pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation and preparation method thereof

Abstract

The application discloses a pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation and a preparation method thereof. The hydrogel is prepared from carboxymethyl chitosan, cinnamaldehyde and polyacrylic acid, generates imine bonds with pH responsiveness through Schiff base reaction, and introduces polyacrylic acid to improve mechanical properties. The preparation process is simple, and the hydrogel has excellent water retention performance, flexible mechanical fitting property and pH-responsive controlled release characteristics. In a weakly acidic microenvironment of postharvest strawberries, the hydrogel can intelligently trigger the controlled release of cinnamaldehyde, effectively inhibit the growth of microorganisms, synergistically maintain the hardness of strawberries, and slow down the weight loss and degradation of vitamin C. Experimental results show that the hydrogel significantly prolongs the shelf life of strawberries, and a specific formula combination (such as 4.5% CMCS, 1:1 aldehyde-ammonia ratio and 30 DEG C crosslinking) exhibits the best preservation effect, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of hydrogel materials technology, and in particular to a pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation and its preparation method. Background Technology

[0002] Strawberries, due to their high water content and rich nutrients, are highly susceptible to microbial infection and mechanical damage after harvest, leading to spoilage and severely limiting their shelf life. Cinnamaldehyde, the main active ingredient in cinnamon essential oil, is safe and environmentally friendly. It exerts its antibacterial effect through multiple pathways, including disrupting the integrity of microbial cell membranes and inducing the accumulation of reactive oxygen species. However, cinnamaldehyde has drawbacks such as high volatility, poor photothermal stability, and low water solubility, making its release rate uncontrollable when applied directly, thus hindering long-term preservation. Therefore, constructing a suitable carrier system to achieve the stabilization and controlled release of cinnamaldehyde is crucial for expanding its application in fruit preservation. Hydrogels, with their three-dimensional network structure, offer unique advantages in loading and protecting active substances. Carboxymethyl chitosan (CMCS), a water-soluble derivative of chitosan, contains abundant amino groups in its molecular chain, serving as active sites for Schiff base reactions. Studies have shown that the imine bonds (C=N) formed through the Schiff base reaction between the amino groups on CMCS and the aldehyde groups in cinnamaldehyde molecules exhibit pH-responsive properties. These bonds can undergo hydrolytic cleavage in weakly acidic environments, resulting in the responsive release of the loaded substance. This characteristic closely aligns with the actual situation of pH reduction in the packaging microenvironment due to respiration during post-harvest fruit storage, providing a new approach for developing intelligent responsive preservation materials.

[0003] Furthermore, existing carboxymethyl chitosan-based hydrogels generally suffer from insufficient mechanical properties, limiting their practical application in fruit preservation. Polyacrylic acid (PAA), an anionic polyelectrolyte, can form physical crosslinks with carboxymethyl chitosan through hydrogen bonding and van der Waals forces, potentially improving the mechanical strength of hydrogels.

[0004] Therefore, this invention provides a pH-responsive cinnamaldehyde / carboxymethyl chitosan composite hydrogel, its preparation method, and its application. The aim is to construct a dynamic cross-linking network through Schiff base reaction and introduce polyacrylic acid to improve mechanical properties, thereby achieving on-demand and controllable release of antibacterial agents during post-harvest storage of fruits, and thus effectively extending the shelf life of fruits. Summary of the Invention

[0005] One objective of this invention is to provide a pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation, comprising an aqueous solution of carboxymethyl chitosan, an ethanolic solution of cinnamaldehyde, and an aqueous solution of polyacrylic acid; wherein the amino group of the carboxymethyl chitosan reacts with the aldehyde group of cinnamaldehyde through a Schiff base reaction to generate a pH-responsive imine bond, and the hydrogel has a three-dimensional network structure.

[0006] Preferably, the mass ratio of polyacrylic acid to carboxymethyl chitosan is 1:1.5.

[0007] Preferably, the molar ratio of the aldehyde group in cinnamaldehyde to the amino group in carboxymethyl chitosan is 1~1.5:1~1.5.

[0008] The second objective of this invention is to provide a method for preparing a pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation, comprising the following steps: S1. Add carboxymethyl chitosan to water, stir to dissolve, and then cool to room temperature to obtain a carboxymethyl chitosan aqueous solution; S2. Cinnamaldehyde is dissolved in anhydrous ethanol to prepare cinnamaldehyde ethanol solution; S3. Under stirring conditions, the cinnamaldehyde ethanol solution is slowly added dropwise to the carboxymethyl chitosan aqueous solution to carry out a Schiff base reaction. S4. Add polyacrylic acid solution to the reacted mixture, continue to stir evenly, transfer to a mold and let stand to form a gel, thus obtaining pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation.

[0009] Preferably, in step S1, the mass-volume concentration of carboxymethyl chitosan in the carboxymethyl chitosan aqueous solution is 3.5% to 4.5%.

[0010] Preferably, the temperature for stirring and dissolving in step S1 is 35°C.

[0011] Preferably, in step S2, the volume ratio of anhydrous ethanol to cinnamaldehyde in the cinnamaldehyde ethanol solution is 3:2.

[0012] Preferably, the Schiff base reaction in step S3 is carried out at a temperature of 30-40°C for 10-15 minutes.

[0013] Preferably, the mass fraction of the polyacrylic acid solution in step S4 is 10%, and the settling time is 24 hours.

[0014] Preferably, the hydrogel satisfies any one of the following parameter combinations: (1) The concentration of carboxymethyl chitosan is 4.0%, the molar ratio of aldehyde group in cinnamaldehyde to amino group in carboxymethyl chitosan is 1.5:1, and the crosslinking temperature is 30℃; (2) The concentration of carboxymethyl chitosan is 4.5%, the molar ratio of aldehyde group in cinnamaldehyde to amino group in carboxymethyl chitosan is 1:1, and the crosslinking temperature is 30℃; (3) The concentration of carboxymethyl chitosan is 3.5%, the molar ratio of aldehyde group in cinnamaldehyde to amino group in carboxymethyl chitosan is 1.5:1, and the crosslinking temperature is 40℃.

[0015] The beneficial effects of this invention are: This invention constructs a pH-responsive hydrogel through the Schiff base reaction of carboxymethyl chitosan and cinnamaldehyde, and builds a physical cross-linking network through physical cross-linking effects such as hydrogen bonds and van der Waals forces between polyacrylic acid and carboxymethyl chitosan, thereby improving the mechanical strength and water retention performance of the hydrogel. At the same time, when the hydrogel is used for strawberry preservation, the weakly acidic environment generated by the postharvest respiration of the fruit triggers the hydrolysis of imine bonds, significantly accelerating the release rate of cinnamaldehyde, continuously inhibiting microbial growth, slowing down fruit weight loss and softening, and thus effectively extending the shelf life of perishable fruits such as strawberries. Attached Figure Description

[0016] Figure 1 These are stress-strain curves of the hydrogels prepared in Examples 1-9 of this invention; wherein Figure 1 From top to bottom, a represents Examples 3, 2, and 1. Figure 1 The numbers b from top to bottom are 5, 4, and 6. Figure 1 c is 7, 9, and 8 from top to bottom; Figure 2 The bar chart shows the water content of the hydrogels prepared in Examples 1-9 of this invention. Figure 3 shows the change in mass of the hydrogels prepared in Examples 1-9 of the present invention over time under different pH conditions; wherein Figure 3(a) shows the change in mass of the hydrogel over time under pH 7.0 conditions, and Figure 3(b) shows the change in mass of the hydrogel over time under pH 5.0 conditions. Figure 4 shows line graphs of cinnamaldehyde release concentration over time at different pH conditions for the hydrogels prepared in Examples 1-9 of this invention; Figure 4(a) is a line graph of cinnamaldehyde release concentration over time at pH 5.0, and Figure 4(b) is a line graph of cinnamaldehyde release concentration over time at pH 7.0. Figure 5 Infrared curves of the hydrogels prepared in Examples 1-9 of this invention; Figure 6 Thermogravimetric curves of the hydrogels prepared in Examples 1-9 of this invention; Figure 7 The XRD patterns are those of the hydrogels prepared in Examples 8, 6, and 3 of this invention. Figure 8 shows SEM images of the hydrogels prepared in Examples 8, 6, and 3 of this invention; wherein Figure 8(a) is an SEM image of the hydrogel prepared in Example 3, Figure 8(b) is an SEM image of the hydrogel prepared in Example 6, and Figure 8(c) is an SEM image of the hydrogel prepared in Example 8. Figure 9 The images show a comparison of the appearance changes of strawberries with and without hydrogels prepared according to preferred embodiments 8, 6, and 3; where, Figure 9(a) is a comparison diagram of the changes in the appearance of strawberries in Example 6. Figure 9 (b) is a comparison diagram of the changes in the appearance of strawberries in Example 8. Figure 9 (c) is a comparison diagram of the appearance changes of strawberries in Example 3; Figure 10 Linear graphs showing the weight loss changes of strawberries with hydrogels prepared in preferred embodiments 8, 6, and 3 and strawberries without hydrogels. Figure 11 Line graphs showing the change in hardness between strawberries with and without hydrogels prepared using preferred embodiments 8, 6, and 3. Figure 12 Line graphs showing the pH changes over time for strawberries with and without hydrogels prepared in preferred embodiments 8, 6, and 3. Figure 13 Line graphs showing the change in vitamin C content over time for strawberries with and without hydrogels prepared using preferred embodiments 8, 6, and 3. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] The carboxymethyl chitosan (CMCS) of this invention, with a degree of substitution ≥90% and a molecular weight of approximately 200,000, was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the cinnamaldehyde (Cin), with a purity ≥98%, was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; the polyacrylic acid (PAA), with a molecular weight of 250,000 Da and a solid content of 98%, was purchased from Zhengzhou Erqi District Guangfu Chemical Products Store; the anhydrous ethanol (analytical grade) was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; and the experimental water was deionized water.

[0019] Preparation of pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation: A pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation comprises an aqueous solution of carboxymethyl chitosan, an ethanolic solution of cinnamaldehyde, and an aqueous solution of polyacrylic acid. The amino groups of the carboxymethyl chitosan and the aldehyde groups of cinnamaldehyde react via a Schiff base reaction to generate pH-responsive imine bonds. The hydrogel has a three-dimensional network structure.

[0020] A method for preparing pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation includes the following steps: S1. Add carboxymethyl chitosan to deionized water and stir in a water bath at 35°C until completely dissolved. Then cool to room temperature to obtain a carboxymethyl chitosan aqueous solution with a mass-volume concentration of 3.5~4.5% (w / v). S2. Cinnamaldehyde is dissolved in anhydrous ethanol, wherein the volume ratio of ethanol to cinnamaldehyde is 3:2, to prepare a cinnamaldehyde ethanol solution. S3. Under high-speed stirring conditions (800~1000r / min), the cinnamaldehyde ethanol solution is slowly and uniformly added dropwise to the carboxymethyl chitosan aqueous solution at a rate of 0.5~1mL / min, and the Schiff base crosslinking reaction is carried out at a reaction temperature of 30~40℃ for 10~15min; wherein the molar ratio of aldehyde group in cinnamaldehyde to amino group in carboxymethyl chitosan is 1:1.5~1:1.5. S4. Add polyacrylic acid aqueous solution to the reaction mixture. Add 10% polyacrylic acid aqueous solution at a mass ratio of 1:1.5 to the mass ratio of polyacrylic acid aqueous solution to carboxymethyl chitosan aqueous solution. Continue stirring for 30 minutes, then transfer to a mold and let stand for 24 hours to form a gel, thus obtaining pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation.

[0021] Nine examples were designed based on three factors: CMCS mass volume concentration (3.5%, 4.0%, 4.5%), aldehyde-ammonia molar ratio (1:1.5, 1:1, 1.5:1), and crosslinking temperature (30℃, 35℃, 40℃). The specific examples are shown in Table 1 below.

[0022] Table 1

[0023] The hydrogels prepared in Examples 1-9 were divided into two portions. One portion was refrigerated at 4°C for performance testing, and the other portion was freeze-dried and sealed for structural characterization.

[0024] Experimental Example 1: Relevant performance and characterization tests were performed on Examples 1-9: I. Mechanical Property Testing: Compression tests were conducted on the hydrogel using a texture analyzer (CT3, Brookfield, USA). The sample was a cylinder with a diameter of 10 mm and a height of 10 mm. Test parameters: initial speed 1.0 mm / s, speed during test 1.0 mm / s, speed after test 1.0 mm / s, target compression height 4 mm, trigger force 5 g, and 3 parallel samples per group. Load-deformation data were recorded, and stress-strain curves were plotted, as shown below. Figure 1As shown, all samples exhibited typical hydrogel compression behavior, which can be divided into three stages: the elastic deformation zone, the yield plateau zone, and the densification zone. Example 1 (3.5% + 1:1.5 + 30℃) had the highest hardness (21.07 kPa), followed by Example 8 (4.5% + 1:1 + 30℃) (19.59 kPa), while Examples 4 and 7 both had a hardness of 18.20 kPa. Except for Examples 5 (6.66 kPa) and 9 (7.51 kPa), the hardness of the remaining samples was 11.62 kPa or higher.

[0025] II. Water Content Test: The water content of the hydrogel was determined using the drying-to-constant-weight method. Fresh samples were weighed (wet weight, W0), dried in an oven at 60℃ until constant weight, and then weighed (dry weight, W0). d Moisture content is calculated as (W0-W) d ) / W0×100% calculation, with 3 parallel samples per group, results are as follows Figure 2 As shown, the water content of all nine samples was above 91%, indicating that the prepared hydrogel has excellent water retention properties. High water content is a typical characteristic of hydrogels, which is beneficial for maintaining the humidity balance of the packaging microenvironment during fruit preservation and preventing the fruit from wilting due to water loss.

[0026] III. Swelling Performance Test: The dried samples were placed in pH 5.0 and pH 7.0 buffer solutions and immersed at a constant temperature of 25℃. They were weighed (Wt) at 1, 2, 4, 8, 12, 24, and 48 h, with three replicates per group. The swelling rate was calculated as (Wt-W0) / W0×100%. Figure 3 shows the swelling changes of the nine groups of samples over 48 hours under pH 5.0 and pH 7.0 conditions. This experiment determined the swelling kinetics within 48 h; some samples had not yet reached complete swelling equilibrium at 48 h. The mass change under pH 7.0 conditions is shown in Figure 3. Figure 3a As shown, all nine hydrogel samples exhibited continuous water absorption and swelling characteristics within 48 h, with their mass continuously increasing and no obvious swelling equilibrium being reached. This behavior indicates that in a neutral environment, the Schiff base cross-linked network of the hydrogel remains stable, and water molecules slowly enter the network interior through diffusion, exhibiting typical continuous water absorption characteristics.

[0027] Mass change at pH 5.0 is as follows Figure 3bAs shown, the mass changes of all hydrogel samples exhibited a typical pattern of initial increase followed by decrease, which contrasts sharply with the continuous increase observed under pH 7.0 conditions. This "increase followed by decrease" phenomenon is essentially a competition between two processes: water absorption and swelling, and network degradation. In the initial stage, the rate of water molecule diffusion into the network is faster than the rate of network degradation, leading to an increase in hydrogel mass. With the continued influence of the weakly acidic environment, imine bonds gradually hydrolyze and break, and the cross-linked network begins to disintegrate. When the rate of network degradation exceeds the rate of water absorption, the mass begins to decrease. When degradation reaches a certain extent, some soluble polymer chains or cross-linked fragments detach from the gel matrix and enter the solution, further reducing the mass, even to below the initial dry weight.

[0028] The swelling of hydrogels varied depending on their formulation. Example 8 (4.5% + 1:1 + 30℃): After 48 hours, the swelling rate remained positive (8.51%), and the mass was not lower than the initial dry weight. This indicates that the network degradation rate was very slow, the water absorption process dominated throughout 48 hours, and the swelling equilibrium period was relatively long, making it suitable for long-term preservation. Example 7 (4.5% + 1:1.5 + 40℃): After 48 hours, the swelling rate dropped to -27.5%, and the mass was far lower than the initial dry weight. This indicates that the network degradation rate was very fast, the network was severely damaged before 48 hours, and the swelling equilibrium period was relatively short. This may be because excessive network disintegration led to the early release or loss of cinnamaldehyde.

[0029] IV. pH Response Release Test: The release concentration of cinnamaldehyde was determined by UV-Vis spectrophotometry. A standard curve (0–6 μg / mL, detection wavelength 298 nm) was prepared using cinnamaldehyde standards. Cinnamaldehyde-loaded hydrogels were placed in 50 mL of pH 5.0 and pH 7.0 buffer solutions and released at 25°C. Samples were taken at 1, 2, 4, 8, 12, 24, and 48 h, diluted 128 times, and the absorbance was measured to calculate the release concentration. pH response release performance is the core indicator for formula screening in this study. Nine groups of hydrogel samples were placed in pH 5.0 and pH 7.0 buffer solutions, and the concentration of cinnamaldehyde in the release medium was measured at 1, 2, 4, 8, 12, 24, and 48 h. The release concentration-time curves are shown in Figure 4. The release concentration of all samples at pH 5 was significantly higher than that at pH 7. At pH 7, the release amounts of the nine samples ranged from 192 to 597 mg / L, with a relatively slow release rate. At pH 5, the release amounts ranged from 268 to 814 mg / L, with a significantly faster release rate. This comparison fully demonstrates the pH-responsive release characteristics of the hydrogel: the acidic environment triggers the hydrolytic breakage of imine bonds, the disintegration of the cross-linked network, and the accelerated release of cinnamaldehyde.

[0030] Release curve characteristic analysis: Based on the trend of the release curves within 48 hours, the 9 groups of samples can be divided into two categories: Category 1 (long release period, continuously rising curve): Includes Examples 9, 6, 8, 3, 2, and 5. The release curves of these six samples maintained an upward trend throughout 48 hours, without a clear plateau. This indicates that their network degradation rate is slow or the degradation process is continuous, resulting in a long release period; the release endpoint may not have been reached even after 48 hours.

[0031] The second category (short release period, curve tending to flatten): includes Examples 1, 4, and 7. The release curves of these three groups of samples tended to flatten within 12-48 hours. This indicates that its network degradation rate is relatively fast, and the release is basically completed before 48 hours, resulting in a short release period and a limited total release amount.

[0032] V. Fourier Transform Infrared Spectroscopy (FTIR, Nicolet iS10, Thermo Fisher Scientific, USA) was used to analyze the chemical structure of the freeze-dried hydrogel samples. The samples were mixed with KBr at a 1:100 ratio and ground until homogeneous. Transparent thin films were then pressed using a tablet press. The scanning range was 4000–400 cm⁻¹, the resolution was set to 4 cm⁻¹, and 32 scans were performed, with air used as the background to subtract interference. By comparing the characteristic absorption peaks of the samples, the formation of imine bonds (C=N) in the Schiff base reaction and the physical cross-linking effects such as hydrogen bonding and van der Waals forces between PAA and CMCS were verified. The infrared spectra of the 9 groups of hydrogel samples are shown below. Figure 5 As shown in the figure, the characteristic absorption peaks of all samples are basically consistent, indicating that the chemical structure of the hydrogel did not change significantly within the formulation range investigated by the orthogonal experiment. All samples exhibit a broad and strong absorption peak at 3200–3500 cm⁻¹, attributed to the stretching vibrations of OH and NH, reflecting the skeletal characteristics of carboxymethyl chitosan. All samples show a distinct characteristic absorption peak at 1700–1720 cm⁻¹, attributed to the stretching vibration of C=O in the carboxyl group of polyacrylic acid (PAA). Pure carboxymethyl chitosan does not exhibit this characteristic peak in this region, proving that PAA has been successfully incorporated into the hydrogel network. A distinct absorption peak appears in the 1630–1650 cm⁻¹ range, attributed to the stretching vibration of the imine bond (C=N) formed by the Schiff base reaction. This peak is not present in the original spectrum of carboxymethyl chitosan. This confirms that a Schiff base reaction successfully occurred between the aldehyde group of cinnamaldehyde and the amino group of carboxymethyl chitosan, forming a dynamic imine bond. The imine bond can be hydrolyzed and broken under acidic conditions, which is the structural basis for achieving pH-responsive release.

[0033] VI. Thermogravimetric analysis (TGA, STA-2500 simultaneous thermal analyzer, Netzsch, Germany) was used to evaluate the thermal stability of the hydrogels. 5–10 mg of the freeze-dried sample was weighed and placed in an alumina crucible, and heated from room temperature to 600 °C at a heating rate of 10 °C / min. The mass loss as a function of temperature (TG curve) was recorded, and each stage of weight loss was analyzed. The thermogravimetric analysis curves of the 9 groups of hydrogel samples are shown below. Figure 6 As shown in the figure, the TG curves of all samples basically overlap, indicating that the thermal decomposition behavior is relatively consistent, suggesting that different formulations have little impact on the thermal stability of the hydrogel. The first stage (room temperature ~ 200℃): This is a slow weight loss stage, with a weight loss rate of approximately 10%. The mass loss in this stage is mainly due to the evaporation of physically adsorbed free water and bound water in the hydrogel network.

[0034] The second stage (approximately 200℃~300℃): This is the most significant weight loss stage, with the curve dropping sharply and a weight loss rate as high as 20%~30%. This is the region where the hydrogel polymer skeleton begins to decompose. The third stage (>300℃): The weight loss rate slows down again, and the curve flattens out. This stage is the process of further decomposition and carbonization of the polymer skeleton, ultimately forming stable carbonaceous residues. The thermogravimetric analysis curves show that Example 8 (4.5% + 1:1 + 30℃) has the best thermal stability, followed by Example 5 > Example 3 > Example 6.

[0035] Using pH-responsive release performance as the core evaluation criterion, and combining mechanical properties, water content, and swelling properties as auxiliary evaluation criteria, optimal formulations for antibacterial experiments were screened. Examples 3, 6, and 8 were identified as three optimal formulations for subsequent XRD and SEM testing.

[0036] Experimental Example 2: Characterization tests were performed on preferred embodiments 3, 6, and 8. I. X-ray diffraction (XRD, GNR APD 2000PRO, GNR Italy) analysis of the crystalline structure of the hydrogel. The freeze-dried hydrogel sample was ground into a fine powder and placed in the groove of a dedicated XRD glass sample holder. The testing conditions were: tube voltage 40 kV, current adjusted to 40 mA, scanning range 2θ = 3°–50°, and scanning speed 2° / min. The degree of crystallinity and amorphous structure of the hydrogel were determined by analyzing the position, intensity, and shape of the diffraction peaks. Results are shown in [Figure number missing]. Figure 7 As shown: Figure 7The XRD patterns of three hydrogel samples with optimized formulations are shown. As can be seen from the figures, all three samples exhibit a broad, diffuse peak near 2θ≈20°, indicating that the prepared hydrogels are predominantly amorphous. This is because the original ordered crystalline structure of carboxymethyl chitosan is disrupted after carboxymethylation modification, and the introduction of Schiff base crosslinking and PAA further inhibits the ordered arrangement of molecular chains. The peak shapes of the three samples are basically consistent, indicating that different formulations have little impact on the crystal structure of the hydrogel. This predominantly amorphous structure facilitates the diffusion and penetration of water molecules within the gel network, providing a structural basis for the hydrogel's good swelling properties and the release of cinnamaldehyde.

[0037] II. Observation of the microstructure of the hydrogel using scanning electron microscopy (SEM, Sigma 300, ZEISS, Germany). Cross-sections of the freeze-dried hydrogel samples were obtained, sputter-coated with gold, and observed under an accelerating voltage of 5 kV at a working distance of approximately 8–10 mm. Observations were made at magnifications of 20x (scale bar 500 µm), 35x (scale bar 300 µm), 20x (scale bar 200 µm), and 100x (scale bar 100 µm), focusing on the three-dimensional network structure, internal morphology, and network density of the hydrogel. The results are shown in Figure 8: Figure 8 shows the SEM cross-sectional morphology of the three preferred formulation hydrogel samples, observed under accelerating voltage of 5 kV, scale bar of 300 µm, magnification of 35x and 500 µm, and magnification of 20x. As can be seen from the figure, all three samples formed a good three-dimensional network structure, but the network density differed significantly. Observing the overall morphology under a 500 µm scale, it was found that the hydrogel formed a complete three-dimensional network system with an overall lamellar structure. Example 3: Sample a (3.5% + 1.5:1 + 40℃) had the most loose network structure, with large interlayer spacing and numerous voids in the field of view. Example 6: Sample b (4.0% + 1.5:1 + 30℃) had a relatively dense network structure, with increased lamellar thickness and decreasing voids. Example 8: Sample c (4.5% + 1:1 + 30℃) had the most dense network structure, with tightly stacked lamellars and almost no visible voids in the field of view.

[0038] Further observation of local details under a 300 µm scale made the above differences even clearer. Example 3: Sample a has a loose lamellar structure with large interlayer gaps; Example 6: Sample b has a more compact lamellar structure with significantly reduced interlayer gaps; Example 8: Sample c has highly dense lamellar structures with almost complete interlayer fusion, exhibiting a continuous network morphology. The differences in the network structure of the three samples are mainly attributed to polymer concentration and crosslinking conditions. Example 8: Sample c, using the highest concentration and a moderate molar ratio, achieved the most complete crosslinking, forming the densest three-dimensional network. Furthermore, the cross-sections of all three samples were relatively smooth, with no obvious phase separation observed, indicating good compatibility among CMCS, Cin, and PAA. This three-dimensional network structure provides a structural basis for water absorption and cinnamaldehyde release.

[0039] Experiment Example 3: Strawberry Preservation Experiment To further illustrate the preservation effect of this invention on strawberries, three preferred formulations—Example 6 (4.0% + 1.5:1 + 30℃), Example 8 (4.5% + 1:1 + 30℃), and Example 3 (3.5% + 1.5:1 + 40℃)—were selected for strawberry preservation application experiments. Strawberries without added hydrogel served as a blank control group. Two identical formulations and a blank control group were prepared for each formulation; one group was used for weight loss testing, and the other was used to test firmness, pH, and vitamin C. Three replicates were prepared for each formulation. The experiment was conducted at room temperature.

[0040] I. Changes in the appearance of strawberries The appearance changes of strawberries during storage were recorded using a periodic photography method. Each day, at the same time and under the same light conditions, strawberries in each group were photographed with a mobile phone. Changes in appearance quality, such as surface color, gloss, degree of wilting, and mold, were observed and recorded. By comparing the appearance differences between the control group and each hydrogel-treated group, the preservation effect of the hydrogel on strawberries was visually evaluated.

[0041] Changes in the appearance of strawberries during storage, such as Figure 9 As shown in the photos, it is clear that the strawberries in each group showed varying degrees of quality deterioration as storage time increased. Example 6: Group A maintained a good appearance, bright color, and plump fruit for the first three days; slight wilting and softening began on the fourth day; browning and mold appeared on the fifth day, and mold increased further on the sixth day. However, its overall appearance was better than Group C and the control group. Example 8: Group B maintained a good appearance, fresh color, and plump fruit for the first four days; softening began on the fifth day; mold and browning began on the sixth day. Example 3: Group C maintained a good appearance and freshness for the first three days; blackening began on the fourth day; severe mold on the fifth day, but less than the control group; and severe mold on the sixth day. The control group had a good appearance for the first two days; softening and browning began on the third day; mold appeared on the fourth day; and severe mold on the fifth and sixth days.

[0042] Comparing the appearance changes of each group, the preservation effect of Example 8: Group b (4.5%+1:1+30℃) was the most significant, and the strawberries maintained a good appearance quality at the end of the storage period; Example 6: Group a was the second best, and Example 3: Group c also showed a better preservation effect than the blank group.

[0043] II. Strawberry weight loss determination The weight loss rate of strawberries during storage was determined using a weighing method. The initial weight (W0) of each group of strawberries was recorded as the weight on day 1. The weight (W0) of each group of strawberries was measured at the same time each day. t After weighing each day, the sample is returned to its original position without being removed or damaged. Measurements are continued until the end of storage. Weight loss rate = (W0 - W) t The change in weight loss rate of strawberries during storage is shown as follows: (W0*100%) Figure 10 As shown in the figure, the weight loss rate of strawberries in each group increased with the extension of storage time. It was found that in Example 8, group b had the lowest weight loss rate because it released a higher concentration of cinnamaldehyde and, as indicated by the swelling rate, a longer release period. In Example 6, group a was the second lowest, with the highest release concentration but a shorter release period. In Example 3, group c had the lowest release concentration and the shortest release period compared to the previous two groups, resulting in the highest weight loss rate, but still better than the control group.

[0044] III. Strawberry firmness test: The hardness change of strawberries during storage was determined using a texture analyzer. Since the strawberries showed only slight rot in the first two days, hardness was tested on the third day. Thereafter, one strawberry was taken from each sample group daily (three replicates per formulation), and a compression test was performed using a 12.7 mm probe. Test parameters were set as follows: test speed 1.0 mm / s, target distance 8 mm, trigger force 5 g. The maximum peak force during compression was recorded as the hardness value (g). Three replicates were set for each group, and the average value was taken. The hardness results are shown below. Figure 11 As shown: Figure 11As can be seen, the firmness of strawberries in all groups decreased with prolonged storage time, but the rate of firmness decrease varied significantly among different treatment groups. The control group experienced the fastest firmness decrease, with only 120 g remaining on the second day. Due to the lack of antibacterial protection, a large number of microorganisms grew and multiplied, and combined with the strawberry's own respiration and cell wall degrading enzyme activity, it softened rapidly. In Example 3, the firmness of group c also decreased rapidly (226 g remaining on the second day). This group released less hydrogel (646 mg / L), which was insufficient to effectively inhibit microbial growth and enzyme activity. In Example 6, group a released the most (814 mg / L), but its firmness retention was not as good as in Example 8, group b (390 g remaining on the second day). This is because, according to the swelling test results, its release period was slightly shorter than that of group b. Group b maintained the best firmness because its release amount was larger (669 mg / L) and its release time was longer, allowing it to inhibit microbial growth and cell wall degrading enzyme activity for a longer period, thereby minimizing the softening of the strawberries. The order of firmness retention from strongest to weakest among the treatment groups was: Example 8 > Example 6 > Example 3 > Control.

[0045] IV. pH Measurement of Strawberries The pH changes of strawberries during storage were measured using a PHS-3C pH meter. One strawberry was taken from each sample group daily, crushed in a mortar and pestle, centrifuged, and the supernatant was used as the strawberry stock solution. An appropriate amount of the strawberry stock solution was diluted with deionized water at a 1:1 ratio, and the pH value was measured using a pH meter. Three replicates were set up for each group, and the average value was taken. The pH changes of strawberries during storage are shown below. Figure 12 As shown in the figure, the pH of strawberries in each group first increased and then decreased with the extension of storage time, but the magnitude of change and the turning point of the different treatment groups were significantly different.

[0046] Analysis of the reasons for pH changes in strawberries: The content of organic acids in strawberries directly affects the pH value. In the early stage of storage, strawberry respiration consumes organic acids, and the pH gradually rises. As the storage time increases, microorganisms multiply and produce organic acids through metabolism. At the same time, as strawberries age, cell structure is damaged, organic acids are released, and the pH begins to decrease.

[0047] The control group experienced the most dramatic pH changes: the pH rose steadily to 3.96 from day 1 to 3, and then rapidly dropped to 3.25 from day 4 to 6. This indicates that the strawberries in the control group had vigorous respiration in the early stages, resulting in a large consumption of organic acids; in the later stages, rapid microbial reproduction and metabolic acid production led to a sharp drop in pH, indicating that the fruit had already severely rotted.

[0048] Comparing the three experimental groups, the pH fluctuation was greatest in Example 3 (group c), followed by Example 6 (group a), and least in Example 8 (group b), remaining relatively stable throughout the storage period. This is because group b had a higher hydrogel release concentration and a longer release period, which continuously inhibited microbial growth and organic acid metabolism, thereby maintaining pH stability. The pH stability ranking of the experimental groups was: Example 8: group b > Example 6: group a > Example 3: group c > control group, consistent with the results of appearance observation, weight loss rate, and hardness.

[0049] V. Determination of Vitamin C Content in Strawberries: The vitamin C content of strawberries during storage was determined using ultraviolet-visible spectrophotometry. First, the ascorbic acid standard curve was determined. Strawberries were taken from each sample group daily, crushed in a mortar and pestle, centrifuged, and the supernatant was used as the strawberry stock solution. An appropriate amount of the stock solution was diluted at a suitable ratio, and the absorbance was measured at 265 nm. The vitamin C content (mg / 100g) was calculated based on the standard curve. Three replicates were set up for each group, and the average value was taken. The changes in vitamin C content of strawberries during storage are shown below. Figure 13 As shown in the figure, the vitamin C content of strawberries in all groups decreased with prolonged storage time. The vitamin C content of the control group decreased the fastest. This is because without the protection of antibacterial agents, microorganisms multiplied rapidly, their respiratory metabolism was vigorous, and vitamin C was quickly oxidized and consumed.

[0050] Example 3: The vitamin C content in group c also decreased rapidly, reaching 18.5 mg / 100g on day 6 (a decrease of 55.4%). The hydrogel release concentration in this group was lower than that in groups a and b, and its performance in effectively inhibiting vitamin C degradation needs to be improved.

[0051] Example 6: Group a had the highest release concentration (814 mg / L), but the vitamin C retention effect was similar to that of Group b. This may be related to the relatively short release period of Group a, which weakened the antibacterial ability in the later stage and accelerated the degradation of vitamin C.

[0052] Example 8: Group b had a high release concentration (669 mg / L) and a long release period, which could continuously inhibit microbial growth and strawberry respiratory metabolism throughout the entire storage period, and slow down the oxidative degradation of vitamin C.

[0053] Three selected optimal formulations—Example 6 (4.0% + 1.5:1 + 30℃), Example 8 (4.5% + 1:1 + 30℃), and Example 3 (3.5% + 1.5:1 + 40℃)—were applied to strawberry preservation. The preservation effects of each formulation were evaluated by measuring changes in appearance, weight loss, hardness, pH, and vitamin C content of the strawberries during storage. Considering all indicators, group b hydrogel showed the best preservation effect, exhibiting the best performance in weight loss, hardness, pH stability, and appearance retention. Example 6: group a was second best, with vitamin C retention comparable to Example 8: group b. Example 3: group c was better than the blank group but weaker than the other two. The results indicate that the prepared cinnamaldehyde-carboxymethyl chitosan hydrogel has good potential for preservation applications, with Example 8 (4.5% + 1:1 + 30℃) being the optimal solution for strawberry preservation.

[0054] When the three formulations were applied to strawberry preservation, Example 8 (4.5% + 1:1 + 30℃) showed the best preservation effect, followed by Example 6, and Example 3 was better than the control group. Example 6, which had the highest release concentration, did not have the best preservation effect, while Example 8, with the longest release period, performed best, indicating that sustained release capability is more important than instantaneous high concentration.

[0055] This experiment systematically investigated the effects of carboxymethyl chitosan concentration, aldehyde-amino molar ratio, and crosslinking temperature on the hydrogel properties. The preparation process was optimized through orthogonal experiments, and nine groups of samples were systematically tested. The optimized formulation was ultimately applied to strawberry preservation. Mechanical testing revealed that the hardness of the samples in Example 7 exceeded 10 kPa, meeting the basic requirements for preservation. However, the formulation with the best mechanical properties did not necessarily have the best preservation effect, indicating that release performance is crucial while meeting basic strength requirements. Swelling tests showed that the hydrogel initially increased and then decreased at pH 5.0, confirming that the acidic environment induced the hydrolysis of imine bonds, which is the structural basis for pH-responsive release. Release tests showed that Examples 6 and 9 had the highest release concentration (814 mg / L), followed by Example 8 (669 mg / L), and Example 3 was third (646 mg / L). The swelling rate of Example 8 remained positive, and the release period was long. The swelling rate of Example 6 was -14.8%, indicating high release efficiency but a slightly shorter release period compared to Example 8. Fourier transform infrared spectroscopy confirmed the occurrence of the Schiff base reaction.

[0056] When the three formulations were applied to strawberry preservation, Example 8 (4.5% + 1:1 + 30℃) showed the best preservation effect, followed by Example 6, and Example 3 was better than the control group. Example 6, which had the highest release concentration, did not have the best preservation effect, while Example 8, which had the longest release period, performed best, indicating that sustained release capability is more important than instantaneous high concentration.

[0057] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation, characterized in that, The hydrogel comprises an aqueous solution of carboxymethyl chitosan, an ethanolic solution of cinnamaldehyde, and an aqueous solution of polyacrylic acid; the amino groups of the carboxymethyl chitosan and the aldehyde groups of cinnamaldehyde react via a Schiff base reaction to form pH-responsive imine bonds, and the hydrogel has a three-dimensional network structure.

2. The pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation according to claim 1, characterized in that, The mass ratio of the polyacrylic acid aqueous solution to the carboxymethyl chitosan aqueous solution is 1:1.

5.

3. The pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation according to claim 1, characterized in that, The molar ratio of the aldehyde group in cinnamaldehyde to the amino group in carboxymethyl chitosan is 1~1.5:1~1.

5.

4. A method for preparing a pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Add carboxymethyl chitosan to water, stir to dissolve, and then cool to room temperature to obtain a carboxymethyl chitosan aqueous solution; S2. Cinnamaldehyde is dissolved in anhydrous ethanol to prepare cinnamaldehyde ethanol solution; S3. Under stirring conditions, the cinnamaldehyde ethanol solution is slowly added dropwise to the carboxymethyl chitosan aqueous solution to carry out the Schiff base crosslinking reaction; S4. Add polyacrylic acid aqueous solution to the reaction mixture, continue to stir evenly, transfer to a mold and let stand to form a gel, thus obtaining pH-responsive cinnamaldehyde carboxymethyl chitosan hydrogel for strawberry preservation.

5. The preparation method according to claim 4, characterized in that, In step S1, the mass-volume concentration of carboxymethyl chitosan in the carboxymethyl chitosan aqueous solution is 3.5%~4.5%.

6. The preparation method according to claim 4, characterized in that, The temperature for stirring and dissolving in step S1 is 35°C.

7. The preparation method according to claim 4, characterized in that, In step S2, the volume ratio of anhydrous ethanol to cinnamaldehyde in the cinnamaldehyde ethanol solution is 3:

2.

8. The preparation method according to claim 4, characterized in that, In step S3, the crosslinking temperature of the Schiff base reaction is 30~40℃, and the reaction time is 10~15min.

9. The preparation method according to claim 4, characterized in that, In step S4, the mass fraction of the polyacrylic acid solution is 10%, and the settling time is 24 hours.

10. The preparation method according to claim 4, characterized in that, The hydrogel satisfies any one of the following combinations of parameters: (1) The concentration of carboxymethyl chitosan is 4.0%, the molar ratio of aldehyde group in cinnamaldehyde to amino group in carboxymethyl chitosan is 1.5:1, and the Schiff base reaction crosslinking temperature is 30℃; (2) The concentration of carboxymethyl chitosan is 4.5%, the molar ratio of aldehyde group in cinnamaldehyde to amino group in carboxymethyl chitosan is 1:1, and the Schiff base reaction crosslinking temperature is 30℃. (3) The concentration of carboxymethyl chitosan is 3.5%, the molar ratio of aldehyde group in cinnamaldehyde to amino group in carboxymethyl chitosan is 1.5:1, and the Schiff base reaction crosslinking temperature is 40℃.

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