Optimization of the fermentation process of Cordyceps sobolifera Se-CEPS and its anti-tumor activity in vivo
© The Author(s). 2016
Received: 17 May 2016
Accepted: 14 June 2016
Published: 23 June 2016
Cordyceps sobolifera (C. sobolifera) isolated from cicadae was used as the starting fungus to produce selenium-enriched C. sobolifera extracellular polysaccharide (Se-CEPS). An orthogonal experimental design based on a single-factor experiment was used to optimize the C. sobolifera fermentation conditions, including the potato juice, peptone, and KH2PO4 concentrations. Ultraviolet (UV) and infrared (IR) analyses of CEPS and Se-CEPS were conducted, as well as an in vivo anti-tumor analysis.
Under optimal conditions (i.e., 40 potato juice, 0.4 KH2PO4, and 0.5 % peptone), the fermentation yield of Se-CEPS was 5.64 g/L. UV and IR spectra showed that Se-CEPS contained a characteristic absorption peak of a selenite Se = O double bond, demonstrating the successful preparation of Se-CEPS. Activity tests showed that Se-CEPS improved the immune organ index, serum cytokine content, and CD8+ and CD4+ T lymphocyte ratio in colon cancer CT26 tumor-bearing mice, thereby inhibiting tumor growth. When combined with 5-FU, Se-CEPS reduced the toxicity and enhanced the function of 5-FU.
The result of these experiments indicated that orthogonal experimental design is a promising method for the optimization of Se-CEPS production, and the Se-CEPS from C. sobolifera can improve the anti-tumor capacity of mice.
KeywordsCordyceps sobolifera Extracellular polysaccharide Anti-tumor Fermentation
Cordyceps sobolifera is a rare and unique medicinal fungus that exhibits characteristics of both animals and plants. C. sobolifera, a well-known and valued traditional Chinese medicine, is an entomogenous fungal species that is parasitic on wing-less cicada nymphs . Modern medical research and applications have shown that C. sobolifera exhibits various functions such as enhancing immunity [2, 3], having anti-aging and anti-fatigue activities , having anti-tumor activity , improving renal function [5–8], and providing nourishment and strength . C. sobolifera and Cordyceps sinensis belong to the same insect fungi complex and contain a similar active ingredient; C. sinensis has been widely harvested, and the natural supplies have been markedly depleted. Therefore, C. sobolifera is often regarded as a substitute for C. sinensis. The growth of C. sobolifera requires a specific ecological environment and host insects. Moreover, the harvesting of C. sobolifera has also become extensive, leading to a steady decline of available sources. Therefore, the use of artificially cultivated C. sobolifera mycelium to replace natural C. sobolifera has emerged as a future option for its development. In recent years, there have been extensive investigations and reports on C. sinensis, but reports on C. sobolifera are rare. Because C. sobolifera and C. sinensis have similar chemical compositions, the medicinal value of the two species is similar, providing the theoretical basis for the substitution of C. sinensis with C. sobolifera [9–11].
Selenium is an essential trace element that is necessary for maintaining the normal physiological metabolism of the human body . Most diseases of the human body, such as anemia, coronary heart disease, diabetes, and cancer, are related to a lack of selenium [13, 14]. Research has shown that organic selenium is more effective and safer than inorganic selenium as a dietary supplement  and that the biological activity of selenium polysaccharide is markedly higher than that of selenium or polysaccharide alone . Selenium polysaccharide is an organic selenium compound composed of selenium and biological polysaccharide, and it exhibits numerous biological effects, such as antioxidation, anti-tumor, immunity enhancement, and blood lipid reduction activities [17, 18].
Results and discussion
Orthogonal test for optimization of C. sobolifera extracellular polysaccharide (Se-CEPS) fermentation conditions
Factors and levels of fermentation condition
Potato juice (%) A
Peptone (%) B
KH2PO4 (%) C
Results and analysis of orthogonal experiment design in submerged fermentation conditions of C. sobolifera
3.48 ± 0.27
5.22 ± 0.39
4.35 ± 0.41
5.17 ± 0.38
5.61 ± 0.40
4.73 ± 0.26
3.64 ± 0.22
4.97 ± 0.29
4.23 ± 0.21
Analysis of variance
Potato juice (A)
UV spectra of sodium selenite and polysaccharides
The infrared spectra of CEPS and Se-CEPS
Effects of Se-CEPS on tumor growth, immune organ index, and body weight in tumor-bearing mice
Effects of Se-CEPS on the body weight, tumor growth in CT26-bearing mice (n = 8, *p < 0.05 vs model group)
20.34 ± 2.13
1.34 ± 0.31
16.21 ± 1.86*
0.47 ± 0.16*
20.27 ± 0.98
1.27 ± 0.28
20.04 ± 2.01
1.03 ± 0.19
20.13 ± 1.46
0.91 ± 0.33
20.72 ± 2.14
1.06 ± 0.25
20.41 ± 0.79
0.82 ± 0.14
20.42 ± 1.68
0.64 ± 0.09*
Se-CEPS + 5-FU group
200(Se-CEPS) + 20(5-FU)
18.15 ± 2.17*
0.36 ± 0.17*
Effects of Se-CEPS on immune organ index in CT26-bearing mice (n = 8, *p < 0.05 vs model group, #p < 0.05 vs 5-FU group)
Spleen index (mg/g)
Thymus index (mg/g)
9.18 ± 0.74
4.12 ± 0.29
5.34 ± 0.52
2.51 ± 0.27
9.22 ± 0.49
4.52 ± 0.44
9.28 ± 0.98
4.78 ± 0.31
9.45 ± 0.72
5.11 ± 0.37
9.41 ± 0.63
5.14 ± 0.42
10.18 ± 0.47
5.58 ± 0.28*
10.92 ± 0.32*
5.97 ± 0.31*
Se-CEPS + 5-FU group
200(Se-CEPS) + 20(5-FU)
8.04 ± 0.49*#
3.73 ± 0.25*#
Effect of Se-CEPS on the content of cytokines in the serum of tumor-bearing mice
Effects of polysaccharides on CD8+ and CD4+ T cell counts in the spleen of tumor-bearing mice
Effects of Se-CEPS on the percentage of CD8+ and CD4+ T cells among total T lymphocytes in CT26-bearing mice (n = 8, *p < 0.05 vs model group, #p < 0.05 vs 5-FU group)
34.59 ± 3.07
20.64 ± 1.43
1.68 ± 0.14
22.86 ± 1.35*
17.37 ± 1.09
1.32 ± 0.31*
34.64 ± 2.89
20.67 ± 0.84
1.68 ± 0.17
35.22 ± 2.42
20.89 ± 1.78
1.69 ± 0.20
35.73 ± 3.77*
21.03 ± 1.24*
1.70 ± 0.34*
35.04 ± 3.13
20.81 ± 1.17
1.68 ± 0.27
37.38 ± 2.962*
21.39 ± 0.92*
1.75 ± 0.21*
43.46 ± 4.83*
21.76 ± 1.58*
2.00 ± 0.28*
Se-CEPS + 5-FU group
200(Se-CEPS) + 20(5-FU)
31.25 ± 2.71*#
19.23 ± 1.30*#
1.63 ± 0.19*#
In the tumor-bearing mouse model, Se-CEPS intragastric administration significantly improved the immune organ index values and TNF-α and IL-2 levels in the serum, increased the proportion of cytotoxic T lymphocytes, and inhibited the growth of the transplanted tumors. In addition, Se-CEPS enhanced the anti-tumor activity of 5-FU and reduced the damage to the immune organs of the mice. 5-FU is an important chemotherapy drug for cancer treatment; however, the drug can cause severe bone marrow suppression, infection, hair loss, vomiting, and other serious side effects, and it can even endanger the patient’s life. The combination of polysaccharide and chemotherapy can improve the antitumor activity of chemotherapeutic drugs and reduce their side effects .
The body’s immune function, including the two aspects of cellular immunity and humoral immunity, is closely related to the occurrence and development of tumors. In particular, cell immunity has a primary role in tumor clearance . Tumors are known to induce the production of inhibitory T cells and inhibitory macrophages, which can inhibit the production of IL-2 lymphocytes and promote tumor growth [23, 24]. To a certain extent, IL-2 activity reflects the body’s immune monitoring function, and its main biological activity involves promoting the proliferation of T lymphocytes and NK cells, differentiation and proliferation of B cells, and production of antibodies, among other functions . Therefore, IL-2 has an important role in anti-tumor immunity . IL-2 is an important immune regulatory protein that positively promotes a variety of immune cell activities . For example, IL-2 can induce the differentiation of cytotoxic T lymphocytes and lymphokine-activated killer cells , which are both crucial in killing tumor cells . The cytokine TNF-α has a variety of biological activities . This cytokine can directly kill tumor cells, induce the apoptosis of tumor cells, and participate in the resistance to infection by bacteria, viruses, and parasites. Moreover, TNF-α can induce cell differentiation and promote mononuclear cells or T cells to secrete a variety of cytokines . Mature T lymphocytes can be divided into two subsets: CD4+ and CD8+ [32, 33]. CD4+ cells are T helper cells that aid in secreting numerous cytokines and enhance the killing effect of CD8+ cells in tumors . CD8+ cells are cytotoxic and suppressor T cells and act as important effector cells . The lack of CD8+ and CD4+ T lymphocytes can lead to a weak immune response against tumors . In general, our results demonstrate that Se-CEPS not only inhibits the formation of CT26 colon carcinoma but also improves the levels of IL-2 and TNF-α in the serum of mice. The results show that the effects of Se-CEPS on tumor inhibition and cell immune function and the secretion of IL-2 and TNF-α in the CT26 colon cancer mouse model are relevant. The level of CD8+ in the spleen of tumor-bearing mice was significantly increased by Se-CEPS treatment, indicating that Se-CEPS promotes the proliferation and activation of CD8+ T lymphocytes, directly kills tumor cells, and exerts an anti-tumor effect. Radiotherapy and chemotherapy are the most commonly used treatments for tumors, but they cause the inhibition of immune function and other serious side effects. The ability of Se-CEPS to enhance immune activity indicates its considerable potential for use in the treatment of tumors in the future.
The results demonstrate the optimal fermentation conditions for the production of extracellular selenylated polysaccharide from C. sobolifera mycelium. The ultraviolet and infrared spectral analyses showed that selenium was successfully enriched in the extracellular polysaccharide. Moreover, activity tests showed that Se-CEPS improved the immune organ index of CT26 tumor-bearing mice and increased the TNF-α and IL-2 levels in serum. In addition, Se-CEPS improved the proportions of CD8+ and CD4+ T lymphocytes in the spleen, thereby inhibiting tumor growth. When combined with 5-FU, Se-CEPS improved the antitumor activity and reduced the side effects of the drug.
C. sobolifera was collected and isolated from the Zhejiang Anji Bamboo Garden. CT26 colon carcinoma (CT26) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The 5-fluorouracil (5-FU) were purchased from Sigma (USA). Mouse ELISA kits for Interleukin 2 (IL-2) and tumor necrosis factor-α (TNF-α) were supplied by 4A Biotech Co. Ltd. (China). Fluorescein isothiocyanate (FITC)-conjugated anti-Mouse CD4 and phycoerythrin (PE)-conjugated anti-Mouse CD8 monoclonal antibodies were provided by eBioscience (USA). All other chemicals and reagents used were of analytical grade.
The seed medium contained the following: potato extract, 20 %; glucose, 4 %; peptone, 0.2 %; yeast extract, 0.2 %; KH2PO4, 0.1 %; and MgSO4, 0.05 %. It was sterilized at 121 °C for 20 min.
The submerged fermentation medium contained the following: potato extract, 20 %; glucose, 4 %; peptone, 0.4 %; MgSO4, 0.05 %; KH2PO4, 0.1 %; yeast extract, 0.2 %; and vitamin B1, 0.002 %. It was sterilized at 121 °C for 20 min.
Preparation and culture of the seed
A small activation inoculum was inoculated into a 250 mL Erlenmeyer flask. The volume was 100 mL, and it was shaken and cultured for at 26 °C for 3 d at 160 rpm. An 8 % (v/w) inoculum was transferred to 50 mL of sterilized fermentation medium in a 250 mL flask and then cultured for 120 h under the same conditions. The content of extracellular polysaccharide was measured in the fermentation broth after centrifugation at 4000 rpm for 15 min. All treatments were conducted three times.
Extraction of extracellular polysaccharides
After centrifugation of the fermentation broth at 4000 rpm for 15 min, the supernatant was obtained. The supernatant was mixed with three volumes of 95 % ethanol (v/v), stirred vigorously, and left overnight at 4 °C. The precipitated polysaccharides were centrifuged at 8,000 × g for 15 min, and the supernatant was discarded. The polysaccharide precipitates were washed three times with 70 % ethanol and lyophilized to a constant weight in vacuo.
Optimization of the fermentation medium
Potato juice (20–60 %), peptone (0.2–1.0 %), and KH2PO4 (0.2–0.6 %) were examined using a three-factor and three-level L9 (33) orthogonal array design. In addition, analysis of variance (ANOVA) was used to evaluate the statistical significance of the effects of the individual factors on Se-CEPS production. The yield of Se-CEPS was further evaluated to confirm the production rate of target compounds based on the optimized conditions.
Construction of the tumor bearing mouse model
After CT26 cells were grown to an 80 to 90 % fusion rate, the cells were digested in trypsin and collected. The cell concentration was adjusted to 5 × 106/mL using phosphate-buffered saline (PBS). Seventy-two BALB/c mice were used in the study. The healthy mice were purchased from the Laboratory Animal Center of Zhejiang University (Institutional Animal Welfare and Ethics Committee of Zhejiang University, China). This study was approved by the Animal Ethics Committee of Zhejiang University. A 75 % alcohol solution was used to disinfect the armpit of the right arm of the mice, and a 0.2 mL CT26 cell suspension was then inoculated subcutaneously. On the seventh day after the inoculation of tumor cells and after a 80–90 mm3 tumor had formed at the inoculation site, the mice were randomly divided into nine groups: the control group (saline group), Se-CEPS or CEPS high dose group (200 mg/kg/d), Se-CEPS or CEPS middle dose group (100 mg/kg/d), Se-CEPS or CEPS low dose group (50 mg/kg/d), Se-CEPS + 5-FU group (200 mg/kg/d Se-CEPS + 20 mg/kg/d 5-FU), and positive control group (5-FU: 20 mg/kg/d). The intragastric administration of the drug was performed once a day for 14 d.
Measurement of polysaccharides in the fermentation broth
The extracellular polysaccharides were measured using the phenol sulfuric acid method .
Measurement of selenium content
Selenium was measured in selenium-enriched polysaccharides using a PerkinElmer AAanalvst800 atomic absorption spectrophotometer.
Ultraviolet spectrum analysis of Se-CEPS and CEPS
Ten percent Se-CEPS and CEPS solutions were prepared using ultrapure water as a blank control, and ultraviolet scanning analysis was conducted in the range of 200–400 nm.
Infrared spectrum analysis of Se-CEPS and CEPS
The infrared (IR) spectra were recorded using the KBr-disc method with a Fourier transform infrared (FTIR) spectrometer (Tensor27 Fourier transform infrared spectrometer; Bruker, Germany) in the range of 400–4,000 cm−1.
Measurement of the tumor inhibition rate, spleen index, and thymus index
Determination of serum cytokine levels by the ELISA assay
The levels of IL-2 and TNF-α in the serum of the mice from each group were determined using commercial mouse ELISA kits according to the manufacture’s protocols.
CD8+ T and CD4+ T lymphocyte analysis
Mice were killed by cervical dislocation, and their spleens were removed. One milliliter PBS was added to each spleen, and the spleen was teased apart into a single cell suspension by passing it through a 3 mL syringe. The suspension was subjected to red blood cell lysis and 1000 rpm centrifugation for 10 min and then washed with sterile PBS once. The suspension was combined with 1.5 μL FITC-CD3, 3.75 μL anti-CD8, or anti-CD4 and then incubated in the dark at 4 °C for 30 min. A 0.05 % sodium azide PBS solution was used to wash away the unbound antibody, and the cells were resuspended in 200 μL 2.0 fetal bovine serum and 0.05 % sodium azide PBS buffer. BD FACSVerse flow cytometry was performed, and the data were obtained and analyzed using FACSuite software.
All data are expressed as the mean ± standard error. SPSS17.0 statistical software (SPSS Inc. 233 South Wacker Drive, 11th Floor, Chicago) was used for single factor analysis of variance (one-way ANOVA). A p value < 0.05 indicates a statistically significant difference.
5-FU, 5-fluoro-2,4(1 h, 3 h)pyrimidinedione; ANOVA, analysis of variance; C. sobolifera, extracellular polysaccharide; C. sobolifera, Cordyceps sobolifera; CD4+, Cluster of Differentiation 4; CD8+, Cluster of Differentiation 8; CT26, Colonic adenocarcinoma cell; ELISA, Enzyme-linked immuno sorbent assay; IL-2, Interleukin-2; IR, infrared; CEPS, C. sobolifera extracellular polysaccharide; PBS, phosphate-buffered saline; Se-CEPS, selenium-enriched; TNF-α, Tumor Necrosis Factor; UV, Ultraviolet
The authors would like to acknowledge science and technology department of Zhejiang province. Great appreciation is given to all members of our laboratory for their enthusiastic participation in the research.
This work was supported by a key project funded by science and technology department of Zhejiang province (No. 2013C02028). The funding supported all of the research including the design, experiments, data collection and analysis, and writing of the manuscript.
Availability of data and materials
Authors do not wish to share their data now because these data has not still been published and we are going to write a new paper by refining new results based upon these data.
SY conceived of the study and its design, carried out the fermentation studies, participated in the anti-tutor activity research and drafted the manuscript. HZ carried out the immunoassays, participated in the anti-tutor activity research, performed the statistical analysis and helped to draft the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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