Skip to main content
SpringerLink
Log in

Influence of fermentation conditions on polysaccharide production and the activities of enzymes involved in the polysaccharide synthesis of Cordyceps militaris

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The influence of different fermentation conditions on intracellular polysaccharide (IPS) production and activities of the phosphoglucomutase (PGM), UDPG-pyrophosphorylase (UGP), phosphoglucose isomerase (PGI), UDPG-dehydrogenase (UGD), and glucokinase (GK) implicated in metabolite synthesis in Cordyceps militaris was evaluated. The highest IPS production (327.57 ± 6.27 mg/100 mL) was obtained when the strain was grown in the optimal medium containing glucose (40 g · L−1), beef extract (10 g · L−1), and CaCO3 (0.5 g · L−1), and the initial pH and temperature were 7 and 25 °C, respectively. The activities of PGM, UGP, and PGI were proved to be influenced by the fermentation conditions. A strong correlation between the activities of these enzymes and the production of IPS was found. The transcription level of the pgm gene (encoding PGM) was 1.049 times and 1.467 times compared to the ugp gene and pgi gene (encoding UGP and PGI), respectively, in the optimal culture medium. This result indicated that PGM might be the highly key enzyme to regulate the biosynthesis of IPS of C. militaris in a liquid-submerged culture. Our study might be helpful for further research on the pathway of polysaccharide biosynthesis aimed to improve the IPS production of C. militaris.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chen C, Luo SS, Li Y, Sun YJ, Zhang CK (2004) Study on antioxidant activity of three Cordyceps sp. by chemiluminescence. Shanghai J Tradit Chin Med 38(7):53–55

    Google Scholar 

  • Choi SB, Park CH, Choi MK, Jun DW, Park S (2004) Improvement of insulin resistance and insulin secretion by water extracts of Cordyceps militaris, Phellinus linteus, and Paecilomyces tenuipes in 90% pancreatectomized rats. Biosci Biotech Bioch 68:2257–2264

    Article  CAS  Google Scholar 

  • Cui JD (2015) Biotechnological production and applications of Cordyceps militaris, a valued traditional Chinese medicine. Crit Rev Biotechnol 35(4):475–484

    Article  PubMed  Google Scholar 

  • Cui JD, Jia SR (2010) Optimization of medium on exopolysaccharides production in submerged culture of Cordyceps militaris [J]. Food Sci Biotechnol 19(6):1567–1571

    Article  CAS  Google Scholar 

  • Cui JD, Yuan LQ (2011) Optimization of culture conditions on mycelial grown in submerged culture of Cordyceps militaris. Int J Food Eng 7:1–11

    Article  Google Scholar 

  • Cui JD, Zhang YN (2012) Evaluation of metal ions and surfactants effect on cell growth and exopolysaccharide production in two-stage submerged culture of Cordyceps militaris. Appl Biochem Biotechnol 168:1394–1404

    Article  CAS  PubMed  Google Scholar 

  • Daran JM, Dallies N, Thines-Sempoux D, Paquet V, Francois J (1995) Genetic and biochemical characterization of the UGP1 gene encoding the UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae. Eur J Biochem 233:520–530

    Article  CAS  PubMed  Google Scholar 

  • Degeest B, Vuyst LD (2000) Correlation of activities of the enzymes a-phosphoglucomutase, UDP-galactose 4-epimerase and UDP-pyrophosphorylase with exopolysaccharide biosynthesis by Streptococcus thermophilus LY03. Appl Environ Microb 66:3519–3527

    Article  CAS  Google Scholar 

  • Delattre C, Vijayalaksmi MA (2009) Monolith enzymatic microreactor at the frontier of glycomic toward a new route for the production of bioactive oligosaccharides. J Mol Catal B Enzym 60(3–4):97–105

    Article  CAS  Google Scholar 

  • Delattre C, Pierre G, Gardarin C, Traikia M, Eiboutachfaiti R, Isogai A, Michaud P (2015) Antioxidant activities of a polyglucuronic acid sodium salt obtained from TEMPO-mediated oxidation of xanthan. Carbohyd Polym 116:34–41

    Article  CAS  Google Scholar 

  • Dong JZ, Lei C, Ai XR, Wang Y (2012) Selenium enrichment on Cordyceps militaris Link and analysis on its main active components. Appl Biochem Biotechnol 166:1215–1224

    Article  CAS  PubMed  Google Scholar 

  • Duan XH, Chi ZM, Wang L, Wang XH (2008) Influence of different sugars on pullulan production and activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase involved in pullulan synthesis in Aureobasidium pullulans Y68. Carbohyd Polym 73:587–593

    Article  CAS  Google Scholar 

  • Dubois M, Gilles KA, Hamilton JK (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–353

    Article  CAS  Google Scholar 

  • Elisavet K, George E, Diomi M, Evangelos T, Dimitris G, Paul C (2011) Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum. Enzyme Microb Tech 48:217–224

    Article  Google Scholar 

  • Fan DD, Wang W, Zhong JJ (2012) Enhancement of cordycepin production in submerged cultures of Cordyceps militaris by addition of ferrous sulfate. Biochem Eng J 60:30–35

    Article  CAS  Google Scholar 

  • Garcia-Rocha M, Roca A, Iglesia NDL, Baba O, Fernandez-Novell JM, Ferrer JC (2001) Intracellular distribution of glycogen synthase and glycogen in primary cultured rat hepatocytes. Biochem J 357:17–24

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grobben GJ, Smith MR, Sikkema J, De Bont JAM (1996) Influence of fructose and glucose on the production of exopolysaccharides and the activities of enzymes involved in the sugar metabolism and the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772. Appl Microbiol Biot 46:279–284

    Article  CAS  Google Scholar 

  • Hou AI, Meng QF, An JS, Zhu K, Feng Y, Teng LR (2008) Isolation and purification of polysaccharides from Cordyceps minlitaris and its inhibition on the proliferation of rat glomerular mesangial cells. Chem Res Chinese U 5(5):584–587

    Article  Google Scholar 

  • Huang L, Li QZ, Chen YY, Wang XF, Zhou XW (2009) Determination and analysis of cordycepin and adenosine in the products of Cordyceps spp. Afr J Microbiol Res 3(12):957–961

    CAS  Google Scholar 

  • Jiang LF, Wu SJ, Kim JM (2011) Effect of different nitrogen sources on activities of UDPG-pyrophosphorylase involved in pullulan synthesis and pullulan production by Aureobasidium pullulans. Carbohyd Polym 86:1085–1088

    Article  CAS  Google Scholar 

  • Kim SW, Hwang HJ, Xu CP, Na YS, Song SK, Yun JW (2002) Influence of nutritional conditions on mycelial growth and exopolysaccharide production in Paecilomyces sinclairii. Lett Appl Microbiol 34:389–393

    Article  CAS  PubMed  Google Scholar 

  • Kim SW, Hwang HJ, Xu CP, Sung JM, Choi JW, Yun JW (2003) Optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by Cordyceps militaris C738. J Appl Microbiol 94:120–126

    Article  PubMed  Google Scholar 

  • Kothari D, Delatter C, Goyal A (2015) Bioactive isomalto-oligosaccharides synthesized from leuconostoc mesenteroides nrrl B-1426 dextransucrase with colon cancer cells inhibiting and functional food additive properties. Int J Food Nutr Sci 4(4):37–46

    Google Scholar 

  • Kwon JS, Lee JS, Shin WC, Lee KE, Hong EK (2009) 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

    Article  CAS  Google Scholar 

  • Lee JS, Hong EK (2011) Immunostimulating activity of the polysaccharides isolated from Cordyceps militaris [J]. Int J Immunopath Ph 11:1226–1233

    CAS  Google Scholar 

  • Levander F, Radstrom P (2001) Requirement for phosphoglucomutase in exopolysaccharide biosynthesis in glucose- and lactose-utilizing Streptococcus thermophilus. Appl Environ Microbiol 67(6):2734–2738

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lin YW, Chiang BH (2008) Anti-tumor activity of the fermentation broth of Cordyceps militaris cultured in the medium of Radix astragali. Process Boichem 43:244–250

    Article  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using realtime quantitative PCR and the 2-△△Ct method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Martínez LI, Piattoni CV, Garay SA, Rodrígues DE, Guerrero SA, Iglesias AA (2011) Redox regulation of UDP-glucose pyrophosphorylase from Entamoeba histolytica. Biochimie 93:260–268

    Article  PubMed  Google Scholar 

  • Mellal M, Jaffrin MY, Ding LH, Delattre C, Michaud P, Courtois J (2008) Separation of oligoglucuronans of low degrees of polymerization by using a high shear rotating disk filtration module. Sep Purif Technol 60(1):22–29

    Article  CAS  Google Scholar 

  • Methacanon P, Madla S, Kirtikara K, Prasitsil M (2005) Structural elucidation of bioactive fungi-derived polymers. Carbohyd Polym 60:199–203

    Article  CAS  Google Scholar 

  • Pan SK, Yao DR, Chen J, Wu SJ (2013) Influence of controlled pH on the activity of UDPG-pyrophosphorylase in Aureobasidium pullulans. Carbohyd Polym 92:629–632

    Article  CAS  Google Scholar 

  • Papagianni M (2004) Fungal morphology and metabolite production in submerged mycelial processes. Biotechnol Adv 22:189–259

    Article  CAS  PubMed  Google Scholar 

  • Park JP, Kim SW, Hwang HJ (2001) Optimization of submerged culture conditions for the mycelia growth and exo-biopolymer production by Cordyceps militaris [J]. Lett Appl Microbiol 33:76–81

    Article  CAS  PubMed  Google Scholar 

  • Park JP, Kim SW, Hwang HJ, Cho YJ, Yun JW (2002) Stimulatory effect of plant oils and fatty acids on the exo-biopolymer production in Cordyceps militaris. Enzyme MicrobTech 31:250–255

    Article  CAS  Google Scholar 

  • Park JP, Kim SW, Hwang HJ, Yun JW (2004) Optimization of submerged culture conditions for the mycelial growth and exobiopolymer. Process Biochem 39:2241–2247

    Article  Google Scholar 

  • Park SE, Kim J, Lee YW (2009) Antitumor activity of water extracts from Cordyceps militaris in NCI-H460 cell xenografted nude mice [J]. J Acupunct Meridian Stud 2:294–300

    Article  PubMed  Google Scholar 

  • Paterson RR (2008) Cordyceps: a traditional Chinese medicine and another fungal therapeutic biofactory? Phytochemistry 69(7):1469–1495

    Article  CAS  PubMed  Google Scholar 

  • Petit C, Grill JP, Maazouzi N, Marczak R (1991) Regulation of polysaccharide formation by Streptococcus thermophilus in batch and fed-batch cultures. Appl Microbiol Biot 36:216–221

    Article  CAS  Google Scholar 

  • Qian N, Stanley GA, Hahn-Hägerdal B, Rådström P (1994) Purification and characterisation of two phosphoglucomutases from Lactococcus lactis subsp. lactis and their regulation in maltose- and glucose-utilizing cells. J Bacteriol 176:5304–5311

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rachmawati R, Kinoshita H, Nihira T (2013) Establishment of transformation system in Cordyceps militaris by using integration vector with benomyl resistance gene. Procedia Environ Sci 17:142–149

    Article  CAS  Google Scholar 

  • Saad N, Delattre C, Urdaci M, Schmitter JM, Bressollier P (2013) An overview of the last advances in probiotic and prebiotic field. LWT-Food Sci Technol 50(1):1–16

    Article  CAS  Google Scholar 

  • Schiller JG, Bowser AM, David SF (1973) Partial purification and properties of UDPG dehydrogenase from Escherichia coli. BBA- Enzymology 293(1):1–10

    CAS  Google Scholar 

  • Shih IL, Tsai KL, Hsieh C (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 

  • Shih I, Chang S, Chen Y (2010) Cultivation of Cordyceps militaris in solid and liquid culture. J Am Diet Assoc 110(9):A51

    Article  Google Scholar 

  • Shingel KI (2004) Current knowledge on biosynthesis, biological activity, and chemical modification of the exopolysaccharide pullulan. Carbohyd Res 339:447–460

    Article  CAS  Google Scholar 

  • Tang YJ, Zhong JJ (2002) Exopolysaccharide biosynthesis and related enzyme activities of the medicinal fungus, Ganoderma lucidum, grown on lactose in a bioreactor. Biotechnol Lett 24:1023–1026

    Article  CAS  Google Scholar 

  • Tavernier ML, Petit E, Delattre C, Courtois B, Courtois J, Strancar A, Michaud P (2008) Production of oligoglucuronans using a monolithic enzymatic microreactor. Carbohyd Res 343(15):2687–2691

    Article  CAS  Google Scholar 

  • Velasco SE, Yebra MJ, Monedero V, Ibarburu I, Dueñas MT, Irastorza A (2007) Influence of the carbohydrate source on β-glucan production and enzyme activities involve d in sugar metabolism in Pediococcus parvulus 2.6. Int J Food Microbiol 115:325–334

    Article  CAS  PubMed  Google Scholar 

  • Won SY, Park EH (2005) Anti-inflammatory and related pharmacological activities of cultured mycelia and fruiting bodies of Cordyceps militaris. J Ethnopharmacol 96:555–561

    Article  PubMed  Google Scholar 

  • Xiao JH, Chen DX, Liu JW, Liu ZL, Wan WH, Fang N, Xiao Y, Qi Y, Liang ZQ (2004) Optimization of submerged culture requirements for the production of mycelial growth and exo-polysaccharide by Cordyceps jiangxiensis JXPJ 0109. J Appl Microbiol 96:1105–1116

    Article  CAS  PubMed  Google Scholar 

  • Xiao JH, Xiao DM, Xiong Q, Liang ZQ, Zhong JJ (2010) Nutritional requirements for the hyperproduction of bioactive exopolysaccharides by submerged fermentation of the edible medicinal fungus Cordyceps taii. Biochem Eng J 49:241–249

    Article  CAS  Google Scholar 

  • Xu CP, Kim SW, Hwang HJ, Yun JW (2002) Application of statistically based experimental designs for the optimization of exopolysaccharide production by Cordyceps militaris NG3. Biotechnol Appl Biochem 36:127–131

    Article  CAS  PubMed  Google Scholar 

  • Yang FC, Liau CB (1998) The influence of environmental conditions on polysaccharide formation by Ganoderma lucidum in submerged cultures. Process Biochem 33(5):547–553

    Article  CAS  Google Scholar 

  • Yang L, Zhang L (2009) Chemical structural and chain conformational characterization of some bioactive polysaccharides isolated from natural sources. Carbohyd Polym 76:349–361

    Article  CAS  Google Scholar 

  • Yang FC, Ke YF, Kuo SS (2000) Effect of fatty acids on the mycelial growth and polysaccharide formation by Ganoderma lucidum in shake flask cultures. Enzyme Microb Tech 27:295–301

    Article  CAS  Google Scholar 

  • Yang S, Jin L, Ren XD, Lu JH, Meng QF (2014) Optimization of fermentation process of Cordyceps militaris and antitumor activities of polysaccharides in vitro. J Food Drug Anal 22(4):468–476

    Article  CAS  Google Scholar 

  • Yu R, Yang W, Song L (2007) Structural characterization and antioxidant activity of a polysaccharide from the fruiting bodies of cultured Cordyceps militaris [J]. Carbohyd Polym 70:430–436

    Article  CAS  Google Scholar 

  • Zhang M, Cui SW, Cheung PCK, Wang Q (2007) Antitumor polysaccharides from mushrooms: a review on their isolation process, structural characteristics and antitumor activity. Trends Food Sci Tech 18:4–19

    Article  Google Scholar 

  • Zheng P, Xia YL, Xiao GH, Xiong CH, Hu X, Zhang SW, Zheng HJ, Huang Y, Zhou Y (2011) Genome sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional Chinese medicine. Genome Biol 12(11):287–302

    Google Scholar 

  • Zhou X, Gong Z, Su Y, Lin J, Tang K (2009) Cordyceps fungi: natural products, pharmacological functions and developmental products. J Pharm Pharmacol 61:279–291

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the National Spark Key Program of China (2015GA610001), the Foundation of Tianjin University of Science and Technology (Nos. 20120106), the International Science and Technology Cooperation Program of China (2013DFA31160), and the Foundation of Tianjin Educational Committee (20090604).

Author information

Authors and Affiliations

  1. Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, People’s Republic of China

    Zhen-Yuan Zhu, Xiao-Cui Liu, Feng-Ying Dong, Ming-Zhu Guo, Xiao-Ting Wang & Zheng Wang

  2. Tianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center, Tianjin, 300457, People’s Republic of China

    Zhen-Yuan Zhu

  3. Institut Parisien de Chimie Moléculaire, Université Pierre et Marie Curie-Paris 6, UMR CNRS 8232, 4 place Jussieu, 75005, Paris, France

    Yong-Min Zhang

Authors
  1. Zhen-Yuan Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiao-Cui Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Feng-Ying Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ming-Zhu Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiao-Ting Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zheng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yong-Min Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zhen-Yuan Zhu.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical statement

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

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhu, ZY., Liu, XC., Dong, FY. et al. Influence of fermentation conditions on polysaccharide production and the activities of enzymes involved in the polysaccharide synthesis of Cordyceps militaris . Appl Microbiol Biotechnol 100, 3909–3921 (2016). https://doi.org/10.1007/s00253-015-7235-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-015-7235-4

Keywords

Search

Navigation