Edible fungi are typical heterotrophic organisms. How to more efficiently convert and utilize organic matter such as lignin and cellulose, along with other nutrient components, remains the core challenge in the development and technological innovation of the edible fungus industry^[1]. As an essential source of nutrients for the growth and development of edible fungi, cultivation substrates provide carbon sources, nitrogen sources, and various trace elements. The type, origin, and processing methods of substrates directly determine the growth cycle, yield, nutritional quality, and economic efficiency of edible fungi. With China's high-quality economic development and accelerated industrial restructuring, the application of cultivation substrates faces several new challenges: first, frequent price fluctuations in bulk substrates; second, under increasingly stringent environmental policies, the stable supply of sawdust-based substrates faces substantial uncertainty, while alternative substrate sources remain limited and slow to adopt; third, with the promotion of technologies such as straw returning to fields, corn silage, and perennial rice, the supply of straw-based substrates may further decrease in the future; fourth, the biological conversion efficiency of current substrates remains generally low, leaving room for improvement in resource utilization; fifth, factory-scale production models have driven substantial growth in production capacity, making spent mushroom substrate (SMS) treatment and substrate resource utilization an increasingly prominent issue. Consequently, the ongoing development of the edible fungus industry faces the risk of forced adjustments to baseline formulations. Researching novel substrate formulas and matching cultivation technologies that feature lower costs and higher yields has become a vital factor for the sustainable development of the industry.

To address these challenges, the author systematically summarizes research progress in the field of edible fungus cultivation substrates in China based on core journal literature from Web of Science and CNKI published since 2015. This review explores research hotspots, development trends, and future directions, aiming to provide theoretical references for in-depth studies and the high-quality, sustainable development of the edible fungus industry.

1. Application of Novel Substrates

1.1 Tree Branch and Wood Chips

Sawdust is the primary substrate source for wood-decaying fungi. Current research on sawdust substrates has expanded from hardwood sawdust typically derived from Fagaceae species (such as oak, Liquidambar, beech, and Fagus) to fruit tree sawdust including apple, mulberry, peach, kiwi, Persian walnut, pear, jujube, wild jujube, and grape branch chips. It also extends to economic timber sawdust such as camellia, tea branch, mulberry branch, rubberwood, Sichuan pepper, goji branch, vitex, yellow willow, Moringa, eucalyptus bark, and eucalyptus chips. Furthermore, research on the application of oleaginous coniferous wood chips like Cryptomeria and spruce in Pleurotus ostreatus cultivation provides theoretical backing for the development of wood resources rich in functional components like tannins, pectins, and total flavonoids^[2].

1.2 Crop Stalks and Straw

Crop stalks such as rice straw, wheat straw, and corn stalks are rich in cellulose and hemicellulose, serving as major substrate sources for straw-rotting fungi like Agaricus bisporus, Volvariella volvacea, and Stropharia rugosoannulata. The primary research trend shows a transition from traditional grain crop straws (rice, corn, sorghum) to economic crop stalks (rapeseed, potato, soybean, hemp, ramie, cassava, tea stalk, wormwood) as well as vegetable crop stalks and leaves (asparagus, ginger, stem mustard).

1.3 Food Processing By-products

Substrates derived from food processing by-products have expanded from traditional oilseed residues like wheat bran and soybean meal to various seed meals, husks, and pomace/dregs. Seed meals mainly include cottonseed meal, castor seed meal, rapeseed meal, peony seed meal, and Sichuan pepper seed meal. Seed husk research has expanded from common materials like coffee husks and peanut shells to regional resources including betel nut shells, mountain jujube pits, grape seeds, lotus seed shells, and Gorgon nut shells. Food processing residues mainly comprise white spirit distillery dregs, brewer's spent grain, vinegar residue, water chestnut residue, Rosa roxburghii pomace, sea buckthorn pomace, coffee grounds, and tea residue. Additionally, sugar mill filter mud is recognized as a viable supplementary material for quick-acting carbon sources^[3].

1.4 Medicinal Plants and Herbal Residues

Medicinal plant substrates primarily originate from post-harvest plant residues, such as Evodia rutaecarpa branches, honeysuckle stems/leaves, Codonopsis pilosula stems/leaves, Eucommia ulmoides leaves and bark, Platycodon grandiflorus, Perilla frutescens stalks, and cinnamon wood processing by-products. Although pharmaceutical residues (herb dregs) are non-traditional substrates, relevant research has increased significantly in recent years, mainly focusing on single herb residues, Chinese herbal formula residues, and Western pharmaceutical residues. Examples include using Astragalus residue for Hericium erinaceus^[4], Polygonum cuspidatum residue for Pleurotus eryngii^[5]; utilizing residues from 6 Chinese patent medicines (Huoxiang Zhengqi Oral Liquid, Jinqiancao Granules, Jizhi Syrup, Dabaidu Capsules, Bajheng Mixture, Huangqin Granules) for Pleurotus cornucopiae^[6], Jizhi Syrup residue for Stropharia rugosoannulata^[7], and VC Yinqiao Tablets formula residue for Coprinus comatus^[8-9]; as well as Oxytetracycline Western pharmaceutical residue for Pleurotus eryngii^[10]. Medicinal plants and herb residue substrates hold immense potential for cultivating fruiting bodies with specific nutritional or functional properties, though their safety still requires systematic verification.

1.5 Other Substrates

Juncao (fungus grass) substrates represent an emerging domain in edible fungus substrate research. Common Juncao materials include Cenchrus fungigraminus (Giant Juncao), elephant grass, Miscanthus floridulus, Neyraudia reynaudiana, and alfalfa, while Miscanthus lutarioriparius and Arundo donax serve as promising regional resources. Notably, significant microscopic morphological differences in Lentinula edodes mycelia were observed between fresh and dried Juncao culture media, offering a new dimension for substrate research^[11].

Casing soil material research focuses mainly on loam, sandy soil, peat soil, wheat field soil, paddy soil, garden soil, spent mushroom compost soil, reed residue, and pond mud, which are commonly applied in the cultivation of straw-rotting fungi like Agaricus bisporus, Oudemansiella pseudoradicata, Coprinus comatus, and Stropharia rugosoannulata.

2. Substrate Physicochemical Indicators and Processing Methods

2.1 Physicochemical Indicators

Particle size, carbon-to-nitrogen ratio (C/N), moisture content, aeration, and pH are crucial physicochemical indicators affecting edible fungus cultivation. Proper adjustment of these metrics can enhance yield and quality. Substrate particle size and aeration directly influence fungal growth and yield. Kong et al.^[12] found through Pleurotus ostreatus fermented substrate trials with different particle sizes that small-particle substrates had lower water-soluble organic carbon content than large-particle ones, but contained higher ammonia content and mold-inhibiting substances. Smaller substrate particle sizes resulted in lower compost temperatures during the mycelial running period, lower contamination rates, and higher biological efficiency. Further metabolomic research demonstrated that adding small corn cobs (D₅₀ = 0.5 cm) versus large corn cobs (D₅₀ = 1.5 cm) in fermented substrates caused significant differences in microbial metabolites, with 464 and 201 differential metabolites identified in positive (POS) and negative (NEG) ionization modes, respectively^[13]. Yu et al.^[14] pointed out that wood chip particle size significantly affected single mushroom weight, mushroom shape, biological conversion rate, and production cycle in industrial Lentinula edodes production. Correlation analysis by He et al.^[15] revealed that the aeration of cultivation substrates and casing materials directly impacted fruiting time and yield in Stropharia rugosoannulata, with oxygenated water or magnetized water showing potential to improve agronomic traits and increase yield. Xiao et al.^[16] applied magnetized water during substrate mixing and humidification for Flammulina filiformis, enhancing the activities of extracellular laccase, polyphenol oxidase, carboxymethyl cellulase, and hemicellulase, which shortened the production cycle, lowered bag contamination rates, and increased single-bottle yield by over 2 g. Moderately alkalized substrates favor higher fruiting body yield and quality; Zhang et al.^[17] suggested this may be linked to alkalization boosting fungal laccase activity.

2.2 Substrate Processing Methods

Substrate processing methods primarily comprise substrate compression, substrate fermentation, and sterilization techniques.

(1) Substrate Compression: Research focuses on the impact of various compression techniques on mycelial growth and yield. Geng et al.^[18] used compressed cattle manure and wheat straw as primary materials to cultivate Agaricus bisporus, finding that a compression density of 500 kg/m³ significantly saved cultivation space and costs while increasing yield. Bao et al.^[19] employed a mechanical pressing device using hydro-thermal shaping to prepare Auricularia heimuer bags with Quercus mongolica sawdust as the main component. Results showed that at a bulk density of 0.72 g/cm³, mycelial growth was fastest, dry matter decomposition and utilization were highest, fruiting occurred earlier, ear-like fruiting bodies were longer and thicker, and firmness and rehydration rates decreased. Additionally, Qi et al.^[20] utilized biomass briquetting machinery to compress grape branch chips into granular substrate pellets for Pleurotus ostreatus, significantly boosting yield.

(2) Substrate Fermentation: Involves two main avenues. First, mold inhibition mechanisms. Cui et al.^[21] observed that extracts from fermented corn cob substrates disrupted the mycelial cell walls and membranes of Trichoderma viride and Penicillium griseofulvum, causing intracellular leakage and inhibiting growth and spore germination. Second, fermentation process optimization. Huang et al.^[22] explored loading parameters for Volvariella volvacea tunnel fermentation; Zha et al.^[23] analyzed changes in pH, electrical conductivity, moisture content, and nitrogen content during fermentation; Wang et al.^[24] studied the effects of tunnel fermentation technology on Agaricus bisporus mycelial growth and substrate degradation.

(3) Substrate Sterilization: Studies cover conventional high-pressure, step-up high-pressure, atmospheric-pressure (autoclaving/steaming), and short-duration high-pressure sterilization processes. Chen et al.^[25] discovered that increasing atmospheric sterilization frequency led to decreases in aeration porosity, moisture content, and pH in Pleurotus geesteranus bags, with sterilization frequency negatively correlated with full-bag colonization time and biological efficiency. Yang et al.^[26] found that step-up high-pressure sterilization outperformed conventional methods by boosting the activities of 3 ligninolytic enzymes (laccase, manganese peroxidase, lignin peroxidase) and 4 cellulolytic enzymes (filter paperase, endoglucanase, β-glucosidase, hemicellulase) in Hypsizygus marmoreus mycelia, while also increasing average cluster yield.

3. Effects of Exogenous Factors on Fungal Growth

3.1 Mineral Elements

Mineral element levels and compositions in substrates directly affect fruiting body yield and nutritional profile. Key functions include maintaining cellular structure, regulating extracellular enzymes, and counteracting heavy metal toxicity^[27].

(1) Selenium (Se): Adding sodium selenite to substrates has been widely studied for biofortified cultivation in Pleurotus ostreatus, Agaricus bisporus, Stropharia rugosoannulata, Flammulina filiformis, Hypsizygus marmoreus, and Pleurotus eryngii. Studies show that sodium selenite treatment yielded higher Se recovery rates in F. filiformis fruiting bodies compared to selenomethionine and sodium selenate^[28]. Sodium selenite treatment significantly increased Se content, improved nutritional quality, and enhanced antioxidant capacity in S. rugosoannulata^[29-30].

(2) Calcium (Ca): Calcium is a structural component of cell walls and essential for cell membrane stability, participating in the regulation of mycelial turgor and enzymatic activity. Adding appropriate amounts of calcium carbonate not only supplies essential calcium but also neutralizes acidic substances generated during substrate fermentation or mycelial growth. Wu et al.^[31] noted that in A. bisporus cultivation, Ca levels dropped significantly during composting and mycelial growth stages (most notably in casing soil), indicating Ca actively participates in mycelial metabolism and enzyme activity; during the harvest stage, Ca, carbon, and oxygen levels stabilized across casing and compost layers.

(3) Zinc (Zn): Zinc sulfate is commonly added for regulation. In Pholiota nameko, supplementing substrates with zinc sulfate significantly boosted activities of filter paperase, carboxymethyl cellulase, hemicellulase, and β-glucosidase during mycelial growth, showing yield-enhancing effects^[32].

(4) Other Minerals: Sun et al.^[33] screened strontium-enriched Auricularia heimuer cultivars by adding varying concentrations of strontium chloride to substrates; Lin et al.^[34] added 0.5 g/L sodium citrate to PDB liquid medium, significantly enhancing Volvariella volvacea mycelial biomass and shortening the incubation period. Zhu et al.^[35] observed that co-supplementing yeast powder and copper sulfate synergistically enhanced laccase activity in Pleurotus eryngii fermentation broth. Wang et al.^[36] confirmed that exogenous addition of MnSO₄, Na₂SeO₃, CaSO₄, and FeSO₄ significantly boosted mycelial growth rate, biomass, and antioxidant enzyme activities (e.g., SOD, CAT) in V. volvacea.

Furthermore, metabolic interactions between mineral elements occur in mycelial cells. For instance, phosphorus or sulfur deficiencies in compost can reduce the uptake rate of sodium selenite in F. filiformis^[37], while adding zinc sulfate helps reduce cadmium accumulation in Lentinula edodes mycelia^[38]. Zhang et al.^[39] discovered that under manganese stress, Pisolithus tinctorius and Suillus luteus could solubilize insoluble soil potassium by secreting oxalic acid and hydrogen ions. Recent studies increasingly focus on synergistic interactions and comprehensive regulatory mechanisms among multiple mineral elements.

3.2 Organic Acids

Certain organic acids show positive effects on fungal strain rejuvenation, fruiting body yield, and nutritional improvement. Kong et al.^[40] compared the rejuvenation effects of 20 L-amino acids on degenerated Volvariella volvacea strains. Treatments with serine (Ser), alanine (Ala), valine (Val), leucine (Leu), and proline (Pro) all promoted mycelial growth and biomass accumulation while increasing mycelial polysaccharides, proteins, flavonoids, and polyphenols, effectively inhibiting reactive oxygen species (ROS) accumulation and enhancing antioxidant enzyme activities. Further research indicated that Ser treatment significantly increased methionine (Met), cysteine (Cys), and Ser contents, as well as Mg, Cu, and Zn mineral levels in mycelia^[41]. Gong et al.^[42] found exogenous citric acid and arginine enhanced arginine synthesis pathways in the TCA cycle of Hypsizygus marmoreus mycelial cells, boosting fruiting body yield. Zhang et al.^[43] supplemented substrates with kojic acid, which elevated laccase and cellulase activities in H. marmoreus mycelia during reproductive growth, thereby improving lignocellulose utilization and overall yield. Wang et al.^[44] demonstrated that adding specific concentrations of salicylic acid to media favored mycelial growth in Macrocybe gigantea.

3.3 Carbohydrates

Carbohydrates act as carbon sources that effectively stimulate mycelial growth. Liu et al.^[45] found that sucrose, fructose, mannitol, and trehalose all enhanced aerial mycelial density, growth rate, and biomass in degenerated Volvariella volvacea strains, while increasing polysaccharide and protein contents, suppressing ROS accumulation, and enhancing SOD and POD activities. Cheng et al.^[46] also reported that exogenous mannitol largely restored filter paperase, endoglucanase, laccase, and manganese peroxidase activities in degenerated V. volvacea strains. Yang et al.^[47] indicated that trehalose supplementation stimulated mycelial growth and biomass in Stropharia rugosoannulata and Hypsizygus marmoreus, exerting strong regulatory control over cellulase and laccase activities. Furthermore, trial results by Liu et al.^[48] confirmed that low concentrations of exogenous trehalose alleviated heat stress inhibition on mycelial growth, highlighting trehalose as a promising substrate additive for non-biotic stress protection.

3.4 Botanical Extracts

Adding specific botanical extracts to substrates provides multiple benefits, including antibacterial protection, accelerated cell growth, enhanced phenolic metabolism, and elevated antioxidant activity. Incorporating a mixed garlic-ginger extract into Pleurotus ostreatus media exerted strong antibacterial effects, shortened pinheading time, and produced mushrooms with firmer texture and smaller caps^[49]. Wild apricot shell wood vinegar effectively suppressed bacterial brown spot disease in P. ostreatus while accelerating mycelial growth^[50]. Garlic and Platycladus orientalis extracts added to Coprinus comatus substrates exhibited marked antibacterial action against Penicillium and Rhizopus, boosting yield, shortening flushing intervals, and improving cap-to-stem ratios^[51]. Bao et al.^[52] found that betel nut receptacle water extracts significantly increased antioxidant activity in P. ostreatus, Pleurotus citrinopileatus, and Flammulina filiformis mycelia. Wu et al.^[53] reported that adding 8% (w/w) tea branch extract accelerated mycelial growth and enhanced phenolic metabolic turnover and hydroxyl radical scavenging capacity. Fermented corn cob extract (10-day fermentation) similarly improved P. ostreatus mycelial growth rate and biomass, while boosting ATPase, succinate dehydrogenase, alkaline phosphatase, laccase, and protease activities; microscopic analysis revealed a marked increase in mitochondria and vesicular structures within fungal cells^[54].

3.5 Other Exogenous Factors

Plant growth regulators modulate fungal metabolism, promote mycelial growth, and increase enzyme activities. For instance, IAA, NAA, 6-BA, KT-30, gibberellin, and sodium humate all demonstrated growth-promoting effects on Agaricus balchaschensis mycelia^[55]. Adding 2.0 mL of β-oligoacid per 1 kg of dry substrate promoted mycelial growth across Pleurotus eryngii, Coprinus comatus, Ganoderma lucidum, and Hericium erinaceus^[56]. Moreover, chlorophenoxyacetic acid, NAA, and zeatin have been applied in liquid spawn cultures and hold potential as substrate additives.

Enzyme preparations offer dual benefits of boosting yield and improving fruit body marketability. Zhang et al.^[57] added a composite enzyme formulation (comprising enzymes, gene expression inducers, and enzyme activators) to Lentinula edodes substrates, accelerating full-bag colonization by 15 days compared to control. During the color-turning stage, cellulase, protease, and amylase activities rose significantly, accompanied by a 21.99% increase in biological efficiency. Xin et al.^[58] reported that supplementing compost prior to primary fermentation with 1% acidic cellulase, 1% neutral cellulase, and 0.5% hemicellulase (w/w) significantly enlarged bud/primordium diameter in Volvariella volvacea.

Fungal stipe bases and spent substrate extracts applied as exogenous nutrient solutions show great potential. Yin et al.^[59] mixed stipe extracts from Lentinula edodes, Pleurotus eryngii, and Hypsizygus nebrodensis with soil and applied it to ring-cut substrate bags of Pleurotus nebrodensis. The L. edodes stipe extract treatment increased yield by 54.6% over control while enhancing protein and nitrate contents. Zhang et al.^[60] sprayed Flammulina filiformis spent substrate extract onto Agaricus bisporus casing layers, finding higher AAS, CS, SRC, EAAI, BV, and NI scores in fruiting bodies compared to control, demonstrating that spent substrate extracts can upgrade protein nutritional quality.

4. Microbial Community Dynamics in Cultivation Substrates

4.1 Isolation, Identification, and Screening of Functional Strains from Fermented Substrates

Bacterial communities in fermented substrates exhibit high diversity, with dominant taxa and relative abundances shifting markedly across fermentation stages. A decline in dominant group abundance can lead to fermentation failure, whereas the absence of fruiting-stage specific taxa can trigger abnormal fruiting. Consequently, isolating, identifying, and screening functional strains with antibacterial, growth-promoting, or detoxifying capabilities to optimize fermentation is a major research focus. 16S rRNA gene sequencing has been widely applied in these investigations^[61].

Zhang et al.^[62] isolated 94 bacterial strains from Pleurotus ostreatus fermented substrates across different periods, identifying 11 strains capable of simultaneously inhibiting Trichoderma and promoting P. ostreatus growth. Further study revealed that several growth-promoting strains possessed strong IAA synthesis capacity^[63]. Cui et al.^[64] observed that aflatoxin levels during P. ostreatus substrate fermentation were negatively correlated with the relative abundance of Paenibacillus and Luteimonas, suggesting these genera may participate in aflatoxin degradation. Wu et al.^[65] isolated a bacterial consortium agent from distillery dregs. Adding this agent to P. ostreatus fermented substrates accelerated carbohydrate metabolic rates, increased substrate temperature, and extended the thermophilic phase. Furthermore, fungal ergosterol content, lignocellulose degradation rates, and associated enzyme activities exceeded control levels, encouraging faster mycelial colonization.

4.2 Casing Soil Microbial Community Structure and Dominant Group Shifts

Casing soil microorganisms play pivotal roles in mycelial growth, primordium formation, and yield enhancement in straw-rotting fungi. Significant variations exist in microbial abundance and dominant taxa across different flushing cycles. Wang et al.^[66] discovered that during the 1st flush of Agaricus bisporus, dominant genera included Sphingomonas, Dongia, and Achromobacter; by the 3rd flush, dominant taxa shifted to Norank, Pseudomonas, Flavobacterium, and Brevundimonas. Furthermore, broad interactions occur among casing soil communities, as confirmed by Yang et al.^[67] in Phlebopus portentosus. Researchers have also isolated specific functional strains from casing layers; Hua et al.^[68] isolated Rhizobium strain CACMS001 from soil around Wolfiporia cocos sclerotia, which promoted mycelial growth in both W. cocos and Armillaria gallica while significantly boosting xylanase activity in A. gallica.

5. Extracellular Enzymes and Substrate Degradation Patterns

5.1 Correlation Between Extracellular Enzyme Activity and Fruiting Body Yield

Substrate nutrient utilization by edible fungi primarily involves macromolecular degradation (lignin, cellulose, hemicellulose, proteins), mineral absorption (N, P, K), and small-molecule metabolism (amino acids, peptides, lipids, vitamins, phenylpropanoids, polyketides, pyran acids/pyrones, phytohormones, antibacterial agents). Degradation of macromolecular organic matter relies on extracellular enzymes secreted by fungi to break down polymers into absorbable units. Thus, extracellular enzyme activities correlate closely with mycelial growth rate and fruiting body yield. Xie et al.^[69] found across different substrates that lignocellulose degradation rates and lignocellulolytic enzyme activities in Pleurotus eryngii correlated positively with biological efficiency. Lin et al.^[70] showed that fresh yield of Hypsizygus marmoreus correlated positively with carboxymethyl cellulase, xylanase, filter paperase, and amylase activities. Liu et al.^[71] noted that high-yielding batches of Volvariella volvacea grown on waste cotton substrates displayed significantly higher carboxymethyl cellulase and xylanase activities than low-yielding batches. Cai et al.^[72] also reported positive correlations between cellulase/xylanase activities and Agaricus bisporus yield.

5.2 Developmental Dynamics of Extracellular Enzyme Activities

Extracellular enzyme activities show distinct stage-specific patterns during fungal development. Lei et al.^[73] observed that filter paperase, carboxymethyl cellulase, β-glucosidase, amylase, and hemicellulase activities in Pleurotus citrinopileatus cell cultures initially increased before declining, maintaining elevated levels through the young mushroom stage. Conversely, laccase and peroxidase activities peaked during early mycelial growth and decreased with prolonged cultivation. Yue et al.^[74] found that carboxymethyl cellulase, pectinase, filter paperase, and hemicellulase in Hericium erinaceus reached activity peaks during fruiting body development, whereas amylase, pectinase, and peroxidase were most active during mycelial vegetative growth. Li et al.^[75] measured laccase activity across mycelial, knotting, primordium, young mushroom, and mature fruiting body stages in Pleurotus eryngii; laccase activity at the mature fruiting stage was significantly higher than all preceding stages, which showed minimal variation among themselves.

5.3 Influence of Carbon Sources on Extracellular Enzyme Activity

Carbon source types and inclusion ratios substantially influence extracellular enzyme activities. Compared to media containing cottonseed hulls, wood chips, and corn cobs, wheat bran-containing media stimulated higher laccase activity in Auricularia cornea^[76]. Pleurotus ostreatus grown on fermented cottonseed hulls exhibited higher carboxymethyl cellulase, laccase, and neutral protease activities than when grown on corn cob substrates^[77]. Substrate formulation ratios also drive activity shifts. Chen et al.^[78] demonstrated that during mycelial growth of Auricularia heimuer, a 70% corn cob inclusion produced maximum lignin peroxidase activity, 50% produced peak manganese peroxidase activity, and 30% yielded highest carboxymethyl cellulase activity. Increasing corn cob proportions decreased laccase activity while elevating N-acetyl-β-D-glucosaminidase; β-1,3-glucanase, β-glucosidase, and endo-β-1,4-glucanase activities initially rose before declining. Notably, research has begun exploring carbon-source regulation of extracellular enzymes at the molecular level; Xiao et al.^[79] conducted systematic gene expression analyses related to cellulose and hemicellulose degradation in Sparassis latifolia.

6. Relationship Between Cultivation Substrates and Fruiting Body Quality

6.1 Effects of Different Substrates on Nutritional Components

Studies typically cultivate a target species across multiple substrates, evaluating nutritional parameters such as protein, amino acids, total sugars, polysaccharides, polysaccharide-peptides, and dietary fiber to determine substrate impacts on nutritional value and guide novel substrate formulation. On one hand, amino acid content and composition are heavily investigated. Hu^[80] cultivated Pleurotus ostreatus using Juncao combined with lotus seed shells and lotus receptacles, producing fruiting bodies with diverse and abundant amino acids and fatty acids. Lin et al.^[81] added fresh Juncao to cottonseed hull substrates, significantly boosting amino acid levels in Tremella fuciformis. Ke et al.^[82] used spent Flammulina filiformis and Pleurotus eryngii substrates as primary components for Agaricus bisporus, achieving superior amino acid composition and flavor amino acid profiles compared to traditional straw formulas. On the other hand, research increasingly targets specific functional nutrients to develop specialized mushroom products. Wen et al.^[83] revealed that elevating substrate nitrogen (e.g., urea) enhanced γ-aminobutyric acid (GABA) accumulation across multiple species. Adding sea buckthorn wood chips, pomace, or leaves significantly elevated total flavonoid content in Pholiota nameko^[84]. Cassava stalk supplementation raised trehalose levels in Auricularia heimuer, P. ostreatus, and P. citrinopileatus^[85]. Incorporating Astragalus stems and leaves enriched P. ostreatus fruiting bodies with bioactive compounds such as terpenoids and flavonoids^[86].

6.2 Effects of Different Substrates on Flavor Compounds

Cultivation substrates differentially impact volatile flavor compounds, taste-active amino acids, and non-volatile flavor components in fruiting bodies. Substrates rich in taste-active amino acids can increase corresponding amino acid levels in mushrooms via mycelial absorption. For instance, Pleurotus cornucopiae grown on herbal medicine residues contained higher umami and sweet amino acid levels than those grown on sorghum husks^[87]. Substrates also alter volatile compound profiles and promote specific aromatic synthesis. Yu et al.^[88] analyzed major volatiles including isovaleraldehyde, hexanal, 1-octen-3-ol, methanethiol, 2-pentylfuran, and dimethyl sulfide in Volvariella volvacea, finding significant aroma differences across formulations: spent Pleurotus eryngii substrate produced the best aroma quality, whereas spent Flammulina filiformis substrate scored lowest. Comparative trials by Yin et al.^[89] demonstrated that cottonseed hull substrates favored flavor ester formation in Pleurotus species, whereas hardwood sawdust favored free amino acid accumulation. Substrates also influence non-volatile taste components; Yu et al.^[90] observed that cottonseed hull substrates yielded optimal hydrolyzed amino acid profiles in V. volvacea, while rice straw substrates yielded highest soluble sugar alcohols and organic acids. Li et al.^[91] partially replaced wheat bran with fermented corn steep liquor in P. eryngii cultivation, achieving higher soluble sugar alcohols, organic acids, and 5'-nucleotides than the control group.

7. Spent Mushroom Substrate (SMS) Reutilization Research

7.1 Types of Spent Substrates and Utilization Approaches

In terms of spent substrate types, domestic research focuses heavily on industrial spent substrates from Pleurotus eryngii, Flammulina filiformis, Hypsizygus marmoreus, and Hypsizygus tephroleucus, as well as spent materials from large-scale traditional species like Lentinula edodes and Auricularia heimuer. Emerging waste streams, including spent Morchella nutrient bags, spent Gastrodia elata timber, and spent Cordyceps militaris rice media, are also receiving growing attention.

Regarding utilization pathways, the predominant approach uses wood-decaying fungal SMS to cultivate straw-rotting species, usually blended with fresh raw materials. Certain spent substrates, such as spent Tremella timber and A. heimuer SMS, can partially substitute fresh sawdust for wood-decaying species. Beyond serving as bulk substrate, most SMS materials supply carbon and nitrogen; spent C. militaris rice media, for instance, serves as an excellent nitrogen supplement^[92]. In addition to crushing and mixing fresh SMS directly, Zhang et al.^[93] applied four modification treatments (alkaline reaction, smother-fermentation, baking, ultrasonication) to SMS, successfully improving F. filiformis yield and quality under SMS-based cultivation.

7.2 Efficacy Evaluation and Formula Optimization

Safety evaluation is the primary concern when re-using SMS for mushroom production. Chen et al.^[94] added spent Pleurotus eryngii substrate to cultivate Pleurotus ostreatus, P. eryngii, and Flammulina filiformis, confirming that As, Hg, Pb, and Cd heavy metal levels in fruiting bodies remained strictly within national safety limits.

Current research hotspots focus on screening optimal SMS formulas by comprehensively evaluating mycelial growth, yield performance, nutritional profiles, and economic returns. Wen et al.^[95] investigated varying inclusion rates of industrial F. filiformis SMS on P. ostreatus bag cultivation, identifying optimal inclusion ratios. Li et al.^[96] substituted 60% (w/w) waste cotton with spent P. eryngii and Agaricus bisporus substrates, shortening the Volvariella volvacea production cycle by 1.6 days while increasing yields by 15% and 17%, respectively. Chen et al.^[97] found that replacing 30% straw with spent P. eryngii substrate achieved maximum yield in Agaricus subrufescens, with crude protein, amino acid, and polysaccharide levels outperforming traditional formulas while cutting raw material costs by over 35%.

8. Future Prospects

Over the past decade, research on edible fungus cultivation substrates in China has yielded fruitful outcomes, demonstrating clear trends toward diversified material sources, varied processing methods, and composite formulation strategies. Understanding of microbial community succession during fermentation, extracellular enzymatic degradation mechanisms, and substrate impacts on nutritional and flavor compounds has deepened significantly. Future research should prioritize the following directions:

(1) In-depth deciphering of substrate component mechanisms. Research will focus on molecular mechanisms governing fungal growth and development responses to substrate components, particularly regulatory networks controlling substrate degradation, nutrient transport, and secondary metabolite synthesis^[98].

(2) Rapid development and commercial application of specialized commercial substrates. As industrial production advances and supply chains specialize, commercial substrate manufacturers will accelerate the development of high-efficiency, safe, functional substrates, particularly specialized compound formulations with customized additives. Concurrently, modifying substrates for non-traditional habitats like alpine areas, deserts, and saline-alkali lands will become a vital research focus.

(3) Expanding microbial community structure and function studies in substrates and casing soil. While current studies focus on growth-promoting and antibacterial mechanisms of beneficial microbes during specific growth stages, future work will adopt broader ecological perspectives to map inter-microbial interactions, functional redundancy, and regulatory networks for precise microbial manipulation.

(4) Establishing ecological risk and biosafety evaluation systems for novel substrates. As novel materials enter substrate formulations, evaluating their potential impacts on ecosystems and human health becomes essential. A systematic evaluation framework is urgently needed to assess ecological safety, nutritional quality, and potential toxicological effects, ensuring high yields without compromising environmental safety or human health^[99].

(5) Circular recycling technologies for spent substrates and substrate re-use. With expanding capacity, improving resource efficiency and reducing costs are top industry priorities. Exploring multi-cycle substrate re-use, revealing physicochemical evolution across successive uses, and determining impacts on mushroom yield and quality will provide theoretical and technical support for a green, low-carbon, and sustainable industry.

(6) Development and deployment of intelligent substrate management systems. Leveraging IoT, big data, and artificial intelligence, developing self-learning, species-adapted intelligent substrate management systems will enable real-time tracking, monitoring, and precise control across substrate sourcing, processing, nutritional analysis, and degradation monitoring, representing a key path for industrial facility upgrades^[100].