CN116391864A

CN116391864A - Method for assembling nano-embedded lutein by pH (potential of hydrogen) synergistic ethanol-induced protein, protein-based lutein nanoparticle and application

Info

Publication number: CN116391864A
Application number: CN202310243522.3A
Authority: CN
Inventors: 唐传核; 颜斌
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2023-03-14
Filing date: 2023-03-14
Publication date: 2023-07-07

Abstract

The invention discloses a method for assembling nano-embedded lutein by utilizing pH synergistic ethanol-induced protein, protein-based lutein nano-particles and application thereof. The preparation method comprises the following steps: adjusting the pH of the protein dispersion to be alkaline, fully stirring, and dripping the lutein-absolute ethyl alcohol solution into the alkaline protein solution; during the process of evaporating and removing ethanol, the unfolded alkaline protein is folded to ensure that lutein is encapsulated in a protein hydrophobic cavity; and (3) in the process of adjusting the pH to be neutral, the unfolded protein is subjected to secondary folding and rearrangement, and lutein is subjected to secondary encapsulation, so that the protein-based lutein nanoparticle with compact structure is finally formed. The method has the advantages of simple equipment, simple and convenient steps, safety, greenness, easily available raw materials, economy and high efficiency; the prepared protein-based lutein nano-particles remarkably improve the water dispersibility, the heat stability, the light stability, the storage stability, the digestion stability and the bioavailability of lutein; therefore, the method has wide development prospect in industries such as pigment, feed, food, medicine and the like.

Description

Method for assembling nano-embedded lutein by pH (potential of hydrogen) synergistic ethanol-induced protein, protein-based lutein nanoparticle and application

Technical Field

The invention belongs to the technical field of food bioactive substance nano-embedding, and particularly relates to a method for assembling nano-embedded lutein by utilizing pH to cooperate with ethanol to induce protein, protein-based lutein nano-particles and application.

Background

Lutein and zeaxanthin isomer thereof are oxygen-containing carotenoids other than provitamin A, contain very polyunsaturated double bonds in the structure, and have excellent oxidation resistance. They can act to prevent age-related maculopathy and cataract by antioxidant effect and blue-wave filtering effect. As with most hydrophobic bioactive substances, lutein is poorly soluble in water, can be dissolved in organic solvents such as ethanol, ethyl acetate, dichloromethane and the like, is extremely sensitive to light and heat, and has low bioavailability, thus severely limiting the application thereof in the food field.

In order to solve the problems, the lutein is usually encapsulated by microcapsule, nanoparticle, emulsion and other systems at home and abroad, and ethanol is usually used as a solvent to introduce the lutein. Zhao et al (Physicochemical Properties of Lutein-Loaded Microcapsules and Their Uptake via Caco-2 Monolayers [ J ]. Molecular, 2018,23 (7): 1805) drop lutein-ethanol solution into sodium caseinate-ethanol solution after heat treatment at 60deg.C for 5min, high-speed dispersion at 10000rpm for 4min, sieving with 100 mesh sieve to remove insoluble large particles, and spray drying to obtain microcapsule with encapsulation rate of 89.95%, wherein lutein retention rate is 79.83% after dark treatment at 25deg.C for 5d, and microcapsule particles are generally larger and biological potency is 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 into whey protein isolate-dextran-chondroitin sulfate ternary nanoparticle solution, cavitation of ultrasonic wave was utilized to promote migration of lutein molecules into nanoparticle, encapsulation rate could reach 94.07%, average particle size of nanoparticle loaded with lutein was 150nm, but preparation process involved heating, ice bath, ultrasonic treatment, steps were complicated and equipment was expensive.

The patent CN 113143885A is prepared by dissolving lutein in 95% ethanol solution and preparing lutein particles with the particle size of 100-1000nm by using a supercritical emulsification technology, wherein the average particle size can be below 400nm under the preferred condition, but the supercritical emulsification technology cannot realize large-scale industrial application at present. The patent CN 114468295A is characterized in that an phycocyanin solution synthesized based on Mannich reaction and a lutein ethanol solution are mixed and stirred for 24 hours in equal volume, ethanol is evaporated to obtain nano particles loaded with lutein, the size is 370+/-50 nm, the embedding rate is only 74.42%, and the particles are large and have low embedding rate. The patent CN 111938157A adds lutein-absolute ethyl alcohol solution into chickpea protein isolate-stevioside composite system, uses a probe type ultrasonic breaker to carry out ultrasonic treatment for 6-10min and volatilizes the ethyl alcohol to obtain lutein nano emulsion with the average particle size of 195.1nm, and also has the problem of expensive equipment.

The problem of large particle size, low embedding efficiency and low retention rate exists in the particles prepared by the lutein embedding technology with ethanol as a solvent. This is because ethanol as a polar organic solvent can change the natural conformation of the protein molecule, so that the protein is unfolded to expose a large number of hydrophobic groups, and hydrophobic active substances dissolved in ethanol can be combined with the protein to form a complex through hydrophobic interaction; at the same time, however, ethanol also has the effect of promoting aggregation of proteins, which on the one hand results in a particle size which is far greater than that of the native protein and on the other hand in a decrease in the entrapment efficiency of the hydrophobic active substance with increasing ethanol concentration, which mechanism is revealed by Liu et al (Novel so beta-conglycinin nanoparticles by ethanol-assisted disassembly and reassembly: outstanding nanocarriers for hydrophobic nutraceuticals [ J ]. Food Hydrocolloids,2019, 91:246-255.). The common solution is to crush, squeeze and shear the particles by ultrasonic, high pressure and micro-jet to reduce the size of the particles, promote the migration of lutein molecules into the particles to improve the encapsulation efficiency, but all the defects of expensive equipment and high cost are accompanied.

Therefore, the related lutein embedding technology patent with simple equipment, convenient and fast method, high embedding efficiency and smaller size is not published at present.

The patent CN 113080452A encapsulates the curcumin in the protein by utilizing the characteristics of increased water solubility of curcumin phenolic hydroxyl ionization and protein unfolding and even dissociation into subunits under the alkaline pH condition, realizes high-efficiency loading of the curcumin, and improves the water solubility, stability and bioavailability of the 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 recombinant particle size formed after pH adjustment of treated sodium caseinate to pH 7 at pH=12 was significantly smaller than the protein particle size without alkali treatment. Therefore, the pH induced protein self-assembly not only can realize efficient embedding of the hydrophobic active substance, but also can prepare the recombinant particles with smaller size, and the preparation process is simple, safe, low in cost and low in energy consumption.

In summary, the invention provides a method for assembling nano-embedded lutein by pH cooperated with ethanol induced protein, which is based on the following principle: under the alkaline pH condition, the protein is far away from the isoelectric point, the electrostatic repulsive force among alkaline protein molecules is increased along with the increase of the pH value, so that the protein is unfolded or even dissociated Cheng Yaji, hydrophobic groups in the protein molecules are exposed to a hydrophilic environment, at the moment, self-assembled units are obviously reduced, and the protein dispersion liquid becomes clear and transparent; ethanol is used as an inducer to promote the alkaline dissociated proteins to be further unfolded, the exposure degree of hydrophobic groups is increased, the hydrophobic effect among the alkaline dissociated proteins is also enhanced, loose and larger aggregates are formed, and meanwhile, the ethanol is used as a solvent to introduce lutein, so that the lutein can be combined with the hydrophobic groups fully exposed by the alkaline dissociated proteins through hydrophobic interaction to form a compound, and the solution becomes turbid; in the process of removing the ethanol, fully developed alkaline dissociated proteins start to fold back, lutein is encapsulated in subunits for the first time or is combined with the surfaces of protein molecules, loose structures become converged, originally aggregated alkaline dissociated protein particles return to a state of being separated from each other when the ethanol is not added, the size of the particles is obviously reduced, and the solution becomes clear again; and (3) in the process of adjusting the pH to be neutral, the unfolded or dissociated protein is continuously folded and recombined, lutein is subjected to secondary encapsulation, the loose structure becomes compact, and finally the protein-based lutein nano-particle similar to the natural protein structure is formed.

The method has the advantages of simple equipment, simple and convenient process and low cost, and the prepared protein-based lutein nano-particles can realize high-efficiency encapsulation of lutein and can also improve the water solubility, stability and bioavailability of lutein. Is suitable for large-scale industrial production and processing, and has certain application prospect in the fields of pigment, feed, food, medicine and the like.

Disclosure of Invention

In view of the drawbacks and deficiencies of the prior art, a first aspect of the present invention is directed to a method for assembling nano-embedded lutein by pH-synergistic ethanol-induced protein. The method utilizes protein to fold or dissociate into subunits under alkaline pH condition to expose a large amount of hydrophobic groups in the protein to the surface of the protein; then introducing lutein-absolute ethanol solution, wherein ethanol is used as an inducer to promote the protein to be unfolded and aggregated, and is used as a solvent to introduce lutein to form a complex with the protein through hydrophobic interaction; removing ethanol by evaporation to enable the unfolded alkaline dissociated protein to be folded for the first time, enabling lutein to be subjected to first embedding, and weakening the characteristic that ethanol is utilized to promote protein particles to gather by electrostatic repulsive force, so that the particle size is obviously reduced; and finally, the pH value is adjusted back to enable the unfolded or dissociated protein to carry out secondary folding and recombination, and lutein is subjected to secondary embedding at the same time, so that the protein-based lutein nanoparticle similar to the natural protein structure is formed.

The purpose of the second aspect of the invention is to prepare the protein-based lutein nanoparticle which is easy to disperse, high in water solubility, high in embedding rate, high in stability and high in bioavailability by the method.

The third aspect of the present invention is aimed at realizing commercial application of the above protein-based lutein nanoparticle.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

(1) The pH of the protein dispersion is adjusted to be in an alkaline state, and the protein is fully stirred to be unfolded or dissociated into subunits, so that the particle size is obviously reduced, and the solution becomes clear and transparent; then adding a certain volume ratio of lutein-absolute ethyl alcohol solution, enabling the alkaline dissociated protein to be further unfolded through alcohol-water interaction by the aid of the ethyl alcohol, fully stirring to enable the lutein to form a stable compound with the protein through hydrophobic interaction, and meanwhile, enhancing the hydrophobic interaction among alkaline dissociated protein particles to form a loose aggregate with larger size, so that the solution becomes turbid.

(2) The ethanol in the mixed solution is removed by rotary evaporation, the unfolded alkaline dissociation proteins are gradually overlapped, the loose structure starts to be converged, lutein is subjected to first embedding, the aggregated alkaline protein particles return to a state of being separated from each other when no ethanol is added, the particle size is obviously reduced, and the solution becomes clear again.

(3) Regulating pH of the mixed solution to be neutral, carrying out rearrangement of subunits separated from each other originally by electrostatic repulsive force, carrying out secondary overlapping and recombination on protein, carrying out secondary embedding on lutein, enabling a loose structure to become compact, and adding water with the same volume as ethanol removed by evaporation to obtain the protein-based lutein nano-particles.

Further, the protein in the step (1) is at least one of whey protein, serum protein, ovalbumin, lysozyme, beta-lactoglobulin, sodium caseinate, legume 7S globulin, legume 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.

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

Further, the pH range of the protein dispersion liquid in the step (1) is adjusted to be 10-12, and the stirring time is 30-120min; preferably, the pH of the regulatory protein dispersion is in the range of 11-12 and the stirring time is 60-120min.

Further, the concentration of the lutein-absolute ethanol solution in the step (1) is 0.5-1.5mg/mL.

Further, the lutein-absolute ethyl alcohol solution is added in the step (1) in a volume ratio of 10-70% of the mixed solution, and the stirring time is 30-120min; preferably, the volume ratio of the lutein-absolute ethyl alcohol solution is 40% -60% of the mixed solution, the stirring time is 60-120min, more preferably, the volume ratio of the lutein-absolute ethyl alcohol solution is 40% -50% of the mixed solution, and the stirring time is 60-120min.

Further, the rotary evaporation vacuum degree in the step (2) is 0-0.1MPa, the heating temperature is 35-45 ℃, and the rotating speed is 80-120rpm.

Further, the pH value of the step (3) is adjusted to 6.8-8.0; preferably, step (3) adjusts the pH to 6.8-7.2.

The invention provides protein-based lutein nanoparticle prepared by the method for assembling nano-embedded lutein by utilizing the pH synergistic ethanol-induced protein.

The invention provides an application of protein-based lutein nano particles prepared by a method for assembling nano-embedded lutein by utilizing pH synergistic ethanol-induced protein in preparation of products, and further, the products are any one of (1) - (4): (1) The pigment can be used as a colorant for producing foods such as cakes and candies to give orange-yellow appearance; (2) Food can be directly used as dietary supplement or used as auxiliary material to produce lutein-reinforced food such as functional beverage, etc.; (3) The medicine can be used as an auxiliary material to produce medicines for relieving eye fatigue; (4) The feed can be used as chicken feed to enrich xanthophyll in egg.

The principle of the invention is as follows: lutein is a type of oxygen-containing carotenoid which is not provitamin A, is insoluble in water, but is soluble in ethanol, and has multiple hydrophobic sites in lutein molecules, and can be combined with protein through hydrophobic interaction. The hydrophobic sites of the native protein are located inside the molecule, while the structure of the protein is maintained by hydrogen bonding, hydrophobic interactions, and electrostatic forces. It has been found that both ethanol and pH can alter the natural structure of proteins, allowing proteins to unfold or dissociate, especially in alkaline pH conditions, certain proteins dissociate Cheng Yaji, thus exposing much of the originally buried hydrophobic groups to the surface of the protein molecule; in the presence of ethanol, the alkaline dissociated proteins on the one hand further unfold exposing more hydrophobic sites causing aggregation of the particles and on the other hand forming complexes with lutein by hydrophobic interactions; in the process of removing ethanol, the alkaline dissociating proteins with loose structures are subjected to first folding, part of lutein is embedded in the alkaline dissociating proteins, and the aggregated alkaline dissociating protein particles return to a state of being separated from each other when no ethanol is added; in the process of adjusting the pH to be neutral, the unfolded or dissociated protein molecules are subjected to secondary folding and recombination, lutein on the surface of the protein can be further embedded, and finally, new particles similar to the structure of the natural protein are formed.

In conclusion, the protein-based lutein nanoparticle prepared by assembling nano-embedded lutein through pH and ethanol-induced protein can realize efficient embedding of lutein, solve the defects of poor water dispersibility, poor stability and low bioavailability of lutein, and improve the stability of lutein products in the food processing process and finally the effective absorption of the lutein products in human bodies.

The preparation method has the advantages of simple and safe preparation process, low cost and low energy consumption, can realize efficient encapsulation, and can be applied to various occasions.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention provides a method for assembling nano-embedded lutein by utilizing pH synergistic ethanol induced protein for the first time, which has the advantages of simple equipment, simple and convenient steps, safety, greenness, low cost and low energy consumption, is suitable for large-scale industrial production and processing, meets the food safety requirements in the production process and materials, is suitable for pigment, food, medicine and feed industries, and has wide application prospect.

(2) According to the method for assembling nano-embedded lutein by utilizing the pH and alcohol to induce the protein, the characteristics of the natural structure of the protein can be changed by utilizing the alcohol and the pH, so that the protein is unfolded, dissociated and recombined twice, the lutein is efficiently encapsulated, the characteristic of promoting protein aggregation by utilizing electrostatic repulsive force to weaken the alcohol, and finally the protein-based lutein nano-particle with compact structure and smaller size is formed.

(3) According to the method for assembling nano-embedded lutein by utilizing the pH synergistic alcohol to induce the protein, the size of the formed protein-based lutein nano-particles can be regulated and controlled by changing the pH, the protein concentration, the alcohol concentration and the time of pH synergistic alcohol induction.

(4) The protein-based lutein nanoparticle prepared by adopting pH to cooperate with ethanol to induce protein to assemble nano-embedded lutein can improve the water dispersibility, the thermal stability, the light stability, the storage stability and the bioavailability of lutein.

Drawings

FIG. 1 is a solution appearance diagram of the protein-based lutein nanoparticle prepared in examples 1-4;

FIG. 2 is a graph showing particle size distribution of protein-based lutein nanoparticles prepared from different proteins in example 1;

FIG. 3 is a powder appearance diagram of the protein-based lutein nanoparticle in the form of solution prepared from different proteins in example 1 after vacuum freeze drying;

FIG. 4 is a graph showing particle size distribution of protein-based lutein nanoparticles prepared under different pH conditions in example 2;

FIG. 5 is a graph showing particle size distribution of protein-based lutein nanoparticles prepared under different protein concentration conditions in example 3;

FIG. 6 is a graph showing lutein retention rate after heat treatment of lutein-absolute ethanol solution and protein-based lutein nanoparticles prepared under different protein concentration conditions in example 3 at 80 ℃;

FIG. 7 is a graph showing the lutein retention rate of the lutein-based nanoparticle prepared under the condition of different protein concentrations in example 3 after the lutein-absolute ethanol solution is irradiated with ultraviolet light;

FIG. 8 is a graph showing particle size distribution of protein-based lutein nanoparticles prepared under different ethanol concentration conditions in example 4 and comparative example 1;

FIG. 9 is a transmission scanning electron microscope image of the protein-based lutein nanoparticle prepared in example 4 and comparative example 1 at 40% ethanol concentration;

FIG. 10 is a graph showing the lutein retention rate after storage of the protein-based lutein nanoparticles and lutein-anhydrous ethanol solution in the form of solution prepared under different ethanol concentration conditions at 35℃in the example 4 in the absence of light;

FIG. 11 is a graph showing the retention of lutein after simulated gastric digestion and/or intestinal digestion of protein-based lutein nanoparticles and lutein-absolute ethanol solution in the form of solutions prepared under different ethanol concentration conditions in example 4;

FIG. 12 is a statistical plot of lutein retention and bioavailability of lutein in the form of solutions prepared under different ethanol concentration conditions in example 4 after simulated gastric digestion and/or intestinal digestion of lutein-anhydrous ethanol solution.

Detailed Description

The invention is further illustrated by the following specific examples of implementation.

It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

The experimental methods in each of the following examples, in which specific conditions are not noted, are generally performed under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents and the like used in this example are commercially available ones unless otherwise specified.

Example 1

Method 1: the protein-based lutein nanoparticle prepared by a method for assembling nano-embedded lutein by utilizing pH and ethanol to induce protein comprises the following steps:

(1) Whey protein isolate was dispersed in water to an initial concentration of 16.7mg/mL, the pH of the dispersion was adjusted to 12.0 with 4M NaOH solution, and magnetic stirring was continued at 1000rpm/min for 120min at room temperature to allow the protein to be fully unfolded or dissociated and the solution became clear and transparent. 1.5mg/mL lutein-absolute ethanol solution is dripped into the alkaline dissociating protein dispersion liquid to ensure that the absolute ethanol concentration reaches 40% (v/v) of the mixed liquid, the pH value is maintained at 12.0 during the period, the final concentration of the alkaline dissociating protein is 10mg/mL, and the magnetic stirring is continued at the speed of 1000rpm/min for 30min at room temperature. Under the action of ethanol, the alkaline dissociative protein is further unfolded to fully expose hydrophobic groups, on one hand, the alkaline dissociative protein forms a complex with lutein through hydrophobic interaction, on the other hand, the alkaline dissociative protein is aggregated into loose large particles, and the solution becomes turbid.

(2) Removing the ethanol in the mixed solution in the step (1) by a rotary evaporator, gradually overlapping the unfolded alkaline dissociation proteins, starting to converge the loose structure, allowing lutein to undergo first embedding, returning the aggregated alkaline protein particles to a state of being separated from each other when no ethanol is added, reducing the particle size, and clarifying the solution again. The rotary evaporation vacuum degree is 0.05MPa, the heating temperature is 40 ℃, and the rotating speed is 100rpm.

(3) And (3) regulating the pH value of the mixed solution obtained in the step (2) to 7.0 by using a 2M hydrochloric acid solution, rearranging subunits separated from each other by electrostatic repulsive force, carrying out protein second overlapping, carrying out secondary embedding on lutein, enabling a loose structure to become compact, adding water with the same volume as ethanol removed by evaporation, and enabling the final concentration of protein to be 10mg/mL, thus obtaining the protein-based lutein nanoparticle.

Method 2: the method for preparing the protein-based lutein nanoparticle by combining pH with ethanol to induce protein to assemble nano-embedded lutein is the same as the method 1 in the embodiment, and the difference is that the protein in the step (1) is sodium caseinate.

Method 3: the method for preparing protein-based lutein nanoparticle by combining pH and ethanol to induce protein assembly nano-embedded lutein is the same as the method 1 in the embodiment, and the difference is that the protein in the step (1) is soybean protein isolate.

Example 2

(1) The isolated soy protein was dispersed in water to an initial concentration of 16.7mg/mL, the pH of the dispersion was adjusted to 10.0 with 4M NaOH solution, and magnetic stirring was continued at 1000rpm/min for 120min at room temperature to allow the protein to be fully unfolded or dissociated and the solution became clear and transparent. 1.5mg/mL of lutein-absolute ethanol solution is dripped into the alkaline dissociating protein dispersion liquid to ensure that the absolute ethanol concentration reaches 40% (v/v) of the mixed liquid, the pH value is maintained at 10.0 during the period, the final concentration of the alkaline dissociating protein is 10.0mg/mL, and the magnetic stirring is continued at the speed of 1000rpm/min for 60min at room temperature. Under the action of ethanol, the alkaline dissociative protein is further unfolded to fully expose hydrophobic groups, on one hand, the alkaline dissociative protein forms a complex with lutein through hydrophobic interaction, on the other hand, the alkaline dissociative protein is aggregated into loose large particles, and the solution becomes turbid.

(3) And (3) regulating the pH value of the mixed solution obtained in the step (2) to 7.0 by using a 2M hydrochloric acid solution, rearranging subunits separated from each other by electrostatic repulsive force, carrying out protein second overlapping, carrying out secondary embedding on lutein, enabling a loose structure to become compact, adding water with the same volume as ethanol removed by evaporation, and enabling the final concentration of protein to be 10.0mg/mL, thus obtaining the protein-based lutein nanoparticle.

Method 2: the preparation method of the protein-based lutein nanoparticle prepared by combining pH with ethanol to induce protein assembly nano-embedded lutein is the same as the method 1 in the embodiment, and the difference is that the pH in the step (1) is 11.0.

Method 3: the protein-based lutein nanoparticle prepared by a method for assembling nano-embedded lutein by utilizing pH and ethanol to induce protein is different from the method 1 in the embodiment only in that the pH in the step (1) is 12.0.

Example 3

(1) The isolated soy protein was dispersed in water to an initial concentration of 8.3mg/mL, the pH of the dispersion was adjusted to 11.0 with 4M NaOH solution, and magnetic stirring was continued at 1000rpm/min for 120min at room temperature to allow the protein to be fully unfolded or dissociated and the solution became clear and transparent. 1.5mg/mL of lutein-absolute ethanol solution is dripped into the alkaline dissociating protein dispersion liquid to ensure that the absolute ethanol concentration reaches 40% (v/v) of the mixed liquid, the pH value is maintained at 11.0 during the period, the final concentration of the alkaline dissociating protein is 5.0mg/mL, and the magnetic stirring is continued at the speed of 1000rpm/min for 90min at room temperature. Under the action of ethanol, the alkaline dissociative protein is further unfolded to fully expose hydrophobic groups, on one hand, the alkaline dissociative protein forms a complex with lutein through hydrophobic interaction, on the other hand, the alkaline dissociative protein is aggregated into loose large particles, and the solution becomes turbid.

(3) And (3) regulating the pH value of the mixed solution obtained in the step (2) to 7.0 by using a 2M hydrochloric acid solution, rearranging subunits separated from each other by electrostatic repulsive force, carrying out protein second overlapping, carrying out secondary embedding on lutein, enabling a loose structure to become compact, adding water with the same volume as ethanol removed by evaporation, and enabling the final concentration of protein to be 5.0mg/mL, thus obtaining the protein-based lutein nanoparticle.

Method 2: the protein-based lutein nanoparticle prepared by the method for assembling nano-embedded lutein by utilizing pH and ethanol to induce protein is the same as the method 1 in the embodiment, and the difference is that the initial protein concentration in the step (1) is 20.8mg/mL, and the final protein concentration in the step (1) and the step (3) is 12.5mg/mL.

Method 3: the protein-based lutein nanoparticle prepared by the method for assembling nano-embedded lutein by utilizing pH and ethanol to induce protein is the same as the method 1 in the embodiment, and the difference is that the initial protein concentration in the step (1) is 33.3mg/mL, and the final protein concentration in the step (1) and the step (3) is 20.0mg/mL.

Method 4: the protein-based lutein nanoparticle prepared by the method for assembling nano-embedded lutein by utilizing pH and ethanol to induce protein is the same as the method 1 in the embodiment, and the difference is that the initial protein concentration in the step (1) is 41.7mg/mL, and the final protein concentration in the step (1) and the step (3) is 25.0mg/mL.

Example 4

(1) The isolated soy protein was dispersed in water to an initial concentration of 16.7mg/mL, the pH of the dispersion was adjusted to 12.0 with 4M NaOH solution, and magnetic stirring was continued at 1000rpm/min for 120min at room temperature to allow the protein to be fully unfolded or dissociated and the solution became clear and transparent. 1.5mg/mL of lutein-absolute ethyl alcohol solution is dripped into the alkaline dissociating protein dispersion liquid to ensure that the absolute ethyl alcohol concentration reaches 40% (v/v) of the mixed liquid, the pH value is maintained at 12.0 during the period, the final concentration of the alkaline dissociating protein is 10.0mg/mL, and the magnetic stirring is continuously carried out at the speed of 1000rpm/min for 120min at room temperature. Under the action of ethanol, the alkaline dissociative protein is further unfolded to fully expose hydrophobic groups, on one hand, the alkaline dissociative protein forms a complex with lutein through hydrophobic interaction, on the other hand, the alkaline dissociative protein is aggregated into loose large particles, and the solution becomes turbid.

Method 2: the preparation method of the protein-based lutein nanoparticle prepared by the method of combining pH with ethanol to induce protein to assemble nano-embedded lutein is the same as the method 1 in the embodiment, and the difference is that the ethanol concentration in the step (1) is 50% (v/v) of the mixed solution.

Method 3: the preparation method of the protein-based lutein nanoparticle prepared by the method of combining pH with ethanol to induce protein to assemble nano-embedded lutein is the same as the method 1 in the embodiment, and the difference is that the ethanol concentration in the step (1) is 60% (v/v) of the mixed solution.

Comparative example 1

Method 1: a protein-based lutein nanoparticle prepared by ethanol induction comprises the following steps:

(1) The isolated soy protein was dispersed in water to an initial concentration of 16.7mg/mL, adjusted to pH 7.0 with 4M NaOH solution and magnetically stirred at 1000rpm/min for 120min at room temperature. 1.5mg/mL lutein-absolute ethanol solution is dripped into the protein dispersion liquid to ensure that the ethanol concentration reaches 40% (v/v) of the mixed liquid, the final concentration of the protein is 10.0mg/mL, and the magnetic stirring is continuously carried out for 120min at the speed of 1000rpm/min at room temperature. Under the action of ethanol, the protein gradually unfolds to expose hydrophobic groups, forming a complex with lutein through hydrophobic interaction on the one hand, and aggregating into loose large particles on the other hand, so that the solution becomes turbid.

(2) Removing ethanol in the mixed solution in the step (1) by a rotary evaporator, gradually overlapping the unfolded protein to form a compact structure, and heating at 40 ℃ and rotating at 100rpm under the rotary evaporation vacuum degree of 0.05 MPa. The pH was adjusted to 7.0 with 2M hydrochloric acid solution, and water was added in the same volume as the ethanol removed by evaporation, so that the final concentration of isolated soy protein was 10mg/mL.

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

Method 3: the preparation method of the protein-based lutein nanoparticle prepared by ethanol induction is the same as that of the method 1 in the embodiment, and the difference is that the ethanol concentration in the step (1) is 60% (v/v) of the mixed solution.

Effect examples

1. Particle size, PDI and lyophilization yield of the protein-based lutein nanoparticles obtained in example 1

The protein-based lutein nanoparticle prepared from whey protein isolate, sodium caseinate and soy protein isolate in example 1 was diluted to a protein concentration of 1mg/mL, the experiment was repeated three times, and the hydrated particle size and PDI (Polymer dispersity index, polymer dispersibility index, used to describe the polymer molecular weight distribution) were measured using a malvern nanoparticle size and potential analyzer, and the results are shown in table 1 and fig. 2.

In the appearance diagram in fig. 1 (a), the turbidity of the solution prepared by whey protein isolate is highest, and in fig. 2, the particle size of the whey protein isolate lutein nanoparticle is in obvious bimodal distribution, which indicates that more association and aggregation of protein exist, on one hand, because ethanol with a certain concentration has the effect of promoting aggregation of protein molecules; on the other hand, whey protein isolate is relatively weak in the ability to bind lutein through hydrophobic interactions due to lack of sufficient hydrophobic sites in the structure, strong surface hydrophilicity, weak amphipathic subunits, etc., and the proteins are more easily bound to each other, so that larger particles are easily formed. The soy protein isolate and the sodium caseinate are more easily combined with lutein because of having good amphipathic property and more hydrophobic sites, the lutein is encapsulated in the recombinant protein, the average particle size of the recombinant particles is smaller, and the solution prepared from the soy protein isolate and the sodium caseinate in the figure 1 (a) has the characteristics of clarity and transparency. Although the particle size of the soy protein isolate based lutein nanoparticles is slightly larger than the particles prepared from sodium caseinate, the polydispersity index is minimal and the soy protein isolate is more widely available and readily available than sodium caseinate and is therefore widely used in the examples of this patent.

The protein-based lutein nanoparticle solution prepared from the different proteins in example 1 was freeze-dried to give pale yellow powder as shown in fig. 3. In all the dried powders, no crystalline particles of lutein were found, which fully suggests that lutein in the protein-based lutein nanoparticles is efficiently entrapped and that lutein is relatively stable in protein binding.

The freeze-drying yield is calculated according to the formula (1), wherein W ₂ To the mass of the granules obtained after drying, W ₁ The results are shown in Table 1 for the mass of the non-evaporable particles contained in the formulation (i.e., protein and lutein). The protein-based lutein nano particles have higher yield after vacuum freeze drying, and the yield is more than 80 percent.

TABLE 1 average particle size, PDI, and lyophilization yield of nanoparticles prepared in example 1

2. Particle size, PDI, encapsulation efficiency and loading of the protein-based lutein nanoparticle obtained in example 2

The appearance of the protein-based lutein nanoparticle in the form of solution obtained in example 2 is shown in fig. 1 (b), and the solution is 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, and in alkaline state, the average particle size of the obtained nanoparticles gradually decreases with increasing pH, the average particle size of the prepared nanoparticles is maximum at ph=10, and is 157.5±9.0nm, and when pH is 11-12, the average particle sizes of the particles are relatively close, 134.2±5.4nm and 127.1±5.7nm, respectively. This is because the greater the pH in alkaline condition, the higher the extent of protein unfolding and dissociation, the more sites available for hydrophobic binding to lutein, and the smaller and denser the recombinant particle structure formed. As can also be seen from fig. 4, the larger the pH in the alkaline state, the larger the area ratio of the small particle diameter peak, and the whole particle diameter distribution map moves in the direction of smaller particle diameter.

The encapsulation efficiency and the load of the protein-based lutein nanoparticle in the form of solution obtained in example 2 were measured, the experiment was repeated three times, and the calculation was performed according to formulas (2) and (3), and the results are shown in table 2, in which the encapsulation efficiency has a significant negative correlation with the particle size. In an alkaline state, as the pH is increased, the more the protein is unfolded and dissociated, the more sites can be subjected to hydrophobic binding with lutein; after the pH is adjusted back to 7, the more lutein is encapsulated in the protein, the higher the encapsulation efficiency is, and the load capacity is increased under the condition of the same protein concentration.

Table 2 average particle diameter, PDI, encapsulation efficiency and load of nanoparticles prepared in example 2

3. Particle size, PDI, heat treatment stability and light treatment stability of the protein-based lutein nanoparticle obtained in example 3

The appearance of the protein-based lutein nanoparticle in the form of solution prepared in example 3 is shown in fig. 1 (c), and the turbidity of the solution increases with the increase of the protein concentration. The hydrated particle size and PDI of the protein-based lutein nanoparticles prepared in example 3 at different protein concentrations are shown in Table 3 and FIG. 5. With the increase of the protein concentration, the average particle size and PDI of the protein-based lutein nanoparticle prepared by the method of combining pH with ethanol to induce protein to assemble nano-embedded lutein are firstly reduced and then increased, and when the protein concentration is 12.5mg/mL, the average particle size and PDI are minimum, namely 129.2+/-1.8 nm and 0.351+/-0.004 nm respectively; when the protein concentration is as high as 25.0mg/mL, although the particle size of particles formed by protein unfolding or dissociation is obviously reduced under the condition of pH=12, the protein concentration is increased from 25.0mg/mL to 41.7mg/mL after ethanol is removed, the probability of collision among protein molecules is increased in the process of adjusting the pH to 7, large particles are more easily formed, even if distilled water is supplemented to restore the protein concentration to 25.0mg/mL, the size of the self-assembled nano particles cannot be changed, and the average particle size of the protein-based lutein nano particles prepared under the protein concentration is 235.7+/-2.4 nm; when the protein concentration is as low as 5.0mg/mL, the lutein content required to be loaded per unit protein is increased, so that the aggregation degree among proteins is increased, and the average particle size of the formed nano particles is correspondingly increased and is 151.8+/-3.6 nm.

TABLE 3 average particle diameter and PDI of nanoparticles prepared in example 3

7mL of the protein-based lutein nanoparticle solution prepared in the embodiment 3 and 7mL of the 1mg/mL lutein-absolute ethyl alcohol solution are respectively taken and placed in a water bath kettle at 80 ℃ for 4 hours for heat treatment, the lutein content is measured at 0h, 0.5h, 1h, 2h and 4h, the experiment is repeated three times, and the lutein retention rate is calculated according to the formula (4), and the result is shown in figure 6. After heat treatment for 4 hours at 80 ℃, the retention rate of lutein in the protein-based lutein nano-particles provided by the embodiment is over 80 percent, and the retention rate of free lutein is only 9.97+/-0.43% under the same condition, which indicates that the protein-based lutein nano-particles provided by the embodiment can obviously improve the thermal stability of lutein.

7mL of the protein-based lutein nanoparticle solution prepared in the embodiment 3 with different protein concentrations and 7mL of 1mg/mL of lutein-absolute ethanol solution are respectively taken and irradiated under 254nm and 365nm ultraviolet light, the lutein content is measured in 0h, 2h, 4h, 8h and 24h, the experiment is repeated three times, and the lutein retention rate is calculated according to the formula (4), and the result is shown in figure 7. After 24h ultraviolet irradiation, the retention rate of lutein in the protein-based lutein nano-particles provided by the embodiment is over 70 percent, and the retention rate of free lutein is only 1.58 percent under the same condition, which indicates that the protein-based lutein nano-particles provided by the embodiment can obviously improve the illumination stability of lutein.

4. Particle size, PDI, long-term storage stability, in vitro digestion stability and bioavailability of the protein-based lutein nanoparticles obtained in example 4

The hydrated particle size and PDI of the protein-based lutein nanoparticles prepared by different ethanol concentrations in example 4 and comparative example 1 are shown in Table 4 and FIG. 8, and the average particle size of the protein-based lutein nanoparticles prepared by the method for assembling nano-embedded lutein by pH-synergistic ethanol-induced protein under the same ethanol concentration condition is smaller than that of the nanoparticles prepared by ethanol-induced alone.

The average particle size (123.3+ -1.9 nm) of the protein-based lutein nanoparticle prepared in example 4 was smaller than that of comparative example 1 (167.8+ -2.4 nm) under the condition of 40% ethanol concentration; the same rule is also adopted when the concentration of ethanol is 50%, namely, the average particle size (162.7+/-4.8 nm) of the protein-based lutein nano-particles prepared in the example 4 is smaller than that of the comparative example 1 (382.0 +/-21.3 nm); when the ethanol concentration was increased to 60%, the nanoparticle size (269.3.+ -. 0.9 nm) prepared in example 4 was still smaller than that of comparative example 1 (405.3.+ -. 16.8 nm).

As can be seen from fig. 8, compared with the nanoparticles prepared by ethanol induction alone, the particle size distribution diagram of the nanoparticles prepared by the method for assembling nano-embedded lutein by using the pH-collaborative ethanol-induced protein moves toward a smaller particle size, and when the ethanol concentration is 40% and 50%, the particle size of the nanoparticles shows a unimodal distribution, which is consistent with smaller PDI, indicating that the method for assembling nano-embedded lutein by using the pH-collaborative ethanol-induced protein can obtain protein-based lutein nanoparticles with smaller particle size and more uniform distribution. This is probably because under ph=12 conditions, the protein is sufficiently unfolded and even dissociated into smaller size subunits under alkaline conditions than induced by ethanol alone, and although the protein forms loose, larger aggregates in the presence of ethanol, the aggregates return to a state of separation from each other when ethanol is not added after rotary evaporation to remove ethanol, and the particle size is significantly smaller; the cells in which self-assembly occurs are smaller in size so that the resulting particles are also smaller and more uniform.

As can be seen from table 4 and fig. 8, as the ethanol concentration gradually increases, the particle size of the prepared nanoparticles also gradually increases. This is because ethanol has an effect of promoting aggregation of proteins, and the higher the concentration of ethanol, the more remarkable the aggregation of proteins, and as can be seen from (d) of FIG. 1, the solution becomes clear and transparent when the concentration of ethanol is 40% -50%, and becomes cloudy when the concentration of ethanol is increased to 60%.

TEM observation was performed on the protein-based lutein nanoparticles prepared under the condition of 40% ethanol concentration in example 4 and comparative example 1, and as shown in FIG. 9, the microscopic morphology of the nanoparticles provided in example 4 is in a regular circle, and the size is smaller and the distribution is more uniform than that of comparative example 1.

As can be seen from table 4, the protein-based lutein nanoparticle prepared by the method for assembling nano-embedded lutein by using the pH-synergistic ethanol-induced protein shows higher encapsulation efficiency and loading capacity, and the encapsulation efficiency of example 1 is higher than 90% and higher than that of comparative example 1. The method is characterized in that the protein is subjected to secondary unfolding and secondary folding in the preparation method of the protein assembled nano-embedded lutein by the pH synergistic ethanol induction, when the protein is in a pH=12 condition, the protein molecules are greatly unfolded or even dissociated Cheng Yaji, and the exposure degree of the hydrophobic groups is increased; when the lutein-absolute ethyl alcohol solution is introduced into the system, the protein structure is further unfolded by the ethyl alcohol, lutein and fully unfolded alkaline protein are quickly combined through hydrophobic interaction, lutein is encapsulated in a hydrophobic cavity of the alkaline protein for the first time in the process of removing the folding of the ethyl alcohol protein, the protein is folded and recombined again in the process of adjusting the pH value, the loose structure becomes compact, and the lutein originally positioned on the surface of the protein is encapsulated inside, so that the encapsulation rate is improved. When the ethanol concentration and the protein concentration are consistent, the higher the encapsulation efficiency is, the higher the load is.

Table 4 average particle diameter, PDI, encapsulation efficiency and load of nanoparticles prepared in example 4 and comparative example 1

5mL of the solution of the protein-based lutein nanoparticle prepared in the example 4 and 5mL of the free lutein-absolute ethyl alcohol solution are taken respectively, the solution is stored for 45 days in a dark place at 35 ℃ in an incubator, the lutein content is measured at 0d,7d,15d and 45d, the experiment is repeated three times, and the long-time storage retention rate of lutein is calculated according to the formula (4). As can be seen from FIG. 10, the retention rate of free lutein after being stored at 35 ℃ for 7d is only 4.60+/-0.16%, and the retention rate of lutein in the nano-particles provided in example 4 is above 90%, and the retention rate of lutein after 45d is also above 70%, which indicates that the protein-based lutein nano-particles provided in the example can remarkably improve the long-term storage stability of lutein.

A simulated in vitro digestion experiment was performed on 5mL of the protein-based lutein nanoparticle solution prepared in example 4 and 5mL of 1mg/mL lutein-absolute ethanol solution to determine the digestion stability and bioavailability thereof, and the experiment was repeated three times, and the results are shown in FIG. 11 and FIG. 12. After 120 minutes of simulated gastric digestion, the average retention of lutein in the form of solution (40%, 50%, 60%) was 97%, 94%, 93%, respectively, while the retention of free lutein was 88%; after digestion for 120 min in simulated small intestine, the retention of lutein in the protein-based lutein nanoparticles in solution form is above 85%, while the retention of free lutein is reduced to 35%. After the whole digestion process is finished, the average value of lutein bioavailability (the content of lutein in the supernatant after 10000g of digestion solution is centrifuged for 30min reflects the bioavailability) of the protein-based lutein nano particles (40% ethanol concentration, 50% ethanol concentration and 60% ethanol concentration) in the form of the solution prepared in the embodiment 4 is 38%, 32% and 30%, and the bioavailability of free lutein is only 4%. The protein-based lutein nanoparticle provided in the embodiment 4 can obviously improve the digestion stability and bioavailability of lutein, can be used as a raw material or an auxiliary material to be added into medicines and foods, and has great market potential.

Claims

1. The method for assembling the nano-embedded lutein by utilizing the pH and ethanol to induce the protein is characterized by comprising the following steps of:

(1) The pH of the protein dispersion is adjusted to be in an alkaline state, and the protein is fully stirred to be unfolded or dissociated into subunits, so that the particle size is obviously reduced, and the solution becomes clear and transparent; then adding a lutein-absolute ethyl alcohol solution, enabling the alkaline dissociated protein to be further unfolded through alcohol-water interaction, fully stirring to enable lutein to form a stable compound with the protein through hydrophobic interaction, and meanwhile, enhancing the hydrophobic interaction among alkaline dissociated protein particles to form a loose aggregate with larger size, so that the solution becomes turbid;

(2) Removing ethanol in the mixed solution by rotary evaporation, gradually overlapping the unfolded alkaline dissociation proteins, starting to converge the loose structure, enabling lutein to undergo first embedding, enabling the aggregated alkaline protein particles to return to a state of being separated from each other when no ethanol is added, remarkably reducing the particle size, and clarifying the solution again;

2. The method for assembling nano-embedded lutein by utilizing pH-coordinated ethanol-induced protein according to claim 1, wherein in the step (1), the protein is at least one of whey protein, serum protein, ovalbumin, lysozyme, beta-lactoglobulin, sodium caseinate, legume 7S globulin, legume 11S globulin, soy protein isolate and pea protein isolate.

3. The method for assembling nano-embedded lutein by utilizing pH to cooperate with ethanol to induce protein according to claim 1, wherein in the step (1), the protein concentration is 1-60mg/mL.

4. The method for assembling nano-embedded lutein by utilizing pH to cooperate with ethanol to induce protein according to claim 1, wherein in the step (1), the pH of the protein dispersion liquid is in the range of 10-12.

5. The method for assembling nano-embedded lutein by utilizing pH to cooperate with ethanol to induce protein according to claim 1, wherein in the step (1), the protein is fully stirred to be unfolded or dissociated into subunits, and the stirring time is 30-120min.

6. The method for assembling nano-embedded lutein by utilizing pH synergistic alcohol induced protein according to claim 1, wherein in the step (1), the volume ratio of the added lutein-absolute alcohol solution is 10% -70% of the mixed solution, and the stirring time is 30-120min.

7. The method for assembling nano-embedded lutein by utilizing pH to cooperate with ethanol to induce protein according to claim 1, wherein in the step (2), the rotary evaporation vacuum degree is 0-0.1MPa, the heating temperature is 35-45 ℃, and the rotating speed is 80-120rpm.

8. The method for assembling nano-embedded lutein by utilizing pH to cooperate with ethanol induced protein according to claim 1, wherein in the step (3), the pH is adjusted to be 6.8-8.0.

9. The protein-based lutein nanoparticle prepared by the preparation method of any one of claims 1 to 8.

10. The use of the protein-based lutein nanoparticle of claim 9: pigments, foods, medicines or feeds.