Isolation and Characterization of a Novel Fungus, Rhizopus arrhizus MNQW, for Effective Biodegradation and Detoxification of Zearalenone

Isolation and Characterization of a Novel Fungus, Rhizopus arrhizus MNQW, for Effective Biodegradation and Detoxification of Zearalenone | 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 Isolation and Characterization of a Novel Fungus, Rhizopus arrhizus MNQW, for Effective Biodegradation and Detoxification of Zearalenone Dan He, Yunfan Shan, Han Qiu, Gang Wang, Junqiang Hu, Yuzhuo Wu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8695845/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Apr, 2026 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted 9 You are reading this latest preprint version Abstract Zearalenone (ZEN) is a prevalent estrogenic mycotoxin in cereals and feedstuffs, posing persistent risks to feed safety and animal health. In this study, a food-grade filamentous fungus, Rhizopus arrhizus MNQW, was isolated from a traditional rice starter ( Xiaoqu ) and systematically evaluated for its ZEN biodegradation capacity and practical applicability. Strain MNQW efficiently removed over 98% of 5 mg/L ZEN in minimal salt medium within 36 h under mild fermentation-compatible conditions. Subcellular fractionation and inhibition assays indicated that ZEN degradation was predominantly mediated by heat- and protease-sensitive intracellular enzymes. UPLC–MS/MS analysis revealed only transient formation of α-zearalenol at trace levels, followed by complete detoxification without accumulation of estrogenically active intermediates. In vitro bioassays using estrogen receptor-positive (ER-positive) MCF-7 and HepG2 cells confirmed that the final degradation products exhibited neither estrogenic activity nor cytotoxicity. Importantly, application of MNQW in solid-state fermentation of ZEN-contaminated maize flour (3 mg/kg) achieved approximately 97% toxin removal within 36 h while simultaneously improving nutritional quality, including increased crude protein, vitamin B 2 , folate, and beneficial fatty acids. Whole-genome analysis identified multiple oxidoreductase- and hydrolase-encoding genes potentially involved in ZEN biotransformation. Collectively, these findings demonstrate that R. arrhizus MNQW represents a safe, efficient, and application-ready microbial candidate for detoxification and value-added processing of ZEN-contaminated feed materials. Zearalenone Rhizopus arrhizus Biodegradation Solid-state fermentation Mycotoxin detoxification Food safety Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin predominantly produced by several Fusarium spp. and frequently contaminates cereals such as maize, wheat, and barley (Zinedine et al. 2007 ). Owing to its chemical stability during storage and processing, ZEN can persist throughout the food and feed chain, contributing to sustained exposure and resulting in reproductive disorders, hepatotoxicity, immune dysfunction, and potential carcinogenic risks in animals, thereby posing concerns for human health (Minervini & Dell’Aquila. 2008; Yang et al. 2025 ). Moreover, ZEN metabolites, particularly α-zearalenol (α-ZOL), may exhibit comparable or even higher estrogenic activity than the parent compound, further intensifying its toxicological hazards (Rizvi et al. 2025 ; Zinedine et al. 2007 ). To mitigate these risks, regulatory authorities have implemented stringent measures to reduce human exposure to ZEN. In the European Union, maximum levels of 100–350 µg/kg have been established for cereals and cereal-based products, while the Joint FAO/WHO Expert Committee on Food Additives (JECFA) has established a tolerable daily intake (TDI) of 0.25 µg/kg body weight per day (EFSA Panel on Contaminants in the Food Chain. 2011). Current detoxification strategies for ZEN include physical adsorption, chemical oxidation, and microbial degradation. Physical and chemical approaches can reduce ZEN content, but often impair nutritional value and sensory quality or generate undesirable by-products (Pankaj et al., 2018 ; Qi et al., 2016 ). In contrast, microbial degradation represents an environmentally friendly strategy that operates under mild conditions with high specificity, while preserving nutritional and organoleptic properties (Gari & Abdella. 2023; Hamad et al. 2023 ; Murtaza et al. 2022 ). Although various bacteria (e.g. Bacillus , Lactobacillus ) and fungi (e.g. Clonostachys rosea , yeasts) have been reported to exhibit ZEN-transforming capabilities, food-grade strains with high degradation efficiency and suitability for industrial fermentation remain scarce (Gari & Abdella. 2023; Ruiz, 2025 ; Yang et al. 2017 ). The fungal genus Rhizopus is widely utilized in food fermentation and is designated as generally recognized as safe (GRAS) by the U.S. Food and Drug Administration. Species of Rhizopus secrete a broad range of extracellular enzymes, including hydrolases and oxidoreductases, which enable the transformation of diverse substrates and render them promising candidates for mycotoxin bioremediation (Vellozo-Echevarría et al. 2024 ). Despite this potential, the degradation of ZEN by Rhizopus spp. has not yet been systematically investigated. In this study, we isolated and identified a novel GRAS fungal strain, Rhizopus arrhizus MNQW, from a traditional rice fermentation starter ( Xiaoqu ). Its growth characteristics, ZEN degradation kinetics, and the localization of active ZEN-degrading components were comprehensively investigated, and the toxicity of the degradation products was evaluated using in vitro cell culture models. Furthermore, the application of MNQW in the solid-state fermentation of ZEN-contaminated maize flour was explored, with a particular focus on the impact of biodegradation on nutritional quality. Collectively, this study provides a new microbial resource and mechanistic insights for the safe and effective bioremediation of ZEN in food and feed matrices. 2. Materials and Methods 2.1. Chemicals, media, and reagents Zearalenone (ZEN, ≥ 98%) was purchased from Sigma-Aldrich (USA). Tryptone and yeast extract were obtained from Thermo Fisher Scientific (USA). Dipotassium hydrogen phosphate (K 2 HPO 4 ), potassium dihydrogen phosphate (KH 2 PO 4 ), sodium chloride (NaCl), ammonium chloride (NH 4 Cl), and magnesium sulfate heptahydrate (MgSO 4 ·7H 2 O) were supplied by Xilong Scientific Co., Ltd. (China). Agar powder was obtained from Solarbio Science & Technology Co., Ltd. (Shanghai, China). TaKaRa Ex Taq polymerase and the EZ‑10 Bacterial Genomic DNA Extraction Kit were purchased from Takara Bio Inc. (Japan) and Sangon Biotech Co., Ltd. (Shanghai, China), respectively. All solvents used for high-performance liquid chromatography (HPLC) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analyses were of HPLC grade. 2.2. Screening and isolation of ZEN-degrading strains 2.2.1. Sample source Fermented rice starter ( Xiaoqu ) was obtained from Angel Yeast (China) in January 2023. Upon arrival, the sample was stored at room temperature in a dry environment and used within one week for subsequent experiments. 2.2.2. Enrichment and primary screening Five grams of Xiaoqu were suspended in 50 mL sterile water and shaken at 180 rpm for 4–6 h. Subsequently, 1 mL of the supernatant was inoculated into 9 mL of minimal salt medium (MSM) supplemented with 10 mg/L ZEN as the selective pressure (Gari & Abdella. 2023). Cultures were incubated at 30℃, with agitation at 180 rpm for 5 days, followed by three consecutive subculturing cycles. Residual ZEN concentrations were quantified using HPLC, and samples exhibiting ZEN-degrading activity were selected for further isolation. 2.2.3. Isolation of single strains Enriched cultures (1 mL) were transferred into 9 mL of MSM containing 10 mg/L ZEN, with uninoculated flasks containing MSM supplemented with ZEN serving as controls. Cultures were incubated at 30°C and 180 rpm for 5 days. Cultures exhibiting high ZEN‑degrading activity were serially diluted (10 − 4 –10 − 7 ) and spread onto potato dextrose agar (PDA) plates. Following incubation at 30℃ for 2–5 days, morphologically distinct colonies were isolated and individually evaluated for ZEN degradation, leading to the identification of a high‑efficiency strain, designated MNQW. 2.2.4. HPLC analysis of ZEN degradation Culture broth (500 µL) was extracted with an equal volume of HPLC‑grade ethyl acetate, vortexed thoroughly for 5 min, and centrifuged at 10,000 rpm for 5 min. The organic phase was collected, and the extraction was repeated three times. The combined organic phases were evaporated to dryness under nitrogen at 40–50℃ for approximately 45 min. The residue was reconstituted in 300 µL of HPLC-grade methanol, vortexed for 5 min, filtered through a 0.22 µm polytetrafluoroethylene (PTFE) syringe filter, and transferred into amber HPLC vials. ZEN concentrations were determined using an HPLC system equipped with an Agilent ZORBAX SB-C18 column (250 × 4.6 mm, 5 µm). The mobile phase consisted of methanol/water (4:1, v/v) at a flow rate of 0.8 mL/min, with an injection volume of 10 µL and UV detection at 236 nm (Pascari et al., 2023 ). The degradation rate (%) was calculated according to the following equation: where A sample and A control represent the peak areas of ZEN in treated and control samples, respectively. 2.3. Identification of strain MNQW 2.3.1. Morphological characterization MNQW was cultured on PDA at 30℃ for 2 days, and colony color, surface texture, margin, and mycelial morphology were recorded. For light microscopy, sterile coverslips were inserted into fresh PDA plates, and 7 mm agar plugs from the colony edges were placed approximately 1 cm from the coverslips, with five replicates. Plates were incubated at 30℃ Hyphae on the coverslips were then examined under a light microscope (EVO-LS10, Sartorius, Beijing, China). For scanning electron microscopy (SEM), MNQW was inoculated on PDA, and the mycelia were fixed with 2.5% glutaraldehyde at 4℃. Hyphal morphology was subsequently observed using a scanning electron microscope (EVO-LS10, Carl Zeiss, Germany) to provide detailed structural information. 2.3.2. Molecular identification Genomic DNA of MNQW was extracted using the EZ-10 Bacterial Genomic DNA Kit (Sangon, China). The internal transcribed spacer (ITS) region was amplified using the universal fungal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') with TaKaRa Ex Taq polymerase. PCR products were verified on 1% agarose gel electrophoresis, purified, quantified, and subjected to Sanger sequencing (Sangon, China). ITS sequences were compared against NCBI GenBank using BLAST for taxonomic identification. A phylogenetic tree was constructed using the neighbor-joining method based on ITS sequences from MNQW and reference strains. Bootstrap analysis with 1,000 replications was conducted to assess branch support. Whole-genome sequencing of MNQW was performed on an Illumina NovaSeq 6000 platform using a paired-end library with an insert size of 400 bp, following the manufacturer’s instructions. 2.4. Growth and ZEN degradation characterization of strain MNQW The growth of MNQW was evaluated by inoculating 7 mm PDA agar plugs onto PDA plates and incubating at 30℃ in the dark for 3–5 days. Colony diameters were measured every 3 h using the cross method to generate growth curves, with five replicates per condition. The effects of temperature (15–40℃) and initial pH (4.0–10.0) were assessed under identical conditions. Additionally, the influence of carbon sources (starch, maize flour, lactose, glucose, maltose, sucrose), and nitrogen sources (urea, yeast extract, peptone, NaNO 3 , NH 4 Cl, (NH 4 ) 2 SO 4 ) on growth was investigated using MSM-based solid media, with each factor added at 1% of the medium and five replicates per treatment (Tai et al. 2020 ). To assess the effect of pH on ZEN degradation, MNQW agar plugs were inoculated into 20 mL of MSM containing 5 mg/L ZEN with initial pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0 and 9.0. Cultures were incubated at 35℃ with agitation at 180 rpm for 36 h. To evaluate temperature effects, MSM containing 5 mg/L ZEN (pH 5.0) was incubated at 25, 30, 35, 40 and 45℃ (pH 5.0) under otherwise identical conditions. Residual ZEN was extracted and quantified by HPLC as described in Section 2.2.4 . All experiments were performed in triplicate to assess the tolerance and degradation performance of MNQW under different environmental conditions. 2.5. Localization of ZEN-degrading activity A 7 mm agar plug of MNQW was inoculated into 100 mL of potato dextrose broth (PDB) and incubated at 35℃ with agitation at 180 rpm for 3 days. The culture was centrifuged at 8,000 rpm for 10 min, and the resulting supernatant was filtered through a 0.22 µm membrane to obtain the cell-free culture supernatant (CFS). Mycelial pellets were washed twice with phosphate-buffered saline (PBS), blotted dry, rapidly frozen in liquid nitrogen, and ground into a fine powder. The powder was resuspended in pre‑chilled PBS (pH 7.0), centrifuged at 12,000 rpm for 10 min at 4℃, and filtered through a 0.22 µm membrane to obtain the mycelial extract. Aliquots (500 µL) of both CFS and mycelial extract were subjected to heat inactivation (100℃, 10 min) or proteinase K treatment (37℃, 1 h), followed by incubation with 5 mg/L ZEN for 24 h. Residual ZEN was subsequently quantified by HPLC. These treatments were conducted to evaluate the involvement of proteinaceous components in ZEN degradation, consistent with enzyme-mediated mycotoxin degradation mechanisms (Liu et al. 2023 ; Wang et al. 2017 ). 2.6. Analysis of ZEN degradation products ZEN degradation products were prepared under the previously determined optimal conditions. Briefly, strain MNQW was inoculated into 10 mL of MSM (pH 5.5) supplemented with ZEN at a final concentration of 5 µg/mL and incubated at 35℃. Uninoculated MSM containing the same concentration of ZEN served as the control. All experiments were conducted in triplicate. Samples were collected at 24 h intervals, and ZEN together with its degradation products were extracted according to the procedure described in Section 2.2.4 . The extracted samples were initially analyzed by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) using a SCIEX Triple Quad™ 3500 system (SCIEX, USA) equipped with an ACE UltraCore Super C18 column (3.0 × 150 mm, 2.5 µm). Compound annotation was performed using TraceFinder and Discovery software, following previously reported analytical strategies for mycotoxin degradation products (Ji et al. 2023 ). 2.7. Cytotoxicity assessment MCF-7 and HepG2 cells were seeded at 5 × 10 4 cells/well in 96-well plates and incubated for 12 h at 37℃ with 5% CO 2 . MCF-7 cells were cultured in phenol-red-free Dulbecco’s modified Eagle medium (DMEM) supplemented with charcoal-dextran-treated fetal bovine serum (FBS), whereas HepG2 cells were cultured in standard DMEM. Cells were treated with ZEN (10 − 9 to 10 − 5 M for MCF-7 and 1 to 25 mg/L for HepG2) or the corresponding degradation products for 24 h. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and calculated as follows: where A 1 and A 2 represent the absorbances of the treatment and control groups, respectively. 2.8. Application in ZEN-contaminated maize flour ZEN-contaminated maize flour (400 mg/kg), prepared from Fusarium -inoculated cracked maize, was mixed with uncontaminated commercial maize flour to achieve a final ZEN concentration of 3 mg/kg. The mixture was dried at 65℃ to constant weight and stored at 4℃. MNQW spores were harvested from V8 agar cultures grown at 35℃ for 3–4 days and adjusted to 10 7 CFU/mL. For degradation assays, 8 g portions of maize flour were placed in 90-mm Petri dishes and inoculated with 2 mL of spore suspension; controls received 2 mL of sterile PBS. Samples were incubated at 35℃, and moisture was maintained by spraying 3 mL PBS every 3 h. Subsamples were collected at 12, 24, 36, and 48 h. Additional experiments evaluated the effects of temperature (18–40℃) and inoculum levels (10 5 –10 9 CFU/mL). Nutritional composition of MNQW-fermented maize flour—including moisture, starch, crude protein, crude fat, amino acids, and vitamins B 1 and B 2 —was analyzed according to Chinese National Standards (GB/T 6435 − 2006, GB 5009.9–2016, GB/T 6432 − 2018, GB/T 6433 − 2006, GB/T 18246 − 2000, GB/T 14700 − 2018, GB/T 14701 − 2019). ZEN was extracted from maize flour using 20 mL of 80% acetonitrile containing 0.1% formic acid and filtered prior to LC–MS/MS analysis (Sulyok et al. 2006 ). Quantification was performed using an Agilent Eclipse XDB-C18 column (2.1 × 150 mm, 3.5 µm, 40℃) under gradient elution with 0.1% formic acid–water (A) and methanol (B) at 0.4 mL/min (Leeman et al. 2025 ). ZEN degradation efficiency was calculated as: where C control and C sample denote ZEN concentrations in the control and treatment samples, respectively. 2.9. Statistical analysis All experiments were performed in triplicate. Data are presented as mean ± standard error (SE). Statistical analyses were conducted using one-way ANOVA followed by Tukey’s multiple comparison test in OriginPro software (OriginLab, USA). Differences were considered statistically significant at p < 0.05. 3. Results and Discussion 3.1. Screening and characterization of the ZEN-degrading strain MNQW ZEN is a non-steroidal estrogenic mycotoxin frequently detected in cereals and cereal-derived products, exerting its toxicity primarily through competitive binding to estrogen receptors (ERs) and subsequent endocrine disruption (Chen et al., 2025 ; el-Sharkawy et al. 1991 ). Chronic dietary exposure to ZEN has been associated with reproductive disorders and an increased risk of hormone-dependent tumors (Singh et al. 2024 ). Conventional detoxification strategies, including physical adsorption and chemical treatment, often suffer from limited efficiency, nutrient loss, or secondary contamination (Lach & Kotarska. 2024). Microbial degradation, in contrast, has emerged as a promising alternative due to its substrate specificity, mild reaction conditions, and environmental compatibility (Sun et al. 2023 ; Xu et al. 2022 ). Screening of microbial isolates obtained from cereal- and soil-derived samples across different regions of China revealed pronounced variability in ZEN-degrading capacity (Table S1 ). Among the tested isolates, a filamentous fungal strain designated MNQW, isolated from a traditional fermented rice starter ( Xiaoqu ), exhibited the highest degradation efficiency, removing approximately 98% of 5 mg/L ZEN in mineral salt medium within 72 h at 30℃ (Fig. 1 A). In contrast, the remaining isolates showed only moderate or limited degradation activities, with efficiencies generally below 60%. Compared with previously reported ZEN-degrading bacteria and fungi, including Bacillus spp. and Geobacillus spp., which often require lower toxin concentrations or extended incubation periods to achieve comparable removal rates, MNQW demonstrated superior degradation performance under relatively stringent conditions (Liu et al. 2023 ; Sun et al. 2024 ). These results identify MNQW as a highly efficient ZEN-degrading fungus with strong potential for application in food and feed detoxification. Based on morphological features, ITS rRNA gene sequencing, and whole-genome analysis, MNQW was identified as R. arrhizus . The strain formed dense, cotton-like aerial mycelia with compact gray-brown colonies, unbranched sporangiophores, and ellipsoidal sporangiospores, consistent with typical species descriptions (Fig. 1 B–D). ITS sequence analysis revealed 99.84% similarity to R. arrhizus (MH865594.1) with 93% bootstrap support in the phylogenetic tree, confirming its taxonomic assignment (Fig. 1 E, Table S2 ). Whole-genome sequencing yielded a 39.19 Mb draft genome comprising 12,651 predicted protein-coding genes, with functional annotation performed against the NCBI non-redundant protein database (NR), Swiss-Prot, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and the Carbohydrate-Active enZYmes database (CAZy) databases (Fig. 1 F). Notably, genes encoding oxidoreductases, hydrolases, and CAZymes—enzyme families frequently implicated in xenobiotic transformation and fungal secondary metabolism—were identified, suggesting a versatile enzymatic system potentially responsible for ZEN biotransformation. R. arrhizus is a ubiquitous filamentous fungus widely distributed in soil and plant-associated environments and is known for rapid growth, low nutritional requirements, and high environmental adaptability. The species has been extensively exploited for production of organic acids and industrial enzymes, such as lipases and amylases, and its mycelial biomass has demonstrated adsorption and immobilization capacities for hazardous compounds, highlighting its bioremediation potential (Guo et al. 2024 ; Meng et al. 2025 ). Despite these advantages, reports on ZEN biodegradation by R. arrhizus remain scarce. Most ZEN-degrading fungi reported to date belong to genera such as Aspergillus spp., Trichoderma spp., and Clonostachys spp., with only limited evidence implicating R. arrhizus in ZEN transformation (el-Sharkawy et al. 1991 ). Therefore, the identification of R. arrhizus MNQW as a highly efficient ZEN-degrading strain expands the known functional repertoire of this species and provides new insights into its potential application in the detoxification of contaminated food and feed. 3.2. Growth characteristics and degradation kinetics of strain MNQW under varied environmental conditions The adaptability of microorganisms to environmental conditions is a critical determinant of their practical applicability in food and feed systems. Most food and feed processing environments are mildly acidic; however, many previously reported ZEN-degrading microorganisms exhibit optimal activity under neutral to alkaline conditions, which substantially limits their industrial applicability (Sun et al. 2024 ). Therefore, evaluating the growth behavior and degradation performance of strain MNQW under varied environmental conditions is essential. The growth curve of MNQW exhibited a short lag phase during the initial 16 h, followed by a pronounced exponential phase from 16 to 31 h, before entering the stationary phase (Fig. 2 A). Strain MNQW demonstrated remarkable environmental adaptability, with optimal growth observed at temperatures ranging from 35 to 40℃ and within a mildly acidic pH range of 5.0–6.0 (Fig. 2 B–C). Carbon source profiling revealed a clear preference for complex substrates, particularly maize flour, over simple sugars (Fig. 2 D). Although maize flour is not fully soluble in liquid media, it forms a stable suspension and can be gradually hydrolyzed and assimilated through extracellular enzymatic activities, a process that closely resembles substrate utilization in practical feed fermentation systems. Regarding nitrogen sources, yeast extract supported the highest biomass accumulation; notably, however, MNQW also exhibited robust growth when sodium nitrate (NaNO 3 ) was supplied as the sole nitrogen source (Fig. 2 E), highlighting its potential for cost-effective industrial application. Consistent with its favorable growth characteristics, MNQW displayed efficient ZEN degradation kinetics, achieving approximately 98% removal of 5 mg/L ZEN within 36 h (Fig. 2 F). The degradation rate peaked during the exponential growth phase and gradually declined as the substrate concentration decreased. Temperature exerted a significant influence on degradation efficiency, with maximal activity (approximately 96%) observed between 35 and 40℃, followed by a sharp decline at 45℃ (Fig. 2 G). Similarly, pH profiling revealed optimal degradation at pH 5.5, with sustained activity across a range of 4.5–6.0 (Fig. 2 H). The close alignment between optimal growth conditions and ZEN degradation performance underscores the metabolic efficiency of MNQW under mildly acidic environments. This physiological trait is particularly advantageous for feed applications, where organic acids such as fumaric acid—a major fermentation product of R. arrhizus—are commonly incorporated as acidifiers (Kuenz et al. 2023 ). Such acidic conditions not only suppress undesirable microbial growth but also enhance feed palatability, especially in swine production systems (Xu et al. 2022 ). Combined with its ability to utilize inexpensive nitrogen sources such as NaNO 3 and complex agricultural substrates, MNQW exhibits strong economic and practical potential for large-scale mycotoxin detoxification, distinguishing it from previously reported fungal strains with more limited environmental tolerance (Guo et al. 2024 ; Meng et al. 2025 ). 3.3. Subcellular localization and metabolite identification of ZEN degradation To localize the ZEN-degrading activity in strain MNQW, the degradation capacities of different cellular fractions were evaluated. As shown in Fig. 3 A, the culture supernatant exhibited limited ZEN degradation activity (10.31%), whereas the intracellular extract retained a substantially higher degradation efficiency (88.23%) within 12 h. Heat treatment of the intracellular extract markedly reduced the degradation efficiency to 10.64%, and subsequent proteinase K treatment almost completely abolished the activity. These results indicate that the ZEN-degrading activity of strain MNQW is predominantly associated with intracellular proteinaceous components, suggesting an enzyme-mediated transformation process. This intracellular localization presents a significant practical advantage for feed applications, as it minimizes the risk of enzyme leakage during processing, thereby enhancing safety compared to strains relying on extracellular enzymes (Liu et al. 2024 ). The metabolic fate of ZEN during incubation with strain MNQW was further investigated using UPLC-MS/MS analysis. Among the detected compounds, α-ZOL was identified as the only ZEN-related intermediate metabolite. The identity of α-ZOL was confirmed by comparison of its retention time and mass spectral characteristics with those of an authentic α-ZOL standard (Fig. 3 B). Quantitative analysis revealed that α-ZOL accumulated only transiently, reaching a maximum level corresponding to 1.32% of the initial ZEN concentration, and was no longer detectable after 7 days of incubation (Fig. 3 C). These results indicate that α-ZOL did not persist as a stable transformation product during ZEN degradation by strain MNQW. Several additional differential compounds, including 3-pyridinemethanol, adenosine, acetyl-L-carnitine, and triphenyl phosphate, were detected in the metabolite profiles of the MNQW culture. These compounds are considered endogenous metabolites of strain MNQW and showed no apparent structural relationship with ZEN. Notably, no other ZEN-derived products with characteristic UV absorption were detected under the analytical conditions employed, suggesting that a substantial proportion of ZEN was transformed into products not readily detectable by UV-based UPLC-MS/MS analysis. However, the chemical structures and biological activities of the final transformation products require further investigation. 3.4. Cytotoxicity and estrogenic activity of ZEN degradation products The detoxification efficacy of strain MNQW was further evaluated using in vitro bioassays targeting both estrogenic activity and cytotoxicity. Considering the estrogen-like effects of ZEN at low concentrations, ER-positive human breast cancer cells (MCF-7) were selected to assess residual estrogenic activity, while human hepatocellular carcinoma cells (HepG2) were employed to evaluate cytotoxicity, reflecting the primary metabolic target organ of ZEN in vivo . In MCF-7 cells, cell proliferation was first quantified using the MTT assay (Fig. 4 A). As expected, native ZEN induced a typical biphasic response, characterized by significant proliferative stimulation at low concentrations and growth inhibition at higher concentrations, consistent with its well-documented estrogenic properties. In contrast, treatment with MNQW degradation products did not induce any significant proliferative response across the tested concentration range (10 − 9 –10 − 5 mol/L). Notably, the degradation products represent a complex mixture; therefore, exposure levels were normalized based on the initial molar concentration of ZEN prior to degradation, rather than individual metabolite molarity, a strategy commonly adopted in microbial mycotoxin detoxification studies (Guo et al. 2024 ; Wu et al. 2025 ). Morphological observations further supported the quantitative results (Fig. 4 B). Cells treated with 17β-estradiol (E2) or low concentrations of ZEN exhibited increased cell density and partial multilayer growth, whereas cells exposed to ZEN degradation products maintained clear boundaries and a typical monolayer morphology comparable to the untreated control, indicating the absence of detectable estrogenic stimulation. The cytotoxicity of ZEN and its degradation products was subsequently assessed in HepG2 cells. MTT assays revealed a marked, concentration-dependent reduction in cell viability following ZEN exposure, whereas cells treated with degradation products maintained significantly higher viability at corresponding exposure levels (Fig. 4 C). Consistently, microscopic examination showed extensive cell damage and loss of adhesion in ZEN-treated cells, while cells exposed to degradation products retained intact morphology and normal growth patterns (Fig. 4 D). Although α-ZOL, a metabolite reported to exhibit equal or higher estrogenic potency than ZEN, was transiently detected during the degradation process, no estrogenic or cytotoxic effects were observed for the final degradation products in either cell model. This suggests that α-ZOL did not accumulate to biologically relevant levels and was further transformed by strain MNQW. Collectively, these results demonstrate that MNQW-mediated degradation of ZEN effectively eliminates both estrogenic activity and cytotoxicity, underscoring the potential of strain MNQW for safe and practical application in mycotoxin detoxification in feed systems (Wang et al. 2025 ). 3.5. Detoxification of ZEN-contaminated maize flour via solid-state fermentation Approximately 70% of maize produced in China is utilized for animal feed, making the development of effective detoxification strategies for mycotoxin-contaminated cereals essential to ensure feed safety. In this study, the practical detoxification performance of R. arrhizus MNQW was evaluated using ZEN-contaminated maize flour under solid-state fermentation conditions. As shown in Fig. 5 A, ZEN levels in maize flour (initial concentration 3 mg/kg) decreased continuously during fermentation with strain MNQW and were completely eliminated within 36 h, whereas no significant change was observed in the uninoculated control. Importantly, α-ZOL, a metabolite with higher estrogenic potency than ZEN, was not detected throughout the entire 72-h fermentation period (Fig. 5 B), indicating that MNQW mediates a safe detoxification pathway without accumulation of hazardous intermediates. To facilitate practical application, key fermentation parameters were optimized. The highest detoxification efficiency was achieved at 35°C with an inoculum level of 10 8 CFU/mL, resulting in more than 97% ZEN degradation within 36 h (Fig. 5 C–D). Increasing the temperature to 45°C markedly reduced degradation efficiency, consistent with the growth characteristics of MNQW, while excessive inoculum levels promoted premature sporulation, adversely affecting the sensory quality of the fermented maize flour. These results highlight the importance of balancing detoxification efficiency with product quality and process feasibility. Compared with previously reported bacterial or yeast-based detoxification systems, MNQW exhibits several notable advantages, including rapid and complete ZEN removal, food-grade safety, compatibility with solid-state fermentation, and the absence of estrogenically active by-products. Given that Rhizopus species are widely used in food and feed fermentation and recognized as safe microorganisms, MNQW represents a promising candidate for industrial-scale detoxification of ZEN-contaminated feed materials (Acs-Szabo et al. 2025 ; Podgórska-Kryszczuk et al. 2022 ; Zhou et al. 2024 ). 3.6. Nutritional enhancement of maize flour through MNQW fermentation Under optimized fermentation conditions, solid-state fermentation with R. arrhizus MNQW markedly improved the nutritional quality of maize flour (Table 1 ). Specifically, crude protein content increased by 2.66%, accompanied by pronounced changes in B-vitamin composition and a substantial restructuring of the fatty acid profile. This simultaneous enhancement of detoxification efficiency and nutritional quality represents a dual functional benefit that is rarely reported for ZEN-degrading microorganisms (Uwineza et al. 2024 ). The increase in crude protein can be attributed to microbial biomass accumulation and a substrate concentration effect associated with an overall mass reduction of 8.25% during fermentation. Notable alterations were also observed in the vitamin profile: although vitamin B 1 decreased, vitamin B 2 and folate levels increased substantially, resulting in a more balanced B-vitamin composition. Balanced B-vitamin intake is particularly relevant for livestock with limited capacity for endogenous vitamin retention. Fermentation also significantly reshaped the fatty acid profile. Oleic acid content increased by 12.32%, whereas linoleic acid decreased by 7.19%. At the class level, total monounsaturated fatty acids (MUFA) increased markedly, saturated fatty acids (SFA) increased moderately, and polyunsaturated fatty acids (PUFA) decreased slightly. This redistribution of the SFA–MUFA–PUFA balance indicates that MNQW fermentation systematically restructures the lipid matrix rather than simply altering individual fatty acids, which may contribute to improved oxidative stability, feed palatability, and energy utilization efficiency. Detailed fatty acid composition before and after fermentation is provided in Table S3 . Table 1 Changes in nutritional composition and major fatty acid classes of maize flour after solid-state fermentation with R. arrhizus strain MNQW (36 h, 35℃). Component Before fermentation After fermentation Change Major nutrients Corn meal weight (%) 100.00 91.75 -8.25 Crude Protein (%) 6.56 9.22 + 2.66 Starch (%) 74.10 51.60 -22.50 Vitamin B 1 (mg/kg) 9.20 4.87 -4.33 Vitamin B 2 (mg/kg) 1.68 5.45 + 3.77 Folate (mg/kg) 378.98 390.45 + 11.47 Major fatty acids (%) C16:0 (Palmitic acid) 15.03 22.88 + 7.85 C18:0 (Stearic acid) 2.55 5.75 + 3.20 C18:1n9c (Oleic acid) 27.44 39.76 + 12.32 C18:2n6c (Linoleic acid) 49.67 42.48 -7.19 C18:3n3 (α-Linolenic acid) 1.31 0.39 -0.92 C18:3n6 (γ-Linolenic acid) 0.00 1.40 + 1.40 Fatty acid classes ∑SFA 19.47 33.48 + 14.01 ∑MUFA 27.98 46.40 + 18.42 ∑PUFA 51.19 45.53 -5.66 ∑USFA 79.18 91.93 + 12.75 Note: Values are expressed as percentages of total fatty acids (for fatty acids) or absolute content (for nutrients). The “Change” column represents the difference (After fermentation − Before fermentation). ∑SFA, total saturated fatty acids; ∑MUFA, total monounsaturated fatty acids; ∑PUFA, total polyunsaturated fatty acids; ∑USFA, total unsaturated fatty acids (∑USFA = ∑MUFA + ∑PUFA, not mutually exclusive). Detailed fatty acid composition is provided in Table S3. These compositional changes are consistent with the metabolic characteristics of R. arrhizus , which secretes α-amylase and lipase during fermentation, promoting starch hydrolysis and lipid remodeling (Anigboro et al. 2020 ; Terefe et al. 2021 ). Collectively, these findings demonstrate that MNQW fermentation simultaneously detoxifies ZEN-contaminated maize flour and enhances its nutritional quality by increasing protein content, optimizing fatty acid distribution, and improving B-vitamin balance, highlighting the strong potential of MNQW as a sustainable, food-grade microbial agent for the bioprocessing of contaminated feed materials. 4. Conclusion This study reports the isolation and comprehensive characterization of a novel food-grade fungal strain, R . arrhizus MNQW, from a traditional fermented rice starter ( Xiaoqu ), and demonstrates its high efficiency for ZEN biodegradation. Morphological, molecular, and genomic analyses confirmed its taxonomic identity and revealed the presence of multiple oxidoreductase- and hydrolase-encoding genes that may contribute to ZEN transformation. Strain MNQW rapidly degraded more than 98% of ZEN (5 mg/L) under mildly acidic conditions (35℃, pH 5.5), predominantly via heat-labile intracellular proteinaceous components, indicating an enzyme-mediated degradation mechanism. UPLC–MS/MS analysis showed that α-ZOL was only transiently formed at trace levels and did not accumulate during the degradation process. Importantly, the final degradation products exhibited no detectable estrogenic activity in ER-positive MCF-7 cells and no apparent cytotoxicity in HepG2 cells under the tested conditions, demonstrating effective biological detoxification of ZEN. Application of MNQW in ZEN-contaminated maize flour under solid-state fermentation resulted in rapid and complete detoxification, achieving approximately 97% ZEN removal within 36 h without the formation of estrogenically active intermediates. In parallel, MNQW fermentation significantly improved the nutritional quality of maize flour by increasing crude protein content, enhancing vitamin B 2 and folate levels, and favorably restructuring the fatty acid profile, thereby conferring a dual benefit of detoxification and nutritional enhancement. Although MNQW shows considerable promise as a safe and efficient microbial agent for controlling ZEN contamination in cereal-based feed materials, further studies are warranted to identify the key enzymes involved, elucidate the underlying genetic determinants, and validate its efficacy and safety in vivo . Overall, these findings highlight the strong potential of R. arrhizus MNQW for sustainable mycotoxin detoxification and value-added bioprocessing in feed systems. Declarations CRediT authorship contribution statement Dan He: Conceptualization, Methodology, Investigation, Validation, Formal analysis, Data curation, Writing – original draft, Writing – review & editing, Visualization. Yunfan Shan: Formal analysis, Methodology, Writing – review & editing. Han Qiu: Methodology, Investigation, Formal analysis, Data curation. Gang Wang: Supervision, Writing – review & editing. Junqiang Hu: Writing – review & editing. Yuzhuo Wu: Validation. Keke Ji: Methodology. Hao Xu: Validation. Yin-Won Lee: Supervision, Writing – review & editing. Jianhong Xu: Resources, Funding acquisition, Project administration, Supervision, Writing – review & editing. Xin Yan: Supervision, Writing – review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This study was funded by the National Key R&D Program of China (2023YFD1301004), the National Natural Science Foundation of China (32372454), the Jiangsu Province Science and Technology Support Program (BE2022377), and the Jiangsu Agricultural Science and Technology Program (CX(23)1002). Additional support was provided by the National Special Project for Agro-product Safety Risk Evaluation of China (GJFP20240102) and the State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products (No. 2021DG700024-KF202513). 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Detailed fatty acid composition of maize flour before and after solid-state fermentation with R. arrhizus strain MNQW (36 h, 35℃). Cite Share Download PDF Status: Published Journal Publication published 18 Apr, 2026 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted Editorial decision: Revision requested 02 Mar, 2026 Reviews received at journal 25 Feb, 2026 Reviews received at journal 24 Feb, 2026 Reviewers agreed at journal 04 Feb, 2026 Reviewers agreed at journal 04 Feb, 2026 Reviewers invited by journal 03 Feb, 2026 Editor assigned by journal 28 Jan, 2026 Submission checks completed at journal 28 Jan, 2026 First submitted to journal 25 Jan, 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. 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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-8695845","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":585903939,"identity":"e5b61705-0dcb-4b19-9523-147c31771957","order_by":0,"name":"Dan He","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"He","suffix":""},{"id":585903946,"identity":"09f14fa5-4c4d-4606-aec2-934cd42f505d","order_by":1,"name":"Yunfan Shan","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yunfan","middleName":"","lastName":"Shan","suffix":""},{"id":585903948,"identity":"9987ecf1-07ef-40a9-99d4-56d9aa052234","order_by":2,"name":"Han Qiu","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Qiu","suffix":""},{"id":585903950,"identity":"2134508b-0be0-45fc-8bcf-5235b69eaa97","order_by":3,"name":"Gang Wang","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Wang","suffix":""},{"id":585903951,"identity":"7778a8a1-a393-44f6-a489-5b2e9f8220b1","order_by":4,"name":"Junqiang Hu","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Junqiang","middleName":"","lastName":"Hu","suffix":""},{"id":585903952,"identity":"31a7895c-8af6-4c45-a1d7-49f7184f0886","order_by":5,"name":"Yuzhuo Wu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuzhuo","middleName":"","lastName":"Wu","suffix":""},{"id":585903954,"identity":"438988e2-3490-4113-8252-05ad0cdf1e00","order_by":6,"name":"Keke Ji","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Keke","middleName":"","lastName":"Ji","suffix":""},{"id":585903956,"identity":"291973c1-b53b-492c-93a7-dbcde2f9ffd7","order_by":7,"name":"Hao Xu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Xu","suffix":""},{"id":585903959,"identity":"52871cae-159c-46b9-8137-74402916c78c","order_by":8,"name":"Yon-won Lee","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yon-won","middleName":"","lastName":"Lee","suffix":""},{"id":585903964,"identity":"b5177d0f-bc4f-4f96-b437-2e0cf7875a38","order_by":9,"name":"Jianhong Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIie2PsWrCUBSGDwSS5UrWODTP8MsFteDDnCBkEhF8gU6Z0j0vITg63nBpuwRcM3TQxU2xW4WWVmNd762b4P2Wc4b/4+cncjhuE1ZEqvmwxyC+TukUk1T+t+mseGKvE2sWNa/VYfEe98KZmg7gMQX6ZW5S2gVz+Vxt5GOxYzmCPyaRprVJCSNm1cp0Mq8rHBUxpUh0jYp/VMrvi9JHlDzZlFOLblqWOSQBdqWdr1g/ZFqiFujkYOnbtuBtNPzYZjrGsuri8+snDgP9alSIBJ9vxD6adeb4iUD9rVLeyp52OByOe+QX+OdSCgqNM5UAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Jianhong","middleName":"","lastName":"Xu","suffix":""},{"id":585903969,"identity":"31ee31ce-4579-41b0-a665-afa3f8607c2b","order_by":10,"name":"Xin Yan","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Yan","suffix":""}],"badges":[],"createdAt":"2026-01-26 02:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8695845/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8695845/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11274-026-04963-5","type":"published","date":"2026-04-18T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101931656,"identity":"a21b453d-2fa1-4b44-b00e-2d2f596e3977","added_by":"auto","created_at":"2026-02-05 07:42:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":222592,"visible":true,"origin":"","legend":"\u003cp\u003eScreening, morphological, molecular, and genomic characterization of the ZEN-degrading strain MNQW. \u003cstrong\u003e(A)\u003c/strong\u003e Representative HPLC chromatograms showing degradation of 5 mg/L ZEN in MSM by strain MNQW after 72 h at 30 ℃; \u003cstrong\u003e(B)\u003c/strong\u003e Colony morphology of strain MNQW on PDA after 48 h at 30 ℃; \u003cstrong\u003e(C)\u003c/strong\u003e Sporangial morphology observed by light microscopy (scale bar = 20 μm); \u003cstrong\u003e(D)\u003c/strong\u003e SEM images showing hyphal and sporangiophore structures (scale bar = 20 μm); \u003cstrong\u003e(E)\u003c/strong\u003e ITS rRNA gene-based identification of strain MNQW: agarose gel electrophoresis of the amplified ITS region (\u003cstrong\u003ea\u003c/strong\u003e, M, DL-2000 DNA marker; lane 1, MNQW) and neighbor-joining phylogenetic tree (\u003cstrong\u003eb\u003c/strong\u003e) showing taxonomic placement within \u003cem\u003eR. arrhizus\u003c/em\u003e (bootstrap values \u0026gt;50% from 1,000 replications are shown at branch nodes); \u003cstrong\u003e(F)\u003c/strong\u003e Circular genome map of strain MNQW. The draft genome is 39.19 Mb and contains 12,651 predicted protein-coding genes, including oxidoreductases, hydrolases, and CAZymes potentially involved in ZEN biotransformation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8695845/v1/d52e66fe35cc935953400b3a.png"},{"id":101931682,"identity":"38d8e837-7f45-4d29-a281-522264f29f3f","added_by":"auto","created_at":"2026-02-05 07:42:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108283,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth and ZEN degradation kinetics of \u003cem\u003eR. arrhizus\u003c/em\u003e MNQW under varied environmental conditions. \u003cstrong\u003e(A) \u003c/strong\u003eGrowth curve on PDA at 30°C showing lag, exponential, and stationary phases; \u003cstrong\u003e(B–C) \u003c/strong\u003eEffects of incubation temperature and initial pH on colony growth diameter (mm); \u003cstrong\u003e(D–E) \u003c/strong\u003eEffects of carbon and nitrogen sources on biomass accumulation; maize flour and yeast extract supported optimal growth; \u003cstrong\u003e(F) \u003c/strong\u003eTime-course degradation of 5 mg/L ZEN in MSM showing approximately 98% removal within 36 h; \u003cstrong\u003e(G–H) \u003c/strong\u003eEffects of incubation temperature and initial pH on ZEN degradation efficiency (%) after 36 h. Data represent mean ± SD(n = 5); different letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8695845/v1/3805de4989739620a9bdb8f6.png"},{"id":101931643,"identity":"374d0492-d015-414a-9ead-5b4f9b5bd946","added_by":"auto","created_at":"2026-02-05 07:42:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":183633,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular localization of ZEN-degrading activity and identification of the intermediate metabolite in \u003cem\u003eR. arrhizus\u003c/em\u003eMNQW. \u003cstrong\u003e(A)\u003c/strong\u003e ZEN degradation efficiencies of different cellular fractions after 12 h incubation: culture supernatant (A), heat-inactivated culture supernatant (B), proteinase K–treated culture supernatant (C), intracellular extract obtained by cell disruption (D), heat-inactivated intracellular extract (E), and proteinase K–treated intracellular extract (F); \u003cstrong\u003e(B)\u003c/strong\u003e UPLC–MS/MS chromatograms of α-ZOL detected in the ZEN degradation system (a) and an authentic α-ZOL standard (1 μg/mL) (b); \u003cstrong\u003e(C)\u003c/strong\u003e Time-course profiles of ZEN degradation and transient accumulation followed by complete disappearance of α-ZOL during incubation with strain MNQW. Data are presented as mean ± SD (n = 3); different letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8695845/v1/aa60b3fa3343507780ea7e29.png"},{"id":101931644,"identity":"787d2049-e0eb-4bcc-88c3-d2b7a551e195","added_by":"auto","created_at":"2026-02-05 07:42:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":208665,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity and estrogenic activity of ZEN and its degradation products by strain MNQW. \u003cstrong\u003e(A)\u003c/strong\u003e Relative proliferation of ER-positive MCF-7 cells after 48 h exposure to ZEN, E2, or MNQW degradation products at ZEN-equivalent concentrations, determined by the MTT assay; \u003cstrong\u003e(B)\u003c/strong\u003eRepresentative phase-contrast images of MCF-7 cells corresponding to treatments in panel A; \u003cstrong\u003e(C)\u003c/strong\u003e Relative viability of HepG2 cells after 48 h exposure to ZEN or MNQW degradation products assessed by the MTT assay; \u003cstrong\u003e(D)\u003c/strong\u003eRepresentative phase-contrast images of HepG2 cells corresponding to treatments in panel C. Exposure levels for degradation products were normalized based on the initial molar concentration of ZEN prior to degradation. Data are presented as mean ± SD (n = 3); different letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8695845/v1/3b4190164cea62a5b851fecc.png"},{"id":102295023,"identity":"21922b26-4507-447a-b0e3-7408ab009b21","added_by":"auto","created_at":"2026-02-10 10:07:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":134861,"visible":true,"origin":"","legend":"\u003cp\u003eDetoxification of ZEN-contaminated maize flour by \u003cem\u003eR.arrhizus\u003c/em\u003e MNQW under solid-state fermentation. \u003cstrong\u003e(A)\u003c/strong\u003eTime-course of ZEN degradation in maize flour (initial concentration: 3 mg/kg) during solid-state fermentation with strain MNQW; \u003cstrong\u003e(B)\u003c/strong\u003e Representative UPLC–MS/MS chromatograms showing the absence of α-ZOL in the fermentation system, with comparison to an authentic α-ZOL standard (a) and the ZEN peak detected in the uninoculated control (b); \u003cstrong\u003e(C)\u003c/strong\u003e Effect of spore inoculum level (number of spores) on ZEN degradation efficiency after 36 h of fermentation; \u003cstrong\u003e(D)\u003c/strong\u003e Effect of fermentation temperature on ZEN degradation efficiency after 36 h. Data are presented as mean ± SD (n = 3); different letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8695845/v1/42ab4d05f942ddf4b1bc1436.png"},{"id":107350722,"identity":"545c90d0-1269-4680-8d5e-de0d1269b9b5","added_by":"auto","created_at":"2026-04-20 16:01:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1237260,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8695845/v1/f57dd6de-7222-47df-b902-71a9ab433397.pdf"},{"id":101931648,"identity":"5b0d2267-0b80-46c7-9469-411a4a90e4ac","added_by":"auto","created_at":"2026-02-05 07:42:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1.\u003c/strong\u003e Degradation efficiency of ZEN by microbial isolates obtained from different sources in China.\u003c/p\u003e","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8695845/v1/5e06d331c68319c1b7c746d3.docx"},{"id":101931634,"identity":"3e99264b-f00d-429d-99e0-0a1d2deb9e2c","added_by":"auto","created_at":"2026-02-05 07:41:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11425,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2. \u003c/strong\u003eITS nucleotide sequence of \u003cem\u003eR.arrhizus\u003c/em\u003e strain MNQW.\u003c/p\u003e","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8695845/v1/11f33d02c182db1d68d96c66.docx"},{"id":101931654,"identity":"d800057a-0347-44db-906a-e8695d9eb9e3","added_by":"auto","created_at":"2026-02-05 07:42:04","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14745,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3. \u003c/strong\u003eDetailed fatty acid composition of maize flour before and after solid-state fermentation with \u003cem\u003eR. arrhizus\u003c/em\u003e strain MNQW (36 h, 35℃).\u003c/p\u003e","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8695845/v1/218ef6f5db759c9445919b2f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation and Characterization of a Novel Fungus, Rhizopus arrhizus MNQW, for Effective Biodegradation and Detoxification of Zearalenone","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eZearalenone (ZEN) is a non-steroidal estrogenic mycotoxin predominantly produced by several \u003cem\u003eFusarium\u003c/em\u003e spp. and frequently contaminates cereals such as maize, wheat, and barley (Zinedine et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Owing to its chemical stability during storage and processing, ZEN can persist throughout the food and feed chain, contributing to sustained exposure and resulting in reproductive disorders, hepatotoxicity, immune dysfunction, and potential carcinogenic risks in animals, thereby posing concerns for human health (Minervini \u0026amp; Dell\u0026rsquo;Aquila. 2008; Yang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Moreover, ZEN metabolites, particularly α-zearalenol (α-ZOL), may exhibit comparable or even higher estrogenic activity than the parent compound, further intensifying its toxicological hazards (Rizvi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zinedine et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). To mitigate these risks, regulatory authorities have implemented stringent measures to reduce human exposure to ZEN. In the European Union, maximum levels of 100\u0026ndash;350 \u0026micro;g/kg have been established for cereals and cereal-based products, while the Joint FAO/WHO Expert Committee on Food Additives (JECFA) has established a tolerable daily intake (TDI) of 0.25 \u0026micro;g/kg body weight per day (EFSA Panel on Contaminants in the Food Chain. 2011).\u003c/p\u003e \u003cp\u003eCurrent detoxification strategies for ZEN include physical adsorption, chemical oxidation, and microbial degradation. Physical and chemical approaches can reduce ZEN content, but often impair nutritional value and sensory quality or generate undesirable by-products (Pankaj et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Qi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In contrast, microbial degradation represents an environmentally friendly strategy that operates under mild conditions with high specificity, while preserving nutritional and organoleptic properties (Gari \u0026amp; Abdella. 2023; Hamad et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Murtaza et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although various bacteria (e.g. \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e) and fungi (e.g. \u003cem\u003eClonostachys rosea\u003c/em\u003e, yeasts) have been reported to exhibit ZEN-transforming capabilities, food-grade strains with high degradation efficiency and suitability for industrial fermentation remain scarce (Gari \u0026amp; Abdella. 2023; Ruiz, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe fungal genus \u003cem\u003eRhizopus\u003c/em\u003e is widely utilized in food fermentation and is designated as generally recognized as safe (GRAS) by the U.S. Food and Drug Administration. Species of \u003cem\u003eRhizopus\u003c/em\u003e secrete a broad range of extracellular enzymes, including hydrolases and oxidoreductases, which enable the transformation of diverse substrates and render them promising candidates for mycotoxin bioremediation (Vellozo-Echevarr\u0026iacute;a et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Despite this potential, the degradation of ZEN by \u003cem\u003eRhizopus\u003c/em\u003e spp. has not yet been systematically investigated.\u003c/p\u003e \u003cp\u003eIn this study, we isolated and identified a novel GRAS fungal strain, \u003cem\u003eRhizopus arrhizus\u003c/em\u003e MNQW, from a traditional rice fermentation starter (\u003cem\u003eXiaoqu\u003c/em\u003e). Its growth characteristics, ZEN degradation kinetics, and the localization of active ZEN-degrading components were comprehensively investigated, and the toxicity of the degradation products was evaluated using \u003cem\u003ein vitro\u003c/em\u003e cell culture models. Furthermore, the application of MNQW in the solid-state fermentation of ZEN-contaminated maize flour was explored, with a particular focus on the impact of biodegradation on nutritional quality. Collectively, this study provides a new microbial resource and mechanistic insights for the safe and effective bioremediation of ZEN in food and feed matrices.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Chemicals, media, and reagents\u003c/h2\u003e\n \u003cp\u003eZearalenone (ZEN, \u0026ge;\u0026thinsp;98%) was purchased from Sigma-Aldrich (USA). Tryptone and yeast extract were obtained from Thermo Fisher Scientific (USA). Dipotassium hydrogen phosphate (K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e), potassium dihydrogen phosphate (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), sodium chloride (NaCl), ammonium chloride (NH\u003csub\u003e4\u003c/sub\u003eCl), and magnesium sulfate heptahydrate (MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO) were supplied by Xilong Scientific Co., Ltd. (China). Agar powder was obtained from Solarbio Science \u0026amp; Technology Co., Ltd. (Shanghai, China). TaKaRa Ex Taq polymerase and the EZ‑10 Bacterial Genomic DNA Extraction Kit were purchased from Takara Bio Inc. (Japan) and Sangon Biotech Co., Ltd. (Shanghai, China), respectively. All solvents used for high-performance liquid chromatography (HPLC) and liquid chromatography\u0026ndash;tandem mass spectrometry (LC\u0026ndash;MS/MS) analyses were of HPLC grade.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Screening and isolation of ZEN-degrading strains\u003c/h2\u003e\n \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.1. Sample source\u003c/h2\u003e\n \u003cp\u003eFermented rice starter (\u003cem\u003eXiaoqu\u003c/em\u003e) was obtained from Angel Yeast (China) in January 2023. Upon arrival, the sample was stored at room temperature in a dry environment and used within one week for subsequent experiments.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.2. Enrichment and primary screening\u003c/h2\u003e\n \u003cp\u003eFive grams of \u003cem\u003eXiaoqu\u003c/em\u003e were suspended in 50 mL sterile water and shaken at 180 rpm for 4\u0026ndash;6 h. Subsequently, 1 mL of the supernatant was inoculated into 9 mL of minimal salt medium (MSM) supplemented with 10 mg/L ZEN as the selective pressure (Gari \u0026amp; Abdella. 2023). Cultures were incubated at 30℃, with agitation at 180 rpm for 5 days, followed by three consecutive subculturing cycles. Residual ZEN concentrations were quantified using HPLC, and samples exhibiting ZEN-degrading activity were selected for further isolation.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.3. Isolation of single strains\u003c/h2\u003e\n \u003cp\u003eEnriched cultures (1 mL) were transferred into 9 mL of MSM containing 10 mg/L ZEN, with uninoculated flasks containing MSM supplemented with ZEN serving as controls. Cultures were incubated at 30\u0026deg;C and 180 rpm for 5 days. Cultures exhibiting high ZEN‑degrading activity were serially diluted (10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u0026ndash;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e) and spread onto potato dextrose agar (PDA) plates. Following incubation at 30℃ for 2\u0026ndash;5 days, morphologically distinct colonies were isolated and individually evaluated for ZEN degradation, leading to the identification of a high‑efficiency strain, designated MNQW.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.4. HPLC analysis of ZEN degradation\u003c/h2\u003e\n \u003cp\u003eCulture broth (500 \u0026micro;L) was extracted with an equal volume of HPLC‑grade ethyl acetate, vortexed thoroughly for 5 min, and centrifuged at 10,000 rpm for 5 min. The organic phase was collected, and the extraction was repeated three times. The combined organic phases were evaporated to dryness under nitrogen at 40\u0026ndash;50℃ for approximately 45 min. The residue was reconstituted in 300 \u0026micro;L of HPLC-grade methanol, vortexed for 5 min, filtered through a 0.22 \u0026micro;m polytetrafluoroethylene (PTFE) syringe filter, and transferred into amber HPLC vials. ZEN concentrations were determined using an HPLC system equipped with an Agilent ZORBAX SB-C18 column (250 \u0026times; 4.6 mm, 5 \u0026micro;m). The mobile phase consisted of methanol/water (4:1, v/v) at a flow rate of 0.8 mL/min, with an injection volume of 10 \u0026micro;L and UV detection at 236 nm (Pascari et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). The degradation rate (%) was calculated according to the following equation:\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n \u003cp\u003ewhere \u003cem\u003eA\u003c/em\u003e\u003csub\u003esample\u003c/sub\u003e and \u003cem\u003eA\u003c/em\u003e\u003csub\u003econtrol\u003c/sub\u003e represent the peak areas of ZEN in treated and control samples, respectively.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Identification of strain MNQW\u003c/h2\u003e\n \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\n \u003ch2\u003e2.3.1. Morphological characterization\u003c/h2\u003e\n \u003cp\u003eMNQW was cultured on PDA at 30℃ for 2 days, and colony color, surface texture, margin, and mycelial morphology were recorded. For light microscopy, sterile coverslips were inserted into fresh PDA plates, and 7 mm agar plugs from the colony edges were placed approximately 1 cm from the coverslips, with five replicates. Plates were incubated at 30℃ Hyphae on the coverslips were then examined under a light microscope (EVO-LS10, Sartorius, Beijing, China). For scanning electron microscopy (SEM), MNQW was inoculated on PDA, and the mycelia were fixed with 2.5% glutaraldehyde at 4℃. Hyphal morphology was subsequently observed using a scanning electron microscope (EVO-LS10, Carl Zeiss, Germany) to provide detailed structural information.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n \u003ch2\u003e2.3.2. Molecular identification\u003c/h2\u003e\n \u003cp\u003eGenomic DNA of MNQW was extracted using the EZ-10 Bacterial Genomic DNA Kit (Sangon, China). The internal transcribed spacer (ITS) region was amplified using the universal fungal primers ITS1 (5\u0026apos;-TCCGTAGGTGAACCTGCGG-3\u0026apos;) and ITS4 (5\u0026apos;-TCCTCCGCTTATTGATATGC-3\u0026apos;) with TaKaRa Ex Taq polymerase. PCR products were verified on 1% agarose gel electrophoresis, purified, quantified, and subjected to Sanger sequencing (Sangon, China). ITS sequences were compared against NCBI GenBank using BLAST for taxonomic identification. A phylogenetic tree was constructed using the neighbor-joining method based on ITS sequences from MNQW and reference strains. Bootstrap analysis with 1,000 replications was conducted to assess branch support. Whole-genome sequencing of MNQW was performed on an Illumina NovaSeq 6000 platform using a paired-end library with an insert size of 400 bp, following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Growth and ZEN degradation characterization of strain MNQW\u003c/h2\u003e\n \u003cp\u003eThe growth of MNQW was evaluated by inoculating 7 mm PDA agar plugs onto PDA plates and incubating at 30℃ in the dark for 3\u0026ndash;5 days. Colony diameters were measured every 3 h using the cross method to generate growth curves, with five replicates per condition. The effects of temperature (15\u0026ndash;40℃) and initial pH (4.0\u0026ndash;10.0) were assessed under identical conditions. Additionally, the influence of carbon sources (starch, maize flour, lactose, glucose, maltose, sucrose), and nitrogen sources (urea, yeast extract, peptone, NaNO\u003csub\u003e3\u003c/sub\u003e, NH\u003csub\u003e4\u003c/sub\u003eCl, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) on growth was investigated using MSM-based solid media, with each factor added at 1% of the medium and five replicates per treatment (Tai et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eTo assess the effect of pH on ZEN degradation, MNQW agar plugs were inoculated into 20 mL of MSM containing 5 mg/L ZEN with initial pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0 and 9.0. Cultures were incubated at 35℃ with agitation at 180 rpm for 36 h. To evaluate temperature effects, MSM containing 5 mg/L ZEN (pH 5.0) was incubated at 25, 30, 35, 40 and 45℃ (pH 5.0) under otherwise identical conditions. Residual ZEN was extracted and quantified by HPLC as described in Section \u003cspan class=\"InternalRef\"\u003e2.2.4\u003c/span\u003e. All experiments were performed in triplicate to assess the tolerance and degradation performance of MNQW under different environmental conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Localization of ZEN-degrading activity\u003c/h2\u003e\n \u003cp\u003eA 7 mm agar plug of MNQW was inoculated into 100 mL of potato dextrose broth (PDB) and incubated at 35℃ with agitation at 180 rpm for 3 days. The culture was centrifuged at 8,000 rpm for 10 min, and the resulting supernatant was filtered through a 0.22 \u0026micro;m membrane to obtain the cell-free culture supernatant (CFS). Mycelial pellets were washed twice with phosphate-buffered saline (PBS), blotted dry, rapidly frozen in liquid nitrogen, and ground into a fine powder. The powder was resuspended in pre‑chilled PBS (pH 7.0), centrifuged at 12,000 rpm for 10 min at 4℃, and filtered through a 0.22 \u0026micro;m membrane to obtain the mycelial extract. Aliquots (500 \u0026micro;L) of both CFS and mycelial extract were subjected to heat inactivation (100℃, 10 min) or proteinase K treatment (37℃, 1 h), followed by incubation with 5 mg/L ZEN for 24 h. Residual ZEN was subsequently quantified by HPLC. These treatments were conducted to evaluate the involvement of proteinaceous components in ZEN degradation, consistent with enzyme-mediated mycotoxin degradation mechanisms (Liu et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Analysis of ZEN degradation products\u003c/h2\u003e\n \u003cp\u003eZEN degradation products were prepared under the previously determined optimal conditions. Briefly, strain MNQW was inoculated into 10 mL of MSM (pH 5.5) supplemented with ZEN at a final concentration of 5 \u0026micro;g/mL and incubated at 35℃. Uninoculated MSM containing the same concentration of ZEN served as the control. All experiments were conducted in triplicate. Samples were collected at 24 h intervals, and ZEN together with its degradation products were extracted according to the procedure described in Section \u003cspan class=\"InternalRef\"\u003e2.2.4\u003c/span\u003e. The extracted samples were initially analyzed by ultra-performance liquid chromatography\u0026ndash;tandem mass spectrometry (UPLC\u0026ndash;MS/MS) using a SCIEX Triple Quad\u0026trade; 3500 system (SCIEX, USA) equipped with an ACE UltraCore Super C18 column (3.0 \u0026times; 150 mm, 2.5 \u0026micro;m). Compound annotation was performed using TraceFinder and Discovery software, following previously reported analytical strategies for mycotoxin degradation products (Ji et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. Cytotoxicity assessment\u003c/h2\u003e\n \u003cp\u003eMCF-7 and HepG2 cells were seeded at 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well in 96-well plates and incubated for 12 h at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. MCF-7 cells were cultured in phenol-red-free Dulbecco\u0026rsquo;s modified Eagle medium (DMEM) supplemented with charcoal-dextran-treated fetal bovine serum (FBS), whereas HepG2 cells were cultured in standard DMEM. Cells were treated with ZEN (10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M for MCF-7 and 1 to 25 mg/L for HepG2) or the corresponding degradation products for 24 h. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and calculated as follows:\u003c/p\u003e\n \u003cdiv id=\"Equb\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ewhere \u003cem\u003eA\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e and \u003cem\u003eA\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e represent the absorbances of the treatment and control groups, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8. Application in ZEN-contaminated maize flour\u003c/h2\u003e\n \u003cp\u003eZEN-contaminated maize flour (400 mg/kg), prepared from \u003cem\u003eFusarium\u003c/em\u003e-inoculated cracked maize, was mixed with uncontaminated commercial maize flour to achieve a final ZEN concentration of 3 mg/kg. The mixture was dried at 65℃ to constant weight and stored at 4℃. MNQW spores were harvested from V8 agar cultures grown at 35℃ for 3\u0026ndash;4 days and adjusted to 10\u003csup\u003e7\u003c/sup\u003e CFU/mL.\u003c/p\u003e\n \u003cp\u003eFor degradation assays, 8 g portions of maize flour were placed in 90-mm Petri dishes and inoculated with 2 mL of spore suspension; controls received 2 mL of sterile PBS. Samples were incubated at 35℃, and moisture was maintained by spraying 3 mL PBS every 3 h. Subsamples were collected at 12, 24, 36, and 48 h. Additional experiments evaluated the effects of temperature (18\u0026ndash;40℃) and inoculum levels (10\u003csup\u003e5\u003c/sup\u003e\u0026ndash;10\u003csup\u003e9\u003c/sup\u003e CFU/mL). Nutritional composition of MNQW-fermented maize flour\u0026mdash;including moisture, starch, crude protein, crude fat, amino acids, and vitamins B\u003csub\u003e1\u003c/sub\u003e and B\u003csub\u003e2\u003c/sub\u003e\u0026mdash;was analyzed according to Chinese National Standards (GB/T 6435\u0026thinsp;\u0026minus;\u0026thinsp;2006, GB 5009.9\u0026ndash;2016, GB/T 6432\u0026thinsp;\u0026minus;\u0026thinsp;2018, GB/T 6433\u0026thinsp;\u0026minus;\u0026thinsp;2006, GB/T 18246\u0026thinsp;\u0026minus;\u0026thinsp;2000, GB/T 14700\u0026thinsp;\u0026minus;\u0026thinsp;2018, GB/T 14701\u0026thinsp;\u0026minus;\u0026thinsp;2019).\u003c/p\u003e\n \u003cp\u003eZEN was extracted from maize flour using 20 mL of 80% acetonitrile containing 0.1% formic acid and filtered prior to LC\u0026ndash;MS/MS analysis (Sulyok et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). Quantification was performed using an Agilent Eclipse XDB-C18 column (2.1 \u0026times; 150 mm, 3.5 \u0026micro;m, 40℃) under gradient elution with 0.1% formic acid\u0026ndash;water (A) and methanol (B) at 0.4 mL/min (Leeman et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e). ZEN degradation efficiency was calculated as:\u003c/p\u003e\n \u003cdiv id=\"Equc\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ewhere \u003cem\u003eC\u003c/em\u003e\u003csub\u003econtrol\u003c/sub\u003e and \u003cem\u003eC\u003c/em\u003e\u003csub\u003esample\u003c/sub\u003e denote ZEN concentrations in the control and treatment samples, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e\n \u003cp\u003eAll experiments were performed in triplicate. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). Statistical analyses were conducted using one-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison test in OriginPro software (OriginLab, USA). Differences were considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Screening and characterization of the ZEN-degrading strain MNQW\u003c/h2\u003e\n \u003cp\u003eZEN is a non-steroidal estrogenic mycotoxin frequently detected in cereals and cereal-derived products, exerting its toxicity primarily through competitive binding to estrogen receptors (ERs) and subsequent endocrine disruption (Chen et al., \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e; el-Sharkawy et al. \u003cspan class=\"CitationRef\"\u003e1991\u003c/span\u003e). Chronic dietary exposure to ZEN has been associated with reproductive disorders and an increased risk of hormone-dependent tumors (Singh et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Conventional detoxification strategies, including physical adsorption and chemical treatment, often suffer from limited efficiency, nutrient loss, or secondary contamination (Lach \u0026amp; Kotarska. 2024). Microbial degradation, in contrast, has emerged as a promising alternative due to its substrate specificity, mild reaction conditions, and environmental compatibility (Sun et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eScreening of microbial isolates obtained from cereal- and soil-derived samples across different regions of China revealed pronounced variability in ZEN-degrading capacity (Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Among the tested isolates, a filamentous fungal strain designated MNQW, isolated from a traditional fermented rice starter (\u003cem\u003eXiaoqu\u003c/em\u003e), exhibited the highest degradation efficiency, removing approximately 98% of 5 mg/L ZEN in mineral salt medium within 72 h at 30℃ (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). In contrast, the remaining isolates showed only moderate or limited degradation activities, with efficiencies generally below 60%. Compared with previously reported ZEN-degrading bacteria and fungi, including \u003cem\u003eBacillus\u003c/em\u003e spp. and \u003cem\u003eGeobacillus\u003c/em\u003e spp., which often require lower toxin concentrations or extended incubation periods to achieve comparable removal rates, MNQW demonstrated superior degradation performance under relatively stringent conditions (Liu et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sun et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). These results identify MNQW as a highly efficient ZEN-degrading fungus with strong potential for application in food and feed detoxification.\u003c/p\u003e\n \u003cp\u003eBased on morphological features, ITS rRNA gene sequencing, and whole-genome analysis, MNQW was identified as \u003cem\u003eR. arrhizus\u003c/em\u003e. The strain formed dense, cotton-like aerial mycelia with compact gray-brown colonies, unbranched sporangiophores, and ellipsoidal sporangiospores, consistent with typical species descriptions (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;D). ITS sequence analysis revealed 99.84% similarity to \u003cem\u003eR. arrhizus\u003c/em\u003e (MH865594.1) with 93% bootstrap support in the phylogenetic tree, confirming its taxonomic assignment (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE, Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). Whole-genome sequencing yielded a 39.19 Mb draft genome comprising 12,651 predicted protein-coding genes, with functional annotation performed against the NCBI non-redundant protein database (NR), Swiss-Prot, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and the Carbohydrate-Active enZYmes database (CAZy) databases (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). Notably, genes encoding oxidoreductases, hydrolases, and CAZymes\u0026mdash;enzyme families frequently implicated in xenobiotic transformation and fungal secondary metabolism\u0026mdash;were identified, suggesting a versatile enzymatic system potentially responsible for ZEN biotransformation.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eR. arrhizus\u003c/em\u003e is a ubiquitous filamentous fungus widely distributed in soil and plant-associated environments and is known for rapid growth, low nutritional requirements, and high environmental adaptability. The species has been extensively exploited for production of organic acids and industrial enzymes, such as lipases and amylases, and its mycelial biomass has demonstrated adsorption and immobilization capacities for hazardous compounds, highlighting its bioremediation potential (Guo et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e; Meng et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e). Despite these advantages, reports on ZEN biodegradation by \u003cem\u003eR. arrhizus\u003c/em\u003e remain scarce. Most ZEN-degrading fungi reported to date belong to genera such as \u003cem\u003eAspergillus\u003c/em\u003e spp., \u003cem\u003eTrichoderma\u003c/em\u003e spp., and \u003cem\u003eClonostachys\u003c/em\u003e spp., with only limited evidence implicating \u003cem\u003eR. arrhizus\u003c/em\u003e in ZEN transformation (el-Sharkawy et al. \u003cspan class=\"CitationRef\"\u003e1991\u003c/span\u003e). Therefore, the identification of \u003cem\u003eR. arrhizus\u003c/em\u003e MNQW as a highly efficient ZEN-degrading strain expands the known functional repertoire of this species and provides new insights into its potential application in the detoxification of contaminated food and feed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Growth characteristics and degradation kinetics of strain MNQW under varied environmental conditions\u003c/h2\u003e\n \u003cp\u003eThe adaptability of microorganisms to environmental conditions is a critical determinant of their practical applicability in food and feed systems. Most food and feed processing environments are mildly acidic; however, many previously reported ZEN-degrading microorganisms exhibit optimal activity under neutral to alkaline conditions, which substantially limits their industrial applicability (Sun et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, evaluating the growth behavior and degradation performance of strain MNQW under varied environmental conditions is essential.\u003c/p\u003e\n \u003cp\u003eThe growth curve of MNQW exhibited a short lag phase during the initial 16 h, followed by a pronounced exponential phase from 16 to 31 h, before entering the stationary phase (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Strain MNQW demonstrated remarkable environmental adaptability, with optimal growth observed at temperatures ranging from 35 to 40℃ and within a mildly acidic pH range of 5.0\u0026ndash;6.0 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;C). Carbon source profiling revealed a clear preference for complex substrates, particularly maize flour, over simple sugars (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). Although maize flour is not fully soluble in liquid media, it forms a stable suspension and can be gradually hydrolyzed and assimilated through extracellular enzymatic activities, a process that closely resembles substrate utilization in practical feed fermentation systems. Regarding nitrogen sources, yeast extract supported the highest biomass accumulation; notably, however, MNQW also exhibited robust growth when sodium nitrate (NaNO\u003csub\u003e3\u003c/sub\u003e) was supplied as the sole nitrogen source (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE), highlighting its potential for cost-effective industrial application.\u003c/p\u003e\n \u003cp\u003eConsistent with its favorable growth characteristics, MNQW displayed efficient ZEN degradation kinetics, achieving approximately 98% removal of 5 mg/L ZEN within 36 h (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). The degradation rate peaked during the exponential growth phase and gradually declined as the substrate concentration decreased. Temperature exerted a significant influence on degradation efficiency, with maximal activity (approximately 96%) observed between 35 and 40℃, followed by a sharp decline at 45℃ (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG). Similarly, pH profiling revealed optimal degradation at pH 5.5, with sustained activity across a range of 4.5\u0026ndash;6.0 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e\n \u003cp\u003eThe close alignment between optimal growth conditions and ZEN degradation performance underscores the metabolic efficiency of MNQW under mildly acidic environments. This physiological trait is particularly advantageous for feed applications, where organic acids such as fumaric acid\u0026mdash;a major fermentation product of R. arrhizus\u0026mdash;are commonly incorporated as acidifiers (Kuenz et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Such acidic conditions not only suppress undesirable microbial growth but also enhance feed palatability, especially in swine production systems (Xu et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Combined with its ability to utilize inexpensive nitrogen sources such as NaNO\u003csub\u003e3\u003c/sub\u003e and complex agricultural substrates, MNQW exhibits strong economic and practical potential for large-scale mycotoxin detoxification, distinguishing it from previously reported fungal strains with more limited environmental tolerance (Guo et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e; Meng et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Subcellular localization and metabolite identification of ZEN degradation\u003c/h2\u003e\n \u003cp\u003eTo localize the ZEN-degrading activity in strain MNQW, the degradation capacities of different cellular fractions were evaluated. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, the culture supernatant exhibited limited ZEN degradation activity (10.31%), whereas the intracellular extract retained a substantially higher degradation efficiency (88.23%) within 12 h. Heat treatment of the intracellular extract markedly reduced the degradation efficiency to 10.64%, and subsequent proteinase K treatment almost completely abolished the activity. These results indicate that the ZEN-degrading activity of strain MNQW is predominantly associated with intracellular proteinaceous components, suggesting an enzyme-mediated transformation process. This intracellular localization presents a significant practical advantage for feed applications, as it minimizes the risk of enzyme leakage during processing, thereby enhancing safety compared to strains relying on extracellular enzymes (Liu et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe metabolic fate of ZEN during incubation with strain MNQW was further investigated using UPLC-MS/MS analysis. Among the detected compounds, \u0026alpha;-ZOL was identified as the only ZEN-related intermediate metabolite. The identity of \u0026alpha;-ZOL was confirmed by comparison of its retention time and mass spectral characteristics with those of an authentic \u0026alpha;-ZOL standard (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Quantitative analysis revealed that \u0026alpha;-ZOL accumulated only transiently, reaching a maximum level corresponding to 1.32% of the initial ZEN concentration, and was no longer detectable after 7 days of incubation (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results indicate that \u0026alpha;-ZOL did not persist as a stable transformation product during ZEN degradation by strain MNQW.\u003c/p\u003e\n \u003cp\u003eSeveral additional differential compounds, including 3-pyridinemethanol, adenosine, acetyl-L-carnitine, and triphenyl phosphate, were detected in the metabolite profiles of the MNQW culture. These compounds are considered endogenous metabolites of strain MNQW and showed no apparent structural relationship with ZEN. Notably, no other ZEN-derived products with characteristic UV absorption were detected under the analytical conditions employed, suggesting that a substantial proportion of ZEN was transformed into products not readily detectable by UV-based UPLC-MS/MS analysis. However, the chemical structures and biological activities of the final transformation products require further investigation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Cytotoxicity and estrogenic activity of ZEN degradation products\u003c/h2\u003e\n \u003cp\u003eThe detoxification efficacy of strain MNQW was further evaluated using \u003cem\u003ein vitro\u003c/em\u003e bioassays targeting both estrogenic activity and cytotoxicity. Considering the estrogen-like effects of ZEN at low concentrations, ER-positive human breast cancer cells (MCF-7) were selected to assess residual estrogenic activity, while human hepatocellular carcinoma cells (HepG2) were employed to evaluate cytotoxicity, reflecting the primary metabolic target organ of ZEN \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eIn MCF-7 cells, cell proliferation was first quantified using the MTT assay (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). As expected, native ZEN induced a typical biphasic response, characterized by significant proliferative stimulation at low concentrations and growth inhibition at higher concentrations, consistent with its well-documented estrogenic properties. In contrast, treatment with MNQW degradation products did not induce any significant proliferative response across the tested concentration range (10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u0026ndash;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol/L). Notably, the degradation products represent a complex mixture; therefore, exposure levels were normalized based on the initial molar concentration of ZEN prior to degradation, rather than individual metabolite molarity, a strategy commonly adopted in microbial mycotoxin detoxification studies (Guo et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wu et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e). Morphological observations further supported the quantitative results (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Cells treated with 17\u0026beta;-estradiol (E2) or low concentrations of ZEN exhibited increased cell density and partial multilayer growth, whereas cells exposed to ZEN degradation products maintained clear boundaries and a typical monolayer morphology comparable to the untreated control, indicating the absence of detectable estrogenic stimulation.\u003c/p\u003e\n \u003cp\u003eThe cytotoxicity of ZEN and its degradation products was subsequently assessed in HepG2 cells. MTT assays revealed a marked, concentration-dependent reduction in cell viability following ZEN exposure, whereas cells treated with degradation products maintained significantly higher viability at corresponding exposure levels (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Consistently, microscopic examination showed extensive cell damage and loss of adhesion in ZEN-treated cells, while cells exposed to degradation products retained intact morphology and normal growth patterns (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\n \u003cp\u003eAlthough \u0026alpha;-ZOL, a metabolite reported to exhibit equal or higher estrogenic potency than ZEN, was transiently detected during the degradation process, no estrogenic or cytotoxic effects were observed for the final degradation products in either cell model. This suggests that \u0026alpha;-ZOL did not accumulate to biologically relevant levels and was further transformed by strain MNQW. Collectively, these results demonstrate that MNQW-mediated degradation of ZEN effectively eliminates both estrogenic activity and cytotoxicity, underscoring the potential of strain MNQW for safe and practical application in mycotoxin detoxification in feed systems (Wang et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Detoxification of ZEN-contaminated maize flour via solid-state fermentation\u003c/h2\u003e\n \u003cp\u003eApproximately 70% of maize produced in China is utilized for animal feed, making the development of effective detoxification strategies for mycotoxin-contaminated cereals essential to ensure feed safety. In this study, the practical detoxification performance of \u003cem\u003eR. arrhizus\u003c/em\u003e MNQW was evaluated using ZEN-contaminated maize flour under solid-state fermentation conditions. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, ZEN levels in maize flour (initial concentration 3 mg/kg) decreased continuously during fermentation with strain MNQW and were completely eliminated within 36 h, whereas no significant change was observed in the uninoculated control. Importantly, \u0026alpha;-ZOL, a metabolite with higher estrogenic potency than ZEN, was not detected throughout the entire 72-h fermentation period (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating that MNQW mediates a safe detoxification pathway without accumulation of hazardous intermediates.\u003c/p\u003e\n \u003cp\u003eTo facilitate practical application, key fermentation parameters were optimized. The highest detoxification efficiency was achieved at 35\u0026deg;C with an inoculum level of 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, resulting in more than 97% ZEN degradation within 36 h (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;D). Increasing the temperature to 45\u0026deg;C markedly reduced degradation efficiency, consistent with the growth characteristics of MNQW, while excessive inoculum levels promoted premature sporulation, adversely affecting the sensory quality of the fermented maize flour. These results highlight the importance of balancing detoxification efficiency with product quality and process feasibility.\u003c/p\u003e\n \u003cp\u003eCompared with previously reported bacterial or yeast-based detoxification systems, MNQW exhibits several notable advantages, including rapid and complete ZEN removal, food-grade safety, compatibility with solid-state fermentation, and the absence of estrogenically active by-products. Given that \u003cem\u003eRhizopus\u003c/em\u003e species are widely used in food and feed fermentation and recognized as safe microorganisms, MNQW represents a promising candidate for industrial-scale detoxification of ZEN-contaminated feed materials (Acs-Szabo et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e; Podg\u0026oacute;rska-Kryszczuk et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhou et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6. Nutritional enhancement of maize flour through MNQW fermentation\u003c/h2\u003e\n \u003cp\u003eUnder optimized fermentation conditions, solid-state fermentation with \u003cem\u003eR. arrhizus\u003c/em\u003e MNQW markedly improved the nutritional quality of maize flour (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Specifically, crude protein content increased by 2.66%, accompanied by pronounced changes in B-vitamin composition and a substantial restructuring of the fatty acid profile. This simultaneous enhancement of detoxification efficiency and nutritional quality represents a dual functional benefit that is rarely reported for ZEN-degrading microorganisms (Uwineza et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). The increase in crude protein can be attributed to microbial biomass accumulation and a substrate concentration effect associated with an overall mass reduction of 8.25% during fermentation. Notable alterations were also observed in the vitamin profile: although vitamin B\u003csub\u003e1\u003c/sub\u003e decreased, vitamin B\u003csub\u003e2\u003c/sub\u003e and folate levels increased substantially, resulting in a more balanced B-vitamin composition. Balanced B-vitamin intake is particularly relevant for livestock with limited capacity for endogenous vitamin retention. Fermentation also significantly reshaped the fatty acid profile. Oleic acid content increased by 12.32%, whereas linoleic acid decreased by 7.19%. At the class level, total monounsaturated fatty acids (MUFA) increased markedly, saturated fatty acids (SFA) increased moderately, and polyunsaturated fatty acids (PUFA) decreased slightly. This redistribution of the SFA\u0026ndash;MUFA\u0026ndash;PUFA balance indicates that MNQW fermentation systematically restructures the lipid matrix rather than simply altering individual fatty acids, which may contribute to improved oxidative stability, feed palatability, and energy utilization efficiency. Detailed fatty acid composition before and after fermentation is provided in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eChanges in nutritional composition and major fatty acid classes of maize flour after solid-state fermentation with \u003cem\u003eR. arrhizus\u003c/em\u003e strain MNQW (36 h, 35℃).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eComponent\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBefore fermentation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter fermentation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChange\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMajor nutrients\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCorn meal weight (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e91.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-8.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrude Protein (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e+\u0026thinsp;2.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStarch (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e74.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e51.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-22.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVitamin B\u003csub\u003e1\u003c/sub\u003e (mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVitamin B\u003csub\u003e2\u003c/sub\u003e (mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e+\u0026thinsp;3.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFolate (mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e378.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e390.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e+\u0026thinsp;11.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMajor fatty acids (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC16:0 (Palmitic acid)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e+\u0026thinsp;7.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC18:0 (Stearic acid)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e+\u0026thinsp;3.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC18:1n9c (Oleic acid)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e+\u0026thinsp;12.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC18:2n6c (Linoleic acid)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e49.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e42.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-7.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC18:3n3 (\u0026alpha;-Linolenic acid)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC18:3n6 (\u0026gamma;-Linolenic acid)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e+\u0026thinsp;1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFatty acid classes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026sum;SFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e+\u0026thinsp;14.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026sum;MUFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e+\u0026thinsp;18.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026sum;PUFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e51.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-5.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026sum;USFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e91.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e+\u0026thinsp;12.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eNote:\u0026nbsp;\u003c/strong\u003eValues are expressed as percentages of total fatty acids (for fatty acids) or absolute content (for nutrients). The \u0026ldquo;Change\u0026rdquo; column represents the difference (After fermentation \u0026minus; Before fermentation). \u0026sum;SFA, total saturated fatty acids; \u0026sum;MUFA, total monounsaturated fatty acids; \u0026sum;PUFA, total polyunsaturated fatty acids; \u0026sum;USFA, total unsaturated fatty acids (\u0026sum;USFA = \u0026sum;MUFA + \u0026sum;PUFA, not mutually exclusive). Detailed fatty acid composition is provided in Table S3.\u003c/p\u003e\n \u003cp\u003eThese compositional changes are consistent with the metabolic characteristics of \u003cem\u003eR. arrhizus\u003c/em\u003e, which secretes \u0026alpha;-amylase and lipase during fermentation, promoting starch hydrolysis and lipid remodeling (Anigboro et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Terefe et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Collectively, these findings demonstrate that MNQW fermentation simultaneously detoxifies ZEN-contaminated maize flour and enhances its nutritional quality by increasing protein content, optimizing fatty acid distribution, and improving B-vitamin balance, highlighting the strong potential of MNQW as a sustainable, food-grade microbial agent for the bioprocessing of contaminated feed materials.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study reports the isolation and comprehensive characterization of a novel food-grade fungal strain, \u003cem\u003eR\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;arrhizus\u003c/em\u003e MNQW, from a traditional fermented rice starter (\u003cem\u003eXiaoqu\u003c/em\u003e), and demonstrates its high efficiency for ZEN biodegradation. Morphological, molecular, and genomic analyses confirmed its taxonomic identity and revealed the presence of multiple oxidoreductase- and hydrolase-encoding genes that may contribute to ZEN transformation. Strain MNQW rapidly degraded more than 98% of ZEN (5 mg/L) under mildly acidic conditions (35℃, pH 5.5), predominantly via heat-labile intracellular proteinaceous components, indicating an enzyme-mediated degradation mechanism. UPLC\u0026ndash;MS/MS analysis showed that \u0026alpha;-ZOL was only transiently formed at trace levels and did not accumulate during the degradation process. Importantly, the final degradation products exhibited no detectable estrogenic activity in ER-positive MCF-7 cells and no apparent cytotoxicity in HepG2 cells under the tested conditions, demonstrating effective biological detoxification of ZEN. Application of MNQW in ZEN-contaminated maize flour under solid-state fermentation resulted in rapid and complete detoxification, achieving approximately 97% ZEN removal within 36 h without the formation of estrogenically active intermediates. In parallel, MNQW fermentation significantly improved the nutritional quality of maize flour by increasing crude protein content, enhancing vitamin B\u003csub\u003e2\u003c/sub\u003e and folate levels, and favorably restructuring the fatty acid profile, thereby conferring a dual benefit of detoxification and nutritional enhancement.\u0026nbsp;Although MNQW shows considerable promise as a safe and efficient microbial agent for controlling ZEN contamination in cereal-based feed materials, further studies are warranted to identify the key enzymes involved, elucidate the underlying genetic determinants, and validate its efficacy and safety \u003cem\u003ein vivo\u003c/em\u003e. Overall, these findings highlight the strong potential of \u003cem\u003eR. arrhizus\u003c/em\u003e MNQW for sustainable mycotoxin detoxification and value-added bioprocessing in feed systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDan He:\u003c/strong\u003e Conceptualization, Methodology, Investigation, Validation, Formal analysis, Data curation, Writing – original draft, Writing – review \u0026amp; editing, Visualization.\u0026nbsp;\u003cstrong\u003eYunfan Shan:\u003c/strong\u003e Formal analysis, Methodology, Writing – review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eHan Qiu:\u003c/strong\u003e Methodology, Investigation, Formal analysis,\u0026nbsp;Data curation.\u0026nbsp;\u003cstrong\u003eGang Wang:\u003c/strong\u003e Supervision, Writing – review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eJunqiang Hu:\u003c/strong\u003e Writing – review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eYuzhuo Wu:\u003c/strong\u003e Validation.\u0026nbsp;\u003cstrong\u003eKeke Ji:\u003c/strong\u003e Methodology.\u0026nbsp;\u003cstrong\u003eHao Xu:\u003c/strong\u003e Validation.\u0026nbsp;\u003cstrong\u003eYin-Won Lee:\u003c/strong\u003e Supervision, Writing – review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eJianhong Xu:\u003c/strong\u003e Resources, Funding acquisition, Project administration, Supervision, Writing – review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eXin Yan:\u003c/strong\u003e Supervision, Writing – review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Key R\u0026amp;D Program of China (2023YFD1301004), the National Natural Science Foundation of China (32372454), the Jiangsu Province Science and Technology Support Program (BE2022377), and the Jiangsu Agricultural Science and Technology Program (CX(23)1002). Additional support was provided by the National Special Project for Agro-product Safety Risk Evaluation of China (GJFP20240102) and the State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products (No. 2021DG700024-KF202513).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcs-Szabo L, Pfliegler WP, Kov\u0026aacute;cs S, Ad\u0026aacute;csi C, R\u0026aacute;cz HV, Horv\u0026aacute;th E, Papp LA, Murvai KP, Kir\u0026aacute;ly S, Mikl\u0026oacute;s I, P\u0026eacute;ter G, Pusztahelyi T, P\u0026oacute;csi I. (2025) Striking mycotoxin tolerance and zearalenone elimination capacity of the decaying wood associated yeast Sugiyamaella novakii (Trichomonascaceae). 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Food Chem Toxicol. 45(1):1-18.https://doi.org/10.1016/j.fct.2006.07.030\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Zearalenone, Rhizopus arrhizus, Biodegradation, Solid-state fermentation, Mycotoxin detoxification, Food safety","lastPublishedDoi":"10.21203/rs.3.rs-8695845/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8695845/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eZearalenone (ZEN) is a prevalent estrogenic mycotoxin in cereals and feedstuffs, posing persistent risks to feed safety and animal health. In this study, a food-grade filamentous fungus, \u003cem\u003eRhizopus arrhizus\u003c/em\u003e MNQW, was isolated from a traditional rice starter (\u003cem\u003eXiaoqu\u003c/em\u003e) and systematically evaluated for its ZEN biodegradation capacity and practical applicability. Strain MNQW efficiently removed over 98% of 5 mg/L ZEN in minimal salt medium within 36 h under mild fermentation-compatible conditions. Subcellular fractionation and inhibition assays indicated that ZEN degradation was predominantly mediated by heat- and protease-sensitive intracellular enzymes. UPLC\u0026ndash;MS/MS analysis revealed only transient formation of α-zearalenol at trace levels, followed by complete detoxification without accumulation of estrogenically active intermediates. \u003cem\u003eIn vitro\u003c/em\u003e bioassays using estrogen receptor-positive (ER-positive) MCF-7 and HepG2 cells confirmed that the final degradation products exhibited neither estrogenic activity nor cytotoxicity. Importantly, application of MNQW in solid-state fermentation of ZEN-contaminated maize flour (3 mg/kg) achieved approximately 97% toxin removal within 36 h while simultaneously improving nutritional quality, including increased crude protein, vitamin B\u003csub\u003e2\u003c/sub\u003e, folate, and beneficial fatty acids. Whole-genome analysis identified multiple oxidoreductase- and hydrolase-encoding genes potentially involved in ZEN biotransformation. Collectively, these findings demonstrate that \u003cem\u003eR. arrhizus\u003c/em\u003e MNQW represents a safe, efficient, and application-ready microbial candidate for detoxification and value-added processing of ZEN-contaminated feed materials.\u003c/p\u003e","manuscriptTitle":"Isolation and Characterization of a Novel Fungus, Rhizopus arrhizus MNQW, for Effective Biodegradation and Detoxification of Zearalenone","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-05 07:40:27","doi":"10.21203/rs.3.rs-8695845/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-02T12:05:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T10:43:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-24T22:47:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"222595134325730566584730597407827950606","date":"2026-02-04T18:48:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33388705673947248675141454337548364352","date":"2026-02-04T17:23:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-03T11:31:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-28T09:53:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-28T07:29:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2026-01-26T02:47:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4830d239-6a78-407d-acd0-b901d250032d","owner":[],"postedDate":"February 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:00:30+00:00","versionOfRecord":{"articleIdentity":"rs-8695845","link":"https://doi.org/10.1007/s11274-026-04963-5","journal":{"identity":"world-journal-of-microbiology-and-biotechnology","isVorOnly":false,"title":"World Journal of Microbiology and Biotechnology"},"publishedOn":"2026-04-18 15:57:28","publishedOnDateReadable":"April 18th, 2026"},"versionCreatedAt":"2026-02-05 07:40:27","video":"","vorDoi":"10.1007/s11274-026-04963-5","vorDoiUrl":"https://doi.org/10.1007/s11274-026-04963-5","workflowStages":[]},"version":"v1","identity":"rs-8695845","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8695845","identity":"rs-8695845","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}