Preparation of Double-Crosslinked Zein-Carboxymethyl Chitosan Nanoparticles for Improving the Thermal Stability of Polyphenols

Abstract

The present invention discloses the preparation of double-crosslinked zein-carboxymethyl chitosan nanoparticles for improving the thermal stability of polyphenols, belonging to the field of food thermal processing. The preparation method of the thermally stable double-crosslinked nanoparticles is to covalently crosslink zein with tannic acid and then use it to prepare tannic acid-crosslinked zein-carboxymethyl chitosan nanoparticles loaded with quercetin. Adding Ca<supgt;2+< / supgt; can improve the crosslinking degree between tannic acid-carboxymethyl chitosan and carboxymethyl chitosan molecules in the zein nanoparticles to enhance the compactness of the structure, so that during the thermal processing after the formation of nanoparticles, the stability of the nanoparticle structure can be maintained, ultimately protecting the encapsulated quercetin inside and increasing the retention rate of quercetin in thermal processing. The method of the present invention is simple, green, pollution-free, and low-energy-consuming, and the prepared nanoparticles can improve the thermal stability of quercetin and can be used as natural additives in thermally processed foods.

Description

Technical Field

[0001] The invention relates to preparation of double-crosslinked zein-carboxymethyl chitosan nanoparticles for improving the thermal stability of polyphenols, and belongs to the technical field of food additives. Background Art

[0002] Quercetin is a common flavonol compound with antioxidant, antibacterial, anti-inflammatory, anti-allergic, antihypertensive, antilipidemic, and immunomodulatory functions. However, quercetin has low bioavailability, poor water solubility, and is extremely unstable under light and heat conditions, which greatly limits its use in food thermal processing. The most commonly used thermal processing methods in food production include steaming, blanching, hot extrusion, and thermal sterilization. Heat treatment is one of the most important methods for improving food quality and extending the shelf life of food. Therefore, it is necessary to design a colloidal delivery system to protect the thermal stability of quercetin in steamed foods to improve its retention rate under high temperature environments.

[0003] However, most of the colloidal delivery systems prepared so far are designed based on the gastrointestinal delivery of the human body and are not suitable for use in food thermal processing. The colloidal delivery system is one of the most commonly used methods for improving the stability of polyphenols. It is of great guiding significance to cross-link the raw materials used to prepare the colloidal delivery system to improve its thermal stability in food thermal processing. At present, common cross-linking methods include physical cross-linking, chemical cross-linking and enzyme cross-linking. The cross-linking method can change or modify the structure of components such as proteins and polysaccharides to improve their performance, ultimately achieving the purpose of expanding the application range of the colloidal delivery system and improving food quality. However, the interaction formed by using only one cross-linking is weak and easily destroyed. The enzyme is easily inactivated during the enzyme cross-linking process, and the process is complex and costly.

[0004] At present, polysaccharide coating is often added to improve the stability of the delivery system, but when heated, the polysaccharide coating is easily separated from the core part, causing the internal encapsulated material to escape and lose its activity due to heating. There is a lack of research on modifying the raw materials at the structural level to improve the application of the delivery system in the thermal processing of steamed food, especially the double cross-linking of multiple components of the system. Summary of the invention

[0005] In order to solve the above problems, the present invention adopts tannic acid and Ca 2+ The structure of protein and polysaccharide was modified by double cross-linking, that is, tannic acid was first used to cross-link and modify zein to prepare a covalent complex, so that the protein molecules were more tightly bound and the escape of quercetin molecules during cooking was reduced. The phenolic hydroxyl group of tannic acid grafted in the quercetin-loaded zein-tannic acid nanoparticles at pH 6 was negatively charged. Ca 2+Acting as a salt bridge to connect the carboxyl group of the amphoteric polyelectrolyte carboxymethyl chitosan and the phenolic hydroxyl group of tannic acid, achieving more carboxymethyl chitosan through Ca 2+ The interaction with tannic acid adsorbed on the surface of the composite rather than relying solely on the electrostatic interaction of the raw materials. On the other hand, Ca 2+ It can form a network structure by combining with the carboxyl group of carboxymethyl chitosan, which can better bind the protein nanoparticles inside. In addition, there are amino groups in carboxymethyl chitosan, which are partially protonated under acidic conditions and carry positive charges. The protonated amino groups can combine with the negatively charged areas in zein through electrostatic interactions. Finally, a stable zein-tannic acid-Ca 2+ -carboxymethyl chitosan structure, thereby improving their binding tightness and strength to protect the internally encapsulated quercetin. 2+ Cross-linked networks with complementary properties can be created to create a strong and tough delivery system to improve the stability of quercetin: the network formed by tannic acid cross-linked proteins is soft and elastic, which helps to maintain structural integrity during deformation, and the second network is strong and fragile, which can effectively dissipate energy. Ultimately, the purpose of reducing leakage and overflow of quercetin under cooking conditions is achieved. The method is simple, green and highly feasible. The retention rate of quercetin in the double-crosslinked nanoparticles after cooking at 90°C for 30 minutes can be increased by 28.6% compared with the single zein nanoparticles, 20.6% compared with the existing technology (adding polysaccharide coating), and 9.3% compared with single cross-linking (tannic acid cross-linking).

[0006] The first object of the present invention is to provide a method for preparing polyphenol-ion double cross-linked nanoparticles, the method comprising the following steps:

[0007] (1) dispersing zein in an ethanol solution, stirring to obtain a zein solution, and adjusting the pH;

[0008] (2) dispersing tannic acid in an ethanol solution, stirring to obtain a tannic acid solution, and adjusting the pH;

[0009] (3) adding tannic acid solution dropwise to the zein solution, stirring and mixing, adjusting pH, fully contacting with oxygen, reacting, dialyzing, and drying;

[0010] (4) the freeze-dried and cross-linked zein is stirred and dissolved in an ethanol solution to obtain a solution A;

[0011] (5) adding quercetin to solution A and stirring until completely dissolved to obtain solution B;

[0012] (6) adding solution B dropwise into the carboxymethyl chitosan solution and stirring thoroughly to obtain solution C;

[0013] (7) Add CaCl to solution C 2 solution, stirring and mixing to react, and removing ethanol to obtain solution D;

[0014] (8) The pH of solution D was adjusted, and the evaporated ethanol was replenished with deionized water of the corresponding pH, and free quercetin was removed by centrifugation.

[0015] In one embodiment of the present invention, the alcohol-soluble protein in step (1) is zein.

[0016] In one embodiment of the present invention, the concentration of the ethanol solution in step (1) is 75% to 85%;

[0017] In one embodiment of the present invention, the concentration of tannic acid in step (2) is 0.6-4 mg / ml; the stirring time in step (2) is 2-3 h; the rotation speed is 600-900 rpm;

[0018] In one embodiment of the present invention, the pH in step (2) refers to adjusting the pH to 9-12, and the reaction time is 20-24 hours. The volume fraction of the ethanol solution in step (2) is 75%-85%.

[0019] In one embodiment of the present invention, the dialysis process in step (3) is performed by replacing deionized water every 4 to 6 hours, the temperature range is 10° C. to 25° C., and the dialysis time is 24 to 48 hours.

[0020] In one embodiment of the present invention, the drying method in step (2) is vacuum freeze drying.

[0021] In one embodiment of the present invention, the volume fraction of the ethanol solution in step (3) is 75% to 85%; the rotation speed in step (3) is 600 to 900 rpm; the pH in step (3) is 9 to 12, and the reaction time is 20 to 24 hours. The molecular weight of the dialysis bag used in the dialysis in step (3) is 14000 Da; the dialysis time is 24 to 48 hours; the drying method in step (3) is vacuum freeze drying.

[0022] In one embodiment of the present invention, the volume fraction of the ethanol solution in step (4) is 75% to 85%.

[0023] In one embodiment of the present invention, the stirring time in step (4) is 2 to 3 hours; the rotation speed is 600 to 900 rpm;

[0024] In one embodiment of the present invention, the polyphenol in step (5) is quercetin, and the concentration of quercetin is 0.1 mg / ml to 0.2 mg / ml. The ratio of tannic acid cross-linked zein to polyphenol in step (5) is 10:1 to 20:1; preferably, the stirring time in step (5) is 2 to 3 hours; the rotation speed is 600 to 900 rpm;

[0025] In one embodiment of the present invention, the concentration of the carboxymethyl chitosan solution in step (6) is 0.25 mg / ml to 5 mg / ml. Preferably, the mass ratio of the carboxymethyl chitosan to the cross-linked zein in step (6) is 1:10 to 2:1; preferably, the stirring time in step (6) is 1 to 1.5 hours, and the rotation speed is 800 to 900 r / min;

[0026] In one embodiment of the present invention, the carboxymethyl chitosan solution is prepared by adding carboxymethyl chitosan into deionized water and stirring at a speed of 600-900 rpm overnight until the water is completely dissolved and hydrated to obtain a 1.67 mg / ml carboxymethyl chitosan solution.

[0027] In one embodiment of the present invention, the CaCl 2 The concentration of the solution is 4-12 mM, and the rotation speed of step (7) is 600-900 rpm; the pH adjusted in step (7) is 6-6.5; the centrifugal speed in step (8) is 3000-4000 r / min, and the centrifugal time is 5-15 min.

[0028] In one embodiment of the present invention, the method for removing ethanol in step (8) is rotary evaporation, the temperature of rotary evaporation is 35-45°C, the rate of rotary evaporation is 40-60rpm, and the time of rotary evaporation is 10-15min.

[0029] The present invention also provides polyphenol-ion method double cross-linked nanoparticles prepared by the above method.

[0030] The second object of the present invention is to provide a preparation method for improving the thermal stability of functional polyphenols.

[0031] The third object of the present invention is to provide an application of the double cross-linked nanoparticles described above in steamed food.

[0032] The present invention provides a method for improving the thermal stability of polyphenols, characterized in that the method comprises the following steps: (1) dispersing zein in an ethanol solution, stirring to obtain a zein solution, and adjusting the pH;

[0033] (2) dispersing tannic acid in an ethanol solution, stirring to obtain a tannic acid ethanol solution, and adjusting the pH;

[0034] (3) adding tannic acid ethanol solution dropwise to the zein solution obtained in step (1), adjusting the pH and fully contacting with oxygen, stirring to react, dialyzing, and drying;

[0035] (4) stirring and dissolving the dried zein obtained in step (3) in an ethanol solution to obtain a solution A;

[0036] (5) adding polyphenol to solution A and stirring until polyphenol is completely dissolved to obtain solution B;

[0037] (6) dropping solution B dropwise into the carboxymethyl chitosan solution and stirring to obtain solution C;

[0038] (7) Add CaCl to solution C 2 solution, stirring and mixing to obtain solution D;

[0039] (8) The pH of solution D was adjusted, ethanol was removed by rotary evaporation, and the evaporated ethanol was replenished with deionized water of corresponding pH, and free polyphenols were removed by centrifugation.

[0040] In one embodiment of the present invention, the concentration of the zein solution in step (1) is 20-40 mg / ml;

[0041] In one embodiment of the present invention, the volume fraction of the ethanol solution in step (1) is 75% to 85%; the pH in step (1) is 9 to 12;

[0042] In one embodiment of the present invention, the polyphenols include but are not limited to quercetin.

[0043] In one embodiment of the present invention, the concentration of the tannic acid solution in step (2) is 0.6-4 mg / ml;

[0044] In one embodiment of the present invention, the volume fraction of the ethanol solution in step (2) is 75% to 85%;

[0045] In one embodiment of the present invention, the pH in step (3) is 9 to 12, and the reaction time is 20 to 24 hours;

[0046] In one embodiment of the present invention, the molecular weight of the dialysis bag used in the dialysis in step (3) is 14000Da; the dialysis time is 24 to 48 hours;

[0047] In one embodiment of the present invention, the drying method in step (3) is vacuum freeze drying.

[0048] In one embodiment of the present invention, the ratio of the tannic acid cross-linked zein and polyphenols in step (5) is 10:1 to 20:1;

[0049] In one embodiment of the present invention, the stirring time in step (5) is 2 to 3 hours; the rotation speed is 600 to 900 rpm;

[0050] In one embodiment of the present invention, the mass ratio of the carboxymethyl chitosan to the cross-linked zein in step (6) is 1:10 to 2:1;

[0051] In one embodiment of the present invention, the stirring time in step (6) is 1 to 1.5 hours, and the rotation speed is 800 to 900 r / min;

[0052] In one embodiment of the present invention, the CaCl 2 The concentration of the solution is 4-12 mM;

[0053] In one embodiment of the present invention, the pH adjusted in step (8) is 6 to 6.5;

[0054] In one embodiment of the present invention, the method of removing ethanol in step (9) is rotary evaporation, the temperature of the rotary evaporation is 35 to 45° C., the rate of the rotary evaporation is 40 to 60 r / min, and the time of the rotary evaporation is 10 to 15 min;

[0055] In one embodiment of the present invention, the centrifugal speed in step (8) is 3000-4000 r / min, and the centrifugal time is 5-15 min.

[0056] The present invention provides the application of the zein-carboxymethyl chitosan nanoparticles or the method in preparing food and medical supplies.

[0057] Beneficial Effects

[0058] (1) The present invention first uses tannic acid to chemically cross-link zein, and then uses Ca 2+ The negatively charged tannic acid in protein was cross-linked with the carboxyl groups in carboxymethyl chitosan. In addition, the electrostatic interaction between the negatively charged hydroxyl groups in tannic acid and the protonated amino groups in carboxymethyl chitosan, as well as the hydrogen bonding and hydrophobic interactions between the zein-tannic acid covalent complex and carboxymethyl chitosan, resulted in the preparation of a protein-polyphenol-Ca-containing composite. 2+ -High-strength nanoparticles of the three-dimensional cross-linked network of polysaccharides are used to improve the stability of quercetin during thermal processing, overcoming the defects of the existing delivery system that is unstable during thermal processing and cannot protect the internal quercetin.

[0059] (2) The application of the double cross-linked heat-stable nanoparticles in steamed food increases the retention rate of quercetin by 28.6% compared with zein alone, by 20.6% compared with the prior art (adding a coating), and by 9.3% compared with single cross-linking (tannic acid cross-linking). This greatly expands the scope of use of quercetin in food processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a schematic diagram of the principle of the present invention.

[0061] Figure 2 It is the retention rate of quercetin in the nanoparticles of Examples 1 to 5 of the present invention and Comparative Examples 1 to 4 after the quercetin is cooked at 90° C. for 30 min.

[0062] Figure 3 This is the thermogravimetric analysis of the nanoparticles of Examples 1 to 5 of the present invention and Comparative Examples 1 to 4.

[0063] Figure 4 This is a Fourier transform infrared spectrum of Example 4 of the present invention and the raw materials of the prepared double-crosslinked nanoparticles.

[0064] Figure 5 This is a scanning electron microscope image of the double-crosslinked composite nanoparticles of Example 4 of the present invention. DETAILED DESCRIPTION

[0065] The present invention is further described below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0066] The detection methods involved in the following embodiments are as follows:

[0067] Determination of retention rate after heating:

[0068] 10 mL of sample was placed in a test tube and incubated in a water bath at 90 °C for 30 minutes. The samples before and after heating were centrifuged at 10,000 r / min for 10 minutes, and the supernatant was diluted with ethanol solution. The quercetin content in the ethanol solution was determined at a wavelength of 374 nm using a UV-Vis spectrophotometer (UV-5200, Metash, China). A suitable calibration curve was determined to calculate quercetin, and the retention rate after heating was calculated using the following formula.

[0069]

[0070] Thermal stability analysis:

[0071] Weigh 2-3 mg of sample and place it in a crucible, then heat it from 30°C to 600°C at a constant rate of 10°C / min, with the nitrogen flow rate controlled at 20 ml / min. Draw a thermogravimetric curve based on the obtained data.

[0072] Particle size determination:

[0073] The particle size of the nanoparticles was measured using a Malvern particle size and zeta potential analyzer (Malvern Instruments Ltd.).

[0074] Infrared spectrum test:

[0075] The FTIR spectra of the samples were measured by FTIR spectrometer. The samples were mixed with KBr powder, ground and pressed into thin slices, which were then analyzed by FTIR spectrometer. The wave number range was 400-4000cm -1 .

[0076] Scanning electron microscopy:

[0077] The sample was diluted 10 times with deionized water of pH 6, dropped on the silicon wafer and allowed to air dry naturally. The silicon wafer was then glued to the conductive adhesive and sprayed with gold. It was then magnified at 30k and 60k and observed and photographed.

[0078] Example 1: Tannic acid and CaCl 2 Effect of double cross-linked zein-carboxymethyl chitosan nanoparticles on their thermal stability

[0079] The specific steps are as follows:

[0080] (1) dispersing zein in a 75% ethanol solution, stirring at 600 rpm for 2 h to obtain a 40 mg / ml zein ethanol solution, and adjusting the pH to 9;

[0081] (2) Preparation of tannic acid ethanol solution: Add tannic acid to 75% ethanol solution and stir to mix. Stir at 600 rpm for 2 h to obtain a tannic acid ethanol solution with a final concentration of 2 mg / ml, and adjust the pH to 9.

[0082] (3) Add the tannic acid ethanol solution obtained in step (2) dropwise to the zein solution obtained in step (1), adjust the pH to 9, place the solution in a brown bottle (sealed with tin foil and pierced with a syringe on the surface of the tin foil), and then place it in a magnetic stirrer and stir at 500 rpm to allow it to fully contact with oxygen (exposed to the air) for 24 hours, place the obtained solution in a dialysis device, place the dialysis device in deionized water and dialyze for 48 hours (in order to obtain a dialysate with a molecular weight of 25,000 Da to 45,000 Da), and after the dialysate is freeze-dried, tannic acid covalently cross-linked zein is obtained.

[0083] (4) The freeze-dried tannic acid-zein covalent complex was dissolved in 75% ethanol and stirred at 600 rpm for 3 h until dissolved to obtain a 10 mg / ml tannic acid-zein covalent complex solution.

[0084] (5) After adding quercetin to the tannic acid-zein covalent complex solution obtained in step (4), stirring at 600 rpm for 3 h until it is completely dissolved, wherein the concentration of quercetin added is 0.5 mg / ml.

[0085] (6) Preparation of carboxymethyl chitosan solution: Carboxymethyl chitosan was added to deionized water and stirred at 900 rpm overnight until it was completely dissolved and hydrated to obtain a 1.67 mg / ml carboxymethyl chitosan solution.

[0086] (7) The solution obtained in step (5) was dripped dropwise into the carboxymethyl chitosan solution obtained in step (6) (volume ratio of 1:3), and stirred at 900 rpm for 1 h.

[0087] (8) Add CaCl to the solution obtained in step (7) 2 solution (4mM / ml), so that the final system contains CaCl 2 The concentration was 0.2 mM / ml, and the mixture was quickly stirred and mixed, and then the pH was adjusted to 6, and the ethanol was removed by rotary evaporation (40°C, -0.01 MPa), and finally the evaporated ethanol was supplemented with deionized water. Free quercetin was removed by centrifugation (3000 rpm / min, 10 min).

[0088] The retention rate of quercetin in the double-crosslinked nanoparticles prepared in this example is as follows Figure 2 shown.

[0089] Example 2

[0090] The specific implementation method is the same as that of Example 1, except that the CaCl 2 The added concentration was 6 mM / ml, so that the concentration in the final system was 0.3 mM / mL. The other conditions were consistent with Example 1, and the double-crosslinked nanoparticles for improving the thermal stability of polyphenols were obtained.

[0091] Example 3

[0092] The specific implementation method is the same as that of Example 1, except that the CaCl 2 The added concentration of the solution was 8 mM / ml, so that the concentration in the final system was 0.4 mM / mL. The other conditions were consistent with Example 1, and the double-crosslinked nanoparticles for improving the thermal stability of polyphenols were obtained.

[0093] Example 4

[0094] The specific implementation method is the same as that of Example 1, except that the CaCl 2The added concentration of the solution was 10 mM / ml, so that the concentration in the final system was 0.5 mM / mL. The other conditions were consistent with those in Example 1, thereby obtaining the double-crosslinked nanoparticles for improving the thermal stability of polyphenols.

[0095] Example 5

[0096] The specific implementation method is the same as that of Example 1, except that the CaCl 2 The added concentration of the solution was 12 mM / ml, so that the concentration in the final system was 0.6 mM / mL. The other conditions were consistent with Example 1, and the double-crosslinked nanoparticles for improving the thermal stability of polyphenols were obtained.

[0097] Example 6

[0098] The specific implementation method is the same as Example 1, except that the concentration of the tannic acid ethanol solution in step (2) of Example 1 is adjusted to 0.67 mg / ml, and the other steps are consistent with Example 1 to obtain the double-crosslinked nanoparticles for improving the thermal stability of polyphenols.

[0099] Example 7

[0100] The specific implementation method is the same as Example 1, except that the concentration of the tannic acid ethanol solution in step (2) of Example 1 is adjusted to 4 mg / ml, and the other steps are consistent with Example 1 to obtain the double-crosslinked nanoparticles for improving the thermal stability of polyphenols.

[0101] Comparative Example 1: Effect of pure zein on its thermal stability

[0102] The specific steps are as follows:

[0103] (1) dispersing zein in a 75% ethanol solution and stirring at 600 rpm for 3 h to obtain a 10 mg / ml zein solution;

[0104] (2) adding quercetin to the zein solution and stirring at 600 rpm for 1 h until dissolved, wherein the concentration of quercetin is 0.5 mg / ml;

[0105] (3) The solution obtained in (2) was dripped dropwise into deionized water at a volume ratio of 1:3, stirred at 900 rpm for 1 h, and then rotary evaporated (40°C, -0.01 MPa) to remove ethanol, and the evaporated ethanol was replenished with deionized water, and centrifuged (3000 r / min, 10 min) to remove free quercetin.

[0106] Comparative Example 2: Effect of Tannic Acid Covalently Cross-linked Zein Nanoparticles on Their Thermal Stability

[0107] The specific steps are as follows:

[0108] (1) dispersing zein in a 75% ethanol solution, stirring at 600 rpm for 2 h to obtain a 40 mg / ml zein ethanol solution, and adjusting the pH to 9;

[0109] (2) Preparation of tannic acid ethanol solution: Add tannic acid to 75% ethanol solution and stir to mix. Stir at 600 rpm for 2 h to obtain a tannic acid ethanol solution with a final concentration of 2 mg / ml, and adjust the pH to 9.

[0110] (3) Add the tannic acid ethanol solution obtained in step (2) dropwise to the zein solution obtained in step (1), adjust the pH to 9, place the solution in a brown bottle (sealed with tin foil and pierced with a syringe on the surface of the tin foil), and then place it in a magnetic stirrer and stir at 500 rpm to allow it to fully contact oxygen and react for 24 hours. Place the obtained solution in a dialysis device, place the dialysis device in deionized water and dialyze for 48 hours (in order to obtain a dialysate with a molecular weight of 25,000 Da to 45,000 Da), and freeze-dry the dialysate to obtain tannic acid covalently cross-linked zein.

[0111] (3) The freeze-dried tannic acid covalently cross-linked zein was dissolved in a 75% ethanol solution and stirred at 600 rpm for 3 h until dissolved to obtain a 10 mg / ml tannic acid-zein covalent complex solution.

[0112] (4) adding quercetin to the tannic acid-zein covalent complex solution obtained in step (2), stirring at 600 rpm for 1 h until the solution is completely dissolved, wherein the concentration of quercetin is 0.5 mg / ml.

[0113] (5) The solution in step (4) was dripped dropwise into deionized water at a volume ratio of 1:3, stirred at 900 rpm for 1 h, and the ethanol was removed by rotary evaporation (40° C., −0.01 MPa), the evaporated ethanol was replenished with deionized water, and free quercetin was removed by centrifugation (3000 rpm, 10 min).

[0114] Comparative Example 3: Effect of Zein-Carboxymethyl Chitosan Nanoparticles on Its Thermal Stability

[0115] The specific steps are as follows:

[0116] (1) dispersing zein in a 75% ethanol solution and stirring at 600 rpm for 3 h to obtain a 10 mg / ml zein ethanol solution;

[0117] (2) After adding quercetin to the zein solution, stir at 600 rpm for 1 h until it is completely dissolved, wherein the concentration of quercetin is 0.5 mg / ml.

[0118] (3) Preparation of carboxymethyl chitosan solution: Carboxymethyl chitosan was added to deionized water and stirred at 900 rpm until completely dissolved to obtain a 1.67 mg / ml carboxymethyl chitosan solution.

[0119] (4) The solution in step (2) was dripped dropwise into the carboxymethyl chitosan solution (1.67 mg / ml) at a volume ratio of 1:3, stirred at 900 rpm for 1 h, and then the pH was adjusted to 6. The ethanol was removed by rotary evaporation (40°C, -0.01 MPa), and the evaporated ethanol was supplemented with deionized water (pH 6), and the free quercetin was removed by centrifugation (3000 rpm, 10 min).

[0120] Comparative Example 4: Effect of Tannic Acid Covalently Cross-linked Zein-Carboxymethyl Chitosan Nanoparticles on Their Thermal Stability The specific steps are as follows:

[0121] (1) dispersing zein in a 75% ethanol solution, stirring at 600 rpm for 2 h to obtain a 40 mg / ml zein ethanol solution, and adjusting the pH to 9;

[0122] (2) Preparation of tannic acid ethanol solution: Add tannic acid to 75% ethanol solution and stir to mix. Stir at 600 rpm for 2 h to obtain a tannic acid ethanol solution with a final concentration of 2 mg / ml, and adjust the pH to 9.

[0123] (3) Add the tannic acid ethanol solution obtained in step (2) dropwise to the zein solution obtained in step (1), adjust the pH to 9, place the solution in a brown bottle (sealed with tin foil and pierced with a syringe on the surface of the tin foil), and then place it in a magnetic stirrer and stir at 500 rpm to allow it to fully contact oxygen and react for 24 hours. Place the obtained solution in a dialysis device, place the dialysis device in deionized water and dialyze for 48 hours (in order to obtain a dialysate with a molecular weight of 25,000 Da to 45,000 Da), and freeze-dry the dialysate to obtain tannic acid covalently cross-linked zein.

[0124] (4) The freeze-dried tannic acid covalently cross-linked zein was dissolved in a 75% ethanol solution and stirred at 600 rpm for 3 h until dissolved to obtain a 10 mg / ml tannic acid-zein covalent complex solution.

[0125] (5) adding quercetin to the tannic acid-zein covalent complex solution obtained in step (4), and stirring at 600 rpm for 2 h until the solution is completely dissolved, wherein the concentration of quercetin added is 0.5 mg / ml.

[0126] (6) Preparation of carboxymethyl chitosan solution: Carboxymethyl chitosan was added to deionized water and stirred at 900 rpm until completely dissolved to obtain a 1.67 mg / ml carboxymethyl chitosan solution.

[0127] (7) The solution in step (5) was dripped into the carboxymethyl chitosan solution at a volume ratio of 1:3, stirred at 900 rpm for 1 h, the pH was adjusted to 6, and the ethanol was removed by rotary evaporation (40°C, -0.01 MPa), and finally the evaporated ethanol was supplemented with deionized water.

[0128] Experimental results:

[0129] The performance of the polyphenol-loaded nanoparticles obtained in Examples 1 to 5 and Comparative Examples 1 to 4 was tested. The test results are shown in Table 1. Figure 2 to Figure 4 .

[0130] 1. Particle size test results

[0131] Table 1 shows the particle size test results of the nanoparticles of Comparative Examples 1 to 4 and Examples 1 to 5.

[0132] sample Particle size (nm) Comparative Example 1 248.00±3.07 Comparative Example 2 136.67±11.30 Comparative Example 3 315.33±37.91 Comparative Example 4 199.80±3.12 Example 1 274.00±20.07 Example 2 245.37±1.02 Example 3 224.40±2.25 Example 4 221.73±1.91 Example 5 218.57±4.04 Example 6 255.80±17.68 Example 7 621.83±24.63

[0133] It can be seen from Table 1 that Comparative Example 2 is smaller than Comparative Example 1, indicating that tannic acid cross-links the zein molecules to make them more tightly bound. The particle size of Comparative Example 4 is smaller than that of Comparative Example 3, indicating that compared with zein, zein-tannic acid can have stronger hydrogen bonds, electrostatic interactions and hydrophobic interactions with carboxymethyl chitosan. Examples 1 to 7 can all form nanoparticles, and the particle sizes of Examples 1 to 6 are in the range of 200 to 300 nm. The particle size of Example 7 is 621.83 nm, and the particles formed are relatively large. With the addition of CaCl 2 With the increase of concentration, the particle size of nanoparticles gradually decreases, which indicates that tannic acid and Ca 2+ The cross-linked network structure between carboxymethyl chitosan and zein-tannic acid makes the nanoparticles more compact and makes it difficult for the encapsulated quercetin to escape.

[0134] 2. Thermal stability testing

[0135] Table 2 shows the retention rate of quercetin in the nanoparticles of Comparative Examples 1 to 4 and Examples 1 to 7 after heating and the results of thermogravimetric analysis of the nanoparticles

[0136] sample Retention rate (%) Final remaining mass fraction (%) Comparative Example 1 66.98±11.2 16.05 Comparative Example 2 86.31±2.01 17.89 Comparative Example 3 75.03±0.73 9.13 Comparative Example 4 88.87±1.32 28.10 Example 1 86.91±1.72 29.11 Example 2 90.44±9.00 18.47 Example 3 92.37±1.55 29.14 Example 4 95.65±1.74 28.64 Example 5 84.59±2.70 26.31 Example 6 78.53±2.57 24.53 Example 7 88.80±4.92 27.8

[0137] As can be seen from the table, the retention rates of quercetin in the double-crosslinked nanoparticles of Example 2, Example 3, and Example 4 after heating are 90.44%, 92.37%, and 95.65%, respectively, which are higher than all the comparative examples, and the retention rate of quercetin in Example 4 is higher than the retention rates of Example 2 and Example 3; thermogravimetric analysis shows that the residual mass fraction (the mass fraction of the remaining residue after burning) of Example 3 and Example 4 is 29.14% and 28.64%, which are higher than all the comparative examples. In summary, Example 4 has the best protection effect on quercetin and the best thermal stability.

[0138] Figure 4 is the FTIR graph of the double cross-linked nanoparticles of Example 4. Figure 1 It can be seen that carboxymethyl chitosan is at 1530~1660cm -1 and 1400~1451cm -1 The peaks observed in the range of 16.54 to 18.30 cm-1 may be related to the vibration of carboxylic anions. The characteristic peak of zein is at 1657.92 cm-1. -1 (Amide I) and 1536.82 cm -1 (amide II, -CN and -NH). The peaks of the zein-tannic acid covalent complex obtained after tannic acid cross-linking changed at amide I and amide II, indicating that zein formed a covalent bond with the amino group of tannic acid. Finally, Example 4 -1 The sharp characteristic peak of quercetin was not observed in the spectrum of , which is because quercetin was encapsulated in the double-cross-linked nanoparticles in an amorphous form. These results indicate that hydrogen bonds, amide bonds, hydrophobic interactions, and electrostatic interactions are involved in the formation of double-cross-linked nanoparticles.

[0139] Figure 5 The scanning electron microscope images of the double-crosslinked nanoparticles of Example 4 at different magnifications show that many carboxymethyl chitosans are distributed on the surface of the particles. The nanoparticles present a spherical structure and the particle size is about 200 nm.

[0140] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A double cross-linked zein-carboxymethyl chitosan nanoparticle for improving the thermal stability of quercetin, characterized in that: The preparation method of the zein-carboxymethyl chitosan nanoparticles comprises the following steps: (1) dispersing zein in an ethanol solution, stirring to obtain a zein solution with a zein concentration of 40 mg / mL, and adjusting the pH to 9-12; (2) dispersing tannic acid in an ethanol solution, stirring to obtain a tannic acid ethanol solution with a tannic acid concentration of 2 mg / mL, and adjusting the pH; (3) adding tannic acid ethanol solution dropwise to the zein solution obtained in step (1), adjusting the pH and fully contacting with oxygen, stirring to react, dialyzing, and drying; (4) stirring and dissolving the dried zein obtained in step (3) in an ethanol solution to obtain a solution A having a tannic acid-zein covalent complex concentration of 10 mg / mL; (5) Add quercetin to solution A and stir until completely dissolved to obtain solution B with a quercetin concentration of 0.5 mg / mL; (6) Add solution B dropwise into a carboxymethyl chitosan solution having a carboxymethyl chitosan concentration of 1.67 mg / mL at a volume ratio of 1:3, and stir to obtain solution C; (7) Add CaCl2 solution to solution C, stir and mix to obtain solution D with a CaCl2 concentration of 0.5 mM; (8) The pH of solution D was adjusted, ethanol was removed by rotary evaporation, and the evaporated ethanol was replenished with deionized water of corresponding pH. Free polyphenols were removed by centrifugation to prepare double-cross-linked zein-carboxymethyl chitosan nanoparticles for improving the thermal stability of polyphenols.

2. The zein-carboxymethyl chitosan nanoparticles according to claim 1, characterized in that: The volume fraction of the ethanol solution in step (1) is 75% to 85%.

3. The zein-carboxymethyl chitosan nanoparticles according to claim 1 or 2, characterized in that: The volume fraction of the ethanol solution in step (2) is 75% to 85%; the pH in step (3) is 9 to 12, and the reaction time is 20 to 24 h; the molecular weight of the dialysis bag used in the dialysis in step (3) is 14000 Da; the dialysis time is 24 to 48 h; and the drying method in step (3) is vacuum freeze drying.

4. The zein-carboxymethyl chitosan nanoparticles according to claim 1 or 2, characterized in that: The stirring time in step (5) is 2 to 3 h, and the rotation speed is 600 to 900 rpm; the stirring time in step (6) is 1 to 1.5 h, and the rotation speed is 800 to 900 r / min.

5. The zein-carboxymethyl chitosan nanoparticles according to claim 1 or 2, characterized in that: The pH value adjusted in step (8) is 6-6.5; the method for removing ethanol in step (8) is rotary evaporation, the temperature of rotary evaporation is 35-45°C, the speed of rotary evaporation is 40-60 r / min, and the time of rotary evaporation is 10-15 min; the centrifugal speed in step (8) is 3000-4000 r / min, and the centrifugal time is 5-15 min.

6. A method for improving the thermal stability of quercetin, characterized in that: The method comprises the following steps: (1) dispersing zein in an ethanol solution, stirring to obtain a zein solution with a zein concentration of 20-40 mg / mL, and adjusting the pH to 9-12; (2) dispersing tannic acid in an ethanol solution, stirring to obtain a tannic acid ethanol solution with a tannic acid concentration of 2 mg / mL, and adjusting the pH; (3) adding tannic acid ethanol solution dropwise to the zein solution obtained in step (1), adjusting the pH and fully contacting with oxygen, stirring to react, dialyzing, and drying; (4) stirring and dissolving the dried zein obtained in step (3) in an ethanol solution to obtain a solution A having a tannic acid-zein covalent complex concentration of 10 mg / mL; (5) Add quercetin to solution A and stir until completely dissolved to obtain solution B with a quercetin concentration of 0.5 mg / mL; (6) Add solution B dropwise into a 1.67 mg / mL carboxymethyl chitosan solution at a volume ratio of 1:3, and stir to obtain solution C; (7) Add CaCl2 solution to solution C, stir and mix to obtain solution D with a CaCl2 concentration of 0.5 mM; (8) Adjust the pH of solution D, remove ethanol by rotary evaporation, replenish the evaporated ethanol with deionized water of the corresponding pH, and remove free polyphenols by centrifugation.

7. The method according to claim 6, characterized in that The volume fraction of the ethanol solution in step (1) is 75% to 85%; the volume fraction of the ethanol solution in step (2) is 75% to 85%; the pH in step (3) is 9 to 12, and the reaction time is 20 to 24 h; the molecular weight of the dialysis bag used in the dialysis in step (3) is 14000 Da; the dialysis time is 24 to 48 h; the drying method in step (3) is vacuum freeze drying.

8. The method according to claim 7, characterized in that The stirring time in step (5) is 2 to 3 h, and the rotation speed is 600 to 900 rpm; the stirring time in step (6) is 1 to 1.5 h, and the rotation speed is 800 to 900 r / min.

9. The method according to claim 8, characterized in that The pH value adjusted in step (8) is 6-6.5; the method for removing ethanol in step (8) is rotary evaporation, the temperature of rotary evaporation is 35-45°C, the speed of rotary evaporation is 40-60 r / min, and the time of rotary evaporation is 10-15 min; the centrifugal speed in step (8) is 3000-4000 r / min, and the centrifugal time is 5-15 min.

10. Use of the zein-carboxymethyl chitosan nanoparticles according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9 in the preparation of food or medical supplies.

Images