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Carbon Source Affects Synthesis, Structures, and Activities of Mycelial Polysaccharides from Medicinal Fungus Inonotus obliquus

  • He, Huihui (State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University) ;
  • Li, Yingying (State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University) ;
  • Fang, Mingyue (State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University) ;
  • Li, Tiantian (State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University) ;
  • Liang, Yunxiang (State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University) ;
  • Mei, Yuxia (State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University)
  • Received : 2021.02.04
  • Accepted : 2021.04.19
  • Published : 2021.06.28

Abstract

The effects of various carbon sources on mycelial growth and polysaccharide synthesis of the medicinal fungus Inonotus obliquus in liquid fermentation were investigated. After 12-d fermentation, mycelial biomass, polysaccharide yield, and polysaccharide content were significantly higher in Glc+Lac group (glucose and lactose used as combined carbon source) than in other groups. Crude polysaccharides (CIOPs) and the derivative neutral polysaccharides (NIOPs) were obtained from mycelia fermented using Glc, fructose (Fru), Lac, or Glc+Lac as carbon source. Molecular weights of four NIOPs (termed as NIOPG, NIOPF, NIOPL, and NIOPGL) were respectively 780.90, 1105.00, 25.32, and 10.28 kDa. Monosaccharide composition analyses revealed that NIOPs were composed of Glc, Man, and Gal at different molar ratios. The NIOPs were classified as α-type heteropolysaccharides with 1→2, 1→3, 1→4, 1→6 linkages in differing proportions. In in vitro cell proliferation assays, viability of RAW264.7 macrophages was more strongly enhanced by NIOPL or NIOPGL than by NIOPG or NIOPF, and proliferation of HeLa or S180 tumor cells was more strongly inhibited by NIOPG or NIOPGL than by NIOPF or NIOPL, indicating that immune-enhancing and anti-tumor activities of NIOPs were substantially affected by carbon source. qRT-PCR analysis revealed that expression levels of phosphoglucose isomerase (PGI) and UDP-Glc 4-epimerase (UGE), two key genes involved in polysaccharide synthesis, varied depending on carbon source. Our findings, taken together, clearly demonstrate that carbon source plays an essential role in determining structure and activities of I. obliquus polysaccharides by regulating expression of key genes in polysaccharide biosynthetic pathway.

Keywords

Acknowledgement

This study was supported by the Natural Science Foundation of Hubei Province, China (Grant No. 2020CFB527), Key Research and Development Program of Hubei Province, China (Grant No. 2020BAB095) and Fundamental Research Funds for the Central Universities of China (Grant No. 2662019PY066). The authors are grateful to Dr. S. Anderson for English editing of the manuscript.

References

  1. Wei ZH, Chen N, Li YJ, Fan QL, Yu T-F, Wang K-X, et al. 2018. Glucose fed-batch integrated dissolved oxygen control strategy enhanced polysaccharide, total triterpenoids and inotodiol production in fermentation of a newly isolated Inonotus obliquus strain. Process Biochem. 66: 1-6. https://doi.org/10.1016/j.procbio.2018.01.006
  2. Wold CW, Kjeldsen C, Corthay A, Rise F, Christensen BE, Duus JO, et al. 2018. Structural characterization of bioactive heteropolysaccharides from the medicinal fungus Inonotus obliquus (Chaga). Carbohydr. Polym. 185: 27-40. https://doi.org/10.1016/j.carbpol.2017.12.041
  3. Lee KH, Kim H, Oh SH, Hwang JH, Yu KW. 2017. Immunomodulating Activity of crude polysaccharide from Inonotus obliquus sclerotia by fractionation including MeOH reflux. Korean J. Food Nutr. 30: 96-104. https://doi.org/10.9799/ksfan.2017.30.1.096
  4. Kim YO, Park HW, Kim JH, Lee JY, Moon SH, Shin CS. 2006. Anti-cancer effect and structural characterization of endopolysaccharide from cultivated mycelia of Inonotus obliquus. Life Sci. 79: 72-80. https://doi.org/10.1016/j.lfs.2005.12.047
  5. Wang C, Li W, Chen Z, Gao X, Yuan G, Pan Y, et al. 2018. Effects of simulated gastrointestinal digestion in vitro on the chemical properties, antioxidant activity, α-amylase and α-glucosidase inhibitory activity of polysaccharides from Inonotus obliquus. Food Res. Int. 103: 280-288. https://doi.org/10.1016/j.foodres.2017.10.058
  6. Zhang CJ, Guo JY, Cheng H, Li L, Liu Y, Shi Y, et al. 2020. Spatial structure and anti-fatigue of polysaccharide from Inonotus obliquus. Int. J. Biol. Macromol. 151: 855-860. https://doi.org/10.1016/j.ijbiomac.2020.02.147
  7. Liu P, Xue J, Tong SS, Dong WX, Wu PP. 2018. Characterization and hypoglycaemic activities of two polysaccharides from Inonotus obliquus. Molecules 23:1948. https://doi.org/10.3390/molecules23081948
  8. Chen HJ, Chen YS, Liu SL, Liou BK, Chen CS. 2020. The influence of submerged fermentation of Inonotus obliquus with control atmosphere treatment on enhancing bioactive ingredient contents. Appl. Biochem. Biotechnol. 191: 412-425. https://doi.org/10.1007/s12010-020-03273-2
  9. Wang MY, Zhao ZZ, Zhou X, Hu JR, Xue J, Liu X, et al. 2019. Simultaneous use of stimulatory agents to enhance the production and hypoglycaemic activity of polysaccharides from Inonotus obliquus by submerged fermentation. Molecules 24: 4400. https://doi.org/10.3390/molecules24234400
  10. Li H, Wu J, Chen Q. 2019. Effect of farnesol, a Kind of quorum sensing molecule on the production of triterpenoids and betulinic acid in the submerged fermentation of Inonotus obliquus. J. Chin. Inst. Food Sci. Technol. 19: 171-176.
  11. Xu X, Quan L, Shen M. 2015. Effect of chemicals on production, composition and antioxidant activity of polysaccharides of Inonotus obliquus. Int. J. Biol. Macromol. 77: 143-150. https://doi.org/10.1016/j.ijbiomac.2015.03.013
  12. Dubois M, Gilles KA, Hamilton JK, F. S. 1956. Colorimetric method for determiation of sugars and related substances. Anal. Chem. . 28: 350-356. https://doi.org/10.1021/ac60111a017
  13. Kruger NJ. 1994. The Bradford Method for Protein Quantitation. In: Walker J.M. (eds) Basic Protein and Peptide Protocols. Methods in Molecular Biology, vol. 32. Humana Press.
  14. Wang T, He H, Liu X, Liu C, Liang Y, Mei Y. 2019. Mycelial polysaccharides of Lentinus edodes (shiitake mushroom) in submerged culture exert immunoenhancing effect on macrophage cells via MAPK pathway. Int. J. Biol. Macromol. 130: 745-754. https://doi.org/10.1016/j.ijbiomac.2019.03.023
  15. Zhang J, Mei Z, Huang X, Ding Y, Liang Y, Mei Y. 2020. Inhibition of Maillard reaction in production of low-molecular-weight chitosan by enzymatic hydrolysis. Carbohydr. Polym. 236: 116059. https://doi.org/10.1016/j.carbpol.2020.116059
  16. Zhang Y, Wang Z, Li D, Zang W, Zhu H, Wu P, et al. 2018. A polysaccharide from Antrodia cinnamomea mycelia exerts antitumor activity through blocking of TOP1/TDP1-mediated DNA repair pathway. Int. J. Biol. Macromol. 120: 1551-1560. https://doi.org/10.1016/j.ijbiomac.2018.09.162
  17. Kong F, Cao M, Sun P, Liu W, Mao Y. 2015. Selection of reference genes for gene expression normalization in Pyropia yezoensis using quantitative real-time PCR. J. Appl. Phycol. 27: 1003-1010. https://doi.org/10.1007/s10811-014-0359-6
  18. Chang CJ, Lin CS, Lu CC, Martel J, Ko YF, Ojcius DM, et al. 2015. Ganoderma lucidum reduces obesity in mice by modulating the composition of the gut microbiota. Nat. Commun. 6: 7489. https://doi.org/10.1038/ncomms8489
  19. Mei YX, Chen HX, Zhang J, Zhang XD, Liang YX. 2013. Protective effect of chitooligosaccharides against cyclophosphamide-induced immunosuppression in mice. Int. J. Biol. Macromol. 62: 330-335. https://doi.org/10.1016/j.ijbiomac.2013.09.038
  20. Tang YJ, Zhu LL, Li DS, Mi ZY, Li HM. 2008. Significance of inoculation density and carbon source on the mycelial growth and Tuber polysaccharides production by submerged fermentation of Chinese truffle Tuber sinense. Process Biochem. 43: 576-586. https://doi.org/10.1016/j.procbio.2008.01.021
  21. West TP. 2011. Effect of carbon source on polysaccharide production by alginate-entrapped Aureobasidium pullulans ATCC 42023 cells. J. Basic Microb. 51: 673-677. https://doi.org/10.1002/jobm.201100048
  22. Ding Z, Jia SR, Han PP, Yuan NN, Tan N. 2013. Effects of carbon sources on growth and extracellular polysaccharide production of Nostoc flagelliforme under heterotrophic high-cell-density fed-batch cultures. J. Appl. Phycol. 25: 1017-1021. https://doi.org/10.1007/s10811-012-9928-8
  23. Wasser SP, Elisashvili VI, Tan K-K. 2003. Effects of carbon and nitrogen sources in the medium on Tremella mesenterica Retz.:Fr. (Heterobasidiomycetes) growth and polysaccharide production. Int. J. Med. Mushrooms 5: 49-56. https://doi.org/10.1615/InterJMedicMush.v5.i1.70
  24. Hwang HJ, Kim SW, Choi JW, Yun JW. 2003. Production and characterization of exopolysaccharides from submerged culture of Phellinus linteus KCTC 6190. Enzyme Microb. Technol. 33: 309-319. https://doi.org/10.1016/S0141-0229(03)00131-5
  25. Troxler LJ, Werren JP, Schaffner TO, Mostacci N, Vermathen P, Vermathen M, et al. 2019. Carbon source regulates polysaccharide capsule biosynthesis in Streptococcus pneumoniae. J. Biol. Chem. 294: 17224-17238. https://doi.org/10.1074/jbc.RA119.010764
  26. Xu L, Lu Y, Cong Y, Zhang P, Han J, Song G, et al. 2019. Polysaccharide produced by Bacillus subtilis using burdock oligofructose as carbon source. Carbohydr. Polym. 206: 811-819. https://doi.org/10.1016/j.carbpol.2018.11.062
  27. Peng L, Li J, Liu Y, Xu ZH, Wu JY, Ding ZY, et al. 2016. Effects of mixed carbon sources on galactose and mannose content of exopolysaccharides and related enzyme activities in Ganoderma lucidum. RSC Adv. 6: 39284-39291. https://doi.org/10.1039/C6RA04798J
  28. Han PP, Yao SY, Guo RJ, Shen SG, Yan RR, Tan ZL, et al. 2017. The relationship between monosaccharide composition of extracellular polysaccharide and activities of related enzymes in Nostoc flagelliforme under different culture conditions. Carbohydr. Polym. 174: 111-119. https://doi.org/10.1016/j.carbpol.2017.05.093
  29. Kim SK, Lee CG, Yun HS. 2003. Heavy metal adsorption characteristics of extracellular polysaccharide produced by Zoogloea ramigera grown on various carbon sources. J. Microbiol. Biotechnol. 13: 745-750.
  30. Wang Q, Wang F, Xu Z, Ding Z. 2017. Bioactive mushroom polysaccharides: a review on monosaccharide composition, biosynthesis and regulation. Molecules 22: 955. https://doi.org/10.3390/molecules22060955
  31. Zhu ZY, Liu XC, Dong FY, Guo MZ, Wang XT, Wang Z, et al. 2016. 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. https://doi.org/10.1007/s00253-015-7235-4
  32. Wei ZH, Liu L, Guo XF, Li YJ, Hou BC, Fan QL, et al. 2016. Sucrose fed-batch strategy enhanced biomass, polysaccharide, and ganoderic acids production in fermentation of Ganoderma lucidum 5.26. Bioprocess Biosyst. Eng. 39: 37-44. https://doi.org/10.1007/s00449-015-1480-x
  33. Peng L, Qiao SK, Xu ZH, Guan F, Ding ZY, Gu ZH, et al. 2015. Effects of culture conditions on monosaccharide composition of Ganoderma lucidum exopolysaccharide and on activities of related enzymes. Carbohydr. Polym. 133: 104-109. https://doi.org/10.1016/j.carbpol.2015.07.014