A lutein-loaded composite modified wheat gliadin nanoparticle, preparation and use thereof

Abstract

The present invention belongs to the technical field of processing zein-loaded drug nanoparticles of wheat, and particularly relates to a lutein-loaded composite modified zein nanoparticle of wheat, a preparation thereof and uses. The composite modified zein nanoparticle of wheat is modified by enzymatic hydrolysis combined with dextran grafting. Among them, the dextran grafting adopts a low temperature-high temperature cycling method, which significantly improves the grafting degree and has a lower degree of browning. The lutein-loaded composite modified zein nanoparticle of wheat adopts a pH folding combined with ultrasonic method, which significantly improves the encapsulation rate of lutein. The obtained nanoparticles can significantly improve the light and heat stability of the nanoparticles and the stability in the gastrointestinal environment, and are suitable for improving the oral utilization rate of lutein, and are used for preparing oral drugs, oral health foods, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of processing zein-loaded drug nanoparticles, and particularly relates to a lutein-loaded composite modified zein nanoparticle, a preparation and a use thereof. Background Art

[0002] Lutein is a natural flavonoid compound widely present in daily diet, and has functions such as antioxidant, antibacterial and anti-inflammatory effects. However, defects such as poor stability, low solubility and low bioavailability of lutein limit the application of lutein in the fields of health care and food. For this reason, a variety of loading systems have been developed, such as solid dispersions, microemulsions, liposomes, polymer nanoparticles, etc. to improve the stability, solubility and bioavailability of lutein. However, the protection effects of such carriers on lutein vary greatly, and there are potential safety hazards after being put into commercial production, which is not conducive to further promotion. In addition, the preparation cycle of various carriers is relatively long, the steps are cumbersome, and the operation is complex, which is not conducive to commercial continuous production.

[0003] Biomacromolecules in food matrices, such as proteins, can be used as carriers for loading lutein. However, the natural properties of proteins themselves have deficiencies. To improve their loading and protection properties, proteins are often modified, such as enzymatic modification or chemical modification (glycosylation or phosphorylation modification, etc.). Patent document CN114159409A provides a zein nanoparticle loaded with lutein. The zein is modified by enzymatic hydrolysis combined with glycosylation modification (glucosamine modification), which can improve the stability of lutein. The glycosylation modification requires the aid of transglutaminase.

[0004] Research shows that in the glycosylation modification of zein, the harmful products of polysaccharide modification are lower than those of monosaccharides. However, when using polysaccharide-modified zein to load substances such as quercetin, rutin, ferulic acid, chlorogenic acid, emodin, capsaicin, lutein, etc., the polysaccharide-modified zein is usually modified under strong alkaline and high temperature conditions, while substances such as lutein have poor stability under strong alkaline or high temperature conditions, making it a new technical problem how to effectively load unstable substances such as quercetin and lutein with polysaccharide-modified zein (Analysis of influencing factors for the formation of harmful products during the glycosylation modification of wheat zein, Food Science, 2017). In addition, currently when using dextran grafted zein, the grafting degree is often low (lower than 20%, mostly about 10%), such as The influence of dextran molecular weight on the structure and emulsifying properties of zein grafts (Zhao Chengbin et al., Transactions of the Chinese Society of Agricultural Engineering, 2018, etc.; Dong Yanjiao), and the loading rate is low, usually about 3-6%.

[0005] Therefore, improving the polysaccharide modification process of zein, enhancing the polysaccharide modification effect of zein and the loading effect on unstable substances are still the directions that need to be optimized at present. Summary of the Invention

[0006] In view of the above technical problems, the object of the present invention is to provide lutein-loaded composite modified wheat gliadin nanoparticles, preparations and uses thereof. To achieve the above object, the technical solution of the present invention is as follows:

[0007] A kind of lutein-loaded composite modified wheat gliadin nanoparticles, the preparation method thereof comprises the following steps:

[0008] S1 Preparation of enzymatically hydrolyzed wheat gliadin: Disperse wheat gliadin in water, add alkaline protease for enzymatic hydrolysis, centrifuge to collect the supernatant, and freeze-dry to obtain enzymatically hydrolyzed wheat gliadin;

[0009] S2 Grafting of enzymatically hydrolyzed wheat gliadin with dextran: Disperse enzymatically hydrolyzed wheat gliadin in water to prepare an aqueous solution of enzymatically hydrolyzed wheat gliadin. According to the mass ratio of enzymatically hydrolyzed wheat gliadin to dextran of (1-2):1, add dextran. First, stir and react in a water bath at 60°C at a rotation speed of 100xg-300xg for 20 min; then stir and react in a water bath at 80°C at a rotation speed of 400xg-700xg for 10 min, regarded as a low temperature-high temperature cycle; after performing 3-10 low temperature-high temperature cycle reactions, the reaction ends, cool, adjust the pH to 7.0, centrifuge and collect the supernatant, perform ultrafiltration centrifugation in an ultrafiltration centrifuge tube to remove unreacted enzymatically hydrolyzed wheat gliadin and dextran, collect the ultrafiltered clear liquid, and freeze-dry to obtain composite modified wheat gliadin;

[0010] S3 Preparation of drug-loaded nanoparticles: Disperse composite modified wheat gliadin in water to prepare an aqueous solution of composite modified wheat gliadin. Add lutein according to the mass ratio of composite modified wheat gliadin to lutein of 10:1, adjust the pH to 11.0, ultrasonicate at 100W-300W for 2min-4min, adjust the pH to 7.0, centrifuge to remove unencapsulated lutein, collect the supernatant, and freeze-dry to obtain lutein-loaded composite modified wheat gliadin nanoparticles.

[0011] Preferably, in step S2, the molecular weight of the dextran is 2 kDa-100 kDa. Further preferably, the molecular weight of the dextran is 6 kDa

[0012] Preferably, in step S1, the reaction conditions for the enzymatic hydrolysis are: the addition amount of alkaline protease is 1000 U / g wheat gliadin-7000 U / g wheat gliadin; the enzymatic hydrolysis temperature is 40°C-60°C; the enzymatic hydrolysis pH is 8-10; the enzymatic hydrolysis time is 30 min-240 min. Further preferably, in step S1, the reaction conditions for the enzymatic hydrolysis are that the addition amount of alkaline protease is 3000 U / g wheat gliadin; the enzymatic hydrolysis temperature is 55°C; the enzymatic hydrolysis pH is 8.5; the enzymatic hydrolysis time is 60 min.

[0013] Preferably, in step S1, the centrifugation conditions are centrifugation at 4°C, 6000 xg to 12000 xg for 5 min to 60 min; More preferably, in step S1, the centrifugation is at 8000 xg for 20 min.

[0014] Preferably, in step S2, in the enzymatically hydrolyzed gliadin aqueous solution, the mass fraction of enzymatically hydrolyzed gliadin is 1%; in step S3, in the composite modified gliadin aqueous solution, the mass fraction of composite modified gliadin is 1%.

[0015] Preferably, in step S2, the mass ratio of enzymatically hydrolyzed gliadin to dextran is 1:1.

[0016] Preferably, in step S2, the number of low-temperature - high-temperature cyclic reactions is 6 to 8 times.

[0017] Preferably, in step S2, the ultrafiltration centrifugation conditions are: the molecular weight cut-off of the ultrafiltration centrifugal tube is 30 kDa, centrifugation at 4°C, 4000 xg for 20 min.

[0018] Preferably, in step S2, the stirring speed in the 60°C water bath is 200 xg, and the stirring speed in the 80°C water bath is 500 xg.

[0019] Preferably, in step S3, the ultrasonic power is 200 W.

[0020] Preferably, in step S3, the ultrasonic time is 4 min.

[0021] Preferably, in step S3, the centrifugation conditions are: centrifugation at 4°C, 8000 xg for 10 min.

[0022] An oral preparation is made from the aforementioned lutein-loaded composite modified gliadin nanoparticles and excipients.

[0023] Preferably, the excipient is water.

[0024] Use of the aforementioned lutein-loaded composite modified gliadin nanoparticles or oral preparation in the preparation of drugs or health foods.

[0025] Advantages of the present invention:

[0026] (1) In the present invention, gliadin is used as the raw material, and its production raw material gluten is a by-product of wheat starch separation and processing, with a low price.

[0027] (2) Aiming at the problems of poor solubility of gliadin and weak encapsulation ability in the aqueous phase system, the present invention adopts the method of enzymatic hydrolysis combined with glycosylation reaction to improve the structure and functional properties of gliadin; and constructs nanoparticles loaded with lutein, improving the encapsulation effect and stability of lutein.

[0028] (3) The present invention adopts a low-temperature - high-temperature cyclic heating method for the dextran glycosylation reaction of gliadin, which improves the grafting degree of protein and polysaccharide, weakens the degree of browning, and is beneficial to improving the drug loading capacity (encapsulation rate and loading rate) of nanoparticles and improving the appearance and process stability of the preparation. Among them, the mass ratio of modified gliadin to lutein can be as low as 10:1, and the encapsulation rate of lutein can reach 75 - 82%, that is, the loading rate of lutein can reach 7.5 - 8.2% (the loading rate of lutein is the percentage of encapsulated lutein / modified gliadin).

[0029] (4) The present invention combines the pH folding method with the ultrasonic method to prepare lutein-loaded nanoparticles, replacing the traditional long-time stirring method with a short-time ultrasonic process, saving the preparation time and improving the preparation efficiency. Description of the Drawings

[0030] Figure 1 It is the transmission electron microscopy image and particle size distribution diagram of lutein-loaded composite modified wheat gliadin nanoparticles;

[0031] Figure 2 It is the result diagram of the influence of storage time on the degradation degree of lutein;

[0032] Figure 3 It is the result diagram of the influence of light exposure time on the degradation degree of lutein;

[0033] Figure 4 It is the result diagram of the influence of heating time on the degradation degree of lutein;

[0034] Figure 5 It is the result diagram of the influence of in vitro digestion on the bioavailability of lutein; Detailed Embodiments

[0035] The following combines specific embodiments and drawings to further elaborate on the present invention. The processes, conditions, reagents, and experimental methods for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and well-known common sense in the art, and the present invention has no particularly restricted content.

[0036] The reagents used in the following embodiments of the present invention are as follows:

[0037] Alkaline protease is purchased from Shanghai Yuanye Bio-Technology Co., Ltd.;

[0038] Dextran is purchased from Macklin Co., Ltd. with a molecular weight of 6 kDa;

[0039] Artificial gastric juice is purchased from Regene Biotech;

[0040] Gluten is purchased from Shanghai Xintai Industrial Co., Ltd.;

[0041] Lutein lyophilized powder (purity 80%) was purchased from Shanghai Xianding Biotechnology Co., Ltd.

[0042] The detection method involved in the present invention is as follows:

[0043] 1. Determination of degree of hydrolysis

[0044] The pH-state method was adopted to calculate the degree of hydrolysis (DH) of the protease hydrolysate based on the volume of NaOH consumed during the reaction. The specific calculation formula is as follows:

[0045]

[0046] In the formula, B represents the volume of NaOH consumed (mL), N B represents the concentration of NaOH (mol / L), M p represents the mass of the added protein (g), h tot represents the theoretical amount of peptide bonds per unit mass of protein (8.03 mmol / g), and α represents the degree of dissociation of amino acids, which can be calculated using the following formula. The pK value is obtained according to the Gibbs-Helmholz equation:

[0047]

[0048] 2. Determination of grafting degree

[0049] The grafting degree was determined by the o-phthalaldehyde method (OPA). 80 mg of o-phthalaldehyde (OPA) was completely dissolved in 2 mL of methanol and mixed with 50 mL of 0.1 mol / L sodium tetraborate buffer (pH 9.7), 200 μL of β-mercaptoethanol, and 5 mL of 200 mg / mL sodium dodecyl sulfate (SDS) solution. The volume of the solution was made up to 100 mL with distilled water and prepared freshly for use, which was the OPA reagent used. When determining the grafting degree, 100 μL of the sample solution (5 mg / mL) was thoroughly mixed with 4 mL of the OPA reagent, placed in a constant temperature water bath at 35 °C in the dark, and reacted for 2 min. The absorbance of the sample at 340 nm was measured, using distilled water as the blank solution.

[0050]

[0051] In the formula, A represents the absorbance of the modified gliadin at 340 nm, and A t represents the absorbance of the sample at 340 nm.

[0052] 3. Determination of browning degree

[0053] The protein solution was diluted with a 0.1% SDS solution by mass concentration to make the protein mass concentration 0.2%, and the absorbance value A was measured at a wavelength of 420 nm 420, the absorbance is proportional to the browning of the protein.

[0054] 4. Determination of encapsulation efficiency

[0055] Prepare lutein solutions with concentrations of 10, 15, 20, 25, and 30 μg / mL using methanol, measure the absorbance of the samples at 445 nm, and plot a standard curve. Mix the precipitate (containing unencapsulated lutein) after centrifugation in the preparation step of the drug-loaded nanoparticles with 10 mL of methanol, vortex for 5 min in the dark, centrifuge at 4 °C and 10000 xg for 30 min, and take the supernatant for measurement. Determine the lutein content using high-performance liquid chromatography (U3000, Thermo Fisher Scientific, America). The specific chromatographic conditions are as follows: The chromatographic column is a C30 column (4.6 mm × 250 mm, 5 μm), the column temperature is 35 °C; the mobile phase: methanol / water (volume ratio 95:5, containing 0.1% dibutylhydroxytoluene), isocratic elution is used, the flow rate is 1.0 mL / min, the detection wavelength is 445 nm, and the injection volume is 50 μL.

[0056] Calculate the lutein concentration according to the standard curve. The encapsulation efficiency of lutein is calculated according to the following formula:

[0057]

[0058] 5. Determination of lutein stability

[0059] Mix 1 mL of a 4 mg / mL lutein methanol solution with 24 mL of deionized water as free lutein (i.e., the control without nanoparticle loading).

[0060] (1) Determination of storage stability

[0061] At 4 °C, store the same volume of free lutein and lutein-loaded nanoparticle solution together for 30 d, and measure the remaining lutein content in the solution every 6 d. Evaluate the storage stability of lutein by measuring the change in lutein content.

[0062] (2) Determination of photostability

[0063] At room temperature, irradiate the same volume of free lutein and lutein-loaded nanoparticle solution in an HR-XD-150 xenon lamp chamber for 180 min. The light intensity of the lamp tube is 0.5 W / m 2 , and the distance between the sample and the lamp tube is fixed at 40 cm. Take a certain amount of sample every 30 min and measure the remaining lutein content. Evaluate the photostability of lutein by measuring the change in lutein content.

[0064] (3) Determination of thermal stability

[0065] At 90 °C, the same volume of free lutein and lutein-loaded nanoparticles solution were heated together for 180 min. A certain amount of samples was taken every 30 min and immediately cooled to room temperature to measure the remaining lutein content. The thermal stability of lutein was evaluated by measuring the change in lutein content.

[0066] The method for measuring the remaining lutein content was as follows: 1 mL of free lutein / lutein-loaded nanoparticles solution was mixed with 4 mL of methanol, vortexed for 5 min in the dark, centrifuged at 4 °C and 10,000 xg for 30 min, and the supernatant was taken for measurement. High-performance liquid chromatography was used to measure the lutein content in the solution, and the measurement method was the same as that for the encapsulation efficiency. The calculation method for the lutein retention rate was as follows:

[0067]

[0068] 5. Simulating in vitro digestion

[0069] 20 mL of lutein-loaded nanoparticles and free lutein solution were mixed with 20 mL of simulated gastric juice (mainly containing 3.2 mg / mL pepsin, 2 mg / mL NaCl, and 7 mL / L concentrated hydrochloric acid), and the pH of the solution was adjusted to 2.0 using 1 mol / L HCl. The mixture was stirred at 37 °C and 300 xg for 120 min to simulate the gastric digestion process (SGF). After gastric digestion, the pH of the solution was adjusted to 7.5 for intestinal digestion.

[0070] 20 mL of simulated intestinal fluid (prepared by mixing 6.8 mg / mL K2HPO4, 0.8 mg / mL trypsin, 20 mg / mL bile extract, and 8.775 mg / mL NaCl) was pre-prepared. The mixture after gastric digestion was mixed with an equal volume of simulated intestinal fluid, the pH of the solution was adjusted to 7.5 using 1 mol / L NaOH, and it was stirred at 37 °C and 300 xg for 120 min to simulate the intestinal digestion process (SIF). After the intestinal digestion was completed, the digestive fluid was placed in a boiling water bath for 10 min to inactivate the digestive enzymes. During the simulation of gastric and intestinal digestion, 3 mL of digestive fluid was collected every 30 min for subsequent measurement.

[0071] The collected gastric digestive juice and intestinal digestive juice were heated at 90 °C for 5 min to inactivate the digestive enzymes. The digestive juice was cooled to room temperature and centrifuged at 10000 xg for 30 min, and the micellar phase was taken for determination. The total amount of lutein in the micellar phase was determined by the SDS dissociation method. 2 mL of SDS (1.5 mol / L) was mixed with 0.5 mL of the micellar phase and vortexed for 2 min. The total volume of each mixture was diluted to 10 mL with deionized water and allowed to stand at room temperature for 30 min. The absorbance of the supernatant at 446 nm was measured by a UV-2700 ultraviolet-visible spectrophotometer, and the lutein concentration was calculated according to the standard curve. The bioavailability of lutein was calculated by the following formula:

[0072]

[0073] The preparation method of the gliadin used in the examples of the present invention is as follows:

[0074] The wheat gluten was mixed with dichloromethane at a material-liquid ratio of 1 g:7 mL, continuously stirred for 2 h, and the dichloromethane was removed by vacuum filtration. The above degreasing process was repeated twice. The defatted gluten was mixed with an ethanol aqueous solution with a volume fraction of 70% at a material-liquid ratio of 1 g:10 mL, stirred for 3 h, centrifuged at 4 °C and 9000 xg for 10 min, the supernatant was collected, the ethanol was removed by rotary evaporation, and the obtained suspension was freeze-dried to obtain gliadin freeze-dried powder.

[0075] The preparation method of the enzymatically hydrolyzed gliadin used in the examples of the present invention is as follows:

[0076] The gliadin freeze-dried powder was mixed with distilled water to prepare a 1% (w / w) gliadin dispersion. Based on the mass of the gliadin freeze-dried powder, 3000 U / g of alkaline protease was added, and the mixture was enzymatically hydrolyzed at 55 °C and pH 8.5 for 60 min. The supernatant was centrifuged and collected, and enzymatically hydrolyzed gliadin was obtained by freeze-drying.

[0077] Example 1: Effect of low-temperature-high-temperature cycle times on the grafting of enzymatically hydrolyzed gliadin

[0078] Mix the enzymatically hydrolyzed gliadin with distilled water to prepare an aqueous solution of modified gliadin with a mass fraction of 1%. Add dextran so that the mass ratio of modified gliadin to dextran is 1:1. Adjust the pH to 8.0 and stir for 2 h, then carry out continuous cyclic glycosylation reaction. The specific operation process is as follows: First, place the mixed solution in a water bath at 60 °C and stir at a speed of 200xg for 20 min; Second, place the solution in a water bath at 80 °C and react at a speed of 500xg for 10 min, which is regarded as one cycle. After 3 - 8 cycles, immediately cool after the reaction ends, adjust the pH of the sample solution to 7.0, centrifuge and collect the supernatant. Centrifuge the supernatant in an ultrafiltration centrifuge tube with a cut-off molecular weight of 30 kDa at 4 °C and 4000xg for 20 min to remove unreacted modified gliadin and dextran. Collect the ultrafiltered clear liquid and freeze-dry to obtain composite modified gliadin nanoparticles.

[0079] Table 1 Effects of the number of cycles on the grafting degree and browning degree of composite modified gliadin

[0080]

[0081]

[0082] Note: Lowercase letters (a - f) represent significant differences in the effects of the number of cycles on the grafting degree and browning degree of composite modified gliadin

[0083] As shown in Table 1, with the increase of the number of cycles, the grafting degree of dextran is significantly improved. When the number of cycles reaches 6 or more, the grafting degree of dextran exceeds 20%. And the browning degree is still lower than 0.07%.

[0084] The grafting degree increases with the increase of the number of reaction cycles and reaches the highest (up to 24.4%) at 7 cycles. It can be seen that using low temperature and low stirring speed can prevent protein aggregation, but glycosylation reaction can still occur. The second step of the cycle requires heating at 80 °C for 10 min. The short heating time can prevent excessive protein aggregation and accelerate the glycosylation reaction, thereby significantly improving the grafting efficiency.

[0085] Example 2 Effects of reaction temperature and time on the grafting of enzymatically hydrolyzed gliadin

[0086] To investigate the effects of the temperature and time of the low temperature - high temperature cycle on the grafting of enzymatically hydrolyzed gliadin, the following reference samples were prepared for control experiments:

[0087] Control 1: Continuously carry out glycosylation reaction at 60 °C: Mix enzymatically hydrolyzed gliadin with distilled water to prepare an aqueous solution of modified gliadin with a mass fraction of 1%. Add dextran so that the mass ratio of modified gliadin to dextran is 1:1. Adjust the pH to 8.0 and stir together for 2 h. Place the mixed solution in a water bath at 60 °C and continuously stir at a speed of 500 xg for 1 - 5 h. Immediately cool after the reaction ends, adjust the pH of the sample solution to 7.0, centrifuge and collect the supernatant. Centrifuge the supernatant in an ultrafiltration centrifuge tube with a cut-off molecular weight of 30 kDa at 4 °C and 4000 xg for 20 min to remove unreacted modified gliadin and dextran. Collect the ultrafiltered clear liquid and freeze-dry it to obtain the composite modified gliadin.

[0088] Control 2: Continuously carry out glycosylation reaction at 80 °C: Mix enzymatically hydrolyzed gliadin with distilled water to prepare an aqueous solution of modified gliadin with a mass fraction of 1%. Add dextran with a molecular weight of 6 kDa so that the mass ratio of modified gliadin to dextran is 1:1. Adjust the pH to 8.0 and stir together for 2 h. Place the mixed solution in a water bath at 60 °C and continuously stir at a speed of 500 xg for 1 - 5 h. Immediately cool after the reaction ends, adjust the pH of the sample solution to 7.0, centrifuge and collect the supernatant. Centrifuge the supernatant in an ultrafiltration centrifuge tube with a cut-off molecular weight of 30 kDa at 4 °C and 4000 xg for 20 min to remove unreacted modified gliadin and dextran. Collect the ultrafiltered clear liquid and freeze-dry it to obtain the composite modified gliadin.

[0089] Table 2 Effects of different reaction temperatures and times on the grafting degree and browning degree of composite modified gliadin

[0090]

[0091] Note: Lowercase letters (a - e) represent significant differences in the effects of different reaction temperatures and times on the grafting degree and browning degree of composite modified gliadin.

[0092] As shown in Table 2, through comparison, it is found that when continuously carrying out glycosylation reaction at 60 °C and 80 °C, the maximum grafting degree is reached at 4 h, and the grafting degree is lower than 20%. In addition, continuously reacting at a high temperature of 80 °C leads to a relatively large browning degree of the product (close to 0.1%), generating too many intermediate substances, which may affect the properties and results of the protein. As shown in Table 1, the low-temperature - high-temperature cycle effectively inhibits the occurrence of browning and is an efficient and beneficial reaction method.

[0093] Example 3 Preparation of lutein-loaded composite modified gliadin nanoparticles

[0094] The composite modified wheat gliadin used in this example was prepared according to Example 1, and the number of cycles was 7 times.

[0095] (1) Determination of ultrasonic power

[0096] Mix the composite modified wheat gliadin with distilled water to prepare an aqueous solution of composite modified wheat gliadin with a mass fraction of 1%; add a certain amount of lutein to the solution to make its mass concentration 0.1%, and adjust the pH to 11.0. Use an ultrasonic device to ultrasonically treat the solution, set the ultrasonic power to 100 - 400 W, ultrasonic time 4 min, and then adjust the pH to 7.0. Centrifuge the sample at 4 °C and 8000 xg for 10 min to remove unencapsulated lutein. Collect the supernatant and freeze-dry to obtain lutein-loaded composite modified wheat gliadin nanoparticles.

[0097] Table 3 Effects of different ultrasonic powers on the encapsulation efficiency of lutein

[0098]

[0099]

[0100] Note: Lowercase letters (a - d) represent significant differences in the encapsulation efficiency of lutein under different ultrasonic powers

[0101] As shown in Table 3 (ultrasonic power of 0 in Table 3 means no ultrasonic treatment but conventional stirring at 500 xg for 4 min), increasing the ultrasonic power can significantly improve the encapsulation efficiency of lutein. When the ultrasonic power is 200 W, the encapsulation efficiency is the highest. When the ultrasonic power increases to 400 W, the encapsulation efficiency decreases. Therefore, taking the encapsulation efficiency of lutein as the evaluation index, the preferred ultrasonic power is 100 - 300 W, and the best is 200 W.

[0102] (2) Determination of ultrasonic time

[0103] Mix the composite modified wheat gliadin with distilled water to prepare an aqueous solution of composite modified wheat gliadin with a mass fraction of 1%; add a certain amount of lutein to the solution to make its mass concentration 0.1%, and adjust the pH to 11.0. Use an ultrasonic device to ultrasonically treat the solution, set the ultrasonic power to 200 W, ultrasonic time 1 - 5 min, and then adjust the pH to 7.0. Centrifuge the sample at 4 °C and 8000 xg for 10 min to remove unencapsulated lutein. Collect the supernatant and freeze-dry to obtain lutein-loaded composite modified wheat gliadin nanoparticles.

[0104] Table 4 Effects of different ultrasonic times on the encapsulation efficiency of lutein

[0105] Ultrasound time (min) Entrapment efficiency (%) 0 <![CDATA[51.3±0.2 d > 1 <![CDATA[73.4±0.2 c > 2 <![CDATA[78.5±0.1 b > 3 <![CDATA[80.2±0.1 a > 4 <![CDATA[81.3±0.5 a > 5 <![CDATA[74.3±0.2 c >

[0106] Note: The lowercase letters (a - d) represent significant differences in the encapsulation efficiency of lutein at different ultrasonic times.

[0107] As shown in Table 4 (in Table 4, the ultrasonic power is 0, that is, ultrasonic treatment is not used, but conventional stirring at 500 xg for 4 min is adopted), with the extension of ultrasonic time, the encapsulation efficiency of lutein increases. When the ultrasonic time is 4 min, the encapsulation efficiency is the highest. However, when the ultrasonic time increases to 5 min, the encapsulation efficiency decreases significantly. Therefore, taking the encapsulation efficiency of lutein as the evaluation index, the preferred ultrasonic time is 2 - 4 min, and the best is 4 min.

[0108] According to the experimental results, when the ultrasonic power is 200 W and the ultrasonic time is 4 min, the encapsulation efficiency of lutein is 81.3 ± 0.5%. The transmission electron microscopy image and particle size distribution diagram of lutein - loaded composite modified wheat gliadin nanoparticles are as Figure 1 shown. The nanoparticles present a uniformly dispersed spherical structure, and their particle sizes are between 50 - 100 nm.

[0109] As a control, the present invention carried out 2 groups of control experiments:

[0110] Control 1: Mix the composite modified wheat gliadin with distilled water to prepare an aqueous solution of composite modified wheat gliadin with a mass fraction of 1%; add a certain amount of lutein to the solution to make its mass concentration 0.1%, adjust the pH to 11.0, stir at a speed of 500 xg for 4 min, and then adjust the pH to 7.0. Centrifuge the sample at 4°C and 8000 xg for 10 min to remove unencapsulated lutein. Collect the supernatant and freeze - dry it to obtain lutein - loaded composite modified wheat gliadin nanoparticles.

[0111] Control 2: Mix the composite modified wheat gliadin with distilled water to prepare an aqueous solution of composite modified wheat gliadin with a mass fraction of 1%; add a certain amount of lutein to the solution to make its mass concentration 0.1%, adjust the pH to 7.0, and use an ultrasonic device to ultrasonically treat the solution. The ultrasonic power is set to 200 W and the ultrasonic time is 4 min. Centrifuge the sample at 4°C and 8000 xg for 10 min to remove unencapsulated lutein. Collect the supernatant and freeze - dry it to obtain lutein - loaded composite modified wheat gliadin nanoparticles.

[0112] It was determined that the encapsulation efficiency of lutein obtained by Control 1 was 51.3 ± 0.2%, and that obtained by Control 2 was 30.2 ± 0.5%, both of which were significantly lower than the encapsulation efficiency of the product obtained by the preparation method of the present invention (81.3 ± 0.5%). This shows that simply using pH shift or physical methods has limited effect on improving the lutein loading rate, and deeper treatment of the protein itself is required.

[0113] Example 4: Environmental Stability of Lutein

[0114] The lutein-loaded composite modified gliadin nanoparticles of this example were prepared by the method of Example 3 (ultrasonic power 200 W, ultrasonic time 4 min; adjust the pH to 11.0 before ultrasonic treatment and adjust the pH to 7.0 after ultrasonic treatment). Take the lutein-loaded composite modified gliadin nanoparticle solution and free lutein solution (preparation: mix 1 mL of lutein methanol solution with a concentration of 4 mg / mL with 24 mL of deionized water as the free lutein solution, that is, the control without nanoparticle loading) and conduct stability investigation under the same conditions.

[0115] Figure 2 The influence of storage time on the stability of lutein at 4 °C is shown. As shown in the figure, the stability of free lutein gradually decreases with the prolongation of time. After storing for 30 days, the retention rate of lutein in the control group (that is, the content of lutein retained in the sample) decreased significantly, dropping to 50.3 ± 0.6%. The lutein-loaded composite modified gliadin nanoparticles can increase the lutein retention rate, reaching 96.4 ± 0.9%. The results show that the nanoparticles prepared by composite modified gliadin have a good encapsulation effect on lutein and can protect lutein from the influence of the environment.

[0116] Due to the presence of polyphenols and unsaturated double bonds in the lutein structure, it is prone to decomposition under ultraviolet light irradiation. Figure 3 The influence of light irradiation time on the stability of lutein is shown. As shown in the figure, after irradiating for 180 min, the retention rate of lutein is 65.2 ± 3.2%, and the retention rate of lutein in the nanoparticles is 82.4 ± 2.5%. The results show that the nanoparticle loading system protects lutein from ultraviolet radiation to a great extent.

[0117] Figure 4 The influence of different heating times on the stability of lutein at 90 °C is shown. As shown in the figure, after heating at 90 °C for 180 min, the retention rate decreased to 35.2 ± 3.2%. However, the nanoparticles can effectively improve the thermal stability of lutein, and the retention rate of lutein is 63.2 ± 0.9%. The results show that encapsulating lutein in nanoparticles is an effective means to improve its thermal stability, mainly due to the presence of more hydrogen bonds in the nanoparticles.

[0118] In summary, the composite modified gliadin nanoparticles can achieve efficient encapsulation of lutein and maintain its stability in different environments.

[0119] Example 5: Analysis of the Influence of Nanoparticles on the Bioavailability of Lutein

[0120] The lutein-loaded composite modified wheat gliadin nanoparticles solution of this example was prepared by the method of Example 3 (ultrasonic power 200 W, ultrasonic time 4 min. Before ultrasonic treatment, the pH was adjusted to 11.0, and after ultrasonic treatment, the pH was adjusted to 7.0). The free lutein solution was used as a control (prepared by the same method as in Example 4).

[0121] During intestinal digestion, hydrophobic bioactive components can combine with bile salts and lipids to form mixed micelles, and the hydrophobic bioactive substances dissolved in the micellar phase are absorbed through small intestinal cells. In this invention, the bioavailability of lutein was evaluated by measuring the lutein remaining in the micellar phase at the end of in vitro simulated gastrointestinal digestion.

[0122] The lutein-loaded composite modified wheat gliadin nanoparticles and lutein were subjected to continuous gastric (2 h) and intestinal (2 h) digestion to determine the effect of nanoparticle encapsulation on the bioavailability of lutein.

[0123] As Figure 5 shown, after continuous gastric (2 h) and intestinal (2 h) digestion, the bioavailability of free lutein was only 19.4 ± 0.6%, and the bioavailability of lutein in the nanoparticles was 50.9 ± 1.0%. The results proved that encapsulating lutein in nanoparticles significantly improved the absorption and utilization of lutein in the intestine, and the improvement effect of the composite modified wheat gliadin nanoparticles was better.

[0124] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A lutein-loaded composite modified wheat gliadin nanoparticle, characterized in that, The preparation method of the nanoparticles comprises the following steps: S1 Preparation of enzymatically hydrolyzed gliadin: Gliadin is dispersed in water, alkaline protease is added for enzymatic hydrolysis, the supernatant is collected by centrifugation, and freeze-dried to obtain enzymatically hydrolyzed gliadin; S2 Grafting of glucan onto enzymatically hydrolyzed gliadin: The enzymatically hydrolyzed gliadin is dispersed in water to prepare an aqueous solution of enzymatically hydrolyzed gliadin. According to the mass ratio of enzymatically hydrolyzed gliadin to glucan of (1-2):1, glucan is added. First, it is stirred and reacted in a water bath at 60 °C at a rotation speed of 100xg - 300 xg for 20 min; then it is stirred and reacted in a water bath at 80 °C at a rotation speed of 400xg - 700 xg for 10 min, regarded as a low-temperature - high-temperature cycle; after 6 - 8 low-temperature - high-temperature cycle reactions, the reaction ends, it is cooled, the pH is adjusted to 7.0, centrifuged and the supernatant is collected, ultrafiltration centrifugation is carried out in an ultrafiltration centrifugal tube to remove unreacted enzymatically hydrolyzed gliadin and glucan, the ultrafiltered clear liquid is collected, freeze-dried to obtain composite modified gliadin; S3 Preparation of drug-loaded nanoparticles: The composite modified gliadin is dispersed in water to prepare an aqueous solution of composite modified gliadin. According to the mass ratio of composite modified gliadin to lutein of 10:1, lutein is added, the pH is adjusted to 11.0, ultrasonicated at 100W - 300W for 4 min, the pH is adjusted to 7.0, centrifuged to remove unencapsulated lutein, the supernatant is collected, freeze-dried to obtain lutein-loaded composite modified gliadin nanoparticles.

2. The lutein-loaded composite modified wheat gliadin nanoparticles according to claim 1, characterized in that, In step S1, the reaction conditions for the enzymatic hydrolysis are: the addition amount of alkaline protease is 1000U / g gliadin - 7000U / g gliadin; the enzymatic hydrolysis temperature is 40 °C - 60 °C; the enzymatic hydrolysis pH is 8 - 10; the enzymatic hydrolysis time is 30 min - 240 min.

3. A lutein-loaded composite modified wheat gliadin nanoparticle according to claim 1, characterized in that, In step S1, the centrifugation conditions are 4 °C, centrifugation at 6000xg - 12000xg for 5 min - 60 min.

4. A lutein-loaded composite modified gliadin nanoparticle according to claim 1, wherein In step S2, the molecular weight of the glucan is 2 kDa - 100 kDa.

5. A lutein-loaded composite modified wheat gliadin nanoparticle according to claim 1, characterized in that, In step S2, in the aqueous solution of enzymatically hydrolyzed gliadin, the mass fraction of enzymatically hydrolyzed gliadin is 1%; in step S3, in the aqueous solution of composite modified gliadin, the mass fraction of composite modified gliadin is 1%.

6. The lutein-loaded composite modified wheat gliadin nanoparticles according to claim 1, wherein In step S2, the mass ratio of enzymatically hydrolyzed gliadin to glucan is 1:

1.

7. A lutein-loaded composite modified wheat gliadin nanoparticle according to claim 1, wherein In step S2, the conditions for ultrafiltration centrifugation are: the molecular weight cut-off of the ultrafiltration centrifugal tube is 30 kDa, 4 °C, centrifugation at 4000xg for 20 min.

8. A lutein-loaded composite modified wheat gliadin nanoparticle according to claim 1, wherein In step S2, the stirring speed in the 60 °C water bath is 200xg, and the stirring speed in the 80 °C water bath is 500xg.

9. A lutein-loaded composite modified wheat gliadin nanoparticle according to claim 1, wherein In step S3, the ultrasonic power is 200W.

10. A lutein-loaded composite modified wheat gliadin nanoparticle according to claim 1, characterized in that, In step S3, the ultrasonic time is 4 min.

11. A lutein-loaded composite modified wheat gliadin nanoparticle according to claim 1, characterized in that, In step S3, the centrifugation conditions are: 4 °C, centrifugation at 8000xg for 10 min.

12. An oral preparation, characterized in that, It is made from the lutein-loaded composite modified gliadin nanoparticles according to any one of claims 1 to 11 and excipients.

Images