Study on nutritional components in the cultivation mediums of cordyceps militaris

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  GRADUATION THESIS STUDY ON NUTRITIONAL COMPONENTS IN THE CULTIVATION MEDIUMS OF CORDYCEPS MILITARIS Student Name : NGUYEN THI THUY Student Code : 620636 Class : K62CNSHE Supervisior : NGUYEN VAN GIANG Assoc Prof Hanoi – 2022 COMMITMENT I hereby declare that all the data and the results that I have provided in this thesis are truthful, accurate, and not used in any other reports I also assure that any help in this thesis has been greatly appreciated thankful and the information cited has been acknowledged Hanoi, May, 2022 Student Nguyen Thi Thuy i ACKNOWLEDGEMENTS First and foremost, I have to thank the Vietnam National University of Agriculture for giving me a chance to study in a professional environment that provided me the sufficient knowledge throughout four and a half academic years I would like to express my endless thanks and gratitude to my supervisor Assoc Prof Nguyen Van Giang His kindly support and continuous advice went through the process of completion of my thesis Without his motivation and instructions, the thesis would have been impossible to be done effectively I would like to thanks to everyone at Vietnam Institute of Medicinal Herbs for their encouragement and guidance for my thesis Last but not least, my special thanks approve to my parents and my friends for their endless love, and care and have most assistance and motivation during the process of completed graduate thesis Hanoi, May, 2022 Student, Nguyen Thi Thuy ii ABSTRACT Cordyceps militaris is an entomopathogenic fungus with many medicinal values similar to Cordyceps sinensis and has been used for a long time in traditional medicines Different from Cordyceps sinensis, which has very low mass production and only grows in natural environment, the fungus Cordyceps militaris can be farmed in artificial conditions Therefore, collecting of technical information and researches will help promoting the production of Cordyceps militaris in Vietnam to satisfy the demand of national and abroad markets This study evaluated the role of Cordyceps militaris in modern medicine based on summing up the scientific research results, as well as assessing the biological characteristics and the effects of the cultivating conditions for Cordyceps militaris to develop farming processes in our country, which will bring essential economic benefits for local producers Results from scientific publications showed that there had been many researches confirming the potential applications of C.militaris in illness treatment, also it was widely used in the modern pharmaceutical industry, such as cancer treatment, immunomodulatory, impaired liver and kidney function, etc (Shonkor, et al., 2010; Shin, et al., 2009) For the production of C.militaris in artificial conditions, the strict control of environmental conditions such as fungal seeds, temperature, humidity, light, and nutrients was found as essential to maintain the yield and quality C.militaris iii CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii ABSTRACT iii LIST OF ABBREVIATIONS vi LIST OF TABLES vii LIST OF FIGURES viii I INTRODUCTION .1 1.1 Introduction 1.2 Research purpose 1.3 Location and time studies II LITERATURE REVIEW 2.1 General introduction to Cordyceps militaris(C.militaris) 2.1.1 Classification and description of C.militaris 2.1.2 The life cycle of C.militaris 2.1.3 Nutritional value of Cordyceps 2.1.4 Medicinal value of C.militaris 2.2.Research situation of C.militaris 2.2.1 Research situation of C.militaris in the world 2.2.2 Research situation of C.militaris in Vietnam 10 III MATERIALS AND METHODS 12 3.1 Materials and equipment 12 3.1.1 Materials 12 3.1.2 Equipment 13 3.2 Methods 13 3.2.1 Culture conditions 13 3.2.2 Substrate preparation and cultivation 14 IV RESULTS AND DISCUSSION 16 iv 4.1 Effect of different propagation solid media composition on the growth rate and characteristics of C.militaris VTV.1 strain 16 4.2 Effects of different liquid medium components on the propagation of C.militaris VTV.1 strain 18 4.3 Cultivation characteristics of C.militaris VTV1 strain on different nutrient medium 21 V CONCLUSIONS 28 VI REFERENCES 29 v LIST OF ABBREVIATIONS Abbreviations Explaination CME Cordyceps militaris Extract CMP Cordyceps militaris prote NAA Naphthaleneacetic acid CS-Cr Cordyceps sinensis crysta WE HPLC Water extract High-performance liquid chromatography et al Et alia vi LIST OF TABLES Table Summary of various pharmacological and therapeutic effects of Cordyceps sp Table Level propagation medium 12 Table Level propagation medium 12 Table Production medium 13 Table The growth and characteristics of the mycelia was different on nutrient media 16 Table The growth and characteristics of the fruiting body was different on nutrient medium 23 vii LIST OF FIGURES Figure 2.1 Natural (A) and cultivated (B) C.militaris Figure 4.1 C.militaris VTVT1 strain in different medium on the 5th days 17 Figure 4.2 C.militaris VTVT1 strain in medium F1 and F2 on the 5th days 18 Figure 4.3 C.Militaris VTVT1 strain in liquid mediums on the 7th days 19 Figure 4.4 The mycelial dry weight of Cordyceps militaris cultivated in submerged culture 20 Figure 4.5 C.Militaris VTV strain was cultured in 15 different production mediums after 90 days 21 Fig 4.6.Characteristics of the fruiting body of Cordyceps militaris strain cultured in 15 mediums; A Length of fruiting body (Cm); B Weight of fruiting body (g/box); C Weight of dried fruiting body (g) 25 Fig 4.7 Cordycepin content of C.militaris VTV.1 strain cultured in mediums 26 viii I INTRODUCTION 1.1 Introduction Cordyceps spp mushrooms, classified into the Ascomycota group, have a long tradition of use as a natural agent in Asian ethnomedicine because of their adaptogenic and tonic effects and their ability to reduce fatigue and stimulate the immune system in humans Cordyceps is a broad term describing a group of ascomycetous fungi that have carved out a niche as endoparasites primarily of arthropods and also as symbionts of the ascomycete truffle genus Elaphomyces So far, 540 species have been reported as cordyceps The term Cordyceps is derived from the Latin words, ‘cord’ and ‘ceps’, respectively meaning ‘club’ and ‘head’ Among the numerous, approximately 500, species of Cordyceps, most scientific research has been conducted on Cordyceps sinensis, which is included in the Chinese Pharmacopoeia (2015), and its adenosine concentration is considered as the main quality indicator Considering several complications regarding C.sinensis, the species C.militaris has attracted more attention from scientists and the industry over recent years C.sinensis is an authorized ingredient in food and dietary supplements in the European Union (EU), and it is included in European Commission (EC) and European Food Safety Authority (EFSA) documents such as the Novel Foods Catalogue, Compendium of Botanicals Health claims for C.sinensis have been proposed in the past, such as that C.sinensis possesses antioxidant properties (400–800 mg/day), stimulates the immune system and increases performance and endurance during exercise, Because of the limited resources of C.sinensis, endemic occurrence, unique growth pattern and biology of its life cycle in the ecosystem, the longterm process of symbiosis between the fungus and the larva (from autumn to spring) is time-consuming, and the harvesting process of this species from its natural site results in a very high price Obtaining this species from the natural As reported by Kim et al (2003), during submerged cultivation of C.militaris, the cells formed mainly pellets at the initial stage and then maintained this growth form almost constant during the entire fermentation period Several parameters such as diameter, circularity, roughness, and compactness are normally estimated to characterize pellet morphology (Park et al.,2002) Among these characteristics, pellet size is found to be one of the most important parameters The pellet size greatly affects the transportation of oxygen and nutrients into the core region of pellet cells The lack of oxygen and nutrients in the center leads to the death of large pellet cells Thus, pellets with a Mycelial dry weight [g/l] small size are advantageous (Turlo , 2014) 1.1 5.4 5.5 5.9 4.7 4.5 4.9 1.2 2.1 2.2 3.1 3.2 Mediums Figure 4.4 The mycelial dry weight of Cordyceps militaris cultivated in submerged culture Under submerged culture conditions, dry biomass weight ranged from 4.5g/l to 5.9g/l The highest biomass production was obtained for media 2.1 at days after inoculation with 5.9 g/l, and the lowest biomass production was obtained for media 3.1 with 4.5 g/l The large dry biomass weight of the fungus is due to a large number of mycelial formed after the fermentation process 20 Therefore, the medium with a large dry biomass weight will be good for the development of fruiting bodies From the Fig 4.4 , select mediums with mycelial dry weight greater than (media 1.1, 1.2, 2.1) to experiment 4.3 Cultivation characteristics of C.militaris VTV1 strain on different nutrient medium Inoculated mycelia (pellets) from selected liquid media( 1.1; 1.2; 2.1.), where they best grow, into different producing media In total ,there are 15 mediums ( 1.1.1; 2.1.1; 3.1.1; 4.1.1; 5.1.1; 1.1.2; 2.1.2; 3.1.2; 4.1.2; 5.1.2; 1.2.1; 2.2.1; 3.2.1; 4.2.1; 5.2.1), respectively Each medium was cultured in repeats times (total 75 boxes) For the artificial cultivation of C militaris, cottonseed shells, cob particles ,brown rice, German millet, and millet (Kim et al., 2010) could be used as the basal substrate and nutritional sources Compared to other substrates, brown rice, German millet, and millet exhibited higher stromata lengths of approximately 50 mm(Kim et al., 2010) Brown rice showed the highest yield of fruiting bodies with 6–7 g of fresh weight per bottle (Kim et al., 2010) Thus, brown rice as the main substrate was used in the present study Figure 4.5 C.Militaris VTV strain was cultured in 15 different production mediums after 90 days 21 When culturing fungi on different nutrient media, the growth and development of fruiting bodies are also different That difference is evident in the time the mycelium spread to the surface of the medium, the length of the fruiting bodies, and the weight (dry and fresh) of each box From the table 6, it shows that the speed of spreading to the surface of the medium is from 4-7 days, the fastest is in medium 2.2.1( days) This is because the composition and nutrient content in different environments are different The medium used for the cultivation of C.militaris is very important in determining the quality and quantity of the fruiting bodies The nutrient-rich environment will give the mushroom fruiting body a high growth rate Results Fig 7A shows that the fruit body length increases gradually over time The length of the fruiting body is one of the most important morphological characteristics that affect the commercial value of C.militaris As reported by Kim et al (2010), higher lengths with fewer fruiting bodies often lead to the reduced weight of the fruiting body and thus affect commercial value At 30 days after culture, fruiting bodies in all media were 0.5 to 3.0 cm low After 60 days, the fruiting body of the fungus developed high, in which medium 1.1.2 gave the highest fruit body length of 6.2 cm, medium 1.1.1 have the lowest fruit body length of 2.5 cm After 90 days of cultivation, medium 1.1.2 and 2.2.1 has the highest length of fruiting body of 9.3 cm and 8.7 cm, respectively At this stage, almost the fruiting bodies reach the optimal index because, after 90 days of culture with suitable temperature and humidity conditions, the fungus will give the best fruiting body length In addition to the criterion of fruiting body length, the weight of C.militaris fungus is considered to be quite important because based on this index, it will be estimated how many grams of fruiting body weight will be obtained from a known amount of initial substrate For the media in this study, the fresh fruiting body weight of C.militaris mushroom ranged from 42 to 57.3 22 g/box, the highest in medium 1.1.2 and the dry weight of mushrooms ranges from 5.3-7.27 g, the highest in medium 1.1.2 Table The growth and characteristics of the fruiting body was different on nutrient medium Culture media 1.1.1 2.1.1 3.1.1 4.1.1 5.1.1 1.1.2 2.1.2 3.1.2 4.1.2 5.1.2 1.2.1 2.2.1 3.2.1 4.2.1 5.2.1 Time for Length of fruiting body (Cm) the mycelia 30 days 60 days 90 days to fully cover the surface 7 7 6 5 7 0.5 0.8 1.2 1.0 1.5 3.0 1.0 1.5 2.0 2.0 0.8 1.8 0.8 1.5 0.5 2.5 4,5 4 6,2 4.5 5.5 4.5 3.5 4.2 4,2 3,5 Weight of fruiting body (g/box) 5.6 8.3 8.6 9.3 7.3 8.5 8.0 6.3 8.3 8.7 7.3 8.6 6.6 42.7 42 50.7 51.3 55.7 57.3 52.3 52.2 47.8 43.2 45.2 56.5 47.8 55.5 44.4 Weight of dried fruiting body (g) 5.47 5.3 6.87 6.63 7.23 7.27 6.03 7.14 6.93 5.63 5.77 7.09 6.76 6.98 5.6 To ensure both yield and quality, mediums that produced the highest fruiting body length and weight were selected to test cordycepin content.From the table 6, culture mediums has the best fruiting body length and yield are - Media 1.1.2 ( Length of fruiting body: 9.3cm; Weight of fruiting body (g/box): 57.3 ; Weight of dried fruiting body (g): 7.27) - Media 2.2.1 (Length of fruiting body: 8.7 cm; Weight of fruiting body (g/box): 56.5 ; Weight of dried fruiting body (g): 7.09) 23 - Media 3.1.2 (Length of fruiting body: 8.5 cm; Weight of fruiting body (g/box): 52.2 ; Weight of dried fruiting body (g): 7.14) - Media 4.2.1 (Length of fruiting body: 8.6 cm; Weight of fruiting body (g/box): 55.5 ; Weight of dried fruiting body (g): 6.98) - Media 5.1.1 (Length of fruiting body: 8.6 cm; Weight of fruiting body Length of fruiting body (Cm) (g/box): 55.7 ; Weight of dried fruiting body (g): 7.23) A 12 10 30 days 60 days 24 90 days 1.1.1 2.1.1 3.1.1 4.1.1 5.1.1 1.1.2 2.1.2 3.1.2 4.1.2 5.1.2 1.2.1 2.2.1 3.2.1 4.2.1 5.2.1 Weight of fruiting body (g/box) 70 60 50 40 30 20 10 Weight of dried fruiting body (g) B C Mediums 1.1.1 2.1.1 3.1.1 4.1.1 5.1.1 1.1.2 2.1.2 3.1.2 4.1.2 5.1.2 1.2.1 2.2.1 3.2.1 4.2.1 5.2.1 1.1.1 2.1.1 3.1.1 4.1.1 5.1.1 1.1.2 2.1.2 3.1.2 4.1.2 5.1.2 1.2.1 2.2.1 3.2.1 4.2.1 5.2.1 Mediums Fig 4.6.Characteristics of the fruiting body of Cordyceps militaris strain cultured in 15 mediums; A Length of fruiting body (Cm); B Weight of fruiting body (g/box); C Weight of dried fruiting body (g) Cordycepin content of C.militaris VTV.1 strain cultured in mediums 25 Cordycepin, a well-known biomarker of the genus Cordyceps (Ghatnur et al., 2015; Guo et al 2016), has been widely used as a bioactive compound in immunological, hepatic, renal, and cardiovascular disorders and as an anticancer agent (Jin Y et al., 2018).The content of cordycepin in C.militaris is related to Cordycepin content( mg/g) several factors such as culture condition, growth stage, and in gredients of the 4.5 3.5 2.5 1.5 0.5 4.37 3.69 2.74 2.95 3.17 1.1.2 2.2.1 3.1.2 4.2.1 5.1.1 mediums Fig 4.7 Cordycepin content of C.militaris VTV.1 strain cultured in mediums medium Thus, to enhance the production yield of cordycepin in C.militaris culture, numerous studies have been conducted to optimize culture conditions C.militaris strains cultivated in silkworm exhibited the highest cordycepin concentration (4.17 ± 1.66 mg/g) followed by brown rice medium (2.98 ± 1.41 mg/g), while C.militaris cultivated in PDB showed the least content of cordycepin with 1.08 ± 0.73 mg/g (Kang et al., 2017) This may be because the silkworm pupae medium contains a high concentration of proteins and low carbohydrate content (Lee et al., 2017; Guo et al., 2016) C.militaris cultivated on different media could produce cordycepin in amounts ranging from to 14 g (Cho et al., 2010) In the present study, the cordycepin content of the C.militaris 26 VTV.1 strain cultured in mediums ranged from 2.73 - 4.37 mg/g, the highest in medium 4.2.1 From the researched culture media formulas, that medium 4.2.1 was the most suitable for the growth of the C militaris VTV.1 strain to achieve the best yield and quality 27 V CONCLUSIONS The best propagation medium levels I for C.militaris VTV.1 is medium 1:N1 The best propagation medium levels II for C.militaris VTV.1 is the medium 2: N1 + 20gr silkworm pupae/L and the time of incubation is days Substrate medium with components: 30gr brown rice + 10 gr bean sprouts + 10 gr silkworm pupae is the optimal medium for growing Cordyceps militaris 28 VI REFERENCES Chen, D (1995) "Effects of JinShuiBao capsule on the quality of life of patients with heart failure." Journal of Administration of Traditional Chinese Medicine 5(1995): 40-43 Chu, H.-L., et al (2011) "Protective effect of Cordyceps militaris against high glucose-induced oxidative stress in human umbilical vein endothelial cells." Food chemistry 129(3): 871-876 Cho S.M., Park H.J., Seo G.-S., Hong J.-D (2010) Effect of medias composition on the Cordycepin and content Nutritional Components of Cordyceps militaris Korean J Mycol 37(2): 161–166 Das, S K., Masuda, M., Sakurai, A., & Sakakibara, M (2010) Medicinal uses of the mushroom Cordyceps militaris: current state and prospects Fitoterapia 81(8), 961-968 Davis, R Michael; Sommer, Robert; Menge, John A (2012) Field Guide to Mushrooms of Western North America Berkeley: University of California Press pp 381–382 Dong, J.Z., Lei, C., Ai, X.R & Wang Y., (2012) Selenium enrichment on Cordyceps militaris Link and analysis on its main active components Appl Biochem Biotechnol 166: 1215-1224 Ghatnur S., Parvatam G., Balaraman M (2015) Culture conditions for production of biomass, adenosine, and cordycepin from Cordyceps sinensis CS1197: Optimization by desirability function method Pharmacogn Mag 11(44): 448 Gu, Y.X., Wang, Z.S., Li, S.X & Yuan, Q.S (2007) Effects of multiple factors on accumulation of nucleosides and bases in Cordyceps militaris Food Chem, 102, 1304–9 Guo M., Guo S., Huaijun Y., Bu N, Dong C.-H (2016) Comparison of major bioactive compounds of the caterpillar medicinal mushroom, Cordyceps militaris (Ascomycetes), fruiting bodies cultured on wheat substrate and pupae Int J Med Mushrooms 18(4): 327–336 10.Hajek, A E., & St Leger, R J (1994) Interactions between fungal pathogens and insect hosts Annual review of entomology, 39(1), 293-322 11.Hsu, C., et al (2002) The mechanism of Cordyceps sinensis on steroidogenesis in normal mouse Leydig cells Biology of Reproduction, SOC STUDY REPRODUCTION 1603 MONROE ST, MADISON, WI 53711-2021 USA 12.Hur, H (2008) "Chemical ingredients of Cordyceps militaris." Mycobiology 36(4): 233-235 13.Jiang, X.L & Sun, Y (1999) The determination of active components in various Cordyceps militaris strains Acta Edulis Fungi, 6, 47–50 29 14.Jeong, M.-H., et al (2012) "Effect of cordycepin purified from Cordyceps militaris on Th1 and Th2 cytokines in mouse splenocytes." Journal of microbiology and biotechnology 22(8): 1161-1164 15.Jin, C.-Y., et al (2008) "Induction of apoptosis by aqueous extract of Cordyceps militaris through activation of caspases and inactivation of Akt in human breast cancer MDA-MB-231 Cells." Journal of microbiology and biotechnology 18(12): 1997-2003 16.Jin, L.Y., Du, S.T & Ma, L 2009 Optimization on mathematical model of basic medium of Cordyceps militaris cultivation J Northwest A F Univ (Nat Sci Ed) 37(11):175–179 17.Jin J., Zhong C., Qin Y., Cai Y., Zhen L et al (2017) "A new cordycepinproducing caterpillar fungus Ophiocordyceps xuefengensis with artificial infection to the host, cultivation of mycelia and stromata" FEMS Microbiol Lett 364(20): 1–8 18 Jin Y., Meng X., Qiu Z., Su Y., Yu P., Qu P (2018) Anti-tumor and antimetastatic roles of cordycepin, one bioactive compound of Cordyceps militaris Saudi J Biol Sci 25(5): 991–995 19.Jo, W S., et al (2010) "The anti-inflammatory effects of water extract from Cordyceps militaris in murine macrophage." Mycobiology 38(1): 46-51 20.Kamble V.R and Agre D.G., 2012.Reinvestigation of insect parasite fungus Cordyceps militaris from Maharashtra Bionano Frontier 5(2):224-225 21.Kamp, A M., & Bidochka, M J (2002) Conidium production by insect pathogenic fungi on commercially available agars Letters in Applied Microbiology, 35(1), 74-77 22.Kang N., Lee H.H., Park I., Seo Y.S (2017) Development of high cordycepin-producing 23.Kim, Ho Gyoung, et al (2010) "Cordyceps pruinosa extracts induce apoptosis of HeLa cells by a caspase dependent pathway." Journal of ethnopharmacology 128(2): 342-351 24.Kim S., Shrestha B., Sung G., Han S., Sung J (2010) Optimum conditions for artificial fruiting body formation of Cordyceps cardinalis Mycobiology 38(2): 133–136 25.Kim S.W., Xu C.P., Hwang H.J., Choi J.W., Kim C.W., Yun J.W (2003) Production and characterization of exopolysaccharides from an enthomopathogenic fungus Cordyceps militaris NG3 Biotechnol Prog 19(2): 428–435 26.Kobayasi, Y (1982) Keys to the taxa of the genera Cordyceps and Torrubiella Transactions of the mycological Society of Japan, 23, 329-364 27.Kuo, M.-C., et al (2007) "Immunomodulatory effect of exo-polysaccharides from submerged cultured Cordyceps sinensis: enhancement of cytokine 30 synthesis, CD11b expression, and phagocytosis." Applied microbiology and biotechnology 75(4): 769-775 28.Lan, L (2002) "Study on effect of Cordyceps sinensis and artemisinin in preventing recurrence of lupus nephritis." Chinese Journal of Integrated Traditional and Western Medicine 8(2): 89-89 29.Lee, E J., et al (2010) "Cordycepin suppresses TNF‐alpha‐induced invasion, migration and matrix metalloproteinase‐9 expression in human bladder cancer cells." Phytotherapy Research 24(12): 1755-1761 30.Lee H.H., Kang N., Park I., Park J., Kim I et al (2017) Characterization of newly bred Cordyceps militaris strains for higher production of cordycepin through HPLC and URPPCR analysis J Microbiol Biotechnol 27(7): 1223–1232 31.Lee, S.-J., et al (2009) "Cordycepin causes p21WAF1-mediated G2/M cellcycle arrest by regulating c-Jun N-terminal kinase activation in human bladder cancer cells." Archives of biochemistry and biophysics 490(2): 103109 32.Li, S., et al (2011) "RP-HPLC determination of ergosterol in natural and cultured Cordyceps." Chin J Mod Appl Pharm 18: 297-299 33.Li, T and W Li (2009) "Impact of polysaccharides from Cordyceps on antifatigue in mice." Scientific Research and Essays 4(7): 705-709 34.Li, Y., et al (1993) "Effect of Cordyceps sinensis on erythropoiesis in mouse bone marrow." Chinese Medical Journal 106(04): 313-316 35.Lim, L.T., Lee, C.Y & Chang, E.T., 2012 Optimization of solid state culture conditions for the production of adenosine, cordycepin, and D-mannitol in fruiting bodies of medicinal caterpillar fungus Cordyceps militaris (L.: Fr.) Link (Ascomycetes), Int J Med Mushrooms, 2: 181-187 36.Lin, W.-H., et al (2007) "Improvement of sperm production in subfertile boars by Cordyceps militaris supplement." The American journal of Chinese medicine 35(04): 631-641 37.L.T Nguyen, V.V Le, B.T.T Nguyen et al (2020)."Cultural characteristics and cordycepin production of some Cordyceps militaris strains under artificial cultivation conditions" Journal of Biotechnology, Computational Biology and Bionanotechnology 101(2).135–145 38.MA Kaymakci, E G (2022) "Promising potential pharmaceuticals from the genus cordyceps for COVID-19 treatment: A review study" Bezmialem Science 39.Manabe, N., et al (2000) "Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism and blood flow in dietary hypoferric anaemic mice." British Journal of Nutrition 83(2): 197204 31 40.Nakamura, K., et al (1999) "Inhibitory effect of Cordyceps sinensis on spontaneous liver metastasis of Lewis lung carcinoma and B16 melanoma cells in syngeneic mice." The Japanese Journal of Pharmacology 79(3): 335341 41.Nan, J.-X., et al (2001) "Antifibrotic effect of extracellular biopolymer from submerged mycelial cultures ofCordyceps militaris on liver Fibrosis induced by Bile duct ligation and scission in rats." Archives of Pharmacal Research 24(4): 327-332 42.Ng, T and H Wang (2005) "Pharmacological actions of Cordyceps, a prized folk medicine." Journal of Pharmacy and Pharmacology 57(12): 1509-1519 43.apoptosis in MA-10 mouse Leydig tumor cells." Evidence-Based Complementary and Alternative Medicine 2011 44.Park, B T., et al (2009) "Antifungal and anticancer activities of a protein from the mushroom Cordyceps militaris." The Korean Journal of Physiology & Pharmacology 13(1): 49-54 45.Park J.P., Kim Y.M., Kim S.W., Hwang J., Cho Y.J et al (2002) Effect of aeration rate on the mycelial morphology and exo -biopolymer production in Cordyceps militaris Process Biochem 37(11): 1257–1262 46.Rao, Y K., et al (2010) "Constituents isolated from Cordyceps militaris suppress enhanced inflammatory mediator's production and human cancer cell proliferation." Journal of Ethnopharmacology 131(2): 363-367 47.Reis, F.S., Barros, L., Calhelha, R.C., 2013 The methanolic extract of Cordyceps militaris (L.) Link fruiting body shows antioxidant, antibacterial, antifungal and antihuman tumor cell lines properties Food Chem Toxicol, 62, 91–8 48.Phạm Quang Thu, Lê Thị Xuân Nguyễn Mạnh Hà, 2012 Nghiên cứu đặc điểm sinh học hệ sợi nuôi cấy khiết chủng nấm Đông trùng hạ thảo Cordyceps militaris(L.:FrLink) Tạp chí Viện khoa học lâm nghiệp Việt Nam, 5: 28-39 49.Sari, N., Suparmin, A., Kato, T., & Park, E Y (2016) Improved cordycepin production in a liquid surface culture of Cordyceps militaris isolated from wild strain Biotechnology and Bioprocess Engineering, 21(5), 595-600 50.Shonkor Kumar, D A S., FUJIHARA, S., MASUDA, M., & SAKURAI, A (2010) Efficient Production of Anticancer Agent Cordycepin by Repeated Batch Culture of Cordyceps militaris Mutant In Proceedings of the World Congress on Engineering and Computer Science (Vol 2) 51.Shin, S., Lee, S., Kwon, J., Moon, S., Lee, S., Lee, CK, & Kim, K (2009) Cordycepin ngăn chặn biểu gen điều hòa bệnh tiểu đường cách ức chế trình viêm lipopolysaccharide gây đại thực bào Mạng lưới miễn dịch , (3), 98-105 32 52.Shrestha, B., et al (2012) "Fruiting body formation of Cordyceps militaris from multi-ascospore isolates and their single ascospore progeny strains." Mycobiology 40(2): 100-106 53.Shrestha, B., Kim, H.K., Sung, G.H., 2004a Bipolar heterothallism, a principal mating system of Cordyceps militaris in vitro Biotechnol Bioprocess Eng 9:440–446 54.Shrestha, B., Park, Y.J, Han, S.K., 2004b Instability in in vitro fruiting of Cordyceps militaris J Mushroom Sci Prod 2:140–144 55.Shrestha, B., Choi, S.K, Kim, H.K., 2005a Genetic analý sis of pigmentation in Cordyceps militaris Mycobiology 33:125–130 56.Shrestha, B, Han, S.K., Lee, W.H 2005b Distribution and in vitro fruiting of Cordyceps militaris in Korea Mycobiology 33:178–181 57.Sugar, A M and R P McCaffrey (1998) "Antifungal activity of 3′deoxyadenosine (cordycepin)." Antimicrobial agents and chemotherapy 42(6): 1424-1427 58.Sung, Gi-Ho, et al (2011) "Cultural characteristics of Ophiocordyceps heteropoda collected from Korea." Mycobiology 39: 1-6 59.Sung, J.M, Park, Y.J, Lee, J.O 2006a Effect of preservation periods and subcultures on fruiting body formation of Cordyceps militaris in vitro Mycobiology 34:196–199 60.Sung, J.M, Park, Y.J, Lee, J.O 2006b Selection of superior strains of Cordyceps militaris with enhanced fruiting body productivity Mycobiology 34:131–137 61.Thakur, A., et al (2011) "Pro-apoptotic effects of Paecilomyces hepiali, a Cordyceps sinensis extract on human lung adenocarcinoma A549 cells in vitro." Journal of Cancer Research and Therapeutics 7(4): 421 62.Tong, Y.K, Kuang, T., Wu, Y.X, 1997 Comparison of components of Cordyceps mycelium and natural Cordyceps sinensis Shi Pin Yan JiuYu Kai Fa, 18, 40–42 (in Chinese) 63.Trigo, P., et al (1971) "The effects of cordycepin on malaria parasites." Transactions of the Royal Society of Tropical Medicine and Hygiene 65(4): 514-520 64.Trịnh Thị Xuân Lê Tuấn Anh,( 2016) Nghiên cứu môi trường thích hợp cho sản xuất thể nấm dược liệu Cordyceps militaris Tạp chí khoa học trường Đại học Cần Thơ Số chuyên đề: Nông nghiệp (Tập 3): 88-92 65.Turlo J (2014) The biotechnology of higher fungi - current state and perspectives Folia Biol Oecol 10: 49–65 66.Wang, B.S., Lee, Ch.P., Chen, Z.T., 2012b Comparison of the hepatoprotective activity between cultured Cordyceps militaris and natural Cordyceps sinensis J Funct Foods, 4, 489–95 33 67.Wen, T.C., Li, G.R., Kang, J.C., Kang, C., Hyde, K.D., 2014.Optimization of Solid-state Fermentation for Fruiting Body Growth and Cordycepin Production by Cordyceps militari Chiang Mai J Sci, 41(4): 858-872 68.Yang, B.-K., et al (2000) "Production of exo-polymers by submerged mycelial culture of Cordyceps militaris and its hypolipidemic effect." Journal of microbiology and biotechnology 10(6): 784-788 69.Yang, F., et al (2009) "Analysis of sterols and fatty acids in natural and cultured Cordyceps by one-step derivatization followed with gas chromatography–mass spectrometry." Journal of Pharmaceutical and Biomedical analysis 49(5): 1172-1178 70.Yoo, H.-S., et al (2004) "Effects of Cordyceps militaris extract on angiogenesis and tumor growth." Acta Pharmacologica Sinica 25(5): 657665 71.Yun, Y.-H., et al (2003) "Anti-diabetic effects of CCCA, CMESS, and cordycepin from Cordyceps militaris and the immune responses in streptozotocin-induced diabetic mice." Natural Product Sciences 9(4): 291298 72.Zhang, Q., et al (2004) "Induction of HL-60 apoptosis by ethyl acetate extract of Cordyceps sinensis fungal mycelium." Life sciences 75(24): 29112919 73.Zhao, C.Y., Li, H., Zhang, M 2006 Optimization on conditions of artificial cultivation of Cordyceps militaris J Shenyang Agric Univ 37:209–212 74.Zhu, J.-S., et al (1998) "The scientific rediscovery of a precious ancient Chinese herbal regimen: Cordyceps sinensis Part II." The Journal of Alternative and Complementary Medicine 4(4): 429-457 34