Edible mushroom fresh-keeping method based on modified atmosphere packaging and static magnetic field

Abstract

The invention discloses an edible mushroom fresh-keeping method based on modified atmosphere packaging cooperating with a static magnetic field, and belongs to the technical field of edible mushroom postharvest fresh-keeping, and the edible mushroom fresh-keeping method comprises the following steps: S1, pre-cooling the freshly harvested edible mushrooms; s2, putting the pre-cooled edible mushrooms into a packaging box, filling the packaging box with controlled atmosphere gas, and sealing the packaging box with a film; s3, static magnetic field treatment is applied to the edible mushrooms subjected to modified atmosphere packaging in the step S2; s4, the environment temperature is controlled to be 4 + / -1 DEG C in the whole process. According to the method, the postharvest quality, including sensory quality, physical quality and nutritional quality, of the stropharia rugoso-annulata can be comprehensively kept. The fresh-keeping mechanism is illuminated from the physiological and biochemical level, that is, the activity of antioxidant enzymes such as SOD, CAT, APX and the like is remarkably improved, reactive oxygen species (ROS) are effectively removed, the content of membrane lipid peroxidation products MDA is reduced, and the integrity of a cell membrane structure is maintained. At the same time, it is revealed from the molecular biology level that aging is delayed from the source by regulating and controlling gene expression of energy metabolism, an anti-oxidation system and related metabolic pathways.

Description

Technical Field

[0001] This invention relates to the field of postharvest preservation technology for edible fungi, and in particular to a method for preserving edible fungi based on modified atmosphere packaging and a static magnetic field. Background Technology

[0002] Giant king mushroom ( Stropharia rugosoannulata The large-cap mushroom, also known as the wrinkled cap mushroom, is loved by consumers for its rich nutrition and delicious taste. However, after harvesting, the large-cap mushroom has a vigorous respiration rate, tender tissue, and high water content, making it extremely prone to quality deterioration such as cap opening, softening, browning, and spoilage. Its shelf life is very short, usually only 1-3 days, which seriously restricts its commodity circulation and commercial value.

[0003] Currently, the main methods for preserving *Stropharia carinata* include low-temperature storage, modified atmosphere packaging (MAP), and chemical preservative coating. While low-temperature storage is effective, it is energy-intensive, and low temperatures alone cannot completely inhibit its physiological metabolism. MAP, by adjusting the O2 and CO2 ratio within the packaging, can inhibit respiration and microbial growth to some extent, but its effect on inhibiting browning and maintaining texture is limited when used alone. Chemical preservatives pose potential safety hazards and raise concerns about consumer acceptance.

[0004] Static magnetic field (SMF), as a non-thermal, residue-free physical preservation technology, has shown application potential in the field of fruit and vegetable preservation in recent years. Studies have shown that SMF can affect the permeability of biofilms, regulate enzyme activity, and inhibit microbial growth, thereby delaying the senescence of fruits and vegetables. However, there is currently no systematic research or practice on the synergistic application of modified atmosphere packaging and SMF technology in the preservation of *Stropharia masticata*. Existing technologies lack an integrated method that can efficiently, greenly, and safely address the problems of post-harvest browning, rot, softening of texture, and nutrient loss in *Stropharia masticata*. Summary of the Invention

[0005] This invention aims to solve the above problems and provides a method for preserving edible fungi based on modified atmosphere packaging and a synergistic static magnetic field, comprising the following steps: S1: Pre-cooling treatment for freshly harvested edible fungi; S2: Place the pre-cooled edible fungi in a packaging box, fill the packaging box with modified atmosphere gas and seal it; S3: Apply a static magnetic field to the edible fungi that have completed modified atmosphere packaging in S2; S4: The ambient temperature is controlled at 4±1℃ throughout the process.

[0006] Furthermore, the pre-cooling process in step S1 involves pre-cooling the fresh edible fungi in a cold storage at a temperature of 4°C and a relative humidity of 85-90% for more than two hours.

[0007] Furthermore, the modified atmosphere gas in step S2 is a low-oxygen mixture with a gas composition of 5% O2, 10% CO2, and 85% N2.

[0008] Furthermore, the intensity of the static magnetic field treatment in step S3 is between 2 mT and 10 mT. Preferably, the static magnetic field intensity is 4 mT.

[0009] Furthermore, the edible fungus is a climacteric edible fungus or an edible fungus prone to browning. A more preferred edible fungus is *Stropharia macrocarpa*.

[0010] Furthermore, the method of this invention can control the browning index of *Stropharia masticata* to below 43.4% and the decay rate to below 31.34% when stored at 4±1℃ for 10 days. The modified atmosphere packaging combined with the static magnetic field treatment can significantly maintain the firmness, elasticity, and moisture content of *Stropharia masticata*, and inhibit the decline in soluble protein, vitamin C, and total phenolic content. The modified atmosphere packaging combined with the static magnetic field treatment can significantly increase the activity of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in *Stropharia masticata*, and significantly reduce the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2). The modified atmosphere packaging combined with the static magnetic field treatment upregulates energy metabolism-related genes. SrATP5C , SrARF6 , SrAACS and antioxidant-related genes SrCAT , SrAPX The expression of [something] was downregulated, while [something] associated with browning and oxidation was also downregulated. SrSOD , SrPOD , SrPPO Gene expression regulates the metabolic pathways of *Agaricus bisporus* at the transcriptional level, thus delaying aging.

[0011] Based on the same invention, this invention also provides an edible fungus preservation system for implementing the above method, comprising: A modified atmosphere packaging unit is used to create and maintain a specific gaseous environment; A static magnetic field generating unit is used to generate a static magnetic field of 1 mT to 100 mT within the modified atmosphere packaging unit. A temperature control unit is used to maintain the system ambient temperature between 0°C and 10°C.

[0012] Based on the above technical solutions, the present invention also provides an application of modified atmosphere packaging combined with static magnetic field treatment in the preparation of preservatives or preservation devices for extending the shelf life of edible fungi.

[0013] The present invention has the following beneficial effects: 1. This invention is the first to organically combine modified atmosphere packaging (MAP) and static magnetic field technology for the preservation of king oyster mushrooms, with the two technologies producing a significant synergistic effect. Experiments have shown that the synergistic treatment of "MAP + magnetic field" is significantly better than either MAP alone or magnetic field alone in inhibiting browning, reducing rot rate, and maintaining firmness and elasticity.

[0014] 2. The method of the present invention can maintain the post-harvest quality of giant king mushrooms in all aspects, including sensory quality (color, shape), physical quality (hardness, elasticity, water holding capacity) and nutritional quality (soluble protein, vitamin C, total phenols).

[0015] 3. This invention elucidates its preservation mechanism from a physiological and biochemical perspective, namely, by significantly increasing the activity of antioxidant enzymes such as SOD, CAT, and APX, it effectively scavenges reactive oxygen species (ROS), reduces the content of MDA, a product of membrane lipid peroxidation, and maintains the integrity of the cell membrane structure. Simultaneously, from a molecular biology perspective, it reveals that it regulates energy metabolism (such as... SrATP5C ), antioxidant systems (such as SrCAT , SrAPX It can delay aging at its root by inhibiting gene expression in the ribosome and related metabolic pathways (such as the TCA cycle).

[0016] 4. No chemical preservatives are added during the entire preservation process. It relies on physical methods, leaving no residue and causing no pollution, which meets the requirements of modern food industry for green, safe and healthy products.

[0017] 5. The method described in this invention has clear parameters, is simple to operate, and is easy to integrate and apply in cold chain logistics and commercial storage, and has broad industrialization prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other embodiments can be derived from the provided drawings without creative effort.

[0019] Figure 1 The effect of modified atmosphere packaging combined with static magnetic field on the appearance of *Stropharia macrocarpa*; Figure 2 The effect of modified atmosphere packaging and synergistic static magnetic field on the browning index of Pleurotus ostreatus; Figure 3 The effect of modified atmosphere packaging combined with static magnetic field on the decay rate of Pleurotus ostreatus; Figure 4 The effect of modified atmosphere packaging combined with static magnetic field on moisture distribution of Pleurotus ostreatus, where A is the control group, B is the magnetic field group, C is the modified atmosphere packaging, and D is the modified atmosphere packaging combined with magnetic field. Figure 5 The effect of modified atmosphere packaging combined with static magnetic field on the moisture content of Pleurotus ostreatus; Figure 6 The effect of modified atmosphere packaging and synergistic static magnetic field on the relative conductivity of *Stropharia macrocarpa*. Figure 7 The effects of modified atmosphere packaging and synergistic static magnetic field on the elasticity (A) and hardness (B) of *Stropharia macrocarpa*. Figure 8 Effects of modified atmosphere packaging and synergistic static magnetic field on the soluble protein (A), total phenols (B), and vitamin C (C) content of *Stropharia macrocarpa*. Figure 9 The effect of modified atmosphere packaging and synergistic static magnetic field on the MDA content of *Stropharia macrocarpa*. Figure 10 Effects of modified atmosphere packaging and synergistic static magnetic field on the content of superoxide anion (A) and hydrogen peroxide (B) in *Schefflera heptaphylla*. Figure 11 The effect of modified atmosphere packaging combined with static magnetic field on ROS distribution in *Schefflera heptaphylla*. A represents fresh mushrooms, B represents the control group for 10 days, C represents modified atmosphere packaging for 10 days, D represents static magnetic field for 10 days, and E represents modified atmosphere packaging combined with static magnetic field for 10 days. Figure 12 The effect of modified atmosphere packaging and synergistic static magnetic field on DPPH free radicals in *Stropharia macrocarpa*. Figure 13 Effects of modified atmosphere packaging and synergistic static magnetic field on the activities of SOD (A), CAT (B), APX (C), and POD (D) enzymes in *Agaricus bisporus*. Figure 14 Effects of modified atmosphere packaging and synergistic static magnetic field on the PPO(A) and LOX(B) enzyme activities of *Agaricus macrocarpa*. Figure 15 PCA analysis of transcriptome data; Figure 16 : Clustering heatmap of differential gene expression; Figure 17 Volcano map of differentially expressed genes; Figure 18 GO enrichment analysis of differentially expressed genes; Figure 19 KEGG enrichment analysis of differentially expressed genes; Detailed Implementation The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. The following embodiments are only for illustrative purposes and are not intended to limit the scope of the present invention in any way. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are conventional methods. Unless otherwise specified, the materials and reagents used in the present invention are commercially available. Furthermore, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0020] Example 1. Test materials The king oyster mushrooms were purchased from Shandong Nongfa Group. King oyster mushrooms of uniform size, free from disease, of good quality, and without mechanical damage were selected for the experiment.

[0021] 2. Test methods 2.1 Processing methods for St. Peter's Basil Fresh king oyster mushrooms were pre-cooled in a cold storage at 4℃ and 85-90% relative humidity for more than two hours to remove field heat. King oyster mushrooms of uniform size, free from disease, of good quality, and without mechanical damage were selected, randomly divided into four groups, and placed in modified atmosphere packaging boxes.

[0022] The four groups of large king oyster mushrooms were processed as follows: The control group was placed in a modified atmosphere packaging box without sealing. The magnetic field assembly is placed in a 4 mT magnetic field device without being sealed. After the modified atmosphere packaging unit is filled with gas (5% O2, 10% CO2, 85% N2), it is sealed. Modified atmosphere packaging + magnetic field assembly: After being filled with gas (5% O2, 10% CO2, 85% N2), sealed, and placed in a 4 mT static magnetic field device.

[0023] The storage temperature was 4±1℃. Photos were taken and samples were collected at 0, 2, 4, 6, 8, and 10 days.

[0024] 2.2 Determination of color difference and decay rate Browning parameters of *Stropharia macrocarpa* were determined using a colorimeter. Each sample was tested in triplicate, and the average value was recorded. The L*, a*, and b* values ​​were recorded. The L* value represents lightness / darkness; a higher value indicates a brighter color, and vice versa. The a* and b* values ​​represent different colors: a* represents the intensity of red-green (positive for red, negative for green); b* represents the intensity of yellow-blue (positive for yellow, negative for blue). The browning calculation formula is as follows: BI=100×[(a*+1.75L*) / (5.645L*-3.012b*)-0.31] / 0.172 The decay status of *Stropharia carinata* mushrooms is comprehensively evaluated based on their softening, shrinkage, browning, and off-odor. Mushrooms that have lost their edible value are considered rotten. The formula for calculating the decay rate is: Rot rate (%) = (Number of rotten mushrooms / Total number of mushrooms) × 100% 2.3 Determination of moisture distribution, relative conductivity, hardness, elasticity, and soluble solids content Moisture distribution was determined using pulsed CPMG sequences in a low-field nuclear magnetic resonance (LF-NMR) instrument. Samples were placed in NMR glass tubes, and the testing temperature was set to 32°C. CPMG decay curves were fitted using multi-exponential fitting and the SRIT algorithm, and NMR parameters were obtained using MultiExp Inv analysis software. Imaging was performed using magnetic resonance imaging (MRI) in an MRI analyzer, followed by pseudocolor analysis using MultiExp Inv software.

[0025] The method for measuring relative conductivity is as follows: cut a 0.3 cm section of the stipe of a large-cap mushroom on the same day. 3 Take 5g of the mixture from the left and right sides and put it into a 50ml centrifuge tube. Add 20ml of redistilled water and measure the conductivity with a conductivity meter. Measure the conductivity a second time after 1 hour. Then place the tube in a water bath and boil for 15 minutes. After cooling to room temperature, shake well and measure again. Repeat the test 3 times for each sample group.

[0026] When measuring the elasticity index, the stipe of the large-cap mushroom was taken. The elasticity was measured using a cylindrical probe with a diameter of 10 mm. The test parameters were: initial force of 0.5 N, compression deformation of 30%, and moving speed of 150 mm / min.

[0027] For hardness measurement, a needle probe was used, with an insertion depth of 1 cm. Three parallel samples were tested for each treatment group, and the elasticity and hardness of *Agaricus macrocarpa* were obtained based on the test results.

[0028] Soluble solids content was determined using a multifunctional refractometer, following the method described by Cao Jiankang et al. (2007). Samples of *Agaricus bisporus* were ground in a mortar, and the resulting liquid was filtered through gauze for determination of soluble solids content.

[0029] 2.4 Determination of soluble protein content, vitamin C content, and total phenols Soluble protein content was determined using the Coomassie brilliant blue method. Bovine serum albumin was used as the standard curve for calculation. Soluble protein content is expressed in μg / g FW.

[0030] The ascorbic acid content of *Agaricus bisporus* was determined using the 2,6-dichlorophenolindophenol method (Cao et al., 2007). 2g of mushroom sample was homogenized with 2% HCl, centrifuged, and the supernatant was collected. After decolorization, the sample to be tested was obtained. The sample was titrated with 2,6-dichlorophenolindophenol solution against the standard ascorbic acid solution and a blank solution, respectively, until a faint red color appeared for 30 seconds and did not fade. The ascorbic acid content is expressed as the mass of ascorbic acid per gram of *Agaricus bisporus*, in mg / g FW.

[0031] Total phenol content was determined using the Folin-Ciocalteu method (Wang et al., 2021c). Gallic acid was used as the standard for total phenol content. Content is expressed in mg / g FW.

[0032] 2.5 Measurement of MDA, H2O2, superoxide anion, and ROS images The MDA determination method (Zhu et al., 2019) is as follows: Take 2.0 g of mushroom sample, add 10 mL of 100 g / L trichloroacetic acid, homogenize, centrifuge, take 2 mL of the supernatant, add 2 mL of 6 g / L thiobarbituric acid, mix thoroughly, seal and boil for 15 min, cool, centrifuge, and take the supernatant to measure the absorbance at 450, 532, and 600 nm on a spectrophotometer. The MDA content is expressed as μmol / g FW.

[0033] The H2O2 content was determined using the method described by Zhu et al. (2021), with slight modifications. 3.0 g of mushroom sample was weighed, and 5 mL of pre-cooled acetone was added. The mixture was ground in an ice bath and centrifuged at 13,000 rpm at 4 °C for 13 min. 1 mL of the supernatant was collected and added to 0.1 mL of 5% titanium sulfate solution and 0.2 mL of concentrated ammonia. The resulting complex was then centrifuged at 13,000 rpm at 4 °C for 13 min. The precipitate was dissolved in 3 mL of sulfuric acid solution. Finally, the OD value was measured at 420 nm. A standard curve was plotted using H2O2, and the H2O2 content was expressed as μmol / g FW.

[0034] O 2-The formation rate was determined using a slightly modified method described by Zhu et al. (2021). 3.0 g of *Agaricus macrocarpa* sample was added to 5 mL of 0.1 mM PBS solution (pH 7.8) containing 100 μM EDTA. The mixture was ground on ice and centrifuged at 12,000 rpm for 20 min at 4 °C. 1 mL of the supernatant was collected, and 1 mL of hydroxylamine hydrochloride was added. After thorough mixing, the mixture was incubated in a 40 °C water bath for 1 h. Then, 1 mL of p-aminobenzenesulfonic acid and 1 mL of α-naphthylamine were added, mixed, and allowed to develop color for 10 min before measuring the OD value at 540 nm. A nitrite standard curve was plotted using NaNO₂ solution. The superoxide anion formation rate was expressed as μmol / min / g FW. Each sample was tested three times.

[0035] The distribution of ROS in *Agaricus bisporus* was detected using laser confocal scanning microscopy (Zhang et al., 2015). The mushrooms were sliced ​​and incubated in 50 μM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) solution in the dark for 70 min to allow the probe to fully bind to ROS. The samples were washed three times with phosphate buffer to remove excess probe. The samples were observed using laser confocal microscopy at an excitation wavelength of 485 nm and an emission wavelength of 510 nm.

[0036] 2.6 Determination of Antioxidant Capacity Related Indicators The DPPH radical scavenging ability was assessed using the method of Ma et al. (Ma et al., 2022), with slight modifications. Two g of *Agaricus macrocarpa* sample was weighed, added to 10 mL of 80% ethanol solution, homogenized, and centrifuged at 4 °C and 10000 r / min for 15 min. The supernatant was collected for later use. The solution was then centrifuged in 1.5 mL of a 2×10⁻⁶ column. –4 Add 1.0 mL of sample solution to 1 mol / L DPPH solution, then add 1.0 mL of 80% ethanol. Incubate at room temperature in the dark for 25 min, and record the absorbance at 517 nm as A1. Then, replace the DPPH solution with double-distilled water, following the same steps, and record the absorbance as A2. For the blank group, replace the sample solution with 80% ethanol, and record the absorbance as A0. The DPPH scavenging rate is calculated using the following formula: DPPH radical scavenging rate (%) = [A0 - (A1 - A2)] × 100 / A0 Catalase (CAT) activity was determined using the method described by Zhu et al. (2019). One unit of CAT activity was defined as a change of 0.01 μL in absorbance per minute at 240 nm, expressed as U / g FW.

[0037] Ascorbate peroxidase (APX) activity was measured in samples of *Agaricus bisporus* using a kit provided by Beijing Solarbio Science & Technology Co., Ltd.

[0038] Superoxide dismutase (SOD) activity was determined using the method described by Song et al. (2020). One unit of SOD activity was defined as a change in absorbance of 1 μL per minute at 560 nm, and the results were expressed as U / g FW.

[0039] Peroxidase (POD) activity was determined using the method described by Zhu et al. (2019). One unit of POD activity was defined as an increase of 0.01 μL of absorbance per minute at 470 nm, and the result was expressed as U / g FW.

[0040] Polyphenol oxidase (PPO) activity was determined using the method described by Zhu et al. (2019). One unit of PPO activity was defined as an increase in absorbance of 0.01 μL per minute at 410 nm. Results were expressed as U / g FW.

[0041] Lipoxygenase (LOX) activity was determined using the method described by Song et al. (2020). One unit of LOX enzyme activity was defined as an increase of 0.001 units of absorbance per minute at 234 nm. These enzyme activities were expressed as U / g FW.

[0042] 2.7 Transcriptome Measurement and Analysis To ensure sample accuracy and reduce systematic errors, samples of *Agaricus bisporus* (six-day-old mushrooms) in consistent condition were collected after thorough mixing. Three biological replicates were performed for each treatment, and samples were sent to Genomics Technology Co., Ltd. for transcriptome sequencing and analysis. Principal component analysis (PCA) was performed on the samples based on expression levels using the DESeq R package. Differentially expressed genes (DEGs) were analyzed using DESeq2 software. Gene expression volcano plots were generated using the ggplots2r package. DEG cluster heatmaps were visualized using Genesis software. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genetics and Genomes (KEGG) enrichment analysis were performed on differentially expressed genes using the clusterProfiler R package.

[0043] 2.8 Data Statistics and Analysis Each group underwent three independent biological replicates. Data are presented as mean ± standard deviation, and significance was determined using Duncan's multiple analysis in SPSS Statistics 26. Graphs were plotted using GraphPad Prism 9.

[0044] 3 Results and Analysis 3.1 Effects of Modified Atmosphere Packaging and Synergistic Static Magnetic Field on the Sensory Quality of *Stropharia macrocarpa* like Figure 1 As shown, both the control group and the magnetic field group of *Stropharia masticata* mushrooms experienced water loss, drying, and shrinkage during storage, resulting in reduced mushroom weight and a significant decrease in volume. In the modified atmosphere packaging group, *Stropharia masticata* mushrooms began to open their caps on the sixth day, with the stipe in contact with the cap starting to thin and elongate. By the tenth day, the mushrooms in the package had an off-odor, felt soft and rotten to the touch, and exuded yellow sap; the stipe showed signs of detaching from the cap. However, the *Stropharia masticata* mushrooms treated with modified atmosphere packaging and magnetic field showed no significant changes. On the tenth day after opening, the mushrooms softened slightly, but no yellow sap was exuded. Modified atmosphere packaging and magnetic field treatment maintained the better texture and shape of the *Stropharia masticata* mushrooms.

[0045] like Figure 2 As shown in the browning index line graph, the browning of the control group of *Stropharia masticata* was severely aggravated on day 6, far exceeding that of the other three groups, with a browning index of 68.6% by day 10. The browning index of mushrooms treated with modified atmosphere packaging and magnetic field therapy was 43.4% by day 10. Modified atmosphere packaging combined with magnetic field therapy inhibited browning in *Stropharia masticata*, which is beneficial for extending the shelf life of mushrooms.

[0046] like Figure 3 As shown, during storage, the control group and the magnetic field group exhibited the fastest decay rates of *Stropharia masticata*, with similar numerical values, reaching 66.64% and 60.54% respectively on day 10. The modified atmosphere packaging (MAP) combined with magnetic field treatment resulted in the lowest decay rate, at only 31.34% on day 10. The ordinary MAP group had a decay rate of 42.07% on day 10. MAP combined with magnetic field treatment effectively inhibited the decay of *Stropharia masticata*. Magnetic field treatment can extend the storage period of fruits and vegetables by reducing their respiration rate and water flow.

[0047] 3.2 Effects of Modified Atmosphere Packaging and Synergistic Static Magnetic Field on the Physical Properties of *Stropharia macrocarpa* Figure 4 As shown, the free water content of mushrooms in both the control group and the magnetic field group decreased, indicating that moisture was gradually and rapidly lost during storage. Figure 5This finding is confirmed by the gradual bluening of the color in the graph, indicating a gradual decrease in moisture content. This is a result of the intense respiration and transpiration of *Stropharia masticata* after harvest. Modified atmosphere packaging (MAP) treatment resulted in better moisture retention in the *Stropharia masticata*, but significant loss of non-flowing water. This is because the humid environment inside the MAP treatment inhibits transpiration and controls water loss. The non-flowing water content in the MAP-treated mushrooms decreased. In an environment with near-saturated external humidity (RH > 95%), the water gradient between the inside and outside of cells decreased, limiting water diffusion. Simultaneously, changes in cell membrane permeability caused non-flowing water to convert into bound water. The MAP combined with magnetic field treatment group showed better control of non-flowing water within the *Stropharia masticata*, with less loss of free water.

[0048] like Figure 6 As shown, the relative conductivity of all groups of *Stropharia macrocarpa* gradually increased during storage, with the control group showing the fastest increase. The relative conductivity of modified atmosphere packaging combined with magnetic field treatment remained at a low level, indicating that modified atmosphere packaging combined with magnetic field treatment suppressed the increase in relative conductivity.

[0049] like Figure 7 As shown, the elasticity of fresh *Stropharia masticata* was 88.2%, and its firmness was 2.46 gf. During storage, the elasticity and firmness of *Stropharia masticata* gradually decreased. On day 10, the elasticity of *Stropharia masticata* treated with modified atmosphere packaging and magnetic field was 70%, compared to only 58% and 51% in the control group and magnetic field group, respectively. The firmness of *Stropharia masticata* treated with modified atmosphere packaging and magnetic field on day 10 was 1.21 gf, compared to 0.61 gf in the control group and 0.98 gf in the magnetic field group. Therefore, modified atmosphere packaging combined with magnetic field treatment can effectively inhibit the decrease in elasticity and firmness, maintaining the good textural state of *Stropharia masticata*.

[0050] 3.3 Effects of Modified Atmosphere Packaging and Synergistic Static Magnetic Field on Nutrients in *Stropharia macrocarpa* like Figure 8 As shown, the soluble protein content gradually decreased during storage, but modified atmosphere packaging combined with magnetic field treatment maintained a consistently high level of soluble protein in *Stropharia masticata*. *Stropharia masticata* treated with modified atmosphere packaging and magnetic field treatment had a higher vitamin C content during storage. The total phenolic content fluctuated significantly, peaking on day 6 and remaining at its highest level thereafter. This indicates that modified atmosphere packaging combined with magnetic field treatment is effective in maintaining the total phenolic content of *Stropharia masticata*.

[0051] 3.4 Effects of Modified Atmosphere Packaging and Synergistic Static Magnetic Field on the Reactive Oxygen State of *Stropharia macrocarpa* Depend on Figure 9It is evident that the MDA content in the control group experienced a surge on day 4 and remained at a high level thereafter. The modified atmosphere packaging (MAP) combined with magnetic field treatment consistently resulted in the lowest MDA content in *Agaricus bisporus*, reaching 31.7% less than the control group by day 10 of storage. MAP combined with magnetic field treatment effectively inhibited lipid peroxidation of *Agaricus bisporus* cell membranes, mitigated cell membrane structural damage, and maintained cell membrane integrity and stability, thereby inhibiting MDA generation and accumulation. Furthermore, MAP treatment reduced the oxygen concentration in the environment, slowing the generation rate of reactive oxygen species (ROS), while magnetic field treatment may have further enhanced the activity of antioxidant enzymes (such as SOD and POD) within the mushroom cells, synergistically improving the mushroom's own antioxidant defense capabilities. The combined effect significantly reduced the accumulation level of MDA during storage.

[0052] Figure 10 It is evident that the H2O2 content and superoxide anion production rate of *Stropharia masticata* treated with modified atmosphere packaging and magnetic field remained at low levels throughout the storage period. Reactive oxygen species (ROS) accumulation in *Stropharia masticata* after modified atmosphere packaging and magnetic field treatment was detected using a fluorescent probe. Figure 11 As shown, during the 10-day storage period, compared with the control group, the modified atmosphere packaging combined with magnetic field group showed weaker reactive oxygen species (ROS) fluorescence intensity in *Agaricus bisporus*. This combined treatment effectively reduced the respiratory metabolic intensity of the mushrooms during storage, decreasing the production and accumulation of intracellular ROS. These results are attributed to the regulatory effect of magnetic fields on free radical generation, and the combination with modified atmosphere packaging significantly enhances antioxidant capacity, thereby delaying quality deterioration.

[0053] 3.5 Effects of modified atmosphere packaging and synergistic static magnetic field on the antioxidant capacity of *Stropharia macrocarpa* Figure 12 The results showed that the DPPH free radical scavenging rate of *Agaricus macrocarpa* gradually decreased in all four experimental groups during storage. The DPPH free radical scavenging rate of fresh *Agaricus macrocarpa* was 81.03%. In the control group, it decreased to 15.39% on day 10. The modified atmosphere packaging combined with magnetic field treatment only decreased to 44.36% on day 10. Modified atmosphere packaging combined with magnetic field treatment may have induced the accumulation of natural antioxidants such as phenols, flavonoids, polysaccharides, and ascorbic acid in the mushrooms, enhancing the overall antioxidant capacity of the mushrooms and effectively maintaining the DPPH free radical scavenging capacity during storage. Magnetic field treatment may have activated the activity of antioxidant-related enzymes in the mushrooms, improving the efficiency of ROS scavenging and reducing the loss of antioxidants in the mushrooms by free radicals, thus maintaining a high DPPH free radical scavenging capacity.

[0054] like Figure 13As shown in -A, the SOD activity of the four treatment groups of *Agaricus bisporus* showed a trend of first increasing and then decreasing during the storage period. The difference became more obvious from day 6. The *Agaricus bisporus* in the modified atmosphere packaging and magnetic field treatment group had the highest SOD activity, which was 15.6% higher than the control group by the last day of storage.

[0055] like Figure 13 As shown in Figure B, the CAT enzyme activity of *Stropharia masticata* increased during storage, and modified atmosphere packaging combined with magnetic field treatment resulted in even higher CAT enzyme activity. During storage, the activities of SOD, APX, and CAT enzymes were significantly higher than the control. Modified atmosphere packaging combined with magnetic field treatment significantly improved the antioxidant enzyme activity of *Stropharia masticata*, enhanced its antioxidant capacity, and to some extent formed a self-protective mechanism against oxidative damage, maintaining reactive oxygen species balance and delaying aging through antioxidant activity.

[0056] Changes in LOX enzyme activity during storage period, such as Figure 14 As shown, the LOX enzyme activity of the control group of *Stropharia masticata* gradually decreased over 10 days, while the activity of the mushrooms in the modified atmosphere packaging and static magnetic field treatment group gradually increased in the first 6 days, and then began to decrease. PPO activity fluctuated significantly during storage, showing a trend of first increasing and then decreasing. The peak activity of the control group of *Stropharia masticata* occurred on day 2, while the peak activity of the modified atmosphere packaging and magnetic field treatment group occurred on day 8. The modified atmosphere packaging and magnetic field treatment delayed the peak time of PPO activity. LOX catalyzes the oxidation of unsaturated fatty acids in cell membranes; increased activity generates a large number of free radicals and reactive oxygen species, accelerating oxidative stress. PPO catalyzes the formation of quinone intermediates from polyphenols; these substances further polymerize to form brown or brownish substances, exacerbating browning. Free radicals and ROS generated by LOX further activate PPO activity, intensifying the browning reaction. The intermediates produced by PPO-catalyzed oxidation exacerbate membrane lipid damage and enhance LOX activity, creating a negative feedback loop that accelerates quality deterioration. Magnetic field treatment can activate the antioxidant enzyme system in *Agaricus bisporus*, enhance ROS scavenging capacity, and alter the oxidative environment required for LOX and PPO catalytic reactions, thereby indirectly increasing their activities. Magnetic field treatment further alters the activities of LOX and PPO by maintaining cell membrane lipid structure stability, reducing the rate of membrane lipid peroxidation, and decreasing the accumulation of intracellular oxidative products.

[0057] 3.6 Transcriptomic Analysis of Pleurotus ostreatus under Modified Atmosphere Packaging and Synergistic Static Magnetic Field Treatment PCA analysis results show that ( Figure 15 The variances of PC1 and PC2 were 28.62% and 11.97, respectively. The groups were close to each other within each group and separated between groups, showing good reproducibility within each group and statistically significant differences between the modified atmosphere packaging combined with magnetic field treatment and the control group.

[0058] Figure 16 Cluster heatmap analysis revealed significant differences in gene expression between the modified atmosphere packaging / magnetic field treatment and the control group. Genes within each group exhibited similar expression patterns, forming distinct clusters. The expression patterns of variables within different clusters were consistent, while significant differences were observed between clusters. This indicates that the *Agaricus bisporus* treated with modified atmosphere packaging and static magnetic field showed significant changes by day 6 compared to the control group.

[0059] Figure 17 The differentially expressed gene volcano plot shows the specific number of differentially expressed genes. Green represents genes that are significantly upregulated, totaling 2,636; red represents genes that are significantly downregulated, totaling 2,457; and gray represents genes that are not significantly expressed, totaling 6,984.

[0060] Figure 18 The GO enrichment analysis results show that the red areas represent significantly enriched pathways in biological processes, including gene expression, peptide metabolism, and translation; the green areas represent significantly enriched cellular components, including ribosomes, ribonucleoprotein complexes, organelles, and the inner mitochondrial membrane; and the blue areas represent significantly enriched pathways in molecular function, including ribosome structural components, transporter protein activity, DNA binding, and catalytic or electron transport pathways. Based on these results, modified atmosphere packaging combined with magnetic field treatment significantly regulates protein synthesis and transcription / translation processes. During cellular metabolism, the metabolism and synthesis of nitrogenous compounds and peptides were significantly affected, and modified atmosphere packaging combined with magnetic field treatment significantly enhanced cellular protein synthesis pathways.

[0061] Figure 19 In the KEGG enrichment analysis, the most significantly enriched pathways were ribosomes, oxidative phosphorylation, and the tricarboxylic acid cycle, all of which play important roles in bioenergy metabolism and protein synthesis. In addition, the enriched nicotinic acid and nicotinamide metabolism, as well as purine and pyrimidine metabolism, may participate in energy metabolism to some extent. Higher energy levels help maintain cell membrane stability, enhance cellular self-repair capabilities, and prevent cell dehydration, aging, and oxidation. The metabolic pathways and secondary metabolite synthesis pathways showed the highest number of enriched genes, suggesting that *Agaricus bisporus* may therefore generate more natural antioxidants, delaying oxidative browning.

[0062] The above results indicate that the modified atmosphere packaging and magnetic field combined with the effects of the magnetic field make the Pleurotus ostreatus more active in physiological processes such as cell growth and proliferation and resistance to environmental stress than the control group. A large amount of nucleotides, amino acids and secondary metabolites are being synthesized, which helps maintain the integrity of cell structure, delay post-harvest oxidative damage and delay the aging of mushrooms.

[0063] 3.7 qPCR validation of related genes Table 1 shows the qPCR validation results of the relevant genes. Compared with the control group, the mushrooms treated with modified atmosphere packaging and magnetic field showed significantly higher growth rates. SrSOD , SrPOD , SrPPO The expression level was downregulated. SrLOX , SrCAT , SrAPX Expression levels were significantly upregulated. This change suggests that magnetic field treatment may, to some extent, inhibit the expression of enzymes related to oxidation reactions, thereby slowing down the browning process in mushrooms. Simultaneously, antioxidant-related genes ( SrCAT and SrAPX Upregulation of ) helps enhance the cell's ability to scavenge reactive oxygen species and maintain cell homeostasis. SrLOX Significant upregulation of these genes is associated with enhanced cell membrane stability. Magnetic field treatment reduces membrane lipid peroxidation by regulating the lipoxygenase pathway, thereby improving the quality retention of mushrooms during storage. (Energy metabolism-related genes) SrATP5C , SrARF6 and SrAACS The modified atmosphere packaging and magnetic field treatment also showed a significant upward trend. SrATP5C As an important component of the mitochondrial ATP synthase complex, increased expression of this gene helps improve the efficiency of ATP production in cells; ADP-related enzyme gene SrARF6 Closely related to the regulation of energy signaling pathways and metabolic regulation, its upregulation reflects the positive response of cells to external stimuli; AMP-related genes SrARF6 It plays a crucial role in providing energy substrates. These results indicate that magnetic field-assisted modified atmosphere treatment not only plays a positive role in antioxidation but also enhances cellular energy metabolism activity, providing ample energy support for the synthesis of antioxidant enzymes and cellular repair processes, thereby delaying tissue decay.

[0064] Table 1. Changes in the expression levels of related genes

[0065] In summary, by comparing the control group, the modified atmosphere packaging group alone, the magnetic field packaging group alone, and the "modified atmosphere packaging + magnetic field" synergistic treatment group, the effects of different treatments on the post-harvest storage quality of *Stropharia carinata* were systematically evaluated. Both modified atmosphere packaging and magnetic field packaging alone inhibited browning and decay of *Stropharia carinata* to some extent, but the synergistic treatment of "modified atmosphere packaging + magnetic field" showed the best effect, with significantly lower decay rate and browning index than other groups, indicating that the two preservation methods have a synergistic effect in delaying the deterioration of appearance quality.

[0066] Compared to single modified atmosphere or static magnetic field treatments, the synergistic treatment of "modified atmosphere + static magnetic field" more effectively maintains the firmness and elasticity of *Agaricus bisporus* and inhibits the accumulation of reactive oxygen species (ROS). The synergistic treatment group outperformed other treatment groups in sensory quality (such as color and morphology), delaying browning and softening processes. It also effectively slowed the degradation rate of nutrients such as soluble proteins, vitamin C, and polyphenols, maintaining a high nutritional level. Simultaneously, by enhancing the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), it significantly reduced the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2), alleviating oxidative damage.

[0067] Transcriptomic analysis showed that synergistic treatment inhibited respiratory metabolic intensity and maintained cell membrane integrity by differentially regulating the expression of energy metabolism-related genes (such as glycolysis and the tricarboxylic acid cycle pathways), antioxidant enzyme system genes (such as SOD and CAT encoding genes), and cell membrane stability genes (such as lipid metabolism-related genes), thereby delaying the aging process. Energy metabolism assays showed that synergistic treatment ensured cellular energy homeostasis by maintaining ATP content and energy charge levels, thus preventing quality deterioration caused by energy deficit.

[0068] The results of this invention demonstrate the significant synergistic effect of static magnetic field and modified atmosphere packaging in extending the shelf life and maintaining the quality of king oyster mushrooms. It reduces oxidative damage and microbial spoilage, and also has obvious advantages in maintaining nutrition and texture, providing an important basis for the subsequent development of more efficient and green integrated preservation technologies.

[0069] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preserving edible fungi based on modified atmosphere packaging and a synergistic static magnetic field, characterized in that, Includes the following steps: S1: Pre-cooling treatment for freshly harvested edible fungi; S2: Place the pre-cooled edible fungi in a packaging box, fill the packaging box with modified atmosphere gas and seal it; S3: Apply a static magnetic field to the edible fungi that have completed modified atmosphere packaging in S2; S4: The ambient temperature is controlled at 4±1℃ throughout the process.

2. The method for preserving edible fungi based on modified atmosphere packaging and synergistic static magnetic field according to claim 1, characterized in that, The pre-cooling process in step S1 involves pre-cooling the fresh edible fungi in a cold storage at a temperature of 4℃±1℃ and a relative humidity of 85-90% for more than two hours.

3. The method for preserving edible fungi based on modified atmosphere packaging and synergistic static magnetic field according to claim 1, characterized in that, The modified atmosphere gas in step S2 is a low-oxygen mixture with a gas composition of 5% O2, 10% CO2, and 85% N2.

4. The method for preserving edible fungi based on modified atmosphere packaging and synergistic static magnetic field according to claim 1, characterized in that, The intensity of the static magnetic field treatment in step S3 is 2 mT to 10 mT.

5. The method for preserving edible fungi based on modified atmosphere packaging and synergistic static magnetic field according to claim 1, characterized in that, The edible fungi mentioned are either climacteric edible fungi or edible fungi prone to browning.

6. The method for preserving edible fungi based on modified atmosphere packaging and synergistic static magnetic field according to claim 5, characterized in that, The edible fungus is *Stropharia macrocarpa*.

7. A mushroom preservation system for implementing the method of any one of claims 1-6, characterized in that, include: A modified atmosphere packaging unit is used to create and maintain a specific gaseous environment; A static magnetic field generating unit is used to generate a static magnetic field of 1 mT to 100 mT within the modified atmosphere packaging unit. A temperature control unit is used to maintain the system ambient temperature between 0°C and 10°C.

8. The application of modified atmosphere packaging combined with static magnetic field treatment in the preparation of preservatives or preservation devices for extending the shelf life of edible fungi.

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