PREPARATION OF DUAL CROSS-LINKED ZEIN-CARBOXYMETHYL CHITOSAN NANOPARTICLES FOR IMPROVING THERMAL STABILITY OF POLYPHENOL

Abstract

Disclosed is preparation of dual cross-linked zein-carboxymethyl chitosan nanoparticles for improving the thermal stability of polyphenol. After covalently cross-linked zein, tannic acid is used to prepare tannic acid cross-linked zein-carboxymethyl chitosan nanoparticles loaded with quercetin, Ca.sup.2+ is then added to increase a degree of crosslinking between the tannic acid and the carboxymethyl chitosan, as well as between molecules of the carboxymethyl chitosan in the zein nanoparticles to make the structure tighter, such that structural stability of the nanoparticles can be maintained during the thermal processing after the nanoparticles are formed, the quercetin encapsulated inside is protected well, and a retention rate of quercetin during the thermal processing is improved. The method provided in the present disclosure is simple, green, pollution-free and low energy consumption, and the prepared nanoparticles can improve the thermal stability of quercetin, and can be used as a natural additive for thermal processing of food.

Claims

1. A dual cross-linked zein-carboxymethyl chitosan nanoparticle for improving thermal stability of quercetin, wherein a preparation method for the zein-carboxymethyl chitosan nanoparticle 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 pH of the zein solution 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 pH of the tannic acid-ethanol solution; (3) adding the tannic acid-ethanol solution dropwise to the zein solution obtained in the step (1), adjusting pH to make the solution fully exposed to oxygen, and mixing for reaction, dialysis and drying to obtain zein; (4) dissolving the dried zein obtained in the step (3) in an ethanol solution to obtain a solution A with a tannic acid-zein covalent complex concentration of 10 mg/mL; (5) adding quercetin to the solution A and stirring until completely dissolved to obtain a solution B with a quercetin concentration of 0.5 mg/mL; (6) adding the solution B dropwise to a carboxymethyl chitosan solution with a carboxymethyl chitosan concentration of 1.67 mg/mL at a volume ratio of 1:3, and stirring thoroughly to obtain a solution C; (7) adding a CaCl.sub.2 solution to the solution C, stirring and mixing for reaction to obtain a solution D with a CaCl.sub.2 concentration of 0.5 mM; and (8) adjusting pH of the solution D, removing ethanol through rotary evaporation, making up the evaporated ethanol with deionized water of the corresponding pH, and centrifuging to remove free polyphenol to obtain the dual cross-linked zein-carboxymethyl chitosan nanoparticle for improving thermal stability of quercetin.

2. The zein-carboxymethyl chitosan nanoparticle according to claim 1, wherein a volume fraction of the ethanol solution in the step (1) is 75%-85%.

3. The zein-carboxymethyl chitosan nanoparticle according to claim 1 or 2, wherein a volume fraction of the ethanol solution in the step (2) is 75%-85%; the pH in the step (3) is 9-12, and the reaction lasts for 20-24 hours; a dialysis bag used for the dialysis in the step (3) has a molecular weight of 14000 Da; the dialysis lasts for 24-48 hours; and a method for the drying in the step (3) is vacuum freeze drying.

4. The zein-carboxymethyl chitosan nanoparticle according to claim 1 or 2, wherein the stirring in the step (5) lasts for 2-3 hours at a rotation speed of 600-900 rpm; and the stirring in the step (6) lasts for 1-1.5 hours at a rotation speed of 800-900 rpm.

5. The zein-carboxymethyl chitosan nanoparticle according to claim 1 or 2, wherein the pH in the step (8) is adjusted to 6-6.5; a method for removing the ethanol in the step (8) is rotary evaporation, and the rotary evaporation is performed at a temperature of 35-45 C. at a rate of 40-60 rpm for 10-15 minutes; and the centrifugation in the step (8) is performed at 3000-4000 rpm for 5-15 minutes.

6. A method for improving thermal stability of quercetin, comprising 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 pH of the zein solution 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 pH of the tannic acid-ethanol solution; (3) adding the tannic acid-ethanol solution dropwise to the zein solution obtained in the step (1), adjusting pH to make the solution fully exposed to oxygen, and mixing for reaction, dialysis and drying to obtain zein; (4) dissolving the dried zein obtained in the step (3) in an ethanol solution to obtain a solution A with a tannic acid-zein covalent complex concentration of 10 mg/mL; (5) adding quercetin to the solution A and stirring until completely dissolved to obtain a solution B with a quercetin concentration of 0.5 mg/mL; (6) adding the solution B dropwise to a carboxymethyl chitosan solution with a concentration of 1.67 mg/mL at a volume ratio of 1:3, and stirring thoroughly to obtain a solution C; (7) adding a CaCl.sub.2 solution to the solution C, stirring and mixing for reaction to obtain a solution D with a CaCl.sub.2 concentration of 0.5 mM; and (8) adjusting pH of the solution D, removing ethanol through rotary evaporation, making up the evaporated ethanol with deionized water of the corresponding pH, and centrifuging to remove free polyphenol.

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

8. The method according to claim 7, wherein the stirring in the step (5) lasts for 2-3 hours at a rotation speed of 600-900 rpm; and the stirring in the step (6) lasts for 1-1.5 hours at a rotation speed of 800-900 rpm.

9. The method according to claim 8, wherein the pH in the step (8) is adjusted to 6-6.5; a method for removing the ethanol in the step (8) is rotary evaporation, and the rotary evaporation is performed at a temperature of 35-45 C. at a rate of 40-60 rpm for 10-15 minutes; and the centrifugation in the step (8) is performed at 3000-4000 rpm for 5-15 minutes.

10. The zein-carboxymethyl chitosan nanoparticle according to any one of claims 1-5 or the method according to any one of claims 6-9 in preparing food and pharmaceutical products.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 is a schematic principle diagram according to the present disclosure.

[0066] FIG. 2 shows retention rates of quercetin in nanoparticles obtained in Examples 1-7 and Comparative Examples 1-7 according to the present disclosure after cooking at 90 C. for 30 minutes.

[0067] FIG. 3 shows thermogravimetric analysis results of nanoparticles obtained in Examples 1-7 and Comparative Examples 1-7 according to the present disclosure.

[0068] FIG. 4 is a Fourier transform infrared spectrometer image of raw material of dual cross-linked nanoparticles obtained in Example 4 according to the present disclosure.

[0069] FIG. 5. is a scanning electron microscope image of dual cross-linked nanoparticles obtained in Example 4 according to the present disclosure.

DETAILED DESCRIPTIONS OF THE EMBODIMENTS

[0070] The present disclosure will be described in further detail below in conjunction with specific embodiments, but the implementation modes of the present disclosure are not limited thereto.

[0071] The detection methods used in the following examples are as follows:

Determination of Retention Rate after Heating

[0072] 10 mL of a sample under test is placed in a test tube and incubating in a water bath at 90 C. for 30 minutes; the sample under test before and after heating is centrifuged at 10,000 rpm for 10 minutes, collecting supernatant and diluting the supernatant with an ethanol solution; a content of quercetin in the ethanol solution is measured at a wavelength of 374 nm using a UV-Vis spectrophotometer (UV-5200, Metash, China); and a suitable calibration curve is measured to calculate a retention rate of the quercetin, and a retention rate after heating is calculated according to the following formula.

[00001]$\begin{array}{cc}\mathrm{Retention}\mathrm{rate}\left(\%\right)=\frac{\mathrm{Content}\mathrm{of}\mathrm{quercetin}\mathrm{after}\mathrm{heating}}{\mathrm{Content}\mathrm{of}\mathrm{quercetin}\mathrm{before}\mathrm{heating}}100\%& \left(1\right)\end{array}$

Analysis of Thermal Stability

[0073] 2-3 mg of the sample is weighed and placed in a crucible, which is heated from 30 C. to 600 C. at a constant rate of 10 C./minute, with a nitrogen flow rate controlled at 20 mL/minute; and a thermogravimetric curve is plotted according to the obtained data.

Determination of Particle Size

[0074] Particle sizes of nanoparticles are measured using a Malvern particle size and Zeta-potential analyzer (Malvern Instruments Ltd.).

Infrared Spectrum Test

[0075] A Fourier transform infrared (FTIR) spectrum of the sample is measured using an FTIR spectrometer, the sample is mixed with KBr powder, ground and pressed to prepare into a thin slice, which is then placed in the FTIR spectrometer for analysis, with a wavenumber range of 400-4000 cm.sup.1.

Scanning Electron Microscopy

[0076] The sample is diluted 10 times with deionized water at pH 6 and dropped onto a silicon wafer for natural air drying; and the silicon wafer is attached to conductive adhesive, sprayed with gold, and then observed at magnifications of 30k and 60k, and photographed.

EXAMPLE 1: INFLUENCE OF TANNIC ACID AND CACL.SUB.2 .DUAL CROSS-LINKED ZEIN-CARBOXYMETHYL CHITOSAN NANOPARTICLES ON THERMAL STABILITY OF QUERCETIN

[0077] Specific steps were as follows: [0078] (1) zein was dispersed in 75% ethanol solution and stirred at 600 rpm for 2 hours to obtain 40 mg/ml zein ethanol solution, and a pH value was adjusted to 9; [0079] (2) tannic acid was added to 75% ethanol solution and stirred at 600 rpm for 2 hours to obtain a tannic acid ethanol solution a final concentration of 2 mg/ml, and a pH value was adjusted to 9; [0080] (3) the tannic acid ethanol solution obtained in the step (2) was added dropwise to the zein ethanol solution obtained in the step (1) (at a volume ratio of 1:1) to obtain a solution, a pH value was adjusted to 9, the solution was added in a brown bottle (sealed with tin foil and punctured with a syringe on a surface of the tin foil), and then placed in a magnetic stirrer for stirring at 500 rpm to ensure sufficient contact with oxygen (exposed to air) for further reaction for 24 hours, the resulting solution was transferred into a dialysis device, the dialysis device was placed in deionized water for 48 hours (to obtain dialysate with a molecular weight ranging from 25,000 Da-45,000 Da); and the dialysate was freeze-dried to obtain tannic acid covalently cross-linked zein; [0081] (4) the freeze-dried tannic acid covalently cross-linked zein was dissolved in 75% ethanol and stirred at 600 rpm for 3 hours until completely dissolved to obtain 10 mg/ml tannic acid covalently cross-linked zein solution; [0082] (5) quercetin was added to the tannic acid covalently cross-linked zein solution obtained in the step (4) and stirred at 600 rpm for 3 hours until completely dissolved to obtain a solution, where a concentration of the quercetin was 0.5 mg/ml; [0083] (6) carboxymethyl chitosan was added to deionized water and stirred at 900 rpm overnight until completely dissolved and hydrated to obtain 1.67 mg/ml carboxymethyl chitosan solution; [0084] (7) the solution obtained in the step (5) was added dropwise to the carboxymethyl chitosan solution obtained in the step (6) (at a volume ratio 1:3) to obtain a solution, and the solution was stirred at 900 rpm for 1 hour to obtain a dispersion; and [0085] (8) CaCl.sub.2 solution (4 mM/ml) was added to the solution obtained in the step (7) to make a concentration of CaCl.sub.2 in a final system up to 0.2 mM/ml, the solution was rapidly stirred and mixed, a pH value was adjusted to 6, ethanol was removed through rotary evaporation (40 C., 0.01 Mpa), deionized water was added to make up the evaporated ethanol, and free quercetin was removed by centrifugation (3000 rpm, 10 minutes).

[0086] A retention rate of quercetin in the dual cross-linked nanoparticles prepared in this example was shown in FIG. 2.

EXAMPLE 2

[0087] Specific implementation mode was the same as that in Example 1, except that a concentration of CaCl.sub.2 added in the step (8) in Example 1 was 6 mM/ml to make a concentration of CaCl.sub.2 in a final system up to 0.3 mM/ml, and other steps were consistent with those in Example 1 to obtain dual cross-linked nanoparticles capable of improving the thermal stability of quercetin.

EXAMPLE 3

[0088] Specific implementation mode was the same as that in Example 1, except that a concentration of CaCl.sub.2 added in the step (8) in Example 1 was 8 mM/ml to make a concentration of CaCl.sub.2 in a final system up to 0.4 mM/ml, and other steps were consistent with those in Example 1 to obtain dual cross-linked nanoparticles capable of improving the thermal stability of quercetin.

EXAMPLE 4

[0089] Specific implementation mode was the same as that in Example 1, except that a concentration of CaCl.sub.2 added in the step (8) in Example 1 was 10 mM/ml to make a concentration of CaCl.sub.2 in a final system up to 0.5 mM/ml, and other steps were consistent with those in Example 1 to obtain dual cross-linked nanoparticles capable of improving the thermal stability of quercetin.

EXAMPLE 5

[0090] Specific implementation mode was the same as that in Example 1, except that a concentration of CaCl.sub.2 added in the step (8) in Example 1 was 12 mM/ml to make a concentration of CaCl.sub.2 in a final system up to 0.6 mM/ml, and other steps were consistent with those in Example 1 to obtain dual cross-linked nanoparticles capable of improving the thermal stability of quercetin.

EXAMPLE 6

[0091] Specific implementation mode was the same as that in Example 1, except that a concentration of the tannic acid ethanol solution in the step (2) in Example 1 was 0.67 mg/ml, and other steps were consistent with those in Example 1 to obtain dual cross-linked nanoparticles capable of improving the thermal stability of quercetin.

EXAMPLE 7

[0092] Specific implementation mode was the same as that in Example 1, except that a concentration of the tannic acid ethanol solution in the step (2) in Example 1 was 4 mg/ml, and other steps were consistent with those in Example 1 to obtain dual cross-linked nanoparticles capable of improving the thermal stability of quercetin.

COMPARATIVE EXAMPLE 1: INFLUENCE OF PURE ZEIN ON THERMAL STABILITY OF QUERCETIN

[0093] Specific steps were as follows: [0094] (1) zein was dispersed in 75% ethanol solution and stirred at 600 rpm for 3 hours to obtain 10 mg/ml zein ethanol solution; [0095] (2) quercetin was added to the zein solution and stirred at 600 rpm for 1 hour until completely dissolved to obtain a solution, where a concentration of the quercetin was 0.5 mg/ml; and [0096] (3) the solution obtained in the step (2) was added dropwise to deionized water at a volume ratio of 1:3, and then stirred at 900 rpm for 1 hour, ethanol was removed through rotary evaporation (40 C., 0.01 Mpa), deionized water was added to make up the evaporated ethanol, and free quercetin was removed by centrifugation (3000 rpm, 10 minutes).

COMPARATIVE EXAMPLE 2: INFLUENCE OF TANNIC ACID COVALENTLY CROSS-LINKED ZEIN ON THERMAL STABILITY OF QUERCETIN

[0097] Specific steps were as follows: [0098] (1) zein was dispersed in 75% ethanol solution and stirred at 600 rpm for 2 hours to obtain 40 mg/ml zein ethanol solution, and a pH value was adjusted to 9; [0099] (2) tannic acid was added to 75% ethanol solution and stirred at 600 rpm for 2 hours to obtain a tannic acid ethanol solution a final concentration of 2 mg/ml, and a pH value was adjusted to 9; [0100] (3) the tannic acid ethanol solution obtained in the step (2) was added dropwise to the zein ethanol solution obtained in the step (1) to obtain a solution, a pH value was adjusted to 9, the solution was added in a brown bottle (sealed with tin foil and punctured with a syringe on a surface of the tin foil), and then placed in a magnetic stirrer for stirring at 500 rpm to ensure sufficient contact with oxygen for further reaction for 24 hours, the resulting solution was transferred into a dialysis device, the dialysis device was placed in deionized water for 48 hours (to obtain dialysate with a molecular weight ranging from 25,000 Da to 45,000 Da); and the dialysate was frozen and dried to obtain tannic acid covalently cross-linked zein. [0101] (4) the freeze-dried tannic acid covalently cross-linked zein was dissolved in 75% ethanol solution and stirred at 600 rpm for 3 hours until completely dissolved to obtain 10 mg/ml tannic acid covalently cross-linked zein solution; [0102] (5) quercetin was added to the tannic acid covalently cross-linked zein solution obtained in the step (4) and stirred at 600 rpm for 1 hours until completely dissolved to obtain a solution, where a concentration of the quercetin was 0.5 mg/ml; and [0103] (6) the solution obtained in the step (5) was added dropwise to deionized water at a volume ratio of 1:3, and then stirred at 900 rpm for 1 hour, ethanol was removed through rotary evaporation (40 C., 0.01 Mpa), deionized water was added to make up the evaporated ethanol, and free quercetin was removed by centrifugation (3000 rpm, 10 minutes).

COMPARATIVE EXAMPLE 3: INFLUENCE OF ZEIN-CARBOXYMETHYL CHITOSAN NANOPARTICLES ON THERMAL STABILITY OF QUERCETIN

[0104] Specific steps were as follows: [0105] (1) zein was dispersed in 75% ethanol solution and stirred at 600 rpm for 3 hours to obtain 10 mg/ml zein ethanol solution; [0106] (2) quercetin was added to the zein ethanol solution and stirred at 600 rpm for 1 hour until completely dissolved to obtain a solution, where a concentration of the quercetin was 0.5 mg/ml; [0107] (3) carboxymethyl chitosan was added to deionized water and stirred at 900 rpm overnight until completely dissolved to obtain 1.67 mg/ml carboxymethyl chitosan solution; [0108] (4) the solution obtained in the step (2) was added dropwise to the 1.67 mg/ml carboxymethyl chitosan solution obtained in the step (3) at a volume ratio of 1:3, and then stirred at 900 rpm for 1 hour, a pH value was adjusted to 6, ethanol was removed through rotary evaporation (40 C., 0.01 Mpa), deionized water (pH 6) was added to make up the evaporated ethanol, and free quercetin was removed by centrifugation (3000 rpm, 10 minutes).

COMPARATIVE EXAMPLE 4: EFFECT OF TANNIC ACID COVALENTLY CROSS-LINKED ZEIN-CARBOXYMETHYL CHITOSAN NANOPARTICLES ON THERMAL STABILITY OF QUERCETIN

[0109] Specific steps were as follows: [0110] (1) zein was dispersed in 75% ethanol solution and stirred at 600 rpm for 2 hours to obtain 40 mg/ml zein ethanol solution, and a pH value was adjusted to 9; [0111] (2) tannic acid was added to 75% ethanol solution and stirred at 600 rpm for 2 hours to obtain tannic acid ethanol solution a final concentration of 2 mg/ml, and a pH value was adjusted to 9; [0112] (3) the tannic acid ethanol solution obtained in the step (2) was added dropwise to the zein ethanol solution obtained in the step (1) to obtain a solution, a pH value was adjusted to 9, the solution was added in a brown bottle (sealed with tin foil and punctured with a syringe on a surface of the tin foil), and then placed in a magnetic stirrer for stirring at 500 rpm to ensure sufficient contact with oxygen for further reaction for 24 hours, the resulting solution was transferred into a dialysis device, the dialysis device was placed in deionized water for 48 hours (to obtain dialysate with a molecular weight ranging from 25,000 Da-45,000 Da); and the dialysate was frozen and dried to obtain tannic acid covalently cross-linked zein; [0113] (4) the freeze-dried tannic acid covalently cross-linked zein was dissolved in 75% ethanol solution and stirred at 600 rpm for 3 hours until completely dissolved to obtain 10 mg/ml tannic acid covalently cross-linked zein solution; [0114] (5) quercetin was added to the tannic acid covalently cross-linked zein solution obtained in the step (4) and stirred at 600 rpm for 2 hours until completely dissolved to obtain a solution, where a concentration of the quercetin was 0.5 mg/ml; [0115] (6) carboxymethyl chitosan was added to deionized water and stirred at 900 rpm overnight until completely dissolved to obtain 1.67 mg/ml carboxymethyl chitosan solution; [0116] (7) the solution obtained in the step (5) was added dropwise to the carboxymethyl chitosan solution obtained in the step (6) at a volume ratio of 1:3, and then stirred at 900 rpm for 1 hour, a pH value was adjusted to 6, ethanol was removed through rotary evaporation (40 C., 0.01 Mpa), and deionized water was added to make up the evaporated ethanol.

COMPARATIVE EXAMPLE 5: INFLUENCE OF GENIPIN AND CACL.SUB.2 .DUAL CROSS-LINKED ZEIN-CARBOXYMETHYL CHITOSAN NANOPARTICLES ON THERMAL STABILITY OF QUERCETIN

[0117] (1) Zein was dispersed in 75% ethanol solution and stirred at 600 rpm for 3 hours to obtain 10 mg/ml zein ethanol solution; [0118] (2) quercetin was added to the zein ethanol solution and stirred at 600 rpm for 1 hour until completely dissolved to obtain a solution, where a concentration of the quercetin was 0.5 mg/ml; [0119] (3) carboxymethyl chitosan was added to deionized water and stirred at 900 rpm overnight until completely dissolved to obtain 1.67 mg/ml carboxymethyl chitosan solution; [0120] (4) the solution obtained in the step (2) was added dropwise to the carboxymethyl chitosan solution obtained in the step (3) at a volume ratio of 1:3, and then stirred at 900 rpm for 1 hour, a pH value was adjusted to 6, and ethanol was removed through rotary evaporation (40 C., 0.01 Mpa) to obtain a dispersion; [0121] (5) 2 ml genipin solution (5 mmol/L) was added to the dispersion obtained in the step (4) and stirred at 900 rpm to obtain genipin cross-linked zein-carboxymethyl chitosan nanoparticles loaded with the quercetin; and [0122] (6) CaCl.sub.2 solution was added to the nanoparticle dispersion obtained in the step (5) to make a concentration of CaCl.sub.2 in a final system up to 0.4 mM/ml, the dispersion was rapidly stirred at 900 rpm, ethanol was removed through rotary evaporation (40 C., 0.01 Mpa), deionized water was added to make up the evaporated ethanol and solution, free quercetin was removed by centrifugation (3000 rpm, 10 minutes) to obtain the genipin and CaCl.sub.2 dual cross-linked zein-carboxymethyl chitosan nanoparticles loaded with the quercetin.

COMPARATIVE EXAMPLE 6: INFLUENCE OF KCL AND CACL.SUB.2 .DUAL CROSS-LINKED ZEIN-CARBOXYMETHYL CHITOSAN NANOPARTICLES ON THERMAL STABILITY OF QUERCETIN

[0123] (1) Zein was dispersed in 75% ethanol solution and stirred at 600 rpm for 3 hours to obtain 10 mg/ml zein ethanol solution; [0124] (2) quercetin was added to the zein ethanol solution and stirred at 600 rpm for 1 hour until completely dissolved to obtain a solution, where a concentration of the quercetin was 0.5 mg/ml; [0125] (3) carboxymethyl chitosan was added to deionized water and stirred at 900 rpm overnight until completely dissolved to obtain 1.67 mg/ml carboxymethyl chitosan solution; [0126] (4) the solution obtained in the step (2) was added dropwise to the carboxymethyl chitosan solution obtained in the step (3) (at a volume ratio 1:3) to obtain a solution, and the solution was stirred at 900 rpm for 1 hour to obtain a dispersion; and

[0127] (5) KCl solution (4 mM/ml) and CaCl.sub.2 solution (4 mM/ml) were added to the dispersion obtained in the step (4) to make concentrations of KCl and CaCl.sub.2 in a final system both up to 0.2 mM/ml, the dispersion was rapidly stirred and mixed, a pH value was adjusted to 6, ethanol was removed through rotary evaporation (40 C., 0.01 Mpa), deionized water was added to make up the evaporated ethanol, and free quercetin was removed by centrifugation (3000 rpm, 10 minutes) to obtain the KCl and CaCl.sub.2 dual cross-linked zein-carboxymethyl chitosan nanoparticles.

COMPARATIVE EXAMPLE 7: INFLUENCE OF GLUTAMYLTRANSFERASE AND CACL.SUB.2 .DUAL CROSS-LINKED ZEIN-CARBOXYMETHYL CHITOSAN NANOPARTICLES ON THERMAL STABILITY OF QUERCETIN

[0128] (1) Zein was dispersed in 75% ethanol solution and stirred at 600 rpm for 3 hours to obtain 10 mg/ml zein ethanol solution; [0129] (2) quercetin was added to the zein ethanol solution and stirred at 600 rpm for 1 hour until completely dissolved to obtain a solution, where a concentration of the quercetin was 0.5 mg/ml; [0130] (3) carboxymethyl chitosan was added to deionized water and stirred at 900 rpm overnight until completely dissolved to obtain 1.67 mg/ml carboxymethyl chitosan solution; [0131] (4) the solution obtained in the step (2) was added dropwise to the carboxymethyl chitosan solution obtained in the step (3) at a volume ratio of 1:3, and then stirred at 900 rpm for 1 hour, a pH value was adjusted to 6, and ethanol was removed through rotary evaporation (40 C., 0.01 Mpa) to obtain a dispersion; [0132] (5) 30 U/g glutamyltransferase was added to the dispersion obtained in the step (4) and reacted in a water bath at a constant temperature of 50 C. for 60 minutes, then treated at 75 C. for 15 minutes to inactivate enzyme, and the dispersion was stirred at 600 rpm to obtain glutamyltransferase-cross-linked quercetin-loaded zein-carboxymethyl chitosan nanoparticles loaded with the quercetin; and [0133] (6) CaCl.sub.2 solution was added to the nanoparticle dispersion obtained in the step (5) to make a concentration of CaCl.sub.2 in a final system up to 0.4 mM/ml, the dispersion was rapidly stirred at 900 rpm, ethanol was removed through rotary evaporation, deionized water was added to make up the evaporated ethanol and solution, free quercetin was removed by centrifugation (3000 rpm, 10 minutes) to obtain the glutamyltransferase and CaCl.sub.2 dual cross-linked zein-carboxymethyl chitosan nanoparticles loaded with the quercetin.

EXPERIMENTAL RESULTS

[0134] The nanoparticles loaded with the polyphenol obtained in Examples 1-7 and Comparative Examples 1-7 were subjected to performance test, and test results were shown in Table 1, FIGS. 2-4.

1. Particle Size Test Results

TABLE-US-00001 TABLE 1 Particle size test results of nanoparticles obtained in Examples 1-7 and Comparative Examples 1-7 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 Comparative Example 5 319.23 7.57 Comparative Example 6 230.30 11.23 Comparative Example 7 232.00 3.32 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

[0135] As can be seen from Table 1, the particle size of nanoparticles obtained in Comparative Example 2 was smaller than that of Comparative Example 1, indicating that the tannic acid cross-linked with the zein molecules could reach tighter binding. The particle size of nanoparticles obtained in Comparative Example 4 was smaller than that of Comparative Example 3, indicating that compared with the zein, the zein-tannic acid had stronger hydrogen bonds, electrostatic interaction, and hydrophobic interaction with the carboxymethyl chitosan. Examples 1-7 could generate nanoparticles, and the particle sizes of nanoparticles obtained in Examples 1-6 ranged from 200-300 nm. The particle size of nanoparticles obtained in Example 7 was 621.83 nm, forming relatively larger particles. As the concentration of CaCl.sub.2 increased, the particle size of nanoparticles gradually decreased, indicating that the network structure between the carboxymethyl chitosan and the zein-tannic acid formed by cross-linking tannic acid and Ca.sup.2+ could make the nanoparticles tighter, thereby preventing the encapsulated quercetin from escaping.

2. Test of Thermal Stability

[0136] Table 2 shows the retention rates of quercetin after heating and the thermogravimetric analysis results of nanoparticles from Comparative Examples 1-7 and Examples 1-7, as shown in Table 2 and FIG. 3.

TABLE-US-00002 TABLE 2 Retention rate and thermogravimetric analysis results Retention Mass fraction of Sample rate (%) final residue (%) 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 Comparative Example 5 87.80 2.45 26.67 Comparative Example 6 74.12 1.76 15.22 Comparative Example 7 69.54 5.08 19.74 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 after heating of the dual cross-linked nanoparticles in Examples 2, 3, and 4 were 90.44%, 92.37%, and 95.65%, respectively, higher than those of all Comparative Examples, and the retention rate of quercetin in Example 4 was higher than those of Examples 2 and 3. Compared with Comparative Examples 1-3, the retention rates and mass fraction of final residue (the mass fraction of final residue after burning) in Examples 3 and 4 were significantly higher than those in Comparative Examples 1-3, indicating that the protective effects of the tannic acid and CaCl.sub.2 dual cross-linked nanoparticles for quercetin was higher than that of the uncross-linked nanoparticles. Compared with Comparative Examples 5-7, the retention rates of quercetin in Examples 3 and 4 were higher than those in Comparative Examples 5-7, indicating that the protection effects of the tannic acid and CaCl.sub.2 dual cross-linked nanoparticles for quercetin were greater than that of other dual cross-linking methods. Compared with Comparative Example 4, the retention rates of quercetin in Examples 3 and 4 were higher, indicating that the protection effects of the tannic acid and CaCl.sub.2 dual cross-linked nanoparticles for quercetin are greater than the tannic acid cross-linked nanoparticles. The thermogravimetric analysis showed that the mass fraction of residue in Examples 3 and 4 (the mass fraction of final residue after burning) were 29.14% and 28.64%, respectively, higher than those of all the Comparative Examples. To sum up, Example 4 provided the best protection effects for quercetin and exhibited the best thermal stability.

[0138] FIG. 4 is an FTIR spectrum of the dual cross-linked nanoparticles obtained in Example 4. As can be seen from FIG. 1, peaks observed in ranges of 1530-1660 cm.sup.1 and 1400-1451 cm.sup.1 for carboxymethyl chitosan are likely related to the vibration of carboxylic acid anions. The characteristic peaks of zein were observed at 1657.92 cm.sup.1 (amide I) and 1536.82 cm.sup.1 (amide II, CN and NH). The peaks of the zein-tannic acid covalent complex obtained after cross-linking with the tannic acid changed at amide I and amide II, indicating that a covalent bond formed between the amino groups of zein and tannic acid. Finally, Example 4, the sharp characteristic peaks of quercetin were not observed in the spectrum range of 750-1750 cm.sup.1 in Example 4, this was because the quercetin was encapsulated in the dual cross-linked nanoparticles in an amorphous form. The results indicated that hydrogen bonding, amide bonds, hydrophobic interactions, and electrostatic interactions were all involved in the formation of the dual cross-linked nanoparticles.

[0139] FIG. 5 is a scanning electron microscope image of the dual cross-linked nanoparticles obtained in Example 4 at different magnifications. As can be seen from the image, a great number of carboxymethyl chitosan were distributed on a surface of the nanoparticles, and the nanoparticles had a spherical structure, with a particle size of about 200 nm.

[0140] Although the present disclosure has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present disclosure. Those skilled in the art can make various modifications and variations without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure should be defined by the claims.