Domestication Cultivation and Nutritional Analysis of Hericium coralloides

Domestication Cultivation and Nutritional Analysis of Hericium coralloides | 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 Domestication Cultivation and Nutritional Analysis of Hericium coralloides Yun Li, Jiarong Cai, Xiaomin Li, Xin Hu, Junli Zhang, Xiaopin Wu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5897250/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract [Objective] : To isolate and identify a wild strain resembling Hericium coralloides (strain SH001) collected from the wild, explore its biological characteristics, and investigate the cultivation, nutritional composition, and antioxidant and anticancer activities of its fruiting body polysaccharides. [Methods] : The strain was identified based on its morphological characteristics and ITS sequence analysis. The biological properties were assessed by evaluating mycelial growth under varying conditions, including different carbon and nitrogen sources, temperatures, and pH values. Nutritional analysis of the fruiting body was conducted using Kjeldahl nitrogen determination, Soxhlet extraction, and light scattering detection methods. The antioxidant potential of the polysaccharides was evaluated through assays measuring DPPH, ABTS, OH radical scavenging activities, and iron ion reduction capacity. The anticancer effects of the polysaccharides on HepG 2 liver cancer cells and MDA-MB-468 breast cancer cells were assessed using the MTT assay. [Results] : The strain was identified as Hericium coralloides . The optimal growth conditions were found to be 30°C, pH 7, fructose as the preferred carbon source, and yeast extract as the optimal nitrogen source. Nutritional analysis revealed that the fruiting body was rich in protein (15.4 g/100 g dry weight), dietary fiber (34.7 g/100 g dry weight), and minerals, while being low in fat (3.5 g/100 g dry weight). The most abundant amino acids were glutamic acid, followed by aspartic acid. The polysaccharides exhibited significant antioxidant activity, with ABTS scavenging comparable to that of Vitamin C, achieving a clearance rate of 96.95% at concentrations between 0.25–5 mg/mL. At a concentration of 5 mg/mL, the DPPH and OH radical scavenging activities reached their peak (83.77% and 67.31%, respectively), along with the highest iron ion reducing capacity (FRAP value: 4.43 mmol/L). Polysaccharides also exhibited notable anticancer activity, inhibiting HepG2 liver cancer cells and MDA-MB-468 breast cancer cells, with IC50 values of 3.896 mg/mL and 2.561 mg/mL, respectively. [Conclusion] : This study demonstrates that wild Hericium coralloides can be successfully cultivated in vitro. The fruiting bodies possess substantial nutritional value, and the polysaccharides extracted from them show promising antioxidant and anticancer activities, particularly against HepG 2 liver cancer cells and MDA-MB-468 breast cancer cells. Hericium coralloides biological characteristics nutritional composition antioxidant properties culture conditions domestication and cultivation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Hericium coralloides (Scop.) Pers., a member of the Basidiomycota phylum, Agaricomycetes class, Russulales order, and Hericiaceae family, is closely related to Hericium erinaceus (Bu.) Pers.(Li et al. 2015). This species, resembling coral in appearance, features a bright white coloration with a narrow base and coral-like branching, with fine branches densely covered with spines. H. coralloides is primarily found on the trunks or decayed wood of broad-leaved trees in Northeast, Northwest, and Southwest China (Mao et al. 2000). As a rare edible and medicinal fungus, it is valued for its substantial nutritional and pharmacological properties. The genus Hericium is one of the most renowned sources of edible and medicinal fungi in China. In recent years, it has been extensively utilized in the development of new pharmaceutical products. The mycelial polysaccharides from this genus have demonstrated a wide range of bioactivities, including anti-tumor, immune-enhancing, anti-coagulation, and cholesterol-lowering effects. Notably, the polysaccharides of Hericium erinaceus are well-known for significantly boosting immune function and exhibiting anti-cancer properties. Recent studies have shown that Hericium coralloides can alleviate Alzheimer’s disease and cognitive dysfunction by activating the Nrf2 signaling pathway and modulating the gut microbiota (Guan et al. 2024; Guan Y et al. 2023). Moreover, its metabolic products contain 18 amino acids, contributing to tonic, digestive, and therapeutic functions, such as in the treatment of neurosis and gastric ulcers. Additionally, H. coralloides is rich in various bioactive polysaccharides, with proven anti-tumor, immune-boosting, antioxidant, anti-coagulant, and cholesterol-lowering effects (Wu et al. 2019; Tabibzadeh 2022; Williams et al. 2023; Zhang et al. 2019; Cheng et al. 2018). Among these, β-glucan, the most abundant polysaccharide, is often referred to as "immune gold" due to its potent biological and pharmacological properties, leading to its widespread use in pharmaceuticals, health products, and the food industry. Due to its appealing appearance and rich nutritional and medicinal values, Hericium coralloides has gained considerable popularity among consumers in recent years, with promising prospects for industrial development. Like other edible fungi, such as black fungus, shiitake mushrooms, and reishi, H. coralloides is a wood-decaying fungus that primarily utilizes lignocellulose as its nutritional source. Research into selecting superior strains and developing novel cultivation substrates for H. coralloides has made significant strides, resulting in technological breakthroughs. Large-scale production of this highly nutritious and delicious fungus is anticipated in the near future, offering it to a broader consumer base. However, there is limited research on the utilization of wild H. coralloides resources. This study aims to investigate the optimal cultivation conditions for the mycelium of H. coralloides , develop liquid inoculum, and successfully induce fruiting. The nutritional composition of the resulting fruiting bodies is analyzed, and the bioactivity of polysaccharides extracted from these bodies is explored, providing valuable insights for the future development and commercial exploitation of Hericium coralloides. Material and methods Experimental Materials The experimental material was a wild fruiting body sample, numbered SH001, collected from Qinduo Town, Bomi County, Tibet Autonomous Region, from which mycelium was obtained after tissue separation. Main Reagents Maltose, mannose, fructose, soluble starch, sucrose, glucose, tryptone, yeast extract, beef extract, copper sulfate, potassium sulfate, anhydrous ether, anhydrous ethanol, acetic acid, sulfuric acid, sodium acetate, sodium hydroxide, potassium nitrate, ammonium nitrate, urea, magnesium sulfate, vitamin B 1 , potassium dihydrogen phosphate, hydrochloric acid, 2-mercaptoethanol, and other reagents (analytical grade, purchased from China National Pharmaceutical Group Corporation).D3390-01 Fungal DNA Kit (OMEGA Bio-Tek, USA); DPPH, ABTS, TPTZ, and ascorbic acid (Sigma-Aldrich, USA).Cell culture medium (Hyclone, USA).Fetal bovine serum (Gibco, USA). Experimental Culture Medium Enriched PDA Medium:200 g of peeled potatoes, 20 g of glucose, 5 g of peptone, 2 g of K 2 HPO 4 , 1.5 g of MgSO 4 , 10 mg of vitamin B 1 , 20 g of agar, and 1 L of distilled water, adjusted to natural pH. Liquid Inoculum Culture Medium:200 g of peeled potatoes, 20 g of fructose, 2 g of ammonium sulfate, 1.5 g of magnesium sulfate, 2 g of potassium dihydrogen phosphate, 10 mg of vitamin B1, and 1 L of distilled water, pH 5.0. Substrate for Cultivation:60% hardwood sawdust, 20% cottonseed hulls, 18% wheat bran, 1% lime, and 1% sugar, mixed in the appropriate proportions with water to achieve a final moisture content of 60%. Morphological Identification of Strain SH001 The morphological characteristics of both the fruiting body and the mycelium of strain SH001 were examined. The observations were made with reference to the work of Li et al. (2015). Molecular Identification of Strain SH001 The mycelium DNA of strain SH001 was extracted using the OMEGA Fungal DNA Extraction Kit. The extracted DNA was used as a template for PCR amplification with the universal fungal primers ITS1/ITS4, following the amplification and sequencing protocols described by Liu et al. (Liu et al. 2020). After amplification, 3 µL of the PCR product was subjected to 1% agarose gel electrophoresis, and a single bright band was observed. The remaining PCR product was then sent to Fuzhou Baijing Biotechnology Co., Ltd. for sequencing. The resulting ITS sequence was submitted to the NCBI Nucleotide Database ( http://www.ncbi.nlm.nih.gov ) for BLAST analysis. High-homology ITS sequences were downloaded, and a phylogenetic tree was constructed using MEGA 11 software to determine the taxonomic classification of the strain. Biological Characterization of Strain SH001 Effect of Carbon Sources on Mycelial Growth Different carbon sources, including glucose, sucrose, fructose, maltose, mannose, and starch, were used to replace glucose in the enriched PDA medium, while keeping all other components constant. Activated fungal cultures were inoculated by making a 5 mm hole at the edge of the agar plates, and small fungal blocks were transferred to the center of the plates containing different carbon source media (9 cm diameter). The plates were incubated in the dark at 25°C. The colony diameter was measured using the "cross" method. Each treatment was performed in five replicates. The mycelial growth rate (mm/day) was calculated as: Mycelial Growth Rate (mm/day)= (Colony Diameter (mm) − Inoculum Diameter (mm))/ Growth Duration (days)​. (1) Effect of Nitrogen Sources on Mycelial Growth Different nitrogen sources, including urea, yeast extract, ammonium sulfate, beef extract, peptone, and ammonium nitrate, were used to replace peptone in the enriched PDA medium, with all other components remaining constant. Fungal blocks were inoculated at the center of 9 cm diameter plates containing media with different nitrogen sources. The procedure was carried out as described for the effect of carbon sources on mycelial growth. Effect of pH on Mycelial Growth Enriched PDA medium was used to investigate the effect of pH on mycelial growth. The pH was adjusted to values of 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 using 1.0 mol/L NaOH and 1.0 mol/L HCl solutions. The procedure was conducted as described for the effect of carbon sources on mycelial growth. Effect of Temperature on Mycelial Growth Enriched PDA medium was used to investigate the effect of temperature on mycelial growth. Following inoculation, the plates were incubated in constant temperature incubators at 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C under dark conditions. The procedure was performed as described for the effect of carbon sources on mycelial growth. Domestication and Cultivation Trials of Hericium coralloides Preparation of Liquid Mycelial Culture The liquid mycelial culture medium was aliquoted into Erlenmeyer flasks, with 100 mL per flask, and sterilized in a high-temperature, high-pressure autoclave for future use. Under a sterile laminar flow hood, PDA agar plugs (7 mm in diameter) were inoculated into shake flasks, with six plugs per flask, and sealed with stoppers. The flasks were then incubated in the dark at 23°C with shaking at 160 rpm. cottonseed hulls were fully soaked in water for 12 hours, then thoroughly drained. A mixture of fermented wood sawdust, pre-moistened cottonseed hulls, wheat bran, lime, and white sugar was prepared in appropriate proportions. Water was added incrementally and mixed thoroughly to ensure the substrate absorbed sufficient moisture, maintaining a moisture content of approximately 60%. The pH was adjusted to 5.0. The substrate was aliquoted into cultivation bags, with 900 g (wet weight) per bag, and sterilized in an autoclave at 121°C for 3 hours. After cooling to room temperature, the bags were transferred to a sterile laminar flow hood, where liquid mycelial culture was carefully inoculated, with each flask inoculating five bags. Following inoculation, the bags were placed in a mycelial growth chamber and incubated at 22–25°C under dark conditions for mycelial colonization. After the mycelium has fully colonized the substrate and undergone a one-week maturation period, the bags were promptly transferred to the fruiting room for bag opening and fruiting induction. The temperature was maintained at 18–20°C, and the relative humidity was increased to 95%. Once the primordia appeared, the temperature was adjusted to 20–23°C to promote further maturation and differentiation of the fruiting bodies. Nutritional Composition Analysis Moisture Content Determination The moisture content was determined using the direct drying method. An appropriate amount of fruiting body was weighed (W₁) in a pre-weighed dish (W₀), dried at 70°C until constant weight was achieved, cooled in a desiccator, and reweighed (W₂). The calculation formula is as follows: Moisture Content （g/100g）= [（W₁−W₂）/(W₁−W₀)]×（100g/100g）. （2) Protein Content Determination The protein content was measured according to the Kjeldahl method as referenced by Amin R et al. (2018). The fruiting body was weighed and digested with a potassium sulfate-copper sulfate catalyst and concentrated sulfuric acid, heated at 380–420°C until a clear solution was obtained. After cooling, the mixture was alkalinized, distilled, and the released ammonia was absorbed in boric acid. The solution was titrated with 0.01mol/L HCl standard solution to the endpoint, and the volume difference was recorded. Blank controls and instrument calibration were performed. The calculation formula is as follows: Nitrogen content (g/100g) = [(Vsample -Vblank )×C×14.01/msample]×100. ( 3 ) Protein content = Nitrogen content × 6.25. ( 4 ) Ash Content Determination Ash content was determined using the high-temperature incineration method as described by Uffelman CN et al. (2023). An appropriate amount of sample (precisely 0.001 g) was weighed into a pre-weighed crucible, carbonized until smokeless, and incinerated in a muffle furnace at 550°C for 4–6 hours until gray-white ash was obtained. The crucible was cooled to 200°C in the furnace, transferred to a desiccator, and weighed at room temperature. The incineration was repeated until constant weight was achieved (weight difference ≤ 0.5 mg). Blank correction and desiccator hygroscopicity control were performed.The calculation formula is as follows: Ash content (g/100g) = [(ash + crucible weight − crucible weight)/sample mass]×100. ( 5 ) Fat Content Determination Fat content was measured following the Soxhlet extraction method as outlined by Amin R et al. (2018). The sample was crushed, dried, homogenized, and packed into filter paper thimbles, which were then placed in a Soxhlet extractor. Excess diethyl ether was added, and the system was refluxed for 10 hours to dissolve fats. After extraction, the solvent was evaporated, and the fat-containing flask was dried to constant weight. The fat mass was measured gravimetrically. Fat Content (g/100g) = [fat mass/sample mass×100]. ( 6 ) Total Sugar Content Determination The sample was ground and passed through an 80-mesh sieve, and 5 g was weighed into a 100 mL volumetric flask. After adding 50 mL of water to dissolve the sample, petroleum ether was used for defatting, and the residue was transferred back to the volumetric flask. Subsequently, 5 mL of zinc acetate solution (21.9 g/100 mL) and 5 mL of potassium ferrocyanide solution (10.6 g/100 mL) were added, followed by magnetic stirring for 30 min. The solution was then diluted to the mark with water. After filtration through dry filter paper, the initial filtrate was discarded, and the subsequent filtrate was passed through a 0.45 µm membrane for further analysis. A sugar-specific amino column (250 × 4.6 mm, 5 µm) was used for separation, with a mobile phase of acetonitrile-water (70:30, v/v) at a flow rate of 1.0 mL/min and a column temperature of 40°C. An evaporative light scattering detector (ELSD) was employed with a drift tube temperature of 85°C and a nitrogen pressure of 350 kPa. A standard curve was constructed using glucose, fructose, sucrose, maltose, and lactose standards (0–10 mg/mL), and the total sugar content was calculated according to the formula. Total sugar content (g/100g) = =[(ρ − ρ 0 )×V×n/(m×1000)]×100 (ρ: concentration of the sample solution, mg/mL; V: volume, mL; n: dilution factor; m: sample mass, g) ( 7 ) Dietary Fiber Content Determination Dietary fiber content was quantified using the enzymatic gravimetric method as referenced by Phillips KM et al. (2021). The fruiting body was crushed and sequentially hydrolyzed with α-amylase, protease, and glucosidase (pH 8.2, 37°C) to remove starch and proteins. The residue was precipitated with ethanol, filtered into a pre-weighed sintered glass crucible, washed with 78% ethanol and acetone, dried to constant weight at 105°C, and weighed. Dietary fiber content (g/100g) = [(residue mass − blank)/sample mass]×100. ( 8 ) Sodium Content Determination Sodium content was determined using flame atomic emission spectrometry as per Moniruzzaman M et al. (2014). The sample was precisely weighed (0.001 g), digested with nitric acid via microwave, diluted to volume, and filtered. Sodium standard solutions were prepared, and emission intensity was measured at 589 nm using flame atomic emission spectroscopy (FAES) to construct a calibration curve. The sample solution was analyzed identically. Sodium content (g/100g) = [(measured concentration×dilution factor×0.1)/sample mass]×100. ( 9 ) Amino Acid Content Determination Amino acid content was analyzed using an automatic amino acid analyzer, as referenced by Purkiewicz A et al. (2023). The sample was hydrolyzed with 6 mol/L hydrochloric acid at 110°C for 24 h, diluted to volume, centrifuged, and filtered. The filtrate was injected into an amino acid analyzer, separated via ion-exchange chromatography, and subjected to post-column derivatization with ninhydrin. Peak areas of individual amino acids were detected. Calibration curves were generated using standard amino acid solutions. Total amino acid content (g/100g) = [∑(concentration of each amino acid×dilution factor×0.1)/sample mass]×100. ( 10 ) Three parallel experiments were conducted for each analysis, and the average values of the respective indicators were calculated. Exploration of the Bioactivity of Hericium coralloides Polysaccharides Preparation of Polysaccharides from Hericium coralloides Fruiting Bodies The method described by Smiderle FR et al. (2011) was slightly modified as follows: After freeze-drying, the fruiting bodies were ground into powder and sieved through an 80-mesh screen for use. A ratio of 1:20 (g:mL) of the sample to 75% ethanol was added, followed by ultrasonic treatment for 2 hours. The filtrate was removed by suction filtration. Then, a ratio of 1:30 (g:mL) of the sample to distilled water was added and extracted in a 90°C water bath for 2 hours. The extraction was repeated twice, and the extracts were combined. The extract was concentrated in a rotary evaporator to one-third of its original volume. Anhydrous ethanol was added at a 4:1 (v/v) ratio, and the mixture was left at 4°C overnight for alcohol precipitation. The solution was centrifuged at 5,000 rpm for 10 minutes, and the precipitate was collected. The precipitate was re-dissolved in water and mixed with an equal volume of Sevage solution (chloroform:n-butanol = 4:1), followed by stirring and centrifugation at 10,000 rpm for 3 minutes. The supernatant was collected, and the process was repeated until no protein or other impurities were present. The polysaccharide solution was then dialyzed for 48 hours against running water, and freeze-dried to a constant weight, yielding the crude polysaccharide freeze-dried powder from the fruiting bodies. Chemical Antioxidant Activity of Hericium coralloides Fruiting Body Polysaccharides Hydroxyl Radical Scavenging Activity Assay The method described by Ding L et al. (2021) was slightly modified as follows: Polysaccharide solutions with concentrations of 0.025, 0.05, 0.25, 0.5, 1, 2, and 5 mg/mL, along with vitamin C (VC) solutions, were accurately prepared. In a 96-well plate, 75 µL of polysaccharide solution at each concentration was added to the respective wells. Subsequently, 15 µL of FeSO 4 solution (9 mmol/L), salicylic acid-ethanol solution (9 mmol/L), and H 2 O 2 solution (8.8 mmol/L) were added sequentially to each well. The plate was gently shaken to ensure proper mixing. Finally, 100 µL of distilled water was added to each well. The plate was then incubated in a 37°C water bath for 30 minutes, and absorbance at 510 nm was measured, recorded as Ay. Vitamin C was used as a positive control. The reaction system for the blank group was prepared by replacing the polysaccharide sample with distilled water, and the absorbance was recorded as Ao. For the control group, distilled water was used instead of the H 2 O 2 solution, and the absorbance was recorded as Ap. Five replicates were performed for each concentration, and the average values were calculated.The calculation formula is as follows: Hydroxyl radical scavenging rate (%) = [1 − (Ay − Ap) / Ao] × 100 ( 11 ) ABTS Radical Scavenging Activity Assay The method described by Miller et al.(1993) was slightly modified as follows: A 5 mL aliquot of 7.0 mmol/L ABTS solution and 88 µL of 2.45 mmol/L potassium persulfate aqueous solution were mixed and kept in the dark at room temperature for 12–16 hours to prepare the ABTS + stock solution. The stock solution was then diluted with distilled water, and its absorbance at 734 nm was measured using a spectrophotometer to adjust the absorbance to 0.70 ± 0.023, which was used as the working ABTS + solution, prepared fresh for each use. In a 96-well plate, 100 µL of polysaccharide solutions at concentrations of 0.025, 0.05, 0.25, 0.5, 1, 2, and 5 mg/mL, along with the ABTS + working solution, were added to each well and mixed thoroughly. The plate was incubated in the dark at 25°C for 20 minutes, and absorbance at 734 nm was measured using a microplate reader (denoted as Ay). Vitamin C (VC) was used as a positive control. For the blank group, the polysaccharide sample was replaced with distilled water, and the absorbance was recorded as Ao. In the control group, distilled water was used instead of the ABTS + solution, and the absorbance was recorded as Ap. Five replicates were performed for each concentration. The calculation formula is as follows: ABTS Radical Scavenging Rate (%) = [1 − (Ay − Ap) / Ao] × 100 ( 12 ) DPPH Radical Scavenging Activity Assay The method described by Saiga A et al.(2003) was slightly modified as follows: Aliquots of 100 µL of polysaccharide solutions (0.025, 0.05, 0.25, 0.5, 1, 2, and 5 mg/mL) and 0.2 mmol/L DPPH solution were added sequentially to a 96-well plate. The plate was gently shaken to ensure thorough mixing, and the reaction was allowed to proceed in the dark at room temperature for 30 minutes. Absorbance at 517 nm was then measured and recorded as Ay. Vitamin C (VC) was used as a positive control. The blank group reaction system was prepared by replacing the polysaccharide sample with absolute ethanol, and the absorbance was recorded as Ao. In the control group, absolute ethanol was used instead of the DPPH solution, and the absorbance was recorded as Ap. Five replicates were performed for each concentration. The DPPH radical scavenging activity was calculated using the following formula: DPPH Radical Scavenging Activity (%) = [1 − (Ay − Ap) / Ao] × 100 ( 13 ) Ferric Ion Reducing Antioxidant Power (FRAP) Assay The method described by Benzie et al. (1996) was slightly modified as follows: A FRAP working solution was prepared by mixing 0.3 mol/L acetate-acetic acid buffer (pH 3.6), 0.02 mol/L FeCl3 solution, and 0.01 mol/L TPTZ solution in a volume ratio of 10:1:1, and used fresh. Aliquots of 0.5 mL of FeSO4 solutions at concentrations of 0.025, 0.1, 0.15, 0.2, 0.4, 0.5, 0.8, 1.0, and 1.5 mmol/L were added to 3.0 mL of the FRAP working solution. The mixtures were thoroughly mixed and incubated at 37°C for 15 minutes. Absorbance at 593 nm was measured to construct a standard curve. The same procedure was used to determine the absorbance of each reaction system. Specifically, for the reaction mixtures containing polysaccharide solutions at concentrations of 0.025, 0.05, 0.25, 0.5, 1, 2, and 5 mg/mL, combined with the FRAP working solution, the absorbance at 593 nm was recorded as Ay. In the blank and control groups, distilled water replaced the polysaccharide sample and the FRAP working solution, with the corresponding absorbance values recorded as Ao and Ap, respectively. The FRAP value was calculated by determining the difference between Ay, Ao, and Ap, and then referring to the standard curve to obtain the corresponding FeSO4 concentration. Toxicity of Hericium coralloides Polysaccharides on Different Cancer Cell The HepG 2 and MDA-MB-231 cancer cell lines were cultured in cell culture medium at 37°C with 5% CO 2 in a humidified incubator for subculturing. Upon reaching the logarithmic growth phase and achieving 80–90% confluence, the cells were harvested for plating. The original culture medium was discarded, and the cells were washed three times with PBS. Trypsin was then added to digest the cells for 30 seconds, followed by the addition of fresh culture medium to terminate the digestion. The cell suspension was centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cell pellet was resuspended in culture medium to ensure even dispersion. The cell density was adjusted to 1×10 ^5 cells/mL. A 200 µL aliquot of the cell suspension was added to each well of a 96-well plate, with 200 µL of PBS added to the outermost wells as blanks. The plate was incubated at 37°C with 5% CO 2 for cell attachment. Once the cells had fully adhered, the original medium was removed, and treatment with polysaccharide was administered. The treatment groups included a polysaccharide group, a blank group, and a control group. The polysaccharide group received 200 µL of culture medium containing polysaccharides at different concentrations (0.025, 0.05, 0.25, 0.5, 1, 2, and 5 mg/mL). The control group received 200 µL of drug-free culture medium, and the blank group consisted of medium without cells, serving as the zero control. Each group was set up with five replicate wells. The plate was incubated at 37°C with 5% CO 2 for 24 hours. After incubation, the culture medium was discarded, and 100 µL of culture medium containing 10% MTT was added to each well. The plate was incubated for an additional 4 hours. Afterward, the medium was removed, and 150 µL of dimethyl sulfoxide (DMSO) was added to each well. The plate was gently shaken until the purple-brown precipitate was completely dissolved, and the absorbance at 490 nm was measured using a microplate reader. Cell viability at different polysaccharide concentrations was calculated using the following formula, and the IC50 value was determined. Cell viability (%) = [(OD of polysaccharide group - OD of blank group) / (OD of control group - OD of blank group)] × 100. ( 14 ) Results Identification of Wild Hericium coralloides Strain Morphological Identification A wild Hericium coralloides specimen was collected from a broadleaf forest in Milin County, Nyingchi City, Tibet. The wild fruiting body was white, with a long and thick stalk, and coral-like branches. The cap surface was rough, exhibiting radial grooves that extended outward up to 8 cm and reached a width of 10 cm and a height of 5 cm. Upon comparison, these characteristics were consistent with the description of Hericium coralloides in the "Illustrated Guide to Major Fungal Resources in China" (Li et al. 2015). Molecular Identification : The ITS sequence of strain SH001 (NCBI accession number: PQ094906) was compared for homology using GenBank. The results showed that the ITS sequence of strain SH001 shared 98.93% homology with the reported sequence of Hericium coralloides (MG735348). Using Russula lutea as the outgroup, ITS sequences of other Hericium species were included, and a phylogenetic tree was constructed using the neighbor-joining method. The tree (Fig. 2 ) indicated that strain SH001 grouped closely with Hericium coralloides and showed a considerable genetic distance from other Hericium species. Based on the combined results from both morphological observation and molecular biology identification, the tested strain SH001 was confirmed to be Hericium coralloides . Effects of Culture Conditions on H. coralloides Mycelial Growth Optimization of Culture Conditions Carbon Source: The mycelium of Hericium coralloides SH001 can grow on various carbon sources, but significant differences in growth were observed. The mycelium exhibited the fastest growth when cultured on fructose, showing a white, dense, and robust appearance, with optimal growth performance. In contrast, starch resulted in the poorest growth, significantly lower than the control group. The growth rates, in descending order, were: fructose (10.15 mm/d) > glucose (8.81 mm/d) > mannitol (7.61 mm/d) > maltose (5.63 mm/d) > sucrose (3.53 mm/d) > starch (1.31 mm/d) (Fig. 3 A-a). Overall, fructose is identified as the most suitable carbon source for the mycelial growth of Hericium coralloides . Nitrogen Source: The mycelium of SH001 was capable of growth on various nitrogen sources, with noticeable differences in growth performance. The fastest growth occurred when cultured with yeast extract, and the growth rates were ranked from highest to lowest as follows: yeast extract (9.85 mm/d) > ammonium nitrate (8.25 mm/d) > peptone (8.23 mm/d) > ammonium sulfate (7.93 mm/d) > beef extract (7.78 mm/d) (Fig. 3 B-b). No growth was observed when urea was used as the nitrogen source. Overall, yeast extract was the most effective nitrogen source for promoting robust, dense, and healthy mycelial growth in Hericium coralloides . pH: The mycelium of Hericium coralloides SH001 can grow within a pH range of 5.0 to 10.0. The fastest growth rate was observed at pH 5, with a growth rate of 8.71 mm/d. No significant difference in growth rates was found between pH 6 and pH 7 (P > 0.05), though the mycelium at pH 7 appeared denser and more compact than at pH 5.0. The growth rate at pH 5.0 was significantly slower compared to pH 9 and pH 10 (P < 0.01). Based on the growth rate and overall performance, pH 7 was found to be the most suitable for optimal mycelial growth. Temperature: Hericium coralloides SH001 does not tolerate low temperatures and cannot grow at 15°C. The growth rate of the mycelium increased progressively with temperature. The growth rates, from highest to lowest, were: 30°C > 35°C > 40°C > 25°C > 20°C > 15°C. The mycelium grew fastest at 30°C (8.40 mm/d) and 35°C (7.10 mm/d), followed by 40°C (6.25 mm/d) (Fig. 3 D-d). At 30°C, the mycelium was the densest and exhibited well-defined edges, while at 35°C, the mycelium was also dense, but with slightly less defined edges. In conclusion, the optimal temperature for mycelial growth of Hericium coralloides SH001 is 30°C. Domestication of H. coralloides After 28 days, the mycelium had fully colonized the bags. Following an additional 10 days under optimal environmental conditions, pale yellow primordia began to appear. The temperature was maintained between 20 and 23°C, with humidity exceeding 90%. Ten days after the bags were opened, the fruiting bodies reached maturity and were harvested. The average fresh weight of the first flush of fruiting bodies per bag was 249.07 g, with a second flush also emerging. The growth conditions of the cultivated fruiting bodies are illustrated in Fig. 4 . As shown, both the fruiting bodies and mycelium of Hericium coralloides SH001 were white (Figs. 4 ). The fruiting bodies displayed coral-like branches, which further branched into small twigs, from which dense, small spines developed. Nutrient Content of Subentity Nutrient Content of H. coralloides Subentity Routine nutritional analysis (Table 1 ) showed that the contents of crude protein, ash, fat, total sugars, dietary fiber, and sodium in Hericium coralloides were 15.4 g/100 g dry weight, 6.8 g/100 g dry weight, 3.5 g/100 g dry weight, 1.6 g/100 g dry weight, 34.7 g/100 g dry weight, and 10.0 mg/100 g dry weigh t , respectively. Although the protein content of Hericium cor-alloides is lower than that of its congeneric species Hericium erinaceus , it still falls within the typical range (8–24%) observed in eight common edible mushrooms (shiitake, oyster, enoki, white button, velvet, king oyster, tea tree, and black fungus) (Li et al. 2021). The fat content is within the general range (2–4%) for edible mushrooms (Zheng et al. 2016), and is lower than that found in Hericium erinaceus . The crude ash content in wild edible mushrooms, which consists of inorganic salts and heavy metals, serves as an indicator of the heavy metal content in the soil of the mushroom's growing environment (Wen et al. 2021) and the con-tribution of minerals to nutritional value. The ash content of Hericium coralloides is comparable to that of Hericium erinaceus . May provide nutritional advantages, such as a rich content of minerals. The crude fiber content of Hericium coralloides is slightly higher than that of the common mushroom Pleurotus eryngii (Han et al., 2015). with significant variations in fiber content across different edible mushrooms (Chen et al. 2015). Sodium, an essential trace mineral for humans, cannot be synthesized in the body and must be obtained through food or water. It plays a vital role in muscle and nerve tissue. The sodium content of Hericium coralloides is 10.0 mg/100 g, suggesting that supplementation with trace minerals via fungi could be beneficial. Table 1 Content of nutritional components in fruit body of Hericium coralloide. Nutrient composition Cotent (g·100-1g-1 dry weight) Moisture* (g/100g) (fresh) Hericium coralloides 91.68 Hericium erinaceus (Cohen et al. 2014) - Moisture** (g/100g) (dry) Carbohydrate(g/100g) 10.0 64.3 6.2 61.1 Crude protein (g/100g) 15.4 ± 1.2 20.08 Ash (g/100g) 6.8 ± 0.8 6.8 Fat (g/100g) 3.5 ± 0.5 5.1 Total sugar (g/100g) 1.6 ± 0.2 - Na (mg/100g) Dietary fiber (g/100g) 10.0 ± 1.5 34.7 ± 0.7 - - Energy (kcal/100g) 280.1 374 * Represents the fresh weight measurement. **Represented as Total Carbohydrates. Amino Acids The radar chart in Fig. 5 illustrates the amino acid composition of Hericium coralloides . The concentration of medicinal amino acids was the highest (4.69 g/100 g dry weight), followed by sweet-tasting amino acids (2.81 g/100 g dry weight), umami amino acids (2.56 g/100 g dry weight), bitter amino acids (2.40 g/100 g dry weight), and neutral amino acids (0.61 g/100 g dry weight). The sweet-tasting amino acids primarily contribute to the flavor of Hericium coralloides , though the presence of sweet and salty compounds often masks the bitterness, ultimately defining the flavor profile. The most abundant amino acids are glutamic acid, followed by aspartic acid, which enhance the umami flavor of the mushroom. Arginine, a medicinal amino acid, is also essential for children's healthy development. Overall, the amino acid profile and composition align with that of a high-quality protein source. Fifteen amino acids were detected in the Hericium coralloides samples, with cysteine (Cys), methionine (Met), and tryptophan (Trp) not detected. This suggests that either Hericium coralloides contains no measurable levels of Cys, Met, and Trp, or that their concentrations are too low to be detected by the instrumentation. The ratio of essential amino acids to total amino acids in Hericium coralloides was 0.32, while the ratio of essential to non-essential amino acids was 0.47. The E/T and E/N values are close to the ideal values for high-quality proteins recommended by the Food and Agriculture Organization/World Health Organization (approximately 0.40 and ≥ 0.60, respectively) (Pellett et al. 1990). Table 2 Content of amino acids in fruitbodies of Hericium coralloides Antioxidant Activity of H. coralloides Polysaccharide The antioxidant capacity of Hericium coralloides polysaccharides is illustrated in Fig. 6 . As shown in Fig. 6 A, at a concentration of 0.25 mg/mL, the ABTS radical scavenging rate reached 96.95%, which is comparable to the positive control group. Within the concentration range of 0.25–5 mg/mL, the scavenging activity was similar to that of ascorbic acid (VC), demonstrating significant antioxidant activity with an EC50 value of 0.04 mg/mL. Figure 6 B indicates that as the concentration increased, the difference in DPPH radical scavenging activity between the sample and the positive control (VC) gradually decreased. The DPPH radical scavenging ability exhibited a dose-dependent enhancement, reaching a peak of 83.77% at 5 mg/mL, with an EC50 value of 1.417 mg/mL. In Fig. 6 C, at polysaccharide concentrations ranging from 0 to 1 mg/mL, the scavenging ability against hydroxyl radicals did not differ significantly from that of the VC group. However, as the concentration increased, the scavenging rate of hydroxyl radicals increased progressively, although it remained significantly lower than that of VC. The highest scavenging rate, 67.31%, was achieved at a concentration of 5 mg/mL, with an EC50 value of 2.655 mg/mL. Figure 6 D demonstrates that, with increasing polysaccharide concentration, the ferric ion reducing power positively correlated with concentration, reaching a maximum FRAP value of 4.43 mmol/L at 5 mg/mL. However, at the same concentration, the reducing power was significantly lower than that of the VC group. In Vitro Anticancer Activity of H. coralloides Polysaccharides Fungi are valuable biological resources (Dávila et al. 2020), and with advancements in science and technology, the medicinal potential of fungal polysaccharides has attracted increasing attention. Fungal polysaccharides are non-cytotoxic compounds that do not exhibit harmful side effects on normal cells (Chang et al. 2022). They have demonstrated the ability to inhibit cancer cell proliferation (Li et al. 2020) and possess antioxidant properties. To assess the anticancer effects of Hericium coralloides polysaccharides, an MTT assay was conducted to evaluate their toxicity on HepG2 and MDA-MB-468 cells. The results revealed that Hericium coralloides polysaccharides inhibited the growth of both cancer cell lines to some extent. A clear dose-dependent relationship was observed between polysaccharide concentration and cell viability. As the concentration of polysaccharides increased, the inhibitory effect on cancer cell growth became more pronounced. At a concentration of 5 mg/mL, the lowest cell viability was observed. The IC50 values for HepG2 and MDA-MB-468 cells were 3.896 mg/mL and 2.561 mg/mL, respectively, indicating that Hericium coralloides polysaccharides exerted a stronger inhibitory effect on MDA-MB-468 cells compared to HepG2 cells. Discussion Traditional fungal classification has primarily been based on the observation and description of morphological traits, which group fungi into different categories according to these features (Justo et al. 2017). However, fruiting bodies may exhibit polymorphism influenced by environmental factors and nutritional conditions. In contrast, the ITS rDNA region evolves at a relatively rapid rate, displaying considerable sequence polymorphism. Furthermore, its short length facilitates amplification and sequencing, making it widely utilized in fungal taxonomy (Zhou et al. 2016). In this study, a combination of morphological observation and ITS sequence-based molecular identification was employed to characterize a wild strain (SH001) from Tibet. The results showed that the ITS sequence of this strain closely resembled that of strain MG735348.1, establishing a close phylogenetic relationship with Hericium erinaceum , and confirming its identification as Hericium coralloides . Additionally, the study observed that the strain exhibited accelerated mycelial growth at pH 5 and 30°C when yeast extract was used as the nitrogen source and fructose as the carbon source. Early studies have explored high-yield cultivation techniques for Hericium coralloides . Huang et al. (2022) found that a substrate consisting of 88% cottonseed hulls, 10% wheat bran, 1% gypsum, and 1% malic acid could increase the yield of Hericium coralloides to 150–200 g (Zhuang et al. 2024). Zhang et al. (2024) conducted domestication and cultivation research on two wild Hericium coralloides strains from the Xiao Xing'anling region, demonstrating that a substrate containing 83% broadleaf sawdust, 15% wheat bran, 1% gypsum, and 1% sucrose achieved a fruiting body yield of 136.5 g, with a biological efficiency of 63.4% (Zhang et al. 2024). Through interviews with local farmers in Fujian, we selected suitable cultivation substrates, increasing the yield of Hericium coralloides to 249 g. This provides a new reference for cultivating Hericium coralloides in regions with similar climatic conditions to Fujian. This study found that the fruiting bodies of Hericium coralloides contain 26.3% protein (dry weight basis). Although the protein content of Hericium coralloides is lower than that of its congeneric species Hericium erinaceus , it still falls within the typical range (8–24%) observed in eight common edible mushrooms (shiitake, oyster, enoki, white button, velvet, king oyster, tea tree, and black fungus) and is significantly comparable to legumes (Sar et al. 2020).Additionally, the fruiting bodies are rich in 15 amino acids, including six essential ones, with a ratio of essential amino acids to total amino acids of 0.32. The low content of total sugars and lipids makes this fungus a low-sugar, low-fat food source, providing valuable insights for the development of H. coralloides -based products. The bioactivity of H. coralloides extracellular polysaccharides (EPS) was also examined. Preliminary results demonstrated that H. coralloides EPS exhibits notable antioxidant activity. The EC50 values were 1.417 mg/mL for DPPH, 0.04 mg/mL for ABTS, and 2.655 mg/mL for hydroxyl radical scavenging. Similarly, Tabibzadeh et al. (2022) investigated the bioactivity of H. coralloides polysaccharides collected in Iran, confirming their antioxidant properties, with EC50 values of 4.12 mg/mL for DPPH, 17.0 mg/mL for FRAP, and 2.83 mg/mL for ABTS. In comparison, the polysaccharides from our successfully domesticated and cultivated H. coralloides exhibited superior antioxidant activity. Within a concentration range of 0.25–5 mg/mL, the DPPH and ABTS radical scavenging effects were comparable to those of ascorbic acid (VC). Experimental evidence suggests that polysaccharides from the Hericium genus exhibit significant anticancer potential (Hyder et al. 2021). For instance, polysaccharides from H. erinaceus have shown anticancer activity against human liver cancer (HepG2), breast cancer (MCF-7), and colon cancer (HCT116) cells (Hetland et al. 2020). These polysaccharides effectively inhibit the proliferation and colony formation of SGC-7901 cells by inducing apoptosis in the S phase and arresting the cell cycle (Zan et al. 2015). In line with these findings, our study demonstrates that H. coralloides EPS is capable of inducing apoptosis in human liver cancer (HepG2) and triple-negative breast cancer (MDA-MB-468) cells. In this research, we isolated and identified a wild Hericium coralloides strain from Tibet, China. By optimizing the cultivation substrate, we significantly enhanced the yield of H. coralloides fruiting bodies. The analysis of the fruiting bodies' nutritional composition, along with the antioxidant and anticancer properties of its polysaccharides, underscores the potential of H. coralloides as a promising dietary supplement for cancer therapy. However, further studies are necessary to investigate its time-dependent effects on cancer cell elimination and to validate its efficacy in vivo. This study holds important implications for the large-scale conservation of wild fungal germplasm and the expansion of the global edible fungal resource database. Additionally, it contributes to the economic exploration of wild fungal germplasm and offers valuable insights for the future development of cancer therapies, potentially making a significant contribution to human health. Declarations Ethics Statement : Not applicable to this work. Author Contributions : J.S.F. and X.H. designed the eperiments; Y.L. and J.R.C. preparedthema-terials; J.R.C. and Y.L.carriedouttheexperiments; X.M.L. and J.L.Z. analyzed the data and worte the manuscript.X.P.W revised the manuscript.All authors have read and agreed the published version of the manuscript. Funding : This work was supported by Key Development Project of Science and Technology Department of Tibet, grant number XZ202301ZY0013N and XZ202001ZY0041N. Data Availability Statement : The specimens and strains and their data were deposited at Mycological Research Center, College of Life Sciences, College of Life Sciences, Fujian Agriculture and Forestry University. Conflicts of Interest : The authors declare no conflict of intere. References Li Y, Li T-H,Yang z-l, (2015) Atlas of Chinese Macrofungal Resources [M]. Zhongyuan Peasant Publishing House 76, 430. Mao X-L, (2000). Large Fungi of China [M]. Henan Science and Technology Publishing House. Guan Y, Wang C, Li L, et al (2024) Structural characterization of hericium coralloides polysaccharide and its neuroprotective function in alzheimer’s disease. Int J Biol Macromol 277:133865. https://doi.org/10.1016/j.ijbiomac.2024.133865 Guan Y, Shi D, Wang S, et al (2023) Hericium coralloides ameliorates alzheimer’s disease pathologies and cognitive disorders by activating Nrf2 signaling and regulating gut microbiota. Nutrients 15:3799. https://doi.org/10.3390/nu15173799 Wu F, Zhou L-W, Yang Z-L, et al (2019) Resource diversity of chinese macrofungi: edible, medicinal and poisonous species. Fungal Diversity 98:1–76. https://doi.org/10.1007/s13225-019-00432-7 Tabibzadeh F Antioxidant activity and cytotoxicity of exopolysaccharide from mushroom. Biomass Convers Biorefin Antioxidant activity and cytotoxicity of exopolysaccharide from mushroom Hericium coralloides in submerged fermentation. Biomass Convers Biorefin. 2022 Oct 18:1-11. Williams LM, Berthon BS, Stoodley IL, et al (2023) Medicinal mushroom extracts from hericium coralloides and trametes versicolor exert differential immunomodulatory effects on immune cells from older adults In vitro. Nutrients 15:2227. https://doi.org/10.3390/nu15092227 Zhang J, Zhang J, Zhao L, et al (2019) Antioxidant and anti-aging activities of ethyl acetate extract of the coral tooth mushroom, hericium coralloides (agaricomycetes). Int J Med Mushrooms 21:561–570. https://doi.org/10.1615/IntJMedMushrooms.2019030840 Cheng Y-F,Han A-L,Yun S-J, et al (2018). Hypocholesterolemic Effects and Mechanisms of Polysaccharides from Hericium coralloides [J] Journal of Nutrition, 40(02):172-176. Liu C-Y,Hao X-Y,Lin P-L, et al (2020) Identification of a Wild Cordyceps cicadae Strain and Comparison of Its Intracellular and Extracellular Polysaccharides' Anti-Liver Cancer Activity [J] Journal of Northwest A&F University (Natural Science Edition) 48(12):117-126. Amin R, Rahman SS, Hossain M, Choudhury N (2018) Physicochemical and microbiological qualities’ assessment of popular bangladeshi mango fruit juice. Open Microbiol J 12:135–147. https://doi.org/10.2174/1874285801812010135 Uffelman CN, Doenges KA, Armstrong ML, et al (2023) Metabolomics profiling of white button, crimini, portabella, lion’s mane, maitake, oyster, and shiitake mushrooms using untargeted metabolomics and targeted amino acid analysis. Foods (Basel Switz) 12:2985. https://doi.org/10.3390/foods12162985 Chen H, Chen H, Lu H, et al (2021) Carbohydrate analysis of mortierella alpina by colorimetry and HPLC-ELSD to reveal accumulation differences of sugar and lipid. Biotechnol Lett 43:1289–1301. https://doi.org/10.1007/s10529-021-03120-2 Phillips KM, McGinty RC, Couture G, et al (2021) Dietary fiber, starch, and sugars in bananas at different stages of ripeness in the retail market. PLOS One 16:e0253366. https://doi.org/10.1371/journal.pone.0253366 Moniruzzaman M, Chowdhury MAZ, Rahman MA, et al (2014) Determination of mineral, trace element, and pesticide levels in honey samples originating from different regions of Malaysia compared to manuka honey. Biomed Res Int 2014:359890. https://doi.org/10.1155/2014/359890 Purkiewicz A, Stasiewicz M, Nowakowski JJ, Pietrzak-Fiećko R (2023) The influence of the lactation period and the type of milk on the content of amino acids and minerals in human milk and infant formulas. Foods (Basel Switz) 12:3674. https://doi.org/10.3390/foods12193674 Smiderle FR, Ruthes AC, Van Arkel J, et al (2011) Polysaccharides from agaricus bisporus and agaricus brasiliensis show similarities in their structures and their immunomodulatory effects on human monocytic THP-1 cells. BMC Complement Altern Med 11:58. https://doi.org/10.1186/1472-6882-11-58 Ding L, Zhang X, Zhang J (2021) Antioxidant activity In vitro guided screening and identification of flavonoids antioxidants in the extract from tetrastigma hemsleyanum diels et gilg. Int J Anal Chem 2021:1–11. https://doi.org/10.1155/2021/7195125 Miller NJ, Rice-Evans C, Davies MJ, et al (1993) A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci (Lond Engl: 1979) 84:407–412. https://doi.org/10.1042/cs0840407 Saiga A, Tanabe S, Nishimura T (2003) Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment. J Agric Food Chem 51:3661–3667. https://doi.org/10.1021/jf021156g Benzie IF, Strain JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem 239:70–76. https://doi.org/10.1006/abio.1996.0292 Li T, Lu S-J, Sun J-M, et al (2021) Nutritional Composition Analysis and Evaluation of 26 Commonly Sold Edible Mushrooms [J]. Journal of China Edible Fungi.40(12):66-72.DOI:10.13629/j.cnki.53-1054.2021.12.012. Zheng H-Y,Wang T-T,Xie Y-J, et al (2016) Nutritional Analysis of 16 Edible Mushrooms and Algae-Based Foods [J]. Journal of China Edible Fungi. 35(06):69-72.DOI:10.13629/j.cnki.53-1054.2016.06.017. Wen C-Y,Xu-M,Yang Y-L, et al (2021) Research Progress on the Nutritional Composition and Value Evaluation of Wild Edible Mushrooms in China [J]. Journal of China Edible Fungi.40(11):1-10+20.DOI:10.13629/j.cnki.53-1054.2021.11.001. Chen X,Wang J,Li H-J ,et al (2015) Basic Composition and Nutritional Value of Bachu Mushrooms [J]. Agricultural Products Processing. (09):46-48. Han C, Lin K, He Y, et al. Study on the extraction of dietary fiber from deep processing residue of Pleurotus eryngii and its monosaccharide composition. Food Science and Technology, 2015, 40(12): 208-213+217. doi: 10.13684/j.cnki.spkj.2015.12.042. Cohen N, Cohen J, Asatiani MD, et al (2014) Chemical composition and nutritional and medicinal value of fruit bodies and submerged cultured mycelia of culinary-medicinal higher basidiomycetes mushrooms. Int J Med Mushrooms 16:273–291. https://doi.org/10.1615/IntJMedMushr.v16.i3.80 Pellett PL, Young VR (1990) Commentary: joint FAO/WHO expert consultation on protein quality evaluation bethesda, MD, USA, 4–8 december 1989. Ecology of Food and Nutrition 24:297–303. https://doi.org/10.1080/03670244.1990.9991147 Dávila G LR, Murillo A W, Zambrano F CJ, et al (2020) Evaluation of nutritional values of wild mushrooms and spent substrate of Lentinus crinitus (L.) Fr. Heliyon 6:e03502. https://doi.org/10.1016/j.heliyon.2020.e03502 Chang,X.N.;Chen, X.F.;Gong, P.;Liu, M.;Wang, M.R.;Wang, X.J, et al (2022) Structural characterization,in vitro anti-oxidative effect and hypoglycemic activity of lentinan from Rongshui county.China Food Additives 33, 1-9 Li,Y.L.;jiang, M.;Tan, M.q, et al (2020) Research Progress on Pharmacological Value of Polysaccharides from Edible Fungi,Journal of Ningxia Agriculture and Forestry Science and Technology 61, 24-26+39. Justo A, Miettinen O, Floudas D, et al (2017) A revised family-level classification of the polyporales (basidiomycota). Fungal Biol 121:798–824. https://doi.org/10.1016/j.funbio.2017.05.010 Zhou J-L, Zhu L, Chen H, Cui B-K (2016) Taxonomy and phylogeny of polyporus group melanopus (polyporales, basidiomycota) from China. PLOS One 11:e0159495. https://doi.org/10.1371/journal.pone.0159495 Zhuang L, Sun Z-H, Yu S-T, et al. (2024) Breeding of a New Variety of Hericium coralloides ‘ZLsh-1’. Mycological Journal. 43: 155–162. https://doi.org/10.13346/j.mycosystema.240121 Zhang P, Shi L, Yu H-Y, et al. (2024) Domestication and Cultivation of Two Wild Hericium coralloides Strains from the Xiaoxing'anling Region. Northern Horticulture 110–114. Sar T, Larsson K, Fristedt R, et al (2022) Demo-scale production of protein-rich fungal biomass from potato protein liquor for use as innovative food and feed products. Food Biosci 47:101637. https://doi.org/10.1016/j.fbio.2022.101637 Tabibzadeh F, Alvandi H, Hatamian-Zarmi A, et al (2022) Antioxidant activity and cytotoxicity of exopolysaccharide from mushroom hericium coralloides in submerged fermentation. Biomass Convers Biorefin 1–11. https://doi.org/10.1007/s13399-022-03386-0 Hyder MS, Dutta SD (2021) Mushroom-derived polysaccharides as antitumor and anticancer agent: a concise review. Biocatal Agric Biotechnol 35:102085. https://doi.org/10.1016/j.bcab.2021.102085 Hetland G, Tangen J-M, Mahmood F, et al (2020) Antitumor, anti-inflammatory and antiallergic effects of agaricus blazei mushroom extract and the related medicinal basidiomycetes mushrooms, hericium erinaceus and grifola frondosa: a review of preclinical and clinical studies. Nutrients 12:1339. https://doi.org/10.3390/nu12051339 Zan X, Cui F, Li Y, et al (2015) Hericium erinaceus polysaccharide-protein HEG-5 inhibits SGC-7901 cell growth via cell cycle arrest and apoptosis. Int J Biol Macromol 76:242–253. https://doi.org/10.1016/j.ijbiomac.2015.01.060 Cite Share Download PDF Status: Posted Version 1 posted 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-5897250","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451991599,"identity":"a08bf299-faf0-43ea-b5c5-91cb330949d5","order_by":0,"name":"Yun Li","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Li","suffix":""},{"id":451991600,"identity":"721bc765-52c5-497c-92d4-2bb3823e8d80","order_by":1,"name":"Jiarong Cai","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Jiarong","middleName":"","lastName":"Cai","suffix":""},{"id":451991601,"identity":"83792262-383e-4940-bc91-ece4ad62b1b6","order_by":2,"name":"Xiaomin Li","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xiaomin","middleName":"","lastName":"Li","suffix":""},{"id":451991602,"identity":"c67aad91-268f-4450-b182-b415c61ec190","order_by":3,"name":"Xin Hu","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Hu","suffix":""},{"id":451991603,"identity":"3fef88f8-34c2-4e6a-b004-baf38e5f5767","order_by":4,"name":"Junli Zhang","email":"","orcid":"","institution":"Tibet Academy of Agricultural and Animal Husbandry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Junli","middleName":"","lastName":"Zhang","suffix":""},{"id":451991604,"identity":"4ea23eb9-a0f0-4cbf-9f57-c023ce21c906","order_by":5,"name":"Xiaopin Wu","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xiaopin","middleName":"","lastName":"Wu","suffix":""},{"id":451991605,"identity":"b80c1b7c-3e0f-487b-bc39-6b8d648fb886","order_by":6,"name":"Junsheng Fu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIie3PPQrCMBTA8ZRCskS7FgS9whPBDyx6lZRCJgdvYCcnD6AXcO4Rog87Ca4FHQShc0eHDsYPHJuOgvlDeMv7kYQQm+0HYzEReih9XKUKCCZGwtWX0HC3nsuoDiEfwnvIi70TG4kv8ta8vLQH3lZgAMolDA+JgcjWZpn3RuubwhlcmoRLmVWR6ZM0YgyTLBWa5C7xeb+SvG7hJS6S7Ag4BHTieoSigNMKkNQi/BqNG0vsJhkVuxXIiJr+wtksPOuHdeCEWNzLYOIxTCuJRuI9/c+k1evPmHpPT5l3bTab7T97ADbZUvIj6waxAAAAAElFTkSuQmCC","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Junsheng","middleName":"","lastName":"Fu","suffix":""}],"badges":[],"createdAt":"2025-01-24 16:39:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5897250/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5897250/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82232285,"identity":"85fb21b9-7d53-4f99-a30d-319d679b4f8b","added_by":"auto","created_at":"2025-05-08 06:19:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":332928,"visible":true,"origin":"","legend":"\u003cp\u003eAbout the Abstract Diagram of \u003cem\u003eH. coralloides.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5897250/v1/7d1c1241381aed9168256725.png"},{"id":82232287,"identity":"084625de-002c-4504-ae04-163f3945f8a2","added_by":"auto","created_at":"2025-05-08 06:19:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":186643,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Ecological Photo of\u003cem\u003e H. coralloides\u003c/em\u003e. (B) Phylogeny based on ITS sequence construction.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5897250/v1/7ae02f5a6b8a1b1a14368422.png"},{"id":82232297,"identity":"e92807ea-9b5c-4968-9b65-018155c8a7ee","added_by":"auto","created_at":"2025-05-08 06:19:52","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1013005,"visible":true,"origin":"","legend":"\u003cp\u003eBiological properties of Hericium coralloides. (A) Growth trend diagram. (B) Mycelial growth rate chart. On the bar chart, the same letters indicate no significant difference, while different letters indicate a significant difference (P \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5897250/v1/398d65795f36b008f4472b63.jpeg"},{"id":82233441,"identity":"7c0faae6-48db-4614-a448-4d64f0785fc8","added_by":"auto","created_at":"2025-05-08 06:35:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":460164,"visible":true,"origin":"","legend":"\u003cp\u003e(A) \u003cem\u003eHericium coralloides\u003c/em\u003e are full of mycelium. (B-F) Artificial do mestication of subentity.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5897250/v1/434bed3a150d7164a0fa88f6.png"},{"id":82232294,"identity":"a9cf10c8-8e76-4019-8432-ec5d656515f6","added_by":"auto","created_at":"2025-05-08 06:19:52","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":272417,"visible":true,"origin":"","legend":"\u003cp\u003eRandom forest map of amino acids in \u003cem\u003eHericium coralloides\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5897250/v1/80baebae1446c87036d52ef7.jpeg"},{"id":82232288,"identity":"c52018ce-a187-4139-9056-bcf48de3bf6d","added_by":"auto","created_at":"2025-05-08 06:19:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":117723,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant activity in vitro of polysaccharide from \u003cem\u003eHericium coralloides\u003c/em\u003e. (A) DPPH free radical clearance ability. (B) ABTS free radical clearance ability. (C) OH free radical clearance ability. (D) Ferric reducing antioxidant power. VC is a positive control.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5897250/v1/f5ae26deb622901fc1551076.png"},{"id":82233440,"identity":"c6d260d2-8b8a-4d25-97f7-f267ef79941f","added_by":"auto","created_at":"2025-05-08 06:35:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":26673,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of polysaccharides of \u003cem\u003eHericium coralloides\u003c/em\u003e on activities of differernt cancer cells. (A) HepG2 cell inhibition. (B) MDA-MB-468 cell inhibition.Compared to the control group (polysaccharide concentration is 0 mg/mL), *P\u0026lt;0.05, **P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5897250/v1/b0037b0df2a071f3e29aa902.png"},{"id":89586412,"identity":"42b9de75-412e-46aa-9a9a-659ba7b0e90b","added_by":"auto","created_at":"2025-08-21 15:13:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3743726,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5897250/v1/34ddb987-d39e-4b82-99a9-1a60133edf92.pdf"}],"financialInterests":"","formattedTitle":"Domestication Cultivation and Nutritional Analysis of Hericium coralloides","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eHericium coralloides\u003c/em\u003e (Scop.) Pers., a member of the Basidiomycota phylum, Agaricomycetes class, Russulales order, and Hericiaceae family, is closely related to \u003cem\u003eHericium erinaceus\u003c/em\u003e (Bu.) Pers.(Li et al. 2015). This species, resembling coral in appearance, features a bright white coloration with a narrow base and coral-like branching, with fine branches densely covered with spines. \u003cem\u003eH. coralloides\u003c/em\u003e is primarily found on the trunks or decayed wood of broad-leaved trees in Northeast, Northwest, and Southwest China (Mao et al. 2000). As a rare edible and medicinal fungus, it is valued for its substantial nutritional and pharmacological properties.\u003c/p\u003e \u003cp\u003eThe genus Hericium is one of the most renowned sources of edible and medicinal fungi in China. In recent years, it has been extensively utilized in the development of new pharmaceutical products. The mycelial polysaccharides from this genus have demonstrated a wide range of bioactivities, including anti-tumor, immune-enhancing, anti-coagulation, and cholesterol-lowering effects. Notably, the polysaccharides of \u003cem\u003eHericium erinaceus\u003c/em\u003e are well-known for significantly boosting immune function and exhibiting anti-cancer properties. Recent studies have shown that \u003cem\u003eHericium coralloides\u003c/em\u003e can alleviate Alzheimer\u0026rsquo;s disease and cognitive dysfunction by activating the Nrf2 signaling pathway and modulating the gut microbiota (Guan et al. 2024; Guan Y et al. 2023). Moreover, its metabolic products contain 18 amino acids, contributing to tonic, digestive, and therapeutic functions, such as in the treatment of neurosis and gastric ulcers. Additionally, \u003cem\u003eH. coralloides\u003c/em\u003e is rich in various bioactive polysaccharides, with proven anti-tumor, immune-boosting, antioxidant, anti-coagulant, and cholesterol-lowering effects (Wu et al. 2019; Tabibzadeh 2022; Williams et al. 2023; Zhang et al. 2019; Cheng et al. 2018). Among these, β-glucan, the most abundant polysaccharide, is often referred to as \"immune gold\" due to its potent biological and pharmacological properties, leading to its widespread use in pharmaceuticals, health products, and the food industry.\u003c/p\u003e \u003cp\u003eDue to its appealing appearance and rich nutritional and medicinal values, \u003cem\u003eHericium coralloides\u003c/em\u003e has gained considerable popularity among consumers in recent years, with promising prospects for industrial development. Like other edible fungi, such as black fungus, shiitake mushrooms, and reishi, \u003cem\u003eH. coralloides\u003c/em\u003e is a wood-decaying fungus that primarily utilizes lignocellulose as its nutritional source. Research into selecting superior strains and developing novel cultivation substrates for \u003cem\u003eH. coralloides\u003c/em\u003e has made significant strides, resulting in technological breakthroughs. Large-scale production of this highly nutritious and delicious fungus is anticipated in the near future, offering it to a broader consumer base. However, there is limited research on the utilization of wild \u003cem\u003eH. coralloides\u003c/em\u003e resources.\u003c/p\u003e \u003cp\u003eThis study aims to investigate the optimal cultivation conditions for the mycelium of \u003cem\u003eH. coralloides\u003c/em\u003e, develop liquid inoculum, and successfully induce fruiting. The nutritional composition of the resulting fruiting bodies is analyzed, and the bioactivity of polysaccharides extracted from these bodies is explored, providing valuable insights for the future development and commercial exploitation of \u003cem\u003eHericium coralloides.\u003c/em\u003e\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Materials\u003c/h2\u003e \u003cp\u003eThe experimental material was a wild fruiting body sample, numbered SH001, collected from Qinduo Town, Bomi County, Tibet Autonomous Region, from which mycelium was obtained after tissue separation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMain Reagents\u003c/h3\u003e\n\u003cp\u003eMaltose, mannose, fructose, soluble starch, sucrose, glucose, tryptone, yeast extract, beef extract, copper sulfate, potassium sulfate, anhydrous ether, anhydrous ethanol, acetic acid, sulfuric acid, sodium acetate, sodium hydroxide, potassium nitrate, ammonium nitrate, urea, magnesium sulfate, vitamin B\u003csub\u003e1\u003c/sub\u003e, potassium dihydrogen phosphate, hydrochloric acid, 2-mercaptoethanol, and other reagents (analytical grade, purchased from China National Pharmaceutical Group Corporation).D3390-01 Fungal DNA Kit (OMEGA Bio-Tek, USA); DPPH, ABTS, TPTZ, and ascorbic acid (Sigma-Aldrich, USA).Cell culture medium (Hyclone, USA).Fetal bovine serum (Gibco, USA).\u003c/p\u003e\n\u003ch3\u003eExperimental Culture Medium\u003c/h3\u003e\n\u003cp\u003eEnriched PDA Medium:200 g of peeled potatoes, 20 g of glucose, 5 g of peptone, 2 g of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1.5 g of MgSO\u003csub\u003e4\u003c/sub\u003e, 10 mg of vitamin B\u003csub\u003e1\u003c/sub\u003e, 20 g of agar, and 1 L of distilled water, adjusted to natural pH.\u003c/p\u003e \u003cp\u003eLiquid Inoculum Culture Medium:200 g of peeled potatoes, 20 g of fructose, 2 g of ammonium sulfate, 1.5 g of magnesium sulfate, 2 g of potassium dihydrogen phosphate, 10 mg of vitamin B1, and 1 L of distilled water, pH 5.0.\u003c/p\u003e \u003cp\u003eSubstrate for Cultivation:60% hardwood sawdust, 20% cottonseed hulls, 18% wheat bran, 1% lime, and 1% sugar, mixed in the appropriate proportions with water to achieve a final moisture content of 60%.\u003c/p\u003e\n\u003ch3\u003eMorphological Identification of Strain SH001\u003c/h3\u003e\n\u003cp\u003eThe morphological characteristics of both the fruiting body and the mycelium of strain SH001 were examined. The observations were made with reference to the work of Li et al. (2015).\u003c/p\u003e\n\u003ch3\u003eMolecular Identification of Strain SH001\u003c/h3\u003e\n\u003cp\u003eThe mycelium DNA of strain SH001 was extracted using the OMEGA Fungal DNA Extraction Kit. The extracted DNA was used as a template for PCR amplification with the universal fungal primers ITS1/ITS4, following the amplification and sequencing protocols described by Liu et al. (Liu et al. 2020). After amplification, 3 \u0026micro;L of the PCR product was subjected to 1% agarose gel electrophoresis, and a single bright band was observed. The remaining PCR product was then sent to Fuzhou Baijing Biotechnology Co., Ltd. for sequencing. The resulting ITS sequence was submitted to the NCBI Nucleotide Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for BLAST analysis. High-homology ITS sequences were downloaded, and a phylogenetic tree was constructed using MEGA 11 software to determine the taxonomic classification of the strain.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBiological Characterization of Strain SH001\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eEffect of Carbon Sources on Mycelial Growth\u003c/h2\u003e \u003cp\u003eDifferent carbon sources, including glucose, sucrose, fructose, maltose, mannose, and starch, were used to replace glucose in the enriched PDA medium, while keeping all other components constant. Activated fungal cultures were inoculated by making a 5 mm hole at the edge of the agar plates, and small fungal blocks were transferred to the center of the plates containing different carbon source media (9 cm diameter). The plates were incubated in the dark at 25\u0026deg;C. The colony diameter was measured using the \"cross\" method. Each treatment was performed in five replicates.\u003c/p\u003e \u003cp\u003eThe mycelial growth rate (mm/day) was calculated as:\u003c/p\u003e \u003cp\u003eMycelial Growth Rate (mm/day)= (Colony Diameter (mm)\u0026thinsp;\u0026minus;\u0026thinsp;Inoculum Diameter (mm))/ Growth Duration (days)​. (1)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eEffect of Nitrogen Sources on Mycelial Growth\u003c/h3\u003e\n\u003cp\u003eDifferent nitrogen sources, including urea, yeast extract, ammonium sulfate, beef extract, peptone, and ammonium nitrate, were used to replace peptone in the enriched PDA medium, with all other components remaining constant. Fungal blocks were inoculated at the center of 9 cm diameter plates containing media with different nitrogen sources. The procedure was carried out as described for the effect of carbon sources on mycelial growth.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffect of pH on Mycelial Growth\u003c/h2\u003e \u003cp\u003eEnriched PDA medium was used to investigate the effect of pH on mycelial growth. The pH was adjusted to values of 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 using 1.0 mol/L NaOH and 1.0 mol/L HCl solutions. The procedure was conducted as described for the effect of carbon sources on mycelial growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Temperature on Mycelial Growth\u003c/h2\u003e \u003cp\u003eEnriched PDA medium was used to investigate the effect of temperature on mycelial growth. Following inoculation, the plates were incubated in constant temperature incubators at 15\u0026deg;C, 20\u0026deg;C, 25\u0026deg;C, 30\u0026deg;C, 35\u0026deg;C, and 40\u0026deg;C under dark conditions. The procedure was performed as described for the effect of carbon sources on mycelial growth.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDomestication and Cultivation Trials of\u003c/b\u003e \u003cb\u003eHericium coralloides\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Liquid Mycelial Culture\u003c/h2\u003e \u003cp\u003eThe liquid mycelial culture medium was aliquoted into Erlenmeyer flasks, with 100 mL per flask, and sterilized in a high-temperature, high-pressure autoclave for future use. Under a sterile laminar flow hood, PDA agar plugs (7 mm in diameter) were inoculated into shake flasks, with six plugs per flask, and sealed with stoppers. The flasks were then incubated in the dark at 23\u0026deg;C with shaking at 160 rpm.\u003c/p\u003e \u003cp\u003ecottonseed hulls were fully soaked in water for 12 hours, then thoroughly drained. A mixture of fermented wood sawdust, pre-moistened cottonseed hulls, wheat bran, lime, and white sugar was prepared in appropriate proportions. Water was added incrementally and mixed thoroughly to ensure the substrate absorbed sufficient moisture, maintaining a moisture content of approximately 60%. The pH was adjusted to 5.0. The substrate was aliquoted into cultivation bags, with 900 g (wet weight) per bag, and sterilized in an autoclave at 121\u0026deg;C for 3 hours. After cooling to room temperature, the bags were transferred to a sterile laminar flow hood, where liquid mycelial culture was carefully inoculated, with each flask inoculating five bags. Following inoculation, the bags were placed in a mycelial growth chamber and incubated at 22\u0026ndash;25\u0026deg;C under dark conditions for mycelial colonization.\u003c/p\u003e \u003cp\u003eAfter the mycelium has fully colonized the substrate and undergone a one-week maturation period, the bags were promptly transferred to the fruiting room for bag opening and fruiting induction. The temperature was maintained at 18\u0026ndash;20\u0026deg;C, and the relative humidity was increased to 95%. Once the primordia appeared, the temperature was adjusted to 20\u0026ndash;23\u0026deg;C to promote further maturation and differentiation of the fruiting bodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eNutritional Composition Analysis\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eMoisture Content Determination\u003c/h2\u003e \u003cp\u003eThe moisture content was determined using the direct drying method. An appropriate amount of fruiting body was weighed (W₁) in a pre-weighed dish (W₀), dried at 70\u0026deg;C until constant weight was achieved, cooled in a desiccator, and reweighed (W₂). The calculation formula is as follows:\u003c/p\u003e \u003cp\u003eMoisture Content （g/100g）= [（W₁−W₂）/(W₁−W₀)]×（100g/100g）. （2)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eProtein Content Determination\u003c/h2\u003e \u003cp\u003eThe protein content was measured according to the Kjeldahl method as referenced by Amin R et al. (2018). The fruiting body was weighed and digested with a potassium sulfate-copper sulfate catalyst and concentrated sulfuric acid, heated at 380\u0026ndash;420\u0026deg;C until a clear solution was obtained. After cooling, the mixture was alkalinized, distilled, and the released ammonia was absorbed in boric acid. The solution was titrated with 0.01mol/L HCl standard solution to the endpoint, and the volume difference was recorded. Blank controls and instrument calibration were performed. The calculation formula is as follows:\u003c/p\u003e \u003cp\u003eNitrogen content (g/100g) = [(Vsample -Vblank )\u0026times;C\u0026times;14.01/msample]\u0026times;100. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eProtein content\u0026thinsp;=\u0026thinsp;Nitrogen content \u0026times; 6.25. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAsh Content Determination\u003c/h2\u003e \u003cp\u003eAsh content was determined using the high-temperature incineration method as described by Uffelman CN et al. (2023). An appropriate amount of sample (precisely 0.001 g) was weighed into a pre-weighed crucible, carbonized until smokeless, and incinerated in a muffle furnace at 550\u0026deg;C for 4\u0026ndash;6 hours until gray-white ash was obtained. The crucible was cooled to 200\u0026deg;C in the furnace, transferred to a desiccator, and weighed at room temperature. The incineration was repeated until constant weight was achieved (weight difference\u0026thinsp;\u0026le;\u0026thinsp;0.5 mg). Blank correction and desiccator hygroscopicity control were performed.The calculation formula is as follows:\u003c/p\u003e \u003cp\u003eAsh content (g/100g) = [(ash\u0026thinsp;+\u0026thinsp;crucible weight\u0026thinsp;\u0026minus;\u0026thinsp;crucible weight)/sample mass]\u0026times;100. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFat Content Determination\u003c/h2\u003e \u003cp\u003eFat content was measured following the Soxhlet extraction method as outlined by Amin R et al. (2018). The sample was crushed, dried, homogenized, and packed into filter paper thimbles, which were then placed in a Soxhlet extractor. Excess diethyl ether was added, and the system was refluxed for 10 hours to dissolve fats. After extraction, the solvent was evaporated, and the fat-containing flask was dried to constant weight. The fat mass was measured gravimetrically.\u003c/p\u003e \u003cp\u003eFat Content (g/100g) = [fat mass/sample mass\u0026times;100]. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTotal Sugar Content Determination\u003c/h2\u003e \u003cp\u003eThe sample was ground and passed through an 80-mesh sieve, and 5 g was weighed into a 100 mL volumetric flask. After adding 50 mL of water to dissolve the sample, petroleum ether was used for defatting, and the residue was transferred back to the volumetric flask. Subsequently, 5 mL of zinc acetate solution (21.9 g/100 mL) and 5 mL of potassium ferrocyanide solution (10.6 g/100 mL) were added, followed by magnetic stirring for 30 min. The solution was then diluted to the mark with water. After filtration through dry filter paper, the initial filtrate was discarded, and the subsequent filtrate was passed through a 0.45 \u0026micro;m membrane for further analysis. A sugar-specific amino column (250 \u0026times; 4.6 mm, 5 \u0026micro;m) was used for separation, with a mobile phase of acetonitrile-water (70:30, v/v) at a flow rate of 1.0 mL/min and a column temperature of 40\u0026deg;C. An evaporative light scattering detector (ELSD) was employed with a drift tube temperature of 85\u0026deg;C and a nitrogen pressure of 350 kPa. A standard curve was constructed using glucose, fructose, sucrose, maltose, and lactose standards (0\u0026ndash;10 mg/mL), and the total sugar content was calculated according to the formula.\u003c/p\u003e \u003cp\u003eTotal sugar content (g/100g) = =[(ρ\u0026thinsp;\u0026minus;\u0026thinsp;ρ\u003csub\u003e0\u003c/sub\u003e)\u0026times;V\u0026times;n/(m\u0026times;1000)]\u0026times;100 (ρ: concentration of the sample solution, mg/mL; V: volume, mL; n: dilution factor; m: sample mass, g) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDietary Fiber Content Determination\u003c/h2\u003e \u003cp\u003eDietary fiber content was quantified using the enzymatic gravimetric method as referenced by Phillips KM et al. (2021). The fruiting body was crushed and sequentially hydrolyzed with α-amylase, protease, and glucosidase (pH 8.2, 37\u0026deg;C) to remove starch and proteins. The residue was precipitated with ethanol, filtered into a pre-weighed sintered glass crucible, washed with 78% ethanol and acetone, dried to constant weight at 105\u0026deg;C, and weighed.\u003c/p\u003e \u003cp\u003eDietary fiber content (g/100g) = [(residue mass\u0026thinsp;\u0026minus;\u0026thinsp;blank)/sample mass]\u0026times;100. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSodium Content Determination\u003c/h2\u003e \u003cp\u003eSodium content was determined using flame atomic emission spectrometry as per Moniruzzaman M et al. (2014). The sample was precisely weighed (0.001 g), digested with nitric acid via microwave, diluted to volume, and filtered. Sodium standard solutions were prepared, and emission intensity was measured at 589 nm using flame atomic emission spectroscopy (FAES) to construct a calibration curve. The sample solution was analyzed identically.\u003c/p\u003e \u003cp\u003eSodium content (g/100g) = [(measured concentration\u0026times;dilution factor\u0026times;0.1)/sample mass]\u0026times;100. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eAmino Acid Content Determination\u003c/h2\u003e \u003cp\u003eAmino acid content was analyzed using an automatic amino acid analyzer, as referenced by Purkiewicz A et al. (2023). The sample was hydrolyzed with 6 mol/L hydrochloric acid at 110\u0026deg;C for 24 h, diluted to volume, centrifuged, and filtered. The filtrate was injected into an amino acid analyzer, separated via ion-exchange chromatography, and subjected to post-column derivatization with ninhydrin. Peak areas of individual amino acids were detected. Calibration curves were generated using standard amino acid solutions.\u003c/p\u003e \u003cp\u003eTotal amino acid content (g/100g) = [\u0026sum;(concentration of each amino acid\u0026times;dilution factor\u0026times;0.1)/sample mass]\u0026times;100. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThree parallel experiments were conducted for each analysis, and the average values of the respective indicators were calculated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExploration of the Bioactivity of\u003c/b\u003e \u003cb\u003eHericium coralloides\u003c/b\u003e \u003cb\u003ePolysaccharides\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of Polysaccharides from\u003c/b\u003e \u003cb\u003eHericium coralloides\u003c/b\u003e \u003cb\u003eFruiting Bodies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe method described by Smiderle FR et al. (2011) was slightly modified as follows: After freeze-drying, the fruiting bodies were ground into powder and sieved through an 80-mesh screen for use. A ratio of 1:20 (g:mL) of the sample to 75% ethanol was added, followed by ultrasonic treatment for 2 hours. The filtrate was removed by suction filtration. Then, a ratio of 1:30 (g:mL) of the sample to distilled water was added and extracted in a 90\u0026deg;C water bath for 2 hours. The extraction was repeated twice, and the extracts were combined. The extract was concentrated in a rotary evaporator to one-third of its original volume. Anhydrous ethanol was added at a 4:1 (v/v) ratio, and the mixture was left at 4\u0026deg;C overnight for alcohol precipitation. The solution was centrifuged at 5,000 rpm for 10 minutes, and the precipitate was collected. The precipitate was re-dissolved in water and mixed with an equal volume of Sevage solution (chloroform:n-butanol\u0026thinsp;=\u0026thinsp;4:1), followed by stirring and centrifugation at 10,000 rpm for 3 minutes. The supernatant was collected, and the process was repeated until no protein or other impurities were present. The polysaccharide solution was then dialyzed for 48 hours against running water, and freeze-dried to a constant weight, yielding the crude polysaccharide freeze-dried powder from the fruiting bodies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChemical Antioxidant Activity of\u003c/b\u003e \u003cb\u003eHericium coralloides\u003c/b\u003e \u003cb\u003eFruiting Body Polysaccharides\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eHydroxyl Radical Scavenging Activity Assay\u003c/h2\u003e \u003cp\u003eThe method described by Ding L et al. (2021) was slightly modified as follows: Polysaccharide solutions with concentrations of 0.025, 0.05, 0.25, 0.5, 1, 2, and 5 mg/mL, along with vitamin C (VC) solutions, were accurately prepared. In a 96-well plate, 75 \u0026micro;L of polysaccharide solution at each concentration was added to the respective wells. Subsequently, 15 \u0026micro;L of FeSO\u003csub\u003e4\u003c/sub\u003e solution (9 mmol/L), salicylic acid-ethanol solution (9 mmol/L), and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution (8.8 mmol/L) were added sequentially to each well. The plate was gently shaken to ensure proper mixing. Finally, 100 \u0026micro;L of distilled water was added to each well. The plate was then incubated in a 37\u0026deg;C water bath for 30 minutes, and absorbance at 510 nm was measured, recorded as Ay. Vitamin C was used as a positive control. The reaction system for the blank group was prepared by replacing the polysaccharide sample with distilled water, and the absorbance was recorded as Ao. For the control group, distilled water was used instead of the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution, and the absorbance was recorded as Ap. Five replicates were performed for each concentration, and the average values were calculated.The calculation formula is as follows:\u003c/p\u003e \u003cp\u003eHydroxyl radical scavenging rate (%) = [1 \u0026minus; (Ay\u0026thinsp;\u0026minus;\u0026thinsp;Ap) / Ao] \u0026times; 100 (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eABTS Radical Scavenging Activity Assay\u003c/h2\u003e \u003cp\u003eThe method described by Miller et al.(1993) was slightly modified as follows: A 5 mL aliquot of 7.0 mmol/L ABTS solution and 88 \u0026micro;L of 2.45 mmol/L potassium persulfate aqueous solution were mixed and kept in the dark at room temperature for 12\u0026ndash;16 hours to prepare the ABTS\u0026thinsp;+\u0026thinsp;stock solution. The stock solution was then diluted with distilled water, and its absorbance at 734 nm was measured using a spectrophotometer to adjust the absorbance to 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023, which was used as the working ABTS\u0026thinsp;+\u0026thinsp;solution, prepared fresh for each use. In a 96-well plate, 100 \u0026micro;L of polysaccharide solutions at concentrations of 0.025, 0.05, 0.25, 0.5, 1, 2, and 5 mg/mL, along with the ABTS\u0026thinsp;+\u0026thinsp;working solution, were added to each well and mixed thoroughly. The plate was incubated in the dark at 25\u0026deg;C for 20 minutes, and absorbance at 734 nm was measured using a microplate reader (denoted as Ay). Vitamin C (VC) was used as a positive control. For the blank group, the polysaccharide sample was replaced with distilled water, and the absorbance was recorded as Ao. In the control group, distilled water was used instead of the ABTS\u0026thinsp;+\u0026thinsp;solution, and the absorbance was recorded as Ap. Five replicates were performed for each concentration. The calculation formula is as follows:\u003c/p\u003e \u003cp\u003eABTS Radical Scavenging Rate (%) = [1 \u0026minus; (Ay\u0026thinsp;\u0026minus;\u0026thinsp;Ap) / Ao] \u0026times; 100 (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eDPPH Radical Scavenging Activity Assay\u003c/h2\u003e \u003cp\u003eThe method described by Saiga A et al.(2003) was slightly modified as follows: Aliquots of 100 \u0026micro;L of polysaccharide solutions (0.025, 0.05, 0.25, 0.5, 1, 2, and 5 mg/mL) and 0.2 mmol/L DPPH solution were added sequentially to a 96-well plate. The plate was gently shaken to ensure thorough mixing, and the reaction was allowed to proceed in the dark at room temperature for 30 minutes. Absorbance at 517 nm was then measured and recorded as Ay. Vitamin C (VC) was used as a positive control. The blank group reaction system was prepared by replacing the polysaccharide sample with absolute ethanol, and the absorbance was recorded as Ao. In the control group, absolute ethanol was used instead of the DPPH solution, and the absorbance was recorded as Ap. Five replicates were performed for each concentration. The DPPH radical scavenging activity was calculated using the following formula:\u003c/p\u003e \u003cp\u003eDPPH Radical Scavenging Activity (%) = [1 \u0026minus; (Ay\u0026thinsp;\u0026minus;\u0026thinsp;Ap) / Ao] \u0026times; 100 (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eFerric Ion Reducing Antioxidant Power (FRAP) Assay\u003c/h2\u003e \u003cp\u003eThe method described by Benzie et al. (1996) was slightly modified as follows: A FRAP working solution was prepared by mixing 0.3 mol/L acetate-acetic acid buffer (pH 3.6), 0.02 mol/L FeCl3 solution, and 0.01 mol/L TPTZ solution in a volume ratio of 10:1:1, and used fresh. Aliquots of 0.5 mL of FeSO4 solutions at concentrations of 0.025, 0.1, 0.15, 0.2, 0.4, 0.5, 0.8, 1.0, and 1.5 mmol/L were added to 3.0 mL of the FRAP working solution. The mixtures were thoroughly mixed and incubated at 37\u0026deg;C for 15 minutes. Absorbance at 593 nm was measured to construct a standard curve. The same procedure was used to determine the absorbance of each reaction system. Specifically, for the reaction mixtures containing polysaccharide solutions at concentrations of 0.025, 0.05, 0.25, 0.5, 1, 2, and 5 mg/mL, combined with the FRAP working solution, the absorbance at 593 nm was recorded as Ay. In the blank and control groups, distilled water replaced the polysaccharide sample and the FRAP working solution, with the corresponding absorbance values recorded as Ao and Ap, respectively. The FRAP value was calculated by determining the difference between Ay, Ao, and Ap, and then referring to the standard curve to obtain the corresponding FeSO4 concentration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eToxicity of\u003c/b\u003e \u003cb\u003eHericium coralloides\u003c/b\u003e \u003cb\u003ePolysaccharides on Different Cancer Cell\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe HepG\u003csub\u003e2\u003c/sub\u003e and MDA-MB-231 cancer cell lines were cultured in cell culture medium at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator for subculturing. Upon reaching the logarithmic growth phase and achieving 80\u0026ndash;90% confluence, the cells were harvested for plating. The original culture medium was discarded, and the cells were washed three times with PBS. Trypsin was then added to digest the cells for 30 seconds, followed by the addition of fresh culture medium to terminate the digestion. The cell suspension was centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cell pellet was resuspended in culture medium to ensure even dispersion. The cell density was adjusted to 1\u0026times;10\u003csup\u003e^5\u003c/sup\u003e cells/mL. A 200 \u0026micro;L aliquot of the cell suspension was added to each well of a 96-well plate, with 200 \u0026micro;L of PBS added to the outermost wells as blanks. The plate was incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for cell attachment. Once the cells had fully adhered, the original medium was removed, and treatment with polysaccharide was administered. The treatment groups included a polysaccharide group, a blank group, and a control group. The polysaccharide group received 200 \u0026micro;L of culture medium containing polysaccharides at different concentrations (0.025, 0.05, 0.25, 0.5, 1, 2, and 5 mg/mL). The control group received 200 \u0026micro;L of drug-free culture medium, and the blank group consisted of medium without cells, serving as the zero control. Each group was set up with five replicate wells. The plate was incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours. After incubation, the culture medium was discarded, and 100 \u0026micro;L of culture medium containing 10% MTT was added to each well. The plate was incubated for an additional 4 hours. Afterward, the medium was removed, and 150 \u0026micro;L of dimethyl sulfoxide (DMSO) was added to each well. The plate was gently shaken until the purple-brown precipitate was completely dissolved, and the absorbance at 490 nm was measured using a microplate reader. Cell viability at different polysaccharide concentrations was calculated using the following formula, and the IC50 value was determined.\u003c/p\u003e \u003cp\u003eCell viability (%) = [(OD of polysaccharide group - OD of blank group) / (OD of control group - OD of blank group)] \u0026times; 100. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification of Wild\u003c/b\u003e \u003cb\u003eHericium coralloides\u003c/b\u003e \u003cb\u003eStrain\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMorphological Identification\u003c/strong\u003e \u003cp\u003eA wild \u003cem\u003eHericium coralloides\u003c/em\u003e specimen was collected from a broadleaf forest in Milin County, Nyingchi City, Tibet. The wild fruiting body was white, with a long and thick stalk, and coral-like branches. The cap surface was rough, exhibiting radial grooves that extended outward up to 8 cm and reached a width of 10 cm and a height of 5 cm. Upon comparison, these characteristics were consistent with the description of \u003cem\u003eHericium coralloides\u003c/em\u003e in the \"Illustrated Guide to Major Fungal Resources in China\" (Li et al. 2015).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular Identification\u003c/b\u003e: The ITS sequence of strain SH001 (NCBI accession number: PQ094906) was compared for homology using GenBank. The results showed that the ITS sequence of strain SH001 shared 98.93% homology with the reported sequence of \u003cem\u003eHericium coralloides\u003c/em\u003e (MG735348). Using \u003cem\u003eRussula lutea\u003c/em\u003e as the outgroup, ITS sequences of other \u003cem\u003eHericium\u003c/em\u003e species were included, and a phylogenetic tree was constructed using the neighbor-joining method. The tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) indicated that strain SH001 grouped closely with \u003cem\u003eHericium coralloides\u003c/em\u003e and showed a considerable genetic distance from other \u003cem\u003eHericium\u003c/em\u003e species. Based on the combined results from both morphological observation and molecular biology identification, the tested strain SH001 was confirmed to be \u003cem\u003eHericium coralloides\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Culture Conditions on H. coralloides Mycelial Growth\u003c/h2\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003eOptimization of Culture Conditions\u003c/h2\u003e \u003cdiv id=\"Sec30\" class=\"Section4\"\u003e \u003ch2\u003eCarbon Source:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe mycelium of \u003cem\u003eHericium coralloides\u003c/em\u003e SH001 can grow on various carbon sources, but significant differences in growth were observed. The mycelium exhibited the fastest growth when cultured on fructose, showing a white, dense, and robust appearance, with optimal growth performance. In contrast, starch resulted in the poorest growth, significantly lower than the control group. The growth rates, in descending order, were: fructose (10.15 mm/d)\u0026thinsp;\u0026gt;\u0026thinsp;glucose (8.81 mm/d)\u0026thinsp;\u0026gt;\u0026thinsp;mannitol (7.61 mm/d)\u0026thinsp;\u0026gt;\u0026thinsp;maltose (5.63 mm/d)\u0026thinsp;\u0026gt;\u0026thinsp;sucrose (3.53 mm/d)\u0026thinsp;\u0026gt;\u0026thinsp;starch (1.31 mm/d) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-a). Overall, fructose is identified as the most suitable carbon source for the mycelial growth of \u003cem\u003eHericium coralloides\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eNitrogen Source:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe mycelium of SH001 was capable of growth on various nitrogen sources, with noticeable differences in growth performance. The fastest growth occurred when cultured with yeast extract, and the growth rates were ranked from highest to lowest as follows: yeast extract (9.85 mm/d)\u0026thinsp;\u0026gt;\u0026thinsp;ammonium nitrate (8.25 mm/d)\u0026thinsp;\u0026gt;\u0026thinsp;peptone (8.23 mm/d)\u0026thinsp;\u0026gt;\u0026thinsp;ammonium sulfate (7.93 mm/d)\u0026thinsp;\u0026gt;\u0026thinsp;beef extract (7.78 mm/d) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-b). No growth was observed when urea was used as the nitrogen source. Overall, yeast extract was the most effective nitrogen source for promoting robust, dense, and healthy mycelial growth in \u003cem\u003eHericium coralloides\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003epH:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe mycelium of \u003cem\u003eHericium coralloides\u003c/em\u003e SH001 can grow within a pH range of 5.0 to 10.0. The fastest growth rate was observed at pH 5, with a growth rate of 8.71 mm/d. No significant difference in growth rates was found between pH 6 and pH 7 (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), though the mycelium at pH 7 appeared denser and more compact than at pH 5.0. The growth rate at pH 5.0 was significantly slower compared to pH 9 and pH 10 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Based on the growth rate and overall performance, pH 7 was found to be the most suitable for optimal mycelial growth.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eTemperature:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cem\u003eHericium coralloides\u003c/em\u003e SH001 does not tolerate low temperatures and cannot grow at 15\u0026deg;C. The growth rate of the mycelium increased progressively with temperature. The growth rates, from highest to lowest, were: 30\u0026deg;C\u0026thinsp;\u0026gt;\u0026thinsp;35\u0026deg;C\u0026thinsp;\u0026gt;\u0026thinsp;40\u0026deg;C\u0026thinsp;\u0026gt;\u0026thinsp;25\u0026deg;C\u0026thinsp;\u0026gt;\u0026thinsp;20\u0026deg;C\u0026thinsp;\u0026gt;\u0026thinsp;15\u0026deg;C. The mycelium grew fastest at 30\u0026deg;C (8.40 mm/d) and 35\u0026deg;C (7.10 mm/d), followed by 40\u0026deg;C (6.25 mm/d) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-d). At 30\u0026deg;C, the mycelium was the densest and exhibited well-defined edges, while at 35\u0026deg;C, the mycelium was also dense, but with slightly less defined edges. In conclusion, the optimal temperature for mycelial growth of \u003cem\u003eHericium coralloides\u003c/em\u003e SH001 is 30\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eDomestication of \u003cem\u003eH. coralloides\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eAfter 28 days, the mycelium had fully colonized the bags. Following an additional 10 days under optimal environmental conditions, pale yellow primordia began to appear. The temperature was maintained between 20 and 23\u0026deg;C, with humidity exceeding 90%. Ten days after the bags were opened, the fruiting bodies reached maturity and were harvested. The average fresh weight of the first flush of fruiting bodies per bag was 249.07 g, with a second flush also emerging. The growth conditions of the cultivated fruiting bodies are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. As shown, both the fruiting bodies and mycelium of \u003cem\u003eHericium coralloides\u003c/em\u003e SH001 were white (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The fruiting bodies displayed coral-like branches, which further branched into small twigs, from which dense, small spines developed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eNutrient Content of Subentity\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eNutrient Content of \u003cem\u003eH. coralloides\u003c/em\u003e Subentity\u003c/div\u003e \u003cp\u003eRoutine nutritional analysis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) showed that the contents of crude protein, ash, fat, total sugars, dietary fiber, and sodium in \u003cem\u003eHericium coralloides\u003c/em\u003e were 15.4 g/100 g dry weight, 6.8 g/100 g dry weight, 3.5 g/100 g dry weight, 1.6 g/100 g dry weight, 34.7 g/100 g dry weight, and 10.0 mg/100 g dry weigh\u003cem\u003et\u003c/em\u003e, respectively. Although the protein content of \u003cem\u003eHericium cor-alloides\u003c/em\u003e is lower than that of its congeneric species \u003cem\u003eHericium erinaceus\u003c/em\u003e, it still falls within the typical range (8\u0026ndash;24%) observed in eight common edible mushrooms (shiitake, oyster, enoki, white button, velvet, king oyster, tea tree, and black fungus) (Li et al. 2021). The fat content is within the general range (2\u0026ndash;4%) for edible mushrooms (Zheng et al. 2016), and is lower than that found in \u003cem\u003eHericium erinaceus\u003c/em\u003e. The crude ash content in wild edible mushrooms, which consists of inorganic salts and heavy metals, serves as an indicator of the heavy metal content in the soil of the mushroom's growing environment (Wen et al. 2021) and the con-tribution of minerals to nutritional value. The ash content of \u003cem\u003eHericium coralloides\u003c/em\u003e is comparable to that of \u003cem\u003eHericium erinaceus\u003c/em\u003e. May provide nutritional advantages, such as a rich content of minerals. The crude fiber content of \u003cem\u003eHericium coralloides\u003c/em\u003e is slightly higher than that of the common mushroom \u003cem\u003ePleurotus eryngii\u003c/em\u003e (Han et al., 2015). with significant variations in fiber content across different edible mushrooms (Chen et al. 2015). Sodium, an essential trace mineral for humans, cannot be synthesized in the body and must be obtained through food or water. It plays a vital role in muscle and nerve tissue. The sodium content of \u003cem\u003eHericium coralloides\u003c/em\u003e is 10.0 mg/100 g, suggesting that supplementation with trace minerals via fungi could be beneficial.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eContent of nutritional components in fruit body of \u003cem\u003eHericium coralloide.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNutrient composition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCotent (g\u0026middot;100-1g-1 dry weight)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoisture* (g/100g)\u0026nbsp;(fresh)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHericium\u0026nbsp;coralloides\u003c/em\u003e\u003c/p\u003e \u003cp\u003e91.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHericium erinaceus\u003c/em\u003e (Cohen et al. 2014)\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoisture** (g/100g) (dry)\u003c/p\u003e \u003cp\u003eCarbohydrate(g/100g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003cp\u003e64.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.2\u003c/p\u003e \u003cp\u003e61.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude\u0026nbsp;protein (g/100g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh (g/100g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFat (g/100g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u0026nbsp;sugar (g/100g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNa\u0026nbsp;(mg/100g)\u003c/p\u003e \u003cp\u003eDietary\u0026nbsp;fiber (g/100g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003cp\u003e34.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnergy (kcal/100g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e280.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e374\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e* Represents the fresh weight measurement. **Represented as Total Carbohydrates.\u003c/p\u003e\n\u003ch3\u003eAmino Acids\u003c/h3\u003e\n\u003cp\u003eThe radar chart in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e illustrates the amino acid composition of \u003cem\u003eHericium coralloides\u003c/em\u003e. The concentration of medicinal amino acids was the highest (4.69 g/100 g dry weight), followed by sweet-tasting amino acids (2.81 g/100 g dry weight), umami amino acids (2.56 g/100 g dry weight), bitter amino acids (2.40 g/100 g dry weight), and neutral amino acids (0.61 g/100 g dry weight). The sweet-tasting amino acids primarily contribute to the flavor of \u003cem\u003eHericium coralloides\u003c/em\u003e, though the presence of sweet and salty compounds often masks the bitterness, ultimately defining the flavor profile. The most abundant amino acids are glutamic acid, followed by aspartic acid, which enhance the umami flavor of the mushroom. Arginine, a medicinal amino acid, is also essential for children's healthy development. Overall, the amino acid profile and composition align with that of a high-quality protein source.\u003c/p\u003e \u003cp\u003eFifteen amino acids were detected in the \u003cem\u003eHericium coralloides\u003c/em\u003e samples, with cysteine (Cys), methionine (Met), and tryptophan (Trp) not detected. This suggests that either \u003cem\u003eHericium coralloides\u003c/em\u003e contains no measurable levels of Cys, Met, and Trp, or that their concentrations are too low to be detected by the instrumentation. The ratio of essential amino acids to total amino acids in \u003cem\u003eHericium coralloides\u003c/em\u003e was 0.32, while the ratio of essential to non-essential amino acids was 0.47. The E/T and E/N values are close to the ideal values for high-quality proteins recommended by the Food and Agriculture Organization/World Health Organization (approximately 0.40 and \u0026ge;\u0026thinsp;0.60, respectively) (Pellett et al. 1990).\u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTable\u0026nbsp;2 Content of amino acids in fruitbodies of \u003cem\u003eHericium coralloides\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e \u003cp\u003e\u003cimg 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\" width=\"360\" height=\"407\"\u003e\u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant Activity of \u003cem\u003eH. coralloides\u003c/em\u003e Polysaccharide\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe antioxidant capacity of Hericium coralloides polysaccharides is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, at a concentration of 0.25 mg/mL, the ABTS radical scavenging rate reached 96.95%, which is comparable to the positive control group. Within the concentration range of 0.25\u0026ndash;5 mg/mL, the scavenging activity was similar to that of ascorbic acid (VC), demonstrating significant antioxidant activity with an EC50 value of 0.04 mg/mL. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB indicates that as the concentration increased, the difference in DPPH radical scavenging activity between the sample and the positive control (VC) gradually decreased. The DPPH radical scavenging ability exhibited a dose-dependent enhancement, reaching a peak of 83.77% at 5 mg/mL, with an EC50 value of 1.417 mg/mL. In Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, at polysaccharide concentrations ranging from 0 to 1 mg/mL, the scavenging ability against hydroxyl radicals did not differ significantly from that of the VC group. However, as the concentration increased, the scavenging rate of hydroxyl radicals increased progressively, although it remained significantly lower than that of VC. The highest scavenging rate, 67.31%, was achieved at a concentration of 5 mg/mL, with an EC50 value of 2.655 mg/mL. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD demonstrates that, with increasing polysaccharide concentration, the ferric ion reducing power positively correlated with concentration, reaching a maximum FRAP value of 4.43 mmol/L at 5 mg/mL. However, at the same concentration, the reducing power was significantly lower than that of the VC group.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003eIn Vitro Anticancer Activity of \u003cem\u003eH. coralloides\u003c/em\u003e Polysaccharides\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFungi are valuable biological resources (D\u0026aacute;vila et al. 2020), and with advancements in science and technology, the medicinal potential of fungal polysaccharides has attracted increasing attention. Fungal polysaccharides are non-cytotoxic compounds that do not exhibit harmful side effects on normal cells (Chang et al. 2022). They have demonstrated the ability to inhibit cancer cell proliferation (Li et al. 2020) and possess antioxidant properties.\u003c/p\u003e \u003cp\u003eTo assess the anticancer effects of \u003cem\u003eHericium coralloides\u003c/em\u003e polysaccharides, an MTT assay was conducted to evaluate their toxicity on HepG2 and MDA-MB-468 cells. The results revealed that \u003cem\u003eHericium coralloides\u003c/em\u003e polysaccharides inhibited the growth of both cancer cell lines to some extent. A clear dose-dependent relationship was observed between polysaccharide concentration and cell viability. As the concentration of polysaccharides increased, the inhibitory effect on cancer cell growth became more pronounced. At a concentration of 5 mg/mL, the lowest cell viability was observed. The IC50 values for HepG2 and MDA-MB-468 cells were 3.896 mg/mL and 2.561 mg/mL, respectively, indicating that \u003cem\u003eHericium coralloides\u003c/em\u003e polysaccharides exerted a stronger inhibitory effect on MDA-MB-468 cells compared to HepG2 cells.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTraditional fungal classification has primarily been based on the observation and description of morphological traits, which group fungi into different categories according to these features (Justo et al. 2017). However, fruiting bodies may exhibit polymorphism influenced by environmental factors and nutritional conditions. In contrast, the ITS rDNA region evolves at a relatively rapid rate, displaying considerable sequence polymorphism. Furthermore, its short length facilitates amplification and sequencing, making it widely utilized in fungal taxonomy (Zhou et al. 2016). In this study, a combination of morphological observation and ITS sequence-based molecular identification was employed to characterize a wild strain (SH001) from Tibet. The results showed that the ITS sequence of this strain closely resembled that of strain MG735348.1, establishing a close phylogenetic relationship with \u003cem\u003eHericium erinaceum\u003c/em\u003e, and confirming its identification as \u003cem\u003eHericium coralloides\u003c/em\u003e. Additionally, the study observed that the strain exhibited accelerated mycelial growth at pH 5 and 30\u0026deg;C when yeast extract was used as the nitrogen source and fructose as the carbon source.\u003c/p\u003e \u003cp\u003eEarly studies have explored high-yield cultivation techniques for \u003cem\u003eHericium coralloides\u003c/em\u003e. Huang et al. (2022) found that a substrate consisting of 88% cottonseed hulls, 10% wheat bran, 1% gypsum, and 1% malic acid could increase the yield of \u003cem\u003eHericium coralloides\u003c/em\u003e to 150\u0026ndash;200 g (Zhuang et al. 2024). Zhang et al. (2024) conducted domestication and cultivation research on two wild \u003cem\u003eHericium coralloides\u003c/em\u003e strains from the Xiao Xing'anling region, demonstrating that a substrate containing 83% broadleaf sawdust, 15% wheat bran, 1% gypsum, and 1% sucrose achieved a fruiting body yield of 136.5 g, with a biological efficiency of 63.4% (Zhang et al. 2024). Through interviews with local farmers in Fujian, we selected suitable cultivation substrates, increasing the yield of \u003cem\u003eHericium coralloides\u003c/em\u003e to 249 g. This provides a new reference for cultivating \u003cem\u003eHericium coralloides\u003c/em\u003e in regions with similar climatic conditions to Fujian.\u003c/p\u003e \u003cp\u003eThis study found that the fruiting bodies of \u003cem\u003eHericium coralloides\u003c/em\u003e contain 26.3% protein (dry weight basis). Although the protein content of \u003cem\u003eHericium coralloides\u003c/em\u003e is lower than that of its congeneric species \u003cem\u003eHericium erinaceus\u003c/em\u003e, it still falls within the typical range (8\u0026ndash;24%) observed in eight common edible mushrooms (shiitake, oyster, enoki, white button, velvet, king oyster, tea tree, and black fungus) and is significantly comparable to legumes (Sar et al. 2020).Additionally, the fruiting bodies are rich in 15 amino acids, including six essential ones, with a ratio of essential amino acids to total amino acids of 0.32. The low content of total sugars and lipids makes this fungus a low-sugar, low-fat food source, providing valuable insights for the development of \u003cem\u003eH. coralloides\u003c/em\u003e-based products.\u003c/p\u003e \u003cp\u003eThe bioactivity of \u003cem\u003eH. coralloides\u003c/em\u003e extracellular polysaccharides (EPS) was also examined. Preliminary results demonstrated that \u003cem\u003eH. coralloides\u003c/em\u003e EPS exhibits notable antioxidant activity. The EC50 values were 1.417 mg/mL for DPPH, 0.04 mg/mL for ABTS, and 2.655 mg/mL for hydroxyl radical scavenging. Similarly, Tabibzadeh et al. (2022) investigated the bioactivity of \u003cem\u003eH. coralloides\u003c/em\u003e polysaccharides collected in Iran, confirming their antioxidant properties, with EC50 values of 4.12 mg/mL for DPPH, 17.0 mg/mL for FRAP, and 2.83 mg/mL for ABTS. In comparison, the polysaccharides from our successfully domesticated and cultivated \u003cem\u003eH. coralloides\u003c/em\u003e exhibited superior antioxidant activity. Within a concentration range of 0.25\u0026ndash;5 mg/mL, the DPPH and ABTS radical scavenging effects were comparable to those of ascorbic acid (VC).\u003c/p\u003e \u003cp\u003eExperimental evidence suggests that polysaccharides from the \u003cem\u003eHericium\u003c/em\u003e genus exhibit significant anticancer potential (Hyder et al. 2021). For instance, polysaccharides from \u003cem\u003eH. erinaceus\u003c/em\u003e have shown anticancer activity against human liver cancer (HepG2), breast cancer (MCF-7), and colon cancer (HCT116) cells (Hetland et al. 2020). These polysaccharides effectively inhibit the proliferation and colony formation of SGC-7901 cells by inducing apoptosis in the S phase and arresting the cell cycle (Zan et al. 2015). In line with these findings, our study demonstrates that \u003cem\u003eH. coralloides\u003c/em\u003e EPS is capable of inducing apoptosis in human liver cancer (HepG2) and triple-negative breast cancer (MDA-MB-468) cells.\u003c/p\u003e \u003cp\u003eIn this research, we isolated and identified a wild \u003cem\u003eHericium coralloides\u003c/em\u003e strain from Tibet, China. By optimizing the cultivation substrate, we significantly enhanced the yield of \u003cem\u003eH. coralloides\u003c/em\u003e fruiting bodies. The analysis of the fruiting bodies' nutritional composition, along with the antioxidant and anticancer properties of its polysaccharides, underscores the potential of \u003cem\u003eH. coralloides\u003c/em\u003e as a promising dietary supplement for cancer therapy. However, further studies are necessary to investigate its time-dependent effects on cancer cell elimination and to validate its efficacy in vivo.\u003c/p\u003e \u003cp\u003eThis study holds important implications for the large-scale conservation of wild fungal germplasm and the expansion of the global edible fungal resource database. Additionally, it contributes to the economic exploration of wild fungal germplasm and offers valuable insights for the future development of cancer therapies, potentially making a significant contribution to human health.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e: Not applicable to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: J.S.F. and X.H. designed the eperiments; Y.L. and J.R.C. preparedthema-terials; J.R.C. and Y.L.carriedouttheexperiments; X.M.L. and J.L.Z. analyzed the data and worte the manuscript.X.P.W revised the manuscript.All authors have read and agreed the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This work was supported by Key Development Project of Science and Technology Department of Tibet, grant number XZ202301ZY0013N and XZ202001ZY0041N.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e: The specimens and strains and their data were deposited at Mycological Research Center, College of Life Sciences, College of Life Sciences, Fujian Agriculture and Forestry University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e: The authors declare no conflict of intere.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi Y, Li T-H,Yang z-l, (2015) Atlas of Chinese Macrofungal Resources [M]. Zhongyuan Peasant Publishing House 76, 430. \u003c/li\u003e\n\u003cli\u003eMao X-L, (2000). Large Fungi of China [M]. Henan Science and Technology Publishing House.\u003c/li\u003e\n\u003cli\u003eGuan Y, Wang C, Li L, et al (2024) Structural characterization of\u003cem\u003e hericium coralloides\u003c/em\u003e polysaccharide and its neuroprotective function in alzheimer\u0026rsquo;s disease. Int J Biol Macromol 277:133865. https://doi.org/10.1016/j.ijbiomac.2024.133865\u003c/li\u003e\n\u003cli\u003eGuan Y, Shi D, Wang S, et al (2023) Hericium coralloides ameliorates alzheimer\u0026rsquo;s disease pathologies and cognitive disorders by activating Nrf2 signaling and regulating gut microbiota. Nutrients 15:3799. https://doi.org/10.3390/nu15173799\u003c/li\u003e\n\u003cli\u003eWu F, Zhou L-W, Yang Z-L, et al (2019) Resource diversity of chinese macrofungi: edible, medicinal and poisonous species. Fungal Diversity 98:1\u0026ndash;76. https://doi.org/10.1007/s13225-019-00432-7\u003c/li\u003e\n\u003cli\u003eTabibzadeh F Antioxidant activity and cytotoxicity of exopolysaccharide from mushroom. Biomass Convers Biorefin Antioxidant activity and cytotoxicity of exopolysaccharide from mushroom \u003cem\u003eHericium coralloides \u003c/em\u003ein submerged fermentation. Biomass Convers Biorefin. 2022 Oct 18:1-11.\u003c/li\u003e\n\u003cli\u003eWilliams LM, Berthon BS, Stoodley IL, et al (2023) Medicinal mushroom extracts from hericium coralloides and trametes versicolor exert differential immunomodulatory effects on immune cells from older adults In vitro. Nutrients 15:2227. https://doi.org/10.3390/nu15092227\u003c/li\u003e\n\u003cli\u003eZhang J, Zhang J, Zhao L, et al (2019) Antioxidant and anti-aging activities of ethyl acetate extract of the coral tooth mushroom, hericium coralloides (agaricomycetes). Int J Med Mushrooms 21:561\u0026ndash;570. https://doi.org/10.1615/IntJMedMushrooms.2019030840\u003c/li\u003e\n\u003cli\u003eCheng Y-F,Han A-L,Yun S-J, et al (2018). Hypocholesterolemic Effects and Mechanisms of Polysaccharides from \u003cem\u003eHericium coralloides\u003c/em\u003e [J] Journal of Nutrition, 40(02):172-176.\u003c/li\u003e\n\u003cli\u003eLiu C-Y,Hao X-Y,Lin P-L, et al (2020) Identification of a Wild \u003cem\u003eCordyceps cicadae\u003c/em\u003e Strain and Comparison of Its Intracellular and Extracellular Polysaccharides\u0026apos; Anti-Liver Cancer Activity [J] Journal of Northwest A\u0026amp;F University (Natural Science Edition) 48(12):117-126.\u003c/li\u003e\n\u003cli\u003eAmin R, Rahman SS, Hossain M, Choudhury N (2018) Physicochemical and microbiological qualities\u0026rsquo; assessment of popular bangladeshi mango fruit juice. Open Microbiol J 12:135\u0026ndash;147. https://doi.org/10.2174/1874285801812010135\u003c/li\u003e\n\u003cli\u003eUffelman CN, Doenges KA, Armstrong ML, et al (2023) Metabolomics profiling of white button, crimini, portabella, lion\u0026rsquo;s mane, maitake, oyster, and shiitake mushrooms using untargeted metabolomics and targeted amino acid analysis. Foods (Basel Switz) 12:2985. https://doi.org/10.3390/foods12162985\u003c/li\u003e\n\u003cli\u003eChen H, Chen H, Lu H, et al (2021) Carbohydrate analysis of mortierella alpina by colorimetry and HPLC-ELSD to reveal accumulation differences of sugar and lipid. Biotechnol Lett 43:1289\u0026ndash;1301. https://doi.org/10.1007/s10529-021-03120-2\u003c/li\u003e\n\u003cli\u003ePhillips KM, McGinty RC, Couture G, et al (2021) Dietary fiber, starch, and sugars in bananas at different stages of ripeness in the retail market. PLOS One 16:e0253366. https://doi.org/10.1371/journal.pone.0253366\u003c/li\u003e\n\u003cli\u003eMoniruzzaman M, Chowdhury MAZ, Rahman MA, et al (2014) Determination of mineral, trace element, and pesticide levels in honey samples originating from different regions of Malaysia compared to manuka honey. Biomed Res Int 2014:359890. https://doi.org/10.1155/2014/359890\u003c/li\u003e\n\u003cli\u003ePurkiewicz A, Stasiewicz M, Nowakowski JJ, Pietrzak-Fiećko R (2023) The influence of the lactation period and the type of milk on the content of amino acids and minerals in human milk and infant formulas. Foods (Basel Switz) 12:3674. https://doi.org/10.3390/foods12193674\u003c/li\u003e\n\u003cli\u003eSmiderle FR, Ruthes AC, Van Arkel J, et al (2011) Polysaccharides from agaricus bisporus and agaricus brasiliensis show similarities in their structures and their immunomodulatory effects on human monocytic THP-1 cells. BMC Complement Altern Med 11:58. https://doi.org/10.1186/1472-6882-11-58\u003c/li\u003e\n\u003cli\u003eDing L, Zhang X, Zhang J (2021) Antioxidant activity In vitro guided screening and identification of flavonoids antioxidants in the extract from tetrastigma hemsleyanum diels et gilg. Int J Anal Chem 2021:1\u0026ndash;11. https://doi.org/10.1155/2021/7195125\u003c/li\u003e\n\u003cli\u003eMiller NJ, Rice-Evans C, Davies MJ, et al (1993) A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci (Lond Engl: 1979) 84:407\u0026ndash;412. https://doi.org/10.1042/cs0840407\u003c/li\u003e\n\u003cli\u003eSaiga A, Tanabe S, Nishimura T (2003) Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment. J Agric Food Chem 51:3661\u0026ndash;3667. https://doi.org/10.1021/jf021156g\u003c/li\u003e\n\u003cli\u003eBenzie IF, Strain JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of \u0026ldquo;antioxidant power\u0026rdquo;: the FRAP assay. Anal Biochem 239:70\u0026ndash;76. https://doi.org/10.1006/abio.1996.0292\u003c/li\u003e\n\u003cli\u003eLi T, Lu S-J, Sun J-M, et al (2021) Nutritional Composition Analysis and Evaluation of 26 Commonly Sold Edible Mushrooms [J]. Journal of China Edible Fungi.40(12):66-72.DOI:10.13629/j.cnki.53-1054.2021.12.012.\u003c/li\u003e\n\u003cli\u003eZheng H-Y,Wang T-T,Xie Y-J, et al (2016) Nutritional Analysis of 16 Edible Mushrooms and Algae-Based Foods [J]. Journal of China Edible Fungi. 35(06):69-72.DOI:10.13629/j.cnki.53-1054.2016.06.017.\u003c/li\u003e\n\u003cli\u003eWen C-Y,Xu-M,Yang Y-L, et al (2021) Research Progress on the Nutritional Composition and Value Evaluation of Wild Edible Mushrooms in China [J]. Journal of China Edible Fungi.40(11):1-10+20.DOI:10.13629/j.cnki.53-1054.2021.11.001.\u003c/li\u003e\n\u003cli\u003eChen X,Wang J,Li H-J ,et al (2015) Basic Composition and Nutritional Value of Bachu Mushrooms [J]. Agricultural Products Processing. (09):46-48.\u003c/li\u003e\n\u003cli\u003eHan C, Lin K, He Y, et al. Study on the extraction of dietary fiber from deep processing residue of Pleurotus eryngii and its monosaccharide composition. Food Science and Technology, 2015, 40(12): 208-213+217. doi: 10.13684/j.cnki.spkj.2015.12.042.\u003c/li\u003e\n\u003cli\u003eCohen N, Cohen J, Asatiani MD, et al (2014) Chemical composition and nutritional and medicinal value of fruit bodies and submerged cultured mycelia of culinary-medicinal higher basidiomycetes mushrooms. Int J Med Mushrooms 16:273\u0026ndash;291. https://doi.org/10.1615/IntJMedMushr.v16.i3.80\u003c/li\u003e\n\u003cli\u003ePellett PL, Young VR (1990) Commentary: joint FAO/WHO expert consultation on protein quality evaluation bethesda, MD, USA, 4\u0026ndash;8 december 1989. Ecology of Food and Nutrition 24:297\u0026ndash;303. https://doi.org/10.1080/03670244.1990.9991147\u003c/li\u003e\n\u003cli\u003eD\u0026aacute;vila G LR, Murillo A W, Zambrano F CJ, et al (2020) Evaluation of nutritional values of wild mushrooms and spent substrate of Lentinus crinitus (L.) Fr. Heliyon 6:e03502. https://doi.org/10.1016/j.heliyon.2020.e03502\u003c/li\u003e\n\u003cli\u003eChang,X.N.;Chen, X.F.;Gong, P.;Liu, M.;Wang, M.R.;Wang, X.J, et al (2022) Structural characterization,in vitro anti-oxidative effect and hypoglycemic activity of lentinan from Rongshui county.China Food Additives 33, 1-9 \u003c/li\u003e\n\u003cli\u003eLi,Y.L.;jiang, M.;Tan, M.q, et al (2020) Research Progress on Pharmacological Value of Polysaccharides from Edible Fungi,Journal of Ningxia Agriculture and Forestry Science and Technology 61, 24-26+39.\u003c/li\u003e\n\u003cli\u003eJusto A, Miettinen O, Floudas D, et al (2017) A revised family-level classification of the polyporales (basidiomycota). Fungal Biol 121:798\u0026ndash;824. https://doi.org/10.1016/j.funbio.2017.05.010\u003c/li\u003e\n\u003cli\u003eZhou J-L, Zhu L, Chen H, Cui B-K (2016) Taxonomy and phylogeny of polyporus group melanopus (polyporales, basidiomycota) from China. PLOS One 11:e0159495. https://doi.org/10.1371/journal.pone.0159495\u003c/li\u003e\n\u003cli\u003eZhuang L, Sun Z-H, Yu S-T, et al. (2024) Breeding of a New Variety of \u003cem\u003eHericium coralloides\u003c/em\u003e \u0026lsquo;ZLsh-1\u0026rsquo;. Mycological Journal. 43: 155\u0026ndash;162. https://doi.org/10.13346/j.mycosystema.240121\u003c/li\u003e\n\u003cli\u003eZhang P, Shi L, Yu H-Y, et al. (2024) Domestication and Cultivation of Two Wild \u003cem\u003eHericium coralloides\u003c/em\u003e Strains from the Xiaoxing\u0026apos;anling Region. Northern Horticulture 110\u0026ndash;114. \u003c/li\u003e\n\u003cli\u003eSar T, Larsson K, Fristedt R, et al (2022) Demo-scale production of protein-rich fungal biomass from potato protein liquor for use as innovative food and feed products. Food Biosci 47:101637. https://doi.org/10.1016/j.fbio.2022.101637\u003c/li\u003e\n\u003cli\u003eTabibzadeh F, Alvandi H, Hatamian-Zarmi A, et al (2022) Antioxidant activity and cytotoxicity of exopolysaccharide from mushroom\u003cem\u003e hericium coralloides\u003c/em\u003e in submerged fermentation. Biomass Convers Biorefin 1\u0026ndash;11. https://doi.org/10.1007/s13399-022-03386-0\u003c/li\u003e\n\u003cli\u003eHyder MS, Dutta SD (2021) Mushroom-derived polysaccharides as antitumor and anticancer agent: a concise review. Biocatal Agric Biotechnol 35:102085. https://doi.org/10.1016/j.bcab.2021.102085\u003c/li\u003e\n\u003cli\u003eHetland G, Tangen J-M, Mahmood F, et al (2020) Antitumor, anti-inflammatory and antiallergic effects of agaricus blazei mushroom extract and the related medicinal basidiomycetes mushrooms, hericium erinaceus and grifola frondosa: a review of preclinical and clinical studies. Nutrients 12:1339. https://doi.org/10.3390/nu12051339\u003c/li\u003e\n\u003cli\u003eZan X, Cui F, Li Y, et al (2015) \u003cem\u003eHericium erinaceus\u003c/em\u003e polysaccharide-protein HEG-5 inhibits SGC-7901 cell growth via cell cycle arrest and apoptosis. Int J Biol Macromol 76:242\u0026ndash;253. https://doi.org/10.1016/j.ijbiomac.2015.01.060\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hericium coralloides, biological characteristics, nutritional composition, antioxidant properties, culture conditions, domestication and cultivation ","lastPublishedDoi":"10.21203/rs.3.rs-5897250/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5897250/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003e[Objective]\u003c/strong\u003e: To isolate and identify a wild strain resembling \u003cem\u003eHericium coralloides\u003c/em\u003e (strain SH001) collected from the wild, explore its biological characteristics, and investigate the cultivation, nutritional composition, and antioxidant and anticancer activities of its fruiting body polysaccharides.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[Methods]\u003c/strong\u003e: The strain was identified based on its morphological characteristics and ITS sequence analysis. The biological properties were assessed by evaluating mycelial growth under varying conditions, including different carbon and nitrogen sources, temperatures, and pH values. Nutritional analysis of the fruiting body was conducted using Kjeldahl nitrogen determination, Soxhlet extraction, and light scattering detection methods. The antioxidant potential of the polysaccharides was evaluated through assays measuring DPPH, ABTS, OH radical scavenging activities, and iron ion reduction capacity. The anticancer effects of the polysaccharides on HepG\u003csub\u003e2\u003c/sub\u003e liver cancer cells and MDA-MB-468 breast cancer cells were assessed using the MTT assay.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[Results]\u003c/strong\u003e: The strain was identified as \u003cem\u003eHericium coralloides\u003c/em\u003e. The optimal growth conditions were found to be 30°C, pH 7, fructose as the preferred carbon source, and yeast extract as the optimal nitrogen source. Nutritional analysis revealed that the fruiting body was rich in protein (15.4 g/100 g dry weight), dietary fiber (34.7 g/100 g dry weight), and minerals, while being low in fat (3.5 g/100 g dry weight). The most abundant amino acids were glutamic acid, followed by aspartic acid. The polysaccharides exhibited significant antioxidant activity, with ABTS scavenging comparable to that of Vitamin C, achieving a clearance rate of 96.95% at concentrations between 0.25–5 mg/mL. At a concentration of 5 mg/mL, the DPPH and OH radical scavenging activities reached their peak (83.77% and 67.31%, respectively), along with the highest iron ion reducing capacity (FRAP value: 4.43 mmol/L). Polysaccharides also exhibited notable anticancer activity, inhibiting HepG2 liver cancer cells and MDA-MB-468 breast cancer cells, with IC50 values of 3.896 mg/mL and 2.561 mg/mL, respectively. \u003cstrong\u003e[Conclusion]\u003c/strong\u003e: This study demonstrates that wild \u003cem\u003eHericium coralloides\u003c/em\u003e can be successfully cultivated in vitro. The fruiting bodies possess substantial nutritional value, and the polysaccharides extracted from them show promising antioxidant and anticancer activities, particularly against HepG\u003csub\u003e2 \u003c/sub\u003eliver cancer cells and MDA-MB-468 breast cancer cells.\u003c/p\u003e","manuscriptTitle":"Domestication Cultivation and Nutritional Analysis of Hericium coralloides","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-08 06:19:47","doi":"10.21203/rs.3.rs-5897250/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"019147ae-0d9a-42e8-ae98-cb991c0bd530","owner":[],"postedDate":"May 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-21T15:05:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-08 06:19:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5897250","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5897250","identity":"rs-5897250","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}