Prebiotic, Antioxidant, and Immunomodulatory Properties of Acidic Exopolysaccharide From Marine Rhodotorula RY1801

In this study, an extracellular acidic polysaccharide (EAPS) from marine Rhodotorula sp. RY1801 was extracted, and its biological properties were investigated. EAPS is mainly composed of monosaccharides, including mannose, rhamnose, glucose, galactose, and fucose, had an average molecular weight of 5.902 × 107 Da. The results indicated that EAPS can promote the growth of Lactobacillus acidophilus and L. acidophilus plantarum. EAPS is capable of scavenging both superoxide anion and hydroxyl radicals in vitro. The highest scavenging rate of superoxide anion and hydroxyl radicals is 29 and 84%, respectively. Using in vivo model, we found that the EAPS can expand the lifespan and increase the disease resistance of Caenorhabditis elegans against Klebsiella pneumoniae infection via the DAF-2/DAF-16 pathway. These results suggested that EAPS from marine Rhodotorula sp. RY1801 could promote the growth of beneficial bacteria and can be used as an antioxidant and immunomodulator, which had considerable potential in the food and health industry.


INTRODUCTION
The marine Rhodotorula RY1801 (CGMCC 15980) used in this study was isolated from the South Yellow Sea, Yancheng, China by the laboratory in the previous study (14). Bacteria used in this study include Lactobacillus acidophilus CGMCC1.2686, Lactobacillus plantarum ATCC8014, Escherichia coli OP50, and Klebsiella pneumoniae ATCC70063. Rhodotorula RY1801 were grown in Martin Broth at 30 • C. E. coli and K. pneumoniae were grown in Luria-Bertani (LB) broth at 37 • C with 120 rpm for 18-24 h. L. acidophilus and L. plantarum were grown in de Man, Rogosa, and Sharpe Broth (MRS Broth) at 37 • C under anaerobic conditions. For long-term storage, bacteria were preserved at −70 • C in LB containing 20% (v/v) glycerol.
Caenorhabditis elegans N2 (Bristol) was propagated under standard conditions, synchronized by hypochlorite bleaching, and cultured on nematode growth medium (NGM) at 16-20 • C using E. coli strain OP50 as a standard food source.

Materials and Chemicals
The Martin Broth, MRS, and NB mediums were purchased from Aoboxing Bio-Tech (Beijing, China). The Trizol Reagent was obtained from Thermo Fisher Scientific (Waltham, MA, USA). The One-Step TB Green PrimeScript TM RT-PCR Kit (Perfect Real-Time) was bought from TaKaRa (Shiga, Japan). The pyrogallol was purchased from Sangon Biotech (Shanghai, China) and the sodium salicylate from Sinopharm Chemical Reagent (Shanghai, China). Diethylaminoethyl (DEAE)-cellulose 52 and Sephadex G-200 were purchased from Fujian Phygene Biological Technology Co., Ltd. (Fujian, China).

Extraction, Purification, and Characteristics of EAPS
Crude polysaccharides were extracted via previously reported methods (11). The fermentation broth was collected by centrifugation at 10,000 × g for 10 min. The cell-free supernatants were mixed with four volumes of 95% ethanol, stirred vigorously, and kept overnight at 4 • C, and then centrifuged (10,000 × g, 10 min). The precipitate was dissolved in distilled water, and deproteinated by the Sevag method (chloroform: N-butanol at a 4:1) (15), followed by concentration, dialysis (cut-off 3,500 Da), and lyophilization. The crude EPS were obtained. The crude EPS was then purified using an anion exchange chromatography column (1.6 × 30 cm) with DEAE-cellulose 52 (11). The neutral fraction was first eluted with distilled water at 1 ml/min and then the acidic fractions were eluted with linear NaCl gradients (0-0.4 M) at 1 ml/min. The eluates were collected by an automatic collector (2 ml/tube). Collected fractions eluted with distilled water were obtained as extracellular neutral polysaccharides (ENPS). The fractions eluted with NaCl solution were obtained as an EAPS. The EAPS was then purified using a size exclusion chromatography column (1.5 × 60 cm) with Sephadex-G200. Gradient elution was conducted with distilled water at a flow rate of 0.3 ml/min, and the eluates were collected 1.5 ml/tube by an automatic collector. Collected fractions eluted with distilled water were concentrated and freeze-dried to obtain higher purity polysaccharides. The phenol-sulfuric acid method was used to determine the total sugar content of the eluent (16).
The molecular weights of EAPS were determined on highperformance gel permeation chromatography (HPGPC) via an Agilent-1200 high-performance liquid chromatographic (HPLC) system matched with a TSK gel G4000 PWxl column (7.8 × 300 mm), column temperature 30 • C, and detected using a differential refraction index detector (RID) at 35 • C as described by Zhang et al. (17).

Prebiotic Effect of EAPS
We used acid polysaccharide EAPS to investigate its growthpromoting effects on L. acidophilus and L. plantarum (19). Basic MRS containing different concentrations of fructooligosaccharides (FOS) (Hefei Bomei Biotechnology Co., Hefei, China) and EAPS were used to test the prebiotic activity. About 1 × 10 6 CFU/ml bacterial culture was added to the medium and incubated at 37 • C for 48 h. The bacterial count was determined using the plate counting method, three independent experiments were performed, and the bacteria count was rescored as log 10 CFU/ml.

Hydroxyl Radical Scavenging Activity
The hydroxyl radical scavenging activities of EAPS were determined according to the method by Wang et al. (20) with slight modification. About 1 ml of polysaccharide solution was mixed with 0.5 ml 1.5 mM FeSO 4 , 0.35 ml 6 mM H 2 O 2 , 0.15 ml 20 mM sodium salicylate, and then incubated at 37 • C for 1 h. Then, the A 510 value was detected, and the hydroxyl radical scavenging ability was calculated following the formula: where A 1 is the absorbance of the hydroxylated salicylate complex with the test sample, and A 0 is with replacement distilled water. Ascorbic acid was used as a positive control in the same concentrations as that of the sample.

Superoxide Anion Radical Scavenging Activity
We used the pyrogallol autoxidation method to determine the superoxide anion radical scavenging ability of EAPS (21 ) × 100% Ascorbic acid was used as a positive control in the same concentrations as that of the sample.

Determination of Mean Lifespan of C. elegans
The influence of EAPS on the longevity of C. elegans was determined as described previously by Yaguchi et al. (22). Sixty synchronized nematodes were transferred to NGM plates spread with E. coli OP50 containing different concentrations of EAPS and 5-fluorodeoxyuridine and cultured at 20 • C. The number of nematode survivors was counted daily, and the survival rate was calculated until all nematodes died.

Resistance Against K. pneumoniae Infection
The protective effect of EAPS on K. pneumoniae infection was tested (23). Plates with different concentrations of 0.6 mg/ml EAPS and 5-fluorodeoxyuridine were prepared by spreading 100 µl of overnight cultured K. pneumoniae ATCC70063. The plates

Gene
Primer Sequence were incubated at 37 • C for 18-24 h to grow the bacterial lawn. E. coli OP50 plates were used as the negative control. Twenty synchronized L4 nematodes were transferred to each plate and incubated at 20 • C, the number of worms was recorded for 96 h by visual observation of plates under a microscope. Each group had three replicates.

RNA Isolation and Quantitative Real-Time PCR
Nematodes fed with E. coli OP50 or K. pneumoniae were harvested and washed with sterile M9 solution two times, and total mRNA was isolated using the TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The primer sequences used in this study are shown in Table 1 (24). The quantitative real-time PCR (qRT-PCR) was performed using the Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) with the One-Step TB Green PrimeScript TM RT-PCR Kit (TaKaRa Shiga, Japan), the reverse transcription portion was 42 • C, 5 min, 95 • C, 10 s, and then the PCRs were carried out following conditions: 95 • C at 5 s, 60 • C at 34 s and cycled 40 times. Relative expression levels were calculated using the 2 − CT threshold cycle method. The control gene act-2 was used to normalize the expression data of genes.

Statistical Analysis
The experiments were repeated three times. Data analysis and mapping using the Graphpad 8.3 software. Statistical differences between groups were determined using one-way ANOVA. The level of p < 0.05 was considered to be statistically significant, and the level of p < 0.01 was considered to be statistically extremely significant.

Isolation, Purification, and Characterization of EAPS
The extracellular polysaccharide was obtained from marine Rhodotorula RY1801 by alcohol precipitation, dialysis, and lyophilization. DEAE-cellulose 52 anion exchange chromatography was performed, and one neutral polysaccharide fraction and one acidic polysaccharide fraction were obtained, the EPS in each concentration was shown in Figure 1A. Fractions 3-8 were defined as ENPS, fractions 22-30 as EAPS. We purified EAPS using a Sephadex G200 gel column, and the fractions (10-19 tubes) were collected ( Figure 1B), dialyzed, and lyophilized to obtain the purified EAPS for the following analysis.
The molecular weight of the EAPS was determined by using HPGPC. As shown in Figure 2A, two relatively homogeneous fractions were obtained at the retention time of 49.784 and 58.629 min. The molecular of the dominant component (peak 2) was estimated to be 5.902 × 10 7 Da. In contrast, the molecular of the small peak was estimated to be 2.340 × 10 5 Da. The biological activities of polysaccharides depending on the molecular weight and monosaccharide composition. Previously found that polysaccharides with a molecular weight of about 50-200 kDa have biological activities (25).
The monosaccharide composition of EAPS was analyzed by pre-column derivatization HPLC. It was found that EAPS is mainly composed of mannose, rhamnose, glucose, galactose, and fucose compared with standard spectra (Figure 2B). The molar ratio mannose:rhamnose:glucose:galactose:fucose is 44.9:1.0:3.7:8.0:4.6. Some researchers found that glucose and mannose are the major bioactive components in polysaccharides, which play essential roles in biological activities (26,27). In this study, we found mannose is the main monosaccharide in EPAS. Therefore, the EPAS from marine Rhodotorula RY1801probably expresses some biological activities because of the high composition of mannose.
Several Rhodotorula spp. are capable of producing EPS with different monosaccharide compositions but mainly consist of mannose (27). Rhodotorula minuta has been confirmed as a safe source of EPS (28). Exopolysaccharide from Rhodotorula mucilaginosa CICC 33013 was composed of galactose, arabinose, glucose, and mannose (11). EPS of R. minuta ATCC 10658 consisted of mannose and glucose at a molar ratio of about 1.4:7.7 (29). The compositions of exopolysaccharides from a new cold-adapted R. mucilaginosa sp. GUMS16 are glucose and mannose at molar ratio of 5.7:1 (30). The monosaccharide composition of the exopolysaccharide produced by Rhodotorula acheniorum MC was 92.8% mannose and 7.2% glucose (31). In this study, we found the monosaccharide compositions of marine red yeast Rhodotorula RY1801 EPSs are mainly mannose, rhamnose, glucose, galactose, and fucose.

Prebiotic Effects of EAPS
We used EAPS as supplemental carbon sources to investigate its growth promotion effects on two probiotic stains, L. acidophilus and L. plantarum. EAPS showed prebiotic activity, which can promote the growth of L. acidophilus and L. plantarum (Figures 3A,B). FOS as the positive control also showed a significant promotion of L. acidophilus and L. plantarum growth. Various polysaccharides have been reported to show prebiotic activity, such as rapeseed polysaccharides can promote the growth of Bifidobacterium bifidum, Bifidobacterium adolescentis, L. acidophilus, and Bifidobacterium infantis (32). Soluble polysaccharides of palm kernel cake were found to support the growth performance of L. plantarum ATCC 8014 and L. rhamnosus ATCC 53103 (33). A long linear chain of dextran produced by Weissella cibaria enhanced the relative amount of Prevotella and Bacteroides (34). Polysaccharides isolated from Grateloupia filicina and Eucheuma spinosum can significantly promote the growth of Bifidobacterium (35). Lycium barbarum polysaccharide can support the growth of L. acidophilus and B. longum. After feeding mice with L. barbarum polysaccharide, sequencing of the intestinal flora of mice revealed that the abundance of the phyla Proteobacteria and Firmicutes was increased, which reduced the abundance of the phylum Bacteroidetes (36). In this study, the results demonstrated that EAPS promotes the growth of L. acidophilus and L. plantarum in a dose-dependent manner. These results could be explained by those polysaccharides can be used as a carbon and energy source by the microbial population.

Assay of Antioxidant Activities in vitro of EAPS
Hydroxyl radical is known to be the most harmful of active oxygens to living organisms. It can cause oxidative damage to amino acids, proteins, nucleic acids, and lipids (37). We used the salicylic acid method to determine the scavenging ability of EAPS to hydroxyl radicals. As shown in Figure 4, at 0.2 and 0.4 mg/ml, the scavenging rates of EAPS were 32 and 48%, respectively. As the concentration increased, so did the scavenging rate. The hydroxyl free radical scavenging of EAPS reached a maximum of 84% at a concentration of 1 mg/ml. A similar trend in the percent inhibition was also produced by ascorbic acid as the positive control.
Superoxide anion radical is one of the active oxygen free radicals generated in the human body, which plays an important role in several human diseases, such as lipid peroxidation, carcinogenesis, cancer, etc. In this study, we used the pyrogallol autoxidation method to determine the ability of EAPS to scavenge the superoxide anion radical. As shown in Figure 5, scavenging rates of superoxide anion radical by EAPS were low at concentrations of 0.2 mg/ml (0.5%) and 0.4 mg/ml (1.4%). At the concentration of 0.8 mg/ml, the clearance reached a maximum of 29%. Likewise, ascorbic acid, which was used as a positive control, also shown a concentration-dependent superoxide anion radical activity, and its activity is higher than EAPS.
Usually, the DPPH, OH, and O 2 -scavenging ratios were used to indicate the antioxidant capacity of polysaccharides in vitro. Various polysaccharides from different sources have shown antioxidant capacity (38). In this study, we found that EAPS derived from Rhodotorula RY1801 showed certain antioxidant capacity in a dose-dependent manner. Polysaccharides from Epimedium acuminatum Franch showed excellent ability to scavenge hydroxyl radicals and superoxide radicals, and it may be related to reactive hydroxyl groups in polysaccharides and uronic acid content, respectively (39). Extracellular and intracellular polysaccharides from Fomitopsis pinicola showed antioxidant activities in a dose-dependent manner (40). In vitro, marine red yeast polysaccharides obtained from Rhodosporidium paludigenum showed weak antioxidant activity compared with Vitamin C (41). Exopolysaccharides produced by L. sanfranciscensis Ls-1001 had high antioxidant activity and had a significant effect on the removal of ABTS free radicals (42). The antioxidant capacity of different polysaccharides mainly depends on their physicochemical features, including the molecular weight and monosaccharide composition (43).

Effects of EAPS on the Lifespan of C. elegans
In this study, we found that the mean lifespan of nematodes in the EAPS 0.6 and 0.8 mg/ml groups was 11.23 ± 0.22 and 11.95 ± 0.20 days, respectively, an increase of 11.19 and 18.32% compared to the control group. As shown in Figure 6, no nematodes died in all groups in the first 2 days. On day 3, two nematodes died in the control group, only one nematode died in the EAPS 0.6 mg/ml group, and no nematode died in the EAPS 0.8 mg/ml group. In the EAPS 0.8 mg/ml group, only till day 5, the nematode began to die. Compared to the other groups, the initial death of EAPS 0.08 mg/ml was delayed by 2 days. On day 11, the survival rate of the control group was only 50%, whereas the other groups were >60%. Over time, all nematodes in the control group died on day 14. All nematodes in the EAPS 0.6 mg/ml group died on day 15. Finally, the group EAPS 0.8 mg/ml had no living nematodes on day 16. Overall, the nematodes of the EAPS 0.8 mg/ml group lived the longest. There have been many previous studies on the effects of various foods on the life of nematodes. These foods include some prebiotics and even water extracts of natural plants (44,45). However, it is rare to see polysaccharides used in life tests of nematodes, especially those derived from microorganisms. In this experiment, the EPSs were derived from the Rhodotorula RY1801 that was independently isolated and  identified by the laboratory. When applied to nematodes, it was found that EAPS showed a certain life extension effect. EAPS showed a certain antioxidant capacity in vitro. In this experiment, the survival rate of nematodes was also improved to some extent, which seems to coincide. The life extension effect of EAPS maybe because of the antioxidant activity and prebiotic activity on gut microbiota which can promote the healthy function of the host intestinal system. The same results were found in other polysaccharides. The lifespan extension effect of polysaccharides from Rehmannia glutinosa may be related to its antioxidant activity (46). Polysaccharides (HWPs) from Chlorella vulgaris significantly increased the lifespan of C. elegans under oxidative stress induced by hydrogen peroxide (47). Some other studies showed that polysaccharides can modulate gut microbiota and then promote the longevity of C. elegans (48,49).

EAPS Attenuate the Pathogenicity of K. pneumoniae in C. elegans
Caenorhabditis elegans provides an excellent model for studying host-bacteria interactions. We detected the protective effect of EAPS on C. elegans against K. pneumoniae infection. EAPS increased the survival of the infected C. elegans in a concentration-dependent manner (Figure 7). Polysaccharides can be used by beneficial bacteria, such as Bifidobacterium and Lactobacillus, and inhibit the growth of pathogenic bacteria. Microbial exopolysaccharides have been shown to stimulate the immune system and increase the disease-resistant of the host (50). A recent study suggests that polysaccharides from Sophora moorcroftiana seeds significantly extend the life span, improved reproduction, and increased the stress resistance and antimicrobial capacity of C. elegans (51).

Relative Gene Expression in C. elegans
The DAF-2/DAF-16 pathway is related to the lifespan regulation of nematodes (52). In this study, we examined the following genes related to nematode immunity and longevity: daf-2, daf-16, dbl-1 pmk-1, sek-1, and sma-3 genes were used to investigate the effect of the EAPS on nematodes. The qPCR results are shown in Figure 8, the expression of daf-16, dbl-1 pmk-1, sek-1, and sma-3 genes in the nematodes was significantly downregulated after K. pneumoniae infection. The relevant genes were re-examined after different concentrations of EAPS-treated infected nematodes, and daf-2 gene expression was extremely significantly downregulated in the EAPS-treated infected nematode group compared to the infected nematode group and control. The daf-16 gene, which was significantly downregulated in infected nematodes, rebounded significantly after treatment with EAPS, particularly in the EAPS 0.8 mg/ml group. Similarly, the sam-3 gene, which is downregulated in infected nematodes, was significantly regressed in the EAPS 0.8 FIGURE 8 | Relative expression of immunity and longevity-related genes in infected nematodes treated with EAPS. All tests were performed in triplicate and repeated at least once, and the results were expressed by their means ± SD. Statistical significance of the treatment effects was determined by Duncan's multiple range t-test (**p < 0.01 and ***p < 0.001). mg/ml group. But the expression of the other three genes, sek-1, pmk-1, and dbl-1, was further suppressed after treatment with EAPS.
The nematode C. elegans is now a major model organism in biology research, such as lifespan, bacterial infection, and host innate immune. Although C. elegans may not much resemble humans, the molecules that regulate the immune response in these two organisms prove to be quite similar (53). Nematodes lack adaptive immunity and rely on innate immunity to respond to external threats, mainly including four conserved immune signaling pathways (54): (1) transforming growth factor (TGF)-β pathway; (2) the p38 mitogen-activated protein kinase (MAPK) pathway; (3) programmed cell death pathway; and (4) DAF-2/DAF-16 pathway. In the TGF-β pathway, dbl-1 is responsible for encoding the TGF-b receptor. The combination of the two causes phosphorylation of SMAD proteins (composed of sma-2, sma-3, and sma-4) and related antibacterial genes thereby activated (55). Sek-1 and mek-1 are related to the innate immunity of nematodes in the p38 MAPK pathway. They activate pmk-1, but the pathway has not been elucidated. The DAF-16 enhances the resistance to stress in C. elegans, which is important for aging. However, DAF-2 inhibits the production of DAF-16. In this experiment, infected nematodes were treated with EAPS and the daf-16 gene was significantly regressed. Correspondingly, the daf-2 gene was significantly downregulated. These results suggested that disease resistance of nematode is diminished after infection with K. pneumoniae. The nematode resistance was repaired to some extent after treatment with EAPS. It can be hypothesized that EAPS plays a role in the DAF-2/DAF-16 pathway of C. elegans and may increase DAF-16 production by inhibiting DAF-2 expression to increase nematode resistance and prolong life. Besides, treatment with EAPS upregulated the sma-3 gene that was significantly downregulated after nematode infection and was not significantly different from the control group, which may indicate that the antibacterial capacity of C. elegans was restored. However, the previous literature reported differently whether dbl-1, an upstream gene that activates the sma-3 gene in the TGF-β pathway, was further suppressed and whether EAPS is involved in other immune signaling pathways in nematodes and initiates expression of the sma-3 gene. Downregulation of other genes pmk-1 and sek-1 may imply that EAPS does not act through the p38 MAPK pathway.

CONCLUSIONS
In this study, we obtained water-extractable EAPS from marine Rhodotorula sp. RY1801 by DEAE-cellulose 52 anion exchange chromatography and Sephadex G200 chromatography. Results indicated that EAPS showed a growth promotion effect on the growth of L. acidophilus and L. plantarum. EAPS all possessed antioxidant activity in superoxide anion and hydroxyl radicals in vitro, respectively. EAPS could expand the lifespan and increase the disease resistance of C. elegans against K. pneumoniae infection. EAPS from marine Rhodotorula sp. RY1801 was found to be excellent microbial sources of prebiotics, antioxidants, and immunomodulators for health promotion. The EAPS exhibited great potential to be developed into a valuable additive for the food and pharmaceutical industries.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
WC and ZW: conceptualization. YZ: methodology and data curation. ZW: software and writing the original draft preparation and review and editing. ZW and YZ: validation. ZW and YJ: formal analysis. YZ and YJ: investigation. WC: resources, visualization, supervision, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.