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Article

Formula Screening and Optimization of Physical and Chemical Properties for Cultivating Flammulina filiformis Using Soybean Straw as Substrate

by
Ruixiang Sun
1,2,†,
Jiandong Han
2,†,
Peng Yang
2,
Shude Yang
3,
Hongyan Xie
2,
Jin Li
2,
Chunyan Huang
2,
Qiang Yao
2,
Qinghua Wang
4,
He Li
5,
Xuerong Han
1,* and
Zhiyuan Gong
2,*
1
International Cooperation Research Center of China for New Germplasm Breeding of Edible Mushrooms, Jilin Agricultural University, Changchun 130118, China
2
State Key Laboratory of Nutrient Use and Management, Key Laboratory of Wastes Matrix Utilization, Ministry of Agriculture and Rural Affairs, Shandong Academy of Agricultural Sciences, Jinan 250100, China
3
School of Horticulture, Ludong University, Yantai 264000, China
4
Yijun Agricultural Development Co., Ltd., Zibo 255000, China
5
Hinggan League Institute of Agricultural and Husbandry Sciences, Inner Mongolia Innovation Center of Biological Breeding Technology, Ulan Hot City 137400, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(8), 947; https://doi.org/10.3390/horticulturae11080947
Submission received: 16 July 2025 / Revised: 6 August 2025 / Accepted: 8 August 2025 / Published: 11 August 2025
(This article belongs to the Special Issue Advances in Propagation and Cultivation of Mushroom)
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Abstract

Recently, there has been a growing interest in using agricultural and forestry residues to cultivate Flammulina filiformis. However, there is limited research on cultivating F. filiformis with soybean straw as a substrate. This study systematically optimized the cultivation formula for F. filiformis using soybean straw as the raw substrate and explored the effects of the water content, carbon-to-nitrogen ratio (C/N ratio), substrate particle size, and substrate loading on its growth and development. By replacing corncob, wheat bran, and soybean hulls with soybean straw and increasing the proportion of rice bran, the cultivation formula for growing F. filiformis was optimized. We found that the maximum fruiting body yield of 405 g (330 g dry substrate per bottle) and a biological efficiency of 122.73% were achieved using a substrate mixture of 25% soybean straw, 20% corncob, 20% cottonseed hull, 25% rice bran, 8% wheat bran, 1% CaCO3, and 1% shellfish powder. The yield and biological efficiency of fruiting bodies cultivated on the substrate containing 25% soybean straw did not show significant differences compared to the control group. However, the cultivation formula containing 25% soybean straw yielded F. filiformis with significantly higher levels of amino acids, essential amino acids, and fat. These findings suggest that the 25% soybean straw substrate formulation can serve as a viable alternative to the control formulation for the cultivation of F. filiformis, although variations in the nutritional composition exist. Based on this optimized formula, an optimal biological efficiency can be achieved with a substrate-to-water ratio of 1:1.7, a wet substrate loading amount of 940 g (in a 1250 mL cultivation bottle), and a soybean straw particle size range of 6–8 mm. The optimal C/N ratio for cultivating F. filiformis using soybean straw ranges from 27:1 to 32:1. Additionally, orthogonal experiments revealed that the nitrogen content significantly affected the fruiting body yield, stipe length, and stipe diameter, while the water content mainly affected the pileus diameter, pileus thickness, and number of fruit bodies. Under defined conditions (dry substrate loading volume of 337 g (in a 1250 mL cultivation bottle), a substrate-to-water ratio of 1:1.6, and a C/N ratio of 26:1), the maximum yield and biological efficiency per bottle reached 395 g and 117.21%, respectively. Our findings indicate that the F. filiformis cultivation using soybean straw as the raw substrate exhibits a promising performance and extensive application potential.

1. Introduction

Wild Flammulina filiformis occurs in clusters on the trunks or stumps of broadleaf trees, such as mountain poplars, willows, and elms, typically during the period from late fall to early spring. These mushrooms are characterized by their light brown to brown caps and straw-colored stipes [1]. F. filiformis is considered the most significant species within the genus Flammulina and has garnered particular attention in East Asia [2]. F. filiformis is considered one of the four most popular edible mushrooms globally. It is renowned for its excellent taste and is rich in protein, amino acids, and other essential nutritional components, as well as bioactive substances [3]. Furthermore, it exhibits a range of health benefits, including antioxidant, anti-inflammatory, cholesterol-lowering, immune-regulatory, and anti-tumor properties [3,4,5]. As a white rot fungus, F. filiformis can secrete enzymes such as cellulase, hemicellulase, and ligninase, which break down cellulose and lignin into smaller molecules for absorption and utilization [6]. The optimal pH range for the mycelial growth of F. filiformis is 5.0–6.5 [7,8], with a suitable growth temperature ranging from 18 to 25 °C, a carbon dioxide concentration below 0.3–0.5%, and a culture substrate humidity level between 50% and 80%. There are notable differences in growth conditions between the mycelium colonization stage and the mycelial reproductive phase. After scratching the surface layer, the temperature should be kept at 14–16 °C and the humidity at 90–95%. Once the primordia have formed, the temperature should be regulated at 3–5 °C, and the light exposure should be maintained at 500–1000 Lx. When the young mushrooms reach a height of around 3 cm, the temperature should be maintained at 5 °C and the humidity adjusted to 75–80% until harvesting [1,2].
Currently, mushrooms can be cultivated using various substrates, including cottonseed hulls, sawdust, bagasse, corncobs, and wheat straw [9,10,11]. In cultivating F. filiformis, the primary raw substrates are corn cobs, cottonseed hulls, rice bran, wheat bran, soybean hulls, and soybean meal. With the rapid expansion of China’s edible mushroom industry, the prices of traditional cultivation substrates, such as wood chips, cottonseed hulls, and bran, have been rising, causing supply issues. Traditional cultivation substrates fail to meet the market demand [12], leading to higher production costs and lower profits. To improve profitability, it is essential to explore new raw substrate sources and optimize production formulas.
Crop straw is abundant in cellulose, hemicellulose, and lignin, making it both a form of agricultural waste and a valuable biomass resource [13]. In China, various types of crop straw are available, including soybean straw, cotton straw, rice straw, corn straw, peanut straw, and others [14]. Annually, China produces over 1 billion tons of crop straw; however, only approximately 200 million tons are utilized effectively [15]. Incorporating crop straw into edible fungus cultivation can elevate the value of straw; reduce production costs; and deliver economic, social, and ecological benefits. Soybean straw exhibits a relatively high nutrient content, with approximately 1.42% nitrogen [16]. Additionally, it is characterized by a substantial yield and ease of collection. The feasibility of using soybean straw for the cultivation of F. filiformis requires further investigation.
The choice of substrate significantly affects the yield and quality of mushrooms [17,18]. Changing the culture substrate’s composition alters its physicochemical properties, such as the water retention capacity and air permeability, which in turn affect the mycelial growth rate, fruiting body yield, and the overall growth cycle of the mushroom. In this study, soybean straw was employed for cultivating F. filiformis, with an investigation into optimal conditions to enhance the fruiting body yield and quality. This was achieved by examining the proportion of soybean straw in relation to cultivation factors, including the water content, substrate loading amount, particle size, and C/N ratio. Additionally, an orthogonal experiment was conducted to evaluate the effects of different factors on both the yield and agronomic traits of F. filiformis. This research offers valuable insights for optimizing F. filiformis production.

2. Substrates and Methods

2.1. Inoculum Source and Spawn Preparation

The F. filiformis strain for this study was obtained from Shandong Youshuo Biotechnology Co., Ltd., located in Jining, China. The strain is currently preserved under the accession number FfWRY-18 at the Institute of Agricultural Resources and Environment, Shandong Academy of Agricultural Sciences. The mycelium presents a velvety texture, appears pure white in color, is relatively dense, and exhibits well-defined margins. The mycelium adheres tightly to the surface of the culture medium, with a limited amount of aerial hyphae present (Figure 1).
The stock culture was maintained on PDA (Potato Dextrose Agar) medium, and the spawn was prepared using a mix of 18% wheat bran, 80% sawdust, and 2% lime. Spawns were incubated in the dark at 25 °C for 30 days and subsequently stored at 4 °C for future use. The liquid medium was formulated as sucrose(Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) 20 g/L, soybean meal 3.3 g/L, magnesium sulfate(Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) 0.66 g/L, and potassium dihydrogen phosphate(Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) 0.66 g/L. Soybean meal should be sieved using a 300-mesh sieve to ensure a particle size of less than 0.05 mm. Liquid medium was sterilized and accessed to 3 pieces of strain blocks of 8 mm diameter. After that, the medium was placed in a shaker at 165 rpm and 23 °C for 7 days before use. The solid medium was employed for strain preservation, while the liquid medium was used for inoculating the culture substrate.

2.2. Substrate Source

Soybean straw, cottonseed hulls, corn cobs, wheat bran, rice bran, soybean hulls, CaCO3, and scallop shell powder were purchased from Shandong Youshuo Biotechnology Co., Ltd., located in Jining, China.

2.3. The Optimization of the Formula with Different Soybean Straw Proportions

The raw substrate compositions for different formulations are presented in Table 1. Raw substrates were first weighed according to the designed proportions, transferred into a mixer, and stirred for 20 min. Water was then added at a substrate-to-water ratio of 1:1.65, followed by an additional 40 min of thorough mixing. The substrate was then transferred into 1250 mL polypropylene bottles. During filling, each bottle was loaded with approximately 880 g of wet substrate (equivalent to 330 g dry substrate), with consistent pressure during bottling. After bottling, five holes were punched in each bottle, residual substrate adhering to the bottle rim was cleaned, and the substrates were sterilized. Each sterilized bottle was inoculated with 30 mL of liquid spawn and incubated in a dark incubation chamber maintained at 18 °C with controlled CO2 levels lower than 0.3%. Once fully colonized by the mycelium, the surface layer (0.3–0.5 cm) was scraped off, and the cultures were subsequently maintained at 14 °C with 90–92% relative humidity (RH). Lighting was initiated on the third day. Upon the appearance of primordia, RH was slightly reduced to 80%. Once fruiting bodies elongated to 2–3 cm above the bottle rim, a paper collar was added, and the temperature was adjusted at 8–12 °C until harvest. Three replicates were conducted for each treatment, with each replicate consisting of 12 bottles to ensure statistical reliability.

2.4. The Optimization of the Substrate-to-Water Ratio in the Culture Substrate

A screening test was conducted to evaluate the effects of different substrate-to-water ratios on the growth and development of F. filiformis, using the aforementioned optimized screening formulation as the test formulation and a control formulation consisting of 30% corn cobs, 20% cottonseed hulls, 20% rice bran, 25% wheat bran, 3% soybean hulls, 1% CaCO3, and 1% scallop shell powder. Using the test formulation as the base formulation, three substrate-to-water ratio gradients (1:1.6, 1:1.7, and 1:1.8) were set and designated as formula 1, formula 2, and formula 3, respectively. Each treatment comprised 32 bottles. Substrate mixing and post-inoculation management procedures were consistent with those described previously.

2.5. Optimization of Substrate Loading Volume in Culture Substrate

Screening tests for optimal loading were conducted using a test formulation consisting of 25% soybean straw, 20% corn cobs, 20% cottonseed hulls, 25% rice bran, 8% wheat bran, 1% CaCO3, and 1% scallop shell powder, while the control formulation comprised 30% corn cobs, 20% cottonseed hulls, 20% rice bran, 25% wheat bran, 3% soybean hulls, 1% CaCO3, and 1% scallop shell powder. The test formulation served as the base formulation, with a substrate-to-water ratio fixed at 1:1.7. Three substrate loading gradients (900 g, 920 g, and 940 g per 1250 mL bottle) were established and designated as formula 1, formula 2, and formula 3, respectively. All other experimental conditions were consistent with those in the water content optimization experiment.

2.6. Optimization of Soybean Straw Particle Size

Using a test formulation consisting of 25% soybean straw, 20% corn cobs, 20% cottonseed hulls, 25% rice bran, 8% wheat bran, 1% CaCO3, and 1% scallop shell powder, the particle size of the crushed soybean straw was evaluated using three sieve sizes: 6 mm, 8 mm, and 10 mm. These were designated as formula 1, formula 2, and formula 3, respectively. A control formulation comprising 30% corn cobs, 20% cottonseed hulls, 20% rice bran, 25% wheat bran, 3% soybean hulls, 1% light calcium carbonate, and 1% scallop shell powder was included for comparison. Substrate mixing and post-inoculation management procedures were consistent with those described previously.

2.7. Optimization of Carbon-to-Nitrogen (C/N) Ratio

By modifying the proportion of raw substrates in the formula, different C/N ratio treatments were established (the detailed formulations are presented in Table 2). Water was added to substrate at a fixed substrate-to-water ratio of 1:1.65, and the pressure was consistently maintained when loading the substrate. Substrate mixing and post-inoculation management procedures were consistent with those described previously.

2.8. Three-Factor Four-Level Orthogonal Test

Based on the results of preliminary experiments, a three-factor, four-level orthogonal experimental design was conducted using IBM SPSS Statistics 27 software to examine the effects of key factors on the growth and developmental characteristics of F. filiformis (Table 3). The factors considered in this study included substrates’ loading volume, moisture content, and C/N ratios.

2.9. Indicators for Test Measurements

Throughout the cultivation cycle, mycelial growth dynamics were monitored for each treatment group. Contaminated bottles were promptly identified and discarded. Key parameters, including mycelial growth rate and full mycelial colonization time (the duration from inoculation to complete substrate colonization), were recorded. At harvest, the weight of the fruiting bodies per bottle was measured. For each treatment, six representative bottles (with uniform Flammulina filiformis growth) were selected to record the number of mushroom roots. Fresh fruiting bodies from each treatment were collected for further analysis. The yield (g/bottle) was calculated as the fresh weight of mushrooms harvested during the first flush from each bottle. Biological efficiency (BE) was determined as the percentage of the fresh weight of mushrooms per bottle divided by the dry weight of the corresponding substrate, multiplied by 100%. Additionally, agronomic traits of individual fruiting bodies were evaluated, including fruiting body count, pileus thickness, pileus diameter, stipe length, and stipe diameter.

2.10. Main Nutritional Qualities and Assay of Amino Acid Composition

Dried fruiting bodies from each treatment were pulverized, passed through a 90-mesh sieve, and stored at 4 °C for subsequent analysis. The contents of crude protein, crude fat, total sugar, and ash were measured. The assay was performed with reference to the methods of Filipa S [19] and Lu et al. [20]. The amino acid composition of dried fruiting bodies was analyzed using an automatic amino acid analyzer (L-8800, Hitachi, Japan) [21]. Specific methods were carried out with reference to Han et al. [22].

2.11. Statistical Analysis

One-way ANOVA, conducted using SPSS (v.25.0), tested the significance of different treatments, with p < 0.05 indicating statistical significance.
The agronomic traits, yield, and biological efficiency indicators of F. filiformis were analyzed for correlations using R-4.1.2 software, and the results were visualized using the corrplot function.

3. Results

3.1. Nutrient Contents of Different Substrates

Our research group previously analyzed the nutritional composition of the culture substrate and found that the soybean straw exhibited a higher nitrogen content compared to the corn straw, rice straw, and cotton straw, reaching 1.42%. Furthermore, this nitrogen level exceeded that of the corncob, a commonly used traditional substrate. Given its high nutrient content and rich cellulose composition, soybean straw serves as an appropriate substrate for mushroom cultivation (Table 4).

3.2. The Influence of Different Soybean Straw Proportions on the Growth and Development of F. filiformis

The effects of different soybean straw formulations on the growth and development of F. filiformis are shown in Table 5. No significant differences were observed in mycelial growth dynamics among treatments, with the mycelial full colonization time ranging from 23 to 24 days—1–2 days longer than that of the control group. When the soybean straw proportion was between 30% and 35%, the growth rate of the fruiting body was significantly reduced compared to the control group (p < 0.05), whereas the diameter of the pileus was significantly larger than that of other treatment groups (p < 0.05). When the soybean straw proportion was 25%, no significant differences were observed between the substrate group and the control group in terms of the yield (405.02 g/bottle) or biological efficiency (122.73%) (Table 5). All other treatments showed a significantly lower yield and biological efficiency than those of the control group (p < 0.05) (Figure 2).
When the soybean straw was added at 25%, the protein in the fruiting bodies was not significantly different from the control, the fat content was significantly higher than that of the control (p < 0.05), and the total sugar and ash were significantly lower than that of the control (p < 0.05) (Table 6). The contents of valine, isoleucine, phenylalanine, proline, essential amino acids, and total amino acids were significantly higher than that of the control group (p < 0.05), and the content of alanine was significantly lower compared with that of the control group (p < 0.05); the contents of the rest of the kinds of amino acids were not significantly different (Table 6 and Table S1).

3.3. Effects of Different Substrate-to-Water Ratios on the Growth and Development of F. filiformis

Table 7 illustrates the impacts of different substrate-to-water ratios on the growth and development of F. filiformis. A positive correlation was observed between the substrate-to-water ratio and the mycelial colonization time: as the ratios increased, the time required for the mycelium to fully colonize the bottle was prolonged. The yield and biological efficiency of the fruiting bodies exhibited a unimodal trend, first increasing and then decreasing with rising substrate-to-water ratios. When the substrate-to-water ratio ranged from 1:1.6 to 1:1.7, the per-bottle yield was slightly higher compared to the control group, though the difference was not statistically significant. Notably, at a substrate-to-water ratio of 1:1.7, the biological efficiency of the fruiting bodies was significantly greater than that of the control group by two percentage points (p < 0.05) (Figure 3).

3.4. The Effect of Different Substrate Loading Volumes on the Growth and Development of F. filiformis

The effects of varying substrate loading volumes on the growth and development of F. filiformis are presented in Table 8. As the loading volume increased, the mycelial full colonization time was prolonged across treatments, ranging from 21 to 23 days. The per-bottle yield exhibited an upward trend with the increase in the substrate loading volume. The biological efficiency of the fruiting bodies initially increased and then decreased. Notably, the highest biological efficiency was observed at a substrate loading volume of 940 g/bottle, peaking at 125.77%. This value was 5.22 percentage points higher than that of the control group and was significantly higher than other formulas (p < 0.05) (Figure 4).

3.5. Effects of Soybean Straw Partical Sizes of Soybean Straw on the Growth and Development of F. filiformis

The influence of different soybean straw particle sizes on the growth and development of F. filiformis is summarized in Table 9. As the particle size of the soybean straw increases, significant variations are observed in the water absorption characteristics of the raw substrates. The mycelial full colonization time for all treatments ranged from 21 to 22 days. Specifically, at a particle size of 8 mm, the average mycelial full colonization time is 21, which is approximately 0.6 days shorter than treatments with 6 mm and 10 mm particle sizes. Both the weight of the fruiting body and biological efficiency demonstrate a decreasing trend as the soybean straw particle size increases. Notably, the fruiting body weight in the 10 mm particle size treatment group is significantly lower compared to the control group. The highest biological efficiency, reaching 108.84%, is achieved in the 6 mm particle size treatment group, showing no significant difference from the control group or the 8 mm particle size treatment group. Overall, the soybean straw with particle sizes ranging from 6 to 8 mm yields an optimal performance for F. filiformis cultivation (Figure 5).

3.6. The Impact of Varying C/N Ratios in Soybean Straw-Based Substrates on the Growth and Development of F. filiformis

The influence of varying C/N ratios in soybean straw-based substrates on the growth and development of F. filiformis is summarized in Table 10. The average mycelial growth rate initially increased and then decreased within the C/N ratio range of 22:1 to 42:1, with the peak growth rate of 3.18 mm/day observed at a C/N ratio of 32:1, which was significantly higher than those of other treatments within this range (p < 0.05). Within the C/N ratio range of 42:1 to 57:1, the mycelial growth rate exhibited an upward trend, likely due to the improved air permeability of the culture substrate facilitated by the five-point perforation of the culture bottles, enabling the mycelium to fully colonize the substrate within 19 to 21 days. The per-bottle yield and biological efficiency generally followed a unimodal trend. At C/N ratios of 27:1 and 32:1, the fruiting body yields were 366.75 g and 354.50 g, with corresponding biological efficiencies of 107.14% and 103.56%, respectively, which are both significantly higher than those of other treatments (p < 0.05). When the C/N ratio exceeded 42:1, the fruiting body yield and biological efficiency were relatively low, with a biological efficiency ranging from 54% to 60% (Figure 6).

3.7. The Results of a Three-Factor, Four-Level Experiment Investigating the Effects of the Water Content, Substrate Loading Amount, and C/N Ratio as Key Factors

The orthogonal experiment results demonstrate that the highest per-bottle yield of 395 g/bottle was achieved under the following conditions: a dry substrate loading volume of 337 g/bottle, a substrate-to-water ratio of 1:1.6, and a C/N ratio of 26:1. Based on the average values at various levels of each parameter, the theoretically optimal combination is determined as follows: a dry substrate loading of 329 g per bottle, a substrate-to-water ratio of 1:1.6, and a C/N ratio of 26:1 (Table 11).
The results of the range analysis reveal that the C/N ratio is the most significant factor influencing the yield of fruiting bodies, followed by the moisture content, while the substrate loading has the least impact. For the stipe length, the carbon/nitrogen ratio remains the most influential factor, followed by the moisture content, with the substrate loading having the least effect. Regarding the stipe diameter, the C/N ratio is still the most influential factor, followed by the amount of substrate, while the moisture content has the least impact. For the pileus diameter, the pileus thickness, and the number of fruiting bodies, the moisture content is the most significant factor, followed by the amount of substrate, whereas the C/N ratio has the least influence (Figure 7).

3.8. Correlation Analysis Between F. filiformis Fruit Body Yield, Biological Efficiency, and Agronomic Traits

A Pearson correlation analysis was performed to examine the relationships among the yield, biological efficiency, and key agronomic traits. The results indicated that both the yield and biological efficiency of the fruiting bodies were significantly and positively correlated with the stipe diameter, the stipe length, and the number of fruiting bodies. Additionally, the stipe diameter exhibited a significant positive correlation with the stipe length. Furthermore, the stipe diameter, the stipe length, and the number of fruiting bodies were found to be significantly and positively intercorrelated (Figure 8).

4. Discussion

The composition of the cultivation substrate plays a significant role in influencing the mushroom growth, yield, and quality [23,24], making it a critical determinant in mushroom production. The selection of an appropriate cultivation substrate should be tailored to the specific type of mushroom being cultivated.
In previous studies, various researchers have investigated the cultivation of F. filiformis using different substrate formulations. Lu et al. reported a biological efficiency of 98.6% using a mixture consisting of 88% cottonseed husk, 10% broiler concentrate, 1% sugar, and 1% lime [25]. Ji et al. obtained a biological efficiency of 73% with a composition containing 88% maize straw, 5% wheat bran, 5% maize flour, 1% sucrose, and 1% lime [26]. Harith et al. cultivated F. filiformis using a combination of 25% paddy straw and 75% palm empty fruit bunches, resulting in a biological efficiency of 185% [27]. Sangkaew et al. attained a biological efficiency of 129.60% by applying a substrate blend of 25.90% sunflower meal, 50.37% corn cob, and 23.73% rice bran [28]. Xie et al. reported that using a substrate formula composed of 50% ramie stalks, 20% cottonseed hulls, 25% wheat bran, 2% calcium carbonate, and 4% corn starch the biological efficiency of F. filiformis reached 119% [29]. The type and proportion of raw materials significantly influence the biological efficiency of F. filiformis. This effect is primarily attributed to the nutritional composition inherent to the substrate. Appropriate increases in the proportion of the supplementation can enhance the overall yield. The price of soybean straw is comparable to that of corn cobs. When crushed, soybean straw forms uniform granules, exhibiting excellent water absorption and air permeability when used as a substrate for edible fungi cultivation. In contrast, the particles obtained from crushing corn and rice straws are uneven in size and demonstrate an inferior air permeability and a lower loading capacity as substrates. Compared to corn and rice straws, soybean straw possesses a higher nitrogen content and superior physical characteristics, making it a more suitable material for use as a substrate in edible fungi production. The utilization of soybean straw in the commercial cultivation of F. filiformis holds significant potential for improving the resource efficiency of agricultural byproducts and enhancing the economic returns associated with F. filiformis production.
Variations in the water content, loading substrate volumes, and raw substrate particle sizes within the substrate can alter its aeration properties, thereby affecting the mycelial growth rates and fruiting body development [30,31,32,33,34].
Nutrients are transported from the mycelium to the fruiting bodies through stable water-mediated transport [35]. The optimal water addition ratio is intricately linked to the substrate composition, as different formulas exhibit varying water-holding capacities. An insufficient water content may retard mycelial growth and reduce the fruiting body yield, while excessive water can lead to waterlogging at the bottom of the culture container, restricting oxygen diffusion, inhibiting mycelial growth in lower substrate layers, and negatively impacting the overall yield. In this study, an optimal substrate-to-water ratio of 1:1.7 (corresponding to 67.7% moisture content) was determined. Moonmoon et al. [36] and Ryu et al. [37] recommended maintaining the moisture content between 65% and 68% for Pleurotus eryngii cultivation, which closely aligns with the moisture requirements for F. filiformis cultivation. Beyond this ratio, a reduced fruiting body height and yield were observed.
An appropriate substrate loading volume can enhance the biological efficiency. Insufficient loading improves aeration, promoting mycelial growth, but increases the risk of substrate detachment from the bottle wall during colonization, leading to the emergence of premature fruiting bodies at the substrate edges. Conversely, excessive substrate loading impairs aeration, decreases mycelial activity, slows the mycelial growth rate, and impairs the mycelial quality. These findings underscore the importance of balancing the loading volume to maintain optimal aeration and mycelial activity.
The particle size of raw substrates influences the substrate porosity and aeration, thereby affecting microbial oxygen utilization [23]. The particle size of various raw substrates can influence the rate at which the mycelium decomposes and utilizes these substrates, thereby affecting the mushroom yield. Research findings demonstrate that as the particle size of the soybean straw increases, both the weight of the fruiting bodies and the biological efficiency exhibit a downward trend. Specifically, when the particle size of the soybean straw is approximately 10 mm, the yield of F. filiformis fruiting bodies and the biological efficiency are notably lower compared to the control group. Larger particles increase the interstitial space, enhancing oxygen transfer but reducing the surface area available for microbial nutrition and water transfer processes [38]. Conversely, excessively small particles may compact the substrate, limiting aeration and reducing the yield, which is consistent with previous reports [39]. Thus, an optimal particle size (6–8 mm) balances the porosity and surface area to maximize the nutrient utilization and yield.
Different mushroom species exhibit distinct requirements for the C/N ratio of the culture substrate. For instance, the optimal C/N ratio for Agaricus bisporus is 19:1 [40], whereas Lentinula edodes, Ganoderma lucidum, and Volvariella volvacea thrive at 30–35:1, 70–80:1, and 40–60:1, respectively [17,41,42,43]. Different C/N ratios influence the secretion of extracellular enzymes by mycelia and the degradation rate of lignocellulose [29,44]. High-nitrogen substrates have been shown to enhance the lignin degradation in the substrate [45]. Moreover, different C/N ratios affect the nutritional components (e.g., protein, amino acids, and polysaccharides) and flavor substances of fruiting bodies [22,46], highlighting its role in both the yield and quality optimization. Studies indicate that F. filiformis mycelia demonstrate a better degradation performance on low C/N ratio substrates compared to high C/N ratio substrates [27,29].
Orthogonal experiments clarified the hierarchical influences of key factors on the F. filiformis yield and agronomic traits. Through orthogonal experiments, we have elucidated the primary and secondary relationships among various factors. Based on these experimental findings, targeted adjustments to specific agronomic traits can be implemented to enhance the overall quality of the fruiting bodies.
In this study, soybean straw was utilized as a raw material to partially substitute corncobs and wheat bran, with an increased proportion of rice bran incorporated into the formulation. This study confirmed the feasibility of using soybean straw as an alternative substrate material, providing a reference for optimizing the cultivation formula for F. filiformis. Based on the selected soybean straw-based formulation, a systematic analysis was conducted on key factors including the moisture content, carbon-to-nitrogen ratio, substrate loading volumes, and particle size in the substrate. The effects of these factors on the growth and development of F. filiformis were evaluated. Through an orthogonal experimental design, the primary and secondary influences of the substrate moisture content, substrate loading volumes, and carbon-to-nitrogen ratio on the yield, biological efficiency, and agronomic traits were investigated. In practical production, it is recommended to optimize the formula based on the carbon-to-nitrogen ratio of the substrate in order to enhance the yield. Agronomic traits, such as the diameter of pileus and the length of stipe, can be regulated by adjusting the moisture content and substrate loading volumes, thereby improving the appearance quality of F. filiformis. These findings hold significant implications for the industrial-scale cultivation of F. filiformis.

5. Conclusions

The cultivation of F. filiformis using a substrate consisting of 25% soybean straw, 20% corn cobs, 20% cottonseed hulls, 25% rice bran, 8% wheat bran, 1% CaCO3, and 1% shell powder achieved the highest yield and biological efficiency (122.73%). The fat content, essential amino acid content, and total amino acid content of the fruiting bodies cultivated using the formulation containing 25% soybean straw were significantly higher than those in the control group, whereas the total sugar and ash contents were significantly lower. Based on this optimized formula, an ideal biological efficiency was achieved under the following conditions: a substrate-to-water ratio of 1:1.7, a substrate loading volume of 940 g/bottle (in a 1250 mL cultivation bottle), a soybean straw particle size range of 6–8 mm, and a C/N ratio ranging from 27:1 to 32:1. The carbon-to-nitrogen ratio of the culture substrate exerts the most significant influence on the yield of F. filiformis fruiting bodies, as well as the length and diameter of the stipe. Meanwhile, the moisture content of the culture substrate predominantly affects the diameter and thickness of the pileus and the number of fruiting bodies. It is feasible to cultivate F. filiformis using soybean straw as a primary substrate, while no significant differences in the yield or quality were observed compared to the conventional control group formula. Adjusting the carbon/nitrogen ratio of the culture substrate to ensure the yield of fruiting bodies and regulating the moisture content to improve their appearance quality can effectively enhance both the yield and overall quality of F. filiformis. This study serves as a valuable reference for the cultivation of F. filiformis using soybean straw and holds significant importance for related research and applications.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11080947/s1: Table S1: Effects of different formula on amino acid content in F. filiformis fruiting bodies; Table S2: Effects of different substrate-to-water ratio on the agronomic characters of F. filiformis using soybean straw as main substrate; Table S3: Effects of different substrate loading volume on F. filiformis agronomic traits; Table S4: Effects of different particle sizes of soybean straw on the agronomic traits of F. filiformis; Table S5: Effects of different C/N ratios on F. filiformis agronomic characters; Table S6: Orthogonal Test Combinations and Results; Table S7: Effects of different formulations on the revenue of F. filiformis; and Table S8: Carbon-Nitrogen Ratio Formulas in Orthogonal Experiments.

Author Contributions

Conceptualization, X.H. and Z.G.; Methodology, J.H.; Software, C.H.; Validation, H.X.; Formal analysis, S.Y. and Q.W.; Investigation, Q.Y. and H.L.; Resources, Z.G. and X.H.; Data curation, R.S. and J.H.; Writing—original draft preparation, R.S., J.H. and S.Y.; Writing—review and editing, R.S. and J.H.; Visualization, P.Y. and J.L.; Supervision, J.H.; Project administration, Z.G.; Funding acquisition, Z.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the earmarked fund for China Agriculture Research System (CARS-20-5), the Key R&D Program of Shandong Province, China (2024LZGC016, 2022TZXD0024), the key supported regions in Shandong Province in 2024 to introduce urgently needed and scarce talent projects—research and application project on the cultivation of excellent germplasm and efficient factory production technology for Grifola frondosa—as well as the 2024 Science and Technology Support Project of the Inner Mongolia Innovation Center of Biological Breeding Technology (2024NSZC01).

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

Author Qinghua Wang was employed by the company Yijun Agricultural Development Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Horticulturae 11 00947 g001
Figure 1. An image of the mycelial growth of the F. filiformis’s test strains.
Figure 1. An image of the mycelial growth of the F. filiformis’s test strains.
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Figure 2. The growth of F. filiformis treated with different contents of soybean straw. CK: control group; D-1 to D-4 denote the respective treatments involving the addition of 20%, 25%, 30%, and 35% soybean straw.
Figure 2. The growth of F. filiformis treated with different contents of soybean straw. CK: control group; D-1 to D-4 denote the respective treatments involving the addition of 20%, 25%, 30%, and 35% soybean straw.
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Figure 3. Growth status of F. filiformis under different substrate-to-water ratios. CK: control group; 1–3 correspond to treatments with substrate-to-water ratios of 1:1.6, 1:1.7, and 1:1.8, respectively.
Figure 3. Growth status of F. filiformis under different substrate-to-water ratios. CK: control group; 1–3 correspond to treatments with substrate-to-water ratios of 1:1.6, 1:1.7, and 1:1.8, respectively.
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Figure 4. Growth state of fruiting body treated with different loading volumes. CK: control group; treatments 1–3 correspond to loading volume of 920 g/bottle, 940 g/bottle, and 960 g/bottle, respectively.
Figure 4. Growth state of fruiting body treated with different loading volumes. CK: control group; treatments 1–3 correspond to loading volume of 920 g/bottle, 940 g/bottle, and 960 g/bottle, respectively.
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Figure 5. Growth status of fruiting body treated by adding soybean straw with different particle sizes. CK: control group; 1–3 correspond to soybean straw particle size treatments of 6 mm, 8 mm, and 10 mm, respectively.
Figure 5. Growth status of fruiting body treated by adding soybean straw with different particle sizes. CK: control group; 1–3 correspond to soybean straw particle size treatments of 6 mm, 8 mm, and 10 mm, respectively.
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Figure 6. Growth status of fruiting body treated with different C/N ratios. Treatments 1 to 8 correspond to substrates with carbon-to-nitrogen ratios of 22:1, 27:1, 32:1, 37:1, 42:1, 47:1, 52:1, and 57:1, respectively.
Figure 6. Growth status of fruiting body treated with different C/N ratios. Treatments 1 to 8 correspond to substrates with carbon-to-nitrogen ratios of 22:1, 27:1, 32:1, 37:1, 42:1, 47:1, 52:1, and 57:1, respectively.
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Figure 7. The growth status of F. filiformis with different treatments. The specific conditions corresponding to the various treatments are presented in Table S6.
Figure 7. The growth status of F. filiformis with different treatments. The specific conditions corresponding to the various treatments are presented in Table S6.
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Figure 8. Correlation analysis among different indicators.
Figure 8. Correlation analysis among different indicators.
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Table 1. Formulas for F. filiformis cultivation using soybean straw as carbon source.
Table 1. Formulas for F. filiformis cultivation using soybean straw as carbon source.
FormulasRaw Substrate Composition (%)
xSoybean StrawCorncobCottonseed HullsRice BranWheat BranSoybean HullsCaCO3Scallop Shell Powder
CK030202025311
D-1202720238011
D-2252020258011
D-3301320278011
D-435620298011
Table 2. Test formulations with different C/N ratios.
Table 2. Test formulations with different C/N ratios.
Raw Substrate TypeFormulas
12345678
Soybean straw25.1025.1025.1025.1025.1015.689.920
Corncob18.8418.8223.7740.7656.6170.5081.2791.20
Rice husk32.2041.8841.8332.2016.3511.886.876.86
Cottonseed hulls6.186.186.1800000
Wheat bran15.746.181.1800000
CaCO30.870.870.870.870.870.870.870.87
Scallop shell powder1.071.071.071.071.071.071.071.07
Carbon to nitrogen ratio22:127:132:137:142:147:152:157:1
Table 3. Orthogonal experimental design.
Table 3. Orthogonal experimental design.
LevelFactor
Loading Volume
(Dry Substrate Weight) (g) 		Substrate-to-Water RatioCarbon to Nitrogen Ratio
13291:1.432:1
23371:1.630:1
33451:1.828:1
43531:1.926:1
Note: the loading amount, moisture content, and C/N ratio are, respectively, factor A, factor B, and factor C.
Table 4. Determination of nutrient content in substrates.
Table 4. Determination of nutrient content in substrates.
Substrate TypeTotal Nitrogen (%)Ash (%)Hemicellulose (%)Cellulose (%)Lignin (%)
Corncob0.66 [1]2.96 [1]40.9927.764.34
Cottonseed hulls1.52 [1]4.84 [1]10.4536.3027.91
Corn straw1.22 [1]8.13 [1]28.7128.187.89
Rice straw0.90 [1]11.43 [1]29.6329.694.94
Soybean straw1.42 [1]5.49 [1]19.8629.3210.42
Cotton straw1.14 [1]4.22 [1]19.1332.3218.54
Soybean hulls1.34----
Wheat bran2.77----
Rice bran1.75----
Note: - = no tests.
Table 5. Effects of different contents of soybean straw on the growth and development of F. filiformis.
Table 5. Effects of different contents of soybean straw on the growth and development of F. filiformis.
FormulasMycelial Colonization Cycle (d)The Fruiting Body Height (mm) *The Diameter of Pileus (cm) **Total Yield (g/bottle)Biological Efficiency (%)
CK22155.02 ± 4.51 a7.22 ± 1.20 b408.00 ± 13.02 a123.64 ± 3.95 a
D-123156.13 ± 7.83 a8.50 ± 1.52 b389.07 ± 15.53 b117.90 ± 4.71 b
D-223154.16 ± 4.91 a8.63 ± 2.11 b405.02 ± 10.06 a122.73 ± 3.05 a
D-324148.06 ± 8.90 b8.91 ± 2.53 a383.35 ± 9.65 b116.17 ± 2.92 b
D-424146.21 ± 5.82 b9.12 ± 1.80 a369.82 ± 14.32 c112.07 ± 4.34 c
Note: Different lowercase letters represent significant differences at p < 0.05; *: the fruiting body height was measured at the date of harvesting in the control group; **: the date of measuring the pileus diameter was the harvest date of each treatment.
Table 6. Effects of different formulas on the nutritional quality of fruiting bodies.
Table 6. Effects of different formulas on the nutritional quality of fruiting bodies.
FormulaAdd Straw TypesProtein (%)Fat (%)Total Sugar (%)Ash (%)
CK-18.62 ± 0.12 a1.84 ± 0.06 b33.01 ± 1.61 a7.14 ± 0.11 a
D-225% Soybean straw18.21 ± 0.15 a2.20 ± 0.08 a28.13 ± 1.42 b6.01 ± 0.09 b
Note: Different lowercase letters represent significant differences at p < 0.05. CK: control group; D-2: treatment group with 25% soybean straw content.
Table 7. Influence of substrate-to-water ratios on F. filiformis growth and development.
Table 7. Influence of substrate-to-water ratios on F. filiformis growth and development.
FormulasSubstrate-to-Water RatioWet Substrate Loading Volume (g/bottle)Dry Substrate Loading Volume (g/bottle)Mycelial Colonization Cycle (d)Total Yield (g)Biological Efficiency (%)
CK1:1.70950351.8523424.14 ± 7.48 a120.55 ± 1.96 b
11:1.60930357.6920428.05 ± 11.30 a119.67 ± 2.95 b
21:1.70950351.8521431.20 ± 11.32 a122.55 ± 3.04 a
31:1.80960342.8622391.18 ± 25.35 b114.09 ± 6.52 c
Note: different lowercase letters represent significant differences at p < 0.05.
Table 8. Effect of substrate loading volume on F. filiformis growth and development.
Table 8. Effect of substrate loading volume on F. filiformis growth and development.
FormulasSubstrate-to-Water RatioWet Substrate Loading Volume (g/bottle)Dry Substrate Loading Volume (g/bottle)Mycelial Colonization Cycle (d)Total Yield (g)Biological Efficiency (%)
CK1:1.70950351.8523424.14 ± 7.05 b120.55 ± 2.01 b
11:1.70920332.1421404.18 ± 11.16 c121.69 ± 3.36 b
21:1.70940342.8622431.20 ± 11.32 ab125.77 ± 3.30 a
31:1.70960353.5723434.58 ± 8.52 a122.91 ± 2.41 b
Note: different lowercase letters represent significant differences at p < 0.05.
Table 9. Effects of different soybean straw particle sizes on the growth and development of F. filiformis.
Table 9. Effects of different soybean straw particle sizes on the growth and development of F. filiformis.
FormulasSubstrate-to-Water RatioThe Particle Size of Soybean Straw (mm)Wet Substrate Loading Volume (g/bottle)Dry Substrate Loading Volume (g/bottle)Mycelial Colonization Cycle (d)Total Yield (g)Biological Efficiency (%)
CK1:1.7-920340.7421.43 ± 1.08 ab384.13 ± 12.44 a112.73 ± 3.65 a
11:1.76920340.7421.85 ± 0.37 a370.86 ± 13.70 ab108.84 ± 4.02 ab
21:1.78920340.7421.21 ± 1.23 b363.02 ± 12.23 ab106.54 ± 2.59 ab
31:1.710910337.0421.88 ± 0.33 a349.70 ± 11.86 b103.77 ± 3.52 b
Note: different lowercase letters represent significant differences at p < 0.05.
Table 10. Effects of different C/N ratios on the growth and development of F. filiformis using soybean straw as the main substrate.
Table 10. Effects of different C/N ratios on the growth and development of F. filiformis using soybean straw as the main substrate.
FormulasCarbon to Nitrogen RatioWet Substrate Loading Volume (g/bottle)Dry Substrate Loading Volume (g/bottle)Average Daily Growth Rate of Mycelium (mm/d)Mycelial Colonization Cycle (d)Total Yield (g)Biological Efficiency (%)
122:1875336.542.82 ± 0.07 d21.00 ± 0.82 a302.92 ± 13.86 b90.01 ± 4.12 c
227:1890342.312.94 ± 0.16 cd21.00 ± 1.00 a366.75 ± 7.37 a107.14 ± 2.15 a
332:1890342.313.18 ± 0.22 a19.80 ± 0.84 ab354.50 ± 8.33 a103.56 ± 2.43 ab
437:1810311.543.03 ± 0.11 bc19.80 ± 0.84 ab307.80 ± 4.36 b98.80 ± 1.40 b
542:1680261.543.00 ± 0.08 bc20.29 ± 1.11 ab177.53 ± 11.56 c67.88 ± 4.42 d
647:1670257.693.05 ± 0.18 abc20.50 ± 1.97 ab153.22 ± 8.54 d59.46 ± 3.31 e
752:1590226.923.09 ± 0.17 ab19.00 ± 1.73 b135.45 ± 10.50 de59.69 ± 4.63 e
857:1590226.923.14 ± 0.15 ab20.00 ± 0.63 ab123.30 ± 14.50 e54.34 ± 6.39 e
Note: different lowercase letters represent significant differences at p < 0.05.
Table 11. The influence of various factors on the yield and agronomic traits of F. filiformis fruiting bodies.
Table 11. The influence of various factors on the yield and agronomic traits of F. filiformis fruiting bodies.
IndexDifferent Influencing Factors, LevelsAverage Values at Different Factors and LevelsIndexDifferent Influencing Factors, LevelsAverage Values at Different Factors and Levels
Total yieldK1353.22351.67367.62Biological efficiencyK1103.69103.22111.74
K2363.48371.34365.22K2106.64108.97108.37
K3362.25367.35355.33K3106.30107.88102.99
K4374.87363.47365.66K4110.06106.62103.59
R21.6519.6712.30R6.385.758.75
Stipe length K1150.87152.73152.91The diameter of stipeK12.993.002.93
K2149.79153.75152.11K23.033.012.92
K3150.08149.68151.67K32.802.903.06
K4154.98149.58149.05K42.982.882.89
R5.194.183.86R0.230.130.17
Pileus diameterK19.018.708.61The thickness of pileusK12.862.752.79
K28.999.339.34K22.862.932.84
K39.048.789.13K32.792.932.85
K49.519.739.47K42.892.812.92
R0.511.030.86R0.100.180.13
Fruiting body NosK1477.17533.50466.34
K2473.92531.67459.58
K3502.67441.50496.92
K4496.17443.25527.08
R28.7591.9967.5
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Sun, R.; Han, J.; Yang, P.; Yang, S.; Xie, H.; Li, J.; Huang, C.; Yao, Q.; Wang, Q.; Li, H.; et al. Formula Screening and Optimization of Physical and Chemical Properties for Cultivating Flammulina filiformis Using Soybean Straw as Substrate. Horticulturae 2025, 11, 947. https://doi.org/10.3390/horticulturae11080947

AMA Style

Sun R, Han J, Yang P, Yang S, Xie H, Li J, Huang C, Yao Q, Wang Q, Li H, et al. Formula Screening and Optimization of Physical and Chemical Properties for Cultivating Flammulina filiformis Using Soybean Straw as Substrate. Horticulturae. 2025; 11(8):947. https://doi.org/10.3390/horticulturae11080947

Chicago/Turabian Style

Sun, Ruixiang, Jiandong Han, Peng Yang, Shude Yang, Hongyan Xie, Jin Li, Chunyan Huang, Qiang Yao, Qinghua Wang, He Li, and et al. 2025. "Formula Screening and Optimization of Physical and Chemical Properties for Cultivating Flammulina filiformis Using Soybean Straw as Substrate" Horticulturae 11, no. 8: 947. https://doi.org/10.3390/horticulturae11080947

APA Style

Sun, R., Han, J., Yang, P., Yang, S., Xie, H., Li, J., Huang, C., Yao, Q., Wang, Q., Li, H., Han, X., & Gong, Z. (2025). Formula Screening and Optimization of Physical and Chemical Properties for Cultivating Flammulina filiformis Using Soybean Straw as Substrate. Horticulturae, 11(8), 947. https://doi.org/10.3390/horticulturae11080947

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