Chlorogenic acid-entrapped zein-chitosan composite nanoparticles as well as preparation method and application of chlorogenic acid-entrapped zein-chitosan composite nanoparticles
By encapsulating chlorogenic acid in zein-chitosan composite nanoparticles, the problem of poor stability of chlorogenic acid in fruit and vegetable preservation was solved, achieving effective inhibition of Botrytis cinerea and improving the preservation effect of fruits.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI ACAD OF AGRI SCI
- Filing Date
- 2026-01-06
- Publication Date
- 2026-04-10
AI Technical Summary
In existing technologies, chlorogenic acid, as a plant-derived antibacterial agent, suffers from poor stability, easy degradation, and low solubility, which affects its application effect in fruit and vegetable preservation.
Chlorogenic acid was encapsulated in zein-chitosan composite nanoparticles and prepared by antisolvent method. The nanoparticles had a particle size of about 120 nm and a stable PDI value of about 0.22, which improved the stability and antibacterial effect of chlorogenic acid.
It significantly inhibits the germination of Botrytis cinerea spores, improves the preservation effect of fruits, and the nano system has good stability, especially at pH 2.0~6.0, providing a more stable in vitro antibacterial effect.
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Figure CN121817262A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a chlorogenic acid-encapsulated zein-chitosan composite nanoparticle, its preparation method, and its application, belonging to the field of agricultural product preservation technology. Background Technology
[0002] Botrytis cinerea (B. cinerea) is the pathogen that causes gray mold in plants. Due to its wide host range and high variability, it limits the growth and yield of many economic and horticultural crops. Gray mold can infect more than 1,400 plant species worldwide, and can occur at different developmental stages and in different parts of agricultural products, leading to significant economic losses. Botrytis cinerea is widely recognized in the field of plant pathology as the second most impactful fungus on agricultural production. This fungus not only damages plants in the field, causing large amounts of rotten fruit at harvest, but the conidia latent on the fruit can also cause widespread decay and spoilage during post-harvest transportation and storage. Thiabendazole, phenylpyrrole, benzoyl, and flunioxime are currently widely used chemical fungicides for controlling gray mold. These fungicides may remain in fruits and vegetables, affecting food safety and hindering environmental sustainability. In recent years, many natural plant extracts have been explored as effective alternatives to traditional chemical antibacterial agents due to their low toxicity levels and favorable ecological properties. Chlorogenic acid, a derivative of hydroxycinnamic acid, is the main phenolic acid detected in peach fruit. Extensive research has been conducted on the antioxidant properties of chlorogenic acid, revealing its potential anti-inflammatory, glycemic, and cardioprotective effects. In addition to its antioxidant properties, increasing research indicates that chlorogenic acid has significant antibacterial efficacy against various plant pathogens related to horticulture and agriculture. Previous studies have reported that chlorogenic acid can enhance the resistance of peach fruit to *Penicillium expansum* by regulating the activity of key defense-related enzymes and the expression of genes related to the salicylic acid signaling pathway.
[0003] Chlorogenic acid (CGA) is a plant-derived extract, belonging to the polyphenol class, and is a bioactive component. Phenolic compounds possess significant antioxidant and antibacterial properties; however, they suffer from poor stability, easy degradation, and low solubility, resulting in relatively low bioavailability in most cases. Adding plant-derived phenolic compounds to food significantly affects their physicochemical properties, stability, solubility, and bioavailability. Nanoencapsulation technology can prevent the degradation of phenolic compounds because it improves their stability to external factors such as light, oxygen, and pH. Nanoencapsulation also prevents interactions between polyphenols and other food components. Zein, an amphiphilic high-molecular-weight biopolymer extracted from corn storage protein, is rich in nonpolar amino acids such as leucine, alanine, and proline, and is soluble in high-concentration ethanol aqueous solutions. This invention prepares a nano-antibacterial agent encapsulated with chlorogenic acid, developing a preservation method that effectively inhibits postharvest gray mold in peaches and kiwifruit, providing a reference for their application in fruits and vegetables. Experiments have shown that the zein-chitosan-chlorogenic acid (ZCC) nanoparticle dispersion system can be applied to peach and kiwifruit fruits, significantly (P<0.05) inhibiting the growth of Botrytis cinerea on the fruits, and is expected to become a novel antibacterial agent with high stability. Summary of the Invention
[0004] The purpose of this invention is to provide zein-chitosan composite nanoparticles loaded with chlorogenic acid, which can significantly inhibit the germination of Botrytis cinerea spores and exhibit a more stable in vitro antibacterial effect.
[0005] The technical solution adopted in this invention is as follows: A method for preparing chlorogenic acid-loaded zein-chitosan composite nanoparticles includes the following steps: 1) Dissolve zein in an ethanol solution, then add chlorogenic acid and mix thoroughly in the dark; 2) The solution obtained in step 1) is added dropwise to an aqueous solution of acetic acid containing chitosan, and the mixture is stirred to remove ethanol from the system, thereby obtaining a zein-chitosan composite nanoparticle dispersion system loaded with chlorogenic acid.
[0006] Preferably, the ratio of zein:chlorogenic acid:chitosan is 5:1:1, which is a mass ratio.
[0007] Preferably, the ethanol solution in step 1) is a 70% ethanol solution.
[0008] Preferably, in step 1), the concentration of zein in the ethanol solution is 60%-90%.
[0009] Preferably, in step 1), after dissolving zein in an ethanol solution, centrifuging to remove the precipitate, adding chlorogenic acid and mixing evenly, centrifuging again to remove the precipitate.
[0010] Preferably, in step 2), the volume fraction of acetic acid in the aqueous acetic acid solution is 1-2%.
[0011] Preferably, in step 2), the concentration of chitosan in the aqueous acetic acid solution is 2 mg / ml.
[0012] Preferably, the reaction is stirred for 1-2 hours in step 2).
[0013] Preferably, in step 2), vacuum rotary evaporation is used to remove ethanol from the system at 40-45 °C.
[0014] The chlorogenic acid-loaded zein-chitosan composite nanoparticles were prepared by the above method.
[0015] And the application of the aforementioned chlorogenic acid-encapsulated zein-chitosan composite nanoparticles in food preservation.
[0016] Preferably, the food is fresh fruit.
[0017] Beneficial Effects: This invention successfully prepared a zein-chitosan-chlorogenic acid (ZCC) nanoparticle dispersion system via an antisolvent method. The nanoparticle size was approximately 120 nm, the PDI value was stable at around 0.22, the particle size distribution was uniform, and the nanosystem exhibited high stability at pH values of 2.0–6.0. ZCC significantly (P < 0.05) inhibited the germination of *Botrytis cinerea* spores, demonstrating a more stable in vitro antibacterial effect. This study provides a new approach for developing antibacterial agents based on plant extracts and offers a reference for the application prospects of CGA treatment in harvested fruits and vegetables. Attached Figure Description
[0018] Figure 1 : Flowchart of the nanoparticle preparation process of the present invention.
[0019] Figure 2 : Particle size and dispersion coefficient (A), potential (B), and particle size scan (C) of the prepared nanoparticles.
[0020] Figure 3 : Chlorogenic acid standard curve (A), encapsulation efficiency and loading rate of the sample (B), turbidity (C), physical image (D).
[0021] Figure 4 Electron micrographs of ZC and ZCC after freeze-drying and natural drying, respectively.
[0022] Figure 5pH stability of ZC and ZCC.
[0023] Figure 6 Ultraviolet (A), fluorescence (B), Fourier transform infrared (C), and X-ray diffraction (D) of ZC and ZCC.
[0024] Figure 7 Effects of ZC and ZCC on Botrytis cinerea spore germination.
[0025] Figure 8 The antagonistic effect of ZCC on botrytis cinerea inoculation of peach fruit.
[0026] Figure 9 The effects of ZCC treatment on fruit storage resistance and quality.
[0027] Figure 10 The effect of ZCC on the preservation effect of kiwifruit. Detailed Implementation
[0028] The present invention will be further described below with reference to the embodiments, but the description of the embodiments does not limit the scope of protection of the present invention in any way.
[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.
[0030] Unless otherwise specified, all substances or instruments used in the following examples can be obtained from conventional commercial sources.
[0031] Comparative Example 1: Preparation of a chlorogenic acid-free Zein-CS (ZC) nanoparticle dispersion system 500 mg of zein was dissolved in 50 mL of 70% ethanol solution. The solution was magnetically stirred at 600 r / min at room temperature until fully dissolved, then centrifuged to remove insoluble substances (4000 × g, 10 min). The clarified solution was then added dropwise to 50 mL of 1% (v / v) acetic acid aqueous solution containing 2 mg / mL chitosan, while maintaining magnetic stirring at 600 r / min for 1 h to ensure uniform dispersion. Finally, the ethanol was removed from the system using a vacuum rotary evaporator at 40 °C, yielding a chlorogenic acid-free Zein-CS nanoparticle (ZC) dispersion, which was stored at 4 °C.
[0032] Example 1: Preparation of chlorogenic acid-loaded zein-chitosan composite nanoparticles like Figure 1 As shown, 500 mg of zein was dissolved in 50 mL of 70% ethanol solution. After thorough dissolution and centrifugation, 10, 50, and 100 mg of chlorogenic acid were added respectively, and the mixtures were thoroughly mixed in the dark. The mixtures were then centrifuged at 1000 g for 10 minutes to remove any insoluble impurities. Under dark conditions, the clarified centrifuged solution was added dropwise to 50 mL of 1% (v / v) acetic acid aqueous solution containing 2 mg / mL chitosan, while maintaining magnetic stirring at 600 r / min for 1 h to ensure uniform dispersion. Finally, the ethanol was removed from the system using a vacuum rotary evaporator at 40 °C to obtain a chlorogenic acid-loaded Zein-CS nanoparticle (ZCC) dispersion.
[0033] The properties of the ZC particles in Comparative Example 1 and the ZCC particles in Example 1 were tested (including particle size, potential, polydispersity index, encapsulation and loading rates, turbidity, pH stability, microstructure, potential molecular forces, crystal diffraction information and crystallization state of chlorogenic acid, etc.), and the results are as follows: Figure 2-6 As shown.
[0034] like Figure 2 As shown, with the increase of CGA addition, the average particle size of the nanosystem increased from 120 nm to 130 nm, and the PDI value stabilized at around 0.22, indicating that the nanodispersion system had a uniform particle size distribution and was relatively stable. In the nanodispersion system, if the ζ-potential of the nanosystem is high, the electrostatic repulsion between particles will be correspondingly enhanced. This electrostatic repulsion can effectively prevent the nanosystem from agglomerating in the solution, thereby improving the stability of the entire system.
[0035] from Figure 3 As can be seen, the encapsulation efficiency and loading rate of CGA in the nano-dispersion system were detected by utilizing the characteristic peak of CGA at 324 nm in the ultraviolet light (3A). For example, the encapsulation efficiency and loading rate of CGA in the nano-dispersion system were detected by utilizing the characteristic peak of CGA at 324 nm in the ultraviolet light. Figure 3 As shown in Figure B, the encapsulation efficiencies in the nanosystem after adding 10, 50, and 100 mg of CGA were 50.52%, 15.53%, and 50.59%, respectively. The loading rates were 0.84%, 1.29%, and 8.43%, respectively. With increasing CGA concentration, the encapsulation efficiencies initially decreased and then increased, while the loading rates gradually increased.
[0036] The change in turbidity was similar to the change in particle size; turbidity was directly proportional to the amount of CGA added, with ZCC100 exhibiting the highest turbidity, which corresponds to its larger particle size. Figure 3 C and 3D).
[0037] from Figure 4 It can be seen that ZC contains more particulate matter than ZCC. Figure 4 A and Figure 4 B), and is relatively rough. During the drying process, small particles may be adsorbed onto large particles due to the high viscosity of CS. At the same time, molecular chain entanglement and degradation may also be caused by magnetic stirring, leading to agglomeration.
[0038] After natural drying, ZC scanning electron microscopy revealed numerous scattered spherical nanostructures, with the particles adhering to each other and forming clusters. Figure 4 C). ZCC observed granular but relatively coarse material coating the tightly packed flakes. Figure 4 (D) During the drying process, free CS in the dispersion acts as a bridge, connecting other complex particles and thus initiating bridging flocculation. This bridging flocculation further leads to particle aggregation and ultimately the formation of a thin film.
[0039] from Figure 5 It can be seen that the nanosystem is more adaptable to acidic conditions. The results show that the nanosystem is relatively stable at pH values between 2.0 and 6.0. ZCC exhibits even greater stability than ZC.
[0040] from Figure 6 It can be seen that CGA and Zein interact to form a complex ( Figure 6 A); Single Zein shows peaks around 304 nm and 348 nm, while ZCC with added CGA only shows a peak at 386 nm. This is mainly due to fluorescence quenching caused by the binding of tyrosine and tryptophan with CGA. Figure 6 B). In ZCC, the characteristic absorption band of CGA almost disappears, possibly because the encapsulation of the nanocarrier restricts the extension of chemical groups on the CGA. Figure 6 C). XRD patterns were used to analyze the crystal diffraction information and crystallization state of CGA in the nanosystem. Zein, as a biomacromolecule, exhibits an amorphous crystalline structure, displaying two distinct broad peaks. CS has two humps, with diffraction peaks at 2θ = 11.5° and 20.7°. CGA shows multiple sharp diffraction peaks between 5° and 50°, indicating a highly crystalline state. However, when encapsulated by Zein, no sharp diffraction peaks were observed in its highly crystalline form. Figure 6(D) This result reveals that CGA loses its original crystal structure in the nanosystem, possibly because the interaction between CGA and Zein inhibits CGA crystallization. Characterization of the ZCC nanosystem by fluorescence UV spectroscopy, FTIR, and XRD showed that hydrophobic interactions and hydrogen bonds exist between CGA and Zein, inhibiting CGA crystallization and causing it to exist in an amorphous form within the nanosystem, confirming that CGA is encapsulated within Zein.
[0041] Example 2 Evaluation of the antibacterial properties of nanoparticles The minimum inhibitory concentration (MIC) of zein-chitosan (ZC) was determined to be 1 / 2 ZC and the MIC of zein-chitosan-chlorogenic acid (ZCC) was determined to be 1 / 4 ZCC (100 mg) using the half-dilution method. Figure 7 As shown, in the germination experiment of *Botrytis cinerea* spores, when spores germinated in the control group, the germination rate in the nano-dispersion system ZC was significantly lower (P < 0.05) than that in the control group and the free chlorogenic acid group, while the spore germination rate in ZCC was 0%. With the increase of culture time, the spore germination rates in the control group (water and free chlorogenic acid) increased, but there was no significant difference; however, the spores in the ZCC group still did not germinate. Figure 7 The four treatment groups were: (1) Control, which was sprayed with water as a control group; (2) CGA, which was sprayed with the corresponding free CGA; (3) ZC, which was sprayed with a nano-dispersion system without CGA; and (4) ZCC, which was sprayed with a nano-dispersion system containing CGA.
[0042] Example 3 Performance evaluation of nanoparticles in peach fruit preservation Select peaches that are uniform in size, healthy, free from pests and diseases, and without mechanical damage to their surface. Disinfect the fruit surface with 2% sodium hypochlorite for 2 minutes, then rinse the fruit with water and place it on a clean table to dry. Subsequently, the fruit is divided into a control group and a ZCC group (100mg ZCC was used in subsequent experiments). The control group received no treatment, while the ZCC group had ZCC sprayed on the fruit surface and dried in the dark.
[0043] Using a sterile inoculation needle, make two evenly spaced holes, 3 mm deep and 3 mm wide, at the relative equatorial region of the peach fruit. After allowing the hole openings to air dry naturally, inoculate with 10 μL of 10 5 spores mL -1 Botrytis cinerea spore suspension was prepared. All infected fruits were sealed in polyethylene bags and kept at an ambient temperature of 25 ± 1 ℃ and a relative humidity of 85 ~ 90%. The diameter of lesions on each fruit was measured at 0, 1, 3, and 5 days. The results are as follows: Figure 8As shown, by day 5, the growth of Botrytis cinerea in the ZCC-treated group was inhibited, significantly lower than that in the control group. Throughout the storage period, ZCC treatment inhibited Botrytis cinerea spore development; by day 5, the spore diameter in the control group was 30.68 mm, while that in the ZCC-treated group was only 16.69 mm.
[0044] Example 4: Application of nanoparticles in food preservation Store peaches inoculated with gray mold at room temperature (25℃). Figure 9 As shown, during the storage period, the diameter of lesions on fruits in different treatment groups showed a continuous increasing trend; from 0 to 3 days of storage, gray mold could grow normally in fruits of both the ZCC treatment group and the control group, with no significant difference; from 3 to 7 days, the a* value increased sharply, and the color difference a* value of fruits in the ZCC treatment group was significantly higher than that of fruits in the control group; from 1 to 9 days, the color difference b* value of fruits in the ZCC treatment group was significantly higher than that of the control group; throughout the storage period, the respiration intensity of fruits in the treatment group was significantly higher than that of the control group; the soluble solids content of fruits in the ZCC treatment group was relatively stable; by the 9th day, the rot rate of fruits in the control group was as high as 33.33%, while no rot occurred in fruits in the ZCC treatment group.
[0045] Kiwifruit inoculated with gray mold should be stored at room temperature (25℃). Figure 10 As shown, the diameter of lesions in all fruits inoculated with Botrytis cinerea continued to increase during 5 days of storage at 25 ℃. After 3 days of storage, the CGA, ZC, and ZCC groups showed significant differences (P < 0.05) compared with the control. On day 5, the ZCC group significantly (P < 0.05) inhibited the growth of Botrytis cinerea in kiwifruit, but there was no significant difference between the ZC and CGA groups.
Claims
1. A method for preparing zein-chitosan composite nanoparticles loaded with chlorogenic acid, characterized in that, Includes the following steps: 1) Dissolve zein in an ethanol solution, then add chlorogenic acid and mix thoroughly in the dark; 2) The solution obtained in step 1) is added dropwise to an aqueous solution of acetic acid containing chitosan, and the mixture is stirred to remove ethanol from the system, thereby obtaining a zein-chitosan composite nanoparticle dispersion system loaded with chlorogenic acid.
2. The preparation method according to claim 1, characterized in that: The ratio of zein, chlorogenic acid, and chitosan is 5:1:
1.
3. The preparation method according to claim 1, characterized in that: The ethanol solution in step 1) is an ethanol solution with a concentration of 60-90%.
4. The preparation method according to claim 1, characterized in that: In step 1), after dissolving zein in ethanol solution, centrifuge to remove the precipitate, add chlorogenic acid, mix well, and then centrifuge again to remove the precipitate.
5. The preparation method according to claim 1, characterized in that: In step 2), the volume fraction of acetic acid in the aqueous solution is 1-2%.
6. The preparation method according to claim 1, characterized in that: Stir the reaction for 1-2 hours in step 2).
7. The preparation method according to claim 1, characterized in that: In step 2), vacuum rotary evaporation is used to remove ethanol from the system at 40-45℃.
8. The chlorogenic acid-loaded zein-chitosan composite nanoparticles prepared by the method of any one of claims 1-7.
9. The application of the chlorogenic acid-loaded zein-chitosan composite nanoparticles as described in claim 8 in food preservation.
10. The application according to claim 9, characterized in that, The food in question is fresh fruit.