Preparation method and application of protein-based lutein nanoparticles

Abstract

The invention discloses a method for pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein and protein-based lutein nanoparticles and applications. The preparation method comprises: adjusting the pH of the protein dispersion to alkaline, fully stirring, and dripping the lutein-anhydrous ethanol solution into the alkaline protein solution; during the process of evaporating and removing the ethanol, the unfolded alkaline protein is folded back so that the lutein is encapsulated in the protein hydrophobic cavity; during the process of adjusting the pH to neutral, the unfolded protein is folded back and rearranged for the second time, and the lutein undergoes a second encapsulation, and finally forms a densely structured protein-based lutein nanoparticles. This method has simple equipment, simple steps, safety and greenness, easy-to-obtain raw materials, and is economical and efficient; the prepared protein-based lutein nanoparticles significantly improve the water dispersibility, thermal stability, light stability, storage stability, digestion stability and bioavailability of lutein; therefore, it has broad development prospects in industries such as pigments, feed, food and medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-encapsulation of food bioactive substances, and specifically relates to a method for pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein, and protein-based lutein nanoparticles and applications. Background Art

[0002] Lutein and its isomer zeaxanthin are a class of oxygen-containing carotenoids that are not provitamin A. They contain many unsaturated double bonds in their structure and have excellent antioxidant properties. They can prevent age-related macular degeneration and cataracts through antioxidant and blue wave filtering effects. Like most hydrophobic bioactive substances, lutein is insoluble in water, soluble in organic solvents such as ethanol, ethyl acetate, and dichloromethane, extremely sensitive to light and heat, and have low bioavailability, which severely limits its application in the food field.

[0003] In order to solve the above problems, lutein is usually encapsulated by microcapsules, nanoparticles and emulsions at home and abroad, and ethanol is often used as a solvent to introduce lutein. Zhao et al. (Physicochemical Properties of Lutein-Loaded Microcapsules and Their Uptake via Caco-2 Monolayers [J]. Molecules, 2018, 23 (7): 1805.) dripped lutein-ethanol solution into sodium caseinate-ethanol solution after heat treatment at 60°C for 5 minutes, dispersed it at 10000 rpm for 4 minutes, and then passed through a 100-mesh sieve to remove insoluble large particles, and then spray-dried it to obtain microcapsules with an encapsulation rate of 89.95%. However, after dark treatment at 25°C for 5 days, the lutein retention rate was only 79.83%, and the microcapsule particles were generally large, and the biological efficacy was not high. Dai et al. (Stable Nanoparticles Prepared by Heating Electrostatic Complexes of Whey Protein Isolate-Dextran Conjugate and Chondroitin Sulfate [J]. Journal of Agricultural and Food Chemistry, 2015, 63 (16): 4179-4189) added lutein-ethanol solution to whey protein isolate-dextran-chondroitin sulfate ternary nanoparticle solution and used the cavitation effect of ultrasound to promote the migration of lutein molecules into the nanoparticles. The encapsulation efficiency reached 94.07%, and the average particle size of the lutein-loaded nanoparticles was 150 nm. However, the preparation process involved heating, ice bath, and ultrasonic treatment, and the steps were complicated and the equipment was expensive.

[0004] Patent CN 113143885 A dissolves lutein in 95% ethanol solution and uses supercritical emulsification technology to prepare lutein microparticles with a particle size of 100-1000nm. Under optimal conditions, the average particle size can reach below 400nm, but supercritical emulsification technology cannot currently be used in large-scale industrial applications. Patent CN 114468295 A mixes equal volumes of phycocyanin solution synthesized based on the Mannich reaction with lutein ethanol solution and stirs for 24h, then evaporates ethanol to obtain nanoparticles loaded with lutein, with a size of 370±50nm and an embedding rate of only 74.42%. The particles are large and the embedding rate is not high. Patent CN 111938157 A adds lutein-anhydrous ethanol solution to a chickpea protein isolate-stevioside composite system, uses a probe-type ultrasonic crusher for 6-10min ultrasonic treatment, and then evaporates ethanol to obtain a lutein nanoemulsion with an average particle size of 195.1nm. The equipment is also expensive.

[0005] Looking at the particles prepared by lutein encapsulation technology using ethanol as solvent, there are problems such as large particle size, low encapsulation efficiency and low retention rate. This is because ethanol, as a polar organic solvent, can change the natural conformation of protein molecules, causing the protein to unfold and expose a large number of hydrophobic groups. Hydrophobic active substances dissolved in ethanol can bind to proteins through hydrophobic interactions to form complexes; but at the same time, ethanol also promotes protein aggregation. On the one hand, the final particle size is much larger than that of natural proteins. On the other hand, the encapsulation efficiency of hydrophobic active substances decreases with the increase of ethanol concentration. Liu et al. (Novel soy beta-conglycinin nanoparticles by ethanol-assisted disassembly and reassembly: Outstanding nanocarriers for hydrophobic nutraceuticals [J]. Food Hydrocolloids, 2019, 91: 246-255.) revealed this mechanism. A common solution is to use ultrasound, high pressure and microfluidics to crush, extrude and shear the particles to reduce the size of the particles and promote the migration of lutein molecules into the particles to improve the encapsulation efficiency. However, these methods are accompanied by the defects of expensive equipment and high cost.

[0006] Therefore, there is no patent published on the lutein encapsulation technology with simple equipment, convenient method, efficient encapsulation and small size.

[0007] Patent CN 113080452 A uses the characteristics of curcumin phenolic hydroxyl ionization under alkaline pH conditions to increase water solubility and protein unfolding or even dissociation into subunits, encapsulates curcumin inside the protein, achieves efficient loading of curcumin, and improves the water solubility, stability and bioavailability of curcumin. Pan et al. (pH-driven encapsulation of curcumin in self-assembled casein nanoparticles for enhanced dispersibility and bioactivity [J]. Soft Matter, 2014, 10 (35): 6820-6830.) found that the recombinant particle size formed after the sodium caseinate treated under pH = 12 was adjusted to pH 7 was significantly smaller than the protein particle size that was not treated with alkali. It can be seen that pH-induced protein self-assembly can not only achieve efficient embedding of hydrophobic active substances, but also prepare recombinant particles of smaller size, and the preparation process is simple, safe, low-cost and low-energy.

[0008] In summary, the present invention proposes a method for pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein, the principle of which is as follows: under alkaline pH conditions, the protein is far away from the isoelectric point, and the electrostatic repulsion between alkaline protein molecules increases with the increase of pH, resulting in protein unfolding or even dissociation into subunits, and the hydrophobic groups inside the protein molecules are exposed to the hydrophilic environment. At this time, the self-assembled units become significantly smaller, and the protein dispersion becomes clear and transparent; ethanol acts as an inducer to promote the further unfolding of the alkaline dissociated protein, and the exposure degree of the hydrophobic groups increases accordingly, and the hydrophobic interaction between the alkaline dissociated proteins is also enhanced to form loose and larger aggregates. At the same time, ethanol acts as a solvent to introduce lutein, Lutein is able to combine with the fully exposed hydrophobic groups of alkaline dissociated proteins through hydrophobic interactions to form a complex, and the solution becomes turbid; in the process of removing ethanol, the fully unfolded alkaline dissociated proteins begin to fold back, and lutein is encapsulated inside the subunit or bound to the surface of the protein molecule for the first time, the loose structure becomes convergent, and the originally aggregated alkaline dissociated protein particles return to the state of separation from each other when no ethanol is added, the size of the particles is significantly reduced, and the solution becomes clear again; in the process of adjusting the pH back to neutral, the unfolded or dissociated proteins continue to fold back and reorganize, and lutein undergoes a second encapsulation, and the loose structure becomes dense, eventually forming protein-based lutein nanoparticles similar to the natural protein structure.

[0009] The method has simple equipment, simple process and low cost. The prepared protein-based lutein nanoparticles can achieve efficient encapsulation of lutein and improve the water solubility, stability and bioavailability of lutein. It is suitable for large-scale industrial production and processing and has certain application prospects in the fields of pigments, feed, food, medicine and so on. Summary of the invention

[0010] In view of the shortcomings and deficiencies of the prior art, the first aspect of the present invention aims to provide a method for pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein. The method utilizes the unfolding or dissociation of proteins into subunits under alkaline pH conditions to expose a large number of hydrophobic groups inside the proteins to the protein surface; then introduces a lutein-anhydrous ethanol solution, ethanol is used as an inducer to promote protein unfolding and aggregation, and lutein is introduced as a solvent to form a complex with the protein through hydrophobic interaction; ethanol is removed by evaporation to make the unfolded alkaline dissociated protein fold back for the first time, and lutein undergoes the first embedding, while electrostatic repulsion is used to weaken the property of ethanol to promote protein particle aggregation, so that the particle size is significantly reduced; finally, the pH is adjusted to allow the unfolded or dissociated protein to fold back and reorganize for the second time, and lutein also undergoes the second embedding, forming protein-based lutein nanoparticles similar to the natural protein structure.

[0011] The purpose of the second aspect of the present invention is to prepare a type of protein-based lutein nanoparticles that are easily dispersible, highly water-soluble, have a high embedding rate, high stability and high bioavailability through the above method. The protein-based lutein nanoparticles in the present invention are prepared from protein and lutein dissolved in anhydrous ethanol, and the protein is an alkaline protein.

[0012] The third aspect of the present invention aims to realize the commercial application of the above-mentioned protein-based lutein nanoparticles.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] (1) The pH of the protein dispersion is adjusted to an alkaline state, and the solution is stirred thoroughly to allow the protein to unfold or dissociate into subunits, the particle size is significantly reduced, and the solution becomes clear and transparent; then a certain volume ratio of lutein-anhydrous ethanol solution is added, and ethanol further unfolds the alkaline dissociated protein through alcohol-water interaction. The solution is stirred thoroughly to allow lutein to form a stable complex with the protein through hydrophobic interaction. At the same time, the hydrophobic interaction between the alkaline dissociated protein particles is enhanced to form larger, loose aggregates, and the solution becomes turbid.

[0015] (2) The ethanol in the above-mentioned mixed solution is removed by rotary evaporation, and the unfolded alkaline dissociated protein gradually folds back, the loose structure begins to converge, the lutein undergoes the first embedding, and the aggregated alkaline protein particles return to the state of being separated from each other when no ethanol is added. The particle size is significantly reduced, and the solution becomes clear again.

[0016] (3) The pH of the mixed solution is adjusted to neutral. The subunits that were originally separated from each other due to electrostatic repulsion are rearranged, the protein is folded back and reorganized for the second time, and the lutein undergoes a second embedding. The loose structure becomes dense, and water with the same volume as the ethanol removed by evaporation is added to obtain protein-based lutein nanoparticles.

[0017] Furthermore, the protein in step (1) is at least one of whey protein, serum protein, ovalbumin, lysozyme, β-lactoglobulin, sodium caseinate, bean 7S globulin, bean 11S globulin, soy protein isolate and pea protein isolate; preferably, the protein is at least one of soy protein isolate, sodium caseinate and whey protein isolate.

[0018] Furthermore, the protein concentration in step (1) is 1-60 mg / mL; preferably, the protein concentration is 5-30 mg / mL; more preferably, the protein concentration is 5-20 mg / mL.

[0019] Furthermore, the pH range of the protein regulating dispersion in step (1) is 10-12, and the stirring time is 30-120 min; preferably, the pH range of the protein regulating dispersion is 11-12, and the stirring time is 60-120 min.

[0020] Furthermore, the concentration of the lutein-anhydrous ethanol solution in step (1) is 0.5-1.5 mg / mL.

[0021] Furthermore, the volume ratio of the lutein-anhydrous ethanol solution added in step (1) is 10%-70% of the mixed solution, and the stirring time is 30-120 minutes; preferably, the volume ratio of the lutein-anhydrous ethanol solution added is 40%-60% of the mixed solution, and the stirring time is 60-120 minutes, and more preferably, the volume ratio of the lutein-anhydrous ethanol added is 40%-50% of the mixed solution, and the stirring time is 60-120 minutes.

[0022] Furthermore, in step (2), the vacuum degree of rotary evaporation is 0-0.1 MPa, the heating temperature is 35-45° C., and the rotation speed is 80-120 rpm.

[0023] Furthermore, in step (3), the pH is adjusted to 6.8-8.0; preferably, in step (3), the pH is adjusted to 6.8-7.2.

[0024] The present invention provides protein-based lutein nanoparticles prepared by the method of pH-coordinated ethanol-induced protein assembly and nano-encapsulation of lutein.

[0025] The present invention provides an application of protein-based lutein nanoparticles prepared by a method for pH-coordinated ethanol-induced protein assembly of nano-encapsulated lutein in the preparation of products. Further, the product is any one of (1)-(4): (1) a pigment, which can be used as a colorant in the production of cakes, candies and other foods to give them an orange-yellow appearance; (2) a food, which can be directly used as a dietary supplement, or as an auxiliary material to produce lutein-fortified foods such as functional beverages; (3) a medicine, which can be used as an auxiliary material to produce medicines for relieving eye fatigue; (4) a feed, which can be used as chicken feed to enrich eggs with lutein.

[0026] The principle of the present invention is that lutein, as a type of oxygen-containing carotenoid that is not provitamin A, is insoluble in water but soluble in ethanol. Lutein molecules have multiple hydrophobic sites that can bind to proteins through hydrophobic interactions. The hydrophobic sites of natural proteins are located inside the molecules, and the structure of proteins is maintained by hydrogen bonds, hydrophobic interactions and electrostatic forces. Studies have found that both ethanol and pH can change the natural structure of proteins, causing them to unfold or dissociate. In particular, under alkaline pH conditions, some proteins can dissociate into subunits. Therefore, the hydrophobic groups originally buried inside are exposed to a large number of protein molecule surfaces. In the presence of ethanol, the alkaline-dissociated proteins, on the one hand, further unfold and expose more hydrophobic sites, causing particle aggregation, and on the other hand, form complexes with lutein through hydrophobic interactions. During the removal of ethanol, the loosely structured alkaline-dissociated proteins undergo the first folding, and part of the lutein is embedded inside the alkaline-dissociated proteins, and the aggregated alkaline-dissociated protein particles return to the state of separation from each other when no ethanol is added. In the process of adjusting the pH back to neutral, the unfolded or dissociated protein molecules undergo a second folding and reorganization, and the lutein located on the protein surface can be further embedded, and finally new particles with a structure similar to that of natural proteins are formed.

[0027] In summary, protein-based lutein nanoparticles prepared by pH-synergistic ethanol-induced protein assembly nano-encapsulation of lutein can achieve efficient encapsulation of lutein, solve the shortcomings of lutein's poor water dispersibility, poor stability and low bioavailability, and improve the stability of lutein products in the food processing process and ultimately its effective absorption in the human body.

[0028] The preparation process of the invention is simple, safe, low-cost, low-energy-consuming and can achieve efficient encapsulation, and can be applied to various occasions.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention proposes for the first time a method for pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein, which involves simple equipment and simple steps, is safe, green, low-cost, and low-energy, suitable for large-scale industrial production and processing, and the production process and materials meet food safety requirements. It is suitable for the pigment, food, medicine, and feed industries, and has broad application prospects.

[0031] (2) The method of pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein proposed in the present invention utilizes the characteristics of ethanol and pH that can change the natural structure of proteins, causing the proteins to unfold, dissociate and reassemble twice, thereby achieving efficient encapsulation of lutein, and utilizing electrostatic repulsion to weaken the characteristics of ethanol promoting protein aggregation, ultimately forming protein-based lutein nanoparticles with compact structure and small size.

[0032] (3) The method of pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein proposed in the present invention can regulate the size of the formed protein-based lutein nanoparticles by changing the pH, protein concentration, ethanol concentration, and the time of pH-coordinated ethanol induction.

[0033] (4) The protein-based lutein nanoparticles prepared by the present invention using pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein can improve the water dispersibility, thermal stability, light stability, storage stability and bioavailability of lutein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a picture of the appearance of the solution of protein-based lutein nanoparticles prepared in Example 1-4;

[0035] Figure 2 is a particle size distribution diagram of protein-based lutein nanoparticles prepared from different proteins in Example 1;

[0036] Figure 3 This is a powder appearance diagram of the solution-based protein-based lutein nanoparticles prepared from different proteins in Example 1 after vacuum freeze-drying;

[0037] Figure 4 is a particle size distribution diagram of protein-based lutein nanoparticles prepared under different pH conditions in Example 2;

[0038] Figure 5 is a particle size distribution diagram of protein-based lutein nanoparticles prepared under different protein concentration conditions in Example 3;

[0039] Figure 6 is a graph showing the lutein retention rate of the solution-based protein-based lutein nanoparticles and the lutein-anhydrous ethanol solution prepared under different protein concentration conditions in Example 3 after heat treatment at 80°C;

[0040] Figure 7 is a graph showing the lutein retention rate of the solution-based protein-based lutein nanoparticles and the lutein-anhydrous ethanol solution prepared under different protein concentration conditions in Example 3 after being irradiated with ultraviolet light;

[0041] Figure 8 is a particle size distribution diagram of protein-based lutein nanoparticles prepared under different ethanol concentration conditions in Example 4 and Comparative Example 1;

[0042] Fig. 9 is a transmission scanning electron microscopic image of the protein-based lutein nanoparticles prepared when the ethanol concentration in Example 4 and Comparative Example 1 is 40%;

[0043] Fig.10 is a graph showing the lutein retention rate of the protein-based lutein nanoparticles in the form of solutions and the lutein-anhydrous ethanol solution prepared under different ethanol concentration conditions in Example 4 after being stored at 35° C. in the dark;

[0044] Fig.11 is a graph showing the retention rate of lutein in the form of a solution of protein-based lutein nanoparticles and lutein-anhydrous ethanol solution prepared under different ethanol concentration conditions in Example 4 after simulated gastric digestion and / or small intestinal digestion;

[0045] Fig.12 It is a statistical graph of the retention rate and bioavailability of lutein of the protein-based lutein nanoparticles and lutein-anhydrous ethanol solutions in the form of solutions prepared under different ethanol concentrations in Example 4 after simulated gastric digestion and / or small intestinal digestion. DETAILED DESCRIPTION

[0046] The present invention will be further described below by means of specific implementation examples.

[0047] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0048] The experimental methods without specific conditions in the following examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The materials and reagents used in the present examples are commercially available unless otherwise specified.

[0049] Example 1

[0050] Method 1: A method for preparing protein-based lutein nanoparticles by pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein, comprising the following steps:

[0051] (1) Disperse whey protein isolate in water to an initial concentration of 16.7 mg / mL, adjust the pH of the dispersion to 12.0 with 4M NaOH solution, and continue magnetic stirring at 1000 rpm / min for 120 min at room temperature to fully unfold or dissociate the protein, and the solution becomes clear and transparent. Drop a 1.5 mg / mL lutein-anhydrous ethanol solution into the alkaline dissociated protein dispersion to make the anhydrous ethanol concentration reach 40% (v / v) of the mixed solution, maintain the pH at 12.0, and the final concentration of the alkaline dissociated protein is 10 mg / mL. Continue magnetic stirring at 1000 rpm / min for 30 min at room temperature. Under the action of ethanol, the alkaline dissociated protein further unfolds to fully expose the hydrophobic group, on the one hand, forms a complex with lutein through hydrophobic interaction, and on the other hand, aggregates into loose large particles, and the solution becomes turbid.

[0052] (2) The ethanol in the mixed solution of step (1) is removed by a rotary evaporator, the unfolded alkaline dissociated protein gradually folds back, the loose structure begins to converge, the lutein undergoes the first embedding, the aggregated alkaline protein particles return to the state of being separated from each other when no ethanol is added, the particle size decreases, and the solution becomes clear again. The vacuum degree of the rotary evaporation is 0.05 MPa, the heating temperature is 40° C., and the rotation speed is 100 rpm.

[0053] (3) The pH of the mixed solution obtained in step (2) is adjusted to 7.0 with a 2M hydrochloric acid solution. The subunits separated from each other by electrostatic repulsion are rearranged, the protein is folded back for the second time, and the lutein undergoes a second embedding. The loose structure becomes dense, and water with the same volume as the ethanol removed by evaporation is added to make the final protein concentration 10 mg / mL, thereby obtaining the protein-based lutein nanoparticles.

[0054] Method 2: Protein-based lutein nanoparticles prepared by a pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein. The preparation method is the same as method 1 in this embodiment, except that the protein in step (1) is sodium caseinate.

[0055] Method 3: Protein-based lutein nanoparticles prepared by a pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein. The preparation method is the same as method 1 in this embodiment, except that the protein in step (1) is soy protein isolate.

[0056] Example 2

[0057] Method 1: A method for preparing protein-based lutein nanoparticles by pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein, comprising the following steps:

[0058] (1) Soy protein isolate was dispersed in water to an initial concentration of 16.7 mg / mL, and the pH of the dispersion was adjusted to 10.0 with 4M NaOH solution. The dispersion was continuously stirred with magnetic force at 1000 rpm / min for 120 min at room temperature to fully unfold or dissociate the protein, and the solution became clear and transparent. A 1.5 mg / mL lutein-anhydrous ethanol solution was dropped into the alkaline dissociated protein dispersion to make the anhydrous ethanol concentration reach 40% (v / v) of the mixed solution, and the pH was maintained at 10.0 during the period. The final concentration of the alkaline dissociated protein was 10.0 mg / mL, and the dispersion was continuously stirred with magnetic force at 1000 rpm / min for 60 min at room temperature. Under the action of ethanol, the alkaline dissociated protein further unfolded to fully expose the hydrophobic group, on the one hand, forming a complex with lutein through hydrophobic interaction, and on the other hand, aggregating into loose large particles, and the solution became turbid.

[0059] (2) The ethanol in the mixed solution of step (1) is removed by a rotary evaporator, the unfolded alkaline dissociated protein gradually folds back, the loose structure begins to converge, the lutein undergoes the first embedding, the aggregated alkaline protein particles return to the state of being separated from each other when no ethanol is added, the particle size decreases, and the solution becomes clear again. The vacuum degree of the rotary evaporation is 0.05 MPa, the heating temperature is 40° C., and the rotation speed is 100 rpm.

[0060] (3) The pH of the mixed solution obtained in step (2) is adjusted to 7.0 with a 2M hydrochloric acid solution. The subunits separated from each other by electrostatic repulsion are rearranged, the protein is folded back for the second time, and the lutein undergoes a second embedding. The loose structure becomes dense, and water with the same volume as the ethanol removed by evaporation is added to make the final protein concentration 10.0 mg / mL, thereby obtaining the protein-based lutein nanoparticles.

[0061] Method 2: Protein-based lutein nanoparticles prepared by a pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein. The preparation method is the same as method 1 in this embodiment, except that the pH in step (1) is 11.0.

[0062] Method 3: Protein-based lutein nanoparticles prepared by a method of pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein. The preparation method is the same as method 1 in this embodiment, except that the pH in step (1) is 12.0.

[0063] Example 3

[0064] Method 1: A method for preparing protein-based lutein nanoparticles by pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein, comprising the following steps:

[0065] (1) Soy protein isolate was dispersed in water to an initial concentration of 8.3 mg / mL, and the pH of the dispersion was adjusted to 11.0 with 4M NaOH solution. The dispersion was continuously stirred with magnetic force at 1000 rpm / min for 120 min at room temperature to fully unfold or dissociate the protein, and the solution became clear and transparent. A 1.5 mg / mL lutein-anhydrous ethanol solution was dropped into the alkaline dissociated protein dispersion to make the anhydrous ethanol concentration reach 40% (v / v) of the mixed solution, during which the pH was maintained at 11.0. The final concentration of the alkaline dissociated protein was 5.0 mg / mL, and the dispersion was continuously stirred with magnetic force at 1000 rpm / min for 90 min at room temperature. Under the action of ethanol, the alkaline dissociated protein further unfolded to fully expose the hydrophobic groups, on the one hand, forming a complex with lutein through hydrophobic interaction, and on the other hand, aggregating into loose large particles, and the solution became turbid.

[0066] (2) The ethanol in the mixed solution of step (1) is removed by a rotary evaporator, the unfolded alkaline dissociated protein gradually folds back, the loose structure begins to converge, the lutein undergoes the first embedding, the aggregated alkaline protein particles return to the state of being separated from each other when no ethanol is added, the particle size decreases, and the solution becomes clear again. The vacuum degree of the rotary evaporation is 0.05 MPa, the heating temperature is 40° C., and the rotation speed is 100 rpm.

[0067] (3) The pH of the mixed solution obtained in step (2) is adjusted to 7.0 with a 2M hydrochloric acid solution. The subunits separated from each other by electrostatic repulsion are rearranged, the protein is folded back for the second time, and the lutein undergoes a second embedding. The loose structure becomes dense, and water with the same volume as the ethanol removed by evaporation is added to make the final protein concentration 5.0 mg / mL, thereby obtaining the protein-based lutein nanoparticles.

[0068] Method 2: Protein-based lutein nanoparticles prepared by a pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein. The preparation method is the same as method 1 in this embodiment, except that the initial protein concentration in step (1) is 20.8 mg / mL, and the final protein concentration in steps (1) and (3) is 12.5 mg / mL.

[0069] Method 3: Protein-based lutein nanoparticles prepared by a pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein. The preparation method is the same as method 1 in this embodiment, except that the initial protein concentration in step (1) is 33.3 mg / mL, and the final protein concentration in steps (1) and (3) is 20.0 mg / mL.

[0070] Method 4: Protein-based lutein nanoparticles prepared by a pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein. The preparation method is the same as method 1 in this embodiment, except that the initial protein concentration in step (1) is 41.7 mg / mL, and the final protein concentration in steps (1) and (3) is 25.0 mg / mL.

[0071] Example 4

[0072] Method 1: A method for preparing protein-based lutein nanoparticles by pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein, comprising the following steps:

[0073] (1) Soy protein isolate was dispersed in water to an initial concentration of 16.7 mg / mL, and the pH of the dispersion was adjusted to 12.0 with 4M NaOH solution. The dispersion was continuously stirred at 1000 rpm / min for 120 min at room temperature to fully unfold or dissociate the protein, and the solution became clear and transparent. A 1.5 mg / mL lutein-anhydrous ethanol solution was dropped into the alkaline dissociated protein dispersion to make the anhydrous ethanol concentration reach 40% (v / v) of the mixed solution, and the pH was maintained at 12.0. The final concentration of the alkaline dissociated protein was 10.0 mg / mL, and the dispersion was continuously stirred at 1000 rpm / min for 120 min at room temperature. Under the action of ethanol, the alkaline dissociated protein further unfolded to fully expose the hydrophobic group, on the one hand, forming a complex with lutein through hydrophobic interaction, and on the other hand, aggregating into loose large particles, and the solution became turbid.

[0074] (2) The ethanol in the mixed solution of step (1) is removed by a rotary evaporator, the unfolded alkaline dissociated protein gradually folds back, the loose structure begins to converge, the lutein undergoes the first embedding, the aggregated alkaline protein particles return to the state of being separated from each other when no ethanol is added, the particle size decreases, and the solution becomes clear again. The vacuum degree of the rotary evaporation is 0.05 MPa, the heating temperature is 40° C., and the rotation speed is 100 rpm.

[0075] (3) The pH of the mixed solution obtained in step (2) is adjusted to 7.0 with a 2M hydrochloric acid solution. The subunits separated from each other by electrostatic repulsion are rearranged, the protein is folded back for the second time, and the lutein undergoes a second embedding. The loose structure becomes dense, and water with the same volume as the ethanol removed by evaporation is added to make the final protein concentration 10.0 mg / mL, thereby obtaining the protein-based lutein nanoparticles.

[0076] Method 2: Protein-based lutein nanoparticles prepared by a pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein. The preparation method is the same as method 1 in this embodiment, except that the ethanol concentration in step (1) is 50% (v / v) of the mixed solution.

[0077] Method 3: Protein-based lutein nanoparticles prepared by a pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein. The preparation method is the same as method 1 in this embodiment, except that the ethanol concentration in step (1) is 60% (v / v) of the mixed solution.

[0078] Comparative Example 1

[0079] Method 1: An ethanol-induced preparation of protein-based lutein nanoparticles, comprising the following steps:

[0080] (1) Soy protein isolate was dispersed in water to an initial concentration of 16.7 mg / mL, and its pH was adjusted to 7.0 with 4M NaOH solution. The mixture was continuously stirred at 1000 rpm / min for 120 min at room temperature. A 1.5 mg / mL lutein-anhydrous ethanol solution was added dropwise to the protein dispersion to make the ethanol concentration reach 40% (v / v) of the mixture. The final protein concentration was 10.0 mg / mL. The mixture was continuously stirred at 1000 rpm / min for 120 min at room temperature. Under the action of ethanol, the protein gradually unfolded to expose the hydrophobic groups. On the one hand, it formed a complex with lutein through hydrophobic interaction, and on the other hand, it aggregated into loose large particles, and the solution became turbid.

[0081] (2) The ethanol in the mixed solution of step (1) is removed by a rotary evaporator, and the unfolded protein is gradually folded back to form a compact structure. The vacuum degree of the rotary evaporation is 0.05 MPa, the heating temperature is 40° C., and the rotation speed is 100 rpm. The pH is adjusted to 7.0 with a 2M hydrochloric acid solution, and water with the same volume as the ethanol removed by evaporation is added to make the final concentration of the soy protein isolate 10 mg / mL.

[0082] Method 2: A protein-based lutein nanoparticle prepared by ethanol induction, the preparation method is the same as method 1 in this comparative example, the only difference is that the ethanol concentration in step (1) is 50% (v / v) of the mixed solution.

[0083] Method 3: A protein-based lutein nanoparticle prepared by ethanol induction, the preparation method is the same as method 1 in this comparative example, the only difference is that the ethanol concentration in step (1) is 60% (v / v) of the mixed solution.

[0084] Effect Example

[0085] 1. Particle size, PDI and freeze-drying yield of protein-based lutein nanoparticles obtained in Example 1

[0086] The solution of protein-based lutein nanoparticles prepared by using whey protein isolate, sodium caseinate and soy protein isolate in Example 1 was diluted to a protein concentration of 1 mg / mL. The experiment was repeated three times. The hydrated particle size and PDI (Polymer dispersity index, which is a polymer dispersity index used to describe the molecular weight distribution of polymers) were measured using a Malvern nanoparticle size and potentiometric analyzer. The results are shown in Tables 1 and Figure 2 shown.

[0087] Figure 1 In the appearance diagram (a), it can be seen that the solution prepared by whey protein isolate has the highest turbidity. Figure 2 The particle size of whey protein isolate-based lutein nanoparticles showed a clear bimodal distribution, indicating that there were more protein associations and aggregations. On the one hand, a certain concentration of ethanol has the effect of promoting the aggregation of protein molecules; on the other hand, whey protein isolate has a relatively weak ability to bind to lutein through hydrophobic interactions due to the lack of sufficient hydrophobic sites in its structure, strong surface hydrophilicity, and weak subunit amphiphilicity. Proteins are more likely to bind to each other, so larger particles are easily formed. Soy protein isolate and sodium caseinate are easier to bind to lutein because of their good amphiphilic properties and more hydrophobic sites. Lutein is encapsulated inside the recombinant protein, and the average particle size of the recombinant particles is smaller. Figure 1 The solution prepared from soy protein isolate and sodium caseinate in (a) is clear and transparent. Although the particle size of soy protein isolate-based lutein nanoparticles is slightly larger than that of the particles prepared from sodium caseinate, the polydispersity index is the smallest, and soy protein isolate is more widely available and easier to obtain than sodium caseinate, so it is widely used in the embodiments of this patent.

[0088] The protein-based lutein nanoparticle solution prepared from different proteins in Example 1 was freeze-dried to obtain Figure 3 The light yellow powder shown. No lutein crystal particles were found in all the dried powders, which fully demonstrated that the lutein in the protein-based lutein nanoparticles was efficiently embedded and the lutein was stably bound to the protein.

[0089] The freeze-drying yield was calculated according to formula (1), where W2 is the mass of the particles obtained after drying, and W1 is the mass of the particles contained in the formula that cannot be evaporated (i.e., protein and lutein). The results are shown in Table 1. The yield of protein-based lutein nanoparticles after vacuum freeze drying is relatively high, all above 80%.

[0090]

[0091] Table 1 Average particle size, PDI and freeze-drying yield of the nanoparticles prepared in Example 1

[0092]

[0093] 2. Particle size, PDI, encapsulation efficiency and loading of protein-based lutein nanoparticles obtained in Example 2

[0094] The appearance of the protein-based lutein nanoparticles in the form of a solution obtained in Example 2 is shown in FIG. Figure 1 As shown in (b), the solutions are all clear and transparent. The hydrated particle size and PDI of the protein-based lutein nanoparticles prepared under different pH conditions in Example 2 are shown in Table 2. Under alkaline conditions, as the pH increases, the average particle size of the obtained nanoparticles gradually decreases. The average particle size of the nanoparticles prepared at pH = 10 is the largest, which is 157.5±9.0nm. When the pH is 11-12, the average particle size of the particles is relatively close, which are 134.2±5.4nm and 127.1±5.7nm, respectively. This is because the higher the pH in alkaline conditions, the higher the degree of protein unfolding and dissociation, the more sites that can undergo hydrophobic binding with lutein, and the recombinant particle structure formed is smaller and denser. Figure 4 It can also be seen that under alkaline conditions, the higher the pH, the greater the area proportion of the small particle size peak, and the particle size distribution graph moves toward smaller particle sizes as a whole.

[0095] The encapsulation efficiency and loading of the protein-based lutein nanoparticles in solution obtained in Example 2 were determined. The experiment was repeated three times and calculated according to formulas (2) and (3). The results are shown in Table 2. There is an obvious negative correlation between the encapsulation efficiency and the particle size. Under alkaline conditions, as the pH increases, the degree of protein unfolding and dissociation increases, and the number of sites that can hydrophobically bind to lutein increases; after the pH is adjusted back to 7, the more lutein is encapsulated in the protein, the higher the encapsulation efficiency, and under the same protein concentration conditions, the loading amount also increases accordingly.

[0096]

[0097] Table 2 Average particle size, PDI, encapsulation efficiency and loading of the nanoparticles prepared in Example 2

[0098]

[0099] 3. Particle size, PDI, thermal treatment stability and light treatment stability of protein-based lutein nanoparticles obtained in Example 3

[0100] The appearance of the protein-based lutein nanoparticles in the form of a solution prepared in Example 3 is as follows Figure 1 As shown in (c), the turbidity of the solution increases with the increase of protein concentration. The hydrated particle size and PDI of protein-based lutein nanoparticles prepared at different protein concentrations in Example 3 are shown in Tables 3 and Figure 5As shown. With the increase of protein concentration, the average particle size and PDI of protein-based lutein nanoparticles prepared by the method of pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein first decreased and then increased. When the protein concentration was 12.5 mg / mL, the average particle size and PDI were the smallest, 129.2±1.8 nm and 0.351±0.004, respectively. When the protein concentration was as high as 25.0 mg / mL, although the particle size formed by protein unfolding or dissociation at pH=12 was significantly reduced, the protein concentration increased from 25.0 mg / mL to 41.7 mg / mL after removing ethanol. During the process of adjusting the pH to 7, the probability of collision between protein molecules increased, making it easier to form large particles. Even if distilled water was added to restore the protein concentration to 25.0 mg / mL, the size of the self-assembled nanoparticles could not be changed. The average particle size of the protein-based lutein nanoparticles prepared at this protein concentration was 235.7±2.4 nm. When the protein concentration was as low as 5.0 mg / mL, the content of lutein required to be loaded per unit protein increased, which increased the degree of aggregation between proteins and the average particle size of the formed nanoparticles also increased accordingly, which was 151.8±3.6 nm.

[0101] Table 3 Average particle size and PDI of the nanoparticles prepared in Example 3

[0102]

[0103] 7 mL of the protein-based lutein nanoparticles prepared at different protein concentrations in Example 3 and 7 mL of 1 mg / mL lutein-anhydrous ethanol solution were respectively placed in an 80°C water bath for 4 h of heat treatment. The lutein content was measured at 0 h, 0.5 h, 1 h, 2 h, and 4 h. The experiment was repeated three times, and the lutein retention rate was calculated according to formula (4). The results are as follows: Figure 6 After heat treatment at 80°C for 4 hours, the retention rate of lutein in the protein-based lutein nanoparticles provided in this embodiment was above 80%, while the retention rate of free lutein under the same conditions was only 9.97±0.43%, indicating that the protein-based lutein nanoparticles provided in this embodiment can significantly improve the thermal stability of lutein.

[0104]

[0105] 7 mL of the protein-based lutein nanoparticle solution prepared at different protein concentrations in Example 3 and 7 mL of 1 mg / mL lutein-anhydrous ethanol solution were respectively irradiated under 254 nm and 365 nm ultraviolet light, and the lutein content was measured at 0 h, 2 h, 4 h, 8 h, and 24 h. The experiment was repeated three times, and the lutein retention rate was calculated according to formula (4). The results are as follows: Figure 7After 24 hours of ultraviolet light irradiation, the retention rate of lutein in the protein-based lutein nanoparticles provided in this embodiment is above 70%, while the retention rate of free lutein under the same conditions is only 1.58%, indicating that the protein-based lutein nanoparticles provided in this embodiment can significantly improve the light stability of lutein.

[0106] 4. Particle size, PDI, long-term storage stability, in vitro digestion stability and bioavailability of protein-based lutein nanoparticles obtained in Example 4

[0107] The hydrated particle size and PDI of protein-based lutein nanoparticles prepared at different ethanol concentrations in Example 4 and Comparative Example 1 are shown in Tables 4 and Figure 8 As shown, under the same ethanol concentration conditions, the average particle size of protein-based lutein nanoparticles prepared by the method of pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein is smaller than the average particle size of nanoparticles prepared by ethanol induction alone.

[0108] Under the condition of 40% ethanol concentration, the average particle size of the protein-based lutein nanoparticles prepared in Example 4 (123.3±1.9nm) is smaller than that in Comparative Example 1 (167.8±2.4nm); when the ethanol concentration is 50%, the same rule also applies, that is, the average particle size of the protein-based lutein nanoparticles prepared in Example 4 (162.7±4.8nm) is also smaller than that in Comparative Example 1 (382.0±21.3nm); when the ethanol concentration is increased to 60%, the particle size of the nanoparticles prepared in Example 4 (269.3±0.9nm) is still smaller than that in Comparative Example 1 (405.3±16.8nm).

[0109] from Figure 8 It can be seen that compared with the nanoparticles prepared by ethanol induction alone, the particle size distribution of the nanoparticles prepared by the method of pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein moves towards a smaller particle size, and when the ethanol concentration is 40% and 50%, the particle size of the nanoparticles presents a single peak distribution, which is consistent with the smaller PDI, indicating that the method of pH-coordinated ethanol-induced protein assembly nano-encapsulation of lutein can obtain protein-based lutein nanoparticles with smaller particle size and more uniform distribution. This may be because under pH = 12, compared with the self-assembly induced by ethanol alone, the protein is fully unfolded or even dissociated into subunits with smaller particle size under alkaline conditions. Although the protein will form loose and larger aggregates in the presence of ethanol, after the ethanol is removed by rotary evaporation, the aggregates return to the state of separation when no ethanol is added, and the particle size is significantly reduced; the self-assembled units are smaller in size, making the final particles smaller and more uniform.

[0110] From Table 4 and Figure 8It can be seen that as the ethanol concentration gradually increases, the particle size of the prepared nanoparticles also gradually increases. This is because ethanol has the effect of promoting protein aggregation. The higher the ethanol concentration, the more obvious the protein aggregation. Figure 1 It can also be seen in (d) that when the ethanol concentration is 40%-50%, the solution is clear and transparent, and when the ethanol concentration increases to 60%, the solution becomes turbid.

[0111] The protein-based lutein nanoparticles prepared under 40% ethanol concentration in Example 4 and Comparative Example 1 were observed by TEM. Fig. 9 As shown, the microscopic morphology of the nanoparticles provided in Example 4 presents a regular circle, and the size is smaller than that of Comparative Example 1, and the distribution is more uniform.

[0112] As can be seen from Table 4, the protein-based lutein nanoparticles prepared by the method of pH-coordinated ethanol-induced protein assembly nano-encapsulated lutein show high encapsulation efficiency and loading capacity. The encapsulation efficiency of Example 1 is greater than 90%, which is higher than that of Comparative Example 1. This is because the protein undergoes secondary unfolding and secondary folding in the preparation method of pH-coordinated ethanol-induced protein assembly nano-encapsulated lutein. When the protein is under the condition of pH=12, the protein molecule unfolds to a great extent and even dissociates into subunits, and the degree of exposure of the hydrophobic group increases; when the lutein-anhydrous ethanol solution is introduced into the system, ethanol further unfolds the protein structure, and lutein and the fully unfolded alkaline protein are quickly combined through hydrophobic interactions. In the process of removing the ethanol protein folding, the lutein is first encapsulated in the hydrophobic cavity of the alkaline protein. In the process of adjusting the pH, the protein folds back and reorganizes again, and the loose structure becomes dense, encapsulating the lutein originally located on the surface of the protein inside, so that the encapsulation efficiency is improved. When the ethanol concentration is consistent with the protein concentration, the higher the encapsulation rate, the correspondingly higher the loading capacity.

[0113] Table 4 Average particle size, PDI, encapsulation efficiency and loading of nanoparticles prepared in Example 4 and Comparative Example 1

[0114]

[0115] 5 mL of the protein-based lutein nanoparticle solution prepared in Example 4 and 5 mL of 1 mg / mL free lutein-anhydrous ethanol solution were taken and stored in a 35°C constant temperature box in the dark for 45 days. The lutein content was measured at 0 days, 7 days, 15 days, and 45 days. The experiment was repeated three times, and the lutein long-term storage retention rate was calculated according to formula (4). Fig.10It can be seen that the retention rate of free lutein after storage at 35°C for 7 days is only 4.60±0.16%, while the lutein retention rate in the nanoparticles provided in Example 4 is above 90%, and the lutein retention rate after 45 days is also greater than 70%, indicating that the protein-based lutein nanoparticles provided in this example can significantly improve the long-term storage stability of lutein.

[0116] 5 mL of the protein-based lutein nanoparticle solution prepared in Example 4 and 5 mL of 1 mg / mL lutein-anhydrous ethanol solution were used to perform a simulated in vitro digestion experiment to determine the digestion stability and bioavailability. The experiment was repeated three times. The results are as follows: Fig.11 and Fig.12 As shown. After 120 minutes of simulated gastric digestion, the average values ​​of the retention rates of lutein in the solution-based protein-based lutein nanoparticles (40% ethanol concentration, 50% ethanol concentration, 60% ethanol concentration) were 97%, 94%, 93%, respectively, while the retention rate of free lutein was 88%; after 120 minutes of simulated small intestinal digestion, the retention rates of lutein in the solution-based protein-based lutein nanoparticles were all above 85%, while the retention rate of free lutein was reduced to 35%. After the entire digestion process was completed, the average values ​​of the lutein bioavailability (the content of lutein in the supernatant after centrifugation of the digestive fluid at 10000g for 30 minutes) of the solution-based protein-based lutein nanoparticles (40% ethanol concentration, 50% ethanol concentration, 60% ethanol concentration) prepared in Example 4 were 38%, 32%, 30%, respectively, while the bioavailability of free lutein was only 4%. This indicates that the protein-based lutein nanoparticles provided in Example 4 can significantly improve the digestion stability and bioavailability of lutein, and can be added to medicines and foods as raw materials or auxiliary materials, and have great market potential.

Claims

1. A method for preparing protein-based lutein nanoparticles, characterized in that: The following steps are involved: (1) dispersing the protein in water to an initial concentration of 1-60 mg / mL and a pH range of 10-12, stirring the protein to unfold or dissociate into subunits, and the solution becomes clear and transparent to obtain an alkaline dissociated protein dispersion, and the stirring time is 30-120 min; then adding a lutein-anhydrous ethanol solution, wherein the ethanol further unfolds the alkaline dissociated protein through alcohol-water interaction, and stirring the lutein to form a stable complex with the protein through hydrophobic interaction, thereby enhancing the hydrophobic interaction between the alkaline dissociated protein particles and aggregating into loose large particles, and the solution becomes turbid to obtain a mixed solution; the volume ratio of the lutein-anhydrous ethanol solution added is 10%-70% of the mixed solution, and the stirring time is 30-120 min; (2) removing ethanol from the mixed solution by rotary evaporation, the unfolded alkaline dissociated protein folds back, the loose structure begins to converge, the lutein undergoes the first embedding, the aggregated alkaline dissociated protein particles return to the state of being separated from each other when no ethanol is added, the particle size decreases, and the solution becomes clear again; the vacuum degree of the rotary evaporation is 0-0.1 MPa, the heating temperature is 35-45° C., and the rotation speed is 80-120 rpm; (3) The pH of the mixed solution is adjusted to neutral, and the subunits that were originally separated from each other due to electrostatic repulsion are rearranged, the protein is folded back and reorganized for the second time, and the lutein undergoes a second embedding, and the loose structure becomes dense. Water with the same volume as the ethanol removed by evaporation is added to obtain protein-based lutein nanoparticles; In step (1), the protein is at least one of whey protein, serum protein, ovalbumin, lysozyme, β-lactoglobulin, sodium caseinate, bean 7S globulin, bean 11S globulin, soy protein isolate and pea protein isolate.

2. Protein-based lutein nanoparticles are prepared by the preparation method described in claim 1.

3. The protein-based lutein nanoparticles according to claim 2 are used in: food or feed.

4. The protein-based lutein nanoparticles according to claim 2 are used for: pigments.

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