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Article

Utilizing Agrobyproducts: Potential Alternative Substrates for Cultivation of Lentinula edodes

by
Zhiguo Zhou
1,
Guohui Cheng
2,3,
Wenjie Chen
1,
Chunyan Xie
1,
Guisen Hou
1 and
Xiaoya An
1,3,*
1
Department of Life Science, Langfang Normal University, Langfang 065000, China
2
Department of Plant Protection, Shenyang Agricultural University, Shenyang 110866, China
3
Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Changchun 130118, China
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(5), 245; https://doi.org/10.3390/fermentation11050245
Submission received: 24 March 2025 / Revised: 19 April 2025 / Accepted: 27 April 2025 / Published: 30 April 2025
(This article belongs to the Section Industrial Fermentation)
Browse Figures
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Abstract

:
This study evaluated six agrobyproducts (oak, jujube, apple, pear, peach, black locust) as alternative substrates for Lentinula edodes cultivation to mitigate oak dependency. Twelve substrate formulations were tested, including individual and mixed sawdust combinations. Results demonstrated successful mycelial colonization across all treatments, with treatment PAS (78% pear sawdust, 20% wheat bran, 1.5% gypsum, and 0.5% lime) exhibiting the fastest mycelial growth (4.70 mm/day) and full colonization in 105 days. Treatment BLS (78% black locust sawdust, 20% wheat bran, 1.5% gypsum, and 0.5% lime) achieved the highest biological efficiency (97.26%) and productivity (0.85 kg/bag). Nutrient analysis revealed substrate-specific enhancements: PAS maximized vitamin C (4.88 mg/100 g) and iron, while PAS + OS (39% peach sawdust, 39% oak sawdust, 20% wheat bran, 1.5% gypsum, and 0.5% lime) elevated protein (3.88%), phosphorus, and zinc. PCA highlighted distinct nutritional profiles for BLS- and jujube-based mushrooms. Correlation analyses identified the third (r = 0.838) and fourth flushes (r = 0.922) as critical for total yield, with selenium and zinc significantly linked to growth rates. Black locust and peach substrates outperformed or complemented oak, offering sustainable alternatives. These findings underscore the potential of agrowaste utilization to reduce ecological strain while maintaining high yields and nutritional quality, aligning with global agricultural sustainability goals.

1. Introduction

Lentinula edodes, commonly known as “Xianggu” in China, is one of the most widely cultivated and consumed mushroom species worldwide [1,2]. It is unique for its delightful aroma and distinctive flavor, and its popularity is growing due to its high nutritional and medicinal benefits [3,4]. Lentinula edodes is rich in protein, polysaccharides, vitamins, and minerals. Scientific studies have suggested its therapeutic potential in preventing and treating various diseases, particularly cancer management, immune regulation, thrombosis prevention, and cholesterol control [4,5].
China boasts a long history of cultivating L. edodes, dating back over 800 years [6]. It is now the largest producer and exporter of these mushrooms in the world, with total production exceeding 13 million tons [7,8]. The top four provinces in China for edible fungi production are Henan, Fujian, Heilongjiang, and Hebei (China Edible Fungi Association. Available online: https://mp.weixin.qq.com/s/aFSu1OVNjR8-Q-KVhR_iNw, accessed on 24 December 2024). Farmers mainly cultivate L. edodes on substrates such as sawdust, wheat bran, lime, and gypsum. For many years, oak (Quercus spp.) has been the preferred substrate for mushroom cultivation due to its high nutrient content and availability [9,10]. However, increased demand for oak and other timber has caused imbalances in forest ecosystems and higher production costs due to dwindling resources [11]. Thus, researchers have begun exploring alternative wood waste substrates, which can help reduce reliance on traditional timber while promoting waste material utilization [12,13,14,15].
Farmers usually select sawdust based on local availability, but not all tree species are suitable for cultivation [16]. Coniferous trees contain high levels of tannins, terpenoids, flavonoids, and alkaloids, which inhibit mycelial growth in edible fungi. In contrast, broadleaf-derived sawdust exhibits high compatibility for cultivating L. edodes, owing to its favorable lignin–cellulose composition [17]. However, the biological efficiency and nutritional quality vary depending on the tree species used [18,19,20]. Yu et al. [21] reported that substrate composition (e.g., wheat straw, oak sawdust, or corncob) critically influences the biological efficiency of Lentinula cultivation, with optimized corncob-based formulations achieving up to 80% efficiency. Wang et al. [22] reported that substrate formulations incorporating 10–30% tangerine sawdust significantly influenced the biological efficiency (63–71%) and essential amino acid profiles of L. edodes fruiting bodies. Feng et al. [23] demonstrated that substituting 63.2% oak with apricot sawdust in L. edodes cultivation maximized yield and optimized yield and increased fruiting body polysaccharide content. The crude polysaccharide content of the fruiting body increased significantly with higher apricot sawdust addition. Notably, spatial heterogeneity in substrate nutrient composition altered fruiting body polysaccharide levels even under identical genetic and formulation conditions, underscoring the need for localized substrate optimization [24].
Hebei Province is a key agricultural and forestry province in China. By the end of 2022, the area of economic forests had reached 10,158 thousand hectares, among which the combined proportion of apple, pear, peach, jujube, oak, and locust trees was 56.75%. The annual felling volume is approximately 8.28–9.74 million tons, generating about 4–7 million tons of waste [25,26,27]. If 20% of these waste materials are used for L. edodes cultivation, with the biological efficiency maintained at 70%, this could annually yield at least 56 thousand tons of fresh mushrooms each year, generating an economic output of CNY 336 million.
This study focuses on utilizing the abundant forest waste resources in Hebei Province for mushroom cultivation. We tested six wood substrates: jujube, apple, pear, peach, black locust, and oak. These woods were used individually or in combination to create 12 different formulas for mushroom production. We closely monitored and recorded the growth status, yield, and mushroom nutrient composition. The findings of this study are significant for addressing the problem of forest biomass waste and protecting ecological balance.

2. Materials and Methods

2.1. Experimental Treatments

Six distinct lignocellulosic substrates were comparatively analyzed, comprising oak sawdust (os), jujube sawdust (js), apple sawdust (as), pear sawdust (ps), peach sawdust (pas), and black locust sawdust (bls). The wood materials were locally sourced from Hebei Province, China, mechanically chipped to a 0.2–0.4 cm particle size, and pretreated sequentially through 72 h of water immersion (ambient temperature) followed by 30 d of natural air-drying [20]. Each substrate formulation (detailed in Table 1) incorporated supplementary constituents including wheat bran, gypsum, and corn flour. The sawdust required premoistening before bag preparation. Within 24 h of prewetting, wheat bran (wb), lime, gypsum, and corn flour were added according to each specific formula. The moisture content of the mixture was adjusted to 55–60%, with a pH range of 5–6 [21]. Subsequently, the substrates were packed into polyethylene bags with dimensions of 16 cm (diameter) × 55 cm (height) and a thickness of 0.05–0.07 mm, each containing 2.5 kg of substrate. A total of 500 bags were prepared for each formulation. Sterilization was performed at 100 °C under atmospheric pressure for 24 h, after which the bags were allowed to cool to ambient temperature before transfer to a sterile environment.

2.2. Spawning and Incubation

The substrate treatments were inoculated at four equidistant points on the same side in a sterile room using 8 cm3 spawn of the L. edodes 0912 strain (obtained from Qingxin Edible Mushroom Co., Ltd., Xian County, Cangzhou City, China). After inoculation, the bags were arranged in a “#” shape and spaced approximately 50 cm apart in the culture room. The room temperature was maintained between 20 °C and 25 °C, with 60% relative humidity, and the environment was kept in complete darkness. During the incubation period, the mycelial growth was closely monitored, and the time required for complete colonization of each bag was recorded. Additionally, the weight changes of the bags were tracked throughout the growth process. Bags that completed color change ahead of schedule were removed, stored in a cold room (4 °C), and uniformly placed on shelves for fruiting.

2.3. Fruiting and Harvest

Fruiting was initiated upon complete mycelial colonization of the substrate, followed by implementation of appropriate fruiting management protocols. The relative humidity in the growing room was maintained at 80–90% through regular misting of water on both the floor and the substrate bags. Mushrooms were harvested at the juvenile stage, characterized by a cap diameter of 4 cm and before the rupture of the partial veil. Harvesting was conducted over five successive flushes, and the freshly harvested mushrooms were weighed immediately on the collection day. The total yield, along with the biological efficiency (BE), was subsequently calculated.
B E = W e i g h t   o f   f r e s h   m u s h r o o m s W e i g h t   o f   d r y   s u b s t r a t e × 100 %

2.4. Analytical Tests on Mushrooms

We collected mushrooms from the first flush of L. edodes to evaluate their nutritional value. The vitamin C (VC) content was quantified using the 2,6-dichloroindophenol method, as described by Tibuhwa [22]. Moisture and dry matter were accurately assessed using the direct-drying method, following the procedures outlined by Sebaaly et al. [23]. Protein content was determined through the Kjeldahl nitrogen method, in accordance with the protocol established by Reis et al. [24]. The acid–base method, as specified in the “GB/T 5009.10-2003” National Food Safety standard, was employed to measure crude fiber. Selenium (Se) levels were quantitatively analyzed using the fluorometric method [25]. For β-carotene analysis, high-performance liquid chromatography (HPLC) was utilized, following the methodologies referenced by Anubhuti et al. [26] and Reis et al. [24]. Soluble solids were measured using the refractometric method to determine the total soluble solids in fruits and vegetables. The elemental composition, including calcium (Ca), potassium (K), phosphorus (P), manganese (Mn), zinc (Zn), magnesium (Mg), iron (Fe), selenium (Se), and copper (Cu), was thoroughly analyzed using inductively coupled plasma atomic emission spectroscopy (ICP-AES). This analysis adhered to the “GB/T 14609-2008” National Food Safety standard, which primarily addresses the determination of mineral elements in vegetables, fruits, and vegetable-derived products.

2.5. Statistical Analysis

Mycelial growth parameters (colonization period and growth rate) and weight dynamics (initial, colonized, and weight loss) were evaluated in six independent groups, each comprising 50 bags. Fruiting body yield from the first five flushes was analyzed across three 50-bag experimental groups. Nutritional and elemental compositions were determined with five biological replicates. Following Z-score normalization of all experimental data, Pearson correlation analyses were conducted in two distinct matrices: (1) examining relationships between colonization period and six yield characteristics (Flush 1–5 and total yield), and (2) evaluating interactions among two physiological parameters (weight loss, growth rate), one yield component (total yield), and five nutritional profiles (dry matter, protein, crude fiber, soluble solids, VC) along with nine mineral elements (K, P, Mg, Ca, Cu, Fe, Mn, Zn, Se). Principal component analysis (PCA) was subsequently conducted on the 12 treatments using these 15 nutritional and elemental traits as variables, with data suitability confirmed by Kaiser–Meyer–Olkin (KMO > 0.6) and Bartlett’s sphericity tests (p < 0.001).

3. Results

3.1. Mycelial Growth and Development of L. edodes Under Different Treatments

All substrates supported robust mycelial colonization of the L edodes strain 0912, although growth rates varied significantly (Figure 1A). As shown in Figure 1A, formula PAS exhibited the fastest mycelial growth at 4.70 mm/day, achieving full colonization in 105 days—19% faster than oak (OS), which took 114 days. Treatments BLS and PS demonstrated moderate growth rates, ranging from 4.34 to 4.38 mm/day, while JS lagged behind at 3.77 mm/day. The mixed formula (adding oak) did not necessarily improve the growth rate of mushroom strain 0912. There was no significant difference in mycelial growth rate between AS + OS and AS or between PS and PS + OS. Meanwhile, that of JS + OS was significantly higher than that of JS, and that of BLS + OS was significantly higher than that of BLS. This indicates that peach wood and pear wood are more similar to oak than black locust and jujube wood.
The weight loss of cultivation bags indicates substrate utilization by mycelia, thus providing a key metric for formulation assessment. Compared with the initial weight, the bag weight in all treatments significantly decreased when filled with the mycelium (Figure 1B). Throughout the growth process, the weight of the bags displayed a similar trend across all treatments, with the total weight loss ranging from 0.14 kg to 0.3 kg. Notably, BLS + OS experienced the least weight loss (0.14 kg). However, PAS showed the most reduction (0.3 kg), followed by BLS and JS, which indicated that 0912 had better nutrition absorption in peach wood. As the mycelium degraded and absorbed the substrate, the weight of the bags continued to decrease. All formulations showed a low contamination rate during cultivation. Contamination rates remained uniformly low (≤4%), confirming the suitability of the substrates.

3.2. Effect of Treatment on the Yield of L. edodes

We collected data from the first five flushes of L. edodes, and the yield is presented in Figure 2. The mushrooms grown under different treatments exhibited significant differences in yield (p < 0.05). The total output produced by the BLS and OS treatments was significantly higher than that of the other treatments, yielding 42.55 kg and 40.33 kg per 50 bags, respectively, with biological efficiencies (BEs) of 97.26% and 92.42%. In contrast, there was no significant difference in total output among the JS, JS + OS, PS + OS, PAS + OS, OS + corn, AS, PS, and AS + OS treatments, as the yields ranged from 34.2 kg to 37.71 kg per 50 bags. However, the PS treatment yielded significantly less than the other treatments, producing only 32.9 kg per 50 bags, which is 22.7% lower than the BLS treatment, with a biological efficiency of 75.2%. The analysis of single-flush weight indicated that the production weight across all combinations followed a similar trend, initially increasing and then decreasing (Figure 2). The second, third, and fourth flushes were the primary contributors to the total output, with the highest weight obtained from the third flush.

3.3. Effect of Treatment on the Nutritional Value of L. edodes

As illustrated in Figure 3A (Table S1), the results concerning the nutritional composition of mushrooms revealed a significant increase in vitamin C (VC) content across all treatments compared to AS (p < 0.05). Treatment PAS exhibited the highest VC value, measuring 4.88 mg/100 g, with no significant differences compared to JS, OS, OS + corn, JS + OS, and PAS + OS. In terms of crude fiber content, treatments PAS + OS, PAS, BLS + OS, and BLS demonstrated a significant increase compared to OS (1.29%). Notably, PAS + OS had the highest crude fiber value at 1.43%. Conversely, AS, AS + OS, and PS + OS did not differ significantly from one another but showed a significant decrease when compared to OS and OS + corn (p < 0.05). Regarding soluble solids, substrates utilizing pear and black locust sawdust (treatments PS, PS + OS, BLS, and BLS + OS) exhibited higher levels, with PS reaching the highest value at 13.5%, which was statistically significant compared to other treatments (p < 0.05). The results indicated that the choice of substrate had a notable impact on the nutritional profile of L. edodes, with implications for both cultivation practices and the potential utilization of these mushrooms in different applications.
Protein content analysis indicated that all treatments, except for BLS + OS, had protein levels that were either higher than or equivalent to those of OS, with PAS + OS demonstrating the highest protein value at 3.88%. The analysis of dry matter revealed that BLS + OS had the lowest content at 12.62%, while PAS exhibited the highest at 13.60%, followed by OS and BLS. Notably, β-carotene was not detected in mushrooms cultivated with the provided formulations.
Lentinula edodes contains several minerals, including Ca, K, P, Mn, Zn, Mg, Fe, Se, and Cu (Figure 3B, Table S2). Among these, K exhibited the highest concentration, with the highest level observed in the BLS + OS and BLS treatments, ranging from 4420 to 4450 mg/kg (p < 0.05). Zinc displayed the greatest variability across treatments, with the PAS + OS treatment showing the highest concentration at 15.93 mg/kg. Additionally, mushrooms cultivated on PAS + OS exhibited the highest levels of P and Mn, at 0.13 mg/kg and 2.59 mg/kg, respectively (p < 0.05). Furthermore, the PAS treatment had the highest Fe content, while oak-based formulations (OS and OS + corn) demonstrated the lowest iron levels (p < 0.05).
The incorporation of jujube, apple, and pear wood into oak substrates improved the Ca content in mushrooms, with the highest concentration observed in the JS + OS treatment at 95.29 mg/kg and the lowest in the BLS + OS treatment at 45.60 mg/kg (p < 0.05). The Mg content in the AS + OS treatment was significantly higher than in other treatments, while the BLS + OS treatment recorded the lowest Mg content at 148.32 mg/kg, followed by the oak-based formulas. Substrates composed of pear and apple wood (PS, AS, AS + OS, and PS + OS) demonstrated the highest Cu concentrations. Although Se content was low across all mushrooms, jujube wood substrates significantly elevated the Se levels, reaching up to 1.68 × 10−2 mg/kg, while oak and peach wood exhibited notably lower Se levels.

3.4. Correlation Between Various Paraments of L. edodes Grown on Different Formulas

Pearson correlation coefficient matrix (Figure 4, Table S3) assessed the relationships between various characteristics and their contributions to mushroom production (* p < 0.05, ** p < 0.01, *** p < 0.001, representing the significance levels of correlations). Significant positive correlations were found between protein and weight loss (r = 0.576*), protein and P (r = 0.835***), protein and Cu (r = 0.681*), protein and Fe (r = 0.655*), and protein and Zn (r = 0.632*). Additionally, Zn demonstrated a positive correlation with mycelial growth rate (r = 0.76**), P (r = 0.805**), crude fiber (r = 0.589*), and Cu (r = 0.615*). Significant negative correlations were observed between Ca and mycelial growth rate (r = −0.650*), Se and growth rate (r = −0.825**), Se and crude fiber (r = −0.806**), Cu and total yield (r = −0.677*), Cu and VC (r = −0.758**), and Fe and total yield (r = −0.585*). Total output positively correlated with the weight of the first four flushes, but not with the fifth (r = 0.356) (Figure 4, Table S4). The third (r = 0.838***) and fourth (r = 0.922****) flushes significantly influenced overall production, with the third flush strongly correlating with both the first and second flushes, particularly the second.

3.5. Principal Component Analysis Among Nutrient Composition of L. edodes

To understand the nutritional components of L. edodes with the effects of treatments, we conducted a principal component analysis (PCA) on the measured indices. As shown in Table 2, six of the fourteen principal components (PC1 to PC6) accounted for approximately 91.30% of the variance among the traits. PC1 exhibited the highest variance at 32.30%, followed by PC2 at 21.34%, PC3 at 17.74%, PC4 at 10.77%, PC5 at 6.04%, and PC6 at 4.11%. Table 2 details the variance percentages associated with each principal component’s eigenvalues. PC1, with an eigenvalue of 4.52, was primarily related to the nutrient value of L. edodes. Table 3 and the eigenvector matrix indicate that PC1 is associated with proteins, P, Zn, Cu, and crude fiber. PC2 is linked to Se, Ca, and Mg. PC3 focused on dry matter, Fe, and VC, while Mn, Ca, and Mg are significant in PC4. PC5 is related to soluble solids, dry matter, P, Ca, and VC, and PC6 is strongly connected to K, Ca, Se, and VC.
Figure 5 illustrates significant differences in the nutritional components of mushrooms cultivated on various substrates. Among all nutrients, protein, P, Zn, crude fiber, Cu, and Fe are closely associated with nutritional value. The following pairs are closer in proximity: OS vs. OS + corn, PAS vs. PAS + OS, PS + OS vs. AS + OS, indicating notable similarities. Similarly, BLS + OS is closer in proximity to treatments OS and OS + corn, demonstrating that BLS + OS is more similar to OS and OS + corn than to BLS, although all four formulas are in the same quadrant. In contrast, JS, AS, PS, BLS, and JS + OS each exhibit distinct characteristics. While each formulation retains its unique traits, JS and JS + OS are more similar to each other than to other formulations. Additionally, BLS demonstrates greater specificity, as indicated by its separate distribution.

4. Discussion

The findings of this study demonstrate that locally available substrates, such as peach, black locust, and jujube sawdust, can serve as viable alternatives to oak for shiitake mushroom cultivation, thereby alleviating pressure on forest ecosystems. While all substrates supported successful mycelial colonization, significant variations in growth rates and nutritional profiles underscore the critical role of substrate composition in shaping cultivation outcomes. Notably, PAS achieved the fastest mycelial growth (4.70 mm/day), completing colonization in 105 days—19% faster than OS (114 days). This aligns with Chen’s [28] reported range (30–120 days) but exceeds the 72-day colonization period observed by Sebaaly et al. [29], likely due to strain-specific adaptations [30] or differences in substrate pretreatment protocols.
Growth rate variations may be attributed not only to physical substrate properties but also to phytochemical interactions. In our study, jujube wood’s slower colonization rate corroborates Chai’s [31] findings and likely reflects inhibitory effects of its secondary metabolites, such as terpenoids and flavonoids [32,33]. Conversely, peach wood’s phytochemicals [34] may enhance mycelial proliferation, though its resinous content likely explains yield reductions at higher inclusion ratios (>34%) [35]. Metabolites found in other wood types, such as pear and apple, have been shown to enhance the nutritional value of mushrooms, as noted by Endara et al. [36] and Huang et al. [37]. This highlights the delicate balance between substrate suitability and inhibitory compounds.
The differences in chemical composition among different wood substrates are critical factors influencing the nutrition of shiitake mushrooms. Research has indicated that the content and composition of cellulose, hemicellulose, and lignin in wood directly affect the nutritional absorption capacity of shiitake mushrooms [38]. For instance, the inclusion of peach and pear woods significantly enhanced the soluble solids and protein content, aligning with findings by Zhang et al. [39]. Mushrooms grown in pear and apple wood exhibited higher Cu content, consistent with Yu et al. [40]. Principal component analysis (PCA) further confirmed the uniqueness of BLS- and jujube-derived mushrooms, emphasizing substrate-driven metabolic specialization. Notably, mixed substrates (e.g., PAS + OS) often enhance multiple nutrients, suggesting potential synergistic effects. These results are consistent with studies showing that the initial carbon-to-nitrogen ratio in the substrate significantly impacts the nutritional composition of mushrooms [41,42,43,44]. However, the absence of β-carotene across all treatments highlights a gap in understanding how substrates influence fat-soluble vitamins. Future studies could explore pretreatment methods or substrate blends to address this limitation.
The absorption of fiber and structural carbohydrates in the substrate is crucial for maximizing mushroom production [45]. Among the twelve formulations examined, BLS emerged as a standout alternative, achieving 97.26% biological efficiency and surpassing oak, aligning with the findings of Cai et al. [46]. In contrast, Wang and colleagues used black locust sawdust to cultivate shiitake (“Senyuan 135” and “Tingxiang 18”) and revealed that these two strains had lower yields than those grown on oak substrates [47]. This discrepancy may reflect strain-specific enzymatic adaptations [41], and L. edodes 0912 likely expresses robust ligninolytic systems, enabling efficient degradation of BLS’s recalcitrant lignocellulose. Supporting this, PAS substrates exhibited the highest weight loss (0.30 kg/bag), correlating with elevated zinc levels (r = 0.76)—a known cofactor for lignin peroxidase (LiP) and manganese peroxidase (MnP) [48]. Szczepkowski [49] demonstrated that pruning residues from fruit trees (peach, pear, and apple) can be valuable substrates for mushroom cultivation. However, in this study, the yield of formulations with peach wood (PS and PS + OS) decreased compared with OS. This observation is consistent with Tian’s [35] discovery that adding more than 34% peach wood could reduce shiitake production. A similar phenomenon has been reported in Auricularia cultivation [49]. The reduced yield may be attributed to the tendency of peach trees to produce colloidal resin [34,50,51], and the impact may vary depending on the specific strain used. Despite this, peach wood can still serve as an auxiliary substrate in mushroom cultivation. However, potential issues such as residues of heavy metals and pesticides in apple wood need to be carefully considered during its use [40].
Mushrooms use their enzymatic systems to degrade wood materials during growth [48,52]. The composition of different substrates may affect the type and levels of degradation enzymes produced by shiitake mushrooms, thus influencing their nutrient acquisition and growth rate. The best oak alternatives possess comparable physical and chemical properties [53]. Although L. edodes is reported to be grown on several agricultural byproducts and forest residues, such as ground wheat straw [54,55], mango branches [56], kiwi branches [57], hazelnut husks [20], and corncob and vine pruning waste [21], it seems that no other materials can be equivalent to oak, so most of them serve as cooperators. Future work should quantify lignin peroxidase (LiP) and manganese peroxidase (MnP) activities across substrates to map degradation pathways. The degradation was also closely related to the specific strain [58], and breeding new germplasms suitable for diverse substrates is also a way to solve the contradiction between mushrooms and forests and realize the sustainable development of agriculture.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11050245/s1, Table S1: Nutritional quality of shiitake mushrooms cultivated on different substrate formulations; Table S2: Elemental composition of shiitake mushrooms cultivated on different substrate formulations; Table S3: Correlation among different paraments (pooled value); Table S4: Correlation among total yield, filling time, and single flush.

Author Contributions

Conceptualization, Z.Z. and X.A.; methodology, Z.Z. and G.C.; software, Z.Z. and G.C.; validation, Z.Z. and G.C.; formal analysis, G.C.; investigation, resources, Z.Z., W.C., C.X. and G.H.; data curation, Z.Z. and G.C.; Writing—original draft: Z.Z.; writing—review and editing, X.A.; visualization, Z.Z. and G.C.; supervision, X.A.; funding acquisition, X.A. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Hebei Province Capacity Enhancement Construction Project for Edible and Medicinal Fungi Science Popularization Demonstration Base (grant number 24452901K) from the Ministry of Science and Technology of Hebei Province, China and the Development of Substrates for Edible Fungi Cultivation Using Plantation Forest Residues (grant number 2024013035) from Langfang science and Technology Bureau project.

Informed Consent Statement

Not applicable.

Data Availability Statement

The authors declare that all data supporting the findings of this study are available within the article.

Acknowledgments

The authors thank the farmers for providing tree materials and Qingxin Edible Mushroom Co., Ltd., Cangzhou, China, for providing mushroom strains.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Mycelial growth and development of Lentinula edodes under different treatments. (A) The line graph illustrates the mycelial growth rate and the time required for the mycelium to completely colonize a bag using different substrates for the Lentinula edodes strain 0912. The color of the area between the two lines represents the time taken for full-bag colonization; lighter colors (white area) indicate shorter time, while darker colors signify longer times. Results are expressed as the mean ± standard deviation (SD) (n = 6). Statistical significance was assessed by one-way ANOVA with Bonferroni’s post hoc test (α = 0.05), with significant differences denoted by lowercase letters (a, b, c, d, e). Mycelial growth vigor is categorized as follows: “++++” (heavily dense), “+++” (dense). (B) The bar charts depict changes in bag weight for different substrates. The black bars indicate the final bag weight after complete mycelial colonization (overfilled state), while the hatched bars represent the corresponding weight loss during cultivation. A dotted reference line at 2.5 kg marks the initial weight for comparison. A two-tailed Student’s t-test (α = 0.05) was employed for paired comparisons between initial and colonized weights (** p < 0.01, *** p < 0.001, **** p < 0.0001). Weight loss for the 12 formulations was analyzed using one-way ANOVA followed by Bonferroni’s post hoc test (α = 0.05), with significant differences denoted by lowercase letters (a, b, c, d).
Figure 1. Mycelial growth and development of Lentinula edodes under different treatments. (A) The line graph illustrates the mycelial growth rate and the time required for the mycelium to completely colonize a bag using different substrates for the Lentinula edodes strain 0912. The color of the area between the two lines represents the time taken for full-bag colonization; lighter colors (white area) indicate shorter time, while darker colors signify longer times. Results are expressed as the mean ± standard deviation (SD) (n = 6). Statistical significance was assessed by one-way ANOVA with Bonferroni’s post hoc test (α = 0.05), with significant differences denoted by lowercase letters (a, b, c, d, e). Mycelial growth vigor is categorized as follows: “++++” (heavily dense), “+++” (dense). (B) The bar charts depict changes in bag weight for different substrates. The black bars indicate the final bag weight after complete mycelial colonization (overfilled state), while the hatched bars represent the corresponding weight loss during cultivation. A dotted reference line at 2.5 kg marks the initial weight for comparison. A two-tailed Student’s t-test (α = 0.05) was employed for paired comparisons between initial and colonized weights (** p < 0.01, *** p < 0.001, **** p < 0.0001). Weight loss for the 12 formulations was analyzed using one-way ANOVA followed by Bonferroni’s post hoc test (α = 0.05), with significant differences denoted by lowercase letters (a, b, c, d).
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Figure 2. Yields of Lentinula edodes 0912 as influenced by different treatments. Left: The line plot illustrates the total production (kg per 50 bags) and biological efficiency (BE) of 12 different formulations. Results are expressed as the mean ± standard deviation (SD) (n = 3). Statistical significance was evaluated using one-way ANOVA, followed by Bonferroni’s post hoc test (α = 0.05), with significant differences denoted by lowercase letters (a, b, c, d, e). Right: The stacked bar chart displays the mushroom weight of a single flush for the 12 formulations. Different colors represent distinct single flushes. Results are expressed as the mean ± SD (n = 3), and the total height of each bar indicates the total production of 50 bags for each formulation. Statistical significance was analyzed using one-way ANOVA, followed by Bonferroni’s post hoc test (α = 0.05), with significant differences denoted by lowercase letters (a, b, c, d, e, f).
Figure 2. Yields of Lentinula edodes 0912 as influenced by different treatments. Left: The line plot illustrates the total production (kg per 50 bags) and biological efficiency (BE) of 12 different formulations. Results are expressed as the mean ± standard deviation (SD) (n = 3). Statistical significance was evaluated using one-way ANOVA, followed by Bonferroni’s post hoc test (α = 0.05), with significant differences denoted by lowercase letters (a, b, c, d, e). Right: The stacked bar chart displays the mushroom weight of a single flush for the 12 formulations. Different colors represent distinct single flushes. Results are expressed as the mean ± SD (n = 3), and the total height of each bar indicates the total production of 50 bags for each formulation. Statistical significance was analyzed using one-way ANOVA, followed by Bonferroni’s post hoc test (α = 0.05), with significant differences denoted by lowercase letters (a, b, c, d, e, f).
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Figure 3. Nutritional value of Lentinula edodes growing in different treatments. (A) The line plot illustrates the nutritional content of L. edodes strain 0912 cultivated on 12 different treatments. (B) The line plot illustrates the trace elements of L. edodes strain 0912 cultivated on 12 treatments. Results are expressed as the mean ± standard deviation (SD) (n = 5). Statistical significance was assessed using one-way ANOVA, followed by Bonferroni’s post hoc test (α = 0.05), with significant differences denoted by lowercase letters (a, b, c, d, e, f, g, h, i, j).
Figure 3. Nutritional value of Lentinula edodes growing in different treatments. (A) The line plot illustrates the nutritional content of L. edodes strain 0912 cultivated on 12 different treatments. (B) The line plot illustrates the trace elements of L. edodes strain 0912 cultivated on 12 treatments. Results are expressed as the mean ± standard deviation (SD) (n = 5). Statistical significance was assessed using one-way ANOVA, followed by Bonferroni’s post hoc test (α = 0.05), with significant differences denoted by lowercase letters (a, b, c, d, e, f, g, h, i, j).
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Figure 4. Correlation relationships among different parameters of Lentinula edodes. Correlation analysis was performed to evaluate the relationships among nutritional components, growth rate, yield, and weight loss of mushrooms from the 12 formulations. Upper right panel: A heat map shows the correlations between weight loss, mycelial growth rate, total production, and nutrient content. Lower left panel: A heat map shows the correlations between bag filling time and yield. Green squares represent negative correlations, red squares represent positive correlations, and the darker the color, the stronger the correlation. An asterisk (“*”) denotes p < 0.05, two asterisks (“**”) denote p < 0.01, and three asterisks (“***”) denote p < 0.001.
Figure 4. Correlation relationships among different parameters of Lentinula edodes. Correlation analysis was performed to evaluate the relationships among nutritional components, growth rate, yield, and weight loss of mushrooms from the 12 formulations. Upper right panel: A heat map shows the correlations between weight loss, mycelial growth rate, total production, and nutrient content. Lower left panel: A heat map shows the correlations between bag filling time and yield. Green squares represent negative correlations, red squares represent positive correlations, and the darker the color, the stronger the correlation. An asterisk (“*”) denotes p < 0.05, two asterisks (“**”) denote p < 0.01, and three asterisks (“***”) denote p < 0.001.
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Figure 5. Principal component analysis of nutrient composition of Lentinula edodes.
Figure 5. Principal component analysis of nutrient composition of Lentinula edodes.
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Table 1. List of substrates and contents of each formula.
Table 1. List of substrates and contents of each formula.
TreatmentsFormulas
OSos (78%) + gypsum (1.5%) + lime (0.5%) + wb (20%)
JSjs (78%) + gypsum (1.5%) + lime (0.5%) + wb (20%)
ASas (78%) + gypsum (1.5%) + lime (0.5%) + wb (20%)
PSps (78%) + gypsum (1.5%) + lime (0.5%) + wb (20%)
PASpas (78%) + gypsum (1.5%) + lime (0.5%) + wb (20%)
BLSbls (78%) + gypsum (1.5%) + lime (0.5%) + wb (20%)
JS + OSjs (39%) + os (39%) + gypsum (1.5%) + lime (0.5%) + wb (20%)
AS + OSas (39%) + os (39%) + gypsum (1.5%) + lime (0.5%) + wb (20%)
PS + OSps (39%) + os (39%) + gypsum (1.5%) + lime (0.5%) + wb (20%)
PAS + OSpas (39%) + os (39%) + gypsum (1.5%) + lime (0.5%) + wb (20%)
BLS + OSbls (39%) + os (39%) + gypsum (1.5%) + lime (0.5%) + wb (20%)
OS + cornos (78%) + gypsum (1.5%) + lime (0.5%) + wb (17%) + corn meal (3%)
Note: Treatment codes (left column) represent complete substrate formulations (e.g., OS = oak sawdust-based formulation, JS = jujube sawdust-based formulation). Formula abbreviations (right column) indicate raw materials (e.g., os = oak sawdust, js = jujube sawdust, wb = wheat bran). All formulations contain fixed supplements: 1.5% gypsum + 0.5% lime + wb (17–20%).
Table 2. Eigenvalues, variance, and cumulative Eigenvalues of nutrition value.
Table 2. Eigenvalues, variance, and cumulative Eigenvalues of nutrition value.
Principal ComponentEigenvaluePercentage of Variance (%)Cumulative (%)
PC14.5226132.3043632.30436
PC22.9870821.3362653.64062
PC32.4830117.735871.37641
PC41.5071610.7654382.14184
PC50.84596.0421388.18397
PC60.574934.1066392.29061
PC70.464353.316895.6074
PC80.266741.9052797.51268
PC90.174991.2499698.76264
PC100.098940.7067399.46937
PC110.047010.3357899.80515
PC120.012950.0924899.89763
PC130.011210.0800799.9777
PC140.003120.0223100
Table 3. Principal Components for nutrition of Lentinula edodes.
Table 3. Principal Components for nutrition of Lentinula edodes.
VariablePC1PC2PC3PC4PC5PC6
Dry matter0.20389−0.140180.418760.253610.025660.06756
Protein0.408490.128980.08017−0.170520.10091−0.01931
Crude fiber0.24918−0.432840.17533−0.0644−0.052540.28786
Soluble solids0.02587−0.05909−0.517030.004370.461530.19259
Vitamin C−0.2049−0.292010.33642−0.180670.330490.31522
K0.06958−0.25247−0.335580.1914−0.589870.51645
P0.418910.07810.042910.084010.335960.18874
Mg0.261280.3240.206670.32944−0.234380.01762
Ca−0.164540.330750.209210.412820.258790.41309
Cu0.340370.24149−0.305730.018810.06887−0.13456
Fe0.275140.278350.21911−0.342−0.226280.09427
Mn0.0687−0.26009−0.037850.650750.07019−0.34826
Zn0.3953−0.14864−0.20695−0.020620.142370.15541
Se−0.242090.42173−0.142420.07706−0.017610.36059
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Zhou, Z.; Cheng, G.; Chen, W.; Xie, C.; Hou, G.; An, X. Utilizing Agrobyproducts: Potential Alternative Substrates for Cultivation of Lentinula edodes. Fermentation 2025, 11, 245. https://doi.org/10.3390/fermentation11050245

AMA Style

Zhou Z, Cheng G, Chen W, Xie C, Hou G, An X. Utilizing Agrobyproducts: Potential Alternative Substrates for Cultivation of Lentinula edodes. Fermentation. 2025; 11(5):245. https://doi.org/10.3390/fermentation11050245

Chicago/Turabian Style

Zhou, Zhiguo, Guohui Cheng, Wenjie Chen, Chunyan Xie, Guisen Hou, and Xiaoya An. 2025. "Utilizing Agrobyproducts: Potential Alternative Substrates for Cultivation of Lentinula edodes" Fermentation 11, no. 5: 245. https://doi.org/10.3390/fermentation11050245

APA Style

Zhou, Z., Cheng, G., Chen, W., Xie, C., Hou, G., & An, X. (2025). Utilizing Agrobyproducts: Potential Alternative Substrates for Cultivation of Lentinula edodes. Fermentation, 11(5), 245. https://doi.org/10.3390/fermentation11050245

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