Beta-carotene-loaded alcoholized Pickering emulsion as well as preparation method and application thereof

Abstract

The invention provides beta-carotene loaded alcoholized Pickering emulsion as well as a preparation method and application thereof, and belongs to the technical field of food processing. The preparation method comprises the following steps: firstly, preparing a milk protein-prolamin composite nanoparticle solution; alcoholizing the polysaccharide solution, adjusting the pH value, and mixing the polysaccharide solution with the lactoprotein-prolamin composite nanoparticle solution to obtain a protein-polysaccharide composite nanoparticle solution; the oil phase solution of the beta-carotene and the solution of the protein-polysaccharide composite particles are subjected to shearing and homogenizing treatment, and the alcoholized Pickering emulsion loaded with the beta-carotene is obtained. Protein-polysaccharide composite particles in the prepared emulsion are firmly and irreversibly adsorbed on an oil-water interface to form a compact interface layer, and sufficient steric hindrance and electrostatic repulsive force are provided, so that the embedding rate and light and heat stability of beta-carotene are improved, the diffusion rate of an oxidant to an oil phase is effectively inhibited, and the stability of beta-carotene is improved. The stability and the biological accessibility of the beta-carotene are improved.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and in particular to a Pickering emulsion loaded with alcoholized beta-carotene and a preparation method and application thereof. Background Art

[0002] In daily life, some medicines, skin care products, and foods are related to ethanol-water systems and their emulsions, and the consumption of ethanol-containing emulsions such as cream liqueur, eggnog, and ethanol-containing ice cream is gradually increasing. Therefore, ethanol-water system emulsions should be studied for subsequent development and utilization. Emulsions are thermodynamically unstable systems, and the interface between the dispersed phase and the continuous phase needs to be modified to improve the stability of the emulsion. However, the presence of ethanol will change the emulsion environment such as emulsion density, dielectric constant, surface tension, etc., and affect the emulsification properties of the emulsifier and the stability of the protein, thereby increasing the complexity of interface modification.

[0003] During the interface modification process, a single emulsifier has limitations in stabilizing the emulsion. When the continuous phase environment and external conditions change, unstable phenomena such as gravity separation, flocculation, and aggregation are prone to occur. The ability to stabilize the emulsion can be improved by compounding different components such as proteins, sugars, small molecule emulsifiers, and polyphenols. Compared with a single emulsifier, the complex has a unique structure and physical and chemical properties. The emulsion stabilized by the complex has a stronger anti-interference ability and can resist the adverse effects of extreme pH, extreme temperature, and high ionic strength.

[0004] At the same time, ethanol-containing emulsions can be used as delivery systems for physiologically active ingredients to promote the subsequent development of functional alcoholic foods and beverages. β-Carotene is a natural food colorant that can be converted into vitamin A after being metabolized by the human body, thereby maintaining the health of the eye and epithelial tissue. At the same time, the physiological activity of β-carotene has gradually attracted attention. It can be antioxidant, anti-inflammatory, regulate the immune system, and reduce the risk of chronic diseases. However, the human body cannot synthesize β-carotene on its own and must obtain sources from the outside world. However, β-carotene is sensitive to environmental factors such as light, heat, oxygen, and pH value, and is prone to oxidative degradation. In addition, β-carotene itself is insoluble in water and has low bioaccessibility. These shortcomings limit the application of β-carotene, and it is necessary to construct a suitable delivery system to improve the stability and bioaccessibility of β-carotene.

[0005] β-Carotene is an important lipophilic carotenoid with a bright orange-red color, which is found in many vegetables and fruits. β-Carotene has high antioxidant activity and vitamin A activity. Intake of β-carotene can prevent certain cancers, inhibit the occurrence of cardiovascular diseases, reduce metabolic syndrome, prevent eye diseases such as cataracts, and is beneficial to health. However, β-carotene is highly hydrophobic, insoluble in water, slightly soluble in oil at room temperature, and has low bioaccessibility (<5%). In addition, since the β-carotene molecule has 11 conjugated double bonds, the structure is extremely unstable and is easily degraded and isomerized under light, oxygen, free radicals, metal ions and high temperature conditions. These limit the practical application of β-carotene in the food industry. In order to improve the storage stability and bioaccessibility of β-carotene, it is necessary to establish an effective β-carotene delivery system. Currently, common delivery systems include emulsions, nanoparticles, solid lipid particles, microcapsules, etc.

[0006] Pickering emulsions have been widely used in the food and pharmaceutical industries due to their superior rheological properties, irreversible interfacial absorption and anti-agglomeration properties. Before forming the Pickering emulsion by shear homogenization, the lipophilic components are first incorporated into the oil phase. During the high-speed shearing process, the particles are irreversibly adsorbed on the oil-water interface to form a strong interfacial film, which can provide a physical barrier for the bioactive substances in the oil phase, thereby effectively improving the solubility, stability and bioaccessibility of the lipophilic bioactive substances.

[0007] However, the stability and bioaccessibility of β-carotene still need to be further improved. Summary of the invention

[0008] In view of this, the present invention provides a method for preparing a Pickering emulsion loaded with alcoholized β-carotene, which can improve the stability and bioaccessibility of β-carotene.

[0009] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0010] A method for preparing a β-carotene-loaded alcoholized Pickering emulsion comprises the following steps:

[0011] S10: mixing the alcohol-soluble protein solution with the milk protein solution, adjusting the pH value, and preparing a milk protein-alcohol-soluble protein composite nanoparticle solution;

[0012] S20: alcoholizing the polysaccharide solution, adjusting the pH value, and then mixing the polysaccharide solution with the milk protein-alcohol-soluble protein composite nanoparticle solution to obtain a protein-polysaccharide composite particle solution;

[0013] S30: First, prepare an oil-phase solution of β-carotene, and then subject it to shear homogenization with a protein-polysaccharide composite particle solution to obtain a Pickering emulsion loaded with β-carotene alcoholized.

[0014] Preferably, the zein in step S10 is at least one of zein from corn, zein from wheat, and zein from sorghum;

[0015] The solvent of the zein solution is an ethanol aqueous solution with an ethanol volume fraction of 55% - 90%;

[0016] In the zein solution, the ratio of zein to the solvent is 1 g: 50 - 150 mL.

[0017] Preferably, the milk protein in step S10 is at least one of lactoferrin, lactoglobulin, and α-lactalbumin;

[0018] The milk protein solution includes milk protein and water, and the ratio of milk protein to water is 1 g: 200 - 400 mL.

[0019] Preferably, the method for preparing the milk protein-zein composite nanoparticle solution in step S10 includes the following steps: Drop the zein solution into the milk protein solution, stir at a speed of 600 rpm - 700 rpm for 20 min - 60 min, and adjust the pH value to obtain a milk protein-zein composite nanoparticle solution;

[0020] The pH value of the milk protein-zein composite nanoparticle solution is 4.0 - 5.0, and the volume concentration of ethanol is not higher than 15%;

[0021] The average particle size of the milk protein-zein composite nanoparticles is 100 nm - 120 nm;

[0022] In the milk protein-zein composite nanoparticles, the mass ratio of milk protein to zein is (1 - 2):(1 - 3).

[0023] Preferably, the polysaccharide in step S20 is at least one of propylene glycol alginate (PGA), high-methoxyl pectin, and hyaluronic acid;

[0024] The polysaccharide solution is composed of polysaccharide and water, and the ratio of polysaccharide to water is 1 g:(80 - 90) mL;

[0025] The steps of the alcoholization treatment are: Drop anhydrous ethanol into the polysaccharide solution and stir for 10 - 12 h for alcoholization treatment; The volume ratio of the polysaccharide solution to anhydrous ethanol is 80 - 90:10 - 20.

[0026] The reagent used to adjust the pH value is an alkaline or acidic solution; the pH value is 4.0 to 5.0;

[0027] Preferably, in the protein-polysaccharide composite particle solution in step S20, the mass ratio of protein to polysaccharide is 1-2:1-3.

[0028] The pH value of the solution of the protein-polysaccharide composite particles is 4.0-5.0, and the volume concentration of ethanol is not higher than 15%.

[0029] Preferably, the method for preparing the oil phase solution of β-carotene in step S30 comprises the following steps: dispersing the β-carotene oil suspension in the oil phase material to form the oil phase solution of β-carotene;

[0030] The concentration of β-carotene in the β-carotene oil suspension is not less than 30%;

[0031] The oil phase material is at least one of medium chain triglycerides, sunflower oil, corn oil and soybean oil.

[0032] Preferably, the preparation method of the alcoholized Pickering emulsion loaded with β-carotene in step S30 comprises the following steps: adding the oil phase solution of β-carotene to the solution of protein-polysaccharide composite particles, and performing high-speed shear mixing at a speed of 13000rpm to 15000rpm for 3min to 10min to obtain the alcoholized Pickering emulsion loaded with β-carotene; the volume ratio of the oil phase solution of β-carotene to the solution of protein-polysaccharide composite particles is 20%-70%:30% to 80%;

[0033] The pH value of the alcoholized Pickering emulsion loaded with beta-carotene is 4.0-5.0, and the ethanol concentration is not higher than 15%.

[0034] The present invention also provides a Pickering emulsion loaded with alcoholized β-carotene prepared by the above method;

[0035] In the alcoholized Pickering emulsion loaded with β-carotene, the mass concentration of β-carotene is 0.4% to 0.6%;

[0036] The volume fraction of the oil phase solution in the alcoholized Pickering emulsion loaded with beta-carotene is 20% to 70%.

[0037] The present invention also provides an application of the alcoholized Pickering emulsion loaded with beta-carotene. The alcoholized Pickering emulsion loaded with beta-carotene is used in beverages, functional foods and medicines.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The invention provides a method for preparing a β-carotene-loaded alcoholized Pickering emulsion. After preparing milk protein-alcohol-soluble protein composite nanoparticles, polysaccharides are introduced to obtain a solution of protein-polysaccharide composite particles; then, an oil phase solution of β-carotene is mixed with the alcoholized protein-polysaccharide composite particle solution, so that β-carotene is loaded onto the alcoholized protein-polysaccharide composite particles, and an alcoholized protein-polysaccharide composite particle-loaded β-carotene-loaded alcoholized Pickering emulsion is obtained. The preparation process of the invention is simple and suitable for industrial large-scale production and application. In the prepared β-carotene-loaded alcoholized Pickering emulsion, the protein-polysaccharide composite particles are firmly and irreversibly adsorbed on the oil-water interface to form a dense interface layer, and provide sufficient steric hindrance and electrostatic repulsion, which is conducive to improving the embedding rate, light and heat stability of β-carotene. At the same time, the high-viscosity emulsion system effectively inhibits the diffusion rate of the pro-oxidant to the oil phase, thereby improving the storage stability of β-carotene. Therefore, Pickering emulsion stabilized by protein-polysaccharide composite particles effectively improved the stability and bioaccessibility of loaded β-carotene.

[0040] The prepared Pickering emulsion loaded with alcoholized beta-carotene is a Pickering emulsion stably loaded with alcoholized beta-carotene by protein-polysaccharide composite particles, wherein the protein-polysaccharide composite particles can be firmly and irreversibly adsorbed on the oil-water interface to form a dense interface layer, provide sufficient steric hindrance and electrostatic repulsion, and improve the embedding rate, light and heat stability of beta-carotene. At the same time, the high-viscosity emulsion system can effectively inhibit the diffusion rate of pro-oxidants to lipids, thereby improving the stability and bioaccessibility of beta-carotene.

[0041] The alcoholized Pickering emulsion loaded with beta-carotene prepared by the present invention has good loading stability for beta-carotene and high bioaccessibility, and beta-carotene has high antioxidant activity and vitamin A activity. Intake of beta-carotene can prevent certain cancers, inhibit the occurrence of cardiovascular diseases, reduce metabolic syndrome, prevent eye diseases such as cataracts, and is beneficial to health. Therefore, the alcoholized Pickering emulsion loaded with beta-carotene of the present invention can be widely used in the fields of beverages, functional foods, medicines, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The test graphs of droplet volume-average diameter and ζ-potential value of Pickering emulsion loaded with β-carotene alcohol with different oil phase volume fractions;

[0043] Figure 2 The test graph of the Turbiscan stability index of Pickering emulsions loaded with β-carotene alcohol with different oil phase volume fractions;

[0044] Figure 3 The frequency scanning results of Pickering emulsions loaded with β-carotene alcohol with different oil phase volume fractions;

[0045] Figure 4 The apparent viscosity results of Pickering emulsions loaded with β-carotene alcohol with different oil phase volume fractions;

[0046] Figure 5 The temperature scanning results of Pickering emulsions loaded with β-carotene alcohol with different oil phase volume fractions;

[0047] Figure 6 The CLSM results of Pickering emulsions loaded with β-carotene alcohol with different oil phase volume fractions;

[0048] Figure 7 The β-carotene encapsulation rate results of the β-carotene-loaded Pickering emulsions with different oil phase volume fractions;

[0049] Figure 8 The results of photostability of β-carotene in Pickering emulsions loaded with β-carotene alcohol at different oil phase volume fractions;

[0050] Fig. 9 The thermal stability results of β-carotene in Pickering emulsions loaded with β-carotene alcohol at different oil phase volume fractions;

[0051] Fig.10 The thermal stability test diagram of Pickering emulsions loaded with β-carotene alcohol with different oil phase volume fractions;

[0052] Fig.11 The droplet volume-average diameter ion stability results of Pickering emulsions loaded with β-carotene alcohol at different oil phase volume fractions;

[0053] Fig.12 The ζ-potential value and ionic stability results of Pickering emulsions loaded with β-carotene alcohol with different oil phase volume fractions;

[0054] Fig.13 The storage stability results of the alcoholized Pickering emulsions loaded with β-carotene at different oil phase volume fractions at 37°C;

[0055] Fig.14 The storage stability results of Pickering emulsions loaded with β-carotene and alcoholized with different oil phase volume fractions at 55°C;

[0056] Fig.15 The storage stability results of β-carotene in Pickering emulsions loaded with β-carotene alcohol at 37°C are shown in Figure 2.

[0057] Fig.16 The storage stability results of β-carotene in Pickering emulsions loaded with β-carotene alcohol at 55℃ are shown in Figure 2.

[0058] Fig.17 The bioaccessibility results of β-carotene in Pickering emulsions loaded with β-carotene and alcoholized with different oil phase volume fractions. DETAILED DESCRIPTION

[0059] The present invention provides a method for preparing a β-carotene-loaded alcoholized Pickering emulsion, comprising the following steps:

[0060] S10: mixing the alcohol-soluble protein solution with the milk protein solution, adjusting the pH value, and preparing a milk protein-alcohol-soluble protein composite nanoparticle solution;

[0061] S20: alcoholizing the polysaccharide solution, adjusting the pH value, and then mixing the polysaccharide solution with the milk protein-alcohol-soluble protein composite nanoparticle solution to obtain a protein-polysaccharide composite particle solution;

[0062] S30: First, an oil phase solution of β-carotene is prepared, and then the oil phase solution and the protein-polysaccharide composite particle solution are subjected to shear homogenization treatment to obtain a Pickering emulsion loaded with alcoholized β-carotene.

[0063] Furthermore, the alcohol-soluble protein in step S10 is at least one of zein, wheat alcohol-soluble protein and kajirin; these proteins can be dissolved in a relatively high concentration of ethanol aqueous solution, and a solution of milk protein-alcohol-soluble protein composite nanoparticles can be quickly prepared by an anti-solvent precipitation method.

[0064] In a specific embodiment of the present invention, the solvent of the alcohol-soluble protein solution is an ethanol aqueous solution with an ethanol volume fraction of 55% to 90%; under this concentration condition, alcohol-soluble protein has a higher solubility. Specifically, the concentration (v / v) of the ethanol aqueous solution can be 55%, 60%, 70%, 80%, or 90%.

[0065] In a specific embodiment of the present invention, in the alcohol-soluble protein solution, the ratio of alcohol-soluble protein to solvent is 1g: 50-150mL. Under this ratio, the alcohol-soluble protein solution has a more suitable concentration, which is conducive to the subsequent preparation of a solution of milk protein-alcohol-soluble protein composite nanoparticles through a mixing process. Specifically, the ratio of alcohol-soluble protein to solvent can be 1g: 50mL, 1g: 60mL, 1g: 80mL, 1g: 100mL, 1g: 120mL, 1g: 150mL.

[0066] More specifically, the alcohol-soluble protein is zein. Zein is soluble in 55%-90% (v / v) ethanol aqueous solution. By changing the solvent environment through the anti-solvent precipitation method, the molecular conformation of zein can be regulated to form nanoparticles, which have the advantages of being easy to obtain, green, and biocompatible. In the anti-solvent, lipophilic molecules can be embedded in the hydrophobic core of the nanoparticles, improving the environmental stability and bioaccessibility of β-carotene. In addition, the nanoparticles have good interfacial properties and are irreversibly adsorbed on the oil-water interface to form a solid interfacial layer, which is beneficial to protecting the lipophilic functional factors in the oil phase.

[0067] Furthermore, the milk protein in step S10 is at least one of lactoferrin, lactoglobulin, and α-lactalbumin; after these milk proteins are complexed with alcohol-soluble proteins, they can provide sufficient steric hindrance and electrostatic repulsion to enhance the stability of the composite particles, thereby improving the ability of the particles to deliver active factors.

[0068] More specifically, milk proteins include lactoferrin. Lactoferrin belongs to transferrin and has multiple biological activities such as antioxidant activity, antibacterial activity, promotion of wound healing, etc., and has broad application prospects in the food field. Since single zein particles have high surface hydrophobicity and are exposed to high ionic strength, high temperature and pH near the isoelectric point, zein nanoparticle delivery carriers are prone to aggregation and precipitation, limiting their application in food. The complex of lactoferrin and zein, that is, the lactoferrin layer formed on the periphery of the nanoparticles, provides sufficient steric hindrance and electrostatic repulsion, enhances the stability of the composite particles, and thereby improves the ability of the particles to deliver active factors.

[0069] In a specific embodiment of the present invention, the milk protein solution includes milk protein and water, and the ratio of milk protein to water is 1g: 200-400mL. At this ratio, milk protein has better solubility, and the concentration of the milk protein solution is conducive to the subsequent preparation of a solution of milk protein-alcohol-soluble protein composite nanoparticles by anti-solvent means. Specifically, the milk protein solution may include a ratio of 1g: 200mL, 1g: 250mL, 1g: 300mL, 1g: 350mL, 1g: 400mL.

[0070] Further, the preparation method of the lactoprotein-alcohol-soluble protein composite nanoparticle solution described in step S10 comprises the following steps: dropping the alcohol-soluble protein solution into the lactoprotein solution, stirring for 20min to 60min at a speed of 600rpm to 700rpm, adjusting the pH value, and obtaining a lactoprotein-alcohol-soluble protein composite nanoparticle solution; the method is conducive to forming a solution of lactoprotein-alcohol-soluble protein composite nanoparticles with smaller particle size, better thermal stability, and better colloidal stability. In addition, lactoprotein is a water-soluble protein and cannot be dissolved in a high-concentration ethanol-water system, while alcohol-soluble protein can only be dissolved in an ethanol-water system (such as 55%-90% ethanol). By an anti-solvent precipitation method, a solution containing alcohol-soluble protein is dropped into a solution of lactoprotein, and the polarity of the system is increased to form a solution of lactoprotein-alcohol-soluble protein composite nanoparticles. Specifically, the rotation speed can be 600rpm, 650rpm, 700rpm, etc., and the stirring time can be 20min, 30min, 40min, 50min, 60min, etc.

[0071] In a specific embodiment of the present invention, the pH value of the lactoprotein-alcohol-soluble protein composite nanoparticle solution is 4.0-5.0, and the volume concentration of ethanol is not higher than 15%; on the one hand, the pH of beverage products is usually around 2.5-5.0, and the pH value of the solution is 4-5, which is beneficial to its application performance. On the other hand, when the pH value is 4-5, the lactoprotein-alcohol-soluble protein composite nanoparticles are complete and have good stability. Too high pH close to the isoelectric point of alcohol-soluble protein (pH value is 6.0) is not conducive to particle stability, and too low pH will cause lactoferrin denaturation and reduce its biological activity. In addition, as the ethanol concentration increases, the alcohol-soluble protein particles will gradually dissolve, the edges will be blurred, and it will be difficult to form complete lactoprotein-alcohol-soluble protein composite nanoparticles. However, the reduction of ethanol concentration will not affect the formation of nanoparticles. In order to broaden the application market of low-alcohol beverages and low-alcohol milk beverages, the ethanol concentration of the solution of lactoprotein-alcohol-soluble protein composite nanoparticles is not higher than 15%. Specifically, the pH value of the solution of the lactoprotein-prolamin composite nanoparticles can be 4.0, 4.2, 4.5, 4.6, 4.8, 5.0

[0072] In a specific embodiment of the present invention, the average particle size of the lactoprotein-alcohol-soluble protein composite nanoparticles is 100nm to 120nm; more specifically, the average particle size is 110.9±8.4nm to 112.9±3.6nm. The lactoprotein-alcohol-soluble protein composite nanoparticles have a small particle size and high system uniformity, which is conducive to improving their stability and application performance.

[0073] In a specific embodiment of the present invention, in the lactoprotein-alcohol-soluble protein composite nanoparticles, the mass ratio of lactoprotein to alcohol-soluble protein is (1-2): (1-3). Under this ratio, it is beneficial to improve the stability of the lactoprotein-alcohol-soluble protein composite nanoparticles and obtain a solution of lactoprotein-alcohol-soluble protein composite nanoparticles with a smaller particle size and good stability. Specifically, in the lactoprotein-alcohol-soluble protein composite nanoparticles, the mass ratio of lactoprotein to alcohol-soluble protein can be 1:1, 1:2, 1:3, 2:1, 2:3.

[0074] Furthermore, the polysaccharide in step S20 is at least one of propylene glycol alginate (PGA), high methoxy pectin, and hyaluronic acid; these polysaccharides can load and deliver β-carotene, improve the physical stability of the emulsion by enhancing the viscosity of the emulsion and the spatial network structure of the emulsion, and improve the storage stability and biological activity of β-carotene.

[0075] In a specific embodiment of the present invention, the polysaccharide solution is composed of polysaccharide and water, and the ratio of the polysaccharide to water is 1g:(80-90)mL; the step of the alcoholization treatment is: dropwise adding anhydrous ethanol to the polysaccharide solution, stirring for 10-12h for alcoholization treatment; the volume ratio of the polysaccharide solution to anhydrous ethanol is 80-90:10-20; the reagent used for adjusting the pH value is an alkaline or acidic solution; the pH value is 4.0-5.0; after dissolving the polysaccharide in water, the method adds ethanol for alcoholization treatment, and adds an alkaline or acidic solution to adjust the pH value, ensuring that the ethanol concentration and pH value of the polysaccharide solution match the ethanol concentration of the solution of the milk protein-alcohol-soluble protein composite nanoparticles. Specifically, the ratio of polysaccharide to water is 1g:80mL, 1g:83mL, 1g:85mL, 1g:88mL, 1g:90mL, the added volume of anhydrous ethanol can be 10mL, 13mL, 15mL, 18mL, 20mL, the alkaline solution can be 1M sodium hydroxide, ammonia water, potassium hydroxide, the acidic solution can be 1M hydrochloric acid, citric acid and malic acid; the pH value can be 4.0, 4.2, 4.5, 4.6, 4.8, 5.0.

[0076] More specifically, the polysaccharide is propylene glycol alginate. Propylene glycol alginate (PGA) can be dissolved in an ethanol aqueous solution. The pH of the polysaccharide solution after dissolving in a 15% (v / v) ethanol aqueous system is close to 4.0, which can reduce the introduction of ions. In addition, propylene glycol alginate has good surface activity and thickening properties. In loading and delivering β-carotene, PGA can improve the physical stability of the emulsion by enhancing the viscosity of the emulsion and the spatial network structure of the emulsion, thereby improving the stability of the active substance in the oil phase. In addition, the addition of PGA can provide a large amount of negative charge and greater steric hindrance for protein particles, thereby inhibiting protein particle aggregation, improving particle stability, and promoting the embedding, protection and slow release of bioactive substances.

[0077] Furthermore, in the protein-polysaccharide composite particle solution of step S20, the mass ratio of protein to polysaccharide is 1-2:1-3. The protein-polysaccharide composite particles formed by the above ratio of protein to polysaccharide can be more stably and irreversibly adsorbed on the oil-water interface to form a dense interface layer, and provide sufficient steric hindrance and electrostatic repulsion, which is more conducive to improving the embedding rate, light and thermal stability of β-carotene. Exemplarily, in the solution of protein-polysaccharide composite particles, the mass ratio of the total mass of protein to the mass of polysaccharide can be 1:1, 1:2, 1:3, 2:1, 2:3

[0078] In a specific embodiment of the present invention, the pH value of the solution of the protein-polysaccharide composite particles is 4.0-5.0, and the volume concentration of ethanol is not higher than 15%. Under this condition, the protein-polysaccharide composite particles have better stability, can load β-carotene more stably, and are conducive to improving the embedding rate, light and heat stability of β-carotene, and improving the storage stability and bioaccessibility of β-carotene.

[0079] Furthermore, the method for preparing the oil phase solution of β-carotene in step S30 comprises the following steps: dispersing the β-carotene oil suspension in the oil phase material to form the oil phase solution of β-carotene;

[0080] In a specific embodiment of the present invention, the concentration of β-carotene in the β-carotene oil suspension is not less than 30%, thereby ensuring the content of β-carotene.

[0081] In a specific embodiment of the present invention, the oil phase material is at least one of medium chain triglycerides, sunflower oil, corn oil, and soybean oil. These oil phase materials have good solubility for β-carotene, are not easily oxidized, and are colorless and tasteless. More specifically, the oil phase material includes medium chain triglycerides.

[0082] Furthermore, the preparation method of the alcoholized Pickering emulsion loaded with β-carotene in step S30 comprises the following steps: adding an oil phase solution of β-carotene to a solution of protein-polysaccharide composite particles, wherein the oil phase accounts for 20%-70% (v / v), and performing high-speed shear mixing at a speed of 13000 rpm to 15000 rpm for 3 min to 10 min to obtain the alcoholized Pickering emulsion loaded with β-carotene;

[0083] In a specific embodiment of the present invention, the pH value of the alcoholized Pickering emulsion loaded with β-carotene is 4.0-5.0, and the ethanol concentration is not higher than 15%. Under this condition, not only the stability and bioaccessibility of β-carotene in the emulsion can be improved, but also the alcoholized Pickering emulsion loaded with β-carotene can be applied to ordinary beverages, alcoholic beverages, alcoholic milk beverages and other products, thus expanding the application prospects of the emulsion.

[0084] More specifically, the pH value of the alcoholized Pickering emulsion loaded with β-carotene is 4.0, the ethanol concentration is not higher than 15%, the mass concentration of β-carotene is 0.5%, and the volume fraction of the oil phase is 60%.

[0085] The invention also provides a beta-carotene alcoholized Pickering emulsion prepared by the method.

[0086] In a specific embodiment of the present invention, the mass concentration of β-carotene in the alcoholized Pickering emulsion loaded with β-carotene is 0.4% to 0.6%; in this case, the loaded β-carotene has better stability and biological activity. Specifically, the mass concentration of β-carotene in the alcoholized Pickering emulsion loaded with β-carotene can be 0.4%, 0.5%, or 0.6%.

[0087] In a specific embodiment of the present invention, the volume fraction of the oil phase solution in the alcoholized Pickering emulsion loaded with β-carotene is 20% to 70%. The oil phase in the alcoholized Pickering emulsion loaded with β-carotene includes an oily solvent and an oil phase material in a β-carotene oil suspension. The content of the oil phase makes the alcoholized Pickering emulsion loaded with β-carotene have better physicochemical properties, rheological properties, and microstructure, and improves the storage stability and bioaccessibility of β-carotene. Specifically, the volume fraction of the oil phase in the alcoholized Pickering emulsion loaded with β-carotene can be 20%, 30%, 40%, 50%, 60%, or 70%.

[0088] More specifically, the volume fraction of the oil phase solution in the alcoholized Pickering emulsion loaded with β-carotene is 60%. At this time, the physical stability of the alcoholized Pickering emulsion stably loaded with β-carotene by the protein-polysaccharide composite particles is the best.

[0089] The prepared Pickering emulsion loaded with alcoholized beta-carotene includes a Pickering emulsion stably loaded with alcoholized beta-carotene by protein-polysaccharide composite particles, wherein the protein-polysaccharide composite particles can be firmly and irreversibly adsorbed on the oil-water interface to form a dense interface layer, provide sufficient steric hindrance and electrostatic repulsion, and improve the embedding rate, light and heat stability of beta-carotene. In addition, the high-viscosity emulsion system can effectively reduce the diffusion rate of the pro-oxidant to the oil phase, and improve the stability and bioaccessibility of beta-carotene.

[0090] In the alcoholized Pickering emulsion loaded with β-carotene, the mass concentration of β-carotene is 0.4% to 0.6%; the volume fraction of the oil phase is 50% to 70%. In this case, the storage stability and bioaccessibility of β-carotene can be better improved, broadening the application prospects of the alcoholized Pickering emulsion loaded with β-carotene. By controlling the volume of the oil phase, the droplet size and the number of charges on the interface film are adjusted, thereby optimizing the stability of the emulsion. With the increase of the oil phase, the interaction between the droplets and the particles is enhanced, prompting the protein-polysaccharide composite particles to form a dense network structure between adjacent droplets, inhibiting the flocculation and aggregation of the droplets. In addition, the droplets with high stacking density increase the viscosity of the system and restrict the movement of the droplets.

[0091] The present invention also provides an application of the alcoholized Pickering emulsion loaded with beta-carotene. The alcoholized Pickering emulsion loaded with beta-carotene is used in beverages, functional foods and medicines.

[0092] The alcoholized Pickering emulsion loaded with β-carotene prepared by the present invention has good loading stability for β-carotene and high bioaccessibility, and β-carotene has high antioxidant activity and vitamin A activity. Intake of β-carotene can prevent certain cancers, inhibit the occurrence of cardiovascular diseases, reduce metabolic syndrome, prevent eye diseases such as cataracts, and is beneficial to health. Therefore, the alcoholized Pickering emulsion loaded with β-carotene in the embodiment of the present invention can be widely used in the fields of beverages, functional foods, medicines, etc.

[0093] Specifically, the alcoholized Pickering emulsion loaded with β-carotene can be applied to ordinary beverages, alcoholic beverages, alcoholic milk beverages and other products.

[0094] The present invention will be further described below in conjunction with the embodiments.

[0095] Example 1

[0096] A method for preparing a β-carotene-loaded alcoholized Pickering emulsion, comprising the following steps:

[0097] S10: Preparation of LF-Zein composite nanoparticle solution: First, weigh 1g of corn alcohol-soluble protein (Zein) and dissolve it in 100mL of 60% ethanol aqueous solution to obtain Zein dispersion; weigh 1g of lactoferrin (LF) and dissolve it in 300mL of aqueous solution, and stir them on a magnetic stirrer for 1h until the sample is fully dissolved to obtain LF aqueous solution; then add the Zein dispersion dropwise into the LF aqueous solution, and stir at 650rpm for 30min to form LF-Zein composite nanoparticles (the final ethanol concentration is 15% (v / v)); finally, adjust the sample to pH 4.0 with 0.1M hydrochloric acid (HCl) solution to obtain LF-Zein composite nanoparticle solution;

[0098] S20: Preparation of LF-Zein-PGA composite particle solution: Weigh 1g of propylene glycol alginate (PGA) and dissolve it in 85mL of deionized water, stir overnight, and after it is fully dissolved, add 15mL of anhydrous ethanol to the PGA aqueous solution, stir for 12h for alcoholization, and then adjust the sample to pH 4.0 with 1M NaOH solution to obtain a PGA solution; mix the PGA solution with the LF-Zein composite nanoparticle solution in a volume ratio of 1:1, adjust the pH to 4.0, and obtain a LF-Zein-PGA (abbreviated as LZ-PGA) composite particle solution, and place it in a 4°C refrigerator for the next analysis;

[0099] S30: Preparation of Pickering emulsion loaded with β-carotene alcoholization: 1 g of β-carotene oil suspension (purchased from Zhejiang Xinchang Pharmaceutical Co., Ltd., specification 30%, the same below) was dispersed in 11 mL, 17 mL, 23 mL, 29 mL, 35 mL, and 41 mL of medium chain triglycerides (MCT), respectively, and dissolved at 140°C for 10 s to form an oil phase. Then, the oil phase containing β-carotene at different levels was slowly added to the LZ-PGA composite particle solution (ethanol content was 15% (v / v)), and a high-speed shearing machine was used for high-speed shearing at 13600 rpm for 4 min to prepare Pickering emulsions with different oil phase volume fractions (20%, 30%, 40%, 50%, 60%, 70%, v / v), and the total volume of the emulsion was fixed at 20 mL. The β-carotene content in the final alcoholized Pickering emulsion was 0.5% (w / v).

[0100] The following experimental tests were performed on the β-carotene loaded alcoholized Pickering emulsion prepared in Example 1:

[0101] Experiment 1: Droplet size test: The particle size of the Pickering emulsion samples loaded with β-carotene alcoholization with different oil phase volume fractions was measured using a laser scattering particle size analyzer LS230, that is, the volume-average diameter (D 4,3 ). Among them, the refractive indexes of MCT and 15% (v / v) ethanol water are 1.52 and 1.34.

[0102] Experiment 2, ζ-potential test: The ζ-potential of the Pickering emulsion droplets loaded with β-carotene alcohol was measured using a Malvern particle size analyzer. The emulsion was diluted 1000 times with 15% (v / v) ethanol aqueous solution (pH 4.0) to avoid multiple scattering effects. After 120 seconds of equilibrium, data were collected. All measurements were repeated three times.

[0103] The droplet size and ζ-potential results of Pickering emulsions loaded with β-carotene alcohol with different oil content (20%-70% (v / v)) are shown in Figure 2. Figure 1 As shown, different letters a, b, c, d, e, f indicate significant differences (P<0.05). It can be seen that the oil phase content has a significant effect on the emulsion particle size. When the oil phase content increases from 20% (v / v) to 60% (v / v), the droplet size decreases from 14.46±0.68μm to 11.25±0.13μm. There are two main reasons: (i) LZ-PGA composite particles are adsorbed at the oil-water interface and form a barrier, inhibiting coalescence to form large droplets. As the oil phase content increases, the oil phase continuously compresses the ethanol-water phase space, and the particles on the droplet surface connect adjacent droplets through non-covalent bonds to form a dense spatial network, which restricts the movement of droplets. (ii) When the oil phase content is low, there may be excessive unadsorbed particles in the mobile phase, exhaustion flocculation occurs, the emulsion droplet size is larger, and the emulsion stability decreases. When the oil phase content continued to increase to 70% (v / v), the droplet size increased slightly (15.46±0.36μm). This was attributed to the fact that the LZ-PGA composite particles could not completely cover the droplet surface. Under the influence of van der Waals forces and hydrophobicity, the droplets were prone to flocculation and aggregation, resulting in reduced emulsion stability.

[0104] In addition, the ζ-potentials of all β-carotene-loaded Pickering emulsions are negative, because the LZ-PGA composite particles carry negative charges. As the oil phase content increases (20%-60% (v / v), the droplet ζ-potential decreases from -29.73±1.06mV to -35.67±0.93mV. The greater the absolute value of the emulsion potential, the stronger the electrostatic repulsion between the emulsion droplets, which effectively inhibits the droplet aggregation and improves the stability of the emulsion system. It is generally believed that when the droplet ζ-potential is lower than -30mV, the emulsion system has a strong anti-flocculation and aggregation ability.

[0105] In summary, the droplet size and the amount of charge on the interfacial film can be adjusted by controlling the volume of the oil phase, thereby optimizing the stability of the emulsion. When the oil phase content is 60% (v / v), the Pickering emulsion of β-carotene stably loaded by LZ-PGA composite particles shows good physical stability.

[0106] Experiment 3, physical stability test of emulsion: The physical stability of the Pickering emulsion loaded with β-carotene stabilized by composite particles was evaluated using the Turbiscan multifunctional stability analyzer. 20 mL of the Pickering emulsion loaded with β-carotene was transferred to a glass bottle. At 25°C, the sample was scanned from the bottom to the top using near-infrared light. The scan was performed every 15 minutes for 24 hours, and the backscattered light change data (ΔBS) of the sample was obtained. The Turbiscan Stability Index (TSI) was calculated using TowerSoft software (Ver. 2.0.0.9), as shown in formula (1):

[0107]

[0108] In formula (1): i is the backscattered light value measured each time, χ bs is the average backscattered light value, and n is the number of measurements.

[0109] The time-dependent curves of the stability index TSI of Pickering emulsions loaded with β-carotene and alcoholized with different oil phase contents (20%-70% (v / v)) are shown in Figure 2. Figure 2 As shown. Figure 2It can be seen that after 24 hours, all sample curves are basically stable, and the TSI values ​​are 21.86 (LZ-PGA-20%), 17.47 (LZ-PGA-30%), 14.71 (LZ-PGA-40%), 8.45 (LZ-PGA-50%), 2.72 (LZ-PGA-60%), and 8.59 (LZ-PGA-70%). The lower the TSI value of the emulsion, the higher the stability of the emulsion. When the oil content is 60% (v / v), the emulsion TSI value (24h) is the smallest, and the rate of change of the TSI curve over time is the smallest. This is because the increase in the oil phase content enhances the interaction between droplets and particles, prompting the LZ-PGA composite particles to form a dense network structure between adjacent droplets. This is conducive to capturing emulsion droplets, restricting droplet flow, and delaying the rate of droplet flocculation and coalescence, thereby improving the stability of the emulsion. In addition, when the oil phase content continued to increase, TSI increased instead, probably because the protein-polysaccharide composite particles were not sufficient to completely cover the droplets, causing the Pickering emulsion droplets loaded with β-carotene to coalesce and the emulsion stability to decrease.

[0110] Experiment 4, Rheological properties test of emulsion frequency scanning: The rheological properties of Pickering emulsion loaded with β-carotene alcohol were measured using a HAAKE IQAir rheometer and a P35 / Ti parallel plate (35mm). Before the measurement, the emulsion was deposited on the plate for 3 minutes of equilibrium, and the interval was set to 1.00mm. First, a large amplitude oscillatory strain scanning mode was selected in the strain range of 0.01%-100%, and the frequency was fixed at 1Hz to determine the linear viscoelastic range (LVR) of the sample. The strain value was selected to be 0.1%, and the emulsion sample was subjected to an oscillatory frequency sweep in the linear frequency range of 0.1-10Hz, and the changes in the storage modulus (G') and loss modulus (G") with frequency were recorded. The shear rate was changed from 0.1s -1 Increase to 100s -1 , a steady-state shear flow test was conducted on the sample, and the variation of the apparent viscosity (η) with the shear rate was recorded. The rheological behavior of the emulsion was analyzed using the Power Law (Ostwald de-Waele) model, as shown in formula (2):

[0111] η=Kγ n-1 (2)

[0112] In formula (2), η is the apparent viscosity of the emulsion (Pa·s), K is the consistency coefficient (Pa·sn), n is the flow behavior index (dimensionless quantity), and γ is the shear rate (s -1 ). All measurements were performed at 25°C.

[0113] The frequency scanning results of Pickering emulsions loaded with β-carotene alcohol with different oil content are shown in Figure 2. Figure 3 As shown. Figure 3 It can be seen that in the range of 0.1-10 Hz, the G' of all samples is one order of magnitude higher than G", indicating that the Pickering emulsion stabilized by LZ-PGA composite particles loaded with β-carotene alcohol has solid-like properties. In addition, the emulsion G' remains relatively stable at the test frequency, which is attributed to the strong interaction between the adsorbed molecules on the interfacial adsorption layer, forming a weak gel network structure, giving the emulsion a certain strain resistance. With the increase of oil phase content, G' first increases and then decreases, and the LZ-PGA-60% sample has the highest G' value. This is because the relative density of the emulsion droplets increases, which is conducive to the formation of a more solid three-dimensional network structure around the droplets, reduces the frequency of droplet collisions, and improves the physical stability of the emulsion.

[0114] The apparent viscosity test of Pickering emulsions loaded with β-carotene alcohol with different oil phase volume fractions is shown in the figure below. Figure 4 ,from Figure 4 It can be seen that with the increase of shear rate, the apparent viscosity of all Pickering emulsions decreases. Therefore, Pickering emulsions loaded with β-carotene alcohol with different oil contents are all non-Newtonian pseudoplastic fluids. Under the action of external shear, the emulsion structure is destroyed. When the destruction speed is faster than the structural rearrangement speed, the viscosity decreases rapidly.

[0115] The dependence of the apparent viscosity of the emulsion on the shear rate conforms to the PowerLaw model, and the fitting results are shown in Table 1. The parameter "K" represents the consistency coefficient, which reflects the degree to which the flow of the emulsion is affected by resistance. "n" is the flow behavior index, which reflects the ability of the emulsion to resist shear deformation. Among them, the n values ​​of all emulsions are less than 1, which again shows that the emulsion is a typical pseudoplastic fluid.

[0116] Table 1

[0117] sample Consistency coefficient (K, Pa·sn) Flow behavior index (n) <![CDATA[R 2 ]]> LZ-PGA-20% <![CDATA[21.77±2.40 e ]]> <![CDATA[0.026±0.006 a ]]> 0.971 LZ-PGA-30% <![CDATA[29.44±0.95 d ]]> <![CDATA[0.105±0.017 c ]]> 0.997 LZ-PGA-40% <![CDATA[31.71±0.78 c ]]> <![CDATA[0.093±0.013 c ]]> 0.998 LZ-PGA-50% <![CDATA[36.86±1.31 b ]]> <![CDATA[0.178±0.018 b ]]> 0.996 LZ-PGA-60% <![CDATA[39.06±2.07 ab ]]> <![CDATA[0.254±0.028 a ]]> 0.985 LZ-PGA - 70% <![CDATA[41.26±1.16 a ]]> <![CDATA[0.265±0.015 a ]]> 0.995

[0118] In Table 1, different letters (a, b, c, d, e) in each column indicate significant differences (P < 0.05).

[0119] From the above test results, it can be seen that with the increase of oil phase content (20%-60% (v / v)), the K value increases from 21.77±2.40Pa·sn to 29.44±0.95Pa·sn, 32.71±0.78Pa·sn, 36.86±1.30Pa·sn, 39.06±2.07Pa·sn, and 41.26±1.16Pa·sn. The Pickering emulsion loaded with β-carotene alcoholization exhibits higher viscosity at higher oil phase content. On the one hand, the higher the oil phase content, the more droplets per unit volume, which makes the droplets accumulate more tightly and the interaction is enhanced, thereby causing the viscosity of the system to increase. On the other hand, the droplet size of the Pickering emulsion loaded with β-carotene alcoholization decreases with the increase of oil phase content, and the droplets are densely distributed, resulting in worse system fluidity and increased K value. The higher the viscosity of the emulsion, the more conducive it is to inhibit the movement, sedimentation, and coalescence of the droplets, thereby improving the stability of the emulsion. However, when the oil phase content is increased to 70% (v / v), the viscosity decreases, which is not conducive to the stability of the emulsion.

[0120] Experiment 5, Rheological property test of emulsion temperature scanning: set the temperature from 25°C to 90°C, the heating rate is 5°C / min, the strain value is 0.1%, the scanning frequency is 1Hz, the temperature of the emulsion sample is scanned, and the changes of storage modulus (G') and loss modulus (G") with temperature are recorded.

[0121] Test results such as Figure 5 As shown. Figure 5 It can be seen that within the temperature test range, the G' of all samples is higher than G", which indicates that the Pickering emulsion loaded with β-carotene stabilized by LZ-PGA always maintains a solid-like behavior. In addition, in the range of 25℃-90℃, the G' and G" of the samples hardly change with increasing temperature, indicating that the sample emulsions have good heat resistance. This is attributed to the formation of strongly adsorbed particles and thick interfacial films, which are conducive to maintaining the stability of the emulsion network structure during heating. The Pickering emulsion loaded with β-carotene stabilized by LZ-PGA-60% has the strongest thermal stability and has good application prospects in the food field.

[0122] Experiment 6, emulsion micro-interface structure test: Confocal scanning laser microscopy (CLSM) can visualize the interface structure of the Pickering emulsion loaded with β-carotene alcohol. The oil droplets were stained with Nile red dye (0.1% (w / v)), the protein was stained with Nile blue dye (0.1% (w / v)), and the PGA was stained with Calcofluor White dye. 5 μL of Nile red dye, 5 μL of Nile blue dye, and 10 μL of Calcofluor White dye were added to 1 mL of Pickering emulsion sample. The stained emulsion droplets were added to the concave confocal microscope slide, gently covered with a coverslip, and observed under a 100× oil lens. Three laser excitations were used: 488 nm (Nile red), 633 nm (Nile blue), and 405 nm (Calcofluor White).

[0123] CLSM images of Pickering emulsions loaded with β-carotene at different oil content (20%-70% (v / v)) are shown in Figure 2. Figure 6 As shown, A is a combined fluorescence image; B is an oil phase fluorescence image; C is a protein fluorescence image; and D is a polysaccharide fluorescence image. MCT oil was stained with Nile red (green), protein was stained with Nile blue A (red), and PGA was stained with Calcofluor White (blue). The droplet interface of all samples was covered with red protein and blue polysaccharide circles. This shows that the Pickering emulsions stably loaded with β-carotene alcoholization by LZ-PGA composite particles are all ethanol water-in-oil emulsions, and no phase inversion occurs. LZ-PGA composite particles are adsorbed on the droplets to form a dense interface layer and provide a large steric hindrance for the droplets, which is conducive to stabilizing the Pickering emulsion loaded with β-carotene alcoholization. When the oil phase content is low (20%-30% (v / v)). The droplet size is larger and the distribution is more discrete. When the oil phase content increases from 40% (v / v) to 60% (v / v), the droplet density continues to increase and the droplet size decreases. In particular, the droplet size in the LZ-PGA-60% sample is the smallest and the distribution is dense and uniform. Smaller droplets have a higher specific surface area, which can provide more binding sites for particles, which is conducive to the adsorption of particles on the interface. At the same time, the distance between droplets is shortened, and the interaction between particles is enhanced, which promotes the formation of a cross-linked network on the interface, restricts the movement of droplets, and effectively inhibits droplet flocculation and coalescence. This is consistent with the rheological results. When the oil phase content is 70% (v / v), the particle size of the emulsion droplets becomes larger, because too much oil phase will cause the droplets to coalesce. In addition, a shared interface appears between the droplets, which are easy to squeeze each other, resulting in polygonal deformation of the interface. Therefore, some droplets have irregular shapes.

[0124] Experiment 7, β-carotene content determination: All emulsions were diluted to an appropriate concentration, and then a mixed reagent (anhydrous ethanol: n-hexane = 1:3 (v / v)) was used to extract the β-carotene embedded in the diluted emulsion. The extraction was repeated three times, and the upper extracts were combined. The absorbance was measured at 450nm using a UV-1800 spectrophotometer. The β-carotene content was calculated with reference to the β-carotene standard curve measured under the same conditions (y = 0.2724x-0.0011, R2 = 0.9994). The β-carotene encapsulation efficiency (EE) was calculated as shown in formula (3):

[0125]

[0126] In formula (3): the mass of embedded β-carotene refers to the concentration of β-carotene contained in the emulsion droplets. The total amount of β-carotene added refers to the concentration of β-carotene added when preparing the sample.

[0127] The above test results are as follows Figure 7 As shown in the figure, different letters a, b, c, d, e indicate significant differences (P<0.05). Figure 7 It can be seen that, overall, the Pickering emulsion loaded with β-carotene alcoholization has a certain embedding capacity for β-carotene. The LZ-PGA composite particles are firmly and irreversibly adsorbed on the oil-water interface, forming a dense interface layer, which promotes the successful embedding of β-carotene in the droplets. As the oil phase content gradually increases (20%-70% (v / v)), the embedding of β-carotene first increases and then decreases. The Pickering emulsion loaded with LZ-PGA-60% β-carotene alcoholization has the highest β-carotene embedding rate (93.71±0.45%). On the one hand, the emulsion with an oil content of 60% (v / v) has the smallest droplet size and the largest droplet density, and can load more β-carotene during the emulsification process. On the other hand, the denser the network structure of the Pickering emulsion loaded with β-carotene alcoholization and the greater the system viscosity, the more effectively it can inhibit droplet coalescence and oil phase precipitation, thereby improving the β-carotene embedding rate.

[0128] Experiment 8, emulsion light stability test: Take 10mL of fresh β-carotene-loaded Pickering emulsion and put it into a transparent glass bottle, and then put it into a light box (0.35W / m2, 35℃) for 5h. The experiment fixed sampling at 0h, 1h, 2h, 3h, 4h, and 5h, measured the β-carotene concentration, calculated the retention rate as formula (4), and plotted the β-carotene retention rate against the light exposure time. The results are shown in Figure 4. Figure 8 .

[0129]

[0130] In formula (4): C t Refers to the β-carotene content after treatment time t; C 0 is the initial β-carotene content.

[0131] from Figure 8 It can be seen that the retention rate of β-carotene in the alcoholized Pickering emulsion loaded with β-carotene prepared by LZ-PGA composite particles at different oil phase contents depends on the UV irradiation time. The results show that the loss rate of β-carotene free in MCT is close to 50% after 1 h of light irradiation. The retention rate of β-carotene in the emulsion is significantly higher than that of the MCT control, indicating that the alcoholized Pickering emulsion loaded with β-carotene stabilized by LZ-PGA composite particles has a protective effect on β-carotene. There are two main reasons for this: (i) LZ-PGA composite particles cover the surface of the droplets to form a hard and thick interface layer. This spatial barrier physically limits the diffusion of pro-oxidants to the surface of the droplets and the contact with β-carotene inside, which is beneficial to improve the photostability of β-carotene. (ii) Proteins contain aromatic amino acid residues, which have the ability to absorb ultraviolet rays and protect β-carotene from photodegradation and isomerization.

[0132] In order to further explore the effect of oil content on the protective effect of β-carotene, a first-order kinetic model (ln(C / C 0 )=-k t ) was used to evaluate the photodegradation kinetics of β-carotene, and the degradation rate constant (k), half-life (t 1 / 2 , h) as shown in formula (5) and formula (6):

[0133]

[0134] In formula (5): C 0 , C t represent the initial content of β-carotene and the content at irradiation time t, respectively.

[0135] The calculated degradation rate constants and half-lives are shown in Table 2:

[0136] Table 2

[0137] sample <![CDATA[k(h -1 )]]> <![CDATA[t 1 / 2 (h)]]> <![CDATA[R 2 ]]> MCT control <![CDATA[0.4863±0.0035 a ]]> <![CDATA[1.421±0.010 f ]]> 0.981 LZ-PGA-20% MCT <![CDATA[0.2520±0.0025 b ]]> <![CDATA[2.753±0.003 e ]]> 0.972 LZ-PGA-30% MCT <![CDATA[0.2363±0.0007 c ]]> <![CDATA[2.942±0.009 d ]]> 0.972 LZ-PGA-40% MCT <![CDATA[0.1834±0.0208 d ]]> <![CDATA[3.779±0.043 c ]]> 0.983 LZ-PGA-50% MCT <![CDATA[0.1758±0.009 e ]]> <![CDATA[3.942±0.021 b ]]> 0.984 LZ-PGA-60% MCT <![CDATA[0.1553±0.0026 f ]]> <![CDATA[4.415±0.076 a ]]> 0.992 LZ-PGA-70% MCT <![CDATA[0.1867±0.038 d ]]> <![CDATA[3.677±0.076 c ]]> 0.990

[0138] In Table 2, different letters (a, b, c, d, e, f) in the same column indicate significant differences (P < 0.05).

[0139] From the above test results, we can see that the linear fitting result R 2>0.97, which proves that the degradation process of β-carotene basically conforms to the first-order kinetic model, which is consistent with other literature. The smaller the reaction rate k, the higher the t 1 / 2 The larger the value, the more obvious the protective effect. As the oil phase content increases (20%-70% (v / v)), the k values ​​are 0.2520, 0.2363, 0.1834, 0.1758, 0.1553, and 0.1867, respectively. When the oil phase content is 60% (v / v), the k value reaches the minimum and the t value reaches the maximum (4.415±0.076h), which is higher than the free β-carotene (1.421±0.010h). This is because LZ-PGA-60% has the largest apparent viscosity, which effectively inhibits the diffusion of pro-oxidants into the oil phase and reduces the molecular migration rate, thereby alleviating the oxidative degradation of β-carotene. At the same time, the isomerization rate of β-carotene also decreases with the increase of the emulsion viscosity. In addition, the research results also show that the stability of the emulsion has an effect on the protective effect of β-carotene. As the oil content increases, the particle size of the emulsion decreases, the net charge increases, and the droplet flocculation and aggregation are effectively inhibited, reducing the release of the embedded β-carotene, thereby achieving the purpose of protecting β-carotene. In summary, the LZ-PGA-60% sample has the best photostabilization effect on β-carotene.

[0140] Experiment 9, emulsion thermal stability test: To evaluate the thermal stability of the alcoholized Pickering emulsion loaded with β-carotene stabilized by LZ-PGA composite particles, the effect of heating treatment on the emulsion droplet size and β-carotene retention rate was investigated.

[0141] On the one hand, after 12 hours of preparing the alcoholized Pickering emulsion loaded with β-carotene, the sample was heated in a water bath (80° C.) for 1 hour. After cooling to 25° C., the droplet size of the emulsion was measured.

[0142] The above heat treatments have different effects on different emulsions, e.g. Fig. 9As shown, different capital letters (A, B, C...) indicate that there are significant differences between different groups at the same time / ion concentration (P<0.05). Different lowercase letters (a, b, c...) indicate that there are significant differences at different times / ion concentrations in the same group (P<0.05). When the oil phase content is 20% (v / v) and 30% (v / v), the Pickering emulsion loaded with β-carotene alcoholization increases the particle size after heat treatment, from 14.46±0.68μm, 13.61±0.42μm to 16.46±0.47μm, 14.83±0.13μm, respectively. Heat treatment increases the droplet movement rate, promotes its flocculation and aggregation, resulting in an increase in droplet particle size, thereby reducing the stability of the emulsion. As the oil phase content continues to increase, the particle size of the Pickering emulsion loaded with β-carotene alcoholization does not change significantly after heat treatment. This is because the thermal stability of the emulsion mainly depends on the particles providing strong electrostatic repulsion and large steric hindrance to the droplets. When the oil phase content is 40%-60% (v / v), the ζ-potential value of the Pickering emulsion droplets loaded with β-carotene stabilized by LZ-PGA composite particles is higher, the electrostatic repulsion between the droplets is larger, the droplet aggregation is inhibited, and the emulsion has better thermal stability. In addition, the rheological results show that the emulsion has a harder network structure and higher viscosity, which helps to inhibit the movement of droplets and reduce the possibility of collision, thereby improving the thermal stability of the emulsion. When the oil phase content continues to increase to 70% (v / v), the emulsion particle size increases. This shows that excessive oil will reduce the thermal stability of the emulsion on the contrary. This is because the number of particles loaded per unit droplet surface is reduced, resulting in a decrease in the rigidity of the interface film and a decrease in resistance to heat treatment.

[0143] On the other hand, 12 hours after the preparation of the alcoholized Pickering emulsion loaded with β-carotene, the sample was heated in a water bath (80°C) for 4 hours. Samples were taken every 1 hour, cooled to 25°C, and the β-carotene retention rate was measured.

[0144] The retention rate of β-carotene in the β-carotene-loaded Pickering emulsion changes with heating time. Fig.10As shown in the figure, different capital letters (A, B, C...) indicate that there are significant differences between different groups at the same time / ion concentration (P<0.05). Different lowercase letters (a, b, c...) indicate that there are significant differences at different times / ion concentrations within the same group (P<0.05). After 4 hours of heat treatment, the retention rate of β-carotene in the alcoholized Pickering emulsion stably loaded with β-carotene by LZ-PGA composite particles is higher than that in free MCT (10.56±0.91%). The improvement of β-carotene thermal stability may be due to the following reasons: (i) polysaccharides form composite particles with proteins through electrostatic interactions, which is beneficial to improve the thermal stability of proteins, and further promotes LZ-PGA composite particles to form a thick thermal insulation physical barrier around the droplets, effectively delaying the thermal degradation rate of β-carotene in emulsified oil droplets. (ii) Lactoferrin can not only chelate iron ions, but also act as a free radical scavenger, thereby inhibiting the occurrence and transmission of oxidation reactions. As the oil content increased (20%-60% (v / v)), the retention rate of β-carotene gradually increased from 24.70±1.31% to 34.31±1.25%, 38.74±1.01%, 45.01±0.92%, and 53.92±1.23%. The results showed that high viscosity solutions and biopolymer networks reduce the rate of heat penetration and transfer. Therefore, as the oil content increased, the emulsion network strength and viscosity increased, which was beneficial to alleviate the heat-induced degradation of β-carotene.

[0145] In summary, the thermal stability of the emulsion is consistent with that of β-carotene. The thermal stability of the emulsion containing LZ-PGA-60% is the best, and its thermal protection effect on β-carotene is the best.

[0146] Experiment 10, emulsion ion stability test: 12 hours after the preparation of the Pickering emulsion loaded with β-carotene alcoholization, the sample was mixed with different masses of potassium chloride (KCl) powder for 2 hours until it was completely dissolved. The final KCl concentrations were 10mM, 50mM, and 100mM, respectively. After storage for 12 hours, the droplet size and ζ-potential of the Pickering emulsion loaded with β-carotene alcoholization were measured.

[0147] The droplet size of the Pickering emulsion loaded with β-carotene stabilized by LZ-PGA composite particles was determined by adding different concentrations of salt ions. Fig.11 The ζ-potential results are shown in Fig.12 As shown, Fig.11 and Fig.12In the table, different capital letters (A, B, C...) indicate that there are significant differences (P<0.05) between different groups at the same time / ion concentration. Different lowercase letters (a, b, c...) indicate that there are significant differences (P<0.05) at different times / ion concentrations within the same group. Fig.11 It can be seen that with the addition of salt ions, the surface charge of the emulsion droplets is reduced. This is attributed to the electrostatic shielding effect of salt ions. Salt particles can accumulate on the surface of the droplets, play an electrostatic shielding role, and weaken the electrostatic repulsion between the droplets. If the attraction between the droplets (hydrophobic interaction, van der Waals force) is greater than the repulsive interaction, droplet aggregation will occur, resulting in reduced system stability.

[0148] from Fig.12 From the particle size results, the addition of salt ions did not cause significant changes. This may be because the LZ-PGA composite particles formed a thick interface layer, providing greater steric hindrance, inhibiting the droplets from approaching each other, so the emulsion showed good salt resistance. This is because the adsorption layer formed by the protein-polysaccharide composite particles on the interface is thicker and has greater steric hindrance, which effectively inhibits the flocculation and aggregation of droplets and improves the ionic stability of the emulsion.

[0149] Based on the above research results, it can be inferred that steric repulsion plays a major role in the process of LZ-PGA composite particles stabilizing emulsions.

[0150] Experiment 11, emulsion storage stability test: The Pickering emulsion loaded with β-carotene alcohol was placed in a constant temperature and humidity chamber at 37°C or 55°C for 12 days. The retention rate of β-carotene (the effect on the β-carotene content in the Pickering emulsion loaded with β-carotene alcohol) and the average particle size of the emulsion droplets were measured on the 0th, 3rd, 6th, 9th and 12th days. The test results are shown in Figure 11. Figures 13-16 As shown in the figure, different capital letters (A, B, C...) indicate that there are significant differences between different groups at the same time (P<0.05). Different lowercase letters (a, b, c...) indicate that there are significant differences at different times in the same group (P<0.05).

[0151] in Fig.13 is the average particle size after storage at 37°C; Fig.14 is the average particle size after storage at 55°C; Fig.15 is the retention rate after storage at 37°C; Fig.16 is the retention rate after storage at 55°C;

[0152] from Fig.13It can be seen that after storage at 37℃ for 12 days, the droplet size of all emulsions showed different degrees of change. The droplet size of LZ-PGA-20% increased from 14.46±0.69μm to 27.38±1.06μm, that of LZ-PGA-30% increased from 13.61±0.42μm to 21.49±0.69μm, and that of LZ-PGA-40% increased from 12.80±0.19 The LZ-PGA-50% increased from 12.17±0.14μm to 17.36±0.90μm, the LZ-PGA-60% increased from 11.24±0.14μm to 18.38±1.43μm, and the LZ-PGA-70% increased from 15.46±0.40μm to 26.90±2.99μm. During storage, flocculation and coalescence occurred continuously between the droplets, resulting in the formation of larger droplets and reduced emulsion stability.

[0153] from Fig.14 It can be seen that the droplet size of all emulsions changes greatly with the increase of storage time. After storage for 12 days, the droplet size of LZ-PGA-20% emulsion increases to 56.31±1.82μm, showing obvious instability. As the oil phase content increases, the particle size change of the Pickering emulsion loaded with β-carotene alcoholization decreases. As the oil phase content increases, the droplet density increases. The tightly packed droplets are conducive to the formation of a dense network structure, so it is not easy to demulsify and aggregate during high-temperature storage, forming a Pickering emulsion loaded with β-carotene alcoholization with long-term stability. The surface negative charge of LZ-PGA-50% and 60% droplets is relatively high, and the strong electrostatic repulsion can inhibit the interaction between droplets and improve the stability of the emulsion. However, further increasing the oil phase content (70% (v / v)) will reduce the storage stability of the emulsion.

[0154] from Fig.15 It can be seen that after storage at 37℃ for 12 days, the retention rates of β-carotene in LZ-PGA-50% and LZ-PGA-60% are still 90.98±1.26% and 90.54±0.91%. This result shows that the Pickering emulsion loaded with β-carotene alcohol has a good protective effect on β-carotene (37℃). This is because the LZ-PGA composite particles form a dense interfacial film that effectively isolates the oxidant. At the same time, the increase in the oil phase content is conducive to enhancing the strength of the emulsion network structure, thereby improving the physical stability of the emulsion and the protective effect on β-carotene.

[0155] from Fig.16It can be seen that after storage at 55°C for 12 days, the retention rate of β-carotene in all samples was lower than that at 37°C. Higher storage temperature accelerates the isomerization and oxidative degradation of β-carotene. However, the emulsions obtained with different oil phase contents have different degrees of β-carotene loss. As the oil phase content increases (20%-70% (v / v)), the retention rates of β-carotene are 47.53±2.70%, 53.43±1.00%, 73.66±1.51%, 86.09±1.70%, 87.16±1.18%, and 63.67±0.82%, respectively. This is consistent with the stability of the emulsion. The loss of β-carotene in the Pickering emulsion loaded with β-carotene alcoholization under low oil phase is large because of its low viscosity and network structure strength. During high-temperature storage, the droplets tend to flocculate, aggregate, and even phase separate, which increases the possibility of interaction between β-carotene and pro-oxidants and reduces the storage stability of β-carotene.

[0156] Experiment 12, emulsion β-carotene bioaccessibility test:

[0157] 1. Simulation of in vitro digestion process: This experiment uses a two-stage gastrointestinal model to simulate the human gastrointestinal tract (GIT) conditions.

[0158] Gastric phase: The β-carotene-loaded Pickering emulsion was mixed with an equal volume of simulated gastric fluid (SGF) containing 3.2 mg / mL pepsin and 2 mg / mL NaCl, and the pH of the simulated gastric fluid was adjusted to 2.0 using 0.1 M HCl. The mixture was placed in a light-proof water bath shaker (37°C, 150 rpm) and stirred for 2 h. The mixture was then adjusted to pH 7.0 to terminate the digestion of SGF.

[0159] Small intestinal phase: The above gastric phase digestion fluid was mixed with simulated intestinal fluid (SIF) in equal volumes. SIF consisted of 12 mg / mL bile salt, 2 mg / mL trypsin, 3.2 mg / mL lipase, 8.8 mg / mL NaCl, 6.8 mg / mL KH 2 PO 4 Composition. Adjust the pH of the simulated intestinal fluid to 7.0. Place the mixed sample in a light-proof water bath shaker (37°C, 150 rpm) and stir for 4 hours to simulate small intestinal digestion.

[0160] 2. Bioaccessibility of β-carotene: After digestion of gastrointestinal fluid, the digestive fluid was centrifuged at 18000 rpm and 4°C for 60 min, the micellar phase containing β-carotene was collected, the β-carotene content was determined, and the bioaccessibility was calculated as shown in formula (7):

[0161]

[0162] In formula (7), C micelle refers to the β-carotene content in the micelle phase, and C emulsion refers to the β-carotene content in the initial emulsion.

[0163] The bioaccessibility of β-carotene refers to the part of the ingested β-carotene that is released into the gastrointestinal cavity and dissolved in the mixed micelles that are available for absorption. In the simulated gastrointestinal digestion process, the emulsion droplets stabilized by LZ-PGA composite particles are hydrolyzed by proteases and lipases, releasing β-carotene while producing monoacylglycerol and free fatty acids. These lipid digestion products combine with bile salts to form mixed micelles, which can dissolve β-carotene and pass through the mucosal layer into the small intestinal epithelial cells. After these micelles are absorbed by cells, they undergo structural reorganization and form chylomicrons together with β-carotene, which enter the blood, tissues, and organs through lymphatic transport, thereby exerting biological activity. Therefore, improving the bioaccessibility of β-carotene is conducive to enhancing the absorption and utilization of β-carotene by the human body.

[0164] The bioaccessibility test results of β-carotene in Pickering emulsions loaded with β-carotene alcohol with different oil content are shown in Figure 2. Fig.17 As shown, different letters a, b, c, d and e indicate significant differences (P<0.05). The study showed that, unlike the MCT control (4.51±0.28%), the Pickering emulsion stably loaded with β-carotene effectively improved the bioaccessibility of β-carotene. This is because the specific surface area formed by the MCT control is small, which reduces the contact with bile salts and lipase, and is not conducive to micelle formation. When the oil phase content gradually increases (20%-50% (v / v)), the bioaccessibility of β-carotene increases from 9.79±0.18% to 16.11±0.26%. This is because the droplet size is an important factor affecting the bioaccessibility of the active ingredient. As the oil phase content increases, the emulsion droplet size decreases. Smaller droplets have a larger specific surface area, are easily digested by proteases and lipases, and can produce more micelles, which is beneficial for delivering β-carotene to intestinal epithelial cells.

[0165] The above examples of the present invention respectively explore the effects of different oil phase contents (20%-70% (v / v)) on the physicochemical properties, rheological properties, physicochemical stability, microstructure and bioaccessibility of the β-carotene-loaded Pickering emulsions of LF-Zein-PGA composite particles loaded with β-carotene alcohol. In summary, the test results show that:

[0166] (1) Based on the particle size, ζ-potential, physical stability and rheological properties of the emulsion, the optimal oil phase content for preparing a stable β-carotene-loaded alcoholized Pickering emulsion was 60% (v / v).

[0167] (2) With the increase of oil phase, the interaction between droplets and particles is enhanced, which prompts LZ-PGA composite particles to form a dense network structure between adjacent droplets, inhibiting droplet flocculation and coalescence. In addition, the high packing density of droplets increases the viscosity of the system and restricts the movement of droplets. Therefore, the thermal stability, ionic stability and storage stability of the Pickering emulsion loaded with β-carotene alcoholization are improved.

[0168] (3) The LZ-PGA composite particles are firmly and irreversibly adsorbed on the oil-water interface, forming a dense interface layer and providing sufficient steric hindrance and electrostatic repulsion, which is beneficial to improving the embedding rate, light and thermal stability of β-carotene. At the same time, the high viscosity emulsion system effectively inhibits the diffusion of pro-oxidants into the oil phase, thereby improving the storage stability of β-carotene.

[0169] (4) Higher oil content and smaller droplet size are beneficial to improving the bioaccessibility of β-carotene.

[0170] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a β-carotene-loaded alcoholized Pickering emulsion, characterized in that: The following steps are involved: S10: mixing the alcohol-soluble protein solution with the milk protein solution, adjusting the pH value, and preparing a milk protein-alcohol-soluble protein composite nanoparticle solution; S20: alcoholizing the polysaccharide solution, adjusting the pH value, and then mixing the polysaccharide solution with the milk protein-alcohol-soluble protein composite nanoparticle solution to obtain a protein-polysaccharide composite particle solution; S30: First, an oil phase solution of β-carotene is prepared, and then the oil phase solution and the protein-polysaccharide composite particle solution are subjected to shear homogenization treatment to obtain a Pickering emulsion loaded with alcoholized β-carotene.

2. The method for preparing the β-carotene-loaded alcoholized Pickering emulsion according to claim 1, characterized in that: The alcohol-soluble protein in step S10 is at least one of zein, wheat alcohol-soluble protein and kafirin; The solvent of the alcohol-soluble protein solution is an ethanol aqueous solution with an ethanol volume fraction of 55% to 90%; In the alcohol-soluble protein solution, the ratio of alcohol-soluble protein to solvent is 1 g: 50-150 mL.

3. The method for preparing the alcoholized Pickering emulsion loaded with β-carotene according to claim 1, characterized in that: The milk protein in step S10 is at least one of lactoferrin, lactoglobulin and α-lactalbumin; The milk protein solution includes milk protein and water, and the ratio of the milk protein to water is 1g:200-400mL.

4. The method for preparing the alcoholized Pickering emulsion loaded with β-carotene according to claim 1, characterized in that: Step S10: The method for preparing the milk protein-alcohol-soluble protein composite nanoparticle solution comprises the following steps: dropping the alcohol-soluble protein solution into the milk protein solution, stirring at a speed of 600 rpm to 700 rpm for 20 min to 60 min, adjusting the pH value, and obtaining the milk protein-alcohol-soluble protein composite nanoparticle solution; The pH value of the milk protein-prolamin composite nanoparticle solution is 4.0-5.0, and the volume concentration of ethanol is not higher than 15%; The average particle size of the milk protein-prolamin composite nanoparticles is 100 nm to 120 nm; In the milk protein-alcohol-soluble protein composite nanoparticles, the mass ratio of milk protein to alcohol-soluble protein is (1-2): (1-3).

5. The method for preparing the alcoholized Pickering emulsion loaded with β-carotene according to claim 1, characterized in that: The polysaccharide in step S20 is at least one of propylene glycol alginate (PGA), high methoxy pectin, and hyaluronic acid; The polysaccharide solution is composed of polysaccharide and water, and the ratio of polysaccharide to water is 1g: (80-90)mL; The alcoholization treatment step is: dropwise adding anhydrous ethanol into the polysaccharide solution, stirring for 10 to 12 hours for alcoholization treatment; the volume ratio of the polysaccharide solution to anhydrous ethanol is 80 to 90:10 to 20. The reagent used to adjust the pH value is an alkaline or acidic solution; the pH value is 4.0-5.

0.

6. The method for preparing the alcoholized Pickering emulsion loaded with β-carotene according to claim 1, characterized in that: In the protein-polysaccharide composite particle solution in step S20, the mass ratio of protein to polysaccharide is 1-2:1-3. The pH value of the solution of the protein-polysaccharide composite particles is 4.0-5.0, and the volume concentration of ethanol is not higher than 15%.

7. The method for preparing the alcoholized Pickering emulsion loaded with β-carotene according to claim 1, characterized in that: The method for preparing the oil phase solution of β-carotene in step S30 comprises the following steps: dispersing the β-carotene oil suspension in the oil phase material to form the oil phase solution of β-carotene; The concentration of β-carotene in the β-carotene oil suspension is not less than 30%; The oil phase material is at least one of medium chain triglycerides, sunflower oil, corn oil and soybean oil.

8. The method for preparing the alcoholized Pickering emulsion loaded with β-carotene according to claim 1, characterized in that: Step S30 The method for preparing the alcoholized Pickering emulsion loaded with β-carotene comprises the following steps: adding the oil phase solution of β-carotene to the solution of protein-polysaccharide composite particles, and performing high-speed shear mixing at a rotation speed of 13000rpm to 15000rpm for 3min to 10min to obtain the alcoholized Pickering emulsion loaded with β-carotene; the volume ratio of the oil phase solution of β-carotene to the solution of protein-polysaccharide composite particles is 20%-70%:30% to 80%; The pH value of the alcoholized Pickering emulsion loaded with beta-carotene is 4.0-5.0, and the ethanol concentration is not higher than 15%.

9. A Pickering emulsion loaded with alcoholized β-carotene, characterized in that: Prepared according to the method according to any one of claims 1 to 8; In the alcoholized Pickering emulsion loaded with β-carotene, the mass concentration of β-carotene is 0.4% to 0.6%; The volume fraction of the oil phase solution in the alcoholized Pickering emulsion loaded with beta-carotene is 20% to 70%.

10. The use of the alcoholized Pickering emulsion loaded with β-carotene according to claim 9, characterized in that: The Pickering emulsion loaded with beta-carotene alcohol is used in beverages, functional foods and medicines.

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