Chitosan / lysozyme amyloid protein fiber composite microneedle based on ethephon controlled release and application of chitosan / lysozyme amyloid protein fiber composite microneedle in on-demand ripening acceleration of picked fruits

Abstract

The invention discloses a chitosan / lysozyme amyloid protein fiber composite microneedle based on ethephon controlled release and application of the composite microneedle in on-demand ripening acceleration of harvested fruits, and belongs to the technical field of agricultural product after-harvest treatment. The composite microneedle is composed of chitosan, lysozyme amyloid protein fibers and ethephon, the mass ratio of the chitosan to the lysozyme amyloid protein fibers is 5: 5, and the ethephon loading capacity is that 131.13 [mu] L of a 40% ethephon solution is added to every 17.5 mL of a 1% chitosan solution. The microneedle can be inserted into the epidermis of picked fruits to realize on-demand slow release of ethephon and promote ripening of the fruits, meanwhile, the lysozyme amyloid protein fiber provides an antibacterial effect, the aging process of the fruits after ripening is effectively delayed, the infection rate of colletotrichum gloeosporioides is reduced, and the ethephon residue in the fruits is remarkably lower than the national limited standard. The method solves the problems of non-uniform fruit ripening, excessive softening and potential toxicity caused by a traditional ethephon ripening method, and realizes integration of batch on-demand ripening and preservation of picked fruits.

Description

Technical Field

[0001] This invention belongs to the field of agricultural chemistry and biomaterials technology, specifically relating to a chitosan / lysozyme amyloid protein fiber composite microneedles based on ethephon controlled release and its application in postharvest fruit ripening on demand. Background Technology

[0002] With the continuous development of postharvest preservation and intelligent control technologies for agricultural products, precise ripening of climacteric fruits (such as mangoes, bananas, and kiwis) has become a key link in reducing postharvest losses and enhancing commercial value. Ethylene, as the core plant hormone regulating fruit ripening, has its artificial release agent, ethephon, widely used in agricultural production. However, traditional ethephon application methods mainly rely on spraying or soaking, which are one-time, non-targeted, and extensive operations. This easily leads to simultaneous ripening, concentrated aging, and even rotting of fruits, severely restricting the flexibility of the supply chain and market responsiveness. In addition, ethephon has a certain degree of toxicity, and conventional application methods can easily lead to excessive chemical residues in the peel and even the pulp, posing potential food safety hazards. It may also cause phytotoxicity or excessive softening due to excessively high local concentrations, affecting fruit quality.

[0003] Existing technologies attempt to slow down the release rate of ethephon by loading it onto natural polymers such as chitosan to construct a sustained-release system. While such films can extend the ethylene release time to some extent, the release behavior remains passive, continuous, and uncontrollable, failing to achieve precise "on-demand" control. Furthermore, the film only forms a two-dimensional contact with the fruit surface, resulting in low penetration efficiency and difficulty in ensuring uniform ripening. Although chitosan possesses certain antibacterial properties, its inhibitory effect on major postharvest pathogenic fungi (such as anthracnose) is limited. While composite coatings of chitosan and natural lysozyme can enhance preservation, they completely lack ripening function and cannot construct a three-dimensional structure with penetrating capabilities due to insufficient mechanical properties. It is noteworthy that lysozyme can self-assemble into amyloid fibrils (LAF) under specific acid-heat conditions. This structure possesses both high specific surface area and excellent mechanical strength, but its application in agricultural controlled-release carrier systems has not yet been explored.

[0004] While microneedle technology has been widely applied in transdermal drug delivery, its design logic is strictly based on the characteristics of human skin, making it difficult to adapt to the dense, hard, and non-self-repairing structure of fruit peels. More importantly, current technologies have not used microneedles for "transdermal delivery of plant growth regulators" nor have they integrated them with information labeling functions, lacking user-friendly solutions for retail terminals.

[0005] Therefore, there is an urgent need to develop a multifunctional composite microneedle system that integrates press-triggered ethephon controlled release, potent synergistic antibacterial properties, extremely low chemical residues, and biodegradability, in order to overcome the current technical bottlenecks of uneven ripening of postharvest fruits in batches, short shelf life, and safety risks. Summary of the Invention

[0006] The purpose of this invention is to provide a chitosan / lysozyme amyloid protein fiber composite microneedles based on ethephon controlled release and its application in postharvest fruit ripening on demand, to achieve single-fruit-level, on-demand triggered ethephon release, while simultaneously activating antibacterial preservation mechanisms, significantly reducing chemical residues, extending shelf life, and supporting information labeling and retail integration.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a chitosan / lysozyme amyloid fiber composite microneedle based on ethephon controlled release. The composite microneedle is composed of chitosan, lysozyme amyloid fiber and ethephon, wherein the mass ratio of chitosan to lysozyme amyloid fiber is 5:5, and the ethephon loading is 131.13 μL of 40% ethephon solution added to every 17.5 mL of 1% chitosan solution.

[0008] Furthermore, the lysozyme amyloid protein fiber is prepared by the following method: 1.5g of lysozyme is dissolved in 75mL of distilled water, concentrated hydrochloric acid is added dropwise to adjust the pH to 2, and the mixture is stirred at 200rpm and 90℃ for 24h to obtain a lysozyme protein-amyloid protein solution, which is then freeze-dried to obtain lysozyme amyloid protein fiber.

[0009] Furthermore, the composite microneedles are prepared by the following method: 1g of chitosan is dissolved in 100mL of 1% acetic acid solution, 17.5mL of chitosan solution is taken and mixed with the prepared dried lysozyme amyloid protein fibers in a 5:5 ratio, 131.13μL of 40% ethephon solution is added to form a composite solution, and after ultrasonic degassing, it is injected into a latex microneedle mold, pressed at room temperature under a vacuum pressure of 0.1MPa for 2h, the pressing is repeated twice, and then dried and demolded at 40℃ to obtain the final product.

[0010] Furthermore, the mechanical strength of a single composite microneedle is greater than 1.4 N / needle.

[0011] The present invention provides a label constructed from the composite microneedles, wherein the composite microneedles are hidden on the back of the label, and the label can display information about the fruit.

[0012] This invention provides an application of the aforementioned label in post-harvest fruit ripening on demand.

[0013] Furthermore, the application involves directly attaching the label to the surface of the fruit, pressing it to allow microneedles to penetrate the fruit's epidermis, thereby achieving the slow release of ethephon, enabling the fruit to reach an edible state within 3 days, while simultaneously delaying the softening process after the fruit has ripened.

[0014] Furthermore, the fruit is a mango, banana, kiwi, or star fruit.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention loads ethephon onto chitosan and lysozyme protein-starch fiber composite microneedles to achieve on-demand slow release of ethephon, which can rapidly induce ripening on the fruit epidermis after harvest, while delaying senescence after ripening and improving the disease resistance of the fruit. The operation is simple and easy to achieve batch ripening of the fruit.

[0016] (2) While achieving efficient ripening, the present invention also has excellent antibacterial and preservation functions and safety, effectively delaying the quality deterioration process after fruit ripening, and significantly improving the commercial value and food safety of the harvested fruit.

[0017] (3) The microneedles provided by the present invention adopt a patch label design, which can hide the microneedles and not affect the appearance and sales of the fruit. At the same time, it can carry fruit information (such as variety, harvest date and maturity level), which facilitates circulation management and commercial application.

[0018] (4) This invention is applicable to a variety of postharvest fruits, including mango, banana, star fruit and kiwi, and has wide applicability and application prospects.

[0019] In summary, this invention achieves precise controlled release of ethephon through microneedle technology, solving the problems of uneven fruit ripening, excessive softening, and potential toxicity caused by traditional ethephon ripening methods. It provides a safe, efficient, and practical technical solution for on-demand ripening and preservation of postharvest fruits. Attached Figure Description

[0020] Figure 1 The images show the finished composite microneedles of the present invention (A), SEM scanning electron microscope images (B) of composite microneedles with different proportions, and mechanical property diagrams (C).

[0021] Figure 2 The images show the Fourier transform infrared (FTIR) spectrum (A), X-ray crystal diffraction (XRD) spectrum (B), and thermal stability analysis (TGA:C and DTG:D) of the composite microneedles of this invention.

[0022] Figure 3 X-ray photoelectron spectroscopy (XPS) of chitosan / lysozyme amyloid protein fiber composite microneedles of the present invention (total spectrum: A and Cl2p peak of composite microneedles: B).

[0023] Figure 4 The graphs show the antibacterial activity (AB) of the composite microbes of this invention against Escherichia coli and Staphylococcus aureus, and the antibacterial activity (CD) against anthrax.

[0024] Figure 5 Image showing the appearance changes of mango fruits under different treatments (CK: untreated mango, ETH: mango soaked in ethephon, CLE: mango treated with composite microneedles).

[0025] Figure 6 The graph shows the changes in color (AC), soluble solids content (D), firmness (E), and decay index (F) of mango fruits under different treatments.

[0026] Figure 7 Figures showing the appearance of anthracnose lesions on mango fruits treated with ethephon immersion and compound microneedles (A), lesion diameter (B), and ethephon residue (C).

[0027] Figure 8 Images of the appearance and starch staining of different post-harvest fruits ripened by composite microneedles (banana: A, star fruit: B, and kiwi: C).

[0028] Figure 9 The ripening effect of compound microbes on banana, star fruit and kiwi fruit - color difference change (AF) and firmness change (GI). Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0030] Example 1: Preparation of composite microneedles Dissolve 1.5 g of lysozyme in 75 mL of distilled water, add concentrated hydrochloric acid to adjust the pH of the solution to 2, and stir on a magnetic stirrer at 200 rpm and 90℃ for 24 h to obtain a lysozyme amyloid fibrillary (LAF) solution, which is then freeze-dried for later use.

[0031] Dissolve 1 g of chitosan (CS) in 100 mL of 1% acetic acid solution. Take 17.5 mL of CS solution and mix it with dried LAF in a 5:5 ratio to obtain a composite solution. Then add 131.13 μL of 40% ethephon solution and mix evenly to form an ethephon-loaded CS / LAF composite solution (CLE).

[0032] After ultrasonic degassing, the composite solution was injected into a pre-made latex microneedle mold and pressed at 0.1 MPa vacuum pressure for 2 hours at room temperature. The pressing was repeated twice. After pressing, the mold was placed in a 40℃ forced-air drying oven for drying and demolding to obtain chitosan / lysozyme amyloid protein fiber / ethephon composite microneedles (CLE).

[0033] The resulting CLE composite microneedles are yellow and transparent in appearance, and have a highly distributed needle array. Figure 1 -A). Secondly, SEM (scanning electron microscopy) observation of the microstructure of CLE composite microneedles with different proportions revealed that pure chitosan microneedles had a complete array distribution, with high needle body formation rate and integrity. However, the doping of lysozyme amyloid fibers caused changes in the chitosan structure. At a ratio of 3 / 7, the matrix of the composite microneedles showed a large number of protrusions, and the needle body was severely fractured. Figure 1 -B). CLE composite microneedles exhibit a similar appearance and needle arrangement to pure chitosan, with a single needle diameter of approximately 549 micrometers. Furthermore, the single-needle force of CLE composite microneedles is above 1.4 N / needle, significantly higher than that of chitosan microneedles (-B). Figure 1 -C), indicating that lysozyme amyloid protein fibers can significantly enhance the mechanical properties of chitosan microneedles, which is beneficial for chitosan composite microneedles to penetrate the fruit epidermis.

[0034] Analysis of the Fourier transform infrared spectrum (FTIR) revealed that at 3400 cm⁻¹ −1 The peak at this location belongs to the stretching vibration of the N single bond H, and the peak gradually increases, which can be reasonably considered as the gradual increase of hydrogen bonding interaction between lysozyme amyloid filaments and chitosan. No new characteristic peaks were found in the control composite microneedles, indicating that no chemical interaction occurred between lysozyme amyloid filaments and chitosan. Figure 2 -A). Furthermore, characteristic groups of ethephon were observed in the FTIR spectrum, indicating that ethephon was successfully encapsulated in microneedles.

[0035] Analysis of X-ray diffraction (XRD) patterns revealed that both pure CS and LAF exhibited a broad peak in the 15°-25° range. The peak intensity of pure chitosan was significantly higher than that of lysozyme amyloid fibers, indicating that chitosan has stronger crystallinity. However, when chitosan and lysozyme amyloid fibers were mixed to construct CL microneedles, the position and intensity of the main peak of the composite microneedles did not change significantly compared to lysozyme amyloid fibers, indicating that chitosan did not affect the secondary structure of lysozyme amyloid fibers. Figure 2 -B).

[0036] Analysis of thermogravimetric analysis (TGA) and differential thermogravimetric (DTG) curves revealed that CLE composite microneedles exhibited higher heat loss compared to pure lysozyme amyloid fibers. This was primarily due to the more flexible network structure of the composite microneedles and their higher moisture content. Furthermore, the weight loss of the loaded microneedles before 100°C was greater than that of the control microneedles, mainly due to the thermal decomposition of ethephon. Ethephon decomposes rapidly at high temperatures, disrupting the network structure of the microneedles. Figure 2 -C、 Figure 2 -D).

[0037] Analysis of the X-ray photoelectron spectroscopy (XPS) spectrum revealed characteristic C1s, O1s, and N1s peaks in all CLE composite microneedle samples, indicating the presence of amino and hydroxyl groups. Furthermore, both CL and CLE composite microneedles exhibited ethephon-specific Cl2p and P2p peaks at 198 eV and 134 eV, respectively, suggesting that ethephon can create active sites in the lysozyme amyloid fibrous structure through hydrogen bonding. Figure 3 -A、 Figure 3 -B).

[0038] Example 2: Antibacterial performance test of CLE composite microneedles First, the bacterial culture concentration was enriched to 10 by cell culture. 5 -10 6 CFU / mL of *Escherichia coli* and *Staphylococcus aureus* were cultured. 1 mL of the bacterial culture was then added to 10 mL of LB broth, along with one chitosan composite microneedle tablet. The mixture was co-cultured at 37°C for 12 hours. After incubation, the bacterial cultures of each component were centrifuged and diluted with sterile physiological saline before being plated. The bacterial inhibition effect of the chitosan composite microneedle co-culture was calculated based on the number of colonies.

[0039] Simultaneously, the morphology of bacteria co-cultured in each component of the membrane was observed using scanning electron microscopy. Specifically, *E. coli* and *Staphylococcus aureus* cultured in each component were washed with sterile physiological saline and then fixed with glutaraldehyde for 12 hours. Subsequently, the immobilized bacteria were separated by centrifugation and washed and dehydrated sequentially with 30%, 50%, 70%, 90%, and 100% ethanol solutions. Finally, the treated samples were dried in a forced-air drying oven, and the morphological changes of each group of bacteria were observed using scanning electron microscopy.

[0040] Furthermore, the inhibitory activity of chitosan-based microneedles on Bacillus anthracis was further tested. First, chitosan microneedles were sterilized by irradiation under UV light for 30 minutes, then dissolved in sterile water and subsequently mixed into PDA medium in the same proportion to prepare resistance plates. Bacillus anthracis bacterial cakes cultured for 2-5 days, approximately 7 mm in diameter, were placed in the center of the microneedle-doped PDA medium, sealed with sealing film, and incubated at 25°C. After one week of incubation, the microneedle-treated Bacillus anthracis culture dishes were removed, and the growth diameter of the Bacillus anthracis was measured. Similarly, the microstructure of Bacillus anthracis was characterized using SEM microscopic observation methods for bacteria.

[0041] The antibacterial performance test results of CLE composite microneedles showed that Escherichia coli and Staphylococcus aureus in the control group and the pure chitosan microneedle treatment group had high activity, and the bacteria grew densely on the plates with a bacterial growth index in the range of 6-10. Figure 4 -A (a, b, c and d), Figure 4 -B). However, with the addition of lysozyme amyloid fibrils, the inhibitory activity of chitosan microbes on both bacteria increased by more than 60%. Secondly, SEM scanning electron microscopy revealed that the control group ( Figure 4 -Aa、 Figure 4 Both *Escherichia coli* and *Staphylococcus aureus* (CLE) exhibited high integrity. Although chitosan microneedle treatment reduced bacterial growth, its bactericidal effect was limited, with only slight wrinkling in the microstructure of both bacteria. Surprisingly, CLE microneedles showed stronger damaging effects on both bacteria, producing numerous wrinkles and ruptures on the bacterial surface. This is mainly because the positively charged residues of lysozyme amyloid filaments bind to the negatively charged surfaces of the bacteria, causing electrostatic interactions in the bacterial cell membrane, leading to cell membrane lysis and ultimately bacterial death.

[0042] Similarly, in the inhibitory activity against anthrax, the anthrax spores in the control group covered the entire plate after one week of culture, while the anthrax spores treated with chitosan microneedles spread to half of the plate. This indicates that the cationic charge of chitosan has a certain inhibitory effect on anthrax. Figure 4 -C、 Figure 4 -D). Importantly, CLE microneedles exhibit significant inhibitory activity against Bacillus anthracis, maintaining low spore growth even after one week of culture, with the diameter of the Bacillus anthracis reduced by more than three times compared to the chitosan microneedle treatment group. Figure 4 -Ce). Secondly, the microscopic morphology of anthrax bacteria under each treatment group was observed using scanning electron microscopy (SEM), such as... Figure 4As shown in Figure -Cf, the mycelial surface of the control group was plump and smooth with intact morphology, indicating normal growth and good structure. In contrast, the surface of the treated group showed obvious shrinkage, depressions, and folds, with severe cell wall collapse, indicating that the treatment disrupted the integrity of the anthracnose cell membrane, leading to cell structural damage. Therefore, CLE microbes exhibit high inhibitory activity against both bacteria and fungi, and their application to harvested fruits and vegetables can effectively inhibit microbial infection, thereby delaying fruit decay and spoilage.

[0043] Example 3: Application of CLE composite microneedles in mango ripening and preservation Forty mangoes of uniform size, ripeness, and free from mechanical damage and pests were selected. Mango tags constructed using CLE microneedles were inserted into the surface of the fruit and pressed for 5 minutes to ensure complete penetration of the needles. Simultaneously, 40 mangoes were placed in each of the following groups: a treatment group soaked in 1000 ppm ethephon solution and an untreated control group.

[0044] During storage, the appearance, color difference, soluble solids content, and decay index of each group of fruits were recorded every 3 days. The color difference was obtained by testing three points in the middle of the fruit with a colorimeter, while the soluble solids content was obtained by testing with a digital display saccharimeter. The decay index of the fruit was assessed according to the degree of decay (0:0%, 1:0-20%, 2:20-40%, 3:40-60%, 4:60-80%, 5:80-100%).

[0045] The results showed that the fruit in the control group remained greenish-blue after 3 days of storage, with high firmness and a total soluble solids (TSS) content of approximately 8%, indicating that it had not yet reached the edible ripe stage. In contrast, the fruit in both the CLE microneedle group and the ethephon treatment group showed obvious color change, with the peel gradually turning yellow and the firmness decreasing to approximately 5-8 N. The TSS content in both groups exceeded 14%, indicating that the fruit was ripe and edible. By the 9th day of storage, the fruit in the CLE microneedle group was still about 3 times firmer than the ethephon treatment group, the TSS content remained at approximately 16%, and the decay index was about 40% lower than that of the ethephon group. Figure 5 , Figure 6 This is mainly attributed to the fact that CLE microneedles can achieve slow release and localized uniform diffusion of ethephon, allowing ethylene to be released stably in the fruit tissue, thus avoiding excessive stimulation and quality deterioration caused by high concentrations of ethephon. Therefore, CLE microneedles not only effectively promote fruit ripening, but also demonstrate superior preservation effects in delaying decay and maintaining fruit quality.

[0046] Example 4: Inhibitory effect of CLE composite microbes on anthrax and detection of ethephon residues Six mango fruits from each of the CLE microneedle treatment group and the ethephon immersion treatment group were collected. A channel approximately 10 μm deep was drilled in the middle of the fruit's equator, and 10 μL of anthracnose suspension was injected using a pipette. The diameter of the lesions on the fruit surface was observed every 3 days during storage for each group.

[0047] Meanwhile, samples of peel and pulp were taken from the wounds of mango fruits inserted with CLE microneedles, while samples of mango fruits soaked in ethephon were taken along the equator of the fruit.

[0048] Method for detecting ethephon residues in mango fruit: Weigh a certain amount of mango sample and place it in a 5 mL centrifuge tube. Add 1 mL of methanol / hydrochloric acid aqueous solution (1:1, pH 3–4) and extract by ultrasonication at 200 W for 10 min. After centrifugation at 5000 rpm for 3 min, transfer the supernatant to a 2 mL polyethylene centrifuge tube and slowly purge with nitrogen at 35 °C until the methanol evaporates. Add 0.5 mL of anhydrous diethyl ether, vortex for 3 min and let stand for 10 min. Transfer the ether layer to another tube and repeat the ether extraction once more. Combine the liquids and dry them under nitrogen at 35 °C. Add 100 μL of toluene and vortex for 30 s. Then add 10 μL of the derivatization reagent MTBSTFA, vortex for 30 s, and seal and react in a 60 °C water bath for 30 min. Make up to 1 mL with toluene, vortex to mix, and filter through a 0.22 μm organic phase filter membrane for GC-MS / MS analysis. The internal standard was deuterated styrene prepared in methanol (25 μg / mL, CAS: 19361-62-7). The sample was then placed in a headspace vial, shaken at 60°C for 15 min, extracted with SPME fiber at 60°C for 30 min, and then thermally desorbed (260°C, 5 min) using the GC injector. The chromatographic column was an HP-5MS (30 m × 0.25 mm × 0.25 μm), the carrier gas was high-purity He (99.999%), and the flow rate was 1.0 mL / min. The injection mode was split injection (split ratio 25:1). The temperature program was: initial 50°C, held for 5 min; increased at 7°C / min to 230°C, held for 3 min; then increased at 40°C / min to 320°C, held for 5 min. Mass spectrometry conditions: EI ion source, positive ion mode, electron energy 70 eV; ion source temperature 240℃, quadrupole temperature 160℃; acquisition mode SIM, monitoring ions 74 and 112.1.

[0049] The results showed that after inoculation with anthracnose, obvious lesions appeared on the fruit after 3 days of storage; by 6 days of storage, the diameter of the lesions in the ethephon-immersed group was approximately 1.27 cm, while that in the CLE microneedle group was only 0.63 cm, demonstrating a significant antibacterial effect. With prolonged storage, the lesions in the ethephon-immersed group continued to spread, while the lesions in the CLE microneedle-treated group expanded significantly more slowly, maintaining a smaller diameter throughout, indicating that CLE microneedles can effectively inhibit the infection and spread of pathogens. Figure 7 -A、 Figure 7 -B).

[0050] Testing revealed that almost no ethephon residue was detected in the peel and pulp of fruits ripened with CLE microneedles (detection limit 0.133 ng / g), far below the national limit (2 μg / g). In contrast, the ethephon-soaked group showed ethephon residues of 75.64 ± 5.90 ng / g in the peel and 1.21 ± 0.40 ng / g in the pulp. While these residue levels still meet national standards, they are significantly higher than those in the CLE microneedle group. Figure 7 -C).

[0051] Example 5: Evaluation of the ripening effect of CLE composite microbes on various fruits To verify the effectiveness of CLE microbes on postharvest fruits, bananas, star fruit, and kiwifruit were selected as experimental subjects. The appearance, color difference, and firmness of the fruits were recorded. For bananas and kiwifruit, starch was stained with iodine-potassium iodide to observe fruit ripening.

[0052] The results showed that CLE microneedling treatment significantly promoted the ripening process of bananas, star fruit, and kiwifruit. CLE microneedling exhibited a high ripening effect on post-harvest bananas, star fruit, and kiwifruit. Within 5 days after treatment, all three fruits completed a significant ripening process, with a significant decrease in starch content accompanied by a large accumulation of sugar, rapidly bringing the fruits to an edible state. Figure 8 This indicates that CLE microneedles can effectively regulate the internal metabolism of fruits, promote the conversion of starch into sugar, and thus accelerate the ripening process.

[0053] Furthermore, under CLE microneedling treatment, the peel color of bananas and star fruit changed significantly, with brightness (L), redness (a), and yellowness (b) all increasing significantly, indicating a marked improvement in fruit maturity. Figure 9 -A、 Figure 9 -B、 Figure 9 -C、 Figure 9 -D、 Figure 9 -E、 Figure 9 -F). Meanwhile, the firmness of the three fruits decreased rapidly under the action of CLE microneedles, all dropping below 1 N after 5 days (-F). Figure 9 -G、 Figure 9 -H、 Figure 9 -I), which fully demonstrates that CLE microneedles can transform fruit from physiological maturity to an edible state in a relatively short period of time.

[0054] In summary, the CLE microneedles provided by this invention possess excellent mechanical properties, antibacterial activity, and biodegradability, and can effectively insert into fruit tissue. By slowly releasing ethephon, these microneedles promote fruit ripening to an edible state within 3 days, while significantly delaying decay and maintaining quality stability. Their ability to inhibit disease occurrence and the significant reduction in ethephon residues jointly ensure the safety and controllability of the ripening and preservation process. Furthermore, this technology accelerates the improvement of fruit appearance quality (such as color and firmness) and promotes internal physiological metabolism (such as starch degradation and sugar accumulation), enabling postharvest fruits such as bananas, star fruit, and kiwifruit to reach ideal edible quality in a short period. These results fully demonstrate the application potential of CLE microneedles in the ripening and quality management of various fruits, providing a reliable basis for expanding their application to the postharvest management of other fruits.

Claims

1. Ethylene glycol controlled release based chitosan / lysozyme amyloid fibril composite microneedle characterized in that, The composite microneedle is composed of chitosan, lysozyme amyloid fiber and ethephon, wherein the mass ratio of chitosan to lysozyme amyloid fiber is 5:5, and the loading amount of ethephon is 131.13 μL of 40% ethephon solution added per 17.5 mL of 1% chitosan solution.

2. The composite microneedle of claim 1, wherein, The lysozyme amyloid fiber is prepared by the following method: 1.5 g of lysozyme is dissolved in 75 mL of distilled water, concentrated hydrochloric acid is added dropwise to adjust the pH to 2, and stirring is carried out at a rotation speed of 200 rpm and a temperature of 90°C for 24 h to obtain a lysozyme protein-amyloid fiber solution, and lysozyme amyloid fibers are obtained after freeze-drying.

3. The composite microneedle of claim 2, wherein, The composite microneedle is prepared by the following method: 1 g of chitosan is dissolved in 100 mL of 1% acetic acid solution, 17.5 mL of the chitosan solution is taken, and the dry lysozyme amyloid fibers prepared in claim 2 are mixed uniformly at a ratio of 5:5, 131.13 μL of 40% ethephon solution is added to form a composite solution, the solution is degassed by ultrasonic treatment, and then the solution is injected into a latex microneedle mold, and the mold is pressed at room temperature under a vacuum pressure of 0.1 MPa for 2 h, the pressing is repeated twice, and then the mold is dried at a temperature of 40°C to obtain the composite microneedle.

4. The composite microneedle of claim 1, wherein, The single-needle mechanical strength of the composite microneedle is greater than 1.4 N / needle.

5. A label constructed of the composite microneedle of claim 1, wherein The composite microneedle is hidden on the back of the label, and the label can display information of the fruit.

6. Use of the label of claim 5 in on-demand ripening of postharvest fruits.

7. Use according to claim 6, characterized in that, The use is: the label is directly attached to the surface of the fruit, the microneedle is inserted into the fruit skin by pressing, the slow release of ethephon is realized, the fruit reaches an edible state within 3 days, and the softening process of the fruit after ripening is delayed.

8. Use according to claim 6, characterized in that, The fruit is mango, banana, kiwi or citron.

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