Mechanism of High-Voltage Electrostatic Field Treatment in Maintaining the Postharvest Quality of Agaricus bisporus

Abstract

This study utilized high-voltage electrostatic field (HVEF) treatment combined with cold storage to preserve Agaricus bisporus, characterized by high water content and susceptibility to browning, cap opening, and mechanical injury. Key quality indicators, such as surface and flesh color, weight loss, respiration rate, hardness, and soluble solids, were monitored to determine optimal HVEF intensities. Transcriptomic, physiological, and biochemical analyses were used to reveal the underlying preservation mechanisms. This study demonstrates that high-voltage electrostatic field (HVEF) treatment at 30 kV m⁻¹ combined with cold storage effectively delays browning, weight loss, and respiration rate in A. bisporus while maintaining color, texture, and flavor. Transcriptomic analysis revealed that HVEF modulates key metabolic pathways, including ATP synthesis, fatty acid metabolism, and redox enzyme activity, leading to reduced ATP levels, suppressed respiration, and delayed senescence. Additionally, the treatment enhances antioxidant capacity through increased ascorbic acid (AsA) and glutathione (GSH) levels, while decreasing malondialdehyde (MDA) content and membrane electrical conductivity, thereby preserving membrane integrity. The suppression of polyphenol oxidase (PPO) and peroxidase (POD) activities reduces pigment formation and browning. Furthermore, the active metabolism of osmoprotectants such as proline improves cold resistance. These findings provide a mechanistic basis for HVEF-based preservation strategies for A. bisporus, supporting its application in postharvest technology.

1. Introduction

Agaricus bisporus, commonly known as the button mushroom, is extensively cultivated worldwide. Currently, China stands as the largest producer and consumer of this species. Rich in polysaccharides, phenolic compounds, proteins, dietary fibers, vitamins, and other bioactive constituents, A. bisporus has garnered widespread recognition in both medicinal and health-promoting applications within domestic and international markets [1,2]. Since the 1950s, China has implemented large-scale cultivation of the species, reaching a production volume of 1.5625 million tons by 2023, ranking as the seventh most cultivated edible mushroom after Lentinula edodes (13.04 million tons), Auricularia auricula-judae (black fungus, 7.15 million tons), Pleurotus spp. (oyster mushrooms, 6.13 million tons) and so on [3]. Different from dried and storable varieties such as shiitake mushroom and black fungus, A. bisporus primarily relies on fresh sales or canning processes, making postharvest preservation techniques critical to industry profitability. Postharvest metabolic activity in A. bisporus persists following substrate harvesting; due to rapid metabolism, high respiration rates, and significant water loss, the mushroom deteriorates swiftly after harvest. Manifestations of spoilage include browning, weight reduction, softening of tissue, cap opening, and fungal contamination, all of which severely compromise its commercial value [4,5]. As a specialty poverty alleviation crop in certain regions, A. bisporus significantly contributes to rural economic development in China. However, extending shelf life and minimizing postharvest losses remain key challenges for industry development of A. bisporus [6].

Currently, preservation methods for A. bisporus primarily encompass biological, chemical, and physical approaches [7]. Physical and chemical preservation such as irradiation, pulsed electric fields, the application of antimicrobial agents, coatings, ozone treatment, and electrolyzed water can effectively inactivate microbial activity and influence physical properties of A. bisporus, including texture, color, and weight loss [8]. Among these, low-temperature storage is the most widely employed method, with research indicating that the optimal preservation conditions for A. bisporus are at 0–2 °C and 90% relative humidity, under which the shelf life can reach 5–7 days [9]. Additionally, various chemical agents such as citric acid have been widely utilized in the preservation of edible mushrooms. For example, treatment with 2.5% citric acid enables A. bisporus to remain stable under refrigerated storage for 12 days. Likewise, biobased preservation strategies, such as native catechins combined with branched starch, CaCl₂, and NaCl have also been explored. This mixture, when sprayed onto the surface of A. bisporus to form a coating, can preserve the mushroom for 12 days when stored at 4 °C [10]. However, excessive use of chemical preservatives raises food safety concerns and does not fundamentally address the quality deterioration during storage, highlighting the need for safer and more effective preservation strategies.

A high-voltage electrostatic field (HVEF) refers to a voltage with no current flow or variation during the experimental process. To generate a uniform electric field, parallel plate electrodes are typically employed; due to field enhancement, the electric field between the two electrodes remains equal, except at the edges of the electrodes [11]. This technique has been employed in food technology applications, including drying, thawing, extending shelf-life of fruit and vegetable, refrigeration, freezing, extraction and so on [12]. HVEF has been applied in the preservation of jujube [13], strawberry [14], cherry tomato [15], spinach [16], broccoli [17]. As a food preservation technology, HVEF effectively inhibits microbial growth and modulates the activity of various enzymes, thereby delaying the natural ripening process of fruits and vegetables while maintaining or even enhancing their intrinsic nutritional quality and sensory attributes [18]. This non-thermal processing technique can be regarded as a form of non-biological stress, which activates the antioxidant defense systems in produce and suppresses spoilage enzymes such as PPO, lipoxygenase (LOX), pectin methylesterase (PME), polygalacturonase (PG), and cellulase (Cel). Consequently, it positively influences textural properties like hardness and crispness, color, electrical conductivity, antioxidant compound levels, and microstructural integrity, while reducing electrolyte leakage, MDA content, and browning degree. Additionally, this method increases the concentrations of fructose, glucose, and sucrose, and decreases the production of carbon dioxide (CO₂) and hydrogen peroxide (H₂O₂) [18]. Therefore, HVEF represents a residue-free preservation method that does not contribute to environmental pollution, offers ease of use, and conserves resources. In previous study of Yan et al. [19], HVEF can reduce the loss of firmness and whiteness, inhibit the accumulation of malondialdehyde, delay the loss of total phenolics, inactivate polyphenol oxidase (PPO), and effectively maintain the postharvest quality of A. bisporus. However, the parameters for HVEF treatment of A. bisporus require further optimization, and the evaluation period should be extended accordingly. Additionally, the mechanisms underlying HVEF induced preservation of A. bisporus warrant more in-depth investigation.

This study focuses on A. bisporus, systematically investigated the effects and underlying mechanisms of HVEF treatment on postharvest preservation quality. Key evaluation parameters include weight loss rate, hardness, changes in cap surface and flesh coloration, and respiration intensity. Initially, the suitability of HVEF treatments at various intensities for postharvest preservation of A. bisporus is analyzed, and the optimal treatment intensity is determined. Subsequently, transcriptomic sequencing was employed to elucidate the molecular mechanisms by which HVEF influences mushroom preservation. Further, by assessing ATP content, relative membrane electrical conductivity, activities of key glycolytic enzymes, MDA levels, the study clarifies the inhibitory effects of HVEF on respiratory metabolism. Additionally, the mechanisms by which HVEF suppressed surface browning are explored through measurements of GSH, AsA, PPO and POD activity. This research aims to provide a theoretical foundation and data support for the development of efficient postharvest HVEF treatment protocols and the construction of an HVEF preservation technology system for A. bisporus.

2. Materials and Methods

2.1. Materials and Equipment

2.1.1. A. Bisporus Samples

The A. bisporus samples were sourced from Yisheng Biotechnology Co., Ltd., Fenxixian, Shanxi Province, China (Coordinates: 111.585051° E, 36.619463° N). Uniformly shaped and sized samples without mechanical damage, browning, or pest and disease infestation were selected as experimental materials and harvested. Immediately after collection, the samples were transported to a cold storage facility and pre-cooled at 0 °C. Following overnight pre-cooling, the samples were processed accordingly.

2.1.2. High-Voltage Electrostatic Field Apparatus

As illustrated in Figure S1, two 2 mm thick steel plates were positioned in parallel at specified distances (20 cm, 30 cm, 60 cm) to generate a uniform electric field. The plates were connected to the positive and negative terminals of a high-voltage power supply, creating an electric field between them. To measure the electrostatic field, we employed an electrostatic voltmeter to monitor in real time the voltage applied by the power supply between the two plates, thereby indirectly determining the electric field strength between them. The voltmeter was pre-calibrated using a standard reference to ensure measurement accuracy. During the measurement, the two terminals of the voltmeter were securely connected to the two plates. The electric field intensity was calculated based on the applied voltage (6 kV) and the distance between plates (E = V/d), yielding theoretical values of 10 kV m⁻¹ (20 cm), 20 kV m⁻¹ (30 cm), and 30 kV m⁻¹ (60 cm).

2.2. Sample Treatment in the High-Voltage Electrostatic Field and Sampling Protocol

Based on preliminary experimental results, four groups of samples were designated: the control group and three treatment groups exposed to 10 kV m⁻¹, 20 kV m⁻¹, and 30 kV m⁻¹, respectively, within the HVEF apparatus. Each treatment session lasted 60 min, and this duration was strictly maintained for all subsequent repeated treatments conducted every 3 days according to their groupings. After completing all treatments, the samples were stored at 0 °C in a cold storage. Periodic assessments of relevant indicators were conducted every 3 days. The mushroom caps, after removal of the outer skin, were cut into small pieces, flash-frozen in liquid nitrogen, and stored in labeled self-sealing bags at −80 °C for future use (Figure S1).

2.3. Selection of Parameters for High-Voltage Electrostatic Field Treatment

2.3.1. Colorimetric

The lightness was assessed referring the method of Yan et al. [19], with certain modifications. Changes in surface color (L value) of A. bisporus caps were periodically measured using an SMY-2000ST colorimeter (Beijing Shengmingyang Technology Development Co., Ltd., Beijing, China). Fifteen mushrooms were randomly selected from each sample group and marked accordingly; 3 measurement points were designated on each mushroom surface. Additionally, 15 mushrooms were sliced longitudinally to assess internal color variations.

2.3.2. Weight Loss Rate

The weight loss rate was evaluated following the methodology of Liu et al. [20], with specific modifications. For each treatment, 30 samples were divided into 3 groups of 10. The mass of each group was measured using an electronic balance every 3 days to calculate weight loss rate, using the formula:

weight loss rate (%) = (m₁ − m₂)/m₁ × 100

where m₁ is the initial mass of A. bisporus on 0 day (g), and m₂ is the mass on sampling days (day 0, 3, 6, 9, 12, 15, 18) (g).

2.3.3. Respiratory Intensity

Respiratory intensity was measured using Liu et al.’s [20] method with modifications. From each group, 30 samples were randomly selected and divided into 3 subgroups of 10. Samples were placed in sealed containers with a capacity of 4500 mL. Using a portable O₂/CO₂ analyzer, the difference in CO₂ concentration before and after 2 h was measured. Respiratory intensity was calculated as:

Respiratory intensity (mg kg⁻¹·h⁻¹) = (V₁ − V₂)/(T × m) × n × 1.96 × 1000

where n is the CO₂ concentration difference (%), V₁ is the container volume (L), V₂ is the sample volume (L), m is the fresh weight of the sample (kg), and T is the measurement duration (hours).

2.3.4. Soluble Solids Content

Soluble solids content was quantified employing M. Adibian et al.’s [21] methodology with specified modifications. Approximately 20 g of mushroom tissue was wrapped in gauze, pressed, and filtered to extract juice. The filtrate was placed on the handheld refractometer for measurement. Each sample was tested 3 times.

2.3.5. Hardness Assessment

The hardness was determined with the method of Yan et al. [19] with some modifications. First, the stipe was excised, and a 2 mm thick epidermal layer was removed from the cap surface. The geometric center of the cap was selected as the testing site. Pre-treated samples were placed on a testing platform, and a 5 mm cylindrical flat-ended probe was used for measurement. The specific experimental parameters were set as follows: a load cell range of 100 N, a trigger force of 0.5 N, a testing speed of 60 mm min⁻¹, and a compression displacement of 50% of the sample’s original height. The experiment was designed according to a completely randomized block design, with each treatment group comprising 3 biological replicates, and 5 samples were tested per replicate.

2.4. RNA-Seq

2.4.1. Total RNA Extraction

Samples consisted of 0-day control and 0-day 30 kV m⁻¹ HVEF-treated A. bisporus, with 3 biological replicates per condition. Samples were pulverized in liquid nitrogen using a mortar and pestle. 1 g of the sample was subjected to total RNA isolation following the protocol of the Polysaccharides & Polyphenolics-rich Plant Total RNA Kit (Hangzhou Simgen Biological Reagents Development Co., Ltd., Hangzhou, China). RNA concentration and purity were determined via spectrophotometry, and integrity was verified through agarose gel electrophoresis. High-quality RNA samples were stored at −80 °C for downstream applications.

2.4.2. cDNA Synthesis

Total RNA was reverse transcribed into complementary DNA (cDNA) utilizing the PrimeScript™ RT reagent Kit with gDNA Eraser (Beijing Quanshijin Biotechnology Co., Ltd., Beijing, China) according to the manufacturer’s instructions.

2.4.3. Transcriptome Sequencing

Transcriptome sequencing and preliminary bioinformatic analyses were conducted by Beijing Novozymes Biotechnology Co., Beijing, China.

2.5. Measurement of Respiratory Metabolism-Related Indicators

2.5.1. ATP Content

Accurately weigh 1.0 g of A. bisporus tissue and add an appropriate volume of cold double-distilled water at a ratio of 1:9 (w:v). Homogenize the mixture using a mortar under ice bath conditions to prepare a 10% tissue homogenate suspension. Incubate the suspension in a water bath at 100 °C for 10 min, then immediately vortex at 2000 rpm for 1 min. Centrifuge the sample at 3500 rpm in a refrigerated centrifuge (4 °C) for 10 min, and collect the clear supernatant for subsequent ATP quantification. Following the protocol of the ATP assay kit (Nanjing Jiancheng, Nanjing, China), add the specified reagents and measure absorbance at OD₆₃₆ using a NanoDop One C ultra-micro UV-Vis spectrophotometer (TealMeo SeaCo, Houston, TX, USA).

2.5.2. 6-Phosphogluconate Dehydrogenase Activity

Precisely weigh 0.1 g of the biological specimen, add 1 mL of extraction buffer, and homogenize in an ice bath to obtain a 10% tissue homogenate. Centrifuge the homogenate at 8000× g at 4 °C for 10 min, then collect the supernatant for subsequent enzymatic activity analysis. Follow the protocol provided by the 6-Phosphogluconate Dehydrogenase activity assay kit (Beijing Solarbio Technology Co., Ltd., Beijing, China), and determine absorbance at OD₃₄₀ using a NanoDop One C ultra-micro UV-Vis spectrophotometer (TealMeo SeaCo, Houston, TX, USA).

2.6. Determination of Antioxidant Properties and Browing Related Indicators

2.6.1. Ascorbic Acid (AsA) Content

A 1.0 g sample was weighed and mixed with 9 times its weight of 0.9% saline solution to prepare a 10% homogenate, using an ice bath for uniform grinding. The homogenate was centrifuged at 2500 rpm for 10 min, and the supernatant was used for analysis. Following the manufacturer’s instructions for the Ascorbic Acid test kit (Nanjing Jiancheng), absorbance was measured at OD₅₃₆ using a NanoDop One C ultra-micro UV-Vis spectrophotometer (TealMeo SeaCo, Houston, TX, USA).

2.6.2. Glutathione Content

Construction of the standard curve involved preparing glutathione solutions at varying concentrations. These standards were sequentially mixed with the specified reagents as per the protocol, and their absorbance values were measured. For sample preparation, precisely weigh 0.1 g of the dual-spored mushroom tissue, add 1 mL of homogenization medium, and perform ice bath grinding. Subsequently, centrifuge the homogenate at 12,000 rpm for 10 min to obtain the supernatant. Following the instructions of the Reduced Glutathione (GSH) Content Assay Kit (Beijing Solarbio Technology Co., Ltd., Beijing, China), add the appropriate reagents to the supernatant and measure the absorbance at OD₄₁₂ using a NanoDop One C ultra-micro UV-Vis spectrophotometer (TealMeo SeaCo, Houston, TX, USA).

2.6.3. Cell Membrane Relative Conductivity

Six mushrooms per group were cut into pieces and placed into 3 separate centrifuge tubes, each containing 25 mL of distilled water. After incubation for 60 min, the electrical conductivity of the mixture was measured and recorded as P₁. The samples were then heated in a boiling water bath for 30 min, cooled to room temperature, and the conductivity was measured again, recorded as P₂. The relative membrane conductivity was calculated as:

Relative conductivity (%) = [(P₁ − P₀)/(P₂ − P₀)] × 100%.

where P₀ is the conductivity of distilled water.

2.6.4. Malondialdehyde (MDA)

A precise 1.0 g sample of the mushroom was weighed and homogenized with an appropriate volume of PBS in a 1:9 ratio (w:v) using a mortar under ice bath conditions to produce a 10% tissue homogenate. The homogenate was centrifuged at 3500 rpm for 10 min at 4 °C, and the supernatant was collected for biochemical analysis. Following the protocol of the MDA assay kit (Nanjing Jiancheng), absorbance was measured at OD₅₃₂ using a NanoDop One C ultra-micro UV-Vis spectrophotometer (TealMeo SeaCo, Houston, TX, USA).

2.6.5. Enzymatic Activity of Polyphenol Oxidase (PPO)

Precisely weigh 0.2 g of A. bisporus, add 1 mL of extraction solvent, and homogenize in an ice-cooled environment. Centrifuge the homogenate at 8000 rpm at ambient temperature for 10 min; collect the supernatant for subsequent spectrophotometric analysis. Follow the protocol outlined in the PPO Assay Kit (Nanjing Jiancheng) to process the sample, and determine absorbance at OD₄₂₀ using a NanoDop One C ultra-micro UV-Vis spectrophotometer (TealMeo SeaCo, Houston, TX, USA).

2.6.6. Enzymatic Activity of Peroxidase (POD)

Sample was homogenized with 8 mL saline after excision along the central axis. From 2 g of homogenate, slurry was prepared on ice, centrifuged at 4000 rpm for 10 min, and supernatant collected for POD activity measurement using a commercial kit (Beijing Solarbio, Beijing, China), and was measured at OD₄₂₀ using a NanoDop One C ultra-micro UV-Vis spectrophotometer (TealMeo SeaCo, Houston, TX, USA). Three replicates were performed, and mean activity was calculated.

2.7. Statistical Analyses

Using Microsoft Excel 2021 for data organization. Statistical significance among treatment groups at different storage durations was assessed with SPSS 27.0. Graphical representations were generated using GraphPad Prism 9.5.

3. Results

3.1. Identification of Optimal Parameters for High-Voltage Electrostatic Field Treatment

3.1.1. Lightness

As illustrated in Figure 1A, the epidermal color difference across all treatment groups generally exhibited a decreasing trend based on measurements. On day 18, the 10 and 20 kV treatment groups showed a sharp and significant reduction in color difference, markedly lower than the control group. The 30 kV m⁻¹ treatment group displayed an upward trend in L-value, with its L-value exceeding that of the control by 0.98. Visual inspection (Figure 1C) indicated that from day 15 onward, the epidermal coloration in the 30 kV m⁻¹ group was notably superior to that of other treatments. As shown in Figure 1B, the L-value of the mushroom flesh experienced a precipitous decline on day 12. Furthermore, on days 15 and 18, the L-value in the 30 kV m⁻¹ group was significantly lower than in other groups, with a difference of 0.92 compared to the control.

3.1.2. Weight Loss and Respiratory Intensity

As shown in Figure 2A, with the extension of storage time, the weight loss rate of all groups exhibited a linear upward trend; however, treatment with HVEF delayed the increase in weight loss rate. The respiration intensity across all groups generally followed a pattern of initial increase followed by decrease (Figure 2B). The respiration peaks for the control, 10, 20, and 30 kV m⁻¹ treatment groups appeared on days 9, 12, 12, and 15, respectively, indicated that the 30 kV m⁻¹ treatment delayed the onset of respiratory peaks. Furthermore, on days 6, 9, 12, and 18, the respiration rates of the 30 kV m⁻¹ group were significantly lower than those of the other groups, demonstrating that the 30 kV HVEF treatment effectively suppressed the respiratory activity of A. bisporus. As illustrated in Figure 2C, the soluble solids content across all groups exhibited an overall decreasing trend, with no significant differences observed between groups at different time (p < 0.05), indicated that HVEF treatment does not induce changes in the flavor profile of A. bisporus. The hardness of all groups generally declines with increasing storage duration (Figure 2D). Notably, the groups treated at 30 kV m⁻¹ on days 3, 6, and 9 maintain higher hardness levels compared to other groups, suggested that 30 kV m⁻¹ treatment effectively preserved the hardness of A. bisporus during the early storage period.

3.2. GO and KEGG Enrichment via Treatment with 30 kV m⁻¹ HVEF

GO enrichment analysis was performed on all differentially expressed genes (DEGs) in the A. bisporus subjected to control and 30 kV m⁻¹ HVEF treatment. The DEGs were categorized into three major functional groups: biological process (Figure 3A), molecular function (Figure 3B), and cellular component (Figure 3C). Notably, processes such as ATP biosynthetic process, ATP metabolic process, NAD binding, cofactor metabolic process, and cofactor biosynthetic process were likely associated with respiratory activity in the A. bisporus. Structural constituents of the cell wall, fungal-type cell wall were related to cell wall integrity and composition. The oxidoreductase activity may be linked to browning phenomena in A. bisporus. Subcategories such as cellular response to stress and response to stress were indicative of the mushroom’s mechanisms for environmental stress adaptation.

Furthermore, KEGG pathway enrichment analysis, as shown in Figure 3D, revealed that pathways including biosynthesis of cofactors, ascorbate and aldarate metabolism, cysteine and methionine metabolism, biosynthesis of secondary metabolites, glycolysis/gluconeogenesis, oxidative phosphorylation, biosynthesis of amino acids, arginine and proline metabolism, fatty acid degradation, valine, leucine and isoleucine degradation, valine, leucine and isoleucine biosynthesis and glutathione metabolism were intimately connected to the respiratory processes, antioxidant defenses, browning, and cold resistance of A. bisporus.

3.3. Effect of HVEF on Respiratory Metabolism of A. bisporus

In Figure 4A, treatment with 30 kV m⁻¹ HVEF significantly activated the expression of ATPase-related genes, indicated there was a reduction in ATP content. As illustrated in Figure 4B, overall, except on day 6, the ATP levels in the 30 kV m⁻¹ HVEF treatment group remained significantly lower than those in the control group. The greatest difference was observed on day 18, with ATP content in the 30 kV m⁻¹ HVEF group being 2.07 μmol g⁻¹ lower than the control. As shown in Figure 4C, throughout the storage, except on day 9, the G6PD activity in the 30 kV m⁻¹ HVEF group was consistently lower than that in the control; on days 3, 6, 12, 15, and 18, enzyme activity in the treatment group was reduced by 222.9 U g⁻¹, 128.6 U g⁻¹, 199.33 U g⁻¹, 248.62 U g⁻¹, and 176.7 U g⁻¹ respectively compared to the control.

3.4. Effect of HVEF on Antioxidant Properties and Browning of A. bisporus

As shown in Figure 5A,B, exposure to 30 kV m⁻¹ HVEF markedly influenced the expression patterns of GSH and AsA metabolism-related genes in A. bisporus. Specifically, within the GSH metabolic pathway, genes involved in synthesis were significantly upregulated, while those associated with degradation were suppressed, resulted in an accumulation of GSH in the mushroom tissue (Figure 5C), this accumulation was evidenced by significantly higher GSH levels in the treated samples on days 9 and 15 of storage compared to controls. In terms of AsA metabolism, the expression of biosynthetic genes was similarly markedly enhanced, leading to a significant accumulation of AsA in the treated mushrooms (Figure 5D); notably, after 30 kV m⁻¹ HVEF treatment, AsA content was significantly higher than controls throughout the storage period, except on days 0 and 3. The MDA content, as measured in Figure 5E, further demonstrated that 30 kV m⁻¹ HVEF treatment significantly reduced MDA levels on days 3, 9, 12, and 15. Additionally, the relative electrical conductivity during days 6 to 18 of storage was significantly lower in the treated group compared to controls (Figure 5F). Regarding the enzymatic activities of PPO and POD, the results indicated that PPO activity was significantly lower in the treated samples on days 3, 6, and 9 (Figure 5G). Although POD activity generally showed an increasing trend, a sharp decline was observed in the control group on day 12. Post-treatment, POD activity was significantly reduced in the treated samples on days 0, 3, 6, 9, and 15 compared to controls (Figure 5H).

3.5. Effect of HVEF on Cold Resistance of A. bisporus

KEGG pathway enrichment analysis of differentially expressed genes (Figure 6) revealed that HVEF treatment significantly activated amino acid biosynthesis pathways in A. bisporus. Notably, genes involved in the biosynthesis of valine, leucine, and isoleucine—encoding acetolactate synthase I (EC: 2.2.1.6), ketol-acid reductoisomerase (EC: 1.1.1.86), branched-chain amino acid aminotransferase (EC: 2.6.1.42), 2-isopropylmalate synthase (EC: 2.3.3.13), 3-isopropylmalate dehydratase (EC: 4.2.1.33), and 3-isopropylmalate dehydrogenase (EC: 1.1.1.85)—were each significantly upregulated. Additionally, six genes encoding enzymes involved in β-alanine synthesis, one gene encoding glutamine synthetase (EC: 6.3.1.2), which is directly involved in glutamine biosynthesis, and one gene encoding proline iminopeptidase (EC: 3.4.11.5), directly related to proline synthesis, exhibited marked upregulation following high-voltage electrostatic field treatment.

4. Discussion

4.1. Comparative Evaluation of HVEF-Refrigeration Synergy in A.bisporus Preservation

A. bisporus (button mushroom) exhibits heightened sensitivity to physical damage and microbial invasion due to the absence of a protective keratinized layer on its surface. Post-harvest, this vulnerability leads to a series of quality deterioration phenomena, including water loss, enzymatic browning, textural softening, off-odor development, and nutrient leaching. Under conventional storage conditions, its shelf life is markedly limited: at ambient temperature (20–25 °C), preservation lasts only 1–3 days, whereas refrigeration (0–2 °C) extends this period to 5–7 days. Nonetheless, the brief shelf life remains a critical factor constraining its economic value. To address this challenge, postharvest preservation techniques—encompassing physical, chemical, and thermal treatments—have been developed as essential strategies to maintain mushroom quality and prolong shelf life [8]. Among these, HVEF technology has demonstrated considerable potential due to its one-time investment, long-term usability, absence of chemical residues, and environmental friendliness [22]. HVEF is a non-thermal, low-energy process that generates corona discharge between electrodes, producing an electric wind that inhibits microbial proliferation and biochemical reactions on the surface of fruits and vegetables. This technology is particularly suitable for postharvest preservation, as it can suppress oxidative browning and tissue softening, preserve color and texture, and inhibit microbial growth, thereby effectively extending shelf life [23]. This study systematically investigated the applicability and underlying mechanisms of HVEF combined with refrigeration for postharvest preservation of A. bisporus, utilizing transcriptomic analysis and physiological-biochemical assessments to validate regulatory effects. The metabolic dynamics of fruit and vegetable during storage are reflected by respiration intensity [24], while weight loss—driven by respiration, transpiration, and microbial activity—serves as a key indicator of freshness, moisture retention, and shelf life [25]. Experimental findings indicate that the synergistic application of 30 kV m⁻¹ HVEF and refrigeration markedly prolongs shelf life by mitigating weight loss and respiration rate, while enhancing colorimetric attributes and textural hardness without adversely affecting organoleptic properties. The proposed mechanism involves the generation of a high concentration of negative ions during storage, which suppresses metabolic enzymatic activity and sustains a reduced respiration rate, thereby decelerating quality degradation [26]; this phenomenon is consistent with prior observations in HVEF treatment of Brassica oleracea var. acephala, where HVEF exposure diminishes respiratory activity [22].

4.2. Energy Metabolism as One of the Core Mechanisms of HVEF in the Preservation of A.bisporus

The primary benefits of HVEF corona discharge stem from its ionization of molecules in ambient air, generating reactive species such as ozone, negative air ions, and free radicals. These active components exert their effects through multiple mechanisms: they inhibit or eradicate surface pathogenic microorganisms on fruits and vegetables; concurrently, they decrease the stomatal aperture of epidermal cells and modulate carbohydrate metabolic pathways [23]. Compared to previous studies that primarily focused on microbial suppression and surface quality maintenance, this work delves deeper into the metabolic underpinnings of HVEF’s efficacy. Specifically, we found that the applied HVEF not only influences overall metabolic responses but also markedly suppresses ATP degradation in A. bisporus. Notably, as studies of Ko et al. [27] shown that the electric field applied to biological tissues not only influences overall metabolic responses but also markedly suppresses ATP degradation, thereby positively impacting processes such as delaying ATP breakdown, protein denaturation, microbial proliferation, and oxidative deterioration. Furthermore, this study identifies a novel regulatory role of HVEF in energy metabolism: it effectively interrupts the acceleration of respiratory rate by reducing ATP synthesis and inhibiting the activity of glucose-6-phosphate dehydrogenase (G6PD), thereby disrupting energy metabolism pathways associated with respiratory enhancement. This mechanistic insight offers a more comprehensive view of HVEF’s multifaceted preservation capabilities.

4.3. Antioxidants as One of the Core Mechanisms of HVEF in the Preservation of A. bisporus

Antioxidant capacity refers to the ability of antioxidant molecules or composites to eliminate or neutralize chemical free radicals present in the environment [28]. Reactive oxygen species (ROS), as critical intracellular signaling molecules, participate in regulating signal transduction cascades and play essential roles in various physiological processes such as cell development and differentiation. However, in cellular redox homeostasis, an imbalance characterized by ROS levels exceeding antioxidant defenses (ROS > antioxidants) results in oxidative damage to lipids, proteins, and DNA; conversely, a balanced state (ROS = antioxidants) maintains normal cell differentiation, growth, and overall homeostasis. Notably, an excessive shift toward antioxidant dominance (ROS < antioxidants) may suppress cellular proliferation and immune responses, indicating that the dynamic regulation of redox balance is crucial. Certain phytochemicals function as biological antioxidants, thereby supporting the organism’s defense mechanisms against reactive oxygen species (ROS) [29]. Existing research corroborates that application of high-voltage electrostatic fields (HVEF) can augment the antioxidant capacity in horticultural commodities. For instance, cherry tomatoes exposed to HVEF demonstrate increased textural firmness, elevated AsA concentration, and enhanced antioxidant activity, concurrently exhibiting decreased microbial contamination and improved microstructural integrity [15]. Further investigations indicate that HVEF induces biophysical effects such as membrane perforation in plant cells, thereby stimulating the biosynthesis of secondary metabolites—including AsA, carotenoids, and phenolic compounds—and consequently enhancing plant resilience to abiotic stresses (e.g., oxidative damage) and biotic stresses. This mechanism indirectly fortifies the antioxidant defense system [15]. In this study, HVEF treatment directly increased levels of AsA and GSH, while significantly decreasing MDA content and membrane relative electrical conductivity, effectively maintaining cell membrane integrity. These results are consistent with prior HVEF-based interventions, which reduce MDA levels and boost antioxidant enzyme activities through a physical mechanism—disruption of membrane lipid structures—thereby decreasing electrolyte leakage caused by increased membrane permeability and delaying cellular senescence.

4.4. Delaying Browning as One of the Core Mechanisms of HVEF in the Preservation of A. bisporus

Browning is one of the central issues affecting post-harvest quality deterioration in A. bisporus, primarily triggered by phenolic oxidation mediated by PPO and POD [30]. Existing studies on HVEF treatment highlight its multi-dimensional mechanisms in delaying browning in fruits and vegetables. For example, in fresh-cut potatoes, HVEF has been shown to suppress the activities of PPO, POD, and tyrosinase, while concurrently reducing the levels of total phenols, tyrosine, and H₂O₂ during storage. This dual-action strategy—targeting both enzymatic activity and substrate availability, effectively interrupts the enzymatic browning pathway at critical steps [31]. Notably, this study reveals that the preservative effects of HVEF in mushrooms are primarily attributed to its direct suppression of PPO and POD enzymatic activities. This targeted intervention disrupts the phenolic oxidation cascade by inhibiting the key enzymatic steps, thereby reducing melanin formation and significantly delaying browning.

4.5. Cold Resistance as One of the Core Mechanisms of HVEF in the Preservation of A. bisporus

Cold storage of A. bisporus, such as storage at 0 °C, is a critical measure for extending its shelf life; however, low-temperature stress may induce cellular damage, including membrane lipid peroxidation and other physiological abnormalities [8]. Amino acids serve as precursor molecules for the biosynthesis of various bioactive compounds in eukaryotic organisms, potentially influencing stress resistance through mechanisms such as elevating intracellular metabolite concentrations and modulating secondary metabolite synthesis. In plants, osmoprotectants such as proline and glycine serve as key compatible solutes that typically accumulate significantly under abiotic stresses like extreme temperatures, playing a central role in mitigating adverse effects. Notably, proline exhibits pronounced protective functions: it stabilizes proteins and cellular membranes, scavenges free radicals, and acts as an osmolyte to maintain cellular osmotic balance, thereby sustaining turgor pressure and ensuring normal growth and cellular function under stress conditions [32]. In response to low-temperature stress, the expression of glutamine synthetase (GS) is also of considerable importance. GS catalyzes the synthesis of glutamine from glutamate and ammonium ions; its activity serves as a vital physiological indicator of plant cold tolerance, directly reflecting the degree of cold resistance [33]. Furthermore, the type I antifreeze proteins (AFPs), characterized by high alanine content (over 60% alanine in the sequence), protect cold-blooded organisms from freezing damage by inhibiting ice crystal growth, further underscoring alanine’s critical role in cold adaptation [34]. The leucine-rich repeat (LRR) protein family, extensively involved in fundamental plant metabolism and signal transduction, also functions in abiotic stress responses [35]. Proteins containing valine-glutamine (VQ) motifs act as transcriptional co-regulators, with their unique molecular features facilitating a bridging role between plant stress resistance, immune responses, and growth and development [36]. This study demonstrates that HVEF treatment directly activates the metabolic pathways of osmoprotectants (e.g., proline, glutamine, leucine, isoleucine, alanine, and valine) in A. bisporus, thereby mitigating membrane damage caused by low-temperature stress and significantly enhancing the cold adaptation capacity of mushroom cells. While previous research has shown that HVEF improves cold resistance in maize seeds [37], the current findings reveal a distinct pathway in A. bisporus: the direct modulation of osmoprotectant metabolism. This mechanistic divergence highlights the adaptability of HVEF in inducing stress tolerance across diverse biological systems, with the present study specifically uncovering its role in fine-tuning osmotic balance through targeted metabolic activation, underscoring a novel mechanism of HVEF in cold adaptation.

Current preservation techniques for A. bisporus predominantly rely on chemical treatments and low-temperature storage. In contrast, the HVEF treatment employed in this study leverages a multi-faceted synergistic mechanism—encompassing respiratory metabolism regulation, antioxidant activity enhancement, browning inhibition, and cold resistance improvement, offering a novel approach for comprehensive postharvest preservation.

5. Conclusions

This study confirmed that 30 kV m⁻¹ HVEF combined with cold storage effectively delays weight loss and respiration in A. bisporus and preserves epidermal and flesh color, hardness, and flavor, offering a viable physical postharvest preservation strategy. Subsequently, Transcriptomic analysis demonstrated that 30 kV m⁻¹ HVEF treatment modulates key metabolic pathways, including energy metabolism, fatty acid oxidation, redox enzyme activity, and stress response mechanisms. The treatment effectively inhibits ATP synthesis and G6PD activity, reducing respiratory rate, while simultaneously enhancing antioxidant compounds such as AsA and glutathione. This dual action decreases membrane lipid peroxidation and permeability, preserving cellular membrane integrity. Additionally, HVEF suppresses phenolic oxidation mediated by PPO and POD, thereby delaying enzymatic browning. The improved cold resistance is closely linked to the activation of osmoprotectants like proline, which mitigates membrane damage under low-temperature stress, collectively contributing to delayed senescence. This study elucidates HVEF’s regulatory effects on A. bisporus’s physiological and biochemical indices and reveals its preservation mechanism via molecular pathways, providing a scientific basis for high-voltage electrostatic field technology. The results support extending edible fungi shelf life and reducing postharvest losses. Future work should optimize HVEF parameters and evaluate its applicability across mushroom varieties for large-scale cold chain application.