Preparation method of pH-responsive chitosan-citral-p-coumaric acid zinc complex with anti-vibrio and antioxidant activities

Abstract

The present invention provides a preparation method of a pH-responsive chitosan-citral-p-coumaric acid zinc complex with anti-vibrio and antioxidant activity, belonging to the field of biomedicine. The preparation method of the present invention is: a citral solution is slowly added dropwise to a chitosan dispersion, heated and stirred; after the system is stabilized, a chitosan-citral dispersion is obtained by reaction under supercritical carbon dioxide conditions; a p-coumaric acid solution is slowly added dropwise to a zinc salt solution, heated and stirred to react, and concentrated to dryness, and then a solvent is added to produce a precipitate, the precipitate is removed, and further concentrated to obtain a p-coumaric acid zinc solution. The obtained chitosan-citral dispersion is slowly added dropwise to the obtained p-coumaric acid zinc solution, heated and stirred evenly; after the system is stabilized, a precipitate is obtained by reaction under supercritical carbon dioxide conditions, and a chitosan-citral-p-coumaric acid zinc complex is obtained after solvent washing and drying. The complex has anti-vibrio, antioxidant activity and pH responsiveness.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a method for preparing a pH-responsive chitosan-citral-zinc p-coumarate complex with anti-vibrio and antioxidant activities. Background Art

[0002] The configurations of natural citral mainly include α-citral (nerol) and β-citral (geranial), and their sources are mainly galangal oil and Litsea cubeba oil. Litsea cubeba is a kind of forest-source spice plant unique to my country, with abundant resources. The content of natural citral in its volatile oil can be as high as 70% or more. Therefore, the development and utilization of natural citral is of great significance for improving the resource utilization rate and added value of Litsea cubeba. Citral has broad application prospects in the fields of food, daily chemicals, medicine, etc., and has multiple functions such as flavor enhancement, antibacterial and pheromone. However, citral is volatile and has active chemical properties, and it is easy to undergo redox reactions to generate substances such as geranic acid or geraniol / nerol. Therefore, improving the stability of citral is a key issue in its application.

[0003] Chitosan (CTS) is a cationic alkaline aminopolysaccharide with good biocompatibility, non-toxicity, non-antigenicity and biodegradability, and is an ideal drug carrier material. Chitosan also has multiple biological activities such as antibacterial, antiviral and antitumor. The amino group on chitosan can react with the aldehyde group on citral to form a chitosan-citral crosslink; p-coumaric acid is a common plant phenolic compound with good antioxidant and antibacterial properties. The amino group and hydroxyl group on chitosan and the carboxyl group and hydroxyl group on p-coumaric acid can form complexes with a variety of metal ions (such as Ca, Zn, Fe, Cu, etc.) to form organic metal salts. Zinc salts are a type of commonly used food and feed additives, such as zinc acetate, zinc sulfate, zinc citrate, basic zinc chloride, zinc glycinate, etc., which have the functions of preservation, freshness preservation and stability, and are also an effective zinc nutritional enhancer. Supercritical fluid (commonly used carbon dioxide) has the special ability to dissolve other substances. In addition to being used for extraction and separation, it can also be applied to chemical reactions under supercritical conditions, and has the advantages of high reaction efficiency, low reaction temperature, oxygen isolation, and environmentally friendly solvents. The present invention uses chitosan, citral, p-coumaric acid and zinc salt as main raw materials, and uses Schiff base reaction and complex reaction to prepare a chitosan-citral-p-coumaric acid zinc complex with pH responsiveness under supercritical carbon dioxide conditions. The complex has anti-vibrio and antioxidant activities, and can effectively improve the stability and functionality of citral. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the present invention proposes to use chitosan, citral, p-coumaric acid and zinc salt as raw materials to prepare a chitosan composite under supercritical carbon dioxide conditions to increase the antibacterial and antioxidant active groups of chitosan. The prepared chitosan composite has excellent anti-vibrio, antioxidant and pH responsive functions.

[0005] In order to solve the above problems, the technical solution adopted by the present invention is as follows:

[0006] A preparation method of a pH-responsive chitosan-citral-zinc p-coumarate complex with anti-vibrio and antioxidant activities comprises the following steps: dropping a chitosan-citral dispersion into a zinc p-coumarate solution, heating and stirring the mixture evenly, reacting the mixture under supercritical carbon dioxide conditions after the system is stable, washing the mixture several times with a large amount of solvent after the system generates a stable yellow or dark yellow precipitate, and drying the mixture to obtain the chitosan-citral-zinc p-coumarate complex.

[0007] Furthermore, the mass fraction of the chitosan-citral dispersion is 0.5-17.5wt.%; the preparation method of the chitosan-citral dispersion is: slowly dropwise adding a 0.5-25wt.% citral solution into a 0.5wt.%-10wt.% chitosan dispersion, stirring evenly under a heating condition of 40-60°C and after the system is stable, reacting for 10-60min under supercritical carbon dioxide conditions at 30-60°C and 10-30MPa, and obtaining the chitosan-citral dispersion when the system becomes a stable light yellow or yellow color; wherein the solvent used for the citral solution is ethanol, ethyl acetate or acetone; and the solvent used for the chitosan dispersion is ethanol, ethyl acetate or acetone.

[0008] Furthermore, the mass fraction of the chitosan-citral dispersion is 10wt.%; the preparation method of the chitosan-citral dispersion is: slowly dropwise adding a 15wt.% citral ethanol solution into a 5wt.% chitosan ethanol dispersion, stirring evenly under a heating condition of 50°C and after the system is stable, reacting for 40 minutes under supercritical carbon dioxide conditions at 45°C and 25MPa, and obtaining a 10wt.% chitosan-citral dispersion when the system becomes a stable light yellow or yellow color.

[0009] Furthermore, the mass fraction of the zinc p-coumarate solution is 0.5-25wt.%; the preparation method of the zinc p-coumarate solution is: slowly dropwise adding a 0.5-25wt.% p-coumaric acid solution into a 0.5-25wt.% zinc salt solution, and stirring the reaction for 0.5-3h under heating conditions of 30-60°C, adding 2-15 times the volume of methanol or ethanol solution to the solution after the reaction is completed to produce precipitation, removing the precipitation, and further concentrating the solution to obtain a zinc p-coumarate solution of a certain concentration; wherein the type of solvent used for the p-coumaric acid solution is ethanol, ethyl acetate or acetone; the type of zinc salt is zinc chloride, basic zinc chloride, zinc sulfate, zinc acetate, zinc citrate, zinc lactate or zinc gluconate.

[0010] Furthermore, the mass fraction of the zinc p-coumarate solution is 10wt.%; the preparation method of the zinc p-coumarate solution is: slowly dropwise adding an ethanol solution containing 10wt.% of p-coumaric acid into a zinc sulfate solution containing 10wt.%, stirring and reacting at 45°C for 1h, adding 10 times the volume of ethanol solution to the solution after the reaction is completed to produce a precipitate, removing the precipitate, and further concentrating the solution to obtain a zinc p-coumarate solution of a certain concentration.

[0011] Furthermore, the temperature of the heating and stirring is 40-60°C; the temperature of the reaction under supercritical carbon dioxide conditions is 30-60°C and the pressure is 10-30MPa; the reaction time is 10-60min; and the solvent is ethanol, ethyl acetate or acetone.

[0012] Furthermore, the temperature of the heating and stirring is 50° C.; the temperature of the reaction under supercritical carbon dioxide conditions is 45° C. and the pressure is 25 MPa; the reaction time is 40 min; and the solvent is ethanol.

[0013] Furthermore, the preparation method of the pH-responsive chitosan-citral-zinc p-coumarate complex having anti-Vibrio and antioxidant activities comprises the following steps:

[0014] (1) First, a 15 wt.% citral ethanol solution is added dropwise to a 5 wt.% chitosan ethanol dispersion, and stirred evenly under a heating condition of 50° C. until the system is stable, and then reacted under supercritical carbon dioxide conditions at a temperature of 45° C. and a pressure of 25 MPa for 40 min, until the system becomes a stable light yellow or yellow color, thus obtaining a 10 wt.% chitosan-citral dispersion;

[0015] (2) slowly dropping a 10 wt.% p-coumaric acid ethanol solution into a 10 wt.% zinc sulfate solution, stirring and reacting at 45° C. for 1 h, adding 10 times the volume of ethanol solution to the solution to generate a precipitate after the reaction is completed, removing the precipitate, and further concentrating the solution to obtain a p-coumaric acid zinc solution of a certain concentration;

[0016] (3) Finally, the 10 wt.% chitosan-citral dispersion obtained in step (1) is slowly added dropwise to the 10 wt.% zinc p-coumarate obtained in step (2), and stirred evenly under heating at 50°C. After the system is stable, the mixture is reacted under supercritical carbon dioxide conditions at a temperature of 45°C and a pressure of 25 MPa for 40 minutes, until the color of the system deepens to form a stable yellow or dark yellow precipitate, and the precipitate is subsequently washed several times with a large amount of ethanol and dried to obtain a chitosan-citral-zinc p-coumarate complex.

[0017] Furthermore, the pH-responsive chitosan-citral-zinc p-coumarate complex with anti-Vibrio and antioxidant activities prepared by the method has good dispersibility in water, a particle size distribution range of 650-1054 nm, and a distribution coefficient of 0.301; good stability, no color change, no agglomeration, and no deterioration within three months of natural placement at room temperature; and good pH responsiveness and stable release of zinc ions in an acidic environment.

[0018] Furthermore, the chitosan-citral-zinc p-coumarate complex is used in the field of anti-Vibrio and antioxidant activity. The chitosan-citral-zinc p-coumarate complex has a significant inhibitory effect on Vibrio parahaemolyticus, with a minimum inhibitory concentration of 128 μg / mL; it has good antioxidant activity, and the half-elimination concentration is 676.8 μg / mL (DPPH method).

[0019] Compared with the prior art, the advantages of this application are:

[0020] (1) The present invention uses chitosan-citral-p-coumaric acid and zinc salt as main raw materials, utilizes Schiff base reaction and complex reaction, and prepares a chitosan-citral-calcium salt complex under supercritical conditions, with a particle size distribution range of 650-1054nm and a distribution coefficient of 0.301.

[0021] (2) The composite prepared by the present invention has good stability, good water dispersibility, and obvious biological activity. The dosage form can not only meet the requirements of scientific research related biological activity experiments, but also can be added to solid, fluid, semi-fluid and other related preparations or products with high water content, and has a wide range of applications.

[0022] (3) The main raw materials used in the present invention meet the requirements of Chinese food additive raw materials, the solvents used are all low-toxic solvents, the preparation method is simple, the process stability is good, and it can be applied to the fields of food, medicine, etc.

[0023] (4) The composite prepared by the present invention has good pH responsiveness, antivibrio activity and antioxidant activity, and can be developed into food or feed additives, antibacterial agents, etc.; and the composite has a rich zinc content and can also be used as an animal zinc nutritional supplement. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The surface morphology of the chitosan-citral-p-coumaric acid zinc complex is shown in the scanning electron microscope image; Figure 1 A is a 2500-fold magnification image; Figure 1 B is a picture magnified 10000 times;

[0025] Figure 2 is the particle size distribution diagram of chitosan-citral-zinc p-coumarate complex in solution;

[0026] Figure 3 This is the analysis diagram of zinc ion release of chitosan-citral-p-coumaric acid zinc complex in acid environment (pH responsiveness);

[0027] Figure 4 This is a scanning electron microscopic image of the inhibitory effect of chitosan-citral-zinc p-coumarate complex on Vibrio parahaemolyticus;

[0028] Figure 5 This is an analysis chart of the antioxidant activity of chitosan-citral-zinc p-coumarate complex. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0030] Example 1

[0031] A method for preparing a pH-responsive chitosan-citral-zinc p-coumarate complex with anti-vibrio and antioxidant activities includes the preparation of a chitosan-citral dispersion, a zinc p-coumarate solution, and a chitosan-citral-zinc p-coumarate.

[0032] (1) The preparation of chitosan-citral dispersion is as follows:

[0033] Slowly add 10 mL of 15 wt.% citral ethanol solution into 10 mL of 5 wt.% chitosan ethanol dispersion, heat at 50°C, stir evenly and wait for the system to stabilize, then react for 40 minutes under supercritical carbon dioxide conditions at 45°C and 25 MPa, and obtain a 10 wt.% chitosan-citral dispersion when the system turns into a stable light yellow or yellow color.

[0034] (2) The preparation of zinc p-coumarate solution is as follows:

[0035] 10 mL of 10 wt.% p-coumaric acid ethanol solution was slowly added dropwise to 10 mL of 10 wt.% zinc sulfate solution, and the mixture was stirred and reacted at 45°C for 1 h. After the reaction was completed, 10 times the volume of ethanol solution was added to the solution to produce a precipitate. The precipitate was removed and the solution was further concentrated to obtain a 10 wt.% p-coumaric acid zinc solution.

[0036] (3) The preparation of chitosan-citral-zinc p-coumarate is as follows:

[0037] 10 mL of the chitosan-citral dispersion containing 10 wt.% obtained in step (1) is slowly added dropwise to the 10 wt.% zinc p-coumarate obtained in step (2), and stirred evenly under heating at 50° C. and after the system is stable, the reaction is carried out under supercritical carbon dioxide conditions at a temperature of 45° C. and a pressure of 25 MPa for 40 minutes, and the color of the system deepens to form a stable yellow or dark yellow precipitate, and then the precipitate is washed three times with a large amount of ethanol, and dried to obtain a chitosan-citral-zinc p-coumarate complex.

[0038] Example 2

[0039] A method for preparing a pH-responsive chitosan-citral-zinc p-coumarate complex with anti-vibrio and antioxidant activities includes the preparation of a chitosan-citral dispersion, a zinc p-coumarate solution, and a chitosan-citral-zinc p-coumarate.

[0040] (1) The preparation of chitosan-citral dispersion is as follows:

[0041] Slowly add 10 mL of 25 wt.% citral ethanol solution into 10 mL of 10 wt.% chitosan ethanol dispersion, heat at 60°C, stir evenly and wait for the system to stabilize, then react for 60 minutes under supercritical carbon dioxide conditions at 60°C and 30 MPa, and obtain a 17.5 wt.% chitosan-citral dispersion when the system turns into a stable light yellow or yellow color.

[0042] (2) The preparation of zinc p-coumarate solution is as follows:

[0043] 10 mL of 25 wt.% p-coumaric acid ethanol solution was slowly added dropwise to 10 mL of 25 wt.% zinc acetate solution, and the mixture was stirred and reacted at 60°C for 3 h. After the reaction was completed, 15 times the volume of ethanol solution was added to the solution to produce a precipitate. The precipitate was removed and the solution was further concentrated to obtain a 25 wt.% p-coumaric acid zinc solution.

[0044] (3) The preparation of chitosan-citral-zinc p-coumarate is as follows:

[0045] 10 mL of the chitosan-citral dispersion containing 17.5 wt.% obtained in step (1) is slowly added dropwise to the 25 wt.% zinc p-coumarate obtained in step (2), and stirred evenly under heating conditions at 60° C. and after the system is stable, the reaction is carried out under supercritical carbon dioxide conditions at a temperature of 60° C. and a pressure of 30 MPa for 60 min, and the color of the system deepens to form a stable yellow or dark yellow precipitate, and then the precipitate is washed 5 times with a large amount of ethanol, and dried to obtain a chitosan-citral-zinc p-coumarate complex.

[0046] Example 3

[0047] A method for preparing a pH-responsive chitosan-citral-zinc p-coumarate complex with anti-vibrio and antioxidant activities includes the preparation of a chitosan-citral dispersion, a zinc p-coumarate solution, and a chitosan-citral-zinc p-coumarate.

[0048] (1) The preparation of chitosan-citral dispersion is as follows:

[0049] Slowly add 10 mL of 0.5 wt.% citral ethanol solution into 10 mL of 0.5 wt.% chitosan ethanol dispersion, heat at 40°C, stir evenly and wait for the system to stabilize, then react for 10 minutes under supercritical carbon dioxide conditions at 30°C and 10 MPa, and obtain 0.5 wt.% chitosan-citral dispersion when the system turns into a stable light yellow or yellow color.

[0050] (2) The preparation of zinc p-coumarate solution is as follows:

[0051] 10 mL of 0.5 wt.% p-coumaric acid ethanol solution was slowly added dropwise to 10 mL of 0.5 wt.% zinc citrate solution, and the reaction was stirred for 0.5 h under heating conditions at 30°C. After the reaction was completed, 2 times the volume of ethanol solution was added to the solution to produce a precipitate, the precipitate was removed, and the solution was further concentrated to obtain a 0.5 wt.% p-coumaric acid zinc solution.

[0052] (3) The preparation of chitosan-citral-zinc p-coumarate is as follows:

[0053] 10 mL of the chitosan-citral dispersion containing 0.5 wt.% obtained in step (1) is slowly added dropwise to the 0.5 wt.% zinc p-coumarate obtained in step (2), and stirred evenly under heating at 40° C. and after the system is stable, react for 10 minutes under supercritical carbon dioxide conditions at 30° C. and a pressure of 10 MPa, until the color of the system deepens to form a stable yellow or dark yellow precipitate, and then the precipitate is washed three times with a large amount of ethanol and dried to obtain a chitosan-citral-zinc p-coumarate complex.

[0054] Example 4

[0055] A method for preparing a pH-responsive chitosan-citral-zinc p-coumarate complex with anti-vibrio and antioxidant activities includes the preparation of a chitosan-citral dispersion, a zinc p-coumarate solution, and a chitosan-citral-zinc p-coumarate.

[0056] (1) The preparation of chitosan-citral dispersion is as follows:

[0057] Slowly add 20 mL of 1 wt.% citral ethanol solution into 20 mL of 10 wt.% chitosan ethanol dispersion, heat at 50°C, stir evenly and wait for the system to stabilize, then react for 20 minutes under supercritical carbon dioxide conditions at 40°C and 20 MPa, and obtain a 5.5 wt.% chitosan-citral dispersion when the system turns into a stable light yellow or yellow color.

[0058] (2) The preparation of zinc p-coumarate solution is as follows:

[0059] 20 mL of 5 wt.% p-coumaric acid ethanol solution was slowly added dropwise to 20 mL of 5 wt.% zinc lactate solution, and the mixture was stirred and reacted at 30°C for 3 h. After the reaction was completed, 5 times the volume of ethanol solution was added to the solution to produce a precipitate. The precipitate was removed and the solution was further concentrated to obtain a 5 wt.% zinc p-coumarate solution.

[0060] (3) The preparation of chitosan-citral-zinc p-coumarate is as follows:

[0061] 20 mL of the chitosan-citral dispersion containing 5.5 wt.% obtained in step (1) is slowly added dropwise to the 5 wt.% zinc p-coumarate obtained in step (2), and stirred evenly under heating conditions at 50° C. and after the system is stable, the reaction is carried out under supercritical carbon dioxide conditions at a temperature of 40° C. and a pressure of 20 MPa for 30 minutes, and the color of the system deepens to form a stable yellow or dark yellow precipitate, and then the precipitate is washed three times with a large amount of ethanol, and dried to obtain a chitosan-citral-zinc p-coumarate complex.

[0062] Example 5

[0063] A method for preparing a pH-responsive chitosan-citral-zinc p-coumarate complex with anti-vibrio and antioxidant activities includes the preparation of a chitosan-citral dispersion, a zinc p-coumarate solution, and a chitosan-citral-zinc p-coumarate.

[0064] (1) The preparation of chitosan-citral dispersion is as follows:

[0065] Slowly add 20 mL of 25 wt.% citral ethanol solution into 20 mL of 3 wt.% chitosan ethanol dispersion, heat at 50°C, stir evenly and wait for the system to stabilize, then react for 40 minutes under supercritical carbon dioxide conditions at 40°C and 30 MPa, and obtain a 14 wt.% chitosan-citral dispersion when the system turns into a stable light yellow or yellow color.

[0066] (2) The preparation of zinc p-coumarate solution is as follows:

[0067] 20 mL of 20 wt.% p-coumaric acid ethanol solution was slowly added dropwise to 20 mL of 10 wt.% zinc chloride solution, and the mixture was stirred and reacted at 40°C for 1 h. After the reaction was completed, 10 times the volume of ethanol solution was added to the solution to produce a precipitate. The precipitate was removed and the solution was further concentrated to obtain a 15 wt.% p-coumaric acid zinc solution.

[0068] (3) The preparation of chitosan-citral-zinc p-coumarate is as follows:

[0069] 20 mL of the chitosan-citral dispersion containing 14 wt.% obtained in step (1) is slowly added dropwise to the 15 wt.% zinc p-coumarate obtained in step (2), and stirred evenly under heating conditions at 50° C. and after the system is stable, the reaction is carried out under supercritical carbon dioxide conditions at a temperature of 40° C. and a pressure of 30 MPa for 30 min, and the color of the system deepens to form a stable yellow or dark yellow precipitate, and then the precipitate is washed 5 times with a large amount of ethanol, and dried to obtain a chitosan-citral-zinc p-coumarate complex.

[0070] Example 6

[0071] A method for preparing a pH-responsive chitosan-citral-zinc p-coumarate complex with anti-vibrio and antioxidant activities includes the preparation of a chitosan-citral dispersion, a zinc p-coumarate solution, and a chitosan-citral-zinc p-coumarate.

[0072] (1) The preparation of chitosan-citral dispersion is as follows:

[0073] Slowly add 20 mL of 10 wt.% citral ethanol solution into 20 mL of 10 wt.% chitosan ethanol dispersion, heat at 50°C, stir evenly and wait for the system to stabilize, then react for 30 minutes under supercritical carbon dioxide conditions at 50°C and 20 MPa, and obtain a 10 wt.% chitosan-citral dispersion when the system turns into a stable light yellow or yellow color.

[0074] (2) The preparation of zinc p-coumarate solution is as follows:

[0075] 20 mL of 5 wt.% p-coumaric acid ethanol solution was slowly added dropwise to 20 mL of 10 wt.% zinc gluconate solution, and the mixture was stirred and reacted at 40°C for 2 h. After the reaction was completed, 5 times the volume of ethanol solution was added to the solution to produce a precipitate. The precipitate was removed and the solution was further concentrated to obtain a 7.5 wt.% zinc p-coumarate solution.

[0076] (3) The preparation of chitosan-citral-zinc p-coumarate is as follows:

[0077] 20 mL of the chitosan-citral dispersion containing 10 wt.% obtained in step (1) is slowly added dropwise to 7.5 wt.% of zinc p-coumarate obtained in step (2), and stirred evenly under heating conditions at 50° C. and after the system is stable, the mixture is reacted under supercritical carbon dioxide conditions at 40° C. and a pressure of 20 MPa for 50 min, until the color of the system deepens to form a stable yellow or dark yellow precipitate, and the precipitate is subsequently washed three times with a large amount of ethanol and dried to obtain a chitosan-citral-zinc p-coumarate complex.

[0078] Example 7

[0079] The chitosan-citral-p-coumaric acid zinc complexes obtained in the above Examples 1-6 were respectively tested for particle size distribution (including distribution coefficient, laser particle size test), pH responsiveness and stable zinc ion release (concentration of zinc ions continuously released per unit time in simulated gastric fluid pH = 1.5), anti-Vibrio activity (double dilution method) and antioxidant activity (DPPH method). The test results are shown in Table 1 below.

[0080] Table 1: Comparison of performance test results of chitosan-citral-p-coumaric acid zinc composites obtained in Examples 1-6

[0081]

[0082] Specifically, it has good dispersibility in water, with a particle size distribution range of 620 to 1500 nm and a distribution coefficient of 0.28 to 0.40; it has good stability, with no color change, no agglomeration, and no deterioration within three months of natural placement at room temperature; it has good pH responsiveness and stable release of zinc ions in an acidic environment; it has a significant inhibitory effect on Vibrio parahaemolyticus, with a minimum inhibitory concentration of 128 to 512 μg / mL; it has good antioxidant activity, with half-elimination concentrations of 640 to 710 μg / mL (DPPH method).

[0083] According to the data in Table 1 above, in the particle size distribution test results, the particle sizes of Examples 1, 3, 4 and 6 are smaller than those of Examples 2 and 5, and the distribution is more concentrated, that is, the dispersibility in water is better; in the pH responsiveness and zinc ion stable release test results, the maximum zinc ion release rate of each embodiment is close, and the zinc ion release rate of Examples 1, 2, 3 and 6 reaches 80%, and the time used is significantly higher than the values ​​of Examples 4 and 5, which means that the release of the first four is more stable than that of the latter two; in the anti-Vibrio test results, the minimum inhibitory concentration of Examples 1, 2, 5 and 6 is lower than that of Examples 3 and 4, which means that the anti-Vibrio properties of the first four are stronger than those of the latter two; in the antioxidant test results, the order of half-clearance concentration from small to large (antioxidant from large to small) is Example 5≈Example 2>Example 4≈Example 3≈Example 1>Example 6.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a pH-responsive chitosan-citral-p-coumaric acid zinc complex having anti-Vibrio and antioxidant activities, characterized in that: The chitosan-citral dispersion with a mass fraction of 0.5-17.5wt.% is added dropwise to the zinc p-coumarate solution with a mass fraction of 0.5-25wt.%, and the mixture is heated and stirred evenly. After the system is stable, the mixture is reacted under supercritical carbon dioxide conditions. After the system generates a stable yellow or dark yellow precipitate, the mixture is washed several times with a solvent and dried to obtain a chitosan-citral-zinc p-coumarate complex. The preparation method of the chitosan-citral dispersion is as follows: slowly dropwise adding a 0.5-25wt.% citral solution into a 0.5wt.%-10wt.% chitosan dispersion, stirring evenly under a heating condition of 40-60°C and after the system is stable, reacting for 10-60min under supercritical carbon dioxide conditions at 30-60°C and 10-30MPa, and obtaining the chitosan-citral dispersion when the system turns into a stable light yellow or yellow color; wherein the solvent used for the citral solution is ethanol, ethyl acetate or acetone; and the solvent used for the chitosan dispersion is ethanol, ethyl acetate or acetone; The preparation method of the p-coumaric acid zinc solution is as follows: slowly dropwise adding a 0.5-25wt.% p-coumaric acid solution into a 0.5-25wt.% zinc salt solution, stirring and reacting for 0.5-3h under a heating condition of 30-60°C, adding 2-15 times the volume of methanol or ethanol solution into the solution after the reaction to generate precipitation, removing the precipitation, and further concentrating the solution to obtain a p-coumaric acid zinc solution of a certain concentration; wherein the type of solvent used for the p-coumaric acid solution is ethanol, ethyl acetate or acetone; and the type of zinc salt is zinc chloride, basic zinc chloride, zinc sulfate, zinc acetate, zinc citrate, zinc lactate or zinc gluconate.

2. The method for preparing the pH-responsive chitosan-citral-zinc p-coumarate complex with anti-vibrio and antioxidant activities according to claim 1, characterized in that: The chitosan-citral dispersion has a mass fraction of 10 wt. %. The preparation method of the chitosan-citral dispersion is as follows: slowly dropwise adding a 15 wt. % citral ethanol solution into a 5 wt. % chitosan ethanol dispersion, stirring evenly under a heating condition of 50° C. and after the system is stable, reacting for 40 minutes under supercritical carbon dioxide conditions at 45° C. and 25 MPa, and obtaining the chitosan-citral dispersion when the system turns into a stable light yellow or yellow color.

3. The method for preparing the pH-responsive chitosan-citral-zinc p-coumarate complex with anti-vibrio and antioxidant activities according to claim 1, characterized in that: The mass fraction of the zinc p-coumarate solution is 10wt.%; the preparation method of the zinc p-coumarate solution is: slowly dropwise adding an ethanol solution containing 10wt.% of p-coumaric acid into a zinc sulfate solution containing 10wt.%, stirring and reacting for 1h under heating conditions of 45°C, concentrating to dryness after the reaction is completed, then adding 10 times the volume of ethanol solution to generate precipitation, removing the precipitation, and further concentrating the solution to obtain a zinc p-coumarate solution of a certain concentration.

4. The method for preparing the pH-responsive chitosan-citral-zinc p-coumarate complex with anti-Vibrio and antioxidant activities according to claim 1, characterized in that: The heating and stirring temperature is 40-60°C; the reaction temperature under supercritical carbon dioxide conditions is 30-60°C and the pressure is 10-30MPa; the reaction time is 10-60min; and the solvent is ethanol, ethyl acetate or acetone.

5. The method for preparing the pH-responsive chitosan-citral-zinc p-coumarate complex having anti-Vibrio and antioxidant activities according to claim 4, characterized in that: The heating and stirring temperature is 50° C.; the reaction temperature under supercritical carbon dioxide conditions is 45° C. and the pressure is 25 MPa; the reaction time is 40 min; and the solvent is ethanol.

6. The method for preparing the pH-responsive chitosan-citral zinc p-coumarate complex having anti-Vibrio and antioxidant activities according to any one of claims 1 to 5, characterized in that: The steps are as follows: (1) firstly, a 15 wt.% citral ethanol solution is dripped into a 5 wt.% chitosan ethanol dispersion, and the mixture is stirred evenly under a heating condition of 50° C. and after the system is stable, the mixture is reacted under supercritical carbon dioxide conditions at 45° C. and 25 MPa for 40 minutes, and a 10 wt.% chitosan-citral dispersion is obtained when the system turns into a stable light yellow or yellow color; (2) an ethanol solution containing 10 wt.% p-coumaric acid is slowly dripped into a zinc sulfate solution containing 10 wt.%, and the mixture is stirred and reacted for 1 hour under a heating condition of 45° C. After the reaction is completed, the mixture is concentrated to dryness, and then 10 times the volume of ethanol solution to produce a precipitate, remove the precipitate, and further concentrate the solution to obtain a zinc p-coumarate solution of a certain concentration; (3) finally, slowly dropwise adding the 10 wt.% chitosan-citral dispersion obtained in step (1) to the 10 wt.% zinc p-coumarate obtained in step (2), heating at 50° C., stirring evenly, and after the system is stable, reacting at 45° C. under supercritical carbon dioxide for 40 minutes, until the color of the system deepens to generate a stable yellow or dark yellow precipitate, then washing the precipitate with a large amount of ethanol for several times, and obtaining a chitosan-citral-zinc p-coumarate complex after drying.

7. A pH-responsive chitosan-citral-zinc p-coumarate complex having anti-Vibrio and antioxidant activities prepared according to any one of claims 1 to 6, characterized in that: The chitosan-citral-p-coumaric acid zinc complex has good dispersibility in water, with a particle size distribution range of 650-1054nm and a distribution coefficient of 0.301; it has good stability, with no color change, no agglomeration, and no deterioration within three months of natural storage at room temperature; in an acidic environment, it has good pH responsiveness and stable release of zinc ions.

8. The use of the chitosan-citral-zinc p-coumarate complex according to claim 7 in the field of anti-Vibrio and antioxidant activities, characterized in that: Chitosan-citral-zinc p-coumarate complex has a significant inhibitory effect on Vibrio parahaemolyticus, with a minimum inhibitory concentration of 128μg / mL; it has good antioxidant activity, with a half-elimination concentration of 676.8μg / mL DPPH method.

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