Study on Optimization of Culture Conditions for Wild Isaria cicadae Miquel Liquid Strain

Chunqin XIE，Zheng CAO，Junmin FAN，Junqi XU，Jun JIA*

Biological Engineering Department，Jiangsu Polytechnic College of Agriculture and Forestry，Jurong 212400，China

Abstract  ［Objectives］ To study the effect of different culture conditions on the growth of wild I.cicadae Miq.liquid strain.［Methods］ The I.cicadae Miq.strain was inoculated into the liquid culture medium with different carbon sources，nitrogen sources，micronutrients and pH，cultured in the constant temperature shaking incubator with rotating speed of 120 r / min at 19℃ ，and the mycelium pellet diameter，densi- ty and weight were compared between different treatments. ［Results］ The results showed that the optimum components of I.cicadae Miq.strain liquid included soluble starch，milk powder and vitamin B12 ，and the optimum pH was 5. 0 － 6. 0.［Conclusions］ Soluble starch was the most suitable carbon source for the culture medium of I.cicadae Miq.liquid strain; milk powder was the most suitable nitrogen source for the culture medium of I. cicadae Miq. liquid strain; the most suitable pH was 5. 0 － 9. 0 for the mycelial growth of I. cicadae Miq. ; the formu- la and mixture ratio of the optimum culture medium for the growth of liquid strain were determined.

Key words  Wild Isaria cicadae Miquel，Liquid strain，Culture medium

Received: October 14，2017    

Accepted: January 3，2018

Supported by " Six Talents Peaks" Project in Jiangsu Province in 2015 ( NY-024) ;  Project of Jiangsu Polytechnic College of Agriculture and Forestry ( 2014KJ28) .

* Corresponding author. E-mail: xiechunqin@ 163.com

1   Introduction

Isaria cicadae Miquel is the complex of fungus stroma parasitizing the cicada nymphs and nymph bodies［1］，and it is in the Cordycip- itaceae family.  The Cordyceps cicadae ( Miq. )  Massee and Paecilomyces cicadae Samson are the synonym of I.  cicadae Miq.［2］. The wild I. cicadae Miq. resources are scarce，and u- sing the artificial cultivation techniques，the wild resources can be turned into the medicinal resources for sustainable use，integrating protection and utilization.  The artificially cultivated I.  cicadae Miq. does not contain heavy metals，and is much safer than the natural Cordyceps sinensis.

The main active substances of I. cicadae Miq. are similar to the main active substances of Cordyceps sinensis［3 － 4］，and the main com- ponents include mannitol，Cordyceps polysaccharide CT-4［5］，D-man- nitol［6］，myriocin ( ISP-1) ［7 － 12］，ergosterol，ergosterol peroxide［13 － 14］， cyclic peptide compound［13］，galactomannan C-3［5］ and cordycepic acid ( mannitol) ，etc. ，having a good therapeutic effect on a variety of diseases，including adjuvant treatment of diabetes［14］.

The main production methods of I. cicadae Miq. strain in- clude liquid and solid culture［6］，and the liquid strain has a short growth cycle，so as to facilitate the large-scale production［16］，but at present，the main method of producing I. cicadae Miq. liquid strain is based on Cordyceps［17 － 18］，and there are few studies on the nutrient and component mixture ratio for the production of I. cicadae Miq. liquid strain. The main research work in this experiment was to study the effect of the optimal carbon sources，nitrogen sources，micronutrients as well as the initial pH value of culture medium on the mycelial growth of liquid strain of the wild I. cicadae Miq. in Jurong，in order to provide high quality prove- nances for the standardized，large-scale and industrialized artifi- cial cultivation of I. cicadae Miq.

2   Materials and methods

2. 1  Materials

2. 1. 1  Source of strain. The I. cicadae Miq. strain was obtained by Jiangsu Polytechnic College of Agriculture and Forestry and Jiangsu Institute of Edible Mushrooms from the wild I. cicadae Miq. collected from Wawu Mountain Forest Farm in Jiangsu Ju- rong using the tissue separation method.

2. 1. 2  Reagents. The test reagents used are shown in Table 1.

2. 1. 3    Instruments. DHZ-DA constant temperature oscillator，PL203 Mettler-Toledo electronic balance， PRX-250A constant temperature incubator，superclean bench，YX-400A high-pressure steam sterilizer，and other commonly used tools provided by Jiang- su Institute of Edible Mushrooms.

2. 2  Methods  

By measuring the effect of adding different car- bon sources，nitrogen sources，micronutrients to the culture liquid as well as the pH value of the culture liquid on the mycelium ball density，mycelium pellet diameter and biomass of wild I. cicadae Miq. liquid［19］，we selected the optimum carbon sources，nitrogen sources，micronutrients and initial pH values for liquid culture， and made test comparison on the mixture ratio of the selected opti- mum carbon sources，nitrogen sources and micronutrients in the culture medium，so as to optimize the liquid culture medium for- mula of wild I. cicadae Miq.

2. 2. 1  Basic culture medium formula. The basic culture medium formula of wild I. cicadae Miq. liquid strain: glucose 20 g; pep- tone 10 g; potassium dihydrogen phosphate 0. 5 g; magnesium sul- fate 1 g; vitamin B1 10 tablets; water 1 000 mL.

2. 2. 2  Optimization of the carbon source culture medium formula. In the test，the same amount of maltose，sucrose，lactose and soluble starch was used to replace the glucose in basal medium as a carbon source，respectively，and other components and mixture ratio remained constant in the basal culture medium［18 － 19］.

2. 2. 3  Optimization of the nitrogen source culture medium formula. The same amount of yeast extract，milk powder，urea，ammonium chloride was used to replace the peptone in the basal culture medium as a nitrogen source，respectively，and other components and mixture ratio remained constant in the basal culture medium［18 － 19］.

2. 2. 4   Optimization of the micronutrient culture medium formu- la. The same amount of vitamin B2 ，vitamin B6 ，and vitamin B12 was used to replace the vitamin B1 in the basal culture medium， and other components and mixture ratio remained constant in the basal culture medium.

2. 2. 5  Optimization of pH value of culture medium. By the ex- perimental comparison of 2. 2. 2 － 2. 2. 4，the best carbon source， nitrogen source and micronutrient were added to the basal culture in the same amount，and other components and mixture ratio re- mained unchanged. The liquid culture medium was made up，and the citric acid ( 2 mol / L) and NaOH ( 1 mol / L) were used to ad- just the pH value of liquid culture medium to 1 － 10. After 40 min of conventional autoclaving，it was cooled for later use［19］.

2. 2. 6   Optimization of the best culture medium formula. Based on the above experiments，the same amount of the best carbon source，nitrogen source and micronutrient was used as replacement in the basic culture medium in 2. 2. 1，and this formula was the optimal basic culture medium ( CK) . Under the same culture con- ditions，the nutrition in the CK formula was halved ( formula I) ， increased 2 times ( formula II) ，increased 4 times ( formula III) ， increased 6 times ( formula IV) ，to measure the mycelial growth traits of I. cicadae Miq. in each formula，so as to select the best mixture ratio of components of the culture medium.

2. 2. 7    Preparation of the liquid culture medium. According to the conventional liquid culture medium preparation method［20］， the prepared liquid culture medium was dispensed into a triangular flask，based on 100 mL/ bottle ( 500 mL flask) ，and it was sealed with a special sealing film for tissue culture，and after 40 min of conventional autoclaving，it was cooled for later use.

2. 2. 8  Inoculation and culture. According to the requirements of aseptic operation，two fungus cakes with the diameter of 0. 5 cm were inoculated into each bottle. After inoculation，it was immedi- ately put into the 19℃ constant temperature incubator for 24 h of static culture，and then put into the shaker with the rotating speed of 120 r / min for continued shaking culture.

2. 2. 9   Determination items and methods. ( i) Determination of mycelium pellet diameter of liquid strain. 48 hours after inocula- tion，it was determined，and then it was measured once every 24 h. In each measurement，the liquid should be taken in the ul- tra-clean bench，and 3 mL of culture medium was transferred to the culture dish padded with absorbent paper using a pipette. 30 mycelium pellets were randomly selected in a row and the total length was measured with a vernier caliper［20］，and the average di- ameter of each mycelium pellet was calculated. ( ii) Determina- tion of mycelium pellet density of liquid strain. Measurement time and the method of taking liquid were as the above. 3 mL of culture solution was taken and put into the culture dish padded with col- ored absorbent paper，and the counting process was used to calcu- lated the number of mycelium pellets in 1 mL of culture solution.  ( iii) Determination of mycelial biomass of liquid strain. Measure- ment time and the method of taking liquid were as the above. 3 mL of culture solution was taken and put into the culture dish padded with filter paper to stand still for 60 s，and all the myceli- um pellets were transferred to the dried culture dish，and placed on an electronic balance to measure the weight. ( iv) Determina- tion of pH of liquid culture medium. In the ultra-clean bench，in an aseptic manner，the culture solution was placed in the pH me- ter with the sterile pipette to measure and record the pH.

3   Results and analysis

3. 1   Effect of different carbon sources on the mycelial growth of I. cicadae Miquel in liquid culture  

Carbon is the most important nutrition of fungi，and it is not only the carbohy- drate，but also the basic element of protein. At the same time，it is also an important source of energy.  There were significant differences in the growth of I. cicadae Miq. mycelium when differ- ent carbon sources were added to the culture medium ( Table 2) . Among the five carbon sources，the mycelial growth was the best with soluble starch as the liquid culture medium，and the mycelial growth and mycelium density under the soluble starch liquid cul- ture medium were 14 and 4 times the mycelial growth and myceli- um density under the basal culture medium and the glucose culture medium. 3 － 4 d after the liquid strain culture was the most pro- ductive period of mycelial growth ( Fig. 1) ，and in terms of the size of liquid mycelial growth in the five carbon source culture media，it was in the order of soluble starch ＞ lactose ＞ maltose ＞ glucose ＞ sucrose.

Note: The picture from left to right is glucose，maltose，sucrose，lactose，soluble starch.

Fig. 1  The mycelial growth of Isaria cicadae Miquel in the liquid culture medium of different carbon sources on the 4th d

3. 2   Effect of different nitrogen sources on the mycelial growth of I. cicadae Miquel  

There was a significant difference in the effect of five nitrogen sources on the mycelial growth of I. ci- cadae Miq. in liquid culture. The mycelial growth was the best with milk powder as nitrogen source. The mycelium pellet density of yeast nitrogen and peptone organic nitrogen was high，but the diameter of mycelium pellet was inconsistent. The mycelium pellet diameter of milk powder and peptone was higher than the mycelium pellet diameter of other nitrogen sources，and there was almost no mycelial growth in the culture medium with urea as the nitrogen source ( Fig. 2) .

In the process of culture，the color of the culture solution also changed. The yeast extract showed a reddish brown color and the milk powder culture solution exhibited a pink color ( Fig. 3) .

Fig. 2  Effect of different nitrogen sources on the mycelial growth of Isaria cicadae Miquel

Note: The picture from left to right is peptone，yeast extract，milk powder，urea and ammonium chloride.

Fig. 3  The mycelial growth of Isaria cicadae Miquel in the liquid culture medium of different nitrogen sources on the 4th d

3. 3  Effect of different trace elements on the mycelial growth of I. cicadae Miquel  

B vitamins，as micronutrients，have a sig- nificant impact on the growth of I. cicadae Miq. Adding some B vitamins to the liquid culture medium can promote the mycelial growth. In the culture media of vitamin B1 ，vitamin B2 ，vitamin B6 ，vitamin B12 ，the mycelial growth of I. cicadae Miq. in the culture medium supplemented with vitamin B12 was significantly larger than the mycelial growth of vitamin B1 ，vitamin B2 ，vitamin B6 ( Fig. 4) .

In the process of I. cicadae Miq. liquid strain culture，on the 4th d，there was no discoloration in vitamin B1 culture medium， while the culture medium of the other three vitamins was brown to red ( Fig. 5) .

3. 4  Effect of different initial pH of the liquid culture medi- um on the mycelial growth of I. cicadae Miquel  

In the for- mula with the initial pH of liquid culture medium at 1. 0 － 10. 0，there were some differences in the mycelial growth of I. cicadae Miq. The mycelial growth of I. cicadae Miq. with the initial pH of 5. 0 － 9. 0 was significantly better than the mycelial growth of I. cicadae Miq. with other pH ranges. When pH was 5. 0 － 6. 0， the density of mycelium pellets was the highest，the mycelial growth was maximal，but the diameter of mycelium pellets was moderate ( Table 3) .

Fig. 4  Effect of different trace elements in the culture medium on the mycelial growth of Isaria cicadae Miquel

Note: The pictures from left to right are vitamin B1 ，vitamin B2 ，vitamin B6 ，and vitamin B12 .

Fig. 5  The mycelial growth of Isaria cicadae Miquel in the liquid culture medium of different micronutrients on the 4th d

3. 5  Optimization of the best culture medium of liquid strain 

In the liquid culture medium，different supplementing amounts of the best carbon sources，nitrogen sources and micronutrients had a significant effect on the mycelial growth of I. cicadae Miq. ，and the mycelial growth was the best in the optimal basal medium ( CK) ( Fig. 6) ，while the mycelial growth was the worst in the halved culture medium，and the mycelium pellet density was only one-eighth of that in the optimum culture medium on the 5th d of culture ( Fig. 7) .

Fig. 6  Effect of the optimum nutrients supplemented to the culture medium on the mycelial growth of Isaria cicadae Miquel

Fig. 7  The mycelial growth of Isaria cicadae Miquel in the liquid culture medium of the optimal nutrition on the 4th d of culture

4   Conclusions

4 . 1   Soluble starch as the most suitable carbon source for the culture medium of I. cicadae Miquel liquid strain 

Under the condition of constant N source and trace element in the basic culture medium，the I. cicadae Miq. was cultured with dif- ferent C sources.  The utilization rate of soluble starch was the best，and the mycelial growth rate was the highest，higher than that of glucose，sucrose，maltose and lactose as C sources. In the commercial production of I. cicadae Miq. ，the soluble starch can be used as the C source.

4. 2  Milk powder as the most suitable nitrogen source for the culture medium of I. cicadae Miquel liquid strain  

In the case of constant C source and growth factors in the basal culture medi- um，the I. cicadae Miq. was cultured with different N sources， and the utilization rate of milk powder was the best. The mycelial growth rate was the highest，higher than that of peptone，ammoni- um chloride，yeast extract and urea as N sources. In the commer- cial production of I. cicadae Miq. ，the milk powder can be used as the N source.

4. 3  Vitamin B12 as the most suitable nutrient for the culture medium of I. cicadae Miquel liquid strain  

In the case of con- stant C source and N source in the basal culture medium，the I. cicadae Miq. was cultured with different growth factors，the utili- zation rate of vitamin B12 was the highest，and the mycelial growth rate was the highest，higher than that of vitamin B1 ，vitamin B2 ， vitamin B6 as the nutrients. In the commercial production of I. ci- cadae Miq. ，the vitamin B12 can be used as the nutrient.

4. 4   5. 0 － 9. 0 as the most suitable pH for the mycelial growth of I. cicadae Miquel  

In the case of constant basal cul- ture medium formula，the initial natural pH of was adjusted，to ad- just the initial pH of culture medium to 1 － 10，and when the ini- tial pH was 5. 0 － 9. 0，the mycelial growth rate was the highest， and when the pH was 5. 0 － 6. 0，the mycelial growth rate was higher than under other pH values. In the commercial production， when the pH of culture liquid of I. cicadae Miq. was controlled to be 5. 0 to 6. 0，it was conducive to the mycelial growth and devel- opment of I. cicadae Miq.

4. 5  The formula and mixture ratio of the optimum culture medium for the growth of liquid strain  

By comparing the effects of adding different carbon sources，nitrogen sources and mi- cronutrients to the culture medium on the growth of liquid strain of I. cicadae Miq. ，it was found that the best culture medium formula was soluble starch 20 g; milk powder 10 g; potassium dihydrogen phosphate 0. 5 g; magnesium sulfate 1 g; vitamin B12 10 tablets; water 1 000 mL. In the mycelium growth process of I. cicadae Miq. ，the pH value of fermentation liquid of liquid culture medium was reduced，and the appropriate initial pH value of culture medi- um was 5. 0 － 6. 0，indicating that the acidic environment in the liquid fermentation was conducive to the mycelial growth.

On the 3rd to 4th d of mycelial culture of I. cicadae Miq. liq- uid strain，it was the most vigorous mycelial growth period，and at this point，the diameter of mycelium was moderate，the density was high，and the mycelial growth was the fastest，so it was the best inoculation period for the liquid fermentation and culture. Therefore，in the commercial cultivation of I. cicadae Miq. ，the mycelium pellet cultured for 3 － 4 d can be used as strain to obtain high yield.

References

［1］ TENG Y，SONG YL，GUAN ZH. Progress in artificial culture of cicadas［J］.  Nei Mongol Journal of Traditional Chinese Medicine，2012，31 (12) : 111 － 112. ( in Chinese) .

［2］ DONG CH，LI WJ，LI ZZ. Cordyceps industry in China: current status， challenges and perspectives ———Jinhu declaration for cordyceps industry development［J］. Mycosystema，2016，35( 1) : 1 － 15. ( in Chinese) .

［3］YU SJ，CHAI XY，FAN MZ. Analysis of nutritional and bioactive compo- nents of solid-state fermented substrate of Isaria cicadae［J］. Food and Machinery，2015，31( 1) : 155 － 158. ( in Chinese) .

［4］ CHEN C，XU JW，SUN XJ，et al. Study on the isolation and bioactivity of cicadas in Maoshan area［J］. Jiangsu Agricultural Sciences，2015，43 (4) : 347 － 352. ( in Chinese) .

［5］ KIHO T，NAGAI K，MIYAMOTO I，et al. Polysaccharides in fungi. XXV. Biological activities of two galactomannans from the inseclrbody portion of Chan hua ( fungus: Cordyceps cicadae) ［J］. Yakugaku Zasshi， 1990，110: 86 － 288.

［6］ WEN X. The selection of cicadas and the optimization of liquid fermenta- tion process［D］. Changsha: Hunan Agricultural University，2013. ( in Chinese) .

［7］ FUJITA T，INOUE K，YAMAMOTO S，et al. Fungal metabolites. Part I. A potent immunosuppressive activity found in Isaria sinclairii metabolite［J］. The Journal of Antibiotics，1994，47: 208 － 215.

［8］ PASSER SP. Medicinal mushrooms as a source of antitumor and immuno- modulating polysaccharides［J］. Applied Microbiology and Biotechnology， 2002，60: 258 － 274.

［9］ HOJJATI MR，LIZ Q，ZHOU HW，et al.  Efect of myriocin on plasma sphingolipid metabolism and atherosclerosis in Apo E-deficient mice［J］. Journal of Biological Chemistry，2005，280: 10284 － 10289.

［10］ PARK TS，ROSEBURY W，KINDT EK，et al. Serine palmitoyltrans- ferase inhibitor myriocin induces the regression of atherosclerotic plaques in hyperlipidemic Apo E-deficient mice［J］. Pharmaceutical Research， 2008，58: 45 － 51.

［11］ GLAROS EN，KIM WS，RYE KA，et al. Inhibition of atherosclerosis by the serine palmitoyl transferase inhibitor myriocin is associated with re- duced plasma glycosphingolipid concentration［J］. Biochemical Pharma- cology，2007，73: 1340 － 1346.

［12］PARK TS，PANEK RL，REKTIIEL MD，et al. Modulation of lipoprotein metabolism by inhibition of sphingomyelin synthesis in Apo E knockout mice［J］. Atherosclerosis，2006，189: 264 － 272.

［13］ KUO YC，LIN LC，DON MJ，et al. Cyclodesipeptide and dioxomorpho- line derivatives isolated from the insect-body portion of the fungus Cordyceps cicadae［J］.  Journal of the Chinese Medical Association， 2002，13: 209 － 219.

［14］ KUO YC，WENG SC，CHOU CJ，et al. Activation and proliferation sig- nals in primary human T lymphocytes inhibited by ergosterol peroxide i- solated from Cordyceps cicadae［J］. British Journal of Pharmacology， 2003，140: 895 － 906.

［15］ HE YH，HE XQ，YANG Y，et al. The research progress of cicadas in treating diabetic nephropathy［J］. Heilongjiang Medicine Journal，2012， 25 ( 6) : 868 － 869. ( in Chinese) .

［16］ YANG HD. To promote the production of liquid bacteria to change the production of edible fungi［J］. Modern Agricultural Science and Tech- nology，2009( 11) : 93. ( in Chinese) .

［17］ WANG Y，YU RM，TONG ZQ，et al. The determination of optimal cul- ture conditions and biodynamics for Cordyceps militaris［J］.  Chinese Wild Plant Resources，2003，22( 4) : 56 － 60. ( in Chinese) .

［18］ WANG W. The research and application of the bacteria of edible fungus liquid［D］. Zhejiang: Jiangsu University，2005. ( in Chinese) .

［19］MA RX，LIU H. Study on the screening of liquid spawn medium and ex- plore of culture conditions of Pleurotus eryngii［J］. Chinese Agricultural Science Bulletin，2011，27( 7) : 452 － 456. ( in Chinese) .

［20］ ZHENG TT. On the optimization for deep fermentation of Cordyceps mili- taris and content analysis of its active substance［D］. Xi’an: Northwest University，2006. ( in Chinese) .