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Optimization of culture conditions and medium components for the production of mycelial biomass and exo-polysaccharides with Cordyceps militaris in liquid culture

Abstract

Both crude exo-biopolymers and mycelial biomass, produced by liquid culture of Cordyceps species, are believed to possess several potential health benefits. As a result of its known biological activities, Cordyceps militaris has been extensively characterized in regards to potential medicinal applications. However, optimized liquid culture conditions for enhanced polysaccharide productivity have yet to be developed, which is a necessary step for industrial applications. Therefore, in this study, the liquid culture conditions were optimized for maximal production of mycelial biomass and exo-polysaccharide (EPS) by C. militaris. The effects of medium composition, environmental factors, and C/N ratio were investigated. Among these variables 80 g, glucose; 10 g, yeast extract; 0.5 g, MgSO4·7H2O; and 0.5 g, KH2PO4 in 1 L distilled water were found to be the most suitable carbon, nitrogen, and mineral sources, respectively. The optimal temperature, initial pH, agitation, and aeration were determined to be 24°C, uncontrolled pH, 200 rpm, and 1.5 vvm, respectively. Under these optimal conditions, mycelial growth in shake flask cultures and 5 L jar bioreactors was 29.43 and 40.60 g/L, respectively, and polysaccharide production in shake flask cultures and 5 L jar bioreactors was 2.53 and 6.74 g/L, respectively.

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References

  1. Dalmo, R. A. and J. Boqwald (2008) Beta-glucans as conductors of immune symphonies. Fish Shellfish Immunol. 25: 384–396.

    Article  CAS  Google Scholar 

  2. Kiho, T., A. Yamane, J. Hui, S. Usui, and S. Ukai (1996) Polysaccharides in fungi. XXXVI. Hypoglycemic activity of polysaccharide (CF-F30) from the cultural mycelium of Cordyceps sinensis and its effect on glucose metabolism in mouse liver. Biol. Pharm. Bull 19: 294–296.

    CAS  Google Scholar 

  3. Yang, L. Y., A. Chen, Y. C. Kuo, and C. Y. Lin (1999) Efficacy of a pure compound H1-A extracted from Cordyceps sinensis on autoimmune disease of MRL lpr/lpr mice. J. Lab. Clin. Med. 134: 492–500.

    Article  CAS  Google Scholar 

  4. Park, C., S. H. Hong, J. Y. Lee, G. Y. Kim, B. T. Choi, Y. T. Lee, D. I. Park, Y. M. Park, Y. K. Jeong, and Y. H. Choi (2005) Growth inhibition of U937 leukemia cells by aqueous extract of Cordyceps militaris through induction of apoptosis. Oncol. Rep. 13: 1211–1216.

    Google Scholar 

  5. Nakamura, K., Y. Yamaguchi, S. Kagota, Y. M. Kwon, K. Shinozuka, and M. Kunitomo (1999) Inhibitory effects of Cordyceps sinensis on spontaneuous liver metastasis of Lewis lung carcinoma and B16 melanoma cells on syngeneic mice. Jpn. J. Pharmacol. 79: 335–341.

    Article  CAS  Google Scholar 

  6. Yang, B. K., J. Y. Ha, S. C. Jeong, S. Das, J. W. Yun, Y. S. Lee, J. W. Choi, and C. H. Song (2000) Production of exo-polymers by submerged mycelial culture of Cordyceps militaris and its hypolipidemic effect. J. Microbiol. Biotechnol. 10: 784–788.

    CAS  Google Scholar 

  7. Yu, R., L. Song, Y. Zhao, W. Bin, L. Wang, H. Zhang, Y. Wu, Y. Ye, and X. Yao (2004) Isolation and biological properties of polysaccharide CPS-1 from cultured Cordyceps militaris. Fitoterapia 75: 465–472.

    Article  CAS  Google Scholar 

  8. Yu, R., W. Yang, L. Song, C. Yan, Z. Zhang, and Y. Zhao (2007) Structural characterization and antioxidant activity of a polysaccharide from the fruiting bodies of cultured Cordyceps militaris. Carbohydr. Polymers 70: 430–436.

    Article  CAS  Google Scholar 

  9. Ohta, A. (1990) A new medium for mycelial growth of mycorrhizal fungi. Trans. Mycol. Soc. Jpn. 31: 323–334.

    CAS  Google Scholar 

  10. Jeong, G. T., J. C. Woo, and D. H. Park (2007) Effect of plant growth regulators on growth and biosynthesis of phenolic compounds in genetically transformed hairy roots of Panax ginseng C. A. Meyer. Biotechnol. Bioprocess Eng. 12: 86–91.

    Article  CAS  Google Scholar 

  11. Shih, I. L., K. L. Tsai, and C. Hsieh (2007) Effects of culture conditions on the mycelial growth and bioactive metabolite production in submerged culture of Cordyceps militaris. Biochem. Eng. J. 33: 193–201.

    Article  CAS  Google Scholar 

  12. Mao, X. B., T. Eksriwong, S. Chauvatcharin, and J. J. Zhong (2005) Optimization of carbon source and carbon/nitrogen ratio for cordycepin production by submerged cultivation of medicinal mushroom Cordyceps militaris. Process Biochem. 40: 1667–1672.

    Article  CAS  Google Scholar 

  13. Tang, Y. J. and J. J. Zhong (2002) Fed-batch fermentation of Ganoderma lucidium for hyperproduction of polysaccharide and ganoderic acids. Enzym. Microbial. Technol. 31: 20–28.

    Article  CAS  Google Scholar 

  14. Min, B. J., Y. S. Park, S. W. Kang, Y. S. Song, J. H. Lee, C. W. Park, C. W. Kim, and S. W. Kim (2007) Statistical optimization of medium components for the production of xylanase by Aspergillus niger KK2 in submerged cultivation. Biotechnol. Bioprocess Eng. 3: 302–307.

    Article  Google Scholar 

  15. Shin, H. J., C. J. Kim, and S. B. Kim (2007) Optimization of culture medium for rifamycin SV production by Amycolatopsis mediterranei MM2 using statistical designs. Biotechnol. Bioprocess Eng. 4: 457–461.

    Article  Google Scholar 

  16. Choi, J. H., T. M. Ha, Y. H. Kim, and Y. D. Rho (1996) Studies on the main factors affecting the mycelial growth of Phellinus linteus. Kor. J. Mycol. 24: 214–222.

    Google Scholar 

  17. Choi, J. H., U. T. Lee, S. Y. Kim, D. K. Oh, and J. H. Kim (1998) Optimization of culture conditions for production of a high viscosity polysaccharide, methylan, by Methylobacterium organophilum from methanol. Kor. J. Appl. Microbiol. Biotechnol. 26: 244–249.

    CAS  Google Scholar 

  18. Park, K. S. and J. S. Lee (1991) Optimization of media composition and culture conditions for the mycelial growth of Coriolus versicolor and Lentinus edodes. Kor. J. Biotechnol. Bioeng. 6: 91–98.

    Google Scholar 

  19. Catley, B. J. (1971) Utilization of carbon sources by Pullularia pullulans for the elaboration of extracellular polysaccharides. Appl. Environ. Microbiol. 22: 641–649.

    CAS  Google Scholar 

  20. Catley, B. J. (1971) Role of pH and nitrogen limitation in the elaboration of the extracellular polysaccharide pullulan by Pullularia pullulans. Appl. Environ. Microbiol. 22: 650–654.

    CAS  Google Scholar 

  21. Heald, P. J. and B. Kristiansen (1985) Synthesis of polysaccharide by yeast-like forms of Aureobasidium pullulans. Biotechnol. Bioeng. 27: 1516–1519.

    Article  CAS  Google Scholar 

  22. Salleh, M. M., L. S. Tsuey, and A. B. Ariff (2008) The profile of enzymes relevant to solvent production during direct fermentation of sago starch by Clostridium saccharobutylicum P262 utilizing different pH control strategies. Biotechnol. Bioprocess Eng. 1: 33–39.

    Article  CAS  Google Scholar 

  23. Cho, E. J., J. Y. Oh, H. Y. Chang, and J. W. Yun (2006) Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis. J. Biotechnol. 127: 129–140.

    Article  CAS  Google Scholar 

  24. Chen, W., Z. Zhao, S. F. Chen, and Y. Q. Li (2008) Optimization for the production of exopolysacchsaride from Fomes fomentarius in submerged culture and its antitumor effect in vitro. Bioresour. Technol. 99: 3187–3194.

    Article  CAS  Google Scholar 

  25. Pokhrel, C. P. and S. Ohga (2007) Submerged culture conditions for mycelial yield and polysaccharide production by Lyophyllum decastes. Food Chem. 105: 641–646.

    Article  CAS  Google Scholar 

  26. Kim H. K., J. C. Cheong, H. Y. Chang, G. P. Kim, D. Y. Cha, and B. J. Moon (1997) The artificial cultivation of Pleurotus eryngii (I). Investigation of mycelial growth conditions. Kor. J. Mycol. 25: 305–310.

    Google Scholar 

  27. Hashimoto, K. and Z. Takahshi (1974) Studies on the growth of Pleurotus ostreatus. Mush. Sci. 9: 585–593.

    Google Scholar 

  28. Hong, J. S. and K. H. Kang (1983) Fruit-body formation of Pleurotus florida on the synthetic medium. Kor. J. Mycol. 11: 121–128.

    CAS  Google Scholar 

  29. Kim, M. K., I. Y. Lee, J. H. Ko, Y. H. Rhee, and Y. H. Park (1999) Higher intracellular levels of uridinemonophosphate under nitrogen-limited conditions enhance metabolic flux of curdlan synthesis in Agrobacterium species. Biotechnol. Bioeng. 62: 317–323.

    Article  CAS  Google Scholar 

  30. Reeslev, M. and B. Jensen (1995) Influence of Zn2+ and Fe3+ on polysaccharide production and mycelium/yeast dimorphism of Aureobasidium pullulans in batch cultivation. Appl. Microbiol. Biotechnol. 42: 910–915.

    Article  CAS  Google Scholar 

  31. Pilz, F., G. Auling, D. Stephan, U. Rau, and F. Wagner (1991) A high affinity Zn2+ uptake system controls growth and biosynthesis of an extracellular, branched β-1,3-β-1,6-glucan in Sclerotium rolfsii ATCC 15205. Exp. Mycol. 15: 181–192

    Article  CAS  Google Scholar 

  32. Tang, Y. J., L. L. Zhu, R. S. Liu, H. M. Li, D. S. Li, and Z. Y. Mi (2008) Quantitative response of cell growth and Tuber polysaccharides biosynthesis by medicinal mushroom Chinese truffle Tuber sinense to metal ion in culture medium. Bioresour. Technol. 99: 7606–7615.

    Article  CAS  Google Scholar 

  33. Dreventon, E., M. Frederic, L. Jacqueline, B. Daniel, and C. Lionen (1994) Effect of mixing and mass transfer conditions on gellan production by Aureomonas elodea. J. Ferment. Bioeng. 77: 642–649.

    Article  Google Scholar 

  34. Herbst, H., A. Shumpe, and W. D. Deckwer (1992) Xanthan production in stirred tank fermenters: oxygen transfer and scale-up. Chem. Eng. Technol. 15: 425–434.

    Article  CAS  Google Scholar 

  35. Lee, J. Y., S. W. Kang, and S. W. Kim (2008) Relationship between agitation speed and the morphological characteristics of Verticillium lecanii CS-625 during spore production. Biotechnol. Bioprocess Eng. 13: 1–6.

    Article  CAS  Google Scholar 

  36. Park, J. P., Y. M. Kim, S. W. Kim, H. J. Hwang, Y. J. Cho, Y. S. Lee, C. H. Song, and J. W. Yun (2002) Effect of agitation intensity on the exo-biopolymer production and mycelial morphology in Cordyceps militaris. Appl. Microbiol. Biotechnol. 36: 465–468.

    Google Scholar 

  37. Stasinopoulos, S. J. and Seviour, R. J. (1992) Exopolysaccharide production by Acremonium persicinum in stirred-tank and air-lift fermentors. Appl. Microbiol. Biotechnol. 36: 465–468.

    Article  CAS  Google Scholar 

  38. Rau, U., E. Gura, E. Olszewski, and F. Wagner (1992) Enhanced glucan formation of filamentous fungi by effective mixing, oxygen limitation, and fed-batch processing. J. Indus. Microbiol. 9: 19–26.

    Article  CAS  Google Scholar 

  39. Cui, J. and Y. Chisti (2003) Polysaccharopeptides of Coriolus versicolor physiological activity, uses, and production. Biotechnol. Adv. 21: 109–122.

    Article  CAS  Google Scholar 

  40. Lawford, H. and J. Rousseau (1989) Effects of oxygen on the rate of β-1,3-glucan microbial exopolysaccharide production. Biotechnol. Lett. 11: 125–130.

    Article  CAS  Google Scholar 

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Correspondence to Eock Kee Hong.

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Kwon, J.S., Lee, J.S., Shin, W.C. et al. Optimization of culture conditions and medium components for the production of mycelial biomass and exo-polysaccharides with Cordyceps militaris in liquid culture. Biotechnol Bioproc E 14, 756–762 (2009). https://doi.org/10.1007/s12257-009-0024-0

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  • DOI: https://doi.org/10.1007/s12257-009-0024-0

Keywords

  • Cordyceps militaris
  • exo-polysaccharide
  • mycelial growth
  • carbon-to-nitrogen ratio