Background
The phytosterol is an alcohol compound taking cyclopentane perhydro-phenanthrene as a framework, is white crystal powder or particles at normal temperature, is odorless and tasteless, has a melting point of 130-170 ℃, is insoluble in water, strong acid and strong alkali, and is slightly soluble in organic solvents such as acetone, absolute ethyl alcohol, diethyl ether, benzene, chloroform, petroleum ether and the like at normal temperature. Phytosterols are present in almost all vegetable foods, such as grains, legumes, vegetables, fruits and nuts, in amounts that vary significantly from plant to plant, with the greatest amount being found in oil crops. At present, the phytosterol has been proved to have various physiological functions of cholesterol reduction, cardiovascular disease reduction, cancer resistance, inflammation resistance, oxidation resistance, atherosclerosis resistance and the like, but the bioavailability of the phytosterol in a human body is extremely low due to the characteristics of being insoluble in water and slightly soluble in an organic solvent, so that the application of the phytosterol in foods or medicines is limited.
In order to solve the problem of poor water solubility of the phytosterol, the phytosterol can meet daily requirements of human beings, and at present, a nanoparticle technology is generally adopted to improve the water solubility of the phytosterol. The nanoparticle technology for embedding the bioactive substances has the advantages of site-directed targeting delivery and efficient cell absorption, and can also improve the solubility, stability and bioavailability of the embedded bioactive substances. For example, patent CN105410934a discloses a water-soluble protein-phytosterol nanoparticle and preparation and application thereof, and the phytosterol nanoparticle is prepared by embedding a water-soluble protein such as sodium caseinate, soy isolate protein or whey protein concentrate and freeze-drying the embedded phytosterol, although the patent improves the water solubility of the phytosterol to a certain extent, the particle size of the phytosterol nanoparticle prepared by the method is larger, and the water solubility, bioavailability, embedding rate, dispersibility, re-solubility and the like of the phytosterol nanoparticle are all required to be further improved.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a phytosterol nano-preparation, a preparation method and application thereof, and the prepared phytosterol nano-preparation has high embedding rate, good resolubility and dispersibility and high bioavailability, and can well improve the application of phytosterol in foods and medicines.
In order to achieve the technical effects, the invention adopts the following technical scheme:
the invention provides a preparation method of a phytosterol nano-preparation, which comprises the following steps:
(1) Dissolving maltodextrin and protein in deionized water to obtain a water phase for later use;
(2) Adding the phytosterol and the emulsifying agent into ethanol, and stirring until the phytosterol and the emulsifying agent are completely dissolved to obtain an organic phase for later use;
(3) Mixing the aqueous phase in the step (1) with the organic phase in the step (2), and then sequentially carrying out homogenization and ultrasonic treatment to obtain mixed emulsion;
(4) And (3) removing ethanol in the mixed emulsion in the step (3), and then performing freeze drying to obtain the plant sterol nano preparation.
Further, the protein of step (1) is ovotransferrin.
Further, the mass ratio of the maltodextrin to the protein in the step (1) is 1:1-8, and the mass volume ratio of the total amount of the maltodextrin and the protein to the deionized water is 1-3mg/mL.
Further, the dissolving method in the step (1) is to add maltodextrin into deionized water, stir until the maltodextrin is completely dissolved, then add protein under stirring, and stir until the maltodextrin is completely dissolved.
Further, the plant sterol in the step (2) is soybean plant sterol, the emulsifier comprises a natural emulsifier or a synthetic emulsifier, preferably one or more of tween 40, castor oil and span 20, and the emulsifier has the advantages of good emulsifying function property, safety and reliability.
Further, the mass ratio of the plant sterol to the emulsifier in the step (2) is 1:1-5, and the mass volume ratio of the emulsifier to the ethanol is 10-50mg/mL.
Further, the stirring temperature in the step (2) is 80-100 ℃, and the stirring time is 1-5min.
Further, the volume ratio of the water phase to the organic phase in the step (3) is 10:1-3, the homogenizing rotating speed is 3000-10000rpm, the homogenizing time is 3-8min, the ultrasonic power is 400W, and the ultrasonic time is 5-15min.
Further, the ethanol is removed in the step (4) by rotary evaporation, the temperature of the rotary evaporation is 40-50 ℃, the vacuum degree is 0.04-0.1MPa, the mixed emulsion with the ethanol removed is diluted to the mass before the rotary evaporation by deionized water before the freeze drying, and the freeze drying time is 24-48 hours.
The phytosterol nanometer preparation prepared by the preparation method is provided.
The invention also provides application of the phytosterol nano-preparation in preparation of functional foods and medicines.
Compared with the prior art, the invention has the following beneficial effects: the invention adopts maltodextrin and the egg transferrin as wall materials to embed the phytosterol, wherein the egg transferrin has better interfacial activity, can be used as a natural emulsifier to improve the emulsifying effect on the phytosterol, thereby reducing the particle size of the nano-preparation of the phytosterol, and simultaneously has various biological characteristics such as antioxidation, antibiosis, anti-tumor, anti-inflammatory and the like; the maltodextrin has the characteristics of good emulsifying effect and thickening effect, good solubility, excellent filling effect, difficult moisture absorption, difficult agglomeration, good carrier property, good stability and the like, can improve the dispersibility of the phytosterol nano-preparation, and simultaneously contains a large amount of polysaccharide bioactive substances, and trace elements such as calcium, iron and the like and mineral substances beneficial to human bodies.
In addition, the prepared nano phytosterol preparation has high embedding rate, good re-solubility and dispersibility and high bioavailability, and can well improve the application of the phytosterol in foods and medicines.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the invention, are within the scope of the invention.
The test methods or test methods described in the following examples are all conventional methods unless otherwise specified; the starting materials and auxiliaries, unless otherwise specified, are obtained commercially from conventional sources or are prepared in conventional manner.
Example 1
The preparation method of the phytosterol nano preparation comprises the following specific steps:
(1) Adding maltodextrin into deionized water, stirring until the maltodextrin is completely dissolved, slowly adding egg transferrin under a stirring state, and stirring and dissolving uniformly to obtain a water phase, wherein the mass ratio of the maltodextrin to the egg transferrin is 1:4, and the mass volume ratio of the total amount of the maltodextrin and the egg transferrin to the deionized water is 2mg/mL;
(2) Adding soyasterol, tween 40 and castor oil into ethanol, and magnetically stirring at 100deg.C for 2min to fully dissolve to obtain organic phase, wherein the mass ratio of soyasterol to emulsifier (Tween 40 and castor oil) is 1:4, the mass ratio of Tween 40 to castor oil is 1:1, and the mass volume ratio of emulsifier (Tween 40 and castor oil) to ethanol is 25mg/mL;
(3) Mixing the aqueous phase in the step (1) with the organic phase in the step (2), wherein the volume ratio of the aqueous phase to the organic phase is 10:1, then carrying out homogenization treatment under the condition of 5000rpm, wherein the homogenization time is 5min, and then carrying out ultrasonic treatment under the condition of 400W, wherein the ultrasonic time is 10 mm, so as to obtain mixed emulsion;
(4) And (3) placing the mixed emulsion in a distillation flask, performing rotary evaporation at 45 ℃ and a vacuum degree of 0.09MPa to remove ethanol, diluting the product after rotary evaporation to the mass before rotary evaporation by deionized water, and performing freeze drying for 24 hours to obtain the phytosterol nano-preparation.
Example 2
The preparation method of the phytosterol nano preparation comprises the following specific steps:
(1) Adding maltodextrin into deionized water, stirring until the maltodextrin is completely dissolved, slowly adding egg transferrin under a stirring state, and stirring and dissolving uniformly to obtain a water phase, wherein the mass ratio of the maltodextrin to the egg transferrin is 1:4, and the mass volume ratio of the total amount of the maltodextrin and the egg transferrin to the deionized water is 2mg/mL;
(2) Adding soyasterol and Tween 40 into ethanol, and magnetically stirring at 100deg.C for 2min to fully dissolve to obtain organic phase, wherein the mass ratio of soyasterol and Tween 40 is 1:3, and the mass volume ratio of Tween 40 and ethanol is 20mg/mL;
(3) Mixing the aqueous phase in the step (1) with the organic phase in the step (2), wherein the volume ratio of the aqueous phase to the organic phase is 10:1, then carrying out homogenization treatment under the condition of 5000rpm, wherein the homogenization time is 5min, and then carrying out ultrasonic treatment under the condition of 400W, wherein the ultrasonic time is 10 mm, so as to obtain mixed emulsion;
(4) And (3) placing the mixed emulsion in a distillation flask, performing rotary evaporation at 45 ℃ and a vacuum degree of 0.09MPa to remove ethanol, diluting the product after rotary evaporation to the mass before rotary evaporation by deionized water, and performing freeze drying for 24 hours to obtain the phytosterol nano-preparation.
Example 3
The preparation method of the phytosterol nano preparation comprises the following specific steps:
(1) Adding maltodextrin into deionized water, stirring until the maltodextrin is completely dissolved, slowly adding egg transferrin under a stirring state, and stirring and dissolving uniformly to obtain a water phase, wherein the mass ratio of the maltodextrin to the egg transferrin is 1:2, and the mass volume ratio of the total amount of the maltodextrin and the egg transferrin to the deionized water is 2mg/mL;
(2) Adding soybean phytosterol, tween 40 and span 20 into ethanol, and magnetically stirring at 100deg.C for 2min to fully dissolve to obtain organic phase, wherein the mass ratio of soybean phytosterol to emulsifier (tween 40 and span 20) is 1:3, the mass ratio of tween 40 to span 20 is 2:1, and the mass volume ratio of emulsifier (tween 40 and span 20) to ethanol is 20mg/mL;
(3) Mixing the aqueous phase in the step (1) with the organic phase in the step (2), wherein the volume ratio of the aqueous phase to the organic phase is 10:1, then carrying out homogenization treatment under the condition of 5000rpm, wherein the homogenization time is 5min, and then carrying out ultrasonic treatment under the condition of 400W, wherein the ultrasonic time is 10 mm, so as to obtain mixed emulsion;
(4) And (3) placing the mixed emulsion in a distillation flask, performing rotary evaporation at 45 ℃ and a vacuum degree of 0.09MPa to remove ethanol, diluting the product after rotary evaporation to the mass before rotary evaporation by deionized water, and performing freeze drying for 24 hours to obtain the phytosterol nano-preparation.
Comparative example 1
The difference from example 3 is that the ovotransferrin in step (1) is replaced with soy protein isolate and the rest of the procedure is the same as example 3.
Comparative example 2
The difference from example 3 is that maltodextrin in step (1) was omitted and the rest of the steps were the same as in example 3.
The phytosterol nanoformulations prepared in examples 1-3 and comparative examples 1-2 were tested for performance using the following test methods:
1) The embedding rate of the prepared phytosterol nano-preparation is measured by adopting a gas chromatography method, wherein the embedding rate is = (1-the amount of surface phytosterol/the amount of total phytosterol) multiplied by 100%;
2) Measuring the particle size and PDI of the prepared phytosterol nano-preparation by using a particle size potentiometer;
3) The method for measuring the bioavailability comprises the following steps: obtaining a phytosterol nano-preparation digestive juice according to an in vitro digestion model in Fei-Ping Chen,Bian-Sheng Li,Chuan-He Tang.Nanocomplexation between curcumin and soy protein isolate:influence on curcumin stability/bioaccessibility and in vitro protein digestibility.Journal of Agricultural and Food Chemistry,2015,63(13),3559-3569., taking 1mL of the phytosterol nano-preparation digestive juice into a centrifuge tube, centrifuging for 30min at 10000g and 10 ℃, taking supernatant to determine the amount of the phytosterol, and the bioavailability = the amount of the phytosterol in the supernatant/the amount of the total phytosterol multiplied by 100%;
4) Method for measuring solubility: dissolving a certain amount of the phytosterol nano-preparation in water to enable a sample to reach a supersaturated state, sufficiently oscillating for 10min, oscillating for 24h at 37 ℃ by a shaking table, centrifuging for 10min at 10000rpm, taking supernatant, and measuring the phytosterol content in the supernatant by gas chromatography-mass spectrometry, wherein the solubility=the phytosterol content (mg)/the solution volume (mL) in the supernatant.
The test results are shown in the following table.
The experimental data show that the plant sterol nano-preparation prepared in the examples 1-3 has good embedding rate, re-solubility, dispersibility and bioavailability, and can effectively improve the water solubility of plant sterols.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.