FENG Li-guo, XU Ning, HUANG Xiao-hui, DENG Zhao-li, XIA Yi-liang, ZHOU Nan

Institute of Hunan Edible Fungi, Changsha 410013, PRC

Abstract In this paper, the liquid seed medium of Grifola frondosa was optimized by single factor and orthogonal experiments using the shake flask culture method. The single factor experiments results showed that soybean powder and maltose were the optimal nitrogen and carbon sources, and MgSO4 was the optimal inorganic salt. The orthogonal experiment results showed that the optimal combination of liquid seed culture medium for Grifola frondosa was: 20% potato, 0.75% soybean powder, 5% maltose, 0.1% KH2PO4, 0.05% MgSO4, 0.001% VB1. Using the optimized medium, shaking culture for 10 d under the inoculum amount of 5%, the temperature of 28°C, and the rotation speed of 150 r/min, the dry weight of mycelium exceeded 1.322 8 g/100mL.

Key words Grifola frondosa; Medium; Single factor experiments; Orthogonal experiments

1. Introduction

Grifola frondosa(Fr.) S. F. Gray is a kind of edible fungus used for both food and medicine, belonging to Basidiomycotina, Hymenomycetes, Aphyllophorales, Polyporaceae, and Ramalina[1-2]. It has the characteristics of crisp and tender meat, delicious taste and unique flavor. Because it is rich in essential amino acids, vitamins and minerals, it has obvious effects on inhibiting hypertension, obesity, preventing cirrhosis and diabetes, anti-aging and improving immunity.

Industrialized and scaled production ofGrifola frondosahave realized in China. At present, researches onGrifola frondosamainly focused on the extraction of polysaccharide and peptides, as well as their function. However, researches concerning liquid culture ofGrifola frondosaare extremely scarce[3-9]. Liquid strain has the advantages of fast flow, good dispersity, more germination points, rapid fungus development and early mycelium coverage[10-12]. In order to further improve the production capacity of liquid strain ofGrifola frondosaand lay the foundation for study on the fermentation of liquid strain ofGrifola frondosa, this study optimized the liquid seed medium ofGrifola frondosaby shake flask culture method, and explored the effects of carbon sources, nitrogen sources and inorganic salts on the growth ofGrifola frondosamycelium.

2. Materials and Methods

2.1. Materials

2.1.1. StrainGrifola frondosawas used as test strain, provided by microbiology laboratory of Jishou University.

2.1.2. Main reagent

Sucrose, glucose, soluble starch, (NH4)2SO4, CaCl2, MgSO4, MnCl2, KH2PO4and VB1were all analytical reagent made in China. Peptone, beef extract and maltose were all purchased from Beijing Aobaxing Biotechnology Co. Ltd. Soybean flour and potato were market products.

2.1.3. Culture medium

Slant medium: potato 20%, glucose 4%, peptone 0.5%, KH2PO40.1%, MgSO40.05%, VB10.001%, agar 2%.

Liquid seed medium before optimization: potato 20%, glucose 4%, peptone 0.5%, KH2PO40.1%, MgSO40.05%, VB10.001%.

Carbon source basal medium: glucose 4%, KH2PO40.1%, MgSO40.05%, VB10.001%.

Nitrogen source basal medium: peptone 0.5%, KH2PO40.1%, MgSO40.05%, VB10.001%.

Mineral basal medium: glucose 4%, peptone 0.5%, KH2PO40.1%, VB10.001%.

2.2. Methods

2.2.1. Activation of culture

The strain were activated and cultured at 28℃ in an incubator, then cut into uniform pieces and inoculated into fresh PDA slant medium, cultured at 28℃ in an incubator for 10 d. Slant section of mycelium was inoculated into plating medium and also cultured at 28℃ in an incubator for 10 d.

2.2.2. Seed cultivation

The slant strain was activated and cut into pieces, then inoculated into conical flask containing 100 mL liquid medium, cultured on a constant temperature shaker at 28℃ and 150 r/min. When mycelium grew vigorously and more, it can be used as liquid strain in single factor experiment.

2.2.3. Single factor experiment

2.2.3.1. Carbon source

Based on basal medium, 4% of maltose, glucose, sucrose and soluble starch were used as carbon sources respectively to prepare liquid medium. The liquid medium was placed in 250 mL conical flask, 100 mL per bottle, each carbon source was repeated for 3 times, a total of 12 bottles were sterilized at 121℃ for 0.5 h.

5% liquid strain was inoculated into conical flask and cultured on a constant temperature shaker at 28℃ and 150 r/min for 10 d. Then conical flask was then taken out and mycelium was filtered with filter paper, it was dried to constant weight at 60℃, and it was weighed repeatedly for 3 times and the average value was taken. The average dry weight of the 3 bottles of mycelium was taken as the final result, which was calculated as g/100mL. The optimal carbon source was determined according to the final weight of the mycelium.

2.2.3.2. Nitrogen source

Based on basal medium, 0.5% of soybean powder, peptone, beef extract, (NH4)2SO4were used as nitrogen sources respectively to prepare liquid medium. The liquid medium was placed in 250 mL conical flask, 100 mL per bottle, each nitrogen source was repeated for 3 times, a total of 12 bottles were sterilized at 121℃ for 0.5 h.

5% liquid strain was inoculated into conical flask and cultured on a constant temperature shaker at 28℃ and 150 r/min for 10 d. The conical flask was then taken out and mycelium was filtered with filter paper, it was dried to constant weight at 60℃, it was weighed repeatedly for 3 times and the average value was taken. The average dry weight of the 3 bottles of mycelium was taken as the final result, which was calculated as g/100mL. The optimal nitrogen source was determined according to the final weight of the mycelium.

2.2.3.3. Inorganic salt

Based on basal medium, 0.05% of CaCl2、MgSO4and MnCl2were used as inorganic salt respectively to prepare liquid medium. The liquid medium was placed in 250 mL conical flask, 100 mL per bottle, each inorganic salt was repeated for 3 times, a total of 12 bottles were sterilized at 121℃ for 0.5 h.

5% liquid strain was inoculated into conical flask and cultured on a constant temperature shaker at 28℃ and 150 r/min for 10 d. Then the conical flask was taken out and mycelium was filtered with filter paper, it was dried to constant weight at 60℃, it was weighed repeatedly for 3 times and the average value was taken. The average dry weight of the 3 bottles of mycelium was taken as the final result, which was calculated as g/100mL. The optimal inorganic salt was determined according to the final weight of the mycelium.

2.2.4. Orthogonal experiment

Based on single factor experiment, the optimal carbon source, nitrogen source and inorganic salt were selected to conduct L9(34)orthogonal experiment. 9 levels design were shown in Table 1.

Table 1 The factors and levels of orthogonal experiment

5% liquid strain was inoculated into conical flask and cultured on a constant temperature shaker at 28℃ and 150 r/min for 10 d. Then the conical flask was taken out and mycelium was filtered with filter paper, it was dried to constant weight at 60℃, it was weighed repeatedly for 3 times and the average value was taken. The average dry weight of the 3 bottles of mycelium was taken as the final result, which was calculated as g/100mL. The optimal medium combination was determined according to the final weight of the mycelium and variance analysis results.

3. Results and Analysis

3.1. Effects of carbon sources on the growth of Grifola frondosa mycelium

4% of maltose, glucose, sucrose and soluble starch 4 carbon sources were added into basal medium, respectively. As shown in Fig. 1, all four carbon sources could be used byGrifola frondosaand maltose had the highest content of mycelium.

Fig. 1 Effects of carbon sources on the growth of Grifola frondosa mycelia

3.2. Effects of nitrogen sources on the growth of

Grifola frondosamycelium

Soybean powder, peptone, beef extract and (NH4)2SO44 nitrogen sources were added into basal medium, respectively. It can be seen from Fig. 2 that all four nitrogen sources could be used byGrifola frondosaand mycelium cultured with soybean powder was the most. Compared with the other three nitrogen sources, soybean powder was widely available and inexpensive.

Fig. 2 Effects of nitrogen sources on the growth of Grifola frondosa mycelia

3.3. Effects of inorganic salt on the growth of Grifola frondosa mycelium

CaCl2、MgSO4and MnCl23 inorganic salts were added into basal medium, respectively. It can be seen from Fig. 3 that all three inorganic salts could used byGrifola frondosa. CaCl2had the worst utilization effect and MgSO4had the best utilization effect.

Fig. 3 Effects of inorganic salts on the growth of Grifola frondosa mycelia

3.4. Orthogonal experiment result

It can be seen from Table 2, the optimal combination of liquid seed culture medium forGrifola frondosawas A3B3C1, namely 5% maltose, 0.75% soybean powder, 0.05% MgSO4. Using the optimized medium, shaking culture for 10 d at the inoculum amount of 5%, the temperature of 28°C,and the rotation speed of 150 r/min, the dry weight of mycelium exceeded 1.322 8 g/100mL. Variance analysis was performed according to the results in Table 2 and the results were shown in Table 3. As shown in Table 2 and 3, maltose and soybean powder had significant effects on the growth of mycelium, MgSO4had no significant effects on the growth of mycelium. The effects of 3 factors on the the growth of mycelium was in the order: soybean powder> maltose>MgSO4.

Table 2 Orthogonal experiment results

Table 3 Analysis of variance

4. Conclusion

The optimal combination of liquid seed culture medium forGrifola frondosawas determined by single factor experiment and orthogonal experiment, namely 20% potato, 0.75% soybean powder, 5% maltose, 0.1% KH2PO4, 0.05% MgSO4, 0.001% VB1, nature pH value. Shaking culture for 10 d at the inoculum amount of 5%, the temperature of 28°C, and the rotation speed of 150 r/min, the dry weight of mycelium exceeded 1.322 8 g/100mL. When maltose was used as carbon source,Grifola frondosagrew best, whereas its price was a little more expensive than other carbon sources. The optimal nitrogen source was soybean powder, which had a lower price compared to peptone, beef extractetc. CaCl2、MgSO4and MnCl23 inorganic salts had no significant effects on the growth ofGrifola frondosa, therefore appropriate level of MgSO4was selected.Grifola frondosacultured by optimized medium had small mycelium pellets and large density, which not only provided excellent medium for the strain ofGrifola frondosa, but also laid the foundation for the large area cultivation ofGrifola frondosaand liquid fermentation culture for the realization of industrial production.