Thymol nano-preservation agent with reversible conversion of hydrophilicity and hydrophobicity in response to temperature

Abstract

The application discloses a temperature-responsive reversible conversion of hydrophilic and hydrophobic thymol nano-preservation agent, and belongs to the technical field of pesticides. The nano-preservation agent is composed of temperature-responsive block polymers, thymol and metal ions, forms regular rod-shaped nanoparticles, and has an average particle size of 185-225 nm and an average Zeta potential of +40.8 mV to +44.4 mV. The nano-preservation agent presents a hydrophobic state at 35-40 DEG C, enhances the adhesion to the wax layer of the fruit skin, presents a hydrophilic state at 20-25 DEG C, reduces the adhesion, and realizes water washing and desorption. The application further discloses a preparation method of the nano-preservation agent and application of the nano-preservation agent in postharvest antibacterial preservation of fruits. The nano-preservation agent utilizes the natural temperature difference between postharvest storage and room temperature cleaning, realizes intelligent response of high-temperature adhesion preservation and low-temperature easy washing, and realizes low residue while ensuring efficient preservation.

Description

Technical Field

[0001] This invention relates to the field of pesticide technology, specifically to a temperature-responsive thymol nanopreservative with reversible hydrophilicity-hydrophobicity conversion. Background Technology

[0002] Statistics show that approximately 50% of fresh fruit worldwide is lost annually due to post-harvest decay, resulting in a massive waste of resources. The main causes of post-harvest decay are high water content, active respiration, and susceptibility to microbial contamination during storage, transportation, and sales. Currently, surface coating with chemical preservatives (such as soaking or spraying) remains one of the primary methods for extending fruit shelf life, as it does not rely on energy-intensive cold chain storage and effectively maintains the freshness and nutritional quality of the fruit. However, most fruits are covered with a natural hydrophobic barrier composed of cuticle and wax, making it difficult for water-soluble preservatives to spread effectively and deposit evenly on the fruit peel, resulting in insufficient preservation efficiency.

[0003] To improve the adhesion of preservatives to hydrophobic surfaces, researchers have developed various advanced preservation systems in recent years, including functional films, nano-preservatives, and bio-based coatings. These technologies extend the shelf life of fruits to some extent by enhancing the affinity between preservatives and fruit peels and prolonging the release period. However, existing technologies still face a long-standing core contradiction: to achieve efficient preservation during storage and transportation, preservatives need to form strong adhesion to the fruit peel surface; but this strong adhesion makes it difficult to completely wash off the preservatives before consumption, resulting in chemical residues and posing food safety risks. Some studies have attempted to use edible and biocompatible materials to prepare preservatives to reduce the potential harm of residues to the human body. However, this method only indirectly alleviates the risk and does not truly solve the problem of high residues. Furthermore, its formulation often depends on specific material systems, resulting in poor stability and universality, making widespread application difficult. Most traditional nano-preservatives fail to fully utilize temperature characteristics to regulate their adhesion and residue effects on fruit peels, and no preservatives optimized for different temperature conditions have yet been developed in current technology.

[0004] Therefore, in order to address the above problems, the present invention provides a temperature-responsive hydrophobic reversible thymol nanopreservative to solve the technical problem of balancing preservation effect and residue safety in existing fruit preservation technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a temperature-responsive, reversibly hydrophilic-hydrophobic nanopreservative of thymol. By encapsulating thymol and metal ions with a temperature-responsive block polymer to form regular rod-shaped nanoparticles, this nanopreservative exhibits a hydrophobic state at 35-40°C to enhance adhesion to the waxy layer of fruit peel, and a hydrophilic state at 20-25°C to reduce adhesion to the waxy layer of fruit peel. This achieves highly efficient adhesion preservation during post-harvest storage of fruit and low-residue desorption during pre-consumption washing, thus solving the technical problem of balancing preservation effect and residue safety in existing fruit preservation technologies.

[0006] The objective of this invention is achieved through the following technical solution: A temperature-responsive, reversibly hydrophilic-hydrophobic nanopreservative is disclosed. The nanopreservative comprises thymol and metal ions encapsulated by a temperature-responsive block polymer, forming regular rod-shaped nanoparticles. The nanopreservative exhibits a hydrophobic state at 35-40°C, enhancing adhesion to the waxy layer of fruit peel; and a hydrophilic state at 20-25°C, reducing adhesion to the waxy layer and enabling water-washable desorption. The average particle size of the nanoparticles is 185-225 nm, and the average Zeta potential is +40.8 mV to +44.4 mV. This technology utilizes a temperature-responsive block polymer that undergoes a hydrophilic-hydrophobic transition at high temperatures, enhancing the hydrophobicity of the nano-preservative and improving its affinity for the waxy layer of fruit peel, thus achieving efficient adhesion and retention. During low-temperature, room-temperature washing, the polymer undergoes a hydrophobic-hydrophilic transition, increasing the hydrophilicity of the nano-preservative surface and reducing its affinity for the waxy layer of fruit peel, thereby achieving rapid desorption and low residue. The reversible switching between adhesion and desorption can be achieved by adjusting the temperature, thus simultaneously meeting the dual needs of post-harvest preservation and pre-consumption washing.

[0007] Preferably, the temperature-responsive block polymer is PDMAEMA-b-PCL, PNIPAM-b-PCL, or POEGMA-b-PCL; the metal ion is Fe. 3+ Zn 2+ or Cu 2+ The metal ions coordinate with the phenolic hydroxyl groups in the thymol. POEGMA-b-PCL exhibits excellent biocompatibility and temperature responsiveness, while PDMAEMA-b-PCL and PNIPAM-b-PCL also possess temperature-sensitive properties. Through coordination with the phenolic hydroxyl groups of thymol, the metal ions effectively improve the encapsulation efficiency of the active ingredients and the structural stability of the nanoparticles, ensuring that the nano-preservative maintains its regular rod-like shape during storage and use.

[0008] Preferably, at 20°C, the average number of hydrogen bonds in the nano-preservative is 164.8, and the radius of gyration is 2.904 nm; at 40°C, the average number of hydrogen bonds is 155.2, and the radius of gyration is 2.649 nm. These parameters indicate that as the temperature increases, the number of hydrogen bonds inside the nano-preservative decreases, the molecular chain conformation shrinks, and the radius of gyration decreases, thereby causing its surface to change from hydrophilic to hydrophobic. This microstructural change is the intrinsic mechanism by which the nano-preservative achieves temperature-responsive reversible hydrophilic-hydrophobic conversion.

[0009] This application also claims a method for preparing the above-mentioned nano-preservative, comprising the following steps: S1, dissolving thymol and a temperature-responsive block polymer in tetrahydrofuran to prepare a stream 1, wherein the concentration of thymol in stream 1 is 1.0 mg / mL and the concentration of the temperature-responsive block polymer is 0.2 mg / mL; S2, dissolving a metal chloride (FeCl3, ZnCl2 or CuCl2) in deionized water to prepare a stream 2, wherein the concentration of the metal chloride in stream 2 is 5 mM; S3, simultaneously injecting equal volumes of stream 1 and stream 2 into a mixer for mixing at an injection flow rate of 30 mL / min to obtain a nanoparticle mixture; S4, dialyzing the nanoparticle mixture obtained in step S3 in ultrapure water for 12-14 hours to remove tetrahydrofuran, free thymol, and metal ions, thereby obtaining the temperature-responsive hydrophobic reversible thymol nano-preservative. This method employs microfluidic co-assembly technology, where polymers, thymol, and metal ions self-assemble through instantaneous mixing to form regular rod-shaped nanoparticles. It offers advantages such as ease of operation, mild conditions, and good batch-to-batch reproducibility. The dialysis step effectively removes organic solvents and small molecule impurities, ensuring the purity and safety of the nano-preservative.

[0010] This application also claims a method for preserving fruit using the aforementioned nano-preservative, comprising the following steps: applying the nano-preservative to the fruit surface at 35-40°C by immersion or spraying for 28-33 seconds, utilizing the high-temperature hydrophobic properties of the nano-preservative to enhance its adhesion and retention on the waxy layer of the fruit peel, thus inhibiting microbial infection; before consumption, washing the fruit in water at 20-25°C, utilizing the low-temperature hydrophilic properties of the nano-preservative to reduce its adhesion to the fruit peel, allowing the nano-preservative to detach from the fruit surface. This method fully utilizes the natural temperature difference between the post-harvest storage temperature and the room temperature washing temperature, achieving an intelligent response behavior of "high-temperature adhesion preservation and low-temperature easy detachment," is simple to operate, requires no additional energy consumption, and is suitable for large-scale commercial applications.

[0011] Preferably, the fruit is blueberry, cherry tomato, strawberry, or cherry; the residue ratio of the nano-preservative after washing in water at 20-25°C is less than 16%. After washing with deionized water at room temperature, the residue ratio of the nano-preservative on the surface of blueberries, cherry tomatoes, strawberries, and cherries is all less than 16%, and can be reduced to as low as about 10.8%, indicating that the nano-preservative has good washability and significantly reduces the risk of chemical residue.

[0012] Preferably, the survival rate of the nano-preservative against human bronchial epithelial cells is 62.5% and against human oral keratinocytes is 58.2% at a concentration of 40 μg / mL. Cell viability was determined using the MTT assay; even at a high concentration of 40 μg / mL, the survival rate of both types of human cells was above 50%, indicating that the nano-preservative exhibits low toxicity to human cells, possesses good biocompatibility, and can be safely applied in the food industry.

[0013] Preferably, the nano-preservative maintains over 95% of the surface integrity of blueberries, cherry tomatoes, strawberries, or cherries during a 30-day storage period. After treatment with the nano-preservative, blueberries showed no obvious rot within 30 days, cherry tomatoes had smooth skin without mold spots within 14 days, strawberries showed less than 2% mold area within 7 days, and cherries basically maintained their original shape within 14 days. In contrast, the untreated control group showed severe rot, shriveling, or mold, indicating that the nano-preservative has universal and highly effective preservation capabilities for a variety of fruits.

[0014] This application also claims the use of the aforementioned nano-preservative in the preparation of an antibacterial product for inhibiting Staphylococcus aureus or Escherichia coli on the surface of fruit, wherein the nano-preservative exhibits a bactericidal rate of over 89% against both Staphylococcus aureus and Escherichia coli. After co-culturing the nano-preservative with bacterial culture, the bactericidal rates against Staphylococcus aureus and Escherichia coli reached 89.1% and 89.9%, respectively. Scanning electron microscopy analysis results show that the nano-preservative exerts its bactericidal effect by disrupting the integrity of the bacterial cell membrane, causing wrinkling, depressions, and ruptures on the bacterial surface.

[0015] This application also claims the application of the aforementioned nano-preservative in postharvest fruit preservation. The nano-preservative is coated onto the fruit surface at 35-40°C, making it hydrophobic to adhere to the waxy layer of the fruit peel. Then, it is washed with water at 20-25°C to make it hydrophilic and detach from the fruit surface. This application method fully utilizes the temperature-responsive characteristics of the nano-preservative. During postharvest storage, high temperature enhances adhesion for long-term preservation; before consumption, room-temperature washing achieves low-residue desorption, balancing preservation effectiveness and food safety.

[0016] The working mechanism of this invention is as follows: This invention utilizes the temperature-responsive properties of temperature-responsive block polymers, combined with the coordination complexation of thymol and metal ions, to construct regular rod-shaped nanoparticles. When the temperature rises to 35-40℃, the polymer segments change from a hydrophilic to a hydrophobic state, increasing the hydrophobicity of the nano-preservative surface, reducing the number of hydrogen bonds, shrinking the radius of gyration, and generating a strong hydrophobic interaction with the waxy layer of the fruit peel, achieving efficient adhesion and long-lasting preservation. When the temperature drops to 20-25℃, the polymer segments return to a hydrophilic state, increasing surface hydrophilicity and reducing the affinity with the waxy layer of the fruit peel, allowing for rapid desorption by room temperature washing, achieving low residue. Metal ions coordinate and complex with the phenolic hydroxyl groups of thymol, improving the encapsulation rate and structural stability; thymol exerts its bactericidal effect by disrupting the integrity of bacterial cell membranes. This invention utilizes the natural temperature difference between post-harvest storage and room temperature washing to achieve an intelligent response of "high-temperature adhesion preservation and low-temperature easy washing," solving the technical challenge of balancing preservation effect and residue safety.

[0017] Due to the application of the above technical solution, the present invention has the following beneficial effects compared with the prior art: 1. This invention uses thymol as the active ingredient and utilizes temperature-responsive block polymers to encapsulate thymol and metal ions. It achieves desorption by enhancing adhesion through high-temperature hydrophobicity and desorption through low-temperature hydrophilicity, thus solving the technical problem of balancing preservation effect and residue safety.

[0018] 2. This invention uses microfluidic co-assembly technology to prepare nano-preservatives. The operation is simple and the batch repeatability is good. The obtained nanoparticles have a particle size of 185~225nm and a potential of +40.8mV~+44.4mV, and have good dispersibility and stability.

[0019] 3. This invention has a highly efficient preservation effect on a variety of fruits such as blueberries, cherry tomatoes, strawberries, and cherries. The fruit surface integrity rate remains above 95% within 30 days, and the residual proportion is less than 16% after washing with water at room temperature.

[0020] 4. The present invention has a bactericidal rate of over 89% against Staphylococcus aureus and Escherichia coli, and exhibits low toxicity to human bronchial epithelial cells and oral keratinocytes.

[0021] 5. This invention utilizes the natural temperature difference between post-harvest storage and room temperature washing of fruits, without requiring additional energy consumption, and achieves an intelligent response of "high-temperature adhesion preservation and low-temperature easy washing off". Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, some of the drawings in the following description are some embodiments of the present invention. For those skilled in the art, other drawings can be made based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram illustrating the function of the nano-preservative prepared in Example 1 of the present invention; Figure 2 These are TEM images and particle size statistics of the nano-preservative prepared in Example 1 of this invention; Figure 3 This is a diagram showing the Zeta potential and particle size of the nano-preservative prepared in Example 1 of this invention under cyclic changes at 25℃~40℃; Figure 4 This is a graph showing the contact angle and liquid retention of the nano-preservative prepared in Example 1 of this invention on blueberry skin at different temperatures; Figure 5 These are SEM images of the nano-preservative prepared in Example 2 of this invention on blueberry peels at different temperatures; Figure 6 This is a comparison chart of the percentage of fluorescence retention area on blueberry skin at different temperatures for the nano-preservative prepared in Example 1 of this invention; Figure 7 This is a diagram showing the bactericidal effect of the nano-preservative prepared in Example 1 of this invention on Staphylococcus aureus and Escherichia coli. Figure 8 These are scanning electron microscope images comparing the effects of the nano-preservative prepared in Example 1 of this invention on Staphylococcus aureus and Escherichia coli after treatment; Figure 9 This is a comparison chart showing the preservation ability of the nano-preservative prepared in Example 1 of this invention on apple slices; Figure 10 This is a comparison chart showing the preservation capabilities of the nano-preservative prepared in Example 1 of this invention on blueberries, cherry tomatoes, strawberries, and cherries; Figure 11 This is a graph showing the residual proportions of the nano-preservative prepared in Example 3 of this invention on blueberries, cherry tomatoes, strawberries, and cherries after washing. Figure 12 This is a graph showing the toxicity of the nano-preservative prepared in Example 1 of this invention to human oral keratinocytes and human bronchial epithelial cells. Detailed Implementation

[0024] To provide a clearer understanding of the technical features, objectives, and effects of this invention, specific implementation schemes are now described in detail.

[0025] The present invention will be further described below with reference to embodiments, but the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0026] Example 1 See appendix Figure 1 -Appendix Figure 4 Appendix Figure 6 -Appendix Figure 10 and appendix Figure 12 This embodiment provides a temperature-responsive thymol nanopreservative with reversible hydrophilicity-hydrophobicity conversion, the preparation method of which includes the following steps: S1. Thymol and POEGMA-b-PCL are dissolved in tetrahydrofuran to prepare a stream 1, wherein the concentration of thymol in the stream 1 is 1.0 mg / mL and the concentration of POEGMA-b-PCL is 0.2 mg / mL. S2. Dissolve FeCl3 in deionized water to prepare stream 2, wherein the concentration of FeCl3 in stream 2 is 5 mM; S3. Inject equal volumes of stream 1 and stream 2 into a mixer for mixing at a flow rate of 30 mL / min to obtain a nanoparticle mixture. S4. The nanoparticle mixture obtained in step S3 is dialyzed in ultrapure water for 14 hours to remove tetrahydrofuran, free thymol, and Fe. 3+ The temperature-responsive hydrophilic-hydrophobic reversible thymol nanopreservative was obtained (see...). Figure 1 ).

[0027] Transmission electron microscopy analysis of the prepared nano-preservatives showed that they possessed good dispersibility and a uniform rod-like structure (see [link to study]). Figure 2 The particle size and potential of the above-mentioned nano-preservatives under temperature cycling were analyzed using Zetasizer. The results showed that the particle size increased with increasing temperature, while the potential decreased with increasing temperature. Furthermore, the particle size and potential exhibited reversible changes during temperature cycling (see [link to Zetasizer]). Figure 3 ).

[0028] The nano-preservative prepared in Example 1 was dripped onto the skin of fresh blueberries at different temperatures (25°C, 32°C, and 40°C), and the change in contact angle was measured using a contact angle meter. Blueberries were then immersed in the nano-preservative solution for 30 seconds and removed until no more water dripped; the difference in mass before and after immersion was measured to calculate the liquid retention. The results are as follows: Figure 4As shown, the contact angles of the nano-preservative on the blueberry skin at 25℃, 32℃, and 40℃ were 89.9°, 75.2°, and 59.4°, respectively, indicating that the wettability of the nano-preservative on the blueberry skin significantly increased with increasing temperature. The liquid holding capacity measurement results showed that the liquid holding capacity of the nano-preservative at 40℃ was 14.1 mg / cm³. 3 The level was significantly higher than the 5.3 mg / cm² in the 25℃ group. 3 This indicates that the nano-preservative has a stronger affinity for the waxy coating of fruit peel at high temperatures and has a stronger liquid retention capacity.

[0029] Fresh blueberries were immersed in the nano-preservative solution prepared in Example 1 at 25℃, 32℃, and 40℃ for 30 seconds respectively, and then removed until no water dripped. A portion of the blueberry skin was carefully removed, dried completely at the corresponding temperature, and then analyzed by scanning electron microscopy. Figure 5 As shown, the blueberry skin at 25℃ is smooth with only a few scattered rod-shaped nanoparticles; however, as the temperature rises, the blueberry skin shows increasingly dense rod-shaped nanoparticles that evenly cover the skin surface, indicating that the nano preservative can effectively deposit and remain on the blueberry skin at high temperatures.

[0030] Using Staphylococcus aureus and Escherichia coli as indicator organisms, the bactericidal properties of the nano-preservative were investigated. Bacterial suspensions in the logarithmic growth phase were co-cultured for 6 hours with sterile water (control group) and the nano-preservative prepared in Example 1 (preservative group), respectively. The resulting cultures were then spread onto LB agar plates and incubated at 37°C for 12 hours. Colony growth was observed, and the bactericidal rate was calculated. Figure 7 As shown, the bacterial colonies on the sterile water plate were densely and evenly distributed, while only sporadic colonies were observed on the plate treated with the nano-preservative. Quantitative analysis showed that the nano-preservative achieved bactericidal rates of 89.1% and 89.9% against Staphylococcus aureus and Escherichia coli, respectively, demonstrating its strong antibacterial ability.

[0031] After co-culturing Staphylococcus aureus and Escherichia coli bacterial suspensions with sterile water and the nano-preservative prepared in Example 1 above for 6 hours, the bacterial cells were resuspended in 2.5% glutaraldehyde, fixed overnight at 4°C, and then treated sequentially with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol solutions for 15 minutes each before being dropped onto a silicon wafer for scanning electron microscopy analysis. Figure 8 As shown, bacteria treated with sterile water have smooth surfaces and intact morphology, exhibiting typical spherical or rod-shaped forms; while bacteria treated with nano-preservatives show obvious wrinkling, depressions, and ruptures on their surfaces, and the integrity of the bacterial membrane is destroyed, indicating that nano-preservatives exert their bactericidal effect by disrupting the structure of bacteria.

[0032] Fresh apples were cut into evenly sized pieces and soaked in deionized water and the nano-preservative prepared in Example 1 for 30 seconds respectively. After being removed and air-dried, they were stored at 25°C and 70% relative humidity for 7 days, and the changes in appearance were recorded by taking photos. Figure 9 As shown, apple pieces soaked in deionized water showed severe browning and surface shrinkage after 7 days, and even mold, rendering them inedible. In contrast, apple pieces treated with nano-preservatives maintained their original color and plump shape after 7 days, with almost no browning, indicating that nano-preservatives have excellent antioxidant and preservation effects.

[0033] Fresh blueberries, cherry tomatoes, strawberries, and cherries were soaked and dried separately in deionized water and the nano-preservative prepared in Example 1 above at the appropriate temperatures for fruit ripening (40°C for blueberries, cherry tomatoes, and cherries; 30°C for strawberries). They were then stored under simulated sales conditions (15°C, 50% relative humidity for blueberries, cherry tomatoes, and strawberries; 25°C, 60% relative humidity for cherries). The extent of rot was regularly photographed and the proportion of intact skin area was statistically analyzed. Figure 10 As shown, after 30 days of storage, the surface rotten area of ​​blueberries in the deionized water group reached 39.2%, while no obvious rottenness was observed on the surface of blueberries in the nano-preservative treatment group. After 14 days of storage, the surface rotten area of ​​cherry tomatoes in the deionized water group reached 50.7%, while the cherry tomatoes in the nano-preservative treatment group had smooth skin and no mold spots. After 7 days of storage, the surface rotten area of ​​strawberries in the deionized water group reached 49.9%, while the strawberries in the nano-preservative treatment group showed mold spots in less than 2% of the area and the flesh remained intact. After 14 days of storage, the surface of cherries in the deionized water group showed obvious dents, shriveling, and mold spots, while the cherries in the nano-preservative treatment group basically maintained their original shape. These results indicate that nano-preservatives have a universal and highly effective preservation ability for a variety of fruits.

[0034] Human bronchial epithelial cells (BEAS-2B) and human oral keratinocytes (HOK) were used as cell models. After co-culturing with different concentrations (5, 10, 20, 40 μg / mL) of the nano-preservative prepared in Example 1 for 24 hours, cell viability was determined using the MTT assay. BEAS-2B or HOK cells in the logarithmic growth phase were seeded at a density of 8000 cells / well in 96-well plates with 100 μL of culture medium per well. After the cells had fully adhered to the bottom of the wells, the original culture medium was removed, and 100 μL of fresh culture medium containing different concentrations of the nano-preservative was added. Six replicates were set up for each condition, with the condition without nanoparticle culture medium serving as a blank control. After incubating the 96-well plate in an incubator for 24 hours, 150 μL of pre-prepared 5 mg / mL MTT working solution was added to each well. The plate was then returned to the incubator and incubated in the dark for 4 hours. The culture medium was then aspirated, and 150 μL of dimethyl sulfoxide was added to each well to dissolve the generated blue-purple formazan crystals. The plates were shaken thoroughly on a shaker, and the cell viability in each well was measured using a microplate reader. Figure 12 As shown, cell viability gradually decreased with increasing concentration of the nano-preservative. However, at a high concentration of 40 μg / mL, the viability of BEAS-2B cells was 62.5%, and that of HOK cells was 58.2%, both exceeding 50%. This indicates that the nano-preservative exhibits low toxicity to both types of human cell lines and demonstrates good biocompatibility.

[0035] Example 2 See appendix Figure 5 This embodiment provides a temperature-responsive thymol nanopreservative with reversible hydrophilicity-hydrophobicity conversion, the preparation method of which includes the following steps: S1. Thymol, POEGMA-b-PCL and 5-isothiocyanate fluorescein were dissolved in tetrahydrofuran, with thymol concentration of 1.0 mg / mL, POEGMA-b-PCL concentration of 0.2 mg / mL and 5-isothiocyanate fluorescein concentration of 0.1 mg / mL, as stream 1. S2. Dissolve FeCl3 in ultrapure water to a concentration of 5 mM, and use it as stream 2; S3. Inject equal volumes of stream 1 and stream 2 into a mixer for mixing at a flow rate of 30 mL / min to obtain a nanoparticle mixture. S4. The nanoparticle mixture obtained in step S3 is dialyzed in ultrapure water for 14 hours to remove tetrahydrofuran, free thymol, and Fe. 3+ By combining 5-isothiocyanofluorescein, a temperature-responsive hydrophilic-hydrophobic reversible thymol nanopreservative loaded with 5-isothiocyanofluorescein was obtained.

[0036] Fresh blueberries were immersed in the 5-isothiocyanate-loaded nano-preservative solution prepared in Example 2 at 25℃, 32℃, and 40℃ respectively. After 30 seconds, they were removed until no water dripped. A portion of the blueberry skin was carefully removed, dried completely at room temperature, and analyzed by confocal microscopy. The percentage of fluorescent area on the blueberry skin was calculated using ImageJ to assess the retention of the nano-preservative on the blueberry skin as the temperature increased. Figure 5 As shown, when the temperature is low, the fluorescent area accounts for only about 6.0% of the total area, but as the temperature increases, the proportion of the fluorescent area increases significantly, reaching 33.2% at 40℃. This indicates that as the temperature increases, the amount of nano-preservatives retained on the blueberry skin increases, which is consistent with the analysis of scanning electron microscopy and liquid holding capacity experiments.

[0037] Example 3 See appendix Figure 11 This embodiment provides a temperature-responsive thymol nanopreservative with reversible hydrophilicity-hydrophobicity conversion, the preparation method of which includes the following steps: S1. Thymol, POEGMA-b-PCL and indocyanine green were dissolved in tetrahydrofuran, with thymol concentration of 1.0 mg / mL, POEGMA-b-PCL concentration of 0.2 mg / mL and indocyanine green concentration of 0.1 mg / mL, as stream 1. S2. Dissolve FeCl3 in ultrapure water to a concentration of 5 mM, and use it as stream 2; S3. Inject equal volumes of stream 1 and stream 2 into a mixer for mixing at a flow rate of 30 mL / min to obtain a nanoparticle mixture. S4. The nanoparticle mixture obtained in step S3 is dialyzed in ultrapure water for 14 hours to remove tetrahydrofuran, free thymol, and Fe. 3+ Indocyanine green was used to obtain a temperature-responsive hydrophilic-hydrophobic reversible thymol nanopreservative loaded with indocyanine green.

[0038] Fresh blueberries, cherry tomatoes, strawberries, and cherries were immersed in the indocyanine green-loaded nano-preservative solution prepared in Example 3 above at the appropriate fruit ripening temperatures (40°C for blueberries, cherry tomatoes, and cherries; 30°C for strawberries). After 30 seconds, the fruits were removed and dried, and the fluorescence intensity was calculated using an image processing unit (IPU) and a two-zone imaging system. The fruits were then immersed in deionized water at room temperature, and after half an hour, they were naturally dried and observed again using an IPU and the fluorescence intensity was calculated using ImageJ. The residue of the nano-preservative after washing was calculated by statistically analyzing the two fluorescence intensities. Figure 11As shown, the fluorescence signal of the nano preservative on the fruit surface was significantly reduced after washing, all dropping to below 16%, indicating that the nano preservative has extremely low residue after washing with water at room temperature and has good washability.

[0039] Comparative Example 1 This comparative example is based on Example 1 above. The similarities with Example 1 will not be repeated. The difference between this comparative example and Example 1 is that no metal ions are added in this comparative example. That is, in step S2, deionized water is used as stream 2 and no metal chlorides are added.

[0040] Testing revealed that the nanoparticles prepared in this comparative example had uneven particle size distribution, significantly lower encapsulation efficiency than those in Example 1, and poorer structural stability.

[0041] Comparative Example 2 This comparative example is based on Example 1 above. The similarities with Example 1 will not be repeated. The difference between this comparative example and Example 1 is that no temperature-responsive block polymer is added in this comparative example. That is, in step S1, only thymol is dissolved in tetrahydrofuran and POEGMA-b-PCL is not added.

[0042] Testing revealed that this comparative sample was unable to form regular rod-shaped nanoparticles, and thymol rapidly aggregated and precipitated in water, failing to achieve temperature-responsive adhesion and desorption.

[0043] The nano-preservatives prepared in the above examples and comparative examples were subjected to performance testing. The test items included: particle size, zeta potential, sterilization rate against Staphylococcus aureus, sterilization rate against Escherichia coli, surface integrity rate of blueberries after 30 days, residual content after room temperature water washing, and BEAS-2B cell viability (40 μg / mL). The test results are shown in Table 1.

[0044] Table 1

[0045] As can be seen from the test results in Table 1, the nano-preservatives prepared in Examples 1 to 3 all have regular rod-shaped structures, suitable particle size ranges, and high positive Zeta potentials. They all achieved bactericidal rates of over 89% against Staphylococcus aureus and Escherichia coli, maintained a blueberry fruit surface integrity rate of over 95% during a 30-day storage period, and had a residue ratio of less than 12% after room temperature washing. Furthermore, they exhibited low toxicity to human bronchial epithelial cells. Example 1 (using POEGMA-b-PCL and Fe...) 3+ It has the best overall performance.

[0046] Comparative Example 1, lacking the addition of metal ions, resulted in a decreased encapsulation rate and poor structural stability of the nanoparticles, failing to form stable nanoparticles. Consequently, its sterilization rate and preservation effect were significantly lower than those of Example 1, and the residual proportion after room temperature water washing was as high as 35.6%. Comparative Example 2, lacking the addition of a temperature-responsive block polymer, was unable to form nanoparticles. Thymol rapidly aggregated and precipitated in water, failing to achieve temperature-responsive adhesion and desorption, resulting in the worst preservation effect.

[0047] The above results show that the present invention utilizes a temperature-responsive block polymer to encapsulate thymol and metal ions to form a nano-preservative, which enhances adhesion through high-temperature hydrophobicity and achieves desorption through low-temperature hydrophilicity, thus ensuring high-efficiency preservation while achieving low residue, demonstrating excellent technical effects.

[0048] In summary, the temperature-responsive hydrophobic reversible thymol nanopreservative constructed in this invention utilizes the natural temperature difference between postharvest high temperature (40℃) and room temperature washing (25℃) to achieve intelligent responsive behavior of "strong adhesion at high temperatures and easy desorption at low temperatures." This nanopreservative has shown significant preservation effects in various fruits such as blueberries, strawberries, cherry tomatoes, and cherries, effectively inhibiting fruit rot and mold growth, extending shelf life, and maintaining a fruit peel surface integrity rate of over 95% during long-term storage. Furthermore, the residue rate of this nanopreservative after washing various fruits at room temperature is below 16%, and it exhibits low toxicity to human bronchial and oral epithelial cells, demonstrating excellent biocompatibility. This invention provides a highly efficient, safe, universally applicable, and washable intelligent nanostrategy for postharvest fruit preservation.

[0049] The embodiments described above are merely illustrative of more specific and detailed implementations of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A temperature-responsive thymol nanopreservative with reversible hydrophilic-hydrophobic conversion, characterized in that, The nano-preservative is composed of temperature-responsive block polymers encapsulating thymol and metal ions to form nanoparticles with a regular rod-shaped structure. The nano-preservative exhibits a hydrophobic state at 35-40℃, enhancing its adhesion to the waxy layer of the fruit peel; and a hydrophilic state at 20-25℃, reducing its adhesion to the waxy layer of the fruit peel and enabling water washing and desorption. The nanoparticles have an average particle size of 185~225nm and an average Zeta potential of +40.8mV~+44.4mV.

2. The temperature-responsive hydrophobic reversible thymol nanopreservative according to claim 1, characterized in that, The temperature-responsive block polymer is PDMAEMA-b-PCL, PNIPAM-b-PCL, or POEGMA-b-PCL; the metal ion is Fe. 3+ Zn 2+ or Cu 2+ The metal ions coordinate with the phenolic hydroxyl groups in the thymol.

3. The temperature-responsive hydrophilic-hydrophobic reversible thymol nanopreservative according to claim 1, characterized in that, At 20°C, the average number of hydrogen bonds in the nano-preservative is 164.8, and the radius of gyration is 2.904 nm; at 40°C, the average number of hydrogen bonds in the nano-preservative is 155.2, and the radius of gyration is 2.649 nm.

4. A method for preparing a temperature-responsive hydrophobic reversible thymol nanopreservative as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Thymol and temperature-responsive block polymer are dissolved in tetrahydrofuran to prepare a stream 1, wherein the concentration of thymol in stream 1 is 1.0 mg / mL and the concentration of temperature-responsive block polymer is 0.2 mg / mL. S2. Dissolve the metal chloride in deionized water to prepare stream 2, wherein the concentration of the metal chloride in stream 2 is 5 mM; S3. Inject equal volumes of stream 1 and stream 2 into a mixer for mixing at a flow rate of 30 mL / min to obtain a nanoparticle mixture. S4. The nanoparticle mixture obtained in step S3 is dialyzed in ultrapure water for 12-14 hours to remove tetrahydrofuran, free thymol, and metal ions, thereby obtaining the temperature-responsive hydrophobic reversible thymol nanopreservative.

5. A method for preserving fruit using a temperature-responsive, hydrophobic-reversible thymol nanopreservative as described in any one of claims 1-3, characterized in that, Includes the following steps: The nano-preservative is applied to the surface of fruit at 35-40°C by soaking or spraying for 28-33 seconds. The high temperature and hydrophobic properties of the nano-preservative enhance its adhesion and retention on the waxy layer of the fruit peel, thereby inhibiting microbial infection. Before consumption, the fruit is washed in water at 20-25°C. The low-temperature hydrophilic properties of the nano-preservative reduce its adhesion to the fruit peel, allowing the nano-preservative to detach from the fruit surface.

6. The method according to claim 5, characterized in that, The fruit is blueberry, cherry tomato, strawberry or cherry; the residue of the nano preservative after washing in water at 20-25℃ is less than 16%.

7. The method according to claim 5, characterized in that, The nano-preservative showed a survival rate of 62.5% against human bronchial epithelial cells and 58.2% against human oral keratinocytes at a concentration of 40 μg / mL.

8. The method according to claim 5, characterized in that, The nano-preservative maintains over 95% of the surface integrity of blueberries, cherry tomatoes, strawberries, or cherries during a 30-day storage period.

9. The application of a temperature-responsive hydrophilic-hydrophobic reversible thymol nanopreservative as described in any one of claims 1-3 in the preparation of an antibacterial product for inhibiting Staphylococcus aureus or Escherichia coli on the surface of fruit, wherein the nanopreservative achieves a bactericidal rate of over 89% against both Staphylococcus aureus and Escherichia coli.

10. The application of a temperature-responsive hydrophilic-hydrophobic reversible thymol nanopreservative as described in any one of claims 1-3 in postharvest fruit preservation, characterized in that, The nano-preservative is coated onto the surface of the fruit at 35-40°C, making the nano-preservative hydrophobic so that it adheres to the waxy layer of the fruit peel. Then, it is washed with water at 20-25°C to make the nano-preservative hydrophilic so that it can be desorbed from the fruit surface.

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