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Results

Morphological identification

On PDA medium, the colonies were round with neat edges. They were flocculent in the early stages and powdery in the later stages. The colonies were light yellow on the front and the back. Aerial hyphae and vegetative hyphae were present (Figure 1). Based on morphology, the strain was preliminarily identified as mold.

FIGURE 1

Frontal and reverse colony morphology of the strain S1 after culturing on a PDA medium plate at 30◦C for 144 h.

Molecular systematics and phylogenetic analysis

A phylogenetic tree of the S1 sequence was constructed (Figure 2). The sequence for strain S1 (Genbank Accession no. MW188645) had the highest similarity with C. cicadae strain minfu13 (99.82%). Therefore, the strain should be classified as C. cicadae. In combination with the morphology and molecular systematics results, it was determined that the strain was C. cicadae and it was named S1.

FIGURE 2

Phylogenetic tree based on strain S1 sequence.

Single-factor test results

Figures 3A–G shows that when the concentration of sodium selenite was 0.1 g/L, the maximum yield of extracellular polysaccharides in C. cicadae was 3.97 g/L. As the concentration of sodium selenite increased, the polysaccharide yield decreased; therefore, the optimum concentration of sodium selenite was 100 mg/L. Among the five different carbon sources selected, when the carbon source was sucrose, the yield of polysaccharide was the highest (2.95 g/L); this was significantly higher than with the other four carbon sources. Sucrose is composed of the monosaccharides glucose and fructose, both of which are hexoses and can participate directly in the glycolysis and pentose phosphate pathways in the fermentation process of C. cicadae. This improved the carbon conversion efficiency of the reaction process and thereby improved the yield of extracellular polysaccharides (27). However, a further increase in the sucrose concentration inhibited the growth of bacteria. Too much sugar increased bacterial respiration and decreased dissolved oxygen in the fermentation broth, resulting in insufficient oxygen to meet the metabolic demands of the bacteria and affecting the synthesis of metabolites, including polysaccharides (28). Therefore, the optimum carbon source concentration was 8%.

Among the five different nitrogen sources, the highest yield of polysaccharide was 7.42 g/L after the addition of yeast powder, which was significantly higher than with the other nitrogen sources. Yeast powder is an organic nitrogen source, which is more conducive to improving the yield of medicinal fungal polysaccharides than inorganic nitrogen sources. Organic nitrogen sources are rich in proteins, polypeptides, free amino acids, and a small amount of fat, trace elements, and auxin, which are conducive to the growth of bacteria and the synthesis of various metabolites (29). When the concentration of yeast powder was 60 g/L, the maximum yield of polysaccharide was 7.85 g/L. The reason of yeast powder as the best nitrogen source may be that it can provide the necessary factors for microbial growth and can affect those enzyme activities involving the biosynthesis pathway of polysaccharide. However, as the concentration of yeast powder increased, the yield of polysaccharide decreased. This may have been because the higher concentrations of the nitrogen source led to the accumulation of nitrogen in the later stages of fermentation, which inhibited the synthesis of polysaccharides. Therefore, the optimum concentration of the nitrogen source was 60 g/L.

The addition of an appropriate amount of Mg2+ and K+ to the culture medium significantly promoted the yield of extracellular polysaccharide. Figures 3F,G show that when the concentration of Mg2+ was 1 g/L, the concentration of extracellular polysaccharide was the highest (7.81 g/L). As the Mg2+ concentration increased, the yield of extracellular polysaccharide decreased. Therefore, the optimum Mg2+concentration was 1 g/L. K+ significantly promoted the production of extracellular polysaccharide by C. cicadae and the polysaccharide yield was the highest (7.75 g/L) when the concentration of K2HPO4 was 5 g/L. Metal ions can function as enzyme activators during the growth and polysaccharide production of C. cicadae, which is beneficial to bacterial growth and metabolism. However, high concentrations of Mg2+ and K+ are toxic to C. cicadae cells and negatively affect their growth and development.

In summary, the single-factor results for the C. cicadae liquid fermentation medium determined that the optimal conditions are as follows: 0.1 g/L sodium selenite, 80 g/L sucrose, 60 g/L yeast powder, 1 g/L MgSO4·7H2O, and 5 g/L K2 HPO4.

FIGURE 3

The effect of carbon source (A), sucrose concentration (B), nitrogen source (C), yeast powder concentration (D), magnesium ion concentration (E), potassium ion concentration (F), and sodium selenite concentration (G) on C. cicadae polysaccharide yield.

Plackett Burman test results

The Plackett Burman test was used to determine the three factors that had the greatest impact on the polysaccharide yield from the liquid fermentation of C. cicadae. The test results are shown in Supplementary Table S1. Yeast powder and sucrose had a significant effect on the polysaccharide yield (P < 0.01), as did dipotassium hydrogen phosphate (P< 0.05). The order of significance was X2 (yeast powder) > X1 (sucrose) > X3 (K2HPO4). Therefore, the three factors with significant influence—yeast powder (X2), sucrose (X1) and K2HPO4 (X3)—were selected as the model factors for the subsequent response surface analysis.

Response surface analysis

The response surface analysis results of the interaction among sucrose, yeast powder, and potassium hydrogen phosphate are shown in Supplementary Table S2. Minitab17 was used to analyse the results and a linear equation between the polysaccharide content (Y) and sucrose (X1), yeast powder (X2) and K2HPO4 (X3) was obtained: Y = 8.354-−0.019 X1 + 0.017 X2 + 0.047 X3−2.387 X∗1 X1−1.931 X∗2 X2−1.654 X∗3 X3−0.168 X∗1 X2−0.174 X∗1 X3 + 0.303 X∗2 X3(R2 = 0.9724).

A variance analysis was conducted to determine the reliability of this equation, with the results shown in Supplementary Table S3. The model value of F = 19.60 (P < 0.01) indicated that the model had a significant impact on the test results. The mismatch term of F = 0.28 (P > 0.05) had no significant impact on the test results. In the primary term, factor X2 had the greatestinfluence on the test results and the F values were in the order X2 > X1 > X3. X12, X22 and X32 in the secondary term had a significant impact, while no factors in the interactive term had a significant impact, indicating that there was no interaction between the factors. The adjusted model R2 value of 0.9228 indicated that 92.28% of the experimental results could be explained by the model.

This model was applied to predict the optimal medium composition. Taking one of the three factors as the central value and observing the three-dimensional response diagram of the remaining two factors, the influence of each factor on the response value was analyzed. The results are shown in Figures 4A–C. According to the Minitab17 results, when Y was maximal (8.3540 g/L), X1 =0, X2 =0.0101, X3 =0.0101, sucrose = 79.9936 g/L, yeast powder = 60.0058 g/L, and K2HPO4 = 5.0153 g/L. For practical considerations, these values were revised to 80 g/L sucrose, 60 g/L yeast powder, and 5 g/L K2HPO4 for subsequent experiments. The average experimental yield of C. cicadae polysaccharide was 8.0942 g/L, which differed by only 3.1% fromthe predicted value. Therefore, thismodel has practical reference value.

FIGURE 4

Curved surface plot and curved graph: polysaccharide yield, sucrose, and yeast powder (A); polysaccharide yield, dipotassium hydrogen phosphate, and sucrose (B); polysaccharide yield, dipotassium hydrogen phosphate, and yeast powder (C).

Purification and separation of PACI

After extraction of the crude polysaccharides from the fermentation broth by alcohol precipitation, PACI was separated using a DEAE-52 cellulose column and Sephadex G-100 gel column. According to the Figure 5A, there were three main fractions existed in it at the beginning of the purification process. The two fractions were then concentrated and named PACI-1 and PACI-2, respectively. After drying, the amount of PACI-2 was very small so was not studied further.

FIGURE 5

Structural analysis of PACI-1. DEAE-52 anion exchange elution curve (A). G-100 gel elution curve (B). High-performance liquid chromatography spectrum (C). Monosaccharide composition (D). IR spectrum (E). CD spectrum (F). AFM images of PACI-1 (G).

Molecular weight and monosaccharide

composition

The average molecular weight of PACI-1 was determined by HPLC. The HPLC spectrum contained a single symmetrical peak (Figure 5C), which indicated that the polysaccharide PACI-1 obtained after separation and purification had high purity and homogeneity. The calibration curve obtained from the dextran standard was log Mw = −0.2422 X + 8.7833 (R2 = 0.9974). According to calculations based on the standard curve, the molecular weight of PACI-1 was 9.95 × 103 Da. PACI-1 was determined to be a homopolysaccharide composed of fructose (Figure 5D).

Infrared spectroscopy

The FT-IR spectrum identified the following structural characteristics of the polysaccharide (Figure 5E). The peak from 3,500 to 3,100cm−1 corresponded to the stretching vibrations of O-H and C-H, which are characteristic absorption peaks of sugars. The peak at 3,430 cm−1 indicated that the polysaccharide had obvious intermolecular hydrogen bonding (30). The peaks between 3,000 and 2,800 cm−1 corresponded to the stretching vibrations of carbohydrate C-H moieties and the absorption peak at 2,938 cm−1 corresponded to stretching vibrations of C-H (31). The absorption peak at 1,379 cm−1 corresponded to the deformation of =CH2 and the peak at 1,353 cm−1 corresponded to C-H bending vibrations. The absorption peak at 1,066 cm−1 may have been due to the overlap of ring vibrations with the tensile vibrations of the C-OHside group and vibrations of the C-O-C glycosidic bond, indicating the presence of pyranose (32). Additionally, the stretching vibration of the pyran skeleton was also detected at 600 cm−1 and the peak at 870 cm−1 indicated the presence of b-glycosidic bonds (33).

Circular dichroism

The CD spectrum of PACI-1 comprised a large positive peak at 195nm with a positive cotton effect, which was due to the presence of C-O and O-H groups in the polysaccharide molecule (Figure 5F). There was a negative cotton effect at 240nm. The presence of both positive and negative cotton peaks between 190 and 250nm was indicative of molecular asymmetry; that is, PACI-1 could readily form curls, folds, and three-strand spiral structures in aqueous solution (34).

Atomic force microscopy

AFM images were obtained to determine the surface morphology and roughness of PACI-1. In AFM, as the height is not affected by the “widening effect,” only the length and width change at differentmeasurement positions. Therefore, the height can be used as a reference to determine the true diameters of molecules. The height of PACI-1 was about 88.68nm, much higher than 15–50nm, indicating that PACI-1 had a threestrand helical structure (35). This was consistent with the CD results. Additionally, the height of PACI-1 wasmuch higher than that of monosaccharide chains without selenium modification (0.1–1.0nm) (36).

As seen in Figure 5E, PACI-1 had an irregular polymer particle morphology. No irregular linear molecular chain conformations were observed. This phenomenon indicated that there was cross-linking of the PACI-1 polysaccharide chains to form polymer particles. This may have been due to van der Waals forces between the polysaccharide chains or hydrogen bond interactions between the molecules, causing the polysaccharide chains to intertwine and form a spherical structure (34).

NMR spectroscopy

1H NMR can be used to determine the configuration of glycosidic bonds in polysaccharides (Figure 6). Generally, the a proton signal of a-type glycosidic bonds is at 5–6 ppm and the b proton signal of b-type glycosidic bonds is at 3–5 ppm (37). The 1HNMR spectrum contained a peak at 4.89 ppm, indicating that PACI-1 may contain b-type glycosidic bonds. Additionally, the main heterocephalic carbon proton signal of CPA-1 was at 3–4 ppm, indicating that PACI-1mainly contained b-configured pyranoside bonds. This conclusion was the same as that obtained using infrared spectroscopy.

FIGURE 6

1H-NMR spectrum of PACI-1.

In vitro antioxidant activity of PACI-1

The scavenging ability of PACI-1 for DPPH and ABTS free radicals was studied with Vitamin C as the positive control. The DPPH radical scavenging ability of PACI-1 is shown in Figure 7A. The free radical scavenging ability of PACI-1 increased as the polysaccharide concentration increased. At 8 mg/mL, the DPPH scavenging rate of PACI-1 reached 56.46%, which was higher than that of FVR-1 (47.38%), a natural polysaccharide component from Flammulina velutipes (38). As shown in Figure 7B, the scavenging ability of PACI-1 for ABTS free radicals had an obvious concentration dependence as the polysaccharide concentration increased. The IC50 value for the scavenging of ABTS free radicals by PACI-1 was 8.45 mg/mL, which was greater than the ABTS radical scavenging ability of the natural polysaccharide PKP-E-1-1 isolated from Pinus koraiensis (IC50 = 1.52 × 103 mg/mL) (33). These results confirmed the antioxidant capacity of PACI-1, which increased as the concentration of PACI-1 increased.