Xiao-Cui Liuab,
Hongran Liacd,
Tong Kangacd,
Zhen-Yuan Zhu*acd,
Ying-Liang Liue,
Hui-Qing Sunacd and
Li-chao Panacd
aState Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin 300457, PR China. E-mail: zhyuanzhu@tust.edu.cn; Fax: +86 22 60601437; Tel: +86 22 60601437
bKey Laboratory of Food Bio-technology, School of Food and Bioengineering, Xihua University, Chengdu 610039, P. R. China
cKey Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, PR China
dCollege of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China
eSchool of Life Sciences, Guizhou Normal University, Guiyang, Guizhou 550001, China
First published on 10th June 2019
This study investigates the effect of fermentation conditions on the structure and anti-tumor activity of intracellular polysaccharides (IPS) of Cordyceps gunnii (C. gunnii) in submerged fermentation. The environmental and nutritional conditions are determined in a shaker flask by a single factor test. The inhibition of IPS on S180 cells was as an optimization index. The results show that the optimal fermentation conditions of C. gunnii are an initial pH value of 6, a temperature of 25 °C, a rotation speed of 150 rpm, 4% glucose, and 1.0% peptone. Under these conditions, the macro molecular weight (Mw) polysaccharide content and anti-tumor activity of IPS are significantly higher than that in the basal culture medium. GC, HPGPC, periodate oxidation-Smith degradation, NMR, and FT-IR determine the structural characteristics of CPS-JC and CPS-YH (pure IPS cultured in basal culture medium and optimal culture medium, respectively). The results indicate that CPS-JC is mainly composed of α-D-glucans, whereas CPS-YH primarily contain α-D-glucans with a trace amount of β-D-glucans.
Since the fruiting bodies of wild C. gunnii are rare and expensive, an effective way to obtain large quantities of polysaccharides from Cordyceps is from the mycelium via liquid submerged fermentation.17,18 Fermentation is a useful way to produce bioactive substances with health-promoting properties. The fermentation conditions are crucial to the production of bioactive materials since they are directly related to secondary metabolite biosynthesis and cell proliferation.19,20 Moreover, the fermentation conditions affect the activity of metabolites. Lin and Chiang reported that C. militaris samples cultured in Radix astragali displayed higher anti-tumor activity than those cultured in the synthetic medium.21 These results proved that the fermentation conditions played a significant role in the anti-tumor benefits of polysaccharides, and have an essential effect on structural variability. The research results of Kai et al. indicated that the monosaccharide composition of hetero-polysaccharides was highly dependent on the type of monosaccharide obtained from the fermentation medium.22 However, no notable studies currently exist involving the relationship between fermentation conditions, anti-tumor activity, and the structure of C. gunnii polysaccharides subjected to submerged.
Sarcoma 180 (S180) cell line is commonly used to establish the model of transplanted tumor in mice, because it lacks major histocompatibility complex molecules and is not easy to be rejected by recipient mice. And there is no specific requirement for the recipient mouse species. In addition, S180 ascites sarcoma cells are easier to subculture, and have higher survival rate. Therefore, researchers usually select S180 cells to investigate the anti-tumor activity of some substances from medicinal fungi. Shin et al. reported that the fruiting bodies polysaccharide of Paecilomyces japonica, a new type of Cordyceps spp., had antitumor activity on the S180 tumor cells.23 Huang et al. reported that the water-soluble polysaccharides (Pi-PCM0 to Pi-PCM2) from Poria cocos all exhibited anti-tumor activities in vivo (Sarcoma 180 solid tumor implanted 1 in BALB/c mice).24 Our previous research showed that polysaccharides extracted by different methods from C. gunnii mycelia have significant inhibition ability toward S180 sarcoma cells.25 MTT assay is widely used to detect the promoting cell proliferative activity and cytotoxicity of some natural active ingredients. This method is simple to operate, safe to use, low cost, and can achieve high detection sensitivity by controlling experimental conditions. Wei et al. reported that polysaccharide isolated from Dendrobium officinale had anti-tumor activity on HepG-2 cells by MTT assay.26 The growth inhibitory rates of the crude polysaccharides to human gastric cancer cells were evaluated in vitro by MTT assay.3 The polysaccharide of strain FK1 was extracted from the mycelium free supernatant by boiling water method and evaluated for antitoxicity effect on two human cancer cell lines: HeLa cell line and lymphoblastoid cell line (LCL) by MTT method.27 The antitumor activities of Ganoderma lucidum polysaccharide to the human breast cancer cell (MDA-MB-231) in vitro was evaluated by MTT assay.16 Hence, in this paper we took the S180 ascites sarcoma cells and MTT assay to investigate the anti-tumor activity of polysaccharide from mycelium of C. gunnii in vitro. Different analysis types to determine anti-tumor activity such as morphological, genetic or molecular analysis need be further studied.
This study utilizes different environmental and nutritional conditions to cultivate C. gunnii and investigate the anti-tumor activity of the fermentation products. The purpose is to obtain the optimal culture medium suitable for the formation of polysaccharides possessing higher anti-tumor activity. Furthermore, the structural characteristics of CPS-JC and CPS-YH were compared using a combination of chemical and instrumental analysis, such as acid hydrolysis, GC, HPGPC, periodate oxidation-Smith degradation, NMR, and FT-IR.
The standard monosaccharides (D-glucose, D-xylose, D-galactose, L-rhamnose, D-mannose, and D-arabinose), Sephadex G-100 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used in this study were of analytical grade.
The flask culture experiments were conducted in 250 mL flasks containing 100 mL of the basal culture medium (sucrose 40 g L−1, peptone 5 g L−1, KH2PO4 3 g L−1, MgSO4·7H2O 1.5 g L−1, distilled water, initially natural pH) after being inoculated with 8% (v/v) of the seed liquid.
A single factor-screening test was used to investigate each factor of the optimal medium,28 and to ascertain the optimal culture conditions (environmental conditions and nutrition requirements) for determining the IPS displaying the highest anti-tumor activity. First, the anti-tumor activity of IPS was considered as optimization indexes to select the optimal environmental conditions including initial pH value, temperature, and rotation speed. Then, based on these established environmental conditions, the anti-tumor activity of IPS was used as an additional indicator to select the optimal nutritional requirements including carbon source types, nitrogen source types, as well as their concentration levels. Finally, the mycelium of C. gunnii was cultured in the basal culture medium and the optimal medium, respectively. The cultured mycelia from one sample were freeze-dried after being separated from the medium and thoroughly rinsed with a large amount of distilled water.29 Moreover, the crude polysaccharides extracted from the two types of mycelium were named JC and YH, respectively.
Group | IPS production (mg/100 mL) | Macro Mw (%) | Medium Mw (%) | Low Mw (%) |
---|---|---|---|---|
a Experiments were performed in triplicate and results were presented as mean ± SD; *p < 0.05, **p < 0.01 vs. maximum IPS production. | ||||
Initial pH | ||||
pH = 5 | 84.01 ± 3.01* | 78.919 | 10.9574 | 10.123 |
pH = 6 | 95.47 ± 2.92 | 82.717 | 5.972 | 11.311 |
pH = 7 | 80.63 ± 4.24* | 72.554 | 13.2682 | 14.178 |
pH = 8 | 66.21 ± 3.96** | 81.747 | 8.476± | 9.786 |
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Temperature | ||||
20 °C | 86.41 ± 4.36* | 84.548 | 6.440 | 9.012 |
25 °C | 97.36 ± 4.35 | 89.910 | 6.560 | 2.529 |
30 °C | 67.08 ± 3.46** | 80.449 | 9.009 | 5.726 |
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Rotation speed | ||||
100 rpm | 89.45 ± 4.37 | 82.956 | 11.0790 | 4.264 |
150 rpm | 90.74 ± 4.21 | 85.312 | 8.197 | 3.246 |
180 rpm | 87.39 ± 3.79 | 73.838 | 9.835 | 8.146 |
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Carbon source types | ||||
Sucrose | 83.25 ± 4.03* | 76.146 | 9.821 | 14.033 |
Glucose | 93.00 ± 6.84 | 77.766 | 12.2883 | 9.737 |
Soluble starch | 81.47 ± 3.97* | 54.546 | 42.1617 | 3.293 |
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Nitrogen source types | ||||
Peptone | 98.87 ± 4.77 | 87.057 | 0 | 12.943 |
Yeast extract powder | 85.59 ± 3.36* | 66.016 | 13.7327 | 20.252 |
NaNO3 | 56.05 ± 4.47** | 73.252 | 17.7811 | 8.967 |
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Carbon source concentrations | ||||
1% | 87.61 ± 4.53* | 66.356 | 19.5361 | 14.108 |
2% | 62.45 ± 3.74** | 66.654 | 20.1321 | 13.214 |
3% | 93.98 ± 4.51* | 54.083 | 16.7526 | 29.166 |
4% | 138.78 ± 5.24 | 71.046 | 14.5347 | 14.420 |
5% | 32.64 ± 3.15** | 65.861 | 19.8571 | 14.281 |
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Nitrogen source concentrations | ||||
0.5% | 67.27 ± 2.46** | 75.104 | 10.425 | 14.471 |
1.0% | 130.41 ± 4.79 | 86.531 | 4.336 | 5.133 |
1.5% | 67.12 ± 2.31** | 77.616 | 3.690 | 17.695 |
2.0% | 58.09 ± 2.36** | 80.019 | 8.140 | 11.841 |
2.5% | 80.51 ± 1.89* | 79.668 | 4.587 | 15.745 |
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Crude IPS | ||||
JC | 99.63 ± 3.16* | 73.070 | 16.253 | 10.677 |
YH | 115.27 ± 4.28 | 82.326 | 15.2471 | 2.427 |
A medium pH level has a seemingly significant impact on cell membrane function, cell morphology and structure, as well as the uptake of various nutrients, and product biosynthesis in all the environmental factors.34 As shown in Table 1, the highest macro Mw polysaccharide of IPS reached 82.717% when the pH value of fermentation medium was 6.0, and the IPS production reached (95.47 ± 2.92 mg/100 mL) and was higher (10–20) mg/100 mL than other samples in the pH group. Additionally, the IPS cultured at pH 6.0 exhibited a higher inhibition ratio at about 60% in the S180 cells at a concentration of 400 μg mL−1 (Fig. 1A). The results indicated that pH 6.0 was the optimal pH value to promote the initial growth of IPS displaying the highest anti-tumor activity.
S180 cells, were exposed to three different fermentation temperatures (20 °C, 25 °C, and 30 °C) in shake flask cultures to evaluate anti-tumor activity (Fig. 1B). There is no significant difference of inhibition ratio between 25 °C and 30 °C at the concentration of 200 μg mL−1 and 400 μg mL−1. However, the IPS obtained a relatively higher inhibition ratio at 25 °C than that at 30 °C for 400 μg mL−1 concentration. At the same culture temperature, macro Mw polysaccharide content and IPS production of C. gunnii both reached the highest level at 89.91% and 97.36 ± 4.35 mg/100 mL, respectively (Table 1). This result corresponded with the findings of existing literature that the optimal cultivation temperature for Cordyceps mycelia in a submerged fermentation culture medium appeared to be 25 °C.21
It is well-known that oxygen supply is closely related to the formation and accumulation of bioactive metabolites in medicinal fungal submerged cultivation processes.35 Rotation speeds of the shaking table can directly affect the oxygen capacity of the fermented liquid. Therefore, the effects of rotation speeds (100 rpm, 150 rpm, and 180 rpm) on the IPS production and activity were also investigated. As shown in Table 1, the IPS production and macro Mw polysaccharide content at a rotation speed of 150 rpm exhibited a relatively higher level than that at the other two rotation speeds. Additionally, anti-tumor activity of IPS reached the highest levels at a rotation speed of 150 rpm for 200 μg mL−1 and 400 μg mL−1 concentrations (Fig. 1C). In comparison with other rotation speeds, 150 rpm was a good candidate for the rotation speed for its suitable dissolved oxygen amount and low machine loss. Therefore, 150 rpm was the best choice of rotation speed.
Carbon sources exerted considerable influence on the production and compositions of polysaccharides in fungi.36 To determine a suitable carbon source for the production of C. gunnii with a higher anti-tumor activity IPS, various carbon sources such as sucrose, glucose, and soluble starch were individually utilized at a concentration of 40 g L−1 in the basal medium. As shown in Table 1, the maximum IPS production (93.00 ± 6.84 mg/100 mL) was obtained by using glucose as the carbon source in the basic medium, and was significantly higher than sucrose and soluble starch (*p < 0.05). This result does not correspond to the nutritional requirements of other species of Cordyceps in submerged cultures. Hsieh et al. reported that sucrose could significantly affect the mycelium growth of C. sinensis.37 The fact that carbon sources improved the production of polysaccharides might be attributed to energy provision for the fungi growth, and the accumulation of polysaccharides. Moreover, the macro Mw polysaccharide content and anti-tumor activity of IPS (Table 1 and Fig. 1D) in the glucose group appeared to be higher than in other carbon source groups. Moreover, the promoting effect was evident in S180 cell growth when soluble starch was used as the carbon source. The reasons might be that the starch was not completely dissolved in the medium. Compared with other carbon sources, glucose was a good candidate for the carbon source due to its ease-of-use and low cost. Therefore, glucose was selected as the primary carbon source for subsequent experiments.
The nitrogen sources are essential factors for mycelium growth and polysaccharide production.38 The results (Table 1) showed that the maximum IPS production (98.87 ± 4.77 mg/100 mL) and macro Mw polysaccharide content 87.057% were obtained by using peptone as a nitrogen source. The effects of nitrogen sources on the anti-tumor activity of IPS in C. gunnii during submerged cultivation are shown in Fig. 1E. IPS cultured in an inorganic nitrogen source (NaNO3) medium exhibited relatively lower anti-tumor activity than the samples cultured in an organic nitrogen source (peptone, yeast extract powder) medium. This result is consistent with previous suggestions that most basidiomycetes prefer complex organic nitrogen sources to promote their growth in submerged cultures,39 and possibly for certain essential amino acid(s) that were barely synthesized from inorganic nitrogen sources in the cultivation of active substances.40 Peptone yielded the best IPS with higher anti-tumor activity compared with all other organic sources.
Additionally, in order to obtain the optimal carbon source and nitrogen source concentration, C. gunnii was cultivated with various levels of glucose (i.e. 1%, 2%, 3%, 4%, and 5%, w/v), and peptone concentrations (i.e. 0.5%, 1.0%, 1.5%, 2.0% and 2.5%, w/v). As shown in Table 1, the highest IPS production and macro Mw polysaccharide content were obtained at 4% glucose (138.78 ± 5.24 mg/100 mL; 71.046%) and 1.0% peptone (130.41 ± 4.79 mg/100 mL; 86.531%), respectively. As shown in Fig. 1F and G, considering the four different concentration levels, IPS exhibited significantly higher anti-tumor activity when the glucose concentration was at 4%, and the nitrogen source concentration was at 1.0%. These results indicated that a higher glucose concentration and a lower peptone concentration were beneficial to the production and anti-tumor activity of IPS. These findings correspond with previous studies that high concentrations of glucose were beneficial to the mycelial formation.41
In this study, the polysaccharide at lower concentrations exhibited proliferative effect on S180 cells. The result was coincided with the report in the literature that extracellular polysaccharide (Pi-PCM0) from Poria cocos mycelia exhibit proliferative effect on human MCF-7 cells at the lower concentration of 50 μg mL−1.42 Peng et al. reported that the heteropolysaccharide coded as EPF1 was obtained from the crude extracellular polysaccharide of Ganoderma tsugae mycelium also exhibit proliferative effect on Sarcoma 180 cells at the lower concentration of 0.005 g L−1.43 It implied that some polysaccharide at lower concentration did not have toxicity to tumor cells, which need to be explained by further research.
Therefore, all the fermentation conditions (including environmental conditions and nutritional conditions) significantly affected the production, Mw distribution, and anti-tumor activity of IPS in C. gunnii in submerged fermentation. Wang et al. reported that the cytokine nitric oxide (NO) can inhibit of a variety of tumors, and polysaccharides with a Mw above 100 kDa could significantly promote the release of NO.44 Therefore, the fermentation conditions influenced the Mw of polysaccharides, and related to the anti-tumor activity. Moreover, the results of this study indicated that polysaccharides with a larger Mw displayed higher anti-tumor activity when they were cultured in different fermentation mediums.
Considering all the available results, the optimal culture process of C. gunnii was determined at an initial pH value of 6, a temperature of 25 °C, a rotation speed of 150 rpm, 4% glucose, and 1.0% peptone. The crude IPS fermented in the optimized medium was defined as YH, while the crude IPS fermented in the basal medium was defined as JC.
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Fig. 3 Thin-Layer Chromatography (TLC) of CPS-JC and CPS-YH (A) after acid hydrolysis; the gas chromatography (GC) of derivatives CPS-JC (B) and CPS-YH (C). |
The monosaccharide compositions of CPS-JC and CPS-YH are shown in Fig. 3B and C, respectively. The results indicated that CPS-JC and CPS-YH were both primarily composed of D-mannose, D-glucose, and D-galactose in the molar ratio of 1.18:
4.39
:
1.30 and 2.37
:
4.56
:
2.23, respectively, with a trace amount of other sugars.
The IR spectra for the CPS-JC and CPS-YH samples are shown in Fig. 4A and B. The broad stretching peaks at 3373 cm−1 (CPS-JC) and 3418 cm−1 (CPS-YH) were ascribed to hydroxyl groups with stretching vibration, while the weak absorption peaks at 2925 cm−1 (CPS-JC) and 2933 cm−1 (CPS-YH) were characteristic of C–H stretching vibration. Additionally, the bands at 1653 cm−1 (CPS-JC) and 1648 cm−1 (CPS-YH) exhibited a strong absorption peak indicative of CO stretching vibration. Therefore, the three bands above were characteristic IR absorption peaks of polysaccharides. Furthermore, strong absorption in the range of 1200–1000 cm−1 was apparent, suggesting that the monosaccharides in CPS-JC and CPS-YH both displayed a pyranose ring.45 The characteristic absorption bands at 854 cm−1 indicated that CPS-JC contained α-glycosidic linkages. However, CPS-YH exhibited no visible characteristic absorption peaks at 850 cm−1. These results suggest that CPS-JC could be a type of alpha-pyran polysaccharide. However, further study is required to establish the exact configuration of CPS-YH.
The results of NMR analysis involving 1H-NMR and 13C-NMR experiments were used to assign the chemical shifts of the sugar residues.46 The 1H NMR spectrum of CPS-JC (Fig. 5A) showed that the anomeric proton resonance exceeded 4.95 ppm, and is attributed to α-pyranose. There were three primary anomeric H at δ5.374–δ4.964 ppm, indicating that CPS-JC was mainly composed of three types of sugar. Moreover, the 13C NMR spectrum of CPS-JC (Fig. 5C) displayed no signal at a low field from 160–180 ppm, which indicated that no uronic acid was present. Furthermore, three anomeric C at δ97–δ106 ppm were evident, and the peak at 99.76 ppm corresponded to C-1 of α-D-pyranose, which was in accordance with the analysis of the IR spectrum. The 1H NMR spectrum (Fig. 5B) and 13C NMR spectrum (Fig. 5D) of CPS-YH displayed similar signals when compared with CPS-JC. However, a weak signal at 106 ppm indicated that β-D-glycosidic linkage might be present in CPS-YH.
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Fig. 5 1H NMR spectra of CPS-JC (A) and CPS-YH (B); 13C NMR spectra of CPS-JC (C) and CPS-YH (D) in D2O; GC chromatograms of the Smith degradation of CPS-JC (E) and CPS-YH (F). |
The periodate oxidation of CPS-JC and CPS-YH consumed 1.96 × 10−4 mol and 2.04 × 10−4 mol of NaIO4, respectively. No formic acid was observed, indicating the absence of 1→6 linked saccharides in the two samples. After Smith degradation analysis of the oxidation in CPS-JC (Fig. 5E) and CPS-YH (Fig. 5F), glycerol and a trace amount of erythritol were found in the degradation products using GC analysis. The molar ratio of glycerol and erythritol was 0.9164:
0.0836 and 0.7686
:
0.2314, respectively in CPS-JC and CPS-YH. The relatively high content of glycerol was indicative of 1→2 linkage content in the main chain or branch. The substantially lower content of erythritol suggested the 1→4 linkages. The results from the periodate oxidation-Smith degradation indicated that 1→4, 1→2 linkages or 1→3 linkages might exist in CPS-JC and CPS-YH. These results implied that different fermentation conditions did not affect the linkage of carbohydrate chains.
Based on the TLC, GC, IR, NMR, and periodate oxidation-Smith degradation results, CPS-JC and CPS-YH might contain 1→4 linked Glcp residues, as well as some 1→2 linked, 1→3 linked Glcp residues. However, CPS-JC only had α-D-configuration, while CPS-YH had both α-D- and β-D-configurations.
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