it can be 2min, 2min10s, 2min 20s, 2min 30s, 2min 40s, 2min 50s, 3min, etc.

[0052] In some embodiments of the present invention, the heating temperature of the heating and stirring in step S20 is 60-100°C, for example, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, etc. In some specific embodiments of the present invention, the heating temperature of the heating in step S20 is preferably 65-95°C. Further, the heating method, stirring speed and stirring time of the heating and stirring can be adjusted according to the amount of materials and the mixing state of the materials during actual operation. The stirring conditions set for small-scale laboratory preparation and industrial mass production may be different. The specific heating method and stirring conditions are based on achieving visible mixing of the materials. Specifically, taking the small-scale preparation in the laboratory as an example, in some embodiments of the present invention, the heating method of the heating and stirring includes water bath heating, which is more evenly heated, and requires simple equipment and is energy-saving; the heating time of the heating and stirring is 15 to 25 minutes, for example, it can be 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes and the like. In some specific embodiments of the present invention, the heating time of the heating and stirring is preferably 20 minutes; the stirring rate of the stirring heating is 200 to 300 rpm, for example, it can be 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, 260 rpm, 270 rpm, 280 rpm, 290 rpm, 300 rpm and the like.

[0053] Furthermore, the inventors have found through experimental studies that the solubility of phytosterols varies with concentration and temperature. In order to increase the embedding rate of phytosterols and to better improve the bioavailability and functional activity of phytosterols, the inventors have found through experiments that when phytosterols are encapsulated with pumpkin seed oil bodies, the extraction solvent of the pumpkin seed oil bodies, the amount of phytosterols added to the oil body diluent, and the heating temperature after the phytosterols and the oil body diluent are uniformly mixed (i.e., the heating temperature in step S20), the above three parameters can have the following preferred combinations:

[0054] (1) The extraction solvent of pumpkin seed oil body is NaHCO3 solution, the added mass of phytosterol is 10% of the mass of oil body diluent, and the heating temperature after phytosterol and oil body diluent are evenly mixed is 75°C;

[0055] (2) The extraction solvent of pumpkin seed oil body is Tris-HCl solution, the added mass of phytosterol is 10% of the mass of oil body diluent, and the heating temperature after phytosterol and oil body diluent are evenly mixed is 75°C;

[0056] (3) The extraction solvent of pumpkin seed oil body is PBS solution, the added mass of phytosterol is 10% of the mass of oil body diluent, and the heating temperature after phytosterol and oil body diluent are evenly mixed is 85°C.

[0057] There are many ways to implement the cooling in step S30. The plant sterol oil body mixture can be placed in a low temperature environment of -2 to 4°C, such as a refrigerator set at a temperature of -2 to 4°C, or in an ice water bath, etc. The specific method can be selected according to the amount of materials. Similarly, taking a small amount of laboratory preparation as an example, in some embodiments of the present invention, the cooling method in step S30 includes ice water bath cooling, that is, the plant sterol oil body mixture after heating is immediately placed in an ice water bath until it is completely cooled, which is simple, fast, and more energy-saving.

[0058] Similarly, the centrifugal operating conditions described in step S30 can be adjusted according to the amount of material, and the centrifugal conditions set for laboratory small-scale production and industrial mass production may be different. Specifically, taking laboratory small-scale production as an example, in some embodiments of the present invention, in step S30: the centrifugal speed of the centrifugation is 4000-8000rpm, for example, it can be 4000rpm, 4500rpm, 5000rpm, 5500rpm, 6000rpm, 6500rpm, 7000rpm, 7500rpm, 8000rpm, etc., and the centrifugal time of the centrifugation is 10-30min, for example, it can be 10min, 12min, 14min, 15min, 16min, 18min, 20min, 22min, 24min, 25min, 26min, 28min, 30min, etc.

[0059] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0060] Example 1

[0061] (1) Select pumpkin seed oil body extracted with NaHCO3 solution as extraction solvent, add 10 times the mass of distilled water to dilute it, and then heat it in a water bath at 75°C for 2 minutes to obtain pumpkin seed oil body dilution;

[0062] (2) adding 10% phytosterols by weight to the pumpkin seed oil body dilution, stirring at a rate of 250 rpm for 5 min, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonically treating at a power of 400 W for 3 min, and then heating and stirring in a water bath at 75° C. for 20 min at a stirring rate of 250 rpm to obtain a phytosterol oil body mixed solution;

[0063] (3) The phytosterol oil body mixture that has been heated in a water bath in step (2) is taken out, immediately cooled in an ice water bath, and then centrifuged at 4000 rpm for 20 min. The upper paste is taken to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is obtained.

[0064] Example 2

[0065] (1) Select pumpkin seed oil body extracted with NaHCO3 solution as extraction solvent, add 10 times the mass of distilled water to dilute it, and then heat it in a water bath at 75°C for 2 minutes to obtain pumpkin seed oil body dilution;

[0066] (2) adding 10% phytosterols by weight to the pumpkin seed oil body dilution, stirring at a rate of 250 rpm for 5 min, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonic treatment is performed at a power of 400 W for 3 min, and then heated and stirred in a 95° C. water bath for 20 min at a stirring rate of 250 rpm to obtain a phytosterol oil body mixed solution;

[0067] (3) The phytosterol oil body mixture that has been heated in a water bath in step (2) is taken out, immediately cooled in an ice water bath, and then centrifuged at 4000 rpm for 20 min. The upper paste is taken to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is obtained.

[0068] Example 3

[0069] (1) Select pumpkin seed oil bodies extracted with Tris-HCl solution as the extraction solvent, add 10 times the mass of distilled water to dilute it, and then place it in a water bath at 75° C. and heat it for 2 minutes to obtain a pumpkin seed oil body dilution solution;

[0070] (2) adding 10% phytosterols by weight to the pumpkin seed oil body dilution, stirring at a rate of 250 rpm for 5 min, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonically treating at a power of 400 W for 3 min, and then heating and stirring in a water bath at 75° C. for 20 min at a stirring rate of 250 rpm to obtain a phytosterol oil body mixed solution;

[0071] (3) The phytosterol oil body mixture that has been heated in a water bath in step (2) is taken out, immediately cooled in an ice water bath, and then centrifuged at 4000 rpm for 20 min. The upper paste is taken to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is obtained.

[0072] Example 4

[0073] (1) Select pumpkin seed oil bodies extracted with Tris-HCl solution as the extraction solvent, add 10 times the mass of distilled water to dilute it, and then place it in a water bath at 75° C. and heat it for 2 minutes to obtain a pumpkin seed oil body dilution solution;

[0074] (2) adding 10% phytosterols by weight to the pumpkin seed oil body dilution, stirring at a rate of 250 rpm for 5 min, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonic treatment is performed at a power of 400 W for 3 min, and then heated and stirred in a 95° C. water bath for 20 min at a stirring rate of 250 rpm to obtain a phytosterol oil body mixed solution;

[0075] (3) The phytosterol oil body mixture that has been heated in a water bath in step (2) is taken out, immediately cooled in an ice water bath, and then centrifuged at 4000 rpm for 20 min. The upper paste is taken to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is obtained.

[0076] Example 5

[0077] (1) Pumpkin seed oil bodies extracted with PBS solution as the extraction solvent were selected, 10 times the mass of distilled water was added thereto for dilution, and then placed in a 75° C. water bath for heating for 2 min to obtain a pumpkin seed oil body dilution solution;

[0078] (2) adding 10% phytosterols by weight to the pumpkin seed oil body dilution, stirring at a rate of 250 rpm for 5 min, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonically treating at a power of 400 W for 3 min, and then heating and stirring in a water bath at 75° C. for 20 min at a stirring rate of 250 rpm to obtain a phytosterol oil body mixed solution;

[0079] (3) The phytosterol oil body mixture that has been heated in a water bath in step (2) is taken out, immediately cooled in an ice water bath, and then centrifuged at 4000 rpm for 20 min. The upper paste is taken to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is obtained.

[0080] Example 6

[0081] (1) Pumpkin seed oil bodies extracted with PBS solution as the extraction solvent were selected, 10 times the mass of distilled water was added thereto for dilution, and then placed in a 75° C. water bath for heating for 2 min to obtain a pumpkin seed oil body dilution solution;

[0082] (2) adding 10% phytosterols by weight to the pumpkin seed oil body dilution, stirring at a rate of 250 rpm for 5 min, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonic treatment is performed at a power of 400 W for 3 min, and then heated and stirred in a 95° C. water bath for 20 min at a stirring rate of 250 rpm to obtain a phytosterol oil body mixed solution;

[0083] (3) The phytosterol oil body mixture that has been heated in a water bath in step (2) is taken out, immediately cooled in an ice water bath, and then centrifuged at 4000 rpm for 20 min. The upper paste is taken to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is obtained.

[0084] Example 7

[0085] (1) Select pumpkin seed oil body extracted with NaHCO3 solution as extraction solvent, add 2 times the mass of distilled water to dilute it, and then heat it in a water bath at 70°C for 1 minute to obtain pumpkin seed oil body dilution;

[0086] (2) adding 1% by weight of phytosterols to the pumpkin seed oil body dilution, stirring at a rate of 200 rpm for 3 min, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonic treatment is performed at a power of 200 W for 2 min, and then heated and stirred in a water bath at 60° C. for 25 min at a stirring rate of 200 rpm to obtain a phytosterol oil body mixed solution;

[0087] (3) The phytosterol oil body mixture that has been heated in a water bath in step (2) is taken out, immediately placed in an ice water bath for cooling, and then centrifuged at a speed of 5000 rpm for 30 minutes, and the upper paste is taken to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is prepared.

[0088] Example 8

[0089] (1) Pumpkin seed oil body extracted with NaHCO3 solution as extraction solvent was selected, 4 times the mass of distilled water was added thereto for dilution, and then placed in a 72°C water bath and heated for 1 min 30 s to obtain a pumpkin seed oil body dilution solution;

[0090] (2) adding 5% by weight of phytosterols to the pumpkin seed oil body dilution, stirring at a rate of 220 rpm for 3 min 30 s, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonically treating at a power of 250 W for 2 min 20 s, and then heating and stirring in a water bath at 65° C. for 23 min at a stirring rate of 280 rpm to obtain a phytosterol oil body mixed solution;

[0091] (3) The phytosterol oil body mixture that has been heated in a water bath in step (2) is taken out, immediately cooled in an ice water bath, and then centrifuged at 6000 rpm for 25 min. The upper paste is taken to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is obtained.

[0092] Example 9

[0093] (1) Pumpkin seed oil body extracted with NaHCO3 solution as extraction solvent was selected, 6 times the mass of distilled water was added thereto for dilution, and then placed in a 78°C water bath for heating for 1 min 20 s to obtain a pumpkin seed oil body dilution solution;

[0094] (2) adding 15% of phytosterols by weight to the pumpkin seed oil body dilution, stirring at a rate of 270 rpm for 4 min, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonic treatment is performed at a power of 300 W for 2 min 30 s, and then heated and stirred in a water bath at 80° C. for 18 min at a stirring rate of 220 rpm to obtain a phytosterol oil body mixed solution;

[0095] (3) The phytosterol oil body mixture that has been heated in a water bath in step (2) is taken out, immediately cooled in an ice water bath, and then centrifuged at 7000 rpm for 15 min. The upper paste is taken to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is obtained.

[0096] Example 10

[0097] (1) Pumpkin seed oil body extracted with NaHCO3 solution as extraction solvent was selected, 8 times the mass of distilled water was added thereto for dilution, and then placed in a water bath at 80°C for 1 min 40s to obtain a pumpkin seed oil body dilution solution;

[0098] (2) adding phytosterols accounting for 20% of the weight of the pumpkin seed oil body dilution, stirring at a rate of 300 rpm for 4 min 30 s, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonic treatment is performed at a power of 350 W for 2 min 40 s, and then placed in a 100° C. water bath and heated and stirred for 15 min at a stirring rate of 300 rpm to obtain a phytosterol oil body mixed solution;

[0099] (3) The phytosterol oil body mixture that has been heated in a water bath in step (2) is taken out, immediately cooled in an ice water bath, and then centrifuged at 8000 rpm for 10 min. The upper paste is taken to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is obtained.

[0100] Comparative Example 1

[0101] (1) Selecting pumpkin seed oil bodies extracted with NaHCO3 solution as an extraction solvent, adding 10 times the mass of distilled water thereto for dilution, to obtain a pumpkin seed oil body dilution solution;

[0102] (2) adding 10% by weight of phytosterols to the pumpkin seed oil body dilution, stirring at a rate of 250 rpm for 5 min, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonically treating at a power of 400 W for 3 min, and then stirring at a rate of 250 rpm for 20 min to obtain a phytosterol oil body mixed solution;

[0103] (3) The phytosterol oil body mixture obtained in step (2) is centrifuged at 4000 rpm for 20 min, and the upper paste is collected to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is obtained.

[0104] Comparative Example 2

[0105] (1) selecting pumpkin seed oil bodies extracted with a Tris-HCl solution as an extraction solvent, adding 10 times the mass of distilled water thereto for dilution, and obtaining a pumpkin seed oil body dilution solution;

[0106] (2) adding 10% by weight of phytosterols to the pumpkin seed oil body dilution, stirring at a rate of 250 rpm for 5 min, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonically treating at a power of 400 W for 3 min, and then stirring at a rate of 250 rpm for 20 min to obtain a phytosterol oil body mixed solution;

[0107] (3) The phytosterol oil body mixture obtained in step (2) is centrifuged at 4000 rpm for 20 min, and the upper paste is collected to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is obtained.

[0108] Comparative Example 3

[0109] (1) selecting pumpkin seed oil bodies extracted with PBS solution as an extraction solvent, adding 10 times the mass of distilled water thereto for dilution, and obtaining a pumpkin seed oil body dilution solution;

[0110] (2) adding 10% by weight of phytosterols to the pumpkin seed oil body dilution, stirring at a rate of 250 rpm for 5 min, and after the phytosterols are completely immersed in the pumpkin seed oil body dilution, ultrasonically treating at a power of 400 W for 3 min, and then stirring at a rate of 250 rpm for 20 min to obtain a phytosterol oil body mixed solution;

[0111] (3) The phytosterol oil body mixture obtained in step (2) is centrifuged at 4000 rpm for 20 min, and the upper paste is collected to obtain pumpkin seed oil bodies loaded with phytosterols, that is, the phytosterol delivery system is obtained.

[0112] Comparative Example 4

[0113] Free phytosterols: Use anhydrous ethanol to prepare a 10% phytosterol solution.

[0114] The plant sterol delivery system provided in the above embodiments and the plant sterol delivery system and free sterol solution provided in the comparative example were tested respectively. The test items included the phytosterol embedding rate test, bioavailability test, and functional activity test, wherein the functional activity test included antioxidant activity test and cholesterol-lowering activity test. The test methods and results are as follows:

[0115] 1. Plant sterol embedding rate test

[0116] Anhydrous ethanol was added to the final system obtained in the embodiment and the comparative example, free phytosterols were extracted and the content was detected, and the embedding rate of phytosterols was calculated.

[0117] 2. Plant sterol bioavailability test

[0118] The final systems obtained in Example and Comparative Example 4 were placed in simulated gastrointestinal digestive fluid for 4 hours, and an in vitro release test was performed to test the bioavailability of phytosterols.

[0119] 3. Test of antioxidant activity of phytosterols

[0120] The final systems obtained in Example and Comparative Example 4 were placed in test tubes, and their antioxidant properties were tested using DPPH and ABTS kits.

[0121] 4. Phytosterol cholesterol-lowering activity test

[0122] The final system obtained in Example and Comparative Example 4 was placed in cholesterol micelles and subjected to a 2-h digestion experiment to test the cholesterol-lowering activity of phytosterols.

[0123] The test results are shown in Table 1 below.

[0124] Table 1 Test data of embodiments and comparative examples

[0125]

[0126]

[0127] The following conclusions can be drawn from Table 1:

[0128] (1) Without heat treatment (Comparative Examples 1-3), phytosterols were hardly embedded; however, after heat treatment in the examples, the embedding rate was significantly increased, up to 96.63%, indicating that the method provided by the present invention can significantly increase the embedding rate of phytosterols in plant oils and fats. In addition, since comparative examples 1-3 were almost unable to embed phytosterols, no subsequent testing was required.

[0129] (2) Compared with the control examples, the bioavailability of phytosterols in the embodiments is significantly improved, among which the bioavailability of phytosterols in Example 3 is the highest (75.74%), indicating that the method provided by the present invention can significantly improve the bioavailability of phytosterols. It can be seen from Examples 1-6 that the preparation method provided by the present invention can make the bioavailability of phytosterols better by changing the parameters.

[0130] (3) Compared with Comparative Example 4, the DPPH radical scavenging rate and ABTS cation radical scavenging rate of the embodiments are significantly improved, among which Examples 1 and 5 have higher scavenging rates, indicating that the method provided by the present invention can significantly improve the antioxidant activity of phytosterols. It can be seen from Examples 1-6 that the preparation method provided by the present invention can make the antioxidant activity of phytosterols better by changing the parameters.

[0131] (4) Compared with Comparative Example 4, the cholesterol concentration in the digestive juice after the reaction of the embodiment is significantly reduced, and the cholesterol-lowering activity is significantly improved, among which the cholesterol-lowering activity of Example 3 is the best, indicating that the method provided by the present invention can significantly improve the cholesterol-lowering activity of plant sterols. It can be seen from Examples 1-6 that the preparation method provided by the present invention can make the cholesterol-lowering activity of plant sterols better by changing the parameters.

[0132] 5. Anti-inflammatory activity test of phytosterols

[0133] The final systems obtained in Examples 1, 3, 5 and Comparative Example 4 were diluted to a certain concentration and cultured in the RAW264.7 macrophage inflammation model for 24 h. The release of NO, IL-6 and TNF-α was tested to evaluate the anti-inflammatory activity of phytosterols. The release of NO, IL-6 and TNF-α in the cell inflammation model was as follows: Figure 2 , Figure 3 and Figure 4 shown.

[0134] Depend on Figure 2 , Figure 3 and Figure 4 It can be seen that compared with Comparative Example 4, the systems prepared in Examples 1, 3 and 5 showed stronger inhibitory effects on pro-inflammatory factors at different concentrations, and the release rates of NO, IL-6 and TNF-α were significantly reduced, achieving better anti-inflammatory effects, indicating that the method provided by the present invention can significantly improve the anti-inflammatory activity of phytosterols.

[0135] In summary, the preparation method of the phytosterol delivery system provided by the present invention can significantly improve the bioavailability and functional activity of phytosterols, and the scheme provided by the present invention is highly inclusive, and can specifically improve a certain functional activity of phytosterols for phytosterol delivery systems prepared with different solvents, different temperatures, and different concentrations.

[0136] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for preparing a phytosterol delivery system, characterized in that: The preparation method of the phytosterol delivery system comprises the following steps: Diluting the vegetable oil with water and preheating it to obtain an oil dilution liquid; Adding phytosterols to the oil body dilution liquid, subjecting to initial stirring and ultrasonic treatment, and then heating and stirring to obtain a phytosterol oil body mixed liquid; The phytosterol oil body mixture is cooled, and then centrifuged to obtain the upper paste to obtain the phytosterol-loaded oil body, that is, the phytosterol delivery system is prepared.

2. The method for preparing the phytosterol delivery system according to claim 1, characterized in that: In the step of diluting the vegetable oil with water and preheating the oil to obtain an oil dilution solution: The vegetable fat bodies include pumpkin seed fat bodies.

3. The method for preparing the phytosterol delivery system according to claim 2, characterized in that: The extraction solvent of the pumpkin seed oil body includes any one of NaHCO3 solution, PBS solution and Tris-HCl solution.

4. The method for preparing the phytosterol delivery system according to claim 1, characterized in that: In the step of diluting the vegetable oil with water and preheating the oil to obtain an oil dilution solution: The mass of the water is 2 to 10 times the mass of the vegetable oil.

5. The method for preparing the phytosterol delivery system according to claim 1, characterized in that: In the step of diluting the vegetable oil with water and preheating the oil to obtain an oil dilution solution: The preheating method includes water bath heating, the preheating temperature is 70-80° C., and the heating time is 1-2 min.

6. The method for preparing the phytosterol delivery system according to claim 1, characterized in that: In the step of adding phytosterols to the oil body dilution liquid, subjecting the mixture to initial stirring and ultrasonic treatment, and then heating and stirring to obtain a phytosterol oil body mixed liquid: The mass of the phytosterol is 1-20% of the mass of the oil body diluent.

7. The method for preparing the phytosterol delivery system according to claim 1, characterized in that: In the step of adding phytosterols to the oil body dilution liquid, subjecting the mixture to initial stirring and ultrasonic treatment, and then heating and stirring to obtain a phytosterol oil body mixed liquid: The stirring rate of the initial stirring is 200-300 rpm, and the stirring time is 3-5 min.

8. The method for preparing the phytosterol delivery system according to claim 1, characterized in that: In the step of adding phytosterols to the oil body dilution liquid, subjecting the mixture to initial stirring and ultrasonic treatment, and then heating and stirring to obtain a phytosterol oil body mixed liquid: The ultrasonic power of the ultrasonic treatment is 200-400W, and the ultrasonic time is 2-3min.

9. The method for preparing the phytosterol delivery system according to claim 1, characterized in that: In the step of adding phytosterols to the oil body dilution liquid, subjecting the mixture to initial stirring and ultrasonic treatment, and then heating and stirring to obtain a phytosterol oil body mixed liquid: The heating method of the heating and stirring includes water bath heating, the heating temperature of the heating and stirring is 60-100° C., and the heating time is 15-25 minutes.

10. The method for preparing the phytosterol delivery system according to claim 1, characterized in that: The phytosterol oil body mixture is cooled, and then centrifuged to obtain the upper paste to obtain the phytosterol-loaded oil body, that is, the step of preparing the phytosterol delivery system: The cooling method includes ice water bath cooling.

11. The method for preparing the phytosterol delivery system according to claim 1, characterized in that: The phytosterol oil body mixture is cooled, and then centrifuged to obtain the upper paste to obtain the phytosterol-loaded oil body, that is, the step of preparing the phytosterol delivery system: The centrifugal speed of the centrifugation is 4000-8000 rpm, and the centrifugal time is 10-30 min.

Images