A strongly absorbent water-soluble carotenoid and its preparation method

Abstract

The present invention relates to a strongly absorbable water-soluble carotenoid and a preparation method thereof, specifically including the following steps: (1) preparation of water-soluble chitosan; (2) preparation of carotenoid-fatty acid mixture; (3) preparation of alkaline electrolyte water; (4) preparation of carotenoid-protein complex by electrolyzed water coupling ultrasonic technology; (5) preparation of chitosan-protein-fatty acid-carotenoid complex; (6) obtaining of water-soluble carotenoid. The present invention innovatively combines the alkaline electrolyzed water coupling ultrasonic technology and the non-covalent self-assembly technology to prepare a strongly absorbable water-soluble carotenoid. Compared with the untreated carotenoid, the carotenoid has good water solubility and stability, and the intestinal absorption efficiency is increased by 5 to 10 times. The preparation process of the present invention is simple, has a high embedding rate, good safety, and the product has good stability and high absorption rate, and can be widely used as a nutritional fortifier in the food system and is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, in particular to a highly absorbable water-soluble carotenoid and a preparation method thereof. Background Art

[0002] Many studies have shown that carotenoids have multiple functional properties such as anti-oxidation, anti-inflammatory, anti-obesity and prevention of cardiovascular diseases, and have broad application prospects in the food industry. However, as fat-soluble nutritional active substances, carotenoids have disadvantages such as poor water solubility, instability under light / heat and acid-base conditions, and low bioavailability. Therefore, how to simultaneously improve the solubility, processing stability and bioavailability of carotenoids is a technical problem that needs to be solved in the development of food nutritional preparations or functional foods using carotenoids.

[0003] At present, researchers at home and abroad have begun to use microemulsion carriers, nanoemulsion carriers, Pickering emulsions, and nanocomposite carriers to embed carotenoids. In order to produce relatively small diameter particles, large loading capacity, and good stability, some high-energy methods with strong destructive power and the use of chemical reagents are usually used: (1) There are safety hazards, strong acids and alkalis and organic solvent residues are added to the product; (2) It is difficult to absorb, and the delivery carrier is released too quickly or incompletely in the intestine, causing intestinal absorption disorders; (3) The production efficiency is low, and there is a large loss of carotenoids during the production process.

[0004] Natural macromolecules are green, environmentally friendly, and widely available. In recent years, they have been widely used in delivery carrier wall materials. However, carotenoids are heat-sensitive and easily oxidized, and their structure and activity will be destroyed to a certain extent at high temperatures. In addition, natural macromolecules such as proteins and polysaccharides are good culture media for microorganisms, which are very easy to breed microorganisms and cause corruption and deterioration. High-temperature sterilization or the addition of preservatives is usually required to increase the storage time of food. Alkaline electrolyzed water is water with electrolytes added to it. After being placed in a certain electrolysis device and treated by an electric field, the pH value, effective chlorine concentration, redox potential and other indicators of the water are changed, and alkaline electrofunctional water with certain functions is produced. Alkaline electrolyzed water has a high redox potential, a certain antibacterial effect, strong permeability, high safety and low cost. As an electro-generated functional water, the essence of electrolyzed water is still water. After being exposed to the air and contacted with light, oxygen and organic matter, its pH value and ORP have no residual properties, and all indicators gradually approach those of the original water. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the existing traditional nanoemulsion and the technical problem of low absorption rate, and to provide a highly absorbent water-soluble carotenoid and a preparation method thereof.

[0006] The present invention prepares water-soluble carotenoid microparticles by coupling alkaline electrolyzed water with ultrasonic self-assembly technology, which can not only overcome the defects of traditional nanoemulsions, without the need to add additional bacteriostatic agents and stabilizers, but also avoid using chemical reagents such as organic solvents, strong acids / bases, and surfactants with potential food safety hazards, and there is no residue of any toxic and harmful substances, fully ensuring the safety of the product; at the same time, high-pressure homogenization, high-speed shearing and other unit operations are not required during the production process, the production cost is low, and it is suitable for industrial production.

[0007] Compared with other nanoparticles, the water-soluble carotenoid microparticles prepared by the present invention can be sterilized without high temperature treatment, and the microparticle powder is also very stable, which is convenient for storage and transportation; compared with protein carriers, introducing glycosylated modified chitosan can delay the release of carotenoids in the gastrointestinal tract, and coupled with good absorption promotion effect, it has a wide range of uses in the fields of food and biomedicine.

[0008] The specific scheme is as follows:

[0009] A preparation method of strongly absorbable water-soluble carotenoids, comprising the following specific steps:

[0010] (1) Preparation of water-soluble chitosan powder: Mix chitosan, acetic acid solution and reducing sugar, carry out Maillard reaction, adjust the pH after the reaction and carry out drying treatment to obtain the water-soluble chitosan powder;

[0011] (2) Preparation of carotenoid-fatty acid mixture: Mix carotenoids, ethanol and fatty acids evenly to dissolve the carotenoids to obtain the carotenoid-fatty acid mixture;

[0012] (3) Preparation of alkaline electrolyte water: Using carbonate as the electrolyte, ionize the aqueous solution of the electrolyte, and obtain alkaline electrolyte water at the anode;

[0013] (4) Preparation of carotenoid-fatty acid-protein complex aqueous solution by electrolyzed water coupling ultrasonic technology: Mix protein powder with the alkaline electrolyte water to obtain a protein solution; then add the carotenoid-fatty acid mixture to the protein solution to obtain a mixed solution; subsequently, ultrasonically treat the mixed solution to cause self-assembly of the carotenoids, fatty acids and proteins in the mixed solution, and after the reaction, leave it open at room temperature to obtain the carotenoid-fatty acid-protein complex aqueous solution;

[0014] (5) Preparation of chitosan - protein - fatty acid - carotenoid complex: Mix the water - soluble chitosan powder prepared in step (1) with the carotenoid - fatty acid - protein complex aqueous solution prepared in step (4), and react. Through non - covalent self - assembly, chitosan binds to the protein shell of the complex to obtain the chitosan - protein - fatty acid - carotenoid complex aqueous solution;

[0015] (6) Obtaining water - soluble carotenoid dry powder: Dry the chitosan - protein - fatty acid - carotenoid complex aqueous solution to obtain the water - soluble carotenoid dry powder.

[0016] Optionally, the reducing sugar in step (1) includes at least one of glucose, fructose, galactose, xylose or mannose;

[0017] Optionally, after mixing chitosan, acetic acid solution and reducing sugar, the mass ratio of chitosan to reducing sugar in the solution is (1 - 2):(1 - 3), and the pH of the reaction system after mixing is 5.0 - 6.0;

[0018] Optionally, the temperature of the Maillard reaction is 60 - 70 °C and the time is 48 - 96 hours.

[0019] Optionally, the carotenoid in step (2) includes at least one of lutein, zeaxanthin, fucoxanthin, lycopene, β - carotene or capsanthin;

[0020] Optionally, the fatty acid includes at least one of myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid or docosahexaenoic acid;

[0021] Optionally, the mass ratio of the carotenoid to the fatty acid is 1:(3 - 5).

[0022] Furthermore, the ionization in step (3) is carried out at a voltage of 220V for 15 - 30 min continuously. The pH value of the obtained alkaline electrolyte water is 10.0 - 13.0, and the oxidation - reduction potential is - 670 - 860 mV.

[0023] Furthermore, the mass ratio of the carotenoid to the protein powder in the mixed solution in step (4) is (0.5 - 1.5):(15 - 25);

[0024] Preferably, the mass ratio of the carotenoid to the protein powder in the mixed solution is (0.8 - 1.2):(18 - 22);

[0025] More preferably, the mass ratio of the carotenoid to the protein powder in the mixed solution in step (4) is 1:20;

[0026] Optionally, the concentration of the protein is 5-15% by mass, and the ultrasonic treatment is carried out at a power of 300-500 W for 3-5 minutes to obtain an aqueous solution of the chitosan-protein-fatty acid-carotenoid complex, wherein the embedding rate of the carotenoid is ≥95%;

[0027] Optionally, after the reaction, it is left open at room temperature to make the pH of the system 7-8, and a clear aqueous solution of the carotenoid-protein complex is obtained.

[0028] Furthermore, the drying treatment in step (6) refers to spray drying or freeze drying after vacuum concentration.

[0029] The present invention also protects a water-soluble carotenoid dry powder prepared by the above method.

[0030] Furthermore, the particle size of the water-soluble carotenoid dry powder after redissolving in deionized water is 100-500 nm.

[0031] Furthermore, after heating the water-soluble carotenoid dry powder at 80-120 °C for 10-30 minutes, the retention rate of the carotenoid is ≥90%;

[0032] Furthermore, the absorption efficiency of the water-soluble carotenoid dry powder is increased by 5-10 times compared with the raw material carotenoid. Beneficial effects:

[0033] In the present invention, the water-soluble carotenoid is prepared by an alkaline electrolyzed water-ultrasonic coupling technology. Due to the strong reduction potential of alkaline electrolyzed water, it can effectively avoid the oxidation and degradation of carotenoids during the preparation process; by coupling alkaline electrolyzed water with ultrasonic technology, the embedding rate of carotenoids can be significantly increased, making the embedding rate ≥95%.

[0034] Moreover, the present invention uses water-soluble chitosan as the particle shell to achieve the controlled release of carotenoids during digestion. The present invention first uses the Maillard reaction modification technology to increase the pH dissolution range of chitosan, and then uses the non-covalent self-assembly technology to cover the surface of carotenoid particles with chitosan, which can effectively improve the stability of carotenoids during gastrointestinal digestion and achieve the effect of intestinal targeted slow release.

[0035] Furthermore, the present invention selectively introduces fatty acid components that promote the intestinal absorption of carotenoids, which can improve the absorption of carotenoids by the human body, thereby giving full play to the nutritional function of carotenoids existing in the human body.

[0036] In summary, a water-soluble carotenoid provided by the present invention is a product with good water solubility, strong stability, and high absorption rate. Its preparation method has a simple technical route, does not require high-pressure homogenization and high-speed shearing processing, has low production costs, and is suitable for large-scale industrial production in the market. Detailed implementation manners

[0037] The preferred implementation manners of the present invention will be described in more detail below. Although the preferred implementation manners of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the implementation manners described herein. For those not specified in the examples regarding specific technologies or conditions, the technologies or conditions described in the literature in the field or according to the product specifications shall be followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase. In the following examples, unless otherwise specified, "%" refers to weight percentage, and "parts" refers to parts by weight.

[0038] The measurement methods in the following examples are as follows:

[0039] The digestion stability, controlled release effect, and bioaccessibility of water-soluble lutein were analyzed using the standardized method of in vitro simulated food digestion by Brodkorb et al. (Brodkorb et al., Nature Protocols, 2019, 14: 991-1014).

[0040] Example 1

[0041] Chitosan powder was dissolved in an acetic acid solution (acetic acid concentration is 1% by volume), stirred evenly to ensure complete dissolution, the concentration of chitosan was 0.5% by mass, and then xylose was added in a mass ratio of 2:1. After mixing evenly, under the conditions of 65 °C and pH 5.0, the Maillard reaction was carried out for 96 hours. After the reaction, the pH was adjusted to neutral, and then spray-dried to obtain water-soluble chitosan powder. Lutein powder was dissolved in a small amount of ethanol solution, and then oleic acid was added in a mass ratio of 1:3 and stirred evenly to obtain a lutein fatty acid dispersion solution; 10.0 g of bovine serum albumin powder was dissolved in alkaline electrolyte water (pH 10.0, redox potential -670 mV), and stirred thoroughly to dissolve to obtain an alkaline protein electrolyte aqueous solution (the concentration of protein was 10% by mass); then the lutein-fatty acid mixture was added to the protein solution so that the mass ratio of lutein to protein was 1:20; ultrasonic treatment was carried out at a power of 300 W for 5 minutes to make lutein, fatty acid, and protein undergo uniform self-assembly; it was left open at room temperature to allow the pH of the reaction system to naturally return to 7.6, and a clear aqueous solution of lutein-fatty acid-protein complex was obtained. A part of the prepared water-soluble chitosan powder was added to the aqueous solution of lutein-fatty acid-protein complex so that the mass ratio of lutein, fatty acid, protein, and water-soluble chitosan was 1:3:20:1, mixed well and allowed to stand for 30 minutes, and chitosan was combined with the protein shell of the complex through non-covalent self-assembly to obtain an aqueous solution of chitosan-protein-fatty acid-lutein complex (the entrapment efficiency of lutein was 97.9%); spray-dried, or freeze-dried after concentration under reduced pressure to obtain water-soluble lutein dry powder.

[0042] The physicochemical properties, gastrointestinal digestion stability, controlled release effect, and bioaccessibility of the water-soluble lutein dry powder were further investigated: after it was redissolved in deionized water, a clear and transparent solution was formed, and the particle size was 100.0 nm; the retention rate of water-soluble lutein microparticles after being heated at 80 °C for 30 min was 90.6%. The results showed that the retention rate of lutein after simulated gastric digestion was 90.8%, and the retention rate after simulated intestinal digestion was 86.6%; after simulated gastrointestinal digestion, the release rate of lutein was 97.4%, and the bioaccessibility was increased by 21.7 times.

[0043] Example 2

[0044] Dissolve chitosan powder in acetic acid solution (acetic acid concentration is 1% by volume), stir evenly to make it fully dissolve, the concentration of chitosan is 0.5% by mass, then add glucose in a mass ratio of 1:1, mix evenly and carry out Maillard reaction for 72 hours under the conditions of 65 °C and pH 5.5. After the reaction, adjust the pH to neutral and spray dry to obtain water-soluble chitosan powder. Dissolve zeaxanthin powder in a small amount of ethanol solution, and then add linoleic acid in a mass ratio of 1:3 and stir evenly to obtain a zeaxanthin fatty acid dispersion solution; dissolve 10.0 g of bovine serum albumin powder in alkaline electrolyte water (pH 11.0, redox potential -706 mV), stir well to dissolve to obtain an alkaline protein electrolyte aqueous solution (protein concentration is 10% by mass); then add the zeaxanthin-fatty acid mixture to the protein solution so that the mass ratio of zeaxanthin to protein is 1:20; ultrasonically treat for 4 minutes at a power of 350 W to make zeaxanthin, fatty acid, and protein undergo uniform self-assembly; leave it open at room temperature to make the pH of the reaction system naturally return to 7.0 to obtain a clear aqueous solution of zeaxanthin-fatty acid-protein complex. Add a part of the prepared water-soluble chitosan powder to the aqueous solution of zeaxanthin-fatty acid-protein complex so that the mass ratio of zeaxanthin, fatty acid, protein, and water-soluble chitosan is 1:3:20:2, mix well and let it stand for 30 minutes, and make chitosan bind to the protein shell of the complex through non-covalent self-assembly to obtain an aqueous solution of chitosan-protein-fatty acid-zeaxanthin complex (zeaxanthin entrapment rate is 95.0%); spray dry, or carry out freeze-drying after concentration under reduced pressure to obtain water-soluble zeaxanthin dry powder.

[0045] Further investigate the physicochemical properties, gastrointestinal digestion stability, controlled release effect, and bioaccessibility of the water-soluble zeaxanthin dry powder: After redissolving it in deionized water, a clear and transparent solution is formed with a particle size of 156 nm; the retention rate of water-soluble zeaxanthin microparticles after being heated at 100 °C for 15 min is 90.0%. The results show that the retention rate of zeaxanthin after simulated gastric digestion is 93.2%, and the retention rate after simulated intestinal digestion is 89.5%; after simulated gastrointestinal digestion, the release rate of zeaxanthin is 96.5%, and the bioaccessibility is increased by 22.2 times.

[0046] Example 3

[0047] Dissolve chitosan powder in acetic acid solution (acetic acid concentration is 1% by volume), stir evenly to make it fully dissolved, the concentration of chitosan is 0.5% by mass, then add fructose according to the mass ratio of 1:2, mix evenly, and under the conditions of 65 °C and pH 5.5, carry out Maillard reaction for 72 hours. After the reaction, adjust the pH to neutral, and spray-dry to obtain water-soluble chitosan powder. Dissolve fucoxanthin powder in a small amount of ethanol solution, and then add myristic acid according to the mass ratio of 1:4 and stir evenly to obtain a fucoxanthin fatty acid dispersion solution; dissolve 10.0 g of bovine serum albumin powder in alkaline electrolyte water (pH 12.0, redox potential -736 mV), stir fully to dissolve, and obtain an alkaline protein electrolyte aqueous solution (the concentration of protein is 10% by mass); then add the fucoxanthin-fatty acid mixture to the protein solution so that the mass ratio of fucoxanthin to protein is 1:20; ultrasonically treat for 3.5 minutes at a power of 400 W to make fucoxanthin, fatty acid, and protein undergo uniform self-assembly; place it open at room temperature to make the pH of the reaction system naturally return to 8.0 to obtain a clear aqueous solution of fucoxanthin-fatty acid-protein complex. Add a part of the prepared water-soluble chitosan powder to the aqueous solution of fucoxanthin-fatty acid-protein complex so that the mass ratio of fucoxanthin, fatty acid, protein, and water-soluble chitosan is 1:4:20:3, mix well and let it stand for 30 minutes, and make chitosan bind to the protein shell of the complex through non-covalent self-assembly to obtain an aqueous solution of chitosan-protein-fatty acid-fucoxanthin complex (the embedding rate of fucoxanthin is 97.4%); spray-dry, or carry out freeze-drying after concentration under reduced pressure to obtain water-soluble fucoxanthin dry powder.

[0048] Further investigate the physical and chemical properties, gastrointestinal digestion stability, controlled release effect, and bioaccessibility of this water-soluble fucoxanthin dry powder: After redissolving it in deionized water, a clear and transparent solution is formed, and the particle size is 200.0 nm; the retention rate of water-soluble fucoxanthin particles after being heated at 100 °C for 15 min is 91.5%. The results show that the retention rate of fucoxanthin after simulated gastric digestion is 92.2%, and the retention rate after simulated intestinal digestion can reach 85.4%; after simulated gastrointestinal digestion, the release rate of fucoxanthin is 98.0%, and the bioaccessibility is increased by 19.8 times.

[0049] Example 4

[0050] Chitosan powder was dissolved in an acetic acid solution (acetic acid concentration was 1% by volume), stirred evenly to fully dissolve it, the concentration of chitosan was 0.5% by mass, and then galactose was added at a mass ratio of 1:3. After mixing evenly, Maillard reaction was carried out at 65 °C and pH 6.0 for 48 hours. After the reaction ended, the pH was adjusted to neutral, and then spray-dried to obtain water-soluble chitosan powder. Lycopene powder was dissolved in a small amount of ethanol solution, and then palmitic acid was added at a mass ratio of 1:4 and stirred evenly to obtain a lycopene fatty acid dispersion solution; 10.0 g of bovine serum albumin powder was dissolved in alkaline electrolyte water (pH 13.0, redox potential -860 mV), and stirred fully to dissolve it to obtain an alkaline protein electrolyte aqueous solution (protein concentration was 10% by mass); then the lycopene-fatty acid mixture was added to the protein solution so that the mass ratio of lycopene to protein was 1:20; ultrasonic treatment was carried out at a power of 500 W for 3 minutes to make lycopene, fatty acid, and protein undergo uniform self-assembly; it was placed open at room temperature to make the pH of the reaction system naturally return to 7.5, and a clear aqueous solution of lycopene-fatty acid-protein complex was obtained. The prepared water-soluble chitosan powder was added to the aqueous solution of lycopene-fatty acid-protein complex so that the mass ratio of lycopene, fatty acid, protein, and water-soluble chitosan was 1:4:20:5, mixed well and allowed to stand for 30 minutes, and chitosan was combined with the protein shell of the complex through non-covalent self-assembly to obtain an aqueous solution of chitosan-protein-fatty acid-lycopene complex (lycopene encapsulation rate was 96.7%); spray-dried, or freeze-dried after concentration under reduced pressure, to obtain water-soluble lycopene dry powder.

[0051] The physicochemical properties, gastrointestinal digestion stability, controlled release effect, and bioaccessibility of the water-soluble lycopene dry powder were further investigated: after it was redissolved in deionized water, a clear and transparent solution was formed, and the particle size was 398 nm; the retention rate of the water-soluble lycopene microparticles after being heated at 120 °C for 10 min was 93.7%. The results showed that the retention rate of lycopene after simulated gastric digestion was 95.6%, and the retention rate after simulated intestinal digestion was 92.8%; after simulated gastrointestinal digestion, the release rate of lycopene was 96.3%, and the bioaccessibility was increased by 25.8 times.

[0052] Example 5

[0053] Dissolve chitosan powder in acetic acid solution (acetic acid concentration is 1 vol%), stir evenly to make it fully dissolved, the concentration of chitosan is 0.5 wt%, then add mannose in a mass ratio of 1:2, mix evenly and carry out Maillard reaction at 65 °C and pH 6.0 for 48 hours. After the reaction, adjust the pH to neutral and spray dry to obtain water-soluble chitosan powder. Dissolve capsanthin powder in a small amount of ethanol solution, then add stearic acid in a mass ratio of 1:5 and stir evenly to obtain a capsanthin fatty acid dispersion solution; dissolve 10.0 g of bovine serum albumin powder in alkaline electrolyte water (pH 11.0, redox potential -706 mV), stir well to dissolve to obtain an alkaline protein electrolyte aqueous solution (protein concentration is 10 wt%); then add the capsanthin-fatty acid mixture to the protein solution so that the mass ratio of capsanthin to protein is 1:20; sonicate for 5 minutes at a power of 300 W to make capsanthin, fatty acid, and protein undergo uniform self-assembly; leave it open at room temperature to make the pH of the reaction system naturally return to 7.4 to obtain a clear capsanthin-fatty acid-protein complex aqueous solution. Add a part of the prepared water-soluble chitosan powder to the capsanthin-fatty acid-protein complex aqueous solution so that the mass ratio of capsanthin, fatty acid, protein, and water-soluble chitosan is 1:5:20:4, mix well and let it stand for 30 minutes, and bind chitosan to the protein shell of the complex through non-covalent self-assembly to obtain a chitosan-protein-fatty acid-capsanthin complex aqueous solution (capsanthin encapsulation rate is 95.4%); spray dry, or carry out freeze-drying after concentration under reduced pressure to obtain water-soluble capsanthin dry powder.

[0054] Further investigate the physical and chemical properties, gastrointestinal digestion stability, controlled release effect, and bioaccessibility of the water-soluble capsanthin dry powder: After redissolving it in deionized water, a clear and transparent solution is formed with a particle size of 500 nm; the retention rate of the water-soluble capsanthin particles after being heated at 100 °C for 15 min is 92.3%. The results show that the retention rate of capsanthin after simulated gastric digestion is 93.7%, and the retention rate after simulated intestinal digestion is 90.3%; after simulated gastrointestinal digestion, the release rate of capsanthin is 96.9%, and the bioaccessibility is increased by 23.4 times.

[0055] Example 6

[0056] Dissolve chitosan powder in acetic acid solution (acetic acid concentration is 1% by volume), stir evenly to fully dissolve it, with the chitosan concentration being 0.5% by mass. Then add mannose in a mass ratio of 1:2, mix evenly, and carry out the Maillard reaction at 65 °C and pH 6.0 for 48 hours. After the reaction, adjust the pH to neutral and spray-dry to obtain water-soluble chitosan powder. Dissolve β-carotene powder in a small amount of ethanol solution, and then add docosahexaenoic acid in a mass ratio of 1:5 and stir evenly to obtain a β-carotene fatty acid dispersion solution; dissolve 10.0 g of bovine serum albumin powder in alkaline electrolyte water (pH 11.0, redox potential -706 mV), stir fully to dissolve, and obtain an alkaline protein electrolyte aqueous solution (protein concentration is 10% by mass); then add the β-carotene-fatty acid mixture to the protein solution so that the mass ratio of β-carotene to protein is 1:20; carry out ultrasonic treatment at a power of 300 W for 5 minutes to make β-carotene, fatty acid, and protein undergo uniform self-assembly; place it open at room temperature to allow the pH of the reaction system to naturally return to 7.4, and obtain a clear aqueous solution of β-carotene-fatty acid-protein complex. Add a part of the prepared water-soluble chitosan powder to the aqueous solution of β-carotene-fatty acid-protein complex so that the mass ratio of β-carotene, fatty acid, protein, and water-soluble chitosan is 1:5:20:4, mix well and let it stand for 30 minutes, and make chitosan bind to the protein shell of the complex through non-covalent self-assembly to obtain an aqueous solution of chitosan-protein-fatty acid-β-carotene complex (β-carotene encapsulation rate is 97.2%); spray-dry, or carry out freeze-drying after concentration under reduced pressure to obtain water-soluble β-carotene dry powder.

[0057] Further investigate the physicochemical properties, gastrointestinal digestion stability, controlled release effect, and bioaccessibility of the water-soluble β-carotene dry powder: After redissolving it in deionized water, a clear and transparent solution is formed with a particle size of 460 nm; the retention rate of the water-soluble β-carotene particles after being heated at 100 °C for 15 min is 93.5%. The results show that the retention rate of β-carotene after simulated gastric digestion is 92.5%, and the retention rate after simulated intestinal digestion is 89.7%; after simulated gastrointestinal digestion, the release rate of β-carotene is 96.4%, and the bioaccessibility is increased by 21.6 times.

[0058] Example 7

[0059] Refer to the specific operation method of Example 1, the difference is only that in this example, soy protein is used to prepare water-soluble carotenoid dry powder.

[0060] Example 8

[0061] Referring to the specific operation method of Example 1, the difference is only that casein is used to prepare the water-soluble carotenoid dry powder in this example.

[0062] Example 9

[0063] Referring to the specific operation method of Example 1, the difference is only that the Maillard reaction conditions in this example are 60 °C and pH 6.0, and the mass ratio of lutein to protein is 0.5:15. The specific operation is as follows:

[0064] Dissolve chitosan powder in an acetic acid solution (acetic acid concentration is 1% by volume), stir evenly to make it fully dissolve, the concentration of chitosan is 0.5% by mass, then add xylose in a mass ratio of 2:1, mix evenly and carry out the Maillard reaction at 60 °C and pH 6.0 for 96 hours. After the reaction, adjust the pH to neutral and spray-dry to obtain water-soluble chitosan powder. Dissolve lutein powder in a small amount of ethanol solution, then add oleic acid in a mass ratio of 1:3 and stir evenly to obtain a lutein fatty acid dispersion solution; dissolve 10.0 g of bovine serum albumin powder in alkaline electrolyte water (pH 10.0, redox potential -670 mV), stir well to dissolve to obtain an alkaline protein electrolyte aqueous solution (protein concentration is 5% by mass); then add the lutein-fatty acid mixture to the protein solution to make the mass ratio of lutein to protein 0.5:15; ultrasonically treat at a power of 300 W for 5 minutes to make lutein, fatty acid, and protein undergo uniform self-assembly; leave it open at room temperature to make the pH of the reaction system naturally return to 7.6 to obtain a clear aqueous solution of lutein-fatty acid-protein complex. Add a part of the prepared water-soluble chitosan powder to the aqueous solution of lutein-fatty acid-protein complex to make the mass ratio of lutein, fatty acid, protein, and water-soluble chitosan 0.5:1.5:15:1, mix well and let it stand for 30 minutes, and make chitosan bind to the protein shell of the complex through non-covalent self-assembly to obtain an aqueous solution of chitosan-protein-fatty acid-lutein complex; spray-dry, or carry out freeze-drying after concentration under reduced pressure to obtain water-soluble lutein dry powder.

[0065] Example 10

[0066] Referring to the specific operation method of Example 1, the difference is only that the Maillard reaction conditions in this example are 70 °C and pH 5.0, and the mass ratio of lutein to protein is 1.5:25. The specific operation is as follows:

[0067] Dissolve chitosan powder in acetic acid solution (acetic acid concentration is 1% by volume), stir evenly to fully dissolve it, the concentration of chitosan is 0.5% by mass, then add xylose in a mass ratio of 2:1, mix evenly, and carry out Maillard reaction for 96 hours under the conditions of 70 °C and pH 5.0. After the reaction, adjust the pH to neutral, and spray-dry to obtain water-soluble chitosan powder. Dissolve lutein powder in a small amount of ethanol solution, and then add oleic acid in a mass ratio of 1:3 and stir evenly to obtain a lutein fatty acid dispersion solution; dissolve 10.0 g of bovine serum albumin powder in alkaline electrolyte water (pH 10.0, redox potential -670 mV), stir well to dissolve it, and obtain an alkaline protein electrolyte aqueous solution (the concentration of protein is 15% by mass); then add the lutein-fatty acid mixture to the protein solution so that the mass ratio of lutein to protein is 1.5:25; carry out ultrasonic treatment for 5 minutes at a power of 300 W to make lutein, fatty acid, and protein undergo uniform self-assembly; place it open at room temperature to make the pH of the reaction system naturally return to 7.6 to obtain a clear aqueous solution of lutein-fatty acid-protein complex. Add a part of the prepared water-soluble chitosan powder to the aqueous solution of lutein-fatty acid-protein complex so that the mass ratio of lutein, fatty acid, protein, and water-soluble chitosan is 1.5:4.5:25:1, mix well and let it stand for 30 minutes, and make chitosan bind to the protein shell of the complex through non-covalent self-assembly to obtain an aqueous solution of chitosan-protein-fatty acid-lutein complex; spray-dry it, or carry out freeze-drying after concentration under reduced pressure to obtain water-soluble lutein dry powder.

[0068] Example 11

[0069] Refer to the specific operation method of Example 10

[0070] The method is only different in that the mass ratio of lutein to protein in this example is 0.8:18.

[0071] Example 12

[0072] Refer to the specific operation method of Example 10, the difference is only that the mass ratio of lutein to protein in this example is 1.2:22.

[0073] Example 13 (Absorption effect evaluation)

[0074] Test samples: Unencapsulated carotenoids and the water-soluble carotenoid dry powders obtained in Examples 1-6.

[0075] Experimental animals: SPF-grade female ICR mice, weighing 15 - 20 g, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. The composition of the basal diet for mice: carbohydrates 64%, protein 21%, fat 4%, fiber 5%, water 6%. The above feed was purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Breeding room temperature: 23 ± 2 °C, relative humidity 55 ± 5%.

[0076] Animal grouping and treatment method: After 1 week of adaptive feeding, the mice were randomly divided into 6 groups with 9 mice in each group, namely the blank group (gavage with deionized water), the groups of unembedded carotenoids (lutein, zeaxanthin, fucoxanthin, lycopene, β-carotene, capsanthin), and the groups of water-soluble carotenoid dry powders obtained from Examples 1 - 6. The water-soluble carotenoid dry powder was dissolved in deionized water to prepare a test solution (unembedded carotenoids required ethanol for solubilization). Each mouse was gavaged with 200 μL of the test solution or deionized water each time. After 4 hours, the mouse eyeballs were removed, and the changes in the contents of carotenoids and their metabolites in plasma were analyzed by high-performance liquid chromatography (HPLC) method (Huang et al., Food Hydrocolloids, 2022, 123: 107152). The total contents of carotenoids and their metabolites in plasma and the serum response concentration were calculated according to the standard curve method, so as to reflect the intestinal absorption efficiency of carotenoids.

[0077] The alkaline electrolyzed water - ultrasonic coupling technology of the present invention for preparing water-soluble carotenoid nanoparticles can effectively improve the intestinal absorption efficiency of carotenoids. The specific experimental results are shown in Table 1.

[0078] Table 1. Promotion effect of molecular self-assembly on the absorption effect of carotenoids

[0079] Grouping Total response concentration of serum fucoxanthin (pmol / mL) Blank group Not detected Lutein (unembedded) 26.98 Zeaxanthin (unembedded) 31.54 Fucoxanthin (unembedded) 51.67 Lycopene (unembedded) 10.56 β-Carotene (unembedded) 15.82 Capsanthin (unembedded) 12.35 Example 1 (water-soluble lutein) 184.54 Example 2 (water-soluble zeaxanthin) 206.89 Example 3 (water-soluble fucoxanthin) 258.35 Example 4 (water-soluble lycopene) 106.20 Example 5 (water-soluble β-carotene) 121.02 Example 6 (water-soluble capsanthin) 105.22

[0080] The total response concentration of carotenoids in the sera of mice in each treatment group was analyzed by HPLC, and the results are shown in Table 1. The results indicated that after oral gavage of unencapsulated lutein, zeaxanthin, fucoxanthin, lycopene, β-carotene, and capsanthin, the total serum response concentrations were only 26.98 pmol / mL, 31.54 pmol / mL, 51.67 pmol / mL, 10.56 pmol / mL, 15.82 pmol / mL, and 12.35 pmol / mL, respectively. When encapsulated by the technology of the present invention, the absorption effect was significantly improved, and the total serum response concentrations of lutein, zeaxanthin, fucoxanthin, lycopene, β-carotene, and capsanthin were increased by 6.84-fold, 6.55-fold, 5.00-fold, 10.06-fold, 7.65-fold, and 8.52-fold, respectively. It was demonstrated that the preparation of water-soluble carotenoid nanoparticles by the alkaline electrolyzed water-ultrasound coupling technology of the present invention could effectively improve the intestinal absorption efficiency of carotenoids.

[0081] In Comparative Example 1, no Maillard reaction was carried out on chitosan.

[0082] Referring to the specific operation method of Example 1, the difference was only that the chitosan powder was directly used in this comparative example.

[0083] The specific steps were as follows:

[0084] Dissolve lutein powder in a small amount of ethanol solution, and then add oleic acid in a mass ratio of 1:3 and stir evenly to obtain a lutein fatty acid dispersion solution; dissolve 10.0 g of bovine serum albumin powder in alkaline electrolyte water (pH 10.0, redox potential -670 mV), stir well to dissolve, and obtain an alkaline protein electrolyte aqueous solution (the protein concentration is 10% by mass); then add the lutein-fatty acid mixture to the protein solution so that the mass ratio of lutein to protein is 1:20; perform ultrasonic treatment for 5 minutes at a power of 300 W to enable uniform self-assembly of lutein, fatty acid, and protein; place it open to the air at room temperature to adjust the pH of the reaction system to 7.6, and obtain an aqueous solution of lutein-fatty acid-protein complex. Add chitosan powder to the aqueous solution of lutein-fatty acid-protein complex so that the mass ratio of lutein, fatty acid, protein, and chitosan is 1:3:20:1, mix well and let stand for 30 minutes. The unmodified chitosan without Maillard reaction precipitated directly because it was insoluble in water. Centrifuge to obtain an aqueous solution of protein-fatty acid-lutein complex (the encapsulation rate of lutein was 90.6%); perform spray drying, or perform freeze drying after vacuum concentration to obtain lutein dry powder.

[0085] Further investigate the physicochemical properties, gastrointestinal digestion stability, controlled release effect and bioaccessibility of the water-soluble lutein dry powder: After redissolving it in deionized water, the formed solution has a particle size of 85 nm; the retention rate of the water-soluble lutein microparticles after being treated at 100 °C for 15 min is 68.4%. The results show that the retention rate of lutein after simulated gastric digestion is 65.2%, and the retention rate after simulated intestinal digestion is 41.3%; after simulated gastrointestinal digestion, the release rate of lutein is 98.2%, and the bioaccessibility is increased by 4.5 times.

[0086] In Comparative Example 2, the alkaline electrolyzed water was replaced with an alkaline aqueous solution

[0087] Operate according to the specific method of Example 1, and the main difference is only that in this comparative example, an alkaline aqueous solution (sodium hydroxide aqueous solution) is used to prepare the water-soluble lutein dry powder. The specific steps are as follows:

[0088] Dissolve the chitosan powder in an acetic acid solution (acetic acid concentration is 1% by volume), stir evenly to make it fully dissolve, the concentration of chitosan is 0.5% by mass, then add xylose according to a mass ratio of 2:1, mix evenly and carry out the Maillard reaction at 65 °C and pH 5.0 for 96 hours. After the reaction, adjust the pH to neutral and spray dry to obtain the water-soluble chitosan powder. Dissolve the lutein powder in a small amount of ethanol solution, then add oleic acid according to a mass ratio of 1:3 and stir evenly to obtain a lutein fatty acid dispersion solution; dissolve 10.0 g of bovine serum albumin powder in a sodium hydroxide aqueous solution (pH 10.0), stir well to dissolve to obtain an alkaline protein aqueous solution (protein concentration is 10% by mass); then add the lutein-fatty acid mixture to the protein solution so that the mass ratio of lutein to protein is 1:20; ultrasonically treat for 5 minutes at a power of 300 W to obtain a lutein-fatty acid-protein complex aqueous solution. Add a part of the prepared water-soluble chitosan powder to the lutein-fatty acid-protein complex aqueous solution so that the mass ratio of lutein, fatty acid, protein, and water-soluble chitosan is 1:3:20:1, mix well and let stand for 30 minutes, and make the chitosan bind to the protein shell of the complex through non-covalent self-assembly to obtain a chitosan-protein-fatty acid-lutein complex aqueous solution (lutein entrapment rate is 78.4%); spray dry, or carry out freeze-drying after concentration under reduced pressure to obtain the water-soluble lutein dry powder.

[0089] Further investigate the physicochemical properties, gastrointestinal digestion stability, controlled release effect and bioaccessibility of the water-soluble lutein dry powder: After redissolving it in deionized water, a clear and transparent solution is formed with a particle size of 102 nm; the retention rate of the water-soluble lutein microparticles after being treated at 100 °C for 15 min is 70.5%. The results show that the retention rate of lutein after simulated gastric digestion is 71.2%, and the retention rate after simulated intestinal digestion is 62.4%; after simulated gastrointestinal digestion, the release rate of lutein is 96.2%, and the bioaccessibility is increased by 5.3 times.

[0090] It can be seen from Comparative Examples 1-2 that the alkaline aqueous solution used in the present invention does not have the characteristic of strong reduction potential and cannot effectively avoid the oxidative degradation of carotenoids during the preparation process; even when the alkaline aqueous solution is coupled with the ultrasonic technology, the encapsulation rate of carotenoids can only reach 78.4%. The too low encapsulation rate caused by oxidative degradation damage also directly affects the stability and bioavailability of the product during gastrointestinal digestion. It can be seen from Comparative Example 2 that when the alkaline electrolyzed water is replaced with ordinary alkaline water (other preparation conditions are the same), the retention rates after gastric and intestinal digestion are reduced from 90.8% and 86.6% to 71.2% and 62.4% respectively, and the improvement effect of bioaccessibility is also reduced from an increase of 21.7 times to an increase of 5.3 times.

[0091] On the other hand, under the condition of not using the Maillard reaction technology to improve the solubility of chitosan, chitosan cannot undergo molecular self-assembly with proteins, fatty acids and carotenoids. It can be seen from Comparative Example 1 that when chitosan cannot bind to the surface of the lutein-fatty acid-protein complex, the encapsulation rate of lutein also decreases, and its encapsulation rate drops from 97.9% to 90.6%; at the same time, due to the loss of the protective effect of the chitosan outer shell, the heating stability also decreases, and the retention rate after being treated at 100 °C for 15 min drops from 90.6% to 70.5%. More importantly, without the protection of the chitosan outer shell, the gastrointestinal digestion stability and bioaccessibility of water-soluble lutein also significantly decrease: the retention rates after gastric and intestinal digestion are reduced from 90.8% and 86.6% to 65.2% and 41.3% respectively, and the improvement effect of bioaccessibility is also reduced from an increase of 21.7 times to an increase of 4.5 times.

[0092] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0093] In addition, it should be noted that, among the various specific technical features described in the above specific embodiments, they can be combined in any appropriate manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0094] In addition, any combination can be made among various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should equally be regarded as the content disclosed by the present invention.

Claims

1. A preparation method of a strongly absorbent water-soluble carotenoid, characterized in that: It includes the following specific steps: (1) Preparation of water-soluble chitosan powder: Chitosan, acetic acid solution, and reducing sugar are mixed to carry out the Maillard reaction. After the reaction, the pH is adjusted and dried to obtain the water-soluble chitosan powder; after mixing chitosan, acetic acid solution, and reducing sugar, the mass ratio of chitosan to reducing sugar in the solution is (1-2):(1-3), and the pH of the reaction system after mixing is 5.0-6.0; the temperature of the Maillard reaction is 60-70 °C, and the time is 48-96 hours; (2) Preparation of carotenoid-fatty acid mixture: Carotenoid, ethanol, and fatty acid are mixed evenly to dissolve the carotenoid to obtain the carotenoid-fatty acid mixture; (3) Preparation of alkaline electrolyte water: Using carbonate as the electrolyte, the aqueous solution of the electrolyte is ionized, and alkaline electrolyte water is prepared at the anode; the ionization is carried out at a voltage of 220 V for 15-30 min continuously, and the pH value of the obtained alkaline electrolyte water is 10.0-13.0, and the redox potential is -670--860 mV; (4) Preparation of carotenoid-fatty acid-protein complex aqueous solution by electrolyzed water coupling ultrasonic technology: Protein powder is mixed with the alkaline electrolyte water to obtain a protein solution, and the concentration of protein in the protein solution is 5-15% by mass; then the carotenoid-fatty acid mixture is added to the protein solution to obtain a mixed solution; subsequently, the mixed solution is ultrasonically treated to cause self-assembly of the carotenoid, fatty acid, and protein in the mixed solution. After the reaction, it is left open at room temperature to obtain the carotenoid-fatty acid-protein complex aqueous solution; the mass ratio of carotenoid to protein powder in the mixed solution is 1:20; the ultrasonic treatment is carried out at a power of 300-500 W for 3-5 minutes to obtain the carotenoid-fatty acid-protein complex aqueous solution, wherein the encapsulation rate of the carotenoid is ≥95%; the protein powder is bovine serum albumin powder; (5) Preparation of chitosan-protein-fatty acid-carotenoid complex: The water-soluble chitosan powder prepared in step (1) is mixed with the carotenoid-fatty acid-protein complex aqueous solution prepared in step (4) to react, and chitosan is combined with the protein shell of the complex through non-covalent self-assembly to obtain a chitosan-protein-fatty acid-carotenoid complex aqueous solution; (6) Obtaining of water-soluble carotenoid dry powder: The chitosan-protein-fatty acid-carotenoid complex aqueous solution is dried to obtain the water-soluble carotenoid dry powder.

2. The preparation method of a strongly absorbent water-soluble carotenoid according to claim 1, characterized in that: The reducing sugar in step (1) includes at least one of glucose, fructose, galactose, xylose, or mannose.

3. The preparation method of a strongly absorbent water-soluble carotenoid according to claim 1, characterized in that: The carotenoid in step (2) includes at least one of lutein, zeaxanthin, fucoxanthin, lycopene, β-carotene, or capsanthin.

4. The preparation method of a strongly absorbent water-soluble carotenoid according to claim 3, characterized in that: The fatty acids include at least one of myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid or docosahexaenoic acid.

5. The preparation method of a strongly absorbable water-soluble carotenoid according to claim 3, characterized in that: The mass ratio of the carotenoid to the fatty acid is 1:(3-5).

6. The preparation method of a strongly absorbent water-soluble carotenoid according to claim 1, characterized in that: In step (4), after the reaction, it is left open at room temperature to make the pH of the system 7-8, and a clear aqueous solution of the carotenoid-fatty acid-protein complex is obtained.

7. The preparation method of a strongly absorbent water-soluble carotenoid according to claim 1, characterized in that: The drying treatment in step (6) refers to spray drying or freeze drying after concentration under reduced pressure.

8. A water-soluble carotenoid dry powder prepared by the method according to any one of claims 1-7.