Ethanol-promoted antibacterial hydrogel

Abstract

The invention discloses ethanol-promoted bacteriostatic hydrogel which is prepared by the following steps: (1) taking sodium alginate, N-isopropylacrylamide and methacrylated hyaluronic acid as substrate materials, adding glycerol and distilled water, uniformly mixing, adding acetic acid to adjust the pH value to be neutral, adding a photoinitiator, uniformly mixing, and performing ultraviolet curing to obtain primary gel; and (2) soaking the primary gel in an ethanol solution containing plant antibacterial components for secondary crosslinking, and cleaning with absolute ethyl alcohol to obtain the ethanol-promoted antibacterial hydrogel. The preparation method is simple and easy to implement, and the hydrogel is long in bacteriostatic action time and remarkable in bacteriostatic effect.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogel production, and particularly relates to an ethanol-promoted antibacterial hydrogel. Background Art

[0002] Essential oils are volatile aromatic substances extracted from plants by methods such as steam distillation, pressing, cold soaking, solvent extraction, microwave-assisted extraction of essential oils, or supercritical fluid extraction. These essential oils not only have specific aromas, but more importantly, essential oils have certain antibacterial, anti-inflammatory, and antioxidant properties, making them highly concerned in the fields of biomedicine, food industry, cosmetics, hygiene products, etc. However, problems such as strong aroma, high sensitivity, and volatile instability of essential oils limit their applications in certain fields (such as agriculture, food, pharmaceuticals, etc.).

[0003] Researchers have actively explored ways to overcome the limitations of essential oils in applications through advanced encapsulation technologies. Commonly used encapsulation systems, such as lipid nanoparticles, thin films, emulsifying gels, and oil-in-water emulsions, although improving the stability and utilization rate of essential oils to a certain extent, still have certain limitations, such as complex preparation processes, high costs, or certain irritation to the skin. The hydrogel system has shown great potential in the field of essential oil encapsulation due to its unique advantages. By forming a three-dimensional network structure, the hydrogel can provide a stable and safe encapsulation environment for essential oils, effectively avoiding direct contact between essential oils and the external environment, thereby prolonging their biological activity. In addition, the crosslinking degree and composition of the hydrogel can be flexibly adjusted according to different application requirements to achieve effective control and release of essential oil components. Compared with traditional encapsulation methods, the hydrogel system not only has better biocompatibility and stability, but also can significantly reduce the irritation of essential oils to the skin, while improving the utilization rate and persistence of essential oils, bringing new breakthroughs and development opportunities to the field of essential oil encapsulation.

[0004] Despite the high attention paid to essential oils due to their excellent biological activities, their inherent hydrophobic characteristics limit their direct application in hydrophilic matrices, which may not only lead to a significant reduction in the biological activities of essential oils, but also often require the use of high concentrations of essential oils to achieve the desired effects.

[0005] Since most essential oils are alcohol-soluble rather than water-soluble, when loading essential oils into hydrogels, most of them need to go through complex emulsification and nanosization processes. CN119499439A discloses a preparation method of a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil, including: (1) using 4-formylphenylboronic acid as a cross-linking agent to form Schiff base and borate ester bonds with carboxymethyl chitosan and two hyaluronic acids with different molecular weights respectively, preparing a skin surface wound-filling and dual-dynamic covalent cross-linked adhesive hydrogel, and freeze-drying for later use; (2) preparing microcapsules of frankincense essential oil by the emulsification-coacervation method and freeze-drying for later use; (3) mixing the freeze-dried powders of the hydrogel and the frankincense essential oil microcapsules, adding deionized water or PBS and stirring to redissolve to form a pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules. This method requires preparing microcapsules of frankincense essential oil and then incorporating them into the hydrogel, which is cumbersome to operate and the effect is not good. At present, there is still no report on the research of using ethanol as a medium to encapsulate essential oils in hydrogels and simultaneously promoting the cross-linking of hydrogels. Summary of the Invention

[0006] The purpose of the present invention is to provide an ethanol-promoted antibacterial hydrogel, which is simple and easy to prepare, has a long antibacterial action time and a significant antibacterial effect.

[0007] The technical solution adopted by the present invention to solve its technical problems is: An ethanol-promoted antibacterial hydrogel is prepared by the following method: (1) Using sodium alginate, N-isopropylacrylamide and methacrylated hyaluronic acid as base materials, adding glycerol and distilled water, mixing evenly, adding acetic acid to adjust the pH to neutral, adding a photoinitiator, mixing evenly, and ultraviolet curing to obtain a primary gel; (2) Immersing the primary gel in an ethanol solution containing plant antibacterial components for secondary cross-linking, and washing with absolute ethanol to obtain an ethanol-promoted antibacterial hydrogel.

[0008] In the present invention, different plant antibacterial components (essential oils) are directly dissolved in ethanol, and through the immersion of the hydrogel in ethanol, the antibacterial substances are quickly loaded into the hydrogel.

[0009] The present invention is a phase change-regulated hydrogel, NIPAM is a phase change material, NIPAM is highly swollen below the LCST (about 32 °C), forming a loose porous network, and the release of antibacterial components is relatively fast, which is suitable for rapid antibacterial during cold chain and room temperature storage; when the environmental temperature rises to the LCST, the hydrogel undergoes a phase change, the volume shrinks, and the network structure becomes dense, preventing the release of antibacterial substances, avoiding excessive loss, and prolonging the fresh-keeping period. The present invention can perform secondary strengthening on the hydrogel through ethanol to promote the cross-linking of the hydrogel structure.

[0010] The mass ratio of sodium alginate:N-isopropylacrylamide:methacryloylated hyaluronic acid = 10 - 30:10 - 30:1.

[0011] The ratio of sodium alginate:N-isopropylacrylamide:methacryloylated hyaluronic acid:glycerol:distilled water:photoinitiator is 100 mg:200 mg:10 mg:0.5 mL:2 mL:5 mg.

[0012] In the ethanol solution containing plant antibacterial components, the plant antibacterial component is thymol, and the composition ratio of the ethanol solution containing plant antibacterial components is: 1.5 - 2 mg of thymol, 2.5 mL of absolute ethanol.

[0013] In the ethanol solution containing plant antibacterial components, the plant antibacterial component is one or more of eugenol, carvacrol, rosemary essential oil, cinnamaldehyde, p-anisaldehyde, and the composition ratio of the ethanol solution containing plant antibacterial components is: 1.0 - 1.5 μL of plant antibacterial component, 2.5 mL of absolute ethanol.

[0014] The time for secondary crosslinking is 30 - 60 min.

[0015] The photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (photoinitiator LAP).

[0016] A fresh-keeping and transportation method for fruits and vegetables, wherein the ethanol-promoted antibacterial hydrogel is laid at the bottom of the fruits and vegetables transportation box, and then the fruits and vegetables are loaded for fresh-keeping transportation.

[0017] The beneficial effects of the present invention are: In the prior art, antibacterial materials are constructed through a complex preparation process, but the antibacterial effect and action time of the obtained antibacterial materials are limited; while in the present invention, the hydrogel still has a large antibacterial zone 5 days after being prepared and formed, with an obvious antibacterial effect.

[0018] In the present invention, different plant antibacterial components (essential oils) are directly dissolved in ethanol, and through the immersion of the hydrogel in ethanol, the antibacterial substances are quickly loaded into the hydrogel, and at the same time, ethanol can also secondarily strengthen the hydrogel and promote the crosslinking of the hydrogel structure. Description of the Drawings

[0019] Figure 1 are SEM images of different substrate materials; Figure 2 are gel formation comparison diagrams of gelatin, sodium alginate, and gum arabic; Figure 2 A is the ultraviolet light crosslinking process of the hydrogel, Figure 2 B is the state of the hydrogel after being treated with ethanol before (the upper two rows) and after (the lower two rows) ultraviolet light crosslinking, Figure 2C is the weight ratio of the hydrogel prepared by photo-crosslinking followed by alcohol strengthening to the weight of the original hydrogel. Figure 2 D is the hydrogel prepared by photo-crosslinking followed by alcohol strengthening. Figure 3 is the influence of the proportion of the substrate material on the antibacterial performance of the hydrogel; A is the antibacterial zone experiment, and B is the co-culture experiment. Figure 4 is the characteristic analysis diagram of the plant antibacterial component and its loaded hydrogel. Figure 4 A is the ultraviolet full-wavelength scanning diagram of the plant antibacterial component. Figure 4 B is the antibacterial effect diagram of the ethanol-promoted hydrogel loaded with different plant antibacterial components. Figure 5 is the comparison diagram of FTIR and XPS of hydrogels with different treatments. Figure 5 A is the FTIR diagram of hydrogels with different treatments. Figure 5 B is the XPS C 1s spectrum diagram of the hydrogel. Figure 5 C is the XPS N 1s spectrum diagram of the hydrogel. Figure 5 D is the XPS O 1s spectrum diagram of the hydrogel. Figure 6 are the TG and DSC curves of the hydrogel prepared by photo-crosslinking and the hydrogel prepared by photo-crosslinking followed by alcohol strengthening. Specific Embodiments

[0020] The technical solutions of the present invention will be further specifically described below through specific embodiments.

[0021] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0022] Example 1 An ethanol-promoted antibacterial hydrogel is prepared by the following method: (1) Using sodium alginate, N-isopropylacrylamide and methacrylated hyaluronic acid as the substrate materials, adding glycerol and distilled water, mixing evenly, adding acetic acid to adjust the pH to neutral, adding a photoinitiator (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate), mixing evenly, and ultraviolet curing to obtain a primary gel; the ratio of sodium alginate: N-isopropylacrylamide: methacrylated hyaluronic acid: glycerol: distilled water: photoinitiator is 100 mg: 200 mg: 10 mg: 0.5 mL: 2 mL: 5 mg.

[0023] (2) Soak the primary gel in an ethanol solution containing a plant antibacterial component for secondary crosslinking for 50 min. After washing with absolute ethanol, an ethanol-promoted antibacterial hydrogel is obtained. The plant antibacterial component is thymol, and the composition ratio of the ethanol solution containing the plant antibacterial component is: 1.8 mg of thymol and 2.5 mL of absolute ethanol.

[0024] Example 2 An ethanol-promoted antibacterial hydrogel is prepared by the following method: (1) Using sodium alginate, N-isopropylacrylamide and methacrylated hyaluronic acid as base materials, add glycerol and distilled water. After mixing, add acetic acid to adjust the pH to neutral, add a photoinitiator (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate), mix well, and cure under ultraviolet light to obtain a primary gel; the ratio of sodium alginate:N-isopropylacrylamide:methacrylated hyaluronic acid:glycerol:distilled water:photoinitiator is 300 mg:100 mg:10 mg:0.5 mL:2 mL:5 mg.

[0025] (2) Soak the primary gel in an ethanol solution containing a plant antibacterial component for secondary crosslinking for 30 min. After washing with absolute ethanol, an ethanol-promoted antibacterial hydrogel is obtained. The plant antibacterial component is thymol, and the composition ratio of the ethanol solution containing the plant antibacterial component is: 1.5 mg of thymol and 2.5 mL of absolute ethanol.

[0026] Example 3 An ethanol-promoted antibacterial hydrogel is prepared by the following method: (1) Using sodium alginate, N-isopropylacrylamide and methacrylated hyaluronic acid as base materials, add glycerol and distilled water. After mixing, add acetic acid to adjust the pH to neutral, add a photoinitiator (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate), mix well, and cure under ultraviolet light to obtain a primary gel; the ratio of sodium alginate:N-isopropylacrylamide:methacrylated hyaluronic acid:glycerol:distilled water:photoinitiator is 100 mg:300 mg:10 mg:0.5 mL:2 mL:5 mg.

[0027] (2) Soak the primary gel in an ethanol solution containing a plant antibacterial component for secondary crosslinking for 60 min. After washing with absolute ethanol, an ethanol-promoted antibacterial hydrogel is obtained. The plant antibacterial component is thymol, and the composition ratio of the ethanol solution containing the plant antibacterial component is: 2 mg of thymol and 2.5 mL of absolute ethanol.

[0028] Example 4 An ethanol-promoted antibacterial hydrogel is prepared by the following method: (1) Using sodium alginate, N-isopropylacrylamide, and methacryloylated hyaluronic acid as the base materials, add glycerol and distilled water. After mixing evenly, add acetic acid to adjust the pH to neutral. Then add a photoinitiator (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate), mix well, and perform UV curing to obtain a primary gel. The ratio of sodium alginate:N-isopropylacrylamide:methacryloylated hyaluronic acid:glycerol:distilled water:photoinitiator is 100 mg:200 mg:10 mg:0.5 mL:2 mL:5 mg.

[0029] (2) Immerse the primary gel in an ethanol solution containing plant antibacterial components for secondary cross-linking for 60 minutes. After washing with absolute ethanol, an ethanol-promoted antibacterial hydrogel is obtained. The plant antibacterial component is eugenol, and the composition ratio of the ethanol solution containing plant antibacterial components is: 1.25 μL of plant antibacterial component and 2.5 mL of absolute ethanol.

[0030] Example 5 The difference between this example and Example 4 is that the plant antibacterial component is carvacrol, and the composition ratio of the ethanol solution containing plant antibacterial components is: 1 μL of plant antibacterial component and 2.5 mL of absolute ethanol.

[0031] Example 6 The difference between this example and Example 4 is that the plant antibacterial component is rosemary essential oil.

[0032] Example 7 The difference between this example and Example 4 is that the plant antibacterial component is cinnamaldehyde, and the composition ratio of the ethanol solution containing plant antibacterial components is: 1.5 μL of plant antibacterial component and 2.5 mL of absolute ethanol.

[0033] Example 8 The difference between this example and Example 4 is that the plant antibacterial component is p-anisaldehyde.

[0034] Example 9 A fresh-keeping transportation method for fruits and vegetables. Lay the ethanol-promoted antibacterial hydrogel on the bottom of the fruits and vegetables transportation box, and then load the fruits and vegetables for fresh-keeping transportation.

[0035] Experimental research 1 Materials and methods 1.1 Materials Methacryloylated hyaluronic acid (HAMA), N-isopropylacrylamide (NIPAM), acetic acid, ethanol, glycerol; Macromolecular framework substances: gelatin, chitosan, sodium alginate, arabic gum, PAM; Plant antibacterial components: thymol, eugenol, carvacrol, rosemary essential oil, cinnamaldehyde, p-anisaldehyde.

[0036] 1.2 Method 1.2.1 Preparation of ethanol-promoted antibacterial hydrogel 100 mg of gelatin, chitosan, sodium alginate, arabic gum, PAM were respectively mixed with 200 mg of NIPAM, 10 mg of HAMA, and 0.5 mL of glycerol in 2 ml of distilled water and mixed evenly. After adding 10 uL of acetic acid to adjust the pH value of the solution to neutral, the mixed solution was ultrasonically treated for 30 min. Subsequently, 0.5 mL of LAP solution (a solution prepared by dissolving 10 mg of LAP in 1 mL of distilled water) was added, and irradiated with ultraviolet light at 365 nm for 15 min to achieve preliminary crosslinking. 1.875 mg of thymol, 1.25 µL of eugenol, 1.25 µL of carvacrol, 1.25 µL of rosemary essential oil, 1.25 µL of cinnamaldehyde, and 1.25 µL of p-anisaldehyde were respectively dissolved in 2.5 mL of absolute ethanol to prepare an ethanol solution containing plant antibacterial components. The preliminarily crosslinked hydrogel material was soaked in 2.5 mL of the ethanol solution containing essential oil for 1 h to achieve secondary crosslinking. Subsequently, it was repeatedly rinsed with absolute ethanol 3 - 5 times to remove the free essential oil, and the finally obtained hydrogel was used for subsequent material characterization and function verification.

[0037] 2.2.2 Material characterization The morphology and microstructure of gelatin, chitosan, sodium alginate, arabic gum, PAM and the above macromolecular substances after mixing with NIPAM were observed using a scanning electron microscope (SEM, model Sigma 300, Carl Zeiss AG, Germany). And macromolecular substances with larger pore sizes and neatly arranged and dense structures were preliminarily selected for the preparation of sustained-release hydrogel materials.

[0038] According to the crosslinking speed of the hydrogel, the response degree to different temperatures, and the change in weight after ethanol replacement, macromolecular substances that can sensitively respond to temperature changes were further screened.

[0039] Thymol was loaded into hydrogels prepared with different proportions of base materials. Through the antibacterial zone and co-culture, the optimal ratio of the base materials was jointly determined. Specifically: Sodium alginate, NIPAM and HAMA were prepared into hydrogels according to the mass ratios of 20:10:1 (i.e., 200mg:100mg:10mg), 10:20:1, 10:10:2, 10:10:1, 30:10:1, 10:30:1 and 10:10:3 according to the method of 1.2.1. The hydrogels were uniformly prepared into circular fragments using a punching machine with a diameter of 15 mm. After sterilization with ultraviolet light at 365 nm, the circular fragments were combined with those containing 10 5 CFU mL -1The bacterial suspension was co-cultured at 37 °C for 3 h. After 3 h, the bacterial suspension was diluted 10-fold in phosphate buffer and shaken thoroughly. Then, 100 μL of the resulting mixture was added to the medium and incubated in a bacterial incubator at 37 °C for 12 h. Subsequently, the resulting bacterial colonies were counted. This process was for screening the proportion of the substrate material by the co-culture method. 100 μL of the bacterial suspension containing 10 5 CFU mL -1 was evenly spread on the LB agar medium. Circular hydrogel fragments with a diameter of 15 mm were placed on the spread medium. After culturing at 37 °C for 3 h, the size of the antibacterial zone was observed. The appropriate proportions of sodium alginate, phase change material, and HAMA were screened through co-culture and antibacterial zone tests.

[0040] Using the full-wavelength scanning function of an ultraviolet spectrophotometer, the absorption peaks of thymol, eugenol, carvacrol, rosemary essential oil, cinnamaldehyde, and p-anisaldehyde were determined.

[0041] The action time and effect of the antibacterial hydrogels loaded with different essential oils were studied through antibacterial zone tests.

[0042] The chemical composition of the materials was analyzed using a Fourier transform infrared spectrometer (FTIR, model Nicolet iS20, Thermo Scientific, USA) and an X-ray photoelectron spectrometer (XPS, model Thermo Kalpha, Thermo Fisher Scientific, USA) to determine whether the functional substances were successfully loaded onto the substrate materials.

[0043] The thermogravimetric and differential thermal information of the materials was evaluated using a synchronous thermal analyzer (TG-DSC, model STA 449 F3, Netzsch, Germany) to determine the thermal stability of the materials.

[0044] 2 Results and Discussion 2.1 Selecting the substrate according to the pore size (able to load more antibacterial substances) In this study, we selected 5 common polymer substances (gelatin, chitosan, sodium alginate, arabic gum, PAM) to form the framework of the hydrogel with the phase change material NIPAM. According to SEM ( Figure 1As can be seen, whether gelatin is a pure substance or combined with NIPAM, it shows a dense pore structure and good stability; the open pore structure of sodium alginate is conducive to the penetration of essential oils, and its stability is enhanced after combination with NIPAM; the pore arrangement of gum arabic is regular, and the pores become dense after combination with NIPAM, which may further improve its loading capacity, making it a potential candidate material for preparing essential oil-loaded hydrogels. The structural improvement of chitosan after combination with NIPAM is not obvious, and the structure of PAM becomes loose after combination with NIPAM, which may not be conducive to the stable loading of essential oils. Through screening, it is preliminarily concluded that gelatin, sodium alginate and gum arabic are suitable candidate materials for preparing essential oil-loaded hydrogels.

[0045] 2.2 Select substrates according to the crosslinking speed and temperature response (turning white) Through systematic screening of the substrate materials suitable for preparing hydrogels by photocrosslinking and alcohol strengthening, it is found that gelatin, sodium alginate and gum arabic can all crosslink with HAMA and NIPAM under the irradiation of LAP photoinitiator and 365 nm ultraviolet light to form a three-dimensional network hydrogel ( Figure 2 ). Among them, the hydrogel systems based on gelatin and gum arabic show excellent photoinitiation efficiency, while the photocrosslinking speed of sodium alginate-based hydrogels is slower ( Figure 2 A), and the resulting gel network structure is relatively loose ( Figure 2 A&2B). Subsequently, the preliminarily formed hydrogels were immersed in an equal volume of ethanol for 1 h. After that, the weights of gelatin, sodium alginate and gum arabic-based hydrogels decreased by 23.39%, 13.94% and 46.12% respectively. Moreover, the originally incompletely formed sodium alginate hydrogel achieved secondary crosslinking quickly through ethanol soaking, forming a more compact and stable gel structure ( Figure 2C&2D). For the gelatin and gum arabic-based hydrogels with the action of ethanol, the strong polarity of ethanol may disrupt the hydrogen bonds of the triple helix structure of gelatin, and at the same time induce the molecular chain relaxation and rearrangement of the branched polysaccharide of gum arabic, resulting in the dissociation of physical entanglement nodes; while for the hydrogels prepared from sodium alginate, although its structure is relatively loose during the initial forming, the addition of ethanol may change the microenvironment inside the hydrogel, enabling the originally incompletely cross-linked sodium alginate molecular chains to have the opportunity to rearrange and undergo cross-linking reactions under the action of ethanol, thus forming a more stable and dense gel structure. In addition, gelation usually occurs due to the cross-linking or enhanced interaction between macromolecular chains. Ethanol can promote the interaction between macromolecular chains by changing the solvent environment, thereby realizing the transformation from sol to gel. The presence of ethanol also significantly increases the viscosity, yield stress and shear thinning characteristics of the solution, showing a unique function of regulating gel behavior. The secondary cross-linking achieved by ethanol soaking in the present invention not only improves the structural stability of the sodium alginate hydrogel, but also endows it with better application potential. In order to load antibacterial substances into the hydrogel through ethanol subsequently, sodium alginate was finally selected as the substrate for ethanol-strengthened hydrogel.

[0046] 2.3 Determination of the proportion of substrate materials When the proportions of sodium alginate, NIPAM and HAMA are different, the structural stability and tightness of the formed hydrogel will be different, which will lead to differences in the ability of the hydrogel to load essential oils. In order to intuitively judge the influence of the proportion of substrate materials on the ability of the hydrogel to load antibacterial essential oils, we set up 7 antibacterial tests with different proportions to determine the optimal proportion of substrate materials. The antibacterial zone test found that ( Figure 3 A): When the content of sodium alginate is higher than that of the phase change material, the hydrogel will undergo structural collapse near the lower critical solution temperature (LCST) of the phase change material, and when the temperature returns to room temperature, this situation does not improve, resulting in the rapid loss of antibacterial substances and a decrease in antibacterial effect. When the content of NIPAM is higher than that of sodium alginate, the hydrogel strengthened by ethanol undergoes a phase change at LCST, the volume shrinks, and the network structure becomes dense, and the release of antibacterial substances decreases, but the hydrogels in the 10:20:1 and 10:30:1 groups still have antibacterial zones, indicating that they have an inhibitory effect on the growth of Escherichia coli. Combining with the co-culture test ( Figure 3 B) found that the antibacterial hydrogel prepared with the ratio of sodium alginate, NIPAM and HAMA of 10:20:1 produced the least total number of colonies after co-culture with Escherichia coli. Therefore, 10:20:1 was determined as the optimal proportion of substrate materials.

[0047] 2.4 Research on antibacterial effect We selected sodium alginate as the macromolecular framework, and set the ratio of sodium alginate, NIPAM, and HAMA to be 10:20:1 to prepare the hydrogel. After ultraviolet crosslinking, it was soaked in absolute ethanol containing 0.75 mg / ml thymol, 0.5 μl / ml eugenol, 0.5 μl / ml carvacrol, 0.5 μl / ml rosemary essential oil, 0.5 μl / ml cinnamaldehyde, and 0.5 μl / ml p-anisaldehyde for 1 h to prepare an ethanol-promoted antibacterial hydrogel and study its antibacterial effect. Through UV full-wavelength scanning, the absorption peaks of thymol, eugenol, carvacrol, cinnamaldehyde, and p-anisaldehyde were found to be 276 nm, 281 nm, 276 nm, 286 nm, and 276 nm respectively. Since the content of rosemary essential oil cannot be quickly detected by ultraviolet spectrophotometer ( Figure 4 A), therefore, the action time and effect of the antibacterial hydrogel were detected through antibacterial experiments next ( Figure 4 B). The research showed that cinnamaldehyde, rosemary essential oil, eugenol, and p-anisaldehyde could all inhibit the growth of Escherichia coli when they were first loaded into the hydrogel. Among them, cinnamaldehyde had the best antibacterial effect, followed by rosemary essential oil. The antibacterial zones of eugenol and p-anisaldehyde were smaller and the antibacterial effects were limited. Thymol and carvacrol basically had no antibacterial zones. We speculated that cinnamaldehyde, rosemary essential oil, etc. not only entered the hydrogel successfully through physical action but also might be able to chemically react with the hydrogel base material (chain crosslinking, double bond formation, etc.); while thymol and carvacrol might have a reduced release rate due to the phase change of the hydrogel at 37°C, resulting in a denser structure and an insignificant antibacterial effect. The antibacterial effect of cinnamaldehyde was still obvious within 120 h after preparation and shaping, and that of rosemary essential oil was obvious within 60 h, but the antibacterial effect also gradually weakened with time basically. Since the hydrogel material basically dried out after 120 h. Therefore, only the antibacterial effect of the hydrogel loaded with essential oil within 120 h was observed. The results showed that through the post-treatment step of ethanol soaking, not only could the crosslinking of the hydrogel structure be promoted, but also plant antibacterial substances such as cinnamaldehyde, rosemary essential oil, eugenol, and p-anisaldehyde could be successfully loaded into the hydrogel to exert antibacterial efficacy.

[0048] 2.5 FTIR and XPS 2.5.1 FTIR It was found that there were two situations in the FTIR spectra of the hydrogels prepared with sodium alginate, NIPAM, HAMA, and LAP photoinitiator ( Figure 5 A). The first situation: The FTIR spectrum of the hydrogel prepared only by ultraviolet crosslinking contained 2 absorption peaks, which were located at 3263.9 cm -1 (stretching vibration of O-H bond) and 1631.0 cm -1 (stretching vibration of C=O bond) respectively. 3263.9 cm-1 The absorption peak at [1631.0 cm⁻¹] may originate from the carboxyl groups in sodium alginate, HAMA, or the hydroxyl groups in water molecules. -1 The absorption peak at [1631.0 cm⁻¹] corresponds to the C=O bonds in the carboxyl groups, methacrylate groups of sodium alginate and NIPAM, and possibly the cross-linking reaction products. The second case: The FTIR spectrum of the hydrogel prepared by ultraviolet light cross-linking and alcohol strengthening contains 6 absorption peaks, which are respectively at 3283.9 cm⁻¹ -1 (stretching vibration of O-H bond), 2979.4 cm⁻¹ -1 (C-H stretching vibration), 1647.7 cm⁻¹ -1 (stretching vibration of C=C double bond, C=O), 1088.9 cm⁻¹ -1 (stretching vibration of C-O bond), 1048.8 cm⁻¹ -1 (stretching vibration of C-O bond), 878.1 cm⁻¹ -1 (substituents of aromatic hydrocarbons), indicating the presence of carboxyl groups, amide groups, methacrylate groups, carbon-carbon double bonds, benzoyl groups, and hydroxyl groups in the sample. There are only two absorption peaks in the hydrogel formed by photo-crosslinking. After further strengthening treatment with ethanol, due to the interaction or chemical reaction between ethanol and the components in the hydrogel, new chemical bonds or functional groups are introduced, resulting in an increase in the number of absorption peaks in the FTIR spectrum. Specifically, as a solvent, ethanol may interact with the hydroxyl groups in the hydrogel to form hydrogen bonds, causing a shift or intensity change in the stretching vibration absorption peak of the O-H bond; promoting the continued cross-linking reaction of the unreacted hydroxyl groups in the hydrogel to form more chemical bonds such as ester bonds; reacting with the carboxyl groups in sodium alginate to form ester bonds (C-O-C bonds); and may also promote the polymerization reaction between methacrylate groups to form more carbon-carbon single bonds or double bonds.

[0049] After loading cinnamaldehyde and eugenol into the hydrogel prepared by photo-crosslinking and alcohol strengthening, only the displacement or intensity change of the FTIR absorption peak occurs. When the hydrogel loaded with essential oils (bacteriostatic components) is placed at 60 °C, the FTIR absorption peak is the same as that of the hydrogel prepared only by photo-crosslinking, with only two absorption peaks at approximately the same position. The reason is that the high temperature causes changes in the hydrogel structure, weakens the release or interaction of essential oil molecules, masking or disappearing multiple absorption peaks originally introduced by alcohol strengthening treatment, and restoring the spectral characteristics of the hydrogel prepared by photo-crosslinking. These changes reflect the influence and alteration of ethanol treatment on the chemical structure of the hydrogel.

[0050] 2.5.2 XPS Based on the analysis of the evolution of chemical bonds in the hydrogel by XPS, the correlation mechanism between the material structure and properties can be systematically elaborated: In the C 1s spectrum of the directly photo-crosslinked hydrogel ( Figure 5B), the broad peak (with the maximum intensity) of C-C / C-H bonds at 284.8 eV indicates that the system is based on a hydrophobic carbon chain (such as the isopropyl group of NIPAM and the methacrylate backbone of HAMA), while the weak peaks at 287.9 eV (O-C=O, carboxyl / ester group), 286.7 eV (C-O, ether bond / hydroxyl group), and 283.6 eV (pollutant) reflect the carboxylate of sodium alginate, the hydroxyl group of glycerol, and potential residual impurities. After ethanol strengthening, the C-O peak shifted from 286.7 eV to 286.3 eV to the left, and the intensity of the O-C=O peak with enhanced hydrogen bonding at 532.7 eV in the O1s spectrum increased significantly, indicating that ethanol optimized the compactness of the gel network by promoting the protonation of carboxylic acid groups (-COO⁻ → -COOH) and the formation of intermolecular hydrogen bonds; at the same time, the increase in the intensity of the C-C peak (284.8 eV) may be due to the aggregation and exposure of hydrophobic segments (such as NIPAM) in ethanol, and the shift of the 287.9 eV peak to 287.6 eV further supports the protonation reaction of carboxylic acid groups. The evolution of the N 1s spectrum further verified the crosslinking mechanism ( Figure 5 C): The double peaks at 399.9 eV (amide group of NIPAM) and 398.5 eV (residual primary amine) in the directly photo-crosslinked gel were simplified to a single peak at 399.5 eV after alcohol strengthening, indicating that the amide group participated in network crosslinking through hydrogen bonds or covalent bonds; the decrease in the intensity of the 399.6 eV peak after loading cinnamaldehyde was due to the physical shielding of the amide group by hydrophobic molecules, and the further decrease in the intensity of the 399.7 eV peak caused by high-temperature phase transition indicated that the temperature-sensitive NIPAM chain contracted during the phase transition, burying the amide group deeper into the hydrophobic micro-region and reducing the XPS detection sensitivity. In the O 1s spectrum ( Figure 5 D), multiple low-intensity peaks (532.2 eV O-C=O, 531.2 eV C-O, 530.0 eV pollutant) of the photo-crosslinked gel evolved into a double peak at 532.7 eV (strongly hydrogen-bonded carboxylic acid oxygen) and 531.1 eV (ether bond oxygen) after ethanol strengthening, indicating that ethanol significantly enhanced the interaction of carboxylic acid groups through protonation and hydrogen bonding, and loading cinnamaldehyde and high-temperature phase transition did not change the oxygen chemical state, indicating that the two mainly affected the gel structure through physical effects (such as hydrophobic encapsulation and chain rearrangement). In summary, the photo-crosslinking-ethanol strengthening strategy synergistically improved the mechanical stability of the gel through chemical bond reconstruction (carboxylic acid protonation, hydrogen bond densification) and hydrophobic effects; while cinnamaldehyde loading and low-temperature phase transition changed the sustained-release behavior of antibacterial components and the temperature-controlled response performance by regulating the exposure degree of functional groups and molecular arrangement, providing a chemical basis for the design of multifunctional hydrogels.

[0051] 2.6 TG and DSC Figure 6Among them, the black curve represents the hydrogel prepared by photocrosslinking, and the red curve represents the TG curve of the hydrogel prepared by photocrosslinking followed by alcohol strengthening. During the heating process from room temperature (30 °C) to 800 °C, the photocrosslinked hydrogel undergoes three weight loss processes, with weight losses of -33.23%, -20.54%, and -39.39% respectively. During this process, the hydrogel experiences the processes of losing adsorbed water and bound water, decomposition of small molecule organic substances or uncrosslinked polymer chain segments, and pyrolysis or carbonization of the polymer network structure. The hydrogel prepared by photocrosslinking followed by alcohol strengthening undergoes two weight loss processes, with weight losses of -47.42% and -44.33% respectively. During this process, the hydrogel experiences the processes of volatilization or decomposition of alcohols or other small molecule organic substances, and pyrolysis or carbonization of the polymer network structure. And it is found that at the same temperature, the weight of the hydrogel after secondary crosslinking is always higher than that of the photocrosslinked hydrogel, and the weight loss rate of the former is generally lower than that of the latter. The reason is that after the hydrogel is secondarily crosslinked, more crosslinking points are formed between the polymer chain segments, making the network structure more compact and stable. This compact network structure not only improves the mechanical strength and heat resistance of the hydrogel, but also reduces the volatilization or decomposition of small molecule components and polymer chain segments during the heating process. Therefore, under the same heating conditions, the secondarily crosslinked hydrogel can maintain a higher weight and a lower weight loss rate. In the DSC curve, the hydrogel only subjected to photocrosslinking has a melting peak at 28.73 °C, while the melting peak of the hydrogel prepared by photocrosslinking followed by alcohol strengthening advances to 8.59 °C, indicating that the thermal stability of the hydrogel after photocrosslinking followed by alcohol strengthening is reduced. It may be that alcohol molecules penetrate into the network structure of the hydrogel and interact with the base material, such as forming hydrogen bonds or changing the original intermolecular forces, affecting the thermal stability of the material. It may also be that alcohol strengthening promotes the further crosslinking of some incompletely crosslinked parts, or causes the deconstruction of some crosslinked structures, affecting the thermal stability of the hydrogel.

[0052] The above-described embodiments are only a preferred solution of the present invention and do not impose any form of limitation on the present invention. There are other variations and modifications without exceeding the technical solutions recorded in the claims.

Claims

1. An ethanol-promoted antibacterial hydrogel, characterized in that: Prepared by the following method: (1) Sodium alginate, N-isopropylacrylamide and methacryloyl hyaluronic acid are used as base materials, glycerol and distilled water are added, mixed, acetic acid is added to adjust the pH to neutral, a photoinitiator is added, mixed, and UV-cured to obtain a primary gel; (2) The primary gel is immersed in an ethanol solution containing plant antibacterial components for secondary crosslinking, and then washed with anhydrous ethanol to obtain an ethanol-promoted antibacterial hydrogel.

2. The ethanol-promoted antibacterial hydrogel according to claim 1, characterized in that: The mass ratio of sodium alginate: N-isopropylacrylamide: methacryloyl hyaluronic acid = 10-30:10-30:

1.

3. An ethanol-promoted antibacterial hydrogel according to claim 1 or 2, characterized in that: The ratio of sodium alginate: N-isopropylacrylamide: methacryloyl hyaluronic acid: glycerol: distilled water: photoinitiator is 100 mg: 200 mg: 10 mg: 0.5 mL: 2 mL: 5 mg.

4. The ethanol-promoted antibacterial hydrogel according to claim 1, characterized in that: The ethanol solution containing plant antibacterial components is thymol, and the composition ratio of the ethanol solution containing plant antibacterial components is: 1.5-2 mg thymol and 2.5 mL anhydrous ethanol.

5. The ethanol-promoted antibacterial hydrogel according to claim 1, characterized in that: The ethanol solution containing plant antibacterial components comprises one or more of eugenol, carvacrol, rosemary essential oil, cinnamaldehyde and p-anisaldehyde, and the composition ratio of the ethanol solution containing plant antibacterial components is: 1.0-1.5 μL of the plant antibacterial components and 2.5 mL of anhydrous ethanol.

6. The ethanol-promoted antibacterial hydrogel according to claim 1, characterized in that: The secondary cross-linking time is 30-60min.

7. The ethanol-promoted antibacterial hydrogel according to claim 1, characterized in that: The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

8. A method for preserving and transporting fruits and vegetables, characterized in that: The ethanol-promoted antibacterial hydrogel according to claim 1 is laid on the bottom of a fruit and vegetable transport box, and then the fruits and vegetables are loaded and transported fresh.

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