Optimisation of the production of a selenium-enriched polysaccharide from Cordyceps cicadae S1 and its structure and antioxidant activity

Wanwan Zhuansun1, Jun Xu2, Hengzhao Liu2, Ying Zhao2, Lulu Chen2, Shufang Shan2, Shiqin Song3,4, Haoyu Zhang1, Tingting Dong1, Huawei Zeng1* and Qinxiang Xu2*

1 Anhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, School of Life Sciences, Huaibei Normal University, Huaibei, China, 2 R&D Center of Anhui Kouzi Distillery Co., Ltd, Huaibei, China, 3 Key Laboratory of Se-enriched Products Development and Quality Control, Ministry of Agriculture and Rural Affairs, Ankang, China, 4 National-Local Joint Engineering Laboratory of Se-enriched Food Development, Ankang R&D Center for Se-enriched Products, Ankang, China

The fermentation medium of a newly identified Cordyceps cicadae S1 was optimized by response surface methodology, with the optimal medium containing sucrose (80 g/L), yeast powder (60 g/L), KH2PO4 (5 g/L), MgSO4·7H2O (1 g/L) and Na2SeO3  (0. 1 g/L). Under these conditions, the extracellular polysaccharide yield was 8.09 g/L. A novel selenium-enriched polysaccharide (PACI-1) was isolated from Cordyceps cicadae, purified and identified as a homofructose polysaccharide with a low average molecular weight of 9.95 × 103  Da. The fine structure of PACI-1  was analyzed   using NMR, CD, and AFM. Additionally, the in vitro antioxidant results showed that the PACI-1 had stronger antioxidant capacity than natural polysaccharides. These results provided a candidate strain for producing selenium polysaccharide and a new polysaccharide from C. cicadae, which showed good antioxidant activity.

KEYWORDS

Cordyceps cicadae,  selenium-enriched, polysaccharide, response surface optimization, antioxidation activity

OPEN ACCESS

EDITED BY

Junxiang Zhu,Qingdao Agricultural University, China

REVIEWED BY

Kai Yang,Zhejiang University of Technology, China 

Xiaolong Ji,Zhengzhou University of Light Industry, China

*CORRESPONDENCE

Huawei Zeng Huaweizeng@163.com Qinxiang Xu xuqinxiang@kouzi.cn

SPECIALTY SECTION

This article was submitted to Nutrition and Food Science Technology, a section of the journal Frontiers in Nutrition

RECEIVED 30 August 2022

ACCEPTED 28 September 2022

PUBLISHED 20 October 2022

CITATION

Zhuansun W, Xu J, Liu H, Zhao Y, Chen L, Shan S, Song S, Zhang H, Dong T, Zeng H and Xu Q (2022) Optimisation of the production of a selenium-enriched polysaccharide from Cordyceps cicadae S1 and its structure and antioxidant activity. Front. Nutr. 9:1032289.

doi: 10.3389/fnut.2022.1032289

COPYRIGHT

© 2022 Zhuansun, Xu, Liu, Zhao, Chen, Shan, Song, Zhang, Dong, Zeng and Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Introduction

Cordyceps cicadae, which belongs to the Clavicipitaceous family and Paecilomyces genus, is an entomogenous fungus (1). This medicinal fungus produces numerous primary metabolites in its fermentation liquid, including polysaccharides, proteins, nucleotides, adenosine, and ergosterol (2–4). Polysaccharides, the most abundant component in C. cicadae fermentation broth, have good antioxidant activity (5). Polysaccharides have been investigated to fulfill the purpose of raising their uses for antioxidant activity with removing free radicals (6, 7), thereby reducing the risk of ischaemic and cardiovascular diseases, Alzheimer’s disease, cancer, Parkinson’s disease etc. (8).

Selenium is an essential trace element for humans and animals. It can only be obtained from food and cannot be synthesized independently by the body. The daily recommended intake for adults is 60 µg (9). Selenium is a cofactor of more than 30 enzymes and a key component of selenoproteins. Selenium affects the functions of several body systems, including the central nervous system, endocrine system, immune system, and cardiovascular system  (10).  In  the  body, organic selenium compounds enhance immunity, exert anti-aging effects, prevent cardiovascular and cerebrovascular diseases, and inhibit cancer cell metastasis (11, 12). Today, billions of people worldwide live in selenium-deficient areas, mainly in China, New Zealand, and Europe (13). Accordingly, there has been increasing research attention on selenium compounds. Because inorganic selenium compounds have the disadvantages of high toxicity and unstable biological activity (14), obtaining high-yield organic selenium compounds is a research hotspot.

Selenium polysaccharides are a good selenium supplement as they combine the biological activities of selenium and polysaccharides. It has been reported that fungi can use inorganic selenium sources in the culture medium to convert exopolysaccharides into selenium-enriched exopolysaccharides in the process of liquid fermentation (15). For example, Alvandi added Na2SeO3 to the liquid fermentation medium of Fomes fomentarius to produce extracellular selenium polysaccharide. The modified extracellular selenium polysaccharide showed significant antioxidant activity and good antibacterial activity (16). In the present study, the medicinal fungus C. cicadae was taken as the research subject. Sodium selenite was added to its fermentation medium and the biotransformation  method was used to synthesize  selenium  polysaccharide  with  the  aim of obtaining a high-yield organic selenium  compound. The experiment was divided into two stages. First, the components of the selenium-containing C. cicadae medium were optimized by response surface methodology  to  obtain the maximum yield of selenium polysaccharide. Second, the polysaccharide in  the  fermentation  medium  was  isolated  and purified and its structure was determined by high- performance liquid chromatography (HPLC), atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FT-IR), circular dichroism (CD), and nuclear magnetic resonance spectroscopy (NMR). Furthermore, its biological activity was studied, and the relationship between its structure and biological activity was preliminarily analyzed.

Materials and methods

Materials

The Cordyceps cicadae S1 strain was screened and collected in the laboratory. Potato dextrose agar (PDA) was purchased from Best Biotechnology Co., Ltd. (Hangzhou, China); 1,1- diphenyl-2-pyridyl hydrazide (DPPH) and 2,2’-diazo-bis-3- ethylbenzothiazoline-6-sulphonic acid (ABTS) were purchased from Zhiji Biotechnology Co., Ltd. (Shanghai, China). The dialysis membranes, DEAE-52 cellulose anion exchange column, and Sephadex G-100 were purchased from Shanghai Qite Analytical Instrument Co., Ltd. (Shanghai, China). Other chemical reagents  and  solvents  were  of  analytical  grade  and provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Identification of strains and determination of optimal medium conditions

Identification of strains 

The S1 strain  was  isolated  from  Cordyceps  cicadae  in the laboratory and inoculated  into  PDA  medium  by  the  plate scribing method. C. cicadae was cultured in a 28◦C incubator for 5 days and the colony morphology was observed. DNA extraction, PCR amplification, fragment  purification,  and sequencing were conducted as previously reported (17). The  obtained  sequences  were  compared  by  BLAST  and  the phylogenetic tree was constructed using MEGA-X. The phylogenetic relationship between the  isolated  strains  and  the near-source strains registered in the  GenBank  database was analyzed.

Microbial cultivation

After 5 days of culture, the strain was used as the seed    and inoculated into the seed  culture  medium  (glucose  50  g/L, maltose 50 g/L, yeast powder 3 g/L, K2HPO4 3 g/L, MgSO4·7H2O 0.2 g/L and pH 5.5), then cultured at 28◦C with shaking (180 rpm) for 3 days. The cultured seed was inoculated into the liquid fermentation medium (fructose 20 g/L, yeast powder  20  g/L,  K2HPO4  1  g/L,  MgSO4·7H2O  3  g/L,  sodium selenite  0.05  g/L) at 10%  (v/v).  The liquid  fermentation was carried out at 28◦C and 180 rpm for 3 days.

Extraction and characterization of polysaccharides

The polysaccharide in the fermentation broth was extracted according to the method published by Qiao et al. (18). The carbohydrate content was determined by the phenol–sulfuric acid method with glucose as the standard. (19).

One-factor-at-a-time  experiments

The fructose (carbon source) in the initial fermentation medium was successively replaced with glucose, maltose, lactose, and sucrose. The yeast powder (nitrogen source) in the initial fermentation medium was successively replaced with potassium nitrate, ammonium sulfate, beef extract, and peptone. The initial concentrations of both the carbon and nitrogen sources were 20 g/L. The carbon source and nitrogen source were tested at 20, 40, 60, 80, and 100 g/L. The concentration of MgSO4·7H2O was tested at 0, 1, 2, and 3 g/L. The concentration of K2HPO4 was tested at 0, 1, 3, 5, 7, and 9 g/L. The concentration of sodium selenite was tested at 0, 0.025, 0.050, 0.075, 0.10, and 0.125 g/L. Subsequently, the seed liquid was inoculated into the designed fermentation medium and cultured for 3 d under each set of fermentation conditions. Determination of the best condition for each tested parameters using the content of extracellular polysaccharide as an indicator.

Plackett-Burman (P-B) design

In the single-factor test, the influences of the concentration of sucrose, yeast powder, dipotassium hydrogen phosphate, potassium ions and magnesium ions were determined. Positive (+1) and negative (−1) levels of these five influencing factors were trialed, with the positive level of  each  factor  being  twice the negative level. The Plackett-Burman test was created in Minitab17 and repeated three times to determine the significance of each factor on the culture conditions (20).

Box-Behnken design

Based on the results of the P-B experiment, taking sucrose (X1), yeast powder (X2), and K2HPO4 (X3) as the independent variables and the yield of C. cicadae as response value (Y), response surface optimisation modeling was performed. The design of each factor level is shown in Table 1.

Extraction and purification of extracellular polysaccharide from C. cicadae S1

 The method of Xu et al. (21) was used with modification. 1,000 mL of C. cicadae fermentation broth was concentrated   to 200 mL at 55◦C using a rotary evaporator (Yarong re-  2000b, Shanghai, China), then precipitated with absolute ethanol (600 mL) at 4◦C for 12 h and centrifuged at 4,000 rpm for     10 min to collect the precipitate. The precipitate was dissolved in 100 mL of ultrapure water and the protein was removed from the solution using Sevag reagent (1-butanol/trichloromethane, 1:4 v/v). The remaining polysaccharide solution was dialysed using a 3,500 Da dialysis bag for 48 h and then precipitated with three times the volume of absolute ethanol for 12 h. The precipitate was freeze-dried in a freeze dryer (BoYiKang FD-1A-50, Beijing, China) to obtain the crude polysaccharide (PACI). 

PACI was redissolved in pure water to a final concentration of 0.1 g/mL and then purified using a DEAE-52 column (2.6 × 30 cm) which was equilibrated with pure water. Then, 3 mL of the PACI solution was loaded onto the column, which was eluted with pure water and a step gradient of 0.1 to 0.3 M sodium chloride at a flow rate of 0.5 mL/min. Eluent (6 mL) was automatically collected in each tube. Trace detection was performed using the phenol-sulfuric acid method. PACI was divided into two peaks and the main fractions of each peak were freeze-dried to obtain a solid powder. A solution of the main fractions was purified using a Sephadex G-100 column (2.6 × 50 cm) with pure water as the eluent at a flow rate of 0.5 mL/min. Then, the main fraction was filtered using 8,000 Da molecular mass membranes to desalt. The quantity of polysaccharide was determined by the phenol-sulfuric acid method. After freeze- drying, one fraction (PACI-1) was obtained.

Structural analysis of PACI-1

Molecular weight (Mw) determination

The uniformity and molecular weight of PACI-1 were determined by HPLC (Agilent 1260 series, Agilent Technologies, USA) with an evaporative light-scattering detector (ELSD) and ultrahydrogel 250 column (7.8 × 300 nm, Waters Corp., USA). The injection volume was 20 µL of 2 mg/mL sample solution, the mobile phase was ultrapure water, the flow rate was 1 mL/min, and the column temperature was 35◦C. The T-series dextran standard was used to construct the standard curve, which was used to determine the molecular weight of PACI-1 (3).

Monosaccharide composition analysis

The monosaccharide composition of PACI-1 was determined by HPLC with  PMP  pre-column  derivatisation,  as previously reported (22). PACI-1 (10 mg) was hydrolysed with 2 M trifluoroacetic acid solution at 120◦C for 6 h, and the excess trifluoroacetic acid solution was removed by a rotary evaporator. At 70◦C, the obtained hydrolysate was derived with 0.5 M PMP in methanol and 0.3 M aqueous NaOH solution for 30 min, then neutralized with 0.3 M HCl. The derivatives were separated by HPLC equipped with a ZORBAX eclipse XDB-C18 column  (4.6  ×  250 mm)  and  a  DAD  detector.  The column temperature was 30◦C, the mobile phase was acetonitrile and ammonium acetate, and the flow rate was 1.0 mL/min. FT-IR spectroscopy Dried PACI-1 (1 mg) was mixed with 100 mg of dried KBr and pressed into a disk. FT-IR spectra were recorded in the range of 4,000–500 cm−1 (Spectrum 100 FT-IR, Thermo Fisher Scientific, USA) (23).

Circular dichroism (CD)

The CD spectrum (J-815, JASCO, Japan) of 0.5 mg/mL polysaccharide was recorded with a 1 cm path length. The CD spectrum accumulated at a rate of 50  nm/min  from  190 to 400 nm. The bandwidth was 2.5 nm in this range (24).

Molecular morphology observation by AFM

Atomic force microscopy (AFM) was employed to observe the molecular morphology of PACI-1. PACI-1 was dissolved in pure water (1 × 10−3 mg/mL), dried on freshly cleaved mica in a dryer for 2 h and then observed using AFM (Hitachi High-Tech company, China) (25).

NMR spectroscopy

The PACI-1 (20 mg) were re-dissolved in 0.5 mL D2O, and then trans- ferred into 5 mm NMR tube for testing. The analysis was performed using an NMR analyzer. (VANCE-600, Bruker Inc., Rheinstetten, Germany)(23).

Antioxidant activity of PACI-1

DPPH scavenging activity

The DPPH radical scavenging ability of PACI-1 was determined using the method of Sharma et al. (26) with modification. 2 mL DPPH was added to PACI-1 solutions (2 mL) of different concentrations (2, 4, 6, 8, 10 mg/mL). After reaction in the dark for 30 min, the absorbance value (Aj) was measured at 517 nm. The DPPH free radical solution was replaced with absolute ethanol to determine Ai and the PACI-1 solution was replaced with absolute ethanol to determine Ac. Ascorbic acid was used as the positive control. Each test was performed in triplicate. The equation to calculate DPPH radical scavenging capacity is:

Scavenging rate (%) = [1 − (Aj × Ai)/Ac] × 100

ABTS scavenging activity

The ABTS radical scavenging activity of PACI-1 was determined according to the method of Zeng (8). ABTS (4 mL) and potassium persulphate solution were added to PACI-1 sample solutions (400 µL) of different concentrations (1, 2, 4, 6, 8, 10 mg/mL). The mixture was shaken evenly and allowed to react at room temperature for 6 min. Then, the absorbance was measured at 734 nm to determine A0. Distilled water was used as the blank and ascorbic acid was used as the positive control to determine A1. Each test was performed in triplicate.

The equation to calculate ABTS radical scavenging capacity is:

Scavenging rate (%) = (A0 × A1) /A1 × 100

Statistical analysis

All results are expressed as the mean value of at least three replicates ± standard deviation (S.D.). The data obtained were subjected to One-way analysis of variance (ANOVA) using IBM SPSS Statistics 21. A p-value of < 0.05 was considered statistically significant.