{"paper_id":"03be772d-3ca2-4c4a-aea8-921af74503e1","body_text":"Impact of slightly acidic electrolyzed water in combination with ultrasound on safety and quality of Fresh-cut Agaricus bisporus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Impact of slightly acidic electrolyzed water in combination with ultrasound on safety and quality of Fresh-cut Agaricus bisporus Limei Wang, wuriliga Bai, Yong Lian, Rui Shi, Yuanyuan Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9230235/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Fresh-cut Agaricus bisporus is highly susceptible to browning and microbial spoilage during refrigerated storage. This study evaluated slightly acidic electrolyzed water (SAEW) and ultrasound (US), applied individually and in combination, as green washing–preservation strategies for fresh-cut mushrooms. Sliced samples were treated with distilled water (CK), SAEW (70 mg/L available chlorine), US (100 W, 40 kHz), or combined SAEWUS, packaged in PA/PE bags, and stored at 4°C for 10 days. Quality changes were assessed by color parameters, browning-related enzyme activities (PPO, POD), lipid oxidation (MDA), antioxidant components and capacities, and microbial counts. In addition, microbial community succession in CK and SAEWUS samples was analyzed using 16S rRNA sequencing. Compared with single treatments, SAEWUS most effectively delayed browning, maintained lower a * values, inhibited PPO and POD activities, and reduced oxidative damage. The combined treatment also better preserved antioxidant levels and achieved a greater reduction in aerobic plate counts during storage. Microbial profiling further indicated that SAEWUS moderated spoilage-associated microbial succession and improved community stability. Overall, the synergistic application of SAEW and ultrasound represents an effective, residue-free approach to enhance quality retention and extend the refrigerated shelf life of fresh-cut mushrooms. Slightly Acidic Electrolyzed Water Ultrasound Fresh-cut Microbial decontamination Agaricus bisporus Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Fresh-cut edible fungi, particularly Agaricus bisporus , have gained significant popularity in the ready-to-eat and pre-prepared food markets in recent years due to their low calories, high protein content, abundant dietary fiber, and flavor compounds (Kalac, 2016 ; Roupas et al., 2012 ). However, Agaricus bisporus is inherently fragile and has a high moisture content, which makes it prone to cellular structural damage during fresh-cut processing (Jin et al., 2025 ; Saltveit, 2000 ; Xu et al., 2021 ; L. Zhang et al., 2017 ). This damage accelerates the oxidation of polyphenolic compounds, browning reactions, and microbial proliferation, significantly reducing its commercial value and shelf life (Liang et al., 2024 ; Saravanakumar et al., 2021 ). Moreover, as consumer demand shifts toward minimally processed foods with clean-label preservation methods, the lack of effective and residue-free sanitation strategies for fresh-cut mushrooms has become a critical bottleneck in scaling up industrial production and cold-chain distribution (Parvathy Nayana et al., 2025 ). Therefore, developing a green, safe, and effective cleaning and preservation system to slow down quality deterioration during low-temperature storage is one of the key technical challenges in the commercialization and industrialization of fresh-cut edible fungi. Various physical and chemical disinfection techniques have been explored to address these challenges, including pulsed electric field, irradiation, high-pressure processing, ultraviolet radiation, ozone treatment, and cold plasma techniques (Onwude et al., 2017 ). Among these, slightly acidic electrolyzed water (SAEW) stands out as a widely used preservation technology for fresh-cut foods, such as fruits, vegetables, and seafood, due to its broad-spectrum antimicrobial properties, ease of preparation, and low toxic residue (Huang et al., 2024 ; Issa-Zacharia, 2023 ; C. Zhang et al., 2016 ). SAEW is typically generated through the electrolysis of dilute hydrochloric acid or sodium chloride and has moderate pH (5.0–6.5), suitable chlorine concentration, and a high oxidation-reduction potential (Akther et al., 2023 ). These properties allow SAEW to achieve non-thermal sterilization by disrupting microbial cell membranes and interfering with their enzyme systems. Compared with traditional chemical disinfectants such as sodium hypochlorite, SAEW offers a milder antimicrobial mechanism with minimal impact on the nutritional and sensory qualities of fresh-cut produce (Du et al., 2024 ). Previous studies have shown that SAEW effectively reduces microbial load and delays respiration rate and browning of fresh-cut carrots, strawberries, apples, and other fruits and vegetables (Ding et al., 2015 ; Koide et al., 2011 ; Tango et al., 2017 ). However, when processing fresh-cut Agaricus bisporus , which has a loose structure and high polyphenolic oxidation activity, the proteins and phenolic compounds exuded from the cut surfaces may react with the chlorine in SAEW, leading to reduced sterilization efficiency(Gil et al., 2009 ). Additionally, SAEW is sensitive to temperature and storage conditions, which can result in decreased stability. More importantly, SAEW has limited ability to suppress enzymatic browning, thus making it insufficient as a standalone strategy for controlling browning reactions in mushrooms. Ultrasound (US), as a typical non-thermal physical preservation technique, has shown great potential in food cleaning, sterilization, and structural regulation in recent years (Chemat et al., 2011; Fan et al., 2021). The core mechanism of US is the cavitation effect, which generates microjets and high-pressure pulses that enhance the contact efficiency between the treatment liquid and the food surface, disrupt microbial biofilms, and promote the diffusion and penetration of the treatment liquid into the food tissue, thereby improving overall cleaning and sterilization effectiveness (Irazoqui et al., 2019 ; Song et al., 2024 ). Previous studies have demonstrated that US can effectively increase the microbial removal rate in fresh-cut fruits and vegetables, slow down the respiration rate, delay browning, and prevent texture deterioration, thus extending shelf life (Jiang et al., 2025 ; J. Wang et al., 2024 ). However, the preservation effect of US is influenced by processing parameters and food matrix characteristics, and improper control may cause structural damage, moisture loss, and color deterioration (Khaire et al., 2022 ); these adverse effects may be even more pronounced when processing products such as fresh-cut Agaricus bisporus , which have fragile tissues and are highly susceptible to browning. Furthermore, although numerous studies have investigated ultrasound-assisted washing in fruits and vegetables, its mechanistic interaction with mushroom-specific physiology—high porosity, rapid enzymatic oxidation, and weak tissue coherence—remains largely unexplored, leaving a significant knowledge gap. Moreover, the preservation mechanism of US in this type of high-moisture, delicate food still lacks systematic research. Therefore, this study aims to investigate the preservation effect of combined US and SAEW treatment on fresh-cut Agaricus bisporus . We propose a composite preservation strategy that combines the chemical antimicrobial mechanism of SAEW and the physical enhancement function of US, applied to the storage system of fresh-cut Agaricus bisporus , which is prone to browning, has high moisture content, and is structurally sensitive. By sampling at multiple time points, we systematically evaluate the effects of different treatments on the sensory quality, physicochemical properties, and microbial stability of Agaricus bisporus during storage. The results of this study will provide technical support for the commercialization and cold-chain circulation of fresh-cut edible fungi products and offer theoretical and practical guidance for the efficient integration of green sterilization and preservation technologies in perishable food applications. 2. Materials and Methods 2.1. Sample collection and pretreatment Fresh Agaricus bisporus samples were purchased from Meitong Market (Hohhot, China). After removing visible soil and debris, mushrooms with uniform color and appearance, free from mechanical damage or microbial infection, were selected for the experiments. The samples were sliced into pieces of approximately 0.5-1.0 cm thickness and randomly divided into four groups, each subjected to a different washing treatment: (i) the control group (CK) was immersed in distilled water for 5 min; (ii) the slightly acidic electrolyzed water group (SAEW) was washed with SAEW containing 70 mg/L available chlorine for 5 min; (iii) the ultrasound group (US) was treated at 100 W and 40 kHz for 5 min; and (iv) the combined treatment group (SAEWUS) underwent ultrasonic treatment (100 W, 40 kHz, 5 min) followed by washing with SAEW (70 mg/L, 5 min). All treatments were conducted at 5°C to minimize tissue metabolism and prevent heat-induced denaturation. Although ultrasound was applied prior to SAEW washing, the cavitation-induced microstructural loosening and removal of surface barriers may enhance the subsequent accessibility and efficacy of active chlorine species during SAEW treatment. After treatment, samples were air-dried in a sterile environment under laminar airflow until no visible surface moisture remained. Approximately 80 g of mushrooms were placed in PA/PE composite bags (non-vacuum sealed) and stored at 4°C for subsequent analyses. 2.2. Physicochemical, antioxidant, enzymatic, and microbiological analyses 2.2.1 Color measurement Color parameters of fresh-cut Agaricus bisporus were measured using a colorimeter (CR-20, Konica Minolta Co., Japan). The CIE Lab* color system was used, where L* indicates lightness, a* denotes the red–green axis, and b* denotes the yellow–blue axis. 2.2.2 Ascorbic acid and Total phenolic content Ascorbic acid content was determined by titration with 2,6-dichloroindophenol (DCIP) solution and expressed as mg per 100 g fresh weight (mg/100 g FW). Total phenolic content was determined using the Folin–Ciocalteu colorimetric method and expressed as grams of gallic acid equivalents per kilogram fresh weight (mg·g − 1 FW). 2.2.3 Antioxidant capacity (DPPH, Superoxide, and Hydroxyl radicals) DPPH radical scavenging activity was determined using a DPPH–ethanol solution. Extracts were mixed with 0.1 mmol/L DPPH and incubated for 30 min at room temperature, and absorbance was measured at 517 nm. Scavenging activity was calculated using Eq. (1): $$\\:\\begin{array}{c}DPPH\\:free\\:radical\\:clearance\\:\\left(\\%\\right)=\\frac{{A}_{0}-{A}_{S}}{{A}_{0}}\\times\\:100\\#\\left(1\\right)\\end{array}$$ Where \\(\\:{A}_{0}\\) and \\(\\:{A}_{S}\\) represent the absorbance of the control and the sample, respectively. Superoxide anion radical clearance was determined using a nitroblue tetrazolium (NBT) reduction system, and absorbance was measured at 320 nm. Results were expressed as inhibition percentage. Hydroxyl radical scavenging rate was determined using a Fenton reaction system by measuring absorbance at 510 nm, and results were expressed as percentage inhibition of hydroxyl radicals. 2.2.4 Malondialdehyde (MDA) content MDA content was determined using the thiobarbituric acid (TBA) colorimetric method. Absorbance was measured at 450, 532, and 600 nm, and MDA content was calculated according to Eq. (2): $$\\:\\begin{array}{c}MDA\\left(nmol·{g}^{-1}\\:FW\\right)=\\frac{\\left[6.45\\times\\:\\left({A}_{532}-{A}_{600}\\right)-0.56\\times\\:{A}_{450}\\right]\\times\\:V}{{V}_{S}\\times\\:m}\\#\\left(2\\right)\\end{array}$$ Where \\(\\:V\\) is the total reaction volume (mL); \\(\\:{V}_{S}\\) is the volume of sample extract used (mL); and \\(\\:m\\) is the mass of the mushroom sample (g). 2.2.5 Enzyme activities (PPO and POD) Polyphenol oxidase (PPO) and peroxidase (POD) activities were determined using colorimetric methods. Extracts were prepared in phosphate buffer containing 0.5% (w/v) PVP. PPO activity was assayed using catechol by measuring the increase in absorbance at 410 nm, while POD activity was assayed using guaiacol and H 2 O 2 at 470 nm. Activities were expressed as the change in absorbance per minute per gram fresh weight (min − 1 ·g − 1 FW). 2.2.6 Soluble protein and Soluble sugar Soluble protein content was determined using the Bradford method, and absorbance was measured at 595 nm. Results were expressed as mg·g − 1 FW. Soluble sugar content was determined using the anthrone colorimetric method, and absorbance was measured at 485 nm. Results were expressed as percentage of fresh weight (% FW). 2.2.7 Aerobic plate count Aerobic plate count was determined using the plate count method. Serial dilutions were plated on plate count agar and incubated at 37°C for 48 h. Results were expressed as logarithmic colony-forming units per gram (lg CFU/g). 2.2.8 Microbial community analysis CK and SAEWUS groups were selected for microbial profiling to represent the most contrasting quality deterioration trajectories. To characterize microbial succession during storage and to compare the microbial dynamics under contrasting quality trajectories, microbial diversity analysis was performed for CK group and SAEWUS group samples at days 0, 2, 5, and 8. The V4 region of the bacterial 16S rRNA gene was amplified using the universal primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT). PCR products were purified, quantified, and used to construct sequencing libraries, which were sequenced on the Illumina MiSeq platform (Novogene Co., Beijing, China). Sequencing raw reads were subjected to standard preprocessing, including barcode removal, primer trimming, and paired-end merging, before entering the downstream bioinformatics workflow. Chloroplast and mitochondrial sequences were retained for relative abundance analysis to reflect overall sequencing composition but were not interpreted as active bacterial taxa. 2.3 Data processing Unless otherwise specified, all physicochemical measurements were performed in triplicate. Data visualization was conducted using Origin 2021, and statistical tests were performed using SPSS 26. One-way ANOVA followed by Duncan’s multiple range test was used to determine significant differences among treatments (p < 0.05). For microbial community analysis, raw 16S sequencing reads were processed using QIIME 1.9.1, including quality filtering, read merging, chimera removal, OTU clustering at 97% similarity, taxonomic annotation against the SILVA database, and calculation of α- and β-diversity indices. 3. Results and Discussion 3.1 Effects of Different Treatments on Browning-Related Parameters of Fresh-Cut Agaricus bisporus Browning development in fresh-cut Agaricus bisporus was strongly influenced by the pretreatments, as reflected by changes in color attributes and biochemical indicators (Fig. 1 ; Fig. S1 ). The a* value increased continuously during storage, with the CK group already showing the highest values among all treatments from day 2 onward and reaching 8.37 on day 10, indicating rapid red–brown discoloration (Fig. 1 A). In contrast, the SAEW and US groups moderated the increase in a* value, while the SAEWUS group exhibited the strongest inhibition, maintaining significantly lower values such as 4.65 on day 4 and 5.39 on day 8 ( p < 0.05). This behavior suggests that the combined treatment more effectively delayed red–brown discoloration, and this effect is likely due to the synergistic action of SAEW-mediated oxidative inactivation of surface microorganisms and ultrasound-induced cavitation, which improves mass transfer at the tissue–solution interface and facilitates the removal or dilution of browning precursors. Changes in L* and b* further supported these findings. L* decreased in all samples during storage (Fig. S1 A), with the CK group showing the most rapid decline, from 72.09 on day 0 to 51.25 on day 10. The SAEW and SAEWUS groups effectively preserved L* , with the SAEWUS group maintaining significantly higher L* values ( p < 0.05), including 64.53 on day 6. Conversely, b* values increased throughout storage (Fig. S1 B), reflecting progressive yellow–brown pigment formation. The CK group exhibited the steepest rise, reaching 16.39 on day 6, whereas the SAEWUS group consistently maintained significantly lower b* values after day 4 ( p < 0.05), including 14.46 on day 10. These improvements in L* and b* indicate reduced enzymatic oxidation of phenolic substrates and improved preservation of cut-surface tissue integrity under the SAEWUS group. The enzymatic indicators PPO and POD showed trends consistent with the color changes (Fig. 1 B,C). The CK group maintained the significantly highest activities of both PPO and POD throughout storage, reaching peak values of 15.59 min − 1 ·g − 1 FW and 0.040 min − 1 ·g − 1 FW on day 4 and day 10, respectively, thereby accelerating enzymatic browning. The SAEW and US groups reduced these enzyme activities to varying extents, whereas the SAEWUS group achieved the strongest inhibition, with PPO and POD activities of 9.43 min − 1 ·g − 1 FW and 0.018 min − 1 ·g − 1 FW at the corresponding time points. Ultrasound-induced cavitation may transiently disrupt enzyme conformation and reduce catalytic efficiency, while the SAEW group can oxidize key sulfhydryl or amino groups required for enzyme activity. The combined treatment likely amplifies these effects by enhancing the SAEW group penetration into surface tissues through ultrasound-enhanced microstreaming and localized mechanical disruption. MDA content increased steadily in all groups (Fig. 1 D), with the CK group showing the fastest accumulation and reaching 1.82 nmol·g − 1 FW on day 10. All pretreatments slowed the rise in MDA, and the SAEWUS group consistently maintained the lowest levels, such as 0.99 nmol·g − 1 FW on day 2 and 1.57 nmol·g − 1 FW on day 10, indicating reduced oxidative stress and delayed tissue deterioration. Overall, the SAEWUS group most effectively delayed browning by mitigating color deterioration ( a* , L* , b* ), inhibiting PPO and POD activities, and reducing membrane lipid peroxidation. These synergistic benefits likely result from the combined antimicrobial, oxidative, and cavitation-induced effects of the SAEW and US, which collectively limit browning substrates and weaken the enzymatic drivers of discoloration. To further elucidate the mechanistic basis underlying these macroscopic quality changes, beyond the observed suppression of PPO and POD activities, the inhibitory effects of the combined SAEWUS treatment can be further interpreted from a structural and reaction-level perspective. PPO and POD are copper-containing oxidoreductases whose catalytic activity strongly depends on the integrity of metal-binding sites and the redox state of key amino acid residues, such as histidine, cysteine, and tyrosine (Liang et al., 2024 ; Xu et al., 2021 ). Slightly acidic electrolyzed water, characterized by a high oxidation-reduction potential and the presence of active chlorine species, may oxidize sulfhydryl and imidazole groups located near the catalytic center, thereby reducing enzyme–substrate affinity and catalytic efficiency (Gil et al., 2009 ; Issa-Zacharia, 2023 ). Meanwhile, ultrasound treatment generates localized cavitation, microstreaming, and transient pressure fluctuations, which can induce reversible conformational loosening or partial unfolding of enzyme structures, further limiting enzymatic accessibility to phenolic substrates (Khaire et al., 2022 ). When combined, ultrasound is likely to enhance the interaction between SAEW and enzyme molecules at the cut surface by reducing boundary layer resistance and promoting mass transfer, resulting in more efficient enzyme inactivation than either treatment alone. This complementary action at both the chemical and physical levels provides a plausible explanation for the markedly lower PPO and POD activities and the reduced accumulation of lipid peroxidation products observed under SAEWUS treatment. 3.2 Effects of Different Treatments on Antioxidant Capacity and Redox-Related Physicochemical Attributes of Fresh-Cut Agaricus bisporus As shown in Fig. 2 , non-enzymatic antioxidants and redox-related parameters exhibited distinct, treatment-dependent temporal patterns during cold storage, indicating coordinated yet heterogeneous redox responses among treatments and reflecting the differential effectiveness of the pretreatments in mitigating oxidative deterioration. The superior preservation of non-enzymatic antioxidants under the SAEWUS treatment also reflects a more stable intracellular redox environment during storage. Ascorbic acid and phenolic compounds serve as primary redox buffers in mushroom tissues and are rapidly depleted under excessive oxidative stress (Hu et al., 2022 ; Saltveit, 2000 ; Xu et al., 2021 ). In untreated samples, accelerated membrane lipid peroxidation and enzymatic oxidation likely increased reactive oxygen species (ROS) accumulation, resulting in continuous consumption of antioxidant pools. In contrast, the combined SAEWUS treatment effectively mitigated oxidative pressure by simultaneously suppressing enzymatic browning reactions and limiting microbial proliferation, both of which are major sources of ROS generation in fresh-cut mushrooms (Gil et al., 2009 ). Ultrasound-enhanced mass transfer may further facilitate the removal of oxidized metabolites from the tissue surface, while SAEW reduces microbial-derived oxidative stress at the cut interface. Together, these effects contribute to a more balanced redox state, thereby slowing antioxidant depletion and maintaining higher radical-scavenging capacity throughout storage. Ascorbic acid content followed a characteristic “sharp decrease–partial recovery–final decline” trajectory during storage (Fig. 2 A). A rapid decrease occurred by day 2 due to intense oxidative consumption immediately after cutting, with the CK group dropping to 37.32 mg·100g − 1 FW, whereas the US and SAEWUS groups retained significantly higher values 40.77 and 44.06 mg·100g − 1 FW ( p < 0.05). From day 2 to day 6, a partial rebound was observed, most prominently under the SAEWUS group, which maintained the highest levels with 44.18 mg·100g − 1 FW on day 4 and 45.59 mg·100g − 1 FW on day 6, followed by the US and SAEW groups, while the CK group remained substantially lower throughout this period. This temporary recovery likely reflects transient metabolic adjustment and reduced oxidative pressure under the pretreatments. After day 6, ascorbic acid declined again in all treatments, with CK group showing the significantly fastest depletion and decreasing to 24.52 mg·100g − 1 FW on day 10 ( p < 0.05). The SAEW, US and particularly SAEWUS groups significantly slowed this late-stage loss, with the SAEWUS group consistently retaining the highest ascorbic acid levels up to 30.58 mg·100g − 1 FW, indicating strong protection against oxidative degradation. Total phenolic content displayed a different temporal pattern, characterized by rapid early accumulation followed by continuous depletion (Fig. 2 B). On day 2, total phenols increased significantly as part of the wound response, with the US group showing the significantly highest level at 6.58 mg·g − 1 FW ( p < 0.05). This value was significantly higher than those of the CK group (5.56 mg·g − 1 FW) and the SAEW group (4.93 mg·g − 1 FW), while the SAEWUS group showed an intermediate increase to 5.86 mg·g − 1 FW. After day 2, phenolic content declined steadily in all groups, reflecting progressive consumption as substrates for enzymatic and non-enzymatic oxidation. The CK group displayed the fastest depletion, whereas all pretreatments slowed this decline. By day 6 and day 10, the SAEWUS group maintained the significantly highest phenolic levels, recorded at 4.45 mg·g − 1 FW and 3.39 mg·g − 1 FW ( p < 0.05), respectively. This pattern is consistent with the inhibition of PPO/POD-mediated phenolic oxidation shown in Fig. 1 , indicating more effective retention of phenolic antioxidants under the SAEWUS group. DPPH scavenging capacity exhibited a clear pattern of early stimulation followed by continuous decline (Fig. 2 C). By day 2, cutting stress induced a short-lived elevation in scavenging capacity. The CK group rose to 14.92%, while the pretreatment groups reached values between 14.01% and 14.89%, with no significant differences among treatments at this stage ( p > 0.05), suggesting enhanced release or activation of readily extractable antioxidants. After this initial increase, DPPH clearance decreased progressively in all treatments. The CK group declined most rapidly, falling to 5.55% on day 4 and further decreasing to 2.59% by day 10. The pretreatments slowed this reduction, with the SAEWUS group consistently maintaining the highest scavenging capacity, reaching 8.83% on day 4 and 3.75% on day 10 ( p < 0.05). This correspondence between metabolite retention and functional scavenging supports the interpretation that SAEWUS stabilized the low-molecular-weight antioxidant pool. Superoxide anion scavenging capacity showed a clear fluctuation over storage time (Fig. 2 D). A slight decline occurred by day 2 across all groups, with no significant differences ( p > 0.05), but all treatments exhibited a pronounced rebound on day 4. At this point, CK group increased to 60.86%, whereas the SAEW, US, and SAEWUS groups reached significantly higher values ( p < 0.05) of 65.87%, 65.04%, and 67.34%, respectively, indicating that the pretreatments enhanced the superoxide-scavenging response more effectively than CK group. After day 4, superoxide clearance gradually decreased in all groups, with CK group consistently maintaining the lowest activity throughout the remainder of storage. The SAEWUS group showed the strongest preservation effect, maintaining the highest capacities ( p < 0.05) at multiple stages (day 6, 8, and 10), further confirming its superior support of the enzymatic superoxide-detoxification system. Hydroxyl radical scavenging showed a transient early rise followed by a sharp decline and partial late-stage stabilization (Fig. 2 E). By day 2, the US and SAEWUS groups exhibited a notable increase with 42.24% and 51.61%, significantly higher ( p < 0.05) than the CK and SAEW groups at 33.33% and 37.09%, likely reflecting ultrasound-induced release of bound antioxidants or activation of redox enzymes. After day 2, scavenging capacity decreased rapidly across all treatments. CK group showed the lowest values throughout mid-storage, with 17.84% at day 4 and 9.84% at day 6. SAEW and US groups maintained moderately higher levels, whereas the SAEWUS group demonstrated higher or comparable values at most time points. By day 10, pretreatments again showed an advantage, with values of 9.29%, 9.01%, and 10.66%, whereas CK group dropped to 6.01% ( p < 0.05), confirming that SAEWUS more effectively mitigated oxidative intensification and preserved hydroxyl-radical-scavenging activity. Soluble protein content followed a clear “early increase then continuous decline” pattern (Fig. 2 F). By day 2, the pretreatment groups showed a marked increase in soluble protein (1.48–1.58 mg·g − 1 FW), whereas the CK group exhibited a slight decrease to 1.35 mg·g − 1 FW. This divergence suggests that without protective pretreatment, the CK group samples experienced more rapid membrane injury, reducing the pool of extractable cytosolic proteins, while the increases observed in the treated groups may be associated with altered extractability of cytosolic proteins after cutting and pretreatment and increased extractability of cytosolic proteins immediately after cutting. After day 2, soluble protein declined steadily in all treatments. The CK group decreased most sharply, reaching 1.06 mg·g − 1 FW by day 4 and falling to 0.47 mg·g − 1 FW by day 10 ( p < 0.05). The SAEW and US groups slowed this decline, maintaining intermediate levels of 0.60–0.69 mg·g − 1 FW, while the SAEWUS group consistently retained the highest protein content throughout storage, maintaining 1.54 mg·g − 1 FW on day 4 and 0.77 mg·g − 1 FW on day 10. These protein-retention outcomes align with the reduced MDA accumulation reported in Fig. 1 , further confirming that SAEWUS effectively suppresses oxidative denaturation and preserves cellular integrity. Overall, the SAEWUS group most effectively stabilized the antioxidant system by maintaining higher levels of ascorbic acid and phenolics, enhancing free-radical scavenging capacity (DPPH, O 2 − ·, ·OH) and slowing oxidation-related protein degradation. Collectively, this treatment imposed the most comprehensive redox-stabilizing effect during storage, consistent with its superior inhibition of browning and oxidative injury reported in Fig. 1 . In this context, the synergistic effect observed in the SAEWUS treatment should not be regarded as a simple additive outcome of two preservation methods. SAEW and ultrasound act at distinct yet complementary levels: SAEW primarily exerts chemical antimicrobial and oxidative modulation effects, whereas ultrasound functions as a physical enhancer by intensifying liquid-solid interactions and weakening diffusion barriers at the tissue surface (Irazoqui et al., 2019 ; Song et al., 2024 ). Through cavitation-induced microstreaming and transient microstructural disturbance, ultrasound likely increases the accessibility of active chlorine species to microbial cells, enzymes, and oxidation-sensitive substrates. This parallel rather than hierarchical interaction allows each treatment to maintain its intrinsic preservation function while amplifying overall efficacy, explaining why the combined treatment consistently outperformed the single treatments across multiple quality attributes (Y. Wang et al., 2025 ). 3.3 Effects of Different Treatments on Soluble Sugars and Microbial Quality of Fresh-Cut Agaricus bisporus Soluble sugar content and aerobic bacterial counts displayed tightly coupled, treatment-dependent trends during storage, reflecting the interplay between endogenous carbohydrate metabolism and microbial activity. Soluble sugars declined sharply by day 2 across all groups, with the CK group showing the greatest decrease to 0.96%, whereas the SAEW, US and SAEWUS groups maintained slightly higher levels between 0.97% and 0.99% (Fig. 3 A). From day 2 to day 6, a partial recovery occurred, most evident in the SAEWUS group, which increased to 1.08% on day 4 and 1.12% on day 6, while the US and SAEW groups exhibited moderate rebounds and CK group remained consistently lower. During late storage, soluble sugars declined again. CK group showed the steepest reduction, falling to 0.87% by day 10 ( p < 0.05), whereas the pretreatments slowed this loss and the SAEWUS group retained the highest residual sugar content at 1.03%. These sugar-preservation outcomes suggest that the SAEWUS group may reduce carbohydrate depletion by mitigating both physiological consumption and microbial utilization. Aerobic bacterial growth accelerated progressively during storage but at markedly different rates among treatments (Fig. 3 B). The CK group exhibited the fastest proliferation, rising to 3.02 lg CFU/g by day 2 and reaching 6.36 lg CFU/g by day 10, values that were significantly higher ( p < 0.05) than those of all pretreatment groups at the corresponding time points. In contrast, the SAEW, US and SAEWUS groups suppressed microbial growth throughout storage, maintaining significantly lower ( p < 0.05) counts of 5.03–5.18 lg CFU/g at the end of the period. Among the pretreatments, the SAEWUS group consistently achieved the strongest inhibition, consistent with its combined antimicrobial effect of slightly acidic electrolyzed water and the cavitation-enhanced disruption of microbial cells and attachment structures. Overall, the coordinated preservation of soluble sugars and suppression of aerobic bacteria demonstrates that the SAEWUS group most effectively maintained metabolic stability and microbial quality of fresh-cut Agaricus bisporus during cold storage. 3.4 Effects of Different Treatments on Microbial Diversity and Structure of Fresh-Cut Agaricus bisporus Based on taxonomic annotation, bacterial community profiles were summarized across multiple taxonomic levels, among which phylum- and genus-level compositions were emphasized due to their dominant contribution to community structure and functional relevance. Comparative analysis of community composition revealed distinct succession patterns between CK and SAEWUS samples during storage, indicating that the combined treatment reshaped microbial assembly rather than merely reducing overall microbial load. These compositional shifts provide a mechanistic explanation for the observed differences in microbial growth and sugar utilization between treatments. At the phylum level, the bacterial communities of fresh-cut Agaricus bisporus exhibited pronounced treatment- and storage-dependent shifts (Fig. 4 A). The initial microbiota (a0d) showed a relatively balanced structure, with Proteobacteria accounting for approximately one-third of the total community, accompanied by substantial proportions of Firmicutes, Bacteroidota, Cyanobacteria, and Actinobacteriota, indicating a diverse baseline community prior to storage. In the CK group, microbial succession was characterized by a progressive dominance of Proteobacteria, although the temporal pattern was not strictly monotonic. Proteobacteria increased sharply by day 2, followed by a slight relative decrease at day 5, and ultimately became overwhelmingly dominant by day 8, accounting for nearly the entire community. This late-stage enrichment coincided with a marked depletion of Firmicutes, Bacteroidota, and Cyanobacteria, reflecting a severe simplification of the microbial structure typically associated with advanced spoilage. In contrast, the SAEWUS-treated samples displayed a markedly different succession trajectory. Although Proteobacteria remained a major component, its relative abundance was consistently lower than that in the corresponding CK samples at all storage stages. Notably, the SAEWUS group exhibited a pronounced enrichment of Cyanobacteria at day 5, accompanied by a concomitant reduction in Proteobacteria, resulting in a more evenly distributed community structure. By day 8, Proteobacteria increased again but did not reach the overwhelming dominance observed in the CK group, while Firmicutes and Bacteroidota were better retained. These phylum-level differences suggest that SAEWUS treatment altered not only the abundance but also the succession pattern of dominant microbial groups, prompting further examination of key spoilage-associated genera. At the genus level, pronounced differences in microbial succession were observed between the CK and SAEWUS treatments during storage (Fig. 4 B). In the initial sample (a0d), Pseudomonas accounted for only a minor fraction of the total community, and the microbiota was dominated by a large proportion of low-abundance and unclassified taxa, indicating a relatively undisturbed baseline state. In the CK group, Pseudomonas exhibited a rapid but non-monotonic expansion. Its relative abundance increased sharply by day 2, reaching the highest level among CK samples, followed by a gradual decline at day 5 and a further reduction by day 8. This fluctuation suggests that although Pseudomonas responded rapidly to the favorable post-cutting environment, prolonged storage led to progressive community destabilization rather than sustained dominance of a single spoilage genus. Concurrently, the increase in unclassified taxa and the reduction of other identifiable genera indicate a fragmented and deterioration-prone microbial structure at the late storage stage. In contrast, the SAEWUS-treated samples showed a distinctly altered succession pattern. The relative abundance of Pseudomonas remained consistently lower than that in the corresponding CK samples at all time points. Notably, at day 5, Pseudomonas was strongly suppressed, while sequences annotated as unidentified chloroplasts and mitochondria accounted for a large proportion of the total reads, likely reflecting a marked reduction in bacterial load and a proportional enrichment of host-derived sequences rather than active microbial proliferation. By day 8, Pseudomonas abundance increased again but did not reach the peak levels observed in the CK group at earlier stages.These genus-level patterns further indicate that SAEWUS delayed the dominance of typical spoilage-associated taxa, which is expected to influence overall microbial diversity and community complexity. Alpha diversity analysis based on the Shannon index revealed pronounced differences in microbial community complexity between treatments during storage (Fig. 4 C). The initial samples (a0d) exhibited the highest Shannon values, reflecting a highly diverse baseline microbiota composed of multiple low-abundance taxa. In the CK group, microbial diversity decreased sharply after cutting, with a marked reduction in Shannon index already observed at day 2, followed by a further decline at day 5 and reaching the lowest level by day 8. This progressive loss of diversity indicates a rapid microbial succession toward a simplified, spoilage-dominated community structure under untreated conditions. In contrast, the SAEWUS-treated samples maintained significantly higher microbial diversity at the early storage stage. At day 2, the Shannon index of the SAEWUS group remained comparable to that of a0d and was substantially higher than that of the CK group, suggesting that the combined treatment effectively restrained early dominance of specific taxa and preserved community complexity. Although a temporary decrease in Shannon index was observed at day 5 in the SAEWUS group, microbial diversity recovered at day 8, showing a clear divergence from the continuously declining trend observed in CK samples. To further evaluate whether these diversity differences were accompanied by shifts in overall community structure, beta diversity analysis was performed using PCoA. Principal coordinates analysis (PCoA) based on Bray–Curtis dissimilarity revealed temporal shifts in the bacterial community structure during storage (Fig. 4 D). Samples did not form completely discrete clusters, indicating moderate rather than drastic community differentiation, which is consistent with the short storage period and non-thermal pretreatments applied. Nevertheless, a clear tendency of divergence between CK and SAEWUS samples was observed along the PC1 axis as storage progressed. CK samples exhibited a larger dispersion over time, suggesting a more pronounced restructuring of the microbial community, whereas SAEWUS-treated samples showed relatively constrained distribution, particularly at day 5 and day 8. Taken together, the beta diversity results corroborate the compositional and diversity analyses, demonstrating that SAEWUS treatment moderated microbial succession dynamics and contributed to improved microbial stability during cold storage. From a functional perspective, the moderated microbial succession observed under SAEWUS treatment is particularly relevant to quality preservation in fresh-cut mushrooms. Rather than eliminating the microbial community entirely, the combined treatment appeared to delay the rapid dominance of spoilage-associated taxa and preserve higher microbial diversity during early and mid-storage stages. Such stabilization may reduce metabolic fluctuations typically associated with advanced spoilage (Parvathy Nayana et al., 2025 ; Shi et al., 2022 ). This interpretation is consistent with the slower decline in soluble sugars and the restrained increase in aerobic plate counts observed in SAEWUS-treated samples. Therefore, the microbial modulation induced by SAEWUS contributes not only to microbial safety but also to metabolic stability, providing an additional explanation for its superior performance in maintaining overall quality. 4. Conclusion The combined SAEWUS group treatment provided the most comprehensive preservation of fresh-cut Agaricus bisporus quality during cold storage. It markedly delayed browning, maintaining an a* value of 6.19 on day 10 compared with 8.37 in the CK group, and reduced PPO and POD activities by roughly 15–45%. Lipid oxidation was also minimized, with MDA levels reaching only 1.57 nmol·g − 1 FW by day 10, lower than the 1.82 nmol·g − 1 FW measured in the CK group. Mid-storage antioxidant retention was substantially improved under the SAEWUS group, which preserved 45.59 mg·100g − 1 FW of ascorbic acid and 4.45 mg·g − 1 FW of phenolics on day 6, both clearly higher than the corresponding values in the CK group. Microbial quality followed the same trend; by day 10, aerobic counts in the SAEWUS group were approximately 1.4 log CFU/g lower than those in the CK group. These advantages highlight the complementary roles of slightly acidic electrolyzed water and ultrasound, which act at chemical and physical levels, respectively, to enhance preservation efficiency. Given its residue-free nature, operational simplicity, and compatibility with low-temperature processing, SAEWUS represents a promising green pretreatment strategy for industrial fresh-cut mushroom processing and cold-chain distribution. Declarations Author Contributions Limei Wang : Resources, Writing–original draft. Bai wuriliga : Data curation, Investigation. Yong Lian : Software, Methodology. Rui Shi : Validation. Yuanyuan Zhang : Investigation. Xiuli Liu : Methodology, Software. Mu Jia : Investigation. Tao Sun : Review, Editing & Supervision. Funding Hohhot Science and Technology Plan Project Task (2023-Agriculture-10),Science and Technology Plan of Inner Mongolia Autonomous Region (2025YFHH0275) Clinical trial number: not applicable. Ethics approval and consent to participate: not applicable. Consent for publication: not applicable. Conflicts of Interest The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper. Data Availability Statement The raw 16S rRNA gene amplicon sequencing data generated in this study have been deposited in the Genome Sequence Archive (GSA), National Genomics Data Center (NGDC), China National Center for Bioinformation (CNCB) under accession number CRA041143. The data are currently accessible via the following direct link: https://ngdc.cncb.ac.cn/gsa/s/8548vdJZ. All other data supporting the findings of this study are available within the article and its Supplementary Information files. 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Slightly acidic electrolyzed water combined with chemical and physical treatments to decontaminate bacteria on fresh fruits. Food Microbiol. 2017;67:97–105. https://doi.org/10.1016/j.fm.2017.06.007 . Wang J, Cui Y, Zhang M, Wang L, Aihaiti A, Maimaitiyiming R. Pulsed-control plasma-activated water: An emerging technology to assist ultrasound for fresh-cut produce washing. Ultrason Sonochem. 2024;102:106739. https://doi.org/10.1016/j.ultsonch.2023.106739 . Wang Y, Zhang Z, Zhang W, Zhang P, Zhang B, Zhang Z, Yang H. Synergistic Inactivation of Listeria monocytogenes and Inhibition of Softening in Blueberries by Combined Ultrasound and CaCl2 Slightly Acidic Electrolyzed Water. Food Control. 2025;111838. https://doi.org/10.1016/j.foodcont.2025.111838 . Xu D, Gu S, Zhou F, Hu W, Feng K, Chen C, Jiang A. Mechanism underlying sodium isoascorbate inhibition of browning of fresh-cut mushroom (Agaricus bisporus). Postharvest Biol Technol. 2021;173:111357. https://doi.org/10.1016/j.postharvbio.2020.111357 . Zhang C, Cao W, Hung Y-C, Li B. Disinfection effect of slightly acidic electrolyzed water on celery and cilantro. Food Control. 2016;69:147–52. https://doi.org/10.1016/j.foodcont.2016.04.039 . Zhang L, Li S, Wang A, Li J, Zong W. Mild heat treatment inhibits the browning of fresh-cut Agaricus bisporus during cold storage. LWT - Food Sci Technol. 2017;82:104–12. https://doi.org/10.1016/j.lwt.2017.04.035 . Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 19 May, 2026 Reviewers agreed at journal 15 May, 2026 Reviews received at journal 15 May, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers invited by journal 21 Apr, 2026 Editor invited by journal 16 Apr, 2026 Editor assigned by journal 15 Apr, 2026 Submission checks completed at journal 13 Apr, 2026 First submitted to journal 13 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-9230235\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":630667760,\"identity\":\"759da13b-1bef-4fba-a4d7-b02dcd390e45\",\"order_by\":0,\"name\":\"Limei Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Inner Mongolia Academy of Agricultural \\u0026 Animal Husbandry Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Limei\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":630667761,\"identity\":\"97419e55-6a09-4c5d-86bb-5e194b6b81ce\",\"order_by\":1,\"name\":\"wuriliga Bai\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Inner Mongolia Academy of Agricultural \\u0026 Animal Husbandry Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"wuriliga\",\"middleName\":\"\",\"lastName\":\"Bai\",\"suffix\":\"\"},{\"id\":630667762,\"identity\":\"ef2405d9-65c6-442c-8919-eef75a21179f\",\"order_by\":2,\"name\":\"Yong Lian\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Inner Mongolia Academy of Agricultural \\u0026 Animal Husbandry Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yong\",\"middleName\":\"\",\"lastName\":\"Lian\",\"suffix\":\"\"},{\"id\":630667764,\"identity\":\"3afc0f90-e077-4441-9fd4-74fc8aea77e3\",\"order_by\":3,\"name\":\"Rui Shi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Inner Mongolia Academy of Agricultural \\u0026 Animal Husbandry Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Rui\",\"middleName\":\"\",\"lastName\":\"Shi\",\"suffix\":\"\"},{\"id\":630667768,\"identity\":\"d460a060-6c9d-415a-9c02-23e5225ef47e\",\"order_by\":4,\"name\":\"Yuanyuan Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Inner Mongolia Academy of Agricultural \\u0026 Animal Husbandry Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yuanyuan\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":630667769,\"identity\":\"ae2f428a-0974-4109-a1c8-6c26d53d5eef\",\"order_by\":5,\"name\":\"Xiuli Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Inner Mongolia Academy of Agricultural \\u0026 Animal Husbandry Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xiuli\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"},{\"id\":630667770,\"identity\":\"65ba5822-b12f-47c1-8697-eb6352d65fdb\",\"order_by\":6,\"name\":\"Mu Jia\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Inner Mongolia Academy of Agricultural \\u0026 Animal Husbandry Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mu\",\"middleName\":\"\",\"lastName\":\"Jia\",\"suffix\":\"\"},{\"id\":630667771,\"identity\":\"2ad96ad1-1e0f-4773-93b4-da6af56ac27f\",\"order_by\":7,\"name\":\"Tao Sun\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYNACAyBmb2x8+IFI9YwNYC08h5uNJYjXAgIS6W0CPMSol5+Re/wBQ8E2eYObD9sYJBjs5HQbCGgxuJGXCHTYbcMNtxPbHhQwJBubHSCkRSLHEKSFEail3UCC4UDiNkJa5GdAtNhvuHmwTYKHGC0MNyBaEjfcYCRSi8GZN4YzgFqSZ55JBAayARF+kW/PMfjA8Oe2bd/x4w8ffqiwkyOoBQSY/yAsJUL5KBgFo2AUjALCAADZG0SQJysBMgAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Inner Mongolia Academy of Agricultural \\u0026 Animal Husbandry Sciences\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Tao\",\"middleName\":\"\",\"lastName\":\"Sun\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-03-26 06:54:00\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-9230235/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-9230235/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":108246931,\"identity\":\"997ac4d6-7527-4443-b578-70827bee076a\",\"added_by\":\"auto\",\"created_at\":\"2026-05-01 00:28:18\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":119716,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffects of different treatments on browning-related parameters of fresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e during cold storage. (A) \\u003cem\\u003ea*\\u003c/em\\u003e value; (B) polyphenol oxidase (PPO) activity; (C) peroxidase (POD) activity; (D) malondialdehyde (MDA) content. Different letters indicate significant differences among treatments (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9230235/v1/69ee363a0d2322357e6f58d8.png\"},{\"id\":108491634,\"identity\":\"c2a774fd-588c-4429-8bf4-dbaa1bda68ff\",\"added_by\":\"auto\",\"created_at\":\"2026-05-05 09:54:58\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":182976,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffects of different treatments on antioxidant components and radical scavenging activities of fresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e during cold storage.(A) Ascorbic acid (VC); (B) Total phenolic content (TPC); (C) DPPH radical scavenging activity; (D) Superoxide anion radical clearance; (E) Hydroxyl radical scavenging activity; (F) Soluble protein content. Different letters indicate significant differences among treatments (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9230235/v1/06996839587de4ef3899d566.png\"},{\"id\":108246928,\"identity\":\"cdcfccbc-0c37-4b73-b69e-e875d4f36a5a\",\"added_by\":\"auto\",\"created_at\":\"2026-05-01 00:28:18\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":50974,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eChanges in soluble sugar content and aerobic bacterial count of fresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e under different treatments during cold storage.(A) soluble sugar content; (B) aerobic plate count. Different letters indicate significant differences among treatments (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9230235/v1/d3797c3fdd5aa1b0fab54e5b.png\"},{\"id\":108491755,\"identity\":\"7e4899cc-4300-4216-8a0f-efd598dbf6c2\",\"added_by\":\"auto\",\"created_at\":\"2026-05-05 09:55:30\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":71986,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffects of different treatments on microbial community composition and diversity of fresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e during cold storage.(A) Relative abundance of bacterial communities at the phylum level; (B) Relative abundance of dominant bacterial genera; (C) Alpha diversity indices (Shannon and Chao1); (D) Principal coordinate analysis (PCoA) based on Bray–Curtis distances.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9230235/v1/aed887a7f7904a0a36335001.png\"},{\"id\":108495027,\"identity\":\"b9cbdf97-fc15-4d3e-9c78-f0d9ceb13377\",\"added_by\":\"auto\",\"created_at\":\"2026-05-05 10:08:28\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":685801,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9230235/v1/06b53ecd-0aa3-4cf5-9f9d-717d7f840028.pdf\"},{\"id\":108246927,\"identity\":\"f3d4ef7c-fce9-4c7c-9091-92b6e3d065e3\",\"added_by\":\"auto\",\"created_at\":\"2026-05-01 00:28:18\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":72267,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementarymaterial.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9230235/v1/25eba5622e51a9953befdcff.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Impact of slightly acidic electrolyzed water in combination with ultrasound on safety and quality of Fresh-cut Agaricus bisporus\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eFresh-cut edible fungi, particularly \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e, have gained significant popularity in the ready-to-eat and pre-prepared food markets in recent years due to their low calories, high protein content, abundant dietary fiber, and flavor compounds (Kalac, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Roupas et al., \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). However, \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e is inherently fragile and has a high moisture content, which makes it prone to cellular structural damage during fresh-cut processing (Jin et al., \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e; Saltveit, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2000\\u003c/span\\u003e; Xu et al., \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; L. Zhang et al., \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). This damage accelerates the oxidation of polyphenolic compounds, browning reactions, and microbial proliferation, significantly reducing its commercial value and shelf life (Liang et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Saravanakumar et al., \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Moreover, as consumer demand shifts toward minimally processed foods with clean-label preservation methods, the lack of effective and residue-free sanitation strategies for fresh-cut mushrooms has become a critical bottleneck in scaling up industrial production and cold-chain distribution (Parvathy Nayana et al., \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e). Therefore, developing a green, safe, and effective cleaning and preservation system to slow down quality deterioration during low-temperature storage is one of the key technical challenges in the commercialization and industrialization of fresh-cut edible fungi.\\u003c/p\\u003e \\u003cp\\u003eVarious physical and chemical disinfection techniques have been explored to address these challenges, including pulsed electric field, irradiation, high-pressure processing, ultraviolet radiation, ozone treatment, and cold plasma techniques (Onwude et al., \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Among these, slightly acidic electrolyzed water (SAEW) stands out as a widely used preservation technology for fresh-cut foods, such as fruits, vegetables, and seafood, due to its broad-spectrum antimicrobial properties, ease of preparation, and low toxic residue (Huang et al., \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Issa-Zacharia, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; C. Zhang et al., \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). SAEW is typically generated through the electrolysis of dilute hydrochloric acid or sodium chloride and has moderate pH (5.0\\u0026ndash;6.5), suitable chlorine concentration, and a high oxidation-reduction potential (Akther et al., \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). These properties allow SAEW to achieve non-thermal sterilization by disrupting microbial cell membranes and interfering with their enzyme systems. Compared with traditional chemical disinfectants such as sodium hypochlorite, SAEW offers a milder antimicrobial mechanism with minimal impact on the nutritional and sensory qualities of fresh-cut produce (Du et al., \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Previous studies have shown that SAEW effectively reduces microbial load and delays respiration rate and browning of fresh-cut carrots, strawberries, apples, and other fruits and vegetables (Ding et al., \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Koide et al., \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e; Tango et al., \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). However, when processing fresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e, which has a loose structure and high polyphenolic oxidation activity, the proteins and phenolic compounds exuded from the cut surfaces may react with the chlorine in SAEW, leading to reduced sterilization efficiency(Gil et al., \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). Additionally, SAEW is sensitive to temperature and storage conditions, which can result in decreased stability. More importantly, SAEW has limited ability to suppress enzymatic browning, thus making it insufficient as a standalone strategy for controlling browning reactions in mushrooms.\\u003c/p\\u003e \\u003cp\\u003eUltrasound (US), as a typical non-thermal physical preservation technique, has shown great potential in food cleaning, sterilization, and structural regulation in recent years (Chemat et al., 2011; Fan et al., 2021). The core mechanism of US is the cavitation effect, which generates microjets and high-pressure pulses that enhance the contact efficiency between the treatment liquid and the food surface, disrupt microbial biofilms, and promote the diffusion and penetration of the treatment liquid into the food tissue, thereby improving overall cleaning and sterilization effectiveness (Irazoqui et al., \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Song et al., \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Previous studies have demonstrated that US can effectively increase the microbial removal rate in fresh-cut fruits and vegetables, slow down the respiration rate, delay browning, and prevent texture deterioration, thus extending shelf life (Jiang et al., \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e; J. Wang et al., \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). However, the preservation effect of US is influenced by processing parameters and food matrix characteristics, and improper control may cause structural damage, moisture loss, and color deterioration (Khaire et al., \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e); these adverse effects may be even more pronounced when processing products such as fresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e, which have fragile tissues and are highly susceptible to browning. Furthermore, although numerous studies have investigated ultrasound-assisted washing in fruits and vegetables, its mechanistic interaction with mushroom-specific physiology\\u0026mdash;high porosity, rapid enzymatic oxidation, and weak tissue coherence\\u0026mdash;remains largely unexplored, leaving a significant knowledge gap. Moreover, the preservation mechanism of US in this type of high-moisture, delicate food still lacks systematic research.\\u003c/p\\u003e \\u003cp\\u003eTherefore, this study aims to investigate the preservation effect of combined US and SAEW treatment on fresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e. We propose a composite preservation strategy that combines the chemical antimicrobial mechanism of SAEW and the physical enhancement function of US, applied to the storage system of fresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e, which is prone to browning, has high moisture content, and is structurally sensitive. By sampling at multiple time points, we systematically evaluate the effects of different treatments on the sensory quality, physicochemical properties, and microbial stability of \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e during storage. The results of this study will provide technical support for the commercialization and cold-chain circulation of fresh-cut edible fungi products and offer theoretical and practical guidance for the efficient integration of green sterilization and preservation technologies in perishable food applications.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1. Sample collection and pretreatment\\u003c/h2\\u003e \\u003cp\\u003eFresh \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e samples were purchased from Meitong Market (Hohhot, China). After removing visible soil and debris, mushrooms with uniform color and appearance, free from mechanical damage or microbial infection, were selected for the experiments. The samples were sliced into pieces of approximately 0.5-1.0 cm thickness and randomly divided into four groups, each subjected to a different washing treatment: (i) the control group (CK) was immersed in distilled water for 5 min; (ii) the slightly acidic electrolyzed water group (SAEW) was washed with SAEW containing 70 mg/L available chlorine for 5 min; (iii) the ultrasound group (US) was treated at 100 W and 40 kHz for 5 min; and (iv) the combined treatment group (SAEWUS) underwent ultrasonic treatment (100 W, 40 kHz, 5 min) followed by washing with SAEW (70 mg/L, 5 min). All treatments were conducted at 5\\u0026deg;C to minimize tissue metabolism and prevent heat-induced denaturation. Although ultrasound was applied prior to SAEW washing, the cavitation-induced microstructural loosening and removal of surface barriers may enhance the subsequent accessibility and efficacy of active chlorine species during SAEW treatment.\\u003c/p\\u003e \\u003cp\\u003eAfter treatment, samples were air-dried in a sterile environment under laminar airflow until no visible surface moisture remained. Approximately 80 g of mushrooms were placed in PA/PE composite bags (non-vacuum sealed) and stored at 4\\u0026deg;C for subsequent analyses.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. Physicochemical, antioxidant, enzymatic, and microbiological analyses\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.2.1 Color measurement\\u003c/h2\\u003e \\u003cp\\u003eColor parameters of fresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e were measured using a colorimeter (CR-20, Konica Minolta Co., Japan). The CIE Lab* color system was used, where \\u003cem\\u003eL*\\u003c/em\\u003e indicates lightness, \\u003cem\\u003ea*\\u003c/em\\u003e denotes the red\\u0026ndash;green axis, and \\u003cem\\u003eb*\\u003c/em\\u003e denotes the yellow\\u0026ndash;blue axis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.2.2 Ascorbic acid and Total phenolic content\\u003c/h2\\u003e \\u003cp\\u003eAscorbic acid content was determined by titration with 2,6-dichloroindophenol (DCIP) solution and expressed as mg per 100 g fresh weight (mg/100 g FW).\\u003c/p\\u003e \\u003cp\\u003eTotal phenolic content was determined using the Folin\\u0026ndash;Ciocalteu colorimetric method and expressed as grams of gallic acid equivalents per kilogram fresh weight (mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.2.3 Antioxidant capacity (DPPH, Superoxide, and Hydroxyl radicals)\\u003c/h2\\u003e \\u003cp\\u003eDPPH radical scavenging activity was determined using a DPPH\\u0026ndash;ethanol solution. Extracts were mixed with 0.1 mmol/L DPPH and incubated for 30 min at room temperature, and absorbance was measured at 517 nm. Scavenging activity was calculated using Eq.\\u0026nbsp;(1):\\u003cdiv id=\\\"Equa\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equa\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:\\\\begin{array}{c}DPPH\\\\:free\\\\:radical\\\\:clearance\\\\:\\\\left(\\\\%\\\\right)=\\\\frac{{A}_{0}-{A}_{S}}{{A}_{0}}\\\\times\\\\:100\\\\#\\\\left(1\\\\right)\\\\end{array}$$\\u003c/div\\u003e\\u003c/div\\u003e\\u003c/p\\u003e \\u003cp\\u003eWhere \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{A}_{0}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e and \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{A}_{S}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e represent the absorbance of the control and the sample, respectively.\\u003c/p\\u003e \\u003cp\\u003eSuperoxide anion radical clearance was determined using a nitroblue tetrazolium (NBT) reduction system, and absorbance was measured at 320 nm. Results were expressed as inhibition percentage.\\u003c/p\\u003e \\u003cp\\u003eHydroxyl radical scavenging rate was determined using a Fenton reaction system by measuring absorbance at 510 nm, and results were expressed as percentage inhibition of hydroxyl radicals.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.2.4 Malondialdehyde (MDA) content\\u003c/h2\\u003e \\u003cp\\u003eMDA content was determined using the thiobarbituric acid (TBA) colorimetric method. Absorbance was measured at 450, 532, and 600 nm, and MDA content was calculated according to Eq.\\u0026nbsp;(2):\\u003cdiv id=\\\"Equb\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equb\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:\\\\begin{array}{c}MDA\\\\left(nmol\\u0026middot;{g}^{-1}\\\\:FW\\\\right)=\\\\frac{\\\\left[6.45\\\\times\\\\:\\\\left({A}_{532}-{A}_{600}\\\\right)-0.56\\\\times\\\\:{A}_{450}\\\\right]\\\\times\\\\:V}{{V}_{S}\\\\times\\\\:m}\\\\#\\\\left(2\\\\right)\\\\end{array}$$\\u003c/div\\u003e\\u003c/div\\u003e\\u003c/p\\u003e \\u003cp\\u003eWhere \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:V\\\\)\\u003c/span\\u003e\\u003c/span\\u003e is the total reaction volume (mL); \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{V}_{S}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e is the volume of sample extract used (mL); and \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:m\\\\)\\u003c/span\\u003e\\u003c/span\\u003e is the mass of the mushroom sample (g).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.2.5 Enzyme activities (PPO and POD)\\u003c/h2\\u003e \\u003cp\\u003ePolyphenol oxidase (PPO) and peroxidase (POD) activities were determined using colorimetric methods. Extracts were prepared in phosphate buffer containing 0.5% (w/v) PVP. PPO activity was assayed using catechol by measuring the increase in absorbance at 410 nm, while POD activity was assayed using guaiacol and H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e at 470 nm. Activities were expressed as the change in absorbance per minute per gram fresh weight (min\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.2.6 Soluble protein and Soluble sugar\\u003c/h2\\u003e \\u003cp\\u003eSoluble protein content was determined using the Bradford method, and absorbance was measured at 595 nm. Results were expressed as mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW.\\u003c/p\\u003e \\u003cp\\u003eSoluble sugar content was determined using the anthrone colorimetric method, and absorbance was measured at 485 nm. Results were expressed as percentage of fresh weight (% FW).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.2.7 Aerobic plate count\\u003c/h2\\u003e \\u003cp\\u003eAerobic plate count was determined using the plate count method. Serial dilutions were plated on plate count agar and incubated at 37\\u0026deg;C for 48 h. Results were expressed as logarithmic colony-forming units per gram (lg CFU/g).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.2.8 Microbial community analysis\\u003c/h2\\u003e \\u003cp\\u003eCK and SAEWUS groups were selected for microbial profiling to represent the most contrasting quality deterioration trajectories. To characterize microbial succession during storage and to compare the microbial dynamics under contrasting quality trajectories, microbial diversity analysis was performed for CK group and SAEWUS group samples at days 0, 2, 5, and 8. The V4 region of the bacterial 16S rRNA gene was amplified using the universal primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT). PCR products were purified, quantified, and used to construct sequencing libraries, which were sequenced on the Illumina MiSeq platform (Novogene Co., Beijing, China). Sequencing raw reads were subjected to standard preprocessing, including barcode removal, primer trimming, and paired-end merging, before entering the downstream bioinformatics workflow. Chloroplast and mitochondrial sequences were retained for relative abundance analysis to reflect overall sequencing composition but were not interpreted as active bacterial taxa.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Data processing\\u003c/h2\\u003e \\u003cp\\u003eUnless otherwise specified, all physicochemical measurements were performed in triplicate. Data visualization was conducted using Origin 2021, and statistical tests were performed using SPSS 26. One-way ANOVA followed by Duncan\\u0026rsquo;s multiple range test was used to determine significant differences among treatments (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). For microbial community analysis, raw 16S sequencing reads were processed using QIIME 1.9.1, including quality filtering, read merging, chimera removal, OTU clustering at 97% similarity, taxonomic annotation against the SILVA database, and calculation of α- and β-diversity indices.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results and Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1 Effects of Different Treatments on Browning-Related Parameters of Fresh-Cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e\\u003c/h2\\u003e \\u003cp\\u003eBrowning development in fresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e was strongly influenced by the pretreatments, as reflected by changes in color attributes and biochemical indicators (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e; Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e). The \\u003cem\\u003ea*\\u003c/em\\u003e value increased continuously during storage, with the CK group already showing the highest values among all treatments from day 2 onward and reaching 8.37 on day 10, indicating rapid red\\u0026ndash;brown discoloration (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). In contrast, the SAEW and US groups moderated the increase in \\u003cem\\u003ea*\\u003c/em\\u003e value, while the SAEWUS group exhibited the strongest inhibition, maintaining significantly lower values such as 4.65 on day 4 and 5.39 on day 8 (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). This behavior suggests that the combined treatment more effectively delayed red\\u0026ndash;brown discoloration, and this effect is likely due to the synergistic action of SAEW-mediated oxidative inactivation of surface microorganisms and ultrasound-induced cavitation, which improves mass transfer at the tissue\\u0026ndash;solution interface and facilitates the removal or dilution of browning precursors. Changes in \\u003cem\\u003eL*\\u003c/em\\u003e and \\u003cem\\u003eb*\\u003c/em\\u003e further supported these findings. \\u003cem\\u003eL*\\u003c/em\\u003e decreased in all samples during storage (Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eA), with the CK group showing the most rapid decline, from 72.09 on day 0 to 51.25 on day 10. The SAEW and SAEWUS groups effectively preserved \\u003cem\\u003eL*\\u003c/em\\u003e, with the SAEWUS group maintaining significantly higher \\u003cem\\u003eL*\\u003c/em\\u003e values (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), including 64.53 on day 6. Conversely, \\u003cem\\u003eb*\\u003c/em\\u003e values increased throughout storage (Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eB), reflecting progressive yellow\\u0026ndash;brown pigment formation. The CK group exhibited the steepest rise, reaching 16.39 on day 6, whereas the SAEWUS group consistently maintained significantly lower \\u003cem\\u003eb*\\u003c/em\\u003e values after day 4 (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), including 14.46 on day 10. These improvements in \\u003cem\\u003eL*\\u003c/em\\u003e and \\u003cem\\u003eb*\\u003c/em\\u003e indicate reduced enzymatic oxidation of phenolic substrates and improved preservation of cut-surface tissue integrity under the SAEWUS group.\\u003c/p\\u003e \\u003cp\\u003eThe enzymatic indicators PPO and POD showed trends consistent with the color changes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB,C). The CK group maintained the significantly highest activities of both PPO and POD throughout storage, reaching peak values of 15.59 min\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW and 0.040 min\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW on day 4 and day 10, respectively, thereby accelerating enzymatic browning. The SAEW and US groups reduced these enzyme activities to varying extents, whereas the SAEWUS group achieved the strongest inhibition, with PPO and POD activities of 9.43 min\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW and 0.018 min\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW at the corresponding time points. Ultrasound-induced cavitation may transiently disrupt enzyme conformation and reduce catalytic efficiency, while the SAEW group can oxidize key sulfhydryl or amino groups required for enzyme activity. The combined treatment likely amplifies these effects by enhancing the SAEW group penetration into surface tissues through ultrasound-enhanced microstreaming and localized mechanical disruption. MDA content increased steadily in all groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD), with the CK group showing the fastest accumulation and reaching 1.82 nmol\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW on day 10. All pretreatments slowed the rise in MDA, and the SAEWUS group consistently maintained the lowest levels, such as 0.99 nmol\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW on day 2 and 1.57 nmol\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW on day 10, indicating reduced oxidative stress and delayed tissue deterioration.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eOverall, the SAEWUS group most effectively delayed browning by mitigating color deterioration (\\u003cem\\u003ea*\\u003c/em\\u003e, \\u003cem\\u003eL*\\u003c/em\\u003e, \\u003cem\\u003eb*\\u003c/em\\u003e), inhibiting PPO and POD activities, and reducing membrane lipid peroxidation. These synergistic benefits likely result from the combined antimicrobial, oxidative, and cavitation-induced effects of the SAEW and US, which collectively limit browning substrates and weaken the enzymatic drivers of discoloration.\\u003c/p\\u003e \\u003cp\\u003eTo further elucidate the mechanistic basis underlying these macroscopic quality changes, beyond the observed suppression of PPO and POD activities, the inhibitory effects of the combined SAEWUS treatment can be further interpreted from a structural and reaction-level perspective. PPO and POD are copper-containing oxidoreductases whose catalytic activity strongly depends on the integrity of metal-binding sites and the redox state of key amino acid residues, such as histidine, cysteine, and tyrosine (Liang et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Xu et al., \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Slightly acidic electrolyzed water, characterized by a high oxidation-reduction potential and the presence of active chlorine species, may oxidize sulfhydryl and imidazole groups located near the catalytic center, thereby reducing enzyme\\u0026ndash;substrate affinity and catalytic efficiency (Gil et al., \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e; Issa-Zacharia, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Meanwhile, ultrasound treatment generates localized cavitation, microstreaming, and transient pressure fluctuations, which can induce reversible conformational loosening or partial unfolding of enzyme structures, further limiting enzymatic accessibility to phenolic substrates (Khaire et al., \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). When combined, ultrasound is likely to enhance the interaction between SAEW and enzyme molecules at the cut surface by reducing boundary layer resistance and promoting mass transfer, resulting in more efficient enzyme inactivation than either treatment alone. This complementary action at both the chemical and physical levels provides a plausible explanation for the markedly lower PPO and POD activities and the reduced accumulation of lipid peroxidation products observed under SAEWUS treatment.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2 Effects of Different Treatments on Antioxidant Capacity and Redox-Related Physicochemical Attributes of Fresh-Cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e\\u003c/h2\\u003e \\u003cp\\u003eAs shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, non-enzymatic antioxidants and redox-related parameters exhibited distinct, treatment-dependent temporal patterns during cold storage, indicating coordinated yet heterogeneous redox responses among treatments and reflecting the differential effectiveness of the pretreatments in mitigating oxidative deterioration.\\u003c/p\\u003e \\u003cp\\u003eThe superior preservation of non-enzymatic antioxidants under the SAEWUS treatment also reflects a more stable intracellular redox environment during storage. Ascorbic acid and phenolic compounds serve as primary redox buffers in mushroom tissues and are rapidly depleted under excessive oxidative stress (Hu et al., \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Saltveit, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2000\\u003c/span\\u003e; Xu et al., \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). In untreated samples, accelerated membrane lipid peroxidation and enzymatic oxidation likely increased reactive oxygen species (ROS) accumulation, resulting in continuous consumption of antioxidant pools. In contrast, the combined SAEWUS treatment effectively mitigated oxidative pressure by simultaneously suppressing enzymatic browning reactions and limiting microbial proliferation, both of which are major sources of ROS generation in fresh-cut mushrooms (Gil et al., \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). Ultrasound-enhanced mass transfer may further facilitate the removal of oxidized metabolites from the tissue surface, while SAEW reduces microbial-derived oxidative stress at the cut interface. Together, these effects contribute to a more balanced redox state, thereby slowing antioxidant depletion and maintaining higher radical-scavenging capacity throughout storage.\\u003c/p\\u003e \\u003cp\\u003eAscorbic acid content followed a characteristic \\u0026ldquo;sharp decrease\\u0026ndash;partial recovery\\u0026ndash;final decline\\u0026rdquo; trajectory during storage (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). A rapid decrease occurred by day 2 due to intense oxidative consumption immediately after cutting, with the CK group dropping to 37.32 mg\\u0026middot;100g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW, whereas the US and SAEWUS groups retained significantly higher values 40.77 and 44.06 mg\\u0026middot;100g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). From day 2 to day 6, a partial rebound was observed, most prominently under the SAEWUS group, which maintained the highest levels with 44.18 mg\\u0026middot;100g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW on day 4 and 45.59 mg\\u0026middot;100g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW on day 6, followed by the US and SAEW groups, while the CK group remained substantially lower throughout this period. This temporary recovery likely reflects transient metabolic adjustment and reduced oxidative pressure under the pretreatments. After day 6, ascorbic acid declined again in all treatments, with CK group showing the significantly fastest depletion and decreasing to 24.52 mg\\u0026middot;100g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW on day 10 (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). The SAEW, US and particularly SAEWUS groups significantly slowed this late-stage loss, with the SAEWUS group consistently retaining the highest ascorbic acid levels up to 30.58 mg\\u0026middot;100g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW, indicating strong protection against oxidative degradation. Total phenolic content displayed a different temporal pattern, characterized by rapid early accumulation followed by continuous depletion (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). On day 2, total phenols increased significantly as part of the wound response, with the US group showing the significantly highest level at 6.58 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). This value was significantly higher than those of the CK group (5.56 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW) and the SAEW group (4.93 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW), while the SAEWUS group showed an intermediate increase to 5.86 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW. After day 2, phenolic content declined steadily in all groups, reflecting progressive consumption as substrates for enzymatic and non-enzymatic oxidation. The CK group displayed the fastest depletion, whereas all pretreatments slowed this decline. By day 6 and day 10, the SAEWUS group maintained the significantly highest phenolic levels, recorded at 4.45 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW and 3.39 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), respectively. This pattern is consistent with the inhibition of PPO/POD-mediated phenolic oxidation shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, indicating more effective retention of phenolic antioxidants under the SAEWUS group.\\u003c/p\\u003e \\u003cp\\u003eDPPH scavenging capacity exhibited a clear pattern of early stimulation followed by continuous decline (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). By day 2, cutting stress induced a short-lived elevation in scavenging capacity. The CK group rose to 14.92%, while the pretreatment groups reached values between 14.01% and 14.89%, with no significant differences among treatments at this stage (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05), suggesting enhanced release or activation of readily extractable antioxidants. After this initial increase, DPPH clearance decreased progressively in all treatments. The CK group declined most rapidly, falling to 5.55% on day 4 and further decreasing to 2.59% by day 10. The pretreatments slowed this reduction, with the SAEWUS group consistently maintaining the highest scavenging capacity, reaching 8.83% on day 4 and 3.75% on day 10 (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). This correspondence between metabolite retention and functional scavenging supports the interpretation that SAEWUS stabilized the low-molecular-weight antioxidant pool. Superoxide anion scavenging capacity showed a clear fluctuation over storage time (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). A slight decline occurred by day 2 across all groups, with no significant differences (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05), but all treatments exhibited a pronounced rebound on day 4. At this point, CK group increased to 60.86%, whereas the SAEW, US, and SAEWUS groups reached significantly higher values (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) of 65.87%, 65.04%, and 67.34%, respectively, indicating that the pretreatments enhanced the superoxide-scavenging response more effectively than CK group. After day 4, superoxide clearance gradually decreased in all groups, with CK group consistently maintaining the lowest activity throughout the remainder of storage. The SAEWUS group showed the strongest preservation effect, maintaining the highest capacities (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) at multiple stages (day 6, 8, and 10), further confirming its superior support of the enzymatic superoxide-detoxification system. Hydroxyl radical scavenging showed a transient early rise followed by a sharp decline and partial late-stage stabilization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE). By day 2, the US and SAEWUS groups exhibited a notable increase with 42.24% and 51.61%, significantly higher (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) than the CK and SAEW groups at 33.33% and 37.09%, likely reflecting ultrasound-induced release of bound antioxidants or activation of redox enzymes. After day 2, scavenging capacity decreased rapidly across all treatments. CK group showed the lowest values throughout mid-storage, with 17.84% at day 4 and 9.84% at day 6. SAEW and US groups maintained moderately higher levels, whereas the SAEWUS group demonstrated higher or comparable values at most time points. By day 10, pretreatments again showed an advantage, with values of 9.29%, 9.01%, and 10.66%, whereas CK group dropped to 6.01% (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), confirming that SAEWUS more effectively mitigated oxidative intensification and preserved hydroxyl-radical-scavenging activity.\\u003c/p\\u003e \\u003cp\\u003eSoluble protein content followed a clear \\u0026ldquo;early increase then continuous decline\\u0026rdquo; pattern (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eF). By day 2, the pretreatment groups showed a marked increase in soluble protein (1.48\\u0026ndash;1.58 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW), whereas the CK group exhibited a slight decrease to 1.35 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW. This divergence suggests that without protective pretreatment, the CK group samples experienced more rapid membrane injury, reducing the pool of extractable cytosolic proteins, while the increases observed in the treated groups may be associated with altered extractability of cytosolic proteins after cutting and pretreatment and increased extractability of cytosolic proteins immediately after cutting. After day 2, soluble protein declined steadily in all treatments. The CK group decreased most sharply, reaching 1.06 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW by day 4 and falling to 0.47 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW by day 10 (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). The SAEW and US groups slowed this decline, maintaining intermediate levels of 0.60\\u0026ndash;0.69 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW, while the SAEWUS group consistently retained the highest protein content throughout storage, maintaining 1.54 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW on day 4 and 0.77 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW on day 10. These protein-retention outcomes align with the reduced MDA accumulation reported in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, further confirming that SAEWUS effectively suppresses oxidative denaturation and preserves cellular integrity.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eOverall, the SAEWUS group most effectively stabilized the antioxidant system by maintaining higher levels of ascorbic acid and phenolics, enhancing free-radical scavenging capacity (DPPH, O\\u003csub\\u003e2\\u003c/sub\\u003e\\u003csup\\u003e\\u0026minus;\\u003c/sup\\u003e\\u0026middot;, \\u0026middot;OH) and slowing oxidation-related protein degradation. Collectively, this treatment imposed the most comprehensive redox-stabilizing effect during storage, consistent with its superior inhibition of browning and oxidative injury reported in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003eIn this context, the synergistic effect observed in the SAEWUS treatment should not be regarded as a simple additive outcome of two preservation methods. SAEW and ultrasound act at distinct yet complementary levels: SAEW primarily exerts chemical antimicrobial and oxidative modulation effects, whereas ultrasound functions as a physical enhancer by intensifying liquid-solid interactions and weakening diffusion barriers at the tissue surface (Irazoqui et al., \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Song et al., \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Through cavitation-induced microstreaming and transient microstructural disturbance, ultrasound likely increases the accessibility of active chlorine species to microbial cells, enzymes, and oxidation-sensitive substrates. This parallel rather than hierarchical interaction allows each treatment to maintain its intrinsic preservation function while amplifying overall efficacy, explaining why the combined treatment consistently outperformed the single treatments across multiple quality attributes (Y. Wang et al., \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3 Effects of Different Treatments on Soluble Sugars and Microbial Quality of Fresh-Cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e\\u003c/h2\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eSoluble sugar content and aerobic bacterial counts displayed tightly coupled, treatment-dependent trends during storage, reflecting the interplay between endogenous carbohydrate metabolism and microbial activity. Soluble sugars declined sharply by day 2 across all groups, with the CK group showing the greatest decrease to 0.96%, whereas the SAEW, US and SAEWUS groups maintained slightly higher levels between 0.97% and 0.99% (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). From day 2 to day 6, a partial recovery occurred, most evident in the SAEWUS group, which increased to 1.08% on day 4 and 1.12% on day 6, while the US and SAEW groups exhibited moderate rebounds and CK group remained consistently lower. During late storage, soluble sugars declined again. CK group showed the steepest reduction, falling to 0.87% by day 10 (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), whereas the pretreatments slowed this loss and the SAEWUS group retained the highest residual sugar content at 1.03%. These sugar-preservation outcomes suggest that the SAEWUS group may reduce carbohydrate depletion by mitigating both physiological consumption and microbial utilization.\\u003c/p\\u003e \\u003cp\\u003eAerobic bacterial growth accelerated progressively during storage but at markedly different rates among treatments (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB). The CK group exhibited the fastest proliferation, rising to 3.02 lg CFU/g by day 2 and reaching 6.36 lg CFU/g by day 10, values that were significantly higher (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) than those of all pretreatment groups at the corresponding time points. In contrast, the SAEW, US and SAEWUS groups suppressed microbial growth throughout storage, maintaining significantly lower (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) counts of 5.03\\u0026ndash;5.18 lg CFU/g at the end of the period. Among the pretreatments, the SAEWUS group consistently achieved the strongest inhibition, consistent with its combined antimicrobial effect of slightly acidic electrolyzed water and the cavitation-enhanced disruption of microbial cells and attachment structures. Overall, the coordinated preservation of soluble sugars and suppression of aerobic bacteria demonstrates that the SAEWUS group most effectively maintained metabolic stability and microbial quality of fresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e during cold storage.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.4 Effects of Different Treatments on Microbial Diversity and Structure of Fresh-Cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e\\u003c/h2\\u003e \\u003cp\\u003eBased on taxonomic annotation, bacterial community profiles were summarized across multiple taxonomic levels, among which phylum- and genus-level compositions were emphasized due to their dominant contribution to community structure and functional relevance. Comparative analysis of community composition revealed distinct succession patterns between CK and SAEWUS samples during storage, indicating that the combined treatment reshaped microbial assembly rather than merely reducing overall microbial load. These compositional shifts provide a mechanistic explanation for the observed differences in microbial growth and sugar utilization between treatments.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eAt the phylum level, the bacterial communities of fresh-cut Agaricus bisporus exhibited pronounced treatment- and storage-dependent shifts (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). The initial microbiota (a0d) showed a relatively balanced structure, with Proteobacteria accounting for approximately one-third of the total community, accompanied by substantial proportions of Firmicutes, Bacteroidota, Cyanobacteria, and Actinobacteriota, indicating a diverse baseline community prior to storage. In the CK group, microbial succession was characterized by a progressive dominance of Proteobacteria, although the temporal pattern was not strictly monotonic. Proteobacteria increased sharply by day 2, followed by a slight relative decrease at day 5, and ultimately became overwhelmingly dominant by day 8, accounting for nearly the entire community. This late-stage enrichment coincided with a marked depletion of Firmicutes, Bacteroidota, and Cyanobacteria, reflecting a severe simplification of the microbial structure typically associated with advanced spoilage. In contrast, the SAEWUS-treated samples displayed a markedly different succession trajectory. Although Proteobacteria remained a major component, its relative abundance was consistently lower than that in the corresponding CK samples at all storage stages. Notably, the SAEWUS group exhibited a pronounced enrichment of Cyanobacteria at day 5, accompanied by a concomitant reduction in Proteobacteria, resulting in a more evenly distributed community structure. By day 8, Proteobacteria increased again but did not reach the overwhelming dominance observed in the CK group, while Firmicutes and Bacteroidota were better retained. These phylum-level differences suggest that SAEWUS treatment altered not only the abundance but also the succession pattern of dominant microbial groups, prompting further examination of key spoilage-associated genera.\\u003c/p\\u003e \\u003cp\\u003eAt the genus level, pronounced differences in microbial succession were observed between the CK and SAEWUS treatments during storage (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). In the initial sample (a0d), Pseudomonas accounted for only a minor fraction of the total community, and the microbiota was dominated by a large proportion of low-abundance and unclassified taxa, indicating a relatively undisturbed baseline state. In the CK group, Pseudomonas exhibited a rapid but non-monotonic expansion. Its relative abundance increased sharply by day 2, reaching the highest level among CK samples, followed by a gradual decline at day 5 and a further reduction by day 8. This fluctuation suggests that although Pseudomonas responded rapidly to the favorable post-cutting environment, prolonged storage led to progressive community destabilization rather than sustained dominance of a single spoilage genus. Concurrently, the increase in unclassified taxa and the reduction of other identifiable genera indicate a fragmented and deterioration-prone microbial structure at the late storage stage. In contrast, the SAEWUS-treated samples showed a distinctly altered succession pattern. The relative abundance of Pseudomonas remained consistently lower than that in the corresponding CK samples at all time points. Notably, at day 5, Pseudomonas was strongly suppressed, while sequences annotated as unidentified chloroplasts and mitochondria accounted for a large proportion of the total reads, likely reflecting a marked reduction in bacterial load and a proportional enrichment of host-derived sequences rather than active microbial proliferation. By day 8, Pseudomonas abundance increased again but did not reach the peak levels observed in the CK group at earlier stages.These genus-level patterns further indicate that SAEWUS delayed the dominance of typical spoilage-associated taxa, which is expected to influence overall microbial diversity and community complexity.\\u003c/p\\u003e \\u003cp\\u003eAlpha diversity analysis based on the Shannon index revealed pronounced differences in microbial community complexity between treatments during storage (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC). The initial samples (a0d) exhibited the highest Shannon values, reflecting a highly diverse baseline microbiota composed of multiple low-abundance taxa. In the CK group, microbial diversity decreased sharply after cutting, with a marked reduction in Shannon index already observed at day 2, followed by a further decline at day 5 and reaching the lowest level by day 8. This progressive loss of diversity indicates a rapid microbial succession toward a simplified, spoilage-dominated community structure under untreated conditions. In contrast, the SAEWUS-treated samples maintained significantly higher microbial diversity at the early storage stage. At day 2, the Shannon index of the SAEWUS group remained comparable to that of a0d and was substantially higher than that of the CK group, suggesting that the combined treatment effectively restrained early dominance of specific taxa and preserved community complexity. Although a temporary decrease in Shannon index was observed at day 5 in the SAEWUS group, microbial diversity recovered at day 8, showing a clear divergence from the continuously declining trend observed in CK samples. To further evaluate whether these diversity differences were accompanied by shifts in overall community structure, beta diversity analysis was performed using PCoA.\\u003c/p\\u003e \\u003cp\\u003ePrincipal coordinates analysis (PCoA) based on Bray\\u0026ndash;Curtis dissimilarity revealed temporal shifts in the bacterial community structure during storage (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD). Samples did not form completely discrete clusters, indicating moderate rather than drastic community differentiation, which is consistent with the short storage period and non-thermal pretreatments applied. Nevertheless, a clear tendency of divergence between CK and SAEWUS samples was observed along the PC1 axis as storage progressed. CK samples exhibited a larger dispersion over time, suggesting a more pronounced restructuring of the microbial community, whereas SAEWUS-treated samples showed relatively constrained distribution, particularly at day 5 and day 8.\\u003c/p\\u003e \\u003cp\\u003eTaken together, the beta diversity results corroborate the compositional and diversity analyses, demonstrating that SAEWUS treatment moderated microbial succession dynamics and contributed to improved microbial stability during cold storage.\\u003c/p\\u003e \\u003cp\\u003eFrom a functional perspective, the moderated microbial succession observed under SAEWUS treatment is particularly relevant to quality preservation in fresh-cut mushrooms. Rather than eliminating the microbial community entirely, the combined treatment appeared to delay the rapid dominance of spoilage-associated taxa and preserve higher microbial diversity during early and mid-storage stages. Such stabilization may reduce metabolic fluctuations typically associated with advanced spoilage (Parvathy Nayana et al., \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e; Shi et al., \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). This interpretation is consistent with the slower decline in soluble sugars and the restrained increase in aerobic plate counts observed in SAEWUS-treated samples. Therefore, the microbial modulation induced by SAEWUS contributes not only to microbial safety but also to metabolic stability, providing an additional explanation for its superior performance in maintaining overall quality.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Conclusion\",\"content\":\"\\u003cp\\u003eThe combined SAEWUS group treatment provided the most comprehensive preservation of fresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e quality during cold storage. It markedly delayed browning, maintaining an \\u003cem\\u003ea*\\u003c/em\\u003e value of 6.19 on day 10 compared with 8.37 in the CK group, and reduced PPO and POD activities by roughly 15\\u0026ndash;45%. Lipid oxidation was also minimized, with MDA levels reaching only 1.57 nmol\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW by day 10, lower than the 1.82 nmol\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW measured in the CK group. Mid-storage antioxidant retention was substantially improved under the SAEWUS group, which preserved 45.59 mg\\u0026middot;100g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW of ascorbic acid and 4.45 mg\\u0026middot;g\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e FW of phenolics on day 6, both clearly higher than the corresponding values in the CK group. Microbial quality followed the same trend; by day 10, aerobic counts in the SAEWUS group were approximately 1.4 log CFU/g lower than those in the CK group. These advantages highlight the complementary roles of slightly acidic electrolyzed water and ultrasound, which act at chemical and physical levels, respectively, to enhance preservation efficiency. Given its residue-free nature, operational simplicity, and compatibility with low-temperature processing, SAEWUS represents a promising green pretreatment strategy for industrial fresh-cut mushroom processing and cold-chain distribution.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eLimei Wang\\u003c/strong\\u003e: Resources, Writing\\u0026ndash;original draft. \\u003cstrong\\u003eBai wuriliga\\u003c/strong\\u003e: Data curation, Investigation. \\u003cstrong\\u003eYong Lian\\u003c/strong\\u003e: Software, Methodology. \\u003cstrong\\u003eRui Shi\\u003c/strong\\u003e: Validation. \\u003cstrong\\u003eYuanyuan Zhang\\u003c/strong\\u003e: Investigation. \\u003cstrong\\u003eXiuli Liu\\u003c/strong\\u003e: Methodology, Software.\\u003cstrong\\u003e\\u0026nbsp;Mu Jia\\u003c/strong\\u003e: Investigation.\\u003cstrong\\u003e\\u0026nbsp;Tao Sun\\u003c/strong\\u003e: Review, Editing \\u0026amp; Supervision.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eHohhot Science and Technology Plan Project Task (2023-Agriculture-10),Science and Technology Plan of Inner Mongolia Autonomous Region (2025YFHH0275)\\u003c/p\\u003e\\n\\u003cp\\u003eClinical trial number: not applicable.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eEthics approval and consent to participate: not applicable.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eConsent for publication: not applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflicts of Interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability Statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe raw 16S rRNA gene amplicon sequencing data generated in this study have been deposited in the Genome Sequence Archive (GSA), National Genomics Data Center (NGDC), China National Center for Bioinformation (CNCB) under accession number CRA041143. The data are currently accessible via the following direct link: https://ngdc.cncb.ac.cn/gsa/s/8548vdJZ. All other data supporting the findings of this study are available within the article and its Supplementary Information files.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAkther S, Islam MR, Alam M, Alam MJ, Ahmed S. Impact of slightly acidic electrolyzed water in combination with ultrasound and mild heat on safety and quality of fresh cut cauliflower. Postharvest Biol Technol. 2023;197:112189. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.postharvbio.2022.112189\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.postharvbio.2022.112189\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChemat F, Khan MK. 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Mild heat treatment inhibits the browning of fresh-cut Agaricus bisporus during cold storage. LWT - Food Sci Technol. 2017;82:104\\u0026ndash;12. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.lwt.2017.04.035\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.lwt.2017.04.035\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"discover-food\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"discoverfood\",\"sideBox\":\"Learn more about [Discover Food](https://www.springer.com/44187)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Discover Food\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Discover Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Slightly Acidic Electrolyzed Water, Ultrasound, Fresh-cut, Microbial decontamination, Agaricus bisporus\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-9230235/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-9230235/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eFresh-cut \\u003cem\\u003eAgaricus bisporus\\u003c/em\\u003e is highly susceptible to browning and microbial spoilage during refrigerated storage. This study evaluated slightly acidic electrolyzed water (SAEW) and ultrasound (US), applied individually and in combination, as green washing\\u0026ndash;preservation strategies for fresh-cut mushrooms. Sliced samples were treated with distilled water (CK), SAEW (70 mg/L available chlorine), US (100 W, 40 kHz), or combined SAEWUS, packaged in PA/PE bags, and stored at 4\\u0026deg;C for 10 days. Quality changes were assessed by color parameters, browning-related enzyme activities (PPO, POD), lipid oxidation (MDA), antioxidant components and capacities, and microbial counts. In addition, microbial community succession in CK and SAEWUS samples was analyzed using 16S rRNA sequencing. Compared with single treatments, SAEWUS most effectively delayed browning, maintained lower \\u003cem\\u003ea\\u003c/em\\u003e* values, inhibited PPO and POD activities, and reduced oxidative damage. The combined treatment also better preserved antioxidant levels and achieved a greater reduction in aerobic plate counts during storage. Microbial profiling further indicated that SAEWUS moderated spoilage-associated microbial succession and improved community stability. Overall, the synergistic application of SAEW and ultrasound represents an effective, residue-free approach to enhance quality retention and extend the refrigerated shelf life of fresh-cut mushrooms.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Impact of slightly acidic electrolyzed water in combination with ultrasound on safety and quality of Fresh-cut Agaricus bisporus\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-05-01 00:28:14\",\"doi\":\"10.21203/rs.3.rs-9230235/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewerAgreed\",\"content\":\"152243081993764777346735265029275159281\",\"date\":\"2026-05-19T09:34:46+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"246660194502058052406112824668967691891\",\"date\":\"2026-05-15T15:20:08+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-15T08:18:55+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"38158693976568627787198292812023115646\",\"date\":\"2026-04-24T00:48:36+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-04-21T14:36:25+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2026-04-16T13:59:58+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-04-15T09:07:24+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-04-14T01:26:13+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Discover Food\",\"date\":\"2026-04-14T01:20:14+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"discover-food\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"discoverfood\",\"sideBox\":\"Learn more about [Discover Food](https://www.springer.com/44187)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Discover Food\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Discover Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"57dbbe4c-9d0b-48f0-98bf-6478b8093497\",\"owner\":[],\"postedDate\":\"May 1st, 2026\",\"published\":true,\"recentEditorialEvents\":[{\"type\":\"reviewerAgreed\",\"content\":\"152243081993764777346735265029275159281\",\"date\":\"2026-05-19T09:34:46+00:00\",\"index\":47,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"246660194502058052406112824668967691891\",\"date\":\"2026-05-15T15:20:08+00:00\",\"index\":46,\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-15T08:18:55+00:00\",\"index\":44,\"fulltext\":\"\"}],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-05-01T00:28:14+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-05-01 00:28:14\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-9230235\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-9230235\",\"identity\":\"rs-9230235\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}