Statistical Optimization of Biomass and Extracellular Polysaccharide Production by the Wild <i>Helvella lacunosa</i> Mycelium in Liquid-State Fermentation Using a Pine Needles Extract Medium

Guo, Shang; Xu, Li-Na; Li, Yan-Ting; Guo, Wei-Wei; Guo, Xiao-Fei; Hong, Sha-Sha

doi:https://doi.org/10.1155/2023/3591787

Journal of Chemistry

On this page

Abstract Introduction Materials and Methods Results and Discussion Conclusion Data Availability Ethical Approval Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 3591787 | https://doi.org/10.1155/2023/3591787

Statistical Optimization of Biomass and Extracellular Polysaccharide Production by the Wild Helvella lacunosa Mycelium in Liquid-State Fermentation Using a Pine Needles Extract Medium

Shang Guo,¹Li-Na Xu,¹Yan-Ting Li,¹Wei-Wei Guo,¹Xiao-Fei Guo,¹and Sha-Sha Hong¹

Academic Editor: Vijay Kumar Garlapati

Received07 Jan 2023

Revised11 Apr 2023

Accepted17 Apr 2023

Published28 Apr 2023

Abstract

The objective of this study was using a statistical model to optimize the medium formula and liquid culturing conditions, which was employed to evaluate the potency of pine needles extract as nutritional component in basal medium for culturing Helvella lacunose in order to enhancing the production of mycelia and extracellular polysaccharides (EPS). The most proper carbon and nitrogen source, various fermentation parameters (i.e., initial pH in medium, fermentation time, and temperature) were investigated by single factor test. Based on the experimentation, the optimal concentrations of carbon and nitrogen sources were analyzed by response surface methodology (RSM). The results showed that the optimal medium formula and liquid culturing conditions were 1000 mL pine needles extract, 33.56 g glucose, 4.11 g peptone, 4.06 g YE, 1 g KH₂PO₄, 1 g NaHCO₃, 0.5 g MgSO₄, 0.01 g vitamin B₁ for the accumulation of mycelial biomass (at pH 6.0, 25°C culturing of 7 days), and EPS (at pH 7.0, 25°C culturing of 10 days). Under optimized medium, the experimental production of mycelia and EPS were 11.78 g/L and 223.16 mg/L, which were 10.52 and 4.85 times than that of control (1.12 ± 0.52 g/L and 46.08 ± 1.18 mg/L, respectively). This data implied that statistical model is very useful tools for optimizing media components and fermentation parameters, and the screened media components and parameters have reference value for further large-scale production.

1. Introduction

Helvella lacunosa belongs to Helvel laceae and Helvella of Pezizales [1]. The species has black pileus and white handle and commonly named “Mu Er” mushroom for its shape looks like Agaric [2]. It has good taste and has the high oxidation ability in radical scavenging activity [3]. It has long been prescribed to prevent and treat various human diseases in northern region of China. Since 1987, H. lacunosa have attracted a great deal of attention because its fruiting bodies contain a variety of bioactive substances, such as polysaccharides, triterpenes, helvellisin, proteins, and other small molecule compounds [4–6]. In addition, the usage of H. lacunosa is without any toxicity [5]. Due to the special growth environment and excessive excavation, H. lacunose is in short supply. Since H. lacunose has the greatest potential for exploitation in antidiabetes, the study of it has aroused great interest of researchers. Researchers hope to cultivate H. lacunose artificially. In that case, much more consumers would have the chance to enjoy the benefits of H. lacunose [7].

As with other edible fungi, the cultivation of H. lacunose includes solid cultivation and submerged fermentation. Many researchers think that liquid submerged fermentation (LSF) is a better system for producing metabolic substance than cultivating the fruit body of mushrooms [8–10]. Such as Hu et al. [11], he implied that the mycelia of H. lacunosa had same biological activity as the fruit body. Compared with the cultivation of fruit bodies, submerged culture has many advantages, such as confined space, shorter time, easier control, and higher content of bioactive metabolites [12]. Among the process of fermentation, nitrogen and carbon sources of medium had an important role in promoting the growth of mycelia [13, 14]. Many reports showed that the natural materials have the potential to be substrate of fungal medium [10–13], which gives us enlightenment that natural materials may have the potential ability to cultivate H. lacunosa. On the other hand, fermentation parameters (such as, medium volume, fermentation time and temperature, pH in medium, and rotation speed) also have important effects on the production of polyphenolics, polysaccharide, etc [15]. Nowadays, few studies are conducted on submerged fermentation to produce mycelium and EPS of H. lacunosa.

Pinus massoniana, belongs to pine that grow everywhere in China. Study showed that the pine needles of Pinus massoniana have lots of phytochemicals [16]. Reports showed that the extracts from pine needles had strong inhibitory effect on various bacteria [17] and hydroxyl radical activity [18]. Aims of our study were to explore the proper culturing condition of LSF for the accumulation of mycelium and EPS by H. lacunosa using a statistically-based experimental design. First, a one-factor-at-a-time method was designed to explore the influence of nutrient element (i.e. carbon and nitrogen sources) and environmental factors (i.e. temperature, time, and pH) on the accumulation of mycelium and EPS. Subsequently, the optimal additive amount of carbon and nitrogen source was analyzed by RSM. This work will help in developing a more efficient and dependable culture conditions for mycelial biomass and extracellular polysaccharide production with H. lacunosa.

2. Materials and Methods

2.1. Microorganisms and Media

H. lacunose strain was preserved in our laboratory. The strain was routinely grown on potato agar dextrose (PDA) at 25°C for regular subculture and maintained on PDA slants at 4°C for a maximum of 3 months. The basal medium for the submerged fermentation of H. lacunosa consisted of 1000 mL pine needles extract (cooking 100 g pine needles for 30 min in 1 L of tap water), 20 g sucrose, 1 g KH₂PO₄, 0.5 g MgSO₄, and 0.01 g vitamin B₁, pH 7.0.

2.2. Submerged Fermentation

The strain of H. lacunosa was cultured in a 250 mL shaken flasks (150 mL medium) inoculated with 4 discs (8 mm) of PDA (25°C, 120 rpm, 7 days).

2.3. Determination of Mycelial Biomass and EPS Yield

Centrifuging the fermentation substrate at 8000 rpm for 12 min and then drying at 85°C to constant weight to detect the biomass [13]. The production of EPS detected according to the phenol-sulfuric method [13, 19].

2.4. One-Factor-at-a-Time Experiment

One-factor-at-a-time methods were designed to detect the relationship between biomass (EPS) and each composition (i.e., carbon sources: glucose, sucrose, and maltose and nitrogen sources: peptone, yeast extract, and (NH₄)₂SO₄) in substrate. Meanwhile, three process parameters (fermentation time, fermentation temperature, and initial pH of media) were routinely investigated in 250 ml shake-flask at 150 rpm.

2.5. Optimization of Pine Needles Extract Medium by RSM

RSM was built (two-level, three variables) depend on the experimentation of one-factor-at-a-time methods (Table 1). Finally, the Design Expert 8.0.6 (USA) and the Box–Behnken Design (BBD) were used to optimize the proper addition of carbon and nitrogen resource in medium. The effect between the value and three selected variables (x₁: Glucose, x₂: Yeast Extract, and x₃: Peptone) was approximated by a second order polynomial equation.where Y refers to the response, β₀, β_i, β_ii, and β_ij are constant and x_i and x_j (i = 1, 3; j = 1, 3; i ≠ j) stands for the average of the coded value of the ith test variable. All trials were performed three times.

2.6. Validation Test

To verify the optimization results derived from equations (2) and (3), five replicates were conducted under the optimized medium. Mycelial biomass and EPS yield were determined routinely after the fermentation for verifying whether the experimental values were in agreement with the ones predicted by the model and whether the model was satisfactory and accurate.

2.7. Experimentation and Analysis

All experiments contained three parallel tests. Analysis of variance (ANOVA) for the experimental data and model coefficients was calculated by means of Design-Expert Program software (version 8.0.6, Stat-Ease Inc., Minneapolis, USA). Significant differences () were conducted using the SPSS version 16.0 (SPSS Inc., Chicago, IL, USA).

3. Results and Discussion

3.1. Effects of Nutrient Addition on Mycelium and EPS Production

Effects of initial pH of media, fermentation time and temperature on the accumulation of mycelium of H. lacunose were investigated (Figure 1). Figure 1(a) showed that the biomass contents varied from temperature and got its highest value (1.07 g/L) at 25°C. This phenomenon showed that high temperature would restrict the biomass activity and damage its structure. Active mycelial growth was observed on the same medium at pH between 3.0 and 7.0 in the shake flasks (Figure 1(b)). Those results implied that the proper pH for accumulation of mycelium was 6.0–7.0. Within this pH scale range, H. lacunosa grew well and formed into a regular mycelial pellet. As to the initial pH, investigators have different opinions, mainly common in neutral [20] and acidic pH value [21]. Based on the above results, we chose pH 6.0 as initial pH condition for further optimization of medium composition for H. lacunosa growth and production of bioactive components. Meanwhile, the relationship between fermentation time and the accumulation of mycelium of H. lacunose were studied (Figure 1(c)). Results showed that when the culturing time ranged from 1 to 9 days, the biomass content rose and then declined sharply as the time increased. In view of H. lacunosa growing into stationary phases and accumulating metabolites, the suitable incubation time of H. lacunosa was confirmed on 7 d for further study. Our results are same as reported research [22].

(a)

(b)

(c)

Effects of fermentation parameter (such as pH, fermentation time, and temperature) on the EPS of H. lacunose were shown in Figures 2(a)–2(c). Figure 2(a) showed that the contents of EPS enhanced sharply when fermentation temperature range from 5 to 25°C, but decreased when temperature reached over 25°C. There are many papers showing that temperature act as a key role in the hydrolysis extraction process. However, higher temperature may inactivate the enzyme and adversely affect the solubility of the solvent and the target extracts [23, 24].

(a)

(b)

(c)

Figure 2(b) illustrated that the investigated range of initial pH in medium from 3 to 10. Results showed that the EPS contents varied from pH and got its highest value (38.08 mg/L) at 7.0. When fermentation time prolong from 3 to 10 days, the EPS content increased significantly and then decreased noticeably according to the time increased (Figure 2(c)). This phenomenon would be implied that the accumulation of metabolites in mycelium enhanced sharply during the stable period. So we chose 10 days for culturing H. lacunosa to obtain more EPS in our paper.

3.2. Effects of Medium Ingredient on Mycelial Biomass and EPS Yield

Effects of supplementary nutritive factor on the accumulation of mycelium and EPS of H. lacunosa were presented in Tables 2 and 3. Those results indicated that glucose would be the most proper carbon source for the accumulation of mycelium of H. lacunose. Also, among these nitrogen sources (peptone, yeast extract, and (NH₄)₂SO₄), peptone gave the highest EPS yield and biomass, followed by YE (Table 2).

As shown in Table 3, small changes in additive amount of carbon and nitrogen sources would have a significant effect on the growth of H. lacunose. When adding 40 g/L glucose in the substrate, mycelia biomass production would reach 4.42 g/L, 3.95 times than in the control (). When adding 4 g/L YE in the substrate, mycelia biomass production would reach 4.38 g/L, 3.91 times than in the control (). When adding 3 g/L peptone in the substrate, mycelia biomass production would reach 4.28 g/L, 3.82 times than in the control (). Similarly, supplementation of 40 g/L glucose, 5.0 g/L peptone, and 4.0 g/L YE to the basal medium would cause sharp increase in the production of EPS (). It may imply that the carbon and nitrogen sources have involved in some metabolic processes of fungi and improved the production of biomass [19].

3.3. Optimization of Biomass and EPS Production by RSM

Effects of three ingredients on biomass and EPS are presented in Figures 3 and 4. The experimental data are shown in Table 4. The equations obtained from regression analysis for biomass and EPS are as follows:

(a)

(b)

(c)

(d)

(e)

(f)

(a)

(b)

(c)

(d)

(e)

(f)

To assess whether or not the quadratic model was significant, the ANOVA of the model was examined (Tables 5 and 6). The results showed , which indicated the high significance and reliability of the polynomial model for correlating the experimental results. It showed that the small alteration of the concentration in peptone, glucose, and YE would influence the yield of mycelia and EPS.

The relationship between the addition of peptone, YE, and glucose on the accumulation of mycelium from H. lacunosa in LSF by pine needles extract is displayed in Figure 3. The figure shows the 3D plot with data obtained by equation (2). Those results implied that the interaction between factors B and C was stronger (Figures 3(e) and 3(f)) than the effects of factor A and C (Figures 3(c) and 3(d)) or factor A and B (Figures 4(a) and 4(b)) on biomass production. As analysis in the same way as used above, it can be seen that the interaction between factor B and C was stronger (Figures 4(e) and 4(f)) than the effects between factor A and C (Figures 4(c) and 4(d)), or factor A and B (Figures 3(a) and 3(b)) on the EPS production. Finally, the proper addition of peptone, YE, and glucose were calculated by BBD. The proper values of the 3 factors were found to be 33.56 g glucose, 4.11 g peptone, and 4.06 g YE. And this model forecasted that the maximum value of mycelial and EPS were 11.60 g/L and 217.78 mg/L, respectively.

3.4. Validation Test

The validation test was done according to the results of Tables 4, 5, and 6. Due to the optimized medium, the experimental value of mycelium and EPS were 11.78 g/L and 223.16 mg/L, which were 10.52 and 4.85 times than that of control (1.12 ± 0.52 g/L and 46.08 ± 1.18 mg/L, respectively). The experimentation and the predicted data have litter difference, which suggested that the model for predicting mycelial biomass and EPS were adequate. The feasibility of H. lacunose LSF of pine needles extract was validated methodologically in our study.

RSM is a powerful method to design experiments and build models. At present, RSM is becoming more and more popular in optimizing the medium nutrition components. Zhai and Han [13] reported that after optimizing the medium with RSM, the biomass and intracellular polysaccharides production of Agaricus blazei was increased. Also, Liu et al. [19] and Dwibedi et al. [25] reported that RSM would be an effective method to optimize the medium and provide optimum condition for fungi culturing. The results suggested that the liquid fermentation has the potential to get more mycelia of edible fungus. In our optimized experiment, the mycelia biomass was 8.83 times of control, which illuminated that the submerged fermentation can be used to produce mycelia of H. lacunosa.

The above results implied that while suitable carbon and nitro resources were added to the medium, the amount of EPS yield is related to the mycelia biomass of H. lacunose in our study. Zhai and Han [13] also pointed out that the polysaccharides production was positively correlated with the biomass in Agaricus blazei. But in the study of Hsieh et al. [8], he found that the EPS production was not correlated with the growth of G. lucidum CCRC 36041 in LSF. These results showed that different strains had different metabolic characteristics and there seemed no lag phase between the primary metabolism and the secondary metabolism in fungus.

Pine needles contain lots of secondary phytochemicals [16]. Study showed that, the extract of pine needles would show results against bacteria [17], reduce DNA oxygen damage, scavenging free radicals [18], and dietary inclusion of fermented pine (Pinus densiflora) needle extract could improve laying performance and the antioxidant capacity of eggs [26]. In our study, the production of extracellular polysaccharide would be increased when the culture substrate contain the water extraction of pine needles. Studies show that extracellular polysaccharide of H. lacunose had a strong ability for antioxidant effects and exploitation in antidiabetes [4, 7]. In summary, pine needles would be a potential resource as the basal medium for H. lacunose under liquid fermentation.

The nutritional values and taste components of H. lacunosa have been studied [27]. In addition to their nutritional value, H. lacunosa mycelia are of high active substance [11]. This implied that mycelia of H. lacunosa could be processed into many special functional foods. In this case, the extraction process of its nutrition ingredient in mycelia with pine needles of H. lacunosa should be developed.

4. Conclusion

A statistical model was successfully employed to evaluate the effects of critical medium components on biomass and EPS production via the submerged fermentation of H. lacunosa with pine needles extracts. Under the optimized medium and conditions, the production of mycelium and EPS of H. lacunosa were 11.78 g/L and 223.16 mg/L, which were 10.52 and 4.85 folds higher than the control, respectively. The results showed that these methods used in our study were very useful for optimizing the parameter in submerged fermentation for H. lacunose to get more mycelium and EPS production.

Data Availability

All data are included within the article.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the author.

Conflicts of Interest

All authors declare that they have no conflicts of interest.

Acknowledgments

The authors are grateful to the support and facilities given by Shanxi Youth Science Research Project (202103021223160); Doctoral Research Project of Shanxi Agricultural University (2021BQ45); Shanxi Province Excellent Doctoral Work Award-Scientific Research Project (SXBYKY2021086); Research Innovation Team Special Project of Shanxi Academy of Agricultural Sciences (YGC2019TD03); Shanxi Province Scientific and Technological Achievements Transformation Guide Special Project (201904D131042), and Key Research and Development Project of Shanxi Province (202102140601016).

References

F. Landeros, T. Iturriaga, A. Rodríguez, G. Vargas-Amado, and L. Guzmán-Dávalos, “Advances in the phylogeny of Helvella (Fungi: ascomycota), inferred from nuclear ribosomal LSU sequences and morphological data,” Revista Mexicana de Biodiversidad, vol. 86, no. 4, pp. 856–871, 2015.
View at: Publisher Site | Google Scholar
F. L. Dai, in China Fungi Club, science press, Beijing, China, 1979.
Q. Z. Zheng, “A delicious fungus needed to be developed urgently-the research of Helvella lacunosa,” Edible fungi, vol. 1, no. 6, pp. 1-2, 1987.
View at: Google Scholar
Z. B. Zhou, H. Zeng, and J. P. Fu, “Radical scavenging activity of crude polysaccharides extracted from Helvella leucopus fruit bodies,” Acta Edulis Fungi, vol. 1, pp. 43–46, 2009.
View at: Google Scholar
G. Q. Zhang, H. X. Wang, X. Q. Zhang, and T. Ng, “Helvellisin, a novel alkaline protease from the wild ascomycete mushroom Helvella lacunose,” Journal of Bioscience and Bioengineering, vol. 109, no. 1, pp. 20–24, 2010.
View at: Publisher Site | Google Scholar
L. P. Teng, H. Zeng, and H. B. Wang, “Study on the deproteinization technology of crude polysaccharide from Helvella leucopus,” Journal of Henan Agricultural Sciences, vol. 42, no. 9, pp. 138–137, 2013.
View at: Google Scholar
Q. I. Zhao, B. Tolgor, Y. Zhao, Z. Yang, and K. D. Hyde, “Species diversity within the Helvella crispa group (ascomycota: helvellaceae) in China,” Phytotaxa, vol. 239, no. 2, pp. 130–142, 2015.
View at: Publisher Site | Google Scholar
C. Hsieh, M. H. Tseng, and C. J. Liu, “Production of polysaccharides from Ganoderma lucidum (ccrc 36041) under limitations of nutrients,” Enzyme and Microbial Technology, vol. 38, no. 1-2, pp. 109–117, 2006.
View at: Publisher Site | Google Scholar
M. Kawagoe, M. Ito, K. Naoe, and H. Noda, “Effects of temperature change on submerged culture of Flammulina velutipes in an external-loop airlift bubble column fermentor,” Journal of Chemical Engineering of Japan, vol. 45, no. 9, pp. 651–654, 2012.
View at: Publisher Site | Google Scholar
P. W. Mao, L. D. Li, Y. L. Wang, X. H. Bai, and X. W. Zhou, “Optimization of the fermentation parameters for the production of Ganoderma lucidum immunomodulatory protein by Pichia pastoris,” Preparative Biochemistry & Biotechnology, vol. 50, no. 4, pp. 357–364, 2020.
View at: Publisher Site | Google Scholar
J. J. Hu, J. M. Wang, and G. W. Zhang, “Changes of extra-cellular enzyme activity and reducing sugar consistency during the submerged culture of Helvella crispa,” Journal of Northwest Forestry University, vol. 33, no. 11, pp. 94–98, 2005.
View at: Google Scholar
L. Yan, W. N. Yuan, and X. W. Zhou, “Optimization of submerged culture conditions for the mycelial biomass and bioactive metabolites production by Cordyceps militaris,” Journal of Pure and Applied Microbiology, vol. 8, no. 6, pp. 4245–4254, 2014.
View at: Google Scholar
F. H. Zhai and J. R. Han, “Mycelial biomass and intracellular polysaccharides yield of edible mushroom Agaricus blazei, produced in wheat extract medium,” LWT-food science and technology, vol. 66, pp. 15–19, 2016.
View at: Publisher Site | Google Scholar
D. D. Romero, H. V. Rivera, B. Reyes, J. I. A. Reyes, and A. R. M. Campos, “Isolation and purification of ectomycorrhizal fungus Helvella lacunosa in different culture media,” Tropical and Subtropical Agroecosystems, vol. 16, no. 1, pp. 51–59, 2013.
View at: Google Scholar
Y. Liu, M. Y. Jin, P. W. Mao, W. R. Cong, W. N. Yuan, and X. W. Zhou, “Statistical optimization of polysaccharides Production by the Lingzhi or Reishi medicinal mushroom Ganoderma lucidum (agaricomycetes) in solid-state fermentation using highland barley grains,” International Journal of Medicinal Mushrooms, vol. 22, no. 11, pp. 1033–1041, 2020.
View at: Publisher Site | Google Scholar
A. A. Kadam, S. G. Singh, and K. Kirtiraj, “Chitosan based antioxidant films incorporated with pine needles (Cedrus deodara) extract for active food packaging applications,” Food Control, vol. 124, no. 1, pp. 123–126, 2021.
View at: Google Scholar
Y. S. Kim and D. H. Shin, “Volatile components and antibacterial effects of pine needle (Pinus densiflora S. and Z.) extracts,” Food Microbiology, vol. 22, no. 1, pp. 37–45, 2005.
View at: Publisher Site | Google Scholar
A. Tajwar, J. Ghulam, N. C. Arshad, S. A. Muhammad, A. Rukhsanda, and A. Rizwan, “Terpenes and phenolics in alcoholic extracts of pine needles exhibit biocontrol of weeds (Melilotus albus and Asphodelus tenuifolius) and insect-pest (Plutella xylostella),” Journal of King Saud University Science, vol. 4, no. 34, pp. 1–6, 2022.
View at: Google Scholar
G. Q. Liu, Y. Zhao, X. L. Wang, and Z. Chao-Yang, “Response surface methodology for optimization of polysaccharides extraction by mild alkaline hydrolysis from fruiting body of medicinal mushroom, Ganoderma lucidum,” Journal of Medicinal Plants Research, vol. 5, no. 10, pp. 382–385, 2011.
View at: Google Scholar
J. A. Cheng, H. Huang, Z. H. Zheng, Y. J. Huang, and W. J. Su, “A study on liquid fermentation of Cordyceps militaris,” Journal of Jimei University, vol. 6, no. 3, pp. 219–223, 2001.
View at: Google Scholar
H. Cheng, W. Guo, M. C. Chang, J. L. Meng, and J. Yang, “Study of optimization on liquid fermentation conditions of Cordyceps militaris mycelium,” Journal of Shanxi Agricultural University (Society Science Edition), vol. 31, no. 1, pp. 66–72, 2011.
View at: Google Scholar
X. C. Liu, H. Li, T. Kang et al., “The effect of fermentation conditions on the structure and anti-tumor activity of polysaccharides from Cordyceps gunnii,” RSC Advances, vol. 9, no. 32, pp. 18205–18216, 2019.
View at: Publisher Site | Google Scholar
M. Y. Lung and P. C. Huang, “Optimization of exopolysaccharide production from Armillaria mellea in submerged cultures,” Letters in Applied Microbiology, vol. 50, no. 2, pp. 198–204, 2010.
View at: Publisher Site | Google Scholar
L. N. Xu, S. Guo, and S. W. Zhang, “Effects of solid-state fermentation with three higher fungi on the total phenol contents and antioxidant properties of diverse cereal grains,” FEMS Microbiology Letters, vol. 365, no. 16, pp. 132–140, 2018.
View at: Publisher Site | Google Scholar
V. Dwibedi, S. K. Rath, R. Prakas, and S. Saxena, “Response surface statistical optimization of fermentation parameters for resveratrol production by the endophytic fungus Arcopilus aureus and its tyrosinase inhibitory activity,” Biotechnology Letters, vol. 43, no. 3, pp. 1–5, 2021.
View at: Google Scholar
S. K. Kim, “Effect of dietary supplementation of fermented pine needle extract on productive performance, egg quality, and serum lipid parameters in laying hens,” Animals, vol. 11, no. 5, pp. 2–11, 2021.
View at: Google Scholar
Q. Q. Wang and Tologor, “Effect of selected parameters on the yield of crude Helvella lacunosa fruit body extracts and evaluation of anti-oxidative activity,” Acta Edulis Fungi, vol. 23, no. 2, pp. 49–51, 2016.
View at: Google Scholar

Copyright

Copyright © 2023 Shang Guo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

32

Downloads

30

Citations