Preparation method and application of plasma-activated water-functionalized composite hydrogels

Abstract

This invention provides a method for preparing and applying a plasma-activated water functionalized composite hydrogel, belonging to the field of food preservation technology. The technical solution uses plasma-activated water as the functional material, carboxymethyl chitosan and sodium alginate as the matrix, and citric acid as the crosslinking agent to prepare a PAW composite hydrogel that can slowly release active substances and extend shelf life. This invention combines the antibacterial and preservative properties of PAW with the slow-release function of the hydrogel. Compared to directly treating button mushrooms with PAW, the composite hydrogel avoids the one-time rapid release of active substances, prolonging the antibacterial and quality control time and reducing the adverse effects caused by direct liquid contact with button mushrooms. Through the synergistic effect of the two, the occurrence of postharvest water loss, browning, softening, and spoilage of button mushrooms is significantly reduced, extending shelf life, improving marketability and quality stability, thereby achieving a milder, more stable, and longer-lasting preservation effect.

Description

Technical Field

[0001] This invention relates to the field of food preservation technology, specifically to a method for preparing and applying plasma-activated water-functionalized composite hydrogels. Background Technology

[0002] Button mushrooms are not only an important edible fungus in my country, but also one of the most commercially cultivated, widely distributed, and highest-yielding edible fungi in the world. Their mycelium and fruiting bodies are mostly white, with thick caps and short, straight stems, possessing edible, nutritional, and economic value. Button mushrooms are high in protein, low in fat and calories, and rich in vitamins, dietary fiber, unsaturated fatty acids, minerals, and polyphenols, exhibiting potential health effects such as antioxidant properties, lipid-lowering effects, anti-tumor effects, improvement of gut microbiota, and intervention in abnormal glucose metabolism.

[0003] Benefiting from the popularization of health concepts and the versatility of button mushrooms in various catering options, their global production has gradually increased, making them the world's largest edible fungus producer with a 26% market share, indicating a promising future for the industry. Against this backdrop, developing safe, green, and sustainable post-harvest preservation technologies for button mushrooms is of great significance for improving product quality and promoting the green development of the industry. The post-harvest quality deterioration of button mushrooms is the result of both their own aging process and external environmental stress. Their tissues are soft and tender, with extremely high water content (85%–95%) and a lack of dense protective structure on the surface. Therefore, post-harvest respiration and transpiration are very vigorous, making them highly susceptible to environmental stress. Furthermore, mechanical damage such as collisions and compression during distribution makes button mushrooms vulnerable to various bacteria and fungi, leading to browning, off-odors, and spoilage. This results in a significant decline in appearance, flavor, and nutritional value, shortened shelf life, and reduced commercial value.

[0004] Currently, the preservation methods for button mushrooms fall into three main categories: physical methods involving environmental control and packaging improvement; chemical treatments such as fungicides, acids, and plant essential oils; and biological strategies such as antagonistic microorganisms. Physical preservation is simple to operate and cost-effective, but it is energy-intensive and requires sophisticated equipment. Chemical preservation, such as ethylene inhibitors, is highly effective, but carries the risk of chemical residues. Biological preservation, primarily using biological preservatives and extracts, is green, harmless, and environmentally friendly, but its basic applications are still insufficient. The industry urgently needs a scalable, easy-to-operate, low-cost, safe, and environmentally friendly preservation technology to delay aging, reduce post-harvest losses, and enhance commercial value.

[0005] Plasma-Activated Water (PAW) is a green functional water formed by reacting water with cold plasma (CP). It is rich in active components such as reactive oxygen and nitrogen, exhibiting certain antibacterial, antioxidant, and deterioration-delaying effects. However, direct spraying or immersion of samples with PAW still has limitations: the active ingredients have poor stability, release rapidly, and are prone to decay, resulting in a limited preservation time; liquid treatment increases free water on the surface of the fungi, easily inducing tissue softening, browning, or microbial growth; and it is difficult to achieve a continuous, mild, and controllable preservation effect during packaging and distribution. Therefore, how to extend the action time of PAW's active components, improve its stability and application adaptability are key issues in promoting the practical application of PAW in the preservation of button mushrooms. Summary of the Invention

[0006] The first technical problem to be solved by the present invention is that the preservation effect of the method of directly spraying or soaking button mushrooms with PAW is not ideal.

[0007] The second technical problem to be solved by this invention is: how to develop a scalable, easy-to-operate, low-cost, safe and environmentally friendly mushroom preservation technology.

[0008] To achieve the above technical objectives, the present invention adopts the following technical solution: A method for preparing plasma-activated water-functionalized composite hydrogels includes the following steps: 1) The bubble spark discharge reactor was immersed in deionized water and treated at a working voltage of 70~80 volts and a current of 1.9~2.1 amperes. PAW was prepared by selecting a plasma activation time of 14~16 minutes to obtain PAW15. 2) Dissolve CMCS in PAW15 buffer and stir until completely dissolved; add SA and continue stirring until a homogeneous precursor solution is obtained; inject the resulting solution into a mold, crosslink with CA to form a hydrogel, and dry to constant weight.

[0009] Preferably, the amount of deionized water used in step 1) is 1.5L.

[0010] Preferably, the operating voltage in step 1) is 75 volts.

[0011] Preferably, the current in step 1) is 2 amperes.

[0012] Preferably, in step 1), during the processing, the discharge parameters are monitored using a high-voltage probe, a current probe, and an oscilloscope.

[0013] Preferably, a plasma activation time of 15 minutes is selected in step 1) to prepare PAW.

[0014] Preferably, in step 2), the ratio of CMCS, PAW15 buffer, and SA is 1.6 g: 100 ml: 0.4 g.

[0015] Preferably, in step 2), the stirring until completely dissolved is performed at 500 revolutions per minute at room temperature; and the drying to constant weight is performed at room temperature.

[0016] Based on the above technical solutions, the present invention further provides the application of the above-mentioned plasma-activated water functionalized composite hydrogel for the preservation of edible fungi.

[0017] Preferably, the edible fungus is Agaricus bisporus.

[0018] This invention provides a method for preparing and applying plasma-activated water functionalized composite hydrogels. The technical solution uses plasma-activated water (PAW) as the functional material, carboxymethyl chitosan (CMCS) and sodium alginate (SA) as the matrix, and citric acid (CA) as the crosslinking agent to prepare a PAW composite hydrogel that can sustainably release active substances and extend its storage period.

[0019] This invention uses PAW as a preservative. Its preparation process does not require the addition of additional chemical preservatives, has low residual risk after treatment, is environmentally friendly, and can also solve the problem of insufficient uniformity or over-treatment of irregular samples caused by plasma treatment.

[0020] Hydrogels possess excellent water absorption, water retention, loading, and sustained-release capabilities. Active ingredients can be loaded and released in a controlled manner through blending, cross-linking incorporation, or adsorption-swelling. Their soft, cushioning properties also protect food products from impacts and compression during transportation, reducing mechanical damage. Replacing ordinary water with polyunsaturated glycol (PAW) in hydrogel preparation not only immobilizes or embeds the active components within the hydrogel network structure but also leverages the hydrogel's porous structure and water-retention properties to achieve sustained-release of components. Furthermore, PAW influences the hydrogel's cross-linking behavior, pore structure, and mechanical properties, altering its dense microstructure and enhancing its structural stability, adsorption loading capacity, and sustained-release performance. This improves the hydrogel's suitability for packaging, storage, and logistics of button mushrooms.

[0021] This invention combines the antibacterial and preservative properties of PAW (polydioxanone) with the slow-release function of hydrogel. Compared to directly treating button mushrooms with PAW, the composite hydrogel avoids the rapid release of active substances in a single step, extending the antibacterial and quality control time and reducing the adverse effects caused by direct liquid contact with button mushrooms. Through the synergistic effect of the two, postharvest dehydration, browning, softening, and spoilage of button mushrooms are significantly reduced, extending shelf life, improving marketability and quality stability, thereby achieving a gentler, more stable, and longer-lasting preservation effect. Attached Figure Description

[0022] Figure 1 (a) is a graph showing the pH changes of PAW at different activation times; Figure 1 (b) is a graph showing the changes in ORP of PAW at different activation times; Figure 1 (c) is a graph showing the change in conductivity of PAW at different activation times.

[0023] Figure 2 (a) shows the FT-IR spectra of CMCS, SA, CA and hydrogel; Figure 2 (b) shows the XRD patterns of CMCS, SA, CA and hydrogel; Figure 2 (c) is the TGA curve of the hydrogel; Figure 2 (d) is the DTG curve of the hydrogel.

[0024] Figure 3 This is a SEM micrograph of the hydrogel surface.

[0025] Figure 4 (a) is a graph showing the water content of the hydrogel; Figure 4 (b) is a graph showing the expansion rate of the hydrogel; Figure 4 (c) is a graph showing the water retention rate of the hydrogel.

[0026] Figure 5 These are rheological property analysis diagrams for PCS0 and PCS15.

[0027] Figure 6 (a) is a graph showing the porosity of the hydrogel; Figure 6 (b) is a graph showing the oxygen permeability of the hydrogel; Figure 6 (c) is a graph showing the water vapor permeability of the hydrogel.

[0028] Figure 7 (a) is a graph showing the experimental results of the ABTS free radical scavenging activity of the hydrogel; Figure 7 (b) is a graph showing the experimental results of the hydrogel's DPPH free radical scavenging activity.

[0029] Figure 8 (a) is an image of the inhibition zone of the hydrogel; Figure 8 (b) is a graph showing the diameter of the inhibition zone of the hydrogel; Figure 8 (c) is a photograph of bacterial colonies after the hydrogel came into contact with the sample; Figure 8 (d) is a graph showing the viable colony counts of Escherichia coli and Staphylococcus aureus after the hydrogel came into contact with the sample.

[0030] Figure 9 (a) is a graph showing the weight loss rate of button mushrooms at different times after treatment; Figure 9 (b) is a graph showing the soluble solids (TSS) content of mushrooms treated at different times; Figure 9 (c) is a graph showing the browning degree (BI) of button mushrooms at different times after treatment. Detailed Implementation

[0031] The specific embodiments of the present invention will be described in detail below. To avoid excessive and unnecessary detail, well-known structures or functions will not be described in detail in the following embodiments. The approximate language used in the following embodiments is for quantitative purposes, indicating that a certain degree of variation in quantity is permissible without changing the basic function. Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0032] 1. Preparation of Materials and Reagents 1.1 Material Selection Carboxymethyl chitosan (CMCS), sodium alginate (SA), and citric acid (CA) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China); 1,1-diphenyl-2-trinitrohydrazine (DPPH) was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China); and strains of Escherichia coli and Staphylococcus aureus were provided by the College of Agricultural Engineering and Food Science, Shandong University of Technology (Zibo, China).

[0033] Fresh button mushrooms were purchased from Zhangdian District, Zibo City, Shandong Province, China. Samples were immediately transported to the laboratory and stored in a refrigerator at 4°C before use.

[0034] 1.2 PAW Preparation The plasma was prepared using a plasma reaction system consisting of a bubble spark discharge reactor, a high-voltage power supply (CTP 2000K), an air pump, and an electrical diagnostic system. During the preparation process, the reactor was immersed in 1.5L of deionized water. Discharge parameters were monitored using a high-voltage probe, a current probe, and an oscilloscope at a working voltage of 75V and a current of 2.0±0.1A. A plasma activation time of 15 minutes (PAW15) was selected for PAW preparation.

[0035] 1.3 Preparation of PAW Functionalized Hydrogels Dissolve 1.6 g of CMCS in 100 mL of deionized water or PAW15 buffer and stir at 500 rpm at room temperature until completely dissolved. Add 0.4 g of SA and continue stirring under the same conditions until a homogeneous precursor solution is obtained. Inject the resulting solution into a mold and crosslink with CA to form a hydrogel. The hydrogels prepared with deionized water and PAW are labeled PCS0 and PCS15, respectively. After drying at room temperature to constant weight, store the solutions.

[0036] 2 Experimental Methods 2.1 Physicochemical properties of PAW Component concentration determination: nitrate After pretreatment at 220 Concentration is calculated from ultraviolet absorption spectra at different wavelengths; use Griess The method uses sulfonamides and N-(1-naphthyl)ethylenediamine hydrochloride as colorimetric reagents, at 540 °C. Absorbance was measured at a wavelength; hydrogen peroxide ( The concentration of dissolved ozone was determined using a commercially available test kit; The concentration was measured using a UV-Vis spectrophotometer at 610. Record the absorbance at the wavelength.

[0037] pH value, oxidation-reduction potential (ORP), and conductivity were measured: The freshly treated PAW was measured using a calibrated pH meter, ORP electrode, and conductivity meter, respectively.

[0038] 2.2 Microstructure characterization of composite hydrogels Functional groups and molecular interactions: at 4000–500 Band recording of Fourier transform infrared spectrum (FT-IR).

[0039] Crystal structure characteristics: as shown by X-ray diffraction (XRD) patterns. Scan within the range.

[0040] Thermogravimetric analysis: In a nitrogen atmosphere, the temperature was raised from room temperature to 600°C at a constant rate, and the decomposition of the sample was analyzed by the corresponding derivative thermogravimetric analysis (DTG) curve.

[0041] Microstructure: After gold plating to enhance conductivity by sputtering, the sample was placed in the chamber of a scanning electron microscope for observation.

[0042] 2.3 Macroscopic physicochemical properties of composite hydrogels Mechanical properties: The thickness and tensile strength of the samples were measured. ) and elongation at break ( ). , Calculate according to the following standard formula:

[0043]

[0044] In the formula, Indicates the tensile force (N) at fracture; The original cross-sectional area of ​​the sample obtained by measurement ( ); The length of the membrane sample at the time of fracture ( ); The original length of the membrane sample ( ).

[0045] Water absorption and swelling properties: The water content of the samples was measured ( ), swelling ratio ( ) and water retention ( First, record the initial mass. The sample was then dried in a 40°C oven until constant weight, cooled to room temperature, and then weighed. The dried sample was then immersed in distilled water until it reached swelling equilibrium. After removing the surface moisture, the mass was recorded. Finally, the sample was dried again at 40℃, cooled, and weighed, and the weight was recorded as follows. .

[0046] Moisture content ( ), swelling ratio ( ) and water retention ( Calculate using the following formula:

[0047]

[0048]

[0049] Rheological properties: First, a strain scan was performed at a constant frequency of 1 Hz within a strain range of 0.1% to 10% to determine the linear viscoelastic region (LVR); then, a scan was performed within a frequency range of 0.1-10 Hz under a fixed strain of 0.1% to record the storage modulus. ' ) and loss modulus ( " (This varies with the oscillation frequency.)

[0050] Porosity Regarding barrier properties: To calculate porosity, the volume of the hydrogel must first be calculated using the following formula. :

[0051] In the formula, The side length is ( ); Hydrogel thickness The sample was then immersed in anhydrous ethanol, and the initial mass was recorded. After standing for 30 minutes, remove the ethanol and remove excess ethanol. Record the mass of the remaining ethanol. Calculate according to the following formula :

[0052] In the formula, The mass of ethanol absorbed ( ); .

[0053] Barrier performance is mainly measured by oxygen permeability (OP) and water vapor permeability (WVP): To measure OP, the deoxidizer mixture and hydrogel are first placed in a weighing bottle, sealed, and the initial mass is recorded. Then store at 100% relative humidity and 25°C for 48 hours. Record the quality after the storage period. And calculate OP:

[0054] In the formula, For time ( ); The effective area of ​​the hydrogel ( ).

[0055] To determine WVP, the hydrogel was first sealed in a container with 50... The conical neck of the distilled water flask was then further sealed with a paraffin film. The flask was placed in an oven at 3°C ​​for 24 hours, and the weight loss was recorded. And calculate WVP:

[0056] In the formula, For thickness ( ); For the permeation area ( ); For time ( ); For partial pressure difference (0.1) ).

[0057] 2.4 Application of composite hydrogels in food preservation Antioxidant activity: Includes ABTS radical and DPPH radical scavenging experiments, where radical scavenging activity is calculated according to the following formula:

[0058] In the formula, This represents the absorbance of the control group; This indicates the absorbance of the sample.

[0059] Antibacterial activity: Includes inhibition zone test and plate count test. In the inhibition zone test, *Escherichia coli* and *Staphylococcus aureus* were subjected to an inhibition zone of 37°C and 150°C. Incubate in LB broth for 8 hours under the specified conditions, then dilute to approximately [amount missing]. After CFU / mL, take 50 The sample was evenly spread on LB agar plates. The hydrogel sample was then placed on the agar surface and incubated at 37°C for 12 hours before the diameter of the inhibition zone was measured.

[0060] To determine the antibacterial rate, the hydrogel sample was mixed with a bacterial suspension ( (CFU / mL) at 37℃, 150 Mixed for 1 hour under the specified conditions, followed by a series of dilutions, then 100g was taken. Equal portions of the sample were spread onto LB agar plates and incubated at 37°C for 12 hours. The antibacterial efficiency was calculated by recording the number of colonies.

[0061] Storage experiment: Fresh button mushrooms of the same batch, uniform size, good appearance, and stable physiological state were selected. They were first soaked in CMCS-SA for 30 seconds, and then immersed in CA-distilled water and CA-PAW15 for 10 seconds respectively, forming a uniform hydrogel coating on the surface of the button mushrooms. Uncoated button mushrooms were used as the control group (CK), while the distilled water treatment group (CA-DW) and the hydrogel treatment group (PCS0, PCS15) were used as experimental groups. Key quality parameters such as weight loss, hardness, browning degree, and total soluble solids (TSS) were measured for each group during storage.

[0062] 3. Research Results and Analysis 3.1 Physicochemical properties of PAW like Figure 1 As shown in (a), the pH value of PAW decreased significantly with increasing treatment time, with a marked decrease in the first 5 minutes, followed by a gradual stabilization. In contrast, the ORP value increased significantly. Figure 1 (b) The trend is particularly evident in the first 15 minutes, then stabilizes. Similarly, the conductivity of PAW also shows a continuous increasing trend with the extension of plasma treatment time. Figure 1 (c)), from approximately 0.5 Rising to nearly 33 .

[0063] Based on the data in Table 1, Figure 1 (c) The rapid increase within 5 to 15 minutes is mainly due to the formation of a large amount of substances such as... 、 、 Ions. In addition, like hydrogen peroxide ( ),ozone( The concentrations of components such as [list of components] reached 26.50 ± 0.78 after 15 minutes. 1.34±0.44 ,in The highest concentration indicates that hydrogen peroxide is one of the main long-lived reactive oxygen species generated during plasma-liquid interactions. While the measurable concentration of dissolved ozone is relatively lower... However, ozone is considered a strong oxidant and plays an important role in the oxidation properties of PAW.

[0064] Table 1. Concentration of main active substances in PAW at different activation times

[0065] 3.2 Microstructure characterization of composite hydrogels Figure 2 (a) Shows the FT-IR spectra of CMCS, SA, CA, and PCS0 and PCS15. PCS0 and PCS15 show significant changes compared to the spectra of CMCS, SA, and CA, specifically around 3400. The broad peak at that point broadens and shifts slightly, while the characteristic peak related to the carboxyl group (around 1600) becomes more prominent. and 1400 The slight shift suggests that the interaction between CMCS, SA and CA molecules may have been enhanced.

[0066] Figure 2 In (b), the diffraction patterns of CMCS and SA are relatively broad and lack sharp peaks, indicating that they are mainly amorphous structures with disordered molecular chains. CA, on the other hand, shows multiple sharp diffraction peaks within the scanning range, indicating its highly crystalline nature. The diffraction patterns of PCS0 and PCS15 show broad and relatively smooth diffraction peaks, with the disappearance of crystalline diffraction peaks. This indicates that CA molecules have successfully integrated into the hydrogel network through intermolecular interactions with CMCS and SA, forming an amorphous three-dimensional network structure.

[0067] like Figure 2 As shown in (c), both hydrogels exhibit similar TGA trends, indicating that they have similar thermal degradation processes: first, slight loss occurs at about 150°C; then, at about 180-320°C, major weight loss occurs due to the release of volatile products; finally, above about 320°C, the remaining organic components gradually decompose to form stable carbonaceous residues. Figure 2 The DTG curve shown in (d) demonstrates that both samples exhibit a significant degradation peak at approximately 200°C, indicating that the degradation rate reaches its maximum at this temperature.

[0068] Compared to PCS0, PCS15 exhibits slightly enhanced hydroxyl stretching vibration peaks and a slight shift in carboxyl-related peaks in FT-IR; its XRD pattern shows a slightly smoother diffraction curve; its thermal degradation rate is slightly lower, and its DTG peak is more gradual. These characteristics indicate that the active ingredients in PAW can enhance the intermolecular interactions within the CMCS, SA, and CA hydrogel networks, thereby forming a more compact hydrogel structure.

[0069] like Figure 3 As shown, PCS0 and PCS15 exhibit significant microscopic differences. At low magnification ( Figure 3 (a) and 3(c)), PCS0 is relatively regular and layered, with a smooth and continuous surface structure. PCS15, on the other hand, is more uniform and dense, with finer structural features; at higher magnification ( Figure 3 In (b) and 3(d)), PCS0 exhibits a layered structure with a low crosslinking density. In contrast, PCS15 exhibits a dense and wrinkled microstructure. This demonstrates that the active ingredient in PAW can enhance the intermolecular interactions between CMCS, SA, and CA, forming a more compact and uniform network structure.

[0070] 3.3 Macroscopic physicochemical properties of composite hydrogels exist Figure 4 As shown in (a), both hydrogels have high water content, with PCS15 slightly lower than PCS0. This indicates that PAW has a limited impact on the initial water-holding capacity of the hydrogels, and the hydrophilic functional groups of the polymer remain essentially unchanged. However, the swelling ratio of PCS15 is lower than that of PCS0. Figure 4 (b) This is mainly because its denser, more cross-linked network structure restricts the permeation and diffusion of water molecules. Conversely, PCS15 has significantly higher water retention than PCS0 ( Figure 4 (c) This indicates that the various active ingredients in PAW reduce the rate of water migration and evaporation, proving that PCS15 is more effective in retaining moisture.

[0071] Table 2 shows that the thicknesses of PCS0 and PCS15 are not significantly different, indicating that PAW has no significant effect on the hydrogel thickness. However, when PAW is used instead of deionized water, the TS value of the hydrogel increases by approximately 63%, and the EAB value rises to 52.40 ± 4.76%. This indicates that the flexibility and deformability of PCS15 are significantly improved, with stronger intermolecular interactions and a denser structure. The simultaneous positive changes in TS and EAB values ​​also suggest that the internal structure of PCS15 is more uniform and ordered.

[0072] Table 2. Thickness, tensile strength and elongation at break of hydrogels

[0073] Figure 5The rheological properties of the hydrogel were evaluated using strain and frequency scanning measurements. Strain scanning showed ( Figure 5 (ab)), PCS0 and PCS15 both exhibit viscoelastic liquid properties in the 1-10% strain range (i.e., > The precursor system is primarily viscous before gelation. Frequency scan results ( Figure 5 (cd) further reveals the viscoelastic response characteristics of the precursor solution: 'and "All increase with increasing frequency and across the entire frequency range" 'Greater than This indicates that a preliminary network structure already exists before complete gelation.

[0074] Figure 6 (ac) shows the porosity, oxygen permeability (OP), and water vapor permeability (WVP) of the hydrogel. Compared with PCS0, PCS15 has a lower porosity, which means that its internal void space is reduced and the crosslinking density is increased, indicating that the introduction of PAW forms a more dense and tightly packed network structure. This structural difference is further reflected in the OP test results. Figure 6 (b) The OP value of PCS15 is lower than that of PCS0, mainly because the denser network structure and lower porosity limit gas transport and increase diffusion resistance within the hydrogel matrix. A similar trend was also observed in water vapor permeability. Figure 6 (c): The WVP of PCS15 was significantly reduced, indicating that the PAW-treated hydrogel could more effectively block water penetration.

[0075] 3.4 Application of composite hydrogels in food preservation The results of the ABTS radical and DPPH radical scavenging experiments are as follows: Figure 7 As shown in (ab), both experiments demonstrate that PCS15 exhibits significantly more radical scavenging activity, indicating that its antioxidant capacity is enhanced after incorporation with PAW. This enhancement is mainly due to the active ingredient introduced by PAW and the enhanced intermolecular interactions within the hydrogel. The denser, more compact structure helps stabilize the active functional groups and increases the likelihood that these functional groups will participate in radical scavenging reactions.

[0076] The antibacterial test results of the hydrogel are as follows: Figure 8 As shown in (ab), no inhibition zone was observed in the control group, while both PCS0 and PCS15 showed significant inhibitory effects on bacteria. For *Escherichia coli* and *Staphylococcus aureus*, the inhibition zone diameter of PCS15 was significantly larger than that of PCS0, indicating a stronger antibacterial effect. Figure 8In (cd), the antibacterial properties of both were further verified by viable colony counting: bacteria grew densely in the control group, PCS0 reduced the number of colonies to some extent, while PCS15 significantly reduced the number of colonies. These results further confirm that PCS15 induces oxidation reactions through the active components in PAW, damages the cell membrane, and interferes with intracellular components such as proteins and nucleic acids, ultimately leading to bacterial inactivation.

[0077] In a 15-day storage experiment, the PAW composite hydrogel (PCS15) exhibited the best preservation performance for button mushrooms. Figure 9 In (a) and (c), the loss rate and browning degree of each treatment group increased with prolonged storage time. The PCS15 group consistently maintained the lowest levels in both weight loss and browning index: the loss rate on day 15 was only 6.8%, and the browning index BI was 24.1, significantly better than other treatments. This indicates that the PAW composite hydrogel coating can form an effective physical barrier through its dense network structure, reducing browning. Furthermore, PAW can enhance the water retention of the hydrogel, and its active substances can inhibit the activity of polyphenol oxidase, thus delaying browning and reducing water loss overall.

[0078] The TSS content in each group generally showed an M-shaped fluctuation trend. Figure 9 (b) This is mainly due to a combination of factors, including water loss and concentration in the early stage of storage of button mushrooms, respiratory consumption in the middle stage, and cell tissue lysis in the later stage. Compared with the control group, each treatment group reduced the fluctuation of TSS to a certain extent. The button mushrooms treated with PAW composite hydrogel could maintain relative stability during the storage period, which indicates that PAW composite hydrogel can effectively delay the respiratory metabolism and nutrient consumption of button mushrooms and maintain the stability of their nutritional quality.

[0079] 4. Research Conclusions The above experiments evaluated the physicochemical properties of PAW, the microscopic and macroscopic structural characteristics of the composite hydrogel, and its preservation performance on button mushrooms. The results showed that PAW, by altering the system's composition and properties, enhanced the intermolecular interactions of the hydrogel, increasing the tensile strength by approximately 63%. It significantly improved water retention while reducing gas permeability, effectively preventing loss and browning of button mushrooms due to transpiration and respiration. This research indicates that this composite system provides a safe and long-lasting preservation method for button mushrooms and offers new ideas and theoretical basis for the application of plasma-activated water in post-harvest preservation of edible fungi.

[0080] This invention innovatively applies PAW to the CMCS-CA system hydrogel, utilizing the antibacterial, redox environment regulation, and quality deterioration functions of PAW, along with the flexibility and buffering properties of polysaccharide hydrogels, to organically construct a novel preservation method that can both delay the deterioration of button mushrooms through plasma and solve the problems of short action time and insufficient stability of PAW. This provides a new approach to synergistic preservation that is gentle, uniform, and continuously slow-release for the development of the button mushroom industry.

[0081] This invention innovatively modulates and verifies the hydrogel matrix based on the active components in polysaccharide-based osmotic pressure (PAW). The reactive oxygen species (ROS) and reactive nitrogen species (RNS) in PAW can interact with polysaccharide functional groups, enhancing intermolecular hydrogen bonds and cross-linking, thereby significantly improving the hydrogel's mechanical strength, water retention, and sustained-release properties. This approach allows the hydrogel to maintain good flexibility while becoming more compact, with stronger water-holding capacity, providing a milder and more stable storage environment.

[0082] While the combination of plasma technology and hydrogels has shown great potential in the development of functional materials, its application in food preservation remains relatively limited. Therefore, this invention addresses the industry pain point of post-harvest browning and spoilage in button mushrooms. Building upon existing applications of PAW (polydioxanone) and hydrogels in fruit and vegetable preservation, it innovatively combines the two to develop a composite preservation method suitable for post-harvest storage of button mushrooms. By constructing a PAW-loaded hydrogel system, its dual role in inhibiting post-harvest microbial growth and maintaining storage quality in button mushrooms is explored. This aims to reduce quality deterioration during post-harvest distribution, enhance the commercial value and market competitiveness of button mushrooms, and provide a new technical path and theoretical reference for the green preservation of edible fungi.

[0083] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing plasma-activated water-functionalized composite hydrogels, characterized in that, Includes the following steps: 1) The bubble spark discharge reactor was immersed in deionized water and treated at a working voltage of 70~80 volts and a current of 1.9~2.1 amperes. PAW was prepared by selecting a plasma activation time of 14~16 minutes to obtain PAW15. 2) Dissolve CMCS in PAW15 buffer and stir until completely dissolved; add SA and continue stirring until a homogeneous precursor solution is obtained; The obtained solution was injected into a mold, crosslinked with CA to form a hydrogel, and dried to constant weight.

2. The preparation method of plasma-activated water-functionalized composite hydrogel according to claim 1, characterized in that, The amount of deionized water used in step 1) is 1.5L.

3. The preparation method of plasma-activated water-functionalized composite hydrogel according to claim 1, characterized in that, The operating voltage mentioned in step 1) is 75 volts.

4. The method for preparing plasma-activated water-functionalized composite hydrogel according to claim 1, characterized in that, The current mentioned in step 1) is 2 amperes.

5. The method for preparing plasma-activated water-functionalized composite hydrogel according to claim 1, characterized in that, Step 1) During the processing, discharge parameters are monitored using a high-voltage probe, a current probe, and an oscilloscope.

6. The method for preparing plasma-activated water-functionalized composite hydrogel according to claim 1, characterized in that, In step 1), a plasma activation time of 15 minutes was selected to prepare PAW.

7. The method for preparing plasma-activated water-functionalized composite hydrogel according to claim 1, characterized in that, In step 2), the ratio of CMCS, PAW15 buffer, and SA is 1.6 g: 100 ml: 0.4 g.

8. The method for preparing plasma-activated water-functionalized composite hydrogel according to claim 1, characterized in that, In step 2), the stirring until completely dissolved is performed at 500 revolutions per minute at room temperature; the drying to constant weight is performed at room temperature.

9. The application of the plasma-activated water-functionalized composite hydrogel according to any one of claims 1 to 8 for the preservation of edible fungi.

10. The application according to claim 9, characterized in that, The edible fungus is Agaricus bisporus.

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